Immunohistochemical support for three putative transmitters in one neuron: Coexistence of 5-hydroxytryptamine, substance p- and thyrotropin releasing hormone-like immunoreactivity in medullary neurons projecting to the spinal cord

Using indirect immunofluorescence histochemistry, in part combined with the elution and restaining technique of Tramu, Pillez & Leonardelli (1978), the distribution of 5-hydroxytryptamine (5-HT), thyrotropin releasing hormone (TRH) and substance P immunoreactive neurons has been studied in the medulla oblongata and spinal cord of normal and colchicine-treated rats. Evidence was obtained that at least some cell bodies in the medullary raphe nuclei and adjacent areas contained all three compounds, 5-HT, TRH and substance P. Other cell bodies in the same areas may contain two or only one of these three putative transmitters. Alternatively, the intraneuronal levels of one or two of the substances may be too low to be detected with the present technique, in spite of the fact that colchicine treatment was used to elevate peptide levels in the cell somata. In a quantitative evaluation the proportion of 5-HT, TRH and substance P neurons was calculated at different levels and in different nuclei of the medulla oblongata. Out of all immunoreactive neurons, there were approximately twice as many 5-HT (56%) as TRH (23%) and substance P (21%) cells respectively, and this relation was also found in several major subnuclei, such as the nucleus raphe magnus and nucleus raphe obscurus. In the 'arcuate' region very high proportions of 5-HT cells (about 60-80%) were observed with only few substance P cells (2-12%). The 'parapyramidal' and 'paraolivar' regions, which include the nucleus interfascicularis hypoglossi, had more substance P (26-36%) than TRH (15-17%) cells. The most 'even' distribution was observed in the nucleus raphe pallidus (5-HT: 43%; TRH: 32%; substance P: 25%). The evaluation also indicated how the respective cell type (5-HT, TRH and substance P cells) distributed between the different subnuclei. Thus, at rostral levels the 'suprapyramidal' region contained a large proportion (about 30%) of the total numbers of counted 5-HT, TRH and substance P cells, respectively. Furthermore, the nucleus raphe magnus contained a large part (about 30%) of the TRH and substance P cells, but a smaller fraction (about 20%) of the 5-HT cells. Analysis of adjacent sections at regular intervals confirmed the overall quantitative evaluation. Generally, the distribution of 5-HT, TRH and substance P cells were roughly parallel. An exception was the midportion of the rostral medulla oblongata, where 5-HT cells were very numerous. Of particular interest was the fact that, especially in the nucleus raphe pallidus, there were in several series almost the same number of 5-HT, TRH and substance P cells, supporting the view that many cells in this nucleus contained all these compounds. In the spinal cord overlapping networks of 5-HT, TRH and substance P immunoreactive fibres were observed in the ventral horn. The number of 5-HT immunoreactive fibres seemed higher than the TRH and substance P immunoreactive ones. After treatment with the neurotoxins 5,6- or 5,7-dihydroxytryptamine there was an almost complete disappearance of all three types of fibres in the ventral horn, further supporting the occurrence of the two peptides in 5-HT neurons, either both of them together in the same 5-HT neuron or each of them in separate 5-HT neurons. It is, however, important to note that there are, in all probability, 5-HT neurons in the lower medulla oblongata which contain neither TRH nor substance P. Furthermore, in other brain regions there is no certain correlation between the distribution patterns of 5-HT, TRH and substance P immunoreactive cells. The results are consistent with the coexistence of 5-HT, TRH and substance P in neurons of the medulla oblongata that project to the spinal cord. Some neurons may contain detectable levels only of 5-HT and substance P, others only of 5-HT and TRH, while others contain all three substances. It can, however, not be excluded that some neurons contain only one of these compounds or that other combinations exist.