Temperature-dependent differences in mouse gut motility are mediated by stress.

Researchers have advocated elevating mouse housing temperatures from the conventional ~22 °C to the mouse thermoneutral point of 30 °C to enhance translational research. However, the impact of environmental temperature on mouse gastrointestinal physiology remains largely unexplored. Here we show that mice raised at 22 °C exhibit whole gut transit speed nearly twice as fast as those raised at 30 °C, primarily driven by a threefold increase in colon transit speed. Furthermore, gut microbiota composition differs between the two temperatures but does not dictate temperature-dependent differences in gut motility. Notably, increased stress signals from the hypothalamic-pituitary-adrenal axis at 22 °C have a pivotal role in mediating temperature-dependent differences in gut motility. Pharmacological and genetic depletion of the stress hormone corticotropin-releasing hormone slows gut motility in stressed 22 °C mice but has no comparable effect in relatively unstressed 30 °C mice. In conclusion, our findings highlight that colder mouse facility temperatures significantly increase gut motility through hormonal stress pathways.

Gpr37 modulates the severity of inflammation-induced GI dysmotility by regulating enteric reactive gliosis.

The enteric nervous system (ENS) is contained within two layers of the gut wall and is made up of neurons, immune cells, and enteric glia cells (EGCs) that regulate gastrointestinal (GI) function. EGCs in both inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS) change in response to inflammation, referred to as reactive gliosis. Whether EGCs restricted to a specific layer or region within the GI tract alone can influence intestinal immune response is unknown. Using bulk RNA-sequencing and in situ hybridization, we identify G-protein coupled receptor Gpr37 , as a gene expressed only in EGCs of the myenteric plexus, one of the two layers of the ENS. We show that Gpr37 contributes to key components of LPS-induced reactive gliosis including activation of NF-kB and IFN-y signaling and response genes, lymphocyte recruitment, and inflammation-induced GI dysmotility. Targeting Gpr37 in EGCs presents a potential avenue for modifying inflammatory processes in the ENS.

Enteric Nervous System Striped Patterning and Disease: Unexplored Pathophysiology.

The enteric nervous system (ENS) controls gastrointestinal (GI) motility, and defects in ENS development underlie pediatric GI motility disorders. In disorders such as Hirschsprung's disease (HSCR), pediatric intestinal pseudo-obstruction (PIPO), and intestinal neuronal dysplasia type B (INDB), ENS structure is altered with noted decreased neuronal density in HSCR and reports of increased neuronal density in PIPO and INDB. The developmental origin of these structural deficits is not fully understood. Here, we review the current understanding of ENS development and pediatric GI motility disorders incorporating new data on ENS structure. In particular, emerging evidence demonstrates that enteric neurons are patterned into circumferential stripes along the longitudinal axis of the intestine during mouse and human development. This novel understanding of ENS structure proposes new questions about the pathophysiology of pediatric GI motility disorders. If the ENS is organized into stripes, could the observed changes in enteric neuron density in HSCR, PIPO, and INDB represent differences in the distribution of enteric neuronal stripes? We review mechanisms of striped patterning from other biological systems and propose how defects in striped ENS patterning could explain structural deficits observed in pediatric GI motility disorders.

Comparison of wholemount dissection methods for neuronal subtype marker expression in the mouse myenteric plexus.

BACKGROUND: Accurately reporting the identity and representation of enteric nervous system (ENS) neuronal subtypes along the length of the gastrointestinal (GI) tract is critical to advancing our understanding of ENS control of GI function. Reports of varying proportions of subtype marker expression have employed different dissection techniques to achieve wholemount muscularis preparations of myenteric plexus. In this study, we asked whether differences in GI dissection methods could introduce variability into the quantification of marker expression. METHODS: We compared three commonly used methods of ENS wholemount dissection: two flat-sheet preparations that differed in the order of microdissection and fixation and a third rod-mounted peeling technique. We also tested a reversed orientation variation of flat-sheet peeling, two step-by-step variations of the rod peeling technique, and whole-gut fixation as a tube. We assessed marker expression using immunohistochemistry, genetic reporter lines, confocal microscopy, and automated image analysis. KEY RESULTS AND CONCLUSIONS: We found no significant differences between the two flat-sheet preparation methods in the expression of calretinin or neuronal nitric oxide synthase (nNOS) as a proportion of total neurons in ileum myenteric plexus. However, the rod-mounted peeling method resulted in decreased proportion of neurons labeled for both calretinin and nNOS. This method also resulted in decreased transgenic reporter fluorescent protein (tdTomato) for substance P in distal colon and choline acetyltransferase (ChAT) in both ileum and distal colon. These results suggest that labeling among some markers, both native protein and transgenic fluorescent reporters, is decreased by the rod-mounted mechanical method of peeling. The step-by-step variations of this method point to mechanical manipulation of the tissue as the likely cause of decreased labeling. Our study thereby demonstrates a critical variability in wholemount muscularis dissection methods.

Loss of ASD-related molecule Cntnap2 affects colonic motility in mice.

Gastrointestinal (GI) symptoms are highly prevalent among individuals with autism spectrum disorder (ASD), but the molecular link between ASD and GI dysfunction remains poorly understood. The enteric nervous system (ENS) is critical for normal GI motility and has been shown to be altered in mouse models of ASD and other neurological disorders. Contactin-associated protein-like 2 (Cntnap2) is an ASD-related synaptic cell-adhesion molecule important for sensory processing. In this study, we examine the role of Cntnap2 in GI motility by characterizing Cntnap2's expression in the ENS and assessing GI function in Cntnap2 mutant mice. We find Cntnap2 expression predominately in enteric sensory neurons. We further assess in vivo and ex vivo GI motility in Cntnap2 mutants and show altered transit time and colonic motility patterns. The overall organization of the ENS appears undisturbed. Our results suggest that Cntnap2 plays a role in GI function and may provide a molecular link between ASD and GI dysfunction.

Spiral NeuroString: High-Density Soft Bioelectronic Fibers for Multimodal Sensing and Stimulation.

Bioelectronic fibers hold promise for both research and clinical applications due to their compactness, ease of implantation, and ability to incorporate various functionalities such as sensing and stimulation. However, existing devices suffer from bulkiness, rigidity, limited functionality, and low density of active components. These limitations stem from the difficulty to incorporate many components on one-dimensional (1D) fiber devices due to the incompatibility of conventional microfabrication methods (e.g., photolithography) with curved, thin and long fiber structures. Herein, we introduce a fabrication approach, ‶spiral transformation″, to convert two-dimensional (2D) films containing microfabricated devices into 1D soft fibers. This approach allows for the creation of high density multimodal soft bioelectronic fibers, termed Spiral NeuroString (S-NeuroString), while enabling precise control over the longitudinal, angular, and radial positioning and distribution of the functional components. We show the utility of S-NeuroString for motility mapping, serotonin sensing, and tissue stimulation within the dynamic and soft gastrointestinal (GI) system, as well as for single-unit recordings in the brain. The described bioelectronic fibers hold great promises for next-generation multifunctional implantable electronics.

Spontaneous enteric nervous system activity precedes maturation of gastrointestinal motility.

Spontaneous neuronal network activity is essential in development of central and peripheral circuits, yet whether this is a feature of enteric nervous system development has yet to be established. Using ex vivo gastrointestinal (GI) motility assays with unbiased computational analyses, we identify a previously unknown pattern of spontaneous neurogenic GI motility. We further show that this motility is driven by cholinergic signaling, which may inform GI pharmacology for preterm patients.

Anatomical and functional maturation of the mid-gestation human enteric nervous system.

Immature gastrointestinal motility impedes preterm infant survival. The enteric nervous system controls gastrointestinal motility, yet it is unknown when the human enteric nervous system matures enough to carry out vital functions. Here we demonstrate that the second trimester human fetal enteric nervous system takes on a striped organization akin to the embryonic mouse. Further, we perform ex vivo functional assays of human fetal tissue and find that human fetal gastrointestinal motility matures in a similar progression to embryonic mouse gastrointestinal motility. Together, this provides critical knowledge, which facilitates comparisons with common animal models to advance translational disease investigations and testing of pharmacological agents to enhance gastrointestinal motility in prematurity.

Loss of ASD-Related Molecule Cntnap2 Affects Colonic Motility in Mice.

Gastrointestinal (GI) symptoms are highly prevalent among individuals with autism spectrum disorder (ASD), but the molecular link between ASD and GI dysfunction remains poorly understood. The enteric nervous system (ENS) is critical for normal GI motility and has been shown to be altered in mouse models of ASD and other neurological disorders. Contactin-associated protein-like 2 (Cntnap2) is an ASD-related synaptic cell-adhesion molecule important for sensory processing. In this study, we examine the role of Cntnap2 in GI motility by characterizing Cntnap2's expression in the ENS and assessing GI function in Cntnap2 mutant mice. We find Cntnap2 expression predominately in enteric sensory neurons. We further assess in-vivo and ex-vivo GI motility in Cntnap2 mutants and show altered transit time and colonic motility patterns. The overall organization of the ENS appears undisturbed. Our results suggest that Cntnap2 plays a role in GI function and may provide a molecular link between ASD and GI dysfunction.

Single-cell transcriptomic landscape of the developing human spinal cord.

Understanding spinal cord assembly is essential to elucidate how motor behavior is controlled and how disorders arise. The human spinal cord is exquisitely organized, and this complex organization contributes to the diversity and intricacy of motor behavior and sensory processing. But how this complexity arises at the cellular level in the human spinal cord remains unknown. Here we transcriptomically profiled the midgestation human spinal cord with single-cell resolution and discovered remarkable heterogeneity across and within cell types. Glia displayed diversity related to positional identity along the dorso-ventral and rostro-caudal axes, while astrocytes with specialized transcriptional programs mapped into white and gray matter subtypes. Motor neurons clustered at this stage into groups suggestive of alpha and gamma neurons. We also integrated our data with multiple existing datasets of the developing human spinal cord spanning 22 weeks of gestation to investigate the cell diversity over time. Together with mapping of disease-related genes, this transcriptomic mapping of the developing human spinal cord opens new avenues for interrogating the cellular basis of motor control in humans and guides human stem cell-based models of disease.

Single-cell multiome sequencing clarifies enteric glial diversity and identifies an intraganglionic population poised for neurogenesis.

The enteric nervous system (ENS) consists of glial cells (EGCs) and neurons derived from neural crest precursors. EGCs retain capacity for large-scale neurogenesis in culture, and in vivo lineage tracing has identified neurons derived from glial cells in response to inflammation. We thus hypothesize that EGCs possess a chromatin structure poised for neurogenesis. We use single-cell multiome sequencing to simultaneously assess transcription and chromatin accessibility in EGCs undergoing spontaneous neurogenesis in culture, as well as small intestine myenteric plexus EGCs. Cultured EGCs maintain open chromatin at genomic loci accessible in neurons, and neurogenesis from EGCs involves dynamic chromatin rearrangements with a net decrease in accessible chromatin. A subset of in vivo EGCs, highly enriched within the myenteric ganglia and that persist into adulthood, have a gene expression program and chromatin state consistent with neurogenic potential. These results clarify the mechanisms underlying EGC potential for neuronal fate transition.

Comparison of wholemount dissection methods for neuronal subtype marker expression in the mouse myenteric plexus.

Background: Accurately reporting the identity and representation of enteric nervous system (ENS) neuronal subtypes along the length of the gastrointestinal (GI) tract is critical to advancing our understanding of ENS control of GI tract function. Reports of varying proportions of subtype marker expression have employed different dissection techniques to achieve wholemount muscularis preparations of myenteric plexus. In this study we asked whether differences in GI dissection methods could introduce variability into the quantification of marker expression. Methods: We compared three commonly used methods of ENS wholemount dissection: two flat-sheet preparations that differed in the order of microdissection and fixation as well as a rod-mounted peeling technique. We assessed marker expression using immunohistochemistry, genetic reporter lines, confocal microscopy, and automated image analysis. Key Results and Conclusions: We found no significant differences between the two flat-sheet preparation methods in the expression of calretinin, neuronal nitric oxide synthase (nNOS), or somatostatin (SST) in ileum myenteric plexus. However, the rod-mounted peeling method resulted in decreased marker labeling for both calretinin and nNOS. This method also resulted in decreased transgenic reporter fluorescent protein (tdTomato) for substance P in ileum and choline acetyltransferase (ChAT) in both ileum and distal colon. These results suggest that labeling among some markers, both native protein and transgenic fluorescent reporters, is decreased by the rod-mounted mechanical method of peeling, demonstrating a critical variability in wholemount muscularis dissection methods.

Transcription factor gene Pea3 regulates erectile function during copulation in mice.

Male mice with homozygous loss of function mutations of the transcription factor gene Pea3 (Pea3 null) are infertile due to their inability to inseminate females, however the specific deficits in male sexual behaviors that drive this phenotype are unknown. Here, the copulatory behavior of male mice (Pea3 null and control) with hormonally primed ovariectomized females was monitored via high-speed and high-resolution digital videography to assess for differences in female-directed social behaviors, gross sexual behaviors (mounting, thrusting), and erectile and ejaculatory function. Pea3 null male mice exhibit greatly reduced erectile function, with 44% of males displaying no visible erections during copulation, and 0% achieving sustained erections. As such, Pea3 null males are incapable of intromission and copulatory plug deposition, despite displaying largely normal female-directed social behaviors, mounting behaviors, and ejaculatory grasping behavior. Additionally, the organization and timing of thrusting behaviors is impaired in Pea3 null males. Our results show that the transcription factor gene Pea3 regulates the ability to achieve and maintain erections during copulation in mice.

Regional cytoarchitecture of the adult and developing mouse enteric nervous system.

The organization and cellular composition of tissues are key determinants of their biological function. In the mammalian gastrointestinal (GI) tract, the enteric nervous system (ENS) intercalates between muscular and epithelial layers of the gut wall and can control GI function independent of central nervous system (CNS) input.1 As in the CNS, distinct regions of the GI tract are highly specialized and support diverse functions, yet the regional and spatial organization of the ENS remains poorly characterized.2 Cellular arrangements,3,4 circuit connectivity patterns,5,6 and diverse cell types7-9 are known to underpin ENS functional complexity and GI function, but enteric neurons are most typically described only as a uniform meshwork of interconnected ganglia. Here, we present a bird's eye view of the mouse ENS, describing its previously underappreciated cytoarchitecture and regional variation. We visually and computationally demonstrate that enteric neurons are organized in circumferential neuronal stripes. This organization emerges gradually during the perinatal period, with neuronal stripe formation in the small intestine (SI) preceding that in the colon. The width of neuronal stripes varies throughout the length of the GI tract, and distinct neuronal subtypes differentially populate specific regions of the GI tract, with stark contrasts between SI and colon as well as within subregions of each. This characterization provides a blueprint for future understanding of region-specific GI function and identifying ENS structural correlates of diverse GI disorders.

Unraveling the complex genetics of neural tube defects: From biological models to human genomics and back.

Neural tube defects (NTDs) are a classic example of preventable birth defects for which there is a proven-effective intervention, folic acid (FA); however, further methods of prevention remain unrealized. In the decades following implementation of FA nutritional fortification programs throughout at least 87 nations, it has become apparent that not all NTDs can be prevented by FA. In the United States, FA fortification only reduced NTD rates by 28-35% (Williams et al., 2015). As such, it is imperative that further work is performed to understand the risk factors associated with NTDs and their underlying mechanisms so that alternative prevention strategies can be developed. However, this is complicated by the sheer number of genes associated with neural tube development, the heterogeneity of observable phenotypes in human cases, the rareness of the disease, and the myriad of environmental factors associated with NTD risk. Given the complex genetic architecture underlying NTD pathology and the way in which that architecture interacts dynamically with environmental factors, further prevention initiatives will undoubtedly require precision medicine strategies that utilize the power of human genomics and modern tools for assessing genetic risk factors. Herein, we review recent advances in genomic strategies for discovering genetic variants associated with these defects, and new ways in which biological models, such as mice and cell culture-derived organoids, are leveraged to assess mechanistic functionality, the way these variants interact with other genetic or environmental factors, and their ultimate contribution to human NTD risk.

Proprioception revisited: where do we stand?

Originally referred to as 'muscle sense', the notion that skeletal muscle held a peripheral sensory function was first described early in the 19th century. Foundational experiments by Sherrington in the early 20th century definitively demonstrated that proprioceptors contained within skeletal muscle, tendons, and joints are innervated by sensory neurons and play an important role in the control of movement. In this review, we will highlight several recent advances in the ongoing effort to further define the molecular diversity underlying the proprioceptive sensorimotor system. Together, the work summarized here represents our current understanding of sensorimotor circuit formation during development and the mechanisms that regulate the integration of proprioceptive feedback into the spinal circuits that control locomotion in both normal and diseased states.

COUNTEN, an AI-Driven Tool for Rapid and Objective Structural Analyses of the Enteric Nervous System.

The enteric nervous system (ENS) consists of an interconnected meshwork of neurons and glia residing within the wall of the gastrointestinal (GI) tract. While healthy GI function is associated with healthy ENS structure, defined by the normal distribution of neurons within ganglia of the ENS, a comprehensive understanding of normal neuronal distribution and ganglionic organization in the ENS is lacking. Current methodologies for manual enumeration of neurons parse only limited tissue regions and are prone to error, subjective bias, and peer-to-peer discordance. There is accordingly a need for robust, and objective tools that can capture and quantify enteric neurons within multiple ganglia over large areas of tissue. Here, we report on the development of an AI-driven tool, COUNTEN (COUNTing Enteric Neurons), which is capable of accurately identifying and enumerating immunolabeled enteric neurons, and objectively clustering them into ganglia. We tested and found that COUNTEN matches trained humans in its accuracy while taking a fraction of the time to complete the analyses. Finally, we use COUNTEN's accuracy and speed to identify and cluster thousands of ileal myenteric neurons into hundreds of ganglia to compute metrics that help define the normal structure of the ileal myenteric plexus. To facilitate reproducible, robust, and objective measures of ENS structure across mouse models, experiments, and institutions, COUNTEN is freely and openly available to all researchers.

Single-cell transcriptomic analysis of the adult mouse spinal cord reveals molecular diversity of autonomic and skeletal motor neurons.

The spinal cord is a fascinating structure that is responsible for coordinating movement in vertebrates. Spinal motor neurons control muscle activity by transmitting signals from the spinal cord to diverse peripheral targets. In this study, we profiled 43,890 single-nucleus transcriptomes from the adult mouse spinal cord using fluorescence-activated nuclei sorting to enrich for motor neuron nuclei. We identified 16 sympathetic motor neuron clusters, which are distinguishable by spatial localization and expression of neuromodulatory signaling genes. We found surprising skeletal motor neuron heterogeneity in the adult spinal cord, including transcriptional differences that correlate with electrophysiologically and spatially distinct motor pools. We also provide evidence for a novel transcriptional subpopulation of skeletal motor neuron (γ*). Collectively, these data provide a single-cell transcriptional atlas ( http://spinalcordatlas.org ) for investigating the organizing molecular logic of adult motor neuron diversity, as well as the cellular and molecular basis of motor neuron function in health and disease.

Chandelier Cells Swipe Right for L1CAM.

Establishing a functional neuronal circuit requires not only synapsing with the right cell type, but also targeting the right subcellular compartment. In this issue of Neuron, Tai et al. (2019) identify the cell adhesion molecule L1CAM as integral to the mechanism by which chandelier cells establish subcellular compartment-specific innervation of pyramidal neurons in the mammalian cerebral cortex.

Sensory and descending motor circuitry during development and injury.

Proprioceptive sensory input and descending supraspinal projections are two major inputs that feed into and influence spinal circuitry and locomotor behaviors. Here we review their influence on each other during development and after spinal cord injury. We highlight developmental mechanisms of circuit formation as they relate to the sensory-motor circuit and its reciprocal interactions with local spinal interneurons, as well as competitive interactions between proprioceptive and descending supraspinal inputs in the setting of spinal cord injury.