Circadian rhythms in colonic function.

A rhythmic expression of clock genes occurs within the cells of multiple organs and tissues throughout the body, termed "peripheral clocks." Peripheral clocks are subject to entrainment by a multitude of factors, many of which are directly or indirectly controlled by the light-entrainable clock located in the suprachiasmatic nucleus of the hypothalamus. Peripheral clocks occur in the gastrointestinal tract, notably the epithelia whose functions include regulation of absorption, permeability, and secretion of hormones; and in the myenteric plexus, which is the intrinsic neural network principally responsible for the coordination of muscular activity in the gut. This review focuses on the physiological circadian variation of major colonic functions and their entraining mechanisms, including colonic motility, absorption, hormone secretion, permeability, and pain signalling. Pathophysiological states such as irritable bowel syndrome and ulcerative colitis and their interactions with circadian rhythmicity are also described. Finally, the classic circadian hormone melatonin is discussed, which is expressed in the gut in greater quantities than the pineal gland, and whose exogenous use has been of therapeutic interest in treating colonic pathophysiological states, including those exacerbated by chronic circadian disruption.

Role of circadian rhythms and melatonin in bladder function in heath and diseases.

The circadian system modulates all visceral organ physiological processes including urine storage and voiding. The "master clock" of the circadian system lies within suprachiasmatic nucleus of the hypothalamus while "peripheral clocks" are found in most peripheral tissue and organs, including the urinary bladder. Disruptions of circadian rhythms can cause organ malfunction and disorder or exacerbate pre-existing ones. It has been suggested that nocturia, which develops mostly in the elderly, could be a circadian-related disorder of the bladder. In the bladder, many types of gap junctions and ion channels in the detrusor, urothelium and sensory nerves are likely under strict local peripheral circadian control. The pineal hormone, melatonin, is a circadian rhythm synchroniser capable of controlling a variety of physiological processes in the body. Melatonin predominantly acts via the melatonin 1 and melatonin 2 G-protein coupled receptors expressed in the central nervous system, and many peripheral organs and tissues. Melatonin could be beneficial in the treatment of nocturia and other common bladder disorders. The ameliorating action of melatonin on bladder function is likely due to multiple mechanisms which include central effects on voiding and peripheral effects on the detrusor and bladder afferents. More studies are warranted to determine the precise mechanisms of circadian rhythm coordination of the bladder function and melatonin influences on the bladder in health and diseases.

Endocannabinoids, anandamide and 2-AG, regulate mechanosensitivity of mucosal afferents in the Guinea pig bladder.

Bladder afferents play a crucial role in urine storage and voiding, and conscious sensations from the bladder. Endocannabinoids, anandamide (AEA) and 2-arachidonolylglycerol (2-AG), are endogenous ligands of G-protein coupled cannabinoid receptors 1 and 2 (CB1 and CB2) found in the CNS and peripheral organs. They also have off-target effects on some ligand- and voltage-gated channels. The aim of this study is to determine the role of AEA and 2-AG in regulation of mechanosensitivity of probable nociceptive neurons innervating the bladder - capsaicin-sensitive mucosal afferents. The activity of these afferents was determined by ex vivo single unit extracellular recordings in the guinea pig bladder. A stable analogue of anandamide, methanandamide (mAEA) evoked initial excitatory response of mucosal afferents followed by potentiation of their responses to mechanical stimulation. In the presence of TRPV1 antagonist (AMG9810), mAEA's effect on mechanosensitivity switched from excitatory to inhibitory. The inhibitory effect of mAEA is due to activation of both CB1 and CB2 cannabinoid receptors since it was abolished by combined application of selective CB1 (NESS0327) and CB2 (SR144528) antagonists. 2-AG application evoked a brief excitation of mucosal afferents, without potentiation of their mechanosensitivity, followed by the inhibition of their responses to mechanical stimulation. CB2 receptor antagonist, SR144528 abolished the inhibitory effect of 2-AG. Our data indicated that anandamide and 2-AG have opposite effects on mechanosensitivity of mucosal capsaicin-sensitive afferents in the guinea pig bladder; mAEA potentiated while 2-AG inhibited responses of mucosal afferents to mechanical stimulation. These findings are important for understanding of the role of endocannabinoids in regulating bladder sensation and function.

TRPV1 and TRPM8 antagonists reduce cystitis-induced bladder hypersensitivity via inhibition of different sensitised classes of bladder afferents in guinea pigs.

BACKGROUND AND PURPOSE: Interstitial cystitis (=painful bladder syndrome) is a chronic bladder syndrome characterised by pelvic and bladder pain, urinary frequency and urgency, and nocturia. Transient receptor potential (TRP) channels are an attractive target in reducing the pain associated with interstitial cystitis. The current study aims to determine the efficacy of combination of TRP vanilloid 1 (TRPV1) and TRP melastatin 8 (TRPM8) channel inhibition in reducing the pain associated with experimental cystitis in guinea pigs. EXPERIMENTAL APPROACH: A novel animal model of non-ulcerative interstitial cystitis has been developed using protamine sulfate/zymosan in female guinea pigs. Continuous voiding cystometry was performed in conscious guinea pigs. Ex vivo "close-to-target" single unit extracellular recordings were made from fine branches of pelvic nerves entering the guinea pig bladder. Visceromotor responses in vivo were used to determine the effects of TRP channel antagonists on cystitis-induced bladder hypersensitivity. KEY RESULTS: Protamine sulfate/zymosan treatment evoked mild inflammation in the bladder and increased micturition frequency in conscious animals. In cystitis, high threshold muscular afferents were sensitised via up-regulation of TRPV1 channels, high threshold muscular-mucosal afferents were sensitised via TRPM8 channels, and mucosal afferents by both. Visceromotor responses evoked by noxious bladder distension were significantly enhanced in cystitis and were returned to control levels upon administration of combination of low doses of TRPV1 and TRPM8 antagonists. CONCLUSIONS AND IMPLICATIONS: The data demonstrate the therapeutic promises of combination of TRPV1 and TRPM8 antagonists for the treatment of bladder hypersensitivity in cystitis.

The T-type calcium channel Ca V 3.2 regulates bladder afferent responses to mechanical stimuli.

ABSTRACT: The bladder wall is innervated by a complex network of afferent nerves that detect bladder stretch during filling. Sensory signals, generated in response to distension, are relayed to the spinal cord and brain to evoke physiological and painful sensations and regulate urine storage and voiding. Hyperexcitability of these sensory pathways is a key component in the development of chronic bladder hypersensitivity disorders including interstitial cystitis/bladder pain syndrome and overactive bladder syndrome. Despite this, the full array of ion channels that regulate bladder afferent responses to mechanical stimuli have yet to be determined. Here, we investigated the role of low-voltage-activated T-type calcium (Ca V 3) channels in regulating bladder afferent responses to distension. Using single-cell reverse-transcription polymerase chain reaction and immunofluorescence, we revealed ubiquitous expression of Ca V 3.2, but not Ca V 3.1 or Ca V 3.3, in individual bladder-innervating dorsal root ganglia neurons. Pharmacological inhibition of Ca V 3.2 with TTA-A2 and ABT-639, selective blockers of T-type calcium channels, dose-dependently attenuated ex-vivo bladder afferent responses to distension in the absence of changes to muscle compliance. Further evaluation revealed that Ca V 3.2 blockers significantly inhibited both low- and high-threshold afferents, decreasing peak responses to distension, and delayed activation thresholds, thereby attenuating bladder afferent responses to both physiological and noxious distension. Nocifensive visceromotor responses to noxious bladder distension in vivo were also significantly reduced by inhibition of Ca V 3 with TTA-A2. Together, these data provide evidence of a major role for Ca V 3.2 in regulating bladder afferent responses to bladder distension and nociceptive signalling to the spinal cord.

Melatonin inhibits muscular-mucosal stretch-sensitive bladder afferents via the MT2 receptors.

Melatonin is a circadian rhythm regulator capable of controlling a variety of physiological processes in the body. It predominantly acts via the melatonin 1 (MT1) and MT2 receptors expressed in the CNS neurons and peripheral organs and tissues. Melatonin can modulate urinary bladder function, however, to date it is not known if melatonin can regulate activity of sensory neurons innervating the bladder. Bladder afferents play an important role in urine storage and voiding. Therefore, this study aims to determine if melatonin can regulate mechanosensitivity of 2 major classes of sensory neurons in the guinea pig bladder: stretch-insensitive mucosal and low threshold stretch-sensitive muscular-mucosal afferents. The effects of melatonin on the mechanosensitivity of mucosal and muscular-mucosal afferents were measured ex vivo using single unit extracellular recording. Melatonin did not affect the responses of mucosal afferents to stroking of their receptive fields but did concentration-dependently, significantly inhibit 69% of muscular-mucosal afferents responses to stroking and bladder stretch. This inhibitory effect was not affected by the MT1 receptor antagonist, S26131 but was blocked by the selective MT2 receptor antagonists, K-185 and 4-P-PDOT. Forskolin significantly potentiated the responses of muscular-mucosal afferents to stroking and stretch, which were prevented by melatonin. These findings demonstrate a direct inhibitory effect of melatonin on the mechanosensitivity of low threshold stretch-sensitive muscular-mucosal bladder afferents acting via MT2 receptors, which is independent from its action on detrusor muscle. This may have important clinical implications for the treatment of many common bladder disorders including nocturia.

Disengaging spinal afferent nerve communication with the brain in live mice.

Our understanding of how abdominal organs (like the gut) communicate with the brain, via sensory nerves, has been limited by a lack of techniques to selectively activate or inhibit populations of spinal primary afferent neurons within dorsal root ganglia (DRG), of live animals. We report a survival surgery technique in mice, where select DRG are surgically removed (unilaterally or bilaterally), without interfering with other sensory or motor nerves. Using this approach, pain responses evoked by rectal distension were abolished by bilateral lumbosacral L5-S1 DRG removal, but not thoracolumbar T13-L1 DRG removal. However, animals lacking T13-L1 or L5-S1 DRG both showed reduced pain sensitivity to distal colonic distension. Removal of DRG led to selective loss of peripheral CGRP-expressing spinal afferent axons innervating visceral organs, arising from discrete spinal segments. This method thus allows spinal segment-specific determination of sensory pathway functions in conscious, free-to-move animals, without genetic modification.

Time-of-day dependent changes in guinea pig bladder afferent mechano-sensitivity.

The voiding of urine has a clear circadian rhythm with increased voiding during active phases and decreased voiding during inactive phases. Bladder spinal afferents play a key role in the regulation of bladder storage and voiding, but it is unknown whether they exhibit themselves a potential circadian rhythm. Therefore, this study aimed to determine the mechano- and chemo- sensitivity of three major bladder afferent classes at two opposite day-night time points. Adult female guinea pigs underwent conscious voiding monitoring and bladder ex vivo single unit extracellular afferent recordings at 0300 h and 1500 h to determine day-night modulation of bladder afferent activity. All guinea pigs voided a higher amount of urine at 1500 h compared to 0300 h. This was due to an increased number of voids at 1500 h. The mechano-sensitivity of low- and high-threshold stretch-sensitive muscular-mucosal bladder afferents to mucosal stroking and stretch was significantly higher at 1500 h compared to 0300 h. Low-threshold stretch-insensitive mucosal afferent sensitivity to stroking was significantly higher at 1500 h compared to 0300 h. Further, the chemosensitivity of mucosal afferents to N-Oleoyl Dopamine (endogenous TRPV1 agonist) was also significantly increased at 1500 h compared to 0300 h. This data indicates that bladder afferents exhibit a significant time-of-day dependent variation in mechano-sensitivity which may influence urine voiding patterns. Further studies across a 24 h period are warranted to reveal potential circadian rhythm modulation of bladder afferent activity.

Endocannabinoids in Bladder Sensory Mechanisms in Health and Diseases.

The recent surge in research on cannabinoids may have been fueled by changes in legislation in several jurisdictions, and by approval for the use of cannabinoids for treatment of some chronic diseases. Endocannabinoids act largely, but not exclusively on cannabinoid receptors 1 and 2 (CBR1 and CBR2) which are expressed in the bladder mainly by the urothelium and the axons and endings of motor and sensory neurons. A growing body of evidence suggests that endocannabinoid system constitutively downregulates sensory bladder function during urine storage and micturition, under normal physiological conditions. Similarly, exogenous cannabinoid agonists have potent modulatory effects, as do inhibitors of endocannabinoid inactivation. Results suggest a high potential of cannabinoids to therapeutically ameliorate lower urinary tract symptoms in overactive bladder and painful bladder syndromes. At least part of this may be mediated via effects on sensory nerves, although actions on efferent nerves complicate interpretation. The sensory innervation of bladder is complex with at least eight classes identified. There is a large gap in our knowledge of the effects of endocannabinoids and synthetic agonists on different classes of bladder sensory neurons. Future studies are needed to reveal the action of selective cannabinoid receptor 2 agonists and/or peripherally restricted synthetic cannabinoid receptor 1 agonists on bladder sensory neurons in animal models of bladder diseases. There is significant potential for these novel therapeutics which are devoid of central nervous system psychotropic actions, and which may avoid many of the side effects of current treatments for overactive bladder and painful bladder syndromes.

CB2 cannabinoid receptor agonist selectively inhibits the mechanosensitivity of mucosal afferents in the guinea pig bladder.

Bladder afferents play a pivotal role in bladder function such as urine storage and micturition as well as conscious sensations such as urgency and pain. Endocannabinoids are ligands of cannabinoid 1 and 2 (CB1 and CB2) receptors but can influence the activity of a variety of G protein-coupled receptors as well as ligand-gated and voltage-gated channels. It is still not known which classes of bladder afferents are influenced by CB1 and CB2 receptor agonists. This study aimed to determine the role of CB2 receptors in two major classes of afferents in the guinea pig bladder: mucosal and muscular-mucosal. The mechanosensitivity of these two classes was determined by an ex vivo extracellular electrophysiological recording technique. A stable analog of endocannabinoid anandamide, methanandamide (mAEA), potentiated the mechanosensitivity of mucosal bladder afferents in response to stroking. In the presence of a transient receptor potential vanilloid 1 antagonist (capsazepine), the effect of mAEA switched from excitatory to inhibitory. A selective CB2 receptor agonist, 4-quinolone-3-carboxyamide (4Q3C), significantly inhibited the mechanosensitivity of mucosal bladder afferents to stroking. In the presence of a CB2 receptor antagonist, the inhibitory effect of 4Q3C was lost. mAEA and 4Q3C did not affect responses to stretch and/or mucosal stroking of muscular-mucosal afferents. Our findings revealed that agonists of CB2 receptors selectively inhibited the mechanosensitivity of capsaicin-sensitive mucosal bladder afferents but not muscular-mucosal afferents. This may have important implications for understanding of the role of endocannabinoids in modulating bladder function and sensation in health and diseases.NEW & NOTEWORTHY This article describes, for the first time, to our knowledge, the direct inhibitory effect of cannabinoid 2 receptor agonists on guinea pig mucosal bladder afferents. The cannabinoid 2 receptor is involved in pain and inflammation, suggesting that this may be a viable target for treatment of bladder disorders such as cystitis.

Characterization of putative interneurons in the myenteric plexus of human colon.

BACKGROUND: The enteric nervous system contains multiple classes of neurons, distinguishable by morphology, immunohistochemical markers, and projections; however, specific combinations differ between species. Here, types of enteric neurons in human colon were characterized immunohistochemically, using retrograde tracing combined with multiple labeling immunohistochemistry, focussing on non-motor neurons. METHODS: The fluorescent carbocyanine tracer, DiI, was applied to the myenteric plexus in ex vivo preparations, filling neurons projecting within the plexus. Limits of projection lengths of motor neurons were established, allowing them to be excluded from the analysis. Long ascending and descending interneurons were then distinguished by labeling for discriminating immunohistochemical markers: calbindin, calretinin, enkephalin, 5-hydroxytryptamine, nitric oxide synthase, and substance P. These results were combined with a previous published study in which nitric oxide synthase and choline acetyltransferase immunoreactivities were established. KEY RESULTS: Long ascending neurons (with projections longer than 8 mm, which excludes more than 95% motor neurons) formed four types, in descending order of abundance, defined by immunoreactivity for: (a) ChAT+/ENK+, (b) ChAT+/ENK+/SP+, (c) ChAT+/Calb+, and (d) ChAT+/ENK+/Calb+. Long descending neurons, up to 70 mm long also formed at least four types, distinguished by immunoreactivity for (a) NOS + cells (without ChAT), (b) ChAT+/NOS+, (c) ChAT+/Calret+, and (d) ChAT+/5HT + cells (with or without NOS). CONCLUSIONS AND INFERENCES: Long interneurons, which do not innervate muscularis externa, are likely to coordinate neural activity over distances of many centimeters along the colon. Characterizing their neurochemical coding provides a basis for understanding their roles, investigating their connectivity, and building a comprehensive account of human colonic enteric neurons.

Morphological and neurochemical characterisation of anterogradely labelled spinal sensory and autonomic nerve endings in the mouse bladder.

The bladder is innervated by axons of sympathetic and parasympathetic efferent nerves, and by spinal afferent neurons. The objective was to characterise anatomically and immunohistochemically the terminal endings of sensory and autonomic motor nerve endings in wholemount preparations of the mouse bladder. We used both anterograde labelling of pelvic and hypogastric nerves ex vivo and anterograde labelling from lumbosacral dorsal root ganglia (DRG) in vivo in male and female mice. These were combined with immunohistochemistry for major markers of sensory, sympathetic and parasympathetic nerves. Selective labelling of spinal afferent endings following dextran biotin-labelling from DRGs in vivo showed no co-localisation of VAChT or TH in sensory terminals in the detrusor and suburothelial plexus. Biotinamide was applied ex vivo to nerve trunks arising in the pelvic ganglion and running towards the bladder. Among the filled axons, 38% of detrusor fibres and 47% of suburothelial axons were immunoreactive for calcitonin-gene related peptide (CGRP). Vesicular acetylcholine transporter (VAChT) immunoreactivity was present in 26% of both detrusor and suburothelial axons. For tyrosine hydroxylase (TH), the proportions were 15% and 17%, respectively. Three major morphological types of CGRP-immunoreactive nerve endings were distinguished in the bladder wall: simple, branching and complex. VAChT-immunoreactive parasympathetic axons had simple and branching endings; TH immunoreactive axons all had simple morphologies. Our findings revealed that different subtypes of sensory and autonomic nerve endings can be reliably identified by combining anterograde labelling ex vivo with specific immunohistochemical markers, although morphologically some of these types of endings were indistinguishable.

Roles of three distinct neurogenic motor patterns during pellet propulsion in guinea-pig distal colon.

KEY POINTS: Enteric neural circuits enable isolated preparations of guinea-pig distal colon to propel solid and fluid contents by a self-sustaining neuromechanical loop process. In addition there are at least three neural mechanisms which are not directly involved in propulsion: cyclic motor complexes, transient neural events and distal colon migrating motor complexes. In excised guinea-pig colon we simultaneously recorded high resolution manometry, video-imaging of colonic wall movements and electrophysiological recordings from smooth muscle, which enabled us to identify mechanisms that underlie the propulsion of colonic content. The results show that the intermittent propulsion during emptying of the multiple natural faecal pellets is due to the intermittent activation of cyclic motor complexes and this is facilitated by transient neural events. Loss or dysfunction of these activities is likely to underlie disordered gastrointestinal transit. ABSTRACT: It is well known that there are different patterns of electrical activity in smooth muscle cells along different regions of the gastrointestinal tract. These different patterns can be generated by myogenic and/or neurogenic mechanisms. However, what patterns of electrical activity underlie the propulsion of natural faecal content remains unknown, particularly along the large intestine, where large quantities of water are reabsorbed and semi-solid faeces form. In this study, we developed a novel approach which enables for the first time the simultaneous recording of high resolution intraluminal manometry, electrophysiology from the smooth muscle, and spatio-temporal video imaging of colonic wall movements. Using this approach we were able to reveal the nature of enteric neuromuscular transmission and patterns of motor activity responsible for the movement of content. Three distinct neurogenic patterns of electrical activity were recorded even in the absence of propulsive movement. These were the cyclic motor complexes (CMCs), the transient neural events (TNEs) and the slowly propagating distal colonic migrating motor complexes (DCMMCs). We present evidence that the initiation of pellet propulsion is due to a cyclic motor complex (CMC) occurring oral to the pellet. Furthermore, we discovered that the intermittent propulsion of natural faecal pellets is generated by intermittent activation of CMCs; and this propulsion is facilitated by hexamethonium-sensitive TNEs. However, TNEs were not required for propulsion. The findings reveal the patterns of electrical activity that underlie propulsion of natural colonic content and demonstrate that propulsion is generated by a complex interplay between distinct enteric neural circuits.

Characterization of projections of longitudinal muscle motor neurons in human colon.

BACKGROUND: The enteric nervous system contains inhibitory and excitatory motor neurons which modulate smooth muscle contractility. Cell bodies of longitudinal muscle motor neurons have not been identified in human intestine. METHODS: We used retrograde tracing ex vivo with DiI, with multiple labeling immunohistochemistry, to characterize motor neurons innervating tenial and inter-tenial longitudinal muscle of human colon. KEY RESULTS: The most abundant immunohistochemical markers in the tertiary plexus were vesicular acetylcholine transporter, nitric oxide synthase (NOS), and vasoactive intestinal polypeptide (VIP). Of retrogradely traced motor neurons innervating inter-tenial longitudinal muscle, 95% were located within 6mm oral or anal to the DiI application site. Excitatory motor neuron cell bodies, immunoreactive for choline acetyltransferase (ChAT), were clustered aborally, whereas NOS-immunoreactive cell bodies were distributed either side of the DiI application site. Motor neurons had small cell bodies, averaging 438 + 18µm2 in cross-sectional area, similar for ChAT- and NOS-immunoreactive subtypes. Motor neurons innervating the tenia had slightly longer axial projections, with 95% located within 9mm. ChAT-immunoreactive excitatory motor neurons to tenia were clustered aborally, whereas NOS-immunoreactive inhibitory motor neurons had both ascending and descending projections. VIP immunoreactivity was rarely present without NOS immunoreactivity in motor neurons. CONCLUSIONS AND INFERENCES: Tenial and inter-tenial motor neurons innervating the longitudinal muscle have short projections. Inhibitory motor neurons have less polarized projections than cholinergic excitatory motor neurons. Longitudinal and circular muscle layers are innervated by distinct local populations of excitatory and inhibitory motor neurons. A population of human enteric neurons that contribute significantly to colonic motility has been characterized.

Functional changes in low- and high-threshold afferents in obstruction-induced bladder overactivity.

Neural mechanisms of lower urinary tract symptoms in obstruction-induced bladder overactivity remain unclear. We made the first single unit recordings from different types of spinal afferents to determine the effects of bladder outlet obstruction in guinea pigs. A model of gradual bladder outlet obstruction in male guinea pigs was used to produce overactive bladder. Conscious voiding was assessed in metabolic cages, and micturition was recorded in anesthetized guinea pigs in vivo. Single unit extracellular recordings were made ex vivo from spinal afferent nerves in flat sheet preparations of the bladder. Guinea pigs with partially obstructed bladders showed a significant increase in conscious voiding frequency compared with sham-operated guinea pigs. Also, nonvoiding contractions increased significantly in both frequency and amplitude. Although spontaneous firing of low-threshold bladder afferents was increased, their stretch-induced firing was reduced. The proportion of capsaicin-sensitive low-threshold afferents increased in obstructed bladders. Interestingly, spontaneous and stretch-induced firing were both significantly increased in high-threshold afferents after obstruction. In summary, sensory signaling increased in the obstructed bladder during the filling phase. This is largely mediated by low-threshold stretch-sensitive afferents that are activated by increased local nonvoiding contractions. Increased spontaneous firing by high-threshold afferents also contributes. Our findings revealed a complex effect of bladder outlet obstruction on different types of bladder afferents that needs consideration for potential therapeutic targeting of lower urinary tract symptoms in obstruction-induced bladder overactivity.

Translating peripheral bladder afferent mechanosensitivity to neuronal activation within the lumbosacral spinal cord of mice.

Primary afferent neurons transduce distension of the bladder wall into action potentials that are relayed into the spinal cord and brain, where autonomic reflexes necessary for maintaining continence are coordinated with pathways involved in sensation. However, the relationship between spinal circuits involved with physiological and nociceptive signalling from the bladder has only been partially characterised. We used ex vivo bladder afferent recordings to characterise mechanosensitive afferent responses to graded distension (0-60 mm Hg) and retrograde tracing from the bladder wall to identify central axon projections within the dorsal horn of the lumbosacral (LS) spinal cord. Labelling of dorsal horn neurons with phosphorylated-MAP-kinase (pERK), combined with labelling for neurochemical markers (calbindin, calretinin, gamma aminobutyric acid, and parvalbumin) after in vivo bladder distension (20-60 mm Hg), was used to identify spinal cord circuits processing bladder afferent input. Ex vivo bladder distension evoked an increase in primary afferent output, and the recruitment of both low- and high-threshold mechanosensitive afferents. Retrograde tracing revealed bladder afferent projections that localised with pERK-immunoreactive dorsal horn neurons within the superficial laminae (superficial dorsal horn), dorsal gray commissure, and lateral collateral tracts of the LS spinal cord. Populations of pERK-immunoreactive neurons colabelled with calbindin, calretinin, or gamma aminobutyric acid, but not parvalbumin. Noxious bladder distension increased the percentage of pERK-immunoreactive neurons colabelled with calretinin. We identified LS spinal circuits supporting autonomic and nociceptive reflexes responsible for maintaining continence and bladder sensations. Our findings show for the first time that low- and high-threshold bladder afferents relay into similar dorsal horn circuits, with nociceptive signalling recruiting a larger number of neurons.

Identifying unique subtypes of spinal afferent nerve endings within the urinary bladder of mice.

Spinal afferent neurons are responsible for the transduction and transmission of noxious (painful) stimuli and innocuous stimuli that do not reach conscious sensations from visceral organs to the central nervous system. Although the location of the nerve cell bodies of spinal afferents is well known to reside in dorsal root ganglia (DRG), the morphology and location of peripheral nerve endings of spinal afferents that transduce sensory stimuli into action potentials is poorly understood. The individual nerve endings of spinal afferents that innervate the urinary bladder have never been unequivocally identified in any species. We used an anterograde tracing technique developed in our laboratory to selectively label only spinal afferents. Mice were anesthetized and unilateral injections of dextran-amine made into lumbosacral DRGs (L5-S2). Seven to nine days postsurgery, mice were euthanized, the urinary bladder removed, then fresh-fixed and stained for immunoreactivity to calcitonin-gene-related-peptide (CGRP). Four distinct morphological types of spinal afferent ending in the bladder were identified. Three types existed in the detrusor muscle and one major type in the sub-urothelium and urothelium. Most nerve endings were located in detrusor muscle where the three types could be identified as having: "branching", "simple", or "complex" morphology. The majority of spinal afferent nerve endings were CGRP-immunoreactive. Single spinal afferent axons bifurcated many times upon entering the bladder and developed varicosities along their axon terminal endings. We present the first morphological identification of spinal afferent nerve endings in the mammalian urinary bladder.

Spinal afferent nerve endings in visceral organs: recent advances.

Spinal afferent neurons play a major role in detection and transduction of painful stimuli from internal (visceral) organs. Recent technical advances have made it possible to visualize the endings of spinal afferent axons in visceral organs. Although it is well known that the sensory nerve cell bodies of spinal afferents reside within dorsal root ganglia (DRG), identifying their endings in internal organs has been especially challenging because of a lack of techniques to distinguish them from endings of other extrinsic and intrinsic neurons (sympathetic, parasympathetic, and enteric). We recently developed a surgical approach in live mice that allows selective labeling of spinal afferent axons and their endings, revealing a diverse array of different types of varicose and nonvaricose terminals in visceral organs, particularly the large intestine. In total, 13 different morphological types of endings were distinguished in the mouse distal large intestine, originating from lumbosacral DRG. Interestingly, the stomach, esophagus, bladder, and uterus had less diversity in their types of spinal afferent endings. Taken together, spinal afferent endings (at least in the large intestine) appear to display greater morphological diversity than vagal afferent endings that have previously been extensively studied. We discuss some of the new insights that these findings provide.

Conscious voiding during bladder obstruction in guinea pigs correlates with contractile activity of isolated bladders.

PURPOSE: There are many hypotheses accounting for detrusor overactivity; however, the exact mechanisms are still incompletely understood. We used a model of bladder outlet obstruction in male guinea pigs as a way to produce detrusor overactivity. The objective was to determine whether changes in voiding of obstructed guinea pigs correlates with specific changes in contractile activity of their isolated bladders in vitro. MATERIAL AND METHODS: Conscious voiding activity of sham-operated and obstructed animals was measured in metabolic cages. Contractile activity (spontaneous or evoked by distension, electrical field stimulation or cholinergic agonists) was recorded via a pressure transducer in the isolated bladders in vitro. RESULTS: The frequency of conscious voiding increased (while voiding volume decreased) in the obstructed group, compared with the sham-operated group, 4 weeks after surgical intervention. In comparison to the sham-operated animals, the bladders from the obstructed guinea pigs were enlarged and inflamed, their frequency of spontaneous contractions was higher, while the amplitudes of electrical field stimulation (EFS)-induced contractions and bladder compliance were lower. Changes in conscious voiding during obstruction were significantly associated with alterations in structural parameters (bladder weight, thickness and histological damage score) and functional contractile parameters (frequency of spontaneous contractions, amplitude of EFS-induced contractions and bladder compliance) of their isolated bladders. CONCLUSIONS: Our findings revealed significant association between conscious voiding and structural and contractile activity changes of the isolated bladders in obstruction. The data suggest that change in contractile activity of the bladder itself is a major contributor to obstruction-induced bladder overactivity.

Neurochemical characterization of extrinsic nerves in myenteric ganglia of the guinea pig distal colon.

Extrinsic nerves to the gut influence the absorption of water and electrolytes and expulsion of waste contents, largely via regulation of enteric neural circuits; they also contribute to control of blood flow. The distal colon is innervated by extrinsic sympathetic and parasympathetic efferent and spinal afferent neurons, via axons in colonic nerve trunks. In the present study, biotinamide tracing of colonic nerves was combined with immunohistochemical labeling for markers of sympathetic, parasympathetic, and spinal afferent neurons to quantify their relative contribution to the extrinsic innervation. Calcitonin gene-related peptide, vesicular acetylcholine transporter, and tyrosine hydroxylase, which selectively label spinal afferent, parasympathetic, and sympathetic axons, respectively, were detected immunohistochemically in 1 ± 0.5% (n = 7), 15 ± 4.7% (n = 6), and 24 ± 4% (n = 7) of biotinamide-labeled extrinsic axons in myenteric ganglia. Immunoreactivity for vasoactive intestinal polypeptide, nitric oxide synthase, somatostatin, and vesicular glutamate transporters 1 and 2 accounted for a combined maximum of 14% of biotinamide-labeled axons in myenteric ganglia. Thus, a maximum of 53% of biotinamide-labeled extrinsic axons in myenteric ganglia were labeled by antisera to one of these eight markers. Viscerofugal neurons were also labeled by biotinamide. They had distinct morphologies and spatial distributions that correlated closely with their immunoreactivity for nitric oxide synthase and choline acetyltransferase. As reported for the rectum, nearly half of all extrinsic nerve fibers to the distal colon lack the key immunohistochemical markers commonly used for their identification. Their abundance may therefore have been significantly underestimated in previous immunohistochemical studies.