Whole-cell patch-clamp in vitro

A single slice is transferred to a submerged recording chamber (2.5–3 ml/min, 33.5°C), on the stage

of an upright microscope equipped for infrared video microscopy (Hamamatsu, Tokyo), allowing direct visualization of the recorded neurons.

ACSF composition is (in mM): 126 NaCl, 2.5 KCl,1.2 MgCl₂, 1.2 NaH2PO4, 2.4 CaCl2, 10 glucose and 25 NaHCO3. The solution is saturated with a mixture of O2/CO2

Neurons were recorded in whole cell patch-clamp configuration using 1.5-mm borosilicate glass electrodes (3–4 MOhm), pulled with a vertical puller (PP-83 Narishige) and filled with (in mM): K-gluconate (135), KCl (10), HEPES (10), MgCl₂(2), EGTA (0.1), CaCl₂ (0.05), Mg₂-ATP (4), Na₃-GTP (0.3), Phosphocreatine-Na₂(10), adjusted to pH 7.3 with KOH. Membrane currents were

recorded in voltage-clamp mode at a holding potential of -60 mV, using a differential amplifier (Multiclamp 700B, Molecular Devices, LLC., 3860 N First Street San Jose, CA 95134 USA). Signals were filtered at 3 kHz, and digitized at 10 kHz.