Simultaneous use of retrograde fluorescent tracers and fluorescence histochemistry for convenient and precise mapping of monoaminergic projections and collateral arrangements in the CNS.

A procedure is described for the use of fluorescent retrograde tracers in conjunction with monamine fluorescence histochemistry for single or double labeling studies of the projections of identified catecholamine or indoleamine neurons in the CNS. A number of fluorescent tracers, including Evans Blue, bisbenzimide, DAPI, 'True Blue', 'Granular Blue' and propidium iodide, have been tested and characterized microspectrofluorometrically. Of these, 'True Blue', propidium iodide and Evans Blue were found to have the most suitable properties to be studied concomitant with the monoamine fluorphores. A combination of two of these tracers--'True Blue' fluorescing blue and propidium iodide or Evans Blue fluorescing orange to red--and the catecholamines and indoleamines fluorescing yellow-green and yellow to brownish yellow, respectively, makes possible simultaneous use of four different fluorescent markers in one and the same section. The present procedure has great practical advantages over available HRP techniques and should in modified form also be applicable to other types of transmitter-specific neuronal tracing based on immunocytochemistry or enzyme histochemistry.