Stereotypic morphology of glutamatergic synapses on identified muscle cells of Drosophila larvae

Abstract

The distribution and morphology of glutamatergic synapses on Drosophila bodywall muscle fibers were examined at the single-synapse level using immunocytochemistry and electrophysiology. We find that glutamate-immunoreactive motor endings innervate the entire larval bodywall musculature, with each muscle fiber receiving at least one glutamatergic ending. The innervation is initiated at stereotyped locations on each muscle fiber from where moderately branched varicose nerve processes project over the internally facing muscle surface. Individual muscle fibers have distinct stereotypic patterns of nerve endings that occupy characteristic regions on the cell surface. The muscle-specific branching pattern of motor endings is reiterated by segmentally homologous fibers. Two morphological types of innervating nerve processes can be distinguished by their bouton size distributions: (1) Type I processes, which have localized branching and a broad size distribution of relatively large varicosities ranging up to 8 microns (mean diameter, 3.1 +/- 1.6 microns; +/- SD, n = 521), and (2) thinner Type II processes, which have a narrower distribution of small varicosities with a mean diameter of only 1.4 +/- 0.6 microns (+/- SD, n = 214). Immunoelectron microscopy with peroxidase-labeled second antibody demonstrates that the varicosities are surrounded by a subsynaptic reticulum, that they contain immunoreactive vesicles of about 30-50 nm, and thus probably represent synaptic release sites. By iontophoretic application of glutamate we mapped the responsive sites on the muscle surface and found an excellent correspondence between transmitter sensitivity and the patterns of endings as described by immunocytochemistry. In contrast to our finding of numerous glutamate iontophoresis-sensitive sites, we did not detect any aspartate-responsive muscles. These data provide strong new evidence for glutamate being an endogenous transmitter at the Drosophila larval neuromuscular junction.

Drosophila glutamate receptor RNA expression in embryonic and larval muscle fibers

“…Electrophysiological studies have demonstrated that functional glutamate receptors are concentrated at synaptic sites both in embryonic and in larval muscle fibers (Jan and Jan, 1976;Johansen et al, 1989;Broadie and Bate, 1993a). Because glutamate receptors remain diffusely distributed on embryonic muscle fibers that are not innervated, innervation is necessary for localizing glutamate receptors to synaptic sites (Broadie and Bate, 1993b).…”

“…We dissected third instar larvae, prepared whole mount preparations of their body-wall muscles, and determined the distribution of DGluR-I1 mRNA expression by in situ hybridization. The arrangement of the individual larval muscle fibers (Bate, 1990) and their pattern of innervation has been described in detail (Johansen et al, 1989). These studies demonstrate that most muscle fibers are innervated over a substantial portion of their cell surface whereas the innervation of muscle fibers 6 and 7 is more restricted (Johansen et al, 1989;Broadie and Bate, 1993a).…”

“…The arrangement of the individual larval muscle fibers (Bate, 1990) and their pattern of innervation has been described in detail (Johansen et al, 1989). These studies demonstrate that most muscle fibers are innervated over a substantial portion of their cell surface whereas the innervation of muscle fibers 6 and 7 is more restricted (Johansen et al, 1989;Broadie and Bate, 1993a). The nerve terminals on muscles 6 and 7 arborize within the central region of the muscle and do not extend to the anterior 20% or the posterior 30% of the muscle cell surface.…”

“…The receptor for the excitatory neurotransmitter glutamate a t Drosophila neuromuscular synapses is localized to synaptic sites (Jan and Jan, 1976;Johansen et al, 1989;Broadie and Bate, 1993a; see also Takeuchi and Takeuchi, 19751, and recent studies indicate that mechanisms which lead to accumulation of glutamate receptors at synaptic sites may be shared with those which lead to accumulation of AChRs at vertebrate neuromuscular synapses (Broadie and Bate, 1993a,b). The utility of genetic approaches for studying development in Drosophila raises the possibility that molecules which are required for clustering glutamate receptors might be identified by genetic screens in Drosophila and lead to identification of related molecules that are important for synapse formation in vertebrates.…”

Ultrastructure of neuromuscular junctions in Drosophilal: Comparison of wild type and mutants with increased excitability

“…The most characterized of these transmitters is glutamate, which, as in most invertebrate neuromuscular junctions, is believed to be the main excitatory transmitter (Jan and Jan, 1976b;Johansen et al, 1989a;Broadie and Bate, 1993). Immunocytochemical studies indicate that this transmitter is ubiquitously expressed at synaptic terminals that innervate all body wall muscles (Johansen, 1989b).…”