Alterations in responsiveness to ethanol and neurotrophic substances in fetal septohippocampal neurons following chronic prenatal ethanol exposure

DEVELOPMENTAL BRAIN RESEARCH

ELSEVIER

Developmental

Brain

Research

85 (1995)

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Research report

Alterations in responsiveness to ethanol and neurotrophic substances in fetal septohippocampal neurons following chronic prenatal ethanol exposure Marieta Barrow Heaton Unicersity

of Florida

Brain Institute, Uniuersity of Florida

* , Michael Paiva, Douglas J. Swanson, Don W. Walker

Department of Neuroscience, Center for Neurobiological Sciences, Center for Alcohol College of Medicine and DVA Medical Center, Gainesville, FL 32610.0244, USA Accepted

4 October

Research,

1994

Abstract Pregnant Long-Evans rats were maintained on three diets: a liquid diet in which ethanol accounted for 35539% of the total calories, a similar diet with the isocaloric substitution of sucrose for ethanol, and a lab chow control diet. At gestation day 18, the fetuses were taken and cultures of septal and hippocampal neurons prepared. Neuronal survival and neurite outgrowth were compared in cultures from the three diet groups, using the following media supplements: ethanol (1.2, 1.8 or 2.4 g/dl), neurotrophic factors (nerve growth factor [NGFI with the septal cultures, basic fibroblast growth factor [bFGF] with the hippocampal cultures), or ethanol plus neurotrophic factors. Both the septal and hippocampal neurons responded to ethanol in a dose-dependent manner. The neurons from both populations from fetuses which had been exposed prenatally to ethanol, however, tolerated considerably higher ethanol concentrations before decreases in survival or outgrowth were seen. These ethanol-exposed neuronal populations were also less responsive to neurotrophic factors: in hippocampal cultures, process outgrowth was significantly enhanced by bFGF in control but not ethanol-derived cultures, and in septal and hippocampal cultures, the neurotrophic factors significantly ameliorated ethanol neurotoxicity in control cultures, but not in those from the ethanol-exposed fetuses. The possible relevance of these observations to the fetal alcohol syndrome is discussed. Keywords: Fetal Septohippocampal

alcohol syndrome; system

Ethanol;

Ethanol

neurotoxicity;

1. Introduction

Ethanol has been shown to produce neurotoxic effects both in the adult and in the developing nervous system. The constellation of anomalies seen following prenatal ethanol exposure has been termed the fetal alcohol syndrome [251 (FAS). These abnormalities include facial dysmorphia, hyperactivity, altered muscle tone, attention deficits, memory and learning defects, and lowered IQ [48]. A number of central nervous system (CNS) regions have been shown in rodent animal models to be selectively vulnerable to prenatal and perinatal ethanol treatment, most notably the cerebel-

* Corresponding of Florida College

Center, Gainesville,

author. Department of Neuroscience, University of Medicine, PO Box 100244, Health Science FL 32610-0244, USA. Fax: (1) (904) 392-8347.

0165-3806/95/$09.50 0 1995 El sevier SSDI 0165-3806(94)00180-4

Science

B.V.

All rights

reserved

Neurotrophic

factor;

NGF;

bFGF;

Neuroprotection;

lum [17,32,55], cerebral cortex [1,36,37], hippocampus [2,7,54], and the septal region [44,49]. It has been hypothesized that alterations in the septohippocampal system underlie many of the behavioral and cognitive impairments seen in FAS. In attempts to identify mechanisms underlying ethanol neurotoxicity, a number of investigators in recent years have utilized the tissue culture procedure. Using this procedure, it is possible to view the direct effects of ethanol on neuronal survival and differentiation, without the complications produced by other non-optimal conditions which may occur as a consequence of prenatal ethanol treatment (e.g. hypoxia/ischemia, hypoglycemia, the presence of ethanol metabolites, etc.). In recent studies, we investigated neuronal ethanol responsiveness using cultured peripheral and central nervous system populations (embryonic chick dorsal root ganglion [DRGI cells [201;

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fetal rat septal and hippocampal neurons [221). Survival of each of these populations is relatively refractory to ethanol concentrations considerably in excess of physiological levels, although process elaboration is compromised at much lower levels. We have also shown, both in DRGs and in septohippocampal neurons, that neurotrophic factors can serve to provide amelioration with respect to ethanol neurotoxicity 120,221. In the present study, we wished to assess ethanol and neurotrophic factor responsiveness of CNS populations from animals which had been exposed to ethanol during gestation, and to determine the relative effectiveness of neurotrophic factors in modulating observed ethanol neurotoxicity. Prior characterizations of neuronal populations from animals exposed to ethanol in vivo have demonstrated a number of cellular alterations resulting from early ethanol exposure. These alterations include atypical physiological and pharmacological responsiveness [15,16,46], reduced plating efficiency [3], delays in cellular maturation [30], and changes in neurotransmitter synthesis [38]. The current investigation was intended to extend these earlier observations, in assessing direct ethanol influences on neuronal survival and differentiation following chronic prenatal ethanol exposure, and neurotrophic factor responsiveness. For these determinations, we cultured fetal rat septal and hippocampal neurons from lab chow animals, and from animals which had been maintained on a liquid diet containing either ethanol, or isocaloric quantities of sucrose.

2. Materials

and methods

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weight-matched group E partner consumed the previous day. The ethanol or sucrose diets were initiated on gestation day one (the day after insemination), and were continued until the animals were sacrificed. 2.2. Cell culture

preparations

The medial septal region (containing the medial septal and the diagonal band of Broca nuclei) and the hippocampus were dissected out under aseptic conditions from gestation day 18 (G18) fetuses from chow-fed (C), sucrose liquid diet-fed (S), or ethanol liquid diet-fed (E) dams, collected in balanced salt solution (BSS), then placed in 2 ml of 0.9% sterile phosphate-buffered saline (PBS) with 0.15 ml trypsin (2.5%; Gibco) and 0.10 ml deoxyribonuclease I (DNAse; Sigma) in a water bath (37°C) for 15-20 min. After this time, the buffer-trypsin solution was removed and replaced with either a modified Leibowitz L-15 medium, containing 1% penicillin/streptomycin, 0.7% fungizone, 1% glutamine, 5% heat-inactivated-horse serum (HS) and 0.5% fetal bovine serum (FBS; septal region), or with Eagle’s Minimum Essential Medium (MEM) with 10 mM sodium bicarbonate, 1 mM L-glutamine, 1 mM sodium pyruvate, 20 mM potassium chloride, 1% glucose, penicillin/streptomycin and fungizone, as above, and 10% FBS (hippocampal region). The serum acted to terminate the trypsinization. The tissues were then dissociated by gentle trituration using a flame-narrowed borosilicate pipette. The dissociates were centrifuged briefly, and the medium removed from the pellet and replaced with 1 ml of fresh medium. The cells were plated in 22-mm-diameter plastic culture wells which had been coated with collagen (type I from rat tail) and polyornithine (0.5 mg/ml and 0.04 mg/ml, respectively). Plating density averaged 35,000-39,000 neurons/cm’. In addition, in some instances, cells from both populations were plated at a lower density with approximately 22,000 cells/cmz, since previous studies have shown that certain cultured CNS neurons are increasingly dependent on neurotrophic factors (NTFs) as plating density is reduced 113,181. In experiments in which bFGF was to be used with hippocampal cultures (see below). 2 Kg/ml of the mitotic inhibitor cytosine arabinoside (ara-C) was added to the medium in order to inhibit proliferation of non-neuronal cells fostered by the growth factor. The cultures were incubated at 37”C, with 5% CO,-95% air.

2.1. Subjects

2.3. Experimental

Long-Evans hooded rats, purchased from Charles River Co., were bred in our facility. Nulliparous females were individually placed each evening with a male until a vaginal smear indicated day 1 of pregnancy. They were then individually housed and maintained on a 07.00-19.00 light cycle. The females were matched according to age and weight, and assigned to one of three treatment groups: one received an ethanol-containing liquid diet in which ethanol accounted for 35-39% of the total caloric intake. The second group received the identical diet except for the isocaloric substitution of sucrose for ethanol. The liquid diets were prepared by the mixture of a stock ethanol solution or an isocaloric sucrose solution with Sustacal (Mead Johnson). Both diets were additionally fortified with Vitamin Diet Fortification Mixture and Salt Mixture (ICN Nutritional Biochemicals). The diets contain 1.3 kcal/ml and provide several times the daily requirement of all essential vitamins and nutrients [53]. The third group received Lab Chow ad libitum (a control for possible non-specific effects of the liquid diet). Blood ethanol concentrations were determined using a microenzymatic assay (Sigma ethanol kit no. 332-UV). Evening blood ethanol levels ranged from 112-254 mg/dl, averaging 161 rt 18 mg/dl (as measured on gestation day 18). The animals receiving the sucrose control (S) diet were individually pair-fed with animals in the ethanol (E) group by giving each group S animal the volume of diet that its age and

The cells from each region from C, S or E fetuses were grown in one of three culture conditions: (1) control, in which the neurons were cultured with basic medium; (2) ethanol, in which varying concentrations of ethanol were added to the basic medium; and (3) ethanol plus supplementation, in which ethanol of varying concentrations and neurotrophic factors (NGF or bFGF) were added to the basic medium. Ethanol concentrations used were 1.2, 1.8 and 2.4 g/dl. In preliminary studies intended to empirically define the ethanol tolerance levels with respect to these populations, concentrations of 150, 300, and 600 mg/dl were also applied. Since these concentrations had no discernible effects on survival or process extension in either population, they were not included in subsequent analyses. Additional supplements used were NGF (20 rig/ml; a generous gift from Dr. Eugene Johnson), or bFGF (30 rig/ml; from Upstate Biotechnology). These substances were chosen because of their demonstrated relevance to the neuronal populations of interest: NGF has been shown. both in vivo and in vitro, to have survival and growth-promoting effects on the cholinergic hippocampal-projecting septal neurons [18,29]. Basic FGF has been shown to provide similar neurotrophic support for cultured hippocampal neurons [13,51,52]. Ethanol and NTFs were added at the time of plating. Control and experimental cultures and other cultures in which comparisons were to be made (e.g. ethanol and ethanol plus NTFs) were always grown

conditions

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and analyzed simultaneously, so that apparent differences would not arise as a result of slight variations in culture conditions. A minimum of four replicate cultures were used for each condition.

3. Results

2.4. Culture

The rate of ethanol evaporation in our preparations was determined for each of the three concentrations used (1.2, 1.8 and 2.4 g/dl). Evaporation resulted in loss of 53 to 60% of the initial ethanol present by 12 h, from 71-77% by 24 h, and approximately 95% by 48 h. The ethanol remaining was still considerable, however, particularly at the higher concentrations. For example, with an initial ethanol concentration of 2.4 g/dl, 960 mg/dl k 18.9 remained at 12 h, 552 f 10.7 mg/dl at 24 h, and 120 &- 15.7 mg/dl at 48 h. This ethanol exposure paradigm, then, consists of the cultures being initially challenged with target concentrations, followed by a declining presence over subsequent h in culture. Similar ethanol administration in vivo has been likened to the ‘binge drinking’ consumption pattern, and has proven to be more deleterious than chronic exposure at lower concentration [4,40].

analysis

procedures

An etched glass coverslip containing an array of 0.6 x0.6 mm squares identified by a sequence of letters and numbers (Bellco) was attached to the bottom of the tissue culture wells, so that specified regions could be assessed and re-assessed in each preparation. The number of neurons within 16 such regions, randomly chosen, was counted at 4 h after plating, and counts within these same regions were made at 24, 48 and 72 h post-plating. At all but the initial time point, the number of neurons with processes was also assessed. The number of neurons surviving was expressed as a percent of those counted initially in each well, and the number of cells with processes was expressed as a percent of the total neurons present on each day viewed. Neuronal viability was determined by their rounded, phasebright appearance, and in some cases was confirmed by the trypan blue exclusion procedure. All analyses were made using a Nikon inverted phase contrast microscope fitted with a temperature controlled chamber which maintained the cultures at incubation temperature. Statistical analyses were made via a one-way Analysis of Variance (ANOVA), with the Fisher’s protected least significant difference (PLSD) post-hoc test, and by the t-test.

2.5. Acerylcholinesterase

hisrochemistry

In some experiments, sister cultures of septal neurons, grown simultaneously with the cultures from which cell counts were obtained, were grown on glass coverslips, and were subjected to acetylcholinesterase (AChEI histochemical staining after the 96-h time point. This procedure was performed in order to determine whether the cholinergic subpopulation of the septal region was differentially affected by certain manipulations. AChE has been shown to be a reliable marker for cholinergic basal forebrain neurons. Hefti et al. [23], for example, found that all ChAT-positive neurons in cultures prepared from fetal rat septum were co-stained for AChE, while only 6% of the AChE-positive cells were ChAT-negative. Control and NGF-containing cultures from C, S and E groups were subjected to this procedure. For this staining, the coverslips were washed with phosphate-buffered saline (PBS) and fixed for 5 min in 10% formalin. They were then washed in buffered saline, and incubated for 72 h in 0.1 M acetate buffer with 2.4 mM acetylthiocholine iodide, 3 mM cupric sulfate, 5 mM sodium citrate, 0.5 mM potassium ferricyanide, and 1% gelatin (to prevent the diffusion of the reaction product). After incubation, the gelatin was dissolved, and the cultures were rinsed and exposed to Tris-buffered diaminobenzidine (0.5 mg/ml) for intensification (S-20 min). They were then mounted on slides, and both stained and unstained cells were counted, using a Zeiss microscope. Counts were made of all cells encountered in sequential fields in two perpendicular bands through the medial plane of the coverslips (a total of approximately 40 fields), at a magnification of 200X. From 5-11 preparations were counted for each condition assessed (control cultures and cultures supplemented with 20 rig/ml NGF from C, S and E fetuses).

2.6. Ethanol

assays

In order to assess the rate of ethanol evaporation from the culture wells, samples of the three ethanol concentrations used were taken at 12-, 24- and 48-h intervals, and subjected to a microenzymatic assay to determine remaining ethanol concentrations.

3.1. Ethanol

evaporation

3.2. General characteristics diet conditions

rates

of cultures from

the three

There were no obvious morphological differences in either septal or hippocampal cultures from C, S or E fetuses, and no differences were detected in the initial plating characteristics of the three groups (i.e. the number of aliquoted cells adhering to the substrate), Subsequent survival and process extension across the 72 h culture period for control (non-supplemented) cultures were also comparable for the three diet conditions, with no appreciable differences seen in any measure at any time point. These baseline survival and differentiative profiles are presented in Fig. 1. 3.3. Effects of prenatal ethanol responsiveness: septal cultures

exposure

on ethanol

Septal neurons responded to ethanol in the culture medium in a dose-dependent manner. The lowest concentration used (1.2 g/d11 produced no significant decrements in neuronal survival in any experimental group, and neurite outgrowth was affected only after 72 h in vitro in the cultures from C and S fetuses (P < 0.04 and 0.03, respectively), but not in the E groups, when the raw data was compared statistically to concurrent controls via the t-test (neurons with processes in C groups = 79.9 + 9% controls; in S groups = 81 k 4.5% controls; in E groups = 103 k 20% controls). The ANOVA did not reveal overall differences in process outgrowth between the three diet groups at this ethanol concentration, although the post hoc test indicated that the difference between the C and E groups at 72 h approached significance (P <

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0.06). When 1.8 g/d1 ethanol was applied to cultures from C and S animals, neuronal survival was significantly depressed after 48 and 72 h in vitro, and neurite outgrowth was inhibited at all observations points, when comparisons were made to concurrent, non-ethanol controls. When cultures from ethanol-treated animals were exposed to this ethanol concentration, however, neuronal survival and process elaboration were not significantly affected, and resembled these measures seen in control cultures, particularly at the 24 and 48 h assessment points. The ANOVA applied to the survival data revealed a significant diet effect at the 24 and 48 h time points (P < 0.02 and 0.05, respectively), and the SePtal --

Neurons 1

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post-hoc test indicated that survival in the E groups exceeded that in both C and S groups at 24 and 48 h. By 72 h, these differences were not statistically significant. The ANOVA applied to the outgrowth data also indicated a significant diet effect at all three time points assessed (P < 0.008 at 24 h; P < 0.006 at 48 h; P < 0.0001 at 72 h), with the post-hoc test showing that outgrowth in the E groups surpassed that seen in both C and S groups at all three time points. These data are presented in Fig. 2A (note that the data in this and subsequent figures are expressed as percent concurrent controls). When the highest ethanol concentration was used (2.4 g/dl), both survival and process outgrowth were significantly depressed at most time points in all three diet groups, with no significant diet effects seen. These results are presented in Fig. 2B. At both ethanol concentrations in all three diet groups, neuronal survival tended to decline slightly across days in vitro (DIV), while process outgrowth was relatively stable across time. There was no significant recovery or rebound effect, in terms of enhanced differentiation of remaining cells at the later days in culture, despite the fact that the ethanol in the medium was dissipating during this time. 3.4. Effects of prenatal ethanol exposure responsiueness: hippocampal cultures

I 24 hrs

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Time in Culture Fig. 1. Neuronal survival and neurite outgrowth across three days in vitro in control, non-supplemented septal and hippocampal cultures from G18 fetal rats. The fetuses came from dams maintained throughout gestation on lab chow (Chow), a liquid diet containing ethanol (Ethanol), or a liquid diet with the isocaloric substitution of sucrose for ethanol (Sucrose). Initial neuronal counts were made at 4 h after plating, and the survival data is expressed as the percent of this number remaining at the subsequent evaluation points. Neurons with processes represents the percent of surviving neurons on each day with processes. There are no significant differences between the three diet groups in either measure at any time point.

on ethanol

At the lowest ethanol concentration (1.2 g/dl), there were no significant effects on survival or fiber growth in the hippocampal cultures from any diet group. When 1.8 g/d1 ethanol was applied, however, responsiveness closely paralleled that seen in the septal cultures: both survival and process elaboration were depressed in C and S groups, but were considerably less affected in the E groups (neurite outgrowth was significantly below that of controls only at 24 h, P < 0.01). These results are presented in Fig. 3A. In assessing the survival data, the ANOVA did not indicate strong diet effects, although the post-hoc test showed that survival in the E groups tended to be greater than that in C and S groups after 72 h in vitro. The ANOVA indicated a significant main effect of diet on process outgrowth at both 48 and 72 h in culture (P < 0.05 at each time point), and the post-hoc test revealed that outgrowth in the E groups significantly surpassed that seen in either C or S groups at these time points (Fig. 3A). In the E groups at this ethanol concentration, process outgrowth increased across DIV. This neurite elaboration was significantly greater at 48 and 72 h than at 24 h (P < 0.051, while that seen in C and S groups remained stable. The increase in differentiation in the E groups might be attributable to a rebound following the gradual depletion of the ethanol from the culture medium, although such an effect might be expected to be mani-

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fested in the other groups as well. Alternatively, this progressive growth in these E cultures might reflect greater resistance to the depressing effect of the ethanol, or an enhanced capacity to recover from this effect. When the hippocampal cultures were supplemented with 2.4 g/d1 ethanol, neuronal survival was generally similar to that seen with 1.8 g/dl. Process outgrowth, however, was significantly reduced in all groups at all time points. These data are presented in Fig. 3B. At this ethanol concentration, neuronal survival in all three groups was relatively stable across DIV. Process outgrowth tended to increase between 24 and 48 h in culture, then remained stable. This slight enhancement of differentiation by the 48 h time point may, as above, be a consequence of the progressive ethanol evaporation from the culture medium. Curiously, this rebound (between 24 and 48 h) was somewhat greater than that seen with the lower ethanol concentration (1.8 g/dl), although it did not reach statistical significance. 3.5. Effects of prenatal ethanol exposure on neurotrophic factor responsiveness: septal cultures

Neurotrophic factor (NTF) responsiveness of septal neurons from the three diet conditions was assessed in

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three ways: first, cultures of our usual plating density (35-39,000 cells/cm2; considered to be relatively high density by some previous investigators [13]) were grown with and without 20 rig/ml NGF and measures of neuronal survival and differentiation were made as above. Second, lower density cultures (approximately 22,000 cells/cm2) were grown on glass coverslips for 4 days, and then processed for AChE histochemistry, to assess possible differential survival of cholinergic neurons within this mixed medial septal population (plating density has previously been shown to be a determining factor in the NTF-dependence of cultured cholinergic septal neurons [18]). Third, cultures from each diet group were challenged with the two higher ethanol concentrations (1.8 and 2.4 g/d0 with and without NGF in the culture medium (these preparations were grown at our usual, relatively high, plating density). In our previous studies, we had found that NGF appreciably ameliorates ethanol neurotoxicity in this neuronal population [22]. When septal neurons were cultured at relatively high density with NGF, no differences in survival or process outgrowth were detected in any treatment group when comparisons to control, non-NGF-containing cultures were made (data not shown). When lower density cultures were grown with and without NGF

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Fig. 2. Neuronal survival and neurite outgrowth in septal cultures from G18 fetal rats grown in two concentrations of ethanol. The fetuses came from dams maintained throughout gestation on lab chow (Chow), a liquid diet containing ethanol (Ethanol), or a liquid diet with the isocaloric substitution of sucrose for ethanol (Sucrose). The data in this figure were obtained after 24, 48 and 72 h in vitro, and are expressed as percent concurrent non-ethanol control from the appropriate diet condition. (A) Cultures supplemented with 1.8 g/dl ethanol. (B) Cultures supplemented with 2.4g/dl ethanol. a = Significantly different from Chow diet group, P < 0.05; b = significantly different from Sucrose diet group, P < 0.05; c = significantly different from concurrent (non-ethanol) control, P < 0.05; d = significantly different from Chow and Sucrose groups, P < 0.006.

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and subsequently stained histochemically for AChE, no selective rescue of the cholinergic neurons was detected in any experimental group, when the proportions of AChE-positive neurons were compared in NGF and control cultures. The percent of surviving cholinergic neurons in the presence of NGF was also generally similar when comparisons were made across the three diet groups (C, S and E), although significantly more labeled cells were detected in NGF cultures from the C groups than those from the E groups (9.9 k 0.3% in C groups; 9.0 k 0.2% in E groups; P = 0.058). When septal neurons from C and S animals were cultured with 1.8 g/d1 ethanol plus NGF, neuronal survival was unchanged from that seen with ethanol alone, but process outgrowth was consistently greater in the NGF-containing cultures. This difference was significant at all three time points for the S groups (P < 0.04, 0.05 and 0.05), and at 72 h for the C groups (P < 0.04). The survival and process outgrowth seen in NGF-supplemented cultures derived from the ethanol-treated animals, however, did not differ from these same measures in the ethanol-only cultures. This can be explained by the fact that at this ethanol concentration, these measures in E groups were not appre-

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ciably depressed. When cultures were supplemented with 2.4 g/d1 ethanol with and without NGF, neuronal survival was not improved in the NGF-ethanol groups over that seen in the ethanol-only cultures in any diet group, but ethanol neurotoxicity with respect to processoutgrowth was again mitigated in C and S groups. This measure was significantly greater in the NGF-supplemented cultures from these two groups than that seen in their ethanol-only counterparts at most time points (see Table 1 for statistical comparisons). The depressed neurite outgrowth in the E groups, however, was not attenuated by the inclusion of NGF in the culture medium (Table 1). These results for the higher ethanol concentration after 72 h in culture are presented graphically in Fig. 4A. 3.6. Effects of prenatal ethanol exposure on neurotrophic factor responsiveness:hippocampal cultures In order to determine whether chronic prenatal ethanol treatment altered the capacity of hippocampal neurons to respond to relevant NTFs, and whether this prior exposure affects the potential of NTFs to ameliorate ethanol neurotoxicity with respect to this popula-

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Fig. 3. Neuronal survival and neurite outgrowth in hippocampal cultures from G18 fetal rats grown in two concentrations of ethanol. The fetuses came from dams maintained throughout gestation on lab chow (Chow), a liquid diet containing ethanol (Ethanol), or a liquid diet with the isocaloric substitution of sucrose for ethanol (Sucrose). The data in this figure were obtained after 24, 48 and 72 h in vitro, and are expressed as percent concurrent non-ethanol control from the appropriate diet condition. A: cultures supplemented with 1.8 g/d1 ethanol. B: cultures supplemented with 2.4 g/d1 ethanol. a = significantly different from concurrent (non-ethanol) control, P < 0.05; b = significantly different from Chow diet group, P < 0.07; c = significantly different from Sucrose diet group, P < 0.06; d = significantly different from Chow diet group, P < 0.007; e = significantly different from Sucrose diet group, P < 0.04

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Table 1 Statistical comparisons of septal cultures from diet animals * with 2.4 g/d1 ethanol, with and without NGF. Neurite outgrowth in cultures from ethanol animals is not affected by NGF in the culture medium Diet condition

Comparison

Measure/time

P-value

Chow Chow Chow

Etoh vs. Etoh + NGF Etoh vs. Etoh + NGF Etoh vs. Etoh + NGF

Outgrowth Outgrowth Outgrowth

-

24 h 48 h 72 h

ns P < 0.02 P < 0.05

Sucrose Sucrose Sucrose

Etoh vs. Etoh + NGF Etoh vs. Etoh + NGF Etoh vs. Etoh + NGF

Outgrowth Outgrowth Outgrowth

-

24 h 48 h 72 h

P < 0.05 P < 0.05 P < 0.03

Ethanol Ethanol Ethanol

Etoh vs. Etoh + NGF Etoh vs. Etoh + NGF Etoh vs. Etoh + NGF

Outgrowth Outgrowth Outgrowth

-

24 h 48 h 72 h

ns ns ns

* The cultures came from G18 rat fetuses from dams maintained on lab chow (Chow), on a sucrose-containing liquid diet (Sucrose), or on a liquid diet with ethanol substituted for sucrose (Ethanol). Statistical comparisons were made via the t-test.

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parisons). An ANOVA applied to these data indicated a significant diet effect, with the post-hoc test revealing significant differences between S vs. E groups at all three time points, and with values approaching significance for C vs. E groups at 48 and 72 h (Table 3). Septal

Cultures

100

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T

a

Chow

b

80 2 sE

80

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40

20

0

tion, the following experiments were performed: Hippocampal neurons were grown in both relatively highdensity cultures and in lower-density cultures, with and without bFGF, a neurotrophic substance which has been show to provide support for this population [50,52]. The rationale for using the lower-density cultures was the same as that for similar septal preparations, since NTF dependence in cultured hippocampal neurons may also be density dependent (prior studies in which bFGF was shown to enhance survival and differentiation of cultured hippocampal neurons have typically utilized cultures of quite low density, ranging from 2000 to 18,000 cells/sq. cm [13,35,50,521). In addition, hippocampal cultures derived from animals from the three diet conditions were grown in cultures of both low and high plating density with neurotoxic concentrations of ethanol with and without bFGF, a procedure analogous to that implemented with the septal cultures and NGF, again with a rationale similar to that described for the septal preparations (i.e., we previously found bFGF provided neuroprotection against ethanol neurotoxicity with hippocampal preparations [22]). When hippocampal cultures were plated at relatively high initial densities, there were no appreciable differences in survival or process extension in bFGFcontaining cultures, when compared to controls, in any diet condition (data not shown). When low-density cultures were supplemented with bFGF, neuronal survival was not enhanced compared to concurrent controls in cultures from any diet condition. Neurite outgrowth, however, was significantly increased in lowdensity bFGF cultures at most time points from both C and S groups but not from E groups, when compared to concurrent controls (see Table 2 for statistical com-

2.4g/di

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2.4gldl + NGF

Neuronal Survival

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Neurite Outgrowth

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=

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Neuronal Survival

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2.4g/dl + bFGF

Neurite Outgrowth

Fig. 4. Neuronal survival and neurite outgrowth in septal and hippocampal cultures from G18 fetal rats grown with ethanol, or ethanol + neurotrophic factors. The fetuses came from dams maintained throughout gestation on lab chow (Chow), a liquid diet containing ethanol (Ethanol), or a liquid diet with the isocaloric substitution of sucrose for ethanol (Sucrose). The data in this figure were obtained after 72 h in vitro, and are expressed as percent concurrent non-ethanol control from the appropriate diet condition. A: septal cultures, grown at relatively high initial plating density, supplemented with 2.4 g/d1 ethanol, or 2.4 g/d1 ethanol + 20 rig/ml NGF. B: hippocampal cultures, grown at relatively low initial plating density, supplemented with 2.4 g/d1 ethanol, or 2.4 g/d1 ethanol + 30 rig/ml bFGF. a = significantly different from Chow diet group with 2.4 g/d1 ethanol, P < 0.05; b = significantly different from Sucrose diet group with 2.4 g/d1 ethanol, P < 0.05; c = significantly different from Chow and Sucrose diet groups with 2.4 g/dl ethanol, P < 0.01.

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Table 2 Statistical comparisons of low-density hippocampal cultures diet animals * grown with bFGF. Neurite outgrowth in cultures ethanol animals is not affected by bFGF in the culture medium Measure/time

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Diet condition

Comparison

Chow Chow Chow

Con vs. bFGF Con vs. bFGF Con vs. bFGF

Outgrowth Outgrowth Outgrowth

-

24 h 48 h 72 h

P < 0.06 ns P < 0.05

Sucrose Sucrose Sucrose

Con vs. bFGF Con vs. bFGF Con vs. bFGF

Outgrowth Outgrowth Outgrowth

-

24 h 48 h 72 h

P < 0.025 P < 0.002 P < 0.002

Ethanol Ethanol Ethanol

Con-vs. bFGF Con vs. bFGF Con vs. bFGF

Outgrowth Outgrowth Outgrowth

-

24 h 48 h 72 h

ns ns ns

Measure/time

Svs. E Svs. E S vs. E

Outgrowth Outgrowth Outgrowth

-

24 h 48 h 72 h

P < 0.04 P < 0.05 P < 0.04

Cvs. Cvs. Cvs.

Outgrowth Outgrowth Outgrowth

-

24 h 48 h 72 h

ns P < 0.07 P < 0.06

E E E

3

120

'E s

100

E 8 $ a

a

a

60 60 40

Survival

Outgrowth

Fig. 5. Neuronal survival and neurite outgrowth in low-density hippocampal cultures from G18 fetal rats grown in medium supplemented with 30 rig/ml bFGF. The fetuses came from dams maintained throughout gestation on lab chow (Chow), a liquid diet containing ethanol (Ethanol), or a liquid diet with the isocaloric substitution of sucrose for ethanol (Sucrose). The data in this figure were obtained after 72 h in vitro, and are expressed as percent concurrent controls from the appropriate diet condition. a= significantly different from non-bFGF controls, P < 0.05; b = different from Chow diet group with bFGF, P < 0.06; c = significantly different from Sucrose diet group with bFGF, P < 0.04.

* The cultures came from G18 rat fetuses from dams maintained on lab chow-(Chow), on a sucrose-containing liquid diet (Sucrose), or on a liquid diet with ethanol substituted for sucrose (Ethanol). Statistical comparisons were made via the t-test.

Comparison

l-13

160

P-value

Table 3 Statistical comparisons of low-density hippocampal cultures diet animals * grown with bFGF: Neurite outgrowth in cultures chow and sucrose animals surpasses that seen in cultures ethanol animals in response to bFGF

85 (1995)

from from from

P-value

were challenged with 1.8 g/d1 ethanol, and ethanol plus bFGF, ethanol-induced alterations in neuronal survival and process outgrowth were not significantly affected by the presence of bFGF in any diet group (data not shown). When 2.4 g/d1 ethanol was used for supplementation, however, the ethanol inhibition of neurite outgrowth was markedly ameliorated by bFGF in cultures from the C and S animals, but not in those from the E animals. This amelioration reached or approached statistical significance at all time points for C and S groups (Table 4). Comparable results were obtained when the cultures were of low initial plating density, with increased process extension in the

* The cultures came from G18 rat fetuses from dams maintained on lab chow (C), on an ethanol-containing liquid diet (El, or.on a liquid diet with sucrose substituted for ethanol (S). Statistical comparisons were made via the ANOVA, followed by the Fisher’s PLSD post-hoc test.

These data from the 72 h time point are shown in Fig. 5. If hippocampal cultures of relatively high density

Table 4 Statistical comparisons of high- and low-density hippocampal cultures from diet animals Neurite outgrowth in cultures from ethanol animals is not affected by bFGF in the culture Diet condition

Comparison

Measure/time

Chow Chow Chow

Etoh vs. Etoh + bFGF Etoh vs. Etoh + bFGF Etoh vs. Etoh + bFGF

Outgrowth Outgrowth Outgrowth

-

Sucrose Sucrose Sucrose

Etoh vs. Etoh + bFGF Etoh vs. Etoh + bFGF Etoh vs. Etoh + bFGF

Outgrowth Outgrowth Outgrowth

Ethanol Ethanol Ethanol

Etoh vs. Etoh + bFGF Etoh vs. Etoh + bFGF Etoh vs. Etoh + bFGF

Outgrowth Outgrowth Outgrowth

* The cultures came from G18 rat fetuses from dams maintained liquid diet with ethanol substituted for sucrose (Ethanol). Statistical

* with medium

2.4 g/d1

ethanol,

with

and without

High-density P-value

Low-density P-value

24 h 48 h 72 h

P < 0.001 P < 0.05 P < 0.08

P < 0.02 P < 0.07 P < 0.01

-

24 h 48 h 72 h

P < 0.06 P < 0.05 P < 0.05

;< 0.06 P < 0.05

-

24 h 48 h 72 h

ns ns ns

ns ns ns

on lab chow (Chow), on a sucrose-containing comparisons were made via the f-test.

liquid

diet (Sucrose),

bFGF:

or on a

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et al. /Developmenral

bFGF-supplemented cultures from C and S animals but not in those from E animals (Table 4). The data from the 72 h time point from these low-density cultures with ethanol with and without bFGF are plotted in Fig. 4B. Note that at this lower plating density, neurite elaboration is considerably more depressed by ethanol than that seen in the higher-density C and S cultures (as depicted in Fig. 31, but not in cultures from the ethanol-exposed fetuses. In comparing the three diet groups in the ethanol-only cultures at 72 h, there were no differences between C and S groups, but outgrowth in the E groups surpassed that in C (P < 0.008) and S groups (P < 0.009). Thus, at this lower plating density, there is an enhanced vulnerability to in vitro ethanol neurotoxicity in cells from control animals, but not in those from ethanol-exposed animals.

4. Discussion This study has demonstrated that chronic prenatal ethanol exposure produces distinct alterations in cultured septohippocampal neurons. These alterations are manifested in two ways: first, the threshold for ethanol neurotoxicity, particularly as measured by neurite expression, is considerably elevated following prenatal ethanol exposure. We found that while process outgrowth in septal neurons from chow and sucrose control animals was disrupted by initial ethanol concentrations of 1.2 g/dl, and in hippocampal neurons by concentrations of 1.8 g/dl, this measure in cells from ethanol-exposed animals required much higher ethanol concentrations before process elaboration was significantly affected (2.4 g/d1 in both populations). Neuronal survival in C and S groups was also compromised by intermediate ethanol concentrations (1.8 g/dl), particularly in septal cultures, but a considerably higher concentration was required before significant cell loss was detected in cultures derived from ethanol-treated fetuses (2.4 g/dl). The second alteration arising as a consequence of prenatal ethanol treatment was a decline in the responsiveness of both septal and hippocampal neurons to neurotrophic factors. Thus, neurite outgrowth in hippocampal neurons from ethanol-exposed animals was not enhanced by bFGF, as it was in cells from C and S control animals, and the neuroprotective effects afforded by NGF and bFGF against ethanol toxicity, seen in control preparations of septal and hippocampal neurons, respectively, were significantly compromised following in vivo ethanol exposure. The failure to detect NTF neuroprotection at the intermediate neurotoxic ethanol concentration (1.8 g/d11 can be attributed to the fact that both neuronal survival and process extension approached control levels in E cultures with this ethanol concentration. This was not the case, how-

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ever, with the 2.4 g/d1 concentration when both measures were depressed in cultures from ethanol-exposed animals, as in control animals. Yet the NTF attenuation of the ethanol toxicity was not achieved in the E groups. We did not find striking differences in the survival of AChE-positive cholinergic septal neurons in cultures from control and ethanol-exposed animals, in comparing cultures with and without NGF (but lacking ethanol). The proportion of cholinergic cells stained was similar across experimental groups, although there were slightly greater numbers of stained cells in the C than in the E groups. In a previous study 1221, we were also unable to elicit specific NGF-mediated survival of the cholinergic septal subpopulation. This lack of specificity likely results from the relatively high plating density of our cultures, since cellular density has been clearly demonstrated to be an important determinant of NGF dependence in cultured cholinergic septal neurons [13,18]. It is notable that the ethanol concentrations required to produce the neurotoxicity seen in the present study considerably exceed levels which have been demonstrated to produce cell loss and various CNS morphological anomalies using in vivo animal models [39,40]. We have observed this phenomenon of high in vitro ethanol tolerance in previous culture studies [20,22], as have other investigators [45,56]. A number of factors could directly or indirectly contribute to the disparity between in vivo and in vitro effects. These include fetal/maternal interactions (e.g. the possible involvement of ethanol metabolites such as acetaldehyde as a concomitant to ethanol neurotoxicity; fetal hypoxic or hypoglycemic insult; diminished placental nutrient transfer), alterations in the cellular microenvironment (e.g. disruption of calcium homeostasis), aberrant intercellular interactions (e.g. initiation of neuronal hyperexcitabilityl, direct or indirect effects on neuronal or glial proliferation or migration, abnormal neuronal/glial interactions, or disruptions in normal critical neurotrophic relationships (e.g. ethanol-induced decreases in the synthesis of or responsiveness to neurotrophic factors). Most likely, in vivo fetal alcohol effects involve a cascade of interactive events. The tissue culture model provides a convenient tool with which to examine many of these possible components, both in isolation and in combinations. Previous characterizations of neurons from animals exposed to ethanol in utero are not numerous, but those studies which have been conducted have reported a variety of cellular alterations as a consequence of ethanol exposure. Shen and Chiodo 1461, for example, studied physiological and pharmacological responsiveness of adult rat nigrostriatal dopaminergic (NSDA) neurons following chronic prenatal ethanol treatment (CPET), with ethanol administered from gestation day 8 (GS) to parturition. Their results sug-

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gested a functional down-regulation of dopamine receptors within the nigrostriatal system, and a change in the firing pattern of the NSDA neurons. Blakley and Fedoroff [3] cultured early (G18) cerebral cortical cells from fetal mice following CPET (Gil-17), using a colony-culture method to study neuronal proliferation. They found the plating efficiency of the cell suspensions from CPET animals was significantly lower than in controls, possibly resulting from a decreased number of proliferating (colony forming) cells. The typical colony size was also reduced in the preparations from the ethanol-exposed fetuses. They interpreted these results as being indicative of functional abnormalities in the ethanol-exposed neurons. In assessing the influence of CPET on subsequent ethanol responsiveness, as in the present study, Okonmah et al. [38] cultured brain tissue from G22 rat fetuses following CPET (G15-21). Assays of choline acetyltransferase (ChAT) from these cultures revealed more than twice the activity of that found in control cultures. When these preparations were challenged with 0.1% ethanol (100 mg/dl) in the culture medium, ChAT activity increased further, particularly in the cultures from the CPET fetuses, where a level more than six times that measured in cultures from animals never exposed to ethanol was achieved. The activity of AChE was also assayed in this study, and the basal levels measured did not differ in cultures derived from control and CPET fetuses. When the cultures were exposed to ethanol, however, AChE activity was greatly enhanced in CPET but not control cultures, reaching a level approximately four times that seen in all other conditions (i.e. control with and without ethanol; CPET without ethanol). Ledig et al. [30] cultured neurons and glia from G15 rat cerebral cortex following CPET (throughout gestation). They found delayed cellular maturation in the populations derived from the CPET fetuses, as assayed by biochemical markers such as enolase and glutamine synthetase. Protein content of the CPET cultures was also diminished by 40%. When 30 mM ethanol (138 mg/dl) was applied to glial cultures, protein content and enzymatic activities decreased in controls, with the maximal effect being within the first week in vitro (the cultures were maintained for 4 weeks). In the cultures from CPET fetuses, however, this effect occurred gradually over the entire culture period, suggesting a lower sensitivity to acute ethanol in the cells from the ethanol-exposed animals, an observation similar to that made in the present study. Gruol and colleagues utilized a tissue culture procedure to mimic the in vivo ethanol exposure paradigm. They exposed cerebellar Purkinje cells to physiological concentrations of ethanol (22 or 44 mM [approximately 100 and 200 mg/dl]) during their primary period of morphological and physiological development (begin-

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ning on G20). Using this protocol, they found that 1-2 weeks of ethanol treatment produced significant increases in membrane input resistance and immature firing patterns in Purkinje cells [14]. In addition, several components of responsiveness to excitatory amino acid receptor agonists were altered following ethanol exposure: Spontaneous firing, agonist-evoked firing, the initial period of activity of the response to the receptor agonist quisqualate, and the inhibitory period of the response to glutamate were all significantly reduced in chronically treated cells, while the inhibitory period of response to kainate was significantly increased [HI. Gruol and Parsons [16] also found that chronic ethanol exposure disrupts voltage-sensitive calcium channels, with the high threshold current being enhanced and the low threshold current being depressed. The results from the present study suggest two additional ways in which prenatal ethanol exposure may affect neuronal populations: an increased ethanol tolerance, as measured by the capacity of neurons to survive and undergo morphological differentiation in the presence of ethanol, and an impaired responsiveness to NTFs. Tolerance to ethanol, which can be defined as an attenuation of biological responsiveness to a given ethanol concentration, is a well-known consequence of chronic ethanol consumption in adults. Recent studies investigating the cellular mechanisms underlying this phenomenon have suggested that alterations in dihydropyridine-sensitive calcium channels, which are up-regulated with chronic ethanol exposure, may be involved in this adaptation. Supportive of this hypothesis is the observation that treatment with dihydropyridine calcium channel antagonists prevents this tolerance effect from being manifested [8,9,33,34]. It will be important in future studies to examine the mechanisms underlying the decreased sensitivity seen in the present study, and to determine whether this effect is enduring. Of possible relevance to this latter point is a study by Randall et al. [42], in which mice were exposed to ethanol prenatally (G8-parturition), and at 24 or 100 days of age, were administered a challenging dose of 3.5 or 4.5 g/kg ethanol. They were subsequently tested for ethanol-induced sleep time. The prenatal-ethanol-treated animals did not differ from non-ethanol-exposed controls in this measure, and therefore did not appear to have increased ethanol tolerance, at least at this level of analysis. The second observation made in the present study, that of disruption in NTF responsiveness following CPET, is consistent with an earlier study from our laboratory [19]. In this study, chick embryos were chronically exposed to ethanol (30 mg/day), beginning on embryonic day 4 (E4). They were sacrificed on E8-9, and dorsal root ganglion (DRG) neurons from these ethanol-treated or saline-treated control embryos were cultured with either NGF or an extract derived

M.B.

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et al. / De~dopmental

from El6 chick forebrain (FBX), which has been shown to provide potent neurotrophic support for this population [21]. We found that the DRGs from the CPET embryos were less viable in the presence of NGF and were less responsive to FBX in terms of neuritic production than were those from saline-treated control embryos. A somewhat similar observation of ethanolinduced disruption of neuronal-NTF interactions was made in a recent study of Rahman et al. [41], also using the embryonic chick FAS model. In this study, embryos were treated with ethanol from El-3, and it was found that while in control embryos, intracerebral administration of NGF significantly elevated CNS ChAT activity, this cholinotrophic effect was abolished in the ethanol-treated embryos. Impaired responsiveness to neurotrophic factors during nervous system development could be an important mechanism underlying many of the anomalies seen in FAS. Neurotrophic interactions have long been thought to be critical to normal development, and investigations using antibody neutralization [ 12,24,43], and more recently, gene deletion techniques [6,5,10, 11,26-28,31,47], have confirmed the importance of these interactions. Altered neurotrophic responsiveness, such as that seen in the present study, could be a consequence of abnormalities in trophic factor receptors or receptor expression, resulting from early ethanol exposure. Recent ‘knockout’ studies have produced mouse mutants lacking genes encoding the receptors involved in signal transduction for several members of the neurotrophin family (i.e. NGF, BDNF and NT-3) [6,27,28,31,47]. The homozygous mutants lacking these receptors (~75, trkA, trkB and trkC) presented with a varied but generally devastating array of dysfunctions. In one study by Davies and colleagues [6], trigeminal ganglion neurons from homozygous transgenics lacking the p75 receptor showed a displaced dose-response to NGF, requiring 3- to 4-fold higher concentrations for a half-maximal response to be elicited. The heterozygous animals were intermediate in NGF responsiveness, compared to the mutants and wild-type animals. Abnormalities in neurotrophic factor receptor expression or function following early ethanol exposure, although doubtlessly not of this magnitude, could contribute to the reduced responsiveness seen in the present study. Such abnormalities could play a major role in the neuroteratology of FAS. In summary, the results of the present study suggest that septal and hippocampal neurons from ethanol-exposed fetuses appear less susceptible to (further?) ethanol-induced damage, tolerating quite high ethanol concentrations before declines in survival and process differentiation are seen. Somewhat paradoxically, these ethanol-exposed neurons also appear to be less responsive to neurotrophic substances, substances which have been shown to have the capacity to mitigate the ethanol

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neurotoxicity [20,22]. It has been well established that rat hippocampal neurons are lost following prenatal and perinatal ethanol exposure [2,54], and several studies suggest neurons of the septal region are similarly affected: preliminary studies from our laboratory, for example, indicate that ChAT activity in the rat septal region is decreased following CPET (unpublished observations), and in mice, prenatal ethanol exposure results in loss of ChAT-positive septal neurons 1441. If these populations are becoming less vulnerable to ethanol as exposure persists, then decreased NTF responsiveness, as it relates to either support, establishing appropriate connectivity, or neuroprotection, may be a crucial link in the complex cascade of interactive events leading to and underlying FAS. It will be important in future studies to specifically address this question.

Acknowledgements This research was supported by a grant from the Alcoholic Beverage Medical Research Foundation, by a Children’s Miracle Network award, by NIAAA Grants AA09128 and AAOO200, and by the Medical Research Service of the Department of Veterans Affairs. Douglas Swanson was supported by the Center for Neurobiological Sciences and by NIAAA fellowship AAO.5332. We thank Douglas Bradley, Cynthia Carroll, Nancy Lee MacLennan, Laura Tonjes and Jason Yockey, for excellent technical assistance in the course of the study.

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[521 Walicke, P.A. and Baird, A., Neurotrophic effects of basic and acidic fibroblast growth factors are not mediated through glial cells, Deu. Brain Rex, 40 (1988) 71-79. [53] Walker, D.W. and Freund, G., Impaired shuttle box avoidance learning following prolonged alcohol consumption in rats, Physiol. Behau., 7 (1971) 773-778. [54] West, J.R., Hamre, K.M. and Cassell, M.D., Effects of ethanol exposure during the third trimester equivalent on neuron number in rat hippocampus and dentate gyrus, Alcoholism; C&z. Exp. Rex, 10 (1986) 190-197. [55] West, J.R., Long-term effects of developmental exposure to alcohol, Neurotoxicology, 7 (1986) 245-256. 1561 Zou, J.-Y., Rabin, R.A. and Pentney, R.J., Ethanol enhances neurite outgrowth in primary cultures of rat cerebellar macroneurons, Dec. Brain Res., 7 (1993) 75-84.