Endocannabinoids regulate enteric neuron-glia networks and visceral hypersensitivity following inflammation through a glial-dependent mechanism.

Acute gastrointestinal (GI) inflammation induces neuroplasticity that produces long-lasting changes in gut motor function and pain. The endocannabinoid system is an attractive target to correct pain and dysmotility, but how inflammation changes endocannabinoid control over cellular communication in enteric neurocircuits is not understood. Enteric glia modulate gut neurons that control motility and pain and express monoacylglycerol lipase (MAGL) which controls endocannabinoid availability. We used a combination of in situ calcium imaging, chemogenetics, and selective drugs to study how endocannabinoid mechanisms affect glial responses and subsequent enteric neuron activity in health and following colitis in Wnt1Cre;GCaMP5g-tdT;GFAP::hM3Dq mice. Trpv1Cre;GCaMP5gtdT mice were used to study nociceptor sensitivity and Sox10CreERT2;Mgllf/f mice were used to test the role of glial MAGL in visceral pain. The data show that endocannabinoid signaling regulates neuro-glial signaling in gut neurocircuits in a sexually dimorphic manner. Inhibiting MAGL in healthy samples decreased glial responsiveness but this effect was lost in females following colitis and converted to an excitatory effect in males. Manipulating CB1 and CB2 receptors revealed further sex differences amongst neuro-glia signaling that were impacted following inflammation. Inflammation increased gut nociceptor sensitivity in both sexes but only females exhibited visceral hypersensitivity in vivo. Blocking MAGL normalized nociceptor responses in vitro and deleting glial Mgll in vivo rescued visceral hypersensitivity in females. These results show that sex and inflammation impact endocannabinoid mechanisms that regulate intercellular enteric glia-neuron communication. Further, targeting glial MAGL could provide therapeutic benefits for visceral nociception in a sex-dependent manner.

Enteric Glia.

Enteric glia are a unique type of peripheral neuroglia that accompany neurons in the enteric nervous system (ENS) of the digestive tract. The ENS displays integrative neural circuits that are capable of governing moment-to-moment gut functions independent of input from the central nervous system. Enteric glia are interspersed with neurons throughout these intrinsic gut neural circuits and are thought to fulfill complex roles directed at maintaining homeostasis in the neuronal microenvironment and at neuroeffector junctions in the gut. Changes to glial functions contribute to a wide range of gastrointestinal diseases, but the precise roles of enteric glia in gut physiology and pathophysiology are still under examination. This review summarizes current concepts regarding enteric glial development, diversity, and functions in health and disease.

Mechanisms of enteric neuropathy in diverse contexts of gastrointestinal dysfunction.

The enteric nervous system (ENS) commands moment-to-moment gut functions through integrative neurocircuitry housed in the gut wall. The functional continuity of ENS networks is disrupted in enteric neuropathies and contributes to major disturbances in normal gut activities including abnormal gut motility, secretions, pain, immune dysregulation, and disrupted signaling along the gut-brain axis. The conditions under which enteric neuropathy occurs are diverse and the mechanistic underpinnings are incompletely understood. The purpose of this brief review is to summarize the current understanding of the cell types involved, the conditions in which neuropathy occurs, and the mechanisms implicated in enteric neuropathy such as oxidative stress, toll like receptor signaling, purines, and pre-programmed cell death.

Early life adversity promotes gastrointestinal dysfunction through a sex-dependent phenotypic switch in enteric glia.

Irritable bowel syndrome and related disorders of gut-brain interaction (DGBI) are common and exhibit a complex, poorly understood etiology that manifests as abnormal gut motility and pain. Risk factors such as biological sex, stressors during critical periods, and inflammation are thought to influence DGBI vulnerability by reprogramming gut-brain circuits, but the specific cells affected are unclear. Here, we used a model of early life stress to understand cellular mechanisms in the gut that produce DGBIs. Our findings identify enteric glia as a key cellular substrate in which stress and biological sex converge to dictate DGBI susceptibility. Enteric glia exhibit sexual dimorphism in genes and functions related to cellular communication, inflammation, and disease susceptibility. Experiencing early life stress has sex-specific effects on enteric glia that cause a phenotypic switch in male glia toward a phenotype normally observed in females. This phenotypic transformation is followed by physiological changes in the gut, mirroring those observed in DGBI in humans. These effects are mediated, in part, by alterations to glial prostaglandin and endocannabinoid signaling. Together, these data identify enteric glia as a cellular integration site through which DGBI risk factors produce changes in gut physiology and suggest that manipulating glial signaling may represent an attractive target for sex-specific therapeutic strategies in DGBIs.

Innervation of adipocytes is limited in mouse perivascular adipose tissue.

Perivascular adipose tissue (PVAT) regulates vascular tone by releasing anticontractile factors. These anticontractile factors are driven by processes downstream of adipocyte stimulation by norepinephrine; however, whether norepinephrine originates from neural innervation or other sources is unknown. The goal of this study was to test the hypothesis that neurons innervating PVAT provide the adrenergic drive to stimulate adipocytes in aortic and mesenteric perivascular adipose tissue (aPVAT and mPVAT), and white adipose tissue (WAT). Healthy male and female mice (8-13 wk) were used in all experiments. Expression of genes associated with synaptic transmission were quantified by qPCR and adipocyte activity in response to neurotransmitters and neuron depolarization was assessed in AdipoqCre+;GCaMP5g-tdTf/WT mice. Immunostaining, tissue clearing, and transgenic reporter lines were used to assess anatomical relationships between nerves and adipocytes. Although synaptic transmission component genes are expressed in adipose tissues (aPVAT, mPVAT, and WAT), strong nerve stimulation with electrical field stimulation does not significantly trigger calcium responses in adipocytes. However, norepinephrine consistently elicits strong calcium responses in adipocytes from all adipose tissues studied. Bethanechol induces minimal adipocyte responses. Imaging neural innervation using various techniques reveals that nerve fibers primarily run alongside blood vessels and rarely branch into the adipose tissue. Although nerve fibers are associated with blood vessels in adipose tissue, they demonstrate limited anatomical and functional interactions with adjacent adipocytes, challenging the concept of classical innervation. These findings dispute the significant involvement of neural input in regulating PVAT adipocyte function and emphasize alternative mechanisms governing adrenergic-driven anticontractile functions of PVAT.NEW & NOTEWORTHY This study challenges prevailing views on neural innervation in perivascular adipose tissue (PVAT) and its role in adrenergic-driven anticontractile effects on vasculature. Contrary to existing paradigms, limited anatomical and functional connections were found between PVAT nerve fibers and adipocytes, underscoring the importance of exploring alternative mechanistic pathways. Understanding the mechanisms involved in PVAT's anticontractile effects is critical for developing potential therapeutic interventions against dysregulated vascular tone, hypertension, and cardiovascular disease.

TRPV4 is expressed by enteric glia and muscularis macrophages of the colon but does not play a prominent role in colonic motility.

Background: Mechanosensation is an important trigger of physiological processes in the gastrointestinal tract. Aberrant responses to mechanical input are associated with digestive disorders, including visceral hypersensitivity. Transient Receptor Potential Vanilloid 4 (TRPV4) is a mechanosensory ion channel with proposed roles in visceral afferent signaling, intestinal inflammation, and gut motility. While TRPV4 is a potential therapeutic target for digestive disease, current mechanistic understanding of how TRPV4 may influence gut function is limited by inconsistent reports of TRPV4 expression and distribution. Methods: In this study we profiled functional expression of TRPV4 using Ca2+ imaging of wholemount preparations of the mouse, monkey, and human intestine in combination with immunofluorescent labeling for established cellular markers. The involvement of TRPV4 in colonic motility was assessed in vitro using videomapping and contraction assays. Results: The TRPV4 agonist GSK1016790A evoked Ca2+ signaling in muscularis macrophages, enteric glia, and endothelial cells. TRPV4 specificity was confirmed using TRPV4 KO mouse tissue or antagonist pre-treatment. Calcium responses were not detected in other cell types required for neuromuscular signaling including enteric neurons, interstitial cells of Cajal, PDGFRα+ cells, and intestinal smooth muscle. TRPV4 activation led to rapid Ca2+ responses by a subpopulation of glial cells, followed by sustained Ca2+ signaling throughout the enteric glial network. Propagation of these waves was suppressed by inhibition of gap junctions or Ca2+ release from intracellular stores. Coordinated glial signaling in response to GSK1016790A was also disrupted in acute TNBS colitis. The involvement of TRPV4 in the initiation and propagation of colonic motility patterns was examined in vitro. Conclusions: We reveal a previously unappreciated role for TRPV4 in the initiation of distension-evoked colonic motility. These observations provide new insights into the functional role of TRPV4 activation in the gut, with important implications for how TRPV4 may influence critical processes including inflammatory signaling and motility.

Sexually Dimorphic Effects of Histamine Degradation by Enteric Glial Histamine N-Methyltransferase (HNMT) on Visceral Hypersensitivity.

Histamine is a neuromodulator that affects gut motility and visceral sensitivity through intrinsic and extrinsic neural pathways, yet the mechanisms regulating histamine availability in these pathways remain poorly understood. Here, we show that enteric glia contribute to histamine clearance in the enteric nervous system (ENS) through their expression of the enzyme histamine N-methyltransferase (HNMT). Glial HNMT expression was initially assessed using immunolabeling and gene expression, and functionally tested using CRISPR-Cas9 to create a Cre-dependent conditional Hnmt ablation model targeting glia. Immunolabeling, calcium imaging, and visceromotor reflex recordings were used to assess the effects on ENS structure and visceral hypersensitivity. Immunolabeling and gene expression data show that enteric neurons and glia express HNMT. Deleting Hnmt in Sox10+ enteric glia increased glial histamine levels and altered visceromotor responses to colorectal distension in male mice, with no effect in females. Interestingly, deleting glial Hnmt protected males from histamine-driven visceral hypersensitivity. These data uncover a significant role for glial HNMT in histamine degradation in the gut, which impacts histamine-driven visceral hypersensitivity in a sex-dependent manner. Changes in the capacity of glia to clear histamines could play a role in the susceptibility to developing visceral pain in disorders of the gut-brain interaction.

Enteric glia promote visceral hypersensitivity during inflammation through intercellular signaling with gut nociceptors.

Inflammation in the intestines causes abdominal pain that is challenging to manage. The terminals of sensory neurons innervating the gut are surrounded by glia. Here, using a mouse model of acute colitis, we found that enteric glia contribute to visceral pain by secreting factors that sensitized sensory nerves innervating the gut in response to inflammation. Acute colitis induced a transient increase in the production of proinflammatory cytokines in the intestines of male and female mice. Of these, IL-1ß was produced in part by glia and augmented the opening of the intercellular communication hemichannel connexin-43 in glia, which made normally innocuous stimuli painful in female mice. Chemogenetic glial activation paired with calcium imaging in nerve terminals demonstrated that glia sensitized gut-innervating nociceptors only under inflammatory conditions. This inflammatory, glial-driven visceral hypersensitivity involved an increased abundance of the enzyme COX-2 in glia, resulting in greater production and release of prostaglandin E2 that activated EP4 receptors on sensory nerve terminals. Blocking EP4 receptors reduced nociceptor sensitivity in response to glial stimulation in tissue samples from colitis-model mice, and impairing glial connexin-43 reduced visceral hypersensitivity induced by IL-1ß in female mice. The findings suggest that therapies targeting enteric glial-neuron signaling might alleviate visceral pain caused by inflammatory disorders.

Stimulator of interferon genes (STING) expression in the enteric nervous system and contributions of glial STING in disease.

BACKGROUND: Appropriate host-microbe interactions are essential for enteric glial development and subsequent gastrointestinal function, but the potential mechanisms of microbe-glial communication are unclear. Here, we tested the hypothesis that enteric glia express the pattern recognition receptor stimulator of interferon genes (STING) and communicate with the microbiome through this pathway to modulate gastrointestinal inflammation. METHODS: In situ transcriptional labeling and immunohistochemistry were used to examine STING and IFNß expression in enteric neurons and glia. Glial-STING KO mice (Sox10CreERT2+/- ;STINGfl/fl ) and IFNß ELISA were used to characterize the role of enteric glia in canonical STING activation. The role of glial STING in gastrointestinal inflammation was assessed in the 3% DSS colitis model. RESULTS: Enteric glia and neurons express STING, but only enteric neurons express IFNß. While both the myenteric and submucosal plexuses produce IFNß with STING activation, enteric glial STING plays a minor role in its production and seems more involved in autophagy processes. Furthermore, deleting enteric glial STING does not affect weight loss, colitis severity, or neuronal cell proportions in the DSS colitis model. CONCLUSION: Taken together, our data support canonical roles for STING and IFNß signaling in the enteric nervous system through enteric neurons but that enteric glia do not use these same mechanisms. We propose that enteric glial STING may utilize alternative signaling mechanisms and/or is only active in particular disease conditions. Regardless, this study provides the first glimpse of STING signaling in the enteric nervous system and highlights a potential avenue of neuroglial-microbial communication.

BQ788 reveals glial ETB receptor modulation of neuronal cholinergic and nitrergic pathways to inhibit intestinal motility: Linked to postoperative ileus.

BACKGROUND AND PURPOSE: ET-1 signalling modulates intestinal motility and inflammation, but the role of ET-1/ETB receptor signalling is poorly understood. Enteric glia modulate normal motility and inflammation. We investigated whether glial ETB signalling regulates neural-motor pathways of intestinal motility and inflammation. EXPERIMENTAL APPROACH: We studied ETB signalling using: ETB drugs (ET-1, SaTX, BQ788), activity-dependent stimulation of neurons (high K+ -depolarization, EFS), gliotoxins, Tg (Ednrb-EGFP)EP59Gsat/Mmucd mice, cell-specific mRNA in Sox10CreERT2 ;Rpl22-HAflx or ChATCre ;Rpl22-HAflx mice, Sox10CreERT2 ::GCaMP5g-tdT, Wnt1Cre2 ::GCaMP5g-tdT mice, muscle tension recordings, fluid-induced peristalsis, ET-1 expression, qPCR, western blots, 3-D LSM-immunofluorescence co-labelling studies in LMMP-CM and a postoperative ileus (POI) model of intestinal inflammation. KEY RESULTS: In the muscularis externa ETB receptor is expressed exclusively in glia. ET-1 is expressed in RiboTag (ChAT)-neurons, isolated ganglia and intra-ganglionic varicose-nerve fibres co-labelled with peripherin or SP. ET-1 release provides activity-dependent glial ETB receptor modulation of Ca2+ waves in neural evoked glial responses. BQ788 reveals amplification of glial and neuronal Ca2+ responses and excitatory cholinergic contractions, sensitive to L-NAME. Gliotoxins disrupt SaTX-induced glial-Ca2+ waves and prevent BQ788 amplification of contractions. The ETB receptor is linked to inhibition of contractions and peristalsis. Inflammation causes glial ETB up-regulation, SaTX-hypersensitivity and glial amplification of ETB signalling. In vivo BQ788 (i.p., 1 mg·kg-1 ) attenuates intestinal inflammation in POI. CONCLUSION AND IMPLICATIONS: Enteric glial ET-1/ETB signalling provides dual modulation of neural-motor circuits to inhibit motility. It inhibits excitatory cholinergic and stimulates inhibitory nitrergic motor pathways. Amplification of glial ETB receptors is linked to muscularis externa inflammation and possibly pathogenic mechanisms of POI.

Enteric Neuromics: How High-Throughput "Omics" Deepens Our Understanding of Enteric Nervous System Genetic Architecture.

Recent accessibility to specialized high-throughput "omics" technologies including single cell RNA sequencing allows researchers to capture cell type- and subtype-specific expression signatures. These omics methods are used in the enteric nervous system (ENS) to identify potential subtypes of enteric neurons and glia. ENS omics data support the known gene and/or protein expression of functional neuronal and glial cell subtypes and suggest expression patterns of novel subtypes. Gene and protein expression patterns can be further used to infer cellular function and implications in human disease. In this review we discuss how high-throughput "omics" data add additional depth to the understanding of established functional subtypes of ENS cells and raise new questions by suggesting novel ENS cell subtypes with unique gene and protein expression patterns. Then we investigate the changes in these expression patterns during pathology observed by omics research. Although current ENS omics studies provide a plethora of novel data and therefore answers, they equally create new questions and routes for future study.

Functional Intraregional and Interregional Heterogeneity between Myenteric Glial Cells of the Colon and Duodenum in Mice.

Enteric glia are a unique population of peripheral neuroglia that regulate homeostasis in the enteric nervous system (ENS) and intestinal functions. Despite existing in functionally diverse regions of the gastrointestinal tract, enteric glia have been approached scientifically as a homogeneous group of cells. This assumption is at odds with the functional specializations of gastrointestinal organs and recent data suggesting glial heterogeneity in the brain and ENS. Here, we used calcium imaging in transgenic mice of both sexes expressing genetically encoded calcium sensors in enteric glia and conducted contractility studies to investigate functional diversity among myenteric glia in two functionally distinct intestinal organs: the duodenum and the colon. Our data show that myenteric glia exhibit regionally distinct responses to neuromodulators that require intercellular communication with neurons to differing extents in the duodenum and colon. Glia regulate intestinal contractility in a region-specific and pathway-specific manner, which suggests regionally diverse engagement of enteric glia in local motor patterns through discrete signaling pathways. Further, functional response profiles delineate four unique subpopulations among myenteric glia that are differentially distributed between the colon and duodenum. Our findings support the conclusion that myenteric glia exhibit both intraregional and interregional heterogeneity that contributes to region-specific mechanisms that regulate digestive functions. Glial heterogeneity adds an unexpected layer of complexity in peripheral neurocircuits, and understanding the specific functions of specialized glial subtypes will provide new insight into ENS physiology and pathophysiology.SIGNIFICANCE STATEMENT Enteric glia modulate gastrointestinal functions through intercellular communication with enteric neurons. Whether heterogeneity exists among neuron-glia interactions in the digestive tract is not understood. Here, we show that myenteric glia display regional heterogeneity in their responses to neuromodulators in the duodenum and the colon, which are functionally distinct organs. Glial-mediated control of intestinal motility is region and pathway specific. Four myenteric glial subtypes are present within a given gut region that are differently distributed between gut regions. These data provide functional and regional insights into enteric circuit specificity in the adult enteric nervous system.

Enteric glial adenosine 2B receptor signaling mediates persistent epithelial barrier dysfunction following acute DSS colitis.

Intestinal epithelial barrier function is compromised in inflammatory bowel disease and barrier dysfunction contributes to disease progression. Extracellular nucleotides/nucleosides generated in gut inflammation may regulate barrier function through actions on diverse cell types. Enteric glia modulate extracellular purinergic signaling and exert pathophysiological effects on mucosal permeability. These glia may regulate inflammation with paracrine responses, theoretically mediated via adenosine 2B receptor (A2BR) signaling. As the cell-specific roles of A2BRs in models of colitis and barrier dysfunction are unclear, we studied glial A2BRs in acute dextran sodium sulfate (DSS) colitis. We performed and validated conditional ablation of glial A2BRs in Sox10CreERT2+/-;Adora2bf/f mice. Overt intestinal disease activity indices in DSS-colitis were comparable between Sox10CreERT2+/-;Adora2bf/f mice and littermate controls. However, ablating glial A2BRs protected against barrier dysfunction following acute DSS-colitis. These benefits were associated with the normalization of tight junction protein expression and localization including claudin-1, claudin-8, and occludin. Glial A2BR signaling increased levels of proinflammatory mediators in the colon and cell-intrinsic regulation of genes including Csf3, Cxcl1, Cxcl10, and Il6. Our studies show that glial A2BR signaling exacerbates immune responses during DSS-colitis and that this adenosinergic cell-specific mechanism contributes to persistent gut epithelial barrier dysfunction.

Astrocyte Cell Surface Antigen 2 and Other Potential Cell Surface Markers of Enteric glia in the Mouse Colon.

Enteric glia regulate gut functions in health and disease through diverse interactions with neurons and immune cells. Intracellular localization of traditional markers of enteric glia such as GFAP, s100b, and Sox10 makes them incompatible for studies that require antigen localization at the cell surface. Thus, new tools are needed for probing the heterogeneous roles of enteric glia at the protein, cell, and functional levels. Here we selected several cell surface antigens including Astrocyte Cell Surface Marker 2 (ACSA2), Cluster of differentiation 9 (CD9), lysophosphatidic acid receptor 1 (LPAR1), and Proteolipid protein 1 (PLP1) as potential markers of enteric glia. We tested their specificity for enteric glia using published single-cell/-nuclei and glia-specific translating mRNA enriched transcriptome datasets, immunolabeling, and flow cytometry. The data show that ACSA2 is a specific marker of mucosal and myenteric glia while other markers are suitable for identifying all subpopulations of enteric glia (LPAR1), glia and immune cells (CD9), or are not suitable for cell-surface labeling (PLP1). These new tools will be useful for future work focused on understanding specific glial functions in health and disease.Summary StatementThis study identifies astrocyte cell surface antigen 2 as a novel marker of myenteric glia in the intestine. This, in combination with other markers identified in this study, could be used for selective targeting of enteric glia.

LPAR1 regulates enteric nervous system function through glial signaling and contributes to chronic intestinal pseudo-obstruction.

Gastrointestinal motility disorders involve alterations to the structure and/or function of the enteric nervous system (ENS) but the causal mechanisms remain unresolved in most cases. Homeostasis and disease in the ENS are processes that are regulated by enteric glia. Signaling mediated through type I lysophosphatidic acid receptors (LPAR1) has recently emerged as an important mechanism that contributes to disease, in part, through effects on peripheral glial survival and function. Enteric glia express LPAR1 but its role in ENS function and motility disorders is unknown. We used a combination of genetic, immunohistochemical, calcium imaging, and in vivo pharmacological approaches to investigate the role of LPAR1 in enteric glia. LPAR1 was enriched in enteric glia in mice and humans and LPA stimulated intracellular calcium responses in enteric glia, subsequently recruiting activity in a subpopulation of myenteric neurons. Blocking LPAR1 in vivo with AM966 attenuated gastrointestinal motility in mice and produced marked enteric neuro- and gliopathy. Samples from humans with chronic intestinal pseudo-obstruction (CIPO), a severe motility disorder, showed reduced glial LPAR1 expression in the colon and ileum. These data suggest that enteric glial LPAR1 signaling regulates gastrointestinal motility through enteric glia and could contribute to severe motility disorders in humans such as CIPO.