Electrophysiological Characterization of CA1 Pyramidal Neurons

A single brain slice was transferred to a recording chamber of an upright microscope (Axioskop 2-FS; Zeiss, Germany) and continuously perfused (3 mL sec-1, 32 °C) with aCSF. Whole-cell patch-clamp recordings were made from the soma of CA1 pyramidal neurons, identified using a magnification of 60x. All recordings were performed with Axon 700B amplifier using a 4 kHz low pass-filter, digitized at 20 kHz with a Digidata 1400 A and computer-saved using Clampex 10.3 (all from Molecular Devices, Sunnyvale, CA). No liquid junction potential correction was applied. Recording electrodes (3–4.5 MΩ) were pulled from thin-wall borosilicate glass tubes (TW150F-4; World Precision Instruments, Germany) and filled with (in mM): 140 CsCl, 1 MgCl2, 10 HEPES, 2.5 QX314-Cl, 4 Mg-ATP (~ 290 mOsm, pH 7.29). The extracellular solution for recording inhibitory currents was aCSF containing (in μM) 10 NBQX (Abcam), 50 D-AP5 (Abcam), 1 CGP55845 (Sigma-Aldrich) and 5 CNO to block the activity of AMPA/kainate, NMDA and GABAB receptors, respectively.
PV_A females were injected intraperitoneally with either vehicle (Veh: saline) or CNO 40 min before slicing. Spontaneous currents (sIPSCs) were recorded in voltage-clamp mode, holding the membrane potential at -70 mV. For analysis, one-minute-long analysis window was scanned for the detection of sIPSCs; single events were detected manually using an amplitude threshold crossing method in Clampfit 10.3 (Molecular Devices, Sunnyvale, CA) and analysed for amplitude, instantaneous frequency and charge transfer; all parameters were tested for time stability using Spearman’s rank order correlation test and segments of events that showed time instability during the experiment were excluded from further analysis. At least 400 events were analyzed for each experiment.