Catecholaminergic innervation of the sympathetic preganglionic cell column of the filefish Stephanolepis cirrhifer.

Nerve fibers immunoreactive for enzymes synthesizing catecholamines were examined in the central autonomic nucleus, a column of sympathetic preganglionic neurons, in the filefish Stephanolepis cirrhifer. Varicose nerve fibers immunoreactive for tyrosine hydroxylase were densely distributed in the rostral part, sometimes in contact with perikarya but were sparse in the caudal part of this nucleus. Fluorescent double labeling distinguished noradrenergic nerve fibers immunoreactive for both tyrosine hydroxylase and dopamine beta hydroxylase, and dopaminergic fibers immunoreactive only for tyrosine hydroxylase. In the brainstem, catecholaminergic neurons were observed in the locus coeruleus, the caudal dorsomedial reticular zone of the medulla, and the area postrema. Double labeling of tyrosine hydroxylase and dopamine beta hydroxylase showed that the neurons in the locus coeruleus were all noradrenergic, and those in the caudal dorsomedial medulla were mostly noradrenergic, whereas the area postrema contained both noradrenergic and dopaminergic neurons. No catecholaminergic neurons were found in the ventral region of the brainstem. After application of DiI to the central autonomic nucleus, retrogradely labeled neurons were seen in the caudal dorsomedial medulla but not in the locus coeruleus or the area postrema. These findings suggest that the sympathetic preganglionic neurons of the filefish may receive noradrenergic axonal projections from neurons in the caudal dorsomedial medulla. In the light of previous studies, inputs of these catecholaminergic fibers to the central autonomic nucleus may be involved in regulation of sympathetic activity of peripheral organs, together with serotoninergic and peptidergic inputs to this nucleus.     

Choline acetyltransferase immunoreactive sympathetic ganglion cells in a teleost,Stephanolepis cirrhifer

The present study showed neurons immunoreactive for choline acetyltransferase (ChAT) in the cranial sympathetic ganglia lying close to the trigeminal-facial nerve complex of the filefish. In these ganglia, less than 1% of ganglion cells were positive for choline acetyltransferase. Choline acetyltransferase-positive neurons were significantly larger than the randomly sampled neurons in this ganglion. The majority of choline acetyltransferase-positive neurons were negative for tyrosine hydroxylase, but many of them were positive for galanin (GAL). Some neurons were positive for both choline acetyltransferase and tyrosine hydroxylase, but these neurons were rarely immunoreactive for dopamine beta hydroxylase, suggesting that they are not adrenergic. In the cranial sympathetic ganglia and the celiac ganglia, many nerve fibers immunoreactive for galanin were seen, and varicose terminals were in contact selectively with neurons negative for both choline acetyltransferase and tyrosine hydroxylase, but not with those positive for choline acetyltransferase or tyrosine hydroxylase. Nerve fibers immunoreactive for choline acetyltransferase were found to be present in contact with the deep layer of chromatophores, which was observed only in the labial region. These results suggest that cholinergic postganglionic neurons are present in the filefish cranial sympathetic ganglia, and that they also contain galanin. As few cholinergic sympathetic neurons express tyrosine hydroxylase and none express dopamine beta hydroxylase, they are unlikely to synthesize noradrenaline or adrenaline.     

Differential distribution of nerve terminals immunoreactive for substance P and cholecystokinin in the sympathetic preganglionic cell column of the filefish Stephanolepis cirrhifer

Immunoreactivity for substance P and cholecystokinin-8 was examined in the nerve fibers in the central autonomic nucleus, a cell column for sympathetic preganglionic neurons, in the filefish Stephanolepis cirrhifer. Substance P-immunoreactive fibers were distributed throughout the entire rostrocaudal extent, but were more abundant in the caudal part of the column, where substance P-immunoreactive varicosities sometimes made contacts with the sympathetic preganglionic neurons. Cholecystokinin-8-immunoreactive fibers were found almost entirely in the rostral part of the column, where a dense network of varicosities was in close apposition to a considerable number of the sympathetic preganglionic neurons. Double labeling immunohistochemistry showed that substance P fibers and cholecystokin-8 fibers were entirely different, and distinct from serotonin-immunoreactive fibers. By using immunoelectron microscopy, synaptic specialization was sometimes observed between the dendrites of preganglionic neurons and varicosities immunoreactive for substance P and cholecystokinin-8. Substance P- and cholecystokinin-8 fibers were seen from the descending trigeminal tract, through the dorsolateral funiculus and the ventral portion of the dorsal horn, to the central autonomic nucleus. After colchicine treatment, substance P-immunoreactive perikarya were found in the cranial and spinal sensory ganglia. These results suggest that the sympathetic preganglionic neurons of the filefish receive innervation by substance P fibers and cholecystokinin fibers, and that the former might be of primary sensory origin. Topographical distribution of cholecystokinin-8-immunoreactive terminals in the central autonomic nucleus along the rostrocaudal extent might underlie the differential regulation of sympathetic activity via a distinct population of sympathetic preganglionic neurons.     

Monoaminergic and peptidergic axonal projections to the vagal motor cell column of a teleost, the filefish Stephanolepis cirrhifer

In an immunohistochemical study, the vagal motor nucleus of a teleost, the filefish Stephanolepis cirrhifer, could be divided into a rostral part and a caudal part, and the former into a dorsolateral group and a ventromedial group. The dorsolateral group consisted of neurons immunoreactive for calcitonin gene-related peptide, whereas the ventrolateral-caudal group was negative for calcitonin gene-related peptide. The latter group was retrogradely labeled after dextran amine injection to the visceral ramus of the vagus nerve, suggesting that it is a general visceral efferent column, made up of parasympathetic preganglionic neurons, whereas the dorsolateral rostral group is a special visceral efferent column. In the general visceral efferent column, a dense concentration of nerve fibers immunoreactive for serotonin, tyrosine hydroxylase, cholecystokinin-8, and substance P, and a small number of fibers immunoreactive for neuropeptide Y was observed. Perikarya in contact with varicose terminals immunoreactive for these substances were frequently seen. In contrast, in the special visceral efferent column, only a moderate concentration of neuropeptide Y-immunoreactive nerve fibers and a sparse distribution of fibers immunoreactive for tyrosine hydroxylase were observed. Perikarya in contact with varicose terminals immunoreactive for these substances were rare. These results suggest that the vagal parasympathetic preganglionic neurons might receive multiple inputs of monoaminergic and peptidergic fibers involved in the regulation of the visceral organs. On the other hand, monoaminergic and peptidergic afferent fibers might be of much less significance in the activity of the special visceral efferent component of the vagus nerve.     

Light microscopic and ultrastructural localization of GABA-like immunoreactive input to retrogradely labeled sympathetic preganglionic neurons

The organization of gamma-aminobutyric acid-like immunoreactive (GABA-LIR) processes was studied within the sympathetic preganglionic neuropil of male Sprague-Dawley rats and pigeons (Columba livia). Sympathetic preganglionic neurons were retrogradely labeled following horseradish peroxidase (HRP) injections into either the adrenal medulla or superior cervical ganglion in rats or into the avian homologue of the mammalian stellate ganglion (paravertebral ganglion 14) in pigeons. GABA-LIR staining was visualized using peroxidase-antiperoxidase (PAP), avidin-biotin complex (ABC), or post-embedding immunogold methods. The pigeon preganglionic neuropil contained a dense network of GABA-LIR processes with punctate swellings that encircled sympathetic preganglionic perikarya within the principal preganglionic cell column (column of Terni) and the nucleus intercalatus spinalis. GABA-LIR spinal neurons were intermingled among HRP-labeled sympathetic preganglionic neurons within the column of Terni and throughout the zona intermedia. In the rat the density of the GABA-LIR processes within the four thoracic sympathetic preganglionic nuclei was less than that observed in the pigeon. Nevertheless, GABA-LIR profiles distinctively dotted preganglionic perikarya within the nuclei intermediolateralis pars principalis and pars funicularis, nucleus intercalatus spinalis and the central autonomic nucleus. GABA-LIR neurons were rarely observed within the nucleus intermediolateralis pars principalis, but were numerous in the zona intermedia and area X. No GABA-LIR spinal neurons in either vertebrate were retrogradely labeled with HRP. The ultrastructural arrangements of GABA-LIR processes within the sympathetic preganglionic neuropils of pigeons and rats were similar. GABA-LIR boutons formed symmetrical synaptic contacts and contained small round electron-lucent vesicles (50 nm) and one to several larger dense-core vesicles (80 nm). GABA-LIR terminals contacted HRP-labeled sympathetic preganglionic perikarya in all spinal nuclear regions in both vertebrates. More frequently, GABA-LIR boutons synapsed on dendrites. Occasionally, axo-axonic configurations were observed; each time only one of the axonal elements was GABA-LIR. Numerous unmyelinated and some thinly myelinated GABA-LIR axons coursed through the sympathetic preganglionic neuropils of both vertebrates. Synapses between GABA-LIR processes were present within the sympathetic preganglionic neuropil of both vertebrates. GABA-LIR dendrites were contacted by unlabeled terminals (predominantly small spherical vesicles with asymmetric synaptic specializations) and GABA-LIR terminals on GABA-LIR dendrites were similar in appearance to those synapsing on sympathetic preganglionic cell bodies and dendrites.     

Stabilization of the discharge rate of sympathetic preganglionic neurons

A characteristic feature of the sympathetic preganglionic neuron (SPN) is the low rate of firing during both tonic and evoked activity. Firing rates between 1 and 2 Hz are typical of tonic activity, and the rates increase only slightly during sustained reflex activation. The low mean firing rate of the SPN may result from mechanisms which depress the excitability of the neuron and /or from a very low synaptic efficacy of its excitatory inputs. In recent years depressant mechanisms, other than baroreceptor inhibition, have been identified which may be involved in the control of SPN firing rate. Some of these mechanisms are spinal. This paper reviews data on 3 depressant mechanisms, namely post-impulse depression, recurrent inhibition and inhibition by myelinated spinal afferents, which are operating within the spinal cord.     

Spinal origin of sympathetic preganglionic neurons in the rat

The segmental distribution of sympathetic preganglionic neurons (SPNs) and dorsal root ganglion cells (DRGs) was studied after Fluoro-gold injections into the major sympathetic ganglia and adrenal gland in rats. A quantitative assessment of the segmental and nuclear locations was made. Four general patterns of innervation were apparent: (1) a large number of SPNs (1000–2000/ganglion) innervate the sympathetic ganglia which control head or thoracic organs and a relatively small number of SPNs (100–400/ganglion) innervate the sympathetic ganglia controlling the gut, kidney, and pelvic organs; this difference in density of innervation probably relates to the level of fine control that can occur in these end organs by the SPNs; (2) the reverse pattern is seen in the DRG labeling where a large number of DRGs were labeled after Fluoro-gold injections into the preaortic ganglia (celiac, superior, and inferior mesenteric) and a small number were labeled after injections into the cervical sympathetic ganglia; (3) the intermediolateral cell column is the main source of SPNs except for the inferior mesenteric ganglion which is innervated predominantly by SPNs originating in the central autonomic nucleus (75%); the lateral funiculus is a source of SPNs mainly for the cervical sympathetic ganglia; and (4) each sympathetic ganglion and the adrenal gland receives a multisegmental SPN and DRG input with one segment being the predominant source of the innervation. The adrenal gland shows an intermediate position in terms of the density of SPN input (800 cells) and dorsal root input (300 cells); it has a widespread segmental input (T₄-T₁₂) with the T₈ segment being the major source.     

Serotonergic excitation of sympathetic preganglionic neurons: a microiontophoretic study

The effects of microiontophoretically applied serotonin on the extracellularly recorded discharges of sympathetic preganglionic neurons (SPNs) were studied in anesthetized cats. Thoracic SPNs were identified on the basis of constancy of antidromic activation and collision. Low ejecting currents of serotonin (5-30 nA) invariably excited spontaneously active SPNs. Serotonin also excited the vast majority of quiescent SPNs, as well as neurons brought to discharge threshold by the excitatory amino acid L-glutamate. A population of SPNs was identified which was insensitive to the excitatory effects of both serotonin and L-glutamate. Iontophoretic or intravenous administration of the putative serotonin antagonists methysergide and metergoline blocked the excitatory effects of serotonin on SPNs. The blockade of the serotonin-induced excitation was not associated with a local anesthetic action of methysergide or metergoline. Methysergide and metergoline also reduced the firing rate of SPNs in intact but not in spinal animals. These data provide strong evidence to support the contention that serotonergic neurons provide a tonic excitatory input to SPNs.     

Development and migration of avian sympathetic preganglionic neurons.

Modern neuronanatomical techniques were used to investigate the development of the avian sympathetic preganglionic cell column in the spinal cord of the chick embryo. [3H]thymidine autoradiography indicated that the majority of these preganglionic, or "Terni column" neurons are generated between stages 18 and 24 (days 2-4). This coincides with the genesis of the somatic motoneurons in the thoracic levels of the cord, and therefore differences in the time of origin cannot explain the divergent fates of these two neuronal populations. Data obtained from short-survival autoradiographic experiments indicated that many early born cells remain close to the ventral region of the ventricular epithelium until day 5 of incubation. Ventral root injections used to label retrogradely neurons projecting an axon into the ventral root (Terni cells and somatic motoneurons) have labeled neurons next to the ventricular epithelium at the same early stages. Thus, it seems likely that some Terni cells, if not all, maintain medial positions and do not migrate laterally to join a common motor column before initiating a dorsal migration. Analysis of a closely staged series of embryos, whose Terni column neurons were retrogradely labeled with wheat germ agglutinin-horseradish peroxidase (WGA-HRP), revealed that between days 5 and 8 of incubation, Terni column neurons migrated dorsally to attain their adult position adjacent to the central canal. These changes in position were reflected in the changing morphology of the Terni column neurons, visualized by the Golgi-like HRP labeling. The positions of the migrating Terni cells differed from those of commissural cells, indicating that these fibers are not the substrate for the dorsal migration. The dorsal migration of Terni column cells was not disrupted by the surgical removal of the sympathetic ganglia, the synaptic targets of these neurons, nor by disruption of spinal afferents. Taken together, these results suggest that the migratory behavior of Terni cells in distinctive when compared to that of somatic motoneurons, and that local and/or intrinsic cues within the spinal cord guide the dorsal migration of Terni column cells.     

Slow EPSP and the depolarizing action of noradrenaline on sympathetic preganglionic neurons

Intracellular recordings were made from sympathetic preganglionic neurons of the lateral horn in slices of cat thoracic cord maintained in vitro. Focal electrical stimulation of the slice evoked, in addition to the already described fast EPSPs, EPSPs of several seconds duration. The slow EPSPs, like the fast EPSPs, were graded with stimulus intensity and were abolished by TTX or low Ca and high Mg superfusion. The slow EPSP decreased in amplitude with membrane hyperpolarization and was nullified at -90 mV but did not reverse with further hyperpolarization. The slow EPSP was abolished by phentolamine or prazosin but not by yohimbine. Noradrenaline NA, 10-50 microM) caused in 30% of neurons a TTX-resistant depolarization. The NA-evoked depolarization had the same characteristics as the slow EPSP with respect to sensitivity to membrane potential and to adrenergic blockers. These results suggest that NA, acting on an alpha 1-receptor, may be the mediator of the slow EPSP evoked in this neuron by focal stimulation.     

Segmental organization of sympathetic preganglionic neurons in the mammalian spinal cord

We have used retrograde transport of horseradish peroxidase to determine the distribution of the preganglionic cell bodies whose axons join particular rami of the thoracic spinal cord in a series of guinea pigs, and in a small number of hamsters and cats. In contrast to other recent studies, our results show that the neurons sending axons to a ramus are confined to a single segment at the corresponding spinal level. This segmental organization supports the idea that the rostro-caudal position of preganglionic cell bodies is one determinant of selective synapse formation between preganglionic axons and sympathetic ganglion cells.     

Sexual dimorphism in sympathetic preganglionic neurons of the rat hypogastric nerve

Horseradish peroxidase (HRP) applied to one hypogastric nerve labelled sensory neurons in T11-L3 dorsal root ganglia (DRG) bilaterally and preganglionic neurons (PGN) in the spinal cord segments T13-L3. An average of 130 small DRG neurons were labelled per animal (male or female). These were concentrated in the L1 + L2 DRGs (92%). About 75% were located ipsilateral to the site of HRP application. Central projections from DRG neurons were noted throughout Lissauer's tract and in the marginal zones (medial and lateral) near the borders of Lissauer's tract. A short projection was also seen extending to the dorsolateral funiculus. More than 90% of the preganglionic neurons were located in segments L1 + L2. Most of these were found in the dorsal commissural nucleus (75%) and most of the remainder were located bilaterally in the intermediolateral columns. Somewhat more intermediolateral neurons were labelled on the ipsilateral side than on the contralateral side. There were a few intercalating neurons and a very few funicular cells. An average of 415 PGNs were labelled in the male animals and 110 in the females, demonstrating a strong sexual dimorphism. No dimorphism was found in the sensory components.     

Migratory pathway of sympathetic preganglionic neurons in normal and reeler mutant mice

Our previous study showed that the migration of sympathetic preganglionic neurons (SPN) in the spinal cord is affected in the reeler mutant. The present study, using morphometric analysis to describe and compare the location of SPN at progressive developmental stages, provides detailed information on how SPN migrate in the presence or absence of the reelin gene. We found that the initial migration (prior to E11.5) of SPN from the neuroepithelium to the ventrolateral spinal cord is similar in both control (wild-type and heterozygous) and reeler mice. However, as development progressed (E12.5-E15.5), SPN in control mice migrated dorsally toward the intermediate lateral spinal cord region, where 80% settled to form the intermediolateral column (IML); the rest migrated medially to locations between the IML and the central canal. In reeler, 80% of SPN migrated dorsomedially to cluster around the central canal, with the rest distributed between the central canal and the intermediate lateral spinal cord region. The present study also examined the relationship among SPN, Reelin, and radial glial fibers in control and reeler mice. Confocal microscopic studies showed that during their initial migration, SPN in both control and reeler mice were closely apposed to radial glial fibers in the ventrolateral spinal cord. The majority of SPN in control mice then migrated dorsolaterally, in a direction perpendicular to radial glial fibers, to form the IML. In contrast, the majority of SPN in reeler migrated in the same orientation as radial glial fibers back toward the central canal, instead of migrating dorsolaterally to form the IML. A possible explanation for these results is that Reelin acts to prevent SPN from back-migration on radial glial fibers toward the central canal.     

Growth cones of chick sympathetic preganglionic neurons in vitro interact with other neurons in a cell-specific manner

The ability of the growth cones of sympathetic preganglionic neurons to recognize the neurons they encounter during their outgrowth and to react to them in a cell-type-specific manner may play a role in guiding them to appropriate targets during development in vivo. In this study, we examined the in vitro growth of sympathetic preganglionic neurons as they interacted with motor neurons, dorsal root ganglion neurons, and sympathetic ganglion neurons. All of these cell types might potentially be encountered by a growing preganglionic axon. The interaction of sympathetic preganglionic growth cones with each cell type was distinct. Sympathetic preganglionic growth cones fasciculated on motor-neuron neurites, collapsed after contact with the cell bodies and neurites of dorsal root ganglion neurons, and grew across the cell bodies and neurites of sympathetic ganglion neurons. These cell-type-specific responses stand in contrast to the collapse and retraction reported to be the most common growth-cone behaviors that result from contact between central and peripheral neurons in vitro and suggest that contact-mediated recognition might be sufficient for growth to and interaction with appropriate targets.     

Evidence for the coexistence of acetylcholine and enkephalin in the sympathetic preganglionic neurons of rats

The localization of the cholinergic neurons in the lower thoracic segments of the spinal cord of rats was examined by a monoclonal antibody against choline acetyltransferase (ChAT). The ChAT-immunoreactive neurons were located in the intermediate as well as anterior gray matters. In the intermediate gray the highest incidence of the immunoreactive neurons was in the nucleus intermediolateralis, followed by the nucleus intercalatus pars paraependymalis and a few immunoreactive neurons were seen in the nucleus intercalatus proprius. In the sequential immunostaining of one and the same section of the spinal cord pretreated with colchicine using the ChAT antibody and a polyclonal antibody against methionine-enkephalin-argynine-glycine-leucine (Met-Enk-Arg-Gly-Leu), substantial numbers of neurons were immunostained simultaneously by the two antibodies in the intermediate gray matter. The present finding gives strong evidence for the coexistence of acetylcholine and enkephalins in, at least, some of the preganglionic neurons projecting their axons to the periphery.     

Light and electron microscopic analyses of intraspinal axon collaterals of sympathetic preganglionic neurons

Experiments were performed in pigeons (Columba livia). Sympathetic preganglionic neurons (SPNs) in the first thoracic spinal cord segment (T1) were identified electrophysiologically using antidromic activation and collision techniques and then intracellularly labeled with horseradish peroxidase (HRP). In 6 of 10 HRP-labeled SPNs, the site of axon origin and intraspinal axonal trajectory could be specified. In 2 of the 6 HRP-labeled axons, the peripherally projecting process branched intraspinally. The presence or absence of SPN intraspinal axonal collateralization did not correlate with parent perikaryal subnuclear location or dendritic alignment. None of the collaterals were recurrent onto the SPN of origin. Light microscopically, the collateral branches appeared to end with punctate, bulbous swellings. The spinal regions of the terminal end swellings for the two axons did not overlap one another. In one instance the entire terminal field was confined within the principal preganglionic cell column (column of Terni). The other axon had collateral branches which terminated in the lateral white matter and in a ventrolateral region of lamina VII. A serial section, electron microscopic reconstructive analysis of the entire intraspinal collateral terminal field within the column of Terni revealed that: (a) the primary collateral process was unmyelinated and arose at a node of Ranvier; (b) after issuance of the collateral branch, the myelinated parent axon continued to increase its myelin wrapping throughout the spinal gray; (c) the bulbous swellings observed light microscopically corresponded to axon terminal boutons and regions of synaptic contact; (d) the axon collateral terminals were exclusively presynaptic to small caliber dendrites and formed only asymmetric specializations; and (e) the collateral terminals contained numerous mitochondria, and densely packed, electron-lucent, spherical vesicles.     

Reelin inhibits migration of sympathetic preganglionic neurons in the spinal cord of the chick

The present study examined the effects of Reelin in the migration of sympathetic preganglionic neurons (SPN) in the spinal cord of the chick. SPN in the chick first migrate from the neuroepithelium to the ventrolateral spinal cord. They then undergo a secondary migration to cluster adjacent to the central canal, forming the column of Terni (CT). During secondary migration, abundant Reelin is found in large areas of the ventral spinal cord; the only areas devoid of Reelin are areas occupied by SPN or somatic motor neurons and the pathway along which SPN migrate. Ectopic expression of Reelin in the pathway of SPN through electroporation of full-length Reelin DNA stopped SPN migration toward their destination. The spatiotemporal pattern of Reelin expression, along with the inhibition of SPN migration by exogenous Reelin, suggests that Reelin functions as a barrier to SPN migration during normal development of the spinal cord.