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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viii, 244 leaves : ill. ; 28 cm.
Rogers, Carl J., 1953-
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Alcohol -- Physiological effect   ( lcsh )
Psychology thesis Ph. D
Dissertations, Academic -- Psychology -- UF
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Thesis (Ph. D.)--University of Florida, 1986.
Includes bibliographical references (leaves 231-243).
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Also available online.
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by Carl J. Rogers.
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This dissertation is dedicated to my wife, Jane,
and to my parents, Carl F. and Sophie Rogers,
for their unending support.


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.


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!





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


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

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

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








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


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

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

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

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


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



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.




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



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


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


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


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


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).


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


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


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


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.,


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


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


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


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,


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


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


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.


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


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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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


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


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


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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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


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).


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


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


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


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


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


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).


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.


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.


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.



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


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


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.


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).


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.


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.


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


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.


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



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


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


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


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.



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


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,


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




25% 50%
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

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).


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.



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).


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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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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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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.


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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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


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.


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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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


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


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


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


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


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


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


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


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.


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.



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


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


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


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?.



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


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).


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