Ro 15-4513 Antagonizes Alcohol-Induced Sedation in Mice Through αβγ2-type GABA(A) Receptors

Abstract

Ethyl alcohol (ethanol) has many molecular targets in the nervous system, its potency at these sites being low compared to those of sedative drugs. This has made it difficult to discover ethanol's binding site(s). There are two putative binding sites at γ-aminobutyric acid (GABA) type A receptor subtypes for the proposed ethanol antagonist Ro 15-4513, the established γ2 subunit-dependent benzodiazepine site and the recently reported δ subunit-dependent Ro 15-4513/ethanol binding site. Here, we aimed at clarifying the in vivo role of Ro 15-4513 at these two sites. We found that the antagonism of ethanol actions by Ro 15-4513 in wildtype mice was dependent on the test: an open field test showed that light sedation induced by 1.5-1.8 g/kg ethanol was sensitive to Ro 15-4513, whereas several tests for ethanol-induced anxiolytic effects showed that the ethanol-induced effects were insensitive to Ro 15-4513. Antagonism of ethanol-induced sedation by Ro 15-4513 was unaffected in GABA(A) receptor δ subunit knockout mice. By contrast, when testing the GABA(A) receptor γ2 subunit F77I knock-in mouse line (γ2I77 mice) with its strongly reduced affinity of the benzodiazepine sites for Ro 15-4513, we found that the ethanol-induced sedation was no longer antagonized by Ro 15-4513. Indeed, γ2I77 mice had only a small proportion of high-affinity binding of [(3)H]Ro 15-4513 left as compared to wildtype mice, especially in the caudate-putamen and septal areas, but these residual sites are apparently not involved in ethanol antagonism. In conclusion, we found that Ro 15-4513 abolished the sedative effect of ethanol by an action on γ2 subunit-dependent benzodiazepine sites.

Keywords: GABAA receptor; Ro 15-4513; alcohol antagonist; ethanol; inverse agonist.

FIGURE 1

Ethanol-induced reduction in total locomotor activity was reversed by pretreatment with Ro 15-4513 in δ−/− mice. Male and female δ−/− and littermate wildtype δ+/+ mice were pretreated with 3 mg/kg Ro 15-4513 (i.p.) or vehicle (Veh) 15 min before administration of 1.5 g/kg ethanol (EtOH; i.p.) and tested 10 min later on a novel arena for 5 min for total locomotor activity analyzed as percent time moving of the total testing time. ^#p < 0.05 compared to vehicle controls within the same genotype (one-way ANOVA followed by Student's t-test). The data of males and females were pooled. n = 5–7.

FIGURE 2

Reversal of ethanol-induced reduction in total locomotor activity by Ro 15-4513 in wildtype mice. Male C57BL/6J mice were co-administered with 1.8 g/kg ethanol (EtOH) and 3 mg/kg Ro15-4513 (or vehicle, Veh) 10 min before being tested on a novel arena for 5 min. (A) Co-treatment with Ro 15-4513 reversed ethanol-induced reduction in total locomotor activity. (B) Co-treatment with Ro 15-4513 attenuated ethanol-induced reduction in rearing. ^##p < 0.01, ^###p < 0.001 compared to vehicle treatment; ^✦✦✦p < 0.001 compared to ethanol treatment (one-way ANOVA followed by Newman–Keuls test). n = 9–17.

FIGURE 3

Ro 15-4513 reversed ethanol-induced reduction in total locomotor activity in wildtype γ2F77 mice, but not in γ2I77 knock-in mice. Male and female γ2I77 and littermate wildtype γ2F77 mice were co-administered with 1.8 g/kg ethanol and 3 mg/kg Ro15-4513 (or vehicle, Veh) 10 min before being tested on a novel arena for 5 min. (A) Ro 15-4513 reversed ethanol-induced reduction in total locomotor activity in the γ2F77, but not in γ2I77 mice. (B) Ro 15-4513 failed to reverse ethanol-induced reduction in rearing both in the γ2F77 and γ2I77 mice. Because three-way ANOVA revealed no sex effect or interaction the data of males and females were pooled. _***p < 0.001 compared to similarly treated γ2F77 mice; ^###p < 0.001 compared to vehicle controls within the same genotype; ✦✦✦p < 0.001 compared to ethanol treatment within the same genotype (one-way ANOVA followed by Newman–Keuls multiple comparison test). n = 10–14.

FIGURE 4

Effects of ethanol and its combination with Ro 15-4513 on elevated plus-maze behavior in wildtype γ2F77 and γ2I77 knock-in mice. Male and female γ2I77 and littermate wildtype γ2F77 mice were co-administered with 1 g/kg ethanol (EtOH) and 3 mg/kg Ro15-4513 (or vehicle, Veh) 10 min before being tested for anxiolytic-like behavior on an elevated plus-maze for 5 min. (A) The percent time spent in the open arms of the total testing time. (B) The number of open-arm entries. (C) The percent time spent in the closed arms of the total testing time. (D) The number of closed-arm entries. (E) Total movements. Because three-way ANOVA revealed no sex effect or interaction the data of males and females were pooled. ^#p < 0.05, ^##p < 0.01 compared to vehicle-treated mice within the same genotype (one-way ANOVA followed by Student's t-test). n = 10–19.

FIGURE 5

Effects of ethanol and its combination with Ro 15-4513 on elevated plus-maze behavior in γ2I77 knock-in mice. Male and female γ2I77 mice were co-administered with 1 g/kg ethanol (EtOH) and 3 mg/kg Ro 15-4513 (or vehicle, Veh) 10 min before being tested on the elevated plus-maze for 5 min. (A) The percent time spent in the open arms of the total testing time (B) The number of open-arm entries. (C) The percent time spent in the closed arms of the total testing time. (D) The number of closed-arm entries. (E) Total movements. Because two-way ANOVA revealed no sex effect or interaction the data of males and females were pooled. ^#p < 0.05, ^##p < 0.01 compared to vehicle-treated mice (one-way ANOVA followed by Student's t-test). n = 24–26.

FIGURE 6

Effects of ethanol and its combination with Ro 15-4513 on elevated plus-maze behavior in δ+/+ and δ−/− mice. Male and female δ−/− and littermate wildtype δ+/+ mice were co-administered (i.p.) with 1 g/kg ethanol (EtOH) and 3 mg/kg Ro15-4513 (or vehicle, Veh) 10 min before being tested for anxiolytic-like behavior on an elevated plus-maze for 5 min. (A) The percent time spent in the open arms of the total testing time. (B) The number of open-arm entries. (C) The percent time spent in the closed arms of the total testing time. (D) The number of closed-arm entries. (E) Total movements. Because three-way ANOVA revealed no sex effect or interaction the data of males and females were pooled. ^#p < 0.05, ^##p < 0.01 compared to vehicle-treated mice within the same genotype (one-way ANOVA followed by Student's t-test). n = 4–7.

FIGURE 7

Residual benzodiazepine site binding of [³H]Ro 15-4513 to brain sections of γ2I77 knock-in mice as compared to the total binding in wildtype γ2F77 mice. (A) Total [³H]Ro 15-4513 binding to horizontal sections from adult wildtype γ2F77 and γ2I77 mice, showing on the left an optimized image for the γ2F77 section, on the right an optimized image for the residual binding to mutant mouse section, and in the middle the same γ2F77 image using the settings optimized for the mutant. The mean percentage values of the residual binding are given for those areas that show significant binding. (B) In situ hybridization signals for specific oligonucleotide probes for mRNAs of the three γ subunit genes in sections from adult γ2I77 mice.

FIGURE 8

Effects of ethanol (1–100 mM) on [³H]Ro 15-4513 binding to horizontal sections from wildtype γ2F77 and γ2I77 knock-in mice. Representative images for both mouse lines have been prepared using identical imaging settings for contrast and brightness. No consistent inhibition of the binding by ethanol was observed. Non-specific binding in the presence of 10 μM flumazenil was negligible at the background level, except for the cerebellar granule cell layer of wildtype brains. In that area, it amounted to about 3% of total binding.