Supplementary Materials [Supplementary Data] bhp156_index. the rats experienced class IV/V seizures
May 15, 2019
Supplementary Materials [Supplementary Data] bhp156_index. the rats experienced class IV/V seizures (Racine 1972). After 3 h of = 26) with a mossy fiber sprouting (see the Supplementary Fig. S1) according to the previously well-described Timm staining (Tauck and Nadler 1985; Represa et al. 1987). Age-matched untreated (na?ve rats, = 29) or treated with scopolamine and diazepam but NaCl (0.9%) instead of pilocarpine (sham rats, = 5) were used as controls (range 3C13 months old; mean age = 5.5 0.5 months old; = 34). Because there was no difference between na?ve and sham rats (not shown), the data were pooled together. Preparation of Hippocampal Slices Animals were deeply anesthetized with chloral hydrate (350 mg/kg, i.p.) and decapitated. The brain was removed rapidly, the Slc3a2 hippocampi were dissected, and transverse 400-M-thick hippocampal slices were cut using HM650V MGCD0103 cost MicroM tissue slicer in a solution containing the following (in mM): 110 choline, 2.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 7 MgCl2, 0.5 CaCl2, and 7 D-glucose (5 C). Slices were then transferred for rest at room heat (1 h) in oxygenated normal artificial cerebrospinal fluid (ACSF) containing the following (in mM): 126 NaCl, 3.5 KCl, 1.2 NaH2PO4, 26 NaHCO3, 1.3 MgCl2, 2.0 CaCl2, and 10 D-glucose, pH 7.4. Patch-Clamp Recordings Whole-cell recordings of dentate gyrus granule cells from chronic epileptic and control rats were obtained using the blind patch-clamp technique in a submerged chamber (ASCF; 30C32 C) using low resistance electrodes (5C8 M). For current-clamp experiments, electrodes were filled with an internal solution containing the following (in mM): 130 KMeSO4, 5 KCl, 5 NaCl, 10 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 2.5 MgATP, 0.3 NaGTP, and 0.5% biocytin, pH 7.25. For voltage-clamp experiments, the internal answer contained the following (in mM): 110 CsF, 20 CsCl, 11 sodium ethylene glycol tetraacetic acid, 10 HEPES, 2 MgCl2, 0.1 CaCl2, 2 MgATP, 0.4 NaGTP, 10 phosphocreatine. Access resistance ranged between 10 and 20 M, and the results were discarded if the access resistance changed by more than 20%. For loose cell-attached patch recordings, pipettes were filled with ACSF. Whole-cell recordings were performed using an Axoclamp 2B and a Multiclamp 700A amplifier (Axon Devices, Molecular Devices, Union City, CA). Data were filtered at 2 kHz, digitized (20 MGCD0103 cost kHz) with a Digidata 1200 and 1322A (Molecular Devices) to a personal computer, and acquired using Axoscope 7.0 and Clampex 9.2 softwares (PClamp, Axon Instruments, Molecular Devices). Signals were analyzed off-line using MiniAnalysis 6.0.1 (Synaptosoft, Decatur, GA), and Clampfit 10.1 (Molecular Devices). AMPA/kainate receptorCmediated EPSPs were isolated in the presence of blockers of N-methyl-D-aspartate (NMDA) (40 M D-APV or 10 M MK801), GABAA (10 M bicuculline), and GABAB (5 M CGP 55845) receptors (Epsztein et al. 2005). Electrical Stimulations Small EPSPs (3C5 mV) were evoked by poor stimulations performed via a bipolar NiCh electrode (50 m diameter, NI-0.7F, Phymep, Paris) positioned either in the inner one-third of the molecular layer of the dentate gyrus to stimulate proximal inputs (PI; i.e., associational/commissural inputs in controls and recurrent mossy fiber MGCD0103 cost inputs in epileptic rats) or in the outer one-third of the molecular layer of the dentate gyrus to stimulate distal perforant inputs (PP) in control and epileptic rats. The stimulus intensity, pulse duration, and frequency were around 30V, 70 s, and 0.2 Hz, respectively. Using these stimulation parameters DGCs usually discharged in single-spike mode and never in burst firing mode. Following action potential, the decay of EPSP.