A light and electron microscopic analysis of the sacral parasympathetic nucleus after labelling primary afferent and efferent elements with HRP

Primary afferent input to the cat sacral parasympathetic nucleus (SPN) has been examined by injury filling sacral dorsal roots, ventral roots, or both with horseradish peroxidase (HRP). Appropriate spinal segments were processed for the demonstration of HRP with diaminobenzidine and prepared for sequential light (LM) and electron (EM) microscopy. At the LM level, a large fascicle of primary afferent fibers was observed passing ventrally along the lateral edge of the dorsal horn into the region of the SPN. Varicosities were seen throughout the course of the axons but were particularly abundant within the SPN. Injury filling of the ventral roots with HRP resulted in a Golgi-like labelling of preganglionic neurons and their dendritic arbors, as well as ventral root afferent fibers. Swellings on both dorsal and ventral root afferent axons were observed in close apposition to labelled preganglionic neurons and their dendrites. At the ultrastructural level, afferent terminals were found to contain clear spherical vesicles; 66% of these terminals also contained at least one dense-cored vesicle. Of particular interest was the presence of labelled dorsal and ventral root afferent terminals synapsing on labelled preganglionic neurons. Preganglionic neurons were also postsynaptic to unlabelled terminals containing clear spherical (79.7%) or pleomorphic vesicles (20.3%). These data indicate that preganglionic neurons receive direct input from several sources, and provide the first demonstration of direct input to these cells from sensory fibers in the dorsal and ventral roots. The connections described in the present study provide interesting and, as yet, unexplored possibilities for sensory and autonomic reflex integration.     

Primary afferent projections from dorsal and ventral roots to autonomic preganglionic neurons in the cat sacral spinal cord: light and electron microscopic observations

HRP applied to cut dorsal and ventral roots of the cat sacral spinal cord labeled afferent axons with swellings in close apposition to labeled preganglionic neurons (PGNs) in the sacral parasympathetic nucleus. Electron microscopy allowed characterization of synaptic contacts between afferents and PGNs. The results suggest that both the dorsal and ventral root afferents can directly activate autonomic preganglionic neurons.     

The role of neuropeptides in the sacral autonomic reflex pathways of the cat

Immunohistochemical and pharmacological studies were conducted to examine the origin and function of peptidergic nerves in the sacral autonomic system of the cat. Leucine-enkephalin (L-Enk) immunoreactivity was identified in nerve terminals in peripheral ganglia on the surface of the urinary bladder and in the parasympathetic nucleus in the sacral spinal cord. In colchicine-treated animals L-Enk was also detected in sacral preganglionic neurons (sPGN) identified by retrograde transport of a fluorescent dye. L-Enk terminals in bladder ganglia are believed to arise from sPGN since the terminals were eliminated by transection of the sacral ventral roots. Pharmacological studies indicated that exogenous as well as endogenously released enkephalins have an inhibitory action at both ganglionic and spinal sites in the sacral outflow to the urinary bladder. Peptides were also associated with afferents nerves in the sacral autonomic system. The distribution of substance P, VIP and cholecystokinin in the sacral dorsal horn paralleled the distribution of visceral afferent projections as demonstrated with HRP techniques. Dye labeling combined with immunohistochemistry revealed that some dorsal root ganglion cells projecting to the pelvic viscera contain substance P or VIP.     

Identification of preganglionic parasympathetic neruons in the sacral spinal cord of the cat

The preganglionic parasympathetic neurons in the sacral spinal cord of the cat have been demonstrated by retrogade changes following section of the pelvic nerve. Transection of the pelvic nerve twice, a week apart, was necessary to produce reliable signs of chromatolysis in the preganglionic neurons. Serial sections of the sacral spinal cord were made and the location of affected cells plotted. The sacral parasympathetic nucleus was located in the intermediate region of S-2 and S-3. The majority of the perikarya were located in the intermediolateral cell column, but a significant number were also found in the intermediomedial column. The distribution of afferent fibers in the pelvic nerve was demonstrated by chromatolysis in cells of the dorsal root ganglia. Retrograde changes were limited to the ganglia of S-2 and S-3 in five cats, while a few cells with chromatolysis were found also in the S-1 ganglion of four cats.     

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.     

Bilateral projections of the pontine micturition center to the sacral parasympathetic nucleus in the rat

Previous work has revealed that pontine micturition center (PMC) neurons send projections to the sacral parasympathetic nucleus (SPN) of the intermediolateral (IML) regions of L6-S1 spinal cord segments in rats. Although unilateral SPN injections will retrogradely label PMC neurons bilaterally, it is not known whether single PMC neurons project bilaterally to the SPN. There may be two different populations of PMC neurons on each side of the brainstem, with both groups independently connecting to the SPNs on opposite sides of the spinal cord. To verify one of these alternatives, a small injection of either rhodamine-labeled latex microspheres or a red fluorescent emulsion was made into the SPN on one side of the cord; a similar injection of either fluorescein-tagged microspheres or a green fluorescent emulsion was made into the other. After at least seven days, the rats were perfused. Inspection of 40 μm cord sections confirmed the similar placement of these injections along the rostrocaudal axis of the cord and that no tracer had spread across midline. Thirty-micron brain sections were examined for filled neurons. Red, green and double labeled neurons were found bilaterally in the PMC, subcoeruleus, and A5 regions. Although some red nucleus cells were also filled, they were only singly labeled and always located contralateral to the injection. Finally, immunohistochemical staining of dopamine-β-hydroxylase (DBH) containing cells confirmed that some labeled cells were also noradrenergic. We therefore conclude that some PMC, subcoeruleus, and A5 neurons send axons to the SPN on both sides of the lumbosacral cord.     

The subdivisions of the intermediolateral nucleus in the sacral spinal cord of the cat

The subdivisions of the sacral intermediolateral nucleus (IML) of the cat have been studied by using a double-labeling technique of retrograde Fluoro-gold (FG) and wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) tracing. The parasympathetic preganglionic neurons (PGNs) that were labeled by the FG injected into the pelvic nerve formed a ‘V’-shaped column known as the sacral parasympathetic nucleus (SPN) in the sacral IML. The neurons that were labeled by the WGA-HRP applied to the lateral parabrachial nucleus (PBL) formed an elongated spindle-shaped column extending throughout the IML of the sacral segments. We designated it by the name of sacral visceral sensory nucleus (SVSN). These findings indicate that the sacral IML of the cat contain two distinct subdivisions, SPN and SVSN.     

Distribution of neurons in the lateral pontine tegmentum projecting to thoracic, lumbar and sacral spinal segments in the cat

The course of descending fibers projecting to the spinal cord and the arrangement of their parent cells located in various nuclei of the dorso-lateral pontine tegmentum were studied using the horseradish peroxidase (HRP) retrograde axonal transport technique. Retrogradely labeled neurons were found in the locus coeruleus (LC), subcoeruleus (SC), K?lliker-Fuse nucleus (KF) and in the lateral parabrachial nucleus (LPB) after HRP injections into various spinal segments. Neurons innervating the thoracic spinal cord were found to be arranged in the ventral portion of the LC and in the entire SC; their axons descended ipsilaterally. Neurons with descending axons to lumbar segments were seen mainly in the ventral portion of the LC and in the medial portion of SC. Most of their axons were also seen to descend ipsilaterally. Neurons projecting to sacral segments occurred in the entire LC and in the medial portion of the SC. Large part of descending fibers crossed the midline at the level of (or near) the termination site. Neurons of all portions of the KF and LPB projected to the thoracic spinal cord only ipsilaterally, while many descending fibers innervating the sacral segments crossed the midline.     

Spinal and brain circuits to motoneurons of the bulbospongiosus muscle: retrograde transneuronal tracing with rabies virus.

Retrograde transneuronal tracing with rabies virus from the left bulbospongiosus muscle (BS) was used to identify the neural circuits underlying its peripheral and central activation. Rats were killed at 2, 3, 4, and 5 days post-inoculation (p.i.). Rabies immunolabelling was combined with immunohistochemical detection of choline acetyltransferase and oxytocin. Virus uptake was restricted to ipsilateral BS motoneurons (2 days p.i.). The onset of transfer (3 days p.i.) visualized interneurons in the dorsal grey commissure (DGC), intermediate zone, and sacral parasympathetic nucleus (SPN), mainly in DGC at L5-S1, and revealed synaptic connections between BS and external urethral sphincter motoneurons. At 4 and 5 days p.i., higher-order interneurons were labelled in other spinal areas and segments. Supraspinal labelling initially involved only Barrington's nucleus, nucleus reticularis magnocellularis, and paragigantocellularis lateralis (4 days p.i.). Later, labelling extended to other populations traditionally associated with control of sexual activity and micturition (periaqueductal grey, paraventricular nucleus, medial preoptic area, prefrontal cortex), but also indicated the intervention of somatic descending motor pathways (vestibulospinal and reticulospinal neurons, "hindlimb" regions of sensorimotor cortex and red nucleus) and cerebellar nuclei in multisynaptic innervation of the labelled motoneurons. Dual color immunofluorescence disclosed multisynaptic links between these motoneurons and thoracolumbar medial sympathetic (choline acetyltransferase-immunoreactive) neurons. In contrast, preganglionic neurons in SPN and most oxytocinergic neurons in paraventricular hypothalamic nucleus remained unlabelled, suggesting that parasympathetic and somatic outflow to pelvic organs are probably controlled by separate interneuronal populations and that oxytocinergic spinal projections are more likely to influence sacral autonomic rather than somatic outflow.     

Neurons in the sacral parasympathetic nucleus that project to the hypothalamus do not also project through the pelvic nerve--a double labeling study combining Fluoro-gold and cholera toxin B in the rat

We recently reported that neurons in the sacral parasympathetic nucleus (SPN) project directly to the hypothalamus. In the present study, we examined the possibility that individual neurons in SPN send both an axon into the pelvic nerve and an ascending projection to the hypothalamus. We used a new double-labeling technique in which two sensitive retrograde tracers (Fluoro-gold and cholera toxin subunit B immunocytochemically stained with rhodamine-labeled antibodies) were combined. The effectiveness of this combination for singly and doubly labeling neurons was established in experiments in which both tracers were injected into overlapping areas of the tongue or ventrobasal thalamus. These injections doubly labeled large numbers of neurons in the hypoglossal or dorsal column nuclei, respectively. In studies of the projections of neurons in the SPN, injection of one tracer into the hypothalamus and the other into the pelvic nerve and/or pelvic ganglion singly labeled many neurons (more than 3300 in the 7 examined cases). However, no SPN neurons were doubly labeled. These findings indicate that the SPN in the rat consists of at least two distinct groups of cells, parasympathetic preganglionic neurons and neurons that project to the hypothalamus.     

The components of the hypogastric nerve in male and female guinea pigs

A quantitative study has been made of the neural components of the hypogastric nerves of male and female guinea pigs using retrograde transport of horseradish peroxidase (HRP) to identify the population of neurones projecting in the nerve trunk, and electronmicroscopic analysis of the myelinated and unmyelinated axons present. Application of HRP to the transected axons of the hypogastric nerve labelled the cell bodies of sensory neurones in lumbar and sacral dorsal root ganglia, preganglionic neurones in the lumbar and sacral spinal cord, and postganglionic neurones in the inferior mesenteric ganglion and in the lumbar paravertebral chain; some ganglion cells of the pelvic plexus were also labelled. The number and distribution of each type of neurone with axons in the hypogastric nerve differed between the sexes: in particular, about twice as many preganglionic axons were present in the male as in the female.     

Location and morphology of parasympathetic preganglionic neurons in the sacral spinal cord of the cat revealed by retrograde axonal transport of horseradish peroxidase

The distribution and morphology of preganglionic neurons in the sacral parasympathetic nucleus (SPN) of the cat have been studied with the horseradish peroxidase (HRP) tracing technique. HRP applied to the cut pelvic nerve was identified in cells located ipsilaterally, primarily in the intermediate gray matter. They formed a column approximately 10 mm long, usually contained within two, but occasionally three, sacral segments. S₂ contained a majority of cells. In transverse sections the SPN had the appearance of an inverted “L.” Cells were medium-sized, oval or spindle-shaped, and transversely oriented. They were distributed among two major components and one minor one: (1) dorsal band (34%) located mainly in lamina V beneath the dorsal horn (cells and dendrites horizontally oriented), (2) lateral band (64%) along the lateral edge of the gray matter in laminae VII through V (cells oriented dorsoventrally with dendrites extending within the nucleus and into the dorsolateral funiculus), and (3) a small group (2%) of cells at the rostral end of the SPN in lamina VII in the middle of the ventral horn. These data coupled with the results of other investigations indicate that the SPN has a viscerotopic organization wherein the colon is innervated primarily by cells in the dorsal band and the urinary bladder is innervated primarily by cells in the lateral band.     

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.     

Bulbospinal respiratory neurons are a source of double synapses onto phrenic motoneurons following cervical spinal cord hemisection in adult rats

The purpose of this study was to determine if the medullary neurons that provide the primary excitatiry drive to phrenic motoneurons (i.e., rostral ventral respiratory group, rVRG) are a source of double synapse formation in the phrenic nucleus after spinal cord hemisection. The axons of rVRG neurons either ipsilateral or contralateral to the hemisection were labeled by injection of a mixture of HRP and WGA-HRP into the rostral ventral respiratory group. Phrenic motoneurons ipsilateral and caudal to the hemisection were labeled by the retrograde transport of HRP. The ultrastructural results indicated that after hemisection, rVRG neurons from both sides of the medulla formed labelled double synapses in the phrenic nucleus.     

Nerve growth factor receptor immunoreactivity in the neuronal perikarya of human sensory and sympathetic nerve ganglia

We examined immunohistochemically the dorsal root ganglia, sympathetic ganglia, spinal cord, ventral and dorsal roots, and sciatic nerves obtained at autopsy from adult humans, using a monoclonal antibody against the human nerve growth factor receptor. We observed labelling in a granular pattern in the neuronal perikarya of dorsal root and sympathetic nerve ganglia. Ventral horn cells and axons were not labelled.     

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.     

Evidence for invasion of regenerated ventral root afferents into the spinal cord of the rat subjected to sciatic neurectomy during the neonatal period

Sectioning the sciatic nerve of experimental animals at the neonatal stage triggers growth of afferent fibers in the ventral root. The present study examined the possibility that the regenerating fiber terminals grow into the spinal cord. The sciatic nerve on one side was cut in neonatal rats. After the rats were fully grown, either an electrophysiological or a histochemical study was performed. The results of electrophysiological experiments showed that stimulation of certain loci in the L5 spinal cord evoked antidromic potentials in the L5 ventral root with a long latency. Various evidence suggests that the long latency potentials are due to activation of C fibers. These C-fiber potentials were on average bigger and were elicited from more numerous loci on the side ipsilateral to the sciatic nerve lesion than on the contralateral side. Furthermore, stimulation of the spinal cord of unoperated normal rats rarely evoked such potentials. For the histochemical study, horseradish peroxidase (HRP) was injected into the L5 spinal cord after cutting the L4-L6 dorsal roots. A lot more cells in the L5 dorsal root ganglion (DRG) on the side ipsilateral to the sciatic nerve lesion were labeled with HRP transported retrogradely through the L5 ventral root than on the contralateral side. Control experiments showed that few DRG cells are labeled with HRP in normal unoperated rats. The combined results of the electrophysiological and histochemical studies suggest invasion of ventral root afferents into the spinal cord, given enough postoperative time. It is not known whether or not these terminals make functional synaptic contacts in the spinal cord.     

Composition and central projections of the pudendal nerve in the rat investigated by combined peptide immunocytochemistry and retrograde fluorescent labelling

The central distribution of the afferent and efferent projections of pelvic striated muscles, the pudendal and sciatic nerves, were systemically investigated in rats by retrograde tracing techniques combined with immunocytochemistry using antibodies to 9 neuropeptides. True Blue was injected into either the pelvic muscles, pudendal or sciatic nerves. Seven days later the spinal cord and dorsal root ganglia (L3-S2 levels) were processed for immunocytochemistry. Injection of tracer into the pelvic muscles labelled dorsomedial, ventral and dorsolateral motoneuron groups of the L6 segment and a few sensory neurons in the respective dorsal root ganglia. Pudendal nerve injection also labelled the same motoneuron groups, 50% of neurons of the retrodorsolateral column and numerous sensory neurons of the dorsal root ganglion. Concomitant labelling of pudendal and sciatic nerves with different fluorescent tracers revealed a small number of double-labelled cells in the dorsal root ganglia but only single-labelled cells in the retrodorsolateral nucleus. Enkephalin-, somatostatin- and neuropeptide Y-containing fibres were particularly abundant in and around dorsomedial and dorsolateral groups as well as the intermediolateral cell column. We conclude that in the rat (a) the pudendal nerve has motor, sensory and autonomic (parasympathetic) components in contrast to the sciatic which is primarily motor and somatosensory, (b) some afferents from these nerves exhibit pre-spinal convergence and (c) dorsomedial and dorsolateral motoneuron groups are homologous to Onuf's nucleus in man.     

Segmental distribution and central projectionsof renal afferent fibers in the cat studied by transganglionic transport of horseradish peroxidase

The segmental and central distributions of renal nerve afferents in adult cats and kittens were studied by using retrograde and transganglionic transport of horseradish peroxidase (HRP). Transport of HRP from the central cut ends of the left renal nerves labeled afferent axons in the ipsilateral minor splanchnic nerves and sensory perikarya in the dorsal root ganglia from T12 to L4. The majority of labeled cells (85%) were located between L1 and L3. A few neurons in the contralateral dorsal root ganglia were also labeled. Labeled cells were not confined to any particular region within a dorsal root ganglion. Some examples of bifurcation of the peripheral and central processes within the ganglion were noted. A small number of preganglionic neurons, concentrated in the intermediolateral nucleus, were also identified in some experiments. In addition, many sympathetic postganglionic neurons were labeled in the renal nerve ganglia, the superior mesenteric ganglion, and the ipsilateral paravertebral ganglia from T12 to L3 Transganglionic transport of HRP labeled renal afferent projections to the spinal cord of kittens from T1 1 to L6, with the greatest concentrations between Ll and L3. These afferents extended rostrocaudally in Lissauer's tract and sent collaterals into lamina I. In the transverse plane, a major lateral projection and a minor medial projection were observed along the outer and inner margins of the dorsal horn, respectively. From the lateral projection many fibers extended medially in laminae V and VI forming dorsal and ventral bundles around Clarke's nucleus. The dorsal bundle was joined by collaterals from the medial afferent projection and crossed to the contralateral side. The ventral bundle extended into lamina VII along the lateroventral border of Clarke's nucleus. Some afferents in the lateral projection could be followed ventrally into the dorsolateral portion of lamina VII in the vicinity of the intermediolateral nucleus. In the contralateral spinal cord, labeled afferent fibers were mainly seen in laminae V and VI These results provide the first anatomical evidence for sites of central termination of renal afferent axons. Renal inputs to regions (laminae I, V, and VI) containing spinoreticular and spinothajamic tract neurons may be important in the mediation of supraspinal cardiovascular reflexes as well as in the transmission of activity from nociceptors in the kidney. In addition, the identification of a bilateral renal afferent projection in close proximity to the thoracolumbar autonomic nuclei is consistent with the demonstration in physiological experiments of a spinal pathway for the renorenal sympathetic reflexes.