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.

Vasoactive intestinal polypeptide and substance P in primary afferent pathways to the sacral spinal cord of the cat.

An analysis of vasoactive intestinal polypeptide immunoreactivity (VIP-IR) and substance P-IR in the cat spinal cord has revealed marked differences in the distribution of the two peptides. While substance P-IR was located at all levels of the cord, VIP-IR was most prominent in the sacral segments in Lissauer's tract and lamina I on the lateral edge of the dorsal horn. VIP-IR was also present in the sacral cord in (1) laminae V, VII, and X, (2) a thin band on the medial side of the dorsal horn, (3) the dorsal commissure, (4) the lateral band of the sacral parasympathetic nucleus, and (5) in a few animals in Onuf's nucleus. In other segments of the spinal cord VIP-IR was much less prominent but was present in Lissauer's tract and laminae I, II, and X. Substance P-IR was more uniformly distributed at all segmental levels in laminae I-III, V, VII, and X and in the dorsal commissure. In ventrolateral lamina I of the sacral spinal cord both VIP-IR and substance P-IR exhibited a distinctive periodic pattern in the rostrocaudal axis. The peptides were associated with bundles of dorsoventrally oriented axons and varicosities spaced at approximately 210-micron intervals center to center along the length of the spinal cord. The bundles in lamina I continued into lamina V where they further divided into smaller bundles that extended medially through laminae V and VII. The most prominent bundles of VIP axons passed ventrally from lateral laminae V and VII to enter lamina X and the ventral part of the dorsal gray commissure. On the other hand the majority of substance P axons in lamina V turned dorsally to join with axons on the medial side of the dorsal horn and to pass into the dorsal part of the dorsal gray commissure. Rostrocaudal VIP axons were present not only in Lissauer's tract but also in dorsolateral lamina I, in the lateral funiculus and in the ependymal cell layer of the central canal. Following unilateral transection of the sacral dorsal roots (2 weeks-22 months) the density of VIP axons and terminals was markedly reduced in ipsilateral Lissauer's tract and lateral laminae I and V; however, no change was detected in lamina X. Sacral deafferentation reduced substance P-IR in the dorsal gray commissure and in lateral laminae I and V.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunohistochemical study of subclasses of olfactory nerve fibers and their projections to the olfactory bulb in the rabbit.

The organization of the olfactory nerve projection to the olfactory bulb was studied immunohistochemically in the rabbit by using monoclonal antibodies (MAbs). Out of 42 MAbs raised against the homogenate of the olfactory bulb, two types of MAbs that strongly stained the olfactory nerve fibers (axons of olfactory receptor cells) were selected and their staining patterns were analysed in detail. MAbs of one type (represented by MAb R2D5) specifically labeled all olfactory receptor cells in the nasal epithelium and all olfactory nerve fibers and their terminal portions in the bulb. The other type of MAbs (represented by MAb R4B12) recognized only a subgroup of olfactory nerve fibers. The R4B12-positive fibers were distributed over the ventrolateral areas but not in the dorsomedial areas of the epithelium. Similarly in the bulb, the R4B12-positive fibers terminated in the glomeruli in the ventrolateral and the caudal regions but not in the dorsomedial region. These results demonstrate for the first time the cellular heterogeneity among olfactory receptor neurons at the molecular level. The segregated distribution of the subtypes of olfactory receptor cell axons both in the epithelium and the bulb indicates a defined topographical organization of the olfactory nerve projection. These results also suggest a functional division between dorsomedial and ventrolateral areas both in the epithelium and the bulb.

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)

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.

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)

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.

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.

Studies on cholecystokinin-containing neuronal pathways in rat cerebral cortex and striatum.

Lesion experiments were performed to investigate the origin of CCK-containing afferents of the striatum. All the subdivisions of the striatum that were investigated seem to receive CCK afferents from dorsolateral and lateral neocortical areas. However, destruction of these cortical areas alone did not reduce CCK-IRC in the striatum. Only after an additional parasagittal severance of the corpus callosum were significant decreases in CCK-IRC of all striatal subdivisions observed. Thus, CCK neurons in ipsilateral midline areas (such as the cingulate cortex) or, more likely, in contralateral cortical areas, seem to project to the striatum of one side. The CCK fibers seem to enter the striatum via the capsula externa, since a lesion of this structure has been shown to diminish the CCK-IRC in the striatum. In addition, the dorsomedial part of the head of the striatum may receive a projection of CCK fibers from the anterior cingulate area. A series of lesions which severed the afferents of structures caudal to the striatum, that is, the amygdaloid complex and the ventral tegmental area plus substantia nigra, did not reduce CCK-IRC in the striatum. Some of these lesions even significantly enhanced CCK-IRC in several subdivisions of the ipsilateral and contralateral striatum. Further studies will be necessary to cast some light on these caudal CCK afferents to the striatum, which are obviously extremely complex.

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.

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.

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.

Chemical characterization of substance P-like immunoreactivity in primary afferent neurones.

Substance P-like immunoreactivity (SPLI) in rat dorsal root ganglia and dorsal spinal cord was characterized by high-performance liquid chromatography followed by radioimmunoassay. In spinal cord and ganglia, respectively, 87% and 64% of SPLI eluted with authentic SP. The remainder of the SPLI in ganglia eluted as a single peak which did not represent the sulphoxide of SP or any of its C-terminal fragments.

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.

Muscarine-binding sites localized to cortical dopamine terminals.

The effects of lesions to the mesocortical dopaminergic system on D2 dopamine receptors and muscarine receptors in the frontal cortex of the rat was examined. Four weeks following 6-hydroxydopamine lesioning of the ventral tegmental area, there was a 26% increase in the number of [3H]spiroperidol sites, and a 13% decrease in the number of [3H]oxotremorine-M sites in the frontal cortex, indicating a development of D2 receptor supersensitivity, as a result of deafferentation, and a loss of acetylcholine sites, as result of terminal degeneration. This demonstrates that in the frontal cortex, as in the striatum and nucleus accumbens, the activity of dopaminergic terminals may be partially modulated by cholinergic inputs.

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.

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 quantitative analysis of the afferent and extrinsic efferent innervation of specific regions of the bladder and urethra in the cat.

Using the fluorescent tracer dyes bisbenzimide, nuclear yellow and fast blue, the possibility of differential innervation of various regions of the bladder and urethra was tested in cats. The dyes were injected into the lateral detrusor, bladder base, and urethra. Fluorescent cell bodies were counted in serial 48 micron sections of dorsal root, inferior mesenteric, sympathetic chain and pelvic plexus ganglia. Several dorsal root ganglia, primarily S2, were the principal source of afferent innervation to all locations injected. The bladder and urethra received significant efferent innervation from both the inferior mesenteric ganglia and sympathetic chain ganglia (particularly L7 to S2) however, pelvic plexus ganglia made only small contribution to the innervation of these areas. The sympathetic chain and inferior mesenteric ganglia contributed equally to the innervation of the detrusor and bladder base but the sympathetic chain made double the contribution of the inferior mesenteric ganglia to the innervation of the urethra. There was a very low incidence (less than 1%) of neurons which projected to more than one injection site.

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.