Chronic benzodiazepine treatment does not alter interactions between positive GABA(A) modulators and flumazenil or pentylenetetrazole in monkeys.

Benzodiazepines are positive modulators of γ-aminobutyric acidA (GABAA) receptors, and when they bind to their site on this receptor complex, they facilitate the actions of GABA, thereby increasing Cl- flux. Other binding sites on GABAA receptors include neuroactive steroid and barbiturate sites, through which drugs can also modulate this receptor complex. Although they act at different binding sites, positive GABAA modulators produce similar acute behavioral effects. In contrast to their acute effects, several differences have been observed between benzodiazepines and neuroactive steroids during chronic treatment. First, tolerance develops to benzodiazepines, including their effects on seizure activity (Gonsalves and Gallager 1987; Loscher et al. 1996) and schedule-controlled behavior (McMahon and France 2002b; Gerak 2009), but tolerance does not develop to those effects of neuroactive steroids (Kokate et al. 1998; Reddy and Rogawski 2000; McMahon and France 2002a). Second, during chronic benzodiazepine treatment, the potency of neuroactive steroids to alter rates of responding does not change, indicating that cross tolerance does not develop to neuroactive steroids (Gerak 2009). Finally, in diazepam-treated monkeys discriminating flumazenil, temporary discontinuation of diazepam treatment results in the emergence of withdrawal signs as well as responding on the flumazenil lever (McMahon et al. 2007), suggesting that monkeys are dependent on diazepam and that flumazenil-lever responding is related to benzodiazepine withdrawal. Regardless of their site of action, positive modulators reverse withdrawal signs, including flumazenil-lever responding; however, relatively smaller doses of neuroactive steroids are needed, as compared to the doses of benzodiazepines (McMahon et al. 2007). Several effects have been observed in vitro that might contribute to differences between benzodiazepines and neuroactive steroids reported in vivo. For example, chronic benzodiazepine treatment uncouples the benzodiazepine binding site from the GABA, barbiturate and neuroactive steroid binding sites (Tietz et al. 1989; Hu and Ticku 1994a; Friedman et al. 1996). Moreover, chronic benzodiazepine treatment decreases mRNA levels for some subunits and increases mRNA levels for others in rat cortex, thereby altering subunit composition and binding sites of GABAA receptors (Heninger et al. 1990; Kang and Miller 1991; Holt et al. 1996). Such changes in binding sites in vitro might contribute to in vivo differences among these drugs during chronic benzodiazepine treatment. If binding sites on GABAA receptors are altered by daily diazepam administration, then interactions between GABAA modulators might also be expected to change for some drug combinations. In addition to binding site selectivity, GABAA modulators can differ on many other dimensions, including efficacy. For example, while positive modulators facilitate the actions of GABA, negative modulators inhibit, and a drug like flumazenil has little impact on its actions. In untreated monkeys, interactions have been studied extensively between benzodiazepines and drugs with lower efficacy that act at benzodiazepine sites, including flumazenil and negative modulators; under those conditions, the lower-efficacy drugs antagonize the effects of benzodiazepines (Paronis and Bergman 1999; Lelas et al. 2000). When drugs acting at different sites are studied in combination, negative modulators, but not flumazenil, attenuate the effects of positive modulators (McMahon and France 2006). If binding sites are altered by chronic diazepam administration, then interactions different from those observed in untreated monkeys would be predicted in diazepam-treated monkeys. The present studies examined drug interactions in two groups of monkeys: one group discriminated the benzodiazepine midazolam and the second group received diazepam daily and discriminated flumazenil. Interactions were examined in both groups by studying flumazenil and pentylenetetrazole, a negative GABAA modulator acting at a third, distinct binding site (Allan and Harris 1986), in combination with midazolam and pregnanolone. Pentylenetetrazole should attenuate the discriminative stimulus effects of midazolam and pregnanolone whereas flumazenil should attenuate the effects of midazolam and not pregnanolone. To study these interactions in diazepam-treated monkeys, responding on the flumazenil lever was established by administering a single dose of either flumazenil or pentylenetetrazole; thereafter, increasing doses of a positive GABAA modulator were administered to reverse flumazenil-lever responding. Shifts in dose-effect curves of the positive modulators were compared between the two groups by examining changes in dose-effect curves as the dose of flumazenil or pentylenetetrazole was increased. Changes in GABAA receptors in untreated and diazepam-treated monkeys might be reflected by quantitative or qualitative differences in interactions between GABAA modulators.