Prolonged cytosolic calcium elevations in growth cones contacting muscle

Encounters by growth cones or neurites of motor neurons with target muscle cells evoke prolonged elevations in the concentrations of neuronal cytosolic free calcium ([Ca(2+)](c)). These calcium elevations are initiated at the point of contact and spread throughout the neuron over a period of tens of minutes. In this study, we addressed how target muscle cells initiate this unique presynaptic response. Primary questions regarding the nature of the muscle signal are whether it is diffusible and whether it must first be induced by a growth cone as part of reciprocal interaction. We addressed whether the signal was strictly target-contact dependent by fixing C2 mouse myotubes with formaldehyde, rinsing extensively and then allowing processes of chick ciliary ganglion neurons to interact with them. We observed frequent sustained elevations in [Ca(2+)](c) in ciliary ganglion processes contacting the fixed myotubes. As a control, ciliary neurons were allowed to interact with fixed myotubes of the S27 variant line. S27 cells were isolated from the parent C2 line on the basis of a defect in glycosaminoglycan biosynthesis and previously shown to be defective in supporting synaptic vesicle localization in contacting neurites. Few elevations in [Ca(2+)](c) were detected in encounters between ciliary processes and fixed S27 cells. In addition, neuron-neuron encounters never elicited prolonged increases in [Ca(2+)](c). These observations demonstrate contact dependence in the neuronal response and rule out reciprocal cellular interactions, diffusible factors or electrical activity in the muscle. The defect in carbohydrate biosynthesis in S27 cells further suggests that cell surface carbohydrates are essential to the signal on the myotube surface that triggers the presynaptic elevation in [Ca(2+)](c). We conclude that growth cone contact with preexisting cell surface structures on target muscle cells induces changes in presynaptic [Ca(2+)](c) that are associated with retrograde signaling, and that proper carbohydrate biosynthesis is required for this signal.