Primary neurons were isolated from the hippocampi of C57BL/6J mice at embryonic stage 16, plated onto PDL-coated coverslips, and cultured in complete medium [Neurobasal 1×, 2% B27, 2 mM GlutaMAX, and gentamicin (2 μg/ml)] until DIV8 to allow the development of extensive synaptic networks. The cultures were treated with selected compounds or 0.1% DMSO for 24 hours before measuring sEPSCs. Whole-cell patch-clamp recordings were performed using an Axon MultiClamp 700B amplifier, a 1440A Digidata digitizer, and data acquisition using pClamp software (Axon Instruments, Foster City, CA). The neurons were first recorded in a current clamp mode to monitor cell health. Only neurons with a resting membrane potential of less than −40 mV were used for further analysis. sEPSC recordings were made at 50 kHz and subsequently filtered at 5 kHz. The membrane potential was held at −70 mV during the recording. The extracellular bath solution for the recordings contained 135 mM NaCl, 10 mM glucose, 3 mM CaCl2, 2 mM KCl, 2 mM MgCl2, and 5 mM Hepes, adjusted pH to 7.4 with NaOH, and to 300 to 315 mOsm with sucrose. Patch pipettes were pulled from borosilicate glass with a micropipette puller (Sutter Instrument Co.) and filled with intracellular solution containing 100 mM K-gluconate, 1.7 mM KCl, 0.6 mM EGTA, 5 mM MgCl2, 10 mM Hepes, 4 mM ATP, and 0.1 mM guanosine triphosphate, adjusted pH to 7.2 with NaOH and to 300 to 315 mOsm with sucrose. Once a neuron was patched, its seal was monitored, and if the transient resistance was less than 100 mΩ, then the recording was not used for analysis. All experiments were performed at room temperature. The frequency and amplitude of sEPSCs were analyzed using the template match search (pClamp) and measured as a percentage of baseline level, calculated from the average of a 5-min baseline recording. The values of the amplitude and frequency of EPSCs for each recording were reported as an average from a 5-min recording period.

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