“Satellite cells” and nerve terminals in the crayfish opener muscle visualized with fluorescent dyes

Abstract

Nerve terminals and associated cells on the muscle's surface were visualized in the crayfish opener muscle with several fluorescent dyes in conjunction with confocal microscopy and conventional fluorescence microscopy. The nerve terminals of the excitatory and inhibitory axons were best seen with 4-diethylaminostyryl-N-methylpyridinium iodide (4-Di-2-Asp). This dye is selectively accumulated in mitochondria, which are numerous both in the axons and in synapse-bearing terminal varicosities. Muscle nuclei were also clearly visualized, because they excluded 4-Di-2-Asp but were stained by acridine orange (AO). A positive attraction between muscle nuclei and nerve terminals was evident by visual inspection and was confirmed by spatial statistics. Additional flat cells on the muscle's surface appeared as bright rings with elongated processes that were often close to or overlapped nearby nerve terminals. The structure of these cells was established by electron microscopy after labeling them with fluorescent polystyrene beads, which could be found over structures on the muscle surface in sections of embedded specimens. The flat surface cells were distinct from peripheral glial cells closely associated with axons and nerve terminals. Nevertheless, spatial statistics showed that the surface cells were grouped near nerve terminals. They occupied a small fraction of the muscle cell's surface. Their functional role has not been determined in crustacean muscles.

Distribution and morphology of inhibitory innervation in crayfish ( Procambarus clarkii ) limb and abdominal muscles

“…In the second procedure, live preparations were first stained for 3-5 min with the vital dye 4-(4-(diethylamino)styryl)-N-methylpyridunium iodide (4-Di-2-Asp; Molecular Probes, Eugene, Ore., USA), diluted to a final concentration of 5 µM in crayfish saline solution (Magrassi et al 1987). Micrographs were taken from an area of interest and the imaged area was marked by leaving behind fluorescent beads (Cooper et al 1995b;Harrington and Atwood 1995). After being washed to remove the dye, the specimens were then fixed and labelled with GABA antibodies as described above, and micrographs were taken from the same terminal region imaged previously, by using the deposited fluorescent beads to identify the previously studied locations.…”

Quantal release at visualized terminals of a crayfish motor axon: Intraterminal and regional differences

“…5 ) , the fluorescence microscope was used in conjunction with a Bio-Rad 600 confocal laser microscope. Further technical information can be found elsewhere (Harrington and Atwood, 1995).…”

“…With the development of fluorescent dyes that can be applied to living nerve terminals without severely injuring them, morphological diversity among endings on crustacean muscle fibers has been frequently observed (Tse et al, 1991;Atwood et al, 1994;Govind et al, 1994;Harrington and Atwood, 1995). The question arises, are the morphological differences among terminals in different locations accompanied by physiological differences, and, if so, what implications would this have for attempts to define the structure and function of a neural system by taking limited samples for physiological or microanatomical analysis?…”

Quantal release at visualized terminals of a crayfish motor axon: Intraterminal and regional differences

“…Preparations so treated continue to release transmitter for the duration of the experiment as in standard solution (Cooper et al, 1995a). Further technical information can be found elsewhere (Harrington and Atwood, 1995). For more detailed morphological work, such as obtaining composite images for illustrating the double labelling (see Fig.…”