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 cytosoli c free calcium ([Ca2+](c)). These calcium elevations are initiated at the p oint of contact and spread throughout the neuron over a period of tens of m inutes. In this study, we addressed how target muscle cells initiate this u nique presynaptic response. Primary questions regarding the nature of the m uscle signal are whether it is diffusible and whether it must first be indu ced by a growth cone as part of reciprocal interaction. We addressed whethe r the signal was strictly target-contact dependent by fixing C2 mouse myotu bes with formaldehyde, rinsing extensively and then allowing processes of c hick ciliary ganglion neurons to interact with them. We observed frequent s ustained elevations in [Ca2+](c) in ciliary ganglion processes contacting t he fixed myotubes. As a control, ciliary neurons were allowed to interact w ith fixed myotubes of the S27 variant line. S27 cells were isolated from th e parent C2 line on the basis of a defect in glycosaminoglycan biosynthesis and previously shown to be defective in supporting synaptic vesicle locali zation in contacting neurites. Pew elevations in [Ca2+](c) were detected in encounters between ciliary processes and fixed S27 cells. In addition, neuron-neuron encounters never elicited prolonged increases in [Ca2+](c). These observations demonstrate contact dependence in the neuron al response and rule out reciprocal cellular interactions, diffusible facto rs or electrical activity in the muscle. The defect in carbohydrate biosynt hesis in S27 cells further suggests that cell :surface carbohydrates are es sential to the signal on the myotube surface that triggers the presynaptic elevation in [Ca2+](c). We conclude that growth cone contact with preexisti ng cell surface structures on target muscle cells induces changes in presyn aptic [Ca2+](c) that are associated with retrograde signaling, and that pro per carbohydrate biosynthesis is required for this signal. Copyright (C) 20 00 S. Karger AG, Basel.