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. Author manuscript; available in PMC: 2008 Oct 12.
Published in final edited form as: Brain Res. 2007 Aug 16;1174:47–52. doi: 10.1016/j.brainres.2007.07.077

Effect of chronic administration of ethanol on the regulation of the δ-subunit of GABAA receptors in the rat brain

CR Marutha Ravindran 1, Ashok K Mehta 1, Maharaj K Ticku 1
PMCID: PMC2278031  NIHMSID: NIHMS32377  PMID: 17854781

Abstract

In the present study, we investigated the effect of chronic ethanol (CE) administration on the polypeptide levels of the δ-subunit of GABAA receptors and [³H]muscimol binding to the immunoprecipitated δ-subunit-containing GABAA receptor assemblies in the rat brain. CE administration resulted a down-regulation of polypeptide levels of the δ-subunit of GABAA receptors in the rat cerebellum and hippocampus, whereas there were no changes in the δ-subunit polypeptide levels in the rat cerebral cortex. Further, CE administration caused a down-regulation of native δ-subunit-containing GABAA receptor assemblies in the rat cerebellum as determined by [³H]muscimol binding to the immunoprecipitated receptor assemblies. These results indicate that the δ-subunit-containing GABAA receptors may play a role in chronic ethanol-induced tolerance and dependence.

Keywords: Chronic ethanol, GABAA receptors, δ-Subunit, Polypeptide levels, Immunoprecipitation, Radioligand binding

1. Introduction

Extensive structural heterogeneity exists among various subtypes of GABAA receptor since native pentameric receptor assemblies are derived from several subunits such as α1–6, β1–3, γ1–3, δ, ε, π, and θ (see Mehta and Ticku, 1999a; Sieghart et al., 1999; Whiting, 1999). Most common subunits-combination for GABAA receptors in the brain is α1β2γ2 (Fritschy et al., 1992). GABAA receptors gene expression is affected by physiological and pathological processes as well as by the drugs that modulate GABAA receptors (Aguayo et al., 2002; Cagetti et al., 2003; Follesa et al., 2003; Sanna et al., 2003). Ethanol is known to modulate the activity of a variety of receptors and ion channels (Lovinger et al., 1989; Lovinger and White 1991; Lovinger, 1999; Lewohl et al., 1999; Mihic, 1999; Narahashi et al., 1999; Woodward, 1999; Walter and Messing, 1999; Weight et al., 1999; Lei et al., 2000; Aguayo et al., 2002; Carta et al., 2003; Davies et al., 2003; Roberto et al., 2003; Gehlert et al., 2007), and role of GABAA receptors in the behavioral effects of ethanol is well documented (see Ticku and Mehta, 1995).

Chronic ethanol (CE) as well as chronic intermittent ethanol (CIE) treatment is known to modulate the expression of the major subunits of GABAA receptors in the brain (Montpied et al., 1991; Mhatre and Ticku, 1992, 1994; Devaud et al., 1995, 1997; see Mehta and Ticku, 1999a; Cagetti et al., 2003; Marutha Ravindran and Ticku, 2006a, 2006b; Marutha Ravindran et al., 2007). Recently, it has been suggested that the GABAA receptor derived from δ-subunit is important site for the pharmacological actions of ethanol (Mihalek et al., 2001; Sundstrom-Poromaa et al., 2002; Cagetti et al., 2003; Wallner et al., 2003; Wei et al., 2004; Hanchar et al., 2004; Glykys et al., 2007). On the other hand, there are also reports contradicting the involvement of the δ-subunit-containing GABAA receptors in the pharmacology of clinically relevant low concentrations of ethanol (Borghese et al., 2006; Yamashita et al., 2006; Casagrande et al., 2007). Recently, we have reported that ethanol (up to 50 mM) does not affect [³H]muscimol binding to the immunoprecipitated δ-subunit-containing GABAA receptor assemblies in the rat cerebellum and hippocampus, thereby suggesting that the native δ-subunit-containing GABAA receptors do not play a major role in the pharmacology of clinically relevant low concentrations of ethanol (Mehta et al., 2007). In the present study, we investigated the effect of CE administration on the polypeptide levels of the δ-subunit of GABAA receptors in the rat cerebellum, hippocampus and cerebral cortex. Further, we investigated the effect of chronic administration of ethanol on the regulation of native δ-subunit-containing GABAA receptor assemblies in the rat cerebellum so as to examine whether δ-subunit-containing GABAA receptors are a potential target site for the chronic ethanol-induced phenomena.

2. Results

2.1. Effect of CE administration on the polypeptide levels of the δ-subunit of GABAA receptors in the rat brain

Western blot experiments revealed that chronic administration of ethanol causes down-regulation of the polypeptide levels of the δ-subunit of GABAA receptors in the rat cerebellum (Fig. 1A–a,c; ANOVA F2,6 = 43.59, p = 0.0003; Dunnett’s multiple comparison p < 0.01) and hippocampus (Fig. 1B–a,c; ANOVA F2,6 = 29.79, p = 0.0008; Dunnett’s multiple comparison p < 0.01). These changes in the polypeptide levels of δ-subunit reverted back to control levels in the ethanol-withdrawn group (48 h) as shown in Fig. 1A and Fig. 1B. However, chronic administration of ethanol did not result any change in the polypeptide levels of the δ-subunit of GABAA receptors in the rat cerebral cortex (Fig. 1C–a,c; ANOVA F2,6 = 1.104, p = 0.39; Dunnett’s multiple comparison p > 0.05). Notably, there was equal intensity of the protein actin band following probing with actin antibody by Western blotting (Fig. 1A–b, Fig. 1B–b and Fig. 1C–b).

Fig. 1.

Fig. 1

Fig. 1

Fig. 1

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Effect of chronic administration of ethanol and its withdrawal on the polypeptide levels of the δ-subunit of GABAA receptors in the rat (A) cerebellum, i.e., CB, (B) hippocampus, i.e., HC, and (C) cerebral cortex, i.e., CC. Panel (a): Western blot for the GABAA receptors δ-subunit polypeptide; panel (b): reprobed with actin; and panel (c): percentage polypeptide levels of the δ-subunit of GABAA receptors. Molecular mass of the δ-subunit of GABAA receptors was found to be 54 kDa in Western blot analysis. Each value is % mean ± S.E.M derived from three different Western blots performed on different days using pooled tissue from 10 rats in each group. % Polypeptide levels were calculated by comparing the intensity of each band with the average control intensity (100%) from three different Western blots as detailed in materials and methods. Lane 1: proteins extracted from control tissues (saline-administered rats); lane 2: proteins extracted from tissues harvested from chronic ethanol-administered rats; lane 3: proteins extracted from tissues harvested from ethanol-withdrawn rats (48 h).

*p < 0.01 as compared to the control group by one-way analysis of variance (ANOVA) followed by Dunnett test.

2.2. Effect of CE administration on the GABAA receptor assemblies derived from δ-subunit in the rat cerebellum

Antiserum for the rat GABAA receptors δ-subunit immunoprecipitated 18.0 ± 1.1% of the GABAA receptor assemblies in the rat cerebellum as determined with [³H]muscimol (Table 1). Chronic administration of ethanol to the rats decreased the percentage immunoprecipitation of the binding activity for the GABAA receptor assemblies derived from δ-subunit in the rat cerebellum as determined with [³H]muscimol (11.1 ± 0.7% vs. 18.0 ± 1.1%, ANOVA F2,15 = 16.67, p = 0.0002, Dunnett’s multiple comparison p < 0.01, Table 1), and this effect reverted to control level following withdrawal of chronic ethanol treatment (48 h) as shown in Table 1.

Table 1.

Effect of chronic administration of ethanol on the quantitative immunoprecipitation of the δ-subunit-containing GABAA receptor assemblies in the rat cerebellum.

Group % Immunoprecipitation of the binding activity
[³H]Muscimol (36 nM)  
   
Control 18.0 ± 1.1
   
Chronic ethanol 11.1 ± 0.7*
   
Ethanol withdrawn (48 h) 17.6 ± 0.8
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The values are mean ± S.E.M. of six individual experiments, each performed in triplicate using the pooled cerebellum from ten rats in each group. Immunoprecipitations were done using 30 µl of the antiserum specific for the rat δ-subunit of GABAA receptors. [³H]Muscimol (36 nM) binding (100%) to the solubilized receptors (pellet+supernatant) of the rat cerebellum was 0.28 ± 0.02 pmol/mg protein.

*

p < 0.01 as compared to the control group by one-way analysis of variance (ANOVA) followed by Dunnett test.

3. Discussion

Role of GABAA receptors in the pharmacology of ethanol has been investigated in detail (see Ticku and Mehta, 1995; Mehta and Ticku, 1999a; Sieghart et al., 1999; Whiting, 1999; Hanchar et al., 2004). The subunit composition and stoichiometry of native GABAA receptors are currently unknown, but the most abundant population of native GABAA receptors in the mammalian brain is believed to be the α1β2γ2 subunit combination (Benke et al., 1991; Fritschy et al., 1992; McKernan and Whiting 1996), and these subunits are present in abundant amount in almost every region of the brain (see Mehta and Ticku 1999a). Chronic administration of ethanol is reported to reduce the levels of α1-, α2-, and α5-subunits mRNA and polypeptide levels in cerebellum and cerebral cortex (Mhatre and Ticku 1992; Devaud et al., 1997; Marutha Ravindran et al., 2007). However, the α4-, γ1-, γ2S- (Devaud et al., 1995), α6- (Mhatre and Ticku 1992) and β2/3-subunits (Mhatre and Ticku 1994) mRNA levels increase in the ethanol-dependent animals. CE as well as CIE exposure of cultured cortical neurons of mice results in up-regulation of the β2-subunit, down-regulation of the α1-subunit and no change in the polypeptide levels of the γ2-subunit of the GABAA receptors (Marutha Ravindran and Ticku, 2006a, 2006b).

Our present study revealed that chronic administration of ethanol results in a down-regulation of the polypeptide levels of the δ-subunit of the GABAA receptors in the rat cerebellum and hippocampus. However, chronic administration of ethanol did not elicit any changes in the polypeptide levels of the δ-subunit of GABAA receptors in the rat cerebral cortex. Our results with CE paradigm are consistent with the report indicating that CIE treatment of rats elicits a decrease in the polypeptide levels of the δ-subunit of the GABAA receptors in hippocampus (Cagetti et al., 2003). On the other hand, there is also a report indicating that CE treatment of cultured cerebellar granule neurons causes a statistically insignificant reduction in the expression of δ-subunit (Follesa et al., 2005). However, these researchers also observed a statistically significant reduction in the expression of δ-subunit in the cultured cerebellar granule neurons at 6 h of ethanol-withdrawal following CE paradigm, which returned to control values at 12 h of ethanol-withdrawal (Follesa et al., 2005). Further, these researchers found that CE paradigm results in an increase in the expression of δ-subunit in cultured hippocampal neurons (Follesa et al., 2005). These differences may be due to different experimental models (intact rats versus cultured neurons). Notably, CE-induced changes in polypeptide levels of δ-subunit of GABAA receptors in our present study reverted back to normal levels in the rat cerebellum and hippocampus following 48 h of ethanol withdrawal.

Consistent with chronic ethanol-induced down-regulation of the polypeptide levels of the GABAA receptors δ-subunit in our study, we also observed a down-regulation of δ-subunit-containing GABAA receptor assemblies in the rat cerebellum as determined by immunoprecipitation followed by [³H]muscimol binding. It is possible that chronic ethanol-induced down-regulation of native δ-subunit-containing GABAA receptor assemblies may be due to altered receptor trafficking, as reported recently in the case of α1-subunit-containing GABAA receptors (Kumar et al., 2003). Down-regulation of the polypeptide levels of the GABAA receptors δ-subunit as well as δ-subunit-containing GABAA receptor assemblies following the chronic administration of ethanol may alter the GABAergic transmission and synaptic responses, thereby leading to tolerance and dependence. Thus our study suggests δ-subunit-containing GABAA receptors as an important potential target for the treatment of chronic ethanol-induced tolerance and dependence.

In summary, CE administration caused a down-regulation of the polypeptide levels of the GABAA receptors δ-subunit in the rat cerebellum and hippocampus. Further, chronic administration of ethanol resulted in a down-regulation of native δ-subunit-containing GABAA receptor assemblies in the rat cerebellum. These CE-induced changes in the δ-subunit-containing GABAA receptors may have important implications in chronic ethanol-induced dependence and tolerance.

4. Experimental procedures

Adult male Sprague-Dawley rats (Harlan, Indianapolis IN, U.S.A.) weighing 200–250 g were maintained at a constant room temperature (22°C) on a 12-h light/12-h dark cycle. All experiments were conducted in accordance with the Declaration of Helsinki and/or with the Guide for the Care and Use of Laboratory Animals, as adopted and promulgated by the National Institutes of Health. Adequate measures were taken to minimize pain or discomfort to the animals. Food and water were available ad libitum. They were divided into three groups: saline control, ethanol-maintained (chronic ethanol-administered) and ethanol-withdrawn rats.

4.1. Chronic administration of ethanol to the rats

The animals were intoxicated by intragastric intubation method for 6 days as described earlier (Majchrowicz 1975; Mhatre et al., 1988; Mehta and Ticku 1999b; Marutha Ravindran et al., 2007). Briefly, at the beginning of the experiment, a priming dose of ethanol of 5 g/kg (20% v/v in normal saline) followed by 9 g/kg (20% v/v in normal saline) over 24 h period in three divided doses was administered orally to all animals for six days. The control rats received normal saline. Chronic ethanol-maintained rats were sacrificed 1 h after the last dose of ethanol and the ethanol-withdrawn rats were sacrificed 48 h after the last dose of ethanol. Different regions of the rat brain were dissected, and tissues were stored at −80°C until use.

4.2. Electrophoresis and immunoblotting

Tissues were homogenized in ice-cold lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 1% Triton X 100, 10 mM NaF, 1 mg/ml bacitracin, 50 mM β-glycerophosphate, 1 mM phenyl methyl sulfonyl fluoride (PMSF), 1 mM N-ethylmaleimide, 1 mM Na3VO4, 10 µg/ml leupeptin). These samples were centrifuged (2300 g, 10 min, 4°C) so as to remove the supernatant containing protein (Bjornstrom et al., 2002; Marutha Ravindran and Ticku, 2006a, 2006b; Marutha Ravindran et al., 2007). Fifty micrograms of protein was boiled in a boiling water-bath for 5 min to denature the proteins, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli, 1970) and transferred to polyvinylidene difluoride membrane followed by treatment with 5% nonfat dry milk (Nestle, Ohio, USA) in Tris-buffered saline containing 0.1% Tween 20. Following this, the membrane was incubated with 1:100 dilution of the antiserum for the GABAA receptors δ-subunit since preliminary experiments revealed that 1:100 dilution of the δ-subunit antiserum yields the intensity of its protein band in a linear range. The membrane was washed several times and peroxidase-coupled secondary antibody (anti-mouse IgG/anti-rabbit IgG; New England Biolabs, USA) was added and incubated for 1 h. Following this, the membrane was washed and specific bands were visualized using super signal west pico chemiluminescent substrate kit (Pierce Biotechnology Inc, USA). Intensities of the bands on the membrane were scanned using a densitometer. Average intensity of the bands from three different Western blots of the control group was considered as 100% and the changes in the intensity of the band in the experimental group were determined as detailed earlier (Marutha Ravindran and Ticku, 2004, 2006a, 2006b; Marutha Ravindran et al., 2007). The data are expressed as % mean ± S.E.M. from three different Western blots performed on different days using pooled tissue from 10 rats in each group. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Dunnett test. Antiserum for the rat GABAA receptors δ-subunit (AA 1–11) was procured from Alpha Diagnostics (San Antonio, TX, USA). We have reported the characterization of this antiserum for the GABAA receptors δ-subunit recently (Mehta et al., 2007). Pre-incubation with the antigenic peptide (AA 1–11; 20 µg/ml) for the δ-subunit of GABAA receptors antibody blocked the immunoprecipitation in control experiments, and molecular mass of the δ-subunit of GABAA receptors was found to be 54 kDa in Western blot analysis (Mehta et al., 2007).

4.3. Immunoprecipitation and [³H]muscimol binding

The rat cerebellum membrane preparation, the GABAA receptors solubilization, immunoprecipitation and [³H]muscimol binding assays were performed as reported in the literature (Khan et al., 1993; Mehta and Ticku, 1999b, 2005). Briefly, the frozen tissue was thawed, homogenized in ice-cold 0.32 M sucrose solution (pH 7.4) and it was then centrifuged at 78,000 g for 30 min at 4°C. The pellet was suspended in 0.32 M sucrose solution (pH 7.4) and kept frozen overnight at −80°C. After thawing, Tris-HCl (50 mM, pH 7.4) was added to the tissue, and it was centrifuged at 30,000 g for 10 min at 4°C. The pellet was resuspended in Tris-HCl buffer (50 mM, pH 7.4), and centrifuged at 30,000 g for 10 min at 4°C. The last step was repeated four more times. The pellet was resuspended in Tris-HCl buffer, and kept frozen overnight at −80°C. After thawing, Tris-HCl buffer was added to the tissue, and the mixture was centrifuged at 78,000 g for 30 min at 4°C. The membranes were then suspended in Tris-HCl (50 mM, pH 7.4), distributed in aliquots, and kept frozen at −80°C until use. GABAA receptors were solubilized in modified radioimmune precipitation assay buffer (RIPA), i.e., solubilization buffer (pH 7.4) containing NaCl (0.137 M), sodium deoxycholate (1% w/v), Triton X-100 (1% v/v), sodium dodecyl sulfate, i.e, SDS (0.1% w/v), Tris (10 mM) and a cocktail of protease inhibitors containing EDTA (1 mM), EGTA (1 mM), benzamidine HCl (2 mM), trypsin inhibitor type 1-S (0.1 mg/ml), bacitracin (0.1 mg/ml) and phenylmethylsulfonyl fluoride (0.3 mM). After incubation for 1 h at 4°C, insoluble material was removed by centrifugation at 100,000 g for 1 h at 4°C. A sample of 400 µl (≈300 µg protein) of the solubilized receptors was incubated overnight at 4°C with 30 µl of the antiserum for the rat GABAA receptors δ-subunit since preliminary experiments revealed that 30 µl of the antiserum elicits maximal immunoprecipitation of the δ-subunit-containing GABAA receptor assemblies. The receptor-antibody complexes were recovered by incubation with protein A-agarose suspension (60 µl of 40% v/v) followed by centrifugation. Immunoprecipitation was quantified by determining the binding of [³H]muscimol (36 nM) to the immunoprecipitated pellet and supernatant. Non-specific radioligand binding was determined with GABA (100 µM). Protein was estimated using Micro BCA™ Protein Assay Kit (Pierce, Rockford IL U.S.A.). The data are expressed as mean ± S.E.M. of six individual experiments, each performed in triplicate using the pooled cerebellum from ten rats in each group. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Dunnett test.

Acknowledgements

This research work was supported by National Institute on Alcohol and Alcohol Abuse (NIAAA) grant AA10552.

Abbreviations

CE

chronic ethanol

CIE

chronic intermittent ethanol

CB

cerebellum

HC

hippocampus

CC

cerebral cortex

Footnotes

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