Axotomy of single fluorescent nerve fibers in developing mammalian spinal cord by photoconversion of diaminobenzidine

Francisco F. De-Miguel, Kenneth J. Muller, William B. Adams, John G. Nicholls

Research output: Contribution to journalArticle

5 Scopus citations

Abstract

A technique has been developed for cutting single nerve fibers in mammalian spinal cord. In the presence of diaminobenzidine (DAB), a laser microbeam was applied to carbocyanine (Dil) stained sensory fibers in cultured spinal cords of the newly born opossum Monodelphis domestica. Digital images of fluorescent fibers were acquired with an intensified video CCD-camera coupled to an image processor. Laser illumination of two spots on a fiber in the presence of 3 mg/ml DAB cut it, so that following DAB wash out, Dil fluorescence did not return after the intermediate segment was bleached. In contrast, when a similar procedure was carried out without DAB, fluorescence of the bleached segment was recovered within minutes in darkness, by dye diffusion from adjacent regions of the uncut fiber. After exposure to DAB, through-conduction of compound action potentials continued in undamaged fibers. The DAB reaction product remained as a dark precipitate, helping to localize the lesion sites. By illuminating a continuous series of spots it was possible to cut whole nerve roots. Fluorescent fibers extended across the cut segment 24 h later. With minor modifications, the procedure described here allows a precise lesioning of single fibers within an intact nervous system.

Original languageEnglish (US)
Pages (from-to)73-79
Number of pages7
JournalJournal of Neuroscience Methods
Volume117
Issue number1
DOIs
StatePublished - May 30 2002

Keywords

  • Axotomy
  • Laser ablation
  • Opossum
  • Regeneration
  • Single fiber
  • Spinal cord

ASJC Scopus subject areas

  • Neuroscience(all)

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