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

Peptide-secreting 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 levels ([Ca2+]i) have been implicated in the 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 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 increases in [Ca2+]i in soma, neurite, and lamellipodium of veiling neurons. Increases were greater 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 by addition of 100 microM Cd2+, a calcium channel blocker. Outgrowth resumed following brief exposures to Cd2+. Branching neurons were unaffected by Cd2+. Cd2+ at lower levels (10 microM) 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 microM) 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.