Distribution of ionic currents in the soma and growing region of an identified peptidergic neuron in defined culture

Abstract

1. Somata and lamellipodia of a distinct type of crustacean peptidergic neuron were isolated by severing the connecting neurite. The whole-cell variation of the patch-clamp technique was then used to study the electrical activity and ionic currents of each part. Neurons were enzymatically isolated from the X-organ of Cardisoma carnifex and cultured in defined medium. The neurons studied were recognizable by their large ovoid somata (approximately 35 microns minor diam) and broad, flat lamellipodium regrown from the remaining neurite. These cells are immunopositive against crustacean hyperglycemic hormone (CHH) antisera. Recordings were made 18-30 h after plating. 2. In current-clamp recordings, 10 of 12 lamellipodia fired overshooting action potentials (mean half width = 8.2 +/- 2.9 ms, mean +/- SD), whereas only two of seven somata did so. Spontaneous activity in isolated somata and lamellipodia was rarely encountered. The action potential in isolated lamellipodia has both Na+ and Ca2+ components, whereas the regenerative activity recorded in isolated somata was predominantly Ca(2+)-based. 3. The inward currents examined under voltage clamp consisted of Na current (INa) and Ca current (ICa). Both currents could be resolved in isolated somata and lamellipodia. INa was completely blocked by tetrodotoxin [TTX (1 microM)]. The INa density in lamellipodia was approximately 4-19 times greater than that of the somata from which they had been separated. In contrast, ICa density in lamellipodia was two to five times smaller than that of somata. The properties of ICa were similar in both somata and lamellipodia, with the exception that ICa in lamellipodia did not recover after large depolarizing prepulses. 4. Two types of outward current were readily identified under voltage clamp. These were the transient 4-aminopyridine-sensitive current and the delayed outward current that was partially sensitive to tetraethylammonium ions. The peak potassium current-density ratio for lamellipodia-somata pairs ranged from 0.5 to 1.7 (mean = 1.2, SD +/- 0.6), suggesting that both channel types are distributed relatively evenly over the cell surface. 5. The differences in current density in the different regions did not correlate with the length of the neurite remaining after separation of the soma and lamellipodium. 6. It is concluded that CHH neurons in the early phase of regeneration in primary culture show a distinct pattern of ion channel distribution. These ion channels are present in new membrane < 18 h after formation.(ABSTRACT TRUNCATED AT 400 WORDS)