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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.

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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.

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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.

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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.

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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.

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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.

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Gulf War Illness (GWI) is a chronic multisymptom illness that includes gastrointestinal disorders. Although the exact etiology of GWI is unknown, exposure to the drug pyridostigmine bromide (PB) is considered a major factor. Exposure to PB drives enteric neuroinflammation, promotes immunosuppression, and alters physiological functions of the colon in the short term but whether exposure to PB is sufficient to promote long term dysfunction is not known. Here, we tested whether exposure to PB is sufficient to drive long term changes that reflect GWI, and whether the endogenous anti-inflammatory mediator palmitoylethanolamide (PEA) is sufficient to reduce the detrimental effects of PB in the gut and brain of mice. Exposure to PB alone was not sufficient to cause major changes in neuromuscular transmission but did drive major changes by altering the effects of PEA. Calcium imaging data show that the mechanisms responsible include a shift in receptor signaling mediated by TRPV1, endocannabinoids, and peroxisome proliferator-activated receptors alpha (PPARα). Additional mechanisms include the development of glial reactivity and changes in enteric neurochemical coding and survival. PB and PEA caused major shifts in pro-inflammatory cytokines/chemokines in the brain and colon that persisted up to 5 months following exposure. Many of the effects of PB and PEA exhibit significant sex differences. Together, these results highlight novel mechanisms whereby PB promotes long-lasting changes in nervous system and immune function by inducing occult neuroplasticity that is revealed by subsequent exposure to unrelated drugs in a sex dependent manner.

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Chronic abdominal pain is the most common gastrointestinal issue and contributes to the pathophysiology of functional bowel disorders and inflammatory bowel disease. Current theories suggest that neuronal plasticity and broad alterations along the brain-gut axis contribute to the development of chronic abdominal pain, but the specific mechanisms involved in chronic abdominal pain remain incompletely understood. Accumulating evidence implicates glial cells in the development and maintenance of chronic pain. Astrocytes and microglia in the central nervous system and satellite glia in dorsal root ganglia contribute to chronic pain states through reactive gliosis, the modification of glial networks, and the synthesis and release of neuromodulators. In addition, new data suggest that enteric glia, a unique type of peripheral glia found within the enteric nervous system, have the potential to modify visceral perception through interactions with neurons and immune cells. Understanding these emerging roles of enteric glia is important to fully understand the mechanisms that drive chronic pain and to identify novel therapeutic targets. In this review, we discuss enteric glial cell signaling mechanisms that have the potential to influence chronic abdominal pain.

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Background & Aims: Tachykinins are involved in physiological and pathophysiological mechanisms in the gastrointestinal tract. The major sources of tachykinins in the gut are intrinsic enteric neurons in the enteric nervous system and extrinsic nerve fibers from the dorsal root and vagal ganglia. Although tachykinins are important mediators in the enteric nervous system, how they contribute to neuroinflammation through effects on neurons and glia is not fully understood. Here, we tested the hypothesis that tachykinins contribute to enteric neuroinflammation through mechanisms that involve intercellular neuron-glia signaling. Methods: We used immunohistochemistry and quantitative real-time polymerase chain reaction, and studied cellular activity using transient-receptor potential vanilloid-1 (TRPV1)tm1(cre)Bbm/J::Polr2atm1(CAG-GCaMP5g,-tdTomato)Tvrd and Sox10CreERT2::Polr2atm1(CAG-GCaMP5g,-tdTomato)Tvrd mice or Fluo-4. We used the 2,4-di-nitrobenzene sulfonic acid (DNBS) model of colitis to study neuroinflammation, glial reactivity, and neurogenic contractility. We used Sox10::CreERT2+/-/Rpl22tm1.1Psam/J mice to selectively study glial transcriptional changes. Results: Tachykinins are expressed predominantly by intrinsic neuronal varicosities whereas neurokinin-2 receptors (NK2Rs) are expressed predominantly by enteric neurons and TRPV1-positive neuronal varicosities. Stimulation of NK2Rs drives responses in neuronal varicosities that are propagated to enteric glia and neurons. Antagonizing NK2R signaling enhanced recovery from colitis and prevented the development of reactive gliosis, neuroinflammation, and enhanced neuronal contractions. Inflammation drove changes in enteric glial gene expression and function, and antagonizing NK2R signaling mitigated these changes. Neurokinin A-induced neurodegeneration requires glial connexin-43 hemichannel activity. Conclusions: Our results show that tachykinins drive enteric neuroinflammation through a multicellular cascade involving enteric neurons, TRPV1-positive neuronal varicosities, and enteric glia. Therapies targeting components of this pathway could broadly benefit the treatment of dysmotility and pain after acute inflammation in the intestine.

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