Whole-cell electrophysiology and data analyses

Voltage-clamp whole-cell recordings were obtained from cultured neurons at room temperature (22° to 25°C). An external solution containing 126 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4·H2O, 26 mM NaCO3, 25 mM glucose, 2 mM MgSO4, and 2 mM CaCl2 (pH adjusted to 7.2 with KOH) was used for the recordings. For recording mEPSCs, glass pipettes with a resistance of 5 to 8 megohms were filled with an internal solution consisting of 120 mM CsMeSO3, 15 mM CsCl, 8 mM NaCl, 0.2 mM EGTA, 10 mM Hepes, 2 mM Mg–adenosine triphosphate (ATP), 0.3 mM Na–guanosine triphosphate, 10 mM tetraethylammonium, and 5 mM QX-314 [5-N-(2,6-dimethylphenylcarbamoylmethyl) triethylammonium bromide] (290 to 300 mOsm). pH was adjusted to 7.3 with CsOH. Neurons were held at a holding potential of −70 mV. In addition, 1 μM tetrodotoxin and 10 μM bicuculline were added to the external recording solution. For recording mIPSCs, glass pipettes were filled with an internal solution consisting of 140 mM CsCl, 0.1 mM GaCl2, 2 mM MgCl2, 10 mM Hepes, 0.5 mM EGTA, 4 mM K-ATP, and 5 mM QX-314 (390 to 300 mOsm). pH was adjusted to 7.3 with CsOH. Neurons were held at a holding potential of −70 mV. In addition, 1 μM tetrodotoxin, 10 μM CNQX, and 50 μM D-AP5 were added. The signals were filtered at 2.9 kHz and digitized at 10 kHz using an EPC-10 amplifier and PatchMaster (v2x53) software (HEKA Elektronic Inc., Germany). Recordings with a pipette access resistance of <20 megohms and <20% changes for the duration of recording were included. The mEPSC and mIPSC recordings were analyzed using Clampfit 10.7 software. The frequency and amplitude were measured in each group.

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