A role for interleukin 17A in IBD-related neuroplasticity.

BACKGROUND: Changes to the structure and function of the innervation of the gut contribute to symptom generation in inflammatory bowel diseases (IBD). However, delineation of the mechanisms of these effects has proven difficult. Previous work on sympathetic neurons identified interleukin (IL)-17A as a novel neurotrophic cytokine. Since IL-17A is involved in IBD pathogenesis, we tested the hypothesis that IL-17A contributes to neuroanatomical remodeling during IBD. METHODS: Immunohistochemistry for tyrosine hydroxylase was used to identify sympathetic axons in mice with dextran sulphate sodium (DSS)-induced colitis and controls. Axon outgrowth from sympathetic neurons in response to incubation in cytokines or endoscopic patient biopsy supernatants was quantified. KEY RESULTS: DSS-induced colitis led to an increase in tyrosine hydroxylase immunoreactivity in the inflamed colon but not the spleen. Colonic supernatants from mice with colitis and biopsy supernatants from Crohn's disease patients increased axon outgrowth from mouse sympathetic neurons compared to supernatants from uninflamed controls. An antibody that neutralized IL-17A blocked the ability of DSS-induced colitis and Crohn's disease supernatants to induce axon extension. CONCLUSIONS AND INFERENCES: These findings identify IL-17A as a potential mediator of neuroanatomical remodeling of the gut innervation during IBD.

Ghrelin receptors as targets for novel motility drugs.

Constipation arises from a multitude of causes, including aging, spinal cord injury (SCI), and dietary issues. The heterogeneity of inciting factors has made the treatment of constipation particularly challenging. Agonists of ghrelin receptors have beneficial effects on delayed gastric emptying, but less is known about their ability to improve colorectal motility. Recent publications indicate that the activation of the ghrelin receptors in the spinal cord can alleviate constipation due to dietary causes, Parkinsonism, and SCI in rodents. Ghrelin-responsive neurons in the intermediolateral cell column of the lumbosacral spinal cord can activate enteric microcircuits that coordinate propulsive colorectal contractions, leading to defecation. Learning more about the properties of neurons in the spinal defecation center and the roles of ghrelin receptors in the defecation reflex will accelerate the development of improved treatments of constipation.

Mouse models of sepsis elicit spontaneous action potential discharge and enhance intracellular Ca2+ signaling in postganglionic sympathetic neurons.

Sepsis is a severe systemic inflammatory disorder that rapidly activates the sympathetic nervous system to enhance catecholamine secretion from postganglionic sympathetic neurons and adrenal chromaffin cells. Although an increase in preganglionic drive to postganglionic sympathetic tissues has been known to contribute to this response for quite some time, only recently was it determined that sepsis also has direct effects on adrenal chromaffin cell Ca2+ signaling and epinephrine release. In the present study, we characterized the direct effects of sepsis on postganglionic sympathetic neuron function. Using the endotoxemia model of sepsis in mice, we found that almost a quarter of postganglionic neurons acquired the ability to fire spontaneous action potentials, which was absent in cells from control mice. Spontaneously firing neurons possessed significantly lower rheobases and fired a greater number of action potentials at twice the rheobase compared to neurons from control mice. Sepsis did not significantly affect voltage-gated Ca2+ currents. However, global Ca2+ signaling was enhanced in postganglionic neurons isolated from 1 to 24 h endotoxemic mice. A similar increase in the amplitude of high-K+-stimulated Ca2+ transients was observed during the cecal ligation and puncture model of sepsis. The enhanced excitability and Ca2+ signaling produced during sepsis likely amplify the effect of increased preganglionic drive on norepinephrine release from postganglionic neurons. This is important, as sympathetic neurons are integral to the anti-inflammatory autonomic reflex that is activated during sepsis.

Release of endogenous opioids during a chronic IBD model suppresses the excitability of colonic DRG neurons.

BACKGROUND: Endogenous opioids are implicated in pain-regulation in chronic inflammatory bowel disease (IBD). We sought to examine whether endogenous opioids suppress the excitability of colonic nociceptive dorsal root ganglia (DRG) neurons during chronic IBD, and if so, whether modulation of underlying voltage-gated K(+) currents was involved. METHODS: The effects of chronic dextran sulfate sodium (DSS) colitis on afferent signaling in mice was studied using patch clamp recordings. Colonic DRG neurons were identified using Fast Blue retrograde labeling and recordings obtained from small DRG neurons (<40 pF). KEY RESULTS: In current-clamp recordings, the rheobase of neurons was increased 47% (P < 0.01) and action potential discharge at twice rheobase decreased 23% (P < 0.05) following incubation in colonic supernatants from chronic DSS mice. ß-endorphin increased 14-fold, and tissue opioid immunoreactivity and expression in CD4+ cells observed by flow cytometry increased in chronic DSS colons. Incubation of naïve neurons in the µ-opioid receptor agonist D-Ala(2), N- MePhe(4), Gly-ol (DAMGO) (10 nM) partially recapitulated the effects of supernatants from DSS mice on rheobase. Supernatant effects were blocked by the µ-opioid receptor antagonist naloxone. In voltage clamp, chronic DSS supernatants and DAMGO increased I(A) K(+) currents. CONCLUSIONS & INFERENCES: The release of endogenous opioids during chronic inflammation in mice suppresses the excitability of nociceptive DRG neurons. Targeting immune cells may provide a novel means of modulating IBD pain.

Axon reflexes evoked by transient receptor potential vanilloid 1 activation are mediated by tetrodotoxin-resistant voltage-gated Na+ channels in intestinal afferent nerves.

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.

The participation of the sympathetic innervation of the gastrointestinal tract in disease states.

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.

Loss of purinergic vascular regulation in the colon during colitis is associated with upregulation of CD39.

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.

Sympathetic vasoconstrictor regulation of mouse colonic submucosal arterioles is altered in experimental 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.

Plasticity of the enteric nervous system during intestinal 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.

Optical mapping system for recording action potential durations in adult mouse left and right atrium.

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.

Inhibition of L-type Ca2+ current by C-type natriuretic peptide in bullfrog atrial myocytes: an NPR-C-mediated effect.

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.

Electrophysiological characteristics distinguish three classes of neuron in submucosal ganglia of the guinea-pig distal colon.

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.

Neurochemical classification of enteric neurons in the guinea-pig distal colon.

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.

Shapes and projections of tertiary plexus neurons of the guinea-pig small intestine.

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.

Origins of cholinergic inputs to the cell bodies of intestinofugal neurons in the guinea pig distal colon.

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.

Correlation of morphology, electrophysiology and chemistry of neurons in the myenteric plexus of the guinea-pig distal colon.

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.

Catenary cultures of embryonic gastrointestinal tract support organ morphogenesis, motility, neural crest cell migration, and cell differentiation.

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.

Electrophysiology, shape, and chemistry of neurons that project from guinea pig colon to inferior mesenteric ganglia.

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.

Identification of the populations of enteric neurons that have NK1 tachykinin receptors in the guinea-pig small intestine.

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.