Requirement of alpha5-GABAA receptors for the development of tolerance to the sedative action of diazepam in mice

Abstract

Despite its pharmacological relevance, the mechanism of the development of tolerance to the action of benzodiazepines is essentially unknown. The acute sedative action of diazepam is mediated via alpha1-GABA(A) receptors. Therefore, we tested whether chronic activation of these receptors by diazepam is sufficient to induce tolerance to its sedative action. Knock-in mice, in which thealpha1-,alpha2-,alpha3-, oralpha(5)-GABA(A) receptors had been rendered insensitive to diazepam by histidine-arginine point mutation, were chronically treated with diazepam (8 d; 15 mg x kg(-1) x d(-1)) and tested for motor activity. Wild-type, alpha2(H101R), and alpha3(H126R) mice showed a robust diminution of the motor-depressant drug action. In contrast, alpha5(H105R) mice failed to display any sedative tolerance. alpha1(H101R) mice showed no alteration of motor activity with chronic diazepam treatment. Autoradiography with [3H]flumazenil revealed no change in benzodiazepine binding sites. However, a decrease in alpha5-subunit radioligand binding was detected selectively in the dentate gyrus with specific ligands. This alteration was observed only in diazepam-tolerant animals, indicating that the manifestation of tolerance to the sedative action of diazepam is associated with a downregulation of alpha5-GABA(A) receptors in the dentate gyrus. Thus, the chronic activation of alpha(5)-GABA(A) receptors is crucial for the normal development of sedative tolerance to diazepam, which manifests itself in conjunction with alpha1-GABA(A) receptors.

Figure 1.

Motor activity in wild-type and point-mutated mice subjected to an 8 d chronic diazepam treatment regimen (15 mg · kg^-1 · d^-1). a, In wild-type mice, the test dose of diazepam (10 mg/kg) was equally effective in decreasing motor activity when given either acutely or 18 hr after a chronic vehicle treatment. Mice chronically treated with diazepam showed levels of motor activity similar to those of mice chronically treated with vehicle in response to either diazepam or vehicle (H = 22.958; p < 0.001; n = 6-7 mice per group). b, Inα₁(H101R) mice, there was no overall effect of the different treatment conditions on motor activity (H = 5.914; not significant; n = 6 mice per group). However, an increased motor activity was seen in animals chronically treated with vehicle in response to diazepam (p < 0.01 compared with Veh-Veh; Mann-Whitney test). c, The test dose of diazepam decreased motor activity in animals chronically treated with the vehicle but not inα₂(H101R) (H = 14.942; p < 0.001; n = 8 mice per group) and α₃(H126R) (H = 12.194; p < 0.002; n = 8 mice per group) mice treated with diazepam. In α₅(H105R) mice, the same diazepam test dose depressed motor activity in animals chronically treated with either vehicle or diazepam (H = 12.005; p < 0.002; n = 8 mice per group). Results are given as means ± SE. ⁺⁺p < 0.01 versus Veh-Veh; Mann-Whitney test.

Figure 2.

Autoradiography of [³H]flumazenil binding after cessation of an 8 d treatment regimen with vehicle or diazepam (15 mg · kg^-1 · d^-1) and administration of the test dose of diazepam (10 mg/kg) in wild-type, α₁(H101R), and α₂(H101R) mice. Regardless of the genotype, the chronic diazepam treatment did not alter [³H]flumazenil binding compared with chronic vehicle or acute diazepam, as quantified for three regions involved in motor control and for the hippocampal formation. Standards and the differential [³H]flumazenil binding profile related to the point mutation are also presented in representative transverse sections from animals chronically treated with vehicle. The different binding levels in the mutant mice reflect the loss of diazepam binding to the mutated subunit. Results are expressed in mean ± SD nanocuries per milligram of protein; n = 4-7 mice per group.

Figure 3.

Alteration of α₅-GABA_A receptor binding in dentate gyrus after cessation of an 8 d treatment regimen with vehicle or diazepam (15 mg · kg^-1 · d^-1) assessed autoradiographically with [³H] RY 80 and [³H]L655708 in wild-type mice (a, b) and with [³H]RY80 in α₂(H101R) (c) and α₁(H101R) (d) mice. Standards and representative transverse sections from mice chronically treated with vehicle are shown. a, [³H]RY80 binding levels were decreased in wild-type mice chronically treated with diazepam. b, A similar decrease in [³H]L655708 binding level was observed in the same wild-type mice. c, In α₂(H101R) mice, only animals chronically treated with diazepam showed a significant reduction of [³H]RY80 binding. d, In α₁(H101R) mice, no alteration in [³H]RY80 binding was seen, regardless of the chronic drug treatment. Results are given as mean ± SD nanocuries per milligram of protein. ⁺⁺p < 0.01 versus Veh-Veh; ^**p < 0.01 versus Veh-Diaz; Mann-Whitney test.