Vagal afferent innervation of smooth muscle in the stomach and duodenum of the mouse: morphology and topography

Intraganglionic laminar endings (IGLEs) and intramuscular arrays (IMAs), the two putative mechanoreceptors that the vagus nerve supplies to the gastrointestinal smooth muscle, have been characterized almost exclusively in the rat. To provide normative inventories of these afferents for the mouse, the authors examined the endings in the stomach and small intestine of three strains used as backgrounds for gene manipulations (i.e., C57, 129/SvJ, and WBB6). Animals received nodose ganglion injections of wheat germ agglutinin-horseradish peroxidase or dextran-tetramethylrhodamine conjugated to biotin. The horseradish peroxidase tissue was processed with tetramethylbenzidine and was used to map the distributions and densities of the two endings; the dextran material was counterstained with c-Kit immunohistochemistry to assess interactions between intramuscular arrays and interstitial cells of Cajal. IGLEs and IMAs constituted the vagal innervation of mouse gastric and duodenal smooth muscle. IGLE morphology and distributions, with peak densities in the corpus-antrum, were similar in the three strains of mice and comparable to those observed in rats. IMAs varied in complexity from region to region but tended to be simpler (fewer telodendria) in mice than in rats. IMAs were most concentrated in the forestomach and sphincters in mice, as in rats, but the topographic distributions of the endings varied both between strains of mice (subtly) and between species (more dramatically). IMAs appeared to make appositions with both interstitial cells and smooth muscle fibers. This survey should make it practical to assay the effects of genetic (e.g., knockout) and experimental (e.g., regeneration) manipulations affecting visceral afferents and their target tissues.     

Vagal afferent innervation of the atria of the rat heart reconstructed with confocal microscopy

We have used confocal microscopy to analyze the vagal afferent innervation of the rat heart. Afferents were labeled by injecting 1,1'-dioleyl-3,3,3',3'-tetramethylindocarbocyanine methanesulfonate (DiI) into the nodose ganglia of animals with prior supranodose de-efferentations, autonomic ganglia were stained with Fluoro-gold, and tissues were examined in whole mounts. Distinctively different fiber specializations were observed in the epi-, myo-, and endocardium: Afferents to the epicardium formed complexes associated with cardiac ganglia. These ganglia consisted of four major ganglionated plexuses, two on each atrium, at junctions of the major vessels with the atria. Ganglionic locations and sizes (left > right) were consistent across animals. In addition to principal neurons (PNs), significant numbers of small intensely fluorescent (SIF) cells were located in each of these plexuses, and vagal afferents provided dense pericellular varicose endings around the SIF cells in each ganglionic plexus, with few if any terminations on PNs. In the myocardium, vagal afferents formed close contacts with cardiac muscles, including conduction fibers. In the endocardium, vagal fibers formed “flower-spray” and “end-net” terminals in connective tissue. With three-dimensional reconstruction of confocal optical sections, a novel polymorphism was seen: Some fibers had one or more collaterals ending as endocardial flower sprays and other collaterals ending as myocardial intramuscular endings. Some unipolar or pseudounipolar neurons within each cardiac ganglionic plexus were retrogradely labeled from the nodose ganglia. In conclusion, vagal afferents form a heterogeneity of differentiated endings in the heart, including structured elements which may mediate chemoreceptor function, stretch reception, and local cardiac reflexes. J. Comp. Neurol. 381:1-17, 1997. © 1997 Wiley-Liss, Inc.     

Vagal afferent innervation of the lower esophageal sphincter

To supply a fuller morphological characterization of the vagal afferents innervating the lower esophageal sphincter (LES), specifically to label vagal terminals in the tissues forming the LES in the gastroesophageal junction, the present experiment employed injections of dextran biotin into the nodose ganglia of rats. Four types of vagal afferents innervated the LES. Clasp and sling muscle fibers were directly and prominently innervated by intramuscular arrays (IMAs). Individual IMA terminals subtended about 16° of arc of the esophageal circumference, and, collectively, the terminal fields were distributed within the muscle ring to establish a 360° annulus of mechanoreceptors in the sphincter wall. 3D morphometry of the terminals established that, compared to sling muscle IMAs, clasp muscle IMAs had more extensive arbors and larger receptive fields. In addition, at the cardia, local myenteric ganglia between smooth muscle sheets and striated muscle bundles were innervated by intraganglionic laminar endings (IGLEs), in a pattern similar to the innervation of the myenteric plexus throughout the stomach and esophagus. Finally, as previously described, the principle bundle of sling muscle fibers that links LES sphincter tissue to the antropyloric region of the lesser curvature was innervated by exceptionally long IMAs as well as by unique web ending specializations at the distal attachment of the bundle. Overall, the specialized varieties of densely distributed vagal afferents innervating the LES underscore the conclusion that these sensory projections are critically involved in generating LES reflexes and may be promising targets for managing esophageal dysfunctions.     

Vagal postganglionic innervation of the canine sinoatrial node

Differential, selective distribution of parasympathetic, postganglionic innervation to the atrioventricular nodal (AVN) region of the canine heart was recently described. Ablation of parasympathetic pathways to the AVN by disruption of the epicardial fat pad at the junction of the inferior vena cava and inferior left atrium did not interfere with normal vagal control of the sinoatrial node (SAN) function. In sharp contrast, surgical dissection of the fat pad overlying the right pulmonary vein-left atrial junction interrupted the major right and left vagal inputs to the SAN region. The pulmonary vein fat pad (PVFP) in the dog heart is triangular in shape with roughly equilateral dimensions of approximately 1 cm, its base extending from superior to inferior veins, and its apex extending nearly to the sinus nodal artery as it courses rostrally in the sulcus terminalis. Careful dissection of smaller fat pads around the circumference of the pulmonary veins and particularly over the rostral-dorsal surfaces of the right superior pulmonary vein and adjacent right atrium, completed SAN parasympathetic denervation. Care in making these dissections left the vagal supply to the AVN region essentially intact, and preserved the sympathetic supplies to both SAN and AVN regions. Autonomic ganglia, varying in size from 1 or 2 cells to 80-100 cells, were found scattered throughout the ventral PVFP (overlying and surrounding the right pulmonary vein-left atrial junction). The ganglia were generally imbedded in fatty connective tissue, although they commonly rested very close to, or were loosely surrounded by epicardial muscle. Ganglia were also found in smaller fat pads on the dorsal surfaces of the atrium between the azygos and the right superior pulmonary vein.     

Vagal afferent innervation of the rat fundic stomach: morphological characterization of the gastric tension receptor.

Although the gastric tension receptor has been characterized behaviorally and electrophysiologically quite well, its location and structure remains elusive. Therefore, the vagal afferents to the rat fundus (forestomach or nonglandular stomach) were anterogradely labeled in vivo with injections of the carbocyanine dye Dil into the nodose ganglia, and the nerves and ganglia of the enteric nervous system were labeled in toto with intraperitoneal Fluorogold injection. Dissected layers and cryostat cross sections of the fundic wall were mounted in glycerin and analyzed by means of conventional and laser scanning confocal microscopy. Particularly in the longitudinal, and to a lesser extent in the circular, smooth muscle layers, Dil-labeled fibers and terminals were abundant. These processes, which originated from fibers coursing through the myenteric ganglia and connectives, entered either muscle coat and then ran parallel to the respective muscle fibers, often for several millimeters. They ran in close association with the Fluorogold-labeled network of interstitial cells of Cajal, upon which they appeared to form multiple spiny appositions or varicosities. In the myenteric plexus, two different types of afferent vagal structures were observed. Up to 300 highly arborizing endings forming dense accumulations of small puncta similar to the esophageal intraganglionic laminar endings (Rodrigo et al., '75 Acta Anat. 92:79-100) were found in the fundic wall ipsilateral to the injected nodose ganglion. They often covered small clusters of myenteric neurons or even single isolated ganglion cells (mean = 5.8 neurons) and tended to extend throughout the neuropil of the ganglia. In a second pattern, fine varicose fibers with less profuse arborizations innervated mainly the central regions of myenteric ganglia. Camera lucida analyses established that single vagal afferent fibers had separate collaterals in both a smooth muscle layer and the myenteric ganglia. Finally, Dil-labeled afferent vagal fibers were also found in the submucosa and mucosa. Control experiments in rats with supranodose vagotomy as well as rats with Dil injections directly in the distal cervical vagus ruled out the possibility of colabeling of afferent fibers of passage. In triple labeling experiments, in conjunction with Dil labeling of afferents and Fluorogold labeling of enteric neurons, the carbocyanine dye DiA was injected into the dorsal motor nucleus of the vagus to anterogradely label the efferent vagal fibers and terminals. The different distributions and morphological characteristics of the vagal afferents and efferents could be simultaneously compared. In some instances the same myenteric ganglion was apparently innervated by an afferent laminar ending and an efferent terminal.(ABSTRACT TRUNCATED AT 400 WORDS)     

Vagal afferent innervation of the proximal gastrointestinal tract mucosa: chemoreceptor and mechanoreceptor architecture

The vagus nerve supplies low-threshold chemo- and mechanosensitive afferents to the mucosa of the proximal gastrointestinal (GI) tract. The absence of a full characterization of the morphology and distributions of these projections has hampered comprehensive functional analyses. In the present experiment, dextran (10K) conjugated with tetramethylrhodamine and biotin was injected into the nodose ganglion and used to label the terminal arbors of individual vagal afferents of both rats and mice. Series of serial 100-μm thick sections of the initial segment of the duodenum as well as the pyloric antrum were collected and processed with diaminobenzidine for permanent tracer labeling. Examination of over 400 isolated afferent fibers, more than 200 from each species, indicated that three vagal afferent specializations, each distinct in morphology and in targets, innervate the mucosa of the proximal GI tract. One population of fibers, the villus afferents, supplies plates of varicose endings to the apical tips of intestinal villi, immediately subjacent to the epithelial wall. A second type of afferent, the crypt afferent, forms subepithelial rings of varicose processes encircling the intestinal glands or crypts, immediately below the crypt-villus junction. Statistical assessment of the isolated fibers indicated that the villus arbors and the crypt endings are independent, issued by different vagal afferents. A third vagal afferent specialization, the antral gland afferent, arborizes along the gastric antral glands and forms terminal concentrations immediately below the luminal epithelial wall. The terminal locations, morphological features, and regional distributions of these three specializations provide inferences about the sensitivities of the afferents.     

Vagal and spinal afferent innervation of the rat esophagus: A combined retrograde tracing and immunocytochemical study with special emphasis on calcium-binding proteins

Vagal afferent neurons contain a variety of neurochemical markers and neuroactive substances, most of which are present also in dorsal root ganglion cells. To test for the suitability of the calcium-binding protein calretinin as a specific marker for vagal afferent fibers in the periphery, immunocytochemistry for this protein was combined with retrograde tracing. Nerve fibers in the rat esophagus, as well as vagal and spinal sensory neurons innervating the esophagus, were investigated for co-localization of calretinin with calbindin, calcitonin gene-related peptide, and NADPH diaphorase. The results indicated that calretinin immunocytochemistry demonstrates neuronal structures known as vagal afferent from other studies, in particular intraganglionic laminar endings. A few enteric neurons whose distribution was unrelated to intraganglionic laminar endings also stained for calretinin. Strikingly, calretinin immunoreactivity was absent from spinal afferent neurons innervating the rat esophagus. In intraganglionic laminar endings and nodose ganglion cells calretinin was highly co-localized with calbindin but not with calcitonin gene-related peptide. On the other hand, calbindin was also found in spinal afferents to the esophagus where it was co-localized with calcitonin gene-related peptide. Vagal afferent neurons innervating the esophagus were never positive for NADPH diaphorase. Thus, calretinin appears to be a more specific marker for vagal afferent structures in the esophagus than calbindin, which is expressed by both vagal and spinal sensory neurons. Calretinin immunocytochemistry may be utilized as a valuable tool for investigations of subpopulations of vagal afferents in certain viscera. J. Comp. Neurol. 398:289–307, 1998. © 1998 Wiley-Liss, Inc.     

Vagal stimulation-induced gastric acid secretion in the anesthetized rat

The dynamics of gastric acid secretion induced by electrical stimulation of the cervical vagus in the anesthetized rat were investigated using a continuous collection-titration system permitting high temporal resolution. Stimulation with pulse rates of 2 or 4 impulses/s (pps) produced maximal gastric acid responses with small cardiovascular effects. With continuous stimulation, secretion was sustained for at least 1 h. Frequency-response profiles suggested that the parietal cells are innervated predominantly by fine-caliber C-fibers. Continuous stimulation was 3 times as effective as stimulation in bursts of higher frequencies. The minimal latency for the onset of secretion was 2.6 min at 4 pps, however, one- and two-min stimulations still produced proportionate but delayed secretory responses. It is concluded that, with low frequency cervical vagus stimulation, the rat stomach preparation described and employed in the present experiment is a useful model for further studies on the interaction of neural and humoral factors on gastric acid secretion.     

Catecholamine innervation of the rat hypoglossal nucleus

The catecholamine innervation of the hypoglossal nucleus (XII) was investigated immunocytochemically by comparing the distribution patterns of tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) in the rat. Numerous TH- and DBH-positive profiles were found throughout XII, while only occasional PNMT immunoreactivity was observed. Significantly, the distribution patterns of TH and DBH immunoreactivity were coextensive with the most intense staining found ventromedially along the caudal half of XII. We conclude, therefore, that the catecholamine innervation of XII is largely noradrenergic, and that motoneurons innervating the genioglossi muscles, the principal protrusors of the tongue, are the primary targets of this input.     

Afferent innervation of the rat incisor pulp

The present investigation was carried out to find out if there are any afferent nerves in rat incisor pulp and also to measure the conduction velocities of the nerves in this tissue. Electrodes were placed in the labial crown of the lower incisor of rats and electromyogram electrodes were placed in the anterior belly of the digastric muscle. Electrical stimulation of the incisor pulp gave rise to short- and long-latency digastric responses. Pulpal transection abolished the long-latency response confirming that it was due to pulpal afferent fibers, whereas the short-latency response remained and was due to stimulus spread to extrapulpal afferent fibers. With a similar set up, recordings were made from the pulp while the trigeminal ganglion was stimulated electrically. Recordings were made both before and after pulpal transection. A compound action potential was recorded with a latency indicating a conduction velocity of about 2 m/s. Further experiments were carried out in which the inferior alveolar nerve was exposed close to the mandibular foramen by a lateral approach, cut centrally, and placed on electrodes. Recordings were made from the tooth during electrical stimulation of the nerve and from the nerve during stimulation of the tooth, both before and after pulpal transection. From the results it was calculated that the conduction velocities of the nerves were 1.4 m/s inside the pulp and about 22 m/s outside.     

Orchidectomy affects the innervation of the rat thymus

To assess a putative role of the neural pathways in transfer of information from the gonads to the thymus, adult AO rats were orchidectomized (ORX) or sham ORX (controls); sacrificed 1, 3, 7, or 15 days later and their thymi were analyzed for: (a) the concentrations of noradrenaline (NA), dopamine (DA) and serotonin (5-HT) and distribution of monoamine-containing nerve profiles and (b) the acetylcholinesterase (AChE) activity and distribution of AChE-containing nerve profiles. Three days after the castration, an elevation in the level of both catecholamines, reflecting an increase in the overall intensity of nerve fibers autofluorescence, was found. Seven days post castration neither NA nor DA concentration differed from the appropriate control values, while 15 days after the surgery the concentration of NA was lower than that in the controls, most likely, due to diminished density of noradrenergic nerve profiles. In both the rats sacrificed 7 and 15 days after orchidectomy the concentration of 5-HT was reduced as result of a decrease in the density of 5-HT-containing autofluorescent cells. The activity of AChE was depressed one day after the surgery; then increased, so that 3 days post castration its value was higher than that in the sham ORX. After this increase, AChE activity decreased being, at postoperative day 7 and 15, lower than that in the controls. It seems that this decrease in AChE activity reflected, not only a reduction in the density of AChE-containing nerve fibers, but also a decrease in the density of AChE positive cells. Thus, the results indicate that orchidectomy can evoke changes in the T-cell maturation altering modulatory influences on this process coming via neural route, as well as those coming from the mast cells and AChE positive epithelial cells which constitute important component of the thymus microenvironment.     

Noradrenergic innervation of the villi of rat jejunum

Using fluorescent histochemical methods it has been shown that the villi of the jejunum are innervated by noradrenergic fibres. Varicose fibres originating in the plexus associated with the arterioles of the submucosa run close to the central lacteal and extend to the villus tip. It is suggested that the nerves may be involved in the control of absorption although exactly which structures in the villi are innervated remains unclear.     

Substance P innervation of the rat hippocampal formation

Light and electron microscopic substance P (SP) immunostaining was performed on hippocampal sections of colchicine-pretreated, control, untreated fimbria-fornix-transected (5 days), as well as perforant path-stimulated Sprague-Dawley rats fixed in 5% acrolein. Numerous SP-immunoreactive neurons could be observed in the stratum oriens of the Ammon's horn and subiculum, fewer were seen in the dentate hilar area and stratum radiatum of CA2 and CA3, and even fewer were seen at the border between the CA1 strata radiatum and the lacunosum moleculare of CA1 subfield. A higher dose of colchicine resulted in SP immunoreactivity in a large population of granule cells and mossy axon terminals. The entire CA2 region, the stratum oriens of CA1, CA3, and the subiculum were densely innervated by SP-containing axon terminals. A homogeneous SP innervation was found in the stratum radiatum of CA1. Only a few SP fibers were seen adjacent to the granule cell layer. Symmetric axosomatic contacts were seen between SP-containing boutons and somata in the stratum oriens of the Ammon's horn. However, throughout the hippocampal formation, the majority of SP-containing axons formed axodendritic symmetric synapses. A dense population of SP-immunoreactive boutons that formed axodendritic asymmetric synapses was observed in the strata oriens and radiatum of the CA3a and CA2 regions, and a few were found in the supragranular and subgranular layers of the dentate gyrus. Fimbria-fornix transection resulted in a marked loss of SP fibers in the strata oriens, pyramidale, and radiatum of the CA3a and CA2 subfields. In perforant pathway-stimulated animals, a population of granule cells and a large number of mossy axon terminals were immunoreactive for SP. These observations suggest two sources of SP innervation to the hippocampal formation: one arising from intrinsic sources (interneurons and granule cells) and one arising from extrinsic sources, most likely the supramammillary region. J. Comp. Neurol. 384:41–58, 1997. © 1997 Wiley-Liss, Inc.     

Noradrenaline neuron innervation of the neocortex in the rat

Noradrenaline innervation of the rat neocortex is studied by glyoxylic acid histochemistry and radioisotopic biochemical analysis. The data indicate that all neocortical areas receive a noradrenergic innervation which is identical in organization but varies in density from area to area. Radioisotopic analysis of catecholamines in the cortical areas studied reveals only the presence of significant levels of noradrenaline. Unilateral locus coeruleus ablation greatly diminishes ipsilateral noradrenaline content and fiber innervation in all neocortical areas studied. Detailed histochemical analysis reveals a diffuse plexus-like arrangement of noradrenaline fibers, with each cortical layer having a distinctive pattern of innervation. Single noradrenergic fibers enter layer VI of cortex and branch at all levels to undergo extensive collateralization. Terminal horizontal branching in the molecular layer results in the most dense fiber plexus of all cortical layers. This pattern of noradrenaline innervation is similar to that of other non-specific afferent systems innervating neocortex.     

Noradrenergic sympathetic innervation of the spleen: III. Development of innervation in the rat spleen

The ontogeny of noradrenergic innervation and its compartmental development were studied in the rat spleen using glyoxylic acid histofluorescence and high-performance liquid chromatography (HPLC). Noradrenergic nerves were present at birth in bundles adjacent to the splenic artery and vein. On days 1-3, fluorescent profiles largely were associated with the vasculature and with the perivascular zone. By day 6, these fibers formed increasingly elaborate and tortuous plexuses around the central arteries and their branches. By day 10, fibers were present along the marginal sinus and extended into the developing marginal zone. Between day 10 and day 13 the largest increase in norepinephrine (NE) levels (per mg protein) were noted, and the periarteriolar lymphatic sheath (PALS) achieved its adult form, with increased innervation of the parenchyma. In contrast, the venous/trabecular system developed relatively late. The first trabecular fibers were evident at day 10, and the capsule was not innervated until day 13. From 13 days to adulthood, there was a gradual refinement and extension of existing patterns with no change in NE levels as measured by HPLC (per mg protein), suggesting that the innervation was keeping pace with rapid increases in spleen growth. The pattern of growth and development for noradrenergic nerves in the PALS remarkably parallels changes in T cell compartmentation during this period. We propose that norepinephrine is available for interaction with T cells at the earliest stages of development and could play a role in such processes as lymphocyte packing and the onset of immunocompetence.     

Vagal modulation of nociception is mediated by adrenomedullary epinephrine in the rat

Vagal afferent activity modulates mechanical nociceptive threshold and inflammatory mediator-induced hyperalgesia, effects that are mediated by the adrenal medulla. To evaluate the role of epinephrine, the major hormone released from the adrenal medulla, the beta2-adrenergic receptor antagonist ICI 118,551 was chronically administered to vagotomized rats and epinephrine to normal rats. In vagotomized rats, chronic administration of ICI 118,551 markedly attenuated vagotomy-induced enhancement of bradykinin hyperalgesia but had no effect on nociceptive threshold. In normal rats, chronic epinephrine had the opposite effect, enhancing bradykinin hyperalgesia. Like vagotomy-, epinephrine-induced enhancement of hyperalgesia developed slowly, taking 14 days to reach its peak. Vagotomy induced a chronic elevation in plasma concentrations of epinephrine. We suggest that ongoing activity in vagal afferents inhibits the release of epinephrine from the adrenal medulla. Chronically elevated levels of epinephrine, occurring after vagotomy, desensitize peripheral beta2-adrenergic receptors and lead to enhancement of bradykinin hyperalgesia. The ability of prolonged elevated plasma levels of epinephrine to sensitize bradykinin receptors could contribute to chronic generalized pain syndromes.     

Origin of cingulate cortex cholinergic innervation in the rat

The relative contribution of the n. diagonal band and thalamic nuclei to the cholinergic innervation of the cingulate cortex was examined. Lesions were placed in the n. diagonal band, anterior thalamus, and medial thalamus of rats, and changes in choline acetyltransferase in discrete regions of the cingulate cortex were determined. The n. diagonal band lesion produced a large decrease in choline acetyltransferase activity while the thalamic lesions produced no significant change in activity.     

Postnatal development of the efferent innervation of the rat cochlea

The postnatal development of the efferent innervation of the rat cochlea was studied by intracochlear injection of the fluorescent retrograde neuronal traces Diamidino yellow and Fast blue. Injections were performed on adult rats and on neonatal rats ranging from 0 to 8 postnatal days. It was found that the total number of neurones labelled in the brainstem after intracochlear injection was not significantly different in the newborn rat, compared to the adult. On the basis of cell body location and laterality of projections, there was a clear separation into lateral and medial efferent systems at the earliest postnatal age studied (PO). Evidence was also found in the newborn for a tonotopicity in the lateral system projection similar to that in the adult. Differences between the newborn and adult were a slight but significantly greater number of bilaterally-projecting cells in the newborn, and the presence in the newborn of a small number of cells located in the lateral superior olivary nucleus contralateral to their target cochlea. These were extremely rare in the adult brainstem. Evidence was found for the occurrence of postnatal neuronal death in nuclei of origin of both efferent systems. It is suggested that although the overall extent and general organization of the efferent projection to the cochlea in the rat appears to be established at birth, regressive changes are occurring during the postnatal shaping and maturation of this brainstem-to-cochlea pathway.     

Organization of the sympathetic postganglionic innervation of the rat heart

The origins and organization of cardiac sympathetic postganglionic nerves in the rat were identified in the present investigation. The retrograde tracer, Diamidino Yellow, was injected into the right or left ventricles to label somata in the sympathetic chain. Analysis of all sympathetic ganglia from superior cervical ganglion through the 10th thoracic ganglion indicated that the postganglionic innervation of the rat cardiac ventricles originates bilaterally. The majority of these somata were located in the middle and inferior cervical ganglia (middle cervical-stellate ganglion complex) (approximately 92% of all labelled cells), with lesser contributions from the superior cervical and 4th through 6th thoracic ganglia. To confirm and further quantitate these findings, the middle cervical-stellate ganglion complex was removed (MC-S ganglionectomy) bilaterally or ipsilaterally from the left or right sides, and regional cardiac norepinephrine concentration (left and right atrial appendages and left and right ventricles) was analysed 7 or 28 days later. At both times after bilateral MC-S ganglionectomy, regional cardiac norepinephrine was reduced by 89% to 100%, indicating the removal of almost all cardiac noradrenergic cells of origin and possibly fibers of passage. The results of unilateral MC-S ganglionectomy experiments indicated that the atrial appendages and the left ventricle receive bilateral innervation from the middle cervical-stellate ganglion complex. However, the left middle cervical-stellate ganglion complex appears to contribute a majority of the norepinephrine to the right ventricle. Furthermore, between 7 and 28 days after contralateral MC-S ganglionectomy, atrial appendages, but not ventricles, display significant recovery of norepinephrine content. The present data demonstrate: (1) a bilateral locus of origin of cardiac sympathetic postganglionic neurons, limited longitudinally to cervical through mid-thoracic ganglia, and (2) the ability of the cardiac postganglionic innervation to regenerate after partial denervation. These results demonstrate anatomical evidence for significant bilateral integration of cardiac sympathetic activity at the level of the sympathetic ganglion in the rat.     

Sympathetic innervation of the supratentorial dura mater of the rat

The origin, density, and distribution of sympathetic nerve fibers in the supratentorial dura mater of the rat were examined in detail in the current study by using wheat germ agglutinin horseradish peroxidase (WGA-HRP) retrograde tracing procedures, glyoxylic acid-induced fluorescence, and dopamine beta-hydroxylase (DBH) immunocytochemical staining of dural whole mount preparations. Application of WGA-HRP to the superior sagittal sinus and adjacent areas of the supratentorial dura mater labeled numerous neurons in each of the left and right superior cervical ganglia. Glyoxylic acid and DBH immunocytochemical staining of fixed dural whole mount preparations revealed prominent plexuses of sympathetic nerves about the middle meningeal artery and its branches, about the superior sagittal and transverse sinuses, and "free" within the dura mater, i.e., apparently unassociated with any vasculature. Significantly, in all of these areas, the density of sympathetic innervation revealed in this study was considerably greater than that previously demonstrated by other workers. An impressive population of mast cells also was observed within the dura mater of the glyoxylic acid-treated preparations. The majority of these cells were perivascular; however, a significant number were also present within the dura unrelated to the vasculature, and occasional cells were seen in close apposition to fluorescent sympathetic nerve fibers. Taken together, the identification of a robust sympathetic plexus and prominent mast cell population associated with a dura mater that also receives significant sensory projections from the trigeminal system raises interest regarding the functional interactions of these elements. These observations warrant further consideration regarding their role in the pathogenesis of vascular headache and head pain.