Visualization of efferent retinal projections by immunohistochemical identification of cholera toxin subunit B

The present study describes the use of cholera toxin subunit B as an anterograde and retrograde neuronal tracer for studying retinal projections of the rat, mouse, gerbil, and hamster. The tracer was pressure injected in the posterior chamber of the eye and the labeled neurons were identified using an avidin-biotin immunoperoxidase technique using diaminobenzidine as chromagen. Doses of 3-8 microliters (30-80 micrograms) cholera toxin subunit B and a survival for 24 h resulted in an optimal transport of the tracer in all rodent species investigated. The cholera toxin subunit B-containing retinal efferents were effectively stained and yielded the presence of axons with delicate boutons on passage and nerve endings. Smooth and thick fibers were also observed, indicating a distinction between passing and terminating axons, respectively. Immunoreactive axons were observed in the hypothalamus, thalamus, ad mesencephalon, and the precise distribution of positive nerves could be identified in counterstained sections, some of them as delicate endings in apposition to neuronal surfaces. Labeled cell bodies were observed in the oculomotor nucleus and the pretectum, indicating that the tracer is transported retrogradely as well. Because the tracer is identified immunohistochemically, the retinofugal and retinopetal pathways can be mapped more precisely, perhaps in combination with immunohistochemical detection of other antigens.