Genetic essential tremor in gamma-aminobutyric acidA receptor alpha1 subunit knockout mice

Abstract

Essential tremor is the most common movement disorder and has an unknown etiology. Here we report that gamma-aminobutyric acidA (GABA(A)) receptor alpha1-/- mice exhibit postural and kinetic tremor and motor incoordination that is characteristic of essential tremor disease. We tested mice with essential-like tremor using current drug therapies that alleviate symptoms in essential tremor patients (primidone, propranolol, and gabapentin) and several candidates hypothesized to reduce tremor, including ethanol; the noncompetitive N-methyl-D-aspartate receptor antagonist MK-801; the adenosine A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA); the GABA(A) receptor modulators diazepam, allopregnanolone, and Ro15-4513; and the L-type Ca2+ channel antagonist nitrendipine. Primidone, propranolol, and gabapentin reduced the amplitude (power) of the pathologic tremor. Nonsedative doses of ethanol eliminated tremor in mice. Diazepam, allopregnanolone, Ro15-4513, and nitrendipine had no effect or enhanced tremor, whereas MK-801 and CCPA reduced tremor. To understand the etiology of tremor in these mice, we studied the electrophysiological properties of cerebellar Purkinje cells. Cerebellar Purkinje cells in GABA(A) receptor alpha1-/- mice exhibited a profound loss of all responses to synaptic or exogenous GABA, but no differences in abundance, gross morphology, or spontaneous synaptic activity were observed. This genetic animal model elucidates a mechanism of GABAergic dysfunction in the major motor pathway and potential targets for pharmacotherapy of essential tremor.

Figure 1

GABA_A receptor α1^–/– mice exhibit essential-like tremor. Normal physiologic tremor (wide range of low-power frequencies, 25–40 Hz) was observed in α1^+/+, α1^+/–, and α1^–/– mice. Knockout mice also exhibited pathologic tremor (small range of high-power frequencies, 17–21 Hz) characteristic of essential tremor. Representative voltage tracings and Fourier transformation of tremor-induced displacement is shown for α1^+/+ (A), α1^+/– (B), and α1^–/– (C) mice. (A) α1^+/+ mice exhibit a tremor with a mean maximal power of 2.6 × 10¹¹ ± 3.2 × 10¹¹ N and a mean frequency of 32.1 ± 0.6 Hz (n = 16); (B) α1^+/– mice, mean maximal power of 6.0 × 10¹¹ ± 1.1 × 10¹¹ N and mean frequency of 35.0 ± 1.9 Hz (n = 5); (C) α1^–/– mice, mean maximal power of 24.3 × 10¹¹ ± 4.2 × 10¹¹ N and mean frequency of 19.3 ± 0.9 Hz (n = 13). Tremor is plotted as the voltage generated upon displacement of the transducer as a function of time (seconds). The power of individual frequencies that contribute to the overall tremor was determined by Fourier transformation of the voltage trace over time.

Figure 2

Inhibition of essential-like tremor in GABA_A receptor α1^–/– mice by ethanol and MK-801. (A) Ethanol (0.25–2.5 g/kg) inhibited the essential-like tremor (approximately 19 Hz) in α1^–/– mice in a dose-dependent manner with an ED₅₀ of 0.35 mg/kg reaching a maximal inhibition of 100% (n = 5–11 per genotype). Ethanol (0.25 g/kg) increased physiological tremor (approximately 32 Hz) in α1^+/+ mice but lacked any effect at higher doses. (B) MK-801 (0.01–0.2 mg/kg) reduced the approximately 19 Hz tremor power in α1^–/– mice with an ED50 of 0.024 mg/kg reaching a maximal inhibition of 65–80% (n = 5–6). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3

GABA_A receptor α1^–/– mice exhibit normal Purkinje cell number and morphology but complete loss of both spontaneous mIPSCs and exogenous GABA inhibition of whole-cell voltage-clamp electrophysiological responses. (A) Calbindin staining of Purkinje cell number and morphology in cerebellum of 8-month-old α1^+/+ and α1^–/– mice. Magnification, ×400. (B) Similar spontaneously active Purkinje cells were found in both α1^+/+ (22 of 29 penetrations) and α1^–/– (28 of 36 penetrations) mice. Mean rate for α1^–/– mice (26.7 ± 3.8) did not differ from that of α1^+/+ mice (32.4 ± 5.5). (C) Spontaneous mIPSCs recorded over a 30-second period from a Purkinje cell mechanically dissociated from an α1^+/+ and α1^–/– mouse. Spontaneous postsynaptic picrotoxin-sensitive currents with amplitude greater than 50 pA and fall-times greater than 4 ms were recorded from 10 of 16 α1^+/+ mice and 0 of 14 α1^–/– mice. (D) Whole-cell voltage-clamp recordings were obtained from mechanically dissociated cerebellar Purkinje cells from α1^+/+ and α1^–/– mice. GABA was applied to the neurons by a U-tube. GABA (3, 30, and 100 μM) gated a concentration-dependent inward current when applied to Purkinje cells from α1^+/+ mice, but no current was gated in α1^–/– Purkinje cells. (E) Mean GABA-gated currents from cerebellar Purkinje cells mechanically isolated from α1^+/+ or α1^–/– mice. There was a statistically reliable concentration-related increase in GABA response in the α1^+/+ mice (*P < 0.001) but no effect of GABA in the α1^–/– mice (P > 0.1) (n = 6–11 neurons per group).