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Updated: Mar 19, 2026
Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo
Published on: June 26, 2013
RetroDISCO: Clearing technique to improve quantification of retrograde labeled motor neurons of intact mouse spinal
Emilija Žygelytė1, Megan E Bernard1, Joy E Tomlinson1
1Department of Clinical Sciences, Cornell College of Veterinary Medicine, Cornell University, Ithaca, NY, United States.
Background:
Quantification of the number of axons reinnervating a target organ is often used to assess regeneration after peripheral nerve repair, but because of axonal branching, this method can overestimate the number of motor neurons regenerating across an injury. Current methods to count the number of regenerated motor neurons include retrograde labeling followed by cryosectioning and counting labeled motor neuron cell bodies, however, the process of sectioning introduces error from potential double counting of cells in adjacent sections.
New Method:
We describe a method, retroDISCO, that optically clears whole mouse spinal cord without loss of fluorescent signal to allow imaging of retrograde labeled motor neurons using confocal microscopy.
Results:
Complete optical clearing of spinal cords takes four hours and confocal microscopy can obtain z-stacks of labeled motor neuron pools within 3-5min. The technique is able to detect anticipated differences in motor neuron number after cross-suture and conduit repair compared to intact mice and is highly repeatable.
Comparison With Existing Method:
RetroDISCO is inexpensive, simple, robust and uses commonly available microscopy techniques to determine the number of motor neurons extending axons across an injury site, avoiding the need for labor-intensive cryosectioning and potential double counting of motor neuron cell bodies in adjacent sections.
Conclusions:
RetroDISCO allows rapid quantification of the degree of reinnervation without the confounding produced by axonal sprouting.
