Anterograde and retrograde effects of synapse formation on calcium currents and neurite outgrowth in cultured leech neurons

Abstract

The aim of our experiments has been to analyse how formation of chemical synapses affects the distribution of calcium (Ca$^{2+}$) currents and neurite outgrowth of leech Retzius cells. Previous results showed that Ca$^{2+}$ currents measured in the initial process or `stump' of postsynaptic cells were significantly smaller than those in corresponding sites on presynaptic neurons. In the present experiments, neurons were plated together in close apposition as pairs or as triads, with the tip of one Retzius cell touching the soma of another. Ca$^{2+}$ currents from selected areas of the neuronal surfaces were measured by loose-patch recording before and after the formation of chemically mediated synaptic connections, which developed in about 8 h. With three cells arranged in a row, the last of the series, which was purely postsynaptic (i.e. with no target), also showed a dramatic reduction in Ca$^{2+}$ currents in its initial segment, compared with the currents seen in either the first cell (purely presynaptic) or the second cell of the chain (which was both postsynaptic to the first cell and presynaptic to the third). This suggests that retrograde as well as anterograde effects on Ca$^{2+}$ currents occurred as a result of synapse formation: the Ca$^{2+}$ currents in the middle cell did not decrease although a synapse had been formed on it. To test for additional consequences of synapse formation, neurite outgrowth was measured in postsynaptic cells and in single cells plated on an extract of extracellular matrix containing laminin (ECM-laminin). After 48 h, the total length of neurite outgrowth in postsynaptic cells was only about one third of that in single cells. These results show that the establishment of a chemical synapse between two cells produces characteristic changes in membrane properties and sprouting; the nature of these changes depends on whether the neuron itself has formed synapses on another cell and whether or not synapses have formed upon it.