Remote optogenetic control of the enteric nervous system and brain-gut axis in freely-behaving mice enabled by a wireless, battery-free optoelectronic device.

Wireless activation of the enteric nervous system (ENS) in freely moving animals with implantable optogenetic devices offers a unique and exciting opportunity to selectively control gastrointestinal (GI) transit in vivo, including the gut-brain axis. Programmed delivery of light to targeted locations in the GI-tract, however, poses many challenges not encountered within the central nervous system (CNS). We report here the development of a fully implantable, battery-free wireless device specifically designed for optogenetic control of the GI-tract, capable of generating sufficient light over large areas to robustly activate the ENS, potently inducing colonic motility ex vivo and increased propulsion in vivo. Use in in vivo studies reveals unique stimulation patterns that increase expulsion of colonic content, likely mediated in part by activation of an extrinsic brain-gut motor pathway, via pelvic nerves. This technology overcomes major limitations of conventional wireless optogenetic hardware designed for the CNS, providing targeted control of specific neurochemical classes of neurons in the ENS and brain-gut axis, for direct modulation of GI-transit and associated behaviours in freely moving animals.

BAP1 is required prenatally for differentiation and maintenance of postnatal murine enteric nervous system.

Epigenetic regulatory mechanisms are underappreciated, yet are critical for enteric nervous system (ENS) development and maintenance. We discovered that fetal loss of the epigenetic regulator Bap1 in the ENS lineage caused severe postnatal bowel dysfunction and early death in Tyrosinase-Cre Bap1fl/fl mice. Bap1-depleted ENS appeared normal in neonates; however, by P15, Bap1-deficient enteric neurons were largely absent from the small and large intestine of Tyrosinase-Cre Bap1fl/fl mice. Bowel motility became markedly abnormal with disproportionate loss of cholinergic neurons. Single-cell RNA sequencing at P5 showed that fetal Bap1 loss in Tyrosinase-Cre Bap1fl/fl mice markedly altered the composition and relative proportions of enteric neuron subtypes. In contrast, postnatal deletion of Bap1 did not cause enteric neuron loss or impaired bowel motility. These findings suggest that BAP1 is critical for postnatal enteric neuron differentiation and for early enteric neuron survival, a finding that may be relevant to the recently described human BAP1-associated neurodevelopmental disorder.

Mesenchymal cells regulate enteric neural crest cell migration via RET-GFRA1b trans-signaling.

During early development, the enteric nervous system forms from the migration of enteric neural crest cells (ENCCs) from the foregut to the hindgut, where they undergo proliferation and differentiation facilitated by interactions with enteric mesenchymal cells (EMCs). This study investigates the impact on ENCC migration of EMC-ENCC communication mediated by GFRA1b expressed in EMCs. GFRA1-expressing cells in day 11-12 (E11-12) mouse embryos differentiated into smooth muscle cells from E12 onwards. Observations at E12-13.5 revealed high levels of GFRA1 expression on the anti-mesenteric side of the hindgut, correlating with enhanced ENCC migration. This indicates that GFRA1 in EMCs plays a role in ENCC migration during development. Examining GFRA1 isoforms, we found high levels of GFRA1b, which lacks amino acids 140-144, in EMCs. To assess the impact of GFRA1 isoforms on EMC-ENCC communication, we conducted neurosphere drop assays. This revealed that GFRA1b-expressing cells promoted GDNF-dependent extension and increased neurite density in ENCC neurospheres. Co-culture of ENCC mimetic cells expressing RET and GFRA1a with EMC mimetic cells expressing GFRA1a, GFRA1b, or vector alone showed that only GFRA1b-expressing co-cultured cells sustained RET phosphorylation in ENCC-mimetic cells for over 120 min upon GDNF stimulation. Our study provides evidence that GFRA1b-mediated cell-to-cell communication plays a critical role in ENCC motility in enteric nervous system development. These findings contribute to understanding the cellular interactions and signaling mechanisms that underlie enteric nervous system formation and highlight potential therapeutic targets for gastrointestinal motility disorders.

The role of galanin in the progression and prognosis of colorectal cancer: the unfinished story.

The paper presents a summary of immunohistochemical (IHC) and biochemical investigations on the presence of galanin (Gal), one of the neuropeptides abundant in the enteric nervous systems, and three types of its receptors (GalR1-3) in colorectal cancer (CRC) tissue and non-involved colon wall and their associations with clinical-pathological data of the CRC patients. We were the first to morphologically demonstrate the presence of endogenous Gal in CRC sections and measure its content in homogenates of tumor tissue and dissected compartments of unchanged colon wall. The prominent atrophy of myenteric plexuses displaying Gal immunoreactivity (Gal-Ir) located close to the tumor invasion was found to be accompanied by higher Gal content in the tumor-adjacent muscularis externa than in tumor-distant tissue. In further studies for the first time, we demonstrated by the IHC technique the presence of the GalR1-3 receptors in the CRC tumors and the colon mucosa and found that higher GalR3-Ir in the tumor tissue correlated with longer overall survival of CRC patients. Furthermore, we discovered that lower GalR1 expression in submucosal plexuses located near the tumor correlated with a better prognosis in patients with CRC. These findings suggest that GalR1 could be considered as a novel therapeutic target in CRC. In conclusion, our morphological investigations provided novel data documenting the involvement of Gal and its receptors in the progression of CRC and showed the usefulness of the IHC technique for the prognosis of CRC patients.

Phosphatase and Tensin Homolog Inhibition in Proteolipid Protein 1-Expressing Cells Stimulates Neurogenesis and Gliogenesis in the Postnatal Enteric Nervous System.

Phosphatase and tensin homolog (Pten) is a key regulator of cell proliferation and a potential target to stimulate postnatal enteric neuro- and/or gliogenesis. To investigate this, we generated two tamoxifen-inducible Cre recombinase murine models in which Pten was conditionally ablated, (1) in glia (Plp1-expressing cells) and (2) in neurons (Calb2-expressing cells). Tamoxifen-treated adult (7-12 weeks of age; n = 4-15) mice were given DSS to induce colitis, EdU to monitor cell proliferation, and were evaluated at two timepoints: (1) early (3-4 days post-DSS) and (2) late (3-4 weeks post-DSS). We investigated gut motility and evaluated the enteric nervous system. Pten inhibition in Plp1-expressing cells elicited gliogenesis at baseline and post-DSS (early and late) in the colon, and neurogenesis post-DSS late in the proximal colon. They also exhibited an increased frequency of colonic migrating motor complexes (CMMC) and slower whole gut transit times. Pten inhibition in Calb2-expressing cells did not induce enteric neuro- or gliogenesis, and no alterations were detected in CMMC or whole gut transit times when compared to the control at baseline or post-DSS (early and late). Our results merit further research into Pten modulation where increased glia and/or slower intestinal transit times are desired (e.g., short-bowel syndrome and rapid-transit disorders).

Pathophysiology and Management of Postoperative Ileus in Adults and Neonates: A Review.

Postoperative ileus (POI) is caused by enteric neural dysfunction and inflammatory response to the stress of surgery as well as the effect of anesthetics and opioid pain medications. POI results in prolonged hospital stays, increased medical costs, and diminished enteral nutrition, rendering it a problem worth tackling. Many cellular pathways are implicated in this disease process, creating numerous opportunities for targeted management strategies. There is a gap in the literature in studies exploring neonatal POI pathophysiology and treatment options. It is well known that neonatal immune and enteric nervous systems are immature, and this results in gut physiology which is distinct from adults. Neonates undergoing abdominal surgery face similar surgical stressors and exposure to medications that cause POI in adults. In this review, we aim to summarize the existing adult and neonatal literature on POI pathophysiology and management and explore applications in the neonatal population.

Characterizing enteric neurons in dopamine transporter (DAT)-Cre reporter mice reveals dopaminergic subtypes with dual-transmitter content.

The enteric nervous system (ENS) comprises a complex network of neurons whereby a subset appears to be dopaminergic although the characteristics, roles, and implications in disease are less understood. Most investigations relating to enteric dopamine (DA) neurons rely on immunoreactivity to tyrosine hydroxylase (TH)-the rate-limiting enzyme in the production of DA. However, TH immunoreactivity is likely to provide an incomplete picture. This study herein provides a comprehensive characterization of DA neurons in the gut using a reporter mouse line, expressing a fluorescent protein (tdTomato) under control of the DA transporter (DAT) promoter. Our findings confirm a unique localization of DA neurons in the gut and unveil the discrete subtypes of DA neurons in this organ, which we characterized using both immunofluorescence and single-cell transcriptomics, as well as validated using in situ hybridization. We observed distinct subtypes of DAT-tdTomato neurons expressing co-transmitters and modulators across both plexuses; some of them likely co-releasing acetylcholine, while others were positive for a slew of canonical DAergic markers (TH, VMAT2 and GIRK2). Interestingly, we uncovered a seemingly novel population of DA neurons unique to the ENS which was ChAT/DAT-tdTomato-immunoreactive and expressed Grp, Calcb, and Sst. Given the clear heterogeneity of DAergic gut neurons, further investigation is warranted to define their functional signatures and decipher their implication in disease.

Sacral Neural Crest-Independent Origin of the Enteric Nervous System in Mouse.

BACKGROUND & AIMS: The enteric nervous system (ENS), the gut's intrinsic nervous system critical for gastrointestinal function and gut-brain communication, is believed to mainly originate from vagal neural crest cells (vNCCs) and partially from sacral NCCs (sNCCs). Resolving the exact origins of the ENS is critical for understanding congenital ENS diseases but has been confounded by the inability to distinguish between both NCC populations in situ. Here, we aimed to resolve the exact origins of the mammalian ENS. METHODS: We genetically engineered mouse embryos facilitating comparative lineage-tracing of either all (pan-) NCCs including vNCCs or caudal trunk and sNCCs (s/tNCCs) excluding vNCCs. This was combined with dual-lineage tracing and 3-dimensional reconstruction of pelvic plexus and hindgut to precisely pinpoint sNCC and vNCC contributions. We further used coculture assays to determine the specificity of cell migration from different neural tissues into the hindgut. RESULTS: Both pan-NCCs and s/tNCCs contributed to established NCC derivatives but only pan-NCCs contributed to the ENS. Dual-lineage tracing combined with 3-dimensional reconstruction revealed that s/tNCCs settle in complex patterns in pelvic plexus and hindgut-surrounding tissues, explaining previous confusion regarding their contributions. Coculture experiments revealed unspecific cell migration from autonomic, sensory, and neural tube explants into the hindgut. Lineage tracing of ENS precursors lastly provided complimentary evidence for an exclusive vNCC origin of the murine ENS. CONCLUSIONS: sNCCs do not contribute to the murine ENS, suggesting that the mammalian ENS exclusively originates from vNCCs. These results have immediate implications for comprehending (and devising treatments for) congenital ENS disorders, including Hirschsprung's disease.

Brain-first vs. body-first Parkinson's disease: An update on recent evidence.

We recently proposed a new disease model of Parkinson's disease - the a-Synuclein Origin site and Connectome model. The model posits that the initial pathology starts either in the olfactory bulb or amygdala leading to a brain-first subtype, or in the enteric nervous system leading to a body-first subtype. These subtypes should be distinguishable early in the disease course on a range of imaging, clinical, and neuropathological markers. Here, we review recent original human studies, which tested the predictions of the model. Molecular imaging studies were generally in agreement with the model, whereas structural imaging studies, such as MRI volumetry, showed conflicting findings. Most large-scale clinical studies were supportive, reporting clustering of relevant markers of the body-first subtype, including REM-sleep behavior disorder, constipation, autonomic dysfunction, neuropsychiatric symptoms, and cognitive impairment. Finally, studies of a-synuclein deposition in antemortem and postmortem tissues revealed distribution of pathology, which generally supports the model.

Peripheral neuronal activation shapes the microbiome and alters gut physiology.

The gastrointestinal (GI) tract is innervated by intrinsic neurons of the enteric nervous system (ENS) and extrinsic neurons of the central nervous system and peripheral ganglia. The GI tract also harbors a diverse microbiome, but interactions between the ENS and the microbiome remain poorly understood. Here, we activate choline acetyltransferase (ChAT)-expressing or tyrosine hydroxylase (TH)-expressing gut-associated neurons in mice to determine effects on intestinal microbial communities and their metabolites as well as on host physiology. The resulting multi-omics datasets support broad roles for discrete peripheral neuronal subtypes in shaping microbiome structure, including modulating bile acid profiles and fungal colonization. Physiologically, activation of either ChAT+ or TH+ neurons increases fecal output, while only ChAT+ activation results in increased colonic contractility and diarrhea-like fluid secretion. These findings suggest that specific subsets of peripherally activated neurons differentially regulate the gut microbiome and GI physiology in mice without involvement of signals from the brain.

Autologous cell transplantation for treatment of colorectal aganglionosis in mice.

Neurointestinal diseases cause significant morbidity and effective treatments are lacking. This study aimes to test the feasibility of transplanting autologous enteric neural stem cells (ENSCs) to rescue the enteric nervous system (ENS) in a model of colonic aganglionosis. ENSCs are isolated from a segment of small intestine from Wnt1::Cre;R26iDTR mice in which focal colonic aganglionosis is simultaneously created by diphtheria toxin injection. Autologous ENSCs are isolated, expanded, labeled with lentiviral-GFP, and transplanted into the aganglionic segment in vivo. ENSCs differentiate into neurons and glia, cluster to form neo-ganglia, and restore colonic contractile activity as shown by electrical field stimulation and optogenetics. Using a non-lethal model of colonic aganglionosis, our results demonstrate the potential of autologous ENSC therapy to improve functional outcomes in neurointestinal disease, laying the groundwork for clinical application of this regenerative cell-based approach.

The critical role of muscularis macrophages in modulating the enteric nervous system function and gastrointestinal motility.

Macrophages are the originators of inflammatory compounds, phagocytic purifiers in their local environment, and wound healing protectors in oxidative environments. They are molded by the tissue milieu they inhabit, with gastrointestinal (GI) muscularis macrophages (MMs) being a prime example. MMs are located in the muscular layer of the GI tract and contribute to muscle repair and maintenance of GI motility. MMs are often in close proximity to the enteric nervous system, specifically near the enteric neurons and interstitial cells of Cajal (ICCs). Consequently, the anti-inflammatory function of MMs corresponds to the development and maintenance of neural networks in the GI tract. The capacity of MMs to shift from anti-inflammatory to proinflammatory states may contribute to the inflammatory aspects of various GI diseases and disorders such as diabetic gastroparesis or postoperative ileus, functional disorders such as irritable bowel syndrome, and organic diseases such as inflammatory bowel disease. We reviewed the current knowledge of MMs and their influence on neighboring cells due to their important role in the GI tract.

Spatial gene expression profile of Wnt-signaling components in the murine enteric nervous system.

Introduction: Wnt-signaling is a key regulator of stem cell homeostasis, extensively studied in the intestinal crypt and other metazoan tissues. Yet, there is hardly any data available on the presence of Wnt-signaling components in the adult enteric nervous system (ENS) in vivo. Methods: Therefore, we employed RNAscope HiPlex-assay, a novel and more sensitive in situ hybridization technology. By amplifying target specific signals, this technique enables the detection of low abundance, tightly regulated RNA content as is the case for Wnt-signaling components. Additionally, we compared our data to previously published physiological single cell RNA and RiboTag-based RNA sequencing analyses of enteric gliosis using data-mining approaches. Results: Our descriptive analysis shows that several components of the multidi-mensional regulatory network of the Wnt-signaling pathway are present in the murine ENS. The transport and secretion protein for Wnt-ligands Wntless as well as canonical (Wnt3a and Wnt2b) and non-canonical Wnt-ligands (Wnt5a, Wnt7a, Wnt8b and Wnt11) are detectable within submucosal and myenteric plexus. Further, corresponding Frizzled receptors (Fzd1, Fzd3, Fzd6, and Fzd7) and regulatory signaling mediators like R-Spondin/DKK ligands are present in the ENS of the small and large intestine. Further, data mining approaches revealed, that several Wnt-related molecules are expressed by enteric glial cell clusters and are dynamically regulated during the inflammatory manifestation of enteric gliosis. Discussion: Our results suggest, that canonical and non-canonical Wnt-signaling has a much broader impact on the mature ENS and its cellular homeostasis in health and inflammation, than previously anticipated.

Agrin Inhibition in Enteric Neural Stem Cells Enhances Their Migration Following Colonic Transplantation.

Regenerative cell therapy to replenish the missing neurons and glia in the aganglionic segment of Hirschsprung disease represents a promising treatment option. However, the success of cell therapies for this condition are hindered by poor migration of the transplanted cells. This limitation is in part due to a markedly less permissive extracellular environment in the postnatal gut than that of the embryo. Coordinated interactions between enteric neural crest-derived cells (ENCDCs) and their local environment drive migration along the embryonic gut during development of the enteric nervous system. Modifying transplanted cells, or the postnatal extracellular environment, to better recapitulate embryonic ENCDC migration could be leveraged to improve the engraftment and coverage of stem cell transplants. We compared the transcriptomes of ENCDCs from the embryonic intestine to that of postnatal-derived neurospheres and identified 89 extracellular matrix (ECM)-associated genes that are differentially expressed. Agrin, a heparin sulfate proteoglycan with a known inhibitory effect on ENCDC migration, was highly over-expressed by postnatal-derived neurospheres. Using a function-blocking antibody and a shRNA-expressing lentivirus, we show that inhibiting agrin promotes ENCDC migration in vitro and following cell transplantation ex vivo and in vivo. This enhanced migration is associated with an increased proportion of GFAP + cells, whose migration is especially enhanced.

Differences in enteric neuronal density in the NSE-Noggin mouse model across institutes.

The enteric nervous system (ENS) is a large and complex part of the peripheral nervous system, and it is vital for gut homeostasis. To study the ENS, different hyper- and hypo-innervated model systems have been developed. The NSE-Noggin mouse model was described as one of the few models with a higher enteric neuronal density in the colon. However, in our hands NSE-Noggin mice did not present with a hyperganglionic phenotype. NSE-Noggin mice were phenotyped based on fur appearance, genotyped and DNA sequenced to demonstrate transgene and intact NSE-Noggin-IRES-EGFP construct presence, and RNA expression of Noggin was shown to be upregulated. Positive EGFP staining in the plexus of NSE-Noggin mice also confirmed Noggin protein expression. Myenteric plexus preparations of the colon were examined to quantify both the overall density of enteric neurons and the proportions of enteric neurons expressing specific subtype markers. The total number of enteric neurons in the colonic myenteric plexus of transgenic mice did not differ significantly from wild types, nor did the proportion of calbindin, calretinin, or serotonin immunoreactive myenteric neurons. Possible reasons as to why the hyperinnervated phenotype could not be observed in contrast with original studies using this mouse model are discussed, including study design, influence of microbiota, and other environmental variables.

Updates and Challenges in ENS Cell Therapy for the Treatment of Neurointestinal Diseases.

Neurointestinal diseases represent a significant challenge in clinical management with current palliative approaches failing to overcome disease and treatment-related morbidity. The recent progress with cell therapy to restore missing or defective components of the gut neuromusculature offers new hope for potential cures. This review discusses the progress that has been made in the sourcing of putative stem cells and the studies into their biology and therapeutic potential. We also explore some of the practical challenges that must be overcome before cell-based therapies can be applied in the clinical setting. Although a number of obstacles remain, the rapid advances made in the enteric neural stem cell field suggest that such therapies are on the near horizon.

Optogenetic Activation of Cholinergic Enteric Neurons Reduces Inflammation in Experimental Colitis.

BACKGROUND & AIMS: Intestinal inflammation is associated with loss of enteric cholinergic neurons. Given the systemic anti-inflammatory role of cholinergic innervation, we hypothesized that enteric cholinergic neurons similarly possess anti-inflammatory properties and may represent a novel target to treat inflammatory bowel disease. METHODS: Mice were fed 2.5% dextran sodium sulfate (DSS) for 7 days to induce colitis. Cholinergic enteric neurons, which express choline acetyltransferase (ChAT), were focally ablated in the midcolon of ChAT::Cre;R26-iDTR mice by local injection of diphtheria toxin before colitis induction. Activation of enteric cholinergic neurons was achieved using ChAT::Cre;R26-ChR2 mice, in which ChAT+ neurons express channelrhodopsin-2, with daily blue light stimulation delivered via an intracolonic probe during the 7 days of DSS treatment. Colitis severity, ENS structure, and smooth muscle contractility were assessed by histology, immunohistochemistry, quantitative polymerase chain reaction, organ bath, and electromyography. In vitro studies assessed the anti-inflammatory role of enteric cholinergic neurons on cultured muscularis macrophages. RESULTS: Ablation of ChAT+ neurons in DSS-treated mice exacerbated colitis, as measured by weight loss, colon shortening, histologic inflammation, and CD45+ cell infiltration, and led to colonic dysmotility. Conversely, optogenetic activation of enteric cholinergic neurons improved colitis, preserved smooth muscle contractility, protected against loss of cholinergic neurons, and reduced proinflammatory cytokine production. Both acetylcholine and optogenetic cholinergic neuron activation in vitro reduced proinflammatory cytokine expression in lipopolysaccharide-stimulated muscularis macrophages. CONCLUSIONS: These findings show that enteric cholinergic neurons have an anti-inflammatory role in the colon and should be explored as a potential inflammatory bowel disease treatment.

Genetic regulation of enteric nervous system development in zebrafish.

The enteric nervous system (ENS) is a complex series of interconnected neurons and glia that reside within and along the entire length of the gastrointestinal tract. ENS functions are vital to gut homeostasis and digestion, including local control of peristalsis, water balance, and intestinal cell barrier function. How the ENS develops during embryological development is a topic of great concern, as defects in ENS development can result in various diseases, the most common being Hirschsprung disease, in which variable regions of the infant gut lack ENS, with the distal colon most affected. Deciphering how the ENS forms from its progenitor cells, enteric neural crest cells, is an active area of research across various animal models. The vertebrate animal model, zebrafish, has been increasingly leveraged to understand early ENS formation, and over the past 20 years has contributed to our knowledge of the genetic regulation that underlies enteric development. In this review, I summarize our knowledge regarding the genetic regulation of zebrafish enteric neuronal development, and based on the most current literature, present a gene regulatory network inferred to underlie its construction. I also provide perspectives on areas for future zebrafish ENS research.

Stroke Alters the Function of Enteric Neurons to Impair Smooth Muscle Relaxation and Dysregulates Gut Transit.

BACKGROUND: Gut dysmotility is common after ischemic stroke, but the mechanism underlying this response is unknown. Under homeostasis, gut motility is regulated by the neurons of the enteric nervous system that control contractile/relaxation activity of muscle cells in the gut wall. More recently, studies of gut inflammation revealed interactions of macrophages with enteric neurons are also involved in modulating gut motility. However, whether poststroke gut dysmotility is mediated by direct signaling to the enteric nervous system or indirectly via inflammatory macrophages is unknown. METHODS AND RESULTS: We examined these hypotheses by using a clinically relevant permanent intraluminal midcerebral artery occlusion experimental model of stroke. At 24 hours after stroke, we performed in vivo and ex vivo gut motility assays, flow cytometry, immunofluorescence, and transcriptomic analysis. Stroke-induced gut dysmotility was associated with recruitment of muscularis macrophages into the gastrointestinal tract and redistribution of muscularis macrophages away from myenteric ganglia. The permanent intraluminal midcerebral artery occlusion model caused changes in gene expression in muscularis macrophages consistent with an altered phenotype. While the size of myenteric ganglia after stroke was not altered, myenteric neurons from post-permanent intraluminal midcerebral artery occlusion mice showed a reduction in neuronal nitric oxide synthase expression, and this response was associated with enhanced intestinal smooth muscle contraction ex vivo. Finally, chemical sympathectomy with 6-hydroxydopamine prevented the loss of myenteric neuronal nitric oxide synthase expression and stroke-induced slowed gut transit. CONCLUSIONS: Our findings demonstrate that activation of the sympathetic nervous system after stroke is associated with reduced neuronal nitric oxide synthase expression in myenteric neurons, resulting in impaired smooth muscle relaxation and dysregulation of gut transit.

Characterization of mitochondria-associated ER membranes in the enteric nervous system under physiological and pathological conditions.

Alterations in endoplasmic reticulum (ER)-mitochondria associations and in mitochondria-associated ER membrane (MAM) behavior have been reported in the brain in several neurodegenerative diseases. Despite the emerging role of the gut-brain axis in neurodegenerative disorders, the biology of MAM in the enteric nervous system (ENS) has not previously been studied. Therefore, we set out to characterize the MAM in the distal colon of wild-type C57BL/6J mice and senescence-accelerated mouse prone 8 (SAMP8), a mouse model of age-related neurodegeneration. We showed for the first time that MAMs are widely present in enteric neurons and that their association is altered in SAMP8 mice. We then examined the functions of MAMs in a primary culture model of enteric neurons and showed that calcium homeostasis was altered in SAMP8 mice when compared with control animals. These findings provide the first detailed characterization of MAMs in the ENS under physiological conditions and during age-associated neurodegeneration. Further investigation of MAM modifications in the ENS in disease may provide valuable information about the possible role of enteric MAMs in neurodegenerative diseases.NEW & NOTEWORTHY Our work shows for the first time the presence of contacts between endoplasmic reticulum and mitochondria in the enteric neurons and that the dynamic of these contacts is affected in these cells from an age-related neurodegeneration mouse model. It provides new insights into the potential role of enteric mitochondria-associated endoplasmic reticulum membrane in neurodegenerative disorders.