Regular and photodamage-enhanced remodelling in vitally stained frog and mouse neuromuscular junctions

Abstract

Repeatedin 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, stainedin 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 μm, retraction to 5–52 μm, 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 inin 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-α-bungarotoxin, unusually large (>10 μm) terminal sprouts and retraction with gain and 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.