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 (I-Ca) in mature neurons cultured in defined medium and derived from the principal neurosecretory system of decapod crustaceans, the X-orga n-sinus gland, After 1 day in culture, X-organ neurons of the crab Car disoma carnifex showed vigorous outgrowth characterized either by the production of broad lamellipodia (veils) or, from smaller somata, a br anching morphology, The neurons developing veils (veilers) had a large I-Ca (approximately 650 pA) and I-Ca current density (approximately 5 mu A cm(-2)),while other types of neuron had little or no I-Ca. This distinction between the two types was still present after 5-6 days in culture, However, morphologies observed after additional outgrowth, wh en correlated with the I-Ca responses, allowed four groups to be disti nguished: (1) veilers and (2) branching veilers, which developed from veilers and had a similar I-Ca density (approximately 3 mu A cm(-2)); and, developing from the 1 day branchers, (3) spiny branchers or (4) s mall cells (I-Ca density approximately 0.8 mu A cm(-2)). Immunoreactiv ity indicative of the presence of crustacean hyperglycemic hormone was found in all veilers and branching veilers tested, while molt-inhibit ing hormone reactivity, when observed, was seen in cells having a robu st I-Ca density (greater than or equal to 1.2 mu A cm(-2)). Normalized average current-voltage curves for each morphological group were exam ined for changes with increasing time in culture, The curves were cons istent with the I-Ca being produced by a population of high-voltage-ac tivated Ca2+ channels whose properties are biophysically indistinguish able 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 I-Ca density, This indi cated that net addition of Ca2+ channels did not match the addition of new membrane under our culturing conditions.