Benzodiazepine‐site pharmacology on GABAA receptors in histaminergic neurons

Abstract

BACKGROUND AND PURPOSEThe histaminergic tuberomamillary nucleus (TMN) of the posterior hypothalamus controls the cognitive aspects of vigilance which is reduced by common sedatives and anxiolytics. The receptors targeted by these drugs in histaminergic neurons are unknown. TMN neurons express nine different subunits of the GABAA receptor (GABAAR) with three α-(α1, α2 and α5) and two γ-(γ1, γ 2) subunits, which confer different pharmacologies of the benzodiazepine-binding site. EXPERIMENTAL APPROACHWe investigated the actions of zolpidem, midazolam, diazepam, chlordiazepoxide, flumazenil (Ro15-1788) and methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM) in TMN neurons using mouse genetics, electrophysiological and molecular biological methods. KEY RESULTSWe find the sensitivity of GABAAR to zolpidem, midazolam and DMCM significantly reduced in TMN neurons from γ2F77I mice, but modulatory activities of diazepam, chlordiazepoxide and flumazenil not affected. Potencies and efficacies of these compounds are in line with the dominance of α2-and α1-subunit containing receptors associated with γ2-or γ1-subunits. Functional expression of the γ1-subunit is supported by siRNA-based knock-down experiments in γ2F77I mice. CONCLUSIONS AND IMPLICATIONSGABAAR of TMN neurons respond to a variety of common sedatives with a high affinity binding site (γ2F77I) involved. The γ1-subunit likely contributes to the action of common sedatives in TMN neurons. This study is relevant for understanding the role of neuronal histamine and benzodiazepines in disorders of sleep and metabolism. LINKED ARTICLESThis article is part of a themed issue on Histamine Pharmacology Update. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2013.170.issue-1 AbbreviationsChlordiazepoxide, 7-chloro-2-methylamino-5-phenyl-3H-1,4-benzodiazepine-4-oxide; Diazepam, 7-chloro-1,3-dihydro-1-methyl-5-phenyl-1,4-benzodiazepin-2(3H)-one; Div, days in vitro; DMCM, Flumazenil,imidazo [1,5-a][1,4]diazepine-3-carboxylate; GABAAR, GABA receptor type A; HE, hepatic encephalopathy; KI, knock-in; MEA, microelectrode array; Midazolam, 8-chloro-6-(2-fluorophenyl)-1-methyl-4H-imidazo [1,5-a] [1,4]benzodiazepine; RAMH, R-α-methylhistamine; TMN, tuberomamillary nucleus; WT, wild type; Zolpidem, N,pyridin-3-yl)acetamide IntroductionMost frequently used drugs for the treatment of insomnia and anxiety are acting at the benzodiazepine-site of GABAA receptors (GABAAR). These receptors are pentameric assemblies of subunits that form a central ion channel (in mammals: α1-6, β1-3, γ1-3, δ, π, ρ1-3, ε, θ). The GABAbinding pocket is formed at the α/β subunit interface, whereas the modulatory benzodiazepine binding site is located at the α/γ interface (Sigel, 2002) in the subunits -β-α-β-α (Baumann et al., 2002;Farrant and Nusser, 2005). Nineteen known GABAAR subunits co-assemble with a restricted number of preferred combinations. For example, the prevailing GABAAR type in the hypothalamus, striatum and amygdala is composed of α2, β3 and γ2-subunits . T...

Ethanol inhibits histaminergic neurons in mouse tuberomammillary nucleus slices via potentiating GABAergic transmission onto the neurons at both pre- and postsynaptic sites

“…The GABAergic synapses formed on histaminergic TMN neurons express at least nine different GABA A receptor subunits: α1, α2, α5, β1, β2, β3, γ1, γ2, and ε [46] . Although the γ1-subunit of GABA A receptors likely contributes to the action of common sedatives in TMN neurons [17] , Wafford et al reported that the α1β1γ2 GABA A receptor subunit combination, which forms a type-1 benzodiazepine receptor, allowed ethanol enhancement of GABA responses, whereas other GABA A receptor subunit combinations did not, even at high ethanol concentrations [47] . Hence, the specific GABA A receptor subtype responsible for the effect of ethanol in the TMN remains to be examined in further studies.…”

“…However, whether ethanol affects the activity of histaminergic neurons in the TMN remained unknown. Given that GABA A and GABA B receptors are present in GABAergic synapses on histaminergic TMN neurons [16,17] , we hypothesized that ethanol may alter GAB-Aergic transmission in the TMN.…”

Zolpidem is a potent stoichiometry-selective modulator of α1β3 GABAA receptors: evidence of a novel benzodiazepine site in the α1-α1 interface

“…Like many benzodiazepines, zolpidem exhibits sedative and hypnotic effects via α1 containing receptors, but unlike benzodiazepines, zolpidem reverses cognitive and motor deficits in neuropathological states, improves motor function in patients with Parkinson’s disease, progressive supranuclear palsy and stroke 13 14 15 16 17 . Studies also show that there are residual effects with zolpidem in the γ2 F77I mouse where binding by zolpidem to α(+)-γ2(−) is attenuated 18 19 . Further, zolpidem can modulate tonic GABA currents in the rat dorsal motor nucleus of the vagus 20 and primary motor cortex 15 , implicating extrasynaptic receptors containing α5 and δ subunits.…”

Fast and Slow Inhibition in the Visual Thalamus Is Influenced by Allocating GABAA Receptors with Different γ Subunits

“…As well as removing zolpidem sensitivity, the γ2F77I point mutation results in DMCM insensitivity (Buhr et al, 1997; Ogris et al, 2004; Leppä et al, 2005). Nevertheless, DMCM will act as an inverse agonist at γ3 subunit-containing GABA A Rs (Herb et al, 1992; Kerti-Szigeti et al, 2014), while DMCM will potentiate γ1-containing GABA A Rs (Puia et al, 1991; Khom et al, 2006; May et al, 2013). In control cells, 10 μM DMCM significantly enhanced the average sIPSC charge transfer by 39.8 ± 15.59% ( n = 7) due to a combined action on the peak amplitude and decay of sIPSCs (paired t -test, P = 0.004; Figure 4D).…”