Sprague-Dawley rats (n = 16) were used throughout the study and were treated in accordance with the guidelines set by the National Institute of Health and Dartmouth Medical School for the humane treatment of animals. Rats were subjected to 58–60 flurothyl-induced seizures from postnatal (P) day 1 to 10 (5–6 seizures per day) using previously described methods from our laboratory (Liu et al., 1999
; Isaeva et al., 2006
). Control rats were handled and treated in the same manner but not exposed to flurothyl.
At P20–30 rats were deeply anaesthetized with isoflurane and decapitated, brains were removed, and brain slices were sectioned (Leica 1000S vibroslicer, Leica Microsystems, Nussloch GmbH, Germany) at a thickness of 500 µm in cold (4°C) dissecting solution of the following composition (mM): sucrose 250, KCl 2, CaCl2 0.5, MgCl2 7, NaHCO3 26, NaH2PO4 1.2 and glucose 11 (pH=7.4). Slices were then stored in warm (32°C) oxygenated (95% O2, 5% CO2) artificial cerebrospinal fluid (ACSF) of the following composition (mM): NaCl 126, KCl 3.5, CaCl2 2.0, MgCl2 1.3, NaHCO3 25, NaH2PO4 1.2 and glucose 11 (pH 7.3) for at least 1.5 h before use.
sIPSCs and mIPSCs were recorded from visually identified (Olympus BX51WI, Japan layer 2/3 (L2/3) pyramidal cells of somatosensory cortex using patch-clamp technique in whole-cell configuration. For recordings of sIPSCs the slice was continuously superfused with oxygenated ACSF at 32°C. mIPSCs were recorded in the presence of tetrodotoxin (TTX, 1 µM). Patch electrodes were pulled (Narishige, PC-10, Japan) from borosilicate glass capillaries (GC150F-15, Clark Electromedical Instruments) and had an impedance of 2–3 MO when filled with a solution of the following composition (in mM): Cs-gluconate 117.5, CsCl 17.5, NaCl 8, HEPES 10, EGTA 10, Na3GTP 0.2, and MgATP 2 (pH 7.3). Whole-cell recordings were made using an Axopatch 200B amplifier (Axon Instruments). A liquid junction potential was subtracted from the recorded membrane potentials. The series resistances were compensated on 80–90%. Recordings were digitized online with the PClamp 7 software (Axon Instruments) using a Digidata 1322A acquisition board (Axon Instruments). Spontaneous events were analyzed with the MiniAnalysis (version 6.0.7, Synaptosoft Inc., Decatur, GA), Clampfit (Axon Instruments) and Origin 7.0 (Microcal Software, Northampton, MA, USA) software. The amplitude and interevent interval of sIPSCs and mIPSCs were estimated for every single event then combined in two groups (control and flurothyl-treated) and averaged. Rise time and half-width of IPSCs were estimated and averaged for each cell and the mean values were averaged and compared for control and flurothyl-treated rats. All data are expressed as the mean ± SE. The statistical significance of differences in amplitude and kinetic parameters of IPSC were evaluated using the Student's t test. To compare the interevent interval of IPSC in groups we used the Kolmogorov–Smirnov (K-S) test. SR95531 was obtained from Tocris (Ellisville, MO, USA). All other chemicals were purchased from Sigma (St. Louis, MO, USA).