Mouse strains
All experiments on mice were performed following approval by the Institutional Animal Care and Use Committee at Duke University Medical Center under the protocol A212-21-10. Mice were group-housed in Duke University’s Division of Laboratory Animal Resources, where they were kept on a 12-h light–dark cycle (0700–1900) with access to water and standard mouse chow (Purina 5001) ad libitum, unless otherwise indicated. The facility maintained an ambient temperature of 18–23 °C and humidity of 40–60%. Male and female adult mice aged 6–20 weeks were used in all experiments. The following experimental mouse strains were purchased, received or bred in-house and used directly: C57BL6/J (JAX 000664), PYY–GFP13, CCK–GFP61, Pyycre62, NeuroD1-Cre (JAX 028364), loxP-STOP-loxP c***ette (LSL)_tdTomato (JAX 007914), LSL_Halo-YFP (JAX 014539), LSL_ChR2-tdTomato (JAX 012567), LSL_Salsa6f (JAX 031968), Tlr5fl (JAX 028599), Myd88fl (JAX 008888), B6.Cg-Snap25tm3.1Hze/J (JAX 025111) and Fostm2.1(icre/ERT2)Luo/J (JAX 030323). The following double-transgenic mouse strains were bred in-house: Pyycre;Salsa6f, NeuroD1-Cre_Salsa6f, Pyycre;Halo-YFP and Pyycre;ChR2-tdTomato. Pyycre mice were also bred to floxed Tlr5fl/fl (floxed exon 4) and Myd88fl/fl (floxed exon 3) mice to generate the following conditional knockout mice: Pyycre;Tlr5fl/fl and Pyycre;Myd88fl/fl.
Dissociation and isolation of single intestinal epithelial cells
Colons and small intestines of mice were dissociated for qPCR (PYY–GFP), calcium imaging (Pyycre;Salsa6f) or sequencing (CCK–GFP and PYY–GFP) as previously described1. In brief, the entire colon or proximal one-third of the small intestine was removed, flushed with cold PBS and cut into 2–3-mm sections. Tissue was rinsed with cold PBS and then shaken in 1.5 mM EDTA in PBS for 30 min before a 15-min incubation at 37 °C. The epithelial layer was then mechanically detached from the muscle layer by shaking in cold PBS. Following centrifugation at 800 r.p.m. (Eppendorf 5702 RH; rotor A-4-38), the pellet was resuspended and incubated in HBSS (Gibco) with dispase and collagenase for 10 min at 37 °C. Samples were then centrifuged (800 r.p.m.), filtered twice through a 70-μm and 40-μm filter, and resuspended in L15 medium (5% FBS, 10 μl ml−1 10 mM HEPES, 2,000 U ml−1 penicillin–streptomycin and 100 μl of 1,700 U ml−1 DNAse) to produce a single-cell suspension for further ***ysis. For whole-epithelial-layer ***yses, first the pellet was resuspended in lysis buffer and further processed for RNA extraction.
Dissociation and isolation of single nodose neurons
Nodose neurons of mice were dissociated for qPCR (C57BL/6J), calcium imaging (NeuroD1-Cre;Salsa6f) or sequencing (C57BL/6J) as previously described1. In brief, nodose ganglia were dissected and immediately placed into 500 μl of ganglia dissociation solution containing 10 mM HEPES, 1× glutamine, 1× N2 supplement, 1× B27 supplement, 0.5 μg ml−1 nerve growth factor and 55 μg ml−1 liberase (Roche, 5401054001) in Advanced DMEM/F-12. Following digestion, ganglia were rinsed twice with PBS, mechanically dissociated in dissociation solution, and filtered through a 70-μm cell strainer. The dissociated solution was then carefully laid on a density gradient of 500 μl 12% and 500 μl 28% Percoll (Sigma) and centrifuged for 10 min at 2,900g at room temperature. Once centrifugation was complete, the top 700 μl was removed, and 700 μl of fresh dissociation solution was added. Cells were then centrifuged for 15 min at 2,900g, and the final pellet was resuspended in 500 μl PBS plus 0.04% BSA.
RNA sequencing
RNA was extracted from single-cell suspensions using the Single Cell RNA Purification Kit (Norgen). All samples were ***essed with a Bio***yzer (Agilent), and only samples with RNA integrity number scores >8.0 were used for downstream ***ysis. Given the rarity of the cells, Single-cell RNA barcoding sequencing was used to generate libraries. Libraries were sequenced on an Illumina NextSeq 500. STAR was used with the mm10 mouse reference genome to align reads, and count tables were generated using featureCounts. Pairwise comparisons between genes from the PYY–GFP+ and PYY–GFP− groups were made using DESeq2. Gene ontology ***yses were conducted using topGO.
qPCR
The colonic epithelium of PYY–GFP mice was dissociated, and cells were sorted as described above. An equal number of GFP+ and GFP− cells were collected directly into lysis buffer. Whole epithelium was dissociated from Pyycre;Tlr5fl/fl and Tlr5fl/fl control littermates and collected into lysis buffer as described above. Nodose was dissected and flash-frozen in liquid nitrogen. RNA was extracted on the basis of the manufacturer’s protocol using the RNeasy Micro Plus Kit (Qiagen no. 74034). Spleen and whole-colon tissues were collected and homogenized in TRIzol reagent (Thermo Fisher,15596026), and RNA was extracted per the manufacturer’s protocol. cDNA was produced per the manufacturer’s protocol using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, 4368814). The following TaqMan probes were used for transcript identification: Pyy (Mm00520716_g1), 18s (Mm03928990_g1), Tlr1 (Mm00446095_m1, Mm01208874_m1), Tlr2 (Mm00442346_m1, Mm01213946_g1), Tlr3 (Mm01207404_m1), Tlr4 (Mm00445273_m1), Tlr5 (Mm00546288_s1), Actb (Mm02619580_g1), Tnf (Mm00443258_m1), Il1b (Mm00434228_m1), Il6 (Mm00446190_m1), Tjp1 (Mm01320638), Tjp2 (Mm00495620_m1), Ocln (Mm00500910_m1) and Cldn (Mm01342184). qPCR was run on a StepOnePlus System (Thermo Fischer), using TaqMan Fast Universal PCR Master Mix (Applied Biosystems no. 4352042) according to the manufacturer’s protocol. Transcription rate was determined as 2−ΔCt or compared as fold change using 2−ΔΔCt. All values are reported as mean ± s.e.m.
In situ hybridization with immunofluorescence
NeuroD1_tdTomato and PYY–GFP mice were transcardially perfused with PBS for 3 min followed by 4% PFA for 3 min at a rate of 600 µl min−1. The entire intestine was collected, opened lengthwise and divided into different sections: proximal, middle and distal third of the small intestine; and proximal and distal halves of the large intestine. Intestinal tissue was then rolled with the proximal end in the centre, and post-fixed in 4% PFA for 24 h. Nodose ganglia and dorsal root ganglia at the levels of L5, L6 and S1 were also dissected bilaterally. Neuronal ganglia were rinsed in PBS and post-fixed in 4% PFA for 24 h. Tissue was then dehydrated in 10% sucrose for 1 h and 30% sucrose for at least 12 h. Samples were embedded in OCT (VWR) and stored at −80 °C. Tissue was sectioned onto slides at 16 μm using a cryostat. RNA detection was performed using the RNAscope Multiplex Fluorescent Reagent Kit v2 Assay (ACD). In brief, tissue slides were baked for 30 min at 60 °C, and post-fixed in 10% neutral buffered formalin (VWR) for 60 min before being washed in PBS twice (Sigma). Slides were then dehydrated using successive alcohol washes of 50%, 70% and 100%, and a second 100% of ethanol for 5 min each. Slides were then incubated with hydrogen peroxide for 10 min before undergoing target retrieval using RNAscope reagents in a steamer. Slides were submerged into the RNAscope target retrieval solution at >99 °C for 5 min. Slides were then treated with protease III for 30 min at 40 °C before subsequent hybridization and amplification steps per the manufacturer’s instructions. The probes used were all purchased from ACD: Mm-Tlr5 (catalogue no. 468888), Mm-Pyy-C3 (catalogue no. 420681) and Mm-Npy2r (catalogue no. 515431). Hybridization signal was detected using Opal dyes (Akoya Biosciences) at a dilution of 1:1,500. Tissue was then blocked in 10% donkey serum (Jackson ImmunoResearch) for 1 h. Tissue was then incubated with primary antibody dissolved in antibody dilution solution (PBS with 1% BSA and 0.0025% Triton X-100) for 24 h at 4 °C, followed by 1 h at room temperature. Primary antibodies and dilutions were as follows: Rb-anti-PYY (1:250; gift from the Liddle laboratory), Rb-anti-PGP9.5 (1:500; Abcam: ab27053), Gt-anti-serotonin (1:500; Abcam, ab66047) and CHK-anti-GFP (1:500; Abcam: ab13970). Following primary antibody incubation, tissue was washed in 0.05% Tween-20 in TBS buffer (TBST) and then incubated with secondary antibody in antibody dilution solution for 1 h at room temperature: Dk-anti-Rb-488 (1:250, catalogue no. 711-546-152), Dk-anti-Rb-Cy3 (1:250, catalogue no. 11-166-152), Dk-anti-Ck-488 (1:250, catalogue no. 703-546-155), Dk-anti-Gt-647 (1:500, catalogue no. 705-606-147), all from Jackson ImmunoResearch. Tissue was then washed with TBST, stained with DAPI (1:4,000) for 10 min, washed in TBST, and mounted using Fluoro-Gel with Tris buffer (Electron Microscopy Sciences). Imaging was carried out on a Zeiss 880 Airyscan inverted confocal microscope. Images were adjusted for brightness and contrast using ImageJ (Fiji V.2.9.0). In each region of the intestine, the 50 most proximal cells were ***ysed from a total of n = 3 mice. Cells with >2 puncta within the cell body were considered positive for the gene. Control slides using the negative control probes (ACD) were used to ensure that background staining was <3 puncta per cell. Counts are presented as the mean percentage of co-localization ± s.e.m.
Assessment of basal phenotypes
Pyycre;Tlr5fl/fl, Pyycre;Myd88fl/fl and their Cre-negative littermates were weighed following weaning, and every week thereafter until 3 months of age. Mice were then euthanized, and colon length, colon weight and spleen weight were measured. Colonic tissues were flushed with cold PBS, incubated in 10% neutral buffered formalin (VWR) for ≥24 h at 4 °C, dehydrated in a graded series of ethanol and embedded in paraffin. Tissues were sectioned and stained with haematoxylin and eosin (Abcam, ab245880) per the manufacturer’s protocol and imaged for ***essment of developmental and immune phenotypes.
Measurement of food intake
Age-matched Pyycre;Tlr5fl/fl and littermates were placed into clean cages with food hoppers. The hoppers are designed to minimize the ability of mice to remove entire pellets. The weight of the food in the hopper was recorded at both the start and end of a 24-h period. Mice were then returned to their home cage for at least 2 days before repeating the test twice more. The mean of three separate testing days for each individual mouse is reported.
Detailed feeding, activity and meal pattern ***ysis
Age- and block-matched Pyycre;Tlr5fl/fl, Pyycre;Myd88fl/fl and their Cre-negative littermates were placed in a custom-built PhenoMaster behavioural phenotyping system for 10 days (TSE Systems). The first week was considered an acclimation period, and all meal pattern ***yses were performed on the last 3 days within the system. The PhenoMaster was programmed (software version 6.6.9) to automatically maintain a light cycle (07:00 lights on; 19:00 lights off), temperature control (22 °C) and humidity control (40%). The PhenoMaster holds 12 clear cages, in which animals were singly housed. Cages were industrially washed, and bedding (ALPHA-dri) was replaced weekly. Animals were provided with standard mouse chow (Purina 5001) and reverse-osmosis water ad libitum. All cages also housed an enrichment device, which also served to weigh the animals. Food hopper, water bottle and weigh container were attached to weight sensors (TSE). Food intake, water intake and weight were automatically measured every 5 s to the nearest 0.01 g. For drinking measurements, a 10-s smoothing interval with a maximum raw ***og-to-digital conversion count difference of 40,000 was permitted. For weight measurements, a 15-s smoothing interval with a 15-g threshold and a maximum raw ***og-to-digital conversion count difference of 1,000,000 was permitted. Intake was measured every 5 s and binned every minute for ***yses unless otherwise indicated. Animal activity was determined by beams crossed in the x and y planes and was collected with a 100-Hz scan rate. Unless otherwise indicated, all activity, food intake and water intake measurements were binned in 1-min intervals for ***ysis. Data were corrected for minor fluctuations by only permitting a monotonically increasing function for both food and water intake: values that represented negative food intake were replaced by the most recent value. Meal size, frequency and timing were defined on the basis of parameters within the PhenoMaster system. Inter-meal intervals were required to be >10 min. Only meals of size 0.1–1 g and rate <0.25 g min−1 were included in the meal pattern ***ysis.
Fasting blood glucose and oral glucose tolerance test
Blood glucose was measured in age-matched Pyycre;Tlr5fl/fl and Tlr5fl/fl mice following an overnight fast of 18 h. For oral glucose tolerance test, a separate cohort of mice were food- and water-deprived for 5 h. Then, mice were gavaged with sucrose (2 g kg−1 body weight in sterile PBS). Blood glucose was measured (True Metrix 60 Blood Glucose Meter) after the deprivation, and 15, 30, 60, 90 and 120 min following gavage.
Fecal lipocalin-2 measurements
Colonic inflammation was ***essed by ***aying for fecal lipocalin-2 using an ELISA (Ray Biotech). Stool samples were collected from age-matched Pyycre;Tlr5fl/fl and Cre-negative controls during the beginning of the light cycle. Fecal samples were reconstituted in PBS to a final concentration of 100 mg ml−1 and vortexed for 5 min to obtain a homogeneous mixture. Fecal matter was then centrifuged for 10 min at 14,000g. Supernatants were collected and stored at −80 °C. Lipocalin-2 levels were ***essed following the manufacturer’s instructions, and optical density was measured at 450 nm (Tecan Infinite 200 Pro).
Colonic myeloperoxidase ***ay
Neutrophil activity in tissue was ***essed by testing for the enzymatic activity of myeloperoxidase using a colorimetric kit (Abcam). A 2–3-mm segment of the distal colon from age- and block-matched Pyycre;Tlr5fl/fl and littermates was dissected and weighed. Tissue was then washed in PBS, and mechanically homogenized in the lysis buffer provided in the kit. Tissue was then freeze–thawed twice and sonicated. Myeloperoxidase activity was ***essed following the manufacturer’s protocol and normalized to the tissue m***, and optical density was measured at 450 nm (Tecan Infinite 200 Pro).
Serum hormone measurement
Age-matched Pyycre;Tlr5fl/fl and Cre-negative controls were fasted for 15 h and then fed ad libitum for 2 h. Following the re-feeding period, mice were euthanized, and serum was collected. DPP-4 inhibitor and aprotinin were added to serum samples to prevent peptide degradation. Total PYY3-36 (RayBiotech) and total GLP-1 (Alpco) levels were ***essed using an ELISA per the manufacturer’s protocols.
Stool flagellin ***ay
Flagellin levels were ***essed using HEK-Blue-mTLR5 cells (Invivogen). Stool was collected from age-matched wild-type, Pyycre;Tlr5fl/fl and Cre-negative controls during the start of the light cycle, following ad libitum feeding, an 18-h overnight fast or an 18-h overnight fast followed by a flagellin enema (1 μg ml−1 in 100 μl). Fecal material was resuspended in PBS to a final concentration of 100 mg ml−1. Solutions were mechanically homogenized and vortexed for 5 min to form a suspension. Samples were then centrifuged at 8,000g for 2 min, and serum was collected and either immediately ***essed or stored at −20 °C for later testing. Serial dilutions of the solution were placed onto the HEK-TLR5 cells, and purified S. typhimurium flagellin (Invivogen) was used to generate a standard curve. After 18 h of stimulation, cell culture supernatant was applied to QUANTI-Blue medium and incubated for 30 min at 37 °C. QUANTI-Blue alkaline phosphatase activity was then read at 620 nm (Tecan Infinite 200 Pro).
Calcium imaging of dissociated cells
For neurons, NeuroD1-Cre_Salsa6f nodose neurons were dissociated as described above. Neurons were plated on 12-mm coverslips and placed in a 37 °C incubator overnight. Neuronal medium included: 1× GlutaMAX, 10 mM HEPES, 200 U ml−1 penicillin–streptomycin, 1× N2 supplement, 1× B27 supplement and 10 ng ml−1 nerve growth factor in Advanced DMEM/F-12. Cells were imaged 2–3 days after plating. For enteroendocrine cells, Neurod1Cre_Salsa6f cells were dissociated as described above and fluorescence-sorted (BD FACSAria), selecting for tdTomato+ fluorescent cells. Cells were then plated on coverslips coated with 2.5% Matrigel (Corning no. 356231). Enteroendocrine cells were imaged 2–6 h after plating. Cells were washed twice in imaging buffer (120 mM NaCl, 3 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM HEPES, 10 mM glucose; 305 mOsm ± 3 mOsm) and placed in the dark for 5 min until they reached room temperature. Coverslips were then placed in the recording chamber of a Zeiss Examiner Z1 and imaged with a Hamamatsu camera (Orca-flash4.0; C11440) using the Zeiss ZEN Blue software package. GCaMP6f emission images were obtained using 570-nm excitation. Images were collected at 1.5-s intervals with a 100-ms exposure time. Each recording was 180 s long, with a stimulus perfused between 30 and 60 s. Imaging buffer was continuously perfused (about 2 ml min−1) over the coverslips throughout the imaging session. Each coverslip underwent four recordings: buffer, test stimulus, repeat of the test stimulus, and KCl. Each recording session concluded with 50 mM KCl as an activity control (KCl concentration was achieved by substituting for NaCl, and not an addition of more KCl). A response to KCl was defined as a ratio >10% increase above baseline. Cells that did not reach this KCl threshold were not included in ***yses. A 5-min wash-out period with continuous perfusion of imaging buffer was carried out between the two test stimuli. For experiments involving the TLR5 inhibitor, TH1020 was added to the well to reach a final concentration of 1 μM following the wash-out period. Buffer flow was stopped, and cells were incubated with the inhibitor for 10 min before restarting flow and retesting with the stimulus.
Analysis
Fluorescence values for each individual cell were calculated as the mean fluorescence intensity in a user-defined region of interest on Fiji software. Intracellular calcium changes were then calculated as ∆F/F = (F – Fo)/Fo, in which Fo is the average intensity of the cell within the first 15 s. Ratiometric values were then normalized to the peak KCl response. A positive response was defined as an increase in ratio >10% above baseline.
PYY release ***ay
Colons from wild-type and knockout mice were dissected, flushed with cold PBS, opened lengthwise and cut into pieces of about 1 cm. Tissue pieces were incubated on ice in PBS for 2 h before incubation with 1.5 mM EDTA on ice for 30 min, and then 37 °C for 15 min. Crypts were detached by shaking in cold PBS, pelleted at 100g and plated on 12-mm coverslips coated with 2.5% Matrigel (Corning no. 356231). Crypts were then incubated in 50% L-WRN Buffer (ATCC) with 10 μM Y-27632 (Enzo) for 16 h before stimulation. Crypts were then stimulated with buffer (120 mM NaCl, 3 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM HEPES, 10 mM glucose), 100 ng ml−1 flagellin in buffer, or a mixture of 1 μM IBMX (Sigma) and 1 nM forskolin (Sigma) in buffer for 30 min at 37 °C. Supernatant was collected, and the crypts were then incubated in lysis buffer for 30 min at 4 °C. Lysate was collected, and both lysate and supernatant were centrifuged for 10 min at 13,000g to remove insoluble material, and stored at −20 °C for up to 2 weeks. PYY concentration in samples was ***essed using the PYY ELISA Kit (Ray BioTech) following the manufacturer’s protocol. Each experimental condition was run in duplicate on every plate. A PYY standard was run for every plate. Absorbance at 450 nm was measured on a plate reader (Tecan Infinite 200 Pro). PYY amount and concentration was calculated using the standard curve. PYY release was calculated as supernatant/(supernatant + lysate).
Vagus nerve recordings
Whole-nerve recordings were performed in wild-type mice, Pyycre;Halo-YFP mice, Pyycre;ChR2 mice, Pyycre;Tlr5fl/fl mice, Pyycre;Myd88fl/fl mice and negative genetic controls. Whole-nerve electrophysiology recordings of the cervical vagus nerve were performed as previously reported4. A 20-gauge gavage needle with two connected tubes for PBS perfusion and stimulant delivery was surgically inserted through the caeblock wall into the proximal colon. A perfusion exit incision was made just proximal to the rectum for colon. Fecal pellets were gently expressed from the colon using cotton applicators. PBS was constantly perfused through the isolated intestinal region at about 400 μl min−1 as a baseline and volume pressure control. Stimulation conditions were applied after recording 2 min of baseline activity. During stimulation conditions, PBS perfusion was continuous, and 200 μl of 2 µg ml−1 flagellin was perfused over 1 min using a syringe pump (Fusion 200, Chemyx). As a positive control to activate the vagus nerve from the colon lumen, intralipid (7%, Sigma) was infused.
Data acquisition
Extracellular voltage was recorded as previously described4. The raw data were ***ysed using Spike Tailor, a custom MATLAB software (Mathworks) script4. Spikes were detected using a threshold detected on the basis of root-mean-square noise. The firing rate was calculated using a Gaussian kernel smoothing algorithm in 200-ms bins.
Optogenetic inhibition and stimulation
The optoelectronic colon fibre was threaded along the gavage needle into the colonic lumen. MicroLED stimulation was applied simultaneously with nutrient infusion. The microLED was pulsed for 1 min at 40 Hz, 5 V peak and 20% duty cycle (473 nm, 532 nm).
Pharmacologic inhibition
Following recording of a pre-inhibitor response, the inhibitor was delivered over 1 min (10 μl g−1 BIIE-0246) and allowed to incubate for 10 min before re-infusion of flagellin.
Data ***ysis
Stimulation response was quantified as the maximum firing rate after stimulation (stimulant conditions) or during recording (baseline). Each trial served as its own control by normalizing the firing rate to the pre-stimulus baseline firing rate (first 2 min of recording). Throughout experiments, intralipid response was used as a positive control. For all nutrient and laser stimulation conditions, data were excluded if an intralipid response was not seen throughout the recording session. Maximum firing rate and area under the curve were ***ysed across stimulation condition.
Optoelectronic colon fibre fabrication
The preform ***embly for optoelectronic graded-index fibres began with moulding polystyrene-block-poly(ethene-co-butadiene)-block-polystyrene (SEBS) pellets (Kraton G1657M) into desired geometrical patterns in a CNC machined inverse aluminium mould at 200 °C for 8 h under vacuum. The top layer defined the hollow square channels (3.6 mm × 3.6 mm × 30 cm) with a pitch size of 4 mm for hosting the interconnect microwires. The SEBS convergence channels were subsequently lined with a U-shaped PC layer that had a wall thickness of 1 mm and channel size of 1.6 mm × 1.6 mm produced by standard CNC machining process. This preform was consolidated in an oven (130 °C, 45 min) and subsequently drawn into metres-long microscale fibres at a size reduction ratio of 40–45, while simultaneously feeding three spools of 40-μm Ag–Cu microwires.
Fibre device fabrication
Fabrication of graded-index fibre device began with dissolving away the SEBS layer in the distal 1 cm of fibre (8.5 cm total length) in dichloromethane for 10 min, which exposed the interconnect microwires. The interconnects were subsequently soldered onto male header pins that were ***embled inside a custom 3D-printed box (5 mm × 7 mm × 0.5 mm) and secured using UV curable epoxy. About 0.5 cm of the interconnect microwires was exposed by low-end machining with a razor blade at the distal end of the fibre under an optical microscope, followed by mounting of blue (473-nm emission maxima) and green (532-nm emission maxima) InxGa1−xN microLED chips (CREE TR2227 and SR2130) using two-part silver epoxy (Epo-tek). Subsequently, a 10-μm layer of vapour-deposited parylene-C defined the bio-fluid barrier layer. Finally, the device was encapsulated in an approximately 50–100-μm layer of medical-grade silicone by inserting the fibre device in a PTFE tubing with an inner diameter of 0.8 mm, which acted as a sacrificial mould. The silicone mixture was filled and cured in the tubing, and subsequently the tubing was cut open to yield a silicone-coated soft device. The final fibre had an overall length of 8.5 cm with three green and blue microLEDs hosted on the distal 2 cm of the fibre at a separation of 1 cm.
In vivo two-photon calcium imaging and compartment ***ysis of temporal activity by fluorescence in situ hybridization
Snap25-FosTRAP-tdTomato triple-transgenic mice were used to generate landmark tdTomato+ vagal nodose neurons for compartment ***ysis of temporal activity by fluorescence in situ hybridization (catFISH) ***ysis through targeted recombination in active populations (TRAP) at least 10 days before calcium imaging as previously described57. In brief, 6-h-fasted mice were given intragastric infusions of nutrients (500 μl; 100 μl min−1) 30 min before dark cycle onset. At 3 h after stimulus delivery, mice were injected with 4-hydroxytamoxifen (30 mg kg−1, intraperitoneal; Sigma) to induce tdTomato expression in a subpopulation of nutrient-responsive vagal sensory neurons, allowing for post hoc landmarking and alignment. Chow was returned 3 h after 4-hydroxytamoxifen injection. For calcium imaging, mice were fasted overnight (18 h) and maintained under continuous anaesthesia (isoflurane–oxygen) on a heating pad to sustain body temperature throughout the procedure. An abdominal incision was first made in the anaesthetized mice to expose the caeblock and colon. A 20-gauge gavage needle with dual tubing for PBS perfusion and stimulant delivery was surgically inserted through the caecal wall into the proximal colon and secured with a suture. The rectum was severed to create an exit for fluid drainage. Fecal pellets were gently expelled from the colon using cotton applicators. Next, an incision (about 2 cm) was made above the sternum and below the jaw. The carotid artery and vagus nerve were exposed by separating the salivary glands. Retractors were used to pull the sternomastoid, omohyoid and posterior belly of the digastric muscle aside to visualize the nodose ganglion. The vagus nerve was transected just above the nodose ganglion, which was carefully separated from the hypoglossal nerve and small adjacent branches. The vagus nerve was then dissected away from the carotid artery and surrounding tissues. The right nodose ganglion was gently positioned on a stable imaging platform consisting of a 5-mm-diameter coverslip attached to a metal arm fixed to a magnetic base. Surgical silicone adhesive (Kwik-Sil, WPI) was applied to immobilize the vagus nerve on the coverslip, and the nodose ganglion, immersed in Dulbecco’s modified Eagle medium (Corning), was covered with a second coverslip before imaging.
Stimulus perfusion
Perfusions were performed using a precision pump connected to silicone tubing filled with PBS, flagellin (2 µg ml−1) or 7% intralipid. PBS (1,000 µl min−1) was continuously perfused throughout the recording. Baseline neuronal activity was recorded for 30 s, followed by a 2-min flagellin infusion (333 µl min−1) and an additional 2 min of recording post-infusion.
In mice in which responses to both flagellin and intralipid were tested, the colon was flushed with 10 ml of PBS after flagellin perfusion to remove residual flagellin through the exit port. A second baseline activity recording was then taken, followed by a 2-min intralipid perfusion (333 µl min−1) and another 2-min post-perfusion recording.
Imaging
In vivo imaging was performed using a two-photon microscope (Bruker) equipped with a galvanometer for image acquisition and a piezo objective combined with a galvo/resonant scanner, enabling image capture at 29 frames per second (Prairie View v.5.7). The microscope was set up for in vivo conditions with a Somnosuite (isoflurane) anaesthesia device coupled to a homeothermic control warming pad (Kent Scientific) and a programmable syringe pump (Harvard Apparatus PHD 2000) for nutrient perfusion into the gut. Imaging was conducted using a 16× water-immersion upright objective.
RNAscope and alignment
For nodose catFISH ***ysis, we adapted a previously reported protocol for registration of vagal neurons between in vivo calcium imaging and RNAscope fluorescent in situ hybridization images59. After in vivo imaging, immobilized nodose ganglia were immediately fixed in 4% PFA for 2 h and then placed in 30% sucrose for 1 h before being embedded in OCT and frozen at −20 °C. Nodose ganglia were then sliced into 10-µm sections, and an RNAscope ***ay was performed to probe for Npy2r mRNA expression as described above, without slide baking, post-fixation and target retrieval. To align RNAscope sections to in vivo imaging z-stacks, ‘guidepost’ neurons were used as positional landmarks for mapping between in vivo and sectioned images. In brief, tdTomato+ neurons visible in both RNAscope sections and in vivo planes were identified as landmark reference points and were manually paired using the BigWarp tool within the Fiji BigDataViewer plugin. Using this tool, RNAscope sections were transformed and aligned to corresponding planes within the in vivo imaging z-stack, allowing visualization of NPY2R+ neurons overlaid on GCaMP6s-fluorescent neurons. Cells with ambiguous or unsuccessful alignment were not used for further ***ysis.
Quantification of neural activity
GCaMP6s fluorescence changes were quantified by outlining regions of interest (ROIs), each corresponding to a single cell throughout the imaging session. For nodose images processed using catFISH alignment, ROIs were selectively generated around NPY2R+ cells. Pixel intensities in ROIs (average across pixels) were calculated frame by frame using ImageJ and exported to Excel for ***ysis. The z-score for each neuron was calculated by subtracting the mean baseline fluorescence (over a 30-s period) from the fluorescence intensity at each time point and dividing by the standard deviation of the baseline fluorescence. This normalized value represents the number of standard deviations from the baseline fluorescence. A neuron was considered responsive if: the peak GCaMP6s fluorescence reached a z-score of ≥2.5, and the mean GCaMP6s fluorescence was ≥2.5 above the baseline mean for at least 5 s during or after infusion. Neurons without baseline activity were excluded from the ***ysis.
Food intake behavioural system
Mice were acclimated to enema two times before the start of the test. Tests were started 3 h into the start of the light cycle. Mice were fasted overnight for 18 h. At the start of the test, mice received a 100-µl enema of 1 µg ml−1 flagellin or PBS and were then placed into a clean cage. A pre-weighed pellet of standard rodent chow (5001 Purina) was then introduced to the cage, and then weighed at 20, 40, 60 and 180 min following the enema. At the end of the test, mice were returned to their home cages. The start of all test sessions was separated by at least 48 h. For pharmacological inhibition of TLR5, C57BL/6J wild-type mice were acclimated to enema, a baseline response to flagellin was established, and then mice received enemas of 1 μg ml−1 flagellin or PBS combined with 10 μM TH1020 (Sigma). Food intake was then measured as described above.
For pharmacologic inhibition of Y2 receptors with intraperitoneal injections, C57BL/6J wild-type mice were acclimated to enema and intraperitoneal injections two times before the start of the test. At the start of the test, mice received an intraperitoneal injection with 2 ng kg−1 BIIE-0246 or vehicle and were then placed into a clean cage. After 10 min, mice received a 100-μl enema of 1 μg ml−1 flagellin or PBS. Food intake was then measured as described above.
Germ-free mice
Age- and block-matched germ-free C57BL/6J wild-type mice were transferred from the Duke Gnotobiotic core in sterile, individual cages. Tests were started 3 h into the start of the light cycle. Mice were fasted overnight for 18 h. At the start of the test, mice received a 100-μl enema of PBS, followed by an enema 1 μg ml−1 flagellin 7 days later. Food intake was measured as described above.
Crunch Master behavioural system
Female and male, 8-week-old, wild-type C57BL/6J mice were habituated for 1 h a day for 2 days in the behavioural device before the test session. Mice were food-deprived 18 h before the test session, which was performed during the light phase of the mice. Preceding the test session, mice were weighed and subsequently administered a 100-μl enema of either 1 μg ml−1 flagellin or PBS. Immediately following the enema administration, the mice were placed into the acrylic box and allowed to feed ad libitum for 1 h. After the test session, the mice were returned to their home cages.
Behavioural device
An acrylic box (37 cm × 27 cm) was equipped with a microphone (FIFINE T-669) attached to one wall. To enhance sound recording, 12 small drill holes (0.5 cm diameter) were incorporated into the wall near the microphone. A video camera (Kayeton Technology, model KYT-U400-MCS2812R01) was positioned 57 cm beneath the acrylic box to capture a bottom-up view of the mouse’s feeding behaviour. On the same wall as the microphone, a single standard chow food pellet was glued to a plastic lid that was then affixed to the wall. The weight of the food pellet was measured both before and after the test session to calculate food intake, with adjustments made for any food spillage. The audio and video data were recorded using the OBS 29.1.3 software.
Audio processing
Audio recordings were converted into .ogg format. Initial recordings were captured at a sampling frequency of 44.1 kHz, and then subsampled to 2,205 Hz for power spectrogram computation. The biting frequency was determined to lie between 400 and 1,000 Hz, the absolute power spectrum was averaged, and a band-p*** filter between 400 and 1,000 Hz was applied to accurately identify biting frames. From the filtered signal, a threshold of 0.5 standard deviations above the mean was calculated. Each audio frame exceeding this threshold was binarized to 1 (indicating a potential bite), whereas those below were set to 0 (non-bite). This procedure further helped to eliminate false bite events. The binarized signal was then used to compute the bite start and end. A bite was defined as a sequence of binarized audio frames (with values of 1) separated by pauses in feeding (values of 0) longer than 10 s. These pauses were referred to as inter-bite intervals. A minimum of three consecutive audio frames with a value of 1 was required to be considered a bite.
Video processing
To ensure accurate bite identification, video snippets were automatically generated for each potential bite event. These snippets were then reviewed and validated by a human observer. Only the video snippets depicting correctly identified bites were included in the subsequent ***ysis. All video snippets utilized in this study are available in the Supplementary Information. The original video footage was recorded in .mkv format at a frame rate of 30 frames per second.
Statistics and reproducibility
We performed statistical ***yses using R (3.1) and JMP Pro (SAS, version 16), unless otherwise indicated. Data were evaluated for normality using a Q–Q plot. For normally distributed data, ANOVA was used and Tukey HSD post hoc testing was performed when applicable. For behaviour studies, we used a repeated-measures ANOVA to account for each individual, followed by post hoc paired Student’s t-tests. For data not normally distributed, means were evaluated by Kruskal–Wallis test with non-parametric comparisons using the Wilcoxon method. For other studies, comments on statistical tests performed are included throughout the Methods and in the figure legends. All error bars and shaded regions represent s.e.m. unless otherwise indicated. Sample size was not predetermined using power ***yses. Standardized randomization was not performed for in vitro or in vivo experiments. All behavioural studies were counterbalanced across age and block to control for variables including position in cage and order effect. Experimenters were not blinded to treatment condition, genotype or outcome.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
