2.3. Electrophysiology recording
For an electrophysiological experiment, cultured neurons were put in the upright microscope perfusion tank with extracellular bath solution (in mM): 125 NaCl, 2 KCl, 3 CaCl2, 1 MgCl2, 10 HEPES, 30 Glucose, pH=7.3, 300 mOsm. Pipettes had a resistance of 2-7 MΩ when filled with internal solution (in mM): 135 potassium gluconate, 8 NaCl, 10 HEPES, 4 Mg-ATP, 0.4 Na-GTP, 0.6 MgCl2, 0.1 CaCl2, pH=7.25, 295 mOsm. After establishing whole-cell configuration, cell capacitance and access resistance were routinely compensated. All experiments were conducted in a whole-cell patch-clamp configuration. For the recordings of action potentials, a switch to the current clamp was performed. A current was injected to have membrane potentials around -60 mV, then step currents from -40 pA to 50 pA were injected to elicit action potentials by 10 sweeps. Pipettes were prepared by a P-1000 micropipette puller (Sutter Instruments, Hofheim, Germany). Recordings were performed using an EPC10 USB patch-clamp amplifier (HEKA Elektronik, Lambrecht, Germany), data were digitized at 10 kHz.