Whole Cell Recording
Cells were plated at 105 per 35 mm culture dish. Attached cells were washed three times with bath solution at room temperature. The bath solution contained 130 mM NaCl, 10 mM HEPES, 34 mM dextrose, 1.5 mM CaCl2, 0.5 mM MgSO4, and 1.3 mM KH2PO4, adjusted to pH 7.35. The pipette solution for the whole-cell recordings contained 30 mM KCl, 90 mM NaCl, 2 mM MgCl2, 0.1 mM CaCl2, 1.1 mM EGTA, 10 mM HEPES, and 30 mM dextrose plus 2 mM DA, adjusted to pH 7.35. Patch electrodes were pulled from quartz pipettes on a P-2000 puller (Sutter Instruments, Novato, CA) and filled with the pipette solution. Whole-cell currents were recorded using an Axopatch 200B with a low-pass Bessel filter set at 1000 Hz. After establishing the whole-cell configuration, we waited 10 min to allow perfusion of the intracellular milieu with the pipette solution. Current-voltage relationships were generated using a voltage step (1 s) protocol ranging from -120 to +100 mV separated by 20 mV from a holding potential of -20 mV. Current and oxidative (amperometric) signals were measured simultaneously. Data were recorded and analyzed off-line using the software pCLAMP 8 from Axon Instruments (Union City, CA).
A carbon fiber electrode connected to a second amplifier (Axopatch 200B) was attached to the plasma membrane of the cell held at +700 mV for all experiments unless noted otherwise. The carbon fiber electrodes (ProCFE; fiber diameter is 5 μm) were obtained from Axon Instruments. Oxidative (amperometric) current-voltage relationship was generated as above. As described previously (Khoshbouei et al., 2003), unlike the usual amperometric calibration, which requires conversion to concentration, we report the current directly without considering the effective volume. Thus, our requirements are a defined baseline and our data represent a lower limit to the DA efflux because some transmitter is lost to the bulk solution. The amperometric currents were low-pass filtered at 100 Hz. Data were recorded and analyzed off-line using the software pCLAMP 8 from Axon Instruments. Current-voltage relations were generated by plotting against the test voltage the values of the amperometric currents between 800 and 1000 ms after the step.