Simultaneous visualization and electrophysiology of corneal A-delta and C fiber afferents

Fluorescent staining of neuronal elements has become an important tool in neuroscience research; however, investigations have been limited by the toxic effects produced by most dyes, especially following excitation by strong epifluorescent illumination. The present study investigated effects produced by the methylpyridinium fluorescent dyes, 4-di-1-ASP and 4-di-2-ASP, on electrophysiologic responses from corneal A-delta and C fiber afferents. Rabbit corneal tissue was isolated and maintained in vitro to facilitate staining, visualization, and electrophysiologic recording of corneal nerves. Nerve fibers were selectively stained by the dyes and could be followed from their point of entry in small nerve bundles at the cornea-sclera border to individual free nerve ending terminals in the corneal epithelium. Neither the dyes nor epifluorescence, alone or in combination, produced any statistically significant changes (P greater than 0.1; t test) in spontaneous discharge activity, spike amplitude, spike dV/dt or stimulus evoked activity in A-delta and C fibers. Epifluorescent visualization of corneal afferents and simultaneous electrophysiologic recording will permit detailed investigations of sensory transduction processes and structure-function relationships in mammalian sensory nerves.