Measuring Spontaneous Neuronal Activity

Whole-cell recordings were made from cortical neurons (14 DIV), which were treated with DMSO or diazepam for 72 h as described before above. Patch pipettes (resistance 8-10 MΩ) were pulled from borosilicate glass tubing and filled with an internal solution containing (mM): 144 K-gluconate, 3 MgCl₂, 0.2 EGTA, 2 Na₂-ATP, 0.2 Na₂-GTP, 10 HEPES pH 7.2-7.4, 300 mOsm. Spontaneous activity of the neurons was recorded in current clamp mode (SEC 05L/H, NPI electronics, Tamm, Germany), in the presence of TTX (1 μM), D-AP5 (50 μM) and CNQX (20 μM, all from Tocris). Synaptic potentials recorded were amplified, low-pass filtered at 2 kHz, and digitized at 5 kHz using a CED 1401 interface and data acquisition programme, Signal 4.04 (Cambridge Electronic Design, Cambridge, UK), and analysed offline using Signal. Single sweep amplitudes were measured from the baseline to the peak of the IPSP, and selected for analysis if greater than 0.05 mV 19 (link). Statistical analysis was conducted using the Student’s t test and data plotted using OriginPro 9.1.