Hydrogen peroxide preferentially activates capsaicin-sensitive high threshold afferents via TRPA1 channels in the guinea pig bladder.

BACKGROUND AND PURPOSE: There is increasing evidence suggesting that ROS play a major pathological role in bladder dysfunction induced by bladder inflammation and/or obstruction. The aim of this study was to determine the effect of H2 O2 on different types of bladder afferents and its mechanism of action on sensory neurons in the guinea pig bladder. EXPERIMENTAL APPROACH: 'Close-to-target' single unit extracellular recordings were made from fine branches of pelvic nerves entering the guinea pig bladder, in flat sheet preparations, in vitro. KEY RESULTS: H2 O2 (300-1000 µM) preferentially and potently activated capsaicin-sensitive high threshold afferents but not low threshold stretch-sensitive afferents, which were only activated by significantly higher concentrations of hydrogen peroxide. The TRPV1 channel agonist, capsaicin, excited 86% of high threshold afferents. The TRPA1 channel agonist, allyl isothiocyanate and the TRPM8 channel agonist, icilin activated 72% and 47% of capsaicin-sensitive high threshold afferents respectively. The TRPA1 channel antagonist, HC-030031, but not the TRPV1 channel antagonist, capsazepine or the TRPM8 channel antagonist, N-(2-aminoethyl)-N-[[3-methoxy-4-(phenylmethoxy)phenyl]methyl]thiophene-2-carboxamide, significantly inhibited the H2 O2 -induced activation of high threshold afferents. Dimethylthiourea and deferoxamine did not significantly change the effect of H2 O2 on high threshold afferents. CONCLUSIONS AND IMPLICATIONS: The findings show that H2 O2 , in the concentration range detected in inflammation or reperfusion after ischaemia, evoked long-lasting activation of the majority of capsaicin-sensitive high threshold afferents, but not low threshold stretch-sensitive afferents. The data suggest that the TRPA1 channels located on these capsaicin-sensitive afferent fibres are probable targets of ROS released during oxidative stress.

Activation of intestinal spinal afferent endings by changes in intra-mesenteric arterial pressure.

KEY POINTS: A major class of mechano-nociceptors to the intestine have mechanotransduction sites on extramural and intramural arteries and arterioles ('vascular afferents'). These sensory neurons can be activated by compression or axial stretch of vessels. Using isolated preparations we showed that increasing intra-arterial pressure, within the physiological range, activated mechano-nociceptors on vessels in intact mesenteric arcades, but not in isolated arteries. This suggests that distortion of the branching vascular tree is the mechanical adequate stimulus for these sensory neurons, rather than simple distension. The same rises in pressure also activated intestinal peristalsis in a partially capsaicin-sensitive manner indicating that pressure-sensitive vascular afferents influence enteric circuits. The results identify the mechanical adequate stimulus for a major class of mechano-nociceptors with endings on blood vessels supplying the gut wall; these afferents have similar endings to ones supplying other viscera, striated muscle and dural vessels. ABSTRACT: Spinal sensory neurons innervate many large blood vessels throughout the body. Their activation causes the hallmarks of neurogenic inflammation: vasodilatation through the release of the neuropeptide calcitonin gene-related peptide and plasma extravasation via tachykinins. The same vasodilator afferent neurons show mechanical sensitivity, responding to crushing, compression or axial stretch of blood vessels - responses which activate pain pathways and which can be modified by cell damage and inflammation. In the present study, we tested whether spinal afferent axons ending on branching mesenteric arteries ('vascular afferents') are sensitive to increased intravascular pressure. From a holding pressure of 5 mmHg, distension to 20, 40, 60 or 80 mmHg caused graded, slowly adapting increases in firing of vascular afferents. Many of the same afferent units showed responses to axial stretch, which summed with responses evoked by raised pressure. Many vascular afferents were also sensitive to raised temperature, capsaicin and/or local compression with von Frey hairs. However, responses to raised pressure in single, isolated vessels were negligible, suggesting that the adequate stimulus is distortion of the arterial arcade rather than distension per se. Increasing arterial pressure often triggered peristaltic contractions in the neighbouring segment of intestine, an effect that was mimicked by acute exposure to capsaicin (1 µm) and which was reduced after desensitisation to capsaicin. These results indicate that sensory fibres with perivascular endings are sensitive to pressure-induced distortion of branched arteries, in addition to compression and axial stretch, and that they contribute functional inputs to enteric motor circuits.

Novel spinal pathways identified by neuronal c-Fos expression after urethrogenital reflex activation in female guinea pigs.

Pudendal nerve-spinal pathways are involved in urethrogenital sensation, pain and sexual activity. However, details of these pathways and their modulation are unclear. We examined spinal pathways activated by the urethrogenital reflex (UGR) and visualized by c-Fos immunoreactivity in reflexly activated neurons within spinal cord. In anesthetized female guinea pigs, a balloon was inserted into the urethra and inflated with short-repeat or long-continuous distension to activate the UGR. A second balloon recorded reflex contractions of the vagina and uterus. Two control groups had either no balloon or a vaginal balloon (VB) only. Ninety minutes after UGR activation, c-Fos immunoreactivity in L3 and S2 spinal segments was examined. Reflex activated c-Fos immunoreactivity also was investigated in some animals with acute spinal transections at either L4 or T12 levels. There was no significant difference in spinal c-Fos expression between the control groups. Short-repeat distension reliably induced a UGR and a two- to threefold increase in c-Fos-expressing neurons throughout dorsal, intermediate and lateral spinal gray matter at S2 and about twofold increase in superficial dorsal horn at L3. T12 transection had little effect on c-Fos expression at either spinal level. However, after L4 transection, UGR generation was associated with a four- to sixfold increase in c-Fos-expressing neurons in lateral horn (LH) and central canal areas at S2, and but only 20-30% increase at L3. Thus, UGR activates preganglionic neurons projecting to pelvic viscera in both sacral and lumbar spinal cord. The reflex also must activate ascending and descending spinal inhibitory circuits that suppress c-Fos-expression in neurons at both sacral and lumbar spinal levels.

Targeted electrophysiological analysis of viscerofugal neurons in the myenteric plexus of guinea-pig colon.

Enteric viscerofugal neurons are mechanosensory interneurons that form the afferent limb of intestino-intestinal reflexes involving prevertebral sympathetic neurons. Fast synaptic inputs to viscerofugal neurons arise from other enteric neurons, but their sources are unknown. We aimed to describe the origins of synaptic inputs to viscerofugal neurons by mapping the locations of their cell bodies within the myenteric plexus. Viscerofugal neuron somata were retrogradely traced with 1,1'-didodecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (DiI) from colonic nerve trunks and impaled with microelectrodes, in longitudinal muscle/myenteric plexus preparations of the guinea-pig distal colon (39 impalements, n=14). Thirty-eight viscerofugal neurons were uni-axonal and had the electrophysiological characteristics of myenteric S-neurons; one neuron was multipolar with AH-neuron electrophysiological characteristics. Depolarizing current pulses evoked either single- or multiple action potentials in viscerofugal neurons (range 1-25 spikes, 500 ms, 100-900 pA, 21 cells). Electrical stimulation of internodal strands circumferential to viscerofugal neurons evoked fast excitatory postsynaptic potentials (EPSPs) in 19/24 cells. Focal pressure-ejection of the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP, 10 µm) directly onto viscerofugal nerve cell bodies evoked large depolarizations and action potentials (23 ± 10 mV, latency 350 ± 230 ms, 21/22 cells). DMPP was then focally applied to multiple sites, up to 3mm from the recorded viscerofugal neuron, to activate other myenteric S-neurons. In a few sites in myenteric ganglia, DMPP evoked repeatable fast EPSPs in viscerofugal neurons (latency 300 ± 316 ms, 38/394 sites, 10 cells). The cellular sources of synaptic inputs to viscerofugal neurons were located both orally and aborally (19 oral, 19 aboral), but the amplitude of oral inputs was consistently greater than aboral inputs (13.1 ± 4.3 mV vs. 10.1 ± 4.8 mV, respectively, p<0.05, paired t-test, n=6). Most impaled viscerofugal neurons were nitric oxide synthase (NOS) immunoreactive (20/27 cells tested). Thus, the synaptic connections onto viscerofugal neurons within the myenteric plexus suggest that multiple enteric neural pathways feed into intestino-intestinal reflexes, involving sympathetic prevertebral ganglia.

Identification and mechanosensitivity of viscerofugal neurons.

Enteric viscerofugal neurons are interneurons with cell bodies in the gut wall; they project to prevertebral ganglia where they provide excitatory synaptic drive to sympathetic neurons which control intestinal motility and secretion. Here, we studied the mechanosensitivity and firing of single, identified viscerofugal neurons in guinea-pig distal colon. Flat sheet preparations of gut were set up in vitro and conventional extracellular recordings made from colonic nerve trunks. The nicotinic agonist, 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) (1mM), was locally pressure ejected onto individual myenteric ganglia. In a few ganglia, DMPP promptly evoked firing in colonic nerves. Biotinamide filling of colonic nerves revealed that DMPP-responsive sites corresponded to viscerofugal nerve cell bodies. This provides a robust means to positively identify viscerofugal neuron firing. Of 15 single units identified in this way, none responded to locally-applied capsaicin (1 µM). Probing with von Frey hairs at DMPP-responsive sites reliably evoked firing in all identified viscerofugal neurons (18/18 units tested; 0.8-5 mN). Circumferential stretch of the preparation increased firing in all 14/14 units (1-5 g, p<0.05). Both stretch and von Frey hair responses persisted in Ca(2+)-free solution (6 mM Mg(2+), 1mM EDTA), indicating that viscerofugal neurons are directly mechanosensitive. To investigate their adequate stimulus, circular muscle tension and length were independently modulated (BAY K8644, 1 µM and 10 µM, respectively). Increases in intramural tension without changes in length did not affect firing. However, contraction-evoked shortening, under constant load, significantly decreased firing (p<0.001). In conclusion, viscerofugal neuron action potentials contribute to recordings from colonic nerve trunks, in vitro. They provide a significant primary afferent output from the colon, encoding circumferential length, largely independent of muscle tension. All viscerofugal neurons are directly mechanosensitive, although they have been reported to receive synaptic inputs. In short, viscerofugal neurons combine interneuronal function with length-sensitive mechanosensitivity.

Viscerofugal neurons recorded from guinea-pig colonic nerves after organ culture.

BACKGROUND: Enteric viscerofugal neurons provide cholinergic synaptic inputs to prevertebral sympathetic neurons, forming reflex circuits that control motility and secretion. Extracellular recordings of identified viscerofugal neurons have not been reported. METHODS: Preparations of guinea pig distal colon were maintained in organotypic culture for 4-6 days (n = 12), before biotinamide tracing, immunohistochemistry, or extracellular electrophysiological recordings from colonic nerves. KEY RESULTS: After 4-6 days in organ culture, calcitonin gene-related peptide and tyrosine hydroxylase immunoreactivity in enteric ganglia was depleted, and capsaicin-induced firing (0.4 µmol L(-1) ) was not detected, indicating that extrinsic sympathetic and sensory axons degenerate in organ culture. Neuroanatomical tracing of colonic nerves revealed that viscerofugal neurons persist and increase as a proportion of surviving axons. Extracellular recordings of colonic nerves revealed ongoing action potentials. Interestingly, synchronous bursts of action potentials were seen in 10 of 12 preparations; bursts were abolished by hexamethonium, which also reduced firing rate (400 µmol L(-1) , P < 0.01, n = 7). DMPP (1,1-dimethyl-4-phenylpiperazinium; 10(-4) mol L(-1) ) evoked prolonged action potential discharge. Increased firing preceded both spontaneous and stretch-evoked contractions (χ(2) = 11.8, df = 1, P < 0.001). Firing was also modestly increased during distensions that did not evoke reflex contractions. All single units (11/11) responded to von Frey hairs (100-300 mg) in hexamethonium or Ca(2+) -free solution. CONCLUSIONS & INFERENCES: Action potentials recorded from colonic nerves in organ cultured preparations originated from viscerofugal neurons. They receive nicotinic input, which coordinates ongoing burst firing. Large bursts preceded spontaneous and reflex-evoked contractions, suggesting their synaptic inputs may arise from enteric circuitry that also drives motility. Viscerofugal neurons were directly mechanosensitive to focal compression by von Frey hairs.

Spontaneous release of acetylcholine from autonomic nerves in the bladder.

BACKGROUND AND PURPOSE: Bladder contractility is regulated by intrinsic myogenic mechanisms interacting with autonomic nerves. In this study, we have investigated the physiological role of spontaneous release of acetylcholine in guinea pig and rat bladders. EXPERIMENTAL APPROACH: Conventional isotonic or pressure transducers were used to record contractile activity of guinea pig and rat bladders. KEY RESULTS: Hyoscine (3 micromol x L(-1)), but not tetrodotoxin (TTX, 1 micromol x L(-1)), reduced basal tension, distension-evoked contractile activity and physostigmine (1 micromol x L(-1))-evoked contractions of the whole guinea pig bladder and muscle strips in vitro. omega-Conotoxin GVIA (0.3 micromol x L(-1)) did not affect physostigmine-induced contractions when given either alone or in combination with omega-agatoxin IVA (0.1 micromol x L(-1)) and SNX 482 (0.3 micromol x L(-1)). After 5 days in organotypic culture, when extrinsic nerves had significantly degenerated, the ability of physostigmine to induce contractions was reduced in the dorso-medial strips, but not in lateral strips (which have around 15 times more intramural neurones). Most muscle strips from adult rats lacked intramural neurones. After 5 days in culture, physostigmine-induced or electrical field stimulation-induced contractions of the rat bladder strips were greatly reduced. In anaesthetized rats, topical application of physostigmine (5-500 nmol) on the bladder produced a TTX-resistant tonic contraction that was abolished by atropine (4.4 micromol x kg(-1) i.v.). CONCLUSIONS AND IMPLICATIONS: The data indicate that there is spontaneous TTX-resistant release of acetylcholine from autonomic cholinergic extrinsic and intrinsic nerves, which significantly affects bladder contractility. This release is resistant to blockade of N, P/Q and R type Ca(2+) channels.

TNBS-induced inflammation modulates the function of one class of low-threshold rectal mechanoreceptors in the guinea pig.

The effects of trinitrobenzene sulfonic acid (TNBS)-induced inflammation on specialized, low-threshold, slowly adapting rectal mechanoreceptors were investigated in the guinea pig. Under isoflurane anesthesia, 300 microl saline or TNBS (15 mg/ml) in 30% ethanol was instilled 7 cm from the anal sphincter. Six or 30 days later, single unit extracellular recordings were made from rectal nerve trunks in flat-sheet in vitro preparations attached to a mechanical tissue stretcher. TNBS treatment caused macroscopic ulceration of the rectal mucosa at 6 days, which fully resolved by 30 days. Muscle contractility was unaffected by TNBS treatment. At 6 days posttreatment, responses of low-threshold rectal mechanoreceptors to circumferential stretch were increased, and the proportion of afferents responding with von Frey hair thresholds

Intraganglionic laminar endings are mechano-transduction sites of vagal tension receptors in the guinea-pig stomach.

1. Distension-sensitive vagal afferent fibres from the cardiac region of the guinea-pig stomach were recorded extracellularly, then filled with biotinamide, using an anterograde tracing technique. 2. Most of the stretch-sensitive units of the guinea-pig stomach (41 out of 47; number of animals N = 26) had low thresholds (less than 1 mm) to circumferential stretch and showed slow adaptation. Twenty of these units fired spontaneously under resting conditions (mean: 1.9 +/- 0.3 Hz, n = 20, N = 14). 3. Adaptation of firing during slow or maintained stretch correlated closely with accommodation of intramural tension, but tension-independent adaptation was also present. 4. Nicardipine (3 microM) with hyoscine (3 microM) reduced stretch-evoked firing of gastric vagal afferents, by inhibiting smooth muscle contraction. Gadolinium (1 mM) blocked distension-evoked firing. 5. Focal stimulation of the stomach muscle wall with a von Frey hair (0.4 mN) identified one to six punctate receptive fields in each low threshold vagal distension-sensitive afferent. These were marked on the serosal surface of the stomach wall. 6. Anterograde filling of recorded nerve trunks revealed intraganglionic laminar endings (IGLEs) within 142 +/- 34 microm (n = 38; N = 10) of marked receptive fields. The mean distance from randomly generated sites to the nearest IGLE was significantly greater (1500 +/- 48 microm, n = 380, N = 10, P < 0.0001). Viscerofugal nerve cell bodies, intramuscular arrays and varicose axons were not associated with receptive fields. The results indicate that IGLEs are the mechanotransduction sites of low threshold, slowly adapting vagal tension receptors in the guinea-pig upper stomach.

Propagating contractions of the circular muscle evoked by slow stretch in flat sheets of guinea-pig ileum.

Flat sheet preparations of guinea-pig ileum were stretched circumferentially and the propagation of circular muscle contractions along the preparation was investigated. Slow stretch, at 100 microm s-1, of a 50-mm long flat sheet of intestine, evoked circular muscle contraction orally, which propagated, without decrement, for up to 30 mm. This occurred despite circular muscle shortening being prevented, and in the absence of propulsion of contents. Thus, propagation in this flat sheet preparation could not explained on the basis of neuro-mechanical interactions, as previously proposed. Irrespective of the length of preparations, contraction amplitude decreased significantly in the most aboral 10-15 mm of intestine. This was not due to descending inhibitory pathways, but was associated with interruption of ascending excitatory pathways near the aboral end. Slow waves were not detected in circular muscle cells in any preparation (n=8). Smooth muscle action potentials evoked in circular muscle cells, in the presence of tetrodotoxin (TTX, 0.6 micromol L-1), did not propagate for more than 1 mm in the longitudinal axis. Propagation of circular muscle activity, evoked by slow stretch of flat sheet preparations, reveals the presence of a mechanism other than myogenic spread or the neuro-mechanical interactions previously proposed to account for propagation; the nature of this mechanism remains to be determined.

Transduction sites of vagal mechanoreceptors in the guinea pig esophagus.

Extrinsic afferent neurons play an essential role in both sensation and reflex control of visceral organs, but their specialized morphological peripheral endings have never been functionally identified. Extracellular recordings were made from fine nerve trunks running between the vagus nerve and esophagus of the guinea pig. Mechanoreceptors, which responded to esophageal distension, fired spontaneously, had low thresholds to circumferential stretch, and were slowly adapting. Calibrated von Frey hairs (0.12 mN) were used to probe the serosal surface at 100-200 sites, which were mapped on a video image of the live preparation. Each stretch-sensitive unit had one to three highly localized receptive fields ("hot spots"), which were marked with Indian ink applied on the tip of the von Frey hair. Recorded nerve trunks were then filled anterogradely, using biotinamide in an artificial intracellular solution. Receptive fields were consistently associated with intraganglionic laminar endings (IGLEs) in myenteric ganglia, but not with other filled neuronal structures. The average distance of receptive fields to IGLEs was 73 +/- 14 microm (24 receptive fields, from 12 units; n = 5), compared to 374 +/- 17 microm for 240 randomly generated sites (n = 5; p < 0.001). After maintained probing on a single receptive field, spontaneous discharge of units was inhibited, as were responses to distension. During adapted discharge to maintained distension, interspike intervals were distributed in a narrow range. This indicates that multiple receptive fields interact to encode mechanical distortion in a graded manner. IGLEs are specialized transduction sites of mechanosensitive vagal afferent neurons in the guinea pig esophagus.

An electrophysiological study of developmental changes in the innervation of the guinea-pig taenia coli.

A sucrose-gap technique was used to study the development of neuromuscular transmission in the taenia coli of fetal, 1- to 2-day-old, 3- to 4-week-old and 3-month-old guinea-pigs. In addition, the effects of exogenous, alpha,beta-methylene adenosine 5'-triphosphate (ATP), noradrenaline, vasoactive intestinal polypeptide (VIP) and carbachol were examined. Taking the response to a single pulse of electrical field stimulation as the index of a developed neuromuscular junction, it was apparent that the non-adrenergic inhibitory system arose before, and matured more quickly than, the cholinergic system. The inhibitory system was present by 8 weeks of gestation in some fetuses, but, in contrast, excitatory cholinergic transmission was not seen until birth. As evidenced by responses to carbachol, alpha,beta-methylene ATP and VIP, cholinergic, purinergic and VIP receptors were present on the smooth muscle at the earliest ages studied. No changes in sensitivity to these agents were noted throughout development, although in adults the level of the maximum responses increased.

Studies of the inhibitory non-adrenergic neuromuscular transmission in the smooth muscle of the normal human intestine and from a case of Hirschsprung's disease.

A modified sucrose-gap method was used to study both non-adrenergic inhibitory neuromuscular transmission and effects of adenosine 5'-triphosphate (ATP) on isolated smooth muscle preparations from the human intestine. It was found that non-adrenergic inhibition in the circular smooth muscle layer was of larger amplitude than in the longitudinal layer. Study of the ionic mechanisms underlying non-adrenergic inhibition indicated that an increase in K+ conductance was responsible for the generation of non-adrenergic inhibitory junction potentials (IJPs). The results suggest that the inhibitory actions of the endogenous neurotransmitter and exogenous ATP are due to increases in Ca2+-dependent K+ conductance. The K+-channel blockers tetraethylammonium and 4-aminopyridine had no effect on IJPs or ATP, while apamin slightly decreased both the amplitude of the IJP and the hyperpolarization of the circular smooth muscle caused by ATP. These results are consistent with the purinergic hypothesis of non-adrenergic inhibition. In addition to inhibitory purinoceptors, the existence of excitatory purinoceptors was identified in the longitudinal muscle, activation of which probably caused an increase in Na+-conductance. The excitatory purinoceptor-mediated contraction in the longitudinal muscle from the constricted region of large intestine from patients with Hirschsprung's disease was greater than that found in control specimens. It is possible that excitatory purinoceptors play a role in the pathophysiology of Hirschsprung's disease.