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Client Article | Nat. Commun. | Zhi Zhang’s Team from the University of Science and Technology of China Reveals the Neural Circuit Mechanism of "Pain Inhibiting Pain"

Release time:2025-03-27 14:40:09
In humans and animals, pain in one area can suppress pain perception in another, a phenomenon known as conditioned pain modulation (CPM) in humans and diffuse noxious inhibitory control (DNIC) in animals. While previous studies have focused on the role of brainstem-to-spinal cord descending projections in DNIC, the mechanisms by which the cerebral cortex regulates "pain inhibiting pain" remain unclear.

On February 21, 2025, the research team led by Zhi Zhang and Xia Zhu from the University of Science and Technology of China, in collaboration with Wenjuan Tao’s team from Anhui Medical University, published a study in Nature Communications titled "Intra-somatosensory cortical circuits mediating pain-induced analgesia."
The study reveals a feedforward inhibitory circuit from S2 to S1HL and a cross-hemispheric circuit from contralateral S2 to S2 and then to S1HL, which respectively mediate ipsilateral and contralateral pain-induced analgesia.

 

Part 1: Pain in Different Body Regions Induces Analgesia

To determine whether pain can suppress pain in other body regions in mice, an inflammatory pain model was first established by injecting Complete Freund's Adjuvant (CFA, 1 mg/ml, 10 μl) into the left hind paw. Various noxious stimuli, such as pinching and capsaicin injection, were then applied. The results showed that stimulating the left forepaw, right forepaw, or cheek significantly inhibited pain-related behaviors in the left hind paw, increasing the pain threshold. Specifically, the von Frey test detected an elevated pain threshold (i.e., increased mechanical sensitivity threshold), the hot plate test showed a prolonged latency of the licking response, the brush test recorded a decrease in dynamic pain scores, and the spontaneous pain test indicated a reduction in spontaneous pain scores. This effect gradually diminished within 20 minutes after the removal of the clamping stimulus (Figure 1a-b). Moreover, the analgesic effect faded over time after stimulus removal and was dependent on stimulus intensity. For instance, a low concentration of capsaicin (1 μg/10 μl) failed to effectively increase the pain threshold, whereas capsaicin at 5 μg/10 μl and 10 μg/10 μl produced significant effects (Figure 1c-h). Additionally, the study ruled out the influence of attention distraction on the experimental results, confirming that "pain-induced analgesia" is a genuine neural regulatory phenomenon.

Figure 1: Noxious Stimulation in Different Body Regions Induces Analgesia in the Mouse Hind Paw

Part 2: Increased Neuronal Activity in the Secondary Somatosensory Cortex During Pain-Induced Analgesia

To verify whether a specific neuronal ensemble exists within the somatosensory cortex that processes pain from different body regions and mediates pain-induced analgesia, the researchers crossed FosCreERT2 (Fos-TRAP2) mice, which express tamoxifen-inducible Cre recombinase, with Ai14-tdTomato reporter mice, generating the FosTRAP2:Ai14 mouse line. After intraperitoneal injection of 4-hydroxytamoxifen (4-OHT), capsaicin was injected into the left forepaw, right forepaw, or cheek of the mice. Two weeks later, the mice were sacrificed, and tdTomato expression in whole-brain slices was examined to indicate neuronal activity.

In CFA-treated mice injected with capsaicin, compared to the control group, tdTomato expression significantly increased in the right secondary somatosensory cortex (S2), the right primary somatosensory cortex forelimb region (S1FL), and the left contralateral S2 (contra-S2) after left forepaw capsaicin injection, while tdTomato expression significantly decreased in the right primary somatosensory cortex hindlimb region (S1HL) (Figure 2a-c). Similarly, in CFA mice receiving capsaicin injection in the right forepaw, tdTomato expression increased significantly in the contralateral S1FL, S2, and contra-S2, but decreased in S1HL. In CFA mice receiving capsaicin injection in the cheek, tdTomato expression significantly increased in the insular cortex (IC), the barrel field region of the primary somatosensory cortex (S1BF), S2, and contra-S2, while it decreased in S1HL.

These findings indicate that neurons in S2 expressing c-Fos (labeled by TRAP) were activated across all three pain stimulation sites, with the activated S2 neurons mainly concentrated in layers 2–4 (Figure 2d-g). Conversely, c-Fos expression in S1HL neurons was significantly reduced, suggesting that the somatosensory cortex (S2 and S1) is involved in mediating pain-induced analgesia.
Figure 2: Activity-Dependent Labeling of Neurons During Pain-Induced Analgesia
 
To investigate whether the same population of neurons is activated during analgesia induced by pain in different body regions, the researchers used FosTRAP2:Ai14 CFA mice to capture S2 neurons activated by capsaicin injection in the right forepaw or cheek. These mice then underwent c-Fos immunohistochemical staining to label S2 neurons activated by capsaicin injection in the left forepaw. The results showed that approximately 70% of S2 neurons activated by capsaicin injection in the left and right forepaws overlapped, while approximately 72% overlapped between the left forepaw and cheek stimulation. Additionally, saline injection experiments ruled out the possibility that S2 neuron activation resulted from increased baseline activity.

In summary, noxious stimuli from different body regions activated the same population of neurons in S2 (Figure 3h-l).

Figure 3: The Same Population of S2 Neurons is Activated During Pain in Different Body Regions
 

Part 3: S2 Mediates Pain-Induced Analgesia by Inhibiting S1HL-Glu Neuronal Activity

To investigate the potential role of S2 and S1HL neurons in pain-induced analgesia, researchers employed microendoscopic imaging technology to simultaneously analyze the activity patterns of S2 and S1HL glutamatergic neurons (S1HL-Glu) by monitoring calcium responses. They injected an adeno-associated virus (AAV-DIO-GCaMP6m) expressing Cre-dependent calcium indicator GCaMP6m into the S2 region of FosTRAP2 mice and AAV-CaMKII-GCaMP6m into the S1HL region. Gradient refractive index (GRIN) lenses were then implanted at the corresponding sites (Figure 4a).

Following capsaicin or saline injection into the left forepaw of mice, results showed that in CFA mice injected with capsaicin, the TRAP-labeled S2 neurons exhibited a significant increase in Ca²⁺ transient frequency and mean z-score activity (Figure 4b-c). In contrast, S1HL-Glu neurons showed a significant decrease in Ca²⁺ transient frequency and mean z-score activity (Figure 4d-e).

Figure 4: Calcium Activity of S2 and S1HL-Glu Neurons During Pain-Induced Analgesia

 
To further explore this mechanism, researchers used chemogenetic methods, injecting a Cre-dependent hM4Di-mCherry virus (AAV-DIO-hM4Di-mCherry) into the S2 region of FosTRAP2 mice to inhibit TRAP-labeled S2 neurons. Simultaneously, they injected AAV-CaMKII-GCaMP6m into S1HL to monitor S1HL-Glu neuronal activity (Figure 5f). The results demonstrated that inhibiting S2 neurons abolished the capsaicin-induced reduction in Ca²⁺ transient frequency and mean z-score activity in S1HL-Glu neurons (Figure 5g-h) and blocked pain-induced analgesia in the hind paw. Conversely, chemogenetic activation of S2 neurons significantly increased the pain threshold in CFA mice (Figure 5i-j).

These findings suggest that S2 mediates pain-induced analgesia across different body sites by inhibiting the activity of S1HL-Glu neurons.

Figure 5: Chemogenetic Inhibition of S2 Neurons Blocks Pain-Induced Analgesia

 

Part 4: S2→S1HL Feedforward Inhibitory Circuit Regulates Analgesia

Using a cell-type-specific anterograde monosynaptic tracing system, researchers injected FosTRAP2 mice’s S2 region with Cre-dependent TK-GFP virus (AAV-DIO-TK-EGFP). Three weeks later, they administered HSV-ΔTK-tdTomato virus (Figure 6a). tdTomato+ signals were detected in multiple regions, including the primary motor cortex, various areas of the primary somatosensory cortex, and the contralateral S2. These signals were primarily distributed in layer 4 of S1HL, with approximately 30% colocalizing with glutamate-specific antibodies and 65% colocalizing with GABA-specific antibodies (Figure 6b-c). This indicates that TRAPed S2 neurons mainly project to GABAergic neurons in layer 4 of S1HL.

To further investigate connectivity, researchers used a cell-type-specific retrograde monosynaptic tracing strategy, injecting Cre-dependent helper viruses into the S1HL region of CaMKII-Cre mice. Three weeks later, they administered rabies virus (RV-EnvA) (Figure 6d). Numerous DsRed-labeled neurons colocalizing with glutamatergic markers were identified in S2. Similar findings were observed in GAD2-Cre mice (Figure 6e-g),
confirming that both S1HL-glu and S1HL-gaba neurons receive direct input from S2.

Figure 6: Tracing the S2→S1HL Circuit

To assess functional connectivity, researchers injected AAV-DIO-ChR2-mCherry into the S2 region of FosTRAP2 mice and AAV-CaMKII-GFP into S1HL (Figure 7h-i), recording optogenetically evoked postsynaptic currents in S1HL neurons. Under a holding potential of -70mV, light stimulation of S2 fibers reliably induced excitatory postsynaptic currents (EPSCs) in S1HL-glu neurons, which were blocked by the AMPA receptor antagonist DNQX. At 0mV holding potential, light stimulation also induced inhibitory postsynaptic currents (IPSCs), which were also DNQX-sensitive, with IPSCs exhibiting a longer latency than EPSCs (Figure 7j-k). These findings suggest that S1HL-glu neurons receive input from local S1HL-gaba interneurons, both of which are directly innervated by S2.

Additionally, after injecting relevant viruses into the S1HL region of GAD2-Cre mice (Figure 7l-m), blue light stimulation of local S1HL-gaba neurons significantly increased IPSCs in S1HL-glu neurons, an effect that was blocked by the GABA receptor antagonist bicuculline (Figure 7n-o). This further confirmed the existence of the S2→S1HL-gaba→S1HL-glu feedforward inhibitory circuit.

Finally, chemogenetic inhibition experiments were conducted by injecting AAV-DIO-hM4Di-mCherry into the S2 region of FosTRAP2 mice and implanting a cannula in the S1HL region (Figure 7p). The results showed that silencing the S2-S1 circuit abolished the increase in hind paw pain threshold, while motor function remained unaffected (Figure 7q).
This indicates that noxious stimulation of the left forepaw activates the S2→S1HL-gaba→S1HL-glu feedforward inhibitory circuit, mediating pain-induced analgesia without impairing motor function.

Figure 7: The S2→S1HL-gaba→S1HL-glu Feedforward Inhibitory Circuit Mediates Pain-Induced Analgesia

Part 5: Contralateral S2→S2→S1HL Circuit Mediates Contralateral Pain Inhibition

Previous studies have shown that somatosensory processing in mammals is lateralized, meaning sensory information from one side of the body is primarily processed in the contralateral hemisphere of the brain. It is known that harmful stimuli to the right forepaw (on the contralateral body side) can suppress pain-related behavior in the left hindpaw, and the harmful information from the right side of the body is encoded by the contralateral hemisphere. Based on this, researchers hypothesized that contralateral S2 may play a role in mediating the S2→S1HL circuit, possibly contributing to the pain inhibition induced by harmful stimulation of the right forepaw in CFA mice. To test this hypothesis, the researchers employed a three-step retrograde tracing strategy. First, retro-AAV-hSyn-Cre virus was injected into the S1HL of C57 mice, and Cre-dependent helper virus was injected into S2. Three weeks later, RV-ΔG-DsRed virus was injected into S2 (Figure 8a). After the injections, DsRed+ signals were detected in cortical, thalamic, and other brain regions, as well as in contralateral S2 (colocalized with glutamate antibody) (Figures 8b-d). This result suggests that the S2→S1HL circuit indeed receives direct neural input from contralateral S2. The researchers further injected AAV-hSyn-Cre virus into the contralateral S2 of C57 mice and AAV-DIO-YPet virus into S2, using the CLARITY technique to render the brain translucent and enable whole-brain imaging (Figures 8e-f), observing projections of S2 neurons. The results showed significant connectivity between S2 and contralateral S2, as well as projections to the ipsilateral S1 and primary motor cortex (M1) (Figures 8g-h), with numerous subcortical projections, indicating that S2 neurons interact with contralateral S2 and control S1HL.

Figure 8: Tracing Contralateral S2→S2→S1HL Circuit


Next, the researchers examined the neuronal activity of contralateral S2, S2, and S1HL-glu during contralateral pain-induced analgesia. They injected AAV-DIO-GCaMP6m and AAV-CaMKII-GCaMP6m viruses into contralateral S2, S2, and S1HL of FosTRAP2 mice (Figure 9i), using microendoscope imaging to monitor calcium responses. After capsaicin injection into the right forepaw of the mice, the transient frequency and average z-score activity of contralateral S2 and S2 neurons significantly increased, while these indicators significantly decreased in S1HL-glu neurons (Figures 9j-o).

Figure 9: Calcium Activity of Contralateral S2, S2, and S1HL-glu Neurons during Contralateral Pain-Induced Analgesia

 
Further, chemical genetic viral inhibition of contralateral S2 neurons was performed, and the activity of S2 and S1HL-glu neurons was monitored via AAV-DIO-GCaMP6m and AAV-CaMKII-GCaMP6m viruses (Figure 10a). The results showed that after inhibiting contralateral S2 neurons, the increased activity of S2 neurons and the decreased activity of S1HL-glu neurons in CFA FosTRAP2 mice, induced by capsaicin injection into the right forepaw, were blocked (Figures 10b-e). This indicates that the contralateral S2→S2→S1HL pathway is activated during contralateral pain-induced analgesia. To explore whether the contralateral S2→S2→S1HL pathway is necessary for contralateral pain-induced analgesia, the researchers injected AAV-DIO-hM4Di-mCherry or AAV-DIO-mCherry viruses into contralateral S2 of FosTRAP2 mice and implanted a cannula in S2 for chemogenetic inhibition experiments. The results showed that after inhibition of contralateral S2 neurons, the increase in S2 neuron activity and the decrease in S1HL-glu neuron activity induced by capsaicin injection into the right forepaw were blocked, and the increase in the pain threshold of the hindpaw was also abolished (Figures 10f-g). This confirms that the contralateral S2→S2→S1HL circuit is activated during contralateral pain-induced analgesia and is essential for this process.

Figure 10: Contralateral S2→S2→S1HL Circuit Mediates Contralateral Pain-Induced Analgesia


Summarize

This study for the first time clearly identified a neural circuit in the somatosensory cortex that mediates "pain inhibiting pain," expanding our understanding of pain relief mechanisms. From a basic research perspective, it reveals the functional heterogeneity of different layers of S2 neurons in pain modulation and emphasizes the importance of the S2-S1 connection in coordinating pain perception across different body parts. From a clinical application standpoint, it provides potential targets for the development of new pain therapies. In the future, precise modulation of these neural circuits may offer more effective ways to alleviate the suffering of chronic pain patients.
 

The viral tools used in this study are available through Brain Case Biotech:

Product Types          Product TypesProduct Types


HSV Anterograde Mono- Synapse Tracing System

rAAV-Ef1α-DIO-EGFP-T2A-TK
HSV-△TK-LSL-tdTomato



RV Retrograde Mono-Synapse Tracing System

rAAV-Ef1α-DIO-RVG
rAAV-EF1α-DIO-EGFP-T2A-TVA
RV-EnvA-△G-DsRed


Recombinase 

retro-AAV-hSyn-Cre
AAV2/1-hSyn-Cre


Fluorescent Protein 

rAAV-EF1a-DIO-Ypet-2A-mGFP
rAAV-Ef1α-DIO-mCherry


Optogenetics Chemogenetics 

rAAV-Ef1α-DIO-hChR2(H134R)-mCherry
rAAV-Ef1α-DIO-hM4D(Gi)-mCherry


Chemogenetics 

AAV-DIO-GCaMP6m
AAV-CaMKII-GCaMP6m

*Link to article 👇👇
Intra-somatosensory cortical circuits mediating pain-induced analgesia | Nature Communications

 

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