CHANGES IN ETHANOL PREFERENCE BY RATS TREATED WITH ...

Alcohol & Alcoholism Vol. 36, No. 4, pp. 309–313, 2001

CHANGES IN ETHANOL PREFERENCE BY RATS TREATED WITH γ1 AND γ2 GABAA RECEPTOR SUBUNIT ANTISENSE OLIGODEOXYNUCLEOTIDES EWA MALATYNSKA1,2*, WANDA DYR3, PAWEL KRZASCIK3,4 and WOJCIECH KOSTOWSKI3,4 Indiana University School of Medicine, Departments of 1Pharmacology & Toxicology and 2Psychiatry, Evansville, IN 47712, USA, 3Institute of Psychiatry and Neurology, Department of Pharmacology and Physiology of the Nervous System, 02957 Warsaw and 4Medical Academy, Department of Experimental and Clinical Pharmacology, 00-927 Warsaw, Poland (Received 12 October 2000; in revised form 22 January 2001; accepted 5 February 2001) Abstract — Micro-injections (10 nmol/day over 5 days) of antisense oligodeoxynucleotides (aODNs) to γ-aminobutyric acid A (GABAA) receptor α1 and γ2 subunits reduce the mRNA for these subunits in rat brain. In this study, the effects of α1 and γ2 subunit aODNs on rat alcohol preference were investigated. Reduction of the α1 subunit mRNA decreased, whereas reduction of the γ2 subunit mRNA increased, ethanol intake in rats.

hypothesis by investigating the effect of α1 and γ2 GABAA receptor subunit knockdown by antisense oligodeoxynucleotides (aODNs) on voluntary ethanol consumption.

INTRODUCTION The GABAA receptor–chloride ionophore complex mediates much of the pharmacological activity of ethanol (for review see Little, 1991; McBride and Li, 1998; Kostowski and Bienkowski, 1999; Mehta and Ticku, 1999). However, the mechanism of this mediation and its influence on alcohol preference behaviour are not completely understood. Biochemical studies have suggested the involvement of GABAA receptors in the mechanism of alcohol preference. GABAA receptor agonists, such as muscimol and 4,5,6,7tetrahydroisoxazolo[5,4- c]pyridin-3-ol (THIP), can increase voluntary ethanol intake and decrease withdrawal syndromes (Smith et al., 1992; Boyle et al., 1993; Tomkins et al., 1994). GABAA receptor antagonists such as bicuculline and SR95531 (Hyytia and Koob, 1995; Nowak et al., 1998) and benzodiazepine receptor inverse agonists such as Ro15-4513 (June et al., 1998) can conversely decrease ethanol intake and increase withdrawal syndromes (Becker and Anton, 1989). The GABAA receptor is a pentameric protein complex consisting of two α two β and one γ, δ or ρ subunit that combine using different subtypes of these subunits to form the chloride ionophore (for review see Rabow et al., 1995; Mehta and Ticku, 1999). Following chronic ethanol administration, the levels of the mRNA and/or polypeptides of α1, α2, α3 and α5 subunits were reduced, whereas those of α4, α6, β2/3, γ1 and γ2S subunits were elevated and γ2L, γ3 and δ subunits were unchanged in the rat cerebral cortex and/or cerebellum (for review see Mehta and Ticku, 1999). The participation by the α6, β3, γ2S, or γ2L subunits in many of the effects of ethanol, such as anxiolysis, ataxia, tolerance, dependence and withdrawal has been studied using GABAA receptor subunit gene knockout mice (for review see Mehta and Ticku, 1999). However, these studies did not include measurement of ethanol intake or preference for these mutant mice. We postulate that the molecular composition of the GABAA receptor, already known to be important for mediating the effects of alcohol on the central nervous system, might contribute to the behavioural preference for ethanol consumption. In the experiments described here, we have directly tested this

MATERIALS AND METHODS Antisense nucleotides Antisense oligodeoxynucleotides were designed from sequences of rat GABAA receptor subunits (Gene Bank accession numbers: α1, L08490; γ2, L08497). This was described elsewhere (Malatynska et al., 2000a,b). The sequences were: (1) α1 subunit antisense ODN (ORF): 673–690: 5′-CTGAACAATTCCAGAGTC-3′; (2) α1 subunit missense ODN (ORF): 673–690: 5′-CTCTTATCGCGAAAACAG-3′; (3) γ2 subunit antisense ODN 28–45 (ORF): 5′-AGAGTAGACTGTGCTTCC-3′; (4) γ 2 subunit antisense ODN did not differentiate between γ 2S and γ2L subtypes. Animals Adult (250–280 g) male WHP (Warsaw High-Preferring) rats from the generation F16 were used in the present study. WHP rats voluntarily drink at least 5 g of ethanol/kg/24 h (Bisaga and Kostowski, 1993; Dyr et al., 1999). WHP rats were housed individually in stainless steel cages in a room with constant temperature (20 ± 2°C), humidity (60–65%) and a 12-h light/12-h dark cycle (lights on 08.00). Rats had free access to granulated food and fluids. Tap water and 10% (v/v) aqueous ethanol solution were presented in two calibrated tubes attached to the front of each cage. Micro-injections The animals were placed in a Stoelting stereotaxic apparatus and the right cerebral ventricle was implanted with stainless steel guide cannulae. All cannulations were performed with rats under ketamine anaesthesia (75 mg/kg, i.p.). The stereotaxic coordinates were: A = –0.8 mm; L = 1.5 mm from Bregma; 2.7 mm past the surface of the skull. Rats were allowed to recover for 10 days after surgery to ensure that ethanol drinking returned to the pre-surgery level. The aODNs were injected into the right cerebral ventricle of the rat brain once a day at a dose of 10 nmol/4 µl/day for 5 days. The aODNs were dissolved in water. The same volume of vehicle

*Author to whom correspondence should be addressed. 309

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(control) was injected into the control animals. The duration of one injection was 5 min to avoid osmotic effects. Previously we have shown that the results of micro-injections of missense ODNs and water did not differ (e.g. on mRNA level or on GABA-stimulated 36Cl–). Thus, in this study, we used sterile water micro-injected rats as controls. Verification of the cannula placement At the end of the experiment, the rats were anaesthetized with ketamine and micro-injected with Methylene Blue (1 µl) 1 min before decapitation. The brain of each animal was removed, sectioned on a freezing microtome and histological placements of cannulae were identified using the atlas of Pellegrino et al. (1979). Ethanol and water intake measurement The free choice intake of tap water or the 10% ethanol solution in tap water was measured in the graduated drinking tubes and converted to a value in ml/kg/24 h of absolute ethanol equivalents. The ethanol intake was measured during the 4 weeks before surgery. It was also measured after surgery at 24 h before micro-injections, to ascertain that it remained at the pre-surgery (basal) level and 24 h after each micro-injection (aODNs related level). The basal levels of ethanol and water intakes were normalized to 100% and then measured every day 24 h after each micro-injection. The results were calculated for each rat as a percentage of the basal ethanol intake and then the mean ± SEM were calculated for the group. Food intake measurement Food intake was measured for individual rats in g/kg/24 h. The food intake was measured during the four weeks before surgery (range 50–60 g/kg/24 h) and at the basal level a week after surgery (range 45–62 g/kg/24 h). The 24-h food intake was then measured after each micro-injection. The results were calculated for each rat as a percentage of the basal level of food intake that was normalized to 100% and then the mean ± SEM were calculated for the group. Statistical analysis The effect of micro-injections with α1 and γ2 aODNs on ethanol intake was compared to water-injected controls using 2-way analysis of variance (ANOVA) performed with StatView IITM software (Abacus Concepts, Inc. Berkeley, CA, USA). The dependent measure for two-way ANOVA was the level of ethanol intake and the independent measures were treatment groups and treatment time. The results of the ANOVA were followed by post hoc analysis using Fisher’s test. The effects of micro-injections with α1 and γ2 aODNs on water and food intakes were compared to water-injected controls using one-way ANOVA. The dependent measure for one-way ANOVA was the level of water or food intake, and the independent measures were treatment groups. The results from ANOVA were followed by use of the t-test with equal or unequal variances using Excel Version 5.0 for the Macintosh. RESULTS All animals in the study drank ethanol before and after surgery. The amount of ingested ethanol was in the range

5.18–9.20 g/kg/24 h before the surgery and 5.23–9.60 g/kg/ 24 h after the surgery. Micro-injections of the α1 or γ2 subunit of the GABAA receptor aODNs (10 nmol/day for 5 days) altered ethanol intake (Fig. 1). The treatment group effect was significant at P = 0.001; F = 7.1 (ANOVA). The post hoc Fisher’s test revealed a significant effect of α1 and γ2 subunits of the GABAA receptor aODNs at P = 0.03 and P = 0.05, respectively, as compared with controls. Micro-injections of the α1 subunit of the GABAA receptor aODNs (10 nmol/day for 5 days) resulted in a treatment-dependent decrease in ethanol intake (Fig. 1). The results significantly differed from ethanol intake by water micro-injected rats (controls) by the fourth or fifth day of treatment. The ethanol intake for α1 subunit micro-injected rats was 64.5 ± 13.7% on the fourth day and 59.5 ± 12.6% on the fifth day when normalized to the initial ethanol intake (94.3 ± 3.8 ml/24 h/kg) by these rats. The ethanol intake for water micro-injected rats was 97.7 ± 8.7% on the fourth day and 85.7 ± 8.1% on the fifth day when normalized to the initial ethanol intake (73.5 ± 4.6 ml/24 h/kg). These data were significantly different for the fourth and fifth days of micro-injections with P = 0.02 and P = 0.04, respectively (t-test). Rats micro-injected with aODNs encoding for the γ 2 subunit fragment of the GABAA receptor (10 nmol/day for 5 days) showed a significant increase in ethanol intake on the fifth day relative to controls (Fig. 1). The ethanol intake for γ2 subunit aODNs micro-injected rats was 125 ± 24% on the fifth day when normalized to the initial ethanol intake (74.4 ± 5.0 ml/24 h/kg) by these rats. These data were significantly different from water micro-injected rats at P = 0.05 (t-test).

Fig. 1. Free choice ethanol intake. Male rats were micro-injected with water (j), α1 (d) or γ2 (m) γ-aminobutyric acid A receptor subunit antisense deoxynucleotides (aODNs) (10 nmol/4 µl/day for 5 days). The basal initial values for ethanol intake in water, α1 or γ2 subunit aODN-treated animals were 73.5 ± 4.6, 94.3 ± 3.8 and 74.4 ± 5.0 ml/24 h/kg, respectively. The results are presented as a mean percentage of the basal ethanol intake ± SEM. The numbers of animals were 11 in the control group, eight in the α1 aODNs group and seven in the γ2 aODNs group. *Significantly different from controls (water micro-injected rats) at P < 0.05 (t-test).

GABAA RECEPTOR SUBUNITS AND ALCOHOL CONSUMPTION

Water intake by the same group of rats was inversely related to ethanol intake, being increased after micro-injections of aODNs for α1 subunit of the GABAA receptor and decreased after micro-injections of aODNs for γ2 subunit of the GABAA receptor (Fig. 2). A significant group effect (aODNs versus water) was observed for γ2 subunit aODN treatment at (P = 0.03; F = 3.9; ANOVA). However the t-test following ANOVA did not reach statistical significance between the aODN treatment groups and the controls. The initial values (before micro-injections) for water intake were 13.4 ± 7.9, 36.2 ± 12.6 and 10.0 ± 4.4 ml/24 h/kg for GABAA receptor α1 and γ2 subunit aODNs and water micro-injected rats, respectively. There was no statistically significant difference between these initial values. The high value for group 2 animals is due to three out of seven rats exhibiting very high water intakes. There was a significant (P = 0.04; F = 3.85) treatment group (α1 or γ2 subunit aODNs or water-treated animals) effect on food intake as revealed by one-way ANOVA. This was followed by a significant decrease (P < 0.05) in food intake after micro-injections of aODNs for γ2 subunit of the GABAA receptor as compared with controls (Fig. 3). The absolute initial values for food intake in these groups were 58.6 ± 4.8 and 45.9 ± 4.5 g/24 h/kg, respectively. There was no difference in food intake by control (water micro-injected) rats and rats treated with α1 GABAA receptor aODNs. However, a tendency for increased food intake can be noticed after microinjections of aODNs for α1 subunit of the GABAA receptor as compared with controls. The absolute initial value for food intake in this group was 41.3 ± 6.2 g/24 h/kg. DISCUSSION The major finding of this study was that genetically selected WHP rats treated intracerebroventricularly with aODNs derived from α1 subunit of the GABAA receptor had decreased ethanol intake after 4–5 days of treatment, whereas aODNs encoding for the γ2 subunit of the GABAA receptor increased ethanol

Fig. 2. Free choice water drinking. Male rats were micro-injected with water, α1, or γ2 γ-aminobutyric acid A receptor subunit antisense deoxynucleotides (aODNs) at a dose of 10 nmol/4 µl/day for 5 days. The basal values (before micro-injections) for water intake were 13.4 ± 7.9, 36.2 ± 12.6 and 10.0 ± 4.4 ml/24 h/kg respectively. The results are presented as a mean percentage of the basal water intake ± SEM. The number of animals in each group was the same as described for ethanol intake in the legend to Fig. 1.

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Fig. 3. Food intake. Food intake in three groups of rats, micro-injected with water, α1 or γ2 γ-aminobutyric acid A receptor subunit antisense deoxynucleotides (aODNs) at a dose of 10 nmol/4 µl/day for 5 days. The basal initial values for food intake in α1 or γ2 subunit aODN- or water-treated animals were 58.6 ± 4.8, 45.9 ± 4.5 and 41.3 ± 6.2 g/24 h/kg, respectively. The results are presented as a mean percentage of the basal food intake ± SEM. The number of animals in each group was the same as described for ethanol intake in the legend to Fig. 1. *Significantly different from controls (water micro-injected rats) at P < 0.05 (t-test).

intake after 5 days of micro-injections, as compared to watertreated controls. These findings support a conclusion that GABAA receptors with these subunits are involved in voluntary ethanol intake in WHP rats. The water and food intakes by each group of rats had a tendency to be inversely related to their ethanol intake. The former were increased after micro-injections of aODNs for α1 subunit of the GABAA receptor and decreased after microinjections of aODNs for γ2 subunit of the GABAA receptor. However, only the decrease in food intake after micro-injections with γ2 GABAA receptor subunits reached statistical significance. This effect may result from caloric substitution of food by ethanol (Colombo, 1997) or may indicate the role for the γ2 subunit in food intake (Sugrue, 1987; Leonhardt et al., 1999). The decrease of specific subunit mRNA or protein level of the GABAA receptors was demonstrated for α1, 4, 6, γ2 subunits (Brussaard and Baker, 1995; Karle et al., 1995; Zhu et al., 1996; Smith et al., 1998; Zhao et al., 1998). We have previously used aODNs for the α1 and γ2 GABAA receptor subunits to demonstrate a 30–40% decrease of mRNA for α1 and γ2 subunits as well as a 40–50% decrease in specific [3H]GABA and [3H]flunitrazepam binding, respectively (Malatynska et al., 2000a,b). In this study, we have shown that lowering the level of the α1 subunit of the GABAA receptor in the rat cerebral cortex decreased voluntary ethanol intake in WHP. Other investigators have shown that chronic treatment with ethanol (5–14 days) decreases the mRNA and protein levels of the α1 subunit of the GABAA receptor in the rat cerebral cortex. Furthermore, the level of α1 subunit returns to baseline following the cessation of the ethanol treatment (for review see Mehta and Ticku, 1999). These results suggest that the α1 subunit of the GABAA receptor could mediate a preference for alcohol, whereas tolerance to the effects of alcohol may occur through a downregulation of the subunit. This suggestion is supported by studies on cell lines stably expressing different α subunits accompanied by the same β and γ subunits, showing that, after treatment with ethanol,

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muscimol stimulated 36Cl– uptake in L(tk–) cells stably expressing α1β3γ2S, but failed to do so in L(tk–) cells stably expressing α6β3γ2S (Harris et al., 1997). GABAA receptors in the rat cerebral cortex and in the hippocampus consist of many subunit variations. The predominant GABAA receptor subunit combination in cerebral cortex is α1β2γ2, whereas in the hippocampus the α5β1γ2 (or complexes where the α2 and α3 subunits substitute for α5) predominates (for review see Rabow et al., 1995). Many investigators observed ethanol enhancement of the response to GABA in the rat cerebral cortex (Reynolds and Prasad, 1991; Proctor et al., 1992) whereas the majority of investigators failed to observe this effect in the rat hippocampus (Carlen et al., 1985; Gage and Robertson, 1985; Mancillas et al., 1986; Siggins et al., 1987). The present finding indicates that animals with reduced levels of the γ 2 subunit of the GABAA receptor in the cerebral cortex have increased ethanol consumption. It was shown by other investigators that chronic ethanol exposure increases mRNA and/or protein level for γ2 subunit of the GABAA receptor and that this increased level returns to normal after cessation of ethanol exposure (Devaud et al., 1996). The mechanism by which the reduced level of the γ2 subunit of the GABAA receptor could increase alcohol intake remains unclear. One possible explanation is that low levels of γ2 subunit are associated with changes in the aversive and/or rewarding effects of ethanol. Notably, picrotoxin administration has been reported to attenuate ethanol-induced conditioned taste aversion (Smith et al., 1989). In summary, our study shows that rat ethanol preference behaviour is sensitive to the changes in GABAA receptor subunit composition. It is inhibited by low levels of α1 subunits and is potentiated by reduced levels of the γ2 subunit. These findings may suggest that GABAA receptors mediate either the rewarding or aversive effects of ethanol that contribute to voluntary ethanol consumption. Acknowledgements — The authors thank Drs Richard Knapp and Godfrey Tunnicliff for reading the manuscript and for helpful suggestions.

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