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Human neuropeptide FF2 (hFF2) receptor has been postulated to mediate central autonomic regulation by virtue of its ability to bind with high affinity to many amidated neuropeptides. In the present immunohistochemical study, we identified hFF2 positive neurons in the forebrain and medulla oblongata of individuals, who died suddenly of mechanical trauma or hypothermia. Morphologically, these neurons demonstrated features identified with both projection neurons and interneurons. In the forebrain, the highest density of hFF2 expressing neurons was observed in the anterior amygdaloid area and dorsomedial hypothalamic nucleus, especially in its caudal part. A lesser density of hFF2 neurons was identified in the ventromedial hypothalamic nucleus, lateral and posterior hypothalamic areas whereas few cells were visualized in the paraventricular hypothalamic nucleus, perifornical nucleus, horizontal limb of the diagonal band, ventral division of the bed nucleus of the stria terminalis, nucleus basalis of Meynert and ventral tegmental area. In the medulla, significant numbers of hFF2 neurons were observed in the dorsal motor nucleus of vagus and to a lesser extent in the area of catecholaminergic cell groups, A1/C1. These data provide first immunohistochemical evidence of hFF2 localization in the human brain, which is consistent with that reported for tissue distribution of FF2 mRNA and FF2 binding sites within the brain of a variety of mammalian species. The distribution of hFF2 may help in identifying the role of amidated neuropeptides in the human brain within the context of central autonomic and neuroendocrine regulation.

Regional distribution of prolactin-releasing peptide (PrRP) in the human brain was studied by radioimmunoassay. The antiserum raised against human PrRP-31 in a rabbit was used in the assay, which showed 100% cross reaction with PrRP-20 and no significant cross reaction with other peptides. The highest concentrations of immunoreactive-PrRP were found in hypothalamus (912 +/- 519 fmol/g wet weight, n = 6, mean +/- SEM), followed by medulla oblongata (496 +/- 136 fmol/g wet weight) and thalamus (307 +/- 117 fmol/g wet weight). On the other hand, immunoreactive-PrRP was not detected in frontal lobe or temporal lobe (<50 fmol/g wet weight). Sephadex G50 column chromatography of the immunoreactive-PrRP in the hypothalamus and medulla oblongata showed three immunoreactive peaks; one peak eluting in the position of PrRP-20, one eluting in the position of PrRP-31 and one eluting earlier. Reverse phase high-performance liquid chromatography (HPLC) of these brain tissue extracts showed a peak eluting in the position of PrRP-20 and PrRP-31. The present study has shown for the first time the presence of immunoreactive-PrRP in the human brain. The immunoreactive-PrRP levels in the human hypothalamus were, however, lower than the levels of other neuropeptides with prolactin-releasing activity, such as thyrotropin-releasing hormone and vasoactive intestinal polypeptide.

Gastric adaptive relaxation is a vago-vagal reflex, probably involving the site of interface of vagal afferents and efferents in the dorsal vagal complex of the medulla. Previous studies have shown that both substance P and nitric oxide in the dorsal vagal complex decrease intragastric pressure. The purpose of this study is, firstly, to localize NK1 tachykinin receptor immunoreactive (ir) staining in the dorsal vagal complex and, secondly, to determine its anatomical relationship to nitrergic cells in the dorsal motor nucleus of the vagus. Sections were stained by avidin-biotin immunocytochemistry using antiserum to NK1 receptor alone or combined with NADPH-diaphorase histochemistry. In the nucleus tractus solitarius, NK1 receptor-ir varicosities were moderately dense in the medial subnucleus, but sparse in the centralis and gelatinosus subnuclei. In the dorsal motor nucleus of the vagus, NK1 receptor-ir staining in cell bodies and fibers was present throughout, with a markedly dense varicose fiber and cell body staining in a lateral column of the rostral portion of the nucleus. NADPH-diaphorase staining is most marked in cell bodies in the same region of the dorsal motor nucleus of the vagus. In dual-stained sections, there was complete overlap of NADPH-diaphorase and NK1 receptor-ir stain; however, the markers were very rarely colocalized within the same vagal motor neurons. Ipsilateral vagotomy almost completely abolished NK1r-ir staining in vagal motor neurons. We conclude that, in the dorsal motor nucleus of the vagus, NK1 receptor is synthesized by a population of vagal motor neurons which are in close anatomical proximity to, but separate from, nitrergic neurons. Based on these observations, substance P-mediated gastric relaxation in this region is unlikely to be via activation of nitrergic vagal preganglionic neurons. In the nucleus tractus solitarius, the NK1 receptor and NADPH-diaphorase stain are not codistributed in subnuclei mediating gastric and esophageal control. Therefore, substance P and nitric oxide may mediate their respective gastrointestinal effects via separate afferent pathways.

[125I]beta-endorphin bound to high affinity (Kd = 0.25 nM) receptors in the caudal dorsomedial medulla of rats with a Bmax of 97 fmol/mg protein. The relative potency for displacement of [125I]beta-endorphin binding was: beta-endorphin(1-31) > beta-endorphin(1-27) > DAMGO > naloxone > N-acetyl-beta-endorphin(1-31) > U50488 > DPDPE. The Bmax for [3H]DAMGO binding was 81 fmol/mg protein, indicating that most [125I]beta-endorphin binding corresponds to mu-opioid receptors. [3H]DAMGO binding was not influenced by lesioning noradrenergic nerve terminals in the caudal dorsomedial medulla. Our findings indicate that beta-endorphin interacts primarily with mu-opioid receptors in the caudal dorsomedial medulla. These receptors are not affected by noradrenergic denervation.

Endomorphin-1 (Tyr-Pro-Trp-Phe-NH2) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) are endogenous ligands that have greater affinity and selectivity for the mu-opiate receptor than any other known mammalian peptide. A polyclonal antiserum, screened for specificity to endomorphin-2 by immunodot-blot assay and preabsorption controls, was used for localization of this peptide. Immunocytochemistry performed on the brainstem, spinal cord, and sensory ganglia of rats by the avidin-biotin-peroxidase method revealed a continuous dense aggregation of endomorphin-2-like immunoreactive varicose fibers in the superficial laminae of the dorsal horn of the medulla and spinal cord. Immunoreactive fibers were detected in the dorsal root as well as within the dorsal root ganglia. The results suggest that endomorphin-2 is synthesized in primary sensory neurons in ganglia, transported to the superficial dorsal horn, and released near neurons expressing mu receptors. Its distribution appears to represent a functional unit likely to be associated with modulation of nociceptive stimuli.

The coexistence of neurokinin A (NKA) with substance P (SP) and serotonin (5-HT) in ventral medullary neurons of the parapyramidal region and nucleus raphe pallidus of the rat was studied using multiple immunofluorescence labeling. Nearly all of the NKA-immunoreactive (IR) cells in the parapyramidal region and raphe pallidus were SP-IR nd 5-HT-IR, whereas about 70% of the SP-IR neurons and about 60% of the 5-HT-IR neurons contained NKA-IR. There were no apparent differences in the patterns of coexistence between parapyramidal and raphe pallidus neurons. NKA-IR neurons, which colocalized SP-IR and 5-HT-IR, were studied for projections to the lumbar and thoracic spinal cord by use of retrograde transport of fluorescent tracer. Whereas about 50% of the retrogradely labeled neurons of the parapyramidal region and raphe pallidus contained NKA-IR, nearly all of the NKA-IR neurons projected to the thoracic and lumbar spinal cord. In addition, some NKA-IR neurons in the ventral medulla were retrogradely labeled with tracer from localized injections into the thoracic intermediolateral cell column. In summary, this study demonstrated that NKA-IR is colocalized with SP-IR in bulbospinal serotonergic neurons of the parapyramidal region and raphe pallidus, which are known to regulate sensory, motor, and autonomic activities of the spinal cord.

The distribution of neurokinin B (NKB) was determined by immunocytochemistry with antisera directed toward its amino terminus. Immunoreactive perikarya were detected in the main and accessory olfactory bulbs, cortical regions, the olfactory tubercle, the bed nucleus of the stria terminalis, the diagonal band of Broca, the nucleus accumbens, the septum, the neostriatum, several hypothalamic nuclei, the superior colliculus, the central gray, the substantia nigra, the medullary reticular formation, and the external cuneate nucleus. The distribution of NKB-containing perikarya revealed by immunocytochemistry was similar to the distribution of protachykinin B-containing cells previously visualized by in situ hybridization. Immunoreactive nerve fibers and terminals were detected in all major subdivisions of the brain. The levels of NKB measured by radioimmunoassay were highest in the hypothalamus. The distribution of NKB in the rat brain was similar to the distribution of substance P; however, there were several regions where the two distributions were clearly different.