5-HT2C receptors in the glutamatergic neurons of dorsal raphe nucleus orchestrate the comorbidity of pain and anxiety in mice

Abstract

Background

Pain-anxiety comorbidity represents a prevalent clinical concern. This study aims to investigate the molecular mechanisms underlying the comorbidities via focusing on the activity of dorsal raphe nucleus (DRN) glutamatergic neurons and the functional role of their 5-hydroxytryptamine 2C (5-HT2C) receptors in relation to gut microbiota.

Methods

We established a Complete Freund’s Adjuvant (CFA)-induced pain-anxiety comorbidity model in mice and systematically investigated the role of the brain-gut axis in the comorbidity using behavioral phenotyping, molecular biology, pharmacological/chemogenetic modulation, and gut microbiota profiling.

Results

Heightened activity in the glutamatergic neurons of the DRN was found in mice with comorbidity. Chemogenetic activation of DRN glutamatergic neurons replicated the comorbid phenotype in naïve mice, while the selective inhibition of DRN glutamatergic neurons effectively reversed the behavioral and physiological impairments induced by CFA. Notably, a significant upregulation in the protein levels of 5-HT2C receptors in the DRN was detected in the comorbid state. Bidirectional manipulation of 5-HT2C receptors in the DRN glutamatergic neurons bidirectionally regulates neuronal excitability and comorbid phenotypes: agonism or overexpression exacerbates comorbidity, while antagonism or knockdown attenuates CFA-induced deficits.

Conclusions

These findings uncover the role of 5-HT2C receptors in DRN glutamatergic neurons in pain-anxiety comorbidity, thereby presenting novel targets for potential therapeutic interventions.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12916-026-04620-6.

Background

Chronic pain is clinically defined as nociceptive signaling that persists beyond 3 months following an initial injury or pathological process [1]. This condition represents a substantial public health challenge, with epidemiological studies estimating that approximately one-fifth of adults experience debilitating symptoms linked to functional limitations and diminished quality of life [2]. Chronic pain exhibits a significant comorbidity with psychiatric manifestations, and emerging evidence demonstrates that approximately 40% of adults with persistent nociceptive disorders present clinically validated anxiety symptoms [3]. Anxiety disorders conversely amplify pain intensity and duration, creating a vicious cycle that complicates clinical management [4]. Current mechanistic studies often separately investigate chronic pain and anxiety with limited exploration of their comorbidity [5, 6]. Addressing pain-anxiety comorbidity is crucial for elucidating shared pathological mechanisms and developing novel therapeutic strategies that effectively alleviate pain while minimizing adverse side effects.

The dorsal raphe nucleus (DRN), situated ventromedial to the periaqueductal gray matter, is a fan-shaped midline structure. The DRN comprises heterogeneous neuronal populations, including serotonergic, dopaminergic, gamma-aminobutyric acid-ergic (GABAergic), and glutamatergic neurons [7]. Those neurons form complex intrinsic microcircuits and project broadly to other brain regions, critically regulating pain perception and neuropsychiatric disorders [8–11]. Emerging evidence highlights the DRN-related circuit as a key mediator of pain-anxiety comorbidity [12]. Notably, the functional contributions of glutamatergic neurons remain understudied. As the primary source of central serotonin, the DRN also expresses multiple 5-hydroxytryptamine (5-HT) receptors [13], among which 5-hydroxytryptamine 2 C (5-HT2C) receptors regulate neural network dynamics via neurotransmitter release and serve as therapeutic targets for neuropsychiatric drugs [14, 15]. However, their role in pain-anxiety comorbidity remains unexplored.

The brain-gut axis, a bidirectional communication system involving gut-to-brain afferents and brain-to-gut efferents, integrates gut microbiota as a key mediator. Gut microbes interact with the brain directly via signaling molecules or indirectly through the brain-gut axis, while the brain modulates microbial composition through neural and endocrine pathways [16]. Distinct perturbations in the gut microbiota play an important role in gastrointestinal disorders, neuropsychiatric diseases, immune dysfunction, and metabolic syndromes [17–19]. Recent evidence suggests that an abnormality in gut microbiota contributes to chronic pain [20, 21], as also demonstrated in our prior studies using diverse pain models [22–24]. Gut microbiota disturbances are further linked to reduced neurogenesis and abnormal neuroinflammation, which are critical drivers for anxiety [17, 25]. However, mechanistic insights into how gut microbiota directly mediates pain-anxiety comorbidity, particularly under specific regulation of brain region, remain elusive.

In the this study, we established a Complete Freund’s Adjuvant (CFA)-induced pain-anxiety comorbidity model and systematically investigated the role of 5-HT2C receptors in DRN glutamatergic neurons through the brain-gut axis using multimodal approaches: behavioral phenotyping, molecular biology, pharmacological/chemogenetic modulation, and gut microbiota profiling. In this regard, we aimed to unravel novel therapeutic targets for comorbid pain and anxiety disorders.

Methods

Animals

Male C57BL/6 J mice (Beijing Charles River Laboratory Animal Breeding Co. Ltd.) aged 7–8 weeks were adopted in this study. All mice were housed 4 or 5 per cage under a 12-h light/dark cycle with food and water available ad libitum. All procedures strictly followed the Guide for the Care and Use of Laboratory Animals and received approval from the Institutional Animal Care and Use Committee of Nanjing Medical University, Nanjing, Jiangsu.

A total of 351 mice were utilized in the study, with each mouse serving as an independent experimental unit. Twenty-four mice were excluded due to off-target virus vector expression, health issues, or experimental failures. Sample sizes for each group were determined based on our previously published studies [6, 22–24]. Corresponding control groups were established for every experiment. Mice were randomly assigned to groups using an online randomization tool (https://www.random.org/lists/). To minimize the influence of confounding variables, measures such as staggered cross-group measurements and scheduled rotation of cage positions among different groups were implemented. All experiments were conducted as blind studies, wherein the personnel responsible for carrying out the procedures, assessing outcomes, and analyzing data were unaware of the group allocations.

Chronic pain model

Complete Freund’s Adjuvant (CFA)-induced chronic inflammatory pain was performed as described previously [6]. Briefly, CFA (10 µL) was delivered subcutaneously to the left hind paw’s plantar surface. Saline (10 µL) was injected as the control.

Behavioral tests

Pain threshold measurement

Paw withdrawal latency (PWL) to thermal stimuli was quantified using a calibrated radiant heat apparatus as described previously [26]. Briefly, mice underwent a 30–60-min acclimatization period in individual polyethylene chambers positioned on a temperature-controlled glass surface. The infrared emitter intensity was calibrated to achieve baseline latencies of 10–15 s, with a safety cutoff at 20 s to prevent tissue injury. Following habituation, the left hind paw plantar surface was exposed to focused thermal stimulation. PWL was defined as the time interval between heat onset and paw withdrawal behaviors (flinching/flicking), measured in triplicate with 5 min intervals.

Paw withdrawal thresholds (PWT) to a mechanical stimulus was assessed using an optimized up-down paradigm as described previously [27]. Mice were positioned on elevated wire-mesh platforms (30–60 min acclimation) before testing. Von Frey filaments (North Coast, USA) were applied perpendicularly to the plantar surface of the left hind paw for 3 s. Starting with a 0.4 g filament, subsequent stimuli were selected based on withdrawal responses: increased force for negative reactions, decreased force for positive withdrawals. After five sequential trials (5 min rest intervals), the 50% PWT was calculated using the Bonin formula to minimize inter-trial variability.

Open field test (OFT)

The OFT was performed as described previously [28]. Briefly, mice were individually placed in a sanitized acrylic arena (40 × 40 × 35 cm) under standardized conditions. The center zone is a 20 × 20 cm area in the center part of the chamber. After 75% ethanol disinfection and 60 s environmental adaptation, behavior was monitored for 300 s. Relative parameters were automatically quantified through video tracking using YHTSData (Wanhan Yihong Technology Co., Ltd, China).

Elevated plus maze test (EPMT)

The EPMT apparatus comprised two opposing open arms (30 × 5 cm) and two enclosed arms (30 × 5 cm × 15 cm) elevated 70 cm above ground, forming a cruciform configuration with a central junction (5 × 5 cm). After 75% ethanol disinfection, mice were positioned in the central zone facing an open arm under controlled conditions. Exploratory behavior was recorded for 300 s. Relative parameters were automatically quantified through video tracking using EMPData (Wanhan Yihong Technology Co., Ltd, China).

Immunofluorescent staining (IF)

Following anesthesia via intraperitoneal injection of 1% pentobarbital sodium (45 mg/kg), cardiac perfusion was initiated with physiological saline, succeeded by 4% paraformaldehyde (PFA) infusion. Harvested brain tissues were post-fixed in 4% PFA overnight, followed by cryoprotection through immersion in 30% sucrose at 4 ℃ until complete submersion. Coronal Sects. (40 µm thickness) were prepared using a freezing microtome (Leica), then subjected to three 10 min PBS washes. After blocking with 0.3% BSA (2 h, room temperature), sections were exposed overnight at 4 °C to primary antibodies targeting c-fos (Stock #AB208942, Abcam, 1:500; Stock #2250S, Cell Signaling Technology 1:200), 5-HT (Stock #AB6336, Abcam, 1:50), and 5-HT2C (Stock #SC17797, Santa Cruz Biotechnology, 1:50). Subsequent PBST rinses (3 × 10 min) preceded incubation with fluorescent-conjugated secondary antibodies for 2 h at room temperature. Nuclear counterstaining was achieved using DAPI (Stock #AB104139, Abcam), with immediate coverslipping post-staining. Fluorescent signals were visualized with a Leica Thunder microscope, and image analysis was conducted using the manufacturer’s software.

Stereotaxic surgery and viral injection

Following anesthesia induction with intraperitoneal 1% pentobarbital sodium (40 mg/kg), animals were immobilized in a stereotaxic apparatus (stock #68,804, RWD Life Science, China). Craniotomy was performed to access target nuclei: dorsal raphe nucleus (DRN; coordinates AP − 4.25 mm, ML 0 mm, DV − 3.3 mm). Using a 33-gauge Hamilton syringe connected to an automated pump, 150–200 nL viral suspension was infused at 0.1 nL/min. Post-injection stabilization (8 min) preceded needle withdrawal. Viral expression was permitted for 21 days prior to experimental procedures. All adeno-associated viruses (AAVs) were purchased from Tailool, Brain VTA, and OBIO Technology, including the following: AAV2/9-hSyn-Cre, AAV2/9-CaMKII-Cre, AAV2/9-vglut2-Cre, AAV2/9-vgat1-Cre, AAV2/9-DIO-EGFP–5HT2C, AAV2/9-DIO-EGFP-scramble, AAV2/9-DIO-EGFP-5HT2C-shRNA, AAV −2/9-DIO-mCherry, AAV2/9-hSyn-DIO-hM3Dq(Gq)-mCherry, and AAV2/9-hSyn -hM4Di(Gi)-mCherry.

Chemogenetic and pharmacological manipulation

To inhibit or activate DRN neurons, stereotaxic viral injections were performed. Following a 21-day incubation period, animals underwent 1 h habituation in the testing room. Intraperitoneal administration of CNO (1.5 mg/kg; MedChemExpression) preceded behavioral assessments for chemogenetic modulation.

RS102221 and MK212 obtained from MedChemExpress (China) were used to selectively antagonize/activate 5-HT2C receptors, respectively. They were dissolved in 0.9% saline. In the intracerebral injection experiments, doses of RS102221 (0.1 µg) and MK212 (5.0 µg) were injected into DRN (coordinates AP –4.25 mm, ML 0 mm, DV –3.3 mm) as described above. All mice were allowed a 24-h postoperative recovery period in standard housing cages following stereotaxic surgery before proceeding to behavioral testing or fecal sample collection. In the systemic administration experiments, RS102221 and MK212 (1 mg/kg) were administered intraperitoneally to mice, and relevant behavioral changes were assessed 30 min and 24 h post-injection.

Western blot

Western blot was conducted following established electrophoretic protocols. Tissue lysates (40 µg/sample) were resolved by SDS-PAGE and electrophoretically transferred to PVDF membranes. Membranes underwent dual-probe immunoreaction with mouse monoclonal anti-5-HT2C (Stock #SC17797, Santa Cruz Biotechnology, 1:200) and rabbit polyclonal β-actin (Stock #66,009, Proteintech, 1:2000) antibodies in 4 °C overnight incubation. Following TBST washes, membranes were exposed to HRP-conjugated species-matched secondary antibodies (Beyotime, 1:2000) for 1 h at ambient temperature. Chemiluminescent detection was achieved using NBT/BCIP substrate (Sigma 72,091), with band intensity quantified via ImageJ analysis suite.

16S rRNA analysis of fecal samples

Fresh fecal specimens were immediately aliquoted into sterile tubes and cryopreserved at − 80 °C until processing. The composition of gut microbes was detected by 16S rRNA sequencing (OEbiotech Co., Ltd., Shanghai, China). Microbial genomic DNA was isolated using the A DNeasy PowerSoil kit (Qiagen, Hilden, Germany). Targeted amplification of bacterial 16S rRNA gene hypervariable regions V3-V4 was achieved with universal primers pairs (343F: 5′-TACGGRAGGCAGCAG-3′; 798R:5′-AGGGTATCTAATCCT−3′). Both primers contained Illumina sequencing adapters and unique barcodes. Purified amplicons were quantified and sequenced on an Illumina NovaSeq6000 with two paired-end read cycles of 250 bases each. (Illumina Inc., San Diego, CA; OEBiotech Company; Shanghai, China). Following adapter removal, paired-end reads underwent quality filtering to remove low-quality sequences, with subsequent noise reduction and paired-read assembly. All representative reads were annotated and blasted against Silva database Version 138 (or Unite) (16 s/18 s/ITS rDNA) using q2-feature-classifier with the default parameters.The microbial diversity in samples was estimated using the alpha diversity that include Chao1 index and Shannon index. The Unifrac distance matrix performed by QIIME software was used for unweighted Unifrac Principal coordinates analysis (PCoA) and phylogenetic tree construction. The 16S rRNA gene amplicon sequencing and analysis were conducted by OE Biotech Co., Ltd. (Shanghai, China) [24].

Statistical analysis

Statistical analyses were conducted with Prism 8.0 (GraphPad Software, Inc., USA). All data were analyzed with a two-tailed test and represented as mean ± SEM. For intergroup comparisons, normally distributed data with equal variances were analyzed using the unpaired t test, while data with unequal variances were evaluated via Welch’s t test with adjusted degrees of freedom. Non-normally distributed data were subjected to the Mann–Whitney U test. Multigroup comparisons employed two-way analysis of variance (ANOVA), followed by Sidak’s post hoc comparison. One-way ANOVA followed by Bonferroni posttest was used for four group comparisons. Associations between different variables were quantified using simple linear regression. Data are considered to be statistically significant if P < 0.05.

Microbiota-behavior correlation analysis: All raw data were normalized to a percentage scale (0%–100%) based on the maximum and minimum values within each group using Prism 8.0 (GraphPad Software, Inc., USA). The normalized data were then subjected to Pearson correlation analysis in Prism 8.0. Heatmaps were constructed using the resulting Pearson r values, with blue indicating negative correlations and orange indicating positive correlations. Color intensity reflected the strength of the correlations.

Results

CFA administration induces chronic pain and anxiety-like behaviors

To investigate the mechanisms underlying the comorbidity of chronic pain and anxiety, we established a CFA-induced chronic inflammatory model. Paw withdrawal threshold (PWT) and thermal withdrawal latency (PWL) were measured to assess pain sensitivity, while locomotor function and anxiety-like behaviors were evaluated through open field test (OFT) and elevated plus maze test (EPMT) (Fig. 1A). Compared to controls, CFA mice exhibited significantly decreased PWT and PWL (Fig. 1B and C) at 1, 3, and 5 days after the CFA administration. Three days post-CFA was selected as the representative time point of chronic pain for subsequent behavioral analyses. No statistical difference in the total distance of movement was found in CFA mice (Fig. 1D and E), indicating unaffected locomotor function. However, compared to the control mice, CFA mice exhibited decreased time and distance proportion traveled in the central zone during OFT (Fig. 1F and G). In EPMT, CFA mice showed markedly reduced time in open arms and fewer open-arm entries, suggesting anxiety-like behaviors (Fig. 1H–J). Linear regression analysis exhibited positive correlations between pain and anxiety indices in OFT (Fig. 1K) and EPMT (Fig. 1L). The mice’s pain thresholds, as well as their exploratory behaviors in the open field and elevated plus maze tests, had returned to pre-injection levels 7 days after CFA injection (Additional file 2: Fig. S1). These findings demonstrate that the CFA model effectively induces concurrent chronic pain and anxiety-like behaviors, aligning with our prior report [6].

Fig. 1.

CFA administration induces chronic pain and anxiety-like behaviors associated with increased neural activity in the DRN, and chemogenetic activation of DRN neurons recapitulates this comorbidity in naïve mice. A Timeline of CFA/Saline injection, pain and anxiety-like behavioral tests. B PWT before and after CFA/Saline injection. C PWL before and after CFA/Saline injection. D–G OFT 3 days after CFA/Saline injection. D Representative trajectories of OFT exploration. E Total distance. F Time spent in the OF center. G Percentage of distance spent in the OF center. H–J EPMT 3 days after CFA/Saline injection. H Representative trajectories of EPMT exploration. I Time spent in the open arms. J Frequency of open arm entries. K Left: correlations between PWT and center duration. Right: correlations between PWT and center distance. L Left: correlations between PWT and time in open arm. Right: correlations between PWT and entries in open arms. M Representative immunofluorescent images of c-fos expression in the DRN. Scale bars: 200 µm. N Quantification of fluorescent intensity of c-fos expression. O Left: timeline of AAV/CNO/Saline injection and behavioral tests. Right: diagram of AAV injection and representative photomicrographs of the DRN site. Scale bars: 200 µm. P PWT 20 min after CNO/Saline injection. Q–T OFT 20 min after CNO/Saline injection. Q Representative trajectories of OFT exploration. R Total distance. S Time spent in the OF center. T Percentage of distance spent in the OF center. U–W EPMT 20 min after CNO/Saline injection. U Representative trajectories of EPMT exploration. V Time spent in the open arms. W Frequency of open arm entries. Data are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant. CFA, Complete Freund’s Adjuvant; CNO, clozapine N-oxide; DRN, dorsal raphe nucleus; EPMT, elevated plus maze test; Sal, saline; OFT, open field test; PWL, paw withdrawal latency to thermal stimuli; PWT, paw withdrawal thresholds to mechanical stimuli. Further details of statistical data analysis are shown in Additional file 1: Table S1

DRN glutamatergic neuronal activity contributes to pain-anxiety comorbidity

To elucidate the neural mechanisms underlying pain-anxiety comorbidity, we focused on the DRN, a region closely associated with pain and/or negative affect [9, 11, 12]. Immunofluorescence staining revealed significantly increased c-fos expression in the DRN neurons of CFA mice compared to controls (Fig. 1M and N), indicating enhanced DRN neuronal activity in CFA mice. Chemogenetic activation of DRN neurons through viral vector-mediated expression of hM3Dq (Gq) in naïve mice (Fig. 1O) produced comorbid phenotypes, as evidenced by the reduced PWT, preserved locomotor function, decreased central zone duration/distance in the OFT, and diminished open arm exploration in the EPMT following the clozapine-N-oxide (CNO) administration (Fig. 1P–W).

To further identify the neuronal subtypes in the DRN regulating comorbidity, we employed a Cre-loxP viral expression system to label glutamatergic neurons in the DRN using the vesicular glutamate transporter 2 (vglut2) promoter (Fig. 2A). Immunofluorescence staining of the DRN across groups revealed a significant increase in the number of c-fos⁺/vglut2⁺ co-labeled neurons in CFA mice compared to the saline group (Fig. 2A–C). In contrast, co-labeling immunofluorescence showed markedly fewer c-fos⁺ neurons co-expressing vesicular GABA transporter1 (vgat1) or 5-HT in the CFA group relative to vglut2⁺ neurons (Additional file 2: Fig. S2). These results indicate that vglut2-positive glutamatergic neurons, but not the vgat1-positive GABAergic or 5-HT-positive serotonergic neurons in the DRN, are activated in the status of pain-anxiety comorbidity.

Fig. 2.

Chemogenetic manipulation of DRN glutamatergic neurons affects the comorbidity of pain and anxiety-like behaviors. A Timeline of AAV/CFA/Saline injection and immunofluorescent staining. B Representative immunofluorescent images of c-fos expression in DRN glutamatergic neurons. Scale bars: 200 µm. C Quantification of fluorescent intensity of co-labeled (c-fos⁺ + vglut2.⁺) cells. D Left: timeline of AAV/CNO/Saline injection and behavioral tests. Right: representative photomicrographs of the DRN site. Scale bars: 200 µm. E PWT 20 min after CNO/Saline injection. F–I OFT 20 min after CNO/Saline injection. F Representative trajectories of OFT exploration. G Total distance. H Time spent in the OF center. I Percentage of distance spent in the OF center. J–L EPMT 20 min after CNO/Saline injection. J Representative trajectories of EPMT exploration. K Time spent in the open arms. L Frequency of open arm entries. M Left: timeline of AAV/CNO/CFA/Saline injection and behavioral tests. Right: representative photomicrographs of the DRN site. Scale bars: 200 µm. N PWT 20 min after CNO/Saline injection. O–R OFT 20 min after CNO/Saline injection. O Representative trajectories of OFT exploration. P Total distance. Q Time spent in the OF center. R Percentage of distance spent in the OF center. S–U EPMT 20 min after CNO/Saline injection. S Representative trajectories of EPMT exploration. T Time spent in the open arms. U Frequency of open arm entries. Data are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant. CFA, Complete Freund’s Adjuvant; CNO, clozapine N-oxide; DRN, dorsal raphe nucleus; EPMT, elevated plus maze test; IF, immunofluorescent staining; Sal, saline; OFT, open field test; PWT, paw withdrawal thresholds to mechanical stimuli. Further details of statistical data analysis are shown in Additional file 1: Table S1

We next investigated the role of DRN glutamatergic neuronal activity in regulating this comorbidity. Using chemogenetic approaches, we specifically activated vglut2⁺ glutamatergic neurons in the DRN (Fig. 2D). Following verification of viral expression (Fig. 2D), we demonstrated that the activation of DRN glutamatergic neurons significantly reduced mechanical pain thresholds (Fig. 2E), decreased center zone duration and distance traveled in the OFT (Fig. 2F–I), and reduced open-arm time and entries in the EPMT (Fig. 2J–L) without impairing motor function (Fig. 2G). These results mirror the behavioral phenotypes observed in the CFA model.

Conversely, chemogenetic inhibition of vglut2⁺ neurons in the DRN, validated by viral expression (Fig. 2M) and reduced c-fos (Additional file 2: Fig. S3), did not alter motor, pain, or anxiety-like behaviors under physiological conditions (Fig. 2N–U). However, this manipulation robustly reversed CFA-induced pain-anxiety comorbidity without affecting locomotor function (Fig. 2N–U). We also inhibited DRN neurons using the CaMKII promoter (Additional file 2: Fig. S4A and B), a widely used marker for glutamatergic populations. Similar to vglut2-promoted inhibition of the glutamatergic neurons in the DRN, CaMKII-based inhibition reversed CFA-induced comorbidity behaviors without motor side effects (Additional file 2: Fig. S4). Together, these findings establish glutamatergic neurons as the pivotal cellular substrate in the DRN for regulating pain-anxiety comorbidity.

Anatomical localization and functional effects of 5-HT2C receptor in DRN glutamatergic neurons

The 5-HT2C receptor is densely distributed in the DRN and critically involved in regulating neuronal activity, pain processing, and anxiety-related pathophysiological mechanisms. To clarify its role in pain-anxiety comorbidity, we examined protein expression of 5-HT2C receptors in the DRN. Our results revealed that CFA administration significantly increased the expression of 5-HT2C receptors in the DRN (Fig. 3A and Additional file 2: Fig. S5). We next performed immunofluorescence staining to anatomically localize the 5-HT2C receptors in the DRN and observed abundant expression of this receptor across glutamatergic (Fig. 3B and C), GABAergic (Additional file 2: Fig. S6A and B), and serotonergic (Additional file 2: Fig. S6C and D) neurons. Given the role of glutamatergic neurons in pain-anxiety comorbid behaviors, we focused on the function and expression of 5-HT2C receptors specifically within the DRN glutamatergic populations. To probe functional relevance, we stereotactically injected the receptor-specific agonist MK212 into the DRN to pharmacologically activate 5-HT2C receptors (Fig. 3D) and subsequently assessed the behavioral results (Fig. 3E–L). MK212-treated mice exhibited equal motor function (Fig. 3G), reduced central zone activity in the OFT (Fig. 3F–I), and diminished open-arm exploration in the EPMT, reflected by shorter time spent in open arms and fewer open-arm entries (Fig. 3J–L). More importantly, systemic administration of MK212 did not alter pain thresholds or anxiety-related behaviors in naïve mice at either 30 min or 24 h post-injection (Additional file 2: Fig. S7).

Fig. 3.

DRN glutamatergic 5-HT2C receptor upregulation promotes comorbid pain and anxiety, mimicked by pharmacological agonism or cell-specific overexpression. A Protein levels of 5-HT2C in the DRN. B Representative immunofluorescent images of c-fos expression in DRN glutamatergic neurons. Scale bars: 200 µm. C Percentage of DRN glutamatergic neurons that express 5-HT2C. D Left: timeline of Saline/MK212 (5-HT2C receptor agonist) injection and behavioral tests. Right: representative photomicrographs of needle track. Scale bars: 200 µm. E PWT 1 day after Saline/MK212 injection. F–I OFT 1 day after Saline/MK212 injection. F Representative trajectories of OFT exploration. G Total distance. H Time spent in the OF center. I Percentage of distance spent in the OF center. J–L EPMT 1 day after Saline/MK212 injection. J Representative trajectories of EPMT exploration. K Time spent in the open arms. L Frequency of open arm entries. M Left: timeline of AAV injection, behavioral tests and IF. N Representative immunofluorescent images of c-fos expression in DRN glutamatergic neurons. Scale bars: 200 µm. O Quantification of fluorescent intensity of co-labeled (c-fos⁺ + vglut2.⁺) cells. P PWT 21 days after AAV injection. Q–T OFT 21 days after AAV injection. Q Representative trajectories of OFT exploration. R Total distance. S Time spent in the OF center. T Percentage of distance spent in the OF center. U–W EPMT 21 days after AAV injection. U Representative trajectories of EPMT exploration. V Time spent in the open arms. W Frequency of open arm entries. Data are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant. 5-HT2C, 5-hydroxytryptamine receptor 2 C; CFA, Complete Freund’s Adjuvant; CNO, clozapine N-oxide; DRN, dorsal raphe nucleus; EPMT, elevated plus maze test; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IF, immunofluorescent staining; Sal, saline; OFT, open field test; PWT, paw withdrawal thresholds to mechanical stimuli. Further details of statistical data analysis are shown in Additional file 1: Table S1

To examine the effects of receptor overexpression, we utilized a Cre-loxP strategy to selectively increase 5-HT2C receptor expression in DRN glutamatergic neurons. Behavioral analysis following viral expression (Fig. 3M and N) and function (Additional file 2: Fig. S8A and B) validation revealed that the overexpression induced behavioral phenotypes of pain-anxiety comorbidity (Fig. 3M–W) analogous to the CFA model. Subsequent immunofluorescence staining of the DRN showed a marked increase in the count of c-fos⁺ and vglut2⁺ co-labeled neurons in the overexpression group compared to the controls (Fig. 3N), suggesting that aberrant function and expression of 5-HT2C receptors in DRN glutamatergic neurons may promote the pain-anxiety comorbidity through enhancing neuronal excitability.

To further delineate the role of 5-HT2C receptors in DRN glutamatergic neurons in pain-anxiety comorbidity, we pharmacologically antagonized the receptor or virally knocked down its expression and assessed subsequent effects on neuronal excitability and behaviors (Fig. 4). Intracerebral pharmacological antagonism (RS102221) (Fig. 4A) did not alter motor function (Fig. 4D), pain sensitivity (Fig. 4B), or anxiety-like behaviors (Fig. 4C - I) under physiological conditions (Fig. 4B–I, left), but significantly reversed CFA-induced pain-anxiety comorbidity (Fig. 4B–I, right) without affecting locomotor activity (Fig. 4D). However, systemic administration of RS102221 did not alter pain-related or anxiety-like behaviors of mice at either 30 min or 24 h post-injection (Additional file 2: Fig. S9). Using a 5-HT2C-shRNA combined with the Cre-loxP system to selectively knockdown 5-HT2C receptor in DRN glutamatergic neurons (Fig. 4J and Additional file 2: Fig. S8C and D), we observed consistent behavioral outcomes: no changes in saline mice but partial reversal of CFA-induced pain-anxiety comorbid behaviors (Fig. 4K–R), again independent of motor effects (Fig. 4M). Immunofluorescence analysis further confirmed a reduction in the number of c-fos⁺/vglut2⁺ co-labeled neurons in the DRN of knockdown mice (Fig. 4S and T). These results demonstrate that the expression and function of 5-HT2C receptors in DRN glutamatergic neurons critically regulate pain-anxiety comorbidity, likely through mechanisms involving modulation of neuronal excitability.

Fig. 4.

Pharmacological antagonism of DRN 5-HT2C receptors reverses CFA-induced comorbid phenotypes, while cell-specific knockdown of the receptor in glutamatergic neurons further alleviates comorbidity by increasing neuronal activity. A Left: timeline of Saline/RS102221 (5-HT2C receptor antagonist) injection and behavioral tests. Right: representative photomicrographs of needle track. Scale bars: 200 µm. B PWT 1 day after Saline/RS injection. C–F OFT 1 day after Saline/RS injection. C Representative trajectories of OFT exploration. D Total distance. E Time spent in the OF center. F Percentage of distance spent in the OF center. G–I EPMT 1 day after Saline/RS injection. G Representative trajectories of EPMT exploration. H Time spent in the open arms. I Frequency of open arm entries. J Timeline of AAV/Saline/CFA injection, behavioral tests and IF. K PWT 21 days after AAV injection. L–O OFT 21 days after AAV injection. L Representative trajectories of OFT exploration. M Total distance. N Time spent in the OF center. O Percentage of distance spent in the OF center. P–R EPMT 21 days after AAV injection. P Representative trajectories of EPMT exploration. Q Time spent in the open arms. R Frequency of open arm entries. S Representative immunofluorescent images of c-fos expression in DRN glutamatergic neurons. Scale bars: 200 µm. T Quantification of fluorescent intensity of co-labeled (c-fos⁺ + vglut2.⁺) cells. Data are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant. 5-HT2C, 5-hydroxytryptamine receptor 2 C; CFA, Complete Freund’s Adjuvant; CNO, clozapine N-oxide; DRN, dorsal raphe nucleus; EPMT, elevated plus maze test; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IF, immunofluorescent staining; Sal, saline; OFT, open field test; PWT, paw withdrawal thresholds to mechanical stimuli. Further details of statistical data analysis are shown in Additional file 1: Table S1

Pharmacological activation of 5-HT2C receptors in DRN alters the α-diversity and β-diversity of gut microbiota

To further investigate the involvement of 5-HT2C receptors in DRN in mediating the gut-brain axis, we collected fecal samples from mice 24–48 h post-MK212 injection and performed 16S rRNA sequencing analysis. Sankey diagrams revealed significant alterations in the distribution of dominant microbial taxa at both phylum and genus levels following DRN 5-HT2C receptor activation (Fig. 5A). The heat map revealed marked variances in the relative abundances of the top 15 gut microbiota at the genus level between groups (Fig. 6B). While no significant differences were observed in Chao1 estimates between groups (Fig. 5C), MK212-treated mice exhibited lower Shannon indices (Fig. 5D), indicating diminished α-diversity in gut microbiota. Principal coordinates analysis (PCoA) further demonstrated significant β-diversity alterations induced by MK212 treatment (Fig. 5E). These findings collectively suggest that pharmacological activation of 5-HT2C receptors in DRN exerts modulatory effects on gut microbial diversity and compositional architecture.

Fig. 5.

Pharmacological activation of 5-HT2C receptors in the DRN alters gut microbiota diversity and expression profiles. A Sankey diagram of phylum and genus levels (from left to right) between the control and MK212 groups. B Heat map of different genus levels between the control and MK212 groups. C α-diversity represented by Chao 1. D α—diversity represented by Shannon Index. E β-diversity represented PCoA-Bray–Curtis analysis of gut bacteria (PC1 versus PC2). F–Q Relative abundance of phylum Actinobacteriota, phylum Proteobacteria, genus Alistipes, genus Alloprevotella, genus Anaerotruncus, genus Parabacteroides, genus Parasutterella, genus Prevotellaceae NK3B31 group, genus Tuzzerella, species Bacteroides thetaiotaomicron g Bacteroides, species Burkholderiales bacterium g Parasutterella, species Parabacteroides goldsteinii g Parabacteroides. Data are shown as mean ± SEM. n = 10 per group. *P < 0.05, **P < 0.01,***P < 0.001. Con, control group. Data are shown as mean ± SEM. *P < 0.05; ns, not significant. Con, control group. Further details of statistical data analysis are shown in Additional file 1: Table S1

Fig. 6.

Correlations between behaviors and gut bacteria. A The heat map of correlations between behaviors and differential bacteria at different levels. B Correlations between PWT and the relative abundance of phylum Actinobacteriota. C Correlations between center distance in OFT and the relative abundance of phylum Actinobacteriota. D Correlations between PWT and the relative abundance of phylum Proteobacteria. E Correlations between entries in open arms in EPMT and the relative abundance of phylum Proteobacteria. F Correlations between PWT and the relative abundance of genus Alistipes. G Correlations between time in open arms in EPMT and the relative abundance of genus Alistipes. H Correlations between PWT and the relative abundance of genus Tuzzerella. I Correlations between entries in open arms in EPMT and the relative abundance of genus Tuzzerella. J Correlations between PWT and the relative abundance of species Burkholderiales bacterium g Parasutterella. K Correlations between entries in open arms in EPMT and the relative abundance of species Burkholderiales bacterium g Parasutterella. L Correlations between PWT and the relative abundance of Parabacteroides goldsteinii g Parabacteroides. M Correlations between center duration in OFT and the relative abundance of Parabacteroides goldsteinii g Parabacteroides. EPMT, elevated plus maze test; OFT, open field test; PWT, paw withdrawal thresholds to mechanical stimuli. Further details of statistical data analysis are shown in Additional file 1: Table S1

Analysis of gut microbiota abundance and its correlation with comorbid behaviors following 5-HT2C receptor activation in the DRN

We subsequently quantified gut microbiota abundance at phylum, genus, and species levels following MK212 treatment. At the phylum level, MK212-treated mice exhibited decreased Actinobacteriota and increased Proteobacteria compared to controls (Fig. 5F and G). Genus-level analysis revealed reduced Alistipes and Alloprevotella, alongside elevated Anaerotruncus, Parabacteroides, Parasutterella, Prevotellaceae NK3B31 group, and Tuzzerella in the MK212 group (Fig. 5H–N). At the species level, MK212 treatment increased the relative abundance of Bacteroides thetaiotaomicron g acteroides, Burkholderiales bacterium g Parasutterella, and Parabacteroides goldsteinii g Parabacteroides (Fig. 5O–Q).

To establish potential functional correlations between microbial shifts and behavioral phenotypes, we generated a correlation heatmap comparing microbial abundance with normalized pain-anxiety behavioral scores (Fig. 6A). Among the 12 microbial taxa, 3 demonstrated positive correlations (warm color) with pain thresholds and exploratory behaviors (classified as beneficial bacteria), while 9 (cool color) exhibited negative correlations (classified as detrimental bacteria). Subsequent linear regression analysis identified phylum Actinobacteriota (Fig. 6B and C) and genus Alistipes (Fig. 6F and G) as beneficial bacteria strongly associated with behavioral scores. Conversely, phylum Proteobacteria (Fig. 6D and E), genus Tuzzerella (Fig. 6H and I), and species Burkholderiales bacterium g Parasutterella/Parabacteroides goldsteinii g Parabacteroides (Fig. 6J–M) emerged as detrimental taxa exhibiting negative correlations with pain-anxiety indices. These distinct gut microbiota profiles may serve as key intermediaries in the DRN 5-HT2C receptor-mediated pain-anxiety comorbidity (Fig. 7).

Fig. 7.

Graphical abstract. Schematic shows the role of 5-HT2CR in DRN glutamatergic neurons in pain-anxiety comorbidity via microbiota remodeling. 5-HT2CR, 5-hydroxytryptamine receptor 2 C receptor; DRN, dorsal raphe nucleus

Discussion

While chronic pain and anxiety disorders have been extensively investigated as separate entities, the mechanistic underpinnings of their comorbidity remain poorly understood. Here, we demonstrate that DRN glutamatergic neurons drive the comorbidity via a 5-HT2C receptor-dependent mechanism, thereby identifying it as a potential therapeutic target. Emerging evidence reveals a pathogenic synergy between chronic nociceptive sensitization and anxiety-related psychopathology. Recent epidemiological data reveal that 40.2% of adults with persistent nociceptive conditions exhibit validated anxiety symptomatology [3, 5]. In this study, behavioral analysis confirmed that the widely used CFA-induced chronic inflammatory pain model concurrently exhibits anxiety-like behaviors in mice. While the conventional understanding places the critical window for anxiety-like behaviors at 7–14 days post-CFA, their emergence as early as 1–3 days has been documented [29–31], consistent with our prior observation of comorbidity [6]. This study therefore selected the 3-day timepoint to investigate pain-anxiety comorbidity. It is also noteworthy that in the assessment of anxiety-like behaviors using the OFT and EPMT, no change was observed in the total distance. This finding effectively excludes the potential interference of pain-induced hypoactivity or generalized motivational deficits with the reductions in center entries or time spent in the open arms, thereby allowing for a clearer interpretation of the behavioral phenotype associated with pain-anxiety comorbidity.

The neural circuitry mechanisms underlying pain-anxiety comorbidity involve dynamic imbalances across multiple brain regions [32–37]. Emerging evidence highlights the DRN as a critical hub. The DRN integrates nociceptive signals via multi-receptor systems [38–41], neuroimmune interactions [42], and circuits [43], while its engagement in anxiety pathogenesis involves complex neurotransmitter crosstalk and molecular pathways [10, 38, 44, 45]. Regarding comorbidity, electroacupuncture alleviates CFA-induced anxiety by reversing anterior cingulate cortex (ACC)–DRN glutamatergic circuit dysfunction [12], while co-activation of ventrolateral periaqueductal gray (vlPAG)–DRN GABAergic neurons exacerbates mechanical hypersensitivity and anxiety [46]. Our studies revealed increased neuronal excitability (c-fos⁺) in DRN under CFA-induced comorbidity, and chemogenetic activation of DRN neurons induces comorbid-like behaviors in naïve mice. While serotonergic and GABAergic neurons dominate current research, DRN glutamatergic subpopulation remain unexplored. Recent studies reveal that VGluT3^DRN—DA^VTA activation alleviates neuropathic pain and comorbid anhedonia via glutamate release [11], suggesting critical roles of DRN glutamatergic projections in pain-negative affect comorbidity. Our results from cell subtype localization and bidirectional chemogenetic regulation further indicate that glutamatergic neurons, rather than 5-HT or GABAergic neurons, contribute to pain-anxiety comorbidity behaviors. This regulatory role of non-serotonergic neurons in the DRN represents a substantial addition to the current understanding in this field.

Serotonin (5-HT) receptors are widely distributed throughout the central nervous system (CNS) and peripheral tissues, playing crucial roles in various physiological and pathological processes [47, 48]. The 5-HT2C receptor subtype, highly expressed in the CNS, is pivotal in negative affect and pain perception [15, 49]. Studies indicate that 5-HT2C receptor antagonism enhances the antidepressant and anxiolytic effects of selective serotonin reuptake inhibitors (SSRIs) [50]. Local knockdown of 5-HT2C receptors in the basal lateral amygdala reduced anxiety- and depression-like behaviors and elevated sensory thresholds in spinal nerve ligation (SNL) rats [51]. These findings align with our experimental data: intracerebral 5-HT2C receptor agonist administration induced pain-anxiety comorbidity in naïve mice, while pharmacological inhibition of 5-HT2C receptors reversed CFA-induced comorbid phenotypes. Notably, our study found that a single intracerebral administration of the 5-HT2C receptor agonist MK212 or antagonist RS102221 could still alter pain-anxiety comorbidity behaviors in mice 24 h later. Since the direct central pharmacological action of such drugs typically lasts only a few hours, we speculate that these prolonged effects are likely mediated by neural plasticity changes within the DRN or its related projections, along with associated signaling pathway adaptations rather than acute pharmacological actions. Studies [52] have shown that hippocampal microinjection of a 5-HT2C receptor antagonist can depolarize the resting membrane potential, reduce rheobase and medium afterhyperpolarization (mAHP), and increase the action potential (AP) half-width, thereby modulating neuronal excitability, altering neurotransmitter release, and influencing emotion-related behaviors. Similarly, microinjection of MK212 or RS102221 into the DRN may induce changes in 5-HT2C receptor-related signaling pathways [53], leading to alterations in the electrophysiological properties of relevant neurons in the DRN. This could subsequently cause long-term adaptations in downstream projections [10, 54], thereby resulting in the long-term regulation of pain-anxiety comorbidity. Furthermore, systemic administration of MK212 has been reported to reverse stress-induced impairment of hippocampal long-term potentiation (LTP) [55], and modulation of signaling pathways in the DRN can partially restore impaired LTP maintenance in the hippocampus of anxious mice [56]. Thus, the change of DRN-related LTP plasticity may also contribute to the observed long-term behavioral effects. Interestingly, systemic administration of either MK212 or RS102221 at 1 mg/kg did not alter pain- or anxiety-related behaviors in mice, which is consistent with a previous report [57]. The study noted that intraperitoneal injection of a high dose of MK212 (4.0 mg/kg) suppressed locomotor activity, while a moderate dose (2.0 mg/kg) induced anxiety-like behaviors, suggesting that the behavioral effects of 5‑HT2C receptor agonists or antagonists may be dose-dependent.

In addition to pharmacological modulation of DRN 5-HT2C receptor function, bidirectional manipulation of receptor expression on glutamatergic neurons in the DRN also influences comorbid behaviors. More importantly, previous studies have reported that dysregulation of 5-HT2C can alter the excitability of neurons [49, 51, 58]. Our findings further demonstrate that overexpression of 5-HT2C receptors on DRN glutamatergic neurons enhances neuronal excitability, whereas targeted knockdown attenuates CFA-induced hyperexcitability. We therefore propose that 5-HT2C receptors may bidirectionally regulate comorbid behaviors likely through modulating excitability changes in DRN glutamatergic neurons.

Emerging evidence from gut-brain axis research has established a critical link between gut microbiota and neurological functions [19, 20, 59]. However, whether gut microbiota links pain and anxiety to mediate their comorbidity remains unclear. Our study demonstrates that in a pharmacologically induced pain-anxiety comorbidity model, the abundance of multiple gut microbiota species consistently correlates with both pain and anxiety behaviors in mice. Beyond microbial ecosystem balance, these alterations in gut microbiota may influence pain-anxiety comorbidity through neuroimmune modulation and synaptic regulation via bacterial metabolites such as short-chain fatty acids (SCFAs) [60, 61]. In addition, alterations in microbial abundance induced by DRN 5-HT2C receptor activation may subsequently influence feeding [62], stress [63], sex hormone [64], and inflammatory responses [65], ultimately modulating brain function. Notably, alterations in gut microbiota and comorbid behaviors persisted 24 h after MK-212 administration. We speculate that 5-HT2C receptor modulators may initially act centrally to modulate the autonomic nervous system and hypothalamic–pituitary–adrenal (HPA) axis, thereby inducing changes in the intestinal environment and microbial composition. The altered microbiota might subsequently generate metabolites and signaling molecules that feedback to central functions via circulation, establishing a bidirectional gut-brain communication loop. Further validation through gut microbiota-targeting experiments, such as antibiotic treatment, fecal microbiota transplantation, and vagotomy, is necessary.

This study has several limitations. Firstly, while our pharmacological findings demonstrate long-term regulatory effects of 5-HT2C receptor modulation on comorbidity behaviors, whether acute pharmacological actions contribute to these sustained effects remains unexplored. Secondly, although numerous studies [66–70], like ours, have employed a vglut2 promoter to restrict viral expression to glutamatergic neurons, this approach still carries risks of nonspecificity due to factors such as uncontrolled promoter leakage. Therefore, further experiments are needed to more definitively characterize promoter specificity and solidify the conclusions regarding the role of DRN glutamatergic neurons. Thirdly, current understanding of the role of gut microbiota remains at a relatively preliminary stage. To fully establish that DRN glutamatergic neuronal receptors bidirectionally regulate pain-anxiety comorbidity via the gut-brain axis, future experiments should incorporate extensive gut-to-brain explorations, including microbiota transplantation, serotonergic depletion, or vagus nerve transection.

Conclusions

Our study suggests that hyperactivation of DRN glutamatergic neurons is pivotal to pain-anxiety comorbidity, as evidenced by bidirectional chemogenetic manipulation. Importantly, 5-HT2C receptors on these neurons are significantly upregulated under comorbid conditions and bidirectionally modulate neuronal excitability and behavioral deficits. These findings suggest 5-HT2C receptors in DRN glutamatergic neurons as a dual therapeutic target for pain-anxiety comorbidity.

Abbreviations

5-HT

5-Hydroxytryptamine

5-HT2C

5-Hydroxytryptamine 2C

5-HTR

5-Hydroxytryptamine receptor

ACC

Anterior cingulate cortex

CFA

Complete Freund’s Adjuvant

CNO

Clozapine-N-oxide

CNS

Central nervous system

DA

Dopamine

DRN

Dorsal raphe nucleus

EPMT

Elevated plus maze test

GABA

Gamma-aminobutyric acid

HPA

Hypothalamic-pituitary-adrenal

IF

Immunofluorescent staining

LTP

Long-term potentiation

MC4R

Melanocortin-4 receptor

MOR

Mu-opioid receptor

OFT

Open field test

PCoA

Principal coordinates analysis

PWL

Paw withdrawal latency

PWT

Paw withdrawal thresholds

SCFAs

Short-chain fatty acids

SNL

Spinal nerve ligation

SSRIs

Selective serotonin reuptake inhibitors

vgat1

Vesicular gamma-aminobutyric acid transporter1

vglut2

Vesicular glutamate transporter 2

vlPAG

Ventrolateral periaqueductal gray

VTA

Ventral tegmental area

Authors’ contributions

QZ, LX and CZ: Conceptualization, Project administration, Methodology, Formal analysis, Visualization, Writing–original draft. SY and XZ: Investigation, Project administration, Writing–original draft. YW and ZW: Investigation, Writing–original draft, Visualization. CH, DW and LY: Investigation, Validation, Software, Writing–original draft. CY, SH and RJ: Investigation, Conceptualization, Resources, Writing & editing, Supervision. All authors read and approved the final manuscript.

Funding

This study was supported by grants from the National Natural Science Foundation of China (82571395, and 82271254 to C.Y., 82301444 to Q.Z., 82401453 to S.H., 82401469 to X.Z., 82191279 to C.H., 82201420 to D.W.), Innovative and Entrepreneurial Team of Jiangsu Province (JSSCTD202144 to C.Y.), Natural Science Foundation of Jiangsu Province (BK20240054 to C.Y., BK20230741 to S.H., BK20210975 to C.H.), Excellent postdoctoral program of Jiangsu Province (2023ZB599 to S.H.), China Postdoctoral Science Foundation (2023M731409 to S.H., 2023M741467 to Q.Z.) and Wu Jieping Medical Foundation (320.6750.2024-15-81 to Q.Z., 310.6750.2024-15-82 to S.H.).

Data availability

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

Declarations

Ethics approval and consent to participate

All procedures strictly followed the Guide for the Care and Use of Laboratory Animals and received approval (No. IACUC-2407061) from the Institutional Animal Care and Use Committee of Nanjing Medical University, Nanjing, Jiangsu.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.