A new method for simultaneous demonstration of anterograde and retrograde connections in the brain: co-injections of biotinylated dextran amine and the beta subunit of cholera toxin

In studying reciprocally connected brain networks, it is advantageous to us e techniques that allow simultaneous visualization of both efferent and aff erent connections from a single injection site. We report on a new techniqu e to achieve this using pressure injections of a mixture of biotinylated de xtran amine (BDA) and the beta subunit of cholera toxin (Ctb). Adult male h amsters (n = 12) received 20-30-nl injections of either a 1:1 mixture of BD A (Sigma, 10%) and Ctb (List Biological, 0.5%), or each tracer by itself, i nto the medial amygdala. Adult female sheep (n = 4) received 200-300 nl of the combined tracer into the A15 region of the hypothalamus. After 1 (hamst er) or 2 weeks' (sheep) survival, animals were perfused with 4% paraformald ehyde. Sections were double-labeled, first for BDA histochemistry using nic kel-enhanced DAB, then for Ctb using a PAP technique and unenhanced DAB. In all animals, combined injections resulted in clear and consistent patterns of both anterograde and retrograde labeling. Ctb immunoreactivity was dist inct and easily distinguished from BDA labeling. There was no evidence for loss of sensitivity of either tracer due to the combined delivery; no diffe rences were seen between combined or single tracer injections in numbers of retrogradely-labeled cells or in the distribution of anterogradely-labeled fibers. In summary, the combined delivery of BDA and Ctb is an easy and re liable technique for simultaneous afferent and efferent tract tracing in bo th small and large animals; it could potentially be combined with immunocyt ochemistry to determine the neurochemical content of labeled cells or fiber s. (C) 1999 Elsevier Science B.V. All rights reserved.