Cover ArticleResearch Articles, Systems/Circuits

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

Luisa Seguella, Jonathon L. McClain, Giuseppe Esposito and Brian D. Gulbransen
Journal of Neuroscience 16 November 2022, 42 (46) 8694-8708; https://doi.org/10.1523/JNEUROSCI.2379-20.2022

Abstract

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.

Introduction

Enteric glia are a large population of developmentally and functionally unique peripheral neuroglia associated with neurons in the enteric nervous system (Rao et al., 2015; Gulbransen and Christofi, 2018). Enteric glia are mainly localized within the ganglionated myenteric and submucosal plexuses but are also associated with enteric nerve fibers that extend across all layers of the gut wall. Bidirectional cross talk among enteric glia, neurons, and other neighboring cells modulates gastrointestinal functions including neurotransmission, gut motility, intestinal barrier function, defense against pathogens, and local immune and inflammatory responses (Ibiza et al., 2016; Delvalle et al., 2018a,b; Grubišić et al., 2018; Kulkarni et al., 2018; Valès et al., 2018). Changes in enteric glial phenotype and function are, therefore, considered important in the development of gastrointestinal pathologies and gut–brain signaling disorders (Rao et al., 2017; Seguella et al., 2019; Seguella et al., 2021).

Most research to date has approached enteric glia as a homogeneous population of cells. However, subpopulations of enteric glia display differing morphologies, localizations within the gut wall (Hanani and Reichenbach, 1994; Gulbransen and Sharkey, 2012; Seguella and Gulbransen, 2021), protein expression profiles (Nasser et al., 2006a,b), and calcium responses to ATP within the colon (Boesmans et al., 2015). This suggests that substantial glial heterogeneity existing within a given gut region could underlie functional specializations (Valès et al., 2018). Further, differing microenvironments within or between gut regions could promote functional glial specializations that contribute to distinct functions in the specific subcellular compartments (Khakh and Sofroniew, 2015; Chai et al., 2017; Baghdadi et al., 2022). In support, recent data also show that enteric neurons display distinct transcriptional profiles that are dictated, in part, by environmental cues (Muller et al., 2020). Enteric neuron–glia signaling is also synapse, cell, and circuit specific, which likely functions to tune intestinal motor activity in distinct intestinal organs (Ahmadzai et al., 2021).

Here, we addressed the issue of local and regional functional heterogeneity among myenteric glia in two functionally distinct regions of the intestine: the duodenum and the colon. Our goal was to test how glia contribute to the regional diversity of enteric circuits in these organs by decoding similar extracellular signals but in distinct environments. Although the myenteric plexus coordinates motor functions throughout the intestine, the motor innervation of the duodenum is specialized to coordinate motility with gastric emptying and hormonal regulation of food intake, while the colon mainly exhibits propagating peristaltic contractions and simple motor patterns (Konomi et al., 2002). Further, the primary excitatory and inhibitory neurotransmitters involved in enteric reflexes are conserved between gut regions, but many of the specific transmitters and neuromodulators differ. Based on these differences, we hypothesized that the functional specializations of these two organs promote functional heterogeneity among myenteric glia reflected by differential responses to purinergic neuromodulators, a prominent intercellular signal by which enteric neurons and glia communicate and coordinate intestinal motility in the colon, and cholecystokinin (CCK), a neuropeptide involved in a hormonal-dependent feedforward loop driven by duodenal enteric glia. We studied functional glial activity in both regions using transgenic mice that express genetically encoded calcium sensors in enteric neurons and/or glia and performed contractility studies in isolated intestinal specimens to assess how glia regulate motor patterns. Our data show that myenteric glia exhibit distinct response profiles to neuromodulators that require intercellular signaling with enteric neurons to differing extents in the duodenum and colon. Regional heterogeneity in glial responses contributes to functional diversity in mechanisms that control intestinal contractility. Response profiles delineate four unique subpopulations among myenteric glia that are differently distributed between gut regions. These results suggest extensive interregional and intraregional diversity among mature myenteric glia similar to the emerging functional and regional heterogeneity of neuroglia in the CNS (Lanjakornsiripan et al., 2018; Batiuk et al., 2020; Clarke et al., 2021). Although further investigation is required to better understand the functional consequences of enteric glial diversity on intestinal function, our results provide new insight into the mechanisms that regulate gastrointestinal physiology and pathophysiology, and highlight a greater degree of complexity in enteric neural circuits than previously appreciated.

Materials and Methods

Animals.

All experiments involving animals were conducted according with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and were approved by the Michigan State University Institutional Animal Care and Use Committee. Male and female C57BL/6 mice between 8 and 10 weeks of age were used for contractility experiments (The Jackson Laboratory; https://www.jax.org/strain/000664). Transgenic mice expressing the genetically encoded Ca2+ indicator GCaMP5g in enteric glia (Sox10CreERT2+/−;PC::G5-tdT+/−; hereafter referred to as Sox10CreERT2;GCaMP5g-tdT (https://www.jax.org/strain/024477); The Jackson Laboratory; McClain and Gulbransen, 2017) or under the transcriptional control of the Wnt1 promotor (Wnt1Cre2+/−;PC::G5-tdT+/−; hereafter referred to as WntCre2;GCaMP5g-tdT) were bred in-house and were generated by crossing Sox10CreERT2 mice (Laranjeira et al., 2011) or Wnt1Cre2 mice (129S4.Cg-E2f1Tg(Wnt1-cre)2Sor/J (https://www.jax.org/strain/022137); The Jackson Laboratory; RRID:IMSR_JAX:022137) with PC::G5-tdTomato (PC::G5-tdT) mice (Gee et al., 2014; catalog #02447, The Jackson Laboratory; B6;129S6-Polr2aTn(pb-CAG-GCaMP5g,-tdTomato)Tvrd/J; RRID:IMSR_JAX:024477), respectively. All double transgenic mice were maintained as heterozygous for both Cre (Sox10CreERT2+/−; Wnt1Cre2+/−) and the floxed allele (PC::G5-tdT+/−). CreERT2 activity was induced in Sox10CreERT2;GCaMP5g-tdT mice by feeding the animals with chow containing tamoxifen citrate (400 mg/kg) for 1 week followed by 1 week of normal chow before use. Mice of both sexes were used for experiments when they reached 8–12 weeks of age. Mice were maintained in a temperature-controlled environment on a 12 h light/dark cycle with access to acidified water and a minimal phytoestrogen diet (Diet Number 2919, Envigo) ad libitum. Genotyping was performed by Transnetyx.

Contractility studies.

Isometric muscle tension recordings were performed in longitudinally oriented intestinal segments of the duodenum and colon from wild-type mice under passive tension. One centimeter tissue segments were mounted in a tissue bath with oxygenated Krebs solution at 37°C (Nasser et al., 2006a,b). Each tissue strip was attached to an isometric force transducer, and data were charted with LabChart 8 software (ADInstruments) as described in the study by Fried et al. (2017). Tissue segments were equilibrated for 30 min under 1 × g initial tension, and passive tension was recorded. Tissue health was verified by an initial bethanechol (BCH; 10 μm; cholinergic muscarinic agonist-induced contraction). After being rinsed and stabilized, ADP (100 μm) or CCK (100 nm) were bath applied preinhibition and postinhibition of glial metabolism with the drug sodium fluoroacetate (FA; 5 mm) for 1 h. Basal tension was unaffected by FA when compared with a timed control (data not shown). Contractility studies were performed in normal Krebs buffer consisted of the following (in mm): 117 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgCl2, 1.2 NaH2PO4, 25 NaHCO3, and 11 glucose. Data are presented as the percentage of baseline.

Circular muscle myenteric plexus whole-mount preparation.

Segments of duodenum and colon were immediately removed from killed Sox10CreERT2;GCaMP5g-tdT and Wnt1Cre2;GCaMP5g-tdT mice and placed in ice-cold DMEM/Ham's F-12 nutrient mixture (Thermo Fisher Scientific) supplemented with 3 μm nicardipine and 1 μm scopolamine to inhibit smooth muscle contractions. The full-thickness tissues were then opened along the mesenteric border and pinned flat with mucosa facing up in Sylgard-coated Petri dishes (Dow Corning). The mucosa was removed by cutting at the level of the lamina propria while separating the layers with forceps. Tissues were then flipped, and the longitudinal muscle and serosa were removed by microdissection to obtain live whole mounts with intact myenteric plexus lying atop the preparation.

Ca2+ imaging.

Live circular muscle myenteric plexus whole mounts from Sox10CreERT2;GCaMP5g-tdT and WntCre2;GCaMP5g-tdT mice were incubated in DMEM for 45 min at 37°C before starting experiments. Fluorescence imaging was conducted using an upright fixed-stage microscope (model BX51WI, Olympus) fitted with a 40× water-immersion objective (LUMPlan N; 0.8 numerical aperture) and a λ DG-4 Plus Xenon light source (Sutter Instrument). GCaMP5g fluorescence was excited by light passed through a 485 nm, 20 nm bandpass filter and detected by reflected light passing through a 515 nm long-pass filter. The tdT fluorescence was excited by light passed through a 535 nm, 20 nm bandpass filter and detected by reflected light passing through a 610 nm, 75 nm bandpass emission filter. Glial cells were identified by tdT fluorescence in Sox10CreERT2;GCaMP5g-tdT samples and by both tdT fluorescence and morphology in WntCre2;GCaMP5g-tdT samples, as previously described (Ahmadzai et al., 2021). Images of GCaMP5g fluorescence were acquired at a rate of 5 frames/s with a Neo sCMOS camera controlled by MetaMorph software (Molecular Devices). Whole mounts were continually superfused with 37°C Krebs buffer consisting of the following (in mm): 121 NaCl, 5.9 KCl, 2.5 CaCl2, 1.2 MgCl2, 1.2 NaH2PO4, 10 HEPES, 21.2 NaHCO3, 1 pyruvic acid, and 8 glucose, with pH adjusted to 7.4 with NaOH, with 3 μm nicardipine and 1 μm scopolamine at a flow rate of 2–3 ml/min. Drugs (100 μm ADP and 100 nm CCK) were diluted in Krebs buffer and bath applied for 30 s. Tissue was washed with Krebs buffer for at least 5 min before testing the second drug in the same ganglion. Pretreatments by tetrodotoxin (TTX; 300 nm; 3 min of incubation) and FA (5 mm; 2 h of incubation before Ca2+ imaging) were performed to test enteric glial responses when neuronal activity was blocked or glial metabolism was impaired, respectively.

Electrical field stimulation.

Electrical field stimulation (EFS) was used to drive broad neuronal depolarization within colonic neuron–glia networks. In these experiments, platinum electrodes were placed on either side of the preparation and tissues were stimulated by a GRASS SD9 Electrical Stimulator with +70 V voltage (10 Hz) using a single pulse or multiple trains of pulses for 3 s (see Fig. 4a, experimental design).

Chemicals and reagents.

ADP (100 μm; McClain and Gulbransen, 2017), FA (5 mm; McClain and Gulbransen, 2017), TTX (300 nm; McClain et al., 2015), tamoxifen-free base, ionomycin (2 μm; Sundaresan et al., 2017), and chemicals for Krebs buffer and immunohistochemistry were purchased from Sigma-Aldrich. DMEM was purchased from Thermo Fisher Scientific. CCK peptide (100 nm; Sundaresan et al., 2017) was purchased from Anaspec. Bethanechol chloride was purchased from Millipore.

Experimental design and statistical analysis.

Glial Ca2+ imaging data were analyzed with MetaMorph software (Molecular Devices) and regions of interest (ROIs) were drawn around enteric glial cells and their filaments that we identified by tdT fluorescence and morphology within a ganglion. The relative fluorescence intensity was measured, and traces were analyzed using GraphPad Prism 6 (GraphPad Software) and R software. Heatmaps and traces show the individual glial responses, while the average magnitude is expressed as the average change in fluorescence (ΔF/F0) over time or its fold change for tdT-positive responding glia (tdT+) in a given gut region or for a specific glial response profile (Fried and Gulbransen, 2015). The distribution of the amplitude (ΔF/F0) of glial Ca2+ responses to ADP and CCK were also plotted under baseline or TTX conditions for each glial response profile. Ionomycin (2 μm) was used to stimulate maximal GCaMP responses, and these data were used to normalize glial responses in different regions of the intestine. Cells were considered responsive when the normalized fluorescence signal overcame baseline plus three times the SD. The average number of glial cells that responded to ADP or CCK in the duodenum and the colon is expressed as a percentage of tdT+ responding glia or its fold change. The number of responses for glial cells was expressed as the average number of peaks that rose above baseline plus three times the SD from 1 s after drug application to the following 70 s.

Neuronal Ca2+ responses were analyzed with SparkAn 5.5.6.0 software (Adrian D. Boven and Mark T. Nelson, Department of Pharmacology, College of Medicine, University of Vermont, Burlington, Vermont). Briefly, the tdT channel images were overlapped on the relative videos to confirm the identity and location of glial cells and generate corresponding neuronal ROIs. In these two-channel videos, ROIs were manually drawn around enteric neurons, and Ca2+ responses were then measured as the ΔF/F0 over time. The baseline fluorescence (F0) was determined by averaging several images without activity as a continuous baseline, and the individual baseline was subtracted for each peak (100 ms before peak as a mean of 3 points). Neurons with ΔF/F0 = 1 were considered not responsive. Final traces were analyzed based on their frequency (in hertz), amplitude (ΔF/F0), rise time (10−90%; s), duration at one-half amplitude (s), and t1/2 (s), and data are presented as the mean ± SD.

All images were processed with either Imaris 9.5.1 (Bitplane) or Adobe Photoshop CS6 (Adobe Systems) to segment and overlay channels. Images are representative of labeling performed on tissue from a minimum of three mice. Data were analyzed using GraphPad Prism 6 and are shown as the mean ± SEM or mean ± SD, as appropriate. Ca2+ responses among different glial profiles of response and within a given profile were analyzed by one-way ANOVA with Bonferroni's post-test or unpaired t test, and neuronal Ca2+ responses were analyzed by two-way ANOVA with Sidak's multiple-comparisons test, as appropriate, with p < 0.05 considered to be statistically significant.

Data availability.

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Results

Regional heterogeneity among glial responses to neuromodulators

Myenteric glia surround neurons within myenteric ganglia and modulate gut functions through bidirectional signaling with neurons (Gulbransen et al., 2010; McClain et al., 2015). While the primary excitatory and inhibitory neurotransmitters involved in enteric reflexes are conserved between gut regions, many of the specific transmitters and neuromodulators differ. Given that myenteric glia sense transmitters released during synaptic communication, we hypothesized that myenteric glia residing in different gut regions exhibit differential responsiveness to local neuromodulators. We tested this hypothesis by recording enteric glial Ca2+ responses in whole-mount preparations of myenteric plexus taken from the duodenum and colon of Sox10CreERT2;GCaMP5g-tdT mice (McClain and Gulbransen, 2017). We measured the changes in intracellular Ca2+ concentrations in individual glial cells driven by the purinergic transmitter ADP (100 μm) and the neuropeptide CCK (100 nm).

Comparable proportions of myenteric glia responded to ADP and CCK within a given gut region (duodenum: ADP, 64%; CCK, 47%; n = 212 glial cells, eight ganglia, four mice; colon: ADP, 91%; CCK, 79%; n = 174 glial cells, four ganglia, three mice), but more glia responded to ADP in the colon than in the duodenum (t(384) = 2.083, p = 0.0379, unpaired t test; Fig. 1a). A similar trend is displayed by Ca2+ response amplitude with larger peak Ca2+ responses to ADP and CCK in the colon than duodenum (0.39 ± 0.02 ΔF/F0 ADP colon vs 0.13 ± 0.30 ΔF/F0 ADP duodenum, t(384) = 6.826, p < 0.0001; 0.080 ± 0.007 ΔF/F0 CCK colon vs 0.050 ± 0.005 ΔF/F0 CCK duodenum, t(384) = 3.514, p = 0.0005, unpaired t test; Fig. 1b), although the CCK and ADP responses were more similar in magnitude in the duodenum than in the colon (ADP vs CCK in the duodenum and colon, respectively: t(210) = 2.410, p = 0.0167 vs t(172) = 11.73, p < 0.0001; unpaired t test; Fig. 1b). ADP and CCK each evoked numerous individual glial Ca2+ responses in the duodenum that were comparable in terms of magnitude (Fig. 1b,d–i) and with the same frequency (Fig. 1c–i, Movies 1, 2). In contrast, ADP elicited large Ca2+ responses in myenteric glia in the colon that were ∼10-fold larger than the small, higher-frequency Ca2+ transients induced by CCK (t(172) = 3.537, p = 0.0005, unpaired t test; Fig. 1b,c,j–o, Movies 3, 4). Glial Ca2+ responses to both neuromodulators occurred at higher frequency in the duodenum than in the colon (ADP: t(384) = 5.264, p < 0.0001; CCK: t(384) = 2.205, p = 0.0285; unpaired t test; Fig. 1c). Switching drug order had no effect on glial Ca2+ responses to ADP and CCK, and a 5 min interstimulus wash was sufficient to obtain repeatable response (Fig. 2; Gulbransen and Sharkey, 2009; Fried and Gulbransen, 2015; Delvalle et al., 2018a,b). Together, these data show that regional heterogeneity exists among myenteric glia in terms of responsiveness to neuromodulators.

Figure 1.
Figure 1.

Myenteric glia exhibit regional heterogeneity in the responses to ADP and CCK. a, The percentage of glial cells (tdT+) that responded (or not) through a Ca2+ transients to ADP (100 μm) or CCK (100 nm) in the duodenum and the colon (t(384) = 2.083, p = 0.0379). b, Average peak Ca2+ response driven by ADP or CCK in the duodenum and colon (duodenum and colon, respectively: CCK vs ADP: t(210) = 2.410, p = 0.0167 vs t(172) = 11.73, p < 0.0001; ADP or CCK between gut regions: t(384) = 6.826, p < 0.0001 vs t(384) = 3.514, p = 0.0005 for ADP or CCK between gut regions, respectively). ΔF/F0, change in fluorescence over the time. c, Number of responses per tdT+ glia when challenged with ADP or CCK (from 1 s after drug application to the following 70 s; CCK vs ADP in the colon: t(172) = 3.537, p = 0.0005; for ADP or CCK between gut regions, respectively: t(384) = 5.264, p < 0.0001 vs t(384) = 2.205, p = 0.0285). d–f, Representative traces of Ca2+ responses in enteric glial cells evoked by exposure to ADP (blue traces) or CCK (black traces) in a myenteric ganglion of the duodenum show comparable glial responses in term of magnitude (10% ΔF/F0 for either response) and frequency. g–i, Representative images of enteric glia (identified by tdT fluorescence, red) responding to ADP or CCK in the duodenum. j–l, Representative traces of Ca2+ levels in enteric glia within a myenteric ganglion of the colon exposed to ADP (blue traces) or CCK (black traces) show different glial responses in terms of magnitude (100% ΔF/F0 for ADP vs 10% ΔF/F0 for CCK) and frequency. m–o, Representative images from a Ca2+ imaging experiment showing tdT+ glia (red tdT fluorescence, left) responding to ADP and CCK in the colon. Images are z-projections of time series images of myenteric glia on stimulation with ADP or CCK (from 1 s after drug application) in which the maximal peak Ca2+ response of responsive cells is falsely colored (see the color scale for absolute values in arbitrary units). See also Figures 2 and 11. Data are analyzed by unpaired t test and expressed as average percentages or responses in n = 212 and n = 174 tdT+ glial cells, from 8 or 4 myenteric ganglia of N = 4 or N = 3 Sox10CreERT2;GCaMP5g-tdT mice (duodenum and colon, respectively). Ionomycin (2 μm) was used to normalize the glial response in the different intestine regions. Scale bar, 20 µm. *p < 0.05, ***p < 0.001, ****p < 0.0001, #p < 0.05, ###p < 0.001, ####p < 0.0001.

Figure 2.
Figure 2.

Glial Ca2+ responses to ADP and CCK do not change after switching the order of drug application. a, The average peak Ca2+ response driven by CCK or ADP in the duodenum and colon of Sox10CreERT2;GCaMP5g-tdT mice. b, Relative percentage of glial cells (tdT+ cells) that responded (or not) to the initial application of CCK (100 nm) followed by ADP (100 μm) in the duodenum and the colon. ΔF/F0, change in fluorescence over the time. Ionomycin (2 μm) was used to normalized glial response in the different intestine regions. *p < 0.05, **p < 0.01, ****p < 0.0001, ##p < 0.01, ####p < 0.0001.

Movie 1.

ADP-evoked Ca2+ response in a ganglion of the duodenum. Stochastic glial Ca2+ transients in response to ADP are observed in the duodenum.

Movie 2.

CCK-evoked Ca2+ response in a ganglion of the duodenum. Ca2+ responses elicited by CCK in the duodenum are characterized by a high frequency, a low magnitude, and a long duration throughout the glial network.

Movie 3.

ADP-evoked Ca2+ response in a ganglion of the colon. ADP drives robust and uniform Ca2+ response among myenteric glia in the colon.

Movie 4.

CCK-evoked Ca2+ response in a ganglion of the colon. CCK-induced glial Ca2+ transients in the colon are periodic and have a low amplitude compared to ADP responses.

Functional heterogeneity in glia contributions to neuromuscular control by ADP and CCK

Glial activity encoded by Ca2+ responses regulates intestinal motor function through interactions with neurons in myenteric circuits (McClain et al., 2014; Delvalle et al., 2018a,b). Our imaging studies show that ADP and CCK evoke distinct glial Ca2+ responses that might underlie organ-specific mechanisms that control local intestinal motor function. To address this hypothesis, we conducted isometric muscle tension recordings in duodenal and colonic segments and used FA (5 mm) to study the extent to which glia contribute to motor responses induced by ADP and CCK (Fig. 3a). CCK did not affect baseline tension in either intestinal region (Fig. 3b,c) and was unaffected by FA (Fig. 3b,c). ADP decreased baseline tension by nearly 50% in the duodenum but had no effect on baseline tension in the colon (t(65) = 6.887, p < 0.0001, unpaired t test; Fig. 3d,e). ADP-evoked relaxation was not changed by FA in the duodenum; however, perturbing glial metabolism with FA in the colon revealed an inhibitory role of ADP in neuromuscular transmission (colon: t(5) = 5.877, p = 0.0042, unpaired t test; Fig. 3d,e). Together, these results show that glia contribute to purinergic neuromuscular control to differing extents in the colon and duodenum. CCK does not appear to have a major effect on motor function in either organ, and glial responsiveness to CCK likely reflects modulatory roles in nonmotor functions.

Figure 3.
Figure 3.

Selective impairment of glial activity by FA affects the isometric muscle tension in a region-specific and pathway-specific manner. a, Experimental paradigm of duodenal and colonic segments stimulated with ADP (100 μm, 10 min) or CCK (100 nm, 10 min) before and after FA application (5 mm, 60 min). b, Representative traces showing the effect of CCK application on duodenal (right) and colonic (left) isometric tension before (top) and after (bottom) FA treatment. c, Summary data of FA-elicited changes from baseline (percentage) in isometric tension induced by CCK in both intestinal regions. d, Representative traces of ADP application duodenal (right) and colonic (left) isometric tension before (top) and after (bottom) FA treatment. e, Summary data of FA-elicited changes from baseline (percentage) in isometric tension induced by ADP in both intestinal regions. BCH (10 μm) was used to verify tissue health. Data are analyzed by paired two-way ANOVA and expressed as average percentages of tension changes from baseline in N =5–7 mice. ***p < 0.001, ****p < 0.0001.

Differential effects of disrupting neuron–glia communication on glial responses to ADP and CCK in the duodenum and colon

Excitatory neurotransmitters released during synaptic communication promote intercellular communication between enteric neurons and glia that functions to tune enteric neurocircuits (Ahmadzai et al., 2021). This is largely blocked by TTX and is impaired by inhibiting glial metabolism with FA (Gulbransen and Sharkey, 2009; McClain and Gulbransen, 2017). Although FA preferentially affects glia, potential direct effects of FA on neurons could confound interpretations in experiments aimed at addressing glial versus neuronal contributions. Therefore, before addressing the question of regional specificity in enteric neuron–glia network responses to ADP and CCK, we determined the potential of FA to affect neuronal activity. For these experiments, neuronal Ca2+ responses evoked by EFS were recorded in the myenteric plexus from the colons of Wnt1Cre;GCaMP5g-tdT mice (Fig. 4a), in which tdT and GCaMP5g are expressed by both enteric neurons and glia (Ahmadzai et al., 2021; Fig. 5). Although we cannot fully discriminate the effects of FA on neurons and glia because of their intimate and mutual communication, responses to EFS in the myenteric plexus are initiated by neurons, which subsequently activate the surrounding electrically inexcitable glia (Ahmadzai et al., 2021). FA reduced neuron response frequency (Fig. 4b), magnitude (Fig. 4c), and the number of neurons that responded to the second single pulse (Fig. 4g). The onset of the neuronal Ca2+ responses to both single pulses (Fig. 4a, points 1 and 2) was slowed (Fig. 4d), with prolonged peak responses following the first single pulse (Fig. 4e). In addition, FA reduced neuronal response frequencies and slowed response rise times on the second train of pulses (Fig. 4a,b,d, point 4) and prolonged responses that occurred after the first train of pulses (Fig. 4a,e, point 3). These data suggest that FA impacts distinct aspects of the neuronal Ca2+ responses to EFS and the differences observed among the subsequent stimulations might involve both a direct effect of FA on neurons and an indirect effect on neurons because of glial activity impairment.

Figure 4.
Figure 4.

Effect of fluoroacetate on neuronal Ca2+ responses to electrical field stimulation in the colon. a, Schematic diagram illustrating how a broad neuronal depolarization was driven within the myenteric networks by EFS (+70 V, 10 Hz) single pulse (1 s; points 1 and 2), or train of pulses (3 s; points 3 and 4). b–g, Frequency (b), amplitude (c), rise time (10–90%; d), duration at half-amplitude (in seconds; e), half-time (in seconds; f), and number of responding neurons (percentage; g) were measured and averaged for each EFS response. Three hundred eighty-two and 254 neurons were studied in untreated and FA-treated (FA: 5 mm, 2 h) tissues, respectively, with 1–2 ganglia used per mouse in 3 Wnt1Cre2;GCaMP5g-tdT mice. See also Figure 5. Data are analyzed by two-way ANOVA and Sidak's multiple-comparisons test. *p < 0.05, **p < 0.01, ****p < 0.0001.

Figure 5.
Figure 5.

Representative images of dTomato expression in colonic myenteric plexuses of Wnt1Cre2;GCaMP5g-tdT mice. The optogenetic probe tdT is expressed by both enteric neurons and glia in Wnt1Cre2;GCaMP5g-tdT mice. However, neurons and glia are easily differentiated in this line based on cell morphology and tdT expression, which is high in glia and nearly undetectable in neurons.

Given the extensive nature of bidirectional communication between enteric neurons and glia, it is possible that glial responses to ADP and CCK involve intercellular communication with neurons in addition to direct stimulation. We tested the extent to which glial responses to ADP and CCK in the duodenum and colon require intercellular communication with neurons by reducing neuronal activity with TTX (300 nm) and glial activity with FA (5 mm) in Sox10CreERT2;GCaMP5g-tdT mice. In the duodenum, FA decreased the percentage of tdT+ glia responding to ADP by nearly 70% but had no effect on the percentage of cells responding to CCK (FA: F(2, 640) = 6.957, p = 0.0004, one-way ANOVA; Fig. 6a), while TTX had no significant effect on the number of glia responding to either ADP or CCK. The magnitude of ADP-evoked glial Ca2+ responses in the duodenum was reduced by 36% in the presence of TTX, and by 70% in the presence of FA (TTX and FA: F(2,316) = 51.18, p < 0.0001, one-way ANOVA; Fig. 6b). In contrast, the magnitude of glial Ca2+ responses to CCK was potentiated by 139% in the presence of TTX, but was not affected by FA (TTX: F(2,465) = 14.19, p < 0.0001, one-way ANOVA; Fig. 6b). In the colon, ∼25% fewer tdT+ glia responded to ADP in tissues treated with either TTX or FA (TTX: F(2,534) = 2.626, p = 0.0731; FA: F(2,534) = 2.626, p = 0.1250; one-way ANOVA; Fig. 6c). Similarly, 58% fewer cells responded to CCK after incubation with FA, but TTX had no effect on the percentage of glia responding to CCK (FA: F(2,534) = 6.284, p = 0.0067, one-way ANOVA; Fig. 6c). Despite fewer myenteric glia responding to ADP after treatment with either FA or TTX in the colon, those that still responded exhibited 27% larger peak Ca2+ responses in FA-treated tissues and 67% smaller peak Ca2+ responses in TTX-treated tissues (TTX: F(2,462) = 32.04, p < 0.0001; FA: p = 0.0133; one-way ANOVA; Fig. 6d). These results are in agreement with prior work showing that FA reduces the numbers of responsive myenteric glia and potentiates Ca2+ responses in cells that still exhibit responses (McClain and Gulbransen, 2017). FA also increased the magnitude of glial Ca2+ responses to CCK in cells that still exhibited a response by 60%, while TTX had no significant effect on CCK responses (FA: F(2,416) = 10.22, p < 0.0001, one-way ANOVA; Fig. 6d). These data show that glial responses to ADP involve intercellular signaling with neurons in the duodenum and colon. Glial responses to CCK involve neurons in the duodenum but appear to be independent of communication with neurons in the colon. Thus, purinergic signals seem consistent across intestinal regions, while responses to gut peptides likely involve distinct signaling pathways along the intestine.

Figure 6.
Figure 6.

Neuron–glia communication disruption impairs glial responses to ADP and CCK differently in the duodenum and colon. a, b, Effects of TTX (300 nm) and FA (5 mm) on the percentage of tdT+ glia responding to ADP (100 μm) and CCK (100 nm; ADP vs untreated tissues: F(2,640) = 6.957, p = 0.0004; a) and corresponding peak [Ca2+]i responses in the myenteric plexus of duodenum (ADP vs untreated tissues: F(2,316) = 51.18, p < 0.0001; CCK vs untreated tissues: F(2,465) = 14.19, p < 0.0001; b). c, d, The percentage of tdT+ glia still responding to ADP (100 μm; TTX vs untreated tissues: F(2,534) = 2.626, p = 0.0731; FA vs untreated tissues: p = 0.1250; one-way ANOVA) and CCK (100 nm; FA vs untreated tissues: F(2,534) = 6.284, p = 0.0067; one-way ANOVA; c) and relative Ca2+ transients after TTX (300 nm; for ADP vs untreated tissues: F(2,462) = 32.04, p < 0.0001) or FA (5 mm) exposure in the colon (for ADP vs untreated tissues: F(2,462) = 32.04, p = 0.0133; CCK vs untreated tissues: F(2,416) = 10.22, p < 0.0001; d). ΔF/F0, change in fluorescence over the time. Data are shown as the fold change for responses to ADP and CCK in untreated tissues (before TTX or FA incubation). ADP-induced and CCK-induced responses in TTX-treated tissues were recorded in n = 212 and n = 174 tdT+ cells, from 8 or 4 ganglia of N = 4 or N = 3 Sox10CreERT2;GCaMP5g-tdT mice (duodenum and colon, respectively), while ADP and CCK glial responses after FA exposure were assessed in n = 219 and n = 189 tdT+ glial cells, from 8 or 4 ganglia of N = 4 or N = 3 mice (duodenum and colon, respectively). Ionomycin (2 μm) was used to normalize the glial response in the different gut regions. Data are analyzed by one-way ANOVA and Bonferroni's post hoc test. *p < 0.1, ***p < 0.001, ****p < 0.0001, °°p < 0.01, °°°°p < 0.0001.

Myenteric glia exhibit regional and local heterogeneity in their Ca2+ response profiles to neuromodulators

Enteric neurocircuits are designed to control organ-specific functions, which include digestion and nutrient absorption via segmentation in the upper small intestine and propulsion in the large intestine (Konomi et al., 2002; Huizinga et al., 2014; Spencer et al., 2016; Fung and Vanden Berghe, 2020). Neurocircuit complexity differs throughout the intestine (Li et al., 2019), and glial heterogeneity exists within even a defined region of gut (Boesmans et al., 2019; Ahmadzai et al., 2021; Baghdadi et al., 2022). We tested the concept that intraregional and interregional heterogeneity exists among myenteric glia in the duodenum and colon by examining response profiles in a cell-by-cell analysis of Ca2+ responses elicited by ADP and CCK in Wnt1Cre2;GCaMP5g-tdT mice (Fig. 7). Figure 7 shows representative examples of recordings from myenteric ganglia in the duodenum (Fig. 7a) and colon (Fig. 7b), where glial Ca2+ responses are color coded based on time to highlight glia exhibiting differential response profiles. In this study, we defined a “high response” as a peak fluorescence value >3 SDs above the average baseline Ca2+ level measured within a given cell for a fixed recording duration (100 s). Lower values than 3 SDs were considered “low responses.” Glial cells that exhibited similar responses in terms of magnitude were included in the same response profile, and the individual Ca2+ responses to ADP and CCK were averaged within each profile. Based on their responsiveness, we were able to identify the following four distinct response profiles of myenteric glia: ADPhigh/CCKhigh, in which both stimuli elicited high glial Ca2+ transients (profile 1); ADPhigh/CCKlow, glial cells that only displayed high responses to ADP (profile 2); CCKhigh/ADPlow, subset of glial cells with high responses to CCK only (profile 3); and ADPlow/CCKlow, glia that displayed low responses to either neuromodulator (profile 4). In the duodenum, the subset of glia within profile 1 displayed average Ca2+ responses comparable to either neuromodulator, while a markedly higher peak Ca2+ response to ADP or CCK was exhibited by glial profile 2 and profile 3, respectively (profile 2: t(220) = 4.921, p < 0.0001; profile 3: t(273) = 9.647, p < 0.0001; unpaired t test; Fig. 7c, Table 1). Comparing the Ca2+ responses to ADP and CCK responses between glial profiles that responded to the same neuromodulator (profile 1 vs profile 2 for ADP responses, profile 1 vs profile 3 for CCK responses) revealed that only CCK evoked significantly diverse responses in the duodenum, where greater Ca2+ responses to CCK were observed in cells exhibiting profile 1 than cells characterized by profile 3 (t(2112) = 2.153, p = 0.0335, unpaired t test; Fig. 7d). No significant differences were observed between different glial profiles responding to ADP. Glia exhibit a fairly even distribution among response profiles in the duodenum without any individual profile dominating (25% for profile 1; 13% for profile 2; 42% for profile 3; 20% for profile 4; n = 221 tdT+ glial cells, n = 6 ganglia; N = 4 mice; Fig. 7e, Table 1).

View this table:
Table 1.

Glial profiles of response changes after TTX treatment

Figure 7.
Figure 7.

Regional heterogeneity between glial subtypes identified by response properties. Ca2+ imaging reveals four profiles of response to ADP and CCK regionally heterogenic in myenteric glia. a, b, Temporal color-coded images of cells (enteric neurons and glia) responding to 30 s of application of ADP (100 μm) or CCK (100 nm) in a myenteric ganglion of the duodenum and colon of Wnt1Cre2;GCaMP5g-tdT mice, respectively. The four different profiles of Ca2+ response identified in myenteric glia are shown on the tdTomato pictures (red tdT fluorescence, left). A representative glial cell belonging to each of the four different response profiles [(1) ADPhigh/CCKhigh, (2) ADPhigh/CCKlow, (3) CCKhigh/ADPlow, and (4) ADPlow/CCKlow] is indicated by a white circle and enlarged in the bottom panels. Pictures are z-projections of time series images (30 s each) in which the maximal peak Ca2+ response to ADP and CCK is shown at different times in responsive cells (temporal color coded, see color scale). Scale bar, 20 µm. c, d, Average peak Ca2+ responses evoked by ADP and CCK in the duodenum of Wnt1Cre2;GCaMP5g-tdT mice are compared per each glial profile (profile 2: t(220) = 4.921, p < 0.0001; profile 3: t(273) = 9.647, p < 0.0001; c) and among different profiles of response (CCK profile 1 vs profile 3: t(2112) = 2.153, p = 0.0335; d). e, The percentage of tdT+ myenteric glia belonging to the different response profiles in the duodenum. ΔF/F0, change in fluorescence over the time. f, g, Average ADP-induced and CCK-induced peaks of amplitude are compared per each profile of response (profile 1: t(282) = 6.369, p < 0.0001; profile 2: t(244) = 7.671, p < 0.0001; f) and among glial profiles (ADP profile 1 vs profile 2: t(2127) = 2.457, p = 0.0154) in the colon of Wnt1Cre2;GCaMP5g-tdT (g). h, Quantification of the number of tdT+ myenteric glial cells for each profile of response in the colon (F(3,20) = 7.921: profile 3 vs profile 1: p = 0.0063; profile 4 vs profile 1: p = 0.0052; F(3,20) = 7.921: profile 3 vs profile 1: p = 0.0115; profile 4 vs profile 1: p = 0.0095). ΔF/F0, change in fluorescence over the time. Note that the amplitude (ΔF/F0) scale differs between different gut regions. c–h, Data are analyzed by unpaired t test (c, d, f, g, graphs) or one-way ANOVA (e, h, graphs) and expressed as average percentages or responses in n = 221 and n = 194 tdT+ glial cells, from 6 or 4 myenteric ganglia of N = 4 or N = 3 Wnt1Cre2;GCaMP5g-tdT mice (duodenum and colon, respectively). Ionomycin (2 μm) was used to normalize glial response in the different gut regions. Scale bar, 20 µm. *p < 0.1, **p < 0.01, ****p < 0.0001, °p < 0.1, °°p < 0.01.

In the colon, average peak Ca2+ responses to ADP were significantly larger in glial profiles 1 and 2 than CCK responses, while glial profile 3 displayed a significant lower response to ADP compared with CCK (profile 1: t(282) = 6.369, p < 0.0001; profile 2: t(244) = 7.671, p < 0.0001; profile 3: t(233) = 2.398, p = 0.0193; unpaired t test; Fig. 7f, Table 1). In contrast to the duodenum, only ADP responses differed between ADP-responding glial profiles in the colon (profile 1 vs profile 2: t(2127) = 2.457, p = 0.0154, unpaired t test; Fig. 7g), but not CCK-evoked Ca2+ transients (profile 1 vs profile 3). Less heterogeneity among glial responses was observed in the colon than in the duodenum, and >87% of enteric glia displayed only two profiles of responses: profile 1 and profile 2 (profile 1, 45%; profile 2, 42%; profile 3, 7%; profile 4, 6%; n =194 tdT+ glial cells, n = 4 ganglia, N = 3 mice; profile 3: F(3,20) = 7.921, p = 0.0063; profile 4 vs profile 1: p = 0.0052; F(3,20) = 7.921; profile 3: p = 0.0115; profile 4 vs profile 2: p = 0.0095; one-way ANOVA; Fig. 7h, Table 1).

These data reveal heterogeneity among glial responsiveness within the myenteric plexus and suggest that a diverse scale of glial heterogeneity exists in the different intestinal regions similar to the regional differences observed in the innervation of intestinal circular muscle (Konomi et al., 2002).

Contribution of neural signaling to glial response profiles

Glial responses to neuromodulators involve intercellular communication with neurons to differing extents in the colon and duodenum (Fig. 6). Therefore, we blocked most neuronal synaptic signaling with TTX to investigate how intercellular communication with enteric neurons contributes to the various glial response profiles observed (duodenum: Fig. 8b,d–f; colon: Fig. 8h,j–l). TTX altered glial responsiveness to neuromodulators and shifted the distribution of cells within glial response profiles in both intestinal regions (Fig. 8a,b,g,h). TTX also altered glial response amplitude distributions (Fig. 8c,d,i,j). These differences are more apparent when viewed as an amplitude heatmap showing the TTX-induced changes in glial Ca2+ responses to ADP and CCK for each enteric glial cell analyzed (Fig. 8e,k). TTX had diverse effects on glial responses within and between profiles (Fig. 8f,l). Changes in intracellular Ca2+ activity were observed in each glial profile in the duodenum and colon after the application of TTX; however, the most striking differences within a given profile occurred in the duodenal response profiles. Glia characterized by duodenal profiles 2 and 3 exhibited smaller Ca2+ responses in the presence of TTX (Fig. 9, Table 2). CCK responses were lower on average in both profiles 2 and 4, but TTX increased CCK responses in profile 3 (Fig. 9, Table 2). In the colon, only cells exhibiting profile 1 response characteristics displayed a significant reduction in Ca2+ responses to ADP and CCK in the presence of TTX (Fig. 9, Table 2). Together, these data indicate that functionally defined enteric glial subtypes possess regionally distinct physiological responses to neuromodulators that involve intercellular signaling with neurons. Each glial cell exhibits an intrinsic ability to respond to neuromodulators that is affected by changes in the surrounding neural environment, highlighting a potential enteric glial plasticity in “adapting” their responses to the same cue under distinct conditions.

View this table:
Table 2.

Significant differences among glial Ca2+ responses to the same neuromodulator within each response profile before and after TTX (300 nm) treatment in the duodenum and colon

Figure 8.
Figure 8.

Neuron–glia communication disruption by TTX changes the Ca2+ transient properties in each of the four glial profiles of response in the duodenum and colon. a, g, The percentage of myenteric glia defined as (1) ADPhigh/CCKhigh, (2) ADPhigh/CCKlow, (3) CCKhigh/ADPlow, and (4) ADPlow/CCKlow, in the duodenum (a) and colon (g) before TTX treatment, respectively. b, h, The fraction (percentage) of these populations that displayed a different response profile under TTX (300 nm) condition in the duodenum (b) and colon (h), respectively. c, d, i, j, Distribution of the amplitude (ΔF/F0) of glial Ca2+ responses to ADP and CCK under baseline (c, i) or TTX (d, j) conditions for each glial response profile in the duodenum and colon, respectively. Each dot represents a single cell. Cells with similar response profiles were shown by the same color. ΔF/F0, change in fluorescence over the time. e, k, Heatmaps of amplitude show the TTX-induced changes in glial Ca2+ responses to ADP and CCK for every single enteric glial cell in the duodenum and colon, respectively. Glial cells that displayed the same responses profiles, before (–TTX, left) and after (+TTX, right) the treatment with TTX (300 nm), were grouped with the same color coding used in a and g (colored side columns). Intensity of amplitude: yellow, low amplitude; orange, medium amplitude; red, high amplitude. f, l, Average peak Ca2+ responses to ADP and CCK are shown for each glial response profile under baseline and TTX (300 nm) conditions in the duodenum and colon, respectively. Significant differences among profiles are shown in Tables 1 and 2, and Figure 9. Color coding from a and g was retained. Note that the amplitude (ΔF/F0) scale differs between different gut regions. Data are expressed as average percentages or responses in n = 221 and n = 194 tdT+ glial cells, from 6 or 4 myenteric ganglia of N = 4 or N = 3 Wnt1Cre2;GCaMP5g-tdT mice (duodenum and colon, respectively). Ionomycin (2 μm) was used to normalized glial response in the different gut regions.

Figure 9.
Figure 9.

TTX-induced (300 nm) changes in glial Ca2+ responses to ADP (100 μm) and CCK (100 nm) in the four glial response profiles identified in the duodenum and colon. a–d, Changes in amplitude (ΔF/F0) are shown for each glial cell (for the duodenum, a; for the colon, c) and profile (for the duodenum, b; for the colon, d) under baseline (–) and TTX (+) conditions. Note that the amplitude (ΔF/F0) scale differs between different gut regions. Data are expressed as average responses in n = 221 and n = 194 tdT+ glial cells, from 6 or 4 myenteric ganglia of N = 4 or N = 3 Wnt1Cre2;GCaMP5g-tdT mice (duodenum and colon, respectively). Ionomycin (2 μm) was used to normalize the glial response in the different gut regions.

Discussion

Bidirectional communication between enteric neurons and glia tunes regionally distinct homeostatic functions in the digestive tract (Grubišić and Gulbransen, 2017). It is generally accepted that enteric neurons exhibit functional heterogeneity, but whether the same is true for glia is not known. Here, we show that differences in how myenteric glia respond to ADP and CCK in the duodenum and colon are produced by differing mechanisms of communication with neurons. Multiple glial response profiles expand the complexity of signaling mechanisms in differing intestinal regions. Each glial response profile differs with regard to the extent of intercellular communication with enteric neurons and intrinsic responsiveness. Together, our data show complex interregional and intraregional functional diversity among neuron–glia networks that reflect the known differences in motor patterns and enteric neurocircuitry between intestinal regions (Fig. 10).

Figure 10.
Figure 10.

Schematic model of the enteric glia heterogeneity that underlies the distinct responsiveness to ADP and CCK in the duodenum and colon. Myenteric glia exhibit distinct response profiles to ADP and CCK in the duodenum and colon. Response profiles delineate four unique subpopulations among myenteric glia that are differently distributed between gut regions.

Regional heterogeneity among myenteric glia is reflected by differences in responsiveness to neurotransmitters such as ADP and CCK. Purinergic signaling is a prominent mechanism of intercellular communication between enteric neurons and glia within the myenteric plexus of the colon (Gulbransen and Sharkey, 2009), but little is known about glial purinergic signaling in the small intestine. In particular, ADP rapidly increases intracellular Ca2+ levels to a greater extent in enteric glial cells than in enteric neurons in the colon (Fried and Gulbransen, 2015), and this functional diversity between enteric neurons and glia could underlie a distinct expression and/or sensitivity of the purinergic receptors and different cell-specific signaling pathways (Fig. 11). Likewise, CCK is a key mediator of signaling along the gut–brain axis (Varga et al., 2004) that stimulates intracellular Ca2+ transients in duodenal glia (Sundaresan et al., 2017), but whether this capacity is conserved among other populations of enteric glia is not known. In agreement with prior work (Gulbransen et al., 2012; Brown et al., 2016), we found that ADP drives a robust and uniform Ca2+ response among myenteric glia in the colon, but we only observed stochastic glial Ca2+ transients in response to ADP in the duodenum. Similarly, Ca2+ responses elicited by CCK in the duodenum are characterized by high frequency, low magnitude, and a long duration throughout the glial network (duodenum: Fig. 1d–f; colon: Fig. 1j–l; Movies 2, 4). CCK-induced glial Ca2+ transients in the colon are, conversely, periodic and have a low amplitude compared with ADP responses. These differences in glial responses to neurotransmitters could contribute to differences in motor patterns between the duodenum and colon similar to regional differences that have been revealed in distinct segments of the colon (Nezami and Srinivasan, 2010; Li et al., 2019; Nestor-Kalinoski et al., 2022).

Figure 11.
Figure 11.

Visual comparison between duodenal and colonic glial responses to ADP and CCK. Temporal color-coded images of responding enteric neurons and glia to 30 s of application of ADP (100 μm) and CCK (100 nm) in a myenteric ganglion of the duodenum and colon of Wnt1Cre2;GCaMP5g-tdT mice, respectively. Representative pictures are z-projections of time series images (30 s for each one) in which the maximal peak Ca2+ responses to ADP or CCK are shown at different times in responsive cells (temporal color coded; see color scale). Scale bar, 20 µm.

Enteric glia are committed to specific neural networks that control intestinal motility through purinergic and cholinergic signaling (McClain et al., 2015; Ahmadzai et al., 2021). Enteric glia recruited by purinergic signaling constrain activity within descending inhibitory networks in the proximal colon. In agreement, we observed a marked relaxation induced by ADP when glial activity was altered by FA in the colon (Fig. 3). This could indicate that glial-mediated tonic inhibition of inhibitory motor neurons is engaged by ADP to repress the activity of descending neural pathways. It is also possible that impairing glial activity reduced excitatory neuromuscular tone to reveal direct inhibitory effects on P2Y1 receptors expressed by PDGF receptor α fibroblast-like cells or intestinal smooth muscle (Sanders, 2016). ADP-mediated relaxation in the duodenum was not affected by FA and could indicate a lesser role of glial purinergic signaling in motor responses in this organ.

Glial purinergic signaling is important to modulate neuronal activity, intestinal motor patterns, and neuroinflammatory responses in the myenteric plexus of the colon (Gulbransen et al., 2012; McClain et al., 2014; Brown et al., 2016; Grubišić et al., 2018). Interestingly, duodenal glia display the ability to synthesize and release gastrin, and to detect it through CCK B receptors (Sundaresan et al., 2017). Given that glia respond to CCK and ADP through similar guanine nucleotide binding protein α subunit Gq class (Gq) receptor pathways that lead to Ca2+ responses, it is conceivable that CCK might function to modulate enteric circuits through effects on glia. Our data suggest that myenteric glia contribute to responses evoked by CCK to a greater extent in the colon than in the duodenum, and to responses evoked by ADP to a comparable extent in either intestinal region, although the impact of glial responses to ADP on intestinal motility differs between these two regions. Presumably, the different functional roles and regional specializations of different intestinal organs promote neuron–glia networks with specific connections and characteristics, which use diverse mediators and signaling pathways.

Enteric neuron–glia communication modulates gastrointestinal reflexes, gut–brain signaling, neuroinflammation, and neuroimmune interactions (Brown et al., 2016; Chow and Gulbransen, 2017; Gulbransen, 2017). We found that disrupting neuron–glia signaling with TTX or FA impaired glial Ca2+ responses to ADP in either intestinal region, while glial CCK responses were altered by TTX in the duodenum and by FA in the colon (Fig. 6). Together, these results suggest that purinergic signaling between enteric neurons and glia is consistent throughout the gastrointestinal tract, while region-specific signaling could occur via gut peptides.

The concept of glial diversity is gaining considerable traction from work showing that astrocyte subtypes have distinct morphologies, physiologies, and unique transcriptional profiles (Lanjakornsiripan et al., 2018; Batiuk et al., 2020; Clarke et al., 2021). These specialized glial subtypes contribute to the formation of region-specific neuronal circuits and specific brain functions (Sofroniew and Vinters, 2010; Verkhratsky and Nedergaard, 2018; Hu et al., 2019). Our data indicate that glial diversity also contributes to region-specific enteric neurocircuitry in the digestive tract. We speculate that the greater complexity of enteric circuitry could mirror more complex motor patterns and lead to greater local diversity among enteric glia. In support, we found four distinct response profiles among myenteric glia that are differently distributed between the duodenum and colon (Fig. 7). Glia exhibited a fairly even distribution among different response profiles without any individual profile dominating the duodenum, while only two glial profiles were mainly observed in the colon. Less heterogeneity in the colon than in the duodenum supports the view that neuronal circuit complexity dictates glial functional diversity, possibly via glial plasticity (Valès et al., 2018). This also supports recent evidence showing that specific glial subtypes and/or signaling selectively contribute to the development of distinct pathologic conditions that affect the intestine (Brown et al., 2016; Sundaresan et al., 2017; Seguella et al., 2020), thereby prompting toward the development of new therapies that selectively target distinct glial subpopulation.

In conclusion, our results show that myenteric glia display distinct profiles of responses to purinergic neuromodulators and CCK that are heterogeneous between the colon and duodenum. The intraregional and interregional glial heterogeneity involves dynamic intercellular communication with enteric neurons and could contribute to the region-specific mechanisms that regulate digestive functions. Glial heterogeneity adds additional complexity in peripheral neurocircuits, highlighting the importance of studying the unique subtypes of enteric glia to understand their involvement in gastrointestinal physiology and pathophysiology.

Footnotes

  • This work was supported by the National Institutes of Health (Grants R01-DK-103723 and R01-DK-120862, to B.D.G.), the MSU Foundation (to B.D.G.), and a University of Rome “La Sapienza” PhD fellowship (to L.S.).

  • The authors decalre no competing financial interests.

  • Correspondence should be addressed to Brian D. Gulbransen at gulbrans{at}msu.edu

References

  1. ↵
    2. Ahmadzai MM,
    3. Seguella L,
    4. Gulbransen BD
    (2021) Circuit-specific enteric glia regulate intestinal motor neurocircuits. Proc Natl Acad Sci U S A 118:e2025938118.
    OpenUrlAbstract/FREE Full Text
  2. ↵
    2. Baghdadi MB,
    3. Ayyaz A,
    4. Coquenlorge S,
    5. Chu B,
    6. Kumar S,
    7. Streutker C,
    8. Wrana JL,
    9. Kim TH
    (2022) Enteric glial cell heterogeneity regulates intestinal stem cell niches. Cell Stem Cell 29:86–100.e6. pmid:34727519
    OpenUrlPubMed
  3. ↵
    2. Batiuk MY,
    3. Martirosyan A,
    4. Wahis J,
    5. de Vin F,
    6. Marneffe C,
    7. Kusserow C,
    8. Koeppen J,
    9. Viana JF,
    10. Oliveira JF,
    11. Voet T,
    12. Ponting CP,
    13. Belgard TG,
    14. Holt MG
    (2020) Identification of region-specific astrocyte subtypes at single cell resolution. Nat Commun 11:1220. pmid:32139688
    OpenUrlCrossRefPubMed
  4. ↵
    2. Boesmans W,
    3. Lasrado R,
    4. Vanden Berghe P,
    5. Pachnis V
    (2015) Heterogeneity and phenotypic plasticity of glial cells in the mammalian enteric nervous system. Glia 63:229–241. pmid:25161129
    OpenUrlCrossRefPubMed
  5. ↵
    2. Boesmans W,
    3. Hao MM,
    4. Fung C,
    5. Li Z,
    6. Van den Haute C,
    7. Tack J,
    8. Pachnis V,
    9. Vanden Berghe P
    (2019) Structurally defined signaling in neuro-glia units in the enteric nervous system. Glia 67:1167–1178. pmid:30730592
    OpenUrlCrossRefPubMed
  6. ↵
    2. Brown IA,
    3. McClain JL,
    4. Watson RE,
    5. Patel BA,
    6. Gulbransen BD
    (2016) Enteric glia mediate neuron death in colitis through purinergic pathways that require connexin-43 and nitric oxide. Cell Mol Gastroenterol Hepatol 2:77–91. pmid:26771001
    OpenUrlCrossRefPubMed
  7. ↵
    2. Chai H,
    3. Diaz-Castro B,
    4. Shigetomi E,
    5. Monte E,
    6. Octeau JC,
    7. Yu X,
    8. Cohn W,
    9. Rajendran PS,
    10. Vondriska TM,
    11. Whitelegge JP,
    12. Coppola G,
    13. Khakh BS
    (2017) Neural circuit-specialized astrocytes: transcriptomic, proteomic, morphological, and functional evidence. Neuron 95:531–549.e9. doi:10.1016/j.neuron.2017.06.029 pmid:28712653
    OpenUrlCrossRefPubMed
  8. ↵
    2. Chow AK,
    3. Gulbransen BD
    (2017) Potential roles of enteric glia in bridging neuroimmune communication in the gut. Am J Physiol Gastrointest Liver Physiol 312:G145–G152. pmid:28039160
    OpenUrlCrossRefPubMed
  9. ↵
    2. Clarke BE,
    3. Taha DM,
    4. Tyzack GE,
    5. Patani R
    (2021) Regionally encoded functional heterogeneity of astrocytes in health and disease: a perspective. Glia 69:20–27. doi:10.1002/glia.23877
    OpenUrlCrossRefPubMed
  10. ↵
    2. Delvalle NM,
    3. Dharshika C,
    4. Morales-Soto W,
    5. Fried DE,
    6. Gaudette L,
    7. Gulbransen BD
    (2018a) Communication between enteric neurons, glia, and nociceptors underlies the effects of tachykinins on neuroinflammation. Cell Mol Gastroenterol Hepatol 6:321–344. doi:10.1016/j.jcmgh.2018.05.009 pmid:30116771
    OpenUrlCrossRefPubMed
  11. ↵
    2. Delvalle NM,
    3. Fried DE,
    4. Rivera-Lopez G,
    5. Gaudette L,
    6. Gulbransen BD
    (2018b) Cholinergic activation of enteric glia is a physiological mechanism that contributes to the regulation of gastrointestinal motility. Am J Physiol Gastrointest Liver Physiol 315:G473–G483. doi:10.1152/ajpgi.00155.2018 pmid:29927320
    OpenUrlCrossRefPubMed
  12. ↵
    2. Fried DE,
    3. Gulbransen BD
    (2015) In situ Ca2+ imaging of the enteric nervous system. J Vis Exp (95):52506.
  13. ↵
    2. Fried DE,
    3. Watson RE,
    4. Robson SC,
    5. Gulbransen BD
    (2017) Ammonia modifies enteric neuromuscular transmission through glial γ-aminobutyric acid signaling. Am J Physiol Gastrointest Liver Physiol 313:G570–G580. doi:10.1152/ajpgi.00154.2017 pmid:28838986
    OpenUrlCrossRefPubMed
  14. ↵
    2. Fung C,
    3. Vanden Berghe P
    (2020) Functional circuits and signal processing in the enteric nervous system. Cell Mol Life Sci 77:4505–4522. doi:10.1007/s00018-020-03543-6 pmid:32424438
    OpenUrlCrossRefPubMed
  15. ↵
    2. Gee JM,
    3. Smith NA,
    4. Fernandez FR,
    5. Economo MN,
    6. Brunert D,
    7. Rothermel M,
    8. Morris SC,
    9. Talbot A,
    10. Palumbos S,
    11. Ichida JM,
    12. Shepherd JD,
    13. West PJ,
    14. Wachowiak M,
    15. Capecchi MR,
    16. Wilcox KS,
    17. White JA,
    18. Tvrdik P
    (2014) Imaging activity in neurons and glia with a Polr2a-based and cre-dependent GCaMP5G-IRES-tdTomato reporter mouse. Neuron 83:1058–1072. pmid:25155958
    OpenUrlCrossRefPubMed
  16. ↵
    2. Grubišić V,
    3. Gulbransen BD
    (2017) Enteric glia: the most alimentary of all glia. J Physiol 595:557–570.
    OpenUrlCrossRefPubMed
  17. ↵
    2. Grubišić V,
    3. Verkhratsky A,
    4. Zorec R,
    5. Parpura V
    (2018) Enteric glia regulate gut motility in health and disease. Brain Res Bull 136:109–117. pmid:28363846
    OpenUrlPubMed
  18. ↵
    2. Gulbransen BD
    (2017) Enteric glia: the origin of duodenal gastrinomas? Gastroenterology 153:1473–1475. doi:10.1053/j.gastro.2017.10.029 pmid:29100851
    OpenUrlCrossRefPubMed
  19. ↵
    2. Gulbransen BD,
    3. Christofi FL
    (2018) Are we close to targeting enteric glia in gastrointestinal diseases and motility disorders? Gastroenterology 155:245–251. pmid:29964042
    OpenUrlPubMed
  20. ↵
    2. Gulbransen BD,
    3. Sharkey KA
    (2009) Purinergic neuron-to-glia signaling in the enteric nervous system. Gastroenterology 136:1349–1358. pmid:19250649
    OpenUrlCrossRefPubMed
  21. ↵
    2. Gulbransen BD,
    3. Sharkey KA
    (2012) Novel functional roles for enteric glia in the gastrointestinal tract. Nat Rev Gastroenterol Hepatol 9:625–632. doi:10.1038/nrgastro.2012.138 pmid:22890111
    OpenUrlCrossRefPubMed
  22. ↵
    2. Gulbransen BD,
    3. Bains JS,
    4. Sharkey KA
    (2010) Enteric glia are targets of the sympathetic innervation of the myenteric plexus in the guinea pig distal colon. J Neurosci 30:6801–6809. doi:10.1523/JNEUROSCI.0603-10.2010 pmid:20463242
    OpenUrlAbstract/FREE Full Text
  23. ↵
    2. Gulbransen BD,
    3. Bashashati M,
    4. Hirota SA,
    5. Gui X,
    6. Roberts JA,
    7. MacDonald JA,
    8. Muruve DA,
    9. McKay DM,
    10. Beck PL,
    11. Mawe GM,
    12. Thompson RJ,
    13. Sharkey KA
    (2012) Activation of neuronal P2X7 receptor-pannexin-1 mediates death of enteric neurons during colitis. Nat Med 18:600–604. pmid:22426419
    OpenUrlCrossRefPubMed
  24. ↵
    2. Hanani M,
    3. Reichenbach A
    (1994) Morphology of horseradish peroxidase (HRP)-injected glial cells in the myenteric plexus of the guinea-pig. Cell Tissue Res 278:153–160. doi:10.1007/BF00305787 pmid:7954696
    OpenUrlCrossRefPubMed
  25. ↵
    2. Hu X,
    3. Qin S,
    4. Huang X,
    5. Yuan Y,
    6. Tan Z,
    7. Gu Y,
    8. Cheng X,
    9. Wang D,
    10. Lian X-F,
    11. He C,
    12. Su Z
    (2019) Region-restrict astrocytes exhibit heterogeneous susceptibility to neuronal reprogramming. Stem Cell Rep 12:290–304. pmid:30713039
    OpenUrlPubMed
  26. ↵
    2. Huizinga JD, et al
    . (2014) The origin of segmentation motor activity in the intestine. Nat Commun 5:3326. doi:10.1038/ncomms4326 pmid:24561718
    OpenUrlCrossRefPubMed
  27. ↵
    2. Ibiza S,
    3. García-Cassani B,
    4. Ribeiro H,
    5. Carvalho T,
    6. Almeida L,
    7. Marques R,
    8. Misic AM,
    9. Bartow-McKenney C,
    10. Larson DM,
    11. Pavan WJ,
    12. Eberl G,
    13. Grice EA,
    14. Veiga-Fernandes H
    (2016) Glial-cell-derived neuroregulators control type 3 innate lymphoid cells and gut defence. Nature 535:440–443. pmid:27409807
    OpenUrlCrossRefPubMed
  28. ↵
    2. Khakh BS,
    3. Sofroniew MV
    (2015) Diversity of astrocyte functions and phenotypes in neural circuits. Nat Neurosci 18:942–952. pmid:26108722
    OpenUrlCrossRefPubMed
  29. ↵
    2. Konomi H,
    3. Meedeniya AC,
    4. Simula ME,
    5. Toouli J,
    6. Saccone GT
    (2002) Characterization of circular muscle motor neurons of the duodenum and distal colon in the Australian brush-tailed possum. J Comp Neurol 443:15–26. doi:10.1002/cne.10094 pmid:11793344
    OpenUrlCrossRefPubMed
  30. ↵
    2. Kulkarni S,
    3. Ganz J,
    4. Bayrer J,
    5. Becker L,
    6. Bogunovic M,
    7. Rao M
    (2018) Advances in enteric neurobiology: the “brain” in the gut in health and disease. J Neurosci 38:9346–9354. doi:10.1523/JNEUROSCI.1663-18.2018 pmid:30381426
    OpenUrlAbstract/FREE Full Text
  31. ↵
    2. Lanjakornsiripan D,
    3. Pior BJ,
    4. Kawaguchi D,
    5. Furutachi S,
    6. Tahara T,
    7. Katsuyama Y,
    8. Suzuki Y,
    9. Fukazawa Y,
    10. Gotoh Y
    (2018) Layer-specific morphological and molecular differences in neocortical astrocytes and their dependence on neuronal layers. Nat Commun 9:1623. pmid:29691400
    OpenUrlCrossRefPubMed
  32. ↵
    2. Laranjeira C,
    3. Sandgren K,
    4. Kessaris N,
    5. Richardson W,
    6. Potocnik A,
    7. Vanden Berghe P,
    8. Pachnis V
    (2011) Glial cells in the mouse enteric nervous system can undergo neurogenesis in response to injury. J Clin Invest 121:3412–3424. pmid:21865647
    OpenUrlCrossRefPubMed
  33. ↵
    2. Li Z,
    3. Hao MM,
    4. Van den Haute C,
    5. Baekelandt V,
    6. Boesmans W,
    7. Vanden Berghe P
    (2019) Regional complexity in enteric neuron wiring reflects diversity of motility patterns in the mouse large intestine. eLife 8:e42914. doi:10.7554/eLife.42914
    OpenUrlCrossRef
  34. ↵
    2. McClain JL,
    3. Gulbransen BD
    (2017) The acute inhibition of enteric glial metabolism with fluoroacetate alters calcium signaling, hemichannel function, and the expression of key proteins. J Neurophysiol 117:365–375. doi:10.1152/jn.00507.2016
    OpenUrlCrossRefPubMed
  35. ↵
    2. McClain JL,
    3. Grubišić V,
    4. Fried D,
    5. Gomez-Suarez RA,
    6. Leinninger GM,
    7. Sévigny J,
    8. Parpura V,
    9. Gulbransen BD
    (2014) Ca2+ responses in enteric glia are mediated by connexin-43 hemichannels and modulate colonic transit in mice. Gastroenterology 146:497–507.e1. pmid:24211490
    OpenUrlCrossRefPubMed
  36. ↵
    2. McClain JL,
    3. Fried DE,
    4. Gulbransen BD
    (2015) Agonist-evoked Ca2+ signaling in enteric glia drives neural programs that regulate intestinal motility in mice. Cell Mol Gastroenterol Hepatol 1:631–645. pmid:26693173
    OpenUrlCrossRefPubMed
  37. ↵
    2. Muller PA,
    3. Matheis F,
    4. Schneeberger M,
    5. Kerner Z,
    6. Jové V,
    7. Mucida D
    (2020) Microbiota-modulated CART+ enteric neurons autonomously regulate blood glucose. Science 370:314–321. doi:10.1126/science.abd6176
    OpenUrlAbstract/FREE Full Text
  38. ↵
    2. Nasser Y,
    3. Ho W,
    4. Sharkey KA
    (2006a) Distribution of adrenergic receptors in the enteric nervous system of the guinea pig, mouse, and rat. J Comp Neurol 495:529–553. pmid:16498685
    OpenUrlCrossRefPubMed
  39. ↵
    2. Nasser Y,
    3. Fernandez E,
    4. Keenan CM,
    5. Ho W,
    6. Oland LD,
    7. Tibbles LA,
    8. Schemann M,
    9. MacNaughton WK,
    10. Rühl A,
    11. Sharkey KA
    (2006b) Role of enteric glia in intestinal physiology: effects of the gliotoxin fluorocitrate on motor and secretory function. Am J Physiol Gastrointest Liver Physiol 291:G912–G927. pmid:16798727
    OpenUrlCrossRefPubMed
  40. ↵
    2. Nestor-Kalinoski A,
    3. Smith-Edwards KM,
    4. Meerschaert K,
    5. Margiotta JF,
    6. Rajwa B,
    7. Davis BM,
    8. Howard MJ
    (2022) Unique neural circuit connectivity of mouse proximal, middle, and distal colon defines regional colonic motor patterns. Cell Mol Gastroenterol Hepatol 13:309–337.e3. doi:10.1016/j.jcmgh.2021.08.016 pmid:34509687
    OpenUrlCrossRefPubMed
  41. ↵
    2. Nezami BG,
    3. Srinivasan S
    (2010) Enteric nervous system in the small intestine: pathophysiology and clinical implications. Curr Gastroenterol Rep 12:358–365. pmid:20725870
    OpenUrlCrossRefPubMed
  42. ↵
    2. Rao M,
    3. Nelms BD,
    4. Dong L,
    5. Salinas-Rios V,
    6. Rutlin M,
    7. Gershon MD,
    8. Corfas G
    (2015) Enteric glia express proteolipid protein 1 and are a transcriptionally unique population of glia in the mammalian nervous system. Glia 63:2040–2057. pmid:26119414
    OpenUrlCrossRefPubMed
  43. ↵
    2. Rao M,
    3. Rastelli D,
    4. Dong L,
    5. Chiu S,
    6. Setlik W,
    7. Gershon MD,
    8. Corfas G
    (2017) Enteric glia regulate gastrointestinal motility but are not required for maintenance of the epithelium in mice. Gastroenterology 153:1068–1081.e7. doi:10.1053/j.gastro.2017.07.002 pmid:28711628
    OpenUrlCrossRefPubMed
  44. ↵
    2. Sanders KM
    (2016) Enteric inhibitory neurotransmission, starting down under. Adv Exp Med Biol 891:21–29. doi:10.1007/978-3-319-27592-5_3 pmid:27379631
    OpenUrlCrossRefPubMed
  45. ↵
    2. Seguella L,
    3. Gulbransen BD
    (2021) Enteric glial biology, intercellular signalling and roles in gastrointestinal disease. Nat Rev Gastroenterol Hepatol 18:571–587. doi:10.1038/s41575-021-00423-7 pmid:33731961
    OpenUrlCrossRefPubMed
  46. ↵
    2. Seguella L,
    3. Capuano R,
    4. Sarnelli G,
    5. Esposito G
    (2019) Play in advance against neurodegeneration: exploring enteric glial cells in gut-brain axis during neurodegenerative diseases. Expert Rev Clin Pharmacol 12:555–564. pmid:31025582
    OpenUrlPubMed
  47. ↵
    2. Seguella L,
    3. Rinaldi F,
    4. Marianecci C,
    5. Capuano R,
    6. Pesce M,
    7. Annunziata G,
    8. Casano F,
    9. Bassotti G,
    10. Sidoni A,
    11. Milone M,
    12. Aprea G,
    13. de Palma GD,
    14. Carafa M,
    15. Pesce M,
    16. Esposito G,
    17. Sarnelli G
    (2020) Pentamidine niosomes thwart S100B effects in human colon carcinoma biopsies favouring wtp53 rescue. J Cell Mol Med 24:3053–3063. pmid:32022398
    OpenUrlPubMed
  48. ↵
    2. Seguella L,
    3. Pesce M,
    4. Capuano R,
    5. Casano F,
    6. Pesce M,
    7. Corpetti C,
    8. Vincenzi M,
    9. Maftei D,
    10. Lattanzi R,
    11. Del Re A,
    12. Sarnelli G,
    13. Gulbransen BD,
    14. Esposito G
    (2021) High-fat diet impairs duodenal barrier function and elicits glia-dependent changes along the gut-brain axis that are required for anxiogenic and depressive-like behaviors. J Neuroinflammation 18:115. doi:10.1186/s12974-021-02164-5 pmid:33993886
    OpenUrlCrossRefPubMed
  49. ↵
    2. Sofroniew MV,
    3. Vinters HV
    (2010) Astrocytes: biology and pathology. Acta Neuropathol 119:7–35. pmid:20012068
    OpenUrlCrossRefPubMed
  50. ↵
    2. Spencer NJ,
    3. Dinning PG,
    4. Brookes SJ,
    5. Costa M
    (2016) Insights into the mechanisms underlying colonic motor patterns. J Physiol 594:4099–4116. pmid:26990133
    OpenUrlCrossRefPubMed
  51. ↵
    2. Sundaresan S,
    3. Meininger CA,
    4. Kang AJ,
    5. Photenhauer AL,
    6. Hayes MM,
    7. Sahoo N,
    8. Grembecka J,
    9. Cierpicki T,
    10. Ding L,
    11. Giordano TJ,
    12. Else T,
    13. Madrigal DJ,
    14. Low MJ,
    15. Campbell F,
    16. Baker AM,
    17. Xu H,
    18. Wright NA,
    19. Merchant JL
    (2017) Gastrin induces nuclear export and proteasome degradation of menin in enteric glial cells. Gastroenterology 153:1555–1567.e15. pmid:28859856
    OpenUrlPubMed
  52. ↵
    2. Valès S,
    3. Touvron M,
    4. Van Landeghem L
    (2018) Enteric glia: diversity or plasticity? Brain Res 1693:140–145. doi:10.1016/j.brainres.2018.02.001 pmid:29425908
    OpenUrlCrossRefPubMed
  53. ↵
    2. Varga G,
    3. Bálint A,
    4. Burghardt B,
    5. D'Amato M
    (2004) Involvement of endogenous CCK and CCK1 receptors in colonic motor function. Br J Pharmacol 141:1275–1284. pmid:15100163
    OpenUrlCrossRefPubMed
  54. ↵
    2. Verkhratsky A,
    3. Nedergaard M
    (2018) Physiology of astroglia. Physiol Rev 98:239–389. doi:10.1152/physrev.00042.2016 pmid:29351512
    OpenUrlCrossRefPubMed
View Abstract
Back to top

In this issue

Email
Print
View Full Page PDF
Citation Tools
Respond to this article
Functional Intraregional and Interregional Heterogeneity between Myenteric Glial Cells of the Colon and Duodenum in Mice
Luisa Seguella, Jonathon L. McClain, Giuseppe Esposito, Brian D. Gulbransen
Journal of Neuroscience 16 November 2022, 42 (46) 8694-8708; DOI: 10.1523/JNEUROSCI.2379-20.2022
Twitter logo Facebook logo Mendeley logo

Jump to section

Keywords

Responses to this article

Respond to this article

Jump to comment:

No eLetters have been published for this article.