Fetal alcohol effects: mechanisms and treatment.

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chair was Edward P. Riley. The presentations were (1) Does alcohol withdrawal contribute to fetal alcohol effects? by Jennifer D. Thomas and Edward P. Riley; (2) Brain damage and neuroplasticity in an animal model of binge alcohol exposure during the "third trimester equivalent," by Charles R. Goodlett, Anna Y. Klintsova, and William T. Greenough; (3) Ganglioside GM1 reduces fetal alcohol effects, by Basalingappa L. Hungund; and (4) Fetal alcohol exposure alters the wiring of serotonin system at mid-gestation, by F. Zhou, Y. Sari, Charles Goodlett, T. Powrozek, and Ting-Kai Li.

Prenatal alcohol exposure retards the migration and development of serotonin neurons in fetal C57BL mice.

Incomplete neural tube fusion (iNTF), induced by alcohol, in midline floor and roof plates was found in our recent study. In this study, serotonin (5-HT) neurons, known to be born entirely in the midline raphe at brainstem, were examined during their development with fetal alcohol exposure. Weight-matched C57BL mice pregnant dams were divided into three groups on E8: one received ethanol via a chocolate Sustacal liquid diet providing 20% ethanol-derived calories as the sole source of nutrients (ALC); the second received an isocaloric Sustacal liquid diet and was pair-fed to individual dams in the ethanol-fed group (PF); the third was fed ad lib rat chow (Chow). Fetal brains were obtained on E15 and were processed for immunostaining of 5-HT and its trophic factor, S100 beta. The ascending 5-HT neurons, in normal development, appear bilaterally near midline on E12, and by E15, as seen in chow and PF groups, migrate from the midline germinal zone laterally and dorsally to their final position with rich fibers. In contrast, in the E15 ALC group, many 5-HT-im neurons were found remaining in the midline germinal region or had migrated, but with under-differentiated, sparse fibers. There were 20--30% fewer 5-HT-im neurons in ALC as compared to PF and Chow. In addition, the number of S100 beta cells was less in ALC as compared with PF and Chow groups. No difference was found between PF and Chow in number of 5-HT-im or S100 beta-im cells. The 5-HT neurons found compromised in migration and differentiation may, in part, stem from failure of access to floor plate or midline tissue induction and the insufficient support by S100 beta. As 5-HT neurons have been implicated for signaling brain maturation, fewer 5-HT neurons may have lasting effects on the development of brain or, if persistent in the adult, profoundly affect adult brain function.

Mechanisms of alcohol-induced damage to the developing nervous system.

Numerous mechanisms likely contribute to the damaging effects of prenatal alcohol exposure on the developing fetus and particularly the developing central nervous system (CNS). The coexistence of a multitude of mechanisms that may act simultaneously or consecutively and differ among various cell types poses particular challenges to researchers. To study alcohol's effects on the fetus more easily, investigators have used animal models and tissue-culture experiments. Such approaches have identified numerous potential mechanisms through which alcohol acts on the fetus, many of which result in cell death by necrosis or apoptosis. Among these mechanisms are increased oxidative stress, damage to the mitochondria, interference with the activity of growth factors, effects on glia cells, impaired development and function of chemical messenger systems involved in neuronal communication, changes in the transport and uptake of the sugar glucose, effects on cell adhesion, and changes in the regulation of gene activity during development.

Impairment in eyeblink classical conditioning in adult rats exposed to ethanol as neonates.

BACKGROUND: Eyeblink classical conditioning is a learning task that engages well-defined neural circuitry in the cerebellum and brainstem. Binge-like exposure to alcohol during the neonatal brain growth spurt in rats produces neurotoxic effects on both the cerebellum and the brainstem. The precise localization of the neural substrates of eyeblink conditioning makes it an ideal task to study functional disruptions in the cerebellum and brainstem caused by early exposure to alcohol. The purpose of this study was to determine whether impairments in eyeblink conditioning caused by neonatal binge exposure to alcohol persist into adulthood, indicative of long-lasting abnormalities in cerebellar and brainstem function. METHODS: Group Ethanol received alcohol doses of 5.25 g/kg/day via intragastric intubation on postnatal days 4-9. Group Sham Intubated underwent the intragastric intubation procedures on postnatal days 4-9 but did not receive any infusions. Group Nonintubated did not receive any intubations. When all rats were at least 3 months old, they were tested in either paired or unpaired eyeblink conditioning. RESULTS: Group Ethanol showed impaired eyeblink conditioning and some abnormalities in conditioned response timing. Control groups did not differ from each other. CONCLUSIONS: The present data indicate that early exposure to alcohol has long-term effects on eyeblink conditioning, perhaps through enduring effects associated with alcohol-induced loss of Purkinje cells of the cerebellum.

Therapeutic motor training ameliorates cerebellar effects of postnatal binge alcohol.

We have used training on complex motor tasks to ameliorate effect of neonatal alcohol exposure. On postnatal days 4-9, alcohol-exposed (AE) rats were given 4.5 g/kg/day of alcohol by artificial rearing; gastrostomy control (GC) rats were given an isocaloric mixture of maltose/dextrin; suckling control (SC) rats were suckled normally. At 6 months of age, animals from the three groups underwent either rehabilitation training on a series of complex motor tasks, motor conditioning on a flat runway, or an inactive home cage condition. Subsequently, animals were either tested on three tests of balance and coordination, or were used for cerebellar morphology. After rehabilitation, but not after motor conditioning, male and female AE rats exhibited significant improvement in independent tests of motor skills. Using unbiased stereological morphological techniques, rehabilitated SC and AE animals were found to exhibit significantly more parallel fiber synapses per Purkinje cell in the paramedian lobule.

Therapeutic effects of complex motor training on motor performance deficits induced by neonatal binge-like alcohol exposure in rats . I. Behavioral results.

The effects of complex motor task learning on subsequent motor performance of adult rats exposed to alcohol on postnatal days 4 through 9 were studied. Male and female Long-Evans rats were assigned to one of three treatments: (1) alcohol exposure (AE) via artificial rearing to 4.5.g kg-1 day-1 of ethanol in a binge-like manner (two consecutive feedings), (2) gastrostomy control (GC) fed isocaloric milk formula via artificial rearing, and (3) suckling control (SC), where pups remained with lactating dams. After completion of the treatments, the pups were fostered back to lactating dams, and after weaning they were raised in standard cages (two-three animals per cage) until they were 6 months old. Rats from each of the postnatal treatments then spent 20 days in one of three conditions: (1) inactive condition (IC), (2) motor control condition (MC) (running on a flat oval track), or (3) rehabilitation condition (RC) (learning to traverse a set of 10 elevated obstacles). After that all the animals were tested on three tasks, sensitive to balance and coordination deficits (parallel bars, rope climbing and traversing a rotating rod). On parallel bars, both male and female rats demonstrated the same pattern of outcomes: AE-IC rats made significantly more mistakes (slips and falls) than IC rats from both control groups. After 20 days of training in the RC condition, there were no differences between AE and both SC and GC animals in their ability to perform on the parallel bars test. On rope climbing, female animals showed a similar pattern of abilities: AE-IC rats were the worst group; exercising did not significantly improve the AE rats' ability to climb, whereas the RC groups (SC, GC and AE) all performed near asymptote and there were no significant differences among three neonatal treatment groups. There was a substantial effect of the male rats' heavier body weight on climbing ability, and this may have prevented the deficits in AE rats behavior from being detected. Nevertheless, male animals from all three postnatal treatments (SC, GC and AE) were significantly better on this task after RC. Female and male rats from all three postnatal groups demonstrated significantly better performance on the rotarod task after 20 days of 'rehabilitation'. These results suggest that complex motor skill learning improves some of the motor performance deficits produced by postnatal exposure to alcohol and can potentially serve as a model for rehabilitative intervention.

Alcohol-induced Purkinje cell loss depends on developmental timing of alcohol exposure and correlates with motor performance.

Several reports indicate that neonatal ethanol exposure induces cerebellar Purkinje and granule cell loss if exposure occurs before postnatal day (PD) 7, and that cerebellar damage may underlie ethanol-induced motor deficits. The present study used an unbiased stereological method, the optical fractionator, to count total cerebellar Purkinje cell number in groups of Sprague-Dawley rats given binge-like ethanol exposure at different neonatal ages. Correlations between Purkinje cell number (of 55-day-old rats) and parallel bar motor performance (previously tested on PD 30-32) were also evaluated. One group was given binge-like exposure to 6.6 g/kg per day of ethanol via artificial rearing on PD 4 and 5 (PD 4/5); a second group on PD 8 and 9 (PD 8/9); and a third group on both PD 4 and 5 and 8 and 9 (Comb). Gastrostomy (CG) and suckle (SC) control groups were also included. Purkinje cells were significantly reduced in all three ethanol-treated groups compared to controls, but the severity of loss was significantly greater in the PD 4/5 and Comb groups (reduced by 42% and 45%, respectively, relative to GC) compared to the PD 8/9 group (reduced by 15%). Across treatment groups, the total cerebellar Purkinje cell number was significantly correlated with successful parallel bar traversal (r = +0.74), supporting the contention that ethanol-induced motor deficits may be associated with cerebellar cell loss. These data confirm the presence of windows of vulnerability of Purkinje cells to neurotoxic effects of binge ethanol treatment, and demonstrate that both the behavioral and neuroanatomical consequences of binge exposure depend on the developmental timing of the exposure.

Binge neonatal alcohol intubations induce dose-dependent loss of Purkinje cells.

Previous work using artificial rearing methods to administer alcohol to neonatal rats identified postnatal days (PD) 4-6 as a period of enhanced vulnerability to alcohol-induced Purkinje cell loss. To develop an alternative to artificial rearing, alcohol was administered to pups in a binge pattern of exposure using acute intubations, and dose-related effects on blood alcohol concentrations (BACs), somatic growth, and cerebellar Purkinje cell survival were assessed. Pups were intubated with alcohol in milk formula, twice a day, 2 h apart, with total daily doses of 4.5, 5.25, or 6.0 g/kg of alcohol. After intubations on PD 4, the blood alcohol concentration (BAC)-time curves systematically increased with increasing dose. Intubation of these doses on PD 4-6 produced significant, dose-dependent reductions in the total number of cerebellar Purkinje cells on PD 10, counted using the stereological optical fractionator. Somatic growth was significantly affected only by the highest dose. These dose manipulations using intubations confirmed that Purkinje cell death systematically increased as a function of BAC profiles within the PD 4-6 window of vulnerability.

Neonatal ethanol exposure impairs eyeblink conditioning in weanling rats.

Eyeblink conditioning depends on an identified brainstem-cerebellar circuit and may be useful in functional studies of early cerebellar damage produced by neurotoxicants. The present study asked whether binge-like neonatal ethanol exposure that damages the cerebellum would also result in eyeblink conditioning deficits. On postnatal day (PND) 23 to PND24, three groups of Long-Evans rat pups were tested for eyeblink conditioning: (1) ETOH, a group that received intragastric administration of 5.25 g/kg/day of ethanol on PND4 through PND9 via artificial rearing; (2) GC, a gastrostomy control group that received calorically matched milk formula on those days; and (3) SC, suckle controls that were reared normally with their dams. Eyeblink conditioning was severely impaired in the ethanol-treated group relative to the GC and SC groups, which did not differ. This impairment did not reflect sensory, motor, or motivational effects of ethanol treatment, because startle responses to the auditory conditioned stimulus and reflexive eyeblink responses to the unconditioned stimulus did not differ across the three treatment groups. These results suggest that neonatal binge ethanol exposure disrupted brain development in a manner that selectively impaired associative processes involved in eyeblink conditioning, consistent with alcohol-induced damage to the brainstem-cerebellar circuit necessary for this form of learning.

Neonatal binge ethanol exposure using intubation: timing and dose effects on place learning.

Neonatal rats were given ethanol using an acute intubation procedure that resulted in daily binge-like exposure with minimal effects on somatic growth. Acquisition of place learning in the Morris water maze was evaluated on postnatal days (PD) 26-31. In Experiment 1, a total of 5.25 g/kg/day of ethanol was administered in two daily intubations on PD 4-6, PD 7-9, or PD 4-9, producing mean peak BACs of 265 mg/dL. Place learning acquisition deficits in a 114-cm-diameter tank were found for the PD 4-9 and PD 7-9 groups, but not the PD 4-6 group. In Experiment 2, either 4.5 or 5.25 g/kg/day of ethanol was administered on PD 7-9 and place learning was tested in a 171-cm-diameter tank. Significant acquisition deficits resulted from the higher dose, and probe trial search patterns for both ethanol groups were significantly less localized than controls. In Experiment 3, no significant effects of either PD 7-9 dose were found on a visible platform task. These findings reveal selective place learning deficits in this intubation model of neonatal binge exposure, and confirm a temporal window of vulnerability to spatial learning deficits during the second neonatal week.

Therapeutic motor training increases parallel fiber synapse number per Purkinje neuron in cerebellar cortex of rats given postnatal binge alcohol exposure: preliminary report.

Because therapeutic approaches to fetal alcohol effects in humans have been rare, this study explored the rehabilitative effect of complex motor training on an animal model of binge drinking in the third trimester of human pregnancy. Neonatal alcohol exposure induces significant and permanent reductions in Purkinje and granule cell number accompanied by impaired motor behavior in rats. The purpose of this study was to determine: (1) whether the motor skill impairment caused by exposure to alcohol in the early postnatal period could be ameliorated by the learning of a set of complex motor tasks that had been demonstrated to cause synaptogenesis in the cerebellar cortex; and (2) the extent to which cerebellar neurons in alcohol-exposed (AE) rats exhibit synaptic plasticity. The AE group was given 4.5 g/kg/day of ethanol from postnatal days 4 to 9 via an artificial rearing procedure producing a mean peak blood alcohol level of 257 mg/dl. Control groups consisted of a gastrostomy control (GC) group, that received an isocaloric mixture of maltose/dextrin instead of ethanol, and a suckle control (SC) group, that was reared normally by dams. At approximately 6 months of age, animals from the three groups were assigned either to a rehabilitation condition (RC; that received 10 days of training on the motor tasks) or to an inactive condition (IC; where rats stayed in isolation in their cages). Although SC rats were significantly faster to complete the course in the first 5 days of training, there were no differences in ability to perform among animals from all three groups-SC, GC, and AE--at the end of the training period. Unbiased stereological techniques were used to obtain estimates of the number of parallel fiber synapses/Purkinje cell within the cerebellar paramedian lobule. Results showed that the RC rats from the SC and AE groups had significantly more synapses/Purkinje cell than corresponding IC animals. These data demonstrate that rehabilitative intervention (complex motor training) can improve motor performance impaired by postnatal alcohol exposure and that surviving Purkinje neurons retain the capacity for synaptic plasticity.

Binge-like alcohol exposure of neonatal rats via intragastric intubation induces both Purkinje cell loss and cortical astrogliosis.

Binge-like alcohol exposure in neonatal rats on postnatal days 4 to 9 via artificial rearing results in a well-documented transient astrogliosis in the cerebral cortex. A recent study, which replicated the astrogliosis using artificial rearing, found that alcohol administered via daily exposure cycles in a vapor inhalation chamber on postnatal days 4 to 9 failed to elicit the effect, thus suggesting that the gliosis was an interactive effect of the artificial rearing administration and not specific to alcohol. The present study evaluated the effects in an intragastric intubation model that replicated the dosing parameters of the artificial rearing while avoiding the stress of surgery and extended maternal separation. In coronal frozen sections through parietal cortex labeled immunohistochemically for glial fibrillary acidic protein, the pups exposed to alcohol by intubation had a significantly greater density of glial fibrillary acidic protein-positive astrocytes per unit volume, compared with littermate controls intubated with a maltose-dextrin formula; alcohol also induced fibrillary hypertrophy of the labeled astrocytes. In the cerebellum, alcohol induced a significant reduction in Purkinje cell number as determined using the optical disector method. These outcomes extend previous findings that neonatal binge alcohol exposure induces acute cortical astrogliosis and Purkinje cell loss, and confirm that the alcohol-induced astrogliosis is not an artifact of artificial rearing.

Alcohol-induced Purkinje cell loss with a single binge exposure in neonatal rats: a stereological study of temporal windows of vulnerability.

Previous research has shown that the early neonatal period of rats is one of enhanced vulnerability to cerebellar Purkinje cell loss associated with binge-like alcohol exposure, with a prominent sensitive period during the first neonatal week. In this study, an unbiased count of the total number of Purkinje cells was obtained using the stereological optical fractionator, in groups of rats given a single binge-like alcohol exposure either during the most vulnerable neonatal period [postnatal day (PD) 4] or during a later, less vulnerable period (PD 9). Using artificial rearing methods, rats were given 6.6 g/kg of alcohol either on PD 4 or on PD 9, delivered as a 15% (v/v) solution in milk formula on two consecutive feedings of the designated day. Control groups included an artificially reared gastrostomy control and a normally reared suckle control. The mean peak blood alcohol concentrations were not different between the PD 4 and PD 9 alcohol groups, averaging 374 and 347 mg/dl, respectively. The rats were perfused on PD 27. A uniform random sample of sections was obtained from serial frozen sections through the cerebellum, stained with thionin, and Purkinje cells were counted from a uniform random sample of locations on each section with the three-dimensional optical fractionator. The number of Purkinje cells in the suckle control and gastrostomy control groups did not differ from each other, averaging 3.94 (+/- 0.19) and 3.58 (+/- 0.22) x 10(5) cells, respectively. Binge exposure on PD 4 induced significant cell loss (mean of 2.05 +/- 0.20 x (10(5) Purkinje cells), whereas binge exposure on PD 9 did not induce significant Purkinje cell loss (3.70 +/- 0.39 x 10(5) Purkinje cells). These findings confirm that a single neonatal binge alcohol exposure produces pathological Purkinje cell loss, provided that it occurs during the period of enhanced vulnerability coinciding with the early stages of dendritic outgrowth.

Temporal determinants of neonatal alcohol-induced cerebellar damage and motor performance deficits.

The timing and duration of alcohol exposure was manipulated in neonatal rats by using a "binge" model of alcohol exposure during the "third trimester equivalent." Groups of Sprague-Dawley rats were exposed to binges via artificial rearing on postnatal days (PD) 4-9, on PD 4-6 or on PD 7-9, which produced peak blood alcohol concentrations representative of human alcohol abusers (approximately 250 mg/dl). Motor performance was assessed using parallel bar traversal on PD 42-44, and total Purkinje cell numbers were determined by using the 3-dimensional stereological optical fractionator method. PD 4-9 binge exposure induced the most severe Purkinje cell loss (to 68% of controls) and PD 4-6 binge exposure also produced significant loss (to 86% of controls), whereas PD 7-9 binge exposure had no significant effect (98% of controls). Unexpectedly, all three alcohol treatments resulted in significant impairments on the parallel bar task. The time of exposure during the early neonatal period in rats strongly influences the degree of Purkinje cell loss, but Purkinje cell loss is not necessary for the alcohol-induced motor performance deficits. Both neuromorphological and neurobehavioral assessments are needed for a full description of alcohol-related neurodevelopmental disorders.

Behavioral deficits induced by bingelike exposure to alcohol in neonatal rats: importance of developmental timing and number of episodes.

The importance of the timing and number of episodes of bingelike alcohol exposure in neonatal rats on subsequent behavioral outcomes was evaluated with a parallel bar task and a spatial conditional alternation task. Different groups of Sprague-Dawley rat pups were exposed to alcohol delivered via artificial rearing procedures either on postnatal Days (PD) 4 and 5, on PD 8 and 9, or on both PD 4/5 and 8/9 (Combined), producing daily peak blood alcohol concentrations around 400 mg/dl. Controls included an artificially reared group and a normally reared group. Exposure during PD 4/5 produced significantly more severe motor deficits and significantly more severe reductions in cerebellar and brainstem weights than did exposure on PD 8/9. Combined exposure produced greater deficits on these measures than either of the limited exposures. Significant deficits in the acquisition rates for conditional alternation were found only with the Combined exposure, although both the PD 8/9 and Combined groups committed significantly more within-trial errors. All three alcohol treatments produced significant and comparable reductions in forebrain weight. The type and severity of behavioral and neural deficits induced by neonatal bingelike alcohol exposure depend on the timing and number of exposures.

Sex differences in vulnerability to developmental spatial learning deficits induced by limited binge alcohol exposure in neonatal rats.

The two main objectives of this study were (1) to replicate previous findings that 6 days of binge-like exposure to alcohol during the neonatal brain growth spurt induces significant place learning deficits in juvenile rats and (2) to determine whether more limited (3-day) binge-like exposure during the neonatal period induces place learning deficits and whether the effects depend on the developmental timing of the exposure. Using artificial rearing methods and a split-litter experimental design, groups of male and female neonatal rats were given binge-like exposure to 4.5 g/kg/day of ethanol in milk formula either on Postnatal Days (PD) 4-6, PD 7-9, or PD 4-9, which yielded mean peak blood alcohol concentrations of 230-260 mg/dl. Controls included an artificially reared gastrostomy control group (GC) given an isocaloric milk formula diet on PD 4-9 and a suckle control group reared normally by lactating dams. Acquisition of place learning in the Morris spatial navigation task was trained for 6 consecutive days beginning on PD 26; a probe trial was given at the end of the sixth day. As expected, both males and females given alcohol on PD 4-9 had significant deficits in acquisition and probe trial performance relative to SC and GC groups. Males given the PD 7-9 exposure had significant place learning deficits which were as severe as with the full 6-day exposure. The PD 4-6 exposure in males produced only a nonsignificant trend toward slower acquisition. Females were not significantly affected by either 3-day exposure. The latter phase of the neonatal brain growth spurt appears to constitute a sex-specific period of enhanced vulnerability to alcohol-induced developmental spatial learning deficits.

NMDA prevents alcohol-induced neuronal cell death of cerebellar granule cells in culture.

Neuronal cell loss is one of the most debilitating effects of alcohol exposure during development of the nervous system. In this study, primary cultures of neuronal cells (cerebellar granule cells) were used to examine mechanisms of alcohol-induced neuronal cell death. Previously, we established that (Pantazis et al., Alcohol Clin Exp Res 17:1014-1021, 1993): (1) alcohol exposure caused neuronal cell death in cultures of cerebellar granule cells and this cell loss was both time-dependent and dose-dependent; and (2) the vulnerability of cerebellar granule cells to alcohol-induced loss changed with the length of time the cells were in culture before initiating alcohol exposure-that is, younger cultures (1 day in vitro) were much more susceptible to alcohol-induced neuronal cell death than older cultures (4 or 7 days in vitro). The primary goal of the present study was to examine the potential role of the NMDA receptor in alcohol-induced death of cerebellar granule cells in culture. Experiments were performed to test the hypothesis that the alcohol-induced death of cerebellar granule cells can be prevented or reduced by NMDA treatment. Our results indicate that stimulation of the NMDA receptor has a neuroprotective effect and can significantly reduce the alcohol-induced neuronal cell death of newly established cerebellar granule cell cultures. This neuroprotective effect of NMDA is blocked by 2-amino-5-phosphonovalerate, a competitive inhibitor of the NMDA receptor, confirming that this neuroprotective effect is mediated via the NMDA receptor. This is the first report that alcohol's neurotoxic effect can be ameliorated by activation of the NMDA receptor.

Developing rat Purkinje cells are more vulnerable to alcohol-induced depletion during differentiation than during neurogenesis.

This study compared the extent of cerebellar Purkinje cell depletion induced by administering alcohol to rats during two temporally distinct periods of Purkinje cell development--neurogenesis and early differentiation. One group received alcohol (5 g/kg/day) during and shortly after Purkinje cell neurogenesis (gestational days 13-18) via oral intubation of pregnant dams. A second group received alcohol (2.5 g/kg/day) during early Purkinje cell differentiation (postnatal days 4-9) via artificial rearing of pups. The two alcohol treatment protocols were designed to match the cyclic daily blood alcohol profiles of the two groups as closely as possible. Pair-fed intubated controls, artificially reared gastrostomy controls, and normally reared ad lib/suckle controls were also evaluated. Mean peak blood alcohol concentrations (BACs) were 266 mg/dl for the intubated pregnant dams and 205 mg/dl for the pups exposed postnatally. Purkinje cell profiles were counted from single, 2-microns-thick midsaggital sections on postnatal day 10. Alcohol exposure during neurogenesis resulted in no significant change in Purkinje cell profile densities. Exposure during differentiation produced significant reductions in Purkinje cell profile densities, predominantly in the early maturing regions of the vermis (lobules I-IV and IX-X). These results indicate that Purkinje cells are more vulnerable to alcohol-induced population depletion during differentiation than during neurogenesis.

Vulnerability of cerebellar granule cells to alcohol-induced cell death diminishes with time in culture.

This study examined the effects of alcohol exposure on the viability of cerebellar granule cells in culture. Continuous alcohol exposure, starting 1 day after the cultures were established, significantly reduced granule cell numbers, even with a single day of exposure to an alcohol concentration as low as 100 mg/dl. The depletion of cerebellar granule cells by alcohol was concentration-dependent (greater loss of cells at higher alcohol concentrations) and duration-dependent (greater loss of cells at longer exposure durations). The loss of granule cells also depended on the number of days the granule cells were in culture before alcohol exposure. Alcohol was significantly more effective in reducing the cell numbers of newly established granule cell cultures (1 day in vitro) compared with older cultures (4 or 7 days in vitro). Cell cycle analysis established that the cerebellar granule cells did not proliferate in culture, indicating that alcohol exposure did not reduce cell numbers by interfering with cell proliferation in this system. Instead, alcohol-induced killing of the granule cells was the most likely mechanism to account for the depletion of granule cells in vitro. Granule cell cultures are a useful in vitro model system to study the cellular and molecular aspects of neuronal cell depletion associated with fetal alcohol exposure. The potential role of the N-methyl-D-aspartate receptor in this alcohol-induced neuronal cell death is discussed.

Transient cortical astrogliosis induced by alcohol exposure during the neonatal brain growth spurt in rats.

The astrocyte response to central nervous system injury induced by neonatal alcohol exposure was evaluated using radioimmunoassay and immunocytochemistry of glial fibrillary acidic protein (GFAP). Rat pups were exposed to alcohol on postnatal days 4 through 9 via artificial rearing. Alcohol solutions were administered as one of the following treatments: 10.2% (v/v) in two feedings (4.5 g/kg/day), 5.1% (v/v) in four feedings (4.5 g/kg/day), or 2.5% (v/v) in 12 feedings (6.6 g/kg/day), producing mean blood alcohol concentrations (BACs) of approximately 300, 180, and 50 mg/dl, respectively. Littermates were included as gastrostomy controls (GC) and suckle controls (SC). On postnatal day 10, GFAP concentration increased as a function of BAC, and the 10.2% alcohol treatment significantly and dramatically increased GFAP in the cortex (325% of SC). GFAP immunocytochemistry revealed frequent loci of heavily labeled reactive astrocytes surrounding small cortical blood vessels in the 10.2% group. In addition, a generalized increase in GFAP immunoreactivity was present in the deep layers of the cortex in all alcohol groups, marked by astrocytic fibrillary hypertrophy and increased density. Three-dimensional counts in layer V of parietal cortex using confocal microscopy indicated that the density of GFAP-labeled astrocytes of the 10.2% group was twice that of controls. The layer V gliosis was observable even at low BACs, while gliosis around the vasculature occurred only with high BACs. By postnatal day 15, the astroglial effects were no longer evident. These transient astroglial reactions likely constitute an important aspect of cortical pathophysiology resulting from binge alcohol exposure during the brain growth spurt of the third trimester equivalent.