Agonist-Evoked Ca2+ Signaling in Enteric Glia Drives Neural Programs That Regulate Intestinal Motility in MiceSummary

Background & Aims: Gastrointestinal motility is regulated by enteric neural circuitry that includes enteric neurons and glia. Enteric glia monitor synaptic activity and exhibit responses to neurotransmitters that are encoded by intracellular calcium (Ca2+) signaling. What role evoked glial responses play in the neural regulation of gut motility is unknown. We tested how evoking Ca2+ signaling in enteric glia affects the neural control of intestinal motility. Methods: We used a novel chemogenetic mouse model that expresses the designer receptor hM3Dq under the transcriptional control of the glial fibrillary acidic protein (GFAP) promoter (GFAP::hM3Dq mice) to selectively trigger glial Ca2+ signaling. We used in situ Ca2+ imaging and immunohistochemistry to validate this model, and we assessed gut motility by measuring pellet output and composition, colonic bead expulsion time, small intestinal transit time, total gut transit time, colonic migrating motor complex (CMMC) recordings, and muscle tension recordings. Results: Expression of the hM3Dq receptor is confined to GFAP-positive enteric glia in the intestines of GFAP::hM3Dq mice. In these mice, application of the hM3Dq agonist clozapine-N-oxide (CNO) selectively triggers intracellular Ca2+ responses in enteric glia. Glial activation drove neurogenic contractions in the ileum and colon but had no effect on neurogenic relaxations. CNO enhanced the amplitude and frequency of CMMCs in ex vivo preparations of the colon, and CNO increased colonic motility in vivo. CNO had no effect on the composition of fecal matter, small intestinal transit, or whole gut transit. Conclusions: Glial excitability encoded by intracellular Ca2+ signaling functions to modulate excitatory enteric circuits. Selectively triggering glial Ca2+ signaling might be a novel strategy to improve gut function in motility disorders. Keywords: Autonomic, Chemogenetic, Enteric Nervous System, Intestine, Gu

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

Mineralocorticoid Receptor Antagonism Prevents Obesity-Induced Cerebral Artery Remodeling and Reduces White Matter Injury in Rats

Objective: Midlife obesity is a risk factor for dementia development. Obesity has also been linked to hyperaldosteronism, and this can be modeled in rats by high fat (HF) feeding from weaning. Aldosterone, or activation of the mineralocorticoid receptor (MR) causes cerebrovascular injury in lean hypertensive rats. We hypothesized that rats fed a HF diet would show inward middle cerebral artery (MCA) remodeling that could be prevented by MR antagonism. We further proposed that the cerebral artery remodeling would be associated with white mater injury. Methods: Three-week-old male Sprague-Dawley rats were fed a HF diet ± the MR antagonist canrenoic acid (Canr) for 17 weeks. Control rats received normal chow (control NC). MCA structure was assessed by pressure myography. Results: The MCAs from HF fed rats had smaller lumens and thicker walls when compared to arteries from control NC rats; Canr prevented the MCA remodeling associated with HF feeding. HF feeding increased the mRNA expression of markers of cell proliferation and vascular inflammation in cerebral arteries and Canr treatment prevented this. White mater injury was increased in the rats fed the HF diet and this was reduced by Canr treatment. The expression of doublecortin, a marker of new and immature neurons was reduced in HF fed rats, and MR antagonism normalized this. Conclusions: These data suggest that HF feeding leads to MR dependent remodeling of the MCA and this is associated with markers of dementia development

Enteric Glia Mediate Neuron Death in Colitis Through Purinergic Pathways That Require Connexin-43 and Nitric OxideSummary

Background & Aims: The concept of enteric glia as regulators of intestinal homeostasis is slowly gaining acceptance as a central concept in neurogastroenterology. Yet how glia contribute to intestinal disease is still poorly understood. Purines generated during inflammation drive enteric neuron death by activating neuronal P2X7 purine receptors (P2X7R); triggering adenosine triphosphate (ATP) release via neuronal pannexin-1 channels that subsequently recruits intracellular calcium ([Ca2+]i) in surrounding enteric glia. We tested the hypothesis that the activation of enteric glia contributes to neuron death during inflammation. Methods: We studied neuroinflammation in vivo using the 2,4-dinitrobenzene sulfonic acid model of colitis and in situ using whole-mount preparations of human and mouse intestine. Transgenic mice with a targeted deletion of glial connexin-43 (Cx43) [GFAP::CreERT2+/â/Cx43f/f] were used to specifically disrupt glial signaling pathways. Mice deficient in inducible nitric oxide (NO) synthase (iNOSâ/â) were used to study NO production. Protein expression and oxidative stress were measured using immunohistochemistry and in situ Ca2+ and NO imaging were used to monitor glial [Ca2+]i and [NO]i. Results: Purinergic activation of enteric glia drove [Ca2+]i responses and enteric neuron death through a Cx43-dependent mechanism. Neurotoxic Cx43 activity, driven by NO production from glial iNOS, was required for neuron death. Glial Cx43 opening liberated ATP and Cx43-dependent ATP release was potentiated by NO. Conclusions: Our results show that the activation of glial cells in the context of neuroinflammation kills enteric neurons. Mediators of inflammation that include ATP and NO activate neurotoxic pathways that converge on glial Cx43 hemichannels. The glial response to inflammatory mediators might contribute to the development of motility disorders. Keywords: Enteric Nervous System, Hemichannels, Oxidative Stress, Purine

Sexually Dimorphic Effects of Histamine Degradation by Enteric Glial Histamine <i>N</i>-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

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