Localization of NADPH diaphorase in the lumbosacral spinal cord and dorsal root ganglia of the cat

The distribution of NADPH-d activity in the spinal cord and dorsal root ganglia of the cat was studied to evaluate the role of nitric oxide in lumbosacral afferent and spinal autonomic pathways. At all levels of the spinal cord NADPH-d staining was present in neurons and fibers in the superficial dorsal horn and in neurons around the central canal and in the dorsal commissure. In addition, the sympathetic autonomic nucleus in the rostral lumbar segments exhibited prominent NADPH-d cellular staining whereas the parasympathetic nucleus in the sacral segments was not well stained. The most prominent NADPH-d activity in the sacral segments occurred in fibers extending from Lissauer's tract through laminae I along the lateral edge of the dorsal horn to lamina V and the region of the sacral parasympathetic nucleus. These fibers were very similar to VIP-containing and pelvic nerve afferent projections in the same region. They were prominent in the S₁–S₃ segments but not in adjacent segments (L₆–L₇ and Cx₁) or in thoracolumbar and cervical segments. NADPH-d activity and VIP immunoreactivity in Lissauer's tract and the lateral dorsal horn were eliminated or greatly reduced after dorsal-ventral rhizotomy (S₁–S₃), indicating the fibers represent primary afferent projections. A population of small diameter afferent neurons in the L₇–S₂ dorsal root ganglia were intensely stained for NADPH-d. The functional significance of the NADPH-d histochemical stain remains to be determined; however, if NADPH-d is nitric oxide synthase then this would suggest that nitric oxide may function as a transmitter in thoracolumbar sympathetic preganglionic efferent pathways and in sacral parasympathetic afferent pathways in the cat. © 1994 Wiley-Liss, Inc.     

NADPH-diaphorase reveals presumptive sympathetic primary afferents in the developing human spinal cord

Numerous studies have elucidated two visceral afferent pathways in the spinal cord of mammals, the lateral collateral pathway (LCP) and the medial collateral pathway (MCP). The present study utilized NADPH-diaphorase histochemistry to visualize afferent pathways in the developing human thoracolumbar spinal cord. Diaphorase-positive fiber bundles, strikingly similar to the previously defined LCP and MCP, were observed coursing along the lateral and medial aspects of the dorsal horn to the base of the dorsal horn, the intermediate gray, and/or the dorsal commissure. Furthermore, some axons forming the MCP crossed in the dorsal commissure to the contralateral side of the spinal cord. In addition, axons projecting in the LCP often appeared to terminate within clusters of diaphorase-labeled sympathetic preganglionic neurons, supporting the concept that monosynaptic connections may exist between primary afferents and autonomic motor neurons. Thus, nitric oxide may be involved in both afferent and efferent neurons in reflex pathways of the human sympathetic nervous system.     

Mechanisms underlying the pathogenesis of urinary bladder instability - new perspectives for the treatment of reflex incontinence

The urine storage ability of the urinary bladder is markedly impaired following inflammation of the urinary bladder and spinal cord injury because of a hyperexcitability of micturition reflexes. Using two rat models of inflammation-induced bladder overactivity and detrusor hyper-reflexia following spinal cord injury we investigated changes in the neuronal pathways to the urinary bladder which may underlie the development of this instability. Our results suggest that among the factors involved in inflammation-induced bladder instability are significant changes in the expression of the neuropeptides substance P, calcitonin gene-related peptide and galanin at the primary afferent level, as well as of the enzyme neuronal nitric oxide synthase (nNOS) at the afferent and postganglionic efferent level. In the lumbar and sacral spinal cord nNOS-immunoreactivity was depleted from dorsal horn neurones in both cystitis and spinal cord injured rats and from preganglionic parasympathetic neurones after spinal cord injury. Distension of the bladder in chronically spinalized rats elicited c-Fos expression in a significantly greater number of neurones throughout the lumbar and sacral segments than in rats with an intact neuraxis. Thus, under pathological conditions rather complicated changes in the synthesis of neuropeptides and nNOS occur at the primary afferent, spinal cord and postganglionic efferent level that together control the activity of the urinary bladder. Further mechanisms like unmasking of silent synapses and axonal sprouting in the spinal cord might further contribute to an increase in activity in micturition reflex pathways. Local cooling of the dorsal spinal cord at the level L6/S1 with temperatures between 14 and 20 °C proved a simple technique to control the unstable bladder and restore continence in both inflammation-induced detrusor overactivity and detrusor hyperreflexia following spinal cord injury. The effects of cooling are probably the result of a blockade of synaptic transmission within the dorsal cord which eliminates neuronal overactivity. Thus, local spinal cord cooling could offer a new method to treat bladder instability and reflex incontinence.     

Mechanisms underlying the pathogenesis of urinary bladder instability - new perspectives for the treatment of reflex incontinence

The urine storage ability of the urinary bladder is markedly impaired following inflammation of the urinary bladder and spinal cord injury because of a hyperexcitability of micturition reflexes. Using two rat models of inflammation-induced bladder overactivity and detrusor hyper-reflexia following spinal cord injury we investigated changes in the neuronal pathways to the urinary bladder which may underlie the development of this instability. Our results suggest that among the factors involved in inflammation-induced bladder instability are significant changes in the expression of the neuropeptides substance P, calcitonin gene-related peptide and galanin at the primary afferent level, as well as of the enzyme neuronal nitric oxide synthase (nNOS) at the afferent and postganglionic efferent level. In the lumbar and sacral spinal cord nNOS-immunoreactivity was depleted from dorsal horn neurones in both cystitis and spinal cord injured rats and from preganglionic parasympathetic neurones after spinal cord injury. Distension of the bladder in chronically spinalized rats elicited c-Fos expression in a significantly greater number of neurones throughout the lumbar and sacral segments than in rats with an intact neuraxis. Thus, under pathological conditions rather complicated changes in the synthesis of neuropeptides and nNOS occur at the primary afferent, spinal cord and postganglionic efferent level that together control the activity of the urinary bladder. Further mechanisms like unmasking of silent synapses and axonal sprouting in the spinal cord might further contribute to an increase in activity in micturition reflex pathways. Local cooling of the dorsal spinal cord at the level L6/S1 with temperatures between 14 and 20 degrees C proved a simple technique to control the unstable bladder and restore continence in both inflammation-induced detrusor overactivity and detrusor hyperreflexia following spinal cord injury. The effects of cooling are probably the result of a blockade of synaptic transmission within the dorsal cord which eliminates neuronal overactivity. Thus, local spinal cord cooling could offer a new method to treat bladder instability and reflex incontinence.     

NADPH diaphorase in the spinal cord of rats.

To identify spinal neurons that may synthesize nitric oxide, cells and fibers histochemically stained for NADPH diaphorase (a nitric oxide synthase) were studied in the spinal cord of rats. The histochemical reaction gave an image similar to the best Golgi impregnations, staining cells down to their finest processes. Transverse, horizontal, and parasagittal 50 and 100 microns sections were used to follow dendritic and axonal arborizations of stained neurons. Major cell groups were identified in the superficial dorsal horn and around the central canal (at all spinal levels), and in the intermediolateral cell column (at thoracic and sacral levels). Scattered positive cells were also found in deeper dorsal horn, ventral horn, and white matter. In some cases, axons of cells in the dorsal horn could be traced into the white matter; many of these cells resembled neurons projecting to various supraspinal targets. Stained cells in the intermediolateral column, which sent their axons into the ventral root, were presumed to be preganglionic autonomic neurons. Dense plexes of fibers were stained in laminae I and II and in the intermediolateral column. A large number of NADPH diaphorase-positive neurons in the spinal cord appear to be involved in visceral regulation. Fibers of the intermediolateral system had a special relationship with vasculature, suggesting that nitric oxide may help to couple neural activity with regional blood flow in the spinal cord. The abundance of NADPH diaphorase-positive neurons and fibers in the superficial dorsal horn suggests that nitric oxide may also be involved in spinal sensory processing.     

Primary afferent projections of the major splanchnic nerve to the spinal cord and gracile nucleus of the cat

Splanchnic afferent projections to the spinal cord and gracile nucleus were labeled following the application of HRP to the central cut end of the major splanchnic nerve. Labeled afferent fibers were detected in the ipsilateral dorsal column, in Lissauer's tract (LT), in laminae 1, 5, 7, and 10, and in the dorsal gray commissure at T1-T13 levels of the spinal cord. Afferent projections were not identified in laminae 2-4. Collaterals from LT projected ventrally along the lateral and medial margins of the dorsal horn (called lateral and medial pathways, respectively). Afferents in the lateral pathway formed small bundles, spaced rostrocaudally at intervals of 300-1,000 microns, which passed medially at the base of the dorsal horn into laminae 5, 7, and 10 and to the contralateral spinal cord. Some afferents in the lateral pathway projected to the intermediolateral nucleus where labeled sympathetic preganglionic neurons were located. Afferents in the medial pathway entered the lateral aspect of the dorsal column and projected as a group near the midline rostrally to the medulla. The dorsal column pathway terminated in the ventral gracile nucleus in four or five clusters, each occupying a region ranging in size from 0.01-0.1 mm3 and separated in the rostrocaudal axis by distances of 400-800 microns. These clusters were concentrated in the middle and caudal portions of the nucleus below the obex. A comparison of the present results with those from earlier experiments on the central projections of afferent fibers from the heart, kidney, and pelvic organs demonstrates a consistent pattern of visceral afferent termination in the thoracolumbar and sacral segments of the spinal cord. This is not unexpected, since visceral afferent pathways to different organs perform similar functions, such as the transmission of nociceptive information and the initiation of autonomic reflexes.     

Ontogeny of adrenergic fibers in rat spinal cord in relationship to adrenal preganglionic neurons

Adrenergic neurons in the C1 cell group in the rostral ventrolateral medulla oblongata contain epinephrine, as well as its biosynthetic enzyme, phenylethanolamine N-methyltransferase (PNMT). These neurons send axons to regions of the central nervous system known to regulate autonomic function, including the sympathetic preganglionic nuclei of thoracic and upper lumbar spinal cord. Previous studies have shown that PNMT is expressed in neurons located in the medulla oblongata on embryonic day 14; however, the development of the projections from these cells has not been studied. With the aid of high-performance liquid chromatography (HPLC) to determine levels of catecholamines and immunocytochemistry to demonstrate PNMT, the ontogeny of the adrenergic bulbospinal pathway in the embryonic, postnatal, and adult rat has been studied. In addition, the relationship between PNMT-immunoreactive (IR) fibers and retrogradely labeled sympathetic preganglionic neurons projecting to adrenal medulla are described. PNMT-IR fibers were first observed in the caudal medulla oblongata and lateral funiculus of spinal cord on gestational day 15(E15). On E16, PNMT-IR fibers in the thoracic spinal cord were observed in the intermediate gray matter at the level of the lateral horn. Epinephrine was measureable in spinal cord on E20. Both the density of PNMT-IR fibers and the levels of epinephrine increased to a maximum during the second postnatal week and then declined to adult levels. These observations suggest that a period of adrenergic hyperinnervation of spinal sympathetic nuclei occurs during the neonatal period. PNMT-IR terminals in spinal cord were observed, primarily, although not exclusively, in sympathetic nuclei of thoracic cord and parasympathetic nuclei of upper sacral cord. Adrenergic fibers in the intermediolateral nucleus (IML) and the central autonomic nucleus (CAN) dorsal to the central canal were particularly dense during the second postnatal week in both midthoracic and upper sacral segments. In the neonate, a "ladder-like" pattern of PNMT-IR fiber staining was observed which represented transverse fiber bundles connecting IML with CAN and extensive longitundinal fiber bundles along the border of the funiculus in IML. At all spinal levels, adrenergic fibers were also observed adjacent to the ependyma dorsal or lateral to the central canal. The relationship between adrenal preganglionic neurons and PNMT-IR fibers in IML was examined on postnatal days 4, 15, and 60. With retrograde labeling from adrenal medulla, it was demonstrated that PNMT-IR fibers are associated with adrenal preganglionic neurons throughout postnatal development.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Central distribution of afferent and efferent components of the pudendal nerve in cat

Central distribution of afferent and efferent components of the pudendal nerve was examined in the cat by the HRP method after applying HRP to the central cut end of the pudendal nerve. Retrogradely labeled neuronal cell bodies were located primarily in the feline homologue of the Onuf's X nucleus, constituting a slender longitudinal cell column in the ventral horn of the S1 and S2 cord segments. The Onuf's nucleus was present constantly from middle S1 to high S2 cord segments, and occasionally extended rostrally to high S1 or low L7, and caudally to middle S2, low S2, or high S3 cord segments. No sex differences were observed in the distribution pattern, number, and soma size of labeled neurons in the Onuf's nucleus. Transganglionically labeled dorsal root fibers were found to terminate ipsilaterally in the lamina I of the dorsal horn at levels of lower lumbar, sacral, and higher coccygeal cord segments and the gracile nucleus, and bilaterally with an ipsilateral predominance in the dorsal commissural gray and laminae III, IV, V, and VI of the dorsal horn at levels of lower lumbar, sacral, and higher coccygeal cord segments. Some labeled dorsal root fibers appeared to end ipsilaterally in the regions where the sacral parasympathetic preganglionic neurons have been shown to be located.     

Localization of NADPHd-exhibiting neurons in the spinal cord of the rabbit

Segmental and laminar distributions of nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd)-exhibiting neurons were examined in the rabbit spinal cord by using horizontal, sagittal, and transverse sections. A large number of NADPHd-positive neurons in the spinal cord of rabbit appeared to fall into six categories (N1-N6), but others could not be classified. Major cell groups of NADPHd-exhibiting neurons were identified in the superficial dorsal horn and around the central canal at all spinal levels and in the intermediolateral cell column at thoracic and upper lumbar levels. NADPHd-exhibiting neurons of the pericentral region were divided into a thin subependymal cell column containing longitudinally arranged, small bipolar neurons with processes penetrating deeply into the intermediolateral cell column and/or running rostrocaudally in the subependymal layer. The second pericentral cell column located more laterally in lamina X contains large, intensely stained NADPHd-exhibiting neurons with long dendrites radiating in the transverse plane. In the pericentral region (lamina X), close association of NADPHd-exhibiting somata and fibers and mostly longitudinally oriented blood vessels were detected. Neurons of the sacral parasympathetic nucleus, seen in segments S1-S3, exhibited prominent NADPHd cellular staining accompanied by heavily stained fibers extending from Lissauer's tract through lamina I along the lateral edge of the dorsal horn to lamina V. A massive dorsal gray commissure, highly positive in NADPHd staining, was found in segments S1-S3. Scattered positive cells were also found in the deeper dorsal horn, ventral horn, and white matter. Fiberlike NADPHd staining was found in the superficial dorsal horn and pericentral region in all the segments studied. Dense, punctate, nonsomatic NADPHd staining was detected in the superficial dorsal horn, in the pericentral region all along the rostrocaudal axis, and in the nucleus phrenicus (segments C4-C5), nucleus dorsalis (segments Th2-L2), Onuf's nucleus (segments S1-S3), and the dorsal part of the dorsal gray commissure (S1-S3).     

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.     

Spinal cord representation of the micturition reflex

The spinal cord origin and peripheral pathways of the sensory and motor nerves to the urinary bladder were delineated in the cat by stimulating the appropriate nerves near the urinary bladder and recording from the dorsal and ventral rootlets near the spinal cord. The parasympathetic preganglionic neurons originated in the sacral segments of the spinal cord and reached the bladder by way of the pelvic nerve. The preganglionic parasympathetic perikarya to the urinary bladder were distributed over a length of approximately 1.5 segments, centered near the junction of segments S-2 and S-3 in cats with a median arrangement of the lumbosacral plexus. Conduction velocities in preganglionic parasympathetic fibers to the bladder ranged from 46 to 2 M/sec with a mean maximal velocity of 18.2 M/sec. The major sympathetic pathway to the bladder was in the hypogastric nerve. Preganglionic sympathetic fibers originated in the lumbar spinal cord and traveled through the caudal mesenteric ganglion and hypogastric nerve to the urinary bladder. There were both ipsilateral and contralateral preganglionic and afferent fibers in this pathway. The preganglionic sympathetic neurons originated in segments L-2 and L-5. They were usually distributed over approximately 2 full segments centered near the junction of L-3 and L-4 in cats with a median arrangement of the lumbosacral plexus. Neurons involved in the micturition reflex may extend from the rostral end of the L-2 segment to the caudal end of the S-3 segment. The sympathetic preganglionic neurons were usually separated from the somatic and parasympathetic columns by segments L-5 to L-7.     

Estrogen receptor-immunoreactive neurons are present in the female rat lumbosacral spinal cord

Presence of an estrogen receptor is crucial for cells to respond to estrogen; thus, estrogen-responsive neurons should be identifiable by immunohistochemically staining for the estrogen receptor (ER). Even though spinal neurons are involved in sexual behaviors and innervation of genital organs, little information is available about ER-containing neurons in the spinal cord. Consequently, we have undertaken a study of ER-containing neurons in the female rat lumbosacral cord, an area involved in reproductive functions and predicted to contain estrogen-responsive neurons. In addition, since parasympathetic preganglionic neurons in the lumbosacral cord produce nitric oxide (NO), we also sought to determine if ER-immunoreactive (-IR) neurons contain the enzymes for NO production. Finally, we compared the distribution of ER-IR neurons to the presence of uterine cervix-related neurons. Uterine cervix-related neurons were identified by expression of FOS-immunoreactivity after vaginocervical mechanostimulation (VCS). The lumbosacral spinal cords were removed from intact, ovariectomized, and VCS-treated rats and sections stained by immunohistochemistry. ER-IR was present in the nuclei of neurons located predominately in the dorsal one-half of the spinal cord. Specific sites include the dorsal horn, lamina V, the sacral parasympathetic nucleus (SPN) (which contains preganglionic parasympathetic neurons) and extending into the lateral funiculus, and lamina X. Some ER-IR neurons were NADPH-d-positive and were localized in laminae V and VII. FOS-IR neurons had a distribution pattern similar to the distribution of neurons containing ER. The presence of ER neurons in these regions suggest that they are responsive to circulating estrogen. © 1996 Wiley-Liss, Inc.     

Studies on the factors that govern directionality of axonal growth in the embryonic optic nerve and at the chiasm of mice

Preganglionic neurons of the sacral parasympathetic nucleus (SPN) were located almost exclusively (98%) within the L₆-S₁ spinal cord segments. The SPN contained approximately 550 neurons of medium size (10 × 20 μm). These were mainly located in the intermediolateral gray matter and had dendrites that extended into the dorsolateral funiculus, along the lateral marginal zone of the dorsal horn, and medially into the dorsal gray commissure. Labeled dorsal root ganglion cells were almost all located (95%) in the L₆ and S₁ ganglia. An average of approximately 1,500 sensory neurons were found. These were small cells (17 × 25 μm) whose central processes entered Lissauer's tract from which two groups of collaterals emerged: 1) a prominent lateral pathway along the lateral margin of the dorsal horn that extended into the region of the SPN and also into the dorsal gray commissure, 2) a less prominent medial pathway extending around the dorsal margin of the dorsal horn to terminate in the dorsal gray commissure. These two collateral groups formed fiber bundles that were spaced by approximately 100 μm between centers when observed in the horizontal plane. A third afferent bundle, composed of rostrocaudally oriented fibers, was located in the sagittal plane immediately ventral to the central canal. Comparisons are made between the results in rats and the results of similar experiments performed in cats and monkeys.     

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.     

Distribution and origin of corticotropin-releasing factor-immunoreactive axons in the female rat lumbosacral spinal cord.

Corticotropin-releasing factor (CRF) is a neuropeptide traditionally known for its hormonal role in the hypothalamic/pituitary/adrenal stress axis. However, CRF has been reported in axons in sites that may be considered outside of the direct stress axis, e.g., in axons in the lumbosacral spinal cord associated with the micturition response. Whether any of these CRF-immunoreactive axons interacts with uterine-related preganglionic autonomic neurons or projection neurons in the lumbosacral spinal cord is unknown. Thus, immunohistochemistry and retrograde tracing were employed to determine the presence, distribution, and origin of CRF-immunoreactive axons in the L6/S1 spinal cord of the female rat and to ascertain whether these axons are associated with uterine-related neurons. CRF-immunoreactive axons were present in the dorsal horn, medial and lateral collateral pathways, dorsal intermediate gray, laminae VlI and X, and sacral parasympathetic nucleus of the spinal cord. Nitric oxide-synthesizing, i.e., NADPH-d-positive neurons and pseudorabies virus labeled uterine-related neurons were in the sacral parasympathetic nucleus and were closely apposed by CRF-immunoreactive axons. Injection of retrograde tracers (fluorogold or fast blue) into the L6/S1 spinal cord labeled neurons in the hypothalamic paraventricular nucleus and pontine Barrington's nucleus, and some of these neurons were immunoreactive for CRF. This study demonstrates that CRF-immunoreactive axons are present in the L6/S1 spinal cord of the female rat in areas associated with sensory and autonomic processing. Some of these axons originate from the paraventricular nucleus and Barrington's nucleus and are adjacent to uterine-related neurons. These results indicate that CRF may influence neural activity related to the female reproductive system.     

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.     

The distribution and morphology of parasympathetic preganglionic neurons in the cat sacral spinal cord as revealed by horseradish peroxidase applied to the sacral ventral roots

Parasympathetic preganglionic neurons in the sacral parasympathetic nucleus (SPN) of the cat were studied by applying horseradish peroxidase (HRP) to the sacral ventral roots. The results were compared to data from earlier experiments in which these same neurons were labelled by HRP applied to the pelvic nerve at a point much further from the spinal cord. The present experiments have shown. The total number of neurons in the SPN determined by ventral root labelling is equal to the number obtained by pelvic nerve labelling. This indicates that virtually all SPN neurons send their axons into the pelvic nerve. More extensive dendritic projections from SPN neurons were revealed than in the pelvic nerve experiments. In particular, a strong dendritic projection extended within the lateral marginal zone of the dorsal horn close to Lissauer's tract and horizontal dendrites projected well into the contralateral gray matter, some reaching the contralateral sacral parasympathetic nucleus. The neurons labelled via any one ventral root were all contained within a region equal in length to one spinal segment but shifted rostrally by a small amount. Thus, no evidence was obtained for a long intraspinal pathway in which axons of spinal cord neurons entered ventral roots many segments away from their somata.     

Expression of receptors for glial cell line-derived neurotrophic factor family ligands in sacral spinal cord reveals separate targets of pelvic afferent fibers

Nerve growth factor has been proposed to mediate many structural and chemical changes in bladder sensory neurons after injury or inflammation. We have examined the expression of receptors for the glial cell line-derived neurotrophic factor (GDNF) family within sensory terminals located in the sacral spinal cord and in bladder-projecting sacral dorsal root ganglion neurons of adult female Sprague-Dawley rats. Nerve fibers immunolabelled for GFRalpha1 (GDNF receptor), GFRalpha2 (neurturin receptor), or GFRalpha3 (artemin receptor) showed distinct distribution patterns in the spinal cord, suggesting separate populations of sensory fibers with different functions: GFRalpha1-labeled fibers were in outer lamina II and the lateral-collateral pathway and associated with autonomic interneurons and preganglionic neurons; GFRalpha2-labeled fibers were only in inner lamina II; GFRalpha3-labeled fibers were in lamina I, the lateral-collateral pathway, and areas surrounding dorsal groups of preganglionic neurons and associated interneurons. Immunofluorescence studies of retrogradely labelled bladder-projecting neurons in sacral dorsal root ganglia showed that approximately 25% expressed GFRalpha1 or GFRalpha3 immunoreactivity, the preferred receptors for GDNF and artemin, respectively. After cyclophosphamide-induced bladder inflammation, fluorescence intensity of GFRalpha1-positive fibers increased within the dorsal horn, but there was no change in the GFRalpha2- or GFRalpha3-positive fibers. These studies have shown that GDNF and artemin may target bladder sensory neurons and potentially mediate plasticity of sacral visceral afferent neurons following inflammation. Our results have also revealed three distinct subpopulations of sensory fibers within the sacral spinal cord, which have not been identified previously using other markers.