Fig. 4.
Calcium currents (ICa). a. Typical electrophysiological traces of inward calcium currents recorded using a series of depolarizing test steps (250 ms duration, holding potential −67 mV, pulses from −67 to +3 mV, 10 mV increments) in the presence of TTX, 4-AP and TEA (see methods). Note that the calcium currents inactivate during the long test step (scale bars, 50 pA and 50 ms). b. Voltage-dependence of activation and inactivation time constants (τact and τina) of recorded calcium currents (as shown in A, n = 4). To measure τact and τina time constants calcium currents were fitted a single exponential function (start to peak and peak to end respectively) at each holding potential. Both τact and τina were voltage-dependent, becoming faster at more positive potentials (mean τact and τina at −47 mV, 1.27 ms and 114 ms; at −17 mV, 0.62 ms and 16.7 ms respectively). Plotted data were then fitted with a single Boltzmann function to calculate mean V50 (activation, −23.8 mV; inactivation, −39.0 mV) and slope (LVA, −6.9; HVA, −2.6) values (n = 4). c. Average steady-state activation curve (G/Gmax) for calcium currents recorded using a series of depolarising pulses (as shown in A). Normalised conductance plot was fitted with a single Boltzmann function to calculate mean V50 (−26.1 mV) and slope (5.0) values for steady state activation (n = 4). d. Fast voltage-ramp (500 mV/s, −107 to +13 mV) depicting the activation of a calcium currents. Two distinct peaks were identified (both sensitive to cadmium, not shown) that indicate that LVA and HVA calcium currents are expressed on this vlPAG/DRN neuron. Leak subtracted current (dotted line) revealed that LVA calcium currents peaked at around −60 mV and HVA calcium currents peaked at around −15 mV. Leak current reversal under these conditions was −55 mV. e. Steady-state activation curves (G/Gmax) for LVA and HVA calcium currents for the neuron shown in D. Normalised conductance plots were fitted with a single Boltzmann function to calculate V50 (LVA, −63.2 mV; HVA, −22.1 mV) and slope (LVA, 2.6; HVA, 6.7) values for steady-state activation. f. Representative electrophysiological traces recorded in current-clamp mode in normal (2 mM) or zero calcium (see methods) aCSF. Substitution of magnesium with calcium increased spontaneous firing of vlPAG/DRN DA neurons without affecting the firing rate regularity (scale bars, 10 mV and 2 s). g. Bar chart comparison of mean firing frequency before and after replacement of 2 mM calcium for magnesium as shown in F for seven vlPAG/DRN DA neurons. This replacement caused a statistically significant change in firing rate (mean firing frequency in 2 mM calcium, 3.3 Hz; in zero mM calcium, 4.9 Hz, n = 6, *P < 0.05, paired t-test). h. Bar chart comparison of CV-ISI before and after replacement of 2 mM calcium for magnesium as shown in F for seven vlPAG/DRN DA neurons. This replacement did not cause a statistically significant change in CV-ISI (mean CV-ISI in 2 mM calcium, 0.66; in zero mM calcium, 0.40, n = 6, P > 0.05, paired t-test, NS, not significant)