Sensitive Afferents Research Articles

Sensitizing Effects of Chronic Pretreatment with Tumor Necrosis Factor Alpha on Vagal Pulmonary Afferent Sensitivity in Mice

Tumor necrosis factor alpha (TNFα), a pro‐inflammatory cytokine, is known to play an important role in the pathogenesis of allergic asthma. Inhalation of TNFα can also induce airway hyperresponsiveness in healthy human subjects, but underlying mechanisms are not fully understood. A recent study in our lab has demonstrated that TNFα pretreatment induced airway inflammation and a sustained elevation of pulmonary chemoreflex sensitivity (JAP 114: 1536–43, 2013), suggesting a possible involvement of pulmonary C‐fibers, but the direct evidence is lacking. The present study investigated the effects of chronic pretreatment with TNFα on the sensitivity of vagal pulmonary afferents in anesthetized open‐chest mice, using the single‐fiber recording technique. TNFα (10 μg/kg) and vehicle were administered by intra‐tracheal instillation in treated (TNF) and control (Veh) groups, respectively. Our results showed: 1) The peak activity of pulmonary C‐fibers to a bolus right‐atrial injection of capsaicin (Cap; 0.5 μg/kg) was significantly greater in TNF group (6.52 ± 1.34 impulses/s, n=10) than Veh group (2.10 ± 0.56 impulses/s, n=12; p<0.05). 2) The same dose of Cap evoked a burst of discharge (2.44 ± 0.71 impulses/s, n=8) in 75% (6/8) of rapidly adapting receptors (RARs) studied in TNF group, but did not stimulate any of the RARs (0/9) in Veh group. 3) This sensitizing effect of TNFα was further demonstrated in the enhanced response of Ca2+ influx to Cap in isolated vagal pulmonary sensory neurons using the Ca2+ imaging technique, and this effect was completely absent in the mice lacking both TNF receptors (p55/p75‐null mice). In conclusion, chronic pretreatment with TNFα induced a sensitizing effect on the Cap sensitivity in both pulmonary C‐fibers and RARs, and the action was mediated through TNF receptors. These sensitizing effects of TNFα may contribute, at least in part, to the pathogenesis of airway hyperresponsiveness induced by this cytokine.Support or Funding InformationSupported by NIH grants HL‐96914 and UL1TR0000117

MP28-06 COLITIS INDUCES LONG-TERM BLADDER AFFERENT HYPERACTIVITY, WHICH IS ATTENUATED BY CHRONIC ORAL ADMINISTRATION OF THE GUANYLATE CYCLASE-C AGONIST LINACLOTIDE

You have accessJournal of UrologyBladder & Urethra: Anatomy, Physiology & Pharmacology I1 Apr 2016MP28-06 COLITIS INDUCES LONG-TERM BLADDER AFFERENT HYPERACTIVITY, WHICH IS ATTENUATED BY CHRONIC ORAL ADMINISTRATION OF THE GUANYLATE CYCLASE-C AGONIST LINACLOTIDE Luke Grundy, Sonia Garcia-Caraballo, Jessica Maddern, Grigori Rychkov, Gerhard Hannig, Caroline Kurtz, Ada Silos-Santiago, and Stuart M. Brierley Luke GrundyLuke Grundy More articles by this author , Sonia Garcia-CaraballoSonia Garcia-Caraballo More articles by this author , Jessica MaddernJessica Maddern More articles by this author , Grigori RychkovGrigori Rychkov More articles by this author , Gerhard HannigGerhard Hannig More articles by this author , Caroline KurtzCaroline Kurtz More articles by this author , Ada Silos-SantiagoAda Silos-Santiago More articles by this author , and Stuart M. BrierleyStuart M. Brierley More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1056AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Patients suffering from IBS frequently suffer from urological symptoms characteristic of interstitial cystitis/painful bladder syndrome. This viscero-visceral cross-talk has also been described in pre-clinical studies. Acute colitis in rodents is associated with altered bladder cystometry and bladder afferent sensitisation [1,2]. However, it remains to be determined if bladder overactivity persists following the resolution of colitis, in a model of chronic colonic hypersensitivity (CCH), or if reducing colonic nociception is able to alter bladder overactivity. Linaclotide, an FDA approved guanylate cyclase-C (GC-C) agonist, reduces abdominal pain in IBS patients with constipation [3], reverses colonic mechanical hyper-sensitivity in CCH mice, and reduces noxious signalling to the spinal cord in mice in vivo. We hypothesized that oral linaclotide administration may also reduce bladder hypersensitivity. METHODS We investigated healthy C57BL/6J mice and mice with CCH, 28 days after intra-colonic TNBS administration. CCH mice were randomly assigned to either chronic linaclotide (3μg/kg/day) or placebo (water) administration, consisting of a once daily oral gavage for 2 weeks prior to experimentation. In all four groups, micturition pattern analysis was performed by analysing in-vivo natural voiding behavior. Ex-vivo electrophysiological recordings determined bladder afferent and contractile sensitivity to ramp distension. RESULTS Mice with CCH displayed significant changes in voiding frequency (P<0.01) and significant increases in bladder afferent responses to distension (P<0.001), with no changes in muscle compliance. CCH mice treated with linaclotide displayed attenuated bladder afferent sensitivity compared with placebo (p≤0.001). Linaclotide treatment also restored natural voiding behavior (p≤0.05). CONCLUSIONS Mice with CCH also display abnormal bladder voiding behavior and increased bladder afferent excitability, an example of viscero-visceral cross-talk. Chronic oral administration of linaclotide, a gut-restricted GC-C agonist that inhibits colonic nociceptors, reverses these colitis-induced changes in bladder function and sensitivity. Agents that improve abdominal pain may be able to improve urological symptoms through common sensory innervation pathways. 1. Lamb et al, AJPGI, 2006. 2. Ustinova et al, Neurourology and Urodynamics, 2010. 3. Castro et al, Gastroenterology, 2013. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e373 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Luke Grundy More articles by this author Sonia Garcia-Caraballo More articles by this author Jessica Maddern More articles by this author Grigori Rychkov More articles by this author Gerhard Hannig More articles by this author Caroline Kurtz More articles by this author Ada Silos-Santiago More articles by this author Stuart M. Brierley More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MP77-09 CHRONIC PSYCHOLOGICAL STRESS LEADS TO BLADDER HYRALGESIA VIA STRESS INDUCED SENSITIZATION OF C FIBERS AND MECHANORECPTORS

You have accessJournal of UrologyUrodynamics/Lower Urinary Tract Dysfunction/Female Pelvic Medicine: Non-neurogenic Voiding Dysfunction II1 Apr 2016MP77-09 CHRONIC PSYCHOLOGICAL STRESS LEADS TO BLADDER HYRALGESIA VIA STRESS INDUCED SENSITIZATION OF C FIBERS AND MECHANORECPTORS Yunliang Gao, Rong Zhang, Huiyi Chang, and Larissa Rodriguez Yunliang GaoYunliang Gao More articles by this author , Rong ZhangRong Zhang More articles by this author , Huiyi ChangHuiyi Chang More articles by this author , and Larissa RodriguezLarissa Rodriguez More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1923AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Patients affected by overactive bladder and painful bladder syndrome/interstitial cystitis report symptoms exacerbation when stressed. We have shown that anxiety prone animals exposed to chronic psychological water avoidance stress (WAS) develop sustained bladder hyperalgesia. The bladder cooling reflex (BCR) is a segmental reflex believed to be triggered by cold receptors in bladder wall with C fibers signaling. We aimed to evaluate the chronic stress induces changes in BCR and if C fiber sensitization contributes to stress-induced bladder hypersensitivity. METHODS 22 adult female Wistar-Kyoto rats equally divided to 10-day WAS or handled controls. On day 11, animals were evaluated by cystometrogram (CMG) as well as visceromotor reflex (VMR) for bladder sensitivity and pain to saline infusion. CMG and VMR were obtained during bladder infusion at room temperature (RT) and cold (4 degree C) with an open urethral outlet thus allowing the animal to void. CMG and VMR were also obtained during RT isotonic bladder distention (RT-IBD, 10-40 cmH2O) with urethral occlusion. RESULTS During RT infusion, WAS rats had a significant decreases in pressure threshold (PT) and the ratio of VMR threshold/maximum intravesical pressure (IVPmax). VMR duration significantly increased in WAS. BCR induced a more dramatic significant decline in PT and the ratio of VMR threshold/IVPmax in WAS (Fig.1A). No significant differences were found in contraction duration, inter-contraction interval or the VMR area under the curve (AUC) and duration. At RT-IBD (Fig.1B-C), VMR latency in WAS significantly decreased compared to controls. At 20 cmH2O, VMR AUC in WAS significantly increased compared to controls. CONCLUSIONS Chronic psychological stress induces bladder hypersensitivity, which manifests as earlier voiding and VMR. Prolonged VMR duration in WAS correlates with increased bladder sensitivity during voiding. BCR evokes C fibers activation and suggests that stress induced bladder hypersensitivity in functional voiding disorders mediated in part by afferent sensitization. Earlier VMR appearance at RT-IBD suggests a role for mechanoreceptor sensitivity in stress-induced bladder hypersensitivity. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e1018-e1019 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Yunliang Gao More articles by this author Rong Zhang More articles by this author Huiyi Chang More articles by this author Larissa Rodriguez More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Chronic Prostatitis Induces Bladder Hypersensitivity and Sensitizes Bladder Afferents in the Mouse

Chronic prostatitis/chronic pelvic pain syndrome causes symptoms thatinclude the frequent and urgent need to urinate, pain or burning during urination and pain radiating to the back, abdomen and/or colorectum. These bladder symptoms suggest that chronic prostatitis/chronic pelvic pain syndrome is associated with sensitization of adjacent organs, termed cross-organ sensitization. The objective of this study was to determine the extent of 1) changes in immunomodulatory mediators in the prostate and bladder after inflammation ofthe prostate and 2) bladder function and bladder afferent sensitization. Prostate and bladder histology, immunohistochemistry and expression of immunomodulatory targets were examined weekly after zymosan or vehicle was injected in the dorsal lobe of the mouse prostate. Cystometry, bladder and bladder afferent sensitivity were also assessed weekly. Prostate inflammation induced significant up-regulation in proinflammatory and anti-inflammatory cytokines TNF-α (tumor necrosis factor-α) and IL-10 (interleukin-10), growth factor NGF (nerve growth factor), and T-lymphocyte markers FoxP3, CD4 and CD8 in the prostate and the bladder. Notably, prostatitis significantly increased urinary voiding frequency, induced hypersensitivity to bladder distension and sensitized bladder afferents. We also examined sensory (afferent) co-innervation by injecting retrograde tracers DiI and Fast Blue in the bladder wall and the prostate, respectively. This showed that a significant proportion (approximately 17%) of dorsal root ganglion afferent somata contained tracers from the bladder and the prostate. These observations support an afferent contribution to chronic prostatitis/chronic pelvic pain syndrome and cross-organ sensitization from prostate to bladder.

Optogenetic activation of mechanically insensitive afferents in mouse colorectum reveals chemosensitivity.

The sensory innervation of the distal colorectum includes mechanically insensitive afferents (MIAs; ∼25%), which acquire mechanosensitivity in persistent visceral hypersensitivity and thus generate de novo input to the central nervous system. We utilized an optogenetic approach to bypass the process of transduction (generator potential) and focus on transformation (spike initiation) at colorectal MIA sensory terminals, which is otherwise not possible in typical functional studies. From channelrhodopsin2-expressing mice (driven by Advillin-Cre), the distal colorectum with attached pelvic nerve was harvested for ex vivo single-fiber recordings. Afferent receptive fields (RFs) were identified by electrical stimulation and tested for response to mechanical stimuli (probing, stroking, and stretch), and afferents were classified as either MIAs or mechanosensitive afferents (MSAs). All MIA and MSA RFs were subsequently stimulated optically and MIAs were also tested for activation/sensitization with inflammatory soup (IS), acidic hypertonic solution (AHS), and/or bile salts (BS). Responses to pulsed optical stimuli (1-10 Hz) were comparable between MSAs and MIAs whereas 43% of MIAs compared with 86% of MSAs responded tonically to stepped optical stimuli. Tonic-spiking MIAs responded preferentially to AHS (an osmotic stimulus) whereas non-tonic-spiking MIAs responded to IS (an inflammatory stimulus). A significant proportion of MIAs were also sensitized by BS. These results reveal transformation as a critical factor underlying the differences between MIAs (osmosensors vs. inflammatory sensors), revealing a previously unappreciated heterogeneity of MIA endings. The current study draws attention to the sensory encoding of MIA nerve endings that likely contribute to afferent sensitization and thus have important roles in visceral pain.

Sensory function assessment of the human male lower urinary tract using current perception thresholds

To evaluate the feasibility and reliability of current perception threshold (CPT) measurement for sensory assessment of distinct locations in the male lower urinary tract (LUT). Twelve male subjects (>18 years) without LUT symptoms or medical comorbidities were eligible. CPTs were determined twice (interval: 7-20 days) at the bladder dome, trigone and the proximal, membranous, and distal urethra. Square wave electrical stimulation of 3 Hz/0.2 ms and 0.5 Hz/1 ms was applied using a transurethral 8F catheter placed under fluoroscopic control. Bladder volume was kept constant (60 mL) using a second 10F catheter. Repetitive measurements and reliability were assessed by analysis of variance (ANOVA) and intraclass correlation coefficient (ICC). The ANOVA revealed significant main effects for stimulation site (P = 0.008) and type of stimulation (P < 0.001) with lower CPTs for 0.5 Hz/1 ms compared to 3 Hz/0.2 ms. There was no significant effect for visit number (P = 0.061). CPTs were higher for bladder dome than for proximal (0.5 Hz/1 ms: P = 0.022; 3 Hz/0.2 ms: P = 0.022) and distal urethra (0.5 Hz/1 ms: P = 0.026; 3 Hz/0.2 ms: P = 0.030). Reliability of CPT measurements was excellent to good (ICC = 0.67-0.96) except for the bladder dome (5 Hz/1 ms: ICC = 0.45; 3 Hz/0.2 ms: ICC = 0.20) and distal urethra (3 Hz/0.2 ms: ICC = 0.57). CPTs can be reliably detected at different LUT locations. However, alert and compliant subjects are essential. CPTs of LUT may become a complementary assessment method providing information on responsiveness and sensitivity of afferent LUT nerves. This is especially relevant for urethral afferents, which are not covered by standard urodynamic investigations. Neurourol. Urodynam. 36:469-473, 2017. © 2016 Wiley Periodicals, Inc.

Band limited chirp stimulation in vestibular evoked myogenic potentials.

Air conducted vestibular evoked myogenic potentials (VEMP) can be elicited by various low frequency and intense sound stimuli, mainly clicks or short tone bursts (STB). Chirp stimuli are increasingly used in diagnostic audiological evaluations as an effective means to obtain acoustically evoked responses in narrowed or extended frequency ranges. We hypothesized in this study that band limited chirp stimulation, which covers the main sensitivity range of sound sensitive otolithic afferents (around 500Hz), might be useful for application in cervical and ocular VEMP to air conduction. For this purpose we designed a chirp stimulus ranging 250-1000Hz (up chirp). The chirp stimulus was delivered with a stimulus intensity of 100dB nHL in normal subjects (n=10) and patients with otolith involvement (vestibular neuritis) (n=6). Amplitudes of the designed chirp ("CW-VEMP-chirp, 250-1000Hz") were compared with amplitudes of VEMPs evoked by click stimuli (0.1ms) and a short tone burst (STB, 1-2-1, 8ms, 500Hz). CVEMPs and oVEMPs were detectable in 9 of 10 normal individuals. Statistical evaluation in healthy patients revealed significantly larger cVEMP and oVEMP amplitudes for CW-VEMP-chirp (250-1000Hz) stimuli. CVEMP amplitudes evoked by CW-VEMP-chirp (250-1000Hz) showed a high stability in comparison with click and STB stimulation. CW-VEMP-chirp (250-1000Hz) showed abnormal cVEMP and oVEMP amplitudes in patients with vestibular neuritis, with the same properties as click and STB stimulated VEMPs. We conclude that the designed CW-VEMP-chirp (250-1000Hz) is an effective stimulus which can be further used in VEMP diagnostic. Since a chirp stimulus can be easily varied in its properties, in particular with regard to frequency, this might be a promising tool for further investigations.

Dual Modulation of Nociception and Cardiovascular Reflexes during Peripheral Ischemia through P2Y1 Receptor-Dependent Sensitization of Muscle Afferents.

Our current results suggest that P2Y1 modulates heat responsiveness and chemosensation in muscle afferents to play a key role in the development of pain-related behaviors during ischemia. At the same time, under these pathological conditions, the changes in muscle sensory neurons appear to modulate an increase in mean systemic blood pressure after exercise. This is the first report of the potential peripheral mechanisms by which group III/IV muscle afferents can dually regulate muscle nociception and the exercise pressor reflex. These data provide evidence related to the potential underlying reasons for the comorbidity of muscle pain and altered sympathetic reflexes in disease states that are based in problems with peripheral perfusion and may indicate a potential target for therapeutic intervention.

Differences in electrophysiological properties of functionally identified nociceptive sensory neurons in an animal model of cancer-induced bone pain.

BackgroundBone cancer pain is often severe, yet little is known about mechanisms generating this type of chronic pain. While previous studies have identified functional alterations in peripheral sensory neurons that correlate with bone tumours, none has provided direct evidence correlating behavioural nociceptive responses with properties of sensory neurons in an intact bone cancer model.ResultsIn a rat model of prostate cancer-induced bone pain, we confirmed tactile hypersensitivity using the von Frey test. Subsequently, we recorded intracellularly from dorsal root ganglion neurons in vivo in anesthetized animals. Neurons remained connected to their peripheral receptive terminals and were classified on the basis of action potential properties, responses to dorsal root stimulation, and to mechanical stimulation of the respective peripheral receptive fields. Neurons included C-, Aδ-, and Aβ-fibre nociceptors, identified by their expression of substance P. We suggest that bone tumour may induce phenotypic changes in peripheral nociceptors and that these could contribute to bone cancer pain.ConclusionsThis work represents a significant technical and conceptual advance in the study of peripheral nociceptor functions in the development of cancer-induced bone pain. This is the first study to report that changes in sensitivity and excitability of dorsal root ganglion primary afferents directly correspond to mechanical allodynia and hyperalgesia behaviours following prostate cancer cell injection into the femur of rats. Furthermore, our unique combination of techniques has allowed us to follow, in a single neuron, mechanical pain-related behaviours, electrophysiological changes in action potential properties, and dorsal root substance P expression. These data provide a more complete understanding of this unique pain state at the cellular level that may allow for future development of mechanism-based treatments for cancer-induced bone pain.

Transcriptome analysis of trigeminal ganglia following masseter muscle inflammation in rats.

BackgroundChronic pain in masticatory muscles is a major medical problem. Although mechanisms underlying persistent pain in masticatory muscles are not fully understood, sensitization of nociceptive primary afferents following muscle inflammation or injury contributes to muscle hyperalgesia. It is well known that craniofacial muscle injury or inflammation induces regulation of multiple genes in trigeminal ganglia, which is associated with muscle hyperalgesia. However, overall transcriptional profiles within trigeminal ganglia following masseter inflammation have not yet been determined. In the present study, we performed RNA sequencing assay in rat trigeminal ganglia to identify transcriptome profiles of genes relevant to hyperalgesia following inflammation of the rat masseter muscle.ResultsMasseter inflammation differentially regulated >3500 genes in trigeminal ganglia. Predominant biological pathways were predicted to be related with activation of resident non-neuronal cells within trigeminal ganglia or recruitment of immune cells. To focus our analysis on the genes more relevant to nociceptors, we selected genes implicated in pain mechanisms, genes enriched in small- to medium-sized sensory neurons, and genes enriched in TRPV1-lineage nociceptors. Among the 2320 candidate genes, 622 genes showed differential expression following masseter inflammation. When the analysis was limited to these candidate genes, pathways related with G protein-coupled signaling and synaptic plasticity were predicted to be enriched. Inspection of individual gene expression changes confirmed the transcriptional changes of multiple nociceptor genes associated with masseter hyperalgesia (e.g., Trpv1, Trpa1, P2rx3, Tac1, and Bdnf) and also suggested a number of novel probable contributors (e.g., Piezo2, Tmem100, and Hdac9).ConclusionThese findings should further advance our understanding of peripheral mechanisms involved in persistent craniofacial muscle pain conditions and provide a rational basis for identifying novel genes or sets of genes that can be potentially targeted for treating such conditions.

Effects of Src-kinase inhibition in cancer-induced bone pain.

BackgroundBone metastases occur frequently in advanced breast, lung, and prostate cancer, with approximately 70% of patients affected. Pain is a major symptom of bone metastases, and current treatments may be inadequate or have unacceptable side effects. The mechanisms that drive cancer-induced bone pain are not fully understood; however, it is known that there is sensitization of both peripheral bone afferents and central spinal circuits. It is well established that the N-methyl-D-aspartate receptor plays a major role in the pathophysiology of pain hypersensitivity. Inhibition of the non-receptor tyrosine kinase Src controls N-methyl-D-aspartate receptor activity and inhibiting Src reduces the hypersensitivity associated with neuropathic and inflammatory pains. As Src is also implicated in osteoclastic bone resorption, we have investigated if inhibiting Src ameliorates cancer-induced bone pain. We have tested this hypothesis using an orally bioavailable Src inhibitor (saracatinib) in a rat model of cancer-induced bone pain.ResultsIntra-tibial injection of rat mammary cancer cells (Mammary rat metastasis tumor cells -1), but not vehicle, in rats produced hindpaw hypersensitivity to thermal and mechanical stimuli that was maximal after six days and persisted for at least 13 days postinjection. Daily oral gavage with saracatinib (20 mg/kg) beginning seven days after intra-tibial injection reversed the thermal hyperalgesia but not the mechanical allodynia. The analgesic mechanisms of saracatinib appear to be due to an effect on the nervous system as immunoblotting of L2-5 spinal segments showed that mammary rat metastasis tumor cells-1 injection induced phosphorylation of the GluN1 subunit of the N-methyl-D-aspartate receptor, indicative of receptor activation, and this was reduced by saracatinib. Additionally, histology showed no anti-tumor effect of saracatinib at any dose and no significant effect on bone preservation.ConclusionsThis is the first demonstration that Src plays a role in the development of cancer-induced bone pain and that Src inhibition represents a possible new analgesic strategy for patients with bone metastases.

The Scalp Has a Lower Stratum Corneum Function with a Lower Sensory Input than Other Areas of the Skin Evaluated by the Electrical Current Perception Threshold

Many people feel frequent prickling or itching sensations on their scalp. The scalp is an atypical area of the skin since it is normally covered with thick hair and has many sebaceous glands and sweat glands. The scalp often has skin problems that can affect its sensitivity and functions. However, not much is known about stratum corneum function and the neural sensitivity of the scalp. Here we evaluated stratum corneum function and the neural sensitivity of the scalp of 47 normal male individuals in various skin conditions and compared the results to that to the forehead. The neural sensitivity was evaluated by measuring the electrical current perception threshold (CPT). The cutaneous barrier function and stratum corneum moisture-retention ability (MRA) of the scalp were significantly lower than on the forehead, even if there were some scalp problems. Depending on the increase in severity of scalp skin problems, both these skin functional properties and the CPT decreased significantly. However, regardless of its lower functional properties, scalp skin was not significantly lower than that of the forehead. Although the scalp has a low stratum corneum function compared with the forehead and has easily induced skin problems, the scalp skin has less sensitive sensory nerves, resulting in experiencing a worsening of scalp symptoms more easily.

A-kinase anchoring protein 79/150 coordinates metabotropic glutamate receptor sensitization of peripheral sensory neurons.

Glutamate serves as the primary excitatory neurotransmitter in the nervous system. Previous studies have identified a role for glutamate and group I metabotropic receptors as targets for study in peripheral inflammatory pain. However, the coordination of signaling events that transpire from receptor activation to afferent neuronal sensitization has not been explored. Herein, we identify that scaffolding protein A-kinase anchoring protein 79/150 (AKAP150) coordinates increased peripheral thermal sensitivity after group I metabotropic receptor (mGluR5) activation. In both acute and persistent models of thermal somatosensory behavior, we report that mGluR5 sensitization requires AKAP150 expression. Furthermore, electrophysiological approaches designed to record afferent neuronal activity reveal that mGluR5 sensitization also requires functional AKAP150 expression. In dissociated primary afferent neurons, mGluR5 activation increases TRPV1 responses in an AKAP-dependent manner through a mechanism that induces AKAP association with TRPV1. Experimental results presented herein identify a mechanism of receptor-driven scaffolding association with ion channel targets. Importantly, this mechanism could prove significant in the search for therapeutic targets that repress episodes of acute pain from becoming chronic in nature.

The voltage-gated sodium channel NaV 1.9 in visceral pain.

Visceral pain is a common symptom for patients with gastrointestinal (GI) disease. It is unpleasant, debilitating, and represents a large unmet medical need for effective clinical treatments. Recent studies have identified NaV 1.9 as an important regulator of afferent sensitivity in visceral pain pathways to mechanical and inflammatory stimuli, suggesting that NaV 1.9 could represent an important therapeutic target for the treatment of visceral pain. This potential has been highlighted by the identification of patients who have an insensitivity to pain or painful neuropathies associated with mutations in SCN11A, the gene encoding voltage-gated sodium channel subtype 1.9 (NaV 1.9). Here, we address the role of NaV 1.9 in visceral pain and what known human NaV 1.9 mutants can tell us about NaV 1.9 function in gut physiology and pathophysiology.

Activation of colo-rectal high-threshold afferent nerves by Interleukin-2 is tetrodotoxin-sensitive and upregulated in a mouse model of chronic visceral hypersensitivity.

Chronic visceral pain is a defining feature of irritable bowel syndrome (IBS). IBS patients often show alterations in innate and adaptive immune function which may contribute to symptoms. Immune mediators are known to modulate the activity of viscero-sensory afferent nerves, but the focus has been on the innate immune system. Interleukin-2 (IL-2) is primarily associated with adaptive immune responses but its effects on colo-rectal afferent function in health or disease are unknown. Myeloperoxidase (MPO) activity determined the extent of inflammation in health, acute trinitrobenzene-sulfonic acid (TNBS) colitis, and in our post-TNBS colitis model of chronic visceral hypersensitivity (CVH). The functional effects of IL-2 on high-threshold colo-rectal afferents and the expression of IL-2R and NaV 1.7 mRNA in colo-rectal dorsal root ganglia (DRG) neurons were compared between healthy and CVH mice. MPO activity was increased during acute colitis, but subsided to levels comparable to health in CVH mice. IL-2 caused direct excitation of colo-rectal afferents that was blocked by tetrodotoxin. IL-2 did not affect afferent mechanosensitivity in health or CVH. However, an increased proportion of afferents responded directly to IL-2 in CVH mice compared with controls (73% vs 33%; p<0.05), and the abundance of IL-2R and NaV 1.7 mRNA was increased 3.5- and 2-fold (p<0.001 for both) in colo-rectal DRG neurons. IL-2, an immune mediator from the adaptive arm of the immune response, affects colo-rectal afferent function, indicating these effects are not restricted to innate immune mediators. Colo-rectal afferent sensitivity to IL-2 is increased long after healing from inflammation.

TRPA1 mediates amplified sympathetic responsiveness to activation of metabolically sensitive muscle afferents in rats with femoral artery occlusion

Autonomic responses to activation of mechanically and metabolically sensitive muscle afferent nerves during static contraction are augmented in rats with femoral artery occlusion. Moreover, metabolically sensitive transient receptor potential cation channel subfamily A, member 1 (TRPA1) has been reported to contribute to sympathetic nerve activity (SNA) and arterial blood pressure (BP) responses evoked by static muscle contraction. Thus, in the present study, we examined the mechanisms by which afferent nerves' TRPA1 plays a role in regulating amplified sympathetic responsiveness due to a restriction of blood flow directed to the hindlimb muscles. Our data show that 24–72 h of femoral artery occlusion (1) upregulates the protein levels of TRPA1 in dorsal root ganglion (DRG) tissues; (2) selectively increases expression of TRPA1 in DRG neurons supplying metabolically sensitive afferent nerves of C-fiber (group IV); and (3) enhances renal SNA and BP responses to AITC (a TRPA1 agonist) injected into the hindlimb muscles. In addition, our data demonstrate that blocking TRPA1 attenuates SNA and BP responses during muscle contraction to a greater degree in ligated rats than those responses in control rats. In contrast, blocking TRPA1 fails to attenuate SNA and BP responses during passive tendon stretch in both groups. Overall, results of this study indicate that alternations in muscle afferent nerves' TRPA1 likely contribute to enhanced sympathetically mediated autonomic responses via the metabolic component of the muscle reflex under circumstances of chronic muscle ischemia.

Induction of thermal and mechanical hypersensitivity by parathyroid hormone-related peptide through upregulation of TRPV1 function and trafficking.

The neurobiological mechanisms underlying chronic pain associated with cancers are not well understood. It has been hypothesized that factors specifically elevated in the tumor microenvironment sensitize adjacent nociceptive afferents. We show that parathyroid hormone-related peptide (PTHrP), which is found at elevated levels in the tumor microenvironment of advanced breast and prostate cancers, is a critical modulator of sensory neurons. Intraplantar injection of PTHrP led to the development of thermal and mechanical hypersensitivity in both male and female mice, which were absent in mice lacking functional transient receptor potential vanilloid-1 (TRPV1). The PTHrP treatment of cultured mouse sensory neurons enhanced action potential firing, and increased TRPV1 activation, which was dependent on protein kinase C (PKC) activity. Parathyroid hormone-related peptide induced robust potentiation of TRPV1 activation and enhancement of neuronal firing at mild acidic pH that is relevant to acidic tumor microenvironment. We also observed an increase in plasma membrane TRPV1 protein levels after exposure to PTHrP, leading to upregulation in the proportion of TRPV1-responsive neurons, which was dependent on the activity of PKC and Src kinases. Furthermore, co-injection of PKC or Src inhibitors attenuated PTHrP-induced thermal but not mechanical hypersensitivity. Altogether, our results suggest that PTHrP and mild acidic conditions could induce constitutive pathological activation of sensory neurons through upregulation of TRPV1 function and trafficking, which could serve as a mechanism for peripheral sensitization of nociceptive afferents in the tumor microenvironment.

Abstract 139: Renal Afferent Nerve Sensitivity and the Pathophysiology of Salt-sensitive Hypertension

Aim: Altered renal afferent nerve responsiveness contributes to hypertension in the Spontaneously Hypertensive rat. We hypothesized that increased salt-intake differentially impacts the renal afferent nerve sensitivity in salt-resistant vs salt-sensitive rats. Methods: Groups of naïve Sprague-Dawley (SD), Dahl Salt-Resistant (DSR), Dahl Salt-Sensitive (DSS) or SD rats receiving a s.c. norepinephrine (NE:600ng/min) infusion were fed a 0.4% (NS) or 8% NaCl (HS) diet for 14 (SD) or 21 days (DSR &amp; DSS). Following HS intake MAP and plasma NE content were determined. Via a renal pelvis preparation afferent nerve activity was assessed as NE-evoked (6250 pmol) substance P (SP) release (N=5/gp). Results: Salt-resistant phenotypes (SD &amp; DSR) remain normotensive and exhibit HS-evoked suppression of plasma NE and increased renal afferent nerve release of substance P. In contrast in salt-sensitive phenotypes (DSS and NE infused SD) a high salt diet evoked hypertension, increased plasma NE and abolished sodium evoked increased responsiveness of the renal afferent release of substance P. Significance symbols: *p&lt;0.05 vs. resp. NS gp; τ p&lt;0.05 vs. resp. SD Naïve gp. value. Conclusion: These data support a role of the afferent renal nerves in blood pressure regulation during high salt-intake. Increased responsiveness of the renal afferent nerves contributes to the maintenance of normotension in a salt-resistant rat phenotype. Conversely, our data suggest that in salt-sensitive phenotypes excess release of NE triggers impaired activation of the renal afferent nerves, a factor we postulate contributes to the development of salt-sensitive hypertension.

Responses of the human maternal HPA axis to obesity and stress: Implications for offspring behaviour

development, events that increase intestinal permeability, allowing, consequently, the access of luminal antigens to the mucosa. Food components, microorganisms, and toxins can stimulate local responses by activating immune cells and/or directly sensitizing nerve endings. Primary afferent sensitization could affect responsiveness to visceral stimuli and modify neuropeptide release form nerves, consequently leading to neurogenic inflammation. In fact, mast cell proximity to nerves and its correlation with abdominal pain have been identified in IBS patients. Moreover, the presence of higher amount of corticotropin-releasing hormone in mucosal eosinophils in IBS, as compared to healthy controls, in correlation with psychological stress has been identified, enabling local pain signaling and barrier alterations upon eosinophil degranulation. B lymphocytes and plasma cells are also part of the inflammatory infiltrate and display an activated profile in association with major clinical symptoms in IBS. In this presentation Iwill review themain mechanisms associated with pain signaling in IBS with a special focus on stress-induced activation of the mucosal immune system.

Mechanisms of ventilatory control: who is really in charge?

Mechanisms of ventilatory control: who is really in charge?