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The effect of chronic ethanol ingestion on synaptic inhibition in CA1 of the rat

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The effect of chronic ethanol ingestion on synaptic inhibition in CA1 of the rat
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Rogers, Carl J., 1953-
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viii, 244 leaves : ill. ; 28 cm.

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Alcohols ( jstor )
Dendrites ( jstor )
Electrodes ( jstor )
Ethanol ( jstor )
Hippocampus ( jstor )
In vitro fertilization ( jstor )
Interneurons ( jstor )
Pyramidal cells ( jstor )
Rats ( jstor )
Receptors ( jstor )
Alcohol -- Physiological effect ( lcsh )
Dissertations, Academic -- Psychology -- UF
Psychology thesis Ph. D
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 1986.
Bibliography:
Includes bibliographical references (leaves 231-243).
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Also available online.
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Typescript.
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Vita.
Statement of Responsibility:
by Carl J. Rogers.

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THE EFFECT OF CHRONIC ETHANOL INGESTION ON
SYNAPTIC INHIBITION IN CAl OF THE RAT












BY

CARL J. ROGERS


























A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA 1986




















This dissertation is dedicated to my wife, Jane,
and to my parents, Carl F. and Sophie Rogers,
for their unending support.














ACKNOWLEDGEMENTS


The production of this dissertation was made possible by a number of individuals directly and indirectly connected with the experimental work.

Two people, Pat Burnett and Bill Creegan, have been most

instrumental in helping me to get the job done. Pat has helped to smooth the rough edges with my slice work as well as encourage my progress. Bill has been unendingly patient with my continual requests for program modifications and explanations of how he did them. I thank each of you sincerely for your expert help.

Other people have provided help at various times during this long and arduous trek. I thank Larry Ezell, Dot Robinson, Dr. Paul Manis, and Christina King for their time and efforts.

I would especially like to thank Dr. Roger Reep for his insightful thoughts during some of my more challenging moments. His energy and talents are second to none. I also want to thank Dr. Bob Davis for getting me into the hippocampus and supporting me along the way. Dr. and Mrs. Ken Finger have shared their thoughts on a variety of interesting and helpful topics. I thank them for the opportunity to do so.

I would also like to thank Don Walker and Bruce Hunter for their

confidence in my abilities. They have helped to shape this research in many ways. Thanks are also extended to Dr. Chuck Vierck, Dr. Floyd Thompson, Dr. Don Stehouer, and Dr. Marc Branch for their help and encouragement.

iii








Finally, I thank my wife, Jane, for standing with me during the

difficult times and sharing with me the enjoyable moments. She deserves much of the credit for putting this manuscript together as well as for keeping me going.

And, as always, I thank Mom and Dad for their unending support.

We have finally made it!















































iv














TABLE OF CONTENTS


PAGE

ACKNOWLEDGEMENTS...........................iii

ABSTRACT ...............................vii

CHAPTERS

1 GENERAL INTRODUCTION .......................

Foreward.............................1
Behavior............................3
Anatomy.............................5
Physiology...........................6
General Considerations .....................7
The Hippocampal Model and Memory. ...............8
The Normal Human Hippocampal Evidence .. ............9
A Comparison of Hippocampal Deficits with GET Deficits ...10 The Normal Animal Hippocampal Evidence. ...........12
Chronic Ethanol Treatment (CET) and Behavior in Animals .. 14 Summary of Alcohol and Normal Human and Animal Literature. 15 Animal Alcohol Models ......................16
The Normal Hippocampus- -Anatomy and Physiology. ........16
Pharmacology of the Hippocampus. ................27
Rationale and Introduction to Current Experiments .. ......34

2 GENERAL METHODS .........................38

Animals.............................38
Alcohol Diet Regimen......................38
Equipment ...........................40
Basic Procedures........................42
Data Analysis and Interpretation ................44

3 CHARACTERIZATION OF SYNAPTIC INHIBITION IN VIVO .. ... ...48

Introduction..........................48
Methods.............................52
Results.............................57
Discussion...........................73

4 CET AND SYNAPTIC INHIBITION AN IN VLIVO ANALYSIS .. .......87

Introduction..........................87
Methods.............................91
Results.............................95
Discussion...........................131

v







TABLE OF CONTENTS CONTINUED PAGE


5 EFFECT OF GET ON IONTOPHORETIC GABA MEDIATED RESPONSES . 141

Introduction .......... ........................ 141
Methods ............ ........................... .146
Results ............ ........................... .150
Discussion .......... ......................... ..160

6 EFFECT OF CET ON THE IONTOPHORETIC APPLICATION OF BICUCULLINE,
BACLOFEN AND ENKEPHALIN MEDIATED RESPONSES IN CAl ... ...... 168

Introduction .......... ........................ 168
Methods ............ ........................... .170
Results ............ ........................... .173
Discussion .......... ......................... ..205

7 GENERAL DISCUSSION ........ ..................... .219

REFERENCES ............ ............................. .231

BIOGRAPHICAL SKETCH .......... ........................ 244


































vi













Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy


THE EFFECT OF CHRONIC ETHANOL INGESTION ON SYNAPTIC INHIBITION IN CAl OF THE RAT


By


Carl J. Rogers


May 1986


Chairman: Don W. Walker
Major Department: Psychology


Chronic ethanol abuse by humans results in behavioral alterations

as well as anatomical and physiological deficits within the CNS. Animal models of chronic ethanol treatment (CET) using nutritionally controlled diets showed that ethanol itself results in deficits similar to those observed in humans. The hippocampus, a region of the brain implicated in memory processing, has reduced cell populations as well as dendritic spine density after CET. Electrophysiologically, increased population spike amplitudes (PSA) with paired pulse paradigms (PPP) are found without concomitant changes in basic synaptic waveforms suggesting decreased recurrent inhibition (RI). Recently, feedforward inhibition has also been demonstrated in this region.

A nutritionally complete liquid diet containing ethanol or sucrose isocalorically substituted was pair-fed to rats for 20 weeks. Eight weeks after ethanol abstinence, the effects of CET on feedforward and vii








recurrent inhibition were tested using PPP or with four pharmacological agents, gamma-aminobutyric acid (GABA), bicuculline, baclofen, and D-ala(2)-methionine-enkephalinamide (2DA) iontophoretically applied along the dendrites of CAl pyramidal cells.

The PPP with control animals showed that increasing condition pulse current intensity with orthodromic/orthodromic (0/0) stimulation increased the duration of inhibition and always abolished the PSA at short interpulse intervals. Antidromic/orthodromic PPP produced an increased magnitude of inhibition with increasing condition pulse current intensity, however, the PSA was never completely inhibited. These findings suggest an important contribution of feedforward inhibition which, after CET, may be reduced as was RI.

The PPP with in vitro preparations showed CET animals had trends toward increased PSA similar to those found in vivo. Antidromic/ orthodromic results showed significant differences between the two groups suggesting that CET reduced RI.

Responses of CET animals to GABA resulted in trends toward decreased PSA. Bicuculline-mediated PSA were increased after CET suggesting increased GABA receptor sensitivity. Baclofen inhibited PSA but without group differences. Responses to 2DA from CET animals were stimulation site dependent. Stratum radiatum stimulation produced increased PSA whereas stratum oriens stimulation decreased PSA. It is suggested that CET differentially affected the commissural afferents or SO interneurons.

These data continue to reveal detrimental consequences of long term ethanol abuse.

viii














CHAPTER 1
GENERAL INTRODUCTION


Foreword

The consumption of alcohol by individuals in our society and

throughout many regions of the world is a widely accepted and, in many ways, condoned behavior. The effects of acute episodes of alcohol overindulgence are well documented. Increases in response time, errors in judgement and cognitive skill are but a few of the many behavioral effects alcohol imposes upon users. In recent years and months, our federal and state governments have begun to re-assess the costs of alcohol abuse upon society in terms of lost lives and property and to reconsider laws governing the legal responsibilities and consequences of individuals for the behavior they exhibit while under the influence of alcohol. These steps have been taken as a result of increasing alcohol abuse and the concomitant costs to society.

Alcohol abuse is often regarded in terms of single episodic

events. But alcohol abuse is not an issue related only to those periods of active consumption and the behavioral changes that occur within that framework. Instead, and especially from a mental health perspective, alcohol abuse is a lifestyle which carries with it important sociological, psychological and biological components. Although alcohol abuse may begin as a result of sociological, psychological or even biological influences and, in turn, may impart sociological and psychological dysfunction, detrimental biological alterations always occur with prolonged use. These alterations must be better understood in order for

1





2


more effective treatment and preventative programs to be formulated. It is therefore necessary to ask the questions, what are the long term effects of alcohol abuse from behavioral, anatomical, physiological, and biochemical perspectives? Does alcohol permanently alter the way in which we behave, the way in which we think? If a lifestyle of alcohol use and abuse does produce deleterious effects, are there limits beyond which permanent changes occur and, for those individuals who have gone beyond those limits, is there a way to reverse, or at least help to compensate for, those damages? In order to provide effective treatment and to properly educate the public we must know what long term alcohol consumption does and by what means. The cost of this research will surely be minuscule compared to the cost alcohol abuse currently imposes on society in terms of lost cognitive abilities alone. It is estimated that there may be as many as 20 million people in the United States who can be termed problem drinkers (Clark and Midanik, 1982; Malin et al., 1982).

The experiments described herein were designed to provide additional information concerning the effects of long term ethanol abuse on the function of the hippocampus, a vital area of the brain thought to be involved in memory processing. If we can understand why and how ethanol produces deficits in memory function, it may be possible to provide more effective treatment for some of the cognitive dysfunctions of alcoholic patients. Perhaps even more important is the idea that the more substantial the information provided to the general public concerning the effects of ethanol use and abuse, the more likely the public will be to evaluate the consequences of ethanol use in terms of their own physical and mental well-being. Thus, a better understanding of ethanol effects





3


would produce many different avenues of use, from the treatment of the impaired to the willful prevention of the disease itself.



Behavior

The consumption of alcohol in moderate to heavy doses over long periods of time (months to years) has been shown to produce a wide variety of behavioral changes in humans. Long term alcohol consumption does not produce a consistently clear, easily described and universal picture of behavioral, anatomical, or physiological symptomatologies. There may be several avenues of explanation for these inconsistencies of symptomatologies among which are: 1. different genetic populations with different dispositions to the effects of alcohol, 2. the varied and unique individual drinking histories of long term alcohol consumers, and

3. other environmental conditions which are unique such as disease or trauma (Tarter and Alterman, 1984; Parsons and Leber, 1981). Even with the wide variations of symptoms seen in chronic alcohol abusers, reviews of the literature indicate a relatively common set of behavioral and biological alterations (Begleiter et al., 1980; Parsons, 1977; Ron, 1977; Ryan, 1980; Tarter, 1975). These alcohol-induced changes, in turn, provide reasonable indications of which major brain systems may be most severely involved.

The clinical symptoms of long term alcohol abuse include to varying degrees cognitive, memory and motor performance impairments. Deficits have been reported to occur in perceptual, spatial, problem solving and cognitive tasks. However, one of the most persistent and devastating effects of long term ethanol abuse is that of memory loss. Although short term memory is generally reported to be intact, long-term





4


memory processing is often severely impaired. Severe memory deficits are often defined as part of a syndrome named Korsakoff's syndrome (discussed below). Other clinical manifestations of long term alcohol abuse have been associated with nutritional deficits. Brain stem involvement, as evidenced by oculomotor abnormalities, is seen in patients with histories of long term alcohol abuse. However, these abnormalities are often rapidly reversed with thiamine treatment strongly indicating that dietary intake may be disturbed in some patients and thus contribute directly to some of the symptoms observed with alcohol abuse. Long term alcohol exposure also causes irregularities in sleep patterns as well as endocrine function and certain aspects of autonomic nervous system functioning (Kissin, 1979; Wagman and Allan, 1977). It is important to emphasize that long term alcohol abuse has nearly always been shown to produce cognitive impairment and brain damage despite the wide variations found in the symptoms exhibited by alcoholic patients.

One of the most controversial aspects of long term alcohol abuse has centered on the interaction of dietary inadequacies versus direct toxic effects of alcohol on the CNS. This debate has begun to be silenced by carefully controlled human and animal studies where dietary intake has been monitored or controlled. The results of these studies have provided significant evidence that alcohol Per se can produce direct neurotoxic effects. It has been noted that Korsakoff's syndrome has never been found without coincident long-term alcohol abuse (Freund, 1973).

One of the most intensely studied areas of alcohol research has concerned the effect long term alcohol abuse has on memory function. Korsakoff's syndrome represents the most severe form of long term





5


alcohol effects on memory. The major components of Korsakoff's syndrome are: 1. a severe inability to remember current events over periods of time into the future of more than about one minute (anterograde amnesia) beginning with events occurring at the approximate onset of the disease,

2. a limited understanding by the patient of the memory disability and 3. a general state of apathy. Although the ability to remember events prior to the onset of the disease state (retrograde amnesia) is not eliminated, an increasing degradation of memory prior to the onset of the acute phase of the syndrome is seen (Butters and Germak, 1980; Seltzer and Benson, 1974; Marslen-Wilson and Teuber, 1975). Even if alcohol use is eliminated for months or years, memory impairments persist in at least 80 percent of the Korsakoff's syndrome population (Victor et al., 1971).



Anatomy

An increasingly convincing accumulation of neuroanatomical data has been assembled concerning the detrimental effects of long term alcohol consumption on the gross- as well as micro-anatomy of human alcoholics. The most widespread method of gathering human neuroanatomical data has been with computerized tomography scans (CT scans). The results of these procedures have consistently shown in many experiments and with a large sample size that alcoholics of all ages have enlarged cerebral and cerebellar sulci as well as cerebral ventricles. These observations indicate cerebral atrophy (Wilkinson, 1982; Ron, 1977; Ron et al., 1982; Brewer and Perrett, 1971; Cala and Mastaglia, 1981; Epstein et al., 1977; Haug, 1968). Earlier work with pneumoencephalography revealed similar findings. Victor et al. (1971) observed the gross pathology of





6


brains from autopsies of chronic alcoholics and provided a detailed description of brain regions which contained lesions. Among the areas where lesions were more consistently found were the mainmillary bodies, various hypothalamic nuclei, the medial dorsal nucleus of the thalamus, the cerebellum, periventricular regions of the brainstem, the cerebral cortex and the hippocampus. Earlier studies of alcoholic brain material indicated that there was an increased vascularity, a loss of myelin in the cerebral cortex and cerebellum, and a 20 to 40 percent decrease in pyramidal cell number in motor cortex and purkinje cells in the cerebellum (Lynch, 1960). Degenerative changes in the granular layer and dentate nuclei of the cerebellum in alcoholics, a loss of Purkinje cells, neuroglial proliferation and a thickening of blood vessels in alcoholics have been reported after chronic ethanol abuse (cf. Lynch, 1960).



Phys iology

Electrophysiological studies on human alcoholics have relied on the use of electroencephalographic (EEG) and evoked potential recordings. These methodologies have been criticized for their inability to indicate specific underlying brain structure involvement as well as the underlying cellular generators of the recorded potentials. More recent studies, however, in non-alcoholic patients, have increasingly suggested that certain portions of the generated potentials can be isolated to specific underlying structures of the brain. For example, the P300 component of evoked potentials, a commonly used measure in alcoholic patient studies which occurs approximately 300 msecs after stimulus presentation (often a pulse of light), has been convincingly shown to be





7


generated in the hippocampus, an area considered by many to be implicated in memory processing (Begleiter et al., 1980; Halgren et al., 1980).

The evidence from human alcoholics when using EEG recordings

indicated widespread, fast activity when compared to non-alcoholics (Coger et al., 1978). Event related brain potentials have shown that the late P300 components were decreased in amplitude in alcoholics when compared to controls (Begleiter et al., 1980; Porjesz and Begleiter, 1981). The P300 component has been used as an indicator of the evaluation of stimulus significance, "an electrophysiological manifestation of the orienting response" (Begleiter et al., 1980, p. 10). This relation of the P300 component of evoked potentials to stimulus significance is relevant to alcoholics since alcoholics do not differentiate relevant from non-relevant stimuli as do normals (Begleiter et al., 1980). More recently, it has been reported that after ending the consumption of alcohol after long term use, a recovery of the amplitude of the P300 component occurs although never back to control levels (Porjesz and Begleiter, 1981).



General Considerations

A number of problems exist in the above studies from human alcoholics when considering the correlations between variables such as duration of drinking and quantity consumed and the extent of neuropsychological and neuropathological deficits (Parker and Noble, 1980). These variables are not necessarily good predictors of pathology (Ron, 1977). Alcoholics with severe neocortical atrophy may not show appreciable losses in cognitive function while conversely, alcoholics with no grossly apparent





8


CNS damage may exhibit profound psychological impairments (Wilkinson and Garlen, 1980). It is difficult, if not presently impossible, to assume a causal relationship between brain damage and cognitive dysfunction (Parsons and Leber, 1981).

These problems have created an implicit need to better understand and control the relevant variables necessary to obtain reliable relationships between alcohol abuse and brain damage. To do this, better clinical histories of patients would be helpful; however, the accuracy provided can never be completely reliable. An alternative method of gaining a better understanding of the relationships between variables would be to control these variables in the manner of a laboratory experiment. The ethical problems associated with this alternative are numerous. Thus, scientists have developed animal models of chronic alcohol consumption in an effort to determine the effect of alcohol per se on the ONS. Control of variables such as age, drinking history, genetic makeup and nutritional adequacy are then feasible.



The Hippocanwal Model and Memory

Since the most devastating aspect of Korsakoff's syndrome is the profound anterograde amnesia, the study of memory is crucial to an understanding of the alterations which Korsakoff's syndrome produces. Several regions of the CNS have been implicated in memory processing. Of those regions reported to be affected in Korsakoff's syndrome, the temporal stem, the temporal cortex, the medial dorsal thalamus, the mamniillary bodies and the hippocampus have all been considered to be involved in some way with memory processing (Horel, 1978; Victor et al.,





9


1971). Each of these regions would represent a reasonable location in which to begin a search for the effects of Korsakoff's syndrome on memory processing.

The present series of experiments utilize the hippocampus, a

region in which a large amount of literature exists concerning experimental variables related to behavior, anatomy and physiology in the study of memory and other related phenomena. A substantial amount of literature utilizing the hippocampus and the consequent effects of long term ethanol treatment has thus followed.

The hippocampus has emerged as a model system for the study of

memory for several reasons. First, the hippocampus has been strongly implicated in memory processing from human clinical cases (discussed below). Secondly, the hippocampus itself is an important "model" system. It is composed of well-defined cell layers as well as dendritic regions. Inputs and outputs are well characterized and reside within laminae which have little overlap. The anatomical arrangements of the dendrites allow for the summation of electrophysiological potentials which can, in turn, be recorded with relatively simple extracellular recording techniques. Because of the highly laminated structure of the hippocampus, the recorded potentials can be interpreted with a high degree of confidence. It is for all of these reasons that a wealth of literature on the hippocampus exists from different disciplines which further facilitate meaningful interpretation.



The Normal Human Hippocampal Evidence

The evidence for the involvement of the hippocampus in normal human memory emanates from radical procedures used in the mid 1950s to





10


alleviate severe seizure activity in a number of patients (Penfield and Milner, 1958; Scoville and Milner, 1956; Scoville, 1954). Although the seizure activity of these patients was dramatically reduced, severe effects were seen on memory processes. One of these patients, H.M., has been well characterized. Anatomically, the anterior two-thirds of H.M.'s hippocampi were bilaterally removed as well as part of the temporal lobe extending 8 nuns posterior to the temporal poles. The most striking effect of these lesions was an immediate and profound anterograde amnesia. Retrograde amnesia was also found but only for a relatively short (approximately two years) period of time immediately prior to the operation. H.M. could detail his earlier years without problem. Intellectually, H.M.'s intelligence quotient score was not different from normal. H.M.'s short term memory (STM) was also intact. However, he had a profound impairment of long term memory (LTM) including both verbal and non-verbal tasks. Visually and tactually guided mazes were learned only when the number of choices was reduced to be within STM memory span (usually seven to nine choices) and even so H.M. had much more difficulty with tactile mazes (Milner et al., 1968). H.M.'s acquisition performance was worse than normal in these tasks although once learned, he was able to retain the information over long periods of time (Milner et al., 1968).



A Comparison of HiDpocampal Deficits with GET Deficits

A brief comparison of the features of Korsakoff's syndrome with

those of medial temporal resections results in a number of similarities. Both cases exhibit severe anterograde amnesia with both verbal and non-verbal tasks. Pre-lesion (retrograde) memory is intact in both





11


alcoholics and ablated individuals. Korsakoff's syndrome patients tended to exhibit enhanced forgetting when distractor variables were used with STM tasks while H.M., like amnesiacs, showed normal STM decay. Korsakoff's syndrome patients exhibited decreased performance when retrieving information from LTM when an interfering task was included. Hippocampal lesioned patients also appeared to exhibit interference effects although they may be modality specific (DeLuca et al., 1975). These comparisons indicate that although there are some differences in the behavior of these two groups of patients, the similarities are quite strong.

The above similarities are not without important problems. One of the most vexing problems with Korsakoff's syndrome patients is the relatively low correlation found between hippocampal anatomical alterations and the severity of memory problems. Victor et al. (1971) indicated that the hippocampus was bilaterally involved in 36 percent of the cases. The mammillary bodies, however, a structure which receives much of its input from the fornix and which largely emanates from the hippocampus, was much more consistently involved. Still, even the mammillary bodies were not found to be involved in 100 percent of the cases studied. One lesion which has been consistently found in Korsakoff's patients is in the dorsomedial nucleus of the thalamus (Victor et al., 1971). More recently, it has been hypothesized that several different types of amnesia may exist. Although the dorsomedial thalamus may be strongly implicated in the storage, retrieval and encoding of information, the hippocampus may be responsible for specific types of memory processing such as spatial memory (Horel, 1978). Another argument concerning the degree of involvement of these various





12


brain structures reported by Victor et al. (1971) is that they were the result of gross pathological examinations and not histopathological data. Thus, while gross changes were reported, more subtle yet equally devastating consequences of long term alcohol abuse may exist in the microstructure of these and other brain areas. Other investigators have shown the hippocampus to be consistently implicated from autopsy material of human alcoholic patients (Brion, 1969; McLardy, 1975; Miyakawa et al., 1977).

The data presented above suggest that while hippocampal damage may be seen with long term alcohol intake, it is not the most highly implicated structure. However, because the hippocampus has been so thoroughly studied behaviorally, anatomically, and physiologically in the normal animal, it allows for a much more thorough and detailed examination of the effects chronic ethanol treatment may produce than does nearly any other brain structure otherwise thought to be involved in memory processing.



The Normal Animal Hiip~ocampal Evidence

Although the effort to develop an animal model of the human

amnesiac syndrome has been strong, no animal model has yet produced a symptomatology which completely replicates that found with H.M. Instead of performance deficiencies always being found, the animal literature has shown that hippocampectomy can actually produce performance advantages with some tasks. One of the important questions, however, is whether behavioral tasks designed for animal models truly replicate those tests used with human patients. The extent of the hippocampal literature is enormous and has been well reviewed (O'Keefe and Nadel,





13


1978; Douglas, 1967; Isaacson and Pribram, 1975; Seifert, 1984). Therefore, only a brief summary of the more relevant findings is presented below.

The major categories of behavioral testing are summarized as

presented by O'Keefe and Nadel (1978). Hippocampal animals generally exhibited increased reaction to novel stimuli; they are considered hyperactive but at the same time hypoexploratory. Spontaneous alternation studies indicated either random or repetitive behavioral responses. Spatial discrimination after hippocampal lesions proved to be more or less normal as did non-spatial discrimination. However, spatial discrimination reversals were deficient in hippocampal lesioned animals as were non-spatial reversals. Complex maze learning was deficient in nearly all such studies. One way active avoidance tasks were typically normal or showed deficits. Two way active avoidance after hippocampal lesions, however, showed facilitated avoidance. Passive avoidance tests yielded mixed responses, generally responding normal to controls but with occasional deficits on very specific elements of the measures used. Time related tasks such as lever press rates, delayed response, go-no-go, delayed alternation and differential reinforcement of low rates (DRL) with lesioned animals yielded high rates of behavior, hence deficiencies on these tasks. Finally, hippocampal lesioned animals typically showed extinction deficits.

The above results allow for several general observations. These

data indicate that hippocampal lesioned animals can learn but often show deficits in part because of response or strategy perseveration as seen with discrimination reversals or extinction testing tasks. A second point is that tasks which typically rely on spatial or contextual cues





14


are severely affected. This is apparent with maze learning behavior where learning in normal animals takes place quickly. In hippocampal lesioned animals and humans we see that these tasks can be learned but that it takes much longer. It has been suggested that normally an animal uses a spatial-time or contextual coding process for learning these tasks (Hirsch et al., 1978). However, when the hippocampus is damaged, all learning must take place in terms of stimulus-response chains which are more difficult to develop. Finally, it can be seen that tasks which require timing behavior are severely affected. Timing is also thought to be a major aspect of contextual coding. Thus, spatial and time cues produce a context dependent memory. It appears that this memory is severely affected by hippocampal lesions (O'Keefe and Nadel, 1978; Hirsch et al., 1978).



Chronic Ethanol Treatment (CET) and Behavior in Animals

There are many important similarities between the behavioral

changes associated with animals chronically treated with alcohol and animals with hippocampal lesions. Freund (1974) showed that mice, after four months on a liquid diet containing ethanol, were deficient in both the acquisition and retention of a passive avoidance task. One way active avoidance was not affected when tested with hamsters given an ethanol containing diet; however, the ethanol exposure was only for seven days (Harris et al., 1979). Ethanol consuming rats were found to be deficient in the acquisition of Hebb-Williams mazes and in the acquisition and retention of a moving belt task (Fehr et al., 1975). Spontaneous alternation was also shown to be deficient. Tasks requiring timing behavior such as temporal shock discrimination and DRL schedules





15


were impaired (Denoble and Begleiter, 1979; Smith et al., 1979; Walker and Freund, 1973). Temporal single alternation and go-no-go behavioral tasks were also deficient in alcohol treated rats (Walker and Hunter, 1978). Each of these measures of performance is similar to those of the hippocampal lesioned data. Shuttle box avoidance behavior with ethanol treated rats, however, is different from the behavior found with hippocampal lesioned animals. While hippocampal lesions normally facilitate the response to this task, ethanol treatment produced impaired performance (Freund, 1979; Freund and Walker, 1971).



Summary of Alcohol and Normal Human and Animal Literatures

In summary, the similarities found with behavioral testing between Korsakoff's syndrome patients and H.M. appear to be strong. The literatures from hippocampal lesioned animals and CET animals not only provide similarities between hippocampal lesions and CET but also between the human and animal literature. Unfortunately, little detailed human anatomical, electrophysiological or biochemical evidence exists concerning CET effects due to the ethical problems inherent with such human research. Animal studies of CET have, however, begun to provide a wealth of information concerning morphological and functional alterations, especially in the hippocampus and cerebellum. Since the hippocampus, and in particular, the CAl region is important to the studies performed herein, the animal models, anatomy, physiology and pharmacology of the hippocampal CAl region will be considered in the next sections in some detail.





16


Animal Alcohol Models

The use of animal models for chronic alcohol studies brings with it the problem of how to administer the alcohol regimen. Several different methods of alcohol administration have been developed including inhalation (Goldstein and Pal, 1971) and intubation (Majchrowicz, 1975; Pohorecky, 1981; Altshuler, 1981). These methods, however, are not as practical for long term administration as others which require relatively normal feeding procedures. Several animal models do exist which allow ethanol to be administered orally. These methods include the following where ethanol is provided: 1. ad lib as the sole source of fluid with ad lib lab chow also being provided, 2. operant conditioning techniques where an ethanol containing fluid is used as a reward for maintaining drinking behavior (Falk et al., 1972) and 3. an ad lib liquid ethanol-containing diet (Freund, 1969; Lieber and DeCarli, 1973). This liquid diet mixture contains all of the required protein, carbohydrates, vitamin and mineral supplements necessary for healthy development. This last method is currently the most widely used of the animal models (Pohorecky, 1981) and is the method used in the studies completed herein. Each of these methods of alcohol administration has shown that alcohol produces alterations in the behavior, anatomy and electrophysiology of a variety of animals tested.



The Normal HiP~ocampus - Anatomy and Physiology Anatomy of the hippocampus

The hippocampus is a bilateral structure which extends laterally and caudally for several millimeters from a dorsomedial position in the rat brain. It turns ventrally in a wide curve until its ventral tip is





17


extended in a rostral-caudal direction. The hippocampi thus look similar to rams' horns. Each hippocampus consists of two distinct regions, the hippocampus proper or CA region (Cornu Ammonis for its seahorse-like shape) and the dentate gyrus (DG). Each of these areas appears as a C-shaped structure with one facing the other and interlocked. The CA region is further subdivided into four relatively distinct regions (Lorente de No, 1934). The dorsal-most region of the hippocampus and the C-shape is known as CAI (Fig 1-1). It is located next to the subiculum medially and extends laterally to where the hippocampus first turns ventrally. Moving inward on the C-shape is a short CA2 zone followed by a large CA3 region and finally the CA4 area which is located within the two outer blades of the DG. The CA2 zone is generally defined as the zone which distinguishes the regio superior (CAI region) from the regio inferior (CA3 and CA4 regions).

The CA regions consist of several layers. Beginning from the

dorsal surface of the CAI region, the first layer is the alveus, a dense but thin layer of afferent and efferent fibers. Next is the relatively cell-poor molecular layer known as stratum oriens (SO). This layer contains the basilar dendrites of the pyramidal cells. It is followed by a dense but thin layer of pyramidal cells which is called stratum pyramidale (SP). Beneath the pyramidal cell layer is stratum radiatum

(SR) and stratum lacunosum-moleculare (SLM). In the CA3 region of the rat only, an additional laminae is found between the SR and the SP. The apical dendrites of the pyramidal cells project into these last three layers. The majority of afferent fibers to the pyramidal cells project orthogonally to these pyramidal cell dendrites and terminate on them. The afferent fibers which project to CAI originate from several regions





















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20


but especially the ipsilateral and contralateral CA3 regions. These fibers are termed the Schaffer/commissural afferents.

The flow of information through the hippocampus occurs in a

trisynaptic sequence. The major afferent system to the hippocampal complex emanates from the entorhinal cortex and forms the perforant path (or angular bundle). These fibers project onto the granule cells of the DG with a lamellar distribution. The axons of granule cells then project through the hilus to the CA3 pyramidal cells. The axons of CA3 pyramidal cells, in turn, project ipsilaterally as well as contralaterally to the CAI pyramidal cells. The CAI pyramidal cells then project rostrally to the medial septum, anterior thalamus and mammillary bodies and caudally to the subiculum and entorhinal cortex. This last projection actually closes the loop of projections which started in the entorhinal cortex. Each of these connections produces an excitatory influence on the postsynaptic cell (Andersen et al., 1966). A final distinctive feature of the hippocampal circuitry is that each of the major projection cells resides and is preserved within a cross sectional slice or lamellae of the hippocampus (Andersen et al., 1971).

The region of specific interest in the current studies is the CAI pyramidal cell region. The general circuitry has been described above. The microcircuitry within this region is also important and is known to mediate, among other phenomena, the inhibitory control of the pyramidal cell. The pyramidal cell sends its axon through the alveus as described above but also sends an axon collateral to one or more inhibitory interneurons located near or within the pyramidal cell layer. This interneuron, presumably a basket cell, then projects back onto the original as well as a large number of other pyramidal cells thereby





21


inducing relatively widespread inhibition (Andersen et al., 1964a). This feedback type of inhibition is termed recurrent inhibition. Although the definitive circuitry is not yet completely described, feedforward inhibition has been demonstrated in the apical and basilar dendritic regions (Alger et al., 1981; Ashwood et al., 1984; Andersen et al., 1980; Buzsaki, 1984). Several possible anatomical configurations for this circuit are plausible. The best demonstrated configuration, however, is that the Schaffer/commissural afferents terminate on an inhibitory interneuron which resides in SR or SO parallel to the dendrites of the CAI pyramidal cell (Ashwood et al., 1984). These inhibitory interneurons then project to the dendrites of the pyramidal cell (Gall et al., 1981; Schwartzkroin and Mathers, 1978).

The pyramidal cell is the major projection cell of the CAI area. Due to its architectural definition with the afferent fibers being orthogonal to the apical dendrites of these cells, the CAI region offers an exceptional opportunity for the study of both intracellular and extracellular responses. The relative ease of understanding the effects of localized extracellular stimulation and recording in relation to the input and output pathways is unsurpassed elsewhere.



Electrophysiology of the hippocampus: An introduction

Electrophysiological studies of the hippocampus often use

extracellular recording techniques. The ability to use these field potential recordings meaningfully results from the highly organized and relatively homogeneous patterning of the afferent and efferent fibers in this region. The activation of the afferent fibers produces restricted and localized dipole current sources and sinks. When an excitatory





22


synapse is activated, current flows into the cell. This flow of current into the cell is called a current sink. As in any electrical system, current must also flaw out of the system elsewhere. Current will flow out of the cell at what is called the current source and then travel through the intercellular fluid back to the current sink. These current flows can be recorded since a voltage is produced as current flows through the resistive extracellular milieu. Intracellularly, the activation produced at the current sink is seen as a positive voltage. Extracellularly, however, the potential recorded is negative at the sink and positive at the source when stimulating and recording from the same excitatory synaptic region.

In the-CAl region of the hippocampus, the location of the stimulating and recording electrode will affect the characteristic shape of the response. If the stimulating electrode is in SR, a negative-going field response is obtained when recording from the same synaptic region due to the inward flow of current from this excitatory response. If the stimulating electrode is left in SR while the recording electrode is moved into SO in steps, a gradual change in the shape of the response is observed. As the recording electrode approaches the pyramidal cell layer, the response becomes less negative. When the recording electrode passes through the cell layer or thereabout, a response is obtained which is neither positive nor negative in appearance. This point is termed the inversion point and represents the area where the inward flow of current generated by the activation of the apical dendrites equals the outward flow of current. Once the recording electrode is dorsal to the cell layer, the response becomes positive which indicates a current source; current is flowing out of the cell at this point. If the





23


current passed through the stimulating electrode is raised to a level sufficiently high to activate action potentials from a large number of pyramidal cells, a field population spike (PS) becomes superimposed on the field extracellular excitatory post-synaptic potential (EPSP) (Andersen et al., 1971; Lomo, 1971). This PS is seen as a sharp negative going spike (Fig 1-2).

Extracellular recording paradigms in CAl have generally been used to evaluate three relatively distinct kinds of information. The first type of paradigm is designed to test synaptic efficacy and is termed an input/output (1/0) function. This paradigm consists of increasing the stimulation current level in steps in order to determine response characteristics such as EPSP threshold, PS threshold and respective asymptotic levels. The second type of paradigm is designed to test for synaptic plasticity. Briefly, at least two types of response enhancement plasticity are seen in the hippocampus. One is seen as a short lasting increase in the size of the EPSP or PS amplitude in reference to a baseline pulse either with paired pulses or immediately after tetanic stimulation (these phenomena are termed potentiation). This type of potentiation lasts for periods ranging from several milliseconds to seconds (Creager et al., 1980). Another type of potentiation is longer lasting, often minutes to weeks, and is termed long term potentiation. The mechanisms for these two types of potentiation are thought to be different (Dunwiddie and Lynch, 1978). There are several different experimental procedures used to assess or induce these types of potentiation. Paired pulse testing allows for the systematic assessment of inhibition (produced through interneurons) as well as potentiation. In this procedure, two pulses, a condition and a subsequent test pulse, are























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26


successively produced with controlled interpulse intervals (IPI). The second response is typically compared to the first response of the pair or to a baseline control pulse. Two other procedures are also used to measure plasticity; one is frequency potentiation (FP) and the other is long-term potentiation (LTP). FP entails the use of trains of stimulation at specific frequencies generally less than 100 Hz. UP is usually obtained with high frequency (greater than 100 Hz) trains of stimulation. The final type of data collection procedure is designed to examine synaptic distribution. In this procedure, a recording electrode is lowered in discrete steps (25-100 uMs) through the dendritic region of the CAl pyramidal cell usually while stimulating in SR. A profile of the current-sources and sinks can thereby be generated. This technique is called a laminar analysis. Although a laminar analysis can be used to determine current sources and sinks, the resolution obtained is not substantial since current sources and sinks often overlap. If a current source density analysis (CSD) is performed on the laminar profile, a more defined profile can be obtained so that it is theoretically possible to suggest the relative presence, absence and general distribution of synaptic current along the course of the cellular processes (Nicholson and Freeman, 1975). CSD analysis is, in essence, a mathematical transformation of the laminar waveforms which uses the second spatial derivative thus describing rates of change of current flow between recorded levels rather than voltage measures.









Pharmacology of the HiRocaM us 2

Pharmacology of inhibition in the hiRvocampus

The major focus of the experiments in this volume is on the effect of GET on synaptic inhibition. The basic premise for this focus emanates from previous studies suggesting changes in the influence of inhibition in the CAl region of GET animals. The complete details of these findings will be given subsequently. The study of inhibition in CAl of the hippocampus in normal animals has, however, provided an exploding volume of information on different types of inhibition and the underlying pharmacology. This section is designed to provide a brief review of that literature in order to establish a background for the basis of the experiments outlined below.

Recurrent inhibition has been considered to be the dominant type of inhibition in the CAl region (Andersen et al., 1964b). As indicated earlier, this inhibitory circuit consists of the pyramidal cell which projects via axon collaterals to the basket cell. The basket cell, in turn, projects widely to surrounding pyramidal cells as well as to the original "firing" pyramidal cell (Andersen et al., 1964a; Knowles and Schwartzkroin, 1981). An excitatory neurotransmitter, presumably glutamate, is released by the pyramidal cell onto the soma of the inhibitory interneuron (Storm-Mathisen, 1977). The functional effect of recurrent inhibition is profound. If the Schaffer/commissural afferents are stimulated while spontaneous pyramidal cell unit firing is monitored, an initial excitatory response is found followed by a near complete cessation of spontaneous activity for 600 to 700 msecs (Andersen et al., 1964a). With paired pulses, if the second of two pulses is presented within 20 to 80 msecs of the first, the effect of the first





28


pulse is to decrease the amplitude of the population spike on the second or test pulse (in vivo). This decreased PS amplitude is thought to be due to a reduction in the probability that the pyramidal cell and those around it will fire when the second pulse arrives. Paired pulse potentiation overshadows the inhibitory phase after about 80 msecs (Andersen and Lomo, 1970; Dunwiddie et al., 1980). This potentiation phase is thought to be due to a process different from that of inhibition. However, it is also thought that recurrent inhibition may produce a resetting of the fired pyramidal neurons thereby allowing for a more synchronized and simultaneous firing of the pyramidal cells with interpulse periods longer than approximately 80 msecs. This process could account for a portion of the potentiation phase seen with paired pulses (Assaf and Miller, 1978).

More recently, feedforward-like inhibition has begun to be described and characterized in CAl (Alger et al., 1981; Andersen et al., 1978, 1980, 1982). It has been hypothesized that some of the Schaffer/ commissural afferents either terminate on the apical or basilar dendrites of the CAl pyramidal cell and directly release an inhibitory neurotransmitter, gamma-aminobutyric acid (GABA), or more likely, the Schaffer/commissural afferents synapse onto an inhibitory interneuron which, in turn, synapses onto the apical or basilar dendrites or soma of the CAl pyramidal cell (Ashwood et al., 1984; Buzsaki, 1984). The conclusive anatomical evidence for such feedforward inhibitory connections is not yet complete. However, descriptions of interneurons which run parallel to the pyramidal cell apical dendrites do exist in the literature (Gall et al., 1980; Lorente de No, 1934; Schwartzkroin and Mathers, 1978).





29


There are currently at least three major putative neurotransmitter groups which are reported to have major effects on inhibition of the CAI pyramidal cell. These neurotransmitter groups are GABA agonists and antagonists, opiate agonists and antagonists and the benzodiazepines. The following sections provide background for each of these neurotransmitters.



GABA agonists and antagonists and inhibition

Glutamic acid decarboxylase (GAD), the precursor to GABA, and GABA have been found in the hippocampus using immunocytochemical (Barber and Saito, 1976), autoradiographic (Hokfelt and Ljungdahl, 1972) and fluorescence (Wolman, 1971) techniques in a bimodal distribution in CA1. Peaks are seen in the pyramidal cell layer and the molecular layer of SR. Higher levels of GAD are actually seen in stratum lacunosummoleculare than in stratum pyramidale. It has also been shown that if the various afferent pathways to the CAl region are cut, there is no reduction in GAD activity. These data indicate that GAD is not released by the afferent fibers but instead suggest the probable existence of an interneuron (Storm-Mathisen, 1977; Fonnum and Storm-Mathisen, 1978). These findings also suggest the possibility of two populations of GABAergic inhibitory interneurons.

GABA has been demonstrated to mediate recurrent inhibition (cf. Storm-Mathisen, 1977) and feedforward inhibition (Alger and Nicoll, 1980) in the hippocampus. It has been shown in vivo that GABA, applied to the pyramidal cell soma, produced a potentiation of recurrent inhibition (Curtis et al., 1970). It has been suggested that this iontophoretically released effect of GABA occurs as a result of GABA acting on





30


the pyramidal cell soma in a manner similar to that of the recurrent inhibitory interneuron. The relatively specific GABA antagonists, bicuculline and picrotoxin, have furthermore been shown to reduce GABA-induced inhibition (Curtis et al., 1970).

Local application of GABA onto either the pyramidal cell soma or to the basilar dendrites, when recording intracellularly, produced an hyperpolarization-depolarization sequence (Alger and Nicoll, 1980). When GABA was applied to the apical dendrites, a pure depolarizing response was recorded. Both the hyperpolarizing and depolarizing responses produced an inhibitory influence on the pyramidal cell (Alger et al., 1981; Andersen et al., 1978, 1980). Associated with the depolarization of the pyramidal cell apical dendrites was a large conductance increase which presumably accounted for the inhibitory action by shunting the current input. Thus, GABA produced powerful, though regionally specific inhibitory effects on the CAl pyramidal cell. The depolarizing inhibitory effect observed in the apical dendrites is thought to occur as a result of feedforward inhibition while the hyperpolarizing effect was shown to occur as a result of recurrent inhibition.

Another line of evidence concerning the types of inhibition in the CAl region of the hippocampus has emanated from intracellular recordings from CAl pyramidal cells. In these studies, stimulation of either orthodromic SR fibers or antidromic alvear fibers produced two distinct periods of inhibition (Alger and Nicoll, 1982). The first type of inhibition represented what is likely to be the result of classical recurrent inhibition. It can be evoked with either orthodromic or antidromic stimulation. It was seen as a large and sharp hyperpolarizing





31


potential which peaked at about 50 msecs. Immediately following this IPSP was a later hyperpolarizing potential which peaked at a latency from stimulus onset of approximately 200 msecs. This late hyperpolarizing potential could only be evoked with orthodromic stimulation in SR or SO. It cannot be evoked with antidromic stimulation thus ruling out the possibility that it was generated by recurrent interneurons and strongly suggesting it to be the result of feedforward inhibition (Alger and Nicoll, 1982).

The evoked hyperpolarizing potentials recorded above have also been characterized pharmacologically. The late hyperpolarizing potential occurred during the same period as the depolarizing potential which was elicited by localized application of GABA in the apical dendrites. The two different hyperpolarizing potentials (early and late) have been shown to be subserved by two different populations of GABA receptors. The early hyperpolarization has been shown to be mediated by GABA(a) receptors while the late hyperpolarization has been shown to be subserved by GABA(b) receptors. The evidence for this distinction emanated from the finding that the early hyperpolarizing response was antagonized by the GABA antagonist bicuculline. The late hyperpolarizing potential was, however, not antagonized by bicuculline and was therefore said to be bicuculline resistant (Newberry and Nicoll, 1984). Subsequent reports have indicated that baclofen, a GABA agonist, binds to the bicuculline-resistant GABA receptors and directly induced the late hyperpolarization in the pyramidal cell. The response to baclofen was associated with a decrease in neuronal input resistance which may be a result of an increase in potassium conductance (Newberry and Nicoll, 1984; Nicoll and Newberry, 1984). The early IPSP occurred as a result





32


of an increase in the chloride conductance of the cell (Kandel et al., 1961).



The benzodiazepines in the hippocampus

The benzodiazepines have also been reported to influence recurrent inhibition. High affinity binding sites have been found which fulfill many of the criteria of pharmacological receptors. However, the precise location of these receptors has been difficult to determine. A widespread distribution was seen rather than discrete localization. Enhanced binding of benzodiazepines was seen when GABA or a GABA analog was included in the binding assay. Thus, the benzodiazepine receptors appeared to be associated with the GABA receptors in a receptor complex (see Tallman et al., 1980; Ticku, 1983; Greenberg et al., 1984).

The benzodiazepines have been shown to increase the duration of inhibition in the hippocampal CAI pyramidal cell (Adamec et al., 1981; Wolf and Haas, 1977; Tsuchiya and Fukushima, 1978). It was suggested that the benzodiazepines either directly act upon the pyramidal cell soma through benzodiazepine receptors or through a GABA-benzodiazepine receptor complex (Tallman et al., 1980). When GABA was applied to local regions of the CAI pyramidal cell soma or dendrites, hippocampal slices bathed in midazolam, a benzodiazepine, responded with enhanced hyperpolarizations and depolarizations respectively (Jahnsen and Laursen, 1981). Benzodiazepine effects on recurrent inhibition have also been reported to be blocked by the GABA antagonists bicuculline and picrotoxin (Alger et al., 1981; Tallman et al., 1980; Tsuchiya and Fukushima, 1978). These findings suggested that the benzodiazepines may augment recurrent inhibition by either increasing the excitability of





33


interneurons or by increasing the strength of the excitatory afferents to the basket cells.



Opiates in the hippocampus

A third class of pharmacological agents probably interacting with inhibitory processes in the hippocampus is the opiates. Endogenous opiates have been observed in the CAI pyramidal cell region with the use of immunocytochemical techniques (Gall et al., 1981). Two different points of view exist concerning the effects of opiates in CAI (Corrigal, 1983; Haas and Ryall, 1980). It has been shown that electrophysiologically, the administration of [D-Ala(2), d-Leu(5)enkephalin] (DADL) or morphine- in the bathing medium of slices produced increases in the size and duration of EPSPs of dendritic origin without changing the resting membrane properties of the soma, recurrent somatic IPSPs, or dendritic field potentials (Dingledine, 1981; Robinson and Deadwyler, 1980). Three hypotheses have been proposed to account for these findings: 1. that an enhancement of "the electrical coupling between an apparently normal EPSP current generator and a normal spike trigger zone" occurs (Dingledine, 1981, p. 1034), 2. that a form of post-synaptic dendritic inhibition exists which is selectively attenuated by opioid peptides (Dingledine, 1981), and 3. the possibility that a "modulation of excitatory synaptic transmission onto hippocampal dendrites by a postsynaptic action that would entail some amplification mechanism" (Lynch et al., 1981 (no direct evidence supports this hypothesis, Dingledine, 1981, p. 1034)). The second and more widely supported interpretation is that opiate effects are not on the pyramidal cell at all but act by disinhibiting the inhibitory interneuron (Lee et al., 1980; Dunwiddie et al., 1980; Corrigal and Linesman, 1980; Nicoll





34


et al., 1980; Segal, 1977; Zieglgansberger et al., 1979). In either case the net effect of opiate administration is to increase the amplitude of an extracellularly evoked PS.

The administration of morphine to the basal dendrites or soma region of CAl pyramidal cells has been reported to produce membrane depolarization whereas application to the apical dendrites produced a hyperpolarization (Robinson and Deadwyler, 1980). These findings are important in that they are opposite to those found with discrete applications of GABA to these same cell regions. Thus, the differential effect seen with the GABA inhibitory internuerons is mirrored by the opiate data. These findings support an opiate-GABA interaction at CAI pyramidal cells in the hippocampus.



Rationale and Introduction to Current Experiments Chronic alcohol animal studies

Anatomy. CET has been shown to produce alterations in the normal morphology of the rodent hippocampus as well as the cerebellum. Using the Golgi stain, Riley and Walker (1978) reported CAl pyramidal cell basilar dendrites to be severely reduced in overall length and number of branches. The apical dendrites were not as dramatically affected except for the distal extent where arborization was decreased. In addition, the basilar dendritic fields had significantly reduced spine densities. DG granule cell dendrites also had reduced spine densities. A decrease of 15 to 20 percent in the number of CAl and CA3 pyramidal cells, dentate granule cells and cerebellar granule cells has also been reported (Walker et al., 1980; Tavares et al., 1983).




35


Physiology. Electrophysiological data using laminar and current source density (CSD) analysis profiles in the GAl region showed changes in the location of the current sinks after ethanol treatment (Abraham et al., 1982). Normal animals were reported to have two discrete yet overlapping current sinks in the SR of GAl. In GET animals, the more proximal (to the cell layer) current sink was significantly reduced. These data suggested that there was a change in the anatomical distribution of synapses in the CAl region after GET. Abraham et al. (1982) hypothesized that these two current sinks represented a discrete yet overlapping termination of the Schaffer collaterals distally and the commissural fibers proximally. They suggested that the conimissural fibers were preferentially affected. This condition could occur by either the commissural projections being interrupted, a loss of dendritic synapses in the proximal region, or a selective decrease in the efficacy of the synapses onto the proximal apical dendrites.

The normal function of the GAl region has also been shown to be altered by GET. Abraham et al. (1981) reported that although GET did not significantly affect the basic synaptic waveforms, EPSP thresholds, PS thresholds, or I/0 functions, the production of long-term potentiation, nor the basic pattern of response to paired pulse or frequency potentiation, GET did produce enhanced potentiation to paired pulses as well as FP at 5 Hz and 10 Hz stimulation. These findings of enhanced PS potentiation were found in the absence of changes in the field EPSP. It was hypothesized that these results are a function of a change in recurrent inhibitory processes of the GAl region.




36


Anoxia, picrotoxin, and the enkephalins, treatments which appear to decrease inhibition in the hippocampus, also produced enhanced facilitation of the PS amplitudes to paired pulse paradigm (PPP) stimulation (Andersen, 1960; Dunwiddie et al., 1980; Lee et al., 1980). These effects also occur in the absence of significant alterations in the synaptic response to single pulse stimulation. FP findings further support the involvement of recurrent inhibition since FP at 1 Hz resulted in no differences between GET and control animals while both 5 Hz and 10 Hz FP did result in PS differences (Abraham et al., 1981). Since the maximal duration of recurrent inhibition appears to be 600 msecs in CAl (Andersen et al., 1964a), 1 Hz stimulation is too slow to interact with inhibitory processes whereas 5 Hz and 10 Hz stimulation would be expected to interact. More recently, Durand and Carlen (1984) reported results which provided additional support for the hypothesis that GET reduces recurrent inhibition in CAl. They found, using intracellular recordings from GET hippocanipal slices, that only the inhibitory post-synaptic potential (IFS?) amplitudes and afterhyperpolarization durations were significantly different from controls. No differences were found with any of the other membrane characteristics measured.

There has been one interesting biochemical finding potentially related to inhibition in the hippocampus. Freund (1980) found a decrease in the binding of benzodiazepine receptors from GET animals. These findings could be important since there is a strong link between GABA and benzodiazepine receptors with GABA strongly involved in the inhibitory mechanisms of the CAl region.




37


It is apparent that CET affects inhibition in CA1. However, it is not clear whether CET affects recurrent inhibition alone, feedforward inhibition alone or both types of inhibition concurrently. In attempting to determine the mechanisms that may be affected by CET, it is important to know the generality of CET effects on inhibition. Therefore, the basic intent of the experiments reported herein was: 1. to define the nature and parameters of the CET induced changes as well as the characteristics of paired-pulse inhibition and potentiation in CAl in untreated animals and 2. to begin to study the possible pharmacological mechanisms which may underlie these changes. These goals were accomplished through three major series of experiments: 1. characteristics of normal synaptic inhibition -- an in vivo study, 2. characteristics of CET and synaptic inhibition -- an in vitro study, and 3. pharmacological characteristics of CET and synaptic inhibition -- a series of in vitro studies.













CHAPTER 2
GENERAL METHODS


Animals

Long-Evans hooded male rats were purchased from the Charles River

Company for all experiments. The rats were obtained at a body weight of 225 to 250 gms and were 55 to 60 days old prior to beginning the administration of ethanol. Animals were housed in individual stainless steel cages at the University of Florida Animal Resources Center or at the Veterans Administration colony room or building (three different locations). Temperature and humidity were maintained at appropriate levels. Light-dark cycles of twelve hours on and twelve hours off were maintained with the light cycle from 0700 hrs to 1900 hrs daily.



Alcohol Diet Regimen

Animals were maintained on an ethanol or sucrose containing liquid diet. This diet was developed so that experimental and control animals ingested calorically equal, nutritionally complete diets.

The ethanol containing diet provided 35 to 39 percent (8.1 to 9.4 percent ethanol v/v) of the total caloric intake as ethanol. Ethanol or sucrose provided 35 percent of the total caloric intake the first month of the diet regimen. This level was then increased by 1 percent per month in order to provide a relatively constant level of ethanol or sucrose as the animals gained weight. The diets were prepared by combining an equicaloric ethanol- (63.3 percent v/v) or sucrosecontaining 87.0 percent w/v stock solution with Sustacal obtained from 38




39


the Mead-Johnson Company. Both groups of liquid diets were additionally fortified with Vitamin Diet Fortification Mixture (3.0 g/1) and Salt Mixture XIV (5.0 g/1) which were obtained from ICN Nutritional Biochemicals, Cleveland, Ohio. The diets contained 1.3 KCal/ml and provided several times the daily requirement for all essential vitamins and nutrients (Walker and Freund, 1971). Diets were prepared fresh daily and were administered using calibrated glass bottles with stainless steel drinking tubes. Consumption levels of each animal were recorded daily.

Upon arrival at the animal facilities, animals were quarantined for a period of seven days. They were then housed individually and allowed to acclimate to the colony room for one to two weeks or until they reached the appropriate body weight. Next, animals were paired according to their weight. The experimental animals (group E) received ethanol in their daily diet while the control animals (group S) received sucrose substituted isocalorically for the ethanol. A laboratory chow group was not included in these experiments since in the previous 15 years of experiments in this laboratory no differences between sucrose and lab chow control groups were ever found.

Animals in group E were pair fed with animals of group S by giving each animal in group S the volume of diet that its age- and weightmatched group E partner consumed on the previous day. This pair-feeding procedure assured that group E and group S animals ingested identical quantities of calories, vitamins, minerals, proteins, fats and carbohydrates. Differences between group E and group S animals on any dependent variable were then due to ethanol and not nutritional or other




40


variables. A minimum of eight rats were used in each group for all experiments.

In these experiments, group E and group S animals were treated with ethanol or sucrose for 20 weeks. A minimum of eight weeks was allotted after the discontinuation of the alcohol diet prior to beginning any of the electrophysiological procedures. This period of abstinence was included to control for the residual effects of acute alcohol withdrawal. Since the testing of individual preparations consumed an entire day, sampling occurred over an 8 to 24 week period after ethanol discontinuance depending on the initial group size. Individual animal number and group designation were coded to prevent any bias in the placement of recording and stimulating electrodes. Coding was essential due to the response variability observed in recordings from the hippocampus and to prevent unintentional procedural differences between the two groups.



Egui~ment

All recordings were made with glass micropipettes filled with 4M NaCl. Electrodes were chosen to satisfy criteria for tip diameter (1-2 uM), impedance (500K to 1.5 Mohms at 1 KHz) and shaft taper. Concentric bipolar electrodes obtained from Rhodes Medical Instruments (in vivo) or the Frederick Haer Co. (in vitro) were used to activate specific hippocampal afferent or efferent pathways. In all cases, electrodes were controlled with micro-manipulators. Constant current stimulation (duration, 0.1 msec; current, 20-1000 uAs) was used in all procedures. Stimulus pulse timing was controlled with WPI series 1800 timers (.in vivo) or with an Apple computer system using software and hardware designed by the author (in vitro).




41


All recordings were amplified by Grass P511 preamplifiers, filtered (Krohn-Hite) at either .3Hz-lOKHz (field potentials) or 300Hz-lOKHz (extracellular unit activity) and displayed on Tektronix oscilloscopes. All responses were collected on an Apple computer system for subsequent computer analysis. Analog to digital conversion within the computer employed an Interactive Structures Inc. AI13 board. For these experiments, 512 data points were collected during a 28 msecs period. Each data point had a maximum amplitude resolution of one in 4096.

A calibration pulse (1 mV, 2 msecs) was routinely injected immediately prior to each stimulation pulse with a Stoelting GAS calibrator. This calibration pulse not only controlled for alterations in equipment status, but also allowed for uncomplicated adjustment of the gain of the preamplifiers and of the scaling of the data for subsequent computer display and analysis.

Raw data were analyzed for the in vivo experiments on an Apple II

Plus computer system whereas all data from the in vitro experiments were transferred from the Apple computer to an IBM 5150 (PC) for data analysis. All data were stored on 5.25 inch floppy disks. After analyses of the waveforms, all measures were transferred via modem to the North East Regional Data Center (NERDG) computer system for statistical analyses using the Statistical Analysis System (SAS) General Linear Models (GLM) analysis of variance procedures. Graphics were produced using the IBM Displaywriter system with Chartpak software or the IBM 5150 using Lotus Inc.'s Symphony software and a Hewlett Packard 7470A plotter.




42


Basic Procedures

Input/outiput relations

1/0 functions were obtained at the start of each experimental

session by varying the stimulus current from 20 to 1000 uAs with pulse widths at a fixed duration of 0.1 msec. The analysis of the responses generated focused on measures of the latency and amplitude of the EPSP and PS. These measures provided information regarding synaptic response strength. The complete set of current steps used was the following: 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000. These steps were selected in an attempt to more accurately indicate EPSP and PS thresholds as well as asymptotic levels. Additionally, the 1/0 functions were used to normalize stimulus current strength across animals in the later phases of each experiment.



Paired-Rulse paradigm

Stimulus current. An important problem in these experiments was the choice of stimulus current used for the paired pulse series of experimental paradigms. 1/0 curves were used to adjust the stimulus current such that a certain percentage of the PS amplitude at asymptote, up to a maximum of 1000 uAs, was used for the condition or test pulse of paired pulse experiments. This procedure controlled for minor variations in electrode placement and individual animal response variability. If CET altered the basic 1/0 functions, this procedure allowed for the evaluation of synaptic potentiation with baseline responses which fell at the same relative points on the 1/0 curves.




43


Characteristics. When paired shocks are delivered to any of a variety of afferent pathways of the hippocainpus, a characteristic inhibition or potentiation of the test (second) response is observed when the condition and test pulses are separated by a critical time period (Steward et al., 1977). The magnitude of the test response inhibition or facilitation depends upon the inter-pulse-interval (IPI). The typical pattern of the facilitation depends on the response measured. Stimulation of the Schaffer/commissural path while recording in the CAl pyramidal zone produces a characteristic but different response pattern for both the EPSP and the PS. The population EPSP is maximally facilitated at IPIs of 20-30 msecs decreasing at progressively longer intervals. PS amplitude is inhibited at IPIs of 20 to 80 msecs, most likely as a result of inhibitory processes and is facilitated at longer IPIs (> 80 msecs) (Lomo, 1971; Steward et al., 1977; Creager et al., 1980).



Procedures. Paired-pulse inhibition and potentiation were

evaluated by systematically varying the IPI between the condition and test pulse from 20 msecs to 3 secs. Four responses were averaged at each interval tested. The individual intervals tested for all experiments were as follows in msecs: 20, 30, 40, 50, 60, 80, 100, 150, 200, 300, 400, 500, 750, 1000, 1500, 2000, 3000. Paired shocks were delivered at a frequency of 0.1 Hz in the CAl region in the order described above. Paired pulse stimulation current levels in this series of studies used test pulse current levels which were either 25 or 50 percent of the maximal PS response amplitude obtained from mini-I/O curves. Condition pulse current levels were adjusted to 50 percent of




44


the EPSP amplitude at PS threshold (50 EP), PS threshold (0 PS), and to 25, 50, 75 and 100 percent of the PS amplitude at 1/0 asymptotic values. Intensities used for each of the individual experiments are indicated in subsequent sections. Inhibition and potentiation were measured by quantitative comparison of the test response with a baseline response averaged from four single pulse baseline responses recorded immediately prior to each of the paired pulse series.



Data Analysis and Inter2retatio-n

The protocol outlined above was designed to allow for separate

measurement of various population EPSP and PS characteristics. For the EPSP, the following measures were sampled: 1. latency to onset from stimulus artifact and 2. maximal slope of the rising phase of the EPSP calculated either by using a regression equation for all of the data points between the EPSP onset and EPSP peak prior to the PS (all in vivo data) or by using a regression equation for all of the data points 10 percent above the EPSP onset and 10 percent below the EPSP peak prior to the PS (10-90 slope; used for all but the very first series of in vitro data). For the PS, the following measures were sampled: 1. latency to onset of the first PS measured at its maximum negative amplitude from the EPSP onset and 2. rising and falling aspects of up to three different population spikes from the same stimulation pulse. Each PS amplitude was calculated by adding the respective falling and rising phase of the PS spike and dividing by two (Fig 2-1). If multiple PSs were recorded,




















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the PS amplitudes were added to each other. This total PS amplitude was the measure used for all of the PS data analyses. Results were statistically evaluated by two-way analyses of variance with repeated measures. one factor was the experimental treatment (group E or S). The second repeated factor was stimulus current (1/O curves), IPI (PPP), location (iontophoretic application locations) or time (after iontophoretic release). Responses between alcohol and sucrose animals were compared between groups as a percentage change from the baseline of each animal for amplitudes and as a difference score for latency measures. This provided for the normalization of potentially different baselines between the two groups.













CHAPTER 3
CHARACTERIZATION OF SYNAPTIC INHIBITION IN VIVO Introduction

The regulation of pyramidal cell firing in the CAl region of the

hippocampus has long been recognized as an important consideration when attempting to understand, and potentially control, neuronal function or dysfunction of this area. Although there may be a multitude of different and, as of yet, unknown mechanisms by which pyramidal cell firing is regulated, one of the most well documented methods is achieved through an inhibitory interneuron; the now classical recurrent inhibitory circuit found near the pyramidal or granule cell in CAl, CA3 and the dentate gyrus regions of the hippocampus (Cajal, 1968, Lorente de No, 1934). Recurrent inhibition occurs as a result of pyramidal cell firing. An action potential propagates from the pyramidal cell to other major cell types in caudal and rostral directions from the hippocampus. An axon collateral of the pyramidal cell also conducts this action potential to nearby inhibitory basket cells. An excitatory synapse from the pyramidal cell results in the basket cell firing. The basket cell projects widely in a basket-like web to the original as well as many other pyramidal cells. An inhibitory neurotransmitter, GABA, then causes the pyramidal cell to cease firing for a period of time up to 700 msecs (Andersen et al., 1964a, 1964b). More recently, another type of inhibition in the CAl region has been shown to exist which is feedforward in nature. In this system, afferent fibers terminate on an



48




49


inhibitory interneuron or project to both an inhibitory interneuron and pyramidal cell dendrites (Ashwood et al., 1984; Buzsaki, 1984).

Substantial evidence exists for the presence of interneurons

located in the pyramidal cell layer and the SO and SR dendritic areas of the CAI region. Peak concentrations of GAD, an enzyme which catalyzes the formation of GABA, is restricted to interneurons located in the pyramidal cell layer and SLM (Storm-Mathisen, 1977). Immunoreactive staining to GAD showed sparse staining throughout the dendritic regions of both the hippocampal CA regions and the dentate gyrus. A more heavily stained region was found in the distal apical dendrites (Barber and Saito, 1976). These same cells are also observed with cholecystokinin and enkephalin immunoreactive staining (Gall et al., 1981; Harris et al., 1985; Stengaard-Pedersen et al., 1983). Interneurons with dendritic and axonal orientations parallel to the dendritic axis of CAI pyramidal cells have also been described (Cajal, 1968; Lorente de No, 1934; Schwartzkroin and Mathers, 1978). Ashwood et al. (1984) have provided evidence that some of the interneurons in the pyramidal cell region may be feedforward inhibitory since orthodromic activation of the Schaffer collateral/commissural system resulted in some of the inhibitory interneurons firing prior to the pyramidal cell.

The electrophysiological and pharmacological evidence for the

existence of both feedforward and recurrent inhibition is convincing (Alger et al., 1982; Andersen et al., 1980, 1982). However, most studies have utilized intracellular recordings to arrive at these conclusions. While intracellular recordings are most appropriate in defining the existence and details of feedforward and recurrent




50


inhibition, it would also be useful to be able to measure the effects of these two types of inhibition extracellularly.

Recurrent inhibition has often been studied extracellularly using paired pulses delivered from the same stimulation site, usually in stratum radiatum. With this paradigm, the effects of recurrent inhibition and paired-pulse potentiation were thought to be studied. However, with the recent evidence for feedforward inhibition, it is clear that any study of inhibition using this configuration is confounded if the objective of the experiment is the study of recurrent inhibition alone. The use of an antidromic stimulation site for the condition pulse with an orthodromic stimulation site for the test pulse, however, should provide a relatively better extracellular assessment of recurrent inhibition. This technique has been utilized for the study of recurrent inhibition previously (Andersen et al., 1964a; Dingledine and Langmoen, 1980; Haas and Rose, 1982). Thus, recurrent inhibition can be studied relatively independently using extracellular antidromic/orthodromic (A/0) paired pulses. Feedforward inhibition, however, is more difficult to study extracellularly. Recurrent inhibition is almost always activated with orthodromic stimulation unless pharmacological and/or ionic manipulations could be utilized. Because A/0 paired pulses activate recurrent inhibition alone while 0/0 paired pulses activate both recurrent and feedforward inhibition, it is proposed that if the results of 0/0 paired pulses are compared with A/0 paired pulses, the difference, if the amplitude of A/0 responses were compared to the 0/0 responses, may provide a reasonable estimate of feedforward inhibition using extracellular methods. Another possible method by which feedforward inhibition might be studied without recurrent inhibitory influences is




51


with the use of subthreshold condition pulses for the PS of CAl pyramidal cells.

The first goal of this study was an attempt to dissociate recurrent from feedforward inhibition using extracellular recordings. The method used was that of employing paired pulses with two different stimulation configurations: an A/0 and an 0/0 configuration. The 0/0 configuration utilized one stimulating electrode which was placed in the SR of CAl and through which both the condition and test pulse stimuli were emitted. This configuration would activate both the recurrent and feedforward inhibitory mechanisms as well as inducing an EPSP facilitation (observed when recording from the CAl pyramidal cell apical dendritic region). The A/0 configuration utilized two independent stimulation electrodes located in two different pathways. The electrode used for the condition pulse was placed in the alveus to activate antidromically the efferent fibers of the CAl pyramidal cell and thus, to activate orthodromically, the recurrent inhibitory interneuron (normally as a result of pyramidal cell firing). The second stimulating electrode was placed in the SR, as above, for the test pulse stimulation. The antidromic configuration should activate only recurrent inhibitory interneurons with the condition pulse, theoretically leaving the second or test pulse response to be modified predominantly by the effects of recurrent inhibition.

A second goal of these experiments was to more completely describe the parameters of paired pulse inhibition and potentiation. Most studies utilizing paired pulses have used IPIs from 15 to 200 msecs. It has been reported that there are other inhibitory effects which can only be seen with longer interpulse intervals and which may be active for as long as two seconds, although they may be observable only with




52


intracellular techniques (Wigstrom and Gustafsson, 1981). The range of IPIs utilized in these studies was therefore expanded to include IPIs from 20 msecs to 3 secs.

A final goal of these experiments was to better describe the effect of current intensity on the characteristics of paired pulse inhibition and potentiation. In these experiments the effect of varying condition and test pulse current intensity was investigated. While a number of different current intensities have been used previously (Creager et al., 1980; Haas and Rose, 1982; Assaf and Miller, 1978; White et al. 1979; Chaia and Teyler, 1982; Steward et al., 1977), there have been no systematic, parametric analyses of the effect of varying condition and test pulse current intensities with the paired pulse paradigm, especially not with a wide range of IPIs. If different populations of interneurons can be recruited as a function of current intensity as has been suggested (Ashwood et al., 1984), it may be possible to use this parameter to selectively activate feedforward and recurrent types of inhibition. These experiments should therefore provide important information regarding the extracellular correlates of feedforward and recurrent inhibition as well as some of the parametric conditions frequently used in paired pulse studies but which have heretofore lacked comprehensive parametric exploration.



Methods

Animals

Animals used were as described in the general methods except that only sucrose-fed control animals were used.




53


Equipment and techniques for in vivo studies

Animals were initially injected intraperitoneally (IP) with .2 cc of atropine to reduce possible respiratory congestion. Within 10 mins 1.5 gms/Kg of urethane was injected IP for anesthesia. A second injection of 1.0 gms/Kg was given one hour later. Supplemental injections of .05 gms/Kg were then given at intervals of every 1.5 to 2 hrs.

After it was determined that the animal was well anesthetized, it was placed in a Kopf stereotaxic device using ear bars and a nose clamp. An incision was made down the midline of the rat skull and the skin retracted. The skull was then adjusted so that the bregma and lambda sutures were level horizontally with the base of the stereotaxic device. Three burr holes were drilled on the left side of the skull at locations 3.4, 4.4 and 5.4 mms posterior and 2.8 mms lateral to bregma. A concentric bipolar stimulating electrode (Rhodes Medical Instruments) was then lowered to the SR of CAI through the most anterior hole using a hydraulic microdrive. This electrode was used as an orthodromic stimulating electrode. A second stimulating electrode was placed in an antidromic configuration. This electrode was located in the most posterior hole and lowered with a microdrive to the alveus. The recording electrode was lowered through the center hole. This electrode configuration was used since the orientation of the lamellae within the hippocampus is such that an approximate anterior to posterior course is found in the' rat (Andersen et al., 1971).

Precise localization of the electrodes in vivo was performed by

observing the extracellular unit activity as the recording electrode was lowered into the hippocampus. Based upon the characteristic high frequency, complex unit response observed in the pyramidal cell layer,




54


the recording electrode was adjusted to approximately 150 uMs above the region of maximal unit activity. With the recording electrode filtering re-set for field responses, the orthodromic stimulating electrode was lowered into the hippocampus until the evoked response was maximally positive with a superimposed PS (see basic physiology section in general introduction). Obtaining this response required readjusting the recording electrode depth to 150 ta~s above the inversion point until the maximal PS amplitude response was achieved. The antidromic stimulating electrode was then lowered until a short latency, sharply negative-going potential was observed. The antidromic electrode was carefully adjusted to maximize the negative going response without inducing an orthodromic stratum oriens EPSP response. At the end of the recording session, a small lesion was made at the recording site and 400 u~s below. These lesions were used to verify the location of the recording electrode during histological analyses. The sizes of the stimulating electrodes were such that the paths they created were observable without lesions.



Testing procedures

Once the stimulating and recording electrodes were positioned and adjusted for maximal responses, input/output testing was performed for the orthodromically evoked response only. In this procedure, the current intensity delivered by the stimulating electrode was increased in steps from 20 to 1000 uAs as described in the general methods.

After the I/O curves were collected, the paired pulse experiments were begun. Two different groups of animals were used to test the different test pulse current intensities as outlined in Table 3-1. The first group of animals was tested with a test pulse stimulus current




55





TABLE 3-1

PERCENT OF MAXIMAL PS AMPLITUDE ON TEST PULSE



25% 50%
C
O
N 0/0 50% EPSP X X
D O/O 0% PS X
I 0/0 25% PS X X
T 0/0 50% PS X X
I 0/0 75% PS X
0 0/0 100% PS x X
N A/O 0% AS X
A/0 25% AS X X
P A/0 50% AS X X
U A/0 75% AS X
L A/O 100% AS X X
S E


Percent of maximal PS amplitude at PS asymptote, EPSP amplitude at PS threshold, and maximal antidromic spike (AS) amplitude for the condition and test pulses in paired pulse paradigms (in vivo).




56


intensity that produced a PS amplitude 50 percent of the PS amplitude at asymptote while the second group was tested with a stimulus intensity producing 25 percent of the PS amplitude at asymptote. Each of the indicated (Table 3-1) condition pulse current intensities was tested on each animal within the test pulse current intensity level. The order of the individual paired pulse experiments was determined in advance through a random order selection. The IPIs used for all of these experiments were as described in the general methods. IPIs were always presented in the order described.

The current intensity levels used in the paired pulse tests were based on a percentage of the asymptotic amplitude of the PS from brief 1/0 curve functions. For example, 50 percent of the PS amplitude was one of the current intensities tested. The size of the PS was measured from the 1/0 curve at the asymptotic level. The stimulation current level was then adjusted to obtain a PS response amplitude which was half as large as the asymptotic PS response amplitude. This procedure was used in order to control for differences in excitability and asymptotic levels between animals. By using percentages of asymptotic PS response levels, a more accurate comparison between animals was made possible. The different current intensity levels on the condition and test pulse used are shown in Table 3-1.

Data collection and analyses were performed as described in the general methods except that analyses of variance were not performed. The major points to be made in this series of experiments were best addressed descriptively rather than by measures of statistical difference.




57


Results

1/0 relationships

The combined 1/0 curves for the two sets of animals are shown in Fig 3-1. These data are from a total of 22 animals. The PS threshold was 252 uAs + 12.31 (data presented as mean + SEll). The PS response amplitude asymptote was 552 + 41.36 uAs current level.



Characterization of paired Pulse Paradigms in normal animals

The data from these experiments were divided into four major groupings based on the stimulus configuration and the current intensity of the test pulse used. The four groupings were: 1. the 0/0 25 percent test pulse series, 2. the 0/0 50 percent test pulse series, 3. the A/0 25 percent test pulse series, and 4. the A/0 50 percent test pulse series. For all of the superthreshold paired pulse experiments reported herein, the general patterns and amplitudes of the PS inhibition and potentiation are comparable to those previously reported for the CAl and dentate gyrus regions of the hippocampus (Greager et al., 1980; Lomo, 1971; Steward et al., 1977). The EPSP slope data, however, were not similar to these previous reports as discussed subsequently.



0/0 25 Rercent test Rulse series

The 0/0 25 percent test pulse series data showed that increasing the current intensity of the condition pulse, if the current intensity was above PS threshold, produced an increase in the duration of inhibition. When the test pulse PS amplitude was expressed as a percentage of the control response, the PS was almost abolished from 20 to 30 insecs for the 25 percent condition pulse, extending to 50 msecs for the 100




















Figure 3-1. I/O curves for in vivo data. A) Group mean PS
amplitude responses by current level from 20 to 1000 uAs expressed in raw units (mVs); B) Group
mean PS latency measures shown as raw data
(msecs) by current intensity (uAs).








59























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60


percent condition pulse (Fig 3-2A). The 50 percent condition pulse produced a test pulse response pattern which was midway between the 25 percent and 100 percent condition pulse patterns. The total durations of inhibition for the 25, 50 and 100 percent condition pulses were 50, 75 and 120 msecs, respectively. The condition pulse current intensity of 50 percent of the EPS? at PS threshold produced no inhibition of the PS.

The highest levels of potentiation in the 25 percent test pulse

series for each of the condition pulse current intensities were nearly the same in each of these experiments. The highest levels of potentiation were approximately 200 to 250 percent of the control responses. The effects of the different condition pulse current intensities were seen in the time it took to reach the highest level of potentiation as well as the point at which the PS first became potentiated. The condition pulse current intensity of 50 percent of the EPSP at PS threshold became maximally potentiated at an IPI of 40 msecs and subsequently decreased toward control levels. The 25 percent of PS amplitude condition pulse potentiation peaked at 150 msecs, the 50 percent of PS amplitude maximum condition pulse peaked at 200 msecs, and the 100 percent of PS amplitude maximum condition pulse peaked at 300 msecs. All of the experiments in which the condition pulse current intensities were set above PS threshold returned toward control levels together beginning at approximately 400 msecs and ending by 2000 msecs IPIs.

The PS latencies, calculated by measuring the interval from the EPSP onset to the PS maximum, were increased by up to 1.2 msecs with increasing condition pulse current intensities but only at shorter IPIs (Fig 3-3A). These latencies then returned to control levels together by 400 msecs IPIs. PS latencies were calculated from the EPSP onset since





















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65


this controlled for individual animal differences in terms of the EPSP onset as well as the different stimulation current intensity at which it occurred. The PS latency can be used as a measure of pyramidal cell excitability since it generally decreases with increasing PS amplitude.

The EPSP (Fig 3-.4A) was monitored by measuring the slope of the rising phase of the EPSP. These responses showed little change as a result of the different condition pulse current intensities and thus remained near control levels from 20 to 3000 msecs, the entire range of testing. A decrease in the slope of the EPSP was noted at IPIs between 20 and 150 msecs. Previous reports indicate that a facilitation of the EPSP is normally seen with IPIs of 20 to 30 msecs (Creager et al., 1980; Lamo, 1971).



0/0 50 Rercent test pulse series

The 0/0 50 percent test pulse series PS amplitude responses (Fig 3-2B) showed similar patterns for the test response PS amplitude when compared to the 0/0 25 percent data described above. The major difference, however, was that the maximal level of potentiation was lower with the 50 percent test pulse series PS responses. Like the 25 percent test pulse data, increasing the condition pulse current intensity above PS threshold levels produced increasing durations of inhibition in each of the experimental series. Each of the superthreshold condition pulse current intensities produced complete inhibition of the PS amplitude at IPIs of 20 msecs. The maximal PS inhibition was, in general, extended in duration with increasing condition pulse current intensities from 20 msecs at lower condition pulse current intensities to 50 or 60 msecs at 100 percent of the condition pulse PS response amplitude at asymptote.





















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67





















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68


When compared to the 0/0 25 percent test pulse responses, there were no shifts in the duration of inhibition. The point at which the PS amplitude crossed control levels to the potentiation phase ranged from 50 msecs for the condition pulse current level set to PS threshold to approximately 130 msecs for the 100 percent of PS amplitude at asymptote condition pulse current intensity. The condition pulse current intensity of 50 percent of the EPSP at PS threshold, unlike the 0/0 25 percent test pulse equivalent, showed a very brief period of inhibited PS responding at the 20 msecs IPI, but then showed a potentiated PS response at the 30 msecs [PI. As with the 0/0 25 percent test pulse series, the condition pulse current intensity did not affect the maximal level of potentiation. The subthreshold condition pulse PS amplitude was potentiated maximally at the 40 msecs IPI and decreased to control levels thereafter. The superthreshold condition pulse current intensities all reached a peak level between 150 and 200 msecs IPIs and remained near that level until IPIs of approximately 400 msecs at which point they began to return to control levels. Each of the condition pulse series PS responses returned to control levels by 1500 msecs. The maximal level of potentiation observed with the 50 percent test pulse series, however, only reached 140 to 160 percent of control. This maximal potentiation level was in contrast to the 225 percent level observed with the 25 percent test pulse series.

The PS latency data (Fig 3-3B) were similar to the 0/0 25 percent

data. An increase in the latency to a PS was observed with IPIs from 20 to 400 msecs. While the subthreshold condition pulse data showed the smallest difference from control levels, the PS latencies of the superthreshold condition pulses were not apparently influenced by condition pulse current intensity.




69


The slope data (Fig 3-4B) for this series showed a period of inhibition at the 20 msecs IPI which remained slightly inhibited to approximately the 150 msecs IPI. There was no apparent ordering of slope by current intensity except that less inhibition was observed with a subthreshold condition pulse than with a superthreshold condition pulse.



A/0 25 Rercent test pulse series data

The A/0 25 percent of the PS maximum test pulse series (Fig 3-5A)

showed that increasing the current intensity of the antidromic condition pulse increased the magnitude of the inhibition to a greater extent than the duration of inhibition. Unlike the 0/0 experimental series reported above, the A/0 series did not show potentiation. Instead, there appeared to be two phases to the inhibition. There was a short initial period at IPIs of 20 to 30 msecs when inhibition was greatest, followed by a prolonged period where less but continued inhibition was found. The antidromic condition pulse of 25 percent of the maximum antidromic spike amplitude resulted in a test pulse response which was maximally inhibited to 50 percent of the control test response at the 20 msecs IPI. The 50 and 100 percent antidromic condition pulses inhibited the test pulse response to 22 and 14 percent of the control test response, respectively. The IPI at which the 50 percent condition pulse produced its maximum effect was at 20 msecs while the 100 percent condition pulse was maximally inhibited at 30 msecs IPIs. Each of the condition pulse current intensities produced a test pulse response which was inhibited to at least 70 percent of the control response for the duration of the IPIs




















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72


tested. The 50 and 100 percent condition pulse current intensities were inhibited to approximately 50 and 30 percent of control from IPIs of 80 msecs to approximately 300 and 1000 msecs, respectively.

The PS latencies of the A/0 25 percent test pulse series (Fig 3-6A) showed increased latencies from 20 to 400 msecs IPIs. The PS latencies then remained somewhat longer than control responses. A 1.0 msec increase in latency was observed at the 80 msecs IPI for the 100 percent condition pulse current intensity.

The EPSP slopes for these data (Fig 3-7A) showed small fluctuations at IPIs of 20 to 50 msecs but did not vary substantially from control responses thereafter.



A/0 50 percent test Rulse series

The A/0 50 percent of the PS maximum test pulse series PS amplitudes are shown in Fig 3-5B. It can be seen that each increase in condition pulse current intensity produced a greater magnitude of inhibition of the test pulse response. The 0, 25, 50, 75, and 100 percent antidromic conditioning pulses produced maximum inhibition levels to 80, 60, 40, 30, and 20 percent of the control response, respectively. The maximum level of inhibition for each series except the A/0 threshold condition pulse level was reached at the 20 msecs IPI. The threshold condition pulse current intensity produced its maximum level of inhibition at an IPI of 60 msecs. As found with the A/0 25 percent test pulse series, the A/0 50 percent test pulse series resulted in at least two distinct phases of inhibition. Maximal levels of inhibition were found in the early period with IPIs between 20 and 60 msecs while a second phase of inhibition was found between IPIs of 80




73


and 1000 msecs. The A/0 50 percent test pulse series responses, like the A/0 25 percent test pulse series responses, were inhibited for the entire duration of IPIs tested but only to 80 to 90 percent of control levels.

The PS latencies for these experiments are shown in Fig 3-6B. The PS latencies increased from .1 to .4 msec between 20 and 200 msecs IPIs for all of the condition pulse current intensities. The higher the condition pulse current intensity, the longer the latency found to the onset of the PS. Like the PS amplitude responses, the PS latency responses were intermediate between 200 and 1500 msecs IPIs. Like the A/0 25 percent test pulse series, the A/0 50 percent test pulse latency response did not return to control levels with the IPIs tested.

Slope measures are shown in Fig 3-7B. Except for the brief decrease in slope at the 20 msecs IPI seen with the 100 percent condition pulse current intensity, there was only a small decrease in slope observed with the IPIs tested.



Discussion

The effect of varying the current intensity of the condition and test pulse of paired pulses was investigated using both 0/0 and A/0 stimulation configurations. An attempt was made to determine the relative contribution of feedforward and recurrent inhibition with these extracellular paired pulse paradigms. All of the experiments were conducted in CAl using an anesthetized in vivo rat hippocampal preparation.

The general characteristics of the superthreshold PS amplitude

response patterns found with 0/0 paired pulse stimulation for all of the





















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78


superthreshold condition and test pulse current intensities tested were similar to those reported previously (Andersen et al., 1966; Creager et al., 1980; Steward et al., 1977; Lomo, 1971). The subthreshold condition pulse current intensity resulted in little inhibition but shifted the period of maximal potentiation from IPIs of 150 to 400 msecs to IPIs of 20 to 80 msecs. For the 0/0 superthreshold PS amplitude responses, there was an initial period of inhibition at the shorter IPIs which then changed to a period of potentiation at the intermediate length IPIs. The PS latency measures corresponded well with the PS amplitude measures since both yield an index of pyramidal cell excitability. Increasing the current intensity resulted in increased PS latencies at short IPIs since the PS was inhibited for longer periods. The EPSP response patterns, however, were different from those reported previously (Creager et al., 1980; Landfield et al., 1978; Lomo 1971; Steward et al., 1977). The EPSP measure is typically reported as an amplitude at a fixed latency from EPSP onset, the maximal EPSP amplitude or a slope measure of the rising phase of the EPSP (Lomo 1971; McNaughton and Barnes, 1977; White et al., 1979; Creager et al., 1980). Other researchers have reported a period of facilitation of the EPSP measures with 20 to 30 msecs IPIs followed by a gradual decay with a time constant of about 100 msecs (McNaughton, 1982). Slope measures have been utilized in an attempt to reduce the distortion of the EPSP sometimes seen as a result of pyramidal cell or recurrent interneuron firing (Creager et al., 1980). The present slope measures showed changes only at short IPIs, but the changes observed were in the direction of decreased rather than increased slopes. The most likely reason for these response differences was that the present measures were recorded from




79


the pyramidal cell layer rather than in the synaptic zone. Recent testing with recordings from the pyramidal cell layer and from the Schaffer/commissural afferent synaptic zones in our laboratory has confirmed that EPSP slope measures obtained from the synaptic zone match those reported from other laboratories, while EPSP slope measures recorded from the pyramidal cell layer matched the data from the present experiments. These inconsistencies in EPSP measures from the SR synaptic zone versus the pyramidal cell layer may be due to the occurrence of several overlapping events. In the SR region, the EPSP is relatively free of other synaptic influences such as the firing of the pyramidal cell or recurrent inhibitory interneurons. When recording from the pyramidal cell layer, the PS may partially or completely obliterate the EPSP thus making the EPSP measure more difficult to analyze. In addition, the IPSP from the recurrent and/or feedforward inhibitory interneuron may summate with the EPSP (due to the inward flow of current in the SR excitatory region and the outward flow of current from the inhibitory interneurons in the pyramidal cell region) and therefore result in uninterpretable measures. It is our conclusion that the EPSP data recorded from the cell layer do not represent the same phenomena seen in the synaptic zones. It is likely that they reflect a summation of the EPSP and IPSP and therefore it is difficult if not impossible to make any conclusions regarding any alterations that may be occurring in the SR synaptic region. EPSP slope data will therefore not be presented in any of the subsequent chapters.

The A/O paired pulse PS amplitude data at IPIs less than 80 msecs were also similar to those reported by others (Dingledine and Langmoen, 1980). With IPIs of 60 to 80 msecs, however, where the test pulse PS




80


amplitude response was shown to return to control levels in other studies, the current results showed that the return toward baseline levels was different. Instead of the PS amplitude returning directly to control levels, it returned toward control levels by approximately 50 percent at IPIs of 60 to 80 msecs and then very gradually returned to levels closer to control by IPIs of 1000 to 2000 msecs. Even at this point, however, the PS amplitude was inhibited by 10 to 30 percent of control. The A/0 PS latency measures exhibited longer latencies than control responses at short IPIs and subsequently returned toward control levels. Like the PS amplitude responses, however, the PS latency data did not completely return to the control level at any of the IPIs tested. The best characterization of these responses is that A/0 stimulation results in two phases of inhibition. There is an initial, maximal inhibition phase where the greatest magnitude of inhibition is found. This maximal period of inhibition is then followed by a decay phase which was more prolonged than previously noted. This is not consistent with previous reports although most of the evidence has been obtained from hippocampal slice preparations and so the effects of anesthesia may result in the prolonged decay phase.

The 0/0 paired pulse stimulation configurations were seen to

produce near complete inhibition of the PS amplitude response at short IPIs. This occurred despite the EPSP being facilitated at short IPIs (based on reports by others including one using the same preparation, anesthesia levels and stimulating and recording locations as the present investigations but with condition and test pulse current levels subthreshold for evoking PS responses (Abraham et al., 1981)). While increasing the condition pulse current intensity increased the duration




81


of inhibition, the combination of feedforward and recurrent inhibition produced total inhibition of the PS amplitude response even at the current intensity of 25 percent of the PS maximum for the test response. With A/0 stimulation, the PS amplitude was inhibited only to 40 or 50 percent of control levels with 25 percent of the PS maximum condition pulse current intensity. A/0 paired pulse paradigms should predominantly activate recurrent inhibition. 0/0 paired pulse paradigms should activate both recurrent and feedforward inhibition. If the responses of the A/0 paired pulse paradigms are compared with the responses of the 0/0 paired pulse paradigms, it is clear that feedforward inhibition contributes significantly to the level of inhibition seen with 0/0 paired pulse paradigms in spite of facilitated EPSP responses found at IPIs of 20 to 30 msecs.

A more detailed comparison of the A/0 and 0/0 paired pulse paradigm responses revealed a potentially useful method for better characterizing feedforward inhibition in the CAl region of the hippocampus. As described above, 0/0 stimulation produced total inhibition of the PS even with a condition pulse current intensity of 25 percent of the PS amplitude at PS asymptote. The same A/0 condition pulse current intensity resulted in much less inhibition than the 0/0 paradigm (40 percent versus 100 percent inhibition). If the A/0 paired pulse stimulation currents were titrated with decreasing condition pulse current levels from the 25 percent of AS maximum amplitude level to that point at which the A/0 test pulse PS amplitude was just observed (threshold) and then that absolute current intensity was used with the 0/0 configuration, the effect of feedforward inhibition alone may be observed. One of the potential problems with this method is that the effect of the AS current




82


level may not be useful in terms of the separation of recurrent versus feedforward inhibitory influences when applied to the afferent fibers of the SR region. Nonetheless, this methodology may provide a most useful extracellular method by which to study the effects of feedforward inhibition using paired pulses.

Most studies have used a relatively high current intensity on the condition and test pulses when examining different treatment conditions. Different condition and test pulse current intensities may provide substantially different responses since the influence of feedforward and recurrent inhibition may, in effect, change from predominantly feedforward influences with a lower current intensity to more equal contributions from feedforward and recurrent inhibition with a higher current intensity. This conclusion is supported by evidence that indicates that there are two populations of feedforward inhibitory interneurons that have different firing patterns (Ashwood et al., 1984). One group of neurons typically fired more than one action potential with frequent bursts to stimulation which was above PS threshold. These bursts lasted from 30 to 300 msecs. The other group of inhibitory interneurons produced only one action potential even when stimuli were as high as 4.5 times threshold. Increasing the stimulus intensity produced shorter inhibitory interneuron spike latencies. It has also been shown that inhibitory interneurons can be activated at current levels which are subthreshold for a PS (Buszaki and Eidelberg, 1981; Knowles and Schwartzkroin, 1981). Thus, feedforward inhibition may actually prevent pyramidal cell firing when the stimulation current intensity is near threshold levels. The finding of inhibition at the 20 msecs IPI for the




83


0/0 subthreshold condition pulse with 50 percent of PS maximum test pulses can be explained by this subthreshold recruitment of feedforward inhibition.

The use of two 0/0 test pulse current intensities yielded responses which were not substantially different in regard to the effect of different condition pulse current intensities, when the amplitude or duration of inhibition were compared. These results suggest that the intensity of the condition pulse produces significant effects on the inhibitory phase of paired pulse testing without greatly altering the maximal levels of the potentiation phase. The major effect of the two different test pulse current intensities was that two different levels of potentiation were produced. Since it is known that feedforward inhibition can influence the amplitude of the PS with single pulse stimulation (Ashwood et al., 1984; Buszaki, 1984), the best explanation for the lower levels of potentiation found with the higher test pulse current intensity may be that there was a more powerful influence of feedforward inhibition with a higher test pulse current intensity. Recurrent inhibition would not be strongly implicated since the effect of recurrent inhibition on the response to the the two different test pulse current intensities would be the same given the same condition pulse current intensity. The major reason for the difference in maximal potentiation levels most likely emanates from the influence of the test pulse current intensity on the levels of feedforward inhibition.

Another possible explanation for the difference in the maximum

levels of potentiation observed between the 25 percent and 50 percent of the PS amplitude at PS asymptote test pulse current intensities is that there was a ceiling effect. Since the 0/0 PS amplitude percent of




84


control responses were lower with the 50 percent test pulse current intensity than with the 25 percent test pulse, it can be argued that, for paired pulse potentiation, there is a maximum percent change in response size to which the PS can increase. However, if that maximum size is reflected by the asymptotic levels obtained with single pulse stimulation during 1/0 testing, the levels of potentiation could have reached 200 percent of the control response levels since the test pulse current intensity was set to 50 percent of maximum, which they did not. Instead, a maximal level of 140 to 160 percent of control was reached for the 50 percent of PS maximum test pulse series. Similarly, the 25 percent of PS maximum test pulse series could have reached 400 percent of control responses if they had potentiated to the levels reached with high current levels during the I/0 testing. These levels of potentiation argue against the ceiling effect hypothesis as an explanation for differences between the low and higher current intensities on the test pulse of these paired pulse data.

Inhibition in the CAl region of the hippocampus is present ubiquitously. Although recording with single pulse afferent stimulation in the pyramidal cell body layer has often been assumed to be unaffected by inhibition, this premise is likely to be false. It has been demonstrated that inhibition is tonically active in the resting inhibitory interneuron (Alger and Nicoll, 1980) much like it has been shown for excitatory hippocampal pyramidal neurons (Brown et al., 1979). Thus, a background of spontaneous inhibition continuously occurs in the hippocampus against which any incoming stimuli may be modified. Recent characterizations of feedforward inhibition have provided evidence that the feedforward interneuron begins firing prior to the CAl pyramidal cell (Ashwood et




85


al.., 1984; Buzsaki, 1984). This observation implies that feedforward inhibition can therefore influence pyramidal cell firing even with single pulse stimulation. Extracellularly, the amplitude of the PS could be decreased as a function of the feedforward inhibition.

One of the first tests in many extracellular investigations is that of determining the 1/O curve. The 1/O curve provides a good description of the input current strength to response amplitude. It has typically been considered an unencumbered measure and has therefore been used to compare the basic response of one treatment group to another or to a control group. The inference has been that differences signify alterations in the normal functioning of the pyramidal cell. If feedforward inhibition acts prior to pyramidal cell firing and thereby alters the response, then the basic assumption of differences in I/O curves being due to changes in pyramidal cell function may be inaccurate. If different treatment conditions are being tested, it may be that the treatment condition has altered the feedforward inhibitory interneuron which may in turn influence the pyramidal cell firing characteristics. If feedforward interneurons fire differentially to various current intensities, feedforward inhibition may play a major role in determining the sigmoidal shaped I/O curve when tested with different current intensities. The abrupt appearance of the PS may be less reflective of the threshold for the PS Rer se, than it is the point at which the PS finally emerges from the previously overwhelming control of inhibitory mechanisms. Threshold differences between groups may therefore be a function of effects on feedforward inhibition.

These studies suggest that the magnitude of the PS amplitude and latency responses to different current intensities can be carefully




86


governed to recruit different levels and probably ratios of feedforward and recurrent inhibition. Lower current intensities appear to cause a greater predominance of feedforward- rather than recurrent-inhibition. At higher current intensities it is likely that feedforward and recurrent inhibition contribute more equally to the resulting PS amplitudes. Another point indicated by these results is that the current intensity used for the test pulse of paired pulses significantly modifies the magnitude of paired pulse potentiation observed. While the current intensity of the condition pulse influences the early phases of inhibition and the time to reach the maximal level of potentiation, it is the test pulse which most strongly influences the maximum level of potentiation achieved. It is suggested that feedforward inhibition influences the maximal magnitude of paired pulse potentiation rather than recurrent inhibition.

The use of extracellular recording techniques provide for an

examination of cellular events at a more system oriented level than other procedures. The exquisite lamination and arrangement of the hippocampal architecture provides unique opportunities for extracellular studies. The incorporation of new information on the effects of feedforward inhibition should allow for a better understanding as well as improved methodologies in considering what these responses mean and how they can be used to investigate the effects of other treatment conditions.















CHAPTER 4
GET AND SYNAPTIC INHIBITION: AN IN VITRO ANALYSIS Introduction

It has become increasingly clear that chronic ethanol abuse in

humans produces a range of behavioral deficits as well as a range in the severity of those deficits (Tarter and Alterman, 1984; Parsons and Leber, 1981; Parsons, 1977: Ron, 1977; Ryan, 1980). Anatomical studies of human alcoholics have shown general cerebral atrophy and lesions of certain nuclei (Wilkinson, 1982; Ron, 1977, 1982; Cala and Mastaglia, 1981; Brewer and Perrett, 1971; Victor et al., 1971). A major controversy has existed concerning whether alcohol itself or nutritional inadequacies were the cause of these changes (Victor et al., 1971). Animal studies which employed rigorous controls of the dietary intake have, however, provided substantial evidence that chronic ethanol abuse alone caused many of the behavioral, anatomical and functional deficits observed (Walker and Hunter, 1978; Walker and Freund, 1973; Freund and Walker, 1971; Denoble and Begleiter, 1979; Fehr et al., 1975; Tavares et al., 1983; Abraham et al., 1981, 1982, 1984; Durand and Carlen, 1984a, 1984b). Direct evidence of GET effects can be seen in the morphological alterations which have been reported in rats exposed to GET. A 15 to 20 percent decrease in the number of primary cells in the CAl region, the dentate gyrus and the cerebellum have been reported (Walker et al., 1980). CAI pyramidal cell and dentate granule cell spine density has been shown to be decreased (Riley and Walker, 1978). The dendritic branching patterns of these cells have also been shown to be reduced.

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Physiologically, it has been shown that GET produced increased amplitudes of the PS with paired pulses, but no changes in measures of the EPSP or the PS with single pulse stimulation procedures (Abraham et al., 1981). These changes were thought to be a result of a decrease in the efficacy of recurrent inhibition. Intracellular studies of GAl pyramidal cells have also found decreased amplitudes of IPSPs and long lasting after-hyperpolarizations (Durand and Garlen, 1984a). The evidence has thus become powerful concerning GET effects on the hippocampal region. The next set of questions can therefore address the issue of how GET may actually produce these alterations.

The finding that GET produced an increase in the amplitude of the PS of the test pulse of paired pulses without concomitant changes in the EPSP amplitude or slope and without differences found between control and GET animals on the PS of input/output curves led to the hypothesis that the changes in paired-pulse potentiation occurred as a function of a decrease in recurrent inhibition (Abraham et al., 1981). Recurrent inhibition in the GAl region of the hippocampus is a well described regulatory mechanism. Briefly, when the pyramidal cell is caused to fire by inputs to its dendrites, it, in turn, fires an inhibitory interneuron; presumably a basket cell. This interneuron then ramifies widely to the original as well as other pyramidal cells in its plexus and causes a massive cessation of spontaneous firing of the pyramidal cells for up to 600 to 700 msecs (Andersen et al., 1964a, 1964b). Recently, there has been new documentation for a second type of inhibition which operates in conjunction with recurrent inhibition. This type of inhibition is feedforward in nature (Andersen et al., 1982; Alger and Nicoll, 1980, 1982a). It is likely that the anatomical






89


configuration of this system is such that an interneuron is interposed between a fraction of the incoming afferents and the dendrites of both the basilar and apical dendritic regions of CAl pyramidal cells (Ashwood et al., 1984; Buzsaki, 1984; Schwartzkroin and Mathers, 1978).

When using orthodromic paired pulse stimulation of the SR, the

effects of both feedforward and recurrent inhibition are observed since both the feedforward and the recurrent interneuron are activated by afferent stimulation. The previously stated hypothesis concerning GET effects on recurrent inhibition may be incomplete or incorrect since both inhibitory systems were activated by SR stimulation in the studies of Abraham et al., 1981. Therefore, experimental paradigms designed to more selectively activate either feedforward or recurrent inhibition and similar to those used in Chapter 3, were performed in these experiments to help resolve this question.

This set of experiments was performed using the hippocampal slice preparation. There were two major reasons for switching to the in vitro preparation. First, one of the major problems with results from the in vivo experimental series was that the anesthesia used may have produced powerful effects on inhibition in general but on CET effects specifically. Since ethanol may be cross tolerant with barbiturates (Kalant et al., 1971), it is possible that there could be an interaction between the anesthesia and GET. There was little likelihood of this situation however, since the animals were removed from the ethanol containing diet for at least two months prior to testing. The second reason for utilizing the in vitro preparation is that direct pharmacological challenges can be made to the CAl region which are not possible in vivo. While pharmacological manipulations are not a part of the






90


experiments in this section pe e these experiments are crucial to the pharmacological experiments proposed in the following chapter since they lay the groundwork for comparisons between in vivo and in vitro results. It is important to verify that similar patterns of GET effects are found between in vitro and in vivo preparations.

The major negative characteristic of the in vitro technique

emanates from the fact that transverse hippocampal slices have reduced levels of inhibition most probably due to the severance of the longitudinal projections of the recurrent inhibitory interneurons (Teyler, 1980). The result of this decrease in inhibition is that paired pulse response curves do not typically show inhibition with IPIs less than 30 msecs whereas in vivo, inhibition is seen with IPIs as long as 80 to 150 msecs (Dunwiddie et al., 1980). Differences in the levels of inhibition between GET and normal animals should, however, be readily apparent if they exist.

A third question which was posed was whether heterosynaptic afferent paired pulse stimulation could be effectively used to provide additional insight into how and where the inhibitory systems might interact. It has been reported previously that stimulation of the SO or SR afferents with the condition pulse followed by SR or SO afferents, respectively, on the test pulse of paired pulses produced only inhibition (Haas and Rose, 1982; Steward et al., 1977). As indicated earlier A/0 paired pulses result in a moderately clean assessment of recurrent inhibition. Homosynaptic 0/0 paired pulses result in three different mechanisms being activated. Feedforward inhibition, recurrent inhibition and an EPSP facilitation are all seen to occur when stimulating with double pulses in the same afferent region. Heterosynaptic 0/0






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stimulation is similar to homosynaptic 0/0 stimulation in that both feedforward and recurrent inhibition are activated. Since inputs do not originate or impinge on the same pyramidal cell dendritic areas, however, no facilitation of the EPSP occurs. Thus, heterosynaptic 0/0 stimulation offers a way of observing the inhibitory systems of the CAl region without EPSP facilitation or PS potentiation also being induced. This heterosynaptic paired pulse paradigm may therefore provide an additional perspective from which to review the effects of CET on inhibition in CA1.

The three major questions addressed in these experiments are: 1.

Does CET produce alterations in feedforward as well as recurrent inhibition?; 2. Are the in vivo and in vitro preparations comparable as far as the effects of CET are concerned?; and 3. How does heterosynaptic SR/SO stimulation compare with SO/SR paired-pulse stimulation and with SR/SR and A/O paired-pulse stimulation?.



Methods

Animals

Animals used were housed and fed as described in the general methods.



Comments on in vitro technique

All of the experiments performed in this chapter and in the subsequent chapters were performed using the in vitro hippocampal slice technique. This technique has several advantages for the study of recurrent and feedforward inhibition in CAL. One of the most important advantages is the ability to place electrodes visually. Although






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placement of both the recording and stimulating electrodes can be precisely performed in viva, the in vitro experiments in the following chapter made use of microiontophoresis techniques using various pharmacological agents at specific locations within the CAl region. These manipulations would be difficult to perform in vivo. An additional feature in using the slice technique is that anesthetic agents, which have been shown to have powerful effects on hippocanipal inhibition, are not used. All major synaptic pathways are conserved in the hippocampal slice preparation (Teyler, 1980). However, inhibition is reduced since transverse projections of the basket cells are severed. Responses recorded in vivo have been compared with those recorded in vitro. Reports indicate the conservation of nearly all general characteristics between the two preparations (Schwartzkroin, 1981). That the advantages outweigh the disadvantages is attested to by the fact that the majority of the literature concerning recurrent and feedforward inhibition in the hippocainpus have been conducted in vitro (Dingledine and Gjerstad, 1980; Alger et al., 1981; Andersen et al., 1980; Lee et al., 1979; Teyler, 1980).



Annaratus

Commercially obtained tissue chambers were used in these experiments (Frederick Haer and Go). Static recording chambers were used in all experiments. The static chamber consisted of a static pool of artificial CSF which had a piece of filter paper stretched taut on a cylindrical ring on which the brain slice was placed such that diffusion of the artificial CSF occurred from beneath the slice. The static chamber was placed inside of a holding tank which included a d.c. heater




Full Text
230
the course of recovery from anatomical and electrophysiological perspec
tives, it is possible that responses to behavioral tasks which have been
shown to be altered by CET may also recover. These questions are
especially important in light of King's (1984) results.


RAW DATA (MSFC) RAW DATA (MV)
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CURRENT INTENSITY (UAMPS)


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The protocol that Andersen et al., (1982) used was as follows. Two
different stimulating electrodes were placed in both the SO and the SR
afferent zones of the CAl region. The recording electrode was placed in
the pyramidal cell region so that a PS was obtained from stimulation of
either the SO or the SR afferents. A micropipette containing GABA was
successively placed at 10 different locations along the dendritic axis
of the pyramidal cell (see Fig 5-1). A control response using afferent
stimulation from one of the dendritic zones of CAl pyramidal cells was
produced followed by an iontophoretic pulse of GABA after which a second
stimulus (test pulse) was delivered through the same dendritic zone.
The same procedure was then performed using the other stimulating
electrode until all 10 GABA application sites had been tested. The test
pulse after the iontophoresis of GABA was then reported as a percent of
the control pulse obtained 10 seconds before the GABA application. The
results showed two different patterns of responses dependent upon the
stimulation site. The SR stimulation electrode caused a potentiation of
the response when the GABA was applied in the distal SO region. This
potentiation changed to inhibition of the test response when located
more proximal to the pyramidal cell body layer but was still located on
the SO side of the pyramidal cell layer. The inhibition which began in
proximal SO then continued into the SR dendrites approximately 200
microns before it returned toward control levels near the SLM. The
response profile obtained with SO stimulation showed inhibition when
GABA was applied in the SO region which continued through the pyramidal
cell layer. However, within 25 to 50 uMs distal to the pyramidal cell
layer in the proximal SR, there was a decrease in inhibition followed by
a prolonged area of slight potentiation when GABA was applied in the SR


214
and Linesman, 1980; Corrigal, 1983). The effect of enkephalin adminis
tration was similar with both SO and SR stimulation, increasing the PS
amplitude to nearly equal levels. Proximal SO, distal SO and proximal
SR application of the enkephalin (2DA) resulted in comparable levels of
PS potentiation. Distal SR application resulted in minor effects and,
as observed with bicuculline administration, the effects observed can be
explained by diffusion toward the cell body region since the maximal
response amplitude was delayed in time.
The opiates are thought to produce increased PS amplitude responses
by disinhibiting the inhibitory interneurons. This pyramidal cell
response alteration may result from the inhibitory interneurons which
may decrease their firing rate or their release of GABA. Previous work
(Dingledine, 1981), has shown that the enkephalin, DADL, produced the
greatest magnitude of PS amplitude change either in SO or in the
pyramidal cell layer. His data suggested a slightly greater PS ampli
tude response with SO stimulation than with SR stimulation. The time
course of his drug effects are similar to those of the present series.
Ejection of DADL in distal SR resulted in much smaller response ampli
tude increases than were found more proximal to the cell layer and
were comparable to the present results.
The opiates are subserved by at least two physiologically important
classes of receptors, the mu and delta receptors. Both of these binding
sites have been reported in the hippocampus using biochemical techniques
(Chang and Cuatrecasas, 1979). A differential distribution within CAl
has also been reported. Mu receptors were found most concentrated in
the pyramidal cell body region while delta receptors were more diffusely
located in SO, SP and SR (Goodman et al., 1980; Herkenham and Pert,


DATA (MSEC)
104


ACKNOWLEDGEMENTS
The production of this dissertation was made possible by a number
of individuals directly and indirectly connected with the experimental
work.
Two people, Pat Burnett and Bill Creegan, have been most
instrumental in helping me to get the job done. Pat has helped to
smooth the rough edges with my slice work as well as encourage my
progress. Bill has been unendingly patient with my continual requests
for program modifications and explanations of how he did them. I thank
each of you sincerely for your expert help.
Other people have provided help at various times during this long
and arduous trek. I thank Larry Ezell, Dot Robinson, Dr. Paul Manis,
and Christina King for their time and efforts.
I would especially like to thank Dr. Roger Reep for his insightful
thoughts during some of my more challenging moments. His energy and
talents are second to none. I also want to thank Dr. Bob Davis for
getting me into the hippocampus and supporting me along the way. Dr.
and Mrs. Ken Finger have shared their thoughts on a variety of interesting
and helpful topics. I thank them for the opportunity to do so.
I would also like to thank Don Walker and Bruce Hunter for their
confidence in my abilities. They have helped to shape this research in
many ways. Thanks are also extended to Dr. Chuck Vierck, Dr. Floyd
Thompson, Dr. Don Stehouer, and Dr. Marc Branch for their help and
encouragement.
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225
concluding that CET does produce deleterious effects in the normal
functioning of the CAl pyramidal cell. The fact that the increased PS
response amplitude is seen over the range of IPIs discussed implicates
altered functioning of both recurrent and feedforward inhibitory mecha
nisms .
The effect of four putative neurotransmitter agonists or antagonists
were tested along the dendritic axis of the pyramidal neuron. GABA and
baclofen have been reported to produce an inhibition of the PS. GABA
has been shown to mediate both recurrent and feedforward inhibition
(Andersen et al., 1964a, 1982). Recently, it has been suggested that
baclofen may preferentially activate the GABA-sensitive bicuculline-
insensitive receptor, thus providing a GABA agonist which selectively
activates feedforward inhibition. Bicuculline, a GABA antagonist, has
been shown to competitively block GABA receptors. Finally, 2DA, an
opiate agonist, has been shown to result in increased PS amplitudes
possibly as a function of disinhibition.
GABA produced two different response patterns depending on whether
SO or SR afferents were stimulated. With SO stimulation maximal inhibi
tion was observed in the proximal SO and in the pyramidal cell layer.
Responses to GABA at other locations along the pyramidal cell dendrites
with SO stimulation showed potentiated PS responses. SR stimulation
resulted in a larger area of inhibited responses. Inhibition was seen
beginning approximately 200 uMs in SR extending through the pyramidal
cell layer and briefly into SO. Other locations along the dendrites
outside of this region with SR stimulation resulted in potentiated
responses. The magnitude of inhibition was not as great in these
studies as reported by Andersen et al. (1982). The magnitude of


243
Wigstrom, H. and Gustafsson, B. Two types of synaptic facilitation
recorded in pyramidal cells of in vitro hippocampal slices from guinea
pigs. Neurosci. Let., 1981, 26: 73-78.
Wilkinson, D.A. Examination of alcoholics by computed tomography (CT)
scans: A critical review. Alcholism: Clin. Exp. Res., 1982, 6 (1):
31-45.
Wilkinson, D.A. and Carien, P.L. Neurophysiological and neurological
assessment of alcoholism: Discrimination between groups of alcoholics.
J. Stud. Ale., 1980, 41: 129-139.
Wolf, D.L. and Haas, H.L. Effects of diazepines and barbiturates on
hippocampal recurrent inhibition. Arch. Pharm.. 1977, 299: 211-218.
Wolman, M. A fluorescent histochemical procedure for gamma-aminobutyric
acid. Histochemie. 1971, 28: 118-130.
Yamamoto, C. and Mcllwain, H. Electrical activities in thin sections
from the mammalian brain maintained in chemically-defined media in
vitro. J. Neurochem., 1966, 13: 1333-1343.
Zieglgansberger, W., French, E.D., Siggins, G.R. and Bloom, F.E. Opioid
peptides may excite hippocampal pyramidal neurons by inhibiting adjacent
inhibitory interneurons. Science. 1979, 205: 415-417.


Figure 5-2. I/O curves (means) obtained with stimulation from
the stratum radiatum. Data were normalized by
setting the current level at which the PS was
first seen equal to 0 uAs. Data were then
interpolated from that current value such that
interpolated I/O curves were produced with
current levels between zero and 500 uAs. This
procedure controlled for any differences in PS
threshold between groups. A) PS amplitude
responses with stratum radiatum stimulation
normalized between groups. B) PS latency
responses with stratum radiatum stimulation
normalized between groups.


Figure 3-1.
I/O curves for ijn vivo data. A) Group mean PS
amplitude responses by current level from 20 to
1000 uAs expressed in raw units (mVs); B) Group
mean PS latency measures shown as raw data
(msecs) by current intensity (uAs).


Figure 6-11. PS latency responses (means) after enkephalin
iontophoresis (time=0) with stratum radiatum
stimulation. Scales on ordinate represent difference
of PS latency response following bicuculline adminis
tration from control responses obtained prior to
bicuculline administration (test response-control
response) and are independent for each iontophoretic
ejection location. Abcissa represents time in minutes
following iontophoretic ejection of the drug. A) PS
responses with iontophoretic ejection in distal SO. B)
PS responses with iontophoretic ejection in proximal
SO. C) PS responses with iontophoretic ejection in
proximal SR. D) PS responses with iontophoretic
ejection in distal SR.


8
CNS damage may exhibit profound psychological impairments (Wilkinson and
Carien, 1980). It is difficult, if not presently impossible, to assume
a causal relationship between brain damage and cognitive dysfunction
(Parsons and Leber, 1981).
These problems have created an implicit need to better understand
and control the relevant variables necessary to obtain reliable rela
tionships between alcohol abuse and brain damage. To do this, better
clinical histories of patients would be helpful; however, the accuracy
provided can never be completely reliable. An alternative method of
gaining a better understanding of the relationships between variables
would be to control these variables in the manner of a laboratory
experiment. The ethical problems associated with this alternative are
numerous. Thus, scientists have developed animal models of chronic
alcohol consumption in an effort to determine the effect of alcohol per
se on the CNS. Control of variables such as age, drinking history,
genetic makeup and nutritional adequacy are then feasible.
The Hippocampal Model and Memory
Since the most devastating aspect of Korsakoff's syndrome is the
profound anterograde amnesia, the study of memory is crucial to an
understanding of the alterations which Korsakoff's syndrome produces.
Several regions of the CNS have been implicated in memory processing.
Of those regions reported to be affected in Korsakoff's syndrome, the
temporal stem, the temporal cortex, the medial dorsal thalamus, the
mammillary bodies and the hippocampus have all been considered to be
involved in some way with memory processing (Horel, 1978; Victor et al.,


236
Frederickson, R.C.A., Nickander, R., Smithwick, E.L., Shuman, R. and
Norris, F.H. Pharmacological activity of met-enkephalin and analogues
in vitro and in vivo depression of single neuronal activity in specified
brain regions. In Opiates and Endogenous Opioid Peptides. Kosterlitz,
H.W. (ed.). Elsevier, Amsterdam, 1976, 239-246.
Freund, G. Alcohol withdrawal syndrome in mice. Arch. Neurol.. 1969,
21: 315-320.
Freund, G. Chronic central nervous system toxicity of alcohol. Ann.
Rev. Pharmacol.. 1973, 13: 217.
Freund, G. Normal shuttle box avoidance learning after chronic
phenobarbital intoxication in mice. Psvchopharm.. 1974, 40: 199-203.
Freund, G. Effects of chronic alcohol and vitamin-E consumption on
aging pigments and learning performance in mice. Life Sci. 1979, 24:
145-152.
Freund, G. Benzodiazepine receptor loss in brains of mice after chronic
alcohol consumption. Life Sci., 1980, 27: 987-992.
Freund, G. and Walker, D.W. Impairment of avoidance learning by
prolonged ethanol consumption in mice. J. Pharmacol. Exp. Ther.. 1971,
179: 284-292.
Gall, C., Brecha, N., Karten, H.J. and Chang, K-J. Localization of
enkepahlin-like immunoreactivity to identified axonal and neuronal
populations of the rat hippocampus. J. Comp. Neurol.. 1981, 198:
335-350.
Goldstein, D.B. and Pal, N. Alcohol dependence produced in mice by
inhalation of ethanol: Grading the withdrawal reaction. Science.
1971, 172: 288-290.
Goodman, R., Snyder, S.H., Kuhar, M.J. and Young, W.S. Differentiation
of delta and mu opioid receptor localizations by light microscopic
autoradiography. Proc. Natl. Acad. Sci. U.S.A.. 1980, 77: 6239-6243.
Greenberg, D.A., Cooper, E.C., Gordon, A. and Diamond, I. Ethanol and
the GABA-aminobutyric acid-benzodiazepine receptor complex. J.
Neurochem., 1984, 42 (4): 1062-1068.
Haas, H.L. and Rose, G. Long-term potentiation of excitatory synaptic
transmission in the rat hippocampus: The role of inhibitory processes.
J. Physiol., 1982, 329: 541-552.
Haas, H.L. and Ryall, R.W. Is excitation by enkepahlins of hippocampal
neurons in the rat due to presynaptic facilitation or to disinhibition.
J. Physiol., 1980, 308: 315-330.


Finally, I thank my wife, Jane, for standing with me during the
difficult times and sharing with me the enjoyable moments. She deserves
much of the credit for putting this manuscript together as well as for
keeping me going.
And, as always, I thank Mom and Dad for their unending support.
We have finally made it!
iv


41
All recordings were amplified by Grass P511 preamplifiers, filtered
(Krohn-Hite) at either .3Hz-10KHz (field potentials) or 300Hz-10KHz
(extracellular unit activity) and displayed on Tektronix oscilloscopes.
All responses were collected on an Apple computer system for subsequent
computer analysis. Analog to digital conversion within the computer
employed an Interactive Structures Inc. AI13 board. For these experi
ments, 512 data points were collected during a 28 msecs period. Each
data point had a maximum amplitude resolution of one in 4096.
A calibration pulse (1 mV, 2 msecs) was routinely injected immedi
ately prior to each stimulation pulse with a Stoelting CA5 calibrator.
This calibration pulse not only controlled for alterations in equipment
status, but also allowed for uncomplicated adjustment of the gain of the
preamplifiers and of the scaling of the data for subsequent computer
display and analysis.
Raw data were analyzed for the in vivo experiments on an Apple II
Plus computer system whereas all data from the in vitro experiments were
transferred from the Apple computer to an IBM 5150 (PC) for data analy
sis. All data were stored on 5.25 inch floppy disks. After analyses of
the waveforms, all measures were transferred via modem to the North East
Regional Data Center (NERDC) computer system for statistical analyses
using the Statistical Analysis System (SAS) General Linear Models (GLM)
analysis of variance procedures. Graphics were produced using the IBM
Displaywriter system with Chartpak software or the IBM 5150 using Lotus
Inc.'s Symphony software and a Hewlett Packard 7470A plotter.


145
ALV
LM


49
inhibitory interneuron or project to both an inhibitory interneuron and
pyramidal cell dendrites (Ashwood et al., 1984; Buzsaki, 1984).
Substantial evidence exists for the presence of interneurons
located in the pyramidal cell layer and the SO and SR dendritic areas of
the CAl region. Peak concentrations of GAD, an enzyme which catalyzes
the formation of GABA, is restricted to interneurons located in the
pyramidal cell layer and SLM (Storm-Mathisen, 1977). Immunoreactive
staining to GAD showed sparse staining throughout the dendritic regions
of both the hippocampal CA regions and the dentate gyrus. A more
heavily stained region was found in the distal apical dendrites (Barber
and Saito, 1976). These same cells are also observed with
cholecystokinin and enkephalin immunoreactive staining (Gall et al.,
1981; Harris et al., 1985; Stengaard-Pedersen et al., 1983).
Interneurons with dendritic and axonal orientations parallel to the
dendritic axis of CAl pyramidal cells have also been described (Cajal,
1968; Lorente de No, 1934; Schwartzkroin and Mathers, 1978). Ashwood et
al. (1984) have provided evidence that some of the interneurons in the
pyramidal cell region may be feedforward inhibitory since orthodromic
activation of the Schaffer collateral/commissural system resulted in
some of the inhibitory interneurons firing prior to the pyramidal cell.
The electrophysiological and pharmacological evidence for the
existence of both feedforward and recurrent inhibition is convincing
(Alger et al., 1982; Andersen et al., 1980, 1982). However, most
studies have utilized intracellular recordings to arrive at these
conclusions. While intracellular recordings are most appropriate in
defining the existence and details of feedforward and recurrent


102
group on the second I/O curve was 165 uAs + 14.85 SEM and 158 uAs + 5.56
SEM for the sucrose group.
The second set of I/O curves were obtained from the second set of
animals used in the paired pulse experiments. Since one of the primary
objectives was observing the effect of four different stimulation
configurations which included the use of the SO afferent field, I/O
curves were obtained with stimulation in the SR as well as the SO
afferent regions. Fig 4-3 shows the response of alcohol and sucrose-fed
animals with SR stimulation while Fig 4-4 shows the responses of the two
groups with SO stimulation. While no significant differences were seen
between the two groups with stimulation from either the SR or SO
synaptic zone, the alcohol animals tended to have lower PS amplitudes
than the sucrose control animals in this set of animals. The PS
threshold for the alcohol group with SR stimulation was 113.33 7.52
uAs while it was 112.5 +4.96 uAs for the sucrose group. The PS
threshold obtained with SO stimulation was 96.67 + 4.82 uAs for the
alcohol group and was 90.0 + 6.83 uAs for the sucrose group.
Paired pulse results. The paired pulse data collected during these
experiments are similar to the in vitro paired pulse data collected from
other laboratories (Dunwiddie et al., 1980). The general pattern of in
vitro inhibition and potentiation was, however, different from in vivo
preparations (Dunwiddie et al., 1980). In all of the present in vitro
0/0 homosynaptic paired pulse experiments, there was little if any
inhibition seen, even at short IPIs. Instead of a period of inhibition,
potentiation was seen almost immediately. Heterosynaptic paired pulse
experiments showed no potentiation, but did show inhibition and


percent change from control
percent change from control
s 8
8000
8000
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t
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79
the pyramidal cell layer rather than in the synaptic zone. Recent
testing with recordings from the pyramidal cell layer and from the
Schaffer/commissural afferent synaptic zones in our laboratory has
confirmed that EPSP slope measures obtained from the synaptic zone match
those reported from other laboratories, while EPSP slope measures
recorded from the pyramidal cell layer matched the data from the present
experiments. These inconsistencies in EPSP measures from the SR synaptic
zone versus the pyramidal cell layer may be due to the occurrence of
several overlapping events. In the SR region, the EPSP is relatively
free of other synaptic influences such as the firing of the pyramidal
cell or recurrent inhibitory interneurons. When recording from the
pyramidal cell layer, the PS may partially or completely obliterate the
EPSP thus making the EPSP measure more difficult to analyze. In addi
tion, the IPSP from the recurrent and/or feedforward inhibitory inter
neuron may summate with the EPSP (due to the inward flow of current in
the SR excitatory region and the outward flow of current from the
inhibitory interneurons in the pyramidal cell region) and therefore
result in uninterpretable measures. It is our conclusion that the EPSP
data recorded from the cell layer do not represent the same phenomena
seen in the synaptic zones. It is likely that they reflect a summation
of the EPSP and IPSP and therefore it is difficult if not impossible to
make any conclusions regarding any alterations that may be occurring in
the SR synaptic region. EPSP slope data will therefore not be presented
in any of the subsequent chapters.
The A/0 paired pulse PS amplitude data at IPIs less than 80 msecs
were also similar to those reported by others (Dingledine and Langmoen,
1980). With IPIs of 60 to 80 msecs, however, where the test pulse PS


218
commissural fibers terminate selectively on neurons which are in some
way different from the ipsilateral schaffer fibers in their response to
CET.
Alternatively, if methionine- or leucine-enkephalin does not affect
recurrent or feedforward inhibition as indicated by Dingledine (1981),
it may be possible that the opiate effects represent direct actions on
the pyramidal neuron. In this case, it is possible that CET results
either in a loss of receptors per se or a decrease in the sensitivity
of those receptors to increased opiate release. This may occur as a
compensatory mechanism to the hypothesized effect of CET on the
enkephalinergic system or as a function of TIQS (Berger et al., 1982).
Thus, it is possible that with long-term ethanol ingestion a shift in
the sensitivity of the pyramidal cell opiate receptor may occur with SO
afferent activity. Again, this differential sensitivity of CET effects
with SO and SR stimulation may result from differential distribution of
commissural CA3 fibers to SO while ipsilateral fibers project to the SR
region.
The present data suggest that CET may act discriminately on various
aspects of the CAl inhibitory system. They also point out the
importance of testing different afferent systems. The intricacies of
the CAl inhibitory systems are only beginning to be understood. A
complex interaction of various neurotransmitters are involved in the
control of pyramidal cell firing. Although several of these pharmaco
logical systems have been explored in the current manuscript others
have been shown to produce response modifications of the CAl pyramidal
cell by mechanisms not yet well detailed. Consideration of these
systems may provide important clues to a better understanding of CET
effects.


Figure 4-2. I/O curves for Set 1 slice experiments after all
paired pulse stimulation experiments were com
pleted. Data were normalized by setting the
current level at which the PS was first seen
equal to 0 uAs. Data were then interpolated from
that current value such that interpolated I/O
curves were produced with current levels between
zero and 500 uAs. This procedure controlled for
any differences in PS threshold between groups.
A) Normalized PS amplitude responses (means) for
the alcohol and sucrose groups in mVs by current
intensity (uAs). B) Normalized PS latency
responses (means) for the alcohol and sucrose
groups in msecs by current intensity (uAs).


RAW DATA (MSEC) RAW DATA
99


142
procedure was an attempt to directly address whether or not CET
produced alterations in recurrent inhibition. Thus, the hypothesis
concerning decreases in recurrent inhibition as a result of CET
appears to be supported.
While it is apparent that CET produces an effect on recurrent
inhibition, it is probable that CET also affects feedforward inhibition.
Effects on feedforward inhibition have not been easy to dissociate using
the paired pulse technique since feedforward inhibition cannot be
activated without also activating recurrent inhibition and hence con
founding the influence of feedforward inhibition. However, it appears
that the contribution of recurrent versus feedforward inhibition may be
dependent upon the intensity of the condition and test pulse of paired
pulses (see Chapter 3 discussion). In order to better address the
question of feedforward versus recurrent inhibition, other experimental
approaches might provide a better dissociation than does the paired
pulse technique.
Since CET produces deleterious effects on recurrent and feedforward
inhibition in the CAl region of the hippocampus and the inhibitory
mechanisms of this region are GABA mediated, a localized pharmacological
approach to the inhibition question might prove to be helpful. The
application of GABA at discrete locations along the dendritic axis of
the CAl pyramidal cell would provide for both, an indication of the
effect of CET on direct GABAergic modifications of PS responses and
whether the effect of CET on GABA mediated inhibition is greater at the
cell body layer or out in the dendrites. Andersen et al., (1982) used
such a technique which they suggested allowed for the extracellular
localization of recurrent and feedforward (or discriminative) inhibition.


224
The responses observed with heterosynaptic paired pulse stimulation
resulted only in inhibited PS amplitudes and looked much like responses
found after A/0 stimulation. While it has been reported previously that
0/0 heterosynaptic paired pulses resulted in greater durations and
magnitude of inhibition than 0/0 homosynaptic paired pulses (Haas and
Rose, 1982), the responses found in the current studies did not fit this
order. It was suggested that these differences may be a function of
differences in the bathing medium temperature. Haas and Rose (1982)
used a temperature of 32 to 33 degrees centigrade, while the present
studies used temperatures of 34 to 35 degrees centigrade. Small differ
ences in temperature can significantly alter the excitability of the
pyramidal cell (Teyler, 1980).
CET was seen to significantly reduce the magnitude of the decay
phase of recurrent inhibition in vitro. This finding tentatively
supports the conclusion of Abraham et al. (1981). However, it is also
possible that the difference seen between the two groups is a function
of feedforward inhibition. This is suggested since antidromic stimula
tion has been shown to produce maximal levels of inhibition at 50 msecs
post-stimulus (Alger and Nicoll, 1982). The effects of CET were seen
through 400 msecs IPIs. Effects in this time period are more likely a
function of feedforward inhibition since maximal orthodromically acti
vated inhibition is seen at approximately 200 msecs after stimulus
onset. 0/0 homosynaptic paired pulse responses, however, resulted in
maximal group differences at shorter (30 to 80 msecs) IPIs, thus strongly
implicating decrements in recurrent inhibition. The fact that the
trends for increased PS amplitudes were found across each of the 0/0
homosynaptic paired pulse paradigms provides a reasonable basis for


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133
IPIs of 150 or 200 msecs. The higher test pulse current intensity
resulted in lower levels of potentiation as was seen with the in vivo
results reported earlier. The peak levels of potentiation for the
current intensity of 25 percent of the PS amplitude at PS asymptote on
the in vitro test pulse were over 200 percent of control. The test
pulse current intensity of 50 percent of the PS amplitude at PS asymp
tote with the in vitro preparation was under 200 percent potentiation.
The SO homosynaptic paired pulse PS response amplitudes from
control animals showed a similar pattern of potentiation to those
obtained with SR homosynaptic stimulation. Potentiation occurred
abruptly but not until after the 30 msecs IPI. The two stimulation
sites resulted in the peak levels of potentiation occurring by 100 msecs
IPIs. There were two notable differences however. SR stimulation
resulted in a marginally greater level of potentiation but also in
creased to the maximal level more quickly than the SO stimulation site
responses. It is possible that more inhibition resulted from the SO
stimulation site than the SR site since it has been suggested that
commissural afferents may preferentially synapse onto inhibitory
interneurons (Buzsaki, 1984). Furthermore, the SO region has been shown
to have a greater autoradiographic grain count density than SR from
fibers originating in the contralateral CA3 region (Swanson et al.,
1978).
The A/0 paired pulse experiments performed in vitro were very
similar to those performed in vivo. There was an initial, sharp in
crease in the level of inhibition at IPIs of 20 to 30 msecs followed by
a decay phase which, if the current intensity on the condition and test
pulse was high, lasted beyond the range of IPIs tested in vitro but
which returned toward control levels by IPIs of 1000 to 2000 msecs in


114
A/0 paired pulse paradigms -- Set 1
The first set A/O paired pulse PS response amplitudes are shown in
Fig 4-7. The antidromic series in vitro data (Fig 4-7A) with 50 percent
of the antidromic maximum on the condition pulse and 25 percent of the
PS amplitude at PS asymptote on the test pulse compared well with the
in vivo antidromic data (Fig 3-5). The strongest inhibition was seen at
the 20 msecs IPI for both the alcohol and sucrose groups. There were
two apparently different periods of inhibition. One was seen at very
short IPIs of 20 to 30 msecs while the other was much more prolonged and
was seen at IPIs from 40 msecs to 150 or 200 msecs. Although the
alcohol group was somewhat less inhibited (50 to 55 percent) at short
IPIs than was the sucrose group (40 percent), the two groups converged
by the 50 msecs IPI until control levels were reached. The PS latency
data showed (Fig 4-8A) both groups to have longer latencies to the PS
maximum than their respective control responses. The sucrose group also
had longer response times (.3 to .5 msec) than the alcohol group (.1 to
.25 msec) for all of the IPIs shown.
The A/0 paired pulse responses with 100 percent of the AS maximum
and 50 percent of the PS amplitude at PS asymptote on the test pulse are
shown in Fig 4-7B. The alcohol and sucrose groups both revealed a
maximum inhibition level of 40 percent of the control response at the 20
msecs IPI. The alcohol group then returned to near control levels by the
100 msecs IPI. The sucrose group, however, remained inhibited to at
least 70 percent of the amplitude of the control response beyond 400
msecs. This pattern was similar to that seen in vivo (Fig 3-5) where
the test pulse PS amplitude remained inhibited to approximately 70
percent until the IPIs of 1000 to 2000 msecs. The PS amplitude


RAW DATA (MSEC) RAW DATA (MV)
106


232
Altshuler, H.L. Animal models for alcohol research. In Current in
Alcoholism. Vol. VIII, Galanter, M. (ed.). Grue and Stratton, New
York, 1981, 343-357.
Andersen, P. Brain slice work: Some prospects. In Brain Slices.
Dingledine, R. (ed.). Plenum Press, New York, 1984, 375-380.
Andersen, P. Interhippocampal impulses II: Apical dendritic activation
of CAl neurons. Acta Physiol. Scand., 1960, 48: 178-208.
Andersen, P., Bie, B. and Ganes, T. Distribution of GABA sensitive
areas on hippocampal pyramidal cells. Exp. Brain Res., 1982, 45:
357-363.
Andersen, P., Bie, B., Ganes, T. and Laursen, A.M. Two mechanisms for
effects of GABA on hippocampal cells. In Iontophoresis and Transmitter
Mechanisms in the Mammalian Central Nervous System. Ryall, R.W. (ed.).
Elsevier/North-Holland Biomedical Press, 1978, 179-181.
Andersen, P., Blackstad, T.W. and Lomo, T. Location and identification
of excitatory synapses on hippocampal pyramidal cells. Exp. Brain.
Res.. 1966, 1: 236-248.
Andersen, P., Bliss, T.V.P. and Skrede, K.K. Lamellar organization of
hippocampal excitatory pathways. Exp. Brain Res., 1971, 13: 222-238.
Andersen, P., Dingledine, R., Gjerstad, L., Langmoen, I.A. and Laursen,
A.M. Two different responses of hippocampal pyramidal cells to
application of gamma-amino butyric acid. J. Physiol., 1980, 305:
279-296.
Andersen, P., Eccles, J.C. and Loyning, Y. Location of postsynaptic
inhibitory synapses on hippocampal pyramids. J. Neurophvsiol.. 1964a,
27: 592-607.
Andersen, P., Eccles, J.C. and Loyning, Y. Pathway of postsynaptic
inhibition in the hippocampus. J. Neurophvsiol.. 1964b, 27: 608-619.
Andersen. P. and Lomo, T. Mode of control of hippocampal pyramidal cell
discharges. In Neural Control of Behavior. Whalen, R.E. (ed.).
Academic Press, New York, 1970.
Ashwood, T.J., Lancaster, B. and Wheal, H.V. In vivo and in vitro
studies on putative interneurones in the rat hippocampus: Possible
mediators of feed-forward inhibition. Brain Res., 1984, 293: 279-291.
Assaf, S.Y. and Miller, J.J. Neuronal transmission in the dentate
gyrus: Role of inhibitory mechanisms. Brain Res., 1978, 151: 587-592.
Barber, R. and Saito, K. Light microscopic visualization of GAD and
GABA-T in immunocytochemical preparations of rodent CNS. In GABA in
Nervous System Function. Roberts, E., Chase, T.N. and Tower, D.B.
(eds.). Raven Press, New York, 1976, 113-132.


216
point where the maximal PS response amplitude occured. The delay to the
maximal response in each of these graphs (Fig 6-14C and 6-14D) was
probably a function of the diffusion of these drugs. It can be seen
that with SR stimulation the proximal SR iontophoretic site was the most
responsive to 2DA administration. With SO stimulation, the same pattern
held for the sucrose group but not for the alcohol group. This suggests
that the major site of action of the opiates is in proximal SR and that
CET results in a maximally decreased response amplitude to 2DA at this
site.
The finding of trends toward increased PS amplitudes with
iontophoretic application of enkephalin along the dendrites and SR
stimulation after CET when compared to sucrose-fed controls can be
explained by the decreased inhibition found in the CA1 region following
long term ethanol treatment. If 2DA acts by disinhibiting the
inhibitory interneuron, then any condition which has previously reduced
the efficacy or number of these interneurons would be expected to be
further reduced with enkephalin administration. This further reduction
in inhibition would be seen as an increase in the PS amplitude of CET
animals as compared to controls.
The responses to 2DA administration with SO stimulation after CET
were reversed compared to the responses obtained with SR stimulation in
terms of the magnitude of the PS amplitude response by group. The
greatest difference was seen when enkephalin was applied in proximal SR
with SO stimulation. In this case the alcohol group response was
decreased by nearly 400 percentage points. These findings are surpris
ing since no differences by iontophoretic application site or stimula
tion site of this nature have been found with the other pharmacological
agents tested.


97
gms and for the sucrose group was 537.8 + 13.25 gms. The mean alcohol
consumption of the second set of animals was 13.17 + .25 g/Kg. The mean
body weight for the alcohol animals was 541 + 10.55 gms and the sucrose
group was 532 + 15.11 gms.
I/O relationships. Two different sets of animals were used in
performing the experiments reported in this chapter. For the first set
of animals an I/O function was obtained prior to any other experimental
manipulation. A second I/O curve was then obtained after all of the
paired pulse experiments had been completed for the same animals. These
two I/O curves were collected in order to determine whether any gross
changes in pyramidal cell excitability occurred during the course of the
experimental session which was two to four hours in duration. There
were 10 animals in the alcohol treated group as well as 10 animals in
the sucrose-fed control group. The data presented for all of the
subsequent I/O curves were normalized according to the PS onset. Thus,
the first current level at which a PS was observed was normalized to a
"zero" current level. Subsequent data points were obtained by interpo
lating the PS responses to the current intensities as indicated in the
general methods, but only from 0 to 500 uAs. This normalization was
performed in order to control for individual differences between animals
and groups in terms of the PS threshold. As can be seen in Figs 4-1
(pre-paired pulse I/O curve) and 4-2 (post-paired pulse I/O curve) there
were no significant differences between the alcohol and sucrose groups.
The alcohol animals did, however, tend to have slightly higher PS
amplitudes especially at PS asymptote. The PS threshold for the alcohol
group on the initial I/O curve was 159 uAs 12.15 SEM while the sucrose
control group was 181 uAs 18.28 SEM. The PS threshold for the alcohol


Figure 1-2. A CA1 stratum radiatum or stratum oriens evoked extracellular field potential
response recorded in or dorsal to the pyramidal cell layer. The labelled
elements of the waveform are as follows: A, peak of 1 mV (2 msecs) calibration
pulse injected prior to stimulation; B, baseline recording area (for computer
analysis and scaling the distance between the average of x points on A and y
points on B was used to individually scale each waveform for amplitude
measurements); C, stimulus artifact; D, onset of population EPSP (current sink
in distant stratum radiatum, thus seen as current source dorsal to pyramidal
cell layer); E, onset of PS (also, slope was calculated from point D to point
E); F, maximal negative peak of PS (PS latency was measured from point D to
point F); G, post PS peak of EPSP (probably reflects IPSP current sources as
well. Population spike was calculated by adding PS segment between points E and
F and F and G and dividing by two.


Figure 6-12. PS amplitude responses (mean + SEM) after enkephalin
iontophoresis (time=0) with stratum oriens stimulation.
Scales on ordinate represent difference of PS amplitude
response following bicuculline administration from
control responses obtained prior to bicuculline adminis
tration (test response-control response) and are
independent for each iontophoretic ejection location.
Abcissa represents time in minutes following ionto
phoretic ejection of the drug. A) PS responses with
iontophoretic ejection in distal SO. B) PS responses
with iontophoretic ejection in proximal SO. C) PS
responses with iontophoretic ejection in proximal SR.
D) PS responses with iontophoretic ejection in distal
SR.


221
pyramidal cell is closer to threshold as a result of the depolarizing
response. This response has been called "discriminative inhibition"
since it may result in a selective filtering of afferent input
(Andersen et al., 1982).
The finding of increased PS amplitudes and conclusion of decreased
recurrent inhibition by Abraham et al. (1981) raises two important
questions. First, does CET result in alterations in recurrent
inhibition alone or does CET also result in alterations of feedforward
inhibition? The second question is related to how CET may produce
these effects on inhibition from a more mechanistic perspective. What
are the effects, after CET, of different putative transmitter agonists
and antagonists known to influence CA1 inhibition?
The present studies were designed to characterize recurrent and
feedforward inhibition using extracellular techniques. While intra
cellular recording methodologies have been used to assess recurrent and
feedforward inhibition, extracellular recording offers the advantages of
simpler recording protocols as well as the ability to screen different
treatment and testing conditions more easily. The paired pulse tech
nique was initially used with different stimulation configurations in an
effort to dissociate recurrent and feedforward inhibition. The A/0
paired pulse configuration was used to assess the effect of recurrent
inhibition. Activation of the CAl pyramidal cell axons antidromically
by the condition pulse results in an orthodromic activation of the
recurrent inhibitory interneuron. If a test pulse is then administered
a predetermined interval after the condition pulse, the magnitude of the
recurrent inhibition can be assessed. 0/0 homosynaptic paired pulses
result in the activation of both feedforward inhibitory interneurons and
recurrent inhibitory interneurons. Additionally, an EPSP facilitation


130
the control responses by up to .3 msec. The group by IPI interaction
statistic for the PS latency data was significant (F(1,17) 1.93) p =
.01. Subsequent T-test analyses of the individual IPIs for the two
groups showed significant effects as shown with asterisks. The PS
amplitude responses for the SO/SR heterosynaptic experiment are shown in
Fig 4-11C. There were no consistent trends in the data for differences
between the alcohol and sucrose groups, however. The latency data for
this configuration are shown in Fig 4-12C. There was an increase in the
time to PS maximum for both groups by up to .3 msec which was maximal
between 30 and 40 msecs IPIs. The latencies returned to control levels
at longer IPIs. No significant differences between groups were found
however.
A/0 paired pulse paradigm
The final experimental paradigm of this series utilized A/0 stimu
lation with 100 percent of the AS maximum on the condition pulse and 25
percent of the PS amplitude at PS asymptote on the test pulse. The PS
amplitude data (Fig 4-11A) exhibited the normal antidromic response
pattern. There was strong inhibition of the PS response amplitude at
short IPIs (20 to 100 msecs) followed by a gradual return to control
levels. The alcohol group was maximally inhibited to 55 percent of the
control response while the sucrose group was inhibited to 25 percent of
its respective control response. This difference (though not signifi
cant) between the alcohol and sucrose groups was maintained for IPIs
beyond 400 msecs. The PS latency data for this experiment are shown in
Fig 4-12A. An increase in PS latency by up to .4 msec was seen for both
the alcohol and sucrose groups followed by a slow return toward baseline
levels.


14
are severely affected. This is apparent with maze learning behavior
where learning in normal animals takes place quickly. In hippocampal
lesioned animals and humans we see that these tasks can be learned but
that it takes much longer. It has been suggested that normally an
animal uses a spatial-time or contextual coding process for learning
these tasks (Hirsch et al., 1978). However, when the hippocampus is
damaged, all learning must take place in terms of stimulus-response
chains which are more difficult to develop. Finally, it can be seen
that tasks which require timing behavior are severely affected. Timing
is also thought to be a major aspect of contextual coding. Thus,
spatial and time cues produce a context dependent memory. It appears
that this memory is severely affected by hippocampal lesions (O'Keefe
and Nadel, 1978; Hirsch et al., 1978).
Chronic Ethanol Treatment (CET) and Behavior in Animals
There are many important similarities between the behavioral
changes associated with animals chronically treated with alcohol and
animals with hippocampal lesions. Freund (1974) showed that mice, after
four months on a liquid diet containing ethanol, were deficient in both
the acquisition and retention of a passive avoidance task. One way
active avoidance was not affected when tested with hamsters given an
ethanol containing diet; however, the ethanol exposure was only for
seven days (Harris et al., 1979). Ethanol consuming rats were found to
be deficient in the acquisition of Hebb-Williams mazes and in the
acquisition and retention of a moving belt task (Fehr et al., 1975).
Spontaneous alternation was also shown to be deficient. Tasks requiring
timing behavior such as temporal shock discrimination and DRL schedules


Figure 4-5. 0/0 homosynaptic paired pulse PS amplitude responses (means + SEM) with
stimulation through stratum radiatum. All test responses are shown as percent
changes from control responses (test response/control response X 100). A) Test
pulse responses obtained with the condition pulse current intensity set to
obtain an EPSP response 50 percent of the EPSP amplitude at PS threshold for
both the control and sucrose groups. Test pulse response was set to 25 percent
of the PS amplitude at PS asymptote. B) Test pulse responses obtained with 25
percent of the PS amplitude at PS asymptote on both the condition and test
pulses. C) Test pulse responses obtained with the condition pulse adjusted to
50 percent of the PS amplitude at PS asymptote and 25 percent of the PS
amplitude at PS asymptote on the test pulse. D) Test pulse responses obtained
with 50 percent of the PS amplitude at PS asymptote on both the condition and
test pulses.


65
this controlled for individual animal differences in terms of the EPSP
onset as well as the different stimulation current intensity at which it
occurred. The PS latency can be used as a measure of pyramidal cell
excitability since it generally decreases with increasing PS amplitude.
The EPSP (Fig 3-4A) was monitored by measuring the slope of the
rising phase of the EPSP. These responses showed little change as a
result of the different condition pulse current intensities and thus
remained near control levels from 20 to 3000 msecs, the entire range of
testing. A decrease in the slope of the EPSP was noted at IPIs between
20 and 150 msecs. Previous reports indicate that a facilitation of the
EPSP is normally seen with IPIs of 20 to 30 msecs (Creager et al., 1980;
Lomo, 1971).
0/0 50 percent test pulse series
The 0/0 50 percent test pulse series PS amplitude responses (Fig
3-2B) showed similar patterns for the test response PS amplitude when
compared to the 0/0 25 percent data described above. The major differ
ence, however, was that the maximal level of potentiation was lower with
the 50 percent test pulse series PS responses. Like the 25 percent test
pulse data, increasing the condition pulse current intensity above PS
threshold levels produced increasing durations of inhibition in each of
the experimental series. Each of the superthreshold condition pulse
current intensities produced complete inhibition of the PS amplitude at
IPIs of 20 msecs. The maximal PS inhibition was, in general, extended
in duration with increasing condition pulse current intensities from 20
msecs at lower condition pulse current intensities to 50 or 60 msecs at
100 percent of the condition pulse PS response amplitude at asymptote.


125
the sucrose group latencies were only .4 msec shorter than their control
responses. Both groups were close to baseline levels by the 400 msecs
IPI.
0/0 heterosvnantic paired-pulse paradigms -- Set 2
The heterosynaptic 0/0 paired pulse responses are shown in Fig
4-11B and 4-11C. Both experiments utilized condition and test pulse
current intensities set at 25 percent of the PS amplitude at PS asymp
tote. The general pattern of responses to the heterosynaptic stimula
tion was different from that using homosynaptic 0/0 stimulation. The
pattern of responding was more similar to A/0 experiments than to
homosynaptic 0/0 experiments. There was an intial, maximal level of
inhibition to 40 percent of the control response at the 20 msecs IPI
followed by a return to control levels by 60 to 80 msecs IPIs.
The PS amplitude responses for the SR/S0 heterosynaptic 0/0 paired
pulse experiment are shown in Fig 4-11B. Although the difference was
small and not statistically significant, the alcohol group was less
inhibited than was the sucrose group at IPIs from 20 to 200 msecs. The
alcohol group was inhibited 45 to 50 percent of the control response at
an IPI of 20 msecs, while the sucrose group was inhibited 30 to 35
percent at the 20 msecs IPI. Although the alcohol group returned to
control levels by the 60 msecs IPI, the sucrose group remained inhibited
to the 200 msecs IPI. The latency to PS onset data are shown in Fig
4-12B. The alcohol group latency was up to .1 msec longer than the
control response at IPIs from 20 to 50 msecs after which it became
shorter than the control response by up to .2 msec. The alcohol group
PS latency measures remained shorter than control responses beyond 400
msecs IPIs. The sucrose group latencies were consistently longer than


157
GABA iontophoresis with SR stimulation. The effects of CET on
the responses to GABA iontophoresis at ten different locations along
the pyramidal cell dendritic tree are seen in Fig 5-4A. Although none
of the differences were statistically significant, it can be seen that
the CET group mean percent of control PS amplitude measures were more
inhibited in the pyramidal cell region and SR and less potentiated
than sucrose-fed control responses in the SO region. There was a
difference between the two groups of 25 to 40 percentage points for
those locations. The PS latency data (Fig 5-5A) were not statistically
different for the group effect but were for the group by IPI interaction
(F(l,226)-2.13) p>.028. The locations which proved to be statistically
different using follow up T-tests are noted by asterisks.
SO I/O relationships. Long-term ethanol administration did not
result in any significant differences on either the PS amplitude (Fig
5-3a) or PS latency (Fig 5-3b) measures. The PS threshold for the
alcohol group was 124.94 + 24.48 and was 93.57 + 4.49 for the sucrose
group.
GABA iontophoresis with SO stimulation. The effect of CET on the
response to GABA iontophoresis at the ten locations along the
pyramidal cell dendrites using SO stimulation are seen in Fig 5-4B.
In this experiment, the CET responses were more inhibited in the SO
dendritic region and less potentiated in the SR dendritic region than
the sucrose controls. This pattern of responses was very different
from the responses obtained with SR stimulation. With SO stimulation,
responses were only inhibited in the SO region very close to the


241
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10
alleviate severe seizure activity in a number of patients (Penfield and
Milner, 1958; Scoville and Milner, 1956; Scoville, 1954). Although the
seizure activity of these patients was dramatically reduced, severe
effects were seen on memory processes. One of these patients, H.M., has
been well characterized. Anatomically, the anterior two-thirds of
H.M.'s hippocampi were bilaterally removed as well as part of the
temporal lobe extending 8 mms posterior to the temporal poles. The most
striking effect of these lesions was an immediate and profound
anterograde amnesia. Retrograde amnesia was also found but only for a
relatively short (approximately two years) period of time immediately
prior to the operation. H.M. could detail his earlier years without
problem. Intellectually, H.M.'s intelligence quotient score was not
different from normal. H.M.'s short term memory (STM) was also intact.
However, he had a profound impairment of long term memory (LTM) includ
ing both verbal and non-verbal tasks. Visually and tactually guided
mazes were learned only when the number of choices was reduced to be
within STM memory span (usually seven to nine choices) and even so H.M.
had much more difficulty with tactile mazes (Milner et al., 1968).
H.M.'s acquisition performance was worse than normal in these tasks
although once learned, he was able to retain the information over long
periods of time (Milner et al., 1968).
A Comparison of Hippocampal Deficits with CET Deficits
A brief comparison of the features of Korsakoff's syndrome with
those of medial temporal resections results in a number of similarities.
Both cases exhibit severe anterograde amnesia with both verbal and
non-verbal tasks. Pre-lesion (retrograde) memory is intact in both


223
these results were a function of decreased levels of inhibition found
with the slice preparation or as a result of transecting the longitu
dinal inhibitory fibers (Teyler, 1980). It is therefore concluded
that urethane anesthesia was not related to the increased PS response
seen in vivo.
In general, increased condition pulse current intensity resulted in
increased durations of inhibition using 0/0 homosynaptic paired pulses.
Higher test pulse currents resulted in a decreased level of maximal
potentiation. Results from the current in vivo and in vitro experiments
support these conclusions. It is suggested that increasing condition
pulse stimulus intensity resulted in an increased recruitment of
inhibitory interneurons (both recurrent and feedforward) as well as
recruitment of different populations of these interneurons (Buzsaki,
1984). Even the lowest 0/0 superthreshold condition pulse current
intensity, however, resulted in the complete elimination of the PS at
20 msecs IPIs.
Increasing the current intensity of the condition pulse of A70
paired pulses resulted in an increased magnitude of inhibition. The
responses seen with A/0 stimulation consisted of a maximally inhibited
response found at the 20 msecs IPI followed by a prolonged decay phase
lasting hundreds of msecs. The maximal magnitude of inhibition of the
test pulse PS was to 20 percent of the control response. A/0 condition
pulses never resulted in a complete abolishment of the test pulse PS
response. This finding coupled with the 0/0 homosynaptic results
suggests that feedforward inhibition contributes substantially to the
magnitude and duration of inhibition observed with homosynaptic paired
pulse stimulation.


134
vivo. The maximal levels of inhibition found in both the in vivo and in
vitro experiments were at about 20 to 40 percent of the control
response. These findings suggest that there may not be substantial
differences in the responses of recurrent interneurons between in vivo
and in vitro preparations as has been suggested. Instead, it may be
that there is a decrease in the efficacy of feedforward inhibition or
that there is an increased excitability of the pyramidal cells in vitro.
Heterosynaptic paired pulse stimulation provided an additional way
in which to observe the effects of orthodromic paired pulse stimulation
without the effects of paired pulse EPSP facilitation or PS potentiation
superimposed. The pattern as well as the levels of heterosynaptic
paired pulse stimulation are very similar to those found with A/0
stimulation. The major difference between heterosynaptic orthodromic
stimulation and A/0 stimulation is that heterosynaptic stimulation
should set up feedforward inhibition as well as recurrent inhibition on
the condition pulse while A/0 stimulation should predominantly activate
recurrent inhibition. The maximal level of inhibition would therefore
be expected to be greater and for a longer duration with heterosynaptic
stimulation. Haas and Rose (1982) showed that with the in vitro hippo
campal preparation, the levels of inhibition with different stimulation
configurations, fell into an ordered progression. They found that A/0
stimulation produced the least amount and shortest level of inhibition.
Homosynaptic 0/0 stimulation produced more inhibition and for a longer
duration than A/0 stimulation and that heterosynaptic 0/0 stimulation
produced the greatest amount of inhibition and lasted for the longest
period of time. The present results did not follow this pattern.
Instead, A/0 and heterosynaptic 0/0 stimulation produced comparable
levels of inhibition in the present hippocampal slice preparation.


60
percent condition pulse (Fig 3-2A). The 50 percent condition pulse
produced a test pulse response pattern which was midway between the 25
percent and 100 percent condition pulse patterns. The total durations
of inhibition for the 25, 50 and 100 percent condition pulses were 50,
75 and 120 msecs, respectively. The condition pulse current intensity
of 50 percent of the EPSP at PS threshold produced no inhibition of the
PS.
The highest levels of potentiation in the 25 percent test pulse
series for each of the condition pulse current intensities were nearly
the same in each of these experiments. The highest levels of poten
tiation were approximately 200 to 250 percent of the control responses.
The effects of the different condition pulse current intensities were
seen in the time it took to reach the highest level of potentiation as
well as the point at which the PS first became potentiated. The condi
tion pulse current intensity of 50 percent of the EPSP at PS threshold
became maximally potentiated at an IPI of 40 msecs and subsequently
decreased toward control levels. The 25 percent of PS amplitude condi
tion pulse potentiation peaked at 150 msecs, the 50 percent of PS
amplitude maximum condition pulse peaked at 200 msecs, and the 100
percent of PS amplitude maximum condition pulse peaked at 300 msecs.
All of the experiments in which the condition pulse current intensities
were set above PS threshold returned toward control levels together
beginning at approximately 400 msecs and ending by 2000 msecs IPIs.
The PS latencies, calculated by measuring the interval from the
EPSP onset to the PS maximum, were increased by up to 1.2 msecs with
increasing condition pulse current intensities but only at shorter IPIs
(Fig 3-3A). These latencies then returned to control levels together by
400 msecs IPIs. PS latencies were calculated from the EPSP onset since


89
configuration of this system is such that an interneuron is interposed
between a fraction of the incoming afferents and the dendrites of both
the basilar and apical dendritic regions of CAl pyramidal cells (Ashwood
et al., 1984; Buzsaki, 1984; Schwartzkroin and Mathers, 1978).
When using orthodromic paired pulse stimulation of the SR, the
effects of both feedforward and recurrent inhibition are observed since
both the feedforward and the recurrent interneuron are activated by
afferent stimulation. The previously stated hypothesis concerning CET
effects on recurrent inhibition may be incomplete or incorrect since
both inhibitory systems were activated by SR stimulation in the studies
of Abraham et al., 1981. Therefore, experimental paradigms designed to
more selectively activate either feedforward or recurrent inhibition and
similar to those used in Chapter 3, were performed in these experiments
to help resolve this question.
This set of experiments was performed using the hippocampal slice
preparation. There were two major reasons for switching to the in vitro
preparation. First, one of the major problems with results from the in
vivo experimental series was that the anesthesia used may have produced
powerful effects on inhibition in general but on CET effects
specifically. Since ethanol may be cross tolerant with barbiturates
(Kalant et al., 1971), it is possible that there could be an interaction
between the anesthesia and CET. There was little likelihood of this
situation however, since the animals were removed from the ethanol
containing diet for at least two months prior to testing. The second
reason for utilizing the in vitro preparation is that direct pharmaco
logical challenges can be made to the CAl region which are not possible
in vivo. While pharmacological manipulations are not a part of the


51
with the use of subthreshold condition pulses for the PS of CA1 pyrami
dal cells.
The first goal of this study was an attempt to dissociate recurrent
from feedforward inhibition using extracellular recordings. The method
used was that of employing paired pulses with two different stimulation
configurations: an A/0 and an 0/0 configuration. The 0/0 configuration
utilized one stimulating electrode which was placed in the SR of CA1 and
through which both the condition and test pulse stimuli were emitted.
This configuration would activate both the recurrent and feedforward
inhibitory mechanisms as well as inducing an EPSP facilitation (observed
when recording from the CAl pyramidal cell apical dendritic region).
The A/0 configuration utilized two independent stimulation electrodes
located in two different pathways. The electrode used for the condition
pulse was placed in the alveus to activate antidromically the efferent
fibers of the CAl pyramidal cell and thus, to activate orthodromically,
the recurrent inhibitory interneuron (normally as a result of pyramidal
cell firing). The second stimulating electrode was placed in the SR, as
above, for the test pulse stimulation. The antidromic configuration
should activate only recurrent inhibitory interneurons with the condi
tion pulse, theoretically leaving the second or test pulse response to
be modified predominantly by the effects of recurrent inhibition.
A second goal of these experiments was to more completely describe
the parameters of paired pulse inhibition and potentiation. Most
studies utilizing paired pulses have used IPIs from 15 to 200 msecs. It
has been reported that there are other inhibitory effects which can only
be seen with longer interpulse intervals and which may be active for as
long as two seconds, although they may be observable only with


Figure 6-8. PS amplitude responses (means) after baclofen
iontophoresis (time=0) with stratum oriens stimulation.
Scales on ordinate represent difference of PS amplitude
response following bicuculline administration from
control responses obtained prior to bicuculline adminis
tration (test response-control response) and are
independent for each iontophoretic ejection location.
Abcissa represents time in minutes following ionto
phoretic ejection of the drug. A) PS responses with
iontophoretic ejection in distal SO. B) PS responses
with iontophoretic ejection in proximal SO. C) PS
responses with iontophoretic ejection in proximal SR.
D) PS responses with iontophoretic ejection in distal
SR.


73
and 1000 msecs. The A/O 50 percent test pulse series responses, like
the A/0 25 percent test pulse series responses, were inhibited for the
entire duration of IPIs tested but only to 80 to 90 percent of control
levels.
The PS latencies for these experiments are shown in Fig 3-6B. The
PS latencies increased from .1 to .4 msec between 20 and 200 msecs IPIs
for all of the condition pulse current intensities. The higher the
condition pulse current intensity, the longer the latency found to the
onset of the PS. Like the PS amplitude responses, the PS latency
responses were intermediate between 200 and 1500 msecs IPIs. Like the
A/0 25 percent test pulse series, the A/0 50 percent test pulse latency
response did not return to control levels with the IPIs tested.
Slope measures are shown in Fig 3-7B. Except for the brief de
crease in slope at the 20 msecs IPI seen with the 100 percent condition
pulse current intensity, there was only a small decrease in slope
observed with the IPIs tested.
Discussion
The effect of varying the current intensity of the condition and
test pulse of paired pulses was investigated using both 0/0 and A/0
stimulation configurations. An attempt was made to determine the
relative contribution of feedforward and recurrent inhibition with these
extracellular paired pulse paradigms. All of the experiments were
conducted in CAl using an anesthetized in vivo rat hippocampal prepara
tion.
The general characteristics of the superthreshold PS amplitude
response patterns found with 0/0 paired pulse stimulation for all of the


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Zk
Don W. Walker, Chairman
Professor of Psychology and
Neuroscience
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Marc Branch
Professor of Psychology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
%
7/
Floyd Thompson
Associate Professor
Neuroscience
of


50
inhibition, it would also be useful to be able to measure the effects of
these two types of inhibition extracellularly.
Recurrent inhibition has often been studied extracellularly using
paired pulses delivered from the same stimulation site, usually in
stratum radiatum. With this paradigm, the effects of recurrent inhibi
tion and paired-pulse potentiation were thought to be studied. However,
with the recent evidence for feedforward inhibition, it is clear that
any study of inhibition using this configuration is confounded if the
objective of the experiment is the study of recurrent inhibition alone.
The use of an antidromic stimulation site for the condition pulse with
an orthodromic stimulation site for the test pulse, however, should
provide a relatively better extracellular assessment of recurrent
inhibition. This technique has been utilized for the study of recurrent
inhibition previously (Andersen et al., 1964a; Dingledine and Langmoen,
1980; Haas and Rose, 1982). Thus, recurrent inhibition can be studied
relatively independently using extracellular antidromic/orthodromic
(A/0) paired pulses. Feedforward inhibition, however, is more difficult
to study extracellularly. Recurrent inhibition is almost always acti
vated with orthodromic stimulation unless pharmacological and/or ionic
manipulations could be utilized. Because A/0 paired pulses activate
recurrent inhibition alone while 0/0 paired pulses activate both recur
rent and feedforward inhibition, it is proposed that if the results of
0/0 paired pulses are compared with A/0 paired pulses, the difference,
if the amplitude of A/0 responses were compared to the 0/0 responses,
may provide a reasonable estimate of feedforward inhibition using
extracellular methods. Another possible method by which feedforward
inhibition might be studied without recurrent inhibitory influences is


Figure 2-1. A CA1 stratum radiatum or stratum oriens evoked extracellular field potential
response recorded in or dorsal to the pyramidal cell layer. The labelled
elements of the waveform are as follows: A, peak of 1 mV (2 msecs) calibration
pulse injected prior to stimulation; B, baseline recording area (for computer
analysis and scaling the distance between the average of x points on A and y
points on B was used to individually scale each waveform for amplitude
measurements); C, stimulus artifact; D, onset of population EPSP (current sink
in distant stratum radiatum, thus seen as current source dorsal to pyramidal
cell layer); E, onset of PS (also, slope was calculated from point D to point
E); F, maximal negative peak of PS (PS latency was measured from point D to
point F); G, post PS peak of EPSP (probably reflects IPSP current sources as
well. Population spike was calculated by adding PS segment between points E and
F and F and G and dividing by two. G, H and I, second PS detection points. For
analysis, second spike was measured by adding amplitude between G and H and H
and I then dividing by two. This calculated value was then added to calculated
value for first PS.


TABLE OF CONTENTS CONTINUED
PAGE
5 EFFECT OF CET ON IONTOPHORETIC GABA MEDIATED RESPONSES ... 141
Introduction 141
Methods 146
Results 150
Discussion 160
6 EFFECT OF CET ON THE IONTOPHORETIC APPLICATION OF BICUCULLINE,
BACLOFEN AND ENKEPHALIN MEDIATED RESPONSES IN CAl 168
Introduction 168
Methods 170
Results 173
Discussion 205
7 GENERAL DISCUSSION 219
REFERENCES 231
BIOGRAPHICAL SKETCH 244
vi


difference from control difference from control
159
iontophoretic location
iontophoretic location


240
Nicoll, R.A. and Newberry, N.R. A possible postsynaptic, inhibitory
action for GABA(b) receptors on hippocampal pyramidal cells. Neuropharm..
1984, 23 (7B): 849-850.
O'Keefe J. and Nadel, L. The Hippocampus as a Cognitive Map. Oxford:
Clarendon Press, 1978.
Parker, E. and Noble, E. Alcohol and the aging process in social
drinkers. J. Stud. Ale., 1980, 41: 170-178.
Parsons, O.A. Neuropsychological deficits in alcoholics: Facts and
Fancies. Alcoholism Clin. Exp. Res., 1977, 1: 51-56.
Parsons, O.A. and Leber, W.D. The relationship between cognitive
dysfunction and brain damage in alcoholics: Causal, interactive, or
epiphenomenal? Alcoholism: Clin. Exp. Res., 1981, 5 (2): 326-342.
Penfield, W. and Milner, B. Memory deficit produced by bilateral
lesions in the hippocampal zone. A.M.A. Arch. Neurol. Psychol.. 1958,
79: 475-497.
Pohorecky, L.A. Animal analog of alcohol dependence. Fed. Proc.. 1981,
40 (7): 2056-2064.
Porjesz, B. and Begleiter, H. Human evoked brain potentials and
alcohol. Alcoholism: Clin. Exp. Res., 1981, 5: 304-316.
Riley, J.N. and Walker D.W. Morphological alterations in hippocampus
after long-term alcohol consumption in mice. Science. 1978, 201:
646-648.
Robinson, J.H. and Deadwyler, S.A. Morphine excitation: Effects on
field potentials recorded in the in vitro hippocampal slice.
Neuropharm.. 1980, 19: 507-514.
Ron, M.A. Brain damage in chronic alcoholism: A neuropathological
neuroradiological and psychological review. Psych. Med.. 1977, 7:
103-112.
Ron, M.A., Acker, W., Shaw, G.K. and Lishman, W.A. Computerized
tomography of the brain in chronic alcoholism: A survey and follow-up
study. Brain. 1982, 105: 497-514.
Rovira, C., Ben-Ari, Y. and Cherubini, E. Somatic and dendritic actions
of y-aminobutyric acid agonists and uptake blockers in the hippocampus
in vivo. Neurosci.. 1984, 12 (2): 543-555.
Ryan, C. Learning and memory deficits in alcoholics. J. Stud. Ale.,
1980, 41: 437-447.
Schwartzkroin, P.A. To slice or not to slice. In Electrophvsiologv of
Isolated Mammalian CNS Preparations. Kerkut, G.A. and Wheal, W.V.
(eds.). Academic Press, New York, 1981, 15-50.


88
Physiologically, it has been shown that CET produced increased
amplitudes of the PS with paired pulses, but no changes in measures of
the EPSP or the PS with single pulse stimulation procedures (Abraham et
al., 1981). These changes were thought to be a result of a decrease in
the efficacy of recurrent inhibition. Intracellular studies of CAl
pyramidal cells have also found decreased amplitudes of IPSPs and long
lasting after-hyperpolarizations (Durand and Carien, 1984a). The
evidence has thus become powerful concerning CET effects on the hippo
campal region. The next set of questions can therefore address the issue
of how CET may actually produce these alterations.
The finding that CET produced an increase in the amplitude of the
PS of the test pulse of paired pulses without concommitant changes in
the EPSP amplitude or slope and without differences found between
control and CET animals on the PS of input/output curves led to the
hypothesis that the changes in paired-pulse potentiation occurred as a
function of a decrease in recurrent inhibition (Abraham et al., 1981).
Recurrent inhibition in the CAl region of the hippocampus is a well
described regulatory mechanism. Briefly, when the pyramidal cell is
caused to fire by inputs to its dendrites, it, in turn, fires an inhi
bitory interneuron; presumably a basket cell. This interneuron then
ramifies widely to the original as well as other pyramidal cells in its
plexus and causes a massive cessation of spontaneous firing of the
pyramidal cells for up to 600 to 700 msecs (Andersen et al., 1964a,
1964b). Recently, there has been new documentation for a second type of
inhibition which operates in conjunction with recurrent inhibition.
This type of inhibition is feedforward in nature (Andersen et al., 1982;
Alger and Nicoll, 1980, 1982a). It is likely that the anatomical


166
the CAI pyramidal cell. The up-regulation of GABAergic receptors could
be viewed as a compensatory response to the decreased input resulting
from inhibitory interneuronal cell death. This up-regulation, however,
must be opposed by some other process since it would be expected that
paired pulses stimulation would result in a smaller sized PS. A
decreased release of GABA or a loss of GABAergic afferent synapses could
result in such an up-regulation of GABAergic receptors.
Alternatively, the efficacy of other neuro-modulatory systems may
result in changes to the inhibitory systems of the CAl region. It has
been shown with immunocytochemical staining, that enkephalin containing
cells exist throughout the CAl region (Gall et al., 1981). The effect
of opiate peptides on extracellular CAl pyramidal cell responses is to
increase the amplitude of the PS. This increased PS amplitude is
thought to be due to a disinhibition of the inhibitory interneuron. The
CET induced increase of the PS amplitude may therefore represent an
alteration of the enkephalinergic interneurons. The increased response
to iontophoretic GABA application after CET may be related to this
alteration of opioid mechanisms.
The hippocampus has been shown to have the capability for anatomi
cal compensatory changes following CET. King (1984) showed that immedi
ately following 20 weeks of ethanol exposure, there was a trend toward a
relative decrease in spine density in CAl pyramidal cell dendrites
located in SO, SR proximal to the cell body, SR distal to the cell body,
and in the SIM. However, after 20 weeks of ethanol abstinence, there
was a recovery of CAl pyramidal cell spine density in these regions to
levels equal to or above the sucrose-fed controls tested at similar
points in time. Thus, it appears that a major compensatory reorganiza-


Figure 4-8. A/O paired pulse PS latency responses (means) with condition pulse stimulation
through the alveus and test pulse stimulation through stratum radiatum. All
test responses are shown as a difference from control responses (test response -
control response in msecs). A) Test pulse responses obtained with the
antidromic condition pulse current intensity set to obtain a response 50 percent
of the maximum antidromic amplitude. Test pulse response was set to 25 percent
of the PS amplitude at PS asymptote. B) Test pulse responses obtained with 100
percent of the antidromic spike amplitude on the condition pulse and 50 percent
of the PS amplitude at PS asymptote on the test response.


This dissertation is dedicated to my wife, Jane,
and to my parents, Carl F. and Sophie Rogers,
for their unending support.


196
Enkephalin series: SR stimulation
I/O relationships with SR stimulation. See description of I/O
curves as described in Chapter 5. I/O curves for each of the
pharmacological series were combined and presented earlier.
Iontophoresis application with SR stimulation. There were no
statistically significant differences between the alcohol and sucrose
groups for the PS amplitude measure (Fig 6-10) when 2DA application was
in the distal SO with SR stimulation. There was, however, a statisti
cally significant group effect for the PS latency measure (Fig 6-11)
when 2DA was applied in distal SO (F(l,21) 5.81, p .025). The
analyses for the application of 2DA in SO near the cell body layer with
SR stimulation revealed no statistically significant differences between
the two groups nor any interaction effects for PS amplitude or latency
measures. Application of 2DA at sites in SR near the cell body layer or
in distal SR did not result in any differences between groups on either
PS amplitude or PS latency measures.
Enkephalin series: Stratum oriens stimulation
I/O relationships. See description of I/O curves as described in
Chapter 5. I/O curves for each of the pharmacological series were
combined and presented earlier.
Iontophoresis application with SO stimulation. The data for the
application of 2DA at the dendritic locations with SO stimulation are
shown in Fig 6-12 for the PS amplitude data and in Fig 6-13 for the PS
latency data. The most noteworthy observation of these data was that


Figure 6-1. Arrows represent iontophoresis sites for bicuculline,
enkephalin, and baclofen along the dendritic axis of
CA1 pyramidal cells. Note that recordings were of
field responses and that the effects were upon groups
of pyramidal cells rather than single cells. Drugs
were ejected for five seconds with varying currents.
Abbreviations: ALV, alveus; SO, stratum radiatum; SP,
stratum pyramidale; SR, stratum radiatum; SLM, stratum
lacunosum-moleculare.


137
It has been suggested that inhibitory interneurons may respond with
different firing activity depending on the current intensity used
(Ashwood et al., 1984). At lower current intensities, inhibitory
interneurons tended to fire more than one action potential with frequent
bursts to stimulation which was above the PS threshold. Another group
of inhibitory interneurons produced only one action potential even when
stimuli were up to 4.5 times threshold. The present results suggested
that firing to lower stimulation current intensities may not be altered
(since the lower current intensity A/0 paired pulse series CET and
sucrose groups both returned to baseline levels by 150 msecs IPIs).
Higher current intensity stimulation on the condition and test pulse,
which may result in longer duration firing, may be altered in CET
animals. Alternatively, CET may reduce the efficacy of feedforward or
recurrent inhibitory interneurons such that transmitter depletion occurs
rapidly (Andersen et al., 1982).
Although there were no statistically significant differences
between the alcohol and sucrose responses with homosynaptic 0/0 stimula
tion, the trend toward greater paired pulse potentiation from CET
animals occurred consistently. The PS latency responses revealed trends
which supported those found with the PS amplitude responses. In
general, there was a decrease in PS latency when the CET animals were
compared to the sucrose-fed controls.
The heterosynaptic paired pulse experiments did not reveal signifi
cant differences between the CET and sucrose groups on the PS amplitude
responses. However, the trend for the CET animals to be less inhibited
than the sucrose animals continued. The PS latency responses at several
of the IPIs for the CET animals were shown to be significantly different


84
control responses were lower with the 50 percent test pulse current
intensity than with the 25 percent test pulse, it can be argued that,
for paired pulse potentiation, there is a maximum percent change in
response size to which the PS can increase. However, if that maximum
size is reflected by the asymptotic levels obtained with single pulse
stimulation during I/O testing, the levels of potentiation could have
reached 200 percent of the control response levels since the test pulse
current intensity was set to 50 percent of maximum, which they did not.
Instead, a maximal level of 140 to 160 percent of control was reached
for the 50 percent of PS maximum test pulse series. Similarly, the 25
percent of PS maximum test pulse series could have reached 400 percent
of control responses if they had potentiated to the levels reached with
high current levels during the I/O testing. These levels of potentia
tion argue against the ceiling effect hypothesis as an explanation for
differences between the low and higher current intensities on the test
pulse of these paired pulse data.
Inhibition in the CA1 region of the hippocampus is present ubiqui
tously. Although recording with single pulse afferent stimulation in
the pyramidal cell body layer has often been assumed to be unaffected by
inhibition, this premise is likely to be false. It has been demonstrated
that inhibition is tonically active in the resting inhibitory interneuron
(Alger and Nicoll, 1980) much like it has been shown for excitatory
hippocampal pyramidal neurons (Brown et al., 1979). Thus, a background
of spontaneous inhibition continuously occurs in the hippocampus against
which any incoming stimuli may be modified. Recent characterizations of
feedforward inhibition have provided evidence that the feedforward
interneuron begins firing prior to the CAl pyramidal cell (Ashwood et


132
(Dunwiddie et al., 1980; Teyler, 1980). It has, however, been
suggested that the in vitro data may actually be more similar to
non-anesthetized, normal animals than it is usually considered to be
(Dunwiddie, personal communication) since in vivo experiments typically
use barbiturate anesthesia which has been shown to increase the efficacy
of inhibitory systems (Nicoll et al., 1975). With barbiturate
anesthesia, the inhibitory systems are probably more effective than in
the unanesthetized, freely moving animal. Only with the current inten
sity of 50 percent of the PS amplitude at PS asymptote on both the
condition and test pulse of homosynaptic paired pulse stimulation, and
only in the sucrose animals, was any inhibition seen. This finding can
be explained by two independent explanations. First, the sucrose group
would be expected to exhibit more inhibition than the CET group since
CET has been shown to produce trends toward increased PS amplitudes (a
result of decreased inhibition) for nearly all of the present paired
pulse potentiation series. Secondly, if, as proposed in Chapter 3, the
intensity of the test pulse recruits more feedforward interneurons to
fire, hence more inhibition, it is consistent that in this case the
higher test pulse current intensity would be the most likely to show
inhibition in vitro. The inhibition seen here would not, however, be
feedforward alone but a combination of recurrent and feedforward inhibi
tion since the inhibition is seen at short IPIs.
The patterns of paired pulse potentiation were similar between the
in vivo and in vitro preparations except that the IPI at which the
maximal level of potentiation was reached was shorter in vitro. The in
vitro responses were maximally potentiated at 30 to 300 msecs whereas
the in vivo data did not reach comparable levels of potentiation until


229
suggest that SO afferents terminate on a set of interneurons which are
differentially affected by CET.
It has recently been shown that immediately after 20 weeks of CET
there is a decrease in CAl pyramidal cell spine density, but that 20
weeks after ethanol discontinuance the spine density of the pyramidal
cell recovers to levels above controls (King, 1984). These findings
raise one of the most important questions for the present findings. Are
the effects of CET that we are observing the same as the effects we
would see shortly after ethanol discontinuance and are they the same
effects we would see after 20 weeks off of the ethanol diet? Are we
seeing direct effects of ethanol treatment eight to 16 weeks after
ethanol discontinuance or are we seeing the effects of CET after compen
satory reorganization? While lesioned animals undergo the majority of
compensatory changes by 30 days post-lesion (Matthews et al., 1976b),
there is no satisfactory evidence available to indicate whether the
toxicological effects of CET may extend or shorten this recovery period.
These questions are important in regard to the implications for the
present results. If we are in the middle of the compensatory process,
the present findings may have little bearing on what the same responses
might be after just a few weeks more recovery. Alternatively, compensa
tory changes may have ended well before the current observations were
made, in which case the current results take on significant meaning.
Obviously, it is now important to explore the time course of recovery in
detail. It may be possible that different subfields of the hippocampus
have different time courses of recovery or different courses of compen
satory change as well. It is also possible that different types of
neurons have different recovery characteristics. In line with following


172
TABLE 6-1
Cone.
pH
Eject
Retain
Solution
Enkephalin
30 mM
NA
40 nA
20-30 nA
165 mM NaCl
GABA
1 M
3.5
40 nA
5 nA
distilled H20
pH adjusted
Bicuculline
25 mM
NA
40 nA
15-20 nA
165 mM NaCl
Baclofen
75 mM
NA
40 nA
20-30 nA
165 mM NaCl
Drug concentrations, ejection currents, retaining currents,
ejection durations, and pH level for each of the drugs used in these
studies.


23
current passed through the stimulating electrode is raised to a level
sufficiently high to activate action potentials from a large number of
pyramidal cells, a field population spike (PS) becomes superimposed on
the field extracellular excitatory post-synaptic potential (EPSP)
(Andersen et al., 1971; Lomo, 1971). This PS is seen as a sharp nega
tive going spike (Fig 1-2).
Extracellular recording paradigms in CAl have generally been used
to evaluate three relatively distinct kinds of information. The first
type of paradigm is designed to test synaptic efficacy and is termed an
input/output (I/O) function. This paradigm consists of increasing the
stimulation current level in steps in order to determine response
characteristics such as EPSP threshold, PS threshold and respective
asymptotic levels. The second type of paradigm is designed to test for
synaptic plasticity. Briefly, at least two types of response enhance
ment plasticity are seen in the hippocampus. One is seen as a short
lasting increase in the size of the EPSP or PS amplitude in reference to
a baseline pulse either with paired pulses or immediately after tetanic
stimulation (these phenomena are termed potentiation). This type of
potentiation lasts for periods ranging from several milliseconds to
seconds (Creager et al., 1980). Another type of potentiation is longer
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experimental procedures used to assess or induce these types of poten
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inhibition (produced through interneurons) as well as potentiation. In
this procedure, two pulses, a condition and a subsequent test pulse, are


237
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Figure 5-4.
PS amplitude responses (mean + SEM) obtained
after GABA iontophoresis at ten different loca
tions along the dendritic axis of CA1 pyamidal
cells. PS amplitude responses are expressed as a
percent of the control response obtained immedi
ately prior to the iontophoresis of GABA at each
location indicated (GABA (test) response/ control
response X 100). A) GABA mediated PS amplitude
responses obtained with stimulation from the
stratum radiatum. B) GABA mediated PS amplitude
responses obtained with stimulation from the
stratum oriens.


68
When compared to the 0/0 25 percent test pulse responses, there were no
shifts in the duration of inhibition. The point at which the PS ampli
tude crossed control levels to the potentiation phase ranged from 50
msecs for the condition pulse current level set to PS threshold to
approximately 130 msecs for the 100 percent of PS amplitude at asymptote
condition pulse current intensity. The condition pulse current intensity
of 50 percent of the EPSP at PS threshold, unlike the 0/0 25 percent
test pulse equivalent, showed a very brief period of inhibited PS
responding at the 20 msecs IPI, but then showed a potentiated PS re
sponse at the 30 msecs IPI. As with the 0/0 25 percent test pulse
series, the condition pulse current intensity did not affect the maximal
level of potentiation. The subthreshold condition pulse PS amplitude
was potentiated maximally at the 40 msecs IPI and decreased to control
levels thereafter. The superthreshold condition pulse current intensi
ties all reached a peak level between 150 and 200 msecs IPIs and
remained near that level until IPIs of approximately 400 msecs at which
point they began to return to control levels. Each of the condition
pulse series PS responses returned to control levels by 1500 msecs. The
maximal level of potentiation observed with the 50 percent test pulse
series, however, only reached 140 to 160 percent of control. This
maximal potentiation level was in contrast to the 225 percent level
observed with the 25 percent test pulse series.
The PS latency data (Fig 3-3B) were similar to the 0/0 25 percent
data. An increase in the latency to a PS was observed with IPIs from 20
to 400 msecs. While the subthreshold condition pulse data showed the
smallest difference from control levels, the PS latencies of the super
threshold condition pulses were not apparently influenced by condition
pulse current intensity.


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231


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
THE EFFECT OF CHRONIC ETHANOL INGESTION
ON SYNAPTIC INHIBITION IN CAl OF THE RAT
By
Carl J. Rogers
May 1986
Chairman: Don W. Walker
Major Department: Psychology
Chronic ethanol abuse by humans results in behavioral alterations
as well as anatomical and physiological deficits within the CNS. Animal
models of chronic ethanol treatment (CET) using nutritionally controlled
diets showed that ethanol itself results in deficits similar to those
observed in humans. The hippocampus, a region of the brain implicated
in memory processing, has reduced cell populations as well as dendritic
spine density after CET. Electrophysiologically, increased population
spike amplitudes (PSA) with paired pulse paradigms (PPP) are found
without concomitant changes in basic synaptic waveforms suggesting
decreased recurrent inhibition (RI). Recently, feedforward inhibition
has also been demonstrated in this region.
A nutritionally complete liquid diet containing ethanol or sucrose
isocalorically substituted was pair-fed to rats for 20 weeks. Eight
weeks after ethanol abstinence, the effects of CET on feedforward and
vii


80
amplitude response was shown to return to control levels in other
studies, the current results showed that the return toward baseline
levels was different. Instead of the PS amplitude returning directly to
control levels, it returned toward control levels by approximately 50
percent at IPIs of 60 to 80 msecs and then very gradually returned to
levels closer to control by IPIs of 1000 to 2000 msecs. Even at this
point, however, the PS amplitude was inhibited by 10 to 30 percent of
control. The A/0 PS latency measures exhibited longer latencies than
control responses at short IPIs and subsequently returned toward control
levels. Like the PS amplitude responses, however, the PS latency data
did not completely return to the control level at any of the IPIs
tested. The best characterization of these responses is that A/0
stimulation results in two phases of inhibition. There is an initial,
maximal inhibition phase where the greatest magnitude of inhibition is
found. This maximal period of inhibition is then followed by a decay
phase which was more prolonged than previously noted. This is not
consistent with previous reports although most of the evidence has been
obtained from hippocampal slice preparations and so the effects of
anesthesia may result in the prolonged decay phase.
The 0/0 paired pulse stimulation configurations were seen to
produce near complete inhibition of the PS amplitude response at short
IPIs. This occurred despite the EPSP being facilitated at short IPIs
(based on reports by others including one using the same preparation,
anesthesia levels and stimulating and recording locations as the present
investigations but with condition and test pulse current levels sub
threshold for evoking PS responses (Abraham et al., 1981)). While
increasing the condition pulse current intensity increased the duration


93
system, a gas bubbler, a fiber-optic light source, and electrical
connections for grounding and recording from the recording chambers.
Tissue preparation
Experimental and control animals were treated with the same proce
dures since coding of the animals was performed. Well established slice
preparation procedures were used (Andersen et al., 1978; Schwartzkroin,
1981; Langmoen and Andersen, 1981; Teyler, 1980; Alger et al., 1984;
Andersen, 1984; Dingledine et al., 1980; Simmons, 1982; Skrede and
Westgaard, 1971; Yamamoto and Mcllwain, 1966) which allowed for a quick
start up as well as consistency of experimental setups between laborato
ries. Slices were prepared using the method described by Teyler (1980)
with minor modifications. An Haer tissue chopper (modified Mcllwain)
was used to slice the hippocampus into 350-400 uMs thick sections. This
thickness of the sections was selected because it represents a good
compromise between having a core of healthy tissue and having a core
which was too thick to allow for reliable diffusion of the bathing
medium uniformly through the slice. Generally, a layer of tissue on
either side of the cut edges, about 50 to 100 microns, was not viable
due to injury. Thus, a 350 to 400 micron slice provided about 200
microns of viable tissue. After the slices were cut, they were placed
in a transfer dish containing the medium which was used during the
actual recording. The slices were then transferred to the tissue
recording chamber with a large neck glass pipette where they were
incubated for at least 45 minutes prior to use. This incubation period
was necessary for the slice to regain its electrical characteristics


Figure 4-6. 0/0 homosynaptic paired pulse PS latency responses (means) with stimulation
through stratum radiatum. All test responses are shown as a difference from
control responses (test response-control response in msecs). A) Test pulse
responses obtained with the condition pulse current intensity set to obtain an
EPSP response 50 percent of the EPSP amplitude at PS threshold for both the
control and sucrose groups. Test pulse response was set to 25 percent of the PS
amplitude at PS asymptote. B) Test pulse responses obtained with 25 percent of
the PS amplitude at PS asymptote on both the condition and test pulses. C) Test
pulse responses obtained with the condition pulse adjusted to 50 percent of the
PS amplitude at PS asymptote and 25 percent of the PS amplitude at PS asymptote
on the test pulse. D) Test pulse responses obtained with 50 percent of the PS
amplitude at PS asymptote on both the condition and test pulses.


113
potentiated to 300 percent at an IPI of 50 msecs. The alcohol group was
approximately 50 percentage points more potentiated than the sucrose
group from an IPI of 20 msecs to 300 msecs at which point the data from
the two groups converged and returned to baseline levels. The latency
to PS onset data (see Fig 4-6C) showed that the alcohol group had a
shorter latency by .3 msec than did the sucrose group. Both groups had
the shortest latencies with 20 to 40 msecs IPIs and returned to control
levels by 400 msecs.
The 0/0 homosynaptic paired pulse paradigm with the highest current
levels in this series of experiments, 50 percent of the PS amplitude at
PS asymptote on both the condition and test pulses are shown in Fig
4-5D. Following the pattern of the previous superthreshold,
homosynaptic paired pulse results for PS amplitude measures, the 50
percent condition and test pulse current intensity group (see Fig 4-5D)
again showed the alcohol group means to be slightly more potentiated
than the sucrose group. The alcohol group was maximally potentiated
between IPIs of 50 to 60 msecs at 180 to 190 percent of control values.
After the IPI of 80 msecs, the two groups were no longer at separate
levels. The maximal level of potentiation for either of the two groups
with these conditions was lower than that found with the 25 percent test
pulse groups as was shown in vivo. The PS latencies for this experiment
(Fig 4-6D) decreased by .4 to .5 msec at IPIs less than 100 msecs. The
alcohol group, however, did not have significantly different PS latencies
than the sucrose group. Both groups returned to control levels by 200
to 300 msecs IPIs.


226
potentiation, however, was greater than reported by Andersen. These
differences were considered to be due to one of three possibilities.
The first possible reason was a difference in the species used. The
second reason may have been due to a difference in temperature. The
present bath temperature was higher than that used by Andersen et al.
(1982). Finally, the protocol for the iontophoretic ejection of GABA
and the subsequent testing of the GABA response was slightly different
in the present study from that reported by Andersen et al. (1982).
Presently, there was a brief interval between the end of the ejection of
GABA and the stimulation of the afferents. Andersen et al. (1982) had
no such pause, but instead ejected GABA while testing the response.
Bicuculline, iontophoretically applied at four locations along the
pyramidal cell dendrites, resulted in increased PS amplitudes and
multiple spikes with all animals tested. SR or SO afferent stimulation
produced responses which were greatest when bicuculline was applied in
proximal SO and SR. Applications of bicuculline in distal SO and
especially distal SR resulted in smaller levels of potentiation.
Baclofen resulted in inhibited PS response amplitudes at each of
the ejection locations. There was, however, a striking dependence of
the magnitude of the baclofen induced response on the afferent zone
stimulated. SR stimulation resulted in distal SR and proximal SR being
most inhibited. Proximal SO was still strongly inhibited, but distal SO
was only weakly inhibited. With SO stimulation, however, distal SR was
weakly inhibited, proximal SR was moderately inhibited, while both SO
iontophoresis sites were strongly inhibited. These results suggest the
existence of baclofen mediated inhibition at all locations along the
pyramidal cell dendrites. The fact that no potentiation was seen, as


36
Anoxia, picrotoxin, and the enkephalins, treatments which appear to
decrease inhibition in the hippocampus, also produced enhanced facilita
tion of the PS amplitudes to paired pulse paradigm (PPP) stimulation
(Andersen, 1960; Dunwiddie et al., 1980; Lee et al., 1980). These
effects also occur in the absence of significant alterations in the
synaptic response to single pulse stimulation. FP findings further
support the involvement of recurrent inhibition since FP at 1 Hz
resulted in no differences between CET and control animals while both 5
Hz and 10 Hz FP did result in PS differences (Abraham et al., 1981).
Since the maximal duration of recurrent inhibition appears to be 600
msecs in CAl (Andersen et al., 1964a), 1 Hz stimulation is too slow to
interact with inhibitory processes whereas 5 Hz and 10 Hz stimulation
would be expected to interact. More recently, Durand and Carien (1984)
reported results which provided additional support for the hypothesis
that CET reduces recurrent inhibition in CAl. They found, using
intracellular recordings from CET hippocampal slices, that only the
inhibitory post-synaptic potential (IPSP) amplitudes and after
hyperpolarization durations were significantly different from controls.
No differences were found with any of the other membrane characteristics
measured.
There has been one interesting biochemical finding potentially
related to inhibition in the hippocampus. Freund (1980) found a
decrease in the binding of benzodiazepine receptors from CET animals.
These findings could be important since there is a strong link between
GABA and benzodiazepine receptors with GABA strongly involved in the
inhibitory mechanisms of the CAl region.


29
There are currently at least three major putative neurotransmitter
groups which are reported to have major effects on inhibition of the CA1
pyramidal cell. These neurotransmitter groups are GABA agonists and
antagonists, opiate agonists and antagonists and the benzodiazepines.
The following sections provide background for each of these neurotrans
mitters .
GABA agonists and antagonists and inhibition
Glutamic acid decarboxylase (GAD), the precursor to GABA, and GABA
have been found in the hippocampus using immunocytochemical (Barber and
Saito, 1976), autoradiographic (Hokfelt and Ljungdahl, 1972) and fluo
rescence (Wolman, 1971) techniques in a bimodal distribution in CAl.
Peaks are seen in the pyramidal cell layer and the molecular layer of
SR. Higher levels of GAD are actually seen in stratum lacunosum-
moleculare than in stratum pyramidale. It has also been shown that if
the various afferent pathways to the CAl region are cut, there is no
reduction in GAD activity. These data indicate that GAD is not released
by the afferent fibers but instead suggest the probable existence of an
interneuron (Storm-Mathisen, 1977; Fonnum and Storm-Mathisen, 1978).
These findings also suggest the possibility of two populations of
GABAergic inhibitory interneurons.
GABA has been demonstrated to mediate recurrent inhibition (cf.
Storm-Mathisen, 1977) and feedforward inhibition (Alger and Nicoll,
1980) in the hippocampus. It has been shown in vivo that GABA, applied
to the pyramidal cell soma, produced a potentiation of recurrent inhibi
tion (Curtis et al., 1970). It has been suggested that this iontophor-
etically released effect of GABA occurs as a result of GABA acting on


220
More recent evidence has uncovered the existence of another type of
pyramidal cell inhibition. This type of inhibition is activated prior
to the firing of the pyramidal cell itself and has been termed feed
forward inhibition (Alger and Nicoll, 1980, 1982a, 1982b; Andersen et
al., 1978, 1982). The underlying cellular mechanism of recurrent
inhibition has been shown to be a hyperpolarization of the pyramidal
cell body as a function of an increased Cl- conductance. Feedforward
inhibition appears to be more complex in terms of the underlying
mechanisms. There appear at present to be at least three different
mechanisms by which feedforward inhibition is produced. It is likely
that our understanding of the mechanism of inhibition is still in its
infancy and that additional cellular mechanisms will be found.
Briefly, feedforward inhibition has also been shown to result in an
hyperpolarization of the pyramidal cell, but the time course of
maximal effect (200 msecs vs 50 msecs) is later than for antidromically
activated recurrent inhibition (Alger and Nicoll, 1982a). This
hyperpolarizing response has been shown to be subserved by at least
two different receptor populations, a GABA sensitive, bicuculline
sensitive and a GABA sensitive, bicuculline insensitive receptor. It
has been reported that baclofen may preferentially activate the
bicuculline insensitive receptor population (Newberry and Nicoll,
1984). A third receptor type results in a depolarization with
recordings from the pyramidal cell body. This depolarization, however,
results in a current shunt which, in turn, locally inhibits additional
dendritic input. This depolarizing response has unique characteristics
in that it can also result in an "amplification" of additional afferent
input at locations away from the activated, depolarized site since the


39
the Mead-Johnson Company. Both groups of liquid diets were additionally
fortified with Vitamin Diet Fortification Mixture (3.0 g/1) and Salt
Mixture XIV (5.0 g/1) which were obtained from ICN Nutritional
Biochemicals, Cleveland, Ohio. The diets contained 1.3 KCal/ml and
provided several times the daily requirement for all essential vitamins
and nutrients (Walker and Freund, 1971). Diets were prepared fresh
daily and were administered using calibrated glass bottles with stain
less steel drinking tubes. Consumption levels of each animal were
recorded daily.
Upon arrival at the animal facilities, animals were quarantined for
a period of seven days. They were then housed individually and allowed
to acclimate to the colony room for one to two weeks or until they
reached the appropriate body weight. Next, animals were paired accord
ing to their weight. The experimental animals (group E) received
ethanol in their daily diet while the control animals (group S) received
sucrose substituted isocalorically for the ethanol. A laboratory chow
group was not included in these experiments since in the previous 15
years of experiments in this laboratory no differences between sucrose
and lab chow control groups were ever found.
Animals in group E were pair fed with animals of group S by giving
each animal in group S the volume of diet that its age- and weight-
matched group E partner consumed on the previous day. This pair-feeding
procedure assured that group E and group S animals ingested identical
quantities of calories, vitamins, minerals, proteins, fats and carbo
hydrates. Differences between group E and group S animals on any
dependent variable were then due to ethanol and not nutritional or other


185
time after iontophoresis (mins)


17
extended in a rostral-caudal direction. The hippocampi thus look
similar to rams' horns. Each hippocampus consists of two distinct
regions, the hippocampus proper or CA region (Cornu Ammonis for its
seahorse-like shape) and the dentate gyrus (DG). Each of these areas
appears as a C-shaped structure with one facing the other and
interlocked. The CA region is further subdivided into four relatively
distinct regions (Lorente de No, 1934). The dorsal-most region of the
hippocampus and the C-shape is known as CA1 (Fig 1-1). It is located
next to the subiculum medially and extends laterally to where the
hippocampus first turns ventrally. Moving inward on the C-shape is a
short CA2 zone followed by a large CA3 region and finally the CA4 area
which is located within the two outer blades of the DG. The CA2 zone is
generally defined as the zone which distinguishes the regio superior
(CAl region) from the regio inferior (CA3 and CA4 regions).
The CA regions consist of several layers. Beginning from the
dorsal surface of the CAl region, the first layer is the alveus, a dense
but thin layer of afferent and efferent fibers. Next is the relatively
cell-poor molecular layer known as stratum oriens (SO). This layer
contains the basilar dendrites of the pyramidal cells. It is followed
by a dense but thin layer of pyramidal cells which is called stratum
pyramidale (SP). Beneath the pyramidal cell layer is stratum radiatum
(SR) and stratum lacunosum-moleculare (SLM). In the CA3 region of the
rat only, an additional laminae is found between the SR and the SP. The
apical dendrites of the pyramidal cells project into these last three
layers. The majority of afferent fibers to the pyramidal cells project
orthogonally to these pyramidal cell dendrites and terminate on them.
The afferent fibers which project to CAl originate from several regions


Figure 4-11. A/O and O/O Heterosynaptic paired pulse PS amplitude responses (means + SEM).
Note the individual configurations described below. All test responses are
shown as percent changes from control responses (test response/control response
X 100). A) Test pulse responses obtained with antidromic stimulation from the
alveus on the condition pulse and orthodromic stimulation from the stratum
radiatum on the test pulse. The condition pulse current intensity was adjusted
to obtain the maximum (100 percent) sized antidromic spike. The test pulse was
adjusted to 25 percent of the PS amplitude response at PS asymptote. B) Test
pulse responses obtained with condition pulse stimulation through stratum
radiatum and test pulse stimulation through stratum oriens. The condition and
test pulse current intensities were set to obtain a PS amplitude response 25
percent of the PS amplitude at PS asymptote. C) Test pulse responses obtained
with condition pulse stimulation through the stratum oriens and test pulse
stimulation through the stratum radiatum. The condition and test pulse current
intensities were set to obtain a PS amplitude response 25 percent of the PS
amplitude at PS asymptote.


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P. Neuronal loss in hippocampus produced by prolonged ethanol
consumption in rats. Science. 1980, 109: 711-713.
Walker, D.W. and Freund, G. Impairment of timing behavior after
prolonged alcohol consumption in rats. Science. 1973, 182: 597-599.
Walker, D.W. and Freund, G. Impairment of shuttle box avoidance
learning following prolonged alcohol consumption of rats. Physiol.
Behav.. 1971, 7: 773-778.
Walker, D.W. and Hunter, B.E. Short-term memory impairment following
chronic alcohol consumption in rats. Neuronsvchol.. 1978, 16: 545-554.
White, W.F., Nadler, J.V. and Cotman, C.W. Analysis of short-term
plasticity at the perforant path-granule cell synapse. Brain Res..,
1979, 178: 41-53.


Figure 6-5. PS latency responses (means) after bicuculline
iontophoresis (time=0) with stratum oriens stimulation.
Scales on ordinate represent difference of PS latency
response following bicuculline administration from
control responses obtained prior to bicuculline adminis
tration (test response-control response) and are
independent for each iontophoretic ejection location.
Abcissa represents time in minutes following ionto
phoretic ejection of the drug. A) PS responses with
iontophoretic ejection in distal SO. B) PS responses
with iontophoretic ejection in proximal SO. C) PS
responses with iontophoretic ejection in proximal SR.
D) PS responses with iontophoretic ejection in distal
SR.


20
but especially the ipsilateral and contralateral CA3 regions. These
fibers are termed the Schaffer/commissural afferents.
The flow of information through the hippocampus occurs in a
trisynaptic sequence. The major afferent system to the hippocampal
complex emanates from the entorhinal cortex and forms the perforant path
(or angular bundle). These fibers project onto the granule cells of the
DG with a lamellar distribution. The axons of granule cells then
project through the hilus to the CA3 pyramidal cells. The axons of CA3
pyramidal cells, in turn, project ipsilaterally as well as contralaterally
to the CAl pyramidal cells. The CAl pyramidal cells then project
rostrally to the medial septum, anterior thalamus and mammillary bodies
and caudally to the subiculum and entorhinal cortex. This last projection
actually closes the loop of projections which started in the entorhinal
cortex. Each of these connections produces an excitatory influence on
the postsynaptic cell (Andersen et al., 1966). A final distinctive
feature of the hippocampal circuitry is that each of the major projection
cells resides and is preserved within a cross sectional slice or lamellae
of the hippocampus (Andersen et al., 1971).
The region of specific interest in the current studies is the CAl
pyramidal cell region. The general circuitry has been described above.
The microcircuitry within this region is also important and is known to
mediate, among other phenomena, the inhibitory control of the pyramidal
cell. The pyramidal cell sends its axon through the alveus as described
above but also sends an axon collateral to one or more inhibitory
interneurons located near or within the pyramidal cell layer. This
interneuron, presumably a basket cell, then projects back onto the
original as well as a large number of other pyramidal cells thereby


31
potential which peaked at about 50 msecs. Immediately following this
IPSP was a later hyperpolarizing potential which peaked at a latency
from stimulus onset of approximately 200 msecs. This late hyper
polarizing potential could only be evoked with orthodromic stimulation
in SR or SO. It cannot be evoked with antidromic stimulation thus
ruling out the possibility that it was generated by recurrent
interneurons and strongly suggesting it to be the result of feedforward
inhibition (Alger and Nicoll, 1982).
The evoked hyperpolarizing potentials recorded above have also been
characterized pharmacologically. The late hyperpolarizing potential
occurred during the same period as the depolarizing potential which was
elicited by localized application of GABA in the apical dendrites. The
two different hyperpolarizing potentials (early and late) have been
shown to be subserved by two different populations of GABA receptors.
The early hyperpolarization has been shown to be mediated by GABA(a)
receptors while the late hyperpolarization has been shown to be sub
served by GABA(b) receptors. The evidence for this distinction emanated
from the finding that the early hyperpolarizing response was antagonized
by the GABA antagonist bicuculline. The late hyperpolarizing potential
was, however, not antagonized by bicuculline and was therefore said to
be bicuculline resistant (Newberry and Nicoll, 1984). Subsequent
reports have indicated that baclofen, a GABA agonist, binds to the
bicuculline-resistant GABA receptors and directly induced the late
hyperpolarization in the pyramidal cell. The response to baclofen was
associated with a decrease in neuronal input resistance which may be a
result of an increase in potassium conductance (Newberry and Nicoll,
1984; Nicoll and Newberry, 1984). The early IPSP occurred as a result


53
Equipment and techniques for in vivo studies
Animals were initially injected intraperitoneally (IP) with .2 cc
of atropine to reduce possible respiratory congestion. Within 10 mins
1.5 gms/Kg of urethane was injected IP for anesthesia. A second injec
tion of 1.0 gms/Kg was given one hour later. Supplemental injections of
.05 gms/Kg were then given at intervals of every 1.5 to 2 hrs.
After it was determined that the animal was well anesthetized, it
was placed in a Kopf stereotaxic device using ear bars and a nose clamp.
An incision was made down the midline of the rat skull and the skin
retracted. The skull was then adjusted so that the bregma and lambda
sutures were level horizontally with the base of the stereotaxic device.
Three burr holes were drilled on the left side of the skull at locations
3.4, 4.4 and 5.4 mms posterior and 2.8 nuns lateral to bregma. A concen
tric bipolar stimulating electrode (Rhodes Medical Instruments) was then
lowered to the SR of CAl through the most anterior hole using a hydraulic
microdrive. This electrode was used as an orthodromic stimulating
electrode. A second stimulating electrode was placed in an antidromic
configuration. This electrode was located in the most posterior hole
and lowered with a microdrive to the alveus. The recording electrode
was lowered through the center hole. This electrode configuration was
used since the orientation of the lamellae within the hippocampus is
such that an approximate anterior to posterior course is found in the
rat (Andersen et al., 1971).
Precise localization of the electrodes in vivo was performed by
observing the extracellular unit activity as the recording electrode was
lowered into the hippocampus. Based upon the characteristic high
frequency, complex unit response observed in the pyramidal cell layer,


92
placement of both the recording and stimulating electrodes can be
precisely performed in vivo. the in vitro experiments in the following
chapter made use of microiontophoresis techniques using various pharma
cological agents at specific locations within the CAl region. These
manipulations would be difficult to perform in vivo. An additional
feature in using the slice technique is that anesthetic agents, which
have been shown to have powerful effects on hippocampal inhibition, are
not used. All major synaptic pathways are conserved in the hippocampal
slice preparation (Teyler, 1980). However, inhibition is reduced since
transverse projections of the basket cells are severed. Responses
recorded in vivo have been compared with those recorded in vitro.
Reports indicate the conservation of nearly all general characteristics
between the two preparations (Schwartzkroin, 1981). That the advantages
outweigh the disadvantages is attested to by the fact that the majority
of the literature concerning recurrent and feedforward inhibition in the
hippocampus have been conducted in vitro (Dingledine and Gjerstad, 1980;
Alger et al., 1981; Andersen et al., 1980; Lee et al., 1979; Teyler,
1980).
Apparatus
Commercially obtained tissue chambers were used in these experi
ments (Frederick Haer and Co). Static recording chambers were used in
all experiments. The static chamber consisted of a static pool of
artificial CSF which had a piece of filter paper stretched taut on a
cylindrical ring on which the brain slice was placed such that diffusion
of the artificial CSF occurred from beneath the slice. The static
chamber was placed inside of a holding tank which included a d.c. heater


191
differences between the alcohol and sucrose groups for the baclofen
data. In SO, near the cell body layer, there was a trend for the
alcohol group to be slightly more inhibited than the controls.
Baclofen series: Stratum oriens stimulation
I/O relationships. See description of I/O curves as described in
Chapter 5. I/O curves for each of the pharmacological series were
combined and presented earlier.
Iontophoresis application with SO stimulation. There were no
differences between alcohol and sucrose groups nor were there any
interaction effects on either the PS amplitude (Fig 6-8) or the PS
latency (Fig 6-9) measures. The effect of baclofen administration has
not been well documented using extracellular techniques. It can be seen
for the PS amplitude measures, that the magnitude of inhibition is
essentially reversed when stimulating through SO and comparing them with
the responses found with SR stimulation. The largest magnitude of
inhibition was found when baclofen was administered in the same synaptic
region as that being stimulated. Both of the SO application sites,
distal and near to the pyramidal cell body layer, were strongly inhibit
ed when compared to the baclofen administration sites in SR. While
there were no significant differences, there was a suggestion of a trend
for the alcohol group to be more inhibited with SO baclofen application
and SO stimulation. These findings are similar to those found with
baclofen administration near the cell body layer in SO with SR stimula
tion and may relate to the increase in response seen with GABA adminis
tration.


Figure 6-13. PS latency responses (means) after enkephalin
iontophoresis (time=0) with stratum oriens stimulation.
Scales on ordinate represent difference of PS latency
response following bicuculline administration from
control responses obtained prior to bicuculline adminis
tration (test response-control response) and are
independent for each iontophoretic ejection location.
Abcissa represents time in minutes following ionto
phoretic ejection of the drug. A) PS responses with
iontophoretic ejection in distal SO. B) PS responses
with iontophoretic ejection in proximal SO. C) PS
responses with iontophoretic ejection in proximal SR.
D) PS responses with iontophoretic ejection in distal
SR.


3
would produce many different avenues of use, from the treatment of the
impaired to the willful prevention of the disease itself.
Behavior
The consumption of alcohol in moderate to heavy doses over long
periods of time (months to years) has been shown to produce a wide
variety of behavioral changes in humans. Long term alcohol consumption
does not produce a consistently clear, easily described and universal
picture of behavioral, anatomical, or physiological symptomatologies.
There may be several avenues of explanation for these inconsistencies of
symptomatologies among which are: 1. different genetic populations with
different dispositions to the effects of alcohol, 2. the varied and
unique individual drinking histories of long term alcohol consumers, and
3. other environmental conditions which are unique such as disease or
trauma (Tarter and Alterman, 1984; Parsons and Leber, 1981). Even with
the wide variations of symptoms seen in chronic alcohol abusers, reviews
of the literature indicate a relatively common set of behavioral and
biological alterations (Begleiter et al., 1980; Parsons, 1977; Ron,
1977; Ryan, 1980; Tarter, 1975). These alcohol-induced changes, in
turn, provide reasonable indications of which major brain systems may be
most severely involved.
The clinical symptoms of long term alcohol abuse include to
varying degrees cognitive, memory and motor performance impairments.
Deficits have been reported to occur in perceptual, spatial, problem
solving and cognitive tasks. However, one of the most persistent and
devastating effects of long term ethanol abuse is that of memory loss.
Although short term memory is generally reported to be intact, long-term


0rTTCNCC mo*j COnmOL (uSCC)
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200
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300
200
kWTT*^UL3C WTTVAi. (USCC)
<00


Figure 6-14. PS amplitude percent change summary graphs for bicuculline, enkephalin and
baclofen. These graphs are constructed from data which was obtained from the
previous respective graphs 1.5 mins after iontophoretic ejection of the
respective drugs. This time point was chosen because it represented the peak
response size for most of the applications. A) Bicuculline PS amplitude peak
response for each of the different iontophoretic locations and groups with
stratum radiatum stimulation. B) Bicuculline PS amplitude peak response for
each of the different iontophoretic locations and groups with stratum oriens
stimulation. C) Enkephalin PS amplitude peak response for each of the
different iontophoretic locations and groups with stratum radiatum stimulation.
D) Enkephalin PS amplitude peak response for each of the different
iontophoretic locations and groups with stratum oriens stimulation.


78
superthreshold condition and test pulse current intensities tested were
similar to those reported previously (Andersen et al., 1966; Creager et
al., 1980; Steward et al., 1977; Lomo, 1971). The subthreshold condi
tion pulse current intensity resulted in little inhibition but shifted
the period of maximal potentiation from IPIs of 150 to 400 msecs to IPIs
of 20 to 80 msecs. For the 0/0 superthreshold PS amplitude responses,
there was an initial period of inhibition at the shorter IPIs which then
changed to a period of potentiation at the intermediate length IPIs.
The PS latency measures corresponded well with the PS amplitude measures
since both yield an index of pyramidal cell excitability. Increasing
the current intensity resulted in increased PS latencies at short IPIs
since the PS was inhibited for longer periods. The EPSP response
patterns, however, were different from those reported previously
(Creager et al., 1980; Landfield et al., 1978; Lomo 1971; Steward et
al., 1977). The EPSP measure is typically reported as an amplitude at a
fixed latency from EPSP onset, the maximal EPSP amplitude or a slope
measure of the rising phase of the EPSP (Lomo 1971; McNaughton and
Barnes, 1977; White et al., 1979; Creager et al., 1980). Other re
searchers have reported a period of facilitation of the EPSP measures
with 20 to 30 msecs IPIs followed by a gradual decay with a time con
stant of about 100 msecs (McNaughton, 1982). Slope measures have been
utilized in an attempt to reduce the distortion of the EPSP sometimes
seen as a result of pyramidal cell or recurrent interneuron firing
(Creager et al., 1980). The present slope measures showed changes only
at short IPIs, but the changes observed were in the direction of de
creased rather than increased slopes. The most likely reason for these
response differences was that the present measures were recorded from


Figure 4-9. 0/0 paired pulse PS amplitude responses (means + SEM). Note that these experiments
were conducted through two different sets of afferent fibers. All test responses
are shown as a percent of control responses (test response/control response X 100).
A) Test pulse responses obtained with condition and test pulse stimulation through
the stratum oriens. The current intensities on both the condition and test pulse
were adjusted to obtain a PS amplitude response which was 25 percent of the PS
amplitude at PS asymptote. B) Test pulse responses obtained with condition and test
pulse stimulation through the stratum radiatum. The current intensities on both the
condition and test pulse were set to obtain a PS amplitude response which was 25
percent of the PS amplitude at PS asymptote.


83
0/0 subthreshold condition pulse with 50 percent of PS maximum test
pulses can be explained by this subthreshold recruitment of feedforward
inhibition.
The use of two 0/0 test pulse current intensities yielded responses
which were not substantially different in regard to the effect of
different condition pulse current intensities, when the amplitude or
duration of inhibition were compared. These results suggest that the
intensity of the condition pulse produces significant effects on the
inhibitory phase of paired pulse testing without greatly altering the
maximal levels of the potentiation phase. The major effect of the two
different test pulse current intensities was that two different levels
of potentiation were produced. Since it is known that feedforward
inhibition can influence the amplitude of the PS with single pulse
stimulation (Ashwood et al., 1984; Buszaki, 1984), the best explanation
for the lower levels of potentiation found with the higher test pulse
current intensity may be that there was a more powerful influence of
feedforward inhibition with a higher test pulse current intensity.
Recurrent inhibition would not be strongly implicated since the effect
of recurrent inhibition on the response to the the two different test
pulse current intensities would be the same given the same condition
pulse current intensity. The major reason for the difference in maximal
potentiation levels most likely emanates from the influence of the test
pulse current intensity on the levels of feedforward inhibition.
Another possible explanation for the difference in the maximum
levels of potentiation observed between the 25 percent and 50 percent of
the PS amplitude at PS asymptote test pulse current intensities is that
there was a ceiling effect. Since the 0/0 PS amplitude percent of


4
memory processing is often severely impaired. Severe memory deficits
are often defined as part of a syndrome named Korsakoff's syndrome
(discussed below). Other clinical manifestations of long term alcohol
abuse have been associated with nutritional deficits. Brain stem in
volvement, as evidenced by oculomotor abnormalities, is seen in patients
with histories of long term alcohol abuse. However, these abnormalities
are often rapidly reversed with thiamine treatment strongly indicating
that dietary intake may be disturbed in some patients and thus contrib
ute directly to some of the symptoms observed with alcohol abuse. Long
term alcohol exposure also causes irregularities in sleep patterns as
well as endocrine function and certain aspects of autonomic nervous
system functioning (Kissin, 1979; Wagman and Allan, 1977). It is
important to emphasize that long term alcohol abuse has nearly always
been shown to produce cognitive impairment and brain damage despite the
wide variations found in the symptoms exhibited by alcoholic patients.
One of the most controversial aspects of long term alcohol abuse
has centered on the interaction of dietary inadequacies versus direct
toxic effects of alcohol on the CNS. This debate has begun to be
silenced by carefully controlled human and animal studies where dietary
intake has been monitored or controlled. The results of these studies
have provided significant evidence that alcohol per se can produce
direct neurotoxic effects. It has been noted that Korsakoff's syndrome
has never been found without coincident long-term alcohol abuse (Freund,
1973).
One of the most intensely studied areas of alcohol research has
concerned the effect long term alcohol abuse has on memory function.
Korsakoff's syndrome represents the most severe form of long term


233
Begleiter, H., Porjesz, B. and Tenner, M. Neuroradiological and
neurophysiological evidence of brain deficits in chronic alcoholics.
Acta Pschiat. Scand/Alcohol and Brain Res., 1980, 62: 3-13.
Ben-Ari, Y., Krnjevic, K. and Reinhardt, W. Hippocampal seizures and
failure of inhibition. Can. J. Physiol. Pharmacol.. 1979, 57:
1462-2466.
Ben-Ari, Y., Tremblay, E., Riche, D., Ghilini, G. and Naquet, R.
Electrographic, clinical and pathological alterations following systemic
administration of kainic acid, bicuculline and pentetrazole: metabolic
mapping using the deoxyglucose method with special reference to the
pathology of epilepsy. Neurosci. 1981, 6: 1361-1391.
Berger, T., French, E.D., Siggins, G.R., Shier, W.T. and Bloom, F.E.
Ethanol and some tetrahydroisoquinolines alter the discharge of cortical
and hippocampal neurons: Relationship to endogenous opioids.
Pharmacol. Biochem. Behav., 1982, 17: 813-821.
Bird, S.J. and Kuhar, M.J. Iontophoretic application of opiates to the
locus coeruleus. Brain Res., 1977, 177: 523-533.
Bostock, E., Dingledine, R., Xu, G. and Chang, K.-J. Mu opioid
receptors participate in the excitatory effect of opiates in the
hippocampal slice. J. Pharmacol. Exp. Ther., 1984, 231 (3): 512-517.
Bradley, P.B. and Dray, A. Morphine and neurotransmitter substances:
Microiontophoretic study in the rat brain stem. Brit. J. Pharmacol.,
1974, 50: 47-55.
Brewer, C. and Perrett, L. Brain damage due to alcohol consumption: An
air encephalographic, psychometric, and electroencephalographic study.
Br. J. Addict.. 1971, 66: 170-182.
Brion, S. Korsakoff's syndrome: Clinico-anatomic and pathophysiological
considerations. In The Pathology of Memory. Talland, G. and Waugh, N.C.
(eds.). Academic Press, New York, 1969, 29-39.
Brown, T.H., Wong, R.K.S. and Prince, D.A. Spontaneous miniature
synaptic potentials in hippocampal neurons. Brain Res., 1979, 175:
194-199.
Butters, N. and Cermak, L.S. Alcoholic Korsakoff's Syndrome An
Information-processing Approach to Amnesia. Academic Press, New York,
1980.
Buzsaki, G. Feed-forward inhibition in the hippocampal formation.
Prog. Neurobiol., 1984, 22: 131-153.
Buzsaki, G. and Eidelberg, E. Commissural projections to the dentate
gyrus of the rat: Evidence for feed-forward inhibition. Brain Res.,
1981, 230: 346-350.
Cajal, S. Ramon y. The Structure of Ammon's Horn. Charles Thomas,
Springfield, IL, 1968.


176
group. Alcohol animals consumed a mean daily ethanol dosage of 12.87 +
.20 gms/Kg which was comparable to levels consumed in other studies
which showed anatomical and physiological changes as a result. The body
weights of the two groups were as follows: alcohol 542.86 + 7.9,
sucrose 505 10.61.
SR stimulation
I/O relationships with SR stimulation. See description of I/O
curves as described in Chapter 5. I/O curves for each of the
pharmacological series were combined and were presented earlier.
Iontophoresis applications with SR stimulation. Recordings from
distal SO showed that there were no differences between the alcohol
and sucrose control groups for the PS amplitude (Fig 6-2) or the PS
latency (Fig 6-3) responses. There was, however, a group by time
interaction effect for the PS amplitude data (F(l,360) 2.40, p -
.0008). Subsequent T-test analyses of the individual points revealed
the points between 60 and 180 secs to be different. The alcohol group
was more potentiated than the sucrose-fed group. There were no
differences in PS amplitude or PS latency measures in SO near the cell
body layer. Although there were no statistically significant
differences between groups on PS amplitude or PS latency measures near
the cell body layer in SR, there was a significant group by time
interaction for the PS latency measure which revealed the points
between 60 and 150 secs to be different. Furthermore, while the PS
amplitude data for the proximal SR application site were not different
by group, those data approached significance (F(l,17) 3.35, p -


171
Apparatus
The apparatus used for these experiments has been detailed in the
general methods, in vitro methods, and the GABA iontophoresis methods.
No additional equipment has been utilized for these experiments.
Procedures
The protocol for each of these experiments using localized
iontophoretic applications of different pharmacological agents were the
same. The drugs were prepared immediately prior to use and loaded into
individual micropipettes. Only one drug was ever in the slice prepara
tion at any one time. Table 6-1 indicates the drug concentrations,
ejection currents, retaining currents, and ejection durations used to
apply these drugs. A maximum of two drugs were applied to the same
slice successively. Unlike the methodology used for the application of
GABA, the protracted time course of activation for each of these drugs
allowed for only one application of the drug after which the response
was sampled every 30 seconds for ten minutes. As with the GABA series,
each drug was tested using SR and subsequently SO stimulation.
The protocol for each of these experiments was as follows. Prior
to the application of any of the agents, the recording electrode was
positioned as indicated for the GABA experiment. An attempt was made to
obtain good responses with positive-going EPSPs from both the SR and SO
electrodes. In the event that good responses could not be achieved from
both electrodes, preference was given to the response obtained with SR
stimulation. The PS amplitude and latency to the PS measures, however,
were prominent and measurable with both SR and SO stimulation. Once the
recording and stimulation electrodes were positioned, the iontophoretic
pipette was positioned in the slice at the first of the four locations


Figure 4-10. Homosynaptic 0/0 paired pulse PS latency responses (means). Note that these
experiments were conducted through two different sets of afferent fibers. All
test responses are shown as a difference from control responses (test response
- control response in msecs). A) Test pulse responses obtained with condition
and test pulse stimulation through the stratum oriens. The current intensities
on both the condition and test pulse were adjusted to obtain a PS amplitude
response which was 25 percent of the PS amplitude at PS asymptote. B) Test
pulse responses obtained with condition and test pulse stimulation through the
stratum radiatum. The current intensities on both the condition and test pulse
were set to obtain a PS amplitude response which was 25 percent of the PS
amplitude at PS asymptote.


difference from control (msecs)
200
time after iontophoresis (mins)


0rrij^cNCC rnou COumcK. (uscc)
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300
400


Figure 5-5. PS latency responses (means) obtained after GABA
iontophoresis at ten different locations along
the dendritic axis of CA1 pyamidal cells. GABA
mediated PS latency responses are expressed as a
difference from the control response obtained
immediately prior to the iontophoresis of GABA at
each location indicated (GABA (test) response -
control response in msecs). A) GABA mediated PS
latency responses obtained with stimulation from
the stratum radiatum. B) GABA mediated PS
latency responses obtained with stimulation from
the stratum oriens.


25
125
100
75
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25
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CHAPTER 6
THE EFFECT OF CET ON
BICUCULLINE, BACLOFEN AND ENKEPHALIN MEDIATED RESPONSES IN CAl
Introduction
There is now considerable evidence that chronic ethanol abuse in
the human population produces a variety of alterations in the CNS from
changes in behavior (Victor et al., 1971; Ron, 1977; Tarter, 1975), to
lesions of certain nuclei in the brain (Victor et al., 1971), and to
alterations in the electrophysiological functioning of the brain
(Porjesz and Begleiter, 1981). Animal models have provided evidence
that alcohol itself, can produce toxic effects in the brain. These
findings indicated that although malnutrition may induce CNS complica
tions, malnutrition is not responsible for the majority of symptoms
found with long term ethanol abuse. Through the use of animal models,
careful analyses have been made of certain brain regions such as the
hippocampus and cerebellum for alterations in the morphology (Riley and
Walker, 1978; Walker et al., 1980; King, 1984), electrophysiology
(Abraham et al., 1981, 1982; Durand and Carien, 1984a, 1984b) and
neurochemistry (Freund, 1980) following CET. The results of these
studies have shown regionally specific as well as widespread
alterations.
The finding that CET altered synaptic inhibition in the CAl region
of the hippocampus (Abraham et al., 1981) provided an impetus for
studying the underlying transmitter systems known to be involved in
inhibitory processes. The findings from the previous study where GABA
was ejected at discrete locations along the pyramidal cell dendrites
168


44
the EPSP amplitude at PS threshold (50 EP), PS threshold (0 PS), and to
25, 50, 75 and 100 percent of the PS amplitude at I/O asymptotic values.
Intensities used for each of the individual experiments are indicated in
subsequent sections. Inhibition and potentiation were measured by
quantitative comparison of the test response with a baseline response
averaged from four single pulse baseline responses recorded immediately
prior to each of the paired pulse series.
Data Analysis and Interpretation
The protocol outlined above was designed to allow for separate
measurement of various population EPSP and PS characteristics. For the
EPSP, the following measures were sampled: 1. latency to onset from
stimulus artifact and 2. maximal slope of the rising phase of the EPSP
calculated either by using a regression equation for all of the data
points between the EPSP onset and EPSP peak prior to the PS (all in vivo
data) or by using a regression equation for all of the data points 10
percent above the EPSP onset and 10 percent below the EPSP peak prior to
the PS (10-90 slope; used for all but the very first series of in vitro
data). For the PS, the following measures were sampled: 1. latency to
onset of the first PS measured at its maximum negative amplitude from
the EPSP onset and 2. rising and falling aspects of up to three different
population spikes from the same stimulation pulse. Each PS amplitude
was calculated by adding the respective falling and rising phase of the
PS spike and dividing by two (Fig 2-1). If multiple PSs were recorded,


CHAPTER 2
GENERAL METHODS
Animals
Long-Evans hooded male rats were purchased from the Charles River
Company for all experiments. The rats were obtained at a body weight of
225 to 250 gms and were 55 to 60 days old prior to beginning the admin
istration of ethanol. Animals were housed in individual stainless steel
cages at the University of Florida Animal Resources Center or at the
Veterans Administration colony room or building (three different loca
tions) Temperature and humidity were maintained at appropriate levels.
Light-dark cycles of twelve hours on and twelve hours off were main
tained with the light cycle from 0700 hrs to 1900 hrs daily.
Alcohol Diet Regimen
Animals were maintained on an ethanol or sucrose containing liquid
diet. This diet was developed so that experimental and control animals
ingested calorically equal, nutritionally complete diets.
The ethanol containing diet provided 35 to 39 percent (8.1 to 9.4
percent ethanol v/v) of the total caloric intake as ethanol. Ethanol or
sucrose provided 35 percent of the total caloric intake the first month
of the diet regimen. This level was then increased by 1 percent per
month in order to provide a relatively constant level of ethanol or
sucrose as the animals gained weight. The diets were prepared by
combining an equicaloric ethanol- (63.3 percent v/v) or sucrose-
containing 87.0 percent w/v stock solution with Sustacal obtained from
38


162
tude with 0/0 stimulation were often found with IPIs below 100 msecs
where recurrent inhibition has its greatest effect extracellularly. At
intervals beyond 100 msecs, feedforward inhibition appears to produce
the more predominant inhibitory effect (Alger and Nicoll, 1982). CET
resulted in increased PS amplitudes over this entire range of IPIs.
There are a number of influences which mediate the characteristics
of the inhibitory systems in CAl. Increased PS amplitude or decreased
inhibition in CET animals could be explained simply by a decrease in the
number of inhibitory interneurons (Abraham et al., 1981). However, it
is also possible that changes in the efficacy of the GABAergic receptor
population contribute to this CET-induced effect. Alternatively, the
efficacy of other neuro-modulatory systems may also result in changes of
the inhibitory systems of the CAl region. The present experiment was
designed to assess directly, the effects of GABA application at 10
different locations along the dendritic axis of the CAl pyramidal cell.
The pattern of responses to GABA application for SO and SR stimula
tion are similar to Andersen et al. (1982) in several ways. SR stimula
tion produced the greatest region of inhibition with GABA iontophoresis
along the SR dendrites. The pattern of responses to GABA administration
in the present study with SR stimulation was generally parallel to that
reported by Andersen et al. (1982). There was an inhibited response
from distal SR through the pyramidal cell layer and into the proximal SO
region. Responses to GABA applied in distal SO were potentiated in both
this and in the study reported by Andersen et al., (1982). Responses to
GABA application in SLM were near control levels or potentiated in both
studies. SO stimulation with GABA iontophoresis along the dendrites
produced a different pattern of response from that obtained with SR


BIOGRAPHICAL SKETCH
Carl J. Rogers was born on January 4, 1953, in Bridgeport, Conn.,
to Carl Fredrick and Sophie Rogers of Orange, Conn. He married Jane
Walther in August, 1979.
Rogers attended Stratford High School, Stratford, Conn., graduating
in 1971. He then attended Fairfield University in Fairfield, Conn.,
until 1972 when he joined the United States Air Force. While in the
service, Rogers earned his Bachelor of Arts degree in behavioral science
from the University of Maine at Presque Isle. He was honorably
discharged from the Air Force in 1976.
Rogers received a Master of Arts degree in psychology from the
University of Missouri at Kansas City in May 1978. He then worked at
the University of Connecticut prior to starting work on his doctoral
degree at the University of Florida in August 1979. He will receive a
Ph.D. degree in May, 1986. His major research interest has been the
study of long term ethanol effects on the hippocampus, an area of the
brain thought to be involved in memory processing. He has developed a
keen interest in the use of microcomputers in the laboratory and at
home.
He began work on his postdoctoral position at the University of
Michigan during the Fall of 1985. There he is studying the effects of
GABA, benzodiazepine and opiate agonists and antagonists with whole cell
and patch clamp recordings under the supervision of Dr. Robert L.
Macdonald.
244


30
the pyramidal cell soma in a manner similar to that of the recurrent
inhibitory interneuron. The relatively specific GABA antagonists,
bicuculline and picrotoxin, have furthermore been shown to reduce
GABA-induced inhibition (Curtis et al., 1970).
Local application of GABA onto either the pyramidal cell soma or
to the basilar dendrites, when recording intracellularly, produced an
hyperpolarization-depolarization sequence (Alger and Nicoll, 1980).
When GABA was applied to the apical dendrites, a pure depolarizing
response was recorded. Both the hyperpolarizing and depolarizing
responses produced an inhibitory influence on the pyramidal cell (Alger
et al., 1981; Andersen et al., 1978, 1980). Associated with the
depolarization of the pyramidal cell apical dendrites was a large
conductance increase which presumably accounted for the inhibitory
action by shunting the current input. Thus, GABA produced powerful,
though regionally specific inhibitory effects on the CAl pyramidal cell.
The depolarizing inhibitory effect observed in the apical dendrites is
thought to occur as a result of feedforward inhibition while the
hyperpolarizing effect was shown to occur as a result of recurrent
inhibition.
Another line of evidence concerning the types of inhibition in the
CAl region of the hippocampus has emanated from intracellular recordings
from CAl pyramidal cells. In these studies, stimulation of either
orthodromic SR fibers or antidromic alvear fibers produced two distinct
periods of inhibition (Alger and Nicoll, 1982). The first type of
inhibition represented what is likely to be the result of classical
recurrent inhibition. It can be evoked with either orthodromic or
antidromic stimulation. It was seen as a large and sharp hyperpolarizing


DATA (MSEC) RAW DATA (MV)
101


94
which are temporarily lost after sectioning the hippocampus (Teyler,
1980).
Incubating medium
The incubating medium consisted of the following: NaCl (124 mM),
KC1 (5mM), KH(2)P0(4) (1.25mM), MgSo(4) (2mM), CaCl (2mM), NaHCO(3)
(26mM), glucose (lOmM). In addition, the medium was perfused with 95
percent 0(2) and 5 percent C0(2) and maintained at a pH of 7.27 to 7.37.
These constituents of the incubating medium were chosen because they
represented levels which allowed for relatively normal cellular activity
(Teyler, 1980).
Recording chamber
Slices were individually transferred to a recording chamber where
they were placed in contact with the medium described above. Temperature
was maintained at 34 to 35 degrees Centigrade. The atmosphere was
warmed 95 percent 0(2) and 5 percent C0(2). Standard electrophysiolog-
ical methods were used for stimulation and recording as outlined in the
general methods.
Procedures
The procedures used in this set of experiments were similar to
those used in the previous in vivo experiments. All electrode place
ments were, however, done visually. The orthodromic stimulating elec
trode was placed in SR near the CA1-CA2 border. The antidromic stimulat
ing electrode was placed on the alvear fibers lateral to the recording
electrode. Every effort was made to keep the effect of this stimulating


86
governed to recruit different levels and probably ratios of feedforward
and recurrent inhibition. Lower current intensities appear to cause a
greater predominance of feedforward- rather than recurrent-inhibition.
At higher current intensities it is likely that feedforward and recur
rent inhibition contribute more equally to the resulting PS amplitudes.
Another point indicated by these results is that the current intensity
used for the test pulse of paired pulses significantly modifies the
magnitude of paired pulse potentiation observed. While the current
intensity of the condition pulse influences the early phases of inhibi
tion and the time to reach the maximal level of potentiation, it is the
test pulse which most strongly influences the maximum level of potentia
tion achieved. It is suggested that feedforward inhibition influences
the maximal magnitude of paired pulse potentiation rather than recurrent
inhibition.
The use of extracellular recording techniques provide for an
examination of cellular events at a more system oriented level than
other procedures. The exquisite lamination and arrangement of the
hippocampal architecture provides unique opportunities for extracellular
studies. The incorporation of new information on the effects of feed
forward inhibition should allow for a better understanding as well as
improved methodologies in considering what these responses mean and how
they can be used to investigate the effects of other treatment condi
tions .


147
Methods
Animals
The animals used in this experiment were the same as those used in
the second set of animals in the previous experiments and treated as
detailed in the general methods chapter.
Equipment
The equipment used for this experiment was the same as that used
in the previous in vitro experiment. The only major additional piece
of equipment was the Dagan model 6400 iontophoresis system.
Procedures
The treatment of the animals and the preparation and maintenance of
the slice were accomplished as described in the previous experiment and
the general methods sections.
The first data to be collected were I/O curves of the pyramidal
cell responses first with stimulation from the SR and then from the SO
afferents.
GABA was prepared so that a final 1M concentration was obtained.
2.062 gms of GABA were mixed with 20 mis of water with HC1 to achieve
the 1M concentration at a pH of approximately 3.5. This pH level
provided the necessary electrical charge carriers in order to eject the
GABA from the iontophoresis micropipette. New solutions of GABA were
prepared at least weekly. GABA was always refrigerated except when
filling the pipettes.
The protocol for this experiment was modeled after that of Andersen
et al., (1982). The recording electrode was placed in the pyramidal


*CCCNT CMAMCC rHOM CONTROL
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3


Figure 6-4. PS amplitude responses (means + SEM) after bicuculline
iontophoresis (time=0) with stratum oriens stimulation.
Scales on ordinate represent percent change of PS
amplitude responses obtained after bicuculline adminis
tration from control responses obtained immediately
prior to bicuculline administration (bicuculline
test/control x 100) and are independent for each
iontophoretic ejection location. Abcissa represents
time in minutes following iontophoretic ejection of the
drug. A) PS responses with iontophoretic ejection in
distal SO. B) PS responses with iontophoretic ejection
in proximal SO. C) PS responses with iontophoretic
ejection in proximal SR. D) PS responses with ionto
phoretic ejection in distal SR.


160
pyramidal cell body. All of the responses recorded when GABA was
ejected at SR sites were potentiated relative to the control response.
The CET group PS amplitude responses were not different from the
sucrose animals, but the locations where maximum differences were seen
were in distal (to the cell body) SO and distal SR. The PS latency
data are seen in Fig 5-5B. The differences between the alcohol and
sucrose groups were statistically different (F(l,33) 4.19), p =
.048. The PS latencies for the alcohol and sucrose groups were both
shorter than the control responses. The alcohol group latencies to
the PS maximum were slower than the sucrose group as would be expected
if the alcohol group were more inhibited or less potentiated than the
sucrose group. Since decreased PS latency responses have been considered
to be a reflection of increased pyramidal cell excitability, these
differences further support the trend of results with the PS amplitude
measures.
Discussion
The effect of 20 weeks of ethanol exposure was studied on the
electrophysiology of GABA mediated responses in the CA1 region of the
rat hippocampus. The transverse hippocampal slice preparation was used
in order to eliminate any potential confounding due to anesthesia
related issues found in vivo. This issue was important since cross
tolerant effects between ethanol and barbiturates have been reported
(Kalant, 1970). In addition, the slice technique allowed for the
discrete application of GABA, the major inhibitory neurotransmitter in
the CAl pyramidal cell region.


72
tested. The 50 and 100 percent condition pulse current intensities were
inhibited to approximately 50 and 30 percent of control from IPIs of 80
msecs to approximately 300 and 1000 msecs, respectively.
The PS latencies of the A/0 25 percent test pulse series (Fig 3-6A)
showed increased latencies from 20 to 400 msecs IPIs. The PS latencies
then remained somewhat longer than control responses. A 1.0 msec
increase in latency was observed at the 80 msecs IPI for the 100 percent
condition pulse current intensity.
The EPSP slopes for these data (Fig 3-7A) showed small fluctuations
at IPIs of 20 to 50 msecs but did not vary substantially from control
responses thereafter.
A/0 50 percent test pulse series
The A/0 50 percent of the PS maximum test pulse series PS ampli
tudes are shown in Fig 3-5B. It can be seen that each increase in
condition pulse current intensity produced a greater magnitude of
inhibition of the test pulse response. The 0, 25, 50, 75, and 100
percent antidromic conditioning pulses produced maximum inhibition
levels to 80, 60, 40, 30, and 20 percent of the control response,
respectively. The maximum level of inhibition for each series except
the A/0 threshold condition pulse level was reached at the 20 msecs IPI.
The threshold condition pulse current intensity produced its maximum
level of inhibition at an IPI of 60 msecs. As found with the A/0 25
percent test pulse series, the A/0 50 percent test pulse series resulted
in at least two distinct phases of inhibition. Maximal levels of
inhibition were found in the early period with IPIs between 20 and 60
msecs while a second phase of inhibition was found between IPIs of 80


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139
1982). Furthermore, hypoxia has been shown to deleteriously affect
inhibitory interneurons as measured by the paired pulse technique
(Dunwiddie, 1981). Thus, by direct CET induced effects on the
inhibitory interneurons or by CET altering the regional blood flow, it
is possible that the inhibitory interneuronal population is reduced in
number as were the primary cell types in the hippocampus. Since
recurrent inhibitory interneurons are known to project widely, the loss
of only a small percentage of these interneurons may have profound
effects (Walker et al., 1980). If CET produces substantial effects on
feedforward inhibition, it is possible that long, myelinated fibers may
be deleteriously affected by CET as has been shown previously (Dreyfus,
1974).
The present in vitro paired pulse experiments have extended earlier
extracellular studies showing that CET resulted in an increase in the
size of the PS amplitude with paired pulse stimulation. Although there
were no statistically significant differences between CET and sucrose
animals with 0/0 homosynaptic paired pulse stimulation, the CET animals
consistently tended to have increased PS amplitudes. Heterosynaptic
paired pulse stimulation resulted in more equivocal results. However,
the trend was for the CET animals to show less inhibition than sucrose
controls. Finally, A/0 stimulation resulted in significantly reduced
levels of inhibition in CET animals when high condition and test pulse
current intensities were used. Lower current intensities resulted in
CET animals exhibiting trends toward lower levels of inhibition.
The current results continue to support the hypothesis that CET
produces a decrease in the level of recurrent inhibition as suggested by
Abraham et al. (1981). However, it may be that paired pulse


238
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131
Discussion
A nutritionally complete liquid diet was used to produce an animal
model of long term alcohol exposure. This diet has been shown to
produce alterations in the behavior (Walker and Hunter, 1978; Fehr et
al., 1975), anatomy (Walker et al., 1980; Riley and Walker, 1978; King,
1984), physiology (Abraham et al., 1981, 1982, 1984; Durand and Carien,
1984a, 1984b) and biochemistry (Freund, 1980) of animals given similar
durations of ethanol exposure. Electrophysiological evidence from
extracellular studies have shown that the basic characteristics of
single pulse stimulation or of LTP were not changed in CET animals.
However, the PS amplitude was shown to be enhanced to paired pulse or
repetitive stimuli at 5 and 10 Hz without changes in the EPSP (Abraham
et al., 1981). These findings have since been corroborated by intra
cellular recordings from CAl pyramidal cells where it was shown that
only the IPSP amplitude and the late after-hyperpolarization were
reduced in CET animals (Durand and Carien, 1984a). The present in vitro
homosynaptic paired pulse results showed the CET animals to have trends
toward higher levels of potentiation than the sucrose animals. The
nature of these trends were similar to the results found by Abraham et
al. (1981).
The present in vitro paired pulse PS amplitude inhibition data are
not directly comparable to results from similar experimental manipula
tions performed in vivo since there was little inhibition observed in
vitro with homosynaptic stimulation. This difference in the level of
inhibition found between in vivo and in vitro preparations has been
described previously and is thought to result from the severing of the
longitudinal recurrent inhibitory interneuron fiber projections


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43
Characteristics. When paired shocks are delivered to any of a
variety of afferent pathways of the hippocampus, a characteristic
inhibition or potentiation of the test (second) response is observed
when the condition and test pulses are separated by a critical time
period (Steward et al., 1977). The magnitude of the test response
inhibition or facilitation depends upon the inter-pulse-interval (IPI).
The typical pattern of the facilitation depends on the response
measured. Stimulation of the Schaffer/commissural path while recording
in the CAl pyramidal zone produces a characteristic but different
response pattern for both the EPSP and the PS. The population EPSP is
maximally facilitated at IPIs of 20-30 msecs decreasing at progressively
longer intervals. PS amplitude is inhibited at IPIs of 20 to 80 msecs,
most likely as a result of inhibitory processes and is facilitated at
longer IPIs (> 80 msecs) (Lomo, 1971; Steward et al., 1977; Creager et
al., 1980).
Procedures. Paired-pulse inhibition and potentiation were
evaluated by systematically varying the IPI between the condition and
test pulse from 20 msecs to 3 secs. Four responses were averaged at
each interval tested. The individual intervals tested for all
experiments were as follows in msecs: 20, 30, 40, 50, 60, 80, 100, 150,
200, 300, 400, 500, 750, 1000, 1500, 2000, 3000. Paired shocks were
delivered at a frequency of 0.1 Hz in the CAl region in the order
described above. Paired pulse stimulation current levels in this series
of studies used test pulse current levels which were either 25 or 50
percent of the maximal PS response amplitude obtained from mini-I/O
curves. Condition pulse current levels were adjusted to 50 percent of


217
This differential set of responses, seen after CET with SO and SR
stimulation, suggest a differential projection of the commissural
afferents to the basilar versus apical dendrites of the CAl pyramidal
cell. It has been shown that commissural afferents of CA3 pyramidal
cells project more heavily to the basilar dendrites of CAl pyramidal
cells than to the apical dendrites (Swanson et al., 1978). This
projection pattern may result in termination of a different population
of interneuons than do ipsilateral schaffer collateral fibers. If, as
has been suggested, long myelinated fibers are especially prone to CET
(Dreyfus, 1974), it is possible that there is a decreased input to the
enkephalinergic interneurons, hence a decrease in the efficacy. It has
also become common to find peptides co-localized with various other
putative neurotransmitters including GABA. It is therefore possible
that CET reduces a population of interneurons which, given the proper
conditions, may be either inhibitory or excitatory. Finally, if
enkephalin acts by disinhibition, these findings suggest that CET may
result in a decreased sensitivity of the inhibitory interneurons which
receive input from commissural fibers. It has been reported that
acute doses of ethanol activated hippocampal pyramidal neurons (Berger
et al., 1982). It was then hypothesized that this increased response to
ethanol may occur by ethanol itself causing an increased release of
endogenous opiate peptide or by an ethanol-induced formation of
aldehyde-catecholamine condensation products (tetrahydroisoquinolines,
TIQS) which reportedly have opiate-like effects. The important point,
however, is that CET in some way differentially affects the response of
2DA application by afferent stimulation location. This may suggest that


215
1980; Duka et al., 1981). Mu receptors have been shown to be
selective to morphine application whereas the delta receptors were
most selective for leucine- and methionine-enkephalin (Chang and
Cuatrecasas, 1979). Functionally, both morphine agonists and leucine-
or methionine-enkephalins have been shown to produce similar
epileptogenic-like effects in the CAl region of the hippocampus
(Bostock et al., 1984; Lewis et al., 1981).
(D-Ala(2))-met-enkephalinamide used in the present studies would
preferentially activate the delta opiate receptors throughout the
subfields of CAl, it is clear that the largest response changes occurred
in or near the pyramidal cell region and SO. The present findings
suggest either a higher density of delta receptors at the cell layer or
an activation of the mu receptors at the cell layer in addition to the
delta receptors with the iontophoretic concentrations used in this
study.
The effect of CET on enkephalin administration resulted in a
differential PS response amplitude pattern which was dependent upon the
afferent stimulation site as can be seen in Fig 6-14C and Fig 6-14D.
With SR stimulation the CET group had larger PS amplitudes than did the
sucrose controls. With SO stimulation, the reverse was true. The
sucrose group was more potentiated than the CET animals. With
stimulation from either afferent field, the largest sucrose-fed PS
amplitude responses were always at the proximal SR iontophoresis site.
Fig 6-14 shows the PS amplitude response at 1.5 minutes following 2DA
administration for both the alcohol and sucrose groups and in separate
graphs for SR (Fig 6-14C) and SO (Fig 6-14D) stimulation. The time point
chosen (1.5 mins) for each of the drug series plotted was generally the


186
analyses showed the points noted in Fig 6-5 with asterisks as being
different. Finally, there were no differences in either PS total or
PS latency measures with bicuculline application in distal radiatum
with SO stimulation.
Baclofen series: Stratum radiatum stimulation
I/O relationships with SR stimulation. See description of I/O
curves as described in Chapter 5. I/O curves for each of the pharmaco
logical series were combined and presented earlier.
Iontophoresis application with SR stimulation. The data for the
baclofen PS amplitude responses with SR stimulation are shown in Fig
6-6 and in Fig 6-7 for the PS latency measures. There were no group
differences for any of the four sites tested with baclofen
administration and SR stimulation for the PS amplitude measure. The
only group by time interaction found with baclofen administration and
SR stimulation was in SR near the cell body layer (F(1,280) 1.58, p
= .05). Subsequent analyses showed that the individual differences
for the time points were from seven to nine mins after baclofen
application. The general pattern of responses can be seen for the PS
amplitudes in Fig 6-6. With SR stimulation and baclofen
administration, the effect of baclofen on the PS amplitude was most
powerful near the cell body layer in SO and then at both of the
iontophoretic administration sites in SR. Baclofen iontophoresis
actually resulted in the greatest magnitude of inhibition in distal
SR. The duration of inhibition was longest in SO near the cell body
layer. With the exception of the administration of baclofen in SO
near the cell body layer, there were no consistent trends for


150
were tested at each GABA iontophoretic site with both SR and SO stimula
tion. In all cases, at least 30 seconds elapsed before additional GABA
ejections were administered. Once both SR and SO stimulation were
accomplished with the same GABA ejection site, the GABA pipette was
moved and the procedures described above for finding the location of
maximal inhibition were initiated again. The SR and SO stimulations
were carried out and the GABA pipette moved until all 10 locations along
the dendrite were tested. The testing procedure lasted approximately 45
mins to one hr.
Results
The GABA PS results presented below were based on a total sample of
35 rats with 19 rats in the alcohol group and 16 in the sucrose group.
SR I/O relationships. The I/O curves shown in Fig 5-2 and Fig
5-3 represent the overall responses from all of the animals used in
this and the subsequent pharmacological experiments with SR and SO
stimulation, respectively. Since these I/O curves were obtained prior
to any pharmacological testing with both SR and SO stimulation,
pooling the data represented a resource saving measure as well as
providing a stronger statement concerning the I/O measures. There are
29 sucrose and 39 alcohol animals represented in each of the I/O
curves. All subsequent experiments will therefore refer to these I/O
curve data. Chronic alcohol administration did not produce
statistically significant changes in the PS amplitude nor in the PS
latency responses to single pulse stimulation with SR (Fig 5-2)
stimulation. The PS threshold was 134 5.07 and 139.65 8.5 for the
alcohol and sucrose-fed control groups, respectively.


54
the recording electrode was adjusted to approximately 150 uMs above the
region of maximal unit activity. With the recording electrode filtering
re-set for field responses, the orthodromic stimulating electrode was
lowered into the hippocampus until the evoked response was maximally
positive with a superimposed PS (see basic physiology section in general
introduction). Obtaining this response required readjusting the record
ing electrode depth to 150 uMs above the inversion point until the
maximal PS amplitude response was achieved. The antidromic stimulating
electrode was then lowered until a short latency, sharply negative-going
potential was observed. The antidromic electrode was carefully adjusted
to maximize the negative going response without inducing an orthodromic
stratum oriens EPSP response. At the end of the recording session, a
small lesion was made at the recording site and 400 uMs below. These
lesions were used to verify the location of the recording electrode
during histological analyses. The sizes of the stimulating electrodes
were such that the paths they created were observable without lesions.
Testing procedures
Once the stimulating and recording electrodes were positioned and
adjusted for maximal responses, input/output testing was performed for
the orthodromically evoked response only. In this procedure, the
current intensity delivered by the stimulating electrode was increased
in steps from 20 to 1000 uAs as described in the general methods.
After the I/O curves were collected, the paired pulse experiments
were begun. Two different groups of animals were used to test the
different test pulse current intensities as outlined in Table 3-1. The
first group of animals was tested with a test pulse stimulus current


146
region. Andersen et al., (1982) suggested that the responses seen with
SR stimulation reflected feedforward-like inhibition in the SR region
with recurrent inhibition found near the pyramidal cell layer region.
Stimulation with the SO electrode produced what may be SO-induced
feedforward inhibition but may also reflect a large influence of recur
rent inhibition since it has been reported that the recurrent inhibitory
interneurons are located in the pyramidal and proximal SO regions
(Andersen et al., 1964a). SR stimulation probably activates SR feed
forward inhibition while SO activation probably activates SO feedforward
inhibition. Since stimulation of the SO or SR dendritic region does not
apparently activate the recurrent inhibitory mechanisms, SO or SR
stimulation may allow for a relatively clean assessment of SR or SO
feedforward inhibitory effects, but as a function of where GABA is
applied along the dendrites. GABA iontophoresis will activate recurrent
inhibitory interneurons, however SO or SR stimulation alone will not
result in the effects of recurrent inhibition being observed. This
paradigm may therefore allow for a relatively selective investigation of
feedforward and recurrent inhibition along the dendrites of the CAl
pyramidal neuron. In CET animals, it would provide the opportunity to
selectively observe feedforward (in the SR region) and recurrent (in the
cell body region) inhibition in terms of the response to GABA
application on the basis of both the magnitude of the PS response and
the pattern of the PS response. This next experiment utilized this
paradigm.


81
of inhibition, the combination of feedforward and recurrent inhibition
produced total inhibition of the PS amplitude response even at the
current intensity of 25 percent of the PS maximum for the test response.
With A/0 stimulation, the PS amplitude was inhibited only to 40 or 50
percent of control levels with 25 percent of the PS maximum condition
pulse current intensity. A/0 paired pulse paradigms should predomi
nantly activate recurrent inhibition. 0/0 paired pulse paradigms should
activate both recurrent and feedforward inhibition. If the responses of
the A/0 paired pulse paradigms are compared with the responses of the
0/0 paired pulse paradigms, it is clear that feedforward inhibition
contributes significantly to the level of inhibition seen with 0/0
paired pulse paradigms in spite of facilitated EPSP responses found at
IPIs of 20 to 30 msecs.
A more detailed comparison of the A/0 and 0/0 paired pulse paradigm
responses revealed a potentially useful method for better characterizing
feedforward inhibition in the CAl region of the hippocampus. As de
scribed above, 0/0 stimulation produced total inhibition of the PS even
with a condition pulse current intensity of 25 percent of the PS ampli
tude at PS asymptote. The same A/0 condition pulse current intensity
resulted in much less inhibition than the 0/0 paradigm (40 percent
versus 100 percent inhibition). If the A/0 paired pulse stimulation
currents were titrated with decreasing condition pulse current levels
from the 25 percent of AS maximum amplitude level to that point at which
the A/0 test pulse PS amplitude was just observed (threshold) and then
that absolute current intensity was used with the 0/0 configuration, the
effect of feedforward inhibition alone may be observed. One of the
potential problems with this method is that the effect of the AS current


Figure 5-1. GABA iontophoresis sites along the dendritic axis
of CA1 pyramidal cells. Note that recordings
were of field responses and that the GABA effects
were upon groups of pyramidal cells rather than
single cells as depicted. GABA (1M, pH 3.5) was
ejected for five seconds with 40 nAs of current.
Abbreviations: ALV, alveus; SO, stratum oriens;
SP, stratum pyramidale; SR, stratum radiatum;
SLM, stratum lacunosum-moleculare.


206
Carien, 1984a, 1984b) and biochemical (Freund, 1980) changes. These
experiments were designed to more clearly elucidate potential pharmaco
logical mechanisms by which CET produced increased PS amplitudes after
20 weeks of exposure and subsequently at least 8 weeks of being on a
normal rat chow and water diet (Abraham et al., 1981).
The response to GABA administration of CA1 pyramidal cells has been
shown to consist of at least three independent actions. These three
different actions have been reported to be subserved by different
receptor populations. When GABA is applied to the somatic region of the
CAl pyramidal cell, an hyperpolarizing response is found. It has been
shown however, that a subpopulation of these hyperpolarizing receptors
are bicuculline insensitive. The bicuculline sensitive receptors have
been termed GABA(A) receptors while the bicuculline-insensitive recep
tors have been termed GABA(B) receptors. The third receptor sub-type
results in a depolarizing response to GABA and is usually found in the
dendrites rather than in the somal region.
The GABA(A) receptor antagonist, bicuculline, which has been shown
to produce increased PS amplitudes when iontophoretically administered
in the CAl pyramidal cell region, produced similar increases in PS
amplitude responses with CET and sucrose-fed animals. The response
pattern to bicuculline iontophoresis along the dendritic axis was such
that the maximal level of potentiation was in SO near the pyramidal cell
body layer. This pattern held true for stimulation from both the SO and
SR afferents. Bicuculline application in distal SO and proximal SR
resulted in responses which were dependent upon the stimulation site
with each area responding best to its respective afferent input. The


Figure 6-3. PS latency responses (means) after bicuculline
iontophoresis (time=0) with stratum radiatum
stimulation. Scales on ordinate represent difference
of PS latency response following bicuculline adminis
tration from control responses obtained prior to
bicuculline administration (test response-control
response) and are independent for each iontophoretic
ejection location. Abcissa represents time in minutes
following iontophoretic ejection of the drug. A) PS
responses with iontophoretic ejection in distal SO. B)
PS responses with iontophoretic ejection in proximal
SO. C) PS responses with iontophoretic ejection in
proximal SR. D) PS responses with iontophoretic
ejection in distal SR.


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Don Stehouwei/
Assistant Professor of Psychology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Neuroscience
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Bruce Hunter
Associate Professor of
Neuroscience
This dissertation was submitted to the Graduate Faculty of the
Department of Psychology in the College of Liberal Arts and Sciences and
to the Graduate School and was accepted as partial fulfillment of the
requirements for the degree of Doctor of Philosophy.
May, 1986
Dean, Graduate School


time after iontophoresis (mins)
difference from control (msecs)


40
variables. A minimum of eight rats were used in each group for all
experiments.
In these experiments, group E and group S animals were treated with
ethanol or sucrose for 20 weeks. A minimum of eight weeks was allotted
after the discontinuation of the alcohol diet prior to beginning any of
the electrophysiological procedures. This period of abstinence was
included to control for the residual effects of acute alcohol withdrawal.
Since the testing of individual preparations consumed an entire day,
sampling occurred over an 8 to 24 week period after ethanol discon
tinuance depending on the initial group size. Individual animal number
and group designation were coded to prevent any bias in the placement of
recording and stimulating electrodes. Coding was essential due to the
response variability observed in recordings from the hippocampus and to
prevent unintentional procedural differences between the two groups.
Equipment
All recordings were made with glass micropipettes filled with 4M
NaCl. Electrodes were chosen to satisfy criteria for tip diameter (1-2
uM), impedance (500K to 1.5 Mohms at 1 KHz) and shaft taper. Concentric
bipolar electrodes obtained from Rhodes Medical Instruments (in vivo) or
the Frederick Haer Co. (in vitro) were used to activate specific hippo
campal afferent or efferent pathways. In all cases, electrodes were
controlled with micro-manipulators. Constant current stimulation
(duration, 0.1 msec; current, 20-1000 uAs) was used in all procedures.
Stimulus pulse timing was controlled with WPI series 1800 timers (in
vivo) or with an Apple computer system using software and hardware
designed by the author (in vitro).


135
The 0/0 homosynaptic stimulation produced little if any inhibition.
The 0/0 heterosynaptic stimulation paradigms differed in that Haas and
Rose (1982) stimulated two different areas of the SR afferents, whereas
in the present experiments, the SR or SO region was stimulated with the
condition pulse followed by the SO or SR region with the test pulse,
respectively. This difference in the heterosynaptic stimulation para
digm could account for part of the discrepant results. A second
possible difference may be in the stimulation currents used by Haas and
Rose (1982). They used current levels set to 50 percent of the maximum
sized response which would be similar to the present 50 percent of the
PS amplitude at PS asymptote. A final difference and the one which may
provide the best explanation for the discrepancy between the two sets of
data across each of the different stimulation configurations is that
they maintained the bathing medium temperature at 32 to 33.5 degrees
centigrade whereas the present slices were kept at a temperature closer
to 35 degrees. It has been shown that small differences in temperature
can markedly affect the excitability of the hippocampal slice prepara
tion (Teyler, 1980). It should be noted that the results of Haas and
Rose (1982) are different from the results of other investigators in
which the slice technique was used. They showed levels and durations of
0/0 homosynaptic inhibition which were very similar to in vivo results.
Other papers using similar paradigms with the slice technique showed
periods of inhibition extending to 40 or 50 msecs at best (Dunwiddie et
al. 1980). The difference in bathing temperature probably best
explains this difference.
The SR/S0 heterosynaptic experiment, coupled with that of the
homosynaptic SO stimulation data suggest that SO stimulation may result


227
was observed with GABA, indicates a different time course of action, if
not a difference in the site of action, since GABA was never seen to
produce inhibition in distal SR.
The iontophoresis of 2DA along the pyramidal cell dendrites, like
bicuculline, resulted in increased PS amplitudes as well as multiple
spikes in all animals tested. Maximal response increases were seen in
proximal SR and SO although distal SO also resulted in substantially
potentiated responses with both SR and SO stimulation. There was
virtually no effect observed with 2DA administration in distal SR. The
small response seen in distal SR did not reach maximal levels until
three minutes after ejection, indicating this effect was probably a
result of the diffusion of 2DA toward the active cell body region.
These response patterns were similar to those reported previously
(Dingledine, 1981).
GABA iontophoresis resulted in trends for the CET PS amplitudes to
be more inhibited or less potentiated (at various regions along the
pyramidal cell dendritic tree) than sucrose-fed controls. It was
hypothesized that a decrease in afferent projections per se or a de
creased release of GABA would in turn result in an up-regulation
and/or increased sensitivity of GABAergic receptors. In this way,
increased PS amplitudes with paired pulses could be explained as well
as the increased GABAergic response in CET versus sucrose-fed
controls.
Bicuculline administration resulted in increased PS responses for
CET animals when compared to the sucrose-fed controls, especially in
proximal and distal SO. This increased response to bicuculline was
thought to result from the previously hypothesized increase in sensitiv
ity of the GABA receptor. Since bicuculline competitively blocks the


RAW DATA (MSEC) RAW DATA (MV)
152
o 200 400
CURRENT INTENSITY (UAMPS)


210
receptor population which would result in an increased PS response to
bicuculline admininstration since the bicuculline would competitively
overwhelm the release of GABA. However, a decrease in the GABAergic
receptor population would not be consistent with the increased response
found after CET with GABA iontophoresis.
Modifications in the GABA receptor population may occur as a result
of alterations incurred by the benzodiazepine receptor. Freund (1980),
showed that after 20 weeks of CET in mice, there was a decreased number
and affinity of benzodiazepine receptors. Since GABA and benzodiazepine
receptors are reported to occur in a receptor complex with Cl- channels
(Tallman et al., 1980), there may be a structural modification such that
GABA sensitivity is increased after CET.
Iontophoretic application of baclofen along the pyramidal cell
dendrites produced a decrease in the PS amplitude with either SO or SR
stimulation at each of the locations where it was iontophoretically
ejected. There was, however, a stimulation location and iontophoretic
site dependent response to its administration as seen in Fig 6-15A and
Fig 6-15B. With SR stimulation, the greatest levels of inhibition were
seen in proximal SO, proximal SR and distal SR. With SO stimulation,
the greatest levels of inhibition were seen when baclofen was applied in
the proximal and distal SO. These data strongly suggest the presence of
feedforward inhibition along the entire length of the CAl pyramidal cell
dendritic tree. Previous reports using intracellular techniques have
indicated that baclofen inhibition is seen throughout these areas.
Iontophoretic application of baclofen using extracellular recording
techniques has not been reported previously, however.


THE EFFECT OF CHRONIC ETHANOL INGESTION ON
SYNAPTIC INHIBITION IN CAl OF THE RAT
BY
CARL J. ROGERS
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1986

This dissertation is dedicated to my wife, Jane,
and to my parents, Carl F. and Sophie Rogers,
for their unending support.

ACKNOWLEDGEMENTS
The production of this dissertation was made possible by a number
of individuals directly and indirectly connected with the experimental
work.
Two people, Pat Burnett and Bill Creegan, have been most
instrumental in helping me to get the job done. Pat has helped to
smooth the rough edges with my slice work as well as encourage my
progress. Bill has been unendingly patient with my continual requests
for program modifications and explanations of how he did them. I thank
each of you sincerely for your expert help.
Other people have provided help at various times during this long
and arduous trek. I thank Larry Ezell, Dot Robinson, Dr. Paul Manis,
and Christina King for their time and efforts.
I would especially like to thank Dr. Roger Reep for his insightful
thoughts during some of my more challenging moments. His energy and
talents are second to none. I also want to thank Dr. Bob Davis for
getting me into the hippocampus and supporting me along the way. Dr.
and Mrs. Ken Finger have shared their thoughts on a variety of interesting
and helpful topics. I thank them for the opportunity to do so.
I would also like to thank Don Walker and Bruce Hunter for their
confidence in my abilities. They have helped to shape this research in
many ways. Thanks are also extended to Dr. Chuck Vierck, Dr. Floyd
Thompson, Dr. Don Stehouer, and Dr. Marc Branch for their help and
encouragement.
iii

Finally, I thank my wife, Jane, for standing with me during the
difficult times and sharing with me the enjoyable moments. She deserves
much of the credit for putting this manuscript together as well as for
keeping me going.
And, as always, I thank Mom and Dad for their unending support.
We have finally made it!
iv

TABLE OF CONTENTS
PAGE
ACKNOWLEDGEMENTS iii
ABSTRACT vii
CHAPTERS
1 GENERAL INTRODUCTION 1
Foreward 1
Behavior 3
Anatomy 5
Physiology 6
General Considerations 7
The Hippocampal Model and Memory 8
The Normal Human Hippocampal Evidence 9
A Comparison of Hippocampal Deficits with CET Deficits ... 10
The Normal Animal Hippocampal Evidence 12
Chronic Ethanol Treatment (CET) and Behavior in Animals. . 14
Summary of Alcohol and Normal Human and Animal Literature. 15
Animal Alcohol Models 16
The Normal Hippocampus--Anatomy and Physiology 16
Pharmacology of the Hippocampus 27
Rationale and Introduction to Current Experiments 34
2 GENERAL METHODS 38
Animals 38
Alcohol Diet Regimen 38
Equipment 40
Basic Procedures 42
Data Analysis and Interpretation 44
3 CHARACTERIZATION OF SYNAPTIC INHIBITION IN VIVO 48
Introduction 48
Methods 52
Results 57
Discussion 73
4CET AND SYNAPTIC INHIBITION AN IN VIVO ANALYSIS 87
Introduction 87
Methods 91
Results 95
Discussion 131
v

TABLE OF CONTENTS CONTINUED
PAGE
5 EFFECT OF CET ON IONTOPHORETIC GABA MEDIATED RESPONSES ... 141
Introduction 141
Methods 146
Results 150
Discussion 160
6 EFFECT OF CET ON THE IONTOPHORETIC APPLICATION OF BICUCULLINE,
BACLOFEN AND ENKEPHALIN MEDIATED RESPONSES IN CAl 168
Introduction 168
Methods 170
Results 173
Discussion 205
7 GENERAL DISCUSSION 219
REFERENCES 231
BIOGRAPHICAL SKETCH 244
vi

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
THE EFFECT OF CHRONIC ETHANOL INGESTION
ON SYNAPTIC INHIBITION IN CAl OF THE RAT
By
Carl J. Rogers
May 1986
Chairman: Don W. Walker
Major Department: Psychology
Chronic ethanol abuse by humans results in behavioral alterations
as well as anatomical and physiological deficits within the CNS. Animal
models of chronic ethanol treatment (CET) using nutritionally controlled
diets showed that ethanol itself results in deficits similar to those
observed in humans. The hippocampus, a region of the brain implicated
in memory processing, has reduced cell populations as well as dendritic
spine density after CET. Electrophysiologically, increased population
spike amplitudes (PSA) with paired pulse paradigms (PPP) are found
without concomitant changes in basic synaptic waveforms suggesting
decreased recurrent inhibition (RI). Recently, feedforward inhibition
has also been demonstrated in this region.
A nutritionally complete liquid diet containing ethanol or sucrose
isocalorically substituted was pair-fed to rats for 20 weeks. Eight
weeks after ethanol abstinence, the effects of CET on feedforward and
vii

recurrent inhibition were tested using PPP or with four pharmacological
agents, gamma-aminobutyric acid (GABA), bicuculline, baclofen, and
D-ala(2)-methionine-enkephalinamide (2DA) iontophoretically applied
along the dendrites of CA1 pyramidal cells.
The PPP with control animals showed that increasing condition pulse
current intensity with orthodromic/orthodromic (0/0) stimulation
increased the duration of inhibition and always abolished the PSA at
short interpulse intervals. Antidromic/orthodromic PPP produced an
increased magnitude of inhibition with increasing condition pulse
current intensity, however, the PSA was never completely inhibited.
These findings suggest an important contribution of feedforward
inhibition which, after CET, may be reduced as was RI.
The PPP with in vitro preparations showed CET animals had trends
toward increased PSA similar to those found in vivo. Antidromic/
orthodromic results showed significant differences between the two
groups suggesting that CET reduced RI.
Responses of CET animals to GABA resulted in trends toward
decreased PSA. Bicuculline-mediated PSA were increased after CET
suggesting increased GABA receptor sensitivity. Baclofen inhibited PSA
but without group differences. Responses to 2DA from CET animals were
stimulation site dependent. Stratum radiatum stimulation produced
increased PSA whereas stratum oriens stimulation decreased PSA. It is
suggested that CET differentially affected the commissural afferents or
SO interneurons.
These data continue to reveal detrimental consequences of long term
ethanol abuse.
viii

CHAPTER 1
GENERAL INTRODUCTION
Foreword
The consumption of alcohol by individuals in our society and
throughout many regions of the world is a widely accepted and, in many
ways, condoned behavior. The effects of acute episodes of alcohol
overindulgence are well documented. Increases in response time, errors
in judgement and cognitive skill are but a few of the many behavioral
effects alcohol imposes upon users. In recent years and months, our
federal and state governments have begun to re-assess the costs of
alcohol abuse upon society in terms of lost lives and property and to
reconsider laws governing the legal responsibilities and consequences of
individuals for the behavior they exhibit while under the influence of
alcohol. These steps have been taken as a result of increasing alcohol
abuse and the concomitant costs to society.
Alcohol abuse is often regarded in terms of single episodic
events. But alcohol abuse is not an issue related only to those periods
of active consumption and the behavioral changes that occur within that
framework. Instead, and especially from a mental health perspective,
alcohol abuse is a lifestyle which carries with it important sociologi
cal, psychological and biological components. Although alcohol abuse
may begin as a result of sociological, psychological or even biological
influences and, in turn, may impart sociological and psychological
dysfunction, detrimental biological alterations always occur with
prolonged use. These alterations must be better understood in order for
1

2
more effective treatment and preventative programs to be formulated. It
is therefore necessary to ask the questions, what are the long term
effects of alcohol abuse from behavioral, anatomical, physiological, and
biochemical perspectives? Does alcohol permanently alter the way in
which we behave, the way in which we think? If a lifestyle of alcohol
use and abuse does produce deleterious effects, are there limits beyond
which permanent changes occur and, for those individuals who have gone
beyond those limits, is there a way to reverse, or at least help to
compensate for, those damages? In order to provide effective treatment
and to properly educate the public we must know what long term alcohol
consumption does and by what means. The cost of this research will
surely be minuscule compared to the cost alcohol abuse currently imposes
on society in terms of lost cognitive abilities alone. It is estimated
that there may be as many as 20 million people in the United States who
can be termed problem drinkers (Clark and Midanik, 1982; Malin et al.,
1982) .
The experiments described herein were designed to provide additional
information concerning the effects of long term ethanol abuse on the
function of the hippocampus, a vital area of the brain thought to be
involved in memory processing. If we can understand why and how ethanol
produces deficits in memory function, it may be possible to provide more
effective treatment for some of the cognitive dysfunctions of alcoholic
patients. Perhaps even more important is the idea that the more substan
tial the information provided to the general public concerning the
effects of ethanol use and abuse, the more likely the public will be to
evaluate the consequences of ethanol use in terms of their own physical
and mental well-being. Thus, a better understanding of ethanol effects

3
would produce many different avenues of use, from the treatment of the
impaired to the willful prevention of the disease itself.
Behavior
The consumption of alcohol in moderate to heavy doses over long
periods of time (months to years) has been shown to produce a wide
variety of behavioral changes in humans. Long term alcohol consumption
does not produce a consistently clear, easily described and universal
picture of behavioral, anatomical, or physiological symptomatologies.
There may be several avenues of explanation for these inconsistencies of
symptomatologies among which are: 1. different genetic populations with
different dispositions to the effects of alcohol, 2. the varied and
unique individual drinking histories of long term alcohol consumers, and
3. other environmental conditions which are unique such as disease or
trauma (Tarter and Alterman, 1984; Parsons and Leber, 1981). Even with
the wide variations of symptoms seen in chronic alcohol abusers, reviews
of the literature indicate a relatively common set of behavioral and
biological alterations (Begleiter et al., 1980; Parsons, 1977; Ron,
1977; Ryan, 1980; Tarter, 1975). These alcohol-induced changes, in
turn, provide reasonable indications of which major brain systems may be
most severely involved.
The clinical symptoms of long term alcohol abuse include to
varying degrees cognitive, memory and motor performance impairments.
Deficits have been reported to occur in perceptual, spatial, problem
solving and cognitive tasks. However, one of the most persistent and
devastating effects of long term ethanol abuse is that of memory loss.
Although short term memory is generally reported to be intact, long-term

4
memory processing is often severely impaired. Severe memory deficits
are often defined as part of a syndrome named Korsakoff's syndrome
(discussed below). Other clinical manifestations of long term alcohol
abuse have been associated with nutritional deficits. Brain stem in
volvement, as evidenced by oculomotor abnormalities, is seen in patients
with histories of long term alcohol abuse. However, these abnormalities
are often rapidly reversed with thiamine treatment strongly indicating
that dietary intake may be disturbed in some patients and thus contrib
ute directly to some of the symptoms observed with alcohol abuse. Long
term alcohol exposure also causes irregularities in sleep patterns as
well as endocrine function and certain aspects of autonomic nervous
system functioning (Kissin, 1979; Wagman and Allan, 1977). It is
important to emphasize that long term alcohol abuse has nearly always
been shown to produce cognitive impairment and brain damage despite the
wide variations found in the symptoms exhibited by alcoholic patients.
One of the most controversial aspects of long term alcohol abuse
has centered on the interaction of dietary inadequacies versus direct
toxic effects of alcohol on the CNS. This debate has begun to be
silenced by carefully controlled human and animal studies where dietary
intake has been monitored or controlled. The results of these studies
have provided significant evidence that alcohol per se can produce
direct neurotoxic effects. It has been noted that Korsakoff's syndrome
has never been found without coincident long-term alcohol abuse (Freund,
1973).
One of the most intensely studied areas of alcohol research has
concerned the effect long term alcohol abuse has on memory function.
Korsakoff's syndrome represents the most severe form of long term

5
alcohol effects on memory. The major components of Korsakoff's syndrome
are: 1. a severe inability to remember current events over periods of
time into the future of more than about one minute (anterograde amnesia)
beginning with events occurring at the approximate onset of the disease,
2. a limited understanding by the patient of the memory disability and
3. a general state of apathy. Although the ability to remember events
prior to the onset of the disease state (retrograde amnesia) is not
eliminated, an increasing degradation of memory prior to the onset of
the acute phase of the syndrome is seen (Butters and Cermak, 1980;
Seltzer and Benson, 1974; Marslen-Wilson and Teuber, 1975). Even if
alcohol use is eliminated for months or years, memory impairments
persist in at least 80 percent of the Korsakoff's syndrome population
(Victor et al., 1971).
Anatomy
An increasingly convincing accumulation of neuroanatomical data has
been assembled concerning the detrimental effects of long term alcohol
consumption on the gross- as well as micro-anatomy of human alcoholics.
The most widespread method of gathering human neuroanatomical data has
been with computerized tomography scans (CT scans). The results of
these procedures have consistently shown in many experiments and with a
large sample size that alcoholics of all ages have enlarged cerebral and
cerebellar sulci as well as cerebral ventricles. These observations
indicate cerebral atrophy (Wilkinson, 1982; Ron, 1977; Ron et al., 1982;
Brewer and Perrett, 1971; Cala and Mastaglia, 1981; Epstein et al.,
1977; Haug, 1968). Earlier work with pneumoencephalography revealed
similar findings. Victor et al. (1971) observed the gross pathology of

6
brains from autopsies of chronic alcoholics and provided a detailed
description of brain regions which contained lesions. Among the areas
where lesions were more consistently found were the mammillary bodies,
various hypothalamic nuclei, the medial dorsal nucleus of the thalamus,
the cerebellum, periventricular regions of the brainstem, the cerebral
cortex and the hippocampus. Earlier studies of alcoholic brain material
indicated that there was an increased vascularity, a loss of myelin in
the cerebral cortex and cerebellum, and a 20 to 40 percent decrease in
pyramidal cell number in motor cortex and purkinje cells in the cere
bellum (Lynch, 1960). Degenerative changes in the granular layer and
dentate nuclei of the cerebellum in alcoholics, a loss of Purkinje
cells, neuroglial proliferation and a thickening of blood vessels in
alcoholics have been reported after chronic ethanol abuse (cf. Lynch,
1960).
Physiology
Electrophysiological studies on human alcoholics have relied on the
use of electroencephalographic (EEG) and evoked potential recordings.
These methodologies have been criticized for their inability to indicate
specific underlying brain structure involvement as well as the underly
ing cellular generators of the recorded potentials. More recent stud
ies, however, in non-alcoholic patients, have increasingly suggested
that certain portions of the generated potentials can be isolated to
specific underlying structures of the brain. For example, the P300
component of evoked potentials, a commonly used measure in alcoholic
patient studies which occurs approximately 300 msecs after stimulus
presentation (often a pulse of light), has been convincingly shown to be

7
generated in the hippocampus, an area considered by many to be implicat
ed in memory processing (Begleiter et al., 1980; Halgren et al., 1980).
The evidence from human alcoholics when using EEG recordings
indicated widespread, fast activity when compared to non-alcoholics
(Coger et al., 1978). Event related brain potentials have shown that
the late P300 components were decreased in amplitude in alcoholics when
compared to controls (Begleiter et al., 1980; Porjesz and Begleiter,
1981). The P300 component has been used as an indicator of the
evaluation of stimulus significance, "an electrophysiological manifes
tation of the orienting response" (Begleiter et al., 1980, p. 10). This
relation of the P300 component of evoked potentials to stimulus
significance is relevant to alcoholics since alcoholics do not differen
tiate relevant from non-relevant stimuli as do normals (Begleiter et
al., 1980). More recently, it has been reported that after ending the
consumption of alcohol after long term use, a recovery of the amplitude
of the P300 component occurs although never back to control levels
(Porjesz and Begleiter, 1981).
General Considerations
A number of problems exist in the above studies from human alcohol
ics when considering the correlations between variables such as duration
of drinking and quantity consumed and the extent of neuropsychological
and neuropathological deficits (Parker and Noble, 1980). These variables
are not necessarily good predictors of pathology (Ron, 1977). Alcoholics
with severe neocortical atrophy may not show appreciable losses in
cognitive function while conversely, alcoholics with no grossly apparent

8
CNS damage may exhibit profound psychological impairments (Wilkinson and
Carien, 1980). It is difficult, if not presently impossible, to assume
a causal relationship between brain damage and cognitive dysfunction
(Parsons and Leber, 1981).
These problems have created an implicit need to better understand
and control the relevant variables necessary to obtain reliable rela
tionships between alcohol abuse and brain damage. To do this, better
clinical histories of patients would be helpful; however, the accuracy
provided can never be completely reliable. An alternative method of
gaining a better understanding of the relationships between variables
would be to control these variables in the manner of a laboratory
experiment. The ethical problems associated with this alternative are
numerous. Thus, scientists have developed animal models of chronic
alcohol consumption in an effort to determine the effect of alcohol per
se on the CNS. Control of variables such as age, drinking history,
genetic makeup and nutritional adequacy are then feasible.
The Hippocampal Model and Memory
Since the most devastating aspect of Korsakoff's syndrome is the
profound anterograde amnesia, the study of memory is crucial to an
understanding of the alterations which Korsakoff's syndrome produces.
Several regions of the CNS have been implicated in memory processing.
Of those regions reported to be affected in Korsakoff's syndrome, the
temporal stem, the temporal cortex, the medial dorsal thalamus, the
mammillary bodies and the hippocampus have all been considered to be
involved in some way with memory processing (Horel, 1978; Victor et al.,

9
1971). Each of these regions would represent a reasonable location in
which to begin a search for the effects of Korsakoff's syndrome on
memory processing.
The present series of experiments utilize the hippocampus, a
region in which a large amount of literature exists concerning experi
mental variables related to behavior, anatomy and physiology in the
study of memory and other related phenomena. A substantial amount of
literature utilizing the hippocampus and the consequent effects of long
term ethanol treatment has thus followed.
The hippocampus has emerged as a model system for the study of
memory for several reasons. First, the hippocampus has been strongly
implicated in memory processing from human clinical cases (discussed
below). Secondly, the hippocampus itself is an important "model"
system. It is composed of well-defined cell layers as well as dendritic
regions. Inputs and outputs are well characterized and reside within
laminae which have little overlap. The anatomical arrangements of the
dendrites allow for the summation of electrophysiological potentials
which can, in turn, be recorded with relatively simple extracellular
recording techniques. Because of the highly laminated structure of the
hippocampus, the recorded potentials can be interpreted with a high
degree of confidence. It is for all of these reasons that a wealth of
literature on the hippocampus exists from different disciplines which
further facilitate meaningful interpretation.
The Normal Human Hippocampal Evidence
The evidence for the involvement of the hippocampus in normal human
memory emanates from radical procedures used in the mid 1950s to

10
alleviate severe seizure activity in a number of patients (Penfield and
Milner, 1958; Scoville and Milner, 1956; Scoville, 1954). Although the
seizure activity of these patients was dramatically reduced, severe
effects were seen on memory processes. One of these patients, H.M., has
been well characterized. Anatomically, the anterior two-thirds of
H.M.'s hippocampi were bilaterally removed as well as part of the
temporal lobe extending 8 mms posterior to the temporal poles. The most
striking effect of these lesions was an immediate and profound
anterograde amnesia. Retrograde amnesia was also found but only for a
relatively short (approximately two years) period of time immediately
prior to the operation. H.M. could detail his earlier years without
problem. Intellectually, H.M.'s intelligence quotient score was not
different from normal. H.M.'s short term memory (STM) was also intact.
However, he had a profound impairment of long term memory (LTM) includ
ing both verbal and non-verbal tasks. Visually and tactually guided
mazes were learned only when the number of choices was reduced to be
within STM memory span (usually seven to nine choices) and even so H.M.
had much more difficulty with tactile mazes (Milner et al., 1968).
H.M.'s acquisition performance was worse than normal in these tasks
although once learned, he was able to retain the information over long
periods of time (Milner et al., 1968).
A Comparison of Hippocampal Deficits with CET Deficits
A brief comparison of the features of Korsakoff's syndrome with
those of medial temporal resections results in a number of similarities.
Both cases exhibit severe anterograde amnesia with both verbal and
non-verbal tasks. Pre-lesion (retrograde) memory is intact in both

11
alcoholics and ablated individuals. Korsakoff's syndrome patients
tended to exhibit enhanced forgetting when distractor variables were
used with STM tasks while H.M., like amnesiacs, showed normal STM decay.
Korsakoff's syndrome patients exhibited decreased performance when
retrieving information from LTM when an interfering task was included.
Hippocampal lesioned patients also appeared to exhibit interference
effects although they may be modality specific (DeLuca et al., 1975).
These comparisons indicate that although there are some differences in
the behavior of these two groups of patients, the similarities are quite
strong.
The above similarities are not without important problems. One of
the most vexing problems with Korsakoff's syndrome patients is the
relatively low correlation found between hippocampal anatomical altera
tions and the severity of memory problems. Victor et al. (1971)
indicated that the hippocampus was bilaterally involved in 36 percent of
the cases. The mammillary bodies, however, a structure which receives
much of its input from the fornix and which largely emanates from the
hippocampus, was much more consistently involved. Still, even the
mammillary bodies were not found to be involved in 100 percent of the
cases studied. One lesion which has been consistently found in
Korsakoff's patients is in the dorsomedial nucleus of the thalamus
(Victor et al., 1971). More recently, it has been hypothesized that
several different types of amnesia may exist. Although the dorsomedial
thalamus may be strongly implicated in the storage, retrieval and
encoding of information, the hippocampus may be responsible for specific
types of memory processing such as spatial memory (Horel, 1978).
Another argument concerning the degree of involvement of these various

12
brain structures reported by Victor et al. (1971) is that they were the
result of gross pathological examinations and not histopathological
data. Thus, while gross changes were reported, more subtle yet equally
devastating consequences of long term alcohol abuse may exist in the
microstructure of these and other brain areas. Other investigators have
shown the hippocampus to be consistently implicated from autopsy materi
al of human alcoholic patients (Brion, 1969; McLardy, 1975; Miyakawa et
al., 1977).
The data presented above suggest that while hippocampal damage may
be seen with long term alcohol intake, it is not the most highly impli
cated structure. However, because the hippocampus has been so thoroughly
studied behaviorally, anatomically, and physiologically in the normal
animal, it allows for a much more thorough and detailed examination of
the effects chronic ethanol treatment may produce than does nearly any
other brain structure otherwise thought to be involved in memory pro
cessing.
The Normal Animal Hippocampal Evidence
Although the effort to develop an animal model of the human
amnesiac syndrome has been strong, no animal model has yet produced a
symptomatology which completely replicates that found with H.M. Instead
of performance deficiencies always being found, the animal literature
has shown that hippocampectomy can actually produce performance
advantages with some tasks. One of the important questions, however, is
whether behavioral tasks designed for animal models truly replicate
those tests used with human patients. The extent of the hippocampal
literature is enormous and has been well reviewed (O'Keefe and Nadel,

13
1978; Douglas, 1967; Isaacson and Pribram, 1975; Seifert, 1984). There
fore, only a brief summary of the more relevant findings is presented
below.
The major categories of behavioral testing are summarized as
presented by O'Keefe and Nadel (1978). Hippocampal animals generally
exhibited increased reaction to novel stimuli; they are considered
hyperactive but at the same time hypoexploratory. Spontaneous alterna
tion studies indicated either random or repetitive behavioral responses.
Spatial discrimination after hippocampal lesions proved to be more or
less normal as did non-spatial discrimination. However, spatial dis
crimination reversals were deficient in hippocampal lesioned animals as
were non-spatial reversals. Complex maze learning was deficient in
nearly all such studies. One way active avoidance tasks were typically
normal or showed deficits. Two way active avoidance after hippocampal
lesions, however, showed facilitated avoidance. Passive avoidance tests
yielded mixed responses, generally responding normal to controls but
with occasional deficits on very specific elements of the measures used.
Time related tasks such as lever press rates, delayed response,
go-no-go, delayed alternation and differential reinforcement of low
rates (DRL) with lesioned animals yielded high rates of behavior, hence
deficiencies on these tasks. Finally, hippocampal lesioned animals
typically showed extinction deficits.
The above results allow for several general observations. These
data indicate that hippocampal lesioned animals can learn but often show
deficits in part because of response or strategy perseveration as seen
with discrimination reversals or extinction testing tasks. A second
point is that tasks which typically rely on spatial or contextual cues

14
are severely affected. This is apparent with maze learning behavior
where learning in normal animals takes place quickly. In hippocampal
lesioned animals and humans we see that these tasks can be learned but
that it takes much longer. It has been suggested that normally an
animal uses a spatial-time or contextual coding process for learning
these tasks (Hirsch et al., 1978). However, when the hippocampus is
damaged, all learning must take place in terms of stimulus-response
chains which are more difficult to develop. Finally, it can be seen
that tasks which require timing behavior are severely affected. Timing
is also thought to be a major aspect of contextual coding. Thus,
spatial and time cues produce a context dependent memory. It appears
that this memory is severely affected by hippocampal lesions (O'Keefe
and Nadel, 1978; Hirsch et al., 1978).
Chronic Ethanol Treatment (CET) and Behavior in Animals
There are many important similarities between the behavioral
changes associated with animals chronically treated with alcohol and
animals with hippocampal lesions. Freund (1974) showed that mice, after
four months on a liquid diet containing ethanol, were deficient in both
the acquisition and retention of a passive avoidance task. One way
active avoidance was not affected when tested with hamsters given an
ethanol containing diet; however, the ethanol exposure was only for
seven days (Harris et al., 1979). Ethanol consuming rats were found to
be deficient in the acquisition of Hebb-Williams mazes and in the
acquisition and retention of a moving belt task (Fehr et al., 1975).
Spontaneous alternation was also shown to be deficient. Tasks requiring
timing behavior such as temporal shock discrimination and DRL schedules

15
were impaired (Denoble and Begleiter, 1979; Smith et al., 1979; Walker
and Freund, 1973). Temporal single alternation and go-no-go behavioral
tasks were also deficient in alcohol treated rats (Walker and Hunter,
1978). Each of these measures of performance is similar to those of the
hippocampal lesioned data. Shuttle box avoidance behavior with ethanol
treated rats, however, is different from the behavior found with
hippocampal lesioned animals. While hippocampal lesions normally
facilitate the response to this task, ethanol treatment produced im
paired performance (Freund, 1979; Freund and Walker, 1971).
Summary of Alcohol and Normal Human and Animal Literatures
In summary, the similarities found with behavioral testing between
Korsakoff's syndrome patients and H.M. appear to be strong. The litera
tures from hippocampal lesioned animals and CET animals not only provide
similarities between hippocampal lesions and CET but also between the
human and animal literature. Unfortunately, little detailed human
anatomical, electrophysiological or biochemical evidence exists concern
ing CET effects due to the ethical problems inherent with such human
research. Animal studies of CET have, however, begun to provide a
wealth of information concerning morphological and functional altera
tions, especially in the hippocampus and cerebellum. Since the
hippocampus, and in particular, the CAl region is important to the
studies performed herein, the animal models, anatomy, physiology and
pharmacology of the hippocampal CAl region will be considered in the
next sections in some detail.

16
Animal Alcohol Models
The use of animal models for chronic alcohol studies brings with it
the problem of how to administer the alcohol regimen. Several different
methods of alcohol administration have been developed including inhala
tion (Goldstein and Pal, 1971) and intubation (Majchrowicz, 1975;
Pohorecky, 1981; Altshuler, 1981). These methods, however, are not as
practical for long term administration as others which require relative
ly normal feeding procedures. Several animal models do exist which
allow ethanol to be administered orally. These methods include the
following where ethanol is provided: 1. ad lib as the sole source of
fluid with ad lib lab chow also being provided, 2. operant conditioning
techniques where an ethanol containing fluid is used as a reward for
maintaining drinking behavior (Falk et al., 1972) and 3. an ad lib
liquid ethanol-containing diet (Freund, 1969; Lieber and DeCarli, T973).
This liquid diet mixture contains all of the required protein, carbo
hydrates, vitamin and mineral supplements necessary for healthy develop
ment. This last method is currently the most widely used of the animal
models (Pohorecky, 1981) and is the method used in the studies completed
herein. Each of these methods of alcohol administration has shown that
alcohol produces alterations in the behavior, anatomy and electro-
physiology of a variety of animals tested.
The Normal Hippocampus -- Anatomy and Physiology
Anatomy of the hippocampus
The hippocampus is a bilateral structure which extends laterally
and caudally for several millimeters from a dorsomedial position in the
rat brain. It turns ventrally in a wide curve until its ventral tip is

17
extended in a rostral-caudal direction. The hippocampi thus look
similar to rams' horns. Each hippocampus consists of two distinct
regions, the hippocampus proper or CA region (Cornu Ammonis for its
seahorse-like shape) and the dentate gyrus (DG). Each of these areas
appears as a C-shaped structure with one facing the other and
interlocked. The CA region is further subdivided into four relatively
distinct regions (Lorente de No, 1934). The dorsal-most region of the
hippocampus and the C-shape is known as CA1 (Fig 1-1). It is located
next to the subiculum medially and extends laterally to where the
hippocampus first turns ventrally. Moving inward on the C-shape is a
short CA2 zone followed by a large CA3 region and finally the CA4 area
which is located within the two outer blades of the DG. The CA2 zone is
generally defined as the zone which distinguishes the regio superior
(CAl region) from the regio inferior (CA3 and CA4 regions).
The CA regions consist of several layers. Beginning from the
dorsal surface of the CAl region, the first layer is the alveus, a dense
but thin layer of afferent and efferent fibers. Next is the relatively
cell-poor molecular layer known as stratum oriens (SO). This layer
contains the basilar dendrites of the pyramidal cells. It is followed
by a dense but thin layer of pyramidal cells which is called stratum
pyramidale (SP). Beneath the pyramidal cell layer is stratum radiatum
(SR) and stratum lacunosum-moleculare (SLM). In the CA3 region of the
rat only, an additional laminae is found between the SR and the SP. The
apical dendrites of the pyramidal cells project into these last three
layers. The majority of afferent fibers to the pyramidal cells project
orthogonally to these pyramidal cell dendrites and terminate on them.
The afferent fibers which project to CAl originate from several regions

Figure 1-1. Schematic diagram of hippocampus showing dentate gyrus, CA3 and CA1 regions.
Electrode placements for the present experiments are shown. The recording
electrode was positioned within or dorsal to the pyramidal cell layer. The
stimulation electrodes were placed in stratum radiatum at the CA2-CA1 border, in
stratum oriens also at the CA2-CA1 border or in the alveus immediately dorsal or
dorsal and medial to the recording electrode.
Abbreviations: ALV, alveus; COM, commissural fibers; hi, hilus; MF, mossy
fibers; PP, perforant path; SCH, Schaffer collaterals; Sg, stratum granulosum;
SM, stratum moleculare; SO, stratum oriens; SP, stratum pyramidale; SR, stratum
radiatum.


20
but especially the ipsilateral and contralateral CA3 regions. These
fibers are termed the Schaffer/commissural afferents.
The flow of information through the hippocampus occurs in a
trisynaptic sequence. The major afferent system to the hippocampal
complex emanates from the entorhinal cortex and forms the perforant path
(or angular bundle). These fibers project onto the granule cells of the
DG with a lamellar distribution. The axons of granule cells then
project through the hilus to the CA3 pyramidal cells. The axons of CA3
pyramidal cells, in turn, project ipsilaterally as well as contralaterally
to the CAl pyramidal cells. The CAl pyramidal cells then project
rostrally to the medial septum, anterior thalamus and mammillary bodies
and caudally to the subiculum and entorhinal cortex. This last projection
actually closes the loop of projections which started in the entorhinal
cortex. Each of these connections produces an excitatory influence on
the postsynaptic cell (Andersen et al., 1966). A final distinctive
feature of the hippocampal circuitry is that each of the major projection
cells resides and is preserved within a cross sectional slice or lamellae
of the hippocampus (Andersen et al., 1971).
The region of specific interest in the current studies is the CAl
pyramidal cell region. The general circuitry has been described above.
The microcircuitry within this region is also important and is known to
mediate, among other phenomena, the inhibitory control of the pyramidal
cell. The pyramidal cell sends its axon through the alveus as described
above but also sends an axon collateral to one or more inhibitory
interneurons located near or within the pyramidal cell layer. This
interneuron, presumably a basket cell, then projects back onto the
original as well as a large number of other pyramidal cells thereby

21
inducing relatively widespread inhibition (Andersen et al., 1964a).
This feedback type of inhibition is termed recurrent inhibition.
Although the definitive circuitry is not yet completely described,
feedforward inhibition has been demonstrated in the apical and basilar
dendritic regions (Alger et al., 1981; Ashwood et al., 1984; Andersen et
al., 1980; Buzsaki, 1984). Several possible anatomical configurations
for this circuit are plausible. The best demonstrated configuration,
however, is that the Schaffer/commissural afferents terminate on an
inhibitory interneuron which resides in SR or SO parallel to the
dendrites of the CAl pyramidal cell (Ashwood et al., 1984). These
inhibitory interneurons then project to the dendrites of the pyramidal
cell (Gall et al., 1981; Schwartzkroin and Mathers, 1978).
The pyramidal cell is the major projection cell of the CAl area.
Due to its architectural definition with the afferent fibers being
orthogonal to the apical dendrites of these cells, the CAl region offers
an exceptional opportunity for the study of both intracellular and
extracellular responses. The relative ease of understanding the effects
of localized extracellular stimulation and recording in relation to the
input and output pathways is unsurpassed elsewhere.
Electrophvsiology of the hippocampus: An introduction
Electrophysiological studies of the hippocampus often use
extracellular recording techniques. The ability to use these field
potential recordings meaningfully results from the highly organized and
relatively homogeneous patterning of the afferent and efferent fibers in
this region. The activation of the afferent fibers produces restricted
and localized dipole current sources and sinks. When an excitatory

22
synapse is activated, current flows into the cell. This flow of current
into the cell is called a current sink. As in any electrical system,
current must also flow out of the system elsewhere. Current will flow
out of the cell at what is called the current source and then travel
through the intercellular fluid back to the current sink. These current
flows can be recorded since a voltage is produced as current flows
through the resistive extracellular milieu. Intracellularly, the
activation produced at the current sink is seen as a positive voltage.
Extracellularly, however, the potential recorded is negative at the sink
and positive at the source when stimulating and recording from the same
excitatory synaptic region.
In the CAl region of the hippocampus, the location of the stimulat
ing and recording electrode will affect the characteristic shape of the
response. If the stimulating electrode is in SR, a negative-going field
response is obtained when recording from the same synaptic region due to
the inward flow of current from this excitatory response. If the
stimulating electrode is left in SR while the recording electrode is
moved into SO in steps, a gradual change in the shape of the response is
observed. As the recording electrode approaches the pyramidal cell
layer, the response becomes less negative. When the recording electrode
passes through the cell layer or thereabout, a response is obtained
which is neither positive nor negative in appearance. This point is
termed the inversion point and represents the area where the inward flow
of current generated by the activation of the apical dendrites equals
the outward flow of current. Once the recording electrode is dorsal to
the cell layer, the response becomes positive which indicates a current
source; current is flowing out of the cell at this point. If the

23
current passed through the stimulating electrode is raised to a level
sufficiently high to activate action potentials from a large number of
pyramidal cells, a field population spike (PS) becomes superimposed on
the field extracellular excitatory post-synaptic potential (EPSP)
(Andersen et al., 1971; Lomo, 1971). This PS is seen as a sharp nega
tive going spike (Fig 1-2).
Extracellular recording paradigms in CAl have generally been used
to evaluate three relatively distinct kinds of information. The first
type of paradigm is designed to test synaptic efficacy and is termed an
input/output (I/O) function. This paradigm consists of increasing the
stimulation current level in steps in order to determine response
characteristics such as EPSP threshold, PS threshold and respective
asymptotic levels. The second type of paradigm is designed to test for
synaptic plasticity. Briefly, at least two types of response enhance
ment plasticity are seen in the hippocampus. One is seen as a short
lasting increase in the size of the EPSP or PS amplitude in reference to
a baseline pulse either with paired pulses or immediately after tetanic
stimulation (these phenomena are termed potentiation). This type of
potentiation lasts for periods ranging from several milliseconds to
seconds (Creager et al., 1980). Another type of potentiation is longer
lasting, often minutes to weeks, and is termed long term potentiation.
The mechanisms for these two types of potentiation are thought to be
different (Dunwiddie and Lynch, 1978). There are several different
experimental procedures used to assess or induce these types of poten
tiation. Paired pulse testing allows for the systematic assessment of
inhibition (produced through interneurons) as well as potentiation. In
this procedure, two pulses, a condition and a subsequent test pulse, are

Figure 1-2. A CA1 stratum radiatum or stratum oriens evoked extracellular field potential
response recorded in or dorsal to the pyramidal cell layer. The labelled
elements of the waveform are as follows: A, peak of 1 mV (2 msecs) calibration
pulse injected prior to stimulation; B, baseline recording area (for computer
analysis and scaling the distance between the average of x points on A and y
points on B was used to individually scale each waveform for amplitude
measurements); C, stimulus artifact; D, onset of population EPSP (current sink
in distant stratum radiatum, thus seen as current source dorsal to pyramidal
cell layer); E, onset of PS (also, slope was calculated from point D to point
E); F, maximal negative peak of PS (PS latency was measured from point D to
point F); G, post PS peak of EPSP (probably reflects IPSP current sources as
well. Population spike was calculated by adding PS segment between points E and
F and F and G and dividing by two.

B:S07O5050.DT2
WAUEFORM NUMBER: 2
20. MSEC
V
c
F

26
successively produced with controlled interpulse intervals (IPI). The
second response is typically compared to the first response of the pair
or to a baseline control pulse. Two other procedures are also used to
measure plasticity; one is frequency potentiation (FP) and the other is
long-term potentiation (LTP). FP entails the use of trains of stimu
lation at specific frequencies generally less than 100 Hz. LTP is
usually obtained with high frequency (greater than 100 Hz) trains of
stimulation. The final type of data collection procedure is designed to
examine synaptic distribution. In this procedure, a recording electrode
is lowered in discrete steps (25-100 uMs) through the dendritic region
of the CA1 pyramidal cell usually while stimulating in SR. A profile of
the current sources and sinks can thereby be generated. This technique
is called a laminar analysis. Although a laminar analysis can be used
to determine current sources and sinks, the resolution obtained is not
substantial since current sources and sinks often overlap. If a current
source density analysis (CSD) is performed on the laminar profile, a
more defined profile can be obtained so that it is theoretically possible
to suggest the relative presence, absence and general distribution of
synaptic current along the course of the cellular processes (Nicholson
and Freeman, 1975). CSD analysis is, in essence, a mathematical
transformation of the laminar waveforms which uses the second spatial
derivative thus describing rates of change of current flow between
recorded levels rather than voltage measures.

27
Pharmacology of the Hippocampus
Pharmacology of inhibition in the hippocampus
The major focus of the experiments in this volume is on the effect
of CET on synaptic inhibition. The basic premise for this focus ema
nates from previous studies suggesting changes in the influence of
inhibition in the CAl region of CET animals. The complete details of
these findings will be given subsequently. The study of inhibition in
CAl of the hippocampus in normal animals has, however, provided an
exploding volume of information on different types of inhibition and the
underlying pharmacology. This section is designed to provide a brief
review of that literature in order to establish a background for the
basis of the experiments outlined below.
Recurrent inhibition has been considered to be the dominant type of
inhibition in the CAl region (Andersen et al., 1964b). As indicated
earlier, this inhibitory circuit consists of the pyramidal cell which
projects via axon collaterals to the basket cell. The basket cell, in
turn, projects widely to surrounding pyramidal cells as well as to the
original "firing" pyramidal cell (Andersen et al., 1964a; Knowles and
Schwartzkroin, 1981). An excitatory neurotransmitter, presumably
glutamate, is released by the pyramidal cell onto the soma of the
inhibitory interneuron (Storm-Mathisen, 1977). The functional effect of
recurrent inhibition is profound. If the Schaffer/commissural afferents
are stimulated while spontaneous pyramidal cell unit firing is moni
tored, an initial excitatory response is found followed by a near
complete cessation of spontaneous activity for 600 to 700 msecs (Andersen
et al., 1964a). With paired pulses, if the second of two pulses is
presented within 20 to 80 msecs of the first, the effect of the first

28
pulse is to decrease the amplitude of the population spike on the second
or test pulse (in vivo). This decreased PS amplitude is thought to be
due to a reduction in the probability that the pyramidal cell and those
around it will fire when the second pulse arrives. Paired pulse poten
tiation overshadows the inhibitory phase after about 80 msecs (Andersen
and Lomo, 1970; Dunwiddie et al., 1980). This potentiation phase is
thought to be due to a process different from that of inhibition.
However, it is also thought that recurrent inhibition may produce a
resetting of the fired pyramidal neurons thereby allowing for a more
synchronized and simultaneous firing of the pyramidal cells with inter
pulse periods longer than approximately 80 msecs. This process could
account for a portion of the potentiation phase seen with paired pulses
(Assaf and Miller, 1978).
More recently, feedforward-like inhibition has begun to be dej
scribed and characterized in CA1 (Alger et al., 1981; Andersen et al.,
1978, 1980, 1982). It has been hypothesized that some of the Schaffer/
commissural afferents either terminate on the apical or basilar
dendrites of the CA1 pyramidal cell and directly release an inhibitory
neurotransmitter, gamma-aminobutyric acid (GABA), or more likely, the
Schaffer/commissural afferents synapse onto an inhibitory interneuron
which, in turn, synapses onto the apical or basilar dendrites or soma of
the CAl pyramidal cell (Ashwood et al., 1984; Buzsaki, 1984). The
conclusive anatomical evidence for such feedforward inhibitory connec
tions is not yet complete. However, descriptions of interneurons which
run parallel to the pyramidal cell apical dendrites do exist in the
literature (Gall et al., 1980; Lorente de No, 1934; Schwartzkroin and
Mathers, 1978).

29
There are currently at least three major putative neurotransmitter
groups which are reported to have major effects on inhibition of the CA1
pyramidal cell. These neurotransmitter groups are GABA agonists and
antagonists, opiate agonists and antagonists and the benzodiazepines.
The following sections provide background for each of these neurotrans
mitters .
GABA agonists and antagonists and inhibition
Glutamic acid decarboxylase (GAD), the precursor to GABA, and GABA
have been found in the hippocampus using immunocytochemical (Barber and
Saito, 1976), autoradiographic (Hokfelt and Ljungdahl, 1972) and fluo
rescence (Wolman, 1971) techniques in a bimodal distribution in CAl.
Peaks are seen in the pyramidal cell layer and the molecular layer of
SR. Higher levels of GAD are actually seen in stratum lacunosum-
moleculare than in stratum pyramidale. It has also been shown that if
the various afferent pathways to the CAl region are cut, there is no
reduction in GAD activity. These data indicate that GAD is not released
by the afferent fibers but instead suggest the probable existence of an
interneuron (Storm-Mathisen, 1977; Fonnum and Storm-Mathisen, 1978).
These findings also suggest the possibility of two populations of
GABAergic inhibitory interneurons.
GABA has been demonstrated to mediate recurrent inhibition (cf.
Storm-Mathisen, 1977) and feedforward inhibition (Alger and Nicoll,
1980) in the hippocampus. It has been shown in vivo that GABA, applied
to the pyramidal cell soma, produced a potentiation of recurrent inhibi
tion (Curtis et al., 1970). It has been suggested that this iontophor-
etically released effect of GABA occurs as a result of GABA acting on

30
the pyramidal cell soma in a manner similar to that of the recurrent
inhibitory interneuron. The relatively specific GABA antagonists,
bicuculline and picrotoxin, have furthermore been shown to reduce
GABA-induced inhibition (Curtis et al., 1970).
Local application of GABA onto either the pyramidal cell soma or
to the basilar dendrites, when recording intracellularly, produced an
hyperpolarization-depolarization sequence (Alger and Nicoll, 1980).
When GABA was applied to the apical dendrites, a pure depolarizing
response was recorded. Both the hyperpolarizing and depolarizing
responses produced an inhibitory influence on the pyramidal cell (Alger
et al., 1981; Andersen et al., 1978, 1980). Associated with the
depolarization of the pyramidal cell apical dendrites was a large
conductance increase which presumably accounted for the inhibitory
action by shunting the current input. Thus, GABA produced powerful,
though regionally specific inhibitory effects on the CAl pyramidal cell.
The depolarizing inhibitory effect observed in the apical dendrites is
thought to occur as a result of feedforward inhibition while the
hyperpolarizing effect was shown to occur as a result of recurrent
inhibition.
Another line of evidence concerning the types of inhibition in the
CAl region of the hippocampus has emanated from intracellular recordings
from CAl pyramidal cells. In these studies, stimulation of either
orthodromic SR fibers or antidromic alvear fibers produced two distinct
periods of inhibition (Alger and Nicoll, 1982). The first type of
inhibition represented what is likely to be the result of classical
recurrent inhibition. It can be evoked with either orthodromic or
antidromic stimulation. It was seen as a large and sharp hyperpolarizing

31
potential which peaked at about 50 msecs. Immediately following this
IPSP was a later hyperpolarizing potential which peaked at a latency
from stimulus onset of approximately 200 msecs. This late hyper
polarizing potential could only be evoked with orthodromic stimulation
in SR or SO. It cannot be evoked with antidromic stimulation thus
ruling out the possibility that it was generated by recurrent
interneurons and strongly suggesting it to be the result of feedforward
inhibition (Alger and Nicoll, 1982).
The evoked hyperpolarizing potentials recorded above have also been
characterized pharmacologically. The late hyperpolarizing potential
occurred during the same period as the depolarizing potential which was
elicited by localized application of GABA in the apical dendrites. The
two different hyperpolarizing potentials (early and late) have been
shown to be subserved by two different populations of GABA receptors.
The early hyperpolarization has been shown to be mediated by GABA(a)
receptors while the late hyperpolarization has been shown to be sub
served by GABA(b) receptors. The evidence for this distinction emanated
from the finding that the early hyperpolarizing response was antagonized
by the GABA antagonist bicuculline. The late hyperpolarizing potential
was, however, not antagonized by bicuculline and was therefore said to
be bicuculline resistant (Newberry and Nicoll, 1984). Subsequent
reports have indicated that baclofen, a GABA agonist, binds to the
bicuculline-resistant GABA receptors and directly induced the late
hyperpolarization in the pyramidal cell. The response to baclofen was
associated with a decrease in neuronal input resistance which may be a
result of an increase in potassium conductance (Newberry and Nicoll,
1984; Nicoll and Newberry, 1984). The early IPSP occurred as a result

32
of an increase in the chloride conductance of the cell (Kandel et al.,
1961).
The benzodiazepines in the hippocampus
The benzodiazepines have also been reported to influence recurrent
inhibition. High affinity binding sites have been found which fulfill
many of the criteria of pharmacological receptors. However, the precise
location of these receptors has been difficult to determine. A wide
spread distribution was seen rather than discrete localization. En
hanced binding of benzodiazepines was seen when GABA or a GABA analog
was included in the binding assay. Thus, the benzodiazepine receptors
appeared to be associated with the GABA receptors in a receptor complex
(see Tallman et al., 1980; Ticku, 1983; Greenberg et al., 1984).
The benzodiazepines have been shown to increase the duration of
inhibition in the hippocampal CAl pyramidal cell (Adamec et al., 1981;
Wolf and Haas, 1977; Tsuchiya and Fukushima, 1978). It was suggested
that the benzodiazepines either directly act upon the pyramidal cell
soma through benzodiazepine receptors or through a GABA-benzodiazepine
receptor complex (Tallman et al., 1980). When GABA was applied to local
regions of the CAl pyramidal cell soma or dendrites, hippocampal slices
bathed in midazolam, a benzodiazepine, responded with enhanced
hyperpolarizations and depolarizations respectively (Jahnsen and
Laursen, 1981). Benzodiazepine effects on recurrent inhibition have
also been reported to be blocked by the GABA antagonists bicuculline and
picrotoxin (Alger et al., 1981; Tallman et al., 1980; Tsuchiya and
Fukushima, 1978). These findings suggested that the benzodiazepines may
augment recurrent inhibition by either increasing the excitability of

33
interneurons or by increasing the strength of the excitatory afferents
to the basket cells.
Opiates in the hippocampus
A third class of pharmacological agents probably interacting with
inhibitory processes in the hippocampus is the opiates. Endogenous
opiates have been observed in the CAl pyramidal cell region with the use
of immunocytochemical techniques (Gall et al., 1981). Two different
points of view exist concerning the effects of opiates in CAl (Corrigal,
1983; Haas and Ryall, 1980). It has been shown that electrophysio-
logically, the administration of [D-Ala(2), d-Leu(5)enkephalin] (DADL)
or morphine in the bathing medium of slices produced increases in the
size and duration of EPSPs of dendritic origin without changing the
resting membrane properties of the soma, recurrent somatic IPSPs, or
dendritic field potentials (Dingledine, 1981; Robinson and Deadwyler,
1980). Three hypotheses have been proposed to account for these
findings: 1. that an enhancement of "the electrical coupling between an
apparently normal EPSP current generator and a normal spike trigger
zone" occurs (Dingledine, 1981, p. 1034), 2. that a form of
post-synaptic dendritic inhibition exists which is selectively
attenuated by opioid peptides (Dingledine, 1981), and 3. the possibility
that a "modulation of excitatory synaptic transmission onto hippocampal
dendrites by a postsynaptic action that would entail some amplification
mechanism" (Lynch et al., 1981 (no direct evidence supports this
hypothesis, Dingledine, 1981, p. 1034)). The second and more widely
supported interpretation is that opiate effects are not on the pyramidal
cell at all but act by disinhibiting the inhibitory interneuron (Lee et
al., 1980; Dunwiddie et al., 1980; Corrigal and Linesman, 1980; Nicoll

34
et al., 1980; Segal, 1977; Zieglgansberger et al., 1979). In either
case the net effect of opiate administration is to increase the
amplitude of an extracellularly evoked PS.
The administration of morphine to the basal dendrites or soma
region of CA1 pyramidal cells has been reported to produce membrane
depolarization whereas application to the apical dendrites produced a
hyperpolarization (Robinson and Deadwyler, 1980). These findings are
important in that they are opposite to those found with discrete appli
cations of GABA to these same cell regions. Thus, the differential
effect seen with the GABA inhibitory internuerons is mirrored by the
opiate data. These findings support an opiate-GABA interaction at CA1
pyramidal cells in the hippocampus.
Rationale and Introduction to Current Experiments
Chronic alcohol animal studies
Anatomy. CET has been shown to produce alterations in the normal
morphology of the rodent hippocampus as well as the cerebellum. Using
the Golgi stain, Riley and Walker (1978) reported CAl pyramidal cell
basilar dendrites to be severely reduced in overall length and number of
branches. The apical dendrites were not as dramatically affected except
for the distal extent where arborization was decreased. In addition,
the basilar dendritic fields had significantly reduced spine densities.
DG granule cell dendrites also had reduced spine densities. A decrease
of 15 to 20 percent in the number of CAl and CA3 pyramidal cells,
dentate granule cells and cerebellar granule cells has also been report
ed (Walker et al., 1980; Tavares et al., 1983).

35
Physiology. Electrophysiological data using laminar and current
source density (CSD) analysis profiles in the CAl region showed changes
in the location of the current sinks after ethanol treatment (Abraham et
al., 1982). Normal animals were reported to have two discrete yet
overlapping current sinks in the SR of CAl. In CET animals, the more
proximal (to the cell layer) current sink was significantly reduced.
These data suggested that there was a change in the anatomical
distribution of synapses in the CAl region after CET. Abraham et al.
(1982) hypothesized that these two current sinks represented a discrete
yet overlapping termination of the Schaffer collaterals distally and the
commissural fibers proximally. They suggested that the commissural
fibers were preferentially affected. This condition could occur by
either the commissural projections being interrupted, a loss of
dendritic synapses in the proximal region, or a selective decrease in
the efficacy of the synapses onto the proximal apical dendrites.
The normal function of the CAl region has also been shown to be
altered by CET. Abraham et al. (1981) reported that although CET did
not significantly affect the basic synaptic waveforms, EPSP thresholds,
PS thresholds, or I/O functions, the production of long-term
potentiation, nor the basic pattern of response to paired pulse or
frequency potentiation, CET did produce enhanced potentiation to paired
pulses as well as FP at 5 Hz and 10 Hz stimulation. These findings of
enhanced PS potentiation were found in the absence of changes in the
field EPSP. It was hypothesized that these results are a function of a
change in recurrent inhibitory processes of the CAl region.

36
Anoxia, picrotoxin, and the enkephalins, treatments which appear to
decrease inhibition in the hippocampus, also produced enhanced facilita
tion of the PS amplitudes to paired pulse paradigm (PPP) stimulation
(Andersen, 1960; Dunwiddie et al., 1980; Lee et al., 1980). These
effects also occur in the absence of significant alterations in the
synaptic response to single pulse stimulation. FP findings further
support the involvement of recurrent inhibition since FP at 1 Hz
resulted in no differences between CET and control animals while both 5
Hz and 10 Hz FP did result in PS differences (Abraham et al., 1981).
Since the maximal duration of recurrent inhibition appears to be 600
msecs in CAl (Andersen et al., 1964a), 1 Hz stimulation is too slow to
interact with inhibitory processes whereas 5 Hz and 10 Hz stimulation
would be expected to interact. More recently, Durand and Carien (1984)
reported results which provided additional support for the hypothesis
that CET reduces recurrent inhibition in CAl. They found, using
intracellular recordings from CET hippocampal slices, that only the
inhibitory post-synaptic potential (IPSP) amplitudes and after
hyperpolarization durations were significantly different from controls.
No differences were found with any of the other membrane characteristics
measured.
There has been one interesting biochemical finding potentially
related to inhibition in the hippocampus. Freund (1980) found a
decrease in the binding of benzodiazepine receptors from CET animals.
These findings could be important since there is a strong link between
GABA and benzodiazepine receptors with GABA strongly involved in the
inhibitory mechanisms of the CAl region.

37
It is apparent that CET affects inhibition in CAl. However, it is
not clear whether CET affects recurrent inhibition alone, feedforward
inhibition alone or both types of inhibition concurrently. In attempt
ing to determine the mechanisms that may be affected by CET, it is
important to know the generality of CET effects on inhibition. There
fore, the basic intent of the experiments reported herein was: 1. to
define the nature and parameters of the CET induced changes as well as
the characteristics of paired-pulse inhibition and potentiation in CAl
in untreated animals and 2. to begin to study the possible pharmacologi
cal mechanisms which may underlie these changes. These goals were
accomplished through three major series of experiments: 1. characteris
tics of normal synaptic inhibition --an in vivo study, 2. characteristics
of CET and synaptic inhibition --an in vitro study, and 3. pharmacolog
ical characteristics of CET and synaptic inhibition -- a series of in
vitro studies.

CHAPTER 2
GENERAL METHODS
Animals
Long-Evans hooded male rats were purchased from the Charles River
Company for all experiments. The rats were obtained at a body weight of
225 to 250 gms and were 55 to 60 days old prior to beginning the admin
istration of ethanol. Animals were housed in individual stainless steel
cages at the University of Florida Animal Resources Center or at the
Veterans Administration colony room or building (three different loca
tions) Temperature and humidity were maintained at appropriate levels.
Light-dark cycles of twelve hours on and twelve hours off were main
tained with the light cycle from 0700 hrs to 1900 hrs daily.
Alcohol Diet Regimen
Animals were maintained on an ethanol or sucrose containing liquid
diet. This diet was developed so that experimental and control animals
ingested calorically equal, nutritionally complete diets.
The ethanol containing diet provided 35 to 39 percent (8.1 to 9.4
percent ethanol v/v) of the total caloric intake as ethanol. Ethanol or
sucrose provided 35 percent of the total caloric intake the first month
of the diet regimen. This level was then increased by 1 percent per
month in order to provide a relatively constant level of ethanol or
sucrose as the animals gained weight. The diets were prepared by
combining an equicaloric ethanol- (63.3 percent v/v) or sucrose-
containing 87.0 percent w/v stock solution with Sustacal obtained from
38

39
the Mead-Johnson Company. Both groups of liquid diets were additionally
fortified with Vitamin Diet Fortification Mixture (3.0 g/1) and Salt
Mixture XIV (5.0 g/1) which were obtained from ICN Nutritional
Biochemicals, Cleveland, Ohio. The diets contained 1.3 KCal/ml and
provided several times the daily requirement for all essential vitamins
and nutrients (Walker and Freund, 1971). Diets were prepared fresh
daily and were administered using calibrated glass bottles with stain
less steel drinking tubes. Consumption levels of each animal were
recorded daily.
Upon arrival at the animal facilities, animals were quarantined for
a period of seven days. They were then housed individually and allowed
to acclimate to the colony room for one to two weeks or until they
reached the appropriate body weight. Next, animals were paired accord
ing to their weight. The experimental animals (group E) received
ethanol in their daily diet while the control animals (group S) received
sucrose substituted isocalorically for the ethanol. A laboratory chow
group was not included in these experiments since in the previous 15
years of experiments in this laboratory no differences between sucrose
and lab chow control groups were ever found.
Animals in group E were pair fed with animals of group S by giving
each animal in group S the volume of diet that its age- and weight-
matched group E partner consumed on the previous day. This pair-feeding
procedure assured that group E and group S animals ingested identical
quantities of calories, vitamins, minerals, proteins, fats and carbo
hydrates. Differences between group E and group S animals on any
dependent variable were then due to ethanol and not nutritional or other

40
variables. A minimum of eight rats were used in each group for all
experiments.
In these experiments, group E and group S animals were treated with
ethanol or sucrose for 20 weeks. A minimum of eight weeks was allotted
after the discontinuation of the alcohol diet prior to beginning any of
the electrophysiological procedures. This period of abstinence was
included to control for the residual effects of acute alcohol withdrawal.
Since the testing of individual preparations consumed an entire day,
sampling occurred over an 8 to 24 week period after ethanol discon
tinuance depending on the initial group size. Individual animal number
and group designation were coded to prevent any bias in the placement of
recording and stimulating electrodes. Coding was essential due to the
response variability observed in recordings from the hippocampus and to
prevent unintentional procedural differences between the two groups.
Equipment
All recordings were made with glass micropipettes filled with 4M
NaCl. Electrodes were chosen to satisfy criteria for tip diameter (1-2
uM), impedance (500K to 1.5 Mohms at 1 KHz) and shaft taper. Concentric
bipolar electrodes obtained from Rhodes Medical Instruments (in vivo) or
the Frederick Haer Co. (in vitro) were used to activate specific hippo
campal afferent or efferent pathways. In all cases, electrodes were
controlled with micro-manipulators. Constant current stimulation
(duration, 0.1 msec; current, 20-1000 uAs) was used in all procedures.
Stimulus pulse timing was controlled with WPI series 1800 timers (in
vivo) or with an Apple computer system using software and hardware
designed by the author (in vitro).

41
All recordings were amplified by Grass P511 preamplifiers, filtered
(Krohn-Hite) at either .3Hz-10KHz (field potentials) or 300Hz-10KHz
(extracellular unit activity) and displayed on Tektronix oscilloscopes.
All responses were collected on an Apple computer system for subsequent
computer analysis. Analog to digital conversion within the computer
employed an Interactive Structures Inc. AI13 board. For these experi
ments, 512 data points were collected during a 28 msecs period. Each
data point had a maximum amplitude resolution of one in 4096.
A calibration pulse (1 mV, 2 msecs) was routinely injected immedi
ately prior to each stimulation pulse with a Stoelting CA5 calibrator.
This calibration pulse not only controlled for alterations in equipment
status, but also allowed for uncomplicated adjustment of the gain of the
preamplifiers and of the scaling of the data for subsequent computer
display and analysis.
Raw data were analyzed for the in vivo experiments on an Apple II
Plus computer system whereas all data from the in vitro experiments were
transferred from the Apple computer to an IBM 5150 (PC) for data analy
sis. All data were stored on 5.25 inch floppy disks. After analyses of
the waveforms, all measures were transferred via modem to the North East
Regional Data Center (NERDC) computer system for statistical analyses
using the Statistical Analysis System (SAS) General Linear Models (GLM)
analysis of variance procedures. Graphics were produced using the IBM
Displaywriter system with Chartpak software or the IBM 5150 using Lotus
Inc.'s Symphony software and a Hewlett Packard 7470A plotter.

42
Basic Procedures
Input/output relations
I/O functions were obtained at the start of each experimental
session by varying the stimulus current from 20 to 1000 uAs with pulse
widths at a fixed duration of 0.1 msec. The analysis of the responses
generated focused on measures of the latency and amplitude of the EPSP
and PS. These measures provided information regarding synaptic response
strength. The complete set of current steps used was the following:
20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450,
500, 600, 700, 800, 900, 1000. These steps were selected in an attempt
to more accurately indicate EPSP and PS thresholds as well as asymptotic
levels. Additionally, the I/O functions were used to normalize stimulus
current strength across animals in the later phases of each experiment.
Paired-pulse paradigm
Stimulus current. An important problem in these experiments was
the choice of stimulus current used for the paired pulse series of
experimental paradigms. I/O curves were used to adjust the stimulus
current such that a certain percentage of the PS amplitude at asymptote,
up to a maximum of 1000 uAs, was used for the condition or test pulse of
paired pulse experiments. This procedure controlled for minor
variations in electrode placement and individual animal response
variability. If CET altered the basic I/O functions, this procedure
allowed for the evaluation of synaptic potentiation with baseline
responses which fell at the same relative points on the I/O curves.

43
Characteristics. When paired shocks are delivered to any of a
variety of afferent pathways of the hippocampus, a characteristic
inhibition or potentiation of the test (second) response is observed
when the condition and test pulses are separated by a critical time
period (Steward et al., 1977). The magnitude of the test response
inhibition or facilitation depends upon the inter-pulse-interval (IPI).
The typical pattern of the facilitation depends on the response
measured. Stimulation of the Schaffer/commissural path while recording
in the CAl pyramidal zone produces a characteristic but different
response pattern for both the EPSP and the PS. The population EPSP is
maximally facilitated at IPIs of 20-30 msecs decreasing at progressively
longer intervals. PS amplitude is inhibited at IPIs of 20 to 80 msecs,
most likely as a result of inhibitory processes and is facilitated at
longer IPIs (> 80 msecs) (Lomo, 1971; Steward et al., 1977; Creager et
al., 1980).
Procedures. Paired-pulse inhibition and potentiation were
evaluated by systematically varying the IPI between the condition and
test pulse from 20 msecs to 3 secs. Four responses were averaged at
each interval tested. The individual intervals tested for all
experiments were as follows in msecs: 20, 30, 40, 50, 60, 80, 100, 150,
200, 300, 400, 500, 750, 1000, 1500, 2000, 3000. Paired shocks were
delivered at a frequency of 0.1 Hz in the CAl region in the order
described above. Paired pulse stimulation current levels in this series
of studies used test pulse current levels which were either 25 or 50
percent of the maximal PS response amplitude obtained from mini-I/O
curves. Condition pulse current levels were adjusted to 50 percent of

44
the EPSP amplitude at PS threshold (50 EP), PS threshold (0 PS), and to
25, 50, 75 and 100 percent of the PS amplitude at I/O asymptotic values.
Intensities used for each of the individual experiments are indicated in
subsequent sections. Inhibition and potentiation were measured by
quantitative comparison of the test response with a baseline response
averaged from four single pulse baseline responses recorded immediately
prior to each of the paired pulse series.
Data Analysis and Interpretation
The protocol outlined above was designed to allow for separate
measurement of various population EPSP and PS characteristics. For the
EPSP, the following measures were sampled: 1. latency to onset from
stimulus artifact and 2. maximal slope of the rising phase of the EPSP
calculated either by using a regression equation for all of the data
points between the EPSP onset and EPSP peak prior to the PS (all in vivo
data) or by using a regression equation for all of the data points 10
percent above the EPSP onset and 10 percent below the EPSP peak prior to
the PS (10-90 slope; used for all but the very first series of in vitro
data). For the PS, the following measures were sampled: 1. latency to
onset of the first PS measured at its maximum negative amplitude from
the EPSP onset and 2. rising and falling aspects of up to three different
population spikes from the same stimulation pulse. Each PS amplitude
was calculated by adding the respective falling and rising phase of the
PS spike and dividing by two (Fig 2-1). If multiple PSs were recorded,

Figure 2-1. A CA1 stratum radiatum or stratum oriens evoked extracellular field potential
response recorded in or dorsal to the pyramidal cell layer. The labelled
elements of the waveform are as follows: A, peak of 1 mV (2 msecs) calibration
pulse injected prior to stimulation; B, baseline recording area (for computer
analysis and scaling the distance between the average of x points on A and y
points on B was used to individually scale each waveform for amplitude
measurements); C, stimulus artifact; D, onset of population EPSP (current sink
in distant stratum radiatum, thus seen as current source dorsal to pyramidal
cell layer); E, onset of PS (also, slope was calculated from point D to point
E); F, maximal negative peak of PS (PS latency was measured from point D to
point F); G, post PS peak of EPSP (probably reflects IPSP current sources as
well. Population spike was calculated by adding PS segment between points E and
F and F and G and dividing by two. G, H and I, second PS detection points. For
analysis, second spike was measured by adding amplitude between G and H and H
and I then dividing by two. This calculated value was then added to calculated
value for first PS.

B:S07O5050.DT2
,
WAUEFOBK NUMBER: 6
60 MSEC
A
\r*
B
E
A
V
c
F

47
the PS amplitudes were added to each other. This total PS amplitude was
the measure used for all of the PS data analyses. Results were
statistically evaluated by two-way analyses of variance with repeated
measures. One factor was the experimental treatment (group E or S).
The second repeated factor was stimulus current (I/O curves), IPI (PPP),
location (iontophoretic application locations) or time (after
iontophoretic release). Responses between alcohol and sucrose animals
were compared between groups as a percentage change from the baseline of
each animal for amplitudes and as a difference score for latency
measures. This provided for the normalization of potentially different
baselines between the two groups.

CHAPTER 3
CHARACTERIZATION OF SYNAPTIC INHIBITION IN VIVO
Introduction
The regulation of pyramidal cell firing in the CAl region of the
hippocampus has long been recognized as an important consideration when
attempting to understand, and potentially control, neuronal function or
dysfunction of this area. Although there may be a multitude of differ
ent and, as of yet, unknown mechanisms by which pyramidal cell firing is
regulated, one of the most well documented methods is achieved through
an inhibitory interneuron; the now classical recurrent inhibitory
circuit found near the pyramidal or granule cell in CAI, CA3 and the
dentate gyrus regions of the hippocampus (Cajal, 1968, Lorente de No,
1934). Recurrent inhibition occurs as a result of pyramidal cell
firing. An action potential propagates from the pyramidal cell to
other major cell types in caudal and rostral directions from the hippo
campus. An axon collateral of the pyramidal cell also conducts this
action potential to nearby inhibitory basket cells. An excitatory
synapse from the pyramidal cell results in the basket cell firing. The
basket cell projects widely in a basket-like web to the original as well
as many other pyramidal cells. An inhibitory neurotransmitter, GABA,
then causes the pyramidal cell to cease firing for a period of time up
to 700 msecs (Andersen et al., 1964a, 1964b). More recently, another
type of inhibition in the CAl region has been shown to exist which is
feedforward in nature. In this system, afferent fibers terminate on an
48

49
inhibitory interneuron or project to both an inhibitory interneuron and
pyramidal cell dendrites (Ashwood et al., 1984; Buzsaki, 1984).
Substantial evidence exists for the presence of interneurons
located in the pyramidal cell layer and the SO and SR dendritic areas of
the CAl region. Peak concentrations of GAD, an enzyme which catalyzes
the formation of GABA, is restricted to interneurons located in the
pyramidal cell layer and SLM (Storm-Mathisen, 1977). Immunoreactive
staining to GAD showed sparse staining throughout the dendritic regions
of both the hippocampal CA regions and the dentate gyrus. A more
heavily stained region was found in the distal apical dendrites (Barber
and Saito, 1976). These same cells are also observed with
cholecystokinin and enkephalin immunoreactive staining (Gall et al.,
1981; Harris et al., 1985; Stengaard-Pedersen et al., 1983).
Interneurons with dendritic and axonal orientations parallel to the
dendritic axis of CAl pyramidal cells have also been described (Cajal,
1968; Lorente de No, 1934; Schwartzkroin and Mathers, 1978). Ashwood et
al. (1984) have provided evidence that some of the interneurons in the
pyramidal cell region may be feedforward inhibitory since orthodromic
activation of the Schaffer collateral/commissural system resulted in
some of the inhibitory interneurons firing prior to the pyramidal cell.
The electrophysiological and pharmacological evidence for the
existence of both feedforward and recurrent inhibition is convincing
(Alger et al., 1982; Andersen et al., 1980, 1982). However, most
studies have utilized intracellular recordings to arrive at these
conclusions. While intracellular recordings are most appropriate in
defining the existence and details of feedforward and recurrent

50
inhibition, it would also be useful to be able to measure the effects of
these two types of inhibition extracellularly.
Recurrent inhibition has often been studied extracellularly using
paired pulses delivered from the same stimulation site, usually in
stratum radiatum. With this paradigm, the effects of recurrent inhibi
tion and paired-pulse potentiation were thought to be studied. However,
with the recent evidence for feedforward inhibition, it is clear that
any study of inhibition using this configuration is confounded if the
objective of the experiment is the study of recurrent inhibition alone.
The use of an antidromic stimulation site for the condition pulse with
an orthodromic stimulation site for the test pulse, however, should
provide a relatively better extracellular assessment of recurrent
inhibition. This technique has been utilized for the study of recurrent
inhibition previously (Andersen et al., 1964a; Dingledine and Langmoen,
1980; Haas and Rose, 1982). Thus, recurrent inhibition can be studied
relatively independently using extracellular antidromic/orthodromic
(A/0) paired pulses. Feedforward inhibition, however, is more difficult
to study extracellularly. Recurrent inhibition is almost always acti
vated with orthodromic stimulation unless pharmacological and/or ionic
manipulations could be utilized. Because A/0 paired pulses activate
recurrent inhibition alone while 0/0 paired pulses activate both recur
rent and feedforward inhibition, it is proposed that if the results of
0/0 paired pulses are compared with A/0 paired pulses, the difference,
if the amplitude of A/0 responses were compared to the 0/0 responses,
may provide a reasonable estimate of feedforward inhibition using
extracellular methods. Another possible method by which feedforward
inhibition might be studied without recurrent inhibitory influences is

51
with the use of subthreshold condition pulses for the PS of CA1 pyrami
dal cells.
The first goal of this study was an attempt to dissociate recurrent
from feedforward inhibition using extracellular recordings. The method
used was that of employing paired pulses with two different stimulation
configurations: an A/0 and an 0/0 configuration. The 0/0 configuration
utilized one stimulating electrode which was placed in the SR of CA1 and
through which both the condition and test pulse stimuli were emitted.
This configuration would activate both the recurrent and feedforward
inhibitory mechanisms as well as inducing an EPSP facilitation (observed
when recording from the CAl pyramidal cell apical dendritic region).
The A/0 configuration utilized two independent stimulation electrodes
located in two different pathways. The electrode used for the condition
pulse was placed in the alveus to activate antidromically the efferent
fibers of the CAl pyramidal cell and thus, to activate orthodromically,
the recurrent inhibitory interneuron (normally as a result of pyramidal
cell firing). The second stimulating electrode was placed in the SR, as
above, for the test pulse stimulation. The antidromic configuration
should activate only recurrent inhibitory interneurons with the condi
tion pulse, theoretically leaving the second or test pulse response to
be modified predominantly by the effects of recurrent inhibition.
A second goal of these experiments was to more completely describe
the parameters of paired pulse inhibition and potentiation. Most
studies utilizing paired pulses have used IPIs from 15 to 200 msecs. It
has been reported that there are other inhibitory effects which can only
be seen with longer interpulse intervals and which may be active for as
long as two seconds, although they may be observable only with

52
intracellular techniques (Wigstrom and Gustafsson, 1981). The range of
IPIs utilized in these studies was therefore expanded to include IPIs
from 20 msecs to 3 secs.
A final goal of these experiments was to better describe the effect
of current intensity on the characteristics of paired pulse inhibition
and potentiation. In these experiments the effect of varying condition
and test pulse current intensity was investigated. While a number of
different current intensities have been used previously (Creager et al.,
1980; Haas and Rose, 1982; Assaf and Miller, 1978; White et al., 1979;
Chaia and Teyler, 1982; Steward et al., 1977), there have been no
systematic, parametric analyses of the effect of varying condition and
test pulse current intensities with the paired pulse paradigm,
especially not with a wide range of IPIs. If different populations of
interneurons can be recruited as a function of current intensity as has
been suggested (Ashwood et al., 1984), it may be possible to use this
parameter to selectively activate feedforward and recurrent types of
inhibition. These experiments should therefore provide important
information regarding the extracellular correlates of feedforward and
recurrent inhibition as well as some of the parametric conditions
frequently used in paired pulse studies but which have heretofore lacked
comprehensive parametric exploration.
Methods
Animals
Animals used were as described in the general methods except that
only sucrose-fed control animals were used.

53
Equipment and techniques for in vivo studies
Animals were initially injected intraperitoneally (IP) with .2 cc
of atropine to reduce possible respiratory congestion. Within 10 mins
1.5 gms/Kg of urethane was injected IP for anesthesia. A second injec
tion of 1.0 gms/Kg was given one hour later. Supplemental injections of
.05 gms/Kg were then given at intervals of every 1.5 to 2 hrs.
After it was determined that the animal was well anesthetized, it
was placed in a Kopf stereotaxic device using ear bars and a nose clamp.
An incision was made down the midline of the rat skull and the skin
retracted. The skull was then adjusted so that the bregma and lambda
sutures were level horizontally with the base of the stereotaxic device.
Three burr holes were drilled on the left side of the skull at locations
3.4, 4.4 and 5.4 mms posterior and 2.8 nuns lateral to bregma. A concen
tric bipolar stimulating electrode (Rhodes Medical Instruments) was then
lowered to the SR of CAl through the most anterior hole using a hydraulic
microdrive. This electrode was used as an orthodromic stimulating
electrode. A second stimulating electrode was placed in an antidromic
configuration. This electrode was located in the most posterior hole
and lowered with a microdrive to the alveus. The recording electrode
was lowered through the center hole. This electrode configuration was
used since the orientation of the lamellae within the hippocampus is
such that an approximate anterior to posterior course is found in the
rat (Andersen et al., 1971).
Precise localization of the electrodes in vivo was performed by
observing the extracellular unit activity as the recording electrode was
lowered into the hippocampus. Based upon the characteristic high
frequency, complex unit response observed in the pyramidal cell layer,

54
the recording electrode was adjusted to approximately 150 uMs above the
region of maximal unit activity. With the recording electrode filtering
re-set for field responses, the orthodromic stimulating electrode was
lowered into the hippocampus until the evoked response was maximally
positive with a superimposed PS (see basic physiology section in general
introduction). Obtaining this response required readjusting the record
ing electrode depth to 150 uMs above the inversion point until the
maximal PS amplitude response was achieved. The antidromic stimulating
electrode was then lowered until a short latency, sharply negative-going
potential was observed. The antidromic electrode was carefully adjusted
to maximize the negative going response without inducing an orthodromic
stratum oriens EPSP response. At the end of the recording session, a
small lesion was made at the recording site and 400 uMs below. These
lesions were used to verify the location of the recording electrode
during histological analyses. The sizes of the stimulating electrodes
were such that the paths they created were observable without lesions.
Testing procedures
Once the stimulating and recording electrodes were positioned and
adjusted for maximal responses, input/output testing was performed for
the orthodromically evoked response only. In this procedure, the
current intensity delivered by the stimulating electrode was increased
in steps from 20 to 1000 uAs as described in the general methods.
After the I/O curves were collected, the paired pulse experiments
were begun. Two different groups of animals were used to test the
different test pulse current intensities as outlined in Table 3-1. The
first group of animals was tested with a test pulse stimulus current

55
TABLE 3-1
PERCENT OF MAXIMAL PS AMPLITUDE
ON TEST PULSE
c
25%
50%
0
1
1
N
0/0
50%
EPSP |
X |
X
D
0/0
0%
PS |
1
X
I
0/0
25%
PS |
X I
X
T
0/0
50%
PS |
X I
X
I
0/0
75%
PS I
1
X
0
0/0
100%
PS I
x 1
X
N
A/0
0%
AS |
1
X
A/0
25%
AS |
X I
X
P
A/0
50%
AS |
X I
X
U
A/0
75%
AS |
1
X
L
A/0
100%
AS |
X I
X
S
1
1
E
Percent of maximal PS amplitude at PS asymptote, EPSP amplitude at
PS threshold, and maximal antidromic spike (AS) amplitude for the
condition and test pulses in paired pulse paradigms (in vivo).

56
intensity that produced a PS amplitude 50 percent of the PS amplitude at
asymptote while the second group was tested with a stimulus intensity
producing 25 percent of the PS amplitude at asymptote. Each of the
indicated (Table 3-1) condition pulse current intensities was tested on
each animal within the test pulse current intensity level. The order of
the individual paired pulse experiments was determined in advance
through a random order selection. The IPIs used for all of these
experiments were as described in the general methods. IPIs were always
presented in the order described.
The current intensity levels used in the paired pulse tests were
based on a percentage of the asymptotic amplitude of the PS from brief
I/O curve functions. For example, 50 percent of the PS amplitude was
one of the current intensities tested. The size of the PS was measured
from the I/O curve at the asymptotic level. The stimulation current
level was then adjusted to obtain a PS response amplitude which was half
as large as the asymptotic PS response amplitude. This procedure was
used in order to control for differences in excitability and asymptotic
levels between animals. By using percentages of asymptotic PS response
levels, a more accurate comparison between animals was made possible.
The different current intensity levels on the condition and test pulse
used are shown in Table 3-1.
Data collection and analyses were performed as described in the
general methods except that analyses of variance were not performed.
The major points to be made in this series of experiments were best
addressed descriptively rather than by measures of statistical differ
ence .

57
Results
I/O relationships
The combined I/O curves for the two sets of animals are shown in
Fig 3-1. These data are from a total of 22 animals. The PS threshold
was 252 uAs + 12.31 (data presented as mean + SEM). The PS response
amplitude asymptote was 552 41.36 uAs current level.
Characterization of paired pulse paradigms in normal animals
The data from these experiments were divided into four major group
ings based on the stimulus configuration and the current intensity of
the test pulse used. The four groupings were: 1. the 0/0 25 percent
test pulse series, 2. the 0/0 50 percent test pulse series, 3. the A/0
25 percent test pulse series, and 4. the A/0 50 percent test pulse
series. For all of the superthreshold paired pulse experiments reported
herein, the general patterns and amplitudes of the PS inhibition and
potentiation are comparable to those previously reported for the CAl and
dentate gyrus regions of the hippocampus (Creager et al., 1980; Lomo,
1971; Steward et al., 1977). The EPSP slope data, however, were not
similar to these previous reports as discussed subsequently.
0/0 25 percent test pulse series
The 0/0 25 percent test pulse series data showed that increasing
the current intensity of the condition pulse, if the current intensity
was above PS threshold, produced an increase in the duration of inhibi
tion. When the test pulse PS amplitude was expressed as a percentage of
the control response, the PS was almost abolished from 20 to 30 msecs
for the 25 percent condition pulse, extending to 50 msecs for the 100

Figure 3-1.
I/O curves for ijn vivo data. A) Group mean PS
amplitude responses by current level from 20 to
1000 uAs expressed in raw units (mVs); B) Group
mean PS latency measures shown as raw data
(msecs) by current intensity (uAs).

DATA (MSECS) AW DATA (MV
59

60
percent condition pulse (Fig 3-2A). The 50 percent condition pulse
produced a test pulse response pattern which was midway between the 25
percent and 100 percent condition pulse patterns. The total durations
of inhibition for the 25, 50 and 100 percent condition pulses were 50,
75 and 120 msecs, respectively. The condition pulse current intensity
of 50 percent of the EPSP at PS threshold produced no inhibition of the
PS.
The highest levels of potentiation in the 25 percent test pulse
series for each of the condition pulse current intensities were nearly
the same in each of these experiments. The highest levels of poten
tiation were approximately 200 to 250 percent of the control responses.
The effects of the different condition pulse current intensities were
seen in the time it took to reach the highest level of potentiation as
well as the point at which the PS first became potentiated. The condi
tion pulse current intensity of 50 percent of the EPSP at PS threshold
became maximally potentiated at an IPI of 40 msecs and subsequently
decreased toward control levels. The 25 percent of PS amplitude condi
tion pulse potentiation peaked at 150 msecs, the 50 percent of PS
amplitude maximum condition pulse peaked at 200 msecs, and the 100
percent of PS amplitude maximum condition pulse peaked at 300 msecs.
All of the experiments in which the condition pulse current intensities
were set above PS threshold returned toward control levels together
beginning at approximately 400 msecs and ending by 2000 msecs IPIs.
The PS latencies, calculated by measuring the interval from the
EPSP onset to the PS maximum, were increased by up to 1.2 msecs with
increasing condition pulse current intensities but only at shorter IPIs
(Fig 3-3A). These latencies then returned to control levels together by
400 msecs IPIs. PS latencies were calculated from the EPSP onset since

Figure 3-2. PS amplitude responses means for 25 and 50 percent of PS amplitude at asymptote
for the test pulse series with 0/0 stimulation (test response/control response X
100). A) 25 percent test pulse series PS amplitude response with 50 percent of
EPSP, 25, 50 and 100 percent of PS maximum on the condition pulse. B) 50
percent test pulse series PS amplitudes with 50 percent of the EPSP, threshold
(0), 25, 50, 75, and 100 percent of PS maximum on the condition pulse.

ri**v.SC inLkva*. (wC) nTIJva. (usCC)
percent change from control
percent change from control
29

Figure 3-3. PS latency responses (mean) for 25 and 50 percent PS maximum test pulse series
with 0/0 stimulation (test response-control response). A) 25 percent test pulse
series PS latencies with 50 percent of EPSP, 25, 50, and 100 percent of PS
maximum on condition pulse. B) 50 percent test pulse series PS latencies with
50 percent of EPSP, threshold (0), 25, 50, 75, and 100 percent of PS maximum on
condition pulse.

difference from control
ON
-tr
HrtVA*. (wscc)
*^T*^vaJC (wSCC)

65
this controlled for individual animal differences in terms of the EPSP
onset as well as the different stimulation current intensity at which it
occurred. The PS latency can be used as a measure of pyramidal cell
excitability since it generally decreases with increasing PS amplitude.
The EPSP (Fig 3-4A) was monitored by measuring the slope of the
rising phase of the EPSP. These responses showed little change as a
result of the different condition pulse current intensities and thus
remained near control levels from 20 to 3000 msecs, the entire range of
testing. A decrease in the slope of the EPSP was noted at IPIs between
20 and 150 msecs. Previous reports indicate that a facilitation of the
EPSP is normally seen with IPIs of 20 to 30 msecs (Creager et al., 1980;
Lomo, 1971).
0/0 50 percent test pulse series
The 0/0 50 percent test pulse series PS amplitude responses (Fig
3-2B) showed similar patterns for the test response PS amplitude when
compared to the 0/0 25 percent data described above. The major differ
ence, however, was that the maximal level of potentiation was lower with
the 50 percent test pulse series PS responses. Like the 25 percent test
pulse data, increasing the condition pulse current intensity above PS
threshold levels produced increasing durations of inhibition in each of
the experimental series. Each of the superthreshold condition pulse
current intensities produced complete inhibition of the PS amplitude at
IPIs of 20 msecs. The maximal PS inhibition was, in general, extended
in duration with increasing condition pulse current intensities from 20
msecs at lower condition pulse current intensities to 50 or 60 msecs at
100 percent of the condition pulse PS response amplitude at asymptote.

Figure 3-4. EPSP slope responses (means) for 25 and 50 percent of PS maximum test pulse
series with 0/0 stimulation (control slope/test slope X 100). A) 25 percent
test pulse series EPSP slope responses with 50 percent of EPSP, 25, 50 and 100
percent of PS maximum on the condition pulse. B) 50 percent test pulse series
EPSP slope values with 50 percent of EPSP, threshold (0), 25, 50, 75, and 100
percent of PS maximum on condition pulse.

0)Sn) ytAtfiu jrww*Jv (3js) tvawjjw
percent change from control
percent change from control
9

68
When compared to the 0/0 25 percent test pulse responses, there were no
shifts in the duration of inhibition. The point at which the PS ampli
tude crossed control levels to the potentiation phase ranged from 50
msecs for the condition pulse current level set to PS threshold to
approximately 130 msecs for the 100 percent of PS amplitude at asymptote
condition pulse current intensity. The condition pulse current intensity
of 50 percent of the EPSP at PS threshold, unlike the 0/0 25 percent
test pulse equivalent, showed a very brief period of inhibited PS
responding at the 20 msecs IPI, but then showed a potentiated PS re
sponse at the 30 msecs IPI. As with the 0/0 25 percent test pulse
series, the condition pulse current intensity did not affect the maximal
level of potentiation. The subthreshold condition pulse PS amplitude
was potentiated maximally at the 40 msecs IPI and decreased to control
levels thereafter. The superthreshold condition pulse current intensi
ties all reached a peak level between 150 and 200 msecs IPIs and
remained near that level until IPIs of approximately 400 msecs at which
point they began to return to control levels. Each of the condition
pulse series PS responses returned to control levels by 1500 msecs. The
maximal level of potentiation observed with the 50 percent test pulse
series, however, only reached 140 to 160 percent of control. This
maximal potentiation level was in contrast to the 225 percent level
observed with the 25 percent test pulse series.
The PS latency data (Fig 3-3B) were similar to the 0/0 25 percent
data. An increase in the latency to a PS was observed with IPIs from 20
to 400 msecs. While the subthreshold condition pulse data showed the
smallest difference from control levels, the PS latencies of the super
threshold condition pulses were not apparently influenced by condition
pulse current intensity.

69
The slope data (Fig 3-4B) for this series showed a period of
inhibition at the 20 msecs IPI which remained slightly inhibited to
approximately the 150 msecs IPI. There was no apparent ordering of
slope by current intensity except that less inhibition was observed with
a subthreshold condition pulse than with a superthreshold condition
pulse.
A/0 25 percent test pulse series data
The A/0 25 percent of the PS maximum test pulse series (Fig 3-5A)
showed that increasing the current intensity of the antidromic condition
pulse increased the magnitude of the inhibition to a greater extent than
the duration of inhibition. Unlike the 0/0 experimental series reported
above, the A/0 series did not show potentiation. Instead, there appeared
to be two phases to the inhibition. There was a short initial period at
IPIs of 20 to 30 msecs when inhibition was greatest, followed by a
prolonged period where less but continued inhibition was found. The
antidromic condition pulse of 25 percent of the maximum antidromic spike
amplitude resulted in a test pulse response which was maximally inhibited
to 50 percent of the control test response at the 20 msecs IPI. The 50
and 100 percent antidromic condition pulses inhibited the test pulse
response to 22 and 14 percent of the control test response, respectively.
The IPI at which the 50 percent condition pulse produced its maximum
effect was at 20 msecs while the 100 percent condition pulse was maxi
mally inhibited at 30 msecs IPIs. Each of the condition pulse current
intensities produced a test pulse response which was inhibited to at
least 70 percent of the control response for the duration of the IPIs

Figure 3-5. PS amplitudes (means) for 25 and 50 percent of PS maximum test pulse series with
A/0 stimulation (test response/control response X 100) A) 25 percent of test
pulse series PS amplitudes with 25, 50, and 100 percent of the maximum
antidromic spike on the condition pulse. B) 50 percent test pulse series PS
amplitudes with threshold (0), 25, 50, 75, and 100 percent of the maximum
antidromic spike on the condition pulse.

percent change from control percent change from control
oS8!SSISS3SS8¡£KG6S5¡8
Li.

72
tested. The 50 and 100 percent condition pulse current intensities were
inhibited to approximately 50 and 30 percent of control from IPIs of 80
msecs to approximately 300 and 1000 msecs, respectively.
The PS latencies of the A/0 25 percent test pulse series (Fig 3-6A)
showed increased latencies from 20 to 400 msecs IPIs. The PS latencies
then remained somewhat longer than control responses. A 1.0 msec
increase in latency was observed at the 80 msecs IPI for the 100 percent
condition pulse current intensity.
The EPSP slopes for these data (Fig 3-7A) showed small fluctuations
at IPIs of 20 to 50 msecs but did not vary substantially from control
responses thereafter.
A/0 50 percent test pulse series
The A/0 50 percent of the PS maximum test pulse series PS ampli
tudes are shown in Fig 3-5B. It can be seen that each increase in
condition pulse current intensity produced a greater magnitude of
inhibition of the test pulse response. The 0, 25, 50, 75, and 100
percent antidromic conditioning pulses produced maximum inhibition
levels to 80, 60, 40, 30, and 20 percent of the control response,
respectively. The maximum level of inhibition for each series except
the A/0 threshold condition pulse level was reached at the 20 msecs IPI.
The threshold condition pulse current intensity produced its maximum
level of inhibition at an IPI of 60 msecs. As found with the A/0 25
percent test pulse series, the A/0 50 percent test pulse series resulted
in at least two distinct phases of inhibition. Maximal levels of
inhibition were found in the early period with IPIs between 20 and 60
msecs while a second phase of inhibition was found between IPIs of 80

73
and 1000 msecs. The A/O 50 percent test pulse series responses, like
the A/0 25 percent test pulse series responses, were inhibited for the
entire duration of IPIs tested but only to 80 to 90 percent of control
levels.
The PS latencies for these experiments are shown in Fig 3-6B. The
PS latencies increased from .1 to .4 msec between 20 and 200 msecs IPIs
for all of the condition pulse current intensities. The higher the
condition pulse current intensity, the longer the latency found to the
onset of the PS. Like the PS amplitude responses, the PS latency
responses were intermediate between 200 and 1500 msecs IPIs. Like the
A/0 25 percent test pulse series, the A/0 50 percent test pulse latency
response did not return to control levels with the IPIs tested.
Slope measures are shown in Fig 3-7B. Except for the brief de
crease in slope at the 20 msecs IPI seen with the 100 percent condition
pulse current intensity, there was only a small decrease in slope
observed with the IPIs tested.
Discussion
The effect of varying the current intensity of the condition and
test pulse of paired pulses was investigated using both 0/0 and A/0
stimulation configurations. An attempt was made to determine the
relative contribution of feedforward and recurrent inhibition with these
extracellular paired pulse paradigms. All of the experiments were
conducted in CAl using an anesthetized in vivo rat hippocampal prepara
tion.
The general characteristics of the superthreshold PS amplitude
response patterns found with 0/0 paired pulse stimulation for all of the

Figure 3-6. PS latencies (means) for 25 and 50 percent of PS maximum test pulse series with
A/0 stimulation (test response control response). A) 25 percent test pulse
series PS latencies with 25, 50 and 100 percent of the maximum antidromic spike
on the condition pulse. B) 50 percent test pulse series PS latencies with
threshold (0), 25, 50, 75, and 100 percent of the maximum antidromic spike on
the condition pulse.

**n**M. lusic) iwni*ULSC re****. (uUC)
difference from control
difference from control

S

Figure 3-7. EPSP slope (means) for 25 and 50 percent of PS maximum test pulse series with
A/0 stimulation (test slope/control slope X 100). A) 25 percent test pulse
series EPSP slope responses with 25, 50, and 100 percent of maximum antidromic
spike on the condition pulse. B) 50 percent test pulse series EPSP slope
responses with threshold (0), 25, 50, 75, and 100 percent of maximum antidromic
spike on the condition pulse.

percent change from control
percent change from control
s 8
8000
8000
3
V
X
t
i
I
Li

78
superthreshold condition and test pulse current intensities tested were
similar to those reported previously (Andersen et al., 1966; Creager et
al., 1980; Steward et al., 1977; Lomo, 1971). The subthreshold condi
tion pulse current intensity resulted in little inhibition but shifted
the period of maximal potentiation from IPIs of 150 to 400 msecs to IPIs
of 20 to 80 msecs. For the 0/0 superthreshold PS amplitude responses,
there was an initial period of inhibition at the shorter IPIs which then
changed to a period of potentiation at the intermediate length IPIs.
The PS latency measures corresponded well with the PS amplitude measures
since both yield an index of pyramidal cell excitability. Increasing
the current intensity resulted in increased PS latencies at short IPIs
since the PS was inhibited for longer periods. The EPSP response
patterns, however, were different from those reported previously
(Creager et al., 1980; Landfield et al., 1978; Lomo 1971; Steward et
al., 1977). The EPSP measure is typically reported as an amplitude at a
fixed latency from EPSP onset, the maximal EPSP amplitude or a slope
measure of the rising phase of the EPSP (Lomo 1971; McNaughton and
Barnes, 1977; White et al., 1979; Creager et al., 1980). Other re
searchers have reported a period of facilitation of the EPSP measures
with 20 to 30 msecs IPIs followed by a gradual decay with a time con
stant of about 100 msecs (McNaughton, 1982). Slope measures have been
utilized in an attempt to reduce the distortion of the EPSP sometimes
seen as a result of pyramidal cell or recurrent interneuron firing
(Creager et al., 1980). The present slope measures showed changes only
at short IPIs, but the changes observed were in the direction of de
creased rather than increased slopes. The most likely reason for these
response differences was that the present measures were recorded from

79
the pyramidal cell layer rather than in the synaptic zone. Recent
testing with recordings from the pyramidal cell layer and from the
Schaffer/commissural afferent synaptic zones in our laboratory has
confirmed that EPSP slope measures obtained from the synaptic zone match
those reported from other laboratories, while EPSP slope measures
recorded from the pyramidal cell layer matched the data from the present
experiments. These inconsistencies in EPSP measures from the SR synaptic
zone versus the pyramidal cell layer may be due to the occurrence of
several overlapping events. In the SR region, the EPSP is relatively
free of other synaptic influences such as the firing of the pyramidal
cell or recurrent inhibitory interneurons. When recording from the
pyramidal cell layer, the PS may partially or completely obliterate the
EPSP thus making the EPSP measure more difficult to analyze. In addi
tion, the IPSP from the recurrent and/or feedforward inhibitory inter
neuron may summate with the EPSP (due to the inward flow of current in
the SR excitatory region and the outward flow of current from the
inhibitory interneurons in the pyramidal cell region) and therefore
result in uninterpretable measures. It is our conclusion that the EPSP
data recorded from the cell layer do not represent the same phenomena
seen in the synaptic zones. It is likely that they reflect a summation
of the EPSP and IPSP and therefore it is difficult if not impossible to
make any conclusions regarding any alterations that may be occurring in
the SR synaptic region. EPSP slope data will therefore not be presented
in any of the subsequent chapters.
The A/0 paired pulse PS amplitude data at IPIs less than 80 msecs
were also similar to those reported by others (Dingledine and Langmoen,
1980). With IPIs of 60 to 80 msecs, however, where the test pulse PS

80
amplitude response was shown to return to control levels in other
studies, the current results showed that the return toward baseline
levels was different. Instead of the PS amplitude returning directly to
control levels, it returned toward control levels by approximately 50
percent at IPIs of 60 to 80 msecs and then very gradually returned to
levels closer to control by IPIs of 1000 to 2000 msecs. Even at this
point, however, the PS amplitude was inhibited by 10 to 30 percent of
control. The A/0 PS latency measures exhibited longer latencies than
control responses at short IPIs and subsequently returned toward control
levels. Like the PS amplitude responses, however, the PS latency data
did not completely return to the control level at any of the IPIs
tested. The best characterization of these responses is that A/0
stimulation results in two phases of inhibition. There is an initial,
maximal inhibition phase where the greatest magnitude of inhibition is
found. This maximal period of inhibition is then followed by a decay
phase which was more prolonged than previously noted. This is not
consistent with previous reports although most of the evidence has been
obtained from hippocampal slice preparations and so the effects of
anesthesia may result in the prolonged decay phase.
The 0/0 paired pulse stimulation configurations were seen to
produce near complete inhibition of the PS amplitude response at short
IPIs. This occurred despite the EPSP being facilitated at short IPIs
(based on reports by others including one using the same preparation,
anesthesia levels and stimulating and recording locations as the present
investigations but with condition and test pulse current levels sub
threshold for evoking PS responses (Abraham et al., 1981)). While
increasing the condition pulse current intensity increased the duration

81
of inhibition, the combination of feedforward and recurrent inhibition
produced total inhibition of the PS amplitude response even at the
current intensity of 25 percent of the PS maximum for the test response.
With A/0 stimulation, the PS amplitude was inhibited only to 40 or 50
percent of control levels with 25 percent of the PS maximum condition
pulse current intensity. A/0 paired pulse paradigms should predomi
nantly activate recurrent inhibition. 0/0 paired pulse paradigms should
activate both recurrent and feedforward inhibition. If the responses of
the A/0 paired pulse paradigms are compared with the responses of the
0/0 paired pulse paradigms, it is clear that feedforward inhibition
contributes significantly to the level of inhibition seen with 0/0
paired pulse paradigms in spite of facilitated EPSP responses found at
IPIs of 20 to 30 msecs.
A more detailed comparison of the A/0 and 0/0 paired pulse paradigm
responses revealed a potentially useful method for better characterizing
feedforward inhibition in the CAl region of the hippocampus. As de
scribed above, 0/0 stimulation produced total inhibition of the PS even
with a condition pulse current intensity of 25 percent of the PS ampli
tude at PS asymptote. The same A/0 condition pulse current intensity
resulted in much less inhibition than the 0/0 paradigm (40 percent
versus 100 percent inhibition). If the A/0 paired pulse stimulation
currents were titrated with decreasing condition pulse current levels
from the 25 percent of AS maximum amplitude level to that point at which
the A/0 test pulse PS amplitude was just observed (threshold) and then
that absolute current intensity was used with the 0/0 configuration, the
effect of feedforward inhibition alone may be observed. One of the
potential problems with this method is that the effect of the AS current

82
level may not be useful in terms of the separation of recurrent versus
feedforward inhibitory influences when applied to the afferent fibers of
the SR region. Nonetheless, this methodology may provide a most useful
extracellular method by which to study the effects of feedforward
inhibition using paired pulses.
Most studies have used a relatively high current intensity on the
condition and test pulses when examining different treatment conditions.
Different condition and test pulse current intensities may provide
substantially different responses since the influence of feedforward and
recurrent inhibition may, in effect, change from predominantly feed
forward influences with a lower current intensity to more equal contri
butions from feedforward and recurrent inhibition with a higher current
intensity. This conclusion is supported by evidence that indicates that
there are two populations of feedforward inhibitory interneurons that
have different firing patterns (Ashwood et al., 1984). One group of
neurons typically fired more than one action potential with frequent
bursts to stimulation which was above PS threshold. These bursts lasted
from 30 to 300 msecs. The other group of inhibitory interneurons
produced only one action potential even when stimuli were as high as 4.5
times threshold. Increasing the stimulus intensity produced shorter
inhibitory interneuron spike latencies. It has also been shown that
inhibitory interneurons can be activated at current levels which are
subthreshold for a PS (Buszaki and Eidelberg, 1981; Knowles and
Schwartzkroin, 1981). Thus, feedforward inhibition may actually prevent
pyramidal cell firing when the stimulation current intensity is near
threshold levels. The finding of inhibition at the 20 msecs IPI for the

83
0/0 subthreshold condition pulse with 50 percent of PS maximum test
pulses can be explained by this subthreshold recruitment of feedforward
inhibition.
The use of two 0/0 test pulse current intensities yielded responses
which were not substantially different in regard to the effect of
different condition pulse current intensities, when the amplitude or
duration of inhibition were compared. These results suggest that the
intensity of the condition pulse produces significant effects on the
inhibitory phase of paired pulse testing without greatly altering the
maximal levels of the potentiation phase. The major effect of the two
different test pulse current intensities was that two different levels
of potentiation were produced. Since it is known that feedforward
inhibition can influence the amplitude of the PS with single pulse
stimulation (Ashwood et al., 1984; Buszaki, 1984), the best explanation
for the lower levels of potentiation found with the higher test pulse
current intensity may be that there was a more powerful influence of
feedforward inhibition with a higher test pulse current intensity.
Recurrent inhibition would not be strongly implicated since the effect
of recurrent inhibition on the response to the the two different test
pulse current intensities would be the same given the same condition
pulse current intensity. The major reason for the difference in maximal
potentiation levels most likely emanates from the influence of the test
pulse current intensity on the levels of feedforward inhibition.
Another possible explanation for the difference in the maximum
levels of potentiation observed between the 25 percent and 50 percent of
the PS amplitude at PS asymptote test pulse current intensities is that
there was a ceiling effect. Since the 0/0 PS amplitude percent of

84
control responses were lower with the 50 percent test pulse current
intensity than with the 25 percent test pulse, it can be argued that,
for paired pulse potentiation, there is a maximum percent change in
response size to which the PS can increase. However, if that maximum
size is reflected by the asymptotic levels obtained with single pulse
stimulation during I/O testing, the levels of potentiation could have
reached 200 percent of the control response levels since the test pulse
current intensity was set to 50 percent of maximum, which they did not.
Instead, a maximal level of 140 to 160 percent of control was reached
for the 50 percent of PS maximum test pulse series. Similarly, the 25
percent of PS maximum test pulse series could have reached 400 percent
of control responses if they had potentiated to the levels reached with
high current levels during the I/O testing. These levels of potentia
tion argue against the ceiling effect hypothesis as an explanation for
differences between the low and higher current intensities on the test
pulse of these paired pulse data.
Inhibition in the CA1 region of the hippocampus is present ubiqui
tously. Although recording with single pulse afferent stimulation in
the pyramidal cell body layer has often been assumed to be unaffected by
inhibition, this premise is likely to be false. It has been demonstrated
that inhibition is tonically active in the resting inhibitory interneuron
(Alger and Nicoll, 1980) much like it has been shown for excitatory
hippocampal pyramidal neurons (Brown et al., 1979). Thus, a background
of spontaneous inhibition continuously occurs in the hippocampus against
which any incoming stimuli may be modified. Recent characterizations of
feedforward inhibition have provided evidence that the feedforward
interneuron begins firing prior to the CAl pyramidal cell (Ashwood et

85
al., 1984; Buzsaki, 1984). This observation implies that feedforward
inhibition can therefore influence pyramidal cell firing even with
single pulse stimulation. Extracellularly, the amplitude of the PS
could be decreased as a function of the feedforward inhibition.
One of the first tests in many extracellular investigations is that
of determining the I/O curve. The I/O curve provides a good description
of the input current strength to response amplitude. It has typically
been considered an unencumbered measure and has therefore been used to
compare the basic response of one treatment group to another or to a
control group. The inference has been that differences signify altera
tions in the normal functioning of the pyramidal cell. If feedforward
inhibition acts prior to pyramidal cell firing and thereby alters the
response, then the basic assumption of differences in I/O curves being
due to changes in pyramidal cell function may be inaccurate. If differ
ent treatment conditions are being tested, it may be that the treatment
condition has altered the feedforward inhibitory interneuron which may
in turn influence the pyramidal cell firing characteristics. If feed
forward interneurons fire differentially to various current intensities,
feedforward inhibition may play a major role in determining the sigmoidal
shaped I/O curve when tested with different current intensities. The
abrupt appearance of the PS may be less reflective of the threshold for
the PS per se. than it is the point at which the PS finally emerges from
the previously overwhelming control of inhibitory mechanisms. Threshold
differences between groups may therefore be a function of effects on
feedforward inhibition.
These studies suggest that the magnitude of the PS amplitude and
latency responses to different current intensities can be carefully

86
governed to recruit different levels and probably ratios of feedforward
and recurrent inhibition. Lower current intensities appear to cause a
greater predominance of feedforward- rather than recurrent-inhibition.
At higher current intensities it is likely that feedforward and recur
rent inhibition contribute more equally to the resulting PS amplitudes.
Another point indicated by these results is that the current intensity
used for the test pulse of paired pulses significantly modifies the
magnitude of paired pulse potentiation observed. While the current
intensity of the condition pulse influences the early phases of inhibi
tion and the time to reach the maximal level of potentiation, it is the
test pulse which most strongly influences the maximum level of potentia
tion achieved. It is suggested that feedforward inhibition influences
the maximal magnitude of paired pulse potentiation rather than recurrent
inhibition.
The use of extracellular recording techniques provide for an
examination of cellular events at a more system oriented level than
other procedures. The exquisite lamination and arrangement of the
hippocampal architecture provides unique opportunities for extracellular
studies. The incorporation of new information on the effects of feed
forward inhibition should allow for a better understanding as well as
improved methodologies in considering what these responses mean and how
they can be used to investigate the effects of other treatment condi
tions .

CHAPTER 4
CET AND SYNAPTIC INHIBITION: AN IN VITRO ANALYSIS
Introduction
It has become increasingly clear that chronic ethanol abuse in
humans produces a range of behavioral deficits as well as a range in the
severity of those deficits (Tarter and Alterman, 1984; Parsons and
Leber, 1981; Parsons, 1977: Ron, 1977; Ryan, 1980). Anatomical studies
of human alcoholics have shown general cerebral atrophy and lesions of
certain nuclei (Wilkinson, 1982; Ron, 1977, 1982; Cala and Mastaglia,
1981; Brewer and Perrett, 1971; Victor et al., 1971). A major contro
versy has existed concerning whether alcohol itself or nutritional
inadequacies were the cause of these changes (Victor et al., 1971).
Animal studies which employed rigorous controls of the dietary intake
have, however, provided substantial evidence that chronic ethanol abuse
alone caused many of the behavioral, anatomical and functional deficits
observed (Walker and Hunter, 1978; Walker and Freund, 1973; Freund and
Walker, 1971; Denoble and Begleiter, 1979; Fehr et al., 1975; Tavares et
al., 1983; Abraham et al., 1981, 1982, 1984; Durand and Carien, 1984a,
1984b). Direct evidence of CET effects can be seen in the morphological
alterations which have been reported in rats exposed to CET. A 15 to 20
percent decrease in the number of primary cells in the CA1 region, the
dentate gyrus and the cerebellum have been reported (Walker et al.,
1980). CAl pyramidal cell and dentate granule cell spine density has
been shown to be decreased (Riley and Walker, 1978). The dendritic
branching patterns of these cells have also been shown to be reduced.
87

88
Physiologically, it has been shown that CET produced increased
amplitudes of the PS with paired pulses, but no changes in measures of
the EPSP or the PS with single pulse stimulation procedures (Abraham et
al., 1981). These changes were thought to be a result of a decrease in
the efficacy of recurrent inhibition. Intracellular studies of CAl
pyramidal cells have also found decreased amplitudes of IPSPs and long
lasting after-hyperpolarizations (Durand and Carien, 1984a). The
evidence has thus become powerful concerning CET effects on the hippo
campal region. The next set of questions can therefore address the issue
of how CET may actually produce these alterations.
The finding that CET produced an increase in the amplitude of the
PS of the test pulse of paired pulses without concommitant changes in
the EPSP amplitude or slope and without differences found between
control and CET animals on the PS of input/output curves led to the
hypothesis that the changes in paired-pulse potentiation occurred as a
function of a decrease in recurrent inhibition (Abraham et al., 1981).
Recurrent inhibition in the CAl region of the hippocampus is a well
described regulatory mechanism. Briefly, when the pyramidal cell is
caused to fire by inputs to its dendrites, it, in turn, fires an inhi
bitory interneuron; presumably a basket cell. This interneuron then
ramifies widely to the original as well as other pyramidal cells in its
plexus and causes a massive cessation of spontaneous firing of the
pyramidal cells for up to 600 to 700 msecs (Andersen et al., 1964a,
1964b). Recently, there has been new documentation for a second type of
inhibition which operates in conjunction with recurrent inhibition.
This type of inhibition is feedforward in nature (Andersen et al., 1982;
Alger and Nicoll, 1980, 1982a). It is likely that the anatomical

89
configuration of this system is such that an interneuron is interposed
between a fraction of the incoming afferents and the dendrites of both
the basilar and apical dendritic regions of CAl pyramidal cells (Ashwood
et al., 1984; Buzsaki, 1984; Schwartzkroin and Mathers, 1978).
When using orthodromic paired pulse stimulation of the SR, the
effects of both feedforward and recurrent inhibition are observed since
both the feedforward and the recurrent interneuron are activated by
afferent stimulation. The previously stated hypothesis concerning CET
effects on recurrent inhibition may be incomplete or incorrect since
both inhibitory systems were activated by SR stimulation in the studies
of Abraham et al., 1981. Therefore, experimental paradigms designed to
more selectively activate either feedforward or recurrent inhibition and
similar to those used in Chapter 3, were performed in these experiments
to help resolve this question.
This set of experiments was performed using the hippocampal slice
preparation. There were two major reasons for switching to the in vitro
preparation. First, one of the major problems with results from the in
vivo experimental series was that the anesthesia used may have produced
powerful effects on inhibition in general but on CET effects
specifically. Since ethanol may be cross tolerant with barbiturates
(Kalant et al., 1971), it is possible that there could be an interaction
between the anesthesia and CET. There was little likelihood of this
situation however, since the animals were removed from the ethanol
containing diet for at least two months prior to testing. The second
reason for utilizing the in vitro preparation is that direct pharmaco
logical challenges can be made to the CAl region which are not possible
in vivo. While pharmacological manipulations are not a part of the

90
experiments in this section per se. these experiments are crucial to the
pharmacological experiments proposed in the following chapter since they
lay the groundwork for comparisons between in vivo and in vitro results.
It is important to verify that similar patterns of CET effects are found
between in vitro and in vivo preparations.
The major negative characteristic of the in vitro technique
emanates from the fact that transverse hippocampal slices have reduced
levels of inhibition most probably due to the severance of the longitu
dinal projections of the recurrent inhibitory interneurons (Teyler,
1980). The result of this decrease in inhibition is that paired pulse
response curves do not typically show inhibition with IPIs less than 30
msecs whereas in vivo, inhibition is seen with IPIs as long as 80 to 150
msecs (Dunwiddie et al., 1980). Differences in the levels of inhibition
between CET and normal animals should, however, be readily apparent if
they exist.
A third question which was posed was whether heterosynaptic affer
ent paired pulse stimulation could be effectively used to provide
additional insight into how and where the inhibitory systems might
interact. It has been reported previously that stimulation of the SO or
SR afferents with the condition pulse followed by SR or SO afferents,
respectively, on the test pulse of paired pulses produced only inhibi
tion (Haas and Rose, 1982; Steward et al., 1977). As indicated earlier
A/0 paired pulses result in a moderately clean assessment of recurrent
inhibition. Homosynaptic 0/0 paired pulses result in three different
mechanisms being activated. Feedforward inhibition, recurrent inhibi
tion and an EPSP facilitation are all seen to occur when stimulating
with double pulses in the same afferent region. Heterosynaptic 0/0

91
stimulation is similar to homosynaptic 0/0 stimulation in that both
feedforward and recurrent inhibition are activated. Since inputs do not
originate or impinge on the same pyramidal cell dendritic areas,
however, no facilitation of the EPSP occurs. Thus, heterosynaptic 0/0
stimulation offers a way of observing the inhibitory systems of the CAl
region without EPSP facilitation or PS potentiation also being induced.
This heterosynaptic paired pulse paradigm may therefore provide an
additional perspective from which to review the effects of CET on
inhibition in CAl.
The three major questions addressed in these experiments are: 1.
Does CET produce alterations in feedforward as well as recurrent inhibi
tion? ; 2. Are the in vivo and in vitro preparations comparable as far as
the effects of CET are concerned?; and 3. How does heterosynaptic SR/SO
stimulation compare with SO/SR paired-pulse stimulation and with SR/SR
and A/0 paired-pulse stimulation?.
Methods
Animals
Animals used were housed and fed as described in the general
methods.
Comments on in vitro technique
All of the experiments performed in this chapter and in the subse
quent chapters were performed using the in vitro hippocampal slice
technique. This technique has several advantages for the study of
recurrent and feedforward inhibition in CAl. One of the most important
advantages is the ability to place electrodes visually. Although

92
placement of both the recording and stimulating electrodes can be
precisely performed in vivo. the in vitro experiments in the following
chapter made use of microiontophoresis techniques using various pharma
cological agents at specific locations within the CAl region. These
manipulations would be difficult to perform in vivo. An additional
feature in using the slice technique is that anesthetic agents, which
have been shown to have powerful effects on hippocampal inhibition, are
not used. All major synaptic pathways are conserved in the hippocampal
slice preparation (Teyler, 1980). However, inhibition is reduced since
transverse projections of the basket cells are severed. Responses
recorded in vivo have been compared with those recorded in vitro.
Reports indicate the conservation of nearly all general characteristics
between the two preparations (Schwartzkroin, 1981). That the advantages
outweigh the disadvantages is attested to by the fact that the majority
of the literature concerning recurrent and feedforward inhibition in the
hippocampus have been conducted in vitro (Dingledine and Gjerstad, 1980;
Alger et al., 1981; Andersen et al., 1980; Lee et al., 1979; Teyler,
1980).
Apparatus
Commercially obtained tissue chambers were used in these experi
ments (Frederick Haer and Co). Static recording chambers were used in
all experiments. The static chamber consisted of a static pool of
artificial CSF which had a piece of filter paper stretched taut on a
cylindrical ring on which the brain slice was placed such that diffusion
of the artificial CSF occurred from beneath the slice. The static
chamber was placed inside of a holding tank which included a d.c. heater

93
system, a gas bubbler, a fiber-optic light source, and electrical
connections for grounding and recording from the recording chambers.
Tissue preparation
Experimental and control animals were treated with the same proce
dures since coding of the animals was performed. Well established slice
preparation procedures were used (Andersen et al., 1978; Schwartzkroin,
1981; Langmoen and Andersen, 1981; Teyler, 1980; Alger et al., 1984;
Andersen, 1984; Dingledine et al., 1980; Simmons, 1982; Skrede and
Westgaard, 1971; Yamamoto and Mcllwain, 1966) which allowed for a quick
start up as well as consistency of experimental setups between laborato
ries. Slices were prepared using the method described by Teyler (1980)
with minor modifications. An Haer tissue chopper (modified Mcllwain)
was used to slice the hippocampus into 350-400 uMs thick sections. This
thickness of the sections was selected because it represents a good
compromise between having a core of healthy tissue and having a core
which was too thick to allow for reliable diffusion of the bathing
medium uniformly through the slice. Generally, a layer of tissue on
either side of the cut edges, about 50 to 100 microns, was not viable
due to injury. Thus, a 350 to 400 micron slice provided about 200
microns of viable tissue. After the slices were cut, they were placed
in a transfer dish containing the medium which was used during the
actual recording. The slices were then transferred to the tissue
recording chamber with a large neck glass pipette where they were
incubated for at least 45 minutes prior to use. This incubation period
was necessary for the slice to regain its electrical characteristics

94
which are temporarily lost after sectioning the hippocampus (Teyler,
1980).
Incubating medium
The incubating medium consisted of the following: NaCl (124 mM),
KC1 (5mM), KH(2)P0(4) (1.25mM), MgSo(4) (2mM), CaCl (2mM), NaHCO(3)
(26mM), glucose (lOmM). In addition, the medium was perfused with 95
percent 0(2) and 5 percent C0(2) and maintained at a pH of 7.27 to 7.37.
These constituents of the incubating medium were chosen because they
represented levels which allowed for relatively normal cellular activity
(Teyler, 1980).
Recording chamber
Slices were individually transferred to a recording chamber where
they were placed in contact with the medium described above. Temperature
was maintained at 34 to 35 degrees Centigrade. The atmosphere was
warmed 95 percent 0(2) and 5 percent C0(2). Standard electrophysiolog-
ical methods were used for stimulation and recording as outlined in the
general methods.
Procedures
The procedures used in this set of experiments were similar to
those used in the previous in vivo experiments. All electrode place
ments were, however, done visually. The orthodromic stimulating elec
trode was placed in SR near the CA1-CA2 border. The antidromic stimulat
ing electrode was placed on the alvear fibers lateral to the recording
electrode. Every effort was made to keep the effect of this stimulating

95
electrode localized to the alvear fibers without current spread to the
SO dendritic region.
I/O curves were obtained using procedures identical to those
described in the previous chapter except that an additional I/O curve
was also obtained using the SO stimulation site. The IPIs used for all
of the paired pulse experiments were the same as those used in the
previous in vivo experiments. The condition and test pulse current
intensities for this set of experiments are described in Table 4-1.
The in vitro paired pulse data were analyzed in the same manner as
the in vivo data. The PS latency measures from the test pulse responses
were subtracted from single pulse control responses which were obtained
immediately prior to the experimental IPI responses for each experiment.
All amplitude and slope measures were expressed as a percent of the
control response with the control response equal to 100 percent. All
data are described for IPIs only up to 400 msecs. This was done because
the various measures for the alcohol and sucrose animals tended to
return to control levels together and closely resembled the data pre
sented in the previous chapter for the longer IPIs. There were two
separate sets of animals, each containing sucrose and alcohol animals,
used for various aspects of these experiments. They are reported inde
pendently and then compared. Slope data were recorded, but are not
reported since all slope measures were obtained from the cell layer
rather than the synaptic zone (see discussion Chapter 3).
Results
The mean alcohol consumption for the first set of animals was 12.67
-305 g/Kg. The mean body weight for the alcohol animals was 543 +9.9

96
TABLE 4-1
PERCENT OF MAXIMAL PS
AMPLITUDE
ON
TEST
PULSE
Set 1
25%
50%
c
0
Homosynaptic:
1
1
1
N
P
SR/SR 50% EPSP
1 x
1
1
D
U
SR/SR 25% PS
1 x
I
1
I
L
SR/SR 50% PS
1 x
1
1
T
S
SR/SR 50% PS
1
1
X
1
I
E
1
1
1
0
Antidromic:
1
1
1
N
ALV/SR 50% AS
1 X
1
1
ALV/SR 100% AS
1
1
1
1
X
1
1
Set 2
Homosynaptic:
1
1
1
SR/SR 25% PS
1 X
1
1
S0/S0 25% PS
1 X
1
1
1
Antidromic:
1
1
1
1
1
1
ALV/SR 100% AS
1 X
1
1
1
Heterosynaptic:
1
1
1
1
1
1
SR/SO 25% PS
1 X
1
1
SO/SR 25% PS
1 X
1
1
1
1
1
Percent of maximal PS amplitude at PS asymptote, EPSP asmplitude at
PS threshold, and maximal antidromic spike (AS) amplitude for the
condition and test pulses in paired pulse paradigms (in vitro') Note
different stimulation electrode configurations.

97
gms and for the sucrose group was 537.8 + 13.25 gms. The mean alcohol
consumption of the second set of animals was 13.17 + .25 g/Kg. The mean
body weight for the alcohol animals was 541 + 10.55 gms and the sucrose
group was 532 + 15.11 gms.
I/O relationships. Two different sets of animals were used in
performing the experiments reported in this chapter. For the first set
of animals an I/O function was obtained prior to any other experimental
manipulation. A second I/O curve was then obtained after all of the
paired pulse experiments had been completed for the same animals. These
two I/O curves were collected in order to determine whether any gross
changes in pyramidal cell excitability occurred during the course of the
experimental session which was two to four hours in duration. There
were 10 animals in the alcohol treated group as well as 10 animals in
the sucrose-fed control group. The data presented for all of the
subsequent I/O curves were normalized according to the PS onset. Thus,
the first current level at which a PS was observed was normalized to a
"zero" current level. Subsequent data points were obtained by interpo
lating the PS responses to the current intensities as indicated in the
general methods, but only from 0 to 500 uAs. This normalization was
performed in order to control for individual differences between animals
and groups in terms of the PS threshold. As can be seen in Figs 4-1
(pre-paired pulse I/O curve) and 4-2 (post-paired pulse I/O curve) there
were no significant differences between the alcohol and sucrose groups.
The alcohol animals did, however, tend to have slightly higher PS
amplitudes especially at PS asymptote. The PS threshold for the alcohol
group on the initial I/O curve was 159 uAs 12.15 SEM while the sucrose
control group was 181 uAs 18.28 SEM. The PS threshold for the alcohol

Figure 4-1. I/O curves for Set 1 slice experiments prior to
any paired pulse stimulation. Data were
normalized by setting the current level at which
the PS was first seen equal to 0 uAs. Data were
then interpolated from that current value such
that interpolated I/O curves were produced with
current levels between zero and 500 uAs. This
procedure controlled for any differences in PS
threshold between groups. A) Normalized PS
amplitude responses (means) for the alcohol and
sucrose groups in mVs by current intensity (uAs).
B) Normalized PS latency responses (means) for
the alcohol and sucrose groups in msecs by
current intensity (uAs).

RAW DATA (MSEC) RAW DATA
99

Figure 4-2. I/O curves for Set 1 slice experiments after all
paired pulse stimulation experiments were com
pleted. Data were normalized by setting the
current level at which the PS was first seen
equal to 0 uAs. Data were then interpolated from
that current value such that interpolated I/O
curves were produced with current levels between
zero and 500 uAs. This procedure controlled for
any differences in PS threshold between groups.
A) Normalized PS amplitude responses (means) for
the alcohol and sucrose groups in mVs by current
intensity (uAs). B) Normalized PS latency
responses (means) for the alcohol and sucrose
groups in msecs by current intensity (uAs).

DATA (MSEC) RAW DATA (MV)
101

102
group on the second I/O curve was 165 uAs + 14.85 SEM and 158 uAs + 5.56
SEM for the sucrose group.
The second set of I/O curves were obtained from the second set of
animals used in the paired pulse experiments. Since one of the primary
objectives was observing the effect of four different stimulation
configurations which included the use of the SO afferent field, I/O
curves were obtained with stimulation in the SR as well as the SO
afferent regions. Fig 4-3 shows the response of alcohol and sucrose-fed
animals with SR stimulation while Fig 4-4 shows the responses of the two
groups with SO stimulation. While no significant differences were seen
between the two groups with stimulation from either the SR or SO
synaptic zone, the alcohol animals tended to have lower PS amplitudes
than the sucrose control animals in this set of animals. The PS
threshold for the alcohol group with SR stimulation was 113.33 7.52
uAs while it was 112.5 +4.96 uAs for the sucrose group. The PS
threshold obtained with SO stimulation was 96.67 + 4.82 uAs for the
alcohol group and was 90.0 + 6.83 uAs for the sucrose group.
Paired pulse results. The paired pulse data collected during these
experiments are similar to the in vitro paired pulse data collected from
other laboratories (Dunwiddie et al., 1980). The general pattern of in
vitro inhibition and potentiation was, however, different from in vivo
preparations (Dunwiddie et al., 1980). In all of the present in vitro
0/0 homosynaptic paired pulse experiments, there was little if any
inhibition seen, even at short IPIs. Instead of a period of inhibition,
potentiation was seen almost immediately. Heterosynaptic paired pulse
experiments showed no potentiation, but did show inhibition and

Figure 4-3. I/O curves for Set 2 slice experiments before any
paired pulse stimulation experiments were com
pleted. These data are with stratum radiatum
stimulation. Data were normalized by setting the
current level at which the PS was first seen
equal to 0 uAs. Data were then interpolated from
that current value such that interpolated I/O
curves were produced with current levels between
zero and 500 uAs. This procedure controlled for
any differences in PS threshold between groups.
A) Normalized PS amplitude responses (means) for
the alcohol and sucrose groups in mVs by current
intensity (uAs). B) Normalized PS latency
responses (means) for the alcohol and sucrose
groups in msecs by current intensity (uAs).

DATA (MSEC)
104

Figure 4-4. I/O curves for Set 2 slice experiments before any
paired pulse stimulation experiments were com
pleted. These data are with stratum oriens
stimulation. Data were normalized by setting the
current level at which the PS was first seen
equal to 0 uAs. Data were then interpolated from
that current value such that interpolated I/O
curves were produced with current levels between
zero and 500 uAs. This procedure controlled for
any differences in PS threshold between groups.
A) Normalized PS amplitude responses (means) for
the alcohol and sucrose groups in mVs by current
intensity (uAs). B) Normalized PS latency
responses (means) for the alcohol and sucrose
groups in msecs by current intensity (uAs).

RAW DATA (MSEC) RAW DATA (MV)
106

107
otherwise resembled other heterosynaptic 0/0 paired pulse results (Haas
and Rose, 1982). Although the statistical analyses for all of these
experiments showed few statistically significant differences between
alcohol and sucrose data, the general pattern of the alcohol animals was
for higher levels of potentiation and lower levels of inhibition than
found in the sucrose animals. These patterns are similar to those
reported by Abraham et al. (1981).
0/0 homosynaptic paired-pulse paradigms -- Set 1
The 0/0 homosynaptic paired pulse PS amplitude responses obtained
with a current intensity of 50 percent of the EPSP amplitude at PS
threshold set on the condition pulse and 25 percent of the PS amplitude
at PS asymptote set on the test pulse are shown in Fig 4-5A. These data
were very similar to those reported with similar conditions in vivo.
There was an immediate potentiation of the PS which peaked at the 30
msecs IPI for the alcohol data and at the 50 msecs IPI for the sucrose
animals. The maximum levels of potentiation at these intervals were 200
percent for the alcohol animals and 160 percent for the sucrose animals.
Although there were no statistically significant differences, the IPIs
between 20 and 100 msecs showed the greatest differences between the two
groups; the CET group was approximately 20 to 50 percentage points
higher than the sucrose group. The latency measures for these data can
be seen in Fig 4-6A. There is an initial decrease in the latency to the
PS, by .4 msec, for both groups from 20 to 100 msecs IPIs but there were
no differences between groups.
The 0/0 homosynaptic paired pulse PS amplitude responses with the
condition pulse current intensity set to 25 percent of the PS amplitude

Figure 4-5. 0/0 homosynaptic paired pulse PS amplitude responses (means + SEM) with
stimulation through stratum radiatum. All test responses are shown as percent
changes from control responses (test response/control response X 100). A) Test
pulse responses obtained with the condition pulse current intensity set to
obtain an EPSP response 50 percent of the EPSP amplitude at PS threshold for
both the control and sucrose groups. Test pulse response was set to 25 percent
of the PS amplitude at PS asymptote. B) Test pulse responses obtained with 25
percent of the PS amplitude at PS asymptote on both the condition and test
pulses. C) Test pulse responses obtained with the condition pulse adjusted to
50 percent of the PS amplitude at PS asymptote and 25 percent of the PS
amplitude at PS asymptote on the test pulse. D) Test pulse responses obtained
with 50 percent of the PS amplitude at PS asymptote on both the condition and
test pulses.

CCl*T CHANCt mow CONTROi. !< I'ttNCf rOWt CONTROl
0 00 200 300 <00
NTC**ut.*C irtm/Ac (usee)
PCRCCKT CHA/iCC rwOM COfiTWOc ^CKCIXT OWiCC mow COKTKOC.
400
o
vO

Figure 4-6. 0/0 homosynaptic paired pulse PS latency responses (means) with stimulation
through stratum radiatum. All test responses are shown as a difference from
control responses (test response-control response in msecs). A) Test pulse
responses obtained with the condition pulse current intensity set to obtain an
EPSP response 50 percent of the EPSP amplitude at PS threshold for both the
control and sucrose groups. Test pulse response was set to 25 percent of the PS
amplitude at PS asymptote. B) Test pulse responses obtained with 25 percent of
the PS amplitude at PS asymptote on both the condition and test pulses. C) Test
pulse responses obtained with the condition pulse adjusted to 50 percent of the
PS amplitude at PS asymptote and 25 percent of the PS amplitude at PS asymptote
on the test pulse. D) Test pulse responses obtained with 50 percent of the PS
amplitude at PS asymptote on both the condition and test pulses.

0rrij^cNCC rnou COumcK. (uscc)
rcHCtKT cha^cc DrrcwcNCC rwou cokthoc (msec)
0
100
200
IWTTJPUL3C INTIF/AL (USCC)
300
400

112
at PS asymptote and with 25 percent of the PS amplitude at PS asymptote
set on the test pulse are shown in Fig 4-5B. Unlike the in vivo series,
no inhibition was seen at any IPI. The PS amplitude responses for the
alcohol animals were potentiated maximally at IPIs from 30 to 80 msecs
to a level of 300 percent. The sucrose animals were potentiated maxi
mally at an IPI of 60 msecs with the PS amplitude potentiated to 250
percent of the control response. There was approximately a 50
percentage points difference between the alcohol and sucrose groups from
20 to 150 msecs IPIs although this difference was not statistically
significant. The separation seen between groups with the PS amplitude
response was also seen with the latency to the PS measure (Fig 4-6B).
Alcohol animals had shorter latencies to the onset of the PS than did
the sucrose animals although there were no statistically significant
differences. It can be seen that the sucrose group PS latency was
decreased by .4 msec at IPIs of 20 to 40 msecs while the alcohol group
reached its shortest latency to the PS at 40 msecs extending to 80
msecs. The alcohol group PS latency was shortened by .8 msec from the
control response at these IPIs. The two groups were separated by .4
msec from 40 to 200 msecs IPIs.
PS amplitudes for 0/0 homosynaptic paired pulse responses with the
condition pulse current intensity set to 50 percent of the PS amplitude
at PS asymptote and with the test pulse current intensity set to obtain
a response 25 percent of the PS amplitude at PS asymptote are shown in
Fig 4-5C. The alcohol group PS amplitude measures (Fig 4-5C) were
potentiated maximally between 40 and 60 msecs IPIs. The highest levels
of potentiation were approximately 340 to 350 percent of the control
responses. The sucrose group PS amplitude responses were maximally

113
potentiated to 300 percent at an IPI of 50 msecs. The alcohol group was
approximately 50 percentage points more potentiated than the sucrose
group from an IPI of 20 msecs to 300 msecs at which point the data from
the two groups converged and returned to baseline levels. The latency
to PS onset data (see Fig 4-6C) showed that the alcohol group had a
shorter latency by .3 msec than did the sucrose group. Both groups had
the shortest latencies with 20 to 40 msecs IPIs and returned to control
levels by 400 msecs.
The 0/0 homosynaptic paired pulse paradigm with the highest current
levels in this series of experiments, 50 percent of the PS amplitude at
PS asymptote on both the condition and test pulses are shown in Fig
4-5D. Following the pattern of the previous superthreshold,
homosynaptic paired pulse results for PS amplitude measures, the 50
percent condition and test pulse current intensity group (see Fig 4-5D)
again showed the alcohol group means to be slightly more potentiated
than the sucrose group. The alcohol group was maximally potentiated
between IPIs of 50 to 60 msecs at 180 to 190 percent of control values.
After the IPI of 80 msecs, the two groups were no longer at separate
levels. The maximal level of potentiation for either of the two groups
with these conditions was lower than that found with the 25 percent test
pulse groups as was shown in vivo. The PS latencies for this experiment
(Fig 4-6D) decreased by .4 to .5 msec at IPIs less than 100 msecs. The
alcohol group, however, did not have significantly different PS latencies
than the sucrose group. Both groups returned to control levels by 200
to 300 msecs IPIs.

114
A/0 paired pulse paradigms -- Set 1
The first set A/O paired pulse PS response amplitudes are shown in
Fig 4-7. The antidromic series in vitro data (Fig 4-7A) with 50 percent
of the antidromic maximum on the condition pulse and 25 percent of the
PS amplitude at PS asymptote on the test pulse compared well with the
in vivo antidromic data (Fig 3-5). The strongest inhibition was seen at
the 20 msecs IPI for both the alcohol and sucrose groups. There were
two apparently different periods of inhibition. One was seen at very
short IPIs of 20 to 30 msecs while the other was much more prolonged and
was seen at IPIs from 40 msecs to 150 or 200 msecs. Although the
alcohol group was somewhat less inhibited (50 to 55 percent) at short
IPIs than was the sucrose group (40 percent), the two groups converged
by the 50 msecs IPI until control levels were reached. The PS latency
data showed (Fig 4-8A) both groups to have longer latencies to the PS
maximum than their respective control responses. The sucrose group also
had longer response times (.3 to .5 msec) than the alcohol group (.1 to
.25 msec) for all of the IPIs shown.
The A/0 paired pulse responses with 100 percent of the AS maximum
and 50 percent of the PS amplitude at PS asymptote on the test pulse are
shown in Fig 4-7B. The alcohol and sucrose groups both revealed a
maximum inhibition level of 40 percent of the control response at the 20
msecs IPI. The alcohol group then returned to near control levels by the
100 msecs IPI. The sucrose group, however, remained inhibited to at
least 70 percent of the amplitude of the control response beyond 400
msecs. This pattern was similar to that seen in vivo (Fig 3-5) where
the test pulse PS amplitude remained inhibited to approximately 70
percent until the IPIs of 1000 to 2000 msecs. The PS amplitude

Figure 4-7. A/O paired pulse PS amplitude responses (means + SEM) with condition pulse
stimulation through the alveus and test pulse stimulation through stratum
radiatum. All test responses are shown as a percent of control responses (test
response/control response X 100). A) Test pulse responses obtained with the
antidromic condition pulse current intensity set to obtain a response 50 percent
of the maximum antidromic amplitude. Test pulse response was set to 25 percent
of the PS amplitude at PS asymptote. B) Test pulse responses obtained with 100
percent of the antidromic spike amplitude on the condition pulse and 50 percent
of the PS amplitude at PS asymptote on the test response.

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Figure 4-8. A/O paired pulse PS latency responses (means) with condition pulse stimulation
through the alveus and test pulse stimulation through stratum radiatum. All
test responses are shown as a difference from control responses (test response -
control response in msecs). A) Test pulse responses obtained with the
antidromic condition pulse current intensity set to obtain a response 50 percent
of the maximum antidromic amplitude. Test pulse response was set to 25 percent
of the PS amplitude at PS asymptote. B) Test pulse responses obtained with 100
percent of the antidromic spike amplitude on the condition pulse and 50 percent
of the PS amplitude at PS asymptote on the test response.

OirrxACNCC rmou control (mscC)
orrtcxcc rmou cowtkov. (uscc)

119
responses for these two groups were statistically different (F(l,21) =
6.82, p .012). The PS latency measures (Fig 4-8B) showed increased
latencies for both groups. The alcohol group latencies peaked at the 20
msecs IPI with the latency being .5 msec longer than the control
response. The sucrose group peaked at the 40 msecs IPI with the latency
.35 msec longer than the control responses. The latency measures for
both groups then came back toward control levels though they remained
longer than control responses beyond the 400 msecs IPI. PS latencies
were not, however, statistically significant between groups.
Set 2
The remaining experiments were performed in a set of animals
different from those reported above. One of the experiments used the
same condition and test pulse current intensities as that reported above
but provided a control for the additional experiments in this set of
animals. The experiments reported above explored the effect of
different condition and test pulse current intensities oti 0/0 and A/0
paired pulse configurations. This next set of experiments was designed
to explore the effect of four different stimulus configurations each
with similar condition and test pulse current intensities.
0/0 homosvnaptic paired pulse naradipms -- Set 2
Two homosynaptic 0/0 paired pulse paradigms were performed, one
using SR stimulation and the other using SO stimulation. Both of these
experimental paradigms used current intensities adjusted to 25 percent
of the PS amplitude at PS asymptote. Fig 4-9B shows the PS amplitude
responses for the 0/0 paired pulse experiment with SR stimulation.

Figure 4-9. 0/0 paired pulse PS amplitude responses (means + SEM). Note that these experiments
were conducted through two different sets of afferent fibers. All test responses
are shown as a percent of control responses (test response/control response X 100).
A) Test pulse responses obtained with condition and test pulse stimulation through
the stratum oriens. The current intensities on both the condition and test pulse
were adjusted to obtain a PS amplitude response which was 25 percent of the PS
amplitude at PS asymptote. B) Test pulse responses obtained with condition and test
pulse stimulation through the stratum radiatum. The current intensities on both the
condition and test pulse were set to obtain a PS amplitude response which was 25
percent of the PS amplitude at PS asymptote.

NTTAU\.SC INTCWVAi. (uSCC)
400
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300
250
200
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100
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sucrose +
T 1 1 T 1 1 1
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iWTCAPULSC INTT*v*l. (WSCC)

122
There was a higher level of potentiation in the alcohol group at IPIs
between 20 and 150 msecs with the highest level of potentiation at 250
percent of control. The sucrose group was maximally potentiated to 225
percent of control. These differences were not, however, statistically
significant. The PS amplitude responses from the two groups were
virtually overlaid at IPIs from 200 to 400 msecs. The latency data for
this experiment (Fig 4-10B) showed the sucrose group had shorter laten
cies to PS onset than did the alcohol group. The sucrose group laten
cies were up to .7 msec faster than control responses while the alcohol
group was only up to .4 msec faster than their control responses. Both
group latencies were shortest at IPIs of 40 to 60 msecs.
The data for the next experiment were obtained with 0/0 stimulation
through stratum oriens instead of through stratum radiatum. The PS
amplitude data (Fig 4-9A) for the alcohol group were potentiated to
levels of 250 to 300 percent of control at IPIs from 20 to 150 msecs.
These responses returned to control levels beyond the 400 msecs IPI.
The PS amplitude responses for the sucrose group were maximally
potentiated to 200 percent of control at an IPI of 80 msecs. The two
groups remained approximately 50 percentage points away from each other
for IPIs from 20 to 400 msecs. While the differences were not
significant it should be noted that the differences between the alcohol
and sucrose groups with SO stimulation tended to be greater than with SR
stimulation. The PS latency data are shown in Fig 4-10A. The alcohol
group had shorter latencies to the PS onset although the differences
between the alcohol and sucrose groups did not quite reach a
statistically significant level (F(l,17) 3.36) p .083. The alcohol
group had latencies .6 to .8 msec shorter than control responses while

Figure 4-10. Homosynaptic 0/0 paired pulse PS latency responses (means). Note that these
experiments were conducted through two different sets of afferent fibers. All
test responses are shown as a difference from control responses (test response
- control response in msecs). A) Test pulse responses obtained with condition
and test pulse stimulation through the stratum oriens. The current intensities
on both the condition and test pulse were adjusted to obtain a PS amplitude
response which was 25 percent of the PS amplitude at PS asymptote. B) Test
pulse responses obtained with condition and test pulse stimulation through the
stratum radiatum. The current intensities on both the condition and test pulse
were set to obtain a PS amplitude response which was 25 percent of the PS
amplitude at PS asymptote.

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KTtKPULSC KTTXv'AA. (LTSCC)

125
the sucrose group latencies were only .4 msec shorter than their control
responses. Both groups were close to baseline levels by the 400 msecs
IPI.
0/0 heterosvnantic paired-pulse paradigms -- Set 2
The heterosynaptic 0/0 paired pulse responses are shown in Fig
4-11B and 4-11C. Both experiments utilized condition and test pulse
current intensities set at 25 percent of the PS amplitude at PS asymp
tote. The general pattern of responses to the heterosynaptic stimula
tion was different from that using homosynaptic 0/0 stimulation. The
pattern of responding was more similar to A/0 experiments than to
homosynaptic 0/0 experiments. There was an intial, maximal level of
inhibition to 40 percent of the control response at the 20 msecs IPI
followed by a return to control levels by 60 to 80 msecs IPIs.
The PS amplitude responses for the SR/S0 heterosynaptic 0/0 paired
pulse experiment are shown in Fig 4-11B. Although the difference was
small and not statistically significant, the alcohol group was less
inhibited than was the sucrose group at IPIs from 20 to 200 msecs. The
alcohol group was inhibited 45 to 50 percent of the control response at
an IPI of 20 msecs, while the sucrose group was inhibited 30 to 35
percent at the 20 msecs IPI. Although the alcohol group returned to
control levels by the 60 msecs IPI, the sucrose group remained inhibited
to the 200 msecs IPI. The latency to PS onset data are shown in Fig
4-12B. The alcohol group latency was up to .1 msec longer than the
control response at IPIs from 20 to 50 msecs after which it became
shorter than the control response by up to .2 msec. The alcohol group
PS latency measures remained shorter than control responses beyond 400
msecs IPIs. The sucrose group latencies were consistently longer than

Figure 4-11. A/O and O/O Heterosynaptic paired pulse PS amplitude responses (means + SEM).
Note the individual configurations described below. All test responses are
shown as percent changes from control responses (test response/control response
X 100). A) Test pulse responses obtained with antidromic stimulation from the
alveus on the condition pulse and orthodromic stimulation from the stratum
radiatum on the test pulse. The condition pulse current intensity was adjusted
to obtain the maximum (100 percent) sized antidromic spike. The test pulse was
adjusted to 25 percent of the PS amplitude response at PS asymptote. B) Test
pulse responses obtained with condition pulse stimulation through stratum
radiatum and test pulse stimulation through stratum oriens. The condition and
test pulse current intensities were set to obtain a PS amplitude response 25
percent of the PS amplitude at PS asymptote. C) Test pulse responses obtained
with condition pulse stimulation through the stratum oriens and test pulse
stimulation through the stratum radiatum. The condition and test pulse current
intensities were set to obtain a PS amplitude response 25 percent of the PS
amplitude at PS asymptote.

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r\j
3

Figure 4-12. A/O and O/O Heterosynaptic paired pulse PS latency responses (means). Note the
individual configurations described below. All test responses are shown as
differences from control responses (test response control response in msecs).
A) Test pulse responses obtained with antidromic stimulation from the alveus on
the condition pulse and orthodromic stimulation from the stratum radiatum on
the test pulse. The condition pulse current intensity was adjusted to obtain
the maximum (100 percent) sized antidromic spike. The test pulse was adjusted
to 25 percent of the PS amplitude response at PS asymptote. B) Test pulse
responses obtained with condition pulse stimulation through stratum radiatum
and test pulse stimulation through stratum oriens. The condition and test pulse
current intensities were set to obtain a PS amplitude response 25 percent of
the PS amplitude at PS asymptote. C) Test pulse responses obtained with
condition pulse stimulation through the stratum oriens and test pulse
stimulation through the stratum radiatum. The condition and test pulse current
intensities were set to obtain a PS amplitude response 25 percent of the PS
amplitude at PS asymptote.

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iKTCT^ULiC IKTT^Ai. (USCC)
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130
the control responses by up to .3 msec. The group by IPI interaction
statistic for the PS latency data was significant (F(1,17) 1.93) p =
.01. Subsequent T-test analyses of the individual IPIs for the two
groups showed significant effects as shown with asterisks. The PS
amplitude responses for the SO/SR heterosynaptic experiment are shown in
Fig 4-11C. There were no consistent trends in the data for differences
between the alcohol and sucrose groups, however. The latency data for
this configuration are shown in Fig 4-12C. There was an increase in the
time to PS maximum for both groups by up to .3 msec which was maximal
between 30 and 40 msecs IPIs. The latencies returned to control levels
at longer IPIs. No significant differences between groups were found
however.
A/0 paired pulse paradigm
The final experimental paradigm of this series utilized A/0 stimu
lation with 100 percent of the AS maximum on the condition pulse and 25
percent of the PS amplitude at PS asymptote on the test pulse. The PS
amplitude data (Fig 4-11A) exhibited the normal antidromic response
pattern. There was strong inhibition of the PS response amplitude at
short IPIs (20 to 100 msecs) followed by a gradual return to control
levels. The alcohol group was maximally inhibited to 55 percent of the
control response while the sucrose group was inhibited to 25 percent of
its respective control response. This difference (though not signifi
cant) between the alcohol and sucrose groups was maintained for IPIs
beyond 400 msecs. The PS latency data for this experiment are shown in
Fig 4-12A. An increase in PS latency by up to .4 msec was seen for both
the alcohol and sucrose groups followed by a slow return toward baseline
levels.

131
Discussion
A nutritionally complete liquid diet was used to produce an animal
model of long term alcohol exposure. This diet has been shown to
produce alterations in the behavior (Walker and Hunter, 1978; Fehr et
al., 1975), anatomy (Walker et al., 1980; Riley and Walker, 1978; King,
1984), physiology (Abraham et al., 1981, 1982, 1984; Durand and Carien,
1984a, 1984b) and biochemistry (Freund, 1980) of animals given similar
durations of ethanol exposure. Electrophysiological evidence from
extracellular studies have shown that the basic characteristics of
single pulse stimulation or of LTP were not changed in CET animals.
However, the PS amplitude was shown to be enhanced to paired pulse or
repetitive stimuli at 5 and 10 Hz without changes in the EPSP (Abraham
et al., 1981). These findings have since been corroborated by intra
cellular recordings from CAl pyramidal cells where it was shown that
only the IPSP amplitude and the late after-hyperpolarization were
reduced in CET animals (Durand and Carien, 1984a). The present in vitro
homosynaptic paired pulse results showed the CET animals to have trends
toward higher levels of potentiation than the sucrose animals. The
nature of these trends were similar to the results found by Abraham et
al. (1981).
The present in vitro paired pulse PS amplitude inhibition data are
not directly comparable to results from similar experimental manipula
tions performed in vivo since there was little inhibition observed in
vitro with homosynaptic stimulation. This difference in the level of
inhibition found between in vivo and in vitro preparations has been
described previously and is thought to result from the severing of the
longitudinal recurrent inhibitory interneuron fiber projections

132
(Dunwiddie et al., 1980; Teyler, 1980). It has, however, been
suggested that the in vitro data may actually be more similar to
non-anesthetized, normal animals than it is usually considered to be
(Dunwiddie, personal communication) since in vivo experiments typically
use barbiturate anesthesia which has been shown to increase the efficacy
of inhibitory systems (Nicoll et al., 1975). With barbiturate
anesthesia, the inhibitory systems are probably more effective than in
the unanesthetized, freely moving animal. Only with the current inten
sity of 50 percent of the PS amplitude at PS asymptote on both the
condition and test pulse of homosynaptic paired pulse stimulation, and
only in the sucrose animals, was any inhibition seen. This finding can
be explained by two independent explanations. First, the sucrose group
would be expected to exhibit more inhibition than the CET group since
CET has been shown to produce trends toward increased PS amplitudes (a
result of decreased inhibition) for nearly all of the present paired
pulse potentiation series. Secondly, if, as proposed in Chapter 3, the
intensity of the test pulse recruits more feedforward interneurons to
fire, hence more inhibition, it is consistent that in this case the
higher test pulse current intensity would be the most likely to show
inhibition in vitro. The inhibition seen here would not, however, be
feedforward alone but a combination of recurrent and feedforward inhibi
tion since the inhibition is seen at short IPIs.
The patterns of paired pulse potentiation were similar between the
in vivo and in vitro preparations except that the IPI at which the
maximal level of potentiation was reached was shorter in vitro. The in
vitro responses were maximally potentiated at 30 to 300 msecs whereas
the in vivo data did not reach comparable levels of potentiation until

133
IPIs of 150 or 200 msecs. The higher test pulse current intensity
resulted in lower levels of potentiation as was seen with the in vivo
results reported earlier. The peak levels of potentiation for the
current intensity of 25 percent of the PS amplitude at PS asymptote on
the in vitro test pulse were over 200 percent of control. The test
pulse current intensity of 50 percent of the PS amplitude at PS asymp
tote with the in vitro preparation was under 200 percent potentiation.
The SO homosynaptic paired pulse PS response amplitudes from
control animals showed a similar pattern of potentiation to those
obtained with SR homosynaptic stimulation. Potentiation occurred
abruptly but not until after the 30 msecs IPI. The two stimulation
sites resulted in the peak levels of potentiation occurring by 100 msecs
IPIs. There were two notable differences however. SR stimulation
resulted in a marginally greater level of potentiation but also in
creased to the maximal level more quickly than the SO stimulation site
responses. It is possible that more inhibition resulted from the SO
stimulation site than the SR site since it has been suggested that
commissural afferents may preferentially synapse onto inhibitory
interneurons (Buzsaki, 1984). Furthermore, the SO region has been shown
to have a greater autoradiographic grain count density than SR from
fibers originating in the contralateral CA3 region (Swanson et al.,
1978).
The A/0 paired pulse experiments performed in vitro were very
similar to those performed in vivo. There was an initial, sharp in
crease in the level of inhibition at IPIs of 20 to 30 msecs followed by
a decay phase which, if the current intensity on the condition and test
pulse was high, lasted beyond the range of IPIs tested in vitro but
which returned toward control levels by IPIs of 1000 to 2000 msecs in

134
vivo. The maximal levels of inhibition found in both the in vivo and in
vitro experiments were at about 20 to 40 percent of the control
response. These findings suggest that there may not be substantial
differences in the responses of recurrent interneurons between in vivo
and in vitro preparations as has been suggested. Instead, it may be
that there is a decrease in the efficacy of feedforward inhibition or
that there is an increased excitability of the pyramidal cells in vitro.
Heterosynaptic paired pulse stimulation provided an additional way
in which to observe the effects of orthodromic paired pulse stimulation
without the effects of paired pulse EPSP facilitation or PS potentiation
superimposed. The pattern as well as the levels of heterosynaptic
paired pulse stimulation are very similar to those found with A/0
stimulation. The major difference between heterosynaptic orthodromic
stimulation and A/0 stimulation is that heterosynaptic stimulation
should set up feedforward inhibition as well as recurrent inhibition on
the condition pulse while A/0 stimulation should predominantly activate
recurrent inhibition. The maximal level of inhibition would therefore
be expected to be greater and for a longer duration with heterosynaptic
stimulation. Haas and Rose (1982) showed that with the in vitro hippo
campal preparation, the levels of inhibition with different stimulation
configurations, fell into an ordered progression. They found that A/0
stimulation produced the least amount and shortest level of inhibition.
Homosynaptic 0/0 stimulation produced more inhibition and for a longer
duration than A/0 stimulation and that heterosynaptic 0/0 stimulation
produced the greatest amount of inhibition and lasted for the longest
period of time. The present results did not follow this pattern.
Instead, A/0 and heterosynaptic 0/0 stimulation produced comparable
levels of inhibition in the present hippocampal slice preparation.

135
The 0/0 homosynaptic stimulation produced little if any inhibition.
The 0/0 heterosynaptic stimulation paradigms differed in that Haas and
Rose (1982) stimulated two different areas of the SR afferents, whereas
in the present experiments, the SR or SO region was stimulated with the
condition pulse followed by the SO or SR region with the test pulse,
respectively. This difference in the heterosynaptic stimulation para
digm could account for part of the discrepant results. A second
possible difference may be in the stimulation currents used by Haas and
Rose (1982). They used current levels set to 50 percent of the maximum
sized response which would be similar to the present 50 percent of the
PS amplitude at PS asymptote. A final difference and the one which may
provide the best explanation for the discrepancy between the two sets of
data across each of the different stimulation configurations is that
they maintained the bathing medium temperature at 32 to 33.5 degrees
centigrade whereas the present slices were kept at a temperature closer
to 35 degrees. It has been shown that small differences in temperature
can markedly affect the excitability of the hippocampal slice prepara
tion (Teyler, 1980). It should be noted that the results of Haas and
Rose (1982) are different from the results of other investigators in
which the slice technique was used. They showed levels and durations of
0/0 homosynaptic inhibition which were very similar to in vivo results.
Other papers using similar paradigms with the slice technique showed
periods of inhibition extending to 40 or 50 msecs at best (Dunwiddie et
al. 1980). The difference in bathing temperature probably best
explains this difference.
The SR/S0 heterosynaptic experiment, coupled with that of the
homosynaptic SO stimulation data suggest that SO stimulation may result

136
in larger inhibitory effects than SR stimulation. This may be true
since the majority of recurrent inhibitory interneurons appear to reside
in SO or the pyramidal cell layer (Andersen et al., 1964a; Lorento de
No, 1934). It has been suggested that commissural afferents from the
CA3 region may preferentially synapse on CAl feedforward inhibitory
interneurons (Buzsaki, 1984). It has also been demonstrated that the SO
region contains a higher density of CA3 originating commissural fibers
than the SR regions (Swanson et al., 1978). Thus, the SO region may be
an important area for both feedforward and recurrent types of
inhibition.
The use of an antidromic/orthodromic paired pulse paradigm was
aimed at more directly observing the effect of CET on recurrent inhibi
tion. Two different stimulation levels were used to assess the recur
rent inhibitory system, one relatively low and the other relatively
high. No statistically significant differences were found with the
lower condition and test pulse current intensities between groups
although the trend was that the alcohol animals were slightly less
inhibited than the controls. The higher condition and test pulse
current intensities used with A/0 stimulation resulted in significant
differences on the PS amplitude measure between the alcohol and sucrose
groups. The alcohol group, in this case, was initially inhibited to
levels comparable to the sucrose groups at IPIs shorter than 40 msecs.
The maximum levels of inhibition observed at these short IPIs were
similar in both the low and high intensity stimulation conditions. The
nature of the difference between the CET and sucrose groups was that the
alcohol group returned to control levels by the 80 msecs IPI while the
sucrose group remained inhibited to 70 or 80 percent of the control
responses throughout the IPIs tested.

137
It has been suggested that inhibitory interneurons may respond with
different firing activity depending on the current intensity used
(Ashwood et al., 1984). At lower current intensities, inhibitory
interneurons tended to fire more than one action potential with frequent
bursts to stimulation which was above the PS threshold. Another group
of inhibitory interneurons produced only one action potential even when
stimuli were up to 4.5 times threshold. The present results suggested
that firing to lower stimulation current intensities may not be altered
(since the lower current intensity A/0 paired pulse series CET and
sucrose groups both returned to baseline levels by 150 msecs IPIs).
Higher current intensity stimulation on the condition and test pulse,
which may result in longer duration firing, may be altered in CET
animals. Alternatively, CET may reduce the efficacy of feedforward or
recurrent inhibitory interneurons such that transmitter depletion occurs
rapidly (Andersen et al., 1982).
Although there were no statistically significant differences
between the alcohol and sucrose responses with homosynaptic 0/0 stimula
tion, the trend toward greater paired pulse potentiation from CET
animals occurred consistently. The PS latency responses revealed trends
which supported those found with the PS amplitude responses. In
general, there was a decrease in PS latency when the CET animals were
compared to the sucrose-fed controls.
The heterosynaptic paired pulse experiments did not reveal signifi
cant differences between the CET and sucrose groups on the PS amplitude
responses. However, the trend for the CET animals to be less inhibited
than the sucrose animals continued. The PS latency responses at several
of the IPIs for the CET animals were shown to be significantly different

138
from the controls. These PS latency differences further support the
trend for a decrease in the efficacy of inhibition in the CAl pyramidal
cell region.
The current studies using homosynaptic paired pulses and CET
animals were performed with the in vitro preparation and several paired
pulse configurations to extend the findings from earlier studies. The
in vitro preparation was used in an attempt to eliminate any possibility
of an interaction between CET effects and urethane anesthesia which had
been used previously, since it is known that alcohol and the barbitu
rates are cross tolerant (Kalant et al., 1971). The likelihood that
cross tolerant effects existed in previous studies from our lab,
however, were minimal since the animals had been off of the alcohol diet
for at least eight weeks prior to acute electrophysiological testing.
The present results suggest that, while there are considerable
differences in the response patterns observed in vivo versus those
obtained in vitro. urethane anesthesia did not result in any anesthesia
dependent differences between the CET and sucrose-fed controls. The
results obtained in vitro continue to support the previous observation
that CET results in increased PS amplitude responses (Abraham et al.,
1981).
Morphological data from CET animals have shown significant
decreases in pyramidal and granule cell number (Walker et al., 1980).
These CET induced effects may extend to the inhibitory interneurons,
which have not been carefully studied after CET, as well as to the
primary cell types of the hippocampus. It is probable that the
interneurons are at least as, or more prone to functional alterations or
injury and death due to various treatment conditions. It has been shown
that CET altered blood flow to various parts of the brain (Newlin,

139
1982). Furthermore, hypoxia has been shown to deleteriously affect
inhibitory interneurons as measured by the paired pulse technique
(Dunwiddie, 1981). Thus, by direct CET induced effects on the
inhibitory interneurons or by CET altering the regional blood flow, it
is possible that the inhibitory interneuronal population is reduced in
number as were the primary cell types in the hippocampus. Since
recurrent inhibitory interneurons are known to project widely, the loss
of only a small percentage of these interneurons may have profound
effects (Walker et al., 1980). If CET produces substantial effects on
feedforward inhibition, it is possible that long, myelinated fibers may
be deleteriously affected by CET as has been shown previously (Dreyfus,
1974).
The present in vitro paired pulse experiments have extended earlier
extracellular studies showing that CET resulted in an increase in the
size of the PS amplitude with paired pulse stimulation. Although there
were no statistically significant differences between CET and sucrose
animals with 0/0 homosynaptic paired pulse stimulation, the CET animals
consistently tended to have increased PS amplitudes. Heterosynaptic
paired pulse stimulation resulted in more equivocal results. However,
the trend was for the CET animals to show less inhibition than sucrose
controls. Finally, A/0 stimulation resulted in significantly reduced
levels of inhibition in CET animals when high condition and test pulse
current intensities were used. Lower current intensities resulted in
CET animals exhibiting trends toward lower levels of inhibition.
The current results continue to support the hypothesis that CET
produces a decrease in the level of recurrent inhibition as suggested by
Abraham et al. (1981). However, it may be that paired pulse

140
potentiation is more directly influenced by feedforward inhibition as
suggested by the differences seen in the maximal levels of potentiation
with different test pulse current intensities both in vivo and in vitro.
It is therefore possible that the A/0 response differences between
groups, seen with the higher current intensities at the longer IPIs, may
reflect a feedforward induced inhibition of the sucrose-fed control
response PS amplitude at longer IPIs. It may therefore be quite reason
able to suggest that CET may produce decreases in feedforward inhibition
to at least the same extent as for recurrent inhibition. Most generally,
these data indicate that CET produces decreased inhibition (both recurrent
and feedforward) in the CA1 region of the hippocampus. Systematically,
examining the inhibitory systems of the CAl region, through localized
pharmacological challenges, may provide further definitions concerning
the effect of CET on synaptic inhibition in the hippocampus and poten
tially throughout the brain.

CHAPTER 5
EFFECT OF CET ON IONTOPHORETIC GABA MEDIATED RESPONSES
Introduction
The effect of CET on hippocampal electrophysiology has now been
shown repeatedly and in a number of different ways. It has been shown
that when using paired pulse stimulation through one electrode located
in the SR, there is an increase in the amplitude of the PS of CET
animals when compared to sucrose-fed controls (Abraham et al., 1981).
Abraham et al., (1981) hypothesized that the increase in PS amplitude
without changes in the response of the normal synaptic waveform, EPSP or
PS thresholds or I/O functions was due to a decrease in recurrent
inhibition. This hypothesis was given added support from intracellularly
recorded data where it was shown that the only measures that changed in
CET animals were decreased amplitudes of the IPSP and the late after
hyperpolarization in responses from CAl pyramidal cell (Durand and
Carien, 1984a). Results from the previous set of studies in this
manuscript also add support to this hypothesis with the finding of
similar trends for the 0/0 SR data and the extension of those findings
to the SO with 0/0 paired pulse experiments. The A/0 findings where
the condition pulse was delivered to the alvear fibers directly in
order to fire the recurrent inhibitory interneuron, followed by an
orthodromic pulse to the SR afferents, also provided support for the
hypothesis that CET decreased the effect of inhibition in CAl. The
A/0 data showed the CET animals to be inhibited to a significantly
lesser extent than the sucrose-fed control animals. This A/0 testing
141

142
procedure was an attempt to directly address whether or not CET
produced alterations in recurrent inhibition. Thus, the hypothesis
concerning decreases in recurrent inhibition as a result of CET
appears to be supported.
While it is apparent that CET produces an effect on recurrent
inhibition, it is probable that CET also affects feedforward inhibition.
Effects on feedforward inhibition have not been easy to dissociate using
the paired pulse technique since feedforward inhibition cannot be
activated without also activating recurrent inhibition and hence con
founding the influence of feedforward inhibition. However, it appears
that the contribution of recurrent versus feedforward inhibition may be
dependent upon the intensity of the condition and test pulse of paired
pulses (see Chapter 3 discussion). In order to better address the
question of feedforward versus recurrent inhibition, other experimental
approaches might provide a better dissociation than does the paired
pulse technique.
Since CET produces deleterious effects on recurrent and feedforward
inhibition in the CAl region of the hippocampus and the inhibitory
mechanisms of this region are GABA mediated, a localized pharmacological
approach to the inhibition question might prove to be helpful. The
application of GABA at discrete locations along the dendritic axis of
the CAl pyramidal cell would provide for both, an indication of the
effect of CET on direct GABAergic modifications of PS responses and
whether the effect of CET on GABA mediated inhibition is greater at the
cell body layer or out in the dendrites. Andersen et al., (1982) used
such a technique which they suggested allowed for the extracellular
localization of recurrent and feedforward (or discriminative) inhibition.

143
The protocol that Andersen et al., (1982) used was as follows. Two
different stimulating electrodes were placed in both the SO and the SR
afferent zones of the CAl region. The recording electrode was placed in
the pyramidal cell region so that a PS was obtained from stimulation of
either the SO or the SR afferents. A micropipette containing GABA was
successively placed at 10 different locations along the dendritic axis
of the pyramidal cell (see Fig 5-1). A control response using afferent
stimulation from one of the dendritic zones of CAl pyramidal cells was
produced followed by an iontophoretic pulse of GABA after which a second
stimulus (test pulse) was delivered through the same dendritic zone.
The same procedure was then performed using the other stimulating
electrode until all 10 GABA application sites had been tested. The test
pulse after the iontophoresis of GABA was then reported as a percent of
the control pulse obtained 10 seconds before the GABA application. The
results showed two different patterns of responses dependent upon the
stimulation site. The SR stimulation electrode caused a potentiation of
the response when the GABA was applied in the distal SO region. This
potentiation changed to inhibition of the test response when located
more proximal to the pyramidal cell body layer but was still located on
the SO side of the pyramidal cell layer. The inhibition which began in
proximal SO then continued into the SR dendrites approximately 200
microns before it returned toward control levels near the SLM. The
response profile obtained with SO stimulation showed inhibition when
GABA was applied in the SO region which continued through the pyramidal
cell layer. However, within 25 to 50 uMs distal to the pyramidal cell
layer in the proximal SR, there was a decrease in inhibition followed by
a prolonged area of slight potentiation when GABA was applied in the SR

Figure 5-1. GABA iontophoresis sites along the dendritic axis
of CA1 pyramidal cells. Note that recordings
were of field responses and that the GABA effects
were upon groups of pyramidal cells rather than
single cells as depicted. GABA (1M, pH 3.5) was
ejected for five seconds with 40 nAs of current.
Abbreviations: ALV, alveus; SO, stratum oriens;
SP, stratum pyramidale; SR, stratum radiatum;
SLM, stratum lacunosum-moleculare.

145
ALV
LM

146
region. Andersen et al., (1982) suggested that the responses seen with
SR stimulation reflected feedforward-like inhibition in the SR region
with recurrent inhibition found near the pyramidal cell layer region.
Stimulation with the SO electrode produced what may be SO-induced
feedforward inhibition but may also reflect a large influence of recur
rent inhibition since it has been reported that the recurrent inhibitory
interneurons are located in the pyramidal and proximal SO regions
(Andersen et al., 1964a). SR stimulation probably activates SR feed
forward inhibition while SO activation probably activates SO feedforward
inhibition. Since stimulation of the SO or SR dendritic region does not
apparently activate the recurrent inhibitory mechanisms, SO or SR
stimulation may allow for a relatively clean assessment of SR or SO
feedforward inhibitory effects, but as a function of where GABA is
applied along the dendrites. GABA iontophoresis will activate recurrent
inhibitory interneurons, however SO or SR stimulation alone will not
result in the effects of recurrent inhibition being observed. This
paradigm may therefore allow for a relatively selective investigation of
feedforward and recurrent inhibition along the dendrites of the CAl
pyramidal neuron. In CET animals, it would provide the opportunity to
selectively observe feedforward (in the SR region) and recurrent (in the
cell body region) inhibition in terms of the response to GABA
application on the basis of both the magnitude of the PS response and
the pattern of the PS response. This next experiment utilized this
paradigm.

147
Methods
Animals
The animals used in this experiment were the same as those used in
the second set of animals in the previous experiments and treated as
detailed in the general methods chapter.
Equipment
The equipment used for this experiment was the same as that used
in the previous in vitro experiment. The only major additional piece
of equipment was the Dagan model 6400 iontophoresis system.
Procedures
The treatment of the animals and the preparation and maintenance of
the slice were accomplished as described in the previous experiment and
the general methods sections.
The first data to be collected were I/O curves of the pyramidal
cell responses first with stimulation from the SR and then from the SO
afferents.
GABA was prepared so that a final 1M concentration was obtained.
2.062 gms of GABA were mixed with 20 mis of water with HC1 to achieve
the 1M concentration at a pH of approximately 3.5. This pH level
provided the necessary electrical charge carriers in order to eject the
GABA from the iontophoresis micropipette. New solutions of GABA were
prepared at least weekly. GABA was always refrigerated except when
filling the pipettes.
The protocol for this experiment was modeled after that of Andersen
et al., (1982). The recording electrode was placed in the pyramidal

148
cell layer so that good responses were obtained with both SO and SR
stimulation. In the event that a good response (one having a positive
going visible EPSP) could not be obtained, the recording electrode was
placed so that the response with SR stimulation was optimized for the
EPSP with a superimposed PS. For this reason, as well as previously
discussed reasons, the slope data of the SO generated data were not
analyzed. One stimulating electrode was placed in the SR at the CA1-CA2
border while the other stimulating electrode was placed in the SO
afferents. Every effort was made to restrict the stimulating electrodes
from encroaching on the pyramidal cell body layer or onto the alvear
fibers.
The placement of the GABA iontophoresis micropipette at 10
different locations along the CA1 pyramidal dendrites was obtained as
follows. Four different application locations were used in the SO
dendritic region while six application sites were selected in the SR
region (see Fig 5-1). The first application of GABA was made at the
pyramidal cell layer as close to the recording electrode from the SO
side as possible. The next three applications were made by observing
the distance from the recording electrode to the beginning of the alvear
fibers and dividing that region of the SO into three equal distances by
visualization. The same procedure was used in the SR region where the
first application site was as close to the recording electrode as
possible from the SR side. The distance from the recording electrode to
the fissure was then divided into five equal segments by visualization.
This method provided a way by which different distances between the
noted landmarks could be divided such that the same relative region
between different slices and different animals could be controlled. The

149
order of application was the same in all animals. The first application
was at the SO side of the recording electrode working out toward the
alveus and then from the SR side of the recording electrode working out
toward the hippocampal fissure.
The protocol for stimulation and application was as follows. After
the recording and stimulating electrodes had been satisfactorily placed,
the GABA micropipette was situated at the SO side of the recording
electrode. The stimulation current was adjusted prior to lowering the
GABA pipette into the slice so that a response approximately 25 percent
of the PS amplitude at PS asymptote was obtained. The GABA electrode
was then lowered and adjusted in a medial-lateral as well as dorsal-
ventral direction until a maximum level of inhibition was obtained for
the given ejection site with the same GABA ejection current and duration
used in the final procedure. Once all of the adjustments were made, a
control response was obtained by stimulating the SR afferents. Ten
seconds later, GABA was ejected with a cationic current of 40 nAs for 5
seconds. A 5 nA retaining current was used whenever the GABA was not
being ejected. At the end of the 5 second ejection period, another
stimulation pulse was administered to test for the effects of the GABA
administration. These parameters were used since they produce a clear
response change with a relatively quick return to control levels. A
minimum 30 secs delay period was used before a second set of responses
were collected at the same location using exactly the same configura
tion. This second set of responses was then averaged with the first set
and stored. With the iontophoresis pipette still in the same location,
the SO stimulation electrode configuration was tested in the same manner
as described for the SR configuration. Thus, two applications of GABA

150
were tested at each GABA iontophoretic site with both SR and SO stimula
tion. In all cases, at least 30 seconds elapsed before additional GABA
ejections were administered. Once both SR and SO stimulation were
accomplished with the same GABA ejection site, the GABA pipette was
moved and the procedures described above for finding the location of
maximal inhibition were initiated again. The SR and SO stimulations
were carried out and the GABA pipette moved until all 10 locations along
the dendrite were tested. The testing procedure lasted approximately 45
mins to one hr.
Results
The GABA PS results presented below were based on a total sample of
35 rats with 19 rats in the alcohol group and 16 in the sucrose group.
SR I/O relationships. The I/O curves shown in Fig 5-2 and Fig
5-3 represent the overall responses from all of the animals used in
this and the subsequent pharmacological experiments with SR and SO
stimulation, respectively. Since these I/O curves were obtained prior
to any pharmacological testing with both SR and SO stimulation,
pooling the data represented a resource saving measure as well as
providing a stronger statement concerning the I/O measures. There are
29 sucrose and 39 alcohol animals represented in each of the I/O
curves. All subsequent experiments will therefore refer to these I/O
curve data. Chronic alcohol administration did not produce
statistically significant changes in the PS amplitude nor in the PS
latency responses to single pulse stimulation with SR (Fig 5-2)
stimulation. The PS threshold was 134 5.07 and 139.65 8.5 for the
alcohol and sucrose-fed control groups, respectively.

Figure 5-2. I/O curves (means) obtained with stimulation from
the stratum radiatum. Data were normalized by
setting the current level at which the PS was
first seen equal to 0 uAs. Data were then
interpolated from that current value such that
interpolated I/O curves were produced with
current levels between zero and 500 uAs. This
procedure controlled for any differences in PS
threshold between groups. A) PS amplitude
responses with stratum radiatum stimulation
normalized between groups. B) PS latency
responses with stratum radiatum stimulation
normalized between groups.

RAW DATA (MSEC) RAW DATA (MV)
152
o 200 400
CURRENT INTENSITY (UAMPS)

Figure 5-3. I/O curves (means) obtained with stimulation from
the stratum oriens. Data were normalized by
setting the current level at which the PS was
first seen equal to 0 uAs. Data were then
interpolated from that current value such that
interpolated I/O curves were produced with
current levels between zero and 500 uAs. This
procedure controlled for any differences in PS
threshold between groups. A) PS amplitude
responses with stratum radiatum stimulation
normalized between groups. B) PS latency
responses with stratum radiatum stimulation
normalized between groups.

RAW DATA (MSFC) RAW DATA (MV)
154
CURRENT INTENSITY (UAMPS)

Figure 5-4.
PS amplitude responses (mean + SEM) obtained
after GABA iontophoresis at ten different loca
tions along the dendritic axis of CA1 pyamidal
cells. PS amplitude responses are expressed as a
percent of the control response obtained immedi
ately prior to the iontophoresis of GABA at each
location indicated (GABA (test) response/ control
response X 100). A) GABA mediated PS amplitude
responses obtained with stimulation from the
stratum radiatum. B) GABA mediated PS amplitude
responses obtained with stimulation from the
stratum oriens.

percent change from control percent change from control
156
iontophoretic location

157
GABA iontophoresis with SR stimulation. The effects of CET on
the responses to GABA iontophoresis at ten different locations along
the pyramidal cell dendritic tree are seen in Fig 5-4A. Although none
of the differences were statistically significant, it can be seen that
the CET group mean percent of control PS amplitude measures were more
inhibited in the pyramidal cell region and SR and less potentiated
than sucrose-fed control responses in the SO region. There was a
difference between the two groups of 25 to 40 percentage points for
those locations. The PS latency data (Fig 5-5A) were not statistically
different for the group effect but were for the group by IPI interaction
(F(l,226)-2.13) p>.028. The locations which proved to be statistically
different using follow up T-tests are noted by asterisks.
SO I/O relationships. Long-term ethanol administration did not
result in any significant differences on either the PS amplitude (Fig
5-3a) or PS latency (Fig 5-3b) measures. The PS threshold for the
alcohol group was 124.94 + 24.48 and was 93.57 + 4.49 for the sucrose
group.
GABA iontophoresis with SO stimulation. The effect of CET on the
response to GABA iontophoresis at the ten locations along the
pyramidal cell dendrites using SO stimulation are seen in Fig 5-4B.
In this experiment, the CET responses were more inhibited in the SO
dendritic region and less potentiated in the SR dendritic region than
the sucrose controls. This pattern of responses was very different
from the responses obtained with SR stimulation. With SO stimulation,
responses were only inhibited in the SO region very close to the

Figure 5-5. PS latency responses (means) obtained after GABA
iontophoresis at ten different locations along
the dendritic axis of CA1 pyamidal cells. GABA
mediated PS latency responses are expressed as a
difference from the control response obtained
immediately prior to the iontophoresis of GABA at
each location indicated (GABA (test) response -
control response in msecs). A) GABA mediated PS
latency responses obtained with stimulation from
the stratum radiatum. B) GABA mediated PS
latency responses obtained with stimulation from
the stratum oriens.

difference from control difference from control
159
iontophoretic location
iontophoretic location

160
pyramidal cell body. All of the responses recorded when GABA was
ejected at SR sites were potentiated relative to the control response.
The CET group PS amplitude responses were not different from the
sucrose animals, but the locations where maximum differences were seen
were in distal (to the cell body) SO and distal SR. The PS latency
data are seen in Fig 5-5B. The differences between the alcohol and
sucrose groups were statistically different (F(l,33) 4.19), p =
.048. The PS latencies for the alcohol and sucrose groups were both
shorter than the control responses. The alcohol group latencies to
the PS maximum were slower than the sucrose group as would be expected
if the alcohol group were more inhibited or less potentiated than the
sucrose group. Since decreased PS latency responses have been considered
to be a reflection of increased pyramidal cell excitability, these
differences further support the trend of results with the PS amplitude
measures.
Discussion
The effect of 20 weeks of ethanol exposure was studied on the
electrophysiology of GABA mediated responses in the CA1 region of the
rat hippocampus. The transverse hippocampal slice preparation was used
in order to eliminate any potential confounding due to anesthesia
related issues found in vivo. This issue was important since cross
tolerant effects between ethanol and barbiturates have been reported
(Kalant, 1970). In addition, the slice technique allowed for the
discrete application of GABA, the major inhibitory neurotransmitter in
the CAl pyramidal cell region.

161
The duration of exposure to ethanol used in this study has been
shown to result in a 15 to 20 percent loss of granule and pyramidal cell
number in the rat hippocampus (Walker et al., 1980). Decreased branching
of dendritic elements, as well as reduced numbers of dendritic spines in
CAl pyramidal cells of the mouse hippocampus, have also been reported
(Riley and Walker, 1978). Findings from the rat hippocampus have shown
trends toward decreased spine density in CAl immediately following
ethanol withdrawal (King, 1984). These results suggest that there is a
widespread deleterious alteration of hippocampal morphology following
CET. Electrophysiological characteristics of this region of the hippo
campus have also been shown to be altered by long term ethanol
treatment. Abraham et al. (1981) reported that the PS amplitude of
extracellularly evoked paired pulse responses was significantly in
creased in size compared to sucrose-fed controls. This increase in PS
amplitude occurred in the absence of changes in the basic waveforms,
EPSP and PS thresholds, I/O functions or LTP production. It was hypoth
esized that the increased PS amplitude was a function of decreased
recurrent inhibitory influences. Results from the investigations
reported earlier within this manuscript have provided additional evi
dence for CET producing increased PS amplitudes with paired pulse
testing in the CAl region of the hippocampus. The use of additional
paired pulse paradigms have further elucidated the potential contribu
tion of recurrent versus feedforward inhibitory processes to this
increased PS amplitude. While it appears that recurrent inhibition is
altered by CET, it also appears that deficits in feedforward inhibition
may be as profound as those in recurrent inhibitory circuits. This is
suggested by the observation that the greatest effects on the PS ampli-

162
tude with 0/0 stimulation were often found with IPIs below 100 msecs
where recurrent inhibition has its greatest effect extracellularly. At
intervals beyond 100 msecs, feedforward inhibition appears to produce
the more predominant inhibitory effect (Alger and Nicoll, 1982). CET
resulted in increased PS amplitudes over this entire range of IPIs.
There are a number of influences which mediate the characteristics
of the inhibitory systems in CAl. Increased PS amplitude or decreased
inhibition in CET animals could be explained simply by a decrease in the
number of inhibitory interneurons (Abraham et al., 1981). However, it
is also possible that changes in the efficacy of the GABAergic receptor
population contribute to this CET-induced effect. Alternatively, the
efficacy of other neuro-modulatory systems may also result in changes of
the inhibitory systems of the CAl region. The present experiment was
designed to assess directly, the effects of GABA application at 10
different locations along the dendritic axis of the CAl pyramidal cell.
The pattern of responses to GABA application for SO and SR stimula
tion are similar to Andersen et al. (1982) in several ways. SR stimula
tion produced the greatest region of inhibition with GABA iontophoresis
along the SR dendrites. The pattern of responses to GABA administration
in the present study with SR stimulation was generally parallel to that
reported by Andersen et al. (1982). There was an inhibited response
from distal SR through the pyramidal cell layer and into the proximal SO
region. Responses to GABA applied in distal SO were potentiated in both
this and in the study reported by Andersen et al., (1982). Responses to
GABA application in SLM were near control levels or potentiated in both
studies. SO stimulation with GABA iontophoresis along the dendrites
produced a different pattern of response from that obtained with SR

163
stimulation, but again was similar to Andersen et al. (1982). With SO
stimulation, the application of GABA to the proximal regions of SO and
SR, immediately surrounding the pyramidal cell layer, were the only
areas where inhibited or near inhibited responses were found. All other
sites of GABA application to the dendrites resulted in potentiated
responses in both studies.
The mechanism for the potentiation of the response to GABA in the
distal dendrites has not been well studied or discussed. However, it
has been shown that two different responses to GABA application at the
soma or to the dendrites occurs (Andersen et al., 1982; Alger and
Nicoll, 1982). The iontophoresis of GABA at the pyramidal cell results
in an hyperpolarization of the CAl pyramidal cell when recording intra-
cellularly. In the dendritic regions, especially in SR, a depolarizing
response has been found to GABA application. Both of these responses
are inhibitory. The depolarizing dendritic response has been shown to
result in an increased conductance and hence a shunt of the pyramidal
cell dendritic current. However, it has also been indicated that this
shunting response is localized to the point that areas surrounding the
"shunt" area may be more excitable. Andersen et al., (1982) has de
scribed this feedforward type of inhibition as a "discriminative inhibi
tion" since the effects of this type of inhibition may act as a selec
tive filter to afferent input, decreasing the input of one afferent
while accentuating those around it. The increased levels of potentia
tion seen in more distal regions of the pyramidal cell dendrites may
reflect the increased arborization of the dendritic elements, hence, an
inability to apply GABA to the main dendritic branches and thereby
increasing the probability of cellular discharge by decreasing the

164
threshold as a result of the depolarizing response to GABA. It has been
reported that two different types of receptors mediate the hyperpolar
ization and depolarization responses (Alger and Nicoll, 1982).
The major difference between the present results and those of
Andersen et al. (1982) centered on the magnitude of the PS amplitude
response at all locations along the dendritic axis. The present inves
tigation showed relatively little inhibition at those dendritic loca
tions where Andersen's group showed inhibition to 50 percent of the
control response. These results may be due to any of three different
aspects of the experimental protocol. First, the present experiments
were conducted using hippocampal slices from the rat. Andersen et al.
(1982) used guinea pigs. Thus, species differences may account
for some of the differences in the magnitude of the results. The second
difference, however, may better account for the differences seen between
the results of the two experiments. Andersen et al. (1982) collected
their test response during the application of GABA. The present find
ings were obtained 6 to 10 msecs after the GABA ejection current was
turned off. Thus, there may have been a period during which the re
sponse of the pyramidal cells to GABA changed. It was noted in the
present experiments that if, after GABA was ejected, additional test
pulses were given within 5 to 10 secs of the termination of GABA, there
was a marked reversal of the response to GABA. Whereas inhibition was
usually seen immediately after GABA ejection, a test pulse produced 5 to
10 secs after GABA termination resulted in a marked potentiation of the
PS (several hundred percent increase) response to GABA. This post-
GABAergic "fade" response has been described previously (Ben-Ari et al.,
1981, 1979; Rovira et al., 1984). Thus, it is hypothesized that the 6

165
to 10 msecs delay between the time when GABA iontophoresis was terminated
and when the test pulses were administered may have been of a sufficiently
long duration to begin altering the GABAergic response toward an enhanced
response. The third difference between experimental protocols which may
also help to explain the differences found is that Andersen et al.,
(1982) indicated that the temperature of the bathing medium at the slice
was between 32 and 33 degrees centigrade. As indicated earlier, temper
ature differences can significantly affect the excitability of the CAl
pyramidal cell as measured by extracellular PSs (Teyler, 1980). All
other parameters of this experiment were similar to those used by
Andersen et al. (1982).
The predominant effect of CET on GABA modulation of the PS along
the dendritic axis of the pyramidal cell with both SO and SR stimulation
was an apparent increase in the magnitude of inhibition when compared to
the sucrose-fed controls. The responses to GABA application were
inhibited at certain locations along the dendritic axis and potentiated
when compared to control responses at others. CET, however, resulted in
trends toward more inhibition or less potentiation at all locations
tested. The PS latency responses showed differences between the CET and
sucrose-fed controls with SO stimulation which lends additional support
to the findings with PS amplitude responses. These data suggest the
following mechanism; an increase in the efficacy of the GABAergic
receptors and a decreased release of GABA. The present CET response
pattern occurred even though the extracellular response to paired pulses
showed an impairment in the efficacy of the inhibitory interneurons. A
supersensitivity or increased number of GABAergic receptors would be
likely if there was a decreased afferent input to GABAergic receptors in

166
the CAI pyramidal cell. The up-regulation of GABAergic receptors could
be viewed as a compensatory response to the decreased input resulting
from inhibitory interneuronal cell death. This up-regulation, however,
must be opposed by some other process since it would be expected that
paired pulses stimulation would result in a smaller sized PS. A
decreased release of GABA or a loss of GABAergic afferent synapses could
result in such an up-regulation of GABAergic receptors.
Alternatively, the efficacy of other neuro-modulatory systems may
result in changes to the inhibitory systems of the CAl region. It has
been shown with immunocytochemical staining, that enkephalin containing
cells exist throughout the CAl region (Gall et al., 1981). The effect
of opiate peptides on extracellular CAl pyramidal cell responses is to
increase the amplitude of the PS. This increased PS amplitude is
thought to be due to a disinhibition of the inhibitory interneuron. The
CET induced increase of the PS amplitude may therefore represent an
alteration of the enkephalinergic interneurons. The increased response
to iontophoretic GABA application after CET may be related to this
alteration of opioid mechanisms.
The hippocampus has been shown to have the capability for anatomi
cal compensatory changes following CET. King (1984) showed that immedi
ately following 20 weeks of ethanol exposure, there was a trend toward a
relative decrease in spine density in CAl pyramidal cell dendrites
located in SO, SR proximal to the cell body, SR distal to the cell body,
and in the SIM. However, after 20 weeks of ethanol abstinence, there
was a recovery of CAl pyramidal cell spine density in these regions to
levels equal to or above the sucrose-fed controls tested at similar
points in time. Thus, it appears that a major compensatory reorganiza-

167
tion occurs following recovery from CET. The present experiments
utilized animals which were off of the ethanol diet for a minimum of
eight weeks prior to testing. Compensatory changes would certainly be
expected to be well underway within this time frame. An increased
GABAergic response following CET may parallel the compensatory changes
due to CET as measured by golgi impregnated spine density counts from
CA1 pyramidal cells. It is possible that the increased number of spines
on the pyramidal cell may be functionally related to the increased
GABAergic response with iontophoretic GABA application. Alternatively,
the increased PS amplitudes seen in the paired pulses may result from
this compensatory reorganization of the pyramidal cell spines.
The present experiments have provided data which indicate an
increased response of the CA1 pyramidal cell to GABA ejection along its
dendritic axis. These findings were similar with either SO or SR
stimulation even though the response pattern to GABA ejection differed
depending on the dendritic stimulation site. CET animals always showed
more inhibition or less potentiation than pair-fed sucrose controls.
These data were discussed in terms of compensatory changes of dendritic
spines after long-term ethanol treatment as recently reported by King
(1984). The results suggest an increased number or affinity of GABA
receptors on the CAl pyramidal cells after eight weeks of abstinence
from 20 weeks of CET, possibly as a function of decreased GABA release.
Experiments using pharmacological agents which alter inhibition in the
CAl region may provide important additional information concerning CET
effects.

CHAPTER 6
THE EFFECT OF CET ON
BICUCULLINE, BACLOFEN AND ENKEPHALIN MEDIATED RESPONSES IN CAl
Introduction
There is now considerable evidence that chronic ethanol abuse in
the human population produces a variety of alterations in the CNS from
changes in behavior (Victor et al., 1971; Ron, 1977; Tarter, 1975), to
lesions of certain nuclei in the brain (Victor et al., 1971), and to
alterations in the electrophysiological functioning of the brain
(Porjesz and Begleiter, 1981). Animal models have provided evidence
that alcohol itself, can produce toxic effects in the brain. These
findings indicated that although malnutrition may induce CNS complica
tions, malnutrition is not responsible for the majority of symptoms
found with long term ethanol abuse. Through the use of animal models,
careful analyses have been made of certain brain regions such as the
hippocampus and cerebellum for alterations in the morphology (Riley and
Walker, 1978; Walker et al., 1980; King, 1984), electrophysiology
(Abraham et al., 1981, 1982; Durand and Carien, 1984a, 1984b) and
neurochemistry (Freund, 1980) following CET. The results of these
studies have shown regionally specific as well as widespread
alterations.
The finding that CET altered synaptic inhibition in the CAl region
of the hippocampus (Abraham et al., 1981) provided an impetus for
studying the underlying transmitter systems known to be involved in
inhibitory processes. The findings from the previous study where GABA
was ejected at discrete locations along the pyramidal cell dendrites
168

169
showed, by trends in the PS amplitude data that alcohol treatment for 20
weeks produced a more inhibited response to GABA administration when
compared to sucrose animals. The finding that the SO PS latencies were
statistically different provided additional evidence that CET has in
some way altered the efficacy of the pyramidal cell to GABA adminis
trations .
Although GABA is the major transmitter involved in the inhibitory
systems of the CAl region, other pharmacological agents or transmitters
have been shown to modify the pyramidal cell PS, possibly by regulating
the interneurons. At least four pharmacological agents have been shown
to produce alterations in GABA mediated inhibition in the CAl region of
the hippocampus. The GABA antagonists bicuculline or picrotoxin have
been reported to produce a profound decrease in the effect of the
recurrent inhibitory system. Using extracellular recording techniques,
the effect of iontophoretic bicuculline application was to cause an
increase in the size of the PS as well as the number of population
spikes recorded. If the dosage was high, seizure activity was often
found (Curtis et al., 1970). The opiates, including morphine, produced
a response pattern similar to that seen after treatment with GABA
antagonists. There is an increase in the amplitude as well as the
number of population spikes recorded (Lee et al., 1980; Dunwiddie et
al., 1980; Corrigal and Linesman, 1980; Nicoll et al., 1980; Segal,
1977; Zieglgansberger et al., 1979). Although there is disagreement
concerning the mechanisms by which the opiates act (Corrigal, 1983;
Haas and Ryall, 1980), the majority of papers have suggested that
opiates produced the increased PS responses by disinhibiting the
basket cell, an hypothesis consistent with its action in other regions

170
of the nervous system such as locus coeruleus and other brain stem
nuclei (Bird and Kuhar, 1977; Bradley and Dray, 1974; Fredrickson et
al. 1976). Thus, if CET caused an increased production or release of
the putative opiate transmitters in the CAl region, increases in the
PS amplitude could be a result of this action rather than a general
decrease in the efficacy of the inhibitory systems.
Baclofen, a GABA analogue has recently been shown to play a role in
feedforward inhibition. It has been shown that there are two components
to the inhibitory response when recording intracellularly (Alger and
Nicoll, 1982a). The early phase of the response has been shown to be
mediated by recurrent inhibition which is activated by GABA and
antagonized by bicuculline. The late phase, attributed to feedforward
inhibitory influences, has been shown to be bicuculline resistant.
More recently it has been shown that baclofen directly hyperpolarizes
this bicuculline resistant hyperpolarization in a stereoselective
manner (Newberry and Nicoll, 1984; Nicoll and Newberry, 1984). Thus,
baclofen might provide a primary method by which the effect of CET
could be evaluated on the feedforward inhibitory components of CAl
responses.
The following experiments were therefore designed to investigate
the effects of bicuculline, an opiate (D-Ala(2))-met-enkephalinamide
(2DA), and baclofen.
Methods
Animals
Animals were obtained, housed and treated with alcohol and sucrose
as indicated in the general methods section.

171
Apparatus
The apparatus used for these experiments has been detailed in the
general methods, in vitro methods, and the GABA iontophoresis methods.
No additional equipment has been utilized for these experiments.
Procedures
The protocol for each of these experiments using localized
iontophoretic applications of different pharmacological agents were the
same. The drugs were prepared immediately prior to use and loaded into
individual micropipettes. Only one drug was ever in the slice prepara
tion at any one time. Table 6-1 indicates the drug concentrations,
ejection currents, retaining currents, and ejection durations used to
apply these drugs. A maximum of two drugs were applied to the same
slice successively. Unlike the methodology used for the application of
GABA, the protracted time course of activation for each of these drugs
allowed for only one application of the drug after which the response
was sampled every 30 seconds for ten minutes. As with the GABA series,
each drug was tested using SR and subsequently SO stimulation.
The protocol for each of these experiments was as follows. Prior
to the application of any of the agents, the recording electrode was
positioned as indicated for the GABA experiment. An attempt was made to
obtain good responses with positive-going EPSPs from both the SR and SO
electrodes. In the event that good responses could not be achieved from
both electrodes, preference was given to the response obtained with SR
stimulation. The PS amplitude and latency to the PS measures, however,
were prominent and measurable with both SR and SO stimulation. Once the
recording and stimulation electrodes were positioned, the iontophoretic
pipette was positioned in the slice at the first of the four locations

172
TABLE 6-1
Cone.
pH
Eject
Retain
Solution
Enkephalin
30 mM
NA
40 nA
20-30 nA
165 mM NaCl
GABA
1 M
3.5
40 nA
5 nA
distilled H20
pH adjusted
Bicuculline
25 mM
NA
40 nA
15-20 nA
165 mM NaCl
Baclofen
75 mM
NA
40 nA
20-30 nA
165 mM NaCl
Drug concentrations, ejection currents, retaining currents,
ejection durations, and pH level for each of the drugs used in these
studies.

173
along the dendritic axis of the pyramidal cell (Fig 6-1). The first
location was always in SO near the recording electrode located in the
pyramidal cell layer. The second location was in SO approximately
two-thirds of the distance from the cell layer to the alveus. The third
location was in SR near the recording electrode. The placement of the
drug containing pipette near the recording electrode in these
experiments was not as close, by approximately 25 to 50 microns, as that
aimed for in the GABA experiments. The fourth location of the
iontophoretic pipette was in distal SR approximately at the border
between the SR and the SIM. Pharmacological agents were also
administered in the following order: 1. proximal SO, 2. distal SO, 3.
proximal SR, and 4. distal SR-SLM border. Prior to the ejection of the
drug, a single baseline response was obtained. Ten seconds later the
drug to be tested was ejected using the currents outlined in Table 6-1
for five seconds. Responses were then collected every 30 seconds for
the next 10 minutes with afferent stimulation from the SR stimulation
electrode. The entire experiment with the same drug and application
location was then repeated using the SO located stimulation electrode.
The iontophoretic pipette was then moved to the next location and the
procedures repeated until all four application sites were tested with
both SO and SR stimulation pulses. A minimum of 12 minutes separated
the application of any of these agents.
Results
Bicuculline series
The results for the bicuculline series of animals were based on a
total of 21 rats with 12 in the alcohol group and nine in the sucrose

Figure 6-1. Arrows represent iontophoresis sites for bicuculline,
enkephalin, and baclofen along the dendritic axis of
CA1 pyramidal cells. Note that recordings were of
field responses and that the effects were upon groups
of pyramidal cells rather than single cells. Drugs
were ejected for five seconds with varying currents.
Abbreviations: ALV, alveus; SO, stratum radiatum; SP,
stratum pyramidale; SR, stratum radiatum; SLM, stratum
lacunosum-moleculare.

175
AL V
SLM

176
group. Alcohol animals consumed a mean daily ethanol dosage of 12.87 +
.20 gms/Kg which was comparable to levels consumed in other studies
which showed anatomical and physiological changes as a result. The body
weights of the two groups were as follows: alcohol 542.86 + 7.9,
sucrose 505 10.61.
SR stimulation
I/O relationships with SR stimulation. See description of I/O
curves as described in Chapter 5. I/O curves for each of the
pharmacological series were combined and were presented earlier.
Iontophoresis applications with SR stimulation. Recordings from
distal SO showed that there were no differences between the alcohol
and sucrose control groups for the PS amplitude (Fig 6-2) or the PS
latency (Fig 6-3) responses. There was, however, a group by time
interaction effect for the PS amplitude data (F(l,360) 2.40, p -
.0008). Subsequent T-test analyses of the individual points revealed
the points between 60 and 180 secs to be different. The alcohol group
was more potentiated than the sucrose-fed group. There were no
differences in PS amplitude or PS latency measures in SO near the cell
body layer. Although there were no statistically significant
differences between groups on PS amplitude or PS latency measures near
the cell body layer in SR, there was a significant group by time
interaction for the PS latency measure which revealed the points
between 60 and 150 secs to be different. Furthermore, while the PS
amplitude data for the proximal SR application site were not different
by group, those data approached significance (F(l,17) 3.35, p -

Figure 6-2. PS amplitude responses (means + SEM) after bicuculline
iontophoresis (time=0) with stratum radiatum stimulation.
Scales on ordinate axis represent percent change of PS
amplitude responses obtained after bicuculline adminis
tration in comparison to control responses obtained 10
seconds prior to bicuculline administration (bicuculline
test/control x 100) and are independent for each ionto-
phoretic ejection location. Abcissa represents time in
minutes following iontophoretic ejection of the drug.
A) PS response with iontophoretic ejection in distal
SO. B) PS response with iontophoretic ejection in
proximal SO. C) PS response with iontophoretic ejection
in proximal SR. D) PS response with iontophoretic
ejection in distal SR.

500
400
300
200_
100_
500_
C00_
300
200
I00_
300_
200
100_
0
178
D0201
pstot
ALCOHOL
SUCROSE ...
U_xJ_l_L
<1 I "1 "X'-t -l -t
~iiiiir
I 2 3
iiirriiiiii
5 6 7 8 9 10
time after iontophoresis (mins)

Figure 6-3. PS latency responses (means) after bicuculline
iontophoresis (time=0) with stratum radiatum
stimulation. Scales on ordinate represent difference
of PS latency response following bicuculline adminis
tration from control responses obtained prior to
bicuculline administration (test response-control
response) and are independent for each iontophoretic
ejection location. Abcissa represents time in minutes
following iontophoretic ejection of the drug. A) PS
responses with iontophoretic ejection in distal SO. B)
PS responses with iontophoretic ejection in proximal
SO. C) PS responses with iontophoretic ejection in
proximal SR. D) PS responses with iontophoretic
ejection in distal SR.

180
time after iontophoresis (mins )

181
.085). It is reasonable that this near difference be given consider
ation since the PS latency interaction showed differences at several
time points. As can be seen in Fig. 6-2D and Fig 6-3D, there were no
differences seen in distal SR for either of the PS measures.
SO stimulation
I/O relationships with SO stimulation. See description of I/O
curves as described in Chapter 5. I/O curves for each of the
pharmacological series were combined and presented earlier.
Iontophoresis application with SO stimulation. The graphs for
the SO stimulation with bicuculline application at each of the four
different locations along the pyramidal cell dendrites is shown in Fig
6-4 for the PS amplitude measure and in Fig 6-5 for the PS latency
measures. Beginning with the most distal application in SO, there was
a statistically significant group difference with SO stimulation
(F(l,18) 6.21, p = .023). The alcohol treated animals exhibited
increased PS amplitudes. There was also a significant group by time
interaction effect (F(l,360) 3.93, p .0001). Subsequent analyses
revealed the points between one and 4 mins to be different. There
were no effects on the PS latency measures. There were no differences
in either PS amplitude or PS latency measures when bicuculline was
applied at the cell layer but still in SO. There were no differences
for the PS amplitude data when bicuculline was applied in SR near the
cell body region but there were statistically significant group
(F(l,16) = 5.79, p = .028) and group by time interaction effects
(F(l,320) 1.60, p .05) for the PS latency measures. Subsequent

Figure 6-4. PS amplitude responses (means + SEM) after bicuculline
iontophoresis (time=0) with stratum oriens stimulation.
Scales on ordinate represent percent change of PS
amplitude responses obtained after bicuculline adminis
tration from control responses obtained immediately
prior to bicuculline administration (bicuculline
test/control x 100) and are independent for each
iontophoretic ejection location. Abcissa represents
time in minutes following iontophoretic ejection of the
drug. A) PS responses with iontophoretic ejection in
distal SO. B) PS responses with iontophoretic ejection
in proximal SO. C) PS responses with iontophoretic
ejection in proximal SR. D) PS responses with ionto
phoretic ejection in distal SR.

percent change from control
183
?00
time after iontophoresis (mins)

Figure 6-5. PS latency responses (means) after bicuculline
iontophoresis (time=0) with stratum oriens stimulation.
Scales on ordinate represent difference of PS latency
response following bicuculline administration from
control responses obtained prior to bicuculline adminis
tration (test response-control response) and are
independent for each iontophoretic ejection location.
Abcissa represents time in minutes following ionto
phoretic ejection of the drug. A) PS responses with
iontophoretic ejection in distal SO. B) PS responses
with iontophoretic ejection in proximal SO. C) PS
responses with iontophoretic ejection in proximal SR.
D) PS responses with iontophoretic ejection in distal
SR.

185
time after iontophoresis (mins)

186
analyses showed the points noted in Fig 6-5 with asterisks as being
different. Finally, there were no differences in either PS total or
PS latency measures with bicuculline application in distal radiatum
with SO stimulation.
Baclofen series: Stratum radiatum stimulation
I/O relationships with SR stimulation. See description of I/O
curves as described in Chapter 5. I/O curves for each of the pharmaco
logical series were combined and presented earlier.
Iontophoresis application with SR stimulation. The data for the
baclofen PS amplitude responses with SR stimulation are shown in Fig
6-6 and in Fig 6-7 for the PS latency measures. There were no group
differences for any of the four sites tested with baclofen
administration and SR stimulation for the PS amplitude measure. The
only group by time interaction found with baclofen administration and
SR stimulation was in SR near the cell body layer (F(1,280) 1.58, p
= .05). Subsequent analyses showed that the individual differences
for the time points were from seven to nine mins after baclofen
application. The general pattern of responses can be seen for the PS
amplitudes in Fig 6-6. With SR stimulation and baclofen
administration, the effect of baclofen on the PS amplitude was most
powerful near the cell body layer in SO and then at both of the
iontophoretic administration sites in SR. Baclofen iontophoresis
actually resulted in the greatest magnitude of inhibition in distal
SR. The duration of inhibition was longest in SO near the cell body
layer. With the exception of the administration of baclofen in SO
near the cell body layer, there were no consistent trends for

Figure 6-6. PS amplitude responses (means + SEM) after baclofen
iontophoresis (time=0) with stratum radiatum stimula
tion. Scales on ordinate represent difference of PS
amplitude response following bicuculline administration
from control responses obtained prior to bicuculline
administration (test response-control response) and are
independent for each iontophoretic ejection location.
Abcissa represents time in minutes following ionto
phoretic ejection of the drug. A) PS responses with
iontophoretic ejection in distal SO. B) PS responses
with iontophoretic ejection in proximal SO. C) PS
responses with iontophoretic ejection in proximal SR.
D) PS responses with iontophoretic ejection in distal
SR.

time after iontophoresis (mins)
percent change from control

Figure 6-7. PS latency responses (means) after baclofen
iontophoresis (time-0) with stratum radiatum stimu
lation. Scales on ordinate represent difference of PS
latency response following bicuculline administration
from control responses obtained prior to bicuculline
administration (test response-control response) and are
independent for each iontophoretic ejection location.
Abcissa represents time in minutes following ionto
phoretic ejection of the drug. A) PS responses with
iontophoretic ejection in distal SO. B) PS responses
with iontophoretic ejection in proximal SO. C) PS
responses with iontophoretic ejection in proximal SR.
D) PS responses with iontophoretic ejection in distal
SR.

difference from control (msecs)
190

191
differences between the alcohol and sucrose groups for the baclofen
data. In SO, near the cell body layer, there was a trend for the
alcohol group to be slightly more inhibited than the controls.
Baclofen series: Stratum oriens stimulation
I/O relationships. See description of I/O curves as described in
Chapter 5. I/O curves for each of the pharmacological series were
combined and presented earlier.
Iontophoresis application with SO stimulation. There were no
differences between alcohol and sucrose groups nor were there any
interaction effects on either the PS amplitude (Fig 6-8) or the PS
latency (Fig 6-9) measures. The effect of baclofen administration has
not been well documented using extracellular techniques. It can be seen
for the PS amplitude measures, that the magnitude of inhibition is
essentially reversed when stimulating through SO and comparing them with
the responses found with SR stimulation. The largest magnitude of
inhibition was found when baclofen was administered in the same synaptic
region as that being stimulated. Both of the SO application sites,
distal and near to the pyramidal cell body layer, were strongly inhibit
ed when compared to the baclofen administration sites in SR. While
there were no significant differences, there was a suggestion of a trend
for the alcohol group to be more inhibited with SO baclofen application
and SO stimulation. These findings are similar to those found with
baclofen administration near the cell body layer in SO with SR stimula
tion and may relate to the increase in response seen with GABA adminis
tration.

Figure 6-8. PS amplitude responses (means) after baclofen
iontophoresis (time=0) with stratum oriens stimulation.
Scales on ordinate represent difference of PS amplitude
response following bicuculline administration from
control responses obtained prior to bicuculline adminis
tration (test response-control response) and are
independent for each iontophoretic ejection location.
Abcissa represents time in minutes following ionto
phoretic ejection of the drug. A) PS responses with
iontophoretic ejection in distal SO. B) PS responses
with iontophoretic ejection in proximal SO. C) PS
responses with iontophoretic ejection in proximal SR.
D) PS responses with iontophoretic ejection in distal
SR.

25
125
100
75
50
25
125
100
7 5
50
125
100
7 5
50
25
0
193
T0202
pstot
ALCOHOL
SUCROSE ...
1
10
time after iontophoresis (mins)

Figure 6-9. PS latency responses (means) after baclofen
iontophoresis (time=0) with stratum oriens stimulation.
Scales on ordinate represent difference of PS latency
response following bicuculline administration from
control responses obtained prior to bicuculline adminis
tration (test response-control response) and are
independent for each iontophoretic ejection location.
Abcissa represents time in minutes following ionto
phoretic ejection of the drug. A) PS responses with
iontophoretic ejection in distal SO. B) PS responses
with iontophoretic ejection in proximal SO. C) PS
responses with iontophoretic ejection in proximal SR.
D) PS response with iontophoretic ejection in distal
SR.

difference from control (msecs)
195
time after iontophoresis (mins)

196
Enkephalin series: SR stimulation
I/O relationships with SR stimulation. See description of I/O
curves as described in Chapter 5. I/O curves for each of the
pharmacological series were combined and presented earlier.
Iontophoresis application with SR stimulation. There were no
statistically significant differences between the alcohol and sucrose
groups for the PS amplitude measure (Fig 6-10) when 2DA application was
in the distal SO with SR stimulation. There was, however, a statisti
cally significant group effect for the PS latency measure (Fig 6-11)
when 2DA was applied in distal SO (F(l,21) 5.81, p .025). The
analyses for the application of 2DA in SO near the cell body layer with
SR stimulation revealed no statistically significant differences between
the two groups nor any interaction effects for PS amplitude or latency
measures. Application of 2DA at sites in SR near the cell body layer or
in distal SR did not result in any differences between groups on either
PS amplitude or PS latency measures.
Enkephalin series: Stratum oriens stimulation
I/O relationships. See description of I/O curves as described in
Chapter 5. I/O curves for each of the pharmacological series were
combined and presented earlier.
Iontophoresis application with SO stimulation. The data for the
application of 2DA at the dendritic locations with SO stimulation are
shown in Fig 6-12 for the PS amplitude data and in Fig 6-13 for the PS
latency data. The most noteworthy observation of these data was that

Figure 6-10. PS amplitude responses (means + SEM) after enkephalin
iontophoresis (time=0) with stratum radiatum stimulation.
Scales on ordinate represent difference of PS amplitude
response following bicuculline administration from
control responses obtained prior to bicuculline adminis
tration (test response-control response) and are
independent for each iontophoretic ejection location.
Abcissa represents time in minutes following ionto
phoretic ejection of the drug. A) PS responses with
iontophoretic ejection in distal SO. B) PS responses
with iontophoretic ejection in proximal SO. C) PS
responses with iontophoretic ejection in proximal SR.
D) PS responses with iontophoretic ejection in distal
SR.

600
400
200
800
600
400
200
1000
800_
600_
400
200_
400_
200
0
198
10201
pstot
ALCOHOL
SUCROSF ...
~iiir
1 2
iiiiir
3 4 >
~iir
7 6
10
time after iontophoresis (mins)

Figure 6-11. PS latency responses (means) after enkephalin
iontophoresis (time=0) with stratum radiatum
stimulation. Scales on ordinate represent difference
of PS latency response following bicuculline adminis
tration from control responses obtained prior to
bicuculline administration (test response-control
response) and are independent for each iontophoretic
ejection location. Abcissa represents time in minutes
following iontophoretic ejection of the drug. A) PS
responses with iontophoretic ejection in distal SO. B)
PS responses with iontophoretic ejection in proximal
SO. C) PS responses with iontophoretic ejection in
proximal SR. D) PS responses with iontophoretic
ejection in distal SR.

difference from control (msecs)
200
time after iontophoresis (mins)

Figure 6-12. PS amplitude responses (mean + SEM) after enkephalin
iontophoresis (time=0) with stratum oriens stimulation.
Scales on ordinate represent difference of PS amplitude
response following bicuculline administration from
control responses obtained prior to bicuculline adminis
tration (test response-control response) and are
independent for each iontophoretic ejection location.
Abcissa represents time in minutes following ionto
phoretic ejection of the drug. A) PS responses with
iontophoretic ejection in distal SO. B) PS responses
with iontophoretic ejection in proximal SO. C) PS
responses with iontophoretic ejection in proximal SR.
D) PS responses with iontophoretic ejection in distal
SR.

200
600
600
400_
200_
1000
600
600
400
200
800
600
202
10202
pstot
M.COHOI.
SUCROSE ...
t 'H i t
irir-
I 2
T 1 1 1 1 I J I I 1 1 1
1 4 S 6 2 6 ^
time after iontophoresis (mins)
10

Figure 6-13. PS latency responses (means) after enkephalin
iontophoresis (time=0) with stratum oriens stimulation.
Scales on ordinate represent difference of PS latency
response following bicuculline administration from
control responses obtained prior to bicuculline adminis
tration (test response-control response) and are
independent for each iontophoretic ejection location.
Abcissa represents time in minutes following ionto
phoretic ejection of the drug. A) PS responses with
iontophoretic ejection in distal SO. B) PS responses
with iontophoretic ejection in proximal SO. C) PS
responses with iontophoretic ejection in proximal SR.
D) PS responses with iontophoretic ejection in distal
SR.

time after iontophoresis (mins)
difference from control (msecs)

205
the sucrose group was more potentiated under these conditions than the
alcohol group. This was the first condition in which the sucrose
group PS amplitude measure showed increased potentiation or decreased
latencies when compared to the alcohol group. There were no
statistically significant differences between groups for the data
collected with distal SO 2DA application nor with proximal SO
application for either the PS amplitude or the PS latency responses.
The PS latency data collected with 2DA administration in proximal SO
and with SO stimulation showed significant group (F(l,21) = 6.27, p =
.021) and group by time interaction effects (F(l,420) *= 2.27, p -
.0015). The PS amplitude and PS latency measures obtained when 2DA
was applied in SR near the cell body layer revealed no group effects
for either measure but did reveal a statistically significant
interaction of the group by time for the PS amplitude response
(F(l,420) 1.69, p .03). Subsequent analyses of the differences
between groups at the individual time points showed significant
differences between 90 and 180 secs after 2DA iontophoresis. The
application of 2DA in distal SR with SO stimulation did not produce
any group or interaction differences for the PS amplitude or the PS
latency measures.
Discussion
The present series of experiments have utilized three different
pharmacological agents/neurotransmitters or antagonists which have been
shown to affect CAl inhibitory systems. A nutritionally complete liquid
ethanol diet was administered which has previously been shown to produce
anatomical (Walker et al., 1980; Riley and Walker, 1978; King, 1984),
electrophysiological (Abraham et al., 1981, 1982, 1984; Durand and

206
Carien, 1984a, 1984b) and biochemical (Freund, 1980) changes. These
experiments were designed to more clearly elucidate potential pharmaco
logical mechanisms by which CET produced increased PS amplitudes after
20 weeks of exposure and subsequently at least 8 weeks of being on a
normal rat chow and water diet (Abraham et al., 1981).
The response to GABA administration of CA1 pyramidal cells has been
shown to consist of at least three independent actions. These three
different actions have been reported to be subserved by different
receptor populations. When GABA is applied to the somatic region of the
CAl pyramidal cell, an hyperpolarizing response is found. It has been
shown however, that a subpopulation of these hyperpolarizing receptors
are bicuculline insensitive. The bicuculline sensitive receptors have
been termed GABA(A) receptors while the bicuculline-insensitive recep
tors have been termed GABA(B) receptors. The third receptor sub-type
results in a depolarizing response to GABA and is usually found in the
dendrites rather than in the somal region.
The GABA(A) receptor antagonist, bicuculline, which has been shown
to produce increased PS amplitudes when iontophoretically administered
in the CAl pyramidal cell region, produced similar increases in PS
amplitude responses with CET and sucrose-fed animals. The response
pattern to bicuculline iontophoresis along the dendritic axis was such
that the maximal level of potentiation was in SO near the pyramidal cell
body layer. This pattern held true for stimulation from both the SO and
SR afferents. Bicuculline application in distal SO and proximal SR
resulted in responses which were dependent upon the stimulation site
with each area responding best to its respective afferent input. The

207
distal SR iontophoretic site did not respond to either SO or SR stimula
tion in a manner comparable to the other three bicuculline application
sites. It is suggested that the small increase seen at the distal SR
site was a result of the diffusion of bicuculline toward the pyramidal
cell body region.
CET resulted in increased PS amplitudes when compared to the
sucrose-fed control group with both SO and SR stimulation. As can be
seen in Fig 6-14A and Fig 6-14B, the greatest differences between the
two groups were found near the cell body region. These data indicate
that bicuculline produced a greater antagonism of GABA receptors in CET
animals than in controls. Bicuculline has been shown to competitively
antagonize GABA at the receptor level (Cooper et al., 1982). A greater
antagonism of GABA receptors in CET animals might occur as follows. If
the release of GABA is decreased in CET animals due to a loss of
inhibitory interneurons or to a presynaptic alteration of GABA-ergic
synthesis or release, a subsequent up-regulation and/or increase in GABA
receptor sensitivity might be expected. The increased response to GABA
iontophoresis along the dendrites in CA1 in CET animals could be
explained by this sequence of events. Since bicuculline competitively
blocks GABA receptors, a decreased release or availability of GABA
followed by an increase in GABA receptor sensitivity would likely result
in an increased sensitivity to the iontophoretic application of
bicuculline. Thus, if the effects of bicuculline mimic the differential
effects seen with CET and sucrose-fed animals after GABA iontophoresis
but in the opposite direction, an increased response to GABA
administration should result in an increased response to bicuculline
administration. It is also possible that CET may decrease the GABA

Figure 6-14. PS amplitude percent change summary graphs for bicuculline, enkephalin and
baclofen. These graphs are constructed from data which was obtained from the
previous respective graphs 1.5 mins after iontophoretic ejection of the
respective drugs. This time point was chosen because it represented the peak
response size for most of the applications. A) Bicuculline PS amplitude peak
response for each of the different iontophoretic locations and groups with
stratum radiatum stimulation. B) Bicuculline PS amplitude peak response for
each of the different iontophoretic locations and groups with stratum oriens
stimulation. C) Enkephalin PS amplitude peak response for each of the
different iontophoretic locations and groups with stratum radiatum stimulation.
D) Enkephalin PS amplitude peak response for each of the different
iontophoretic locations and groups with stratum oriens stimulation.

Dfij** "rc LOCATION ALONG 0U<0**,rtS
percent change from control
percent change from control
x s
o
o o o
n o a
percent change from control
percent change from control
602
A0201
pstot

210
receptor population which would result in an increased PS response to
bicuculline admininstration since the bicuculline would competitively
overwhelm the release of GABA. However, a decrease in the GABAergic
receptor population would not be consistent with the increased response
found after CET with GABA iontophoresis.
Modifications in the GABA receptor population may occur as a result
of alterations incurred by the benzodiazepine receptor. Freund (1980),
showed that after 20 weeks of CET in mice, there was a decreased number
and affinity of benzodiazepine receptors. Since GABA and benzodiazepine
receptors are reported to occur in a receptor complex with Cl- channels
(Tallman et al., 1980), there may be a structural modification such that
GABA sensitivity is increased after CET.
Iontophoretic application of baclofen along the pyramidal cell
dendrites produced a decrease in the PS amplitude with either SO or SR
stimulation at each of the locations where it was iontophoretically
ejected. There was, however, a stimulation location and iontophoretic
site dependent response to its administration as seen in Fig 6-15A and
Fig 6-15B. With SR stimulation, the greatest levels of inhibition were
seen in proximal SO, proximal SR and distal SR. With SO stimulation,
the greatest levels of inhibition were seen when baclofen was applied in
the proximal and distal SO. These data strongly suggest the presence of
feedforward inhibition along the entire length of the CAl pyramidal cell
dendritic tree. Previous reports using intracellular techniques have
indicated that baclofen inhibition is seen throughout these areas.
Iontophoretic application of baclofen using extracellular recording
techniques has not been reported previously, however.

Figure 6-15. PS amplitude percent change summary graphs for baclofen. These graphs are
constructed from data which was obtained from previous graphs 1.5 mins after
iontophoretic ejection of the respective drugs. This time point was chosen
because it represented the peak response size for most of the applications.
A) Baclofen PS amplitude peak response for each of the different iontophoretic
locations and groups with stratum radiatum stimulation. B) Baclofen PS
amplitude peak response for each of the different iontophoretic locations and
graphs with stratum oriens stimulation.

percent change from control
- *> o a ia i
o O O O O O O O O O O O O o
percent change from control
.SSSSSSgSSSSS
212

213
CET did not result in any significant differences in the PS ampli
tude response when compared to controls. Although the CET animals
showed small trends toward being more inhibited than controls when
baclofen was administered in proximal SR with SR or SO stimulation,
these trends were not conclusive. It is therefore concluded that CET
does not affect baclofen sensitive inhibition.
The finding that baclofen is not affected by CET at first suggests
that feedforward inhibition may not be affected by long term ethanol
treatment. However, as indicated earlier, there are at least two
different feedforward related inhibitory responses. One of these
feedforward responses is characterized by baclofen sensitivity and
bicuculline insensitivity and results in an hyperpolarization of the
intracellularly recorded membrane potential. The other response is a
depolarization of the cell membrane which results in the shunting of
current flow such that it inhibits the pyramidal cell from firing. It
appears that the bicuculline-insensitive receptor population may not be
affected by CET. However, since the paired pulse results presented in
chapter 4 suggest decreased levels of feedforward inhibition, it is
quite possible that these CET-induced alterations are on the
depolarizing sub-population of GABA receptors. The effect of CET on the
depolarizing GABAergic receptors, however, cannot be distinguished with
the present experiments.
The iontophoresis of enkephalin along the CA1 pyramidal cell
dendrites resulted in increased PS amplitudes and multiple population
spikes in all of the animals tested. These results compare well with
previous reports (Dunwiddie et al., 1980; Robinson and Deadwyler, 1980;
Nicoll et al., 1980; Dingledine, 1981; Haas and Ryall, 1980; Corrigan

214
and Linesman, 1980; Corrigal, 1983). The effect of enkephalin adminis
tration was similar with both SO and SR stimulation, increasing the PS
amplitude to nearly equal levels. Proximal SO, distal SO and proximal
SR application of the enkephalin (2DA) resulted in comparable levels of
PS potentiation. Distal SR application resulted in minor effects and,
as observed with bicuculline administration, the effects observed can be
explained by diffusion toward the cell body region since the maximal
response amplitude was delayed in time.
The opiates are thought to produce increased PS amplitude responses
by disinhibiting the inhibitory interneurons. This pyramidal cell
response alteration may result from the inhibitory interneurons which
may decrease their firing rate or their release of GABA. Previous work
(Dingledine, 1981), has shown that the enkephalin, DADL, produced the
greatest magnitude of PS amplitude change either in SO or in the
pyramidal cell layer. His data suggested a slightly greater PS ampli
tude response with SO stimulation than with SR stimulation. The time
course of his drug effects are similar to those of the present series.
Ejection of DADL in distal SR resulted in much smaller response ampli
tude increases than were found more proximal to the cell layer and
were comparable to the present results.
The opiates are subserved by at least two physiologically important
classes of receptors, the mu and delta receptors. Both of these binding
sites have been reported in the hippocampus using biochemical techniques
(Chang and Cuatrecasas, 1979). A differential distribution within CAl
has also been reported. Mu receptors were found most concentrated in
the pyramidal cell body region while delta receptors were more diffusely
located in SO, SP and SR (Goodman et al., 1980; Herkenham and Pert,

215
1980; Duka et al., 1981). Mu receptors have been shown to be
selective to morphine application whereas the delta receptors were
most selective for leucine- and methionine-enkephalin (Chang and
Cuatrecasas, 1979). Functionally, both morphine agonists and leucine-
or methionine-enkephalins have been shown to produce similar
epileptogenic-like effects in the CAl region of the hippocampus
(Bostock et al., 1984; Lewis et al., 1981).
(D-Ala(2))-met-enkephalinamide used in the present studies would
preferentially activate the delta opiate receptors throughout the
subfields of CAl, it is clear that the largest response changes occurred
in or near the pyramidal cell region and SO. The present findings
suggest either a higher density of delta receptors at the cell layer or
an activation of the mu receptors at the cell layer in addition to the
delta receptors with the iontophoretic concentrations used in this
study.
The effect of CET on enkephalin administration resulted in a
differential PS response amplitude pattern which was dependent upon the
afferent stimulation site as can be seen in Fig 6-14C and Fig 6-14D.
With SR stimulation the CET group had larger PS amplitudes than did the
sucrose controls. With SO stimulation, the reverse was true. The
sucrose group was more potentiated than the CET animals. With
stimulation from either afferent field, the largest sucrose-fed PS
amplitude responses were always at the proximal SR iontophoresis site.
Fig 6-14 shows the PS amplitude response at 1.5 minutes following 2DA
administration for both the alcohol and sucrose groups and in separate
graphs for SR (Fig 6-14C) and SO (Fig 6-14D) stimulation. The time point
chosen (1.5 mins) for each of the drug series plotted was generally the

216
point where the maximal PS response amplitude occured. The delay to the
maximal response in each of these graphs (Fig 6-14C and 6-14D) was
probably a function of the diffusion of these drugs. It can be seen
that with SR stimulation the proximal SR iontophoretic site was the most
responsive to 2DA administration. With SO stimulation, the same pattern
held for the sucrose group but not for the alcohol group. This suggests
that the major site of action of the opiates is in proximal SR and that
CET results in a maximally decreased response amplitude to 2DA at this
site.
The finding of trends toward increased PS amplitudes with
iontophoretic application of enkephalin along the dendrites and SR
stimulation after CET when compared to sucrose-fed controls can be
explained by the decreased inhibition found in the CA1 region following
long term ethanol treatment. If 2DA acts by disinhibiting the
inhibitory interneuron, then any condition which has previously reduced
the efficacy or number of these interneurons would be expected to be
further reduced with enkephalin administration. This further reduction
in inhibition would be seen as an increase in the PS amplitude of CET
animals as compared to controls.
The responses to 2DA administration with SO stimulation after CET
were reversed compared to the responses obtained with SR stimulation in
terms of the magnitude of the PS amplitude response by group. The
greatest difference was seen when enkephalin was applied in proximal SR
with SO stimulation. In this case the alcohol group response was
decreased by nearly 400 percentage points. These findings are surpris
ing since no differences by iontophoretic application site or stimula
tion site of this nature have been found with the other pharmacological
agents tested.

217
This differential set of responses, seen after CET with SO and SR
stimulation, suggest a differential projection of the commissural
afferents to the basilar versus apical dendrites of the CAl pyramidal
cell. It has been shown that commissural afferents of CA3 pyramidal
cells project more heavily to the basilar dendrites of CAl pyramidal
cells than to the apical dendrites (Swanson et al., 1978). This
projection pattern may result in termination of a different population
of interneuons than do ipsilateral schaffer collateral fibers. If, as
has been suggested, long myelinated fibers are especially prone to CET
(Dreyfus, 1974), it is possible that there is a decreased input to the
enkephalinergic interneurons, hence a decrease in the efficacy. It has
also become common to find peptides co-localized with various other
putative neurotransmitters including GABA. It is therefore possible
that CET reduces a population of interneurons which, given the proper
conditions, may be either inhibitory or excitatory. Finally, if
enkephalin acts by disinhibition, these findings suggest that CET may
result in a decreased sensitivity of the inhibitory interneurons which
receive input from commissural fibers. It has been reported that
acute doses of ethanol activated hippocampal pyramidal neurons (Berger
et al., 1982). It was then hypothesized that this increased response to
ethanol may occur by ethanol itself causing an increased release of
endogenous opiate peptide or by an ethanol-induced formation of
aldehyde-catecholamine condensation products (tetrahydroisoquinolines,
TIQS) which reportedly have opiate-like effects. The important point,
however, is that CET in some way differentially affects the response of
2DA application by afferent stimulation location. This may suggest that

218
commissural fibers terminate selectively on neurons which are in some
way different from the ipsilateral schaffer fibers in their response to
CET.
Alternatively, if methionine- or leucine-enkephalin does not affect
recurrent or feedforward inhibition as indicated by Dingledine (1981),
it may be possible that the opiate effects represent direct actions on
the pyramidal neuron. In this case, it is possible that CET results
either in a loss of receptors per se or a decrease in the sensitivity
of those receptors to increased opiate release. This may occur as a
compensatory mechanism to the hypothesized effect of CET on the
enkephalinergic system or as a function of TIQS (Berger et al., 1982).
Thus, it is possible that with long-term ethanol ingestion a shift in
the sensitivity of the pyramidal cell opiate receptor may occur with SO
afferent activity. Again, this differential sensitivity of CET effects
with SO and SR stimulation may result from differential distribution of
commissural CA3 fibers to SO while ipsilateral fibers project to the SR
region.
The present data suggest that CET may act discriminately on various
aspects of the CAl inhibitory system. They also point out the
importance of testing different afferent systems. The intricacies of
the CAl inhibitory systems are only beginning to be understood. A
complex interaction of various neurotransmitters are involved in the
control of pyramidal cell firing. Although several of these pharmaco
logical systems have been explored in the current manuscript others
have been shown to produce response modifications of the CAl pyramidal
cell by mechanisms not yet well detailed. Consideration of these
systems may provide important clues to a better understanding of CET
effects.

CHAPTER 7
GENERAL DISCUSSION
Chronic ethanol treatment has been shown to produce alterations
in the behavior (Walker and Hunter, 1978; Walker and Freund, 1973;
Freund and Walker, 1971; Denoble and Begleiter, 1979; Fehr et al.,
1975), anatomy (Walker et al., 1980; Riley and Walker, 1978; McMullen
et al., 1984; King, 1984) and electrophysiology (Abraham et al., 1981,
1982, 1984; Durand and Carien, 1984a, 1984b) of the rodent hippocampus.
Anatomical changes have included a 15 to 20 percent loss of CAl
pyramidal cells as well as dentate gyrus granule cells. Decreased
dendritic spine loss and reduced dendritic branching has also been
found after CET (Riley and Walker, 1978). Electrophysiological
evidence from the CAl region indicated a more constrained synaptic
field in proximal SR dendrites of pyramidal cells with CSD analyses
(Abraham et al., 1982). Each of these anatomical alterations indicates
deleterious effects from long term ethanol ingestion. It should be
noted that these results were obtained at least eight weeks following
the discontinuance of ethanol treatment. They are, therefore, residual
effects.
Functionally, the major finding in the CAl region has been that
CET resulted in increased PS amplitudes with paired pulse testing
(Abraham et al., 1981). It was hypothesized that the increased PS
amplitude was a function of a decrease in the efficacy of recurrent
inhibition. Recurrent inhibition has been shown to have a powerful
regulatory control of CAl pyramidal cell firing once the pyramidal
cell has been activated (Andersen et al., 1964a, 1964b).
219

220
More recent evidence has uncovered the existence of another type of
pyramidal cell inhibition. This type of inhibition is activated prior
to the firing of the pyramidal cell itself and has been termed feed
forward inhibition (Alger and Nicoll, 1980, 1982a, 1982b; Andersen et
al., 1978, 1982). The underlying cellular mechanism of recurrent
inhibition has been shown to be a hyperpolarization of the pyramidal
cell body as a function of an increased Cl- conductance. Feedforward
inhibition appears to be more complex in terms of the underlying
mechanisms. There appear at present to be at least three different
mechanisms by which feedforward inhibition is produced. It is likely
that our understanding of the mechanism of inhibition is still in its
infancy and that additional cellular mechanisms will be found.
Briefly, feedforward inhibition has also been shown to result in an
hyperpolarization of the pyramidal cell, but the time course of
maximal effect (200 msecs vs 50 msecs) is later than for antidromically
activated recurrent inhibition (Alger and Nicoll, 1982a). This
hyperpolarizing response has been shown to be subserved by at least
two different receptor populations, a GABA sensitive, bicuculline
sensitive and a GABA sensitive, bicuculline insensitive receptor. It
has been reported that baclofen may preferentially activate the
bicuculline insensitive receptor population (Newberry and Nicoll,
1984). A third receptor type results in a depolarization with
recordings from the pyramidal cell body. This depolarization, however,
results in a current shunt which, in turn, locally inhibits additional
dendritic input. This depolarizing response has unique characteristics
in that it can also result in an "amplification" of additional afferent
input at locations away from the activated, depolarized site since the

221
pyramidal cell is closer to threshold as a result of the depolarizing
response. This response has been called "discriminative inhibition"
since it may result in a selective filtering of afferent input
(Andersen et al., 1982).
The finding of increased PS amplitudes and conclusion of decreased
recurrent inhibition by Abraham et al. (1981) raises two important
questions. First, does CET result in alterations in recurrent
inhibition alone or does CET also result in alterations of feedforward
inhibition? The second question is related to how CET may produce
these effects on inhibition from a more mechanistic perspective. What
are the effects, after CET, of different putative transmitter agonists
and antagonists known to influence CA1 inhibition?
The present studies were designed to characterize recurrent and
feedforward inhibition using extracellular techniques. While intra
cellular recording methodologies have been used to assess recurrent and
feedforward inhibition, extracellular recording offers the advantages of
simpler recording protocols as well as the ability to screen different
treatment and testing conditions more easily. The paired pulse tech
nique was initially used with different stimulation configurations in an
effort to dissociate recurrent and feedforward inhibition. The A/0
paired pulse configuration was used to assess the effect of recurrent
inhibition. Activation of the CAl pyramidal cell axons antidromically
by the condition pulse results in an orthodromic activation of the
recurrent inhibitory interneuron. If a test pulse is then administered
a predetermined interval after the condition pulse, the magnitude of the
recurrent inhibition can be assessed. 0/0 homosynaptic paired pulses
result in the activation of both feedforward inhibitory interneurons and
recurrent inhibitory interneurons. Additionally, an EPSP facilitation

222
is seen with 0/0 homosynaptic stimulation at short IPIs. Thus, 0/0
homosynaptic stimulation, as used by Abraham et al. (1981) results in
the activation of both recurrent and feedforward inhibition. 0/0
heterosynaptic stimulation, in which two stimulation electrodes are
placed either in two different pyramidal cell afferent zones, such as
the SO or the SR, or in two clearly defined and different regions of the
same afferent region such as the SR, results in the activation of
recurrent and feedforward inhibition without any facilitation of the
EPSP. If the responses of these three different paired pulse
configurations are then compared, it may be possible to determine the
relative contribution of recurrent versus feedforward inhibition.
An additional variable that was used to explore recurrent and
feedforward inhibition was the intensity of the condition and test pulse
of paired pulses. It was thought that different current intensities may
selectively recruit feedforward and recurrent inhibitory interneurons.
A number of different condition and test pulse current intensities were
therefore tested with the paired pulse paradigm.
Finally, paired pulse responses were tested both in vivo and in
vitro. CET induced responses using the in vivo preparation have
always been suspect because urethane anesthesia (a barbiturate) has
been shown to be cross tolerant with ethanol (Kalant et al., 1971).
Cross tolerance has really never been likely in our preparations since
these animals have been off of the ethanol diet for at least two
months prior to any electrophysiological testing. The in vitro
responses after CET resulted in patterns of increased PS amplitude as
was shown in vivo. Although there was a shift in the IPI at which
both CET and sucrose-fed animals were maximally potentiated in vitro.

223
these results were a function of decreased levels of inhibition found
with the slice preparation or as a result of transecting the longitu
dinal inhibitory fibers (Teyler, 1980). It is therefore concluded
that urethane anesthesia was not related to the increased PS response
seen in vivo.
In general, increased condition pulse current intensity resulted in
increased durations of inhibition using 0/0 homosynaptic paired pulses.
Higher test pulse currents resulted in a decreased level of maximal
potentiation. Results from the current in vivo and in vitro experiments
support these conclusions. It is suggested that increasing condition
pulse stimulus intensity resulted in an increased recruitment of
inhibitory interneurons (both recurrent and feedforward) as well as
recruitment of different populations of these interneurons (Buzsaki,
1984). Even the lowest 0/0 superthreshold condition pulse current
intensity, however, resulted in the complete elimination of the PS at
20 msecs IPIs.
Increasing the current intensity of the condition pulse of A70
paired pulses resulted in an increased magnitude of inhibition. The
responses seen with A/0 stimulation consisted of a maximally inhibited
response found at the 20 msecs IPI followed by a prolonged decay phase
lasting hundreds of msecs. The maximal magnitude of inhibition of the
test pulse PS was to 20 percent of the control response. A/0 condition
pulses never resulted in a complete abolishment of the test pulse PS
response. This finding coupled with the 0/0 homosynaptic results
suggests that feedforward inhibition contributes substantially to the
magnitude and duration of inhibition observed with homosynaptic paired
pulse stimulation.

224
The responses observed with heterosynaptic paired pulse stimulation
resulted only in inhibited PS amplitudes and looked much like responses
found after A/0 stimulation. While it has been reported previously that
0/0 heterosynaptic paired pulses resulted in greater durations and
magnitude of inhibition than 0/0 homosynaptic paired pulses (Haas and
Rose, 1982), the responses found in the current studies did not fit this
order. It was suggested that these differences may be a function of
differences in the bathing medium temperature. Haas and Rose (1982)
used a temperature of 32 to 33 degrees centigrade, while the present
studies used temperatures of 34 to 35 degrees centigrade. Small differ
ences in temperature can significantly alter the excitability of the
pyramidal cell (Teyler, 1980).
CET was seen to significantly reduce the magnitude of the decay
phase of recurrent inhibition in vitro. This finding tentatively
supports the conclusion of Abraham et al. (1981). However, it is also
possible that the difference seen between the two groups is a function
of feedforward inhibition. This is suggested since antidromic stimula
tion has been shown to produce maximal levels of inhibition at 50 msecs
post-stimulus (Alger and Nicoll, 1982). The effects of CET were seen
through 400 msecs IPIs. Effects in this time period are more likely a
function of feedforward inhibition since maximal orthodromically acti
vated inhibition is seen at approximately 200 msecs after stimulus
onset. 0/0 homosynaptic paired pulse responses, however, resulted in
maximal group differences at shorter (30 to 80 msecs) IPIs, thus strongly
implicating decrements in recurrent inhibition. The fact that the
trends for increased PS amplitudes were found across each of the 0/0
homosynaptic paired pulse paradigms provides a reasonable basis for

225
concluding that CET does produce deleterious effects in the normal
functioning of the CAl pyramidal cell. The fact that the increased PS
response amplitude is seen over the range of IPIs discussed implicates
altered functioning of both recurrent and feedforward inhibitory mecha
nisms .
The effect of four putative neurotransmitter agonists or antagonists
were tested along the dendritic axis of the pyramidal neuron. GABA and
baclofen have been reported to produce an inhibition of the PS. GABA
has been shown to mediate both recurrent and feedforward inhibition
(Andersen et al., 1964a, 1982). Recently, it has been suggested that
baclofen may preferentially activate the GABA-sensitive bicuculline-
insensitive receptor, thus providing a GABA agonist which selectively
activates feedforward inhibition. Bicuculline, a GABA antagonist, has
been shown to competitively block GABA receptors. Finally, 2DA, an
opiate agonist, has been shown to result in increased PS amplitudes
possibly as a function of disinhibition.
GABA produced two different response patterns depending on whether
SO or SR afferents were stimulated. With SO stimulation maximal inhibi
tion was observed in the proximal SO and in the pyramidal cell layer.
Responses to GABA at other locations along the pyramidal cell dendrites
with SO stimulation showed potentiated PS responses. SR stimulation
resulted in a larger area of inhibited responses. Inhibition was seen
beginning approximately 200 uMs in SR extending through the pyramidal
cell layer and briefly into SO. Other locations along the dendrites
outside of this region with SR stimulation resulted in potentiated
responses. The magnitude of inhibition was not as great in these
studies as reported by Andersen et al. (1982). The magnitude of

226
potentiation, however, was greater than reported by Andersen. These
differences were considered to be due to one of three possibilities.
The first possible reason was a difference in the species used. The
second reason may have been due to a difference in temperature. The
present bath temperature was higher than that used by Andersen et al.
(1982). Finally, the protocol for the iontophoretic ejection of GABA
and the subsequent testing of the GABA response was slightly different
in the present study from that reported by Andersen et al. (1982).
Presently, there was a brief interval between the end of the ejection of
GABA and the stimulation of the afferents. Andersen et al. (1982) had
no such pause, but instead ejected GABA while testing the response.
Bicuculline, iontophoretically applied at four locations along the
pyramidal cell dendrites, resulted in increased PS amplitudes and
multiple spikes with all animals tested. SR or SO afferent stimulation
produced responses which were greatest when bicuculline was applied in
proximal SO and SR. Applications of bicuculline in distal SO and
especially distal SR resulted in smaller levels of potentiation.
Baclofen resulted in inhibited PS response amplitudes at each of
the ejection locations. There was, however, a striking dependence of
the magnitude of the baclofen induced response on the afferent zone
stimulated. SR stimulation resulted in distal SR and proximal SR being
most inhibited. Proximal SO was still strongly inhibited, but distal SO
was only weakly inhibited. With SO stimulation, however, distal SR was
weakly inhibited, proximal SR was moderately inhibited, while both SO
iontophoresis sites were strongly inhibited. These results suggest the
existence of baclofen mediated inhibition at all locations along the
pyramidal cell dendrites. The fact that no potentiation was seen, as

227
was observed with GABA, indicates a different time course of action, if
not a difference in the site of action, since GABA was never seen to
produce inhibition in distal SR.
The iontophoresis of 2DA along the pyramidal cell dendrites, like
bicuculline, resulted in increased PS amplitudes as well as multiple
spikes in all animals tested. Maximal response increases were seen in
proximal SR and SO although distal SO also resulted in substantially
potentiated responses with both SR and SO stimulation. There was
virtually no effect observed with 2DA administration in distal SR. The
small response seen in distal SR did not reach maximal levels until
three minutes after ejection, indicating this effect was probably a
result of the diffusion of 2DA toward the active cell body region.
These response patterns were similar to those reported previously
(Dingledine, 1981).
GABA iontophoresis resulted in trends for the CET PS amplitudes to
be more inhibited or less potentiated (at various regions along the
pyramidal cell dendritic tree) than sucrose-fed controls. It was
hypothesized that a decrease in afferent projections per se or a de
creased release of GABA would in turn result in an up-regulation
and/or increased sensitivity of GABAergic receptors. In this way,
increased PS amplitudes with paired pulses could be explained as well
as the increased GABAergic response in CET versus sucrose-fed
controls.
Bicuculline administration resulted in increased PS responses for
CET animals when compared to the sucrose-fed controls, especially in
proximal and distal SO. This increased response to bicuculline was
thought to result from the previously hypothesized increase in sensitiv
ity of the GABA receptor. Since bicuculline competitively blocks the

228
GABA receptor, any increase in sensitivity to GABA would be expected to
result in an increased sensitivity to bicuculline hence, an increased
block of the GABAergic receptor in CET animals compared to sucrose-fed
controls.
Baclofen did not result in any differences between CET and sucrose-
fed control animals. There was a slight trend at the proximal SO
iontophoretic site with both SO and SR stimulation, for CET animals to
be slightly more inhibited. This finding is similar to that found with
GABA administration.
The administration of 2DA along the pyramidal cell dendrites
resulted in stimulation site dependent differences between CET and
sucrose-fed animals. Stimulation in the SR resulted in increased PS
amplitudes in CET animals when compared to controls. These data could
be explained by the decreased levels of inhibition found after CET.
When 2DA was administered with SO stimulation, the CET animal responses
were decreased by up to 400 percentage points when compared to the
sucrose-fed animals. This response pattern was suggested to be due to
either a loss of enkephalinergic interneurons or to a decreased release
of enkephalin from these interneurons. Furthermore, it would have to be
hypothesized that SO afferents preferentially terminate on this popula
tion of enkephalinergic interneurons and/or that CET preferentially
affects these enkephalinergic interneurons or the afferents projecting
to them. SO afferent fibers have been suggested to preferentially
synapse with interneurons in CAl (Buzsaki, 1984). It is therefore
possible that these interneurons are enkephalinergic or that enkephalin
is co-localized with GABA. Most importantly, however, these results

229
suggest that SO afferents terminate on a set of interneurons which are
differentially affected by CET.
It has recently been shown that immediately after 20 weeks of CET
there is a decrease in CAl pyramidal cell spine density, but that 20
weeks after ethanol discontinuance the spine density of the pyramidal
cell recovers to levels above controls (King, 1984). These findings
raise one of the most important questions for the present findings. Are
the effects of CET that we are observing the same as the effects we
would see shortly after ethanol discontinuance and are they the same
effects we would see after 20 weeks off of the ethanol diet? Are we
seeing direct effects of ethanol treatment eight to 16 weeks after
ethanol discontinuance or are we seeing the effects of CET after compen
satory reorganization? While lesioned animals undergo the majority of
compensatory changes by 30 days post-lesion (Matthews et al., 1976b),
there is no satisfactory evidence available to indicate whether the
toxicological effects of CET may extend or shorten this recovery period.
These questions are important in regard to the implications for the
present results. If we are in the middle of the compensatory process,
the present findings may have little bearing on what the same responses
might be after just a few weeks more recovery. Alternatively, compensa
tory changes may have ended well before the current observations were
made, in which case the current results take on significant meaning.
Obviously, it is now important to explore the time course of recovery in
detail. It may be possible that different subfields of the hippocampus
have different time courses of recovery or different courses of compen
satory change as well. It is also possible that different types of
neurons have different recovery characteristics. In line with following

230
the course of recovery from anatomical and electrophysiological perspec
tives, it is possible that responses to behavioral tasks which have been
shown to be altered by CET may also recover. These questions are
especially important in light of King's (1984) results.

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Wigstrom, H. and Gustafsson, B. Two types of synaptic facilitation
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BIOGRAPHICAL SKETCH
Carl J. Rogers was born on January 4, 1953, in Bridgeport, Conn.,
to Carl Fredrick and Sophie Rogers of Orange, Conn. He married Jane
Walther in August, 1979.
Rogers attended Stratford High School, Stratford, Conn., graduating
in 1971. He then attended Fairfield University in Fairfield, Conn.,
until 1972 when he joined the United States Air Force. While in the
service, Rogers earned his Bachelor of Arts degree in behavioral science
from the University of Maine at Presque Isle. He was honorably
discharged from the Air Force in 1976.
Rogers received a Master of Arts degree in psychology from the
University of Missouri at Kansas City in May 1978. He then worked at
the University of Connecticut prior to starting work on his doctoral
degree at the University of Florida in August 1979. He will receive a
Ph.D. degree in May, 1986. His major research interest has been the
study of long term ethanol effects on the hippocampus, an area of the
brain thought to be involved in memory processing. He has developed a
keen interest in the use of microcomputers in the laboratory and at
home.
He began work on his postdoctoral position at the University of
Michigan during the Fall of 1985. There he is studying the effects of
GABA, benzodiazepine and opiate agonists and antagonists with whole cell
and patch clamp recordings under the supervision of Dr. Robert L.
Macdonald.
244

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Zk
Don W. Walker, Chairman
Professor of Psychology and
Neuroscience
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Marc Branch
Professor of Psychology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
%
7/
Floyd Thompson
Associate Professor
Neuroscience
of

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Don Stehouwei/
Assistant Professor of Psychology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Neuroscience
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Bruce Hunter
Associate Professor of
Neuroscience
This dissertation was submitted to the Graduate Faculty of the
Department of Psychology in the College of Liberal Arts and Sciences and
to the Graduate School and was accepted as partial fulfillment of the
requirements for the degree of Doctor of Philosophy.
May, 1986
Dean, Graduate School



11
alcoholics and ablated individuals. Korsakoff's syndrome patients
tended to exhibit enhanced forgetting when distractor variables were
used with STM tasks while H.M., like amnesiacs, showed normal STM decay.
Korsakoff's syndrome patients exhibited decreased performance when
retrieving information from LTM when an interfering task was included.
Hippocampal lesioned patients also appeared to exhibit interference
effects although they may be modality specific (DeLuca et al., 1975).
These comparisons indicate that although there are some differences in
the behavior of these two groups of patients, the similarities are quite
strong.
The above similarities are not without important problems. One of
the most vexing problems with Korsakoff's syndrome patients is the
relatively low correlation found between hippocampal anatomical altera
tions and the severity of memory problems. Victor et al. (1971)
indicated that the hippocampus was bilaterally involved in 36 percent of
the cases. The mammillary bodies, however, a structure which receives
much of its input from the fornix and which largely emanates from the
hippocampus, was much more consistently involved. Still, even the
mammillary bodies were not found to be involved in 100 percent of the
cases studied. One lesion which has been consistently found in
Korsakoff's patients is in the dorsomedial nucleus of the thalamus
(Victor et al., 1971). More recently, it has been hypothesized that
several different types of amnesia may exist. Although the dorsomedial
thalamus may be strongly implicated in the storage, retrieval and
encoding of information, the hippocampus may be responsible for specific
types of memory processing such as spatial memory (Horel, 1978).
Another argument concerning the degree of involvement of these various


percent change from control
- *> o a ia i
o O O O O O O O O O O O O o
percent change from control
.SSSSSSgSSSSS
212


12
brain structures reported by Victor et al. (1971) is that they were the
result of gross pathological examinations and not histopathological
data. Thus, while gross changes were reported, more subtle yet equally
devastating consequences of long term alcohol abuse may exist in the
microstructure of these and other brain areas. Other investigators have
shown the hippocampus to be consistently implicated from autopsy materi
al of human alcoholic patients (Brion, 1969; McLardy, 1975; Miyakawa et
al., 1977).
The data presented above suggest that while hippocampal damage may
be seen with long term alcohol intake, it is not the most highly impli
cated structure. However, because the hippocampus has been so thoroughly
studied behaviorally, anatomically, and physiologically in the normal
animal, it allows for a much more thorough and detailed examination of
the effects chronic ethanol treatment may produce than does nearly any
other brain structure otherwise thought to be involved in memory pro
cessing.
The Normal Animal Hippocampal Evidence
Although the effort to develop an animal model of the human
amnesiac syndrome has been strong, no animal model has yet produced a
symptomatology which completely replicates that found with H.M. Instead
of performance deficiencies always being found, the animal literature
has shown that hippocampectomy can actually produce performance
advantages with some tasks. One of the important questions, however, is
whether behavioral tasks designed for animal models truly replicate
those tests used with human patients. The extent of the hippocampal
literature is enormous and has been well reviewed (O'Keefe and Nadel,


7
generated in the hippocampus, an area considered by many to be implicat
ed in memory processing (Begleiter et al., 1980; Halgren et al., 1980).
The evidence from human alcoholics when using EEG recordings
indicated widespread, fast activity when compared to non-alcoholics
(Coger et al., 1978). Event related brain potentials have shown that
the late P300 components were decreased in amplitude in alcoholics when
compared to controls (Begleiter et al., 1980; Porjesz and Begleiter,
1981). The P300 component has been used as an indicator of the
evaluation of stimulus significance, "an electrophysiological manifes
tation of the orienting response" (Begleiter et al., 1980, p. 10). This
relation of the P300 component of evoked potentials to stimulus
significance is relevant to alcoholics since alcoholics do not differen
tiate relevant from non-relevant stimuli as do normals (Begleiter et
al., 1980). More recently, it has been reported that after ending the
consumption of alcohol after long term use, a recovery of the amplitude
of the P300 component occurs although never back to control levels
(Porjesz and Begleiter, 1981).
General Considerations
A number of problems exist in the above studies from human alcohol
ics when considering the correlations between variables such as duration
of drinking and quantity consumed and the extent of neuropsychological
and neuropathological deficits (Parker and Noble, 1980). These variables
are not necessarily good predictors of pathology (Ron, 1977). Alcoholics
with severe neocortical atrophy may not show appreciable losses in
cognitive function while conversely, alcoholics with no grossly apparent


52
intracellular techniques (Wigstrom and Gustafsson, 1981). The range of
IPIs utilized in these studies was therefore expanded to include IPIs
from 20 msecs to 3 secs.
A final goal of these experiments was to better describe the effect
of current intensity on the characteristics of paired pulse inhibition
and potentiation. In these experiments the effect of varying condition
and test pulse current intensity was investigated. While a number of
different current intensities have been used previously (Creager et al.,
1980; Haas and Rose, 1982; Assaf and Miller, 1978; White et al., 1979;
Chaia and Teyler, 1982; Steward et al., 1977), there have been no
systematic, parametric analyses of the effect of varying condition and
test pulse current intensities with the paired pulse paradigm,
especially not with a wide range of IPIs. If different populations of
interneurons can be recruited as a function of current intensity as has
been suggested (Ashwood et al., 1984), it may be possible to use this
parameter to selectively activate feedforward and recurrent types of
inhibition. These experiments should therefore provide important
information regarding the extracellular correlates of feedforward and
recurrent inhibition as well as some of the parametric conditions
frequently used in paired pulse studies but which have heretofore lacked
comprehensive parametric exploration.
Methods
Animals
Animals used were as described in the general methods except that
only sucrose-fed control animals were used.


180
time after iontophoresis (mins )


34
et al., 1980; Segal, 1977; Zieglgansberger et al., 1979). In either
case the net effect of opiate administration is to increase the
amplitude of an extracellularly evoked PS.
The administration of morphine to the basal dendrites or soma
region of CA1 pyramidal cells has been reported to produce membrane
depolarization whereas application to the apical dendrites produced a
hyperpolarization (Robinson and Deadwyler, 1980). These findings are
important in that they are opposite to those found with discrete appli
cations of GABA to these same cell regions. Thus, the differential
effect seen with the GABA inhibitory internuerons is mirrored by the
opiate data. These findings support an opiate-GABA interaction at CA1
pyramidal cells in the hippocampus.
Rationale and Introduction to Current Experiments
Chronic alcohol animal studies
Anatomy. CET has been shown to produce alterations in the normal
morphology of the rodent hippocampus as well as the cerebellum. Using
the Golgi stain, Riley and Walker (1978) reported CAl pyramidal cell
basilar dendrites to be severely reduced in overall length and number of
branches. The apical dendrites were not as dramatically affected except
for the distal extent where arborization was decreased. In addition,
the basilar dendritic fields had significantly reduced spine densities.
DG granule cell dendrites also had reduced spine densities. A decrease
of 15 to 20 percent in the number of CAl and CA3 pyramidal cells,
dentate granule cells and cerebellar granule cells has also been report
ed (Walker et al., 1980; Tavares et al., 1983).


difference from control (msecs)
195
time after iontophoresis (mins)


222
is seen with 0/0 homosynaptic stimulation at short IPIs. Thus, 0/0
homosynaptic stimulation, as used by Abraham et al. (1981) results in
the activation of both recurrent and feedforward inhibition. 0/0
heterosynaptic stimulation, in which two stimulation electrodes are
placed either in two different pyramidal cell afferent zones, such as
the SO or the SR, or in two clearly defined and different regions of the
same afferent region such as the SR, results in the activation of
recurrent and feedforward inhibition without any facilitation of the
EPSP. If the responses of these three different paired pulse
configurations are then compared, it may be possible to determine the
relative contribution of recurrent versus feedforward inhibition.
An additional variable that was used to explore recurrent and
feedforward inhibition was the intensity of the condition and test pulse
of paired pulses. It was thought that different current intensities may
selectively recruit feedforward and recurrent inhibitory interneurons.
A number of different condition and test pulse current intensities were
therefore tested with the paired pulse paradigm.
Finally, paired pulse responses were tested both in vivo and in
vitro. CET induced responses using the in vivo preparation have
always been suspect because urethane anesthesia (a barbiturate) has
been shown to be cross tolerant with ethanol (Kalant et al., 1971).
Cross tolerance has really never been likely in our preparations since
these animals have been off of the ethanol diet for at least two
months prior to any electrophysiological testing. The in vitro
responses after CET resulted in patterns of increased PS amplitude as
was shown in vivo. Although there was a shift in the IPI at which
both CET and sucrose-fed animals were maximally potentiated in vitro.


85
al., 1984; Buzsaki, 1984). This observation implies that feedforward
inhibition can therefore influence pyramidal cell firing even with
single pulse stimulation. Extracellularly, the amplitude of the PS
could be decreased as a function of the feedforward inhibition.
One of the first tests in many extracellular investigations is that
of determining the I/O curve. The I/O curve provides a good description
of the input current strength to response amplitude. It has typically
been considered an unencumbered measure and has therefore been used to
compare the basic response of one treatment group to another or to a
control group. The inference has been that differences signify altera
tions in the normal functioning of the pyramidal cell. If feedforward
inhibition acts prior to pyramidal cell firing and thereby alters the
response, then the basic assumption of differences in I/O curves being
due to changes in pyramidal cell function may be inaccurate. If differ
ent treatment conditions are being tested, it may be that the treatment
condition has altered the feedforward inhibitory interneuron which may
in turn influence the pyramidal cell firing characteristics. If feed
forward interneurons fire differentially to various current intensities,
feedforward inhibition may play a major role in determining the sigmoidal
shaped I/O curve when tested with different current intensities. The
abrupt appearance of the PS may be less reflective of the threshold for
the PS per se. than it is the point at which the PS finally emerges from
the previously overwhelming control of inhibitory mechanisms. Threshold
differences between groups may therefore be a function of effects on
feedforward inhibition.
These studies suggest that the magnitude of the PS amplitude and
latency responses to different current intensities can be carefully


207
distal SR iontophoretic site did not respond to either SO or SR stimula
tion in a manner comparable to the other three bicuculline application
sites. It is suggested that the small increase seen at the distal SR
site was a result of the diffusion of bicuculline toward the pyramidal
cell body region.
CET resulted in increased PS amplitudes when compared to the
sucrose-fed control group with both SO and SR stimulation. As can be
seen in Fig 6-14A and Fig 6-14B, the greatest differences between the
two groups were found near the cell body region. These data indicate
that bicuculline produced a greater antagonism of GABA receptors in CET
animals than in controls. Bicuculline has been shown to competitively
antagonize GABA at the receptor level (Cooper et al., 1982). A greater
antagonism of GABA receptors in CET animals might occur as follows. If
the release of GABA is decreased in CET animals due to a loss of
inhibitory interneurons or to a presynaptic alteration of GABA-ergic
synthesis or release, a subsequent up-regulation and/or increase in GABA
receptor sensitivity might be expected. The increased response to GABA
iontophoresis along the dendrites in CA1 in CET animals could be
explained by this sequence of events. Since bicuculline competitively
blocks GABA receptors, a decreased release or availability of GABA
followed by an increase in GABA receptor sensitivity would likely result
in an increased sensitivity to the iontophoretic application of
bicuculline. Thus, if the effects of bicuculline mimic the differential
effects seen with CET and sucrose-fed animals after GABA iontophoresis
but in the opposite direction, an increased response to GABA
administration should result in an increased response to bicuculline
administration. It is also possible that CET may decrease the GABA


ri**v.SC inLkva*. (wC) nTIJva. (usCC)
percent change from control
percent change from control
29


percent change from control percent change from control
156
iontophoretic location


90
experiments in this section per se. these experiments are crucial to the
pharmacological experiments proposed in the following chapter since they
lay the groundwork for comparisons between in vivo and in vitro results.
It is important to verify that similar patterns of CET effects are found
between in vitro and in vivo preparations.
The major negative characteristic of the in vitro technique
emanates from the fact that transverse hippocampal slices have reduced
levels of inhibition most probably due to the severance of the longitu
dinal projections of the recurrent inhibitory interneurons (Teyler,
1980). The result of this decrease in inhibition is that paired pulse
response curves do not typically show inhibition with IPIs less than 30
msecs whereas in vivo, inhibition is seen with IPIs as long as 80 to 150
msecs (Dunwiddie et al., 1980). Differences in the levels of inhibition
between CET and normal animals should, however, be readily apparent if
they exist.
A third question which was posed was whether heterosynaptic affer
ent paired pulse stimulation could be effectively used to provide
additional insight into how and where the inhibitory systems might
interact. It has been reported previously that stimulation of the SO or
SR afferents with the condition pulse followed by SR or SO afferents,
respectively, on the test pulse of paired pulses produced only inhibi
tion (Haas and Rose, 1982; Steward et al., 1977). As indicated earlier
A/0 paired pulses result in a moderately clean assessment of recurrent
inhibition. Homosynaptic 0/0 paired pulses result in three different
mechanisms being activated. Feedforward inhibition, recurrent inhibi
tion and an EPSP facilitation are all seen to occur when stimulating
with double pulses in the same afferent region. Heterosynaptic 0/0


CHAPTER 7
GENERAL DISCUSSION
Chronic ethanol treatment has been shown to produce alterations
in the behavior (Walker and Hunter, 1978; Walker and Freund, 1973;
Freund and Walker, 1971; Denoble and Begleiter, 1979; Fehr et al.,
1975), anatomy (Walker et al., 1980; Riley and Walker, 1978; McMullen
et al., 1984; King, 1984) and electrophysiology (Abraham et al., 1981,
1982, 1984; Durand and Carien, 1984a, 1984b) of the rodent hippocampus.
Anatomical changes have included a 15 to 20 percent loss of CAl
pyramidal cells as well as dentate gyrus granule cells. Decreased
dendritic spine loss and reduced dendritic branching has also been
found after CET (Riley and Walker, 1978). Electrophysiological
evidence from the CAl region indicated a more constrained synaptic
field in proximal SR dendrites of pyramidal cells with CSD analyses
(Abraham et al., 1982). Each of these anatomical alterations indicates
deleterious effects from long term ethanol ingestion. It should be
noted that these results were obtained at least eight weeks following
the discontinuance of ethanol treatment. They are, therefore, residual
effects.
Functionally, the major finding in the CAl region has been that
CET resulted in increased PS amplitudes with paired pulse testing
(Abraham et al., 1981). It was hypothesized that the increased PS
amplitude was a function of a decrease in the efficacy of recurrent
inhibition. Recurrent inhibition has been shown to have a powerful
regulatory control of CAl pyramidal cell firing once the pyramidal
cell has been activated (Andersen et al., 1964a, 1964b).
219


119
responses for these two groups were statistically different (F(l,21) =
6.82, p .012). The PS latency measures (Fig 4-8B) showed increased
latencies for both groups. The alcohol group latencies peaked at the 20
msecs IPI with the latency being .5 msec longer than the control
response. The sucrose group peaked at the 40 msecs IPI with the latency
.35 msec longer than the control responses. The latency measures for
both groups then came back toward control levels though they remained
longer than control responses beyond the 400 msecs IPI. PS latencies
were not, however, statistically significant between groups.
Set 2
The remaining experiments were performed in a set of animals
different from those reported above. One of the experiments used the
same condition and test pulse current intensities as that reported above
but provided a control for the additional experiments in this set of
animals. The experiments reported above explored the effect of
different condition and test pulse current intensities oti 0/0 and A/0
paired pulse configurations. This next set of experiments was designed
to explore the effect of four different stimulus configurations each
with similar condition and test pulse current intensities.
0/0 homosvnaptic paired pulse naradipms -- Set 2
Two homosynaptic 0/0 paired pulse paradigms were performed, one
using SR stimulation and the other using SO stimulation. Both of these
experimental paradigms used current intensities adjusted to 25 percent
of the PS amplitude at PS asymptote. Fig 4-9B shows the PS amplitude
responses for the 0/0 paired pulse experiment with SR stimulation.


69
The slope data (Fig 3-4B) for this series showed a period of
inhibition at the 20 msecs IPI which remained slightly inhibited to
approximately the 150 msecs IPI. There was no apparent ordering of
slope by current intensity except that less inhibition was observed with
a subthreshold condition pulse than with a superthreshold condition
pulse.
A/0 25 percent test pulse series data
The A/0 25 percent of the PS maximum test pulse series (Fig 3-5A)
showed that increasing the current intensity of the antidromic condition
pulse increased the magnitude of the inhibition to a greater extent than
the duration of inhibition. Unlike the 0/0 experimental series reported
above, the A/0 series did not show potentiation. Instead, there appeared
to be two phases to the inhibition. There was a short initial period at
IPIs of 20 to 30 msecs when inhibition was greatest, followed by a
prolonged period where less but continued inhibition was found. The
antidromic condition pulse of 25 percent of the maximum antidromic spike
amplitude resulted in a test pulse response which was maximally inhibited
to 50 percent of the control test response at the 20 msecs IPI. The 50
and 100 percent antidromic condition pulses inhibited the test pulse
response to 22 and 14 percent of the control test response, respectively.
The IPI at which the 50 percent condition pulse produced its maximum
effect was at 20 msecs while the 100 percent condition pulse was maxi
mally inhibited at 30 msecs IPIs. Each of the condition pulse current
intensities produced a test pulse response which was inhibited to at
least 70 percent of the control response for the duration of the IPIs


2
more effective treatment and preventative programs to be formulated. It
is therefore necessary to ask the questions, what are the long term
effects of alcohol abuse from behavioral, anatomical, physiological, and
biochemical perspectives? Does alcohol permanently alter the way in
which we behave, the way in which we think? If a lifestyle of alcohol
use and abuse does produce deleterious effects, are there limits beyond
which permanent changes occur and, for those individuals who have gone
beyond those limits, is there a way to reverse, or at least help to
compensate for, those damages? In order to provide effective treatment
and to properly educate the public we must know what long term alcohol
consumption does and by what means. The cost of this research will
surely be minuscule compared to the cost alcohol abuse currently imposes
on society in terms of lost cognitive abilities alone. It is estimated
that there may be as many as 20 million people in the United States who
can be termed problem drinkers (Clark and Midanik, 1982; Malin et al.,
1982) .
The experiments described herein were designed to provide additional
information concerning the effects of long term ethanol abuse on the
function of the hippocampus, a vital area of the brain thought to be
involved in memory processing. If we can understand why and how ethanol
produces deficits in memory function, it may be possible to provide more
effective treatment for some of the cognitive dysfunctions of alcoholic
patients. Perhaps even more important is the idea that the more substan
tial the information provided to the general public concerning the
effects of ethanol use and abuse, the more likely the public will be to
evaluate the consequences of ethanol use in terms of their own physical
and mental well-being. Thus, a better understanding of ethanol effects


Figure 4-7. A/O paired pulse PS amplitude responses (means + SEM) with condition pulse
stimulation through the alveus and test pulse stimulation through stratum
radiatum. All test responses are shown as a percent of control responses (test
response/control response X 100). A) Test pulse responses obtained with the
antidromic condition pulse current intensity set to obtain a response 50 percent
of the maximum antidromic amplitude. Test pulse response was set to 25 percent
of the PS amplitude at PS asymptote. B) Test pulse responses obtained with 100
percent of the antidromic spike amplitude on the condition pulse and 50 percent
of the PS amplitude at PS asymptote on the test response.


Figure 6-9. PS latency responses (means) after baclofen
iontophoresis (time=0) with stratum oriens stimulation.
Scales on ordinate represent difference of PS latency
response following bicuculline administration from
control responses obtained prior to bicuculline adminis
tration (test response-control response) and are
independent for each iontophoretic ejection location.
Abcissa represents time in minutes following ionto
phoretic ejection of the drug. A) PS responses with
iontophoretic ejection in distal SO. B) PS responses
with iontophoretic ejection in proximal SO. C) PS
responses with iontophoretic ejection in proximal SR.
D) PS response with iontophoretic ejection in distal
SR.


Figure 4-12. A/O and O/O Heterosynaptic paired pulse PS latency responses (means). Note the
individual configurations described below. All test responses are shown as
differences from control responses (test response control response in msecs).
A) Test pulse responses obtained with antidromic stimulation from the alveus on
the condition pulse and orthodromic stimulation from the stratum radiatum on
the test pulse. The condition pulse current intensity was adjusted to obtain
the maximum (100 percent) sized antidromic spike. The test pulse was adjusted
to 25 percent of the PS amplitude response at PS asymptote. B) Test pulse
responses obtained with condition pulse stimulation through stratum radiatum
and test pulse stimulation through stratum oriens. The condition and test pulse
current intensities were set to obtain a PS amplitude response 25 percent of
the PS amplitude at PS asymptote. C) Test pulse responses obtained with
condition pulse stimulation through the stratum oriens and test pulse
stimulation through the stratum radiatum. The condition and test pulse current
intensities were set to obtain a PS amplitude response 25 percent of the PS
amplitude at PS asymptote.


OirrxACNCC rmou control (mscC)
orrtcxcc rmou cowtkov. (uscc)


57
Results
I/O relationships
The combined I/O curves for the two sets of animals are shown in
Fig 3-1. These data are from a total of 22 animals. The PS threshold
was 252 uAs + 12.31 (data presented as mean + SEM). The PS response
amplitude asymptote was 552 41.36 uAs current level.
Characterization of paired pulse paradigms in normal animals
The data from these experiments were divided into four major group
ings based on the stimulus configuration and the current intensity of
the test pulse used. The four groupings were: 1. the 0/0 25 percent
test pulse series, 2. the 0/0 50 percent test pulse series, 3. the A/0
25 percent test pulse series, and 4. the A/0 50 percent test pulse
series. For all of the superthreshold paired pulse experiments reported
herein, the general patterns and amplitudes of the PS inhibition and
potentiation are comparable to those previously reported for the CAl and
dentate gyrus regions of the hippocampus (Creager et al., 1980; Lomo,
1971; Steward et al., 1977). The EPSP slope data, however, were not
similar to these previous reports as discussed subsequently.
0/0 25 percent test pulse series
The 0/0 25 percent test pulse series data showed that increasing
the current intensity of the condition pulse, if the current intensity
was above PS threshold, produced an increase in the duration of inhibi
tion. When the test pulse PS amplitude was expressed as a percentage of
the control response, the PS was almost abolished from 20 to 30 msecs
for the 25 percent condition pulse, extending to 50 msecs for the 100


173
along the dendritic axis of the pyramidal cell (Fig 6-1). The first
location was always in SO near the recording electrode located in the
pyramidal cell layer. The second location was in SO approximately
two-thirds of the distance from the cell layer to the alveus. The third
location was in SR near the recording electrode. The placement of the
drug containing pipette near the recording electrode in these
experiments was not as close, by approximately 25 to 50 microns, as that
aimed for in the GABA experiments. The fourth location of the
iontophoretic pipette was in distal SR approximately at the border
between the SR and the SIM. Pharmacological agents were also
administered in the following order: 1. proximal SO, 2. distal SO, 3.
proximal SR, and 4. distal SR-SLM border. Prior to the ejection of the
drug, a single baseline response was obtained. Ten seconds later the
drug to be tested was ejected using the currents outlined in Table 6-1
for five seconds. Responses were then collected every 30 seconds for
the next 10 minutes with afferent stimulation from the SR stimulation
electrode. The entire experiment with the same drug and application
location was then repeated using the SO located stimulation electrode.
The iontophoretic pipette was then moved to the next location and the
procedures repeated until all four application sites were tested with
both SO and SR stimulation pulses. A minimum of 12 minutes separated
the application of any of these agents.
Results
Bicuculline series
The results for the bicuculline series of animals were based on a
total of 21 rats with 12 in the alcohol group and nine in the sucrose


112
at PS asymptote and with 25 percent of the PS amplitude at PS asymptote
set on the test pulse are shown in Fig 4-5B. Unlike the in vivo series,
no inhibition was seen at any IPI. The PS amplitude responses for the
alcohol animals were potentiated maximally at IPIs from 30 to 80 msecs
to a level of 300 percent. The sucrose animals were potentiated maxi
mally at an IPI of 60 msecs with the PS amplitude potentiated to 250
percent of the control response. There was approximately a 50
percentage points difference between the alcohol and sucrose groups from
20 to 150 msecs IPIs although this difference was not statistically
significant. The separation seen between groups with the PS amplitude
response was also seen with the latency to the PS measure (Fig 4-6B).
Alcohol animals had shorter latencies to the onset of the PS than did
the sucrose animals although there were no statistically significant
differences. It can be seen that the sucrose group PS latency was
decreased by .4 msec at IPIs of 20 to 40 msecs while the alcohol group
reached its shortest latency to the PS at 40 msecs extending to 80
msecs. The alcohol group PS latency was shortened by .8 msec from the
control response at these IPIs. The two groups were separated by .4
msec from 40 to 200 msecs IPIs.
PS amplitudes for 0/0 homosynaptic paired pulse responses with the
condition pulse current intensity set to 50 percent of the PS amplitude
at PS asymptote and with the test pulse current intensity set to obtain
a response 25 percent of the PS amplitude at PS asymptote are shown in
Fig 4-5C. The alcohol group PS amplitude measures (Fig 4-5C) were
potentiated maximally between 40 and 60 msecs IPIs. The highest levels
of potentiation were approximately 340 to 350 percent of the control
responses. The sucrose group PS amplitude responses were maximally


228
GABA receptor, any increase in sensitivity to GABA would be expected to
result in an increased sensitivity to bicuculline hence, an increased
block of the GABAergic receptor in CET animals compared to sucrose-fed
controls.
Baclofen did not result in any differences between CET and sucrose-
fed control animals. There was a slight trend at the proximal SO
iontophoretic site with both SO and SR stimulation, for CET animals to
be slightly more inhibited. This finding is similar to that found with
GABA administration.
The administration of 2DA along the pyramidal cell dendrites
resulted in stimulation site dependent differences between CET and
sucrose-fed animals. Stimulation in the SR resulted in increased PS
amplitudes in CET animals when compared to controls. These data could
be explained by the decreased levels of inhibition found after CET.
When 2DA was administered with SO stimulation, the CET animal responses
were decreased by up to 400 percentage points when compared to the
sucrose-fed animals. This response pattern was suggested to be due to
either a loss of enkephalinergic interneurons or to a decreased release
of enkephalin from these interneurons. Furthermore, it would have to be
hypothesized that SO afferents preferentially terminate on this popula
tion of enkephalinergic interneurons and/or that CET preferentially
affects these enkephalinergic interneurons or the afferents projecting
to them. SO afferent fibers have been suggested to preferentially
synapse with interneurons in CAl (Buzsaki, 1984). It is therefore
possible that these interneurons are enkephalinergic or that enkephalin
is co-localized with GABA. Most importantly, however, these results


Figure 3-7. EPSP slope (means) for 25 and 50 percent of PS maximum test pulse series with
A/0 stimulation (test slope/control slope X 100). A) 25 percent test pulse
series EPSP slope responses with 25, 50, and 100 percent of maximum antidromic
spike on the condition pulse. B) 50 percent test pulse series EPSP slope
responses with threshold (0), 25, 50, 75, and 100 percent of maximum antidromic
spike on the condition pulse.


205
the sucrose group was more potentiated under these conditions than the
alcohol group. This was the first condition in which the sucrose
group PS amplitude measure showed increased potentiation or decreased
latencies when compared to the alcohol group. There were no
statistically significant differences between groups for the data
collected with distal SO 2DA application nor with proximal SO
application for either the PS amplitude or the PS latency responses.
The PS latency data collected with 2DA administration in proximal SO
and with SO stimulation showed significant group (F(l,21) = 6.27, p =
.021) and group by time interaction effects (F(l,420) *= 2.27, p -
.0015). The PS amplitude and PS latency measures obtained when 2DA
was applied in SR near the cell body layer revealed no group effects
for either measure but did reveal a statistically significant
interaction of the group by time for the PS amplitude response
(F(l,420) 1.69, p .03). Subsequent analyses of the differences
between groups at the individual time points showed significant
differences between 90 and 180 secs after 2DA iontophoresis. The
application of 2DA in distal SR with SO stimulation did not produce
any group or interaction differences for the PS amplitude or the PS
latency measures.
Discussion
The present series of experiments have utilized three different
pharmacological agents/neurotransmitters or antagonists which have been
shown to affect CAl inhibitory systems. A nutritionally complete liquid
ethanol diet was administered which has previously been shown to produce
anatomical (Walker et al., 1980; Riley and Walker, 1978; King, 1984),
electrophysiological (Abraham et al., 1981, 1982, 1984; Durand and


Figure 1-1. Schematic diagram of hippocampus showing dentate gyrus, CA3 and CA1 regions.
Electrode placements for the present experiments are shown. The recording
electrode was positioned within or dorsal to the pyramidal cell layer. The
stimulation electrodes were placed in stratum radiatum at the CA2-CA1 border, in
stratum oriens also at the CA2-CA1 border or in the alveus immediately dorsal or
dorsal and medial to the recording electrode.
Abbreviations: ALV, alveus; COM, commissural fibers; hi, hilus; MF, mossy
fibers; PP, perforant path; SCH, Schaffer collaterals; Sg, stratum granulosum;
SM, stratum moleculare; SO, stratum oriens; SP, stratum pyramidale; SR, stratum
radiatum.


165
to 10 msecs delay between the time when GABA iontophoresis was terminated
and when the test pulses were administered may have been of a sufficiently
long duration to begin altering the GABAergic response toward an enhanced
response. The third difference between experimental protocols which may
also help to explain the differences found is that Andersen et al.,
(1982) indicated that the temperature of the bathing medium at the slice
was between 32 and 33 degrees centigrade. As indicated earlier, temper
ature differences can significantly affect the excitability of the CAl
pyramidal cell as measured by extracellular PSs (Teyler, 1980). All
other parameters of this experiment were similar to those used by
Andersen et al. (1982).
The predominant effect of CET on GABA modulation of the PS along
the dendritic axis of the pyramidal cell with both SO and SR stimulation
was an apparent increase in the magnitude of inhibition when compared to
the sucrose-fed controls. The responses to GABA application were
inhibited at certain locations along the dendritic axis and potentiated
when compared to control responses at others. CET, however, resulted in
trends toward more inhibition or less potentiation at all locations
tested. The PS latency responses showed differences between the CET and
sucrose-fed controls with SO stimulation which lends additional support
to the findings with PS amplitude responses. These data suggest the
following mechanism; an increase in the efficacy of the GABAergic
receptors and a decreased release of GABA. The present CET response
pattern occurred even though the extracellular response to paired pulses
showed an impairment in the efficacy of the inhibitory interneurons. A
supersensitivity or increased number of GABAergic receptors would be
likely if there was a decreased afferent input to GABAergic receptors in


difference from control
ON
-tr
HrtVA*. (wscc)
*^T*^vaJC (wSCC)


9
1971). Each of these regions would represent a reasonable location in
which to begin a search for the effects of Korsakoff's syndrome on
memory processing.
The present series of experiments utilize the hippocampus, a
region in which a large amount of literature exists concerning experi
mental variables related to behavior, anatomy and physiology in the
study of memory and other related phenomena. A substantial amount of
literature utilizing the hippocampus and the consequent effects of long
term ethanol treatment has thus followed.
The hippocampus has emerged as a model system for the study of
memory for several reasons. First, the hippocampus has been strongly
implicated in memory processing from human clinical cases (discussed
below). Secondly, the hippocampus itself is an important "model"
system. It is composed of well-defined cell layers as well as dendritic
regions. Inputs and outputs are well characterized and reside within
laminae which have little overlap. The anatomical arrangements of the
dendrites allow for the summation of electrophysiological potentials
which can, in turn, be recorded with relatively simple extracellular
recording techniques. Because of the highly laminated structure of the
hippocampus, the recorded potentials can be interpreted with a high
degree of confidence. It is for all of these reasons that a wealth of
literature on the hippocampus exists from different disciplines which
further facilitate meaningful interpretation.
The Normal Human Hippocampal Evidence
The evidence for the involvement of the hippocampus in normal human
memory emanates from radical procedures used in the mid 1950s to


NTTAU\.SC INTCWVAi. (uSCC)
400
330
300
250
200
iso
100
30
0
alcohol
sucrose +
T 1 1 T 1 1 1
0 100 200 300 400
iWTCAPULSC INTT*v*l. (WSCC)


163
stimulation, but again was similar to Andersen et al. (1982). With SO
stimulation, the application of GABA to the proximal regions of SO and
SR, immediately surrounding the pyramidal cell layer, were the only
areas where inhibited or near inhibited responses were found. All other
sites of GABA application to the dendrites resulted in potentiated
responses in both studies.
The mechanism for the potentiation of the response to GABA in the
distal dendrites has not been well studied or discussed. However, it
has been shown that two different responses to GABA application at the
soma or to the dendrites occurs (Andersen et al., 1982; Alger and
Nicoll, 1982). The iontophoresis of GABA at the pyramidal cell results
in an hyperpolarization of the CAl pyramidal cell when recording intra-
cellularly. In the dendritic regions, especially in SR, a depolarizing
response has been found to GABA application. Both of these responses
are inhibitory. The depolarizing dendritic response has been shown to
result in an increased conductance and hence a shunt of the pyramidal
cell dendritic current. However, it has also been indicated that this
shunting response is localized to the point that areas surrounding the
"shunt" area may be more excitable. Andersen et al., (1982) has de
scribed this feedforward type of inhibition as a "discriminative inhibi
tion" since the effects of this type of inhibition may act as a selec
tive filter to afferent input, decreasing the input of one afferent
while accentuating those around it. The increased levels of potentia
tion seen in more distal regions of the pyramidal cell dendrites may
reflect the increased arborization of the dendritic elements, hence, an
inability to apply GABA to the main dendritic branches and thereby
increasing the probability of cellular discharge by decreasing the


500
400
300
200_
100_
500_
C00_
300
200
I00_
300_
200
100_
0
178
D0201
pstot
ALCOHOL
SUCROSE ...
U_xJ_l_L
<1 I "1 "X'-t -l -t
~iiiiir
I 2 3
iiirriiiiii
5 6 7 8 9 10
time after iontophoresis (mins)


56
intensity that produced a PS amplitude 50 percent of the PS amplitude at
asymptote while the second group was tested with a stimulus intensity
producing 25 percent of the PS amplitude at asymptote. Each of the
indicated (Table 3-1) condition pulse current intensities was tested on
each animal within the test pulse current intensity level. The order of
the individual paired pulse experiments was determined in advance
through a random order selection. The IPIs used for all of these
experiments were as described in the general methods. IPIs were always
presented in the order described.
The current intensity levels used in the paired pulse tests were
based on a percentage of the asymptotic amplitude of the PS from brief
I/O curve functions. For example, 50 percent of the PS amplitude was
one of the current intensities tested. The size of the PS was measured
from the I/O curve at the asymptotic level. The stimulation current
level was then adjusted to obtain a PS response amplitude which was half
as large as the asymptotic PS response amplitude. This procedure was
used in order to control for differences in excitability and asymptotic
levels between animals. By using percentages of asymptotic PS response
levels, a more accurate comparison between animals was made possible.
The different current intensity levels on the condition and test pulse
used are shown in Table 3-1.
Data collection and analyses were performed as described in the
general methods except that analyses of variance were not performed.
The major points to be made in this series of experiments were best
addressed descriptively rather than by measures of statistical differ
ence .


82
level may not be useful in terms of the separation of recurrent versus
feedforward inhibitory influences when applied to the afferent fibers of
the SR region. Nonetheless, this methodology may provide a most useful
extracellular method by which to study the effects of feedforward
inhibition using paired pulses.
Most studies have used a relatively high current intensity on the
condition and test pulses when examining different treatment conditions.
Different condition and test pulse current intensities may provide
substantially different responses since the influence of feedforward and
recurrent inhibition may, in effect, change from predominantly feed
forward influences with a lower current intensity to more equal contri
butions from feedforward and recurrent inhibition with a higher current
intensity. This conclusion is supported by evidence that indicates that
there are two populations of feedforward inhibitory interneurons that
have different firing patterns (Ashwood et al., 1984). One group of
neurons typically fired more than one action potential with frequent
bursts to stimulation which was above PS threshold. These bursts lasted
from 30 to 300 msecs. The other group of inhibitory interneurons
produced only one action potential even when stimuli were as high as 4.5
times threshold. Increasing the stimulus intensity produced shorter
inhibitory interneuron spike latencies. It has also been shown that
inhibitory interneurons can be activated at current levels which are
subthreshold for a PS (Buszaki and Eidelberg, 1981; Knowles and
Schwartzkroin, 1981). Thus, feedforward inhibition may actually prevent
pyramidal cell firing when the stimulation current intensity is near
threshold levels. The finding of inhibition at the 20 msecs IPI for the


Figure 6-10. PS amplitude responses (means + SEM) after enkephalin
iontophoresis (time=0) with stratum radiatum stimulation.
Scales on ordinate represent difference of PS amplitude
response following bicuculline administration from
control responses obtained prior to bicuculline adminis
tration (test response-control response) and are
independent for each iontophoretic ejection location.
Abcissa represents time in minutes following ionto
phoretic ejection of the drug. A) PS responses with
iontophoretic ejection in distal SO. B) PS responses
with iontophoretic ejection in proximal SO. C) PS
responses with iontophoretic ejection in proximal SR.
D) PS responses with iontophoretic ejection in distal
SR.


TABLE OF CONTENTS
PAGE
ACKNOWLEDGEMENTS iii
ABSTRACT vii
CHAPTERS
1 GENERAL INTRODUCTION 1
Foreward 1
Behavior 3
Anatomy 5
Physiology 6
General Considerations 7
The Hippocampal Model and Memory 8
The Normal Human Hippocampal Evidence 9
A Comparison of Hippocampal Deficits with CET Deficits ... 10
The Normal Animal Hippocampal Evidence 12
Chronic Ethanol Treatment (CET) and Behavior in Animals. . 14
Summary of Alcohol and Normal Human and Animal Literature. 15
Animal Alcohol Models 16
The Normal Hippocampus--Anatomy and Physiology 16
Pharmacology of the Hippocampus 27
Rationale and Introduction to Current Experiments 34
2 GENERAL METHODS 38
Animals 38
Alcohol Diet Regimen 38
Equipment 40
Basic Procedures 42
Data Analysis and Interpretation 44
3 CHARACTERIZATION OF SYNAPTIC INHIBITION IN VIVO 48
Introduction 48
Methods 52
Results 57
Discussion 73
4CET AND SYNAPTIC INHIBITION AN IN VIVO ANALYSIS 87
Introduction 87
Methods 91
Results 95
Discussion 131
v


55
TABLE 3-1
PERCENT OF MAXIMAL PS AMPLITUDE
ON TEST PULSE
c
25%
50%
0
1
1
N
0/0
50%
EPSP |
X |
X
D
0/0
0%
PS |
1
X
I
0/0
25%
PS |
X I
X
T
0/0
50%
PS |
X I
X
I
0/0
75%
PS I
1
X
0
0/0
100%
PS I
x 1
X
N
A/0
0%
AS |
1
X
A/0
25%
AS |
X I
X
P
A/0
50%
AS |
X I
X
U
A/0
75%
AS |
1
X
L
A/0
100%
AS |
X I
X
S
1
1
E
Percent of maximal PS amplitude at PS asymptote, EPSP amplitude at
PS threshold, and maximal antidromic spike (AS) amplitude for the
condition and test pulses in paired pulse paradigms (in vivo).


32
of an increase in the chloride conductance of the cell (Kandel et al.,
1961).
The benzodiazepines in the hippocampus
The benzodiazepines have also been reported to influence recurrent
inhibition. High affinity binding sites have been found which fulfill
many of the criteria of pharmacological receptors. However, the precise
location of these receptors has been difficult to determine. A wide
spread distribution was seen rather than discrete localization. En
hanced binding of benzodiazepines was seen when GABA or a GABA analog
was included in the binding assay. Thus, the benzodiazepine receptors
appeared to be associated with the GABA receptors in a receptor complex
(see Tallman et al., 1980; Ticku, 1983; Greenberg et al., 1984).
The benzodiazepines have been shown to increase the duration of
inhibition in the hippocampal CAl pyramidal cell (Adamec et al., 1981;
Wolf and Haas, 1977; Tsuchiya and Fukushima, 1978). It was suggested
that the benzodiazepines either directly act upon the pyramidal cell
soma through benzodiazepine receptors or through a GABA-benzodiazepine
receptor complex (Tallman et al., 1980). When GABA was applied to local
regions of the CAl pyramidal cell soma or dendrites, hippocampal slices
bathed in midazolam, a benzodiazepine, responded with enhanced
hyperpolarizations and depolarizations respectively (Jahnsen and
Laursen, 1981). Benzodiazepine effects on recurrent inhibition have
also been reported to be blocked by the GABA antagonists bicuculline and
picrotoxin (Alger et al., 1981; Tallman et al., 1980; Tsuchiya and
Fukushima, 1978). These findings suggested that the benzodiazepines may
augment recurrent inhibition by either increasing the excitability of


239
Marslen-Wilson, W.D. and Teuber, H.L. Memory for remote events in
anterograde amnesia: Recognition of public figures from news
photographs. Neuroosvch.. 1975, 13: 347-352.
Matthew, D.A., Cotman, C.W. and Lynch, G. An electron microscopic study
of lesion-induced synaptogenesis in the dentate gyrus of the adult rat.
II. Reappearance of morphologically normal contacts. Brain Res.. 1976,
115: 23-41.
McLardy, T. Dentate granule cell sensitivity to proximity of blood
vessels in chronic alcoholism. Int. Res. Comm. Svst. Med. Sci., 1973,
73: 1686.
McMullen, P.A., Saint-Cyr, J.A. and Carien, P.L. Morphological
alterations in rat CAl hippocampal pyramidal cell dendrites resulting
from chronic ethanol consumption and withdrawal. J. Comp. Neurol..
1984, 225: 111-118.
McNaughton, B.L. Long-term synaptic enhancement and short-term
potentiation in rat fascia dentata act through different mechanisms. J.
Physiol., 1982, 324: 249-262.
McNaughton, B.L. and Barnes, C.A. Physiological identification and
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439-454.
Milner, B., Corkin, S. and Teuber, H.L. Further analysis of the
hippocampal amnesic syndrome: Fourteen-year follow-up study of H.M.
Neuropsvchol.. 1968, 6: 215-234.
Miyakawa, T., Hattori, E., Shikai, I., Nagatoshi, K. and Suzuiki, T.
Histopathological changes of chronic alcoholism. Folia Psvchiat.
Neurol. Jap.. 1977, 31: 253-261.
Newberry, N.R. and Nicoll, R.A. Direct hyperpolarizing action of
baclofen on hippocampal pyramidal cells. Nature, 1984, 308 (5958):
450-452.
Newlin, D.B., Golden, C.J., Quaife, M. and Graber, B. Effect of alcohol
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Nicholson, C. and Freeman, J.A. Theory of current-source density
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Nicoll, R.A., Alger, B.E. and Jahr, C.E. Enkepahlin blocks inhibitory
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Nicoll, R.A., Eccles, J.C., Oshima, T. and Rubia, F. Prolongation of
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1975, 258: 625-627.


Figure 6-2. PS amplitude responses (means + SEM) after bicuculline
iontophoresis (time=0) with stratum radiatum stimulation.
Scales on ordinate axis represent percent change of PS
amplitude responses obtained after bicuculline adminis
tration in comparison to control responses obtained 10
seconds prior to bicuculline administration (bicuculline
test/control x 100) and are independent for each ionto-
phoretic ejection location. Abcissa represents time in
minutes following iontophoretic ejection of the drug.
A) PS response with iontophoretic ejection in distal
SO. B) PS response with iontophoretic ejection in
proximal SO. C) PS response with iontophoretic ejection
in proximal SR. D) PS response with iontophoretic
ejection in distal SR.


percent change from control
183
?00
time after iontophoresis (mins)


recurrent inhibition were tested using PPP or with four pharmacological
agents, gamma-aminobutyric acid (GABA), bicuculline, baclofen, and
D-ala(2)-methionine-enkephalinamide (2DA) iontophoretically applied
along the dendrites of CA1 pyramidal cells.
The PPP with control animals showed that increasing condition pulse
current intensity with orthodromic/orthodromic (0/0) stimulation
increased the duration of inhibition and always abolished the PSA at
short interpulse intervals. Antidromic/orthodromic PPP produced an
increased magnitude of inhibition with increasing condition pulse
current intensity, however, the PSA was never completely inhibited.
These findings suggest an important contribution of feedforward
inhibition which, after CET, may be reduced as was RI.
The PPP with in vitro preparations showed CET animals had trends
toward increased PSA similar to those found in vivo. Antidromic/
orthodromic results showed significant differences between the two
groups suggesting that CET reduced RI.
Responses of CET animals to GABA resulted in trends toward
decreased PSA. Bicuculline-mediated PSA were increased after CET
suggesting increased GABA receptor sensitivity. Baclofen inhibited PSA
but without group differences. Responses to 2DA from CET animals were
stimulation site dependent. Stratum radiatum stimulation produced
increased PSA whereas stratum oriens stimulation decreased PSA. It is
suggested that CET differentially affected the commissural afferents or
SO interneurons.
These data continue to reveal detrimental consequences of long term
ethanol abuse.
viii


CHAPTER 1
GENERAL INTRODUCTION
Foreword
The consumption of alcohol by individuals in our society and
throughout many regions of the world is a widely accepted and, in many
ways, condoned behavior. The effects of acute episodes of alcohol
overindulgence are well documented. Increases in response time, errors
in judgement and cognitive skill are but a few of the many behavioral
effects alcohol imposes upon users. In recent years and months, our
federal and state governments have begun to re-assess the costs of
alcohol abuse upon society in terms of lost lives and property and to
reconsider laws governing the legal responsibilities and consequences of
individuals for the behavior they exhibit while under the influence of
alcohol. These steps have been taken as a result of increasing alcohol
abuse and the concomitant costs to society.
Alcohol abuse is often regarded in terms of single episodic
events. But alcohol abuse is not an issue related only to those periods
of active consumption and the behavioral changes that occur within that
framework. Instead, and especially from a mental health perspective,
alcohol abuse is a lifestyle which carries with it important sociologi
cal, psychological and biological components. Although alcohol abuse
may begin as a result of sociological, psychological or even biological
influences and, in turn, may impart sociological and psychological
dysfunction, detrimental biological alterations always occur with
prolonged use. These alterations must be better understood in order for
1


47
the PS amplitudes were added to each other. This total PS amplitude was
the measure used for all of the PS data analyses. Results were
statistically evaluated by two-way analyses of variance with repeated
measures. One factor was the experimental treatment (group E or S).
The second repeated factor was stimulus current (I/O curves), IPI (PPP),
location (iontophoretic application locations) or time (after
iontophoretic release). Responses between alcohol and sucrose animals
were compared between groups as a percentage change from the baseline of
each animal for amplitudes and as a difference score for latency
measures. This provided for the normalization of potentially different
baselines between the two groups.


122
There was a higher level of potentiation in the alcohol group at IPIs
between 20 and 150 msecs with the highest level of potentiation at 250
percent of control. The sucrose group was maximally potentiated to 225
percent of control. These differences were not, however, statistically
significant. The PS amplitude responses from the two groups were
virtually overlaid at IPIs from 200 to 400 msecs. The latency data for
this experiment (Fig 4-10B) showed the sucrose group had shorter laten
cies to PS onset than did the alcohol group. The sucrose group laten
cies were up to .7 msec faster than control responses while the alcohol
group was only up to .4 msec faster than their control responses. Both
group latencies were shortest at IPIs of 40 to 60 msecs.
The data for the next experiment were obtained with 0/0 stimulation
through stratum oriens instead of through stratum radiatum. The PS
amplitude data (Fig 4-9A) for the alcohol group were potentiated to
levels of 250 to 300 percent of control at IPIs from 20 to 150 msecs.
These responses returned to control levels beyond the 400 msecs IPI.
The PS amplitude responses for the sucrose group were maximally
potentiated to 200 percent of control at an IPI of 80 msecs. The two
groups remained approximately 50 percentage points away from each other
for IPIs from 20 to 400 msecs. While the differences were not
significant it should be noted that the differences between the alcohol
and sucrose groups with SO stimulation tended to be greater than with SR
stimulation. The PS latency data are shown in Fig 4-10A. The alcohol
group had shorter latencies to the PS onset although the differences
between the alcohol and sucrose groups did not quite reach a
statistically significant level (F(l,17) 3.36) p .083. The alcohol
group had latencies .6 to .8 msec shorter than control responses while


181
.085). It is reasonable that this near difference be given consider
ation since the PS latency interaction showed differences at several
time points. As can be seen in Fig. 6-2D and Fig 6-3D, there were no
differences seen in distal SR for either of the PS measures.
SO stimulation
I/O relationships with SO stimulation. See description of I/O
curves as described in Chapter 5. I/O curves for each of the
pharmacological series were combined and presented earlier.
Iontophoresis application with SO stimulation. The graphs for
the SO stimulation with bicuculline application at each of the four
different locations along the pyramidal cell dendrites is shown in Fig
6-4 for the PS amplitude measure and in Fig 6-5 for the PS latency
measures. Beginning with the most distal application in SO, there was
a statistically significant group difference with SO stimulation
(F(l,18) 6.21, p = .023). The alcohol treated animals exhibited
increased PS amplitudes. There was also a significant group by time
interaction effect (F(l,360) 3.93, p .0001). Subsequent analyses
revealed the points between one and 4 mins to be different. There
were no effects on the PS latency measures. There were no differences
in either PS amplitude or PS latency measures when bicuculline was
applied at the cell layer but still in SO. There were no differences
for the PS amplitude data when bicuculline was applied in SR near the
cell body region but there were statistically significant group
(F(l,16) = 5.79, p = .028) and group by time interaction effects
(F(l,320) 1.60, p .05) for the PS latency measures. Subsequent


136
in larger inhibitory effects than SR stimulation. This may be true
since the majority of recurrent inhibitory interneurons appear to reside
in SO or the pyramidal cell layer (Andersen et al., 1964a; Lorento de
No, 1934). It has been suggested that commissural afferents from the
CA3 region may preferentially synapse on CAl feedforward inhibitory
interneurons (Buzsaki, 1984). It has also been demonstrated that the SO
region contains a higher density of CA3 originating commissural fibers
than the SR regions (Swanson et al., 1978). Thus, the SO region may be
an important area for both feedforward and recurrent types of
inhibition.
The use of an antidromic/orthodromic paired pulse paradigm was
aimed at more directly observing the effect of CET on recurrent inhibi
tion. Two different stimulation levels were used to assess the recur
rent inhibitory system, one relatively low and the other relatively
high. No statistically significant differences were found with the
lower condition and test pulse current intensities between groups
although the trend was that the alcohol animals were slightly less
inhibited than the controls. The higher condition and test pulse
current intensities used with A/0 stimulation resulted in significant
differences on the PS amplitude measure between the alcohol and sucrose
groups. The alcohol group, in this case, was initially inhibited to
levels comparable to the sucrose groups at IPIs shorter than 40 msecs.
The maximum levels of inhibition observed at these short IPIs were
similar in both the low and high intensity stimulation conditions. The
nature of the difference between the CET and sucrose groups was that the
alcohol group returned to control levels by the 80 msecs IPI while the
sucrose group remained inhibited to 70 or 80 percent of the control
responses throughout the IPIs tested.


42
Basic Procedures
Input/output relations
I/O functions were obtained at the start of each experimental
session by varying the stimulus current from 20 to 1000 uAs with pulse
widths at a fixed duration of 0.1 msec. The analysis of the responses
generated focused on measures of the latency and amplitude of the EPSP
and PS. These measures provided information regarding synaptic response
strength. The complete set of current steps used was the following:
20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450,
500, 600, 700, 800, 900, 1000. These steps were selected in an attempt
to more accurately indicate EPSP and PS thresholds as well as asymptotic
levels. Additionally, the I/O functions were used to normalize stimulus
current strength across animals in the later phases of each experiment.
Paired-pulse paradigm
Stimulus current. An important problem in these experiments was
the choice of stimulus current used for the paired pulse series of
experimental paradigms. I/O curves were used to adjust the stimulus
current such that a certain percentage of the PS amplitude at asymptote,
up to a maximum of 1000 uAs, was used for the condition or test pulse of
paired pulse experiments. This procedure controlled for minor
variations in electrode placement and individual animal response
variability. If CET altered the basic I/O functions, this procedure
allowed for the evaluation of synaptic potentiation with baseline
responses which fell at the same relative points on the I/O curves.


Figure 6-6. PS amplitude responses (means + SEM) after baclofen
iontophoresis (time=0) with stratum radiatum stimula
tion. Scales on ordinate represent difference of PS
amplitude response following bicuculline administration
from control responses obtained prior to bicuculline
administration (test response-control response) and are
independent for each iontophoretic ejection location.
Abcissa represents time in minutes following ionto
phoretic ejection of the drug. A) PS responses with
iontophoretic ejection in distal SO. B) PS responses
with iontophoretic ejection in proximal SO. C) PS
responses with iontophoretic ejection in proximal SR.
D) PS responses with iontophoretic ejection in distal
SR.


16
Animal Alcohol Models
The use of animal models for chronic alcohol studies brings with it
the problem of how to administer the alcohol regimen. Several different
methods of alcohol administration have been developed including inhala
tion (Goldstein and Pal, 1971) and intubation (Majchrowicz, 1975;
Pohorecky, 1981; Altshuler, 1981). These methods, however, are not as
practical for long term administration as others which require relative
ly normal feeding procedures. Several animal models do exist which
allow ethanol to be administered orally. These methods include the
following where ethanol is provided: 1. ad lib as the sole source of
fluid with ad lib lab chow also being provided, 2. operant conditioning
techniques where an ethanol containing fluid is used as a reward for
maintaining drinking behavior (Falk et al., 1972) and 3. an ad lib
liquid ethanol-containing diet (Freund, 1969; Lieber and DeCarli, T973).
This liquid diet mixture contains all of the required protein, carbo
hydrates, vitamin and mineral supplements necessary for healthy develop
ment. This last method is currently the most widely used of the animal
models (Pohorecky, 1981) and is the method used in the studies completed
herein. Each of these methods of alcohol administration has shown that
alcohol produces alterations in the behavior, anatomy and electro-
physiology of a variety of animals tested.
The Normal Hippocampus -- Anatomy and Physiology
Anatomy of the hippocampus
The hippocampus is a bilateral structure which extends laterally
and caudally for several millimeters from a dorsomedial position in the
rat brain. It turns ventrally in a wide curve until its ventral tip is


THE EFFECT OF CHRONIC ETHANOL INGESTION ON
SYNAPTIC INHIBITION IN CAl OF THE RAT
BY
CARL J. ROGERS
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1986


164
threshold as a result of the depolarizing response to GABA. It has been
reported that two different types of receptors mediate the hyperpolar
ization and depolarization responses (Alger and Nicoll, 1982).
The major difference between the present results and those of
Andersen et al. (1982) centered on the magnitude of the PS amplitude
response at all locations along the dendritic axis. The present inves
tigation showed relatively little inhibition at those dendritic loca
tions where Andersen's group showed inhibition to 50 percent of the
control response. These results may be due to any of three different
aspects of the experimental protocol. First, the present experiments
were conducted using hippocampal slices from the rat. Andersen et al.
(1982) used guinea pigs. Thus, species differences may account
for some of the differences in the magnitude of the results. The second
difference, however, may better account for the differences seen between
the results of the two experiments. Andersen et al. (1982) collected
their test response during the application of GABA. The present find
ings were obtained 6 to 10 msecs after the GABA ejection current was
turned off. Thus, there may have been a period during which the re
sponse of the pyramidal cells to GABA changed. It was noted in the
present experiments that if, after GABA was ejected, additional test
pulses were given within 5 to 10 secs of the termination of GABA, there
was a marked reversal of the response to GABA. Whereas inhibition was
usually seen immediately after GABA ejection, a test pulse produced 5 to
10 secs after GABA termination resulted in a marked potentiation of the
PS (several hundred percent increase) response to GABA. This post-
GABAergic "fade" response has been described previously (Ben-Ari et al.,
1981, 1979; Rovira et al., 1984). Thus, it is hypothesized that the 6


**n**M. lusic) iwni*ULSC re****. (uUC)
difference from control
difference from control

S


6
brains from autopsies of chronic alcoholics and provided a detailed
description of brain regions which contained lesions. Among the areas
where lesions were more consistently found were the mammillary bodies,
various hypothalamic nuclei, the medial dorsal nucleus of the thalamus,
the cerebellum, periventricular regions of the brainstem, the cerebral
cortex and the hippocampus. Earlier studies of alcoholic brain material
indicated that there was an increased vascularity, a loss of myelin in
the cerebral cortex and cerebellum, and a 20 to 40 percent decrease in
pyramidal cell number in motor cortex and purkinje cells in the cere
bellum (Lynch, 1960). Degenerative changes in the granular layer and
dentate nuclei of the cerebellum in alcoholics, a loss of Purkinje
cells, neuroglial proliferation and a thickening of blood vessels in
alcoholics have been reported after chronic ethanol abuse (cf. Lynch,
1960).
Physiology
Electrophysiological studies on human alcoholics have relied on the
use of electroencephalographic (EEG) and evoked potential recordings.
These methodologies have been criticized for their inability to indicate
specific underlying brain structure involvement as well as the underly
ing cellular generators of the recorded potentials. More recent stud
ies, however, in non-alcoholic patients, have increasingly suggested
that certain portions of the generated potentials can be isolated to
specific underlying structures of the brain. For example, the P300
component of evoked potentials, a commonly used measure in alcoholic
patient studies which occurs approximately 300 msecs after stimulus
presentation (often a pulse of light), has been convincingly shown to be


161
The duration of exposure to ethanol used in this study has been
shown to result in a 15 to 20 percent loss of granule and pyramidal cell
number in the rat hippocampus (Walker et al., 1980). Decreased branching
of dendritic elements, as well as reduced numbers of dendritic spines in
CAl pyramidal cells of the mouse hippocampus, have also been reported
(Riley and Walker, 1978). Findings from the rat hippocampus have shown
trends toward decreased spine density in CAl immediately following
ethanol withdrawal (King, 1984). These results suggest that there is a
widespread deleterious alteration of hippocampal morphology following
CET. Electrophysiological characteristics of this region of the hippo
campus have also been shown to be altered by long term ethanol
treatment. Abraham et al. (1981) reported that the PS amplitude of
extracellularly evoked paired pulse responses was significantly in
creased in size compared to sucrose-fed controls. This increase in PS
amplitude occurred in the absence of changes in the basic waveforms,
EPSP and PS thresholds, I/O functions or LTP production. It was hypoth
esized that the increased PS amplitude was a function of decreased
recurrent inhibitory influences. Results from the investigations
reported earlier within this manuscript have provided additional evi
dence for CET producing increased PS amplitudes with paired pulse
testing in the CAl region of the hippocampus. The use of additional
paired pulse paradigms have further elucidated the potential contribu
tion of recurrent versus feedforward inhibitory processes to this
increased PS amplitude. While it appears that recurrent inhibition is
altered by CET, it also appears that deficits in feedforward inhibition
may be as profound as those in recurrent inhibitory circuits. This is
suggested by the observation that the greatest effects on the PS ampli-


Figure 3-5. PS amplitudes (means) for 25 and 50 percent of PS maximum test pulse series with
A/0 stimulation (test response/control response X 100) A) 25 percent of test
pulse series PS amplitudes with 25, 50, and 100 percent of the maximum
antidromic spike on the condition pulse. B) 50 percent test pulse series PS
amplitudes with threshold (0), 25, 50, 75, and 100 percent of the maximum
antidromic spike on the condition pulse.


Figure 5-3. I/O curves (means) obtained with stimulation from
the stratum oriens. Data were normalized by
setting the current level at which the PS was
first seen equal to 0 uAs. Data were then
interpolated from that current value such that
interpolated I/O curves were produced with
current levels between zero and 500 uAs. This
procedure controlled for any differences in PS
threshold between groups. A) PS amplitude
responses with stratum radiatum stimulation
normalized between groups. B) PS latency
responses with stratum radiatum stimulation
normalized between groups.


15
were impaired (Denoble and Begleiter, 1979; Smith et al., 1979; Walker
and Freund, 1973). Temporal single alternation and go-no-go behavioral
tasks were also deficient in alcohol treated rats (Walker and Hunter,
1978). Each of these measures of performance is similar to those of the
hippocampal lesioned data. Shuttle box avoidance behavior with ethanol
treated rats, however, is different from the behavior found with
hippocampal lesioned animals. While hippocampal lesions normally
facilitate the response to this task, ethanol treatment produced im
paired performance (Freund, 1979; Freund and Walker, 1971).
Summary of Alcohol and Normal Human and Animal Literatures
In summary, the similarities found with behavioral testing between
Korsakoff's syndrome patients and H.M. appear to be strong. The litera
tures from hippocampal lesioned animals and CET animals not only provide
similarities between hippocampal lesions and CET but also between the
human and animal literature. Unfortunately, little detailed human
anatomical, electrophysiological or biochemical evidence exists concern
ing CET effects due to the ethical problems inherent with such human
research. Animal studies of CET have, however, begun to provide a
wealth of information concerning morphological and functional altera
tions, especially in the hippocampus and cerebellum. Since the
hippocampus, and in particular, the CAl region is important to the
studies performed herein, the animal models, anatomy, physiology and
pharmacology of the hippocampal CAl region will be considered in the
next sections in some detail.


138
from the controls. These PS latency differences further support the
trend for a decrease in the efficacy of inhibition in the CAl pyramidal
cell region.
The current studies using homosynaptic paired pulses and CET
animals were performed with the in vitro preparation and several paired
pulse configurations to extend the findings from earlier studies. The
in vitro preparation was used in an attempt to eliminate any possibility
of an interaction between CET effects and urethane anesthesia which had
been used previously, since it is known that alcohol and the barbitu
rates are cross tolerant (Kalant et al., 1971). The likelihood that
cross tolerant effects existed in previous studies from our lab,
however, were minimal since the animals had been off of the alcohol diet
for at least eight weeks prior to acute electrophysiological testing.
The present results suggest that, while there are considerable
differences in the response patterns observed in vivo versus those
obtained in vitro. urethane anesthesia did not result in any anesthesia
dependent differences between the CET and sucrose-fed controls. The
results obtained in vitro continue to support the previous observation
that CET results in increased PS amplitude responses (Abraham et al.,
1981).
Morphological data from CET animals have shown significant
decreases in pyramidal and granule cell number (Walker et al., 1980).
These CET induced effects may extend to the inhibitory interneurons,
which have not been carefully studied after CET, as well as to the
primary cell types of the hippocampus. It is probable that the
interneurons are at least as, or more prone to functional alterations or
injury and death due to various treatment conditions. It has been shown
that CET altered blood flow to various parts of the brain (Newlin,


percent change from control percent change from control
oS8!SSISS3SS8¡£KG6S5¡8
Li.


Figure 3-2. PS amplitude responses means for 25 and 50 percent of PS amplitude at asymptote
for the test pulse series with 0/0 stimulation (test response/control response X
100). A) 25 percent test pulse series PS amplitude response with 50 percent of
EPSP, 25, 50 and 100 percent of PS maximum on the condition pulse. B) 50
percent test pulse series PS amplitudes with 50 percent of the EPSP, threshold
(0), 25, 50, 75, and 100 percent of PS maximum on the condition pulse.


21
inducing relatively widespread inhibition (Andersen et al., 1964a).
This feedback type of inhibition is termed recurrent inhibition.
Although the definitive circuitry is not yet completely described,
feedforward inhibition has been demonstrated in the apical and basilar
dendritic regions (Alger et al., 1981; Ashwood et al., 1984; Andersen et
al., 1980; Buzsaki, 1984). Several possible anatomical configurations
for this circuit are plausible. The best demonstrated configuration,
however, is that the Schaffer/commissural afferents terminate on an
inhibitory interneuron which resides in SR or SO parallel to the
dendrites of the CAl pyramidal cell (Ashwood et al., 1984). These
inhibitory interneurons then project to the dendrites of the pyramidal
cell (Gall et al., 1981; Schwartzkroin and Mathers, 1978).
The pyramidal cell is the major projection cell of the CAl area.
Due to its architectural definition with the afferent fibers being
orthogonal to the apical dendrites of these cells, the CAl region offers
an exceptional opportunity for the study of both intracellular and
extracellular responses. The relative ease of understanding the effects
of localized extracellular stimulation and recording in relation to the
input and output pathways is unsurpassed elsewhere.
Electrophvsiology of the hippocampus: An introduction
Electrophysiological studies of the hippocampus often use
extracellular recording techniques. The ability to use these field
potential recordings meaningfully results from the highly organized and
relatively homogeneous patterning of the afferent and efferent fibers in
this region. The activation of the afferent fibers produces restricted
and localized dipole current sources and sinks. When an excitatory


107
otherwise resembled other heterosynaptic 0/0 paired pulse results (Haas
and Rose, 1982). Although the statistical analyses for all of these
experiments showed few statistically significant differences between
alcohol and sucrose data, the general pattern of the alcohol animals was
for higher levels of potentiation and lower levels of inhibition than
found in the sucrose animals. These patterns are similar to those
reported by Abraham et al. (1981).
0/0 homosynaptic paired-pulse paradigms -- Set 1
The 0/0 homosynaptic paired pulse PS amplitude responses obtained
with a current intensity of 50 percent of the EPSP amplitude at PS
threshold set on the condition pulse and 25 percent of the PS amplitude
at PS asymptote set on the test pulse are shown in Fig 4-5A. These data
were very similar to those reported with similar conditions in vivo.
There was an immediate potentiation of the PS which peaked at the 30
msecs IPI for the alcohol data and at the 50 msecs IPI for the sucrose
animals. The maximum levels of potentiation at these intervals were 200
percent for the alcohol animals and 160 percent for the sucrose animals.
Although there were no statistically significant differences, the IPIs
between 20 and 100 msecs showed the greatest differences between the two
groups; the CET group was approximately 20 to 50 percentage points
higher than the sucrose group. The latency measures for these data can
be seen in Fig 4-6A. There is an initial decrease in the latency to the
PS, by .4 msec, for both groups from 20 to 100 msecs IPIs but there were
no differences between groups.
The 0/0 homosynaptic paired pulse PS amplitude responses with the
condition pulse current intensity set to 25 percent of the PS amplitude


Figure 4-3. I/O curves for Set 2 slice experiments before any
paired pulse stimulation experiments were com
pleted. These data are with stratum radiatum
stimulation. Data were normalized by setting the
current level at which the PS was first seen
equal to 0 uAs. Data were then interpolated from
that current value such that interpolated I/O
curves were produced with current levels between
zero and 500 uAs. This procedure controlled for
any differences in PS threshold between groups.
A) Normalized PS amplitude responses (means) for
the alcohol and sucrose groups in mVs by current
intensity (uAs). B) Normalized PS latency
responses (means) for the alcohol and sucrose
groups in msecs by current intensity (uAs).


37
It is apparent that CET affects inhibition in CAl. However, it is
not clear whether CET affects recurrent inhibition alone, feedforward
inhibition alone or both types of inhibition concurrently. In attempt
ing to determine the mechanisms that may be affected by CET, it is
important to know the generality of CET effects on inhibition. There
fore, the basic intent of the experiments reported herein was: 1. to
define the nature and parameters of the CET induced changes as well as
the characteristics of paired-pulse inhibition and potentiation in CAl
in untreated animals and 2. to begin to study the possible pharmacologi
cal mechanisms which may underlie these changes. These goals were
accomplished through three major series of experiments: 1. characteris
tics of normal synaptic inhibition --an in vivo study, 2. characteristics
of CET and synaptic inhibition --an in vitro study, and 3. pharmacolog
ical characteristics of CET and synaptic inhibition -- a series of in
vitro studies.


Figure 3-6. PS latencies (means) for 25 and 50 percent of PS maximum test pulse series with
A/0 stimulation (test response control response). A) 25 percent test pulse
series PS latencies with 25, 50 and 100 percent of the maximum antidromic spike
on the condition pulse. B) 50 percent test pulse series PS latencies with
threshold (0), 25, 50, 75, and 100 percent of the maximum antidromic spike on
the condition pulse.


213
CET did not result in any significant differences in the PS ampli
tude response when compared to controls. Although the CET animals
showed small trends toward being more inhibited than controls when
baclofen was administered in proximal SR with SR or SO stimulation,
these trends were not conclusive. It is therefore concluded that CET
does not affect baclofen sensitive inhibition.
The finding that baclofen is not affected by CET at first suggests
that feedforward inhibition may not be affected by long term ethanol
treatment. However, as indicated earlier, there are at least two
different feedforward related inhibitory responses. One of these
feedforward responses is characterized by baclofen sensitivity and
bicuculline insensitivity and results in an hyperpolarization of the
intracellularly recorded membrane potential. The other response is a
depolarization of the cell membrane which results in the shunting of
current flow such that it inhibits the pyramidal cell from firing. It
appears that the bicuculline-insensitive receptor population may not be
affected by CET. However, since the paired pulse results presented in
chapter 4 suggest decreased levels of feedforward inhibition, it is
quite possible that these CET-induced alterations are on the
depolarizing sub-population of GABA receptors. The effect of CET on the
depolarizing GABAergic receptors, however, cannot be distinguished with
the present experiments.
The iontophoresis of enkephalin along the CA1 pyramidal cell
dendrites resulted in increased PS amplitudes and multiple population
spikes in all of the animals tested. These results compare well with
previous reports (Dunwiddie et al., 1980; Robinson and Deadwyler, 1980;
Nicoll et al., 1980; Dingledine, 1981; Haas and Ryall, 1980; Corrigan


28
pulse is to decrease the amplitude of the population spike on the second
or test pulse (in vivo). This decreased PS amplitude is thought to be
due to a reduction in the probability that the pyramidal cell and those
around it will fire when the second pulse arrives. Paired pulse poten
tiation overshadows the inhibitory phase after about 80 msecs (Andersen
and Lomo, 1970; Dunwiddie et al., 1980). This potentiation phase is
thought to be due to a process different from that of inhibition.
However, it is also thought that recurrent inhibition may produce a
resetting of the fired pyramidal neurons thereby allowing for a more
synchronized and simultaneous firing of the pyramidal cells with inter
pulse periods longer than approximately 80 msecs. This process could
account for a portion of the potentiation phase seen with paired pulses
(Assaf and Miller, 1978).
More recently, feedforward-like inhibition has begun to be dej
scribed and characterized in CA1 (Alger et al., 1981; Andersen et al.,
1978, 1980, 1982). It has been hypothesized that some of the Schaffer/
commissural afferents either terminate on the apical or basilar
dendrites of the CA1 pyramidal cell and directly release an inhibitory
neurotransmitter, gamma-aminobutyric acid (GABA), or more likely, the
Schaffer/commissural afferents synapse onto an inhibitory interneuron
which, in turn, synapses onto the apical or basilar dendrites or soma of
the CAl pyramidal cell (Ashwood et al., 1984; Buzsaki, 1984). The
conclusive anatomical evidence for such feedforward inhibitory connec
tions is not yet complete. However, descriptions of interneurons which
run parallel to the pyramidal cell apical dendrites do exist in the
literature (Gall et al., 1980; Lorente de No, 1934; Schwartzkroin and
Mathers, 1978).


95
electrode localized to the alvear fibers without current spread to the
SO dendritic region.
I/O curves were obtained using procedures identical to those
described in the previous chapter except that an additional I/O curve
was also obtained using the SO stimulation site. The IPIs used for all
of the paired pulse experiments were the same as those used in the
previous in vivo experiments. The condition and test pulse current
intensities for this set of experiments are described in Table 4-1.
The in vitro paired pulse data were analyzed in the same manner as
the in vivo data. The PS latency measures from the test pulse responses
were subtracted from single pulse control responses which were obtained
immediately prior to the experimental IPI responses for each experiment.
All amplitude and slope measures were expressed as a percent of the
control response with the control response equal to 100 percent. All
data are described for IPIs only up to 400 msecs. This was done because
the various measures for the alcohol and sucrose animals tended to
return to control levels together and closely resembled the data pre
sented in the previous chapter for the longer IPIs. There were two
separate sets of animals, each containing sucrose and alcohol animals,
used for various aspects of these experiments. They are reported inde
pendently and then compared. Slope data were recorded, but are not
reported since all slope measures were obtained from the cell layer
rather than the synaptic zone (see discussion Chapter 3).
Results
The mean alcohol consumption for the first set of animals was 12.67
-305 g/Kg. The mean body weight for the alcohol animals was 543 +9.9


CHAPTER 5
EFFECT OF CET ON IONTOPHORETIC GABA MEDIATED RESPONSES
Introduction
The effect of CET on hippocampal electrophysiology has now been
shown repeatedly and in a number of different ways. It has been shown
that when using paired pulse stimulation through one electrode located
in the SR, there is an increase in the amplitude of the PS of CET
animals when compared to sucrose-fed controls (Abraham et al., 1981).
Abraham et al., (1981) hypothesized that the increase in PS amplitude
without changes in the response of the normal synaptic waveform, EPSP or
PS thresholds or I/O functions was due to a decrease in recurrent
inhibition. This hypothesis was given added support from intracellularly
recorded data where it was shown that the only measures that changed in
CET animals were decreased amplitudes of the IPSP and the late after
hyperpolarization in responses from CAl pyramidal cell (Durand and
Carien, 1984a). Results from the previous set of studies in this
manuscript also add support to this hypothesis with the finding of
similar trends for the 0/0 SR data and the extension of those findings
to the SO with 0/0 paired pulse experiments. The A/0 findings where
the condition pulse was delivered to the alvear fibers directly in
order to fire the recurrent inhibitory interneuron, followed by an
orthodromic pulse to the SR afferents, also provided support for the
hypothesis that CET decreased the effect of inhibition in CAl. The
A/0 data showed the CET animals to be inhibited to a significantly
lesser extent than the sucrose-fed control animals. This A/0 testing
141


Figure 6-15. PS amplitude percent change summary graphs for baclofen. These graphs are
constructed from data which was obtained from previous graphs 1.5 mins after
iontophoretic ejection of the respective drugs. This time point was chosen
because it represented the peak response size for most of the applications.
A) Baclofen PS amplitude peak response for each of the different iontophoretic
locations and groups with stratum radiatum stimulation. B) Baclofen PS
amplitude peak response for each of the different iontophoretic locations and
graphs with stratum oriens stimulation.


5
alcohol effects on memory. The major components of Korsakoff's syndrome
are: 1. a severe inability to remember current events over periods of
time into the future of more than about one minute (anterograde amnesia)
beginning with events occurring at the approximate onset of the disease,
2. a limited understanding by the patient of the memory disability and
3. a general state of apathy. Although the ability to remember events
prior to the onset of the disease state (retrograde amnesia) is not
eliminated, an increasing degradation of memory prior to the onset of
the acute phase of the syndrome is seen (Butters and Cermak, 1980;
Seltzer and Benson, 1974; Marslen-Wilson and Teuber, 1975). Even if
alcohol use is eliminated for months or years, memory impairments
persist in at least 80 percent of the Korsakoff's syndrome population
(Victor et al., 1971).
Anatomy
An increasingly convincing accumulation of neuroanatomical data has
been assembled concerning the detrimental effects of long term alcohol
consumption on the gross- as well as micro-anatomy of human alcoholics.
The most widespread method of gathering human neuroanatomical data has
been with computerized tomography scans (CT scans). The results of
these procedures have consistently shown in many experiments and with a
large sample size that alcoholics of all ages have enlarged cerebral and
cerebellar sulci as well as cerebral ventricles. These observations
indicate cerebral atrophy (Wilkinson, 1982; Ron, 1977; Ron et al., 1982;
Brewer and Perrett, 1971; Cala and Mastaglia, 1981; Epstein et al.,
1977; Haug, 1968). Earlier work with pneumoencephalography revealed
similar findings. Victor et al. (1971) observed the gross pathology of


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CHAPTER 3
CHARACTERIZATION OF SYNAPTIC INHIBITION IN VIVO
Introduction
The regulation of pyramidal cell firing in the CAl region of the
hippocampus has long been recognized as an important consideration when
attempting to understand, and potentially control, neuronal function or
dysfunction of this area. Although there may be a multitude of differ
ent and, as of yet, unknown mechanisms by which pyramidal cell firing is
regulated, one of the most well documented methods is achieved through
an inhibitory interneuron; the now classical recurrent inhibitory
circuit found near the pyramidal or granule cell in CAI, CA3 and the
dentate gyrus regions of the hippocampus (Cajal, 1968, Lorente de No,
1934). Recurrent inhibition occurs as a result of pyramidal cell
firing. An action potential propagates from the pyramidal cell to
other major cell types in caudal and rostral directions from the hippo
campus. An axon collateral of the pyramidal cell also conducts this
action potential to nearby inhibitory basket cells. An excitatory
synapse from the pyramidal cell results in the basket cell firing. The
basket cell projects widely in a basket-like web to the original as well
as many other pyramidal cells. An inhibitory neurotransmitter, GABA,
then causes the pyramidal cell to cease firing for a period of time up
to 700 msecs (Andersen et al., 1964a, 1964b). More recently, another
type of inhibition in the CAl region has been shown to exist which is
feedforward in nature. In this system, afferent fibers terminate on an
48


Figure 4-4. I/O curves for Set 2 slice experiments before any
paired pulse stimulation experiments were com
pleted. These data are with stratum oriens
stimulation. Data were normalized by setting the
current level at which the PS was first seen
equal to 0 uAs. Data were then interpolated from
that current value such that interpolated I/O
curves were produced with current levels between
zero and 500 uAs. This procedure controlled for
any differences in PS threshold between groups.
A) Normalized PS amplitude responses (means) for
the alcohol and sucrose groups in mVs by current
intensity (uAs). B) Normalized PS latency
responses (means) for the alcohol and sucrose
groups in msecs by current intensity (uAs).


169
showed, by trends in the PS amplitude data that alcohol treatment for 20
weeks produced a more inhibited response to GABA administration when
compared to sucrose animals. The finding that the SO PS latencies were
statistically different provided additional evidence that CET has in
some way altered the efficacy of the pyramidal cell to GABA adminis
trations .
Although GABA is the major transmitter involved in the inhibitory
systems of the CAl region, other pharmacological agents or transmitters
have been shown to modify the pyramidal cell PS, possibly by regulating
the interneurons. At least four pharmacological agents have been shown
to produce alterations in GABA mediated inhibition in the CAl region of
the hippocampus. The GABA antagonists bicuculline or picrotoxin have
been reported to produce a profound decrease in the effect of the
recurrent inhibitory system. Using extracellular recording techniques,
the effect of iontophoretic bicuculline application was to cause an
increase in the size of the PS as well as the number of population
spikes recorded. If the dosage was high, seizure activity was often
found (Curtis et al., 1970). The opiates, including morphine, produced
a response pattern similar to that seen after treatment with GABA
antagonists. There is an increase in the amplitude as well as the
number of population spikes recorded (Lee et al., 1980; Dunwiddie et
al., 1980; Corrigal and Linesman, 1980; Nicoll et al., 1980; Segal,
1977; Zieglgansberger et al., 1979). Although there is disagreement
concerning the mechanisms by which the opiates act (Corrigal, 1983;
Haas and Ryall, 1980), the majority of papers have suggested that
opiates produced the increased PS responses by disinhibiting the
basket cell, an hypothesis consistent with its action in other regions


26
successively produced with controlled interpulse intervals (IPI). The
second response is typically compared to the first response of the pair
or to a baseline control pulse. Two other procedures are also used to
measure plasticity; one is frequency potentiation (FP) and the other is
long-term potentiation (LTP). FP entails the use of trains of stimu
lation at specific frequencies generally less than 100 Hz. LTP is
usually obtained with high frequency (greater than 100 Hz) trains of
stimulation. The final type of data collection procedure is designed to
examine synaptic distribution. In this procedure, a recording electrode
is lowered in discrete steps (25-100 uMs) through the dendritic region
of the CA1 pyramidal cell usually while stimulating in SR. A profile of
the current sources and sinks can thereby be generated. This technique
is called a laminar analysis. Although a laminar analysis can be used
to determine current sources and sinks, the resolution obtained is not
substantial since current sources and sinks often overlap. If a current
source density analysis (CSD) is performed on the laminar profile, a
more defined profile can be obtained so that it is theoretically possible
to suggest the relative presence, absence and general distribution of
synaptic current along the course of the cellular processes (Nicholson
and Freeman, 1975). CSD analysis is, in essence, a mathematical
transformation of the laminar waveforms which uses the second spatial
derivative thus describing rates of change of current flow between
recorded levels rather than voltage measures.


DATA (MSECS) AW DATA (MV
59


140
potentiation is more directly influenced by feedforward inhibition as
suggested by the differences seen in the maximal levels of potentiation
with different test pulse current intensities both in vivo and in vitro.
It is therefore possible that the A/0 response differences between
groups, seen with the higher current intensities at the longer IPIs, may
reflect a feedforward induced inhibition of the sucrose-fed control
response PS amplitude at longer IPIs. It may therefore be quite reason
able to suggest that CET may produce decreases in feedforward inhibition
to at least the same extent as for recurrent inhibition. Most generally,
these data indicate that CET produces decreased inhibition (both recurrent
and feedforward) in the CA1 region of the hippocampus. Systematically,
examining the inhibitory systems of the CAl region, through localized
pharmacological challenges, may provide further definitions concerning
the effect of CET on synaptic inhibition in the hippocampus and poten
tially throughout the brain.


Figure 4-1. I/O curves for Set 1 slice experiments prior to
any paired pulse stimulation. Data were
normalized by setting the current level at which
the PS was first seen equal to 0 uAs. Data were
then interpolated from that current value such
that interpolated I/O curves were produced with
current levels between zero and 500 uAs. This
procedure controlled for any differences in PS
threshold between groups. A) Normalized PS
amplitude responses (means) for the alcohol and
sucrose groups in mVs by current intensity (uAs).
B) Normalized PS latency responses (means) for
the alcohol and sucrose groups in msecs by
current intensity (uAs).


170
of the nervous system such as locus coeruleus and other brain stem
nuclei (Bird and Kuhar, 1977; Bradley and Dray, 1974; Fredrickson et
al. 1976). Thus, if CET caused an increased production or release of
the putative opiate transmitters in the CAl region, increases in the
PS amplitude could be a result of this action rather than a general
decrease in the efficacy of the inhibitory systems.
Baclofen, a GABA analogue has recently been shown to play a role in
feedforward inhibition. It has been shown that there are two components
to the inhibitory response when recording intracellularly (Alger and
Nicoll, 1982a). The early phase of the response has been shown to be
mediated by recurrent inhibition which is activated by GABA and
antagonized by bicuculline. The late phase, attributed to feedforward
inhibitory influences, has been shown to be bicuculline resistant.
More recently it has been shown that baclofen directly hyperpolarizes
this bicuculline resistant hyperpolarization in a stereoselective
manner (Newberry and Nicoll, 1984; Nicoll and Newberry, 1984). Thus,
baclofen might provide a primary method by which the effect of CET
could be evaluated on the feedforward inhibitory components of CAl
responses.
The following experiments were therefore designed to investigate
the effects of bicuculline, an opiate (D-Ala(2))-met-enkephalinamide
(2DA), and baclofen.
Methods
Animals
Animals were obtained, housed and treated with alcohol and sucrose
as indicated in the general methods section.


22
synapse is activated, current flows into the cell. This flow of current
into the cell is called a current sink. As in any electrical system,
current must also flow out of the system elsewhere. Current will flow
out of the cell at what is called the current source and then travel
through the intercellular fluid back to the current sink. These current
flows can be recorded since a voltage is produced as current flows
through the resistive extracellular milieu. Intracellularly, the
activation produced at the current sink is seen as a positive voltage.
Extracellularly, however, the potential recorded is negative at the sink
and positive at the source when stimulating and recording from the same
excitatory synaptic region.
In the CAl region of the hippocampus, the location of the stimulat
ing and recording electrode will affect the characteristic shape of the
response. If the stimulating electrode is in SR, a negative-going field
response is obtained when recording from the same synaptic region due to
the inward flow of current from this excitatory response. If the
stimulating electrode is left in SR while the recording electrode is
moved into SO in steps, a gradual change in the shape of the response is
observed. As the recording electrode approaches the pyramidal cell
layer, the response becomes less negative. When the recording electrode
passes through the cell layer or thereabout, a response is obtained
which is neither positive nor negative in appearance. This point is
termed the inversion point and represents the area where the inward flow
of current generated by the activation of the apical dendrites equals
the outward flow of current. Once the recording electrode is dorsal to
the cell layer, the response becomes positive which indicates a current
source; current is flowing out of the cell at this point. If the




148
cell layer so that good responses were obtained with both SO and SR
stimulation. In the event that a good response (one having a positive
going visible EPSP) could not be obtained, the recording electrode was
placed so that the response with SR stimulation was optimized for the
EPSP with a superimposed PS. For this reason, as well as previously
discussed reasons, the slope data of the SO generated data were not
analyzed. One stimulating electrode was placed in the SR at the CA1-CA2
border while the other stimulating electrode was placed in the SO
afferents. Every effort was made to restrict the stimulating electrodes
from encroaching on the pyramidal cell body layer or onto the alvear
fibers.
The placement of the GABA iontophoresis micropipette at 10
different locations along the CA1 pyramidal dendrites was obtained as
follows. Four different application locations were used in the SO
dendritic region while six application sites were selected in the SR
region (see Fig 5-1). The first application of GABA was made at the
pyramidal cell layer as close to the recording electrode from the SO
side as possible. The next three applications were made by observing
the distance from the recording electrode to the beginning of the alvear
fibers and dividing that region of the SO into three equal distances by
visualization. The same procedure was used in the SR region where the
first application site was as close to the recording electrode as
possible from the SR side. The distance from the recording electrode to
the fissure was then divided into five equal segments by visualization.
This method provided a way by which different distances between the
noted landmarks could be divided such that the same relative region
between different slices and different animals could be controlled. The


167
tion occurs following recovery from CET. The present experiments
utilized animals which were off of the ethanol diet for a minimum of
eight weeks prior to testing. Compensatory changes would certainly be
expected to be well underway within this time frame. An increased
GABAergic response following CET may parallel the compensatory changes
due to CET as measured by golgi impregnated spine density counts from
CA1 pyramidal cells. It is possible that the increased number of spines
on the pyramidal cell may be functionally related to the increased
GABAergic response with iontophoretic GABA application. Alternatively,
the increased PS amplitudes seen in the paired pulses may result from
this compensatory reorganization of the pyramidal cell spines.
The present experiments have provided data which indicate an
increased response of the CA1 pyramidal cell to GABA ejection along its
dendritic axis. These findings were similar with either SO or SR
stimulation even though the response pattern to GABA ejection differed
depending on the dendritic stimulation site. CET animals always showed
more inhibition or less potentiation than pair-fed sucrose controls.
These data were discussed in terms of compensatory changes of dendritic
spines after long-term ethanol treatment as recently reported by King
(1984). The results suggest an increased number or affinity of GABA
receptors on the CAl pyramidal cells after eight weeks of abstinence
from 20 weeks of CET, possibly as a function of decreased GABA release.
Experiments using pharmacological agents which alter inhibition in the
CAl region may provide important additional information concerning CET
effects.


33
interneurons or by increasing the strength of the excitatory afferents
to the basket cells.
Opiates in the hippocampus
A third class of pharmacological agents probably interacting with
inhibitory processes in the hippocampus is the opiates. Endogenous
opiates have been observed in the CAl pyramidal cell region with the use
of immunocytochemical techniques (Gall et al., 1981). Two different
points of view exist concerning the effects of opiates in CAl (Corrigal,
1983; Haas and Ryall, 1980). It has been shown that electrophysio-
logically, the administration of [D-Ala(2), d-Leu(5)enkephalin] (DADL)
or morphine in the bathing medium of slices produced increases in the
size and duration of EPSPs of dendritic origin without changing the
resting membrane properties of the soma, recurrent somatic IPSPs, or
dendritic field potentials (Dingledine, 1981; Robinson and Deadwyler,
1980). Three hypotheses have been proposed to account for these
findings: 1. that an enhancement of "the electrical coupling between an
apparently normal EPSP current generator and a normal spike trigger
zone" occurs (Dingledine, 1981, p. 1034), 2. that a form of
post-synaptic dendritic inhibition exists which is selectively
attenuated by opioid peptides (Dingledine, 1981), and 3. the possibility
that a "modulation of excitatory synaptic transmission onto hippocampal
dendrites by a postsynaptic action that would entail some amplification
mechanism" (Lynch et al., 1981 (no direct evidence supports this
hypothesis, Dingledine, 1981, p. 1034)). The second and more widely
supported interpretation is that opiate effects are not on the pyramidal
cell at all but act by disinhibiting the inhibitory interneuron (Lee et
al., 1980; Dunwiddie et al., 1980; Corrigal and Linesman, 1980; Nicoll


Figure 3-3. PS latency responses (mean) for 25 and 50 percent PS maximum test pulse series
with 0/0 stimulation (test response-control response). A) 25 percent test pulse
series PS latencies with 50 percent of EPSP, 25, 50, and 100 percent of PS
maximum on condition pulse. B) 50 percent test pulse series PS latencies with
50 percent of EPSP, threshold (0), 25, 50, 75, and 100 percent of PS maximum on
condition pulse.


96
TABLE 4-1
PERCENT OF MAXIMAL PS
AMPLITUDE
ON
TEST
PULSE
Set 1
25%
50%
c
0
Homosynaptic:
1
1
1
N
P
SR/SR 50% EPSP
1 x
1
1
D
U
SR/SR 25% PS
1 x
I
1
I
L
SR/SR 50% PS
1 x
1
1
T
S
SR/SR 50% PS
1
1
X
1
I
E
1
1
1
0
Antidromic:
1
1
1
N
ALV/SR 50% AS
1 X
1
1
ALV/SR 100% AS
1
1
1
1
X
1
1
Set 2
Homosynaptic:
1
1
1
SR/SR 25% PS
1 X
1
1
S0/S0 25% PS
1 X
1
1
1
Antidromic:
1
1
1
1
1
1
ALV/SR 100% AS
1 X
1
1
1
Heterosynaptic:
1
1
1
1
1
1
SR/SO 25% PS
1 X
1
1
SO/SR 25% PS
1 X
1
1
1
1
1
Percent of maximal PS amplitude at PS asymptote, EPSP asmplitude at
PS threshold, and maximal antidromic spike (AS) amplitude for the
condition and test pulses in paired pulse paradigms (in vitro') Note
different stimulation electrode configurations.


CHAPTER 4
CET AND SYNAPTIC INHIBITION: AN IN VITRO ANALYSIS
Introduction
It has become increasingly clear that chronic ethanol abuse in
humans produces a range of behavioral deficits as well as a range in the
severity of those deficits (Tarter and Alterman, 1984; Parsons and
Leber, 1981; Parsons, 1977: Ron, 1977; Ryan, 1980). Anatomical studies
of human alcoholics have shown general cerebral atrophy and lesions of
certain nuclei (Wilkinson, 1982; Ron, 1977, 1982; Cala and Mastaglia,
1981; Brewer and Perrett, 1971; Victor et al., 1971). A major contro
versy has existed concerning whether alcohol itself or nutritional
inadequacies were the cause of these changes (Victor et al., 1971).
Animal studies which employed rigorous controls of the dietary intake
have, however, provided substantial evidence that chronic ethanol abuse
alone caused many of the behavioral, anatomical and functional deficits
observed (Walker and Hunter, 1978; Walker and Freund, 1973; Freund and
Walker, 1971; Denoble and Begleiter, 1979; Fehr et al., 1975; Tavares et
al., 1983; Abraham et al., 1981, 1982, 1984; Durand and Carien, 1984a,
1984b). Direct evidence of CET effects can be seen in the morphological
alterations which have been reported in rats exposed to CET. A 15 to 20
percent decrease in the number of primary cells in the CA1 region, the
dentate gyrus and the cerebellum have been reported (Walker et al.,
1980). CAl pyramidal cell and dentate granule cell spine density has
been shown to be decreased (Riley and Walker, 1978). The dendritic
branching patterns of these cells have also been shown to be reduced.
87


91
stimulation is similar to homosynaptic 0/0 stimulation in that both
feedforward and recurrent inhibition are activated. Since inputs do not
originate or impinge on the same pyramidal cell dendritic areas,
however, no facilitation of the EPSP occurs. Thus, heterosynaptic 0/0
stimulation offers a way of observing the inhibitory systems of the CAl
region without EPSP facilitation or PS potentiation also being induced.
This heterosynaptic paired pulse paradigm may therefore provide an
additional perspective from which to review the effects of CET on
inhibition in CAl.
The three major questions addressed in these experiments are: 1.
Does CET produce alterations in feedforward as well as recurrent inhibi
tion? ; 2. Are the in vivo and in vitro preparations comparable as far as
the effects of CET are concerned?; and 3. How does heterosynaptic SR/SO
stimulation compare with SO/SR paired-pulse stimulation and with SR/SR
and A/0 paired-pulse stimulation?.
Methods
Animals
Animals used were housed and fed as described in the general
methods.
Comments on in vitro technique
All of the experiments performed in this chapter and in the subse
quent chapters were performed using the in vitro hippocampal slice
technique. This technique has several advantages for the study of
recurrent and feedforward inhibition in CAl. One of the most important
advantages is the ability to place electrodes visually. Although


difference from control (msecs)
190


Figure 3-4. EPSP slope responses (means) for 25 and 50 percent of PS maximum test pulse
series with 0/0 stimulation (control slope/test slope X 100). A) 25 percent
test pulse series EPSP slope responses with 50 percent of EPSP, 25, 50 and 100
percent of PS maximum on the condition pulse. B) 50 percent test pulse series
EPSP slope values with 50 percent of EPSP, threshold (0), 25, 50, 75, and 100
percent of PS maximum on condition pulse.


Figure 6-7. PS latency responses (means) after baclofen
iontophoresis (time-0) with stratum radiatum stimu
lation. Scales on ordinate represent difference of PS
latency response following bicuculline administration
from control responses obtained prior to bicuculline
administration (test response-control response) and are
independent for each iontophoretic ejection location.
Abcissa represents time in minutes following ionto
phoretic ejection of the drug. A) PS responses with
iontophoretic ejection in distal SO. B) PS responses
with iontophoretic ejection in proximal SO. C) PS
responses with iontophoretic ejection in proximal SR.
D) PS responses with iontophoretic ejection in distal
SR.


27
Pharmacology of the Hippocampus
Pharmacology of inhibition in the hippocampus
The major focus of the experiments in this volume is on the effect
of CET on synaptic inhibition. The basic premise for this focus ema
nates from previous studies suggesting changes in the influence of
inhibition in the CAl region of CET animals. The complete details of
these findings will be given subsequently. The study of inhibition in
CAl of the hippocampus in normal animals has, however, provided an
exploding volume of information on different types of inhibition and the
underlying pharmacology. This section is designed to provide a brief
review of that literature in order to establish a background for the
basis of the experiments outlined below.
Recurrent inhibition has been considered to be the dominant type of
inhibition in the CAl region (Andersen et al., 1964b). As indicated
earlier, this inhibitory circuit consists of the pyramidal cell which
projects via axon collaterals to the basket cell. The basket cell, in
turn, projects widely to surrounding pyramidal cells as well as to the
original "firing" pyramidal cell (Andersen et al., 1964a; Knowles and
Schwartzkroin, 1981). An excitatory neurotransmitter, presumably
glutamate, is released by the pyramidal cell onto the soma of the
inhibitory interneuron (Storm-Mathisen, 1977). The functional effect of
recurrent inhibition is profound. If the Schaffer/commissural afferents
are stimulated while spontaneous pyramidal cell unit firing is moni
tored, an initial excitatory response is found followed by a near
complete cessation of spontaneous activity for 600 to 700 msecs (Andersen
et al., 1964a). With paired pulses, if the second of two pulses is
presented within 20 to 80 msecs of the first, the effect of the first


35
Physiology. Electrophysiological data using laminar and current
source density (CSD) analysis profiles in the CAl region showed changes
in the location of the current sinks after ethanol treatment (Abraham et
al., 1982). Normal animals were reported to have two discrete yet
overlapping current sinks in the SR of CAl. In CET animals, the more
proximal (to the cell layer) current sink was significantly reduced.
These data suggested that there was a change in the anatomical
distribution of synapses in the CAl region after CET. Abraham et al.
(1982) hypothesized that these two current sinks represented a discrete
yet overlapping termination of the Schaffer collaterals distally and the
commissural fibers proximally. They suggested that the commissural
fibers were preferentially affected. This condition could occur by
either the commissural projections being interrupted, a loss of
dendritic synapses in the proximal region, or a selective decrease in
the efficacy of the synapses onto the proximal apical dendrites.
The normal function of the CAl region has also been shown to be
altered by CET. Abraham et al. (1981) reported that although CET did
not significantly affect the basic synaptic waveforms, EPSP thresholds,
PS thresholds, or I/O functions, the production of long-term
potentiation, nor the basic pattern of response to paired pulse or
frequency potentiation, CET did produce enhanced potentiation to paired
pulses as well as FP at 5 Hz and 10 Hz stimulation. These findings of
enhanced PS potentiation were found in the absence of changes in the
field EPSP. It was hypothesized that these results are a function of a
change in recurrent inhibitory processes of the CAl region.


time after iontophoresis (mins)
percent change from control


13
1978; Douglas, 1967; Isaacson and Pribram, 1975; Seifert, 1984). There
fore, only a brief summary of the more relevant findings is presented
below.
The major categories of behavioral testing are summarized as
presented by O'Keefe and Nadel (1978). Hippocampal animals generally
exhibited increased reaction to novel stimuli; they are considered
hyperactive but at the same time hypoexploratory. Spontaneous alterna
tion studies indicated either random or repetitive behavioral responses.
Spatial discrimination after hippocampal lesions proved to be more or
less normal as did non-spatial discrimination. However, spatial dis
crimination reversals were deficient in hippocampal lesioned animals as
were non-spatial reversals. Complex maze learning was deficient in
nearly all such studies. One way active avoidance tasks were typically
normal or showed deficits. Two way active avoidance after hippocampal
lesions, however, showed facilitated avoidance. Passive avoidance tests
yielded mixed responses, generally responding normal to controls but
with occasional deficits on very specific elements of the measures used.
Time related tasks such as lever press rates, delayed response,
go-no-go, delayed alternation and differential reinforcement of low
rates (DRL) with lesioned animals yielded high rates of behavior, hence
deficiencies on these tasks. Finally, hippocampal lesioned animals
typically showed extinction deficits.
The above results allow for several general observations. These
data indicate that hippocampal lesioned animals can learn but often show
deficits in part because of response or strategy perseveration as seen
with discrimination reversals or extinction testing tasks. A second
point is that tasks which typically rely on spatial or contextual cues


0)Sn) ytAtfiu jrww*Jv (3js) tvawjjw
percent change from control
percent change from control
9


149
order of application was the same in all animals. The first application
was at the SO side of the recording electrode working out toward the
alveus and then from the SR side of the recording electrode working out
toward the hippocampal fissure.
The protocol for stimulation and application was as follows. After
the recording and stimulating electrodes had been satisfactorily placed,
the GABA micropipette was situated at the SO side of the recording
electrode. The stimulation current was adjusted prior to lowering the
GABA pipette into the slice so that a response approximately 25 percent
of the PS amplitude at PS asymptote was obtained. The GABA electrode
was then lowered and adjusted in a medial-lateral as well as dorsal-
ventral direction until a maximum level of inhibition was obtained for
the given ejection site with the same GABA ejection current and duration
used in the final procedure. Once all of the adjustments were made, a
control response was obtained by stimulating the SR afferents. Ten
seconds later, GABA was ejected with a cationic current of 40 nAs for 5
seconds. A 5 nA retaining current was used whenever the GABA was not
being ejected. At the end of the 5 second ejection period, another
stimulation pulse was administered to test for the effects of the GABA
administration. These parameters were used since they produce a clear
response change with a relatively quick return to control levels. A
minimum 30 secs delay period was used before a second set of responses
were collected at the same location using exactly the same configura
tion. This second set of responses was then averaged with the first set
and stored. With the iontophoresis pipette still in the same location,
the SO stimulation electrode configuration was tested in the same manner
as described for the SR configuration. Thus, two applications of GABA