Introduction

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The developing brain is extremely sensitive to the effects of ethanol (Abel, 1984; West and Pierce, 1986). Heavy consumption of ethanol during pregnancy can result in a set of profound morphological and neurological aberrations called fetal alcohol syndrome (Lemoine et al., 1968; Jones and Smith, 1973; Jones et al., 1973; Clarren and Smith, 1978). Further, there is a growing appreciation for the concern that moderate ethanol consumption may cause subtle, long-term impairments in the absence of the gross morphological or neurological defects associated with FAS (Shaywitz et al., 1980; Abel, 1984). For example, moderate ethanol exposure in utero causes cognitive deficits in children which may not become apparent until the child is challenged during the educational years (Streissguth et al., 1990; Conry, 1990) and may increase in severity as the child matures (Streissguth et al., 1991, 1994). Recognition of this problem led to the recommendation that the diagnostic classification of fetal alcohol-related defects be expanded to include a new category called alcohol-related neurodevelopmental disorders (ARND; Stratton et al., 1996).

Although the mechanisms underlying cognition and cognitive deficits are complex and involve many systems, increasing evidence indicates that the hippocampal formation (Milner, 1965; DeJong et al., 1969; Olton and Pappas, 1979; Zola-Morgan et al., 1986) and the glutamate neurotransmitter system (Morris et al., 1986; Morris, 1989) participate in the process of memory formation. The neurobiological implications of the subtle, but perhaps more pervasive effects of moderate prenatal ethanol exposure on cognition prompted our investigation of the effects of moderate ethanol consumption during pregnancy on amino acid neurotransmitter systems in hippocampal formation and other brain regions associated with learning in adult offspring. Previous studies suggest that these cognitive deficits may, in part, be linked to multiple abnormalities in hippocampal glutamate neurotransmission of fetal alcohol-exposed offspring, including reduced hippocampal glutamate receptor (Farr et al., 1988a), kainate receptor (Farr et al., 1988b) and NMDA receptor binding (Savage et al., 1991, 1992; Abdollah and Brien, 1995) and functional deficits in hippocampal NMDA neurotransmission (Morrisett et al., 1989; Weaver et al., 1993). In addition to changes in the ionotropic NMDA receptor, we also have observed decreased activation of metabotropic glutamate receptor-stimulated phosphoinositol hydrolysis (Queen et al., 1993). These changes may underlie a diminished capacity to elicit glutamate receptor-dependent electrophysiological phenomena such as long-term potentiation (Swartzwelder et al., 1988; Tan et al., 1990; Sutherland et al., 1997). Furthermore, the neurochemical changes in fetal alcohol-exposed rats may contribute to deficits in animal learning behaviors sensitive to hippocampal glutamate receptor antagonism. Prenatal ethanol-exposed rats exhibit performance deficits in spatial navigation tasks (Blanchard et al., 1987; Kelly et al., 1988; Gianoulakis, 1990) and in one-trial fear-conditioning responses (R. Sutherland, D. Savage, R. McDonald and M. Weisend, unpublished observations). Taken together, these changes suggest that altered NMDA receptor-mediated glutamate neurotransmission is one teratological consequence of prenatal ethanol exposure which contributes to learning disabilities in fetal alcohol-exposed children.

The effect of prenatal ethanol exposure on inhibitory influences of GABA neurotransmission has received less attention thus far. In a single study, specific [³H]muscimol binding to GABA_A receptors was elevated by 11% to 22% in various subfields of the hippocampal formation of fetal ethanol-exposed offspring compared with controls (Savage and Swartzwelder, 1992). These changes were not statistically significant and a subsequent saturation of binding study indicated no change in the total number of specific [³H]muscimol binding sites (B_max). These results suggested that modest elevations in [³H]muscimol binding in prenatal ethanol-exposed offspring may be caused by subtle changes in factors affecting the affinity of GABA_A receptors for binding agonists.

One putative mechanism for subtle alterations in [³H]muscimol binding may be changes in the allosteric modulatory influences that affect the GABA_A receptor affinity and function. It is now well established that several classes of compounds, including various benzodiazepines (Obata et al., 1988; Pritchett et al., 1989), barbiturates (Allan and Harris, 1986a; Olsen et al., 1986), neurosteroids (Gee et al., 1988; Puia et al., 1990) and ethanol (Allan and Harris, 1986b; Aguayo, 1990) exhibit allosteric modulatory influences on GABA_A receptors in different brain regions. Using GABA_A receptor-stimulated ³⁶Cl flux as a marker of GABA_A receptor function, we tested the hypothesis that maternal consumption of moderate amounts of ethanol throughout gestation alters the allosteric modulation of GABA_A receptor function in adult offspring. GABA dose-response curve studies and an assessment of the effects of positive and negative modulators acting either at the benzodiazepine or neurosteroid recognition sites were conducted in the medial frontal cortex, cerebellum and hippocampal formation of adult rat offspring whose mothers had consumed either rat chow ad libitum, pair-fed a 0% ethanol liquid diet or a 5% ethanol liquid diet.

	Materials and Methods

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Ethanol liquid diet paradigm. Five-month-old Sprague-Dawley rat dams (Harlan Industries, Indianapolis, IN) were individually housed in plastic cages in a temperature-controlled room (22°C) on a 16 hr dark:8 hr light schedule (lights off from 5:30 P.M. to 9:30 A.M.). Beginning on day 1 of gestation, rat dams were placed in one of three diet groups. Two of the three diets consisted of a liquid diet based on the Lieber-DeCarli (1982) formulation (BioServ, Frenchtown, NJ). One group received liquid diet containing 5% (v/v) ethanol (26% ethanol-derived calories). This group received 110 ml of 5% ethanol liquid diet at 5:30 P.M. each day. The feeding tubes were removed 16 hr later (at 9:30 A.M. on the next morning), and water bottles were placed on the cages. The other liquid diet group, serving as pair-fed control, was given a 0% ethanol liquid diet (isocalorically equivalent to the 5% ethanol diet) for 16 hr each day. A third diet group had continuous access to Purina breeder block chow and water ad libitum and served as control for the paired feeding technique. Rat dams were maintained on these diets throughout gestation. At birth, all litters were weighed, culled to 10 pups and cross-fostered onto surrogate untreated dams. Rat pups were weaned at 30 days of age and maintained on lab chow and water ad libitum.

Blood ethanol determinations. Maternal blood ethanol concentrations produced by consumption of the 5% ethanol liquid diet were determined in a separate set of dams. The food intake in these additional dams was matched to the daily volume of food ingested by the dams that produced the offspring used in the ³⁶chloride ion flux experiments described below. Blood samples were taken at 11:30 P.M., 6 hr after the introduction of the food tubes. This time point was selected for sampling because peak maternal blood ethanol concentrations occur about an hour after the peak period of liquid diet consumption (data not shown). Blood samples were collected from each dam every other evening during the third week of gestation for a total of three collections per rat dam. A 0.1-ml whole-blood sample was collected from the tail vein and immediately mixed with 0.2 ml of 6.6% perchloric acid and stored frozen at 20°C until assayed. Blood ethanol standards were created by mixing rat whole blood from untreated rats with known amounts of ethanol ranging from 0 to 240 mg/dl and then mixing 0.1-ml aliquots of each standard with perchloric acid and storing the standards frozen with the samples. Blood ethanol samples were assayed by a modification of the method of Lundquist (1959). The ethanol standard curve was linear over the 0 to 240 mg/dl range. Sample blood ethanol values were determined by regression analysis. Because the dams consumed similar quantities of food each night during the third week of gestation, the blood ethanol data collected from all three sessions was combined for all rat dams sampled.

Preparation of brain membranes (microsacs). Brain membrane vesicles (microsacs) were prepared as described by Allan and Harris (1986a). A total of six 5-month-old female offspring (two each from three separate litters) were used from each of the three experimental diet groups. Rats were sacrificed by decapitation, the brain rapidly removed and the medial frontal cortex, cerebellum and hippocampal formation were dissected out on ice. The area identified as the medial frontal cortex included the infralimbic cortex, cingulate cortex, medial, lateral and ventral orbital cortex and agranular insular cortex, according to the Paxinos and Watson atlas (1986). Dissected tissue was homogenized gently by hand with a Dounce homogenizer in assay buffer (145 mM NaCl, 1 mM MgCl₂, 5 mM KCl, 1 mM CaCl₂, 10 mM HEPES, 10 mM glucose, pH 7.5 at 25°C with Tris) containing protease inhibitors (0.05 mg/ml calpain inhibitor I and II, 0.02 mg/ml trypsin inhibitor, 2 µg/ml aprotinin, 1 µg/ml leupeptin). The homogenate was washed twice by centrifugation (700 × g_av for 15 min) and resuspended in assay buffer with protease inhibitors to a final concentration of 1 to 2 mg/ml as determined by a modification of the method of Lowry et al. (1951) as described by Markwell et al. (1981).

GABA_A-stimulated ³⁶chloride ion flux. Uptake of ³⁶chloride ion into brain microsacs was initiated by the addition of assay buffer containing 0.2 mCi/ml ³⁶chloride ion to the microsac preparation and followed for a total of 3 sec. Basal flux was measured as the amount of ³⁶chloride ion taken up in the absence of added GABA. In the GABA dose-response studies, five different concentrations of GABA (0, 10, 25, 50 or 100 µM) were incubated with microsacs. In the allosteric modulation of GABA-stimulated ³⁶chloride ion flux studies, microsacs were incubated with one of four modulatory agents: flunitrazepam (25 nM), FG-7142 (10 nM), alphaxalone (25 µM) or pregnenolone sulfate (25 µM). The concentration of each modulator used was a concentration previously determined to be approximately half-maximally effective (Allan et al., 1991; A. Allan, H. Wu and S. Engel, unpublished observations). GABA (25 µM) was added to tubes containing the positive modulators (flunitrazepam or alphaxalone) and 100 µM GABA was added to the tubes containing the negative modulators (FG-7142 or pregnenolone). Flux was terminated by the addition of assay buffer containing the GABA_A receptor antagonist, bicuculline (75 µM), and the chloride channel antagonist, picrotoxin (100 µM). Samples were immediately filtered under vacuum and counted by liquid scintillation spectroscopy. Each assay condition was run in triplicate. The amount of ³⁶chloride ion flux under basal (no added GABA) condition was subtracted out of all the other determinations. Data are expressed as nanomoles of ³⁶chloride ion per milligram of protein. GABA dose-response data were analyzed by an overall two-factor (3 × 4) ANOVA. Data from the modulation studies were analyzed by one-way ANOVA followed by Student-Neuman-Keuls post hoc tests where appropriate.

	Results

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Fetal ethanol exposure paradigm. Table 1 summarizes rat dam caloric intake, ethanol consumption and maternal blood ethanol concentration data along with the impact of these diets on litter size and offspring birth weight. Rat dams assigned to the pair-fed and 5% ethanol liquid diet group consumed an average of 87 kcal/day (table 1). The rat dams in these groups consumed approximately 92% of the daily caloric requirement for 250-g rat dams (Altman and Dittmer, 1974). This modest reduction in daily caloric intake had no significant effect on offspring outcome measures compared with the ad libitum chow control group (described below).

Dose-dependent GABA-stimulated ³⁶chloride ion flux. Increasing concentrations of added GABA produced a dose-dependent increase in ³⁶chloride ion flux in medial frontal cortex (fig. 1A), cerebellum (fig. 1B) and hippocampal formation (fig. 1C). GABA-stimulated ³⁶chloride ion flux was highest in the medial frontal cortex, followed by the hippocampal formation and the cerebellum. These data are similar to previous studies of ³⁶chloride ion flux measurements (Allan et al., 1991). ³⁶Chloride ion flux continued to increase at 100 µM, preventing calculation of E_max and EC₅₀ values. Concentrations greater than 100 µM were not run because of the limited amount of available tissue. Ideally, full dose-response relationships for these modulatory drugs would have been analyzed. However, we felt that, for this evaluation, the GABA dose-response relationship along with the effects of the modulatory agents should be measured within the same tissue preparation. Thus, we were only able to evaluate the effects of each of the four modulatory agents at a single concentration previously determined to be the half-maximally effective concentration (Allan et al., 1991; A. Allan, H. Wu and S. Engel, unpublished observations).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Effect of prenatal ethanol exposure on GABA stimulation of GABAA receptor-dependent 36chloride ion flux in microsacs prepared from medial frontal cortex (A), cerebellum (B) or hippocampal formation (C). Data points represent the mean ± S.E.M. from six adult rat offspring from each diet treatment group.

Modulation of GABA-stimulated ³⁶chloride flux. Each of the four neuromodulatory agents altered GABA-stimulated ³⁶chloride ion flux in all three brain regions studied in control rats. In the medial frontal cortex and the cerebellum, the positive modulators flunitrazepam and alphaxalone increased ³⁶chloride ion flux to the levels achieved with maximally stimulating concentrations of GABA, whereas the negative modulators FG-7142 and pregnenolone sulfate reduced GABA-stimulated ³⁶chloride ion flux to levels measured in the absence of added GABA (figs. 2 and 3). In the hippocampal formation, the negative modulators had a similar effect on GABA-stimulated ³⁶chloride ion flux. In contrast to the other brain regions, however, the positive modulators produced only a modest increase in GABA-stimulated ³⁶chloride ion flux in the hippocampal formation (fig. 4).

View larger version (35K): [in this window] [in a new window]   Fig. 2.   Effect of prenatal ethanol exposure on benzodiazepine and neurosteroid modulation of GABAA receptor-dependent 36chloride ion flux in medial frontal cortical microsacs. Data points represent the mean ± S.E.M. from six adult rat offspring from each diet treatment group. Asterisks denote data significantly different than both ad libitum (Ad Lib) and pair-fed control diet groups (P < .05; Student-Neuman-Keuls post hoc test).

View larger version (32K): [in this window] [in a new window]   Fig. 3.   Effect of prenatal ethanol exposure on benzodiazepine and neurosteroid modulation of GABAA receptor-dependent 36chloride ion flux in cerebellar microsacs. Data points represent the mean ± S.E.M. from six adult rat offspring from each diet treatment group. Asterisks denote data significantly different than both ad libitum (Ad Lib) and pair-fed control diet groups (P < .05; Student-Neuman-Keuls post hoc test).

View larger version (34K): [in this window] [in a new window]   Fig. 4.   Effect of prenatal ethanol exposure on benzodiazepine and neurosteroid modulation of GABAA receptor-dependent 36chloride ion flux in hippocampal microsacs. Data points represent the mean ± S.E.M. from six adult rat offspring from each diet treatment group. Asterisks denote data significantly different than both ad libitum (Ad Lib) and pair-fed control diet groups (P < .05; Student-Neuman-Keuls post hoc test).

	Discussion

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Prenatal ethanol exposure to relatively moderate maternal blood ethanol concentrations (83 mg/dl) produced long-lasting alterations on the neuromodulatory influences affecting GABA_A receptor-mediated neurotransmission (figs. 2, 3 and 4). These changes occurred without altering the capacity of GABA to stimulate ³⁶chloride ion flux as measured in our 3-sec assay system (fig. 1). Furthermore, these changes occurred in the absence of any of the gross morphological or neurological deficits associated with FAS, which are observed to some degree when rat fetuses or neonates are exposed to higher blood ethanol concentrations (West and Pierce, 1986).

The ³⁶chloride flux studies in hippocampal formation (fig. 1C) are consistent with in vitro receptor autoradiography studies on the impact of prenatal ethanol exposure on [³H]muscimol binding in adult rat offspring (Savage and Swartzwelder, 1992). The [³H]muscimol binding studies revealed no significant change in total GABA_A receptor number, which is consistent with overlapping GABA-stimulated ³⁶chloride ion flux curves among the three diet groups (fig. 1C). When brain sections were incubated with a half-maximally saturating concentration of [³H]muscimol, modest (11-22%) increases in specific [³H]muscimol binding were observed in the hippocampal dentate gyrus stratum moleculare of fetal ethanol-exposed rats compared with ad libitum and pair-fed controls (Savage and Swartzwelder, 1992). In light of the results shown in figure 4, one explanation for a modest [³H]muscimol binding site elevation may be a heightened GABA_A receptor sensitivity to an endogenous positive neuromodulatory substance or a lessened sensitivity to a negative modulator present in the histological section of brain during the binding assay incubation procedure.

The molecular basis for alterations in the modulation of GABA_A receptor function in prenatal ethanol-expose offspring is not known. However, the long-lasting effects of prenatal ethanol exposure in the medial frontal cortex and cerebellum of adult offspring reported in this study are reminiscent of the effects of chronic exposure to ethanol or other modulators of GABA_A receptor function. Chronic administration of ethanol (Morrow et al., 1990; Buck et al., 1991), benzodiazepines (Allan et al., 1992), barbiturates (Yu and Ticku, 1995c) or neurosteroid compounds (Yu and Ticku, 1995a, b) diminishes GABA_A receptor sensitivity to these modulatory agents. Similarly, chronic benzodiazepine administration reduced the apparent coupling between the benzodiazepine agonist site and the chloride channel and increased the apparent coupling between the channel and the inverse agonist site (Allan et al., 1992). Five-day treatment of cerebral cortical cultures with 5-pregnane-3-ol-20-one (5,3-P) decreases GABA-stimulated ³⁶chloride ion flux (Yu and Ticku, 1995a), as well as both 5,3-P and benzodiazepine enhancement of GABA-stimulated ³⁶chloride ion flux (Yu and Ticku, 1995b). Chronic 5,3-P treatment also reduces the stimulation of [³H]flunitrazepam binding by GABA, pentobarbital and 5,3-P. Similar treatment of cortical neurons with chronic pentobarbital also reduces the stimulation of [³H]flunitrazepam binding by GABA, pentobarbital and 5,3-P (Yu and Ticku, 1995c).

Based on the studies described above, there are at least two molecular mechanisms that may contribute to changes in GABA_A receptor sensitivity after chronic drug treatment and perhaps in fetal ethanol-exposed offspring as well. One putative mechanism relates to a differential expression of GABA_A receptor subunits in fetal ethanol-exposed offspring compared with controls. Native brain GABA_A receptors are pentamers comprising two  subunits associated with one or more beta, gamma and/or a delta subunit (Stephenson et al., 1990; Mertens et al., 1993; Khan et al., 1994). Currently, there are six known subtypes of alpha subunits, four beta subunit subtypes and four gamma subunit subtypes, one delta and two rho, with several subtypes displaying slice variants (see Klein and Harris, 1996 for review). More than a dozen heterooligomeric combinations of GABA_A receptors have been found in different brain regions (McKernan and Whiting, 1996). Subunit combination has been an important determinant of GABA_A receptor pharmacology. Benzodiazepines require the presence of the gamma subunit (Pritchett et al., 1989; Ymer et al., 1990; Wafford et al., 1993a, b), whereas modulation by barbiturates or neurosteroids is not influenced by the presence of the gamma subunit (Ymer et al., 1990; Shingai et al., 1991). Recent studies suggest that the presence of the delta subunit reduces neurosteroid modulation of GABA_A receptors (Zhu et al., 1996). Heterogeneity of GABA_A receptor responses to benzodiazepines is determined by the subtype of alpha subunit expressed (Montpied et al., 1988; Pritchett et al., 1989; Pritchett and Seeburg, 1991; Wafford et al., 1993a, b). GABA_A receptors constructed of ₃₂₁ subunits are more sensitive to flunitrazepam, whereas ₂₁ receptors containing alpha-2, alpha-1 or alpha-5 constructs are less sensitive (Costa and Guidotti, 1996). In contrast, GABA_A receptors containing either the alpha-4 or alpha-6 construct have little or no sensitivity to benzodiazepines (Korpi et al., 1993; Sigel et al., 1992).

Based on these observations, one explanation for prenatal ethanol exposure-induced decreases in GABA_A receptor sensitivity to benzodiazepines in medial frontal cortex and cerebellum is an increase in the relative abundance of GABA_A receptors containing alpha-4 or alpha-6 subunits. In forebrain regions, the alpha-4 construct is typically in low abundance relative to alpha-1, alpha-2, alpha-3 and alpha-5 constructs (Wisden et al., 1992), whereas the alpha-6 construct is present in the cerebellar granule cell layer in relatively equal abundance with alpha-1 constructs (Thompson et al., 1992). Morrow and colleagues have shown that chronic ethanol exposure, which decreases GABA_A receptor sensitivity to benzodiazepines (Buck et al., 1991), elevates the expression of alpha-4 constructs relative to alpha-1 constructs in the cerebral cortex (Devaud et al., 1995) and elevates the expression of alpha-6 constructs relative to alpha-1 constructs in the cerebellum (Morrow et al., 1992). An increase in GABA_A receptors containing the alpha-4 or alpha-6 subtypes relative to alpha-1, alpha-2, alpha-3 or alpha-5 could explain the decreased response to flunitrazepam modulation of GABA-mediated ³⁶chloride flux in prenatal ethanol-exposed offspring (figs. 2 and 3). In the medial frontal cortex, where the alpha-3 construct predominates (Wisden et al., 1992), another explanation for diminished GABA_A receptor sensitivity to benzodiazepines may be a reduction in alpha-3 subunits relative to alpha-1, alpha-2 and/or alpha-5 subunits. Conversely, increased flunitrazepam sensitivity of hippocampal GABA_A receptors in fetal ethanol-exposed offspring may be caused by an increased expression of alpha-3 subunits relative to alpha-1, alpha-2 and/or alpha-5 subunits.

The extent to which prenatal ethanol-induced changes in neurosteroid modulation of GABA_A receptors may be a function of differential expression of GABA_A receptor subunits is less clear. Most GABA_A receptor subunits respond to neurosteroids (Lan et al., 1991; Puia et al., 1993), with the possible exception of those containing the delta subunit (Zhu et al., 1996), which indicates that neurosteroid sensitivity does not depend on the expression of certain alpha, beta or gamma subunits. Given the paucity of delta subunit mRNA in the medial frontal cortex (Wisden et al., 1992), it is tempting to speculate that the fetal ethanol exposure-induced reduction in neurosteroid sensitivity in this region (fig. 2) may be caused by increased delta subunit expression. However, the potency and type of allosteric modulation may depend on subunit combination. For example, ₆₁₂ receptors are less sensitive to allopregnenolone than those containing the ₆₁₁ subunit (Puia et al., 1993). Further, neurosteroid enhancement of [³H]muscimol and [³H]flunitrazepam binding varies with the type of alpha subunit present in the GABA_A receptor (Lan et al., 1991) and varies among brain regions (Bureau and Olsen, 1993; Nguyen et al., 1995). Thus, it is possible that alterations in the subtypes of alpha or gamma subunit expressed may play a role in the changes observed in medial frontal cortex (fig. 2) or hippocampal formation (fig. 4).

A second molecular mechanism underlying the changes observed in the medial frontal cortex and the cerebellum of prenatal ethanol-exposed offspring may be a functional uncoupling of the neurosteroid and/or benzodiazepine recognition sites from the GABA recognition site. The molecular basis for such an uncoupling event is unknown and may or may not involve an alteration in GABA_A receptor subunit expression. Conversely, elevated GABA receptor sensitivity to the positive modulators in the hippocampal formation of fetal ethanol-exposed offspring may result from more effective coupling of recognition sites in the presence of positive allosteric modulators and/or less effective coupling in the presence of negative allosteric modulators (fig. 4). An opposite result has been reported after chronic treatment with ethanol or benzodiazepines in adult animals (Buck and Harris, 1990; Allan et al., 1992).

Currently, studies are underway to determine the relative abundance of alpha subunit mRNA along with [³H]flunitrazepam binding studies in fetal ethanol-exposed offspring. Elevations in the relative abundance of alpha-4 subunits (medial frontal cortex) or alpha-6 subunits (cerebellum) along with striking reductions in [³H]flunitrazepam binding in these regions would provide evidence that altered subunit expression plays a predominant role in fetal ethanol exposure-induced alterations in GABA_A receptor pharmacology. Alternatively, little or no change in GABA_A receptor subunit expression or [³H]flunitrazepam binding would suggest a functional uncoupling of GABA_A receptor recognition sites. In addition, it is possible that both of these mechanisms may contribute by different degrees to the changes observed in various brain regions of fetal ethanol-exposed offspring.

The functional implications of these prenatal ethanol exposure-induced changes in GABA_A receptor function are unclear at present. In the hippocampal formation, the heightened influence of positive allosteric modulators relative to negative modulatory influences (fig. 4) may produce a net enhancement of GABA_A receptor-mediated inhibition of local excitatory neuronal circuits. This, in turn, could limit mechanisms underlying activity-dependent changes in synaptic plasticity, such as the hippocampal LTP deficits observed in littermates of the rats used in these ³⁶chloride ion flux studies (Sutherland et al., 1997). A more direct electrophysiological assessment of this interpretation would be an analysis of paired-pulse inhibition and potentiation in the absence and presence of GABA_A receptor modulatory agents. A preliminary in vivo study of paired-pulse inhibition at the perforant path-dentate granule cell synapse has revealed that paired-pulse inhibition in the absence of modulatory agents is unaffected by prenatal ethanol exposure. However, a significantly greater enhancement of paired-pulse inhibition is observed after low doses of either diazepam or alphaxalone in fetal ethanol-exposed offspring compared with pair-fed and ad libitum controls (R. Sutherland, D. Savage, R. McDonald and M. Weisend, unpublished observations).

In contrast to the hippocampal formation, the ability of neurosteroids and/or benzodiazepines to modulate GABA_A receptor-mediated inhibitory influences in other brain regions, such as the medial frontal cortex (fig. 2) or cerebellum (fig. 3), may be severely limited in fetal ethanol-exposed offspring. Although "base-line" GABA_A receptor-mediated neurotransmission may be relatively "normal" in these regions, the range of neural responsiveness to changing physiological or environmental circumstances may be severely diminished. For example, given that some GABA_A receptor subunit complexes are sensitive to metabolites of cortisol or progesterone (see Lambert et al., 1995 for review), some brain regions may not be capable of responding in an appropriate fashion to the presence of stressful situations or during portions of the estrus cycle.

Finally, these results suggest that benzodiazepines, neurosteroids and possibly other centrally active therapeutic agents may produce different effects, or no effects at all, in fetal ethanol-exposed offspring. To date, there are no published studies on the effects of benzodiazepine treatment of children with FAS which might substantiate or refute this hypothesis. However, the results of these experiments raise the possibility of atypical pharmacologic responses by children with FAS to therapeutic agents that directly affect central neurotransmitter receptors.