Electrophysiology. Charge Nurse 1 without assignment during hours of operation Registered Nurse 2:1 Radiological Technologist 1:1
Electrophysiology. Electrophysiological recordings were carried out using the patch clamp technique in the whole cell configuration. Recordings were made in the voltage clamp mode with the voltage of the cell controlled and whole cell currents recorded. Whole cell recording was carried out by forming a gigaseal between the recording electrode and the cell membrane. Data were acquired at 10 kHz with an amplifier (Axopatch 200B with pClamp 8; Axon Instruments, Foster City, CA). Voltage-clamp data were stored on the computer hard disc and analysed off-line (Clampfit 8; Axon Instruments, and Origin 6.1; OriginLab Co., Northampton, MA). Patch pipettes were pulled from borosilicate glass and when filled with pipette solution, had a resistance of between 1.2 and 2.0 MΩ. Typical cell capacitances were between 7 and 25 pF for both STC-1 cells and KRJ-I cells. Access resistances were between 3.0 and 4.0 MΩ and were compensated up to 70%. The holding potential was −80 mV, unless stated otherwise. The I–V relationship was measured by applying voltage pulses to potentials between −100 and +120 mV in 10-mV increments from a holding potential of −80 mV at 0.2 Hz, For calcium currents voltage pulses between −80 and +80mV in 10-mV or 5mV increments were used. To obtain current densities, the current amplitude was normalized to cell membrane capacitance (Cm). From I– V curves the activation threshold was determined as test potential at which the current was 5% of the maximum current. For KRJ-I cells, a voltage ramp between -100mV and +120mV over one second was used in place of the voltage step protocol. For determination of half-maximum activation voltage (V0.5,act) the chord conductance (G) was calculated from the current-voltage curves by dividing the peak current amplitude by its driving force at that respective potential G = I/(V −Vrev), where Vrev is the extrapolated reversal potential, V is the membrane potential, and I is the peak current. The chord conductance was then fitted with a Boltzmann equation: G = Gmax/(1 + e(V0.5,act−Vm)/kact) Where Gmax is the maximum conductance, V0.5,act is the half-maximum activation voltage, Vm is the test potential, and kact is the slope factor of the activation curve. Membrane potential was measured in current clamp mode. Drugs were applied by close local perfusion system that enabled the solution bathing the cell to be changed in less than 1 second.
Electrophysiology. For voltage clamp experiments a cover slip with DRG cell culture was mounted in a chamber on the stage of an inverted microscope. Patch pipettes were pulled from borosilicate glass (Xxxxx XX−150 TF−15) and had resistances of 2.0 to 2.5 M▲ measured in the standard bath solution. Sintered Ag/AgCl electrodes coupled the amplifier input leads to the solutions. To minimize offset caused by low Cl− pipette solutions, the pipette holder (Xxxxxxx et al., 1990) contained a Cl− rich solution at the Ag/AgCl electrode. Giga−seals were made in a microbath of ~75 µl, continuously perfused (~300 µl.min−1) with the standard bath solution (in mM): NaCl 145, KCl 5, CaCl2 2, MgCl2 1, HEPES 10, pH 7.4 (NaOH). The pipette solution contained (mM): Cs− methanesulfonate 103, MgCl2 4, HEPES 9, EGTA 9, (Mg)ATP 4, (tris)GTP 1, (tris)phosphocreatine 14, pH 7.4 (CsOH). After establishment of the whole−cell configuration the barium current was recorded with voltage−step protocols during extracellular perfusion with (mM): TEA−Cl 160, HEPES 10, EGTA 0.1, BaCl2 5, pH 7.4 (TEA−OH). A PC running Clampex 7 (Axon Instruments, Foster City, CA) and a List EPC 7 amplifier provided voltage protocols. Up to 80−90% of the series resistance was compensated. The membrane currents were filtered at 3 kHz in general and at 10 kHz for tail current measurements. Control experiments with an equivalent RC−circuit of the whole−cell showed that current transients with time constants of >100 µs can be reliably measured at 10 kHz filter setting (3−pole Bessel) under our conditions. All currents were leak subtracted using the P/4 method. Membrane capacitance of the selected DRG neurons was 14 ± 3 pF (n = 55). Calcium currents during action− potential clamp were measured under constant perfusion with (mM): TEA−Cl 160, HEPES 10, CaCl2 2, pH 7.4 (TEA−OH). The pipette solution was the same as above in the step voltage−clamp conditions.
