Spatial Distribution of Motor Endplates and its Adaptive Change in Skeletal Muscle

Abstract

Motor endplates (MEPs) are the important interfaces between peripheral nerves and muscle fibers. Investigation of the spatial distribution of MEPs could help us better understand neuromuscular functional activities and improve the diagnosis and therapy of related diseases. Methods: Fluorescent α-bungarotoxin was injected to label the motor endplates in whole-mount skeletal muscles, and tissue optical clearing combined with light-sheet microscopy was used to investigate the spatial distribution of MEPs and in-muscle nerve branches in different skeletal muscles in wild-type and transgenic fluorescent mice. Electrophysiology was used to determine the relationship between the spatial distribution of MEPs and muscle function. Results: The exact three-dimensional distribution of MEPs in whole skeletal muscles was first obtained. We found that the MEPs in the muscle were distributed in an organized pattern of lamella clusters, with no MEPs outside the lamella zone. Each MEP lamella was innervated by one independent in-muscle nerve branch and mediated an independent muscle subgroup contraction. Additionally, the MEPs changed along the lamella clusters after denervation and regained the initial pattern after reinnervation. The integrity and spatial distribution of MEPs could reflect the functional state of muscles. The signal absence of a certain MEP lamella could suggest a problem in certain part of the muscle. Conclusions: The MEP lamella clusters might be the basis of neuromuscular function, and the spatial distribution of MEPs could serve as a testbed for evaluating the functional status of muscle and the therapeutic targeting map related to MEPs.

Multiple retrograde tracing methods compatible with 3DISCO clearing

“…However, better consistency of labelled motor neuron pools in spinal cords was found in mice that smaller muscles were injected, while relatively apparent differences were found in those injecting the larger muscles [20]. According to our recent research, MEPs in limb muscles are distributed as laminar microstructure in a 3D model especially multiple lamination in the multi-head muscles, but not all the rims of MEP laminas reach the surface of muscles forming the MEP line [35]. Injection targeting the full MEP regions may not cover the majority of the MEPs.…”

Three-dimensional visualization of intramuscular innervation in intact adult skeletal muscle by a modified iDISCO method

“…18,26 Adult muscles are necessary objects when studying the muscle development and pathology. 27 But the thickness of fascia surrounding the muscle increased and the tissues become denser with age, the antibody penetration into muscles of adult mice is more difficult than that of young mice. The modified protocol described in this work achieved whole-mount labeling of adult tibialis anterior (about 3-mm thick), which is thicker than muscles such as extensor digitorum longus 28 and lumbricales.…”

Advances in Ex Situ Tissue Optical Clearing

“…Taking natural RI mismatch between muscle fluids and muscle proteins into account, it might be speculated that the crucial advantage of 3DISCO is the removal of inherent heterogeneity of muscle RI and then matching of “dried” muscle proteins (RI = 1.58) by immersion in DBE (RI = 1.56). By coupling electrophysiological examination with Alexa Fluor 647‐conjugated α‐bungarotoxin injection (competitive antagonist of nicotinic acetylcholine receptors), after which the muscles were subjected to 3DISCO clearing, Yin et al . performed detailed study on motor endplates in a variety of mouse muscles, their distribution under physiological conditions and remodeling in the model of denervation and nerve repair.…”