2.5 Whole-cell patch-clamp recording of glutamate currents

CeA slices were sectioned and then incubated in oxygenated aCSF for 30 min at 37°C and then 30 min at room temperature. We recorded from 87 medial CeA neurons that were visualized with infrared differential interference contrast (IR-DIC) optics, a x60 water immersion objective (Olympus BX51WI, Tokyo, Japan) and a CCD camera (EXi Aqua, QImaging, Surrey, BC, Canada). Whole-cell voltage-clamp recordings were performed in gap-free acquisition mode with a sampling rate of 10 kHz and low-pass filtered at 10 kHz, using a Multiclamp 700B amplifier, Digidata 1440A and pClamp 10 software (Molecular Devices, Sunnyvale, CA). Pipettes (3–7 MΩ; King Precision, Claremont, CA) were filled with internal solution (in mM): 145 Kgluconate; 0.5 EGTA; 2 MgCl₂; 10 HEPES; 2 Na-ATP; 0.2 Na-GTP. Spontaneous miniature excitatory postsynaptic currents (mEPSCs) were isolated with the GABA receptor blockers, 30 μM bicuculline and 1 μM CGP 55845A, as well as 0.5 μM tetrodotoxin (TTX). Neurons were clamped at −60 mV and the series resistance was monitored with a 10 mV pulse (experiments with a series resistance >15 MΩ or a >20% change were excluded).