Development of serotonin-induced ion currents in identified embryonic Retzius cells from the medicinal leech (Hirudo medicinalis)

Abstract

Retzius cells and the cutaneous baroreceptive P-cells of adult medicinal leeches (Hirudo medicinalis) respond to 5-HT by Cl- as well as by monovalent cation conductances (Drapeau et al., 1989). However, chemical synaptic connections between Retzius cells (Liu and Nicholls, 1989) as well as Retzius cells and P-cells in culture (Drapeau and Sanchez-Armass, 1988) are mediated by postsynaptic Cl- currents in response to 5-HT release from the presynaptic cell. It was the aim of the present study to find out whether the response, which has been shown to be involved in synaptic transmission or whether the cation current, which has so far only been observed upon extrasynaptic 5-HT application, dominates during embryogenesis. Currents induced by transmitter application with a fast perfusion system were measured with a single-electrode voltage clamp and patch pipettes in whole-cell configuration. Control experiments, performed on Retzius cells from adult leeches after 1 d in culture, showed Cl- currents in response to 5-HT application in all cells investigated. Ninety percent of the cells tested showed additional 5-HT-elicited Na+, K+, or both currents. However, the conductance of cation currents amounted only to 10% of the Cl- currents. Embryonic Retzius cells were identified in dissociated cultures by their violet-blue fluorescence acquired by preincubation of intact ganglia or embryos in culture medium containing the autofluorescent 5-HT analog 5,7-dihydroxytryptamine. The first responses to 5-HT were measured at embryonic day 10 (E10), after voltage-activated Ca2+ and Na+ currents had already developed. 5-HT application induced exclusively Cl- currents until E16. This suggests that the receptor-channel complex, which also mediates synaptic responses, is expressed first in measurable quantities. Hence, there is currently no indication that extrasynaptic receptors dominate in immature cells in this preparation.