Kainic acid injection in NTS evokes hypertension and c-fos expression in spinal cord.

Kainic acid injected into rat nucleus tractus solitarius (NTS) caused a slowly developing hypertension, with a 2-fold increase in Fos-immunoreactive (Fos-IR) nuclei in the area of the presympathetic bulbospinal neurons in the rostral ventrolateral medulla (RVLM) and a widespread activation of sympathetic preganglionic neurons (SPN) in the spinal cord, particularly in the mid to lower thoracic cord. The highest segmental concentration of Fos-IR SPN was in T8, with Fos-IR nuclei increased 12-fold compared with the vehicle injected group. More than 60% of retrogradely labelled sympathoadrenal neurons in T8 were Fos-IR after kainic acid injection, consistent with the 60-fold increases in plasma adrenaline levels observed in these rats.     

Increased c-fos expression in spinal lumbosacral projection neurons and preganglionic neurons after irritation of the lower urinary tract in the rat.

Chemical irritation of the lower urinary tract (LUT) induces c-fos expression in neurons in the lumbosacral (L(6) and S(1)) spinal cord. This study used axonal tracing with fluorescent dyes to identify the types of spinal neurons expressing Fos immunoreactivity (IR) after LUT irritation in the rat. Fos-IR was detected in lateral and medial superficial dorsal horn, the sacral parasympathetic nucleus (SPN) and lamina X around the central canal. Fos-IR was detected in spinal neurons projecting to supraspinal sites (brainstem and hypothalamus), in preganglionic neurons (PGN) and in unlabeled segmental interneurons. A substantial percentage (20%) of dye labeled PGN exhibited Fos-IR after LUT irritation; and a larger percentage (36%) exhibited Fos-IR after electrical stimulation of the pelvic nerve which contains afferent pathways from all of the pelvic organs. The majority (average 55%) of Fos-positive neurons projecting to supraspinal sites were also located in the region of the SPN. A selective distribution of different types of neurons was detected in this region: PGN were located ventral to the spinal projection neurons which in turn were located ventral to the majority of unidentified Fos-positive neurons. The distribution of Fos-positive PGN and projection neurons was similar in spinal intact and spinal transected animals indicating that c-fos expression was mediated by monosynaptic afferent input or input from segmental interneurons and was not due to activation of supraspinal micturition reflex pathways.     

Increased c-fos expression in spinal lumbosacral projection neurons and preganglionic neurons after irritation of the lower urinary tract in the rat

Chemical irritation of the lower urinary tract (LUT) induces c-fos expression in neurons in the lumbosacral (L₆ and S₁) spinal cord. This study used axonal tracing with fluorescent dyes to identify the types of spinal neurons expressing Fos immunoreactivity (IR) after LUT irritation in the rat. Fos-IR was detected in lateral and medial superficial dorsal horn, the sacral parasympathetic nucleus (SPN) and lamina X around the central canal. Fos-IR was detected in spinal neurons projecting to supraspinal sites (brainstem and hypothalamus), in preganglionic neurons (PGN) and in unlabeled segmental interneurons. A substantial percentage (20%) of dye labeled PGN exhibited Fos-IR after LUT irritation; and a larger percentage (36%) exhibited Fos-IR after electrical stimulation of the pelvic nerve which contains afferent pathways from all of the pelvic organs. The majority (average 55%) of Fos-positive neurons projecting to supraspinal sites were also located in the region of the SPN. A selective distribution of different types of neurons was detected in this region: PGN were located ventral to the spinal projection neurons which in turn were located ventral to the majority of unidentified Fos-positive neurons. The distribution of Fos-positive PGN and projection neurons was similar in spinal intact and spinal transected animals indicating that c-fos expression was mediated by monosynaptic afferent input or input from segmental interneurons and was not due to activation of supraspinal micturition reflex pathways.     

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.     

Guanethidine sympathectomy increases substance P concentration in the superior sympathetic ganglion of adult rats

Adult rats received intraperitoneal injections of guanethidine or saline for 5 weeks. Six to 8 weeks following completion of treatment, concentrations of substance P and neuropeptide Y (NPY) were measured by radioimmunoassay in the superior cervical ganglion (SCG) and thoracic spinal cord. The SCG was also immunostained for NPY and substance P. No differences were observed in thoracic spinal cord content of either NPY or substance P. We observed depletion of NPY immunoreactive neurons and NPY levels in the SCG, and pharmacologic evidence of postganglionic denervation in guanethidine-treated rats. In guanethidine-treated rats, there was a marked increase of substance P levels in the SCG, where substance P was localized in fibers, but not cell bodies. Thus, sprouting of substance P-containing sensory fibers in the sympathetic ganglia occurs following postganglionic sympathectomy in adult rats.     

Neuropeptide Y mRNA expression in interneurons in rat spinal cord.

Neuropeptide Y (NPY)-immunoreactive axons are present within the spinal cord. Some of these axons originate from neurons in the brainstem. Other axons arise from within the spinal cord since NPY-immunoreactivity can be detected after complete spinal cord transection. To identify spinal neurons that might express NPY, we localized NPY mRNA in rat spinal cord using in situ hybridization histochemistry. NPY mRNA-containing neurons were localized in the dorsal horn, in medial laminae of the grey matter and in the lateral spinal nucleus in thoracic, lumbar and sacral cord. The location of some of these neurons, and their proximity to sympathetic preganglionic neurons, suggest some NPY-containing interneurons are likely to be involved in spinal as well as supraspinal autonomic reflex pathways.     

Immunohistochemical demonstration of some putative neurotransmitters in the lamprey spinal cord and spinal ganglia: 5-hydroxytryptamine-, tachykinin-, and neuropeptide-Y-immunoreactive neurons and fibers

The distribution of some putative neurotransmitters was investigated in the spinal cord and spinal ganglia of the lamprey, a primitive vertebrate, by using immunohistochemical methods. In the spinal cord a midline row of 5-hydroxytryptamine (5-HT)-immunoreactive neurons was present immediately ventral to the central canal over the entire length of the spinal cord. The ventral processes of these neurons formed a dense ventromedial plexus of varicosities. In the dorsal, lateral, and ventral spinal axon columns, several longitudinal 5-HT fibers were present. After chronic spinal transections the distribution of 5-HT fibers was unchanged; it is therefore concluded that there was no substantial descending 5-HT contribution and that the spinal 5-HT neurons supplied the regional 5-HT innervation. The spinal 5-HT cells sent fibers into the dorsal and ventral roots; 5-HT cell bodies and fibers were also present in the spinal dorsal root ganglia, in their dorsal, ventral, and lateral nerve branches, and in the dorsal and ventral branches of the ventral roots. Neurons and fibers containing peptides of the tachykinin (TK) family (to which, amongst others, substance P belongs) were found in the spinal cord. TK neurons in the spinal cord supplied the local TK innervation, as well as TK fibers in the dorsal and ventral roots. Fibers have been found containing either TK, or 5-HT, or both compounds. Neurons containing neuropeptide-Y (NPY)-immunoreactive material were present in a medial column just dorsal to the central canal. The NPY neurons have longitudinal, mainly descending, fibers that provide the local NPY innervation of the lamprey spinal cord. The present results provide evidence for local spinal systems containing 5-HT, TK, 5-HT and TK, or NPY, but in contrast to mammals, these compounds do not seem to arise from supraspinal neurons.     

Pursuit of the origin of the large myelinated fibers of the anterolateral funiculus in the spinal cord in humans in relation to the pathomechanism in amyotrophic lateral sclerosis

To determine the origin of the large myelinated fibers in the anterolateral funiculus (ALF) in the spinal cord of humans, myelinated fibers in the ALF of the mid-cervical spinal cord were examined quantitatively. Five groups of subjects were examined, consisting of control subjects, patients with cerebral lesions and showing complete degeneration of the unilateral/bilateral pyramis of the medulla oblongata, those with lesions of the pontine tegmentum, those with lesions of the lower cervical spinal cord, and those with thoracic/lumbar lesions. The results indicate that the large myelinated fibers in the ALF of the mid-cervical spinal cord of humans originate from the tegmentum of the brain stem and the lower cervical spinal cord, and not from the cerebrum, or the thoracic or lumbar spinal cord. Thus, they are descending fibers from the brain stem tegmentum and ascending fibers from the lower cervical cord, and not corticospinal tracts or long-ascending fibers from the thoracic or lumbar spinal cord. The origin of the large myelinated fibers in the ALF of the spinal cord in humans, the number of which was severely decreased in patients with amyotrophic lateral sclerosis, is considered to be the long-descending neurons in the brain stem tegmentum and the propriospinal neurons in the spinal cord. Received: 23 December 1998 / Revised, accepted: 29 March 1999     

Glutamate-immunoreactive synapses on retrogradely-labelled sympathetic preganglionic neurons in rat thoracic spinal cord

Retrograde tracing with cholera toxin B subunit (CTB) combined with post-embedding immunogold labelling was used to demonstrate the presence of glutamate-immunoreactive synapses on sympathetic preganglionic neurons that project to the adrenal medulla or to the superior cervical ganglion in rat thoracic spinal cord. At the electron microscope level, glutamate-immunoreactive synapses were found on retrogradely labelled nerve cell bodies and on dendrites of all sizes. Two-thirds of the vesicle-containing axon profiles that were directly apposed to, or synapsed on, CTB-immunoreactive sympathoadrenal neurons were glutamate positive. The proportion of glutamate-immunoreactive contacts and synapses on sympathoadrenal neurons decreased to zero when the anti-glutamate antiserum was absorbed with increasing concentrations of glutamate from 0.1 mM to 10 mM. Double immunogold labelling for glutamate and gamma-aminobutyric acid (GABA) showed that glutamate-immunoreactive profiles did not contain GABA and that GABA-immunoreactive profiles did not contain glutamate. These results suggest that glutamate is the major excitatory neurotransmitter to sympathoadrenal neurons and possibly to other sympathetic preganglionic neurons in the intermediolateral cell column of the spinal cord.     

Growth of central substance P-containing neurons into superior cervical ganglia transplanted in the spinal cord of adult rats

Superior cervical ganglia (SCG) contain substance P-like immunoreactive (SP-IR) fibers but not SP-IR neurons. In the present study, SCG were excised from adult rats and transplanted into the same animal's spinal thoracic cord (Th10). One or two weeks after the operation, SP-IR fibers from the host spinal cord or a higher level had grown and entered the transplanted SCG where they formed direct contacts with SCG neurons. However, these phenomena could not be observed when dorsal root ganglia (L4), which contained numerous SP-IR cells, were transplanted into their own spinal cord (Th10). This suggests that the SP-IR neuron system in the adult is able to grow "new axons' to the grafted tissue to form a "new SP-IR' neuronal circuit when the grafted tissue has lost its own SP-IR input.     

Independent inputs by VGLUT2- and VGLUT3-positive glutamatergic terminals onto rat sympathetic preganglionic neurons

To characterize glutamatergic axon terminals onto sympathetic preganglionic neurons (SPNs), we visualized immunohistochemically three vesicular glutamate transporters (VGLUTs) in the intermediolateral cell column (IML) of rat thoracic spinal cord. VGLUT2 and VGLUT3 immunoreactivities but not VGLUT1 immunoreactivity were distributed in the IML and found in terminals making asymmetric synapses and apposed to dendrites immunopositive for choline acetyltransferase, an SPN marker. VGLUT2 and VGLUT3 immunoreactivities were not co-localized with each other. A population of VGLUT2-immunoreactive but not VGLUT3-immunoreactive terminals were adrenergic or noradrenergic. Some of VGLUT3-immunoreactive but not VGLUT2-immunoreactive terminals contained serotonin. These results indicate at least two independent glutamatergic terminal populations, which include a distinct monoaminergic subpopulation, making excitatory inputs onto SPNs.     

Alpha-2-adrenergic receptors in avian spinal cord: increases in apparent density associated with the sympathetic preganglionic cell column

Vertebrate spinal cord receives a dense and diversified catecholaminergic innervation from brainstem and diencephalon. Within the spinal gray, the densest terminations appear to be within the neuropil surrounding sympathetic preganglionic neurons (SPNs) in thoracic spinal cord. Results of recent iontophoresis investigations showed that several catecholamines and clonidine, an alpha-2 agonist, uniformly inhibited the maintained discharge activity of SPNs [19]. These experiments raised the possibility that the inhibitory effects might be mediated by activation of an alpha-2-adrenergic receptor. The present series of ligand binding studies provide biochemical evidence suggesting the presence of alpha-2-adrenergic receptors in the SPN cell column. Total specific binding (Bmax) of the radiolabeled agonists clonidine (CLO) and para-amino-clonidine (PAC) (at concentrations above and below apparent KDS) was significantly greater in thoracic spinal cord in comparison with cervical spinal cord (P less than 0.001). The elevated levels in thoracic spinal cord were entirely accounted for by increases in apparent receptor density in dorsal horn and the SPN cell column (inclusive of the adjoining intermediate spinal laminae) (P less than 0.005). Adrenergic receptor subtype specificity of [3H]PAC was tested in competitive inhibition experiments. The results confirmed that [3H]PAC is a preferential alpha-2 agonist in thoracic and cervical spinal cord, and indicated the following rank order of potency for its displacement: norepinephrine = yohimbine much greater than prazosin greater than propranolol.     

Distinct subpopulations of cyclic guanosine monophosphate (cGMP) and neuronal nitric oxide synthase (nNOS) containing sympathetic preganglionic neurons in spontaneously hypertensive and Wistar-Kyoto rats

The sympathetic preganglionic neurons (SPN) of the intermediolateral cell column (IML) play a critical role in the maintenance of vascular tone. We undertook a comparative neuroanatomical analysis of neuronal nitric oxide synthase (nNOS) expression in the SPN of the mature normotensive Wistar Kyoto (WKY) and spontaneously hypertensive rat (SHR). The anatomical relationship between nNOS and the NO signaling molecule cyclic guanosine monophosphate (cGMP) was also determined. All animals were male, age > 6 months. Fluorogold (FG) retrograde labeling of SPN (detected with immunohistochemistry) was combined with NADPH-diaphorase histochemistry for NOS in the thoracic spinal cord (T1-11, n = 5 WKY, 5 SHR). There was no difference in the total number of FG-labeled SPN (WKY 6,542 +/- 828, SHR 6,091 +/- 820), but the proportion of FG-labeled cells expressing NOS was significantly less in the SHR (WKY 64.4 +/- 5.1 vs. SHR 55.6 +/- 2.1, P < 0.05). Fluorescence immunohistochemistry for nNOS/cGMP (n = 4 WKY, 4 SHR) was also performed. Confocal microscopy revealed that all nNOS-positive SPN contain cGMP and confirmed a strain-specific anatomical arrangement of SPN cell clusters. A novel subpopulation of cGMP-only cells were also identified. Double labeling for cGMP and choline acetyltransferase (n = 3 WKY, 3 SHR), confirmed these cells as SPN in both WKY and SHR. These results suggest that cGMP is a key signaling molecule in SPN, and that a reduced number of NOS neurons in the SHR may play a role in the increase in sympathetic tone associated with hypertension in these animals.     

Neuropeptide Y in human spinal cord

The distribution of a newly described peptide, neuropeptide Y (NPY) within the human spinal cord has been determined using radioimmunoassay and immunocytochemistry. Higher concentrations were found in the lumbar (49.9 +/- 6.8 pmol/g) and sacral (47.0 +/- 10.6 pmol/g) regions than in the cervical (27.6 +/- 2.7 pmol/g) and thoracic spinal cord (33.8 +/- 5.3 pmol/g). Immunocytochemistry revealed numerous nerve fibers containing NPY in the spinal cord; these were particularly concentrated in the substantia gelatinosa of the dorsal horn. In the ventral spinal cord NPY-containing nerves were sparse becoming more abundant in lumbosacral segments.     

Vasoactive intestinal polypeptide cerebrospinal fluid-contacting neurons of the monkey and cat spinal central canal

Neurons immediately adjacent to the central canal were demonstrated in the cat and monkey to be immunoreactive for the peptide vasoactive intestinal polypeptide (VIP), by means of the peroxidase antiperoxidase method. Most of the cells were found in the thoracic and sacral segments, although a few were present at each level. The thoracic neurons were multipolar and either ependymal or subependymal; they usually had a large, thick dendrite that was oriented radially toward the center of the central canal; this dendrite penetrated through the ependymal layer and ended as a large, fringed podlike process (4-5-microns diameter) along the canal surface in contact with the cerebrospinal fluid (CSF). From the basal surface of the thoracic cell arose several small dendrites and a varicose axon. A few of the thoracic VIP neurons also contained two nuclei. In the sacral cord, the VIP neurons that lie along the central canal were of several types. They were round or multipolar and were either subependymal, within the ependyma, or supraependymal. Many had long dendrites and thin varicose axons stretching for long distances parallel to the cord surface. Other VIP neurons were smaller cells with short, highly branched, varicose processes. Most prominent in the sacral cord of the cat was a massive intricate network of intensely labelled processes extending in parallel along the canal surface. This network contained thick dendrites, highly varicose axons, and small neurons. Electron microscopy demonstrated VIP axons and varicosities containing small round clear vesicles and dense core vesicles. These processes were in desmosomal contact with ependymal cells and in direct contact with the CSF space. VIP processes were also found along the pial surface of the spinal cord at each level. In some cases single axons and bundles of axons arising from the area around the central canal could be traced to terminal fields along the ventral median fissure and the ventral and ventral lateral surface. In summary, the cat and monkey spinal canal is richly innervated by VIP neurons with elaborate processes in contact with the cerebrospinal fluid; further, some of these neurons may also extend axons to the ventral surface of the spinal cord. In these aspects, these cells resemble CSF-containing neurons previously described in lower species.     

Hypothalamic paraventricular axons projecting to the female rat lumbosacral spinal cord contain oxytocin immunoreactivity

Oxytocin-containing axons project from the hypothalamic paraventricular nucleus to the neurohypophysis and thoracic spinal cord to ultimately influence uterine contractions and autonomic activity, respectively. Whether or not oxytocin-immunoreactive axons project to the female rat lumbosacral spinal cord to influence autonomic outflow to pelvic organs has not been investigated. Thus, the present study was designed to investigate the presence, distribution, and origin of oxytocin-immunoreactive axons in the female rat lumbosacral spinal cord. Immunohistochemistry, spinal cord transections, and axonal tracing with Fluorogold, True Blue, and pseudorabies virus were used. Oxytocin-immunoreactive nerve fibers were present in the L6/S1 segments of the spinal cord. Prominent varicose axons were evident throughout the dorsal horn, along the lateral and medial collateral pathways, in the dorsal intermediate gray area, around the central canal in lamina X, and throughout the sacral parasympathetic nucleus. Injection of retrograde tracer into the L6/S1 spinal cord labeled neurons in the hypothalamic paraventricular nucleus. Transection of the thoracic spinal cord eliminated oxytocin-immunoreactive nerve axons in the L6/S1 spinal cord. In addition, transection of the thoracic spinal cord eliminated transport of retrograde axonal tracer from the L6/S1 spinal cord to the paraventricular nucleus. Pseudorabies virus, a transneuronal retrograde tracer, injected into the uterus and cervix marked uterine-related preganglionic neuronal cell bodies in the sacral parasympathetic nucleus and uterine-related neurons in the hypothalamic paraventricular nucleus. Double immuno-labeling of viral-infected spinal cord sections showed oxytocin-immunoreactive axons closely associated with viral labeled uterine-related preganglionic cell bodies of the sacral parasympathetic nucleus. The results of this study revealed that oxytocin-immunoreactive neurons of the hypothalamic paraventricular nucleus project axons to the lumbosacral spinal cord to areas involved in sensory processing and parasympathetic outflow to the uterus.     

Evidence of disynaptic projections from the rostral ventrolateral medulla to the thoracic spinal cord

Sympathetic outflow is regulated by a direct pathway of the rostral ventrolateral reticular formation (rvlm) to the thoracic spinal cord. For the first time, a dual retrograde/anterograde transport technique was used to demonstrate by light microscopy, potential disynaptic pathways from the rvlm to the thoracic spinal cord in the rat. An anterograde tracer, biotinylated dextran amine (BDA) was injected into the rvlm and a retrograde tracer, FluoroGold (FG) deposited into the upper thoracic spinal cord in the same animal. Rostral ventrolateral medullary efferents labeled with BDA were apposed to thoracic reticulospinal neurons labeled with FG in the ventrolateral tegmentum, ipsilateral and contralateral to the injection site in the rvlm. Suggestive evidence was obtained of synaptic interactions with neuronal somata and proximal dendrites. The results support the idea that the rvlm projects to the thoracic cord via disynaptic, intrareticular pathways paralleling the well established monosynaptic projection.     

The termination pattern and postsynaptic targets of rubrospinal fibers in the rat spinal cord: a light and electron microscopic study.

The spinal course, termination pattern, and postsynaptic targets of the rubrospinal tract, which is known to contribute to the initiation and execution of movements, were studied in the rat at the light and electron microscopic levels by using the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L) in combination with calbindin-D28k (CaBP), gamma-aminobutyric acid (GABA), and glycine immunocytochemistry. After injections of PHA-L unilaterally into the red nucleus, labelled fibers and terminals were detected at cervical, thoracic, and lumbar segments of the spinal cord. Most of the descending fibers were located in the dorsolateral funiculus contralateral to the injection site, but axons descending ipsilaterally were also revealed. Rubrospinal axon terminals were predominantly found in laminae V-VI and in the dorsal part of lamina VII at all levels and on both sides of the spinal cord, but stained collaterals were also seen in the ventrolateral aspect of Clark's column and in the ventral regions of lamina VII on both sides. The proportion of axonal varicosities revealed on the ipsilateral side varied at different segments and represented 10-28% of the total number of labelled boutons. Most of the labelled boutons were engaged in synaptic contacts with dendrites. Of the 137 rubrospinal boutons investigated, only 2 were found to establish axosomatic synaptic junctions in the lumbar spinal cord contralateral to the PHA-L injection. With the postembedding immunogold method, 80.8% of dendrites establishing synaptic contacts with rubrospinal terminals did not show immunoreactivity for either GABA or glycine, whereas 19.2% of them were immunoreactive for both amino acids. Rubrospinal axons made multiple contacts with CaBP-immunoreactive neurons in laminae V-VI. Synaptic contacts between rubrospinal terminals and CaBP-immunoreactive dendrites were identified at the electron microscopic level, and all CaBP-containing postsynaptic dendrites investigated were negative for both GABA and glycine. The results suggest that rubrospinal terminals establish synaptic contacts with both excitatory and inhibitory interneurons in the rat spinal cord, and a population of excitatory interneurons receiving monosynaptic rubrospinal input is located in laminae V-VI.