A combined retrograde transport and immunocytochemical staining method for demonstrating the origins of serotonergic projections.

Principle of Technique In recent years considerable interest has been directed toward defining projections of neurons containing specific neurotransmitten substances. As a result, numerous techniques have been developed that use fluorescent retrograde cell markers (2,6,10,1 1,19) on horseradish peroxidase (HRP) (3,4,13,14,17) combined with the fluorescence histochemistry (2, 1 7,19), immunohistofluonescence (6,10), or the unlabeled antibody, peroxidase-antiperoxidase (PAP) method (4,5,14) to identify the projections of neurotransmitter-defined neurons. While these techniques have contributed to the advancement of our basic understanding of the anatomical organization of certain pathways, they are often laborious and inconvenient or the existence of the different cell markers is relatively short-lived. A technique (4) has been developed in our laboratory that combines the retrograde transport of horseradish peroxidase (HRP) or of wheat germ agglutinin conjugated to HRP (WGA-HRP) with the PAP method of Sternberger. This technique can be employed to determine the cells ofonigin ofserotonergic pathways as well as pathways containing other putative neurotransmitter/modulator substances. In the present article, these methods are applied to determining the origin of descending serotonergic pathways to the spinal cord (4,5), but the techniques can be readily adapted to studies ofother systems (15,20). Instruments