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.     

Changes in pituitary adenylate cyclase activating polypeptide expression in urinary bladder pathways after spinal cord injury

These studies examined changes in the pituitary adenylate cyclase activating polypeptide (PACAP) expression in micturition reflex pathways after spinal cord injury (SCI) of various durations. In spinal-intact animals, PACAP immunoreactivity (IR) was expressed in fibers in the superficial dorsal horn in all segmental levels examined (L1, L2, L4-S1). Bladder-afferent cells (35-45%) in the dorsal root ganglia (DRG; L1, L2, L6, S1) from spinal-intact animals also exhibited PACAP-IR. After SCI (6 weeks), PACAP-IR was dramatically increased in spinal segments and DRG (L1, L2, L6, S1) involved in micturition reflexes. The density of PACAP-IR was increased in the superficial laminae (I-II) of the L1, L2, L6, and S1 spinal segments. No changes in PACAP-IR were observed in the L4-L5 segments. Staining was also dramatically increased in a fiber bundle extending ventrally from Lissauer's tract (LT) in lamina I along the lateral edge of the dorsal horn to the sacral parasympathetic nucleus (SPN) in the L6-S1 spinal segments (lateral collateral pathway of Lissauer, LCP). After SCI (range 48 h to 6 weeks), PACAP-IR in cells in the L1, L2, L6, and S1 DRG significantly (P < or = 0.001) increased and the percentage of bladder-afferent cells expressing PACAP-IR also significantly (P < or = 0.001) increased (70-92%). No changes were observed in the L4-L5 DRG. PACAP-IR was reduced throughout the urothelium and detrusor smooth muscle whole mounts after SCI. These studies demonstrate changes in PACAP expression in micturition reflex pathways after SCI that may contribute to urinary bladder dysfunction or reemergence of primitive voiding reflexes after SCI.     

Plasticity in reflex pathways to the lower urinary tract following spinal cord injury

The lower urinary tract has two main functions, storage and periodic expulsion of urine, that are regulated by a complex neural control system in the brain and lumbosacral spinal cord. This neural system coordinates the activity of two functional units in the lower urinary tract: (1) a reservoir (the urinary bladder) and (2) an outlet (consisting of bladder neck, urethra and striated muscles of the external urethra sphincter). During urine storage the outlet is closed and the bladder is quiescent to maintain a low intravesical pressure. During micturition the outlet relaxes and the bladder contracts to promote efficient release of urine. This reciprocal relationship between bladder and outlet is generated by reflex circuits some of which are under voluntary control. Experimental studies in animals indicate that the micturition reflex is mediated by a spinobulbospinal pathway passing through a coordination center (the pontine micturition center) located in the rostral brainstem. This reflex pathway is in turn modulated by higher centers in the cerebral cortex that are involved in the voluntary control of micturition. Spinal cord injury at cervical or thoracic levels disrupts voluntary control of voiding as well as the normal reflex pathways that coordinate bladder and sphincter function. Following spinal cord injury the bladder is initially areflexic but then becomes hyperreflexic due to the emergence of a spinal micturition reflex pathway. However the bladder does not empty efficiently because coordination between the bladder and urethral outlet is lost. Studies in animals indicate that dysfunction of the lower urinary tract after spinal cord injury is dependent in part on plasticity of bladder afferent pathways as well as reorganization of synaptic connections in the spinal cord. Reflex plasticity is associated with changes in the properties of ion channels and electrical excitability of afferent neurons and appears to be mediated in part by neurotrophic factors released in the spinal cord and/or the peripheral target organs.     

Increased NADPH-diaphorase reactivity in bladder afferent pathways following urethral obstruction in guinea pigs

The distribution of reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) reactivity and its alterations after urethral obstruction were examined histochemically in bladder afferent pathways in male guinea pigs. The bladder afferent neurons in L6-S2 dorsal root ganglia (DRG) and their central projections in the corresponding spinal cord segments were identified by retrograde axonal transport following injection of Fast Blue (FB) into the bladder walls. In control animals, a large number of DRG neurons in L6-S2 segments were moderately or weakly stained for NADPH-d, with a small number of them being intensely stained. Following urethral obstruction, NADPH-d reactivity was noticeably increased in the same cells beginning at 24 hours, and was markedly enhanced at 48 hours. Results of cell count showed that the number of intensely stained NADPH-d cells was significantly increased in L6-S2 DRG of the urethral obstructed animals when compared with that of the controls (P < 0.01). In the corresponding spinal cord segments, NADPH-d reactivity in the fibre bundle extending from the Lissauer's tract to the sacral parasympathetic nucleus (SPN) became more pronounced. In sections double labelled for NADPH-d and FB, increased NADPH-d reactivity was detected in the FB-labelled bladder afferent neurons in DRG and their projections in the spinal cord. Present results indicate that neuronal NADPH-d in the bladder afferent pathways is plastic and could be upregulated following urethral obstruction. It is suggested that such alteration may be involved in the processing of nociceptive inputs as a result of overdistension of the bladder after urethral obstruction.     

Anatomical evidence for two spinal 'afferent-interneuron-efferent' reflex pathways involved in micturition in the rat: a 'pelvic nerve' reflex pathway and a 'sacrolumbar intersegmental' reflex pathway

We labeled interneurons in the L1-L2 and L6-S1 spinal cord segments of the rat that are involved in bladder innervation using transneuronal retrograde transport of pseudorabies virus (PRV) in normal animals and in animals with selected nerve transections. Preganglionic neurons were identified using antisera against choline acetyltransferase (ChAT). In some experiments we labelled parasympathetic preganglionic neurons (PPNs) in the L6-S1 spinal cord by retrograde transport of Fluorogold from the major pelvic ganglion. We identified bladder afferent terminals using the transganglionic transport of the anterograde tracer cholera toxin subunit b. We present anatomical evidence for two spinal pathways involved in innervation of the bladder. First, in the intact rat, afferent information from the bladder connects, via interneurons in L6-S1, to the PPNs that provide the efferent innervation of the bladder. The afferent terminals were located mainly in close apposition to interneurons located dorsal to the retrogradely labeled PPNs. Second, using L6-S1 ganglionectomies or L6-S1 ventral root rhizotomies we limited viral transport to the sympathetic pathways innervating the bladder. This procedure also labelled interneurons (but not PPNs) with PRV in the L6-S1 spinal cord in a location very similar to those described in the intact rat. These interneurons also receive bladder afferent terminals but we propose that they project to sympathetic preganglionic neurons, most of which are in the L1-L2 spinal segments. Based on this anatomical evidence, we propose the existence of two spinal reflex pathways involved in micturition: a pathway limited to a reflex arc in the pelvic nerve (presumably excitatory to the detrusor muscle); and a pathway involving the pelvic nerve and sympathetic nerve fibers, some of which may travel in the hypogastric (presumably inhibitory to the detrusor muscle).     

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

This study utilized neuronal c-fos expression to examine the spinal pathways involved in processing nociceptive and non-nociceptive afferent input from the lower urinary tract (LUT) of the urethane-anesthetized rat. C-fos protein was detected immunocytochemically in only a small number of cells (< 2 cells/L6 section) in control animals. However, chemical irritation with 1% acetic acid or mechanical stimulation of the LUT markedly increased the number of c-fos-positive neurons (56-180 cells/L6 section) in four regions of the caudal lumbosacral (L6-S1) spinal cord: medial dorsal horn (MDH), lateral dorsal horn, dorsal commissure (DCM), and sacral parasympathetic nucleus (SPN). Only small numbers of c-fos-positive cells were detected in rostral lumbar segments, a region that is thought to receive nociceptive input from the LUT via afferent pathways in sympathetic nerves. The distribution of c-fos-positive cells in the L6 spinal cord varied according to the stimulus (i.e., urethral catheter, bladder distension, or chemical irritation). Distension of the urinary bladder increased the number of c-fos-positive cells mainly in DCM and SPN regions of the cord. In contrast, irritation of the LUT increased c-fos expression largely in DCM and MDH areas. Spinal cord transection (T8 level) did not alter the c-fos expression induced by a catheter or chemical irritation, indicating that gene expression was mediated by spinal pathways. Denervation experiments showed that c-fos expression was induced by activation of afferent pathways in the pelvic and pudendal nerves. These results suggest that neurons in several regions of the spinal cord are involved in processing afferent input from different parts of the LUT. Neurons in the DCM appear to have an important role since they respond to both nociceptive and non-nociceptive inputs and to visceral (pelvic nerve) and somatic (pudendal nerve) afferent pathways. Thus, these neurons may be involved in the mechanisms of visceral-somatic referred pain.     

Segmental distribution and peptide content of primary afferent neurons innervating the urogenital organs and colon of male rats.

Many visceral afferent neurons contain peptides, which have been proposed as histochemical markers for nerve pathways of particular targets or as transmitter candidates. The former possibility was investigated in the present study. Primary afferent neurons which project to the urinary bladder, distal colon or penis of rats, and the colon of cats were labelled with retrogradely transported fluorescent dyes (Fast Blue, True Blue, or Fluoro Gold). One to six weeks after dye injection into the organs, lumbosacral dorsal root ganglia were removed, treated with colchicine, and processed for immunohistochemical identification of five peptides. Dye-labelled neurons were distributed in an organ-specific manner in the lower lumbosacral ganglia, where colon afferent neurons were almost exclusively found in S1 ganglia, penis neurons primarily in L6, and bladder neurons at both levels. Substance P- (SP), calcitonin gene-related peptide-(CGRP), vasoactive intestinal peptide- (VIP), enkephalin- (ENK), and somatostatin- (SOM) immunoreactivity (IR) were detected in neurons in all lumbosacral ganglia but only some of these peptides were present in a large percentage of labelled neurons. The numbers of peptide-containing neurons innervating each organ were CGRP greater than SP greater than VIP greater than ENK greater than SOM; however some differences were observed in the relative proportions of these neuronal populations between upper lumbar and lower lumbosacral ganglia and between different organs. The major difference seen at the upper lumbar level was amongst the SP-IR neurons, which were common (25-30%) amongst bladder and colon afferent neurons but absent in penis neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibitory control of the urinary bladder in the neonatal rat in vitro spinal cord-bladder preparation

Urinary bladder activity of the neonatal rat is tonically inhibited by neural input from the spinal cord passing through axons in the pelvic nerve. The present study was undertaken to examine the organization of this inhibitory mechanism using in vitro spinal cord-bladder preparations of neonatal rats in which the lumbosacral dorsal roots (DRs) or ventral roots (VRs) were transected. Isovolumetric bladder contractions occurring spontaneously or induced by electrical stimulation of the bladder wall (ES-BW) were measured. In DR transected (DRT) preparations, removal of the spinal cord significantly enhanced (50-59%) the amplitude of spontaneous and ES-BW-evoked bladder contractions; whereas in VR transected (VRT) preparations removal of the spinal cord produced only a small enhancement (6.7-12%). However, in VRT preparations, electrical stimulation of the dorsal roots reduced the amplitude of spontaneous contractions, an effect blocked by a nicotinic ganglionic blocking agent, hexamethonium. In DRT preparations, MK-801 enhanced the amplitude of spontaneous and ES-BW-evoked contractions. These results demonstrate that bladder activity of the neonatal rat is tonically inhibited by input from the lumbosacral spinal cord via parasympathetic pathways in the pelvic nerve. The inhibitory outflow is not dependent upon afferent input to the cord but is facilitated by NMDA glutamatergic transmission in the spinal cord. Antidromic activation of afferent axons also appears to induce inhibition in the bladder via a mechanism involving nicotinic cholinergic receptors. These findings suggest that spinal and peripheral inhibitory mechanisms may play an important role in controlling voiding in the neonatal rat.     

Anatomical evidence for two spinal ‘afferent–interneuron–efferent’ reflex pathways involved in micturition in the rat: a ‘pelvic nerve’ reflex pathway and a ‘sacrolumbar intersegmental’ reflex pathway

We labeled interneurons in the L1–L2 and L6–S1 spinal cord segments of the rat that are involved in bladder innervation using transneuronal retrograde transport of pseudorabies virus (PRV) in normal animals and in animals with selected nerve transections. Preganglionic neurons were identified using antisera against choline acetyltransferase (ChAT). In some experiments we labelled parasympathetic preganglionic neurons (PPNs) in the L6–S1 spinal cord by retrograde transport of Fluorogold from the major pelvic ganglion. We identified bladder afferent terminals using the transganglionic transport of the anterograde tracer cholera toxin subunit b. We present anatomical evidence for two spinal pathways involved in innervation of the bladder. First, in the intact rat, afferent information from the bladder connects, via interneurons in L6–S1, to the PPNs that provide the efferent innervation of the bladder. The afferent terminals were located mainly in close apposition to interneurons located dorsal to the retrogradely labeled PPNs. Second, using L6–S1 ganglionectomies or L6–S1 ventral root rhizotomies we limited viral transport to the sympathetic pathways innervating the bladder. This procedure also labelled interneurons (but not PPNs) with PRV in the L6–S1 spinal cord in a location very similar to those described in the intact rat. These interneurons also receive bladder afferent terminals but we propose that they project to sympathetic preganglionic neurons, most of which are in the L1–L2 spinal segments. Based on this anatomical evidence, we propose the existence of two spinal reflex pathways involved in micturition: a pathway limited to a reflex arc in the pelvic nerve (presumably excitatory to the detrusor muscle); and a pathway involving the pelvic nerve and sympathetic nerve fibers, some of which may travel in the hypogastric (presumably inhibitory to the detrusor muscle).     

Alterations in afferent pathways from the urinary bladder of the rat in response to partial urethral obstruction.

Afferent pathways from the urinary bladder were examined with axonal tracing techniques in normal female Wistar rats and in those with partial urethral ligation. Following injection of wheat germ agglutinin-horseradish peroxidase (HRP) into the bladder wall, HRP was detected in lumbosacral dorsal root ganglion cells and in afferent projections to the L6-S1 spinal cord at sites in laminae I, II, V-VII, and X known to receive visceral afferent input. Partial urethral ligation (6 weeks) produced a sixfold increase in bladder weight and altered the morphology of bladder afferent pathways. Changes included an increase in the average cross-sectional area of labelled neuronal profiles in L6 and S1 dorsal root ganglia in obstructed (766 +/- 378 microns 2, P less than 0.001) compared to control rats (528 +/- 189 mu 2). The cross-sectional area of the largest profiles also increased by approximately 40%. The mean number of labelled dorsal root ganglion cell profiles was similar in ligated (837 +/- 198) and control (883 +/- 352) groups. When compared to control animals the obstructed animals exhibited a 60% increase in the area of the labelled afferent terminal field in the intermediolateral region of the L6-S1 spinal cord. This increased labelling was even more remarkable given that the volume of tracer per bladder weight injected into the hypertrophied bladder was 87% less than controls. These results provide evidence that bladder afferents project to regions of the spinal cord known to regulate micturition and that these afferents can undergo morphological alterations and/or changes in axoplasmic transport in response to urethral ligation. Changes may occur in response to increased target organ mass, increased neural activity, or alterations in the levels or activity of neurotrophic factors.     

Effects of pituitary adenylate cyclase activating polypeptide on lumbosacral preganglionic neurons in the neonatal rat spinal cord

The effects of PACAP-38 on phasic and tonic preganglionic neurons (PGN) in L6 and S1 spinal cord slices from neonatal rats (5--11 days old) were studied using the whole-cell patch clamp technique. PGN were identified by retrograde axonal transport of a fluorescent dye (Fast Blue, 5 microl of 4% solution) injected into the intraperitoneal space 3--7 days prior to the study. Bath application of pituitary adenylate cyclase activating polypeptide (PACAP) (20 nM) increased the frequency of spontaneous excitatory postsynaptic potentials (EPSPs) and spontaneous firing in both types of PGN. PACAP markedly increased the number (200--800%) and frequency of action potentials elicited by depolarizing current pulses in phasic PGN, but had a smaller effect on tonic PGN. PACAP decreased the threshold for action potential generation by approximately 25% in both types of neurons (e.g. -34.0+/-1.5 to -38.4+/-1.7 mV from a holding potential of -50 mV in phasic PGN, P<0.005). PACAP did not affect the duration of the action potential. The amplitude of the spike after hyperpolarization was not changed but the duration was significantly reduced by PACAP from 204.4+/-12.2 to 106.2+/-8.1 ms in tonic but not in phasic PGN. PACAP suppressed a transient outward current that was also suppressed by 4-aminopyridine (0.5 mM). These results coupled with the immunohistochemical identification of a dense collection of PACAP fibers in the region of the PGN, raises the possibility that PACAP may function as an excitatory transmitter in lumbosacral parasympathetic reflex pathways in the neonatal rat.     

Origin of pituitary adenylate cyclase-activating polypeptide (PACAP)-immunoreactive fibers innervating guinea pig parasympathetic cardiac ganglia

The present study investigated the origin of pituitary adenylate cyclase-activating polypeptide (PACAP) -immunoreactive (IR) fibers innervating guinea pig cardiac ganglia. Immunohistochemistry was performed on whole-mounts containing cardiac ganglia, and sections of stellate, nodose, and dorsal root ganglia (DRG, thoracic levels 1-4), and caudal medulla. In control preparations, only 4% of the cardiac neurons were PACAP-IR, although most cardiac ganglion cells were surrounded by a network of PACAP-IR fibers. After 3-7 days in explant culture, the number of PACAP-IR cardiac neurons increased approximately eightfold. However, virtually all PACAP-IR fibers surrounding the cardiac neurons had degenerated, demonstrating that the major source of the PACAP-IR fibers was extrinsic to the cardiac ganglia preparation. PACAP- and choline acetyltransferase (ChAT) immunoreactivity were colocalized in fibers within the stellate ganglia but not within neuropeptide Y (NPY) -IR cell bodies and fibers. PACAP-IR cells and fibers were present in the nodose ganglia. PACAP immunoreactivity also was present in fibers and primarily small neurons in thoracic DRGs. In situ hybridization demonstrated the presence of proPACAP mRNA within neurons in the region of the dorsal motor nucleus of the vagus and nucleus ambiguus. PACAP immunoreactivity was colocalized with ChAT immunoreactivity, but not with NPY immunoreactivity or SP immunoreactivity, in fibers surrounding neurons within cardiac ganglia. We conclude that PACAP-containing fibers innervating the postganglionic parasympathetic neurons in guinea pig cardiac ganglia are primarily preganglionic parasympathetic axons.     

Separate urinary bladder and external urethral sphincter neurons in the central nervous system of the rat: simultaneous labeling with two immunohistochemically distinguishable pseudorabies viruses

This work examines the distribution, in the central nervous system, of virus-labeled neurons from the rat urinary bladder and the external urethral sphincter simultaneously within the same tissue sections. Two immunohistochemically distinct pseudorabies virus strains were injected into male Sprague--Dawley rats (approximately 280 g). One virus was injected into the bladder and the other into the external urethral sphincter. After incubation intervals of 2, 2.5 and 3 days, sections from the spinal cord and brain were treated immunohistochemically to detect cells which were labeled separately by each virus or were labeled by both viruses. The major result of these experiments is that each strain of virus labeled a separate population of neurons and that some neurons were labeled by both strains. In the lumbosacral cord, 3 days post-infection, neurons labeled by virus from the external urethral sphincter were found in Onuf's nucleus, the dorsal gray commissure, and the superficial dorsal horn. Neurons labeled by virus from the urinary bladder were found in the L6--S1 and L1--L2 spinal cord segments within the dorsal gray commissure, the intermediolateral area and the superficial dorsal horn. Double-labeled interneurons were mainly located in the dorsal gray commissure although some were also found in the intermediolateral area and the superficial dorsal horn. In the medulla, external urethral sphincter neurons and bladder neurons and double-labeled neurons were found in the reticular region and the raphe. More rostrally, bladder neurons were located in the pontine micturition center and external urethral sphincter neurons were found in the locus coeruleus and subcoeruleus. A very small number of double-labeled neurons were found in the pontine micturition center and the locus coeruleus or subcoeruleus.     

Estrogen receptor-alpha and neural circuits to the spinal cord during pregnancy

Estrogen receptor immunoreactivity and mRNAs are present in spinal cord neurons in locations that are associated with sensory and autonomic innervation of female reproductive organs. The present study was undertaken to examine the expression of estrogen receptor-alpha in the spinal cord during different stages of pregnancy and to determine whether estrogen receptor-alpha-expressing neurons are related to uterine afferent nerves bringing information to the spinal cord at parturition. Immunohistochemistry showed estrogen receptor-alpha-immunoreactive neurons in the dorsal one-half of the spinal cord, i.e., dorsal horn, dorsal intermediate gray areas (dorsal commissural nucleus), and around the central canal and sacral parasympathetic autonomic nucleus of the lumbosacral spinal cord. Neurons in these areas corresponded topographically to the distribution of central processes of visceral primary afferent neurons (e.g., containing calcitonin gene-related peptide and substance P) that innervate and activate second-order spinal cord neurons (evidenced by their expression of Fos) at parturition. Western blots showed that estrogen receptor-alpha increases in the spinal cord, with a peak at day 20 of gestation, followed by a slight decrease by 2 days postpartum. These studies show that estrogen receptor-alpha is expressed by neurons in autonomic and sensory areas of the lumbosacral spinal cord that have connections with the female reproductive system and that the level of estrogen receptor-alpha changes over the course of pregnancy, which may follow profiles of steroid hormones. Many of these neurons may be involved in processing information related to reproductive organ function, changes during pregnancy, and relays to other CNS centers.     

Inhibitory effect of intrathecal glycine on the micturition reflex in normal and spinal cord injury rats

We examined the influence of lumbosacral glycinergic neurons on the spinobulbospinal and spinal micturition reflexes. Female rats were divided into intact rats, rats with acute injury to the lower thoracic spinal cord (SCI), and rats with chronic SCI. Under urethane anesthesia, isovolumetric cystometry was performed in each group before and after intrathecal (IT) injection of glycine or strychnine into the lumbosacral cord level. The glutamate and glycine levels of the lumbosacral cord were measured after injection of glycine or strychnine in intact and chronic SCI rats. Expression of strychnine-sensitive glycine receptor alpha-1 (GlyR alpha1) mRNA in the lumbosacral cord was also assessed in both rats. In chronic SCI rats, the interval and amplitude of bladder contractions were shorter and smaller when compared with intact rats. IT glycine (0.1-100 microg) prolonged the interval and decreased the amplitude of bladder contractions in both intact rats and chronic SCI rats. IT strychnine (0.01-10 microg) elevated the baseline pressure in intact rats and induced bladder contraction in acute SCI rats. On amino acid analysis, IT glycine (0.01-100 microg) decreased the glutamate level of the lumbosacral cord in intact rats, but not in chronic SCI rats. The glycine level of the lumbosacral cord was 54% lower in chronic SCI rats when compared with intact rats, while the GlyR alpha1 mRNA level did not change after SCI. These results suggest that glycinergic neurons may have an important inhibitory effect on the spinobulbospinal and spinal micturition reflexes at the level of the lumbosacral cord.     

Plasticity of urinary bladder reflexes evoked by stimulation of pudendal afferent nerves after chronic spinal cord injury in cats

Bladder reflexes evoked by stimulation of pudendal afferent nerves (PudA-to-Bladder reflex) were studied in normal and chronic spinal cord injured (SCI) adult cats to examine the reflex plasticity. Physiological activation of pudendal afferent nerves by tactile stimulation of the perigenital skin elicits an inhibitory PudA-to-Bladder reflex in normal cats, but activates an excitatory reflex in chronic SCI cats. However, in both normal and chronic SCI cats electrical stimulation applied to the perigenital skin or directly to the pudendal nerve induces either inhibitory or excitatory PudA-to-Bladder reflexes depending on stimulation frequency. An inhibitory response occurs at 3–10 Hz stimulation, but becomes excitatory at 20–30 Hz. The inhibitory reflex activated by electrical stimulation significantly (P < 0.05) increases the bladder capacity to about 180% of control capacity in normal and chronic SCI cats. The excitatory reflex significantly (P < 0.05) reduces bladder capacity to about 40% of control capacity in chronic SCI cats, but does not change bladder capacity in normal cats. Electrical stimulation of pudendal afferent nerves during slow bladder filling elicits a large amplitude bladder contraction comparable to the contraction induced by distension alone. A bladder volume about 60% of bladder capacity was required to elicit this excitatory reflex in normal cats; however, in chronic SCI cats a volume less than 20% of bladder capacity was sufficient to unmask an excitatory response. This study revealed the co-existence of both inhibitory and excitatory PudA-to-Bladder reflex pathways in cats before and after chronic SCI. However our data combined with published electrophysiological data strongly indicates that the spinal circuitry for both the excitatory and inhibitory PudA-to-Bladder reflexes undergoes a marked reorganization after SCI.     

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

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

Tetrodotoxin-resistant sodium channels Na(v)1.8/SNS and Na(v)1.9/NaN in afferent neurons innervating urinary bladder in control and spinal cord injured rats

Tetrodotoxin-resistant (TTX-R) sodium channels Na(v)1.8/SNS and Na(v)1.9/NaN are preferentially expressed in small diameter dorsal root ganglia (DRG) neurons. The urinary bladder is innervated by small afferent neurons from L6/S1 DRG, of which approximately 75% exhibit high-threshold action potentials that are mediated by TTX-R sodium channels. Following transection of the spinal cord at T8, the bladder becomes areflexic and then gradually hyper-reflexic, and there is an attenuation of the TTX-R sodium currents in bladder afferent neurons. In the present study, we demonstrate that Na(v)1.8 is expressed in both bladder and non-bladder afferent neurons, while Na(v)1.9 is expressed in non-bladder afferent neurons but is rarely observed in bladder afferent neurons. In spinal cord transected rats 28-32 days following transection, there is a decreased expression of Na(v)1.8 sodium channels in bladder afferents, but no change in the expression of Na(v)1.8 in non-bladder afferent neurons. Both bladder and non-bladder afferent neurons exhibit limited increases in Na(v)1.9 expression following spinal cord transection. These results demonstrate that the expression of TTX-R channels in bladder afferent neurons changes after spinal cord transection, and these changes may contribute to the increased excitability of these neurons following spinal cord injury.