OUTGROWTH MORPHOLOGY AND INTRACELLULAR CALCIUM OF CRUSTACEAN NEURONS DISPLAYING DISTINCT MORPHOLOGIES IN PRIMARY CULTURE

Peptide-secreting neurons from crustacean X-organ regenerating in defi ned culture possess different ionic current profiles correlated with t wo distinct morphological types, veiling and branching; voltage-depend ent Ca2+ current is prominent in neurons consistently extending large veils, but is small in neurons that repetitively branch. Intracellular free calcium levels ([Ca2+](i)) have been implicated in the regulatio n of neurite outgrowth underlying the establishment of distinct morpho logies. Here, basal [Ca2+](i) was measured by fura-2 fluorescence rati o imaging from these morphologically distinct neurons and compared. Bo th morphological types can extend out processes over a [Ca2+](i) range (approximately 50 to 300 nM) that is much greater than that reported for neurons of other phyla. Application of high K+ saline led to incre ases in [Ca2+](i) in soma, neurite, and lamellipodium of veiling neuro ns. increases were greater for veiling than branching neurons. These o bservations were consistent with the previous voltage clamp data for c alcium currents. Media altered to perturb [Ca2+](i) were used to asses s the role of [Ca2+](i) in veiling or branching outgrowth programs. Ou tgrowth of veiling cells was arrested by addition of 100 mu M Cd2+, a calcium channel blocker. Outgrowth resumed following brief exposures t o Cd2+. Branching neurons were unaffected by Cd2+ Cd2+ at lower levels (10 mu M) had no effect on outgrowth of either neuronal type, whereas at higher levels (1 mM), outgrowth of both types was arrested. Reduct ion of extracellular sodium to 0.001 of normal concentration stopped v eiling outgrowth, but branching outgrowth continued, although it was l ess robust. Addition of tetrodo-toxin (1 mu M) did not alter outgrowth of either neuronal type relative to controls. Thus, peptidergic neuro ns of differing intrinsic morphologies maintain similar basal [Ca2+](i ) levels under identical culture conditions, yet show differing sensit ivities to manipulations influencing [Ca2+](i) with respect to regener ative outgrowth, but not its form. (C) 1994 John Wiley and Sons, Inc.