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Assessment of ethanol toxicity in the motor system of the chick and the septohippocampal system of the rat and the involvement of neurotrophic factors

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Assessment of ethanol toxicity in the motor system of the chick and the septohippocampal system of the rat and the involvement of neurotrophic factors
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Bradley, Douglas M., 1971-
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English
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x, 178 leaves : ill. ; 29 cm.

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Brain ( jstor )
Embryos ( jstor )
Ethanol ( jstor )
Female animals ( jstor )
Gene expression ( jstor )
Hippocampus ( jstor )
Neurons ( jstor )
Rats ( jstor )
Receptors ( jstor )
Spinal cord ( jstor )
Department of Neuroscience thesis Ph.D ( mesh )
Disease Models, Animal ( mesh )
Dissertations, Academic -- College of Medicine -- Department of Neuroscience -- UF ( mesh )
Ethanol -- toxicity ( mesh )
Fetal Alcohol Syndrome -- chemically induced ( mesh )
Hippocampus -- drug effects ( mesh )
Hippocampus -- embryology ( mesh )
Motor Neurons -- drug effects ( mesh )
Motor Neurons -- embryology ( mesh )
Nervous System -- drug effects ( mesh )
Nervous System -- embryology ( mesh )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1998.
Bibliography:
Includes bibliographical references (leaves 158-177).
Additional Physical Form:
Also available online.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Douglas M. Bradley.

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University of Florida
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29076324 ( ALEPH )
50910814 ( OCLC )

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ASSESSMENT OF ETHANOL TOXICITY IN THE MOTOR SYSTEM OF THE CHICK
AND THE SEPTOHIPPOCAMPAL SYSTEM OF THE RAT AND THE
INVOLVEMENT OF NEUROTROPHIC FACTORS















By

DOUGLAS M. BRADLEY
















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














ACKNOWLEDGMENTS

I would like to acknowledge the help of many people who made completion of this dissertation and doctoral research possible. First and foremost, I would like to thank my wife, Korey, who provided valuable mental support and editing and presentation advice through the years. I also want to thank my advisor, Marieta Heaton, who provided the means for completing this research and provided excellent guidance. My committee, Drs. MacLennan, Shiverick, Streit, and Walker, really helped to make this research better. My father, Dr. Edwin L. Bradley, provided valuable advice on statistics. I want to thank my family, for understanding the time required to complete this degree. I also want to thank the National Science Foundation and NIAAA, for supporting me financially through graduate school. The Department of Neuroscience provided an excellent facility for conducting this research. I also want to thank all of the people in our laboratory who helped in a variety of technical and supportive ways throughout the years. Blaine Moore, has been a friend in addition to giving helpful advice on scientific matters. Francesca Beaman, Steve Farnworth, Kara Kidd, Nancy MacLennan, David Melman, Jean Mitchell, Micheal Paiva, and Leon Williams gave wonderful technical assistance. I also wish to apologize to anyone who is unintentionally omitted from this list.














TABLE OF CONTENTS

page

ACKNOWLEDGMENTS..........................................................ii

LIST OF TABLES ................................................................V

LIST OF FIGURES ............................................................... Vi

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

CHAPTERS

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

Fetal Alcohol Syndrome Background........................................ 1
Chick Embryo.............................................................. 3
Motor System and Ethanol ..................................................5
Motor System and NTFs.................................................... 5
Neuroprotection ............................................................7
Rat Model of FAS.......................................................... 9
Rat Septohippocampal System.............................................. 10
Neurotrophic Factors............................................ I........... 11
Gene Deletion Studies...................................................... 12
NTF Ontogeny in the Hippocampus ........................................ 14
Neurotrophins and Ethanol................................................. 15
Hypotheses ................................................................ 16

2 CHARACTERIZATION OF MOTONEURON SURVI VAL AND CELL
DEATH FOLLOWING ETHANOL EXPOSURE AND CURARE
ADMINISTRATION, AND AFTER THE PERIOD FOR
NATURALLY OCCURRING CELL DEATH....................... 18

Summary................................................................... 18
Introduction................................................................ 18
Methods.................................................................... 21
Results..................................................................... 29
Discussion ................................................................. 38

3 CHARACTERIZATION OFMOTONEURON SURVIVAL FOLLOWING
ETHANOL EXPOSURE AND CONCURRENT TREATMENT
WITH EXOGENOUS GDNF OR BDNF IN THE EMBRYONIC
CHICK SPINAL CORD ............................................ 49

Summary ..................................................................49
Introduction ................................................................50
Materials and Methods..................................................... 55


ill








R e su lts ...................................................................................... 58
D iscu ssio n .................................................................................. 67

4 CHARACTERIZATION OFTHE NEUROTROPHINAND
NEUROTROPHIN RECEPTOR GENE EXPRESSION IN THE HIPPOCAMPUS FOLLOWING CHRONIC TREATMENT AND
EARLY POSTNATAL ETHANOL TREATMENT IN THE RAT ......... 75

S u m m ary .................................................................................... 75
In tro d u ctio n ................................................................................. 76
Materials and Methods .................................................................... 83
R e su lts ...................................................................................... 88
D iscu ssio n ................................................................................. 133

5 CONCLUSIONS AND IMPLICATIONS ............................................ 146

Animal Models ............................................................................ 146
M eth o d s .................................................................................... 147
Hypotheses and Results ................................................................. 150
C o n clu sio n s ............................................................................... 154

REFERENCES .................................................................................... 158

BIOGRAPHICAL SKETCH .................................................................... 178






























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LIST OF TABLES

Table page

2-1. Cell size and spinal cord length .......................................................... 35

2-2. Neurotrophic activity of crude muscle extract ......................................... 37

3-1. Motoneuron Size and Spinal Cord Length .............................................. 59







































v














LIST OF FIGURES

Figure page

2-1. Number of motoneurons in lumbar spinal cord at E12 following treatment ..... 31 from E4 to E11.

2-2. Photomicrographs of coronal sections from the midlumbar region of E12 ..... 34 spinal cords.

2-3. Number of motoneurons in lumbar spinal cord at E16 following ethanol ...... 36 treatment from El0 to EI5.

3-1. Number of motoneurons in the later motor column of the lumbar spinal cord ....... 61 at El6.

3-2. Interaction between ethanol and neurotrophic factors ................................. 62

3-3. Photomicrographs of coronal sections from the midlumbar region of E16 ..... 64 spinal cords.

3-4. High magnification photomicrographs from the midlumbar section of E16 ..... 66 spinal cords.

4-1. Brain weight at P21 of female and male animals following prenatal ethanol ......... 90
exposure.

4-2. Weight gain during postnatal ethanol exposure in male animals .................... 92

4-3. Gross morphological measurements following EPET in male animals at P21 ........ 94 4-4. Brain weight and Brain weight to body weight ratio of EPET male animals ......... 95
at P21.

4-5. Weight gain during postnatal ethanol exposure in female animals .................. 96

4-6. Brain weight and brain weight to body weight ratio in EPET female animals ........ 98 at P21.

4-7. Phosphorimaging view of BDNF Northern blots composed of the .................. 100
hippocampal region from P21 rat brains exposed to ethanol prenatally. 4-8. Relative BDNF 4.4 kb transcript expression in rat hippocampus at P21 ............ 101
following prenatal exposure to ethanol.

4-9. Relative BDNF 1.7 kb transcript gene expression following prenatal ............... 102
exposure in P21 rats.


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4-10. Phosphorimaging view of NT-3 Northern blots composed of the .................. 104
hippocampal region from P21 rat brains exposed to ethanol prenatally. 4-11. Relative NT-3 gene expression following prenatal ethanol exposure in P21 ...... 105 rats.

4-12. Relative trkB active receptor gene expression following prenatal ethanol .......... 106
exposure in P21 rats.

4-13. Relative trkB truncated transcript gene expression following prenatal ............. 107
exposure in P21 rats.

4-14. Phosphorimaging view of trkC Northern blots composed of the ................... 109
hippocampal region from P21 rat brains exposed to ethanol prenatally. 4-15. Relative trkC 14 kb transcript gene expression at P21 in rats exposed to .......... 110
ethanol prenatally.

4-16. Relative trkC 4.7 kb truncated transcript gene expression following ............... 111
prenatal exposure in P21 rats.

4-17. Relative trkC 3.9 kb transcript gene expression following prenatal ................ 112
exposure in P21 rats.

4-18. Phosphorimaging view of cyclophilin Northern blots composed of the ........... 115
hippocampal region from P21 rat brains exposed to ethanol prenatally. 4-19. Phosphorimaging view of BDNF Northern blots from postnatally ................. 118
exposed P21 rats.

4-20. Relative BDNF 4.4 kb transcript gene expression following postnatal ............ 119
exposure in P21 rats.

4-21. Relative BDNF 1.7 kb transcript gene expression following postnatal ............ 120
exposure in P21 rats.

4-22. Phosphorimaging view of NT-3 Northern blots from postnatally exposed ........ 122
P21 rats.

4-23. Relative NT-3 1.5 kb gene expression following postnatal exposure in ........... 123
P21 rats.

4-24. Relative trkB active receptor gene expression following postnatal .................. 124
exposure in P21 rats.

4-25. Relative trkB truncated transcript gene expression following postnatal ............ 125
exposure in P21 rats.

4-26. Phosphorimaging view of trkC Northern blots from postnatally exposed ......... 127
P21 rats.

4-27. Relative trkC 14 kb transcript gene expression in P21 rats following .............. 128
postnatal exposure



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4-28. Relative trkC 4.7 kb truncated transcript gene expression in P21 rats .............. 129
following postnatal exposure. 4-29. Relative trkC 3.9 kb truncated transcript gene expressionin P21 rats .............. 130
following postnatal exposure. 4-30. Phosphorimaging view of cyclophilin Northern blots from postnatally ............ 132
exposed P21 rats.












































viii








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

ASSESSMENT OF ETHANOL TOXICITY IN THE MOTOR SYSTEM OF THE CHICK
AND THE SEPTOHIPPOCAMPAL SYSTEM OF THE RAT AND THE
INVOLVEMENT OF NEUROTROPHIC FACTORS By

Douglas M. Bradley
August 1998

Chairman: Douglas K. Anderson

Major Department: Neuroscience

The research described in this document was undertaken to further the

understanding of the toxic effects that ethanol exerts on the developing nervous system. Fetal alcohol syndrome has been recognized as one of the leading environmentally-induced causes of mental retardation in the western world and continues to be a problem despite education and publicity concerning the dangers of ingesting ethanol-containing beverages during pregnancy. The doctoral research described attempted to ascertain some new properties of ethanol toxicity in the nervous system and to determine ways that these toxic effects could be modulated in living animals. Previous research from other laboratories has suggested that motoneurons of the spinal cord might be susceptible to ethanol's toxic effects. Our laboratory confirmed this finding by administering ethanol to developing chick embryos from embryonic day 4 (E4) to El 1 and assessing the number of motoneurons present in the lumbar spinal cord. Specifically, a reduction in the number of motoneurons present in this population was observed. The present experiments found that embryonic administration of ethanol from El0 to E15 also results in a loss of motoneurons. Further, the neurotrophic activity of muscle from these animals is unchanged from that of control animals. Neuromuscularj unction blocking agents, which prevent naturally occurring cell death of spinal cord motoneurons, have little effect in altering ethanol's toxic effects. Administration of glial cell line-derived neurotrophic factor acted to increase motoneuron number following ethanol administration, but brain-derived neurotrophic factor did not.


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The hippocampus is an important structure of the brain thought to be involved with learning and memory. In a mammalian model of fetal alcohol syndrome, the gene expression of tyrosine receptor kinase C, a neurotrophic factor receptor in the brain, is reduced in the hippocampus of 21-day-old male rats following prenatal ethanol exposure, but is unchanged in the brain of female rats. Appropriate background for understanding this research, as well as the implications of all of these results, is described in the resulting chapters.








































x














CHAPTER 1
BACKGROUND INFORMATION

Fetal Alcohol Syndrome Background
In 1973, Jones and Smith (1973) first described a series of morphological and

cognitive deficits in children and infants of alcoholic mothers which was later termed fetal alcohol syndrome (FAS). Since that time, much evidence has been gathered regarding the effects ethanol exerts in the developing nervous system (Barnes and Walker, 1981; Jones and Smith, 1973; Miller, 1986; Streissguth et al., 1991; West, 1986). FAS is diagnosed in 1-2 out of every 1000 live births in the United States and is characterized by low birth weight, decreased memory and learning, hyperactivity, facial dysmorphia, and lowered IQ (Abel, 1995; Jones and Smith, 1973; Streissguth et al., 1991). Human FAS patients have been analyzed for neuropathology postmortem and this analysis has identified central nervous system (CNS) abnormalities which include disorders of laminae of the cerebral cortex, cerebellar abnormalities, a reduction of dendritic spines on cortical pyramidal cells, hippocampal malformation, and microcephaly (Clarren et al., 1978; Ferrer and Galofre, 1987). Interpretation of these studies is complicated by the fact that most of these infants had related cardiovascular problems (Clarren et al., 1978). Abnormalities in humans can range from physical deficits that are easily distinguished (such as gross microencephaly) to microscopic changes (such as dendritic anomalies in neurons that survived alcohol exposure) that require finer analyses (Ferrer and Galofre, 1987). Motor dysfunction and other behavioral deficits, such as an impairment in sensory and motor functions, are associated with FAS (Streissguth et al., 1983). Additionally, children with FAS are deficient in habituation to redundant stimuli (Church and Gerkin, 1988). What is important





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to note is that as these patients have aged, the deficits have not lessened (Streissguth, 1993).

Specific neuronal populations known to be affected by ethanol in animal models include the cerebellum (Cragg and Phillips, 1985; West, 1986), the septohippocampal system (Barnes and Walker, 1981), cerebral cortex (Miller, 1986), the substantia nigra (Shetty et al., 1993), chief sensory trigeminal nucleus (Miller and Muller, 1989), red nucleus (Zajac et al., 1989), inferior olivary nucleus (Napper and West, 1995), stiatum (Heaton et al., 1996) and motoneurons of the spinal cord (Heaton and Bradley, 1995). Microscopic and molecular changes that have also been observed in animal models following ethanol exposure include decreased dendritic arborization (Davies and Smith, 1981), delayed synaptogenesis (Leonard, 1987), decreased neurotransmitter synthesis (Rawat, 1977; Swanson et al., 1994), changes in connectivity (West et al., 1994), and cell loss (Barnes and Walker, 1981; Bauer-Moffet and Altman, 1975; West et al., 1986). These alterations following ethanol exposure in animals are important because they correlate to deficits observed in human FAS. That is, the neuronal region affected seems to relate to a specific deficiency common to human FAS patients.

The mechanisms of ethanol toxicity in the CNS are not fully understood. Since ethanol can cross the blood brain barrier, it has the ability to directly affect the developing nervous system (West et al., 1994). Ethanol can interact with cellular membranes and proteins and reduce protein synthesis (Zajac and Abel, 1992). Additionally, ethanol has been implicated in producing hypoglycemia (Snyder et al., 1992; West et al., 1994), hypoxia (Mukherjee and Hodgen, 1982), and in increasing oxidative stress (Henderson et al., 1995). All of the above information suggest possible explanations for the toxic effects ethanol exerts on the developing nervous system. The current experiments were designed to test facets of the relationship between neurotrophic factors (NTFs) and ethanol. Important to these studies is the underlying hypothesis that NTFs are involved in FAS neuropathology. The NTF hypothesis for FAS proposes that ethanol exposure results in






3


alterations in the synthesis, availability, delivery, and /or biological activity of normally occurring neurotrophic substances. Further, ethanol may alter the capacity of target neuronal populations to respond to NTFs in a normal fashion. Another important aspect of the NTF hypothesis is the idea that exogenous NTFs may afford some protection to ethanol-susceptible neuronal populations. The current studies sought to determine whether NTF synthesis, availability, and biological activity were affected by ethanol treatment and whether the addition of exogenous factors in vivo could prevent ethanol toxicity in a population known to be vulnerable to ethanol insult. To adequately examine these goals, two animal models were used: the chick embryo and the developing rat. Each model has specific advantages that make it attractive for FAS research and each will be discussed in more detail below. All of the studies detailed in this document are related in that the examine ethanol toxicity as it relates to NTFs. Whether this relationship is in the effect that ethanol has on NTFs or neurotrophic support, or the effect that exogenous NTFs have on ethanol toxicity, the objective is consistent: to understand the manner in which these two types of molecules are related in producing deficits observed in animal models of FAS.

Chick Embryo

Ethanol affects chick embryo development in a manner similar to mammals. Chicks exposed to ethanol prenatally have been shown to exhibit reduced brain size, brain weight, DNA and protein synthesis (Pennington and Kalmus, 1987), and reduced neurotransmitter synthesis (Brodie and Vernadakis, 1990; Swanson et al., 1994). Neuronal populations affected by ethanol exposure in ovo in chick include the cerebellum (Quesada et al., 1990), cerebral cortex (Delphia et al., 1978), and motoneurons of the spinal cord (Heaton and Bradley, 1995). The cerebellum (Marcussen et al., 1994; Smith and Davies, 1990) and cerebral cortex (Miller, 1986) in mammals are also affected by developmental ethanol exposure. While chick motoneurons are susceptible to the toxic effects of ethanol both in culture (Dow and Riopelle, 1985; Heaton and Bradley, 1995) and in vivo (Heaton and Bradley, 1995), they have not been specifically quantified in mammals exposed to ethanol.






4


Some evidence does suggest that there are neuromuscular problems associated with human FAS. Specifically, children exposed to ethanol exhibit motor deficits (Streissguth et al., 1983). More important to the current studies is the fact that ethanol can reduce motoneuron number when administered to chick embryos from embryonic day 4 (E4) to Eli (Heaton and Bradley, 1995). We have hypothesized that this reduction may be dependent on naturally occurring cell death (NOCD) since this period (approximately E6-E9) occurred during the period of ethanol exposure utilized in that study (Pittman and Oppenheim, 1978). During the period of NOCD, nearly half of the original number of motoneurons perish. Curare, as well as other neuromuscular junction blocking agents, have been shown to suspend NOCD in motoneurons presumably by increasing the number of synapses and thereby increasing the availability of target-derived NTFs at the neuromuscular junction (Oppenheim, 1991; Pittman and Oppenheim, 1978). In these experiments, approximately 50% more motoneurons survive in curare-treated embryos than in control embryos.

The chick has several advantages that make it a good choice as a model for FAS. Ethanol can be administered in exact doses to the developing embryo, and only the embryo's liver can remove the ethanol from the bloodstream. However, alcohol dehydrogenase does not begin in the developing chick until around E8 (Wilson et al., 1984). Maternal influences are removed when using the chick embryo. Ethanol is cleared from the bloodstream by the mother in a mammalian system whereas the chick embryo is isolated as it develops. This model allows the investigator to observe direct effects of ethanol without interactions of maternal metabolism interfering. While chick development is different from mammalian gestation, this model allows researchers to study in vivo interactions in a developing organism that are not possible in a mammalian model. Another advantage for the research described in this document is that the chick embryo model has been used to study the ability of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) to regulate NOCD in motoneurons of the chick embryo spinal cord (Oppenheim et al., 1995; Oppenheim et al., 1992). It is important to






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note that these experiments would be impossible to perform in mammals since exogenous NTFs can not be administered individually to developing fetuses. Oppenheim's laboratory has performed experiments using a variety of NTFs to study their effects on NOCD. In these experiments, NTFs are applied directly to the membranes of the developing embryo through windows in the outer egg shell. Replicating these experiments in mammals would require extensive surgical procedures that would undoubtedly have an adverse effect on fetal and maternal survival. Some of the present experiments utilize the chick embryo for all of the advantages described above.

Motor System and Ethanol

There is direct evidence that warrants further investigation into the possible effects that ethanol may exert on motoneurons. In both the human and in animal models, previous studies have determined that ethanol damages developing muscle (Adickes and Shuman, 1983; Nyquist-Battie et al., 1987). In the human, these cases described flaccid, hypotonic neonates which exhibited major muscle structural deficiencies including hypotrophy, dominance of type II fibers, and sarcomeric dysplasia (Adickes and Shuman, 1983). Prenatal exposure to ethanol in the guinea pig resulted in structural malformations of the gastrocnemius muscle including vacuolated sarcoplasmic reticula, enlarged lipid droplets, decreased glycogen, and mitochondrial abnormalities (Nyquist-Battie et al., 1987). Proper muscle fiber maturation is dependent on concurrent development and innervation by motoneurons. Therefore, the possibility exists that deficiencies noted above are due to an underlying effect that ethanol exerts on developing motoneurons (Ishiura et al., 1981). It is equally likely that ethanol may exert some direct effect upon developing muscle. Accordingly, a limited analysis of muscle neurotrophic activity in chick leg muscle is undertaken in the present studies.

Motor System and NTFs

The developing motor system is dependent on many different NTFs for proper

growth. Two of the NTFs that developing motoneurons encounter are BDNF and GDNF.






6


BDNF is a member of the neurotrophin family of NTFs which includes nerve growth factor (NGF), neurotrophin-3 (NT-3), and NT-4/5. BDNF is a 118 amino acid residue polypeptide (Ilag et al., 1994) that forms homodimers to attain its active form and binds with high affinity to tyrosine receptor kinase B (trkB; Klein et al., 1991). Previous research has identified two major pathways that are initiated by autophosphorylation of trk (Stephens et al., 1994; Tolkovsky, 1997). One of these pathways leads to activation of the MAP kinase cascade and may initiate neurite outgrowth, transcription, or cellular hypertrophy (Stephens et al., 1994). The other pathway leads to the activation of akt (a serine/threonine kinase) and may initiate neurite outgrowth, survival, and receptor internalization (Tolkovsky, 1997). Developing skeletal muscle produces BDNF, which is known to support motoneuron survival during development by suspending NOCD in a subset of the developing motoneuron pool and to protect motoneurons of both the chick and rat from degenerating after lesion (Oppenheim et al., 1992; Sendtner et al., 1992; Yan et al., 1992). It is important to not that the addition of recombinant factors such as BDNF does not rescue all motoneurons in the developing motor column. BDNF is also expressed by the hippocampus, adrenal gland, and whole brain during rat development (Maisonpierre et al., 1990).

The other NTF used in the present studies of chick development, GDNF, is a member of the transforming growth factor B superfamily and naturally occurs as a homodimer with a molecular weight of 40-45 kD (each molecule 134 amino acid residues; (Lin et al., 1993). GDNF and its receptors, GDNFRc and c-ret, form a complex that allows c-ret to transduce intracellular signals from GDNF (Jing et al., 1996; Treanor et al., 1996). Prior to binding with c-ret, GDNFR acts as a ligand-binding protein by binding GDNF (Jing et al., 1996). The GDNFRo/GDNF complex then forms a complex with cret--the only molecule of the complex capable of producing intracellular signals (Jing et al., 1996; Rosenthal, 1997; Treanor et al., 1996). The signal transduction pathways initiated by c-ret activation include the MAP kinase pathway (Worby et al., 1996) and the






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Ras/ERK2 pathway (van Weering and Bos, 1997). MAP kinase and ERKs are proteins known to be involved in gene expression (Hazzalin et al., 1997; Mucsi et al., 1996). During rat development, GDNF mRNA is expressed by mesenchymal cells and in developing skeletal muscle beginning at E15, and in developing skin beginning at E17 (Nosrat et al., 1996; Trupp et al., 1995; Wright and Snider, 1996). Peripherally in the rat, GDNF is expressed in the teeth, tongue, retina, nasal cavity, ear, kidney, and gastrointestinal tract during various stages of development (Nosrat et al., 1996). Centrally, GDNF is expressed in the striatum, hippocampus, and cerebellum beginning at E15, and in the trigeminal motor nucleus (E17) and cortex (postnatal day 7). Generally, populations that are responsive to GDNF express c-ret. These populations include substantia nigra dopaminergic neurons (Trupp et al., 1995), spinal motoneurons (Pachnis et al., 1993; Tsuzuki et al., 1995), and certain subpopulations of the peripheral ganglia (Pachnis et al., 1993; Tsuzuki et al., 1995). A small segment of Purkinje neurons does exhibit sensitivity to GDNF early in development before expression of c-ret commences (Nosrat et al., 1997), thus implying that GDNF might have the ability to signal through a receptor other than cret. During chick embryogenesis, c-ret mRNA is expressed in the Wolffian duct and ureteric bud, the enteric, dorsal root, sympathetic and facioacoustic ganglia, and the ventral spinal cord (Schuchardt et al., 1995).

Neuroprotection

NTFs have been shown to protect against insults such as hypoxia, hypoglycemia, and changes in calcium homeostasis. Examples of neuroprotection by polypeptide growth factors include epidermal growth factor protection of whole brain neuronal cultures from anoxia (Pauwels et al., 1989), NGF protection of rat hippocampal and human cortical neurons from hypoglycemia (Cheng and Mattson, 1991), and bFGF prevention of thalamic degeneration following cortical infarction (Yamada et al., 1991). GDNF is particularly potent in protecting neurons from a variety of conditions that normally result in death. Such insults and environmentally-produced conditions include NOCD (Oppenheim et al.,






8


1995), 6-OHDA lesion (Choi-Lundberg et al., 1997; Kearns and Gash, 1995; Tomac et al., 1995), and axotomy (Gimenez y Ribotta et al., 1997; Houenou et al., 1996; Oppenheim et al., 1995). While NOCD is not an insult in the sense that 6-OHDA lesion is, it does result in neuronal death and NTFs such as GDNF do prevent it from proceeding in a subset of the developing motor pool in live animals. Like GDNF, BDNF is effective in providing neuroprotection from events that normally result in neuronal death. However, different neuronal populations are protected by BDNF. For example, BDNF protects against ischemia-induced cell death in rat hippocampal slice cultures (Pringle et al., 1996), and prevents NOCD in some motoneurons (Oppenheim et al., 1992) and apoptotic death in PC12 cells (Jian et al., 1996) and cultured rat cerebellar granule neurons (Kubo et al., 1995). The fact that both GDNF and BDNF provide such potent support for developing and injured neurons suggests that both could protect motoneurons against toxic events produced by ethanol.

Neuroprotection from ethanol has been studied previously, but mostly in culture. Examples of this phenomenon include NGF protection of cultured dorsal root ganglion (DRG) neurons (Heaton et al., 1993) and septal neurons (Heaton et al., 1994) and basic fibroblast growth factor (bFGF) protection of cultured septal and hippocampal neurons (Heaton et al., 1994). Additionally, both NGF and bFGF protect cultured cerebellar granule cells from ethanol-induced cell death (Luo et al., 1997). Neuroprotection afforded by NGF and bFGF was found to require both protein and RNA synthesis which suggests that neuroprotection is related to a signal that the NTF receptor sends to the nucleus of the cell (Luo et al., 1997). GDNF has been shown to protect rat organotypic cultures of cerebellar Purkinje cells from ethanol neurotoxicity (McAlhany et al., 1997). To date, the only in vivo demonstration of NTF neuroprotection from ethanol toxicity is protection of choline acetyltransferase activity by NGF (Brodie et al., 1991). The reason that NTF neuroprotection is important for the study of FAS is that many of the toxic events that are prevented by NTFs in culture are implicated as potential mechanisms for ethanol toxicity in






9


the nervous system. Mechanisms for ethanol toxicity that are suggested by previous research include hypoxia, hypoglycemia, and changes in calcium homeostasis (Alturaet al., 1983; Snyder et al., 1992; Webb et al., 1995). If these insults are indeed responsible for ethanol's toxic effects, NTFs might protect the developing nervous system from damage.

Rat Model of FAS

The rat is the most widely used model in FAS research. However, a caveat of using the rat as a model is the relative gestational period. Rat prenatal development is approximately equivalent to the first two trimesters of human development (Goodlett et al., 1993). A major event of prenatal development in humans is the brain growth spurt (BGS), when many functional synapses are made in the nervous system. In rats, this event occurs postnatally from P4-PlO (West, 1987). Therefore, experiments that wish to mimic third trimester ethanol exposure in humans must incorporate the BGS. This objective requires postnatal ethanol exposure in rats. Exposure to ethanol during the BGS produces deficits that demonstrate the sensitivity of the CNS to ethanol during this period. Postnatal exposure in the rat produces deficits that are different from those seen following prenatal exposure. For example postnatal ethanol exposure can produce loss of cerebellar Purkinje cells (Bonthius and West, 1990; Bonthius and West, 1991; Goodlett and West, 1992; West, 1986; West et al., 1990). Since postnatal ethanol exposure damages neuronal populations known to be damaged in FAS, it does serve as a model for FAS.

A caution of any postnatal exposure paradigm is that maternal metabolism of

ethanol is removed and the subjects are exposed to ethanol in the same manner as adults (i.e., only the neonatal liver removes ethanol from the bloodstream). Alcohol dehydrogenase (ADI-) activity begins in the rat on approximately gestational day 15 (Boleda et al., 1992; Tietjen et al., 1994). Fetal ADH has very low activity in comparison to adult ADH, which suggests that metabolism of ethanol during pregnancy is completed almost entirely by maternal ADH. Between P20 and P39 all subclasses of ADH reach






10


100% of adult activity (IBoleda et at., 1992). Another difficulty is that suckling rats will not readily consume ethanol because their entire diet consists of mother's milk.

Two typical methods for ethanol delivery to newborn rats are artificial rearing (AR) and vapor inhalation, both of which have advantages and disadvantages. AR is a surgical procedure which consists of fitting a neonatal pup with a gastric fistula and tube, maintaining the pups in cups placed in a heated water bath, and feeding the pup an artificial milk solution via the tube and fistula. The advantage of the AR method is that it provides constant nutrition and produces no damage to the mucous membranes of the subject. Major disadvantages of AR are that interaction between mother and pup is removed and that it can be stressful for the neonatal rat. The stress induced by AR produces gliosis in rat cortex (Ryabinin et al., 1995), although gliosis following postnatal exposure via intragastric intubation was observed (Goodlett et al., 1997). In this latter experiment, gliosis was not observed in control animal s (Goodlett et al., 1997). The fact that the AR procedure per se may produce changes in brain structure indicates that results obtained using AR could be difficult to interpret. The ethanol vapor inhalation procedure consists of placing neonatal rats in a sealed chamber that contains circulating air and ethanol vapor. This procedure has been theorized to damage the mucous membranes of the lungs which could interfere with oxygen exchange and general metabolism (Ryabinin et al., 1995). However, no evidence supports this contention and lung damage has not been observed in rats exposed to ethanol vapors (Bauer-Moffet and Altman, 1975). Other methods for delivering ethanol to neonatal rats include delivery of ethanol through mother's milk by limiting the dam's liquid intake to ethanol-containing fluids, and exposing pups to ethanol vapor concurrently with the dam.

Rat Septohippocampal System

Many neuronal populations exhibit some susceptibility to the toxic effects of

ethanol. As was mentioned above, a variety of neuronal types from brain regions such as the cerebellum (Cragg and Phillips, 1985), the septohippocampal system (Barnes and Walker, 1981; West and Pierce, 1986), the cerebral cortex (Miller, 1986), and the






11


oculomotor nucleus (Burrows et al., 1995) are known to be affected in some way by ethanol exposure. The hippocampus is an important structure with regard to memory and learning in humans and animals (Bunsey and Eichenbaum, 1996; Cohen and Squire, 1980). Therefore, damage to the hippocampus observed in the rat following ethanol exposure may correspond to similar damage to the hippocampus in humans. Since learning and memory deficits are a common characteristic of FAS (Abel, 1995; Jones and Smith, 1973; Streissguth et al., 1991), it is not surprising to find that the hippocampus is sensitive to ethanol and exhibits reduced cell number following ethanol exposure (Barnes and Walker, 1981).

Neurotrophic Factors

The neurotrophin family of NTFs plays a valuable role in the development of the nervous system through regulation of neuronal differentiation and survival, and maintenance of basic cellular processes. The neurotrophin family, as noted above, includes NGF (Levi-Montalcini, 1951), BDNF (Leibrock et al., 1989), NT-3 (Maisonpierre et al., 1990), NT-4/5 (Berkemeier et al., 1991; Ip et al., 1992), and neurotrophin-6 (Gotz et al., 1994). The trk family of receptors has been shown to be the high-affinity receptors for the neurotrophins (Martin-Zanca et al., 1990). Trks that bind neurotrophins include trkA (Kaplan et al., 1991; Kaplan et al., 1991), trkB (Klein et al., 1990), and trkC (CordonCardo et al., 1991). TrkA is the preferred receptor for NGF, but will bind both BDNF and NT-3. TrkB is the preferred receptor for BDNF and NT-4/5, but will bind NT-3. TrkC is the preferred receptor for NT-3. The neurotrophins regulate a number of peptides in the rat septohippocampal system, including other neurotrophins (Croll et al., 1994). For example, NGF, BDNF, and NT-3 induce choline acetyltransferase activity (Alderson et al., 1990; Auberger et al., 1987; Gnahn et al., 1983; Nonner et al., 1996); BDNF increases NT-3 activity (Lindholm et al., 1994); and BDNF and NT-3 enhance synaptic transmission in Shaffer collateral-CA 1 hippocampal synapses (Kang and Schuman, 1995). All of these results indicate the importance of the neurotrophins in this brain region.






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Gene Deletion Studies

Studies of gene-deleted "knockout" mice also suggest the importance of

neurotrophins and other NTFs in proper nervous system development. Knockout studies consist of deleting a gene from the mouse genome by homologous recombination (Smithies et al., 1985). Following this procedure, the mice are allowed to develop and are subsequently compared to control mice. All neurotrophin and neurotrophin receptor knockout animals die relatively early in development except for NT-4/5 animals (Conover and Yancopoulos, 1997). Specifically, NGF knockout mice exhibit reduced numbers of superior cervical ganglion, trigeminal ganglion, and DRG neurons (Crowley et al., 1994). BDNF knockout mice have decreased numbers of trigeminal ganglion, geniculate ganglion, nodose-petrosal ganglion, vestibular ganglion, spiral ganglion, and DRG neurons (Conover et al., 1995; Ernfors et al., 1994). NT-3 knockout mice display fewer superior cervical ganglion, trigeminal ganglion, nodose-petrosal ganglion, vestibular ganglion, spiral ganglion, DRG neurons, and spinal cord motoneurons (Ernfors et al., 1995; Kucera et al., 1995). NT-4 knockout mice are similar to BDNF knockouts and have reduced geniculate ganglion, nodose-petrosal ganglion, vestibular ganglion, and DRG neurons (Conover et al., 1995).

Knockouts of receptors of neurotrophins also support the idea that they are

important in proper nervous system development. TrkA knockout mice show the same pattern of neuronal loss that the NGF knockout mice have (Smeyne et al., 1994). TrkB knockouts have reduced numbers of spinal cord and facial motoneurons, and exhibit reduced numbers of trigeminal ganglion, nodose-petrosal ganglion, and DRG neurons (Klein et al., 1993). This result is especially significant in light of the fact that BDNF knockout mice did not exhibit motoneuron deficits. This discrepancy between the two knockout studies suggests that trkB is important for motoneuron development and some other molecule, perhaps NT-3, can bind trkB to promote survival in the absence of BDNF. TrkC knockout mice exhibit reduced numbers of DRG neurons, and completely lack Ia






13


muscle afferents (Klein et al., 1994). All single gene neurotrophin knockout studies observed reduced DRG number. These results support earlier studies that found DRGs respond to many different NTFs and express the receptors for multiple neurotrophins (Buchman and Davies, 1993).

Knockout mice have also been used to study GDNF and c-ret. GDNF-deficient animals exhibit deficits in DRG, sympathetic and nodose neurons, but not in hindbrain noradrenergic or midbrain dopaminergic neurons and completely lack the enteric nervous system, ureters, and kidneys. These animals did display a small yet significant (-20%) loss of spinal cord motoneurons (Moore et al., 1996). C-ret knockout mice do not contain reduced numbers of motoneurons, but do lack enteric nervous system and contain reduced numbers of parasympathetic neurons (Marcos and Pachnis, 1996). These studies demonstrate that NTFs other than the neurotrophins are important to the normal development of the CNS.

While gene-targeting studies have provided invaluable information regarding the action of NTFs and their receptors in the nervous system, they must be interpreted with certain difficulties in mind. Knockout mice are not merely normal animals with one gene conveniently deleted. These organisms possess a number of developmental, physiological, and even behavioral processes that have been altered to compensate for the missing gene (Gerlai, 1997). For example, when one gene is eliminated from an organism, the transcription of other gene products may be altered. This change in transcription could conceivably ameliorate or exacerbate the effects of losing the gene. The many redundancies present in the genome may mask the effects of losing a single gene. Therefore, it would be difficult to determine whether the observed changes in the organism were due to loss of the gene of interest or to the change in genetic background.

The knockout studies relate to the present experiments in that they provide a clue of what would happen to the nervous system should a specific NTF or NTF receptor be removed. Recall that the neurotrophic hypothesis--which is a driving force for this






14

research--suggests that removal or reduction of neurotrophic support would have a deleterious effect upon the developing nervous system. The above evidence from the knockout studies supports this idea. Since some populations are reduced in number in knockout mice, this suggests that a lack of proper neurotrophic support can reduce neuron number. Should ethanol effectively remove or reduce neurotrophic support from a given nervous system population, that population will undoubtedly be adversely affected. To determine whether this change in some aspect of neurotrophic support is indeed occurring, it is logical to examine populations that are known to exhibit cell loss due to ethanol exposure (e.g. the hippocampus). Knockout studies have not recorded any cell loss in the septohippocampal region. The fact that the septohippocampal system is a rich source for a variety of NTFs may provide an explanation for this apparent paradox. If one NTF is eliminated, other NTFs in the background may be able to provide sufficient support for the neurons to continue to survive. The basic idea that NTF support is critical for survival is important for the present studies.

NTF Ontogeny in the Hippocampus

The normal ontogeny of NGF, BDNF, and NT-3 in the developing rat

hippocampus differs. NGF is expressed in rather low levels throughout embryonic development, increases somewhat at birth, and finally achieves its highest levels in the adult. BDNF is virtually undetectable throughout embryonic development, then increases at birth and continues to increase to its highest levels in the adult rat brain. NT-3 has high expression throughout development and decreases in the adult (Maisonpierre et al., 1990). The different temporal expression between individual neurotrophins might help developing neurons achieve correct synapses. The following hypothetical example illustrates this point: The high early expression of NT-3 might promote survival of neurons through a proliferative phase. Then, NGF expression could increase to induce differentiation. Finally, BDNF expression would signal the end of development and thus induce these hypothetical neurons to form synapses. In vivo, neurotrophins have been shown to follow






15


distinct patterns throughout development. Buchman and Davies found that neurotrophins act in sequence during development to promote survival of DRGs (1993). Therefore, if the temporal sequence of neurotrophin expression were to change as a result of ethanol exposure, the normal innervation patterns in the hippocampus--as well as other parts of the developing CNS--could be altered. Such a change could have disastrous effects on the hippocampus and its ability to properly encode new memories.

The ontogeny of trk receptors in the developing brain follows the ontogeny of the neurotrophins. In the septum--a brain structure of which the hippocampus is a target-higher levels of trkA mRNA were detected at 2 and 4 weeks than at 1 weeks of age (Ringstedt et al., 1993). TrkA is not expressed in the hippocampus under normal circumstances in vivo (Martin-Zanca et al., 1990). TrkB is expressed widely in the CNS and is first detectable in the mouse at E8.5 (Klein et al., 1990). TrkB is expressed by the developing hippocampus and expression continues into adulthood (Klein et al., 1990). TrkC mRNA is detectable as early as E7.5 in the nervous system and is expressed at all stages of hippocampal development (Tessarollo et al., 1993). The above information demonstrates that developing hippocampal neurons are responsive to BDNF and NT-3 and that these proteins, plus their receptors, are expressed during prenatal and early postnatal rat development. Thus, all of these proteins were active during the periods of ethanol exposure employed in the present study.

Neurotrophins and Ethanol

Fundamental responses to neurotrophins and production of NTFs are altered

following prenatal ethanol exposure in rat pups. The neurotrophins are not the only NTFs produced by the hippocampus. Other factors, such as bFGF, are synthesized there and could affect these neurons (Ernfors et al., 1990). Cultures of hippocampal neurons derived from rats prenatally exposed to ethanol do not respond to NTFs as well as neurons in control cultures (Heaton et al., 1995b). This result suggests that NTF receptor expression may be decreased in response to prenatal ethanol exposure. However, another logical






16


explanation for this result is that the expression of less active form of NTF receptor has increased relative to a normal form. Truncated trk receptors are similar to normal trk receptors except they lack the catalytic domain that starts the intracellular signal transduction cascade following neurotrophin binding. These receptors are normally expressed in greater abundance than their active counterparts in adult animals. During development their expression increases relative to the active trk receptor until reaching the level of expression found in the adult. Other studies have found altered neurotrophic activity as a result of ethanol exposure. For example, chronic prenatal ethanol treatment (CPET) in the rat increases neurotrophic activity (a gross measure which includes both neurotrophin and other NTF activity) in extracts made from the hippocampus on P21 and cultured on DRG neurons (Heaton et al., 1995c). The increase in neurotrophic activity suggests an increase in NTF expression as a result of prenatal ethanol exposure, but no single NTF is implicated by this study since DRGs respond to a variety of NTFs. Postnatal ethanol exposure reduces neurotrophic activity of P21 hippocampal extracts(Moore et al., 1996), a result opposite to that of prenatal exposure (Heaton et al., 1995c). All of these results suggest that both NTF and receptor might play an essential role in ethanol toxicity.

Hypotheses

As was mentioned previously, all experiments described in this document were

designed to understand some aspect of how ethanol and NTFs are interrelated in producing the deficits observed in models of FAS. The experiments of this project were performed to test the following hypotheses: (1) (a) We hypothesize that ethanol will reduce motoneuron number in the absence NOCD; (b) We hypothesize that ethanol will reduce motoneuron number at period of development that follows the period for NOCD; (2) We hypothesize that exogenous NTFs will provide in vivo protection for motoneurons exposed to ethanol; and (3) We hypothesize that CPET and early postnatal ethanol treatment (EPET) will alter the gene expression of neurotrophins and/or their receptors in the hippocampus of treated rat pups. Analysis of hypothesis la was undertaken to further describe the motoneuron






17


loss observed following ethanol exposure from E4 to El 1 in chick embryos. Specifically, we wanted to determine whether ethanol acted to increase NOCD or provide direct neurotoxicity. Ethanol was shown to reduce motoneuron number during this time period which encompasses the period for NOCD (Heaton and Bradley, 1995). Additionally, neurotrophic content of the developing muscle was analyzed to compare ethanol exposure from El0 to E15 to an earlier study in this laboratory which found that ethanol exposure from E4 to E8 reduced neurotrophic content of developing limb tissue (Heaton and Bradley, 1995). Analyzing neurotrophic content of muscle from embryos exposed to ethanol from ElO to E15 allowed us to relate any deficit in motoneuron number observed in that time period, to any possible change in neurotrophic support. The number of apoptotic cells present during ethanol exposure from E 10 to E15 was analyzed to find whether ethanol exposure from ElO to E15 induced apoptosis among motoneurons. Analysis of hypothesis 2 was executed to determine whether NTFs could modulate ethanol toxicity in vivo. Analysis of hypothesis 3 was performed to ascertain whether ethanol could modulate the genetic expression of NTFs in a living organism. The chapters that follow describe the experiments performed to achieve these hypotheses and provide the results of these analyses. Further, the results are discussed critically with implications for future research and mechanisms for ethanol toxicity suggested.















CHAPTER 2
CHARACTERIZATION OF MOTONEURON SURVIVAL AND CELL DEATH
FOLLOWING ETHANOL EXPOSURE AND CURARE ADMINISTRATION, AND
AFTER THE PERIOD FOR NATURALLY OCCURRING CELL DEATH Summary

The study described below was conducted as a continuation of a previous study in which we found reduced motoneuron number in lumbar spinal cord of the chick embryo following chronic ethanol administration from embryonic day 4 (E4) to Eli1. We sought to determine whether this reduction was due to primary ethanol toxicity or to enhancement of naturally occurring cell death (NOCD) and to determine whether administration of ethanol at a later period of development could also reduce motoneuron number. Earlier studies have shown that curare suspends NOCD in the chick embryo (Pittman and Oppenheim, 1978). By administering both ethanol and curare to these embryos from E4 to Eli1 and examining the lumbar spinal cord on E12, we determined that ethanol was directly toxic to motoneurons and reduced motoneuron number in the absence of NOCD. By administering ethanol from E10 to E15 and examining the lumbar spinal cord on E16, we deter-mined that ethanol can reduce motoneuron number without altering the overall morphology of the spinal cord during more than one stage of chick embryo development. We also determined that ethanol toxicity is not dependent on NOCD. In additional experiments, we demonstrated that ethanol does not affect the neurotrophic content of chick muscle and does not appear to induce apoptosis in developing motoneurons when it is administered from E10 to E15.

Introduction

Over the last two and one-half decades, much evidence has been gathered regarding the effects ethanol exerts in the developing nervous system (Barnes and Walker, 1981;



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Jones and Smith, 1973; Miller, 1986; Streissguth et al., 1991; West, 1986). Fetal alcohol syndrome (FAS) is diagnosed in 1-2 out of every 1000 live births in the United States and is characterized by low birth weight, decreased memory and learning, hyperactivity, facial dysmorphia, and lowered IQ (Abel, 1995; Jones and Smith, 1973; Streissguth et al., 1991). Among heavy drinkers, the incidence of FAS is much greater with a 4.3% diagnosis rate (Abel, 1995). Evidence suggests that these deficits are permanent and do not lessen as the patient ages (Streissguth, 1993). These observations led to the assertion that maternal consumption of ethanol is the leading known cause of mental retardation in the Western Hemisphere (Bonthius and West, 1988).

Many neuronal populations exhibit some susceptibility to the toxic effects of

ethanol. A variety of neuronal types from brain areas such as the cerebellum (Cragg and Phillips, 1985), the septohippocampal system (Barnes and Walker, 1981) (West and Pierce, 1986), the cerebral cortex (Miller, 1986), and the oculomotor nucleus (Burrows et al., 1995) are known to be affected by ethanol exposure. Still other populations that have not been so intensely investigated demonstrate vulnerability to ethanol. These regions include the substantia nigra (Shetty et al., 1993), chief sensory trigeminal nucleus (Miller and Muller, 1989), red nucleus (Zajac et al., 1989), and motoneurons of the spinal cord (Heaton and Bradley, 1995). Of particular interest to the present studies is the fact that motoneurons are affected by ethanol. A previous study from this laboratory found that motoneuron number was reduced by ethanol administration from E4 to El 1 (Heaton and Bradley, 1995). At that time, we hypothesized that ethanol might be exacerbating NOCD. The period for NOCD for motoneurons of the entire spinal cord extends from approximately E6 to E9 (Pittman and Oppenheim, 1978). Motoneuron number is known to peak around E5.5 to E6 (Pittman and Oppenheim, 1978) and proliferation of motoneurons continues until E6 (Hollyday and Hamburger, 1977). During the period of NOCD, nearly half of the original number of motoneurons perish. Curare, as well as other neuromuscular junction blocking agents, have been shown to suspend NOCD in motoneurons (Pittman






20

and Oppenheim, 1978). In these experiments, approximately 50% more motoneurons survive in curare-treated embryos than in control embryos.

In addition to the above, there is more direct evidence that warrants further

investigation into the possible effects that ethanol may exert on motoneurons. Previous research in other laboratories has determined that ethanol damages developing muscle in both the human and in animal models (Adickes and Shuman, 1983; Nyquist-Battie et al., 1987). In the human, these cases described flaccid, hypotonic neonates which exhibited major muscle structural deficiencies including hypotrophy, dominance of type II fibers, and sarcomeric dysplasia (Adickes and Shuman, 1983). Prenatal exposure to ethanol in the guinea pig resulted in structural malformations of the gastrocnemius muscle including vacuolated sarcoplasmic reticula, enlarged lipid droplets, decreased glycogen, and mitochondrial abnormalities (Nyquist-Battie et al., 1987). Since proper muscle fiber maturation is dependent on concurrent development and innervation by motoneurons (Ishiura et al., 1981), the possibility exists that the deficiencies noted in the above cases are due to an underlying effect that ethanol exerts on developing motoneurons.

Previous studies have shown that ethanol affects chick embryo development in a manner similar to both the human and the rat. Chicks have been shown to exhibit reduced brain size, weight, DNA and protein synthesis (Pennington and Kalmus, 1987), and reduced neurotransmitter synthesis (Brodie and Vernadakis, 1990; Swanson et al., 1994) following developmental ethanol exposure. One advantage of using a chick model to study ethanol is its simplicity. Ethanol can be administered in exact doses to the developing embryo. Another advantage is that maternal influences are removed when using the chick embryo. Ethanol is cleared from the bloodstream by both mother and fetus in a mammalian system whereas the chick embryo is isolated as it develops. The chick model allows the investigator to observe direct effects of ethanol without possible interactions of maternal metabolism interfering. The embryos seem to tolerate slight invasions into their environment quite well as long as the underlying membranes are not disrupted. While






21


chick development is clearly different from mammalian gestation, this model allows researchers to study in vivo interactions in a developing organism that are just not possible in a mammalian FAS model.

The present experiments were performed to determine whether ethanol exerted a toxic effect when NOCD is suspended (Curare-Ethanol Coadministration) and whether administering ethanol during a later period of development (Late Exposure)--after the period of cell death--would differ from administration earlier in development. Suspension of NOCD did not hinder ethanol's ability to reduce motoneuron number. This result suggests that ethanol acts by a mechanism other than exacerbation of NOCD, either by direct toxicity, motoneuron loss due to a change in neurotrophic support, or a combination of the two. The Late Exposure study found that ethanol administration from ElO to El5 reduced motoneuron number and that exposure to ethanol did not reduce the neurotrophic activity of chick limb muscle in comparison to Saline treated embryos. This latter analysis was undertaken to compare the results of ethanol exposure from ElO to ElS to an earlier study in this laboratory which found that ethanol exposure from E4 to E8 reduced neurotrophic content of developing limb tissue (Heaton and Bradley, 1995) and to determine if altered neurotrophic support is responsible for the observed motoneuron reduction. Loss of neurotrophic support would suggest a possible mechanism for motoneuron loss due to ethanol exposure at this period of development: reduction of target-derived NTFs. Additionally, an analysis of apoptotic motoneurons in the lumbar spinal cord failed to find evidence that ethanol exposure from ElO to El5 induced apoptosis among motoneurons.

Materials and Methods

Subjects

White Leghorn chick eggs were obtained from the University of Florida Poultry Science Department. Eggs were placed in a Marsh incubator and maintained at 37C and






22


70% relative humidity until R4. At that time, the eggs were moved to a forced draft turning incubator, maintained at the same conditions indicated above, and divided into groups. Curare-ethanol coadministration.

For the Curare-Ethanol Coadministration study, 5 groups were utilized:

Uninjected, Ethanol, Saline, Curare, and CurareEthanol. The data from all Ethanol embryos and 4 of the 10 Saline embryos were obtained from an earlier study (Heaton and Bradley, 1995). Ethanol and saline injection began on 4 and continued daily through Eli1. Curare injection began on E6 and continued daily through Eli1. Ethanol and curare injections were initiated on different days in order to replicate previous research and to coincide curare administration with the onset on NOCD in the lumbar spinal cord (6) (Pittman and Oppenheim, 1978). Since cell death in the lumbar section of the spinal cord does not begin until 6 (Hollyday and Hamburger, 1977; Pittman and Oppenheim, 1978), curare administration before this time point would be without effect. In addition, the present experiment found the combination of curare and ethanol to be quite toxic to the developing embryos; therefore, further loss of embryos was minimized by starting curare administration on 6. All embryos were sacrificed by decapitation on 12, the lumbar section of the spinal cord removed, and prepared for histology. Late exposure study

A separate study was conducted to assess the effects of ethanol during a later

exposure period when motoneuron number is relatively stable. The Late Exposure study had 2 groups: Ethanol and Saline. Embryos received daily injections of either ethanol or saline from 10 to E15. At 16, embryos were removed from the eggs, sacrificed by decapitation, and the lumbar section of the spinal cord removed and prepared for histology. An Uninjected control group was not used in the Late Exposure study because in previous studies, our laboratory has shown that saline injection does not adversely affect embryonic development and has no effect on motoneuron number in the developing chick spinal cord (Heaton and Bradley, 1995).






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Injections

Curare-ethanol coadministration study

As stated above, five experimental groups were used in this study: Ethanol, Saline, Uninjected, Curare, and CurareEthanol. Ethanol and saline injections were administered daily from E4 through Eli1. Ethanol embryos received 150 yl of 20% w/v ethanol (30 mg ethanol per day), dissolved in a 0.9% w/v nonpyrogenic saline vehicle, through a pinhole in the shell into the airspace. Previous work in our laboratory has determined that this concentration of ethanol produces blood ethanol counts that peak at 225 mg/dl by El 1 (Heaton and Bradley, 1995). Saline embryos received 150 jd of the 0.9% wlv nonpyrogenic saline vehicle. Uninjected embryos received no injections, but were handled daily in a manner similar to the other groups. Curare injection, which occurred from E6 through Eli1, involved creating a pinhole directly over the embryo in addition to the pinhole created in the airspace. The airspace was then allowed to shift to a position above the embryo and 150 yl of 16.67 mg/ml tubocurarine chloride (Sigma) was injected into that space above the embryo (2.5 mng curare per day). Curare+Ethanol embryos were given ethanol injections from E4 to El 1 and curare injections from P6 to El I as described above. The embryos were allowed to sit in a Marsh incubator for a period of one hour following ethanol injection on days when two injections were delivered to the same embryo. This delay in injection time was necessary to ensure that the ethanol was absorbed through the inner shell membrane within the airspace before the eggs were turned on their side for the curare administration. Also, the two injections administered in this study represent a significant volume (300 yL) for the embryonic system to incorporate on a daily basis. The delay between injections, therefore, allowed absorption of the volume of ethanol before an equal volume of curare solution was presented to the embryo. Pinholes created by the injection process were sealed with paraffin immediately following injection to prevent evaporation and/or leakage of the ethanol and curare. The eggs were then returned to the turning incubator.






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Late exposure study

Ethanol and saline injections were administered daily from E10 through E15.

Ethanol embryos received 150 y1 of 30% w/v ethanol (45 mng ethanol per day), dissolved in a 0.9% wlv nonipyrogenic saline vehicle, through a pinhole into the airspace. Since embryos at this later stage of development do have the ability to clear ethanol from the bloodstream (Wilson et al., 1984), a larger dose of ethanol was utilized in this study in order to achieve blood ethanol concentrations similar to those observed in embryos exposed to ethanol from E4 to El 1. Peak blood ethanol concentration in this portion of the study ranged from 250 to 300 mg/dl and trough levels were below 30 mg/dl. Saline embryos received 150 y1 of the 0.9% w/v nonpyrogenic saline vehicle. Pinholes created by the injection process were sealed with paraffin following injection to prevent evaporation and/or leakage of the ethanol. The eggs were then returned to the turning incubator, as above.

Dissections and Histological Procedures

Curare-ethanol coadministration study

Embryos of all experimental groups (Ethanol, Saline, Uninjected, Curare, and Curare+Ethanol) were sacrificed by decapitation on E12 and the lumbar section of the spinal cord removed. Following dissection, the E12 spinal cords were placed in Bouin's fixative for 24 hours and then embedded in paraffin. Spinal cords were then cut into 12 pmn coronal sections, mounted onto glass slides, and stained with hematoxylin and eosin. A total of 33 embryos were used in this study: Ethanol (n=6), Saline (n=10), Uninjected (n=6), Curare (n=6), and Curare+Ethanol (n=5). Late exposure study

Embryos of both experimental groups (Ethanol and Saline) were sacrificed by

decapitation on E16 and the lumbar section of the spinal cord removed. The vertebrae of the spinal cords were cut along the dorsal surface to expose the nervous tissue and allow the fixative to adequately penetrate the tissue. Following dissection, the E16 spinal cords






25

were placed in Bouin's Fixative for 14 to 21 days to allow the vertebrae to decalcify (Li et al., 1994). The tissue was then embedded in paraffin, cut into 12 Yim coronal sections, mounted onto glass slides, and stained with hematoxylin and eosin. 7 Ethanol and 6 Saline embryos were used to complete this study.

Motoneuron Counts

Motoneuron counts were completed following methods described previously

(Hamburger, 1975; Heaton and Bradley, 1995). Briefly, a uniform area encompassed by 6 dorsal root ganglia (DRG) was noted on each embryo which ensured that a similar area was counted in each subject. Starting from the most rostral section included in the 6 DRG region, motoneurons in the lateral motor column of one side of every tenth section were marked onto paper using a camera lucida. At 400X magnification, motoneurons were identified in the lateral motor column by their large size, dark cytoplasm, and nucleolus. Laterality was maintained throughout each individual embryo, but chosen at random before beginning the counting process. Previous studies have shown that there is no difference between the number of motoneurons contained in the right and left sides of the spinal cord (Pittman and Oppenheim, 1979). It should also be noted that each embryo was coded so that the experimenter had no knowledge of its experimental treatment until the study was completed. Motoneuron counts reported below are actual counts generated by the above procedure and are not corrected to estimate total motoneuron number of the lumbar spinal cord.

Pre-Cell Death Ethanol Exposure

In order to clarify the results of the Coadministration Study, we administered

ethanol and saline to chicks from E4 to E5 and assessed motoneuron number on E12. This analysis was performed to determine whether ethanol reduces motoneuron number during the time period where ethanol was administered, but curare administration had not yet commenced. Chick eggs were incubated as described previously and placed into two groups: Ethanol and Saline. On E4 and E5 Ethanol embryos received 150 pl of 20% w/v






26


ethanol solution (30 mg) and Saline embryos received 150 J41 of the saline vehicle. No injections were administered from E6 to El 1 and on E12, the embryos were sacrificed by decapitation, the lumbar spinal cord removed, and prepared for histology. Motoneuron Size and Spinal Cord Length Analyses

Motoneuron size and spinal cord length were measured to determine whether

ethanol had altered any general characteristics of the motoneuronal system. Motoneuron size was determined by measuring the diameter of 10 random cells in the same rostralcaudal position of the region of each embryonic spinal cord. In E12 embryos (Coadministration Study) the section exactly 1800 pmn following the beginning of the lumbar spinal cord as determined by the 6 DRG region described above, was sampled. In E16 embryos (Late Exposure Study) the section exactly 2400 pmn following the beginning of the lumbar spinal cord, was sampled. Four embryos from each experimental condition were analyzed for a total of 40 cells per condition. Spinal cord length was determined by counting the number of sections present in each embryo following determination of the boundaries of the lumbar spinal cord by the anatomical methods described previously and multiplying this number by the section thickness (12 Yim). Crude Muscle Extract Study

Extract preparation

In order to determine whether neurotrophic content of chick leg muscle is affected by ethanol exposure during the late exposure period (E1O to E 15), we analyzed the activity of crude muscle extracts on E6 spinal cord cultures. This analysis was undertaken to compare ethanol exposure from ElO to E16 to ethanol exposure from E4 to ES--where a reduction in neurotrophic activity of developing muscle tissue following ethanol exposure was observed. Embryos were treated with ethanol or saline and incubated as described in the Subjects section above. Briefly, Ethanol and Saline embryos received injections of 30% w/v ethanol (45 mg per day) or the saline vehicle, respectively, daily from E10 to E15. On E16, embryos were removed from the egg, sacrificed by decapitation, and the






27


muscle dissected away from the thigh region of each leg. The muscle tissue was flashfrozen on dry ice and then stored at -70'C until extract was prepared. Extract was prepared by homogenizing the tissue for about 15 seconds in F- 12 media (BRL) supplemented with

0.7% fungizone, 1.0% penicillin- streptomycin, and 200 mM glutamine. After homogenization, the extract was centrifuged for 20 minutes at 35,000 rpm at 40C. The supernatant was collected and assayed for protein content (Bradford, 1976). All samples were diluted to 200 pig/ml protein, 10% fetal bovine serum (FBS) added, and then added to mixed spinal cord cultures which were prepared as described below. Spinal cord cultures

Three experimental groups were analyzed in this portion of the study: Ethanol, Saline, and Negative Control (NC). Ethanol cultures consisted of E6 lumbar spinal cord cultures grown in the presence of muscle extract obtained from embryos exposed to ethanol from E10 to E15. Saline cultures were grown in the presence of muscle extract obtained from Saline embryos and NC were grown in the presence of regular culture medium. Regular culture medium consisted of F- 12 media supplemented as described above, with 10% FBS added. The lumbar region of the spinal cord was dissected out of E6 embryos and incubated in 10% v/v trypsin and 5% v/v deoxyribonuclease I in 0.9% nonpyrogenic saline for 20 minutes at 37'C. Cells were disassociated by repeated titration in regular culture medium. The cells were grown in individual wells of 12-well Corning plates coated with 0.5 mg/mI polyornithine. Two hours after the cells were plated initial counts were completed by counting three representative areas from each culture well. Each culture had a Beilco glass slip with an enumerated grid affixed to the bottom. This allowed the experimenter to note the initial areas counted and return to these areas on subsequent days. Neurons were identified by their large size, and rounded, phase-bright appearance. Following 24 and 48 hours in culture, cell counts were again obtained from each culture and in addition, the number of cells expressing a neurite (at least two cell diameters in length) were noted.






28


Assessment of Apoptotic Cells

Following ethanol exposure to embryonic chicks as described previously, spinal cord tissue was examined and apoptotic cells were identified by methods described previously (Homma et al., 1994). Briefly, at 400X magnification cells fitting the following criteria were determined to be undergoing apoptosis. First, cells with chromatin and cytoplasmic condensation--the hallmark of apoptosis--were identified. Since these processes are rather fast, it was unlikely that such cells would be observed. Therefore, a second criterion--where apoptotic debris was identified--was used to make a positive identification of an apoptotic cell. Since this portion of the study utilized an ethanol exposure paradigm that began on ElO and ran through E15, tissue was examined at the following intermediate time points: E12 and E14. Both saline and ethanol-exposed embryos were examined so that a statistical comparison could be made between the two groups. It should be noted, however, that NOCD essentially ends on E9 (Pittman and Oppenheim, 1978). A uniform area encompassed by 6 DRG was noted on each embryo which ensured that a similar area was counted in each subject. Starting from the most rostral section included in the 6 DRG region, apoptotic motoneurons in the lateral motor column of one side of every fifteenth section were noted. This process led to an average of 21 sections being examined per E12 embryo and 30 sections being examined in every E14 embryo. After the entire animal was examined, candidate cells were reexamined to make sure that they truly fit the criteria described above. Both sides of each section were analyzed in order to increase the probability of identifying apoptotic neurons. Even though fewer total sections were analyzed than in the Motoneuron Number analysis, more total area was analyzed per animal since both the right and left side of each section was examined.






29


Statistics

Analysis of variance, Fisher's protected least significant difference post-hoe test, and Student's t-test were performed using the StatView program (Abacus) on a Macintosh computer.

Results

Embryonic Observations

Curare-ethanol coadministration study

Survival varied greatly according to the treatment of each embryonic group. In the Curare group survival was approximately 47%, Saline group survival was 76%, Uninjected group survival was 96%, Ethanol group survival was 4%, and Curare+Ethanol group survival was 0.83%. This latter survival figure indicates that the combination of curare and ethanol was highly toxic to the developing chick embryo. The Curare embryos appeared to be more vascularized, in both the body and the limbs, than the controls. In contrast, the Ethanol and Curare+Ethanol embryos had wider bodies due to extensive bloating from the ethanol treatment. The spinal column in Curare+Ethanol embryos was softer than in the control counterparts and in the Ethanol embryos the lumbar region exhibited a minimal enlargement compared to Saline, Uninjected, and Curare embryos. The profound effects of ethanol treatment were verified by the presence of a green-colored liver in the Ethanol and Curare+Ethanol embryos, compared to the normal brown-colored liver in the remaining three groups. The alteration of the appearance of the liver in animals exposed to ethanol should be investigated further. The pathology of the liver could be analyzed and perhaps related to the death of embryos following ethanol exposure. Collaboration with a pathologist in examining this tissue would be preferable so that an accurate estimation of the method of liver damage can be determined.






30


Late exposure study

Survival between experimental groups was not as variable in the Late Exposure

study as it was in the Coadministration study. Approximately 60% of the Ethanol embryos and 75% of the Saline embryos survived. The livers of the Ethanol embryos were green, thus indicating that ethanol administration did have some general effect. The Saline embryos exhibited the normal brown liver coloration. The Ethanol embryos were not bloated in appearance, as has been observed in earlier studies (Heaton and Bradley, 1995) and the current Coadministration study, perhaps because the embryos of this age have the ability to clear ethanol from the bloodstream (Wilson et at., 1984). Motoneuron Counts

Curare-ethanol coadministration study

Analysis of variance indicated a significant effect due to treatment among these

groups (F=23.061; df=28; p<0.000 1). The number of motoneurons present in the lumbar region of the spinal cord in each of the five experimental groups at E12, could be described as one of three possible outcomes: average, above average, and below average. For these distinctions, "average" is defined to be a number of motoneurons similar to normal or control levels (i.e., Saline or Uninjected); "above average" is defined to be a number of motoneurons significantly higher than average; and "below average" is defined to be a number of motoneurons significantly lower than average. Figure 2-1 displays all of the groups utilized in this study. It should be noted that Figure 2-1 represents the number of motoneurons counted in each embryo and does not attempt to estimate the population of motoneurons in the lumbar spinal cord. Looking at Figure 2-1 from left to right shows how evident the three-tiered outcome of this study was. For example, the Curare group (located at the left in Figure 2- 1) is the only group that displayed above average numbers. In fact, post hoc testing showed that the curare group contained significantly more motoneurons (about 35% more) than all other groups (p<0.0001 for each comparison). The Ethanol group (located at the right in Figure 2- 1) was the only group to exhibit below






31



3000
I Curare
l Curare+Ethanol SEl Uninjected
5 Saline
oa
20- Ethanol
0 2000a


0
0 b

S1000





0


Figure 2-1. Number of motoneurons in lumbar spinal cord at E12 following treatment from E4 to E11. Motoneuron counts are displayed as means + SEM. Data from Curare, Curare+Ethanol, Uninjected, Saline, and Ethanol embryos are displayed. a = significantly more motoneurons than Curare+Ethanol, Uninjected, Saline, and Ethanol (p<0.0001 in all cases). b = significantly less than Curare+Ethanol, Uninjected, and Saline (p<0.005 in all cases).






32

average numbers. This fact is evidenced by the observation that post hoc testing indicated that the Ethanol group contained fewer motoneurons (about 20% fewer) than Saline, Uninjected, and Curare+Ethanol groups (p<0.005 in each comparison). All of the other groups--Saline, Uninjected, and Curare+Ethanol--contained average numbers of motoneurons and there were no significant differences among these groups. The Motoneuron Size and Spinal Cord Length Analyses section below suggests that motoneuron number differences observed in this study are not due to changes in the overall length of the spinal cord.

Figure 2-2 is a collection of photomicrographs obtained from midlumbar segments of Saline, Ethanol, Curare, and Curare+Ethanol embryos. It is apparent that motoneuron number was affected by treatment as the Ethanol section contains the fewest motoneurons. The Saline section contains more than the Ethanol section, but not as many as the Curare section. Finally, the Curare+Ethanol section contains a similar number as the Saline section (refer to Figure 2-2).

Pre-cell death exposure

This analysis was undertaken to determine if ethanol exposure prior to the period for NOCD (E4 to E5) in the spinal cord could affect overall motoneuron number at E12. The T-test indicated no significant difference in the number of motoneurons in the lumbar spinal cord between the Ethanol and Saline embryos. This result suggests that exposure to ethanol before administration of curare, and NOCD, had no adverse effect on the motoneuron population of the lumbar spinal cord. Therefore, results obtained following ethanol exposure from E4 to El 1 are not confounded by the fact that ethanol exposure did not coincide completely with the period for NOCD. Late exposure study.

The results obtained following late ethanol exposure were similar to those obtained when ethanol was administered from E4 to El 1, in that Ethanol embryos exposed from El0 to E16 exhibited a significant reduction in motoneuron number. The length of each






























Figure 2-2. Photomicrographs of coronal sections from the midlumbar region of E12 spinal cords. A. Ethanol, B. Saline, C. Curare, and D. Curare+Ethanol spinal cords. Note that the Curare "bulge" is most pronounced while the Ethanol "bulge" is hardly apparent. Also note the density of motoneurons in the lateral motor column. The Curare contains more and more densely packed motoneurons, while the Ethanol cord contains fewer and less densely packed motoneurons. The Saline and Curare+Ethanol spinal cord sections contain roughly similar numbers of motoneurons. n = 6 Ethanol, 10 Saline, 6 Curare, and 5 Curare+Ethanol







34













R 60






Or I





A 5~

IT,


~y X ~

Sain Etao









w ~A
.t Z'-4










Curare Curare+Ethanol





Figure 2-2.






35

embryo's spinal cord was similar since similar numbers of sections were counted from each embryo and each section is 12ym thick (see section below). The t-test indicated that the E16 Ethanol embryos contained significantly fewer motoneurons (approximately 15%) than did their Saline counterparts (p<0.05). Figure 2-3 displays the data generated in this portion of the study.

Motoneuron Size and Spinal Cord Length Analyses Curare-ethanol coadministration study

Analysis of variance of the motoneuron size data indicted a significant effect due to treatment (F=3.233; df=200; p<0.05). The only significant difference indicated by post hoc testing was that the Curare+Ethanol group contained significantly smaller motoneurons than the Saline (p<0.005), Ethanol (p<0.005), and Curare (p<0.05) groups. There were no other significant differences between any of the other groups. Analysis of variance of spinal cord length found no significant effect due to treatment. Table 2-1 shows the data generated in this portion of the study. Note that even though the difference between the Curare+Ethanol group and the other groups is less than 2 14m, it is significant.


Table 2-1. Cell size and spinal cord length. Coadministration Study
Group Cell Size (ym) Spinal Cord Length (im)
Ethanol 19.025 + 0.404 3940.0 + 95.04
Saline 18.875 + 0.399 3750.0 + 76.92
Uninjected 18.200 + 0.431 3614.9 + 151.80
Curare 18.725 + 0.410 3800.8 + 119.20
Curare+Ethanol 17.275 + 0.326 3993.2 + 68.76

Late Exposure Study
Group Cell Size (/ym) Spinal Cord Length (,um)
Ethanol 20.950 + 0.399 6000.0 + 138.6
Saline 20.650 + 0.408 6060.0 + 187.8

All values are means + SEM. For cell size, n=40 for each group. For spinal cord length Ethanol n=6, Saline n=10, Uninjected n=6, Curare n=6, and Curare+Ethanol n=5.
* denotes significance in comparison to Ethanol (p<0.005), Saline (p<0.005), and Curare (p<0.05).






36


-. 20000

~a

0

lO0


-o
S






Saline Ethanol
Figure 2-3. Number of motoneurons in lumbar spinal cord at E16 following ethanol treatment from El0 to E15. Motoneuron counts obtained in the Late Exposure study are displayed as means + SEM. Data from Ethanol and Saline embryos are displayed. a = significantly fewer motoneurons than Saline (p<0.05).






37


Late exposure study

The t-test indicated that there was no difference in motoneuron size or spinal cord length between the Ethanol and Saline groups. Table 2-1 also contains data generated from this portion of the experiment.

Crude Muscle Extract Study

As mentioned above, both neuronal survival and neurite outgrowth at 24 and 48 hours were observed in this portion of the study. Also recall that muscle tissue was prepared from E16 embryos exposed to ethanol from E10 to E15. Analysis of variance indicated no significant effect due to treatment for survival at either 24 or 48 hours. Likewise, after 24 hours in culture there was no significant effect due to treatment for outgrowth. Following 48 hours in culture, however, there was a significant effect of treatment for outgrowth (F=4.232; df= 18; p<0.05). Post-hoc testing revealed that Ethanol cultures extended significantly more neurites than NC cultures (p<0.05). However, Ethanol extract and Saline extract cultures were not different, thus this result does not indicate that ethanol increases neurotrophic activity in chick limb muscle. Neurite outgrowth at 48 hours in Saline cultures approached statistical significance (p=0.078), which further supports the notion that observations obtained from Ethanol and Saline extract cultures were similar. Table 2-2 displays the results obtained in this portion of the study.


Table 2-2. Neurotrophic activity of crude muscle extract.

Survival (% of Outgrowth (% of
initial counts) surviving cells)
Group 24 Hours 48 Hours 24 Hours 48 Hours
Negative Control 83.4862.861 177.929+3.288 9.143+1.788 12.243+2.095 Saline 86.3141.766 81.443+1.918 11.9712.171 17.557+1.547
Ethanol 88.614+1.840 184.529+1.526 12.400+2.530 20.386+2.309*

All values are means + SEM. n=8 (one culture produced from each of eight embryos) for all groups. denotes significance in comparison to Negative Control (p<0.05).






38

Assessment of Apoptotic Cells

As mentioned above, spinal cords for intermediate time points in the Late Exposure Study (E12 and E14) were assessed for apoptotic cells. Three embryos from the Saline group and three embryos from the Ethanol group were studied as described above. At E12, a total of 2 apoptotic cells were identified among the animals in the Saline group and a total of 3 apoptotic cells were identified among the Ethanol group. As stated above, an average of 21 sections--and both sides of each section--for each animal were examined. Since a total of 5 apoptotic cells were identified at E12, it is unlikely that an analysis of more sections would result in finding significantly more apoptotic motoneurons. Both sides of each section were analyzed for apoptotic cells so that a greater total area was analyzed in this portion of the study than in the Motoneuron Counts section. At E14, a total of 2 apoptotic cells were identified among the animals in the Saline group and a total of

4 apoptotic cells were identified from the Ethanol group (an average of 30 sections were examined in each E14 embryo). These results led to the conclusion that there was no significant difference between the Ethanol and Saline groups in numbers of apoptotic cells when ethanol was administered from ElO to E15. However, the data do not conclusively eliminate apoptosis as a potential mechanism for ethanol in this neuronal population. This possibility will be discussed further below.

Discussion

Curare-Ethanol Coadministration Study

The results of the Curare-Ethanol Coadministration study indicate that ethanol is toxic to developing motoneurons even in the absence of NOCD. This result suggests that ethanol does exhibit a mechanism other than exacerbation of NOCD to developing motoneurons and the change in motoneurons number is not due to a change in spinal cord length. The Curare embryos had significantly more motoneurons than all other groups. This result agrees with prior results from other laboratories (Pittman and Oppenheim, 1979; Pittman and Oppenheim, 1978). The mechanism for curare rescue of motoneurons






39

destined to die of NOCD has been studied previously. Curare increases the number of acetylcholine receptor clusters in developing myofibers (Oppenheim et al., 1989). The implication of this result is that greater numbers of synapses can form and developing motoneurons would then have greater access to target derived NTFs (Oppenheim, 1991). The fact that the Curare+Ethanol group contained significantly fewer motoneurons than the Curare group indicates that ethanol does not reduce motoneuron number by exacerbating NOCD. Since curare administration suspends NOCD, ethanol must cause additional motoneurons to perish by a mechanism other than exacerbation of NOCD. Potential mechanisms for ethanol toxicity are discussed further below. The three-tiered level of motoneuron survival observed in this study also suggests that ethanol and NOCD act independently. The Curare group, which contains that largest number of motoneurons, is not subject to either cell death process. The middle tier contains three groups that are all subject to one of the two cell death processes: Uninjected (NOCD), Saline (NOCD), and Curare+Ethanol (ethanol toxicity). The Ethanol group, which contained the fewest number of motoneurons, was subject to both cell death processes and exhibited an additive effect of motoneuron loss (refer to Figure 2-1).

The timing of ethanol and curare administration for this experiment was designed to coordinate the presence of curare with the onset of NOCD in the lumbar spinal cord. The results indicated that ethanol exposure on E4 and E5 did not reduce motoneuron number. There are two implications of this result: Ethanol exposure prior to curare administration had no adverse effect on the motoneuron population of the lumbar spinal cord and the critical period for ethanol-induced motoneuron death does not include E4 and E5. A possible addition to this study would be a study which further limits exposure time to ethanol and attempts to define a critical period where ethanol exerts its greatest toxic effects on this neuronal population.






40


Late Exposure Study

The results of the Late Exposure study indicate that ethanol has the ability to reduce motoneuron number in the lumbar section of the spinal cord during a later period of development. Since the reduction in motoneuron number during this late exposure period does mimic the reduction observed following ethanol administration from E4 to El1, it is logical to assume that ethanol might have the ability to reduce motoneuron number in the developing chick embryo during any time period of motoneuron development and perhaps during later periods as well. This result is further evidence that ethanol is directly toxic to motoneurons of the embryonic chick because the ethanol exposure took place following the period of NOCD. Additionally, since spinal cord length was unaltered following ethanol treatment, this study suggests that these findings are not an artifact of a change in spinal cord volume.

Additional experiments were conducted to determine whether ethanol exposure during this late period altered the neurotrophic activity of embryonic chick muscle. In a previous study, this laboratory found that neurotrophic activity of chick muscle from limb bud was reduced following ethanol exposure from E4 to E8 (Heaton and Bradley, 1995). However, the current results suggest that there is no difference in muscle neurotrophic activity in embryos treated with ethanol or saline. The results suggest that ethanol does not reduce motoneuron number by decreasing the total amount of neurotrophic support available to the motoneuron population as it does when administered during the earlier stage of development (Heaton and Bradley, 1995). However, it does remain a possibility that individual NTFs produced by muscle are altered in their expression such that one factor was upregulated while another was downregulated. Such a change could alter survival of the NTF dependent motoneuron population if the downregulated factor was critical for survival at the given time period of ethanol administration.






41


General Discussion

The results of the present study offer many possibilities for the action of ethanol in the developing motor system. Since our analyses included motoneuron survival, neurotrophic activity, and apoptotic cells, this study has the ability to question potential mechanisms of ethanol toxicity that are suggested by previous research. Specific areas, as they relate to ethanol toxicity in the developing nervous system, are discussed below. Disruption of NTF support

Ethanol has been shown to affect neurotrophic activity and neuronal responsiveness to NTFs (Heaton et al., 1995b; Heaton et al., 1992; Heaton et al., 1993; Heaton et al., 1994). Since developing motoneurons of the lateral column require neurotrophic support for survival (Oppenheim, 1991), it is possible that ethanol may interfere with the ability of these cells to gain access to NTFs and therefore cause excess cell death. The results of the current experiments are somewhat contradictory. As was mentioned above, extracts made from E16 leg muscle following ethanol exposure from El0 to E15 were significantly increased in neurotrophic activity in comparison to NC cultures. NC cultures were composed of cell cultures grown in regular culture medium, with essentially little exogenous neurotrophic support. Thus, these cultures were negative control groups. Only Saline and Ethanol cultures contained exogenous neurotrophic support. Since NTF activity--as measured by both neurite outgrowth and survival--was not significantly increased in comparison to the positive control group (Saline), it would be erroneous to conclude that ethanol exposure increases neurotrophic activity of developing leg muscle. The current study suggests that late ethanol exposure does not affect the neurotrophic content of chick limb muscle--a result that contrasts with earlier findings that demonstrated ethanol exposure from E4-E8 reduces neurotrophic content of developing limb muscle (Heaton and Bradley, 1995).

The reason for this disparity may lie in the fact that neurotrophic content of

developing limb muscle increases throughout development and peaks at E18 (Thompson






42

and Thompson, 1988). The fact that total neurotrophic activity is nearing its peak at E16 suggests that overall activity is much higher at E16 than at ES. Any gross change in neurotrophic activity at E8 would result in a larger percentage change in activity than a similar change at E16. The implication would be that enough residual neurotrophic activity would remain in E16 muscle to continue to support the spinal cord cultures, whereas the support would be reduced sufficiently to alter the growth of the cultures when ethanol was administered earlier in development. The fact that Saline embryos did not significantly increase neurotrophic activity in comparison to the NC group should be discussed further. The answer may lie in the ontogeny of neurotrophic activity in developing limb muscle. Since this level increases until reaching a peak at E18 (Thompson and Thompson, 1988), the relative amount of neurotrophic factors present in extract is increased in comparison to extracts prepared on E8. Since high levels of NTFs in cultures can be lethal, it is possible that the amount of trophic activity released into the culture medium ceased to be supportive. As was mentioned above, the amount of gross protein, not gross neurotrophic activity, was regulated in these cultures. Therefore, since NTFs are increased relative to overall protein level (Thompson and Thompson, 1988), our cultures may not have been maintained for peak neurotrophic activity.

Another possible explanation for the fact that Saline cultures did not exhibit

significantly greater neurotrophic activity in comparison to the NC is the fact that FBS was used in the culture medium. Since FBS contains NTFs as well as other undefined proteins, it is likely that the NC cultures exhibit growth and survival far above levels that would be present without FBS. Clearly, further experimentation should be completed before concluding that El0 to E15 ethanol exposure in embryonic chick increases neurotrophic activity of developing leg muscle. Trophic support for developing motoneurons is not limited to, but is in a large part provided by, target muscle. Glia in the spinal cord produce NTFs that support developing motoneurons (Arce et al., 1998). However, motoneuron number in the chick has been shown to be regulated by the amount of target muscle






43

present. Specifically, when a limb is removed from a chick embryo, motoneuron number is reduced accordingly (Caldero et al., 1998; Lanser and Fallon, 1987). When a supernumerary limb is grafted onto an embryo, motoneuron number is increased (Hollyday and Hamburger, 1976). Thus, target muscle regulates motoneuron number in a "dosedependent" manner. This relationship has been further confirmed by the removal of varying portions of limb bud from developing chick embryos. In this case the survival of motoneurons was proportional to the amount of limb bud remaining (Lanser and Fallon, 1987).

Response to NTFs

This study found that ethanol did not significantly alter the gross amount of neurotrophic activity produced in target limb muscle in comparison to Saline treated embryos when administered from El0 to E15. The significantly greater neurite outgrowth of the Ethanol group in comparison to the NC group does not provide conclusive evidence that embryonic ethanol exposure increases neurotrophic activity. When this result is combined with the fact the ethanol reduces motoneuron number in the spinal cord during this period, it could be that ethanol has altered the ability of motoneurons to respond to neurotrophic support produced by the target muscle. This change in the ability to respond to NTFs could be achieved by altering retrograde transport capacity, receptor expression, or receptor function. Ethanol is known to hinder retrograde transport in cultured thymocytes (McLane, 1990) and previous research in this laboratory found that prenatal exposure to ethanol in the rat reduced the ability of cultured hippocampal neurons to respond to exogenous NTFs (Heaton et al., 1994). Such a mechanism might occur by ethanol altering expression of NTF receptor genes or by altering the activity of the active receptor.

Ethanol is known to affect certain receptor systems. For example, N-Methyl-Daspartate (NMDA) receptors are a type of glutamate receptor and are involved in long-term potentiation, which has long been thought to be involved in how the hippocampus encodes






44

new memories (Bunsey and Eichenbaum, 1996). Ethanol has been shown to inhibit the flow of ions through NMDA receptors and to block NMDA receptor antagonists from binding to the receptor (Lovinger et al., 1989; Valles et al., 1995). Previous research from this laboratory has implied that ethanol inhibits neuronal ability to respond to NTFs. Specifically, bFGF's ability to promote neurite outgrowth in hippocampal cultures was reduced in those composed of cell from animals exposed prenatally to ethanol (Heaton et al., 1995b). Such a mechanism, inhibition of the NTF/receptor binding system, could be involved in ethanol's toxic effect upon the neuromuscular system. Future experiments will be designed to determine whether such a mechanism is indeed occurring in this system.

Since the neurotrophic content of E16 muscle exposed to ethanol from El0 to El5 is not significantly changed in comparison to Saline exposed embryos, it is likely that ethanol interferes with an individual motoneuron's ability to utilize its neurotrophic support rather than reducing neurotrophic support. In support of this hypothesis, our lab has also found that ethanol disrupts the ability of co-cultures of spinal cord to grow neurites toward limb muscle tissue (Heaton et al., 1995a). This result could also be due to altered NTF receptor function since neurite outgrowth occurs as the growth cone responds to its environment. If the ability of the growth cone to sense its environment were diminished, it would not extend the neurite in a normal manner.

The fact that motoneuron and muscle development proceed concurrently and are interdependent should not be overlooked. Ethanol does reduce the amount of trophic substances produced in muscle when administered early in embryonic development (Heaton and Bradley, 1995) and this further limitation of trophic factors could cause fewer motoneurons than normal to survive. The current results do not support such a hypothesis during late ethanol exposure since ethanol administered from E10 to E15 did not reduce or otherwise alter neurotrophic activity of limb muscle extracts in comparison to the positive control group, Saline. Motoneuron number is reduced following both exposure periods which suggests that NTF developmental activity is not solely responsible for this loss in the






45


embryonic chick, at least during the late exposure period. Since muscle requires motoneuron innervation and activity to develop properly (Ishiura et al., 1981), it is equally possible that a directly toxic effect of ethanol on motoneurons, such as a change in the ability of motoneurons to respond to NTFs as mentioned above, could cause developing muscles to exhibit the malformations noted in both human and animal models of prenatal ethanol exposure (Adickes and Shuman, 1983; Nyquist-Battie et al., 1987). Apoptosis

While the current experiments have been effective in eliminating exacerbation of NOCD as a potential mechanism of ethanol toxicity observed in the developing chick embryo spinal cord, other possible explanations for ethanol toxicity exist. These potential mechanisms could utilize an apoptotic mechanism to achieve cell death. NOCD has been shown to be an apoptotic process that requires the cell to participate actively in its own demise (Columbano, 1995). The term "active" in this context indicates that new RNA and protein synthesis are required for apoptosis to proceed. In fact, NOCD in the spinal cord does require new RNA and protein synthesis (Oppenheim et al., 1989), but some examples of apoptosis in the absence of RNA synthesis have been documented (Kelley et al., 1992). In culture, ethanol has been shown to induce apoptosis in hypothalamic neurons (De et al., 1994), thymocytes (Ewald and Shao, 1993), and cerebellar granule neurons (Bhave and Hoffman, 1997; Liesi, 1997). Previous studies have also implicated chronic ethanol treatment in producing apoptotic cell death in the hippocampus and the cerebellum of adult rats in vivo (Renis et al., 1996; Singh et al., 1995). Additionally, there is evidence that ethanol increases programmed cell death in the cerebellum (Cragg and Phillips, 1985).

The present study did attempt to determine whether ethanol induced apoptosis in motoneurons of the lumbar spinal cord. However, the results did not indicate that such a mechanism was occurring. Saline and Ethanol group spinal cords that were examined at E12 and E14 contained virtually identical, and very few, numbers of motoneurons undergoing apoptosis identified by histological or morphological characteristics. The fact






46


that such cells were not seen does not rule out the possibility that ethanol is inducing apoptosis in spinal cord motoneurons but was not detected with this methodology. As was mentioned above, ethanol exposure from E10 to El5 results in a reduction of approximately 15% in motoneuron number in the lumbar spinal cord. Furthermore, apoptosis is a relatively rapid cellular process that is completed in approximately 3 hours (Bursch et al., 1990) and begins very soon after ethanol exposure (Cragg and Phillips, 1985). To have the best opportunity to observe apoptosis, embryos should have been sacrificed between two and five hours following the ethanol injection on days when this analysis was to occur.

Hypoxia

Another possible mechanism contributing to the toxic effects of ethanol upon

developing motoneurons is hypoxia. Hypoxia, a condition which results from inadequate blood supply, exerts a variety of effects on all organ systems, including the central nervous system (CNS). Previous studies in mammals have found that administration of ethanol causes a decrease in umbilical artery blood flow and a reduction of oxygen delivery (Altura et al., 1983; Jones et al., 1981; Mukherjee and Hodgen, 1982). However, a link between ethanol and hypoxia in the developing chick has not been established. Hypoxia alone has been studied in this animal model and has been shown to reduce the overall vascularity of the chorioallantoic membrane (Strick et al., 1991) and reduce its blood flow (Ar et al., 1991). The fact that the direct relationship between ethanol and hypoxia has not been explored in the chick does not eliminate hypoxia as a potential mechanism in this model. A future goal of this laboratory should be to determine whether or not this relationship exists in developing chick embryos. In the CNS, hypoxic conditions affect hippocampal CA 1 pyramidal neurons and cerebellar Purkinje cells (Auer et al., 1989; Jorgensen and Diemer, 1982). These same neuronal populations are damaged when rats are exposed to ethanol prenatally and postnatally (Barnes and Walker, 1981; Bonthius and West, 1990; Phillips and Cragg, 1982; Pierce et al., 1989). These studies have led to a hypothesis that ethanol-






47


induced hypoxia may cause excitotoxic damage to developing neurons (Michaelis, 1990). If such a mechanism were occurring in response to prenatal ethanol exposure, one would expect to find that ethanol has an effect on Ca2+ homeostasis. In fact, ethanol has been shown to regulate Ca2" homeostasis in cultured neurons (Koike and Tanaka, 1991; Webb et al., 1995). These latter studies do suggest that hypoxia may be involved in ethanol toxicity.

Additional considerations

The motoneuron size findings indicate that for the most part ethanol has no effect on this aspect of motoneuron morphology in the lumbar spinal cord. The fact that Curare+Ethanol embryos contained smaller motoneurons could be due to the combined effect of curare and ethanol which were extremely toxic to the developing embryos. Since survival was poor in this group, it is likely that some general status of the embryo was compromised which could have altered motoneuron size. The finding that ethanol reduces motoneuron number at more than one stage of development suggests that it may have the ability to be toxic to motoneurons throughout chick nervous system development.

The present study found that motoneuron number is reduced following ethanol administration from E4 to El 1 and from ElO to E15. This result is similar to results obtained from other neuronal populations such as cerebellar Purkinje cells which are susceptible to ethanol during a range of time periods (Hamre and West, 1993; Phillips and Cragg, 1982). Specifically, Purkinje cells are reduced in number following both prenatal (Phillips and Cragg, 1982), and postnatal (Hamre and West, 1993; Phillips and Cragg, 1982) ethanol exposure. The similarity in temporal vulnerability between motoneurons and Purkinje cells suggests that some fundamental resemblance between these two populations determines their similar susceptibility to ethanol. To thoroughly and properly investigate the role of ethanol in motoneuron reduction in the chick lumbar spinal cord, completion of a parametric ethanol exposure study will be necessary. Such a study would allow us to determine whether ethanol is directly toxic throughout chick embryonic development or if






48


ethanol is toxic at multiple critical periods. This knowledge would then allow us to better hypothesize mechanisms for ethanol toxicity in this neuronal population.














CHAPTER 3
CHARACTERIZATION OF MOTONEURON SURVIVAL FOLLOWING ETHANOL EXPOSURE AND CONCURRENT TREATMENT WITH EXOGENOUS GDNF OR BDNF IN THE EMBRYONIC CHICK SPINAL CORD Summary

Maternal consumption of ethanol is widely recognized as a leading cause of mental and physical deficits. Many populations of the central nervous system (CNS) are affected by the teratogenic effects of ethanol. Neuroprotection against ethanol has been studied extensively in cell culture models and has also been studied in vivo in response to a variety of neurotoxic events including hypoxia, ischemia, and hypoglycemia. Some neurotrophic factors (NTFs) have been shown to protect against ethanol neurotoxicity in culture. The only in vivo evidence of NTF prevention of ethanol neurotoxicity involved NGF protection of choline acetyltransferase activity in early chick embryos (Brodie et al., 1991). Previous studies in this laboratory have demonstrated that ethanol is toxic to developing chick embryo motoneurons when administered from embryonic day 10 (E10) to E15. Other laboratories have found that developing motoneurons are dependent on glial cell linederived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF). GDNF and BDNF suspend naturally occurring cell death (NOCD) in a subset of developing motoneurons. These factors also rescue motoneurons from axotomy-induced cell death in developing embryos. The concurrent delivery of GDNF with ethanol and BDNF with ethanol was designed to test their ability to provide neuroprotection for this ethanol-sensitive motoneuron population. Analysis of motoneuron number indicated that GDNF, but not BDNF, significantly increased motoneuron number in the developing spinal cord following embryonic ethanol exposure. However, GDNF was not found to




49






50


interact significantly with ethanol. Therefore, GDNF may serve to increase motoneuron number to a level that is significantly greater than in ethanol treated embryos by a mechanism that is independent of ethanol. Further studies should be developed to examine this phenomenon in greater detail and determine whether GDNF does indeed provide protection from ethanol toxicity.

Introduction

As was mentioned previously, much evidence has been gathered regarding the

effects ethanol exerts in the developing nervous system (Barnes and Walker, 1981; Jones and Smith, 1973; Miller, 1986; Streissguth et al., 1991; West, 1986). Fetal alcohol syndrome (FAS) produces CNS deficits that do not lessen as the patient ages (Streissguth, 1993). Postmortem analysis of human FAS neuropathology has identified CNS abnormalities which include disorders of laminae of the cerebral cortex, cerebellar abnormalities, a reduction of dendritic spines on cortical pyramidal cells, hippocampal malformation, and microcephaly (Clarren et al., 1978; Ferrer and Galofre, 1987). Neuronal populations that are known to be affected by ethanol in animal models include the cerebellum (Cragg and Phillips, 1985; West, 1986), the septohippocampal system (Barnes and Walker, 1981), cerebral cortex (Miller, 1986), the substantia nigra (Shetty et al., 1993), chief sensory trigeminal nucleus (Miller and Muller, 1989), red nucleus (Zajac et al., 1989), inferior olivary nucleus (Napper and West, 1995), striatum (Heaton et al., 1996) and motoneurons of the spinal cord (Bradley et al., 1997; Heaton and Bradley, 1995). Some of the microscopic and molecular changes that have been observed following ethanol exposure include decreased dendritic arborization (Davies and Smith, 1981), delayed synaptogenesis (Leonard, 1987), decreased neurotransmitter synthesis (Rawat, 1977; Swanson et al., 1994), changes in connectivity (West et al., 1994), and cell loss (Barnes and Walker, 1981; Bauer-Moffet and Altman, 1975; West et al., 1986).

Ethanol affects chick embryo development in a manner similar to mammals. As was mentioned previously, chicks exposed to ethanol prenatally have been shown to






51


exhibit reduced brain size, brain weight, DNA and protein synthesis (Pennington and Kalmus, 1987), and reduced neurotransmitter synthesis (Brodie and Vemnadakis, 1990; Swanson et al., 1994). An advantage of using a chick model to study ethanol is the fact that ethanol can be administered in exact doses to the developing embryo, and only molecules produced by the embryo itself can remove the ethanol from the embryonic environment. Maternal influences are removed when using the chick embryo as ethanol is cleared from the bloodstream by the mother in a mammalian system. In the chick, the embryo is isolated as it develops. The chick model allows the investigator to observe direct effects of ethanol without possible interactions of maternal metabolism interfering. While chick development is clearly different from mammalian gestation (and this is a caveat of using the chick embryo as a model for FAS), this model allows researchers to study in vivo interactions in a developing organism that are not possible in a mammalian model. This model has been widely used to study the effects of NTFs on the developing motor system (Oppenheim et al., 1995; Oppenheim et al., 1992). NTFs can be administered through windows in the egg shell directly onto the chorioallantoic membrane. Developing chick embryos tolerate slight invasions into their environment as long as the underlying membranes are not disrupted.

The present studies focus on the motor system of the developing chick embryo. Motoneurons have been shown to be susceptible to the toxic effects of ethanol both in culture (Dow and Riopelle, 1985; Heaton et al., 1995b) and in vivo (Bradley et al., 1997; Heaton and Bradley, 1995; Heaton et al., 1995b). Our laboratory has found that ethanol can reduce motoneuron number when administered to chick embryos from E4 to El 1 (H-eaton and Bradley, 1995) and when administered from E10 to E15 (Bradley et al., 1997). This reduction is not dependent on NOCD since curare, an agent which blocks NOCD, does not prevent this ethanol-induced death (Bradley et al., 1997). Furthermore, when the period for NOCD had expired, ethanol still reduced motoneuron number (Bradley et al., 1997).






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Two NTFs important for motoneuron development are BDNF and GDNF. BDNF is a member of the neurotrophin family of NTFs which includes nerve growth factor (NGF), neurotrophin-3, and neurotrophin-4/5. A 118 amino acid residue polypeptide (Ilag et al., 1994), BDNF forms homodimers to attain its active form and binds with high affinity to tyrosine receptor kinase B (trkB; Klein et al., 1991). p75 is the low-affinity binding receptor for all neurotrophins. The role of p75 in mediating neurotrophin-trk binding is unclear. Primary sensory neurons display no biological activity when nerve growth factor binds trkA in the absence of p75 (Verge et al., 1992), while in other cell types the trk receptors can work alone (Klein et al., 1991). BDNF is produced by the developing skeletal muscle and is known to support motoneuron survival during development and to protect motoneurons of both the chick and rat from degenerating after lesion (Oppenheim et al., 1992; Sendtner et al., 1992; Yan et al., 1992). In addition to this expression, BDNF is expressed in the hippocampus, adrenal gland, and in whole brain during rat development (Maisonpierre et al., 1990). In the rat, mRNA for BDNF is expressed in skeletal muscle both prenatally and postnatally (Griesbeck et al., 1995).

GDNF is a member of the transforming growth factor B (TGF-B) superfamily and naturally occurs as a dimer with a molecular weight of 40-45 kD (each molecule 134 amino acid residues; Lin et al., 1993). Recent studies suggest that GDNF and its receptors, GDNFRo and c-ret, form a complex that allows c-ret to transduce the signals from GDNF (Jing et al., 1996; Treanor et al., 1996). In this complex, GDNFRx acts as a ligandbinding protein by binding GDNF (Jing et al., 1996). The GDNFRoc-GDNF complex then forms a complex with c-ret (Jing et al., 1996; Treanor et al., 1996). C-ret is the only molecule of the complex capable of producing intracellular signals (Rosenthal, 1997). During embryogenesis of the rat, GDNF mRNA is expressed by mesenchymal cells and in developing skeletal muscle beginning at E15, and in developing skin beginning at E17 (Nosrat et al., 1996; Trupp et al., 1995; Wright and Snider, 1996). GDNF is also expressed peripherally in the teeth, tongue, retina, nasal cavity, ear, kidney, and






53


gastrointestinal tract during various stages of development (Nosrat et al., 1996). In the CNS, GDNF is expressed in the striatum, hippocampus, and cerebellum beginning at E15, and in the trigeminal motor nucleus (E17) and cortex (postnatal day 7). C-ret is highly expressed in substantia nigra dopaminergic neurons, a population which is protected from 6-hydroxydopamine (6-OHDA) lesion by exogenous GDNF (Trupp et al., 1995). Other populations that are responsive to GDNF express c-ret, including spinal motoneurons (Pachnis et al., 1993; Tsuzuki et al., 1995) and certain subpopulations of the peripheral ganglia (Pachnis et al., 1993; Tsuzuki et al., 1995).

Studies of genetically altered mice, where a specific gene has been deleted from the genome, have added to the knowledge of NTFs and their receptors. Knockout mice have been created to study the relative importance of BDNF, trkB, p75, GDNF, and c-ret. Since the present study is concerned with the development of the motor system, discussion of these animals will be limited to this subject. While BDNF and p75 knockout mice do not display a loss of motoneurons (Jones et al., 1994; Lee et al., 1992), trkB knockout mice do contain reduced motoneuron number (Klein et al., 1993). The situation with regard to GDNF and its receptor is somewhat different. GDNF deficient animals exhibit a small but significant loss of motoneurons (Moore et al., 1996), while c-ret knockout mice do not exhibit reduced numbers (Marcos and Pachnis, 1996). These results suggest that the receptors that actually transduce the signals of BDNF and GDNF are important for proper motoneuron development. Additionally, these results suggest that there may be some redundancy in the NTFs that can activate trkB and c-ret, since GDNF-deficient and BDNF-deficient animals exhibit no deficit in motoneuron number.

Neuroprotection by polypeptide growth factors has been studied extensively in recent years. Examples of neuroprotection include epidermal growth factor protection of whole brain neuronal cultures from anoxia (Pauwels et al., 1989), NGF protection of rat hippocampal and human cortical neurons from hypoglycemia (Cheng and Mattson, 1991), and basic fibroblast growth factor (bFGF) prevention of thalamic degeneration following






54


cortical infarction (Yamada et al., 1991). In the developing nervous system, GDNF has been shown to be potent in protecting neurons from a variety of conditions that normally cause death such as NOCD (Oppenheim et al., 1995), 6-OHDA lesion (Choi-Lundberg et al., 1997; Kearns and Gash, 1995; Tomac et al., 1995), and axotomy (Gimenez y Ribotta et al., 1997; Houenou et al., 1996; Oppenheim et al., 1995). BDNF is also effective in providing neuroprotection from toxic events. For example, BDNF rescues some neurons ischemia-induced cell death in rat hippocampal slice cultures (Pringle et al., 1996). BDNF has also been shown to reduce NOCD in motoneurons (Oppenheim et al., 1992) and apoptotic death in PC 12 cells (Jian et al., 1996) and cultured rat cerebellar granule neurons (Kubo et al., 1995). The fact that both GDNF and BDNF provide such potent support for developing and injured neurons suggests that both could protect motoneurons from ethanol-induced death.

Neuroprotection from ethanol has been demonstrated in culture in previous

research. Previous studies in our laboratory found that NGF can protect cultured dorsal root ganglion (DRG) neurons (Heaton et al., 1993) and septal neurons (Heaton et al., 1994) from ethanol toxicity. bFGF was shown to afford some neuroprotection to cultured septal and hippocampal neurons (Heaton et al., 1994). NGF and bFGF protect cultured cerebellar granule cells from ethanol-induced cell death (Luo et al., 1997). This neuroprotection afforded by NGF and bFGF was found to require both protein and RNA synthesis (Luo et al., 1997). This result suggests that neuroprotection is related to a signal that the NTF receptor sends to the nucleus of the cell. NGF protection of choline acetyl transferase activity from ethanol was the first in vivo demonstration of NTF neuroprotection from ethanol (Brodie et al., 1991). The previous use of the chick embryo in both in vivo NTF (Oppenheim et al., 1995; Oppenheim et al., 1992) and ethanol research (Heaton and Bradley, 1995) makes it an excellent choice for studying ethanolNTF interactions.






55


The objective of the present experiment was to determine whether exogenous NTFs could provide protection for motoneurons exposed to ethanol from El0 through E15. Both GDNF and BDNF are known to be NTFs for developing motoneurons (Henderson et al., 1994; Oppenheim et al., 1995; Oppenheim et al., 1992; Sendtner et al., 1992; Yan et al., 1992). The concurrent delivery of each of these NTFs with ethanol was designed to test the ability of each to provide neuroprotection for this ethanol-sensitive population. Analysis of motoneuron number indicated that GDNF, but not BDNF, resulted in increasing the number of motoneurons present in the lumbar spinal cord following ethanol exposure in the developing lumbar spinal cord.

Materials and Methods

Subjects

White Leghorn chick eggs were obtained from the University of Florida Poultry Science Department. Eggs were placed in a Marsh incubator and maintained at 37C and 70% relative humidity until E4. At that time, the eggs were moved to a forced draft turning incubator, maintained at the same conditions indicated above, and divided into groups. Six experimental groups were used in this study: Ethanol, Saline, GDNF, GDNF+Ethanol, BDNF, and BDNF+Ethanol. Embryos received daily injections of ethanol, saline, a NTF, or a combination of ethanol and an NTF from El0 to El5. At E16, embryos were removed from the eggs, sacrificed by decapitation, and the lumbar section of the spinal cord removed and prepared for histology.

Infections

Ethanol and saline injections were administered daily from El0 through El5.

These dates were chosen to replicate a previous study from this laboratory in which ethanol was shown to reduce motoneuron number in the lumbar spinal cord (Bradley et al., 1997), and because NOCD, which occurs in the chick spinal cord from E5 through E9 (Pittman and Oppenheim, 1978), is completed at this time. Since NOCD is completed when






56


injections begin, any change in motoneuron number observed is attributable to treatment per se and not an interaction of treatment with NOCD. Ethanol embryos received 150 jA of 30% w/v ethanol (45 mg ethanol per day), dissolved in a 0.9% w/v nonpyrogenic saline vehicle, through a pinhole in the shell into the airspace. Previous work in our laboratory has detennined that this concentration of ethanol produces blood ethanol counts that peak between 250 and 300 mg/dl (Bradley et al., 1997). Saline embryos received 150 yl of the

0.9% w/v nonpyrogenic saline vehicle. NTF injection [GDNF (Amgen) or BDNF (Regeneron)], which also occurred from ElO through E15, involved creating a pinhole directly over the embryo in addition to the pinhole created in the airspace. The airspace was then allowed to shift to a position superior to the embryo and 50 jl of 0.2 mg/ml NTF was injected into that space above the embryo (10 yg GDNF or BDNF per day). These levels of GDNF and BDNF administration were previously shown to rescue motoneurons from NOCD without being toxic to the developing embryo (Oppenheim et al., 1995; Oppenheim et al., 1992). GDNF+Ethanol and BDNF+Ethanol embryos were given ethanol injections from El0 to E15 and NTF injections from El0 to E15 as described above. Ethanol injections preceded NTF injections and the embryos were allowed to sit in a Marsh incubator for a period of one hour between injections. This delay in injection time was necessary to ensure that the ethanol was absorbed through the inner shell membrane within the airspace before the eggs were turned on their side for NTF administration. Also, the two injections administered in this study represent a significant volume (200 /,) for the embryonic system to incorporate on a daily basis. The delay between injections, therefore, allowed absorption of the volume of ethanol before the NTF solution was presented to the embryo. Pinholes created by the injection process were sealed with paraffin immediately following injection to prevent evaporation and/or leakage of the solutions. The eggs were then returned to the turning incubator.






57


Dissections and Histological Procedures

Embryos of all experimental groups were sacrificed by decapitation on E16 and the lumbar section of the spinal cord removed. The vertebrae of the spinal cords were cut along the dorsal surface to expose the nervous tissue and allow the fixative to adequately penetrate the tissue. Following dissection, the E16 spinal cords were placed in Bouin's Fixative for 14 to 21 days to allow the vertebrae to decalcify (Li et al., 1994). The tissue was then embedded in paraffin, cut into 12 pm coronal sections, mounted onto glass slides, and stained with hematoxylin and eosin. Motoneuron Size and Spinal Cord Length

Motoneuron size and spinal cord length were measured to determine whether

ethanol alters any general characteristics of the motoneuronal system. Motoneuron size was determined by measuring the diameter of 10 random cells in the same rostral-caudal position of the region of each embryonic spinal cord with an eyepiece micrometer. The section exactly 2400 pm following the beginning of the lumbar spinal cord was sampled. Three embryos from each experimental condition were analyzed for a total of 30 cells per condition. Spinal cord length was determined by counting the number of sections present in each embryo following determination of the boundaries of the lumbar spinal cord by the anatomical methods described previously and multiplying this number by the section thickness (12pym).

Motoneuron Counts

Motoneuron counts were completed following methods described previously

(Hamburger, 1975; Heaton and Bradley, 1995). Briefly, a uniform area encompassed by 6 DRG was noted in each embryo. This procedure ensured that a similar area was counted in each subject. Starting from the most rostral section included in the 6 DRG region, motoneurons in the lateral motor column of one side of every tenth section were marked onto paper using a camera lucida. At 400X magnification, motoneurons were identified in






58


the lateral motor column by their large size, dark cytoplasm, and nucleolus. Laterality was maintained throughout each individual embryo, but chosen at random before beginning the counting process. Previous studies have shown that there is no difference between the number of motoneurons contained in the right and left sides of the spinal cord (Pittman and Oppenheim, 1979). Each embryo was coded so that the experimenter had no knowledge of its experimental treatment until the study was completed. Statistical Analyses

Two-way analysis of variance was performed using SAS version 6.12 on a

pentium computer. When applicable, individual differences between groups were tested using Fisher's protected least significant difference post-hoc analyses. Statistical significance was determined to be p<0.05.

Results

GDNF administration did not seem to harm the embryos since survival was 93% in the GDNF group and 69% in the GDNFEthanol group. These rates compare favorably with control survival rates where 90% of the Saline group and 60% of the Ethanol group survived. BDNF administration had a somewhat different effect upon chick survival. The BDNF group survived at a rate of 82% and the BDNFEthanol group survived at a rate of 65%. These results indicate that the level of GDNF and BDNF administered in this study is not toxic to overall embryonic survival.

Motoneuron Size and Spinal Cord Length

As stated in the Methods section above, motoneuron size and spinal cord length analyses were performed to determine whether treatments utilized in these studies altered the gross morphology of the embryonic spinal cord. Analysis of variance testing indicated that embryos from these experimental groups exhibited no differences in motoneuron size due to treatment (F=0. 13, df=29, p>0.9). That is, the motoneuron size of ethanol-treated animals was unchanged from that of NTF-treated, or control animals. Analysis of variance






59


also revealed that overall lumbar spinal cord length was unaltered by treatment (F=0.44, df=40, p>0.8). There were no significant differences in the length of the spinal cord region counted among the experimental groups. Table 3-1 displays the data from this portion of the study. Taken together, these results suggest that the treatments administered in this study did not adversely affect the basic anatomy of the spinal cord.


Table 3-1. Motoneuron Size and Spinal Cord Length. Group Cell Size (yim) Spinal Cord Length (pm)
Saline 18.6+0.525 6020+ 69.0
Ethanol 18.9 + 0.508 5966 + 137.9
GDNF 18.7 + 0.514 5820 + 154.2
GDNF+Ethanol 18.4 + 0.502 5856 + 148.9
BDNF 18.6+0.433 5904+ 88.2
BDNF+Ethanol 18.6 + 0.438 5900 + 153.1

All values are means + SEM. Motoneuron size was determined by measuring the diameter of 10 random cells in the same rostral-caudal position of the region of three embryonic spinal cords with an eyepiece micrometer. Therefore, n=30 for each group. Spinal cord length was computed by determining the number of sections present in a given spinal cord and then multiplying by section thickness (12 /m). For spinal cord length, Saline n=12, Ethanol n=7, GDNF n=6, GDNF+Ethanol n=5, BDNF n=5, and BDNF+Ethanol n=6. Motoneuron Number

Two way analysis of variance indicated a significant effect due to neurotrophic

factor administration (F=5.645, df=40, p<0.05) and to ethanol treatment (F=8.902, df=40, p<0.005), but not an interaction between neurotrophic factors and ethanol (F=1.708, df=40, p>0. 15). Further analysis limiting the groups to Saline, Ethanol, GDNF, and GDNF+Ethanol found similar results. Analysis of variance found a significant effect due to GDNF treatment (F=9.143, df=29, p<0.01), ethanol treatment (F=6.841, df=29, but not an interaction between the two groups (F=0.786, df=29, p>0.35). Limiting the groups to Saline, Ethanol, BDNF, BDNF+Ethanol did not produce similar results. Analysis of variance found a significant effect due to Ethanol treatment (F=4.381, df=29, p<0.05), but not to BDNF treatment (F=0.671, df=29, p>0.4), or an interaction between ethanol and






60


BDNF (F=2.182, df=29, p>0. 15). Figure 3-1 displays the results of these cell counts. Post hoc testing revealed significant differences among the groups. Specifically, Ethanol embryos contained significantly fewer numbers of motoneurons than GDNF (p<0.0005), GDNF+Ethanol (p<0.05), and Saline (p<0.01) treated embryos. There were no significant differences among the GDNF, GDNF+Ethanol, or Saline groups. Likewise, there were no significant differences among the BDNF, BDNF+Ethanol, Saline, or Ethanol groups. The above results suggest that GDNF significantly increased motoneuron number in a manner that is independent of ethanol. BDNF did not provide significant protection from ethanol. In addition to comparing motoneuron number among the various groups in this study and performing a two way analysis of variance for NTF and ethanol treatment, a contrast analysis was performed to determine if there was an interaction between GDNF and ethanol or between BDNF and ethanol. This analysis found that there was not a significant difference between the difference of GDNF from Ethanol and BDNF from Ethanol (p>0.5). The conclusion of this analysis is that there was no interaction between GDNF and Ethanol or between BDNF and Ethanol. Stated another way, the action of ethanol is independent of the action of either GDNF or BDNF. Figure 3-2 illustrates this conclusion. The slope of the two lines in the figure are not significantly different This result is important because it suggests that the NTFs used in this study are not impaired by the actions of ethanol. This analysis is further evidence that ethanol acts independently of the NTFs used in the present study.

Figure 3-3 and Figure 3-4 display photographs of spinal cords taken from the

animals described in this chapter. Specifically, Figure 3-3 displays photographs of Saline, Ethanol, GDNF, GDNF+Ethanol, BDNF, and BDNF+Ethanol spinal cords at 40x magnification. The overall shape of the spinal cord is altered somewhat by ethanol treatment as fewer motoneurons are present and there is less of a "bulge" on the outer edge of the cord. Figure 3-4 displays 200x magnification views of the same spinal cord






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2000



Ou *
o i



O
1000





0




Saline Ethanol GDNF GDNF+ BDNF BDNF+ Ethanol Ethanol
Figure 3-1. Number of motoneurons in the later motor column of the
lumbar spinal cord at E16. Motoneuron counts are displayed as means + SEM. Data represent actual counts obtained from the lumbar spinal
cords and are not population estimates. All injections were
administered from E10 to E15. = statistical significance in
comparison to Ethanol; saline (p < 0.01), GDNF (p < 0.0005), and
GDNF+Ethanol (p < 0.05). Number of animals used for the experiment
equal to 12 Saline, 7 Ethanol, 6 GDNF, 5 GDNF+Ethanol, 5 BDNF,
and 6 BDNF+Ethanol.






62






1500 GDNF GDNF+Ethanol


BDN
1300
S 100 BDNF+Ethanol
O
Ethanol
1100 1

NTF NTF and Ethanol
Ethanol
Interaction Slope

Figure 3-2. Interaction between ethanol and neurotrophic factors. This figure represents the slope of the interaction between GDNF and ethanol and between BDNF and ethanol. There was not a significant difference between the GDNF group and the BDNF group which suggests that the action of each NTF and ethanol is independent. Number of animals used for the experiment equal to 12 Saline, 7 Ethanol, 6 GDNF, 5 GDNF+Ethanol, 5 BDNF, and 6 BDNF+Ethanol.






























Figure 3-3. Photomicrographs of coronal sections from the midlumbar region of E16 spinal cords. A. Saline, B. Ethanol, C. GDNF, D. GDNF+Ethanol, E. BDNF, and F. BDNF+Ethanol. The only noticeable difference is in the density of the and number of the motoneurons present in the lateral motor column where the Ethanol group appears to have fewer motoneurons present. Number of animals used for the experiment equal to 12 Saline, 7 Ethanol, 6 GDNF, 5 GDNF+Ethanol, 5 BDNF, and 6 BDNF+Ethanol.





64

A









Saline Ethanol






~D





GDNF GDNF+Ethanol


F
"'A V








BDNF BDNF+Ethanol

Figure 3-3.































Figure 3-4. High magnification photomicrographs from the midlumbar section of E16 spinal cords. A. Saline, B. Ethanol, C. GDNF, D. GDNF+Ethanol, E. BDNF, and F. BDNF+Ethanol. This view gives a better perspective of the motor column of each animal. The difference in the number and density of the Ethanol animals is apparent.








66











~f 7
14~























--a







*V .dy{,




SalNc Ethanolano




Figure 3-4.






67

sections. The overall density and number of motoneurons in the lateral motor column again appears reduced in comparison to the other groups.

Discussion
The major finding of this study is that GDNF can significantly increase motoneuron number in the lumbar spinal cord in a manner independent to ethanol (see Figure 3-1). In vivo neuroprotection from ethanol neurotoxicity was not demonstrated in embryos treated with BDNF. The results of the two way analysis of variance indicated a significant effect due to ethanol and NTF treatment. Further analysis of the groups indicated that only GDNF administration had a significant effect upon motoneuron number while BDNF did not. An interaction between either GDNF and ethanol or BDNF and ethanol was not indicated by this powerful statistical test. Thus, the increase in motoneuron number by GDNF is not due to some action of GDNF upon ethanol, but rather GDNF increases motoneuron number in a manner that is independent of ethanol toxicity. Ethanol and NTFs were administered during a period of development when NOCD was complete, but when motoneuron number can be diminished by ethanol exposure. Therefore, NOCD did not provide an added variable for this study. These results support previous research from other laboratories which found that GDNF could protect certain neuronal populations from various neurotoxic events. For example, GDNF protected rat nigral dopamine neurons against 6-OHDA lesion in vivo (Choi-Lundberg et al., 1997; Kearns and Gash, 1995; Tomac et al., 1995). Also, GDNF prevented death of spinal cord motoneurons following axotomy in the chick (Houenou et al., 1996; Oppenheim et al., 1995) and rescued facial motoneurons following axotomy in the rat (Gimenez y Ribotta et al., 1997).

The receptor thought to be responsible for GDNF's activity in the nervous system is c-ret and the high affinity receptor for BDNF is trkB. Recent studies suggest that GDNF, GDNFRoc, and c-ret form a complex that allows c-ret to transduce the signals from GDNF (Jing et al., 1996; Treanor et al., 1996). The ontogeny of the receptors for GDNF and BDNF follow different patterns during the development of the neuromuscular system.






68


C-ret mRNA expression is detectable, albeit very weakly, in chick spinal cord motoneurons as early as E5 and increases throughout development (Nakamura et al., 1996; Schuchardt et al., 1995). By E17, c-ret mRNA expression in the chick is expressed at very high levels in spinal cord motoneurons (Nakamura et al., 1996; Schuchardt et al., 1995). Therefore, c-ret is expressed by the desired target, motoneurons, during the exposure period of the present study (El0 to El5). TrkB mRNA is first detectable at E8 and its level of expression increases throughout development (McKay et al., 1996). At El6, trkB mRNA is highly expressed in the lateral motor column of the spinal cord of the chick (McKay et al., 1996). Therefore, trkB is expressed by motoneurons during the exposure period of the present study. Since both receptors are expressed during the period of exposure used in the present study, another reason must explain the fact that GDNF increases motoneuron number from ethanol toxicity whereas BDNF does not.

In culture chick motoneurons are supported by GDNF (Gouin et al., 1996) whereas they are not supported by BDNF (Arakawa et al., 1990). This is not the case in cultures of rat motoneurons where BDNF does support their growth (Henderson et al., 1993). Even though both GDNF and BDNF prevent NOCD in spinal cord motoneurons, these cells are rescued from NOCD to a greater extent by GDNF than BDNF (Oppenheim et al., 1995; Oppenheim et al., 1992). This fundamental advantage of GDNF over BDNF to support chick motoneurons in culture may explain the results of the present study. The advantage of GDNF to support motoneurons to a greater degree than BDNF is supported by the results of knockout studies. As was mentioned above, GDNF-deficient, but not BDNFdeficient mice exhibit reduced motoneuron number (Jones et al., 1994; Moore et al., 1996). Again, the implication of those studies is that GDNF is required to a greater degree for proper motoneuron development than is BDNF. The present study found that GDNF administration concurrent with developmental ethanol exposure increased motoneuron number in a manner independent of ethanol, while BDNF did not significantly alter ethanol toxicity. This latter portion of the statement is supported by the fact that two way analysis






69


of variance testing did not find a significant effect due to BDNF treatment or an interaction between ethanol and BDNF.

Further experiments using animals genetically altered to overexpress GDNF might provide further information about the nature of the increase in motoneuron number following embryonic ethanol exposure. By administering ethanol to these animals, researchers could examine whether GDNF produced by the animal itself could increase motoneuron number following ethanol exposure. If these animals proved to be more resistant to ethanol insult, it would suggest that some mechanism of the endogenous NTF naturally protects developing motoneurons from ethanol to some extent, provided an interaction between ethanol and the NTF is demonstrated. Obviously, the normal endogenous activity of NTFs in the nervous system does not protect developing chick motoneurons from ethanol toxicity since exposure from ElO to El5 reduces motoneuron number (Bradley et al., 1997).

Another line of inquiry could be into cell death genes and the roles they play in NTF neuroprotection. Ethanol is known to induce apoptosis in culture (Bhave and Hoffman, 1997; De et al., 1994; Ewald and Shao, 1993; Liesi, 1997) and in vivo (Renis et al., 1996; Singh et al., 1995). The bcl-2 family of cell death molecules has been shown to be involved in apoptosis (Boise et al., 1993; Hockenbery et al., 1990). Members of the bcl-2 family include bcl-2, bcl-Xs, bcl-XL, and bax (Boise et al., 1993; Oltavi et al., 1993). Bcl2 is a membrane-associated protein that interferes with apoptotic cell death. Bcl-Xs acts to antagonize bcl-2 activity and promote cell death. Bcl-XL acts in much the same way as bcl2 and bax binds bcl-2 to inhibit its ability to prevent apoptosis (Davies, 1995). To determine whether the bcl-2 family is involved in GDNF increasing motoneuron number in the presence of ethanol, enzyme-linked immunosorbent assay could be used to determine precise levels of these molecules in motoneuron cultures exposed to ethanol and GDNF. The experiments would be designed to determine whether GDNF added to these cultures in the presence of ethanol induces greater expression bcl-2 and bcl-XL--which both prevent






70

apoptosis--or decreases expression of bcl-Xs and bax--which both oppose the protective activity of bcl-2. The results from these proposed experiments would complement the results of the current experiments since they could provide a potential mechanism for neuroprotection, should it happen to be demonstrated in the future, by GDNF. It is important to note that such a mechanism could proceed independent of ethanol since ethanol could conceivably induce apoptosis by another mechanism such as a change in Ca2+ homeostasis (Koike and Tanaka, 1991; Webb et al., 1995).

Evidence linking the roles of NTFs and cell death genes has been examined in

previous studies. Bcl-2 is required for the survival of PC-12 cells dependent on BDNF but not required for survival of CNTF dependent cells (Allsopp et al., 1995). Similarly, bcl-2 expression is required for the survival of NGF-dependent PC- 12 cells (Katoh et al., 1996). Furthermore, NGF has been shown to increase bcl-2 expression in a dose-dependent manner in providing this trophic support for these cultured cells (Katoh et al., 1996). A similar effect is observed in neuronal cells in that cultured trigeminal ganglion and trigeminal mesencephalic neurons are rescued from cell death due to withdrawal of NGF, BDNF, or NT-3 by overexpression of bcl-2 (Allsopp et al., 1993). NTFs are related to and alter expression of proteins that promote cell death. Withdrawal of trophic support did not result in death of axotomized facial motor neurons in bax-deficient mice (Deckwerth et al., 1996). The above examples demonstrate the link between NTFs and cell death genes of the bcl-2 family.

The methodology for determining motoneuron number employed in this study has been used successfully for determining motoneuron number in this laboratory and others (Bradley et al., 1997; Heaton and Bradley, 1995; Oppenheim et al., 1995; Oppenheim et al., 1992; Pittman and Oppenheim, 1979). The results indicate that while ethanol administration does have an adverse effect on the motoneuron population of the lumbar spinal cord, it does not change the overall morphology of the cord. This claim is supported by the fact that lumbar spinal cord length, which is an indicator of spinal cord volume, and






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average motoneuron size are unchanged following ethanol treatment. A previous study from another laboratory also found that administration of exogenous NTFs from E9 to El5 did not alter either motoneuron size or spinal cord length measurements (Qin-Wei et al., 1994). NTFs administered in that study included BDNF, TGF-B, basic fibroblast growth factor, and ten other growth factors (Qin-Wei et al., 1994). The present study supports this finding and has found that exogenous GDNF or BDNF does not alter these relationships since there were no significant differences between any of the experimental groups when motoneuron size and spinal cord length were analyzed (See Table 3-1).

A portion of the current results are somewhat inconsistent with previous research (Oppenheim et al., 1995) in that the present study found that motoneuron number in GDNF-treated embryos did not differ significantly from control embryos. That study found that exogenous GDNF administered from E9 to E15 resulted in significantly more motoneurons in the lateral column of the spinal cord than in control embryos (Oppenheim et al., 1995). A likely explanation for the differences between these two studies is that GDNF injections began on different days. Recall that NOCD continues in the spinal cord through E9 (Pittman and Oppenheim, 1979). By beginning GDNF injections on E9, the earlier study may have rescued some motoneurons that were destined to undergo NOCD and sustained them until E16. The Oppenheim et al. study (1995) found a 12.5% increase in motoneuron number, while the present study found an 8%, but not statistically significant, increase in motoneuron number in comparison to control embryos. Therefore, the absolute difference between the two studies is relatively minimal.

Previous studies led to the hypothesis that ethanol-induced hypoxia may cause excitotoxic damage to developing neurons (Altura et al., 1983; Auer et al., 1989; Barnes and Walker, 1981; Bonthius and West, 1990; Jones et al., 1981; Jorgensen and Diemer, 1982; Michaelis, 1990; Mukherjee and Hodgen, 1982; Pierce et al., 1989). NTFs have been shown to prevent hypoxic/ischemic damage in neurons in research performed in other laboratories. For example, BDNF has been shown to prevent ischemia-induced cell death






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in rat hippocampal slice cultures (Pringle et al., 1996). Even though previous research has not explicitly supported the role of hypoxia in ethanol toxicity, prevention of hypoxia/ischemia is another possible mechanism that could explain GDNF's increase of motoneuron number in the presence of ethanol. Previous research has not searched for a link between ethanol and hypoxia in the developing chick. Hypoxia alone has been studied in this animal model. Hypoxic conditions were found to reduce the overall vascularity of the chorioallantoic membrane (Strick et al., 1991) and to reduce its blood flow (Ar et al., 1991). The fact that the direct relationship between ethanol and hypoxia has not been explored in the chick does not mean hypoxic conditions do not occur in ethanol-exposed chicks. Perhaps a future aim of this research should be to determine whether or not this relationship does exist in developing chick embryos.

As was discussed earlier in the Introduction section of this chapter, the chick embryo exhibits many of the characteristics found in mammalian models of FAS. Morphologically, chick embryos treated with ethanol are smaller than controls and have decreased brain weights (Pennington and Kalmus, 1987). These similarities between avian and mammalian FAS also include molecular changes attributed to ethanol treatment. For example, ethanol decreases kinase activities in whole brain of both the chick and rat (Kruger et al., 1993; Pennington, 1990) and alters cyclic AMP levels in both chick whole brain and in rat striatum (Lucchi et al., 1983; Pennington, 1990). Additionally, neurotransmitter synthesis is altered by ethanol administration in both the chick and rat (Brodie and Vernadakis, 1990; Swanson et al., 1994; Swanson et al., 1995). Other molecular mechanisms attributed to ethanol in culture are potentially occurring in the avian model of FAS. These include those discussed previously such as hypoxia/ischemia (Zajac and Abel, 1992) and apoptosis (Renis et al., 1996; Singh et al., 1995). Other mechanisms implicated in FAS include hypoglycemia (Fisher et al., 1986) and generation of free radicals (Henderson et al., 1995). Such actions could be responsible for the loss of motoneurons in the spinal cord observed following ethanol exposure from El0 to El5






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(Bradley et al., 1997). In addition to NTF protection afforded to neurons from apoptosis and hypoxia/ischemia, NGF protects cultured rat hippocampal and human cortical neurons from hypoglycemic damage (Cheng and Mattson, 1991) and oxidative damage (Mattson and Cheng, 1993). The ability of NTFs to inhibit the processes described above could provide the mechanism behind the current finding that GDNF increased motoneuron number following ethanol exposure. However, the fact that there was not a significant interaction between ethanol and GDNF only allows the following conclusion: GDNF increases motoneuron number in the lumbar spinal cord in a manner independent to ethanol toxicity.

NTFs have proven to be versatile molecules with the ability to sustain neurons when faced with a variety of potentially deadly insults. Now that an increase in motoneuron number by GDNF following ethanol exposure has been demonstrated, additional investigations will need to be conducted to further examine this action of NTFs on developing motoneurons. All of these analyses should be interpreted bearing in mind that the actions of ethanol and the NTFs studied here did not significantly interact. Therefore, their actions may be entirely independent. Other NTFs will be tested for their ability to protect motoneurons from ethanol toxicity. In addition, combinations of NTFs should be tested to determine whether protection is better than when a given NTF is administered alone. Specifically, CNTF and NT-3 are good candidate molecules since they are known to promote motoneuron survival following axotomy or during the period for NOCD (Lo et al., 1995; Yin et al., 1994). Since GDNF increases the number of motoneurons independent of ethanol toxicity, other ethanol-sensitive populations might be increased in number following administration of GDNF, or other NTFs. Clearly, this phenomenon will have to be investigated further to adequately describe the actions of various NTFs are protecting neuronal populations or truly increasing neuron number in a manner independent of the action of ethanol.






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In addition to testing other NTFs to determine whether they can provide

neuroprotection from ethanol toxicity, future studies should attempt to build on results obtained from the present study to develop potential therapies for FAS. GDNF inserted into an adenovirus vector has already proven effective in preventing facial motoneuron death following axotomy (Gimenez y Ribotta et al., 1997). Such a delivery system could prove effective in getting GDNF to spinal cord motoneurons in mammals to increase motoneuron number following ethanol administration. Future research should also focus on determining the mechanism by which this increase in motoneuron number is afforded to motoneurons by GDNF. By introducing toxic insults such as hypoxia/ischemia and hypoglycemia, GDNF's ability to increase motoneuron number in the presence of ethanol should be further defined. Unfortunately, more questions remain to be answered about GDNF in the future than are answered by the present study.














CHAPTER 4
CHARACTERIZATION OF THE NEUROTROPHIN AND NEUROTROPHIN
RECEPTOR GENE EXPRESSION IN THE HIPPOCAMPUS FOLLOWING CHRONIC
TREATMENT AND EARLY POSTNATAL ETHANOL TREATMENT IN THE RAT Summary
Fetal alcohol syndrome (FAS) is caused by maternal consumption of ethanol during pregnancy and was first described more than two and one-half decades ago. In recent years, the thrust of research in this field has been the search for a mechanism of ethanol toxicity. Signal transduction and gene expression studies have allowed researchers to learn about how ethanol affects neuronal populations at the molecular level (Davis-Cox et al., 1996; Gandhi and Ross, 1989; MacLennan et al., 1995; Torres and Horowitz, 1996). Another area that has garnered attention in this field is neurotrophic factors (NTFs) and their ability to affect, and be affected by, ethanol. Previous research has found that ethanol can alter expression of specific genes. Examples of genes regulated by ethanol exposure include insulin-like growth factor I (IGF-I) and IGF-II in rat brain (Breese et al., 1994; Singh et al., 1996). Both of these genes are decreased following ethanol exposure. However, ethanol exposure increases NMDA receptor gene expression in cultured mouse cortical neurons (Hu et al., 1996). The present study attempted to determine the relative expression of brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), tyrosine receptor kinase B (trkB), and trkC in the hippocampus following ethanol exposure during the prenatal or early postnatal period. TrkA was not analyzed because of its extremely low level of expression in the developing hippocampus (Martin-Zanca et al., 1990). Nerve growth factor (NGF), although attempted, was not assessed because it produced signals that were not quantifiable. The results of our analyses indicated that ethanol administration during prenatal development in the rat did not change the genetic expression of BDNF,



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NT-3, and trkB as assessed by quantitative Northern blotting. TrkC expression in male animals, but not female animals, exposed to ethanol prenatally was reduced. Expression of BDNF, NT-3, trkB and trkC was unaffected by postnatal exposure to ethanol during the brain growth spurt (BGS).

Introduction

Maternal consumption of ethanol exerts many effects upon the developing nervous system (Barnes and Walker, 1981; Jones and Smith, 1973; Miller, 1986; Streissguth et al., 1991; West, 1986). FAS continues to be a problem in Western countries and is diagnosed in 1-2 out of every 1000 live births in the United States (Abel, 1995). FAS is characterized by low birth weight, decreased memory and learning, hyperactivity, facial dysmorphia, and lowered IQ (Jones and Smith, 1973; Streissguth et al., 1991). Children born to heavy drinkers experience a higher incidence of FAS with a 4.3% diagnosis rate (Abel, 1995). Previous research suggests that the deficits observed in FAS patients are permanent and do not lessen with age (Streissguth, 1993). Taken together, these observations led to the assertion that maternal consumption of ethanol is the leading known cause of mental retardation in the Western Hemisphere (Bonthius and West, 1988).

Postmortem analysis of human FAS neuropathology has identified central nervous system (CNS) abnormalities which include disorders of laminae of the cerebral cortex, cerebellar abnormalities, a reduction of dendritic spines on cortical pyramidal cells, changes in hippocampal development, and microcephaly (Clarren et al., 1978; Ferrer and Galofre, 1987). A major problem of examining human subjects is that variables such as nutrition and polydrug use are uncontrolled. Animal models have provided controlled exposure to ethanol and have been shown to exhibit CNS deficits and behavioral consequences similar to those observed in humans (Driscoll et al., 1990). Animal models are particularly useful for gaining insight into the effects ethanol exerts on a molecular scale. These models allow researchers to answer questions about ethanol consumption that cannot be answered in human studies due to ethical and practical reasons (West et al., 1994).






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The rat is the most widely used model in FAS research. However, a caveat of using the rat as a model is its gestational period relative to human development. The rat gives birth on what is roughly the equivalent of the beginning of the third trimester in humans (Goodlett et al., 1993). Important events, such as the brain growth spurt (BGS)-where many functional synapses are made in the nervous system--occur in utero during the third trimester in humans and postnatally from P4-Plo in rats (West, 1987). Therefore, experiments that incorporate ethanol exposure during gestation or during the BGS in rats allow researchers to use this as a model of human third trimester ethanol exposure. Exposing rat pups to ethanol postnatally produces deficits that demonstrate the importance of the BGS and the sensitivity of the CNS to ethanol during this period. Similar to prenatal exposure to ethanol in rats, postnatal exposure can produce loss of cerebellar Purkinje cells (Bonthius and West, 1990; Bonthius and West, 1991; Goodlett and West, 1992; West, 1986; West et al., 1990). A problem inherent to any postnatal exposure paradigm is that maternal metabolism of ethanol is removed and the subjects are exposed to ethanol in a more "adult" manner. Another problem is delivery of ethanol. Suckling rats cannot be coerced into readily consuming ethanol because their entire diet consists of mother's milk.

Two of the methods for delivering ethanol to newborn rats are artificial rearing

(AR) and inhalation, both of which have advantages and disadvantages. AR consists of fitting a neonatal pup with a gastric fistula and tube, maintaining the pups in cups placed in a 40'C water bath, and feeding the pup an artificial milk solution via the tube and fistula. The AR method provides constant nutrition and produces no damage to the mucous membranes of the subject, but the interaction between mother and pup is removed. Additionally, AR is a surgical procedure that can be quite stressful for the neonatal rat. The stress induced by AR has been found to produce gliosis in rat cortex (Ryabinin et al., 1995). Recently, the use of intragastric intubation--a less invasive method of neonatal ethanol delivery that allows the pups greater maternal access--also resulted in extensive gliosis in parietal cortex (Goodlett et al., 1997). The fact remains that gastrostomy control






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rats--pups undergoing AR surgery, but receiving no ethanol--exhibit significant gliosis (Ryabinin et al., 1995) and further research should be conducted to determine whether ethanol per se induces gliosis, or whether specific methods of ethanol delivery are responsible. The possibility that the AR procedure in and of itself can produce changes in brain structure indicates that results obtained using AR could be difficult to interpret.

Ethanol vapor inhalation involves placing neonatal rats in a sealed chamber that contains circulating air and ethanol vapor. Inhaling ethanol vapors has been theorized to have the potential to damage the mucous membranes of the lungs which would then interfere with oxygen exchange and general metabolism (Ryabinin et al., 1995). However, no evidence of lung damage has been observed in rats exposed in this manner (BauerMoffet and Altman, 1975). The present study utilized the inhalation procedure because of the problems associated with AR. Additionally, a previous study from this laboratory defined neurotrophic activity in the hippocampus following ethanol inhalation. Therefore, proper continuation of that study requires the use of similar methods of analysis and ethanol delivery. Other methods for delivering ethanol to rats postnatally include concurrent inhalation of mother and pups, direct injection of neonates with ethanol, and delivery of ethanol through mother's milk. This latter method is achieved by substituting water with a 10% ethanol solution. A problem associated with delivery of ethanol through mother's milk is that pups do not receive the same dose of ethanol as the mother due to maternal metabolism.

Mechanisms suggested by previous research may help to explain ethanol's effect on the nervous system (reviewed in West et al., 1994). The ability of ethanol to affect DNA methylation in the developing embryo has implications for FAS research and the present study. Methylation of DNA in eukaryotic cells occurs at the 5' position of cytosine residues and converts them into methylcytosine residues. Repressors and enhancers are then hindered from binding to DNA. The end result is thought to be a change in gene expression (Holliday, 1987). This change, however, can either increase or decrease the






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expression of a given gene since methylation does not selectively interfere with repressors or enhancers. Previous research from the laboratory of Garro found methylation to be decreased in fetal DNA following ethanol exposure to the pregnant dam (1991). Additionally, ethanol has been shown to interfere with the activity and 06-methylguanine transferase. This enzyme is important in repairing DNA and its unfettered activity is crucial for cell survival (Espina et al., 1988). Although these examples have not been shown to be caused by changes in DNA methylation, they do indicate that ethanol has the ability to regulate genetic expression. For example, ethanol has been shown to decrease expression of BDNF mRNA in the hippocampus following chronic exposure to ethanol in adult rats (MacLennan et al., 1995) and increase IGF gene expression in whole brain following prenatal ethanol exposure (Breese et al., 1994). All of these studies indicate a role for ethanol in changing normal cellular biochemistry by affecting genetic expression.

As was mentioned above, learning and memory deficits are a common characteristic of FAS (Abel, 1995; Jones and Smith, 1973; Streissguth et al., 1991). The hippocampus is an important structure with regard to memory and learning in humans and animals (Bunsey and Eichenbaum, 1996; Cohen and Squire, 1980). Thus it is not surprising to find that the hippocampus is sensitive to ethanol and exhibits reduced pyramidal cell number following prenatal ethanol exposure (Barnes and Walker, 1981; Bonthius and West, 1990). Damage to the hippocampus observed in the rat model following ethanol exposure may correspond to similar damage in the hippocampus in humans. The present study analyzed NTF and NTF receptor gene expression in this region because altered neurotrophic activity was implicated in previous studies following both prenatal and postnatal exposure to ethanol (Heaton et al., 1995c; Moore et al., 1996).

The neurotrophin family of NTFs has been shown to play an important role in the development of the CNS and peripheral nervous system (PNS) through involvement in neuronal differentiation, survival, and maintenance of basic cellular processes. The neurotrophin family includes NGF (Levi-Montalcini, 1951), BDNF (Leibrock et al.,






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1989), NT-3 (Maisonpierre et al., 1990), NT-4/5 (Berkemeier et al., 1991; Ip et al., 1992), and neurotrophin-6 (Gotz et al., 1994). The trk family of receptors has been shown to be the high-affinity receptors for the neurotrophins (Martin-Zanca et al., 1990). Trk receptors that interact with neurotrophins include trkA (Kaplan et al., 1991; Kaplan et al., 1991), trkB (Klein et al., 1990), and trkC (Cordon-Cardo et al., 1991). Specifically, trkA is the preferred receptor for NGF, yet to a lesser extent, both BDNF and NT-3 can bind to it. TrkB is the preferred receptor for BDNF and NT-4, but can bind NT-3. And trkC is the preferred receptor for NT-3. As mentioned above, the neurotrophins are important to normal neuronal functioning. They have been shown to regulate a number of peptides-including the expression of other neurotrophins--in the rat septohippocampal system (Croll et al., 1994). For example, NGF, BDNF, and NT-3 induce ChAT activity (Alderson et al., 1990; Auberger et al., 1987; Gnahn et al., 1983; Nonner et al., 1996); BDNF increases NT-3 activity (Lindholm et al., 1994); and BDNF and NT-3 enhance synaptic transmission in Shaffer collateral-CA 1 hippocampal synapses (Kang and Schuman, 1995). This latter result is thought to link neurotrophins to long-term potentitation, the mechanism thought to be partly responsible for hippocampal induction of memory (Bunsey and Eichenbaum, 1996). All of these studies indicate that the neurotrophins are important proteins integral to normal neuronal functioning in the septohippocampal system.

Studies of gene deleted "knockout" mice also suggest the importance of

neurotrophins to proper nervous system development. In totality, the results of these experiments have described deficits in the PNS and CNS. However, in most instances the CNS remains largely intact in these embryos. Specifically, among neurotrophin and neurotrophin receptor knockout mice, the only groups that resulted in statistically significant decrease in CNS neurons were trkB deficient animals (Klein et al., 1993). In the trkB knockouts spinal cord motoneurons and facial motoneurons were reduced (Klein et al., 1993). The effects of the knockouts in the PNS are quite different. All single gene knockout studies have found reduced DRG neuron number (Conover et al., 1995; Crowley






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et al., 1994; Klein et al., 1994; Klein et al., 1993; Smeyne et al., 1994). These results support earlier studies that found DRGs to be sensitive to many NTFs and to express the receptors for multiple neurotrophins (Buchman and Davies, 1993). NGF knockout mice do not survive long postnatally (Conover and Yancopoulos, 1997), and there are reduced numbers of superior cervical ganglion, trigeminal ganglion, and DRG neurons (Crowley et al., 1994). TrkA knockout mice show the same pattern of neuronal loss that the NGF knockout mice have and exhibit high mortality (Smeyne et al., 1994). BDNF knockout mice die soon after birth and have decreased numbers of trigeminal ganglion, geniculate ganglion, nodose-petrosal ganglion, vestibular ganglion, spiral ganglion, and DRG neurons (Conover et al., 1995; Conover and Yancopoulos, 1997). NT-4/5 knockout mice are similar to BDNF knockouts and have reduced geniculate ganglion, nodose-petrosal ganglion, vestibular ganglion, and DRG neurons but do not die early in postnatal life (Conover et al., 1995; Conover and Yancopoulos, 1997). TrkB knockouts exhibit high mortality and have the CNS differences which were mentioned earlier and reduced numbers of trigeminal ganglion, nodose-petrosal ganglion, and DRG neurons (Klein et al., 1993). NT-3 knockout mice expire early in postnatal development display fewer superior cervical ganglion, trigeminal ganglion, nodose-petrosal ganglion, vestibular ganglion, spiral ganglion, and DRG neurons (Conover and Yancopoulos, 1997; Ernfors et al., 1994). TrkC knockout mice have a high mortality rate, do not survive for a very long period of time and exhibit reduced numbers of DRG neurons (Klein et al., 1994). While genetargeting studies are powerful tools for inferring the actions of NTFs and their receptors in the nervous system, these studies are not without their difficulties. Changes in the relative expression of other genes and changes in the genetic background can have a large effect on the development of the knockout animal (Gerlai, 1997). It would be difficult to determine whether the observed changes in the organism were due to loss of the gene of interest or to a change in genetic background. Therefore, knockout studies must be interpreted with these difficulties in mind.






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Important for the present study is the fact that gross neurotrophic responsiveness and activity in the septohippocampal system are changed following prenatal exposure to ethanol. Cultures of hippocampal neurons derived from rats prenatally exposed to ethanol do not respond to basic fibroblast growth factor (bFGF) as well as control cultures (Heaton et al., 1995b). That is, these cultures do not extend neurites as the control cultures do in response to bFGF. Specifically, bFGF does not promote neurite outgrowth in hippocampal cultures derived from ethanol exposed animals to the extent that it does in cultures derived from control animals. This result suggests that NTF receptor expression may be decreased in response to prenatal ethanol exposure. Following chronic prenatal ethanol treatment (CPET) in the rat, neurotrophic activity--which includes both neurotrophin and other NTF activity--is increased in extracts made from the hippocampus on P21 and cultured on DRG neurons (Heaton et al., 1995c). The increase in neurotrophic activity is specific to this region and age of the rat, and suggests an increase in NTF expression as a result of prenatal ethanol exposure. No single NTF is implicated by this study since DRGs respond to a variety of NTFs in vitro. Postnatal ethanol exposure--in contrast to prenatal exposure--produces a reduction in neurotrophic activity of P21 hippocampal extracts (Moore et al., 1996). Taken together, all of these results suggest a role for both the NTF and its receptor in ethanol toxicity. The present study focuses on the neurotrophin family of NTFs and their receptors because these proteins are expressed at their highest levels in the hippocampus and because they have been implicated as important factors for normal septal and hippocampal functioning (Maisonpierre et al., 1990). The neurotrophins are not the only NTFs produced by the hippocampus. Other factors that the hippocampus is responsive to--such as bFGF (Walicke, 1988)--are synthesized there and could affect hippocampal neurons (Ernfors et al., 1990; Riva and Mocchetti, 1991).

The objective of the present study was to determine whether CPET and early postnatal ethanol treatment (EPET) alter the gene expression of neurotrophins in the hippocampus of treated rat pups. Thus, this portion of the study relates to the overall






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scheme of this doctoral research by determining how ethanol affects NTF and NTF receptor gene expression in vivo. In order to specifically determine whether BDNF, NT-3, trkB, and trkC were affected by CPET and EPET, Northern blots were constructed from the hippocampi of treated P21 rats. This age was chosen because previous studies found a change in gross neurotrophic activity following both prenatal and postnatal ethanol exposure that was limited to P21 (Heaton et al., 1995c; Moore et al., 1996). TrkA is expressed at very low levels in the hippocampus and was therefore not examined in this study (Martin-Zanca et al., 1990). While NGF expression is above the threshold of detection for Northern blotting at this age (Maisonpierre et al., 1990), we were unable to examine its expression as the resulting bands on our blots were not quantifiable. Repeated attempts at probing failed to produce usable data. This age (P21) was chosen for the analysis because it coincided with the age of the animals that displayed the alteration of neurotrophic activity following prenatal and postnatal ethanol exposure (Heaton et al., 1995c; Moore et al., 1996). Relative expression of these genes was compared between control and ethanol-exposed animals. Following CPET, male animals exhibited reduced gene expression of trkC while female animals exhibited no significant differences. There were also no significant differences in gene expression in female CPET animals or following EPET.

Materials and Methods

Prenatal Ethanol Exposure

Long-Evans hooded rats originally obtained from Charles River Company were used to establish a breeding colony. Animals were housed individually in plastic cages under controlled temperature and humidity conditions. Nulliparous females were placed in a cage with an experienced male. On the following morning pregnancy was determined by the presence of sperm following vaginal lavage. Animals were placed on one of three diets: Chow, Ethanol, or Sucrose (n=24 for each group). The Chow group was given access to Purina Rat Chow and water ad libitum. The Ethanol group was given free access to an






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ethanol-containing liquid diet in which ethanol comprised 36% of the total caloric intake (ethanol concentration = 8.4% v/v). The Sucrose group was pair-fed the same volume of liquid diet with an isocaloric substitution of sucrose for ethanol. The liquid diet was made from a commercial formula, Sustacal (Mead Johnson), which was supplemented with Vitamin Diet Fortification Mixture (3.0 g/liter) and Salt Mixture (5.0 g/liter; both from ICN Nutritional Biochemicals). The liquid diets contained 1.3 kcal/ml and provided several times the daily requirements of all essential vitamins and nutrients. The additional fortification ensured proper nutritional intake, so that any results obtained from the ethanoltreated animals could be directly attributed to ethanol per se, and not to possible nutritional deficiencies. Ethanol and Sucrose pups were fostered to Chow dams on the day of birth to remove any possible effect the diet might have on the ability of the dam to properly rear the pups. Chow pups were left with their birth mother. Previous experiments in this laboratory have shown that morning blood alcohol levels of the pregnant dams range from 112 mg/dl to 254 mg/dl (Swanson et al., 1995). Postnatal Ethanol Exposure by Inhalation

Pregnant Long-Evans hooded rats obtained from Charles River Company were used for this portion of the study. Dams were fed standard lab chow throughout the experiment, ad libitum. Pups from these litters were placed into one of three groups: Ethanol, control Separated, and control Unseparated (n=24 for each group; these groups will be referred to as Ethanol, Separated, and Unseparated, respectively, for the remainder of this chapter). Ethanol pups never came from a litter that contained another group; however, Separated and Unseparated litters were split so that one dam nursed equal numbers of pups from each group. Ethanol litters were culled to 7 pups on postnatal day

(P4), while Separated/Unseparated litters were culled to 10 pups (Ryabinin et al., 1995). The reduced number of pups in the Ethanol group was done to help eliminate nutritional differences between ethanol-exposed and control pups. Ethanol inhalation occurred from P4 through PlO. Pups were placed in the inhalation chamber on a heating pad at 37'C and






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were allowed to breathe ethanol vapor in a chamber for 2 hours daily. The inhalation chamber consisted of an airtight 10-gallon aquarium fitted with an intake and out-take hose. The intake hose received air flowed into a 1 L Erlenmeyer vacuum flask containing 520 ml 95% ethanol (Aaper) from an aquarium air pump set to pump air at approximately 0.8-1 L/min. Ethanol used in this study, and all studies in this project, was not treated with benzene or any other chemical known to exert detrimental effects upon the nervous system. As the air was forced into the flask it passed through a 1.5 inch air stone submerged in the ethanol. The ethanol-laden vapor was then carried to the chamber. The out-take hose led ethanol vapor from the chamber to a fume hood. Separated pups were placed for 2 hours daily in a similar chamber with the difference being that air was pumped directly into the chamber from the air pump without encountering ethanol. Unseparated animals remained with the nursing dam while the Ethanol and Separated pups were placed in their respective chambers. This paradigm of ethanol exposure resulted in peak blood ethanol counts of approximately 250 mg/dl. Ethanol pups were clearly intoxicated upon removal from the chamber and remained incapacitated, and unable to nurse, for a period of approximately two hours following exposure. In contrast, Separated pups began nursing immediately upon their return to the home cage.

Morphometric Measurements

Prior to sacrifice, all animals were weighed and had crown-rump length

measurements taken. Crown-rump length was defined as the distance from the crown of the skull--defined to be the point directly between the ears--to the base of the tail. The brain of each animal was weighed before the hippocampus was dissected. These measures were taken to provide an estimation of the overall effect that ethanol treatment had on the subjects.

Dis sections

Rats were anesthetized with methoxyflurane (Pittman-Moore) and sacrificed by decapitation on P21. After the brain was removed from the skull, the hippocampus was






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dissected out, wrapped in aluminum foil, and flash-frozen in liquid nitrogen. Endogenous ribonucleases present in the tissue rapidly destroy mRNA present in the brain once a dissection starts. Therefore, mRNA (which happens to be the molecule of interest in the present study) is in danger of being lost if the dissection is not completed with considerable speed. Because of the rapidity with which dissected tissue had to processed and because of the considerable time required to obtain tissue weights, individual weights of the hippocampi were not taken. The tissue was then stored at -70C until RNA was extracted. RNA Extraction

Polyadenylated (poly-A) messenger RNA (mRNA) was extracted from frozen

tissue specimens via the Micro-fastTrack kit (Invitrogen). Poly-A mRNA was stored as a precipitate in 75% ethanol at -70C until the samples were run on an electrophoresis gel. Northern Blots

The procedures used in this study are as previously described (Baek et al., 1994; MacLennan et al., 1994; MacLennan et al., 1995). The amount of mRNA in each sample was assessed by taking the optical density of 1 l of sample in 500 ul dH2O at 260 nm and 280 nm of UV light. The samples were then loaded onto a 1.25% agarose formaldehyde denaturing gel so that each lane contained about 15 pg of poly-A mRNA. To obtain mRNA levels of this magnitude, four animals were used for each lane in each gel. The products were separated using horizontal gel electrophoresis running at 100 V for 30 minutes and then turned down to 25 V and allowed to run overnight. The poly-A mRNA was then transferred to a nylon membrane (ICN). The membrane was then baked in a vacuum oven at 80C for two hours and stored desiccated at room temperature until probed. Northern blots were probed with a cDNA strand encoding one of two neurotrophins (NT-3 or BDNF), one of two neurotrophin receptors (trkB or trkC), or cyclophilin. Cyclophilin mRNA is constituitively expressed and was used to standardize each lane. Previous research has shown that cyclophilin gene expression is not affected by developmental ethanol treatment (Maier et al., 1996). The cDNA strands were labeled with 32p (dCTP






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from Amersham) by random hexamer priming. The BDNF, NT-3, and trkB cDNA probes were a generous gift from Drs. P. Isackson, J.G. Sutcliffe, and S. Whittemore to Drs. Don Walker and A. John MacLennan of this department. The trkC cDNA probe was a generous gift from Dr. Louis Parada to this laboratory. Before starting the entire hybridization procedure, the blots were prewashed for 60 minutes at room temperature in 2X SSC. The blots were prehybridized at 42C for approximately 24 hours in a solution containing 50% formamide, 5X SSC, 5X Denhardt's, 0.5% SDS, 0.05M sodium phosphate, 0.25 mg/ml salmon sperm DNA, and 0.1 mg/mi poly-A. Hybridization was carried out at 42C for approximately 20 hours in the same solution described above with the 32P labeled cDNAs added to the solution. Following hybridization the blots were washed three times in 2X SSC at room temperature for periods of one minute, 30 minutes, and 30 minutes, respectively. The blots were then washed twice at 58C in a solution containing 0. IX SSC and 0.5% SDS for 30 minutes. After this final wash the Northerns were wrapped in plastic wrap and lightly taped to a Molecular Dynamics phosphorimaging cassette for at least 24 hours and analyzed by using the ImageQuant program which computes the density of each band electronically. The exposure time was dependent on the relative expression of the gene being probed. The phosphorimaging cassette is lOX more sensitive to radioactive particles than the x-ray film, but records ambient radiation. The ImageQuant program allows background readings to be subtracted so that radiation from the probe itself can be analyzed. The resulting bands were normalized by dividing each value by the corresponding cyclophilin value.

Stripping and Reprobing

In order to probe the blots for different neurotrophic agents and remove any remaining "P from previous hybridizations, the blots were stripped. The blots were exposed to a solution containing 50% formamide and 0.01M sodium phosphate at 65C for 60 minutes. After stripping, the blots were rinsed in a solution containing 2X SSC and






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0. 1% SIDS for five minutes at room temperature. The blots were then probed as described in the previous section.

Statistical Analyses

Two-way analysis of variance was performed using SAS version 6.12 on a

Pentium computer. Variances were pooled for this analysis since testing revealed that they were not significantly different by gender. When applicable, individual differences between groups were tested using Fisher's protected least significant difference (PLSD) post-hoc analyses. Statistical significance was determined to be p<0.05. Additionally, the Bonferroni/Dunn correction was used to determine if individual differences elucidated by Fisher's PLSD were valid. Statistical significance following the Bonferroni/Dunn correction was p
Results

Morphometric Measurements

Morphometric measurements were obtained to assess the general effect that ethanol had on the development of the animals used in this study. Measurements of body weight, brain weight, and crown-rump length were collected from both prenatally and postnatally treated animals on P21. The postnatally exposed rats were weighed on a daily basis from P4 to PlO to assess their overall growth during the inhalation period. Each group in both the CPET and EPET studies contained 24 animals and sexes were kept separate during all analyses.

CPET

As mentioned above, measurements were taken from both male and female animals on P21. For male animals, analysis of variance found no significant effect due to treatment for body weight, brain weight, or crown-rump length. Female animals had a differing result. Analysis of variance did not find a significant effect due to treatment for body weight or crown-rump length. However, brain weight was significantly affected by






89


treatment (F=6.37, df=71, p<0.005). Post hoc testing reveled that Alcohol animals had significantly smaller brains than both Chow (p<0.005) and Sucrose (p<0.01) animals. Even though the brains were significantly smaller in female animals following CPET, the ratio of brain weight to body weight was not significantly different from the same ratio in control animals. This ratio was also not significantly changed in male animals exposed to ethanol prenatally. Figure 4-1 displays the brain weights obtained from this portion of the study. Since brain weight to body weight ratio was unaffected by CPET, the extent to which brain weight was affected by ethanol exposure is not clear. EPET

Analysis of variance found significant effects due to treatment in both male and female animals in this section of the study. In the interest of clarity, these results will be presented separately.

Male animals. Male animals displayed a significant effect of treatment for weight at P4 (F=7.05, df=71, p<0.005) and PS (F=4.06, df=71, p<0.05). At P4, post hoc testing revealed that Ethanol animals weighed significantly more than both Separated (p<0.05) and Unseparated (p<0.0005) animals. At P5, Ethanol animals were only significantly larger than Unseparated animals (p<0.01). It should be noted that it was not the intent of the researchers to select larger animals in one group over the other. A possible explanation for this difference may lie in the litter size of the relative groups. Since Ethanol groups were set to have 7 pups at the start of inhalation and the control groups (Separated and Unseparated) were combined in one litter, litters containing less than 10 pups were automatically put into the Ethanol group. When both litters contained more than 10 pups, the dam was randomly placed into either a control or Ethanol group. Therefore, the Ethanol groups most likely started, on average, with a smaller litter size. This would make each pup larger on average than pups born to larger litters.

Since there was a difference at the start of treatment, gross initial differences

between the groups were eliminated by analyzing weight gain from day to day. Analysis of






90

2




a














2



















Alcohol Sucrose Chow CONDITION
Figure 4-1. Brain weight at P21 of female and male animals following prenatal ethanol exposure. A. Female brain weight. B. Male brain weight. Measurements are means + SEM. a = Female Ethanol animals are significantly smaller in comparison to Sucrose (p<0.01) and Chow (p<0.005). Animals were fed Ethanol, Sucrose, and Chow diets during gestation as described in the Methods section of this chapter and fostered to chow dams at birth. n = 24 for each group.




Full Text
ASSESSMENT OF ETHANOL TOXICITY IN THE MOTOR SYSTEM OF THE CHICK
AND THE SEPTOHIPPOCAMPAL SYSTEM OF THE RAT AND THE
INVOLVEMENT OF NEUROTROPHIC FACTORS
By
DOUGLAS M. BRADLEY
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

ACKNOWLEDGMENTS
I would like to acknowledge the help of many people who made completion of this
dissertation and doctoral research possible. First and foremost, I would like to thank my
wife, Korey, who provided valuable mental support and editing and presentation advice
through the years. I also want to thank my advisor, Marieta Heaton, who provided the
means for completing this research and provided excellent guidance. My committee, Drs.
MacLennan, Shiverick, Streit, and Walker, really helped to make this research better. My
father, Dr. fid win L. Bradley, provided valuable advice on statistics. I want to thank my
family, for understanding the time required to complete this degree. I also want to thank
the National Science Foundation and NIAAA, for supporting me financially through
graduate school. The Department of Neuroscience provided an excellent facility for
conducting this research. I also want to thank all of the people in our laboratory who
helped in a variety of technical and supportive ways throughout the years. Blaine Moore,
has been a friend in addition to giving helpful advice on scientific matters. Francesca
Beaman, Steve Farnworth, Kara Kidd, Nancy MacLennan, David Melman, Jean Mitchell,
Micheál Paiva, and Leon Williams gave wonderful technical assistance. 1 also wish to
apologize to anyone who is unintentionally omitted from this list.
11

TABLE OF CONTENTS
page
ACKNOWLEDGMENTS ii
LIST OF TABLES v
LIST OF FIGURES vi
ABSTRACT ix
CHAPTERS
1 INTRODUCTION 1
Fetal Alcohol Syndrome Background 1
Chick Embryo 3
Motor System and Ethanol 5
Motor System and NTFs 5
Neuroprotection 7
Rat Model of FAS 9
Rat Septohippocampal System 10
Neurotrophic Factors 11
Gene Deletion Studies 12
NTF Ontogeny in the Hippocampus 14
Neurotrophins and Ethanol 15
Hypotheses 16
2 CHARACTERIZATION OF MOTONEURON SURVIVAL AND CELL
DEATH FOLLOWING ETHANOL EXPOSURE AND CURARE
ADMINISTRATION, AND AFTER THE PERIOD FOR
NATURALLY OCCURRING CELL DEATH 18
Summary 18
Introduction 18
Methods 21
Results 29
Discussion 38
3 CHARACTERIZATION OF MOTONEURON SURVIVAL FOLLOWING
ETHANOL EXPOSURE AND CONCURRENT TREATMENT
WITH EXOGENOUS GDNF OR BDNF IN THE EMBRYONIC
CHICK SPINAL CORD 49
Summary 49
Introduction 50
Materials and Methods 55
iii

Results 58
Discussion 67
4 CHARACTERIZATION OF THE NEUROTROPHIN AND
NEUROTROPHIN RECEPTOR GENE EXPRESSION IN THE
HIPPOCAMPUS FOLLOWING CHRONIC TREATMENT AND
EARLY POSTNATAL ETHANOL TREATMENT IN THE RAT 75
Summary 75
Introduction 76
Materials and Methods 83
Results 88
Discussion 133
5 CONCLUSIONS AND IMPLICATIONS 146
Animal Models 146
Methods 147
Hypotheses and Results 150
Conclusions 154
REFERENCES 158
BIOGRAPHICAL SKETCH 178
iv

LIST OF TAB LES
Table page
2-1. Cell size and spinal cord length 35
2-2. Neurotrophic activity of crude muscle extract 37
3-1. Motoneuron Size and Spinal Cord Length 59
v

LIST OF FIGURES
Figure page
2-1. Number of motoneurons in lumbar spinal cord at E12 following treatment 31
from E4 to Ell.
2-2. Photomicrographs of coronal sections from the midlumbar region of E12 34
spinal cords.
2-3. Number of motoneurons in lumbar spinal cord at E16 following ethanol 36
treatment from E10 to El5.
3-1. Number of motoneurons in the later motor column of the lumbar spinal cord 61
at E16.
3-2. Interaction between ethanol and neurotrophic factors 62
3-3. Photomicrographs of coronal sections from the midlumbar region of E16 64
spinal cords.
3-4. High magnification photomicrographs from the midlumbar section of E16 66
spinal cords.
4-1. Brain weight at P21 of female and male animals following prenatal ethanol 90
exposure.
4-2. Weight gain during postnatal ethanol exposure in male animals 92
4-3. Gross morphological measurements following EPET in male animals at P21 94
4-4. Brain weight and Brain weight to body weight ratio of EPET male animals 95
at P21.
4-5. Weight gain during postnatal ethanol exposure in female animals 96
4-6. Brain weight and brain weight to body weight ratio in EPET female animals 98
at P21.
4-7. Phosphorimaging view of BDNF Northern blots composed of the 100
hippocampal region from P21 rat brains exposed to ethanol prenatally.
4-8. Relative BDNF 4.4 kb transcript expression in rat hippocampus at P21 101
following prenatal exposure to ethanol.
4-9. Relative BDNF 1.7 kb transcript gene expression following prenatal 102
exposure in P21 rats.
vi

4-10. Phosphorimaging view of NT-3 Northern blots composed of the
hippocampal region from P21 rat brains exposed to ethanol prenatally.
104
4-11. Relative NT-3 gene expression following prenatal ethanol exposure in P21 105
rats.
4-12. Relative trkB active receptor gene expression following prenatal ethanol 106
exposure in P21 rats.
4-13. Relative trkB truncated transcript gene expression following prenatal 107
exposure in P21 rats.
4-14. Phosphorimaging view of trkC Northern blots composed of the 109
hippocampal region from P21 rat brains exposed to ethanol prenatally.
4-15. Relative trkC 14 kb transcript gene expression at P21 in rats exposed to 110
ethanol prenatally.
4-16. Relative trkC 4.7 kb truncated transcript gene expression following Ill
prenatal exposure in P21 rats.
4-17. Relative trkC 3.9 kb transcript gene expression following prenatal 112
exposure in P21 rats.
4-18. Phosphorimaging view of cyclophilin Northern blots composed of the 115
hippocampal region from P21 rat brains exposed to ethanol prenatally.
4-19. Phosphorimaging view of BDNF Northern blots from postnatally 118
exposed P21 rats.
4-20. Relative BDNF 4.4 kb transcript gene expression following postnatal 119
exposure in P21 rats.
4-21. Relative BDNF 1.7 kb transcript gene expression following postnatal 120
exposure in P21 rats.
4-22. Phosphorimaging view of NT-3 Northern blots from postnatally exposed 122
P21 rats.
4-23. Relative NT-3 1.5 kb gene expression following postnatal exposure in 123
P21 rats.
4-24. Relative trkB active receptor gene expression following postnatal 124
exposure in P21 rats.
4-25. Relative trkB truncated transcript gene expression following postnatal 125
exposure in P21 rats.
4-26. Phosphorimaging view of trkC Northern blots from postnatally exposed 127
P21 rats.
4-27. Relative trkC 14 kb transcript gene expression in P21 rats following 128
postnatal exposure
vii

4-28. Relative trkC 4.7 kb truncated transcript gene expression in P21 rats 129
following postnatal exposure.
4-29. Relative trkC 3.9 kb truncated transcript gene expressionin P21 rats 130
following postnatal exposure.
4-30. Phosphorimaging view of cyclophilin Northern blots from postnatally 132
exposed P21 rats.
viii

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
ASSESSMENT OF ETHANOL TOXICITY IN THE MOTOR SYSTEM OF THE CHICK
AND THE SEPTOHIPPOCAMPAL SYSTEM OF THE RAT AND THE
INVOLVEMENT OF NEUROTROPHIC FACTORS
By
Douglas M. Bradley
August 1998
Chairman: Douglas K. Anderson
Major Department: Neuroscience
The research described in this document was undertaken to further the
understanding of the toxic effects that ethanol exerts on the developing nervous system.
Fetal alcohol syndrome has been recognized as one of the leading environmentally-induced
causes of mental retardation in the western world and continues to be a problem despite
education and publicity concerning the dangers of ingesting ethanol-containing beverages
during pregnancy. The doctoral research described attempted to ascertain some new
properties of ethanol toxicity in the nervous system and to determine ways that these toxic
effects could be modulated in living animals. Previous research from other laboratories has
suggested that motoneurons of the spinal cord might be susceptible to ethanol’s toxic
effects. Our laboratory confirmed this finding by administering ethanol to developing chick
embryos from embryonic day 4 (E4) to El 1 and assessing the number of motoneurons
present in the lumbar spinal cord. Specifically, a reduction in the number of motoneurons
present in this population was observed. The present experiments found that embryonic
administration of ethanol from E10 to E15 also results in a loss of motoneurons. Further,
the neurotrophic activity of muscle from these animals is unchanged from that of control
animals. Neuromuscular junction blocking agents, which prevent naturally occurring cell
death of spinal cord motoneurons, have little effect in altering ethanol’s toxic effects.
Administration of glial cell line-derived neurotrophic factor acted to increase motoneuron
number following ethanol administration, but brain-derived neurotrophic factor did not.
IX

The hippocampus is an important structure of the brain thought to be involved with learning
and memory. In a mammalian model of fetal alcohol syndrome, the gene expression of
tyrosine receptor kinase C, a neurotrophic factor receptor in the brain, is reduced in the
hippocampus of 21-day-old male rats following prenatal ethanol exposure, but is
unchanged in the brain of female rats. Appropriate background for understanding this
research, as well as the implications of all of these results, is described in the resulting
chapters.
x

CHAPTER 1
BACKGROUND INFORMATION
Fetal Alcohol Syndrome Background
In 1973, Jones and Smith (1973) first described a series of morphological and
cognitive deficits in children and infants of alcoholic mothers which was later termed fetal
alcohol syndrome (FAS). Since that time, much evidence has been gathered regarding the
effects ethanol exerts in the developing nervous system (Barnes and Walker, 1981; Jones
and Smith, 1973; Miller, 1986; Streissguth et ah, 1991; West, 1986). FAS is diagnosed in
1-2 out of every 1000 live births in the United States and is characterized by low birth
weight, decreased memory and learning, hyperactivity, facial dysmorphia, and lowered IQ
(Abel, 1995; Jones and Smith, 1973; Streissguth et al., 1991). Human FAS patients have
been analyzed for neuropathology postmortem and this analysis has identified central
nervous system (CNS) abnormalities which include disorders of laminae of the cerebral
cortex, cerebellar abnormalities, a reduction of dendritic spines on cortical pyramidal cells,
hippocampal malformation, and microcephaly (Clarren et al., 1978; Ferrer and Galofre,
1987). Interpretation of these studies is complicated by the fact that most of these infants
had related cardiovascular problems (Clarren et al., 1978). Abnormalities in humans can
range from physical deficits that are easily distinguished (such as gross microencephaly) to
microscopic changes (such as dendritic anomalies in neurons that survived alcohol
exposure) that require finer analyses (Ferrer and Galofre, 1987). Motor dysfunction and
other behavioral deficits, such as an impairment in sensory and motor functions, are
associated with FAS (Streissguth et al., 1983). Additionally, children with FAS are
deficient in habituation to redundant stimuli (Church and Gerkin, 1988). What is important
1

o
to note is that as these patients have aged, the deficits have not lessened (Streissguth,
1993).
Specific neuronal populations known to be affected by ethanol in animal models
include the cerebellum (Cragg and Phillips, 1985; West, 1986), the septohippocampal
system (Barnes and Walker, 1981), cerebral cortex (Miller, 1986), the substantia nigra
(Shetty etak, 1993), chief sensory trigeminal nucleus (Miller and Muller, 1989), red
nucleus (Zajac et al, 1989), inferior olivary nucleus (Napper and West, 1995), striatum
(Heaton et al., 1996) and motoneurons of the spinal cord (Heaton and Bradley, 1995).
Microscopic and molecular changes that have also been observed in animal models
following ethanol exposure include decreased dendritic arborization (Davies and Smith,
1981), delayed synaptogenesis (Leonard, 1987), decreased neurotransmitter synthesis
(Rawat, 1977; Swanson et al., 1994), changes in connectivity (West et al., 1994), and cell
loss (Barnes and Walker, 1981; Bauer-Moffet and Altman, 1975; West et al., 1986).
These alterations following ethanol exposure in animals are important because they
correlate to deficits observed in human FAS. That is, the neuronal region affected seems to
relate to a specific deficiency common to human FAS patients.
The mechanisms of ethanol toxicity in the CNS are not fully understood. Since
ethanol can cross the blood brain barrier, it has the ability to directly affect the developing
nervous system (West et al., 1994). Ethanol can interact with cellular membranes and
proteins and reduce protein synthesis (Zajac and Abel, 1992). Additionally, ethanol has
been implicated in producing hypoglycemia (Snyder et al., 1992; West et al., 1994),
hypoxia (Mukherjee and Hodgen, 1982), and in increasing oxidative stress (Henderson et
al., 1995). All of the above information suggest possible explanations for the toxic effects
ethanol exerts on the developing nervous system. The current experiments were designed
to test facets of the relationship between neurotrophic factors (NTFs) and ethanol.
Important to these studies is the underlying hypothesis that NTFs are involved in FAS
neuropathology. The NTF hypothesis for FAS proposes that ethanol exposure results in

3
alterations in the synthesis, availability, delivery, and /or biological activity of normally
occurring neurotrophic substances. Further, ethanol may alter the capacity of target
neuronal populations to respond to NTFs in a normal fashion. Another important aspect of
the NTF hypothesis is the idea that exogenous NTFs may afford some protection to
ethanol-susceptible neuronal populations. The current studies sought to determine whether
NTF synthesis, availability, and biological activity were affected by ethanol treatment and
whether the addition of exogenous factors in vivo could prevent ethanol toxicity in a
population known to be vulnerable to ethanol insult. To adequately examine these goals,
two animal models were used: the chick embryo and the developing rat. Each model has
specific advantages that make it attractive for FAS research and each will be discussed in
more detail below. All of the studies detailed in this document are related in that the
examine ethanol toxicity as it relates to NTFs. Whether this relationship is in the effect that
ethanol has on NTFs or neurotrophic support, or the effect that exogenous NTFs have on
ethanol toxicity, the objective is consistent: to understand the manner in which these two
types of molecules are related in producing deficits observed in animal models of FAS.
Chick Embryo
Ethanol affects chick embryo development in a manner similar to mammals. Chicks
exposed to ethanol prenatally have been shown to exhibit reduced brain size, brain weight,
DNA and protein synthesis (Pennington and Kalmus, 1987), and reduced neurotransmitter
synthesis (Brodie and Vernadakis, 1990; Swanson et al., 1994). Neuronal populations
affected by ethanol exposure in ovo in chick include the cerebellum (Quesada et al., 1990),
cerebral cortex (Delphia et al., 1978), and motoneurons of the spinal cord (Heaton and
Bradley, 1995). The cerebellum (Marcussen et al., 1994; Smith and Davies, 1990) and
cerebral cortex (Miller, 1986) in mammals are also affected by developmental ethanol
exposure. While chick motoneurons are susceptible to the toxic effects of ethanol both in
culture (Dow and Riopelle, 1985; Heaton and Bradley, 1995) and in vivo (Heaton and
Bradley, 1995), they have not been specifically quantified in mammals exposed to ethanol.

4
Some evidence does suggest that there are neuromuscular problems associated with human
FAS. Specifically, children exposed to ethanol exhibit motor deficits (Streissguth et al.,
1983). More important to the current studies is the fact that ethanol can reduce motoneuron
number when administered to chick embryos from embryonic day 4 (E4) to El 1 (Heaton
and Bradley, 1995). We have hypothesized that this reduction may be dependent on
naturally occurring cell death (NOCD) since this period (approximately E6-E9) occurred
during the period of ethanol exposure utilized in that study (Pittman and Oppenheim,
1978). During the period of NOCD, nearly half of the original number of motoneurons
perish. Curare, as well as other neuromuscular junction blocking agents, have been shown
to suspend NOCD in motoneurons presumably by increasing the number of synapses and
thereby increasing the availability of target-derived NTFs at the neuromuscular junction
(Oppenheim, 1991; Pittman and Oppenheim, 1978). In these experiments, approximately
50% more motoneurons survive in curare-treated embryos than in control embryos.
The chick has several advantages that make it a good choice as a model for FAS.
Ethanol can be administered in exact doses to the developing embryo, and only the
embryo’s liver can remove the ethanol from the bloodstream. However, alcohol
dehydrogenase does not begin in the developing chick until around E8 (Wilson et al.,
1984). Maternal influences are removed when using the chick embryo. Ethanol is cleared
from the bloodstream by the mother in a mammalian system whereas the chick embryo is
isolated as it develops. This model allows the investigator to observe direct effects of
ethanol without interactions of maternal metabolism interfering. While chick development
is different from mammalian gestation, this model allows researchers to study in vivo
interactions in a developing organism that are not possible in a mammalian model. Another
advantage for the research described in this document is that the chick embryo model has
been used to study the ability of brain-derived neurotrophic factor (BDNF) and glial cell
line-derived neurotrophic factor (GDNF) to regulate NOCD in motoneurons of the chick
embryo spinal cord (Oppenheim et al., 1995; Oppenheim et al., 1992). It is important to

5
note that these experiments would be impossible to perform in mammals since exogenous
NTFs can not be administered individually to developing fetuses. Oppenheim’s laboratory
has performed experiments using a variety of NTFs to study their effects on NOCD. In
these experiments, NTFs are applied directly to the membranes of the developing embryo
through windows in the outer egg shell. Replicating these experiments in mammals would
require extensive surgical procedures that would undoubtedly have an adverse effect on
fetal and maternal survival. Some of the present experiments utilize the chick embryo for
all of the advantages described above.
Motor System and Ethanol
There is direct evidence that warrants further investigation into the possible effects
that ethanol may exert on motoneurons. In both the human and in animal models, previous
studies have determined that ethanol damages developing muscle (Adickes and Shuman,
1983; Nyquist-Battie et al., 1987). In the human, these cases described flaccid, hypotonic
neonates which exhibited major muscle structural deficiencies including hypotrophy,
dominance of type II fibers, and sarcomeric dysplasia (Adickes and Shuman, 1983).
Prenatal exposure to ethanol in the guinea pig resulted in structural malformations of the
gastrocnemius muscle including vacuolated sarcoplasmic reticula, enlarged lipid droplets,
decreased glycogen, and mitochondrial abnormalities (Nyquist-Battie et al., 1987). Proper
muscle fiber maturation is dependent on concurrent development and innervation by
motoneurons. Therefore, the possibility exists that deficiencies noted above are due to an
underlying effect that ethanol exerts on developing motoneurons (Ishiura et al., 1981). It is
equally likely that ethanol may exert some direct effect upon developing muscle.
Accordingly, a limited analysis of muscle neurotrophic activity in chick leg muscle is
undertaken in the present studies.
Motor System and NTFs
The developing motor system is dependent on many different NTFs for proper
growth. Two of the NTFs that developing motoneurons encounter are BDNF and GDNF.

6
BDNF is a member of the neurotrophin family of NTFs which includes nerve growth
factor (NGF), neurotrophin-3 (NT-3), and NT-4/5. BDNF is a 118 amino acid residue
polypeptide (Ilag et al., 1994) that forms homodimers to attain its active form and binds
with high affinity to tyrosine receptor kinase B (trkB; Klein et al., 1991). Previous
research has identified two major pathways that arc initiated by autophosphorylation of trk
(Stephens et al., 1994; Tolkovsky, 1997). One of these pathways leads to activation of the
MAP kinase cascade and may initiate neurite outgrowth, transcription, or cellular
hypertrophy (Stephens et al., 1994). The other pathway leads to the activation of akt (a
serine/threonine kinase) and may initiate neurite outgrowth, survival, and receptor
internalization (Tolkovsky, 1997). Developing skeletal muscle produces BDNF, which is
known to support motoneuron survival during development by suspending NOCD in a
subset of the developing motoneuron pool and to protect motoneurons of both the chick
and rat from degenerating after lesion (Oppenheim et al., 1992; Sendtner et al., 1992; Yan
et al., 1992). It is important to not that the addition of recombinant factors such as BDNF
does not rescue all motoneurons in the developing motor column. BDNF is also expressed
by the hippocampus, adrenal gland, and whole brain during rat development (Maisonpierre
et al., 1990).
The other NTF used in the present studies of chick development, GDNF, is a
member of the transforming growth factor 6 superfamily and naturally occurs as a
homodimer with a molecular weight of 40-45 kD (each molecule 134 amino acid residues;
(Lin et al., 1993). GDNF and its receptors, GDNFR<* and c-ret, form a complex that
allows c-ret to transduce intracellular signals from GDNF (Jing et al., 1996; Treanor et al.,
1996). Poor to binding with c-ret, GDNFR^ acts as a ligand-binding protein by binding
GDNF (Jing et al., 1996). The GDNFRcc/GDNF complex then forms a complex with c-
ret—the only molecule of the complex capable of producing intracellular signals (Jing et al.,
1996; Rosenthal, 1997; Treanor et al., 1996). The signal transduction pathways initiated
by c-ret activation include the MAP kinase pathway (Worby et al., 1996) and the

7
Ras/ERK2 pathway (van Weering and Bos, 1997). MAP kinase and ERKs are proteins
known to be involved in gene expression (Hazzalin et al., 1997; Mucsi et al, 1996).
During rat development, GDNF mRNA is expressed by mesenchymal cells and in
developing skeletal muscle beginning at E15, and in developing skin beginning at E17
(Nosrat et al., 1996; Trupp et al., 1995; Wright and Snider, 1996). Peripherally in the rat,
GDNF is expressed in the teeth, tongue, retina, nasal cavity, ear, kidney, and
gastrointestinal tract during various stages of development (Nosrat et al., 1996). Centrally,
GDNF is expressed in the striatum, hippocampus, and cerebellum beginning at E15, and in
the trigeminal motor nucleus (E17) and cortex (postnatal day 7). Generally, populations
that are responsive to GDNF express c-ret. These populations include substantia nigra
dopaminergic neurons (Trupp et al., 1995), spinal motoneurons (Pachnis et al., 1993;
Tsuzuki et ah, 1995), and certain subpopulations of the peripheral ganglia (Pachnis et al.,
1993; Tsuzuki et al., 1995). A small segment of Purkinje neurons does exhibit sensitivity
to GDNF early in development before expression of c-ret commences (Nosrat et al., 1997),
thus implying that GDNF might have the ability to signal through a receptor other than c-
ret. During chick embryogenesis, c-ret mRNA is expressed in the Wolffian duct and
ureteric bud, the enteric, dorsal root, sympathetic and facioacoustic ganglia, and the ventral
spinal cord (Schuchardt et al., 1995).
Neuroprotection
NTFs have been shown to protect against insults such as hypoxia, hypoglycemia,
and changes in calcium homeostasis. Examples of neuroprotection by polypeptide growth
factors include epidemial growth factor protection of whole brain neuronal cultures from
anoxia (Pauwels et al., 1989), NGF protection of rat hippocampal and human cortical
neurons from hypoglycemia (Cheng and Mattson, 1991), and bFGF prevention of thalamic
degeneration following cortical infarction (Yamada et al., 1991). GDNF is particularly
potent in protecting neurons from a variety of conditions that normally result in death.
Such insults and environmentally-produced conditions include NOCD (Oppenheim et al.,

8
1995), 6-OHDA lesion (Choi-Lundberg et al., 1997; Kearns and Gash, 1995; Tomac et
al., 1995), and axotomy (Giménez y Ribotta et al., 1997; Houenou et al., 1996;
Oppenheim et al., 1995). While NOCD is not an insult in the sense that 6-OHDA lesion is,
it does result in neuronal death and NTFs such as GDNF do prevent it from proceeding in a
subset of the developing motor pool in live animals. Like GDNF, BDNF is effective in
providing neuroprotection from events that normally result in neuronal death. However,
different neuronal populations are protected by BDNF. For example, BDNF protects
against ischemia-induced cell death in rat hippocampal slice cultures (Pringle et al., 1996),
and prevents NOCD in some motoneurons (Oppenheim et al., 1992) and apoptotic death in
PC12 cells (Jian et al., 1996) and cultured rat cerebellar granule neurons (Kubo et al.,
1995). The fact that both GDNF and BDNF provide such potent support for developing
and injured neurons suggests that both could protect motoneurons against toxic events
produced by ethanol.
Neuroprotection from ethanol has been studied previously, but mostly in culture.
Examples of this phenomenon include NGF protection of cultured dorsal root ganglion
(DRG) neurons (Heaton et al., 1993) and septal neurons (Heaton et al., 1994) and basic
fibroblast growth factor (bFGF) protection of cultured septal and hippocampal neurons
(Heaton et al., 1994). Additionally, both NGF and bFGF protect cultured cerebellar
granule cells from ethanol-induced cell death (Luo et al., 1997). Neuroprotection afforded
by NGF and bFGF was found to require both protein and RNA synthesis which suggests
that neuroprotection is related to a signal that the NTF receptor sends to the nucleus of the
cell (Luo et al., 1997). GDNF has been shown to protect rat organotypic cultures of
cerebellar Purkinje cells from ethanol neurotoxicity (McAlhany et al., 1997). To date, the
only in vivo demonstration of NTF neuroprotection from ethanol toxicity is protection of
choline acetyltransferase activity by NGF (Brodie et al., 1991). The reason that NTF
neuroprotection is important for the study of FAS is that many of the toxic events that are
prevented by NTFs in culture are implicated as potential mechanisms for ethanol toxicity in

9
the nervous system. Mechanisms for ethanol toxicity that are suggested by previous
research include hypoxia, hypoglycemia, and changes in calcium homeostasis (Alturaet
al., 1983; Snyder et al., 1992; Webb et al., 1995). If these insults are indeed responsible
for ethanol’s toxic effects, NTFs might protect the developing nervous system from
damage.
Rat Model of FAS
The rat is the most widely used model in FAS research. However, a caveat of
using the rat as a model is the relative gestational period. Rat prenatal development is
approximately equivalent to the first two trimesters of human development (Goodlett et al.,
1993). A major event of prenatal development in humans is the brain growth spurt (BGS),
when many functional synapses are made in the nervous system. In rats, this event occurs
postnatally from P4-P10 (West, 1987). Therefore, experiments that wish to mimic third
trimester ethanol exposure in humans must incorporate the BGS. This objective requires
postnatal ethanol exposure in rats. Exposure to ethanol during the BGS produces deficits
that demonstrate the sensitivity of the CNS to ethanol during this period. Postnatal
exposure in the rat produces deficits that are different from those seen following prenatal
exposure. For example postnatal ethanol exposure can produce loss of cerebellar Purkinje
cells (Bonthius and West, 1990; Bonthius and West, 1991; Goodlett and West, 1992;
West, 1986; West et al., 1990). Since postnatal ethanol exposure damages neuronal
populations known to be damaged in FAS, it does serve as a model for FAS.
A caution of any postnatal exposure paradigm is that maternal metabolism of
ethanol is removed and the subjects are exposed to ethanol in the same manner as adults
(i.e., only the neonatal liver removes ethanol from the bloodstream). Alcohol
dehydrogenase (ADH) activity begins in the rat on approximately gestational day 15
(Boleda et al., 1992; Tietjen et al., 1994). Fetal ADH has very low activity in comparison
to adult ADH, which suggests that metabolism of ethanol during pregnancy is completed
almost entirely by maternal ADH. Between P20 and P39 all subclasses of ADH reach

10
100% of adult activity (Boleda et al., 1992). Another difficulty is that suckling rats will not
readily consume ethanol because their entire diet consists of mother’s milk.
Two typical methods for ethanol delivery to newborn rats are artificial rearing (AR)
and vapor inhalation, both of which have advantages and disadvantages. AR is a surgical
procedure which consists of fitting a neonatal pup with a gastric fistula and tube,
maintaining the pups in cups placed in a heated water bath, and feeding the pup an artificial
milk solution via the tube and fistula. The advantage of the AR method is that it provides
constant nutrition and produces no damage to the mucous membranes of the subject. Major
disadvantages of AR are that interaction between mother and pup is removed and that it can
be stressful for the neonatal rat. The stress induced by AR produces gliosis in rat cortex
(Ryabinin et al., 1995), although gliosis following postnatal exposure via intragastric
intubation was observed (Goodlett et al., 1997). In this latter experiment, gliosis was not
observed in control animals (Goodlett et al., 1997). The fact that the AR procedure per se
may produce changes in brain structure indicates that results obtained using AR could be
difficult to interpret. The ethanol vapor inhalation procedure consists of placing neonatal
rats in a sealed chamber that contains circulating air and ethanol vapor. This procedure has
been theorized to damage the mucous membranes of the lungs which could interfere with
oxygen exchange and general metabolism (Ryabinin et al., 1995). However, no evidence
supports this contention and lung damage has not been observed in rats exposed to ethanol
vapors (Bauer-Moffet and Altman, 1975). Other methods for delivering ethanol to neonatal
rats include delivery of ethanol through mother’s milk by limiting the dam’s liquid intake to
ethanol-containing fluids, and exposing pups to ethanol vapor concurrently with the dam.
Rat Septohippocampal System
Many neuronal populations exhibit some susceptibility to the toxic effects of
ethanol. As was mentioned above, a variety of neuronal types from brain regions such as
the cerebellum (Cragg and Phillips, 1985), the septohippocampal system (Barnes and
Walker, 1981; West and Pierce, 1986), the cerebral cortex (Miller, 1986), and the

11
oculomotor nucleus (Burrows et al., 1995) are known to be affected in some way by
ethanol exposure. The hippocampus is an important structure with regard to memory and
learning in humans and animals (Bunsey and Eichenbaum, 1996; Cohen and Squire,
1980). Therefore, damage to the hippocampus observed in the rat following ethanol
exposure may correspond to similar damage to the hippocampus in humans. Since learning
and memory deficits are a common characteristic of FAS (Abel, 1995; Jones and Smith,
1973; Streissguth et al., 1991), it is not surprising to find that the hippocampus is sensitive
to ethanol and exhibits reduced cell number following ethanol exposure (Barnes and
Walker, 1981).
Neurotrophic Factors
The neurotrophin family of NTFs plays a valuable role in the development of the
nervous system through regulation of neuronal differentiation and survival, and
maintenance of basic cellular processes. The neurotrophin family, as noted above, includes
NGF (Levi-Montalcini, 1951), BDNF (Leibrock et al., 1989), NT-3 (Maisonpierre et al.,
1990), NT-4/5 (Berkemeier et al., 1991; Ip et al., 1992), and neurotrophin-6 (Gotz et al.,
1994). The trk family of receptors has been shown to be the high-affinity receptors for the
neurotrophins (Martin-Zanca et al., 1990). Trks that bind neurotrophins include trkA
(Kaplan et al., 1991; Kaplan et al., 1991), trkB (Klein et al., 1990), and trkC (Cordon-
Cardo et al., 1991). TrkA is the preferred receptor for NGF, but will bind both BDNF and
NT-3. TrkB is the preferred receptor for BDNF and NT-4/5, but will bind NT-3. TrkC is
the preferred receptor for NT-3. The neurotrophins regulate a number of peptides in the rat
septohippocampal system, including other neurotrophins (Croll et al., 1994). For
example, NGF, BDNF, and NT-3 induce choline acetyl transferase activity (Alderson et al.,
1990; Auberger et al., 1987; Gnahn et al., 1983; Nonner et al., 1996); BDNF increases
NT-3 activity (Lindholm et al., 1994); and BDNF and NT-3 enhance synaptic transmission
in Shaffer collateral-CA 1 hippocampal synapses (Kang and Schuman, 1995). All of these
results indicate the importance of the neurotrophins in this brain region.

12
Gene Deletion Studies
Studies of gene-deleted “knockout” mice also suggest the importance of
neurotrophins and other NTFs in proper nervous system development. Knockout studies
consist of deleting a gene from the mouse genome by homologous recombination (Smithies
et al., 1985). Following this procedure, the mice are allowed to develop and are
subsequently compared to control mice. All neurotrophin and neurotrophin receptor
knockout animals die relatively early in development except for NT-4/5 animals (Conover
and Yancopoulos, 1997). Specifically, NGF knockout mice exhibit reduced numbers of
superior cervical ganglion, trigeminal ganglion, and DRG neurons (Crowley et al., 1994).
BDNF knockout mice have decreased numbers of trigeminal ganglion, geniculate ganglion,
nodose-petrosal ganglion, vestibular ganglion, spiral ganglion, and DRG neurons
(Conover et al., 1995; Ernfors et al., 1994). NT-3 knockout mice display fewer superior
cervical ganglion, trigeminal ganglion, nodose-petrosal ganglion, vestibular ganglion,
spiral ganglion, DRG neurons, and spinal cord motoneurons (Ernfors et al., 1995; Kucera
et al., 1995). NT-4 knockout mice are similar to BDNF knockouts and have reduced
geniculate ganglion, nodose-petrosal ganglion, vestibular ganglion, and DRG neurons
(Conover et al., 1995).
Knockouts of receptors of neurotrophins also support the idea that they are
important in proper nervous system development. TrkA knockout mice show the same
pattern of neuronal loss that the NGF knockout mice have (Smeyne et al., 1994). TrkB
knockouts have reduced numbers of spinal cord and facial motoneurons, and exhibit
reduced numbers of trigeminal ganglion, nodose-petrosal ganglion, and DRG neurons
(Klein et al., 1993). This result is especially significant in light of the fact that BDNF
knockout mice did not exhibit motoneuron deficits. This discrepancy between the two
knockout studies suggests that trkB is important for motoneuron development and some
other molecule, perhaps NT-3, can bind trkB to promote survival in the absence of BDNF.
TrkC knockout mice exhibit reduced numbers of DRG neurons, and completely lack la

13
muscle afferents (Klein et al., 1994). All single gene neurotrophin knockout studies
observed reduced DRG number. These results support earlier studies that found DRGs
respond to many different NTFs and express the receptors for multiple neurotrophins
(Buchman and Davies, 1993).
Knockout mice have also been used to study GDNF and c-ret. GDNF-deficient
animals exhibit deficits in DRG, sympathetic and nodose neurons, but not in hindbrain
noradrenergic or midbrain dopaminergic neurons and completely lack the enteric nervous
system, ureters, and kidneys. These animals did display a small yet significant (~20%)
loss of spinal cord motoneurons (Moore et al., 1996). C-ret knockout mice do not contain
reduced numbers of motoneurons, but do lack enteric nervous system and contain reduced
numbers of parasympathetic neurons (Marcos and Pachnis, 1996). These studies
demonstrate that NTFs other than the neurotrophins are important to the normal
development of the CNS.
While gene-targeting studies have provided invaluable information regarding the
action of NTFs and their receptors in the nervous system, they must be interpreted with
certain difficulties in mind. Knockout mice are not merely normal animals with one gene
conveniently deleted. These organisms possess a number of developmental, physiological,
and even behavioral processes that have been altered to compensate for the missing gene
(Gerlai, 1997). For example, when one gene is eliminated from an organism, the
transcription of other gene products may be altered. This change in transcription could
conceivably ameliorate or exacerbate the effects of losing the gene. The many redundancies
present in the genome may mask the effects of losing a single gene. Therefore, it would be
difficult to determine whether the observed changes in the organism were due to loss of the
gene of interest or to the change in genetic background.
The knockout studies relate to the present experiments in that they provide a clue of
what would happen to the nervous system should a specific NTF or NTF receptor be
removed. Recall that the neurotrophic hypothesis—which is a driving force for this

14
research—suggests that removal or reduction of neurotrophic support would have a
deleterious effect upon the developing nervous system. The above evidence from the
knockout studies supports this idea. Since some populations are reduced in number in
knockout mice, this suggests that a lack of proper neurotrophic support can reduce neuron
number. Should ethanol effectively remove or reduce neurotrophic support from a given
nervous system population, that population will undoubtedly be adversely affected. To
determine whether this change in some aspect of neurotrophic support is indeed occurring,
it is logical to examine populations that are known to exhibit cell loss due to ethanol
exposure (e.g. the hippocampus). Knockout studies have not recorded any cell loss in the
septohippocampal region. The fact that the septohippocampal system is a rich source for a
variety of NTFs may provide an explanation for this apparent paradox. If one NTF is
eliminated, other NTFs in the background may be able to provide sufficient support for the
neurons to continue to survive. The basic idea that NTF support is critical for survival is
important for the present studies.
NTF Ontogeny in the Hippocampus
The normal ontogeny of NGF, BDNF, and NT-3 in the developing rat
hippocampus differs. NGF is expressed in rather low levels throughout embryonic
development, increases somewhat at birth, and finally achieves its highest levels in the
adult. BDNF is virtually undetectable throughout embryonic development, then increases
at birth and continues to increase to its highest levels in the adult rat brain. NT-3 has high
expression throughout development and decreases in the adult (Maisonpierre et al., 1990).
The different temporal expression between individual neurotrophins might help developing
neurons achieve correct synapses. The following hypothetical example illustrates this
point: The high early expression of NT-3 might promote survival of neurons through a
proliferative phase. Then, NGF expression could increase to induce differentiation.
Finally, BDNF expression would signal the end of development and thus induce these
hypothetical neurons to form synapses. In vivo, neurotrophins have been shown to follow

15
distinct patterns throughout development. Buchman and Davies found that neurotrophins
act in sequence during development to promote survival of DRGs (1993). Therefore, if the
temporal sequence of neurotrophin expression were to change as a result of ethanol
exposure, the normal innervation patterns in the hippocampus—as well as other parts of the
developing CNS—could be altered. Such a change could have disastrous effects on the
hippocampus and its ability to properly encode new memories.
The ontogeny of trk receptors in the developing brain follows the ontogeny of the
neurotrophins. In the septum—a brain structure of which the hippocampus is a target-
higher levels of trkA mRNA were detected at 2 and 4 weeks than at 1 weeks of age
(Ringstedt et al., 1993). TrkA is not expressed in the hippocampus under normal
circumstances in vivo (Martin-Zanca et al., 1990). TrkB is expressed widely in the CNS
and is first detectable in the mouse at E8.5 (Klein et al., 1990). TrkB is expressed by the
developing hippocampus and expression continues into adulthood (Klein et al., 1990).
TrkC mRNA is detectable as early as E7.5 in the nervous system and is expressed at all
stages of hippocampal development (Tessarollo et al., 1993). The above information
demonstrates that developing hippocampal neurons are responsive to BDNF and NT-3 and
that these proteins, plus their receptors, are expressed during prenatal and early postnatal
rat development. Thus, all of these proteins were active during the periods of ethanol
exposure employed in the present study.
Neurotrophins and Ethanol
Fundamental responses to neurotrophins and production of NTFs are altered
following prenatal ethanol exposure in rat pups. The neurotrophins are not the only NTFs
produced by the hippocampus. Other factors, such as bFGF, are synthesized there and
could affect these neurons (Emfors et al., 1990). Cultures of hippocampal neurons derived
from rats prenatally exposed to ethanol do not respond to NTFs as well as neurons in
control cultures (Heaton et al., 1995b). This result suggests that NTF receptor expression
may be decreased in response to prenatal ethanol exposure. However, another logical

16
explanation for this result is that the expression of less active form of NTF receptor has
increased relative to a normal form. Truncated trk receptors are similar to normal trk
receptors except they lack the catalytic domain that starts the intracellular signal transduction
cascade following neurotrophin binding. These receptors are normally expressed in greater
abundance than their active counterparts in adult animals. During development their
expression increases relative to the active trk receptor until reaching the level of expression
found in the adult. Other studies have found altered neurotrophic activity as a result of
ethanol exposure. For example, chronic prenatal ethanol treatment (CPET) in the rat
increases neurotrophic activity (a gross measure which includes both neurotrophin and
other NTF activity) in extracts made from the hippocampus on P21 and cultured on DRG
neurons (Heaton et al., 1995c). The increase in neurotrophic activity suggests an increase
in NTF expression as a result of prenatal ethanol exposure, but no single NTF is implicated
by this study since DRGs respond to a variety of NTFs. Postnatal ethanol exposure
reduces neurotrophic activity of P21 hippocampal extracts (Moore et al., 1996), a result
opposite to that of prenatal exposure (Heaton et al., 1995c). All of these results suggest
that both NTF and receptor might play an essential role in ethanol toxicity.
Hypotheses
As was mentioned previously, all experiments described in this document were
designed to understand some aspect of how ethanol and NTFs are interrelated in producing
the deficits observed in models of FAS. The experiments of this project were performed to
test the following hypotheses: (1) (a) We hypothesize that ethanol will reduce motoneuron
number in the absence NOCD; (b) We hypothesize that ethanol will reduce motoneuron
number at period of development that follows the period for NOCD; (2) We hypothesize
that exogenous NTFs will provide in vivo protection for motoneurons exposed to ethanol;
and (3) We hypothesize that CPET and early postnatal ethanol treatment (EPET) will alter
the gene expression of neurotrophins and/or their receptors in the hippocampus of treated
rat pups. Analysis of hypothesis la was undertaken to further describe the motoneuron

17
loss observed following ethanol exposure from E4 to El 1 in chick embryos. Specifically,
we wanted to determine whether ethanol acted to increase NOCD or provide direct
neurotoxicity. Ethanol was shown to reduce motoneuron number during this time period
which encompasses the period for NOCD (Heaton and Bradley, 1995). Additionally,
neurotrophic content of the developing muscle was analyzed to compare ethanol exposure
from E10 to E15 to an earlier study in this laboratory which found that ethanol exposure
from E4 to E8 reduced neurotrophic content of developing limb tissue (Heaton and
Bradley, 1995). Analyzing neurotrophic content of muscle from embryos exposed to
ethanol from E10 to E15 allowed us to relate any deficit in motoneuron number observed in
that time period, to any possible change in neurotrophic support. The number of apoptotic
cells present during ethanol exposure from E10 to E15 was analyzed to find whether
ethanol exposure from E10 to E15 induced apoptosis among motoneurons. Analysis of
hypothesis 2 was executed to determine whether NTFs could modulate ethanol toxicity in
vivo. Analysis of hypothesis 3 was performed to ascertain whether ethanol could modulate
the genetic expression of NTFs in a living organism. The chapters that follow describe the
experiments performed to achieve these hypotheses and provide the results of these
analyses. Further, the results are discussed critically with implications for future research
and mechanisms for ethanol toxicity suggested.

CHAPTER 2
CHARACTERIZATION OF MOT ONEURON SURVIVAL AND CELL DEATH
FOLLOWING ETHANOL EXPOSURE AND CURARE ADMINISTRATION, AND
AFTER THE PERIOD FOR NATURALLY OCCURRING CELL DEATH
Summary
The study described below was conducted as a continuation of a previous study in
which we found reduced motoneuron number in lumbar spinal cord of the chick embryo
following chronic ethanol administration from embryonic day 4 (E4) to El 1. We sought to
determine whether this reduction was due to primary ethanol toxicity or to enhancement of
naturally occurring cell death (NOCD) and to determine whether administration of ethanol
at a later period of development could also reduce motoneuron number. Earlier studies
have shown that curare suspends NOCD in the chick embryo (Pittman and Oppenheim,
1978). By administering both ethanol and curare to these embryos from E4 to El 1 and
examining the lumbar spinal cord on E12, we determined that ethanol was directly toxic to
motoneurons and reduced motoneuron number in the absence of NOCD. By administering
ethanol from E10 to E15 and examining the lumbar spinal cord on E16, we determined that
ethanol can reduce motoneuron number without altering the overall morphology of the
spinal cord during more than one stage of chick embryo development. We also determined
that ethanol toxicity is not dependent on NOCD. In additional experiments, we
demonstrated that ethanol does not affect the neurotrophic content of chick muscle and does
not appear to induce apoptosis in developing motoneurons when it is administered from
E10 to E15.
Introduction
Over the last two and one-half decades, much evidence has been gathered regarding
the effects ethanol exerts in the developing nervous system (Barnes and Walker, 1981;
18

19
Jones and Smith, 1973; Miller, 1986; Streissguth et al., 1991; West, 1986). Fetal alcohol
syndrome (FAS) is diagnosed in 1-2 out of every 1000 live births in the United States and
is characterized by low birth weight, decreased memory and learning, hyperactivity, facial
dysmorphia, and lowered IQ (Abel, 1995; Jones and Smith, 1973; Streissguth et al.,
1991). Among heavy drinkers, the incidence of FAS is much greater with a 4.3%
diagnosis rate (Abel, 1995). Evidence suggests that these deficits are permanent and do not
lessen as the patient ages (Streissguth, 1993). These observations led to the assertion that
maternal consumption of ethanol is the leading known cause of mental retardation in the
Western Hemisphere (Bonthius and West, 1988).
Many neuronal populations exhibit some susceptibility to the toxic effects of
ethanol. A variety of neuronal types from brain areas such as the cerebellum (Cragg and
Phillips, 1985), the septohippocampal system (Barnes and Walker, 1981) (West and
Pierce, 1986), the cerebral cortex (Miller, 1986), and the oculomotor nucleus (Burrows et
al., 1995) are known to be affected by ethanol exposure. Still other populations that have
not been so intensely investigated demonstrate vulnerability to ethanol. These regions
include the substantia nigra (Shetty et al., 1993), chief sensory trigeminal nucleus (Miller
and Muller, 1989), red nucleus (Zajac et al., 1989), and motoneurons of the spinal cord
(Heaton and Bradley, 1995). Of particular interest to the present studies is the fact that
motoneurons are affected by ethanol. A previous study from this laboratory found that
motoneuron number was reduced by ethanol administration from E4 to El 1 (Heaton and
Bradley, 1995). At that time, we hypothesized that ethanol might be exacerbating NOCD.
The period for NOCD for motoneurons of the entire spinal cord extends from
approximately E6 to E9 (Pittman and Oppenheim, 1978). Motoneuron number is known to
peak around E5.5 to E6 (Pittman and Oppenheim, 1978) and proliferation of motoneurons
continues until E6 (Hollyday and Hamburger, 1977). During the period of NOCD, nearly
half of the original number of motoneurons perish. Curare, as well as other neuromuscular
junction blocking agents, have been shown to suspend NOCD in motoneurons (Pittman

20
and Oppenheim, 1978). In these experiments, approximately 50% more motoneurons
survive in curare-treated embryos than in control embryos.
In addition to the above, there is more direct evidence that warrants further
investigation into the possible effects that ethanol may exert on motoneurons. Previous
research in other laboratories has determined that ethanol damages developing muscle in
both the human and in animal models (Adickes and Shuman, 1983; Nyquist-Battie et al.,
1987). In the human, these cases described flaccid, hypotonic neonates which exhibited
major muscle structural deficiencies including hypotrophy, dominance of type II fibers, and
sarcomeric dysplasia (Adickes and Shuman, 1983). Prenatal exposure to ethanol in the
guinea pig resulted in structural malformations of the gastrocnemius muscle including
vacuolated sarcoplasmic reticula, enlarged lipid droplets, decreased glycogen, and
mitochondrial abnormalities (Nyquist-Battie et al., 1987). Since proper muscle fiber
maturation is dependent on concurrent development and innervation by motoneurons
(Ishiura et al., 1981), the possibility exists that the deficiencies noted in the above cases are
due to an underlying effect that ethanol exerts on developing motoneurons.
Previous studies have shown that ethanol affects chick embryo development in a
manner similar to both the human and the rat. Chicks have been shown to exhibit reduced
brain size, weight, DNA and protein synthesis (Pennington and Kalmus, 1987), and
reduced neurotransmitter synthesis (Brodie and Vernadakis, 1990; Swanson et al., 1994)
following developmental ethanol exposure. One advantage of using a chick model to study
ethanol is its simplicity. Ethanol can be administered in exact doses to the developing
embryo. Another advantage is that maternal influences are removed when using the chick
embryo. Ethanol is cleared from the bloodstream by both mother and fetus in a mammalian
system whereas the chick embryo is isolated as it develops. The chick model allows the
investigator to observe direct effects of ethanol without possible interactions of maternal
metabolism interfering. The embryos seem to tolerate slight invasions into their
environment quite well as long as the underlying membranes are not disrupted. While

21
chick development is clearly different from mammalian gestation, this model allows
researchers to study in vivo interactions in a developing organism that are just not possible
in a mammalian FAS model.
The present experiments were performed to determine whether ethanol exerted a
toxic effect when NOCD is suspended (Curare-Ethanol Coadministration) and whether
administering ethanol dunng a later period of development (Late Exposure)—after the
period of cell death-would differ from administration earlier in development. Suspension
of NOCD did not hinder ethanol’s ability to reduce motoneuron number. This result
suggests that ethanol acts by a mechanism other than exacerbation of NOCD, either by
direct toxicity, motoneuron loss due to a change in neurotrophic support, or a combination
of the two. The Late Exposure study found that ethanol administration from E10 to El 5
reduced motoneuron number and that exposure to ethanol did not reduce the neurotrophic
activity of chick limb muscle in comparison to Saline treated embryos. This latter analysis
was undertaken to compare the results of ethanol exposure from E10 to E15 to an earlier
study in this laboratory which found that ethanol exposure from E4 to E8 reduced
neurotrophic content of developing limb tissue (Heaton and Bradley, 1995) and to
determine if altered neurotrophic support is responsible for the observed motoneuron
reduction. Loss of neurotrophic support would suggest a possible mechanism for
motoneuron loss due to ethanol exposure at this period of development: reduction of
target-derived NTFs. Additionally, an analysis of apoptotic motoneurons in the lumbar
spinal cord failed to find evidence that ethanol exposure from E10 to E15 induced apoptosis
among motoneurons.
Materials and Methods
Subjects
White Leghorn chick eggs were obtained from the University of Florida Poultry
Science Department. Eggs were placed in a Marsh incubator and maintained at 37°C and

22
70% relative humidity until E4. At that time, the eggs were moved to a forced draft turning
incubator, maintained at the same conditions indicated above, and divided into groups.
Curare-ethanol coadministration
For the Curare-Ethanol Coadministration study, 5 groups were utilized:
Uninjected, Ethanol, Saline, Curare, and Curare+Ethanol. The data from all Ethanol
embryos and 4 of the 10 Saline embryos were obtained from an earlier study (Heaton and
Bradley, 1995). Ethanol and saline injection began on E4 and continued daily through
Ell. Curare injection began on E6 and continued daily through Ell. Ethanol and curare
injections were initiated on different days in order to replicate previous research and to
coincide curare administration with the onset on NOCD in the lumbar spinal cord (E6)
(Pittman and Oppenheim, 1978). Since cell death in the lumbar section of the spinal cord
does not begin until E6 (Hollyday and Hamburger, 1977; Pittman and Oppenheim, 1978),
curare administration before this time point would be without effect. In addition, the
present experiment found the combination of curare and ethanol to be quite toxic to the
developing embryos; therefore, further loss of embryos was minimized by starting curare
administration on E6. All embryos were sacrificed by decapitation on El 2, the lumbar
section of the spinal cord removed, and prepared for histology.
Late exposure study
A separate study was conducted to assess the effects of ethanol during a later
exposure period when motoneuron number is relatively stable. The Late Exposure study
had 2 groups: Ethanol and Saline. Embryos received daily injections of either ethanol or
saline from E10 to E15. At E16, embryos were removed from the eggs, sacrificed by
decapitation, and the lumbar section of the spinal cord removed and prepared for histology.
An Uninjected control group was not used in the Late Exposure study because in previous
studies, our laboratory has shown that saline injection does not adversely affect embryonic
development and has no effect on motoneuron number in the developing chick spinal cord
(Heaton and Bradley, 1995).

23
Injections
Curare-ethanol coadministration study
As stated above, five experimental groups were used in this study: Ethanol, Saline,
Uninjected, Curare, and Curare+Ethanol. Ethanol and saline injections were administered
daily from E4 through Ell. Ethanol embryos received 150]A of 20% w/v ethanol (30 mg
ethanol per day), dissolved in a 0.9% w/v nonpyrogenic saline vehicle, through a pinhole
in the shell into the airspace. Previous work in our laboratory has determined that this
concentration of ethanol produces blood ethanol counts that peak at 225 mg/dl by El 1
(Heaton and Bradley, 1995). Saline embryos received 150 }A of the 0.9% w/v
nonpyrogenic saline vehicle. Uninjected embryos received no injections, but were handled
daily in a manner similar to the other groups. Curare injection, which occurred from E6
through Ell, involved creating a pinhole directly over the embryo in addition to the pinhole
created in the airspace. The airspace was then allowed to shift to a position above the
embryo and 150 ]A of 16.67 mg/ml tubocurarine chloride (Sigma) was injected into that
space above the embryo (2.5 mg curare per day). Curare+Ethanol embryos were given
ethanol injections from E4 to El 1 and curare injections from E6 to El 1 as described above.
The embryos were allowed to sit in a Marsh incubator for a period of one hour following
ethanol injection on days when two injections were delivered to the same embryo. This
delay in injection time was necessary to ensure that the ethanol was absorbed through the
inner shell membrane within the airspace before the eggs were turned on their side for the
curare administration. Also, the two injections administered in this study represent a
significant volume (300 piL) for the embryonic system to incorporate on a daily basis. The
delay between injections, therefore, allowed absorption of the volume of ethanol before an
equal volume of curare solution was presented to the embryo. Pinholes created by the
injection process were sealed with paraffin immediately following injection to prevent
evaporation and/or leakage of the ethanol and curare. The eggs were then returned to the
turning incubator.

24
Late exposure study
Ethanol and saline injections were administered daily from E10 through E15.
Ethanol embryos received 150 pi\ of 30% w/v ethanol (45 mg ethanol per day), dissolved in
a 0.9% w/v nonpyrogenic saline vehicle, through a pinhole into the airspace. Since
embryos at this later stage of development do have the ability to clear ethanol from the
bloodstream (Wilson et al., 1984), a larger dose of ethanol was utilized in this study in
order to achieve blood ethanol concentrations similar to those observed in embryos exposed
to ethanol from E4 to El 1. Peak blood ethanol concentration in this portion of the study
ranged from 250 to 300 mg/dl and trough levels were below 30 mg/dl. Saline embryos
received 150 ]A of the 0.9% w/v nonpyrogenic saline vehicle. Pinholes created by the
injection process were sealed with paraffin following injection to prevent evaporation
and/or leakage of the ethanol. The eggs were then returned to the turning incubator, as
above.
Dissections and Histological Procedures
Curare-ethanol coadministration study
Embryos of all experimental groups (Ethanol, Saline, Uninjected, Curare, and
Curare+Ethanol) were sacrificed by decapitation on E12 and the lumbar section of the
spinal cord removed. Following dissection, the E12 spinal cords were placed in Bouin’s
fixative for 24 hours and then embedded in paraffin. Spinal cords were then cut into 12
ptm coronal sections, mounted onto glass slides, and stained with hematoxylin and eosin. A
total of 33 embryos were used in this study: Ethanol (n=6), Saline (n=10), Uninjected
(n=6), Curare (n=6), and Curare+Ethanol (n=5).
Late exposure study
Embryos of both experimental groups (Ethanol and Saline) were sacrificed by
decapitation on E16 and the lumbar section of the spinal cord removed. The vertebrae of
the spinal cords were cut along the dorsal surface to expose the nervous tissue and allow
the fixative to adequately penetrate the tissue. Following dissection, the E16 spinal cords

25
were placed in Bouin’s Fixative for 14 to 21 days to allow the vertebrae to decalcify (Li et
al., 1994). The tissue was then embedded in paraffin, cut into 12 pirn coronal sections,
mounted onto glass slides, and stained with hematoxylin and eosin. 7 Ethanol and 6 Saline
embryos were used to complete this study.
Motoneuron Counts
Motoneuron counts were completed following methods described previously
(Hamburger, 1975; Heaton and Bradley, 1995). Briefly, a uniform area encompassed by 6
dorsal root ganglia (DRG) was noted on each embryo which ensured that a similar area was
counted in each subject. Starting from the most rostral section included in the 6 DRG
region, motoneurons in the lateral motor column of one side of every tenth section were
marked onto paper using a camera lucida. At 400X magnification, motoneurons were
identified in the lateral motor column by their large size, dark cytoplasm, and nucleolus.
Laterality was maintained throughout each individual embryo, but chosen at random before
beginning the counting process. Previous studies have shown that there is no difference
between the number of motoneurons contained in the right and left sides of the spinal cord
(Pittman and Oppenheim, 1979). It should also be noted that each embryo was coded so
that the experimenter had no knowledge of its experimental treatment until the study was
completed. Motoneuron counts reported below are actual counts generated by the above
procedure and are not corrected to estimate total motoneuron number of the lumbar spinal
cord.
Pre-Cell Death Ethanol Exposure
In order to clarify the results of the Coadministration Study, we administered
ethanol and saline to chicks from E4 to E5 and assessed motoneuron number on E12. This
analysis was performed to determine whether ethanol reduces motoneuron number during
the time period where ethanol was administered, but curare administration had not yet
commenced. Chick eggs were incubated as described previously and placed into two
groups: Ethanol and Saline. On E4 and E5 Ethanol embryos received 150 ]A of 20% w/v

26
ethanol solution (30 mg) and Saline embryos received 150 pil of the saline vehicle. No
injections were administered from E6 to El 1 and on E12, the embryos were sacrificed by
decapitation, the lumbar spinal cord removed, and prepared for histology.
Motoneuron Size and Spinal Cord Length Analyses
Motoneuron size and spinal cord length were measured to determine whether
ethanol had altered any general characteristics of the motoneuronal system. Motoneuron
size was determined by measunng the diameter of 10 random cells in the same rostral-
caudal position of the region of each embryonic spinal cord. In E12 embryos
(Coadministration Study) the section exactly 1800 pim following the beginning of the
lumbar spinal cord as determined by the 6 DRG region described above, was sampled. In
E16 embryos (Late Exposure Study) the section exactly 2400 /on following the beginning
of the lumbar spinal cord, was sampled. Four embryos from each experimental condition
were analyzed for a total of 40 cells per condition. Spinal cord length was determined by
counting the number of sections present in each embryo following determination of the
boundaries of the lumbar spinal cord by the anatomical methods described previously and
multiplying this number by the section thickness (12 pirn).
Crude Muscle Extract Study
Extract preparation
In order to determine whether neurotrophic content of chick leg muscle is affected
by ethanol exposure during the late exposure period (E10 to E15), we analyzed the activity
of crude muscle extracts on E6 spinal cord cultures. This analysis was undertaken to
compare ethanol exposure from E10 to E16 to ethanol exposure from E4 to E8—where a
reduction in neurotrophic activity of developing muscle tissue following ethanol exposure
was observed. Embryos were treated with ethanol or saline and incubated as described in
the Subjects section above. Briefly, Ethanol and Saline embryos received injections of
30% w/v ethanol (45 mg per day) or the saline vehicle, respectively, daily from E10 to
E15. On E16, embryos were removed from the egg, sacrificed by decapitation, and the

27
muscle dissected away from the thigh region of each leg. The muscle tissue was flash-
frozen on dry ice and then stored at -70°C until extract was prepared. Extract was prepared
by homogenizing the tissue for about 15 seconds in F-12 media (BRL) supplemented with
0.7% fungizone, 1.0% penicillin-streptomycin, and 200 mM glutamine. After
homogenization, the extract was centrifuged for 20 minutes at 35,000 rpm at 4°C. The
supernatant was collected and assayed for protein content (Bradford, 1976). All samples
were diluted to 200 /¿g/ml protein, 10% fetal bovine serum (FBS) added, and then added to
mixed spinal cord cultures which were prepared as described below.
Spinal cord cultures
Three experimental groups were analyzed in this portion of the study: Ethanol,
Saline, and Negative Control (NC). Ethanol cultures consisted of E6 lumbar spinal cord
cultures grown in the presence of muscle extract obtained from embryos exposed to ethanol
from E10 to E15. Saline cultures were grown in the presence of muscle extract obtained
from Saline embryos and NC were grown in the presence of regular culture medium.
Regular culture medium consisted of F-12 media supplemented as described above, with
10% FBS added. The lumbar region of the spinal cord was dissected out of E6 embryos
and incubated in 10% v/v trypsin and 5% v/v deoxyribonuclease I in 0.9% nonpyrogemc
saline for 20 minutes at 37°C. Cells were disassociated by repeated titration in regular
culture medium. The cells were grown in individual wells of 12-well Corning plates coated
with 0.5 mg/ml polyomithine. Two hours after the cells were plated initial counts were
completed by counting three representative areas from each culture well. Each culture had a
Bélico glass slip with an enumerated grid affixed to the bottom. This allowed the
experimenter to note the initial areas counted and return to these areas on subsequent days.
Neurons were identified by their large size, and rounded, phase-bright appearance.
Following 24 and 48 hours in culture, cell counts were again obtained from each culture
and in addition, the number of cells expressing a neurite (at least two cell diameters in
length) were noted.

28
Assessment of Apoptotic Cells
Following ethanol exposure to embryonic chicks as described previously, spinal
cord tissue was examined and apoptotic cells were identified by methods described
previously (Homma et al., 1994). Briefly, at400X magnification cells fitting the following
criteria were determined to be undergoing apoptosis. First, cells with chromatin and
cytoplasmic condensation—the hallmark of apoptosis—were identified. Since these
processes are rather fast, it was unlikely that such cells would be observed. Therefore, a
second criterion—where apoptotic debris was identified—was used to make a positive
identification of an apoptotic cell. Since this portion of the study utilized an ethanol
exposure paradigm that began on E10 and ran through E15, tissue was examined at the
following intermediate time points: E12 and E14. Both saline and ethanol-exposed
embryos were examined so that a statistical comparison could be made between the two
groups. It should be noted, however, that NOCD essentially ends on E9 (Pittman and
Oppenheim, 1978). A uniform area encompassed by 6 DRG was noted on each embryo
which ensured that a similar area was counted in each subject. Starting from the most
rostral section included in the 6 DRG region, apoptotic motoneurons in the lateral motor
column of one side of every fifteenth section were noted. This process led to an average of
21 sections being examined per E12 embryo and 30 sections being examined in every E14
embryo. After the entire animal was examined, candidate cells were reexamined to make
sure that they truly fit the criteria described above. Both sides of each section were
analyzed in order to increase the probability of identifying apoptotic neurons. Even though
fewer total sections were analyzed than in the Motoneuron Number analysis, more total
area was analyzed pier animal since both the right and left side of each section was
examined.

29
Statistics
Analysis of variance, Fisher’s protected least significant difference post-hoc test,
and Student’s t-test were performed using the StatView program (Abacus) on a Macintosh
computer.
Results
Embryonic Observations
Curare-ethanol coadministration study
Survival varied greatly according to the treatment of each embryonic group. In the
Curare group survival was approximately 47%, Saline group survival was 76%,
Uninjected group survival was 96%, Ethanol group survival was 4%, and Curare+Ethanol
group survival was 0.83%. This latter survival figure indicates that the combination of
curare and ethanol was highly toxic to the developing chick embryo. The Curare embryos
appeared to be more vascularized, in both the body and the limbs, than the controls. In
contrast, the Ethanol and Curare+Ethanol embryos had wider bodies due to extensive
bloating from the ethanol treatment. The spinal column in Curare+Ethanol embryos was
softer than in the control counterparts and in the Ethanol embryos the lumbar region
exhibited a minimal enlargement compared to Saline, Uninjected, and Curare embryos.
The profound effects of ethanol treatment were verified by the presence of a green-colored
liver in the Ethanol and Curare+Ethanol embryos, compared to the normal brown-colored
liver in the remaining three groups. The alteration of the appearance of the liver in animals
exposed to ethanol should be investigated further. The pathology of the liver could be
analyzed and perhaps related to the death of embryos following ethanol exposure.
Collaboration with a pathologist in examining this tissue would be preferable so that an
accurate estimation of the method of liver damage can be determined.

30
Late exposure study
Survival between experimental groups was not as variable in the Late Exposure
study as it was in the Coadministration study. Approximately 60% of the Ethanol embryos
and 75% of the Saline embryos survived. The livers of the Ethanol embryos were green,
thus indicating that ethanol administration did have some general effect. The Saline
embryos exhibited the normal brown liver coloration. The Ethanol embryos were not
bloated in appearance, as has been observed in earlier studies (Heaton and Bradley, 1995)
and the current Coadministration study, perhaps because the embryos of this age have the
ability to clear ethanol from the bloodstream (Wilson et al., 1984).
Motoneuron Counts
Curare-ethanol coadministration study
Analysis of variance indicated a significant effect due to treatment among these
groups (F=23.061; df=28; pcO.OOOl). The number of motoneurons present in the lumbar
region of the spinal cord in each of the five experimental groups at E12, could be described
as one of three possible outcomes: average, above average, and below average. For these
distinctions, “average” is defined to be a number of motoneurons similar to normal or
control levels (i.e., Saline or Uninjected); “above average” is defined to be a number of
motoneurons significantly higher than average; and “below average” is defined to be a
number of motoneurons significantly lower than average. Figure 2-1 displays all of the
groups utilized in this study. It should be noted that Figure 2-1 represents the number of
motoneurons counted in each embryo and does not attempt to estimate the population of
motoneurons in the lumbar spinal cord. Looking at Figure 2-1 from left to right shows
how evident the three-tiered outcome of this study was. For example, the Curare group
(located at the left in Figure 2-1) is the only group that displayed above average numbers.
In fact, post hoc testing showed that the curare group contained significantly more
motoneurons (about 35% more) than all other groups (pcO.OOOl for each comparison).
The Ethanol group (located at the right in Figure 2-1) was the only group to exhibit below

31
-d
-<—>
tí
tí
o
U
00
tí
O
S-H
tí
tí
o
•*—>
o
s
'o
o
tí
£
3000
2000
1000 -
Figure 2-1. Number of motoneurons in lumbar spinal cord at E12 following
treatment from E4toEll. Motoneuron counts are displayed as means + SEM.
Data from Curare, Curare+Ethanol, Uninjected, Saline, and Ethanol embryos are
displayed, a = significantly more motoneurons than Curare+Ethanol, Uninjected,
Saline, and Ethanol (p<0.0001 in all cases), b = significantly less than
Curare+Ethanol, Uninjected, and Saline (p<0.005 in all cases).

32
average numbers. This fact is evidenced by the observation that post hoc testing indicated
that the Ethanol group contained fewer motoneurons (about 20% fewer) than Saline,
Uninjected, and Curare+Ethanol groups (p<0.005 in each comparison). All of the other
groups-Saline, Uninjected, and Curare+Ethanol—contained average numbers of
motoneurons and there were no significant differences among these groups. The
Motoneuron Size and Spinal Cord Length Analyses section below suggests that
motoneuron number differences observed in this study are not due to changes in the overall
length of the spinal cord.
Figure 2-2 is a collection of photomicrographs obtained from midlumbar segments
of Saline, Ethanol, Curare, and Curare+Ethanol embryos. It is apparent that motoneuron
number was affected by treatment as the Ethanol section contains the fewest motoneurons.
The Saline section contains more than the Ethanol section, but not as many as the Curare
section. Finally, the Curare+Ethanol section contains a similar number as the Saline
section (refer to Figure 2-2).
Pre-cell death exposure
This analysis was undertaken to determine if ethanol exposure prior to the period
for NOCD (E4 to E5) in the spinal cord could affect overall motoneuron number at E12.
The T-test indicated no significant difference in the number of motoneurons in the lumbar
spinal cord between the Ethanol and Saline embryos. This result suggests that exposure to
ethanol before administration of curare, and NOCD, had no adverse effect on the
motoneuron population of the lumbar spinal cord. Therefore, results obtained following
ethanol exposure from E4 to El 1 are not confounded by the fact that ethanol exposure did
not coincide completely with the period for NOCD.
Late exposure study.
The results obtained following late ethanol exposure were similar to those obtained
when ethanol was administered from E4 to El 1, in that Ethanol embryos exposed from
E10 to E16 exhibited a significant reduction in motoneuron number. The length of each

Figure 2-2. Photomicrographs of coronal sections from the midlumbar region of E12
spinal cords. A. Ethanol, B. Saline, C. Curare, and D. Curare+Ethanol spinal cords.
Note that the Curare “bulge” is most pronounced while the Ethanol “bulge” is hardly
apparent. Also note the density of motoneurons in the lateral motor column. The Curare
contains more and more densely packed motoneurons, while the Ethanol cord contains
fewer and less densely packed motoneurons. The Saline and Curare+Ethanol spinal cord
sections contain roughly similar numbers of motoneurons, n = 6 Ethanol, 10 Saline, 6
Curare, and 5 Curare+Ethanol

34
Saline
Ethanol
Curare Curare+Ethanol
Figure 2-2.

35
embryo’s spinal cord was similar since similar numbers of sections were counted from
each embryo and each section is 12/mi thick (see section below). The t-test indicated that
the E16 Ethanol embryos contained significantly fewer motoneurons (approximately 15%)
than did their Saline counterparts (p<0.05). Figure 2-3 displays the data generated in this
portion of the study.
Motoneuron Size and Spinal Cord Length Analyses
Curare-ethanol coadministration study
Analysis of variance of the motoneuron size data indicted a significant effect due to
treatment (F=3.233; df=200; p<0.05). The only significant difference indicated by post
hoc testing was that the Curare+Ethanol group contained significantly smaller motoneurons
than the Saline (p<0.005), Ethanol (p<0.005), and Curare (p<0.05) groups. There were
no other significant differences between any of the other groups. Analysis of variance of
spinal cord length found no significant effect due to treatment. Table 2-1 shows the data
generated in this portion of the study. Note that even though the difference between the
Curare+Ethanol group and the other groups is less than 2 pim, it is significant.
Table 2-1. Cell size and spinal cord length.
Coadministration Study
Group Cell Size {pan) Spinal Cord Length (pim)
Ethanol
19.025 + 0.404
3940.0 + 95.04
Saline
18.875 + 0.399
3750.0 + 76.92
Uninjected
18.200 + 0.431
3614.9+151.80
Curare
18.725 + 0.410
3800.8+ 119.20
Curare+Ethanol
17.275 + 0.326 *
3993.2 + 68.76
Late Exposure Study
Group Cell Size (¡Am) Spinal Cord Length (pmi)
Ethanol
20.950 + 0.399
6000.0 + 138.6
Saline
20.650 + 0.408
6060.0 + 187.8
All values are means + SEM. For cell size, n=40 for each group. For spinal cord length
Ethanol n=6, Saline n=10, Uninjected n=6, Curare n=6, and Curare+Ethanol n=5.
* denotes significance in comparison to Ethanol (p<0.005), Saline (p<0.005), and Curare
(p<0.05).

36
^ 2000
<ü
4—>
tí
tí
O
U
Saline Ethanol
Figure 2-3. Number of motoneurons in lumbar spinal cord at E16 following
ethanol treatment from E10 to E15. Motoneuron counts obtained in the Late
Exposure study are displayed as means + SEM. Data from Ethanol and Saline
embryos are displayed, a = significantly fewer motoneurons than Saline
(p<0.05).

37
Late exposure study
The t-test indicated that there was no difference in motoneuron size or spinal cord
length between the Ethanol and Saline groups. Table 2-1 also contains data generated from
this portion of the experiment.
Crude Muscle Extract Study
As mentioned above, both neuronal survival and neurite outgrowth at 24 and 48
hours were observed in this portion of the study. Also recall that muscle tissue was
prepared from E16 embryos exposed to ethanol from E10 to E15. Analysis of variance
indicated no significant effect due to treatment for survival at either 24 or 48 hours.
Likewise, after 24 hours in culture there was no significant effect due to treatment for
outgrowth. Following 48 hours in culture, however, there was a significant effect of
treatment for outgrowth (F=4.232; df=18; p<0.05). Post-hoc testing revealed that Ethanol
cultures extended significantly more neurites than NC cultures (p<0.05). However,
Ethanol extract and Saline extract cultures were not different, thus this result does not
indicate that ethanol increases neurotrophic activity in chick limb muscle. Neurite
outgrowth at 48 hours in Saline cultures approached statistical significance (p=0.078),
which further supports the notion that observations obtained from Ethanol and Saline
extract cultures were similar. Table 2-2 displays the results obtained in this portion of the
study.
Table 2-2. Neurotrophic activity of crude muscle extract.
Survival (% of Outgrowth (% of
initial counts) surviving cells)
Group 24 Hours 48 Hours 24 Hours 48 Hours
Negative Control
83.486+2.861
77.929+3.288
9.143+1.788
12.243+2.095
Saline
86.314+1.766
81.443+1.918
11.971+2.171
17.557+1.547
Ethanol
88.614+1.840
84.529+1.526
12.400+2.530
20.386+2.309 *
All values are means + SEM. n=8 (one culture produced from each of eight embryos) for
all groups. * denotes significance in comparison to Negative Control (p<0.05).

38
Assessment of Apoptotic Cells
As mentioned above, spinal cords for intermediate time points in the Late Exposure
Study (E12 and E14) were assessed for apoptotic cells. Three embryos from the Saline
group and three embryos from the Ethanol group were studied as described above. At
E12, a total of 2 apoptotic cells were identified among the animals in the Saline group and a
total of 3 apoptotic cells were identified among the Ethanol group. As stated above, an
average of 21 sections—and both sides of each section—for each animal were examined.
Since a total of 5 apoptotic cells were identified at E12, it is unlikely that an analysis of
more sections would result in finding significantly more apoptotic motoneurons. Both
sides of each section were analyzed for apoptotic cells so that a greater total area was
analyzed in this portion of the study than in the Motoneuron Counts section. At E14, a
total of 2 apoptotic cells were identified among the animals in the Saline group and a total of
4 apoptotic cells were identified from the Ethanol group (an average of 30 sections were
examined in each E14 embryo). These results led to the conclusion that there was no
significant difference between the Ethanol and Saline groups in numbers of apoptotic cells
when ethanol was administered from E10 to E15. However, the data do not conclusively
eliminate apoptosis as a potential mechanism for ethanol in this neuronal population. This
possibility will be discussed further below.
Discussion
Curare-Ethanol Coadministration Study
The results of the Curare-Ethanol Coadministration study indicate that ethanol is
toxic to developing motoneurons even in the absence of NOCD. This result suggests that
ethanol does exhibit a mechanism other than exacerbation of NOCD to developing
motoneurons and the change in motoneurons number is not due to a change in spinal cord
length. The Curare embryos had significantly more motoneurons than all other groups.
This result agrees with prior results from other laboratories (Pittman and Oppenheim, 1979;
Pittman and Oppenheim, 1978). The mechanism for curare rescue of motoneurons

39
destined to die of NOCD has been studied previously. Curare increases the number of
acetylcholine receptor clusters in developing myofibers (Oppenheim et al., 1989). The
implication of this result is that greater numbers of synapses can form and developing
motoneurons would then have greater access to target derived NTFs (Oppenheim, 1991).
The fact that the Curare+Ethanol group contained significantly fewer motoneurons than the
Curare group indicates that ethanol does not reduce motoneuron number by exacerbating
NOCD. Since curare administration suspends NOCD, ethanol must cause additional
motoneurons to perish by a mechanism other than exacerbation of NOCD. Potential
mechanisms for ethanol toxicity are discussed further below. The three-tiered level of
motoneuron survival observed in this study also suggests that ethanol and NOCD act
independently. The Curare group, which contains that largest number of motoneurons, is
not subject to either cell death process. The middle tier contains three groups that are all
subject to one of the two cell death processes: Uninjected (NOCD), Saline (NOCD), and
Curare+Ethanol (ethanol toxicity). The Ethanol group, which contained the fewest number
of motoneurons, was subject to both cell death processes and exhibited an additive effect of
motoneuron loss (refer to Figure 2-1).
The timing of ethanol and curare administration for this experiment was designed to
coordinate the presence of curare with the onset of NOCD in the lumbar spinal cord. The
results indicated that ethanol exposure on E4 and E5 did not reduce motoneuron number.
There are two implications of this result: Ethanol exposure prior to curare administration
had no adverse effect on the motoneuron population of the lumbar spinal cord and the
critical period for ethanol-induced motoneuron death does not include E4 and E5. A
possible addition to this study would be a study which further limits exposure time to
ethanol and attempts to define a critical period where ethanol exerts its greatest toxic effects
on this neuronal population.

40
Late Exposure Study
The results of the Late Exposure study indicate that ethanol has the ability to reduce
motoneuron number in the lumbar section of the spinal cord during a later period of
development. Since the reduction in motoneuron number during this late exposure period
does mimic the reduction observed following ethanol administration from E4 to El 1, it is
logical to assume that ethanol might have the ability to reduce motoneuron number in the
developing chick embryo during any time period of motoneuron development and perhaps
during later periods as well. This result is further evidence that ethanol is directly toxic to
motoneurons of the embryonic chick because the ethanol exposure took place following the
period of NOCD. Additionally, since spinal cord length was unaltered following ethanol
treatment, this study suggests that these findings are not an artifact of a change in spinal
cord volume.
Additional experiments were conducted to determine whether ethanol exposure
during this late period altered the neurotrophic activity of embryonic chick muscle. In a
previous study, this laboratory found that neurotrophic activity of chick muscle from limb
bud was reduced following ethanol exposure from E4 to E8 (Heaton and Bradley, 1995).
However, the current results suggest that there is no difference in muscle neurotrophic
activity in embryos treated with ethanol or saline. The results suggest that ethanol does not
reduce motoneuron number by decreasing the total amount of neurotrophic support
available to the motoneuron population as it does when administered during the earlier stage
of development (Heaton and Bradley, 1995). However, it does remain a possibility that
individual NTFs produced by muscle are altered in their expression such that one factor
was upregulated while another was downregulated. Such a change could alter survival of
the NTF dependent motoneuron population if the downregulated factor was critical for
survival at the given time period of ethanol administration.

41
General Discussion
The results of the present study offer many possibilities for the action of ethanol in
the developing motor system. Since our analyses included motoneuron survival,
neurotrophic activity, and apoptotic cells, this study has the ability to question potential
mechanisms of ethanol toxicity that are suggested by previous research. Specific areas, as
they relate to ethanol toxicity in the developing nervous system, are discussed below.
Disruption of NTF support
Ethanol has been shown to affect neurotrophic activity and neuronal responsiveness
to NTFs (Heaton et al., 1995b; Heaton et al., 1992; Heaton et al., 1993; Heaton et al.,
1994). Since developing motoneurons of the lateral column require neurotrophic support
for survival (Oppenheim, 1991), it is possible that ethanol may interfere with the ability of
these cells to gain access to NTFs and therefore cause excess cell death. The results of the
current experiments are somewhat contradictory. As was mentioned above, extracts made
from E16 leg muscle following ethanol exposure from E10 to E15 were significantly
increased in neurotrophic activity in comparison to NC cultures. NC cultures were
composed of cell cultures grown in regular culture medium, with essentially little
exogenous neurotrophic support. Thus, these cultures were negative control groups. Only
Saline and Ethanol cultures contained exogenous neurotrophic support. Since NTF
activity—as measured by both neurite outgrowth and survival—was not significantly
increased in comparison to the positive control group (Saline), it would be erroneous to
conclude that ethanol exposure increases neurotrophic activity of developing leg muscle.
The current study suggests that late ethanol exposure does not affect the neurotrophic
content of chick limb muscle—a result that contrasts with earlier findings that demonstrated
ethanol exposure from E4-E8 reduces neurotrophic content of developing limb muscle
(Heaton and Bradley, 1995).
The reason for this disparity may lie in the fact that neurotrophic content of
developing limb muscle increases throughout development and peaks at E18 (Thompson

42
and Thompson, 1988). The fact that total neurotrophic activity is nearing its peak at E16
suggests that overall activity is much higher at E16 than at E8. Any gross change in
neurotrophic activity at E8 would result in a larger percentage change in activity than a
similar change at E16. The implication would be that enough residual neurotrophic activity
would remain in E16 muscle to continue to support the spinal cord cultures, whereas the
support would be reduced sufficiently to alter the growth of the cultures when ethanol was
administered earlier in development. The fact that Saline embryos did not significantly
increase neurotrophic activity in comparison to the NC group should be discussed further.
The answer may lie in the ontogeny of neurotrophic activity in developing limb muscle.
Since this level increases until reaching a peak at El 8 (Thompson and Thompson, 1988),
the relative amount of neurotrophic factors present in extract is increased in comparison to
extracts prepared on E8. Since high levels of NTFs in cultures can be lethal, it is possible
that the amount of trophic activity released into the culture medium ceased to be supportive.
As was mentioned above, the amount of gross protein, not gross neurotrophic activity, was
regulated in these cultures. Therefore, since NTFs are increased relative to overall protein
level (Thompson and Thompson, 1988), our cultures may not have been maintained for
peak neurotrophic activity.
Another possible explanation for the fact that Saline cultures did not exhibit
significantly greater neurotrophic activity in comparison to the NC is the fact that FBS was
used in the culture medium. Since FBS contains NTFs as well as other undefined proteins,
it is likely that the NC cultures exhibit growth and survival far above levels that would be
present without FBS. Clearly, further experimentation should be completed before
concluding that E10 to E15 ethanol exposure in embryonic chick increases neurotrophic
activity of developing leg muscle. Trophic support for developing motoneurons is not
limited to, but is in a large part provided by, target muscle. Glia in the spinal cord produce
NTFs that support developing motoneurons (Arce et al., 1998). However, motoneuron
number in the chick has been shown to be regulated by the amount of target muscle

43
present. Specifically, when a limb is removed from a chick embryo, motoneuron number
is reduced accordingly (Caldero et ah, 1998; Lanser and Fallon, 1987). When a
supernumerary limb is grafted onto an embryo, motoneuron number is increased (Hollyday
and Hamburger, 1976). Thus, target muscle regulates motoneuron number in a “dose-
dependent” manner. This relationship has been further confirmed by the removal of
varying portions of limb bud from developing chick embryos. In this case the survival of
motoneurons was proportional to the amount of limb bud remaining (Lanser and Fallon,
1987).
Response to NTFs
This study found that ethanol did not significantly alter the gross amount of
neurotrophic activity produced in target limb muscle in comparison to Saline treated
embryos when administered from E10 to E15. The significantly greater neurite outgrowth
of the Ethanol group in comparison to the NC group does not provide conclusive evidence
that embryonic ethanol exposure increases neurotrophic activity. When this result is
combined with the fact the ethanol reduces motoneuron number in the spinal cord during
this period, it could be that ethanol has altered the ability of motoneurons to respond to
neurotrophic support produced by the target muscle. This change in the ability to respond
to NTFs could be achieved by altering retrograde transport capacity, receptor expression,
or receptor function. Ethanol is known to hinder retrograde transport in cultured
thymocytes (McLane, 1990) and previous research in this laboratory found that prenatal
exposure to ethanol in the rat reduced the ability of cultured hippocampal neurons to
respond to exogenous NTFs (Heaton et al., 1994). Such a mechanism might occur by
ethanol altering expression of NTF receptor genes or by altering the activity of the active
receptor.
Ethanol is known to affect certain receptor systems. For example, N-Methyl-D-
aspartate (NMDA) receptors are a type of glutamate receptor and are involved in long-term
potentiation, which has long been thought to be involved in how the hippocampus encodes

44
new memories (Bunsey and Eichenbaum, 1996). Ethanol has been shown to inhibit the
flow of ions through NMDA receptors and to block NMDA receptor antagonists from
binding to the receptor (Lovinger et al., 1989; Valles et ah, 1995). Previous research from
this laboratory has implied that ethanol inhibits neuronal ability to respond to NTFs.
Specifically, bFGF’s ability to promote neurite outgrowth in hippocampal cultures was
reduced in those composed of cell from animals exposed prenatally to ethanol (Heaton et
al., 1995b). Such a mechanism, inhibition of the NTF/receptor binding system, could be
involved in ethanol’s toxic effect upon the neuromuscular system. Future experiments will
be designed to determine whether such a mechanism is indeed occurring in this system.
Since the neurotrophic content of E16 muscle exposed to ethanol from E10 to E15
is not significantly changed in comparison to Saline exposed embryos, it is likely that
ethanol interferes with an individual motoneuron’s ability to utilize its neurotrophic support
rather than reducing neurotrophic support. In support of this hypothesis, our lab has also
found that ethanol disrupts the ability of co-cultures of spinal cord to grow neurites toward
limb muscle tissue (Heaton et al., 1995a). This result could also be due to altered NTF
receptor function since neurite outgrowth occurs as the growth cone responds to its
environment. If the ability of the growth cone to sense its environment were diminished, it
would not extend the neurite in a normal manner.
The fact that motoneuron and muscle development proceed concurrently and are
interdependent should not be overlooked. Ethanol does reduce the amount of trophic
substances produced in muscle when administered early in embryonic development
(Heaton and Bradley, 1995) and this further limitation of trophic factors could cause fewer
motoneurons than normal to survive. The current results do not support such a hypothesis
during late ethanol exposure since ethanol administered from E10 to E15 did not reduce or
otherwise alter neurotrophic activity of limb muscle extracts in comparison to the positive
control group, Saline. Motoneuron number is reduced following both exposure periods
which suggests that NTF developmental activity is not solely responsible for this loss in the

45
embryonic chick, at least during the late exposure period. Since muscle requires
motoneuron innervation and activity to develop properly (Ishiura et al., 1981), it is equally
possible that a directly toxic effect of ethanol on motoneurons, such as a change in the
ability of motoneurons to respond to NTFs as mentioned above, could cause developing
muscles to exhibit the malformations noted in both human and animal models of prenatal
ethanol exposure (Adickes and Shuman, 1983; Nyquist-Battie et al., 1987).
Apoptosis
While the current experiments have been effective in eliminating exacerbation of
NOCD as a potential mechanism of ethanol toxicity observed in the developing chick
embryo spinal cord, other possible explanations for ethanol toxicity exist. These potential
mechanisms could utilize an apoptotic mechanism to achieve cell death. NOCD has been
shown to be an apoptotic process that requires the cell to participate actively in its own
demise (Columbano, 1995). The term “active” in this context indicates that new RNA and
protein synthesis are required for apoptosis to proceed. In fact, NOCD in the spinal cord
does require new RNA and protein synthesis (Oppenheim et al., 1989), but some examples
of apoptosis in the absence of RNA synthesis have been documented (Kelley et al., 1992).
In culture, ethanol has been shown to induce apoptosis in hypothalamic neurons (De et al.,
1994), thymocytes (Ewald and Shao, 1993), and cerebellar granule neurons (Bhave and
Hoffman, 1997; Liesi, 1997). Previous studies have also implicated chronic ethanol
treatment in producing apoptotic cell death in the hippocampus and the cerebellum of adult
rats in vivo (Rems et al., 1996; Singh et al., 1995). Additionally, there is evidence that
ethanol increases programmed cell death in the cerebellum (Cragg and Phillips, 1985).
The present study did attempt to determine whether ethanol induced apoptosis in
motoneurons of the lumbar spinal cord. However, the results did not indicate that such a
mechanism was occurring. Saline and Ethanol group spinal cords that were examined at
E12 and E14 contained virtually identical, and very few, numbers of motoneurons
undergoing apoptosis identified by histological or morphological characteristics. The fact

46
that such cells were not seen does not rule out the possibility that ethanol is inducing
apoptosis in spinal cord motoneurons but was not detected with this methodology. As was
mentioned above, ethanol exposure from E10 to E15 results in a reduction of
approximately 15% in motoneuron number in the lumbar spinal cord. Furthermore,
apoptosis is a relatively rapid cellular process that is completed in approximately 3 hours
(Bursch et al., 1990) and begins very soon after ethanol exposure (Cragg and Phillips,
1985). To have the best opportunity to observe apoptosis, embryos should have been
sacrificed between two and five hours following the ethanol injection on days when this
analysis was to occur.
Hypoxia
Another possible mechanism contributing to the toxic effects of ethanol upon
developing motoneurons is hypoxia. Hypoxia, a condition which results from inadequate
blood supply, exerts a variety of effects on all organ systems, including the central nervous
system (CNS). Previous studies in mammals have found that administration of ethanol
causes a decrease in umbilical artery blood flow and a reduction of oxygen delivery (Altura
et al., 1983; Jones et al., 1981; Mukherjee and Hodgen, 1982). However, a link between
ethanol and hypoxia in the developing chick has not been established. Hypoxia alone has
been studied in this animal model and has been shown to reduce the overall vascularity of
the chorioallantoic membrane (Strick et al., 1991) and reduce its blood flow (Ar et al.,
1991). The fact that the direct relationship between ethanol and hypoxia has not been
explored in the chick does not eliminate hypoxia as a potential mechanism in this model. A
future goal of this laboratory should be to determine whether or not this relationship exists
in developing chick embryos. In the CNS, hypoxic conditions affect hippocampal CA1
pyramidal neurons and cerebellar Purkinje cells (Auer et al., 1989; Jorgensen and Diemer,
1982). These same neuronal populations are damaged when rats are exposed to ethanol
prenatally and postnatally (Barnes and Walker, 1981; Bonthius and West, 1990; Phillips
and Cragg, 1982; Pierce et al., 1989). These studies have led to a hypothesis that ethanol-

47
induced hypoxia may cause excitotoxic damage to developing neurons (Michaelis, 1990).
If such a mechanism were occurring in response to prenatal ethanol exposure, one would
expect to find that ethanol has an effect on Ca2+ homeostasis. In fact, ethanol has been
shown to regulate Ca2+ homeostasis in cultured neurons (Koike and Tanaka, 1991; Webb et
al., 1995). These latter studies do suggest that hypoxia may be involved in ethanol
toxicity.
Additional considerations
The motoneuron size findings indicate that for the most part ethanol has no effect on
this aspect of motoneuron morphology in the lumbar spinal cord. The fact that
Curare+Ethanol embryos contained smaller motoneurons could be due to the combined
effect of curare and ethanol which were extremely toxic to the developing embryos. Since
survival was poor in this group, it is likely that some general status of the embryo was
compromised which could have altered motoneuron size. The finding that ethanol reduces
motoneuron number at more than one stage of development suggests that it may have the
ability to be toxic to motoneurons throughout chick nervous system development.
The present study found that motoneuron number is reduced following ethanol
administration from E4 to El 1 and from E10 to E15. This result is similar to results
obtained from other neuronal populations such as cerebellar Purkinje cells which are
susceptible to ethanol during a range of time periods (Hamre and West, 1993; Phillips and
Cragg, 1982). Specifically, Purkinje cells are reduced in number following both prenatal
(Phillips and Cragg, 1982), and postnatal (Hamre and West, 1993; Phillips and Cragg,
1982) ethanol exposure. The similarity in temporal vulnerability between motoneurons and
Purkinje cells suggests that some fundamental resemblance between these two populations
determines their similar susceptibility to ethanol. To thoroughly and properly investigate
the role of ethanol in motoneuron reduction in the chick lumbar spinal cord, completion of a
parametric ethanol exposure study will be necessary. Such a study would allow us to
determine whether ethanol is directly toxic throughout chick embryonic development or if

48
ethanol is toxic at multiple critical periods. This knowledge would then allow us to better
hypothesize mechanisms for ethanol toxicity in this neuronal population.

CHAPTER 3
CHARACTERIZATION OF MOTONEURON SURVIVAL FOLLOWING ETHANOL
EXPOSURE AND CONCURRENT TREATMENT WITH EXOGENOUS GDNF OR
BDNF IN THE EMBRYONIC CHICK SPINAL CORD
Summary
Maternal consumption of ethanol is widely recognized as a leading cause of mental
and physical deficits. Many populations of the central nervous system (CNS) are affected
by the teratogenic effects of ethanol. Neuroprotection against ethanol has been studied
extensively in cell culture models and has also been studied in vivo in response to a variety
of neurotoxic events including hypoxia, ischemia, and hypoglycemia. Some neurotrophic
factors (NTFs) have been shown to protect against ethanol neurotoxicity in culture. The
only in vivo evidence of NTF prevention of ethanol neurotoxicity involved NGF protection
of choline acetyl transferase activity in early chick embryos (Brodie et al., 1991). Previous
studies in this laboratory have demonstrated that ethanol is toxic to developing chick
embryo motoneurons when administered from embryonic day 10 (E10) to E15. Other
laboratories have found that developing motoneurons are dependent on glial cell line-
derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF).
GDNF and BDNF suspend naturally occurring cell death (NOCD) in a subset of
developing motoneurons. These factors also rescue motoneurons from axotomy-induced
cell death in developing embryos. The concurrent delivery of GDNF with ethanol and
BDNF with ethanol was designed to test their ability to provide neuroprotection for this
ethanol-sensitive motoneuron population. Analysis of motoneuron number indicated that
GDNF, but not BDNF, significantly increased motoneuron number in the developing
spinal cord following embryonic ethanol exposure. However, GDNF was not found to
49

50
interact significantly with ethanol. Therefore, GDNF may serve to increase motoneuron
number to a level that is significantly greater than in ethanol treated embryos by a
mechanism that is independent of ethanol. Further studies should be developed to examine
this phenomenon in greater detail and determine whether GDNF does indeed provide
protection from ethanol toxicity.
Introduction
As was mentioned previously, much evidence has been gathered regarding the
effects ethanol exerts in the developing nervous system (Barnes and Walker, 1981; Jones
and Smith, 1973; Miller, 1986; Streissguth et al., 1991; West, 1986). Fetal alcohol
syndrome (FAS) produces CNS deficits that do not lessen as the patient ages (Streissguth,
1993). Postmortem analysis of human FAS neuropathology has identified CNS
abnormalities which include disorders of laminae of the cerebral cortex, cerebellar
abnormalities, a reduction of dendritic spines on cortical pyramidal cells, hippocampal
malformation, and microcephaly (Garren et al., 1978; Ferrer and Galofre, 1987).
Neuronal populations that are known to be affected by ethanol in animal models include the
cerebellum (Cragg and Phillips, 1985; West, 1986), the septohippocampal system (Barnes
and Walker, 1981), cerebral cortex (Miller, 1986), the substantia nigra (Shetty et al.,
1993), chief sensory trigeminal nucleus (Miller and Muller, 1989), red nucleus (Zajacet
al., 1989), inferior olivary nucleus (Napper and West, 1995), striatum (Heaton et al.,
1996) and motoneurons of the spinal cord (Bradley et al., 1997; Heaton and Bradley,
1995). Some of the microscopic and molecular changes that have been observed following
ethanol exposure include decreased dendritic arborization (Davies and Smith, 1981),
delayed synaptogenesis (Leonard, 1987), decreased neurotransmitter synthesis (Rawat,
1977; Swanson et al., 1994), changes in connectivity (West et al., 1994), and cell loss
(Barnes and Walker, 1981; Bauer-Moffet and Altman, 1975; West et al., 1986).
Ethanol affects chick embryo development in a manner similar to mammals. As
was mentioned previously, chicks exposed to ethanol prenatally have been shown to

51
exhibit reduced brain size, brain weight, DNA and protein synthesis (Pennington and
Kalmus, 1987), and reduced neurotransmitter synthesis (Brodie and Vemadakis, 1990;
Swanson et al., 1994). An advantage of using a chick model to study ethanol is the fact
that ethanol can be administered in exact doses to the developing embryo, and only
molecules produced by the embryo itself can remove the ethanol from the embryonic
environment. Maternal influences are removed when using the chick embryo as ethanol is
cleared from the bloodstream by the mother in a mammalian system. In the chick , the
embryo is isolated as it develops. The chick model allows the investigator to observe direct
effects of ethanol without possible interactions of maternal metabolism interfering. While
chick development is clearly different from mammalian gestation (and this is a caveat of
using the chick embryo as a model for FAS), this model allows researchers to study in vivo
interactions in a developing organism that are not possible in a mammalian model. This
model has been widely used to study the effects of NTFs on the developing motor system
(Oppenheim et al., 1995; Oppenheim et al., 1992). NTFs can be administered through
windows in the egg shell directly onto the chorioallantoic membrane. Developing chick
embryos tolerate slight invasions into their environment as long as the underlying
membranes are not disrupted.
The present studies focus on the motor system of the developing chick embryo.
Motoneurons have been shown to be susceptible to the toxic effects of ethanol both in
culture (Dow and Riopelle, 1985; Heaton et al., 1995b) and in vivo (Bradley et al., 1997;
Heaton and Bradley, 1995; Heaton et al., 1995b). Our laboratory has found that ethanol
can reduce motoneuron number when administered to chick embryos from E4 to El 1
(Heaton and Bradley, 1995) and when administered from E10 to E15 (Bradley et al.,
1997). This reduction is not dependent on NOCD since curare, an agent which blocks
NOCD, does not prevent this ethanol-induced death (Bradley et al., 1997). Furthermore,
when the period for NOCD had expired, ethanol still reduced motoneuron number (Bradley
et al., 1997).

52
Two NTFs important for motoneuron development are BDNF and GDNF. BDNF
is a member of the neurotrophin family of NTFs which includes nerve growth factor
(NGF), neurotrophin-3, and neurotrophin-4/5. A 118 amino acid residue polypeptide (Ilag
et ah, 1994), BDNF forms homodimers to attain its active form and binds with high
affinity to tyrosine receptor kinase B (trkB; Klein et ah, 1991). p75 is the low-affinity
binding receptor for all neurotrophins. The role of p75 in mediating neurotrophin-trk
binding is unclear. Primary sensory neurons display no biological activity when nerve
growth factor binds trkA in the absence of p75 (Verge et ah, 1992), while in other cell
types the trk receptors can work alone (Klein et al., 1991). BDNF is produced by the
developing skeletal muscle and is known to support motoneuron survival during
development and to protect motoneurons of both the chick and rat from degenerating after
lesion (Oppenheim et al., 1992; Sendtner et al., 1992; Yan et al., 1992). In addition to this
expression, BDNF is expressed in the hippocampus, adrenal gland, and in whole brain
during rat development (Maisonpierre et al., 1990). In the rat, mRNA for BDNF is
expressed in skeletal muscle both prenatally and postnatally (Griesbeck et al., 1995).
GDNF is a member of the transforming growth factor B (TGF-B) superfamily and
naturally occurs as a dimer with a molecular weight of 40-45 kD (each molecule 134 amino
acid residues; Lin et al., 1993). Recent studies suggest that GDNF and its receptors,
GDNFRa and c-ret, form a complex that allows c-ret to transduce the signals from GDNF
(Jing et al., 1996; Treanor et al., 1996). In this complex, GDNFRoc acts as aligand-
binding protein by binding GDNF (Jing et al., 1996). The GDNFR^-GDNF complex
then forms a complex with c-ret (Jing et al., 1996; Treanor et al., 1996). C-ret is the only
molecule of the complex capable of producing intracellular signals (Rosenthal, 1997).
During embryogenesis of the rat, GDNF mRNA is expressed by mesenchymal cells and in
developing skeletal muscle beginning at E15, and in developing skin beginning at E17
(Nosrat et al., 1996; Trupp et al., 1995; Wright and Snider, 1996). GDNF is also
expressed peripherally in the teeth, tongue, retina, nasal cavity, ear, kidney, and

53
gastrointestinal tract during various stages of development (Nosrat et al., 1996). In the
CNS, GDNF is expressed in the striatum, hippocampus, and cerebellum beginning at E15,
and in the trigeminal motor nucleus (E17) and cortex (postnatal day 7). C-ret is highly
expressed in substantia nigra dopaminergic neurons, a population which is protected from
6-hydroxydopamine (6-OHDA) lesion by exogenous GDNF (Trupp et al., 1995). Other
populations that are responsive to GDNF express c-ret, including spinal motoneurons
(Pachnis et al., 1993; Tsuzuki et al., 1995) and certain subpopulations of the peripheral
ganglia (Pachnis et al., 1993; Tsuzuki et al., 1995).
Studies of genetically altered mice, where a specific gene has been deleted from the
genome, have added to the knowledge of NTFs and their receptors. Knockout mice have
been created to study the relative importance of BDNF, trkB, p75, GDNF, and c-ret.
Since the present study is concerned with the development of the motor system, discussion
of these animals will be limited to this subject. While BDNF and p75 knockout mice do
not display a loss of motoneurons (Jones et al., 1994; Lee et al., 1992), trkB knockout
mice do contain reduced motoneuron number (Klein et al., 1993). The situation with
regard to GDNF and its receptor is somewhat different. GDNF deficient animals exhibit a
small but significant loss of motoneurons (Moore et al., 1996), while c-ret knockout mice
do not exhibit reduced numbers (Marcos and Pachnis, 1996). These results suggest that
the receptors that actually transduce the signals of BDNF and GDNF are important for
proper motoneuron development. Additionally, these results suggest that there may be
some redundancy in the NTFs that can activate trkB and c-ret, since GDNF-deficient and
BDNF-deficient animals exhibit no deficit in motoneuron number.
Neuroprotection by polypeptide growth factors has been studied extensively in
recent years. Examples of neuroprotection include epidermal growth factor protection of
whole brain neuronal cultures from anoxia (Pauwels et al., 1989), NGF protection of rat
hippocampal and human cortical neurons from hypoglycemia (Cheng and Mattson, 1991),
and basic fibroblast growth factor (bFGF) prevention of thalamic degeneration following

54
cortical infarction (Yamada et al., 1991). In the developing nervous system, GDNF has
been shown to be potent in protecting neurons from a variety of conditions that normally
cause death such as NOCD (Oppenheim et al., 1995), 6-OHDA lesion (Choi-Lundberg et
ah, 1997; Kearns and Gash, 1995; Tomac et ah, 1995), and axotomy (Giménez y Ribotta
et ah, 1997; Houenou et ah, 1996; Oppenheim et ah, 1995). BDNF is also effective in
providing neuroprotection from toxic events. For example, BDNF rescues some neurons
ischemia-induced cell death in rat hippocampal slice cultures (Pringle et ah, 1996). BDNF
has also been shown to reduce NOCD in motoneurons (Oppenheim et ah, 1992) and
apoptotic death in PC12 cells (Jian et ah, 1996) and cultured rat cerebellar granule neurons
(Kubo et ah, 1995). The fact that both GDNF and BDNF provide such potent support for
developing and injured neurons suggests that both could protect motoneurons from
ethanol-induced death.
Neuroprotection from ethanol has been demonstrated in culture in previous
research. Previous studies in our laboratory found that NGF can protect cultured dorsal
root ganglion (DRG) neurons (Heaton et ah, 1993) and septal neurons (Heaton et ah,
1994) from ethanol toxicity. bFGF was shown to afford some neuroprotection to cultured
septal and hippocampal neurons (Heaton et ah, 1994). NGF and bFGF protect cultured
cerebellar granule cells from ethanol-induced cell death (Luo et ah, 1997). This
neuroprotection afforded by NGF and bFGF was found to require both protein and RNA
synthesis (Luo et ah, 1997). This result suggests that neuroprotection is related to a signal
that the NTF receptor sends to the nucleus of the cell. NGF protection of choline acetyl
transferase activity from ethanol was the first in vivo demonstration of NTF
neuroprotection from ethanol (Brodie et ah, 1991). The previous use of the chick embryo
in both in vivo NTF (Oppenheim et ah, 1995; Oppenheim et ah, 1992) and ethanol
research (Heaton and Bradley, 1995) makes it an excellent choice for studying ethanol-
NTF interactions.

55
The objective of the present experiment was to determine whether exogenous NTFs
could provide protection for motoneurons exposed to ethanol from E10 through E15. Both
GDNF and BDNF are known to be NTFs for developing motoneurons (Henderson et al.,
1994; Oppenheim et al., 1995; Oppenheim et al., 1992; Sendtner et al., 1992; Yan et al.,
1992). The concurrent delivery of each of these NTFs with ethanol was designed to test
the ability of each to provide neuroprotection for this ethanol-sensitive population.
Analysis of motoneuron number indicated that GDNF, but not BDNF, resulted in
increasing the number of motoneurons present in the lumbar spinal cord following ethanol
exposure in the developing lumbar spinal cord.
Materials and Methods
Subjects
White Leghorn chick eggs were obtained from the University of Florida Poultry
Science Department. Eggs were placed in a Marsh incubator and maintained at 37°C and
70% relative humidity until E4. At that time, the eggs were moved to a forced draft turning
incubator, maintained at the same conditions indicated above, and divided into groups. Six
experimental groups were used in this study: Ethanol, Saline, GDNF, GDNF+Ethanol,
BDNF, and BDNF+Ethanol. Embryos received daily injections of ethanol, saline, a NTF,
or a combination of ethanol and an NTF from El 0 to E15. At E16, embryos were removed
from the eggs, sacrificed by decapitation, and the lumbar section of the spinal cord
removed and prepared for histology.
Injections
Ethanol and saline injections were administered daily from E10 through E15.
These dates were chosen to replicate a previous study from this laboratory in which ethanol
was shown to reduce motoneuron number in the lumbar spinal cord (Bradley et al., 1997),
and because NOCD, which occurs in the chick spinal cord from E5 through E9 (Pittman
and Oppenheim, 1978), is completed at this time. Since NOCD is completed when

56
injections begin, any change in motoneuron number observed is attributable to treatment
per se and not an interaction of treatment with NOCD. Ethanol embryos received 150 pi of
30% w/v ethanol (45 mg ethanol per day), dissolved in a 0.9% w/v nonpyrogenic saline
vehicle, through a pinhole in the shell into the airspace. Previous work in our laboratory
has determined that this concentration of ethanol produces blood ethanol counts that peak
between 250 and 300 mg/dl (Bradley et al., 1997). Saline embryos received 150 pi of the
0.9% w/v nonpyrogenic saline vehicle. NTF injection [GDNF (Amgen) or BDNF
(Regeneran)], which also occurred from E10 through E15, involved creating a pinhole
directly over the embryo in addition to the pinhole created in the airspace. The airspace was
then allowed to shift to a position superior to the embryo and 50 pi of 0.2 mg/ml NTF was
injected into that space above the embryo (10 pg GDNF or BDNF per day). These levels
of GDNF and BDNF administration were previously shown to rescue motoneurons from
NOCD without being toxic to the developing embryo (Oppenheim et al., 1995; Oppenheim
et al., 1992). GDNF+Ethanol and BDNF+Ethanol embryos were given ethanol injections
from E10 to E15 and NTF injections from E10 to E15 as described above. Ethanol
injections preceded NTF injections and the embryos were allowed to sit in a Marsh
incubator for a period of one hour between injections. This delay in injection time was
necessary to ensure that the ethanol was absorbed through the inner shell membrane within
the airspace before the eggs were turned on their side for NTF administration. Also, the
two injections administered in this study represent a significant volume (200 pi) for the
embryonic system to incorporate on a daily basis. The delay between injections, therefore,
allowed absorption of the volume of ethanol before the NTF solution was presented to the
embryo. Pinholes created by the injection process were sealed with paraffin immediately
following injection to prevent evaporation and/or leakage of the solutions. The eggs were
then returned to the turning incubator.

57
Dissections and Histological Procedures
Embryos of all experimental groups were sacrificed by decapitation on El6 and the
lumbar section of the spinal cord removed. The vertebrae of the spinal cords were cut
along the dorsal surface to expose the nervous tissue and allow the fixative to adequately
penetrate the tissue. Following dissection, the E16 spinal cords were placed in Bouin’s
Fixative for 14 to 21 days to allow the vertebrae to decalcify (Li et al., 1994). The tissue
was then embedded in paraffin, cut into 12 pim coronal sections, mounted onto glass
slides, and stained with hematoxylin and eosin.
Motoneuron Size and Spinal Cord Length
Motoneuron size and spinal cord length were measured to determine whether
ethanol alters any general characteristics of the motoneuronal system. Motoneuron size
was determined by measuring the diameter of 10 random cells in the same rostral-caudal
position of the region of each embryonic spinal cord with an eyepiece micrometer. The
section exactly 2400 pim following the beginning of the lumbar spinal cord was sampled.
Three embryos from each experimental condition were analyzed for a total of 30 cells per
condition. Spinal cord length was determined by counting the number of sections present
in each embryo following determination of the boundaries of the lumbar spinal cord by the
anatomical methods described previously and multiplying this number by the section
thickness (12 ¡¿m).
Motoneuron Counts
Motoneuron counts were completed following methods described previously
(Hamburger, 1975; Heaton and Bradley, 1995). Briefly, a uniform area encompassed by 6
DRG was noted in each embryo. This procedure ensured that a similar area was counted in
each subject. Starting from the most rostral section included in the 6 DRG region,
motoneurons in the lateral motor column of one side of every tenth section were marked
onto paper using a camera lucida. At 400X magnification, motoneurons were identified in

58
the lateral motor column by their large size, dark cytoplasm, and nucleolus. Laterality was
maintained throughout each individual embryo, but chosen at random before beginning the
counting process. Previous studies have shown that there is no difference between the
number of motoneurons contained in the right and left sides of the spinal cord (Pittman and
Oppenheim, 1979). Each embryo was coded so that the experimenter had no knowledge of
its experimental treatment until the study was completed.
Statistical Analyses
Two-way analysis of variance was performed using SAS version 6.12 on a
pentium computer. When applicable, individual differences between groups were tested
using Fisher’s protected least significant difference post-hoc analyses. Statistical
significance was determined to be p<0.05.
Results
GDNF administration did not seem to harm the embryos since survival was 93% in
the GDNF group and 69% in the GDNF+Ethanol group. These rates compare favorably
with control survival rates where 90% of the Saline group and 60% of the Ethanol group
survived. BDNF administration had a somewhat different effect upon chick survival. The
BDNF group survived at a rate of 82% and the BDNF+Ethanol group survived at a rate of
65%. These results indicate that the level of GDNF and BDNF administered in this study
is not toxic to overall embryonic survival.
Motoneuron Size and Spinal Cord Length
As stated in the Methods section above, motoneuron size and spinal cord length
analyses were performed to determine whether treatments utilized in these studies altered
the gross morphology of the embryonic spinal cord. Analysis of variance testing indicated
that embryos from these experimental groups exhibited no differences in motoneuron size
due to treatment (F=0.13, df=29, p>0.9). That is, the motoneuron size of ethanol-treated
animals was unchanged from that of NTF-treated, or control animals. Analysis of variance

59
also revealed that overall lumbar spinal cord length was unaltered by treatment (F=0.44,
df=40, p>0.8). There were no significant differences in the length of the spinal cord
region counted among the experimental groups. Table 3-1 displays the data from this
portion of the study. Taken together, these results suggest that the treatments administered
in this study did not adversely affect the basic anatomy of the spinal cord.
Table 3-1. Motoneuron Size and Spinal Cord Length.
Group Cell Size (pim) Spinal Cord Length ()
Saline
18.6 + 0.525
6020+ 69.0
Ethanol
18.9 + 0.508
5966 + 137.9
GDNF
18.7 + 0.514
5820 + 154.2
GDNF+Ethanol
18.4 + 0.502
5856 + 148.9
BDNF
18.6 + 0.433
5904+ 88.2
BDNF+Ethanol
18.6 + 0.438
5900+ 153.1
All values are means + SEM. Motoneuron size was determined by measuring the diameter
of 10 random cells in the same rostral-caudal position of the region of three embryonic
spinal cords with an eyepiece micrometer. Therefore, n=30 for each group. Spinal cord
length was computed by determining the number of sections present in a given spinal cord
and then multiplying by section thickness (12 pim). For spinal cord length, Saline n=12,
Ethanol n=7, GDNF n=6, GDNF+Ethanol n=5, BDNF n=5, and BDNF+Ethanol n=6.
Motoneuron Number
Two way analysis of variance indicated a significant effect due to neurotrophic
factor administration (F=5.645, df=40, p<0.05) and to ethanol treatment (F=8.902, df=40,
p<0.005), but not an interaction between neurotrophic factors and ethanol (F= 1.708,
df=40, p>0.15). Further analysis limiting the groups to Saline, Ethanol, GDNF, and
GDNF+Ethanol found similar results. Analysis of variance found a significant effect due
to GDNF treatment (F=9.143, df=29, p<0.01), ethanol treatment (F=6.841, df=29, but
not an interaction between the two groups (F=0.786, df=29, p>0.35). Limiting the groups
to Saline, Ethanol, BDNF, BDNF+Ethanol did not produce similar results. Analysis of
variance found a significant effect due to Ethanol treatment (F=4.381, df=29, p<0.05), but
not to BDNF treatment (F=0.671, df=29, p>0.4), or an interaction between ethanol and

60
BDNF (F=2.182, df=29, p>0.15). Figure 3-1 displays the results of these cell counts.
Post hoc testing revealed significant differences among the groups. Specifically, Ethanol
embryos contained significantly fewer numbers of motoneurons than GDNF (p<0.0005),
GDNF+Ethanol (p<0.05), and Saline (p<0.01) treated embryos. There were no significant
differences among the GDNF, GDNF+Ethanol, or Saline groups. Likewise, there were no
significant differences among the BDNF, BDNF+Ethanol, Saline, or Ethanol groups. The
above results suggest that GDNF significantly increased motoneuron number in a manner
that is independent of ethanol. BDNF did not provide significant protection from ethanol.
In addition to comparing motoneuron number among the various groups in this study and
performing a two way analysis of variance for NTF and ethanol treatment, a contrast
analysis was performed to determine if there was an interaction between GDNF and ethanol
or between BDNF and ethanol. This analysis found that there was not a significant
difference between the difference of GDNF from Ethanol and BDNF from Ethanol
(p>0.5). The conclusion of this analysis is that there was no interaction between GDNF
and Ethanol or between BDNF and Ethanol. Stated another way, the action of ethanol is
independent of the action of either GDNF or BDNF. Figure 3-2 illustrates this conclusion.
The slope of the two lines in the figure are not significantly different This result is
important because it suggests that the NTFs used in this study are not impaired by the
actions of ethanol. This analysis is further evidence that ethanol acts independently of the
NTFs used in the present study.
Figure 3-3 and Figure 3-4 display photographs of spinal cords taken from the
animals described in this chapter. Specifically, Figure 3-3 displays photographs of Saline,
Ethanol, GDNF, GDNF+Ethanol, BDNF, and BDNF+Ethanol spinal cords at40x
magnification. The overall shape of the spinal cord is altered somewhat by ethanol
treatment as fewer motoneurons are present and there is less of a “bulge” on the outer edge
of the cord. Figure 3-4 displays 200x magnification views of the same spinal cord

61
Saline Ethanol GDNF GDNF+ BDNF BDNF+
Ethanol Ethanol
Figure 3-1. Number of motoneurons in the later motor column of the
lumbar spinal cord at E16. Motoneuron counts are displayed as means
+ SEM. Data represent actual counts obtained from the lumbar spinal
cords and are not population estimates. All injections were
administered from E10 to E15. * = statistical significance in
comparison to Ethanol; saline (p < 0.01), GDNF (p < 0.0005), and
GDNF+Ethanol (p < 0.05). Number of animals used for the experiment
equal to 12 Saline, 7 Ethanol, 6 GDNF, 5 GDNF+Ethanol, 5 BDNF,
and 6 BDNF+Ethanol.

62
Figure 3-2. Interaction between ethanol and neurotrophic factors. This
figure represents the slope of the interaction between GDNF and ethanol
and between BDNF and ethanol. There was not a significant difference
between the GDNF group and the BDNF group which suggests that the
action of each NTF and ethanol is independent. Number of animals
used for the experiment equal to 12 Saline, 7 Ethanol, 6 GDNF, 5
GDNF+Ethanol, 5 BDNF, and 6 BDNF+Ethanol.

Figure 3-3. Photomicrographs of coronal sections from the midlumbar region of E16
spinal cords. A. Saline, B. Ethanol, C. GDNF, D. GDNF+Ethanol, E. BDNF, and
F. BDNF+Ethanol. The only noticeable difference is in the density of the and number of
the motoneurons present in the lateral motor column where the Ethanol group appears to
have fewer motoneurons present. Number of animals used for the experiment equal to 12
Saline, 7 Ethanol, 6 GDNF, 5 GDNF+Ethanol, 5 BDNF, and 6 BDNF+Ethanol.

64
*«iU:
|P& ó/í ;» XtóaíSrf1^
;
:â– - Vv -.'. '
.
i,«; ', • «Í •'li^. 4,'V*J
Saline
S
â–  â– â– ;â– â–  â– 'â– - ,",v -nil , -V V - :â– â– 
áOT4B&mHM9r>
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SIS «
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i r'i *,> <
•V. • r^r^;W
?* ray**v •< 5|s!.v ¿vsjrri#^
| siV..Y-1 ;v?i'.* {}*¡,v-.f‘( '■?»* v*. v•’ * vtój\ Y$fiiíi'
:v;.- ... ’.:■
GDNF
GDNF+Ethanol
—v ■■ , ■ ' ■;,' ,
■ V '•'••:• .; " • /•«¿‘ '■‘.' ■.’,fJ-.'V
■'■' ■;•"■'.'■■' ■•■líj ;. ■.t,v::';^;.;
;■• « i, kJk
4.V-•;>;:?? ;i.e'•>!,.•' “■ •: ■;.;vi4.i ;
, ' ;g¡¿'
• t ■ ;
' â– /) '
BDNF
Figure 3-3.

Figure 3-4. High magnification photomicrographs from the midlumbar section of E16
spinal cords. A. Saline, B. Ethanol, C. GDNF, D. GDNF+Ethanol, E. BDNF, and
F. BDNF+Ethanol. This view gives a better perspective of the motor column of each
animal. The difference in the number and density of the Ethanol animals is apparent.

66
Saline
. c < *• *
r.>-i'#r-*•'.'.*•;•1 "X .<*.£...v¿(V- ’.1
;rb\.V‘T‘.V:4 ;.•■ ‘
.■: i Q-j? f.. •< ■■!&■'• i
t ,*jiv . ,& « <3f '<* V „ f'f»* •' •
m ' ■ ■ k-: ' • .-• -. .. v: •
* . 1: ,■ • ■•• • '•* "■ ■ * -.'*'•!»?•» • ':
¿t&Xi:** u m **■$
' '>* -.i*-: **" '•.¡•■■i" ■< ír>$,.-i'
4- . .vi- STi •: • • • •• •:.
Ethanol
-'■'V «¿538*affiPK
GDNF+Ethanol
. , •■* .. .
.. * 4,’!» .** i. ' ...V •
'*'*•' *¡ >»Hi. * ^/.v:
BDNF
BDNF+Ethanol
Figure 3-4.

67
sections. The overall density and number of motoneurons in the lateral motor column again
appears reduced in comparison to the other groups.
Discussion
The major finding of this study is that GDNF can significantly increase motoneuron
number in the lumbar spinal cord in a manner independent to ethanol (see Figure 3-1). In
vivo neuroprotection from ethanol neurotoxicity was not demonstrated in embryos treated
with BDNF. The results of the two way analysis of variance indicated a significant effect
due to ethanol and NTF treatment. Further analysis of the groups indicated that only
GDNF administration had a significant effect upon motoneuron number while BDNF did
not. An interaction between either GDNF and ethanol or BDNF and ethanol was not
indicated by this powerful statistical test. Thus, the increase in motoneuron number by
GDNF is not due to some action of GDNF upon ethanol, but rather GDNF increases
motoneuron number in a manner that is independent of ethanol toxicity. Ethanol and NTFs
were administered during a period of development when NOCD was complete, but when
motoneuron number can be diminished by ethanol exposure. Therefore, NOCD did not
provide an added variable for this study. These results support previous research from
other laboratories which found that GDNF could protect certain neuronal populations from
various neurotoxic events. For example, GDNF protected rat nigral dopamine neurons
against 6-OHDA lesion in vivo (Choi-Lundberg et al., 1997; Kearns and Gash, 1995;
Tomac et ah, 1995). Also, GDNF prevented death of spinal cord motoneurons following
axotomy in the chick (Houenou et ah, 1996; Oppenheim et ah, 1995) and rescued facial
motoneurons following axotomy in the rat (Giménez y Ribotta et ah, 1997).
The receptor thought to be responsible for GDNF’s activity in the nervous system
is c-ret and the high affinity receptor for BDNF is trkB. Recent studies suggest that
GDNF, GDNFRoc, and c-ret form a complex that allows c-ret to transduce the signals from
GDNF (Jing et ah, 1996; Treanor et ah, 1996). The ontogeny of the receptors for GDNF
and BDNF follow different patterns during the development of the neuromuscular system.

68
C-ret mRNA expression is detectable, albeit very weakly, in chick spinal cord motoneurons
as early as E5 and increases throughout development (Nakamura et al., 1996; Schuchardt
et al., 1995). By E17, c-ret mRNA expression in the chick is expressed at very high levels
in spinal cord motoneurons (Nakamura et al., 1996; Schuchardt et al., 1995). Therefore,
c-ret is expressed by the desired target, motoneurons, during the exposure period of the
present study (E10 to E15). TrkB mRNA is first detectable at E8 and its level of
expression increases throughout development (McKay et al., 1996). At E16, trkB mRNA
is highly expressed in the lateral motor column of the spinal cord of the chick (McKay et
al., 1996). Therefore, trkB is expressed by motoneurons during the exposure period of the
present study. Since both receptors are expressed during the period of exposure used in
the present study, another reason must explain the fact that GDNF increases motoneuron
number from ethanol toxicity whereas BDNF does not.
In culture chick motoneurons are supported by GDNF (Gouin et al., 1996) whereas
they are not supported by BDNF (Arakawa et al., 1990). This is not the case in cultures of
rat motoneurons where BDNF does support their growth (Henderson et al., 1993). Even
though both GDNF and BDNF prevent NOCD in spinal cord motoneurons, these cells are
rescued from NOCD to a greater extent by GDNF than BDNF (Oppenheim et al., 1995;
Oppenheim et al., 1992). This fundamental advantage of GDNF over BDNF to support
chick motoneurons in culture may explain the results of the present study. The advantage
of GDNF to support motoneurons to a greater degree than BDNF is supported by the
results of knockout studies. As was mentioned above, GDNF-deficient, but not BDNF-
deficient mice exhibit reduced motoneuron number (Jones et al., 1994; Moore et al., 1996).
Again, the implication of those studies is that GDNF is required to a greater degree for
proper motoneuron development than is BDNF. The present study found that GDNF
administration concurrent with developmental ethanol exposure increased motoneuron
number in a manner independent of ethanol, while BDNF did not significantly alter ethanol
toxicity. This latter portion of the statement is supported by the fact that two way analysis

69
of variance testing did not find a significant effect due to BDNF treatment or an interaction
between ethanol and BDNF.
Further experiments using animals genetically altered to overexpress GDNF might
provide further information about the nature of the increase in motoneuron number
following embryonic ethanol exposure. By administering ethanol to these animals,
researchers could examine whether GDNF produced by the animal itself could increase
motoneuron number following ethanol exposure. If these animals proved to be more
resistant to ethanol insult, it would suggest that some mechanism of the endogenous NTF
naturally protects developing motoneurons from ethanol to some extent, provided an
interaction between ethanol and the NTF is demonstrated. Obviously, the normal
endogenous activity of NTFs in the nervous system does not protect developing chick
motoneurons from ethanol toxicity since exposure from E10 to E15 reduces motoneuron
number (Bradley et al., 1997).
Another line of inquiry could be into cell death genes and the roles they play in NTF
neuroprotection. Ethanol is known to induce apoptosis in culture (Bhave and Hoffman,
1997; De et al., 1994; Ewald and Shao, 1993; Liesi, 1997) and in vivo (Renis et al., 1996;
Singh et al., 1995). The bcl-2 family of cell death molecules has been shown to be
involved in apoptosis (Boise et al., 1993; Hockenbery et al., 1990). Members of the bcl-2
family include bcl-2, bcl-Xs, bcl-XL, and bax (Boise et al., 1993; Oltavi et al., 1993). Bcl-
2 is a membrane-associated protein that interferes with apoptotic cell death. Bcl-Xs acts to
antagonize bcl-2 activity and promote cell death. Bcl-XL acts in much the same way as bcl-
2 and bax binds bcl-2 to inhibit its ability to prevent apoptosis (Davies, 1995). To
determine whether the bcl-2 family is involved in GDNF increasing motoneuron number in
the presence of ethanol, enzyme-linked immunosorbent assay could be used to determine
precise levels of these molecules in motoneuron cultures exposed to ethanol and GDNF.
The experiments would be designed to determine whether GDNF added to these cultures in
the presence of ethanol induces greater expression bcl-2 and bcl-X, —which both prevent

70
apoptosis—or decreases expression of bcl-Xs and bax—which both oppose the protective
activity of bcl-2. The results from these proposed experiments would complement the
results of the current experiments since they could provide a potential mechanism for
neuroprotection, should it happen to be demonstrated in the future, by GDNF. It is
important to note that such a mechanism could proceed independent of ethanol since ethanol
could conceivably induce apoptosis by another mechanism such as a change in Ca2+
homeostasis (Koike and Tanaka, 1991; Webb et al., 1995).
Evidence linking the roles of NTFs and cell death genes has been examined in
previous studies. Bcl-2 is required for the survival of PC-12 cells dependent on BDNF but
not required for survival of CNTF dependent cells (Allsopp et al., 1995). Similarly, bcl-2
expression is required for the survival of NGF-dependent PC-12 cells (Katoh et al., 1996).
Furthermore, NGF has been shown to increase bcl-2 expression in a dose-dependent
manner in providing this trophic support for these cultured cells (Katoh et al., 1996). A
similar effect is observed in neuronal cells in that cultured trigeminal ganglion and
trigeminal mesencephalic neurons are rescued from cell death due to withdrawal of NGF,
BDNF, or NT-3 by overexpression of bcl-2 (Allsopp et al., 1993). NTFs are related to
and alter expression of proteins that promote cell death. Withdrawal of trophic support did
not result in death of axotomized facial motor neurons in bax-deficient mice (Deckwerth et
al., 1996). The above examples demonstrate the link between NTFs and cell death genes
of the bcl-2 family.
The methodology for determining motoneuron number employed in this study has
been used successfully for determining motoneuron number in this laboratory and others
(Bradley et al., 1997; Heaton and Bradley, 1995; Oppenheim et al., 1995; Oppenheim et
al., 1992; Pittman and Oppenheim, 1979). The results indicate that while ethanol
administration does have an adverse effect on the motoneuron population of the lumbar
spinal cord, it does not change the overall morphology of the cord. This claim is supported
by the fact that lumbar spinal cord length, which is an indicator of spinal cord volume, and

71
average motoneuron size are unchanged following ethanol treatment. A previous study
from another laboratory also found that administration of exogenous NTFs from E9 to E15
did not alter either motoneuron size or spinal cord length measurements (Qin-Wei et al.,
1994). NTFs administered in that study included BDNF, TGF-B, basic fibroblast growth
factor, and ten other growth factors (Qin-Wei et al., 1994). The present study supports
this finding and has found that exogenous GDNF or BDNF does not alter these
relationships since there were no significant differences between any of the experimental
groups when motoneuron size and spinal cord length were analyzed (See Table 3-1).
A portion of the current results are somewhat inconsistent with previous research
(Oppenheim et al., 1995) in that the present study found that motoneuron number in
GDNF-treated embryos did not differ significantly from control embryos. That study
found that exogenous GDNF administered from E9 to E15 resulted in significantly more
motoneurons in the lateral column of the spinal cord than in control embryos (Oppenheim et
al., 1995). A likely explanation for the differences between these two studies is that
GDNF injections began on different days. Recall that NOCD continues in the spinal cord
through E9 (Pittman and Oppenheim, 1979). By beginning GDNF injections on E9, the
earlier study may have rescued some motoneurons that were destined to undergo NOCD
and sustained them until E16. The Oppenheim et al. study (1995) found a 12.5% increase
in motoneuron number, while the present study found an 8%, but not statistically
significant, increase in motoneuron number in comparison to control embryos. Therefore,
the absolute difference between the two studies is relatively minimal.
Previous studies led to the hypothesis that ethanol-induced hypoxia may cause
excitotoxic damage to developing neurons (Altura et al., 1983; Auer et al., 1989; Barnes
and Walker, 1981; Bonthius and West, 1990; Jones et al., 1981; Jorgensen and Diemer,
1982; Michaelis, 1990; Mukherjee and Hodgen, 1982; Pierce et al., 1989). NTFs have
been shown to prevent hypoxic/ischemic damage in neurons in research performed in other
laboratories. For example, BDNF has been shown to prevent ischemia-induced cell death

72
in rat hippocampal slice cultures (Pringle et al., 1996). Even though previous research has
not explicitly supported the role of hypoxia in ethanol toxicity, prevention of
hypoxia/ischemia is another possible mechanism that could explain GDNF’s increase of
motoneuron number in the presence of ethanol. Previous research has not searched for a
link between ethanol and hypoxia in the developing chick. Hypoxia alone has been studied
in this animal model. Hypoxic conditions were found to reduce the overall vascularity of
the chorioallantoic membrane (Strick et al., 1991) and to reduce its blood flow (Ar et al.,
1991). The fact that the direct relationship between ethanol and hypoxia has not been
explored in the chick does not mean hypoxic conditions do not occur in ethanol-exposed
chicks. Perhaps a future aim of this research should be to determine whether or not this
relationship does exist in developing chick embryos.
As was discussed earlier in the Introduction section of this chapter, the chick
embryo exhibits many of the characteristics found in mammalian models of FAS.
Morphologically, chick embryos treated with ethanol are smaller than controls and have
decreased brain weights (Pennington and Kalmus, 1987). These similarities between avian
and mammalian FAS also include molecular changes attributed to ethanol treatment. For
example, ethanol decreases kinase activities in whole brain of both the chick and rat
(Kruger et al., 1993; Pennington, 1990) and alters cyclic AMP levels in both chick whole
brain and in rat striatum (Lucchi et al., 1983; Pennington, 1990). Additionally,
neurotransmitter synthesis is altered by ethanol administration in both the chick and rat
(Brodie and Vernadakis, 1990; Swanson et al., 1994; Swanson et al., 1995). Other
molecular mechanisms attributed to ethanol in culture are potentially occurring in the avian
model of FAS. These include those discussed previously such as hypoxia/ischemia (Zajac
and Abel, 1992) and apoptosis (Renis et al., 1996; Singh et al., 1995). Other mechanisms
implicated in FAS include hypoglycemia (Fisher et al., 1986) and generation of free
radicals (Henderson et al., 1995). Such actions could be responsible for the loss of
motoneurons in the spinal cord observed following ethanol exposure from E10 to E15

73
(Bradley et al., 1997). In addition to NTF protection afforded to neurons from apoptosis
and hypoxia/ischemia, NGF protects cultured rat hippocampal and human cortical neurons
from hypoglycemic damage (Cheng and Mattson, 1991) and oxidative damage (Mattson
and Cheng, 1993). The ability of NTFs to inhibit the processes described above could
provide the mechanism behind the current finding that GDNF increased motoneuron
number following ethanol exposure. However, the fact that there was not a significant
interaction between ethanol and GDNF only allows the following conclusion: GDNF
increases motoneuron number in the lumbar spinal cord in a manner independent to ethanol
toxicity.
NTFs have proven to be versatile molecules with the ability to sustain neurons
when faced with a variety of potentially deadly insults. Now that an increase in
motoneuron number by GDNF following ethanol exposure has been demonstrated,
additional investigations will need to be conducted to further examine this action of NTFs
on developing motoneurons. All of these analyses should be interpreted bearing in mind
that the actions of ethanol and the NTFs studied here did not significantly interact.
Therefore, their actions may be entirely independent. Other NTFs will be tested for their
ability to protect motoneurons from ethanol toxicity. In addition, combinations of NTFs
should be tested to determine whether protection is better than when a given NTF is
administered alone. Specifically, CNTF and NT-3 are good candidate molecules since they
are known to promote motoneuron survival following axotomy or during the penod for
NOCD (Lo et al., 1995; Yin et al., 1994). Since GDNF increases the number of
motoneurons independent of ethanol toxicity, other ethanol-sensitive populations might be
increased in number following administration of GDNF, or other NTFs. Clearly, this
phenomenon will have to be investigated further to adequately describe the actions of
various NTFs are protecting neuronal populations or truly increasing neuron number in a
manner independent of the action of ethanol.

74
In addition to testing other NTFs to determine whether they can provide
neuroprotection from ethanol toxicity, future studies should attempt to build on results
obtained from the present study to develop potential therapies for FAS. GDNF inserted
into an adenovirus vector has already proven effective in preventing facial motoneuron
death following axotomy (Giménez y Ribotta et al., 1997). Such a delivery system could
prove effective in getting GDNF to spinal cord motoneurons in mammals to increase
motoneuron number following ethanol administration. Future research should also focus
on determining the mechanism by which this increase in motoneuron number is afforded to
motoneurons by GDNF. By introducing toxic insults such as hypoxia/ischemia and
hypoglycemia, GDNF’s ability to increase motoneuron number in the presence of ethanol
should be further defined. Unfortunately, more questions remain to be answered about
GDNF in the future than are answered by the present study.

CHAPTER 4
CHARACTERIZATION OF THE NEUROTROPHIN AND NEUROTROPHIN
RECEPTOR GENE EXPRESSION IN THE HIPPOCAMPUS FOLLOWING CHRONIC
TREATMENT AND EARLY POSTNATAL ETHANOL TREATMENT INTHE RAT
Summary
Fetal alcohol syndrome (FAS) is caused by maternal consumption of ethanol during
pregnancy and was first described more than two and one-half decades ago. In recent
years, the thrust of research in this field has been the search for a mechanism of ethanol
toxicity. Signal transduction and gene expression studies have allowed researchers to learn
about how ethanol affects neuronal populations at the molecular level (Davis-Cox et al.,
1996; Gandhi and Ross, 1989; MacLennan et al., 1995; Torres and Horowitz, 1996).
Another area that has garnered attention in this field is neurotrophic factors (NTFs) and
their ability to affect, and be affected by, ethanol. Previous research has found that ethanol
can alter expression of specific genes. Examples of genes regulated by ethanol exposure
include insulin-like growth factor I (IGF-I) and IGF-II in rat brain (Breese et al., 1994;
Singh et al., 1996). Both of these genes are decreased following ethanol exposure.
However, ethanol exposure increases NMDA receptor gene expression in cultured mouse
cortical neurons (Hu et al., 1996). The present study attempted to determine the relative
expression of brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), tyrosine
receptor kinase B (trkB), and trkC in the hippocampus following ethanol exposure during
the prenatal or early postnatal period. TrkA was not analyzed because of its extremely low
level of expression in the developing hippocampus (Martin-Zanca et al., 1990). Nerve
growth factor (NGF), although attempted, was not assessed because it produced signals
that were not quantifiable. The results of our analyses indicated that ethanol administration
dunng prenatal development in the rat did not change the genetic expression of BDNF,
75

76
NT-3, and trkB as assessed by quantitative Northern blotting. TrkC expression in male
animals, but not female animals, exposed to ethanol prenatally was reduced. Expression of
BDNF, NT-3, trkB and trkC was unaffected by postnatal exposure to ethanol during the
brain growth spurt (BGS).
Introduction
Maternal consumption of ethanol exerts many effects upon the developing nervous
system (Barnes and Walker, 1981; Jones and Smith, 1973; Miller, 1986; Streissguth et al.,
1991; West, 1986). FAS continues to be a problem in Western countries and is diagnosed
in 1-2 out of every 1000 live births in the United States (Abel, 1995). FAS is characterized
by low birth weight, decreased memory and learning, hyperactivity, facial dysmorphia, and
lowered IQ (Jones and Smith, 1973; Streissguth et al., 1991). Children born to heavy
drinkers experience a higher incidence of FAS with a 4.3% diagnosis rate (Abel, 1995).
Previous research suggests that the deficits observed in FAS patients are permanent and do
not lessen with age (Streissguth, 1993). Taken together, these observations led to the
assertion that maternal consumption of ethanol is the leading known cause of mental
retardation in the Western Hemisphere (Bonthius and West, 1988).
Postmortem analysis of human FAS neuropathology has identified central nervous
system (CNS) abnormalities which include disorders of laminae of the cerebral cortex,
cerebellar abnormalities, a reduction of dendritic spines on cortical pyramidal cells, changes
in hippocampal development, and microcephaly (Clarren et al., 1978; Ferrer and Galofre,
1987). A major problem of examining human subjects is that variables such as nutrition
and polydrug use are uncontrolled. Animal models have provided controlled exposure to
ethanol and have been shown to exhibit CNS deficits and behavioral consequences similar
to those observed in humans (Driscoll et al., 1990). Animal models are particularly useful
for gaining insight into the effects ethanol exerts on a molecular scale. These models allow
researchers to answer questions about ethanol consumption that cannot be answered in
human studies due to ethical and practical reasons (West et al., 1994).

77
The rat is the most widely used model in FAS research. However, a caveat of
using the rat as a model is its gestational period relative to human development. The rat
gives birth on what is roughly the equivalent of the beginning of the third trimester in
humans (Goodlett et al., 1993). Important events, such as the brain growth spurt (BGS)—
where many functional synapses are made in the nervous system—occur in útero during the
third trimester in humans and postnatally from P4-P10 in rats (West, 1987). Therefore,
experiments that incorporate ethanol exposure during gestation or during the BGS in rats
allow researchers to use this as a model of human third trimester ethanol exposure.
Exposing rat pups to ethanol postnatally produces deficits that demonstrate the importance
of the BGS and the sensitivity of the CNS to ethanol during this period. Similar to prenatal
exposure to ethanol in rats, postnatal exposure can produce loss of cerebellar Purkinje cells
(Bonthius and West, 1990; Bonthius and West, 1991; Goodlett and West, 1992; West,
1986; West et al., 1990). A problem inherent to any postnatal exposure paradigm is that
maternal metabolism of ethanol is removed and the subjects are exposed to ethanol in a
more “adult” manner. Another problem is delivery of ethanol. Suckling rats cannot be
coerced into readily consuming ethanol because their entire diet consists of mother’s milk.
Two of the methods for delivering ethanol to newborn rats are artificial rearing
(AR) and inhalation, both of which have advantages and disadvantages. AR consists of
fitting a neonatal pup with a gastric fistula and tube, maintaining the pups in cups placed in
a40°C water bath, and feeding the pup an artificial milk solution via the tube and fistula.
The AR method provides constant nutrition and produces no damage to the mucous
membranes of the subject, but the interaction between mother and pup is removed.
Additionally, AR is a surgical procedure that can be quite stressful for the neonatal rat. The
stress induced by AR has been found to produce gliosis in rat cortex (Ryabinin et al.,
1995). Recently, the use of intragastric intubation—a less invasive method of neonatal
ethanol delivery that allows the pups greater maternal access—also resulted in extensive
gliosis in parietal cortex (Goodlett et al., 1997). The fact remains that gastrostomy control

78
rats—pups undergoing AR surgery, but receiving no ethanol—exhibit significant gliosis
(Ryabinin et al., 1995) and further research should be conducted to determine whether
ethanol per se induces gliosis, or whether specific methods of ethanol delivery are
responsible. The possibility that the AR procedure in and of itself can produce changes in
brain structure indicates that results obtained using AR could be difficult to interpret.
Ethanol vapor inhalation involves placing neonatal rats in a sealed chamber that
contains circulating air and ethanol vapor. Inhaling ethanol vapors has been theorized to
have the potential to damage the mucous membranes of the lungs which would then
interfere with oxygen exchange and general metabolism (Ryabinin et al., 1995). However,
no evidence of lung damage has been observed in rats exposed in this manner (Bauer-
Moffet and Altman, 1975). The present study utilized the inhalation procedure because of
the problems associated with AR. Additionally, a previous study from this laboratory
defined neurotrophic activity in the hippocampus following ethanol inhalation. Therefore,
proper continuation of that study requires the use of similar methods of analysis and
ethanol delivery. Other methods for delivering ethanol to rats postnatally include
concurrent inhalation of mother and pups, direct injection of neonates with ethanol, and
delivery of ethanol through mother’s milk. This latter method is achieved by substituting
water with a 10% ethanol solution. A problem associated with delivery of ethanol through
mother’s milk is that pups do not receive the same dose of ethanol as the mother due to
maternal metabolism.
Mechanisms suggested by previous research may help to explain ethanol’s effect on
the nervous system (reviewed in West et al., 1994). The ability of ethanol to affect DNA
methylation in the developing embryo has implications for FAS research and the present
study. Methylation of DNA in eukaryotic cells occurs at the 5' position of cytosine
residues and converts them into methylcytosine residues. Repressors and enhancers are
then hindered from binding to DNA. The end result is thought to be a change in gene
expression (Holliday, 1987). This change, however, can either increase or decrease the

79
expression of a given gene since methylation does not selectively interfere with repressors
or enhancers. Previous research from the laboratory of Garro found methylation to be
decreased in fetal DNA following ethanol exposure to the pregnant dam (1991).
Additionally, ethanol has been shown to interfere with the activity and Os-methylguanine
transferase. This enzyme is important in repairing DNA and its unfettered activity is crucial
for cell survival (Espina et al., 1988). Although these examples have not been shown to be
caused by changes in DNA methylation, they do indicate that ethanol has the ability to
regulate genetic expression. For example, ethanol has been shown to decrease expression
of BDNF mRNA in the hippocampus following chronic exposure to ethanol in adult rats
(MacLennan et al., 1995) and increase IGF gene expression in whole brain following
prenatal ethanol exposure (Breese et al., 1994). All of these studies indicate a role for
ethanol in changing normal cellular biochemistry by affecting genetic expression.
As was mentioned above, learning and memory deficits are a common characteristic
of FAS (Abel, 1995; Jones and Smith, 1973; Streissguth et al., 1991). The hippocampus
is an important structure with regard to memory and learning in humans and animals
(Bunsey and Eichenbaum, 1996; Cohen and Squire, 1980). Thus it is not surprising to
find that the hippocampus is sensitive to ethanol and exhibits reduced pyramidal cell
number following prenatal ethanol exposure (Barnes and Walker, 1981; Bonthius and
West, 1990). Damage to the hippocampus observed in the rat model following ethanol
exposure may correspond to similar damage in the hippocampus in humans. The present
study analyzed NTF and NTF receptor gene expression in this region because altered
neurotrophic activity was implicated in previous studies following both prenatal and
postnatal exposure to ethanol (Heaton et al., 1995c; Moore et al., 1996).
The neurotrophin family of NTFs has been shown to play an important role in the
development of the CNS and peripheral nervous system (PNS) through involvement in
neuronal differentiation, survival, and maintenance of basic cellular processes. The
neurotrophin family includes NGF (Levi-Montalcini, 1951), BDNF (Leibrock et al.,

80
1989), NT-3 (Maisonpierre et al., 1990), NT-4/5 (Berkemeier et al., 1991; Ip et al., 1992),
and neurotrophin-6 (Gotz et al., 1994). The trk family of receptors has been shown to be
the high-affinity receptors for the neurotrophins (Martin-Zanca et al., 1990). Trk receptors
that interact with neurotrophins include trkA (Kaplan et al., 1991; Kaplan et al., 1991),
trkB (Klein et al., 1990), and trkC (Cordon-Cardo et al., 1991). Specifically, trkA is the
preferred receptor for NGF, yet to a lesser extent, both BDNF and NT-3 can bind to it.
TrkB is the preferred receptor for BDNF and NT-4, but can bind NT-3. And trkC is the
preferred receptor for NT-3. As mentioned above, the neurotrophins are important to
normal neuronal functioning. They have been shown to regulate a number of peptides-
including the expression of other neurotrophins-in the rat septohippocampal system (Croll
et al., 1994). For example, NGF, BDNF, and NT-3 induce ChAT activity (Aldersonet
al., 1990; Auberger et al., 1987; Gnahn et al., 1983; Nonner et al., 1996); BDNF increases
NT-3 activity (Lindholm et al., 1994); and BDNF and NT-3 enhance synaptic transmission
in Shaffer collateral-CAl hippocampal synapses (Kang and Schuman, 1995). This latter
result is thought to link neurotrophins to long-term potentitation, the mechanism thought to
be partly responsible for hippocampal induction of memory (Bunsey and Eichenbaum,
1996). All of these studies indicate that the neurotrophins are important proteins integral to
normal neuronal functioning in the septohippocampal system.
Studies of gene deleted “knockout” mice also suggest the importance of
neurotrophins to proper nervous system development. In totality, the results of these
experiments have described deficits in the PNS and CNS. However, in most instances the
CNS remains largely intact in these embryos. Specifically, among neurotrophin and
neurotrophin receptor knockout mice, the only groups that resulted in statistically
significant decrease in CNS neurons were trkB deficient animals (Klein et al., 1993). In
the trkB knockouts spinal cord motoneurons and facial motoneurons were reduced (Klein
et al., 1993). The effects of the knockouts in the PNS are quite different. All single gene
knockout studies have found reduced DRG neuron number (Conover et al., 1995; Crowley

81
et al., 1994; Klein et al., 1994; Klein et al., 1993; Smeyne et al., 1994). These results
support earlier studies that found DRGs to be sensitive to many NTFs and to express the
receptors for multiple neurotrophins (Buchman and Davies, 1993). NGF knockout mice
do not survive long postnatally (Conover and Yancopoulos, 1997), and there are reduced
numbers of superior cervical ganglion, trigeminal ganglion, and DRG neurons (Crowley et
al., 1994). TrkA knockout mice show the same pattern of neuronal loss that the NGF
knockout mice have and exhibit high mortality (Smeyne et al., 1994). BDNF knockout
mice die soon after birth and have decreased numbers of trigeminal ganglion, geniculate
ganglion, nodose-petrosal ganglion, vestibular ganglion, spiral ganglion, and DRG
neurons (Conover et al., 1995; Conover and Yancopoulos, 1997). NT-4/5 knockout mice
are similar to BDNF knockouts and have reduced geniculate ganglion, nodose-petrosal
ganglion, vestibular ganglion, and DRG neurons but do not die early in postnatal life
(Conover et al., 1995; Conover and Yancopoulos, 1997). TrkB knockouts exhibit high
mortality and have the CNS differences which were mentioned earlier and reduced numbers
of trigeminal ganglion, nodose-petrosal ganglion, and DRG neurons (Klein et al., 1993).
NT-3 knockout mice expire early in postnatal development display fewer superior cervical
ganglion, trigeminal ganglion, nodose-petrosal ganglion, vestibular ganglion, spiral
ganglion, and DRG neurons (Conover and Yancopoulos, 1997; Emfors et al., 1994).
TrkC knockout mice have a high mortality rate, do not survive for a very long period of
time and exhibit reduced numbers of DRG neurons (Klein et al., 1994). While gene¬
targeting studies are powerful tools for inferring the actions of NTFs and their receptors in
the nervous system, these studies are not without their difficulties. Changes in the relative
expression of other genes and changes in the genetic background can have a large effect on
the development of the knockout animal (Gerlai, 1997). It would be difficult to determine
whether the observed changes in the organism were due to loss of the gene of interest or to
a change in genetic background. Therefore, knockout studies must be interpreted with
these difficulties in mind.

82
Important for the present study is the fact that gross neurotrophic responsiveness
and activity in the septohippocampal system are changed following prenatal exposure to
ethanol. Cultures of hippocampal neurons derived from rats prenatally exposed to ethanol
do not respond to basic fibroblast growth factor (bFGF) as well as control cultures (Fleaton
et al., 1995b). That is, these cultures do not extend neurites as the control cultures do in
response to bFGF. Specifically, bFGF does not promote neurite outgrowth in
hippocampal cultures derived from ethanol exposed animals to the extent that it does in
cultures derived from control animals. This result suggests that NTF receptor expression
may be decreased in response to prenatal ethanol exposure. Following chronic prenatal
ethanol treatment (CPET) in the rat, neurotrophic activity—which includes both
neurotrophin and other NTF activity—is increased in extracts made from the hippocampus
on P21 and cultured on DRG neurons (Heaton et al., 1995c). The increase in neurotrophic
activity is specific to this region and age of the rat, and suggests an increase in NTF
expression as a result of prenatal ethanol exposure. No single NTF is implicated by this
study since DRGs respond to a variety of NTFs in vitro. Postnatal ethanol exposure—in
contrast to prenatal exposure—produces a reduction in neurotrophic activity of P21
hippocampal extracts (Moore et al., 1996). Taken together, all of these results suggest a
role for both the NTF and its receptor in ethanol toxicity. The present study focuses on the
neurotrophin family of NTFs and their receptors because these proteins are expressed at
their highest levels in the hippocampus and because they have been implicated as important
factors for normal septal and hippocampal functioning (Maisonpierre et al., 1990). The
neurotrophins are not the only NTFs produced by the hippocampus. Other factors that the
hippocampus is responsive to—such as bFGF (Walicke, 1988)—are synthesized there and
could affect hippocampal neurons (Ernfors et al., 1990; Riva and Mocchetti, 1991).
The objective of the present study was to determine whether CPET and early
postnatal ethanol treatment (EPET) alter the gene expression of neurotrophins in the
hippocampus of treated rat pups. Thus, this portion of the study relates to the overall

83
scheme of this doctoral research by determining how ethanol affects NTF and NTF
receptor gene expression in vivo. In order to specifically determine whether BDNF, NT-3,
trkB, and trkC were affected by CPET and EPET, Northern blots were constructed from
the hippocampi of treated P21 rats. This age was chosen because previous studies found a
change in gross neurotrophic activity following both prenatal and postnatal ethanol
exposure that was limited to P21 (Heaton et al., 1995c; Moore et al., 1996). TrkA is
expressed at very low levels in the hippocampus and was therefore not examined in this
study (Martin-Zanca et ah, 1990). While NGF expression is above the threshold of
detection for Northern blotting at this age (Maisonpierre et al., 1990), we were unable to
examine its expression as the resulting bands on our blots were not quantifiable. Repeated
attempts at probing failed to produce usable data. This age (P21) was chosen for the
analysis because it coincided with the age of the animals that displayed the alteration of
neurotrophic activity following prenatal and postnatal ethanol exposure (Heaton et ah,
1995c; Moore et ah, 1996). Relative expression of these genes was compared between
control and ethanol-exposed animals. Following CPET, male animals exhibited reduced
gene expression of trkC while female animals exhibited no significant differences. There
were also no significant differences in gene expression in female CPET animals or
following EPET.
Materials and Methods
Prenatal Ethanol Exposure
Long-Evans hooded rats originally obtained from Charles River Company were
used to establish a breeding colony. Animals were housed individually in plastic cages
under controlled temperature and humidity conditions. Nulliparous females were placed in
a cage with an experienced male. On the following morning pregnancy was determined by
the presence of sperm following vaginal lavage. Animals were placed on one of three diets:
Chow, Ethanol, or Sucrose (n=24 for each group). The Chow group was given access to
Purina Rat Chow and water ad libitum. The Ethanol group was given free access to an

84
ethanol-containing liquid diet in which ethanol comprised 36% of the total caloric intake
(ethanol concentration = 8.4% v/v). The Sucrose group was pair-fed the same volume of
liquid diet with an isocaloric substitution of sucrose for ethanol. The liquid diet was made
from a commercial formula, Sustacal (Mead Johnson), which was supplemented with
Vitamin Diet Fortification Mixture (3.0 g/liter) and Salt Mixture (5.0 g/liter; both from ICN
Nutritional Biochemicals). The liquid diets contained 1.3 kcal/ml and provided several
times the daily requirements of all essential vitamins and nutrients. The additional
fortification ensured proper nutritional intake, so that any results obtained from the ethanol-
treated animals could be directly attributed to ethanol per se, and not to possible nutritional
deficiencies. Ethanol and Sucrose pups were fostered to Chow dams on the day of birth to
remove any possible effect the diet might have on the ability of the dam to properly rear the
pups. Chow pups were left with their birth mother. Previous experiments in this
laboratory have shown that morning blood alcohol levels of the pregnant dams range from
112 mg/dl to 254 mg/dl (Swanson et al., 1995).
Postnatal Ethanol Exposure by Inhalation
Pregnant Long-Evans hooded rats obtained from Charles River Company were
used for this portion of the study. Dams were fed standard lab chow throughout the
experiment, ad libitum. Pups from these litters were placed into one of three groups:
Ethanol, control Separated, and control Unseparated (n=24 for each group; these groups
will be referred to as Ethanol, Separated, and Unseparated, respectively, for the remainder
of this chapter). Ethanol pups never came from a litter that contained another group;
however, Separated and Unseparated litters were split so that one dam nursed equal
numbers of pups from each group. Ethanol litters were culled to 7 pups on postnatal day
(P4), while Separated/Unseparated litters were culled to 10 pups (Ryabinin et al., 1995).
The reduced number of pups in the Ethanol group was done to help eliminate nutritional
differences between ethanol-exposed and control pups. Ethanol inhalation occurred from
P4 through P10. Pups were placed in the inhalation chamber on a heating pad at 37°C and

85
were allowed to breathe ethanol vapor in a chamber for 2 hours daily. The inhalation
chamber consisted of an airtight 10-gallon aquarium fitted with an intake and out-take hose.
The intake hose received air flowed into a 1 L Erlenmeyer vacuum flask containing 520 ml
95% ethanol (Aaper) from an aquarium air pump set to pump air at approximately 0.8-1
L/min. Ethanol used in this study, and all studies in this project, was not treated with
benzene or any other chemical known to exert detrimental effects upon the nervous system.
As the air was forced into the flask it passed through a 1.5 inch air stone submerged in the
ethanol. The ethanol-laden vapor was then carried to the chamber. The out-take hose led
ethanol vapor from the chamber to a fume hood. Separated pups were placed for 2 hours
daily in a similar chamber with the difference being that air was pumped directly into the
chamber from the air pump without encountering ethanol. Unseparated animals remained
with the nursing dam while the Ethanol and Separated pups were placed in their respective
chambers. This paradigm of ethanol exposure resulted in peak blood ethanol counts of
approximately 250 mg/dl. Ethanol pups were clearly intoxicated upon removal from the
chamber and remained incapacitated, and unable to nurse, for a period of approximately
two hours following exposure. In contrast, Separated pups began nursing immediately
upon their return to the home cage.
Morphometric Measurements
Prior to sacrifice, all animals were weighed and had crown-rump length
measurements taken. Crown-rump length was defined as the distance from the crown of
the skull—defined to be the point directly between the ears—to the base of the tail. The brain
of each animal was weighed before the hippocampus was dissected. These measures were
taken to provide an estimation of the overall effect that ethanol treatment had on the
subjects.
Dissections
Rats were anesthetized with methoxyflurane (Pittman-Moore) and sacrificed by
decapitation on P21. After the brain was removed from the skull, the hippocampus was

86
dissected out, wrapped in aluminum foil, and flash-frozen in liquid nitrogen. Endogenous
ribonucleases present in the tissue rapidly destroy mRNA present in the brain once a
dissection starts. Therefore, mRNA (which happens to be the molecule of interest in the
present study) is in danger of being lost if the dissection is not completed with considerable
speed. Because of the rapidity with which dissected tissue had to processed and because of
the considerable time required to obtain tissue weights, individual weights of the
hippocampi were not taken. The tissue was then stored at -70°C until RNA was extracted.
RNA Extraction
Polyadenylated (poly-A) messenger RNA (mRNA) was extracted from frozen
tissue specimens via the Micro-fastTrack kit (Invitrogen). Poly-A mRNA was stored as a
precipitate in 75% ethanol at -70°C until the samples were run on an electrophoresis gel.
Northern Blots
The procedures used in this study are as previously described (Baek et al., 1994;
MacLennan et al., 1994; MacLennan et al., 1995). The amount of mRNA in each sample
was assessed by taking the optical density of 1 pA of sample in 500 ]a\ dH20 at 260 nm and
280 nm of UV light. The samples were then loaded onto a 1.25% agarose formaldehyde
denaturing gel so that each lane contained about 15 pig of poly-A mRNA. To obtain
mRNA levels of this magnitude, four animals were used for each lane in each gel. The
products were separated using horizontal gel electrophoresis running at 100 V for 30
minutes and then turned down to 25 V and allowed to run overnight. The poly-A mRNA
was then transferred to a nylon membrane (ICN). The membrane was then baked in a
vacuum oven at 80°C for two hours and stored desiccated at room temperature until probed.
Northern blots were probed with a cDNA strand encoding one of two neurotrophins (NT-3
or BDNF), one of two neurotrophin receptors (trkB or trkC), or cyclophilin. Cyclophilin
mRNA is constituítively expressed and was used to standardize each lane. Previous
research has shown that cyclophilin gene expression is not affected by developmental
ethanol treatment (Maier et al., 1996). The cDNA strands were labeled with 32P (dCTP

87
from Amersham) by random hexamer priming. The BDNF, NT-3, and trkB cDNA probes
were a generous gift from Drs. P. Isackson, J.G. Sutcliffe, and S. Whittemore to Drs. Don
Walker and A. John MacLennan of this department. The trkC cDNA probe was a generous
gift from Dr. Louis Parada to this laboratory. Before starting the entire hybridization
procedure, the blots were prewashed for 60 minutes at room temperature in 2X SSC. The
blots were prehybridized at 42°C for approximately 24 hours in a solution containing 50%
formamide, 5X SSC, 5X Denhardt’s, 0.5% SDS, 0.05M sodium phosphate, 0.25 mg/ml
salmon sperm DNA, and 0.1 mg/ml poly-A. Hybridization was carried out at 42°C for
approximately 20 hours in the same solution described above with the 32P labeled cDNAs
added to the solution. Following hybridization the blots were washed three times in 2X
SSC at room temperature for periods of one minute, 30 minutes, and 30 minutes,
respectively. The blots were then washed twice at 58°C in a solution containing 0. IX SSC
and 0.5% SDS for 30 minutes. After this final wash the Northerns were wrapped in plastic
wrap and lightly taped to a Molecular Dynamics phosphorimaging cassette for at least 24
hours and analyzed by using the ImageQuant program which computes the density of each
band electronically. The exposure time was dependent on the relative expression of the
gene being probed. The phosphorimaging cassette is 10X more sensitive to radioactive
particles than the x-ray film, but records ambient radiation. The ImageQuant program
allows background readings to be subtracted so that radiation from the probe itself can be
analyzed. The resulting bands were normalized by dividing each value by the
corresponding cyclophilin value.
Stripping and Reprobing
In order to probe the blots for different neurotrophic agents and remove any
remaining32? from previous hybridizations, the blots were stripped. The blots were
exposed to a solution containing 50% formamide and 0.01M sodium phosphate at 65°C for
60 minutes. After stripping, the blots were rinsed in a solution containing 2X SSC and

88
0.1% SDS for five minutes at room temperature. The blots were then probed as described
in the previous section.
Statistical Analyses
Two-way analysis of variance was performed using SAS version 6.12 on a
Pentium computer. Variances were pooled for this analysis since testing revealed that they
were not significantly different by gender. When applicable, individual differences
between groups were tested using Fisher’s protected least significant difference (PLSD)
post-hoc analyses. Statistical significance was determined to be p<0.05. Additionally, the
Bonferroni/Dunn correction was used to determine if individual differences elucidated by
Fisher’s PLSD were valid. Statistical significance following the Bonferroni/Dunn
correction was p<0.137.
Results
Morphometric Measurements
Morphometric measurements were obtained to assess the general effect that ethanol
had on the development of the animals used in this study. Measurements of body weight,
brain weight, and crown-rump length were collected from both prenatally and postnatally
treated animals on P21. The postnatally exposed rats were weighed on a daily basis from
P4 to P10 to assess their overall growth during the inhalation period. Each group in both
the CPET and EPET studies contained 24 animals and sexes were kept separate during all
analyses.
CPET
As mentioned above, measurements were taken from both male and female animals
on P21. For male animals, analysis of variance found no significant effect due to treatment
for body weight, brain weight, or crown-rump length. Female animals had a differing
result. Analysis of variance did not find a significant effect due to treatment for body
weight or crown-rump length. However, brain weight was significantly affected by

89
treatment (F=6.37, df=71, p<0.005). Post hoc testing reveled that Alcohol animals had
significantly smaller brains than both Chow (p<0.005) and Sucrose (pxO.Ol) animals.
Even though the brains were significantly smaller in female animals following CPET, the
ratio of brain weight to body weight was not significantly different from the same ratio in
control animals. This ratio was also not significantly changed in male animals exposed to
ethanol prenatally. Figure 4-1 displays the brain weights obtained from this portion of the
study. Since brain weight to body weight ratio was unaffected by CPET, the extent to
which brain weight was affected by ethanol exposure is not clear.
EPET
Analysis of variance found significant effects due to treatment in both male and
female animals in this section of the study. In the interest of clarity, these results will be
presented separately.
Male animals. Male animals displayed a significant effect of treatment for weight at
P4 (F=7.05, df=71, p<0.005) and P5 (F=4.06, df=71, p<0.05). At P4, post hoc testing
revealed that Ethanol animals weighed significantly more than both Separated (p<0.05) and
Unseparated (p<0.0005) animals. At P5, Ethanol animals were only significantly larger
than Unseparated animals (p<0.01). It should be noted that it was not the intent of the
researchers to select larger animals in one group over the other. A possible explanation for
this difference may lie in the litter size of the relative groups. Since Ethanol groups were
set to have 7 pups at the start of inhalation and the control groups (Separated and
Unseparated) were combined in one litter, litters containing less than 10 pups were
automatically put into the Ethanol group. When both litters contained more than 10 pups,
the dam was randomly placed into either a control or Ethanol group. Therefore, the
Ethanol groups most likely started, on average, with a smaller litter size. This would make
each pup larger on average than pups born to larger litters.
Since there was a difference at the start of treatment, gross initial differences
between the groups were eliminated by analyzing weight gain from day to day. Analysis of

90
A
Ofl
Alcohol Sucrose Chow
CONDITION
Figure 4-1. Brain weight at P21 of female and male animals following
prenatal ethanol exposure. A. Female brain weight. B. Male brain weight.
Measurements are means + SEM. a = Female Ethanol animals are
significantly smaller in comparison to Sucrose (p<0.01) and Chow (p<0.005).
Animals were fed Ethanol, Sucrose, and Chow diets during gestation as
described in the Methods section of this chapter and fostered to chow dams at
birth, n = 24 for each group.

91
variance found a significant effect due to treatment for weight gain in male animals from P5
to P6 (F=7.85, df=71, p<0.001), from P8 to P9 (F=4.56, df=71, p<0.05), from P4 to
P10 (F= 13.53, df=71, p<0.0001), and from P10 to P21 (F=7.18, df=70, p<0.005). Post
hoc testing revealed that the weight gain from P5 to P6 was significantly larger in Separated
(p<0.005) and Unseparated (pcO.OOl) animals than in Ethanol animals. Further, from P8
to P9, Unseparated animals gained significantly more weight than Ethanol animals
(p<0.005). Over the entire course of the ethanol inhalation period (from P4 to P10), there
were significant differences. Separated (p<0.05) and Unseparated (0.0001) animals gained
significantly more weight from P4 to P10 than Ethanol animals. Additionally, Unseparated
animals gained significantly more weight than Separated animals (p<0.01). Figure 4-2
displays the results observed in this section of the study. Following the inhalation period,
the Ethanol animals exhibited a growth spurt which effectively “caught them up,” and
allowed them to surpass the other groups. Recall that the number of animals in each litter
was controlled so that the Ethanol group had 7 pups and the Control group had 10 pups.
This was done to give the Ethanol group greater food availability and to control for possible
nutritional differences. Specifically, Ethanol animals gained significantly more weight
from P10 to P21 than both the Separated (pcO.OOl) and Unseparated (p<0.005) animals.
Figure 4-2 also displays these results.
For male animals at P21, analysis of variance found that body weight (F=4.71,
df=70, p<0.05), crown-rump length (F=4.36, df=70, p<0.05), and brain weight
(F=21.39, dl =71, pcO.0001) were all significantly affected by treatment. For body
weight, post hoc testing revealed that Ethanol animals weighed significantly more than both
Separated (p<0.005) and Unseparated (p<0.05) animals. Similarly, Ethanol animals had a
significantly longer crown-rump length than their Separated (p<0.005) counterparts. These
results are not surprising in light of the fact that Ethanol animals gained significantly more
weight from P10 to P21 than both Separated and Unseparated animals. Both Separated
and Unseparated animals had significantly larger brains than Ethanol animals (p<0.0001

92
Figure 4-2. Weight gain during postnatal ethanol exposure in male animals.
Ethanol exposure extended from P4 through P10. Additionally, body weights at
P21 are displayed. Measurements are means + SEM. a = Ethanol animals show
significantly lower weight gain from P5 to P6 in comparison to Separated
(p<0.005) and Unseparated (pcO.OOl) animals, b = Ethanol animals display
significantly lower weight gain from P8 to P9 in comparison to Unseparated
(p<0.005) animals, c = Ethanol animals exhibit significantly lower weight gain
from P4 to P10 in comparison to Separated (p<0.05) and Unseparated (pcO.OOOl)
animals. Also, Separated animals gained significantly less weight than
Unseparated (p<0.01) animals, d = Ethanol animals gain significantly more
weight from P10 to P21 than Separated (PcO.OOl) and Unseparated (p<0.005)
animals. Ethanol, Separated, and Unseparated groups are described in the
Methods section of this chapter, n = 24 for each group.

93
for both comparisons). Unlike prenatally exposed animals, this difference in brain weight
extended to the brain weight to body weight ratio. For this ratio, analysis of variance
found a significant effect due to treatment (F=13.989, df=71, p<0.0001) and post hoc
testing showed that both Separated (pcO.OOOl) and Unseparated (p<0.0005) had
significantly larger ratios than Ethanol animals. Figures 4-3 and 4-4 display the results of
this portion of the study.
Female animals. Following EPET, female animals exhibited some results which
were similar to male animals and some that were not. Specifically, analysis of variance
found that during the period of ethanol exposure, there was a significant effect due to
treatment on P4 (F=8.74, df=70, p<0.0005) and P5 (F=6.52, df=70, p<0.005). No other
days during the exposure period showed any significant differences. At P4, Ethanol
animals were significantly larger than both Separated (pcO.OOl) and Unseparated
(p<0.001) animals. At P5, Ethanol animals were again larger than both Separated
(pcO.Ol) and Unseparated (p<0.005) animals. During the exposure period, female animals
did not exhibit as much variability as the male animals did and on only one day did the
female animals exhibit any effect due to treatment. Analysis of variance found that from P5
to P6 (F=6.52, df=70, p<0.005), the Separated (p<0.0005) and Unseparated (p<0.005)
animals gained more weight than the Ethanol animals. Over the course of the entire
inhalation period (P4-P10), there was also an effect of treatment (F=5.50, df=70, pcO.Ol).
Specifically, the Separated (p<0.05) and Unseparated (p<0.005) animals gained more
weight over the period than the Ethanol animals. After the period of inhalation was
completed until sacrifice (P10 to P21), analysis of variance approached significance
(F=2.96, df=69, p=0.059). Figure 4-5 shows the results found in this section of the
study.
At P21, when the female animals were sacrificed and prepared for dissection, the
only measurement that was affected by treatment was brain weight (F=31.13, df=70,
p<0.0001). Neither body weight nor crown-rump length was affected. Post hoc analysis

94
Ethanol Separated Unseparated
Condition
Figure 4-3. Gross morphological measurements following EPET in male
animals at P21. A. Body weight. B. Crown rump length. Measurements are
means + SEM. Ethanol, Separated, and Unseparated groups are as described
in the Methods section of this chapter, a = significantly larger than Separated
(p<0.005) and Unseparated (p<0.05) animals, b = significantly longer in
comparison to Separated (p<0.005) animals, n = 24 for each group.

95
A
B
o
P¿¡
Ethanol Separated Unseparated
Figure 4-4. Brain weight and Brain weight to body weight ratio of EPET male
animals at P21. A. Brain weight. B. Brain weight to body weight ratio.
Measurements are means + SEM. Ethanol, Separated, and Unseparated groups
are as described in the Methods section of this Chapter, a = significantly smaller
than both Separated (pcO.OOOl) and Unseparated (pcO.OOOl) animals, b =
significantly smaller ratio than Separated (pcO.OOOl) and Unseparated
(p<0.0005) animals. n= 24 for each group.

96
Figure 4-5. Weight gain during postnatal ethanol exposure in female animals.
The period of ethanol exposure extended from P4 through P10. Additionally,
body weight at P21 is displayed. Measurements are means + SEM. Ethanol,
Separated, and Unseparated groups are as described in the Methods section of this
chapter, a = Ethanol animals exhibit significantly less weight gain from P5 to P6
than Separated (p<0.0005) and Unseparated (p<0.005) animals, b = Ethanol
animals display significantly less weight gain than Separated (p<0.05) and
Unseparated (p<0.005) animals, n = 24 for each group.

97
found that Separated (p<0.0001) and Unseparated (p<0.0001) animals had larger brains
than ethanol animals. Brain weight to body weight ratio was affected similarly. Overall,
there was an effect of treatment (F=14.635, df=68, pcO.OOOl) and both the Separated
(p<0.0005) and Unseparated (pcO.OOOl) animals had a larger ratio than the Ethanol
animals. Figure 4-6 displays the results obtained in this section of the study.
Gene Expression of Neurotrophins and Neurotrophin Receptors
Analysis of gene expression in the hippocampus of rats exposed to ethanol
prenatally (CPET) and postnatally (EPET) is the major experiment of this chapter. As was
evidenced by the above data, ethanol treatment did have some effect on the overall growth
of animals, both prenatally and postnatally. This effect did not necessarily translate into a
change in gene expression of neurotrophins or neurotrophin receptors. Briefly, while there
were significant differences in gene expression in male animals following CPET, there
were none found in female animals. There were no significant differences following EPET
in either male or female animals. The specific differences found are outlined below.
CPET
Analysis of variance found that there were no effects due to treatment in female
animals when BDNF, NT-3, trkB, and trkC gene expression was analyzed. Male animals
displayed no significant effect due to treatment when BDNF, NT-3, and trkB gene
expression was analyzed. Figures 4-7, 4-8, and 4-9 display the results of BDNF gene
expression for male and female animals. Figure 4-7 displays the phosphorimaging view of
these blots and Figures 4-8 and 4-9 are bar graphs showing the quantitative data from these
blots. Figures 4-10 and 4-11 exhibit the results of NT-3 gene expression for male and
female animals. Figure 4-10 is the phosphorimaging view of the NT-3 Northern blots and
Figure 4-11 is a bar graph of NT-3 relative gene expression. Figures 4-12 and 4-13 are bar
graphs depicting the gene expression of trkB active and truncated receptors, respectively.
Figures 4-14, 4-15, 4-16, and 4-17 exhibit the results of trkC gene expression for male and
female animals. Figure 4-14 is the phosphorimaging view of the trkC blots and Figures 4-

98
B
03
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-£3
OX)
• l-H
£
O
CQ
o
03
PQ
Ethanol Separated Unseparated
Figure 4-6. Brain weight and brain weight to body weight ratio in EPET female
animals at P21. A. Brain weight. B. Brain weight to body weight ratio.
Measurements are means + SEM. Ethanol, Separated, and Unseparated groups
are as described previously in the Methods section of this chapter, a =
significantly smaller in comparison to Separated (pcO.OOOl) and Unseparated
(p<0.0001) animals, b = significantly smaller ratio in comparison to Separated
(p<0.0005) and Unseparated (p<0.0001) animals, n = 24 for each group.

Figure 4-7. Phosphorimaging view of BDNF Northern blots composed of the
hippocampal region from P21 rat brains exposed to ethanol prenatally. Images are obtained
from Northern blots composed of male and female animals. Each sex is broken down
further into Chow, Sucrose, and Ethanol groups as described previously in the methods
section of this chapter. Two transcripts for BDNF were detected. The larger transcript is
known to be 4.4 kb and the smaller one is known to be 1.7 kb in length. These sizes were
confirmed by comparing the transcripts to the darkest band of the ladder. Number of lanes
used in the analysis equal to 6 for each group.

100
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Figure 4-7.

101
A 0.02
0.02-
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Ethanol Sucrose Chow
Figure 4-8. Relative BDNF 4.4 kb transcript expression in rat hippocampus at
P21 following prenatal exposure to ethanol. A. Female animals. B. Male
animals. Ethanol, Sucrose, and Chow groups are as described previously in
this chapter. Measurements are means + SEM. Two way analysis of variance
revealed no significant differences among the groups. n=6 for each group.

102
Figure 4-9. Relative BDNF 1.7 kb transcript gene expression following prenatal
exposure in P21 rats. A. Female animals. B. Male animals. Ethanol, Sucrose,
and Chow animals are as described in the Methods section of this chapter.
Measurements are means + SEM. Analysis of variance indicated no significant
differences among the groups. n=6 for each group.

Figure 4-10. Phosphorimaging view of NT-3 Northern blots composed of the
hippocampal region from P21 rat brains exposed to ethanol prenatally. Images are obtained
from Northern blots composed of male and female animals. Each sex is broken down
further into Chow, Sucrose, and Ethanol groups as described previously in the methods
section of this chapter. The NT-3 transcript is known to be 1.5 kb in length. This size was
confirmed by comparing the distance relative to the darkest band of the ladder. Number of
lanes used in the analysis equal to 6 for each group.

104
Male
Female
Chow Sucrose Ethanol
Chow Sucrose Ethanol
1.5 kb
.
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Figure 4-10.

105
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Figure 4-11. Relative NT-3 gene expression following prenatal ethanol exposure
inP21rats. A. Female animals. B. Male animals. Ethanol, Sucrose, and Chow
groups are as described in the Methods section of this chapter. Measurements are
means + SEM. Analysis of variance indicated no significant differences among
the groups. n=6 for each group.

106
A 0.0?
Ethanol Sucrose Chow
Figure 4-12. Relative trkB active receptor gene expression following prenatal
ethanol exposure in P21 rats. A. Female rats. B. Male rats. Ethanol, Sucrose,
and Chow groups are as described in the Methods section of this chapter.
Measurements are means + SEM. Analysis of variance indicated no significant
differences among the groups. n=6 for each group.

107
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Figure 4-13. Relative trkB truncated transcript gene expression following
prenatal exposure in P21 rats. A. Female animals. B. Male animals. Ethanol,
Sucrose, and Chow animals are as described earlier in the Methods section of this
chapter. Measurements are means + SEM. Analysis of variance indicated no
significant differences among the groups. n=6 for each group.

Figure 4-14. Phosphonmaging view of trkC Northern blots composed of the hippocampal
region from P21 rat brains exposed to ethanol prenatally. The largest band of the ladder is
1.7 kb in length. Images are obtained from Northern blots composed of male and female
animals. Each sex is broken down further into Chow, Sucrose, and Ethanol groups as
described previously in the methods section of this chapter. The largest band corresponds
to the active form of trkC (14 kb). The two smaller bands are truncated versions of trkC
(4.7 kb and 3.9 kb). Size was confirmed by comparing the bands relative to the darkest
band on the ladder, a = artifact, not a band. Number of lanes used in the analysis equal to
6 for each group.

109
Male
Female
Chow Sucrose Ethanol
Chow Sucrose Ethanol Ladder
Figure 4-14.

110
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Figure 4-15. Relative trkC 14 kb transcript gene expression at P21 in rats
exposed to ethanol prenatally. A. Female animals B. Male animals.
Measurements are means + SEM. Ethanol, Separated, and Unseparated groups
are as described in the Methods section of this chapter, a = significantly lower
expression in comparison to Sucrose (p<0.01) and Chow (p<0.005) animals, n =
6 for each group.

Ill
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Figure 4-16. Relative trkC 4.7 kb truncated transcript gene expression following
prenatal exposure in P21 rats. A. Female animals. B. Male animals. Ethanol,
Sucrose, and Chow animals are as described earlier in the Methods section of this
chapter. Measurements are means + SEM. Analysis of variance indicated no
significant differences among the groups. n=6 for each group.

112
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Figure 4-17. Relative trkC 3.9 kb transcript gene expression following prenatal
exposure in P21 rats. A. Female animals. B. Male animals. Ethanol, Sucrose,
and Chow animals are as described earlier in the Methods section of this chapter.
Measurements are means + SEM. Analysis of variance indicated no significant
differences among the groups. n=6 for each group.

113
15,4-16, and 4-17 are bar graphs for the relative gene expression of the active receptor,
and two truncated receptors, respectively. The phosphorimaging view of the standard,
cyclophilin is displayed in Figure 4-18. Previous research has found that there are three
poly-A transcripts of trkC (Jaber et al., 1994). The probe used to detect trkC in this study
detected all three of these transcripts. The largest transcript corresponded to the active form
of trkC and was approximately 14 kb in length. The two smaller transcripts corresponded
to truncated versions of the trkC receptor and were approximately 3.9 and 4.8 kb in length.
These sizes are confirmed by comparing the size of the trkC bands to those of BDNF and
NT-3. The same probes used in an earlier study were used in the present study
(MacLennan et al., 1995). The BDNF probe detects two BDNF transcripts that are 4.4 kb
and 1.7 kb in length (MacLennan et al., 1995). The NT-3 probe detects an NT-3 transcript
that is 1.5 kb in length. The ladder present in each Northern blot is pictured in each
Northern blot Figure and comparison to the 4.4 BDNF transcript confirms that the two
smaller trkC truncated transcripts are about 3.9 and 4.7 kb in length. The largest trkC
transcript (14 kb) is clearly larger than the largest band in the RNA ladder which is
approximately 9.5 kb in length. In male animals, there was a significant effect due to
treatment in active trkC gene expression (F=7.0, df=17, p<0.01). Post hoc analyses found
that the active trkC transcript was decreased in Ethanol animals in comparison to Chow
(p<0.005) and Sucrose (p<0.01) animals. These comparisons are also significant
following the Bonferroni/Dunn correction since p<0.0137. These reductions were
approximately 20% in both instances.
In addition to analyzing the absolute gene expression, an analysis of the ratios of
the three trkC transcripts was performed. Analysis of variance found that the ratio of the
active trkC transcript to the smaller truncated transcript was affected by treatment (F=4.72,
df=17, p<0.05). Post hoc testing revealed that in both Chow (p<0.05) and Sucrose
(p<0.05) animals, this ratio was 20% larger than in Ethanol animals. However, the
Bonferroni/Dunn correction found this latter difference to not be significant. The difference

Figure 4-18. Phosphorimaging view of cyclophilin Northern blots composed of the
hippocampal region from P21 rat brains exposed to ethanol prenatally. Images are obtained
from Northern blots composed of male and female animals. Each sex is broken down
further into Chow, Sucrose, and Ethanol groups as described previously in the methods
section of this chapter. The transcript is approximately 1.0 kb in size relative to the darkest
band. Number of lanes used in the analysis equal to 6 for each group.

115
Male
Female
Chow Sucrose Ethanol Chow Sucrose Ethanol Ladder
Figure 4-18.

116
in the ratio of active trkC to truncated trkC is probably due to the fact that absolute
expression of active trkC was significantly smaller in the Ethanol group.
EPET
As mentioned above, there were no significant differences in the expression of any
of the genes analyzed following EPET. Additionally, the ratio of expression of various
transcripts was unchanged. Figures 4-19, 4-20, and 4-21 display the results of BDNF
gene expression for male and female animals. Figure 4-19 displays the phosphorimaging
view of these blots and Figures 4-20 and 4-21 are bar graphs showing the quantitative data
from these blots. Figures 4-22 and 4-23 exhibit the results of NT-3 gene expression for
male and female animals. Figure 4-22 is the phosphorimaging view of the NT-3 Northern
blots and Figure 4-23 is a bar graph of NT-3 relative gene expression. Figures 4-24 and 4-
25 are bar graphs depicting the gene expression of trkB active and truncated receptors,
respectively. Figures 4-26, 4-27, 4-28, and 4-29 exhibit the results of trkC gene
expression for male and female animals. Figure 4-26 is the phosphorimaging view of the
blots probed for trkC and Figures 4-27,4-28, and 4-29 are bar graphs depicting the relative
amount of trkC active and truncated receptors, respectively. Figure 4-30 is the
phosphorimaging view of the standard for these experiments, cyclophilin. The fact that
gene expression in these animals is unchanged is surprising in light of the fact that the
gross state of the nervous system—as evidenced by brain weight and brain weight to body
weight ratio-was greatly affected by EPET. Since the BGS—with the hippocampus
sustaining significant neuronal loss—has proven to be a sensitive period for ethanol
exposure (Bonthius and West, 1991; West et al., 1986), more research will be necessary to
determine if perhaps changes in other NTFs are responsible for EPET damage to this
system.

Figure 4-19. Phosphorimaging view of BDNF Northern blots from postnatally exposed
P21 rats. Images are obtained from Northern blots composed of male and female animals.
Each sex is broken down further into Unseparated, Separated, and Ethanol groups as
described previously in the methods section of this chapter. Two transcripts for BDNF
were detected. The larger transcript is known to be 4.4 kb and the smaller one is known to
be 1.7 kb in length. These sizes were confirmed by comparing the transcripts to the
darkest band of the ladder. Number of lanes used in the analysis equal to 6 for each group.

118
Male
Female
Unseparated Separated Ethanol
Unseparated Separated Ethanol
4.4 kb
1.7 kb
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Ladder

119
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Ethanol Separated Unseparated
Figure 4-20. Relative BDNF 4.4 kb transcript gene expression following
postnatal exposure in P21 rats. A. Female animals. B. Male animals. Ethanol,
Separated, and Unseparated animals are as described earlier in the Methods
section of this chapter. Measurements are means + SEM. Analysis of variance
indicated no significant differences among the groups. n=6 for each group.

120
Ethanol Separated Unseparated
Figure 4-21. Relative BDNF 1.7 kb transcript gene expression following
postnatal exposure in P21 rats. A. Female animals. B. Male animals. Ethanol,
Separated, and Unseparated animals are as described earlier in the Methods
section of this chapter. Measurements are means + SEM. Analysis of variance
indicated no significant differences among the groups. n=6 for each group.

Figure 4-22. Phosphorimaging view of NT-3 Northern blots from postnatally exposed
P21 rats. Images are obtained from Northern blots composed of male and female animals.
Each sex is broken down further into Unseparated, Separated, and Ethanol groups as
described previously in the methods section of this chapter. The NT-3 transcript is known
to be 1.5 kb in length. This size was confirmed by comparing the distance relative to the
darkest band of the ladder. Number of lanes used in the analysis equal to 6 for each group.

122
Male
Female
Unseparated Separated Ethanol Ladder
Unseparated Separated Ethanol
Figure 4-22.

123
0.02
A
0.02-
B
Ethanol Separated Unseparated
Figure 4-23. Relative NT-3 1.5 kb gene expression following postnatal exposure
in P21 rats. A. Female animals. B. Male animals. Ethanol, Separated, and
Unseparated animals are as described earlier in the Methods section of this
chapter. Measurements are means + SEM. Analysis of variance indicated no
significant differences among the groups. n=6 for each group.

124
Figure 4-24. Relative trkB active receptor gene expression following postnatal
exposure in P21 rats. A. Female animals. B. Male animals. Ethanol, Separated,
and Unseparated animals are as described earlier in the Methods section of this
chapter. Measurements are means + SEM. Analysis of variance indicated no
significant differences among the groups. n=6 for each group.

125
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Figure 4-25. Relative trkB truncated transcript gene expression following
postnatal exposure in P21 rats. A. Female animals. B. Male animals. Ethanol,
Separated, and Unseparated animals are as described earlier in the Methods
section of this chapter. Analysis of variance indicated no significant differences
among the groups. n=6 for each group.

Figure 4-26. Phosphorimaging view of trkC Northern blots from postnatally exposed P21
rats. Images are obtained from Northern blots composed of male and female animals.
Each sex is broken down further into Unseparated, Separated, and Ethanol groups as
described previously in the methods section of this chapter. The largest band corresponds
to the active form of trkC (14 kb). The two smaller bands are truncated versions of trkC
(4.7 kb and 3.9 kb). Size was confirmed by comparing the bands relative to the darkest
band on the ladder. Number of lanes used in the analysis equal to 6 for each group.

127
Male
Female
Ladder
Figure 4-26.

128
Figure 4-27. Relative trkC 14 kb transcript gene expression in P21 rats following
postnatal exposure A. Female animals. B. Male animals. Ethanol, Separated,
and Unsepara.ted animals are as described earlier in the Methods section of this
chapter. Measurements are means + SEM. Analysis of variance indicated no
significant differences among the groups. n=6 for each group.

129
0.06
0.06
Ethanol Separated Unseparated
Figure 4-28. Relative trkC 4.7 kb truncated transcript gene expression in P21 rats
following postnatal exposure. A. Female animals. B. Male animals. Ethanol,
Separated, and Unseparated animals are as described earlier in the Methods
section of this chapter. Measurements are means + SEM. Analysis of variance
indicated no significant differences among the groups. n=6 for each group.

130
A 0.04
Ethanol Separated Unseparated
Figure 4-29. Relative trkC 3.9 kb truncated transcript gene expressionin P21 rats
following postnatal exposure. A. Female animals. B. Male animals. Ethanol,
Separated, and Unseparated animals are as described earlier in the Methods
section of this chapter. Measurements are means + SEM. Analysis of variance
indicated no significant differences among the groups. n=6 for each group.

Figure 4-30. Phosphorimaging view of cyclophilin Northern blots from postnatally
exposed P21 rats. Images are obtained from Northern blots composed of male and female
animals. Each sex is broken down further into Unseparated, Separated, and Ethanol
groups as described previously in the methods section of this chapter. The transcript is
approximately 1.0 kb in size relative to the darkest band. Number of lanes used in the
analysis equal to 6 for each group.

132
Male
Female
Unseparated Separated Ethanol
Unseparated Separated Ethanol
Ladder
Figure 4-30.

133
Discussion
CPET
The results showed that the overall growth of male and female animals was not
affected by prenatal ethanol exposure. Neither male nor female animals had decreased
body weight at P21 following CPET. Previous studies in this laboratory have found that
CPET produces a decrease in body weight at birth that does not persist through P21
(Swanson et ah, 1995; Swanson et ah, 1996). The current results agree with these
findings. The fact that body weight and crown-rump length were not affected by CPET
suggests that there were not any nutritional differences among the various groups. The
liquid diet used in this study was developed to provide extra nutritional benefits to
counteract any possible deficiencies that ethanol might cause. The growth of the nervous
system—as evidenced by brain weight to body weight ratio—was unaffected by CPET.
Previous studies from this laboratory did not find that brain weight differences persist until
P21 (Swanson et ah, 1995; Swanson et ah, 1996). Also, ethanol was administered at the
same level in both studies and rats from the same breeding colony were used (Swanson et
ah, 1995; Swanson et ah, 1996). Changes, or the lack of a significant change, in brain
weight did not correlate with changes, or a lack of a change, in gene expression in the
genes examined in the present study. Specifically, male animals exposed to ethanol
prenatally did not exhibit a significant change in brain weight or brain to body weight ratio.
This group was the only one found to exhibit a change in gene expression, with trkC being
significantly reduced. Conversely, the significant decrease in brain weight found in female
animals did not correlate with a change in expression of neurotrophin or neurotrophin
receptor genes studied presently. Overall, these results suggest that ethanol has the ability
to affect neurons at the fundamental level of gene expression.
The only significant change in gene expression found in the present study following
ethanol exposure was in trkC expression in male animals following CPET. Further
analysis found that trkC expression was affected in more than one way. In addition to the

134
absolute decrease in trkC active receptor transcript, the ratio of active receptor transcript to
truncated receptor transcript was also decreased following CPET. This latter comparison is
significant using the PLSD analysis, but not significant following the Bonferroni/Dunn
correction. The difference between full length and truncated trkC receptors is important to
this study. A full length trkC receptor is capable of transducing a signal from NT-3 that
has the ability to alter many processes in the cell, including the activity of proteins capable
of regulating genetic transcription. A truncated receptor is like a full length receptor, except
that it lacks a catalytic domain capable of transducing the signal. Developmentally,
truncated receptors are expressed later than their full length counterparts. By adulthood,
truncated receptors become the dominant form (Escandon et al., 1994). The implication of
this change in the ratio of truncated to active receptor is that hippocampal neurons might be
less able to respond to NT-3 in their environment. The ratio of truncated to active receptors
may have important implications for neuronal survival and normal neuronal function. The
possibility exists that the truncated receptors are a dominant negative effect on trk signaling.
That is, the truncated receptors might inhibit the overall function of trk receptors by either
binding NT-3 or associating with active trk receptors. Recall that NT-3 has the ability to
bind trkB and trkA receptors, but with less affinity than to trkC. Thus, NT-3 does not stay
bound as long to these other receptors as it does to trkC. It is also important to note that no
other neurotrophins have the ability to bind to trkC. The most efficient signal that NT-3
produces would occur from binding to trkC. The greater amount of truncated trkC in
comparison to active trkC also suggests that a given NT-3 molecule would be more likely
to encounter a truncated receptor than in a control animal. Thus, a similar amount of NT-3
in the cellular environment would produce less of a signal in these ethanol-treated animals
than in control animals.
The role that the low-affinity neurotrophin receptor, p75, may play in ethanol
neurotoxicity should also be discussed. There is evidence that unbound p75 receptor may
induce apoptosis in neurons. That is, p75 that is not associated with a trk receptor may

135
promote cell death (Kaplan and Miller, 1997). This idea is supported by the fact that NGF
promotes apoptosis in retinal neurons of the chick that express p75, but do not express
trkA (Frade et al., 1996). Furthermore, mice that express the intracellular domain of p75
exhibit widespread CNS and PNS neuronal loss (Majdan et al., 1997). In fact, the level of
cell death induced by p75 seems to be directly proportional to the amount of p75 expressed.
Higher expression of p75 correlated with higher levels of apoptosis in cultured
neuroblastoma cells (Bunone et al., 1997). All of this evidence supports the idea that
unbound p75 may induce cell death in neurons. Since the present study found a reduction
in trkC gene expression, it might be logical to assume that such a reduction would result in
increased levels of unbound p75. As a result, more hippocampal neurons would perish.
The knockout studies do not necessarily support this claim, since NT-3 knockouts are
phenotypically more impaired than trkC knockouts (Conover and Yancopoulos, 1997).
Careful examination of hippocampal pyramidal cell number in knockout animals has not yet
been completed to date. As was mentioned above, few CNS populations are reduced
following neurotrophin and neurotrophin receptor gene deletion. The fact that CNS
populations are not greatly affected could be due to changes in the genetic background of
the knockout animals and not due to the true loss of activity of the neurotrophin or
neurotrophin receptor of interest.
NT-3 expression remains high throughout CNS and hippocampal development
(Maisonpierre et al., 1990). The fact that its expression is so high suggests that it is
important for neuronal development during this time period. Therefore, a change in the
ability of hippocampal neurons to detect the NT-3 signal due to a reduction in available trkC
receptors could alter the survival status, or some other general status of the cell. Since
pyramidal cells that perish due to ethanol exposure are most likely already dead by P21, it
is possible that the observed reduction (P21) in trkC gene expression has little effect on
neuronal loss in the hippocampus. As learning and memory are impaired by developmental
ethanol exposure (Abel, 1995; Jones and Smith, 1973; Streissguth et al., 1991), and

136
learning and memory are controlled by living neurons, it may be that those neurons that
remain do not perform their functions at a normal level. NTFs and NTF receptors have
been linked to long-term potentiation (LTP) in the hippocampus (Bramham et al., 1996;
Kang and Schuman, 1995; Kang and Schuman, 1996). LTP has been postulated as part of
the mechanism for encoding new memories (Bunsey and Eichenbaum, 1996). Therefore, a
reduction in trkC could reduce the ability of hippocampal neurons to carry out LTP and
fonn new memories. This relationship is important to the current study because our
findings suggest that the ability of developing hippocampal granule and pyramidal neurons
to detect this important NTF might be significantly impaired as a result of prenatal ethanol
exposure.
Pyramidal neurons—a population known to express trkC (Chao and McEwen,
1994)—are greatly affected by prenatal ethanol exposure (Barnes and Walker, 1981). It is
possible that the reduction in this population is responsible for the reduction in trkC
expression observed in the present study. It is also important to examine why BDNF, NT-
3, and trkB gene expression are unaffected by ethanol exposure even though there is a
significant loss of cells expressing these proteins in the hippocampus (Barnes and Walker,
1981). Granule cells, pyramidal cells, and glia of the hippocampus all express BDNF,
NT-3, trkB, and trkC (Chao and McEwen, 1994; Condorelli et al., 1995; Dragunow et al.,
1997; Mathem et al., 1997). Using pyramidal cell loss as a potential explanation for the
reduction in trkC expression is refuted by the fact that all NTF and NTF receptor genes
analyzed in this study were standardized by a constituitively-expressed housekeeping gene.
That is, the reduction in pyramidal cells should not selectively reduce trkC since all other
cell types in the region also express trkC. Also, a loss of pyramidal neurons that express
trkC would also result in a loss of an equal number of cyclophilin-expressing neurons.
Therefore, the reduction of trkC observed in our study is a reduction per unit volume of
tissue and not just a gross subtraction due to cell loss.

137
The results of the present study also relate to a previous study that found that
ethanol reduces neurofilament levels in cultured hippocampal neurons (Saunders et al.,
1997). Neurofilaments are necessary for neurite outgrowth (Saunders et al., 1997). Since
neurotrophins promote neurite outgrowth, presumably through their interaction with trk
receptors, the effect observed in the Saunders et al. study could be due to a reduction in
trkC expression (1997). The reduction in trkC would likely result in decreased NT-3
signal. Less neurofilament protein would be produced and as a result, and fewer neurites
would be extended, which would then affect the individual neurons ability to make
synapses and receive neurotrophic support. The results of the present study may explain
the results of some previous studies and may help to answer questions about the
fundamental nature of ethanol toxicity.
EPET
There were more physical differences at P21 following EPET than there were
following CPET. As evidenced by gross body measurements, male animals were affected
to a greater extent by EPET than female animals. Male Ethanol animals actually weighed
more than both Separated and Unseparated animals at P21 (refer to Figure 4-3). This
finding is probably due to the culling procedure employed in this study (ethanol litters were
limited to seven pups and control litters had 10 pups). Following the exposure period,
which ended on P10, the Ethanol pups had greater opportunity to nurse than the control
pups did and gained more weight. Both male and female animals had decreased brain
weight following EPET. This difference was further borne out by the fact that brain weight
to body weight ratio was reduced in both male and female Ethanol animals in comparison to
both Separated and Unseparated animals. The physical differences described above did not
result in a change in the gene expression of any of the genes analyzed in this study.
The fact that gene expression was unchanged in BDNF, NT-3, trkB and trkC
following EPET could indicate that earlier observations of decreased neurotrophic activity
following EPET were due to an NTF other than one of the neurotrophins studied. Other

138
NTFs, such as bFGF, are produced in the hippocampus and are important to the normal
functioning of neurons in this brain region (Emfors et al., 1990). Also different genes are
more or less active at different points during development (Maisonpierre et al., 1990).
Therefore, it is possible that EPET affects other genes or may affect the genes studied to an
extent that is not detectable by methods used in the present study.
General Discussion
Taken together all of the results suggest that ethanol does have the ability to regulate
NTF receptor gene expression to some extent. Specifically, trkC gene expression was
significantly lower in male rats following CPET than in control animals. This study is not
the first to find that ethanol has the ability to alter gene expression. Prenatally, ethanol
reduces IGF-I (Breese et al., 1994; Singh et al., 1996) and IGF-II in rat brain (Singh et al.,
1996). In contrast, IGF-II gene expression is increased in fetal lung tissue (Fatayerji et al.,
1996). Ethanol exposure increases c-jun and junD levels in cultured neuroblastoma cells in
a dose-dependent manner (Ding et al., 1996) and increases NMDA receptor gene
expression in cultured mouse cortical neurons (Hu et al., 1996). The fact that c-jun is
affected by ethanol treatment could be could be important. C-jun is an immediate early
gene and is activated before a change in genetic expression occurs. Immediate early genes
are thought to control growth and differentiation of neurons by regulating other genes
(Abraham et al., 1991). Members of the jun family form dimers and bind to DNA to
regulate transcription (Vogt and Morgan, 1990). It is possible that c-jun and junD may be
responsible for the change in expression of trkC observed in the present study. If these
factors regulate trkC expression negatively, increasing their expression would likely cause
a decrease in trkC gene expression. The fact that ethanol can regulate this gene further
demonstrates that genetic control in a developing neuron can be affected by ethanol
treatment.
Other studies have examined ethanol’s ability to regulate genetic expression
following development. Chronic ethanol treatment in adult rats resulted in a reduction of

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BDNF gene expression in the hippocampus (MacLennan et ah, 1995). This result is in
contrast to the results of the present study. However, the current study is developmental
and should not necessarily correlate with adult chronic ethanol treatment studies because of
the state of the nervous system during ethanol exposure. The developing CNS contains
many critical periods where functional synapses are being formed and where a slight
disruption can result in neuronal loss. The adult nervous system, while vulnerable to
teratogenic effects of ethanol, does not contain an environment in a state of developmental
flux. Therefore, exposure to ethanol during development—whether in útero or postnatal—is
different from adult ethanol treatment. Trk gene expression was not analyzed in the
MacLennan et al. study but has been analyzed following adult ethanol exposure.
Specifically, trkB expression was observed to be upregulated following ethanol exposure
in adult male rats (Baek et al., 1996).
The present experiments were designed to precisely determine whether BDNF,
NT-3, trkB, or trkC gene expression in the hippocampus is altered as a result of ethanol
exposure. NGF gene expression was also attempted, but repeated attempts at probing
failed to yield quantifiable signals. Efforts to obtain results from NGF probing included
longer exposure to the phosphorimaging plates, increase in the amount of probe, and
increase in the amount of radioactivity. Following a lack of success with each of these
potential solutions, NGF probing was abandoned. Previous experiments in our laboratory
have suggested that ethanol exposure does change the amount of neurotrophic activity
present in extracts made from rat hippocampus. These experiments involved culturing
DRG neurons in the presence of these extracts and assessing survival and neunte
outgrowth. Specifically, CPET increased neurotrophic activity of hippocampal extracts at
P21 (Heaton et al., 1995c), and EPET decreased neurotrophic activity of hippocampal
extracts at this same age (Moore et al., 1996). Even though in the present study there was
no change detected in the expression of the neurotrophins studied, these results do not
necessarily disagree with those previous studies from this laboratory. Since other NTFs

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are synthesized in the hippocampus, overexpression of another polypeptide could have
produced the increase in neurotrophic activity observed following CPET.
The age examined in the present study was chosen because previous studies which
analyzed the neurotrophic activity following both CPET (Heaton et al., 1995c) and EPET
ethanol exposure (Moore et al., 1996) both found altered neurotrophic activity at this, and
not any other, age. Gene expression should be examined at other time points to determine
the temporal extent of the decrease in trkC expression. Specifically, trkC and other NTF
and NTF receptor genes should be assessed at time points that are closer to the period of
ethanol exposure, and at time points between the completion of ethanol exposure and P21.
Additionally, analysis should also occur at a time point near maturity to determine if any
affect of developmental ethanol exposure is long-lasting. For prenatally exposed animals
ethanol exposure ends on P0 so the analysis should take place on PI, P7, P14, and P60.
For postnatally exposed animals ethanol exposure runs from P4 to P10. Therefore,
analysis should occur on PIO, P14, and P60. Additionally, analysis during the exposure
period might reveal ongoing effects of ethanol exposure with respect to gene expression.
Also, other brain regions known to be affected by ethanol exposure, such as the cerebellum
(Cragg and Phillips, 1985; West, 1986) and the cerebral cortex (Miller, 1986) should be
examined to determine if ethanol affects NTF or NTF receptor gene expression. The
amount of functional NTF and NTF receptor protein present in the hippocampus should
also be examined. Since a reduction of trkC gene expression does not necessarily correlate
with a reduction in the functional trkC protein, future studies should examine these animals
to determine if this is indeed the case.
The results of the present study may have some bearing on the results of the
neurotrophic activity following prenatal ethanol exposure study performed in this
laboratory (Heaton et al., 1995c). Perhaps NTFs were over-produced to compensate for
the lack of responsiveness in hippocampal neurons due to the decrease in trkC expression.
Previous research has shown that ethanol exposure causes an upregulation of NGF protein

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in rat hippocampus and cortex (Nakano et al., 1996). Other NTFs and NTF receptors have
been found to be upregulated following neuronal injury. These include CNTF following
entorhinal cortex lesion (Lee et al., 1997) and trkA following striatal and basal forebrain
injury. The decrease in trkC gene expression could also be interpreted as a neuronal
response to injury. Previous research has shown that when the hippocampus is injured by
ibotenic acid, what results is a neuron poor, astroglia rich environment (Belluardo et al.,
1995). In this setting, trkC gene expression was significantly reduced. The possibility that
ethanol produces an injury type of response is interesting, but that fact alone would not
explain the mechanism by which ethanol could injure the neurons. One candidate
mechanism for altering gene expression following ethanol exposure is a change in DNA
methylation. As was mentioned above, DNA is modified by methylation at the 5' position
of cytosine residues. This process converts cytosine to methylcytosine and is thought to
interfere with the binding of proteins—repressors or enhancers—to DNA to change gene
expression (Holliday, 1987). This change in methylation can either increase or decrease
the transcription of a given gene and thereby alter the expression since repressors or
enhancers are not selectively affected.
The relationship between cell types found in the hippocampus, the neurotrophin and
neurotrophin receptor genes that they express, and their susceptibility to ethanol may help
explain the present results. Cell types found in the hippocampus include granule cells,
pyramidal neurons, and glia. Hippocampal granule cells express NGF (Lindefors et al.,
1992), BDNF (Mathem et al., 1997), NT-3 (Mathem et al., 1997), trkB (Dragunow et al.,
1997), and trkC (Dragunow et al., 1997). Pyramidal neurons are known to express
BDNF, NT-3, trkB, and trkC (Chao and McEwen, 1994) as well as bFGF (Chao and
McEwen, 1994; Walicke, 1988) and NGF (Lindefors et al., 1992). Glial cells also express
some of the NTFs used in the present study. Specifically, microglia, astroglia, and
oligodendrocytes express NT-3, trkB, and trkC as well as NGF (Condorelli et al., 1995).
Following prenatal ethanol exposure, pyramidal neuron loss is observed in the

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hippocampus (Barnes and Walker, 1981). Further examination of the pyramidal neurons
revealed that synapses and dendritic branching were also greatly affected by ethanol
exposure (Smith and Davies, 1990).
The effect of ethanol exposure on granule neurons is not as clear. Pierce and West
(1987) found that granule cells are increased following postnatal ethanol exposure while the
overall area of the hippocampus was reduced. Another laboratory found a reduction in
mature granule neurons following prenatal exposure (Wigal and Amsel, 1990). Based on
these studies, it appears that granule cells have a specific temporal vulnerability to ethanol
toxicity. The reduction of trkC gene expression observed in the present study could be due
to a reduction of any of the hippocampal cellular populations, but the fact that pyramidal
cells are greatly affected by ethanol exposure suggests these cells might be where the
largest change is occurring. However, pyramidal cells that are affected by ethanol
exposure are most likely already dead by P21. Logically, trkC is probably altered in its
expression in other cell types. As was mentioned above, the loss of neurons from the
hippocampus results in a neuron poor, astroglia rich environment, where trkC is reduced in
expression (Belluardo et al., 1995). A future aim of this research should be to use in situ
hybridization to determine whether trkC expression is affected in one particular cell type in
the hippocampus. That is, is trkC gene expression reduced in pyramidal cells preferentially
over granule cells or glia. Since pyramidal cells are most likely already reduced in number
by P21, such an analysis should take place over a time course that is close in proximity,
and perhaps includes, the period of ethanol exposure. Analyzing the expression of trkC in
situ, and correlating this expression with pyramidal and granule cell counts on the same
day, would possibly determine if trkC expression in this cell population is related to cell
loss. The fact that all of the cell types in the hippocampus express trkC is important,
because it suggests that these populations respond to NT-3. Therefore, a significant
reduction of trkC could greatly affect these neurons. A relatively simple way to determine
if trkC is required for survival of pyramidal neurons in vivo is to count surviving neurons

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in knockout animals. To date, this analysis has not been completed, but such a study
would help to determine if a reduction in trkC causing significant pyramidal cell loss is
physiologically relevant.
The fact that the present study found that female animals were not affected in a
manner similar to male animals in terms of genetic expression by prenatal ethanol exposure
suggests that there is a fundamental difference between male and female animals’ ability to
withstand ethanol insult. While the concept of sex-dependent effects of ethanol is not new,
previous research has been somewhat inconclusive in finding an absolute difference in
susceptibility between male and female animals following ethanol exposure. In addition to
the present results, our laboratory has identified gender differences in the effect that ethanol
had on neurotrophic activity in the hippocampus of postnatally exposed rats (Moore et al.,
1996). Specifically, male animals were affected to a greater degree than females. The
hippocampus has been previously shown to be affected in a sexually dependent manner in
that male rats exhibit a spatial learning deficit that is not found in female animals following
prenatal exposure (Zimmerberg et al., 1991). The interesting result among these studies is
that male animals are affected to a greater degree than female animals in all instances. In
fact, the studies that initially observed pyramidal neuron loss in the hippocampus were
performed using only male animals (Barnes and Walker, 1981; West, 1986). The
implication is that the hippocampus in male animals may be more susceptible to ethanol
insult than it is in female animals. This result—hippocampal impairment—is logical
considering that learning and memory deficits are a common characteristic of FAS (Abel,
1995; Jones and Smith, 1973; Streissguth et al., 1991). The fact that normal hippocampal
functioning is impaired by ethanol treatment may explain the behavioral deficits observed in
FAS. That is, impairment of the septohippocampal system by ethanol is a possible cause
of these behavioral deficiencies.
Other than the hippocampus, brain regions affected in a sexually dependent manner
include the septum and the amygdala. Our laboratory found that female rats exposed to

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ethanol exhibit a greater reduction in septal parvalbumin neurons than their male
counterparts (Moore et al., 1997) and prenatally-exposed male rats contain greater numbers
of cholinergic septal neurons than female ethanol-exposed animals (Swanson et al., 1996).
Thus, both septal studies exhibit a greater effect among female animals. Other laboratories
have discovered gender differences in response to ethanol exposure. DNA production is
significantly reduced in the amygdala of male rats, but unchanged in female animals
following prenatal ethanol exposure (Kelly and Dillingham, 1994). On a macro level male
and female animals respond differently to stress following ethanol exposure in that ethanol-
fed females exhibited increased corticosterone concentrations in comparison to ethanol-fed
males (Giberson et al., 1997; Weinberg, 1992). All of these studies show that ethanol can
affect the sexes differently, but not in a consistent manner. One sex is not preferentially
affected in all instances. The fact that sexual differences are so common underlies the
importance of analyzing sexes separately in ethanol exposure studies.
Other studies that have examined possible gender disparities did not find significant
disparities. For example, the locus coeruleus was affected by ethanol treatment, but no
gender difference was observed (Lu et al., 1997). Alcohol dehydrogenase activity was
found to exhibit no gender differences in a variety of mouse strains (Rao et al., 1997). Our
laboratory found that ChAT activity was not altered in a sexually dependent manner in male
and female animals following ethanol exposure (Swanson et al., 1995). Even though
different sexual responses to ethanol are not present in all studies, the possibility that
ethanol may differently affect male and female animals should continue to be explored
further in future experiments.
The process of exploring the phenomenon of ethanol-NTF interaction in the
nervous system would require the use of tissue culture to investigate signal transduction in
hippocampal cultures. The cultures would have to be grown in serum-free media since
serum contains large amounts of undefined proteins and would greatly complicate analysis.
Large cultures (107 in number or greater) of hippocampal neurons necessary to study signal

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transduction can be obtained. However, the PC 12 immortalized cell line has been used
extensively in studying signal transduction of trk receptors (Stephens et al., 1994). Even
though PC 12 cells have been widely used to study trk signaling, primary hippocampal
cultures would be preferable for this analysis because they are normal neuronal cells and
not merely a model. While the tissue culture environment does lack some of the in vivo
interactions that are important for a complete analysis of any interaction between trk signal
transduction and ethanol-such as glial cell/neuron interactions—it is the only method that
allows specific signaling events to be studied. NT-3 would be the logical NTF to use in
studying these events since its receptor, trkC, is affected by CPET and expressed by this
neuronal population (Chao and McEwen, 1994; Dragunow et al., 1997). While this
analysis would not explain the decrease in trkC gene expression observed following
prenatal ethanol exposure, it would help to further define the role of ethanol toxicity as it
relates to NTFs. This relationship connects all of the studies in this overall document.
The present study investigated the ability of ethanol to regulate BDNF, NT-3, trkB,
and trkC gene expression. Since trkA is not expressed in the hippocampus it was not
examined in this study (Martin-Zanca et al., 1990). NGF, while expressed at a level that is
above the threshold of detection for Northern blotting (Maisonpierre et al., 1990), did not
yield bands that could be quantified. Perhaps a future goal of this study should be to
examine NGF expression with a finer molecular biology technique—such as RNAse
protection assay. Overall, the results of this study indicate that ethanol can alter the
expression of NTF receptor genes when administered prenatally. The results of the present
study should serve as a point of reference in the search for a mechanism of ethanol toxicity.
Since trkC is now known to be regulated by CPET, future researchers should determine
exactly how this gene is affected by ethanol.

CHAPTER 5
CONCLUSIONS AND IMPLICATIONS
Animal Models
The research described in this document was undertaken to describe the relationship
between ethanol and neurotrophic factors (NTLs) in the developing nervous system. Two
animal models were used to carry out this objective. The chick embryo model was used to
determine the effect that ethanol had on the developing motoneuron population of the spinal
cord and to test the ability of NTLs to modulate the toxicity of ethanol in vivo. The rat
animal model was used to determine the effect that ethanol treatment during hippocampal
development had on NTL and NTL receptor gene expression. Each animal model was
selected for a specific advantage it had in completing the proposed studies. The chick was
chosen because ethanol can be administered in exact doses to the developing embryo, and
only molecules produced by the embryo itself remove ethanol from the embryonic
environment. Ethanol is cleared from the bloodstream by the mother in a mammalian
system whereas the chick embryo is isolated as it develops. The fact that maternal
influences are removed does not make the chick ideal for comparisons to human fetal
alcohol syndrome (FAS), but does allow for the examination of direct effects of ethanol on
a developing neuronal population. Additionally, the chick embryo model has been widely
used to study the effects of NTFs on the developing motor system (Oppenheim et al.,
1995; Oppenheim et al., 1992). NTFs can be administered through small holes directly to
the embryo onto the chorioallantoic membrane since the embryos tolerate slight invasions
into their environment quite well as long as the underlying membranes are not disrupted.
The rat was chosen to examine the ability of ethanol to alter gene expression of
NTFs because it is a mammal and ethanol exposure would be similar to that found in
146

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humans. Additionally, the rat is the most widely studied model of FAS research and
exhibits many of the same deficits found in human FAS (Diaz and Samson, 1980; Sherwin
et al., 1981). As a mammal, the rat central nervous system (CNS) compares favorably
with the human and contains the same major structures found in the human CNS.
Additionally, our laboratory has found that prenatal exposure to ethanol increased
neurotrophic activity of hippocampal extracts on postnatal day 21 while postnatal exposure
to ethanol decreased neurotrophic activity of these same extracts (Heaton et al., 1995c;
Moore et al., 1996). The rat has also been used in previous research which found that
chronic ethanol treatment in adult male rats resulted in a decrease in the expression of the
brain-derived neurotrophic factor (BDNF) gene (MacLennan et al., 1995). The adult
exposure utilized in that experiment is different from the prenatal exposure used in the
present study in that adult exposure requires only the subject’s liver to remove ethanol from
the bloodstream. In prenatal exposure, the mother’s liver can remove ethanol from the
bloodstream. Early postnatal exposure in rats, even though the nervous system is
developmentally like a prenatal human nervous system, is like adult exposure. However,
the CNS environment is very different in developing and adult animals. Where the adult
CNS is relatively stable, the developing CNS is undergoing rapid changes that will
ultimately lead to the structures of the adult CNS. During this active stage of development,
the CNS is also susceptible to disruptions that would not normally harm an adult CNS.
Teratogens that act on these neurons can have a long-lasting effect on the development of
the CNS. The studies described previously are related in that they all examine some facet
of ethanol toxicity as it relates to NTFs. The studies examined two sides of one issue:
Methods
The methodology employed with each animal model in the present research was
chosen for specific reasons. Since the present research builds on results from previous
work—both from this laboratory and from outside laboratories—the methods used in this
study were similar to these previous studies. By replicating methods that had been used in

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the past, better comparisons could be made between our results and previous work. For
example, the method for delivering NTFs to embryonic chicks was similar to that used in
studies from Oppenheim’s laboratory (Oppenheim et al., 1995; Oppenheim et al., 1992).
Delivery of ethanol to prenatal and postnatal rats replicated methods used in studies from
our laboratory (Heaton et al., 1995c; Heaton et al., 1996; Moore et al., 1996; Ryabinin et
al., 1995; Swanson et al., 1995; Swanson et al., 1996). Northern blotting techniques were
similar to those used in assessing the effect chronic ethanol treatment had on NTF gene
expression in the adult hippocampus (MacLennan et al., 1995). Thus, it is clear that great
care was taken to ensure that valid comparisons were made between the present studies and
previous ones.
The method used to deliver NTFs to developing chick embryos was ideal in that the
developing embryo was never physically contacted during the procedure. Past experience
in this laboratory has found that an embryo will likely die if the membranes are disturbed.
One disadvantage of our method was that an occasional embryo was lost because of human
error with the injecting procedure. Another disadvantage is that NTFs are administered
systemically and almost certainly exert effects in addition to those described in Chapter 3.
Future experiments will have to determine whether the current evidence that GDNF
increases motoneuron number in a manner independent to ethanol is achieved by protection
of developing motoneurons from ethanol toxicity. The current experiments do not allow a
conclusion of neuroprotection from ethanol toxicity to be drawn. Such an analysis would
require an interaction between ethanol and GDNF. Specifically, experiments will have to
determine whether ethanol directly hinders the toxic mechanism of ethanol in the nervous
system or some other independent mechanism. For example, ethanol is known to affect
Ca2+ homeostasis in cultured neurons (Myers et al., 1984; Webb et al., 1995). The next
step would be to determine whether GDNF can directly prevent such a change from
occurring. In the current study, NTFs were administered through pinholes in the eggshell
and dropped onto the chorioallantoic membrane of developing chick embryos. The fact that

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survival was highest in animals receiving an NTF only suggests that NTF administration
had little adverse effect on embryonic survival. The methods used to deliver ethanol to
developing rats have been used previously in this laboratory and in others. The ethanol-
containing liquid diet fed to pregnant dams has been shown to produce deficits in the
hippocampus while providing nutrition above and beyond control conditions (Barnes and
Walker, 1981). Ethanol inhalation was used to build on a previous study from this
laboratory and because it has been shown to be an effective, nonintrusive method for
delivering ethanol to neonatal rats (Moore et al., 1996; Ryabinin et al., 1995). While
artificial rearing is also effective in delivering precise doses of ethanol to the subjects, the
possible stresses introduced by this procedure can make interpretation of the results
difficult. For these reasons, and those discussed in Chapter 4, ethanol inhalation was used
to simulate ethanol exposure during the brain growth spurt.
Quantitative Northern blotting was used to determine the relative genetic expression
of neurotrophin and neurotrophin receptor mRNA in the hippocampus. Other methods for
detecting mRNA expression, such as RNAse protection assay and reverse transcriptase
polymerase chain reaction, can detect mRNA expression at least about ten times greater
sensitivity (Lee and Costlow, 1987; Sperisen et al., 1992). The variability found when
using these methods is often much greater than that found with Northern blotting. When
genes are readily detected by Northern blotting, analysis of their expression can be
accurately quantified. All of the genes examined in the current study—BDNF,
neurotrophin-3 (NT-3), tyrosine receptor kinase (trk) B, and trkC-are expressed at levels
which make quantitative Northern blotting appropriate and preferable for these experiments
(Maisonpierre et al., 1990). Originally, our aim was to probe for NGF in addition to the
above NTFs and receptors. However, repeated attempts at probing for NGF produced no
quantifiable results. The fact remains that NGF should be expressed at a high enough level
in the hippocampus to be detected by Northern blotting (Maisonpierre et al., 1990).
Perhaps the NGF cDNA probe used in these studies does not share enough homology with

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Long Evans rats to stay bound through the high stringency washes. An NGF cDNA probe
isolated from a rat from our colony might produce quantifiable bands.
Hypotheses and Results
The research chapters of this document described all of the experiments performed
for this study. Each chapter attempted to answer a portion of the hypotheses described in
Chapter 1: (1) (a) We hypothesized that ethanol would reduce motoneuron number in the
absence of naturally occurring cell death (NOCD); (b) We hypothesized that ethanol would
reduce motoneuron number at period of development that follows the period for NOCD; (2)
We hypothesized that exogenous NTFs would provide in vivo protection for motoneurons
exposed to ethanol; and (3) We hypothesized that chronic prenatal ethanol treatment
(CPET) and early postnatal ethanol treatment (EPET) would alter the gene expression of
neurotrophins and/or their receptors in the hippocampus of treated rat pups.
The major findings of Chapter 2 were that ethanol did not exacerbate NOCD in the
developing spinal cord when administered from E4 to Ell; ethanol reduced motoneuron
number when administered from E10 to E15; and ethanol did not affect the neurotrophic
content of muscle when administered from E10 to E15. Thus, experiments described in
Chapter 2 confirmed the hypothesis that ethanol would reduce motoneuron number in the
lumbar spinal cord when NOCD was suspended and when administered from E10 to E15.
These experiments provided other information that was valuable in describing the action of
ethanol upon developing motoneurons. The fact that neurotrophic content of limb muscle
was unchanged suggests that ethanol was not merely altering the amount of NTFs
produced by the motoneuronal targets to reduce their number at this age. However, the
current results do not definitively demonstrate that NTF activity in chick muscle tissue is
unaffected by ethanol exposure from E10 to E15. The major finding of Chapter 2 was that
ethanol can act directly to affect this population. A specific way that ethanol could directly
affect motoneurons is alteration of Ca2+ homeostasis. Changes in Ca2+ levels are linked to
neuronal death (Choi, 1988) and ethanol is known to modulate this delicate system (Gandhi

151
and Ross, 1989; Leslie et al., 1990; Reynolds et al., 1992; Webb et al., 1995). However,
these studies do not demonstrate a causal relationship between ethanol and Ca2+-induced
cell death.
Another possible way ethanol could directly harm neurons is by altering membrane
fluidity. Indeed, ethanol does have the ability to change this important cellular property
(Avdulov et al., 1995; Schroeder et al., 1988; Wood et al., 1989). If the integrity of the
membrane is compromised, a cell is susceptible to changes that could result in death. One
such change could be an alteration of the intracellular ion concentration, since changes in
membrane fluidization can lead to altered activity in the Na+/K+ ATPase (Madsen et al.,
1992). Furthermore, neurons are dependent on proper concentrations of Na+ and K+ to
generate action potentials. Proper synapse formation is dependent on the ability of neurons
to generate activity and access to target-derived NTFs is related to synapse formation (Lu
and Figurov, 1997). Since NTFs promote neuronal survival and maintenance, changes in
membrane fluidization may have far-reaching effects on the survival of developing
neurons. However, there is no evidence that NTFs affect membrane fluidity. Therefore,
experiments that test the ability of NTFs to stabilize or destabilize the cellular membrane
should be performed before attempting to determine whether they can prevent ethanol
disturbing this integral system. Certainly, other possible mechanisms for ethanol toxicity
exist and this brief discussion should not be considered to be comprehensive.
Chapter 3 sought to determine whether motoneuron death due to ethanol in a living
organism could be prevented by NTF treatment. The major result of this chapter was that
GDNF significantly increased motoneuron number in the presence of ethanol. BDNF did
not significantly protect developing motoneurons and did not significantly interact with
ethanol. Thus our hypothesis that GDNF and BDNF would provide in vivo
neuroprotection was not confirmed since neither NTF interacted with ethanol. Previously,
NGF was previously shown to protect against ethanol induced decrease in cholinergic
activity in whole chick (Brodie et al., 1991). GDNF has been shown previously to provide

152
protection against neurotoxic insults. For example, GDNF protects against 6-
hydroxydopamine lesion in the substantia nigra of the rat (Kearns and Gash, 1995) and
protects against ischemia induced injury in rat cortex (Wang et al., 1997). In fact, GDNF
has even been shown to provide protection against ethanol insult in culture. McAlhany et
al. found that GDNF rescued rat organotypic cultures of cerebellar Purkinje cells from
ethanol neurotoxicity (1997).
Above, two possible mechanisms of direct toxicity were explored and tied to
ethanol. NTFs are known to affect some of these same processes and at the same time
promote cell survival. For example, NGF, bFGF, and insulin-like growth factors I and II
(IGF-I and IGF-II) were all shown to prevent neuronal death due to excitotoxicity
presumably by keeping intracellular calcium levels in the cell at sublethal levels (Mattson
and Cheng, 1993). All of these NTFs are members of different NTF families.
Specifically, NGF is a member of the neurotrophin family; bFGF is a member of the
fibroblast growth factor family; and IGF-I and IGF-II are members of the insulin-like
growth factor family. Thus, NTFs from varied families exhibit similar traits of promoting
neuronal survival. GDNF, as a member of the transforming growth factor 8 family, might
also exhibit these characteristics. To date GDNF’s ability to regulate intracellular calcium
has not been investigated. Therefore, given that ethanol can modulate Ca2+ concentration
and NTFs can stabilize Ca2+, GDNF might protect motoneurons by holding Ca2+ at a safe
level. It is important to note that evidence from the current experiments does not suggest an
interaction between ethanol and GDNF. A relatively simple way to test this hypothesis
would be to replicate the above experiments to determine whether GDNF can alter Ca2+
concentration.
The experiments described in Chapter 4 used another method of defining the
relationship between ethanol and NTFs in the developing nervous system to determine
whether ethanol could modulate the genetic expression of neurotrophins, or their receptors,
in the hippocampus. The major findings of Chapter 4 were that CPET in the rat reduced

153
trkC gene expression in male rat hippocampus on P21. The results both confirm and refute
the hypothesis for this chapter in that CPET altered genetic expression of trkC while
postnatal exposure did not alter the expression of any of the genes studied. None of the
neurotrophin genes were altered following prenatal or postnatal exposure. Therefore, that
part of the hypothesis was also refuted by the results of the study. This study is not the
first to demonstrate a change in the in vivo expression of a NTF gene following embryonic
ethanol exposure. Earlier research found that IGF expression was increased in whole rat
brain following prenatal ethanol exposure (Breese et al., 1994; Singh et al., 1996).
However, receptor gene expression was not affected in that study.
Also important was the fact that female and male animals were not similarly affected
by ethanol exposure. This result suggests that there is a fundamental difference between
male and female animals’ ability to withstand ethanol insult. The hippocampus appears to
be more susceptible to ethanol insult in male rats in comparison to female animals.
Specifically, male rats exhibit deficiencies in spatial learning following prenatal ethanol
exposure (Zimmerberg et al., 1991), decreased neurotrophic activity following postnatal
exposure (Moore et al., 1996), and the present findings indicate that prenatal exposure
reduces trkC gene expression. Other brain regions affected in a sex dependent manner
include the septum and the amygdala. Specifically, female rats exposed to ethanol
prenatally exhibit a greater reduction in septal parvalbumin neurons than their male
counterparts (Moore et al., 1997) and prenatally-exposed male rats contain greater numbers
of cholinergic septal neurons than female ethanol-exposed animals (Swanson et al., 1996).
Both studies of the septum display a greater effect of prenatal ethanol exposure on female
animals. In the amygdala, DNA production is reduced in male animals but unchanged in
female animals following prenatal ethanol exposure (Kelly and Dillingham, 1994). These
studies demonstrate the importance of isolating gender in FAS research.

154
Conclusions
One finding of this research was that genetic expression of trkC is reduced
following prenatal exposure to ethanol in rats. Genes that are known to be affected by
ethanol treatment include BDNF (MacLennan et ah, 1995), IGF-I and IGF-II (Breeseet
ah, 1994; Singh et al., 1996), c-jun and junD (Ding et al., 1996), and NMDA receptor (Hu
et al., 1996). In the current experiments, no neurotrophin genes were altered as a result of
prenatal or postnatal ethanol treatment. The fact that the jun family is modulated by ethanol
treatment may help to illuminate a mechanism for ethanol toxicity in the developing nervous
system. Since c-jun binds DNA directly to regulate transcription, it is possible that ethanol
alterations in its activity serve to change the expression of other genes. This relationship
should be explored further in future experiments.
Since both BDNF and trkC gene expression are known to be affected by ethanol
exposure (MacLennan et al., 1995), and trk intracellular signaling activates MAP kinase
(Stephens et al., 1994), it makes sense to examine signal transduction pathways that result
from trk molecules to determine whether ethanol can interfere. Intracellular trk signaling
involves phosphorylation of many different proteins and eventually activates proteins that
are known to alter transcription in the nucleus. Previous research has identified distinct
pathways involved in trk signaling. Both pathways are initiated by autophosphorylation of
tyrosine 490 on the intracellular domain of trk (Stephens et al., 1994; Tolkovsky, 1997).
As mentioned above, one of these pathways leads to activation of the MAP kinase cascade
and is thought to initiate neurite outgrowth, transcription, or cellular hypertrophy (Stephens
et al., 1994). The other pathway leads to akt (a serine/threonine kinase) activation and may
initiate neurite outgrowth, survival, and receptor internalization (Tolkovsky, 1997). Since
neuronal survival is affected by CPET and EPET (Barnes and Walker, 1981; West et al.,
1986), and neurite outgrowth is inhibited by ethanol exposure in culture (Heaton et al.,
1993; Saunders et al., 1997), it is possible that ethanol may specifically disrupt the latter
pathway of the trk intracellular cascade.

155
Future research will determine where the cascade might be affected. Each protein in
the two trkC pathways should be examined for phosphorylation using antibodies for
phosphotyrosine by western blot. Proteins would be obtained by immunoprecipitation of
cell lysates. The time course of the reactions following neurotrophin binding to trk is
relatively short. For example, SHC (a protein in the trkC intracellular cascade with a src
homology domain) is phosphorylated just one minute after neurotrophin is introduced to
the culture medium. Therefore, hippocampal cultures established to study this
phenomenon do not have to be established for a long period before the analysis begins
(Stephens et al., 1994). These future experiments should allow us to further define any
interaction of ethanol and trk receptors.
The results of this doctoral research do suggest some new ideas about the nature of
ethanol neurotoxicity. Since most of the genes studied in Chapter 4 were not affected
significantly by CPET or EPET, ethanol does have the ability to alter the expression of
discrete genes. This suggests that ethanol alters the activity of specific enhancers or
repressors in the nucleus. Perhaps this result is achieved through changes in methylation
of fetal DNA. Above, it was mentioned that methylation serves to prevent enhancers or
repressors from binding to DNA and may alter gene expression (Holliday, 1987) and that
ethanol is known to alter methylation of fetal DNA (Garro et al., 1991). By interfering
with methylation at a specific site in the genome, ethanol may act in ways that are more
specific than had previously been thought.
The aim of all FAS research is to answer the question: “How does ethanol cause its
toxic effects in the nervous system?” By comparing this research to other studies, some
hypotheses can be made. For example, ethanol is now known to affect neurotrophin
receptor gene expression and alter neurotrophic activity in the hippocampus (Heaton et al.,
1995c). NTFs are known to produce a variety of effects in the hippocampus. For
example, BDNF and NGF have been shown to regulate a number of peptides in the rat
hippocampus (Croll et al., 1994). Specifically, BDNF increases NT-3 activity in the

156
hippocampus (Lindholm et al., 1994) while BDNF, NGF, and NT-3 have been shown to
induce choline acetyltransferase (ChAT) activity in the septohippocampal neurons
(Alderson et al., 1990; Auberger et al., 1987; Gnahn et al., 1983; Nonner et al., 1996).
Prenatal ethanol exposure has been shown to slightly reduce ChAT activity in the septum at
P7, while sparing septal neurons from any significant cell death (Swanson et al., 1995;
Swanson et al., 1996). The reduction of ChAT activity was transient in nature and did not
extend to any later ages. In light of the present results, the change in ChAT activity could
be due to the reduction in trkC gene expression if trkC is also reduced in the septum
following ethanol exposure. If less receptor is available for NT-3 to bind, less signal from
trkC will be produced intracellularly. Therefore, less of an effect (in this case ChAT
activity) would result in ethanol exposed animals. Future studies should attempt to
determine if such a relationship does indeed exist.
As was stated above, the results of Chapter 3 are especially important because they
suggest that ethanol can increase motoneuron number during embryonic ethanol exposure.
The implication of these experiments may be a drug treatment for FAS. One issue that
inevitably arises when discussing this subject is delivery of the drug to the population of
interest. The main difficulty is that most NTFs cannot cross the blood brain barrier
(Anderson et al., 1995). Therefore, an NTF must be delivered by direct administration to
the brain (which is invasive) or by some other vector. The use of viral vectors is not
invasive, but the immune system will eventually hinder the process. Other diseases have
been treated with NTFs include Parkinson’s Disease and amyotrophic lateral sclerosis.
Specific NTFs that have been used as potential therapies for Parkinson’s disease include
GDNF (Lapchak et al., 1997) and IGF-I (Festoff et al., 1995). Amyotrophic lateral
sclerosis has been treated with GDNF (Giménez y Ribotta et al., 1997), BDNF (Giménez y
Ribotta et al., 1997), and CNTF (Aebischer et al., 1996; Stambler et al., 1998). While
exhibiting success in animal models of the disease, these therapies have not yet provided a
cure for human subjects. Beyond defining which NTFs are successful in preventing

157
ethanol toxicity, future research should focus on delivering this protection to mammalian
and human test subjects. This goal is important because the aim for conducting this
research is to treat this harmful disorder in humans when prevention has failed. This next
step—developing a therapy or cure—is the only way that research in this field will ultimately
bejudged.

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BIOGRAPHICAL SKETCH
Douglas M. Bradley was born in Birmingham, Alabama, in 1971. He lived there
until 1990 when he entered the University of Florida as a freshman. There he met his wife-
to-be, Korey Rothman, and majored in Neurobiological Sciences. Among the highlights of
his undergraduate days were a year in the Pride of the Sunshine Marching Band, induction
in Phi Beta Kappa, 1st place in the Undergraduate Research Symposium, and highest
honors upon graduation. In the Fall of 1994, he began his tenure in the Department of
Neuroscience. The achievements for which he is most proud include a National Science
Foundation Predoctoral Fellowship, Grinter Predoctoral Fellowship, and his graduation
within four years of beginning this program.
178

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.
A
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, in scope and quality, as a
dissertation for the degree of Doctor of Philosophy.
A. JohlfMacLennan
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.
V,
Kathleen Shiverick
Professor of Pharmacology and Therapeutics
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 sqope and quality, as a
dissertation for the degree of Doctor of Philosophy./
AC/
Wolfgang J. Streit
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.
/{A? Cc/. LcJgJUc^
Don W. Walker
Professor of Neuroscience
This dissertation was submitted to the Graduate Faculty of the College of Medicine
and to the Graduate School and was accepted as
the degree of Doctor of Philosophy.
August 1998
llment of the requirements for
ge of Medicine
Dean, Graduate School




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