CHARACTERIZATION OF CA CURRENT UNDERLYING BURST FORMATION IN LOBSTER CARDIAC GANGLION MOTORNEURONS

The anterior motoneurons of the cardiac ganglion of Homarus americanus were ligated <300 μm from the soma. This removes impulse-generating membrane and sites of synaptic input while preserving the ability of the soma to generate the burst-forming potentials termed 'driver potentials' regenerative, slow (250-ms duration) depolarizations (to -20 mV) in response to brief, depolarizing stimuli. At stimulus intervals corresponding to rates of bursting observed in spontaneously active, intact ganglia (0.3-1.2/s), driver potential amplitude increases with increasing stimulus interval. A two-electrode voltage clamp was used to characterize inward current observable from the ligated neurons in tetrodotoxin (TTX)- tetraethylammonium (TEA)-containing salines. The amplitude of inward current shows a hyperbolic relation to [Ca](o) that is well fitted by a form of the Michealis-Menten equation. Inward current is maintained but not augmented when Ca2+ is replaced by Ba2+ or Sr2+. It is concluded that the inward current, to be referred to as I(Ca), is mediated by voltage-dependent Ca channels. Contamination of I(Ca) by early outward current (I(A)) was evaluated by addition of 4-aminopyridine (4-AP, 4 mM). In the presence of 4-AP, the net inward current is increased and the potential at which maximum I(Ca) occurs is shifted 10 mV more positive. Subtraction of outward currents recorded in Mn2+-containing saline from overall currents in the absence of Mn2+ provided another means to separate inward from outward current. I-V curves from such 'Mn-subtracted' records show I(Ca) approaches a saturating value for steps to -5 mV and more depolarized. The time to peak I(Ca) is voltage dependent. The largest inward currents (up to 240 nA) and minimal time to peak (4 ms) are observed for steps from holding potentials of -50 to -60 mV. Decline of I(Ca) during depolarized steps observed in Mn-subtracted records represents inactivation rather than development of competing outward current. Inactivation is slow and incomplete; the rate and fractional amount of inactivation are not directly voltage dependent. Nonsubtracted responses to 500-ms depolarizations to potentials evoking little outward current show that an initial rapid decline of I(Ca) (τ ~ 40 ms) is followed at ~80 ms by a slower phase of decline (τ ~ 180 ms). With repetitive clamps, the early phase proved labile. Observations of I(Ca) decline from tail currents at -80 mV (~E(k)) at various times up to 300 ms showed that when nonzero apparent asymptotes are subtracted, the rate of decline of current varies little for steps from -20 to 60 mV and was fitted by a decaying exponential (τ ~ 76 ms). Evidence for a Ca-mediated mechanism of inactivation is provided by two-clamp protocols that showed the extent of inactivation of the second response approximated the I(Ca) I-V curve. Also consistent is the markedly reduced inactivation observed in salines in which Ba2+ or Sr2+ replace Ca2+ and in neurons that have been injected with ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N',-tetraacetic acid (EGTA). Recovery of I(Ca) from inactivation showed an initial phase of recovery with τ ~ 700 ms, whereas full recovery required several seconds. This time course echoes that observed from eliciting pairs of driver potential responses in unclamped somata.