Excitatory synaptogenesis between identified Lymnaea neurons requires extrinsic trophic factors and is mediated by receptor tyrosine kinases

Neurotrophic factors have well established roles in neuronal development an d adult synaptic plasticity, but their precise role in synapse formation ha s yet to be determined. This paper provides the first direct evidence that neurotrophic factors in brain conditioned medium (CM) differentially regula te excitatory and inhibitory synapse formation. Somata of identified presyn aptic and postsynaptic neurons were isolated from the CNS of Lymnaea and we re cultured in a soma-soma configuration in the presence (CM) or absence [d efined medium (DM)] of trophic factors. In DM, excitatory synapses did not form. When they were paired in CM or in DM containing Lymnaea epidermal gro wth factor (EGF); however, all presynaptic neurons reestablished their spec ific excitatory synapses, which had electrical properties similar to those seen in vivo. CM-induced formation of excitatory synapses required transcri ption and de novo protein synthesis, as indicated by the observations that synapse formation was blocked by the protein synthesis inhibitor anisomycin and the protein transcription blocker actinomycin D; the CM factor was ina ctivated by boiling. They were also blocked by receptor tyrosine kinase inh ibitors (lavendustin A, genistein, K252a, and KT5926) but not by inactive a nalogs (genistin and lavendustin B), suggesting that the effect was mediate d by receptor tyrosine kinases. These results, together with our previously published data, demonstrate that trophic factors are required for excitato ry, but not inhibitory, synapse formation and extends the role of EGF from cell proliferation, neurite outgrowth, and survival to excitatory synapse f ormation.