Immunohistochemical evidence for the coexistence of substance P, thyrotropin‐releasing hormone, GABA, methionin‐enkephalin, and leucin‐enkephalin in the serotonergic neurons of the caudal raphe nuclei: A dual labeling in the rat

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Abstract
By means of dual immunohistochemical labeling on the same brain section examined with a light microscope, the present study reports the presence with serotonin (5‐hydroxytryptamine; 5‐HT) of gamma‐aminobutyric acid (GABA), substance P (SP), thyro‐tropin‐releasing hormone (TRH), leucin‐enkephalin (LEU‐enk), or methionin‐enkephalin (MET‐enk), within the same neuron in the nuclei raphe magnus, raphe obscurus, and raphe pallidus of the rat. On the one hand, peptides or GABA are detected with specific rabbit antibodies by indirect peroxidase labeling using peroxidase‐conjugated Fab fragments, and on the other, 5‐HT is detected with a rabbit antibody against the BSA‐serotonin conjugate by radio‐immunocytochemistry using [125I]‐labeled protein A. The possible coexistence of TRH and SP in these neurons is also investigated by using peroxidase labeling and radio‐immunocytochemical detection, respectively.In the whole caudal raphe nuclei the proportion of each coexisting peptide with 5‐HT appears in decreasing order as: TRH > SP > MET‐enk # LEU‐enk > GABA. In all instances the level of coexistence differs considerably in B1–B2 vs. B3 cell groups. No SP/TRH dually labeled cells have ever been found in any of the serotonergic nuclei of the caudal raphe.Given the evidence that these raphe nuclei project possibly to the spinal cord, these data constitute an anatomical substrate for the several distinct physiological functions presumably subserved by 5‐HT in the cord, namely the modulation of nociception, motor, and autonomic functions.

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