Search

Botulinum neurotoxin A (BoNT/A) is a potent neurotoxin that silences cholinergic neurotransmission through the cleavage of the synaptic protein SNAP-25. Previous studies have shown that, in addition to its paralytic effects, BoNT/A can inhibit sensory nerve activity. The aim of this study was to identify how BoNT/A inhibits afferent signalling from the bladder. To investigate the role of SNAP-25 cleavage in the previously reported BoNT/A-dependent inhibition of sensory signalling, we developed a recombinant form of BoNT/A with an inactive light chain, rBoNT/A (0), unable to paralyse muscle. We also developed recombinant light chain (LC)-domain-only proteins to better understand the entry mechanisms, as the heavy chain (HC) of the protein is responsible for the internalisation of the light chain. We found that, despite a lack of catalytic activity, rBoNT/A (0) potently inhibited the afferent responses to bladder distension to a greater degree than catalytically active rBoNT/A. This was also clear from the testing of the LC-only proteins, as the inactive rLC/A (0) protein inhibited afferent responses significantly more than the active rLC/A protein. Immunohistochemistry for cleaved SNAP-25 was negative, and purinergic and nitrergic antagonists partially and totally reversed the sensory inhibition, respectively. These data suggest that the BoNT/A inhibition of sensory nerve activity in this assay is not due to the classical well-characterised 'double-receptor' mechanism of BoNT/A, is independent of SNAP25 cleavage and involves nitrergic and purinergic signalling mechanisms.

Key Points: Functional disorders (i.e. interstitial cystitis/painful bladder syndrome and irritable bowel syndrome) are associated with hyperexcitability of afferent nerves innervating the urinary tract and the bowel, respectively. Various non-5-HT 3 receptor mRNA transcripts are expressed in mouse urothelium and exert functional responses to 5-HT. Whilst 5-HT 3 receptors were not detected in mouse urothelium, 5-HT 3 receptors expressed on bladder sensory neurons plays a role in bladder afferent excitability both under normal conditions and in a mouse model of chronic visceral hypersensitivity. These data suggest that the role 5-HT 3 receptors play in bladder afferent signalling warrants further study as a potential therapeutic target for functional bladder disorders., Abstract: Serotonin (5-HT) is an excitatory mediator that in the gastrointestinal (GI) tract plays a physiological role in gut-brain signalling and is dysregulated in functional GI disorders such as irritable bowel syndrome (IBS). Patients suffering from IBS frequently suffer from urological symptoms characteristic of interstitial cystitis/painful bladder syndrome, which manifests due to cross-sensitization of shared innervation pathways between the bladder and colon. However, a direct modulatory role of 5-HT in bladder afferent signalling and its role in colon-bladder neuronal crosstalk remain elusive. The aim of this study was to investigate the action of 5-HT on bladder afferent signalling in normal mice and mice with chronic visceral hypersensitivity (CVH) following trinitrobenzenesulfonic acid-induced colitis. Bladder afferent activity was recorded directly using ex vivo afferent nerve recordings. Expression of 14 5-HT receptor subtypes, the serotonin transporter (SERT) and 5-HT-producing enzymes was determined in the urothelium using RT-PCR. Retrograde labelling of bladder-projecting dorsal root ganglion neurons was used to investigate expression of 5-HT 3 receptors using single cell RT-PCR, while sensory neuronal and urothelial responses to 5-HT were determined by live cell calcium imaging. 5-HT elicited bladder afferent firing predominantly via 5-HT 3 receptors expressed on afferent terminals. CVH animals showed a downregulation of SERT mRNA expression in urothelium, suggesting increased 5-HT bioavailability. Granisetron, a 5-HT 3 antagonist, reversed bladder afferent hypersensitivity in CVH mice. These data suggest 5-HT exerts a direct effect on bladder afferents to enhance signalling. 5-HT 3 antagonists could therefore be a potential therapeutic target to treat functional bladder and bowel disorders., (© 2019 The Authors. The Journal of Physiology © 2019 The Physiological Society.)

Both TRPV1 and P2X receptors present on bladder sensory nerve fibres have been implicated in mechanosensation during bladder filling. The aim of this study was to determine possible interactions between these receptors in modulating afferent nerve activity. In wildtype (TRPV1 +/+ ) and TRPV1 knockout (TRPV1 -/- ) mice, bladder afferent nerve activity, intravesical pressure, and luminal ATP and acetylcholine levels were determined and also intracellular calcium responses of dissociated pelvic DRG neurones and primary mouse urothelial cells (PMUCs). Bladder afferent nerve responses to the purinergic agonist αβMethylene-ATP were depressed in TRPV1 -/- mice (p ≤ 0.001) and also in TRPV1 +/+ mice treated with the TRPV1-antagonist capsazepine (10 µM; p ≤ 0.001). These effects were independent of changes in bladder compliance or contractility. Responses of DRG neuron to αβMethylene-ATP (30 µM) were unchanged in the TRPV1 -/- mice, but the proportion of responsive neurones was reduced (p ≤ 0.01). Although the TRPV1 agonist capsaicin (1 µM) did not evoke intracellular responses in PMUCs from TRPV1 +/+ mice, luminal ATP levels were reduced in the TRPV1 -/- mice (p ≤ 0.001) compared to wildtype. TRPV1 modulates P2X mediated afferent responses and provides a mechanistic basis for the decrease in sensory symptoms observed following resiniferatoxin and capsaicin treatment for lower urinary tract symptoms.

Urodynamic tests are the gold standard for the diagnosis of bladder dysfunction, and the mechanical compliance of the bladder is an important parameter in these tests. The bladder wall has a layered structure, differentially affected by pathology, so knowledge of the contribution and role of these layers and their constituents to overall bladder compliance will enhance interpretation of these clinical tests. In this study we document the functional morphology of the detrusor and lamina propria of the murine bladder wall using a custom in-situ tensile loading system under multiphoton microscopy (MPM) observation in unloaded state and under incremental uniaxial stretch. Features in the stress-stretch curves of bladder samples were then directly related to corresponding MPM images. Collagen organisation across wall depth was quantified using image analysis techniques. The hypothesis that the lamina propria deformed at low strain by unfolding of the rugae and rearranging collagen fibrils was confirmed. A novel 'pocket' feature in the detrusor was observed along with extensive rearrangement of fibrils in two families at different depths, providing higher stiffness at high stretches in the detrusor. The very different deformations of detrusor and lamina propria were accommodated by the highly coiled structure of collagen in the lamina propria. Imaging and mechanical studies presented here allow gross mechanical response to be attributed to specific components of the bladder wall and further, may be used to investigate the impact of microstructural changes due to pathology or aging, and how they impair tissue functionality., Statement of Significance: This article reports the first in-situ multiphoton microscopy observations of microstructural deformation under uniaxial tensile loading of ex vivo bladder. We describe collagen rearrangement through the tissue thickness and relate this directly to the stress-stretch behaviour. We confirm for the first time the unfolding of rugae and realignment of fibrils in the lamina propria during extension and the rapid stiffening as two fibril families in the detrusor are engaged. This technique provides new insight into microstructure function and will enhance understanding of the impact of changes due to pathology or aging., (Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Background: Advanced age is associated with a reduction in clinical visceral pain perception. However, the underlying mechanisms remain largely unknown. Previous studies have suggested that an abnormal interplay between mast cells, enterochromaffin (EC) cells, and afferent nerves contribute to nociception in gastrointestinal disorders. The aim of this study was to investigate how aging affects afferent sensitivity and neuro-immune association in the human bowel., Methods: Mechanical and chemical sensitivity of human bowel afferents were examined by ex vivo afferent nerve recordings. Age-related changes in the density of mast cells, EC cells, sensory nerve terminals, and mast cell-nerve micro-anatomical association were investigated by histological and immune staining., Key Results: Human afferents could be broadly classified into subpopulations displaying mechanical and chemical sensitivity, adaptation, chemo-sensitization, and recruitment. Interestingly human bowel afferent nerve sensitivity was attenuated with age. The density of substance P-immunoreactive (SP-IR) nerve varicosities was also reduced with age. In contrast, the density of ileal and colonic mucosal mast cells was increased with age, as was ileal EC cell number. An increased proportion of mast cells was found in close apposition to SP-IR nerves., Conclusions & Inferences: Afferent sensitivity in human bowel was reduced with advancing age. Augmentation of mast cells and EC cell numbers and the mast cell-nerve association suggest a compensatory mechanism for sensory neurodegeneration., (© 2016 The Authors. Neurogastroenterology & Motility Published by John Wiley & Sons Ltd.)

The incidence of bladder conditions such as overactive bladder syndrome and its associated urinary incontinence is highly prevalent in the elderly. However, the mechanisms underlying these disorders are unclear. Studies suggest that the urothelium forms a 'sensory network' with the underlying innervation, alterations in which, could compromise bladder function. As the accumulation of reactive oxygen species can cause functional alterations with age, the aim of this study was to investigate whether oxidative stress alters urothelial sensory signalling and whether the mechanism underlying the effect of oxidative stress on the urothelium plays a role in aging. Five-month-old(young) and 24-month-old (aged) mice were used. H2O2 , used to induce oxidative stress, resulted in an increase in bladder afferent nerve activity and urothelial intracellular calcium in preparations from young mice. These functional changes were concurrent with upregulation of TRPM8 in the urothelium. Moreover, application of a TRPM8 antagonist significantly attenuated the H2O2 -induced calcium responses. Interestingly, an upregulation of TRPM8 was also found in the urothelium from aged mice, where high oxidative stress levels were observed, together with a greater calcium response to the TRPM8 agonist WS12. Furthermore, these calcium responses were attenuated by pretreatment with the antioxidant N-acetyl-cysteine. This study shows that oxidative stress affects urothelial function involving a TRPM8-mediated mechanism and these effects may have important implications for aging. These data provide an insight into the possible mechanisms by which oxidative stress causes physiological alterations in the bladder, which may also occur in other organs susceptible to aging., (© 2014 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

The prevalence of lower urinary tract storage disorders such as overactive bladder syndrome and urinary incontinence significantly increase with age. Previous studies have demonstrated age-related changes in detrusor function and urothelial transmitter release but few studies have investigated how the urothelium and sensory pathways are affected. The aim of this study was to investigate the effect of ageing on urothelial-afferent signalling in the mouse bladder. Three-month-old control and 24-month-old aged male mice were used. In vivo natural voiding behaviour, sensory nerve activity, urothelial cell function, muscle contractility, transmitter release and gene and protein expression were measured to identify how all three components of the bladder (neural, contractile and urothelial) are affected by ageing. In aged mice, increased voiding frequency and enhanced low threshold afferent nerve activity was observed, suggesting that ageing induces overactivity and hypersensitivity of the bladder. These changes were concurrent with altered ATP and acetylcholine bioavailability, measured as transmitter overflow into the lumen, increased purinergic receptor sensitivity and raised P2X3 receptor expression in the urothelium. Taken together, these data suggest that ageing results in aberrant urothelial function, increased afferent mechanosensitivity, increased smooth muscle contractility, and changes in gene and protein expression (including of P2X3). These data are consistent with the hypothesis that ageing evokes changes in purinergic signalling from the bladder, and further studies are now required to fully validate this idea.

Objective: To investigate the direct effect of onabotulinumtoxinA (OnaBotA) on bladder afferent nerve activity and release of ATP and acetylcholine (ACh) from the urothelium., Materials and Methods: Bladder afferent nerve activity was recorded using an in vitro mouse preparation enabling simultaneous recordings of afferent nerve firing and intravesical pressure during bladder distension. Intraluminal and extraluminal ATP, ACh, and nitric oxide (NO) release were measured using the luciferin-luciferase and Amplex(®) Red assays (Molecular Probes, Carlsbad, CA, USA), and fluorometric assay kit, respectively. OnaBotA (2U), was applied intraluminally, during bladder distension, and its effect was monitored for 2 h after application. Whole-nerve activity was analysed to classify the single afferent units responding to physiological (low-threshold [LT] afferent <15 mmHg) and supra-physiological (high-threshold [HT] afferent >15 mmHg) distension pressures., Results: Bladder distension evoked reproducible pressure-dependent increases in afferent nerve firing. After exposure to OnaBotA, both LT and HT afferent units were significantly attenuated. OnaBotA also significantly inhibited ATP release from the urothelium and increased NO release., Conclusion: These data indicate that OnaBotA attenuates the bladder afferent nerves involved in micturition and bladder sensation, suggesting that OnaBotA may exert its clinical effects on urinary urgency and the other symptoms of overactive bladder syndrome through its marked effect on afferent nerves., (© 2013 The Authors. BJU International © 2013 BJU International.)

Purpose of Review: Lower urinary tract disorders such as overactive bladder syndrome (OABS) and interstitial cystitis/painful bladder syndrome (IC/PBS) are debilitating conditions with serious adverse effects on quality of life. Common to both OABS and IC/PBS are the sensory symptoms of urgency and frequency, implicating the afferent system in the aetiology of these disorders. Thus, understanding the role that afferent pathways play in the function of the lower urinary tract is the focus of much current research. This review aims to provide an insight into the recent advances in this field., Recent Findings: Sensory transduction in the bladder is not only mediated by direct activation of the afferents via a host of receptors and ion channels located on the afferent terminal but also may be attributed to the interplay between the urothelium and the release of urothelially derived mediators. Recent studies provide compelling evidence to support this concept and highlight the complex nature of the bladder afferent system., Summary: Recent studies provide further evidence that afferent control of the bladder may be dependent on integration of excitatory and inhibitory mediators from the urothelium such as ATP and nitric oxide. A number of studies have examined the role cholinergic and adrenergic mechanisms play in bladder afferent function, and several new potential mechanisms involving the cannabinoid receptors and transient receptor potential channels have emerged as areas which warrant further investigation. A better understanding of afferent mechanisms in the bladder will hopefully lead to more effective treatments of lower urinary tract disorders.

Gastrointestinal vagal afferents transmit satiety signals to the brain via both chemical and mechanical mechanisms. There is indirect evidence that these signals may be attenuated in obesity. We hypothesized that responses to satiety mediators and distension of the gut would be attenuated after induction of diet induced obesity. Obesity was induced by feeding a high fat diet (60% kcal from fat). Low fat fed mice (10% kcal from fat) served as a control. High fat fed mice were obese, with increased visceral fat, but were not hyperglycaemic. Recordings from jejunal afferents demonstrated attenuated responses to the satiety mediators cholecystokinin (CCK, 100 nm) and 5-hydroxytryptamine (5-HT, 10 μm), as was the response to low intensity jejunal distension, while responses to higher distension pressures were preserved. We performed whole cell patch clamp recordings on nodose ganglion neurons, both unlabelled, and those labelled by fast blue injection into the wall of the jejunum. The cell membrane of both labelled and unlabelled nodose ganglion neurons was less excitable in HFF mice, with an elevated rheobase and decreased number of action potentials at twice rheobase. Input resistance of HFF neurons was also significantly decreased. Calcium imaging experiments revealed reduced proportion of nodose ganglion neurons responding to CCK and 5-HT in obese mice. These results demonstrate a marked reduction in afferent sensitivity to satiety related stimuli after a chronic high fat diet. A major mechanism underlying this change is reduced excitability of the neuronal cell membrane. This may explain the development of hyperphagia when a high fat diet is consumed. Improving sensitivity of gastrointestinal afferent nerves may prove useful to limit food intake in obesity.

Background: The main treatment for overactive bladder (OAB) is the use of anticholinergic drugs initially believed to inhibit the effect of parasympathetic acetylcholine (ACh) on the detrusor; however, there is now evidence to suggest that anticholinergic drugs could interact with sensory pathways., Objective: Investigate the role of muscarinic receptors and ACh in modulating bladder afferent sensitivity in the mouse., Design, Setting, and Participants: Bladder and surrounding tissue were removed from wild-type male mice, placed in a recording chamber, and continually perfused with fresh oxygenated Krebs solution at 35 degrees C. Bladders were cannulated to allow infusion and intravesical pressure monitoring, and afferent nerve fibres innervating the bladder were dissected and put into a suction electrode for recording., Measurements: Multiunit afferent activity and intravesical pressure were recorded at baseline and during bladder distension. Experiments were conducted in the presence of muscarinic agonists and antagonist or in the presence of the cholinesterase inhibitor physostigmine., Results and Limitations: Blocking muscarinic receptors using atropine (1 microM) had no effect on spontaneous afferent discharge, the afferent response to bladder distension, or on bladder compliance. However, stimulation of muscarinic receptors directly using bethanechol (100 microM) and carbachol (100 microM) or indirectly using physostigmine (10 microM) significantly inhibited the afferent response to bladder distension and concurrently reduced bladder compliance. Furthermore, prior application of nifedipine prevented the changes in bladder tone but did not prevent the attenuation of afferent responses by bethanechol or physostigmine., Conclusions: These data indicate that stimulation of muscarinic receptor pathways can depress sensory transduction by a mechanism independent of changes in bladder tone, suggesting that muscarinic receptor pathways and ACh could contribute to normal or pathologic bladder sensation., (Copyright 2009 European Association of Urology. Published by Elsevier B.V. All rights reserved.)

Background: Glucagon-like peptide-1 (GLP-1) is a major hormone known to regulate glucose homeostasis and gut function, and is an important satiety mediator. These actions are at least in part mediated via an action on vagal afferent neurons. However, the mechanism by which GLP-1 activates vagal afferents remains unknown. We hypothesized that GLP-1 acts on nodose ganglion neuron voltage-gated potassium (KV) channels, increasing membrane excitability., Methods: Employing perforated patch clamp recordings we examined the effects of GLP-1 on membrane properties as well as voltage-gated potassium currents. Extracellular recordings of jejunal afferents were performed to demonstrate the functional relevance of these effects at the nerve terminal., Key Results: Glucagon-like peptide-1 depolarized a subpopulation of nodose neurons. This membrane depolarization was used to identify neurons containing functional GLP-1 receptors. In these neurons, GLP-1 decreased rheobase and broadened the action potential, and increased the number of action potentials elicited at twice rheobase. We identified a GLP-1 sensitive current whose reversal potential shifted in a depolarizing direction when extracellular potassium was increased. We identified two macroscopic K currents, IA, an inactivating current and IK a sustained current. GLP-1 caused inhibition of these currents, IK by 45%, P < 0.05 and IA currents by 52%P < 0.01, associated with a hyperpolarizing shift of steady-state inactivation curves for both currents. In extracellular recordings of jejunal afferents, GLP-1 increased firing rate, the effect blocked by the K(+) channel antagonist 4-AP., Conclusions & Inferences: These experiments indicate that GLP-1 receptor activation results in vagal afferent excitation, due at least in part to inhibition of sustained and inactivating potassium currents. This mechanism may be important in satiety and glucose homeostatic signals arising from the gastrointestinal tract.

Objective: The aim of this study was to determine whether physician offices located in high-minority and low-income neighborhoods have different levels of access to information technology than offices located in lower-minority and higher-income areas., Design: A cross-sectional survey was conducted of pediatrics, family medicine, and general practice offices in Orange County, California. Survey data were linked with community demographic data from the 2000 Census using a geographical information system., Results: Of 307 offices surveyed, 141 responded (46%). Offices located in high-minority and high-poverty areas were as likely to respond as other offices. Among responding offices, 94% had a computer, 77% had Web access, 29% had broadband Internet access, and 53% used computerized scheduling and billing systems. Offices located in minority and low-income communities had equivalent access to each technology. Offices in communities with larger proportions of Hispanics were less likely to have practice Web pages, but other uses of the Internet were not associated with practice location. Offices reported high levels of interest in online clinical systems but also high levels of concern about these systems' usability and confidentiality. Offices with Web access and those with practice management systems expressed greater interest in online clinical systems but also greater levels of concern about usability and confidentiality. These attitudes were equivalent among offices in different communities., Conclusion: Primary care offices located in poor and minority communities in a large, suburban county had high levels of access to and interest in Web-based systems. Physicians' offices may therefore provide a venue for online services aimed at improving health outcomes for poor and minority communities. Research is needed in other geographic regions to determine the generalizability of these findings.

Auditory sensitivity and processing ability were evaluated in a patient who suffered from hyperacusis, difficulty understanding speech, withdrawn depression, lethargy, and hypersensitivity to touch, pressure, and light. Treatment with fluvoxamine and fluoxetine (selective serotonin reuptake inhibitors) reversibly alleviated complaints. Testing while medicated and unmedicated (after voluntary withdrawal from medication for several weeks) revealed no difference in pure-tone thresholds, speech thresholds, word recognition scores, tympanograms, or acoustic reflex thresholds. Medicated SCAN-A (a screening test for central auditory processing disorders) results were normal, and unmedicated results were abnormal. Unmedicated transient otoacoustic emissions and auditory brainstem response waves I, III, and V were significantly larger bilaterally. Uncomfortable loudness levels indicated greater tolerance during the medicated condition. Central processing and vigilance were evaluated with analog-synthesized three-formant consonant-vowel syllables. While medicated, responses to stimuli at each ear revealed well-defined, labeling crossovers of about 90 msec. Vowel identification matched normal subject responses; labeling of /gE/jE/ and /bE/wE/ continua was well defined but all crossover points differed from normals (p < .0001). During unmedicated testing, responses to /gE/jE/ began at medicated levels but approached chance levels for the entire continuum within 10 min; labeling of /bE/wE/ was consistent with medicated responses throughout with earlier than normal crossover points.

Frequency-following responses (FFRs) evoked by 500 c/sec tone bursts 14 msec in duration, presented at 50 dB SL were recorded from Cz--A1 and Cz--A2 electrode derivations using eight subjects with normal bilateral hearing, and eight subjects with profound unilateral hearing loss. Monaural stimulation of either ear in normal subjects, and of the unimpaired ear in hearing-loss subjects, evoked larger responses from the ipsilateral electrode derivation than from the contralateral electrode derivation. Stimulation of the impaired ear in hearing-loss subjects evoked no response. Binaural stimulation in normal-hearing subjects evoked Cz--A1 and Cz--A2 responses of equal magnitude, each larger than either the ipsilateral or contralateral monaural response, but each slightly smaller than the sum of the ipsilateral and contralateral monaural responses. Binaural stimulation in hearing-loss subjects evoked responses equivalent to those obtained monaurally. The results provide evidence of binaural interaction in normal-hearing subjects and indicate that FFR arises from at least two separate symmetric neural sources, possibly by iterative activation of brainstem evoked response (BER) generators.

Using clicks with varying interstimulus intervals and coherent tone-bursts, early components of the auditory evoked potential (brain stem responses) were studied in four patients with confirmed acoustic neuroma. Abnormalities in responses appeared with shorter interstimulus intervals and with tone-bursts delivered monaurally to the involved ear; bilateral alterations occurred in one patient with brain stem displacement. The results indicate that BSR can provide a stable, independent, noninvasive measure of auditory nerve function useful in the early detection of acoustic neuroma. The results contribute to the understanding of the so-called human FFR.