Faster Gastrointestinal Transit, Reduced Small Intestinal Smooth Muscle Tone and Dysmotility in the Nlgn3R451C Mouse Model of Autism.

Individuals with autism often experience gastrointestinal issues but the cause is unknown. Many gene mutations that modify neuronal synapse function are associated with autism and therefore may impact the enteric nervous system that regulates gastrointestinal function. A missense mutation in the Nlgn3 gene encoding the cell adhesion protein Neuroligin-3 was identified in two brothers with autism who both experienced severe gastrointestinal dysfunction. Mice expressing this mutation (Nlgn3R451C mice) are a well-studied preclinical model of autism and show autism-relevant characteristics, including impaired social interaction and communication, as well as repetitive behaviour. We previously showed colonic dysmotility in response to GABAergic inhibition and increased myenteric neuronal numbers in the small intestine in Nlgn3R451C mice bred on a mixed genetic background. Here, we show that gut dysfunction is a persistent phenotype of the Nlgn3 R451C mutation in mice backcrossed onto a C57BL/6 background. We report that Nlgn3R451C mice show a 30.9% faster gastrointestinal transit (p = 0.0004) in vivo and have 6% longer small intestines (p = 0.04) compared to wild-types due to a reduction in smooth muscle tone. In Nlgn3R451C mice, we observed a decrease in resting jejunal diameter (proximal jejunum: 10.6% decrease, p = 0.02; mid: 9.8%, p = 0.04; distal: 11.5%, p = 0.009) and neurally regulated dysmotility as well as shorter durations of contractile complexes (mid: 25.6% reduction in duration, p = 0.009; distal: 30.5%, p = 0.004) in the ileum. In Nlgn3R451C mouse colons, short contractions were inhibited to a greater extent (57.2% by the GABAA antagonist, gabazine, compared to 40.6% in wild-type mice (p = 0.007). The inhibition of nitric oxide synthesis decreased the frequency of contractile complexes in the jejunum (WT p = 0.0006, Nlgn3R451C p = 0.002), but not the ileum, in both wild-type and Nlgn3R451C mice. These findings demonstrate that changes in enteric nervous system function contribute to gastrointestinal dysmotility in mice expressing the autism-associated R451C missense mutation in the Neuroligin-3 protein.

Computational simulations and Ca2+ imaging reveal that slow synaptic depolarizations (slow EPSPs) inhibit fast EPSP evoked action potentials for most of their time course in enteric neurons.

Transmission between neurons in the extensive enteric neural networks of the gut involves synaptic potentials with vastly different time courses and underlying conductances. Most enteric neurons exhibit fast excitatory post-synaptic potentials (EPSPs) lasting 20-50 ms, but many also exhibit slow EPSPs that last up to 100 s. When large enough, slow EPSPs excite action potentials at the start of the slow depolarization, but how they affect action potentials evoked by fast EPSPs is unknown. Furthermore, two other sources of synaptic depolarization probably occur in enteric circuits, activated via GABAA or GABAC receptors; how these interact with other synaptic depolarizations is also unclear. We built a compartmental model of enteric neurons incorporating realistic voltage-dependent ion channels, then simulated fast EPSPs, slow EPSPs and GABAA or GABAC ligand-gated Cl- channels to explore these interactions. Model predictions were tested by imaging Ca2+ transients in myenteric neurons ex vivo as an indicator of their activity during synaptic interactions. The model could mimic firing of myenteric neurons in mouse colon evoked by depolarizing current during intracellular recording and the fast and slow EPSPs in these neurons. Subthreshold fast EPSPs evoked spikes during the rising phase of a slow EPSP, but suprathreshold fast EPSPs could not evoke spikes later in a slow EPSP. This predicted inhibition was confirmed by Ca2+ imaging in which stimuli that evoke slow EPSPs suppressed activity evoked by fast EPSPs in many myenteric neurons. The model also predicted that synchronous activation of GABAA receptors and fast EPSPs potentiated firing evoked by the latter, while synchronous activation of GABAC receptors with fast EPSPs, potentiated firing and then suppressed it. The results reveal that so-called slow EPSPs have a biphasic effect being likely to suppress fast EPSP evoked firing over very long periods, perhaps accounting for prolonged quiescent periods seen in enteric motor patterns.

Neonatal antibiotics have long term sex-dependent effects on the enteric nervous system.

Infants and young children receive the highest exposures to antibiotics globally. Although there is building evidence that early life exposure to antibiotics increases susceptibility to various diseases including gut disorders later in life, the lasting impact of early life antibiotics on the physiology of the gut and its enteric nervous system (ENS) remains unclear. We treated neonatal mice with the antibiotic vancomycin during their first 10 postnatal days, then examined potential lasting effects of the antibiotic treatment on their colons during young adulthood (6 weeks old). We found that neonatal vancomycin treatment disrupted the gut functions of young adult female and male mice differently. Antibiotic-exposed females had significantly longer whole gut transit while antibiotic-treated males had significantly lower faecal weights compared to controls. Both male and female antibiotic-treated mice had greater percentages of faecal water content. Neonatal vancomycin treatment also had sexually dimorphic impacts on the neurochemistry and Ca2+ activity of young adult myenteric and submucosal neurons. Myenteric neurons of male mice were more disrupted than those of females, while opposing changes in submucosal neurons were seen in each sex. Neonatal vancomycin also induced sustained changes in colonic microbiota and lasting depletion of mucosal serotonin (5-HT) levels. Antibiotic impacts on microbiota and mucosal 5-HT were not sex-dependent, but we propose that the responses of the host to these changes are sex-specific. This first demonstration of long-term impacts of neonatal antibiotics on the ENS, gut microbiota and mucosal 5-HT has important implications for gut function and other physiological systems of the host. KEY POINTS: Early life exposure to antibiotics can increase susceptibility to diseases including functional gastrointestinal (GI) disorders later in life. Yet, the lasting impact of this common therapy on the gut and its enteric nervous system (ENS) remains unclear. We investigated the long-term impact of neonatal antibiotic treatment by treating mice with the antibiotic vancomycin during their neonatal period, then examining their colons during young adulthood. Adolescent female mice given neonatal vancomycin treatment had significantly longer whole gut transit times, while adolescent male and female mice treated with neonatal antibiotics had significantly wetter stools. Effects of neonatal vancomycin treatment on the neurochemistry and Ca2+ activity of myenteric and submucosal neurons were sexually dimorphic. Neonatal vancomycin also had lasting effects on the colonic microbiome and mucosal serotonin biosynthesis that were not sex-dependent. Different male and female responses to antibiotic-induced disruptions of the ENS, microbiota and mucosal serotonin biosynthesis can lead to sex-specific impacts on gut function.

Neuroinflammation as an etiological trigger for depression comorbid with inflammatory bowel disease.

Patients with inflammatory bowel disease (IBD) suffer from depression at higher rates than the general population. An etiological trigger of depressive symptoms is theorised to be inflammation within the central nervous system. It is believed that heightened intestinal inflammation and dysfunction of the enteric nervous system (ENS) contribute to impaired intestinal permeability, which facilitates the translocation of intestinal enterotoxins into the blood circulation. Consequently, these may compromise the immunological and physiological functioning of distant non-intestinal tissues such as the brain. In vivo models of colitis provide evidence of increased blood-brain barrier permeability and enhanced central nervous system (CNS) immune activity triggered by intestinal enterotoxins and blood-borne inflammatory mediators. Understanding the immunological, physiological, and structural changes associated with IBD and neuroinflammation may aid in the development of more tailored and suitable pharmaceutical treatment for IBD-associated depression.

Circadian Control of Gastrointestinal Motility.

With the earth's 24-h rotation cycle, physiological function fluctuates in both diurnal and nocturnal animals, thereby ensuring optimal functioning of the body. The main regulator of circadian rhythm is the suprachiasmatic nucleus (SCN), which is considered the main pacemaker or "central clock" of the body. Located in the anterior hypothalamus, the SCN influences the activity of other brain regions, as well as peripheral organs, through the release of melatonin and corticosteroids. The SCN can be entrained by several cues, with light being the major cue. Light information from the retina is received by the SCN via the retinohypothalamic tract. Non-photic cues such as temperature and exercise can also entrain the SCN, while feeding time can entrain the "molecular clock" contained within peripheral tissues. This enables organs such as the gastrointestinal (GI) tract to coordinate function with environmental factors, such as food availability.The GI tract, which has the main functions of receiving and digesting food, and expelling waste, also shows oscillations in its activity during the circadian cycle. While these changes are evident under normal conditions, GI function is affected when normal circadian rhythm is disrupted. Recent reviews have assessed interactions between the central clock and gut clock; as such, this review aims to focus on the presence of endogenous circadian rhythms in the GI tract, with particular focus to changes to gastrointestinal motility.

Molecular Targets to Alleviate Enteric Neuropathy and Gastrointestinal Dysfunction.

Enteric neuropathy underlies long-term gastrointestinal (GI) dysfunction associated with several pathological conditions. Our previous studies have demonstrated that structural and functional changes in the enteric nervous system (ENS) result in persistent alterations of intestinal functions long after the acute insult. These changes lead to aberrant immune response and chronic dysregulation of the epithelial barrier. Damage to the ENS is prognostic of disease progression and plays an important role in the recurrence of clinical manifestations. This suggests that the ENS is a viable therapeutic target to alleviate chronic intestinal dysfunction. Our recent studies in preclinical animal models have progressed into the development of novel therapeutic strategies for the treatment of enteric neuropathy in various chronic GI disorders. We have tested the anti-inflammatory and neuroprotective efficacy of novel compounds targeting specific molecular pathways. Ex vivo studies in human tissues freshly collected after resection surgeries provide an understanding of the molecular mechanisms involved in enteric neuropathy. In vivo treatments in animal models provide data on the efficacy and the mechanisms of actions of the novel compounds and their combinations with clinically used therapies. These novel findings provide avenues for the development of safe, cost-effective, and highly efficacious treatments of GI disorders.

Potent CCR3 Receptor Antagonist, SB328437, Suppresses Colonic Eosinophil Chemotaxis and Inflammation in the Winnie Murine Model of Spontaneous Chronic Colitis.

Eosinophils and their regulatory molecules have been associated with chronic intestinal inflammation and gastrointestinal dysfunctions; eosinophil accumulation in the gut is prominent in inflammatory bowel disease (IBD). The chemokine receptor CCR3 plays a pivotal role in local and systemic recruitment and activation of eosinophils. In this study, we targeted CCR3-ligand interactions with a potent CCR3 receptor antagonist, SB328437, to alleviate eosinophil-associated immunological responses in the Winnie model of spontaneous chronic colitis. Winnie and C57BL/6 mice were treated with SB328437 or vehicle. Clinical and histopathological parameters of chronic colitis were assessed. Flow cytometry was performed to discern changes in colonic, splenic, circulatory, and bone marrow-derived leukocytes. Changes to the serum levels of eosinophil-associated chemokines and cytokines were measured using BioPlex. Inhibition of CCR3 receptors with SB328437 attenuated disease activity and gross morphological damage to the inflamed intestines and reduced eosinophils and their regulatory molecules in the inflamed colon and circulation. SB328437 had no effect on eosinophils and their progenitor cells in the spleen and bone marrow. This study demonstrates that targeting eosinophils via the CCR3 axis has anti-inflammatory effects in the inflamed intestine, and also contributes to understanding the role of eosinophils as potential end-point targets for IBD treatment.

The enteric nervous system undergoes significant chemical and synaptic maturation during adolescence in mice.

Adolescence is a critical period of development. It is very likely that there is significant maturation of the enteric nervous system (ENS) of the gut during this stage of life, especially since there are substantial changes in factors known to influence the ENS including diet and microbiota during this time, but this remains unknown. To examine maturation of the ENS during adolescence, we performed immunohistochemistry using advanced microscopy and analytical methods to compare enteric neurons and glia of the duodenum and colon of mice taken prior to weaning with those of young adult mice. We found significant changes in the architecture of both myenteric and submucosal plexuses and surprisingly found subsets of enteric cells that co-expressed the pan-neuronal marker, Hu, and either glial markers Sox10 or S100ß, not both. About 70% and 35% of all Hu â€‹+ â€‹neurons in the submucous plexus of the young adult duodenum and colon respectively also expressed S100ß. The proportion of Hu+/Sox10 â€‹+ â€‹cells in the duodenal myenteric plexus decreased, while the proportion of Hu+/S100ß+ cells in the colonic submucosal plexus increased during adolescence. In the submucous plexus, there were significant increases in the proportions of vasoactive intestinal peptide+ and choline acetyltransferase â€‹+ â€‹secretomotor neurons, of neurofilament M (NFM)+ neurons in the colon and of calretinin â€‹+ â€‹neurons in the duodenum during adolescence. There were no age-dependent changes in the neurochemistry of various myenteric neuronal subtypes, including those immunoreactive for neuronal nitric oxide synthase (nNOS), Calbindin, Calretinin or NFM. There were significant increases in the somata sizes of Calretinin â€‹+ â€‹submucosal and myenteric neurons, and nNOS â€‹+ â€‹myenteric neurons, and these enteric neurons received significantly more synaptophysin â€‹+ â€‹contacts onto their cell bodies during adolescence. This is the first study showing that enteric neurons and glia in the gut undergo significant changes in their anatomy and chemistry during adolescence. Notably changes in synaptic contacts within the enteric circuitry strongly suggest maturation in gastrointestinal function occurs during this time.

Early life interaction between the microbiota and the enteric nervous system.

Recent studies on humans and their key experimental model, the mouse, have begun to uncover the importance of gastrointestinal (GI) microbiota and enteric nervous system (ENS) interactions during developmental windows spanning from conception to adolescence. Disruptions in GI microbiota and ENS during these windows by environmental factors, particularly antibiotic exposure, have been linked to increased susceptibility of the host to several diseases. Mouse models have provided new insights to potential signaling factors between the microbiota and ENS. We review very recent work on maturation of GI microbiota and ENS during three key developmental windows: embryogenesis, early postnatal, and postweaning periods. We discuss advances in understanding of interactions between the two systems and highlight research avenues for future studies.

Antibiotic exposure postweaning disrupts the neurochemistry and function of enteric neurons mediating colonic motor activity.

The period during and immediately after weaning is an important developmental window when marked shifts in gut microbiota can regulate the maturation of the enteric nervous system (ENS). Because microbiota-derived signals that modulate ENS development are poorly understood, we examined the physiological impact of the broad spectrum of antibiotic, vancomycin-administered postweaning on colonic motility, neurochemistry of enteric neurons, and neuronal excitability. The functional impact of vancomycin on enteric neurons was investigated by Ca2+ imaging in Wnt1-Cre;R26R-GCaMP3 reporter mice to characterize alterations in the submucosal and the myenteric plexus, which contains the neuronal circuitry controlling gut motility. 16S rDNA sequencing of fecal specimens after oral vancomycin demonstrated significant deviations in microbiota abundance, diversity, and community composition. Vancomycin significantly increased the relative family rank abundance of Akkermansiaceae, Lactobacillaceae, and Enterobacteriaceae at the expense of Lachnospiraceae and Bacteroidaceae. In sharp contrast to neonatal vancomycin exposure, microbiota compositional shifts in weaned animals were associated with slower colonic migrating motor complexes (CMMCs) without mucosal serotonin biosynthesis being altered. The slowing of CMMCs is linked to disruptions in the neurochemistry of the underlying enteric circuitry. This included significant reductions in cholinergic and calbindin+ myenteric neurons, neuronal nitric oxide synthase+ submucosal neurons, neurofilament M+ enteric neurons, and increased proportions of cholinergic submucosal neurons. The antibiotic treatment also increased transmission and responsiveness in myenteric and submucosal neurons that may enhance inhibitory motor pathways, leading to slower CMMCs. Differential vancomycin responses during neonatal and weaning periods in mice highlight the developmental-specific impact of antibiotics on colonic enteric circuitry and motility.

Spontaneous calcium waves in the developing enteric nervous system.

The enteric nervous system (ENS) is an extensive network of neurons in the gut wall that arises from neural crest-derived cells. Like other populations of neural crest cells, it is known that enteric neural crest-derived cells (ENCCs) influence the behaviour of each other and therefore must communicate. However, little is known about how ENCCs communicate with each other. In this study, we used Ca2+ imaging to examine communication between ENCCs in the embryonic gut, using mice where ENCCs express a genetically-encoded calcium indicator. Spontaneous propagating calcium waves were observed between neighbouring ENCCs, through both neuronal and non-neuronal ENCCs. Pharmacological experiments showed wave propagation was not mediated by gap junctions, but by purinergic signalling via P2 receptors. The expression of several P2X and P2Y receptors was confirmed using RT-PCR. Furthermore, inhibition of P2 receptors altered the morphology of the ENCC network, without affecting neuronal differentiation or ENCC proliferation. It is well established that purines participate in synaptic transmission in the mature ENS. Our results describe, for the first time, purinergic signalling between ENCCs during pre-natal development, which plays roles in the propagation of Ca2+ waves between ENCCs and in ENCC network formation. One previous study has shown that calcium signalling plays a role in sympathetic ganglia formation; our results suggest that calcium waves are likely to be important for enteric ganglia development.

Optogenetic Demonstration of Functional Innervation of Mouse Colon by Neurons Derived From Transplanted Neural Cells.

BACKGROUND & AIMS: Cell therapy offers the potential to treat gastrointestinal motility disorders caused by diseased or absent enteric neurons. We examined whether neurons generated from transplanted enteric neural cells provide a functional innervation of bowel smooth muscle in mice. METHODS: Enteric neural cells expressing the light-sensitive ion channel, channelrhodopsin, were isolated from the fetal or postnatal mouse bowel and transplanted into the distal colon of 3- to 4-week-old wild-type recipient mice. Intracellular electrophysiological recordings of responses to light stimulation of the transplanted cells were made from colonic smooth muscle cells in recipient mice. Electrical stimulation of endogenous enteric neurons was used as a control. RESULTS: The axons of graft-derived neurons formed a plexus in the circular muscle layer. Selective stimulation of graft-derived cells by light resulted in excitatory and inhibitory junction potentials, the electrical events underlying contraction and relaxation, respectively, in colonic muscle cells. Graft-derived excitatory and inhibitory motor neurons released the same neurotransmitters as endogenous motor neurons-acetylcholine and a combination of adenosine triphosphate and nitric oxide, respectively. Graft-derived neurons also included interneurons that provided synaptic inputs to motor neurons, but the pharmacologic properties of interneurons varied with the age of the donors from which enteric neural cells were obtained. CONCLUSIONS: Enteric neural cells transplanted into the bowel give rise to multiple functional types of neurons that integrate and provide a functional innervation of the smooth muscle of the bowel wall. Circuits composed of both motor neurons and interneurons were established, but the age at which cells are isolated influences the neurotransmitter phenotype of interneurons that are generated.

PARP inhibition in platinum-based chemotherapy: Chemopotentiation and neuroprotection.

Cisplatin, carboplatin and oxaliplatin represent the backbone of platinum therapy for several malignancies including head and neck, lung, colorectal, ovarian, breast, and genitourinary cancer. However, the efficacy of platinum-based drugs is often compromised by a plethora of severe toxicities including sensory and enteric neuropathy. Acute and chronic neurotoxicity following platinum chemotherapy is a major constraint, contributing to dose-reductions, treatment delays, and cessation of treatment. Identifying drugs that effectively prevent these toxic complications is imperative to improve the efficacy of anti-cancer treatment and patient quality of life. Oxidative stress and mitochondrial dysfunction have been highlighted as key players in the pathophysiology of platinum chemotherapy-induced neuropathy. Inhibition of poly(ADP-ribose) polymerase (PARP), a nuclear enzyme activated upon DNA damage, has demonstrated substantial sensory and enteric neuroprotective capacity when administered in combination with platinum chemotherapeutics. Furthermore, administration of PARP inhibitors alongside platinum chemotherapy has been found to significantly improve progression-free survival in patients with breast and ovarian cancer when compared to those receiving chemotherapy alone. This review summarises the current knowledge surrounding mitochondrial damage and oxidative stress in platinum chemotherapy-induced neuropathy and highlights a potential role for PARP in chemopotentiation and neuroprotection.

White paper on guidelines concerning enteric nervous system stem cell therapy for enteric neuropathies.

Over the last 20 years, there has been increasing focus on the development of novel stem cell based therapies for the treatment of disorders and diseases affecting the enteric nervous system (ENS) of the gastrointestinal tract (so-called enteric neuropathies). Here, the idea is that ENS progenitor/stem cells could be transplanted into the gut wall to replace the damaged or absent neurons and glia of the ENS. This White Paper sets out experts' views on the commonly used methods and approaches to identify, isolate, purify, expand and optimize ENS stem cells, transplant them into the bowel, and assess transplant success, including restoration of gut function. We also highlight obstacles that must be overcome in order to progress from successful preclinical studies in animal models to ENS stem cell therapies in the clinic.

Alterations of colonic function in the Winnie mouse model of spontaneous chronic colitis.

The Winnie mouse, carrying a missense mutation in Muc2, is a model for chronic intestinal inflammation demonstrating symptoms closely resembling inflammatory bowel disease (IBD). Alterations to the immune environment, morphological structure, and innervation of Winnie mouse colon have been identified; however, analyses of intestinal transit and colonic functions have not been conducted. In this study, we investigated in vivo intestinal transit in radiographic studies and in vitro motility of the isolated colon in organ bath experiments. We compared neuromuscular transmission using conventional intracellular recording between distal colon of Winnie and C57BL/6 mice and smooth muscle contractions using force displacement transducers. Chronic inflammation in Winnie mice was confirmed by detection of lipocalin-2 in fecal samples over 4 wk and gross morphological damage to the colon. Colonic transit was faster in Winnie mice. Motility was altered including decreased frequency and increased speed of colonic migrating motor complexes and increased occurrence of short and fragmented contractions. The mechanisms underlying colon dysfunctions in Winnie mice included inhibition of excitatory and fast inhibitory junction potentials, diminished smooth muscle responses to cholinergic and nitrergic stimulation, and increased number of α-smooth muscle actin-immunoreactive cells. We conclude that diminished excitatory responses occur both prejunctionally and postjunctionally and reduced inhibitory purinergic responses are potentially a prejunctional event, while diminished nitrergic inhibitory responses are probably due to a postjunction mechanism in the Winnie mouse colon. Many of these changes are similar to disturbed motor functions in IBD patients indicating that the Winnie mouse is a model highly representative of human IBD. NEW & NOTEWORTHY: This is the first study to provide analyses of intestinal transit and whole colon motility in an animal model of spontaneous chronic colitis. We found that cholinergic and purinergic neuromuscular transmission, as well as the smooth muscle cell responses to cholinergic and nitrergic stimulation, is altered in the chronically inflamed Winnie mouse colon. The changes to intestinal transit and colonic function we identified in the Winnie mouse are similar to those seen in inflammatory bowel disease patients.

Changes in Nicotinic Neurotransmission during Enteric Nervous System Development.

Acetylcholine-activating pentameric nicotinic receptors (nAChRs) are an essential mode of neurotransmission in the enteric nervous system (ENS). In this study, we examined the functional development of specific nAChR subtypes in myenteric neurons using Wnt1-Cre;R26R-GCaMP3 mice, where all enteric neurons and glia express the genetically encoded calcium indicator, GCaMP3. Transcripts encoding α3, α4, α7, ß2, and ß4 nAChR subunits were already expressed at low levels in the E11.5 gut and by E14.5 and, thereafter, α3 and ß4 transcripts were the most abundant. The effect of specific nAChR subtype antagonists on evoked calcium activity in enteric neurons was investigated at different ages. Blockade of the α3ß4 receptors reduced electrically and chemically evoked calcium responses at E12.5, E14.5, and P0. In addition to the α3ß4 antagonist, antagonists to α3ß2 and α4ß2 also significantly reduced responses by P10-11 and in adult preparations. Therefore, there is an increase in the diversity of functional nAChRs during postnatal development. However, an α7 nAChR antagonist had no effect at any age. Furthermore, at E12.5 we found evidence for unconventional receptors that were responsive to the nAChR agonists 1-dimethyl-4-phenylpiperazinium and nicotine, but were insensitive to the general nicotinic blocker, hexamethonium. Migration, differentiation, and neuritogenesis assays did not reveal a role for nAChRs in these processes during embryonic development. In conclusion, there are significant changes in the contribution of different nAChR subunits to synaptic transmission during ENS development, even after birth. This is the first study to investigate the development of cholinergic transmission in the ENS.

A sexually dimorphic effect of cholera toxin: rapid changes in colonic motility mediated via a 5-HT3 receptor-dependent pathway in female C57Bl/6 mice.

KEY POINTS: Cholera causes more than 100,000 deaths each year as a result of severe diarrhoea, vomiting and dehydration due to the actions of cholera toxin; more females than males are affected. Cholera toxin induces hypersecretion via release of mucosal serotonin and over-activation of enteric neurons, but its effects on gastrointestinal motility are not well characterized. We found that cholera toxin rapidly and reversibly reduces colonic motility in female mice in oestrus, but not in males or females in prooestrus, an effect mediated by 5-HT in the colonic mucosa and by 5-HT3 receptors. We show that the number of mucosal enterochromaffin cells containing 5-HT changes with the oestrous cycle in mice. These findings indicate that cholera toxin's effects on motility are rapid and depend on the oestrous cycle and therefore can help us better understand differences in responses in males and female patients. ABSTRACT: Extensive studies of the mechanisms responsible for the hypersecretion produced by cholera toxin (CT) have shown that this toxin produces a massive over-activation of enteric neural secretomotor circuits. The effects of CT on gastrointestinal motility, however, have not been adequately characterized. We investigated effects of luminal CT on neurally mediated motor activity in ex vivo male and female mouse full length colon preparations. We used video recording and spatiotemporal maps of contractile activity to quantify colonic migrating motor complexes (CMMCs) and resting colonic diameter. We compared effects of CT in female colon from wild-type and mice lacking tryptophan hydroxylase (TPH1KO). We also compared CMMCs in colons of female mice in oestrus with those in prooestrus. In female (but not male) colon, CT rapidly, reversibly and concentration-dependently inhibits CMMC frequency and induces a tonic constriction. These effects were blocked by granisetron (5-HT3 antagonist) and were absent from TPH1KO females. CT effects were prominent at oestrus but absent at prooestrus. The number of EC cells containing immunohistochemically demonstrable serotonin (5-HT) was 30% greater in female mice during oestrus than during prooestrus or in males. We conclude that CT inhibits CMMCs via release of mucosal 5-HT, which activates an inhibitory pathway involving 5-HT3 receptors. This effect is sex- and oestrous cycle-dependent and is probably due to an oestrous cycle-dependent change in the number of 5-HT-containing EC cells in the colonic mucosa.

The emergence of neural activity and its role in the development of the enteric nervous system.

The enteric nervous system (ENS) is a vital part of the autonomic nervous system that regulates many gastrointestinal functions, including motility and secretion. All neurons and glia of the ENS arise from neural crest-derived cells that migrate into the gastrointestinal tract during embryonic development. It has been known for many years that a subpopulation of the enteric neural crest-derived cells expresses pan-neuronal markers at early stages of ENS development. Recent studies have demonstrated that some enteric neurons exhibit electrical activity from as early as E11.5 in the mouse, with further maturation of activity during embryonic and postnatal development. This article discusses the maturation of electrophysiological and morphological properties of enteric neurons, the formation of synapses and synaptic activity, and the influence of neural activity on ENS development.

Properties of cholinergic and non-cholinergic submucosal neurons along the mouse colon.

Submucosal neurons are vital regulators of water and electrolyte secretion and local blood flow in the gut. Due to the availability of transgenic models for enteric neuropathies, the mouse has emerged as the research model of choice, but much is still unknown about the murine submucosal plexus. The progeny of choline acetyltransferase (ChAT)-Cre × ROSA26(YFP) reporter mice, ChAT-Cre;R26R-yellow fluorescent protein (YFP) mice, express YFP in every neuron that has ever expressed ChAT. With the aid of the robust YFP staining in these mice, we correlated the neurochemistry, morphology and electrophysiology of submucosal neurons in distal colon. We also examined whether there are differences in neurochemistry along the colon and in neurally mediated vectorial ion transport between the proximal and distal colon. All YFP(+) submucosal neurons also contained ChAT. Two main neurochemical but not electrophysiological groups of neurons were identified: cholinergic (containing ChAT) or non-cholinergic. The vast majority of neurons in the middle and distal colon were non-cholinergic but contained vasoactive intestinal peptide. In the distal colon, non-cholinergic neurons had one or two axons, whereas the cholinergic neurons examined had only one axon. All submucosal neurons exhibited S-type electrophysiology, shown by the lack of long after-hyperpolarizing potentials following their action potentials and fast excitatory postsynaptic potentials (EPSPs). Fast EPSPs were predominantly nicotinic, and somatic action potentials were mediated by tetrodotoxin-resistant voltage-gated channels. The size of submucosal ganglia decreased but the proportion of cholinergic neurons increased distally along the colon. The distal colon had a significantly larger nicotinic ion transport response than the proximal colon. This work shows that the properties of murine submucosal neurons and their control of epithelial ion transport differ between colonic regions. There are several key differences between the murine submucous plexus and that of other animals, including a lack of conventional intrinsic sensory neurons, which suggests there is an incomplete neuronal circuitry within the murine submucous plexus.

VPAC1 receptors regulate intestinal secretion and muscle contractility by activating cholinergic neurons in guinea pig jejunum.

In the gastrointestinal tract, vasoactive intestinal peptide (VIP) is found exclusively within neurons. VIP regulates intestinal motility via neurally mediated and direct actions on smooth muscle and secretion by a direct mucosal action, and via actions on submucosal neurons. VIP acts via VPAC1 and VPAC2 receptors; however, the subtype involved in its neural actions is unclear. The neural roles of VIP and VPAC1 receptors (VPAC1R) were investigated in intestinal motility and secretion in guinea pig jejunum. Expression of VIP receptors across the jejunal layers was examined using RT-PCR. Submucosal and myenteric neurons expressing VIP receptor subtype VPAC1 and/or various neurochemical markers were identified immunohistochemically. Isotonic muscle contraction was measured in longitudinal muscle-myenteric plexus preparations. Electrogenic secretion across mucosa-submucosa preparations was measured in Ussing chambers by monitoring short-circuit current. Calretinin(+) excitatory longitudinal muscle motor neurons expressed VPAC1R. Most cholinergic submucosal neurons, notably NPY(+) secretomotor neurons, expressed VPAC1R. VIP (100 nM) induced longitudinal muscle contraction that was inhibited by TTX (1 µM), PG97-269 (VPAC1 antagonist; 1 µM), and hyoscine (10 µM), but not by hexamethonium (200 µM). VIP (50 nM)-evoked secretion was depressed by hyoscine or PG97-269 and involved a small TTX-sensitive component. PG97-269 and TTX combined did not further depress the VIP response observed in the presence of PG97-269 alone. We conclude that VIP stimulates ACh-mediated longitudinal muscle contraction via VPAC1R on cholinergic motor neurons. VIP induces Cl(-) secretion directly via epithelial VPAC1R and indirectly via VPAC1R on cholinergic secretomotor neurons. No evidence was obtained for involvement of other neural VIP receptors.