Whole-mount optical clearing of rabbit tenuissimus muscle for assessment of muscle spindle morphology.

Muscle spindles are essential proprioceptive receptors that provide the sensory foundation for posture, movement coordination, and reflex control. However, their location deep inside muscles and complex three-dimensional architecture have historically limited morphological analysis to labor-intensive techniques such as silver-impregnation, muscle teasing, or serial sectioning followed by volumetric reconstruction. Here, we present a whole-mount optical clearing and multiplex fluorescence imaging workflow that enables in situ three-dimensional visualization of muscle spindles in the rabbit tenuissimus muscle, a muscle uniquely rich in spindles. We combined fluorescent labeling of spindle outer capsule cells, sensory, and motor innervation, including intrafusal γ neuromuscular junctions labeled by α-bungarotoxin, with solvent-based optical clearing. We achieved robust and reproducible imaging of the complete spindle structure using both confocal and lightsheet imaging. Notably, we improved on previous methods and were able, for the first time, to label motor endplates with α-bungarotoxin in a way compatible with solvent-based optical clearing, allowing simultaneous visualization of both extrafusal neuromuscular junctions and fusimotor terminals in intact muscles. The rabbit tenuissimus exhibited a remarkably high spindle density (1,600 ± 217 spindles/g), facilitating quantitative morphometric analyses including capsule length, volume, cross-sectional area, and intrafusal fiber numbers. High-resolution volumetric imaging also revealed the complex organization of Ia annulospiral endings within their native anatomical context. This reproducible workflow overcomes major limitations of conventional spindle histology and provides a powerful platform for investigating muscle spindle development, plasticity, aging, and pathology. More broadly, it establishes the rabbit tenuissimus as an exceptional model for studies of proprioceptive organ structure.