Regular articleExtrasynaptic GABAA channels activated by THIP are modulated by diazepam in CA1 pyramidal neurons in the rat brain hippocampal slice
Introduction
γ-Aminobutyric acid (GABA) is the main inhibitory transmitter in the brain. When it binds to GABAA receptors, chloride conductance is activated. Because of their wide distribution in the brain, GABAA receptors are of major clinical significance and these receptors are the targets of many therapeutic drugs. THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol), also called gaboxadol, is a potent and specific GABAA receptor agonist (Frolund et al., 2002) and is currently in clinical development as a hypnotic. Several studies have shown that THIP promotes deep sleep and sleep maintenance in addition to facilitating sleep initiation Lancel and Faulhaber 1996, Lancel 1997, Faulhaber et al 1997, Lancel and Langebartels 2000, Mathias et al 2001. THIP has also been shown to be neuroprotective when given together with diazepam in a rat cerebral ischemia model of delayed CA1 pyramidal cell death (Johansen and Diemer, 1991).
GABAA receptors are thought to be pentameres. Eightteen different subunits grouped into eight subunit families (α1–6, β1–3, γ1–3, ρ1–2, δ, ε, θ, π) have been identified to date (Barnard et al., 1998). Work on reconstituted receptors indicates that the subunit composition of the receptors affects the apparent potency for THIP and other GABAA receptor agonists. For receptors expressed in Xenopus oocytes and cell lines the apparent affinity ranges from 6 to 350 μM Ebert et al 1994, Ebert et al 1997, Thompson et al 1999, Brown et al 2002. At α1β2/3γ2S GABAA receptors the apparent affinity of THIP is approximately 100 μM Ebert et al 1997, Thompson et al 1999 and similar to the EC50 value of 80 μM determined in whole-cell studies of isolated rat hippocampal CA1 neurons (Sundstrom-Poromaa, 2002). In contrast, an EC50 value of about 6 μM was obtained in the rat cortical wedge preparation (Ebert et al., 2002), at α5β3γ2 receptors expressed in Xenopus oocytes (Ebert et al., 1997), and at α4β3δ containing receptors expressed in cell lines (Brown et al., 2002). The clinically effective concentration of THIP is approximately 1 μM Schultz et al 1981, Madsen et al 1983.
In this study we examined the effects of THIP on GABAA channels in CA1 pyramidal neurons in rat hippocampal brain slices. In the intact neurons THIP activated high-conductance channels (>40 pS, −Vp = 40 mV) with a delay that could be as long as several minutes. Diazepam modulated THIP-activated receptors.
Section snippets
THIP-activated channels
High-resistance seals were obtained on 182 cells in the CA1 pyramidal cell layer of the rat hippocampal slice. In 22 of these patches no channel activity was detected when the pipette solution contained THIP (2–100 μM). In another 12 patches the current–voltage relationship of the channels did not correlate with chloride-permeable channels. The type of channels recorded in the remaining 148 patches could be grouped into two classes: channels that showed rapid opening and closing (flickery
Discussion
The aim of this study was to characterize the effect THIP had on GABAA receptors expressed in neurons in brain tissue from the hippocampus. The recordings were made in cell-attached patches as only in this configuration is the receptor in contact with the intracellular milieu, cytoskeletal elements, and other proteins and compartments that are parts of the normal intracellular environment. In recent years evidence has mounted indicating that the receptors interact not only with extracellular
Slices
Experiments were carried out on neurons in the CA1 region of rat hippocampal slices. The methods used for preparation of slices have been described previously (Birnir et al., 1994). Briefly, a 17- to 21-day-old rat was decapitated and the brain was removed and placed into ice-cold artificial cerebrospinal fluid (ACSF) containing (mM): 124 NaCl, 26 NaHCO3, 3 KCl, 1.3 MgSO4, 2.5 NaH2PO4, 2.5 CaCl2, and 20 glucose. The pH of this solution when equilibrated with a gas mixture containing 95% O2 and
Acknowledgements
We thank Johan Enquist for his participation in the initial experiments in this study. We thank the Swedish Medical Research Council (Y0924), Vetenskapsrådet (K2002-33X-13408-03A), Åke Wibergs Stiftelse, Swärds Stiftelse, Crafoordska Stiftelsen, Segerfalks Stiftelsen, the Medical Faculty Lund University, and Lundbeck AS for financial support.
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