Comparison of Ca2+ currents of peptidergic neurons developing differing morphology with time in culture

The whole-cell patch-clamp technique was used to examine Ca2+ currents (ICa) in mature neurons cultured in defined medium and derived from the principal neurosecretory system of decapod crustaceans, the X-organ-sinus gland. After 1 day in culture, X-organ neurons of the crab Cardisoma carnifex showed vigorous outgrowth characterized either by the production of broad lamellipodia (veils) or, from smaller somata, a branching morphology. The neurons developing veils (veilers) had a large ICa (approximately 650 pA) and ICa current density (approximately 5 microA cm-2) while other types of neuron had little or no ICa. This distinction between the two types was still present after 5-6 days in culture. However, morphologies observed after additional outgrowth, when correlated with the ICa responses, allowed four groups to be distinguished: (1) veilers and (2) branching veilers, which developed from veilers and had a similar ICa density (approximately 3 microA cm-2); and, developing from the 1 day branchers, (3) spiny branchers or (4) small cells (ICa density approximately 0.8 microA cm-2). Immunoreactivity indicative of the presence of crustacean hyperglycemic hormone was found in all veilers and branching veilers tested, while moltinhibiting hormone reactivity, when observed, was seen in cells having a robust ICa density (> or = 1.2 microA cm-2). Normalized average current-voltage curves for each morphological group were examined for changes with increasing time in culture. The curves were consistent with the ICa being produced by a population of high-voltage-activated Ca2+ channels whose properties are biophysically indistinguishable and unaffected by time in culture. The averaged peak current did not change, despite an increase in neuronal surface area as outgrowth proceeded, and this resulted in a reduction of ICa density. This indicated that net addition of Ca2+ channels did not match the addition of new membrane under our culturing conditions.