Sodium- and calcium-dependent excitability of embryonic leech ganglion cells in culture

Voltage-dependent Na+ and Ca2+ 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 7-to 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 Ca2+-dependent inward current. Both types of Na+ currents were resistant to tetrodotoxin (TTX, 0.2–5 μmoll-1). The Ca2+ current was also carried by Ba2+, 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 Ca2+ current. Between days 8 and 14 the density of the fast Na+ current increased from 22±3 to 51±6μAcm-2 (±S.D., N= 11), while the Ca2+ current grew from 10μAcm-2 (N=2) to 15±4μAcm-2 (N =10) during this time.