Deletion of the α1 or β2 Subunit of GABAAReceptors Reduces Actions of Alcohol and Other Drugs

  1. Yuri A. Blednov1,
  2. S. Jung1,
  3. H. Alva1,
  4. D. Wallace1,
  5. T. Rosahl2,
  6. P.-J. Whiting2 and
  7. R. Adron Harris1
  1. 1Waggoner Center for Alcohol and Addiction Research and Section of Neurobiology, University of Texas at Austin, Austin, Texas (Y.A.B., S.J., H.A., D.W., R.A.H.); and 2Neuroscience Research Center, Merck Sharp and Dohme Research Laboratories, Harlow, Essex, United Kingdom (T.R., P.-J.W.).
  1. Dr. Yuri A. Blednov, The University of Texas at Austin Waggoner Center for Alcohol and Addiction Research, 1 University Station, A4800 2500 Speedway MBB 1.124, Austin, TX 78712-0159. E-mail: yablednov{at}mail.utexas.edu
 
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Abstract

Enhancement of the activation of GABAA receptors is a common feature of many sedative and hypnotic drugs, and it is probable that the GABAA receptor complex is a molecular target for these drugs in the mammalian central nervous system. We set out to elucidate the role of the two predominant (α1 and β2) subunits of GABAA receptor in sedative drug action by studying mice lacking these two subunits. Both α1 (−/−) and β2 (−/−) null mutant mice showed markedly decreased sleep time induced by nonselective benzodiazepine, flurazepam, and GABAA agonist, 4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol. The sleep time induced by the β-selective drug etomidate was decreased only in β2 (−/−) knockout mice. In contrast, α1 (−/−) mice were more resistant to the α1-selective drug zolpidem than β2 (−/−) or wild-type animals. Knockout mice of both strains were similar to wild-type mice in their responses to pentobarbital. The duration of loss of the righting reflex produced by ethanol was decreased in male mice for both null alleles compared with wild-type mice, but there were no differences in ethanol-induced sleep time in mutant females. Deletion of either the α1 or β2 subunits reduced the muscimol-stimulated 36Cl− influx in cortical microsacs suggesting that these mutant mice have reduced number of functional brain GABAA receptors. Our results show that removal of either α1 or β2subunits of GABAA receptors produce strong and selective decreases in hypnotic effects of different drugs. Overall, these data confirm the crucial role of the GABAA receptor in mechanisms mediating sedative/hypnotic effects.

GABA, acting via GABAA receptors, is the brain's major inhibitory neurotransmitter system in the central nervous system. Because the ability to enhance the ion channel activation of GABAA receptors is a common feature of many sedative and hypnotic drugs, it is probable that the action on the GABAA receptor complex is a molecular target for these drugs in the mammalian central nervous system (Charney et al., 2001). Nevertheless, many of these drugs, including ethanol and pentobarbital, may have multiple sites of action and the importance of specific GABAA receptors in drug action is not clearly defined.

The GABAA receptor has a pentameric structure, which is formed by the coassembly of subunit polypeptides that exist in a large multigene family (McKernan and Whiting, 1996; Barnard et al., 1998). There are at least 16 different members of the GABAA receptor gene family, including 6α, 3β, 3γ, δ, ε, θ, and π subunits (Whiting et al., 1999). The largest population of GABAA receptors in the rat brain has a subunit composition of α1β2γ2, whereas together α2β3γ2 and α3βγ2/γ3constitute the next most prevalent subtypes (McKernan and Whiting, 1996). GABA affinity is mainly governed by α subunits (Smith et al., 2001), but affinity and efficacy at the benzodiazepine site are influenced by both α and γ subunits (McKernan et al., 1995; Buhr et al., 1996; Wingrove et al., 1997) but not β subunits (Hadingham et al., 1993). The benzodiazepine site occurs at the interface of an α and a γ subunit, with residues in both influencing modulation (Smith and Olsen, 1995). The presence of γ2 confers the classical benzodiazepine pharmacology to GABAA receptors (Pritchett et al., 1989). In contrast, β subunits control loreclezole and etomidate sensitivity (Stevenson et al., 1995). Mouse strains lacking individual GABAA receptor subunits provide insight regarding the role of GABA receptors. Mice lacking the γ2subunit die shortly after birth (Gunther et al., 1995), whereas mice deficient the γ2L (long splice variant) subunit are viable and show small increases in sleep time responses to midazolam and zolpidem, but responses to nonbenzodiazepine agents such as ethanol, etomidate, and pentobarbital are unchanged (Quinlan et al., 2000). β3 null mice (−/−) did not show any changes in sleep times after administration of pentobarbital or ethanol, but they were more resistant to etomidate and midazolam (Quinlan et al., 1998). Mice lacking the α6subunit of the GABAA receptor, which is expressed exclusively in cerebellar granule cells, have no major phenotypic abnormalities (Jones et al., 1997). Mice deficient the δ subunit are also viable but show attenuated sensitivity to neuroactive steroids and epileptic seizures (Mihalek et al., 1999). Thus, deletion of some of the less abundant GABAA receptor subunits reduces the action of some sedative drugs, but they do not provide evidence for a major role for GABA receptors in actions of many sedatives. It is important to note, however, that the only predominant GABAA subunit that has been deleted is the γ2 and this proved lethal.

Recently, mice lacking the most predominant GABAAreceptor subunits, α1 and β2, were successfully generated (Sur et al., 2001; Kralic et al., 2002). Although the mice lack approximately 60% of the total number of brain GABAA receptors, adult α1 (−/−) and β2(−/−) mice do not display major phenotypic abnormalities or spontaneous seizures. α1 (−/−) mice showed overexpression of the α2 and α3 subunit but lack approximately 40% of the GABAA receptors despite this apparent compensation. In contrast, β2 (−/−) mice displayed an equal reduction in all six α subunits and a loss of GABAA receptors of about 50% (Sur et al., 2001).

In this study, we asked how this substantial decrease of benzodiazepine receptors and reduction of expression of different GABA receptor subtypes might affect receptor function by measuring agonist stimulated 36Cl− uptake in brain tissue from these mutant mice. For the behavioral studies, we used the loss of righting reflex and tested a GABAA agonist, 4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol (THIP), a nonselective benzodiazepine, flurazepam, and α1-selective drug, zolpidem, a β-selective drug, etomidate, and two drugs with activities on several channels in addition to GABAA, ethanol and pentobarbital.

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Materials and Methods

Animals.

Null α1 (−/−) and β2 (−/−) allele mice were created using homologous recombination and genotyped as previously described in Sur et al. (2001). Homozygotes of the F2 generation were interbred avoiding any brother-sister mating, and homozygous colonies of α1 (−/−), β2 (−/−) and wild-type (+/+) mice were established. In this study, the mice from the F6 to F7 generations of this interbreeding have been used.

All mice were at least 14 to 18 weeks of age at the time of analysis; within each experiment, all mice were of similar age. Mice were grouped housed (3–5 per cage) under a 12-h light/dark cycle (lights on at 7:00 AM) and provided ad lib access to food and water. All experiments were conducted in an isolated behavioral testing room in the animal facility to avoid external distractions. All mice were allowed to recover for at least 1 week between each drug treatment. Each group of mice, however, was not used for more than for two different drugs, and drug testing was randomized. All experiments were approved by the Institutional Animal Care and Use Committee.

Loss of Righting Reflex.

Sensitivity to ethanol (Aaper Alcohol and Chemical Co., Shelbyville, KT; 3.4, 3.6, and 3.8 g/kg, i.p.), flurazepam (Sigma-Aldrich, St. Louis, MO; 225.0 mg/kg, i.p.), zolpidem tartrate (gift from Dr. J. S. Vedo; Pharmacia, Skokie, IL; 60.0 mg/kg, i.p.), pentobarbital (Sigma/RBI, Natick, MA; 50.0 mg/kg, i.p.), etomidate (40.0 mg/kg, i.p.), and THIP (Sigma/RBI; 55.0 mg/kg, s.c.) was determined using the standard sleep time assay (Kakihana et al., 1966). Ethanol was diluted in 0.9% saline (20.0% w/v) and administered in doses adjusted by injected volumes. Flurazepam, zolpidem, and etomidate were dissolved in 3 to 4 drops of Tween 80 (Sigma-Aldrich) before saline was added and injected at 0.01 ml/g b.wt. Pentobarbital was dissolved in 0.9% saline and injected at 0.01 ml/g b.wt. THIP was dissolved in 0.9% saline and injected at 0.005 ml/g b.wt.

All sleep time experiments were performed between 9:00 AM and 1:00 PM. Mice were injected with the drug, and when they became ataxic, they were placed in the supine position in V-shaped plastic troughs until they were able to right themselves three times within 30 s. Sleep time was defined as the time from being placed in the supine position until they regained their righting reflex. During all sleep time assays, room temperature was 24°C. Mice that failed to lose the righting reflex (misplaced injections) or had a sleep time greater than two standard deviations from the group mean were excluded from the analysis unless otherwise noted.

36Cl− Uptake.

The procedure was carried out according to Allan and Harris (1986). Animals were killed by decapitation, and their brains were removed and placed in ice-cold buffer (145 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM glucose, 1 mM CaCl2, and 10 mM HEPES; adjusted to pH 7.5 with Tris base). The brain was homogenized by hand (10–12 strokes) using a glass-Teflon homogenizer (size C; Thomas Scientific, Swedesboro, NJ) in 5 ml of ice-cold assay buffer. The homogenate was centrifuged at 900g for 15 min. The supernatant was decanted, and the pellet was washed with 10 ml of assay buffer and centrifuged at 900g for 15 min. The final pellet was suspended in ice-cold assay buffer. Aliquots (0.2 ml) of membrane vesicles (0.6–0.8 mg of protein) were incubated for 5 min in a shaking water bath at 30°C. After this incubation, uptake was initiated by the addition of 0.2 ml of 36Cl− (2 mCi/ml assay buffer) (specific activity 12.8 mCi/g of Cl; obtained from ICN, Irvine, CA) containing 1 to 20 μM muscimol (final concentration). Three seconds after the addition36Cl− influx was terminated by the addition of 4 ml of ice-cold assay buffer containing 100 μM picrotoxin, and rapid filtration under vacuum (10 mm Hg) onto a Whatman GF/C glass microfiber filter (Whatman, Clifton, NJ) using a Hoeffer manifold (Hoefer Scientific, San Francisco, CA) was performed. The filters were washed with an 8-ml assay buffer containing 100 μM picotoxin with the manifold towers removed. The amount of radioactivity on the filters was determined by liquid scintillation spectrometry. The amount of36Cl− bound to the filter in the absence of membranes (no tissue blank) was subtracted from all values. Muscimol-dependent influx was defined as the amount of36Cl− taken up while muscimol was present minus the amount of36Cl− taken up in the absence of muscimol. The apparent potency (EC50) and apparent efficacy (Emax) of muscimol was determined by the construction of a dose-response curve using five concentrations of the drug. The modulation of muscimol-stimulated 36Cl−uptake by ethanol (25 mM), pentobarbital (25 μM), and flunitrazepam (0.1 μM) was determined by comparing36Cl− uptake in solutions of each different type of drug containing36Cl− and 0.65 μM of muscimol for wild-type (+/+) cortex and36Cl− and 1.0 μM of muscimol for cortex from α1 (−/−) and β2 (−/−) null mutant mice with solutions containing only 36Cl− and the corresponding muscimol concentrations for each genotype. These concentrations of muscimol induced a similar stimulation of uptake in all three genotypes. Net uptake was defined as the amount of36Cl− uptake in the presence of muscimol, corresponding to each genotype concentration, plus the drug tested minus the36Cl− uptake in the presence of muscimol alone.

Data Analysis.

The results are expressed as mean ± S.E.M. The data are reported in minutes for sleep time. For36Cl− uptake studies,Emax was determined from the dose-response data as the change in36Cl− flux at the maximally effective concentration, and EC50 was determined by linearly transforming the data (sigmoid curve analysis) using the GraphPad computer program (GraphPad Software, Inc., San Diego, CA). Statistical analysis of parameters was made using the Student's t test with Dunnet's correction for multiple comparisons (Winer, 1971) or by two-way ANOVA whenever appropriate. For all statistical comparisons, p ≤ 0.05 was considered significant.

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Results

The duration of loss of righting reflex (sleep time) produced by ethanol was decreased in both null allele males compared with wild-type mice (P < 0.0001, dependence on genotype;P < 0.0001, dependence on dose, two-way ANOVA) (Fig.1a). The residual response to ethanol was similar in α1 and β2null mutant males. There were no differences in ethanol-induced sleep time in mutant females (Fig. 1b), however. Null allele mice of both strains did not differ from wild-type mice in their responses to pentobarbital (Fig. 1, c and d).

Figure 1
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Figure 1

Ethanol- and pentobarbital-induced loss of righting reflex in mice lacking the α1 or β2 subunit of the GABAA receptor. a and c, show data for males. a,n = 10–12 (+/+); n = 10 α1 (−/−); β2 (−/−) = 10 for each dose of ethanol. c, n = 10 for each genotype. b and d, show data for females. b, n = 10–12 (+/+);n = 10 α1 (−/−); β2(−/−) = 10 for each dose of ethanol. d, n = 10 for each genotype. Summary of statistics: a, males showed an effect of genotype (F2,85 = 27.1;P < 0.0001, two-way ANOVA) and dose (F2,85 = 42.1; P < 0.0001, two-way ANOVA). WT, wild-type.

Analysis of flurazepam and THIP-induced loss of righting reflex revealed that α1 (−/−) and β2 (−/−) null mutant mice showed markedly decreased sleep time in comparison with wild-type mice (Fig.2, a and b). Both sexes of mutant mice showed similar changes in response.

Figure 2
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Figure 2

THIP- and flurazepam-induced loss of righting reflex in mice lacking the α1 or β2 subunit of the GABAA receptor. a and c, show data for males. a,n = 6 (+/+); n = 7 α1 (−/−); β2 (−/−) = 8. c,n = 9 (+/+); n = 8 α1 (−/−) and β2 (−/−). b and d, show data for females. b, n = 9 (+/+);n = 7 α1 (−/−); β2(−/−) = 6. d, n = 9 for each genotype. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001 mutant mice are different from wild-type mice (Student's t test with Dunnet's correction for multiple comparisons). #,p < 0.05; ###, p < 0.001 α1 (−/−) null mutant mice are different from β2 (−/−) knockout mice (Student's ttest with Dunnet's correction for multiple comparisons). WT, wild-type.

The duration of sleep time induced by etomidate was significantly decreased only in β2 (−/−) males (Fig.3a) and females (Fig. 3b). α1 (−/−) mice of both sexes did not differ from wild-type animals in their sensitivity to etomidate. In contrast, α1 (−/−) mice were more resistant to zolpidem (60 mg/kg) in comparison with wild-type animals (Fig. 3, c and d). There was no difference in resistance to zolpidem-induced sleep time between β2 (−/−) and α1 (−/−) females (Fig. 3d). On the other hand, β2 (−/−) males showed resistance intermediate between α1 (−/−) null mutant and wild-type males (Fig. 3c).

Figure 3
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Figure 3

Etomidate- and zolpidem-induced loss of righting reflex in mice lacking the α1 or β2 subunit of the GABAA receptor. a and c, show data for males. a,n = 7 (+/+); n = 6 α1 (−/−) and β2 (−/−). c,n = 11 (+/+); n = 12 α1 (−/−); n = 13 β2(−/−). b and d, show data for females. b, n = 9 (+/+); n = 10 α1 (−/−) and β2 (−/−). d, n = 9 (+/+) and α1 (−/−); n = 12 β2(−/−). ∗, p < 0.05; ∗∗,p < 0.01; ∗∗∗, p < 0.001 mutant mice are different from wild-type mice (Student'st test with Dunnet's correction for multiple comparisons). ##, p < 0.01; ###,p < 0.001 α1 (−/−) null mutant mice are different from β2 (−/−) knockout mice (Student's t test with Dunnet's correction for multiple comparisons). WT, wild-type.

To determine whether the deletion of α1 or β2 subunits changed the GABAA receptor function, muscimol-stimulated36Cl− influx was measured in cortical microsacs. Deletion of either the α1 or β2 subunits reduced the response to muscimol (Fig.4a). The muscimol EC50 was approximately 2-fold higher in both mutant strains than in wild-type mice. The deletion of the β2 subunit significantly increased the slope of muscimol response. The maximal effect of muscimol was approximately 25% higher in membranes from wild-type animals compared with the null mutants (Table 1).

Figure 4
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Figure 4

Effects of deletion of the α1 or β2 subunit of the GABAA receptor on muscimol-stimulated chloride flux measured in membranes from cortex. a,n = 11 for each genotype. b to d,n = 12 (+/+); n = 13 α1 (−/−) and β2 (−/−). ∗,p < 0.05 mutant mice are different from wild-type mice (Student's t test with Dunnet's correction for multiple comparisons). #, p < 0.05 α1 (−/−) null mutant mice are different from β2 (−/−) knockout mice (Student's ttest with Dunnet's correction for multiple comparisons).

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Table 1

Function of GABAA receptors in null mutant mice: parameters of muscimol-stimulated 36Cl− uptake

Flunitrazepam enhanced muscimol-stimulated36Cl− uptake equally in microsacs from wild-type and β2 null mutant mice. Potentiation of muscimol response by flunitrazepam, however, was significantly higher in membranes from α1(−/−) mice than from wild-type mice (Fig. 4b). In contrast, there were no differences in potentiation of muscimol response by pentobarbital in the three genotypes (Fig. 4c). Ethanol potentiation of muscimol-stimulated 36Cl−influx was significantly decreased in β2(−/−) mice (Fig. 4d). There were no differences in these ethanol effects between males and females. Ethanol potentiated muscimol-stimulated 36Cl−influx by 1.70 ± 0.16- and 1.69 ± 0.14-fold in wild-type mice, 1.62 ± 0.22- and 1.75 ± 0.23-fold in α1 (−/−) knockout mice, and 1.37 ± 0.07- and 1.15 ± 0.16-fold in β2 null mutant mice (males and females, respectively).

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Discussion

Taken together, these results show that deletion of either the α1 or β2 subunits of GABAA receptor reduced the behavioral effects of a wide variety of sedative hypnotic drugs (summarized in Table2) and indicate that these mutant mice have a reduced number of functional brain GABAAreceptors.

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Table 2

Summary of changes in drug-induced loss of righting reflex in α1 and β2 null mutant mice

The reductions in sleep time produced by the mutations are generally consistent with biochemical studies of brain and recombinant receptors, indicating that THIP and flurazepam affect most GABAA receptors and thus show similar effects of deletion of either the α1 or β2 subunit. For etomidate, subtypes of the β-subunit influence the potency with which etomidate potentiates GABA-evoked currents and the β isoform is a crucial determinant of the GABA-mimetic activity of this compound (Hill-Venning et al., 1997). Concerning the effects of etomidate on recombinant GABAA receptors composed of α1, γ2S, and either β1, β2, or β3 subunits, Sanna et al. (1997) showed that the order of potency after application of this drug was β3 > β2 > β1. In accordance with these findings, the loss of righting reflex produced by etomidate was reduced only in the β2 mutants not in the α1 knockouts.

Zolpidem has a higher affinity for GABAAreceptors containing α1 subunits than for other receptors (Pritchett et al., 1989; Sieghart, 1995), and sleep time produced by this drug was markedly decreased in α1 null mutant mice. Nevertheless, β2 knockout mice also showed a significant decrease in zolpidem-induced sleep time. This is perhaps surprising in view of the findings that there was a complete loss of high-affinity binding sites for zolpidem in α1 (−/−) mice, whereas high-affinity zolpidem binding sites still accounted for 49% of total [3H]flumazenil sites in β2 (−/−) brains (Sur et al., 2001). It should be noted, however, that at the high doses used to produce loss of righting reflex, it is likely that zolpidem is not completely selective for α1-containing receptors.

In view of the clear changes in action of these GABAergic drugs, it was surprising that pentobarbital sleep time was not altered in the mutant mice, but this is consistent with the robust potentiation by pentobarbital of the GABA current in Purkinje cells isolated from these mutant mice (Sur et al., 2001). In addition, deletion of another β subunit, β3, also failed to change pentobarbital sleep time (Quinlan et al., 1998). Pentobarbital has effects on a number of other ion channels (Wartenberg et al., 2001;Yamakura et al., 2001; Bachmann et al., 2002), and it is possible that these targets are more important for pentobarbital-induced loss of righting reflex than the GABAA receptor.

Reduction of alcohol sleep time by deletion of either the α1 or β2 subunit is consistent with the idea that ethanol enhances GABAA receptor function and thereby produces some of its behavioral effects (Harris, 1999). The effect of the null mutations was only seen in male mice, however, suggesting that females may employ additional mechanisms for production of ethanol effects or that the GABAA receptor action of ethanol is less important in female mice. There is evidence for gender differences in alcohol sleep time and genetic influences upon that behavior (DeFries et al., 1989). In addition, neurosteroids derived from progesterone have been implicated in alcohol actions, including loss of righting reflex (Morrow et al., 2001). Thus, actions of ethanol such as loss of righting reflex likely involve multiple targets, including the GABAA receptor.

Results from GABA receptor function measured in cortical membranes by chloride flux are generally consistent with previous biochemical and current behavioral studies of these mice. In particular, both knockout strains showed decreased maximum stimulation of muscimol-stimulated36Cl− uptake. Sur at al. (2001) showed that both the α1 (−/−) and β2 (−/−) mice demonstrated a large (50–70%) widespread loss of GABAA receptors measured by [35S]t-butylbicyclophosphorothionate and [3H]muscimol binding and of benzodiazepine binding sites measured by [3H]flumazenil binding. Nevertheless, the 25% decrease in chloride flux (Emax) that we observed suggests that mutant mice compensate for loss of receptor subunits by either more efficient trafficking of receptors to the membrane surface or greater function of remaining receptors. In addition to the decrease inEmax, the mutant mice also showed an increased muscimol EC50, likely a consequence of subunit substitution in the remaining receptors. The potency of muscimol varies with the type of α subunit incorporated into the GABAA receptor complex in the order α6 > α5 = α2 > α1 > α3 (Ebert et al., 1997) and substitution of α3 for α1 as a possible explanation for the decreased muscimol EC50 in mutant mice. At first glance, the ability to potentiate the36Cl− uptake by several different sedatives does not appear consistent with behavioral effects of these drugs. For example, potentiation of muscimol action by flunitrazepam was not changed in β2 knockout mice and was increased in α1 null mutant mice, despite decreased sleep times in both mutants. It should be noted, however, that we are measuring flunitrazepam effects on the remaining GABAA receptors; thus, these potentiation values do not reflect the loss of GABA receptors. It appears that the reason for the decrease in behavioral actions of flurazepam is the decrease in number of GABAA receptors, not a change in the properties of the remaining receptors. On the other hand, the increase in flunitrazepam potentiation of muscimol action in α1 null mutant mice could reflect overexpression of α2 and α3 subunits in these mice (Sur et al., 2001). It is known that the benzodiazepine potentiation of GABA action is greater at α3β3γ2subunits than at α1 or α5 containing receptors (Smith at al., 2001). In the case of ethanol, however, there is evidence for decreased sensitivity of the remaining receptors, at least in the β2 null mutants.

The majority of gene-targeted mice are developed on a mixed genetic background of inbred mouse strains C57BL/6J and strain 129, and it is useful to compare the responses of wild-type and null allele mice to the pattern of responses found in the parental mouse strains used. In the present study, genes linked to the α1(−/−) and β2 (−/−) mutations would be derived from strain 129/SvEv, whereas genes linked to the wild-type α1 and β2 genes would stem from C57BL/6J. Two recent articles (Simpson et al., 1997;Threadgill et al., 1997) have shown that there is substantial genetic variation among substrains of 129 inbred strain. There are no available data about effects of sedative drugs in the 129/SvEv substrain.Homanics et al. (1999) found that 129/SvJ and C57BL/6J inbred strains have similar hypnotic responses to ethanol and pentobarbital. 129/SvJ mice, however, are markedly less sensitive to the hypnotic effects of midazolam, zolpidem, and propofol (sleep times 30, 65, and 52% of the C57BL/6J values, respectively), whereas 129/SvJ are significantly less resistant to etomidate (30% greater sleep time than C57BL/6J). Because our data show a decrease of ethanol-induced sleep in α1 (−/−) and β2(−/−) males and of etomidate-induced sleep only in β2 (−/−) mice, these effects are most likely the result of mutations and not of flanking genes from the 129/SvEv strain.

Another concern with studies of null mutant mice is compensation (Crawley, 1996; Gerlai, 2001). It is likely that some compensatory changes occur in the brain to allow near normal brain function without α1 or β2 subunits. Our data showing only a 25% decrease in the maximal chloride flux through GABAa receptors in the face of a larger loss of receptor density suggests one type of compensation. Interestingly, very similar results were recently obtained from independently generated α1 knockout mice (Kralic et al., 2002).

In conclusion, these data show that removal of either α1 or β2 subunits of GABAA receptors produce strong and specific decreases effects of different drugs. Overall, these data confirm the crucial role of the GABAA receptor in mechanisms mediating sedative/hypnotic effects.

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Acknowledgments

We thank Dr. Gregg Homanics for helpful discussions and comments on the experiments.

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Footnotes

  • This work was supported by funds from the Texas Commission on Alcohol and Drug Abuse and National Institutes of Health/National Institute on Alcohol Abuse and Alcoholism Grants AA06399 and AA13520.

  • DOI: 10.1124/jpet.102.042960

  • Abbreviations:
    THIP
    4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol
    ANOVA
    analysis of variance
    • Received August 8, 2002.
    • Accepted September 11, 2002.
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