Pcie 6353 data acquisition board

Before imaging and electrophysiology, neurons on coverslips were placed in a glass-bottomed dish filled with customized high-glucose Tyrode’s solution containing (in mmol/L) 125 NaCl, 2.5 KCl, 3 CaCl₂, 1 MgCl₂, 10 HEPES, and 30 glucose (pH 7.3, adjusted to 305–310 mOsm/kg with sucrose). The synaptic blockers NBQX (10 μmol/L), AP-V (25 μmol/L), and gabazine (20 μmol/L, all from Sigma) were added to the buffer for measurements of single-cell electrophysiology. All experiments were performed at room temperature (~22 °C).
Borosilicate glass electrodes (WPI, FL, USA) were pulled to a tip resistance of 2.5–5 MΩ. The electrodes were filled with internal solution containing (in mmol/L) 125 potassium gluconate, 8 NaCl, 0.6 MgCl₂, 0.1 CaCl₂, 1 EGTA, 10 HEPES, 4 Mg-ATP, 0.4 Na₂-GTP (adjusted to pH 7.3 with KOH and 290–300 mOsm/kg with 1 mol/L sucrose). Neurons were clamped in the whole-cell current-clamp mode, and the membrane voltage signal recorded from the patch amplifier (Axopatch 200B, Molecular Devices, CA, USA) was filtered with an internal 5-kHz Bessel filter and digitized at 9681.48 Hz with a National Instruments (TX, USA) PCIe-6353 data acquisition board. During the current stimulation, simultaneous voltage images were acquired at a 20× down-sampling rate (484.07 Hz, 2.0658 ms/frame).