Distribution of vasoactive intestinal peptide- and peptide histidine isoleucine amide-like immunoreactive neurons and fibers in the thoracic spinal cord of the rat.

The distributions of vasoactive intestinal peptide (VIP)- and peptide histidine isoleucine amide (PHI)-immunoreactivities (IR) in the thoracic spinal cord of the rat are described. VIP- and PHI-IR were present in cells and fibers in the lateral spinal nucleus, lamina VII, nucleus proprius, substantia gelatinosa, intermediolateral cell column, and the area around the central canal. The functions of these peptides in the thoracic spinal cord are not known; however, their locations suggest that they are involved in sensory and autonomic functions, among others.

Evidence that some preganglionic sympathetic neurons in the rat contain vasoactive intestinal peptide- or peptide histidine isoleucine amide-like immunoreactivities.

Physiological studies have established that preganglionic sympathetic nerve fibers innervating the rat superior cervical ganglion release a second transmitter, in addition to acetylcholine. Based on pharmacological and histochemical investigations, possible candidates for this non-cholinergic neurotransmitter include vasoactive intestinal peptide and peptide histidine isoleucine amide. For example, previous immunohistochemical studies have demonstrated that antisera raised against both of these peptides stain neural processes in the rat preganglionic cervical sympathetic trunk and in the superior cervical ganglion. In the present study, it was found that, when the cervical sympathetic trunk was ligated, vasoactive intestinal peptide- and peptide histidine isoleucine amide-like immunoreactivities built up on both sides of the ligature. In addition, examination of the thoracic spinal cord in colchicine-treated animals revealed vasoactive intestinal peptide- and peptide histidine isoleucine amide-like immunoreactivies in neuronal cell bodies in the intermediolateral cell column and in the region of the lateral funiculus adjacent to it. In a second group of animals in which retrograde tracing techniques were used, these two regions of the spinal cord were shown to contain most of the cell bodies of the preganglionic neurons that project to the superior cervical ganglion. Smaller numbers of retrogradely labeled neurons were found dorsal to the central canal and in the nucleus intercalatus. When either vasoactive intestinal peptide- or peptide histidine isoleucine amide-like immunostaining and retrograde labeling were examined in the same animals, double-labeled neurons were found in the intermediolateral cell column and in the lateral funiculus. These data demonstrate that vasoactive intestinal peptide- and peptide histidine amide-like immunoreactivities are present in certain of the preganglionic neurons that project to the superior cervical ganglion, supporting the hypothesis that vasoactive intestinal peptide and peptide histidine isoleucine amide are released in the ganglion when these preganglionic neurons are activated.

Evidence for co-existence of thyrotropin-releasing hormone, substance P and serotonin in ventral medullary neurons that project to the intermediolateral cell column in the rat.

The present study was conducted to determine if substance P-, thyrotropin-releasing hormone- and/or serotonin-immunoreactivities coexist in ventral medullary neurons that project to the intermediolateral cell column in the rat. Neurons that projected to the intermediolateral cell column were identified by the presence of retrogradely transported rhodamine bead-labeled microspheres in the cell body after an injection of the microspheres into the intermediolateral cell column of the third thoracic spinal cord segment. Co-existence was determined by using a combination of dual color immunohistochemistry and serial 4-microns sections that were immunostained with different antibodies. Antibodies to substance P, serotonin, and pre-pro-thyrotropin releasing hormone160-169 were used to identify substance P, serotonin and thyrotropin-releasing hormone, respectively. Neurons that contained substance P-, thyrotropin-releasing hormone- and/or serotonin-immunoreactivities and that projected to the intermediolateral cell column were present in the nucleus raphe magnus, the nucleus raphe pallidus, the nucleus reticularis magnocellularis pars alpha, the paragigantocellular reticular nucleus and the parapyramidal region. Neurons that projected to the intermediolateral cell column, in each of these regions, were found to contain each of the following combinations of immunoreactive neurochemicals: substance P and thyrotropin-releasing hormone: substance P and serotonin; thyrotropin-releasing hormone and serotonin; or substance P, thyrotropin-releasing hormone and serotonin. In addition, most of the regions also contained neurons that appeared to contain only one of the neurochemicals and that also projected to the intermediolateral cell column. The greatest number of neurons that projected to the intermediolateral cell column and that also contained two or more co-existing neurochemicals was present in the midline regions. This study demonstrates the presence of neurons in the ventral medulla that project to the intermediolateral cell column and contain three co-existing neurochemicals. This study also demonstrates the use of a new method for the localization of three neurochemicals in single projection-specific neurons.

Enkephalin-immunoreactive neuronal projections from the medulla oblongata to the intermediolateral cell column: relationship to substance P-immunoreactive neurons.

The present study investigated the ventral medullary distribution of enkephalin-immunoreactive neurons that project to the intermediolateral cell column and the relationship of these neurons to substance P-immunoreactive neurons. Neurons that projected to the intermediolateral cell column were identified by the presence of rhodamine-labeled microspheres within the neuronal cell body after an injection of the microspheres into the intermediolateral cell column of the third thoracic spinal cord segment. Enkephalin- and substance P-immunoreactivities were identified by dual-color immunohisto-chemistry. Enkephalin-immunoreactive neurons that projected to the intermediolateral cell column were present in the raphe magnus, the nucleus reticularis magnocellularis pars alpha, the paragigantocellular reticular nucleus, and the parapyramidal region. These neurons were present throughout the rostrocaudal extent of each of these nuclei. However, in the raphe magnus the greatest number was present at more rostral levels of the nucleus. The morphology and distribution of enkephalin-immunoreactive neurons that projected to the intermediolateral cell column were similar to those of enkephalin-immunoreactive neurons that were not observed to contain rhodamine-labeled microspheres. Substance P- and enkephalin-immunoreactive neurons that projected to the intermediolateral cell column were present in similar distributions in each of the nuclei studied, except the raphe magnus. The raphe magnus contained more enkephalin- than substance P-immunoreactive neurons at rostral levels and more substance P-immunoreactive neurons than enkephalin-immunoreactive neurons at caudal levels. Coexistence of substance P- and enkephalin-immunoreactivities in ventral medullary neurons that projected to the intermediolateral cell column was rarely seen. These studies support the hypothesis that ventral medullary enkephalinergic neurons project to the intermediolateral cell column where they could act to modulate preganglionic sympathetic activity.

Localization of vasoactive intestinal peptide- and peptide histidine isoleucine amide-like immunoreactivities in the rat superior cervical ganglion and its nerve trunks.

Electrical stimulation of the preganglionic cervical sympathetic trunk causes an increase in dopa synthesis in the postganglionic neurons in the superior cervical ganglion (SCG). This transsynaptic biochemical effect can be blocked only partially by cholinergic antagonists, suggesting the involvement of a noncholinergic preganglionic sympathetic neurotransmitter(s). A survey of a large number of possible candidates for this neurotransmitter revealed that, in addition to cholinergic agonists, only a small group of peptides (all members of the secretin-glucagon family) stimulated dopa synthesis in the SCG. The effective peptides included vasoactive intestinal peptide (VIP), peptide histidine isoleucine amide (PHI), and secretin. Consequently we looked for the presence of immunoreactivities for these three peptides in the SCG. VIP- and PHI-like immunoreactive fibers were found in the SCG and in its major pre- and postganglionic nerve trunks. The distributions of the two immunoreactivities were very similar. Immunoreactive fibers were seen both singly and in bundles. In some instances, fibers were found apposed to neuronal cell bodies in the ganglion, and occasionally dense plexuses of fibers were found surrounding the neurons. In addition, punctate immunoreactive profiles were found apposed to the neurons in what appeared to be terminal fields. A small number of immunoreactive neuronal cell bodies were also seen in the ganglion. In a few instances, it was possible to establish, in serial sections, that the same cell body was immunostained with both VIP and PHI antisera. No secretin like-immunoreactive fibers or cells were observed. The presence of VIP-like and PHI-like-immunoreactive fibers in the cervical sympathetic trunk and in the SCG strengthens the possibility that these peptides, or a related molecule(s), serve as preganglionic neurotransmitters in this ganglion.

Substance P binding sites in the nucleus tractus solitarius of the cat.

Substance P binding sites in the nucleus tractus solitarius were visualized with receptor autoradiography using Bolton-Hunter [125I]substance P. Substance P binding sites were found to have distinct patterns within the cat nucleus tractus solitarius. The majority of substance P binding sites were present in the medial, intermediate and the peripheral rim of the parvocellular subdivisions. Lower amounts of substance P binding sites were present in the commissural, ventrolateral, interstitial and dorsolateral subdivisions. No substance P binding sites were present in the central region of the parvocellular subdivision or the solitary tract. The localization of substance P binding sites in the nucleus tractus solitarius is very similar to the patterns of substance P immunoreactive fibers previously described for this region. Results of this study add further support for a functional role of substance P in synaptic circuits of the nucleus tractus solitarius.

Somatostatin mRNA: regional variation in hybridization densities in individual neurons.

Somatostatin mRNA is detected by in situ hybridization of 35S-labeled single-stranded cDNA probes to coronal sections of the rat brain that include the periventricular nucleus of the hypothalamus. Features supporting hybridization specificity include its anatomic distribution, the results of studies using multiple cDNA probes, RNAase experiments, competition studies, and correlations with patterns of somatostatin peptide immunostaining in adjacent sections. The hybridization densities vary strikingly from region to region, with highest densities in the periventricular nucleus and more modest levels in areas such as the cerebral cortex and the striatum. On the basis of the results of in situ and immunohistochemical approaches, we suggest that this variation is due to regional differences in the density of hybridization per positive cell, as well as to regional variation in the densities of somatostatinergic perikarya.

Coexistence of enkephalin and dynorphin immunoreactivities in neurons in the dorsal gray commissure of the sixth lumbar and first sacral spinal cord segments in rat.

The dorsal gray commissure (DGC) of the sixth lumbar (L6) and first sacral (S1) spinal cord segments in the rat has been shown to contain several different peptides, including enkephalin (ENK) and dynorphin (DYN). The present study was conducted to determine if DYN and ENK immunoreactivities coexist in neurons in the DGC in L6 and S1. Coexistence was determined using the elution/restaining technique of Tramu in which sections were stained first with anti-DYN then with anti-ENK after elution of the DYN antiserum. The number and location of DYN-immunoreactive cells was compared to the number and location of ENK-immunoreactive cells using photographic negatives. Of 67 ENK-immunoreactive cells counted in 20 sections through L6, 54 also contained DYN immunoreactivity. Of 100 ENK-immunoreactive cells counted in 47 sections through S1, 95 also contained DYN immunoreactivity. No cells with only DYN immunoreactivity were seen. S1 was found to contain a significantly greater number (P less than 0.005) of ENK-immunoreactive cells that also contained DYN than L6. The results of these studies indicate that ENK and DYN immunoreactivities coexist in most, but not all, neurons in the DGC of L6 and S1 and that a difference exists between L6 and S1 in the extent of coexistence of these peptides. Further studies are necessary to determine if coexistence of opioids is limited only to autonomic regions of the nervous system and also to define the functional significance of this coexistence.

An antiserum to locust adipokinetic hormone reveals a novel peptidergic system in the rat central nervous system.

Using an antiserum to locust adipokinetic hormone I, a novel peptidergic system was identified in the rat central nervous system. Immunoreactive fibers were present in the hypothalamic median eminence and periventricular nucleus and the spinal cord dorsal horn, intermediolateral cell column and sacral parasympathetic nucleus. Immunoreactive cells were present in the dorsal gray commissure of lumbosacral spinal cord, the hypothalamic periventricular nucleus and cerebral cortex.

Distribution of neuropeptide Y-like immunoreactivity and its relationship to FMRF-amide-like immunoreactivity in the sixth lumbar and first sacral spinal cord segments of the rat.

The present study was aimed at describing the distribution of neuropeptide Y (NPY)-like immunoreactivity in the sixth lumbar (L6) and first sacral segments (S1) of the rat spinal cord, comparing this distribution to that of FMRF-amide-like immunoreactivity and determining whether NPY- and FMRF-amide-like immunoreactivities are present in the same neurons in the dorsal gray commissure (DGC) in L6 and S1 of the rat spinal cord. For distribution studies tissue from colchicine-treated animals was processed according to the peroxidase-antiperoxidase technique using anti-NPY as the primary antiserum. For co-localization studies serial 5-micron sections were processed for immunofluorescence. Adjacent sections were incubated with either anti-NPY or anti-FMRF-amide as the primary antiserum. The number of immunoreactive cells per section was counted and each section was photographed. The sections were then restained with the other antiserum (i.e., tissue first stained with anti-NPY was stained with anti-FMRF-amide and vice versa), the number of cells per section was recounted, and the sections were rephotographed. NPY-like immunoreactive cells and fibers were identified in the DGC, sacral parasympathetic nucleus, substantia gelatinosa, marginal zone, nucleus proprius, and ventral horn. Every cell in the DGC that contained NPY-like immunoreactivity was found also to contain FMRF-amide-like immunoreactivity, and the distribution of NPY-like immunoreactive fibers was found to be similar, although denser than FMRF-amide-like immunoreactive fibers. The distribution of NPY-like immunoreactivity in L6 and S1 of the rat spinal cord suggests that an NPY-like peptide may be involved in regulation of pelvic viscera, processing of primary afferent information, and motor regulation of pelvic muscles. The presence of NPY- and FMRF-amide-like immunoreactivities in the same neurons in the DGC together with the lack of bona fide FMRF-amide in the rat central nervous system, the presence of NPY in the rat central nervous system, and the cross-reactivity of anti-FMRF-amide with NPY support the hypothesis that the FMRF-amide antiserum recognizes an NPY-like peptide in the rat spinal cord.

Phe-met-arg-phe-amide (FMRF-NH2) inhibits insulin and somatostatin secretion and anti-FMRF-NH2 sera detects pancreatic polypeptide cells in the rat islet.

FMRF-NH2-like immunoreactivity was localized in the pancreatic polypeptide containing cells of the rat islet. FMRF-NH2 was investigated with regard to its effect on insulin, somatostatin and glucagon secretion from the isolated perfused rat pancreas. FMRF-NH2 (1 microM) significantly inhibited glucose stimulated (300 mg/dl) insulin release (p less than 0.005) and somatostatin release (p less than 0.01) from the isolated perfused pancreas. FMRF-NH2 (1 and 10 microM) was without effect on glucagon secretion, either in low glucose (50 mg/dl), high glucose (300 mg/dl), or during arginine stimulation (5 mM). These findings indicate that these FMRF-NH2 antisera recognize a substance in the pancreatic polypeptide cells of the islet which may be capable of modulating islet beta and D cell activity.

The immunohistochemical localization of nine peptides in the sacral parasympathetic nucleus and the dorsal gray commissure in rat spinal cord.

The sixth lumbar and first sacral spinal cord segments in the rat contain parasympathetic preganglionic neurons which innervate the pelvic viscera. There have been few studies, however, which have specifically considered the distribution of putative peptide neurotransmitters in these cord segments. The present paper describes and compares the immunohistochemical distribution of dynorphin (1-8)-, enkephalin-, somatostatin-, cholecystokinin octapeptide-, avian pancreatic polypeptide-, FMRF-NH2-, neurotensin-, and vasoactive intestinal polypeptide-like immunoreactivities in the dorsal gray commissure and sacral parasympathetic nucleus of the sixth lumbar and first sacral spinal cord segments in colchicine-treated rats. Antisera against all of the peptides, except avian pancreatic polypeptide, stained cells in the sacral parasympathetic nucleus. Dynorphin (1-8-), enkephalin-, and substance P-like immunoreactive cells were present in significantly greater numbers than somatostatin-, neurotensin-, cholecystokinin-, FMRF-NH2-, and vasoactive intestinal polypeptide-like immunoreactive cells. All of the antisera also stained fibers in the sacral parasympathetic nucleus in varying densities, and a fiber bundle which extended between the dorsal gray commissure and the sacral parasympathetic nucleus. Antisera against substance P and cholecystokinin stained a bundle of fibers that extended between the dorsal horn and the sacral parasympathetic nucleus. Antisera against somatostatin, cholecystokinin octapeptide, substance P and FMRF-NH2 stained an additional fiber bundle which extended between the lateral edge of the dorsal horn and the dorsal gray commissure. All the remaining antisera, except neurotensin, also stained fibers that extended between the sacral parasympathetic nucleus and the dorsal gray commissure, but in a sparser distribution. Immunoreactive cells were localized to the dorsal gray commissure in sections stained with each of the antisera. Dynorphin (1-8) and enkephalin antisera stained the greatest number of cells, followed by FMRF-NH2, neurotensin, somatostatin and avian pancreatic polypeptide. The smallest number of immunoreactive cells was present in substance P, cholecystokinin and vasoactive intestinal polypeptide immunostained sections. A significant difference was noted between the number of dynorphin, enkephalin, somatostatin, cholecystokinin, avian pancreatic polypeptide, FMRF-NH2, neurotensin and vasoactive intestinal polypeptide immunoreactive cells in the sacral parasympathetic nucleus and dorsal gray commissure.(ABSTRACT TRUNCATED AT 400 WORDS)