GABAA Receptor Expression in the Forebrain of Ataxic Rolling Nagoya Mice

Abstract

The human CACNA1A gene encodes the pore-forming α₁ subunit of Ca_V2.1 (P/Q-type) calcium channels and is the locus for several neurological disorders, including episodic ataxia type 2 (EA2), spinocerebellar ataxia type 6 (SCA6) and Familial Hemiplegic Migraine type 1 (FHM1). Several spontaneous mouse Cacna1a mutant strains exist, among them Rolling Nagoya (tg^rol), carrying the R1262G point mutation in the mouse Cacna1a gene. tg^rol mice display a phenotype of severe gait ataxia and motor dysfunction of the hind limbs. At the functional level, the R1262G mutation results in a positive shift of the activation voltage of the Ca_V2.1 channel and reduced current density. γ-Aminobutyric acid type A (GABA_A) receptor subunit expression depends critically on neuronal calcium influx, and GABA_A receptor dysfunction has previously been described for the cerebellum of tg^rol and other ataxic Cacna1a mutant mice. Given the expression pattern of Ca_V2.1, it was hypothesized that calcium dysregulation in tg^rol might affect GABA_A receptor expression in the forebrain. Herein, functional GABA_A receptors in the forebrain of tg^rol mice were quantified and pharmacologically dissociated using [³H] radioligand binding. No gross changes to functional GABA_A receptors were identified. Future cell type-specific analyses are required to identify possible cortical contributions to the psychomotor phenotype of tg^rol mice.

Keywords: Ataxia; CaV2.1; Cacan1a; Calcium; Gamma aminobutyric receptor type A; Motor dysfunction; P/Q-type calcium channel; Pharmacology; Rolling Nagoya.

Figure 1

[³H] Muscimol binding. (A) [³H] Muscimol binding to wt and tg^rol forebrain membrane homogenates was similar when fitted to a one-site binding curve. (B) Rosenthal transformation was carried out and the Scatchard plot is shown. (C) B_max values were not statistically significant different between wt and tg^rol. Individual values were obtained from fitting a binding curve against the forebrain sample of a single animal. (D) Similarly, no differences in K_D value were obtained.

Figure 2

[³H] Ro15-4513 binding. (A) Total [³H] Ro15-4513 binding was not affected by the tg^rol mutation in Cacna1a. (B) Rosenthal transformation was carried out and the Scatchard plot is shown. (C) B_max values did not differ between wt and tg^rol. (D) Similarly, K_D values were similar between wt and tg^rol. (E) [³H] Ro15-4513 binding in the presence of 10 μM flunitrazepam defined BZ-IS binding sites did not differ between genotypes. (F) BZ-S [³H] Ro15-4513 binding was determined by subtraction of the estimated number of BZ-IS binding sites from total [³H] Ro15-4513 specific binding sites were similar between wt and tg^rol mice. (G–H) B_max and K_D values did not differ between wt and tgrol mice.

Figure 3

[³H] Flumazenil (Ro15-1788) binding. (A) [³H] Flumazenil binding did not reveal any quantitative differences between binding to wt and tg^rol forebrain membrane homogenates. (B) Scatchard plot is shown for illustration. (C–D) B_max and K_D values did not differ between wt and tg^rol mice.