Neural Mechanisms of Adaptation in Chronic Ethanol Exposure and Alcoholism

0145-6008/96/2008-00I 51 A$03.00/0 ALCOHOLISM: CLINICAL AND EXPERIMENTAL RESEARCH

Vol. 20, No. 8 November Supplement 1996

Symposium

Neural Mechanisms of Adaptation in Chronic Ethanol Exposure and Alcoholism

Chaired by Peter Dodd, Clinical Research Laboratory, Royal Brisbane Hospital Research Foundation, Brisbane, Australia

Introduction and GABA, Receptor Pharmacology and Gene Expression in the Alcoholic Human Brain Peter Dodd and Gregory Thomas, Clinical Research Laboratory, Royal Brisbane Hospital Research Foundation, Brisbane, Australia

Structural and Functional Adaptations of GABA, Receptors in Alcohol [Bpendent and Withdrawn Rats Leslie Morrow, Center for Alcohol Studies, University of North Carolina School of Medicine, Chapel Hill, NC,USA

Adaptive Changes in the NMDA Receptor During Chronic Ethanol-Mechanism and Consequences Peter Wilce, Alcohol Research Unit, Department of Biochemistry, University of Queensland, St. Lucia, Australia

Significant Increases in Cerebral Diazepam Binding inhibitor (DBI) and Its mRNA Expression During the Establishment of Alcohol Dependence: Possible Involvement in the Expression of Alcohol Withdrawal Syndrome Kinya Kuriyama and Masashi Katsura, Department of Pharmacology, Kyoto Prefectural University of Medicine, Kyoto, Japan

U s e of Transfected Cells to Study Mechanisms of Alcohol Adaptation R. Adron Harris, Department of Pharmacology, University of Colorado HSC, Denver, CO, USA

INTRODUCTION

From the chairc Dr. Dodd outlined some of the adaptive changes long-term alcohol use produces. These include the development of tolerance, alterations in physiology, addiction, and the processes underlying pathogenesis. In the brain, investigations have been carried out particularly in systems thought to mediate ethanol’s direct effects, especially GABAergic and glutamatergic neurotransmission. Although pathological differences are widespread in the brains of long- term chronic alcoholics, the variations are more marked in certain circumscribed cortical regions than in others. Cases with diseases concomitant on alcoholism, e.g. cirrhosis of the liver or the thiamin deficiency associated with the Wernicke- Korsakoff syndrome, may show more marked effects. Human brain obtained at autopsy shows selective changes in the pharmacology and density of GABA, receptors in susceptible areas. However, while understanding the human brain remains the target, it is essentially not possible to study adaptive mechanisms with tissue obtained at autopsy, and so it is necessary to resort to model systems. A wealth of animal studies has shown that the subunit composition of receptors may be altered in response to drug-mediated changes in isoform gene expression. Various exposure protocols have been used to confirm that ethanol is among the drugs of addiction which can bring about such changes. Recombinant studies demonstrate that subunit composition controls receptor

characteristics. Hence, it might be expected that the observed changes in GABA, receptor pharmacology are brought about by altered gene expression, and that other transmitter systems will also be affected. However, recent work with both animal and human preparations has shown disparities between mRNA and protein expression in a variety of experimental paradigms. This symposium drew together researchers attacking a number of aspects of this problem, with a view to delineating the specificity of the phenomena, and to characterizing the roles of gene expression, post-translational processing, receptor assembly, and related phenomena, in long-term alterations induced by ethanol and/or alcoholism in the central nervous system.

DISCUSSION

Struuctural and Functional Adaptations of GABA, Receptors. Dr. Morrow explored how chronic ethanol administration alters GABA, receptor function. Ethanol shares several phannacologic actions with barbiturates and benzodiazepines, including anxiolytic and sedative activity and, under appropriate circumstances, the development of cross-tolerance and cr~ss-dependence.~~ The similarities in the actions of ethanol, benzodiazepines, and barbiturates further suggest that all three drugs may share some mechanisms of action. Tolerance to the sedative and intoxicating effects of ethanol has been postulated to result from a compensatory decrease in

Alcohol Clin Exp Res, Vol20, No 8, 1996: pp 151A-156A 151A

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GABA-mediated inhibition in the brain.” Recent studies have show that alterations in the function of the GABA, receptor chloride channels may contribute to some of the signs and symptoms of the ethanol-withdrawal syndrome. Chronic exposure of rats to ethanol produces physical dependence and tolerance. These effects are associated with a decrease in the sensitivity of the GABA, receptor-coupled Cl-channel in the cerebral cortex of dependent animals, when blood ethanol levels produced by the ethanol exposure were greater than 150 mg %.% Muscimol-stimulated Wl - uptake was decreased by 26% and pentobarbital-stimulated W1- uptake was decreased by 25% following chronic ethanol inhalation. The ability of ethanol (20 mM) to potentiate muscimol-stimulated Wl - uptake was completely lost in rat cerebrocortical synaptone- urosomes26 and in mouse cerebellar microsacs following chronic ethanol administration. Benzodiazepine enhancement of muscimol-stimulated chloride flux is reduced in the cerebral cortex of mouse microsacs, while the functional efficacy of inverse agonists is enhanced. Behavioral responses to injections of muscimol into the substantia nigra and to subcutan- eous THIP injections are also reduced following chronic ethanol administration. Likewise, chronic ethanol administration reduces electrophysiological responses to GABA in the medial septa1 nucleus and GABA-induced chloride flux in spinal cord cultured neurons.20 Following the completion of ethanol withdrawal, the decrements in muscimol-stimulated

uptake in rat cerebral cortical synaptoneurosomes26 and flunitrazepam potentiation in mouse cortical microsacs are completely reversed, as would be predicted if these neurochemical changes are related to the behavioral state of withdrawal.

Alterations in the density or affinity of brain GABA, /benzodiazepine receptors following chronic ethanol administration have yielded conflicting results and cannot explain the development of tolerance or withdrawal. For example, the density of low-affinity [3H]agonist binding sites in brain is decreased following chronic ethanol exposure in rats or mice, while the ability of GABA to enhance [3H]flunitrazepam binding in brain membranes prepared from ethanol-treated mice is reduced. However, others have failed to find alterations in the number or affinity of GABA, receptors. Chronic ethanol administration does not alter the density of [3H]flunitrazepam or [35S]TBPS binding sites in brain. Recently, Mhaue et aI2O reported an increase in the density of specific binding sites for [3H]Ro15-4513 in rat cortex and cerebellum following chronic ethanol administration, as well as increased sensitivity to its behavioral effects. We have proposed that the alterations in GABA, receptor function observed following chronic ethanol administration are the result of a decrease in the synthesis and expression of subpopulation(s) of GABA, isore~eptors .~~ Chronic ethanol administration may have differential effects on the expression of individual GABA, receptor subunits, accounting for the diverse effects of ethanol on different radioligand binding sites. If ethanol alters the expression of specific populations of GABA, isoreceptors, radioligands which measure all GABA receptors may lack the selectivity to

detect changes in one or more receptor subunits or subpopu- lations of GABA, isoreceptors.

Chronic ethanol administration differentially alters the expression of GABA, receptor subunit mRNAs in the cerebral cortex5*22,25 and ~erebellum.2~ The levels of GABA, receptor a1 subunit mRNAs are reduced while cq subunit mRNAs are increased by approximately equal amounts in cerebral cor te~.~ Likewise in the cerebellum, decreases in GABA, receptor a1 subunit mRNAs and increases in ag subunit mRNA levels are found.% These changes in mRNA levels suggest alterations in the expression of the corresponding proteins that could account for the alterations in receptor function and binding that have been observed. For example, the increases in q and a6 sub unit expression probably explain the increases in [3H]Ro-15- 45 13 binding and sensitivity2O following chronic ethanol administration. The increased expression of cq subunits may underlie the reduced sensitivity to GABA and benzodiazepine agonists% since recombinant GABA receptors with a 4 P 2 ~ subunits are less sensitive to GABA agonists and benzodiazepines than alp2112 receptors. Ethanol dependent and withdrawn rats are also sensitized to the effects of the neurosteroid 3a,5a-THP.45 This effect may be related to the increase in y1 subunit mRNAs following chronic ethanol expo~ure~*~, since y1 subunits convey greater neurosteroid sensitivity in recombinant expression studies.n

The observation that ethanol tolerance is associated with changes in subunit composition also supports the hypothesis that subunit composition influences ethanol sensitivity. Ethanol potentiation of GABA, receptor function is abolished at the cellular level in rats that are ethanol tolerant and dependent. The changes in subunit expression that result from chronic exposure to ethanol may contribute to this reduction in ethanol sensi t i~i ty .~.~ However, alterations in the phosphory- lation state of GABA, receptors may also be involved. Recent studies in recombinant receptors, where subunit expression is controlled by a dexamethazone sensitive promotor, exhibit changes in receptor function that cannot be attributed to alterations in subunit e x p r e ~ s i o n . ~ ~ . ~ ~ In addition, mouse mutants that lack a protein kinase C isoform exhibit a loss of ethanol sensitivity both in vivo and at the cellular level.1° Thus, ethanol tolerance and dependence may also involve second messenger effects on GABA, receptors.

During ethanol withdrawal, dynamic changes in GABA, receptor subunit mRNAs are observed. GABA, receptor al, cq and y1 subunit mRNAs are returning to control levels, while p2 and p3 subunit mRNA levels are increasing compared to both controls and ethanol dependent rats4 At this time point, corresponding changes in protein expression are unlikely since the receptor turnover rate (tlDJ is estimated to be 1-2 days and peptide levels in rats undergoing withdrawal are measured approximately 6 h after those measured in ethanol dependent rats. In fact, GABA, a1 subunit peptide levels are identical in ethanol dependent vs withdrawn rats, despite the fact that mRNA levels are higher in rats undergoing withdrawal (unpublished results). Therefore, GABA, receptor subunit peptide expression during ethanol withdrawal is probably

NEURAL MECHANISMS OF ADAPTATION IN CHRONIC ETHANOL EXPOSURE AND ALCOHOLISM 153A

similar to that found in ethanol dependent rats. We have proposed that alterations in GABA, receptor

subunit composition, in vivo, may result from adaptations to chronic drug exposure.5* This could result in alterations in the functional properties of receptors with no change in the total number of receptors expressed. Hence, GABAA receptors maybe regulated by alterations in subunit expression which result in the assembly of structurally and functionally distinct GABA isoreceptors. This mechanism appears to play a role following chronic exposure to ethanol, and possibly other drugs that modulate GABAA receptors.23 There is ample evidence for alterations in GABA, receptor subunit composition during de~elopment.~~ Similarly, alterations in the subunit expression of nicotinic cholinergic, glutamate and glycine receptors are observed during neuronal development. There- fore, we propose that ligand-gated ion channels may be subject to alterations in subunit composition that modulate receptor function in vivo.

Adaptive Changes in the NMDA Receptor During Chronic Ethanol-Mechanisms and Consequences. Dr. Wilce, outlining the work of Alison Beckmann, Wenbin Chen, Mark Davidson, Philip Hardy, Izuru Matsumoto and himself, discussed the mechanism and consequences of adaptive changes in the NMDA receptor during chronic ethanol exposure.

A working model to account for chemical dependence on ethanol implicates GABAA receptors and the NMDA type of glutamate receptors. Acute ethanol potentiates inhibitory GABAergic receptor function and inhibits excitatory NMDA receptor function. Compensatory changes under chronic treatment could result in upregulation of NMDA receptor function and downregulation of GABAergic function restoring equili- brium in the presence of ethanol but resulting in ethanol withdrawal hyperactivity.

We have examined this model, concentrating on the NMDA receptor, with two experiments. We determined NMDA receptor function by intrahippocampal injection of NMDA in rats 24 h after ethanol withdrawal. There was more intense NMDA-induced behaviours in the ethanol withdrawn animals and morphological damage resulting from 5 nmol of NMDA was significantly greater in the CA3 region of the hippocampus in these animal^.^ In a second study, we investigated the involvement of regions sensitive to the excitotoxin kainic acid in the genesis of ethanol withdrawal behaviours. Chemical ablation of the CA1 subfield of the hippocampus and the pin- form cortex resulted in a significant reduction in the spontan- eous and/or handling-induced behaviours normally observed during Withdrawal. Sensitivity to audiogenically-induced seizures which has been assigned to the GABAergic system in the inferior colliculus was not affected.I8 These data support the hypothesis that ethanol dependence is associated with hyperactivity of the NMDA receptor which becomes apparent during withdrawal and suggest that kainic acid-sensitive limbic struc- tures may play an important role in precipitation of spontane- ous but not audiogenically-induced ethanol withdrawal symptoms.

The native NMDA receptor is probably a pentomeric complex composed of a number of different subunits (NMDARi and NMDA,,Bdcp) with additional 3' and 5' splice variants of the obligate subunit, NMDA R1. The various combinations of subunits and splice variants result in differences in pharmacology, including sensitivity to modulators such as glycine and spermine. We have been unable to detect changes in total NMDA receptor numbers in the hippocampus or cortex by ligand binding assays using [ 3 H ] ~ 8 0 1 . Using ribonuclease protection assays we demonstrated a markedly increased N M D A R z B mRNA expression associated with an increase in NMDARiA splice variant mRNA (not containing the exon 5 transcript) after chronic ethanol treatment, suggesting that the adaptive changes that underlie the process of dependence and withdrawal may result from an alteration in receptor composition rather than in receptor numbers. The presence of the NMDA,, subunit in the receptor particularly associated with NMDARiA increases polyamine potentiation of receptor function, which may indicate an important r6le for polyamine metabolism in dependence and withdrawal.

The increased calcium flux through the upregulated NMDA receptor during withdrawal has two potential consequences; calcium can be excitotoxic and it is a second messenger that induces immediate early gene (IEG) expression through interaction with calcium responsive elements. We have shown that withdrawal hyperactivity evokes expression of the IEGs c- fos and c-jun coding for leucine zipper proteins.19 This group of proteins form dimeric transcription factors binding to the (Activator Protein-1) AP-1 consensus DNA sequence. Similar- ly, expression of egr-1 (also known as Zif268), coding for a zinc finger protein, which causes transactivation through a specific GC-rich DNA element, is also increased during ~ithdrawa1.I~ We have extended these studies to investigate DNA binding activities after withdrawal by electrophoretic mobility shift assays. The constitutive Spl and OCT DNA binding activities along with binding to the DNA consensus sequence for the cyclic AMP response element binding protein were unchanged during withdrawal. Ap-1 binding activity was transiently elevated in the hippocampus and cerebellum 16 h after withdrawal but remained elevated at 72 h after withdrawal in the cerebral cortex. Egr DNA binding activity resolved into a slow migrating activity identified as egr-1 and an unidentified faster migrating activity Binding to the egr DNA consensus sequence was elevated after withdrawal with a timecourse and regional variation similar to the Ap-1 complex. The two egr binding activities altered after withdrawal essentially in parallel.

Activation of these transcription factors during withdrawal suggest initiation of gene expression following the stimulus of withdrawal hyperactivity. Two alternative consequences for this gene expression are proposed. The IEG encoded transcnp- tion factors have been implicated in the cascade of events leading to programmed cell death in the nervous system: Thus, in cells severely affected by withdrawal-induced, NMDA-receptor-stimulated hyperactivity, the withdrawal induced expression of these transcription factors may be an

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important component of the cascade that subsequently leads to excitotoxic delayed cell death. It may be that the induction of IEG expression during withdrawal could be an indication that delayed cell death is an important component of ethanol withdrawal-induced brain damage. Animals subjected to repeated withdrawal develop progressively more severe withdrawal behaviours and alcoholics who have experienced previous de- toxification are more prone to seizures during withdrawal. A “kindling” hypothesis has been proposed to explain the observation of the increasing severity of withdrawal behaviours. In well-studied models of kindling, e.g. electrical stimulation, the events resulting in the kindling response have been traced to the activation of the NMDA receptor, followed by IEG expression and changes in gene expression with subsequent changes in cell phenotype evident in marked changes in cell structure and function?l The changes in gene expression underpinning ethanol withdrawal kindling, may be initiated by the neuronal hyperexcitability activating the IEG-encoded transcription factors.

By a combination of the mechanisms outlined above, the increased intensity of withdrawal behaviour induced by repeated withdrawal may increase the vulnerability to excitotoxic damage, and the repeated cycles of physical dependence and withdrawal may constitute the aetiological basis of widespread ethanol-induced brain impairment and damage.

Diazepam Binding Inhibitor-Possible Role in the Expression of Alcohol Withdrawal. Professor Kuriyama presented recent work from his laboratory conducted with Masashi Katsura on the diazepam binding inhibitor in alcohol dependence and nicotine tolerance.

Diazepam binding inhibitor (DBI) is an endogenous 10 kDa polypeptide with pharmacological properties as an inverse agonist for the binding sites of benzodiazepines (BDZs) and beta-carb~lines.~ DBI and its mRNA are abundantly distributed in the central nervous system (CNS) and peripheral organs, and DBI levels are considered to increase in parallel to the levels of DBI mRNA and its processing products in the cerebral cortex of BDZ tolerant rat? In rats, intracerebroventricular injection of DBI elicits several behavioural changes resembling those observed during EtOH withdrawal in rats.2.8 These data lead to the assumption that EtOH may affect the level of DBI in the brain through modification of BDZ receptor functions. Therefore, in the present study, we examined the effect of EtOH treatment on the expression of DBI mRNA in the brain prepared from EtOH-treated mouse and neurons, and to discuss possible involvement of BDZ receptors in EtOH- induced alterations of the expression of DBI.

In the first series of experiments, we have investigated the time course of changes in the expression of DBI mRNA following the single administration of EtOH (3 g/kg) using solution hybridization procedures including a ribonuclease protection assay. The level of DBI mRNA showed no significant change up to 48 h after the EtOH treament.13 Similarly, single administration of EtOH did not alter the expression of p- actin rnRNA.l3 These results suggest that the single adminis-

tration of EtOH did not induce any alterations in the expression of DBI mRNA.

In the second series of the experiments, we examined the changes in the expression of DBI mRNA in the mouse cerebral cortices after the EtOH inhalation and EtOH withdrawal, Although the contents of poly(A)+ RNAs isolated from the mouse cerebral cortices following EtOH inhalation and EtOH withdrawal were similar to those from the mice not treated with EtOH, the expression of DBI mRNA in the cerebral cortices from EtOH-treated mice elevated to 130% of that in control mice. Moreover, 8 h after the termination of EtOH vapor administration, when the mice showed high score of withdrawal signs, DBI mRNA levels were further accelerated (230 % of control), whereas the expression of p-actin mRNA did not change after any type of EtOH treatment.

The changes in the expression of DBI mRNA in the mouse cerebral cortex after the EtOH withdrawal have been further examined to analyze whether the expression of DBI mRNA is related to the disappearance of the withdrawal signs. The increase in DBI mRNA level induced by EtOH inhalation was accelerated after the removal of mice from EtOH vapor in a time-dependent manner and the maximal expression of DBI mRNA was observed on the 3rd day after EtOH withdrawal, despite the withdrawal signs disappearing within 2 days of the withdrawal of EtOH. Thereafter, this acceleration of DBI mRNA expression observed faded to the control (EtOH non- treated) level over 14 days after EtOH withdrawal.

It is noteworthy that the gradual increase in DBI mRNA expression was observed after the removal of mice from EtOH vapor and that this increase attained its maximal level 3 days after the EtOH withdrawal in spite of the disappearance of withdrawal signs such as increased motor activity. In the abstinence syndrome, several psychiatric symptoms such as anxiety, irritation and insomnia have been reported to be continued after the disappearance of some excitatory motor activities like convulsions. The injection of DBI into the rat cerebral ventricle is reported to increase proconflict action which is considered to reflect anxiety in humans. Moreover, the concentration of DBI in the cerebrospinal fluid from male alcoholics is significantly elevated compared to that from control subjects.28 These data suggest that the increased expression of DBI mRNA may be involved in the generation of behavioural and psychiatric changes observed after the removal of EtOH.

It has been well known that agonists for the central type of BDZ receptors reduce withdrawal signs in alcoholics.1 In animal models, flunitrazepam effectively suppresses proconflict action induced by intracerebroventricular administration of DBI, and the behavioural changes during EtOH withdrawal are enhanced in the presence of inverse agonists for BDZ receptors. Furthermore, DBI is an endogenous substance to produce anxiety and is assumed to be an inverse agonist for BDZ receptors. Taken together, these data suggest that some of the withdrawal signs observed in EtOH-withdrawn subjects may be mediated through the activation of BDZ receptors by DBI. In fact, the up-regulation of binding sites for Ro 15-4513,

NEURAL MECHANISMS OF ADAPTATION IN CHRONIC ETHANOL EXPOSURE AND ALCOHOLISM 155A

which serves as an inverse agonist for BDZ receptors and an antagonist for EtOH, is selectively induced in the cerebral cortex and cerebellum of the rats administered EtOH and in spinal neurons treated with EtOH?O although controversial data that EtOH treatment shows no effect on the binding pa- rameters of the central types of BDZ receptors have also been

We have also examined whether the elevation of DBI mRNA levels induced by EtOH treatment results from events mediated through the stimulation of BDZ receptors by EtOH. Flunitrazepam, an agonist specific for the central type of BDZ receptors, was simultaneously administered with EtOH treatment. Flunitrazepam completely abolished the increase in DBI mRNA expression induced by both EtOH inhalation and EtOH withdrawal, while the administration of flunitrazepam alone showed no effect on the expression of DBI. The expression of p-actin mRNA was not altered by the treatment with both EtOH and flunitrazepam and with flunitrazepam alone.

One of the new findings demonstrated in our study is that the activation of the central type of BDZ receptors by flunitrazepam inhibits the increase in DBI mRNA expression induced by chronic treatment with EtOH, whereas flunitrazepam alone did not exhibit any changes in the expression of DBI mRNA. Based on previously reported data that EtOH exhibits multiple modification on GABAergic functions17 and the results described here, it is assumed that EtOH alters the functions of GABA, receptor complex, especially of BDZ receptors, and that the interaction between EtOH and BDZ receptors may be attributed to the increased expression of DBI mRNA. Consequently, the increase in DBI in association with the increased expression of its mRNA after chronic EtOH treatment may, in turn, cause anxiety and enhancement of seizure susceptibility observed in EtOH- treated and EtOH-withdrawn animals.

In the third series of the experiments, we investigated changes in DBI mRNA in mouse cerebral cortex and primary cultured cerebral cortical neumns following nicotine treatment, one of the other drugs producing drug dependence and/or tolerance. The expression of DBI mRNA was found to be increased significantly in the cerebral cortex of mice subjected to continuous administrations of nicotine. Following chronic exposure of cerebral cortical neurons to nicotine, the level of DBI mRNA significantly increased in a dose- and time-dependent manner, and this increase was abolished by the concomitant exposure of neurons to nicotine and hexamethonium (10 @), an antagonist specific to nicotine acetylcholine receptor .I2

These results strongly suggest that the expression of DBI mRNA in the brain may be elevated by the treatments with alcohol as well as nicotine, possibly via the activation of BDZ receptors and nicotinic acetylcholine receptors, respectively.

Use of Transfected Cells to Study Mechanisms of Alcohol Adaptation. Dr. Harris presented work from his group, carried out by Susan Brozowski and Dr. Ron Klein, showing effects of chronic ethanol treatments on the function of GABAA receptors in stably transfected mouse fibroblast (L)

reported.

cells. This system provides a model and an approach that is much different from those of the previous speakers who studied receptor changes in laboratory animals or humans consuming alcohol. As noted above, the in vivo models show that chronic ethanol treatments alter the expression of various subunits of the GABA, receptor, and it is assumed that the subunit stoichiometry of the receptor is changed by alcohol exposure, and this “subunit substitution” results in observed changes in GABAA receptor function. However, cells that are stably transfected with defined GABAA receptor subunits under the control of a strong (dexamethasone-sensitive) promoter are unlikely to display changes in subunit expression or subunit sub~ti tut ion.~~J~ Thus, chronic ethanol treatment would be expected to have no effect on the GABAA receptors of these cells if all of the effects of these treatments are due to changes in subunit expression.

However, Dr. Harris presented evidence that culture of cells expressing human or bovine GABAA receptor subunits for 1 4 days in the presence of 25-100 mM ethanol produced changes in the function of GABA, receptors. These results are summarized in Table 1 and are compared to results from rodent studies. In brief, chronic exposure to ethanol reduced the efficacy of muscimol (a GABAA agonist) to stimulate -1- uptake (a measure of GABAA receptor function). In addition to the decreased function of the GABA, receptor, modulation of the receptor by ethanol was decreased (tolerance) as was modulation of the receptor by flunitrazepam (cross-tolerance to a benzodiazepine). However, modulation of the receptor by pentobarbital. zinc, or pregnane-01-one was not affected by chronic ethanol treatment. These effects occurred without any change in the affinity or density of binding sites for [3H]flunitrazepam and without changes in levels of subunit mRNAs.16 Chronic treatment of these cells with muscimol or benzodiazepines also produces changes in the function of the GABAA receptor, although these changes are not identical to those produced by ethanol and these three classes of drugs likely have different mechanisms for GABA, receptor adaptation.14.15

Table 1. Changes in GABA, Receptor Function after Ethanol Treatment Test Drug Effect of Chronic Ethanol on Drug Action

Brain Tissue Transfected Cells

MuscimoUGABA Decrease/No effect Decrease Ethanol Decrease Acute (5 min) Decrease (min or days)

Benzodiazepines Decrease (max response) Decrease ( m a response) Pentobarbital No change or decrease No change Pregnan-olone Increase No change DMCWRol5-4513 Increase Decrease

or Chronic (5-1 0 days)

Results for “Brain Tissue“ are summarized from numerous published studies of chronic ethanol treatment of rat and mice as well as cultured neurons; results for “Transfected Cells” are from mouse fibroblasts stably transfected with GABA, receptor subunits and are mostly unpublished. See text and14 for details.

Chronic ethanol treatment produced similar alterations of function of GABAA receptors in cells expressing bovine al+Pl+n~, human a1+f32+1y2~, or human al+f32+ns. This is of interest because only receptors of the first two cell lines are

156A DODD

affected by acute exposure to ethanol. That is, the y2y2~ subunit (together with an a and a p) is required for low dose (10-50 mM) ethanol potentiation of G A B 4 receptor function. * Thus, the chronic, adaptive effect of ethanol (which is seen with all three cell lines) is not a consequence of the acute potentiating action of ethanol (which is seen with only two of the cell t P ) .

SIGNIFICANCE

The mechanisms by which ethanol alters the subunit composition of GABA, and glutamate receptors in vivo without necessarily changing their density etc, yet may alter their function in cells without changing their subunit composition, remains a challenge for alcohol researchers. It is apparent that future progress will be greatly aided by the multi- disciplined approach exemplified by this symposium. Exploration of this puzzle will hopefully be a part of future RSA and ISBRA meetings.

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Neural Mechanisms of Adaptation in Chronic Ethanol Exposure and Alcoholism