Patch-Clamp Recordings

Electrophysiological recordings were performed at room temperature (24–28°C) using oxygenated ACSF. RA and the surrounding tissues were observed using a BX51WI microscope with a DIC-IR video camera (Olympus, Tokyo, Japan). Recording pipettes were fabricated from borosilicate glass (Sutter Instruments, Novato, CA, USA) using a Flaming-Brown puller (P-97, Sutter Instruments) and then filled with a solution containing (in mM): KMeSO₄ 120, NaCl 5, HEPES 10, EGTA 2, QX-314 5, ATP 2, and GTP 0.3 (pH 7.2–7.4, 340 mOsm). The recording pipettes (with a resistance of 4–7 MΩ) were positioned using an integrated motorized control system (Sutter Instruments). Cell-attached and whole-cell recordings were performed using standard techniques. The two cell types in the RA, PNs and GABAergic interneurons, were identified based on their distinct electrophysiological properties(Spiro et al., 1999; Liao et al., 2011). PNs display regular spontaneous firing and generate a time-dependent inward rectification while hyperpolarized using current injection, while interneurons lack spontaneous firing in the resting state (Spiro et al., 1999; Liao et al., 2011). Brain slices were used only once after drug administration for recordings. The junction potentials were corrected before clamping the neurons. Pipette capacitance and series resistance were compensated online using MultiClamp 700B (Molecular Devices, Foster City, CA, USA). Series resistance was monitored at 2 min intervals. Signals were amplified and filtered with the MultiClamp 700B amplifier (low-pass filtered at 10 kHz). The recordings that showed series resistance >20 MΩ or 10% change were excluded from the analysis. Spontaneous excitatory postsynaptic currents (sEPSCs) were isolated by bath application of 150 μM picrotoxin (PTX) to block GABA receptor-mediated inhibitory synaptic currents. For miniature excitatory postsynaptic currents (mEPSCs) recordings, 1 μM tetrodotoxin (TTX) was added into the bath in addition to PTX. The recorded neurons were allowed to stabilize for 3–5 min after the rupturing of the patch. sEPSCs/mEPSCs were recorded for 10–15 min while the membrane potential was held at −70 mV throughout the whole-cell recording. Signals were amplified using MultiClamp 700B.