Whole-cell patch-clamp recordings

Whole-cell patch-clamp recordings were performed on CA1 pyramidal cells as previously described [39]. The pipette solution contained (in mM): 130 Cs-methanesulfonate, 2 NaCl, 20 HEPES, 0.6 EGTA, 5 QX-314, 4 Mg-ATP, and 0.4 GTP (pH ~7.3 and osmolality 280–300 mOsm). During recordings, the slice was placed in a submersion-recording chamber and perfused with ACSF at a constant flow (~2 ml m⁻¹) at room temperature. The perfusion ACSF contained (in mM) 124 NaCl, 3 KCl, 2 CaCl₂, 1 MgCl₂, 26 NaHCO₃, 1.25 NaH₂PO₄, and 10 D-glucose (gassed with 95% O₂/5% CO₂; pH 7.35). Picrotoxin (100 µM) was added to inhibit GABA_A receptor channels. Patch pipette resistances were 3–5 MΩ. Current was recorded at a sampling frequency of 10 kHz and analog filtered at 3 kHz, using an EPC-10 amplifier (HEKA Elektronik, Lambrecht, Germany). The liquid junction potential was both measured and calculated to be about 8 mV and was not corrected for. Series resistance was monitored using a 20 ms 10 mV hyperpolarizing pulse. The series resistance was not allowed to exceed 20 MΩ in whole-cell recordings, or to change more than 20% during an experiment, otherwise the experiment was discarded. For recording of spontaneous AMPAR (−70 mV) and NMDAR (+40 mV) mediated responses, receptor kinetics as well as tonic APV-sensitive currents, we patched pyramidal cells within the CA1 cell layer. Tonic NMDA currents were recorded as the subtracted holding current at a depolarized potential of +40 mV before and after the addition of APV. For recording of evoked EPSCs, Schaffer collateral/commissural afferents were stimulated using 0.2 ms biphasic (negative/positive) constant current pulses (5–60 µA; STG 1002, Multi Channel Systems, Reutlingen, Germany) delivered through an insulated tungsten microelectrode (resistance ~ 0.5 MΩ). Stimulation electrodes were positioned in the stratum radiatum, at least 100 µm from the recorded cell, and synaptic inputs received test pulse stimulation every 5 s. Evoked and spontaneous responses were analyzed off-line using custom-made IGOR Pro (WaveMetrics, Lake Oswego, OR, USA) software. Receptor kinetics (Tau decay) was analyzed using a single exponential offset curve fit within IGOR Pro. Data are expressed as means ± SEM. Statistical significance for independent samples was evaluated using paired t-test or Student’s t-test, unless otherwise indicated. Multiple comparisons were corrected for using the modified Holm-Bonferroni algorithm.