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.

Quantitative Spatial Analysis of Neuroligin-3 mRNA Expression in the Enteric Nervous System Reveals a Potential Role in Neuronal-Glial Synapses and Reduced Expression in Nlgn3R451C Mice.

Mutations in the Neuroligin-3 (Nlgn3) gene are implicated in autism spectrum disorder (ASD) and gastrointestinal (GI) dysfunction, but cellular Nlgn3 expression in the enteric nervous system remains to be characterised. We combined RNAScope in situ hybridization and immunofluorescence to measure Nlgn3 mRNA expression in cholinergic and VIP-expressing submucosal neurons, nitrergic and calretinin-containing myenteric neurons and glial cells in both WT and Nlgn3R451C mutant mice. We measured Nlgn3 mRNA neuronal and glial expression via quantitative three-dimensional image analysis. To validate dual RNAScope/immunofluorescence data, we interrogated available single-cell RNA sequencing (scRNASeq) data to assess for Nlgn3, Nlgn1, Nlgn2 and their binding partners, Nrxn1-3, MGDA1 and MGDA2, in enteric neural subsets. Most submucosal and myenteric neurons expressed Nlgn3 mRNA. In contrast to other Nlgns and binding partners, Nlgn3 was strongly expressed in enteric glia, suggesting a role for neuroligin-3 in mediating enteric neuron-glia interactions. The autism-associated R451C mutation reduces Nlgn3 mRNA expression in cholinergic but not in VIPergic submucosal neurons. In the myenteric plexus, Nlgn3 mRNA levels are reduced in calretinin, nNOS-labelled neurons and S100 ß -labelled glia. We provide a comprehensive cellular profile for neuroligin-3 expression in ileal neuronal subpopulations of mice expressing the R451C autism-associated mutation in Nlgn3, which may contribute to the understanding of the pathophysiology of GI dysfunction in ASD.

Group I Metabotropic Glutamate Receptors Modulate Motility and Enteric Neural Activity in the Mouse Colon.

Glutamate is the major excitatory neurotransmitter in the central nervous system, and there is evidence that Group-I metabotropic glutamate receptors (mGlu1 and mGlu5) have established roles in excitatory neurotransmission and synaptic plasticity. While glutamate is abundantly present in the gut, it plays a smaller role in neurotransmission in the enteric nervous system. In this study, we examined the roles of Group-I mGlu receptors in gastrointestinal function. We investigated the expression of Grm1 (mGlu1) and Grm5 (mGlu5) in the mouse myenteric plexus using RNAscope in situ hybridization. Live calcium imaging and motility analysis were performed on ex vivo preparations of the mouse colon. mGlu5 was found to play a role in excitatory enteric neurotransmission, as electrically-evoked calcium transients were sensitive to the mGlu5 antagonist MPEP. However, inhibition of mGlu5 activity did not affect colonic motor complexes (CMCs). Instead, inhibition of mGlu1 using BAY 36-7620 reduced CMC frequency but did not affect enteric neurotransmission. These data highlight complex roles for Group-I mGlu receptors in myenteric neuron activity and colonic function.

Oxidative Stress-Induced HMGB1 Translocation in Myenteric Neurons Contributes to Neuropathy in Colitis.

High-mobility group box 1 (HMGB1) is a damage-associated molecular pattern released by dying cells to stimulate the immune response. During cell death, HMGB1 is translocated from the nucleus to the cytoplasm and passively released. High levels of secreted HMGB1 are observed in the faeces of inflammatory bowel disease (IBD) patients, indicating its role in IBD pathophysiology and potential as a non-invasive IBD biomarker. HMGB1 is important in regulating neuronal damage in the central nervous system; its pathological activity is intertwined with oxidative stress and inflammation. In this study, HMGB1 expression in the enteric nervous system and its relevance to intestinal neuroinflammation is explored in organotypic cultures of the myenteric plexus exposed to oxidative stimuli and in Winnie mice with spontaneous chronic colitis. Oxidative stimuli induced cytoplasmic translocation of HMGB1 in myenteric neurons in organotypic preparations. HMGB1 translocation correlated with enteric neuronal loss and oxidative stress in the myenteric ganglia of Winnie mice. Inhibition of HMGB1 by glycyrrhizic acid ameliorated HMGB1 translocation and myenteric neuronal loss in Winnie mice. These data highlight modulation of HMGB1 signalling as a therapeutic strategy to reduce the consequences of enteric neuroinflammation in colitis, warranting the exploration of therapeutics acting on the HMGB1 pathway as an adjunct treatment with current anti-inflammatory agents.

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.

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.

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.

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.

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.

Divergent Adaptations in Autonomic Nerve Activity and Neuroimmune Signaling Associated With the Severity of Inflammation in Chronic Colitis.

BACKGROUND: The autonomic nervous system (ANS) is thought to play a critical role in the anti-inflammatory reflex pathway in acute colitis via its interaction with the spleen and colon. Inflammation in the intestine is associated with a blunting of vagal signaling and increased sympathetic activity. As a corollary, methods to restore sympatho-vagal balance are being investigated as therapeutic strategies for the treatment of intestinal inflammation. Nevertheless, it is indefinite whether these autonomic signaling adaptations in colitis are detrimental or beneficial to controlling intestinal inflammation. In this study, models of moderate and severe chronic colitis are utilized to resolve the correlations between sympatho-vagal signaling and the severity of intestinal inflammation. METHODS: Spleens and colons were collected from Winnie (moderate colitis), Winnie-Prolapse (severe colitis), and control C57BL/6 mice. Changes to the size and histomorphology of spleens were evaluated. Flow cytometry was used to determine the expression of adrenergic and cholinergic signaling proteins in splenic B and T lymphocytes. The inflammatory profile of the spleen and colon was determined using a RT-PCR gene array. Blood pressure, heart rate, splanchnic sympathetic nerve and vagus nerve activity were recorded. RESULTS: Spleens and colons from Winnie and Winnie-Prolapse mice exhibited gross abnormalities by histopathology. Genes associated with a pro-inflammatory response were upregulated in the colons from Winnie and further augmented in colons from Winnie-Prolapse mice. Conversely, many pro-inflammatory markers were downregulated in the spleens from Winnie-Prolapse mice. Heightened activity of the splanchnic nerve was observed in Winnie but not Winnie-Prolapse mice. Conversely, vagal nerve activity was greater in Winnie-Prolapse mice compared with Winnie mice. Splenic lymphocytes expressing α1 and ß2 adrenoreceptors were reduced, but those expressing α7 nAChR and producing acetylcholine were increased in Winnie and Winnie-Prolapse mice. CONCLUSIONS: Sympathetic activity may correlate with an adaptive mechanism to reduce the severity of chronic colitis. The Winnie and Winnie-Prolapse mouse models of moderate and severe chronic colitis are well suited to examine the pathophysiology of progressive chronic intestinal inflammation.

In this study we use mouse models of moderate and severe colitis to resolve the relationship between autonomic and neuroimmune signaling with inflammation. Increased expression of cholinergic markers on immune cells correlated with an anti-inflammatory profile in the spleen, consistent with activation of the splenic cholinergic anti-inflammatory pathway in mice with spontaneous chronic colitis. However, enhanced sympathetic signaling occurred in mice with a less severe phenotype of colitis, which could represent an adaptive mechanism to mitigate the progression of intestinal inflammation.

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.

Enteric neuroimmune interactions coordinate intestinal responses in health and disease.

The enteric nervous system (ENS) of the gastrointestinal (GI) tract interacts with the local immune system bidirectionally. Recent publications have demonstrated that such interactions can maintain normal GI functions during homeostasis and contribute to pathological symptoms during infection and inflammation. Infection can also induce long-term changes of the ENS resulting in the development of post-infectious GI disturbances. In this review, we discuss how the ENS can regulate and be regulated by immune responses and how such interactions control whole tissue physiology. We also address the requirements for the proper regeneration of the ENS and restoration of GI function following the resolution of infection.

Nitric Oxide Regulates Estrus Cycle Dependent Colonic Motility in Mice.

Women are more susceptible to functional bowel disorders than men and the severity of their symptoms such as diarrhea, constipation, abdominal pain and bloating changes over the menstrual cycle, suggesting a role for sex hormones in gastrointestinal function. Nitric oxide (NO) is a major inhibitory neurotransmitter in the gut and blockade of nitric oxide synthase (NOS; responsible for NO synthesis) increases colonic motility in male mice ex vivo. We assessed the effects of NOS inhibition on colonic motility in female mice using video imaging analysis of colonic motor complexes (CMCs). To understand interactions between NO and estrogen in the gut, we also quantified neuronal NOS and estrogen receptor alpha (ERα)-expressing myenteric neurons in estrus and proestrus female mice using immunofluorescence. Mice in estrus had fewer CMCs under control conditions (6 ± 1 per 15 min, n = 22) compared to proestrus (8 ± 1 per 15 min, n = 22, One-way ANOVA, p = 0.041). During proestrus, the NOS antagonist N-nitro-L-arginine (NOLA) increased CMC numbers compared to controls (189 ± 46%). In contrast, NOLA had no significant effect on CMC numbers during estrus. During estrus, we observed more NOS-expressing myenteric neurons (48 ± 2%) than during proestrus (39 ± 1%, n = 3, p = 0.035). Increased nuclear expression of ERα was observed in estrus which coincided with an altered motility response to NOLA in contrast with proestrus when ERα was largely cytoplasmic. In conclusion, we confirm a cyclic and sexually dimorphic effect of NOS activity in female mouse colon, which could be due to genomic effects of estrogens via ERα.

scRNA-Seq Reveals New Enteric Nervous System Roles for GDNF, NRTN, and TBX3.

BACKGROUND AND AIMS: Bowel function requires coordinated activity of diverse enteric neuron subtypes. Our aim was to define gene expression in these neuron subtypes to facilitate development of novel therapeutic approaches to treat devastating enteric neuropathies, and to learn more about enteric nervous system function. METHODS: To identify subtype-specific genes, we performed single-nucleus RNA-seq on adult mouse and human colon myenteric plexus, and single-cell RNA-seq on E17.5 mouse ENS cells from whole bowel. We used immunohistochemistry, select mutant mice, and calcium imaging to validate and extend results. RESULTS: RNA-seq on 635 adult mouse colon myenteric neurons and 707 E17.5 neurons from whole bowel defined seven adult neuron subtypes, eight E17.5 neuron subtypes and hundreds of differentially expressed genes. Manually dissected human colon myenteric plexus yielded RNA-seq data from 48 neurons, 3798 glia, 5568 smooth muscle, 377 interstitial cells of Cajal, and 2153 macrophages. Immunohistochemistry demonstrated differential expression for BNC2, PBX3, SATB1, RBFOX1, TBX2, and TBX3 in enteric neuron subtypes. Conditional Tbx3 loss reduced NOS1-expressing myenteric neurons. Differential Gfra1 and Gfra2 expression coupled with calcium imaging revealed that GDNF and neurturin acutely and differentially regulate activity of ∼50% of myenteric neurons with distinct effects on smooth muscle contractions. CONCLUSION: Single cell analyses defined genes differentially expressed in myenteric neuron subtypes and new roles for TBX3, GDNF and NRTN. These data facilitate molecular diagnostic studies and novel therapeutics for bowel motility disorders.

Inhibition of APE1/Ref-1 Redox Signaling Alleviates Intestinal Dysfunction and Damage to Myenteric Neurons in a Mouse Model of Spontaneous Chronic Colitis.

BACKGROUND: Inflammatory bowel disease (IBD) associates with damage to the enteric nervous system (ENS), leading to gastrointestinal (GI) dysfunction. Oxidative stress is important for the pathophysiology of inflammation-induced enteric neuropathy and GI dysfunction. Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a dual functioning protein that is an essential regulator of the cellular response to oxidative stress. In this study, we aimed to determine whether an APE1/Ref-1 redox domain inhibitor, APX3330, alleviates inflammation-induced oxidative stress that leads to enteric neuropathy in the Winnie murine model of spontaneous chronic colitis. METHODS: Winnie mice received APX3330 or vehicle via intraperitoneal injections over 2 weeks and were compared with C57BL/6 controls. In vivo disease activity and GI transit were evaluated. Ex vivo experiments were performed to assess functional parameters of colonic motility, immune cell infiltration, and changes to the ENS. RESULTS: Targeting APE1/Ref-1 redox activity with APX3330 improved disease severity, reduced immune cell infiltration, restored GI function ,and provided neuroprotective effects to the enteric nervous system. Inhibition of APE1/Ref-1 redox signaling leading to reduced mitochondrial superoxide production, oxidative DNA damage, and translocation of high mobility group box 1 protein (HMGB1) was involved in neuroprotective effects of APX3330 in enteric neurons. CONCLUSIONS: This study is the first to investigate inhibition of APE1/Ref-1's redox activity via APX3330 in an animal model of chronic intestinal inflammation. Inhibition of the redox function of APE1/Ref-1 is a novel strategy that might lead to a possible application of APX3330 for the treatment of IBD.

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.

The Role of the Gastrointestinal Mucus System in Intestinal Homeostasis: Implications for Neurological Disorders.

Mucus is integral to gut health and its properties may be affected in neurological disease. Mucus comprises a hydrated network of polymers including glycosylated mucin proteins. We propose that factors that influence the nervous system may also affect the volume, viscosity, porosity of mucus composition and subsequently, gastrointestinal (GI) microbial populations. The gut has its own intrinsic neuronal network, the enteric nervous system, which extends the length of the GI tract and innervates the mucosal epithelium. The ENS regulates gut function including mucus secretion and renewal. Both dysbiosis and gut dysfunction are commonly reported in several neurological disorders such as Parkinson's and Alzheimer's disease as well in patients with neurodevelopmental disorders including autism. Since some microbes use mucus as a prominent energy source, changes in mucus properties could alter, and even exacerbate, dysbiosis-related gut symptoms in neurological disorders. This review summarizes existing knowledge of the structure and function of the mucus of the GI tract and highlights areas to be addressed in future research to better understand how intestinal homeostasis is impacted in neurological disorders.

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.

Colonic dilation and altered ex vivo gastrointestinal motility in the neuroligin-3 knockout mouse.

Gastrointestinal (GI) dysfunction is commonly reported by people diagnosed with autism spectrum disorder (ASD; autism) but the cause is unknown. Mutations in genes encoding synaptic proteins including Neuroligin-3 are associated with autism. Mice lacking Neuroligin-3 (Nlgn3-/- ) have altered brain function, but whether the enteric nervous system (ENS) is altered remains unknown. We assessed for changes in GI structure and function in Nlgn3-/- mice. We found no significant morphological differences in villus height or crypt depth in the jejunum or colon between wildtype (WT) and Nlgn3-/- mice. To determine whether deletion of Nlgn3 affects enteric neurons, we stained for neural markers in the myenteric plexus. Nlgn3-/- mice had similar numbers of neurons expressing the pan-neuronal marker Hu in the jejunum, proximal mid, and distal colon regions. We also found no differences in the number of neuronal nitric oxide synthase (nNOS+) or calretinin (CalR+) motor neurons and interneurons between WT and Nlgn3-/- mice. We used ex vivo video imaging analysis to assess colonic motility under baseline conditions and observed faster colonic migrating motor complexes (CMMCs) and an increased colonic diameter in Nlgn3-/- mice, although CMMC frequency was unchanged. At baseline, CMMCs were faster in Nlgn3-/- mice compared to WT. Although the numbers of neuronal subsets are conserved in Nlgn3-/- mice, these findings suggest that Neuroligin-3 modulates inhibitory neural pathways in the ENS and may contribute to mechanisms underlying GI disorders in autism. Autism Res 2020, 13: 691-701. © 2019 The Authors. Autism Research published by International Society for Autism Research published byWiley Periodicals, Inc. LAY SUMMARY: People with autism commonly experience gut problems. Many gene mutations associated with autism affect neuronal activity. We studied mice in which the autism-associated Neuroligin-3 gene is deleted to determine whether this impacts gut neuronal numbers or motility. We found that although mutant mice had similar gut structure and numbers of neurons in all gut regions examined, they had distended colons and faster colonic muscle contractions. Further work is needed to understand how Neuroligin-3 affects neuron connectivity in the gastrointestinal tract.