Cultured hippocampal neurons (80 × 103 cells/cm2) with pyramidal morphology (DIV 15 to 17) were whole-cell voltage–clamped to −60 mV, at room temperature, in a Mg-free Tyrode’s solution containing 150 mM NaCl, 4 mM KCl, 10 mM glucose, 10 mM Hepes, and 2 mM CaCl2 (pH 7.35) (310 mOsm). To record and isolate NMDAR-mediated mEPSCs, 6-cyano-7-nitroquinoxaline-2,3-dione [CNQX; 10 μM; Tocris (AMPA/kainate receptor antagonist)], bicuculline {10 μM; Tocris [γ-aminobutyric acid type A (GABAA) receptor antagonist]}, tetrodotoxin [TTX; 500 nM; Tocris (blocker of voltage-gated Na+ channels)], and glycine [15 μM; Sigma-Aldrich (co-agonist of NMDARs)] were added to the bath solution (65). The electrode solution had the following composition 115 mM Cs-MeSO3, 20 mM CsCl, 2.5 mM MgCl2, 10 mM Hepes, 0.6 mM EGTA, 4 mM Na2-ATP (adenosine triphosphate), and 0.4 mM Na-GTP (guanosine triphosphate) (pH 7.3) (300 mOsm; Sigma) (66). Where indicated, hippocampal neurons were preincubated with cycloheximide (50 μg/ml) or with vehicle (DMSO) (1:1000 dilution) for 15 min before recording the NMDAR-mediated mEPSCs. When the effect of BDNF was tested, hippocampal neurons were preincubated with the neurotrophin (50 ng/ml) for at least 30 min before recording the NMDAR-mediated mEPSCs. Recording electrodes were made of borosilicate glass capillaries and pulled on a horizontal stage Sutter Instrument P-97 puller (resistances, 3 to 4 megohms). Recordings were made without series resistance compensation. Cells were recorded for a period of 5 min, and the baseline for the analysis of NMDAR-mediated mEPSCs was manually determined as the average current level of silent episodes during a recording. Whole-cell recordings from hippocampal neurons were performed using an Axon CNS, a MultiClamp 700B amplifier, an Axon Digidata 1550 A acquisition board, and pClamp software (version 10.5, Molecular Devices). Signals were filtered at 2.8 Hz and sampled at 25 kHz, and the amplitude of NMDAR-mediated currents was analyzed offline with pClamp software (version 10.5, Molecular Devices).

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