Colocalization of substance P or enkephalin in serotonergic neuronal afferents to the hypoglossal nucleus in the rat

The serotonergic innervation of the hypoglossal nucleus originates from the caudal raphe nuclei. Non-serotonergic neurons in the caudal raphe nuclei also project to the hypoglossal nucleus. We employed a triple-fluorescence technique to determine whether the substance P- or the enkephalin-containing neurons in the caudal raphe nuclei that projected to the hypoglossal nucleus also contained serotonin. Rhodamine latex microspheres were injected into the hypoglossal nucleus, and then serotonin and peptide dual-immunofluorescence was performed to colocalize perikarya containing serotonin, substance P, and rhodamine microspheres; or perikarya containing serotonin, enkephalin, and rhodamine microspheres. Our results demonstrate that most substance P-containing neuronal afferents to the hypoglossal nucleus colocalize serotonin. In contrast, few enkephalin-containing neuronal afferents to the hypoglossal nucleus also contain serotonin. These data suggest that substance P projections to the hypoglossal nucleus are a subset of serotonergic projections and that limited overlap exists between the populations of enkephalinergic and serotonergic neuronal afferents to the hypoglossal nucleus. Either substance P- or enkephalin-containing somata account for a very small proportion of non-serotonergic caudal raphe projections to the hypoglossal nucleus. Finally, these data demonstrate the medial tegmental field origins of the substance P projections and the enkephalin projections to the hypoglossal nucleus.