2.2 Electrophysiology
The composition of solutions (in mM) was: Intracellular solution: 50 CsCl, 10 NaCl, 60 CsF, 20 EGTA, 10 HEPES; pH 7.2 (adjusted with 1 M CsOH). Extracellular solution: 140 NaCl, 4 KCl, 1 MgCl2, 2 CaCl2, 5 D-Glucose and 10 HEPES; pH 7.4 (adjusted with 1 M NaOH). The osmolality of intra- and extracellular solutions was set to ~290 and ~300 mOsm, respectively. Data were sampled at 20 kHz, and filtered at 10 kHz. Experiments were carried out at room temperature.
In manual patch-clamp experiments, whole-cell currents were recorded using an Axopatch 200B amplifier and the pClamp software (Molecular Devices, San Jose, CA). Pipette resistance ranged between 1.5 and 4.0 MΩ. Solution exchange was performed by the ”liquid filament switch” method (Franke et al., 1987; Jonas, 1995), using a Burleigh LSS-3200 ultrafast solution switching system, as described in detail previously (Pesti et al., 2014). The flow rate was set using a DAD-12 solution exchange system (ALA Scientific Instruments Inc., Farmingdale, NY), to 0.2-0.3 ml/min, which corresponded to 5-20 cm/s velocity. Ten to 90% solution exchange rates were between 1 and 3 ms.
In experiments with the Port-a-Patch system (Nanion, Munich, Germany) currents were recorded using an EPC10 plus amplifier, and the PatchMaster software (HEKA Electronic, Lambrecht, Germany). During cell catching, sealing, and whole-cell formation, the PatchControl software (Nanion) commanded the amplifier and the pressure control unit. The resistance of borosilicate chips was 2.0–3.5 MΩ.