Pertussis toxin-sensitive G-protein and protein kinase C activity are involved in normal synapse elimination in the neonatal rat muscle

Individual skeletal muscle fibers in most new-born rodents are innervated a t a single endplate by several motor axons. During the first postnatal week s, the poly neuronal innervation decreases in a process of synaptic elimina tion. Previous studies showed that the naturally occurring serine-protease thrombin mediates the activity-dependent synapse reduction at the neuromusc ular junction (NMJ) in vitro and that thrombin-receptor activation may modu late nerve terminal consolidation through a protein kinase mechanism. To te st whether these mechanisms may be operating in vivo, we applied external t hrombin and its inhibitor hirudin, and several substances affecting the G p rotein-protein kinase C system (GP-PKC) directly over the external surface of the neonatal rat Levator auris longus muscle. Muscles were processed for immunocytochemistry to simultaneously detect acetylcholine receptors (AChR s) and axons for counting the percentage of polyinnervated NMJ, We found th at exogenous thrombin accelerated synapse loss and hirudin blocked axonal r emoval. Phorbol-12-myristate-13-acetate, a potent PKC activator, had a simi lar effect as thrombin, whereas the PKC inhibitors, calphostin C and stauro sporine, prevented axonal removal. Pertussis toxin, an effective blocker of GP function, blocked synapse elimination. These findings suggest that the normal synapse elimination in the neonatal rat muscle may be modulated, at least in part, by the pertussis-sensitive G-protein and PKC activity and th at thrombin could play a role in the postnatal synaptic maturation in vivo. (C) 2001 Wiley-Liss, Inc.