Sodium- and Calcium-dependent Excitability of Embryonic Leech Ganglion Cells in Culture

Voltage-dependent Na and Ca inward currents underlying the action potential in cultured embryonic ganglion cells of the leech Hirudo medicinalis have been investigated using the gigaseal whole-cell current or voltage-clamp technique. Dissociated ganglion cells were isolated from 7to 14-day-old embryos, and maintained in primary culture for up to 5 days. More than 95 % of the cultured cells had voltage-dependent K currents and about 75 % of the cells had voltagedependent inward currents. Action potentials of 60mV amplitude and 4 ms duration, similar to those in embryonic nerve cells in vivo, could be recorded. Three types of inward currents occurred in these cells: (1) an initial Na current, which activated and inactivated rapidly; (2) a second Na current, which activated slowly and persisted during membrane depolarization, showing very little inactivation, and (3) a Ca-dependent inward current. Both types of Na currents were resistant to tetrodotoxin (TTX, 0.2-5 ^mol I"). The Ca current was also carried by Ba, and was blocked by cobalt and cadmium. The fast Na current was first expressed in cells from 8-day-old embryos, 1 day earlier than the Ca current. Between days 8 and 14 the density of the fast Na current increased from 22±3 to 51±6juAcm" ( ± S . D . , N=ll), while the Ca current grew from m" (N=2) to 15±4/MAcnT 2 (W=10) during this time.