Serotonergic projections to the spinal cord but not those to the olfactory bulb also contain substance P. A combined immunocytochemical and autoradiographic study following retrograde axonal transport of [3H]serotonin labeled products

Abstract Co-localization of substance P with serotonin in raphe projection neurons was studied by combining substance P immunocytochemistry and autoradiography following uptake and retrograde axonal transport of [ 3 H]serotonin and/or its products from target areas. In this study, two central pathways in the rat were investigated: the serotonergic projections of the midbrain raphe to the olfactory bulb and those of the medullary raphe that innervate the thoracic spinal cord. Two hours after pargyline pretreatment, injections of 10 −4 M [ 3 H]serotonin were made either into the olfactory bulb or into the spinal cord and respectively 24 or 60 h thereafter, rats were administered with colchicine. After a 24 h survival time, the paraformaldehyde fixed brains were investigated for substance P immunocytochemistry and then treated for light and electron microscopy autoradiography. Combining both methods, we can define on the same tissue sections at least three labeled neuronal populations: substance P immunolabeled neurons, radiolabeled neurons and doubly immuno-radiolabeled neurons. In the midbrain raphe cells as well as in the olfactory bulb nerve terminals, two kinds of labeled profiles were detected: substance P immunoreactive profiles and radiolabeled ones. The radiolabeled cell bodies of the midbrain raphe (403 counted cells) were never reactive to substance P antibodies. Moreover, they were distributed caudally to substance P stained perikarya. In contrast, in the medullary raphe, of the 336 radiolabeled cell bodies 162 were stained after substance P antibody treatment. They represent about 48% of the serotonin radiolabeled neurons projecting to the thoracic spinal cord, where a great number of varicosities were observed immunolabeled, radiolabeled and doubly immuno-radiolabeled in the dorsal horn. At the ultrastructural level, cell bodies and dendritic processes were also doubly labeled. Both labelings were observed over the cytoplasm and some organelles or perikarya. These observations provide a morphological basis to support the hypothesis that substance P can occur within some but not all serotonergic neurons and raise questions about the expression of this peptide in these systems as well as the modes of interaction of these transmitter molecules.