4-(4-diethylaminostyryl)-n-methylpyridinium and tetramethylrhodamine-isothiocyanate

Repeated in vivo observations of vitally stained neuromuscular junctions allow direct monitoring of ongoing structural changes, although, normally occurring changes (remodelling) and those inflicted by the illumination itself (photodamage) need to be dissociated. In frog cutaneus pectoris muscles, stained in vivo with 4Di-2ASP twice within four to five weeks, growth only was observed in 14 out of 92 junctions (in 18 muscles), retraction only in 19, and both features simultaneously in 22 junctions, while 37 junctions showed no changes. The summed growth in a junction amounted to 5-42 microns, retraction to 5-52 microns, while overall changes in synaptic length were absent. This and the simultaneous occurrence of both, growth and retraction within a single junction, indicates junctional remodelling. Similar amounts of remodelling were observed in junctions illuminated for 180, 60 or 10-30 s, indicating that within this range and with the given optical system, blue light and 4Di-2ASP fluorescence were not harmful. Remodelling was not induced by the experimental procedure per se, since in in vitro preparations signs of sprouting (and retraction) were equally frequent in junctions of totally untreated muscles, in junctions vitally stained four to five weeks previously or vitally stained but not light-exposed in the same muscle. In endplates of mouse gluteus maximus muscles double stained with 4Di-2ASP and rhodamine-alpha-bungarotoxin, unusually large (> 10 microns) terminal sprouts and retraction with gain loss of ACh-receptors became prominent 9-29 days following illumination for more than 60 s. Frequently also, muscle fibre damage with local contracture and acute loss of stainability of axon terminals occurred; often followed by muscle fibre denervation. In contrast, no such changes occurred after lower illumination intensities (12% emission filter for green) or shorter exposure times (< 60 s); even at a fourth exposure performed within 53 days. Previously reported smaller changes indicating regular remodelling were not investigated here.

Topics: alpha7 Nicotinic Acetylcholine Receptor; Animals; Artifacts; Axons; Bungarotoxins; Female; Fluorescent Dyes; Light; Male; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Microscopy, Fluorescence; Nerve Tissue Proteins; Neuromuscular Junction; Pyridinium Compounds; Rana pipiens; Receptors, Nicotinic; Rhodamines; Species Specificity; Staining and Labeling; Synapses; Time Factors

The structure of individually identified neuromuscular junctions (NMJs) in mouse lateral gastrocnemius (LG) muscles was studied on 2 or more occasions over 3-6 months. Presynaptic motor nerve terminals and their underlying acetylcholine receptors were stained in living animals with the fluorescent dye 4-(4-diethylaminostyryl)-N-methylpyridinium iodide) and tetramethylrhodamine isothiocyanate-conjugated alpha-bungarotoxin (R alpha BTX), respectively, and visualized by video-enhanced fluorescence microscopy. The overall shape of most NMJs changed very little over this time except for enlargement of some junctions attributable to growth of the animals. A few junctions did, however, change appreciably: over 3-6 months about 15% underwent modifications such as additions to, or losses from, their original configuration. The frequency and extent of changes in LG NMJs were substantially less than in a similar study of NMJs from mouse soleus (Wigston, 1989). These findings, together with those from other laboratories, indicate a correlation between the extent of NMJ remodeling and the fiber-type composition of a muscle: NMJs in muscles consisting of predominantly fast-twitch myofibers appear to undergo less remodeling than NMJs in muscles containing a substantial fraction of slow-twitch fibers. Since fast- and slow-twitch muscles and their motoneurons exhibit strikingly different patterns of electrical activity, these findings suggest that structural remodeling at mammalian NMJs may depend on the amount of impulse activity experienced by motoneurons, their target muscle, or individual synaptic terminals.

Topics: Animals; Bungarotoxins; Female; Fluorescent Dyes; Hindlimb; Male; Mice; Mice, Inbred C57BL; Microscopy, Fluorescence; Muscles; Neuromuscular Junction; Pyridinium Compounds; Rhodamines; Time Factors