Altering the Relative Abundance of GABAA Receptor Subunits Changes GABA- and Ethanol-Responses in Xenopus Oocytes

Background:  Variations in GABRA2 and GABRG3 , genes encoding the α2 and γ3 subunits of the pentameric GABA_A receptor, are associated with the risk of developing alcoholism in adults, conduct disorder at younger ages, and with differences in electroencephalographic power in the β frequency range. The SNPs associated with alcoholism did not alter the coding of these genes, and extensive DNA sequencing of GABRA2 did not find coding changes in the high-risk haplotypes. Therefore, we hypothesize that the associations arise from differences in gene expression.
Methods:  Here we report studies in Xenopus oocytes to examine the functional effects of altering the relative abundance of these 2 receptor subunits on GABA current and response to ethanol, as a model of potential effects of regulatory differences.
Results:  When human α2β2γ3 subunits are co-expressed, increasing the amount of the α2 subunit mRNA increased GABA current; in contrast, increasing the amount of the γ3 subunit decreased GABA currents. Acute ethanol treatment of oocytes injected with a 1:1:1 or 2:2:1 ratio of α2:β2:γ3 subunit mRNAs resulted in significant potentiation of GABA currents, whereas ethanol inhibited GABA currents in cells injected with a 6:2:1 ratio. Overnight treatment with ethanol significantly reduced GABA currents in a manner dependent on the ratio of subunits.
Conclusions:  These studies demonstrate that changes in relative expression of GABA_A receptor subunits alter the response of the resulting channels to GABA and to ethanol.     

Ethanol, Flunitrazepam, and Pentobarbital Modulation of GABAA Receptors Expressed in Mammalian Cells and Xenopus Oocytes

GABA_A receptors composed of human α1β2γ2L, α1β2γ2S, α1 β3γ2S, α6β3γ2S, and αβ3γ3 subunits as well as bovine α1 β1 γ2L and α1β1 subunits were stably expressed in mammalian L(tk^?) cells and transiently expressed in Xenopus oocytes. Effects of muscimol, ethanol, flunitrazepam, and pentobarbital on receptor function were compared for the two expression systems using a ³⁶CI^? flux assay for cells and an electrophysiological assay for oocytes. Muscimol activated all receptors in both expression systems but was more potent for L(tk^?) cells than oocytes; this difference ranged from 2.6–to 26–fold, depending upon subunit composition. The most pronounced differences between receptors and expression systems were found for ethanol. In L(tk^?) cells, low (5–50 mM) concentrations of ethanol potentiated muscimol responses only with receptors containing the γ2L subunit. In oocytes, concentrations of 30–100 mM produced small enhancements for most subunit combinations. Flunitrazepam enhanced muscimol responses for all receptors except α6β3γ2S and α1β1, and this enhancement was similar for both expression systems. Pentobarbital also enhanced muscimol responses for all receptors, and this enhancement was similar for L(tk^?) cells and oocytes, except for α6β3γ2S where the pentobarbital enhancement was much greater in oocytes than cells. The α6β3γ2S receptors were also distinct in that pentobarbital produced direct activation of chloride channels in both expression systems. Thus, the type of expression/assay system markedly affects the actions of ethanol on GABA_A receptors and also influences the actions of muscimol and pentobarbital on this receptor. Differences between these expression systems may reflect posttranslational modifications of receptor subunits.     

Brain Region-Dependent Sensitivity of GABAA Receptor-Mediated Responses to Modulation by Ethanol

Simultaneous extracellular and intracellular electrophysiological recordings were made from the CA1 region of rat hippocampal brain slices during superfusion with ethanol. Ethanol (80 mM) had a biphasic effect on the extracellularly recorded population spike, with an initial increase followed by a significant reduction (38%) in this response, which was maximal 10 to 15 min after the start of ethanol application. Concurrent intracellular recordings in the CA1 showed a small (0.7 mV) hyperpolarization of the resting membrane potential, with no significant change in the input impedance, EPSP, GABAA and GABAB IPSPs, or after hyperpolarization (AHP) following depolarizing current injection. Ethanol reduced the amplitude and duration of depolarizing responses to brief, localized pressure-ejection of N-methyl-D-aspartate (NMDA) onto pyramidal neuron dendrites, but did not affect the GABAA receptor-mediated depolarizing responses to the dendritic application of GABA. In parallel studies, the effect of ethanol on GABA-stimulated 36Cl- flux was measured in microsac preparations from rat hippocampus, cerebellum, and cerebral cortex. Ethanol application caused substantial enhancement of the chloride uptake from cerebellar and cerebral cortical microsacs, but had no effect on 36Cl- influx in hippocampal microsacs. These results suggest that there are important brain region-dependent differences in the sensitivity of the GABAA receptor/chloride channel to modulation by ethanol.     

Modulation of GABAA Receptor Function by Alcohols: Effects of Subunit Composition and Differential Effects of Ethanol

We sought to test the hypotheses that closely related alcohols would have effects on GABA_A receptor function that were not predicted by differences in lipid solubility, and that the subunit structure of the GABA_A receptor would significantly affect the actions of different alcohols. Cloned subunits of human GABA_A receptors were expressed in Xenopus oocytes, and two-electrode voltage-clamp recording was used to quantify the membrane current response to GABA_A in the presence and absence of different alcohols. 1-Butanol and 2-butanol differentially potentiated the response to 20 μM GABA in oocytes expressing the α₁β₂γ_2L and α₂β₂γ_2L, receptor isoforms. In the α₁β₂γ_2L receptor construct, 1-butanol was more potent than 2-butanol to potentiate GABA, receptor function, but 2-butanol had a greater efficacy. In the α₂β₂ receptor construct, 1-butanol and 2-butanol were equipotent, but 2-butanol again had a greater efficacy. In the a2p2 receptor construct, both 1-butanol and 2-butanol produced large potentiations of the current response to 3 μM GABA. The efficacy for butanol potentiation of GABA responses in the absence of a γ_2L subunit was greater, but the potency was greatly reduced. Low concentrations (20 mM) of ethanol potentiated GABA responses in the α₁β₂γ_2L receptor construct. Ethanol potentiation of GABA_A receptor function was completely blocked by the benrodiazepine receptor partial inverse agonist R015-4513 at a concentration (0.5 μM) that did not alter the control GABA response. In contrast, R015-4513 did not block potentiation of GABA_A receptor activity induced by npropanol, 1-butanol, 2-butanol, 1-heptanol, or propofol (2,0-diisopropylphenol). These results suggest that alcohols have specific interactions with GABA_A receptors, and that ethanol may have unique effects not shared by other longer chain alcohols.     

Chronic Ethanol Exposure Increases 3H-GABA Release in Rat Hippocampus by Presynaptic Muscarinic Receptor Modulation

BACKGROUND: Chronic ethanol treatment (CET) for 28 weeks significantly increases electrically-stimulated 3H-GABA release from hippocampal slices. This increase in GABA release may be one of the mechanisms by which CET decreases the magnitude of long-term potentiation (LTP) in the hippocampus. The present study examined whether CET increases GABA release via an alteration in heterologous presynaptic cholinergic regulation. METHODS: Animals were treated with ethanol or sucrose diet for 28 weeks followed by either no withdrawal or a 48-hr withdrawal period. The electrically-stimulated 3H-GABA release from preloaded superfused hippocampal slices of naive and CET rats was measured. RESULTS: Carbachol increased 3H-GABA release in a concentration-dependent manner, and atropine modulated 3H-GABA release in a biphasic concentration-dependent manner. Atropine (10 microM) significantly blocked the effects of carbachol. Oxotremorine, a selective muscarinic receptor agonist, also increased 3H-GABA release. Mecamylamine, a selective nicotinic antagonist, did not modulate 3H-GABA release and did not block the effects of carbachol. The effects of these agents were also tested in rats 0 or 48 hrs after withdrawal from CET. The biphasic effects of atropine were decreased, whereas the facilitating effects of carbachol were significantly increased. There were no changes in the effects of these agents on 3H-acetylcholine release from hippocampal slices of CET rats compared to sucrose-treated rats. CONCLUSION: These results suggest that presynaptic muscarinic receptors facilitate GABA release, whereas nicotinic receptors do not play a significant role in modulating GABA release in hippocampus. CET selectively alters presynaptic muscarinic regulation of GABA release in hippocampus and may help us to further understand the mechanism underlying the disruption of LTP by CET.     

Ethanol Tolerance and Withdrawal Responses in GABAA Receptor Alpha 6 Subunit Null Allele Mice and in Inbred C57BL/6J and Strain 129/SvJ Mice

We have been using a genetic strategy to define the contribution of specific candidate genes, such as those encoding subunits of the γ-aminobutyric acid type A receptor, to various ethanol sensitive responses. We have used the gene knockout approach in mouse embryonic stem cells to create mice in which the gene encoding the α6 subunit of the γ-aminobutyric acid type A receptor is rendered nonfunctional. In the present report, we provide a detailed characterization of several behavioral responses to ethanol in these null allele mice. In a separate series of experiments, behavioral response to ethanol was compared between two inbred strains of mice that are commonly used as background stock in knockout experiments, namely C57BL/6J and Strain 129/SvJ. Wild type (α6^+/+) and homozygous null allele (α6^?/?) mice did not differ to the ataxic effects of ethanol on acute functional tolerance (95.8 ± 8.7 vs. 98.8 ± 5.7 mg/dl ± SEM, respectively). Withdrawal hyperexcitability was assessed following chronic exposure to ethanol vapor (EtOH) or air (CONT) in inhalation chambers in a multiple withdrawal treatment paradigm. At the end of the last treatment cycle, mice were scored for handling induced convulsions (HIC). After adjusting for differences in blood ethanol concentration between genotypes at the end of the final treatment cycle, we observed a greater area under the 24-hr HIC curves in mice treated with ethanol (p < 0.0001) but did not detect an effect of genotype (α6^+/+/CONT 3.1 ± 2.0; α6^?/?/CONT 5.5 ± 2.5; α6^+/+/EtOH 30.1 ± 6.2; α6^?/?/EtOH 33.0 ± 5.8 mean units ± SEM). We also examined these mice for differences in protracted tolerance; at ～26 hr into the final withdrawal cycle, each mouse was injected with ethanol (3.5 mg/g body weight) and sleep time was measured. We detected a significant effect of treatment (p <0.001) with ethanol-treated mice demonstrating signs of tolerance as reflected by a reduction in duration of sleep time. However, effect of genotype was not significant (α6^+/+/CONT 57.4 ± 7.6; α6-/-/CONT 59.0 ± 7.6; 6^+/+/EtOH 34.8 ± 7.4; α6^?/?/EtOH 30.8 ± 5.6 min ± SEM). From these data we conclude that the α6 subunit of the GABA_A-R exerts little if any influence on acute functional tolerance, withdrawal hyperexcitability, or protracted tolerance. Strain 129/SvJ and C57BL/6J mice were also compared for acute functional tolerance and were found not to differ (96.3 ± 4.4 vs. 94.8 ±11.3 mg/dl ± SEM, respectively). Withdrawal hyperexcitability was assessed by comparing the area under the 24 hr HIC curves. Strain 129/SvJ mice displayed a much greater basal HIC response compared to C57BL/6J mice (19.8 ± 4.3 vs. 0.2 ± 0.2 mean units ± SEM, respectively); after adjusting for differences in blood ethanol concentration between strains at the end of the final ethanol treatment cycle, the HIC response was markedly enhanced by ethanol treatment in Strain 129/SvJ mice but not in C57BL/6J mice (50.4 ± 3.1 vs. 9.5 ± 5.4 mean units ± SEM, respectively). The effects of treatment (p <0.0001), strain (p <0.0001), and the interaction of strain with treatment (p < 0.01) were significant. Since many gene knockout mice are maintained on a mixed genetic background of Strain 129/SvJ and C57BL/6J, we conclude that significant differences in tests of withdrawal hyperexcitability may be confounded by the influence of genes that cosegregate with the gene targeted allele.     

Chronic Ethanol Intoxication Induces Differential Effects on GABAA and NMDA Receptor Function in the Rat Brain

The effect of long-term treatment with ethanol was investigated on the function of γ-aminobutyric acid A (GABA_A) and N-methyl-d-as-partic acid (NMDA) receptors. Rats were rendered ethanol-dependent by repeated forced administration of a 20% ethanol solution (12 to 18 g/kg/day po) for 6 days and tested while still intoxicated or at different time intervals after withdrawal. t-[³⁶S]Butylbicyclophosphorothionate (³⁵S-TBPS) binding was increased by 30% in cortical homogenates of rats killed 1 to 3 hr after last ethanol administration, when compared with saline-treated animals. However, GABA-stimulated ³⁶Cl-uptake and its enhancement by flunitrazepam was decreased in the ethanol-treated animals. ³⁵S-TBPS binding and ³⁶Cl-influx measured 9 to 24 hr following the last ethanol injection, when withdrawal signs were present, were unmodified with respect to saline-treated rats. Moreover, the effects of both isoniazid and FG 7142 on ³⁶S-TBPS binding were unchanged in ethanol-dependent rats tested at 1 to 3 and 9 to 24 hr, compared with controls. In contrast, ethanol-withdrawn rats tested at 9 to 24 hr showed a dramatic enhancement in their sensitivity to the convulsant action of isoniazid (50 to 250 mg/kg, sc). The same animals were also more susceptible to the convulsant action of NMDA (0.5 to 5 μg/5 μl/rat intracerebroventricularly) and kainic acid (12 mg/kg, ip), and this effect was paralleled by an enhancement (+25%) in the density of ³H-MK 801 recognition sites in the hippocampus. The increased pharmacological response to both isoniazid and excitatory amino acids was no longer detectable as early as 3 to 6 days of ethanol withdrawal, when most of the withdrawal signs disappeared. Moreover, 6 days after withdrawal we observed a significant reduction of ³H-MK 801 binding in the hippocampl of ethanol-dependent rats compared with controls. These results indicate that, in contrast to acute administration, chronic ethanol intoxication may lead to a reduction of GABA_A receptor function, an effect no longer detectable during withdrawal. On the contrary, we found a good correlation between development of withdrawal symptoms and the increase in ³H-MK 801 binding sites. The latter finding strongly suggests that a functional activation of glutamatergic synapses, rather than a decrease in GABA_A receptor function, is a crucial event during ethanol withdrawal.     

Human GABA, Receptor αl and α3 Subunits Genes and Alcoholism

γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain. GABA effects are largely mediated by binding to the postsynaptic GABA_A receptor, causing the opening of an integral chloride-ion channel. The GABA_A antagonists picrotoxin and bicuculline reduce some ethanol-induced behaviors, such as motor impairment, sedation, and hypnosis. The role of this receptor in alcoholism is further supported by effective alleviation of alcohol withdrawal symptoms by GABA_A agonists. To determine the role of the GABA, receptor (GABR) genes in the development of alcoholism, we have used α₁ and α ₃ simple sequence repeat polymorphisms in a sample of unrelated alcoholics, alcoholic probands with both parents, and psychiatrically normal controls. For the GABRα₁ gene, the differences between allele frequencies, when all alleles were compared together, were not significant between total alcoholics, subtypes of alcoholics, and normal controls. However, for GABRα₃, the differences between total alcoholics and normal controls were significant when all alleles were compared together. The differences between subtypes of alcoholics and normal controls were not significant. The results of haplotype relative risk analysis for both genes, GABRα₁ and GABRα₃, were also negative. It is possible that the sample size in the haplotype relative risk is too small to have power to detect the differences in transmitted versus nontransmitted alleles. There is a need for a replication study in a large family sample, that will allow haplotype relative risk or affected sib-pair analysis.     

Ethanol-Induced Changes in Chloride Flux are Mediated by Both GABAA and GABAB Receptors

Low concentrations of ethanol (10-30 mM) in the presence of a GABAB receptor agonist, baclofen, promoted 36Cl- uptake into membrane vesicles (microsacs) prepared from mouse cortex. Neither ethanol nor baclofen alone altered chloride influx. The GABAB antagonists, phaclofen and 2-hydroxy-saclofen, completely blocked the increase in chloride flux produced by ethanol in the presence of either baclofen or GABA. Ethanol increased the chloride conductance produced by the GABAA agonists muscimol, isoguvacine, imidazolacetic acid and amino-propane sulfonic acid and this action of ethanol was blocked by phaclofen. The specific GABAA antagonist, bicuculline, blocked ethanol-induced increase in chloride flux in the presence of either baclofen or GABA. GABA-activated chloride channels were also studied in Xenopus oocytes expressing mouse brain mRNA. In this preparation, GABA action was enhanced by ethanol, pentobarbital, and diazepam, and 2-hydroxy-saclofen partially antagonized the action of ethanol without altering the effects of pentobarbital or diazepam. These results suggest that ethanol enhancement of GABAA receptor-chloride channel function also requires activation of GABAB receptors.     

Direct Antagonism of Ethanol's Effects On GABAA Receptors by Increased Atmospheric Pressure

Previous studies have shown that exposure to 12 times normal atmospheric pressure of helium-oxygen gas (heliox) directly antagonizes a range of ethanol's acute and chronic behavioral effects. The present study extends the investigation to the biochemical level by investigating the effects of pressure on ethanol-induced potentiation of GABA_A receptor function in mouse membrane vesicles (micro-sacs). Exposure to 12 atmospheric pressure heliox significantly antagonized ethanol (25 to 100 mM) potentiation of GABA-activated ³⁶Cl^- uptake, but did not significantly alter baseline GABA_A receptor function measured by the response of the system to GABA (10 to 100 μM), bicuculline (3 and 100 μM), or picrotoxin (100 μM). These findings add essential support for the hypothesis that hyperbaric exposure is a direct ethanol antagonist that can be used as a tool to help identify ethanol's initial cellular and molecular sites of action that cause its behavioral effects. Taken in context with previous behavioral studies, the present results also provide important new evidence for a cause-effect relationship between ethanol potentiation of GABA_A receptor function and ethanol's anesthetic and behavioral effects.     

GABAA-Receptor δ Subunit Knockout Mice Have Multiple Defects in Behavioral Responses to Ethanol

BACKGROUND: The gamma-aminobutyric acid type A receptors (GABARs) are involved in mediating some of the behavioral effects of beverage alcohol (ethanol). However, the unique pharmacological and behavioral responses conferred by each of the various receptor subunits are not well understood. METHODS: To address the role of the GABAR delta subunit in mediating ethanol responses, gene knockout mice that lack this subunit were tested for a variety of ethanol-induced behavioral responses. RESULTS: Our results indicate that, compared with controls, delta-deficient mice (delta-/-) have (1) reduced ethanol consumption, (2) attenuated withdrawal from chronic ethanol exposure, and (3) reduced anticonvulsant (seizure-protective) effects of ethanol. These mice demonstrate a normal anxiolytic response to ethanol and a normal hypothermic response to ethanol, and they develop both chronic and acute tolerance. CONCLUSIONS: These results further establish the link between GABARs and specific behavioral responses to ethanol and begin to reveal the role of the delta subunit in these responses.     

Acute and Chronic Ethanol Modulate Dopamine D2-Subtype Receptor Responses in Ventral Tegmental Area GABA Neurons

Background: Ventral tegmental area (VTA) γ‐aminobutyric acid (GABA) neurons appear to be critical substrates underlying the acute and chronic effects of ethanol on dopamine (DA) neurotransmission in the mesocorticolimbic system implicated in drug reward. VTA GABA neuron firing rate is reduced by acute ethanol and enhanced by DA via D2 receptor activation. The objective of this study was to evaluate the role of D2 receptors in acute ethanol inhibition of VTA GABA neuron activity, as well as the adaptation of D2 receptors by chronic ethanol consumption. Methods: Using electrophysiological methods, we evaluated the effects of intraperitoneal ethanol on DA activation of VTA GABA neurons, the effects of DA antagonists on ethanol inhibition of their firing rate, as well as adaptations in firing rate following chronic ethanol consumption. Using single cell quantitative RT‐polymerase chain reaction (PCR), we evaluated the expression of VTA GABA neuron D2 receptors in rats consuming ethanol versus pair‐fed controls. Results: In acute ethanol studies, microelectrophoretic activation of VTA GABA neurons by DA was inhibited by acute intraperitoneal ethanol, and intravenous administration of the D2 antagonist eticlopride blocked ethanol suppression of VTA GABA neuron firing rate. In chronic ethanol studies, while there were no signs of withdrawal at 24 hours, or significant adaptation in firing rate or response to acute ethanol, there was a significant down‐regulation in the expression of D2 receptors in ethanol‐consuming rats versus pair‐fed controls. Conclusions: Inhibition of DA activation of VTA GABA neuron firing rate by ethanol, as well as eticlopride block of ethanol inhibition of VTA GABA neuron firing rate, suggests an interaction between ethanol and DA neurotransmission via D2 receptors, perhaps via enhanced DA release in the VTA subsequent to ethanol inhibition of GABA neurons. Down‐regulation of VTA GABA neuron D2 receptors by chronic ethanol might result from persistent DA release onto GABA neurons.     

Alcohol Actions on GABAA Receptors: From Protein Structure to Mouse Behavior

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were R. Adron Harris and Susumu Ueno. The presentations were (1) Protein kinase Cɛ-regulated sensitivity of γ-aminobutyric acid type A (GABA_A) receptors to allosteric agonists, by Robert O. Messing, A. M. Sanchez-Perez, C. W. Hodge, T. McMahon, D. Wang, K. K. Mehmert, S. P. Kelley, A. Haywood, and M. F. Olive; (2) Genetic and functional analysis of a GABA_A receptor γ2 subunit variant: A candidate for quantitative trait loci involved in alcohol sensitivity and withdrawal, by Kari J. Buck and Heather M. Hood; (3) Tryptophan-scanning mutagenesis in GABA_A receptor subunits: Channel gating and alcohol actions, by Susumu Ueno; and (4) Can a single binding site account for actions of alcohols on GABA_A and glycine receptors? by R. Adron Harris, Yuri Blednov, Geoffrey Findlay, and Maria Paola Mascia.     

Effects of Chronic Ethanol Exposure on GABA Receptors and GABAB Receptor Modulation of 3H-GABA Release in the Hippocampus

Chronic ethanol treatment (CET), sufficient for decreasing long-term potentiation (LTP) in rats, also enhances ³H-GABA release from hippocampal slices in these same animals. The mechanism for an increase in GABA release may involve changes in presynaptic receptors. Therefore, we characterized presynaptic autoreceptor modulation of ³H-GABA release in hippocampal slices from control and CET rats. The effects of a GABA_B receptor agonist (baclofen) and antagonist [2-hydroxy (OH)-saclofen] were tested for their ability to modulate electrically stimulated ³H-GABA release from superfused hippocampal slices. Baclofen decreased stimulated release in a dose-dependent manner and 2-OH-saclofen increased release consistent with the existence of presynaptic GABA_B autoreceptors in hippocampus. The GABA_A antagonist bicuculline did not significantly modulate basal or stimulated release. When the effects of baclofen and 2-OH-saclofen were measured in animals 48 hr after withdrawal from CET, presynaptic modulation of release by baclofen and 2-OH-saclofen was decreased. In addition, we examined the density of ³H-baclofen and ³H-bicuculline binding in the hippocampal formation using quantitative autoradiographic techniques. We found that the density of ³H-baclofen binding sites was not affected by CET, whereas the density of ³H-bicuculline binding sites was increased by 28% in ethanol-treated rats. These data may explain how CET increases presynaptic regulation of GABA release from hippocampus that may contribute to the decrease in LTP seen in rats after CET.     

Discriminative Stimulus Function of Ethanol: Role of GABA, Receptors in the Nucleus Accumbens

Because the subjective effects of drugs may be related to abuse potential, this study was conducted to assess the involvement of GABA_A receptor systems in the nucleus accumbens (N Acc) in the discriminative stimulus effects of ethanol. Male Long-Evans rats were trained to discriminate between intraperitoneal (IP) injections of ethanol (1 g/kg) and saline under a fixed-ratio 10 schedule of sucrose (10% w/v) reinforcement. When performance during training sessions met the accuracy criteria (>80%0 correct responding for five consecutive days), an ethanol generalization curve was determined. The rats were then surgically implanted with bilateral stainless-steel guide cannulae aimed at the N ACC. Intra-accumbens (IA) substitution test sessions were conducted during which the direct GABA_Aagonist muscimol (0.01, 0.04, 0.10, and 0.40 μg/μl; IA) was administered in combination with saline (IP). The direct GABA_A, antagonist bicuculline (0.03,0.10, and 0.30 μg/μl; IA) was administered in combination with the training dose of ethanol (1 g/kg, ip). At 10-min postinjection, IA muscimol partially substituted for IP ethanol. However, at 15-min postinjection, muscimol (0.10 μg/μl; IA) fully substituted for IP ethanol. Bicuculline attenuated the discriminative stimulus properties of IP ethanol, but only at doses that significantly decreased response rate. At 10-min postinjection, muscimol (0.01 and 0.04 μg/μl) potentiated (>80%0 ethanol lever responding) the discriminative stimulus properties of a dose of ethanol (0.5 g/kg) that alone produced only partial generalization. These data suggest that ethanol discrimination is mediated centrally and demonstrate that infusions of the GABA_A, agonist muscimol in the N Acc are sufficient to produce the stimulus effects corresponding to a 1.0 g/kg training dose of ethanol. When taken together with data showing that GABA_A receptor activation in the N Acc potentiates the termination of ethanol self-administration, these data suggest that ethanol's discriminative stimulus function may influence its reinforcement function.     

Analysis of the 5-HT2 Receptor Ligands Dimethoxy-4-indophenyl-2-aminopropane and Ketanserin in Ethanol Discriminations

Previous studies have suggested a modulatory role of the 5-HT₂ receptor system in the behavioral effects of ethanol. The present study examined the discriminative stimulus effects of the 5-HT_2A/2C agonist (–)-dimethoxy-4-indophenly-2-aminopropane (DOI) and the 5-HT_2A antagonist ketanserin in rats trained to discriminate either 1.5 g/kg of ethanol from water (intragastrically, n = 7) or 2.0 g/kg of ethanol from water (intragastrically, n = 8). In substitution tests, neither DOI (0.3 to 1.0 mg/kg, ip) nor ketanserin (3.0 to 17.0 mg/kg, ip) produced discriminative stimulus effects similar to either training dose of ethanol, although decreases in rates of responding were significant at the highest doses tested. Likewise, when given in combination with ethanol, neither 5-HT₂ ligand shifted the ethanol-dose response determination in either the 1.5 or 2.0 g/kg ethanol training groups. DOI in combination with ethanol did not alter rates of responding, whereas ketanserin in combination with ethanol significantly decreased response rates. Thus, the 5-HT_2A receptor ligands do not appreciably affect the discriminative stimulus effects of ethanol, in contrast to previous results with 5-HT_1B ligands.     

Action of Ethanol and Zolpidem on γ-Aminobutyric Acid Responses from Cerebellar Purkinje Neurons: Relationship to β-Adrenergic Receptor Input

The observation that cerebellar Purkinje cells contain type-I benzodiazepine-sensitive GABA_A, receptors is consistent with findings in the present work that the majority of Purkinje neurons are sensitive to enhancement of GABA by the type-1 benzodiazepine agonist, zolpidem. Previous work has demonstrated a relation between zolpidem and ethanol enhancement of GABA responses in several brain regions, but had not tested Purkinje neurons. Therefore, given that a majority of Purkinje neurons were found to be sensitive to zolpidem, ethanol would have been expected to enhance GABA responses from this cell type. However, in agreement with earlier electrophysiological studies, ethanol enhanced GABA inhibitory responses from only a small proportion of these cerebellar Purkinje neurons. Rather than enhancement of GABA, local application of ethanol either inhibited or did not affect responses to GABA from a majority of cerebellar-Purkinje neurons. Nonetheless, as previously reported, a portion of the Purkinje neurons initially insensitive to ethanol enhancement of GABA became sensitive to this action of ethanol with co-application of the β-adrenergic agonist, isoproterenol. Thus, these results collectively implicate a β-adrenergic input dependency for ethanol enhancement of GABA from some, but not all, cerebellar Purkinje neurons sensitive to zolpidem. Because a β-adrenergic input did not allow ethanol enhancement of GABA from all Purkinje neurons, future studies should explore the possibility that other auxiliary neural inputs to zolpidem-sensitive cerebellar Purkinje neurons may be required for ethanol enhancement of GABA responsiveness when a β-adrenergic input does not have this action. Likewise, knowing that the action of zolpidem can predict ethanol enhancement of GABA in other brain regions, the present findings suggest that a future determination be made concerning whether zolpidem-sensitive neurons in these other regions of brain require a β-adrenergic or an alternative neural input for ethanol enhancement of GABA responses.