Animals
Eight-week-old female and male C57BL/6J mice were purchased from the Institute of Experimental Animals of Sun Yat-sen University. The mice were housed in groups of five in a specific pathogen-free (SPF) facility under a 12-h light/dark cycle, with ad libitum access to food and water. All experiments were conducted during the light phase, and animals were randomly assigned to the various experimental groups. Both male and female mice were used but their sex was not recorded and so cannot be reported. All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University (approval number: SYSU-IACUC-2024-000134), we have complied with all relevant ethical regulations for animal use. For surgical tumor implantation, mice were anesthetized with a mixture of teletamine, zolazepam (85 mg/kg, ip, Zoletil 50®, BAILAIYUAN, China) and acepromazine (10 mg/kg, ip, MCE, USA) and given meloxicam (5 mg/kg, subcutaneously) for perioperative analgesia. Post-surgical monitoring was conducted daily. Mice were euthanized using CO2 followed by cervical dislocation. Tumor volume was calculated using the formula: volume = 0.5 × length × width × height69. Mice were euthanized when tumor volume exceeded 1.5 cm3 or signs of distress (weight loss >15%, lethargy) were observed, in accordance with humane endpoints.
Establishment of mice with orthotopic colorectal cancer
MC38 cells (ATCC) at 2–3 passages, reaching 70%–80% confluence, were used for orthotopic tumor implantation. Mice were anesthetized as described in the “Animal” section of the Methods. A suspension of 106 MC38 cells in PBS was mixed 1:1 with ice-cold Matrigel (Corning, USA; Cat#354248) to a final volume of 20 µL. Under a stereomicroscope, the colorectal mucosa approximately 1 cm from the anus was gently grasped with blunt forceps, retracted to expose the area, and the cell mixture was injected into the submucosa using an insulin syringe. The needle was held in place for an additional 30 seconds to ensure proper tissue absorption before removal.
Behavioral tests
To evaluate depression-like behaviors, we employed four widely used behavioral paradigms: the novelty-suppressed feeding test (latency to feed), splash test (grooming time), tail suspension test (immobility time), and social interaction test (social preference index). These tests are well-validated indicators of behavioral despair, anhedonia, and social withdrawal in rodent models of depression70,71. Each behavior was analyzed independently to provide a multi-dimensional profile of depression-like behavior in CRC mice. Behavioral tests were conducted 14 days after the establishment of CRC in mice. Prior to testing, the mice were allowed to acclimate to the testing room for at least 2 h. On the first day, the novelty-suppressed feeding test was performed in the morning, followed by the grooming test in the afternoon. On the second day, social interaction testing was conducted, and pain threshold testing was performed on the third day in the morning, with the tail suspension test conducted in the afternoon. The behavioral testing protocols followed our previous study72.
Novelty-suppressed feeding
After food restriction for 24 h, mice were placed into one of the corners of a white plastic box (40 × 40 × 40 cm) with a single food pellet present in the center. The latency to begin food consumption in a 10-m test was measured.
Splash test
The mice were placed in the cage for free movement for 1 min, and then 10% sucrose water was sprayed on the buttock and back of the mice, and the grooming time of the mice within 5 min was recorded.
Social Interaction test
The social interaction test was conducted in a (60 cm × 40 cm × 20 cm) cuboid field, which was divided into three equal-sized rooms by two transparent partitions. In each partition, a square opening (5 cm × 5 cm) was centrally located, and mice had access to each chamber. Two small round iron cages were placed in the left and right ventricles. An unfamiliar mouse was habituated to the room for 10 min before a day of testing to minimize excessive stress. On the day of testing, a test mouse was first explored for 5 min in a field holding an empty wire cage. The stranger mice were then housed in a wire cage, while the other cage contained a toy mouse. The test mice were placed into the field for 5 min. The social interaction index (DI%) was calculated as: [(time spent exploring the unfamiliar mouse – time spent exploring the toy mouse) / total time spent exploring both] ×100.
von Frey test
Mice were placed in a metal mesh cage in a quiet environment and allowed to acclimate for 10–15 min. Von Frey filaments, with forces ranging from 0.008 g to 4 g, were then used to apply pressure to the soles of the mice. A positive response was recorded when the mouse exhibited a nociceptive reaction, such as withdrawal or flinching. The pressure value of the filament that elicited the response was recorded.
Tail suspension test
The mouse was suspended by attaching one end of a 10 cm tape to its tail and the other end securely to a hanging frame in a quiet environment. The mouse was suspended for 5 min, during which the total immobility time, defined as the duration in which the mouse ceased struggling, was recorded.
Colorectal tracer injection in mice
Mice were anesthetized as described in the “Animal” section of the Methods. Vaseline was applied to their eyes to prevent dryness. Under a body microscope, the colorectal mucosa ~1 cm from the anus was gently lifted using blunt forceps, exposed to the field of view, and the tracer was drawn into a Hamilton syringe connected to a syringe pump. The needle was inserted into the colorectal mucosa or inject into the tumor that has been implanted with MC38 for two weeks at a 45° angle, and 1 µL of PRV (PRV-CAG-EGFP, 4.00E + 8 PFU/mL, BrainVTA, China) was injected at three sites. Following the injection, the needle was left in situ for an additional 2 min, and the mice were allowed to recover on a heated pad.
Stereotaxic injection into the brain
Mice were anesthetized as described in the “Animal” section of the Methods. Vaseline was applied to their eyes to prevent dryness. The mice’s head fur was shaved, and the area was sterilized using iodine followed by 75% ethanol. The scalp was incised with surgical scissors, and the head was secured in a stereotaxic apparatus. A heating pad was placed beneath the head to maintain the mouse’s body temperature. The anterior fontanel was used as the reference point, and the coordinates were set to AP: 0.98 mm, ML: ±0.25 mm, DV: −2 mm. Virus was injected through a Hamilton steel needle at a rate of 20 nL/min, with a total volume of 100 nL per side, and the needle was held in place for 5 min to allow for complete infusion. For chemogenetic inhibition of ACC neurons, AAV9-hSyn-hM4Di(Gi)-EGFP (BrainVTA, China) and AAV8-hSyn-hM4Di(Gi)-mCherry (BrainVTA, China) was used, for chemogenetic activation of ACC neurons, AAV9-hSyn-hM3Dq(Gq)-EGFP (BrainVTA, China) was used, to suppress ACC neurons output, AAV9-hSyn-Tettoxlc- EGFP (BrainVTA, China) was used, while the control group received AAV9-hSyn-mCherry (BrainVTA, China) and AAV9-hSyn- EGFP (BrainVTA, China).
Chemogenetic inhibition of ACC neurons
To reduce the excitability of ACC neurons, mice expressing the inhibitory chemogenetic receptor hM4Di in the ACC were injected i.p. with 1 mg/kg compound 21 (C21, Tocris Bioscience, cat# 6422), once per day, starting from the establishment of CRC mice.
Hematoxylin-eosin staining
Mice were euthanized using CO2 followed by cervical dislocation. Open their abdominal cavities, and cut out the colonic and intestinal tissues. Wash the tissues with normal saline, and immediately immerse them in neutral formalin fixative for fixation for 30–50 min. After fixation, rinse with running water for several hours. The tissues are then dehydrated successively with 70%, 80%, and 90% ethanol solutions for 30 min each, followed by two more times of 95% and 100% solutions, each for 20 min. Then, immerse them in a 15-min mixture of pure alcohol and xylene, xylene for 15 min each (until transparent), mixture of xylene and paraffin for 15 min each, and finally paraffin I and paraffin II for dewaxing for 50–60 min each. Then, proceed with embedding and sectioning. Prepare 3–5 µm paraffin sections for staining, and bake the slides in an oven at 65 °C for 2 h. Perform the following steps sequentially: Deparaffinization: Place the sections on a slide rack and immerse them in xylene I for 10 min, xylene II for 10 min, and xylene III for 5 min. Alcohol Hydration: Gradually rehydrate the sections using absolute ethanol I (10 min), absolute ethanol II (10 min), 95% ethanol (5 min), and 70% ethanol (3 min). Hematoxylin Staining: Stain the nuclei with hematoxylin for 3 min, then rinse thoroughly with distilled water to remove excess stain. Differentiation: Differentiate the sections in 1% hydrochloric acid alcohol differentiation solution for 30 s, followed by rinsing under tap water I for 3 min and tap water II for another 3 min. Eosin Staining: Stain the cytoplasm with eosin for 2 min, then rinse with distilled water to remove excess stain. Dehydration: Sequentially dehydrate the sections in 75% ethanol, 85% ethanol, 95% ethanol, and absolute ethanol for 1 min each. Clearing: Immerse the sections in xylene I for 1 min, followed by xylene II for 1 min. Mounting: Apply a suitable amount of neutral resin to the center of the tissue, then gently lower a coverslip onto the slide.
Preparation of immunofluorescence staining samples
After acclimatization in a quiet environment for more than 2 h, mice were anesthetized with a mixture of teletamine, zolazepam (85 mg/kg, ip, Zoletil 50®, BAILAIYUAN, China) and acepromazine (10 mg/kg, ip, MCE, USA) and the hearts were exposed by opening the thoracic cavity using surgical scissors. A needle was used to infuse 1×PBS for 2 min (10 mL /min) through the mouse heart, followed by 4% PFA for 5 min (10 mL /min). The colorectum and brain were removed and placed in 4% PFA at 4 °C overnight. Then they were transferred to 30% sucrose solution and stored at 4 °C until sedimentation. The colorectum and brain was removed from the sugar water and the surface sugar water was carefully blotted dry. They were then embedded in optimal cutting temperature (OCT) embedding agent and flash-frozen and embedded in a microtome at −40 °C. For handling colorectal tissues, the embedded tissue was placed on the sample stage of a freezing microtome with a thickness of 20 μm, and the sections were directly attached to slides and stored in a refrigerator at −80 °C until use. For handling brain tissue, the brain was sectioned continuously at a thickness of 30 μm, placed in PBS for storage, and then prepared for staining.
Immunofluorescence staining of brain slices
The brain slices,30 μm thick, were placed into 24-well plates containing blocking solution and incubated for 2 h at room temperature with blocking solution. After that, the brain slices were incubated in primary antibody (diluted in the blocking buffer) for c-FOS (Cell Signaling Technology, USA; cat#2250S; 1:500) for 48 h at 4 °C and rinsed with 0.4%PBST three times at room temperature for 10 min each time. They were then placed in secondary antibodies (diluted in the blocking buffer) for Anti-rabbit IgG (H + L) Alexa Fluor® 488(Cell Signaling Technology, USA; cat# 4412; 1:500) and incubated at room temperature in the dark for 2 h. Rinse with 0.4%PBST three times at room temperature in the dark for 10 min each time. Finally, the cells were rinsed in 1×PBS for 10 min in the dark. Brain slices were laid flat on slides, allowed to dry, and then sealed with drops of anti-fluorescence quench for observation. NIKON Ti2-E fluorescence microscope (Nikon, Japan) was used.
C-FOS quantification and network analysis
To evaluate neuronal activity and interregional functional connectivity across the brain, we first quantified c-FOS+ neurons in multiple brain regions using ImageJ software (NIH). Regions of interest (ROIs) were manually delineated on fluorescence images according to anatomical landmarks from the Allen Mouse Brain Atlas. The density of C-FOS+ cells was calculated as the number of c-FOS+ nuclei per mm² within each ROI. For each group, we included bilateral counts from 2 to 3 representative coronal sections per region per animal. Subsequently, interregional relationships were assessed by computing the Pearson correlation coefficients (Pearson’s r) between c-FOS+ cell densities of each pair of regions across all animals within each experimental group. These correlation matrices were visualized as heatmaps to provide an overview of pairwise associations. To construct functional brain networks, we selected region pairs showing statistically significant and strong correlations (Pearson’s r > 0.6, p < 0.05) to define edges in the network. Each brain region was represented as a node, and the strength of interregional correlations defined the edges. Graph-theoretical analysis was performed to calculate the global efficiency (GE) of each network, which reflects the average inverse shortest path length among all nodes and is defined as:
$${GE}=frac{1}{Nleft(N-1right)}{sum}_{ine j}frac{1}{{d}_{{ij}}}$$
where N is the number of nodes and d(ij) is the shortest path length between node i and node j. To identify the most influential regions, we implemented a node-deletion strategy: for each node, we recalculated the GE after its removal and computed the change (ΔGE). The top five nodes causing the greatest reductions in GE were defined as core nodes in the network, representing functionally central regions likely critical for maintaining network integration.
Immunofluorescence staining of colorectal frozen sections
Take out the colorectal frozen sections and let them dry at room temperature for 0.5 h (away from light). Then put the sections into a jar with 1×PBS and wash for 5 min to wash out OCT. Dry the edges of the sections with absorbent paper, draw closed circles along the edges of the slides with an immunohistochemical pen, place them flat in a wet box, and add 200 µL blocking solution. After sealing at room temperature for 2 h, tip off the blocking solution, drain the excess water on the filter paper pad, place it flat in the wet box, add 200 µL primary antibody (diluted in the blocking buffer) for VAChT (Synaptic Systems, Germany; cat#139103; 1:500), TH (Cell Signaling Technology, USA; cat#E2L6M; 1:500), CGRP (Cell Signaling Technology, USA; cat#14959; 1:500), VIP (Cell Signaling Technology, USA; cat#63269; 1:500), Ki67 (Cell Signaling Technology, USA; cat#9129; 1:500) and cleaved caspase-3 (Cell Signaling Technology, USA; cat# 9664; 1:500) and incubate at 4 °C for 12 h, and then incubate at room temperature for 4 h. On the second day, the colorectal frozen sections were rinsed with 0.4% PBST three times at room temperature for 10 min each time. Add 200 µL secondary antibody (diluted in the blocking buffer) for Anti-rabbit IgG (H + L) Alexa Fluor® 488 (Cell Signaling Technology, USA; cat# 4412; 1:500) and apply for 2 h at room temperature in the dark, and rinse with 0.4% PBST by shaking for 3 times, 10 min each time. Excess water was removed, DAPI-containing sealed tablets were sealed, NIKON Ti2-E fluorescence microscope (Nikon, Japan) was used.
Protein extraction, and western blot analysis
Tumor samples were immediately transferred to a mortar pre-filled with liquid nitrogen and ground into a fine powder using a pestle in the presence of liquid nitrogen. Subsequently, lysis buffer (Cell Signaling Technology, USA, cat#9803S)) was added to the powdered tissue, and homogenization was continued. The homogenate was centrifuged at 12,000 × g for 20 min at 4 °C, and the supernatant was carefully aspirated and transferred to a new tube. Protein concentration was determined using the BCA assay (Beyotisme, Chian, cat#P0012). For Western blot analysis,30 μg of total protein extract per lane was separated on a denaturing SDS-PAGE gel and subsequently transferred onto a 0.22 µm PVDF membrane using electroblotting. The following primary antibodies were used: Cleaved Caspase-3 Rabbit Antibody (1:1000, Cell Signaling Technology, USA, cat#9664S), Ki-67 Polyclonal antibody (1:500, Proteintech, USA, cat#27309-1-AP), PCNA Polyclonal antibody (1:5000, Proteintech, USA, cat#10205-2-AP), Beta Tublin Polyclonal antibody (1:5000, Proteintech, USA, cat#10094-1-AP) for loading normalization. The secondary antibody, anti-rabbit IgG (1:4000, Cell Signaling Technology, USA, cat#7074S), was used for detection. After incubation with the antibodies, the Immobilon-FL membranes were washed three times with TBST for 5 min each. The membranes were developed using enhanced chemiluminescence (ECL) solution and imaged with a chemiluminescence imaging system.
RNA sequencing
Sequencing samples were obtained from sham-operated control group (Sham+Ctrl-ACC), mice with orthotopic colorectal cancer group (CRC+Ctrl-ACC) and colorectal cancer chemogenetically suppressed ACC neuronal activity group (CRC+Gi-ACC), with 4–7 samples from each female and male group. RNA amplification library building and sequencing were performed by Nuohezhiyuan Inc. One μg of total RNA with a RIN value higher than 7 was used for the next step of amplification and library construction. The NEBNext® Ultra™ RNA Library Prep Kit for Illumina® was used for RNA amplification and cDNA library construction. Libraries were loaded onto an Illumina HiSeq instrument 6000 (USA) for 150 bp double-end sequencing. Raw sequencing reads were filtered to remove adaptor sequences, N-containing reads, and bases of low read quality. The quality of reads was assessed with the use of Fastp, version 0.19.7. The processed sequences were then aligned to the mm9 mouse reference genome (UCSC) using Hisat2 (version 2.0.1). Genes with counts <5 reads in more than 70% of the samples (low expression genes) were excluded from the analysis. DESeq2 package (version 1.40.2) of R was used to standardize the readings and perform the analysis of differentially expressed genes (DEGs). The screening conditions of differentially expressed genes were p < 0.05. Differential gene results were visualized as heat maps and volcano maps using the pheatmap, ggplot2 and ggpubr packages. Gene Set Enrichment Analysis (GSEA) software was used to analyze the differentially expressed Genes in the Kyoto Encyclopedia of Genes and Genomes (GSEA) database. KEGG pathway analysis (c2.cp.kegg.v2023.1.symbols.gmt), molecular function of Gene ontology (MF, c5.go.mf.v2023.1.symbols.gmt), biological process (BP, c5.go.bp.v2023.1.symbols.gmt) and cellular component (CC, c5.go.cc.v2023.1.symbols.gmt).
Statistics and reproducibility
Fluorescence image analysis was performed using image J software (Version 1.53j). GraphPad Prism 8.0 software was used for data analysis and expressed as mean ± standard error (mean ± s.e.m). We conducted the Shapiro–Wilk test to evaluate the normality of the datasets. For datasets which were normally distributed, outliers were identified as being >2 standard deviations from the mean and excluded. For two-group comparisons, two-tailed unpaired Student’s t test was used for normally-distributed datasets, and the Mann–Whitney test was used for non-normally distributed datasets. For multiple-group comparisons, one-way ANOVA with Tukey’s post hoc tests was used, and p < 0.05 was considered as statistically significant. Images were processed using Adobe Illustrator CC software (Version 19, 23.0.2). Group sizes were determined based on prior work and the literature. Each data point represents an independent biological sample, and the number of biological replicates (n) for each experiment is indicated in the figure legends. For all animal studies, each experimental group contained at least [n = 4] independent animals. The mice used in the experiments were randomly assigned to the different experimental groups.