Development of calcitonin gene-related peptide (CGRP) immunoreactivity in relationship to the formation of neuromuscular junctions in Xenopus myotomal muscle.
Immunoreactivity (IR) to calcitonin gene-related peptide (CGRP) has recently been found in chick motoneurons. Bath application of this peptide causes an increase in surface acetylcholine receptor (AChR) density and cAMP level in cultured chick muscle cells. These results suggest a role for this peptide in the formation of synaptic specializations. In this study, we examined the development of CGRP IR in larval Xenopus myotomal muscle in relation to synaptogenesis. Using an antiserum against CGRP, a monoclonal antibody against the p65 synaptic vesicle antigen and a fluorescence conjugate of alpha-bungarotoxin, we followed the development of synaptic specializations with fluorescence microscopy in whole-mount specimens. We found that the postsynaptic specialization in the form of AChR clusters was first detected in stage 22 (24 hour) embryos. The presynaptic specializations, including the synaptic vesicle clusters as evidenced by p65 antibody staining and CGRP IR, were first detected at stage 32 (40 hours). The appearance of these two presynaptic specializations followed the same time course. Subsequently, all three structures, the AChR clusters, CGRP IR, and synaptic vesicle clusters, were colocalized at the neuromuscular junction (NMJ). This shows that CGRP is unlikely to be involved in signaling the development of the postsynaptic apparatus. This premise is further examined in cultures of Xenopus myotomal muscle cells. CGRP at concentrations up to 1 microM did not affect the number of AChR clusters, nor did it interfere with the formation of clusters induced by polycation-coated beads. In contrast, an extract from the basement membrane of Torpedo electric organ promoted the formation of AChR clusters and interfered with the clustering activity of the beads. These results suggest that CGRP, an integral component of the presynaptic specialization, is not involved in signaling synaptogenesis at the NMJ.
Peng
H B
HB
Department of Cell Biology and Anatomy, University of North Carolina, Chapel Hill 27599-7090.
Chen
Q M
QM
de Biasi
S
S
Zhu
D L
DL
D000818:Animals; D015740:Calcitonin Gene-Related Peptide / Q000378:metabolism* / Q000494:pharmacology; D002478:Cells, Cultured; D007814:Larva; D009132:Muscles / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009469:Neuromuscular Junction / Q000378:metabolism* / Q000502:physiology; D011950:Receptors, Cholinergic / Q000378:metabolism*; D014982:Xenopus laevis / Q000254:growth & development / Q000378:metabolism*
D011950:Receptors, Cholinergic; D015740:Calcitonin Gene-Related Peptide