Pelvic Nerve Afferents Research Articles

Mo1844 Selective Agonists of Somatostatin Receptors Type 1 or 2 Prevent Visceral Hyperalgesia Induced by Activation of Peripheral CRF1 Receptors in Mice

G A A b st ra ct s brought a new understanding on the mechanisms of chronic visceral pain. The present study hypothesizes that inflammation of visceral organs cause glial activation, leading to the release of pro-inflammatory cytokines, resulting in hyperalgesia. Therefore, the inhibition of microglial activation can attenuate visceral hyperalgesia. We also hypothesize that glialspecific purinergic P2X7 receptors play a role in glial-neuron cross talk and blocking of these receptors might produce an analgesic effect. Methods: Colonic inflammation was induced inmale Sprague-Dawley rats (n=12) by intracolonic administration of either zymosan (25mg/ml, 0.5ml) or tri-nitrobenzenesulphonic acid (TNBS). Visceral hyperalgesia was determined by measuring the viscero-motor response (VMR) to graded colorectal distension (CRD) prior, 3hr (for zymosan) and 7 days (for TNBS) after induction of colonic inflammation. The effects of minocycline, (a broad-spectrum tetracycline antibiotic and a microglial inhibitor) and brilliant blue G (BBG; a selective P2X7 antagonist) were tested on VMRs to CRD in preand post-inflamed conditions. Electrophysiological studies involving extracellular single-unit recordings were made from CRD-sensitive pelvic nerve afferent (PNA) fibers in naive non-inflamed and TNBS-treated animals and the effect of minocycline (50mg/kg, i.v.) on the responses of PNAs to graded CRD was studied 30 and 60mins following drug administration. Results: Colonic inflammation produced visceral hyperalgesia to CRD. Administration of minocycline (50mg/kg, i.p.) significantly (p<0.05) attenuated the visceral hyperalgesia in both zymosanand TNBS-treated rats, but not in naive non-inflamed rats. Similarly, selective blocking of P2X7 receptors by BBG (50mg/kg i.p.) attenuated VMRs to CRD in TNBS-treated rats, but not in naive rats. In electrophysiology experiments, minocycline significantly (p<0.05) attenuated mechanotransduction of CRD-sensitive PNA (n=6) to CRD, but had no effect on responses of CRD-sensitive PNA (n=4) from naive non-inflamed rats. Conclusions: Activated glial cells plays an important role in initiating and maintaining visceral hyperalgesia in a rat model of post-inflammatory pain. Blocking the function of glial cells by minocycline or by blocking the glial-specific P2X7 receptor can attenuate visceral hyperalgesia. This effect is partly via peripheral actions of these drugs, which might prove to be a vital target for the treatment of visceral hyperalgesia.

Alterations in peripheral purinergic and muscarinic signaling of rat bladder after long-term fructose-induced metabolic syndrome.

We explored the pathophysiologic mechanisms of long-term fructose-induced lower urinary tract symptoms (LUTS) in rats. Male Wistar rats were fed with fructose for 3 or 6 months. Biochemical and transcystometric parameters were compared between fructose-fed and age-matched normal-diet rats. Pelvic nerve and external urethral sphincter-electromyogram activity recordings were performed to investigate fructose effects on neural control of bladders. Mitochondrial structure, ATP and acetylcholine content and purinergic and muscarinic cholinergic receptors were examined. Cytosolic cytochrome C staining by Western blot and immunocytochemistry for mitochondrial injury and PGP 9.5 stain for nerve density were also determined. The fructose-fed rats with higher plasma triglyceride, LDL and fasting glucose levels displayed LUTS with increased frequency and suppressed voiding contractile amplitude in phase 1 and phase 2 duration versus normal-diet control. Fructose feeding altered the firing types in pelvic afferent and efferent nerves and external urethral sphincter-electromyogram activity. Increased mast cell number, disrupted and swollen mitochondria, increased cytosolic cytochrome C stain and expression and decreased nerve density in bladder smooth muscle layers appeared in the fructose-fed rats. Fructose feeding also significantly reduced ATP and acetylcholine content and enhanced protein expression of postsynaptic P(2)X(1), P(2)X(2) and P(2)X(3) purinergic receptors and M(2) and M(3) muscarinic cholinergic receptors expression in the smooth muscles of urinary bladder. Long-term fructose feeding induced neuropathy and myopathy in the urinary bladders. Impaired mitochondrial integrity, reduced nerve density, ATP and acetylcholine content and upregulation of purinergic and muscarinic cholinergic receptors expression may contribute to the bladder dysfunction of fructose-fed animals.

Nonvoiding Activity of the Guinea Pig Bladder

Detrusor nonvoiding contractions occur in up to 70% of healthy individuals. These contractions increase in those with pathological detrusor function and may be associated with afferent activity. We examined nonvoiding contractions in the urethane anesthetized guinea pig bladder and studied the effect of filling rate and intravesical volume. A total of 14 guinea pigs were anesthetized and underwent bladder catheterization at the dome. In 6 guinea pigs bladder infusion was continuous and 2 physiological filling rates were used, including 25 (0.75 HD) and 50 μl per minute (1.5 HD). In another 8 guinea pigs isovolumetric cystometry was done by filling the bladder incrementally and recording at low, medium and high intravesical volume. Nonvoiding contractions were apparent in all animals. Contractions increased in frequency and amplitude as the bladder filled. Different phases were identified. Immediately after a void no nonvoiding contractions were observed, followed by continuous activity, first with small contractions, and later with small and large contractions. Small nonvoiding contractions showed a phasic pattern in frequency while the frequency of large nonvoiding contractions slowly increased or remained stable. The frequency and amplitude of nonvoiding contractions were higher at a faster filling rate and a higher intravesical volume. Nonvoiding contractions are present in the anesthetized guinea pig. Under normal physiological conditions they increase in amplitude and frequency with the increase in the filling rate and in intravesical volume. Small and large nonvoiding contractions differ in frequency pattern and occur at different bladder filling periods. This may illustrate different afferents functioning during bladder filling, which could be important for understanding bladder pathology.

Neonatal cystitis-induced colonic hypersensitivity in adult rats: a model of viscero-visceral convergence

The objective of this study was to determine if neonatal cystitis alters colonic sensitivity later in life and to investigate the role of peripheral mechanisms. Neonatal rats received intravesical zymosan, normal saline, or anesthesia only for three consecutive days [(postnatal (PN) days 14-16)]. The estrous cycle phase was determined prior to recording the visceromotor response (VMR) to colorectal distension (CRD) in adult rats. Eosinophils and mast cells were examined from colon and bladder tissues. CRD- or urinary bladder distension (UBD)-sensitive pelvic nerve afferents (PNAs) were identified and their responses to distension were examined. The relative expression of N-methyl-d-aspartic acid (NMDA)-NR1 subunit in the lumbo-sacral (L6-S1) spinal cord was examined using Western blot. The VMR to CRD (≥10mmHg) in the neonatal zymosan group was significantly higher than control in both the diestrus, estrus phase and in all phases combined. There was no difference in the total number of eosinophils, mast cells or number of degranulated mast cells between groups. The spontaneous firing of UBD, but not CRD-sensitive PNAs from the zymosan-treated rats was significantly higher than the saline-treated control. However, the mechanosensitive properties of PNAs to CRD or UBD were no different between groups (P>0.05). The expression of spinal NR1 subunit was significantly higher in zymosan-treated rats compared with saline-treated rats (P<0.05). Neonatal cystitis results in colonic hypersensitivity in adult rats without changing tissue histology or the mechanosensitive properties of CRD-sensitive PNAs. Neonatal cystitis does result in overexpression of spinal NR1 subunit in adult rats.

Cyclophosphamide induces NR2B phosphorylation-dependent facilitation on spinal reflex potentiation

It is well-established that cyclophosphamide (CYP) can sensitize the pelvic afferent nerve arising from the urinary bladder and therefore induce suprapubic pain. To test the possibility that CYP might mediate the development of visceral hypereflexia/hyperalgesia by facilitating spinal activity-dependent neural plasticity, we compared the pelvic-urethra reflex activity and spinal N-methyl-d-aspartate receptor NR2B subunit (NR2B) phosphorylation in rats treated with vehicle solution and CYP. Compared with vehicle solution, when accompanied by upregulation of phosphorylated NR2B expression in the lumbosacral (L(6)-S(2)) dorsal horn, CYP increased the evoked spikes in spinal reflex potentiation induced by repetitive stimulation (1 stimulation/1 s). Moreover, intraperitoneal pretreatments with N(G)-nitro-l-arginine methyl ester and roscovitine, nitric oxide synthase and cyclin-dependent protein kinase 5 (Cdk5) antagonists, respectively, overwrote CYP-enhanced reflex potentiation and NR2B phosphorylation. When compared with the untreated group, the treatment with small-interfering RNA of NR2B, which decreased the expression of NR2B expression, abolished CYP-dependent reflex facilitation and spinal NR2B phosphorylation. These results suggested that CYP might facilitate spinal reflex potentiation mediated by N-methyl-d-aspartate receptors and participate in the development of visceral hypereflexia/hyperalgesia through nitric oxide- and Cdk5-dependent NR2B phosphorylation at the lumbosacral dorsal horn.

An exploration of the control of micturition using a novel in situ arterially perfused rat preparation.

Our goal was to develop and refine a decerebrate arterially perfused rat (DAPR) preparation that allows the complete bladder filling and voiding cycle to be investigated without some of the restrictions inherent with in vivo experimentation [e.g., ease and speed of set up (30 min), control over the extracellular milieu and free of anesthetic agents]. Both spontaneous (naturalistic bladder filling from ureters) and evoked (in response to intravesical infusion) voids were routinely and reproducibly observed which had similar pressure characteristics. The DAPR allows the simultaneous measurement of bladder intra-luminal pressure, external urinary sphincter–electromyogram (EUS–EMG), pelvic afferent nerve activity, pudendal motor activity, and permits excellent visualization of the entire lower urinary tract, during typical rat filling and voiding responses. The voiding responses were modulated or eliminated by interventions at a number of levels including at the afferent terminal fields (intravesical capsaicin sensitization–desensitization), autonomic (ganglion blockade with hexamethonium), and somatic motor (vecuronium block of the EUS) outflow and required intact brainstem/hindbrain-spinal coordination (as demonstrated by sequential hindbrain transections). Both innocuous (e.g., perineal stimulation) and nociceptive (tail/paw pinch) somatic stimuli elicited an increase in EUS–EMG indicating intact sensory feedback loops. Spontaneous non-micturition contractions were observed between fluid infusions at a frequency and amplitude of 1.4 ± 0.9 per minute and 1.4 ± 0.3 mmHg, respectively and their amplitude increased when autonomic control was compromised. In conclusion, the DAPR is a tractable and useful model for the study of neural bladder control showing intact afferent signaling, spinal and hindbrain co-ordination and efferent control over the lower urinary tract end organs and can be extended to study bladder pathologies and trial novel treatments.

Effects of stimulation of muscarinic receptors on bladder afferent nerves in the in vitro bladder–pelvic afferent nerve preparation of the rat

Effects of a muscarinic receptor agonist oxotremorine-M (oxo-M) on bladder afferent nerve (BAN) activity were studied in an in vitro bladder–pelvic nerve preparation. Distension of the bladder induced rhythmic bladder contractions that were accompanied by multiunit afferent firing. Intravesical administration of 25 and 50 μM oxo-M significantly increased afferent firing from 41 ± 2 spikes/s to 51 ± 4 spikes/s and 60.5 ± 5 spikes/s, respectively, but did not change the maximum amplitude of spontaneous bladder contractions. The afferent nerve firing induced by isotonic distension of the bladder (10–40 cmH 2O) was increased 22–100% by intravesical administration of 50 μM oxo-M. Electrical stimulation on the surface of the bladder elicited action potentials (AP) in BAN. Oxo-M significantly decreased the voltage threshold by 40% (p < 0.05) and increased by 157% (p < 0.05) the area of the AP evoked at a submaximal stimulus intensity. These effects were blocked by intravesical injection of 5 μM atropine methyl nitrate (AMN). Intravesical administration of 5 μM AMN alone did not alter BAN firing or the amplitude of bladder contractions. The facilitatory effects induced by oxo-M on BAN activity were also suppressed (p < 0.05) by intravesical administration of 2′,3′-0-trinitrophenyl-ATP (TNP-ATP) (30 μM). In preparations pretreated with capsaicin (125 mg/kg, s.c.) the facilitatory effects of 50 μM oxo-M on BAN activity were absent. These results suggest that activation of muscarinic receptors facilitates mechano-sensitive, capsaicin-sensitive BAN activity in part by mechanisms involving purinergic receptors located near the luminal surface of the bladder and ATP release which presumably occurs in the urothelium.

Dopaminergic D2 receptors activate PKA to inhibit spinal pelvic-urethra reflex in rats

To clarify the role of descending dopaminergic innervation in reflexive urethral closure, the impacts of dopaminergic D2 receptor (DR2)-selective agonists and antagonists on repetitive stimulation-induced pelvic-to-urethra spinal reflex potentiation (SRP) were tested using in vivo rat preparations. Pelvic afferent nerve test stimulation (TS; 1 pulse/30 s for 30 min) evoked baseline reflex activity with single spikes in the external urethral sphincter electromyogram (EUSE), whereas, repetitive stimulation (RS; 1 pulse/s for 30 min) induced SRP. Intrathecal application of quinelorane dihydrochloride (Q110; 10, 30, and 100 nM, 10 microl, a selective DR2 agonist) dose dependently inhibited the RS-induced SRP. Pretreatment with L135 (100 nM, 10 microL it, a selective DR2 antagonist) antagonized the Q110-dependent inhibition (100 nM, 10 microl it). Intrathecal AMPA (10 microM, 10 microl, a selective glutamatergic AMPA receptor agonist), and NMDA (10 microM, 10 microl, a selective glutamatergic NMDA receptor agonist) reversed the Q110-dependent inhibition. Intrathecal forskolin (100 nM, 10 microl, a PKA activator) prevented the Q110-dependent inhibition that was reversed by CNQX (10 microM, 10 microl it, a selective glutamate AMPA receptor antagonist) and APV (10 microM, 10 microl it , a selective glutamate NMDA receptor antagonist). Our results suggest that DR2 activation, which inactivates intracellular PKA, may be involved in descending dopaminergic inhibition of NMDA/AMPA receptor-dependent SRP at the lumbosacral spinal cord, which is thought to be involved in reflexive urethral closure.

Acute neurosteroids inhibit the spinal reflex potentiation via GABAergic neurotransmission

Recently, we demonstrated a chronic neurosteroid-dependent inhibition of activity-dependent spinal reflex potentiation (SRP), but it remains unclear whether neurosteroids acutely modulate SRP induction. This study shows progesterone as well as two of its 3alpha,5alpha-derivatives, allopregnalonone and 3alpha,5alpha-tetrahydrodeoxycorticosterone (THDOC), to be capable of producing acute GABA(A) receptor (GABA(A)R)-dependent inhibition of SRP. When compared with test simulation (1 stimulation/30 s) of pelvic afferent nerves that evoked a baseline reflex activity in an external urethra sphincter electromyogram, repetitive stimulation (RS; 1 stimulation/1 s) induced SRP characterized by an increase in the evoked activity. Intrathecal progesterone (3-30 muM, 10 microl) at 10 min before stimulation onset dose dependently prevented RS induction. Intrathecal allopregnalonone (10 muM, 10 microl it) and THDOC (10 microM, 10 microl it) also prevented the SRP caused by RS. Pretreatment with the GABA(A)R antagonist bicuculline (10 microM, 10 microl it) at 1 min before progesterone/neurosteroid injection attenuated the inhibition of SRP caused by progesterone, allopregnanolone, and THDOC. Results suggest that progesterone and its neurosteroid metabolites may be crucial to the development of pelvic visceral neuropathic/postinflammatory pain and imply clinical use of neurosteroids, such as allopregnanolone and THDOC, for visceral pain treatment.

Hyaluronic acid ameliorates bladder hyperactivity via the inhibition of H2O2‐enhanced purinergic and muscarinic signaling in the rat

We hypothesize that increased H(2)O(2) in the urinary bladder may affect ATP and acetylcholine release and activate urothelial purinergic and muscarinic signaling consequently leading to hyperactive bladder. We infused 0.3% and 1.5% H(2)O(2) to the urinary bladder to evaluate the voiding function and frequency, pelvic afferent (PANA) and pelvic efferent nerve activity (PENA) simultaneously in the urethane anesthetized rats. We measured ATP and acetylcholine content in the rat urinary bladder under saline or H(2)O(2) stimulation. Hyaluronic acid, muscarinic, and purinergic receptor antagonists were used to evaluate their effects and mechanisms on H(2)O(2)-induced hyperactive bladder. Intravesical H(2)O(2) administration increased the frequency of voiding and the maximal amplitude of intravesical pressure (IVP) in the urethane anesthetized rats. Intravesical H(2)O(2) decreased the threshold level of PANA and PENA for triggering micturition and increased ATP and acetylcholine contents in the H(2)O(2)-treated bladder in the rat. Hyaluronic acid in vitro directly scavenged H(2)O(2) in a dose-dependent manner. Intravesical hyaluronic acid for 30 min attenuated H(2)O(2)-elicited excitatory effects on the frequency of voiding, amplitude of IVP and the sensitization of PANA and PENA in the rats. Hyaluronic acid treatment reduced H(2)O(2)-induced ATP and acetylcholine release in the urinary bladder. Intravesical administration of muscarinic receptor antagonist atropine methyl nitrate (50 microM) or purinergic receptor antagonist PPADS (1 mM) ameliorated H(2)O(2)-induced hyperactive bladder. These results indicate that hyaluronic acid treatment can ameliorate H(2)O(2)-induced bladder hyperactivity possibly via the antioxidant activity and the inhibition of activating purinergic and muscarinic signaling pathway.

Antinociceptive effects of melatonin in a rat model of post-inflammatory visceral hyperalgesia: A centrally mediated process

Previous reports suggest that melatonin may play an important role in visceral nociception and neurogenic inflammation. We aimed to examine the role of melatonin on visceral hypersensitivity and to explore the site of action using a rat model of post-inflammatory visceral hyperalgesia. In all rats, a baseline viscero-motor response (VMR) to graded colorectal distension (CRD; 10–60 mmHg) was recorded prior and 1 week following tri-nitrobenzenesulfonic acid (TNBS) induced colonic inflammation. Melatonin (30, 45 or 60 mg/kg, ip) was given 20 min before testing the VMR in naïve and TNBS-treated rats. Extracellular single-unit recordings were made from CRD-sensitive pelvic nerve afferent (PNA) fibers and lumbosacral (LS) spinal neurons in TNBS-treated animals. The effect of melatonin (60 mg/kg) was examined on responses of PNAs and spinal neurons to graded CRD. In separate experiments, luzindole (non-specific MT 1/MT 2 receptor antagonist) or naltrexone (non-specific opiod receptor antagonist) was injected prior to melatonin. Following TNBS, there was a significant increase in the VMR to CRD compared to baseline. This increase was attenuated by melatonin (60 mg/kg) at pressures >20 mmHg. The same dose of melatonin had no effect on the VMR in naïve animals. In TNBS-treated rats, melatonin significantly attenuated the responses of CRD-sensitive spinal neurons to CRD, but had no effect in spinal transected rats or PNA fibers. Both luzindole and naltrexone blocked melatonin’s effect on the VMR and LS spinal neurons. Results indicate melatonin’s antinociceptive effects are not via a peripheral site of action but rather a supra-spinal process linked to the central opioidergic system.

Differential roles of stretch-sensitive pelvic nerve afferents innervating mouse distal colon and rectum

Information about colorectal distension (i.e., colorectal dilation by increased intraluminal pressure) is primarily encoded by stretch-sensitive colorectal afferents in the pelvic nerve (PN). Despite anatomic differences between rectum and distal colon, little is known about the functional roles of colonic vs. rectal afferents in the PN pathway or the quantitative nature of mechanosensory encoding. We utilized an in vitro mouse colorectum-PN preparation to investigate pressure-encoding characteristics of colorectal afferents. The colorectum with PN attached was dissected, opened longitudinally, and pinned flat in a Sylgard-lined chamber. Action potentials of afferent fibers evoked by circumferential stretch (servo-controlled force actuator) were recorded from the PN. Stretch-sensitive fibers were categorized into the following four groups: colonic muscular, colonic muscular/mucosal, rectal muscular, and rectal muscular/mucosal. Seventy-nine stretch-sensitive PN afferents evenly distributed into the above four groups were studied. Rectal muscular afferents had significantly greater stretch-responses than the other three groups. Virtually all rectal afferents (98%) had low thresholds for response and encoded stimulus intensity into the noxious range without obvious saturation. Most colonic afferents (72%) also had low thresholds (<14 mmHg), but a significant proportion (28%) had high thresholds (>18 mmHg) for response. These high-threshold colonic afferents were sensitized to stretch by inflammatory soup; response threshold was significantly reduced (from 23 to 12 mmHg), and response magnitude significantly increased. These results suggest that the encoding of mechanosensory information differs between colonic and rectal stretch-sensitive PN afferents. Rectal afferents have a wide response range to stretch, whereas high-threshold colonic afferents likely contribute to visceral nociception.

Mechanosensitive Primary Bladder Afferent Activity in Rats With and Without Spinal Cord Transection

Spinal cord injury induces functional and morphological changes in bladder afferent pathways. However, direct evidence for changes in the excitability of afferent nerve activity primarily originating from the bladder has not been clearly demonstrated. Thus, we determined the characteristics of peripheral mechanosensitive bladder afferents in the pelvic nerve and possible afferent changes in Adelta and C fibers after spinal cord injury. Adult female rats were divided into 2 groups, including spinal cord injured and neurologically intact animals. In the spinal cord injury group the spinal cord was transected at Th9 at 4 weeks before functional experiments. For single unit afferent activity monitoring fine filaments were dissected from the L6 dorsal root and bladder afferent fibers were identified. Single unit afferent activity was studied during constant filling with saline. Two afferent patterns were linked to small phasic increases in intravesical pressure during bladder filling, including accelerated and nonaccelerated types. The incidence of the accelerated type was significantly higher in the spinal cord injury group than in the neurologically intact group regarding Adelta and C fibers. However, we found no relationship between conduction velocity and the functional properties of bladder mechanosensitive afferent fibers in neurologically intact or spinal cord injured rats. Results indicate that mechanosensitive bladder afferent activity has several patterns and is facilitated after spinal cord injury, especially in concert with small bladder contractions (micromotions). The functional properties of these individual afferent fibers are not related in an obvious manner to their conduction velocity and, thus, probably the afferent fiber type.

Effect of nicotine on the pelvic afferent nerve activity and bladder pressure in rats

To record afferent nerve activity and bladder pressure in anesthetized male rats and to investigate whether increased afferent nerve activity induced by nicotine is able to evoke reflex bladder contractions. Using continuous infusion cystometrography, bladder pressure was measured via a bladder cannula. Afferent activity was recorded in the uncut L6 dorsal root. Nicotine was injected intra-arterially through a cannula placed near the bifurcation of the internal iliac artery a few minutes after micturition. Nicotine (0.15-1.5 micromol) evoked a marked elevation of afferent discharge without a simultaneous increase in bladder pressure. Bladder contractions appeared about 43 and 19 s after bolus injection of nicotine at 0.45 and 1.5 micromol, respectively. Firing rates of afferent nerves were reduced when the contraction appeared. Continuous infusion of nicotine at 0.75 micromol/min for 20 min evoked marked elevation of afferent discharge, which was maintained during infusion of nicotine and after it had been withdrawn. Repetitive contractions were observed thereafter and disappeared when the L6 dorsal roots were bilaterally resected. A transient increase in afferent discharges induced by bolus injection of nicotine was unable to evoke reflex bladder contraction. Repetitive bladder contractions after withdrawal of continuous nicotine infusion were induced in a reflex manner by the increased afferent activity.

Peripheral and Central P2X3 Receptor Contributions to Colon Mechanosensitivity and Hypersensitivity in the Mouse

Irritable bowel syndrome is characterized by altered sensory qualities, namely discomfort/pain and colorectal hypersensitivity. In mice, we examined the role of P2X(3) receptors in colon mechanosensitivity and intracolonic zymosan-produced hypersensitivity, a model of persistent colon hypersensitivity without colon inflammation. The visceromotor response to colon distension (15-60 mm Hg) was determined before and after intracolonic saline or zymosan (30 mg/mL, 0.1 mL, daily for 3 days) treatment. Colon pathology and intracolonic adenosine triphosphate release was assessed in parallel experiments. To examine P2X(3) receptor contributions to colon mechanosensation and hypersensitivity, electrophysiologic experiments were performed using an in vitro colon-pelvic nerve preparation. Visceromotor responses to distension were significantly reduced in P2X(3)(+/-) and P2X(3)(-/-) mice relative to wild-type mice. Colon hypersensitivity produced by zymosan was virtually absent in P2X(3)(-/-) relative to wild-type or P2X(3)(+/-) mice. Intralumenal release of the endogenous P2X receptor ligand adenosine triphosphate did not differ between wild-type and P2X(3)(-/-) mice or change after intracolonic zymosan treatment. Responses of muscular and muscular-mucosal pelvic nerve afferents to mechanical stretch did not differ between P2X(3)(-/-) and wild-type mice. Both muscular and muscular-mucosal afferents in wild-type mice sensitized to application of an inflammatory soup, whereas only muscular-mucosal afferents did so in P2X(3)(-/-) mice. These results suggest differential roles for peripheral and central P2X(3) receptors in colon mechanosensory transduction and hypersensitivity.

Estrogen-dependent facilitation on spinal reflex potentiation involves the Cdk5/ERK1/2/NR2B cascade in anesthetized rats

Cyclin-dependent kinase-5 (Cdk5), a proline-directed serine/threonine kinase, may alter pain-related neuronal plasticity by regulating extracellular signal-related kinase-1/2 (ERK1/2) activation. This study investigated whether Cdk5-dependent ERK activation underlies the estrogen-elicited facilitation on the repetitive stimulation-induced spinal reflex potentiaton (SRP) that is presumed to be involved in postinflammatory/neuropathic hyperalgesia and allodynia. Reflex activity of the external urethra sphincter electromyogram evoked by pelvic afferent nerve test stimulation (TS; 1 stimulation/30 s for 10 min) and repetitive stimulation (RS; 1 stimulation/1 s for 10 min) was recorded in anesthetized rats. TS evoked a baseline reflex activity, whereas RS produced SRP. Intrathecal (it) beta-estradiol facilitated the repetitive stimulation-induced SRP that was reversed by pretreatment with the estrogen receptor anatogonist ICI 182,780 (10 nM, 10 microl it), Cdk5 inhibitor roscovitine (100 nM, 10 microl it), ERK inhibitor (U-0126; 100 microM, 10 microl it) and N-methyl-D-aspartate (NMDA) NR2B subunit antagonist (Co-101244; 100 nM, 10 microl it). Moreover, ERalpha (propylpyrazoletriol; 100 nM, 10 microl it) and ERbeta (diarylpropionitrile; 100 microM, 10 microl it) agonists both facilitated the SRP, similar to results with a beta-estradiol injection. In association with the facilitated RS-induced SRP, an intrathecal beta-estradiol injection elicited ERK1/2 and NR2B subunit phosphorylation that were both reversed by intrathecal roscovitine and U-0126. These results indicated that the Cdk/ERK cascade, which is activated by ERalpha and ERbeta, may subsequently phosphorylate the NR2B subunit to develop NMDA-dependent postinflammatory hyperalgesia and allodynia to maintain the protective mechanisms of the body.

W1729 Volume Dependent Recruitment of Adaptive Esophageal Peristaltic and Upper Esophageal Sphincter Contractile Reflexes Is Preserved in Sleep State in Healthy Premature Infants

Background: Serotonin (5-HT) plays a major role in gastrointestinal (GI) functions. Of several subtypes of 5-HT receptors, 5-HT3 and 5-HT4 are involved in GI motility and pain. 5-HT4 receptor agonist tegaserod (TEG) has been shown to modulate visceral pain in both humans and animals. However, its underlying mechanism of action remains unknown. Aim: The objective of the present study is to examine the site of action of this drug in colorectal distension (CRD)-induced visceral pain in naive and post-inflamed sensitized rats. Methods: In each of 47 male Sprague-Dawley rats under fully anesthetized state, a bipolar electrode was implanted surgically into the external oblique muscle of the abdomen to measure the visceromotor response (VMR) represented as electrical activity (EMG) of the muscle to graded CRD (10-80mmHg). VMR was recorded in fully awake rats 72 hours following the surgery. To induce colonic inflammation, 0.5ml of tri-nitrobenzene sulfonic acid (TNBS) in 50% ethanol was infused into the colon under the influence of anesthesia and rats were tested 7days after TNBS injection. Drugs were injected either intraperitoneally (ip), intravenously (iv) or subcutaneously (sc). Electrophysiological recordings were made from the S1 sacral dorsal root receiving pelvic nerve innervating the colon. Responses of pelvic afferents to CRD were recorded before and after TEG injection. Results: Rats exhibited linear increasing VMR to graded CRD. TNBS-treated rats exhibited significantly (p<0.05 vs naive) greater VMR suggesting the sensitization following inflammation. High doses of TEG (5 and 10mg/ kg, ip) resulted in severe CNS effect including agitation, motor impairment and sedation (n=4). CNS effects were delayed in atropine (5mg/kg, ip) pretreated rats (n=4). Lower doses (0.5 and 1mg/kg, ip) of TEG did not produce any visible CNS effect. TEG (1mg/kg) significantly attenuated VMR in both naive and post-inflamed rats. Atropine failed to block the effect of TEG, suggesting that visceral analgesia was not mediated via muscariniccholinergic system. Selective 5-HT4 antagonist GR113808 (5mg/kg, sc) significantly reversed the effect of TEG. Similarly, opioid receptor antagonist naloxone (5mg/kg, iv) significantly reversed the effect of TEG. In electrophysiology experiments, TEG (1mg/kg, ip) did not attenuate the mechanotransduction of colonic pelvic nerve afferents in either naive or postinflamed rats. Conclusion: Results suggest that TEG produces visceral analgesia via the activation of central 5-HT4 receptors. Since opioid antagonist naloxone blocks the effect of TEG, it suggests that the central opioidergic system is linked to 5-HT4 mediated visceral analgesia.

Colon mustard oil instillation induced cross-organ reflex sensitization on the pelvic–urethra reflex activity in rats

We investigated the participation of cyclin-dependent kinase-5 (Cdk5)-mediated N-methyl- d-aspartate receptor (NMDAR) NR2B subunit phosphorylation in cross-organ reflex sensitization caused by colon irritation. The external urethral sphincter electromyogram (EUSE) reflex activity evoked by the pelvic afferent nerve test stimulation (TS, 1 stimulation/30 s) and protein expression in the spinal cord and dorsal root ganglion tissue (T13-L2 and L6-S2 ipsilateral to the stimulation) in response to colon mustard oil (MO) instillation were tested in anesthetized rats. When compared with a baseline reflex activity with a single action potential evoked by the TS before the administration of test agents, MO instillation into the descending colon sensitized the evoked activity characterized by elongated firing in the reflex activity in association with increased protein levels of Cdk5, PSD95, and phosphorylated NR2B (pNR2B) but not of total NR2B (tNR2B) in the spinal cord tissue. Both cross-organ reflex sensitization and increments in protein expression were reversed by intra-colonic pretreatments with ruthenium red (a non-selective transient receptor potential vanilloid, TRPV, antagonist), capsaizepine (a TRPV 1-selective antagonist), lidocaine (a nerve conduction blocker) as well as by the intra-thecal pretreatment with APV (a NRMDR antagonist) Co-101244 (a NR2B-selective antagonist) and roscovitine (a Cdk5 antagonist). Moreover, compared with the control group, both the increase in pNR2B and the cross-organ reflex sensitization were attenuated in the si-RNA of NR2B rats. All these results suggested that Cdk-dependent NMDAR NR2B subunit phosphorylation mediates the development of cross-organ pelvic–urethra reflex sensitization caused by acute colon irritation which could possibly underlie the high concurrence of pelvic pain syndrome with irritable bowel syndrome.

TRPV1 receptors on unmyelinated C‐fibres mediate colitis‐induced sensitization of pelvic afferent nerve fibres in rats

Patients with inflammatory bowel disease often suffer from gastrointestinal motility and sensitivity disorders. The aim of the current study was to investigate the role of transient receptor potential of the vanilloid type 1 (TRPV1) receptors in the pathophysiology of colitis-induced pelvic afferent nerve sensitization. Trinitrobenzene sulphate (TNBS) colitis (7.5 mg, 30% ethanol) was induced in Wistar rats 72 h prior to the experiment. Single-fibre recordings were made from pelvic nerve afferents in the decentralized S1 dorsal root. Fibres responding to colorectal distension (CRD) were identified in controls and rats with TNBS colitis. The effect of the TRPV1 antagonist N-(4-tertiarybutylphenyl)-4-(3-chlorophyridin-2-yl)tetrahydropyrazine-1(2H)carboxamide (BCTC; 0.25-5 mg kg(-1)) or its vehicle (hydroxypropyl-beta-cyclodextrin) was tested on the afferent response to repetitive distensions (60 mmHg). Immunocytochemical staining of TRPV1 and NF200, a marker for A-fibre neurons, was performed in the dorsal root ganglia L6-S1. TNBS colitis significantly increased the response to colorectal distension of pelvic afferent C-fibres. BCTC did not significantly affect the C-fibre response in controls, but normalized the sensitized response in rats with colitis. TNBS colitis increased the spontaneous activity of C-fibres, an effect which was insensitive to administration of BCTC. TNBS colitis had no effect on Adelta-fibres, nor was their activity modulated by BCTC. TNBS colitis caused an immunocytochemical up-regulation of TRPV1 receptors in the cell bodies of pelvic afferent NF200 negative neurons. TRPV1 signalling mediates the colitis-induced sensitization of pelvic afferent C-fibres to CRD, while Adelta-fibres are neither sensitized by colitis nor affected by TRPV1 inhibition.

Orexin-A modulates glutamatergic NMDA-dependent spinal reflex potentiation via inhibition of NR2B subunit

Glucose-sensitive neurons in the lateral hypothalamic area produce orexin-A (OxA) as well as orexin-B (OxB) and send their axons to the spinal dorsal horn, which predominantly expresses orexin receptor-1 (OX-1), showing a higher sensitivity to OxA. The purpose of the present study was to assess the effects of OxA on the induction of a novel form of activity-dependent reflex potentiation, spinal reflex potentiation (SRP), in the pelvic-urethral reflex activity. External urethra sphincter electromyogram in response to pelvic afferent nerve test stimulation (TS; 1/30 Hz) or repetitive stimulation (RS; 1 Hz) was recorded in anesthetized rats. TS evoked a baseline reflex activity, whereas RS produced SRP, which was abolished by intrathecal OxA (30 nM, 10 mul). Intrathecal SB-408124 (10 muM, 10 mul), an OX-1 antagonist, reversed the abolition on SRP caused by OxA. Although there is, so far, no NR2A- and NR2B-specific agonist available, N-methyl-d-aspartate (NMDA) reversed the abolition on the RS-induced SRP caused by the co-administration of OxA and Co-101244 (30 nM, 10 mul; an NMDA NR2B subunit antagonist), but it did not reverse the abolition by the co-administration of OxA and PPPA (300 nM, 10 mul; an NMDA NR2A subunit antagonist). In conclusion, the activation of descending orexinergic fibers may inhibit the repetitive afferent input-induced central sensitization of pelvic-urethral reflex activity and urethra hyperactivity, indicating that spinal orexinergic neural transmission may be a novel target for the treatment of patients with neuropathetic or postinflammatory pain of pelvic origin.