RESUMO
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.
RESUMO
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.
Assuntos
Adipócitos , Norepinefrina , Animais , Masculino , Feminino , Adipócitos/metabolismo , Norepinefrina/metabolismo , Norepinefrina/farmacologia , Camundongos , Tecido Adiposo/inervação , Tecido Adiposo/metabolismo , Camundongos Endogâmicos C57BL , Transmissão Sináptica , Tecido Adiposo Branco/inervação , Tecido Adiposo Branco/metabolismo , Camundongos Transgênicos , Sinalização do CálcioRESUMO
Glia in the central nervous system exert precise spatial and temporal regulation over neural circuitry on a synapse-specific basis, but it is unclear if peripheral glia share this exquisite capacity to sense and modulate circuit activity. In the enteric nervous system (ENS), glia control gastrointestinal motility through bidirectional communication with surrounding neurons. We combined glial chemogenetics with genetically encoded calcium indicators expressed in enteric neurons and glia to study network-level activity in the intact myenteric plexus of the proximal colon. Stimulation of neural fiber tracts projecting in aboral, oral, and circumferential directions activated distinct populations of enteric glia. The majority of glia responded to both oral and aboral stimulation and circumferential pathways, while smaller subpopulations were activated only by ascending and descending pathways. Cholinergic signaling functionally specifies glia to the descending circuitry, and this network plays an important role in repressing the activity of descending neural pathways, with some degree of cross-inhibition imposed upon the ascending pathway. Glial recruitment by purinergic signaling functions to enhance activity within ascending circuit pathways and constrain activity within descending networks. Pharmacological manipulation of glial purinergic and cholinergic signaling differentially altered neuronal responses in these circuits in a sex-dependent manner. Collectively, our findings establish that the balance between purinergic and cholinergic signaling may differentially control specific circuit activity through selective signaling between networks of enteric neurons and glia. Thus, enteric glia regulate the ENS circuitry in a network-specific manner, providing profound insights into the functional breadth and versatility of peripheral glia.
Assuntos
Sistema Nervoso Entérico/fisiologia , Motilidade Gastrointestinal/fisiologia , Plexo Mientérico/fisiologia , Neuroglia/fisiologia , Animais , Comunicação Celular , Sistema Nervoso Entérico/citologia , Feminino , Masculino , Camundongos , Plexo Mientérico/citologia , Neuroglia/citologia , Neurônios/citologia , Transdução de SinaisRESUMO
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.
Assuntos
Cálcio , Sistema Nervoso Entérico , Masculino , Feminino , Camundongos , Animais , Cálcio/metabolismo , Neuroglia/metabolismo , Sistema Nervoso Entérico/metabolismo , Colo/fisiologia , Duodeno/metabolismo , Neurotransmissores/metabolismo , Camundongos Transgênicos , Plexo Mientérico/metabolismoRESUMO
Exposure to early life adversity (ELA) in the form of physical and/or psychological abuse or neglect increases the risk of developing psychiatric and inflammatory disorders later in life. It has been hypothesized that exposure to ELA results in persistent, low grade inflammation that leads to increased disease susceptibility by amplifying the crosstalk between stress-processing brain networks and the immune system, but the mechanisms remain largely unexplored. The meninges, a layer of three overlapping membranes that surround the central nervous system (CNS)- dura mater, arachnoid, and piamater - possess unique features that allow them to play a key role in coordinating immune trafficking between the brain and the peripheral immune system. These include a network of lymphatic vessels that carry cerebrospinal fluid from the brain to the deep cervical lymph nodes, fenestrated blood vessels that allow the passage of molecules from blood to the CNS, and a rich population of resident mast cells, master regulators of the immune system. Using a mouse model of ELA consisting of neonatal maternal separation plus early weaning (NMSEW), we sought to explore the effects of ELA on sucrose preference behavior, dura mater expression of inflammatory markers and mast cell histology in adult male and female C57Bl/6 mice. We found that NMSEW alone does not affect sucrose preference behavior in males or females, but it increases the dura mater expression of the genes coding for mast cell protease CMA1 (cma1) and the inflammatory cytokine TNF alpha (tnf alpha) in females. When NMSEW is combined with an adult mild stress (that does not affect behavior or gene expression in NH animals) females show reduced sucrose preference and even greater increases in meningeal cma1 levels. Interestingly, systemic administration of the mast cell stabilizer Ketotifen before exposure to adult stress prevents both, reduction in sucrose preference an increases in cma1 expression in NMSEW females, but facilitates stress-induced sucrose anhedonia in NMSEW males and NH females. Finally, histological analyses showed that, compared to males, females have increased baseline activation levels of mast cells located in the transverse sinus of the dura mater, where the meningeal lymphatics run along, and that, in males and females exposed to adult stress, NMSEW increases the number of mast cells in the interparietal region of the dura mater and the levels of mast cell activation in the sagittal sinus regions of the dura mater. Together, our results indicate that ELA induces long-term meningeal immune gene changes and heightened sensitivity to adult stress-induced behavioral and meningeal immune responses and that these effects could mediated via mast cells.
Assuntos
Anedonia , Mastócitos , Fatores Sexuais , Estresse Psicológico , Animais , Feminino , Masculino , Apresentação de Antígeno , Expressão Gênica , Privação Materna , Meninges , Sacarose , Fator de Necrose Tumoral alfa , Camundongos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: Mood and metabolic disorders are interrelated and may share common pathological processes. Autonomic neurons link the brain with the gastrointestinal tract and constitute a likely pathway for peripheral metabolic challenges to affect behaviors controlled by the brain. The activities of neurons along these pathways are regulated by glia, which exhibit phenotypic shifts in response to changes in their microenvironment. How glial changes might contribute to the behavioral effects of consuming a high-fat diet (HFD) is uncertain. Here, we tested the hypothesis that anxiogenic and depressive-like behaviors driven by consuming a HFD involve compromised duodenal barrier integrity and subsequent phenotypic changes to glia and neurons along the gut-brain axis. METHODS: C57Bl/6 male mice were exposed to a standard diet or HFD for 20 weeks. Bodyweight was monitored weekly and correlated with mucosa histological damage and duodenal expression of tight junction proteins ZO-1 and occludin at 0, 6, and 20 weeks. The expression of GFAP, TLR-4, BDNF, and DCX were investigated in duodenal myenteric plexus, nodose ganglia, and dentate gyrus of the hippocampus at the same time points. Dendritic spine number was measured in cultured neurons isolated from duodenal myenteric plexuses and hippocampi at weeks 0, 6, and 20. Depressive and anxiety behaviors were also assessed by tail suspension, forced swimming, and open field tests. RESULTS: HFD mice exhibited duodenal mucosa damage with marked infiltration of immune cells and decreased expression of ZO-1 and occludin that coincided with increasing body weight. Glial expression of GFAP and TLR4 increased in parallel in the duodenal myenteric plexuses, nodose ganglia, and hippocampus in a time-dependent manner. Glial changes were associated with a progressive decrease in BDNF, and DCX expression, fewer neuronal dendritic spines, and anxiogenic/depressive symptoms in HFD-treated mice. Fluorocitrate (FC), a glial metabolic poison, abolished these effects both in the enteric and central nervous systems and prevented behavioral alterations at week 20. CONCLUSIONS: HFD impairs duodenal barrier integrity and produces behavioral changes consistent with depressive and anxiety phenotypes. HFD-driven changes in both peripheral and central nervous systems are glial-dependent, suggesting a potential glial role in the alteration of the gut-brain signaling that occurs during metabolic disorders and psychiatric co-morbidity.
Assuntos
Encéfalo/metabolismo , Encéfalo/patologia , Depressão/etiologia , Dieta Hiperlipídica/efeitos adversos , Duodeno/patologia , Transtornos Mentais/etiologia , Neuroglia/metabolismo , Animais , Peso Corporal , Duodeno/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Plexo Mientérico/metabolismo , Plexo Mientérico/patologia , Neuroglia/patologia , Neurônios/metabolismo , Neurônios/patologia , Gânglio Nodoso/metabolismo , Gânglio Nodoso/patologiaRESUMO
Early-life adversity contributes to the development of functional bowel disorders later in life through unresolved mechanisms. Here, we tested the hypothesis that early-life adversity alters anatomical and functional interactions between mast cells and enteric glia. The effects of early-life stress were studied using the neonatal maternal separation (NMS) stress mouse model. Anatomical relationships between mast cells and enteric glia were assessed using immunohistochemistry and mast cell reporter mice (Mcpt5Cre;GCaMP5g-tdT). Immunohistochemistry was used to assess the expression of histamine, histamine 1 (H1) receptors, and glial fibrillary acidic protein. Functional responses of glia to mast cell mediators were assessed in calcium imaging experiments using Sox10CreERT2;GCaMP5g-tdT mice and cultured human enteric glial cells. NMS increases mast cell numbers at the level of the myenteric plexus and their proximity to myenteric ganglia. Myenteric glia respond to mediators released by activated mast cells that are blocked by H1 receptor antagonists in mice and humans and by blocking neuronal activity with tetrodotoxin in mouse tissue. Histamine replicates the effects of mast cell supernatants on enteric glia, and NMS increases histamine production by mast cells. NMS reduces glial responses to mast cell mediators in mouse tissue, while potentiating responses in cultured human enteric glia. NMS increases myenteric glial fibrillary acidic protein expression and reduces glial process length but does not cause neurodegeneration. Histamine receptor expression is not altered by NMS and is localized to neurons in mice, but glia in humans. Early-life stress increases the potential for interactions between enteric glia and mast cells, and histamine is a potential mediator of mast cell-glial interactions through H1 receptors. We propose that glial-mast cell signaling is a mechanism that contributes to enteric neuroplasticity driven by early-life adversity.NEW & NOTEWORTHY Early-life adversity places an individual at risk for developing functional gastrointestinal disorders later in life through unknown mechanisms. Here, we show that interactions between mast cells and glia are disrupted by early-life stress in mice and that histamine is a potential mediator of mast cell-glial interactions.
Assuntos
Histamina/fisiologia , Acontecimentos que Mudam a Vida , Mastócitos/fisiologia , Neuroglia/fisiologia , Neurônios/fisiologia , Animais , Animais Recém-Nascidos , Contagem de Células , Células Cultivadas , Quimases/genética , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Antagonistas dos Receptores Histamínicos H1/farmacologia , Humanos , Privação Materna , Camundongos , Camundongos Endogâmicos C57BL , Plexo Mientérico/citologia , Plexo Mientérico/metabolismo , Gravidez , Receptores Histamínicos H1/metabolismo , Estresse Psicológico/fisiopatologiaAssuntos
Eixo Encéfalo-Intestino , Neuroimunomodulação , Animais , Humanos , Sistema Nervoso Autônomo/fisiopatologia , Sistema Nervoso Autônomo/imunologia , Sistema Nervoso Entérico/imunologia , Sistema Nervoso Entérico/fisiopatologia , Microbioma Gastrointestinal/imunologia , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/inervaçãoRESUMO
Gulf War illness (GWI) is a chronic multisymptom disorder that is prominent in Gulf War veterans. Major unexplained symptoms of GWI include functional gastrointestinal disorders and undiagnosed illnesses, including neurologic disorders. Exposure to the antinerve gas drug pyridostigmine bromide (PB) is linked to the development of GWI, but the exact mechanisms remain unclear. Here, we tested the hypothesis that PB alters gut function by disrupting the neural and immune systems of the intestine. We exposed male and female mice to physiologically comparable amounts of PB that match the dose, route, and time frame of exposure experienced by Gulf War veterans and assessed the acute and chronic impacts on gastrointestinal functions, the functional architecture of the enteric nervous system, and immune responses in the gut and brain. Exposure to PB drove acute alterations to colonic motility and structure in both male and female mice that transitioned to chronic changes in gut functions. PB drove acute alterations to enteric neural and glial activity, glial reactivity, and neuron survival with glial reactivity persisting into the chronic phase in male mice. Despite having no effect on colonic permeability, exposure to PB caused major shifts in the expression of proinflammatory cytokines and chemokines in the colon and brain that suggest immunosuppressive effects. Interestingly, immune disruption was still evident in the colon and brain in female animals at 1 mo following exposure to PB. Together, our results show that the paradigm of PB exposure experienced by veterans of the Persian Gulf War contributes to long-lasting pathophysiology by driving enteric neuroinflammation, promoting immunosuppression, and altering functional anatomy of the colon in a sex-dependent manner.-Hernandez, S., Fried, D. E., Grubisic, V., McClain, J. L., Gulbransen, B. D. Gastrointestinal neuroimmune disruption in a mouse model of Gulf War illness.
Assuntos
Inibidores da Colinesterase/toxicidade , Colo/efeitos dos fármacos , Sistema Nervoso Entérico/efeitos dos fármacos , Síndrome do Golfo Pérsico/imunologia , Brometo de Piridostigmina/toxicidade , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/imunologia , Colo/imunologia , Colo/fisiopatologia , Citocinas/metabolismo , Sistema Nervoso Entérico/imunologia , Sistema Nervoso Entérico/fisiopatologia , Feminino , Motilidade Gastrointestinal , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neuroglia/imunologia , Síndrome do Golfo Pérsico/etiologia , Síndrome do Golfo Pérsico/fisiopatologiaRESUMO
ATP is both an important mediator of physiological gut functions such as motility and epithelial function, and a key danger signal that mediates cell death and tissue damage. The actions of extracellular ATP are regulated through the catalytic functions extracellular nucleoside triphosphate diphosphohydrolase-1 (NTPDase1), -2, -3, and -8, which ultimately generate nucleosides. Ectonucleotidases have distinct cellular associations, but the specific locations and functional roles of individual NTPDases in the intestine are still poorly understood. Here, we tested the hypothesis that differential and cell-selective regulation of purine hydrolysis by NTPDase1 and -2 plays important roles in gut physiology and disease. We studied Entpd1 and Entpd2 null mice in health and following colitis driven by 2% dextran sulfate sodium (DSS) administration using functional readouts of gut motility, epithelial barrier function, and neuromuscular communication. NTPDase1 is expressed by immune cells, and the ablation of Entpd1 altered glial numbers in the myenteric plexus. NTPDase2 is expressed by enteric glia, and the ablation of Entpd2 altered myenteric neuron numbers. Mice lacking either NTPDase1 or -2 exhibited decreased inhibitory neuromuscular transmission and altered components of inhibitory junction potentials. Ablation of Entpd2 increased gut permeability following inflammation. In conclusion, the location- and context-dependent extracellular nucleotide phosphohydrolysis by NTPDase1 and -2 substantially impacts gut function in health and disease.NEW & NOTEWORTHY Purines are important mediators of gastrointestinal physiology and pathophysiology. Nucleoside triphosphate diphosphohydrolases (NTPDases) regulate extracellular purines, but the roles of specific NTPDases in gut functions are poorly understood. Here, we used Entpd1- and Entpd2-deficient mice to show that the differential and cell-selective regulation of purine hydrolysis by NTPDase1 and -2 plays important roles in barrier function, gut motility, and neuromuscular communication in health and disease.
Assuntos
Adenosina Trifosfatases/metabolismo , Antígenos CD/metabolismo , Apirase/metabolismo , Colite/tratamento farmacológico , Colo/metabolismo , Sulfato de Dextrana/farmacologia , Animais , Colite/metabolismo , Colo/efeitos dos fármacos , Feminino , Masculino , Camundongos , Camundongos KnockoutRESUMO
Enteric glia play an important neuroprotective role in the enteric nervous system (ENS) by producing neuroprotective compounds such as the antioxidant reduced glutathione (GSH). The specific cellular pathways that regulate glial production of GSH and how these pathways are altered during, or contribute to, neuroinflammation in situ and in vivo are not fully understood. We investigated this issue using immunohistochemistry to localize GSH synthesis enzymes within the myenteric plexus and tested how the inhibition of GSH synthesis with the selective inhibitor l-buthionine sulfoximine impacts neuronal survival and inflammation. Both enteric glia and neurons express the cellular machinery necessary for GSH synthesis. Furthermore, glial GSH synthesis is necessary for neuronal survival in isolated preparations of myenteric plexus. In vivo depletion of GSH does not induce colitis but alters myenteric plexus neuronal phenotype and survival. Importantly, global depletion of glutathione is protective against some macroscopic and microscopic measures of colonic inflammation. Together, our data highlight the heterogeneous roles of GSH in the myenteric plexus of the ENS and during gastrointestinal inflammation. NEW & NOTEWORTHY Our results show that both enteric glia and neurons express the cellular machinery necessary for glutathione (GSH) synthesis and that glial GSH synthesis is necessary for neuronal survival in isolated enteric nervous system (ENS) preparations. In vivo depletion of GSH with the selective inhibitor l-buthionine sulfoximine is not sufficient to induce inflammation but does alter neuronal neurochemical composition and survival. Together, our data highlight novel heterogeneous roles for GSH in the ENS and during gastrointestinal inflammation.
Assuntos
Antioxidantes/metabolismo , Colite/prevenção & controle , Colo/metabolismo , Glutationa/deficiência , Plexo Mientérico/metabolismo , Neurônios/metabolismo , Animais , Butionina Sulfoximina/farmacologia , Morte Celular , Colite/induzido quimicamente , Colite/metabolismo , Colite/patologia , Colo/efeitos dos fármacos , Colo/patologia , Dinitrofluorbenzeno/análogos & derivados , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Glutamato-Cisteína Ligase/antagonistas & inibidores , Glutamato-Cisteína Ligase/metabolismo , Técnicas In Vitro , Masculino , Camundongos Endogâmicos C57BL , Plexo Mientérico/efeitos dos fármacos , Plexo Mientérico/patologia , Neuroglia/metabolismo , Neuroglia/patologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , FenótipoRESUMO
The reflexive activities of the gastrointestinal tract are regulated, in part, by precise interactions between neurons and glia in the enteric nervous system (ENS). Intraganglionic enteric glia are a unique type of peripheral glia that surround enteric neurons and regulate neuronal function, activity, and survival. Enteric glia express numerous neurotransmitter receptors that allow them to sense neuronal activity, but it is not clear if enteric glia monitor acetylcholine (ACh), the primary excitatory neurotransmitter in the ENS. Here, we tested the hypothesis that enteric glia detect ACh and that glial activation by ACh contributes to the physiological regulation of gut functions. Our results show that myenteric enteric glia express both the M3 and M5 subtypes of muscarinic receptors (MRs) and that muscarine drives intracellular calcium (Ca2+) signaling predominantly through M3R activation. To elucidate the functional effects of activation of glial M3Rs, we used GFAP::hM3Dq mice that express a modified human M3R (hM3Dq) exclusively on glial fibrillary acidic protein (GFAP) positive glia to directly activate glial hM3Dqs using clozapine- N-oxide. Using spatiotemporal mapping analysis, we found that the activation of glial hM3Dq receptors enhances motility reflexes ex vivo. Continuous stimulation of hM3Dq receptors in vivo, drove changes in gastrointestinal motility without affecting neuronal survival in the ENS and glial muscarinic receptor activation did not alter neuron survival in vitro. Our results provide the first evidence that GFAP intraganglionic enteric glia express functional muscarinic receptors and suggest that the activation of glial muscarinic receptors contributes to the physiological regulation of functions. NEW & NOTEWORTHY Enteric glia are emerging as novel regulators of enteric reflex circuits, but little is still known regarding the effects of specific transmitter pathways on glia and the resulting consequences on enteric reflexes. Here, we provide the first evidence that enteric glia monitor acetylcholine in the enteric nervous system and that glial activation by acetylcholine is a physiological mechanism that contributes to the functional regulation of intestinal reflexes.
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Acetilcolina/metabolismo , Sistema Nervoso Entérico/metabolismo , Motilidade Gastrointestinal , Neuroglia/metabolismo , Receptor Muscarínico M3/metabolismo , Animais , Cálcio/metabolismo , Sistema Nervoso Entérico/citologia , Sistema Nervoso Entérico/fisiologia , Feminino , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , ReflexoRESUMO
Glia (from Greek γλοία meaning 'glue') pertains to non-neuronal cells in the central (CNS) and peripheral nervous system (PNS) that nourish neurons and maintain homeostasis. In addition, glia are now increasingly appreciated as active regulators of numerous physiological processes initially considered exclusively under neuronal regulation. For instance, enteric glia, a collection of glial cells residing within the walls of the intestinal tract, regulate intestinal motility, a well-characterized reflex controlled by enteric neurons. Enteric glia also interact with various non-neuronal cell types in the gut wall such as enterocytes, enteroendocrine and immune cells and are therefore emerging as important local regulators of diverse gut functions. The intricate molecular mechanisms that govern glia-mediated regulation are beginning to be discovered, but much remains unknown about the functions of enteric glia in health and disease. Here we present a current view of the enteric glia and their regulatory roles in gastrointestinal (GI) (patho)physiology; from GI motility and epithelial barrier function to enteric neuroinflammation.
Assuntos
Intestinos/citologia , Intestinos/fisiologia , Neuroglia/fisiologia , Animais , Motilidade Gastrointestinal , Humanos , Neurônios/fisiologiaRESUMO
KEY POINTS: The role of enteric glial cell activity in the acute regulation of epithelial barrier and secretomotor functions of the intestines under physiological conditions is not clear. We used transgenic mice to modify glial activity and found that enteric glia significantly contribute to the neurogenic ion transport while glial activity does not appear to play a major role in the acute regulation of barrier function. The selective activation of glial activity evoked electrogenic ion transport primarily through neural pathways and was sufficient to drive electrogenic ion transport to an extent equal to the direct activation of neurogenic ion transport. These findings provide novel insight into the cellular mechanisms that control fluid transport homeostasis in the intestine and might provide novel therapeutic avenues for functional diarrheal diseases. ABSTRACT: Enteric glial cells are often implicated in the regulation of epithelial barrier and secretomotor functions of the intestines. But whether glial cell activity regulates these functions acutely under physiological conditions is not clear. We addressed this issue by using transgenic animal models to modify the activity of enteric glia, either reducing glial expression of connexin 43 in Sox10::CreERT2+/- /Cx43f/f mice or activating glial calcium responses in GFAP::hM3Dq mice, and tested the effects on colonic barrier function and electrogenic ion transport in Ussing chambers. We assessed neuronal-dependent and -independent contributions by activating or inhibiting neurogenic activity with veratridine and tetrodotoxin, respectively. Our results show that the reduction of glial Cx43 expression in Sox10::CreERT2+/- /Cx43f/f mice significantly reduced neurogenic ion transport. The selective glial activation in tissues from GFAP::hM3Dq mice evoked electrogenic ion transport to an extent equal to the direct activation of neurogenic ion transport with veratridine and glial driven responses consisted of both tetrodotoxin-sensitive and -insensitive components. The selective glial stimulation did not affect transmural ion conductance or cell-impermeant dye flux but the baseline ion conductance was more variable in Sox10::CreERT2+/- /Cx43f/f tissues. Together, our findings show that glial activity contributes to the regulation of electrogenic ion transport in the intestine through effects on neurons and possibly direct effects on epithelial cells. However, glial activity does not appear to play a major role in the acute regulation of barrier function. These findings provide novel insight into the cellular mechanisms that control fluid transport homeostasis in the intestine.
Assuntos
Colo/metabolismo , Motilidade Gastrointestinal , Absorção Intestinal , Mucosa Intestinal/metabolismo , Neuroglia/metabolismo , Animais , Colo/citologia , Colo/fisiologia , Conexina 43/genética , Conexina 43/metabolismo , Feminino , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Mucosa Intestinal/inervação , Transporte de Íons , Masculino , CamundongosRESUMO
Glia play key roles in the regulation of neurotransmission in the nervous system. Fluoroacetate (FA) is a metabolic poison widely used to study glial functions by disrupting the tricarboxylic acid cycle enzyme aconitase. Despite the widespread use of FA, the effects of FA on essential glial functions such as calcium (Ca2+) signaling and hemichannel function remain unknown. Therefore, our goal was to assess specifically the impact of FA on essential glial cell functions that are involved with neurotransmission in the enteric nervous system. To this end, we generated a new optogenetic mouse model to study specifically the effects of FA on enteric glial Ca2+ signaling by crossing PC::G5-tdTomato mice with Sox10::creERT2 mice. FA did not change the peak glial Ca2+ response when averaged across all glia within a ganglion. However, FA decreased the percent of responding glia by 30% (P < 0.05) and increased the peak Ca2+ response of the glial cells that still exhibited a response by 26% (P < 0.01). Disruption of Ca2+ signaling with FA impaired the activity-dependent uptake of ethidium bromide through connexin-43 (Cx43) hemichannels (P < 0.05) but did not affect baseline Cx43-dependent dye uptake. FA did not cause overt glial or neurodegeneration, but glial cells significantly increased glial fibrillary acid protein by 56% (P < 0.05) following treatment with FA. Together, these data show that the acute impairment of glial metabolism with FA causes key changes in glial functions associated with their roles in neurotransmission and phenotypic changes indicative of reactive gliosis. NEW & NOTEWORTHY: Our study shows that the acute impairment of enteric glial metabolism with fluoroacetate (FA) alters specific glial functions that are associated with the modification of neurotransmission in the gut. These include subtle changes to glial agonist-evoked calcium signaling, the subsequent disruption of connexin-43 hemichannels, and changes in protein expression that are consistent with a transition to reactive glia. These changes in glial function offer a mechanistic explanation for the effects of FA on peripheral neuronal networks.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Conexina 43/metabolismo , Fluoracetatos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Plexo Mientérico/citologia , Neuroglia/efeitos dos fármacos , Difosfato de Adenosina/farmacologia , Compostos de Anilina/farmacologia , Animais , Contagem de Células , Proteína Semelhante a ELAV 3/metabolismo , Proteína Semelhante a ELAV 4/metabolismo , Feminino , Regulação da Expressão Gênica/genética , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Subunidade beta da Proteína Ligante de Cálcio S100/metabolismo , Fatores de Transcrição SOXE/genética , Fatores de Transcrição SOXE/metabolismo , Xantenos/farmacologiaRESUMO
The 2007 Consensus Statement for Standard of Care in Spinal Muscular Atrophy (SMA) notes that patients suffer from gastroesophageal reflux, constipation and delayed gastric emptying. We used two mouse models of SMA to determine whether functional GI complications are a direct consequence of or are secondary to survival motor neuron (Smn) deficiency. Our results show that despite normal activity levels and food and water intake, Smn deficiency caused constipation, delayed gastric emptying, slow intestinal transit and reduced colonic motility without gross anatomical or histopathological abnormalities. These changes indicate alterations to the intrinsic neural control of gut functions mediated by the enteric nervous system (ENS). Indeed, Smn deficiency led to disrupted ENS signaling to the smooth muscle of the colon but did not cause enteric neuron loss. High-frequency electrical field stimulation (EFS) of distal colon segments produced up to a 10-fold greater contractile response in Smn deficient tissues. EFS responses were not corrected by the addition of a neuronal nitric oxide synthase inhibitor indicating that the increased contractility was due to hyperexcitability and not disinhibition of the circuitry. The GI symptoms observed in mice are similar to those reported in SMA patients. Together these data suggest that ENS cells are susceptible to Smn deficiency and may underlie the patient GI symptoms.
Assuntos
Sistema Nervoso Entérico/fisiopatologia , Gastroenteropatias/metabolismo , Trato Gastrointestinal/inervação , Atrofia Muscular Espinal/complicações , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Proteína 2 de Sobrevivência do Neurônio Motor/química , Proteína 2 de Sobrevivência do Neurônio Motor/deficiência , Animais , Modelos Animais de Doenças , Feminino , Esvaziamento Gástrico , Gastroenteropatias/etiologia , Gastroenteropatias/genética , Gastroenteropatias/fisiopatologia , Trato Gastrointestinal/fisiopatologia , Humanos , Masculino , Camundongos , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 2 de Sobrevivência do Neurônio Motor/genéticaRESUMO
Investigating enteric neuromuscular function poses specific challenges that are not encountered in other systems. The gut has a complex cellular composition, and methods to study diverse multicellular interactions during physiological gut functions have been limited. However, new technologies are emerging in optics, genetics, and bioengineering that greatly expand the capabilities to study integrative functions in the gut. In this mini-review, I discuss several areas where the application of these technologies could benefit ongoing efforts to understand enteric neuromuscular function. I specifically focus on technologies that can be applied to study specific cellular networks and the mechanisms that link activity to function.
Assuntos
Sistema Nervoso Entérico/fisiologia , Trato Gastrointestinal/fisiologia , Músculo Liso/fisiologia , Animais , Eletrofisiologia , Sistema Nervoso Entérico/citologia , Sistema Nervoso Entérico/fisiopatologia , Trato Gastrointestinal/inervação , Trato Gastrointestinal/fisiopatologia , Humanos , Músculo Liso/citologia , Músculo Liso/fisiopatologia , OptogenéticaRESUMO
The enteric nervous system (ENS) is a network of neurons and glia that controls ongoing gastrointestinal (GI) functions. Damage or injury to the ENS can lead to functional GI disorders. Current data support the conclusion that many functional GI disorders are caused by an imbalance between gut microbes and the immune system, but how the ENS is involved in these interactions is less understood. Because of the proximity of the ENS to bacteria and other foreign antigens in the GI tract, it is important to prevent the passage of these antigens through the GI epithelium. If any foreign compounds manage to pass through the GI epithelium, an immune response is triggered to prevent injury to the ENS and underlying structures. However, careful modulation of the inflammatory response is required to allow for adequate elimination of foreign antigens while avoiding inappropriate overactivation of the immune system as in autoimmune disorders. Enteric neurons and glial cells are capable of performing these immunomodulatory functions to provide adequate protection to the ENS. We review recent studies examining the interactions between the ENS and the immune system, with specific focus on enteric glial cells and their ability to modulate inflammation in the ENS.
Assuntos
Trato Gastrointestinal/inervação , Neuroglia/fisiologia , Neuroimunomodulação/fisiologia , Neurônios/fisiologia , Animais , Humanos , Inflamação/metabolismoRESUMO
Impaired gut motility may contribute, at least in part, to the development of systemic hyperammonemia and systemic neurological disorders in inherited metabolic disorders, or in severe liver and renal disease. It is not known whether enteric neurotransmission regulates intestinal luminal and hence systemic ammonia levels by induced changes in motility. Here, we propose and test the hypothesis that ammonia acts through specific enteric circuits to influence gut motility. We tested our hypothesis by recording the effects of ammonia on neuromuscular transmission in tissue samples from mice, pigs, and humans and investigated specific mechanisms using novel mutant mice, selective drugs, cellular imaging, and enzyme-linked immunosorbent assays. Exogenous ammonia increased neurogenic contractions and decreased neurogenic relaxations in segments of mouse, pig, and human intestine. Enteric glial cells responded to ammonia with intracellular Ca2+ responses. Inhibition of glutamine synthetase and the deletion of glial connexin-43 channels in hGFAP::CreERT2+/-/connexin43f/f mice potentiated the effects of ammonia on neuromuscular transmission. The effects of ammonia on neuromuscular transmission were blocked by GABAA receptor antagonists, and ammonia drove substantive GABA release as did the selective pharmacological activation of enteric glia in GFAP::hM3Dq transgenic mice. We propose a novel mechanism whereby local ammonia is operational through GABAergic glial signaling to influence enteric neuromuscular circuits that regulate intestinal motility. Therapeutic manipulation of these mechanisms may benefit a number of neurological, hepatic, and renal disorders manifesting hyperammonemia.NEW & NOTEWORTHY We propose that local circuits in the enteric nervous system sense and regulate intestinal ammonia. We show that ammonia modifies enteric neuromuscular transmission to increase motility in human, pig, and mouse intestine model systems. The mechanisms underlying the effects of ammonia on enteric neurotransmission include GABAergic pathways that are regulated by enteric glial cells. Our new data suggest that myenteric glial cells sense local ammonia and directly modify neurotransmission by releasing GABA.