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

Department of Pharmacology, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, mail code 7764, San Antonio, TX 78229-3900, USA.
Behavioural pharmacology (Impact Factor: 2.15). 02/2011; 22(1):49-57. DOI: 10.1097/FBP.0b013e3283425aa0
Source: PubMed


Benzodiazepines and neuroactive steroids are positive c-aminobutyric acid(A) (GABA(A)) modulators acting at distinct binding sites; during benzodiazepine treatment, tolerance develops to many behavioral effects of benzodiazepines, although cross tolerance typically does not develop to neuroactive steroids. To determine whether differential changes in binding sites contribute to these behavioral differences, interactions between GABA(A) modulators were studied in two groups of four monkeys: one otherwise untreated group discriminated 0.178 mg/kg of the benzodiazepine midazolam; the other received 5.6 mg/kg/day of diazepam and discriminated 0.1 mg/kg of flumazenil, which binds to benzodiazepine sites without modulating GABA(A) receptors. In untreated monkeys, flumazenil antagonized midazolam but not the neuroactive steroid pregnanolone, whereas pentylenetetrazole (a negative modulator acting at a third site) antagonized both positive modulators. In diazepam-treated monkeys, 0.1 mg/kg of flumazenil or 32 mg/kg of pentylenetetrazole produced flumazenil-lever responding, which was reversed by midazolam and pregnanolone. As the flumazenil dose increased, larger doses of midazolam, but not pregnanolone, were needed to reverse flumazenil-lever responding. When the pentylenetetrazole dose increased, larger doses of both positive modulators were needed. Thus, interactions between GABA(A) modulators were not different between diazepam-treated and untreated monkeys and do not reveal changes in binding sites that could account for reported differences between benzodiazepines and neuroactive steroids.

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    • "Although absolute potency of drugs acting at benzodiazepine sites can vary across procedures , relative potencies do not change (Lelas et al., 1999; Rowlett et al., 2006). Drug discrimination has been used extensively to examine interactions between GABA A modulators that vary in efficacy and site of action on GABA A receptors (Lelas et al., 2000; McMahon and France, 2005; Gerak and France, 2011; current study), providing an empirical and theoretical framework against which results could be compared and demonstrating that drug discrimination studies are highly reliable and predictable. Finally, under the conditions used in the current study, additive interactions have been observed between positive modulators acting at different sites, and flumazenil has been shown to enhance the effects of pregnanolone, providing a baseline against which the relative enhancement or attenuation of the combined effects of positive modulators can be compared. "
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    ABSTRACT: Adverse effects of benzodiazepines limit their clinical use; these effects might be reduced without altering therapeutic effects by administering other positive GABA(A) modulators (i.e., neuroactive steroids) with benzodiazepines. One concern with this strategy involves reversing these combined effects in case of overdose. The current study examined whether flumazenil can attenuate the combined effects of two benzodiazepines, midazolam and flunitrazepam, and the combined effects of midazolam and the neuroactive steroid pregnanolone, in four monkeys discriminating midazolam. Each positive modulator produced ≥80% midazolam-lever responding. Interactions between midazolam and either flunitrazepam or pregnanolone were additive. Flumazenil antagonized the benzodiazepines when they were administered alone or in combination. Schild analyses yielded slopes that did not deviate from unity, regardless of whether benzodiazepines were administered alone or together; the pA(2) value for flumazenil was 7.58. In contrast, flumazenil enhanced the effects of pregnanolone with 0.32 mg/kg flumazenil shifting the pregnanolone dose-effect curve 2-fold leftward. Flumazenil attenuated the combined effects of midazolam and pregnanolone, although antagonism was not dose-dependent. Thus, the interaction between two benzodiazepines was similar to that of a benzodiazepine and a neuroactive steroid; however, flumazenil more efficiently attenuated a combination of two benzodiazepines compared with a combination of a benzodiazepine and a neuroactive steroid. Although the magnitude of antagonism of a benzodiazepine combined with a neuroactive steroid was reduced, these results support continued exploration of the use of combinations of positive modulators to enhance therapeutic effects while reducing adverse effects.
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    ABSTRACT: Benzodiazepines and neuroactive steroids act at distinct binding sites on γ-aminobutyric acid(A)(GABA(A)) receptorswhere theypositively 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 0.178mg/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 determinedwhen monkeys had not received chlordiazepoxide and when they received 10mg/kg chlordiazepoxide 46hours earlier; for some tests, flumazenil was given before determination of dose-effect curves. Midazolam and pregnanolone produced ≥80% midazolam-lever responding. When administered 46h 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 pA(2) values of 7.46 whenmonkeys had not received chlordiazepoxide and 7.44 when they received chlordiazepoxide 46h 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 sitesaccount for the development of acute tolerance.
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    ABSTRACT: Neuroactive steroids and benzodiazepines can positively modulate GABA by acting at distinct binding sites on synaptic GABAA receptors. Although these receptors are thought to mediate the behavioral effects of both benzodiazepines and neuroactive steroids, other receptors (e.g., extrasynaptic GABAA, N-methyl-D-aspartate (NMDA), σ1, or 5-HT3 receptors) might contribute to the effects of neuroactive steroids, accounting for differences among positive modulators. The current study established the neuroactive steroid pregnanolone as a discriminative stimulus to determine whether actions in addition to positive modulation of synaptic GABAA receptors might contribute to its discriminative stimulus effects. Four rhesus monkeys discriminated 5.6 mg/kg pregnanolone while responding under a fixed-ratio 10 schedule of stimulus-shock termination. Positive modulators acting at benzodiazepine, barbiturate, or neuroactive steroid sites produced ≥80 % pregnanolone-lever responding, whereas drugs acting primarily at receptors other than synaptic GABAA receptors, such as extrasynaptic GABAA, NMDA, σ1, and 5-HT3 receptors, produced vehicle-lever responding. Flumazenil antagonized the benzodiazepines midazolam and flunitrazepam, with Schild analyses yielding slopes that did not deviate from unity and pA2 values of 7.39 and 7.32, respectively. Flumazenil did not alter the discriminative stimulus effects of pregnanolone. While these results do not exclude the possibility that pregnanolone acts at receptors other than synaptic GABAA receptors, they indicate a primary and possibly exclusive role of synaptic GABAA receptors in its discriminative stimulus effects. Reported differences in the effects of benzodiazepines and neuroactive steroids are not due to differences in their actions at synaptic GABAA receptors.
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