The formation of patterns of functionally appropriate chemical synapses is one of the key aspects of nervous system development. I have investigated the cellular interactions that culminate in the formation of an inhibitory synapse between the R and P neurons of the leech. These neurons may be isolated and maintained in culture, where they reform synaptic connections under easily manipulable conditions. An early event in the formation of this synapse is the loss from sites of contact in the postsynaptic P cell of an excitatory response to the transmitter serotonin. The loss of this response was triggered specifically by contact with the presynaptic R neuron, and not by contact with other leech neurons, including other serotonergic neurons. Furthermore, contact with the R neurons of the reproductive ganglia, which do not innervate P cells, was also ineffective in causing the loss of response. This highly specific cellular interaction was prevented by treatment of the R cell surface with the proteolytic enzyme trypsin or with the lectin wheat germ agglutinin (WGA), suggesting involvement of an R cell surface glycoprotein. WGA blocked not only the loss of the excitatory response, but also prevented the formation of the R-P synapse. An antibody library against the R cell generated using a novel phage-display system produced antibodies which bound to subsets of leech neurons, including the R neurons, but none of these was specific for the R cells. I conclude that an early event in the formation of the R-P synapse is the recognition by the P cell of its correct synaptic partner through an R cell-specific surface molecule.

I explored the relationship between neurite outgrowth and the onset of synaptic activity in the central neuropil of the leech embryo. In order to follow changes in early morphology and the onset of synaptic activity in the same identified neuron, whole cell patch clamp recordings and fluorescent dye fills were obtained from dorsal pressure-sensitive (P) cells, the first neurons that could be reliably identified in the early embryo. I followed the development of the P cell from the first extension of neurites to the elaboration of an adult-like arbour. Following the growth of primary neurites, we observed a profuse outgrowth of transient neurites within the neuropil that retracted to form spurs. Following a dormant period, stable secondary branches grew from the spurs and became tipped with terminals. At this time, neurites of the Retzius (R) cell, a known presynaptic partner in the adult, were observed to contact the terminals. Although voltage-dependent currents were seen in the P cell at the earliest stage, spontaneous synaptic activity was only observed when terminals had formed. Spontaneous release was observed before evoked release could be detected from the R cell. The results suggest that transient neurites are formed during an exploratory phase of development, whereas the more precisely timed outgrowth of stable neurites from the spurs signals functional differentiation during synaptogenesis. As spurs have also been observed in neurons of the mammalian brain, they may constitute a primordial synaptic organizer.

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Serotonergic Retzius (R) neurons of the leech Hirudo medicinalis in culture reform inhibitory synapses with pressure sensitive (P) neurons while selectively reducing an extrasynaptic, depolarizing response to serotonin (5-HT) in the P neuron. We have examined if the selection of 5-HT responses is restricted to sites of contact between processes and growth cones of these cells. As measured by intracellular recording at the soma, focal application of 5-HT depolarized uncontacted P cell bodies, neurites and growth cones but not processes contacted by R cells. In patch clamp recordings of the depolarizing channels, application of 5-HT modulated channel activity in uncontacted but not in contacted growth cones. The selection of transmitter responses during synaptogenesis is therefore localized to discrete sites of contact specifically between synaptic partners. / Prior experiments have shown that tyrosine kinases play a crucial part in the selection of responses to 5-HT that occurs in the P cell (Catarsi and Drapeau, 1993). To further examine the mechanism responsible for this change in transmitter responses, we have utilized a monoclonal antibody against phosphotyrosine to determine if tyrosine phosphorylation could be detected in P and R cell pairs placed in contact. Our results revealed bright, punctate cytoplasmic staining in P cells paired with R cells. / Embryonic leeches were used to examine how R to P synaptogenesis proceeds in vivo. By filling the R and P neurons with different fluorescent dyes (Lucifer Yellow and Rhodamine-Dextran), confocal microscopy established that putative contact between neuropilar processes were made as early as 11 days of development. Spontaneous, chloride-dependent synaptic potentials in embryonic P cells similar to those seen in adult P cells were observed as early as day 10 of development.

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