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

J Neurosci Res. 2001 Feb 15;63(4):330-40. doi: 10.1002/1097-4547(20010215)63:4<330::AID-JNR1027>3.0.CO;2-W.

Abstract

Individual skeletal muscle fibers in most new-born rodents are innervated at a single endplate by several motor axons. During the first postnatal weeks, the polyneuronal innervation decreases in a process of synaptic elimination. Previous studies showed that the naturally occurring serine-protease thrombin mediates the activity-dependent synapse reduction at the neuromuscular junction (NMJ) in vitro and that thrombin-receptor activation may modulate nerve terminal consolidation through a protein kinase mechanism. To test whether these mechanisms may be operating in vivo, we applied external thrombin and its inhibitor hirudin, and several substances affecting the G protein-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 (AChRs) and axons for counting the percentage of polyinnervated NMJ. We found that exogenous thrombin accelerated synapse loss and hirudin blocked axonal removal. Phorbol-12-myristate-13-acetate, a potent PKC activator, had a similar effect as thrombin, whereas the PKC inhibitors, calphostin C and staurosporine, 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 that thrombin could play a role in the postnatal synaptic maturation in vivo.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Animals, Newborn
  • Enzyme Inhibitors / pharmacology
  • GTP-Binding Proteins / metabolism*
  • Hemostatics / pharmacology
  • Male
  • Motor Neurons / chemistry
  • Motor Neurons / metabolism
  • Muscle Development
  • Muscle, Skeletal / growth & development
  • Muscle, Skeletal / innervation
  • Naphthalenes / pharmacology
  • Neuromuscular Junction / growth & development*
  • Neuromuscular Junction / physiology*
  • Pertussis Toxin*
  • Presynaptic Terminals / chemistry
  • Presynaptic Terminals / drug effects
  • Presynaptic Terminals / enzymology*
  • Protein Kinase C / antagonists & inhibitors
  • Protein Kinase C / metabolism*
  • Rats
  • Rats, Sprague-Dawley
  • Receptors, Cholinergic / analysis
  • Staurosporine / pharmacology
  • Thrombin / pharmacology
  • Virulence Factors, Bordetella / pharmacology*

Substances

  • Enzyme Inhibitors
  • Hemostatics
  • Naphthalenes
  • Receptors, Cholinergic
  • Virulence Factors, Bordetella
  • Pertussis Toxin
  • Protein Kinase C
  • Thrombin
  • GTP-Binding Proteins
  • Staurosporine
  • calphostin C