Quadruple labeling of brain-stem neurons: a multiple retrograde fluorescent tracer study of axonal collateralization

Four different fluorochromes were injected into adjacent cervical spinal cord segments, 1 unique tracer per segment. Each tracer, Fluoro-Gold, Fast Blue, Diamidino Yellow dihydrochloride and Propidium Iodide, was taken up by axonal terminals and transported intra-axonally in a retrograde direction to the cell bodies. Some, though by no means all, of these axons were stem axons with terminals in 2, 3 or 4 of the injected spinal segments. Hence as many as 4 different fluorescent tracers could be discerned simultaneously within individual neuronal somata of origin using fluorescent microscopy. These results extend the possibilities for multiple interconnection determinations within the central nervous system. Specifically, the potential for individual neurons of a nucleus to project collateral branches of a stem axon to as many as 4 different central nervous system nuclei now can be studied simultaneously using these 4 fluorescent tracers.