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Capsaicin-sensitive nerves mediate axon vasodilator reflexes in the intestine, but the ion channels underlying action potential (AP) propagation are poorly understood. To examine the role of voltage-gated Na(+) channels underlying these reflexes, we measured vasomotor and electrophysiological responses elicited by capsaicin in guinea pig and mouse dorsal root ganglia (DRG) neurons, submucosal arterioles, and mesenteric arteries in vitro. Transient receptor potential vanilloid 1 (TRPV1) agonists dilated guinea pig ileal submucosal arterioles and were blocked by capsazepine and ruthenium red. In double-chamber baths, capsaicin-evoked activation of TRPV1 on proximal perivascular nerves in the left chamber evoked dilations of the distal segment of the submucosal arteriole in the right chamber. Dilations were tetrodotoxin (TTX) (1 microM)-resistant, but reducing extracellular Na(+) (10% solution) or applying the Na(v) 1.8 antagonist A-803467 [5-(4-chlorophenyl-N-(3,5-dimethoxyphenyl)furan-2-carboxamide] (1 microM) in the proximal chamber blocked capsaicin-evoked dilations in the distal chamber (88%; P = 0.01 and 75% and P < 0.02, respectively). In mouse mesenteric arteries, electrical field stimulation and capsaicin (2 microM) evoked dilations that were also TTX-resistant. In perforated patch-clamp recordings, APs in mouse and guinea pig capsaicin-sensitive DRG neurons were TTX-resistant but blocked by 10% extracellular Na(+). When capsaicin-evoked AP conduction was studied in in vitro ileal multiunit afferent nerve preparations, capsaicin responses were elicited in the presence of TTX, whereas distention-evoked responses were almost completely blocked by TTX. Together, these data provide evidence for TTX-resistant AP conduction in extrinsic sensory neurons that innervate guinea pig and mouse intestine and suggest this neural propagation is sufficient to mediate axon reflexes in the intestine.

Purinergic inhibitory neuromuscular transmission plays an important role in the control of intestinal motility. In most tissues this neurotransmission is apamin-sensitive, but recent studies in human colonic circular smooth muscle (CSM) suggest the presence of apamin-insensitive purinergic inhibitory junction potentials (IJPs). The current studies used conventional intracellular recordings on colonic CSM strips to characterize the purinergic IJPs in murine colonic CSM. P2Y1 receptor expression was examined by using reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. The IJP induced by nerve stimulation (NS) of one and four pulses in neuronal nitric-oxide synthase knockout mice consists of an apamin-sensitive and a dominant apamin-resistant component. These are identical to the IJPs in wild-type and CD1 mice in the presence of N(omega)-nitro-l-arginine methyl ester (200 microM) and were significantly inhibited by alpha,beta-methylene ATP (50 microM), an analog of ATP. IJPs were not affected by the P2X receptor antagonist 2',3'-o-(2,4,6-trinitrophenyl)-ATP (10 microM). Furthermore, apamin-resistant IJPs induced by single-pulse NS were abolished by pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (100 microM), a P2 receptor antagonist; 2'-deoxy-N6-methyl adenosine 3,5-diphosphate (MRS-2179; 10 microM), a selective P2Y1 receptor antagonist; and tetrodotoxin (1 microM). Aboral NS induced apamin-sensitive purinergic IJPs, whereas oral and circumferential NS produced apamin-sensitive and -resistant IJPs, with the latter predominating. RT-PCR and immunohistochemistry confirmed the presence of P2Y1 receptors on smooth muscle and in the myenteric plexus. These data suggest that, depending on stimulus location, activation of P2Y1 receptors produces both apamin-sensitive and apamin-resistant IJPs in murine colonic CSM.

Capsaicin-sensitive extrinsic sensory nerves and submucosal vasodilator neurons provide important vasodilator input to submucosal arterioles, but relatively little is known about the signaling between these populations and the sympathetic vasoconstrictor innervation. This study examined whether release of sympathetic purines can modulate dilator nerves. In vitro submucosal preparations from guinea pig ileum were modified to leave the parent mesenteric artery intact so that perivascular sympathetic and extrinsic afferent nerves could be activated by a bipolar stimulating electrode placed on the parent artery, and submucosal vasodilator neurons were activated using focal electrodes placed on submucosal ganglia. The outside diameter of submucosal arterioles was monitored using videomicroscopy, and dilator responses were examined after preconstricting vessels 80-95% with prostaglandin F(2alpha) (400 nM). Mesenteric nerve stimulation evoked a frequency-dependent dilation, with suramin (100 microM) present throughout to inhibit P(2X) receptor-mediated vasoconstrictions. In the presence of guanethidine (10 microM) to inhibit sympathetic purine release, superfusion of ATP (200 nM-6 microM) caused a concentration-dependent inhibition of nerve-evoked dilations. Vasodilations to substance P (10 nM) were not inhibited by ATP in the presence of guanethidine, implicating a presynaptic effect of ATP on neurotransmitter release. The inhibitory effect of ATP was blocked by the adenosine receptor antagonist 8-phenyltheophylline (8-PT; 10 microM). In addition, 8-PT increased the amplitude of nerve-evoked dilations, suggesting a tonic inhibitory effect of adenosine receptors on vasodilator release. Dilations evoked by electrical stimulation of submucosal ganglia were also inhibited almost 50% by ATP (2 microM) and its nonhydrolyzable analog, alpha,beta-methylene-ATP (10 microM). These data suggest that sympathetic varicosities release ATP or a related purine that can act at presynaptic adenosine receptors on extrinsic sensory and submucosal vasodilator neurons to inhibit neurotransmitter release.

Serotonin (5-HT)-containing enterochromaffin (EC) cells of the intestine transduce chemical and mechanical stimuli from the intestinal lumen by releasing 5-HT on to afferent nerve terminals. Dysfunctional mucosal 5-HT signaling has been implicated in heightened visceral sensitivity and altered motility in patients with inflammatory bowel disease and in animal models. Our aim was to characterize the release and uptake of 5-HT in the mouse dextran sulfate sodium (DSS; 5% wt/vol) model of colitis. We made electrochemical recordings and used an ELISA assay to determine mucosal 5-HT release and uptake in untreated mice and mice with DSS-induced colitis. Peak and steady-state 5-HT concentrations were measured before and during blockade of the serotonin reuptake transporter (SERT) with 1 microM fluoxetine. Electrochemical recordings showed that colons from DSS-treated mice had roughly twice the steady-state levels of extracellular 5-HT and compression-evoked 5-HT release compared with untreated mice. Fluoxetine doubled the compression-evoked and steady-state 5-HT levels in control and DSS mice. These data were supported by ELISA assays, which showed enhanced 5-HT release during colitis, by immunohistochemical analyses, which showed increases in EC cell numbers, and by real-time PCR, which identified a decrease in SERT mRNA expression in the mucosa during colitis. These data are the first to demonstrate 5-HT release close to its release site and near its site of action during DSS-colitis. We conclude that DSS-colitis increases 5-HT availability primarily by an increase in the numbers of EC cells and/or of content of 5-HT in these EC cells.

Knowledge of neural circuits, neurotransmitters and receptors involved in the sympathetic regulation of gastrointestinal (GI) function is well established. However, it is only recently that the interaction of sympathetic neurons, and of sympathetic transmitters, with the GI immune system and with gut flora has begun to be explored. Changes in the behaviour of sympathetic nerves when gut function is compromised, for example in ileus and in inflammation, have been observed, but the roles of the sympathetic innervation in these and other pathologies are not adequately understood. In this article, we first review the principal roles of the sympathetic innervation of the GI tract in controlling motility, fluid exchange and gut blood flow in healthy individuals. We then discuss the evidence that there are important interactions of sympathetic transmitters with the gut immune system and enteric glia, and evidence that inflammation has substantial effects on sympathetic neurons. These reciprocal interactions contribute to pathological changes in ways that are not yet clarified. Finally, we focus on inflammation, diabetes and postoperative ileus as conditions in which there is sympathetic involvement in compromised gut function.

1. Inflammatory bowel diseases (IBD) alter the function of the enteric nervous system and the sensory innervation of the gastrointestinal (GI) tract. Less is known about whether IBD also affects the sympathetic nervous system (SNS). Given the importance of the SNS in regulating GI function and possibly immune system activation, the present review examines the evidence of sympathetic dysfunction during IBD and its possible consequences. 2. Sympathetic axons within the GI tract innervate several cell types, including vascular myocytes, enteric neurons and immune cells. The major neurotransmitters released from sympathetic varicosities are noradrenaline, neuropeptide Y and ATP or a related purine. 3. Clinical studies of IBD patients have provided evidence of an association between IBD and axonal or demyelinating neuropathy. Assays of autonomic function suggest that ulcerative colitis and Crohn's disease, the two major forms of IBD, have contrasting effects on sympathetic neural activity. 4. Animal models of IBD have been used to examine the effects of these diseases on sympathetic neurophysiology. A decrease in the release of noradrenaline from sympathetic varicosities in inflamed and uninflamed regions of the GI tract has consistently been reported. Recent findings suggest that the decrease in neurotransmitter release may be due to inhibition of N-type voltage-gated Ca(2+) current in post-ganglionic sympathetic neurons. 5. Interest in the role of the SNS in IBD is rapidly increasing. However, much work needs to be done to enhance understanding of how SNS function is altered during IBD and what contribution, if any, these changes make to pathogenesis.

Inflammation has profound effects on the innervation of affected tissues, including altered neuronal excitability and neurotransmitter release. As Ca(2+) influx through voltage-gated Ca(2+) channels (VGCCs) is a critical determinant of excitation-secretion coupling in nerve terminals, the aim of this study was to characterize the effect of overnight incubation in the inflammatory mediator tumour necrosis factor alpha (TNFalpha; 1 nM) on VGCCs in dissociated neurons from mouse superior mesenteric ganglia (SMG). Voltage-gated Ca(2+) currents (I(Ca)) were measured using the perforated patch clamp technique and the VGCC subtypes present in SMG neurons were estimated based on inhibition by selective VGCC blockers: omega-conotoxin GVIA (300 nM; N-type), nifedipine (10 microM; L-type), and omega-conotoxin MVIIC (300 nM; N-, P/Q-type). We used intracellular Ca(2+) imaging with Fura-2 AM to compare Ca(2+) influx during depolarizations in control and TNFalpha-treated neurons. TNF receptor and VGCC mRNA expression were measured using PCR, and channel alpha subunit (CaV2.2) was localized with immunohistochemistry. Incubation in TNFalpha significantly decreased I(Ca) amplitude and depolarization-induced Ca(2+) influx. The reduction in I(Ca) was limited to omega-conotoxin GVIA-sensitive N-type Ca(2+) channels. Depletion of glial cells by incubation in cytosine arabinoside (5 microM) did not affect I(Ca) inhibition by TNFalpha. Preincubation of neurons with SC-514 (20 microM) or BAY 11-7082 (1 microM), which both inhibit nuclear factor kappaB signalling, prevented the reduction in I(Ca) by TNFalpha. Inhibition of N-type VGCCs following TNFalpha incubation was associated with a decrease in CaV2.2 mRNA and reduced membrane localization of CaV2.2 immunoreactivity. These data suggest that TNFalpha inhibits I(Ca) in SMG neurons and identify a novel role for NF-kappaB in the regulation of neurotransmitter release during inflammatory conditions with elevated circulating TNFalpha, such as Crohn's disease and Guillain-Barré syndrome.

Inflammatory bowel diseases (IBD) are associated with altered neuronal regulation of the gastrointestinal (GI) tract and impairment of norepinephrine release from sympathetic varicosities. The sympathetic innervation of the GI tract modulates motility, blood flow, and secretion, and therefore defective norepinephrine release may contribute to symptom generation in IBD. Accordingly, our aim here was to utilize the mouse model of dextran sulfate sodium (DSS; 5% wt/vol) of IBD to determine how norepinephrine release is reduced during GI inflammation. We hypothesized that the inflammation-induced reduction in norepinephrine release was due to inhibition of voltage-gated Ca(2+) current (I(Ca)) in prevertebral sympathetic neurons. We compared [(3)H]norepinephrine release in the colon and jejunum and I(Ca) amplitude in superior mesenteric ganglion (SMG) neurons from control mice and mice with DSS-induced colitis. Changes to voltage-gated Ca(2+) channels were investigated by fura 2-AM Ca(2+) imaging, perforated patch-clamp electrophysiology, and real-time PCR. Depolarization-induced norepinephrine release from the inflamed colon and uninflamed jejunum was significantly impaired in mice treated with DSS, as was depolarization-induced Ca(2+) influx in SMG neurons. Colitis reduced I(Ca) in SMG neurons by inhibiting omega-conotoxin GVIA (300 nM)-sensitive N-type Ca(2+) channels. The omega-conotoxin GVIA-sensitive component of norepinephrine release was significantly smaller in the colon during colitis. The inhibition of I(Ca) was accompanied by a decrease in mRNA encoding the Ca(2+) channel alpha subunit (CaV 2.2) and a rightward shift in the voltage dependence of activation of I(Ca). These findings suggest that DSS-induced colitis attenuates norepinephrine release in the colon and jejunum due to inhibition of N-type voltage-gated Ca(2+) channels. This may contribute to functional alterations in both inflamed and uninflamed regions of the GI tract during inflammation.

Evidence from patients with inflammatory bowel disease (IBD) and animal models suggests that inflammation alters blood flow to the mucosa, which precipitates mucosal barrier dysfunction. Impaired purinergic sympathetic regulation of submucosal arterioles, the resistance vessels of the splanchnic vasculature, is one of the defects identified during IBD and in mouse models of IBD. We hypothesized that this may be a consequence of upregulated catabolism of ATP during colitis. In vivo and in vitro video microscopy techniques were employed to measure the effects of purinergic agonists and inhibitors of CD39, an enzyme responsible for extracellular ATP catabolism, on the diameter of colonic submucosal arterioles from control mice and mice with dextran sodium sulfate [DSS, 5% (wt/vol)] colitis. Using a luciferase-based ATP assay, we examined the degradation of ATP and utilized real-time PCR, Western blotting, and immunohistochemistry to examine the expression and localization of CD39 during colitis. Arterioles from mice with DSS colitis did not constrict in response to ATP (10 microM) but did constrict in the presence of its nonhydrolyzable analog alpha,beta-methylene ATP (1 microM). alpha,beta-Methylene ADP (100 microM), an inhibitor of CD39, restored ATP-induced vasoconstriction in arterioles from mice with DSS-induced colitis. CD39 protein and mRNA expression was markedly increased during colitis. Immunohistochemical analysis demonstrated that, in addition to vascular CD39, F4/80-immunoreactive macrophages accounted for a large proportion of submucosal CD39 staining during colitis. These data implicate upregulation of CD39 in impaired sympathetic regulation of gastrointestinal blood flow during colitis.

Recent studies suggest that altered neural regulation of the gastrointestinal microvasculature contributes to the pathogenesis of inflammatory bowel disease. Therefore, we employed video microscopy techniques to monitor nerve-evoked vasoconstrictor responses in mouse colonic submucosal arterioles in vitro and examined the effect of 2,4,6-trinitrobenzene sulphonic acid (TNBS) colitis. Nerve stimulation (2-20 Hz) caused frequency-dependent vasoconstrictor responses that were abolished by tetrodotoxin (300 nm) and guanethidine (10 microm). The P2 receptor antagonist suramin (100 microm) or the alpha(1)-adrenoceptor antagonist prazosin (100 nm) reduced the vasoconstriction and the combination of suramin and prazosin completely abolished responses. Nerve-evoked constrictions of submucosal arterioles from mice with TNBS colitis were inhibited by prazosin but not suramin. Superfusion of ATP (10 microm) resulted in large vasoconstrictions in control mice but had no effect in mice with colitis whereas constrictions to phenylephrine (3 microm) were unaffected. P2X(1) receptor immunohistochemistry did not suggest any alteration in receptor expression following colitis. However, Western blotting revealed that submucosal P2X(1) receptor expression was increased during colitis. In contrast to ATP, alphabeta-methylene-ATP (1 microm), which is resistant to catabolism by nucleotidases, constricted control and TNBS arterioles. This indicates that reduced purinergic transmission to submucosal arterioles may be due to increased degradation of ATP during colitis. These data comprise the first description of the neural regulation of mouse submucosal arterioles and identify a defect in sympathetic regulation of the GI vasculature during colitis due to reduced purinergic neurotransmission.

Functional changes induced by inflammation persist following recovery from the inflammatory response, but the mechanisms underlying these changes are not well understood. Our aim was to investigate whether the excitability and synaptic properties of submucosal neurons remained altered 8 wk post-trinitrobenzene sulfonic acid (TNBS) treatment and to determine whether these changes were accompanied by alterations in secretory function in submucosal preparations voltage clamped in Ussing chambers. Mucosal serotonin (5-HT) release measurements and 5-HT reuptake transporter (SERT) immunohistochemistry were also performed. Eight weeks after TNBS treatment, colonic inflammation resolved, as assessed macroscopically and by myeloperoxidase assay. However, fast excitatory postsynaptic potential (fEPSP) amplitude was significantly increased in submucosal S neurons from previously inflamed colons relative to those in control tissue. In addition, fEPSPs from previously inflamed colons had a hexamethonium-insensitive component that was not evident in age-matched controls. AH neurons were hyperexcitable, had shorter action potential durations, and decreased afterhyperpolarization 8 wk following TNBS adminstration. Neuronally mediated colonic secretory function was significantly reduced after TNBS treatment, although epithelial cell signaling, as measured by responsiveness to both forskolin and bethanecol in the presence of tetrodotoxin, was comparable with control tissue. 5-HT levels and SERT immunoreactivity were comparable to controls 8 wk after the induction of inflammation, but there was an increase in glucagon-like peptide 2-immunoreactive L cells. In conclusion, sustained alterations in enteric neural signaling occur following the resolution of colitis, which are accompanied by functional changes in the absence of active inflammation.

Background and Aims: Intestinal inflammation alters neuronal and enteroendocrine signalling, leading to functional adaptations in the inflamed bowel. Human studies have reported functional alterations at sites distant from active inflammation. Our aims were to determine whether neuronal and enteroendocrine signalling are altered in the uninflamed colon during ileitis., Methods: We used neurophysiological, immunohistochemical, biochemical and Ussing chamber techniques to examine the effect of 2,4,6-trinitrobenzene sulphonic acid (TNBS)-induced ileitis on the properties of submucosal neurones, enteroendocrine cells and epithelial physiology of the distal colon of guinea pigs., Results: Three days after TNBS administration, when inflammation was restricted to the ileum, the properties of colonic enteric neurones were altered. Submucosal AH neurones were hyperexcitable and had reduced after hyperpolarisations. S neurones received larger fast and slow excitatory postsynaptic potentials, due to an increase in non-cholinergic synaptic transmission. Despite the absence of inflammation in the colon, we found increased colonic prostaglandin E(2) content in animals with ileitis. Ileitis also increased the number of colonic 5-hydroxytryptamine (5-HT)- and GLP-2-immunoreactive enteroendocrine cells. This was accompanied by an increase in stimulated 5-HT release. Functional alterations in epithelial physiology occurred such that basal short circuit current was increased and veratridine-stimulated ion transport was reduced in the colon of animals with ileitis., Conclusion: Our data suggest that inflammation at one site in the gut alters the cellular components of enteric reflex circuits in non-inflamed regions in ways similar to those at sites of active inflammation. These changes underlie altered function in non-involved regions during episodes of intestinal inflammation.

Inflammation of the gastrointestinal (GI) tract has pronounced effects on GI function. Many of the functions of the GI tract are subject to neural regulation by the enteric nervous system (ENS) and its extrinsic connections. Therefore, it is possible that inflammatory effects on the ENS contribute to altered function during GI inflammation. The reflex circuitry of the ENS is comprised of sensory transducers in the mucosa (enteroendocrine cells), afferent neurons, interneurons and motor neurons. This review focuses on recent data that describe inflammation-induced changes to the ENS and mucosal enteroendocrine cells. Studies of tissues from patients with inflammatory bowel disease (IBD) and from animal models of IBD have demonstrated marked changes in mucosal enteroendocrine cell signaling. These changes, which have been studied most intensely in 5-HT-containing enterochromaffin cells, involve changes in the number of cells, their signaling molecule content or their means of signal termination. Morphological evidence of enteric neuropathy during inflammation has been obtained from human samples and animal models of IBD. The neuropathy can reduce the number of enteric neurons in the inflamed region and is often accompanied by a change in the neurochemical coding of enteric neurons, both in the inflamed region and at distant sites. Electrophysiological recordings have been made from enteric neurons in inflamed regions of the colon of animal models of IBD. These studies have consistently found that inflammation increases excitability of intrinsic primary afferent neurons and alters synaptic transmission to interneurons and motor neurons. These data set the stage for a comprehensive examination of the role of altered neuronal and enteroendocrine cell signaling in symptom generation during GI inflammation.

Intestinal secretion is regulated by submucosal neurones of the enteric nervous system. Inflammation of the intestines leads to aberrant secretory activity; therefore we hypothesized that the synaptic and electrical behaviours of submucosal neurones are altered during colitis. To test this hypothesis, we used intracellular microelectrode recording to compare the excitability and synaptic properties of submucosal neurones from normal and trinitrobenzene sulphonic acid (TNBS)-inflamed guinea-pig colons. Inflammation differentially affected the electrophysiological characteristics of the two functional classes of submucosal neurones. AH neurones from inflamed colons were more excitable, had shorter action potential durations and reduced afterhyperpolarizations. Stimulus-evoked fast and slow excitatory postsynaptic potentials (EPSPs) in S neurones were larger during colitis, and the incidence of spontaneous fast EPSPs was increased. In control preparations, fast EPSPs were almost completely blocked by the nicotinic receptor antagonist hexamethonium, whereas fast EPSPs in inflamed S neurones were only partially inhibited by hexamethonium. In inflamed tissues, components of the fast EPSP in S neurones were sensitive to blockade of P2(X) and 5-HT(3) receptors while these antagonists had little effect in control preparations. Control and inflamed S neurones were equally sensitive to brief application of acetylcholine, ATP and 5-HT, suggesting that synaptic facilitation was due to a presynaptic mechanism. Immunoreactivity for 5-HT in the submucosal plexus was unchanged by inflammation; this indicates that altered synaptic transmission was not due to anatomical remodelling of submucosal nerve terminals. This is the first demonstration of alterations in synaptic pharmacology in the enteric nervous system during inflammation.

Inflammation of the bowel causes structural and functional changes to the enteric nervous system (ENS). While morphological alterations to the ENS are evident in some inflammatory conditions, it appears that relatively subtle modifications to the neurophysiology of enteric microcircuits may play a role in gastrointestinal (GI) dysfunction. These include changes to the excitability and synaptic properties of enteric neurones. The response of the ENS to inflammation varies according to the site and type of inflammation, with the functional consequences depending on the nature of the inflammatory stimulus. It has become clear that inflammation at one site can produce changes that occur at remotes sites in the GI tract. Immunohistochemical data from patients with inflammatory bowel disease (IBD) and animal models indicate that inflammation alters the neurochemical content of some functional classes of enteric neurones. A growing body of evidence supports an active role for enteric glia in neuronal and neuroimmune communication in the GI tract, particularly during inflammation. In conclusion, plasticity of the ENS is a feature of intestinal inflammation. Elucidation of the mechanisms whereby inflammation alters enteric neural control of GI functions may lead to novel treatments for IBD.

An imaging system for di-4-ANEPPS (4-[beta-[2-(di-n-butylamino)-6-naphthylvinyl]pyridinium]) voltage-sensitive dye recordings has been adapted for recording from an in vitro mouse heart preparation that consists of both atria in isolation. This approach has been used to study inter- and intra-atrial activation and conduction and to monitor action potential durations (APDs) in the left and right atrium. The findings from this study confirm some of our previous findings in isolated mouse atrial myocytes and demonstrate that many electrophysiological properties of mouse atria closely resemble those of larger mammals. Specifically, we made the following observations: 1) Activation in mouse atria originates in the sinoatrial node and spreads into the right atrium and, after a delay, into the left atrium. 2) APD in the left atrium is shorter than in the right atrium. 3) Sites in the posterior walls have longer APDs than sites in the atrial appendages. 4) Superfusion of this preparation with 4-aminopyridine and tetraethylammonium resulted in increases in APD, consistent with their inhibitory effects on the K+ currents known to be expressed in mouse atria. 5) The muscarinic agonist carbachol shortened APD in all areas of the preparation, except the left atrial appendage, in which carbachol had no statistically significant effect on APD. These results validate a new approach for monitoring activation, conduction, and repolarization in mouse atria and demonstrate that the physiological and pharmacological properties of mouse atria are sufficiently similar to those of larger animals to warrant further studies using this preparation.

The effects of C-type natriuretic peptide (CNP) on heart rate and ionic currents were demonstrated by recording the ECG from adult mice and performing voltage-clamp experiments on single sinoatrial (SA) node cells isolated from mouse heart. The selective natriuretic peptide type C receptor (NPR-C) agonist cANF (10(-7) M) significantly decreased heart rate in the presence of isoproterenol (5 x 10(-9) M), as indicated by an increase in the R-R interval of ECGs obtained from Langendorff-perfused hearts. Voltage-clamp measurements in enzymatically isolated single pacemaker myocytes revealed that CNP (10(-8) M) and cANF (10(-8) M) significantly inhibited L-type Ca2+ current [ICa(L)]. These findings suggest that the CNP effect on this current is mediated by NPR-C. Further support for an NPR-C-mediated inhibition of ICa(L) in SA node myocytes was obtained by altering the functional coupling between the G protein Gi and NPR-C. In these experiments, a "Gi-activator peptide," which consists of a 17-amino acid segment of NPR-C containing a specific Gi protein-activator sequence, was dialyzed into SA node myocytes. This peptide decreased ICa(L) significantly, suggesting that NPR-C activation can result in a reduction in ICa(L) when CNP is bound and the Gi protein pathway is activated. This effect of CNP appears to be selective for ICa(L), because the hyperpolarization-activated current was unaffected by CNP or cANF. These results provide the first demonstration that CNP has a negative chronotropic effect on heart rate and suggest that this effect is mediated by selectively activating NPR-C and reducing ICa(L) through coupling to Gi protein.

The increasing availability of murine models of the cardiovascular system has created a need for instrumentation and methods for assessing murine cardiovascular function. We have adapted an existing optical mapping system based on voltage-sensitive dyes to record from an isolated mouse atrial preparation. Initial results indicate that our approach is capable of recording action potentials from isolated mouse atria with sufficient signal quality to determine action potential duration (APD). Preliminary observations suggest that gradients in APD exist in the mouse atria and are similar to those observed in the atria of larger mammals. Future work with this technique will provide important information about mouse atrial electrophysiology and how it relates to that of larger mammals.

Single atrial myocytes were isolated from the bullfrog heart and studied under current and voltage clamp conditions to determine the electrophysiological effects of the C-type natriuretic peptide (CNP). CNP (10(-8) M) significantly shortened the action potential and reduced its peak amplitude after the application of isoproteronol (10(-7) M). In voltage clamp studies, CNP inhibited isoproteronol-stimulated L-type Ca2+ current (ICa) without any significant effect on the inward rectifier K+ current. The effects of cANF (10(-8) M), a selective agonist of the natriuretic peptide C receptor (NPR-C), were very similar to those of CNP. Moreover, HS-142-1, an antagonist of the guanylyl cyclase-linked NPR-A and NPR-B receptors did not alter the inhibitory effect of CNP on ICa. Inclusion of cAMP in the recording pipette to stimulate ICa at a point downstream from adenylyl cyclase increased ICa, but this effect was not inhibited by cANF. These results provide the first demonstration that CNP can inhibit ICa after binding to NPR-C, and suggest that this inhibition involves a decrease in adenylyl cyclase activity, which leads to reduced intracellular levels of cAMP.

Consistent differences in K+ currents in left and right atria of adult mouse hearts have been identified by the application of current- and voltage-clamp protocols to isolated single myocytes. Left atrial myocytes had a significantly (P < 0.05) larger peak outward K+ current density than myocytes from the right atrium. Detailed analysis revealed that this difference was due to the rapidly activating sustained K+ current, which is inhibited by 100 muM 4-aminopyridine (4-AP); this current was almost three times larger in the left atrium than in the right atrium. Accordingly, 100 muM 4-AP caused a significantly (P < 0.05) larger increase in action potential duration in left than in right atrial myocytes. Inward rectifier K+ current density was also significantly (P < 0.05) larger in left atrial myocytes. There was no difference in the voltage-dependent L-type Ca2+ current between left and right atria. As expected from this voltage-clamp data, the duration of action potentials recorded from single myocytes was significantly (P < 0.05) shorter in myocytes from left atria, and left atrial tissue was found to have a significantly (P < 0.05) shorter effective refractory period than right atrial tissue. These results reveal similarities between mice and other mammalian species where the left atrium repolarizes more quickly than the right, and provide new insight into cellular electrophysiological mechanisms responsible for this difference. These findings, and previous results, suggest that the atria of adult mice may be a suitable model for detailed studies of atrial electrophysiology and pharmacology under control conditions and in the context of induced atrial rhythm disturbances.

Whole cell current and voltage clamp techniques were used to examine the properties of acetylcholine-sensitive K+ current (IKACh) in myocytes from adult mouse atrium. Superfusion of a maximal dose of carbachol (CCh; 10 microM) caused a substantial increase in K+ current in all myocytes examined. The current-voltage (I-V) relation of maximally activated IKACh exhibited weak inward rectification. Consequently, CCh increased the amount of depolarising current necessary to evoke action potentials (APs), and APs evoked in CCh had significantly shorter durations than control APs (P<0.05). The effects of CCh on K+ current and on AP properties were blocked by the muscarinic receptor antagonist methoctramine (1 microM). ACh (10 microM) activated a K+ current with identical properties to that activated by CCh, as did the A1 receptor agonist adenosine (100 microM). Right atrial myocytes had significantly more IKACh than left atrial myocytes (P<0.05), regardless of whether IKACh was evoked by superfusion of muscarinic or A1 receptor agonists. IKACh current density was significantly higher in SA node myocytes than either right or left atrial myocytes. These data identify a gradient of IKACh current density across the supraventricular structures of mouse heart. This gradient, combined with the heterogeneous distribution of parasympathetic innervation of the atria, may contribute to the proarrhythmic ability of vagal nerve stimulation to augment dispersion of atrial refractoriness.

Intracellular recordings were made from neurons in the submucosal ganglia of the guinea-pig distal colon. The recording electrode contained the intracellular marker biocytin, which was injected into neurons so that their electrophysiological characteristics could be correlated with their shape. Correlations of electrophysiology and shape have not been reported previously for neurons in this region. Three types of neuron were identified on electrophysiological grounds. Neurons of the first type (S neurons) had tetrodotoxin-sensitive soma action potentials, and received fast and slow excitatory synaptic inputs. They had uniaxonal morphologies and may function as secretomotor or possibly vasomotor neurons. The second type (AH neurons) received only slow synaptic input, while the soma action potential had tetrodotoxin-sensitive and -insensitive components with a shoulder on the falling phase and a long-lasting afterhyperpolarisation of the membrane potential following a single action potential. Neurons of this type had multipolar morphologies and provided dense innervation of adjacent submucosal ganglia. These neurons are similar to the submucosal intrinsic primary afferent neurons of the guinea-pig small intestine. The final type of neuron [the low-threshold (LT) neuron] had electrophysiological characteristics that set it apart from those described previously within enteric plexuses. They expressed tetrodotoxin-insensitive voltage-gated soma currents, did not have long-lasting afterhyperpolarisations and received only slow synaptic input. In addition, these neurons were very excitable: they had large input resistances and low thresholds for action potential discharge, and often fired action potentials in the absence of stimulation. Neurons with these characteristics were uniaxonal and thus are likely to be secretomotor or possibly vasomotor neurons. This study has shown that submucosal neurons of the distal colon fall into three distinct types, which can be distinguished by a combination of electrophysiological and morphological criteria.

Previous studies have identified the chemistries, shapes, projections and electrophysiological characteristics of several populations of neurons in the distal colon of the guinea-pig but it is unknown how these characteristics correlate to define the classes of neurons present. We have used double-label immunohistochemical techniques to identify neurochemically distinct subgroups of enteric neurons in this region. On the basis of colocalisation of neurochemical markers and knowledge gained from previous studies of neural projections, 17 classes of neurons were identified. The myenteric plexus contained the cell bodies of 13 distinct types of neurons. Four classes of descending interneurons and three classes of ascending interneurons were identified, together with inhibitory and excitatory motor neurons to both the circular and longitudinal muscle layers. Dogiel type II neurons, which are presumed to be intrinsic primary afferent neurons, were located in myenteric and submucosal ganglia; they were all immunoreactive for choline acetyltransferase and often calbindin and tachykinins. Three classes of secretomotor neurons with cell bodies in submucosal ganglia were defined. Two of these classes were immunoreactive for choline acetyltransferase and the other class was immunoreactive for both vasoactive intestinal peptide and nitric oxide synthase. Some of the secretomotor neurons probably also have a vasomotor function. The neural subtypes defined in the present study are similar in many respects to those found in the small intestine, although differences are evident, especially in populations of interneurons. These differences presumably reflect the differing physiological roles of the two intestinal regions.

The axons of neurons that innervate the longitudinal muscle of the small intestine in small mammals such as rabbit, rat, guinea pig and mouse form a network, the tertiary plexus, against the inner surface of the muscle. In general, because of their substantial overlap, it has not been possible to follow the ramifications of individual axons in the tertiary plexus. In the present work, the longitudinal muscle motor neurons were filled with marker dyes through an intracellular microelectrode, and their morphologies and projections were examined in whole-mount preparations of longitudinal muscle and myenteric plexus. Most neurons that were examined were in the small intestine (ileum and duodenum), but a few were examined in the distal colon. Neurons in all regions had similar morphologies and projections. The cell bodies were amongst the smallest in myenteric ganglia, with major and minor axes of 14 microns and 25 microns (mean, n = 40) in the plane of the myenteric plexus. Each neuron had a single axon that branched extensively in the tertiary plexus, most had multiple lamellar dendrites and a few had filamentous dendrites or a mixture of filamentous and lamellar dendrites. The mean area of muscle covered by an axon and its branches extended 1.6 mm orally to anally and 1.7 mm circumferentially. The area covered was 2.8 +/- 1.9 mm2 (mean +/- SD, n = 23). From the density of occurrence of cell bodies, it can be calculated that each point in the longitudinal muscle is innervated by the processes of about 100 motor neurons and is influenced by electrotonic conduction of signals through the muscle by about 300 motor neurons.

Integration of function between gut regions is mediated by means of hormones and long neuronal reflex pathways. Intestinofugal neurons, which participate in one of these pathways, have cell bodies within the myenteric plexus and project their axons from the gut with the mesenteric nerves. They form excitatory synapses on neurons in prevertebral ganglia that in turn innervate other gut regions. The aim of the present study was to characterise immunohistochemically the synaptic input to intestinofugal neurons. The cell bodies of intestinofugal neurons that project from the distal colon were labelled with Fast Blue that was injected into the inferior mesenteric ganglia. Varicosities surrounding Fast Blue-labelled neurons were analysed for immunoreactivity for the vesicular acetylcholine transporter, vasoactive intestinal peptide, and bombesin. Most intestinofugal neurons were surrounded by nerve terminals immunoreactive for the vesicular acetylcholine transporter; many of these terminals also contained vasoactive intestinal peptide and bombesin immunoreactivity. This combination of markers occurs in axons of descending interneurons. Extrinsic denervation had no effect on the distribution of cholinergic terminals around intestinofugal neurons. A decrease in the number of vesicular acetylcholine transporter and vasoactive intestinal peptide immunoreactive terminals occurred around nerve cells immediately anal, but not oral, to myotomy operations. Consistent with previous physiological studies, it is concluded that intestinofugal neurons receive cholinergic synaptic input from other myenteric neurons, including cholinergic descending interneurons. Thus, intestinofugal neurons are second, or higher, order neurons in reflex pathways, although physiological data indicate that they also respond directly to distension of the gut wall.

Intracellular recordings were made from myenteric neurons of the guinea-pig distal colon to determine their electrical behaviour in response to intracellular current injection and stimulation of synaptic inputs. The recording microelectrode contained the intracellular marker biocytin, which was injected into impaled neurons so that electrophysiology, shape and immunohistochemistry could be correlated. Myenteric neurons in the distal colon were divided into four morphological groups based on their shapes and projections. One group (29 of the 78 that were characterized electrophysiologically, morphologically and immunohistochemically) was the multiaxonal Dogiel type II neurons, the majority (25/29) of which were calbindin immunoreactive. Each of these neurons had an inflection on the falling phase of the action potential that, in 24/29 neurons, was followed by a late afterhyperpolarizing potential (AHP). Slow excitatory postsynaptic potentials were recorded in 20 of 29 Dogiel type II neurons in response to high frequency internodal strand stimulation and two neurons responded with slow inhibitory postsynaptic potentials. Low amplitude fast excitatory postsynaptic potentials occurred in 3 of 29 Dogiel type II neurons. Neurons of the other three groups were all uniaxonal: neurons with Dogiel type I morphology, filamentous ascending interneurons and small filamentous neurons with local projections to the longitudinal or circular muscle or to the tertiary plexus. Dogiel type I neurons were often immunoreactive for nitric oxide synthase or calretinin, as were some small filamentous neurons, while all filamentous ascending interneurons tested were calretinin immunoreactive. All uniaxonal neurons exhibited prominent fast excitatory postsynaptic potentials and did not have a late AHP following a single action potential, that is, all uniaxonal neurons displayed S type electrophysiological characteristics. However, in 6/19 Dogiel type I neurons and 2/8 filamentous ascending interneurons, a prolonged hyperpolarizing potential ensued when more than one action potential was evoked. Slow depolarizing postsynaptic potentials were observed in 20/29 Dogiel type I neurons, 6/8 filamentous ascending interneurons and 8/12 small filamentous neurons. Six of 29 Dogiel type I neurons displayed slow inhibitory postsynaptic potentials, as did 2/8 filamentous ascending interneurons and 4/12 small filamentous neurons. These results indicate that myenteric neurons in the distal colon of the guinea-pig are electrophysiologically similar to myenteric neurons in the ileum, duodenum and proximal colon. Also, the correlation of AH electrophysiological characteristics with Dogiel type II morphology and S electrophysiological characteristics with uniaxonal morphology is preserved in this region. However, filamentous ascending interneurons have not been encountered in other regions of the gastrointestinal tract and there are differences between the synaptic properties of neurons in this region compared to other regions studied, including the presence of slow depolarizing postsynaptic potentials that appear to involve conductance increases and frequent slow inhibitory postsynaptic potentials.

The embryonic gastrointestinal tract develops from a simple tube into a coiled, flexed, and regionalized structure. The changes in gut morphology coincide with the differentiation of multiple cell types in concentric layers, and include colonization by migratory neuron precursors, and the development of gastrointestinal motility. We describe a reliable method for growing embryonic mouse intestine in vitro by the attachment of segments of intestinal tract by their cut ends, with the intervening region suspended in the culture medium. These are termed "catenary cultures." E11-E11.5 mouse midgut, hindgut, or mid- plus hindgut segments were grown in catenary culture for up to 10 days and their growth, morphology, cell differentiation, ability to support neural precursor migration, and contractile activity were assessed. The increase in size of the cultured explants was not large, but morphogenesis proceeded, best exemplified by elongation of the caecum. Cell differentiation also proceeded. In the mucosa, goblet cells differentiated. Muscle layers, characterized by desmin expression, and kit-positive interstitial cells of Cajal differentiated in the correct positions. Where segments initially included neural precursors in a small sub-region, these migrated and proliferated to form uniform neuronal networks throughout the entire explant, and the cells expressed the neuron markers nitric oxide synthase and neuron specific enolase. Gut motility was attained 5-6 days into the culture period, and both contractile- and mixing-type movements were observed. Thus, cell types representative of all three germ layer contributions developed, and in addition, the gut, being mainly free, was able to elongate and bend (unlike on solid support cultures), while retaining its rostrocaudal identity.

Simultaneous immunofluorescence labelling was used to investigate the patterns of colocalisation of the NK1 tachykinin receptor with other neuronal markers, and hence determine the functional classes of neuron that bear the NK1 receptor in the guinea-pig ileum. In the myenteric plexus, 85% of NK1 receptor-immunoreactive (NK1r-IR) nerve cells had nitric oxide synthase (NOS) immunoreactivity and the remaining 15% were immunoreactive for choline acetyltransferase (ChAT). Of the latter group, about 50% were immunoreactive for both neuropeptide Y (NPY) and somatostatin (SOM), and had the morphologies of secretomotor neurons. Many of the remaining ChAT neurons were immunoreactive for calbindin or tachykinins (TK), but not both. These calbindin immunoreactive neurons had Dogiel type II morphology. No NK1r-IR nerve cells in the myenteric plexus had serotonin or calretinin immunoreactivity. In the submucosal ganglia, 84% of NK1r-IR nerve cells had neuropeptide Y immunoreactivity and 16% were immunoreactive for TK. It is concluded that NK1r-IR occurs in five classes of neuron; namely, in the majority of NOS-immunoreactive inhibitory motor neurons, in ChAT/TK-immunoreactive excitatory neurons to the circular muscle, in all ChAT/NPY/SOM-immunoreactive secretomotor neurons, in a small proportion of ChAT/calbindin myenteric neurons, and in about 50% of ChAT/TK submucosal neurons.

Background & Aims: Prevertebral sympathetic ganglia receive inputs from intestinofugal neurons, with cell bodies located in the wall of the bowel. Intestinofugal neurons are part of the afferent limbs of intestino-intestinal reflexes. The aim of this study was to define the properties of intestinofugal neurons using intracellular recordings., Methods: Intestinofugal neurons of the distal colon were retrogradely labeled from the inferior mesenteric ganglia. In whole mounts of the myenteric plexus/longitudinal muscle of the distal colon, labeled neurons were identified by their fluorescence and recordings were made using biocytin-filled electrodes. Labeled nerves were characterized immunohistochemically and morphologically., Results: Intestinofugal neurons were uniaxonal neurons with multiple dendrites that had lamellar expansions. They were immunoreactive for choline acetyltransferase. Stimulation of nerve fiber tracts elicited large-amplitude excitatory postsynaptic potentials in all labeled neurons. Some received spontaneous fast excitatory postsynaptic potentials. Those cells that fired action potentials fired only one or two at the start of a depolarizing current pulse. No intestinofugal neurons had Dogiel type II morphology or a late afterhyperpolarizing potential., Conclusions: Intestinofugal neurons are likely to be activated by other neurons in the gut wall. They are not AH or Dogiel type II neurons. Thus they seem to be second order neurons in afferent pathways of intestino-intestinal reflexes.

The structural relationships between interstitial cells of Cajal (ICC), varicose nerve fibers, and smooth muscle cells in the gastrointestinal tract have led to the suggestion that ICC may be involved in or mediate enteric neurotransmission. We characterized the distribution of ICC in the murine stomach and found two distinct classes on the basis of morphology and immunoreactivity to antibodies against c-Kit receptors. ICC with multiple processes formed a network in the myenteric plexus region from corpus to pylorus. Spindle-shaped ICC were found within the circular and longitudinal muscle layers (IC-IM) throughout the stomach. The density of these cells was greatest in the proximal stomach. IC-IM ran along nerve fibers and were closely associated with nerve terminals and adjacent smooth muscle cells. IC-IM failed to develop in mice with mutations in c-kit. Therefore, we used W/W(V) mutants to test whether IC-IM mediate neural inputs in muscles of the gastric fundus. The distribution of inhibitory nerves in the stomachs of c-kit mutants was normal, but NO-dependent inhibitory neuro-regulation was greatly reduced. Smooth muscle tissues of W/W(V) mutants relaxed in response to exogenous sodium nitroprusside, but the membrane potential effects of sodium nitroprusside were attenuated. These data suggest that IC-IM play a critical serial role in NO-dependent neurotransmission: the cellular mechanism(s) responsible for transducing NO into electrical responses may be expressed in IC-IM. Loss of these cells causes loss of electrical responsiveness and greatly reduces responses to nitrergic nerve stimulation.