Outgrowth morphology and intracellular calcium of crustacean neurons displaying distinct morphologies in primary culture

Peptidesecreting neurons from crustacean X-organ regenerating in defined culture possess different ionic current profiles correlated with two distinct morphological types, veiling and branching; voltage-dependent Ca²⁺ current is prominent in neurons consistently extending large veils, but is small in neurons that repetitively branch. Intracellular free calcium (Ca²⁺]_i) have been implicated in regulation of neurite outgrowth underlying the establishment of distinct morphologies. Here, basal Ca²⁺]_i was measured by fura-2 fluorescence ratio imaging from these morphologically distinct neurons and compared. Both morphological tapes 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 increases in Ca²⁺]_i in soma, neurite, and lamellipodium of veiling neurons. Increase were great for veiling than branching neurons. These observations were consistent with the previous voltage clamp data for calcium currents. Media altered to perturb Ca²⁺]_i were used to assess the role of Ca²⁺]_i in veiling or branching outgrowth programs. Outgrowth of veiling cells was arrested addition of 100 μMCD²⁺, a calcium channel blocker. Outgrowth resumed following brief exposures to Cd²⁺. Branching neurons were unaffected by Cd²⁺. Cd²⁺ at lower levels (10 μM) had no effect on outgrowth of either neuronal type, whereas at higher levels (1 mM), outgrowth of both types was arrested. Reduction of extracellular sodium to 0.001 of normal concentration stopped veiling outgrowth, but branching outgrowth continued, although it was less robust. Addition of tetrodotoxin (1 μM) did not alter outgrowth of either neuronal type relative to controls. Thus, peptidergic neurons of differing intrinsic morphologies maintain similar basal Ca²⁺]_i levels under identical culture conditions, yet show differing sensitivities to manipulations influencing Ca²⁺]_i with respect to regenerative outgrowth, but not its form. 1994 John Wiley & Sons, Inc.