differential effects of chronic ethanol administration on gabaa receptor α1 and α6 subunit mrna...

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Differential Effects of Chronic Ethanol Administration on GABA, Receptor GUI and c~6 Subunit mRNA Levels in Rat Cerebellum

A. LESLIE MORROW,*~~ JAMES S. HERBERT,* AND PASCALE MONTPIED~ *Department of Psychiatry and Center for Alcohol Studies, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599;

and tSection on Molecular Pharmacology, National Institute of Mental Health, Bethesda, Maryland 20892

Received for publication February 10, 1992

Chronic ethanol exposure alters muscimol, pentobarbital, and benzodiazepine agonist and inverse agonist effects on the function of GABA* receptor-gated Cl- channels in the central nervous system (CNS). We have recently shown that prolonged ethanol inhalation reduces the expression of GABA* receptor nl and (~2 subunit mRNAs in the rat cerebral cortex, with no effect on the level of (~3 subunit transcripts, glutamic acid decarboxylase mRNA levels, or poly(A)+ RNA levels. In the present study, rats were administered alcohol by liquid diet for 2 weeks using a pair-fed design. GABA* receptor a subunit mRNA levels were quantified by Northern analysis using specific cRNA probes. GABA* receptor al subunit mRNA levels were reduced in the cerebral cortex to the same extent as previously reported following prolonged ethanol inhalation. In the cerebellum, chronic ethanol ingestion reduced the levels of GABA* receptor rul subunit mRNAs (4.8 and 4.4 kb) by 20-30% and increased the levels of GABA* receptor a6 subunit mRNA (2.7 kb) by 45%. GABA* receptor a2 and (r3 subunit mRNAs were not detected in the cerebellum. Glutamic acid decarboxylase mRNA levels as well as poly(A)+ RNA levels were not significantly altered following chronic ethanol exposure by liquid diet. Acute ethanol administration had no effect on GABA., receptor a6 subunit mRNA levels. However, acute administration of both Ro15-4513 and its vehicle control altered GABA* receptor ar6 subunit mRNA levels in the cerebellum. Since GABA* receptor a6 subunits contain recognition sites for Ro15-4513, an inverse agonist, and an ethanol antagonist, the elevation in the expression of these subunits following chronic ethanol ingestion may account for increased sensitivity to inverse agonists after chronic ethanol administration and possibly contribute to the withdrawal syndrome. These data also suggest that chronic ethanol exposure regulates GABAA receptor gene expression by differential effects on the synthesis of specific subunits of GABA* receptors in the CNS. o 1992 Academic press, I~~.

1 To whom correspondence should be addressed at Department of Psychiatry, CB No. 7175, Med. Res. Bldg. A, UNC School of Medicine, Chapel Hill, NC 27599.

INTRODUCTION

Prolonged administration of ethanol to animals, in- cluding man, results in the development of tolerance and dependence, manifested by a reduction in ethanol’s behavioral effects and by a withdrawal syndrome following abrupt discontinuation (5,6). The molecular and cellular mechanisms underlying the development of tolerance and dependence are not well understood. It is generally ac- knowledged that many of the pharmacological actions of ethanol are mediated, in part, via an augmentation of GABAergic neurotransmission. There is ample biochemical evidence that ethanol augments GABAA receptor- mediated Cl- ion uptake in cerebral cortical synaptoneurosomes (7), cerebellar microsacs (8), and embryonic neurons in culture (9), suggesting that ethanol, like barbiturates and benzodiazepines, may enhance GABAA receptor-mediated Cl- ion conductance. Electrophysiolog- ical studies have confirmed that ethanol enhances GABAA receptor-mediated Cl- conductance, but only in specific brain regions (10-14) or cell populations (15, 16). We, and others, have suggested that the molecular composition of GABAA receptors determines the presence or absence of ethanol sensitivity (2,10,17). This hypothesis has been supported by electrophysiological evidence in frog oocytes, in which the expression of the y2L subunit is required for ethanol potentiation of GABA (18). Thus it appears that ethanol potentiation of GABAA receptors may be limited to very specific subtypes of GABAA receptors that have a unique regional distribution in brain (10).

GABAA benzodiazepine receptors are now known to constitute a family of receptors in the central nervous system (CNS) that are characterized by the presence of multiple distinct drug recognition sites and multiple distinct subunit compositions that constitute distinct GABAA isoreceptors’ (see Seeburg et al. (19) for review). While the actual heteroligomeric protein complexes that are expressed in brain are not known, it is clear that sub-

2The term isoreceptor (analogous to isosyme) refers to receptors composed of multiple protein subunits encoded by different genes that respond to the same neurotransmitter and produce the same biophysical response, albeit with different affinities, kinetics, and drug sensitivities (47).

251 1044-7431/92 $5.00 Copyright 0 1992 by Academic Press, Inc.

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252 MORROW, HERBERT, AND MONTPIED

unit composition determines the pharmacological and functional properties of these isoreceptors (20-22). The expression of the various (Y and y subunit isoforms has been shown to result in GABA* receptors with different pharmacological properties, which may account for the functional heterogeneity of GABAA receptors (17, 20, 21, 23, 24). Measurement of GABA* receptor expression in mammalian brain using radioligand binding techniques is limited by the fact that most GABA* receptor ligands do not distinguish between various GABA* receptor subunits and/or isoreceptors (23, 25). Alterations in the expression of a specific population of GABAA isoreceptors might not be detected using radioligands that are non- selective. Studies of GABAA receptor expression are therefore best conducted with subunit-specific antibodies or subunit-specific nucleic acid probes for the detection of specific mRNAs.

We, and others, have recently shown that chronic ethanol administration to rodents results in alterations in GABA receptor-mediated 36C1- uptake in brain. Chronic ethanol administration reduces muscimol and pentobarbital stimulation of chloride flux in cerebral cortex (26), abolishes ethanol potentiation of GABA-mediated chloride flux in cortex (26) and cerebellum (27), decreases GABA* receptor sensitivity to benzodiazepines, and increases GABA sensitivity to inverse agonists (4). We have postulated that these alterations in GABA* receptor-gated chloride ion channel function may contribute to the ethanol withdrawal syndrome (2, 26). The mechanism(s) responsible for the ethanol-induced changes in GABA* receptor function appears to involve an alteration in the expression of GABA* receptor(s). We have recently reported that chronic ethanol administration to rats by ethanol vapor inhalation selectively decreases the expression of GABAA receptor cul and a2 subunit mRNAs in the cerebral cortex, with no effect on GABA* receptor (~3 subunit mRNAs, glutamic acid decarboxylase mRNAs, or P-actin mRNA levels (2,28). We now report the effects of chronic ethanol administration by liquid diet ingestion using a pair-fed design on GABA* receptor (Y subunit mRNA levels in cortex and cerebellum, where GABA* receptor (~6 subunit mRNAs are detected, and compare these results to the effects of acute ethanol administration. Since Ro15-4513 binding sites have been identified on cerebellar a6 subunits (3), and since the biochemical and behavioral effects of ethanol are blocked, at least in part, by Ro15-4513 (29), the effects of acute Ro15-4513 administration on a6 subunit expression have also been in- vestigated and compared to the effects of acute ethanol administration.

MATERIALS AND METHODS

Ethanol Administration

In the chronic studies, ethanol was administered by a nutritionally complete liquid diet (Dextrose Diet, ICN

Biochemicals), according to the methods of Frye et al. (30, 31). Groups of 20 male rats (150-160 g) were given the liquid diet ad libitum for 3 days in order to acclimate to the diet. Ethanol administration was initiated by the addition of 5% ethanol (v/v) in the liquid diet. After 1 week, the ethanol concentration was increased to 7.5% (v/v) and ethanol intake (g/kg) was monitored daily. Ethanol concentrations were adjusted to maintain healthy liquid diet consumption. Control animals (8- 10/experiment) were pair-fed the identical diet with equicaloric dextrose substituted for ethanol. Water was available ad Zibitum to both groups. Dietary consumption of each animal was measured each day and indi- vidual body weights were recorded every other day throughout the experiments. The typical daily ethanol consumption using this diet was approximately 8-10 g/ kg, producing mean blood ethanol concentrations of 223 + 21 mg/dl at the time of sacrifice. The mean body weights for the control and pair-fed rats were similar at the termination of the experiments (190.1 f 2.6 vs 180.9 + 3.4 for control and ethanol-fed groups, respectively).

In acute studies (8 rats/group), ethanol (2.0 or 4.0 g/ kg) was administered by intraperitoneal injections of a 25% (w/v) solution to minimize discomfort associated with the injection of large volumes of concentrated ethanol. Control animals were injected with an equal vol- ume of saline. Ro15-4513 (10 or 30 mg/kg) was prepared as a suspension in 4% Tween-saline and injected ip. The Tween-saline vehicle and uninjected rats were used as controls. These concentrations of Ro15-4513 were used because they have been demonstrated to prevent and re- verse ethanol intoxication in rats (29,32). Blood ethanol concentrations were determined at the time of euthanasia for all studies. Trunk blood was collected and samples centrifuged at 2500 rpm. The serum was extracted with trichloroacetic acid and ethanol levels were assayed using the NAD-NADH enzyme spectrophotometric method (Sigma Chemical Co.).

Tolerance to ethanol and physical dependence upon ethanol have been demonstrated using this protocol (30, 31). In these studies, physical dependence was established by the presence of tremor and/or audiogenic seizures 6 h following removal of the ethanol diet. Rats treated con- currently with those used for the expression studies (n = 10) were placed individually in an open field. Tremor was scored according to the method of Frye et al. (30), in which rats were observed for tremor and limb extension following inverted elevation above the open field chamber by holding the tail and following 180” rotation around the axis of the tail. The presence of audiogenic seizures was tested by exposure to a loud tone (98 db) for 1 min. The presence of clonic tonic convulsions and wild running behavior was recorded. Tremor and limb extension were observed in all animals following ethanol withdrawal (see Results).

EFFECTS OF CHRONIC ETHANOL EXPOSURE 253

RNA Preparation

Poly(A)+ RNA was prepared using the method of Bad- ley et al. (33). Cerebral cortices or cerebelli from four to five rats/group were pooled and homogenized using a polytron in 10 vol of lysis buffer consisting of 200 mM NaCl, 200 mM Tris-HCl, pH 7.5, 1.5 mM MgClz, 2.0% SDS, and 200 pg/ml proteinase K, added just before use. The lysates were incubated for 1 h at 45°C with frequent inversions and then centrifuged at 1500 rpm for 5 min. The supematant was retained and adjusted to 0.5 M NaCl. The pellets containing protein and cellular debris were discarded. Poly(A)+ RNA was purified from nonadenyl- ated RNA in the lysate supernatant using a modification of the oligo(dT) column chromatography method of (34). Oligo(dT) cellulose was equilibrated by incubating the cellulose (1 mg/200 pg frozen tissue weight) in high salt buffer containing 0.01 M Tris-HCl, pH 7.5, and 0.5 M NaCl, washing the cellulose with sterile water followed by 0.1 M NaOH, and reequilibrating with 0.01 M Tris to pH 7.0. The lysates were incubated with the equilibrated oligo(dT) cellulose for 1 h at room temperature with con- stant rocking and transferred to columns for washing and elution of mRNA. The cellulose in the columns was thor- oughly washed with high salt buffer until all of the poly(A)- RNA was eluted. Poly(A)+ RNA was collected by elution with low salt buffer containing 0.01 M Tris, pH 7.5, and the RNA was precipitated with 100% ethanol (2.5 vol) overnight. The usual yield of poly(A)+ RNA was 0.003-0.005% of the frozen tissue weight.

Northern Analysis

Northern analysis was conducted as previously described (2). Poly(A)+ RNA samples (l-4 pg) were prepared for electrophoresis by denaturation in a formamide/ formaldehyde solution at 68”C, and separated overnight (30 V) in a 1% agarose gel containing 6% formaldehyde. The RNA was transferred by capillary action under pres- sure to nitrocellulose membranes (BRL, 1058HY) for 16 h and cross-linked to nitrocellulose via ultraviolet radia- tion using a UV Stratalinker (Stratagene).

The Northern blots were prehybridized for 1 h at 55°C in 50% formamide, 50 r&f Na2HP04, 5~ standard saline citrate (SSC), 0.1% sodium dodecyl sulfate (SDS), 1 mA4 EDTA, 0.05% Ficoll, 0.05% polyvinylpyrolidone, and 200 pg/ml salmon sperm DNA. The appropriate [32P]CTP- labeled antisense RNA probe (5 X lo5 cpm/ml) was added to the same solution and the hybridization carried out for at least 16 h at 55°C. Following hybridization, the filters were washed three times for lo-20 min in 0.1% SDS and 0.1% SSC, air-dried and exposed to X-ray film (Kodak AR) using intensifying screens. Hybridizations with each probe were conducted using multiple mRNA concentrations and the data analyzed from these experiments were quantified within the linear range of hybridization.

Densitometric measurements of the films were made using a light box (Northern Light Precision Illuminator),

a video camera (Model 4810, Cohu Electronics, Inc.), and image analysis software (Image 1.26 (35)) operated on a Macintosh IIci computer. Densitometric measurements were obtained following calibration employing Kodak calibration step tablet 809ST601 as a standard to control for the nonlinearity of optical density measurements. The variance in optical density measurements between replicate determinations in these experiments was less than 5% and large populations were compared statistically. Statistical comparisons of the data were made using analysis of variance or Student’s t test for paired samples. The data shown represent the mean of two to three separate experiments (n = 8-lO/group/experiment), using at least two replicate gels and three to four replicate or linear determinations in each gel.

[32P]CTP-labeled antisense cRNA probes, synthesized by SP6 or T7 RNA polymerase using linearized cDNAs as templates, were used for hybridization of the various mRNAs. The cDNAs used included the human GABA* receptor LY subunit, which labels rat al, a2, and (~3 subunits, distinguished by size following gel electrophoresis (2,36). This GABA* receptor (Y subunit cRNA probe corresponds to 726 bp of 5’ coding sequence and selectively identifies two rat al transcripts of 4.4 and 4.8 kb, a rat a2 transcript of approximately 8.0 kb, and the rat (~3 transcript of 3.1 kb (2). We have previously shown that this probe fails to hybridize to rat p subunit transcripts under these hybridization conditions (37). The rat GABA* receptor a6 subunit (25) cRNA probe corresponds to a 1.8-kb insert containing the complete coding sequence, which labels an mRNA transcript in rat cerebellum of about 2.7 kb. No (~6 subunit mRNA was detected in cerebral cortex. Glutamic acid decarboxylase (GADG7) (38, 39) and @-actin (40) probes were also synthesized for these experiments and used as controls. As has been previously reported for rat brain, the mRNA transcripts for GAD and P-actin were 3.7 and 1.9 kb, respectively (38, 40).

RESULTS

Chronic Ethanol Ingestion Produces Physical Dependence

Chronic ethanol administration for 14 days via the nutritionally complete liquid diet resulted in the development of physical dependence upon ethanol. Rats withdrawn from ethanol for 6 h were tested for the presence of tremor, piloerection, and the induction of wild running behaviors or audiogenic seizures. Withdrawal signs and symptoms were observed in all rats that were treated con- currently with those used for expression studies and withdrawn from ethanol for the determination of withdrawal severity (Table 1).

GABAA Receptor al Subunit mRNAs Are Reduced in Rat Cerebellum

Chronic ethanol ingestion decreased the expression of GABA* receptor al subunit mRNAs (4.8 and 4.4 kb) ap-


TABLE 1

The Effects of Chronic Ethanol Ingestion following Withdrawal

Behavior Score” % Responses

Tremor score Piloerection Audiogenic seizure Audiogenic wild running

2.75 f 0.13 100.0 N/A 100.0 N/A 50.0 WA 37.5

0 Tremor score was determined according to the method of Frye et al. (30) where 3 indicates the presence of tonic extension and whole body tremor upon inverted elevation from the floor of the observation chamber; 2 indicates the presence of these symptoms only upon 180” rotation with inverted elevation; 1 indicates forelimb extension and tremors following inverted elevation; and 0 indicates the absence of tremor. Data represent the means + SE of eight rats tested 6 h following the removal of the ethanol liquid diet.

proximately 20% in the rat cerebellum with no effect on the levels of GAD or P-actin mRNA (Table 2). Identical blots used for the analysis of GABAA receptor al subunit mRNA levels were used for rehybridization with both GAD and fi-actin. Actin mRNA levels were reduced in one experiment, increased in a second experiment, and unchanged in the third experiment. When the data from all three experiments were expressed as percentage control mRNA and combined, there was no significant effect of chronic ethanol administration. However, it should be noted that @actin mRNA levels were unstable and un- reliable following prolonged ethanol administration.

GABA* Receptor a6 Subunit mRNA Levels Are Increased in Cerebellum

In contrast, chronic ethanol ingestion resulted in a substantial increase in the levels of GABA* receptor (~6 subunit mRNAs in cerebellum (Figs. 1 and 2). While GA- BAA receptor (~1 subunit mRNA levels were reduced about 20%, the level of GABA* receptor a6 subunit mRNAs was increased approximately 50%. Since there was no change in GAD or P-actin mRNA levels, these alterations were deemed specific and reliable. However, in order to

TABLE 2

Effect of Chronic Ethanol Ingestion on GABA* Receptor al Subunit mRNA Levels in the Rat Cerebellum

mRNA

GABA* receptor cul subunit (4.8 kb) GABA* receptor al subunit (4.4 kb) GAD P-actin

Note. *P < 0.05; NS, not significant.

Percentage control mRNA level

81.3 + 5.8* 79.1 + 6.6*

111.0 + 6.8 (NS) 109.3 f 10.1 (NS)

4.8 4.4 a6 4.8 4.4 a6 a 1 Subunit a Subunit/GAD

FIG. 1. The effects of chronic ethanol ingestion (7-9 g/kg/day) on GABAA receptor cul and (~6 subunit mRNA levels in the rat cerebellum. Rats were administered a nutritionally complete liquid diet containing 5-7.5% ethanol or pair-fed the identical diet with equicaloric dextrose substituted for ethanol as described under Materials and Methods. Data were expressed as percentage control mRNA level for each determination. On the right half of the graph, the data for each determination of GABA, receptor cx subunit mRNA were normalized to the value for GAD mRNA in the exact same sample and expressed as percentage control mRNA level. Data are the means + SE from three separate experiments with 8-10 rats/group in each experiment. *P < 0.05; **P < 0.01.

control for the possibility that there were nonspecific effects of ethanol on GABA* receptor mRNA levels, we normalized each data point for the effect of chronic ethanol ingestion on GABA* receptor (Y subunit mRNA levels to the corresponding value for GAD mRNA levels (Fig. 1). This transformation of the data did not alter the interpretation that chronic ethanol ingestion increases GABA* receptor a6 subunit mRNA levels. However, the statistical significance of the effect of chronic ethanol ingestion on the 4.8-kb cul subunit transcript was reversed using this analysis, since there was greater variability in the GAD mRNA levels than in the GABA, receptor CY subunit mRNA levels (see Table 2). No difference in the total amount of poly(A)+ RNA was detected between groups in any experiment.

Acute Ethanol Administration Does Not Alter GABAA Receptor (~6 Subunit mRNA Levels

The increase in GABA, receptor a6 subunit mRNA levels following chronic ethanol ingestion was not observed following acute administration of ethanol (Fig. 3). There was no effect of ethanol (2 or 4 g/kg) or the com- bination of ethanol (2 g/kg) with Ro15-4513 (10 mg/kg) on GABA* receptor (~6 subunit mRNA levels 30 min following injection, when compared to saline-injected control animals. Acute ethanol administration also had no effect on GAD or P-actin mRNA levels (data not shown). In contrast, Ro15-4513 (10 mg/kg) appeared to increase GABA* receptor (~6 subunit mRNA levels compared to those of saline-injected controls. Since Ro15-4513 was


GABAI Receptor a 6 Subunit

2.7 kb

or mRNA turnover. Variable and inconsistent alterations in the levels of /3-actin mRNA in the cerebellum were observed in each of the three experiments, suggesting that P-actin mRNA levels may be unstable following chronic ethanol administration.

P-Actin

GAD67

1.9 kb

3.7 kb

FIG. 2. Chronic ethanol ingestion increases GABA* receptor (~6 subunit mRNA levels in rat cerebellum with no effect on GAD or @- actin mRNA levels. Rats were fed ethanol in a liquid diet as described in Fig. 1 and Northern blots were made using poly(A)+ RNA purified from pooled tissue samples. The data shown are representative blots using four RNA concentrations (0.2, 0.5, 1.0, and 2.0 pg) from each group to quantify the mRNA level in each group, respectively. Lanes l-4 are control samples and lanes 5-8 are samples from ethanol-fed rats. The same blot was rehybridized with each probe as a control for the specificity of the effects of ethanol on GABA, receptor (~6 subunit mRNA levels. There was no effect of ethanol ingestion on the levels of GAD or fl-actin mRNAs. Chronic ethanol ingestion increased the levels of GABA, receptor ~y6 subunit mRNAs to 145.9 f 19.4% of their re- spective controls, P < 0.01.

injected using a Tween-saline vehicle, this possibility was examined more carefully in two further experiments.

The Effects of Acute Rol5-4513 Administration

The effects of acute injections (10 or 30 mg/kg) of Ro15- 4513 on GABA* receptor a6 subunit mRNA levels were compared to uninjected and vehicle-injected control animals (Table 3). Acute Ro15-4513 administration increased GABAA receptor (~6 subunit mRNA levels only when compared to Tween-saline-injected control animals, but not when compared to uninjected control animals (Fig. 4). These effects were dose dependent and were most ap- parent when the data for each determination were normalized to the level of /3-actin mRNA on the same blots. Acute Ro15-4513 administration had no effect on /?-actin mRNA levels in either experiment.

DISCUSSION

The present studies have demonstrated a reduction (20%) in the level(s) of GABA* receptor al subunit mRNAs in the cerebellum of rats exposed to chronic (2 week) ethanol ingestion, with a concomitant increase (45%) in GABA* receptor (~6 subunit mRNA levels. No changes in the absolute levels of poly(A)+ RNA or in the levels of GAD mRNAs were observed, suggesting that the ethanol-induced alterations in (Y subunit mRNAs were

The functional significance of these alterations in GA- BAA receptor subunit mRNAs is unknown; however, Mhatre et al. (41) have reported a 77% increase in the density of specific binding sites for [3H]Ro15-4513 in the rat cerebellum following chronic ethanol administration as well as increased sensitivity to its behavioral effects (42). There is good agreement between the magnitude of the increase in Ro15-4513 binding and the increase in GABAA receptor a6 subunit mRNA levels found in the present study, considering the difference in blood ethanol concentrations produced in these studies (475 vs 223 mg/ dl, respectively). However, alterations in Ro15-4513 binding were also detected in cerebral cortex (41), where GABA* receptor a6 subunit mRNAs are not detected. Therefore, chronic ethanol administration may produce alterations in the expression of other GABAA receptor subunits, labeled by Ro15-4513, in the cerebral cortex or cerebellum. Chronic ethanol administration also increases the effects of Ro15-4513 on GABA receptor-mediated chloride flux and decreases the effects of benzodiazepine agonists (4). Ro15-4513 selectively labels the a6 subunit in the cerebellum (3) and, therefore, the increase in the expression of this subunit coupled with the decrease in the levels of al subunits may produce the alterations in the function and expression of GABA* receptors that have been observed.

We therefore propose that alterations in the expression of various GABA* receptor subunits following chronic ethanol exposure may account for the fact that chronic ethanol administration alters the function of GABAA receptors in biochemical studies. Thus, it seems plausible that chronic ethanol administration results in the expression of relatively more GABAA isoreceptors in the cerebellum composed of a6 subunits and relatively fewer GA-

Saline Control

EtOH 2 UkQ

EtOH EtOH Ro 154513

Ro f5-4513 4 g/kg 10 mg/kQ

FIG. 3. Acute ethanol administration has no effect on the level of GABA,, receptor (~6 subunit mRNAs in rat cerebellum. Rats were administered ethanol as described under Materials and Methods and sacrificed after 30 min. Ro15-4513 was administered 5 min prior to ethanol. The data shown are a representative blot using four RNA concentrations (0.2,0.5, 1.0, and 2.0 pg) for quantification from each treatment group. Hybridization signals were linear with RNA concentration. The same blot was rehybridixed with probe for GAD and fl-actin with similar results followine acute ethanol administration (see text). not the result of nonspecific alterations in transcription ~~~~


TABLE 3

Effects of Acute Vehicle and Ro15-4513 Injections on GABA* Receptor (~6 Subunit mRNA Levels in Rat Cerebellum

Percentage control mRNA level

Treatment group (n) a6 Subunit a6/&actin

Uninjected (9) Tween vehicle (9) Ro15-4513 (10 mg/kg) (11) Ro15-4513 (30 mg/kg) (11)

98.5 f 3.7 98.8 + 2.6 78.4 + 8.1 69.3 f 3.6*

112.2 + 7.8 107.0 f 6.3** 117.5 f 9.8** 136.0 + 14.5**

Note. Rats were administered Ro15-4513 or vehicle by intraperitoneal injection and sacrificed 30 min following the injection. Data are derived from two separate experiments, each using pooled poly(A)+ RNA from eight rats/group, with four to six replicate determinations on two Northern blots for each experiment. The data were expressed as percentage control optical density in order to collapse data from different autoradiograms. The mean of the control values on each autoradiogram was used to determine the percentage control value for each sample. Each blot was subsequently rehybridized with (3-actin probe and the optical density for (~6 mRNA was normalized to the optical density for P-actin mRNA (cY6/p-actin) and expressed as percentage control. AN- OVA, F = 4.635, P < 0.008; Tukey-Kramer HSD, *P < 0.05 compared to uninjected control; **P < 0.05 compared to Tween vehicle control.

BAA isoreceptors in the cerebellum composed of al subunits than control rats. However, mRNA levels are an indirect measure of GABA* receptor subunit expression and alterations in these levels should be interpreted with caution. The development of subunit-specific antibodies for immunohistochemical or Western blot analyses could help delineate whether actual changes in the expression of specific subunit proteins occur in ethanol-treated an-

2.7 kb

Control Uninjected

Veh 4% Tween

Saline

Ro15-4513 10 mg/kg

FIG. 4. Acute Ro15-4513 administration alters GABA* receptor a6 subunit mRNA levels in rat cerebellum compared to Tween-saline vehicle-injected control animals. Rats were administered Ro15-4513 or vehicle by intraperitoneal injection and sacrificed 30 min following the injection. Data are representative of two separate experiments, using triplicate determinations in pooled poly(A)+ RNA (2.5 pg/lane) from eight rats/group, with three to six replicate determinations on two Northern blots for each experiment. There was no effect of Ro15-4513 on GABAA receptor (~6 subunit mRNA levels when compared to unin- jetted control rats (see Table 3).

imals. In the case of a6 subunits, the fact that Ro15-4513 binding sites are also increased following chronic ethanol administration suggests that the alterations in mRNA levels are predictive of alterations in subunit expression. In the case of al subunits, it is important to note that the reduction in these mRNA levels following prolonged ethanol ingestion is consistent with studies showing a similar decrease in the density of low affinity binding sites for [3H]GABA (43) or [3H]muscimol (44) following chronic ethanol administration. Since GABA receptor- gated chloride uptake is mediated by low affinity binding sites (45), these data are also consistent with the reduc- tions in GABA* receptor function that we previously reported (26).

The present studies have confirmed and extended our previous reports (1, 2) that chronic ethanol inhalation reduces the levels of GABA* receptor cul subunit mRNAs in the cerebral cortex, using a different protocol for ethanol administration. The ethanol inhalation method produced higher blood ethanol concentrations and higher mortality than the liquid diet method (no mortality). In addition, the use of a pair-fed design for ethanol administration in the present studies has eliminated the potential effects of nutritional stress that may have been a factor in the ethanol inhalation studies. The fact that both par- adigms produced the same effect on GABA* receptor CY subunit mRNA levels in the cerebral cortex strongly suggests that these effects on GABA* receptor subunit mRNA levels are mediated by ethanol itself and not by artifacts of the mode of ethanol administration. Similar effects on GABA* receptor cul subunit mRNAs in whole brain have also recently been reported using PCR meth- odology for quantification of mRNAs in withdrawal seizure prone mice administered ethanol by liquid diet (46).

Acute ethanol administration (2 and 4 g/kg) to rats via ip injection had no effect on GABA* receptor (~6 subunit mRNA levels in the cerebellum compared to saline-injected controls. We have previously compared uninjected controls with saline-injected controls and found no differences in GABA* receptor cul subunit mRNA levels (unpublished data). In contrast, the benzodiazepine inverse agonist and behavioral ethanol antagonist, Ro15- 4513 (30 mg/kg), altered the levels of GABA* receptor a6 subunit mRNAs in the cerebellum when compared to vehicle-injected control rats. The fact that acute injection of Ro15-4513 produced an effect on the levels of GABA* receptor a6 subunit mRNAs that was similar to the effects of chronic ethanol administration suggests that Ro15- 4513 may promote the functional (and perhaps behavioral) changes associated with chronic ethanol administration. The injection of the Tween vehicle for the Ro15- 4513 had opposite effects on GABA.,, receptor a6 subunit mRNA levels, suggesting that it is very important in acute experiments to control for the potential effects of vehicle injections. These data also suggest that acute administration of toxic drugs, such as ethanol, may exert effects on GABAA receptor mRNA levels that are not adequately


controlled by saline injections. These effects may be re- lated to stress associated with the injection of the toxic agent.

The observation that chronic ethanol administration simultaneously decreases GABA* receptor al subunit mRNA levels and increases GABA* receptor (~6 subunit mRNA levels in the same region of rat brain suggests that substitution of receptor subunit expression may be an important mechanism of regulation of GABA* receptors. Indeed, a similar phenomenon has been observed for GA- BAA receptors during ontogenesis (37). Further studies will be required to determine whether these alterations in GABA* receptor subunit mRNA expression results in the functional alterations in GABA* receptors that have been observed following chronic ethanol administration.

ACKNOWLEDGMENTS

We thank Dr. Steven Paul (NIMH, Bethesda, MD) for the GABA, receptor (~1 subunit cDNA, Dr. Peter Seeburg (University of Heidelberg, Germany) for the (~6 subunit cDNA, Dr. Alan Tobin (UCLA, Los An- geles, CA) for the GADsr cDNA, and Dr. Donna Chikaraschi (Tufts University, Bedford, MA) for the P-actin cDNA used in these experiments. We also thank Heena P. Mehta for excellent technical assistance, Dr. Amir H. Rezvani for assistance with the measurements of ethanol withdrawal symptoms, and Dr. Wendy J. Keir for helpful discussions. This work was supported by PHS Grant AA09013 and the Center for Alcohol Studies. UNC School of Medicine.

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differential effects of chronic ethanol administration on gabaa receptor α1 and α6 subunit mrna...

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