Prototypic GABA(A) receptor agonist muscimol acts preferentially through forebrain high-affinity binding sites

Abstract

Muscimol has been regarded as a universal agonist for all gamma-aminobutyric acid type A receptor (GABA(A)-R) subtypes. However, brain regional distribution of muscimol's high-affinity binding sites greatly differs from those of other binding sites of the GABA(A)-R. To test whether behavioral effects of muscimol correlated with the density of high-affinity [(3)H]muscimol binding, we examined several GABA(A)-R subunit gene-modified mouse lines: alpha1, alpha4, or delta-knockouts (KO), alpha4+delta-double KO, and Thy1.2 promoter-driven alpha6 transgenic mice (Thy1alpha6). We determined the high-affinity [(3)H]muscimol binding in brain sections by quantitative autoradiography and sedative/ataxic effects induced in vivo by muscimol using a constant speed rotarod. alpha4-KO mice had reduced [(3)H]muscimol binding in the caudate-putamen, thalamus, and hippocampus, and were less sensitive to the behavioral impairment by muscimol. Similarly, delta-KO mice also had reduced binding to forebrain regions and a lower behavioral sensitivity to muscimol than their wild-type controls. In contrast, alpha1-KO mice had unaltered behavioral sensitivity to muscimol and unaltered [(3)H]muscimol binding, even though previous studies have demonstrated dramatically reduced binding to various other GABA(A)-R sites in these mice. Finally, Thy1alpha6 mice exhibited increased behavioral sensitivity to muscimol, and to another direct GABA-site agonist gaboxadol, and increased [(3)H]muscimol binding in the cerebral cortex and hippocampus. Thus, the differences in sedative and motor-impairing actions of muscimol in various mouse models correlated with the level of forebrain high-affinity [(3)H]muscimol binding. These data suggest that a small special population of GABA(A)-Rs, most likely extrasynaptic non-alpha1-containing receptors, strongly contributes to the in vivo pharmacological effects of muscimol.

Figure 1

Regional distribution of 15-nM [³H]muscimol binding in representative horizontal brain sections from various mouse lines. (a) α4+δ-KO, α4-KO, and δ-KO compared with their wild-type (WT) brain images, (b) α1-KO and the corresponding WT images, and (c) the Thy1α6 and C57BL/6 WT control images. OB, olfactory bulb; CPu, caudate putamen; Ctx, cerebral cortex; Th, thalamus; Hi, hippocampus; Gr, granule cell layer of the cerebellum.

Figure 2

α4-KO mice have reduced sensitivity to the behavioral effects of muscimol. The fixed speed rotarod measured effects of muscimol on motor performance at 1.5 mg/kg (n=7 KO and 11 WT) (a), 2.0 mg/kg (n=12 KO and 15 WT) (b), and 3.0 mg/kg (n=12 KO and 15 WT) (c). Effect of muscimol was reduced in α4-KO mice (white squares) compared with WT mice (black squares) at 1.5 (p<0.05), 2.0 (p<0.05), and 3.0 mg/kg (p<0.05) (repeated measures ANOVA).

Figure 3

δ-KO mice have reduced sensitivity to the behavioral effects of muscimol. The fixed speed rotarod measured effects of muscimol on motor performance in δ-KO mice (white squares) and WT mice (black squares). At 1.5 mg/kg, there was a significant effect of gender (p<0.05) and therefore, data were split between (a) females and (b) males. Motor impairment by the dose of 1.5 mg/kg in female δ-KO mice was greatly reduced (p<0.01) compared with female WT mice (n=5 KO and 5 WT). Male mice were not affected by muscimol at this dose (n=3 KO and 4 WT). δ-KO mice were less sensitive to the effects of muscimol at (c) 2.0 mg/kg (p<0.001; n=16 KO and 12 WT) and (d) 3.0 mg/kg (p<0.0001; n=16 KO and 12 WT) (repeated measures ANOVA).

Figure 4

α1-KO mice are equally sensitive as WT mice to the behavioral effects of muscimol. The fixed speed rotarod measured effects of muscimol on motor performance in α1-KO mice (white squares) and WT littermate controls (black squares). No differences in genotypes were observed at 2.0 mg/kg ((a), n=10 KO and 12 WT) or 3.0 mg/kg ((b), n=10 KO and 12 WT) (repeated measures ANOVA).

Figure 5

Thy1α6 transgenic mice have increased sensitivity to the ataxic effects of muscimol. The fixed speed rotarod measured the ataxic effects of 0.75, 1.0, or 1.5 mg/kg muscimol in Thy1α6 transgenic mice (white squares) and C57BL/6 WT controls (black squares). Muscimol impaired the performance of neither mouse line at the dose of 0.75 mg/kg (a, b). Because of significant differences between genders at the two higher doses, data were split between females and males. At those doses and between both genders, Thy1α6 mice were more sensitive to muscimol than controls ((c), females, 1.0 mg/kg, p<0.001; (d), males 1.0 mg/kg, p<0.001; (e), females, 1.5 mg/kg, p<0.05; (f), males, 1.5 mg/kg, p<0.05) (repeated measures ANOVA). n=6 per gender and genotype.

Figure 6

Thy1α6 transgenic mice have increased sensitivity to the locomotor stimulating effect of muscimol in the open field test. Male Thy1α6 transgenic and C57BL/6 WT control mice were treated with saline or 0.75 mg/kg muscimol 30 min before being transferred to an open arena. Total locomotor activity (a), time spent in the periphery of the arena (b), and the number of rears (c) were determined for 5 min. Locomotor activity was increased by muscimol in Thy1α6 mice as compared with saline-treated mice (p<0.001) and muscimol-treated WT mice (p<0.001), especially in the periphery of the arena. Muscimol treatment reduced the number of rears in Thy1α6 mice (p<0.01). (Two-way ANOVA followed by Newman–Keuls post hoc test). n=10 males per genotype and treatment.

Figure 7

Thy1α6 transgenic mice have increased sensitivity to the ataxic effects of the GABA-site agonist gaboxadol. The fixed speed rotarod measured the ataxic effects of 6.0 mg/kg gaboxadol in Thy1α6 transgenic mice (white squares) and C57BL/6 WT controls (black squares). There were no significant differences between the genders. Thy1α6 mice were more sensitive to muscimol than controls (p<0.001) (repeated measures ANOVA). n=6 per gender and genotype.