Zolpidem

Padsevonil ((4R)‐4‐(2‐chloro‐2,2‐difluoroethyl)‐1‐{[2‐(methoxymethyl)‐6‐(trifluoromethyl)imidazo[2,1‐b][1,3,4]thiadiazol‐5‐yl]methyl}pyrrolidin‐2‐one) was synthesized at UCB Pharma. Zolpidem was obtained from Tocris (Ref 0655; B5A/91713), GABA from Sigma (Ref A2129; B045K00721), chlordiazepoxide from Sequoia Research (Cat. No. SRP02170c), and Ro15‐4513 from Tocris (Cat. No. 1997). All stock solutions were prepared at a maximum of 10 mmol/L in dimethyl sulfoxide (DMSO, 100%) and stored at −20°C. When required, the compounds were diluted into the extracellular medium yielding a final DMSO concentration of 0.1%‐0.5%.

We used the α5-specific inverse GABA_A agonist [³H]L-655,708 as ligand for specifically labelling α5-containing GABA_A receptors in our specimens³⁴ (link) and examined the same slice-mounted tissue sections (20 µm, kept at −20°C) as used for in situ hybridization (n = 24 controls, 19 with and eight without hippocampal sclerosis, two post-mortem with SE). We washed the sections four times in fresh ice-cold 10 mM Tris, 1 mM EDTA, pH 7.4 (Tris/EDTA) buffer for 30 min and then incubated them in Tris/EDTA containing 2 nM methoxyl [³H]L-655,708 (Amersham. Buckinghamshire, Great Britain, catalog number: 708N) and 10 µM zolpidem (Tocris, catalog number: 0655) to inhibit low affinity binding to other α-subunit containing receptors at 4°C for 2 h. Non-specific binding was assessed by incubating the sections with the same solution containing 10 µM flunitrazepam. Labelled sections were rinsed twice in Tris/EDTA for one min, dipped in distilled water, dried in cold air and then exposed together with [¹⁴C] micro-scales (Amersham International, Amersham, UK) to Hyperfilm-B (RMN 9 Bmax 1562545) films (Amersham International, Amersham, UK) for 10 weeks. The films were developed with Kodak D-19 developer (16%) for 1 min, briefly rinsed in water and subsequently fixed for three min. The films were then digitalized using a Canon 9000 Mark II scanner.