Electrophysiology experiments were performed as described previously (50), with details provided below.

Slice preparation. Mice were deeply anaesthetized with halothane (2-bromo-2-chloro-1,1,1-trifluoroethane, Takeda Pharmaceutical Co. Ltd.) or isoflurane [(2RS)-2-chloro-2-(difluoromethoxy)-1,1,1-trifluoroethane, Pfizer Inc.] and sacrificed by decapitation. The whole brain was quickly removed into an ice-cold cutting solution comprising 200 mM sucrose, 4 mM KCl, 1 mM NaH2PO4, 0.2 mM CaCl2, 10 mM MgCl2, 26.2 mM NaHCO3, 11 mM d-(+)-glucose, 0.1 mM l(+)-ascorbic acid, and 0.5 mM sodium pyruvate, saturated with 95% O2 and 5% CO2. Each brain hemisphere glued on a piece of agar block (4% in saline) was sliced along the longer axis of hippocampus at a thickness of 400 μm, using a microslicer (LinearSlicer Pro7, Dosaka EM Co. Ltd.). Hippocampi were dissected out of whole-brain slices and recovered in a submerged holding chamber filled with a physiological medium [artificial cerebrospinal fluid (aCSF)] comprising 119 mM NaCl, 2.5 mM KCl, 1 mM NaH2PO4, 2.5 mM CaCl2, 1.3 mM MgCl2, 26.2 mM NaHCO3, 11 mM d-(+)-glucose, 0.1 mM l(+)-ascorbic acid, and 0.5 mM sodium pyruvate, continuously bubbled with 95% O2 and 5% CO2. The slices, which had been equilibrated for more than 2 hours, were transferred to an immersion-type recording chamber and superfused with well-bubbled aCSF at a rate of 2.5 ml/min. All physiological experiments were carried out at 25°C.

Patch clamp (acute hippocampal slices). Whole-cell voltage clamp configuration was obtained by a blind access to CA1 pyramidal cell layer with a patch pipette (4 to 7 megohms, Harvard Apparatus). The composition of the pipette solution for EPSCs recording was the following: 122.5 mM Cs gluconate, 17.5 mM CsCl, 10 mM Hepes, 0.2 mM EGTA, 8 mM NaCl, 2 mM Mg-ATP, and 0.3 mM Na3-GTP (pH 7.3; 290 to 300 mOsm). For IPSC recording, Cs gluconate was partially replaced with CsCl as follows: 40 mM Cs gluconate, 100 mM CsCl, 10 mM Hepes, 0.2 mM EGTA, 8 mM NaCl, 2 mM Mg-ATP, and 0.3 mM Na3-GTP (pH 7.3; 290 to 300 mOsm). AMPA receptor– and NMDA receptor–mediated currents (AMPAR- and NMDAR-EPSCs) were recorded at −70 and +40 mV, respectively, in the presence of picrotoxin (100 μM) and CGP55845 (1 μM). NMDAR-EPSCs were recorded after pharmacologic isolation with 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline (NBQX) (20 μM). Schaffer collateral fibers were surgically cut and stimulated at 0.1 Hz. The amplitude of the EPSC was analyzed for the comparison between genotypes. IPSCs mediated by GABAA receptors were recorded under blockade of NBQX (10 μM) and D-AP5 (25 μM). mEPSC and mIPSC were recorded at −70 mV in the presence of tetrodotoxin (TTX; 0.5 μM). Amplitude and frequency for miniature currents were analyzed using Mini Analysis Program version 6 (Synaptosoft Inc.) for the comparison between genotypes. Series resistances were less than 30 megohms, and there was no significant difference in series resistance between experimental groups. The monitored synaptic responses were filtered at 1 kHz using an Axon 200B or 700B amplifier and digitized at 10 kHz using Clampex software of pClamp 9 or 10 suite (Molecular Devices LLC). Membrane potentials in whole-cell patch clamp were represented without being compensated for liquid junction potential.

Patch clamp (primary cultured cortical GABAergic neurons). Whole-cell patch-clamp recordings were carried out using DIV14 to 21 primary cortical GABAergic interneuron cultures placed on the stage of a Zeiss Axio Examiner upright microscope using Axopatch 200B and Multiclamp amplifiers (Axon Instruments). The recording chamber was continuously perfused with an extracellular solution containing 130 mM NaCl, 2.5 mM KCl, 2.2 mM CaCl2, 1.5 mM MgCl2, 10 mM d-glucose, and 10 mM Hepes (pH 7.35; osmolarity adjusted to 290 mOsm). The micropipettes were made from borosilicate glass capillaries, with a resistance in the range of 3 to 5 megohms. The intracellular solution contained 100 mM k-gluconate, 17 mM KCl, 5 mM NaCl, 5 mM MgCl2, 10 mM Hepes, 0.5 mM EGTA, 4 mM ATPK2, and 0.5 mM GTP-Na (pH 7.3; osmolarity adjusted to 280 mOsm). All recordings were performed at room temperature (21° to 24°C). Data were recorded at 10 kHz and analyzed offline. All analysis was performed in Clampfit and Excel.

Drug. Drugs were purchased from the following sources: picrotoxin (28004-71, Nacalai Tesque Inc.; sc-202765, Santa Cruz Biotechnology Inc.), TTX (32775-51, Nacalai Tesque Inc.; 1078, Tocris Bioscience), CGP55845 (1248, Tocris Bioscience), D-AP5 (0106, Tocris Bioscience), and NBQX (0373, Tocris Bioscience).

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