Acute tolerance to chlordiazepoxide qualitatively changes the interaction between flumazenil and pregnanolone and not the interaction between flumazenil and midazolam in rhesus monkeys discriminating midazolam11These studies were supported by United States Public Health Service Grant R01 DA009157 and a Senior Scientist Award [Grant K05 DA17918 (CPF)]. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute on Drug Abuse or the

Abstract

Benzodiazepines and neuroactive steroids act at distinct binding sites on γ-aminobutyric acidA (GABAA) 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 hr before sessions, chlordiazepoxide produced primarily saline-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 pA2 values of 7.46 when monkeys had not received chlordiazepoxide and 7.44 when they received chlordiazepoxide 46 hr 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.

Quantitative pharmacological analyses of the interaction between flumazenil and midazolam in monkeys discriminating midazolam: Determination of the functional half life of flumazenil

“…Monkeys weighed between 7.6 and 9.3 kg and received primate chow (Harlan Teklad, High Protein Monkey Diet, Madison, WI, USA), fruit, and peanuts daily. The female monkeys (subjects HE, JA, LI and NI) had a history of discriminating midazolam from vehicle while responding under a schedule of stimulus-shock termination (Gerak and France, 2011; Zanettini et al, 2013). The male monkey (subject KI) discriminated midazolam from vehicle under a schedule of food presentation immediately before this experiment (Bai et al, 2011).…”

“…At the beginning of the study, monkeys discriminated 0.178 mg/kg midazolam from vehicle while responding under a fixed-ratio 10 schedule of stimulus-shock termination (Gerak and France, 2012; Zanettini et al, 2013). Over the course of the experiment, 0.178 mg/kg midazolam started to decrease response rates in one monkey (subject NI), and the training dose was changed to 0.133 mg/kg for that monkey; midazolam dose-effect curves were not significantly altered by the change in training dose.…”

“…Stimulus control was considered adequate for testing when responses emitted on the correct lever were ≥ 80% of the total responses for the cycle and when no more than 9 responses (i.e., <one fixed ratio) were emitted on the incorrect lever prior completion of first fixed ratio. Before the first test session was conducted under these conditions, monkeys satisfied the criteria for 5 consecutive or 6 of 7 training sessions, which occurred immediately before these studies for subject KI and previously for the other subjects (Zanettini et al, 2013). Thereafter, stimulus control was considered adequate for testing when the criteria were satisfied during two consecutive training sessions.…”

“…Midazolam dose-effect curves were determined by administering increasing doses across cycles. Saline was given at the beginning of the first cycle and midazolam was given during subsequent cycles with the dose increasing in ¼ log unit increments for each cycle (Gerak and France, 2011; Zanettini et al, 2013). On separate occasions, monkeys received an injection of flumazenil (0.01–1 mg/kg) or its vehicle 15, 30, 60, or 120 min before determination of a midazolam dose-effect curve.…”