Immunohistochemical and biochemical analysis of serotonin and substance P colocalization in the nucleus tractus solitarii and associated afferent ganglia of the rat

Abstract

In a previous study of afferent projections to the nucleus tractus solitarii (NTS), it was shown that over half of the retrogradely‐labelled neurons in the nucleus raphe pallidus contained serotonin‐immunoreactivity and over half of these neurons contained substance P‐immunoreactivity, suggesting that these two putative neurotransmitters are colocalized in NTS‐affent neurons. The objectives of the present study were to (1) directly determine if varicosities in the NTS, the area postrema (AP), and the dorsal motor nucleus of the vagus nerve (DMN) do contain both transmitters, (2) determine if primary afferent neurons in the nodose and petrosal ganglia might also colocalize serotonin and substance P, and (3) quantify the amount of substance P that is contained in serotonergic varicosities in the NTS. Distributions and colocalization of substance P and serotonin in the NTS were studied using dual‐color immunohistochemistry, while the quantity of substance P in serotonergic varicosities was assessed by radioimmunoassay (RIA) using micropunches from the NTS of 5,7‐dihydroxytryptamine‐(5,7 DHT—) and vehicle‐treated rats. Varicosities that contained both serotonin‐ and substance P‐immunoreactivity were found in the NTS, the DMN, and the AP. Double‐labelled varicosities were common in the NTS and DMN (i.e., qualitatively similar to the density seen in the hypoglossal nucleus and in the ventral horn of the cervical spinal cord); however, the vast majority of the varicosities in these autonomic areas only displayed immunoreactivity for one or the other of these transmitters. This paucity of doubly‐labelled varicosities, in comparison to the number of singlylabelled varicosities, was reflected in the lack of a significant decrease in substance P levels as determined by RIA of micropunches taken from caudal and intermediate levels of the NTS in 5,7 DHT—and vehicle‐treated rats. At rostral levels of the NTS, however, there was a significant reduction (45%) in substance P levels in 5,7 DHT‐treated rats compared to vehicle‐treated rats. Immunohistochemical evaluation of sections from the rostral NTS of 5,7 DHT‐treated rats also indicated that the density of substance P varicosities was reduced. Immunohistochemical studies of the nodose and petrosal ganglia showed numerous serotonin‐ and substance P‐immunoreactive neurons, but none of the neurons contained immunoreactivity for both transmitters. In conclusion, serotonin and substance P are colocalized in varicosities in the NTS, the DMN, and the AP. Based upon the absence of primary afferent neurons that exhibit colocalization of serotonin and substance P, and previous studies of serotonin‐ and substance P‐containing NTS‐afferent neurons; it appears that the caudal medullary raphe nuclei are the source of varicosities that colocalize serotonin and substance P. While the density of these varicosities in the NTS and DMN are comparable to the density seen in the ventral horn of the spinal cord, this comprises only a small proportion of the varicosities in the NTS and DMN that contain either transmitter alone.