CA2-CALIFORNICA( INFLUX AND ACTIVATION OF A CATION CURRENT ARE COUPLED TO INTRACELLULAR CA2+ RELEASE IN PEPTIDERGIC NEURONS OF APLYSIA)

1. Stimulation of inputs to bag cell neurons in the abdominal ganglion of Aplysia californica causes an increase in their intracellular Ca2 concentration ([Ca2+](i)). We have used thapsigargin, a specific inhi bitor of the endoplasmic reticulum Ca2+ pump, to analyse the effects o f Ca2+ released from intracellular stores on the electrophysiological responses of bag cell neurons. 2. Using digital imaging of fura-2-load ed isolated bag cell neurons we found that thapsigargin rapidly evoked an increase in [Ca2+](i) in somata, with smaller increases in neurite s. Thapsigargin-induced elevation of [Ca2+](i) peaked at about 1 mu M within 5-10 min and then decayed to basal levels by 30 min. 3. Placeme nt of an extracellular vibrating Ca2+-selective microelectrode to with in 1 mu m of somata revealed a relatively large steady-state Ca2+ effl ux. Thapsigargin produced a rapid increase in Ca2+ influx. Changes in Ca2+ flux were not detected at neurites. 4. Thapsigargin produced a sm all depolarization in isolated bag cell neurons in artificial sea wate r (ASW). Sometimes enhanced depolarizations were observed when extrace llular Naf was replaced by TEA or Tris, but not N-methyl-D-glucamine ( NMDG). The depolarization was not blocked by 100 mu M tetrodotoxin (TT X), removal of extracellular Ca2+ (0.5 mM EGTA) or addition of 10 mM C o2+ to the bath solution. 5. In voltage-clamp experiments, thapsigargi n induced an inward current (I-Tg) that was recorded in Ca2+-free medi a containing TEE or Tris substituted for Na+. The apparent reversal po tential of I-Tg was -16.8 +/- 1.2 mV in TEA-ASW. Induction of I-Tg was inhibited in neurons that were microinjected with the Ca2+ chelator B APTA-Dextran(70) or treated with the membrane-permeant analogue BAPTA AM. Activation of I-TG was not observed when Na+ was replaced with NMD G. Manipulation of [Na+](0) and [K+](0) produced shifts in the reversa l potential of I-Tg consistent with the underlying channels being perm eable to both Na+ and K+. 6. Thapsigargin did not alter the amplitude or kinetics of voltage-activated Ba2+ currents, but in some experiment s it did increase the amplitude of a component of out-ward K+ current. 7. Thapsigargin neither induced bag cell neurons within the intact ga nglion to depolarize and fire spontaneously, nor did it alter the freq uency or duration of firing of an electrically stimulated bag cell aft er-discharge. 8. We conclude that thapsigargin-sensitive Ca2+ pools ar e present predominantly in the somata of bag cell neurons. Ca2+ that i s released from thapsigargin-sensitive Ca2+ stores activates a non-sel ective cation current that may help sustain depolarization of the soma ta, but does not by itself trigger an after-discharge.