Citation
Neuronal susceptibility in rat models of developmental ethanol exposure

Material Information

Title:
Neuronal susceptibility in rat models of developmental ethanol exposure descriptions of cellular and molecular alterations
Portion of title:
Descriptions of cellular and molecular alterations
Creator:
Moore, David Blaine, 1972-
Publication Date:
Language:
English
Physical Description:
x, 209 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Brain ( jstor )
Cell death ( jstor )
Cholinergics ( jstor )
Ethanol ( jstor )
Female animals ( jstor )
Messenger RNA ( jstor )
Neurons ( jstor )
Pups ( jstor )
Rats ( jstor )
Septum ( jstor )
Cerebellum -- drug effects ( mesh )
Choline Acetyltransferase -- drug effects ( mesh )
Department of Neuroscience thesis Ph.D ( mesh )
Dissertations, Academic -- College of Medicine -- Department of Neuroscience -- UF ( mesh )
Ethanol -- toxicity ( mesh )
Gene Expression ( mesh )
Gene Expression Regulation ( mesh )
Genes, bcl-2 ( mesh )
Parvalbumins -- drug effects ( mesh )
Prenatal Exposure Delayed Effects ( mesh )
Rats ( mesh )
Research ( mesh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1998.
Bibliography:
Bibliography: leaves 181-208.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by D. Blaine Moore.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
002406584 ( ALEPH )
52003135 ( OCLC )
AMB1536 ( NOTIS )

Downloads

This item has the following downloads:


Full Text










NEURONAL SUSCEPTIBILITY IN RAT MODELS OF
DEVELOPMENTAL ETHANOL EXPOSURE: DESCRIPTIONS OF
CELLULAR AND MOLECULAR ALTERATIONS














By '

D. BLAINE MOORE


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

UNIVERSITY OF FLORIDA


1998






























For Terri and my Mentors, who nurtured my interest in Biology














ACKNOWLEDGMENTS


There are many people to whom I am indebted, especially my wife, Terri, who is

my best friend and best critic. I am also grateful to my mentors, for their continued

support. I am indebted to my thesis advisory committee members, Drs. Marieta Heaton

(chair), Don Walker, Tony Yachnis, Kevin Anderson and John MacLennan, for their

efforts, and to Drs. Doug Anderson and Gerry Shaw for helpful advice. I benefited from

the assistance of Dr. Jim West and Jo Mahoney (Texas A&M University) in learning the

artificial rearing procedure. Likewise, Dr. Bill Farmerie and Regina Shaw (of the

Interdisciplinary Center for Biotechnology Research, UF), as well as Terri Edwards and

Dr. John MacLennan, provided excellent advice on molecular biology. Thanks to Heaton

and Walker lab members over the years, including Mike Paiva, Jeffrey Thinschmidt, Pat

Burnett, Steve Famworth, Leon Williams and Drs. Doug Bradley, Jean Mitchell, Meg

Davis, Mike King, and Doug Swanson. I am thankful for the support of the NIH during

my graduate training, including NIAAA F31 AA0550201 and NIAAA T32 AA07561.














TABLE OF CONTENTS
page


ACKNOW LEDGM ENTS .................................................................................................... iii

LIST OF TABLES ............................................................................................................. vi

LIST OF FIGURES........................................................................................................ vii

ABSTRACT....................................................................................................................... ix

CHAPTERS

1 INTRODUCTION ..........................................................................................................

Ethanol Exposure During Development and Resulting Nervous System
Alterations......................................................................................................................
Differential Temporal and Regional Vulnerability ...........................................................4...
Prenatal Ethanol Exposure and Neuroanatom ical Alterations..........................................5...
Postnatal Ethanol Exposure and Neuroanatom ical Alterations ........................................8...
Program ed Cell Death (PCD) and the bcl-2 Fam ily .......................................................11
Hypotheses Tested .......................................................................................................... 21

2 EFFECTS OF PRENATAL ETHANOL EXPOSURE ON PARVALBUMIN-
IMMUNOREACTIVE GABAERGIC NEURONS IN THE ADULT RAT
MEDIAL SEPTUM AND ANTERIOR CINGULATE CORTEX..............................25

Summ ary ............................................................................................................................25
Introduction........................................................................................................................26
M materials and M ethods.................................................................................................... 30
Results................................................................................................................................37
Discussion ..........................................................................................................................43

3 EFFECTS OF NEONATAL ETHANOL EXPOSURE ON CHOLINERGIC
NEURON S OF THE RAT M EDIAL SEPTUM ....................................................... 70

Summ ary ............................................................................................................................70
Introduction........................................................................................................................71
M ethods..............................................................................................................................73










Results................................................................................................................................80
D iscussion..........................................................................................................................83

4 EFFECTS OF NEONATAL ETHANOL EXPOSURE ON PURKINJE AND
GRANULE CELLS AND BCL-2 FAMILY MRNA LEVELS IN THE RAT
CEREBELLA R V ERM IS......................................................................................... 97

Sum m ary ............................................................................................................................97
Introduction........................................................................................................................98
M materials and M ethods................................................................................................... 102
Results.............................................................................................................................. 110
D iscussion........................................................................................................................ 116

5 CONCLUSIONS AND FUTURE DIRECTIONS................................................... 153

Recapitulation of Results and H ypotheses Tested ........................................................1... 53
Choice of A nim al M odels.............................................................................................1... 59
Choice of Cell Counting M ethods ................................................................................1... 63
O their M ethodological Considerations ..........................................................................1... 64
Future Directions for Developmental Ethanol Research ..............................................1... 65
Conclusion ....................................................................................................................... 179

REFEREN CES ........................................................................................................... 181

BIO G RA PH ICA L SK ETCH ......................................................................................... 209














LIST OF TABLES


Table page

2-1. Postnatal day 60 body and brain weight and brain to body weight ratio ................56

2-2. Number of sections, medial septum area per section, and parvalbumin-
immunoreactive neuronal density in the adult rat MS ............................................56

2-3. Number of sections, mean area per section, and parvalbumin-immunoreactive
neuronal density in the adult rat anterior cingulate cortex......................................57

3-1. Mean body weight, brain weight, and brain weight to body weight ratio of postnatal-
day 60 anim als ........................................................................................................ 92

3-2. Number of sections, medial septum area per section, and choline acetyltransferase-
immunoreactive neuronal density in the adult rat MS ............................................92

4-1. Lobule I cerebellar vermis volume, area, and cell nuclei diameter data at P21
following exposure on P4 only .............................................................................. 133

4-2. Lobule I cerebellar vermis volume, area, and cell nuclei diameter data at P21
follow ing exposure on P4-5 ................................................................................... 133

4-3. Lobule I cerebellar vermis volume, area, and cell nuclei diameter data at P21
follow ing exposure on P7-8 ................................................................................... 133

4-4. Lobule I cerebellar vermis volume, area, and cell nuclei diameter data at P5
follow ing exposure on P4-5 ................................................................................... 134














LIST OF FIGURES


Figure page

2-1. The mean total number of parvalbumin-expressing neurons detected on alternate
sections through the medial septum of postnatal-day 60........................................58

2-2. Photomicrographs of 40 gpm coronal sections through the medial septum of ethanol-
treated (A) and sucrose-treated (B) postnatal-day 60 female rats...........................59

2-3. The mean number of parvalbumin-expressing neurons per section in alternate
sections through the medial septum of postnatal-day 60........................................61

2-4. The mean diameter of parvalbumin-expressing neurons on alternate sections through
the medial septum of postnatal-day 60 rats............................................................. 62

2-5. The mean medial septum (MS) volume of postnatal-day 60 rats exposed throughout
gestation to an ethanol-containing liquid diet, sucrose-containing liquid diet, or lab
chow and w ater. ...................................................................................................... 63

2-6. The mean total number of parvalbumin-expressing (PA+) neurons detected on
alternate sections through the anterior cingulate cortex of postnatal-day 60.............64

2-7. Photomicrographs of 40 pum coronal sections through the anterior cingulate cortex of
ethanol-treated (A) and sucrose-treated (B) postnatal-day 60 rats..........................65

2-8. The mean number of parvalbumin-immunoreactive neurons per section counted in
the anterior cingulate cortex.................................................................................... 67

2-9. The mean diameter of parvalbumin-immunoreactive neurons in the anterior
cingulate cortex of postnatal-day 60 rats. ................................................................ 68

2-10. The mean cingulate gyrus volume of postnatal-day 60 rats exposed throughout
gestation ....................................................................................................................69

3-1. Mean total number of choline acetyltransferase-immunoreactive neurons in the
medial septum of postnatal day 60 rats................................................................... 93









3-2. Mean number of choline acetyltransferase-immunoreactive neurons per section
detected on alternate 40 p.m sections through the medial septum of postnatal day 60
rats..............................................................................................................................9 4

3-3. Mean somatic cross sectional area (pm2) of choline acetyltransferase-
immunoreactive neurons in the medial septum of postnatal day 60 rats................... 95

3-4. The mean medial septum volume of postnatal-day 60 rats.................................... 96

4-1. Effects of ethanol delivered on P4 only on P21 body weight (panel A), P21 brain
weight (panel B) and P21 brain to body weight ratio (panel C). ............................135

4-2. Effects of ethanol delivered on P4-5 on P4, P5, and P21 body weight (panel A),
brain weight (panel B) and brain to body weight ratio (panel C). ..........................137

4-3. Effects of ethanol delivered on P7-8 on P7, P8, and P21 body weight (panel A),
brain weight (panel B) and brain to body weight ratio (panel C). ..........................139

4-4. Ethanol delivered during the first postnatal week reduces mean Purkinje and granule
cell number per section in lobule I of the cerebellar vermis ................................. 141

4-5. Ethanol delivered during the second postnatal week does not reduce mean Purkinje
or granule cell number per section in lobule I of the cerebellar vermis.................143

4-6. Ethanol delivered during the first postnatal week on days 4-5 reduces mean Purkinje
but not granule cell number per section in lobule I of the cerebellar vermis as
determ ined on postnatal day 5 (P5)........................................................................ 144

4-7. mRNAs encoding pro-apoptotic molecules of the bcl-2 family are upregulated
following acute ethanol delivered on postnatal day 4 ........................................... 145

4-8. A further ethanol exposure on P5 does not significantly alter the expression of bcl-2
fam ily m R N A s.. ................................................... .................................................. 147

4-9. Effects of acute ethanol delivered on postnatal day 7 on bcl-2 family gene
expression. ..........................................................................................................149

4-10. An additional ethanol exposure on postnatal day 8 increases mRNAs encoding the
pro-apoptotic m olecule bax.................................................................................... 151














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

NEURONAL SUSCEPTIBILITY IN RAT MODELS OF
DEVELOPMENTAL ETHANOL EXPOSURE: DESCRIPTIONS OF
CELLULAR AND MOLECULAR ALTERATIONS

By

D. Blaine Moore

December 1998

Chairman: Marieta B. Heaton, Ph.D.
Major Department: Neuroscience

Developmental disorders arising from maternal consumption of ethanol during

pregnancy are collectively termed the fetal alcohol syndrome. Ethanol exposure during

development induces abnormalities in particular brain regions, and is known to alter the

expression of particular genes and their protein products. The present body of work

sought to further document neuronal populations in the brain which display vulnerability

to developmental ethanol exposure. A further goal of this work was to investigate cell

death gene expression shortly after ethanol insult in the cerebellum in order to test a

specific hypothesis about the cellular mechanism of ethanol neurotoxicity.

A prenatal exposure model was used to examine long term changes in protein

expression patterns of parvalbumin (a marker for gamma-aminobutyric acid- (GABA)

expressing neurons) in the rat brain. Deficiencies in the mean number of immuno-

positive cells per section were noted in the medial septum (in a sexually dimorphic








manner) and anterior cingulate cortex of ethanol-treated rats. This represents the first

documentation of GABAergic neuronal susceptibility to ethanol in either brain region. A

neonatal exposure model was used to examine long-term changes in the expression

pattern of choline acetyl-transferase (ChAT, a marker for cholinergic neurons) in the

medial septum; no significant ethanol-induced changes in the mean number of immuno-

positive cells per section were noted.

A similar neonatal exposure paradigm was used to document Purkinje and granule

cell numbers in the cerebellar vermis during known periods of ethanol sensitivity and

insensitivity, and to investigate mRNA levels of the bcl-2 family of cell death molecules.

First postnatal week ethanol treatment significantly reduced Purkinje and granule cell

number, while second week exposure did not. bcl-2 family gene expression was

measured in the vermis shortly after ethanol treatment to determine whether alterations in

these genes might correlate with the noted cell death. Transcripts encoding the pro-

apoptotic molecules bax and bcl-xs were up-regulated following both first and second

week exposure. Thus, a positive correlation between altered bcl-2 expression and

cerebellar cell death was not found. Suppression of these pro-apoptotic processes may be

the critical determinant of cerebellar susceptibility. These findings suggest new avenues

of research on the intracellular consequences of such expression changes.















CHAPTER 1
INTRODUCTION


Ethanol Exposure During Development and Resulting Nervous System Alterations


Ethanol's teratogenic actions have been recognized throughout recorded history

and considerable research has defined its deleterious actions (West et al., 1994). With the

recognition of the fetal alcohol syndrome (FAS), and the linkage of ethanol to

malformations in children of alcoholic mothers, Jones and Smith (1973) spurred a

plethora of studies on alcohol-induced fetal abnormalities (West et al., 1994).

Consequently, prenatal ethanol exposure has been shown to result in serious

developmental alterations, including intrauterine growth deficiencies, facial dysmorphias,

mental retardation (Abel, 1984), attention deficiencies and autistic-like syndromes

(Aronson et al., 1997), and lowered IQ (Mattson et al., 1997). Ethanol readily crosses the

placental and blood-brain barriers, diffuses into all aqueous components of the

developing fetus where it can interact with membrane proteins and lipids (Zajac and

Abel, 1992), and has been shown to affect protein synthesis, placental nutrient transport,

fetal glucose availability, fetal oxygen levels, generation of reactive oxygen radicals, and

neurotrophic factor activity (Abel and Hannigan, 1995; Bonthius and West, 1990; Heaton

and Bradley, 1995; Henderson et al., 1995; Mukherjee and Hodgen, 1982; West et al.,

1994; Zajac and Abel, 1992).








Zajac and Abel (1992) have characterized fetal alcohol exposure as "the leading

known cause of mental retardation in the Western world." Indeed, despite this

recognition, the incidence of FAS in the United States has increased six-fold between

1979 and 1993 (Prevention, 1995) to 1.95/1000 live births, and 43.1/1000 live births

among heavy drinkers (Abel and Hannigan, 1995). Sampson et al. (1997) recently

estimated the incidence of FAS and alcohol-related neurodevelopmental disorders

between 1975-1981 in Seattle as 9.1/1000 live births. As such, FAS remains a significant

health problem in the United States. Further research describing the neurodevelopmental

changes induced by ethanol and the cellular and molecular changes induced by ethanol is

needed, especially since the mechanism of ethanol teratogenicity remains unknown.

The toll that ethanol exacts on the development of the central nervous system

(CNS) represents its most potent danger. Children exposed prenatally to ethanol show,

among other abnormalities, reduction in corpus callosum area (Riley et al., 1995) and

malformations in cerebellar structure (Clarren et al., 1978; Sowell et al., 1996;

Wisniewski et al., 1983). Animal models have been developed which recapitulate many

of the neurodevelopmental alterations and behavioral outcomes seen in humans with

FAS, and these models have allowed for the characterization of various nervous system

changes resulting from developmental exposure to ethanol (Hannigan, 1996). While

various model systems have been employed, including the chick (Bradley et al., 1997)

and mouse (Schambra et al., 1990) the most common choice for investigators is the rat.

Rats exposed during embryonic development show long-lived learning

impairment (Clausing et al., 1995), behavioral alterations (Riley, 1990), microencephaly

(West and Pierce, 1986), changes in neuronal proliferation and migration (Miller, 1986;








Miller, 1995b; Miller, 1996), reductions of neuronal number (Barnes and Walker, 1981;

Miller, 1995a; Napper and West, 1995b), alterations in neuronal circuitry (West et al.,

1981), delays in synaptogenesis (Hoff, 1988), permutations in neuromorphological

development (Burrows et al., 1995; Davies and Smith, 1981; Kotkoskie and Norton,

1989), changes in neurochemistry (Black et al., 1995; Swanson et al., 1995), and

alterations in the levels of specific mRNA species (Lee et al., 1997) and specific receptor

molecules such as the high affinity trkA nerve growth factor receptor and the low affinity

neurotrophin receptor p75 (Dohrman et al., 1997).

The present body of work sought to further document neuronal populations in the

brain which display vulnerability to developmental ethanol exposure. Another goal of

this work was to investigate cell death gene expression shortly after ethanol insult in the

cerebellum in order to test a specific hypothesis about the cellular mechanism of ethanol

neurotoxicity. To accomplish these goals the following studies were performed: first, a

prenatal rat exposure model was used to examine long term changes in protein expression

patterns of parvalbumin (a marker for GABAergic neurons) in the rat medial septum and

cingulate cortex by counting parvalbumin-immunoreactive neurons in these structures;

second, a neonatal rat exposure model was used to examine long-term changes in the

expression pattern of choline acetyltransferase (ChAT, a marker for cholinergic neurons)

in the medial septum by counting ChAT-immunoreactive neurons in this region; and

third, a similar neonatal rat exposure paradigm was used to document Purkinje and

granule cell numbers in the cerebellar vermis during known periods of ethanol sensitivity

and insensitivity, and to investigate mRNA levels of the bcl-2 family of cell death

molecules.








Differential Temporal and Regional Vulnerability


An interesting property of ethanol and its effect on the developing CNS is the fact

that particular brain regions are differentially affected in both human FAS and rodent

models of FAS (West and Pierce, 1986). Fortunately, animal models of FAS provide for

rigorously controlled studies of both regional and temporal susceptibilities, and allow for

the identification of the neuroanatomical substrates that underlie FAS. Although the

brain develops throughout the entire prenatal and early postnatal period in humans and

rats, there are both regional and temporal susceptibilities to ethanol. In terms of regional

vulnerability, the cerebral cortex (Miller, 1986), and hippocampus (Barnes and Walker,

1981) have been demonstrated to be severely affected with chronic prenatal ethanol

exposure. In terms of temporal vulnerability, CNS development during the human third-

trimester equivalent is especially tenuous. During this period rapid, global brain

development is occurring and this dynamic phase, which occurs during the human third

trimester, is often termed the brain growth spurt. Although all mammals develop in a

similar manner, the timing of this intensified growth is different across species. The rat,

for example, from which most animal data regarding FAS are derived, undergoes its brain

growth spurt during the first two postnatal weeks, with a peak at postnatal day 4-10 (P4-

10) (Dobbing and Sands, 1979). Ethanol exposure during this vigorous period of brain

development affects both mature and proliferating neurons (West and Pierce, 1986). The

principal neurons of the cerebellar cortex provide a good example, as neonatal exposure

reduces the number of differentiating Purkinje cells as well as proliferating granule cells

(Bonthius and West, 1990), albeit in a temporally restricted manner (see below).








Prenatal Ethanol Exposure and Neuroanatomical Alterations


Experimental studies, utilizing rodent models of FAS, have been successful in

reproducing many of the behavioral and morphological changes found in FAS, and have

described numerous CNS alterations resulting from in utero ethanol exposure. Some of

these include alterations in a variety of developmental processes in the CNS, including

perturbed neuronal generation and migration (Miller, 1986; Miller, 1995b; Miller, 1996),

permutations in neuromorphological development (Burrows et al., 1995; Davies and

Smith, 1981; Kotkoskie and Norton, 1989), changes in neurochemistry (Black et al.,

1995; Swanson et al., 1995), delayed synapse turnover in the hippocampus (Hoff, 1988),

alterations in neuronal number in hippocampus (Barnes and Walker, 1981), and long-

lasting detrimental effects on learning and behavior (Clausing et al., 1995; Riley, 1990).

Septohippocampal system

The septohippocampal (SH) system of basal forebrain afferents and hippocampal

targets has been shown to be sensitive to prenatal ethanol exposure. Reductions in CAl

pyramidal neuronal number (Barnes and Walker, 1981; Wigal and Amsel, 1990),

alterations in hippocampal mossy fiber organization (West et al., 1981) and dendritic

arborization (Davies and Smith, 1981; Smith and Davies, 1990), delays in synaptogenesis

(Hoff, 1988), changes in neurochemistry (Black et al., 1995; Swanson et al., 1995), and

deficits in hippocampal synaptic plasticity (Sutherland et al., 1997) have all been

demonstrated in the rodent SH system following exposure to ethanol in utero. Given the

importance of the hippocampus in learning and memory and the role of the SH system in

generating and maintaining electrical activity in the hippocampus (Dutar et al., 1995), the








possibility exists that ethanol-induced changes in this system can have detrimental effects

on offspring exposed to ethanol prenatally.

The SH system is a pathway of cholinergic and GABAergic fibers originating

from the medial septal (MS) nucleus and the horizontal and vertical limbs of the nucleus

of the diagonal band of Broca (DBB) which synapse on pyramidal neurons, granule cells

and interneurons of the hippocampus (Dutar et al., 1995; Freund and Antal, 1988).

Evidence exists for an ethanol-induced alteration of the cholinergic component of the SH

pathway. Severe abnormalities were noted in the basal forebrain of fetal mice following

an acute ethanol dose at gestational day 7 (G7) (Sulik et al., 1984), and Schambra et al.

(1990) found a reduction in the number of ChAT+ neurons in fetal mice following an

acute ethanol administration on G7. Arendt et al. (1988) reported similar findings in

adult rats following chronic ethanol treatment, although their experimental design suffers

from lack of pair-fed controls.

Studies from our laboratory utilizing a rat model of chronic prenatal ethanol

treatment have shown an ethanol-induced delay in the normal ontogeny of ChAT enzyme

activity in the SH pathway, but have not revealed an effect on ChAT-immunoreactive

neuronal number (Swanson et al., 1995; Swanson et al., 1996). It was a goal of the

present work to determine if the other major population of neurons in the medial septum,

the GABAergic neuronal population, is affected by developmental ethanol exposure.

Thus, studies were performed to test the hypothesis that GABAergic neurons in the SH

pathway are susceptible to chronic prenatal ethanol treatment, in order to determine

whether this subset of neurons in the medial septum is affected by ethanol. To achieve

this aim, an antibody which recognizes parvalbumin (PA), a calcium binding protein








commonly found in GABAergic neurons, was used and PA-immunoreactive neurons

were counted.

PA and the SH system

Although the GABAergic component of the SH projection has not been as

extensively characterized as the cholinergic component, it is an important part of this

system. Approximately 33% of the neurons in the MS/DBB region display

immunoreactivity to PA, an 11.8 kDa member of a Ca2+ binding superfamily of proteins

(Kiss et al., 1990a; Kiss et al., 1990b; McPhalen et al., 1994) commonly found in rapidly-

firing GABAergic neurons where it influences the activity of Ca2+-dependent K+ channels

(Plogmann and Celio, 1993). In rats, PA ontogeny begins in the MS at G21 and coincides

with the beginning of physiological activity such as spontaneous firing and excitatory

synaptic input (Lauder et al., 1986; Solbach and Celio, 1991). The PA-expressing MS

neurons innervate inhibitory interneurons in the hippocampus (Freund and Antal, 1988).

Although CNS regions differ in the extent to which PA and GABA co-localize (Alonso et

al., 1990; Brauer et al., 1991; Kiss et al., 1990a), within the MS nucleus most, if not all,

of the hippocampal-projecting GABAergic neurons are parvalbumin-immunoreactive

(Freund, 1989; Krzywkowski et al., 1995). Thus, PA-immunoreactivity serves as a

reliable marker for hippocampal-projecting GABAergic neurons in the MS nucleus and

identifies a subpopulation of the total GABAergic neuronal pool within the basal

forebrain.

PA and cingulate cortex

Quantitative analyses of neuroanatomical changes in the cerebral cortex following

prenatal ethanol exposure have been conducted and have suggested ethanol-induced








neuroanatomical alterations. Specifically, alterations in the generation and proliferation

of neurons have been noted (Miller, 1986; Miller, 1996). Another goal of the present

work was to determine whether prenatal ethanol exposure alters the neuroanatomy of

limbic cortex by testing the hypothesis that the number of GABAergic intemeurons

expressing PA are altered in the cingulate cortex following prenatal ethanol exposure.

Although little is known about the cingulate cortex, it is clear that the cingulate is a relay

center of the limbic lobe and is important for emotion and memory (Kupfermann, 1991).

PA expression in the cingulate cortex begins during the first postnatal week in rats and

coincides with the functional maturation of cerebral intemeurons (de-Lecea et al., 1995).

The cingulate cortex was chosen to extend the earlier observations in the cerebral

cortex following prenatal ethanol exposure because of the observed behavioral problems

in children with FAS, including poor judgment, distractibility, and difficulty perceiving

social cues (Streissguth et al., 1991). Additionally, many alcoholics who develop

Korsakoff's syndrome have deficiencies in glucose utilization within the cingulate cortex,

potentially contributing to learning and memory defects due to interruption of Papez'

circuitry (Joyce et al., 1994). It is conceivable that alterations in PA expression patterns

might contribute to behavioral anomalies and/or learning and memory deficiencies.

Postnatal Ethanol Exposure and Neuroanatomical Alterations


SH system

The SH system of basal forebrain afferents and hippocampal targets also exhibits

susceptibility to ethanol during neonatal development. While numerous studies have

described the effects of neonatal ethanol exposure on neurons within the hippocampus








(Bonthius and West, 1990; Bonthius and West, 1991; Greene et al., 1992; Pierce and

West, 1987; West and Pierce, 1986) and have documented deficits in spatial learning

following neonatal ethanol exposure (Goodlett and Peterson, 1995; Kelly et al., 1988), the

effect of ethanol exposure during the brain growth spurt on the cholinergic neurons of the

medial septum is unknown. A previous study from our laboratory documented an

ethanol-induced delay in the normal ontogeny of ChAT enzyme activity in the SH

pathway, but did not reveal an effect on ChAT-immunoreactive neuronal number

following chronic prenatal ethanol treatment (Swanson et al., 1995; Swanson et al.,

1996). It was a goal of the present work to determine the long-term effects of ethanol

exposure on the number of ChAT+ neurons in the rat MS when ethanol is delivered

during the brain growth spurt to determine whether this neuronal population is sensitive

to neonatal ethanol exposure.

Cerebellum

Evidence of cerebellar vulnerability to developmental ethanol exposure comes

from human studies demonstrating size reduction in the cerebellar vermis of children

exposed prenatally to ethanol (Sowell et al., 1996). In rodent studies, the cerebellum

displays a pattern of differential temporal susceptibility to ethanol in the brain growth

spurt. This effect has been demonstrated in multiple laboratories utilizing a variety of

ethanol-exposure techniques. Purkinje cell number is reduced following exposure to

ethanol postnatally, during differentiation, but not following exposure to ethanol

prenatally during neurogenesis (Marcussen et al., 1994). Within the postnatal period,

Purkinje cells have been shown to be particularly vulnerable to ethanol in the first

postnatal week (Bauer-Moffett and Altman, 1977; Bonthius and West, 1991; Goodlett








and Eilers, 1997; Pauli et al., 1995; Pierce et al., 1993). Purkinje cells are generated in

the rat cerebellum between embryonic day 14 and 17; the period of Purkinje cell death in

the cerebellum begins late in gestation and peaks in the first postnatal week (Cragg and

Phillips, 1985). Purkinje cell susceptibility to ethanol within the cell death period has

been well documented, with ethanol-accelerated Purkinje cell loss found as early as 12

hours following a postnatal day 3 ethanol insult (Cragg and Phillips, 1985).

Exposure to comparable levels of ethanol in the second postnatal week, however,

has been shown to have no effect on Purkinje cells (Goodlett et al., 1997; Hamre and

West, 1993; Pauli et al., 1995) or very little effect (Thomas et al., 1998). In contrast to

Purkinje cells, granule cells are generated during the rat brain growth spurt (Altman,

1969). However, like Purkinje cells, granule cells display a differential pattern of

susceptibility to ethanol, with loss occurring following ethanol exposure in the first

postnatal week but not in the second postnatal week (Hamre and West, 1993). One aim

of the present work was to determine the pattern of cerebellar neuronal susceptibility in

our laboratory following first and second postnatal ethanol treatment, in order to validate

the model system for use in our laboratory. The main objective, however, was to

determine whether neonatal ethanol treatment altered mRNA levels of members of the

bcl-2 family of cell death regulators in the cerebellar vermis (and thereby influenced the

survival or death of neurons in this region). Experiments were performed in order to test

the specific hypothesis that altered bcl-2 family gene expression ensues following ethanol

exposure.

A role in maintaining cerebellar neurons has previously been demonstrated for the

bcl-2 family. Gillardon et al. (1995) investigated bcl-2 and bax gene expression in the








cerebella of Purkinje-cell-degeneration mice (mutants that lose nearly all of their Purkinje

cells between P22-28 following otherwise normal development). They found that bcl-2

mRNA levels decreased while bax mRNA levels remained unchanged beginning on P22.

In addition, thyroid hormone-induced upregulation of bcl-2 protects early-differentiating

cerebellar granule cells from apoptosis in vitro (Muller et al., 1995), and transgenic mice

overexpressing bcl-2 contain more cerebellar Purkinje and granule cells than controls

(Zanjani et al., 1996; Zanjani et al., 1997). The following section provides background

on the bcl-2 family and its involvement in cell death regulation.

Programmed Cell Death (PCD) and the bcl-2 Family


PCD, a developmental form of apoptotic cell death, is a common process in the

animal kingdom (Ellis et al., 1991). In the vertebrate nervous system the regulation of

neuronal survival is essential for the correct formation of synapses and for the survival of

the appropriate number of neurons (Oppenheim, 1991). Only the most appropriate

connections are maintained, making axon-target interactions maximally efficient and

simultaneously ensuring that cells which are generated in excess, develop poorly, are

functionally inadequate, or are harmful, do not endure in adult organisms (Ellis et al.,

1991). Competition for a limited supply of target-derived neurotrophic factors is thought

to determine which neurons survive the period of naturally occurring PCD (Davies,

1994). The mechanism by which non-essential neurons are eliminated is consistent with

the apoptotic form of cell death (Johnson and Deckwerth, 1993), with morphological

characteristics consisting of chromatin condensation, cell shrinkage, cleavage of DNA

into oligonucleosomal fragments (Edwards et al., 1991), and phagocytosis of dead cells








(Ellis et al., 1991) without induction of the inflammatory response (Columbano, 1995).

Most mammalian cells constituitivly express the proteins essential for the cell death

program (Davies, 1995; Raffet al., 1993).

Cytotoxicity due to ethanol in vitro has been shown to be apoptotic in fetal

hypothalamic neurons (De et al., 1994) and in thymocytes (Ewald and Shao, 1993).

Additionally, ethanol-induced cell death in the cerebellum appears to proceed through an

apoptotic mechanism. Cerebellar neurons undergo apoptosis in vitro and in vivo in

response to ethanol (Bhave and Hoffman, 1997; Liesi, 1997; Renis et al., 1996; Singh et

al., 1995) and ethanol induces nuclear DNA strand breaks in the cerebellum after chronic

adult exposure (Renis et al., 1996).

Almost a decade ago, a gene, bcl-2, was discovered that appeared to modulate

apoptosis. The name bcl-2 is an acronym for B-cell lymphoma/leukemia-2 gene (Reed,

1994) and the identification of this gene family resulted from studies examining the

t(14;18) chromosomal translocation in human follicular non-Hodgkin's B-cell

lymphomas (Tsujimoto et al., 1985). The protein product encoded by the bcl-2 gene, Bcl-

2, is a 25 kDa protein found predominately in mitochondrial membranes (Hockenbery et

al., 1990) but it is also found in endoplasmic reticula and outer nuclear membranes (Akao

et al., 1994). In recent years, a number of new genes similar to bcl-2 have been

characterized and added to the diverse bcl-2 family of genes. These include bcl-xl (Boise

et al., 1993), bcl-xs (Boise et al., 1993), bax (Oltvai et al., 1993), bad (Yang et al., 1995)

al (Lin et al., 1993), mcl-1 (Kozopas et al., 1993), bak (Chittenden et al., 1995), bcl-w

(Gibson et al., 1996), brag-1 (Das et al., 1996), bok (Hsu et al., 1997), and bim (O'connor

et al., 1998). Some members of the bcl-2 gene family serve to inhibit cell death (e.g. bcl-








2, bcl-xl, mcl-1, al) and others have been found to promote cell death (e.g. bcl-xs, bax,

bad, bak, bok).

Bcl-2 (and the similar anti-apoptotic protein Bcl-xl) functionally blocks apoptotic

death in neurons (Allsopp et al., 1993; Garcia et al., 1992) by inhibiting caspase

activation (Shimizu et al., 1996), regulating mitochondrial membrane potential, proton

flux across mitochondrial membranes (Shimizu et al., 1998), and by preserving

mitochondrial outer membrane integrity (Vanderheiden et al., 1997). The pro-apoptotic

molecules of the bcl-2 family (such as the proteins Bad and Bak) function by inhibiting

the ability of the anti-apoptotic molecules of the bcl-2 family to function. They do so by

preventing the necessary homodimerization of the protective molecules through direct

competition for binding to the ligand binding regions of these proteins (Ottilie et al.,

1997). The intracellular mechanisms of bcl-2 family function are described in detail

below.

Expression patterns

Given the identification of these molecules and the fact that apoptosis is known to

occur in the developing nervous system, the possibility that bcl-2 family members and

their protein products might modulate PCD in the nervous system has been explored.

Castren et al. (1994) have shown that bcl-2 mRNA is expressed in high levels in the

prenatal rat neuroepithelium and cortical plate, with a late-prenatal peak and expression

reaching lower adult levels during postnatal development. In situ hybridization also

revealed that bcl-2 expression is retained in the olfactory bulb, hippocampus, pons,

cerebellum, and ependymal cells of the adult rat brain (Castren et al., 1994). Bcl-2

protein is also widely expressed in the developing nervous system of mice, rhesus








monkeys, and humans, especially during embryonic development, but also during the

period of PCD (Merry et al., 1994). Neuroepithelial cells of the ventricular zone,

postmitotic cells of the cortical plate, Purkinje and granule cells of the cerebellum,

hippocampus, and spinal cord all express Bcl-2 (Gleichmann et al., 1998; Merry et al.,

1994).

Expression of bcl-xl follows a similar pattern, with mRNA levels increasing at the

beginning of the PCD period. In the brain, in contrast to Bcl-2, Bcl-xl expression

increases after birth in neurons of the cortex and olfactory bulb, as well as in Purkinje

cells, to reach a high level in the adult brain, suggesting a role for Bcl-xl in the adult CNS

(Frankowski et al., 1995; Sohma et al., 1996). Although previous reports have suggested

that bcl-xs is not expressed in the rat CNS, newer studies using more sensitive techniques

have detected bcl-xs mRNA in the adult rat brain (Dixon et al., 1997; Rouayrenc et al.,

1995). Although the protein product of bcl-xs is able to weakly bind Bcl-2 and Bcl-xl, it

appears that Bcl-xs affects apoptosis by a distinct mechanism that, unlike other family

members, does not involve direct protein interactions with cell death repressor proteins

(Minn et al., 1996). Both long and short forms of bcl-x are also found in cerebellar

granule cells during development (Gleichmann et al., 1998).

bax is a gene in the bcl-2 family whose protein product, Bax, is a 21 kDa protein

with amino acid homology (21%) with Bcl-2 (Oltvai et al., 1993). Bax forms

heterodimers with Bcl-2, Bcl-xl, Mcl-1, and Al (Sedlak et al., 1995), and Bax can

counteract the protective actions of Bcl-2 when overexpressed (Oltvai et al., 1993). It has

been suggested that the ratio of Bcl-2 to Bax may determine survival or death from

apoptosis by serving as a sort of rheostat: when Bcl-2 is in excess, cells survive, but








when Bax is in excess, cells die (Oltvai et al., 1993). bax mRNA has been found in the

developing and adult rat brain. Similarly, in the adult mouse, the Purkinje and granule

cells of the cerebellum, cerebral cortical neurons, and sympathetic neurons all express

Bax protein (Gleichmann et al., 1998; Krajewski et al., 1994; Oltvai et al., 1993;

Vekrellis et al., 1997). While Bax protein expression is high in the neonatal cerebral

cortex and cerebellum, protein levels drop off dramatically after the PCD period,

suggesting that neurons regulate their sensitivity to apoptosis during development by

regulating expression of Bax (Vekrellis et al., 1997).

Consistent with the rheostat hypothesis, the ratio of Bcl-2:Bax correlates with cell

survival in the mature rat hippocampus following global ischemia. bax mRNA and

protein are both constitutively expressed in the ischemia-sensitive CAl hippocampal

neurons, whereas Bcl-2 is not expressed in these neurons (Chen et al., 1996). In

hippocampal region CA3, a population more resistant to ischemia than cells in region

CAl, Bcl-2 protein expression, but not that of Bax, is high (Chen et al., 1996).

Furthermore, high levels of Bax and concomitant low levels of Bcl-2 have been found in

other populations of neurons that are sensitive to cell death induced by ischemia, such as

Purkinje cells (Krajewski et al., 1995). Additional evidence supporting the bcl-2 rheostat

hypothesis comes from studies in mouse brain that have demonstrated up-regulation of

Bax and down-regulation of Bcl-2 associated with kainate-induced apoptosis (Gillardon

et al., 1995), as well as studies from rat that have shown a decrease in the Bcl-2:Bax ratio

in motoneurons following sciatic nerve transaction (Gillardon et al., 1996) and ischemia

(Isenmann et al., 1998). Additional evidence from Dixon et al. (1997) shows that the pro-

apoptotic bcl-xs mRNA is upregulated shortly after global ischemia in rats. Thus, it is








clear that the balance between pro- and anti-apoptotic molecules (at the protein and

mRNA levels) during development and following injury in adulthood determines the

survival of the cell.

Genetically engineered mice

Studies using genetically engineered mice have been fruitful in describing a role

for various members of the bcl-2 gene family in vivo. In transgenic mice overexpressing

Bcl-2 protein in the nervous system, a reduction in developmental cell death of facial

motoneurons and retinal ganglion cells, and a general hypertrophy of the nervous system

are observed (Martinou et al., 1994). Neurons from these animals are more resistant to

ischemia, neurotrophic factor withdrawal, and axotomy (Dubois-Dauphin et al., 1994;

Farlie et al., 1995). Similarly, facial motoneurons from mice overexpressing bcl-xl are

resistant to axotomy during the postnatal period, indicating a role for the bcl-xl gene in

the survival of postnatal CNS neurons (Parsadanian et al., 1998). Studies using

genetically engineered mice lacking the bcl-2 gene have been similarly instrumental in

defining the normal actions of Bcl-2 in vivo. bcl-2 -/- mice live through gestation,

display massive apoptosis in lymphoid organs, and produce gray hair follicles (Veis et al.,

1993). The brains of these animals, however, appear grossly normal at birth although fine

analyses of specific neuronal populations have not been performed. The lack of massive

cell death in neonatal bcl-2 knockouts has been attributed to redundancy, as the bcl-2

family is so large (Motoyama et al., 1995). However, analysis of neuronal populations

after the PCD period reveal loss relative to controls of motoneurons, sympathetic, and

sensory neurons, demonstrating a role for bcl-2 in maintaining these neuronal populations

(Michaelidis et al., 1996).








Mice lacking the bcl-x gene have also been generated, but die around E13. Upon

examination, extensive apoptotic cell death is evident in neurons of the brain, spinal cord,

and dorsal root ganglion (Motoyama et al., 1995), revealing the importance of bcl-x in

embryonic life. bax-deficient mice have been generated and have helped define the

actions of cell-death promoting molecules in particular neuronal populations (Deckwerth

et al., 1996). Early postnatal sympathetic and facial motoneurons from bax knockouts

survive growth factor deprivation and axotomy. Additionally, superior cervical ganglia

and facial nuclei of bax knockouts possess more surviving neurons in vivo. Thus, a role

for bax has been demonstrated in cell death associated with growth factor deprivation and

axotomy (Deckwerth et al., 1996). Shindler et al. (1997) have generated mice deficient in

both bcl-xl and bax. While bax deficiency does not prevent the embryonic lethality of

bcl-xl deficient mice, the double knockout did demonstrate the interplay between these

pro- and anti-apoptotic molecules in vivo. Specifically, bax and bcl-xl deletion produced

less apoptosis in the brainstem and spinal cord when compared with bcl-xl knockouts.

It is important to note that while studies using genetically engineered mice are

interesting and often quite suggestive, they suffer from several caveats. For instance,

overexpression of a particular gene or the creation of a mutant lacking a particular gene

may cause compensatory up- or down- regulation of other gene products which can lead

to developmental alterations in attempts to compensate for the altered gene (Gerlai,

1996). Moreover, mutant mice may differ in non-targeted gene loci and certain

behavioral alterations may be due to differences in genetic background (Gerlai, 1996).

Thus, while suggestive of the role of particular genes, studies utilizing genetically

engineered mice must be analyzed in light of these caveats.








Intracellular mechanisms

Recent evidence has shed light on the intracellular mechanism by which bcl-2

family members operate and has revealed a "double identity" for the family as both ion

channel and adapter protein (Reed, 1997). Bcl-xl, Bcl-2, and Bax have all been shown to

form functional ion channels in lipid membranes (Antonsson et al., 1997; Minn et al.,

1997; Schendel et al., 1997). Also, Bax channel-forming activity is inhibited by Bcl-2

(Antonsson et al., 1997). These ion channels appear to influence cell survival by

regulating the permeability of the intracellular membranes in which they are anchored. In

particular, the Bcl-2 and Bcl-xl membrane complexes inhibit the release of cytochrome C

from mitochondria (Yang et al., 1997), regulate mitochondrial membrane potential and

proton flux across mitochondrial membranes (Shimizu et al., 1998), and help preserve

mitochondrial outer membrane integrity (Vanderheiden et al., 1997). Bax channels, in

contrast, are known to promote the release of cytochrome C from mitochondria (Rosse et

al., 1998).

Cytochrome C is a known activator of the PCD effector molecules, the caspases

(Yang et al., 1997). Permeability transition across mitochondrial membranes appears to

be an early event in PCD, and is related to the activation of the release of cytochrome C

and the resultant activation of the effector phase of PCD (Petit et al., 1996). This

activation is brought about by cytochrome C-dependent cleavage and activation of

caspases 3 (Li et al., 1997) and 9 (Zou et al., 1997). In keeping with the adapter protein

role of the anti-apoptotic molecules, recent evidence shows that Bcl-xl binds to Apaf-1,

the newly discovered mammalian homolog of the C. elegans CED-4 (Zou et al., 1997),

and these two proteins, along with the uncleaved and inactive caspase 9, exist in a ternary








complex (Pan et al., 1998). Cytochrome C release is thought to promote activation of the

caspase cascade by promoting the dissociation of this ternary complex through an

undefined mechanism. Once caspases are activated, they act upon a variety of

intracellular substrates, including inhibitors of caspase-activated deoxyribonuclease

(ICAD). Caspase 3 is known to cleave ICAD and result in the elimination of its normally

inactivating effect on caspase-activated deoxyribonuclease (CAD). CAD functions

downstream in the proteolysis cascade and its reduced inhibition results in DNA

degradation characteristic of apoptotic cell death (Sakahira et al., 1998).

Another piece to this intriguing puzzle has been provided by Yang et al. (1998),

who have shown that CED-4 (and likely the mammalian counterpart Apaf-1) promotes

CED-3 (the C. elegans caspase) processing and activation by promoting the aggregation

of unprocessed CED-3. This "induced proximity" is thought to sequester inactive caspase

proenzymes to increase their local concentration and promote conformational changes

which will increase the likelihood of their activation (Hengartner, 1998), and is brought

about by oligomerization of the CED-4:CED-3 complex (Yang et al., 1998). Thus, an

important role of Bcl-xl and Bcl-2 appears to be to prevent the association of these

proenzymes by preventing CED-4 (and by association, Apaf-1) oligomerization

(Hengartner, 1998). While a similar role for Apaf-1 has not been definitively

demonstrated, this will surely be one of the important future discoveries in this

blossoming field.

Newly uncovered evidence also indicates that the anti-apoptotic molecules of the

bcl-2 family can contribute to the cell's demise under certain intracellular conditions. For

example, if caspase activation proceeds to a critical point, caspases will act on the Bcl-2








and Bcl-xl proteins as substrates (Cheng et al., 1997; Clem et al., 1998). Indeed, caspase

cleavage of these protective molecules converts them into Bax-like death-promoting

molecules. This cleavage is thought to act as a feed-forward mechanism for further

caspase activation, and should ensure cell death.

Besides these important discoveries, several others have demonstrated that

apoptosis interacts with signal transduction intracellularly and that this interaction is

mediated, in part, by the bcl-2 family (Gajewski and Thompson, 1996). The application

of IL-3 to the FL5.12 cell line leads to phosphorylation and inactivation of the pro-

apoptotic molecule Bad. The phosphorylation of Bad leads to its association with the

cytosolic protein 14-3-3, prevents Bad:Bcl-xl heterodimerization, and promotes Bcl-xl

homodimerization, which results in cell survival (Zha et al., 1996). Additionally, Wang

et al. (1996) showed that Bcl-2 could target Raf-1 to mitochondrial membranes which

results in the phosphorylation of Bad.

Two groups independently determined that the protein kinase Akt is also

responsible for growth factor-mediated Bad phosphorylation (Datta et al., 1997; Delpeso

et al., 1997). Akt (also known as protein kinase B, or PKB) is an important intracellular

molecule and its phosphorylation (and activation) is brought about by growth factor-

mediated phosphorylation of PI3 kinase, followed by PI3 kinase phosphorylation of Akt

(Kahn, 1998; Zhou et al., 1997). Thus, it is apparent that a complex interaction between

extracellular growth and survival signals integrates with the complex intracellular biology

of individual neurons to determine whether a particular cell will survive a developmental

process or injury.








Hypotheses Tested


As mentioned previously, neuronal populations are not uniform in their

vulnerability to ethanol, nor are developmental stages uniform in susceptibility to ethanol

treatment. And while a variety of CNS populations have been demonstrated to be

adversely affected by prenatal ethanol exposure, the full extent of nervous system

vulnerability to ethanol is unknown. Likewise, the cellular effects of ethanol exposure,

such as changes in gene expression, are poorly understood. It is with this in mind that the

research reported in this document was undertaken.

Because of the observed learning and memory deficits in children exposed in

utero to ethanol (Streissguth et al., 1991), the SH system is a natural region in which to

extend studies in rodents of neuroanatomy following ethanol treatment. While cell loss in

the hippocampus has been documented following prenatal exposure (Barnes and Walker,

1981), cellular changes in the cholinergic basal forebrain component of the SH system

have not been found after a similar pattern of exposure (Swanson et al., 1996). Thus, it

was an aim of the present work to determine whether the GABAergic component of the

SH pathway is susceptible to prenatal ethanol treatment. Additionally, the cingulate

cortex was analyzed because of the observed behavioral problems in children with FAS

(Streissguth et al., 1991) and the decreased glucose utilization in the cingulate cortex of

alcoholics (Joyce et al., 1994). Thus, the following hypothesis was explored: chronic

prenatal ethanol exposure will lead to alterations in the number of neurons expressing

PA in the adult rat medial septum and cingulate cortex.








Rat studies have also identified the early postnatal period as a developmental time

during which particular CNS structures are sensitive to ethanol's toxic effects. For

example, the Purkinje cells of the cerebellar cortex are reduced in number when ethanol

is delivered during the early neonatal period, but not when ethanol is given in utero

(Marcussen et al., 1994). The SH system is a region which exhibits susceptibility to

ethanol during both the prenatal and the neonatal periods. Neonatal ethanol exposure

disrupts the normal development of the SH system in rodent models of FAS, and much

research has documented neuroanatomical changes in the hippocampus (Bonthius and

West, 1990; Bonthius and West, 1991; Greene et al., 1992; Pierce and West, 1987; West

and Pierce, 1986). However, the effect of neonatal ethanol exposure on the MS has not

been investigated. Because the number of cholinergic neurons of the medial septum are

not altered by prenatal ethanol treatment, it was an objective of the present work to

determine the long-term effects of neonatal ethanol exposure on the cholinergic neurons

in the rat. The following hypothesis was investigated: early postnatal ethanol exposure

will lead to alterations in the number of neurons expressing ChAT in the adult rat CNS.

While descriptions of susceptible neuronal populations following developmental

ethanol exposure has been, and continues to be, a fruitful avenue of research, further

investigation into the molecular consequences of ethanol treatment (e.g. gene expression

changes resulting from ethanol) are warranted. Given the previously described temporal

pattern of Purkinje and granule cell loss in the postnatal cerebellum, a survey of the bcl-2

literature raises the intriguing possibility that the differential temporal teratogenicity of

ethanol on cerebellar cells may be related to changes in the levels of expression of PCD

repressor and inducer genes. It is significant that proteins translated from bcl-2 and bax








mRNA dimerize, and as noted above, cell death or survival depends on the relative

amounts (ratio) of these proteins (Oltvai et al., 1993). Similar cellular survival outcomes

have been linked to the relative ratio of bcl-2 to bax gene expression (Basile et al., 1997;

Chen et al., 1996).

The observation that cerebellar neurons are susceptible to ethanol neurotoxicity as

a function of the timing of the ethanol insult suggests that ethanol may act in vivo to

modulate (upregulate or downregulate) the expression of certain mRNAs of the bcl-2

family and thereby alter susceptibility to ethanol neurotoxicity. Thus, any ethanol-

induced change in bcl-2 family gene expression might disrupt the normal balance of these

proteins in developing neurons and decrease or increase their chance of cell death.

Recent evidence, mentioned above, in addition to the aforementioned expression patterns,

implicates bcl-2 family members in maintaining cerebellar neurons. Purkinje-cell-

degeneration mouse mutants lose nearly all of their Purkinje cells between P22-28.

Gillardon et al. (1995) investigated bcl-2 and bax expression in the cerebella of these

mice and found that bcl-2 mRNA levels decreased while bax mRNA levels remained

unchanged beginning on P22. There was a concomitant reduction of Bcl-2 expressing

Purkinje cells in the mutants compared with wild-types, suggesting a down-regulation of

bcl-2 in Purkinje cells destined to die (Gillardon et al., 1995).

In addition, thyroid hormone-induced upregulation of bcl-2 protects early-

differentiating cerebellar granule cells from apoptosis in vitro (Muller et al., 1995).

Moreover, transgenic mice overexpressing bcl-2 contain more cerebellar Purkinje and

granule cells than controls whether transgene expression was induced in the embryonic or

postnatal periods (Zanjani et al., 1996; Zanjani et al., 1997). Therefore, another goal of








the present work was to test following hypothesis: ethanol-induced alterations in the

expression levels of bcl-2 family PCD genes in the cerebellum contribute to the

cerebellum's relative temporal susceptibility to ethanol neurotoxicity.

The chapters that follow provide a detailed description of methods utilized to test

these hypotheses, and the data collected are presented and fully discussed. Following

these chapters is a concluding chapter that presents interpretations derived from these

data and discusses specific methodological considerations and future directions which the

current data suggest.















CHAPTER 2
EFFECTS OF PRENATAL ETHANOL EXPOSURE ON PARVALBUMIN-
IMMUNOREACTIVE GABAERGIC NEURONS IN THE ADULT RAT MEDIAL
SEPTUM AND ANTERIOR CINGULATE CORTEX


Summary


Exposure of human fetuses to ethanol often results in the fetal alcohol syndrome

(FAS). Animal models of FAS have been developed and have been utilized to examine

the consequences of prenatal ethanol exposure on the central nervous system. While cell

loss in the hippocampus has been documented following prenatal ethanol exposure

(Barnes and Walker, 1981), cellular changes in the cholinergic basal forebrain component

of the septohippocampal (SH) system have not been found after a similar pattern of

ethanol exposure (Swanson et al., 1996). The objective of this study was to determine the

long-term effects of prenatal ethanol exposure on parvalbumin-expressing (PA+)

GABAergic neurons of the rat medial septum and anterior cingulate cortex.

Pregnant Long-Evans rats were maintained on one of three diets throughout

gestation: an ethanol-containing liquid diet in which ethanol accounted for 35% of the

total calories, a similar diet with the isocaloric substitution of sucrose for ethanol, or a lab

chow control diet. Offspring were sacrificed at postnatal-day 60 and their brains were

prepared for parvalbumin immunocytochemistry. Female rats exposed to the ethanol-

containing diet during gestation had 42 % fewer total PA+ neurons in the medial septum

and reduced PA+ cell density when compared to female rats exposed to the sucrose diet.








Ethanol-exposed females also had fewer PA+ neurons per section than sucrose-control

females. Male rats exposed to ethanol did not display a similar change in PA+ neurons or

density. No effect of prenatal diet was found on the area or volume of the medial septum,

or the size of the PA+ neurons therein. As such, prenatal exposure to ethanol appears to

permanently reduce the number of PA+ neurons in the female rat medial septum without

affecting the size of the structure or the size of the neurons.

Due to the observed behavioral problems in children with FAS (Streissguth et al.,

1991) and the fact that alcoholics show decreased glucose utilization in the cingulate

cortex (Joyce et al., 1994), the effect of prenatal ethanol exposure on the number of PA+

GABAergic neurons in the adult rat anterior cingulate cortex was also determined. This

represents the first attempt to document developmental alterations in the cingulate cortex

following ethanol exposure. Rats exposed to the ethanol-containing diet contained 45%

fewer total PA+ neurons in the anterior cingulate cortex, and fewer PA+ neurons per

section compared with sucrose and chow controls. No gender differences were found in

measures of the anterior cingulate. The reduction in PA+ neurons occurred in the absence

of changes in structure area or volume, and occurred in the absence of changes in PA+

neuronal size. Functional implications and possible relations to the fetal alcohol

syndrome are discussed.

Introduction


Exposure of human fetuses to ethanol often results in a constellation of

developmental anomalies which make up FAS. Such developmental perturbations

include pre- and postnatal growth deficiencies, morphological (e.g. craniofacial)








abnormalities and central nervous system (CNS) deficits (Abel, 1995) and these typically

persist into adulthood (Streissguth et al., 1991). The incidence of FAS in the general

obstetric population of the United States is estimated at 1.95 per 1,000 live births (Abel

and Hannigan, 1995) and FAS is thought to be the leading nongenetic cause of mental

retardation (Abel and Sokol, 1986). Experimental studies, utilizing rodent models of

FAS, have been successful in reproducing many of the behavioral and morphological

changes found in FAS, and have described numerous CNS alterations resulting from in

utero ethanol exposure. Alterations in CNS development include changes in neuronal

proliferation and migration (Miller, 1986; Miller, 1995b; Miller, 1996), altered neuronal

and cortical morphology (Burrows et al., 1995; Davies and Smith, 1981; Kotkoskie and

Norton, 1989), changes in receptor density and enzyme ontogeny (Black et al., 1995;

Swanson et al., 1995), delays in synaptogenesis (Hoff, 1988), alterations in neuronal

number (Barnes and Walker, 1981), and long-lasting deficits in learning (Clausing et al.,

1995) and behavior (Riley, 1990). The present study sought to determine the

vulnerability of GABAergic intemeurons expressing parvalbumin in the medial septum

and anterior cingulate cortex following prenatal ethanol exposure.

Medial Septum

Numerous studies investigating the effects of prenatal ethanol exposure on rodents

have determined that the CNS is not uniform in its susceptibility to ethanol. In addition

to differential temporal susceptibility, the CNS exhibits differential regional vulnerability.

The SH system of basal forebrain afferents and hippocampal targets is one region that has

been shown to be sensitive to developmental ethanol exposure. Permanent reductions of

CAl pyramidal neurons (Bames and Walker, 1981; Wigal and Amsel, 1990), alterations








in hippocampal neuronal circuitry (West et al., 1981) and dendritic arborization (Davies

and Smith, 1981; Smith and Davies, 1990), delayed synaptogenesis in the hippocampal

dentate gyrus (Hoff, 1988), changes in hippocampal muscarinic receptors (Black et al.,

1995), and delayed choline acetyltransferase (ChAT) enzyme ontogeny in the basal

forebrain (Swanson et al., 1995) have all been demonstrated in the rodent SH system

following exposure to ethanol in utero. Because of the SH system's role in generating

and maintaining electrical activity in the hippocampus (Dutar et al., 1995), and the

importance of the hippocampus in learning and memory, the possibility exists that

ethanol-induced changes in this system can have detrimental effects on offspring exposed

to ethanol prenatally.

Intemeurons, pyramidal neurons, and granule cells of the hippocampus all receive

synapses from cholinergic and GABAergic fibers originating in the medial septum (MS)

and the horizontal and vertical limbs of the nucleus of the diagonal band of Broca (DBB)

(Dutar et al., 1995; Freund and Antal, 1988). Evidence exists for an ethanol-induced

alteration of the cholinergic component of the SH pathway. Sulik et al. (1984), for

example, noted severe abnormalities in the basal forebrain of fetal mice following an

acute ethanol dose at gestational day 7 (G7). Moreover, Schambra et al. (1990) found a

reduction in the number of ChAT-immunoreactive neurons in fetal mice following an

acute ethanol administration on G7. Similar findings in adult rats were reported

following chronic ethanol treatment (Arendt et al., 1988).

In contrast, studies from our laboratory utilizing a rat model of chronic prenatal

ethanol treatment (CPET) have shown an ethanol-induced disruption of the normal ChAT

ontogeny in the SH pathway, but have not revealed an effect on ChAT-immunoreactive








neuronal number (Swanson et al., 1995; Swanson et al., 1996). The present study sought

to determine whether the GABAergic component of the SH pathway was susceptible to

CPET.

Although not as extensively characterized as the cholinergic component, the

GABAergic component of the SH projection is an important part of this system.

Parvalbumin (PA), an 11.8 kDa member of a Ca2+ binding superfamily of proteins (Kiss

et al., 1990a; Kiss et al., 1990b; McPhalen et al., 1994), is expressed in approximately

33% of the neurons in the MS/DBB region. PA is commonly found in fast-firing

GABAergic neurons where it influences the activity of Ca2+-dependent K+ channels

(Plogmann and Celio, 1993). PA ontogeny begins in the MS of rats at G21 and coincides

with the beginning of physiological activity such as spontaneous firing and excitatory

synaptic input (Lauder et al., 1986; Solbach and Celio, 1991), and the PA-expressing MS

neurons innervate inhibitory intemeurons in the hippocampus (Freund and Antal, 1988).

CNS regions differ in the extent to which PA and GABA co-localize (Alonso et al., 1990;

Brauer et al., 1991; Kiss et al., 1990a), but within the MS nucleus most, if not all, of the

hippocampal-projecting GABAergic neurons are PA+ (Freund, 1989; Krzywkowski et al.,

1995). The present study sought to determine the long-term effects of prenatal exposure

to ethanol on a PA+ subpopulation of GABAergic projection neurons in the SH pathway.

For this determination we performed counts of PA+ neurons in the adult rat MS nucleus

following CPET.

Cingulate Cortex

The present study also sought to determine the vulnerability of GABAergic

intemeurons expressing parvalbumin in the anterior cingulate cortex following CPET.








The ontogeny of PA mRNA and protein in the rat cingulate cortex, and its functional role,

is similar to that noted previously for the medial septum. PA expression begins in the

first postnatal week, and coincides with the functional maturation of cerebral interneurons

in the cingulate (Alcantara et al., 1993; de-Lecea et al., 1995). The cingulate cortex was

chosen for analysis because it is a major relay center of the limbic lobe and is involved in

motor control, attention, emotion, and memory (Kupfermann, 1991; Muir et al., 1996;

Paus et al., 1993; Picard and Strick, 1996). The well documented cognitive and

behavioral impairments in children with FAS, including poor judgment, distractibility,

and hyperkinetic and emotional disorders (Steinhausen et al., 1993; Streissguth et al.,

1991), led us to investigate whether anatomical alterations in the cingulate cortex may

underlie these behavioral defects. Thus, the current study also sought to determine the

long-term effects of prenatal exposure to ethanol on PA+ interneurons of the cingulate

cortex.

Materials and Methods


Subjects and ethanol treatment

Long-Evans hooded rats, purchased from Charles River Co. (Portage, MI), were

housed individually, or in pairs, under controlled temperature and humidity conditions,

and were maintained on a 07:00-19:00 hour light cycle. Nulliparous females were placed

individually with a male overnight until vaginal smear the following morning was

indicative of insemination. This was defined as GO. At this time, females were matched

according to age and weight and assigned to one of three treatment groups from GO-G21:

ethanol, sucrose, or chow. During this time the ethanol group was given free access to an








ethanol-containing liquid diet in which ethanol comprised 35% of the total calories. The

sucrose group was pair-fed the same volume of a similar liquid diet which lacked ethanol

and contained an isocaloric substitution of sucrose for ethanol. The liquid diet was

prepared by mixing a stock ethanol or sucrose solution with Sustacal (Mead Johnson).

Diets were additionally enriched with Vitamin Diet Fortification Mixture and Salt

Mixture (ICN Nutritional Biochemicals). The diets contained 1.3 kcal/ml and have been

shown to provide several times the daily requirement of all essential vitamins and

nutrients (Walker and Freund, 1971). The chow group was given Purina Rodent Chow

and water ad libitum and served as a control for non-specific effects of the liquid diet.

Upon birth (postnatal-day 0 [PO]) pups from an ethanol or sucrose dam were

fostered to chow dams which had given birth at the same time. Litters were then

randomly culled to ten pups, with approximately equal numbers of males and females.

Pups were weaned at P21 and individually housed until perfusion at P60. In order to

avoid litter bias, animals used in this study are representatives from at least eight different

litters, and no more than one individual from each gender was used from a single litter. A

total of 39 animals was used in this study, with approximately equal numbers of males

and females in each diet treatment group (Ethanol, N=13; Sucrose, N=12; Chow, N=14).

Immunocvtochemical procedures

Animals were randomly selected for immunocytochemical staining at P60, an age

which represents adulthood in rats. This age was selected in order to examine the long

term effects of chronic prenatal ethanol treatment on parvalbumin expressing neurons in

the medial septum and cingulate cortex and corresponds to an age used in previous

analyses of prenatal ethanol influences on cholinergic neurons in the medial septum








(Swanson et al., 1996). At P60, animals were euthanized by pentobarbital overdose prior

to transcardial perfusion with phosphate buffered saline (PBS; 0.1 M, with 0.9% sodium

chloride) followed by 10% formalin (in the same PBS). Brains were removed and

equilibrated overnight in a cryoprotectant solution (PBS with 30% sucrose and 15%

ethylene glycol) and frozen at -700C until processing.

Animals of each gender from each diet group were processed for

immunocytochemistry at a given time. This insured against staining differences between

groups resulting from slight procedural differences. Brains were thawed and equilibrated

in a 30% sucrose-PBS solution, and mounted on the frozen stage of a sliding microtome.

Serial coronal sections were cut throughout the basal forebrain at a thickness of 40 pm.

Free-floating sections were immunostained for PA using a monoclonal antibody

(Accurate # 6092). Immunoreactivity was visualized as a blue-black reaction product

using an avidin-biotin conjugate/nickel intensified staining (see below).

Primary incubation with the monoclonal PA antibody (1:2500 in PBS, 0.1%

normal goat serum [NGS], 0.1% Triton X-100, and 0.005% sodium azide) was carried

out at 40C overnight. Sections were then washed with PBS and incubated with

biotinylated anti-mouse IgG (Sigma, B0529; 1:10000 in PBS, 0.1% NGS, 0.1% Triton X-

100, and 0.005% sodium azide) overnight at 4C. Sections were then washed and

incubated with Extravidin-horseradish peroxidase conjugate (Sigma; 1:1000 in PBS)

overnight at 4C. Sections were washed with 0.1 M sodium acetate (pH 7.2) to eliminate

phosphate which can precipitate divalent cations. Sections were then reacted for 3

minutes at room temperature using a developing buffer with 0.8 M sodium acetate, 8 mM

imidazole, 0.5% nickel (II) sulfate, 0.04% 3,3 diaminobenzidine tetrahydrochloride, and








0.005% hydrogen peroxide. Following development, sections were again washed in

sodium acetate buffer, mounted onto slides in PBS, air dried, dehydrated and

coverslipped. Control sections omitting the primary antibody were routinely developed to

ensure that any observed staining was due to PA. Slides from animals of each gender

from the three groups were randomized and coded such that all subsequent analyses were

carried out blind.

MS cell count, area, and volume analysis

PA+ cell counts were conducted bilaterally on alternate sections throughout the

entire rostral-caudal extent of the MS nucleus. The packing density of the DBB nucleus

was too great for accurate cell counts to be made by the image software. Thus, for the

purposes of this study the MS alone was examined and was defined rostrally by the

ventral fusion of the hemispheres (at the level of the genu of the corpus callosum) and

caudally by the decussation of the anterior commissure. The mean number of PA+

neurons per section was derived. Recent data have demonstrated the utility of manual

cell counts (Clarke and Oppenheim, 1995) and show a direct correlation between cell

counts performed manually and cell counts performed with the optical dissector (Hagg et

al., 1997).

Images of PA immunostained sections were captured and digitized using a

RasterOps 24STV video capture board and software on a Macintosh IIvx computer. Low

magnification images (2.5x objective; effective scale 1.59 imn/pixel) were captured in

order to obtain the entire MS region in one image. When capturing each image, the

lighting and contrast enhancement were optimized for identification of individual cells.

Images were digitally processed using the image analysis program NIH Image (freeware








from NIH). To reduce background variation across the image, a digitally defocused

image was created by passing the primary image through a mean filter. The resultant

image was then subtracted from the primary digitized image. This processed image was

then passed through a Laplace filter to enhance edges and separation between cells.

Analysis of individual images was initiated by outlining the region of the MS

nucleus. The area of a section outlined varied depending on the rostral-caudal location of

the individual section. The MS nucleus in rostral sections was defined as the mediodorsal

group of neurons which were separated from the ventrally-located DBB nucleus. In

intermediate sections, where the demarcation between the MS and DBB nuclei is

ambiguous, the MS was defined ventrally by a line perpendicular to midline at the level

of the anterior commissure. In caudal sections, the MS was defined as the medially

located cells dorsal to the anterior commissure. Cells were highlighted interactively by

adjusting the grayscale threshold level to include only objects which were considered

cells. A previous study from our lab has demonstrated that there is a close correlation

(r2= 0.956) between computer-automated and manual counts performed with a

microscope and a drawing tube (Swanson et al., 1996). Given the thickness of each

section (40 pm), the fact that alternate sections were analyzed, and the fact that PA+

neurons in the MS nucleus range from 6-26 pm in diameter (see below), it was not

deemed necessary to perform a split-cell correction on these counts. The program

counted the highlighted objects and measured the area of the outlined region (mm2).

Tissue volume (mm3) was calculated using a modification of the Cavelieri method

(Michel and Cruz, 1988). These measurements were taken in order to determine whether

ethanol treatment changed the size of the structure of interest, and whether changes in the








number of PA+ neurons per section were due to concomitant changes in the size of the

area examined (Peterson et al., 1997). This was done by multiplying the number of 40

p.m sections analyzed for a given animal by the mean area per section for that animal and

section thickness.

Anterior cingulate cell count, area, and volume analysis

The packing density of PA+ neurons in the anterior cingulate cortex was too great

for accurate cell counts to be made by automated imaging software at the magnification

necessary to include the entire region. Therefore, manual counts of PA+ neurons were

performed on every sixth section through the left side from the genu of the corpus

callosum caudally until decussation of the anterior commissure. The anterior cingulate

gyrus was bordered laterally by the cingulum, and ventrally by the corpus callosum

(Paxinos and Watson, 1982). The mean number of PA+ neurons per section was derived

through manual counting of neurons at 400x.

Because our cell count data are expressed as the mean number of cells per section,

it is necessary to demonstrate that any noted change in cells per section is not due to

changes in the volume of the structure being examined (Peterson et al., 1997). Therefore,

the volume of the anterior cingulate gyrus was calculated by a modification of the

Cavaleri method (Michel and Cruz, 1988) to ensure that changes in mean number of PA+

neurons per section as a result of ethanol treatment were not due to changes in the size of

the gyrus. For the volume determination, the mean area of the anterior cingulate gyrus

was determined for each animal by measuring the distance from midline to the cingulum

and the distance from the corpus callosum to the dorsal brain surface at 25x on three

anatomically matched sections with an eyepiece micrometer. These distances were








multiplied and mean area per section was calculated. This area was then multiplied by

the number of sections through the anterior cingulate gyrus and the section thickness to

determine mean cingulate gyrus volume.

Other analyses

The blood ethanol concentration (BEC) in the ethanol group was determined

between 00:00 and 02:00 hours on Gl 8 utilizing the Sigma 333-UV kit. PA+ cell

diameters were determined at 400x utilizing an eyepiece micrometer. Thirty cells from

identical anatomical locations (in the respective structures) for each gender in all groups

were measured. Density of PA+ neurons was determined by dividing the mean number

of cells per section by the mean area (mm2) per section for the respective structures.

Statistical analysis

Statistical analysis was performed with the program StatView (Abacus Concepts,

Berkeley Ca). For each parameter examined, one-way Analysis of Variance (ANOVA)

was used to test for the main effects of diet and gender separately. When appropriate, the

data were further analyzed with the Fisher's Protected Least Significant Difference

(PLSD) post hoc test to determine individual group differences. Because gender

differences were noted for MS measures (following an initial two-way ANOVA for

effects of diet and gender), males and females were analyzed separately. No gender

differences were noted in cingulate measures (following an initial two-way ANOVA for

effects of diet and gender), and so these data were not split by gender for analysis, and

one-way ANOVA was used to test for effects of diet, followed by the PLSD when

appropriate.








Results


BEC, body and brain weight, and brain to body weight ratio measures

BECs in the ethanol group ranged from 150-175 mg/dl (mean = 161 + 18 mg/dl).

This BEC represents a peak or near-peak level, as the nocturnal rats consumed the

majority of the diet after the twelve hour light cycle and blood samples were taken

between 00:00 and 02:00 hours on G18. P60 body weights were taken prior to perfusion

and P60 brain weights were taken following perfusion and are presented in table 2-1,

along with brain to body weight ratios (all tables are located at the end of the chapter).

The ANOVA for P60 body weight revealed no significant effects of diet on P60 body

weight of males or females. A gender difference was noted in P60 body weight for

ethanol [F(1,11) = 24.7; p< 0.0001], sucrose [F(l,10) = 31.4; p< 0.0001], and chow

[F(1,12) = 39.6; p< 0.0001] animals which was expected given the larger size of males at

P60.

The ANOVA for P60 brain weight demonstrated no effect of diet treatment in

males or in females. Likewise, no significant gender differences were noted for brain

weight within ethanol, sucrose, or chow animals.

The ANOVA for brain to body weight ratio showed no effect of diet in males or

females. Gender differences were noted in P60 brain to body weight ratio for ethanol

[F(l,11) = 37.5; p< 0.0001], sucrose [F(1,10) = 41.2; p< 0.0001], and chow [F(1,12) =

38.5; p< 0.0001] animals, which was not surprising given the larger size of male animals

at this age.








Number of sections, MS area per section, and PA+ neuronal density in the adult rat
medial septum

Table 2-2 presents the number of sections, MS area per section, and PA+ neuronal

density of the P60 MS. The number of alternate 40 uim sections through the MS was

determined by counting the number of sections analyzed between the ventral fusion of the

hemispheres (at the level of the genu of the corpus callosum) and the anterior

commissure. The ANOVA showed no effect of diet for either males or females on the

mean number of alternate sections through the MS, nor were there gender differences for

ethanol or chow animals. The sucrose group, however, did show a gender effect on the

mean number of sections with females containing significantly fewer than males

(F[1,10]= 4.511; p< 0.05).

The area of the MS outlined on a given section was computed by the image

software. No significant differences were noted between any group for males or females

in mean area per section nor were there gender differences for ethanol, sucrose, or chow

animals. Density of PA+ neurons (mean number of cells per section/mean area [mm2]

per section) was also calculated, and the ANOVA showed an effect of diet on cell density

in females (F[2,16]= 7.347; p< 0.01) but not in males. The PLSD post-hoc test

determined that cell density in ethanol females was significantly reduced compared to that

of sucrose females (p< 0.01). Neuronal density in chow females was also significantly

reduced compared to sucrose females (p< 0.05). The greater density of PA+ neurons

noted in sucrose females is perhaps due to a non-specific effect of liquid diet (see below

for discussion). A gender difference in the ethanol group was also noted for neuronal

density: neuronal density of females was reduced from that of males (F[1,1 1]= 5.075; p<








0.05). This difference is best described by the observed reduction in total PA+ cell

number, and not by differences in MS area. No gender differences in neuronal density

were noted for the sucrose or chow groups.

Total PA+ neurons in the MS

Figure 2-1 (all figures are located at the end of chapter) presents the mean total

number of PA+ cells counted on alternate sections throughout the P60 MS for animals of

both genders from each diet group. The ANOVA for total number of PA+ cells in the

MS determined a significant effect of diet treatment in females (F[2,16]= 4.351; p< 0.05)

but not in males. The PLSD post-hoc test further revealed that ethanol females had 42 %

fewer total PA+ cells than sucrose females (p< 0.01). A qualitative reduction in cell

number in the MS of ethanol females as compared to sucrose females can be observed in

Figure 2-2. Ethanol females were not different from chow females, nor were chow

females different from sucrose females. Gender differences were noted in the ethanol

group and the ANOVA for total number of PA+ cells in the MS indicated a reduction in

the number of PA+ cells in ethanol females compared to ethanol males (F[1,l 1]= 8.421;

p< 0.05). Gender differences were not noted in sucrose or chow groups.

Number of PA+ neurons per section in the MS

Figure 2-3 displays the mean number of PA+ neurons detected per section for

animals of both genders from each group in the P60 MS. The ANOVA for mean number

of cells per section revealed a significant effect of diet treatment in females (F[2,16]=

7.342; p< 0.01) but not males. The PLSD post-hoc test further determined that ethanol

females had 44 % fewer PA+ cells per section than sucrose females (p< 0.01). Chow

females also displayed a significant reduction in mean PA+ cells per section compared








with sucrose females (p< 0.05), while ethanol and chow females were not different from

each other. The difference between ethanol females and sucrose females is due to a

reduction in total cell number and not to a difference in tissue volume. The difference

noted between chow females and sucrose females is perhaps due to a non-specific effect

of liquid diet and may be related to the aforementioned increase in PA+ neuronal density

noted in sucrose females (see below for discussion). Gender differences were also noted.

The ANOVA showed a reduced mean number of PA+ cells per section in ethanol

females, compared to ethanol males (F[1,11]= 5.063; p< 0.05). This difference was again

due to a reduction in total PA+ cell number, and not to a difference in volume. No gender

differences were noted for the sucrose or chow groups.

Diameter of PA+ MS neurons

PA+ cell diameters were measured for both genders in all groups and are

presented in figure 2-4. No differences were noted between any treatment group for

males or females; nor were gender differences noted for the ethanol, sucrose, or chow

groups.

MS volume determination

Figure 2-5 presents MS volume (mm 3) for the analyzed sections. Volume was

calculated by multiplying the number of 40 pm sections analyzed for a given animal by

the mean area per section for each animal and the section thickness. The resulting

volume was then compared across groups for each gender and between both genders for

each diet treatment. No significant differences were found between diet groups for males

or females. Gender specific effects of tissue volume, however, were noted in sucrose

animals. The ANOVA showed a significant reduction of tissue volume in the sucrose








females compared to sucrose males (F[1,10]= 6.102; p< 0.05). This was likely due to a

decrease in the number of sections for female sucrose animals compared to male sucrose

animals (see table 2-2; p< 0.05). No gender differences in MS volume were noted for the

ethanol or chow groups.

Number of sections, mean area per section, and PA+ neuronal density in the adult rat
anterior cingulate cortex

Table 2-3 presents the number of sections, mean area per section, and PA+

neuronal density in the adult rat cingulate cortex. The number of 40 pm sections through

the anterior cingulate gyrus was determined by counting the number of sections between

the genu of the corpus callosum and the decussation of the anterior commissure. The

ANOVA showed no effect of diet on the mean number of sections through this region.

The area of the gyrus on a given section was determined by measuring the

distance from midline to the cingulum and the distance from the corpus callosum to the

dorsal brain surface at a magnification of 25x on three anatomically matched sections

with an eyepiece micrometer. No significant differences were noted in mean area per

section. The Density of PA+ neurons in the anterior cingulate (mean number of cells per

section/mean area [mm2] per section) was also calculated, and the ANOVA showed an

effect of diet on cell density (F[2,30]= 7.041; p< 0.01). The PLSD post-hoc test revealed

that cell density in the ethanol group was significantly reduced compared to that of the

sucrose (p< 0.01) and chow groups (p< 0.01).

Total PA+ neurons in anterior cingulate

Figure 2-6 presents the total number of PA+ neurons counted throughout the

anterior cingulate cortex. The ANOVA for total number of PA+ neurons in the anterior








cingulate showed a significant effect of diet treatment (F[2,30]= 12.314; p< 0.0001). The

PLSD post hoc test further revealed that ethanol animals had 45% fewer PA+ neurons

than sucrose (p< 0.0001) or chow (p< 0.0001). Figure 2-7 demonstrates a qualitative

reduction of PA+ neurons in the anterior cingulate cortex of adult rats exposed to ethanol

prenatally when compared with sucrose controls.

Number of PA+ neurons per section in the anterior cingulate

Figure 2-8 presents the mean number of PA+ neurons per section in the anterior

cingulate. The ANOVA revealed a significant effect of diet treatment on the mean

number of PA+ neurons per section (F[2,30]= 12.80; p< 0.0001). The PLSD further

determined that ethanol treatment reduced the mean number of PA+ neurons per section

when compared with sucrose (p< 0.001) and chow (p< 0.0001) controls.

PA+ neuronal diameter in the anterior cingulate cortex

PA+ cell diameters were determined with an eyepiece micrometer at 400x. Ten

whole cells from identical anatomical location for each gender in all groups were

measured on three sections for each brain region. The mean PA+ neuronal diameter for

each group was determined and is presented in figure 2-9. No significant differences in

the mean PA+ neuronal diameter were noted.

Anterior cingulate gyrus volume

The mean gyrus volume for each group was determined, and is presented in figure

2-10. No significant effect of treatment on mean volume was noted.








Discussion


The long-term effects of chronic prenatal ethanol exposure on PA+ neurons were

examined in the medial septum and anterior cingulate cortex of adult rats. This pattern of

exposure produced no significant alterations in P60 body weight (although males were

larger than females for all groups examined). Similarly, no significant differences were

noted in P60 brain weight, or brain to body weight ratio as a function of ethanol treatment

(although brain to body weight ratios were higher in females due to the larger size of

males at P60). The lack of effect of chronic prenatal ethanol treatment (CPET) on the

long-term growth of these animals is consistent with other reports from our laboratory

(Swanson et al., 1995; Swanson et al., 1996). This exposure paradigm did, however,

produce significant alterations in PA+ neurons in both regions examined, although a

sexually dimorphic effect was noted for the medial septum. The results for each brain

region are discussed separately below.

Medial Septum

A major conclusion drawn by the current study is that CPET affects the MS in a

sexually dimorphic manner. This study has demonstrated anatomical changes in the

expression of PA in the female rat MS following prenatal ethanol exposure. The

evidence from this study suggests that a reduction in the number of MS neurons

expressing PA occurs in the absence of area, volume, or PA+ cell size effects. Whether

this reduction is due to lower PA levels in existing cells or to a loss of PA+ neurons is

impossible to conclude. Regardless, the data suggest that alterations in PA expression








occur in female rats following CPET and that PA expression in female rats may be

particularly susceptible to the long-term consequences of CPET.

Although male offspring exposed to ethanol in utero were unaffected as adults, a

number of differences were noted in female ethanol-treated animals when compared to

females in the sucrose group. The mean total number of PA+ neurons in the MS nucleus

of adult females was reduced by 42 % following prenatal exposure to ethanol, PA+

neuronal density was reduced in ethanol females, and ethanol females had fewer PA+

neurons per section when compared to sucrose females. There were also gender

differences in the ethanol groups for a number of measures. Female rats exposed to

ethanol in utero contained fewer total PA+ neurons, reduced density of MS PA+ neurons,

and fewer PA+ neurons per section when compared to males. Gender differences in MS

volume in the sucrose group were also noted, with the MS of females significantly

reduced compared to that of sucrose males. As mentioned previously, this difference was

due to a lower number of 40 gpm sections from the MS of female sucrose animals when

compared to sucrose males. This is not surprising since P60 female rats are smaller than

males. This difference was apparently enhanced in the sucrose animals, since neither the

ethanol nor the chow groups displayed MS volume differences.

Differential gender susceptibility of PA+ neurons in the MS following CPET

The finding that PA expression was affected by prenatal ethanol exposure only in

female animals suggests that differences in the hormonal environment of the males and

females influenced PA-immunoreactivity and susceptibility to ethanol. In fact, the SH

system itself is highly sexually dimorphic (Loy, 1986). Specifically, morphological

differences in hippocampal asymmetry (Diamond et al., 1982) and differences in binding








capacity of hippocampal glucocorticoid receptors (Turner and Weaver, 1985) have been

noted in rats. Additionally, Loy and Milner (Loy and Milner, 1980) have found

differences in lesion-induced hippocampal sprouting between male and female rats.

Whereas sprouting in female rats is uniform in the hippocampus following injury,

sprouting in males occurs predominately in the dentate molecular layer. These results

suggest that the CNS response to damage may be dictated by hormonal environment, and

that this response is sexually dimorphic. Perhaps similar differences in hormonal

environment determine sensitivity to ethanol in this region and account for the noted

sexual dimorphism.

Gender differences have been reported in the alcohol literature as well: female

rats are more affected than males in measures of radial arm maze performance following

chronic adult ethanol treatment (Maier and Pohorecky, 1986); Witt et al. (1986) found an

increase in muscarinic receptor binding sites in the hippocampus of ethanol-treated

females but not ethanol-treated males following chronic adult ethanol treatment; Kelly et

al. (Kelly et al., 1988) reported impaired spatial navigation in adult female rats but not

adult male rats following neonatal ethanol exposure. As in the current study, these

reports suggest an increased susceptibility to ethanol's effects in female animals and it

has been suggested that females may be more sensitive than males to ethanol in animal

models (West et al., 1989). However, not all studies have agreed with an increased

susceptibility for females. Goodlett and Peterson (Goodlett and Peterson, 1995), for

example, found increased susceptibility for spatial learning deficits in male rats following

time-limited binge ethanol exposure. Sexually dimorphic effects of prenatal ethanol

treatment have also been noted on daily water consumption, with males (but not females)








consuming more water than pair-fed controls as adults (McGivem et al., 1998). Clearly a

greater focus on potential gender differences in animal models of FAS is warranted in

order to clarify sexual dimorphism in relation to ethanol exposure. Moreover, an

investigation into gender differences in human FAS is warranted, as no sexual

dimorphisms have been reported in human offspring of mothers who drink during

pregnancy.

What specific differences in hormonal environment might account for the

observed sexually dimorphic effect of CPET on PA+ neurons in the female medial

septum? Estradiol, for example, is known to influence the functioning of the cholinergic

component of the SH system through its influence on choline re-uptake in the

hippocampus (Singh et al., 1994). Indeed, removal of estradiol and other ovarian steroids

by ovariectomy decreases cholinergic neurotransmission in the female hippocampus, and

this is reversible by estradiol treatment (Singh et al., 1994). Moreover, estradiol infusion

ameliorates fimbria-fomix lesion-induced decline of ChAT+ neurons in the rat medial

septum (Rabbani et al., 1997). While estradiol is not known to influence the functioning

of the GABAergic component of the SH system, estrogen is known to increase glutamic

acid decarboxylase (the rate limiting enzyme in the synthesis of GABA) mRNA levels in

the female rat brain (McCarthy et al., 1995). Thus, it is possible that an ethanol-induced

decrease in ovarian steroids might account for some of the effects noted in the present

study on GABAergic neurons. Effects of CPET on ovarian steroids would be a useful

avenue of future research and may shed light on this interesting sexually dimorphic effect.








Liquid diet effects on the MS

In addition to the effects of ethanol on the MS of female rats, a non-specific effect

of the liquid diet on females was noted for some measurements. Previous studies

utilizing rodent models of prenatal ethanol exposure have noted an effect of liquid diet

treatment. For example, Swanson et al. (1995) found a stimulatory effect of liquid diet on

ChAT enzymatic activity during the first postnatal week. Additionally, studies

quantifying ChAT+ cell number in the P 14 rat MS found a liquid diet-induced increase in

ChAT+ neuronal number for female sucrose animals, but not ethanol females or male

sucrose animals (Swanson et al., 1996). It was speculated that a possible sucrose diet-

induced stimulation of MS cholinergic development occurred in female rat pups at P14.

The seemingly protective and perhaps stimulatory nature of the liquid diet is probably due

to its high vitamin and mineral content.

The present study indicates similar diet effects for sucrose females. There seems

to be a liquid diet-induced increase in both the density of PA+ neurons in the MS and the

number of PA+ neurons per section in sucrose females. We speculate that a liquid diet-

induced increase in cell density for the sucrose group, similar to the increase in ChAT+

neurons noted by Swanson et al. (Swanson et al., 1996), raised the base level of PA+

neurons in the liquid diet animals to a level greater than that seen in chows. Although

there was not also a significant increase in cell number, the possibility exists that the

period of naturally occurring cell death served to reduce this overall number while density

remained high. The ethanol-treated group may also display this effect of liquid diet, but,

nonetheless, a significant ethanol effect was noted as ethanol females had fewer PA+

neurons. Apparently the effect of prenatal ethanol treatment is sufficient to produce a








significant cellular reduction in ethanol females. The raised baseline due to the liquid

diet, however, may mask a difference between ethanol and chow females.

Functional considerations for the MS

Although CNS regions differ in the extent to which PA and GABA co-localize,

the MS is a region where most, if not all, GABAergic neurons are PA+. This statement is

supported by studies of Freund (Freund, 1989) utilizing anterograde transport from the

septum to the hippocampus. Of the projection neurons identified by the anterograde

transport, all of the axons shown to be immunoreactive for GABA were immunoreactive

for PA. Thus, PA-immunoreactivity serves as a reliable marker for GABAergic

hippocampal projection neurons in the MS nucleus and identifies a subpopulation of the

total GABAergic neuronal pool within the basal forebrain.

As noted previously, in the MS nucleus PA expression begins at G21, after the

appearance of GABAergic neurons at G16 (Lauder et al., 1986; Solbach and Celio, 1991).

The ethanol exposure regimen utilized in the present study encompasses the entire

prenatal period, and thus ethanol was present in CNS tissue during the initiation of PA

expression in MS neurons and the physiological maturation of these cells. Exposure of

MS neurons to ethanol resulted in a long-term reduction in the number of PA+ neurons in

adult females. How might these neurons have been affected by changes in the normal

expression of PA?

The intracellular concentration of PA in neurons (6-45 gM) and the fact that it is

commonly found in fast-firing neurons (Plogmann and Celio, 1993) implicate PA in

buffering excess Ca2+ at presynaptic nerve terminals following rapid trains of action

potentials (Heizmann, 1984). Since excessively high levels of intracellular Ca2+ are








known to initiate cell death, the possibility exists that an ethanol-induced reduction of PA

expression resulted in fewer cells in the females by reducing those neurons' ability to

buffer calcium and suppress the process of cell death. Though neurons expressing normal

levels of PA can survive experimental cerebral ischemia (Nitsch et al., 1989) and fimbria-

fornix transaction (Kermer et al., 1995), any reduction in PA expression due to ethanol

may impede a protective effect. Solodkin et al. (1996) have shown a decrease in PA

immunostaining in parts of the entorhinal cortex which showed Alzheimer's pathologies.

Since PA expression is reduced in the basal forebrain of aged rats (Krzywkowski et al.,

1995) the possibility exists that the vulnerability of the septohippocampal pathway to

degenerative diseases such as Alzheimer's is due to a reduction in PA expression. The

observed reduction in PA+ neurons found following CPET in the current study may be

due to direct loss of neurons by the process of cell death or a reduction in the level of PA

in MS neurons which would preclude detection by immunostaining due to subthreshold

amounts of antigen/epitope. In fact, Kermer et al. (1995) have detected reduced PA

immunoreactivity in the medial septum following fimbria-fornix transaction in the

absence of PA+ neuronal cell death. However, whether cells are lost in our paradigm or

not, the ability of these neurons to function adequately would likely be diminished in

affected animals.

Altered MS function and FAS

A loss of effective GABAergic MS neurons, whether by a reduction in cell

number or a reduction of PA expression, may impede the normal function of the

septohippocampal system in learning and memory. GABAergic neurons in the basal

forebrain are known to control activity of cholinergic neurons in this region (Dudchenko








and Sarter, 1991) and working memory in the rat is disrupted by antagonism of

GABAergic transmission in the septum (Chrobak and Napier, 1992). Smythe et al.

(1992) have shown that both the cholinergic and GABAergic components of the MS are

necessary to influence the electrical activity in the hippocampus. Mechanistically, the

influence of the basal forebrain on hippocampal electrical activity is known to be on theta

cell rhythm (Bland and Bland, 1986) and the GABAergic component of the MS plays a

part in this influence (Allen and Crawford, 1984; Smythe et al., 1992). PA+ MS neurons

are known to depress the activity of inhibitory interneurons in the hippocampus (Freund

and Antal, 1988). This would serve to increase the excitability of the hippocampus by

removing local inhibitory potentials; theta rhythm would be initiated and the excitability

of the principal hippocampal cells involved in long-term potentiation would be increased

(Freund and Antal, 1988). The current study has demonstrated that CPET permanently

reduces the number of PA+ GABAergic neurons in the female rat MS by 42 % in the

absence of area or volume effects. Such a reduction in the number of PA+ GABAergic

neurons could potentially affect the functioning of the septum and hippocampus. Indeed,

some of the observed phenotypes of FAS, including mental retardation and spatial

learning deficits (see Kelly et al., (1988) Goodlett and Peterson (1995) for animal

studies), may be partially explained by a reduced septohippocampal efficacy. This

possibility is supported by the findings of Miettinen et al. (1993) that the decline in aged

rats in spatial learning correlates with a reduction in PA-containing neurons in the

entorhinal cortex. A similar decline in learning in FAS children may also relate to PA

expression in the SH system.








Cingulate Cortex

This investigation represents, to our knowledge, the first demonstration of

neuroanatomical alterations in the cingulate cortex as a result of developmental ethanol

exposure. In fact, this appears to be the first examination of teratogen-induced alterations

in cingulate cortex neuroanatomy, and is a significant contribution to the literature since

there exists a relative paucity of data on the cingulate cortex in normal or pathological

states.

A conclusion of the present study is that CPET permanently reduced the number

of PA+ neurons in the anterior cingulate cortex without altering size of the structure.

These data are similar in their significance to those reported previously for the MS. One

critical difference, however, between the effect of CPET on the anterior cingulate and the

effect of CPET on the MS is the lack of sexually dimorphic effects in the former. The

factors that are responsible for the lack of gender specific effects of CPET on the anterior

cingulate remain unknown. It is possible that the medial septum and anterior cingulate

respond differentially to different hormonal environments. While the medial septum is

known as a sexually dimorphic region (see above), no data exist which point to a similar

dimorphism for the anterior cingulate. Regardless, the overall pattern of ethanol-induced

abnormalities is similar, with the volume of the anterior cingulate unaffected by prenatal

ethanol exposure even though mean total PA+ neuronal number and mean PA+ neurons

per section was affected. Additionally, the size of the PA+ neurons was determined, and

was found to be unaltered by this pattern of in utero exposure. Our results are similar to

those of Kril and Homewood (1993), who found that PA+ neuronal number was








decreased in the frontal cortex of adult rats following chronic ethanol treatment and

thiamin deficiency.

Since the volume of the anterior cingulate was unaffected by treatment, and

because the cells examined showed no decrease in size, it is unlikely that the decline in

mean PA+ neurons per section is a result of an ethanol effect on the size of this area.

Because our cell counts are derived from PA immunostained sections, we cannot

definitively conclude that the reduced number of PA+ neurons is a result of ethanol-

induced cell death. While this is a possibility, it is equally likely that the reduction of

PA+ neurons noted after prenatal ethanol treatment is due to a decreased expression of

PA in living neurons (perhaps as a result of ethanol interfering with PA ontogeny) which

would preclude detection by immunostaining. Krzywkowski et al. (1995) examined PA

immunoreactivity in the septum of aged rats and found a decrease in the number of PA+

neurons without loss of GABAergic neurons, suggesting that PA+ expression levels were

decreased without cell loss. Similar findings have been reported in patients with

Parkinson's disease (Hardman et al., 1996).

Yet another possibility is that ethanol interfered with the generation of neurons

within the anterior cingulate cortex. Evidence from Miller (1986) shows that prenatal

ethanol exposure delays and extends the period of cortical neuronal generation while

reducing the number of neurons and altering the distribution of neurons in the mature

cerebral cortex. It is conceivable that a similar reduction in the generation of cortical

neurons is responsible for our observed decrease in PA+ neuronal number as a result of

ethanol treatment.








Functional considerations for the cingulate

As mentioned previously, the present study examined PA+ neuronal number in

the cingulate cortex following prenatal ethanol treatment because of earlier observations

related to cerebral cortex functioning following ethanol exposure. These include severe

behavioral problems in children with FAS, including poor judgment, distractibility, and

difficulty perceiving social cues (Streissguth et al., 1991). Additionally, many alcoholics

who develop Korsakoff's syndrome have deficiencies in glucose utilization within the

cingulate cortex, potentially contributing to learning and memory defects due to

interruption of Papez' circuitry (Joyce et al., 1994). Papez' circuit is a neuroanatomical

pathway which originates in the hippocampus, proceeds through the fornix to the

mammillary bodies, and then to the anterior thalamic nucleus, cingulate gyrus, entorhinal

cortex, and then back to the hippocampus (Nolte, 1993). Therefore, it is conceivable that

alterations in PA expression patterns might contribute to behavioral anomalies and/or

learning and memory deficiencies.

Because the intracellular concentration of PA in neurons is in the range necessary

for calcium buffering (6-45 jpM) and the fact that it is commonly found in fast-firing

neurons (Plogmann and Celio, 1993), PA appears to be involved in buffering excess Ca2+

at presynaptic nerve terminals following rapid trains of action potentials (Heizmann,

1984). Since excessively high levels of intracellular Ca2+ are known to initiate cell death,

the possibility exists that an ethanol-induced reduction of PA expression resulted in fewer

cells in the ethanol animals by reducing those neurons' ability to buffer calcium and

suppress the process of cell death. Though neurons expressing normal levels of PA can

survive experimental cerebral ischemia (Nitsch et al., 1989) and fimbria-fomix








transaction (Kermer et al., 1995) any reduction in PA expression due to ethanol may

impede a protective effect. Solodkin et al. (1996) have shown a decrease in PA

immunostaining in parts of the entorhinal cortex which showed Alzheimer's pathologies.

Similarly, the number of PA+ neurons is reduced in the prefrontal cortex of

schizophrenics (Beasley and Reynolds, 1997).

While we cannot determine from the present study whether cell death or reduced

PA antigen is responsible for the noted reduction in PA+ neurons, it should be noted that

whatever the mechanism of the ethanol-induced reduction of PA+ neurons, inhibitory

neurotransmission in the cingulate cortex is likely to be altered. For example, PA+

neurons are lost at epileptic foci in animal models of epilepsy (De Felipe et al., 1993).

Moreover, Jacobs et al. (1996) showed that decreased PA immunoreactivity in cortical

freeze lesions of neonatal rats leads to hyperexcitability in adjacent cortex. Such

alterations in inhibitory and excitatory processes have clear functional consequences to

the organism, and likely result in an imbalance in excitation and inhibition in these

cortical regions (Wang et al., 1996). The current findings of decreased number of PA+

neurons in the anterior cingulate cortex of adult rats as a result of prenatal ethanol

exposure may provide a neuroanatomical basis for the well described cognitive and

behavioral impairments in offspring exposed to ethanol in utero.

Conclusions

The major findings of this study are that PA+ neurons in the medial septum and

anterior cingulate cortex are susceptible to prenatal ethanol exposure. In the medial

septum, a sexually dimorphic effect of ethanol was noted, with females more susceptible

than males. No gender specific effects were noted for the anterior cingulate. These






55

reductions in PA+ neurons occurred in the absence of significant ethanol-induced

alterations in the size of the structures examined, the size of the PA+ neurons within these

structures, or the growth of these animals. This represents the first description of ethanol-

induced alterations in PA+ GABAergic neurons in animal models of FAS. Further

research will help to further define developmental windows of GABAergic vulnerability

(for example, neonatal exposure models).









Table 2-1. Postnatal day 60 (P60) body and brain weight and brain to body weight ratio
(br/bd)*
Diet group N P60 body weight (g) P60 brain weight (g) P60 br/bd (%)
Females
Ethanol 8 220 + 2.92 1.32 + 0.02 0.600 + 0.010"
Sucrose 6 216+5.58 1.31 + 0.02 0.606+ 0.021t
Chow 6 224 + 5.06 1.36 + 0.02 0.607 + 0.022t
Males
Ethanol 5 341 + 13.5t 1.34 + 0.05 0.392 + 0.032
Sucrose 6 351 + 5.81t 1.35 + 0.06 0.384 + 0.019
Chow 8 348 + 13.6t 1.39 + 0.02 0.399 + 0.030
*All measures are expressed as mean + S.E.M. No significant differences were noted.
tSignificantly increased, compared with other gender of same group (p< 0.0001).


Table 2-2. Number of sections, medial septum (MS) area per section, and parvalbumin-
immunoreactive (PA+) neuronal density in the adult rat MS*
Diet group N Number of 40 ipm MS area/section Density (PA+
sections (mm2) cells/mm2)
Females
Ethanol 8 15.75 + 0.453 0.722 + 0.066 49.55 + 6.716t'f
Sucrose 6 15.50 + 0.428 0.655 + 0.061 89.38 + 9.700
Chow 6 17.67 + 1.054 0.670 + 0.065 59.87 + 6.237
Males
Ethanol 5 16.20 + 0.374 0.733 + 0.103 78.031 + 12.01
Sucrose 6 17.50 + 0.428 0.778 + 0.061 66.16 + 10.98
Chow 8 16.87 + 0.811 0.733 + 0.049 63.49 + 5.957
*All measures are expressed as mean + S.E.M. Area and volume measures are
representative of approximately 1/2 of the total MS (alternate sections were analyzed; see
materials and methods section for detail).
tSignificantly reduced from sucrose females (p< 0.01).
ISignificantly reduced from ethanol males (p< 0.05).
Significantly reduced from sucrose females (p< 0.05).
TSignificantly reduced from sucrose males (p< 0.05).






57



Table 2-3. Number of sections, mean area per section, and parvalbumin-immunoreactive
(PA+) neuronal density in the adult rat anterior cingulate cortex*
Diet group N Number of 40 pm Mean area/section Density (PA+
sections (mm2) cells/mm2)
Ethanol 13 41.5 +2.56 2.135 + 0.069 395.5 + 67.85f
Sucrose 12 39.8 + 2.56 2.262 + 0.060 669.6 + 54.56
Chow 14 35.8+ 1.34 2.385 + 0.080 697.4 + 61.88
*All measures are expressed as mean + S.E.M.
tSignificantly reduced compared with sucrose (p< 0.01) and chow (p< 0.01).









1200-

1000-

800-


Male


=Z Ethanol
M Sucrose
- Chow


Female


Figure 2-1. The mean total number of parvalbumin-expressing neurons detected on
alternate sections through the medial septum of postnatal-day 60 rats exposed in utero to
one of three diets, ethanol, sucrose, or chow (see materials and methods section for
detail). Data are expressed as mean + SEM. a: significantly reduced compared with
sucrose females (p< 0.01). b: significantly reduced compared with ethanol males (p<
0.05).


a,b

T


600-

400-

200-


0--































Figure 2-2. Photomicrographs of 40 ptm coronal sections through the medial septum of
ethanol-treated (A) and sucrose-treated (B) postnatal-day 60 female rats. Sections are
matched for anatomical location and are representative of their respective group. A
qualitative reduction in cell number for the ethanol females can be noted, as can an
increase in cell density for sucrose females. Scale bar equals 125 tm.
















A



.4 ',
p


'1



t


0..'
a


'I 4

~41 *j~

I,
I *~
I I
dl fl
4. 1'

r
fr ? *



~ Ii

p.
U


)
~


I ft
*4


I


4
6


I,






fr,1

ft


4/



.* *,


^*;(
'1
>. t^ a









3d e
S .
..- .
f :-\.
*; a
*'
'1 i


"'U
A


r
IVA


S

I!. %


4



Ga
q'~* I
1~


*


Ir
'S.
'119
'I
,

*1
6*


-d I


fr


4


4


e


'I


9
V.
* I


.Ip..Y
4W
h ~

I ~


B'
I~ '

es I
I



S! ^


#
fc


dL











=-- Ethanol
M Sucrose
M Chow


75-



50-



Z 25-



0-


Figure 2-3. The mean number of parvalbumin-expressing neurons per section in alternate
sections through the medial septum of postnatal-day 60 rats exposed in utero to one of
three diets, ethanol, sucrose, or chow (see materials and methods section for detail). Data
are expressed as mean + S.E.M. a: significantly reduced compared with sucrose females
(p< 0.01). b: significantly reduced compared with sucrose females (p< 0.05). c:
significantly reduced compared with ethanol males (p< 0.05).


Female


Male












---1 Ethanol
M Sucrose
M Chow


Female


Figure 2-4. The mean diameter of parvalbumin-expressing neurons on alternate sections
through the medial septum of postnatal-day 60 rats exposed in utero to one of three diets,
ethanol, sucrose, or chow (see materials and methods section for detail). Data are
expressed as mean + S.E.M. No significant differences were noted.


p.i
h

II-0


+


0-


Male















a T


=C1 Ethanol
m Sucrose
M Chow


Female


Figure 2-5. The mean medial septum (MS) volume of postnatal-day 60 rats exposed
throughout gestation to an ethanol-containing liquid diet, sucrose-containing liquid diet,
or lab chow and water. Data are expressed as mean + SEM. a: significantly reduced
compared with sucrose males (p< 0.05).


0.75-


2
2
2
0U


0.50-



0.25-


0.00 -


Male











2000-


1000-


__Tr


-IT_


-7-


Ethanol


Sucrose


Chow


Figure 2-6. The mean total number of parvalbumin-expressing (PA+) neurons detected
on alternate sections through the anterior cingulate cortex of postnatal-day 60 rats
exposed in utero to one of three diets, ethanol, sucrose, or chow (see materials and
methods section for detail). Data are expressed as mean + SEM. a: significantly reduced
compared with sucrose (p< 0.0001) and chow (p< 0.0001).
































Figure 2-7. Photomicrographs of 40 p.m coronal sections through the anterior cingulate
cortex of ethanol-treated (A) and sucrose-treated (B) postnatal-day 60 rats. Sections are
matched for anatomical location and are representative of their respective group. A
qualitative reduction in parvalbumin-immunoreactive neuronal number is seen in the
ethanol-treated animal compared with the sucrose control. Scale bar equals 110 upm.






66






A B





67



300-



S200
a


100-



0
Ethanol Sucrose Chow


Figure 2-8. The mean number of parvalbumin-immunoreactive (PA+) neurons per
section counted in the anterior cingulate cortex of postnatal-day 60 rats exposed
throughout gestation to an ethanol-containing liquid diet, sucrose-containing liquid diet,
or lab chow and water. Data are expressed as mean + SEM. a: significantly reduced
compared with sucrose (p< 0.001) and chow animals (p< 0.0001).









20-






o 10-






Ethanol Sucrose Chow



Figure 2-9. The mean diameter of parvalbumin-immunoreactive (PA+) neurons in the
anterior cingulate cortex of postnatal-day 60 rats exposed throughout gestation to an
ethanol-containing liquid diet, sucrose-containing liquid diet, or lab chow and water.
Data are expressed as mean + SEM. No significant differences were noted.






69




4-





1-







0
Ethanol Sucrose Chow


Figure 2-10. The mean cingulate gyms volume of postnatal-day 60 rats exposed
throughout gestation to an ethanol-containing liquid diet, sucrose-containing liquid diet,
or lab chow and water. Data are expressed as mean + SEM. No significant differences
were noted.















CHAPTER 3
EFFECTS OF NEONATAL ETHANOL EXPOSURE ON CHOLINERGIC NEURONS
OF THE RAT MEDIAL SEPTUM


Summary


Developmental ethanol exposure has been known to affect the normal

development of the central nervous system. Studies in animal models have determined

that chronic prenatal ethanol exposure has no effect on the number of cholinergic neurons

in the rat medial septum (Swanson et al., 1996). Since many brain regions exhibit tight

temporal windows of vulnerability to ethanol, the objective of this study was to determine

the long-term effects of chronic neonatal ethanol exposure on the cholinergic neurons in

the medial septum (MS) of the rat. On postnatal day 4 (P4) pups were assigned to one of

three groups: an ethanol-receiving, gastrostomized group (EtOH); a pair-fed,

gastrostomized control group (GC); and a dam-reared suckle control group (SC).

Gastrostomized pups were infused with ethanol-containing or control diet as a 9.1% v/v

solution for two feedings on each day from P4-10. Choline acetyltransferase (ChAT)

immunocytochemistry was analyzed at P60.

Ethanol treatment resulted in long-lasting microencephaly in P60 animals.

Ethanol exposure did not directly reduce mean total ChAT-expressing (ChAT+) neuronal

number, or the mean number of ChAT+ neurons per section. Neither were changes noted

in MS volume, mean area section, or cell density as a result of ethanol treatment.








However, ethanol exposure significantly reduced ChAT+ neuronal size in males

compared with GC males but not SC males. No differences in ChAT+ neuronal size

were noted in females. Thus neonatal ethanol exposure, while producing long-lived

microencephaly and small changes in ChAT+ neuronal size, has no effect on the number

of cholinergic neurons in the adult rat MS, and has no effect on the size of the MS.

Introduction


Fetal alcohol syndrome (FAS) has been well characterized since its description in

1973 (Jones and Smith, 1973). Perinatal growth deficiencies, craniofacial abnormalities,

as well as central nervous system (CNS) dysfunction have been noted in human offspring

exposed to ethanol prenatally (West et al., 1994). In attempts to thoroughly examine and

accurately define the consequences of developmental ethanol exposure, rodent models of

FAS have been developed and extensively utilized. These animal models have identified

susceptible populations of neurons within the CNS and have described periods of

development during which particular populations exhibit heightened vulnerability to the

effects of ethanol (West et al., 1994). Studies in rats have demonstrated that the early

postnatal period, the so-called brain growth spurt and the equivalent of the human third

trimester (Dobbing and Sands, 1979), is a time of vulnerability for particular CNS

regions.

The septohippocampal (SH) system of basal forebrain afferents and hippocampal

targets is one such region in rats that exhibits susceptibility to ethanol during neonatal

development. The SH system is a pathway ofcholinergic and GABAergic fibers

originating from the MS nucleus and the horizontal and vertical limbs of the diagonal








band of Broca (DBB) that influences electrical activity in the hippocampus (Dutar et al.,

1995). These fibers originate from cell bodies in the basal forebrain and, beginning at

embryonic day 20, innervate the hippocampus where they form synapses on hippocampal

pyramidal neurons, granule cells, and intemeurons (Freund and Antal, 1988; Milner et al.,

1983). Cholinergic neurons comprise the major projection of the SH system and first

express the catalytic enzyme choline acetyltransferase (ChAT) on embryonic day 17

(Armstrong et al., 1987; Dutar et al., 1995).

While numerous studies have described the effects of neonatal ethanol exposure

on neurons within the hippocampus (Bonthius and West, 1990; Bonthius and West, 1991;

Greene et al., 1992; Pierce and West, 1987; West and Pierce, 1986) and have documented

deficits in spatial learning following neonatal ethanol exposure (Goodlett and Peterson,

1995; Kelly et al., 1988), the effect of ethanol exposure during the brain growth spurt on

the cholinergic component of the SH system has yet to be characterized. However,

studies examining the effect of chronic adult ethanol exposure on the neurons of the SH

system have been reported and have suggested a decrease in the number of cholinergic

neurons in the MS, though pair-fed controls were not examined (Arendt et al., 1995;

Arendt et al., 1988). Moreover, studies examining the effect of prenatal ethanol exposure

on the SH system in the rat (Swanson et al., 1995; Swanson et al., 1996) and mouse

(Schambra et al., 1990; Sulik et al., 1984) have been completed. When rats were exposed

to ethanol throughout gestation, a reduction in ChAT enzymatic activity was noted in the

MS during the second postnatal week compared with pair-fed controls (Swanson et al.,

1995).








In contrast, no change in the number or morphology of ChAT+ neurons in the MS

was found (Swanson et al., 1996), though earlier experiments in mice had shown the

cholinergic neurons of the basal forebrain to be particularly sensitive to acute prenatal

ethanol exposure (Schambra et al., 1990; Sulik et al., 1984). The objective of the present

study was to determine the long-term effects of ethanol exposure on the cholinergic

neurons in the rat MS when ethanol was delivered during the brain growth spurt, a time

when the cholinergic neurons are more mature and potentially more susceptible than at

the time points examined in the previous prenatal studies (Bonthius and West, 1991).

Ethanol was delivered from P4-10, a period of differentiation and synaptogenesis in the

SH system (Dutar et al., 1995). ChAT+ neuronal number and morphology was examined

in the adult MS of artificially reared ethanol-treated and pair-fed controls as well as in

dam-raised suckle controls.

Methods


Subjects and artificial rearing

Long-Evans hooded rat pups were obtained from nine timed pregnant dams

ordered from Charles River Co. (Portage, MI). Animals were housed with a 07:00-19:00

light cycle under controlled temperature and humidity conditions. At the time of birth

(PO), litters were culled to 12 pups and the pups were randomly assigned to one of three

groups: ethanol-receiving gastrostomized pups (EtOH); pair-fed gastrostomy controls

(GC); and dam-reared suckle controls (SC). Artificial rearing was performed after West

et al. (West et al., 1984). On P4 a gastrostomy feeding tube was surgically implanted into

the EtOH and GC pups. Pups were placed under methoxyfluorane anesthetic while








gastrostomy tubes were inserted down the esophagus into the stomach. The tube was

pulled through a small hole in the stomach and abdominal wall and secured on the outside

by a small plastic washer. Pups were reared individually in plastic cups filled with

bedding and a fur-like material. Cups were floated in a covered, heated aquarium (400C)

and pups were maintained on a 07:00-19:00 light cycle. SC pups were weighed daily

from P4-10. Dams with SC pups always had a total of eight pups to minimize weight

differences between groups.

Gastrostomized pups were infused with a liquid diet containing evaporated milk,

sterile water, soy protein, L-methionine, L-tryptophan, calcium phosphate, deoxycholic

acid, a vitamin mixture, and a mineral mixture (West et al., 1984). Pups received the

milk formula from P4-10 in 12 feeding periods of 20 minutes each. EtOH pups received

ethanol-supplemented formula as a 9.1% v/v solution for the first two feedings on each

day for a total of 4 g/kg/day. GC pups received an isocaloric amount of maltose-dextrin-

supplemented formula for the first two feedings on each day. The remaining 10 daily

feedings consisted of milk formula alone. Gastrostomized pups were weighed daily and

the daily diet consumption volume (in mls) was equivalent to 33% of the mean litter body

weight. The liquid diet was administered to the EtOH and GC pups by connecting the

gastrostomy tubes to a feeding line connected to diet-filled syringes held within a

Stoelting (Wood Dale, IL) programmable infusion pump. On the morning of P11, pups

were disconnected from the pump, feeding tubes were sealed, and they were returned to

their original dam. EtOH and GC pups were accepted by the dams and began nursing

soon after re-introduction to the home cage. At P21, pups were weaned and housed








individually until sacrifice and perfusion at P60. A total of 38 animals were used in this

analysis (EtOH, n = 12; GC, n = 10; SC, n = 16).

Tissue preparation and immunocytochemistry

Animals were selected for immunocytochemical staining at P60, an age which

represents adulthood in rats. This age was selected in order to examine the long-term

effects of neonatal ethanol treatment on ChAT expression in cholinergic neurons of the

medial septum and corresponds to an age used in previous analyses of prenatal ethanol

influences on cholinergic and GABAergic neurons in the medial septum (Moore et al.,

1997; Swanson et al., 1996). On P60, rats were anesthetized by pentobarbital overdose

just prior to perfusion. Animals were perfused transcardially with phosphate buffered

saline (PBS; 0.1M containing 0.9% sodium chloride) followed by 10% formalin (in the

same PBS). The brains were removed and post-fixed overnight in the same fixative. The

brain tissue was equilibrated overnight (or until it sank) in a cryoprotectant solution (PBS

containing 30% sucrose and 15% ethylene glycol) and stored frozen at -700C until

processed for immunocytochemistry.

Brains were prepared for frozen sectioning by thawing and equilibrating with a

30% sucrose-PBS solution. Frozen serial coronal sections throughout the forebrain were

cut at a thickness of 40 pm and processed free-floating. Alternate sections were

immunostained for choline acetyltransferase using a polyclonal antibody (rabbit anti-

human placental ChAT, Chemicon, Temecula, CA). Immunoreactivity was visualized as

a blue-black reaction product using an avidin-biotin conjugate/nickel intensified staining

(see below). Animals from each diet group were processed for immunocytochemistry at a








given time. This ensured against staining differences between groups resulting from

procedural differences.

Endogenous peroxidase activity was quenched by initially treating tissue sections

for 60 min at room temperature (RT) with PBS containing 3% hydrogen peroxide (H202)

and 10% methanol. Sections were pretreated with 0.4% Triton-X100 (T-X) in PBS for 30

min at RT, then blocked for 60 min with PBS containing 3% normal goat serum (NGS)

and 0.1% T-X. Primary incubation with the polyclonal ChAT antibody (1:750 in PBS +

3% NGS and 0.1% T-X) was carried out for 48 hr at 4C. Sections were then washed in

PBS containing 1% bovine serum albumin (BSA) and incubated with biotinylated goat

anti-rabbit IgG (1:1000 in PBS +1% BSA) overnight at 40C. Tissues were washed and

incubated with extravidin-horseradish peroxidase conjugate (Sigma, St. Louis, MO;

1:1000 in PBS + 1% BSA) overnight at 4C. Tissues were washed with 0.1M sodium

acetate (pH 7.2) to eliminate phosphate which can precipitate divalent cations. Sections

were then reacted for 3 min at RT using a developing buffer, pH 7.2, containing 0.8M

sodium acetate, 8 mM imidazole, 0.5% nickel (II) sulfate, 0.04% 3,3 diaminobenzidine

tetrahydrochloride, and 0.005% H202. The chromogen precipitation reaction was stopped

with 0.1M acetate buffer (pH 7.2) containing 0.1% sodium azide. Sections were mounted

in the same acetate buffer, air dried, dehydrated, and coverslipped. Control for

nonspecific staining was carried out in parallel with stained tissues with omission of the

primary antibody. Slides from individual animals were randomized and coded so that all

subsequent analyses were carried out blind with regard to the diet treatment group and

gender.








MS cell count, volume, and area analysis

ChAT-positive cell counts were conducted on alternate sections throughout the

rostral-caudal extent of the MS nucleus. The packing density of the ChAT+ neurons in

the DBB nucleus was too great for accurate cell counts to be made. Thus, for the

purposes of this study the MS alone was examined and was defined rostrally by the

ventral fusion of the hemispheres (at the level of the genu of the corpus callosum) and

caudally by the decussation of the anterior commissure

Images of ChAT immunostained sections were captured and digitized using a

RasterOps 24STV video capture board and software on a Macintosh computer. Low

magnification images (4x objective; effective scale 1.59 itm/pixel) were captured in order

to obtain the entire MS region in one image. When capturing each image, the lighting

and contrast enhancement were optimized for identification of individual cells. Images

were digitally processed using the image analysis program NIH Image (freeware from

NIH, Bethesda MD). To reduce background variation across the image, a digitally

defocused image (created by passing the primary image through a mean filter) was

subtracted from the primary digitized image. This processed image was then passed

through a Laplace filter to enhance edges and separation between cells.

Analysis of individual images was initiated by outlining the region of the MS

nucleus. The area of a section outlined varied depending on the rostral-caudal location of

the individual section. The MS nucleus in rostral sections was defined as the mediodorsal

group of neurons which were separated from the ventrally-located DBB nucleus. In

intermediate sections, where the demarcation between the MS and DBB nuclei is

ambiguous, the MS was defined ventrally by a line perpendicular to midline at the level








of the anterior commissure. In caudal sections, the MS was defined as the medially

located cells dorsal to the anterior commissure. Cells were highlighted interactively by

adjusting the grayscale threshold level to include only objects which were considered

cells. Given the thickness of each section (40 gm), the fact that alternate sections were

analyzed, the fact that ChAT+ neurons in the MS nucleus range from 10-25 um in

diameter, and the fact that an estimation of the total population was not desired, it was not

deemed necessary to perform a split-cell correction on these counts.

The program counted the highlighted objects and measured the area of the

outlined region (mm2). Because the MS is defined by clusters of ChAT+ cell bodies, the

area outlined was determined by the pattern of ChAT staining on each individual section.

MS volume (mm3) was calculated using a modification of the Cavelieri method (Michel

and Cruz, 1988) to ensure that changes in mean number of ChAT+ neurons per section as

a result of ethanol treatment were not due to changes in the size of the structure of interest

(Peterson et al., 1997). This was done by multiplying the number of 40 pim sections

analyzed for a given animal by the mean area per section for that animal and the section

thickness.

ChAT+ cell size determination

Because a previous study from our laboratory (Swanson et al., 1996) had analyzed

ChAT+ neuronal size using the computer instead of simple measurements of cell

diameters, we also chose to use the computer for these purposes so that comparisons

could be made to the previous work. For morphometric analysis, moderate magnification

images (10x objective; effective scale 0.978 pnm/pixel) were captured in order to

maximize the resolution yet minimize the loss of cells out of the focal plane of each








image. Primary images were not processed further prior to morphometric analysis. Cell

size measurements were performed on ChAT+ neurons in the MS nucleus. Three

sections through the MS were regionally matched in rostro-caudal extent for all animals.

25 non-overlapping cells per section were selected at random and measured. The cross

sectional somatic area of individual cells was highlighted interactively by adjusting the

grayscale threshold and was automatically measured by the computer. The program

computed cross-sectional area for each cell and mean somatic area was determined for

each animal.

Other analyses

The peak blood ethanol concentration (BEC) in the ethanol group was determined

on P7 two hours after the second ethanol infusion (Goodlett et al., 1990) utilizing the

Sigma 333-UV kit. Body and brain weights were recorded for individual animals at P60

following perfusion. Density of ChAT+ neurons was determined by dividing the mean

number of cells per section by the mean area (mm2) per section.

Statistical analyses

Statistical analysis was performed with the program StatView (Abacus Concepts,

Berkeley Ca). For each parameter examined, one-way Analysis of Variance (ANOVA)

was used to test for the main effects of diet and gender separately. When appropriate, the

data were further analyzed with the Fisher's Protected Least Significant Difference

(PLSD) post hoc test to determine individual group differences. Because gender

differences were noted for many measures (following an initial two-way ANOVA for

effects of diet and gender), males and females were analyzed separately.








Results


BEC, body and brain weights, and brain/body weight ratios

The mean BEC in EtOH pups two hours following the last ethanol treatment on

P7 was 269 + 23 mg/dl (n=5). Table 3-1 (all tables are located at the end of the chapter)

presents the mean P60 body and brain weights, and brain/body weight ratios (br/bd) for

all diet groups separated by gender. The ANOVA for P60 body weight revealed no

significant effects of diet on P60 body weight of males or females. A gender difference

was noted in P60 body weight for EtOH [F(1,22) = 45.2; p< 0.0001], GC [F(1,14) = 38.5;

p< 0.0001], and SC [F(l,16) = 40.8; p< 0.0001] animals which was expected given the

larger size of males at P60.

The ANOVA for P60 brain weight demonstrated an effect of diet treatment in

males [F(2,18) = 12.3; p< 0.001] and in females [F(2,25) = 6.69; p< 0.01]. The PLSD

revealed that EtOH male brains weighed less than GC male (p< 0.01), and SC male (p<

0.01) brains. EtOH female brains also weighed less than GC female (p< 0.05), and SC

female (p< 0.01) brains. No significant gender differences were noted for brain weight

within EtOH, GC, or SC animals.

The ANOVA for br/bd showed an effect of diet in males [F(2,18) = 5.16; p< 0.05]

and females [F(2,25) = 11.3; p< 0.001]. The PLSD demonstrated that EtOH males had

reduced br/bd compared with GC males (p< 0.01) while EtOH females had reduced br/bd

compared with both GC females (p< 0.001) and SC females (p< 0.001). Gender

differences were also noted in P60 br/bd for EtOH [F(1,22) = 37.5; p< 0.0001], GC








[F(1,14) = 41.2; p< 0.0001], and SC [F(1,16) = 38.5; p< 0.0001] animals, which was not

surprising given the larger size of male animals at this age.

Number of sections, MS area per section, and ChAT+ neuronal density

Table 3-2 presents number of sections, MS area per section, and ChAT+ neuronal

density measurements for each diet group for either gender. The number of alternate 40

gm sections through the MS was determined by counting the number of sections analyzed

between the ventral fusion of the hemispheres (at the level of the genu of the corpus

callosum) and the anterior commissure. The ANOVA showed no effect of diet for either

males or females on the mean number of alternate sections through the MS. Additionally,

no significant gender differences were noted in the number of alternate sections for EtOH,

GC, or SC animals.

As mentioned above, the computer program determined the area of the outlined

MS on each individual section. The mean MS area per section was determined for each

animal. The ANOVA revealed no significant effect of diet in males or females on the

mean MS area per section. Similarly, no significant gender differences were noted in MS

area per section for EtOH, GC, or SC animals.

ChAT+ neuronal density was calculated by dividing the mean number of ChAT+

neurons per section by the mean area per section. The ANOVA demonstrated no

significant effect of diet in males or females on ChAT+ neuronal density. Also, no

significant gender differences were noted in ChAT+ neuronal density for EtOH, GC, or

SC animals.








Cholinergic cell counts in the MS

The total number of ChAT+ neurons counted on alternate sections through the

P60 MS was determined and is presented in figure 3-1 (all figures are located at the end

of the chapter). The ANOVA for total number of ChAT+ cells demonstrated an effect of

diet in males [F(2,18) = 4.45; p< 0.05] but not females. The PLSD further revealed that

EtOH males contained 24 % fewer MS ChAT+ neurons than SC males (p< 0.05). No

significant gender differences were noted in mean total number of ChAT+ neurons for

EtOH, GC, or SC animals.

In order to normalize for volume, the average number of ChAT+ neurons per

section was determined. Figure 3-2 presents the mean number of ChAT+ neurons per

section. The ANOVA for mean number of ChAT+ neurons per section revealed an effect

of diet on males [F(2,18) = 3.34; p< 0.05] but not females. The PLSD further

demonstrated that EtOH males contained 19 % fewer ChAT+ neurons per section than

SC males (p< 0.05), while the 14 % difference between GC males and SC males was not

significant. No significant gender differences were noted in mean ChAT+ cells per

section for EtOH, GC, or SC animals.

Cholinergic neuronal size

Figure 3-3 presents the mean somatic cross sectional area for ChAT+ neurons

throughout the P60 MS. The ANOVA for mean somatic cross sectional area determined

an effect of diet in males [F(2,18) = 5.76; p< 0.05] but not females. The PLSD further

revealed that GC males contained an increased somatic cross sectional area compared

with SC males (p< 0.05), while EtOH males contained a reduced somatic cross sectional








area compared with GC males (p< 0.01). No significant gender differences were noted in

ChAT+ neuronal size for EtOH, GC, or SC animals.

MS volume

MS volume was determined by multiplying the number of 40 Jpm sections

analyzed for a given animal by the mean area per section for that animal. Figure 3-4

presents the mean MS volume of P60 rats. The ANOVA demonstrated no effect of diet

in males or females on the mean number of alternate sections through the MS. No

significant gender differences in MS volume were noted for EtOH, GC, or SC animals.

Discussion


The major conclusion of the present work is that small reductions in the size of

ChAT+ neurons of the medial septum are produced by neonatal ethanol exposure while

changes in the number of cholinergic neurons or size of the MS are not noted. This study

has also determined that long-lasting microencephaly is found in male and female P60

rats exposed to ethanol via artificial rearing from P4-10.

Because EtOH animals showed no difference when compared with GC animals in

total ChAT+ neuronal number or mean number of cholinergic neurons per section,

ethanol exposure did not directly reduce ChAT+ neuronal number. However, since EtOH

males contained fewer cholinergic neurons when compared with SC males it appears that

ethanol in combination with artificial rearing does affect these neurons. No changes were

noted in mean area per section, cell density, or MS volume as a result of ethanol

treatment in males. Female animals similarly exposed to ethanol did not contain a

reduced number of ChAT+ MS neurons and no changes were noted in MS volume, mean








area per section, or cell density. A permanent reduction in the size of ChAT+ neurons

was noted in EtOH males compared with GC males. However, this was concurrent with

an increase in size of cholinergic neurons in GC compared with SC males. Moreover, no

differences in cell size were found in female rats, making it difficult to determine whether

the reduction in ChAT+ cell size in EtOH males is an effect of ethanol on neuronal size

or merely an effect of diet and artificial rearing on GC males. Thus neonatal ethanol

exposure, while producing long-lived microencephaly, has little effect on the cholinergic

neurons of the adult rat MS, as measured by ChAT+ neuronal number and morphology.

Long-term effects of ethanol on brain growth

Microcephaly and microencephaly are hallmarks of FAS as well as neonatal

animal models of FAS (West and Pierce, 1986). Reduced brain size represents one of the

most reproducible features of ethanol-induced alterations in the brain, and the current

study is no exception. Artificial rearing from P4-10 produced no long-lasting deficits in

body weight and by P60 EtOH and GC pups weighed the same as SC pups. Despite the

lack of effect on body weight, EtOH animals of both genders showed reduced brain

weight at P60 compared with GC and SC animals. Brain to body weight ratio (br/bd) was

similarly reduced in both males and females exposed to ethanol. Taken together, the

reduced brain weight of EtOH animals and the reduced br/bd in EtOH animals

demonstrate that neonatal ethanol exposure produced long-lasting microencephaly.

Goodlett et al. (Goodlett et al., 1991) exposed rats from P4-9 in two of the 12 daily

feedings as a 10.2% solution (an exposure paradigm similar to the current experiments).

Exposure to this concentration of ethanol resulted in a peak BEC of 361 mg/dl (compared

with 269 mg/dl in the current study) and produced microencephaly in both genders in








adult animals. The current study demonstrates that even with a lower peak BEC, brain

growth is severely compromised.

The degree of ethanol-induced reduction in brain weight depends directly on the

peak BEC produced. Bonthius and West (1991) provided an illustration of this by

comparing the effects of a 6.6 g/kg/day ethanol treatment spread out over 12 feedings

from P4-11 with the effects of 4.5 g/kg/day ethanol treatment given in just two feedings.

Animals were sacrificed at P90 to observe long-term effects of neonatal ethanol delivery

on brain weight. Interestingly, they found that the lower 4.5 g/kg/day dose produced

profound brain weight and brain to body weight ratio deficiencies, while the 6.6 g/kg/day

group exhibited no such microencephaly. This differential response to varying ethanol

delivery patterns was directly related to peak BEC as the higher dose spread out over 12

feedings produced a peak BEC of only 43 mg/dl while the lower dose condensed into two

feedings produced a peak BEC of 318 mg/dl (Bonthius and West, 1991). Thus, the

pattern of exposure plays a critical role in the severity of ethanol-induced teratogenicity.

Long-term effects of ethanol and artificial rearing on ChAT+ neurons in the MS

Artificial rearing and ethanol exposure produced a decrease in the mean ChAT+

neuronal number per section in EtOH males compared with SC males in the absence of

volume, area, or density changes. Female animals displayed no differences in mean

cholinergic neuronal number per section, volume, area, or density measurements and,

thus, the reduction occurs in a sexually dimorphic manner. Because the reduction in

ChAT+ neuronal number noted in males was between the gastrostomized EtOH group

and the dam-reared SC group (and not the pair-fed GC group) the reduction appears to be

the result of an interaction between ethanol treatment and some characteristic of the








artificial rearing technique rather than the result of ethanol alone. Kelly (1997) has noted

an effect of artificial rearing on the concentration of neurotransmitters in the SH system

of male rats: neurotransmitter concentration was reduced in artificially reared males

independent of ethanol exposure. While the current results do not implicate artificial

rearing in reducing cholinergic neuronal number (the difference between GC and SC

males in mean ChAT+ neurons per section is not significant) they do suggest that ethanol

in combination with artificial rearing can reduce ChAT+ neuronal number. Perhaps an

interaction between ethanol and the stress of the procedure (from invasiveness or

maternal separation) accounts for the reduced cholinergic neuronal number in artificially

reared and ethanol exposed pups.

Developmental ethanol exposure and ChAT+ neuronal number

The current finding that cholinergic neuronal number in the MS is unaffected by

ethanol alone is consistent with previous experiments from our laboratory. Swanson et

al. (1995; 1996) demonstrated reduced ChAT activity in P14 rats chronically exposed to

ethanol in utero. However, analysis of ChAT+ neuronal number in the MS of P14 and

P60 rats revealed no effect of diet treatment on cholinergic cell number. Similarly,

evidence from Heaton et al. (1996) showed that ChAT+ neuronal number is not

significantly affected in the striatum of male or female rats following CPET. A transient

increase in cholinergic neuronal number was noted in the striatum on P14, but this was

reduced to control levels by P60.

While studies from our laboratory (see above) have not indicated an effect of

developmental ethanol exposure on cholinergic neuronal number in the rat MS or

striatum, studies from other groups have demonstrated ethanol-induced alterations in








ChAT+ number, particularly in the MS. For example data suggest that acute prenatal

exposure of mice to ethanol resulted in severe midline anomalies at embryonic day 18,

including loss of basal forebrain neurons (Schambra et al., 1990) as well as loss of septal

neurons and reduced volume at P 15 (Ashwell and Zhang, 1996). However, it should be

noted that these experiments produced high acute BECs (600-700 mg/dl) which were the

result of one or two ethanol exposures early in embryonic development. Thus,

differences in timing and dosage may account for the apparent discrepancy (Swanson et

al., 1996).

Exposure of adult rats to ethanol for 12 or 28 weeks has been reported to reduce

cholinergic neuronal number in the MS (Arendt et al., 1995; Arendt et al., 1988).

However, it should be noted that comparisons were made between rats exposed to ethanol

as a 20% solution of drinking water and rats fed chow and water ad libitum; no pair-fed

controls were examined. Thus, it is possible that the reduction of cholinergic neurons in

the MS is not due to the influence of ethanol alone. For instance, an interaction between

ethanol and malnutrition may explain the noted differences. However, it is conceivable

that chronic ethanol exposure during mature stages may reduce the number of MS

cholinergic neurons, as found by Arendt et al. (1995; 1988). Regardless, the current data

in combination with the Swanson et al. (1996) and Heaton et al. (1996) studies suggests

that cholinergic neuronal number in the MS is not susceptible to moderately-high or

moderate doses of ethanol during early postnatal or prenatal development, respectively.

A future study examining ChAT+ neuronal number after exposure to a higher peak BEC

would determine whether the apparent lack of effect of ethanol on cholinergic neurons is

due to the moderate doses used in these studies.








Developmental ethanol exposure and ChAT+ neuronal size in the MS

Although ChAT+ neuronal counts were not directly affected by ethanol, analysis

of the mean somatic cross sectional area of cholinergic neurons in the P60 MS

demonstrated a diet effect in male animals. Specifically, neuronal size was reduced in

EtOH males compared with GC males. However, GC males contained larger neurons

compared with SC males and no differences were found in females, making it difficult to

determine whether this is an effect of ethanol on cholinergic neuronal size or merely an

effect of diet and artificial rearing on GC males. It is unlikely that the smaller size of

cholinergic neurons in EtOH males is simply because of the noted microencephaly since

the brains of EtOH and SC males were different in size but the mean size of ChAT+

neurons was similar.

It is possible, however, that GC males experienced an increase in somal size in

response to either the stress of the artificial rearing procedure (such as maternal

separation, isolation, or the invasive surgery) or to some component of the maltose-

dextrin containing milk diet. Factors such as nerve growth factor (NGF) can induce

hypertrophy of ChAT+ basal forebrain neurons (Higgins et al., 1989). NGF is also

known to increase the size of cholinergic neurons in cultures of rat septal neurons

(Markova and Isaev, 1992), and is able to reverse axotmy-induced decreases in MS

cholinergic neuronal cell bodies (Hagg et al., 1989). It is conceivable that male GC

animals increased the expression of NGF, as is known to occur following stress (Smith,

1996), resulting in larger cholinergic MS neurons. However, female GC animals were

similarly stressed and did not display hypertrophy of ChAT+ neurons. Perhaps a more

likely explanation is that a constituent of the liquid diet acted in atrophic manner on these








neurons. Whatever the explanation, these data are consistent with data from other

investigators who have reported sexually dimorphic effects on the size of cholinergic

neurons of the MS. For example, neonatal hyperthyroidism is capable of increasing

ChAT+ neuronal size in the MS of male animals while simultaneously decreasing the size

of ChAT+ neurons in females (Westlind-Danielsson et al., 1991).

One other report from our laboratory has examined cholinergic neuronal size at

P60 following chronic prenatal ethanol exposure (Swanson et al., 1996). While the

exposure times were different in the current study, it is useful to compare the present data

with the Swanson et al data. The size of the ChAT+ neurons at P60 is quite similar. For

example, the range of neuronal size in the Swanson et al. study was 90-100 pm2, which is

comparable to the range noted in the current study (80-110 im2). However, in contrast to

the present data, Swanson et al. (1996) found no ethanol-induced alterations in ChAT+

neuronal size, and found no gender differences.

ChAT+ neuronal insusceptibility: possible role of neurotrophic factors

Why is ChAT+ neuronal number apparently unaltered following developmental

ethanol exposure? One possible explanation is the importance of neurotrophic factors in

regulating the development and maintenance of the SH system. The hippocampus is

known to express a rich variety of neurotrophic factors, including NGF, brain-derived

neurotrophic factor (BDNF), neurotrophin-3, and basic fibroblast growth factor (Ernfors

et al., 1990; Maisonpierre et al., 1990). The high affinity receptors for the various

neurotrophins found in the hippocampus, including trk A, trk B, and trk C, are also

present in the developing septum, including the cholinergic neurons of the medial septum

(Ringstedt et al., 1993). Evidence suggests a role for neurotrophins in regulating the








expression of ChAT in the basal forebrain. Intraventricular injection of NGF antibodies

into rat neonates reduces ChAT immunostaining in the septum (Vantini et al., 1989).

Moreover, mice which have been genetically engineered to lack the various neurotrophin

receptors have been generated, and have further defined a role for neurotrophins in

sustaining septal cholinergic neurons in vivo. For example, mice which lack trk A show

reduced acetylcholinesterase activity in septal projection fibers (Smeyne et al., 1994), and

mice which lack BDNF show reduced density of cholinergic neurons in the MS (Jones et

al., 1994).

In vitro studies have also demonstrated a role for neurotrophins in sustaining

septal neurons in normal conditions and in the presence of ethanol. For example, NGF

stimulates ChAT activity, neuritic complexity, increased fiber length, and increased fiber

outgrowth in cultures of septal cholinergic neurons (Hartikka and Hefti, 1988).

Moreover, studies from our laboratory have demonstrated that NGF protects both

cultured dorsal root ganglion neurons and septal neurons from ethanol neurotoxicity

(Heaton et al., 1993; Heaton et al., 1994). It is conceivable that neurotrophic interactions

are preserved in the current study, and that ethanol is unable to produce toxic effects on

cholinergic neurons in the medial septum because these normal interactions are

maintained. A consequence of this maintenance may be growth factor-mediated

induction of protective molecules of the bcl-2 family of cell death molecules which would

prevent ethanol-induced toxicity in this population. Similar mechanisms are known to

operate in cerebellar granule cells during development. For example, Muller et al. (1997)

demonstrated that NGF induces Bcl-2 protein expression and that this induction resulted

in cell survival.




Full Text

PAGE 1

NEURONAL SUSCEPTIBILITY IN RAT MODELS OF DEVELOPMENT AL ETHANOL EXPOSURE: DESCRIPTIONS OF CELLULAR AND MOLECULAR ALTERATIONS By D. BLAINE MOORE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1998

PAGE 2

For Terri and my Mentors who nurtured my interest in Biology

PAGE 3

ACKNOWLEDGMENTS There are many people to whom I am indebted especially my wife, Terri who is my best friend and best critic I am also grateful to my mentors for their continued support I am indebted to my thesis advisory committee members Drs. Marieta Heaton (chair), Don Walker Tony Yachnis Kevin Anderson and John MacLennan, for their efforts and to Drs Doug Anderson and Gerry Shaw for helpful advice. I benefited from the assistance of Dr Jim West and Jo Mahoney (Texas A&M University) in learning the artificial rearing procedure. Likewise Dr. Bill Farmerie and Regina Shaw (of the Interdisciplinary Center for Biotechnology Research UF) as well as Terri Edwards and Dr John MacLennan provided excellent advice on molecular biology. Thanks to Heaton and Walker lab members over the years, including Mike Paiva Jeffre y Thinschmidt Pat Burnett Steve Farnworth, Leon Williams and Drs. Doug Bradley Jean Mitchell, Meg Davis Mike King and Doug Swanson. I am thankful for the support of the NIH during my graduate training including NIAAA F31 AA0550201 and NIAAA T32 AA07561. Ill

PAGE 4

TABLE OF CONTENTS ACKNOWLEDGMENTS .... .. ... .. ... ................. .... ... . ........... .... ........... ... .... ............... .. . iii LIST OF TABLES ......... ...... ............ .......................... ... ................................ ... ............... vi LIST OF FIGURES ............. .............................................................................. ................ vii ABSTRACT ..... .. .. ....... .. .............. ................ ...... ........... .. ... ....... .... ...... .... ........... ..... ix CHAPTERS 1 INTRODUCTION .. ..... ...... ..... ...... ... .... ....... .... .... .............. ......... ..................... ........... 1 Ethanol E x posure During Development and Resulting Nervous System Alterations .. .... ..... ........................................... .... . . ......... .. . ......... ... .... ... ............. ........ 1 Differential Temporal and Regional Vulnerability .............................................................. 4 Prenatal Ethanol E x posure and Neuroanatomical Alterations .... .... .............. ... ........ .... .... ... 5 Postnatal Ethanol E x posure and Neuroanatomical Alterations .. .. ............. ...................... .... 8 Programmed Cell Death ( PCD) and the bcl-2 Famil y ................ .. ........ .. .. .. ............. .......... 11 Hypotheses Tested ......... ..... ......................... ............. ................ ........... ....... ... ............ ... ... 21 2 EFFECTS OF PREN AT AL ETHANOL E XPOSURE ON P ARV ALBUMINIMMUNOREACTIVE GABAERGIC NEURONS IN THE ADULT RAT MEDIAL SEPTUM AND ANTERIOR CINGULATE CORTEX ........................ ..... 25 Summary ...................... .... ...... ...................... .......... . ...... . ........ ...... ..... ..... .... .............. . . 25 Introduction ...... .......... . ............... ............... ... ........ .. .... ................................. ... .. .......... ..... 26 Materials and Methods .... ......... .... ..... ..... ....... ........ ......... ............... ...................... .. ... ... ..... 30 Results ... .................. . .. .. ..... ... ..... .. ... ...... ... ......... ... .. ... ..... .. .. ......... ... ......... ... .... ........ ... 37 Discussion ... . ..... .. ..... .......... .......... ..... ........... ...... ..... ........ ......... ... .. .. ............ ................ 43 3 EFFECTS OF N E ONATAL ETHANOL EXPOSURE ON CHOLIN E RGIC N E URONS O F T H E RA T M E DIAL S E PTUM ..... .... ..... ... .... .... .... ... ... ...................... 70 Summary ......... . . . ... ... ..... ......... ..... . ...... ............. . .... ..... .................... .... .............. ......... 70 Introduction .......... ....... .... ........ ........ .... ................. .... ... ........... ..... ... ... .. ... ...... ...... ........ .... 71 Methods ... .... .......... ... ...... . .. ..... ... ... ...... .. .... .... ........ ........ ... .... .... ....... .... .. .... ................ ... 73 l V

PAGE 5

Results ..... ............. .... ................................ ... ... ... ............ ..... ..... .............. .. ........... ... .. ..... 80 Discussion ......... ................ ... ... ... ...... ... ..................................... ........ ............ ...... ...... .... 83 4 EFFECTS OF NEONATAL ETHANOL EXPOSURE ON PURKINJE AND GRANULE CELLS AND BCL-2 FAMILY MRNA LEVELS IN THE RAT CEREBELLAR VERMIS . ..................... .... ......... . ... .... .. .... ...... ... ............ ......... .. ... .. .. 97 Summary ............ ............ ..... ......................................... ...... . .. ... ... ............. ...... .... ........ . 97 Introduction . ......... ... ... ..................... ......... .. ......... ........ .............. ... ....... .......... .. ... ............ 98 Materials and Methods .... ..... ............. ... ... ......... ............ ............ ........... ...... ................ ... 102 Results .................... ........... .......... ... ............. ................. .......... ..................................... . 110 Discussion ................ ... ... .... ........ ................... ....... .... ........... ..... ............ ..... ..... ........... .... 116 5 CONCLUSIONS AND FUTURE DIRECTIONS ............ ..... ............... .......... ....... ... 153 Recapitulation of R esults and Hypotheses Tested ... ..... ..... ................................... ..... .. ... 153 Choice of Animal Models ..... ..... .. ........ .......... ......... ..... . ... ...... ............ ... ..... .... ..... ........ 159 Choice of Cell Counting Methods .. ......... ..... ............... ............................... ... ......... ... ..... 163 Other Methodological Considerations .................. ............... ...... ........... ............... .... ... 164 Future Directions for Developmental Ethanol R esearc h ... ............... ... .. ........ ............. ... 165 Conclusion .................. ................ ... ............ ............ .... ................ .... ..... ..... .................... ... 179 REFERENCES ... ... . ........ .... ... .. ... .. ................................ ..... .. ......... ....... ............. .... . .... 181 BIOGRAPHICAL SKETCH .... .. ... ....... ..................... ........... ..... ...... .... . .... ...... .. ... ........ 209 V

PAGE 6

LIST OF TABLES 2-1. Postnatal day 60 body and brain weight and brain to body weight ratio ... ...... ......... 56 2-2 Number of sections medial septum area per section and parvalbuminimmunoreactive neuronal density in the adult rat MS ... .................. ....... .. ... ............ 56 2-3. Number of sections mean area per section, and parvalbumin-immunoreactive neuronal density in the adult rat anterior cingulate cortex .......................... .. .... ..... 57 3-1. Mean body weight brain weight and brain weight to body weight ratio of postnatalday 60 animals .......... ..... ...... ............. ....... ...... .. . .. .... .... .... .. .. .... ... .... ... .... ........ .... . 92 3-2 Number of sections medial septum area per section and choline acetyltransferaseimmunoreacti v e neuronal density in the adult rat MS ... . . ... ... ...................... .... .... .. 92 4-1. Lobule I cerebellar vermis volume area and cell nuclei diameter data at P21 following exposure on P4 only .... .... ..... ...... ....... ........ .................. ........................ .133 4-2. Lobule I cerebellar vermis volume area and cell nuclei diameter data at P21 following exposure on P4-5 ... ......... ....... ... ....... ............... . ...... ... .... .............. ....... 133 4-3. Lobule I cerebellar vermis volume area and cell nuclei diameter data at P21 following exposure on P7-8 .................. ...... ....... ..... .................. ... .................... ... 133 4-4. Lobule I cerebellar vermis volume area and cell nuclei diameter data at P5 following ex posure on P4-5 ......... ............ ..... .. ............... ... ... ..... ... ....... . .. ......... . .134 Vl

PAGE 7

LIST OF FIGURES Figure 2-1. The mean total number of parvalbumin-expressing neurons detected on alternate sections through the medial septum of postnatal-day 60 ....... ... .... ... ...... . ................. 58 2-2. Photomicrographs of 40 m coronal sections through the medial septum of ethanoltreated (A) and sucrose-treated (B) postnatal-day 60 female rats ......... ..................... 59 2-3. The mean number of parvalbumin-expressing neurons per section in alternate sections through the medial septum of postnatal-day 60 ......... ................... ............... 61 2 -4 The mean diameter of parvalbumin-expressing neurons on alternate sections through the medial septum of postnatal-day 60 rats ............. ..... ...... .... ............... ......... ..... .... 62 2-5 The mean medial septum (MS) volume of postnatal-day 60 rats exposed throughout gestation to an ethanol-containing liquid diet sucrose-containing liquid diet or lab chow and water. ........ ..................................... ............................................. .............. 63 2-6. The mean total number of parvalbumin-expressing (PA+) neurons detected on alternate sections through the anterior cingulate cortex of postnatal-day 60 ............. 64 27. Photomicrographs of 40 m coronal sections through the anterior cingulate cortex of ethanol-treated (A) and sucrose-treated (B) postnatal-day 60 rats ............................. 65 2-8 The mean number of parvalburnin-immunoreactive neurons per section counted in the anterior cingulate cortex ................................... ....... ......... ............................ ...... 67 2-9. The mean diameter of parvalbumin-imrnunoreactive neurons in the anterior cingulate cortex of postnatal-day 60 rats ............................. ................. ........... . ....... 68 2-10. The mean cingulate gyrus volume of postnatal-day 60 rats exposed throughout gestation ... ........... ... ... .... ... . ............ ... . .......... ..... ... ..... ... ........ . .... ... ...... ... . ........ ... 69 3-1 Mean total number of choline acetyltransferase-irnmunoreactive neurons in the medial septum of postnatal day 60 rats ............. ......................... . . ...... .................... 93 vu

PAGE 8

3-2 Mean number of choline acetyltransferase-irnrnunoreactive neurons per section detected on alternate 40 m sections through the medial septum of postnatal day 60 rats .. .............. ... .. ........... ..... .............. .............. .................... ........ .... ... ...... .............. 94 3-3. Mean somatic cross sectional area (m2 ) of choline acetyltransferaseimmunoreactive neurons in the medial septum of postnatal day 60 rats ........ .. ... .. .. 95 3-4. The mean medial septum volume of postnatal-day 60 rats ....................................... 96 4-1. Effects of ethanol delivered on P4 only on P21 body weight (panel A) P21 brain weight (panel B) and P21 brain to body weight ratio (panel C) ............................. 135 4-2. Effects of ethanol delivered on P4-5 on P4 PS, and P21 body weight (panel A) brain weight (panel B) and brain to body weight ratio (panel C) ............................ 137 4-3. Effects of ethanol delivered on P7-8 on P7 P8 and P21 body weight (panel A) brain weight (panel B) and brain to body weight ratio (panel C) ................... ....... .139 4 4 Ethanol delivered during the first postnatal week reduces mean Purkinje and granule cell number per section in lobule I of the cerebellar vermis ....... ............. ................ 141 4-5. Ethanol delivered during the second postnatal week does not reduce mean Purkinje or granule cell number per section in lobule I of the cerebellar vermis .................. .143 4-6. Ethanol delivered during the fust postnatal week on days 4-5 reduces mean Purkinje but not granule cell number per section in lobule I of the cerebellar vermis as determined on postnatal day 5 (PS) ............................................. .......... ................. 144 4-7. mRNAs encoding pro-apoptotic molecules of the bcl-2 family are upregulated following acute ethanol delivered on postnatal day 4 .... .... .......... ... ...... ............... 145 4-8. A further ethanol exposure on PS does not significantly alter the expression of bcl-2 family mRNAs .... .... ........... ................... . ... ..... . .... ...... .. .... .. ... ........................... ... 147 4-9. E ffects of acute ethanol delivered on postnatal day 7 on bcl-2 family gene expression . .............. .... ............... ................. ....... .............. ...... ........... ........... ........ 149 4-10. An additional ethanol exposure on postnatal day 8 increases mRNAs encoding the pro-apoptotic molecule bax .......... .... .................... ................... .......... ......... ............ 151 VIII

PAGE 9

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 NEURONAL SUSCEPTIBILITY IN RAT MODELS OF DEVELOPMENT AL ETHANOL EXPOSURE: DESCRIPTIONS OF CELLULAR AND MOLECULAR AL TERA TIONS By D Blaine Moore December 1998 Chairman: Marieta B. Heaton Ph.D. Major Department: Neuroscience Developmental disorders arising from maternal consumption of ethanol during pregnancy are collectively termed the fetal alcohol syndrome. Ethanol exposure during development induces abnormalities in particular brain regions and is known to alter the expression of particular genes and their protein products. The present body of work sought to further document neuronal populations in the brain which display vulnerability to developmental ethanol exposure A further goal of this work was to investigate cell death gene expression shortly after ethanol insult in the cerebellum in order to test a specific hypothesis about the cellular mechanism of ethanol neurotoxicity. A prenatal exposure model was used to examine long term changes in protein expression patterns of parvalbumin (a marker for garnma-aminobutyric acid(GABA) expressing neurons) in the rat brain. Deficiencies in the mean number of immuno positive cells per section were noted in the medial septum (in a sexually dimorphic IX

PAGE 10

manner) and anterior cingulate cortex of ethanol-treated rats. This represents the first documentation of GABAergic neuronal susceptibility to ethanol in either brain region. A neonatal exposure model was used to examine long-term changes in the expression pattern of choline acetyl-transferase (ChA T a marker for cholinergic neurons) in the medial septum ; no significant ethanol-induced changes in the mean number of immuno positive cells per section were noted. A similar neonatal exposure paradigm was used to document Purkinje and granule cell numbers in the cerebellar verrnis during known periods of ethanol sensitivity and insensitivity and to investigate mRNA levels of the bcl-2 family of cell death molecules. First postnatal week ethanol treatment significantly reduced Purkinje and granule cell number while second week exposure did not. bcl-2 family gene expression was measured in the vermis shortly after ethanol treatment to determine whether alterations in these genes might correlate with the noted cell death. Transcripts encoding the pro apoptotic molecules bax and bcl-xs were up-regulated following both first and second week exposure. Thus a positive correlation between altered bcl-2 expression and cerebellar cell death was not found. Suppression of these pro-apoptotic processes may be the critical determinant of cerebellar susceptibility These findings suggest new avenues of research on the intracellular consequences of such expression changes. X

PAGE 11

CHAPTER 1 INTRODUCTION Ethanol Exposure During Development and Resulting Nervous System Alterations Ethanol s teratogenic actions have been recognized throughout recorded history and considerable research has defined its deleterious actions (West et al. 1994). With the recognition of the fetal alcohol syndrome (FAS) and the linkage of ethanol to malformations in children of alcoholic mothers Jones and Smith (1973) spurred a plethora of studies on alcohol-induced fetal abnormalities (West et al. 1994). Consequently prenatal ethanol exposure has been shown to result in serious developmental alterations including intrauterine growth deficiencies facial dysmorphias mental retardation (Abel 1984) attention deficiencies and autistic-like syndromes (Aronson et al. 1997) and lowered IQ (Mattson et al. 1997). Ethanol readily crosses the placental and blood-brain barriers diffuses into all aqueous components of the developing fetus where it can interact with membrane proteins and lipids (Zajac and Abel 1992) and has been shown to affect protein synthesis placental nutrient transport fetal g lucose availability fetal oxygen levels generation of reactive oxygen radicals and neurotrophic factor activity (Abel and Hannigan 1995 ; Bonthius and West 1990 ; Heaton and Bradley 1995 ; Hender s on et al. 1995 ; Mukherjee and Hodgen 1982 ; West et al., 1994 ; Z ajac and Abel 1992)

PAGE 12

2 Zajac and Abel (1992) have characterized fetal alcohol exposure as ''the leading known cause of mental retardation in the Western world ." Indeed despite this recognition the incidence of FAS in the United States has increased six-fold between 1979 and 1993 (Prevention 1995) to 1.95/1000 live births and 43. 1/1000 live births among heavy drinkers (Abel and Hannigan 1995). Sampson et al. (1997) recently estimated the incidence of FAS and alcohol-related neurodevelopmental disorders between 1975-1981 in Seattle as 9.1/1000 live births. As such FAS remains a significant health problem in the United States. Further research describing the neurodevelopmental changes induced by ethanol and the cellular and molecular changes induced by ethanol is needed especially since the mechanism of ethanol teratogenicity remains unknown. The toll that ethanol exacts on the development of the central nervous system (CNS) represents its most potent danger. Children exposed prenatally to ethanol show among other abnormalities reduction in corpus callosum area (Riley et al. 1995) and malformations in cerebellar structure (Clarren et al. 1978 ; Sowell et al. 1996; Wisniewski et al. 1983) Animal models have been developed which recapitulate many of the neurodevelopmental alterations and behavioral outcomes seen in humans with FAS and these models have allowed for the characteri z ation of various nervous system changes resulting from developmental exposure to ethanol (Hannigan 1996). While various model systems have been employed including the chick (Bradley et al. 1997) and mouse (Schambra et al. 1990) the most common choice for investigators is the rat. Rats exposed during embryonic development show long-lived learning impairment (Clausin g et al. 1995) behavioral alterations (Riley, 1990) microencephaly (West and Pierce 1986) chan g e s in neuronal proliferation and migration (Miller 1986 ;

PAGE 13

3 Miller 1995b; Miller 1996) reductions of neuronal number (Barnes and Walker 1981; Miller 1995a ; Napper and West 1995b) alterations in neuronal circuitry (West et al. 1981 ) delays in synaptogenesis (Hoff, 1988) permutations in neuromorphological development (Burrows et al. 1995 ; Davies and Smith 1981; Kotkoskie and Norton 1989) changes in neurochemistry (Black et al. 1995; Swanson et al. 1995), and alterations in the levels of specific mRNA species (Lee et al. 1997) and specific receptor molecules such as the high affinity trkA nerve growth factor receptor and the low affinity neurotrophin receptor p75 (Dohrman et al. 1997). The present body of work sought to further document neuronal populations in the brain which display vulnerability to developmental ethanol exposure. Another goal of this work was to investigate cell death gene expression shortly after ethanol insult in the cerebellum in order to test a specific hypothesis about the cellular mechanism of ethanol neurotoxicity. To accomplish these goals the following studies were performed: first a prenatal rat exposure model was used to examine long term changes in protein expression patterns of parvalbumin (a marker for GABAergic neurons) in the rat medial septum and cingulate cortex by counting parvalbumin-immunoreactive neurons in these structures ; second a neonatal rat exposure model was used to examine long-term changes in the expression pattern of choline acetyltransferase (ChA T a marker for cholinergic neurons) in the medial septum by counting ChA T immunoreactive neurons in this region ; and third a similar neonatal rat exposure paradigm was used to document Purkinje and granul e cell numbers in the cerebellar vermis during known periods of ethanol sensitivity and ins e nsitivity and to investigate mRNA levels of the bcl-2 family of cell death molecules.

PAGE 14

4 Differential Temporal and Regional Vulnerability An interesting property of ethanol and its effect on the developing CNS is the fact that particular brain regions are differentially affected in both human FAS and rodent models of FAS (West and Pierce 1986). Fortunately animal models of FAS provide for rigorously controlled studies of both regional and temporal susceptibilities, and allow for the identification of the neuroanatomical substrates that underlie FAS. Although the brain develops throughout the entire prenatal and early postnatal period in humans and rats, there are both regional and temporal susceptibilities to ethanol. In terms of regional vulnerability, the cerebral cortex (Miller 1986) and hippocampus (Barnes and Walker, 1981) have been demonstrated to be severely affected with chronic prenatal ethanol exposure. In terms of tempora l vulnerabi l ity, CNS development during the human third trimester equivalent is especially tenuous During this period rapid global brain development is occurring and this dynamic phase which occurs during the human third trimester is often termed the brain growth spurt Although all mammals develop in a similar manner the timing of this intensified growth is different across species. The rat for example, from which most animal data regarding FAS are derived undergoes its brain growth spurt during the first two postnatal weeks with a peak at postnatal day 4-10 (P410) (Dobbing and Sands 1979) Ethanol exposure during this vigorous period of brain development affects both mature and proliferating neurons (West and Pierce 1986). The principal neurons of the cerebellar cortex provide a good example as neonatal exposure reduces the number of differentiating Purkinje cells as well as proliferating granule cells (Bonthius and West 1990) albeit in a temporally restricted manner (see below).

PAGE 15

5 Prenatal Ethanol Exposure and Neuroanatomical Alterations Experimental studies utilizing rodent models of FAS have been successful in reproducing many of the behavioral and morphological changes found in FAS and have described numerous CNS alterations resulting from in utero ethanol exposure. Some of these include alterations in a variety of developmental processes in the CNS including perturbed neuronal generation and migration (Miller 1986 ; Miller, 1995b; Miller, 1996) permutations in neuromorphological development (Burrows et al. 1995 ; Davies and Smith 1981; Kotkoskie and Norton 1989), changes in neurochemistry (Black et al. 1995; Swanson et al. 1995) delayed synapse turnover in the hippocampus (Hoff 1988) alterations in neuronal number in hippocampus (Barnes and Walker 1981) and long lasting detrimental effects on learning and behavior (Clausing et al. 1995 ; Riley 1990). Septohippocampal system The septohippocampal (SH) system of basal forebrain afferents and hippocampal targets has been shown to be sensitive to prenatal ethanol exposure. Reductions in CAI pyramidal neuronal number (Barnes and Walker 1981; Wigal and Amsel 1990), alterations in hippocampal mossy fiber organization (West et al. 1981) and dendritic arbori z ation (Davie s and Smith 1981; Smith and Davies 1990) delays in synaptogenesis (Hoff 1988) changes in neurochemistry (Black et al. 1995 ; Swanson et al. 1995) and deficits in hippocampal s ynaptic plasticity (Suth e rland et al. 1997) have all been demonstrated in the rodent SH system following exposure to ethanol in utero. Given the importance of the hippocampus in learning and memory and the role of the SH system in g eneratin g and maintainin g electrical activity in the hippocampus (Dutar et al. 1995) the

PAGE 16

6 possibility exists that ethanol-induced changes in this system can have detrimental effects on offspring exposed to ethanol prenatally. The SH system is a pathway of cholinergic and GABAergic fibers originating from the medial septal (MS) nucleus and the horizontal and vertical limbs of the nucleus of the diagonal band of Broca (DBB) which synapse on pyramidal neurons granule cells and intemeurons of the hippocampus (Dutar et al. 1995 ; Freund and Antal 1988). Evidence exists for an ethanol-induced alteration of the cholinergic component of the SH pathway. Severe abnormalities were noted in the basal forebrain of fetal mice following an acute ethanol dose at gestational day 7 (G7) (Sulik et al. 1984) and Schambra et al. (1990) found a reduction in the number of ChA T + neurons in fetal mice following an acute ethanol administration on G7 Arendt et al. (1988) reported similar findings in adult rats following chronic ethanol treatment although their experimental design suffers from lack of pair-fed controls Studies from our laboratory utilizing a rat model of chronic prenatal ethanol treatment have shown an ethanol induced delay in the normal ontogeny of ChA T enzyme activity in the SH pathway but have not revealed an effect on ChATirnrnunoreactive neuronal number (Swanson et al. 1995 ; Swanson et al. 1996). It was a goal of the present work to determine if the other major population of neurons in the medial septum the GABAergic neuronal population is affected by developmental ethanol exposure Thus studies were performed to test the hypothesis that GABAergic neurons in the SH pathwa y are s u s ceptibl e to chronic prenatal ethanol treatment in order to determine whether this subset of neurons in the medial septum is affected by ethanol. To achieve thi s aim an antibody which recogni z es parvalbumin (PA) a calcium binding protein

PAGE 17

7 commonly found in GABAergic neurons was used and PA-immunoreactive neurons were counted PA and the SH system Although the GABAergic component of the SH projection has not been as extensively characterized as the cholinergic component it is an important part of this system. Approximately 33% of the neurons in the MS/DBB region display immunoreactivity to PA, an 11. 8 kDa member of a Ca2+ binding superfamily of proteins (Kiss et al. 1990a ; Kiss et al. 1990b ; McPhalen et al. 1994) commonly found in rapidly firing GABAergic neurons where it influences the activity of Ca2+ -dependent K + channels (Plagmann and Celia 1993 ) In rats PA ontogeny begins in the MS at G21 and coincides with the beginning of physiological activity such as spontaneous firing and excitatory synaptic input (Lauder et al. 1986 ; Solbach and Celia 1991 ) The PA-expressing MS neurons innervate inhibitory interneurons in the hippocampus (Freund and Antal 1988) Although CNS regions differ in the extent to which PA and GABA co-localize (Alonso et al. 1990 ; Brauer et al ., 1991 ; Kiss et al. 1990a) within the MS nucleus most if not all of the hippocampal-projecting GABAergic neurons are parvalburnin-immunoreactive (Freund 1989 ; Krzywkowski et al. 1995). Thus PA-immunoreactivity serves as a reliable marker for hippocampal-projecting GABAer g ic neurons in the MS nucleus and identifies a subpopulation of the total GABAergic neuronal pool within the basal forebrain PA and cingulate cort ex Quantitative analy ses of neuroanatomical changes in the cer e bral cortex following prenatal ethanol e x posur e have been conducted and have suggested ethanol-induced

PAGE 18

8 neuroanatomical alterations. Specifically, alterations in the generation and proliferation of neurons have been noted (Miller, 1986; Miller, 1996). Another goal of the present work was to determine whether prenatal ethanol exposure alters the neuroanatomy of limbic cortex by testing the hypothesis that the number of GABAergic interneurons expressing PA are altered in the cingulate cortex following prenatal ethanol exposure. Although little is known about the cingulate cortex, it is clear that the cingulate is a relay center of the limbic lobe and is important for emotion and memory (Kupfermann, 1991 ). PA expression in the cingulate cortex begins during the first postnatal week in rats and coincides with the functional maturation of cerebral interneurons ( de-Lecea et al., 1995) The cingulate cortex was chosen to extend the earlier observations in the cerebral cortex following prenatal ethanol exposure because of the observed behavioral problems in children with FAS, including poor judgment distractibility and difficulty perceiving social cues (Streissguth et al. 1991 ). Additionally many alcoholics who develop Korsakoff s syndrome have deficiencies in glucose utilization within the cingulate cortex potentially contributing to learning and memory defects due to interruption of Papez circuitry (Joyce et al. 1994). It is conceivable that alterations in PA expression patterns might contribute to behavioral anomalies and/or learning and memory deficiencies Postnatal Ethanol Exposure and Neuroanatomical Alterations SH system The SH system of basal forebrain afferents and hippocampal targets also exhibits susceptibility to ethanol during neonatal development. While numerous studies have described the effects of neonatal ethanol exposure on neurons within the hippocampus

PAGE 19

9 (Bonthius and West 1990 ; Bonthius and West 1991; Greene et al. 1992; Pierce and West 1987 ; West and Pierce 1986) and have documented deficits in spatial learning following neonatal ethanol exposure (Goodlett and Peterson, 1995; Kelly et al., 1988) the effect of ethanol exposure during the brain growth spurt on the cholinergic neurons of the medial septum is unknown. A previous study from our laboratory documented an ethanol-induced delay in the normal ontogeny of ChA T enzyme activity in the SH pathway, but did not reveal an effect on ChAT-imrnunoreactive neuronal number following chronic prenatal ethanol treatment (Swanson et al. 1995 ; Swanson et al. 1996). It was a goal of the present work to determine the long-term effects of ethanol exposure on the number of ChA T + neurons in the rat MS when ethanol is delivered during the brain growth spurt to determine whether this neuronal population is sensitive to neonatal ethanol exposure. Cerebellum Evidence of cerebellar vulnerability to developmental ethanol exposure comes from human studies demonstrating size reduction in the cerebellar vermis of children exposed prenatally to ethanol (Sowell et al. 1996). In rodent studies the cerebellum displays a pattern of differential temporal susceptibility to ethanol in the brain growth spurt. This effect has been demonstrated in multiple laboratories utilizing a variety of ethanol-exposure techniques. Purkinje cell number is reduced following exposure to ethanol postnatally durin g differentiation but not following exposure to ethanol prenatally during neurogenesis (Marcussen et al. 1994). Within the postnatal period Purkinje cells have been shown to b e particularly vuln e rable to ethanol in the first postnatal week (Bauer-Moffett and Altman 1977 ; Bonthius and West 1991 ; Goodlett

PAGE 20

10 and Eilers, 1997; Pauli et al., 1995; Pierce et al., 1993). Purkinje cells are generated in the rat cerebellum between embryonic day 14 and 17 ; the period of Purkinje cell death in the cerebellum begins late in gestation and peaks in the first postnatal week (Cragg and Phillips 1985). Purkinje cell susceptibility to ethanol within the cell death period has been well documented with ethanol-accelerated Purkinje cell loss found as early as 12 hours following a postnatal day 3 ethanol insult (Cragg and Phillips 1985) Exposure to comparable levels of ethanol in the second postnatal week, however, has been shown to have no effect on Purkinje cells (Goodlett et al., 1997; Hamre and West, 1993; Pauli et al. 1995) or very little effect (Thomas et al., 1998). In contrast to Purkinje cells, granule cells are generated during the rat brain growth spurt (Altman, 1969) However, like Purkinje cells, granule cells display a differential pattern of susceptibility to ethanol, with loss occurring following ethanol exposure in the first postnatal week but not in the second postnatal week (Hamre and West, 1993). One aim of the present work was to determine the pattern of cerebellar neuronal susceptibility in our laboratory following first and second postnatal ethanol treatment, in order to validate the model system for use in our laboratory The main objective, however, was to determine whether neonatal ethanol treatment altered mRNA levels of members of the bcl-2 family of cell death regulators in the cerebellar vermis (and thereby influenced the survival or death of neurons in this region). Experiments were performed in order to test the specific hypothesis that altered bcl-2 family gene expression ensues following ethanol exposure. A role in maintaining cerebellar neurons has previously been demonstrated for the bcl-2 family. Gillardon et al. (1995) investigated bcl-2 and bax gene expression in the

PAGE 21

11 cerebella of Purkinje-cell-degeneration mice ( mutants that lose nearly all of their Purkinje cells between P22-28 following otherwise normal development) They found that bcl-2 mRNA levels decreased while bax mRNA levels remained unchanged beginning on P22. In addition thyroid hormone-induced upregulation of bcl-2 protects early-differentiating cerebellar granule cells from apoptosis in vitro (Muller et al. 1995) and transgenic mice overexpressing bcl2 contain more cerebellar Purkinje and granule cells than controls (Zanjani et al. 1996 ; Zanjani et al. 1997). The following section provides background on the bcl-2 family and its involvement in cell death regulation Programmed Cell Death (PCD) and the bcl-2 Family PCD a developmental form of apoptotic cell death is a common process in the animal kingdom (Ellis et al. 1991 ). In the vertebrate nervous system the regulation of neuronal survival is essential for the correct formation of synapses and for the survival of the appropriate number of neurons (Oppenheim 1991 ). Only the most appropriate connections are maintained making axon-target interactions maximally efficient and simultaneously ensuring that cells which are generated in excess develop poorly are functionally inadequate or are harmful do not endure in adult organisms (Ellis et al. 1991) Competition for a limited supply of target-derived neurotrophic factors is thought to determine which neurons survive the period of naturally occurring PCD (Davies 1994) The mechanism b y which non-essential neurons are eliminated is consistent with the apoptotic form of cell death (Johnson and Deckwerth 1993) with morphological characteristics consisting of chromatin cond e nsation cell shrinkag e, cleavage of DNA into oli g onucleo s omal fra g ments (E dward s e t al. 1991 ) and phagocytosis of dead cells

PAGE 22

12 (Ellis et al. 1991) without induction of the inflammatory response (Columbano 1995). Most mammalian cells constituitivly express the proteins essential for the cell death program (Davies 1995 ; Raff et al. 1993) Cytotoxicity due to ethanol in vitro has been shown to be apoptotic in fetal hypothalamic neurons (De et al 1994) and in thymocytes (Ewald and Shao 1993). Additionally ethanol-induced cell death in the cerebellum appears to proceed through an apoptotic mechanism. Cerebellar neurons undergo apoptosis in vitro and in vivo in response to ethanol (Bhave and Hoffman 1997 ; Liesi 1997; Renis et al. 1996 ; Singh et al. 1995) and ethanol induces nuclear DNA strand breaks in the cerebellum after chronic adult exposure (Renis et al. 1996). Almost a decade ago a gene bcl-2 was discovered that appeared to modulate apoptosis The name bcl-2 is an acronym for B-cell lymphoma/leukernia-2 gene (Reed 1994) and the identification of this gene family resulted from studies examining the t(l 4 ; 18) chromosomal trans location in human follicular non-Hodgkin s B-cell lymphomas (Tsujirnoto et al. 1985) The protein product encoded by the bcl-2 gene Bcl2 is a 25 kDa protein found predominately in mitochondrial membranes (Hackenbery et al. 1990) but it is also found in endoplasmic reticula and outer nuclear membranes (Akao et al. 1994). In recent years a number of new genes similar to bcl-2 have been characterized and added to the diverse bcl-2 family of genes. These include bcl-xl (Boise et al. 1993) bcl-xs (Bois e et al. 1993) bax (Oltvai et al. 1993) bad (Yang et al. 1995) al (Lin et al. 1993) mcl-1 (Kozopas et al. 1993) bak (Chittenden et al. 1995) bcl-w (Gib s on e t al., 1996), bra g-1 ( Da s et al. 1996) bok (Hsu et al. 1997) and bim (O connor et al. 1998 ) Some member s o f the bcl2 g ene family serve to inhibit cell death (e .g. bcl-

PAGE 23

13 2 bcl-xl mcl-1 al) and others have been found to promote cell death (e.g. bcl-xs bax bad bak bok). Bcl-2 (and the similar anti-apoptotic protein Bcl-xl) functionally blocks apoptotic death in neurons (Allsopp et al., 1993; Garcia et al. 1992) by inhibiting caspase activation (Shimizu et al., 1996) regulating mitochondrial membrane potential proton flux across mitochondrial membranes (Shimizu et al. 1998) and by preserving mitochondrial outer membrane integrity (Vanderheiden et al. 1997). The pro-apoptotic molecules of the bcl-2 family (s uch as the proteins Bad and Bak) function by inhibiting the ability of the anti-apoptotic molecules of the bcl-2 family to function. They do so by preventing the necessary homodimerization of the protective molecules through direct competition for binding to the ligand binding regions of these proteins (Ottilie et al. 1997) The intracellular mechanisms of bcl-2 family function are described in detail below Expression patterns Given the identification of these molecules and the fact that apoptosis is known to occur in the developing nervous system the possibility that bcl-2 family members and their protein products might modulate PCD in the nervous system has been explored. Castren et al (1994) have shown that bcl-2 mRNA is expressed in high levels in the prenatal rat neuroepithelium and cortical plate with a late-prenatal peak and expression reaching lower adult levels durin g postnatal development. In situ hybridization also revealed that bcl2 expression is retained in the olfactory bulb hippocampus pons cerebellum and ependymal cells of the adult rat brain (Castren et al. 1994 ). Bcl-2 protein is also widely expressed in the developing nervous system o f mice rhesus

PAGE 24

14 monkeys and humans especially during embryonic development but also during the period of PCD (Merry et al. 1994) Neuroepithelial cells of the ventricular zone postmitotic cells of the cortical plate Purkinje and granule cells of the cerebellum hippocampus and spinal cord all express Bcl-2 (Gleichmann et al. 1998; Merry et al. 1994). Expression of bcl-xl follows a simi l ar pattern with mRNA levels increasing at the beginning of the PCD period. In the brain in contrast to Bcl 2, Bcl-xl expression increases after birth in neurons of the cortex and olfactory bulb as well as in Purkinje cells to reach a high level in the adult brain suggesting a role for Bcl-xl in the adult CNS (Frankowski et al. 1995 ; Sohma et al. 1996) Although previous reports have suggested that bcl-xs is not expressed in the rat CNS newer studies using more sensitive techniques have detected bcl xs mRNA in the adult rat brain (Dixon et al. 1997 ; Rouayrenc et al. 1995). Although the protein product of bcl-xs is able to weakly bind Bcl-2 and Bcl-xl it appears that Bcl-xs affects apoptosis by a distinct mechanism that unlike other family member s, does not involv e direct protein interactions with cell death repressor proteins (Minn et al 1996) Both long and short forms of bcl-x are also found in cerebellar granule cells during development (Gleichmann et al. 1998). bax is a g ene in th e bcl-2 family whose protein product Bax is a 21 kDa protein with amino acid homology (21%) with Bcl -2 (Oltvai et al. 1993). Bax forms heterodimers with Bcl-2 Bcl-xl Mel-I and A 1 (Sedlak et al. 1995) and Bax can counteract the prot e ctiv e actions of Bcl-2 when overexpressed (Oltvai et al. 1993) It has b ee n su gge sted th at the ratio of Bcl2 to B ax may determin e survival or death from apoptosis by servin g as a s ort of rheostat : when Bcl2 is in excess cells survive but

PAGE 25

15 when Bax is in excess, cells die (Oltvai et al., 1993). bax mRNA has been found in the developing and adult rat brain. Similarly in the adult mouse, the Purkinje and granule cells of the cerebellum, cerebral cortical neurons, and sympathetic neurons all express Bax protein (Gleichmann et al. 1998; Krajewski et al., 1994; Oltvai et al., 1993; Vekrellis et al. 1997). While Bax protein expression is high in the neonatal cerebral cortex and cerebellum protein levels drop off dramatically after the PCD period, suggesting that neurons regulate their sensitivity to apoptosis during development by regulating expression of Bax (V ekrellis et al., 1997). Consistent with the rheostat hypothesis the ratio of Bcl-2:Bax correlates with cell survival in the mature rat hippocampus following global ischernia. bax mRNA and protein are both constitutively expressed in the ischemia-sensitive CAI hippocampal neurons whereas Bcl-2 is not expressed in these neurons (Chen et al., 1996). In hippocampal region CA3 a population more resistant to ischemia than cells in region CAI, Bcl-2 protein expression, but not that of Bax, is high (Chen et al. 1996). Furthermore high levels of Bax and concomitant low levels of Bcl-2 have been found in other populations of neurons that are sensitive to cell death induced by ischernia, such as Purkinje cells (Krajewski et al. 1995). Additional evidence supporting the bcl-2 rheostat hypothesis comes from studies in mouse brain that have demonstrated up-regulation of Bax and down-regulation of Bcl-2 associated with kainate-induced apoptosis (Gillardon et al. 1995) as well as studies from rat that have shown a decrease in the Bcl-2:Bax ratio in motoneurons following sciatic nerve transection (Gillardon et al. 1996) and ischernia (Isenmann et al. 1998) Additional evidence from Dixon et al ( 1997) shows that the pro apoptotic bcl-xs mRNA is upregulated shortly after global ischernia in rats Thus, it is

PAGE 26

16 clear that the balance between proand anti-apoptotic molecules (at the protein and mRNA levels) during development and following injury in adulthood determines the survival of the cell. Genetically engineered mice Studies using genetically engineered mice have been fruitful in describing a role for various members of the bcl-2 gene family in vivo. In transgenic mice overexpressing Bcl-2 protein in the nervous system a reduction in developmental cell death of facial motoneurons and retinal ganglion cells and a general hypertrophy of the nervous system are observed (Martinou et al. 1994). Neurons from these animals are more resistant to ischemia neurotrophic factor withdrawal and axotomy (Dubois-Dauphin et al. 1994 ; Farlie et al. 1995). Similarly facial motoneurons from mice overexpressing bcl-xl are resistant to axotom y during the postnatal period indicating a role for the bcl-xl gene in the survi v al of postnatal CNS neurons (Parsadanian et al., 1998). Studies using genetically engineered mice lacking the bcl-2 gene have been similarly instrumental in defining the normal actions of Bcl-2 in vivo. bcl-2 / mice live through gestation display massive apoptosis in lymphoid organs and produce gray hair follicles (V eis et al. 1993). The brains of these animals however appear grossly normal at birth although fine analyses of specific neuronal populations have not been performed The lack of massive cell death in neonatal bcl2 knockouts has been attributed to redundancy as the bcl-2 family is so large (Motoyama et al. 1995). However analysis of neuronal populations after the PCD period reveal loss relative to controls of motoneurons sympathetic and s e nsory n e urons d e mon s tratin g a rol e for bcl -2 in m a intaining these n e uronal population s (Michaelidis et al. 1996).

PAGE 27

17 Mice lacking the bcl-x gene have also been generated, but die around E13. Upon examination extensive apoptotic cell death is evident in neurons of the brain, spinal cord, and dorsal root ganglion (Motoyarna et al. 1995) revealing the importance of bcl-x in embryonic life. bax-deficient mice have been generated and have helped define the actions of cell-death promoting molecules in particular neuronal populations (Deckwerth et al. 1996). Early postnatal sympathetic and facial motoneurons from bax knockouts survive growth factor deprivation and axotomy Additionally superior cervical ganglia and facial nuclei of bax knockouts possess more surviving neurons in vivo. Thus a role for bax has been demonstrated in cell death associated with growth factor deprivation and axotomy (Deckwerth et al. 1996). Shindler et al. (1997) have generated mice deficient in both bcl-xl and bax. While bax deficiency does not prevent the embryonic lethality of bcl-xl deficient mice the double knockout did demonstrate the interplay between these proand anti-apoptotic molecules in vivo. Specifically bax and bcl-xl deletion produced less apoptosis in the brainstem and spinal cord when compared with bcl-xl knockouts. It is important to note that while studies using genetically engineered mice are interesting and often quite suggestive, they suffer from several caveats. For instance overexpression of a particular gene or the creation of a mutant lacking a particular gene may cause compensatory upor downregulation of other gene products which can lead to developmental alterations in attempts to compensate for the altered gene (Gerlai 1996). Moreover mutant mice may differ in non-targeted gene loci and certain behavioral alterations may be due to differences in genetic background (Gerlai 1996). Thus while suggestive of the role of particular genes studies utilizing genetically engin e er e d mic e must b e analyz e d in light of these caveats.

PAGE 28

18 Intracellular mechanisms Recent evidence has shed light on the intracellular mechanism by which bcl-2 family members operate and has revealed a "double identity for the family as both ion channel and adapter protein (Reed 1997). Bcl-xl Bcl-2, and Bax have all been shown to form functional ion channels in lipid membranes (Antonsson et al. 1997 ; Minn et al. 1997; Schendel et al. 1997). Also Bax channel-forming activity is inhibited by Bcl-2 (Antonsson et al. 1997) These ion channels appear to influence cell survival by regulating the permeability of the intracellular membranes in which they are anchored. In particular the Bcl-2 and Bcl-xl membrane complexes inhibit the release of cytochrome C from mitochondria (Yang et al. 1997) regulate mitochondrial membrane potential and proton flux across mitochondrial membranes (Shimizu et al., 1998), and help preserve mitochondrial outer membrane integrity (Vanderheiden et al. 1997). Bax channels in contrast are known to promote the release of cytochrome C from mitochondria (Rosse et al. 1998) Cytochrome C is a known activator of the PCD effector molecules, the caspases (Yang et al. 1997). Permeability transition across mitochondrial membranes appears to be an early event in PCD and is related to the activation of the release of cytochrome C and the resultant activation of the effector phase of PCD (Petit et al. 1996). This activation is brought about by cytochrome C-dependent cleavage and activation of caspases 3 (Li et al. 1997) and 9 (Zou et al. 1997). In keeping with the adapter protein role of the anti-apoptotic molecules recent evidence shows that Bcl-xl binds to Apaf-1 the newly discov e red mammalian homolog of the C elegans CED-4 (Zou et al. 1997) and these two proteins along with the uncleaved and inactive caspase 9 exist in a ternary

PAGE 29

19 complex (Pan et al. 1998). Cytochrome C release is thought to promote activation of the caspase cascade by promoting the dissociation of this ternary complex through an undefined mechanism. Once caspases are activated, they act upon a variety of intracellular substrates including inhibitors of caspase-activated deoxyribonuclease (ICAD). Caspase 3 is known to cleave ICAD and result in the elimination of its normally inactivating effect on caspase-activated deoxyribonuclease (CAD). CAD functions downstream in the proteolysis cascade and its reduced inhibition results in DNA degradation characteristic of apoptotic cell death (Sakahira et al., 1998). Another piece to this intriguing puzzle has been provided by Yang et al. ( 1998) who have shown that CED-4 (and likely the mammalian counterpart Apaf-1) promotes CED-3 (the C. elegans caspase) processing and activation by promoting the aggregation of unprocessed CED-3. This induced proximity" is thought to sequester inactive caspase proenzyrnes to increase their local concentration and promote conformational changes which will increase the likelihood of their activation (Hengartner 1998) and is brought about by oligomerization of the CED-4:CED-3 complex (Yang et al., 1998). Thus an important role of Bel-xi and Bcl-2 appears to be to prevent the association of these proenzymes by preventing CED-4 (and by association, Apaf-1) oligomerization (Hengartner 1998). While a similar role for Apaf-1 has not been definitively demonstrated this will surely be one of the important future discoveries in this blossoming field. Newly uncovered evidence also indicates that the anti-apoptotic molecules of the bcl-2 family can contribute to the cell s demise under certain intracellular conditions For example if caspase activation proceeds to a critical point caspases will act on the Bcl-2

PAGE 30

20 and Bcl-xl proteins as substrates (Cheng et al. 1997 ; Clem et al. 1998). Indeed caspase cleavage of these protective molecules converts them into Bax-like death-promoting molecules. This cleavage is thought to act as a feed-forward mechanism for further caspase activation and should ensure cell death. Besides these important discoveries several others have demonstrated that apoptosis interacts with signal transduction intracellularly and that this interaction is mediated in part by the bcl-2 family (Gajewski and Thompson 1996) The application ofIL-3 to the FL5 12 cell line leads to phosphorylation and inactivation of the pro apoptotic molecule Bad. The phosphorylation of Bad leads to its association with the cytosolic protein 14-3-3 prevents Bad:Bcl-xl heterodimerization and promotes Bcl-xl homodimerization which results in cell survival (Zha et al. 1996). Additionally Wang et al. (1996) showed that Bcl-2 could target Raf-1 to mitochondrial membranes which results in the phosphorylation of Bad Two groups independently determined that the protein kinase Akt is also responsible for growth factor-mediated Bad phosphorylation (Datta et al. 1997 ; Delpeso et al. 1997) Akt (also known as protein kinase B or PKB) is an important intracellular molecule and its phosphorylation (and activation) is brought about by growth factor mediated phosphorylation of PB kinase followed by PB kinase phosphorylation of Akt (Kahn 1998 ; Zhou et al. 1997). Thus it is apparent that a complex interaction between extracellular growth and survival signals integrates with the complex intracellular biology of individual neurons to determine whether a particular cell will survive a developmental process or mJury.

PAGE 31

21 Hypotheses Tested As mentioned previously neuronal populations are not uniform in their vulnerability to ethanol nor are developmental stages uniform in susceptibility to ethanol treatment. And while a variety of CNS populations have been demonstrated to be adversely affected by prenatal ethanol exposure the full extent of nervous system vulnerability to ethanol is unknown Likewise the cellular effects of ethanol exposure such as changes in gene expression are poorly understood It is with this in mind that the research reported in this document was undertaken Because of the observed learning and memory deficits in children exposed in utero to ethanol (Streissguth et al., 1991) the SH system is a natural region in which to extend studies in rodents of neuroanatomy following ethanol treatment. While cell loss in the hippocampus has been documented following prenatal exposure (Barnes and Walker 1981 ) cellular changes in the cholinergic basal forebrain component of the SH system have not been found after a similar pattern of exposure (Swanson et al., 1996) Thus it was an aim of the present work to determine whether the GABAergic component of the SH pathway is susceptible to prenatal ethanol treatment. Additionally the cingulate cortex was analyzed because of the observed behavioral problems in children with FAS (Streissguth et al., 1991) and the decreased glucose utilization in the cingulate cortex of alcoholics (Joyce et al. 1994) Thus the following hypothesis was explo red : chronic prenatal ethanol exposure will l ead to alterations in the number of neurons expressing PA in the adult rat medial septum and cingulate cortex.

PAGE 32

22 Rat studies have also identified the early postnatal period as a developmental time during which particular CNS structures are sensitive to ethanol's toxic effects. For example, the Purkinje cells of the cerebellar cortex are reduced in number when ethanol is delivered during the early neonatal period, but not when ethanol is given in utero (Marcussen et al., 1994). The SH system is a region which exhibits susceptibility to ethanol during both the prenatal and the neonatal periods. Neonatal ethanol exposure disrupts the normal development of the SH system in rodent models of FAS, and much research has documented neuroanatomical changes in the hippocampus (Bonthius and West, 1990; Bonthius and West, 1991; Greene et al., 1992 ; Pierce and West, 1987; West and Pierce 1986). However the effect of neonatal ethanol exposure on the MS has not been investigated Because the number of cholinergic neurons of the medial septum are not altered by prenatal ethanol treatment, it was an objective of the present work to determine the long-term effects of neonatal ethanol exposure on the cholinergic neurons in the rat. The following hypothesis was investigated : early postnatal ethanol exposure will lead to alterations in the number of neurons expressing ChAT in the adult rat CNS While descriptions of susceptible neuronal populations following developmental ethanol exposure has been and continues to be, a fruitful avenue of research further investigation into the molecular consequences of ethanol treatment ( e.g. gene expression changes resulting from ethanol) are warranted. Given the previously described temporal pattern of Purkinje and granule cell loss in the postnatal cerebellum a survey of the bcl-2 literature raises the intriguing possibility that the differential temporal teratogenicity of ethanol on cerebellar cells may be related to changes in the levels of expression of PCD repressor and inducer genes It is significant that proteins translated from bcl-2 and bax

PAGE 33

23 mRNA dimerize and as noted above, cell death or survival depends on the relative amounts (ratio) of these proteins (Oltvai et al., 1993). Similar cellular survival outcomes have been linked to the relative ratio of bcl-2 to bax gene expression (Basile et al., 1997; Chen et al., 1996). The observation that cerebellar neurons are susceptible to ethanol neurotoxicity as a function of the timing of the ethanol insult suggests that ethanol may act in vivo to modulate (upregulate or downregulate) the expression of certain mRNAs of the bcl-2 family and thereby alter susceptibility to ethanol neurotoxicity. Thus, any ethanol induced change in bcl-2 family gene expression might disrupt the normal balance of these proteins in developing neurons and decrease or increase their chance of cell death. Recent evidence mentioned above, in addition to the aforementioned expression patterns implicates bcl-2 family members in maintaining cerebellar neurons. Purkinje-cell degeneration mouse mutants lose nearly all of their Purkinje cells between P22-28. Gillardon et al. (1995) investigated bcl-2 and bax expression in the cerebella of these mice and found that bcl-2 mRNA levels decreased while bax mRNA levels remained unchanged beginning on P22. There was a concomitant reduction of Bcl-2 expressing Purkinje cells in the mutants compared with wild-types suggesting a down-regulation of bcl-2 in Purkinje cells destined to die (Gillardon et al. 1995). In addition, thyroid hormone-induced upregulation of bcl-2 protects early differentiating cerebellar granule cells from apoptosis in vitro (Muller et al. 1995) Moreover transgenic mice overexpressing bcl-2 contain more cerebellar Purkinje and granule cells than controls whether transgene expression was induced in the embryonic or postnatal periods (Zanjani et al. 1996 ; Zanjani et al. 1997). Therefore, another goal of

PAGE 34

24 the present work was to test following hypothesis : ethanol-induced alterations in the expression levels of bcl-2 family PCD genes in the cerebellum contribute to the cerebellum s relative temporal susceptibility to ethanol neurotoxicity The chapters that follow provide a detailed description of methods utilized to test these hypotheses and the data collected are presented and fully discussed. Following these chapters is a concluding chapter that presents interpretations derived from these data and discusses specific methodological considerations and future directions which the current data suggest.

PAGE 35

CHAPTER2 EFFECTS OF PRENATAL ETHANOL EXPOSURE ON PARVALBUMIN IMMUNOREACTIVE GABAERGIC NEURONS IN THE ADULT RAT MEDIAL SEPTUM AND ANTERIOR CINGULATE CORTEX Summary Exposure of human fetuses to ethanol often results in the fetal alcohol syndrome (FAS). Animal models of FAS have been developed and have been utilized to examine the consequences of prenatal ethanol exposure on the central nervous system. While cell loss in the hippocampus has been documented following prenatal ethanol exposure (Barnes and Walker 1981 ) cellular changes in the cholinergic basal forebrain component of the septohippocampal (SH) system have not been found after a similar pattern of ethanol exposure (Swanson et al. 1996) The objective of this study was to determine the long-term effects of prenatal ethanol exposure on parvalbumin-expressing (PA+) GABAergic neurons of the rat medial septum and anterior cingulate cortex. Pregnant Long-Evans rats were maintained on one of three diets throughout gestation: an ethanol-containing liquid diet in which ethanol accounted for 35% of the total calories a similar diet with the isocaloric substitution of sucrose for ethanol or a lab chow control diet. Offspring were sacrificed at postnatal-day 60 and their brains were prepared for parvalburnin immunocytochemistry. Female rats exposed to the ethanol containing diet during gestation had 42 % fewer total PA+ neurons in the medial septum and reduced PA + cell density when compared to female rats exposed to the sucrose diet. 25

PAGE 36

26 Ethanol-exposed females also had fewer PA+ neurons per section than sucrose-control females. Male rats exposed to ethanol did not display a similar change in PA + neurons or density No effect of prenatal diet was found on the area or volume of the medial septum or the size of the PA + neurons therein. As such prenatal exposure to ethanol appears to permanently reduce the number of PA+ neurons in the female rat medial septum without affecting the size of the structure or the size of the neurons Due to the observed behavioral problems in children with FAS (Streissguth et al. 1991) and the fact that alcoholics show decreased glucose utilization in the cingulate cortex ( Joyce et al. 1994) the effect of prenatal ethanol exposure on the number of PA + GABAergic neurons in the adult rat anterior cingulate cortex was also determined. This represents the first attempt to document developmental alterations in the cingulate cortex following ethanol exposure. Rats exposed to the ethanol-containing diet contained 45% fewer total PA + neurons in the anterior cingulate cortex and fewer PA + neurons per section compared with sucrose and chow controls. No gender differences were found in measures of the anterior cingulate The reduction in PA + neurons occurred in the absence of changes in structure area or volume and occurred in the absence of changes in PA + neuronal size Functional implications and possible relations to the fetal alcohol syndrome are discussed Introduction Exposure of human fetuses to ethanol often results in a constellation of developmental anomalies which make up FAS Such developmental perturbations include preand postnatal growth deficiencies morphological ( e .g. craniofacial)

PAGE 37

27 abnormalities and central nervous system (CNS) deficits (Abel, 1995) and these typically persist into adulthood (Streissguth et al., 1991 ). The incidence of FAS in the general obstetric population of the United States is estimated at 1.95 per 1,000 live births (Abel and Hannigan 1995) and FAS is thought to be the leading nongenetic cause of mental retardation (Abel and Sokol, 1986). Experimental studies utilizing rodent models of FAS, have been successful in reproducing many of the behavioral and morphological changes found in FAS, and have described numerous CNS alterations resulting from in utero ethanol exposure. Alterations in CNS development include changes in neuronal proliferation and migration (Miller 1986 ; Miller 1995b; Miller, 1996), altered neuronal and cortical morphology (Burrows et al., 1995; Davies and Smith 1981; Kotkoskie and Norton 1989), changes in receptor density and enzyme ontogeny (Black et al. 1995; Swanson et al., 1995) delays in synaptogenesis (Hoff, 1988), alterations in neuronal number (Barnes and Walker 1981) and long-lasting deficits in learning (Clausing et al. 1995) and behavior (Riley 1990). The present study sought to determine the vulnerability of GABAergic intemeurons expressing parvalburnin in the medial septum and anterior cingulate cortex following prenatal ethanol exposure. Medial Septum Numerous studies investigating the effects of prenatal ethanol exposure on rodents have determined that the CNS is not uniform in its susceptibility to ethanol. In addition to differential temporal susceptibility, the CNS exhibits differential regional vulnerability. The SH system of basal forebrain afferents and hippocampal targets is one region that has been shown to be sensitive to developmental ethanol exposure. Permanent reductions of CAI pyramidal neurons (Barnes and Walker, 1981; Wigal and Amsel 1990) alterations

PAGE 38

28 in hippocampal neuronal circuitry (West et al. 1981) and dendritic arborization (Davies and Smith 1981 ; Smith and Davies 1990) delayed synaptogenesis in the hippocampal dentate gyrus (Hoff 1988) changes in hippocampal muscarinic receptors (Black et al. 1995) and delayed choline acetyltransferase (ChA T) enzyme ontogeny in the basal forebrain (Swanson et al., 1995) have all been demonstrated in the rodent SH system following exposure to ethanol in utero Because of the SH system s role in generating and maintaining electrical activity in the hippocampus (Dutar et al., 1995) and the importance of the hippocampus in learning and memory, the possibility exists that ethanol-induced changes in this system can have detrimental effects on offspring exposed to ethanol prenatally Intemeurons pyramidal neurons, and granule cells of the hippocampus all receive synapses from cholinergic and GABAergic fibers originating in the medial septum (MS) and the horizontal and vertical limbs of the nucleus of the diagonal band of Broca (DBB) (Dutar et al. 1995; Freund and Antal, 1988) Evidence exists for an ethanol induced alteration of the cholinergic component of the SH pathway Sulik et al. (1984) for example noted severe abnormalities in the basal forebrain of fetal mice following an acute ethanol dose at gestational day 7 (G7). Moreover Schambra et al. (1990) found a reduction in the number of ChAT-immunoreactive neurons in fetal mice following an acute ethanol administration on G7. Similar findings in adult rats were reported following chronic ethanol treatment (Arendt et al. 1988) In contrast studies from our laboratory utilizing a rat model of chronic prenatal ethanol treatment (CPET) have shown an ethanol-induced disruption of the normal ChA T ontogen y in the SH pathway but have not revealed an effect on ChAT-immunoreactive

PAGE 39

29 neuronal number (Swanson et al. 1995; Swanson et al., 1996). The present study sought to determine whether the GABAergic component of the SH pathway was susceptible to CPET. Although not as extensively characterized as the cholinergic component the GABAergic component of the SH projection is an important part of this system. Parvalburnin (PA) an 11.8 kDa member of a Ca2+ binding superfamily of proteins (Kiss et al. 1990a; Kiss et al. 1990b; McPhalen et al., 1994), is expressed in approximately 33% of the neurons in the MS/DBB region. PA is commonly found in fast-firing GABAergic neurons where it influences the activity of Ca2+ -dependent K + channels (Plogmann and Celio 1993). PA ontogeny begins in the MS of rats at G21 and coincides with the beginning of physiological activity such as spontaneous firing and excitatory synaptic input (Lauder et al. 1986; Solbach and Celio 1991), and the PA-expressing MS neurons innervate inhibitory interneurons in the hippocampus (Freund and Antal 1988) CNS regions differ in the extent to which PA and GABA co-localize (Alonso et al. 1990; Brauer et al. 1991; Kiss et al., 1990a), but within the MS nucleus most if not all of the hippocampal-projecting GABAergic neurons are PA+ (Freund, 1989; Krzywkowski et al ., 1995) The present study sought to determine the long-term effects of prenatal exposure to ethanol on a PA + subpopulation of GABAergic projection neurons in the SH pathway For this determination we performed counts of PA + neurons in the adult rat MS nucleus following CPET Cingulate Cortex The present study also sought to determine the vulnerability of GABAergic interneurons expressing parvalburnin in the anterior cingulate cortex following CPET

PAGE 40

30 The ontogeny of PA mRNA and protein in the rat cingulate cortex and its functional role is similar to that noted previously for the medial septum. PA expression begins in the first postnatal week and coincides with the functional maturation of cerebral intemeurons in the cingulate (Alcantara et al. 1993; de-Lecea et al. 1995). The cingulate cortex was chosen for analysis because it is a major relay center of the limbic lobe and is involved in motor control attention emotion and memory (Kupfermann 1991 ; Muir et al. 1996 ; Paus et al., 1993; Picard and Strick 1996). The well documented cognitive and behavioral impairments in children with FAS including poor judgment, distractibility and hyperkinetic and emotional disorders (Steinhausen et al. 1993 ; Streissguth et al. 1991 ) led us to investigate whether anatomical alterations in the cingulate cortex may underlie these behavioral defects Thus the current study also sought to determine the long-term effects of prenatal exposure to ethanol on PA+ intemeurons of the cingulate cortex Materials and Methods Subjects and ethanol treatment Long-Evans hooded rats purchased from Charles River Co. (Portage MI) were housed individually or in pairs under controlled temperature and humidity conditions and were maintained on a 07:00-19:00 hour light cycle Nulliparous females were placed individually with a male overnight until vaginal smear the following morning was indicative of insemination This was defined as GO. At this time females were matched according to age and weight and assigned to one of three treatment groups from G0-O21: ethanol sucrose or chow. During this time the ethanol group was given free access to an

PAGE 41

31 ethanol-containing liquid diet in which ethanol comprised 35% of the total calories. The sucrose group was pair-fed the same volume of a similar liquid diet which lacked ethanol and contained an isocaloric substitution of sucrose for ethanol. The liquid diet was prepared by mixing a stock ethanol or sucrose solution with Sustacal (Mead Johnson). Diets were additionally enriched with Vitamin Diet Fortification Mixture and Salt Mixture (ICN Nutritional Biochemicals) The diets contained 1.3 kcal/ml and have been shown to provide several times the daily requirement of all essential vitamins and nutrients (Walker and Freund 1971) The chow group was given Purina Rodent Chow and water ad libitum and served as a control for non-specific effects of the liquid diet. Upon birth (postnatal-day O [PO]) pups from an ethanol or sucrose dam were fostered to chow dams which had given birth at the same time. Litters were then randomly culled to ten pups with approximately equal numbers of males and females. Pups were weaned at P21 and individually housed until perfusion at P60. In order to avoid litter bias animals used in this study are representatives from at least eight different litters and no more than one individual from each gender was used from a single litter. A total of 39 animals was used in this study with approximately equal numbers of males and females in each diet treatment group (Ethanol N =13; Sucrose N = 12; Chow, N = 14) Immunocytochemical procedures Animals were randomly selected for immunocytochemical staining at P60, an age which represents adulthood in rats. This age was selected in order to examine the long term effects of chronic prenatal ethanol treatment on parvalbumin expressing neurons in the medial septum and cingulate cortex and corresponds to an age used in previous analyses of prenatal ethanol influences on cholinergic neurons in the medial septum

PAGE 42

32 (Swanson et al., 1996). At P60 animals were euthanized by pentobarbital overdose prior to transcardial perfusion with phosphate buffered saline (PBS; 0.1 M, with 0.9% sodium chloride) followed by 10% formalin (in the same PBS). Brains were removed and equilibrated overnight in a cryoprotectant solution (PBS with 30% sucrose and 15% ethylene glycol) and frozen at 70C until processing. Animals of each gender from each diet group were processed for immunocytochemistry at a given time. This insured against staining differences between groups resulting from slight procedural differences. Brains were thawed and equilibrated in a 30% sucrose-PBS solution, and mounted on the frozen stage of a sliding microtome. Serial coronal sections were cut throughout the basal forebrain at a thickness of 40 m. Free-floating sections were irnmunostained for PA using a monoclonal antibody (Accurate # 6092). Immunoreactivity was visualized as a blue-black reaction product using an avidin-biotin conjugate / nickel intensified staining (see below) Primary incubation with the monoclonal PA antibody (1 :2500 in PBS 0.1 % normal goat serum [NGS], 0.1% Triton X-100 and 0.005% sodium azide) was carried out at 4C overnight. Sections were then washed with PBS and incubated with biotinylated anti-mouse lgG (Sigma, B0529 ; 1: 10000 in PBS 0.1 % NGS, 0.1 % Triton X100 and 0.005% sodium azide) overnight at 4C. Sections were then washed and incubated with Extravidin-horseradish peroxidase conjugate (Sigma ; 1: 1000 in PBS) overnight at 4C. Sections were washed with 0 1 M sodium acetate (pH 7.2) to eliminate phosphate which can precipitate divalent cations. Sections were then reacted for 3 minut es at room temperature using a developing buffer with 0.8 M sodium acetate 8 mM imidazole 0 .5% nickel (11) sulfate 0.04% 3,3 diaminobenzidine tetrahydrochloride and

PAGE 43

33 0 005% hydrogen peroxide. Following development, sections were again washed in sodium acetate buffer, mounted onto slides in PBS air dried, dehydrated and coverslipped. Control sections omitting the primary antibody were routinely developed to ensure that any observed staining was due to PA. Slides from animals of each gender from the three groups were randomized and coded such that all subsequent analyses were carried out blind. MS cell count, area, and volume analysis PA+ cell counts were conducted bilaterally on alternate sections throughout the entire rostral-caudal extent of the MS nucleus. The packing density of the DBB nucleus was too great for accurate cell counts to be made by the image software. Thus, for the purposes of this study the MS alone was examined and was defined rostrally by the ventral fusion of the hemispheres (at the level of the genu of the corpus callosum) and caudally by the decussation of the anterior commissure. The mean number of PA+ neurons per section was derived. Recent data have demonstrated the utility of manual cell counts (Clarke and Oppenheim 1995) and show a direct correlation between cell counts performed manually and cell counts performed with the optical dissector (Hagg et al. 1997). Images of PA immunostained sections were captured and digitized using a RasterOps 24STV video capture board and software on a Macintosh llvx computer. Low magnification images (2.5x objective; effective scale 1.59 m/pixel) were captured in order to obtain the entire MS region in one image. When capturing each image the lighting and contrast enhancement were optimized for identification of individual cells. Images were digitally processed using the image analysis program NIH Image (freeware

PAGE 44

34 from NIH). To reduce background variation across the image, a digitally defocused image was created by passing the primary image through a mean filter. The resultant image was then subtracted from the primary digitized image. This processed image was then passed through a Laplace filter to enhance edges and separation between cells Analysis of individual images was initiated by outlining the region of the MS nucleus The area of a section outlined varied depending on the rostral-caudal location of the individ u al section. The MS nucleus in rostral sections was defined as the mediodorsal group of neurons which were separated from the ventrally-located DBB nucleus In intermediate sections where the demarcation between the MS and DBB nuclei is ambiguous the MS was defined v entrally by a line perpendicular to midline at the l evel of the anterior commissure In caudal sections the MS was defined as the medially located cells dorsal to the anterior commissure. Cells were high l ighted interactively by adjusting the grayscale threshold level to include only objects which were considered cells A pre v ious stud y from our lab has demonstrated that there is a close correlation (r2= 0 956) betwe e n computer-automated and manual counts performed with a microscope and a drawing tube (Swanson et al., 1996) Given the thickness of each section (40 m) th e fact that alternate sections were analy z ed and the fact that PA+ neurons in the MS nucleu s range from 6-26 min diameter ( see below ), it was not deemed necessary to perform a split-cell correction on these counts. The program counted the highli g hted objects and measured the area of the outlined region (mm2 ) T issue volume ( mm3 ) was calculated using a modification of the Cavelieri method (Mich e l and C ruz 1988 ). T h ese measur e m e nt s w e r e tak e n in ord e r to determine wh e ther ethanol treatment chan g ed the siz e of the structure of interest, and whether changes in the

PAGE 45

35 number of PA+ neurons per section were due to concomitant changes in the size of the area e x amined (Peterson et al. 1997 ). This was done by multiplying the number of 40 m sections analy z ed for a given animal by the mean area per section for that animal and section thickness Anterior cingulate cell count, area, and volume analysis The packing density of PA+ neurons in the anterior cingulate cortex was too great for accurate cell counts to be made by automated imaging software at the magnification necessary to include the entire region. Therefore manual counts of PA+ neurons were performed on every si x th section through the left side from the genu of the corpus callosum caudally until decussation of the anterior commissure. The anterior cingulate gyrus was bordered laterally by the cingulum and ventrally by the corpus callosum (Paxinos and Watson 1982). The mean number of PA+ neurons per section was derived through manual counting of neurons at 400 x. Because our cell count data are expressed as the mean number of cells per section it is necessary to demonstrate that any noted change in cells per section is not due to changes in the volume of the structure being examined (Peterson et al. 1997) Therefore the volume of the anterior cingulate gyrus was calculated by a modification of the Cavaleri method (Mich e l and Cruz 1988) to ensure that changes in mean number of PA+ neuron s p e r section as a re s ult of ethanol treatment were not due to changes in the si z e of the gyrus. For the volume determination the mean area of the anterior cingulate gyrus was determined for each animal b y measuring the distance from midline to the cingulurn and th e di s tanc e from the c orpu s callosum to th e dor s al brain s urface at 2 5 x on thre e anatomically matched sections with an eyepiece micrometer. These distances were

PAGE 46

36 multiplied and mean area per section was calculated. This area was then multiplied by the number of sections through the anterior cingulate gyrus and the section thickness to determine mean cingulate gyrus volume Other analyses The blood ethanol concentration (BEC) in the ethanol group was determined between 00:00 and 02:00 hours on G18 utilizing the Sigma 333-UV kit. PA+ cell diameters were determined at 400x utilizing an eyepiece micrometer. Thirty cells from identical anatomical locations (in the respective structures) for each gender in all groups were measured. Density of PA+ neurons was determined by dividing the mean number of cells per section by the mean area (mm2 ) per section for the respective structures. Statistical analysis Statistical analysis was performed with the program StatView (Abacus Concepts Berkeley Ca) For each parameter examined one-way Analysis of Variance (ANOVA) was used to test for the main effects of diet and gender separately. When appropriate the data were furt);ler analyzed with the Fisher s Protected Least Significant Difference (PLSD) post hoc test to determine individual group differences. Because gender differences were noted for MS measures (following an initial two-way ANOV A for effects of diet and gender) males and females were analyzed separately. No gender differences were noted in cingulate measures (following an initial two-way ANOVA for effects of diet and gender) and so these data were not split by gender for analysis and one-way ANOV A was used to test for effects of diet followed by the PLSD when appropriate.

PAGE 47

37 Results BEC, body and brain weight, and brain to body weight ratio measures BECs in the ethanol group ranged from 150-175 mg/dl (mean= 161 18 mg/dl). This BEC represents a peak or near-peak level, as the nocturnal rats consumed the majority of the diet after the twelve hour light cycle and blood samples were taken between 00:00 and 02 : 00 hours on Gl8. P60 body weights were taken prior to perfusion and P60 brain weights were taken following perfusion and are presented in table 2-1, along with brain to body weight ratios (all tables are located at the end of the chapter). The ANOV A for P60 bod y weight revealed no significant effects of diet on P60 body weight of males or females. A gender difference was noted in P60 body weight for ethanol [F(l 11) = 24 7 ; p < 0.0001] sucrose [F(l 10) = 31.4; p< 0 0001] and chow [F(l 12) = 39.6 ; p < 0 0001] animals which was expected given the larger size of males at P60. The ANOV A for P60 brain weight demonstrated no effect of diet treatment in males or in females. Likewise no significant gender differences were noted for brain weight within ethanol sucrose or chow animals. The ANOVA for brain to body weight ratio showed no effect of diet in males or females. Gender differences were noted in P60 brain to body weight ratio for ethanol [F(l 11) = 37.5; p < 0.0001] sucrose [F(l 10) = 41.2 ; p < 0.0001], and chow [F(l 12) = 38.5; p < 0.0001] animals which was not surprising given the larger size of male animals at this age.

PAGE 48

38 Number of sections, MS area per section, and PA+ neuronal density in the adult rat medial septum Table 2-2 presents the number of sections, MS area per section and PA+ neuronal density of the P60 MS The number of alternate 40 m sections through the MS was determined by counting the number of sections analyzed between the ventral fusion of the hemispheres (at the level of the genu of the corpus callosum) and the anterior commissure The ANOV A showed no effect of diet for either males or females on the mean number of alternate sections through the MS nor were there gender differences for ethanol or chow animals The sucrose group however did show a gender effect on the mean number of sections with females containing significantly fewer than males (F[l, 10] = 4 511; p < 0.05). The area of the MS outlined on a given section was computed by the image software. No significant differences were noted between any group for males or females in mean area per section nor were there gender differences for ethanol sucrose or chow animals Density of PA+ neurons (mean number of cells per section/mean area [ mm2 ] per section) was also calculated and the ANOVA showed an effect of diet on cell density in females (F[2 16] = 7.347; p < 0 01) but not in males. The PLSD post-hoc test determined that cell density in ethanol females was significantly reduced compared to that of sucrose females (p < 0.01 ). Neuronal density in chow females was also significantly reduced compared to sucrose females (p < 0.05) The greater density of PA+ neurons noted in sucrose females is perhaps due to a non-specific effect of liquid diet (see below for discussion). A gender difference in the ethanol group was also noted for neuronal density: neuronal density of females was reduced from that of males (F[l, 11] = 5.075 ; p <

PAGE 49

39 0 05). This difference is best described by the observed reduction in total PA+ cell number and not by differences in MS area. No gender differences in neuronal density were noted for the sucrose or chow groups Total PA+ neurons in the MS Figure 2-1 ( all figures are located at the end of chapter) presents the mean total number of PA+ cells counted on alternate sections throughout the P60 MS for animals of both genders from each diet group. The ANOVA for total number of PA+ cells in the MS determined a significant effect of diet treatment in females (F[2 16] = 4 351 ; p< 0.05) but not in males The PLSD post-hoc test further revealed that ethanol females had 42 % fewer total PA+ cells than sucrose females (p< 0.01 ). A qualitative reduction in cell number in the MS of ethanol females as compared to sucrose females can be observed in Figure 2-2. Ethanol females were not different from chow females nor were chow females different from sucrose females. Gender differences were noted in the ethanol group and the ANOVA for total number of PA + cells in the MS indicated a reduction in the number of PA + cells in ethanol females compared to ethanol males (F[l, 11] = 8.421; p< 0.05). Gender differences were not noted in sucrose or chow groups. Number of PA + neurons per section in the MS Figure 2-3 displays the mean number of PA + neurons detected per section for animals of both genders from each group in the P60 MS. The ANOVA for mean number of cells per section re v eal e d a significant effect of diet treatment in females (F[2 16] = 7 342 ; p < 0 01) but not males The PLSD post-hoc test further determined that ethanol females had 44 % fewer PA+ cells per section than sucrose females (p < 0.01) Chow females also display e d a significant reduction in mean PA + cells per section compared

PAGE 50

40 with sucrose females (p< 0.05) while ethanol and chow females were not different from each other. The difference between ethanol fema l es and sucrose females is due to a reduction in total cell number and not to a difference in tissue volume. The difference noted between chow females and sucrose females is perhaps due to a non-specific effect of liquid diet and may be related to the aforementioned increase in PA+ neuronal density noted in sucrose females (see below for discussion). Gender differences were also noted. The ANOV A showed a reduced mean numbe r of P A+ cells per section in ethanol females compared to ethanol males (F[l 11] = 5.063; p< 0.05) This difference was again due to a reduction in total PA+ cell number and not to a difference in volume. No gender differences were noted for the sucrose or chow groups. Diameter of PA + MS neurons PA+ cell diameters were measured for both genders in all groups and are presented in figure 2-4. No differences were noted between any treatment group for males or females ; nor were gender differences noted for the ethanol sucrose or chow groups. MS volume determination Figure 2-5 presents MS volume (mm3 ) for the analyzed sections. Volume was calculated by multiplying the number of 40 m sections analyzed for a given animal by the mean area per section for each animal and the section thickness The resulting volume was then compared across groups for each gender and between both genders for each diet treatment. No significant differences were found between diet groups for males or females. Gender specific effects of tissue volume, however were noted in sucrose animals. The ANOV A showed a significant reduction of tissue volume in the sucrose

PAGE 51

41 females compared to sucrose males (F[l, 10]= 6.102 ; p< 0 05). This was likely due to a decrease in the number of sections for female sucrose animals compared to male sucrose animals (see table 2-2; p< 0.05). No gender differences in MS volume were noted for the ethanol or chow groups Number of sections, mean area per section, and PA+ neuronal density in the adult rat anterior cingulate cortex Table 2-3 presents the number of sections, mean area per section, and PA+ neuronal density in the adult rat cingulate cortex The number of 40 m sections through the anterior cingulate gyrus was determined by counting the number of sections between the genu of the corpus callosum and the decussation of the anterior comrnissure. The ANOV A showed no effect of diet on the mean number of sections through this region The area of the gyms on a given section was determined by measuring the distance from midline to the cingulum and the distance from the corpus callosum to the dorsal brain surface at a magnification of 25x on three anatomically matched sections with an eyepiece micrometer. No significant differences were noted in mean area per section. The Density of PA+ neurons in the anterior cingulate (mean number of cells per section/mean area [mm2 ] per section) was also calculated and the ANOVA showed an effect of diet on cell density (F[2 30] = 7.041; p < 0.01). The PLSD post-hoc test revealed that cell density in the ethanol group was significantly reduced compared to that of the sucrose (p< 0 01) and chow groups (p < 0.01). Total PA+ neurons in anterior cingulate Figure 2-6 presents the total number of PA+ neurons counted throughout the anterior cingulate cortex. The AN OVA for total number of PA + neurons in the anterior

PAGE 52

42 cingulate showed a significant effect of diet treatment (F[2,30]= 12 314; p< 0.0001). The PLSD post hoc test further revealed that ethanol animals had 45% fewer PA+ neurons than sucrose (p< 0.0001) or chow (p< 0 0001). Figure 2-7 demonstrates a qualitative reduction of PA+ neurons in the anterior cingulate cortex of adult rats exposed to ethanol prenatally when compared with sucrose controls Number of PA+ neurons per section in the anterior cingulate Figure 2 8 presents the mean number of PA+ neurons per section in the anterior cingulate. The ANOVA revea l ed a significant effect of diet treatment on the mean number of PA + neurons per section (F[2 30]= 12.80 ; p< 0 0001). The PLSD further determined that ethanol treatment reduced the mean number of PA+ neurons per section when compared with sucrose (p < 0.001) and chow (p < 0.0001) controls. PA+ neuronal diameter in the anterior cingulate cortex PA+ cell diameters were determined with an eyepiece micrometer at 400x. Ten whole cells from identical anatomical location for each gender in all groups were measured on three sections for each brain region The mean PA+ neuronal diameter for each group was determined and is presented in figure 2-9. No significant differences in the mean PA+ neuronal diameter were noted Anterior cingulate gyrus volume The mean gyms volume for each group was determined and is presented in figure 2-10 No significant effect of treatment on mean volume was noted

PAGE 53

43 Discussion The long-term effects of chronic prenatal ethanol exposure on PA+ neurons were examined in the medial septum and anterior cingulate cortex of adult rats This pattern of exposure produced no significant alterations in P60 body weight (although males were larger than females for all groups examined). Similarly no significant differences were noted in P60 brain weight or brain to body weight ratio as a function of ethanol treatment (although brain to body weight ratios were higher in females due to the larger size of males at P60). The lack of effect of chronic prenatal ethanol treatment (CPET) on the long-term growth of these animals is consistent with other reports from our laboratory (Swanson et al. 1995 ; Swanson et al. 1996). This exposure paradigm did however produce significant alterations in PA+ neurons in both regions examined, although a sexually dimorphic effect was noted for the medial septum The results for each brain region are discussed separately below. Medial Septum A major conclusion drawn by the current study is that CPET affects the MS in a sexually dimorphic manner. This study has demonstrated anatomical changes in the expression of PA in the female rat MS following prenatal ethanol exposure. The evidence from this study suggests that a reduction in the number of MS neurons expressing PA occurs in the absence of area volume, or PA+ cell size effects. Whether this reduction is due to lower PA levels in existing cells or to a loss of PA + neurons is impossible to conclude Regardless the data suggest that alterations in PA expression

PAGE 54

44 occur in female rats following CPET and that PA expression in female rats may be particularly susceptible to the long-term consequences of CPET. Although male offspring exposed to ethanol in utero were unaffected as adults, a number of differences were noted in female ethanol-treated animals when compared to females in the sucrose group. The mean total number of PA+ neurons in the MS nucleus of adult females was reduced by 42 % following prenatal exposure to ethanol, PA+ neuronal density was reduced in ethanol females, and ethanol females had fewer PA+ neurons per section when compared to sucrose females. There were also gender differences in the ethanol groups for a number of measures. Female rats exposed to ethanol in utero contained fewer total PA+ neurons reduced density of MS PA+ neurons, and fewer PA+ neurons per section when compared to males. Gender differences in MS volume in the sucrose group were also noted, with the MS of females significantly reduced compared to that of sucrose males. As mentioned previously, this difference was due to a lower number of 40 m sections from the MS of female sucrose animals when compared to sucrose males. This is not surprising since P60 female rats are smaller than males. This difference was apparently enhanced in the sucrose animals, since neither the ethanol nor the chow groups displayed MS volume differences Differential gender susceptibility of PA+ neurons in the MS following CPET The finding that PA expression was affected by prenatal ethanol exposure only in female animals suggests that differences in the hormonal environment of the males and females influenced PA-immunoreactivity and susceptibility to ethanol. In fact, the SH system itself is highly sexually dimorphic (Loy, 1986). Specifically morphological differences in hippocampal asymmetry (Diamond et al., 1982) and differences in binding

PAGE 55

45 capacity ofhippocampal glucocorticoid receptors (Turner and Weaver, 1985) have been noted in rats. Additionally Loy and Milner (Loy and Milner, 1980) have found differences in lesion-induced hippocampal sprouting between male and female rats. Whereas sprouting in female rats is uniform in the hippocampus following injury sprouting in males occurs predominately in the dentate molecular layer. These results suggest that the CNS response to damage may be dictated by hormonal environment, and that this response is sexually dimorphic. Perhaps similar differences in hormonal environment determine sensitivity to ethanol in this region and account for the noted sexual dimorphism. Gender differences have been reported in the alcohol literature as well: female rats are more affected than males in measures of radial arm maze performance following chronic adult ethanol treatment (Maier and Pohorecky 1986); Witt et al. (1986) found an increase in muscarinic receptor binding sites in the hippocampus of ethanol-treated females but not ethanol-treated males following chronic adult ethanol treatment; Kelly et al. (Kelly et al., 1988) reported impaired spatial navigation in adult female rats but not adult male rats following neonatal ethanol exposure As in the current study, these reports suggest an increased susceptibility to ethanol's effects in female animals and it has been suggested that females may be more sensitive than males to ethanol in animal models (West et al. 1989). However not all studies have agreed with an increased susceptibility for females. Goodlett and Peterson (Goodlett and Peterson 1995), for example found increased susceptibility for spatial learning deficits in male rats following time-limited binge ethanol exposure. Sexually dimorphic effects of prenatal ethanol treatment have also been noted on daily water consumption, with males (but not females)

PAGE 56

46 consuming more water than pair-fed controls as adults (McGivem et al. 1998). Clearly a greater focus on potential gender differences in animal models of FAS is warranted in order to clarify sexual dimorphism in relation to ethanol exposure. Moreover, an investigation into gender differences in human FAS is warranted as no sexual dimorphisms have been reported in human offspring of mothers who drink during pregnancy. What specific differences in hormonal environment might account for the observed sexually dimorphic effect of CPET on PA+ neurons in the female medial septum? Estradiol for example is known to influence the functioning of the cholinergic component of the SH system through its influence on choline re-uptake in the hippocampus (Singh et al. 1994). Indeed, removal of estradiol and other ovarian steroids by ovariectomy decreases cholinergic neurotransmission in the female hippocampus, and this is reversible by estradiol treatment (Singh et al. 1994). Moreover estradiol infusion ameliorates fimbria-fornix lesion-induced decline of ChA T + neurons in the rat medial septum (Rabbani et al. 1997) While estradiol is not known to influence the functioning of the GABAergic component of the SH system estrogen is known to increase glutamic acid decarboxylase (the rate limiting enzyme in the synthesis of GABA) mRNA levels in the female rat brain (McCarthy et al. 1995). Thus it is possible that an ethanol-induced decrease in ovarian steroids might account for some of the effects noted in the present study on GABAergic neurons. Effects of CPET on ovarian steroids would be a useful avenue of future research and may shed light on this interesting sexually dimorphic effect.

PAGE 57

47 Liquid diet effects on the MS In addition to the effects of ethanol on the MS of female rats a non-specific effect of the liquid diet on females was noted for some measurements. Previous studies utilizing rodent models of prenatal ethanol exposure have noted an effect of liquid diet treatment. For example, Swanson et al. (1995) found a stimulatory effect ofliquid diet on ChA T enzymatic activity during the first postnatal week. Additionally studies quantifying ChAT+ cell number in the P14 rat MS found a liquid diet-induced increase in ChA T + neuronal number for female sucrose animals, but not ethanol females or male sucrose animals (Swanson et al. 1996). It was speculated that a possible sucrose diet induced stimulation of MS cholinergic development occurred in female rat pups at P14. The seemingly protective and perhaps stimulatory nature of the liquid diet is probably due to its high vitamin and mineral content. The present study indicates similar diet effects for sucrose females. There seems to be a liquid diet-induced increase in both the density of PA+ neurons in the MS and the number of PA + neurons per section in sucrose females. We speculate that a liquid diet induced increase in cell density for the sucrose group similar to the increase in ChA T + neurons noted by Swanson et al. (Swanson et al. 1996) raised the base level of PA + neurons in the liquid diet animals to a level greater than that seen in chows Although there was not also a significant increase in cell number, the possibility exists that the period of naturally occurring cell death served to reduce this overall number while density remained high. The ethanol-treated group may also display this effect of liquid diet but nonetheless a significant ethanol effect was noted as ethanol females had fewer PA + neurons Apparently the effect of prenatal ethanol treatment is sufficient to produce a

PAGE 58

48 significant cellular reduction in ethanol females. The raised baseline due to the liquid diet however may mask a difference between ethanol and chow females. Functional considerations for the MS Although CNS regions differ in the extent to which PA and GABA co localize the MS is a region where most if not all GABAergic neurons are PA + This statement is supported by studies of Freund (Freund 1989) utilizing anterograde transport from the septum to the hippocampus. Of the projection neurons identified by the anterograde transport all of the axons shown to be immunoreactive for GABA were immunoreactive for PA. Thus PA-immunoreactivity serves as a reliable marker for GABAergic hippocampal projection neurons in the MS nucleus and identifies a subpopulation of the total GABAergic neuronal pool within the basal forebrain As noted previously, in the MS nucleus PA expression begins at G21, after the appearance of GABAergic neurons at G 16 (Lauder et al. 1986 ; Solbach and Celio 1991 ). The ethanol exposure regimen utilized in the present study encompasses the entire prenatal period and thus ethanol was present in CNS tissue during the initiation of PA expression in MS neurons and the physiological maturation of these cells. Exposure of MS neurons to ethanol resulted in a long-term reduction in the number of PA + neurons in adult females. How might these neurons have been affected by changes in the normal expression of PA ? The intracellular concentration of PA in neurons ( 6-4 5 M) and the fact that it is commonl y found in fast-firing neurons (Plagmann and Celio 1993) implicate PA in buffering excess Ca2 + at pre s ynaptic nerve terminals following rapid trains of action potentials (Heizmann 1984 ). Since excessively high levels of intracellu l ar Ca2+ are

PAGE 59

49 known to initiate cell death the possibility exists that an ethanol-induced reduction of PA expression resulted in fewer cells in the females by reducing those neurons' ability to buffer calcium and suppress the process of cell death. Though neurons expressing normal levels of PA can survive experimental cerebral ischemia (Nitsch et al., 1989) and fimbria fornix transection (Kermer et al. 1995), any reduction in PA expression due to ethanol may impede a protective effect. Solodkin et al. ( 1996) have shown a decrease in PA immunostaining in parts of the entorhinal cortex which showed Alzheimer's pathologies. Since PA expression is reduced in the basal forebrain of aged rats (Krzywkowski et al. 1995) the possibility exists that the vulnerability of the septohippocampal pathway to degenerative diseases such as Alzheimer's is due to a reduction in PA expression. The observed reduction in PA+ neurons found following CPET in the current study may be due to direct loss of neurons by the process of cell death or a reduction in the level of PA in MS neurons which would preclude detection by immunostaining due to subthreshold amounts of antigen/epitope. In fact Kermer et al. (1995) have detected reduced PA immunoreactivity in the medial septum following fimbria-fornix transection in the absence of PA+ neuronal cell death. However, whether cells are lost in our paradigm or not the ability of these neurons to function adequately would likely be diminished in affected animals. Altered MS function and FAS A loss of effective GABAergic MS neurons whether by a reduction in cell number or a reduction of PA expression may impede the normal function of the septohippocampal system in learning and memory. GABAergic neurons in the basal forebrain are known to control activity of cholinergic neurons in this region (Dudchenko

PAGE 60

50 and Sarter 1991) and working memory in the rat is disrupted by antagonism of GABAergic transmission in the septum (Chrobak and Napier 1992). Smythe et al. (1992) have shown that both the cholinergic and GABAergic components of the MS are necessary to influence the electrical activity in the hippocampus. Mechanistically the influence of the basal fore brain on hippocampal electrical activity is known to be on theta cell rhythm (Bland and Bland 1986) and the GABAergic component of the MS plays a part in this influence (Allen and Crawford, 1984; Smythe et al., 1992) PA+ MS neurons are known to depress the activity of inhibitory interneurons in the hippocampus (Freund and Antal 1988). This would serve to increase the excitability of the hippocampus by removing local inhibitory potentials ; theta rhythm would be initiated and the excitability of the principal hippocampal cells involved in long-term potentiation would be increased (Freund and Antal 1988) The current study has demonstrated that CPET permanently reduces the number of PA+ GABAergic neurons in the female rat MS by 42 % in the absence of area or volume effects. Such a reduction in the number of PA+ GABAergic neurons could potentially affect the functioning of the septum and hippocampus. Indeed, some of the observed phenotypes of FAS including mental retardation and spatial learning deficits (see Kelly et al. (1988) Goodlett and Peterson (I 995) for animal studies) may be partially explained by a reduced septohippocampal efficacy. This possibility is supported by the findings of Miettinen et al. (1993) that the decline in aged rats in spatial learning correlates with a reduction in PA-containing neurons in the entorhinal cortex A similar decline in learning in FAS children may also relate to PA expression in the SH system

PAGE 61

51 Cingulate Cortex This investigation represents to our knowledge the first demonstration of neuroanatomical alterations in the cingulate cortex as a result of developmental ethanol exposure In fact this appears to be the first examination of teratogen induced alterations in cingu l ate cortex neuroanatomy and is a significant contribution to the literature since there exists a relative paucity of data on the cingulate cortex in normal or pathological states A conclusion of the present study is that CPET permanently reduced the number of PA+ neurons in the anterior cingulate cortex without altering size of the structure These data are similar in their significance to those reported previously for the MS. One critical difference however between the effect of CPET on the anterior cingulate and the effect of CPET on the MS is the lack of sexually dimorphic effects in the former. The factors that are responsible for the lack of gender specific effects of CPET on the anterior cingulate remain unknown It is possible that the medial septum and anterior cingulate respond differentially to different hormonal environments While the medial septum is known as a sexually dimorphic region (see above) no data exist which point to a similar dimorphism for the anterior cingulate. Regardless the overall pattern of ethanol-induced abnormalities is similar with the volume of the anterior cingulate unaffected by prenatal ethanol exposure even though mean total PA + neuronal number and mean PA + neurons per section was affected. Additionally the size of the PA + neurons was determined and was found to be unaltered by this pattern of in utero exposure. Our results are similar to those of Kril and Homewood ( 1993 ) who found that PA+ neuronal number was

PAGE 62

52 decreased in the frontal cortex of adult rats following chronic ethanol treatment and thiamin deficiency. Since the volume of the anterior cingulate was unaffected by treatment and because the cells examined showed no decrease in size it is unlikely that the decline in mean PA+ neurons per section is a result of an ethanol effect on the size of this area. Because our cell counts are derived from PA immunostained sections we cannot definitively conclude that the reduced number of PA+ neurons is a result of ethanol induced cell death While this is a possibility it is equally likely that the reduction of PA+ neurons noted after prenatal ethanol treatment is due to a decreased expression of PA in living neurons (perhaps as a result of ethanol interfering with PA ontogeny) which would preclude detection by immunostaining Krzywkowski et al. (1995) examined PA immunoreactivity in the septum of aged rats and found a decrease in the number of PA+ neurons without loss of GABAergic neurons suggesting that PA+ expression levels were decreased without cell loss. Similar findings have been reported in patients with Parkinson s disease ( Hardman et al. 1996) Yet another possibility is that ethanol interfered with the generation of neurons within the anterior cingulate cortex. Evidence from Miller (1986) shows that prenatal ethanol exposure delays and extends the period of cortical neuronal generation while reducing the number of neurons and altering the distribution of neurons in the mature cerebral cortex. It is conceivable that a similar reduction in the generation of cortical neurons is responsible for our ob s erved decrease in PA+ neuronal number as a result of ethanol treatment.

PAGE 63

53 Functional considerations for the cingulate As mentioned previously the present study examined PA+ neuronal number in the cingulate cortex following prenatal ethanol treatment because of earlier observations related to cerebral cortex functioning following ethanol exposure. These include severe behavioral problems in children with FAS including poor judgment, distractibility, and difficulty perceiving social cues (Streissguth et al. 1991) Additionally many alcoholics who develop Korsakoff s syndrome have deficiencies in glucose utilization within the cingulate cortex potentially contributing to learning and memory defects due to interruption of Papez circuitry (Joyce et al., 1994). Papez circuit is a neuroanatomical pathway which originates in the hippocampus proceeds through the fomix to the mammillary bodies and then to the anterior thalamic nucleus cingulate gyrus, entorhinal cortex and then back to the hippocampus (Nolte, 1993). Therefore it is conceivable that alterations in PA expression patterns might contribute to behavioral anomalies and/or learning and memory deficiencies. Because the intracellular concentration of PA in neurons is in the range necessary for calcium buffering (6-45 M) and the fact that it is commonly found in fast-firing neurons (Plogmann and Celio 1993) PA appears to be involved in buffering excess Ca2+ at presynaptic nerve terminals following rapid trains of action potentials (Heizmann 1984). Since excessively high levels of intracellular Ca2+ are known to initiate cell death the possibility exists that an ethanol-induced reduction of PA expression resulted in fewer cells in the ethanol animals by reducing those neurons' ability to buffer calcium and suppress the process of cell death. Though neurons expressing normal levels of PA can survive experimental cerebral ischemia (Nitsch et al., 1989) and fimbria-fornix

PAGE 64

54 transection (Kermer et al. 1995) any reduction in PA expression due to ethanol may impede a protective effect. Solodkin et al. ( 1996) have shown a decrease in PA immunostaining in parts of the entorhinal cortex which showed Alzheimer s pathologies. Similarly, the number of PA+ neurons is reduced in the prefrontal cortex of schizophrenics (Beasley and Reynolds 1997). While we cannot determine from the present study whether cell death or reduced PA antigen is responsible for the noted reduction in PA+ neurons, it should be noted that whatever the mechanism of the ethanol-induced reduction of PA+ neurons, inhibitory neurotransmission in the cingulate cortex is likely to be altered For example, PA+ neurons are lost at epileptic foci in animal models of epilepsy (De Felipe et al. 1993) Moreover Jacobs et al. (1996) showed that decreased PA immunoreactivity in cortical freeze lesions of neonatal rats leads to hyperexcitability in adjacent cortex. Such alterations in inhibitory and excitatory processes have clear functional consequences to the organism and likely result in an imbalance in excitation and inhibition in these cortical regions (Wang et al. 1996) The current findings of decreased number of PA+ neurons in the anterior cingulate cortex of adult rats as a result of prenatal ethanol exposure may provide a neuroanatomical basis for the well described cognitive and behavioral impairments in offspring exposed to ethanol in utero. Conclusions The major findings of this study are that PA+ neurons in the medial septum and anterior cingulate cortex are susceptible to prenatal ethanol exposure. In the medial septum, a sexually dimorphic effect of ethanol was noted, with females more susceptible than males No gender specific effects were noted for the anterior cingulate. These

PAGE 65

55 reductions in PA+ neurons occurred in the absence of significant ethanol-induced alterations in the size of the structures examined the size of the PA+ neurons within these structures or the growth of these animals. This represents the first description of ethanol induced alterations in PA+ GABAergic neurons in animal models of FAS. Further research will help to further define developmental windows of GABAergic vulnerability (for example, neonatal exposure models).

PAGE 66

56 Table 2-1. Postnatal day 60 (P60) body and brain weight and brain to body weight ratio (br/bd)* Diet group N P60 body weight (g) P60 brain weight (g) P60 br/bd (%) Females Ethanol 8 220 + 2.92 1.32 + 0.02 0.600 + 0.0I0t Sucrose 6 216 + 5.58 1.31 + 0.02 0.606 + 0.021 t Chow 6 224 + 5.06 1.36 + 0.02 0 607 + 0 .022t Males Ethanol 5 341 + 13.5t 1.34 + 0 05 0.392 + 0.032 Sucrose 6 351 + 5 .81 t 1.35 + 0.06 0.384 + 0.019 Chow 8 348 + 13.6t 1.39 + 0.02 0.399 + 0.030 All measures are expressed as mean S.E.M. No significant differences were noted. tSignificantly increased compared with other gender of same group (p< 0.0001). Table 2-2. Number of sections medial septum (MS) area per section and parvalbumin imrnunoreactive (PA+) neuronal density in the adult rat MS* Diet group N Number of 40 m MS area/section Density (PA+ sections (mm2) cells /mm2 ) Females Ethanol 8 15.75 + 0.453 0.722 + 0.066 49.55 + 6.716t t Sucrose 6 15.50 + 0.428, 0.655 + 0.061 89.38 + 9.700 Chow 6 17.67 + 1.054 0 670 + 0.065 59.87 + 6.237 Males Ethanol 5 16.20 + 0.374 0.733 + 0.103 78.031 + 12 .01 Sucrose 6 17.50 + 0.428 0.778 + 0.061 66.16 + 10 98 Chow 8 16 87 + 0 811 0 733 + 0.049 63.49 + 5.957 All measures are expressed as mean S.E.M. Area and volume measures are representative of approximately 1/2 of the total MS (alternate sections were analyzed ; see materials and methods section for detail) tSignificantly reduced from sucrose females (p< 0 01) !Significantly reduced from ethanol males (p< 0.05) Significantly reduced from sucrose females (p< 0.05). 1Significantly reduced from sucrose males (p< 0.05).

PAGE 67

57 Table 2-3. Number of sections mean area per section and parvalbumin-immunoreactive (PA +) neuronal density in the adult rat anterior cingulate corte x Diet group N Number of 40 m Mean area/section Density (PA + sections (mm 2 ) cells/mm 2 ) Ethanol 13 41.5 + 2.56 2.135 + 0.069 395.5 + 67 .85t Sucrose 12 39.8 + 2 56 2.262 + 0 060 669 6 + 54 56 Cho w 14 35. 8 + 1 34 2 385 + 0.080 697.4 + 61.88 All measures are expressed as mean S.E.M. t Significantly reduced compared with sucrose (p < 0 01) and chow (p< 0 01).

PAGE 68

58 1200 = C=:JEthanol 0 1000 Sucrose = z -Chow r.. "O 800 0 a,b -..... = 600 ,.Q = e o =u 400 z ..... 200 0 Male Female Figure 2-1. The mean total number of parvalbumin-expressing neurons detected on alternate sections through the medial septum of postnatal-day 60 rats exposed in utero to one of three diets ethanol sucrose or chow (see materials and methods section for detail). Data are expressed as mean SEM. a: significantly reduced compared with sucrose females (p< 0.01). b: significantly reduced compared with ethanol males (p< 0.05).

PAGE 69

Figure 2-2 Photomicrographs of 40 m coronal sections through the medial septum of ethanol-treated (A) and sucrose-treated (B) postnatal-day 60 female rats. Sections are matched for anatomical location and are representative of their respective group. A qualitative reduction in cell number for the ethanol females can be noted as can an increase in cell density for sucrose females Scale bar equals 125 m.

PAGE 70

60 A B

PAGE 71

75 = 0 .... u 50 r,:i = 0 = Z 25 61 a,c 0 .....__...____.__ Male Female C=:JEthanol Sucrose -Chow Figure 2-3. The mean number of parvalbumin-expressing neurons per section in alternate sections through the medial septum of postnatal-day 60 rats exposed in utero to one of three diets ethanol sucrose or chow (see materials and methods section for detail). Data are expressed as mean S E M a: significantly reduced compared with sucrose females (p < 0 01). b: significantl y reduced compared with sucrose females (p < 0.05). c: significantly reduced compared with ethanol males (p < 0.05).

PAGE 72

62 1-, 20 c::JEthanol .... Sucrose e -Chow ... -e = ::t 10 0 1-, ..._ = z < 0 Male Female Figure 2-4. The mean diameter of parvalbumin-expressing neurons on alternate sections through the medial septum of postnatal-day 60 rats exposed in utero to one of three diets ethanol sucrose or chow (see materials and methods section for detail). Data are expressed as mean S.E.M. No significant differences were noted.

PAGE 73

63 0.75 c:JEthanol I") Sucrose e e 0.50 -Chow .._, e = 0.25 rJJ. Female Figure 2-5. The mean medial septum (MS) volume of postnatal-day 60 rats exposed throughout gestation to an ethanol-containing liquid diet, sucrose-containing liquid diet, or lab chow and water. Data are expressed as mean SEM. a: significantly reduced compared with sucrose males (p< 0.05).

PAGE 74

64 2000 < "'O ..... 0 = = Ji. 0 a u .,Q a r,:i 1000 = = z = ..... z 0 Ethanol Sucrose Chow Figure 2-6. The mean total number ofparvalbumin-expressing (PA+) neurons detected on alternate sections through the anterior cingulate cortex of postnatal-day 60 rats exposed in utero to one of three diets ethanol sucrose or chow (see materials and methods section for detail) Data are expressed as mean SEM. a: significantly reduced compared with s ucrose (p < 0 0001) and chow (p < 0 0001)

PAGE 75

Figure 2-7. Photomicrographs of 40 m coronal sections through the anterior cingulate cortex of ethanol-treated (A) and sucrose-treated (B) postnatal-day 60 rats. Sections are matched for anatomical location and are representative of their respective group. A qualitative reduction in parvalbumin-immunoreactive neuronal number is seen in the ethanol-treated animal compared with the sucrose control. Scale bar equals 110 m.

PAGE 76

66 A B

PAGE 77

67 300 < = 0 .... ...... CJ 0 200 -00. -..c a e C. = fl) z = 0 = 100 = ~z 0 Ethano l Sucrose Cho w Figure 2 -8. The mean number of parvalbumin-imrnunoreactive (PA+) neurons per section counted in the anterior cingulate cortex of postnatal-day 6 0 rats exposed throughout gestation to an ethanol-containing liquid diet, sucrose-containing liquid diet or lab chow and water. Data are expressed as mean SEM a: significantly reduced compared with sucrose (p< 0.001) and chow animals (p< 0 0001).

PAGE 78

68 20 ...-I = bl) = u +U < = -10 = ...-I e 0 Ethanol Sucrose Chow Figure 2-9. The mean diameter of parvalbumin-immunoreactive (PA+) neurons in the anterior cingulate cortex of postnatal-day 60 rats exposed throughout gestation to an ethanol-containing liquid diet sucrose-containing liquid diet, or lab chow and water. Data are expressed as mean SEM. No significant differences were noted.

PAGE 79

69 4 r,:i = ~---c., 3 e .,_ e ._. = t=.J) e 2 = ..... = u = 1 0 Ethanol Sucrose Chow Figure 2-10 The mean cingulate gyrus volume of postnatal-day 60 rats exposed throughout gestation to an ethanol-containing liquid diet sucrose-containing liquid diet or lab chow and water. Data are expressed as mean SEM. No significant differences were noted

PAGE 80

CHAPTER3 EFFECTS OF NEONATAL ETHANOL EXPOSURE ON CHOLINERGIC NEURONS OF THE RAT MEDIAL SEPTUM Summary Developmental ethanol exposure has been known to affect the normal development of the central nervous system. Studies in animal models have determined that chronic prenatal ethanol exposure has no effect on the number of cholinergic neurons in the rat medial septum (Swanson et al., 1996) Since many brain regions exhibit tight temporal windows of vulnerability to ethanol the objective of this study was to determine the long-term effects of chronic neonatal ethanol exposure on the cholinergic neurons in the medial septum (MS) of the rat. On postnatal day 4 (P4) pups were assigned to one of three groups: an ethanol-receiving gastrostomized group (EtOH); a pair-fed gastrostomized control group (GC); and a dam-reared suckle control group (SC). Gastrostomized pups were infused with ethanol-containing or control diet as a 9.1 % v / v solution for two feedings on each day from P4-10. Choline acetyltransferase (ChA T) imrnunocytochemistry was analyzed at P60. Ethanol treatment resulted in long-lasting microencephaly in P60 animals. Ethanol exposure did not directly reduce mean total ChA T-expressing (ChA T + ) neuronal number or the mean number of ChA T + neurons per section. Neither were changes noted in MS volume mean area section or cell density as a result of ethanol treatment. 70

PAGE 81

71 However, ethanol exposure significantly reduced ChAT+ neuronal size in males compared with GC males but not SC males No differences in ChA T + neuronal size were noted in females. Thus neonatal ethanol exposure while producing long-lived microencephaly and small changes in ChA T + neuronal size, has no effect on the number of cholinergic neurons in the adult rat MS, and has no effect on the size of the MS. Introduction Fetal alcohol syndrome (FAS) has been well characterized since its description in 1973 (Jones and Smith 1973). Perinatal growth deficiencies, craniofacial abnormalities, as well as central nervous system (CNS) d ysfunctio n have been noted in human offspring exposed to ethanol prenatally (West et al., 1994) In attempts to thoroughly examine and accurately define the consequences of developmental ethanol exposure, rodent models of FAS have been developed and extensively utilized. These animal models have identified susceptible populations of neurons within the CNS and have described periods of development during which particular populations exhibit heightened vulnerability to the effects of ethanol (West et al. 1994). Studies in rats have demonstrated that the early postnatal period the so-called brain growth spurt and the equivalent of the human third trimester (Dobbing and Sands, 1979) is a time of vulnerability for particular CNS reg10ns. The septohippocampal (SH) system of basal forebrain afferents and hippocampal targets is one such region in rats that exhibits s usceptibility to ethanol during neonatal development. The SH system is a pathway of cholinergic and GABAergic fibers originating from the MS nucleus and the horizontal and vertical limbs of the diagonal

PAGE 82

72 band of Broca (DBB) that influences electrical activity in the hippocampus (Dutar et al. 1995). These fibers originate from cell bodies in the basal forebrain and beginning at embryonic day 20 innervate the hippocampus where they form synapses on hippocampal pyramidal neurons granule cells and intemeurons (Freund and Antal 1988; Milner et al. 1983). Cholinergic neurons comprise the major projection of the SH system and first express the catalytic enzyme choline acetyltransferase (ChA T) on embryonic day 17 (Armstrong et al. 1987 ; Dutar et al. 1995). While numerous studies have described the effects of neonatal ethanol exposure on neurons within the hippocampus (Bonthius and West 1990 ; Bonthius and West 1991; Greene et al. 1992 ; Pierce and West 1987 ; West and Pierce 1986) and have documented deficits in spatial learning following neonatal ethanol exposure (Goodlett and Peterson 1995 ; Kelly et al. 1988) the effect of ethanol exposure during the brain growth spurt on the cholinergic component of the SH system has yet to be characterized. However studies examining the effect of chronic adult ethanol exposure on the neurons of the SH system have been reported and have suggested a decrease in the number of cholinergic neurons in the MS though pair-fed controls were not examined (Arendt et al. 1995 ; Arendt et al. 1988). Moreover studies examining the effect of prenatal ethanol exposure on the SH system in the rat (Swanson et al. 1995 ; Swanson et al. 1996) and mouse (Schambra e t al. 1990 ; Sulik et al. 1984) hav e been completed When rats were exposed to ethanol throughout gestation a reduction in ChA T enzymatic activity was noted in the MS during the second postnatal week compared with pair-fed controls ( Swanson et al. 1995).

PAGE 83

73 In contrast no change in the number or morphology of ChA T + neurons in the MS was found (Swanson et al., 1996), though earlier experiments in mice had shown the cholinergic neurons of the basal forebrain to be particularly sensitive to acute prenatal ethanol exposure (Schambra et al. 1990; Sulik et al. 1984) The objective of the present study was to determine the long-term effects of ethanol exposure on the cholinergic neurons in the rat MS when ethanol was delivered durin g the brain growth spurt, a time when the cholinergic neurons are more mature and potentially more susceptible than at the time points examined in the previous prenatal studies (Bonthius and West, 1991 ). Ethanol was delivered from P4-10 a period of differentiation and synaptogenesis in the SH system (Dutar et al. 1995). ChA T + neuronal number and morphology was examined in the adult MS of artificially reared ethanol-treated and pair-fed controls as well as in dam-raised suckle controls. Methods Subjects and artificial rearing Long-Evans hooded rat pups were obtained from nine timed pregnant dams ordered from Charles River Co. (Portage MI) Animals were housed with a 07:00-19:00 light cycle under controlled temperature and humidity conditions. At the time of birth (PO), litters were culled to 12 pups and the pups were randomly assigned to one of three groups : ethanol -rec eiving gastrostomized pups (EtOH) ; pair-fed gastrostomy controls (GC) ; and dam-reared suckle controls (SC). Artificial rearing was performed after West et al. (West et al. 1984). On P4 a gastrostomy feeding tube was surgically implanted into the EtOH and GC pups Pups were placed under methoxyfluorane anesthetic while

PAGE 84

74 gastrostomy tubes were inserted down the esophagus into the stomach. The tube was pulled through a small hole in the stomach and abdominal wall and secured on the outside by a small plastic washer. Pups were reared individually in plastic cups filled with bedding and a fur-like material. Cups were floated in a covered, heated aquarium (40C) and pups were maintained on a 07:00-19 :0 0 light cycle. SC pups were weighed daily from P4-10. Dams with SC pups always had a total of eight pups to minimize weight differences between groups. Gastrostomized pups were infused with a liquid diet containing evaporated milk sterile water, soy protein L-methionine, L-tryptophan, calcium phosphate deoxycholic acid a vitamin mixture and a mineral mixture (West et al. 1984). Pups received the milk formula from P4-10 in 12 feeding periods of20 minutes each. EtOH pups received ethanol-supplemented formula as a 9.1 % v/v solution for the fust two feedings on each day for a total of 4 g/kg/day. GC pups received an isocaloric amount of maltose-dextrin supplemented formula for the first two feedings on each day The remaining 10 daily feedings consisted of milk formula alone. Gastrostomized pups were weighed daily and the daily diet consumption volume (in mls) was equivalent to 33% of the mean litter bod y weight. The liquid diet was administered to the EtOH and GC pups by connecting the gastrostomy tubes to a feeding line connected to diet-filled syringes held within a Stoelting (Wood Dale IL) programmable infusion pump. On the morning of Pl 1 pups were disconnected from the pump feeding tubes were sealed and they were returned to their original dam. EtOH and GC pups were accepted by the dams and began nursing soon after re-introduction to the home cage. At P21, pups were weaned and housed

PAGE 85

75 individually until sacrifice and perfusion at P60. A total of 38 animals were used in this analysis (EtOH n = 12; GC n = 10; SC n = 16). Tissue preparation and immunocytochemistry Animals were selected for immunocytochemical staining at P60 an age which represents adulthood in rats. This age was selected in order to examine the long-term effects of neonatal ethanol treatment on ChA T expression in cholinergic neurons of the medial septum and corresponds to an age used in previous analyses of prenatal ethanol influences on cholinergic and GABAergic neurons in the medial septum (Moore et al. 1997 ; Swanson et al. 1996). On P60 rats were anesthetized by pentobarbital overdose just prior to perfusion. Animals were perfused transcardially with phosphate buffered saline (PBS; 0.lM containing 0.9% sodium chloride) followed by 10% formalin (in the same PBS) The brains were removed and post-fixed overnight in the same fixative The brain tissue was equilibrated overnight ( or until it sank) in a cryoprotectant solution (PBS containing 30% sucrose and 15% ethylene glycol) and stored frozen at -70 C until processed for immunocytochemistry Brains were prepared for frozen sectioning by thawing and equilibrating with a 30% sucrose-PBS solution. Frozen serial coronal sections throughout the forebrain were cut at a thickness of 40 m and processed free-floating Alternate sections were immunostained for choline acetyltransferase using a polyclonal antibody (rabbit anti human placental ChA T Chemicon Temecula CA). Immunoreactivity was visuali z ed as a blue-black reaction product using an avidin-biotin conjugate / nickel intensified staining (se e b e low). Animal s from ea ch diet group were processed for immunocytoch e mistry at a

PAGE 86

76 given time. This ensured against staining differences between groups resulting from procedural differences Endogenous peroxidase activity was quenched by initially treating tissue sections for 60 min at room temperature (RT) with PBS containing 3% hydrogen peroxide (H202 ) and 10% methanol. Sections were pretreated with 0.4% Triton-Xl00 (T-X) in PBS for 30 min at RT then blocked for 60 min with PBS containing 3% normal goat serum (NGS) and 0.1 % T-X. Primary incubation with the polyclona l ChA T antibody (1 :750 in PBS + 3% NOS and 0.1 % T-X) was carried out for 48 hr at 4C. Sections were then washed in PBS containing 1 % bovine serum albumin (BSA) and incubated with biotinylated goat anti-rabbit IgG (1:1000 in PBS + 1% BSA) overnight at 4C. Tissues were washed and incubated with extravidin-horseradish peroxidase conjugate (Sigma St. Louis, MO; 1: 1000 in PBS + 1 % BSA) overnight at 4C. Tissues were washed with 0. lM sodium acetate (pH 7 2) to eliminate phosphate which can precipitate divalent cations. Sections were then reacted for 3 min at RT using a developing buffer pH 7 2 containing 0 .8M sodium acetate 8 mM imidazole 0.5 % nickel (II) sulfate 0 .04% 3 3 diaminobenzidine tetrahydrochloride and 0.005% H202 The chromogen precipitation reaction was stopped with 0. lM acetate buffer (pH 7.2) containing 0.1 % sodium azide. Sections were mounted in the same acetate buffer air dried dehydrated and coverslipped. Control for nonspecific staining was carried out in parallel with stained tissues with omission of the primary antibody Slides from individual anima l s were randomized and coded so that all subsequent analyses were carried out blind with regard to the diet treatment group and gender.

PAGE 87

77 MS cell count, volume, and area analysis ChA T-positive cell counts were conducted on alternate sections throughout the rostral-caudal extent of the MS nucleus. The packing density of the ChAT+ neurons in the DBB nucleus was too great for accurate cell counts to be made Thus for the purposes of this study the MS alone was examined and was defined rostrally by the ventral fusion of the hemispheres (at the level of the genu of the corpus callosum) and caudally by the decussation of the anterior commissure Images of ChA T immunostained sections were captured and digitized using a RasterOps 24STV video capture board and software on a Macintosh computer Low magnification images ( 4x objective ; effective scale 1.59 m/pixel) were captured in order to obtain the entire MS region in one image. When capturing each image, the lighting and contrast enhancement were optimized for identification of individual cells Images were digitally processed using the image analysis program NIH Image (freeware from NIH Bethesda MD) To reduce background variation across the image a digitally defocused image ( created by passing the primary image through a mean filter) was subtracted from the primary digitized image This processed image was then passed through a Laplace filter to enhance edges and separation between cells. Analysis of individual images was initiated b y outlining the region of the MS nucleus. The area of a section outlined varied depending on the rostral-caudal location of th e individual section. The MS nucleus in rostral sections was defined as th e mediodorsal group of neurons which were separated from the ventrall y -located DBB nucleus In intermediat e sections wh e r e the d e marcation betwe e n the MS and DBB nuclei is ambiguous the MS was defined ventrally by a line perpendicular to midline at the level

PAGE 88

78 of the anterior comrnissure. In caudal sections, the MS was defined as the medially located cells dorsal to the anterior commissure. Cells were highlighted interactively by adjusting the grayscale threshold level to include only objects which were considered cells. Given the thickness of each section ( 40 m) the fact that alternate sections were analyzed, the fact that ChA T + neurons in the MS nucleus range from 10-25 m in diameter and the fact that an estimation of the total population was not desired, it was not deemed necessary to perform a split-cell correction on these counts. The program counted the highlighted objects and measured the area of the outlined region (mm2). Because the MS is defined by clusters of ChA T + cell bodies, the area outlined was determined by the pattern of ChA T staining on each individual section. MS volume (mm3 ) was calculated using a modification of the Cavelieri method (Michel and Cruz, 1988) to ensure that changes in mean number of ChA T + neurons per section as a result of ethanol treatment were not due to changes in the size of the structure of interest (Peterson et al. 1997). This was done by multiplying the number of 40 m sections analyzed for a given animal by the mean area per section for that animal and the section thickness. ChA T + cell size determination Because a previous study from our laboratory (Swanson et al. 1996) had analyzed ChA T + neuronal size using the computer instead of simple measurements of cell diameters, we also chose to use the computer for these purposes so that comparisons could be made to the previous work. For morphometric analysis moderate magnification images (1 Ox objective ; effective scale 0 978 m/pixel) were captured in order to maximize the resolution yet minimize the loss of cells out of the focal plane of each

PAGE 89

79 image Primary images were not processed further prior to morphometric analysis. Cell size measurements were performed on ChA T + neurons in the MS nucleus. Three sections through the MS were regionally matched in rostro-caudal extent for all animals. 25 non-overlapping cells per section were selected at random and measured. The cross sectional somatic area of individual cells was highlighted interactively by adjusting the grayscale threshold and was automatically measured by the computer. The program computed cross-sectional area for each cell and mean somatic area was determined for each animal. Other analyses The peak blood ethanol concentration (BEC) in the ethanol group was determined on P7 two hours after the second ethanol infusion (Goodlett et al. 1990) utilizing the Sigma 333-UV kit. Body and brain weights were recorded for individual animals at P60 following perfusion. Density of ChA T + neurons was determined by dividing the mean number of cells per section by the mean area (mm2 ) per section. Statistical analyses Statistical analysis was performed with the program StatView (Abacus Concepts Berkeley Ca) For each parameter e x amined one-way Analysis of Variance (ANOVA) was used to test for the main effects of diet and g ender separately. When appropriate the data were further analy z ed with the Fisher s Protected Least Significant Difference (PLSD) post hoc test to determine individual group differences. Because gender differences were noted for many measures (following an initial two-way ANOV A for effects of diet and gender) males and females were analyzed separately

PAGE 90

80 Results BEC, body and brain weights, and brain/body weight ratios The mean BEC in EtOH pups two hours following the last ethanol treatment on P7 was 269 23 mg/di (n = 5). Table 3-1 (all tables are located at the end of the chapter) presents the mean P60 body and brain weights, and brain/body weight ratios (br/bd) for all diet groups separated by gender. The ANOVA for P60 body weight revealed no significant effects of diet on P60 body weight of males or females. A gender difference was noted in P60 body weight for EtOH [F(l 22) = 45.2; p< 0.0001] GC [F(l,14) = 38.5; p < 0.0001], and SC [F(l 16) = 40.8; p< 0.0001] animals which was expected given the larger size of males at P60. The ANOV A for P60 brain weight demonstrated an effect of diet treatment in males [F(2 18) = 12.3 ; p < 0.001] and in females [F(2 25) = 6 69; p < 0.01]. The PLSD revealed that EtOH male brains weighed less than GC male (p< 0 .01 ) and SC male (p< 0.01) brains. EtOH female brains also weighed less than GC female (p < 0.05), and SC female (p< 0.01) brains. No significant gender differences were noted for brain weight within EtOH GC or SC animals. The ANOVA for br/bd showed an effect of diet in males [F(2 18) = 5.16 ; p < 0.05] and females [F(2 25) = 11.3 ; p< 0 001]. The PLSD demonstrated that EtOH males had reduced br/bd compared with GC males (p < 0.01) while EtOH females had reduced br/bd compared with both GC females (p < 0.001) and SC females (p< 0.001). Gender differences were also noted in P60 br/bd for EtOH [F(l 22) = 37.5; p < 0.0001] GC

PAGE 91

81 [F(l 14) = 41.2 ; p < 0.0001] and SC [F(l, 16) = 38.5; p< 0.0001] animals which was not surprising given the larger size of male animals at this age Number of sections, MS area per section, and ChA T + neuronal density Table 3-2 presents number of sections MS area per section and ChA T + neuronal density measurements for each diet group for either gender. The number of alternate 40 m sections through the MS was determined by counting the number of sections analyzed between the ventral fusion of the hemispheres (at the level of the genu of the corpus callosum) and the anterior commissure. The ANOV A showed no effect of diet for either males or females on the mean number of alternate sections through the MS. Additionally no significant gender differences were noted in the number of alternate sections for E tOH GC or SC animals. As mentioned above the computer program determined the area of the outlined MS on each individual section. The mean MS area per section was determined for each animal. The ANOVA revealed no significant effect of diet in males or females on the mean MS area per section. Similarly, no significant gender differences were noted in MS area per section for EtOH GC, or SC animals. ChA T + neuronal density was calculated by dividing the mean number of ChA T + neurons per section by the mean area per section The ANOV A demonstrated no significant effect of diet in males or females on ChAT+ neuronal density. Also no significant gender differences were noted in ChA T + neuronal density for EtOH, GC, or SC animals

PAGE 92

82 Cholinergic cell counts in the MS The total number of ChA T + neurons counted on alternate sections through the P60 MS was determined and is presented in figure 3-1 (all figures are located at the end of the chapter). The ANOV A for total number of ChA T + cells demonstrated an effect of diet in males [F(2 18) = 4.45 ; p< 0.05] but not females The PLSD further revealed that EtOH males contained 24 % fewer MS ChA T + neurons than SC males (p< 0.05). No significant gender differences were noted in mean total number of ChA T + neurons for EtOH GC, or SC animals. In order to normalize for volume, the average number of ChA T + neurons per section was determined Figure 3-2 presents the mean number of ChA T + neurons per section. The ANOV A for mean number of ChA T + neurons per section revealed an effect of diet on males [F(2 18) = 3.34 ; p < 0.05] but not females The PLSD further demonstrated that EtOH males contained 19 % fewer ChA T + neurons per section than SC males (p < 0.05) while the 14 % difference between GC males and SC males was not significant. No significant gender differences were noted in mean ChA T + cells per section for EtOH, GC or SC animals Cholinergic neuronal size Figure 3-3 presents the mean somatic cross sectional area for ChA T + neurons throughout the P60 MS The ANOVA for mean somatic cross sectional area determined an effect of diet in males [F( 2, 18) = 5 76 ; p < 0 05] but not females. The PLSD further revealed that GC males contained an increased somatic cross sectional area compared with SC males (p < 0 05) while EtOH males contained a reduced somatic cross sectional

PAGE 93

83 area compared with GC males (p < 0.01). No significant gender differences were noted in ChA T + neuronal size for EtOH, GC, or SC animals. MS volume MS volume was determined by multiplying the number of 40 m sections analyzed for a given animal by the mean area per section for that animal. Figure 3-4 presents the mean MS volume of P60 rats. The ANOV A demonstrated no effect of diet in males or females on the mean number of alternate sections through the MS. No significant gender differences in MS volume were noted for EtOH, GC, or SC animals. Discussion The major conclusion of the present work is that small reductions in the size of ChA T + neurons of the medial septum are produced by neonatal ethanol exposure while changes in the number of cholinergic neurons or size of the MS are not noted This study has also determined that long-lasting microencephaly is found in male and female P60 rats exposed to ethanol via artificial rearing from P4-10. Because EtOH animals showed no difference when compared with GC animals in total ChA T + neuronal number or mean number of cholinergic neurons per section ethanol exposure did not directly reduce ChA T + neuronal number. However since EtOH males contained fewer cholinergic neurons when compared with SC males it appears that ethanol in combination with artificial rearing does affect these neurons. No changes were noted in m e an area p e r section cell density or MS volume as a result of ethanol treatment in males. Female animals similarly exposed to ethanol did not contain a reduced number of ChA T + MS neurons and no changes were noted in MS volume mean

PAGE 94

84 area per section or cell density. A permanent reduction in the size of ChA T + neurons was noted in EtOH males compared with GC males. However this was concurrent with an increase in size of cholinergic neurons in GC compared with SC males. Moreover no differences in cell size were found in female rats making it difficult to determine whether the reduction in ChA T + cell size in EtOH males is an effect of ethanol on neuronal size or merely an effect of diet and artificial rearing on GC males Thus neonatal ethanol exposure while producing long-lived microencephaly has little effect on the cholinergic neurons of the adult rat MS as measured by ChAT + neuronal number and morphology Long-term effects of ethanol on brain growth Microcephaly and microencephaly are hallmarks of FAS as well as neonatal animal models of FAS (West and Pierce 1986). Reduced brain size represents one of the most reproducible features of ethanol-induced alterations in the brain and the current study is no exception. Artificial rearing from P4-10 produced no long-lasting deficits in body weight and by P60 E tOH and GC pups weighed the same as SC pups Despite the lack of effect on body weight EtOH animals of both genders showed reduced brain weight at P60 compared with GC and SC animals. Brain to body weight ratio (br/bd) was similarly reduced in both males and females exposed to ethanol. Taken together the reduced brain weight of EtOH animals and the reduced br/bd in EtOH animals demonstrate that neonatal ethanol exposure produced long-lasting microencephaly. Goodlett et al. ( Goodlett et al. 1991) exposed rats from P4 9 in two of the 12 daily feedings as a 10 .2% solution (an exposure paradigm similar to the current experiments). E xposure to this concentr a tion of ethanol resulted in a peak BEC of 361 m g/ di ( compared with 269 mg/dl in the current stud y ) and produced microencephaly in both genders in

PAGE 95

85 adult animals. The current study demonstrates that even with a lower peak BEC, brain growth is severely compromised The degree of ethanol-induced reduction in brain weight depends directly on the peak BEC produced. Bonthius and West (1991) provided an illustration of this by comparing the effects of a 6.6 g/kg/day ethanol treatment spread out over 12 feedings from P4-11 with the effects of 4 5 g/kg/day ethanol treatment given in just two feedings Animals were sacrificed at P90 to observe long-term effects of neonatal ethanol delivery on brain weight. Interestingly they found that the lower 4 5 g/kg/day dose produced profound brain weight and brain to body weight ratio deficiencies while the 6.6 g/kg/day group exhibited no such microencephaly. This differential response to varying ethanol delivery patterns was directly related to peak BEC as the higher dose spread out over 12 feedings produced a peak BEC of only 43 mg/dl while the lower dose condensed into two feedings produced a peak BEC of 318 mg/dl (Bonthius and West 1991 ). Thus, the pattern of exposure plays a critical role in the severity of ethanol-induced teratogenicity. Long-term effects of ethanol and artificial rearing on ChA T + neurons in the MS Artificial rearing and ethanol exposure produced a decrease in the mean ChA T + neuronal number per section in EtOH males compared with SC males in the absence of volume area or density changes. Female animals displayed no differences in mean cholinergic neuronal number per section volume area or density measurements and thus the reduction occurs in a sexually dimorphic manner. Because the reduction in ChA T + neuronal number noted in males was between the gastrostomized EtOH group and the dam-reared SC group (and not the pair-fed GC group) the reduction appears to be the result of an interaction between ethanol treatment and some characteristic of the

PAGE 96

86 artificial rearing technique rather than the result of ethanol alone. Kelly ( 1997) has noted an effect of artificial rearing on the concentration of neurotransmitters in the SH system of male rats: neurotransmitter concentration was reduced in artificially reared males independent of ethanol exposure. While the current results do not implicate artificial rearing in reducing cholinergic neuronal number (the difference between GC and SC males in mean ChA T + neurons per section is not significant) they do suggest that ethanol in combination with artificial rearing can reduce ChA T + neuronal number. Perhaps an interaction between ethanol and the stress of the procedure (from invasiveness or maternal separation) accounts for the reduced cholinergic neuronal number in artificially reared and ethanol exposed pups. Developmental ethanol exposure and ChA T + neuronal number The current finding that cholinergic neuronal number in the MS is unaffected by ethanol alone is consistent with previous experiments from our laboratory. Swanson et al. (1995; 1996) demonstrated reduced ChA T activity in P 14 rats chronically exposed to ethanol in utero. However analysis of ChA T + neuronal number in the MS of P 14 and P60 rats revealed no effect of diet treatment on cholinergic cell number. Similarly evidence from Heaton et al. ( 1996) showed that ChA T + neuronal number is not significantly affected in the striatum of male or female rats following CPET A transient increase in cholinergic neuronal number was noted in the striatum on P 14, but this was reduced to control levels by P60. While studies from our laboratory (see above) have not indicated an effect of developmental ethanol exposure on cholinergic neuronal number in the rat MS or striatum studies from other groups have demonstrated ethanol-induced alterations in

PAGE 97

87 ChA T + number particularly in the MS. For example data suggest that acute prenatal exposure of mice to ethanol resulted in severe midline anomalies at embryonic day 18 including loss of basal fore brain neurons (Schambra et al., 1990) as well as loss of septa! neurons and reduced volume at P 15 (Ashwell and Zhang, 1996). However it should be noted that these experiments produced high acute BECs (600-700 mg/dl) which were the result of one or two ethanol exposures early in embryonic development. Thus differences in timing and dosage may account for the apparent discrepancy (Swanson et al. 1996) Exposure of adult rats to ethanol for 12 or 28 weeks has been reported to reduce cholinergic neuronal number in the MS (Arendt et al. 1995; Arendt et al. 1988). However it should be noted that comparisons were made between rats exposed to ethanol as a 20% solution of drinking water and rats fed chow and water ad libitum; no pair-fed controls were examined. Thus it is possible that the reduction of cholinergic neurons in the MS is not due to the influence of ethanol alone For instance an interaction between ethanol and malnutrition may explain the noted differences. However i t is conceivable that chronic ethanol exposure during mature stages may reduce the number of MS cholinergic neurons as found by Arendt et al. (1995; 1988) Regardless the current data in combination with the Swanson et al. (1996) and Heaton et al. (1996) studies suggests that cholinergic neuronal number in the MS is not susceptible to moderately-high or moderate doses of ethanol during early postnatal or prenatal development respectively A future study examining ChA T + neuronal number after exposure to a higher peak BEC would determine whether the apparent lack of effect of ethanol on choliner g ic neurons is due to the moderate doses used in these studies.

PAGE 98

88 Developmental ethanol exposure and ChA T + neuronal size in the MS Although ChA T + neuronal counts were not directly affected by ethanol analysis of the mean somatic cross sectional area of cholinergic neurons in the P60 MS demonstrated a diet effect in male animals. Specifically neuronal size was reduced in EtOH males compared with GC males. However GC males contained larger neurons compared with SC males and no differences were found in females making it difficult to determine whether this is an effect of ethanol on cholinergic neuronal size or merely an effect of diet and artificial rearing on GC males It is unlikely that the smaller s i ze of cholinergic neurons in EtOH males is simply because of the noted microencephaly since the brains of EtOH and SC males were different in size but the mean size of ChA T + neurons was similar. It is possible however that GC males experienced an increase in soma! size in response to either the stress of the artificial rearing procedure (such as maternal separation isolation or the invasive surgery) or to some component of the maltose dextrin containing milk diet. Factors such as nerve growth factor (NGF) can induce hypertrophy of ChA T + basal forebrain neurons (Higgins et al. 1989) NGF is also known to increase the size of cholinergic neurons in cultures of rat septa! neurons (Markova and Isaev 1992) and is able to reverse axotrny-induced decreases in MS cholinergic neuronal cell bodies (Hagg et al. 1989). It is conceivable that male GC animals increased the expression ofNGF, as is known to occur following stress (Smith 1996) resulting in larger cholinergic MS neurons. However female GC animals were similarly stressed and did not display hypertrophy of ChA T + neurons. Perhaps a more likely explanation is that a constituent of the liquid diet acted in a trophic manner on these

PAGE 99

89 neurons. Whatever the explanation, these data are consistent with data from other investigators who have reported sexually dimorphic effects on the size of cholinergic neurons of the MS. For example, neonatal hyperthyroidism is capable of increasing ChA T + neuronal size in the MS of male animals while simultaneously decreasing the size of ChAT + neurons in females (Westlin d-Danielsson et al. 1991). One other report from our laboratory has examined cholinergic neuronal size at P60 following chronic prenatal ethanol exposure (Swanson et al. 1996). While the exposure times were different in the current study it is useful to compare the present data with the Swanson et al data. The size of the ChAT+ neurons at P60 is quite similar For example the range of neuronal size in the Swanson et al. study was 90-100 m2, which is comparable to the range noted in the current study (80-110 m2). However in contrast to the present data Swanson et al. (1996) found no ethanol-induced alterations in ChA T + neuronal size and found no gender differences. ChA T + neuronal insusceptibility: possible role of neurotrophic factors Why is ChA T + neuronal number apparently unaltered following developmental ethanol exposure? One possible explanation is the importance of neurotrophic factors in regulating the development and maintenance of the SH system. The hippocampus is known to express a rich variety of neurotrophic factors including NGF brain-derived neurotrophic factor (BDNF), neurotrophin-3 and basic fibroblast growth factor (Ernfors et al. 1990 ; Maisonpierre et al. 1990). The high affinity receptors for the various neurotrophins found in the hippocampus including trk A trk B and trk C are also present in the developing septum including the cholinergic neurons of the medial septum (Ringstedt et al ., 1993) Evidence suggests a role for neurotrophins in regulating the

PAGE 100

90 expression of ChA Tin the basal fore brain. Intraventricular injection of NGF antibodies into rat neonates reduces ChA T immunostaining in the septum (V antini et al., 1989). Moreover mice which have been genetically engineered to lack the various neurotrophin receptors have been generated and have further defined a role for neurotrophins in sustaining septal cholinergic neurons in vivo. For example, mice which lack trk A show reduced acetylcholinesterase activity in septal projection fibers (Smeyne et al., 1994 ), and mice which lack BDNF show reduced density of cholinergic neurons in the MS (Jones et al. 1994) In vitro studies have also demonstrated a role for neurotrophins in sustaining septal neurons in normal conditions and in the presence of ethanol. For example, NGF stimulates ChA T activity, neuritic complexity increased fiber length and increased fiber outgrowth in cultures of septal cholinergic neurons (Hartikka and Hefti 1988) Moreover studies from our laboratory have demonstrated that NGF protects both cultured dorsal root ganglion neurons and septa} neurons from ethanol neurotoxicity (Heaton et al. 1993 ; Heaton et al. 1994). It is conceivable that neurotrophic interactions are preserved in the current study, and that ethanol is unable to produce toxic effects on cholinergic neurons in the medial septum because these normal interactions are maintained. A consequence of this maintenance may be growth factor-mediated induction of protective molecules of the bcl-2 family of cell death molecules which would prevent ethanol-induced toxicity in this population. Similar mechanisms are known to operate in cerebellar granule cells during development. For example Muller et al. (1997) demonstrated that NGF induces Bcl-2 protein expression and that this induction resulted in cell survival.

PAGE 101

91 Conclusions The current study and several others from our laboratory have demonstrated little effect of developmental ethanol exposure on cholinergic neurons in the MS. Prenatal ethanol exposure does not significantly reduce ChA T + neuronal number in the basal forebrain (Swanson et al. 1996) or striatum (Heaton et al. 1996). If neither prenatal nor postnatal ethanol exposure in rats affects the cholinergic neurons in the MS then what accounts for the impairment of spatial learning tasks in rats exposed to ethanol neonatally (Goodlett and Peterson 1995; Kelly et al. 1988)? A possible explanation for these previously described spatial learning deficits is that a reduction of GABAergic neurons in the basal forebrain following neonatal ethanol exposure may alter normal learning of spatial tasks. Although the GABAergic component of the SH system has not been as extensively characterized as the cholinergic component, it is known to control activity of cholinergic neurons in the basal forebrain (Dudchenko and Sarter, 1991) and is necessary to influence hippocampal electrical activity (Smythe et al. 1992). A recent study from our laboratory showed a 42% reduction in parvalbumin immunoreactive neurons of the female MS following prenatal ethanol exposure (Moore et al. 1997). Parvalbumin is located within the GABAergic neurons of the MS (Freund 1989; Krzywkowski et al. 1995). Additionally parvalbumin immunoreactive GABAergic neurons of the anterior cingulate cortex are reduced in male and female rats following prenatal ethanol treatment (Moore et al. 1998b ) Future investigations will determine whether these newly discovered neuroanatomical alterations resulting from developmental ethanol exposure can explain the noted cognitive impairments of FAS children.

PAGE 102

92 Table 3-1. Mean body weight brain weight, and brain weight to body weight ratio (br/bd) of postnatal-day 60 (P60) animals exposed to ethanol or control conditions from P4-10* Diet group N P60 body weight ( g) P60 brain weight ( g) P60 br/bd (%) Males EtOH 15 270 + 6.780 1.4 7 + 0.026:t 0.544 + 0.0lOt GC 8 258 + 7.610 1.61 + 0.035 0.626 + 0.024 t SC 8 289 + 14.60 1.65 + 0.019 0.578 + 0.030 t Females EtOH 9 203 + 8.450t 1.44 + 0.032t 0.712 + 0.020 ~ GC 8 182 + 5.770t 1.53 + 0.033 0.842 + 0.018 SC 10 191 + 7.000t 1.59 + 0.029 0.841 + 0.026 All measures are expressed as mean SEM and are representative of nine artificially reared litters. t Significantly reduced compared with other gender of same group (p< 0.0001). t Significantly reduced compared with GC and SC animals of same gender (p < 0.05). Significantly reduced compared with GC males (p < 0.01) Significantly reduced compared with GC and SC females (p< 0.01). Table 3-2. Number of sections medial septum (MS) area per section and choline acetyltransferase-immunoreactive (ChA T +) neuronal density in the adult rat MS of postnatal-day 60 (P60) animals exposed to ethanol or control conditions from P4l 0* Diet group N Number of 40 m MS area/section Density (ChAT+ sections (mrn2) neurons / mrn 2 ) Males EtOH 8 16.4 + 0 625 0.319 + 0.015 216 + 13.2 GC 5 16.8 + 1.020 0.313 + 0.011 230 + 19.8 SC 8 17. 6 + 0.460 0.353 + 0 009 237 + 14.5 Females EtOH 4 16.8 + 0.479 0.345 + 0 018 213 + 7.79 GC 5 16.6 + 0.678 0.358 + 0.017 220 + 13.7 SC 8 16. 6 + 0 625 0.382 + 0.023 220 + 18.9 All measures are expressed as mean SEM and are representative of approximately one-half of the total MS (alternate sections were analy z ed). No significant differences were noted. Animals are a subset of nine artificially reared litters.

PAGE 103

93 2000 c=JEtOH :a -g GC u ..... = -SC 0 = a 0 -u .c rl'.l 1000 e = = 0 z s ~z ..... 0 Male Female Figure 3-1. Mean total number of choline acetyltransferase-immunoreactive (ChAT + ) neurons in the medial septum of postnatal day 60 (P60) rats. Rats were artificially reared from P4-l O and exposed to ethanol (EtOH), artificially reared and pair-fed (GC), or dam reared (SC). Data are expressed as mean SEM. a: significantly reduced compared with SC males (p< 0.05).

PAGE 104

94 100 c::J Et O H = 80 a GC 0 -.... -SC u 5 rJJ 60 = z = 0 = = 40 ~z 20 0 Male Female Figure 3-2. Mean number of choline acetyltransferase-immunoreactive neurons per section detected on alternate 40 m sections through the medial septum of postnatal day 60 (P60) rats Rats were artificially reared from P4l O and exposed to ethanol (EtOH), artificially reared and pair-fed (GC) or dam-reared (SC) Data are expressed as mean SEM. a: significantly reduced compared with SC males (p< 0 05).

PAGE 105

95 140 c,J b c::JEtOH = co: = 0 120 I. GC 0 = .... ..100 -SC ~z a 80 60 I. u u c..,. = 0 40 co: co: 20 0 Male Female Figure 3-3 Mean somatic cross sectional area (m2 ) of choline acetyltransferase immunor e active ( ChA T + ) neurons in the medial septum of postnatal day 60 (P60) rats Rats were artificially reared from P4-10 and exposed to ethanol (EtOH) artificially reared and pair-fed ( GC ), or dam-reared (SC ) Data are expressed as mean SEM. a: significantly reduced compared with GC males (p < 0.01) b: significantly increased compared with SC males (p < 0.05).

PAGE 106

96 0.3 c:JEtO H GC a a 0.2 -SC -a = 0.1 rJl Female Figure 3 -4. The mean medial septum (MS) volume of postnatal-day 60 (P60) rats Rats were artificially reared from P4-10 and exposed to ethanol (EtOH) artificially reared and pair-fed (GC) or dam-reared (SC) Data are expressed as mean SEM No significant differences were noted

PAGE 107

CHAPTER4 EFFECTS OF NEONATAL ETHANOL EXPOSURE ON PURKINJE AND GRANULE CELLS AND BCL 2 FAMILY MRNA LEVELS IN THE RAT CEREBELLAR VERMIS Summary While many neuronal populations exhibit vulnerability to ethanol delivered during critical developmental stages the cellular mechanism of ethanol's toxicity remains unknown. Often neuronal susceptibility within the brain is determined by the timing of the ethanol exposure. The principa l neurons of the cerebellum (the Purkinje and granule cells) provide a good example of this differential temporal vulnerability to ethanol. It has been documented in studies by a number of investigators that first postnatal week ethanol exposure results in substantial Purkinje and granule cell loss while second postnatal week exposure does not (Goodlett and Eilers 1997 ; Hamre and West 1993). The objective of the present work was to test the hypothesis that differential ethanol-induced cerebellar cell death during development is related to ethanol-induced alterations in the expression of bcl-2 family of cell survival and death genes. Rats were exposed to ethanol or control conditions during the neonatal period and transcript levels of bcl-2 family members relative to cyclophilin were determined. Pups exposed in parallel were taken for cerebellar cell counts. Ethanol exposure during the first postnatal week significantly reduced Purkinje and granule cell numbers by postnatal day 21 (P21 ). Acute first postnatal week ethanol exposure up-regulated mRNA 97

PAGE 108

98 transcripts encoding the cell death-promoting molecules bax and bcl-xs as measured on P4. An additional day of exposure on PS resulted in no further alterations in bcl-2 family transcripts likely because Purkinje cell death was detectable as early as PS. To determine whether pro-apoptotic gene expression changes were specific to first postnatal week ethanol neurotoxicity, we examined bcl-2 family rnRNA levels in rats exposed to ethanol during a developmental period of cerebellar insusceptibility the second postnatal week Exposure on P7-8 produced no cerebellar cell death, but also resulted in increased levels of bax though only after two-day ethanol exposure and not after acute exposure on P7 These data implicate altered expression of pro-apoptotic members of the bcl-2 gene family in acute ethanol-mediated cerebellar cell death during the first postnatal week They also suggest that the differential survival of cerebellar neurons following ethanol exposure during more mature developmental stages may be related to more successful suppression of pro-apoptotic processes. Introduction Evidence of cerebellar vulnerability to developmental ethanol exposure comes from human studies demonstrating size reduction in the cerebellar vermis of children exposed prenatally to ethanol (Sowell et al. 1996). Moreover in rat models of fetal alcohol syndrome ethanol exposure during the first postnatal week results in decreased numbers of Purkinje and granule cells in the cerebellum with the cerebellar vermis particularly affected (Goodlett and Eilers, 1997; Hamre and West, 1993). An interesting property of the cerebellar vermis is that it is differentially susceptible to ethanol. That is ethanol exposure to more mature neonates during the second postnatal week does not

PAGE 109

99 result in profound cerebellar neurotoxicity (Goodlett and Eilers 1997; Hamre and West, 1993) While cell loss following ethanol exposure during critical developmental periods is well documented no molecular mechanisms of ethanol-mediated cell death are known There is reason to hypothesize that the bcl-2 family of cell death molecules is involved in ethanol neurotoxicity The bcl-2 family of genes is comprised of cell death regulators and bcl-2 was the first of this family to be discovered In recent years, a number of genes similar to bcl-2 have been found. These include bax and bad as well as bcl-x whose mRNA is alternatively spliced into long (bcl-xl) and short (bcl-xs) forms (Reed 1994). Some members of the bcl-2 gene family serve to inhibit cell death (e.g. bcl-2 bcl-xl al, mcl-1) and others promote cell death ( e.g. bcl-xs, bax bad, bak). It is the intracellular ratio of cell death repressor to cell death effector molecules which determines whether a cell will undergo apoptosis (Oltvai et al. 1993) The suggestion that the bcl-2 family might be involved in ethanol-induced cell death in the cerebellum is supported by the literature. Cell death following ethanol exposure appears to proceed through an apoptotic mechanism and cerebellar neurons undergo apoptosis in vitro and in vivo in response to ethanol (Bhave and Hoffman 1997 ; Liesi 1997 ; Renis et al ., 1996 ; Singh et al. 1995) Furthermore a role in regulating the death and survival of cerebellar neurons has been demonstrated for the bcl-2 family Gillardon et al. ( 1995) investigated bcl-2 and bax gene expression in the cerebella of Purkinje-cell-degeneration mice (mutants that lose nearly all of their Purkinje cells between P22-28 followin g otherwise normal development). They found that bcl-2 mRNA levels decreased while bax mRNA levels remained unchanged beginning on P22

PAGE 110

100 In addition thyroid hormone-induced upregulation of bcl-2 protects early-differentiating cerebellar granule cells from apoptosis in vitro (Muller et al., 1995) and transgenic mice overexpressing bcl-2 contain more cerebellar Purkinje and granule cells than controls whether transgene expression begins embryonically or neonatally (Zanjani et al ., 1996 ; Zanjani et al. 1997). Additionally data from our laboratory have indicated a role for bcl2 in protecting cerebellar neurons in vivo Transgenic mice overexpressing bcl-2 in nervous tissue are resistant to neonatal ethanol-induced Purkinje cell death (Heaton et al. unpublished observation). Although the cerebellar expression patterns of some bcl-2 family genes including bad and bak are unknown many bcl-2 family members including bax bcl-xs bcl-xl and bcl-2 are expressed throughout the brain during development and their distribution includes cerebellar Purkinje and granule cells (Castren et al. 1994 ; Frankowski et al. 1995 ; Krajewski et al. 1994 ; Rouayrenc et al. 1995). Purkinje cells express high le v els of both forms o f the bclx gene in development and adulthood (Dixon et al. 1997 ; Frankowski et al. 1995) high levels ofbax early in development which is down regulated in adults (Krajewski et al. 1995 ; Vekrellis et al. 1997) and low levels of bcl-2 (Hara et al ., 1996). Granule cells express high levels of bcl-2 initially in development but down-regulate it during adulthood ( Castren et al. 1994). The bax gene is known to be expressed in granule cells during development (Gleichmann et al. 1998 ; Miller et al. 1997) as are both forms of the bcl-x gene (Gleichmann et al. 1998) Studies utilizing geneticall y engineered mice have described roles for various members of the bcl-2 gene family in vivo For instance transgenic mice overexpre s sin g bcl2 in the nervous system s how reduced developmental cell death in certain neuronal

PAGE 111

101 populations and show a general hypertrophy of the nervous system (Martinou et al. 1994). Neurons from these animals are better able to withstand ischemic episodes, removal of growth factor support and axotomy (Dubois-Dauphin et al., 1994; Farlie et al. 1995). Additionally mice overexpressing bcl-xl contain facial motoneurons that are resistant to axotomy during the postnatal period (Parsadanian et al. 1998). Mice lacking the bcl-2 gene have been instrumental in defining the normal actions of this gene in vivo. A role for bcl-2 maintenance of motoneurons sympathetic and sensory neurons was indicated through analysis of cell numbers in these regions in bcl-2 knockout mice. After the PCD period bcl-2 knockouts contained fewer neurons in each of these areas relative to controls (Michaelidis et al. 1996). Inactivation of the bcl-x gene is lethal as mice lacking this gene die around E 13. Investigation of fetuses revealed extensive apoptotic cell death in neuronal populations of the brain, spinal cord and dorsal root ganglion (Motoyama et al., 1995) This indicates the importance of bcl-x in embryonic life. Mice lacking the bax gene have also been generated and have determined a role for cell-death promoting molecules in sculpting neuronal populations (Deckwerth et al. 1996) For example sympathetic and facial motoneurons from bax knockouts survive early postnatal growth factor deprivation and axotomy. Moreover the normal process of cell death is disrupted in the superior cervical ganglia and facial nuclei of bax knockouts since these structures possess more surviving neurons after the period of apoptosis (Deckwerth et al. 1996). Given these demonstrated roles for bcl-2 family members in vivo it was speculated that ethanol-induced alterations in the levels of these molecules might influence cell death in the cerebellar vermis.

PAGE 112

102 The objective of the present study was to test the hypothesis that ethanol-induced cerebellar neurotoxicity during development is related to alterations in the expression of bcl-2 family genes. To accomplish this rats were artificially reared and exposed to ethanol or control conditions for one or two days during the neonatal period. Ethanol was delivered during periods of vermis susceptibility (first postnatal week) and for comparison during a period of vermis insusceptibility (second postnatal week). mRNA levels of four bcl-2 family members (with known cerebellar expression) and the internal standard cyclophilin were determined Pups exposed in parallel were sacrificed for cerebellar cell counts Materials and Methods Experimental design First and second postnatal week ethanol neurotoxicity and its relation to bcl-2 gene expression was examined in the rat cerebellar v ermis since this region is particularly vulnerable to ethanol (Goodlett and Eilers 1997 ; Hamre and West 1993) First postnatal week exposure occurred on P4 only or on P4 and PS. Second postnatal week exposure occurred on P7 only or on P7-8. The relati v e le v els of bcl-2 famil y mRNAs were determined two hours after ethanol exposure on each exposure day. For example some pups were killed two hours after exposure on P4 while others were killed on PS following two days of exposure on P4-S. The same is true for pups used for the second postnatal week experiments We e x amined these acute timepoints because we anticipa t ed that gene expression changes would occur rapidly following the ethanol insult. Cereb e llar cell counts w e r e p e rformed at a common tim e, P 2 1 following first or second

PAGE 113

103 week exposure to ensure that differences in exposure times did not contribute to any noted differences in cell death. In order to determine the timecourse of first postnatal week cell death some animals were sacrificed on P5 following ethanol treatment on P4-5 Subjects and ethanol treatment Sprague Dawley rat pups were obtained from timed pregnant dams from Harlan Sprague Dawley (Indianapolis IN). Dams were housed with a 07:00-19:00 light cycle under controlled temperature and humidity conditions. On the morning that ethanol treatment began (P4 or P7) male pups were randomly assigned to one of three groups: ethanol receiving gastrostomized pups (EtOH); pair-fed gastrostomy controls (GC) ; and dam-reared suckle controls ( SC) used to determine whether non-specific effects of artificial rearing are produced. Because our aim was to establish this model system in our hands and since earlier investigations used only male pups we decided to utilize male and not female animals in this study Also on the morning that ethanol treatment began a gastrostomy feeding tube was surgically implanted into the stomach ofEtOH and GC pups under methoxyfluorane anesthetic and secured on the outside by a small plastic washer. Pups were reared individually in plastic cups filled with bedding and a fur-like material. Cups were floated in a covered heated aquarium ( 40C) and pups were maintained on a 07 :00-19 : 00 light cycle. Gastrostomized pups were infused with a liquid diet containing evaporated milk sterile water soy protein L-methionine L-tryptophan calcium phosphate deoxycholic acid a vitamin mixture and a mineral mixture (West et al. 1984). Pups received the

PAGE 114

104 milk formula in feeding periods of 20 minutes each. EtOH pups received ethanol supplemented formula as a 15. 0 % v / v solution for two feedings on each exposure day for a total of 6 6 g/kg/day (the remaining 10 feedings were of milk alone). This pattern of exposure produced a mean blood ethanol concentration (BEC) of 335 41 mg/dl dl as determined 2 hours after the final ethanol treatment on the first day of exposure. These days and this timepoint were chosen to measure BEC because they are known to represent the peak BEC for this pattern of exposure (Goodlett et al., 1990). GC pups received an isocaloric amount of maltose-dextrin-supplemented formula for two feedings on each day (the remaining 10 feedings were of milk alone) Gastrostomized pups were weighed daily and the daily diet consumption volume (in mls) was equivalent to 33 % of the mean litter body weight. The liquid diet was administered to the EtOH and GC pups by connecting the gastrostomy tubes to a feeding line connected to diet-filled syringes held within a Stoelting (Wood Dale IL) programmable infusion pump. Pups acutely exposed to ethanol ( one-day treatment on P4 only or P7 only) received ethanol infusions onl y on the day of surgery Pups exposed for two day s received milk diet alone for the remaining 10 feedings on the first exposure day and received two additional ethanol feedings on the morning of the second exposure day (PS or P8). Pups used for mRNA analysis were sacrificed two hours after the last ethanol feeding on the final exposure day (the day of sacrifice ; P4 PS, P7 or P8) as were pups taken for cell counts and anatomical analysis on PS. Pups exposed in parallel for P21 cell counts and anatomical anal y sis were returned to the dam after oneor two-day ethanol treatment and raised by the dam until sacrifice at P21.

PAGE 115

105 Histological procedures On the day of sacrifice pups were anesthetized and decapitated and the cerebellum was dissected away from the cerebral hemispheres and placed in Bouin's fixative Following fixation specimens were dehydrated cleared and embedded in paraffin. Serial sagittal 6 m sections were then cut through the midline of the vermis mounted onto glass slides and stained with hematoxylin and eosin (H&E). Cerebellar cell counts Manual cell counts of Purkinje and granule cells were performed and expressed as mean cells per section. While stereological cell enumeration has become a common method of cell counting manual counting remains a viable alternative for cell number quantitation (Guillery and Herrup 1997). In fact recent data have demonstrated the utility of manual cell counts (Clarke and Oppenheim 1995) and show a direct correlation between cell counts performed manually and cell counts performed with the optical dissector (Hagg et al. 1997) Criticisms of manual cell counting methods (and data expressed as mean cells per section) are that overprojection and truncation can result from incorrectly identifying cells within a focal plane and that changes in reference volume can bias cell counts (Peterson et al. 1997). The choice of manual cell counting in the present study is appropriate for three reasons: first thin sections were counted and the three chosen sections which were counted were separated by 60 m. Second verrnis reference volume and lobule area were measured to determine whether ethanol treatment altered verrnis size. Third we only wished to compare the mean number of cells per section between groups and did not intend to estimate total cell number (Hagg et al. 1997). An additional justification comes from recent reports that have utilized

PAGE 116

106 stereological techniques to count cerebellar neurons following developmental ethanol treatment (Goodlett and Johnson 1997 ; Napper and West 1995a). These data are consistent with previous findings using manual cell counting and in fact have documented very similar Purkinje and granule cell losses following ethanol exposure (Hamre and West 1993). Prior to counting of Purkinje and granule cells slides were coded and randomized with an identifying number. Cells were counted only on sections from the midline vermis where lobule I and X are closest to each other and deep cerebellar nuclei are absent. Cell counts were performed in the manner of Hamre and West (1993) on three sections separated by 60 m to ensure that the same cell is not counted twice. Purkinje cells in lobule I were counted if they contained a well delineated nuclear membrane distinct nucleolus darkly stained cytoplasm and were clearly Purkinje cells based on location and size The average number of Purkinje cells per section in lobule I was determined for each animal and means recorded for each group. Counts were performed in lobule I because although all cerebellar lobules are susceptible to ethanol the more anterior lobules display heightened vulnerability (Hamre and West 1993) Granule cells were counted within a grid measuring 2.14 mm2 that was placed in the granule layer of lobule I. Counts were performed on three coded midline sections For cell counts on P5 both the external and internal granule cell layers were counted. Anatomical measurements Anatomical measurements were taken in order to determine whether ethanol treatment significantly altered the size of the vermis. Vermis volume was estimated by counting the number of sections where lobules I and X are both visible Lobule I length

PAGE 117

107 was also measured with an eyepiece micrometer to find whether treatment altered the length of the lobule. The diameter of Purkinje and granule cell nuclei was also measured with an eyepiece micrometer to determine whether treatment altered the size of Purkinje or granule cells. Cerebellar cell counts and anatomical measures were performed on 3-6 pups per group for every timepoint investigated. Preparation of antisense RNA probes cDNAs for bcl-2, bcl-xl, bcl-xs, and bax were cloned from an adult Sprague Dawley cerebellum. Total RNA was extracted and reverse transcribed into first strand cDNA using oligo(dt)-primer and reverse transcriptase (Invitrogen cDNA cycle kit Carlsbad CA). Gene-specific PCR primers were designed for regions of low homology based on known rat cDNA sequences for bcl-2 bcl-xl bcl-xs, and bax (Tilly et al. 1 995). First strand cDNA was taken through 30 cycles of PCR amplification and then ligated into the pCR2.1 cloning vector (Invitrogen Carlsbad CA). E. coli competent cells (JMl 09) were transformed with the recombinant plasmid and plated DNA sequencing (both strands) was performed by the DNA Sequencing Core Laboratory Interdisciplinary Center for Biotechnology Research University of Florida to verify clones. Basic local alignment search tool analysis of the four cDNA clones revealed at least 98% nucleotide sequence homology with bcl-2 bcl-xl bcl-xs and bax previously cloned in rat (Tilly et al. 1995). Antisense RNA probes complimentary to bcl-2 bcl-xl, bcl-xs and bax mRNA coding sequences were synthesized in vitro from linearized plasmid templates. Onl y bcl2 family members with a demonstrated expression in the cerebellum were examined. In vitro transcription was carried out at 3 7C for 40 minutes and was followed by treatment

PAGE 118

108 with DNase for 15 minutes (also at 37C). Two phenol-chloroform extractions were performed followed by precipitation with ammonium acetate and ethanol. An aliquot of probe was measured with a scintillation counter to determine counts per minute (CPM) 32P-labeled cyclophilin probe (template from Ambion, Austin, TX) transcribed as described above, was included in each hybridization reaction along with individual bcl-2, bcl-xl bcl-xs and bax probes and served as a standard. The use of cyclophilin was appropriate because its transcription was unaltered in previous studies of mRNA expression following chronic ethanol treatment in adult male rats and embryonic rats (Maclennan et al., 1995; Maier et al., 1996). Solution hybridization and RNase protection The use of a gross technique such as the RNase protection assay to measure relative mRNA levels is appropriate for this study since expression of bcl-2 and other family members is known to be at low to undetectable levels in glia (F rankowski et al. 1995 ; Vyas et al. 1997). Thus any observed changes in relative transcript levels are unlikely to be due to expression changes in non-neuronal cell types Total RNA was extracted from the cerebellar vermis with the RNA STAT-60 extraction kit ( Tel-Test Inc Friendswood, TX) coded and randomized with an identifying number and stored at 80C until hybridization. Samples were prepared for hybridization by precipitating 2 g total RNA and 18 g E coli tRNA (to aid in pelleting) at 20C with ammonium acetate and ethanol for 30 minutes, followed by centrifugation for 15 minutes. After allowing the pellet to dry it was dissolved in 10 l hybridization buffer containing the radiolabled probes of interest diluted at the appropriate CPM (abo ut 100 000 for bcl-2 family probes and 20, 000 for cyclophilin) Each individual probe of the bcl-2 fami l y was hybridi ze d to

PAGE 119

109 sample RNA along with cyclophilin probe Following incubation for 10 minutes at 80C, hybridization was carried out at 45C overnight. The following day each hybridization product was treated with a mixture of RNases A and Tl at 37C for 60 minutes. This served to destroy any incomplete hybrids or single-stranded RNA in the reaction, leaving only double-stranded probe:mRNA hybrids. Proteinase K was used to hydrolyze the RNases. Hybrids were extracted with phenol and chloroform were precipitated at -20C for 30 minutes and were then centrifuged for 15 minutes at 4 C Pellets were dissolved in formamide loading buffer denatured by boiling quickly placed on ice for 30 seconds and were then separated by polyacrylamide gel electrophoresis. After drying gels were exposed to a phosphor imaging screen overnight. Band intensity was read as an optical density (OD) by imaging software. The OD of the bcl-2 family bands was normalized by dividing the OD of the cyclophilin band from each animal. Normalized OD from each probe (bcl-2 bcl-xl bcl xs and bax) was averaged from 6-8 animals from each treatment group (EtOH GC and SC). Other analyses Blood ethanol concentration determined from trunk blood on the day of sacrifice was assayed with the 333-UV microenzymatic assay (Sigma, St. Louis, MO). Statistics were performed with the program Statview (Abacus Concepts Berkeley CA). One-way analysis of variance (ANOV A) was used to determine significant treatment effects Fisher s protected least significant difference (PLSD) post-hoc test was used to determine specific group differences.

PAGE 120

110 Results Body and brain weight measures Body weight brain weight and brain to body weight ratios were determined at the time of sacrifice at all timepoints (P4 PS, P7 P8 and P21) following the various exposure paradigms (see figure 4-1; all figures are located at the end of the chapter) The ANOVA showed that exposure on P4 produced body weight differences at P21 (F[2 7]= 21.594 ; p < 0 001). The PLSD revealed that SC pups weighed significantly more than EtOH (p < 0.01) and GC pups (p< 0.01) but that GC and EtOH pups (the most appropriate comparison) were not different. Similarly exposure on P4 produced brain weight differences at P21 (F[2 7]= 15.115; p< 0 01) The brains of SC pups weighed significantly more than EtOH (p< 0.01) and GC pups (p < 0.01) but GC and EtOH pups were not different. An effect of treatment on brain to body weight ratio was noted at P21 following P4 exposure ( F[2 7] = 4.363; p < 0.05). The brain to body weight ratio of SC pups was significantly greater than EtOH (p < 0 05) and GC pups (p < 0.05) but GC and EtOH pups were not different. Figure 4-2 presents body weight brain weight and brain to body weight ratios taken at P4 PS, and P21 following treatment on P4-5. The ANOVA determined no effect of treatment on body weight at P4 PS, or P21. Brain weight was affected by treatment on P21 (F[2 24] = 8.752 ; p < 0.05) but not on P4 and PS. The post-hoc test found that P21 brain weight was reduced in EtOH pups compared with both GC (p < 0.01) and SC pups (p < 0 .01). Brain to body weight ratios were changed by treatment on PS (F[2 39] = 10.284 ; p < 0 05) but not on P4 and P21. The brain to body weight ratio of SC pups on

PAGE 121

111 PS was greater than that of both GC (p< 0.01) and EtOH (p< 0.01) pups but EtOH and GC pups were not different. Figure 4-3 presents body weight, brain weight and brain to body weight ratios taken at P7 P8 and P21 following treatment on P7-8. The ANOVA showed no effect of treatment on body weight at P7 P8 or P21. Brain weight was influenced by treatment on P8 (F[2 18]= 4.174 ; p< 0 05) and P21 (F[2 13]= 4.392; p< 0.05) but not on P7. The PLSD determined that P8 brain weight was reduced in GC pups compared with SC pups (p < 0.05) but that EtOH and GC pups were not different. P21 brain weight was reduced in EtOH pups compared with SC pups (p < 0.05), although EtOH and GC pups were not different. Brain to body weight ratios were altered by treatment on P8 (F[2, 18]= 12.159 ; p< 0.001) but not on P7 and P21. The brain to body weight ratio of SC pups was greater than that of both GC (p< 0 001) and EtOH (p< 0 001) pups at P8 but EtOH and GC pups were not different. Measurements of vermis size: section number, lobule length and cerebellar cell nuclear diameters Anatomical measurements were taken in order to determine whether ethanol treatment significantly altered the size of lobule I (see tables 4-1 through 4-4 located at the end of the chapter). The AN OVA found that acute ethanol treatment on P4 (table 4-1) did not alter the mean number of sections through the vermis lobule length Purkinje cell nuclear diameter or granule cell nuclear diameter as measured on P21 Two day ethanol treatment from P4-5 produced the only significant volume difference noted at P21 (see table 4-2). A significant effect of ethanol treatment was noted on the mean number of sections through the vermis (F[2 10] = 12.103 ; p < 0.01) The post-hoc test indicated that

PAGE 122

112 EtOH pups had significantly fewer 6 m sections than GC (p< 0 01) and SC (p< 0.01) pups. Lobule length and Purkinje and granule cell nuclear size were unaffected Treatment on P7-8 produced no significant size alterations as measured on P21 (see table 4-3). No effect of treatment was found on section number, lobule length Purkinje or granule cell nuclear diameters. Ethanol treatment on P4-5 (see table 4-4) also did not produce significant differences as measured on PS, and no effect of treatment was seen on section number lobule length or Purkinje cell nuclear diameter. Granule cell nuclear diameters were not determined at this age because the small size of the cells made it impossible to measure with the eyepiece micrometer even at a magnification of 600x. Because no ubiquitous volume alterations were noted ( only the number of vermal sections and not lobule length was altered following P4-5 exposure; no other treatment pattern produced size changes) it is unlikely that our cell counts are biased due to changes in the size of lobule I. For example ethanol exposure on P4-5 produced verrnis size alterations and neurotoxicity as measured on P21 while exposure only on P4 produced no size alterations and y et also produced cerebellar cell death. P21 cerebellar cell counts following first postnatal week ethanol treatment Figure 4-4 shows cerebellar cell counts at P21 in ethanol-treated and control animals following first postnatal week exposure The ANOVA showed a significant effect of treatment on the number of Purkinje cells per section in lobule I whether ethanol was delivered on P4 (F[2 ,7]= 6 737 ; p < 0.05) or P4-5 (F[2 10] = 46.04; p < 0.0001). The PLSD post-hoc test determined that ethanol treatment on P4 reduced the mean number of Purkinje cells per section in EtOH pups (by approximately 50%) compared with both GC (p < 0.05) and SC (p < 0.05) pups. Similarly ethanol treatment on P4-5 reduced the mean

PAGE 123

113 number of Purkinje cells per section in EtOH pups ( by approximately 65%) compared with both GC (p< 0.0001) and SC (p < 0.0001) pups The mean number of granule cells per section was also altered whether ethanol was delivered on P4 (F[2 ,7]= 7.477 ; p < 0.05) or P4-5 (F[2 10]= 5.877; p < 0.05). Ethanol treatment on P4 reduced the mean number of granule cells per section in EtOH pups compared with both GC (p < 0.05) and SC (p < 0.05) pups Similarly ethanol treatment on P4-5 reduced the mean number of granule cells per section in EtOH pups compared with both GC (p < 0 05) and SC (p < 0 05) pups. Therefore oneor two-day ethanol treatment during the fust postnatal week reduces the mean number of Purkinje and granule cells per section. P21 cerebellar cell counts following second postnatal week ethanol treatment Figure 4-5 shows cerebellar cell counts at P21 in ethanol-treated and control animals following second postnatal week exposure a developmental time during which the cerebellar vermis has been shown to be less susceptible to ethanol-mediated neurotoxicity (Hamre and W e st 1993). The ANOVA showed no significant effect of ethanol treatment on the mean number of Purkinje cells per section in lobule I following ethanol delivery on P7-8. Similarly no significant effect of ethanol treatment was noted on the mean number of granule cells per section in lobule I following ethanol delivery on P7-8 Thus ethanol delivered during the second postnatal week did not reduce the mean number of Purkinje cells per section or granule cells per section in lobule I. PS cerebellar cell counts following first postnatal week ethanol treatment In order to resolve the timecourse of ethanol-induced neurotox i city for cerebellar neurons exposed durin g th e first postnatal week we performed cell counts at PS

PAGE 124

114 following ethanol delivery on P4-5 (figure 4-6) The ANOVA determined a significant effect of treatment on the number of Purkinje cells per section in lobule I at PS (F[2 10] = 5.80; p < 0 05) The PLSD post hoc test found that ethanol treatment from P4-5 reduced the mean number of Purkinje cells per section in EtOH pups compared with both GC (p < 0.05) and SC (p < 0.05) pups Granule cells were also counted in the external and internal granule layer on PS. No effect of treatment was found on the number of granule cells per section at PS in the external or internal granule layer (see figure 4 6). Therefore there is a significant loss of Purkinje cells as early as PS following two-day exposure during the first postnatal week. The lack of reduction in granule cell number following ethanol treatment is likely due to the proliferative potential of granule cells during the first postnatal week ( Altman 1969). bcl-2 family mRNA levels following first postnatal week ethanol exposure Figure 4-7 shows bcl-2 family mRNA levels in the vermis, normalized to cyclophilin, following acute ethanol treatment on P4 (pups were sacrificed on P4 two hours after ethanol treatment). The ANOVA found a significant effect of treatment on bax (F[2 21] = 3 929 ; p < 0 05) and bcl-xs (F[2 21] = 10. 001; p < 0 001) mRNA levels but not bcl-xl or bcl-2. The post-hoc test determined that bax was significantly up regulated in EtOH pups compared with GC (p < 0 05) and SC (p < 0.05) pups. bcl-xs was also significantly up-regulated in EtOH pups compared with GC (p < 0 001) and SC pups (p < 0 001) Therefore acute ethanol exposure on P4 significantly up-regulated transcripts encoding pro-apoptotic members of the bcl-2 family specifically bax and bcl-xs without altering transcripts encoding anti-apoptotic members of the bcl-2 family (bcl-xl and bcl-2)

PAGE 125

115 Figure 4-8 shows bcl-2 family mRNA levels normalized to cyclophilin following two-day ethanol treatment on P4-5 (pups were sacrificed on PS two hours after ethanol treatment). No significant effect of treatment on bax bcl-xs bcl-xl or bcl-2 transcripts was noted. Therefore two-day ethanol treatment did not produce further alterations in mRNA levels of members of the bcl-2 family probably because the susceptible Purkinje cells are already dying by PS. bcl-2 family mRNA levels following second postnatal week ethanol exposure For comparison we also examined bcl-2 family gene express ion following ethanol treatment during the second postnatal week This corresponds to a developmental time during which the cerebellar vermis has been shown to be insusceptible to ethanol mediated neurotoxicity (Hamre and West 1993) Figure 4-9 shows bcl-2 family mRNA levels normalized to cyclophilin following acute ethanol treatment on P7 (pups were sacrificed on P7 two hours after ethanol treatment) The ANOV A showed a significant effect of treatment on bcl-xs mRNA levels (F[2 20] = 3.966 ; p < 0 05). The PLSD indicated that EtOH pups contained higher levels of transcripts encoding bcl-xs compared with SC pups (p < 0 .05), but not GC pups (the more appropriate comparison for EtOH pups since both GC and EtOH pups are gastrostornized and reared artificially). However there was no significant effect of treatment on bax bcl-xl or bcl-2. Thus it does not appear that acute exposure during the second postnatal week up-regulates transcripts encoding pro-apoptotic members of the bcl-2 family to the same degree that acute exposure during the first postnatal week (P4) does. Figure 4-10 shows bcl-2 family mRNA levels normalized to c y clophilin following two-day ethanol treatment on P7-8 (pups were sacrificed on P8 two hours after

PAGE 126

116 ethanol treatment) The ANOVA determined that bax (F[2 18] = 12.34; p< 0.001) and bcl-xs (F[2 l 7] = 11.085; p< 0.01) mRNAs were altered by ethanol treatment. The PLSD showed that EtOH pups contained higher levels of transcripts encoding bax compared with GC (p < 0 001) and SC (p < 0 001) pups. The vermis of EtOH pups also contained higher levels of transcripts encoding bcl-xs compared with SC (p< 0.01) pups. EtOH and GC animals (the most critical comparison) were not different but GC pups did contain higher vermal levels of transcripts encoding bcl-xs compared with SC (p < 0.01) indicating an effect of artificial rearing. However no significant effect of ethanol treatment on bcl-xl or bcl-2 mRNAs was found. Therefore two days of ethanol exposure during the second postnatal week can induce pro-apoptotic processes just as acute exposure during the first postnatal week can Discussion This study has shown that mRNAs encoding certain pro-apoptotic members of the bcl-2 family (bax and bcl-xs) are up-regulated in the cerebellar vermis of ethanol exposed neonatal rats. Acute ethanol treatment on P4 induces pro-apoptotic, but not anti apoptotic gene expression and also results in decreased Purkinje and granule cell numbers. An additional exposure on PS does not produce further alterations in this gene expression Ethanol administered during the second postnatal week does not produce cerebellar cell loss and results in the induction of pro-apoptotic (bax) gene expression but onl y after two days of ethanol treatment and not after acute one day exposure as during the earlier period Therefore the differential susceptibility of the developing vermis to ethanol-induced c e ll death i s not directly related to changes in the up-regulation of

PAGE 127

117 mRNAs encoding pro-apoptotic members of the bcl-2 family. Rather, the major difference between first and second postnatal weeks may be the differential ability of cerebellar neurons to respond to ethanol-induced up-regulation of pro-apoptotic gene expression What follows is a discussion of the effects of ethanol exposure during these stages on brain morphology, measurements of vermis size, cerebellar cell counts and finally bcl-2 family mRNA expression Effect of neonatal ethanol treatment on body weight, brain weight, and brain to body weight ratio Acute neonatal ethanol exposure on P4 did not significantly affect the growth of the pups in the present study since P21 body weight in EtOH and GC animals was not different. However a non-specific effect of artificial rearing was noted on P21 body weight following acute P4 exposure as both EtOH and GC animals weighed less than the SC animals. Regardless the fact that EtOH and GC animals did not differ in body weight on P21 demonstrates that this pattern of neonatal ethanol exposure does not, by itself decrease growth. This is due to the fact that EtOH and GC animals are the most appropriate comparison in this neonatal exposure paradigm and these groups were not different. Similar effects were noted for P21 brain weight with the brains of SC animals weighing more than both gastrostomized groups following acute P4 exposure No differences existed between EtOH and GC animals for P21 brain weight following this pattern of exposure. Not surprisingly brain to body weight ratios followed a similar trend with SC animals reduced compared to EtOH and GC due to the increased body weight previously noted. The brain to body weight ratio of EtOH and GC pups was not

PAGE 128

118 different demonstrating that this pattern of acute P4 ethanol exposure does not significantly alter the growth of the brain. Ethanol treatment from P4-S also did not significantly alter the growth of the pups in this study Body weight was not significantly different in any group at P4, PS, or P21 Brain weight was similarly unaffected at P4 and PS, but by P21 a significant decrease was noted in the EtOH group compared with the GC and SC group Thus, two days of ethanol treatment resulted in microencephaly, while only one day of exposure did not. Brain to body weight ratios were unaffected by ethanol treatment as measured on P4 and P21 indicating that the observed microencephaly at P21 was due to the smaller (but not significantly smaller) size ofEtOH animals at P21. The brain to body weight ratio was increased in SC animals on PS. Since EtOH and GC animals did not differ however it appears that ethanol treatment, per se did not alter brain to body weight ratio. Rather a non-specific effect of gastrostomy was noted. Nonetheless the data taken together demonstrate no direct ethanol effect on relative brain growth at any age examined. Ethanol exposure during the second postnatal week from P7-8 also did not alter the growth of either gastrostomized group or the suckle control group Body weight was unaffected by treatment on P7 P8 or P21. Brain weight was unaffected at P7 but on P8 the brains of GC animals weighed significantly less than the SC animals indicating a non-specific effect of gastrostomy on brain weight in these pups. Ethanol treatment did not alter brain weight on P8, as EtOH and GC pups were not different but by P21 the brains of E tOH animal s weighed significantl y less than SC pups but not GC pups. Brain to body wei g ht ratios were unaffected by second postnatal week exposure as measured on

PAGE 129

119 P7 and P21. However this ratio was increased in SC animals on P8 compared with both EtOH and GC animals; EtOH and GC pups were not different. The results presented here for animal growth (body weight) are similar to those reported in Hamre and West (1993) In both studies utilizing a similar neonatal ethanol exposure paradigm no significant differences in body weight were noted between the EtOH and GC group even though the growth of the gastrostornized pups lagged behind that of the SC pups following return to the dam. Unfortunately, Hamre and West did not report brain weight or brain to body weight ratios following the oneor two-day pattern of exposure making comparisons to the current data impossible. Goodlett et al. (1990) however, did report brain weight following a single ethanol exposure on P4 using the same dose as used in the current study They reported significant brain weight declines in EtOH pups as measured on PIO. These data are in contrast to the data presented here as no significant decline was noted in our pups as measured on P21 following similar one-day ethanol exposure. Indeed, in our hands brain weight differences were only noted following two-day ethanol treatment from P4-5 It is possible that pups used in the current study exhibited "catch up" growth following return to the dam which would account for the discrepancy. Neonatal ethanol and the size of the cerebellar vermis and diameter of cell nuclei within the vermis The only significant volume measure that was affected by any pattern of neonatal ethanol exposure (P4 only P4-5 or P7-8) was the mean number of sections through the vermis on P21 following ethanol exposure on P4-5 (lobule length and cell sizes were unaffected) While vermal section number is reduced by P21, measurements on P5

PAGE 130

120 indicate that these size reductions do not occur until after the P5 timepoint. For all other exposure paradigms the size of the vermis was unaffected by treatment. Likewise for all other exposures lobule length and cell size were unaffected. Because we found no universal volume reductions it is unlikely that our cell counts are biased because of changes in the size of the cerebellar vermis. Exposure on P4-5 for instance, produced both size alterations and neurotoxicity while exposure only on P4 produced no size alterations but also resulted in cerebellar cell death. Similarly P4-5 exposure produces cell number reductions at both the P5 and P21 timepoints and the size of the vermis is unchanged at P5 but not P2 l. This trend of fust postnatal week reduction in the size of the vermis is consistent with the trends noted in brain weight, namely that ethanol exposure on P4 only produces neither vermis nor brain weight reductions while two day treatment on P4-5 is capable of inducing these declines It should be noted however that the cell loss noted in this study is independent of changes in either the size of the brain or the size of the vermis in these animals. Unfortunately recent reports which have utilized single day (e.g P4 only) neonatal ethanol exposure have not reported data on cerebellar size (Goodlett and Eilers 1997) and comparisons to our data are impossible Hamre and West (1993) however did report Purkinje cell layer length and found that P4-5 ethanol exposure significantly reduced the length of the Purkinje cell layer at P2 l These results are in contrast to our own data regarding lobule length which was unaffected by this same pattern of exposure. Our data do indicate though a change in the size of the vermis following P4-5 exposure based on the reduced mean number of sections through the vermis (a measure that Hamre

PAGE 131

121 and West did not report). In agreement with our data Hamre and West ( 1993) did not find any size alterations following P7-8 exposure. Other reports have also examined the size of the nuclei of cerebellar neurons following neonatal ethanol exposure notably Hamre and West (1993). In agreement with our data no ethanol-induced alterations in the size of granule cell nuclei were found following ethanol exposure on P4-5 or P7-8. The y did however report an increase in Purkinje cell nuclear diameter following P4-5 or P7-8 exposure. It should be noted however that these diameters were not measured directly but instead were extrapolated from Purkinje cell area measures. Moreover it is difficult to make a convincing case that Purkinje cell diameters are directly altered by ethanol treatment when other studies from this group have not found an effect of similar patterns of ethanol exposure on cerebellar cell diameter (Pierce et al. 1989). The measures for P21 vermis size and cell nuclei size were generally consisten t across experiments utilizing the different ethanol exposure times. While some litter and individual variation was noted it appears that period of exposure had little or no effect on these parameters The mean number of sections and cell nuclear diameters were most similar across experiments while lobule length showed more v ariability. For example the animals used for the P4-5 treatment groups displayed larger lobules than either o f the other groups measured on P21 (note that no group differences were found in these animals) while Purkinje and granule cell nuclear size measurements and sect i on number displa y ed less variabil i ty Not surprisingly the data demonstrate that the vermis size and cell nuclear si z e measurements were smaller for P5 animal s than for P21 animals

PAGE 132

122 Differential temporal vermis susceptibility to developmental ethanol The differential temporal pattern of vermis susceptibility to developmental ethanol exposure noted here is similar to that seen by other investigators (Goodlett and Eilers, 1997 ; Hamre and West, 1993 ; Pauli et al., 1995). The present study demonstrates that oneor two-day ethanol treatment during the first postnatal week reduced the mean number of Purkinje and granule cells per section in lobule I of the vermis as determined on P21. Also we have documented that two-day exposure produced significant Purkinje (but not granule) cell loss as early as P5. In contrast, two days of ethanol treatment during the second postnatal week (from P7-8) did not reduce Purkinje or granule cells per section as measured on P21. Goodlett and colleagues have used similar exposure methods and similar patterns of ethanol exposure to document the tight window of vulnerability of the cerebellar vermis. These investigators have determined that acute P4 exposure produces cerebellar cell loss while exposure on P9 does not (Goodlett and Eilers, 1997) Interestingly in a recent report (Thomas et al. 1998) a slight (approximately 15%) reduction in Purkinje cells following P8-9 exposure was noted These results stand in contrast to results from second postnatal week exposure in the current study which found no significant reduction in Purkinje cells following P7-8 exposure They also differ from an earlier report which showed no Purkinje cell loss following P7-8 exposure (Hamre and West, 1993). Regardless it is obvious that the first postnatal week is a period of heightened vulnerability while exposure in the second postnatal week produces little or no cell loss. Th e timecours e of Purkinj e cell death noted in the present stud y is cons i stent with that noted in another report demonstrating Purkinje cell loss as early as 12 hours

PAGE 133

123 following a P3 ethanol insult (Cragg and Phillips 1985). While our data show that granule cells do not follow this rapid timecourse of cell death this is not surprising. The lack of effect on granule cell numbers at early timepoints may be because cerebellar granule cells maintain their proliferative potential during the neonatal period (Altman 1969) Therefore, direct ethanol-induced granule cell loss may be masked by compensatory proliferation. Another possibility is that fewer granule cells are dying following ethanol exposure at this time before the period of cell death Alternatively granule cells may be experiencing a secondary (and more prolonged) loss due to lost Purkinje cell targets as hypothesized in other studies (Hamre and West 1993). Regardless we find a similar degree of long-term (P21) Purkinje and granule cell loss following first postnatal week ethanol treatment as other groups (Hamre and West 1993 ) and we provide another demonstration of the developing cerebellum s tight window of vulnerability. Because the loss of granule cells appears to be secondary to Purkinje cell death (Hamre and West 1993) the key factor in cerebellar susceptibility is whether Purkinje cells are initially killed by the ethanol insult. Investigators have hypothesized that some property of Purkinje cells in the early stages of differentiation ( or those in less mature stages for example during generation and proliferation) makes them more susceptible to ethanol than more mature neurons (Goodlett and E ilers 1997). Because differentiating Purkinje cells are also in the peak phase of naturally occurring cell death (Cragg and Phillips 1985) we hypothesized that the differential susceptibility might be related to ethanol-induced alterations in bcl-2 family g ene expression. We chose to investigate transcription of bcl2 family genes with demonstrated Purkinje and granule cell

PAGE 134

124 expression during neonatal rodent development specifically bcl-2 bcl-xl, bcl-xs, and bax (Castren et al. 1994 ; Frankowski et al., 1995; Krajewski et al., 1994; Rouayrenc et al., 1995; Vekrellis et al. 1997). Neonatal ethanol and bcl-2 family gene expression during the first postnatal week Ethanol exposure to neonatal rats during the first postnatal week a developmental timepoint corresponding with cerebellar neuronal susceptibility to ethanol, produced an up-regulation of mRNA transcripts encoding pro-apoptotic members of the bcl-2 gene family. Specifically acute ethanol exposure on P4 significantly up-regulated transcripts encoding bax and bcl-xs two hours following the ethanol insult without altering transcripts encoding anti-apoptotic members of the bcl-2 family (bcl-xl and bcl-2). We also examined bcl-2 family transcripts on PS after two-day ethanol treatment from P4-5. This pattern of exposure did not produce further alterations in mRNA levels of any members of the bcl-2 family examined (bax bcl-xs bcl-xl or bcl-2) The lack of significant alterations in bcl-2 family mRNAs on PS following ethanol exposure on P4-5 is presumably because the susceptible Purkinje cells undergo cell death as early as PS (see above). We do not therefore detect any significant alterations in bcl-2 family mRNAs on PS because we are measuring the transcription of bcl-2 family mRNAs in the surviving cells which appear to be able to withstand the ethanol insult without further changes in the expression of bcl-2 family genes One important point to be considered is that the use of the RNase protection assay does not provide for identification of the cell type-specific changes in gene expression. Because the bcl2 fami l y expression in the verrnis includes Purkinje and granule cells future investigations utilizing in situ hybridization will be necessary to determine neuron-

PAGE 135

125 specific changes in gene expression. Thus an alternative explanation for the P5 da t a is that granule cell expression is masking Purkinje cell expression due to the greater numbers of granule cells. In other words Purkinje cell expression could still be altered but granule cell expression is masking these changes Another important consideration comes from the fact that ethanol-induced alterations in bax and bcl-xs are noted on P4 but granule cell death is not detectable until well after this age While Purkinje cell death was evident as early as P5 following P4 or P4-5 exposure, concomitant reductions in granule cells were not noted until P21. Thus granule cell numbers are reduced by oneor two-day ethanol exposure in the first postnatal week but they undergo a prolonged loss suggesting that the noted changes in pro-apoptotic gene expression on P4 do not occur in granule cells and do not positively correlate with granule cell death. What factors then are responsible for the eventual death of granule cells? As discussed above other investigators have noted that granule cell death appears to be secondary to Purkinje cell death following ethanol treatment (Hamre and West 1993). We speculate that granule cell death in our hands is due to the devastating reductions in the Purkinje cell population. As granule cells begin to exit the proliferative stage and enter the differentiation stage fewer Purkinje cell targets are available for synapse formation resulting in increased granule cell death due to lack of target-derived support. Of course it is possible that bax and bcl-xs expression is elevated in granule cells, but that they are better able to survive the elevation perhaps because of elevated anti-apoptotic expression of bcl-2 family members not investigated in this study. Future investigations utilizing in situ hybridization could definitively determine the anatomical

PAGE 136

126 localization of the noted expression changes in pro-apoptotic genes and would be useful for determining whether compensatory changes in anti-apoptotic genes occurred in granule cells A lack of induction of anti-apoptotic genes in granule cells would suggest that loss of targets is responsible for the prolonged loss of granule cells Our data are consistent with reports of altered bcl-2 family expression from other groups working in different models of brain injury. For example studies in mouse brain have shown up-regulation of Bax protein and down-regulation ofBcl-2 protein associated with kainate-induced apoptosis (Gillardon et al. 1995) Similar protein expression changes in Bax and Bcl-2 have been seen in hippocampus and cerebellum following ischemia (Krajewski et al. 1995). Ischemia also up-regulates bcl-xs mRNA in the hippocampus (Dixon et al. 1997). Additionally the Bcl-2:Bax ratio is decreased in rat motoneurons following sciatic nerve transection (Gillardon et al. 1996) and experiments in Purkinje-cell-degeneration mice have determined that a decreased bcl-2:bax ratio (at the mRNA and protein levels) immediately precedes the gradual loss of Purkinje cells (Gillardon et al. 1995). Therefore it is clear that alterations in bcl2 expression both at the message and protein levels correlates positively with cell death in various model systems of brain injury. In order to determine whether the alteration in bcl-2 family gene expression noted in the present study was specific to first postnatal week ethanol neurotoxicity (and positively correlated with ethanol-induced cell death) ethanol was delivered during a developmental period o f cerebellar insusceptibility the second postnatal week Acute e x po s ure on P7 did not produce the same dramatic induction of pro-apoptotic g ene

PAGE 137

127 expression that was noted two hours after treatment on P4. In fact, EtOH and GC pups did not d iffer in mRNA levels for any bcl-2 family member tested Two-day ethanol treatment during the second postnatal week on da ys 7-8, however, induced pro-apoptotic gene expression as measured on P8. Transcripts encoding bax were significantly up-regulated following this pattern of exposure. bcl-xs was induced by artificial rearing (both gastrostomized groups had elevated bcl-xs compared with SC pups) but ethanol treatment, per se, did not significantly alter bcl-xs expression. Nonetheless, the up-regulation of bax after two-day ethanol treatment suggests that an additional ethanol exposure durin g the second postnatal week induces pro-apoptotic gene expression. Therefore, it is not simply a difference in the induction of pro-apoptotic processes following second postnatal week ethanol exposure that is responsible for the difference in cerebellar neuronal loss. Differential cerebellar susceptibility and the bcl-2 family The differential susceptibility of the cerebellar vermis to ethanol during the first and second postnatal week is possibly due to differences in the timecourse of the induction of pro-apoptotic mRNA. Indeed, the present study documents that mRNAs encoding pro-apoptotic members of the bcl-2 family are up-regulated by only one day of ethanol treatment during the first postnatal week whereas two exposures are necessary to induce pro-apoptotic mRNAs during the second week. A more likely difference however may be the ability of cerebellar neurons to respond to the increased transcription of pro-apoptotic genes. We speculate that the critical difference may be better suppression of pro-apoptotic processes, possibly by growth factors, in more mature cerebellar neurons following ethanol exposure in the second postnatal week.

PAGE 138

128 Several neurotrophins including nerve growth factor (NGF) brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) have distinct influences on the survival and differentiation of cerebellar Purkinje and granule cells during development. Neurotrophin receptors are expressed at high levels in rat Purkinje and granule cells during development and both ligands and receptors are regulated in a spatio-temporal manner (Lindholm et al. 1997). For example, only differentiating granule cells (and not proliferating granule cells) express Trk B receptors and respond to BDNF and NT-3 (Gao et al. 1995) Furthermore Purkinje cell expression oftrkA mRNA is detectable at P4 but only attains peak levels during later differentiation around P 10 (W anaka and Johnson 1990). This spatio-temporal pattern of trkA expression correlates with periods of ethanol-induced cerebellar cell death. Notably during the early ontogeny of trkA (before the peak of its expression) Purkinje cells are susceptible to ethanol. However during the peak period of trkA mRNA expression Purkinje cells are not vulnerable to ethanol. Better established neurotrophic processes in the more mature cerebellum may explain the ability of Purkinje cells to withstand an ethanol insult. Growth factor-mediated post-translational modulation ofbcl-2 family members has been demonstrated particularly with regard to the phosphorylation of the pro apoptotic molecule Bad (Datta et al. 1997 ; Gajewski and Thompson 1996 ; Zha et al. 1996) Bad phosphorylation inactivates its death-promoting properties by inhibiting its association with (and inactivation of) anti-apoptotic molecules. Growth factor-mediated activation of PB kinase followed by PB kinase phosporylation and activation of Akt (Kahn 1998) leads to Akt-mediated Bad phosphorylation (Datta et al. 1997 ; Delpeso et al., 1997 ; Zhou et al. 1997). Furthermore endogenous bcl-2 expression plays a role in

PAGE 139

129 the survival of BDNF-dependent neurons (Allsopp et al. 1995) and similar expression is induced by NGF in developing cerebellar granule cells (Muller et al., 1997) The main influence which may mediate differential cerebellar ethanol susceptibility is the ability of target-derived growth factors to suppress pro-apoptotic or possibly enhance anti apoptotic mechanisms following ethanol exposure Therefore an important hypothesis to test is that the key factor governing cerebellar neuronal susceptibility to ethanol is the presence of a target derived growth factor source to suppress pro-apoptotic processes. For example the present study indicates that cerebellar neurons exposed to ethanol during the second postnatal week experience an induction of pro-apoptotic processes but exhibit no cell death It is possible that growth factor-induced phosphorylation of Bad compensates for the concomitant increase in pro-apoptotic mRNAs by enabling sufficient Bcl 2 function. Bcl-2 homodimerization (and functionality) would be favored in the presence of phosphorylated Bad because of cytosolic sequestering of Bad. While we presume from the present study that increased Bax and Bcl-xs will be competing with Bcl-2 for dimerization it is possible that growth factor modulation of Bad enables sufficient homodimerization of anti-apoptotic members of the bcl-2 family to continue to regula t e mitochondrial membrane potential and stave off cell death. Perhaps the lack of growth factor-induced Bad phosphorylation in the first postnatal week along with the ethanol induced induction of pro-death genes would lead to inadequate Bcl-2 function resulting in lost mitochondrial membrane potential release of cytochrome C and caspase activation. The intracellular mechanisms of bcl-2 family function are discussed in detail below.

PAGE 140

130 Intracellular function of bcl-2 family members While the complex intracellular mechanisms of various bcl-2 family members is still being worked out recent evidence has revealed that certain members of the family function as both ion channel and adapter protein (Reed 1997). Anti-apoptotic (Bcl-2 and Bcl-xl) and pro-apoptotic (Bax) members of the bcl-2 family are known to form functional ion channels in lipid membranes (Antonsson et al. 1997 ; Minn et al. 1997; Schendel et al. 1997). In addition, it is clear that the ability of Bax homodimers to form channels is inhibited by Bcl-2 (Antonsson et al. 1997) indicating that competition for dimerization is an important determinant of cell survival. These ion channels influence cell survival by regulating the permeability of the membranes in which they are anchored, and in particular by influencing the release of cytochrome C from mitochondria (Yang et al. 1997) Cytochrome C is a known co-factor for caspase activation (Yang et al., 1997). Disruption of mitochondrial membranes an early event in PCD results in the release of cytochrome C and the activation of the effector phase of PCD (Petit et al. 1996) through cytochrome C-dependent cleavage and activation of caspases 3 (Li et al. 1997) and 9 (Zou et al. 1997) Interestingly recent evidence shows that Bcl-xl (and presumably Bcl2) binds to Apaf-1 the newly discovered mammalian homolog of the C. elegans CED-4 (Zou et al., 1997). These two molecules, along with the uncleaved and inactive caspase 9 exist in a ternary complex (Pan et al. 1998). How cytochrome C release helps to activate this complex remains unknown. Another important role of Bcl-xl and Bcl-2 has been described by Yang et al. (1998) They have shown that CED-4 (and likely Apaf-1) promotes the processing and

PAGE 141

131 activation of CED-3 by promoting the aggregation of unprocessed CED-3 Increasing the local concentration of CED-3 through induced proximity" is thought to sequester inactive caspase proenzyrnes and promote conformational changes which will increase the likelihood of their activation (Hengartner, 1998). This is brought about by oligomerization of the CED-4:CED-3 complex (Yang et al. 1998). Thus another function of anti-apoptotic members of the bcl-2 family appears to be to prevent the activation of these proenzymes by preventing CED-4 oligomerization (Hengartner, 1998) Yet another wrinkle in the comp l icated intracellular process of cell death is the fact that anti-apoptotic molecules of the bcl-2 family can contribute to the cell s demise under certain pathological conditions. If caspase activation proceeds to a critical point they begin to act on the Bcl-2 and Bel-xi proteins as substrates (Cheng et al., 1997; Clem et al. 1998). Caspase cleavage of these protective molecules converts them into Bax like death-promoting molecules Thus cleavage of anti-apoptotic molecules appears to act as a feed-forward mechanism for further caspase activation ensuring cell death. Future investigations should attempt to characterize these intracellular processes following developmental ethanol exposure and the data presented here offer many new avenues of research. For instance, is Bax channel formation enhanced by ethanol? Our data indicate increased bax gene expression and suggest that ethanol would increase Bax homodimerization and channel formation. Other questions include is cytochrome C release increased by ethanol? Are caspases activated by ethanol and are anti apoptotic molecules cleaved by active caspases ?

PAGE 142

132 Conclusion and significance While other reports have demonstrated bcl-2 expression changes in certain brain injury paradigms this study is the first to document such changes following ethanol treatment. The significance of this work is that it suggests new avenues of research on ethanol s teratogenic actions and suggests that suppression of pro-apoptotic processes might be of therapeutic benefit. Recent data from our lab indicate that mice over expressing a bcl-2 transgene in nervous tissue (including cerebellar Purkinje cells) are resistant to ethanol-mediated Purkinje cell death (Heaton et al., unpublished observation). In conclusion, the present data indicate that the modulation of pro-apoptotic processes may have important implications for neuronal death in animal models of developmental ethanol exposure

PAGE 143

133 Table 4-1. Lobule I cerebellar vermis volume, area, and cell nuclei diameter data at P21 following exposure on P4 only Diet group N Section number Lobule length Purkinje nuclear Granule nuclear (mm) diameter (m) diameter (m) EtOH 3 260.3 + 3.667 4.167 + 0.434 27 07 + 0.758 7.37 + 0.315 GC 3 279.0 + 15.00 4.933 + 0.067 28 54 + 1.120 7.16 + 0.467 SC 4 283.3 + 10.41 4.782 + 0.309 28.28 + 1.166 7.75+ 0.185 All measures are expressed as mean SEM. No s1gruficant differences were noted. Table 4-2. Lobule I cerebellar vermis volume area, and cell nuclei diameter data at P2 l following exposure on P4-s* Diet group N Section number Lobule length Purkinje nuclear Granule nuclear (mm) diameter (m) diameter (m) EtOH 6 247.6 + 12.30t 6.445 + 0.672 22.79 + 0.625 7.026 + 0.187 GC 4 338.0 + 15.27 6 .792 + 0 330 21. 80 + 0.834 6.702 + 0.304 SC 3 326.7 + 16.76 7.000 + 0.995 23.46 + 1.155 6.793+ 0.396 All measures are expressed as mean SEM. tS1gruficantly reduced compared wtth GC and SC animals (p< 0.01). Table 4-3. Lobule I cerebellar vermis volume, area and cell nuclei diameter data at P21 following exposure on P7-s* Diet group N Section number Lobule length Purkinje nuclear Granule nuclear (mm) diameter (m) diameter (um) EtOH 5 297.4 + 17.08 4 638 + 0.434 21. 80 + 0.474 7.270 + 0.218 GC 6 284.2 + 12.65 5.675 + 0.449 22.39 + 0.729 7.330 + 0.257 SC 4 312.0 + 18.86 5.070 + 0.458 23.63 + 0.834 6.700 + 0.294 All measures are expressed as mean SEM. No s1gruficant differences were noted.

PAGE 144

134 Table 4-4. Lobule I cerebellar vermis volume, area and cell nuclei diameter data at PS following exposure on P4-s* Diet group N Section number Lobule length Purkinje nuclear (mm) diameter (m) EtOH 6 192.6 + 14.83 3 380 + 0 900 14 75 + 0.375 GC 4 158.0 + 22.90 2. 700 + 1 .250 15.00 + 0.350 SC 3 185.0 + 6.430 2.690 + 1.350 15.25 + 0.400 All measures are expressed as mean SEM. No significant differences were noted.

PAGE 145

Figure 4-1. Effects of ethanol delivered on P4 only on P21 body weight (panel A) P21 brain weight (panel B) and P21 brain to body weight ratio (panel C). Data are expressed as mean SEM. a : significantly increased compared with EtOH and GC pups (p < 0.01) ; b: significantly increased compared with EtOH and GC pups (p < 0.01) c: significantly decreased compared with EtOH and GC pups (p < 0.01)

PAGE 146

136 Ethanol Exposure on P4 Only A Body Weight 75 a --bl) .d -~ 50 ;;.-. "O 0 25 .... M 0 EtOH GC SC B Brain Weight b 1.5 -bJ) .._, --= bJ) -1.0 Q1 = ; ... 0.5 M 0.0 EtOH GC SC C Brain to Body Weight Ratio 3 .d bl) .... .... ~M 2 ;;.-.~ "O -0 0 o=0:: ; '0 EtOH GC SC

PAGE 147

Figure 4-2. Effects of ethanol delivered on P4-5 on P4 PS, and P21 body weight (panel A) brain weight (panel B) and brain to body weight ratio (panel C). Data are expressed as mean SEM. a: significantly decreased compared with EtOH and GC pups (p < 0 01) b: significantly increased compared with EtOH and GC pups (p< 0 01).

PAGE 148

Ethanol Exposure on P4-5 A Body Weight 75 50 ..... -= bJ) o ........... ___._. 138 P4 Body Weight P5 Body Weight P2 l Body Weight B Brain Weight 1.5 1 0 ..... -= bJ) ... 0.5 a 0 0 P4 Brain Weight P5 Brain Weight P2 l Brain Weight C Brain to Body Weight Ratio ..... -= bJ) ; 5 4 -.~ 3 0 ..... =~ 0 ..... = ... co: i.. = 2 0 b P4 Br/ Bd % P5 Br/ Bd % P21 Br /Bd % c::::::J EtO H GC -SC c::::::J EtO H GC -SC c:J EtOH GC -SC

PAGE 149

Figure 4-3. Effects of ethanol delivered on P7-8 on P7 PS, and P21 body weight (panel A) brain weight (panel B) and brain to body weight ratio (panel C) Data are expressed as mean S EM. a: significantly decreased compared with SC pups (p < 0.05). b: significantly decreased compared with SC pups (p < 0.05) c: significantly increased compared with EtOH and GC pups (p < 0.001).

PAGE 150

140 Ethanol Exposure on P7-8 A Body Weight 75 -50 --= bf) 0 ___.__._ P7 Body Weight pg Body Weight P2 l Body Weight B Brain Weight 1.5 -1.0 --= bf) .... 0.5 0 0 P7 Brain Weight pg Brain Weight P2 l Brain Weight C Brain to Body Weight Ratio 5 --= bf) 4 ;,... 0 3 "0 .... o= 0~ 2 = .... "" = C pg Br/Bd % P 2 I Br / Bd % c:::J EtOH GC -SC c::J EtOH GC -SC c::J EtOH GC -SC

PAGE 151

Figure 4-4. Ethanol delivered during the first postnatal week reduces mean Purkinje and granule cell number per section in lobule I of the cerebellar vermis. The mean number of Purkinje and granule cells per section in lobule I of the cerebellar vermis was determined at postnatal day 21 (P21) in 6 m H&E-stained sections following ethanol or control exposure via artificial rearing on P4 (panels A and B) or P4-5 (panels C and D). Exposure to ethanol for one (P4) or two (P4-5) days during the first postnatal week significantly reduced Purkinje and granule cell number in ethanol (EtOH) pups compared with gastrostomized control (GC) and suckle control (SC) pups Data are expressed as mean SEM a: significantly reduced compared with GC and SC (p< 0.05). b: significantly reduced compared with GC and SC (p < 0.0001).

PAGE 152

14 2 Ethano l Exposure on P4 Only A Purkinje C e lls B Granule Cells so ISO .~ c 4Q a C C 0 :, 0 :: C -1 00 ; 30 a ~ -.. c.. ~1!2 C _: C "' 20 ~= id .., .., so ~ u 1 0 0 EtOH GC SC 0 EtOH GC SC Ethanol E x posure on P4-5 C Purkinje Cells D Gran ul e C e ll s so 200 .~ C 40 a C 0 C :: :, 0 C ; 30 ~-.. c.. l!2 100 C _: C "' : 4i 20 ~ = b .., .., ~u ~ u 1 0 0 0 EtOH GC SC EtOH GC SC

PAGE 153

143 Ethanol Exposure on P7-8 A Purkinje Cells B Granule Cells 5 0 .S!, c 40 C 0 :2 :: l3 c.i 30 i:i..~ 2 0 ~u 10 0 200 C = 0 = t: ... ., c., I 00 = "' ~= ., ., ~u EtOH GC S C 0 E tOH GC SC Figure 4-5. Ethanol delivered during the second postnatal week does not reduce mean Purkinje or granule cell number per section in lobule I of the cerebellar vermis. The mean number of Purkinje (panel A) and granule (panel B) cells per section in lobule I of the cerebellar vermis was determined at postnatal day 21 in 6 m H&Estained sections following ethanol or control exposure via artificial rearing on P7-8 Exposure to ethanol during the second postnatal week did not alter mean number of Purkinje or granule cells per section. Ethanol exposed (EtOH) gastrostornized control (GC), suckle control (SC). Data are expressed as mean SEM. No significant differences were noted

PAGE 154

144 Ethanol Exposure on P4 5 A Purkinje Cells B Granule Cells 75 .!, C C 0 :.i! .;:: so ...... = 4,1 g..~ "' =-OI -:i U 25 0 150 c:::J EtOH a ~" -GC "0 -SC :l 100 .. ~'.i .. = .... :;u 50 EtOH GC SC 0 f.GL !GI.. Figure 4-6. Ethanol delivered during the first postnatal week on days 4-5 reduces mean Purkinje but not granule cell number per section in lobule I of the cerebellar vermis as determined on postnatal day 5 (P5). The mean number of Purkinje (panel A) and granule (panel B) cells per section in lobule I of the cerebellar vermis was determined at P5 in 6 m H&E-stained sections following ethanol or control exposure via artificial rearing on P4-5 Exposure to ethanol from P4-5 significantly reduced Purkinje cell number in ethanol (EtOH) pups compared with gastrostomized control (GC) and suckle control (SC) pups while granule cells were not affected Data are expressed as mean SEM a: significantly reduced compared with GC and SC (p < 0 05)

PAGE 155

Figure 4-7 rnRNAs encoding proapoptotic molecules of the bcl 2 family are upregulated following acute ethanol de l ivered on postnatal day 4. rnRNA levels of bax (panel A) bcl-xs (panel B) bcl-xl (panel C), and bcl-2 (panel D), relative to the internal standard cyclophilin were determined in the cerebellar vermis following ethanol or control exposure via artificial rearing on P4. Pups were sacrificed two hours after the final ethanol or control infusion on P4 and the relative rnRNA levels determined via the RNase protection assay. S i gnificant increases in rnRNAs encoding the pro-apoptotic molecules bax and bcl-xs were observed after ethanol exposure (EtOH) on P4 when compared with gastrostomized control (GC) and suckle control (SC) pups. Data are expressed as mean SEM. a : significantly increased compared with GC and SC (p< 0 05) b : significantly increased compared with GC and SC (p < 0.001).

PAGE 156

146 mRNA l eve l s o n P4 F ollo w i ng Acu te P4 Ethanol Exposure A ba x B bcl-xs Q b 0 = a C -= :a Q. Q. 0 0 c:i v >. >. CJ ...... Q Q 0 0 ('l I .Q v .Q EtOH GC SC EtOH GC SC C bclx l D bcl 2 Q 0 = = -:a :a Q. Q. 0 0 c:i CJ >. >. CJ CJ ...... ...... Q Q 0 0 >< N I I c:i .Q EtOH GC SC EtOH G C SC

PAGE 157

Figure 4-8 A further ethanol exposure on PS does not significantly alter the expression of bcl-2 family mRNAs. mRNA levels ofbax (panel A), bcl xs (panel B) bcl-xl (panel C), and bcl-2 (panel D) relative to the internal standard cyclophi l in were determined in the cerebellar vermis following ethanol or control exposure via artificial rearing on P4-S. Pups were sacrificed two hours after the final ethanol or control infusion on PS, and the relative mRNA levels determined via the RNase protection assay. Ethanol exposed (EtOH) gastrostomized control (GC) suckle control (SC) Data are expressed as mean SEM. No significant differences were noted.

PAGE 158

148 mRNA le vels on PS Following P4-S Ethanol Exposure A bax B bcl-xs Q Q 0 0 = .!: i :a C. 0 0 c:i u >, >, u C,I Q ...... Q 0 0 "' I .&:I ] EtOH GC SC EtOH GC SC C bcl-xl D bcl-2 Q Q 0 0 = = :.a :.a C. C. 0 0 c:i >, C,I C,I ...... ...... Q Q 0 0 >< N I I c:i c:i .&:I .&:I EtOH GC SC EtOH GC SC

PAGE 159

Figure 4-9 Effects of acute ethanol delivered on postnatal day 7 on bcl-2 family gene expression rnRNA levels of bax (panel A) bcl-xs (panel B), bcl-xl (panel C) and bcl-2 (panel D ), relative to the internal standard cyclophilin were determined in the cerebellar vermis following ethanol or control exposure via artific i al rearing on P7. Pups were sacrificed two hours after the final ethanol or control infusion on P7, and the relati v e rnRNA levels determined via the RNase protection assay No significant alterations were noted for any transcript between ethanol (EtOH) and gastrostomized control (GC) pups although E tOH and suckle control (SC) pups were significantl y different in bcl-xs expres s ion. Data are expressed as mean SEM. a : significantly reduced compared with EtOH (p < 0.05 ).

PAGE 160

150 m.RNA levels on P7 Following Acute P7 Ethanol Exposure A bax B bcl-xs Q 0 0 C C :c :a C. C. 0 .!: c3 u >. >. u u Q ...... Q 0 0 "' C: ..!. u EtOH GC SC EtOH GC SC C bcl-xl D bcl-2 Q Q 0 0 C C :c .c C. C. 0 0 c3 u >. >. u u ...... Q Q 0 0 N I I u u EtOH GC SC EtOH GC SC

PAGE 161

Figure 4-10. An additional ethanol exposure on postnatal day 8 increases mRNAs encoding the pro-apoptotic molecule bax. mRNA levels of bax (panel A), bcl-xs (pane l B) bcl-xl (panel C) and bcl 2 (panel D), relative to the internal standard cyclophilin were determined in the cerebellar vermis following ethanol or control exposure via artificial rearing on P7-8. Pups were sacrificed two hours after the final ethanol or control infusion on P8 and the relative mRNA levels determined via the RNase protection assay. Significant increases in mRNAs encoding the pro apoptotic molecule bax were observed after ethanol exposure (EtOH) on P7 8 when compared with gastrostomized control (GC) and suckle control (SC) pups. SC pups contained significantly lower levels of mRNAs encoding bcl-xs when compared with EtOH and GC pups. Data are expressed as mean SEM a: significantly increased compared with GC and SC (p < 0 001). b: significantly reduced compared with EtOH (p< 0.05)

PAGE 162

152 mRNAlevels on P8 Following P7-8 Ethanol Expos ure A bax B bclxs Q a Q 0 0 = = :c :a C. C. 0 0 y ..... <.I <.I 2:S --Q 0 0 "' >< I .!:J EtOH GC SC EtOH GC SC C bcl-xl D bcl-2 Q Q 0 0 = = -:c :c C. C. 0 ..5: y <.I ..... ..... <.I <.I ----Q Q 0 0 N I I y <.I .!:J .!:J EtOH GC SC EtOH G C SC

PAGE 163

CHAPTERS CONCLUSIONS AND FUTURE DIRECTIONS Recapitulation of Results and Hypotheses Tested Developmental disorders arising from maternal consumption of ethanol during pregnancy have been described in the clinical literature. An expanding body of work in animals is helping clinicians and basic scientists to better understand these developmental disorders so that prevention and treatment of the debilitating fetal alcohol syndrome may be pursued Because ethanol exposure during development induces abnormal development in particular brain regions and is known to alter the expression of particular genes and their protein products the present body of work sought to further document neuronal populations in the brain which display vulnerability to developmental ethanol exposure Another goal of the present work was to investigate cell death gene expression shortly after ethanol insult in the cerebellum in order to test a specific hypothesis about the cellular mechanism of differential ethanol neurotoxicity in the developing cerebellum. The results presented in the preceding chapters are summarized below along with a recapitulation of the hypotheses that were tested and their relation to the collected data. 153

PAGE 164

154 Chapter 2: parvalbumin (PA) immunoreactivity in the medial septum and cingulate cortex following prenatal ethanol exposure The objective of this study was to determine the long-term effects of prenatal ethanol exposure on parvalbumin-expressing (PA + ) GABAergic neurons of the rat medial septum and anterior cingulate cortex. The hypothesis tested was that chronic prenatal ethanol exposure would lead to alterations in the number of neurons expressing PA in the adult rat medial septum and cingulate cortex. To test this we fed an ethanol-containing liquid diet, a similar diet with the substitution of sucrose for ethanol, or a lab chow control diet to pregnant rat dams. In order to examine the long-term effects of this pattern of exposure we sacrificed offspring of these dams on postnatal-day 60 and prepared their brains for parvalbumin immunocytochemistry. While this exposure paradigm did not produce alterations in the size of the medial septum or the size of the PA + neurons therein sexually dimorphic results were found for PA + neuronal number in this region. Female rats and not males exposed to the ethanol containing diet during gestation had 42 % fewer total PA + neurons in the medial septum compared with sucrose controls. Females also had reduced PA + cell density and fewer PA+ neurons per section when compared to female rats exposed to the sucrose diet. Male rats exposed to ethanol did not display a similar reduction in PA + density or neurons per section. The effect of prenatal ethanol exposure on the number of PA + GABAergic neurons in the adult rat anterior cingulate cortex was also determined. No sexually dimorphic results were found. Male and female rats exposed to the ethanol-containing diet contained 45% fewer total PA + neurons in the anterior cingulate cortex. Fewer PA +

PAGE 165

155 neurons per section were also found in ethanol-treated rats compared with sucrose and chow controls As with the MS, this reduction in PA+ neurons occurred in the absence of changes in the size of the region of interest or the size of PA+ neurons. Thus, our hypothesis was supported by the data, namely that alterations in the number of neurons expressing PA in the adult rat medial septum and cingulate cortex were noted after chronic prenatal ethanol treatment. One interesting aspect of these data was that the medial septum was differentially affected by prenatal ethanol in male and females with female offspring susceptible to reductions in PA+ neurons, and male offspring insusceptible. The anterior cingulate displayed no sexual dimorphism and each gender was uniformly vulnerable to prenatal ethanol. Chapter 3: choline acetyltransferase (ChA T) immunoreactivity in the medial septum following neonatal ethanol exposure The objective of this study was to determine the long-term effects of neonatal ethanol exposure on the cholinergic neurons in the medial septum (MS) of the rat. The hypothesis tested was that early postnatal ethanol exposure would lead to alterations in the number of neurons expressing ChA T in the adult rat MS. To test this we utilized a neonatal ethanol exposure paradigm known as artificial rearing to infuse ethanol containing or control diet from P4-1 O; gastrostomized pups and dam-reared pups were used as controls. ChA T immunocytochemistry performed and the number of immuno-positive cholinergic neurons was determined at P60. Neonatal exposure did not directly reduce cholinergic neuronal number or the mean number of neurons per section. Similarly no changes were noted in MS volume mean area section or cell density as a result of this

PAGE 166

156 pattern of exposure. However the size of cholinergic neurons was reduced in ethanol treated males compared with gastrostomized controls but not suckle control males. No differences in ChA T + neuronal size were noted for females. Ethanol treatment did result in long-lasting microencephaly in P60 animals although cholinergic neurons in the MS were unaffected. Thus neonatal ethanol exposure produces long-lived microencephaly and small changes in ChA T + neuronal size but does not affect the number of cholinergic neurons or the size of the adult rat MS. Therefore our hypothesis was not supported by the data Cholinergic neurons were not vulnerable to ethanol delivered during neonatal development. We did detect a small but statistically significant reduction in ChA T + neuronal size but this was only found in male animals The whole of data from our laboratory regarding the effects of developmental ethanol e x posure on the number of cholinergic neurons in the rat brain are consistent and point towards a lack of effect of ethanol on the number of cholinergic neurons (Swanson et al ., 1996 ; Heaton et al. 1996 ; Moore et al. 1998a) Chapter 4: differential cerebellar neuronal susceptibility and bcl-2 family gene expression following one-or two-day neonatal ethanol treatment The objective of this study was to determine whether ethanol-induced cerebellar cell death during development is relat e d to alterations in the expression of bcl2 family genes. The hypothesis tested was that alterations in the expression levels of bcl-2 famil y PCD ge n es in the cerebellum contribute to th e c e reb e llum s relative temporal susceptibility to ethanol neurotoxicity. To test this a neonata l exposure paradigm similar to that u s ed above was utilized Trans cript levels of bcl-2 family members r e lative to cyclophilin were measured to determine whether ethanol changed the expression o f cell

PAGE 167

157 death genes To establish this method in our hands pups exposed in parallel were taken for cerebellar cell counts to document the pattern of cerebellar cell loss following treatment. Exposure to ethanol during the first postnatal week resulted in significantly reduced Purkinje and granule cell numbers on postnatal day 21 (P21). Two hours after ethanol exposure on P4 transcripts encoding the cell death-promoting molecules bax and bcl-xs were up-regulated. Exposure for an additional day ( on P5) resulted in no further alterations in bcl-2 family transcripts. This is probably because Purkinje cell death was found as early as P5. To determine whether pro-apoptotic gene expression changes were spec i fic to first postnatal week ethanol neurotoxicity we examined bcl-2 family mRNA levels following ethanol treatment during the second postnatal week. This is a period of de v elopment in rats when the cerebellum does not exhibit profound toxicity in the presence of ethanol. While exposure on P7-8 produced no cerebellar cell death as measured on P2 l this pattern of exposure did result in increased levels of bax mRNA This up-regulation was found only after two-day ethanol exposure and not after acute exposure on P7. These data document increased expression bax and bcl-xs after acute ethanol exposure in first postnatal week They also suggest that key factor influencing the differential survival o f cerebellar neurons following ethanol exposure during more mature developmental stages may be related to better suppression of pro-apoptotic processes Thus our hypothesis regarding differential cerebellar vulnerability and altered bcl-2 family gene expression was not supported by the data because neonatal ethanol exposure during both the first and second postnatal weeks produced altered bcl-2 gene expression. Thus the situation is much more complicated that was initially predicted.

PAGE 168

158 What likely accounts for the differential susceptibility, then, may be better suppression of pro-apoptotic processes in cerebellar neurons following ethanol exposure during the second postnatal week. These data are highly useful for the fetal alcohol field in that they provide a new avenue for future investigation and provide the first demonstrations of altered bcl-2 family gene expression following ethanol insult. In fact it will be useful to consider the results from chapter 4 when follow-up investigations are made into neuroanatomical alterations resulting from developmental ethanol in regions such as the medial septum and cingulate cortex. For example changes in the expression of bcl-2 family genes and proteins may occur in these regions following ethanol treatment. Perhaps a similar induction in pro-apoptotic genes underlies the noted reduction in PA+ neurons of the septum and cingulate. Perhaps the lack of effect of ethanol on cholinergic neurons is related to induction of anti-apoptotic genes or, alternatively better suppression of pro-apoptotic gene expression may occur, as seems to be the case with the cerebellum after second postnatal week exposure. Investigations at the protein level using Western blot will determine whether the noted changes in gene expression also occur at the protein level. The finding of increased expression of bax and bcl-xs can be extended to other pro-apoptotic bcl-2 family members such as bad and bak. Anatomical techniques such as in situ hybridization and immunohistochemistry will determine whether both Purkinje and granule cells increase transcription of pro-apoptotic genes and will determine whether changes occur at the same time or whether induction happens first in Purkinje cells and then granule cells. A more elaborate discussion of future directions is found below.

PAGE 169

159 Choice of Animal Models Two rat models of FAS have been utilized to examine the hypotheses put forth in this document. The fust, used for chapter 2 involves the use of a pair-fed liquid diet to examine the effects of chronic prenatal ethanol treatment on PA+ neurons of the medial septum and anterior cingulate cortex. A pair-fed liquid diet was frrst utilized in rat models by Walker and Freund ( 1971 ) and has several advantages over other widely used prenatal exposure models, such as intraperitoneal injection or intubation. The most obvious is the elimination of nutrition as a confounding variable in the interpretation of results. Because the pair-fed animals in this paradigm are fed an isocaloric diet substituting sucrose for ethanol, and they consume the identical amount of diet as ethanol-treated counterparts it is impossible that any observations result from reduced nutrition Offspring from chow-fed dams are also included in our analyses to control for non-specific effects of the highly nutritive liquid diet. The importance of utilizing a pair fed liquid diet in ethanol studies is driven home by an examination of data from other groups who have not utilized this technique The most salient example is the choice of exposure method employed by Arendt et al. (1988) who simply added ethanol to the drinking water of adult rats and used chow-fed animals as controls Clearly nutrition cannot be ruled out in the interpretation of their data. Thus in our prenatal experiments every effort has been made to eliminate the possibility that nutritional deficiencies have contributed to the observed effects One drawback of the highly nutritive liquid diet is that it may lead to non-specific effects (see chapter 2). For example Swanson et al. (1995) found a stimulatory effect of

PAGE 170

160 liquid diet on ChA T enzymatic activity during the first postnatal week. Add i tionally studies quantifying ChAT + cell number in the P14 rat MS found a liquid diet-induced increase in ChA T + neuronal number for female sucrose animals but not ethanol females or male sucrose animals (Swanson et al. 1996). It was speculated that a possible sucrose diet-induced stimulation of MS cholinergic development occurred in female rat pups at P14. The seemingly protective and perhaps stimulatory nature of the liquid diet is probably due to its high vitamin and mineral content. Similar effects were described in chapter 2 and we speculate that a liquid diet induced increase in cell density for the sucrose group raised the base level of PA+ neurons in the liquid diet animals to a level greater than that seen in chows. The ethanol treated group would presumably be exposed to the same factors in the liquid diet. However a significant ethanol effect was noted since ethanol females had fewer PA+ neurons in the MS. Apparently prenatal ethanol treatment was damaging enough to produce a significant cellular reduction in ethanol females even in the presence of the highly nutritive liquid diet. Even with these caveats the use of the liquid diet is necessary to eliminate the possibility that altered nutrition contributes to any observed developmental alterations following prenatal ethanol exposure. The second rat model utilized in this body of work ( chapters 3 and 4 ) is the neonatal exposure model known as artificial rearing. This method of ethanol exposure is used when investigators want to mimic ethanol exposure during the third trimester equivalent in humans (Dobbing and Sands 1979) and has advantages over other neonatal exposure techniques such as vapor inhalation and intubation Precise delivery of diet b y a syringe infusion pump (as with artificial rearing) is especially advantageous when longer

PAGE 171

161 exposure times are utilized because the infusion pump can be programmed to deliver feedings when the investigator is away. With intubation the investigator must administer each feeding a laborious process when multiple feedings are performed daily. Experiments utilizing inhalation are often undermined by the inability of ethanol exposed pups to nurse effectively after treatment (Ryabinin et al. 1995) With artificial rearing post-ethanol feedings of milk alone are administered by the infusion pump and thus no lapses i n nutrition occur. Artificial rearing was frrst introduced by Messer et al. (1969) modified by Hall (1975) and later modified by Samson and Diaz to include studies on ethanol and brain development (Diaz and Samson 1980). West and colleagues have further refined the technique and have developed the most widely used variation of neonatal exposure paradigms (West et al. 1984). Even though this technique is complicated due to surgical implantation of gastric cannulas as well as time consuming and expensive it allows for precise nutritional control and ethanol delivery. Experimental design includes suckle control pups raised normally by the mother gastrostomy-control pups which are artificially reared and fed a milk-based liquid diet and ethanol pups artificially reared and exposed to a similar diet including ethanol. Since both gastrostomy-control and ethanol groups are fed identical amounts of an isocaloric diet ethanol effects can be examined without the confounding variable of nutritional deficits. Disadvantages of artificial rearing do exist, such as labor-intensive surgery and pup care expensive equipment and supplies stress and prolonged isolation from the dam and siblings Another drawback is that the procedure itself can lead to direct effects or can interact with ethanol to produce alterations. For example artificial rearing effects

PAGE 172

162 have been noted on conditioned emotional response i n female rats (Kelly et al., 1991), similar effects were and were noted in the present work (see chapter 3 and 4). We found that artificially reared control pups had increased ChA T + somatic area compared with ethanol-exposed pups and suckle control pups. We also noted an increase in mRNA levels for bcl-xs in gastrostomized controls compared with suckle controls. These effects are likely because of the stress induced by the invasive technique in combination with littermate and maternal separation. Despite these shortcomings, the ability to exactly control the volume of diet delivered and the timing of delivery outweighs these drawbacks and in many experiments artificial rearing effects are not found Ethano l inhalation, another method for neonatal ethanol delivery was first used by Goldstein ( 1972) and enables the non-invasive exposure of neonates. Pups are separated from the mother for an exposure period, and returned to maternal care following treatment. Alternatively some newer techniques do not separate pups from the darn. In these protocols the entire home cage is placed in the vapor chamber (Pal and Alkana, 1997). Ethanol exposure is carried out in a sealed chamber with an ethanol vapor source. Control groups consist of an unseparated group and a control-separated group which is taken from the mother for the same amount of time as the ethanol group but inhales only air. The technique is advantageous since it allows for normal maternal interaction with pups is inexpensive and easy to do, and requires no intensive animal care. However the technique suffers from the need to cull ethanol exposed litters to three or more pups l ess than that of control-separated litters in an effort to eliminate nutritional differences. Moreover the possibility of hypoxia exists, due to both reduced oxygen in the inhal a tion chamber (Ryabinin et al. 1995) Intubation while not extensively used in the past is

PAGE 173

163 gaining in popularity, especially when experimental design includes short exposure periods (Goodlett et al., 1997). However, a drawback ofthis technique is that it requires much handling of pups whereas with artificial rearing, minimal handling is used and pups are fed automatically by the syringe infusion pump Stress is not significantly reduced by intubation since pups must be separated from the dam for the infusion period and the method of administration still involves inserting a feeding tube down the esophagus of the pup. Thus, for the purposes of the experiments described herein artificial rearing was the best choice of neonatal exposure method. Choice of Cell Counting Methods The recent development of stereological cell enumeration methods has created a controversy over the most appropriate and quantitative method for counting neurons. For example the Journal of Comparative Neurology published a commentary stating tha t non-stereological cell estimation methods were assumption based and inappropriate for cell counting (Coggenshall and Lekan 1996) leading Guillery and Herrup ( 1997) to conclude that journal policy is placing a "methodological straitjacket on in v estigators. Criticisms of non-stereological cell counting methods ( and data expressed as mean cells per section) are that overprojection and truncation can result from incorrectl y identifyin g cells within a focal plane and that changes in reference v olume can bias cell counts (Peterson et al. 1997) The use of manual and computer-automated cell counting in the present bod y of work is suitable because the use of r e lativel y thin sections separated a distance which minimized the possibility of overproj e ction Also the s iz e of the structures of inter e st

PAGE 174

164 and the cells therein were always measured to determine whether ethanol treatment affected this parameter. Finally, we did not intend to estimate total cell number and only wished to compare the mean number of cells per section between groups (Hagg et al., 1997). Moreover, Clarke and Oppenheim ( 1995) have demonstrated that the non stereological cell counting methodology employed in the present work is as accurate and reproducible as stereological methods for a variety of neuronal populations Other Methodological Considerations Immunohistochemistry was used in chapters 2 and 3 to examine the pattern of expression of parvalbumin and choline acetyltransferase after ethanol exposure during development. This is the best technique for localizing a specific protein in tissues, so that the anatomical localization of a particular molecule can be determined. Thus, for our purposes of identifying susceptible neuronal populations in structures such as the medial septum and cingulate cortex where many different neuronal populations co-exist the method of irnmunohistochemistry was most advantageous. This was one way to definitively examine only a subset of the entire neuronal populations within these structures (by utilizing specific antibodies rather than a gross staining technique such as a Nissl stain) and thus allowed for the testing of specific hypotheses regarding ethanol's effects on those subsets of neurons. Thus it is important to selectively mark these populations for examination separately because many structures, for example the medial septum display differential neuronal vulnerability to ethanol. For our cerebellar cell counts in chapter 4, the use of a more general stain such as hematoxylin and eosin was

PAGE 175

165 proper because in that instance we were concerned with documenting direct ethanol induced cell death in a structure without differential neuronal susceptibility. The choice of the ribonuclease protection (RP A) assay for measurements of relative mRNA levels in chapter 4 is also a suitable choice especially because of the highly quantitative nature and sensitivity of this technique. This is an important consideration when measuring transcripts of the bcl-2 family since the intracellular ratio of various molecules is an essential determinant of cell survival or death (Oltvai et al. 1993). The RP A is useful in this sense because it allows for the detection of small changes in expression that can have a significant influence on this ratio. Moreover the fact that glial cells do not express high levels of these molecules means that there is little chance any observed changes in expression are due to non-neuronal cells (Frankowski et al., 1995 ; Vyas et al. 1997) Drawbacks for the use of RP A in this investigation include the lack of anatomical localization of the mRN A changes and the lack of information at the protein level. However the ease of quantitation and the ability to detect subtle gen e expression changes provided by the RP A make it an appropriate choice for this study, which desired to determine relative levels of these transcripts following ethanol treatment. Future Directions for Developmental Ethanol Research The data presented herein are useful in that they add to the current knowledge of the range of CNS defects resulting from developmental ethanol exposure and suggest many future studies. What follows is an attempt to extend the discussion of the data reported here by listing future directions that are suggested from the data

PAGE 176

166 Further documentation of susceptible neuronal populations Because the nervous system is not uniform in its vulnerability to developmental ethanol exposure an important aspect of fetal alcohol research has been, and will continue to be to identify susceptible neuronal populations. This is necessary because the neuroanatomical substrates which underlie FAS must be identified before the syndrome can be adequately understood The present body of work has demonstrated that PA + neuronal number is reduced in the medial septum (in females but not males) and anterior cingulate cortex (in both genders). ChA T + neurons were not affected in the medial septum following neonatal ethanol exposure although cell size alterations were noted. Furthermore we have replicated the observations of others that Purkinje and granule cells of the cerebellar vermis are decreased in number by neonatal ethanol treatment in the first but not second postnatal week. One experiment suggested by the current data is to examine PA+ neuronal number in the anterior cingulate cortex of animals exposed to ethanol during neonatal development. This study would nicely complement the examination of PA + neuronal number in the medial septum following chronic prenatal ethanol treatment and would lend insight into whether these GABAergic neurons exhibit susceptibility only during the prenatal period or if they are also vulnerable during the neonatal period Another informative study would be to examine PA+ neuronal number in the hippocampus and cerebellum (after prenatal and neonatal ethanol exposure) since both structures contain many GABAergic neurons It is conceivable that similar ethanol-induced alterations in PA + neuronal number in these CNS areas might underlie the neuropathology of developmental ethanol disorders Because no investigation has determined whether the

PAGE 177

167 major excitatory neurons of the CNS are affected by ethanol analysis o f glutamatergic neurons (when specific markers are developed) especially in neocortical regions would be useful following ethanol exposure during various developmental periods. All of these experiments could be completed with the use of the previously described techniques of immunohistochemistry followed by cell counts. Functional studies Because our data indicate that a subpopulation of GABAergic neurons expressing parvalbumin is reduced in number following developmental ethanol exposure investigators should look for the functional consequences of this reduction. For example investigators should record synaptic potentials from inhibitory intemeurons in the rat hippocampus to determine whether their activity is altered following ethanol exposure. The GABAergic projections neurons from the medial septum depress the activity of these hippocampal intemeurons normally thereby exciting the pyramidal neurons involved with long term potentiation (Freund and Antal 1988) Ethanol exposure may depress the excitability of the hippocampus and this might give insight into the mechanism of ethanol-induced learning and memory dysfunction. Examining the excitation and inhibition of the anter ior cingulate with ex tracellular recording could monitor functional alterations in this cortical region. Cortical lesions are known to result in decreased numbers of parvalbumin immunoreactive neurons lead i ng to hyperexcitability of adjacent cortical areas (Jacobs et al. 1996). Similar release of inhibition may underlie the reductions noted in the present work. Similarly the input o f the cin g ulate into Papez circuitry could be monitored by recording in the ent orhinal cortex. Investigations such as this could help to further define the neuroana t omical

PAGE 178

168 changes which underlie the learning and memory problems in children with fetal alcohol syndrome. A variety of imaging studies are suggested by the data presented in this body of work. For example positron emission tomography with radio-labeled glucose would be useful to determine whether glucose utilization is altered in the basal forebrain, cingulate cortex hippocampus or entorhinal cortex of children with FAS. The noted reduction in parvalbumin immunoreactive neurons in the present study suggests that normal glucose utilization might be changed This is especially possible because of the role of inhibitory intemeurons and projection neurons in regulating the excitation of other regions. For example hippocampal excitability (and glucose utilization) might be depressed in ethanol-exposed children due to a decline in inhibitory input from the medial septum on inhibitory hippocampal neurons. Investigation into the involvement ofbcl-2 family in PA+ neuronal susceptibility and ChA T + neuronal insusceptibility The results from chapter 4 on increased expression of pro apoptotic mRNAs of the bcl-2 family raises the possibility that similar changes occur in the medial septum and/or cingulate cortex following developmental ethanol exposure For example induction of pro-apoptotic genes might precede the noted reduction in PA+ neurons in the medial septum and cingulate cortex. Other pro-apoptotic bcl-2 family members should be investigated besides bax and bcl-xs such as bad and bak, as different brain regions may utilize different cell death molecules The lack of effect of developmental ethanol exposure on cholinergic neurons might be related to decreased expression of death genes increased expression of survival genes or successful post-translational squelching of cell

PAGE 179

169 death processes (see below). Double-labeling with antibodies to both PA or ChA T and various bcl-2 family members followed by fluorescence microscopy, would allow investigators to examine bcl-2 family expression in subpopulations of neurons. Further investigation into the cellular and molecular mechanism of ethanol-induced neurotoxicity While the descriptive studies outlined in chapters 2 and 3 (and the future experiments suggested above) are useful for identifying novel populations of neurons that exhibit vulnerability to developmental ethanol exposure further investigation into the cellular and molecular mechanisms of ethanol-induced neurotoxicity is warranted Data presented in chapter 4 attempted to address this issue by investigating bcl-2 family gene expression following neonatal ethanol exposure. The noted induction of pro-apoptotic gene expression following both first postnatal week and second postnatal week ethanol exposure suggested that the differential survival of cerebellar neurons following neonatal exposure may be related to better suppression of these pro-apoptotic processes. The following are suggestions to increase our understanding of how these pro-apoptotic processes are induced by ethanol to identify key downstream players in ethanol-induced cell death and to attempt to identify the key differences in more mature cerebellar neurons which decrease their sensitivity to ethanol. Many of the suggestions utilize the cerebellum due to the fact that the principal neuronal types the Purkinje and granule cells are susceptible to ethanol the fact that it has been clearly demonstrated to be a structure which displays considerable vulnerability to developmental ethanol exposure and the fact that it is useful for both in vivo and in vitro studies. Still other experiments involve the use of transfected cell lines and the use of dominant-negative mutations to

PAGE 180

170 determine a molecule's involvement in ethanol-induced cell death. Further investigations into the cellular and molecular mechanisms of ethanol neurotoxicity will benefit the fetal alcohol field by identifying molecular targets for therapeutic intervention in ethanol neurotoxicity. The following are lines of investigation that the data presented in chapter 4 suggest. Pro-apoptotic gene expression and p53 The present investigation documented increased expression of the pro-apoptotic genes bax and bcl-xs following neonatal ethanol exposure. One of the first experiments which should be done is to extend these investigations to other pro-apoptotic members of the bcl 2 gene family such as bad and bak. This could be accomplished at the mRNA level with the RNase protection assay (for comparison to the present study) and at the protein level with Western blot. Another future direction is to determine what intracellular factors are responsible for the noted induction in pro-apoptotic mRNAs Induction and expression of the tumor suppressor gene p53 is known to up-regulate bax gene expression (Mitry et al. 1997 ; Xiang et al. 1998) leading to apoptotic cell death. Additionally phorbol ester-mediated activation of protein kinase C (PKC) blocks nitric oxide-induced apoptosis through suppression of p53 activation and decreased levels of Bax protein (Messmer and Brune 1997) Intriguingly preliminary data from our laboratory indicate that PKC activity is decreased in the cerebellar vermis two hours after ethanol treatment in vivo (Davis et al. unpublished observation) These data suggest that a similar role for reduced PKC activity may lead to p53 activation and increased bax/bcl xs gene e xpre s sion following ethanol exposure This could be investigated in vivo with

PAGE 181

171 phospho-specific antibodies for p53 activation and analysis of bax/bcl-xs gene expression following ethanol treatment. It would be of interest to determine whether an induction of p53 following reduced PKC activity is sufficient to increase bax and/or bcl-xs mRNAs An in vitro cerebellar model system utilizing primary cultures of cerebellar neurons could be used for this purpose. For example can phorbol esters block ethanol-induced apoptosis of cultured cerebellar cells and does this lead to decreased p53 and Bax accumulation? p53 activation could be examined by the use of antibodies specific to phosphorylated p53 with standard Western blot techniques and extraction with protease and phosphatase inhibitors. Cell lines could be transfected with dominant negative mutants of p53, and bax and bcl xs mRNA production monitored to directly determine a role for p53 in ethanol-induced apoptosis. A role for p53 would be indicated by amelioration of ethanol-induced cell death in cells transfected with the mutant (inactive) constructs. If cell death still ensued upon ethanol treatment then other transcription factors would be implicated Many members of the bcl-2 family contain consensus sequences for NF-kB, so it is possible that this transcription factor is involved in bax and bcl-xs mRNA upregulation ( Dixon et al. 1997). For example does activation and nuclear localization of NF-kB result from ethanol treatment? If so, does ethanol-induced activation of NF-kB lead to increased mRNA levels of bax and bcl-xs? Do dominant negative mutants of NF-kB inhibit ethanol-induced cell death? Evidence from Dixon et al. (Dixon et al. 1997) indicates that the induction of bcl-xs gene expression following ischemia occurs along with increased NF-kB activation and nuclear translocation. A similar correlation may be found with ethanol-induced increases in bax and bcl-xs gene expression in our in vivo

PAGE 182

172 cerebellar model system. These questions could be answered with phosph-specific antibodies to investigate NF-kB activation with standard Western techniques and sample homogenization in the presence of protease and phosphatase inhibitors Immunoflorescence and confocal microscopy could be used to investigate nuclear translocation of NF-kB. An in vitro model system with cell lines stably transfected with dominant-negative constructs of NF-kB could be used to definitively demonstrate its involvement in ethnaol-induced up-regulation of bax and bcl-xs genes. mRNA levels of varioius bcl-2 family members can be investigated with the described methods ( see chapter 4) for the RNase protection assay. Downstream of the bcl-2 family: caspases and cytochrome C Another direction suggested by the noted increase in bax and bcl-xs gene expression is to look at caspase induction and activation following developmental ethanol exposure. This could be done in vivo for example by examining caspase 3 protein levels and proteolytic activity in the cerebellar vermis in the acute phases following neonatal ethanol exposure The fact that we saw significant gene expression changes two hours after the end of the ethanol insult suggests that this would be the timepoint to start looking for increased levels of caspases and increased activation. One could confirm caspase involvement by determining whether particular caspase substrates are cleaved such as poly (ADP-ribose) polymerase (PARP), and whether ethanol treatment in vitro or in vivo results in the increased production of the 89 kDa fragment characteristic of P ARP cleavage (Duriez and Shah 1997 ) Another experiment would be to determ i ne whether caspase inhibitors such as YV AD and D E VD can inhibit ethanol-induced cell death in cultures of cerebellar granule cells It is conceivable that the difference between first and

PAGE 183

173 second postnatal week suppression of pro-apoptotic processes occurs at the level of caspase activation Thus investigations should compare the cellular changes in caspases after first or second postnatal week exposure. Because release of cytochrome C is an important early event in the activation of caspases (Li et al., 1997) measurements of the cytoplasmic concentration of cytochrome C in cerebellar granule cell cultures would be useful following ethanol treatment. bcl-2 family dimerization and post-translational modification Members of the bcl-2 family are capable of homoand hetero-dimerization via BH3 domains (bcl-2 homology domain 3) present on all family members (Zha et al. 1996 ; Zha et al., 1997). Homodimerization of anti-apoptotic members promotes cell survival while homodimerization of pro-apoptotic molecules promotes cell death (Reed 1997) Because the data presented in the present work only address the induction of pro apoptotic processes (as measured by gene expression) and do not address translational or post-translational issues it would be important to describe the pattern of bcl-2 family protein levels and dimerization state and phosphorylation status following ethanol treatment in the first and second postnatal week. The subject of bcl2 protein levels can be easily addressed with the Western blot technique Levels of phosphorylated bcl-2 family members can be described with phospho-specific antibodies and the use of protease and phosphatase inhibitors during protein extraction. Dimerization can be investigated with co-immunoprecipitation followed by immunodetection with antibodies to proteins known to utilize the BH3 domain for dimerization All of these experiments can be performed in vivo and thus can utilize the in vi v o cerebellar vermis following acute ethanol administration in the first

PAGE 184

174 postnatal week. Comparisons of bcl-2 family protein levels phosphorylation and dimerization after second postnatal week ethanol treatment would also be enlightening, since this pattern of exposure was capable of inducing pro-apoptotic processes but not cell death For example is the phosphorylation or dimerization different following these patterns of exposure? Can post-translational modification counteract the up-regulation of these molecules and inhibit cell death? JNK.s and post-translational modification of anti-apoptotic molecules Mitogen-acitvated protein kinases (MAPKs) are important intracellular mediators of extracellular survival, growth, differentiation and stress signals (Elion, 1998). Multiple MAPK cascades exist in cells, and control such disparate processes as differentiation, growth and stress response. The c-Jun N terminal kinase / stress activated protein kinase (JNK/SAPK) sub-family of MAPKs is one example that is important in intracellular processing of extracellular stress signals. There is reason to suspect that a stressor such as ethanol would activate this the JNK/SAPK pathway. Preliminary data from our laboratory indicate that ethanol delivered in vivo causes an induction of JNK activity (as indicated by increased phosphorylation of the p54 JNK isoform) two hours after ethanol treatment on P4 in the cerebellar vermis (Davis et al., unpublished observation). Because of the noted link between extracellular-derived factors and post translational modification of bcl-2 family members (Kahn, 1998) it is of considerable importance for investigators of ethanol neurotoxicity to determine whether the intracellular effectors of these extracellular signals are altered by ethanol. For example are neurons responding to stress signa ls initiated by the presence of high ethanol

PAGE 185

175 concentrations by activating JNK/SAPK cascades? Moreover, are these activated kinases acting on bcl-2 family members as substrates to alter their functioning? The noted induction of JNK activity following in vivo ethanol treatment might have profound implications for bcl-2 family members, as they are known substrates of activated JNK. In particular, the anti-apoptotic Bcl-2 protein is phosphorylated by p54 JNK, and MAPK specific phosphatases block Bcl-2 phosphorylation when given concurrently with JNK (Maundrell et al., 1997). The significance of increased Bcl 2 phosphorylation appears to be the inactivation of Bcl-2's anti-apoptotic function (Maundrell et al. 1997). Thus, an experiment of future importance would be to determine whether Bcl-2 and other bcl-2 family members are post-translationally modified by the noted p54 JNK activation. This could be accomplished with phospho specific antibodies (serine and threonine residues) to bcl-2 family members on Western blots and could be done in vivo or in vitro. A follow-up experiment in vitro would be to inhibit p54 JNK activity with phosphatases to determine whether this blocks the ethanol induced, (and possibly p54 dependent) bcl-2 family member phosphorylation. Comparisons of ethanol-induced changes in JNKs following exposure in the fust and second postnatal week would be illuminating. It is possible that differential regulation of bcl-2 family members by JNKs is responsible for the differential suppression of pro apoptotic processes following ethanol exposure. Upstream mediators of bcl-2 family function: PI3K and Akt As mentioned earlier an important recent discovery has been that extracellular factors can influence the function ofbcl-2 family members (Datta et al., 1997; Kahn, 1998; Zha et al., 1996). For example it was known that growth factors could stimulate

PAGE 186

176 PB kinase and its downstream target Akt (Zhou et al. 1997) but the mechanism of Aktinduced cell survival was unknown. Then two groups discovered that growth factor activation of PB kinase and Akt resulted in the phosphorylation of the pro-apoptotic bcl-2 member Bad (Datta et al. 1997; Zha et al. 1996) Bad phosphorylation inactivated its death-promoting activity and resulted in its accumulation in the cytosol away from Bcl-2 or Bcl-xl (Zha et al. 1996). Therefore, it has been definitively demonstrated that extracellular signals influence the function of bcl-2 family members and that this influences the survival of the cell. Although similar modifications have not been described for Bax or Bcl-xs (molecules whose transcripts were up-regulated after neonatal ethanol exposure) it is possible that these mechanisms exist. Another line of work would be to investigate the mediators of cell life and death which function upstream of the bcl-2 family to determine whether ethanol produces any alterations at these levels. For example does ethanol treatment lead to decreased PB kinase and/or Akt activation and decreased Bad phosphorylation? Preliminary data from our laboratory indicate that Akt activation and Bad phosphorylation are decreased in the cerebellar vermis two hours after ethanol treatment in the first postnatal week (Davis et al. unpublished observations). Given that second postnatal week ethanol exposure does not produce cell death one question that arises is whether or not PB kinase activation is preserved following second postnatal week ethanol exposure and is Bad phosphorylation similarly preserved? Can differences in the temporal availability of growth factors account for this differential toxicity? These questions can be answered with phospho specific antibodies and standard Western blot techniques following protein extraction with protease and phosphatase inhibitors

PAGE 187

177 Growth-factor suppression of pro-apoptotic processes : extrinsic modulation of intracellular conditions favoring cell death The most likely mediator of the differential toxicity noted in the cerebellar vermis seems to be better suppression of pro-apoptotic processes induced by ethanol by growth factors. This is an attractive hypothesis because neurotrophins are known influence the survival and differentiation of cerebellar Purkinje and granule cells developmentally. Additionally the ontogeny of neurotrophins and their cognate receptors in the cerebellum is consistent with this hypothesis. Neurotrophin receptors are expressed at high levels in rat Purkinje and granule cells during development and the expression of neurotrophin ligands and receptors is regulated in a spatio-temporal manner (Lindholm et al., 1997) For instance Trk B receptors are only found on differentiating granule cells (and not proliferating granule cells) and only granule cells in the process of differentiation respond to BDNF or NT-3 (Gao et al. 1995) Furthermore although ontogeny begins on P4 the peak period of Purkinje cell expression of trkA mRNA does not occur untilPlO during the later stages of Purkinje cell differentiation (Wanaka and Johnson, 1990). Alternatively other as yet undiscovered growth factor pathways may also interact with the bcl-2 family and might play a role in differential ethanol-induced cell death. Whether PB kinase and Akt are the mediators of the suppression of ethanol-induced pro-apoptotic processes r e mains to be s ee n and it is possible that other pathways are involved. However the known influence of oth e r growth factors on this pathway makes it an appropriate place to start. Activation of these pathwa y s might result in post-translational modification of Bax or Bclx s protein perhaps inactivating their death-promoting properti e s or modification of Bad mi g ht b e invol ved.

PAGE 188

178 Therefore, an important hypothesis to test is that the activation of intracellular growth factor signaling processes, which would presumably decrease cell death by suppression of pro-apoptotic processes is a key factor governing cerebellar neuronal susceptibility to ethanol. In our model system, second postnatal week ethanol exposure produces an induction of pro-apoptotic processes without concomitant cerebellar cell death It is conceivable the increase in cell death mRNAs is counteracted by growth factor-derived phosphorylation of these or other molecules, such as Bad. This would presumably enable anti-apoptotic members of the bcl-2 family to function in their normal capacity to regulate mitochondrial membrane potential. The ethanol-induced induction of pro-death genes in combination with a decline in growth factor-derived Bad phosphorylation in the first postnatal week, may lead to impaired anti-apoptotic processes leading to lost mitochondrial membrane potential, release of cytochrome C, and caspase activation This hypothesis can be tested descriptively by documenting Akt activation and Bad phosphorylation following acute second postnatal week. Unchanged or increased levels of phosphorylated Bad, Bax, or Bcl-xs, along with unchanged or increased levels of active Akt following second week exposure would provide support for this hypothesis. Comparisons should be made to the first postnatal week as well. As mentioned previously Akt activation and Bad phosphorylation are both decreased in the vermis after first postnatal week ethanol exposure (Davis et al. unpublished observation). These studies should be extended to include Bax and Bcl-xs Analysis of Akt activation Providing growth factors in vitro to early neonatal cerebellar neurons along with ethanol and examining cell death and pro-apoptotic molecule phosphorylation would determine

PAGE 189

179 whether growth factor addition could protect early neurons from cell death induced by ethanol. Also direct effects of growth factors on pro-apoptotic (and anti-apoptotic) gene expression could be examined in vitro. Conclusion Despite the fact the FAS has been recognized since 1973 (Jones and Smith, 1973; Jones et al. 1973) the incidence of ethanol-induced developmental abnormalities continues to increase (Prevention, 1995) and remains 20 times higher in the United States than in Europe (Abel 1995). Thus, alcohol-related neurodevelopmental disorders constitute a serious health problem, and research on the neuroanatomical substrates underlying these disorders and the mechanisms through which ethanol acts as a teratogen is essential if treatments for these defects are to be discovered. With these goals in mind, the research described in this document was pursued. The present work identified the population of GABAergic neurons expressing parvalbumin as one that is affected by developmental ethanol exposure. Parvalburnin immunoreactive neurons were decreased in number in the medial septum (in a sexually dimorphic manner) and anterior cingulate cortex (without sexual dimorphism). This work also determined that cholinergic neurons are not susceptible to neonatal exposure during a more mature developmental timepoint. Another advance made by this body of work was to open up new lines of research on the molecular mechanisms of developmental ethanol neurotoxicity. This was accomplished by the determination that pro-apoptotic gene expression is up-regulated in the cerebellar vermis following neonatal ethanol exposure New research on the consequences of this induction and the potential

PAGE 190

180 of growth factors to ameliorate this up-regulation will potentially identify molecular targets for inhibiting ethanol-induced cell death. Therefore the data generated in the current study are useful in that they not only identify a novel population of neurons affected by developmental ethanol treatment but they also suggest new avenues of research on the molecular mechanism of ethanol teratogenicity Investigators will certainly continue to pursue this and other lines of work in hopes of better understanding the developmental alterations induced by ethanol and in hopes of developing treatments to prevent these disorders in alcoholic mothers.

PAGE 191

REFERENCES Abel E L. ( 1984 ). Fetal alcohol syndrome and fetal alcohol effects (New York : Plenum Press). Abel E. L. (1995). An update on incidence of FAS FAS is not an equal opportunity birth defect. Neurotoxicol Teratol 17, 437-443. Abel E. L. and Hannigan J. H. (1995). Maternal risk factors in fetal alcohol syndrome provocative and permissive influences. Neurotoxicol Teratol 17, 445-462. Abel E. L. and Sokol R. J. (1986). Fetal alcohol syndrome is now leading cause of mental retardation [letter] Lancet 2 1222. Akao, Y., Otsuki Y. Kataoka, S ., Ito Y. and Tsujimoto Y. (1994). Multiple subcellular localization of bcl-2 detection in nuclear outer membrane endoplasmic reticulum membrane and mitochondrial membranes Cancer Res 54, 2468-2471. Alcantara S. Ferrer I. and Soriano E. (1993). Postnatal development ofparvalbumin and calbindin D28K immunoreactivities in the cerebral cortex of the rat. Anat. Embryo!. Berl. 188 63-73. Allen C N. and Crawford I. L. (1984). GABAergic agents in the medial septal nucleus affect hippocampal theta rhythm and acetylcholine utilization Brain Res. 32 2, 261 -267. Allsopp T E ., Kiselev S ., Wyatt S. and Davies A. M. (1995). Role of Bcl-2 in the brain-derived neurotrophic factor survival response Eur J Neurosci 7, 1266-1272 Allsopp T. E., Wyatt S., Paterson H F ., and Davies A. M. (1993). The proto-oncogene bcl-2 can selectively rescue neurotrophic factor-dependent neurons from apoptosis. Cell 7 3 295-307. 181

PAGE 192

182 Alonso J. R. Covenas R ., Lara J. and Aijon J. (1990) Distribution ofparvalbumin immunoreactivity in the rat septa} area. Brain Res. Bull. 24 41-48. Altman J. (1969) Autoradiographic and histological studies of postnatal neurogenesis. 3. Dating the time of production and onset of differentiation of cerebellar microneurons in rats J Comp Neurol 136 269-293 Antonsson B. Conti F. Ciavatta A ., Montessuit S. Lewis S., Martinou I., Bernasconi L. Bernard A., Mermod J. J. Mazzei G ., Maundrell K. Gambale F. Sadoul R ., and Martinou J.C. (1997). Inhibition of Bax channel-forming activity by Bcl2 Science 277, 370-372. Arendt T. Bruckner M. K ., Krell T. Pagliusi S. Kruska L., and Heumann R. (1995) Degeneration of rat cholinergic basal forebrain neurons and reactive changes in nerve growth factor expression after chronic neurotoxic injury--11 Reactive expression of the nerve growth factor gene in astrocytes. Neuroscience 65, 647-659. Arendt T ., Henning D. Gray J. A. and Marchbanks R. (1988). Loss of neurons in the rat basal forebrain cholinergic projection system after prolonged intake of ethanol. Brain Res Bull. 2 1 563-569 Armstrong D. M ., Bruce G. Hersh L.B., and Gage F H (1987) Development of cholinergic neurons in the septal / diagonal band complex of the rat. Dev Brain Res 36 249-256 Aronson M. Hagberg B. and Gillberg C (1997). Attention deficits and autistic spectrum problems in children exposed to alcohol during gestation : a follow-up study D e v Med Child Neurol 39 583-587. Ashwell K. W. and Zhang L. L. (1996). Forebrain hypoplasia following acute prenatal ethanol exposure quantitative analysis of effects on specific forebrain nuclei. Pathology 28 161-166. Barnes D E. and Walker D. W. (1981) Prenatal ethanol exposure permanently reduces the number of pyramidal neurons in rat hippocampus Brain Res 2 27, 333-340.

PAGE 193

183 Basile D P. Liapis, H. and Hammerman M. R. (1997) Expression ofbcl-2 and bax in regenerating rat renal tubules following ischemic injury Amer J Physiol-Renal Physiol 41, F640-F647. Bauer-Moffett C. and Altman J. (1977). The effect of ethanol chronically administered to preweanling rats on cerebellar development a morphological study. Brain Res 119 249-68. Beasley C. L., and Reynolds G. P. (1997). Parvalbumin-immunoreactive neurons are reduced in the prefrontal cortex of schizophrenics Schizophr Res 24, 349-355. Bhave S. V and Hoffinan P L. (1997) Ethanol promotes apoptosis in cerebellar granule cells by inhibiting the trophic effect ofNMDA. J Neurochem 68, 578-586 Black A. C. Jr. Goolsby, L. W., Cohen G. A. and Young H. E. (1995). Effects of prenatal ethanol exposure on the hippocampal neurochemistry of a lbino rats at 90 days of postnatal age. Am. J. Obstet. Gynecol. 1 7 3 514-519 Bland S. K. and Bland B. H (1986). Medial septal modulation ofhippocampal theta cell discharges. Brain Res. 3 7 5 102-116. Boise L. H. Gonzalez G.-M ., Postema C. E., Ding L. Lindsten T. Turka, L. A. Mao X. Nunez G ., and Thompson C. B. (1993) bcl-x a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death. Cell 74, 597-608. Bonthius D J ., and West J R. (1990). Alcohol-induced neuronal loss in developing rats increased brain damage with binge exposure Alcohol Clin Exp Res 1 4, 10 7-118. Bonthius D J. and West J. R. (1991). Permanent neuronal deficits in rats exposed to alcohol during the brain growth spurt Teratology 44, 147-163 Bradle y D M., Beaman, F D. Moore D B. and Heaton, M. B (1997). Ethanol influences on the chick embryo spinal cord motor system .2. Effect s of neuromuscular blockade and period of exposure. J Neurobiol 3 2, 684-694.

PAGE 194

184 Brauer K. Schober A. Wolff, J. R. Winkelmann E ., Luppa, H. Luth, H.J. and Bottcher H. (1991). Morphology of neurons in the rat basal forebrain nuclei: comparison between NADPH-diaphorase histochemistry and immunohistochemistry of glutamic acid decarboxylase choline acetyltransferase somatostatin and parvalbumin J. Hirnforsch. 32 1-17. Burrows R. C. Shetty A. K., and Phillips D E. (1995). Effects of prenatal alcohol exposure on the postnatal morphology of the rat oculomotor nucleus. Teratology 51, 318328. Castren E. Ohga Y ., Berzaghi M. P. Tzimagiorgis G Thoenen, H., and Lindholm D. (1994) bcl-2 messenger RNA is localized in neurons of the developing and adult rat brain Neuroscience 61, 165-177. Chen J. Zhu, R. L. Nakayama M. Kawaguchi K. Jin K. Stetler R. A. Simon, R. P ., and Graham S. H. (1996). Expression of the apoptosis-effector gene Bax, is up-regulated in vulnerable hippocampal CAI neurons following global ischemia J Neurochem 67, 6471. Cheng E. H. Y. Kirsch D G ., Clem R. J. Ravi R. Kastan M. B. Bedi A. Ueno, K. and Hardwick J M (1997) Conversion ofBcl-2 to a Bax-like death effector by caspases. Science 2 7 8 1966-1968 Chittenden T. Flemington C. Houghton A. B. Ebb, R G ., Gallo G. J. Elangovan B. Chinnadurai, G ., and Lutz R J. (1995). A conserved domain in Bak, distinct from BHl and BH2, mediates cell death and protein binding functions. EMBO Journal 14, 55895596 Chrobak J. J. and Napier T. C. (1992) Antagonism of GABAergic transmission within the septum disrupt s working/episodic memory in the rat. Neurosci e nce 47, 833-841. Clark e, P. G. H. and Oppenheim R. W. (1995) Neuronal death in vertebrate development: in vivo methods. Meth. Cell Biol. 46 2 277-2321. Clarren S K. Alvord E C ., Jr. Sumi S. M ., Streissguth A P. and Smith, D. W (1978) Brain malformations relat e d to pre natal exposure to ethanol. J Pediatr 92, 64-67.

PAGE 195

185 Clausing P. Ferguson S A. Holson, R.R., Allen, R.R., and Paule M. G. (1995). Prenatal ethanol exposure in rats: long-lasting effects on learning Neurotoxicol. Teratol. 17, 545 552. Clem, R. J. Cheng, E. H. Y. Karp C. L. Kirsch D. G. Ueno, K. Takahashi A. Kastan M. B. Griffin D. E ., Earnshaw, W C. Veliuona, M A. and Hardwick, J.M. (1998) Modulation of cell death by Bcl-x(L) through caspase interaction. Proc Natl Acad Sci USA 95, 554-559 Coggenshall R. E and Lekan, H. A. (1996). Methods for determining numbers of cells and synapses: a case for more uniform standards ofreview. J. Comp. Neurol. 364 6-15 Columbano, A ( 1995). Cell death current difficulties in discriminating apoptosis from necrosis in the context of pathological processes in vivo. J Cell Biochem 58 181-190. Cragg B ., and Phillips S (1985) Natural loss of Purkinje cells during development and increased loss with alcohol. Brain Res 3 2 5, 151-160. Das, R. Reddy E P. Chatterjee D ., and Andrews, D. W. ( 1996). Identification of a novel Bcl-2 related gene BRAG-I, in human glioma. Oncogene 12 947-951. Datta S. R. Dudek, H. Tao, X. Masters S. Fu, H A ., Gotoh, Y. and Greenberg M. E. (1997). Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell 91, 2 312 41. Davies A. M. (1995 ) The Bcl-2 family of proteins and the regulation of neuronal survival. Trends Neurosci 18 355-358. Davies A. M (1994). The role of neurotrophins in the developing nervous system. J Neurobiol 2 5 1334-1348. Davies, D. L., and Smith, D. E. (1981). A Golgi study of mouse hippocampal CAI pyramidal neurons following perinatal ethanol exposure. Neurosci. Lett. 26 49-54.

PAGE 196

186 De, A., Boyadjieva, N. I., Pastorcic M., Reddy B. V. and Sarkar, D. K. (1994). Cyclic AMP and ethanol interact to control apoptosis and differentiation in hypothalamic beta endorphin neurons. J Biol Chem 269 26697-26705 Deckwerth, T. L., Elliott J. L., Knudson, C. M ., Johnson E. M., Jr. Snider W D. and Korsmeyer, S. J. (1996) BAX is required for neuronal death after trophic factor deprivation and during development. Neuron 17, 401-411. De Felipe J ., Garcia S.-R. Marco P., del R.-M. R. Pulido P. and Cajal y .-C. S (1993) Selective changes in the microorganization of the human epileptogenic neocortex revealed by parvalbumin immunoreactivity. Cereb. Cortex 3 39-48. De-Lecea L., del-Rio J. A., and Soriano E. (1995) Developmental expression of parvalbumin mRNA in the cerebral cortex and hippocampus of the rat. Brain Res. Mol. Brain. Res. 32 1-13. Delpeso L., Gonzalezgarcia M. Page, C., Herrera R. and Nunez, G (1997) Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt. Science 2 7 8 687-689. Diamond M C ., Murphy G. M. J. Akiyama K. and Johnson R. E (1982) Morphologic hippocampal asymmetry in male and female rats Exp Neurol 7 6 553-565. Diaz, J., and Samson, H. H. (1980). Impaired brain growth in neonatal rats exposed to ethanol. Science 208 751-753. Dixon, E P. Stephenson D T. Clemens, J A. and Little S. P. (1997). Bcl-X-short is elevated following severe global ischemia in rat brains Brain Res 77 6 222-229. Dobbing J. and Sands J (1979). Comparative aspects of the brain growth spurt. E arly Hum Dev 3 79-83. Dohrman, D. P ., West J. R. and Pantazis N J. (1997). E thanol reduces expression of the nerve growth factor receptor but not nerve growth factor protein levels in the neonatal rat cerebellum. Alcohol Clin Exp Res 21, 882-893.

PAGE 197

187 Dubois-Dauphin M. Frankowski, H. Tsujimoto Y. Huarte J and Martinou, J.C. (1994). Neonatal motoneurons overexpressing the bcl-2 protooncogene in transgenic mice are protected from axotomy-induced cell death. Proc Natl Acad Sci US A 91, 33093313. Dudchenko P. and Sarter M. (1991) GABAergic control of basal forebrain cholinergic neurons and memory. Behav. Brain Res. 42 33-41. Duriez P. J ., and Shah G. M (1997). Cleavage of poly(ADP-ribose) polymerase: a sensitive parameter to study cell death. Biochem Cell Biol 7 5 337-349. Dutar P. Bassant M. H. Senut M. C. and Lamour, Y. (1995). The septohippocampal pathway : structure and function of a central cholinergic system Physiol. Rev. 7 5 393427. E dwards S. N., Buckmaster A. E ., and Tolkovsky A. M (1991). The death programme in cultured sympathetic neurones can be suppressed at the posttranslational level by nerve growth factor c y clic AMP, and depolarization. J Neurochem 57, 2140-2143 E lion E A. (1998) Routing MAP kinase cascades. Science 281, 1625-1626. E llis R. E Yuan J Y. and Horvitz H. R. (1991). Mechanisms and functions of cell death. Annu Rev Cell Biol 7, 663-698 Ernfors P ., Lonnerberg P ., A y er-LeLievre C. and Persson H. (1990) Development and regional expression of basic fibroblast grwoth factor mRNA in the rat central nervous system J. Neurosci. Res. 27, 10-15 Ewald S J. and Shao H (1993) Ethanol increases apoptotic cell death ofthymocytes in vitro. Alcohol C lin Exp R es 17, 3593 65. F arli e, P. G ., Drin gen R. R ee s S M ., Kannourakis G. and Bernard 0. (1995) bcl-2 transgene expression can protect neurons against developmental and induced cell death. Proc Natl Acad Sci U S A 92, 4397-4401.

PAGE 198

188 Frankowski H. Missotten M ., Fernandez P.A., Martinou I., Michel P. Sadoul R. and Martinou J. C (1995) Function and expression of the Bcl-x gene in the developing and adult nervous s y stem. Neuroreport 6 1917-1921. Freund T. F. (1989 ) GABAergic septohippocampal neurons contain parvalburnin. Brain Res. 4 7 8 375-381. Freund T. F ., and Antal M. (1988). GABA-containing neurons in the septum control inhibitory interneurons in the hippocampus. Nature 336 170-173 Gajewski, T. F ., and Thompson, C. B. (1996). Apoptosis meets signal transduction elimination of a BAD influence [comment]. Cell 87, 589-592 Gao, W. Q. Zheng J. L. and Karihaloo M. (1995). Neurotrophin-4 / 5 (NT-4 / 5) and brain-derived neurotrophic factor (BDNF) act at later stages of cerebellar granule cell differentiaton. J Neurosci 15 2656-2667 Garcia I. Martinou I., Tsujimoto Y. and Martinou J. C. (1992 ) Prevention of programmed cell death of sympathetic neurons by the bcl-2 proto-oncogene Science 2 58, 302-304 Gerlai R. (1996). Gene-targ e ting studies of mammalian behavior: is it the mutation or the background genotype ? TINS 19 177-181. Gibson L. Holmgreen S. P ., Huang, D. C. Bernard 0., Copeland N. G. Jenkins N. A., Sutherland G. R ., Baker, E ., Adams, J.M., and Cory, S. (1996) bcl-w a nov el member of the bcl2 family promotes cell survival. Oncogene 13 665-675. Gillardon F. Baurle J ., Wickert H. Grosser C -U. and Zimmermann, M ( 1995). Differential regulation of bcl-2 bax c-fos junB, and krox2 4 e x pression in the c e rebellum of Purkinje cell degeneration mutant mice J Neurosci Res 41, 708-715 Gillardon F ., Klimaschewski L. Wickert H. Krajewski S. Reed, J.C., and Zimmermann, M ( 1996) Expression pattern of candidate c e ll death effector proteins Bax, Bcl-2 Bcl-X and c-Jun in sensory and motor neurons followin g sciatic nerve transcection in the rat. Brain Res 7 39 244-250.

PAGE 199

189 Gillardon F., Wickert H. and Zimmermann M. (1995). Up-regulation of bax and down regulation of bcl-2 is associated with kainate-induced apoptosis in mouse brain. Neurosci Lett 192 85-88. Gleichmann M. Beinroth S. Reed, J. C. Krajewski S ., Schultz J. B., Wullner U. Klockgether T. and Weller M. (1998) Potassium-deprivation induced apoptodis of cerebellar granule neurons: cytochrome c relaease in the absence of altered expression of bcl-2 family proteins. Cell Physiol. Biochem 8, 194-201. Goldstein D. B (1972). Relationship of alcohol dose to intensity of withdrawal signs in mice. J. Pharm. Exp. Therap. 180 203-215. Goodlett C R ., and Eilers A. T. (1997). Alcohol-induced Purkinje cell loss with a single binge exposure in neonatal rats: A stereological study of temporal windows of vulnerability. Alcohol Clin Exp Res 21, 738-744 Goodlett C.R., and Johnson T. B (1997). Neonatal binge ethanol exposure using intubation : Timing and dose effects on place learning. Neurotoxicol Teratol 19 435-446. Goodlett C. R. Marcussen B. L., and West J. R. (1990). A single day of alcohol exposure during the brain growth spurt induces brain weight restriction and cerebellar Purkinje cell loss. Alcohol 7, 107-114. Goodlett C.R., and Peterson S. D (1995). Sex differences in vulnerability to developmental spatial learning deficits induced by limited binge alcohol exposure in neonatal rats. Neurobiol. Learn. Mem. 64 265-275 Goodlett C.R., Peterson S D ., Lundahl K. R. and Pearlman A. D. (1997). Binge-like alcohol exposure of neonatal rats via intragastric intubation induces both Purkinje cell loss and cortical astrogliosis Alcohol Clin E x p Res 21, 1010-1017 Goodlett C R ., Thomas J D ., and West J R. (1991). Long-term deficits in cerebellar growth and rotarod performance of rats following "binge-like" alcohol exposure during the neonatal brain growth spurt. Neurotoxicol Teratol 13, 69-74.

PAGE 200

190 Greene P. L., Diaz-Grandados, J. L. and Amsel A. (1992) Blood ethanol concentration from early postnatal exposure: effects on memory-based learning and hippocampal neuroanatomy in infant and adult rats. Behavior. Neurosci. 106, 51-61. Guillery R. W ., and Herrup K. (1997). Quantification without pontification: choosing a method for counting objects in sectioned tissues. J. Comp. Neurol. 386 2-7. Hagg T., Fass-Holmes, B. Vahising H. L. Manthorpe, M. Connor J.M., and Varon S. (1989). Nerve growth factor (NGF) reverses axotomy-induced decreases in choline acetyltransferase, NGF receptor and size of medial septum cholinergic neurons Brain Res 505 29-38 Hagg, T. Van der Zee C. E E. M., Ross G. M., and Riopelle R. J. (1997). Basal forebrain neuronal loss in mice lacking neurotrophin receptro p75Technical Comment Resonse. Science 277 838-839. Hall W. (1975). Weaning and growth of artificially reared rat. Science 190 302-304. Hamre K. M. and West J. R (1993). The effects of the timing of ethanol exposure during the brain growth spurt on the number of cerebellar Purkinje and granule cell nuclear profiles. Alcohol Clin Exp Res 17, 610-622 Hannigan J. H. (1996). What research with animals is telling us about alcohol-related neurodevelopmental deosroder. Parmacol. Biochem. Behav 55 489-499 Hara A. Hirose Y. Wan g, A., Yoshimi N., Tanaka T., and Mori H. (1996). Localization of Bax and Bcl-2 proteins regulators of programmed cell death in the human central nervous system. Virchows Arch 429 249-253. Hardman C. D. McRitchi e, D. A., Halliday G M ., Cartwright H. R., and Morris J. G (1996). Substantia nigra pars riticulata neurons in parkinson's disease. Neurodegeneration 5 49 55 Hartikka J ., and Hefti, F. (1988). Development of septal cholinergic neurons in culture : plating density and glial cells modlulate effects ofNGF on surviva l fiber growth and expression of transmitter-specific enzymes J. Neurosci. 8, 2967-2985.

PAGE 201

191 Heaton M B. and Bradley D. M. (1995). Ethanol influences on the chick embryo spinal cord motor system analyses of motoneuron cell death, motility and target trophic factor activity and in vitro analyses of neurotoxicity and trophic factor neuroprotection J Neurobiol 26 47-61. Heaton M. B. Paiva, M. Swanson, D. J. and Walker, D W. (1993) Modulation of ethanol neurotoxicity by nerve growth factor. Brain Res. 620 78-85 Heaton M B ., Paiva, M. Swanson, D. J ., and Walker, D W. (1994). Responsiveness of cultured septal and hippocampal neurons to ethanol and neurotrophic substances J. Neurosci. Res. 39 305-318. Heaton M B. Swanson, D. J. Paiva M. and Walker, D. W (1996). Influence of prenatal ethanol exposure on cholinergic development in the rat striatum. J. Comp. Neurol. 364 113-120. Heizmann C W. (1984) Parvalbumin an intracellular calcium-binding protein ; distribution properties and possible roles in mammalian cells. Experientia 40 910-921. Henderson G. I., Devi, B G ., Perez A. and Schenker S. (1995). In utero ethanol exposure elicits oxidative stress in the rat fetus. Alcohol Clin Exp Res 19, 714720. Hengartner M. ( 1998). Death by crowd control. Science 281, 1298-1299. Higgins G A., Koh, S ., Chen K S. and Gage F H (1989) NGF induction ofNGF receptor gene expression and cholinergic neuronal hypertrophy within the basal forbrain of the adult rat. Neuron 3 247-356 Hockenbery D ., Nunez, G ., Milliman C. Schreiber R. D. and Korsmeyer S. J. (1990 ) Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature 34 8, 334-336 Hoff, S F ( 1988) Synaptogenesis in the hippocampal dentate gyrus: effects of in utero ethanol exposure. Brain Res. Bull. 21, 47-54.

PAGE 202

192 Hsu, S Y., Kaipia, A., Mcgee, E., Lomeli, M., and Hsueh, A. J. W. (1997). Bok is a pro apoptotic Bcl-2 protein with restricted expression in reproductive tissues and heterodimerizes with selective anti-apoptotic Bcl-2 family members. Proc Natl Acad Sci USA 94 12401-12406 Isenmann, S. Stoll G. Schroeter, M., Krajewski, S. Reed J C ., and Bahr M. (199 8) Differential regulation of Bax, Bcl-2 and Bcl-X proteins in focal cortical ischemia in the rat. Brain Pathol 8, 49-62. Jacobs K. M. Gutnick M. J., and Prince, D. A. (1996) Hyperexcitability in a model of cortical maldevelopment. Cereb. Cortex 6, 514-523. Johnson E. M ., Jr., and Deckwerth, T. L. (1993) Molecular mechanisms of developmental neuronal death. Annu Rev Neurosci 16 31-46. Jones, K. L., and Smith D W. (1973). Recognition of the fetal alcohol syndrome in early infancy Lancet 2 999-1001. Jones K. L., Smith D. W. Ulleland C. N. and Streissguth A. P. (1973). Pattern of malformation in offspring of chronic alcoholic mothers. Lancet 1, 1267-1271. Jones K. R. Farinas I., Backus C ., and Reichardt L. F (1994). Targeted disruption of the BDNF gene perturbs brain and sensory neuron development but not motor neuron development. Cell 76, 989-999. Joyce, E. M ., Rio D. E., Ruttimann U. E. Rohrbaugh J. W. Martin P.R., Rawlings R. R. and Eckardt, M. J. (1994). Decreased cingulate and precuneate glucose utilization in alcoholic Korsakoffs syndrome. Psychiatry Res 54 225 -239 Kahn A. (1998) The connection between PB kinase Akt, and Bad controls apoptosis M S-Med Sci 14 61-62. Kelly S. J. Goodlett, C.R., Hulsether, S. A., and West J R. (1988). Impa i red spatial navigation in adult female but not adult male rats exposed to alcohol during the brain growth spurt. Behav Brain Res 27, 247-257.

PAGE 203

193 Kelly, S. J. Mahoney J C ., Randich A. and West J. R. (1991) Indices of stress in rats effects of sex, perinatal alcohol and artificial rearing. Physiol Behav 49, 751-756. Kelly, S. J., and Tran, T. D. (1997). Alcohol exposure during development alters social recognition and social communication in rats. Neurotoxicol Teratol 19 383-389. Kenner, P. Naumann, T., Bender R. and Frotscher M. (1995). Fate ofGABAergic septohippocampal neurons after fimbria-fomix transection as revealed by in situ hybridization for glutamate decarboxylase mRNA and parvalburnin immunocytochemistry. J. Comp. Neurol. 362 385-399. Kiss J. Patel A. J. Baimbridge K. G., and Freund T F. (1990a) Topographical localization of neurons containing parvalburnin and choline acetyltransferase in the medial septum-diagonal band region of the rat. Neuroscience 36 61-72 Kiss J. Patel A. J. and Freund T. F (1990b). Distribution of septohippocampal neurons containing parvalburnin or choline acetyltransferase in the rat brain. J. Comp. Neurol. 298, 362-372. Kotkoskie L. A. and Norton, S. (1989). Morphometric analysis of developing rat cerebral cortex following acute prenatal ethanol exposure. Exp. Neurol. 106 283-288. Kozopas K. M. Yang T. Buchan, H. L. Zhou, P. and Craig R W. (1993). MCLl, a gene expressed in programmed myeloid cell differentiation has sequence similarity to BCL2. Proc Natl Acad Sci US A 90, 3516-3520. Krajewski S. Krajewska, M., Shabaik A ., Miyashita, T., Wang, H. G. and Reed, J.C. (1994) Immunohistochemical determination of in vivo distribution of Bax, a dominant inhibitor ofBcl-2. Am J Pathol 145, 1323-1336 Krajewski S Mai, J K. Krajewska M ., Sikorska M. Mossakowski M J and Reed, J C (1995) Upregu lation of bax protein levels in neurons following cerebral ischemia. J Neurosci 15, 6364-6376.

PAGE 204

194 Kril J. J. and Homewood J. (1993). Neuronal changes in the cerebral cortex of the rat following alcohol treatment and thiamin deficiency. J. Neuropathol. Exp. Neurol. 52 586-593. Krzywkowski P. De-Bilbao, F. Senut M. C. and Lamour Y. (1995). Age-related changes in parvalburninand GABA-immunoreactive cells in the rat septum. Neurobiol. Aging 16 29-40. Kupfermann I. (1991). Hypothalamus and limbic system: peptidergic neurons homeostasis and emotional behavior in Principles of Neural Sciecne Third Edition E R. Kandel J H. Schwartz and T. M. Jessell, eds. (Norwalk: Appleton and Lange) Lauder J.M., Han V. K. Henderson P., Verdoom T. and Towle A. C (1986 ) Prenatal ontogeny of the GABAergic system in the rat brain: an immunocytochemical study. Neuroscience 19, 465-493 Lee I. J. Soh Y. J. and Song B. J. (1997). Molecular characterization of fetal alcohol syndrome using mRNA differential display. Biochem Biophys Res Commun 240 309313 Li P. Nijhawan D. Budihardjo I., Srinivasula S. M ., Ahmad, M. Alnernri E. S. and Wang X. D (1997) Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91, 4 79-489. Liesi P. (1997) E thanol-exposed central neurons fail to migrate and undergo apoptosis. J Neurosci Res 48 439-448. Lin E. Y. Orlofsky A. Berger M. S., and Prystowsky M. B (1993) Characterization of Al, a novel hemopoietic-specific early-response gene with sequence similarity to bcl2. J Immunol 151, 1979-1988. Lindholm D. Hamner S ., and Zirrgiebel U. (1997) Neurotrophins and cerebellar development. Perspect Develop Neurobiol 5 83-94. Loy R. (1986). Sexual dimorphism in the septoh i ppocampal system. In The Hippocampus R L. Isaacson and K. H. Pribram eds. (New York: Plenum) pp. 301-3 21.

PAGE 205

195 Loy R ., and Milner T. A (19 80). Sexual dimorphism in extent of axonal sprouting in rat hippocampus. Science 208 1282-1284 MacLennan, A. J ., Lee N. and Walker, D. W. (1995). Chronic ethanol administration decreases brain-derived neurotrophic factor gene expression in the rat hippocampus Neurosci Lett 197, 105-108. Maier D. M. and Pohorecky, L.A. (1986). The effect of ethanol and sex on radial arm maze performance in rats. Pharmacol Biochem Behav 25, 703-709. Maier S. E. Miller J. A., West J. R., and Sohrabji F. (1996). Prenatal exposure to alcohol decreases BDNF mRNA and granule cell number in the main olfactory bulb in rats. Society for Neuroscience Abstracts 22, 471. Maisonpierre P. C. L., B. Friedman, B., Alderson R. F., Wiegand, S. J. Furth M. E., Lindsay R. M. and Yancopoulous G D. (1990) NT-3 BDNF, and NGF in the developiong rat nervous system: parallel as well as reciprocal patterns of expression. Neuron 5 501-509 Marcussen B L., Goodlett C.R., Mahoney, J.C., and West, J. R. (1994). Developing rat Purkinje cells are more vulnerable to alcohol-induced depletion during differentiation than during neurogenesis. Alcohol 11, 14 7-156 Markov a E. G ., and Isaev N. K. ( 1992). Effects of nerve growth factor on the development of the dendritic system of cholinergic neurons in dissociated culture of the rat septum Biull. Eksp. Biol. Med. 113, 318-320 Martinou J C ., Dubois D.-M. Staple J. K. Rodrigue z, I., Frankowski, H., Missotten M. Albertini P. Talabot, D ., Catsicas S. Pietra C. (1994). Overexpression ofBCL-2 in transgenic mice protects neurons from naturally occurring cell death and experimental ischemia Neuron 13, 1017-1030. Mattson S. N ., Riley E. P. Gramling, L., Delis D. C. and Jones K L. (1997). Heavy prenatal alcohol exposure with or without physical features of fetal alcohol syndrome leads to IQ deficits J Pediatr 131, 718721.

PAGE 206

196 Maundrell K., Antonsson, B ., Magnenat E., Camps, M., Muda M., Chabert, C., Gill ieron, C., Boschert U. Vialknecht, E., Martinou J. C., and Ark.install, S. (1997) Bcl2 undergoes phosphorylation by c-Jun N-terminal kinase / stress-activated protein kinases in the presence of the constitutively active OTP-binding protein Rael. J Biol Chem 2 7 2 25238-25242. McCarthy, M. M., Kaufman, L. C., Brooks, P J., Pfaff D W., and Schwartz-Giblin, S. (1995). Estrogen modulation of mRNA levels for the two forms of glutamic acid decarbo xylase (GAD) in female rat brain. J. Comp. Neurol. 360 685-697. McGivem, R F., Ervin M. G., McGeary, J., Somes, C., and Handa, R. J. (1998). Prenatal ethanol exposure induces a sexually dimorphic effect on daily water consumption in prepubertal and adult rats (in process citation). Alcohol Clin Exp Res 22, 868-875 McPhalen C. A. Sielecki A. R., Santarsiero B. D. and James M. N (1994). Refined crystal structure of rat parvalbumin a mammalian alpha-lineage parvalbumin at 2.0 A resolution J Mol. Biol. 235, 718-732. Merry D. E., Veis D. J., Hickey W. F., and Korsmeyer S. J. (1994) bcl-2 protein expression is widespread in the developing nervous system and retained in the adult PNS Development 120 301-311. Messer M. Thoman E. B. Galofre A., Dallman T. and Dallman, P .R. (1969) Artificial feeding of infant rats by continuous gastric infusion. J Nutr 98, 404-410. Messmer U. K. and Brune B. (1997). Attenuation of p53 expression and Bax down regulation during phorbol ester mediated inhibition of apoptosis. Br J Pharmacol 121 625-634. Michaelidis T. M. Sendtner, M. Cooper J. D. Airaksinen M. S., Holtmann, B. Meyer M., and Thoenen H. (1996). Inactivation of bcl2 results in progressive degeneration of motoneurons sympathetic and sensory neurons during early postnatal development. Neuron 17, 75-89. Michel R. P. and Cruz 0.-L. M. (1988) Application of the Cavalieri principle and vertical sections method to estimation of lung volume and pleural surface area. J Microsc. 150 117-136

PAGE 207

197 Miettinen R. Sirvio, J. Riekkinen P., Sr., Laakso M. P. Riekkinen M. and Riekkinen P., Jr. (1993). Neocortical hippocampal and septal parvalbumin-and somatostatin containing neurons in young and aged rats: correlation with passive avoidance and water maze performance. Neuroscience 53, 367-378 Miller M. W (1986). Effects of alcohol on the generation and migration of cerebral cortical neurons Science 233 1308-1311. Miller M. W. (1995a). Effect of pre-or postnatal exposure to ethanol on the total number of neurons in the principal sensory nucleus of the trigeminal nerve cell proliferation and neuronal death. Alcohol Clin Exp Res 19 1359-1363. Miller M W (1995b). Generation of neurons in the rat dentate gyrus and hippocampus : effects of prenatal or postnatal treatment with ethanol. Alcohol. Clin. Exp. Res 19 15001509. Miller M W (1996 ). Limited ethanol exposure selectively alters the proliferation of precursor cells in the cerebral cortex. Alcohol. Clin. Exp. Res. 20 139-144. Miller T. M. Moulder K. L., Knudson C M. Creedon D J. Deshmukh, M ., Korsmeyer S. J. and Johnson E M. (1997). Bax deletion further orders the cell death pathway in cerebellar granule cells and suggests a caspase-independent pathway to cell death J Cell Biol 139 205-217. Milner T. A. Loy R. and Amaral D. G (1983). An anatomical study of the development of the septo-hippocampal projection in the rat. Dev Brain Res 8 343-371. Minn, A. J. Boise L. H ., and Thompson, C B (1996 ) Bcl-x(S) anatagonizes the protective e ffects ofBcl-x(L). J Biol Chem 271 6306-631 2 Minn, A. J. Vele z, P. Schendel S. L., Liang H. Muchmore S. W. Fesik S. W. Fill M. and Thompson, C. B. (1997) Bcl-x(L) forms an ion channel in synthetic lipid membranes Nature 3 8 5 353-357.

PAGE 208

198 Mitry R.R., Sarraf, C. E. Wu, C G. Pignatelli M. and Habib N A. (1997) Wild-type p53 induces apoptosis in Hep3B through up-regulation ofbax expression. Lab Invest 77, 369-378. Moore D B. Lee, P. Paiva, M., Walker, D. W. and Heaton, M B. (1998a). Effec t s of Neonatal Ethanol Exposure on Cholinergic Neurons of the Rat Medial Septum. Alcohol I15 219-226. Moore D B. Quintero M. Ruygrok A. C ., Walker D W. and Heaton, M. B. (1998b) Prenatal ethanol exposure reduces parvalbumin-immunoreactive GABAergic neuronal number in the adult rat cingulate cortex. Neurosci. Lett 249 25-28. Moore D B ., Ruygrok A. C. Walker D W. and Heaton M. B. (1997). Effects of prenatal ethanol exposure on parvalbumin-expressing GABAergic neurons in the adult rat medial septum Alcohol Clin Exp Res 21, 849-856 Motoyama N. Wang, F. Roth, K. A., Sawa, H. Nakayama, K. Nakayama, K., Negishi I., Senju S ., Zhang, Q ., Fujii, S. (1995). Massive cell death of immature hematopoietic cells and neurons in Bcl-x-deficient mice. Science 267, 1506-1510. Muir, J L., Everitt B. J. and Robbins T W (1996). The cerebral cortex of the rat and visual attention function dissociable effects of mediofrontal cingulate anterior dorsolateral and parietal cortex lesions on a five-choice serial reaction time task. Cereb Cortex 6 4 70-481. Mukherjee, A B. and Hodgen, G D. (1982). Maternal ethanol exposure induces transient impairment of umbilical circulation and fetal hypoxia in monkeys. Science 2 1 8, 700-702 Muller Y., Rocchi E. Lazaro, J.B., and Clos J. (1995). Thyroid hormone promotes BCL-2 expression and prevents apoptosis o f e arly differentiating cerebellar granule neurons. Int J Dev Neurosci 13, 871-885. Muller Y. Tangre K ., and Clos J (1997). Autocrine regulation o f apoptosis and bcl2 expression by nerve growth factor in early differentiatin g c e rebellar granule neurons involves low affinity neurotrophin receptor. N eurochem Int 31, 177-191.

PAGE 209

199 Napper R. M., and West, J. R. ( 1 995a). Permanent neuronal cell loss in the cerebellum of rats exposed to continuous low blood alcohol levels during the brain growth spurt a stereological investigation J Comp Neurol 362 283 292 Napper R M. and West, J R (1995b ) Permanent neuronal cell loss in the inferior olive of adult rats exposed to alcohol during the brain growth spurt: a stereological investigation. Alcohol. Clin. Exp. Res 19, 1321 1326. Nitsch C. Scotti A. Sommacal A., and Kalt, G. (1989) GABAergic hippocampal neurons resistant to ischemia-induced neuronal death contain the Ca2(+)-binding protein parvalbumin. Neurosci. Lett. 105, 263-268. Nolte J. ( 1993 ) The human brain Third Edition (St. Louis: Mosby-Year Book Inc). O connor L. Strasser, A., Oreilly L.A., Hausmann, G., Adams J.M., Cory, S. and Huang D. C. S. (1998) Bim: a novel member of the Bcl-2 family that promotes apoptosis. EMBO J 17, 384-395. Oltvai Z. N. Milliman C. L., and Korsmeyer S. J. (1993). Bcl-2 heterodimerizes in vivo with a conserved homolog Bax, that accelerates programmed cell death. Cell 7 4 609 619. Oppenheim R. W. (1991). Cell death during development of the nervous system. Annu Rev Neurosci 14 453-501. Ottilie S ., Diaz J L., Home, W., Chang J., Wang Y. Wilson G. Chang S., Weeks S. Fritz L. C ., and Oltersdorf T. (1997). Dimerization properties of human BAD Identification of a BH-3 domain and analysis of its binding to mutant BCL-2 and BCL-X L proteins. J Biol Chem 2 7 2 30866-30872. Pal N. and Alkana R. L. (1997). Use of inhalation to study the effect of ethanol and ethanol dependence on neonatal mouse development without maternal separation: A preliminary study. Life Sci 61, 1269-1281 Pan G. H. Orourke K., and Dixit V. M. (1998). Caspase-9 Bcl-X-L and Apaf-1 form a ternary complex J Biol Chem 2 7 3 5841-5845.

PAGE 210

200 Parsadanian A. S. Cheng Y., Kellerpeck C.R., Holtzman D M. and Snider, W D. (1998). Bcl-x(L) is an antiapoptotic regulator for postnatal CNS neurons. J Neurosci 18, 1009-1019. Pauli, J ., Wilce P. and Bedi, K. S. (1995). Acute exposure to alcohol during early postnatal life causes a deficit in the total number of cerebellar Purkinje cells in the rat. J Comp Neurol 360 506-512. Paus T. Petrides, M ., Evans, A. C. and Meyer E. (1993). Role of the human anterior cingulate cortex in the control of oculomotor manual and speech responses: a positron emission tomography study. J. Neurophysiol. 7 0 453-469 Paxinos G. and Watson, C. (1982). The rat brain in stereotaxic coordinates: Academic Press). Peterson D. A. Leppert J. T. Lee K. F ., and Gage F. H. (1997). Basal forebrain neuronal loss in mice lacking neurotrophin receptor p75-Technical Comment. Science 277, 837-838. Petit P X. Susin S A. Zamzami N. Mignotte B ., and Kroemer G. (1996) Mitochondria and programmed cell death back to the future FEBS Lett 396 7-13. Picard N. and Strick P. L. (1996). Motor areas of the medi al wall : a review of their location and functional activation. Cereb. Cortex 6 342-353 Pierce, D.R., Goodlett C R. and West J. R. (1989) Differential neuronal loss following early postnatal alcohol exposure. Teratology 4 0 113-126. Pierce D. R. Serbus D. C. and Light, K. E. (1993). Intragastric intubation of alcohol during postnatal development of rats results in selective cell loss in the cerebellum Alcohol Clin Exp Res 17, 1 2 75-1280. Pierce D.R., and West J. R. (1987). Differential deficits in regional brain growth induced by postnatal alcohol. Neurotoxicol Teratol 9 1 2 9-141.

PAGE 211

201 Plogmann D., and Celio M. R. (1993). Intracellular concentration of parvalbumin in nerve cells Brain Res 600 273-279 Prevention Center for Disease Control (1995). Update : trends in fetal alcohol syndrome United States 1979 1993. Morbidity and Mortality Weekly Report 4 4 249 251. Rabbani 0., Panickar K. S Rajakumar, G., King M. A. Bodor N. Meyer E. M. and Simpkins J. W (1997). 17 beta-estradiol attenuates fibrial lesion-induced decline of ChA T immunoreactive neurons in the rat medial septum. Exp. Neurol. 146 179-186 Raff M C., Barres B A., Burne J F., Coles H S ., Ishizaki Y. and Jacobson M. D. (1993) Programmed cell death and the control o f cell survival lessons from the nervous system Science 262 695-700 Reed J.C. ( 1994) Bcl-2 and the regulation of programmed cell death. J Cell Biol 1 24, 1-6. Reed J. C. (1997). Doubl e identity for proteins of the Bcl-2 family. Nature 387, 773-776. Renis M., Calabrese V. Russo A. Calderone A. Barcellona M. L., and Rizza V. (1996) Nucl e ar DNA strand breaks during ethanol-induced oxidative stress in rat brain FEBS L e tt 390 153-156 Riley E P. (1990). The long-term behavioral effects of prenatal alcohol exposure in rats. Alcohol. Clin. E xp. R e s 14 670-673 Riley E P. Mattson S. N ., Sowell E. R., Jernigan T. L. Sobe l, D. F ., and Jones K. L. (1995) Abnormalities of the corpus callosum in children prenatally ex posed to alcohol. Alcohol Clin E xp Res 19 1198-1202 Rin g stedt T., Lagercrantz H., and Persson H (1993 ) Ex pression of members of the tr k family in th e d e v e loping postnatal rat brain. Brain Res. De v Brain Res. 72, 119-131.

PAGE 212

202 Rosse T. Olivier, R ., Monney, L., Rager M. Conus, S. Fellay I., Jansen B. and Bomer, C. (1998). Bcl-2 prolongs cell survival after Bax-induced release of cytochrome c. Nature 391, 496-499. Rouayrenc J. F. Boise, L. H. Thompson, C B ., Privat A. and Patey G (1995). Presence of the long and the short forms of Bcl-X in several human and murine tissues. C R Acad Sci ill 318 537-540. Ryabinin A. E. Cole, M ., Bloom, F. E. and Wilson, M C. (1995). Exposure of neonatal rats to alcohol by vapor inhalation demonstrates specificity of microcephaly and Purkinje cell loss but not astrogliosis. Alcohol Clin Exp Res 19, 784-791. Sakahira, H. Enari M ., and Nagata, S. (1998). Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis. Nature 391, 96-99. Sampson P. D. Streissguth A. P ., Bookstein F. L., Little R. E. Clarren S. K. Dehaene P. Hanson, J. W. and Graham, J.M. (1997). Incidence of fetal alcohol syndrome and prevalence of alcohol-related neurodevelopmental disorder. Teratology 56 317-326. Schambra, U. B. Lauder J.M., Petrusz P. and Sulik K. K. (1990). Development of neurotransmitter systems in the mouse embryo following acute ethanol exposure: a histological and immunocytochemical study. Int. J. Dev. Neurosci. 8 507 522 Schendel S. L., Xie, Z. H. Montal M. 0., Matsuyama S. Montal M. and Reed, J. C (1997) Channel formation by antiapoptotic protein Bcl-2. Proc Natl Acad Sci USA 94 5113-5118. Sedlak T. W. Oltvai Z. N Yang E ., Wang, K. Boise L. H. Thompson, C. B. and Korsmeyer S. J. (1995). Multiple Bcl-2 family members demonstrate selective dimeri z ations with Bax. Proc Natl Acad Sci U S A 92, 7834-7838 Shimizu S E guchi, Y. Kamiike, W ., Funahashi Y. Mignon, A. Lacronique V. Matsuda, H. and Tsujimoto Y. (1998). Bcl-2 prevents apoptotic mitochondrial dysfunction by regul a ting proton flux Proc Natl Acad Sci USA 95 1455-1459.

PAGE 213

203 Shimizu S. Eguchi Y. Kamiike, W., Matsuda, H. and Tsujimoto Y. (1996). Bcl-2 expression prevents activation of the ICE protease cascade Oncogene 12, 2251-2257. Shindler, K. S ., Latham C. B., and Roth, K. A. (1997). Bax deficiency prevents the increased cell death of immature neurons in bcl-x-deficient mice J Neurosci 17, 31123119. Singh M. Meyer E. M. Millard, W. J., and Simpkins J. W. (1994). Ovarion steroid deprivation results in a reversible learning impairment and compromised cholinergic function in female sprague-dawley rats Brain Res. 644 305-312 Singh N. P. Lai H. and Khan A. (1995). Ethanol-induced single-strand DNA breaks in rat brain cells. Mutat Res 345 191-196 Smeyne R. J., Klein R ., Schnapp A. Long L. K ., Bryant S. Lewin A., Lira S A., and Barbacid B. (1994) Severe sensory and sympathetic neuropathies in mice carrying a disrupted trk/ngfreceptor gene. Nature 368 246-249. Smith D. E. and Davies D. L. (1990). Effect of perinatal administration of ethanol on the CAI pyramidal cell of the hippocampus and Purkinje cell of the cerebellum : an ultrastructural survey. J. Neurocytol. 19, 708-717. Smith M A (1996). Hippocampal vulnerability to stress and aging: possible role of neurotrophic factors. Behav. Brain.Res 7 8 25-36. Smythe J. W. Colom L. V. and Bland B. H. (1992). The extrinsic modulation of hippocampal theta depends on the coactivation of cholinergic and GABA-ergic medial septal inputs. Neurosci Biobehav Rev. 16 289-308 Sohma 0., Miz u g uchi M. Takashima S. Yamada M. Ikeda K ., and Ohta, S. (1996) High expression of Bcl-x protein in the developing human cerebellar cortex J. Neurosci. Res 43, 175-182. Solbach S ., and Celio M R. (1991). Ontogeny of the calcium binding protein parvalbumin in the rat nervous system Anat. Embryo!. Berl. 184 103-124.

PAGE 214

204 Solodkin A. Veldhuizen S. D., and Van-Hoesen G W (1996) Contingent vulnerability of entorhinal parvalburnin-containing neurons in Alzheimer's disease. J. N eurosci 16 3311-21. Sowell E. R ., Jernigan T L., Mattson, S N. Riley, E P Sobel D. F. and Jones K. L. (1996). Abnormal development of the cerebellar vermis in children prenatally exposed to alcohol size reduction in lobules 1-V. Alcohol Clin Exp Res 20 31-34 Steinhausen H C ., Willms, J. and Spohr, H. L. (1993). Long-term psychopathological and cognitive outcome of children with fetal alcohol syndrome. J Am Acad Child Adolesc Psychiatry 32 990-994. Streissguth A. P. Aase J M. Clarren S. K. Randels S. P. LaDue R. A. and Smith D. F (1991). Fetal alcohol syndrome in adolescents and adults [see comments]. JAMA 265 1961-1967. Sulik K. K. Lauder J. M ., and Dehart, D. B (1984). Brain malformations in prena t al mice following acute maternal ethanol administration. Int. J Devi. Neuroscience 2 203214 Sutherland R. J. Mcdonald R. J. and Savage, D D. (1997). Prenatal exposure to moderate levels of ethanol can have long-lasting effects on hippocampal synaptic plasticity in adult offspring. Hippocampus 7, 232-238. Swanson D. J. King M.A., Walker D. W. and Heaton M. B. (1995). Chronic prenatal ethanol exposure alters the normal ontogeny of choline acetyltransferase activity in the rat septohippocampal system Alcohol Clin Exp Res 19, 1252-1260. Swanson D. J. Tonjes L., King M A. Walker D. W. and Heaton M B (1996) Influence of chronic prental ethanol on cholinergic neurons of the setptohippocampal system. J. Comp. Neurol. 364 104 112 Thomas, J D ., Goodlett C.R., and West J R. (1998). Alcohol-induced Purkinje cell loss d e pends on dev e lopmental timing of alcohol e x posure and correlates with motor performance. Brain Res Dev Brain Res 105 159-66

PAGE 215

205 Tilly J L., Tilly K. I. Kenton, M. L. and Johnson A. L. (1995). Expression of members of the bcl-2 gene family in the immature rat ovary equine chorionic gonadotropin mediated inhibition of granulosa cell apoptosis is associated with decreased bax and constitutive bcl-2 and bcl-xlong messenger ribonucleic acid levels. Endocrinology 136 232-241. Tsujimoto Y. Cossman J. Jaffe E. and Croce C. M (1985) Involvement of the bcl 2 gene in human follicular lymphoma. Science 228, 1440 1443. Turner B B and Weaver D. A. (1985). Sexual dimorphism of glucocorticoid binding in rat brain. Brain Res 343 16-23. Vanderheiden M G ., Chandel N S. Williamson E K. Schumacker P. T. and Thompson C. B. (1997). Bcl-x(L) regulates the membrane potential and volume homeostasis of mitochondria Cell 91, 627 63 7. Veis D. J. Sorenson C M. Shutter J. R. and Korsmeyer S. J. (1993). Bcl 2-deficient mice demonstrate fulminant lymphoid apoptosis polycystic kidneys and hypopigmented hair. Cell 7 5 229-240. Vekrellis K. McCarthy M J ., Watson A. Whitfield J ., Rubin L. L., and Ham, J. (1997). Bax promotes neuronal cell death and is downregulated during the development of the nervous system. Development 12 4, 123 9-1249. Vyas S. Javoyagid F. Herrero M. T ., Strada 0., Boissiere F ., Hibner U ., and Agid Y. (1997) Expression ofbcl-2 in adult human brain regions with special reference to neurodegenerative disorders. J Neurochem 69 223-231. Walker D. W. and Freund G. (1971). Impairment of shuttle box avoidance learning following prolonged alcohol consumption in rats. Physiol. Behav. 7, 773-778 Wanaka A ., and Johnson E M. (1990) Developmental study of nerve growth factor receptor mRNA expression in the postnatal rat cerebellum Brain Res Deve Brain Res 55 288-292

PAGE 216

206 Wang H. G., Rapp, U. R., and Reed J.C. (1996). Bcl-2 targets the protein kinase Raf-1 to mitochondria [see comments]. Cell 87 629-38. Wang X. H., Jenkins, A. 0., Choi, L., and Murphy E. H (1996) Altered neuronal distribution of parvalbumin in anterior cingulate. Exp Brain Res 112 359-371. West J. R., Chen, W. J. and Pantazis, N. J. (1994) Fetal alcohol syndrome: the vulnerability of the developing brain and possible mechanisms of damage. Metab Brain Dis 9 291-322. West J. R., Goodlett, C.R., Bonthius, D J. and Pierce D.R. (1989). Manipulating peak blood alcohol concentrations in neonatal rats review of an animal model for alcohol related developmental effects. Neurotoxicology 10, 347-365. West J. R., Hamre, K. M., and Pierce, D.R. (1984) Delay in brain growth induced by alcohol in artificially reared rat pups. Alcohol 1 213-222 West J. R ., Hodges C. A., and Black, A. C ., Jr. (1981) Prenatal exposure to ethanol alters the organization ofhippocampal mossy fibers in rats. Science 211 957-959. West J. R., and Pierce D .R. (1986). Perinatal alcohol exposure and neuronal damage. In Alcohol and Brain Development J. R. West, ed. (New York: Oxford University Press) pp 121-157 Westlind-Danielsson A., Gould E., and McEwen B. S. (1991). Thyroid hormone causes sexually distinct neurochemical and morphological alterations in rat septal-diaganol band neurons. J. Neurochem 56, 119-128. Wigal T. and Amsel A. (1990). Behavioral and neuroanatomical effects of prenatal postnatal or combined exposure to ethanol in weanling rats. Behav. Neurosci. 104 116126. Wisniewski K ., Dambska M., Sher J. H., and Qazi Q. (1983). A clinical neuropathological study of the fetal alcohol syndrome Neuropediatrics 14, 197-201.

PAGE 217

207 Witt E. D., C R. M. and Hanin I. (1986). Sex differences in muscarinic receptor binding after chronic ethanol administration in the rat. Psychopharmacology Berl 90, 537542 Xiang H ., Kinoshita Y. Knudson C. M. Korsmeyer S. J. Schwartzkroin P.A., and Morrison R. S (1998). Bax involvement in p53-mediated neuronal cell death. J Neurosci 18, 1363-1373 Yang E. Zha J. Jockel J., Boise, L. H. Thompson C. B ., and Korsmeyer S J. (1995). Bad, a heterodimeric partner for Bel-XL and Bcl-2 displaces Bax and promotes cell death Cell 80 285-291. Yang J., Liu X. Bhalla K., Kim C. N. lbrado A. M. Cai J ., Peng T. I., Jones D P. and Wang X (1997). Prevention of apoptosis by Bcl-2 release of cytochrome c from mitochondria blocked [see comments]. Science 2 7 5 1129-1132. Yang X ., Chang, H Y., and Baltimore D. (1998) Essential role of CED-4 oligomerization in CED-3 activation and apoptosis. Science 281, 1355-1357. Zajac C S., and Abel E. L. (1992). Animal models of prenatal alcohol exposure. Int J Epidemiol 21, S24 S32. Zanjani H. S., Vogel M. W., Delhaye, B -N ., Martinou J C. and Mariani J. (1996) Increased cerebellar Purkinje cell numbers in mice overexpressing a human bcl-2 transgene. J Comp Neurol 3 7 4 332-341. Zanjani H. S. Vogel M. W. Delhaye B.-N. Martinou J.C., and Mariani J. (1997). Increased inferior olivary neuron and cerebellar granule cell numbers in transgenic mice overexpressing the human bcl-2 gene. J Neurobiol 32 502-516. Zha H., Aime S -C., Sato T. and Reed, J C. (1996) Proapoptotic protein Bax heterodimerizes with Bcl-2 and homodimerizes with Bax via a novel domain (BH3) distinct from BHl and BH2 J Biol Chem 2 7 1 7440-7444. Zha J.P., Harada H., Osipov K., Jockel J. Waksman G. and Korsmeyer S. J. (1997) BH3 domain of BAD is required for heterodimerization with BCL-X-L and pro-apoptotic activity. J Biol Chem 272, 24101-24104

PAGE 218

208 Zha, J P ., Harada H., Yang E. Jockel J. and Korsmeyer S. J. (1996). Serine phosphorylation of death agonist BAD in response to survival factor results in binding t o 14-3-3 not BCL-X(L) [see comments]. Cell 87, 619-628. Zhou S Y. Baltimore D ., Cantley L. C ., Kaplan D.R., and Franke T F (1997). Interleukin 3-dependent survival by the Akt protein kinase Proc Natl Acad Sci USA 94, 11345-11350. Zou, H ., Henzel W. J. Liu X. S. Lutschg A. and Wang, X D. ( 1997 ) Apaf-1 a human protein homologous to C-elegans CED-4 participates in cytochrome c-dependent activation of caspase-3 Cell 90 405-413

PAGE 219

BIOGRAPHICAL SKETCH David Blaine Moore a Florida native was born in Jacksonville on July 3 1972. He attended Fletcher High School in Neptune Beach until 1990, where he developed an interest in science. He studied biology at the University of North Florida until 1993, and met his future wife Terri Edwards there. His graduate studies began in the Neuroscience Department at the University of Florida College of Medicine in 1994. His Ph.D. research on animal models of ethanol-induced brain injury during development was performed in the laboratory of Dr. Marieta Heaton and in collaboration with Dr. Don Walker. In graduate school, Blaine was the grateful recipient of an NIAAA predoctoral fellowship and an individual NRSA. 209

PAGE 220

I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate in scope and quality, as a dissertation for the degree of Doctor of Philosophy. ?SY\_~ ~Marieta B. Heaton Chair Professor of Neuroscience I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate scope and quality, as a dissertation for the degree of Doctor of Philosophy. rson Associate Professor of Neuroscience I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. ~&.1C~--< Associate Professor of Neuroscience I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy ~w.(,t!MADon W. Walker Professor of Neuroscience I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Anthony T. is Associate Professor of Pathology Immunology and Laboratory Medicine

PAGE 221

This dissertation was submitted to the Graduate Faculty of the College of Medicine and to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. December 1998 Dean College of Medicine Dean Graduate School

PAGE 222

UNIVERSITY OF FLORIDA II I II IIIIII Ill I l l lllll l llll I I IIIIII I III III I I I llll 111111111111111 3 1262 08554 3386


xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID E7RG3X0XY_X8JNMX INGEST_TIME 2012-03-13T14:58:10Z PACKAGE AA00009002_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES