Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Jul;366(1):145-157.
doi: 10.1124/jpet.118.249250. Epub 2018 May 2.

Evidence That Sedative Effects of Benzodiazepines Involve Unexpected GABA A Receptor Subtypes: Quantitative Observation Studies in Rhesus Monkeys

Affiliations
Free PMC article

Evidence That Sedative Effects of Benzodiazepines Involve Unexpected GABA A Receptor Subtypes: Quantitative Observation Studies in Rhesus Monkeys

Angela N Duke et al. J Pharmacol Exp Ther. .
Free PMC article

Abstract

In nonhuman primates we tested a new set of behavioral categories for observable sedative effects using pediatric anesthesiology classifications as a basis. Using quantitative behavioral observation techniques in rhesus monkeys, we examined the effects of alprazolam and diazepam (nonselective benzodiazepines), zolpidem (preferential binding to α1 subunit-containing GABAA receptors), HZ-166 (8-ethynyl-6-(2'-pyridine)-4H-2,5,10b-triaza-benzo[e]azulene-3-carboxylic acid ethyl ester; functionally selective with relatively high intrinsic efficacy for α2 and α3 subunit-containing GABAA receptors), MRK-696 [7-cyclobutyl-6-(2-methyl-2H-1,2,4-triazol-2-ylmethoxy)-3-(2-flurophenyl)-1,2,4-triazolo(4,3-b)pyridazine; no selectivity but partial intrinsic activity], and TPA023B 6,2'-diflouro-5'-[3-(1-hydroxy-1-methylethyl)imidazo[1,2-b][1,2,4]triazin-7-yl]biphenyl-2-carbonitrile; partial intrinsic efficacy and selectivity for α2, α3, α5 subunit-containing GABAA receptors]. We further examined the role of α1 subunit-containing GABAA receptors in benzodiazepine-induced sedative effects by pretreating animals with the α1 subunit-preferring antagonist β-carboline-3-carboxylate-t-butyl ester (βCCT). Increasing doses of alprazolam and diazepam resulted in the emergence of observable ataxia, rest/sleep posture, and moderate and deep sedation. In contrast, zolpidem engendered dose-dependent observable ataxia and deep sedation but not rest/sleep posture or moderate sedation, and HZ-166 and TPA023 induced primarily rest/sleep posture. MRK-696 induced rest/sleep posture and observable ataxia. Zolpidem, but no other compounds, significantly increased tactile/oral exploration. The sedative effects engendered by alprazolam, diazepam, and zolpidem generally were attenuated by βCCT pretreatments, whereas rest/sleep posture and suppression of tactile/oral exploration were insensitive to βCCT administration. These data suggest that α2/3-containing GABAA receptor subtypes unexpectedly may mediate a mild form of sedation (rest/sleep posture), whereas α1-containing GABAA receptors may play a role in moderate/deep sedation.

Figures

Fig. 1.
Fig. 1.
Dose-dependent sedative behaviors were induced following intravenous injections with the conventional benzodiazepines alprazolam and diazepam (see Table 1 for behavior definitions). The results are shown as mean ± S.E.M. of scores cumulated across the multiple observation periods. Top panels: rest/sleep posture; middle panels: moderate sedation; bottom panels: deep sedation. Note that *P ≤ 0.05, vs. vehicle (V), Bonferroni t tests, n = 4.
Fig. 2.
Fig. 2.
Dose-dependent sedative behaviors differentially induced following intravenous injections of zolpidem and experimental compounds with subtype selectivity. Zolpidem has preferential affinity for α1 subunit-containing GABAA receptors, whereas the three compounds have the following profiles: HZ-166 (selective efficacy for α2 and α3 subunit-containing GABAA receptors, relatively high efficacy as allosteric modulator), MRK-696 (nonselective partial allosteric modulator), TPA-023B (selective efficacy for α2, α3, and α5 subunit-containing GABAA receptors, partial allosteric modulator). All other details as in Fig. 1. Note the absence of moderate sedation data—these ligands did not induce significant changes in this observable behavior. *P ≤ 0.05, vs. vehicle (V), Bonferroni t tests, n = 4.
Fig. 3.
Fig. 3.
Tactile/oral exploration and observable ataxia were altered following intravenous injections with the conventional benzodiazepines alprazolam and diazepam (see Table 1 for definitions). The results are shown as mean ± S.E.M. of scores cumulated across the multiple observation periods. Top panels: Tactile/oral exploration was attenuated by alprazolam and diazepam; bottom panels: Observable ataxia was induced by the highest doses of alprazolam and diazepam. Note that *P ≤ 0.05, vs. vehicle (V), Bonferroni t tests, n = 4.
Fig. 4.
Fig. 4.
Tactile/oral exploration and observable ataxia were altered following intravenous injections with selective compounds. Zolpidem has preferential affinity for α1 subunit-containing GABAA receptors, whereas the three compounds have the following profiles: HZ-166 (selective efficacy for α2 and α3 subunit-containing GABAA receptors, full efficacy allosteric modulator), MRK-696 (nonselective partial allosteric modulator), TPA-023B (selective efficacy for α2, α3, and α5 subunit-containing GABAA receptors, partial allosteric modulator). Other details as in Fig. 3. Top panels: Tactile/oral exploration was dose-dependently enhanced and attenuated by zolpidem (note different scaling on y-axis), but attenuated by all other compounds at the highest dose tested; Bottom panels: Observable ataxia was induced by zolpidem and MRK-696 only. Note that *P ≤ 0.05, vs. vehicle (V), Bonferroni t tests, n = 4.
Fig. 5.
Fig. 5.
Differential effects of pretreatment with βCCT (α1GABAA-preferring antagonist) and flumazenil (nonselective benzodiazepine-site antagonist) on drug-induced sedative behaviors. Data are mean ± S.E.M. of scores cumulated across a test day with multiple observation periods. Multiple doses of βCCT (0.3–3.0 mg/kg, i.v.) and a single dose of flumazenil (“F,” 0.3 mg/kg, i.v.) were administered prior to the session in which peak doses of alprazolam or diazepam were administered (benzodiazepine dose depended on individual dose-response function for each behavioral effect). Top panels: Rest/sleep posture induced by alprazolam (0.1 mg/kg, data point above V, vehicle) or diazepam (3.0 mg/kg, i.v.) was attenuated by flumazenil (F) but not βCCT; middle panels: Moderate sedation was induced by alprazolam (0.3 mg/kg, i.v.) or diazepam (3.0 mg/kg, i.v.) and blocked by both flumazenil (0.3 mg/kg, i.v.) and dose-dependently by βCCT (1.0 and 3.0 mg/kg, i.v.); bottom panels: Deep sedation was induced by the highest doses of alprazolam (1.0 mg/kg) or diazepam (10 mg/kg) and attenuated by flumazenil (0.3 mg/kg) and dose-dependently by βCCT (1.0 and 3.0 mg/kg). Note that *P ≤ 0.05, vs. vehicle (V), Bonferroni t tests, n = 4.
Fig. 6.
Fig. 6.
Effects of pretreatment with βCCT (α1GABAA-preferring antagonist) and flumazenil (“flum,” nonselective benzodiazepine-site antagonist) on sedative behaviors induced by selective compounds. Top panel: Rest/sleep posture induced by HZ-166 (functionally selective α2/3GABAA allosteric modulator) was blocked by flumazenil (0.3 mg/kg, i.v.) but not the highest dose of βCCT tested (3.0 mg/kg, i.v.); bottom panel: Deep sedation induced by zolpidem (α1GABAA-preferring allosteric modulator) was blocked by both flumazenil and the highest dose of βCCT tested. Note that *P ≤ 0.05 vs. HZ-166 or zolpidem alone, Bonferroni t tests, n = 4 monkeys.
Fig. 7.
Fig. 7.
Effects of pretreatment with βCCT (α1GABAA-preferring antagonist) and flumazenil (“F,” nonselective benzodiazepine-site antagonist) on attenuation of species-typical behavior and observable ataxia induced by alprazolam and diazepam. Data are mean ± S.E.M. of scores cumulated across a test day with multiple observation periods. Multiple doses of βCCT (0.3–3.0 mg/kg, i.v.) and a single dose of flumazenil (0.3 mg/kg, i.v.) were administered prior to the session in which peak doses of alprazolam or diazepam were administered (benzodiazepine dose depended on individual dose-response function for each behavioral effect). Top panels: Tactile/oral exploration attenuated by alprazolam (1.0 mg/kg, data point above vehicle “V”) or diazepam (10 mg/kg, i.v.) was attenuated by flumazenil (F) but not βCCT. Note that *P < 0.05 vs. vehicle (V); horizontal dashed lines represent levels of behavior without drug treatment. Bottom panels: Observable ataxia was induced by alprazolam (0.3 mg/kg) or diazepam (3.0 mg/kg) and attenuated by flumazenil (0.3 mg/kg) and dose dependently by βCCT (1.0 and 3.0 mg/kg). Note that *P ≤ 0.05, vs. vehicle (V), Bonferroni t tests, n = 4.
Fig. 8.
Fig. 8.
Antagonism by βCCT and flumazenil of zolpidem- and HZ-166-induced changes in tactile/oral exploration. Other details as in Fig. 7. Left panel: Zolpidem (“Zolp”) at a lower dose (1.0 mg/kg, i.v.) enhanced tactile/oral exploration that was blocked by both flumazenil (“Flum,” 0.3 mg/kg, i.v.) and βCCT (3.0 mg/kg, i.v.); center panel: Zolpidem at a higher dose (10 mg/kg, i.v.) attenuated tactile/oral exploration, which was reversed by flumazenil (0.3 mg/kg, i.v.) but not βCCT (3.0 mg/kg, i.v.); right panel: HZ-166 (30 mg/kg, i.v.) attenuated tactile/oral exploration that was reversed by flumazenil (0.3 mg/kg, i.v.) but not βCCT (3.0 mg/kg, i.v.). Note that *P ≤ 0.05 vs. vehicle (Veh) or “Zolp” condition, Bonferroni t tests, n = 4 monkeys.

Similar articles

See all similar articles

Cited by 5 articles

Publication types

LinkOut - more resources

Feedback