Protracted treatment with diazepam reduces benzodiazepine1 receptor-mediated potentiation of gamma-aminobutyric acid-induced currents in dissociated rat hippocampal neurons

Tolerance to benzodiazepines (BZs) is thought to involve alterations of the gamma-aminobutyric acid (GABA)A receptor as a result of the prolonged occupancy of its modulatory BZ recognition site. We used the whole-cell patch-clamp technique to compare the functional and pharmacological properties of GABAA receptors in acutely dissociated hippocampal neurons from the control or diazepam-tolerant rats. Administration of diazepam (15 mg/kg p.o.) twice a day for 10 days induced tolerance as demonstrated by the decreased potency of acute diazepam i.p. injections to protect against pentylenetetrazole-induced clonictonic convulsions (10.5% of tolerant rats protected by 0.1 mg/kg of diazepam against 55% of nontreated rats, 48 hr after the last dose of the chronic treatment). The specific current induced by 1 microM GABA in acutely dissociated hippocampal neurons 48 hr after withdrawal (10.5 +/- 1.3 microA/cm2) was similar to that observed in the control rats (8.7 +/- 0.8 microA/cm2). The EC50 value for GABA was unchanged by the chronic treatment 6.3 (5.4-7.1) and 7.5 (6.2-8.7) microM in neurons from the control and treated rats, respectively]. The potency of the nonselective allosteric modulator diazepam to stimulate Cl- currents was identical in cells from treated rats EC50 values of 25 (20-30) and 34 (26-41) nM in the control and treated rats, respectively; P < .05], but the potency of the selective BZ1-site ligand zolpidem was decreased EC50 values of 99 (88-111) and 267 (221-313) nM in the control and treated rats, respectively; P < .05]. The maximal potentiation of the GABA-induced current was significantly decreased with diazepam (maximal potentiation: 168.0 +/- 16.2 and 124.0 +/- 8.9% in the control and treated rats, respectively). These results suggest that tolerance to diazepam is accompanied in hippocampal neurons by a decrease in BZ1 binding sites and in the functional coupling of BZ/GABA recognition sites.