A WHOLE-CELL PATCH-CLAMP STUDY ON THE PACEMAKER POTENTIAL IN DOPAMINERGIC-NEURONS OF RAT SUBSTANTIA-NIGRA COMPACTA

A whole-cell patch-clamp recording was obtained from dopamine (DA) neurons (n = 68) in the substantia nigra compacta (SNc) in in vitro slice preparations in order to study the underlying current for pacemaker-like slow depolarization (PLSD) which was considered as a basis for rhythmic firing of DA neurons. SNc DA neurons were identified immunohistochemically after recording. Results demonstrated that: (1) Under current clamped condition in the presence of TTX, DA neurons (n = 5) displayed the oscillation of membrane potential with high threshold spikes. An application of a hyperpolarizing and depolarizing current pulse (at the membrane potential where oscillation was no longer seen) induced a prominent anomalous rectification and pacemaker-like slow depolarization (PLSD), respectively. (2) Under voltage-clamped conditions in the presence of TTX, a command pulse positive to -50 mV from a holding potential of -80 mV induced a persistent Ca2+ current which was usually preceded by either a transient K+ (n = 7) or a transient Ca2+ (n = 4) current recorded with a patch pipette containing potassium gluconate (145 mM). (3) When outward currents were suppressed by 140 mM CsCl and 10 mM EGTA intercellularly applied through the patch pipette, a command pulse positive to -50 to -40 mV induced either a persistent Ca2+ current alone (n = 4) or a persistent Ca2+ current preceded by a transient Ca2+ current (n = 11). (4) The threshold for activation of the persistent Ca2+ current (I-p) was around -60 to -55 mV. The amplitude of I-p produced by a command pulse stepped to -50 mV from a holding potential of -80 mV was -78 +/- 42 pA (n = 23). (5) The threshold for activation of transient Ca2+ current (I-T) was around -70 to -65 mV and inactivated completely at -70 to -65 mV (n = 11). The peak amplitude of I-T evoked at -60 to -55 mV from a holding potential of more negative than - 80 mV was 489 +/- 170 pA (n = 11). (6) The decay time constant of I-T was 28 +/- 12 ms at -60 mV (n = 8) and that of I-p was 2.35 +/- 1.37 s at -50 mV (n = 11) when recorded with a pipette containing 10 mM EGTA and 140 mM CsCl. (7) The decay of I-p was apparently accelerated by decreasing the concentration of EGTA in the pipette solution from 10 to 1 mM. The decay time constant of I-p at -50 mV was 497 +/- 233 ms (n = 7) when recorded with a patch pipette containing 1 mM EGTA and 140 mM CsCl. This was significantly smaller than that recorded with a pipette containing 10 mM EGTA and 140 mM CsCl (P < 0.005, two-tailed t-test). (8) I-V relationship of Ca2+ currents (n = 7) obtained in the extracellular media in which outward currents were suppressed by TEA-Cl (145 mM), showed an inflection of -40 to -30 mV in the I-V curve. The HVA current appeared to be activated from -40 mV and had a component which displayed a prominent voltage-dependent inactivation. (9) Ni2+ (100 mu M, n = 5) and Cd2+ (100 mu M, n = 6) selectively blocked I-T and I-p, respectively. (10) omega-Conotoxin (1 mu M, n = 5) almost completely blocked I-p, but nifedipine (10 mu M, n = 7) could not markedly block I-p. These data indicate that SNc DA neurons display at least three types of calcium currents and that I-p may play a key role in generation of PLSD.