Acute tolerance to chlordiazepoxide qualitatively changes the interaction between flumazenil and pregnanolone and not the interaction between flumazenil and midazolam in rhesus monkeys discriminating midazolam

Abstract

Benzodiazepines and neuroactive steroids act at distinct binding sites on γ-aminobutyric acid(A) (GABA(A)) receptors where they positively modulate GABA, resulting in similar acute behavioral effects. Tolerance to benzodiazepines can develop with repeated treatment; however, cross tolerance to neuroactive steroids does not develop, perhaps due to conformational changes in benzodiazepine, and not neuroactive steroid, binding sites. Three monkeys discriminated 0.178 mg/kg midazolam while responding under a fixed-ratio 10 schedule of stimulus-shock termination. On separate occasions, dose-effect curves for midazolam and pregnanolone were determined when monkeys had not received chlordiazepoxide and when they received 10 mg/kg chlordiazepoxide 46 hours earlier; for some tests, flumazenil was given before determination of dose-effect curves. Midazolam and pregnanolone produced ≥80% midazolam-lever responding. When administered 46 h before sessions, chlordiazepoxide did not produce pregnanolone-lever responding; under those treatment conditions, midazolam dose-effect curves were shifted 2.8-fold rightward and pregnanolone dose-effect curves were not changed. Flumazenil antagonized midazolam; Schild (linear) analyses yielded slopes that were not different from unity and pA(2) values of 7.46 when monkeys had not received chlordiazepoxide and 7.44 when they received chlordiazepoxide 46 h earlier. Flumazenil did not alter the effects of pregnanolone in chlordiazepoxide-treated monkeys. Thus, interactions between flumazenil and midazolam were not qualitatively or quantitatively changed in monkeys acutely tolerant to chlordiazepoxide, suggesting that mechanisms other than alterations of benzodiazepine binding sites account for the development of acute tolerance.

Fig. 1

Discriminative-stimulus effects of midazolam alone and in combination with flumazenil in monkeys that had not received chlordiazepoxide (control; open symbols) and in monkeys that received chlordiazepoxide 46 hr earlier (chlordiazepoxide-treated; filled symbols). Each point represents the mean of 3 monkeys. Two different determinations of the midazolam dose-effect curve obtained in monkeys that did not receive other drugs are shown in this figure (open circles, top and bottom panel); straight lines fitted to these midazolam dose-effect curves were not statistically different. Abscissae, dose in milligrams per kilogram body weight; Ordinate, mean (±1 S.E.M.) percentage of total responding that occurred on the midazolam lever.

Fig. 2

Discriminative-stimulus effects of pregnanolone alone and in combination with flumazenil in monkeys that received chlordiazepoxide 46 hr earlier (chlordiazepoxide-treated). Open circles represent the effects of pregnanolone in the absence of other treatment. Each point represents the mean of 3 monkeys. Abscissae, dose in milligrams per kilogram body weight; Ordinate, mean (± 1 S.E.M.) percentage of total responding that occurred on the midazolam lever.

Fig. 3

Schild plots for flumazenil studied with midazolam in monkeys that had not received chlordiazepoxide (control; open symbols) and in monkeys that received chlordiazepoxide 46 hr earlier (chlordiazepoxide-treated; filled symbols). Raw data are shown in Fig. 1. Abscissa, negative log of the molar dose of flumazenil. Ordinate, log (dose ratio−1).

Fig. 4

Left panel: Effect of flumazenil on pED₅₀ values for midazolam in monkeys that had not received chlordiazepoxide (control; open symbols) and in monkeys that received chlordiazepoxide 46 hr earlier (chlordiazepoxide-treated; filled symbols). Apparent pK_b values and log c were estimated by fitting equation 2 to the data. Abscissa, dose of flumazenil in moles per kilogram. Ordinate, mean (±1 S.E.M.) negative logarithm of midazolam ED₅₀ in moles per kilogram. Right panel: Clark plot of the effects of flumazenil on midazolam pED₅₀ values in non-tolerant and benzodiazepine-tolerant monkeys. Abscissa, logarithm of the dose of flumazenil in molar per kilograms plus K_B; Ordinate, mean (±1 S.E.M.) of the negative logarithm of midazolam ED₅₀ in moles per kilograms.