Abstract
Amphibian spinal neurons exhibit spontaneous elevations of intracellular calcium at early stages of development. The underlying calcium influx involves high-voltage-activated (HVA) currents. To begin to understand how they are triggered, we have studied the biophysical properties and developmental function of low-voltage-activated (LVA) T- type calcium current of neurons cultured from the embryonic neural plate. T current was recorded from young neurons (6–9 hr in vitro) and from mature neurons (18–48 hr in vitro) using whole-cell voltage clamp. For both young and mature neurons, T current has a low threshold and is activated at membrane potentials positive to -60 mV in 2 mM extracellular calcium. The current is maximal at -35 mV with a mean peak amplitude of approximately 50 pA. Nickel blocks both LVA and HVA currents, but the former are 20-fold more sensitive. Amiloride also blocks T current selectively. T current is recorded in 87% of young neurons. This percentage drops to 67% in mature neurons after 1 d in culture and to 35% in mature neurons after 2 d in culture. There are no significant developmental changes in T current threshold, peak density, time course of activation and inactivation, and pharmacological sensitivity to blockers from 6 to 48 hr in culture. Spontaneous transient calcium elevations in young neurons assayed by fluo-3 fluorescence are blocked by nickel or amiloride at concentrations that specifically block T current. T current has the lowest threshold among other inward currents in young neurons. Moreover, mathematical simulations show that T current lowers the threshold of the action potential by 15 mV. We conclude that T current can depolarize cells and trigger action potentials, and constitutes part of the cascade of events leading to spontaneous elevations of intracellular calcium in cultured neurons at early stages of differentiation.
