The distribution of nitric oxide synthase-containing autonomic preganglionic terminals in the rat

Nitric oxide synthase (NOS)-immunoreactivity was co-localised with NADPH diaphorase activity in preganglionic sympathetic neurons and in their terminals in pre- and paravertebral sympathetic ganglia. The density of NOS-containing terminals varied between ganglia. Reactive terminals were densestin the superior cervical, stellate and inferior mesenteric ganglia, where the majority of the neurons were surrounded by reactive fibres, and the coeliac and superior mesenteric ganglia, where about half the postganglionic somata were sorrounded by reactive terminals. Fibres were least abundant in the pelvic ganglia and thoracic and lumbar sympathetic chain ganglia. NOS reactivity did not coincide with the distribution of calcitonin gene related peptide immunoreactivity, a marker for the terminals of NOS-containing sensory neurons in the rat. The distribution of nerve cells and terminals suggests that NOS is present in more than one functional subpopulation of sympathetic preganglionic neurons.     

Calbindin D28K-immunoreactivity identifies distinct subpopulations of sympathetic pre- and postganglionic neurons in the rat

Neurons performing the same function can be identified immunohistochemically because they often share the same neurochemistry. The distribution of calcium-binding proteins, like calbindin, has been used previously to identify functional subpopulations of neurons in many parts of the nervous system. In this study we have investigated the distribution of calbindin D28K-immunoreactivity in subpopulations of sympathetic preganglionic neurons in the intermediolateral nucleus of the rat spinal cord. The majority of calbindin D28K-immunoreactive preganglionic neurons also had co-localised nitric oxide synthase, although a population of preganglionic neurons in the mid- to low thoracic intermediolateral nucleus expressed only calbindin D28K-immunoreactivity. Retrograde-tracing studies showed that calbindin D28K-immunoreactive neurons projected to the superior cervical and stellate ganglia, with smaller numbers of cells projecting to the lumbar sympathetic chain and superior mesenteric ganglia. Very few calbindin D28K-immunoreactive neurons projected to the inferior mesenteric ganglion, and none projected to the adrenal medulla. The distribution of calbindin D28K-immunoreactive terminals and postganglionic neurons in the superior cervical and stellate ganglia was also investigated. Many postganglionic neurons were calbindin D28K-immunoreactive, and most of these lacked neuropeptide Y-immunoreactivity. Calbindin D28K-immunoreactive nerve terminals were common and formed dense pericellular baskets around many postganglionic neurons, including some of those that were calbindin D28K-immunoreactive, but only rarely formed pericellular baskets around neuropeptide Y-immunoreactive neurons. The function of some of the classes of postganglionic neurons that were the target of calbindin D28K-immunoreactive preganglionic terminals was determined by combining immunohistochemistry with retrograde-tracer injections into a range of peripheral tissues. Calbindin D28K-immunoreactive nerve terminals, with co-localised nitric oxide synthase-immunoreactivity, surrounded secretomotor neurons projecting to the submandibular salivary gland and pilomotor neurons projecting to skin, but did not surround neurons projecting to brown fat or vasomotor neurons projecting to the skin, muscle, or salivary glands. J. Comp. Neurol. 386:245–259, 1997. © 1997 Wiley-Liss, Inc.     

Distinct localization and target specificity of galanin-immunoreactive sympathetic preganglionic neurons of a teleost, the filefish Stephanolepis cirrhifer

Immunoreactivity for galanin was examined in the sympathetic preganglionic neurons in the spinal cord, adrenal glands, sympathetic ganglia, and some sensory ganglia of the filefish Stephanolepis cirrhifer. Galanin-immunoreactive neurons were found only in the rostral part, but not in the caudal part of the central autonomic nucleus (a column of sympathetic preganglionic neurons of teleosts). Many galanin-immunoreactive nerve terminals were found in contact with neurons in the celiac ganglia and the cranial sympathetic ganglia on both sides of the body. Most neurons encircled by galanin-immunoreactive nerve fibers were negative for tyrosine hydroxylase. Galanin-immunoreactive nerve fibers were very sparse in the spinal sympathetic paravertebral ganglia. No galanin-immunoreactive nerve fibers were found in the adrenal glands. No sensory neurons of the trigeminal, vagal, or spinal dorsal root ganglia were positive for galanin-immunoreactivity. These results suggest that galanin-immunoreactive sympathetic preganglionic neurons have distinct segmental localization and might project specifically to a population of non-adrenergic sympathetic postganglionic neurons in the celiac and cranial sympathetic ganglia.     

Pituitary adenylate cyclase activating polypeptide-immunoreactive sensory neurons innervate rat adrenal medulla

Rat adrenal chromaffin cells were invested by a dense network of nerve fibers immunoreactive to pituitary adenylate cyclase activating polypeptide-38 (PACAP-IR). Immunohistochemical studies demonstrated the presence of PACAP-IR in nodose and dorsal root ganglion cells, but not in neurons of the intermediolateral cell column and other autonomic nuclei of the thoracic and upper lumbar spinal cord. Somata of the T7 to T12 paravertebral ganglia were PACAP-negative. A few lightly labeled neurons were occasionally noted in the dorsal motor nucleus of the vagus. Injection of the retrograde tracer Fluorogold into the left adrenal medulla 3 days prior to sacrifice resulted in the labeling of a population of neurons in the ipsilateral spinal cord intermediolateral cell column (T1 to L1), ipsilateral and contralateral nodose ganglia and ipsilateral dorsal root ganglia from T7 to T10 inclusive. A small number of lightly labeled somata was occasionally noted in the dorsal motor nucleus of the vagus. Combined retrograde tracing and PACAP immunohistochemistry showed that a population of Fluorogold-containing nodose and dorsal root ganglion cells were also PACAP-positive. Pre-treatment of the rats with capsaicin caused a marked reduction of the PACAP-IR in the adrenal gland as well as in the superficial layers of the dorsal horn and caudal spinal trigeminal nucleus. These findings, in conjunction with the apparent absence of PACAP-IR in spinal sympathetic preganglionic neurons, sympathetic postganglionic neurons, and dorsal motor nucleus of the vagus, raise the possibility that PACAP-IR fibers observed in the adrenal medulla are primarily sensory in origin. As a corollary, catecholamine secretion from chromaffin cells may be modulated by the peptidergic sensory afferents in addition to the cholinergic sympathetic preganglionic nerve fibers.     

Characterization of NADPH diaphorase activity in rat sympathetic autonomic ganglia—effect of diabetes and aging

NADPH-diaphorase histochemistry, which identifies neural sites of nitric oxide production, demonstrated intensely stained nerve terminals surrounding the cell bodies of a subpopulation of neurons in rat prevertebral celiac and superior mesenteric sympathetic ganglia but failed to comparably label terminals in paravertebral superior cervical ganglia or perikarya in any sympathetic ganglion. The superior mesenteric ganglia of aged and diabetic rats, in which synaptic dysplasia (neuroaxonal dystrophy) is prominent, failed to show involvement of diaphorase containing nerve terminals.     

Postganglionic connections between sympathetic ganglia in the solar plexus of the cat demonstrated autoradiographically.

The anatomic substrate for postganglionic interaction between sympathetic ganglia was examined by autoradiography. In anesthetized cats, the left celiac ganglion was surgically isolated using a retroperitoneal approach and injected either hydraulically or electrophoretically with concentrated tritiated proline. Two to twenty-seven days after injection, the celiac and superior mesenteric ganglia, interganglionic connections, adrenal glands, nearby arteries, and splanchnic nerves were removed and prepared for autoradiography. Principal cell bodies in each injection site showed heavy incorporation of radiochemical with little, if any, spread to other regions of the ganglion. Occasionally, labeled processes resembling dendrites or axons were observed emerging from these cells. Small, radioactively labeled fibers streamed from the injected ganglion into the intermediate region between the celiac ganglia. Some of these elements entered “satellite ganglia” in the intermediate zone, and others could be traced to the contralateral celiac ganglion. In both types of ganglia, small fibers formed basket-like structures around the somata of principal cells. Labeled fibers also were observed entering the ipsilateral adrenal gland and terminating on, and around, blood vessels in the adrenal medulla.     

Chemical coding of sympathetic neurons controlling the tarsal muscle of the rat

Sympathetic axons in the upper eyelid and in tissues in the superior retro-orbital space were examined for NPY immunoreactivity. Sympathetic nerve terminals containing co-localised NPY were associated with blood vessels, the conjunctiva and the Meibomian glands. The acini of the Harderian gland completely lacked sympathetic innervation. Sympathetic axons lacking NPY were only found in the tarsal muscle. In addition, a minority of terminals, located in the more proximal part of the tarsal muscle, contained weak immunoreactivity to NPY. Injections of the retrograde tracer, Fast Blue, into the eyelid or retro-orbital space labelled postganglionic somata in the superior cervical ganglion. While many retrogradely labelled somata were immunoreactive for NPY, around half lacked NPY immunoreactivity and so are likely to project to the tarsal muscle. Most of the retrogradely labelled postganglionic somata lacking NPY were surrounded by terminals immunoreactive for met-enkephalin, leu-enkephalin and met-enkephalin arg-gly-leu which were all found to be present in the same nerve terminals. Sectioning the cervico-sympathetic trunk eliminated all enkephalin-immunoreactive pericellular baskets. Many enkephalin-immunoreactive pericellular terminals contained co-localised VAChT, calretinin and calbindin immunoreactivity, but completely lacked nitric oxide synthase immunoreactivity. A second population of nerve terminals that were immunoreactive for nitric oxide synthase also surrounded tarsal muscle-projecting neurons, but these terminals lacked immunoreactivity to enkephalin. Thus, postganglionic neurons projecting to the tarsal muscle are of at least two chemical phenotypes (with or without NPY) and they receive convergent input from at least two populations of preganglionic neurons with distinctive chemical phenotypes.     

由感觉和交感节神经元组成的中枢外反射弧的形态学研究

用免疫细胞化学和辣根过氧化物酶（HRP）逆行追踪法研究了感觉神经节和交感神经节中P物质（SP）样免疫反应物的分布及两种神经节之间的联系。在所有背根节出现大量SP免疫阳性神经元胞体，但在胸交感干节和腹腔-前肠系膜神经节仅有大量的SP阳性纤维和终末，而无阳性胞体。分组切断与腹腔-前肠系膜神经节相联系的神经支后，该节内的SP免疫反应物仅在切断与背根节的联系后明显减少或消失。将HRP注入腹腔神经节，在同侧C8～L3背根节出现大量标记细胞。以上结果表明．在感节中的SP纤维和终末来自背根节初级传入神经元的轴突侧支，提示由初级传人神经元与交感节主细胞在中枢外直接形成了短反射环路。     

Enkephalin-containing neurons in the inferior mesenteric ganglion projecting to the distal colon of cat: evidence from combined retrograde tracing by fluorescent microspheres and immunohistochemistry

Retrograde tracing with rhodamine fluorescent microspheres combined with fluorescein immunolabelling of methionine-enkephalin showed the presence of enkephalin-like material in neurons of the inferior mesenteric ganglion (sympathetic prevertebral ganglion) projecting to the distal colon in cat. Two weeks after injecting the microspheres into the wall of the distal colon, the inferior mesenteric ganglion was dissected out and incubated for 24 hours in a colchicine-containing culture medium in order to facilitate the detection of enkephalins in the soma of ganglion neurons. It was observed that retrogradely labelled ganglion cells contained enkephalin-like immunoreactive material. These ganglion cells corresponded to enkephalin-like postganglionic neurons, the terminals of which were located inside the wall of the distal colon. These enkephalin-like neurons were numerous and scattered throughout the ganglion. Sometimes enkephalin-like immunoreactive fibers, probably originating from spinal preganglionic neurons, ran close to immunoreactive and non-immunoreactive retrogradely labelled ganglion cells. This suggests that enkephalin-like immunoreactive fibers may make synaptic connections with enkephalin-like and non-enkephalin-like postganglionic neurons projecting to the distal colon. The present study establishes for the first time the existence of an enkephalin-like postganglionic pathway to the digestive tract originating from a sympathetic prevertebral ganglion. This finding indicates that the enkephalinergic innervation of the cat digestive tract may have at least two possible sources: (i) the sympathetic prevertebral ganglia; and (ii) the enteric nervous ganglia.     

The afferent and sympathetic components of the lumbar spinal outflow to the colon and pelvic organs in the cat. I. The hypogastric nerve

The cell bodies of the lumbar sensory and sympathetic pre- and postganglionic neurons that project to the pelvic organs in the hypogastric nerve of the cat have been labeled retrogradely with horseradish peroxidase applied to the central end of their cut axons. The numbers, segmental distribution, location, and size of these labeled somata have been determined quantitatively. Afferent and preganglionic cell bodies were located bilaterally in dorsal root ganglia and spinal cord segments L3-L5, with the maximum numbers in L4. Very few cells lay rostral to L3. Afferent cell bodies were generally very small in cross-sectional area relative to the entire population in the dorsal root ganglia. Most of the preganglionic cell bodies lay clustered just medial to the region of the intermediolateral column and extended caudally well beyond its usual limit in the upper part of L4. These neurons were, on the average, larger than the cells of the intermediolateral column itself, with the largest cells lying in the most medial positions. Most of the post-ganglionic somata were in the ipsilateral distal lobe of the inferior mesenteric ganglion, while some (usually less than 10%) lay in accessory ganglia along the lumbar splanchnic nerves and in paravertebral ganglia L3-L5. Postganglionic somata in the inferior mesenteric ganglion were larger than both labeled and unlabeled ganglion cells in the paravertebral ganglia. From the data, it is estimated that about 1,300 afferent neurons, about 1,700 preganglionic neurons, and about 17,000 postganglionic neurons project in each hypogastric nerve in the cat.     

Sympathetic innervation of the upper and lower regions of the uterus and cervix in the rat have different origins and routes

The origins and routes of the postganglionic sympathetic nerve supply to the upper and lower uterus and to the cervix were investigated in the rat by using denervation procedures combined with immunohistochemistry and retrograde tracing. The sympathetic nerve fibers of the upper part of the uterus arise from the ovarian plexus nerve. They mainly originate (90%) from neurons of the suprarenal ganglia (SRG) and of the T10 to L3 ganglia of the paravertebral sympathetic chain. Fluoro-Gold injections into different regions of the upper uterus showed that the SRG neurons mainly provide innervation to the tubal extremity (52%) rather than to the uterine portion below this area (26%). Very few neurons of the celiac ganglion or the aorticorenal ganglia participated in this innervation. Most of the sympathetic innervation of the lower uterus and the cervix (90%) originates from neurons of the paravertebral ganglia T13 to S2, principally at the L2–L4 levels. By using immunocytochemistry, we show that very few tyrosine hydroxylase–positive neurons of the pelvic plexus project to these areas, where they represent only 3% of the sympathetic nerve supply. Again, very few neurons of the inferior mesenteric ganglion (IMG) supply the lower uterus and the cervix. The comparison between retrograde tracing experiments in intact animals and after the removal of the IMG shows that very few sympathetic postganglionic axons from the paravertebral chain pass through the IMG to reach the lower uterus and the cervix. In contrast, these axons mainly project to splanchnic nerves bypassing the IMG to connect with the hypogastric nerves. In addition, some axons supplying the lower uterus follow the superior vesical arteries and then reach the organ. Taken together, these results show that the upper region of the uterus receives a sympathetic innervation that is different in origin and route from that of the lower uterus and the cervix. Such a marked region-specific innervation suggests that nerve control of the myometrial activity may be functionally different between the oviduct and the cervical ends of the uterus. J. Comp. Neurol. 399:403–412, 1998. © 1998 Wiley-Liss, Inc.     

Evidence for coexistence of serotonin and noredrenaline in sympathetic nerves supplying brain vessels of guinea pig

Nerve fibers containing 5-hydroxytryptamine (5-HT) were demonstrated immunohistochemically in the wall of pial vessels associated with the circle of Willis in the guinea pig. The fibers formed a network structure which was more dense in the rostral part of the arterial circle and its branches than in the caudal part. The 5-HT immunoreactive fibers disappeared in all arteries studied after bilateral superior cervical ganglionectomy, and unilateral ganglionectomy eliminated the 5-HT immunoreactivity in the ipsilateral part of the middle cerebral, posterior cerebral and superior cerebellar arteries. Decentralization of the superior cervical ganglion had no effect on the perivascular nerve plexus. Subsequent staining with dopamine-β-hydroxylase (DBH) antiserum following elution of the first antibody revealed that 5-HT was present in the noradrenergic nerve fibers. Small intensive fluorescent cells with positive immunoreaction for 5-HT and DBH, respectively, were located in clusters within the ganglion, which showed no immunohistochemical evidence for the presence of serotonergic neurons. It is concluded that 5-HT is probably not synthesized in truly serotonergic fibers but rather taken up and stored together with noradrenaline in cerebrovascular sympathetic nerves originating in the superior cervical ganglia.     

Sprouting sympathetic fibers form synaptic varicosities in the dorsal root ganglion of the rat with neuropathic injury

Peripheral nerve injury in a rat model (spinal nerve ligation) of neuropathic pain triggers sprouting of sympathetic fibers in the dorsal root ganglion (DRG). This sympathetic sprouting has been suggested as an important underlying mechanism for pain behaviors. To investigate the possibility of functional interaction between sprouted sympathetic fibers and sensory neurons, the present study examined the fine morphology and structural relationship between sympathetic fibers and the DRG neurons by electron microscopy. Sympathetic postganglionic fibers, as identified by electron microscopic immunostaining for tyrosine hydroxylase (TH), were all unmyelinated fibers and some of them ended as growth cones. In addition, many vesicle-containing axonal enlargements (we will refer these as synaptic varicosities) were found in the interstitial space around DRG neurons, and some were enclosed within the satellite cell capsule which surrounded the DRG soma. The presence of sympathetic synaptic varicosities near or in apposition with either the DRG somata or their processes provides a structural basis for possible interactions between sensory neurons and sympathetic fibers in the DRG of neuropathic rats. © 1997 Elsevier Science B.V. All rights reserved.     

Immunohistochemical phenotype of sensory neurons associated with sympathetic plexuses in the trigeminal ganglia of adult nerve growth factor transgenic mice

Following peripheral nerve injury, postganglionic sympathetic axons sprout into the affected sensory ganglia and form perineuronal sympathetic plexuses with somata of sensory neurons. This sympathosensory coupling contributes to the onset and persistence of injury-induced chronic pain. We have documented the presence of similar sympathetic plexuses in the trigeminal ganglia of adult mice that ectopically overexpress nerve growth factor (NGF), in the absence of nerve injury. In this study, we sought to further define the phenotype(s) of these trigeminal sensory neurons having sympathetic plexuses in our transgenic mice. Using quantitative immunofluorescence staining analyses, we show that the invading sympathetic axons specifically target sensory somata immunopositive for several biomarkers: NGF high-affinity receptor tyrosine kinase A (trkA), calcitonin gene-related peptide (CGRP), neurofilament heavy chain (NFH), and P2X purinoceptor 3 (P2X3). Based on these phenotypic characteristics, the majority of the sensory somata surrounded by sympathetic plexuses are likely to be NGF-responsive nociceptors (i.e., trkA expressing) that are peptidergic (i.e., CGRP expressing), myelinated (i.e., NFH expressing), and ATP sensitive (i.e., P2X3 expressing). Our data also show that very few sympathetic plexuses surround sensory somata expressing other nociceptive (pain) biomarkers, including substance P and acid-sensing ion channel 3. No sympathetic plexuses are associated with sensory somata that display isolectin B4 binding. Though the cellular mechanisms that trigger the formation of sympathetic plexus (with and without nerve injury) remain unknown, our new observations yield an unexpected specificity with which invading sympathetic axons appear to target a precise subtype of nociceptors. This selectivity likely contributes to pain development and maintenance associated with sympathosensory coupling.     

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.     

Serotonin inputs to rabbit sympathetic preganglionic neurons projecting to the superior cervical ganglion or adrenal medulla

The input from serotonin-containing nerve fibres to rabbit sympathetic preganglionic neurons projecting to either the superior cervical ganglion or the adrenal medulla was investigated by combining retrograde tracing with the B subunit of cholera toxin and immunocytochemistry for serotonin. There were pronounced rostrocaudal variations in the density of serotonin fibres in the rabbit intermediolateral cell column from T1 to L4; maximum numbers of fibres were found in T3-6 and L3–4 and minimum numbers in T1 and T10–12. By light microscopy, retrogradely labelled sympathetic preganglionic neurons projecting to the superior cervical ganglion or the adrenal medulla received variable densities of close appositions from serotonin-immunoreactive fibres. Some neurons from each population received many close appositions, whereas others received moderate numbers or few appositions. Appositions occurred on the cell bodies, dendrites, and occasionally axons of sympathetic preganglionic neurons. Rare neurons in both groups of retrogradely labelled cells received no appositions from serotonin-containing nerve fibres. At the ultrastructural level, synapses were found between serotonin-positive boutons and sympathetic preganglionic neurons projecting either to the superior cervical ganglion or to the adrenal medulla. These results indicate that, through direct synaptic contacts, serotonin-immunoreactive, presumably bulbospinal, nerve fibres affect the activity of the vast majority of sympathetic preganglionic neurons that send axons either to the superior cervical ganglion or to the adrenal medulla. This serotonin input may be sympathoexcitatory and could mediate increases in sympathetic nerve activity and in the release of catecholamines from the adrenal medulla. © 1995 Wiley-Liss, Inc.     

Axonal branching of canine sympathetic postganglionic cardiopulmonary neurons. A retrograde fluorescent labeling study

In 11 dogs fluorescent retrograde tracers were injected into physiologically identified left-sided sympathetic cardiopulmonary nerves. When two different ipsilateral cardiopulmonary nerves were injected, labeled cells from each injected nerve had overlapping distributions in the middle cervical and stellate ganglia. Most retrogradely labeled neurons were located in the middle cervical ganglion and cranial pole of the stellate ganglion. Following the injection of two different tracers into two different nerves, some neurons in the middle cervical ganglion were retrogradely labeled with two tracers. Double-labeled neurons were rarely found in the stellate ganglion. There were areas within the ganglia in which labeled neurons projected predominantly to one cardiopulmonary nerve. In the thoracic autonomic nervous system Fast Blue was transported most effectively. Bisbenzimide was not transported as well as Fast Blue and Nuclear Yellow was very poorly transported in cardiopulmonary nerves. The results demonstrate that some efferent postganglionic sympathetic neurons project axons into at least two different cardiopulmonary nerves and that an anatomical substrate for axo-axonal reflexes exists in the thoracic sympathetic nervous system.     

The origin of catecholaminergic nerve fibers in the subdiaphragmatic vagus nerve of rat.

It is known that the vagus nerve contains catecholaminergic fibers. However, the origin of these fibers has not been systematically examined. In this study, we addressed this issue using retrograde tracing from the subdiaphragmatic vagus nerve combined with immunocytochemistry. The cervical and thoracic sympathetic trunk ganglia, the nodose ganglia and the dorsal motor nucleus of the vagus nerve were examined following injection of Fluoro-Gold or cholera toxin horseradish peroxidase conjugate into the trunks of the subdiaphragmatic vagus nerve of rats. Numerous retrogradely labeled neurons were seen in the nodose ganglion and the dorsal motor nucleus of the vagus nerve. Very few labeled neurons were found in the sympathetic ganglia (less than 0.06% of the neurons in either superior cervical ganglion or cervicothoracic ganglion were retrogradely labeled). Double labeling with immunofluoresence for catecholamine synthesizing enzymes revealed that: (1) 92% of all Fluoro-Gold retrogradely labeled tyrosine hydroxylase immunoreactive neurons were found in parasympathetic sources (75% in the dorsal motor nucleus of the vagus nerve and 17% in the nodose ganglia), and only 8% in the cervicothoracic sympathetic ganglia; (2) 12% of the retrogradely labeled catecholaminergic neurons in the dorsal motor nucleus of the vagus nerve were also dopamine-beta-hydroxylase immunopositive neurons; (3) 70% of the retrogradely labeled neurons in the sympathetic ganglia were tyrosine hydroxylase immunopositive and 54% of these catecholaminergic neurons contained dopamine-beta-hydroxylase, while 30% of the retrogradely labeled neurons were non-catecholaminergic neurons. These results indicate that catecholaminergic fibers in the abdominal vagus nerve are primarily dopaminergic and of parasympathetic origin, and that only an extremely small number of these fibers, mostly noradrenergic in nature, arise from postganglionic sympathetic neurons.     

Substance P-like immunoreactivity in developing cranial parasympathetic neurons of the rat

Substance P-like immunoreactivity (SPLI) has been observed in cell bodies of fetal cranial parasympathetic ganglia of rat. It first appears at day 16 of gestation at the same time as in cranial sensory ganglia. From day 17 to 21, SPLI neurons constitute most, if not all, submandibular-sublingual and intralingual ganglia, they form 30–40% of otic and pterygopalatine ganglia and numerous such neurons are found in the myenteric plexus of the esophagus as well as in pharyngeal and buccal walls. The immunoreactive material is thinly granular, and its appearance does not change with prenatal development. The immunoreactivity in cell bodies of parasympathetic ganglia decreases at the end of the gestational period, and cannot be evidenced any more in most cells of normal adult ganglia. However, the corresponding SPLI fibers remain intensely immunoreactive. When grafted to rat irides, which have been chemically depleted of intrinsic SPLI fibers, submandibular, otic and pterygopalatine ganglia from pre- or postnatal rats rapidly produce a large amount of SPLI fibers on the iris mimicking the pattern of sensory innervation. This proves the presence of SPLI neurons in adult parasympathetic ganglia, at least in experimental conditions.This study of fetuses and grafts demonstrates the existence of neurons in SPLI parasympathetic cranial ganglia which has been underestimated or ignored previously as a result of observations on adult ganglia. The very large proportion of SPLI neurons in the ganglia of the salivary gland might be of importance for the interpretation of experimental studies on the control of salivation. The presence of SPLI in all three types of peripheral ganglia, sensory, sympathetic and parasympathetic, raises the question of its functional significance in the different compartments of the peripheral nervous system.     

Localization and Axotomy-induced Regulation of the Peptide Secretoneurin in the Rat Superior Cervical Ganglion

This study demonstrates the localization and regulation of a novel neuropeptide of 33 amino acids, secretoneurin (SN), in the rat superior cervical ganglion. Gel filtration chromatography of ganglion proteins followed by a specific radioimmunoassay revealed that SN is the predominant cleavage product of secretogranin II, a member of the chromogranin/secretogranin protein family, in adult ganglia. SN was detected within the majority of nerve endings surrounding postganglionic neurons that were identified by the presence of synaptophysin and, in part, colocalized leu-encephalin. Applying immuno-electronmicroscopy, SN was localized to large dense core vesicles of neuronal and small intensely fluorescent (SIF) cells. In situ hybridization revealed the presence of secretogranin II mRNA in postganglionic neurons and, to a lesser extent, in SIF cells. One week after transection of the postganglionic branches SN levels were not significantly altered; however, a decrease of secretogranin II mRNA was observed in postganglionic neurons but not in SIF cells. After decentralization of the ganglion, SN-immunoreactive nerve terminals disappeared and intraganglionic SN levels were reduced by 70%, indicating the preganglionic origin of SN-positive nerve fibres and varicosities. Secretogranin II mRNA was slightly reduced under this condition. Combined axotomy and decentralization further diminished intraganglionic secretogranin II mRNA, although peptide levels increased significantly above control values under these conditions. Double-labelling immunofluorescence with antibodies against the somatodendritic marker microtubule-associated protein 2 (MAP2) revealed that the increase in SN immunoreactivity was due to an accumulation of SN in axonal processes of postganglionic neurons. SN immunoreactivity was also detected in dissociated neonatal superior cervical ganglion cultures and increased significantly upon treatment with nerve growth factor, the survival and differentiation factor of sympathetic neurons during perinatal development. Co-culture with non-neuronal cells or addition of leukaemia inhibitory factor, a cytokine known to stimulate synthesis of various peptides after nerve transection, did not influence SN immunoreactivity. Therefore, since no fixed relationship between SN and any of the known neuropeptides or neurotransmitters expressed in sympathetic neurons was observed, the expression of this novel peptide appears to be independently regulated.