Different Extrinsic Trophic Factors Regulate Neurite Outgrowth and Synapse Formation between Identified Lymnaea Neurons David W. Munno, 1,2 Melanie A. Woodin, 1 Ken Lukowiak, 1 Naweed I. Syed, 1 Patsy S. Dickinson 2 1 Respiratory and Neuroscience Research Groups, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1 2 Department of Biology, Bowdoin College, Brunswick, Maine 04011 Received 30 November 1999; accepted 21 March 2000 ABSTRACT: The requirement for trophic factors in neurite outgrowth is well established, though their role in synapse formation is yet to be determined. More- over, the issue of whether the trophic factors mediating neurite outgrowth are also responsible for synapse spec- ification has not yet been resolved. To test whether trophic factors mediating neurite outgrowth and syn- apse formation between identified neurons are con- served in two molluscan species and whether these de- velopmental processes are differentially regulated by different trophic factors, we used soma–soma and neu- rite–neurite synapses between identified Lymnaea neu- rons. We demonstrate here that the trophic factors present in Aplysia hemolymph, although sufficient to induce neurite outgrowth from Lymnaea neurons, do not promote specific synapse formation between excitatory partners. Specifically, the identified presynaptic neuron visceral dorsal 4 (VD4) and postsynaptic neuron left pedal dorsal 1 (LPeD1) were either paired in a soma– soma configuration or plated individually to allow neu- ritic contacts. Cells were cultured in either Lymnaea brain-conditioned medium (CM) or on poly-L-lysine dishes that were pretreated with Aplysia hemolymph (ApHM), but contained only Lymnaea defined medium (DM; does not promote neurite outgrowth). In ApHM- coated dishes containing DM, Lymnaea neurons exhib- ited extensive neurite outgrowth, but appropriate exci- tatory synapses failed to develop between the cells. Instead, inappropriate reciprocal inhibitory synapses formed between VD4 and LPeD1. Similar inappropriate inhibitory synapses were observed in Aplysia hemo- lymph-pretreated dishes that contained dialyzed Aplysia hemolymph. These inhibitory synapses were novel and inappropriate, because they do not exist in vivo. A re- ceptor tyrosine kinase inhibitor (Lavendustin A) blocked neurite outgrowth induced by both Lymnaea CM and ApHM. However, it did not affect inappropri- ate inhibitory synapse formation between the neurons. These data demonstrate that neurite outgrowth but not inappropriate inhibitory synapse formation involves re- ceptor tyrosine kinases. Together, our data provide di- rect evidence that trophic factors required for neurite outgrowth are conserved among two different molluscan species, and that neurite extension and synapse specifi- cation between excitatory partners are likely mediated by different trophic factors. © 2000 John Wiley & Sons, Inc. J Neurobiol 44: 20 –30, 2000 Keywords: trophic factors; neurite outgrowth; synapse formation; in vitro; regeneration The role of individual trophic factors in neurite out- growth (Ridgeway et al., 1991; Thoenen, 1995; Schu- man, 1995; Syed et al., 1996; Ghirardi et al., 1996; White, 1998) and synapse formation (Syed et al., 1996; Ghirardi et al., 1996; Feng et al., 1997) has been studied extensively. It is, however, unclear whether the trophic factors involved in the above developmental programs have unique and dedicated function or if they can simul- taneously participate in various different cellular pro- cesses. For instance, whether trophic molecules mediat- ing neurite outgrowth from a select group of neurons are also responsible for synapse specification in the same neurons has not yet been determined. Correspondence to: D. Munno, (dwmunno@ucalgary.ca). Contract grant sponsor: Surdna Undergraduate Research Fel- lowship, Bowdoin College (DWM). Contract grant sponsor: Alberta Heritage Foundation for Med- ical Research, University of Calgary, (DWM). Contract grant sponsor: National Science Foundation (PSD). Contract grant sponsor: Whitehall Foundation (PSD). Contract grant sponsor: Medical Research Council, Canada (NIS, KL). © 2000 John Wiley & Sons, Inc. 20