Nutrients activate distinct patterns of small-intestinal enteric neurons

Fung, Candice; Venneman, Tom; Holland, Amy M.; Martens, Tobie; Alata, Milvia I.; Hao, Marlene M.; Alar, Ceyhun; Obata, Yuuki; Tack, Jan; Sifrim, Alejandro; Pachnis, Vassilis; Boesmans, Werend; Vanden Berghe, Pieter

doi:10.1038/s41586-025-09228-z

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Published: 09 July 2025

Nutrients activate distinct patterns of small-intestinal enteric neurons

Nature volume 644, pages 1069–1077 (2025)Cite this article

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Abstract

The ability to detect and respond appropriately to ingested nutrients is essential for an organism’s survival and to ensure its metabolic demands are met. Nutrient signals from the gut lumen trigger local intestinal reflexes in the enteric nervous system (ENS) to facilitate digestion and absorption^1,2,3,4, but the precise cellular pathways that are involved in the initial neuronal sensory process remain unclear. The extent to which the ENS is capable of discerning different luminal chemicals is also unknown. Here we use calcium imaging to identify specific enteric pathways that are activated in response to luminal nutrients applied to mouse jejunum. Notably, we show that different nutrients activate neurochemically defined ensembles of myenteric and submucosal neurons. Furthermore, we find that enteric neurons are not directly sensitive to nutrients but detect different luminal chemicals through the epithelium, mainly via a serotonin signalling pathway. Finally, our data reveal a spatial distribution of luminal information along the radial axis of the intestine, whereby some signals that originate from the villus epithelium are transmitted first to the myenteric plexus, and then back to the submucosal plexus, which is closer to the lumen.

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Main

Monitoring of ingested nutrients by the gastrointestinal tract is crucial for energy homeostasis. Precise signalling of sensory cues from the intestinal lumen is key for guiding physiologically appropriate responses at not only at the organ but also the behavioural level. For instance, neuropod cells in the intestinal epithelium make specialized connections with the vagus nerve, which enables rapid gut–brain signalling and helps drive the preference for nutritive sugars over non-caloric sweeteners⁵. Sensory information from the gut also forms a key branch of visceral signals transmitted to the nodose ganglion to inform the brain of the body’s overall internal state⁶. However, little is known about how sensory cues, even basic nutrients, are handled at the initial site of contact; namely, by the gut itself and its ENS. Different luminal compositions elicit distinct intestinal behaviours. A nutritious meal produces mixing motor patterns to facilitate digestion and absorption, whereas a non-caloric meal predominantly induces propulsive contractions⁷. When presented with cholera toxin⁸ together with a nutritious fatty acid, the intestine apparently overrides signals to trigger mixing and rapidly evokes propulsive contractions, presumably prioritizing elimination of the diarrhoea-inducing agent⁹. Despite this, how luminal information is communicated to the ENS remains unclear, and the specific enteric pathways that allow the gut to distinguish between different luminal chemicals have yet to be identified.

The ENS comprises a repertoire of intrinsic primary afferent neurons (IPANs, or intrinsic sensory neurons), interneurons and motor neurons organized within two ganglionated plexuses: the submucosal plexus (SMP) and the myenteric plexus (MP)^3,4,10,11. Because the epithelial barrier shields the luminal contents, how the ENS detects luminal cues is unclear. Although previous work has focused on various aspects of mechanosensation of the ingested bolus^12,13, and mucosal chemosensation^14,15,16, less is known about the communication and further processing of these epithelial signals in the ENS plexuses. Here we developed experimental strategies to record ENS activity in response to mucosal stimuli ex vivo and in vivo. We show that chemically distinct signals activate unique combinations of enteric neurons in register with the overlying villi. We uncover a stereotypic sequence of neuronal activation, in which some signals that originate from the villi are transmitted to neurons in the outer MP and then return to the inner SMP. The modular and hierarchical organization of signal-specific neuronal circuits activated by the exposure of villi to distinct chemicals provides a basis for understanding how the gut’s luminal environment elicits meaningful physiological responses.

Villi receive local ENS innervation

To anatomically map the innervation of a villus, we injected a neuronal tracer, DiI, into the tip of a villus. Labelled myenteric and submucosal neurons were mostly in close vicinity to the labelled villus (71 ± 15% of labelled neurons situated within a 558-µm radius of the villus; Fig. 1a–d). This spatial arrangement of enteric neurons relative to the associated villus is in line with the developmental distribution of the related neuroglial progenitor cell lineage in radial columns along the lumen–mucosa axis¹⁷, and is consistent with findings in guinea pigs^18,19. Having established the topographical organization of enteric neurons supplying a villus, we proceeded to examine the functionality of these neurons.

**Fig. 1: ENS responses to mucosal high K⁺.**

Mucosal high K⁺ activates the ENS

Under physiological conditions, the gut epithelium is exposed to a plethora of chemical stimuli, which widely activate enteroendocrine cells (EECs), including serotonin (5-HT)-containing enterochromaffin (EC) cells^20,21,22. A high K⁺ (75 mM) Krebs solution was used to broadly stimulate these electrically excitable EECs^23,24, and the SMP and MP responses were examined in preparations of Wnt1-Cre;R26R-LsL-GCaMP3 (Wnt1|GCaMP3) mouse jejunum (Fig. 1e–l). Mucosal high K⁺ evoked Ca²⁺ transients in a subset of submucosal and myenteric neurons (24 ± 6% and 22 ± 3% of neurons examined, respectively). To further characterize these neurons, imaged preparations were labelled for choline acetyltransferase (ChAT) and vasoactive intestinal peptide (VIP) in the SMP, to distinguish between two major subtypes of submucosal secretomotor and vasodilator neurons²⁵. Responders were mostly cholinergic (ChAT⁺; Fig. 1f–h and Supplementary Table 1). In the MP, we labelled for calbindin, which is a commonly used marker for IPANs, and for nNOS, which is a marker for inhibitory interneurons and motor neurons²⁶. Most myenteric responders were calbindin⁺ and few were nNOS⁺ (Fig. 1j–l and Supplementary Table 2), suggesting that IPANs and downstream nitrergic neurons were activated.

We then assessed whether MP–mucosa nerve connections are necessary for mucosal signal transmission. First, we verified that mucosal high K⁺ could elicit responses in myenteric neurons in intact preparations (Fig. 1m–o). The gut layers were then peeled apart between the circular muscle and the SMP, severing MP–mucosa connections, and the separated layers were replaced. This abolished the responses to mucosal high K⁺ in the ganglia that were imaged previously (Fig. 1p–r), indicating that the effect of high K⁺ was not due to diffusion through the epithelium to the underlying MP. To confirm that the neurons were still viable after peeling, we showed that directly depolarizing the same ganglia evoked robust Ca²⁺ transients in nearly all neurons, including all initial responders (Fig. 1s–u). These data show that MP–mucosa and/or MP–SMP connections are essential for transmitting luminal signals.

Nutrients activate the ENS through the mucosa

To examine the effects of different luminal nutrients on ENS activity, nutrient solutions were applied to the mucosa while imaging the SMP or MP (Fig. 2). Glucose (300 mM; Supplementary Videos 1 and 2), acetate (100 mM; Supplementary Videos 3 and 4) and l-phenylalanine (l-Phe; 100 mM; Supplementary Videos 5 and 6) were selected as a model sugar, short-chain fatty acid and amino acid, respectively. Because glucose (11.5 mM) is present in the Krebs solution, glucose stimulation was considered as the increase from baseline to 300 mM. Krebs perfusion alone did not have any effects, demonstrating that the perfusion pressure applied was not a sufficient mechanical stimulus to evoke mechanosensory responses (Supplementary Tables 1 and 2).

**Fig. 2: ENS responses to mucosal nutrient perfusion.**

In the SMP (Fig. 2a–l), glucose, acetate and l-Phe each activated a comparable percentage of mostly cholinergic (ChAT⁺) neurons (21 ± 4%, 24 ± 7% and 23 ± 3%, respectively; Fig. 2j,k and Supplementary Table 1). Cell size was quantified as another means of classifying responders, because IPANs tend to have type II morphology with somas that are larger than those of other neurons²⁶; this was also comparable between nutrients (Fig. 2l).

In the MP (Fig. 2m–x), glucose (300 mM) evoked responses in 17 ± 6% of neurons, which included calbindin⁺, neuronal nitric oxide synthase⁺ (nNOS⁺) and calbindin⁻nNOS⁻ neurons, whereas acetate (100 mM) and l-Phe (100 mM) elicited responses in 10 ± 2% and 9 ± 2% of neurons, respectively, which included calbindin⁺ and calbindin⁻nNOS⁻ neurons (Fig. 2v,w). The numbers of calbindin⁺ and nNOS⁺ neurons examined were comparable across preparations (Supplementary Table 2). However, the relative proportions of the neurochemical subtypes of responders (P < 0.0001; χ² test) and their cell diameters differed between stimuli (Fig. 2x), confirming that different nutrients activate unique ensembles of myenteric neurons.

To further characterize the molecular identity of nutrient-responsive myenteric neurons, RNAscope was performed, targeting specific populations informed by RNA sequencing (RNA-seq)-based classifications²⁷. We labelled three putative myenteric IPAN populations (distinguished by Nmu, Cck or Nxph2 expression). Nutrient-responsive neurons exhibited mainly Nmu (Extended Data Fig. 1), rarely Cck and never Nxph2 expression (Supplementary Table 3).

To examine the differential response to nutrients, we applied each nutrient to the mucosa sequentially and imaged the same myenteric ganglion. Of the total responders, a small fraction were activated by all or various combinations of nutrients, and more than half responded uniquely to one nutrient (Extended Data Fig. 2); many were acetate-responsive. Because extracellular glucose concentration can influence myenteric neuronal excitability²⁸, glucose exposure might affect subsequent nutrient responses. The potential influence of one nutrient on responses to another should be taken into account when interpreting these data.

Considering nutrient transport or diffusion across the epithelium, we tested whether mucosal sensory nerves are directly nutrient sensitive. Glucose (300 mM), acetate (100 mM) or l-Phe (100 mM) were applied into a single villus, using a micropipette pushed through the epithelium, to target sensory nerve endings. Although 5-HT applied in this manner activates enteric neurons²⁹, this mode of nutrient application was not effective. Local nutrient application onto myenteric ganglia in peeled longitudinal muscle–myenteric plexus (LMMP) preparations also did not elicit neuronal responses (Supplementary Table 4). Thus, mucosal sensory nerves do not directly detect these nutrient solutions, indicating that the epithelium is necessary for initial sensory transduction of nutrient signals.

To verify that the epithelium senses these nutrients, we applied each nutrient solution onto the mucosal surface of jejunal preparations from Villin-cre;R26R-LsL-GCaMP3 (Villin|GCaMP3) mice³⁰. Nutrients and high K⁺ elicited epithelial Ca²⁺ responses (Extended Data Fig. 3 and Supplementary Videos 7–9). Analysing published single-cell and single-nucleus RNA-seq datasets^27,31,32, we found clusters of small-intestinal epithelial cell types expressing genes that encode associated nutrient sensing receptors and transporters, putative neuroactive mediators and vesicular release proteins (Extended Data Fig. 4a).

Consistent with the glucose responses observed, where Ca²⁺ signals were widely distributed throughout the epithelial surface (Supplementary Video 7), Slc5a1 (encoding the sodium–glucose co-transporter 1; SGLT1) and Tas1r2 and Tas1r3 (encoding components of the sweet taste receptor heterodimer T1R2–T1R3) were most highly expressed by enterocytes (the most abundant epithelial cell type). By contrast, acetate and l-Phe responses were confined mainly to distinct clusters of epithelial cells (Supplementary Videos 8 and 9). In accordance with this, expression of Ffar2 (short-chain fatty acid receptor) was highest in EECs and goblet cells, which are constituents of a smaller population of secretory cells sparsely distributed throughout the epithelium. Similarly, expression of Casr (calcium-sensing receptor) and Tas1r1 and Tas1r3 (components of the taste receptor heterodimer T1R1–T1R3), each associated with sensing l-amino acids, was found predominantly in EECs and in goblet cells, respectively. Therefore, epithelial cells involved in nutrient signal transduction might not be limited to EECs, and could include other cell types.

To assess the importance of epithelium–ENS communication in transmitting nutrient information, we tested the effect of disrupting epithelial signalling on neuronal responses. Blocking SGLT1 with mizagliflozin³³ (1 µM) attenuated glucose responses in the MP and SMP (Extended Data Fig. 5), suggesting that SGLT1 has a major role.

Mucosa–ENS signalling involves 5-HT

To identify mediators involved in transducing luminal signals to the ENS, we assessed the ability of molecules to activate enteric neurons through mucosal nerve endings. Putative mucosal mediators (5-HT, ATP, CCK8 and GLP-1; refs. ^34,35) implicated in nutrient signalling were injected into villi (to mimic their basolateral release onto mucosal nerve endings) while imaging the MP (Fig. 3a and Supplementary Table 4).

**Fig. 3: Mucosal nerve endings are sensitive to 5-HT and ATP.**

5-HT–5-HT₃ signalling is considered to be a key mucosal sensory pathway involved in transducing chemical and mechanical stimuli in the gastrointestinal tract^1,13,24,36. 5-HT puffed onto the mucosa stimulates myenteric neurons^37,38. We found that intravillus injection of 5-HT (10 µM) evoked responses in 18 ± 10% myenteric neurons (Fig. 3a–e); many were calbindin⁺ (Fig. 3d). In accordance with our previous findings²⁹, 5-HT acted through 5-HT₃ receptors, because its effect was abolished by ondansetron (10 µM) (Fig. 3e).

ATP is implicated as a mediator in mucosal mechanosensitivity³⁹. Mucosal application of ATP activates myenteric afterhyperpolarization-type neurons in guinea-pig ileum through P2X receptors⁴⁰. Here, intravillus injection of ATP (200 µM) activated 7 ± 2% of myenteric neurons, most of which were calbindin⁺ (Fig. 3f–i). The P2X antagonist TNP-ATP (5 µM) inhibited the response (Fig. 3i).

In the jejunum, intravillus injection of CCK8 (100 nM) or GLP-1 (100 nM) had no effect, suggesting that the responses to intravillus injection of 5-HT or ATP were not due to mechanical stimulation. Considering the enrichment of CCK8-containing I cells in the duodenum and GLP-1-containing L cells in the terminal ileum, we also examined these regions. Unlike what was seen with 5-HT, no responses were observed when CCK8 was injected into duodenal villi or when GLP-1 was injected into terminal ileum villi (Extended Data Fig. 6a–f). GLP-1 might stimulate intestinofugal neurons in the ileum that have mucosal projections³⁵. We could have missed these neurons, because they form only a small proportion of myenteric neurons and tend to be situated along the mesenteric border. We also cannot rule out the possibilities that these mediators target extrinsic mucosal nerve endings or that they modulate activity within the plexus. Indeed, CCK8 or GLP-1 applied onto jejunal LMMP activated some myenteric neurons (Extended Data Fig. 6g–j).

On the basis of an analysis of single-cell and single-nucleus RNA-seq datasets of small-intestinal enteric neurons^27,32, Htr3a and P2rx2 are highly expressed in IPANs, as defined by Calcb, Calb2 and Nmu expression (Extended Data Fig. 4b). This is compatible with our RNAscope data, in which many nutrient responders expressed Nmu (Extended Data Fig. 1). These neurons showed low levels of Glp1r, Cckar, Cckbr and Calb1 expression, despite the fact that calbindin is a commonly used marker for IPANs²⁶. Because the calbindin antibody used might cross-react with calretinin²⁷, calcitonin gene-related peptide (CGRP) was used as an additional IPAN marker. CGRP immunoreactivity is normally observed only in varicosities, so tissues were cultured in colchicine to disrupt axonal transport and concentrate the peptide in the soma before immunostaining⁴¹. In colchicine-treated tissues, 85 ± 11% of CGRP-immunoreactive myenteric neurons were labelled with the calbindin antibody (Extended Data Fig. 7).

Having found that mucosal nerve endings were responsive to 5-HT and ATP, we tested for endogenous 5-HT and ATP release after the application of mucosal high K⁺, using ondansetron (10 µM) and TNP-ATP (5 µM) (Fig. 3j–k). Ondansetron inhibited but did not abolish the response, indicating the activation of a 5-HT–5-HT₃ receptor-independent pathway. On the other hand, TNP-ATP had no effect, demonstrating that ATP acting through P2 receptors is not involved.

Chemosensitive myenteric IPANs

Although mucosal high K⁺ activates calbindin⁺ myenteric neurons, suggesting that these are IPANs, it is unclear whether they respond directly or synaptically. This was addressed using the nicotinic antagonist hexamethonium (Hex, 200 µM) to block the major form of excitatory neurotransmission in the ENS (Fig. 4a,b). The percentage of myenteric neurons activated before and after Hex exposure was comparable, which indicates that responders (observed locally within the field of view, FOV) were activated directly.

**Fig. 4: Mucosal stimulation activates IPANs and synaptic responses.**

With repeated mucosal high K⁺ application, the second stimulation activated significantly more myenteric neurons (Fig. 4a, left). These additional responders were mainly calbindin⁺, nNOS⁺, or calbindin⁻nNOS⁻ neurons with smaller cell bodies than those of the initial responders (Fig. 4a, right), and were probably excitatory and inhibitory motor neurons and/or interneurons. The amplified response with the second mucosal high K⁺ application was inhibited by Hex (Fig. 4b), demonstrating that the additional neurons were recruited through nicotinic neurotransmission.

Because direct and synaptic responses to mucosal high K⁺ were observed, we set out to characterize the myenteric neurons functionally connected to an individual villus with a more localized stimulus using a focal electrode (Fig. 4c–g). Train stimulation (20 Hz, 1 s) in control and Hex conditions elicited reproducible responses in 14 ± 5% of myenteric neurons (Supplementary Table 4). Like the initial mucosal high K⁺ responders, electrical stimulation responders were predominantly calbindin⁺ (Fig. 4d–f). In the Hex condition, a total of 89 ± 7% of initial responders were activated by the second electrical stimulation, whereas the same numbers responded to both stimulations in control conditions (Fig. 4g). The effect of electrical stimulation was therefore mainly direct, with a minor component being synaptic, and 14 ± 5% of myenteric neurons within the FOV functionally innervate a single villus. This is comparable to the numbers of myenteric responders to intravillus 5-HT injection (16 ± 4%), suggesting that most (if not all) mucosal nerve endings are sensitive to 5-HT.

Responses to mucosal high K⁺ in both plexuses were examined simultaneously using spinning disk confocal microscopy (Extended Data Fig. 8). The Ca²⁺ transient peak in the initial myenteric responder typically preceded that of the initial submucosal responder (Extended Data Fig. 8a–c).

Chemosensitive submucosal IPANs

To examine whether submucosal responses to mucosal high K⁺ occur directly or synaptically, we applied Hex (Fig. 4h,i). Repeated mucosal high K⁺ evoked consistent responses in control conditions (Fig. 4h), and these were partly reduced but not abolished by Hex (Fig. 4i), suggesting that non-nicotinic synaptic signalling or direct activation are also involved⁴².

Because submucosal but not myenteric responders to mucosal high K⁺ were Hex sensitive, we assessed the extent to which the SMP receives myenteric inputs. We compared the submucosal responses to electrical stimulation of a single villus with and without the MP. First, a train stimulus (20 Hz, 1 s) was applied to the villus with a focal electrode in an intact preparation, which activated a subset of submucosal neurons (Fig. 4j–l). The same villus (marked by DiI) was then stimulated after removing the MP (Fig. 4m–q), and the response was significantly reduced (Fig. 4o–r). Thus, some submucosal neurons were activated secondarily through the MP. Because responses were still observed after removing the MP, these neurons might be activated directly (IPANs) and/or by antidromic transmission (secretomotor neurons). Repeated electrical stimulation in intact preparations elicited reproducible responses (Fig. 4r).

To determine whether the SMP contains IPANs, we applied nutrients onto mucosa–SMP preparations (Extended Data Fig. 9). Glucose, acetate and l-Phe activated 22 ± 4%, 6 ± 2% and 7 ± 3% of submucosal neurons, respectively, providing evidence for submucosal IPANs. However, fewer neurons were stimulated by acetate and l-Phe in peeled than in full-thickness preparations, indicating that myenteric inputs contribute to submucosal responses to acetate and l-Phe, but not glucose.

Nutrient-induced ENS responses in vivo

Several factors can influence local nutrient responses, including long-range nerve projections within the ENS, extrinsic innervation, blood flow, the presence of mucus, the gut microbiota, and pancreatic and hepatic signals. We examined the integrated system by imaging the ENS in vivo (Fig. 5). As a proof of concept, we luminally applied nutrient solution into an externalized jejunal loop of an anaesthetized mouse and imaged the MP. Consistent with our ex vivo data, acetate (100 mM) acutely activated a subset of myenteric neurons in the jejunal loop in vivo (15 ± 2% of neurons) (Supplementary Video 10); these included calbindin⁺ and nNOS⁺ neurons (6/30 and 13/30 responders, respectively). Because the imaging site is around 5 mm distal of the nutrient perfusion site, downstream motor neurons and interneurons are likely to have been visualized as well as IPANs. Some neurons might be sensitive to distension from perfusion, because luminal Krebs also evoked Ca²⁺ responses⁴³ (in 7 ± 2% of neurons); many were nNOS⁺ (6/9 responders).

**Fig. 5: Myenteric neuronal responses to luminal acetate in the jejunum of a live mouse.**

Discussion

Here, using Ca²⁺ imaging, we have shown that specific enteric pathways are activated by luminal chemicals, and that the ENS can decipher intraluminal contents, with distinct ensembles of neurons responding to different nutrients ex vivo. Similar results were observed when nutrients were applied into an externalized jejunal loop in live mice. Our findings pave the way to a better understanding of how the intestine monitors the luminal chemical environment and how luminal cues shape its functional output. Although further work will be necessary to fully characterize how luminal information is coded and communicated to the ENS, our findings corroborate previous studies that have identified 5-HT as a key molecule mediating signalling between the epithelium and myenteric IPANs through 5-HT₃ receptors^38,44. Because enterocytes and EC cells in the mouse small intestine lack neuropods⁴⁵, their communication with nerve endings must involve another mechanism. Previous work indicates that mucosal 5-HT is likely to act through paracrine signalling⁴⁶. In olfactory sensation, most odours are encoded not by one receptor, but by a pattern of receptors and olfactory receptor neurons⁴⁷. It is likely that different luminal stimuli are similarly encoded by patterns of mediators in addition to 5-HT and depend on combinations of epithelial mediators and corresponding neuronal receptors.

Across all modes of mucosal stimulation, calbindin⁺CGRP⁺ Nmu-expressing myenteric neurons were prominent responders, in line with the initial responders to luminal signals being myenteric IPANs^26,27,48. Downstream of myenteric IPANs are myenteric interneurons, motor neurons and submucosal neurons activated through nicotinic transmission (Extended Data Fig. 10). Studies suggest that IPANs are also present in the mouse SMP, on the basis of neuronal morphology, projection patterns, advillin expression⁴⁹ and RNA-seq data³². Indeed, we observed submucosal responses to nutrients even without the MP, adding functional evidence for submucosal IPANs. Antidromic activation was unlikely, because cholinergic and VIPergic submucosal neurons supplying the mucosa are abundant²⁵, but responders were almost exclusively cholinergic. Mucosal stimulation also activates some submucosal neurons through the MP, in agreement with previous work that showed that submucosal neurons receive nicotinic inputs from myenteric neurons⁵⁰. Myenteric neurons might, in turn, receive inputs from submucosal neurons⁵¹. This bidirectional signalling is likely to be important in coordinating intestinal motility, secretion and blood flow for optimizing digestion and absorption⁵². Collectively, nutrient signals locally activate submucosal and myenteric sensory circuits, as well as motor circuits, and some myenteric neurons converge onto submucosal pathways.

Our study shows that the MP is a key site of initial luminal signal processing and integration, despite the fact that it is physically further away from the lumen than is the SMP. Additional processing occurs in the SMP, in which neurons receive luminal information directly, and via the MP. This spatial organization of sensory circuits might reflect a functional advantage, in which priority is assigned to initiating motility programs and the initial responders are those that are close to the target smooth muscle. The ENS of lower vertebrates, such as zebrafish, comprises a MP layer but lacks a SMP⁵³, suggesting that fine-tuned secretomotor and vasomotor control evolved later. We speculate that myenteric neurons are evolutionarily wired to rapidly process luminal signals, and that this is conserved across species. Further studies will be necessary to determine the full extent of neuronal activation beyond the stimulation site—with regard to not only downstream enteric interneurons and motor neurons, but also extrinsic nerves—and establish how the net activity ultimately translates into complex intestinal behaviours.

Methods

Mice and tissue preparation

Adult C57BL/6, Wnt1-Cre;R26R-LsL-GCaMP3 (Wnt1|GCaMP3) and Villin-cre;R26R-LsL-GCaMP3 (Villin|GCaMP3) mice aged at least six weeks old and of either sex were euthanized by cervical dislocation. The mice used in this study were maintained on a normal diet (Sniff; Soest) and water was provided ad libitum. All procedures were approved by the Animal Ethics Committee of the University of Leuven (Belgium). Wnt1|GCaMP3 mice express the fluorescent Ca²⁺ indicator GCaMP3 in all enteric neurons and glia, whereas GCaMP3 is expressed in the gut epithelium in Villin|GCaMP3 mice^54,55,56.

Five-centimetre pieces of duodenum, jejunum and/or ileum were collected and immersed in Krebs solution (120.9 mM NaCl, 5.9 mM KCl, 1.2 mM MgCl₂, 1.2 mM NaH₂PO₄, 14.4 mM NaHCO₃, 11.5 mM glucose and 2.5 mM CaCl₂) bubbled with 95% oxygen and 5% carbon dioxide. Each intestinal segment was opened by cutting along the mesenteric border and pinned flat with the mucosal side up in a silicone-elastomer-coated dish. The intestinal contents were removed with Krebs washes and any remnants were carefully removed using forceps. Experiments were limited to a maximum of three hours after tissue collection to minimize potential effects of compromised mucosal integrity.

For Ca²⁺ imaging of neuronal activity, either full-thickness preparations of Wnt1|GCaMP3 tissues with all the layers of the intestinal wall intact or microdissected submucosal preparations with the mucosa intact were stretched across an inox ring with the plexus facing up and stabilized with an outer rubber O-ring⁵⁷. By mechanically restricting gut movement in this manner, chemical blockade of smooth-muscle activity (that is, with nifedipine or nicardipine) was not necessary. Up to four ring preparations were obtained from each jejunal segment. Because we cut open the gut tube along the mesenteric border and positioned the anti-mesenteric side within the ring preparation for imaging, intestinofugal neurons, which typically have their cell bodies situated along the mesenteric border and form only a small proportion of total myenteric neurons^58,59,60,61, were probably under-sampled.

In some experiments, the connections between the MP and the submucosa were severed by peeling apart the gut layers between the SMP and the circular muscle. The two separate layers were then positioned back in place and mounted between the rings. For peeled LMMP preparations, the mucosa, SMP and circular muscle were removed by microdissection and the remaining layers were mounted between rings.

For labelling of CGRP, segments of jejunum from C57BL/6 mice were collected and dissected in sterile Krebs. Peeled MP preparations with the mucosa and submucosa removed were cultured overnight at 37 °C (5 % CO₂) in DMEM/F-12 (Thermo Fisher Scientific) containing penicillin (250 U ml⁻¹; Thermo Fisher Scientific), streptomycin (250 µg ml⁻¹; Thermo Fisher Scientific), glutamine, nifedipine (1 µM; Sigma), 2% fetal bovine serum (FBS; Sigma) and colchicine (to concentrate proteins in the neuronal cell soma; 100 µM; Sigma).

DiI labelling to trace enteric neurons innervating a single villus

Ring preparations were placed in a glass-coverslip-bottomed dish and constantly perfused with Krebs solution bubbled with 95% oxygen and 5% carbon dioxide. Preparations were viewed using a 20× objective on a Zeiss Axiovert 200M microscope. DiI (in 100% ethanol) was applied by pressure ejection (Picospritzer II; 10 psi, 2 s; General Valve Corporation) using a micropipette (tip diameter of 10–20 µm) pushed through the epithelial layer and into a single villus. Tissues were then removed from the rings, stretched and pinned flat and fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for two hours at room temperature. This was followed by three 10-min washes in PBS, and tissues were then incubated in PBS at 37 °C for up to three weeks after DiI application.

Live Ca²⁺ imaging of epithelial activity

Villin|GCaMP3 tissues that were stretched and pinned flat with the mucosal side up in a silicone-elastomer-coated dish were continuously superfused with Krebs solution bubbled with 95% oxygen and 5% carbon dioxide throughout the experiment and imaged at room temperature. Imaging was performed using a Leica M165 FC fluorescent stereomicroscope (Leica) fitted with an X-Cite 200DC stabilized fluorescence light source (Lumen Dynamics) and an ORCA-Flash4.0 V3 digital CMOS camera (Hamamatsu). The mucosa was perfused with stimulating solutions using a micropipette tip positioned at the edge of the imaged FOV. Perfusion was achieved through a gravity-assisted tubing system. Recordings were acquired at 2 Hz for a duration of 2 min, and there was at least 5 min between each stimulus application.

Live Ca²⁺ imaging of neuronal activity

Ring preparations were kept at room temperature in a glass-coverslip-bottomed dish and constantly perfused with Krebs solution bubbled with 95% oxygen and 5% carbon dioxide throughout the experiment. Live Ca²⁺ imaging of Wnt1|GCaMP3 ring preparations was performed using either a wide-field or a spinning disk confocal microscopy set-up. Some preparations were imaged using a 20× objective on a Zeiss Axiovert 200M microscope, equipped with a monochromator (Poly V) and a cooled CCD camera (Imago QE) (TILL Photonics). Images were acquired using TILLVISION software (TILL Photonics). A separate set of experiments was performed using an Andor Revolution spinning disk confocal microscope (Andor) equipped with a Nikon 20× lens (NA 0.8). GCaMP3 was excited at 488 nm and 3-dimensional (3D) stacks (21−29 µm) were acquired using a Piezo Z Stage controller.

Mucosal application of nutrient stimuli

Glucose (300 mM), acetate (100 mM) or l-Phe (100 mM) were locally applied for 30 s onto the mucosal surface with a perfusion pipette positioned above the imaged myenteric or SMP, exposing entire villi within the local FOV (445 × 337 μm²) to the nutrient solution. High-K⁺ (75 mM) Krebs solution was applied onto the mucosa for 10 s to broadly depolarize EECs and EC cells, which are electrically excitable. For some experiments, tissues were perfused with various antagonists for 10 min before the second mucosal stimulus. For experiments involving the sequential application of different nutrient solutions to the same ganglia, each nutrient application was separated by 10 min Krebs perfusion and the order of nutrient application was alternated between experiments.

Spritz application of agonist into a single villus

To mimic the basolateral release of mucosal mediators, 5-HT, ATP, CCK8 or GLP-1 were applied by pressure ejection (Picospritzer II; 10 psi, 2 s; General Valve Corporation) using a micropipette (tip diameter of 10–20 µm) pushed through the epithelial layer and into a single villus as previously described²⁹.

Electrical stimulation

To stimulate the nerve endings in a single villus, a train of 300-μs pulses (20 Hz, 1 s, 30 V) was applied using a focal electrode (50-μm-diameter tungsten wire) gently lowered onto the villus tip, coupled to a Grass stimulation unit. The lipophilic tracer DiI (dissolved in ethanol) was applied onto the epithelium of villi by pressure ejection (Picospritzer II; 10 psi, 1 s) as a means of marking and relocating a single villus.

In vivo ENS Ca²⁺ imaging

Adult male Wnt1|GCaMP3 mice aged at least 12 weeks were used for in vivo imaging of the ENS. Mice were fasted for four hours before the surgical procedure. Mice were anaesthetized using isoflurane (2–3%) by inhalation until loss of the pedal withdrawal reflex. Anaesthesia was maintained (around 1.5% isoflurane) throughout the surgical and imaging procedure. Bupivacaine hydrochloride (0.05%) was locally administered to the site of abdominal incision. A laparotomy was then performed and a 3–4-cm jejunal loop with as little gut contents as possible was exposed. The intestinal loop was positioned in a coverslip-bottomed imaging dish filled with Krebs solution containing nifedipine (1 µM) to reduce smooth-muscle contractions. The intestinal segment was further stabilized using a custom-designed 3D-printed biocompatible silicone mesh, which was attached to the coverslip. For the infusion of stimulating solutions into the lumen, a small incision was made along the anti-mesenteric border, avoiding major blood vessels. The tip of a flexible feeding tube (22 ga; Instech) was then inserted through the incision and into the lumen, before sealing off the opening using surgical glue (Vetbond). The feeding tube was then connected to perfusion lines containing Krebs or acetate (100 mM). A second incision was made distal to the intestinal loop positioned in the imaging dish to insert tubing allowing for the outflow of perfused solutions. Wet gauze was placed over the segment of exposed intestine not submerged in the solution of the imaging dish to avoid the tissue drying out. Enteric neuronal activity was imaged using a 20× objective on a Zeiss Axiovert 200M microscope as described above. The mice were euthanized by cervical dislocation after imaging and the imaged jejunal loop was dissected out and fixed for post hoc immunohistochemistry.

Recordings were stabilized in Fiji using the Descriptor-based series registration plug-in⁶² and/or the Landmark Correspondences plug-in (transformation method, moving least squares; transformation class, affine) to overlay the recording onto a template image of the fixed and peeled MP.

Nutrients and drugs

Glucose (300 mM; Millipore) was prepared in distilled water. Acetate (100 mM) was prepared with acetic acid (100 mM; EMSURE) in a modified Krebs solution (in which the concentration of NaCl was reduced to 20 mM) and neutralized using NaOH (1 M). l-Phe (100 mM; Sigma) was also prepared in a modified Krebs solution with the concentration of NaCl adjusted to maintain the osmolarity. The pH of the nutrient solutions was adjusted to 7.4. The concentrations of nutrient solutions were selected to be within the range that is typically ingested. For instance, popular sweetened beverages contain around 300 mM glucose⁶³, vinegar can contain up to 1.17 M acetic acid⁶⁴ and the concentration of l-Phe in beef is around 70 mmol per kg (ref. ⁶⁵).

5-HT (Sigma), ATP (Tocris), GLP-1 (7-36)-amide (Phoenix Pharmaceuticals), CCK8 (PolyPeptide Laboratories), hexamethonium bromide (Sigma), ondansetron (Sigma), TNP-ATP (Tocris) and mizagliflozin (MedChemExpress) were prepared as stock solutions and diluted to the final concentration using standard Krebs solution.

Analysis

Ca²⁺ imaging analyses were performed with custom-written routines in Igor Pro (WaveMetrics)⁶⁶. Regions of interest were drawn over each cell to calculate the average [Ca²⁺]_i signal intensity. Values were then normalized to the baseline fluorescence intensity (F_i/F₀). Responses were considered when the [Ca²⁺]_i signal increased above baseline by at least five times the intrinsic noise. [Ca²⁺]_i peaks were calculated for each response, with the peak amplitude taken as the maximum increase in [Ca²⁺]_i from baseline (ΔF_i/F₀). Image overlays highlighting responding neurons (activity-over-time images) were generated in Igor Pro. Image overlays highlighting active regions of Ca²⁺ activity in Villin-cre;R26R-LsL-GCaMP3 tissues were created in Fiji using the AQuA plug-in⁶⁷. The images presented were adjusted for brightness and contrast. Quantification of the cell diameters of neuronal responders was performed in Fiji.

Immunohistochemistry

After Ca²⁺ imaging experiments, some full-thickness ring preparations were fixed in 4% formaldehyde in PBS for 90 min at room temperature, followed by three 10-min washes in PBS. Fixed tissues were first separated into SMP and MP preparations by microdissection. The SMP was isolated by dissecting the submucosa and mucosa from the underlying muscle and myenteric layers, and the mucosa was then gently scraped off. MP preparations were obtained by removing the circular muscle from the remaining preparation.

Cultured colchicine-treated MP tissues were fixed in 4% formaldehyde in PBS for 60 min at room temperature, followed by three 10-min washes in PBS. The circular muscle was then removed by microdissection before immunostaining.

SMP and MP preparations were then processed for immunohistochemistry. Tissues were incubated first in a blocking solution for two hours at room temperature (4% donkey serum and 0.5% Triton X-100) and then in primary antisera (Supplementary Table 5) for 48–72 h at 4 °C. Tissues were washed in PBS (three 10-min washes) before incubating in secondary antisera (Supplementary Table 5) for two hours at room temperature, and then washed again (three 10-min washes in PBS) before mounting onto slides using Citifluor.

RNAscope

After Ca²⁺ imaging experiments, some full-thickness ring preparations were dissected to isolate the MP layer and then fixed in 4% paraformaldehyde in PBS for 24 h at 4 °C, followed by three 10-min washes in PBS. Fixed myenteric preparations were further dissected to remove the circular muscle by microdissection before further processing for fluorescence in situ hybridization using the RNAscope Multiplex Fluorescent Reagent Kit v2 assay according to the manufacturer’s instructions. Protease III was used to digest the whole-mount preparations. The RNAscope probes (Advanced Cell Diagnostics) used were Nmu-C1 (446831), Cck-C2 (402271) and Nxph2-C3 (870131). After hybridization, tissues were counterstained for DAPI (Invitrogen; 62248) and/or GFP and mounted using VECTASHIELD Antifade Mounting Medium with DAPI (VectorLabs; H-1200-10) or with Citifluor.

Imaging immunolabelled, DiI-labelled or RNAscope-labelled tissues

Labelled preparations were viewed under either an epifluorescence microscope (Olympus BX41) with an Olympus XM10 camera, or a Zeiss Axio Imager M2 with an Axiocam 705 (Zeiss). Images were acquired using Photoshop software or ZEN Blue, and were adjusted for contrast and brightness before overlay and quantification. The Landmark Correspondences plug-in in Fiji (transformation method, moving least squares; transformation class, affine) was used to transform the image of immunolabelled preparations for overlay with the activity-over-time image highlighting responding neurons from Ca²⁺-imaging recordings. Colchicine-treated tissues, DiI-labelled tissues and RNAscope-labelled tissues were also imaged on an LSM 780 confocal microscope (Zeiss) using a water-immersion 25x objective (NA 0.8, Zeiss). Some RNAscope-labelled tissues were imaged on an LSM 880 confocal microscope (Zeiss) using an oil-immersion 40× objective (Zeiss). Confocal micrographs display maximum projections of confocal z-stacks. Images were adjusted for brightness and contrast. Neuronal cell counts and analysis of DiI-labelled preparations were performed in ImageJ–Fiji.

Data and statistics

Data are presented as mean ± s.e.m. At least three mice were used for each set of experiments, unless stated otherwise. Two-sided t-tests were performed. One-way ANOVA or two-way ANOVA followed by a multiple comparisons test was used to determine statistical significance. Where appropriate, the P values presented were adjusted to account for multiple comparisons. P < 0.05 was considered significant. Analyses were performed using GraphPad Prism.

Analysis of RNA-seq data

Single-cell and single-nucleus RNA-seq datasets were downloaded from the Single Cell Portal (SCP44 (ref. ³¹) and SCP1038 (ref. ³²)) and the Sequence Read Archive (SRP135960). The data were analysed in R (v.4.1.2) using the Seurat (v.4.1.0) package. All data were normalized using standard Seurat normalization: for each cell the gene counts were divided by total counts and multiplied by a scaling factor of 10⁴, followed by log transformation. The data were subsequently scaled gene-wise using Seurat’s ScaleData function. The metadata from the original publications were used as cluster and cell-type annotations. Genes relating to nutrient sensing and transport, synaptic release, and neurotransmitters, neuropeptides and hormones, as well as their corresponding receptors, were selected for further downstream analysis. Violin plots for these genes were generated using Seurat’s VlnPlot function with basic parameters.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

All datasets analysed in this study are available in the main text and supplementary information. For Ca²⁺ imaging analysis, the source code and installation instructions can be found at ref. ⁶⁶ ‘Source code 1’ (https://doi.org/10.7554/eLife.42914.039; Ca²⁺ imaging analysis source code) and ‘Source code 2’ (https://doi.org/10.7554/eLife.42914.040; installation instructions and user guide). None of the materials mentioned in this study have use restrictions for which material transfer agreements (MTAs) are required. Source data are provided with this paper.

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Acknowledgements

We thank M. Moons and L. Grammet for technical assistance; P. Van Veldhoven for the Villin-Cre mice; and members of the Laboratory for Enteric Neuroscience (LENS) for advice. This work was supported by grants from KU Leuven (METH/014/05; C.F., P.V.B. and J.T.); the Research Foundation Flanders (FWO) (G012223N (P.V.B.) and W001620N (W.B.)); the Dutch Research Council (NOW) (VIDI 016.196.367; W.B.); the European Union’s Horizon 2020 research and innovation programme H2020-FETPROACT-2018-01, CONNECT (grant agreement 824070; C.F. and P.V.B.) and FAIR CHARM H2020, industrial leadership (ICT) project (grant agreement 101016457; C.F., M.I.A. and P.V.B.). Images were recorded on microscopy equipment in LENS and the Cell and Tissue Imaging Cluster (CIC) supported by the Hercules Foundation (AKUL/11/37, AKUL/09/50 and G093818N; P.V.B. and W.B.).

Author information

Marlene M. Hao
Present address: Department of Anatomy and Physiology, University of Melbourne, Melbourne, Victoria, Australia
Yuuki Obata
Present address: University of Texas Southwestern Medical Center, Dallas, TX, USA

Authors and Affiliations

Laboratory for Enteric Neuroscience (LENS), Translational Research Center for Gastrointestinal Disorders (TARGID), KU Leuven, Leuven, Belgium
Candice Fung, Tom Venneman, Tobie Martens, Milvia I. Alata, Marlene M. Hao, Jan Tack & Pieter Vanden Berghe
Department of Pathology, GROW – Research Institute for Oncology and Reproduction, Maastricht, The Netherlands
Amy M. Holland & Werend Boesmans
Biomedical Research Institute (BIOMED), Hasselt University, Diepenbeek, Belgium
Amy M. Holland & Werend Boesmans
KU Leuven Institute for Single Cell Omics (LISCO), Leuven, Belgium
Ceyhun Alar & Alejandro Sifrim
Laboratory of Multi-omic Integrative Bioinformatics, Center for Human Genetics, KU Leuven, Leuven, Belgium
Ceyhun Alar & Alejandro Sifrim
Nervous System Development and Homeostasis Laboratory, Francis Crick Institute, London, UK
Yuuki Obata & Vassilis Pachnis

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Candice Fung
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Contributions

C.F., P.V.B., W.B. and V.P. contributed to the conception or design of the work. C.F., A.M.H., T.M., T.V., M.I.A., M.M.H., C.A., A.S. and P.V.B. contributed to the acquisition, analysis and/or interpretation of data. C.F. performed the DiI tracing, ex vivo and in vivo imaging and immunolabelling experiments. T.V. assisted with the image registration of in vivo recordings. A.M.H., C.F. and T.M. performed the RNAscope labelling experiments and imaging. C.A. performed the analysis of published RNA-seq datasets. C.F., C.A., V.P., W.B. and P.V.B. contributed to the original draft. C.F., M.M.H., Y.O., J.T., A.S., V.P., W.B. and P.VB. contributed to the revision and editing of the final manuscript.

Corresponding author

Correspondence to Pieter Vanden Berghe.

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Nature thanks Ivan de Araujo, Michael Gershon and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data figures and tables

Extended Data Fig. 1 Many nutrient-responsive myenteric neurons express Nmu.

a–r, Myenteric (GCaMP⁺GFP⁺) neurons which responded to the mucosal application of glucose (300 mM) (n = 2 ganglia, N = 2 mice) (a–f), acetate (100 mM) (n = 4 ganglia; N = 3 mice) (g–l) and l-Phe (100 mM) (n = 6 ganglia; N = 4 mice) (m–r) together with the corresponding RNAscope labelling for Nmu and Cck. Tissues were immunostained with anti-GFP to enhance the GCaMP signal after RNAscope and nuclei were counterstained with DAPI. Scale bars, 20 µm. Ca²⁺ transients in the nutrient-responsive neurons (arrowheads) are also shown (a’,g’,m’). Nutrient solutions were applied from t = 10 s to t = 40 s.

Extended Data Fig. 2 Myenteric responses to sequentially applied nutrients.

a–d, Sequential application of glucose (300 mM) (a,a’), acetate (100 mM) (b,b’) and l-Phe (100 mM) (c,c’) by mucosal perfusion activates common myenteric neurons as well as neurons that respond uniquely to one nutrient—many neurons responded only to acetate (d). n = 706 neurons, N = 24 preparations examined (13 mice). The specific sequence of nutrient application was alternated between experiments. Each nutrient application was separated by 10 min of Krebs perfusion. Nutrient-responsive neurons are marked by arrowheads. Corresponding Ca²⁺ transients in responding neurons are shown in a’–c’, Scale bars, 20 µm. Drawing in a is adapted from ref. ³⁰ (Wiley).

Extended Data Fig. 3 Nutrients elicit defined epithelial Ca²⁺ responses.

a–e’, Representative wide-field images of the mucosal surface of Villin|GCaMP3 mouse jejunum at baseline (t = 0 s) (a–e) and during the application of a’, glucose (300 mM) (n = 3 preparations), b’, acetate (100 mM) (n = 3), c’, l-Phe (100 mM) (n = 3), d’, high K⁺ (75 mM) Krebs (n = 5) to depolarize the mucosa or e’, control Krebs buffer (n = 3). Regions of villi that showed an increase in fluorescence intensity in response to each stimulus are highlighted in blue. For each stimulus, examples of Ca²⁺ transients in selected regions of epithelium (arrowheads) are shown at the bottom (a”–e”). Stimuli were applied from t = 20 s to t = 80 s. The timing and duration of stimulus application are indicated by the blue boxes. Scale bars, 200 µm. Drawing in a is adapted from ref. ³⁰ (Wiley).

Extended Data Fig. 4 Expression of genes involved in luminal nutrient signalling in epithelial cells and enteric neurons of the mouse small intestine.

a,b, Summary of findings extrapolated from single-cell RNA-seq data on small-intestinal epithelium and myenteric neurons sourced from Haber et al.³¹ (a) and Morarach et al.²⁷ (b), respectively. a, Violin plots depicting the differential expression of key genes involved in nutrient signalling across defined epithelial cell types. Genes encoding the sweet taste receptor (T1R2–T1R3) and sodium–glucose co-transporter 1 (SGLT1) (highlighted in blue) are most highly expressed in enterocytes. The free fatty acid receptor 2 (FFAR2; involved in sensing SCFAs e.g. acetate; yellow) is expressed in EECs and goblet cells. Genes encoding the calcium-sensing receptor (CaSR) and T1R1–T1R3, both associated with detecting l-amino acids (e.g. l-Phe; magenta), are expressed in EECs and goblet cells, respectively. EECs also express genes related to synthesis and storage of neuroactive molecules (5-HT, ATP) and peptide hormones (CCK, GLP-1), as well as synaptic release (green). Goblet cells highly express the gene encoding vesicular nucleotide transporter (VNUT; involved in vesicular storage of ATP), along with synaptic protein genes (green). b, Violin plots showing enteric neuronal gene expression of receptors corresponding to key signalling mediators expressed by EECs i.e. 5-HT3 receptors, purinergic P2X2 receptors, CCK receptors (CCKAR and CCKBR) and GLP-1 receptors. 5Ht3a and P2rx2 were most highly expressed in neurons that express the IPAN markers Nmu, Calcb and Calb2 and low levels of Cckar, Cckbr, Glp1r, Calb1 and Nos1 expression. Abbreviations: cholecystokinin, CCK; calcitonin gene-related peptide, CGRP; glucagon-like peptide-1, GLP-1. Drawings are adapted from ref. ³ under a Creative Commons licence CC BY 4.0.

Extended Data Fig. 5 Submucosal and myenteric responses to luminal glucose are partly mediated by SGLT1.

a,b, Repeated mucosal glucose (300 mM) application, separated by 10 min Krebs perfusion, evoked reproducible responses in the (a) SMP (n = 6 FOVs imaged) and (b) MP (n = 6 FOVs imaged) under control conditions. a’, Submucosal (n = 6 preparations) and b’, myenteric (n = 6 preparations) responses to mucosal glucose perfusion were significantly inhibited by mizagliflozin (selective SGLT1 inhibitor, 1 µM; two-sided paired t-test). Drawing in a is adapted from ref. ³⁰ (Wiley).

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Extended Data Fig. 6 Myenteric responses to CCK8, GLP-1 and 5-HT in the small intestine.

a–f, Myenteric responses to intravillus injection of agonists in duodenum (a,c,d) and terminal ileum (b,e,f) full-thickness preparations. While 5-HT (10 µM) evoked neuronal responses in myenteric neurons of both gut regions (d, n = 10 villi examined; f, n = 6 villi; responders indicated by arrowheads), no responses were observed to (c) CCK8 applied into villi of the duodenum (n = 12 villi) or to (e) GLP-1 in the villi of terminal ileum (n = 12 villi). The traces in the panels on the right show changes in fluorescence intensity over time for neurons indicated by filled arrowheads (responders) or empty arrowheads (non-responders). g–j, CCK8 (100 nM) or GLP-1 (100 nM) were also applied directly onto myenteric ganglia in peeled jejunum LMMP preparations by pressure ejection from a micropipette at t = 10 s. g,g’, Two CCK8 applications (separated by a 5 min Krebs washout) evoked reproducible responses in a subset of myenteric neurons (n = 10 FOVs examined). h,h’, Responses to GLP-1 (two applications separated by a 5 min Krebs washout) were less reproducible with only a small percentage of total neurons responding to both applications (n = 8 FOVs examined). i, Some CCK8-responsive neurons as indicated by arrowheads (left, middle) and corresponding Ca²⁺ transients (right). j, Some GLP-1-responsive myenteric neurons as indicated by arrowheads (left, middle) and corresponding Ca²⁺ transients (right). Scale bars, 20 µm. Data are mean ± s.e.m. Drawings in a and g are adapted from ref. ³⁰ (Wiley).

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Extended Data Fig. 7 Myenteric CGRP⁺ neurons in colchicine-treated tissues show calbindin immunoreactivity.

a–c, Confocal images of a colchicine-treated LMMP preparation immunolabelled for a, calcitonin gene-related peptide (CGRP) (cyan), b, calbindin (magenta), and c, Hu (pan-neuronal marker; green). d,e, Merged images illustrating the overlap between CGRP-, calbindin- and/or Hu-immunoreactivity. Scale bars, 50 µm. f, Graph showing the percentage of total Hu⁺ myenteric neurons that are CGRP⁺ and the percentage of total Hu⁺ myenteric neurons that are both CGRP⁺ and calbindin⁺ (n = 10 FOVs assessed; n = 458 Hu⁺ neurons, N = 2 mice). Most CGRP-immunoreactive neuronal cell bodies also show calbindin-immunoreactivity. Data are mean ± s.e.m.

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Extended Data Fig. 8 Timing of myenteric and submucosal responses to mucosal high K⁺.

a–a”, Representative spinning disk confocal microscope recording of Ca²⁺ activity in the MP and SMP in response to mucosal high K⁺ application (from t = 20 s to t = 30 s). Maximum XY projections of the Z stack (25 µm) divided into the (a) MP and (a’) SMP. a”, Maximum XY and XZ projections of the full stack with the MP and SMP depth colour-coded in green and magenta, respectively. Scale bars, 50 µm. Responding neurons are indicated by colour-coded arrowheads. b, Corresponding traces of Ca²⁺ responses in the myenteric (green) and submucosal neurons (magenta) indicated in a–a”, depicted as a change in fluorescence over time. Arrowheads mark the Ca²⁺ peak of the initial responders. c, The Ca²⁺ peak of the first myenteric neuron that responded consistently preceded that of the first submucosal neuron that responded in corresponding preparations (n = 3). d, The percentage of myenteric and submucosal neurons that responded to mucosal high K⁺ (n = 3 preparations). Data are mean ± s.e.m.

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Extended Data Fig. 9 Submucosal responses to nutrients perfused onto the mucosa in peeled preparations.

a–c”, Nutrient responses in peeled mucosa–SMP preparations. a,a’, Mucosal perfusion of glucose (300 mM) activates a subset of submucosal neurons as marked by white arrowheads. Glucose was applied from t = 10 s to t = 40 s. a”, Corresponding Ca²⁺ transients in the responding neurons indicated in the previous panels. b–b”, Mucosal perfusion of acetate (100 mM) activates a subset of submucosal neurons as marked by white arrowheads. Acetate was applied from t = 10 s to t = 40 s. c–c”, Mucosal perfusion of l-Phe (100 mM) activates a subset of submucosal neurons as marked by white arrowheads. Acetate was applied from t = 10 s to t = 40 s. Scale bars, 20 µm. d–f, Nutrient responders in the SMP of full-thickness (FT) preparations versus peeled preparations with the MP removed. While the percentage of submucosal responders to d, glucose were comparable (peeled: n = 8 FOVs examined; FT: n = 5 FOVs), fewer responders were observed in response to e, acetate and f, l-Phe in peeled preparations compared to in FT preparations (two-sided unpaired t-test; acetate, peeled: n = 15 FOVs, FT: n = 7 FOVs; l-Phe, peeled: n = 18 FOVs, FT: n = 10 FOVs). Data are mean ± s.e.m. Drawing in a is adapted from ref. ³⁰ (Wiley).

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Extended Data Fig. 10 Simplified schematic of proposed model for the detection of luminal nutrient information by the ENS of the mouse small intestine.

a, Luminal nutrients are first detected at the mucosal surface by enteroendocrine (EEC) cells, which include EC cells, as well as other epithelial cells such as enterocytes. The basolateral release of 5-HT from EC cells after nutrient activation then stimulates 5-HT₃ receptor (5-HT₃R)-expressing intrinsic sensory nerve endings that supply the villi. Other mucosal mediators that activate intrinsic nerve endings and their corresponding neuronal receptors have yet to be established. Schematic adapted from ref. ³ under a Creative Commons licence CC BY 4.0. b, Intrinsic sensory neurons (IPANs) that project to the mucosa reside in the MP and respond mainly to the luminal stimulus. Interneurons and motor neurons of ascending excitatory and/or inhibitory descending pathways may be activated secondarily through nicotinic transmission. c, IPANs are also found in the SMP and respond directly to luminal stimuli. Other submucosal (secretomotor) neurons respond secondarily through interneurons and/or myenteric sensory neurons, and their responses are mediated by nicotinic transmission.

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Supplementary Video 1

Glucose responses in the submucosal plexus. Recording of submucosal neurons responding to the mucosal perfusion of glucose (300 mM) in a full-thickness jejunal preparation from a Wnt1-cre;R26R-GCaMP3 mouse. Glucose was applied for 30 s.

Supplementary Video 2

Glucose responses in the myenteric plexus. Recording of myenteric neurons responding to the mucosal perfusion of glucose (300 mM) in a full-thickness jejunal preparation from a Wnt1-cre;R26R-GCaMP3 mouse. Glucose was applied for 30 s.

Supplementary Video 3

Acetate responses in the submucosal plexus. Recording of submucosal neurons responding to the mucosal perfusion of acetate (100 mM) in a full-thickness jejunal preparation from a Wnt1-cre;R26R-GCaMP3 mouse. Acetate was applied for 30 s. Some responses in underlying myenteric neurons that are out of focus and located to the right of the submucosal ganglion appear to precede that of the submucosal neurons.

Supplementary Video 4

Acetate responses in the myenteric plexus. Recording of myenteric neurons responding to the mucosal perfusion of acetate (100 mM) in a full-thickness jejunal preparation from a Wnt1-cre;R26R-GCaMP3 mouse. Acetate was applied for 30 s. The response in the initial responding myenteric neuron appears to precede that of the submucosal ganglion that is out of focus and above the myenteric ganglion.

Supplementary Video 5

l-Phe responses in the submucosal plexus. Recording of submucosal neurons responding to the mucosal perfusion of l-phenylalanine (l-Phe; 100 mM) in a full-thickness jejunal preparation from a Wnt1-cre;R26R-GCaMP3 mouse. l-Phe was applied for 30 s.

Supplementary Video 6

l-Phe responses in the myenteric plexus. Recording of myenteric neurons responding to the mucosal perfusion of l-phenylalanine (l-Phe; 100 mM) in a full-thickness jejunal preparation from a Wnt1-cre;R26R-GCaMP3 mouse. l-Phe was applied for 30 s.

Supplementary Video 7

Epithelial glucose responses. Epithelial responses to the perfusion of glucose (300 mM) onto the mucosal surface of a full-thickness jejunal preparation from a Villin-cre;R26R-GCaMP3 mouse. The tips of villi were imaged from a lens positioned above the preparation. Glucose was applied for 1 min.

Supplementary Video 8

Epithelial acetate responses. Epithelial responses to the perfusion of acetate (100 mM) onto the mucosal surface of a full-thickness jejunal preparation from a Villin-cre;R26R-GCaMP3 mouse. The tips of villi were imaged from a lens positioned above the preparation. Acetate was applied for 1 min.

Supplementary Video 9

Epithelial l -Phe responses. Epithelial responses to the perfusion of l-phenylalanine (l-Phe; 100 mM) onto the mucosal surface of a full-thickness jejunal preparation from a Villin-cre;R26R-GCaMP3 mouse. The tips of villi were imaged from a lens positioned above the preparation. l-Phe was applied for 1 min.

Supplementary Video 10

Acetate responses in the myenteric plexus of jejunal loop in vivo. Example Ca²⁺ imaging recording of enteric neuronal activity in response to luminal infusion of acetate (100 mM) solution in a loop of mouse jejunum in vivo. Acetate was applied into the lumen from 30 – 60 s. Frames of the live wide-field recording (green overlay) were mapped onto an image of the fixed and peeled myenteric plexus (greyscale).

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Fung, C., Venneman, T., Holland, A.M. et al. Nutrients activate distinct patterns of small-intestinal enteric neurons. Nature 644, 1069–1077 (2025). https://doi.org/10.1038/s41586-025-09228-z

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Received: 27 January 2023
Accepted: 03 June 2025
Published: 09 July 2025
Issue date: 28 August 2025
DOI: https://doi.org/10.1038/s41586-025-09228-z

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Nutrient sensing and small intestinal enteric neurons
- Katrina Ray
Nature Reviews Gastroenterology & Hepatology (2025)

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Main

Villi receive local ENS innervation

Mucosal high K+ activates the ENS

Nutrients activate the ENS through the mucosa

Mucosa–ENS signalling involves 5-HT

Chemosensitive myenteric IPANs

Chemosensitive submucosal IPANs

Nutrient-induced ENS responses in vivo

Discussion

Methods

Mice and tissue preparation

DiI labelling to trace enteric neurons innervating a single villus

Live Ca2+ imaging of epithelial activity

Live Ca2+ imaging of neuronal activity

Mucosal application of nutrient stimuli

Spritz application of agonist into a single villus

Electrical stimulation

In vivo ENS Ca2+ imaging

Nutrients and drugs

Analysis

Immunohistochemistry

RNAscope

Imaging immunolabelled, DiI-labelled or RNAscope-labelled tissues

Data and statistics

Analysis of RNA-seq data

Reporting summary

Data availability

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Mucosal high K⁺ activates the ENS

Live Ca²⁺ imaging of epithelial activity

Live Ca²⁺ imaging of neuronal activity

In vivo ENS Ca²⁺ imaging