IN-VITRO SYNAPTOGENESIS BETWEEN THE SOMATA OF IDENTIFIED LYMNAEA NEURONS REQUIRES PROTEIN-SYNTHESIS BUT NOT EXTRINSIC GROWTH-FACTORS OR SUBSTRATE ADHESION MOLECULES

Nerve growth factors, substrate and cell adhesion molecules, and prote in synthesis are considered necessary for most developmental programs, including cell proliferation, migration, differentiation, axogenesis, pathfinding, and synaptic plasticity. Their direct involvement in syn apse formation, however, has not yet been fully determined. The neurit e outgrowth that precedes synaptogenesis is contingent on protein synt hesis, the availability of externally supplied growth factors, and sub strate adhesion molecules. It is therefore difficult to ascertain whet her these factors are also needed for synapse formation. To examine th is issue directly we reconstructed synapses between the cell somata of identified Lymnaea neurons. We show that when paired in the presence of brain conditioned medium (CM), mutual inhibitory chemical synapses between neurons right pedal dorsal 1 (RPeD1) and visceral dorsal 4 (VD 4) formed in a soma-soma configuration (86%; n = 50). These synapses w ere reliable and target cell specific and were similar to those seen i n the intact brain. To test whether synapse formation between RPeD1 an d VD4 required de novo protein synthesis, the cells were paired in the presence of anisomycin (a nonspecific protein synthesis blocker). Chr onic anisomycin treatment (18 hr) after cell pairing completely blocke d synaptogenesis between RPeD1 and VD4 (n = 24); however, it did not a ffect neuronal excitability or responsiveness to exogenously applied t ransmitters (n = 7), nor did chronic anisomycin treatment affect synap tic transmission between pairs of cells that had formed synapses (n = 5). To test the growth and substrate dependence of synapse formation, RPeD1 and VD4 were paired in the absence of CM [defined medium; (n = 2 2)] on either plain plastic culture dishes (n = 10) or glass coverslip s (n = 10). Neither CM nor any exogenous substrate was required for sy napse formation. In summary, our data provide direct evidence that syn aptogenesis in this system requires specific, cell contact-induced, de novo protein synthesis but does not depend on extrinsic growth factor s or substrate adhesion molecules.