Evidence for the coexistence of serotonin and substance P in single raphe cells and fiber plexuses: combined immunocytochemistry and autoradiography.

Abstract

Combined immunocytochemistry and autoradiography after in vivo injections of monoclonal antibody to Substance P and 3H-serotonin provide convincing evidence that the soma and processes of a single neuron can contain serotonin and immunoreactivity with another putative peptide transmitter Substance P in a single permanent preparation suitable for study with light and electron microscopy. The serotonin-Substance P cells have uptake systems for low molarity 3H-serotonin that are not affected by reserpine treatment. They are sensitive to serotonin uptake inhibitors and monoamine oxidase inhibitors and the cells are destroyed by 5,6-dihydroxytryptamine. They contain endogenous stores of serotonin detectable by the Falck-Hillarp technique and microspectrofluorimetry and immunofluorescence by antibody to serotonin. Their Substance P content is identified by specific binding with monoclonal antibody, and by animal antisera against Substance P. The raphe pallidus neurons are a mixed population -- some cells have a majority of Substance P immunoreactivity with little detectable serotonin, others have considerable quantities of both Substance P immunoreactivity and serotonin, and still others have only serotonin but are innervated by Substance P immunoreactive processes. These subtle differences in neuronal content of the two compounds are made more obvious by avoiding the use of colchicine, a drug known to inhibit fast axoplasmic transport. Such differences may be an expression of the dynamic or cyclic functions of neurons with multiple putative transmitter substances. Fluctuating levels of one or both substances may depend upon parameters of rhythm, demands for one or the other mediator during specific types or phases of activity. The fundamental scheme of simultaneous injection of multiple labels, one radioactive, another a specific characterized monoclonal antibody for the detection of multiple, separate transmitter systems opens vast avenues for future investigation.