A primary auditory cortex-anterior cingulate cortex circuit underlying cross-modal visceral pain modulation

Domain: neuroscience
Published: 2026-02-04
Editorial outcome: minor_revision

Abstract
Visceral pain represents an unmet clinical need due to the lack of safe and generalizable therapies. Given the analgesic potential of music and the underlying crosstalk between auditory and nociceptive pathways, this study aims to delineate the neural circuits interfacing auditory processing and visceral pain modulation. Using a mouse model of early-life stress, we identify a cortico-cortical circuit linking the primary auditory cortex (Au1) to the anterior cingulate cortex (ACC) that mediates stress-induced visceral pain. Mechanistically, early-life stress suppresses the activity of Au1 inhibitory (GABAergic, Au1GABA) neurons, which disinhibits downstream ACC excitatory (glutamatergic, ACCGlu) neurons, thereby driving visceral hypersensitivity. Notably, music intervention alleviates visceral pain by restoring Au1GABA neuronal activity and suppressing ACCGlu activity. In conclusion, this study unveils the Au1-ACC circuit as a critical hub for stress-induced visceral hypersensitivity and music-induced analgesia, providing a mechanistic foundation for clinical translation of music therapy in visceral pain management.

Introduction
Visceral pain represents a debilitating clinical manifestation associated with a variety of gastrointestinal disorders, affecting more than 20% of the global population and reducing their quality of life1. Despite three decades of research on functional gastrointestinal disorders, particularly irritable bowel syndrome (IBS) characterized by visceral hypersensitivity, the underlying pathogenesis remains incompletely understood, largely due to the inherent heterogeneity of these conditions. Therefore, current therapeutic options are limited and narrowly targeted, such as guanylate cyclase-C agonists linaclotide and plecanatide, 5-HT receptor agonist tegaserod for constipation-predominant IBS (IBS-C), opioid receptor agonist eluxadoline, 5-HT3 receptor antagonist alosetron, and antibiotic rifaximin for diarrhea-predominant IBS (IBS-D)2,3. The limitations of these medications are attributed to their adverse side effects and single-target mechanisms, which consequently restrict their applicability to only a small sub-population of IBS patients. For instance, alosetron was withdrawn due to severe constipation and ischemic colitis; although reintroduced later, its use is strictly limited to female IBS-D patients4. This highlights a significant lack of safe and generalizable treatments for visceral pain management.

The Rome IV criteria introduced in 2016 redefined functional gastrointestinal disorders as disorders of gut-brain interaction, reflecting the evolving understanding of their neurobiological underpinnings. Emerging research now explores central neuromodulation approaches, such as sound, light, electrical and magnetic interventions, in attempts to modulate pain perception. Music therapy has been widely applied to improve mental health, cognitive function and physical wellbeing, given its cost-effectiveness, ease of use and lack of side-effects. Although clinical studies have demonstrated the efficacy of music intervention in alleviating various forms of pain5,6, the underlying neurobiological mechanisms remain elusive, and its specific effects on visceral pain are yet to be revealed.

Sensory hypersensitivity represents a common clinical manifestation across various neuropsychiatric disorders such as autism spectrum disorder, post-traumatic stress disorder, fibromyalgia, sensorineural hearing loss and migraine7. Notably, striking neurological parallels have been noticed between auditory and nociceptive hypersensitivity. Tinnitus (auditory perception occurring without external sounds) and hyperacusis (increased sensitivity to acoustic stimuli) are highly analogous to allodynia and hyperalgesia in pain hypersensitivity. Intriguingly, both auditory and pain hypersensitivity can be triggered by stress8–11, and a clinical correlation between tinnitus and chronic pain has been documented12,13. Furthermore, IBS patients exhibit hyperactivity to auditory stimuli14,15, and conversely, IBS is one of the comorbid conditions of hyperacusis16. This neurobiological convergence implies potential crosstalk between the auditory pathway and the visceral pain pathways. The primary auditory cortex (Au1), serving as the central hub for auditory processing, exhibits functional connectivity across the brain networks to regulate diverse physiological functions. Notably, clinical and preclinical evidence has revealed functional and neuroanatomical disturbance in the auditory cortex of depression patients and preclinical chronic mild stress model17, implying its unexplained role in stress response integration.

This study employed an early-life stress model, neonatal colonic inflammation (NCI), to recapitulate stress-induced visceral hypersensitivity in IBS, and aimed to investigate the role of Au1 in stress response and visceral pain modulation, and to identify the neural circuits linking auditory processing and visceral pain modulation. The findings of this study will shed light on the neurobiological mechanisms underlying cross-modal visceral pain modulation, and expand current therapeutic strategies for visceral pain management.

Results
NCI-induced suppression of Au1GABA neurons drives visceral hypersensitivity
The involvement of Au1 neurons in somatosensory representation remains unclear. An electrophysiological study indicates that Au1 neurons barely respond to tactile stimulation alone, with only approximately 11.6% responding to combined auditory and tactile stimuli18, suggesting limited involvement of the Au1 in direct processing of somatosensory inputs. To determine whether Au1 neurons directly encode visceral nociceptive stimuli, we used in vivo fibre photometry recording to assess their calcium responses to CRD stimuli. In agreement with prior research, our findings revealed that both Au1GABA and Au1Glu neurons exhibited minimal responsiveness to CRD (Fig. 1A–D), suggesting a marginal engagement of the Au1 in direct encoding of visceral pain.

An early-life stress model, NCI (Fig. 1E), was employed to investigate the role of Au1 in stress response integration and visceral pain modulation. Compared to CON mice, visceral pain responses to CRD stimulation were significantly increased in adolescent NCI mice (Fig. 1F&G). To examine the role of Au1 in stress-induced visceral hypersensitivity, spontaneous neuronal activity in the Au1 from adolescent NCI and CON mice was assessed using c-Fos immunofluorescence. The number of c-Fos+ neurons in the Au1 of NCI group was significantly higher than CON group (Fig. 1H&I). To further identify the neuronal subtypes involved in this change, c-Fos colocalization with neuronal marker gamma-aminobutyric acid (GABA, Fig. 1N) or glutamate (Glu, Fig. 1O) was quantified. NCI mice exhibited fewer c-Fos++GABA+ neurons (Fig. 1J) and more c-Fos++Glu+ neurons (Fig. 1K) in the Au1. The proportion of GABAergic neurons in c-Fos+ neurons was decreased in NCI mice (Fig. 1L), while the proportion of glutamatergic neurons was not significantly changed (Fig. 1M), suggesting that Au1GABA may be more vulnerable to early-life stress.

To further determine the role of Au1GABA in stress-induced visceral hypersensitivity, optogenetic and chemogenetic techniques based on AAV vectors were utilized to selectively manipulate the excitability of Au1GABA (Fig. 1P&U). Blue light-induced activation of Au1GABA significantly attenuated visceral pain responses in NCI mice (Fig. 1Q&R) but not CON mice (Fig. 1S&T). In addition, chemogenetic inhibition of Au1GABA on naïve mice significantly reduced visceral pain threshold since Day 1 after CNO injection (Fig. 1U-W). These data suggest that suppression of Au1GABA caused by early-life stress drives visceral hypersensitivity.

Mapping the downstream nuclei of Au1 in visceral pain modulation
Although the Au1 contributed minimally to the encoding of visceral pain, it may shape pain perception by tuning relevant neural network through its projections. Indeed, a recent study has demonstrated that the auditory cortex dampens somatic pain via its descending projections to the thalamus19. To map the downstream projections of Au1, anterograde tracing was performed with AAV2/9-hSyn-EGFP and density of EGFP fluorescence was quantified using ImageJ software (Fig. 2A–C). Axons originating from the Au1 were observed throughout the brain, with prominent innervation in the auditory pathways (medial geniculate nucleus, MGB; inferior colliculus, IC), cerebral cortex (temporal association cortex, TeA; secondary visual cortex lateral area, V2L; contralateral Au1; ACC; insular cortex, ICx;), basal ganglia (caudate putamen, CPu; basolateral amygdaloid nucleus, BLA; claustrum, Cl) and thalamus (ventral posterior thalamic nucleus, VP; posterior thalamic nucleus, PO). Some of these regions have been reported to be implicated in visceral pain modulation by our previous studies, such as the ACC, Cl, ICx, and BLA20–22. Among them, the ACC received relatively more extensive projections from the Au1 (Fig. 2B–C). Furthermore, monosynaptic anterograde tracing using a Cre-DIO system revealed direct projections from the Au1 to the ACC (Fig. 2D). Therefore, we focused on the ACC as a presumed downstream effector of Au1-mediated visceral pain modulation.

ACCGlu neurons encode visceral hypersensitivity in NCI mice
Colocalization analysis indicated that CRD-activated ACC neurons were predominantly glutamatergic (Fig. 2E–G). To examine the functional role of ACCGlu neurons in visceral hypersensitivity, they were optogenetically activated or inhibited in CON and NCI mice, respectively (Fig. 2H). Blue light-induced activation of ACCGlu in CON mice significantly increased visceral pain responses (Fig. 2I–K). Conversely, yellow light-induced inhibition of ACCGlu in NCI mice significantly mitigated visceral pain responses (Fig. 2L–N). In addition, optogenetic inhibition of ACCGlu in CON mice did not change visceral pain responses significantly (Fig. S2A–C). These results collectively demonstrate that ACCGlu neurons play a crucial role in visceral hypersensitivity.

Au1GABA neurons mediate visceral analgesia through enhanced GABAergic output to ACCGlu in synergy with attenuated glutamatergic transmission via Au1Glu
As we have demonstrated the essential role of Au1GABA and ACCGlu in stress-induced visceral hypersensitivity, we further investigated the involvement of the Au1GABA-ACCGlu neural circuitry. Monosynaptic retrograde tracing using RV was performed to identify presynaptic inputs to ACCGlu (Fig. 3A&B). Colocalization analysis of RV+ neurons in the Au1 identified approximately 53.8% as glutamatergic neurons and 28.5% as GABAergic (Fig. 3C–E), suggesting that both Au1GABA and Au1Glu projected to ACCGlu. While emerging evidence has documented the existence of cortico-cortical long-range GABAergic projections, their functional significance remains inadequately explored. To functionally validate our observation from RV tracing, in vivo extracellular recording using optical electrode arrays was performed to record neural activity in conjunction with optogenetic manipulation of Au1GABA projections in the ACC (Fig. 3F). 11 in 71 recorded ACC neurons responded to blue light stimulation with their firing rate attenuated by optogenetic activation of Au1GABA axons in the ACC. This effect was abolished in the presence of a GABAA receptor antagonist bicuculline (1 mg/kg, IP injection, Fig. 3G–H), confirming that the inhibitory effect of blue light was mediated via GABAA receptors and thus indicating the functional existence of GABAergic projections from the Au1 to the ACC. To examine which pathway contributed to modulation of visceral pain, optogenetic manipulation was conducted targeting axonal terminals of Au1GABA and Au1Glu in the ACC, respectively. Optogenetic activation of Au1GABA axonal terminals projecting to the ACC alleviated visceral pain responses in NCI mice (Fig. 3I-K). Interestingly, optogenetic inhibition of axonal terminals in the ACC originating from Au1Glu was used in subsequent experiments unless otherwise stated. To examine whether music induces stress responses in mice, elevated plus maze and open field test were performed to assess the effect of music on anxiety-like behaviours. Compared to CON mice, NCI mice exhibited reduced entries into the open arm, total distance travelled and mean speed, whereas a brief music intervention (15 min) did not alter these anxiety-like behaviours (Fig. S5A–H). In addition, 15-min music intervention did not change serum corticosterone levels (Fig. S5I–J).

Consistent with elevated visceral pain threshold, music significantly attenuated EMG responses to graded CRD stimulation in NCI mice (Fig. 4A–C), while had no effect on CON mice (Fig. 4D–E). To assess the effect of music on Au1 neural activity, in vivo extracellular recording was performed using microelectrode arrays (Fig. 4F). Compared to CON mice, spontaneous firing rate in the Au1 was elevated in NCI mice, and music attenuated Au1 neural activity in NCI mice only (Fig. 4G–H). Given our findings of suppressed Au1GABA activity in NCI mice (Fig. 1L), the impact of music on Au1GABA was assessed using immunofluorescence. Quantitative analysis revealed that music significantly increased the proportion of GABA+ neurons among c-Fos+ neurons in the Au1 of NCI mice (Fig. 4N) while concurrently reducing the ratio of Glu+ neurons in c-Fos+ neurons (Fig. 4O), indicating restoration of the excitation-inhibition balance. This finding aligns with the overall suppression of neural activity during music exposure observed in above electrophysiological experiment. Although music increased activation of both GABAergic and glutamatergic neurons in CON animals (Fig. 4L–M), their equilibrium was not altered (Fig. 4N–O). In addition, neither the NCI stress nor music intervention altered the density of GABA+ neurons and Glu+ neurons (Fig. S6). To functionally verify the role of Au1GABA in music-induced visceral analgesia, AAV vectors with or without yellow light-sensitive eNpHR3.0 were injected into the Au1 of NCI mice (Fig. 4P). In NCI mice injected with control virus, yellow light did not affect music-induced visceral analgesia (Fig. 4Q–R). On the contrary, in NCI mice infected with AAV containing eNpHR, silencing of Au1GABA by yellow light abolished music’s analgesic effect (Fig. 4S–T). These data suggest that music attenuates visceral hypersensitivity by enhancing Au1GABA activity.

Music induces visceral analgesia in NCI mice by suppressing ACCGlu activity
The effect of music on ACC neuronal activity was assessed using c-Fos immunofluorescence. Compared to CON mice, NCI mice exhibited more prominent CRD-induced c-Fos expression in the ACC, which was effectively attenuated by music intervention in NCI but not CON mice (Fig. 5A&B). To further investigate the contribution of ACCGlu neurons to music-induced visceral analgesia, calcium responses of ACCGlu to CRD were assessed using in vivo fibre photometry (Fig. 5C&D). Music significantly attenuated calcium responses to CRD in ACCGlu, as evidenced by reduced AUC and peak of ΔF/F (Fig. 5E–H), whereas no significant effect was observed in CON mice. In agreement with calcium signals, ACC neuronal firings in response to CRD were significantly attenuated by music (Fig. 5I–K). Furthermore, the role of ACCGlu in music-induced visceral analgesia was verified by optogenetic activation of ACCGlu in EMG recordings (Fig. 5L). In NCI mice infected with the control virus lacking blue light-sensitive hChR2, music alleviated CRD-evoked visceral pain responses (Fig. 5M&N). Conversely, in NCI mice injected with AAV vectors containing hChR2, blue light activation of ACCGlu abolished music-induced visceral analgesia (Fig. 5O&P). These observations suggest that music mediates visceral analgesia by suppressing the hyperactivity of ACCGlu.

Music induces visceral analgesia in female NCI mice by modulating the Au1-ACC circuit
To ascertain whether the sex of mice influences music’s analgesic effect on visceral hyperalgesia, we replicated the NCI protocol in female mice (Fig. S7A). As in males, female NCI mice exhibited lower visceral pain threshold than their CON littermates, and music intervention elevated these thresholds in female NCI but not CON mice (Fig. S7C). Furthermore, chemogenetic activation of ACCGlu neurons receiving Au1 input reversed music’s analgesic effect (Fig. S7C). Taken together, music alleviates visceral pain in female mice by modulating the same Au1-ACC circuit as males.

Au1-ACC circuit mediates music-induced somatic analgesia
While our findings thus far have demonstrated that music therapy mitigates visceral pain through modulation of the Au1-ACC circuit, a pivotal question remains: whether this is selective for the visceral pain modality. To strengthen the translational relevance of our study, we adopted the Complete Freund’s Adjuvant (CFA)-induced hind-paw inflammatory pain model for further investigation of somatic pain. We first compared the analgesic dynamics of acute and chronic music interventions between the two modalities. A single 15-min bout of classical music exposure increased visceral pain threshold in NCI mice, and this effect sustained more than 60 min (Fig. 6A); whereas identical acute music intervention increased paw withdrawal threshold in CFA mice after intervention, which diminished within 30 minutes (Fig. 6C). Chronic paradigm consisted of 3 daily sessions of 15-min classical music exposure, with pain thresholds assessed prior to music intervention in the first 3 days. Visceral analgesia emerged on Day 2 and 3 (Fig. 6B), whereas somatic analgesia was only present on day 1 (Fig. 6D). Although the two pain modalities may not be directly comparable, our observations suggest that music appears to produce a more sustained analgesic effect on visceral pain than somatic pain.

To investigate the involvement of Au1GABA neurons and the Au1-ACC circuit in somatic pain modulation, chemogenetic approaches were used to manipulate Au1GABA neurons and ACCGlu neurons receiving Au1 projections (Fig. 6E&H). Chemogenetic activation of Au1GABA neurons mimicked the analgesic effect of music in CFA mice (Fig. 6G). Consistently, chemogenetic activation of ACCGlu neurons innervated by the Au1 reversed music’s analgesic effect (Fig. 6J). These data suggest that the Au1-ACC circuit also mediates music-induced somatic analgesia.

To determine whether music possesses analgesic efficacy in animals subjected to combined early-life and adult stress, CFA injection was performed on NCI and CON mice at 6 weeks of age (Fig. 6K). Only the NCI+CFA group exhibited visceral hyperalgesia, which was relieved by music therapy (Fig. 6L). Conversely, music attenuated somatic pain in both NCI+CFA and CON+CFA mice (Fig. 6M). Taken together, these data suggest that music exerts analgesic efficacy across different pain modalities.

Methods
Animals and ethical approval
Male and female C57BL/6J mice were purchased from the Charles River Laboratories (Beijing, China), and housed at the Experimental Animal Centre of Soochow University under standard conditions (12-h light/dark cycle, temperature of 23–25°C and humidity of 40–60%) with free access to food and water. Mice were given one week to acclimate to the new housing environment prior to any procedures. Male and female mice used in all experiments were 6–10 weeks old and weighed 20–25 g. All experiments were approved by the Animal Care Committee of Soochow University, and conducted in accordance with the guidelines of the International Association for the Study of Pain.

Neonatal colonic inflammation
An early-life stress model, NCI, was used to induce chronic visceral pain as described previously21. Briefly, on postnatal day 10, male or female mouse pups were randomized into NCI and CON groups, and injected with 30 μL of 0.5% acetic acid (diluted in saline) or equal volume of saline, respectively, into the colorectum 1 cm from the anus. Subsequent experiments commenced when the animals reached 6 weeks of age.

Abdominal withdrawal reflex (AWR)
AWR in response to colorectal distension (CRD) was used to evaluate visceral pain threshold21. A homemade balloon (1 cm in length) was lubricated with vaseline and gently inserted into the colorectum of anesthetized mice, with the end of the balloon positioned 0.5 cm from the anus. The catheter of the balloon was securely attached to the base of mouse’s tail, and mice were then placed under transparent acrylic restrainers to acclimate for 1 h. During the AWR test, the balloon catheter was connected to a sphygmomanometer, and the balloon was slowly inflated at a rate of 5 mmHg/sec until a sign of visceral pain (abdominal lifting or back arching) was observed or until a maximum pressure of 40 mmHg was reached. This procedure was repeated five times with 5-min intervals. The readings of distension pressure from the sphygmomanometer were recorded and averaged to determine the colorectal visceral pain threshold.

Electromyography (EMG) recording of visceromotor responses
Electrodes were implanted one week prior to EMG recording. After deep anesthesia induced by isoflurane, the abdominal skin of the mouse was carefully incised to expose underlying muscle tissue. Two electrode wires were implanted into the external oblique muscles on both sides of the abdomen, and were threaded subcutaneously to exit at the neck. The surgical incision was subsequently closed with 5-0 silk suture. Following 1-week recovery, mice were acclimatized in a restrainer for 1 h with a balloon inserted into the colorectum as described above. EMG activity was recorded via MP160 Data Acquisition System and digitized using AcqKnowledge software (BIOPAC Systems Inc., Goleta, CA, USA). EMG responses to graded CRD stimulation (20, 40, 60 and 80 mmHg in duplicate) were examined, with each distension lasting 10 s and a 3-min rest period in between. EMG responses to CRD were analysed as the area under the curve (AUC) of EMG activity during distension (10 s) subtracted by the AUC of baseline immediately prior to distension.

Acoustic interventions
A variety of music genres were chosen from the literature, including classical music (Mozart’s Sonata for Two Pianos, K. 44861), rock music (“Still Loving You” by the Scorpions62) and electronic music (“Breathe” by the Prodigy63). In addition, radio noise (static between radio stations) was used as white noise64. All acoustic interventions were transposed up by one octave (the frequency of each note is doubled) using GarageBand (Version 10.4.7, Apple Inc., Cupertino, CA, USA) in order to better fit the hearing range of mice (1–100 kHz48). Detailed acoustic parameters and frequency distribution of interventions are shown in Table S1 and Fig. S1. All interventions were delivered via a portable audio player on a constant loop with sound pressure level at 50–55 dB in an environment with ambient noise level at 40-45 dB. Additionally, a brief cat meow (75 dB) stimulus was used as a positive control in fibre photometry.

Immunofluorescence
Mice were anaesthetized with xylazine (10 mg/kg, IP injection) and perfused transcardially with saline followed by 4% paraformaldehyde (PFA) in PBS. The brain specimens were carefully removed, post-fixed overnight in 4% PFA and dehydrated successively in 20% and 30% sucrose at 4°C. The brains were embedded in OCT and sectioned into 30 μm-thick coronal slices using a Leica CM3050 S Cryostat (Leica Biosystem, Nussloch, Germany). These slices were collected onto adhesive slides (CITOTEST 188105) and stored at −20°C. For immunofluorescence, the slices were rinsed three times with PBS (each lasting 10 min) and incubated in a blocking buffer (7% normal donkey serum and 0.3% Triton X-100 in PBS) for 1 hour at room temperature. The slices were then incubated with primary antibodies (1:300 dilution) at 4°C for 24 h. Following three-time PBS washes, the sections were incubated with secondary antibodies (1:500 dilution) for 1 h at room temperature in the dark. After three additional PBS washes, the sections were mounted in a medium containing DAPI (Abcam, AB104139). Images were acquired using a fluorescent microscope (BX51, Olympus Corporation, Tokyo, Japan). For quantitative assessment and colocalization analysis, putative cell fragments and dim fluorescence not in the focal plane were excluded. When assessing c-Fos immunofluorescence following CRD or music stimulation, transcardiac perfusion was performed 90 min post-stimulation. Specifically, CRD was applied at 60 mmHg pressure for 5 times, each lasting 20 s separated by 3-min intervals; classical music was played continuously for 30 min following the same protocol as established in other experiments. To enhance the colorblind-friendliness of the fluorescent images, red-green contrast was adjusted to red-cyan or magenta-green colour schemes where necessary.

Stereotaxic surgery
Stereotaxic surgery was performed as previously described65,66. Mice were anesthetized with xylazine (10 mg/kg, IP injection), and firmly secured in a stereotaxic apparatus (RWD, 71000-M, Shenzhen, China). Following disinfection, a precise incision was made in the skull to expose the targeted brain regions (Au1 coordinates: AP −2.6 mm, ML −4.15 mm, DV −2.35 mm; ACC coordinates: AP 1.1 mm, ML −0.35 mm, DV −1.7 mm). Using a microsyringe pump (Legato 130, KD Scientific, Holliston, MA, USA), 200 nL of virus was injected into the targeted regions at a flow rate of 25 nL/min. The needle remained in situ for 15 min to ensure optimal diffusion. All viral injections were performed unilaterally in the right hemisphere of the mouse brain unless otherwise stated. The adeno-associated virus (AAV) and rabies virus (RV) were purchased from BrainVTA (as listed in Table S2).

For fibre photometry recordings and optogenetic manipulation, a ceramic insert (O.D. 200 μm, NA 0.37, length 2.5 mm, ThinkerTech Nanjing Bioscience, Nanjing, China) was implanted into the desired region, ensuring that the tip of the optical fibre was situated 100–200 μm above the viral injection site. This ceramic insert was stabilized on the skull using a cranial nail and dental cement. For local drug administration in the brain, a guide cannula (O.D. 0.48 mm, I.D. 0.34 mm, length 2.5 mm, RWD, Shenzhen, China) was implanted into the desired region. To prevent blockage, a mating cannula (O.D. 0.30 mm, I.D. 0.14 mm, RWD, Shenzhen, China) was inserted into the guide cannula.

Neural tracing
Anterograde tracing was performed via stereotaxic injection of AAV2/9 or AAV2/1 into targeted regions. Monosynaptic retrograde tracing using RV required a two-step injection process: AAV helpers were first injected to express RV glycoprotein and tumour virus A (TVA) receptors, followed by injection of RV-ΔG 3 weeks later. Images of entire coronal slices were acquired by a slide scanner (SLIDEVIEW VS200, Olympus Corporation, Tokyo, Japan). Fluorescence density was analysed using ImageJ software (Version 1.53k, National Institutes of Health, Bethesda, MD, USA). Each experiment was repeated on 3–5 mice.

Fibre photometry recording
In vivo fibre photometry was utilized to record calcium signals of selected neurons using the fluorescent calcium indicator GCaMP6s, which was delivered to targeted regions via AAV vectors. Three weeks after AAV injection and optical fibre implantation, GCaMP6s was excited by a 470-nm laser, with emitted fluorescence captured using a fibre photometry recording system (ThinkerTech Nanjing Bioscience Inc., Nanjing, China). Calcium responses to brief CRD or acoustic stimuli in conscious mice were analysed based on changes in fluorescence (ΔF/F) using custom MATLAB scripts (MATLAB R2017b, MathWorks, Natick, MA, USA). ΔF/F over the 2 s prior to the stimulation (−2 to 0 s) served as the baseline, and ΔF/F during 5 s after the onset of stimulation (0 to 5 s) was used to assess the responses.

Optogenetic and chemogenetic manipulation
To enable optogenetic manipulation of neuronal excitability, targeted neurons were engineered to express light-sensitive proteins via stereotaxic injection of AAV vectors 3 weeks prior to experiments. Humanized channelrhodopsin-2 (hChR2) and enhanced Natronomonas pharaonis halorhodopsin 3.0 (eNpHR3.0) can be activated by blue or yellow light to depolarize or hyperpolarize neurons, respectively. A laser source (Optogenetics-LED-STSI, Prizmatix Ltd., Holon, Israel) connected to an AlphaLab SnR Stimulation and Recording system (Alpha Omega Engineering, Nazareth Illit, Israel) was employed to deliver 5 mW yellow light (589 nm, continuous) or 3 mW blue light (473 nm, 20 Hz, 10 ms pulse width). Yellow light was continuously applied 15 min prior to CRD tests and sustained until the completion of graded CRD stimulation protocol (20, 40, 60 and 80 mmHg in duplicate); in contrast, blue light was synchronized with each 10-s distension only. Similarly, for chemogenetic manipulation, genetically engineered Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) were delivered to targeted regions via AAV vectors 3 weeks prior to experiments. Human M3 muscarinic DREADD receptor coupled to Gq (hM3Dq) and human M4 muscarinic DREADD receptor coupled to Gi/o (hM4Di) were activated by the chemical ligand CNO (3.33 mg/kg, IP injection, CNO-02, BrainVTA). Visceral pain was assessed 30 minutes after CNO injection.

Microelectrode Array
In vivo neural activity was recorded using microwire electrode arrays and CereCube High-Throughput Neural Signal Acquisition System (NeuroXess, Shanghai, China). Electrodes were implanted into the targeted brain regions by stereotaxic surgery as described above. The mice were allowed to recover for one week after surgery. On the day of recording, the mice were briefly anesthetized by isoflurane inhalation and placed in an acrylic restrainer with their electrodes connected to an amplifier headstage (OMNETICS 2409). Neural signals were amplified, processed by a CereCube NSP1 processor, and recorded on a PC running Mind Explorer software (Version 1.5.6, NeuroXess) with sampling rate at 30000 Hz. Spike sorting was performed offline based on V.seek algorithm using Mind Explorer. CRD responses were calculated as mean firing frequency over the 10-s distension period subtracted by the mean firing rate of the 10-s prior to distension. Spontaneous firing rate was averaged using 20 s time windows. Adjustable microwire electrode arrays (KD-MWA-AD-16, Kedou Brain-Computer Technology Co., Ltd., Suzhou, China) were used to record neural activity of the Au1 and the ACC. To record neural activity in conjunction with optogenetic manipulation, optrodes (KD-MWA-F-16) were implanted into the ACC. Additionally, cannulated microelectrode arrays (KD-MWA-A-4) were used to assess neural activity with pharmacological intervention.

Inflammatory pain model and von Frey test
Complete Freund’s Adjuvant (CFA)-induced inflammatory pain was adopted as a somatic pain model. 10 μL CFA was injected into the plantar surface of the right hind paw using a Hamilton syringe. The von Frey test was used to assess mechanical pain threshold on the inflamed paw. The 50% paw withdrawal threshold was determined using the up-down method of Dixon67.

Open field test and elevated plus maze
Open field test and elevated plus maze were performed using the ANY-maze acquisition system and software (Stoelting, Kiel, WI, USA). Mice were acclimated to the testing room 1 day prior to the test. Locomotion was recorded for 5 min in an open-top box (45 × 45 cm), and some key parameters were analysed, including total distance travelled, immobile time, mean speed without rest, and duration spent in the centre. For elevated plus maze, mice were placed at the centre of the apparatus and allowed to explore freely for 5 min. The number of entries into the open arms and time spent in the open arms were reported.

Statistical analyses
All data were expressed as means ± SEM unless otherwise stated. Significant differences were determined by student’s t-test, Mann–Whitney test, and one- or two-way ANOVA with Bonferroni test as appropriate using GraphPad Prism 9.5.0 (GraphPad Software, San Diego, CA, USA). All statistical tests were two-sided. Normality was checked with D’Agostino & Pearson test. When normality assumption was violated, the Mann-Whitney test was used. P < 0.05 was considered statistically significant with significance levels defined as follows: *p < 0.05, **p < 0.01, ***p < 0.001. Experiments and analyses were conducted in a blinded manner.

Key resources
Key resources, including antibodies, viruses and chemicals, are listed in Table S2.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this study, Yu et al. explore the neural circuits involved in visceral pain modulation, specifically focusing on the primary auditory cortex (Au1) and the anterior cingulate cortex (ACC). They created a neonatal colonic inflammation (NCI) model, and showed that the early-life stress disrupted the inhibition-excitation balance in the Au1, leading to ACC hyperactivity by both GABAergic and glutamatergic projections and causing visceral hypersensitivity. Moreover, music intervention could alleviate this hypersensitivity by restoring the inhibition-excitation balance in the Au1 and suppressing ACC hyperactivity. It is an intriguing study that offers insights into the mechanisms behind stress-induced pain and the potential for music therapy in managing visceral pain. However, there are two major issues I outline below, which significantly weaken the authors’ conclusion.\n1. The percentage of GABAergic input from Au1 to ACC is surprisingly high (28.5%). Based on Tomioka et al. 2005, European Journal of Neuroscience, and Tamamaki et al. 2010, Frontiers of Neuroscience, the cortico-cortical long-range GABAergic projection is quite sparse. The only example showing co-localization of mCherry and GABA in Figure 3E is unconvincing since the GABA signal is weak and nontypical compared to nearby signals. The projection intensity in the ACC from Au1 GABAergic neurons is also not presented in Figure 3F. Electrophysiological evidence is needed to demonstrate the property of this projection.\n2. The relationship between the local circuit in Au1 and its projection to the ACC is not well elucidated. In Figure 3N to O, the muscimol application should be able to silence both GABAergic and glutamatergic neurons, so the inhibition of projection in the ACC is not necessary. The excitation induced by activating hM3Dq is mild, and the inhibition induced by muscimol application is strong. Whether the former one can mask the latter one requires electrophysiological evidence.\nMinor points:\n1. Since NCI is the only model adopted in this study, it is inappropriate to claim that this study can demonstrate music intervention is a universal therapy.\n2. Only male animals are adopted. Will there be any difference between male and female subjects?\n3. Figure 1B and C, the responses are quite low to auditory stimulation. The duration of sound and CRD should be marked.\n4. Figure 1D, what does n represent? n = 3 is small.\n5. Figure 1F, the onset time of CRD should be marked, the same as for other similar panels.\n6. Figure 1N and O, the criterion of co-localization should be described, since some potential co-localizations are not marked.\n7. Figure 1M, the insignificance could be caused by a smaller n than in Figure 1L.\n8. Figure 1J and L, why do they have different n? It is presumed that these two panels are from the same set of experiments. Same for Figure 1K and M, Figure 4I and K, and Figure 4J and L.\n9. Figure 1S, why didn't the activation of GABAergic neurons change the control animals’ pain responses? This should at least be discussed.\n10. Figure 2B, the meaning of the arrows should be described.\n11. Figure 2E-G, how was this experiment conducted (e.g. the parameters of CRD and when the c-fos expression was checked after CRD)? Same for all other c-fos experiments.\n12. Figure 2J, Music-CRD should be Blue-CRD. Figure 2M, Music-CRD should be Yellow-CRD.\n13. Figure 2J and M, what is the duration of the laser? Same for other optogenetic experiments.\n14. Figure 2J, will inhibition of ACC excitatory neurons lower CRD responses in control animals, which is related to minor point 9?\n15. Figure 4F and G, it looks that both GABA and glutamate signals are denser in the NCI+Music groups. Should this manipulation change the expression of GABA and glutamate?\n16. line 322, visceral main modulation should be visceral pain modulation.", "reviewer_2": "This is a very interesting and valuable study. The authors established a visceral pain model—neonatal colonic inflammation (NCI)—using an early-life stress paradigm. Music has long been used to relieve stress and induce pleasant emotions. In recent years, its regulatory functions—such as promoting sleep and alleviating pain—have gained increasing attention. However, the precise underlying neural mechanisms remain largely unclear.\nIn this study, Yu et al. demonstrated that classical and electronic music, but not rock music, significantly increased the visceral pain threshold in NCI mice. Through c-Fos staining, viral tracing, and optogenetic and chemogenetic manipulation, the authors showed that GABAergic neurons in the auditory cortex (ACx) were activated and subsequently inhibited glutamatergic neurons in the anterior cingulate cortex (ACC). Visceral pain, such as that seen in irritable bowel syndrome, is often stress-related. Thus, music may offer a unique advantage in treating visceral pain by simultaneously alleviating both emotional and sensory components.\nWhile the findings are exciting, several aspects of the conclusions require further clarification. Below are my major and minor comments:\nMajor Comments\nInvolvement of the Insular Cortex (IC):\nThe ACC is well-documented in pain processing; however, the authors observed dense projections from the ACx to the insular cortex (IC), which is also strongly implicated in both somatic and visceral pain processing. Given the IC's well-established role in interoception and visceral sensation, the authors should justify why they focused on the ACC instead of the IC. Furthermore, it would be valuable to examine whether the ACx–IC pathway plays a role in music-induced visceral analgesia.\nNeed for Electrophysiological Evidence:\nThe authors assessed neural activation in the ACx and ACC using c-Fos staining, a classical but indirect marker of activity. To enhance the strength of the conclusions, electrophysiological validation—particularly in vivo recordings—should be provided to directly assess real-time changes in neuronal activity during music exposure.\nSpecificity of Auditory Cortex Neuronal Activation:\nThe authors reported that Au1 neurons showed minimal responses to tactile stimulation, which is expected. However, in the NCI model, the number of c-Fos–positive neurons in Au1 significantly increased. This raises the question: are the activated neurons in the auditory cortex specifically involved in visceral pain processing? The authors should investigate whether these neurons are also engaged in other pain modalities, such as inflammatory or neuropathic pain.\nPotential Stress-Induced Analgesia:\nAs the authors noted, it is unclear whether mice perceive music similarly to humans. For mice, music may act as a form of sound-induced stress. Given that stress is known to induce analgesia, the authors should examine whether classical and electronic music exposure induces stress responses in mice, which could confound interpretation of the analgesic effects.\nMinor Comments\nPlease replace the images showing co-localization of RV signals with glutamate in Figure 3D to enhance clarity.\nIn Figures 1P and 2J, statistical data for the control group should be included for comparison.", "reviewer_3": "In the manuscript “A Primary Auditory Cortex–Anterior Cingulate Cortex Circuit Underlying Cross-Modal Visceral Pain Modulation,” Yu et al. demonstrated that music therapy produced analgesia in adult mice with visceral pain. They further delineated the underlying circuit and microcircuit mechanisms: music stimulation enhanced the activity of GABAergic neurons in the auditory cortex, which in turn suppressed glutamatergic neurons in both the auditory cortex and the anterior cingulate cortex. Overall, the study is compelling, featuring significant novelty, robust data, and a clear narrative. Nevertheless, I have several concerns that, if addressed, would further improve the quality of the work.\nMajors:\n1. The authors emphasize the analgesic effect of sound on visceral pain. However, the study’s real significance, in my view, is that music therapy produce analgesia in adult mice suffered from early-life stress attack. Indeed, sound stimulation does not relieve CRD-evoked visceral pain in normally reared mice, and the analgesic effect of music is only observed in NCI+CRD animals. This nicely explains why music therapy benefits only a subset of patients in the clinic.\nAnother intriguing question within this framework is whether the analgesic effect of music therapy against combined early-life and adult stress is restricted to visceral pain. The authors propose that neonatal stress reduces the number and activity of GABA neurons in the auditory cortex, and that music exposure in adult mice can rescue the activity of GABA neurons. If this mechanism operates via modulation of GABAergic circuits in the auditory cortex, it may not be exclusive for visceral nociception. The authors should therefore perform an additional experiment: after early-life NCI, deliver a somatic pain challenge (e.g., CFA plantar injection) in adult mice and test whether music therapy still produces analgesia. Given that music engages the auditory cortical GABA network, such an effect is plausible. Besides, the authors named the title of manuscript as ‘…underlying cross-modal … modulation’. However, the whole work did not really represent the cross-model modulation, if they test both visceral and somatic pain, then this statement should be more proper.\nI believe that reorganizing the whole paper outlines and results data around this perspective would substantially enhance the work’s novelty and value and would broaden the therapeutic relevance of their findings..\n2. In a previous Science paper (2022, 377:198–204), the authors show that inhibition of the glutamatergic auditory cortex - thalamus (VP and PO) pathway induces analgesia. They also show that music alleviate the activity of these Glu projecting neurons. This study further demonstrates that music exerts its analgesic effect by directly enhancing the activity of GABAergic neurons in the auditory cortex, thereby indirectly suppressing glutamatergic neurons. These findings provide an important mechanistic complement to the Science paper. This point should be explicitly addressed in the Discussion.\nIn addition, in the Science study, the authors show that 5 dB, rather than 15 dB, white noise produces analgesia, indicating that sound intensity is crucial for the analgesic effect. However, the present work does not address sound intensity at all. Moreover, different music genres differ in efficacy (classical, but not rock, music is analgesic), yet the manuscript offers no convincing explanation. This issue should be addressed by further experiments.\nMinors:\n1. On page 12, line 306, the authors state, “Since Au1 exhibited minimal involvement in direct encoding of visceral pain, it is inferred that the role of Au1 in visceral pain modulation likely depends on its projections to other brain regions encoding visceral pain.” This reasoning is weak. While I agree that probing the downstream projections of Au1 is critical for elucidating the mechanism underlying the auditory cortex–mediated analgesia, the justification cannot be that Au1 itself is poorly activated by visceral pain. If neurons in Au1 do not participate in pain processing, their projections are unlikely to do so either.\n2. In Fig. 2B, the color of the circles indicates different density of EGFP-expressing fibers, but what does the size of the circles represent?\n3. In the model cartoon, the authors depict two balance scales: in the orange one, the GABA-laden side is shown as heavier than the glutamate-laden side, implying the E/I imbalance in the Au induced hypersensitivity of nociception. According to the study’s findings, this should be reversed—the GABA side should actually be lighter."}, "author_response": "Responses to Reviewers’ Comments\nReviewer #1 (Remarks to the Author):\nIn this study, Yu et al. explore the neural circuits involved in visceral pain modulation,\nspecifically focusing on the primary auditory cortex (Au1) and the anterior cingulate\ncortex (ACC). They created a neonatal colonic inflammation (NCI) model, and showed\nthat the early-life stress disrupted the inhibition-excitation balance in the Au1, leading\nto ACC hyperactivity by both GABAergic and glutamatergic projections and causing\nvisceral hypersensitivity. Moreover, music intervention could alleviate this\nhypersensitivity by restoring the inhibition-excitation balance in the Au1 and\nsuppressing ACC hyperactivity. It is an intriguing study that offers insights into the\nmechanisms behind stress-induced pain and the potential for music therapy in\nmanaging visceral pain. However, there are two major issues I outline below, which\nsignificantly weaken the authors’ conclusion.\n1. The percentage of GABAergic input from Au1 to ACC is surprisingly high (28.5%).\nBased on Tomioka et al. 2005, European Journal of Neuroscience, and Tamamaki et al.\n2010, Frontiers of Neuroscience, the cortico-cortical long-range GABAergic projection\nis quite sparse. The only example showing co-localization of mCherry and GABA in\nFigure 3E is unconvincing since the GABA signal is weak and nontypical compared to\nnearby signals. The projection intensity in the ACC from Au1 GABAergic neurons is\nalso not presented in Figure 3F. Electrophysiological evidence is needed to demonstrate\nthe property of this projection.\nThanks for your insightful comments. We agree that cortico-cortical long-range\nGABAergic projections are sparse in terms of density. The proportion of 28.5%\nrepresents the percent of GABAergic input in the total input from the Au1 to the ACC.\nThe two studies you cited above characterized retrograde labeling from somatosensory\nor visceral cortices using GAD67-GFP knock-in mice, particularly GABAergic\nprojections outside the injection sites. In relation with your concern, they reported that\nin several cortical regions, GABAergic projection neurons were accompanied by few\nexcitatory projection neurons. As shown in below figure, red circles (GABAergic\nprojection neurons) outnumber the excitatory projection neurons (grey circles), and this\nmismatch led the authors to propose that “GABAergic projections may be more widely\ndistributed over ipsilateral neocortical areas than excitatory projections” (Tamamaki\nand Tomioka, 2010). As such, the 28.5% of GABAergic input from Au1 to ACC\nreported here is not disproportionately high. This proportion may be the lowest adjacent\nto the tracer injection sites and rises with distance.\n[FIGURE REDACTED]\nFigure 2D\nTamamaki and Tomioka, 2010,\nFrontiers in Neuroscience\nTo address your concern, representative images in Figure 3E have been replaced to\nprovide better visualization of RV-GABA co-localization (see below). We have also\nprovided representative images showing the projection intensity in the ACC from\nAu1GABA and Au1Glu in the revised Figure 3I&L.\nIn addition, we have performed in vivo extracellular recording using optical electrode\narrays to demonstrate this GABAergic projection, as shown in the revised Figure 3F-H.\nOptogenetic activation of Au1GABA axons in the ACC attenuated neural activity in a\nproportion of recorded neurons (11 in 71 neurons), which was abolished in the presence\nof a GABA receptor antagonist bicuculline (1 mg/kg, IP injection). Taken together,\nA\nthese results provide morphological and functional evidence validating the existence of\nGABAergic input from the Au1 to the ACC.\n2. The relationship between the local circuit in Au1 and its projection to the ACC is not\nwell elucidated. In Figure 3N to O, the muscimol application should be able to silence\nboth GABAergic and glutamatergic neurons, so the inhibition of projection in the ACC\nis not necessary. The excitation induced by activating hM3Dq is mild, and the inhibition\ninduced by muscimol application is strong. Whether the former one can mask the latter\none requires electrophysiological evidence.\nThanks for the comments. We agree with the reviewer that muscimol should be able to\nsilence both GABAergic and glutamatergic neurons. Considering mutual inhibition\nbetween different Au1GABA neurons, silencing those inhibitory neurons may cause\ndisinhibition of some Au1GABA neurons. Therefore, we used optogenetic tools to inhibit\nthe Au1GABA projections in the ACC for added precaution. While we admit that the local\nmicrocircuits in the Au1 are more complicated than our simplified schema presented\nhere, the major purpose of this experiment is to demonstrate the functional involvement\nof Au1GABA-Au1Glu-ACCGlu tripartite circuit in visceral pain modulation. To address\nyour concern regarding the counterbalance between muscimol and chemogenic\nactivation, we have conducted supplementary in vivo extracellular recordings. As\nshown in the revised Figure S3A-C, local application of muscimol attenuated firing rate\nof Au1 neurons, and chemogenetic activation of Au1Glu partly reversed the effect of\nmuscimol.\nMinor points:\n1. Since NCI is the only model adopted in this study, it is inappropriate to claim that\nthis study can demonstrate music intervention is a universal therapy.\nThank you for this comment. We have replaced “universal” with “generalizable” in the\nrevised manuscript.\n2. Only male animals are adopted. Will there be any difference between male and\nfemale subjects?\nThanks for this suggestion. We have now replicated some key experiments in female\nmice. Consistent with male mice, NCI-induced visceral hyperalgesia in female mice\nwas relieved by music intervention; furthermore, chemogenetic activation of ACCGlu\nneurons receiving Au1 input reversed music’s analgesic effect(Figure S7). These data\nsuggest that the Au1-ACC circuit also mediates music-induced visceral analgesia in\nfemales.\n3. Figure 1B and C, the responses are quite low to auditory stimulation. The duration\nof sound and CRD should be marked.\nThanks for the comment. The auditory stimulation used here was very brief (1 second).\nThe duration of both sound and CRD have been marked on the revised Figure 1B&C.\n4. Figure 1D, what does n represent? n = 3 is small.\nThanks for the comment. n=3 represents 3 mice. We have increased the sample size to\n6 for revised Figure 1B-D.\n5. Figure 1F, the onset time of CRD should be marked, the same as for other similar\npanels.\nThanks for the comment. For all representative EMG traces, we displayed the entire\n10-second period during CRD stimulation. Although we used the preceding 10-second\nbaseline to calculate CRD responses, the baseline traces were omitted for space\neconomy. This has now been clarified in Figure 1 legend in the revised manuscript:\n“EMG traces in this and all subsequent figures depict the EMG responses during the\nentire 10-second CRD stimulation”.\n6. Figure 1N and O, the criterion of co-localization should be described, since some\npotential co-localizations are not marked.\nThanks for the comment. The criterion has been added to the method section on page\n8, which now reads: “For quantitative assessment and colocalization analysis, putative\ncell fragments and dim fluorescence not in the focal plane were excluded”.\n7. Figure 1M, the insignificance could be caused by a smaller n than in Figure 1L.\nThank you for this comment. We have increased the sample size of c-Fos/glutamate\ncolocalization data set to match that of the c-Fos/GABA colocalization. The proportion\nc-Fos++Glu+/ c-Fos+ is higher in NCI mice (61.33%) than CON (55.99%), but this\ndifference did not reach statistical significance (p = 0.120). We speculate that the\nincrease in total c-Fos+ neurons (the denominator in above proportion) in NCI mice\ndiluted this difference.\n8. Figure 1J and L, why do they have different n? It is presumed that these two panels\nare from the same set of experiments. Same for Figure 1K and M, Figure 4I and K, and\nFigure 4J and L.\nThanks for your careful review. Indeed, the analyses of cell numbers and proportions\nwere based on the same set of experiments. The n values differ because some data sets\nmet outlier criteria (examined by ROUT test) for number metric but not outliers for\nproportion metric. To ensure consistency in the n values, we have excluded any section\nthat qualified as an outlier in either test, and figures have been updated accordingly.\n9. Figure 1S, why didn't the activation of GABAergic neurons change the control\nanimals’ pain responses? This should at least be discussed.\nThanks for your constructive suggestion. It is notable that in the current study,\noptogenetic activation of Au1GABA, optogenetic inhibition of ACCGlu, together with\nmusic therapy did not alter visceral pain responses in CON mice. As discussed in our\ninitial submission, GABAergic neurons may be more vulnerable to stress; and\nconsistently, we observed impaired Au1GABA neuronal activity in NCI mice relative to\nCON mice. As such, it may be inferred that spontaneous activity of Au1GABA in CON\nmice already imposes potent suppression over local and downstream excitatory neurons,\nleaving little room for further inhibition. Therefore, the optogenetic manipulation\nmentioned above or music therapy exhibit little analgesic effect. By contrast, this\ninhibitory brake is compromised in NCI mice, and the same interventions is capable of\nrestoring the excitation-inhibition balance and achieving visceral analgesia.\nFurthermore, this selective relief of visceral pain in NCI but not CON mice offers a\nplausible explanation for why music therapy benefits only a subset of patients in the\nclinic. This has now been fully discussed on page 21 of the revised discussion.\n10. Figure 2B, the meaning of the arrows should be described.\nThanks for the comment. The arrows depict the approximate pathways of projections,\nwhich has been clarified in the revised legend of Figure 2.\n11. Figure 2E-G, how was this experiment conducted (e.g. the parameters of CRD and\nwhen the c-fos expression was checked after CRD)? Same for all other c-fos\nexperiments.\nThank you for this suggestion. The requested information has been incorporated into\nthe revised manuscript on page 8, which now reads: “When assessing c-Fos\nimmunofluorescence following CRD or music stimulation, transcardiac perfusion was\nperformed 90 minutes post-stimulation. Specifically, CRD was applied at 60 mmHg\npressure for 5 times, each lasting 20 seconds separated by 3-minute intervals; classical\nmusic was played continuously for 30 minutes following the same protocol as\nestablished in other experiments”.\n12. Figure 2J, Music-CRD should be Blue-CRD. Figure 2M, Music-CRD should be\nYellow-CRD.\nThanks for pointing out this error. This has been corrected in the revised Figure 2J&M.\n13. Figure 2J and M, what is the duration of the laser? Same for other optogenetic\nexperiments.\nThanks for the comment. Additional methodological details have been added in the\nmethod section on page 9-10: “Yellow light was continuously applied 15 minutes prior\nto CRD tests and sustained until the completion of graded CRD stimulation protocol\n(20, 40, 60 and 80 mmHg in duplicate); in contrast, blue light was synchronized with\neach 10-second distension only”.\n14. Figure 2J, will inhibition of ACC excitatory neurons lower CRD responses in\ncontrol animals, which is related to minor point 9?\nThanks for this suggestion. We have conducted additional experiment to address this\nquestion. Optogenetic silencing of ACC glutamatergic neurons did not change EMG\nresponses to CRD in CON mice (Figure S2). This has been further discussed together\nwith the ineffectiveness of optogenetic activation of Au1GABA and music therapy in\nCON mice on page 21 of the revised manuscript.\n15. Figure 4F and G, it looks that both GABA and glutamate signals are denser in the\nNCI+Music groups. Should this manipulation change the expression of GABA and\nglutamate?\nThanks for the comment. We have re-analyzed the number of GABA+ and glutamate+\nneurons and found no significant differences among all 4 groups (CON, CON+Music,\nNCI, and NCI+Music, Figure S6).\n16. line 322, visceral main modulation should be visceral pain modulation.\nThanks for catching this typo. It has now been corrected.\nReviewer #2 (Remarks to the Author):\nThis is a very interesting and valuable study. The authors established a visceral pain\nmodel—neonatal colonic inflammation (NCI)—using an early-life stress paradigm.\nMusic has long been used to relieve stress and induce pleasant emotions. In recent years,\nits regulatory functions—such as promoting sleep and alleviating pain—have gained\nincreasing attention. However, the precise underlying neural mechanisms remain\nlargely unclear.\nIn this study, Yu et al. demonstrated that classical and electronic music, but not rock\nmusic, significantly increased the visceral pain threshold in NCI mice. Through c-Fos\nstaining, viral tracing, and optogenetic and chemogenetic manipulation, the authors\nshowed that GABAergic neurons in the auditory cortex (ACx) were activated and\nsubsequently inhibited glutamatergic neurons in the anterior cingulate cortex (ACC).\nVisceral pain, such as that seen in irritable bowel syndrome, is often stress-related. Thus,\nmusic may offer a unique advantage in treating visceral pain by simultaneously\nalleviating both emotional and sensory components.\nWhile the findings are exciting, several aspects of the conclusions require further\nclarification. Below are my major and minor comments:\nMajor Comments\nInvolvement of the Insular Cortex (IC):\nThe ACC is well-documented in pain processing; however, the authors observed dense\nprojections from the ACx to the insular cortex (IC), which is also strongly implicated\nin both somatic and visceral pain processing. Given the IC's well-established role in\ninteroception and visceral sensation, the authors should justify why they focused on the\nACC instead of the IC. Furthermore, it would be valuable to examine whether the ACx–\nIC pathway plays a role in music-induced visceral analgesia.\nThank you for highlighting this point. We fully agree that the insular cortex (ICx) plays\na pivotal role in visceral pain processing. In the present study, we focused on the ACC\nrather than ICx because the anterograde tracing from the primary auditory cortex (Au1)\nrevealed denser projections to the ACC compared to the ICx, as depicted in Figure 2B.\nWe suspect that the reviewer might be misled by the shared abbreviation of IC for both\nthe insula cortex and the inferior colliculus (IC), a key region within the canonical\nauditory pathways. To discriminate these two regions, we designated ICx as the\nabbreviation for the insula cortex in Figure 2A&B. We apologize for any\nmisunderstanding this may have caused, and greatly appreciate the valuable suggestion\nregarding the role of ACx-ICx pathway that will undeniably be a focal point of our\nfuture investigations.\nNeed for Electrophysiological Evidence:\nThe authors assessed neural activation in the ACx and ACC using c-Fos staining, a\nclassical but indirect marker of activity. To enhance the strength of the conclusions,\nelectrophysiological validation—particularly in vivo recordings—should be provided\nto directly assess real-time changes in neuronal activity during music exposure.\nThanks for this constructive suggestion. We have conducted in vivo extracellular\nrecording using microelectrode arrays to assess neuronal activity in the Au1 and ACC\nduring music exposure. As shown in the revised Figure 4F-H, spontaneous firing rate\nin the Au1 was elevated in the NCI mice compared to the CON mice, and music\nattenuated Au1 neural activity in the NCI mice only. These findings reinforce the c-Fos\nresults that music enhanced the activity of Au1GABA neurons while suppressing the\nactivity of Au1Glu.\nFurthermore, in vivo extracellular recordings on the ACC have revealed that neuronal\nfiring in response to CRD was significantly increased in NCI versus CON mice, and\nwas attenuated by music exposure (Figure 5I-K). This electrophysiological evidence\ncorroborates our observations in the c-Fos immunofluorescence and fiber photometry.\nSpecificity of Auditory Cortex Neuronal Activation:\nThe authors reported that Au1 neurons showed minimal responses to tactile stimulation,\nwhich is expected. However, in the NCI model, the number of c-Fos–positive neurons\nin Au1 significantly increased. This raises the question: are the activated neurons in the\nauditory cortex specifically involved in visceral pain processing? The authors should\ninvestigate whether these neurons are also engaged in other pain modalities, such as\ninflammatory or neuropathic pain.\nThanks for this insightful comment. We think that the elevated c-Fos signal in the Au1\nof NCI mice most likely reflects stress-induced hyperactivity, rather than a specific\nrepresentation of visceral pain. To test whether Au1GABA neurons and Au1-ACC circuit\nare engaged in other pain modalities, we have performed supplementary experiments\non Complete Freund’s Adjuvant (CFA)-induced inflammatory pain model.\nChemogenetic activation of Au1GABA neurons mimicked the analgesic effect of music\nin CFA mice (Figure 6E-G). Consistently, chemogenetic activation of ACCGlu neurons\nthat receive Au1 projections reversed music’s analgesic effect (Figure 6H-J).\nCollectively, these data suggest that the Au1-ACC circuit also mediates music-induced\nsomatic analgesia.\nPotential Stress-Induced Analgesia:\nAs the authors noted, it is unclear whether mice perceive music similarly to humans.\nFor mice, music may act as a form of sound-induced stress. Given that stress is known\nto induce analgesia, the authors should examine whether classical and electronic music\nexposure induces stress responses in mice, which could confound interpretation of the\nanalgesic effects.\nThanks for the comment. We agree that loud sound may act as a stressor; however, in\nour experiments, the sound pressure level of music was setup at 50-55 dB, marginally\nabove the ambient noise level (40-45 dB). Our initial submission showed that a 15-min\nexposure to classical music did not alter locomotion and anxiety-like behaviors in the\nopen field test and elevated plus maze test (now Figure S5A-H). To extend this\nobservation, we have now measured serum corticosterone levels before and after brief\nmusic exposure, and found no significant difference (Figure S5I-J). Taken together,\nmusic intervention used in this study did not induce stress responses in mice.\nMinor Comments\nPlease replace the images showing co-localization of RV signals with glutamate in\nFigure 3D to enhance clarity.\nThanks for the comment. Representative images in Figure 3D have been replaced.\nIn Figures 1P and 2J, statistical data for the control group should be included for\ncomparison.\nThank you for your comment. In most experiments presented in this study, CON mice\nserve as the control group for NCI mice. When experimental design exclusively\nrequired NCI mice to assess music’s analgesic effect following manipulation of\nAu1GABA (Figure 4P-T) or ACCGlu (Figure 5L-P), a control virus lacking the\noptogenetic elements was used as a control. In Figure 1P-T, CON mice served as the\ncontrol for NCI mice. In Figure 2H-N, optogenetic inhibition of ACCGlu in NCI mice\nprovided a control condition for the converse manipulation (optogenetic activation of\nACCGlu in CON mice). Furthermore, we have conducted supplementary experiments\ndemonstrating that optogenetic inhibition of ACCGlu in CON mice did not alter visceral\npain responses (revised Figure S2), providing an additional control for interpreting the\nresults in Figure 2J-K.\nReviewer #3 (Remarks to the Author):\nIn the manuscript “A Primary Auditory Cortex–Anterior Cingulate Cortex Circuit\nUnderlying Cross-Modal Visceral Pain Modulation,” Yu et al. demonstrated that music\ntherapy produced analgesia in adult mice with visceral pain. They further delineated the\nunderlying circuit and microcircuit mechanisms: music stimulation enhanced the\nactivity of GABAergic neurons in the auditory cortex, which in turn suppressed\nglutamatergic neurons in both the auditory cortex and the anterior cingulate cortex.\nOverall, the study is compelling, featuring significant novelty, robust data, and a clear\nnarrative. Nevertheless, I have several concerns that, if addressed, would further\nimprove the quality of the work.\nMajors:\n1. The authors emphasize the analgesic effect of sound on visceral pain. However, the\nstudy’s real significance, in my view, is that music therapy produce analgesia in adult\nmice suffered from early-life stress attack. Indeed, sound stimulation does not relieve\nCRD-evoked visceral pain in normally reared mice, and the analgesic effect of music\nis only observed in NCI+CRD animals. This nicely explains why music therapy benefits\nonly a subset of patients in the clinic.\nAnother intriguing question within this framework is whether the analgesic effect of\nmusic therapy against combined early-life and adult stress is restricted to visceral pain\nmodality? The authors propose that neonatal stress reduces the number and activity of\nGABA neurons in the auditory cortex, and that music exposure in adult mice can rescue the\nactivity of GABA neurons. If this mechanism operates via modulation of GABAergic\ncircuits in the auditory cortex, it may not be exclusive for visceral nociception. The\nauthors should therefore perform an additional experiment: after early-life NCI, deliver\na somatic pain challenge (e.g., CFA plantar injection) in adult mice and test whether\nmusic therapy still produces analgesia. Given that music engages the auditory cortical\nGABA network, such an effect is plausible. Besides, the authors named the title of\nmanuscript as ‘…underlying cross-modal … modulation’. However, the whole work\ndid not really represent the cross-modal modulation, if they test both visceral and\nsomatic pain, then this statement should be more proper.\nI believe that reorganizing the whole paper outlines and results data around this\nperspective would substantially enhance the work’s novelty and value and would\nbroaden the therapeutic relevance of their findings.\nThanks for your insightful comments. We fully agree that the selective relief of visceral\npain in NCI but not CON mice offers a plausible explanation for why music therapy\nbenefits only a subset of patients in the clinic. This point has now been highlighted in\nthe revised discussion on page 21.\nTo address your question whether the analgesic effect of music against combined early-\nlife and adult stress is restricted to visceral pain modality, we have performed additional\nexperiments on NCI mice challenged with CFA injections at 6 weeks of age. Music\ntherapy produced visceral analgesia on NCI+CFA mice but not CON+CFA mice, while\nit exhibited somatic analgesia in both groups (Figure 6K-M). These data suggest that\nmusic therapy possesses analgesic efficacy across different pain modalities,\nhighlighting its broad translational potential.\n2. In a previous Science paper (2022, 377:198–204), the authors show that inhibition\nof the glutamatergic auditory cortex - thalamus (VP and PO) pathway induces analgesia.\nThey also show that music alleviate the activity of these Glu projecting neurons. This\nstudy further demonstrates that music exerts its analgesic effect by directly enhancing\nthe activity of GABAergic neurons in the auditory cortex, thereby indirectly\nsuppressing glutamatergic neurons. These findings provide an important mechanistic\ncomplement to the Science paper. This point should be explicitly addressed in the\nDiscussion.\nIn addition, in the Science study, the authors show that 5 dB, rather than 15 dB,\nwhite noise produces analgesia, indicating that sound intensity is crucial for the\nanalgesic effect. However, the present work does not address sound intensity at all.\nMoreover, different music genres differ in efficacy (classical, but not rock, music is analgesic),\nyet the manuscript offers no convincing explanation. This issue should be addressed by\nfurther experiments.\nThanks for your comments. Indeed, our findings advance the insights of the Science\npaper by providing an important mechanistic complement that music potentiates the\nactivity of Au1 GABAergic neurons, which in turn suppress local glutamatergic output.\nThis has been highlighted on page 23 in the revised manuscript.\nIn our study, the sound pressure level of music was setup at 50-55 dB, only 10 dB above\nambient noise level (40-45 dB), consistent with that Science paper. As our focus was to\ndissect the underlying neural circuitry mechanism, we did not explore the influence of\nhigher sound pressure level.\nRegarding the differential analgesic outcomes across music genres, we proposed\nacoustic parameters (such as tempo) as an explanation in our initial submission. We\ntherefore have conducted additional experiments to assess the effect of slower classical\nmusic and faster rock music. The Beats per Minute (BPM) of the identical classical\nmusic piece was digitally slowed to 91, while the rock piece was accelerated to 160\n(Table S1). Interestingly, slower classical music lost its analgesic efficacy, whereas\nfaster rock music acquired analgesic effect (Figure S4H&I), suggesting that tempo is a\ncritical parameter for music therapy.\nMinors:\n1. On page 12, line 306, the authors state, “Since Au1 exhibited minimal involvement\nin direct encoding of visceral pain, it is inferred that the role of Au1 in visceral pain\nmodulation likely depends on its projections to other brain regions encoding visceral\npain.” This reasoning is weak. While I agree that probing the downstream projections\nof Au1 is critical for elucidating the mechanism underlying the auditory cortex–\nmediated analgesia, the justification cannot be that Au1 itself is poorly activated by\nvisceral pain. If neurons in Au1 do not participate in pain processing, their projections\nare unlikely to do so either.\nThanks for your insightful comment. We have revised this expression on page 14, which\nnow reads: “Although Au1 contributed minimally to the encoding of visceral pain, it\nmay shape pain perception by tuning relevant neural network through its projections.\nIndeed, a recent study has demonstrated that auditory cortex dampens somatic pain via\nits descending projections to the thalamus”.\n2. In Fig. 2B, the color of the circles indicates different density of EGFP-expressing\nfibers, but what does the size of the circles represent?\nThanks for the comment. The size also indicates the density of EGFP. This has been\nclarified in the legend of Figure 2.\n3. In the model cartoon, the authors depict two balance scales: in the orange one, the\nGABA-laden side is shown as heavier than the glutamate-laden side, implying the E/I\nimbalance in the Au induced hypersensitivity of nociception. According to the study’s\nfindings, this should be reversed—the GABA side should actually be lighter.\nThanks for your suggestion. This has been corrected in the revised Figure 7."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have adequately addressed my concerns. This revised version is ready for publication.", "reviewer_3": "The authors have finished addressing my opinions in the revised version of manuscript. I have no more questions."}, "author_response": ""}]

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