Transmitter localization and vesicle turnover at a serotoninergic synapse between identified leech neurons in culture

Abstract

An electron microscopic study has been made of chemical synapses that develop between identified nerve cells isolated from the CNS of the leech and maintained in culture. Structures resembling synapses were observed in pairs of Retzius cells and P sensory cells at which chemical transmission had been demonstrated by recording with microelectrodes. Vesicle recycling was shown by following the uptake of extracellular markers after stimulation. The membrane separation between the presynaptic Retzius cell (which is known to liberate serotonin) and the postsynaptic P cell was wider in synaptic than in extrasynaptic regions. The Retzius cell contained clusters of clear vesicles apposed to thickenings of the presynaptic membrane. These clear vesicle clusters were capped by a layer of dense core vesicles that did not contact the presynaptic membrane thickenings. Subsynaptic cisternae were found in the postsynaptic cell opposite the presynaptic membrane thickenings. Occasional slight postsynaptic membrane thickenings were seen. Extracellular material was observed within the synaptic cleft. Similar synaptic structures developed between pairs of Retzius cells in culture; even a single Retzius cell was able to form autapses upon itself. Structures resembling transmitter release sites were found in Retzius cells at a distance from any postsynaptic membranes. These are presumed to be locations for the diffuse release of transmitter. Presynaptic structures resembling release sites were never observed in P cells apposed to Retzius cells. Antibody to serotonin (5-HT) labelled with colloidal gold showed serotonin to be localized in the dense core vesicles in Retzius cells. Stimulation of pairs of Retzius and P cells by raised concentrations of K+ resulted in uptake of extracellular markers. Only Retzius cells became labelled. Ferritin was found in cisternae, in dense core vesicles, and in clear vesicles. HRP was found in cisternae and in clear vesicles. Colloidal gold was taken up by coated vesicles and was occasionally found in both clear and dense core vesicles. The uptake of extracellular markers following stimulation was blocked by high Mg++. These results show that structures develop between pairs of cells at which chemical transmission develops and that transmitter release leads to turnover of dense core and clear vesicles.