Ethanol exposure during fetal development is a leading cause of learning disabilities. Studies suggest that it alters learning and memory by permanently damaging the hippocampus. It is generally assumed that this is mediated, in part, via alterations in glutamatergic transmission. Although NMDA receptors are presumed to be the most sensitive targets of ethanol in immature neurons, this issue has not been explored in the developing hippocampus. We performed whole-cell patch-clamp recordings in hippocampal slices from neonatal rats. Unexpectedly, we found that acute ethanol (10 -50 mM) exposure depresses inward currents elicited by local application of exogenous AMPA, but not NMDA, in CA3 pyramidal neurons. These findings revealed a direct effect of ethanol on postsynaptic AMPA receptors. Ethanol significantly decreased the amplitude of both AMPA and NMDA receptor-mediated EPSCs evoked by electrical stimulation. This effect was associated with an increase in the paired-pulse ratio and a decrease in the frequency of miniature EPSCs driven by depolarization of axonal terminals. These findings demonstrate that ethanol also acts at the presynaptic level. -Conotoxin-GVIA occluded the effect of ethanol on NMDA EPSCs, indicating that ethanol decreases glutamate release via inhibition of N-type voltage-gated Ca 2ϩ channels. In more mature rats, ethanol did not affect the probability of glutamate release or postsynaptic AMPA receptor-mediated currents, but it did inhibit NMDA-mediated currents. We conclude that the mechanism by which ethanol inhibits glutamatergic transmission is age dependent and challenge the view that postsynaptic NMDA receptors are the primary targets of ethanol early in development.

Ethanol consumption during development affects the maturation of hippocampal circuits by mechanisms that are not fully understood. Ethanol acts as a depressant in the mature CNS and it has been assumed that this also applies to immature neurons. We investigated whether ethanol targets the neuronal network activity that is involved in the refinement of developing hippocampal synapses. This activity appears during the growth spurt period in the form of giant depolarizing potentials (GDPs). GDPs are generated by the excitatory actions of GABA and glutamate via a positive feedback circuit involving pyramidal neurons and interneurons. We found that ethanol potently increases GDP frequency in the CA3 hippocampal region of slices from neonatal rats. It also increased the frequency of GDP-driven Ca 2+ transients in pyramidal neurons and increased the frequency of GABA A receptormediated spontaneous postsynaptic currents in CA3 pyramidal cells and interneurons. The ethanol-induced potentiation of GABAergic activity is probably the result of increased quantal GABA release at interneuronal synapses but not enhanced neuronal excitability. These findings demonstrate that ethanol is a potent stimulant of developing neuronal circuits, which might contribute to the abnormal hippocampal development associated with fetal alcohol syndrome and alcohol-related neurodevelopmental disorders.

Cerebellar Purkinje neurons (PNs) receive inhibitory GABAergic input from stellate and basket cells, which are located in the outer and inner portions of the molecular layer, respectively. Ethanol (EtOH) was recently shown to increase GABAergic transmission at PNs via a mechanism that involves enhanced calcium release from presynaptic internal stores (J Pharmacol Exp Ther 323: 356 -364, 2007). Here, we further characterized the effect of EtOH on GABA release and assessed its impact on PN excitability. Using whole-cell patch-clamp electrophysiological techniques in cerebellar vermis parasagittal slices, we found that EtOH acutely increases the frequency but not the amplitude or half-width of miniature and spontaneous inhibitory postsynaptic currents (IPSCs). EtOH significantly increased the amplitude and decreased the paired pulse ratio of IPSCs evoked by stimulation in the outer but not inner molecular layer.In current clamp, EtOH decreased both the amplitude of excitatory postsynaptic potentials evoked in PNs by granule cell axon stimulation and the number of action potentials triggered by these events; these effects depended on GABA A receptor activation because they were not observed in presence of bicuculline. Loose-patch cell-attached PN recordings revealed that neither the spontaneous action potential firing frequency nor the coefficient of variation of the interspike interval was altered by acute EtOH exposure. These findings suggest that EtOH differentially affects GABAergic transmission at stellate cell-and basket cell-to-PN synapses and that it modulates PN firing triggered by granule cell axonal input. These effects could be in part responsible for the cerebellar impairments associated with acute EtOH intoxication.The cerebellum is an important target of the acute and chronic actions of ethanol (EtOH) both during development and at maturity. EtOH-induced alterations of balance, speech, motor coordination, and certain cognitive functions are thought to be mediated, in part, by impairment of cerebellar function. In the cerebellar cortex, Purkinje neurons (PNs) have been shown to be EtOH-sensitive. PNs are GABAergic neurons that project to deep cerebellar nuclei and constitute the sole output of the cerebellar cortex. PNs receive two types of excitatory inputs: the mossy-parallel fiber and the climbing fiber systems. Inhibitory inputs to PNs are provided, in part, by two types of interneurons that are located in the molecular layer: basket and stellate cells. Basket cells preferentially innervate axonal-initial segments, whereas stellate cells preferentially innervate dendrites and spines. GABAergic input from these interneurons, as well as PN collaterals, controls the excitability of PNs and is necessary for their normal functioning (Hä usser and Clark, 1997). Synaptic connections between PNs were recently shown to be relatively weak in the parasagittal cerebellar slice preparation (Orduz and Llano, 2007).The effects of EtOH on GABAergic transmission at PNs have been the focus of several studies...

Background: N-methyl-D-aspartate receptors (NMDARs) are glutamate-activated, heterotetrameric ligand-gated ion channels critically important in virtually all aspects of glutamatergic signaling. Ethanol (EtOH) inhibition of NMDARs is thought to mediate specific actions of EtOH during acute and chronic exposure. Studies from our laboratory, and others, identified EtOH-sensitive sites within specific transmembrane (TM) domains involved in channel gating as well as those in subdomains of extracellular and intracellular regions of GluN1 and GluN2 subunits that affect channel function. In this study, we characterize for the first time the physiological and behavioral effects of EtOH on knockin mice expressing a GluN2A subunit that shows reduced sensitivity to EtOH.Methods: A battery of tests evaluating locomotion, anxiety, sedation, motor coordination, and voluntary alcohol intake were performed in wild-type mice and those expressing the GluN2A A825W knock-in mutation. Whole-cell patch-clamp electrophysiological recordings were used to confirm reduced EtOH sensitivity of NMDAR-mediated currents in 2 separate brain regions (mPFC and the cerebellum) where the GluN2A subunit is known to contribute to NMDAR-mediated responses.Results: Male and female mice homozygous for the GluN2A(A825W) knock-in mutation showed reduced EtOH inhibition of NMDAR-mediated synaptic currents in mPFC and cerebellar neurons as compared to their wild-type counterparts. GluN2A(A825W) male but not female mice were less sensitive to the sedative and motor-incoordinating effects of EtOH and showed a rightward shift in locomotor-stimulating effects of EtOH. There was no effect of the mutation on EtOH-induced anxiolysis or voluntary EtOH consumption in either male or female mice.Conclusions: These findings show that expression of EtOH-resistant GluN2A NMDARs results in selective and sex-specific changes in the behavioral sensitivity to EtOH.

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PurposeFetal alcohol syndrome is a devastating condition that causes cell death through many mechanisms. Neurotransmitter receptors, such as glutamate and gamma-aminobutyric acid (GABA) receptors, are altered by alcohol consumption, which may impact brain development. In this work we examined the effects of prenatal alcohol consumption on the fetal guinea pig brain, specifically the cortex and hippocampus. The goal of this work was to evaluate protein expression of ionotropic glutamate receptor subunits NR2A, NR2B, NR1, GLUR1, GLUR2/3, and GLUR6/7 in fetal guinea pigs that were exposed to ethanol in utero.MethodsTimed pregnant Dunkin-Hartley-strain guinea pigs received daily oral administration of one of the following regimens between gestational days (GD) 2 and 65: (1) 4 g ethanol/kg maternal body weight/day (ethanol group), (2) isocaloric sucrose and pair-feeding (sucrose group); or (3) isovolumetric water (water group) with ad libitum access to food and water for all 3 groups. On GD 63-65, dams were euthanized by decapitation under halothane anesthesia and fetuses were removed by cesarean section. The maternal blood ethanol concentration produced by the ethanol regimen was 71 ± 12 mM. Cerebral cortices and hippocampus were analyzed for receptor subunit expression using Western immunoblotting.ResultsIn the cerebral cortex, there was no significant difference in glutamate receptor subunit expression when comparing ethanol to sucrose-exposed fetuses. In the hippocampus, there was a significant decrease of approximately 30% in NR2B expression in the ethanol group when compared to the sucrose-fed dams.ConclusionsHippocampal receptor NR2B expression was decreased in fetuses exposed in utero to ethanol with no significant change in cerebral cortex receptor expression. In contrast, previous work showed increased GLUR2/3 and decreased NR2B cerebral cortex receptor expression in adult guinea pigs exposed to ethanol in utero. These results suggest that ethanol produces age-dependent and region-specific decreases in the expression of NR2B. This action of ethanol could have an impact on synaptic plasticity and contribute to cognitive abnormalities.

the frequency but not the amplitude of miniature EPSCs recorded in the presence of the voltage-gated Na + channel blocker tetrodotoxin (TTX; 500 nM) and SR 95531 (10 µM) that were completely abolished by CGS 19755 (10 µM) and NBQX (3 µM). This effect also was antagonized by AM251 (1 µM). Estradiol benzoate (EB) administered 24 hr prior to experimentation induced a pronounced rightward shift in the dose-response for WIN 55,212-2 to decrease mEPSC frequency. The cannabinoid-induced decrease in mEPSC frequency, and the corresponding modulatory influence of EB, was observed in ARC neurons subsequently identified as glutamate-, ␣-MSH-, or CART-containing cells via immunohistofluorescence. Collectively, these findings indicate that cannabinoids presynaptically inhibit glutamatergic synaptic input onto ARC neurons, including glutamatergic and POMC neurons, and that estrogen attenuates this effect by markedly reducing agonist potency. Results from this study provide great insights into the mechanisms by which cannabinoids and estrogen interact to regulate hypothalamic control of homeostasis.