Anatomy and Neuroscience - Research Publications
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ItemFaster Gastrointestinal Transit, Reduced Small Intestinal Smooth Muscle Tone and Dysmotility in the Nlgn3R451C Mouse Model of Autism(MDPI, 2024-01)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.
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ItemNo Preview AvailableQuantitative 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(MDPI, 2023-07)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 β -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.
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ItemNitric Oxide Regulates Estrus Cycle Dependent Colonic Motility in Mice(FRONTIERS MEDIA SA, 2021-09-29)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.
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ItemA sexually dimorphic effect of cholera toxin: rapid changes in colonic motility mediated via a 5-HT3 receptor-dependent pathway in female C57Bl/6 mice(WILEY, 2016-08-01)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.
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ItemA neuroligin-3 mutation implicated in autism causes abnormal aggression and increases repetitive behavior in mice(BMC, 2015-11-14)BACKGROUND: Aggression is common in patients with autism spectrum disorders (ASD) along with the core symptoms of impairments in social communication and repetitive behavior. Risperidone, an atypical antipsychotic, is widely used to treat aggression in ASD. In order to understand the neurobiological underpinnings of these challenging behaviors, a thorough characterisation of behavioral endophenotypes in animal models is required. METHODS: We investigated aggression in mice containing the ASD-associated R451C (arginine to cysteine residue 451 substitution) mutation in neuroligin-3 (NL3). Furthermore, we sought to verify social interaction impairments and assess olfaction, anxiety, and repetitive and restrictive behavior in NL3(R451C) mutant mice. RESULTS: We show a pronounced elevation in aggressive behavior in NL3(R451C) mutant mice. Treatment with risperidone reduced this aggression to wild-type (WT) levels. Juvenile and adult social interactions were also investigated, and subtle differences in initiation of interaction were seen in juvenile NL3(R451C) mice. No genotype differences in olfactory discrimination or anxiety were observed indicating that aggression was not dependent on altered olfaction, stress response, or social preference. We also describe repetitive behavior in NL3(R451C) mice as assessed by a clinically relevant object exploration task. CONCLUSIONS: The presence of aberrant aggression and other behavioral phenotypes in NL3(R451C) mice consistent with clinical traits strengthen face validity of this model of ASD. Furthermore, we demonstrate predictive validity in this model through the reversal of the aggressive phenotype with risperidone. This is the first demonstration that risperidone can ameliorate aggression in an animal model of ASD and will inform mechanistic and therapeutic research into the neurobiology underlying abnormal behaviors in ASD.