Opiocortin and catecholamine projections to raphe nuclei.

Afferent projections to the nucleus raphe magnus (NRM) and dorsal raphe nucleus (DRN) were identified using retrograde transport of horseradish peroxidase conjugated wheat germ agglutinin (HRP-WGA). Neurons were labeled in important nociceptive regions including periaqueductal gray (PAG), arcuate nucleus, lateral hypothalamus and medial thalamic nuclei following both injections. We have immunocytochemically identified opiocortin/WGA neurons in the arcuate nucleus following NRM and DRN injections. Dual stained catecholamine/WGA perikarya were found in zona incerta, locus coeruleus, substantia nigra, nucleus tractus solitarius and adjacent A2, C2 and C3, lateral paragigantocellular reticular nucleus/C1 and lateral reticular nucleus/A1 following DRN injections and in zona incerta, substantia nigra, nucleus tractus solitarius/A2 and lateral reticular nucleus/A1 after NRM injections. These results provide further evidence for opiocortin and catecholamine modulation of analgesia.

Light and electron microscopic immunocytochemical analysis of the dopamine innervation of the rat visual cortex.

The dopaminergic innervation of the rat primary (area 17) and secondary (areas 18 and 18a) visual cortical areas was examined immunocytochemically using an antiserum directed against dopamine. This innervation was characterized by the differential density of the respective afferents within individual visual areas. Area 18, especially its rostral part, was observed to receive a considerable amount of dopaminergic axons, whereas areas 17 and 18a were sparsely innervated. The innervation of all layers of area 18 seemed to consist to a considerable extent of axonal branches of radial fibres ascending from layer VI to layer I. At the ultrastructural level, dopamine profiles were found to display similar characteristics in all visual areas. Dopamine labelled axon-terminals and axonal varicosities, examined in single and serial ultrathin sections, were seen to form primarily asymmetrical synaptic contacts with dendritic profiles. These observations suggest a 'specific' innervation of cytoarchitectonically distinct cortical regions by dopaminergic axons.

Observations on the projection from the perihypoglossal nuclei to the cerebellar cortex and nuclei in the cat. A retrograde WGA-HRP and fluorescent tracer study.

The origin and distribution of cerebellar cortical and nuclear afferents from the perihypoglossal nuclei have been studied by means of retrograde transport after implants and injections of the wheat germ agglutinin-horseradish peroxidase complex in the cat. The projection reaches all the cerebellar nuclei as well as vermal, intermediate and lateral parts of the cerebellar cortex. It is bilateral with an ipsilateral predominance and originates from all the perihypoglossal nuclei. The majority of the projecting neurons are situated caudally in the nucleus prepositus, while smaller numbers of projecting neurons are located in the rostral part of this nucleus, in the rostral nucleus intercalatus and in the nucleus of Roller. Small and medium-sized spindle-shaped to round cells located throughout the nucleus prepositus and in the rostral nucleus intercalatus have widespread projections, reaching all parts of the cerebellar cortex and nuclei, whereas large multipolar cells located in the caudal ventromedial part of the nucleus prepositus and in the nucleus of Roller have projections only to the flocculus and nodulus and the lateral and intermediate cortices. Retrograde fluorescent double-labelling experiments were made to investigate possible axonal branching of the perihypoglosso-cerebellar fibres. In experiments with injections of rhodamine-B-isothiocyanate (RITC) in the left cerebellar hemisphere and implants of crystalline Fluoro-Gold in the right hemisphere, single- and double-labelled cells were found intermingled throughout the perihypoglossal nuclei. Experiments with cerebellar cortical injections of RITC and implants of crystalline Fluoro-Gold in the underlying nucleus, demonstrated single- and double-labelled cells in the nucleus prepositus and the rostral nucleus intercalatus, while only single-labelled RITC neurons were seen in the group of large neurons in the ventromedial part of the nucleus prepositus and the nucleus of Roller. After injections of RITC in the cerebellar cortex and implants of crystalline Fluoro-Gold in the abducent nucleus on the same side, double-labelled neurons were found only in the rostral nucleus prepositus.

Substance P-like immunoreactivity in the trigeminal sensory nuclei of an infrared-sensitive snake, Agkistrodon blomhoffi.

With the peroxidase-antiperoxidase immunohistochemical method we ascertained the presence of substance P-like immunoreactivity (SPLI) in fibers and cell bodies of the trigeminal sensory system of the pit viper, Agkistrodon blomhoffi. There are a few SPLI fibers each in the principal sensory nucleus and the main neuropil of the lateral descending nucleus (i.e., the infrared sensory nucleus); a moderate number in the descending nucleus; and a large number in the caudal subnucleus, the medial edges of the interpolar subnucleus, and the marginal neuropil of the lateral descending nucleus. About 30% of the cell bodies in the ophthalmic and maxillo-mandibular ganglia show SPLI, and of the two craniocervical ganglia, the proximal ganglion has many more cells with SPLI than the distal ganglion. The SPLI distribution in the common trigeminal sensory system is similar to that of mammals, and suggests that the function of this system is also similar. In the infrared that the function of this system is also similar. In the infrared sensory system, the differing distribution in the main and marginal neuropils suggests separate functions for these two structures in the system.

Characterization of the peptidergic afferent innervation of the stomach in the rat, mouse and guinea-pig.

Retrograde tracing of the fluorescent marker, True Blue, has been used together with immunohistochemistry employing antibodies to substance P, calcitonin gene-related peptide, somatostatin, vasoactive intestinal polypeptide and morphine-modulating peptide to study the afferent innervation of the stomach in rat, mouse and guinea-pig. Up to 85% of spinal afferents to the stomach in all three species contained immunoreactive calcitonin gene-related peptide, and up to 50% contained substance P. In all three species less than 10% of vagal afferents to the stomach reacted with antibodies to calcitonin gene-related peptide, or substance P. Cacitonin gene-related peptide-immunoreactive fibres were found in the myenteric plexus, circular muscle and around submucosal blood vessels in the stomach. In the rat, removal of the coeliac ganglion, splanchnic nerve section, or capsaicin treatment virtually abolished calcitonin gene-related peptide immunoreactivity in the stomach. Capsaicin and splanchnic section also abolished the staining of immunoreactive calcitonin gene-related peptide fibres in the coeliac ganglion. The same treatments abolished substance P staining of fibres around submucosal blood vessels, but in the myenteric plexus and circular smooth muscle there were still abundant immunoreactive fibres, presumably arising from intrinsic cell bodies. No somatostatin-containing visceral afferents could be found, although somatostatin was localized to cell bodies in rat dorsal root ganglia. Immunoreactive vasoactive intestinal polypeptide-containing dorsal root ganglia neurons were not found; although antibodies to morphine-modulatory peptide revealed immunoreactive nerve cell bodies, we were unable to exclude the possibility that this result is attributable to cross reactivity with calcitonin gene-related peptide. These results provide direct evidence that calcitonin gene-related peptide is a marker for a major subset of visceral primary afferent neurons and suggest that this population of spinal afferents makes a major contribution to the total gastric content of calcitonin gene-related peptide.

Calcitonin gene-related peptide-immunoreactive nerve fibers in mandibular periosteum of rat: evidence for primary afferent origin.

Peptidergic neurons may play a role in the local regulation of bone mineralization. The neuropeptide vasoactive intestinal peptide (VIP) increases bone resorption in vitro, while calcitonin gene-related peptide (CGRP) has been shown to inhibit bone resorption in vitro. We have previously reported that sympathetic nerves with VIP-immunoreactivity innervate bone and periosteum. In the present study we sought to determine if CGRP fibers, like VIP fibers, exist in periosteum and what their origin might be. In whole-mount preparations of mandibular periosteum from rat, CGRP- and VIP-immunoreactive (IR) nerve fibers were present as networks within the periosteum. In preparations using two-color immunofluorescence, most CGRP-IR fibers were also immunoreactive for substance P (SP). In rats in which the subperiosteal space subjacent to the mandibular molars was injected with Fast blue or Fluoro-gold, retrogradely labeled cells were seen in ipsilateral trigeminal ganglia, superior cervical ganglia, and nodose ganglia. Individual cells labeled with both CGRP immunoreactivity and retrograde tracer were seen only in the mandibular portion of the trigeminal ganglion. These data suggest that CGRP-IR nerve fibers in periosteum may be of primary afferent origin. Given the reported effects of CGRP on bone mineralization, the present results suggest that primary afferent nerves containing CGRP and SP, as well as sympathetic nerves containing VIP, may play a role in focal bone remodeling.

Nuclear origin of thalamic afferents of the ventral striatum determines their relation to patch/matrix configurations in enkephalin-immunoreactivity in the rat.

The relation of thalamic terminal fibers in the ventral striatum with patch and matrix compartments, as defined by enkephalin-immunohistochemistry, was studied in adjacent or double-stained sections of the rat brain by combining anterograde tracing of Phaseolus vulgaris-leucoagglutinin (PHA-L) and enkephalin staining. Experiments with small PHA-L injections in the dorsal thalamus show that the paraventricular thalamic nucleus projects to the medial nucleus accumbens and rather selectively to the patch compartment in more lateral parts of the nucleus. The central medial thalamic nucleus sends its fibers to more lateral parts of the nucleus accumbens and the ventral part of the caudate-putamen, where these fibers terminate in the matrix compartment.

neuropeptides in long ascending spinal tract cells in the rat: evidence for parallel processing of ascending information.

A study has been made of the involvement of spinal peptidergic neurons in ascending tracts at lumbar-sacral levels in rats, by combining the retrograde transport of a protein-gold complex with immunocytochemistry. Ten neuropeptides have been considered for their presence in the cells of origin of the following six ascending tracts, including some involved in pain transmission: the spinosolitary tract, the medial and lateral spinoreticular tracts, the spinomesencephalic tract, the spinothalamic tract and the postsynaptic dorsal column tract. Although there was overlap in the distribution of several of the types of peptidergic cells and some ascending tract cells only a very small percentage of long ascending tract cells were found to contain neuropeptides. Most (90%) of those peptidergic ascending tract cells, however, were clearly congregated in two distinct spinal regions: the lateral spinal nucleus and the region surrounding the central canal (including lamina X). Ascending tract cells in both of these regions contained a wide variety of neuropeptides. Immunoreactivities for a total of seven different peptides were seen. The lateral spinal nucleus had the highest percentage of neuropeptide containing ascending tract cells; cells of all the four populations of peptidergic neurons lying in this region were involved in supraspinal projections; they stained for vasoactive intestinal polypeptide, bombesin, substance P or dynorphin and their axons projected in the spinomesencephalic, spinoreticular and spinosolitary tracts. The region surrounding the central canal contained bombesin-, enkephalin-, cholecystokinin- and somatostatin-immunoreactive ascending tract cells; these cells were found at the origin of the spinothalamic, spinomesencephalic, spinoreticular and spinosolitary tracts. In this region only the cells staining for substance P were not involved in supraspinal projections. The peptidergic ascending tract cells in other spinal regions were few; they were found in either lamina I or lateral part of lamina V. Ascending tract lamina I cells reacted for dynorphin or vasoactive intestinal polypeptide and their axons projected in the spinosolitary and spinomesencephalic tracts. Ascending tract lamina V cells reacted for somatostatin and were found at the origin of the medial component of the spinoreticular tract. It is proposed that peptidergic ascending tract cells form minor but distinct subgroups within each ascending tract. Each of the ascending tracts are divisible into peptide- and nonpeptide-containing groups of cells which convey information in a parallel fashion.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunocytochemical identification of long ascending peptidergic neurons contributing to the spinoreticular tract in the rat.

In the present study, we examined the peptidergic content of lumbar spinoreticular tract neurons in the colchicine-treated rat. This was accomplished by combining the retrograde transport of the fluorescent dye True Blue with the immunocytochemical labeling of neurons containing cholecystokinin-8, dynorphin A1-8, somatostatin, substance P or vasoactive intestinal polypeptide. After True Blue injections into the caudal bulbar reticular formation, separate populations of retrogradely labeled cells were identified as containing cholecystokinin-like, dynorphin-like, substance P-like or vasoactive intestinal polypeptide-like immunoreactivity. Retrogradely labeled somatostatin-like neurons were not identified in any of the animals examined. Each population of double-labeled cells showed a different distribution in the lumbar spinal cord. The highest yield of double-labeling occurred for cholecystokinin, with 16% of all intrinsic cholecystokinin-like neurons containing True Blue. These double labeled neurons were found predominantly at the border between lamina VII and the central canal region. About 11% of intrinsic vasoactive intestinal polypeptide-like neurons in the lumbar spinal cord were retrogradely labeled from the bulbar reticular formation. These neurons were found mostly in the lateral spinal nucleus, with only a few double-labeled cells located deep in the gray matter. Dynorphin-like double-labeled neurons were localized predominantly near the central canal; a smaller population was also seen in the lateral spinal nucleus. It was found that double-labeled dynorphin-like neurons made up 8% of all intrinsic dynorphin-like neurons. Retrogradely-labeled substance P-like neurons were rare; the few double-labeled neurons were found in the lateral spinal nucleus and lateral lamina V. These findings suggest a significant role for spinal cord peptides in long ascending systems beyond their involvement in local circuit physiology.

Projections of bombesin-like immunoreactive fibers from the rat stomach to the celiac ganglion revealed by a double-labeling technique.

Gastrofugal bombesin (BOM)-like immunoreactive (BOMI) structures in the rat were studied by immunocytochemistry combined with retrograde labeling. Transection of the mesenteric nerve peripheral to the celiac ganglion resulted in the complete disappearance of BOMI nerve terminals, whereas transection of the splanchnic nerves did not alter the immunoreactivity. Injection of biotinylated wheat germ agglutinin into the celiac ganglion labeled several neurons in the myenteric ganglion of the stomach. Simultaneous staining with antiserum against BOM showed that some of them are BOMI-positive. These findings demonstrate that BOMI neurons in the myenteric ganglion of the rat stomach project to the celiac ganglion.

Comparative distribution and cardiovascular actions of substance P and substance K within the nucleus tractus solitarius of rats.

An investigation has been made into the comparative distribution and cardiovascular actions of Substance P (SP) and Substance K (SK) in the nucleus tractus solitarius (NTS) of rats. The two tachykinins were similarly distributed in the NTS region, as determined by radioimmunoassay of microdissected brain nuclei, although SP was present in an approximately 10 fold higher concentration than SK. Unilateral nodose ganglionectomy resulted in a non-significant reduction of tachykinin immunoreactivity in the ipsilateral NTS. Microinjection of synthetic SP into the NTS of urethane anaesthetised rats did not cause any significant changes in mean arterial pressure or heart rate. In contrast, SK (18 pmol) elicited a significant bradycardia which was maximal at 2 min after the injection. The bradycardic response was blocked in rats pre-treated with atropine. These results extend previous studies in which contrasting functional activities of SP and SK were demonstrated in peripheral systems, to indicate that tachykinins may exhibit different functional roles in the brain. SK, unlike SP, may be involved in central cardiovascular control via an action within the NTS.

Galanin-like immunoreactivity in hippocampal afferents in the rat, with special reference to cholinergic and noradrenergic inputs.

The distribution of galanin-like immunoreactivity in the rat hippocampal formation (hippocampus and dentate gyrus) was studied and its origins were determined using various lesioning techniques. Special reference was made to the known cholinergic and noradrenergic hippocampal inputs from the septum-basal forebrain complex and locus coeruleus, both of which have previously been shown to co-contain galanin-like immunoreactivity at the cell body level. Galanin-immunoreactive fibers in the hippocampal formation were of at least three different morphological types: (1) Fine, slender, faintly immunoreactive fibers were seen throughout the hippocampal formation. (2) A strongly fluorescent varicose fiber population was observed mainly in the strata radiatum and oriens of the ventral CA3 region. (3) A population of fine, faint puncta was seen within the granule and pyramidal cell layers throughout the hippocampal formation. Knife cut lesions of the dorsal afferent pathways resulted in almost complete disappearance of all fiber types, except for the ventral fine fibers. Lesions of the fimbria affected mainly the coarse and punctate fiber types, while lesions of the supracallosal striae depleted mainly the fine fibers. Cuts anterior and ventral to the hippocampal formation caused a decrease in ventral fine fibers. Furthermore, lesions of the dorsal bundle caused an almost complete disappearance of the fine fibers in all regions of the hippocampal formation. Neurotoxin lesions of the diagonal band/septal complex resulted in decreases in faintly immunoreactive puncta within the granule cell layer and adjacent fine fibers. It is concluded that most fine galanin-positive fibers originate in the lower brain stem, presumably the locus coeruleus, and appear to reach the hippocampal formation primarily through the supracallosal striae and the ventral route. The fimbria seems to contain a large proportion of the fibers giving rise to the coarse strongly fluorescent innervation, which appears to originate rostral to the pons. The galanin-immunoreactive fibers originating in cholinergic somata of the diagonal band, medial septal nuclei, previously shown to project to the hippocampal formation, seem to give rise to faintly labeled puncta within the granule and pyramidal cell layers, and to a small proportion of the fine fibers bordering the cell layers, as revealed by immunohistochemistry using our antibody.(ABSTRACT TRUNCATED AT 400 WORDS)

Certain basilar pontine afferent systems are GABA-ergic: combined HRP and immunocytochemical studies in the rat.

Injection of the tracer substance wheat germ agglutinin-horseradish peroxidase (WGA-HRP) directly into the basilar pontine nuclei using a ventral surgical approach resulted in the labeling of somata in many areas both rostral and caudal to the basilar pons. Certain of the sections that had been reacted for HRP were also incubated in antiserum prepared against glutamic acid decarboxylase (GAD) and processed according to routine peroxidase anti-peroxidase immunocytochemical procedures. Neuronal somata exhibiting both HRP and GAD reaction products were considered to represent GABA-ergic neurons that provide axonal projections to the basilar pontine nuclei. Such double-labeled neurons were observed within the zona incerta, anterior pretectal nucleus, lateral cerebellar nucleus, perirubral area, and the pontine and medullary reticular formation.

A survey of MabQ113 immunoreactivity in the adult rat brain: differential staining of the lateral and medial habenular nuclei.

Monoclonal antibody mabQ113 recognizes a polypeptide antigen which, in rat cerebellum, is confined exclusively to a subset of Purkinje cells which are organized into parasagittal bands. In this report we have explored the distribution of mabQ113 immunoreactivity in some other regions of the rat brain. The most interesting result was a dramatic differential staining of the habenular complex in which mabQ113 densely and uniformly stained the lateral habenula but did not stain the medial habenula. Within the lateral habenula reaction product is localized primarily in the cellular processes of astrocytes but there is also staining of neighboring neuronal dendritic and axonal profiles. The afferent and efferent tracts of the habenular nuclei are not immunoreactive and there was no systematic difference in staining between the afferent and efferent nuclear groups of the two habenular nuclei. The pattern of mabQ113 immunoreactivity in rat brain is distinct from previously described biochemical differentiation markers of the two nuclei and thus may serve as a useful probe to study habenular anatomy, development and function.

A long ascending pathway of enkephalin-like immunoreactive spinoreticular neurons in the rat.

In the present study, we examined whether some long ascending spinal cord neurons contain enkephalin by combining the retrograde transport of the fluorescent dye True Blue with enkephalin immunocytochemistry. Evidence is presented for the existence of enkephalin in a subpopulation of spinoreticular neurons in the rat located in the central canal region and adjacent gray matter.

The sympathetic and sensory components of the caudal lumbar sympathetic trunk in the cat.

The cell bodies of the lumbar sensory and sympathetic pre- and postganglionic neurons that project in the caudal lumbar sympathetic trunk of the cat have been labeled retrogradely with horseradish peroxidase applied to the central end of their cut axons. The application was made just proximal to the segmental ganglion that sends its gray rami to the L7 spinal nerve, and so identified the sympathetic outflow concerned primarily with the vasculature of the hindlimb and tail. The numbers, segmental distribution, location, and size of the labeled somata have been determined quantitatively. Labeled cell bodies were found ipsilaterally, but the segmental distributions of the different cell types were not matched. Afferent cell bodies lay in dorsal root ganglia L1-L5 (maximum L4), preganglionic cell bodies in spinal segments T10-L5 (maximum L2/3), and postganglionic cell bodies in ganglia L2-L5 (maximum L5). Both numbers and dimensions of labeled dorsal root ganglion cells were variable between experiments (maximum about 1,000); the majority were small relative to the entire population of sensory neurons. Labeled preganglionic cell bodies were located right across the intermediate region of the spinal cord, extending from the lateral part of the dorsolateral funiculus to the central canal. The highest density of labeled neurons lay at the border between the white and gray matter (corresponding to the intermediolateral cell column) with smaller proportions medially in L1-L2, and laterally in caudal L4-L5. Medial preganglionic neurons were generally larger than those lying in lateral positions. From the data, it is estimated that about 650 afferent, about 4,500 preganglionic, and some 2,500 postganglionic neurons project in each lumbar sympathetic trunk distal to the ganglion L5 in the cat.

Immunochemical studies of substance P and cholecystokinin octapeptide recovery in dorsal horn following unilateral lumbosacral ganglionectomy.

Deafferentation of the cat lumbosacral dorsal horn following unilateral ganglionectomy (L2-S3) produced an ipsilateral depletion of substance P (sP) and cholecystokinin octapeptide (CCK) immunoreactivity (IR). The loss of sP IR and CCK IR was most marked at 11 days postlesion, and partial recovery of sP IR and CCK IR was noted in the upper laminae of the dorsal horn ipsilateral to the lesion 14 days after deafferentation. The dorsal horn sP IR and CCK IR distribution and density of staining on the deafferented side were indistinguishable from those on the control side 1 month after ganglionectomy. The pattern and density of methionine-enkephalin (M-ENK) IR was not disrupted by deafferentation, demonstrating that the sP IR and CCK IR depletions were due to the deafferentation and not to nonselective vascular damage. The recovery of sP IR and CCK IR was not affected by midline myelotomy or thoracic cord transections, implying a local origin for the recovered sP IR and CCK IR. Moreover, capsaicin, a primary afferent neurotoxin, depleted sP IR and CCK IR only from the intact side. Thus, these studies indicate that the recovery of two immunochemically identified primary afferent transmitters in the dorsal horn does not involve intraspinal sprouting of primary afferents. Radioimmunoassay (RIA) analysis of the ganglionectomies followed by recovery periods ranging from 7 to 28 days confirmed the depletion and recovery of sP IR, but the RIA indicated a temporal shift in depletion and recovery. Maximal depletion was measured at 21 days and recovery was observed at 28 days after unilateral deafferentation.

Afferents to the lateral reticular nucleus from the oculomotor region. I. The Edinger-Westphal nucleus.

By means of retrograde axonal transport of the wheat germ agglutinin-horseradish peroxidase complex, a projection from the Edinger-Westphal nucleus to the lateral reticular nucleus was demonstrated in the cat. Following small tracer ejections in the main part of the lateral reticular nucleus, a significant number of labelled neurons were found bilaterally throughout the Edinger-Westphal nucleus. Most of the labelled cells were located on the ipsilateral side. The projecting neurons are spindle-shaped to round with a maximum diameter of the cell body between 15 and 30 microns. The findings are discussed in relation to other Edinger-Westphal efferent projections, and some comments are made concerning the cytoarchitecture and delineation of the feline Edinger-Westphal nucleus.

Direct projections to the rat pineal gland via the stria medullaris thalami. An anterograde tracing study by use of horseradish peroxidase.

The possible presence of a direct nervous projection from the paraventricular nucleus (PVN) of the hypothalamus to the pineal gland of the rat was investigated by means of the anterograde neuron-tracing method using horseradish peroxidase. The tracer was injected unilaterally into the PVN and the animals were allowed to survive between 12 and 26 h. Numerous peroxidase-positive fibers were observed, ipsilateral to the injection site, in the stria medullaris thalami and could be followed into the medial habenular nucleus and the habenular commissure. From there, fibers penetrated into the deep pineal gland (lamina intercalaris), and further into the pineal stalk. These data support results of previous investigations describing retrograde labeling of the PVN following intrapineal injections of horseradish peroxidase and are in accordance with recent experiments demonstrating an influence of the PVN on electrical and biochemical activity of the pineal gland.