Effects of Prenatal Ethanol Exposure on Hippocampal Ionotropic-Quisqualate and Kainate Receptors

Previous studies from our laboratories have shown that the consumption of moderate quantities of ethanol by rat dams during pregnancy reduces N-methyl-D-aspartate (NMDA) agonist receptor binding and NMDA-mediated electrophysiological responses in the hippocampal formation of adult offspring. We hypothesized that prenatal ethanol exposure would produce similar effects on receptor number and agonist-mediated responses of two so-called "non-NMDA" subtypes of glutamate receptors, the ionotropic-quisqualate (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-sensitive and the kainate-sensitive receptors. Sprague-Dawley rats were fed either a liquid diet containing 3.35% ethanol, an isocalorically matched liquid diet, or lab chow ad libitum throughout gestation. No significant differences between offspring from these three groups in the agonist concentration-response curves for either AMPA-induced or kainate-induced depolarization of hippocampal CA1 pyramidal neurons were observed. Furthermore, no significant differences in the density of [3H]-AMPA or [3H]-vinylidene kainic acid binding sites in any of the apical dendritic field regions of dorsal or ventral hippocampal formation were observed between the groups. These results indicate that the ionotropic quisqualate and kainate receptors, located in the apical dendritic field regions of the principal hippocampal neurons, are not affected by the same degree of prenatal ethanol exposure, which is known to reduce NMDA receptor binding and function in these same regions.     

Prenatal ethanol exposure reduces mGluR5 receptor number and function in the dentate gyrus of adult offspring

BACKGROUND: Previous studies in our laboratory indicated that metabotropic glutamate receptor (mGluR)-stimulated phosphoinositide hydrolysis is markedly reduced in the hippocampal formation of adult rat offspring whose mothers drank moderate amounts of ethanol during pregnancy. In the present study, we extended these observations by measuring the impact of prenatal ethanol exposure on proteins associated with the mGluR5 receptor-effector system along with two mGluR5 agonist-mediated responses in dentate gyrus of adult offspring. METHODS: Sprague-Dawley rat dams consumed one of three diets throughout gestation: (1) a BioServ liquid diet that contained 5% ethanol (v/v), (2) pair-fed an isocalorically equivalent amount of 0% ethanol liquid diet, or (3) lab chow ad libitum. Microdissected slices of dentate gyrus were prepared from adult female offspring from each diet group and used for (1) Western blot analyses of mGluR5, the G-proteins Galphaq and Galpha11, and phospholipase C-beta1; (2) 2-chloro-5-hydroxyphenylglycine (CHPG)-stimulated growth associated protein 43 (GAP-43) phosphorylation; or (3) CHPG potentiation of electrically evoked [H]-D-aspartate (D-ASP) release from dentate gyrus slices. RESULTS: In tissue prepared from untreated control rats, CHPG produced a dose-dependent increase in phosphate incorporation into GAP-43, with maximal agonist stimulation occurring at 20 microM of CHPG. CHPG produced a quantitatively similar dose-dependent increase in the potentiation of electrically evoked D-ASP release from dentate gyrus slices from untreated controls. Fetal ethanol exposure reduced the amount of dentate gyrus mGluR5 receptor protein by 36% compared with the diet control groups. There were no significant differences between diet groups in the two G-proteins or phospholipase C-beta1 protein. Fetal ethanol exposure reduced CHPG-stimulated GAP-43 phosphorylation to approximately one half the amount of CHPG stimulation observed in the control diet groups. Prenatal ethanol exposure also reduced CHPG potentiation of D-ASP release to a similar degree compared with control. CONCLUSIONS: These results indicate that prenatal exposure to moderate quantities of ethanol reduces mGluR5 expression in the dentate gyrus of adult offspring. Although the subcellular site(s) for reduced mGluR5 expression cannot be discerned from Western blot data, the quantitatively similar effects of prenatal ethanol exposure on mGluR5 agonist stimulation of presynaptically localized GAP-43 phosphorylation and CHPG potentiation of evoked D-ASP release suggest that the presynaptic nerve terminal is one site where prenatal ethanol exposure has reduced mGluR5 receptor number and function. Furthermore, these data implicate these neurochemical alterations as one factor contributing to the hippocampal synaptic plasticity and behavioral deficits that we have observed previously in prenatal ethanol-exposed offspring.     

Prenatal Ethanol Exposure Diminishes Activity-Dependent Potentiation of Amino Acid Neurotransmitter Release in Adult Rat Offspring

Prenatal ethanol exposure has been associated with long-lasting intellectual impairments in children. Previous studies suggest that these deficits are, in part, linked to neurochemical abnormalities that reduce the ability to sustain long-term potentiation (LTP) in hip-pocampal formation of adult offspring. One presynaptic component of LTP that manifests during the first half-hour alter tetanic stimulation is an enhancement of amino acid neurotransmitter release. Given that the onset of enhanced neurotransmitter release correlates temporally with the decay of hippocampal LTP in prenatal ethanol-exposed offspring, we tested the hypothesis that prenatal ethanol exposure reduces tetanus stimulus-induced potentiation of electrically evoked amino acid release in hippocampal slices. Rat dams consumed 1 of 3 diets throughout gestation: (1) a BioServ liquid diet containing 5% (v/v) ethanol (26% ethanol-derived calories) that produces a maternal peak blood ethanol concentration of 83 mg/dl; (2) pair-fed an isocalorically equivalent amount of 0% ethanol liquid diet; or (3) Purina rat chow ad libitum. Hippocampal slices were prepared from adult offspring from each experimental diet group. Neither the amount of hippocampal slice tissue protein nor the incorporation of [³H]- d -aspartate (D-ASP) was affected by prenatal ethanol exposure. Furthermore, spontaneous efflux and electrically evoked D-ASP release were similar among the three diet groups. However, tetanus stimulus-induced potentiation of evoked D-ASP release in prenatal ethanol-exposed offspring was reduced to about one-third of the potentiation of D-ASP release Observed in the control diet groups. These results suggest that prenatal ethanol exposure produces long-lasting deficits in the neurochemical mechanisms responsible for activity-dependent potentiation of amino acid transmitter release without affecting the synaptic machinery responsible for amino acid uptake, storage, and release.     

Prenatal Ethanol Exposure Decreases Hippocampal Mossy Fiber Zinc in 45-Day-Old Rats

The long-term consequences of prenatal ethanol exposure on histochemically detectable hippocampal mossy fiber zinc was examined using a recently developed quantitative histofluorescence procedure. Pregnant Sprague-Dawley rats were maintained throughout gestation on one of three dietary regimens: (a) a liquid diet containing either 3.35% ethanol, (b) an isocalorically matched liquid diet pair-fed to the 3.35% ethanol group, or (c) lab chow ad libitum. At 45 days of age, offspring from each of the three diet groups were sacrificed for determination of hippocampal mossy fiber zinc and zinc analysis of selected tissues by atomic absorption spectroscopy. Hippocampal mossy fiber zinc was reduced by 36% in dorsal and 20% in ventral hippocampal formation stratum lucidum of rats exposed to the 3.35% ethanol diet compared to the offspring of the pair-fed control and ad libitum control dams. No significant differences in zinc:TS-Q histofluorescence were observed between the ad libitum and pair-fed control groups. No significant differences were observed among groups in tissue wet weight or tissue zinc concentration in any of the brain or other body regions analyzed. These results indicate a long lasting prenatal ethanol exposure-induced reduction in hippocampal mossy fiber zinc in the absence of changes in any indices of total body zinc nutriture. These results suggest that prenatal exposure to relatively low blood ethanol levels (30-40 mg/dl) has subtle, yet long-lasting effects in the hippocampal formation, a brain region important in the process of memory consolidation.     

Dose-dependent effects of prenatal ethanol exposure on synaptic plasticity and learning in mature offspring

BACKGROUND: We have observed profound deficits in hippocampal synaptic plasticity and one-trial learning in offspring whose mothers drank moderate quantities of ethanol during pregnancy. In the present study, we examined the question of whether lower maternal blood ethanol concentrations (BECs) could produce functional deficits in offspring. METHODS: Rat dams consumed either a 2%, 3%, or 5% ethanol liquid diet throughout gestation. Three other groups of dams were pair-fed a 0% ethanol liquid diet, and a seventh group consumed lab chow ad libitum. Adult offspring from each diet group were assigned either to studies of evoked [3H]-D-aspartate (D-ASP) release from hippocampal slices or spatial learning studies using the Morris Water Task. RESULTS: Consumption of the 2%, 3%, and 5% ethanol liquid diets produced mean peak maternal BECs of 7, 30 and 83 mg/dL, respectively. Consumption of these ethanol diets had no effect on offspring birthweight, litter size or neonatal mortality. Likewise, evoked D-ASP release from hippocampal slices and performance on a standard version of the Morris Water Task were not affected by prenatal ethanol exposure. By contrast, activity-dependent potentiation of evoked D-ASP release from slices and one-trial learning on a "moving platform" version of the Morris Water Task were markedly reduced in offspring whose mothers consumed the 5% ethanol liquid diet. Intermediate deficits in these two parameters were observed in offspring from the 3% ethanol diet group, whereas offspring from the 2% ethanol diet group were not statistically different than controls. CONCLUSIONS: We conclude that the threshold for eliciting subtle, yet significant learning deficits in offspring prenatally exposed to ethanol is less than 30 mg/dL. This BEC is roughly equivalent to drinking 1 to 1.5 ounces of ethanol per day.     

Neurosteroid modulation of glutamate release in hippocampal neurons: lack of an effect of a chronic prenatal ethanol exposure paradigm

BACKGROUND: Pregnenolone sulfate (PREGS) is a promnesic neurosteroid that is abundantly expressed in the hippocampus of rodents. Studies have shown that the modulation of postsynaptic ligand-gated ion channels by this neurosteroid is impaired in preparations from the brains of fetal ethanol-exposed animals. In this study, we examined whether the presynaptic actions of PREGS also are affected by exposure to ethanol in utero. METHODS: Rat dams were exposed to one of the following diets during pregnancy: (1) 5% ethanol liquid diet, (2) 0% ethanol liquid diet with pair-feeding, and (3) ad libitum controls. We then studied the presynaptic actions of PREGS on (1) alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor-mediated miniature excitatory postsynaptic currents (mEPSCs) recorded from cultured hippocampal neurons in the whole-cell patch-clamp configuration and (2) paired-pulse facilitation of NMDA receptor-dependent excitatory postsynaptic potentials that were intracellularly recorded from CA1 pyramidal neurons in hippocampal slices from adult rats. RESULTS: Chronic prenatal ethanol exposure affected neither basal mEPSC frequency nor its potentiation by PREGS. Basal paired-pulse facilitation (i.e., in the absence of PREGS) was unaffected by fetal ethanol exposure. Chronic prenatal ethanol exposure did not affect the PREGS-induced potentiation of paired-pulse facilitation. CONCLUSIONS: Chronic prenatal ethanol exposure does not affect the basal probability of glutamate release in immature or mature hippocampal neurons. Moreover, the presynaptic actions of the neurosteroid PREGS also are unaffected by this exposure. Given that modulation of glutamate release could have a role in the mechanism of the promnesic actions of this neurosteroid, future studies are warranted to determine whether PREGS can ameliorate learning and memory deficits in fetal ethanol-exposed animals.     

Acamprosate inhibits the binding and neurotoxic effects of trans-ACPD,suggesting a novel site of action at metabotropic glutamate receptors

BACKGROUND: Several reported effects of acamprosate within the glutamatergic system could result from interactions with metabotropic glutamate receptors (mGluRs). The following experiments were performed to determine whether acamprosate could compete with trnas-ACPD (+/--1-aminocyclopentane-trans-1,3-dicarboxylic acid, an equimolecular mixture of 1S, 3R and 1R, 3S-ACPD and an agonist at both group I and group II mGluRs) sensitive binding sites and protect against trans-ACPD-induced neurotoxicity in organotypic hippocampal slice cultures. METHODS: A P2 membrane preparation of cortices, cerebellums, and hippocampi of adult, male Sprague Dawley rats was used to determine the abilities of N-methyl-D-aspartic acid (NMDA) and trans-ACPD to displace [3H]glutamate in both the absence and the presence of the sodium salt of acamprosate (sodium mono N-acetyl homotaurine or Na-acamprosate). A comparison of the effects of 100 microM guanosine 5'-triphosphate on unlabeled glutamate, trans-ACPD, and Na-acamprosate was performed in the same paradigm. For the neurotoxicity studies, organotypic hippocampal slice cultures from male and female 8-day-old neonatal rats were exposed to either 500 microM -ACPD or 50 microM NMDA for 24 hr in normal culture medium containing serum on day 20 in vitro. The effects of Na-acamprosate and 2-methyl-6-(2-phenylethenyl)pyridine (SIB-1893), a noncompetitive antagonist at metabotropic type 5 receptors (mGluR5s), were assessed by determining differences in propidium iodide uptake as compared with neurotoxic challenges alone. RESULTS: Na-acamprosate displaced 31% of [3H]glutamate but did not compete with NMDA for [3H]glutamate binding sites. Na-acamprosate displayed total competition with trans-ACPD. The presence of 100 microM guanosine 5'-triphosphate differentially altered the displacing capabilities of the two mGluR agonists, unlabeled glutamate and trans-ACPD, as compared with Na-acamprosate. Na-acamprosate (200-1000 microM) and SIB-1893 (20-500 microM) both were neuroprotective against trans-ACPD induced neurotoxicity that likely results from mGluR potentiation of NMDARs. In turn, Na-acamprosate and SIB-1893 had no direct effects on NMDA-induced neurotoxicity. CONCLUSIONS: Na-acamprosate demonstrates the binding and functional characteristics that are consistent with a group I mGluR antagonist. The functional similarities between Na-acamprosate and SIB-1893 support an interaction of Na-acamprosate at mGluR5s. The neuroprotective properties of acamprosate and possibly its ability to reduce craving in alcohol-dependent patients may result from its alterations in glutamatergic transmission through mGluRs.     

Effects of Prenatal Ethanol Exposure on Phospholipase C-β1 and Phospholipase A2 in Hippocampus and Medial Frontal Cortex of Adult Rat Offspring

Previous studies in our laboratory using a rat model of fetal alcohol exposure (FAE) suggest that FAE-induced behavioral deficits are, in part, linked to neurochemical and electrophysiological deficits in long-term potentiation (LTP) in the entorhinal cortical perforant path projection to the hippocampal formation. Several findings suggest that signal-activated phospholipase C (PLC) and phospholipase A₂ (PLA₂) are critical to the induction and maintenance of LTP. Thus, alterations in phospholipid metabolism may play a significant role in the LTP deficits observed in FAE offspring. To test this hypothesis, we measured PLC-β1 and PLA₂ activities in the hippocampus and medial frontal cortex of adult rats prenatally exposed to ethanol. PLC-β1 activities were significantly decreased by 20 to 30% in both the hippocampus and medial frontal cortex of FAE rats, compared with ad libitum and pair-fed controls. Total Ca²⁺-dependent PLA₂ activity was 25% lower in the medial frontal cortex of FAE rats, but did not significantly differ from controls in the hippocampal formation. Approximately 30% of the measured activity in both the medial frontal cortex and hippocampal formation of ad libitum and pair-fed animals was associated with an 85 kDa cytosolic PLA₂ form. Cytosolic PLA₂ activities were significantly reduced in both the medial frontal cortex and hippocampal formation of FAE rats, compared with controls. These changes in Ca²⁺-dependent PLA₂ and PLC-β1 activities, coupled with reports of FAE-induced deficits in protein kinase C activity, indicate that prenatal exposure to moderate quantities of ethanol causes profound and long-lasting deficits in the cellular signaling mechanisms associated with activity-dependent synaptic plasticity and memory formation.     

Prenatal ethanol exposure modifies [3H]MK-801 binding to NMDA receptors: spermidine and ifenprodil

BACKGROUND: It has been suggested that abnormalities seen in fetal alcohol syndrome are linked with NMDA receptor malfunction. Our laboratory has previously shown that prenatal ethanol treatment decreases [3H]MK-801 binding density at postnatal day 21, when NMDA receptor subunit protein levels were unaltered. Thus, the focus of the present study was to examine whether prenatal ethanol modifies native NMDA receptor levels. METHODS: Cerebral cortices were taken from offspring born to three treatment groups of pregnant Sprague Dawley(R) rats: an ethanol group given an ethanol liquid diet during the gestational period, a pair-fed control group that received a liquid diet without ethanol, and an ad libitum group fed rat chow and tap water. Western blot studies were carried out at postnatal days 1, 7, 14, and 21 to examine total protein expression of NR1 and NR1b splice variants. NR2 subunit levels were examined by [3H]MK-801 binding studies using spermidine, an endogenous polyamine, and ifenprodil, a selective NR2B antagonist. RESULTS: [3H]MK-801 binding density was significantly reduced in prenatal ethanol-treated groups compared with ad libitum and pair-fed control groups. Spermidine increased [3H]MK-801 binding, although potentiation by spermidine was not significantly different among all three experimental groups. Furthermore, no significant differences in total protein expression of NR1 or NR1b splice variants were observed in cortical membrane homogenates at postnatal days 1 through 21. [3H]MK-801 binding in the presence of ifenprodil showed that prenatal ethanol treatment significantly decreased low-affinity ifenprodil binding. High-affinity ifenprodil binding was reduced in both pair-fed and ethanol-treated groups. CONCLUSIONS: These results suggest that prenatal ethanol treatment reduces [3H]MK-801 binding and that this reduction may be due to a decrease in NR2A subunits.     

Differences in Ethanol Sensitivity of Brain NMDA Receptors of Long-Sleep and Short-Sleep Mice

Long-Sleep (LS) and Short-Sleep (SS) mice, selectively bred mice that differ in the duration of anesthesia produced by an acute dose of ethanol, were used to determine the possible association of differing ethanol sensitivity of brain NMDA receptors with differing sensitivity to the anesthetic effects of ethanol in vivo. NMDA receptor-mediated responses were determined by measurement of l -glutamate-stimulated increases in free intracellular calcium concentration (Ca_l) using the fluorescent indicator for Ca₁, Indo 1, in microsacs (a cell-free brain membrane vesicle preparation) isolated from hippocampi or cerebral cortices of the two mouse lines. In the absence of added drugs, NMDA responses did not differ between the two lines in hippocampal or cerebrocortical microsacs. However, a high concentration of ethanol (200 m_m) inhibited NMDA responses in hippocampal microsacs from LS mice. In contrast, a moderate concentration of ethanol (50 m_m) stimulated NMDA responses in hippocampal microsacs isolated from SS mice. In cerebrocortical microsacs, ethanol inhibited NMDA responses in the two lines to an equivalent degree. MK-801, a noncompetitive blocker of NMDA receptors, blocked NMDA responses at lower concentrations in hippocampal microsacs from LS mice than in SS mice, but produced a similar degree of inhibition of NMDA responses in cerebrocortical microsacs from the two lines. A high concentration of ethanol (200 mm ) increased resting Ca₁ in hippocampal microsacs from LS mice but not in hippocampal microsacs from SS mice, and increased resting Ca_l in cerebrocortical microsacs isolated from both lines of mice equally. The small change in resting Ca, produced by MK-801 in cerebrocortical microsacs did not differ between the two lines. These results show that hippocampal NMDA receptors of LS and SS mice differ in their sensitivity to ethanol, possibly because of differences in allosteric modulation at the MK-801 site or some other site that interacts with the MK-801 site of the NMDA receptor.     

The neurotoxicity induced by ethanol withdrawal in mature organotypic hippocampal slices might involve cross-talk between metabotropic glutamate type 5 receptors and N-methyl-D-aspartate receptors

BACKGROUND: We recently reported that the sodium salt of acamprosate (Na-acamprosate) demonstrates the characteristics of an antagonist at metabotropic glutamate type 5 receptors (mGluR5s) rather than at N-methyl-d-aspartate receptors (NMDARs). Because mGluR5s are able to enhance the function of NMDARs, this interplay may be involved in the dysregulation of glutamatergic transmission during ethanol withdrawal. The following studies use organotypic hippocampal slice cultures at a mature age to investigate the potential for this interplay in the neurotoxicity associated with withdrawal from long-term ethanol exposure. METHODS: At 25 days in vitro, organotypic hippocampal slice cultures prepared from male and female 8-day-old rats were exposed to an initial concentration of 100 mM ethanol for 10 days before undergoing a 24-hr period of withdrawal. The effects of Na-acamprosate; 2-methyl-6-(2-phenylethenyl)pyridine (SIB-1893), a noncompetitive antagonist at mGluR5s; 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester, a noncompetitive antagonist at mGluR1s; dizocilpine (MK-801), a noncompetitive NMDAR antagonist; and staurosporine on the neurotoxicity induced by ethanol withdrawal were assessed by determining differences in propidium iodide uptake. Polypeptide levels of mGluR5s and the NR1 and NR2B subunits of NMDARs were also determined via Western blot analyses after 10 days of ethanol exposure. RESULTS: Significant neurotoxicity was always evident in the CA1 hippocampal region after a 24-hr withdrawal period. This spontaneous neurotoxicity resulted from intrinsic changes induced by the long-term presence of ethanol. Na-acamprosate (200-1000 microM), SIB-1893 (200-500 microM), MK-801 (20 microM), and staurosporine (200 nM) were all neuroprotective. The polypeptide levels of mGluR5s and NR1 and NR2B subunits of NMDARs were all increased after ethanol exposure; however, the increase in mGluR5s did not achieve statistical significance. CONCLUSIONS: From this model of long-term ethanol exposure and withdrawal, the functional interplay between mGluR5s and NMDARs might represent a novel target for the prevention of neurotoxicity associated with ethanol withdrawal.     

Chronic Prenatal Ethanol Exposure Alters the Normal Ontogeny of Choline Acetyltransferase Activity in the Rat Septohippocampal System

In animal models of fetal alcohol syndrome (FAS), the hippocampus has been shown to be especially sensitive to the effects of prenatal ethanol exposure, exhibiting neuronal loss and alterations in neuritic process elaboration. We have characterized the influence of chronic prenatal ethanol treatment (CPET) on the postnatal expression of choline acetyltransferase (ChAT) In the hippocampus and the septal area that contains neurons that provide the primary cholinergic innervation to the hippocampus. On gestation days 1–22, pregnant rats were either fed an ethanol-containing liquid diet, pair-fed a calorically equivalent sucrose-containing diet, or given rat chow ad libitum. In Chow control animals, the ontogenetic progression of ChAT activity in the septal area and hippocampus was characterized by a significant period of upregulation during the 2nd and 3rd postnatal weeks, exhibiting an approximate 5-fold increase (septal area) and 7-fold increase (hippocampus) by postnatal day 21 (P21). At P14, ethanol exposure reduced septal and hippocampal ChAT activity levels, compared with those of pair-fed offspring. ChAT activity reached control levels by P21 in ethanol-exposed pups, suggesting that the earlier decline in activity may reflect a delay in the ontogenetic upregulation. In addition, there was a trend toward increased septal and hippocampal ChAT activities at P1 and P7 in both liquid diet groups. This liquid diet-stimulated increase may mask the effects of ethanol on early postnatal ChAT expression in the septohippocampal system. The results suggest that prenatal ethanol exposure may influence factors that regulate the developmental expression of ChAT in the septohippocampal system. In addition, we found a close relationship between the effects of CPET on litter birthweight and subsequent postnatal body and brain weight measures, but not between litter birthweight and ChAT activity from individual animals. This may imply that the influence of ethanol exposure on ChAT activity is independent of its effects on postnatal brain or body growth. Such an observation of neurochemical dysfunction in the absence of morphological indices frequently used to diagnose alcohol-related deficits could have potential clinical significance, both in the diagnostic and therapeutic realms.     

Alterations in Brain Polyribosomal RNA Translation and Lymphocyte Proliferation in Prenatal Ethanol-Exposed Rats

The long-term effects of prenatal ethanol exposure on the properties of brain polysomes and the proliferative responses of lymphocytes to mitogenic stimulation in adult offspring were assessed. Female Sprague-Dawley rats either ingested the control or 6.6% ethanol-containing Lieber-DeCarli liquid diet during the 3rd trimester of pregnancy. Controls were age-matched and pair-fed. At 42 to 72 days of age, ethanol effects were evaluated on the (1) polysomal properties in the cerebral hemispheres, cerebellum, and hippocampal regions of the brain after translation in a messenger RNA (mRNA)-dependent rabbit reticulocyte lysate system and (2) immunologic functions of lymphocytes cultured from spleen cells by measuring their responses to mitogenic stimulation. Results showed long-term adverse effects of in utero ethanol exposure on the polysomal RNA translation in each of the three brain regions tested with free polysomal mRNAs affected more than the bound polysomal mRNAs. Of these, the hippocampal region appeared to sustain the most injurious effects. In addition, a suppression of the mitogen-induced lymphocyte proliferative responses were present under these conditions. The degree of suppression varied with the specific mitogen used. Data suggest that the ethanol effects on the CNS and lymphocyte proliferation are most possibly irreversible, and in the case of the CNS, a post-translational modification by ethanol is indicated. The reduced lymphocyte responses are suggestive of a possible interference by ethanol of the synthesis of interleukin-2 (IL-2) and/or a reduced binding of IL-2 with its receptor (IL-2 receptors).     

Stability of [3H]MK-801 Binding Sites Following Chronic Ethanol Consumption

Previous work has demonstrated that short periods (1–2 weeks) of exposure to ethanol produce an upregulation of the N-methyl-D-aspartate (NMDA) receptor complex in hippocampus; an alteration that appears to be associated with the development of physical dependence, because a return to control levels occurs over a 24- to 48-hr abstinence period. Prolonged periods of chronic ethanol treatment (CET; 4–8 months of treatment) have been shown to produce severe and permanent alterations in the morphological and functional characteristics of hippocampal pyramidal neurons. Several lines of research have demonstrated that the NMDA receptor complex is involved in excitotoxic cell loss during certain pathological states. On the basis of this evidence, we hypothesized that prolonged ethanol exposure would be accompanied by an enduring increase in NMDA receptors and that NMDA receptor binding in cells surviving CET would be altered. To test this hypothesis, we measured the binding characteristics of the NMDA receptor complex in a variety of brain structures following CET. Animals were fed a nutritionally complete, ethanol-containing diet for 28 weeks and then allowed a 48-hr abstinence period. A control group was fed the same diet, except sucrose was isocalorically substituted for ethanol. We first examined the effect of CET on the binding properties of a noncompetitive antagonist to the NMDA receptor channel, [³H]diclozipene ([³H]MK-801). Next, as an indirect examination of NMDA receptor function, we measured the ability of glutamate to stimulate channel opening and thus [³H]MK-801 binding. In all brain structures examined, neither the K_d nor the B_max of [³H]MK-801 binding to the NMDA receptor was altered following CET. In addition, no effect of treatment was seen on the ability of glutamate to stimulate [³H]MK-801 binding.     

Prenatal Ethanol Exposure Affects Temperature Responses of Adult Rats to Pentobarbital and Diazepam Alone and in Combination with Ethanol

Long-term effects of prenatal alcohol exposure on body temperature responses to pentobarbital and diazepam and to either drug in combination with ethanol were studied in adult rats who were the offspring of dams fed a 5.0% w/v ethanol-containing liquid diet during the last 2 weeks of gestation. Adult offspring of pair-fed and chow-fed dams served as nutritional and normal controls, respectively. Pentobarbital (6.25-25.0 mg/kg) and diazepam (2.5-10.0 mg/kg) produced significantly greater dose-related hypothermic responses in females than males. Following either pentobarbital or diazepam administration female prenatally ethanol-exposed (E) rats responded with a greater fall in body temperature than the controls. Significantly greater hypothermia occurred in both male and female E rats than in controls when ethanol (1.5 g/kg) was administered together with pentobarbital or diazepam. However, the drug combinations did not produce additive effects on body temperature in any prenatal treatment group. Pentobarbital produced acute cross-tolerance to ethanol while diazepam potentiated ethanol's effect. These studies confirm and extend our previous findings of enhanced hypothermic responses to ethanol in adult rats exposed to ethanol in utero and indicate that maternal alcohol consumption produces long-term effects on the central thermoregulatory systems of offspring.     

Influence of Ethanol Consumption on Immune Competence of Adult Animals Exposed to Ethanol In Utero

Ethanol consumption results in significant changes in the immune system of experimental animals and humans. Previous work by ourselves and others has established that in utero exposure to ethanol results in alterations in the immune system of the offspring that persist into adult life. The present study was designed to determine if prenatal exposure to ethanol results in increased vulnerability to the immunosuppressive effects of ethanol consumption in adulthood. Male and female Sprague-Dawley offspring were selected in adulthood from prenatal ethanol (E), pair-fed (PF), and ad libitum-fed control (C) groups, and given either an ethanol-containing liquid diet or were pair-fed an isocaloric liquid diet without ethanol for 30 days. At the end of the 30-day feeding period, lymphocyte responses to the mitogens concanavalin A (Con A) and lipopolysaccharide, and to interleukin-2 (IL-2) were tested using in vitro assays. The results of this study support and extend previous data demonstrating long-term adverse effects of prenatal ethanol exposure on T-cell responses to mitogens, and provide further evidence that deficits seem to be more robust in male than in female offspring. Prenatal E males showed reduced T-lymphocyte proliferation to Con A and T-lymphoblast proliferation to IL-2, compared with their prenatal PF and C counterparts, regardless of whether they were exposed to the ethanol or the control diet in adulthood. In addition, T-lymphoblast proliferation to IL-2 was suppressed in prenatal E, compared with prenatal C, females exposed to control diet in adulthood. This is the first report of a deficit in T-cell aspects of immunity in E females, although it appears that this deficit may have been partially mediated by nutritional effects. A second major finding in this study is that consumption of ethanol diet in adulthood in itself had significant immunosuppressive effects on T-cell responses in both males and females. However, contrary to our expectation, previous exposure to ethanol in utero did not exacerbate the changes in immune responsiveness that were observed after adult ethanol consumption.     

Effects of Prenatal and Early Postnatal Ethanol Exposure on [3H]MK-801 Binding in Rat Cortex and Hippocampus

The effects of prenatal and/or early postnatal exposure to ethanol at high concentrations on N-methyl-D-aspartate (NMDA) receptor number and functioning in the weanling rat were examined. The bingelike exposure protocol was used in an animal model of acute ethanol effects at two critical periods of development. [³H]MK-801 binding parameters for the internal channel phencyclidine site were assessed in the presence of 10 μM glutamate and 10 μ M glycine activation. Four treatment groups were included: (1) animals exposed to ethanol both prenatal and postnatal; (2) animals exposed only prenatal; (3) animals exposed early postnatal only; and (4) control animals with no exposure to ethanol. The results of the [³H]MK-801 binding experiments showed that both prenatal and postnatal exposure to ethanol resulted in a significant decrease in the density of NMDA receptors. In addition, data indicated an apparent increase in the percentage of high-affinity state (open channel state) relative to low-affinity state (close channel state) receptors in the ethanoltreated groups. These results show that both prenatal and postnatal ethanol exposure decrease NMDA receptor density in the cortex and hippocampus. The findings are consistent with previous observations by our laboratory and others that NMDA-mediated calcium influx is reduced in these regions, as well as in whole brain by prenatal ethanol exposure. It is suggested that after ethanol exposure, the remaining functional NMDA receptors might have altered sensitiviity to coagonist activation with an increased probability of channel opening.     

Ethanol exposure in early adolescence inhibits intrinsic neuronal plasticity via sigma-1 receptor activation in hippocampal CA1 neurons

Background: We demonstrated previously that rats exposed to chronic intermittent ethanol (CIE) vapors in early adolescence show increased magnitudes of long‐term potentiation (LTP) of excitatory transmission when recorded at dendritic synapses in hippocampus. Large amplitude LTP following CIE exposure is mediated by sigma‐1 receptors; however, not yet addressed is the role of sigma‐1 receptors in modulating the intrinsic properties of neurons to alter their action potential firing during LTP. Methods: Activity‐induced plasticity of spike firing was investigated using rat hippocampal slice recordings to measure changes in both field excitatory postsynaptic potentials (fEPSPs) and population spikes (pop. spikes) concomitantly at dendritic inputs and soma of CA1 pyramidal neurons, respectively. Results: We observed unique modifications in plasticity of action potential firing in hippocampal slices from CIE exposed adolescent rats, where the induction of large amplitude LTP by 100 Hz stimulations was accompanied by reduced CA1 neuronal excitability––reflected as decreased pop. spike efficacy and impaired activity‐induced fEPSP‐to‐spike (E‐S) potentiation. In contrast, LTP induction in ethanol‐naïve control slices resulted in increased spike efficacy and robust E‐S potentiation. E‐S potentiation impairments emerged at 24 hours after CIE treatment cessation, but not before the alcohol withdrawal period, and were restored with bath‐application of the sigma‐1 receptor selective antagonist BD1047, but not the NMDA receptor antagonist d ‐AP5. Further evidence revealed a significantly shortened somatic fEPSP time course in adolescent CIE‐withdrawn hippocampal slices during LTP; however, paired‐pulse data show no apparent correspondence between E‐S dissociation and altered recurrent feedback inhibition. Conclusions: Results here suggest that acute withdrawal from adolescent CIE exposure triggers sigma‐1 receptors that act to depress the efficacy of excitatory inputs in triggering action potentials during LTP. Such withdrawal‐induced depression of E‐S plasticity in hippocampus probably entails sigma‐1 receptor modulation of 1 or several voltage‐gated ion channels controlling the neuronal input–output dynamics.     

Polyamines contribute to ethanol withdrawal-induced neurotoxicity in rat hippocampal slice cultures through interactions with the NMDA receptor

Background: Several reports demonstrate that withdrawal from long‐term ethanol exposure is associated with significant central nervous system neurotoxicity, produced at least in part by increased activity of N‐methyl‐d‐aspartate receptors (NMDARs). Recent evidence suggests that elevations in the synthesis and release of the polyamines spermidine and spermine, which are known modulators of NMDARs, contribute to the increased activity of the receptor during ethanol withdrawal. Therefore, the goal of this investigation was to examine what role, if any, spermidine and spermine have in the generation of ethanol withdrawal‐induced neurotoxicity. Methods: Neurotoxicity (measured as fluorescence of the cell death indicator propidium iodide, PI), glutamate release (measured by high‐performance liquid chromatography analysis), and polyamine concentrations (by high‐performance liquid chromatography) were measured in rat hippocampal slice cultures undergoing withdrawal from chronic (10 day) ethanol exposure (100 mM). In addition, the effects of the polyamine synthesis inhibitor di‐fluoro‐methyl‐ornithine (DFMO, 0.1–100 nM) and NMDAR polyamine‐site antagonists ifenprodil, arcaine, and agmatine (1 nM‐100 μM) on ethanol withdrawal‐ and NMDA‐induced neurotoxicity were measured. Results: Ethanol withdrawal significantly increased glutamate release (peaking at 18 hr with a 53% increase), increased concentrations of putrescine and spermidine (136% and 139% increases, respectively, at 18 hr), and produced significant cytotoxicity in the CA1 hippocampal region (56% increase in PI staining relative to controls) of the cultures. The cell death produced by ethanol withdrawal was significantly inhibited by ifenprodil (IC₅₀= 14.9 nM), arcaine (IC₅₀= 37.9 nM), agmatine (IC₅₀= 41.5 nM), and DFMO (IC₅₀= 0.6 nM). NMDA (5 μM) significantly increased PI staining in the CA1 region of the hippocampal cultures (365% relative to controls), but ifenprodil, arcaine, agmatine, and DFMO all failed to significantly affect this type of toxicity. Conclusions: These data implicate a role for polyamines in ethanol withdrawal‐induced neurotoxicity and suggest that inhibiting the actions of polyamines on NMDARs may be neuroprotective under these conditions.