ACTIVITY-DEPENDENT INDUCTION OF FUNCTIONAL SECRETORY PROPERTIES AT CULTURED NEUROMUSCULAR SYNAPSES OF HELISOMA

1. The role of activity-dependent mechanisms in target-mediated induct ion of secretory properties was investigated at regenerating neuromusc ular synapses of the American pond snail, Helisoma trivolvis, in cell culture. 2. Identified motoneurons were isolated into cell culture con ditions that promoted neurite outgrowth. Buccal neurons 19 (B19) were cultured alone for 2 days, at which time dissociated muscle fibers wer e manipulated into contact with newly formed neurites. 3. Immediately before the plating of muscle fibers, the sodium channel blocker, tetro dotoxin (TTX), or the acetylcholine receptor antagonist, d-tubocurarin e chloride (curare), was added to the culture dish. After 48 h of expo sure, the inhibitors were removed by repeated dilution of the culture medium and electrophysiological analyses were performed. 4. Cholinocep tive assay cells were manipulated into contact with the presynaptic ne urons to assess secretory properties along neuronal processes. Assay c ells were used to control for variations in postsynaptic sensitivity t hat could result from long-term exposure to activity inhibitors. 5. Th ese analyses demonstrated that inhibition of TTX-sensitive presynaptic activity and inhibition of curare-sensitive postsynaptic activation b oth blocked the induction of excitation-secretion coupling typically i nduced in these motoneurons by appropriate target contact. Neuron B5, which rapidly acquires functional synaptic properties in vitro, was un affected in its secretory function by 48 h of activity inhibition. 6. Acquisition of secretory competence was not suppressed due to a reduct ion in the viability or long-term changes in excitability of the activ ity-inhibited neurons, as indicated by analyses of electrophysiologica l properties. 7. Although target-contact and activity both participate d in the induction of secretory modifications in neuron B19, target-me diated changes did not involve retrograde effects on presynaptic neuro nal excitability. 8. We hypothesize that contact-mediated mechanisms g overn the initiation of presynaptic modifications in B19, however, our data indicate that the acquisition of functional excitation-secretion coupling also involves activity-dependent mechanisms. Although the me chanistic role of activity remains undefined, our results suggest that the activation of the target muscle plays a critical role in a retrog rade signaling pathway underlying maturation of a functional secretory apparatus in target-contacted neuronal processes.