A corticothalamic circuit modulates pain sensitivity and mediates innate fear-induced analgesia in male mice

Domain: biology
Published: 2026-03-13
Editorial outcome: major_revision

Abstract
Fear and pain are two frequently co-occurring states that mammals need to orchestrate to ensure survival. Nevertheless, how the brain dynamically prioritizes between them remains poorly understood. Here, we demonstrate that innate fear suppresses both acute and chronic pain, whereas pain does not reciprocally modulate fear responses in male mice. Using fiber photometry, virus tracing, and electrophysiological approaches, we show that exposure to a fear-inducing odor activates GABAergic neurons in the anterior piriform cortex (APC), which subsequently attenuates pain-associated hyperactivity in the downstream mediodorsal thalamus (MD). Crucially, inhibiting either APCGABA neurons or the APCGABA-MD circuit enhances pain sensitivity and abolishes fear-induced analgesia. Conversely, activation of APCGABA neurons or the APCGABA-MD circuit induces freezing responses and relieves pain, mimicking fear-induced analgesia. These findings unveil a corticothalamic circuit that bidirectionally regulates pain processing and underlies fear-provoked analgesia, offering potential therapeutic avenues for pain management.

Introduction
When facing dynamic physiological and environmental challenges, mammals must rapidly prioritize survival-critical needs to execute appropriate adaptive behaviors1-3. While significant progress has been made in understanding how the central nervous system processes individual states such as fear4, pain5,6, hunger5, and thirst6, the mechanisms by which the brain hierarchically organizes competing survival demands remain poorly understood.

Previous research has reported interactions between pain and other conflicting states such as hunger, thirst, caloric deprivation or acute stressors5-9. In such conflicts, behavioral priorities typically shift from nocifensive response – defined as an organism's defensive response to potentially harmful stimuli, typically involving pain perception and avoidance movements – toward addressing more immediate survival needs5,7-10. Fear, as a fundamental emotion arising from threat, is a frequently encountered state. While conditioned fear-induced analgesia and its neural substrates have been extensively characterized4,11-13, innate fear responses—particularly those elicited by natural predators—remain comparatively unexplored despite their ecological relevance. For most species, predators serve as the primary threat sources. In such cases, survival demands immediate fight-or-flight decisions, requiring temporary suppression of pain processing to facilitate predator confrontation or evasion9,14,15. Surprisingly, little is known to date about the neural mechanisms underlying how the brain makes preferential choices between the intractable conditions of innate fear and pain. In this study, we utilized the odorant molecule of 2-methyl-2-thiazoline (2MT), chemically related to the fox anogenital gland secretion 2,4,5-trimethyl3-thiazoline (TMT), to induce an innate fear state in mice16. Therefore, our study focused on the olfactory system to elucidate ranking mechanisms of innate fear and pain processing.

The piriform cortex (PC) serves as the primary olfactory cortex, receiving odorant information inputs from the upstream olfactory bulb (OB) and lateral olfactory tract (LOT) regions17. This region plays integral roles in olfactory information processing18,19, learning20, memory21,22, and has been implicated in innate fear responses23,24. Anatomically, the PC is subdivided into anterior (APC) and posterior (PPC) regions25.

Here, we demonstrate that innate fear endogenously inhibits pain, whereas pain does not affect fear reactions. We further identify the APC as a node in innate fear-induced analgesia, mediated specifically by a GABAergic projection to the mediodorsal thalamus (MD). Notably, the constitutive activity of both APCGABA neurons and the APCGABA-MD circuit exerts tonic gating of pain perception. Taken together, our findings reveal an olfactory-embedded corticothalamic circuit that bidirectionally modulates pain processing and mediates fear-induced analgesia, while shedding light on potential therapeutic targets for pain management.

Results
Innate fear odor 2MT induces analgesia in mice
To investigate whether innate fear affects both acute and chronic pain, a potent predator-derived odor compound of 2MT which evokes freezing response, a behavioral readout of fear16,26,27, was exposure to mice. Anisole, a neutral odor, served as a control to rule out non-specific responses to olfactory stimulation (Supplementary Fig. 1). Separate cohorts of mice were then subjected to a series of pain sensitivity tests, as described below. We first examined mechanical and thermal sensitivity by von Frey filaments and radiant heat, respectively. Upon 2MT exposure in naïve mice, we observed a remarkable elevation of the paw withdrawal threshold and paw withdrawal latency compared to both water and anisole controls (Fig. 1a), indicating a reduction in basal nociception. By contrast, the pain sensitivity of anisole-exposed mice was unchanged (Fig. 1a).

Next, hot plate and pinprick assays were used to assess acute thermal pain and mechanical pain, respectively. In the hot plate test, exposure to 2MT significantly prolonged the latencies to both jumping and licking and reduced the episodes of these behaviors compared to the water treatment, whereas anisole had no significant effect (Fig. 1b). In the subsequent studies, the two nociceptive behaviors were analyzed as a combined measure. Innate fear odor induced by 2MT also significantly reduced the response frequency to noxious mechanical (pinprick) stimulation of the hind paw (Fig. 1c). Together with the hot plate test results, these findings demonstrate that innate fear odor profoundly alleviates both forms of acute pain.

Finally, we explored 2MT-induced analgesia in models of pathological pain. We found that 2MT, but not a neutral odor, markedly alleviated pain behaviors across multiple models: the nociceptive licking responses in acute inflammatory pain model (Formalin), as well as tactile and thermal hypersensitivity in both complete Freund's adjuvant (CFA)-induced inflammation and spared nerve injury (SNI)-induced neuropathic pain models (Fig. 1d-f).

Since the key behavior readouts of pain sensitivity tests (e.g., increased thresholds, reduced nocifensive responses) rely on motor performance, we investigate whether 2MT exposure could produce motor suppression that might confound the interpretation of analgesic effects. First, the grip strength test was used to evaluate muscle force. We observed no significant difference in grip strength between 2MT-exposed mice and water-exposed controls (Supplementary Fig. 2a). Additionally, high-speed paw kinematics analysis during the pinprick assay revealed comparable paw withdrawal velocity and amplitudes between the two groups (Supplementary Fig. 2b), indicating normal motor output. To further assess motor function, we evaluated motor coordination and balance using the balance beam and rotarod tests. No differences were detected in traversal time (balance beam) or latency-to-fall (rotarod) between 2MT- and water-treated mice (Supplementary Fig. 2c, d). Our results show no differences in traversal time and consistent latency-to-fall between 2MT- and water-exposed mice. These results demonstrate that the observed analgesic effects of 2MT are not attributable to motor impairment.

Altogether, our data demonstrate that an innate fear state can reliably provoke analgesia in both physiological and pathological conditions.

APCGABA neurons respond to innate fear odor 2MT
The piriform cortex (PC) has long been thought to encode olfactory information and odor fear memory18,22. Therefore, we hypothesized that PC neurons might also mediate 2MT-induced analgesia. To test this, we employed TRAP2::Ai14 mice to selectively label neurons activated during 2MT exposure. We found that 2MT exposure paired with the injection of 4-Hydroxytamoxifen (4-OHT) results in substantial expression of tdTomato in the PC. Quantitative analysis showed that the fold change of 2MT-TRAPed tdTomato-positive neurons relative to the water control was significantly greater in the APC than in the PPC (Fig. 2a, b). Based on this pronounced activation, we therefore focused subsequent investigations on the role of the APC in fear-induced analgesia.

To validate whether the Fos-TRAP line works as previously28, we re-exposed 2MT-TRAPed TRAP2::Ai14 mice to either 2MT or anisole. c-Fos immunostaining indicated that approximately 64% of 2MT-TRAPed APC neurons were reactivated by 2MT, compared to only ~23% upon anisole re-exposure (Supplementary Fig. 3), confirming the system's efficacy for activity-dependent labeling.

Next, we employed in vivo fiber photometry to record the real-time population activity of APC neurons in response to 2MT. Following injection of AAV-hSyn-GCaMP6m into the unilateral APC and optical fiber implantation above the injection site (Supplementary Fig. 4a, b), we observed a rapid and robust increase in calcium signal upon 2MT presentation via air puff or cotton swab. In contrast, water or anisole exposure evoked only mild calcium transients (Supplementary Fig. 4c, d). These findings provide additional evidence for APC neurons in processing fear-related olfactory information.

The piriform cortex comprises heterogeneous populations of excitatory and inhibitory neurons. To dissect the specific cell types within the APC mediating fear-induced analgesia, we performed fluorescence in situ hybridization (FISH) to determine whether the 2MT-TRAPed neurons were GABAergic (GAD2-positive) or glutamatergic (CAMKII-positive). Interestingly, upon 2MT exposure, the proportion of GAD2+ cells among the tdTomato-labeled neurons significantly increased compared to the control groups. In contrast, the proportion of CaMKII+ cells remained unchanged (Fig. 2c, d). These data indicate that the fear odor 2MT recruits a larger fraction of APCGABA neurons than does a neutral odor, while APCGLU neurons are equally recruited by both odor types.

To further characterize odor-evoked activation patterns in distinct neuronal populations, we performed in vivo fiber photometry recordings of both inhibitory neurons and excitatory neurons in the APC. We first targeted inhibitory neurons by injecting AAV-DIO-GCaMP6m into the APC of VGAT-Cre mice (Fig. 2e, left). Virus expression and fiber placement were verified upon completion of behavioral tests (Fig. 2e, right). Odor stimuli were delivered via either air puff or cotton swab (Fig. 2f). Each odor was tested across six repeated trials with 1-minute inter-trial intervals. Consistent with our FISH data, fiber photometry revealed a striking difference in odor response profiles. The fear odor 2MT elicited larger calcium transients in inhibitory neurons compared to water exposure, while anisole exposure resulted in only mild changes in calcium signals relative to water controls (Fig. 2g, h). In marked contrast, recordings from excitatory neurons of CAMKII-Cre mice showed that 2MT and anisole exposure elicited calcium transients to a similar extent when compared to water exposure (Fig. 2i-l). Successive odor exposure can cause odorant adaptation in the olfactory receptor cell29, which might in turn affect APC neural activity. However, our trial-by-trial analysis demonstrated stable calcium response magnitudes across six trials of anisole or 2MT exposure (Supplementary Fig. 5). This consistency confirms that the 1-minute inter-trial interval effectively prevents olfactory adaptation.

We further validated these cell-type-specific response patterns in C57 mice through viral-mediated expression of GCaMP6s under either VGAT or CaMKII promoters. Consistent with above Cre-line observations, this population-specific activation dichotomy was observed (Supplementary Fig. 4e-l).

Our findings provide evidence that APCGABA neurons selectively response to the fear odor 2MT.

APCGABA neurons mediate 2MT-induced analgesia
Given that APCGABA neurons exhibit selective tuning to innate fear odor, we hypothesized that these neurons play a crucial role in fear analgesia. To further investigate this, we conducted a series of pain-related tests sequentially in the same cohort of mice following the transient silencing of APCGABA neurons, ranging from mild pain-inducing stimuli (assessing basal nociception) to nerve injury operation. To achieve this, an adeno-associated virus (AAV) carrying the inhibitory hM4Di receptor under the control of the GABAergic neuron promoter (AAV-VGAT-hM4D(Gi)-mCherry) or an AAV-VGAT-mCherry as a control was injected into the bilateral APC of C57 mice. The specificity of the VGAT-promoter driven viruses was further verified in VGAT-Cre mice, which received simultaneous injections of AAV-DIO-EYFP and AAV-VGAT-mCherry into the APC (Supplementary Fig. 6a). We found that approximately 72% of mCherry-positive neurons were also EYFP-positive (Supplementary Fig. 6b, c). Twenty-one days after viral injection, mice received an intraperitoneally (i.p.) injection of clozapine-N-oxide (CNO). Subsequently, the mice were exposed to 2MT and then underwent behavioral testing (Fig. 3a). Post hoc staining showed that the majority of mCherry-expressing neurons were GABA-positive, confirming high specificity of viral targeting (Fig. 3b). Electrophysiological recordings validated the efficacy of the chemogenetic approach, showing that CNO hyperpolarized the resting membrane potential (RMP) and blocked action potential firing in hM4Di-expressing neurons (Fig. 3c). Considering that our behavioral assays necessitate a two-day protocol, we examined whether repeated 2MT exposure affects pain responses. To this end, we analyzed 2MT-induced analgesia on basal nociception from C57 mice over two consecutive days. We did not detect any differences in both mechanical and thermal pain threshold (Supplementary Fig. 7), proving that repeated 2MT exposure across two days does not affect the degree of fear analgesia. Therefore, subsequent behavioral experiments followed a two-day schedule: intraperitoneal saline administration on the first day, followed by CNO on the second day. Notably, chemogenetic inhibition of APCGABA neurons did not affect 2MT-induced freezing behavior, regardless of the saline and CNO treatment order (Supplementary Fig. 8a-c), but significantly attenuated the protective effects of fear on basal nociception, acute thermal pain and chronic neuropathic pain (Fig. 3d-f). Importantly, inhibition of APCGABA neurons did not impair motor performance, as confirmed by grip strength, high-speed paw kinematics, balance beam, rotarod, and OFT assessments (Supplementary Fig. 9a-f), suggesting that changes in the above pain-related tests were not due to motor performance deficit.

Repeated pain testing in the same mice across multiple conditions could alter subsequent sensitivity, introducing possible confounds. To mitigate potential order effects, we implemented a counterbalanced experimental design for the sequence of pain sensitivity tests. In naïve C57 mice, we counterbalanced four test modalities (von Frey, Hargreaves’, hot plate and pinprick tests; Supplementary Fig. 10a), while in nerve-injured and CFA-injected mice, we counterbalanced two sets of pain test (von Frey and Hargreaves’ tests, Supplementary Fig. 10f). We found that baseline sensitivity of all behavioral assays remained stable, independent of testing order. Importantly, the sequence of testing did not affect the magnitude of 2MT-induced analgesia (Supplementary Fig. 10b-e, g, h). Based on these findings, we maintained the same sequential pain testing protocol as used in above chemogenetic inhibition for subsequent neural manipulation experiments.

In our study, we also included a battery of pain sensitivity tests without 2MT exposure but having APCGABA neural inhibition in naïve mice (Supplementary Fig. 11a). Interestingly, silencing APCGABA neurons resulted in both mechanical and thermal hypersensitivity (Supplementary Fig. 11b). Since pain involves more than nocifensive responses, we also assessed its affective component using conditioned place aversion (CPA)30. We found that silencing APCGABA neuron alone produced significant CPA (Supplementary Fig. 11c). These data indicate that the constitutive activity of APCGABA neurons modulates both pain sensation and its associated negative affect.

Due to the hypersensitivity after the silencing of APCGABA neurons, the observed loss of fear analgesia may stem from alterations in baseline pain sensitivity. Furthermore, approximately ~40% of APCGABA neurons were activated by 2MT (Supplementary Fig. 12a-c), manipulating broad APCGABA population could confound the interpretation of results. To specifically dissociate the role of the fear odor-responsive neuronal ensemble from general nociceptive processing, we employed a TRAP2 strategy to selectively target 2MT-activated APC inhibitory neurons. We bilaterally injected either AAV-VGAT-DIO-hM4Di-mCherry or AAV-VGAT-DIO-mCherry into the APC of TRAP2 mice, followed by 2MT exposure paired with 4-OHT injection (Fig. 4a). This approach allowed for the specific chemogenetic inhibition of neurons recruited by the innate fear odor. The virus expression and spillover was verified across included hM4Di-injected mice (Fig. 4b). We found that inhibiting this ensemble attenuated 2MT-induced fear expression (Fig. 4c). Strikingly, although it did not change baseline thermal or mechanical nociception, it significantly attenuated 2MT-induced analgesia (Fig. 4d, e), with no effect on locomotion (Fig. 4g-k). These findings establish a necessary role for 2MT-responsive APCGABA neurons in mediating innate fear-induced analgesia and demonstrate their functional segregation from basal nociceptive processing.

To gain further insight into the contribution of APCGABA neurons to fear analgesia, we explored behavioral consequences of activating these neurons. To this end, we administered an adeno-associated virus (AAV) carrying the excitatory hM3Dq receptor under the control of the VGAT promoter (AAV-VGAT-hM3D(Gq)-mCherry) into the APC region of C57 mice (Fig. 3g). CNO-induced membrane depolarization and neural firing in hM3Dq-expressing neurons verified viral functionality (Fig. 3h). Intriguingly, activation of APCGABA neurons exhibited a resemblance to the behavioral phenotypes observed in the innate fear context. There was a significant increase in freezing response and a decrease in total distance traveled in the 20-minute session (Supplementary Fig. 8d, e). Importantly, both mechanical and thermal nociception were significantly decreased (Fig. 3i). There was also a significant reduction in nocifensive responses towards noxious thermal stimulation in naïve mice (Fig. 3j). Of note, a prominent analgesia in nerve-injured mice was observed (Fig. 3k). To investigate whether activating APCGABA neurons induces pain-relief-seeking behavior, we performed a classic conditional place preference (CPP) test in SNI mice. Indeed, hM3Dq-injected SNI mice displayed a significant greater chamber preference score for the CNO-paired context than did mCherry-injected controls (Fig. 3l). Furthermore, gross or fine motor control, assessed by grip strength, high-speed paw kinematics, balance beam, rotarod tests, along with the total distance traveled in a 5-minute OFT remained unchanged following chemogenetic activation of APCGABA neurons (Supplementary Fig. 9g-k). These results suggest that APCGABA neuron activity is sufficient to produce both fear-like behaviors and analgesia.

Following 2MT exposure (Supplementary Fig. 11d), we assessed pain sensitivity and found that exciting APCGABA neurons further enhanced the fear analgesia. Specifically, this enhancement was observed on mechanical nociception under normal conditions, and on both mechanical allodynia and thermal hypersensitivity in neuropathic pain conditions. However, no further reduction was seen in baseline thermal nociception and acute thermal pain responses (Supplementary Fig. 11e, f), likely due to a ceiling effect from maximal 2MT-induced analgesia.

To complement our inhibition studies, we also employed the TRAP2 strategy to selectively activate 2MT-responsive APC inhibitory neurons (Fig. 4l, m). This manipulation was sufficient to induce both freezing behavior and produce significant analgesia (Fig. 4n-p), again without affecting locomotion assessed by grip strength, high-speed paw kinematics, balance beam and rotarod (Fig. 4q-t). The observed decrease in total distance traveled in the OFT is consistent with the induction of freezing behavior following neural activation (Fig. 4u). These complementary loss- and gain-of-function results collectively demonstrate that 2MT-responsive APCGABA neurons are both necessary and sufficient in mediating innate fear-related analgesia.

Taken together, our data define a critical role for APCGABA neurons in mediating fear-induced analgesia, in addition to their constitutive regulation of basal pain perception.

MD is likely APC’s target to mediate 2MT-induced analgesia in chronic neuropathic pain
To investigate the downstream target of APCGABA neurons that mediate fear-induced analgesia, we conducted anterograde tracing. We injected an AAV-VGAT-mCherry into the unilateral APC of C57 mice to visualize axon projections (Fig. 5a). Given the APC’s location on the ventrolateral cortical surface, near several pain-related regions—including the lateral nucleus accumbens (LatNAc)31,32, ventral pallidum (VP)33, anterior insular cortex (AIC)34, and reticular thalamic nucleus (TRN)35, which are associated with pain and some of which project to the MD35,36, we carefully assessed potential viral spread beyond the APC. Analysis confirmed minimal to no spillover to AIC and TRN (Supplementary Fig. 13). Next, we mapped the projection targets of APCGABA neurons, observing dense neural fibers in the mediodorsal thalamus (MD), ventral lateral nucleus of the thalamus (VL), and substantia nigra pars reticulata (SNR) (Fig. 5b). To validate the functional connectivity of these projections, we injected AAV-VGAT-ChR2 into the APC. After two weeks for expression, we performed whole-cell patch clamp recordings in brain slices containing the MD, VL, or SNR. Blue light stimulation reliably elicited inhibitory postsynaptic currents (IPSCs) in neurons within each of these regions. These IPSCs were abolished by bath application of bicuculline (10 μM), an antagonist of GABA receptors (Fig. 5c), confirming that these projections are GABAergic.

To identify which projection targets of APCGABA neurons mediate fear analgesia, we systematically compared neuronal excitability in each target region. We conducted whole-cell patch clamp recordings from neuropathic (SNI) and sham-control mice. In the MD of SNI mice, we found a significantly increased firing rate and a depolarized membrane potentials (RMP) compared to sham controls (Fig. 4d), indicating enhanced neuronal excitability following nerve injury. In contrast, the excitability of neurons in the VL and SNR remained unchanged (Fig. 4d).

We next investigate whether innate fear could reverse the SNI-induced hyperexcitability of MD neurons. Remarkably, 2MT exposure effectively normalized these pathological changes (Fig. 5e-g), as evidenced by the rescue of firing rate, resting membrane potential, and rheobase. This functional recovery implies a key role for the MD in the interplay between innate fear and neuropathic pain. In addition, 2MT exposure significantly reduced the input resistance (Rin) of MD neurons (Fig. 5h), a change indicative of increased spontaneous inhibitory input. Consistent with this, recordings of spontaneous inhibitory postsynaptic currents (sIPSCs) in MD neurons in SNI mice revealed that 2MT treatment 2MT significantly increased sIPSC frequency without affecting amplitude (Fig. 5i), indicating a presynaptic enhancement of inhibitory transmission onto MD neurons.

Collectively, these findings provide a plausible mechanism by which 2MT exposure strengthens APCGABA→MD inhibition, leading to persistent normalization of MD excitability and alleviation of neuropathic pain.

We further confirmed the monosynaptic nature of APCGABA-MD connection. Light-evoked responses were completely blocked by tetrodotoxin (TTX) and partially restored upon application of 4-aminopyridine (4-AP), a potassium channel blocker (Fig. 5j). The recovered responses were subsequently blocked by bicuculline, confirming a direct, monosynaptic GABAergic pathway.

Inhibition of MD-projecting APCGABA neurons dampens fear impact on pain and SNI-induced neural dysfunction
If the APCGABA-MD pathway plays an essential role in fear analgesia, then inhibition of MD-upstream PCGABA neurons may dampen this effect. To test this hypothesis, we injected AAV2/2-retro-hSyn-Cre into the MD and AAV-VGAT-DIO-hM4D(Gi)-mCherry (or control AAV-VGAT-DIO-mCherry) into the APC of C57 mice. Three weeks later, CNO was administrated intraperitoneally to inhibit MD-upstream APCGABA neurons during 2MT presentation, followed by the same battery of pain tests as described previously (Fig. 6a). Post hoc staining showed that approximately 81% of mCherry-expressing APC neurons were GABA-positive, indicating specific targeting of MD-upstream GABAergic neurons (Fig. 6b). Furthermore, CNO-induced membrane hyperpolarization of hM4Di-expressing APCGABA neurons verified functional inhibition (Fig. 6c). As predicted, chemogenetic inhibition of MD-projecting APCGABA neurons significantly reduced 2MT-induced analgesia across multiple pain modalities, including basal nociception, acute thermal pain, and chronic neuropathic pain (Fig. 6d-f). Of note, this manipulation did not affect fear odor-induced freezing behavior, regardless of the saline and CNO treatment order (Supplementary Fig. 8f-h). Importantly, this loss of analgesia was not due to motor impairment, as evidenced by a comprehensive suits of behavioral assays including grip strength, paw kinematics, balance beam, rotarod and open field tests (Supplementary Fig. 9k-o).

To further define the role of MD-projecting APCGABA neurons in innate fear-induced analgesia, we quantified their activation by the fear odorant 2MT. Our data revealed that approximately 78% of MD-projecting APCGABA neurons were activated by 2MT (Supplementary Fig. 12d-f). This indicates that the vast majority of APC GABAergic cells are activated by fear odorant.

Given that inhibition of MD-upstream APCGABA neurons dramatically attenuated 2MT-induced analgesia under neuropathic pain conditions (Fig. 6f), we next investigated whether this manipulation would prevent the rescue of MD hyperexcitability by 2MT in the chronic pain state. To address this, we performed patch clamp recordings from MD neurons in SNI mice expressing either mCherry or hM4Di in MD-upstream APCGABA neurons. These mice received intraperitoneal CNO injections (30 minutes prior) to either 1-hour 2MT exposure or water exposure. Acute brain slices containing the MD thalamus were then prepared for electrophysiological recordings. In the absence of 2MT, inhibition of MD-projecting APCGABA neurons did not alter SNI-induced MD hyperactivity assessed by firing rate and rheobase, compared with mCherry controls (Fig. 6g-i). This was likely due to a ceiling effect from maximal SNI-induced MD neural hyperactivity. In contrast, while 2MT exposure effectively rescued MD hyperactivity in control (mCherry-expressing) mice, this therapeutic effect was completely abolished in inhibitory hM4Di expressing mice (Fig. 6g-h). This lack of response to 2MT in chemogenetically silenced mice suggests that functional APC-MD GABAergic transmission is essential for reversing SNI-induced MD hyperactivity.

These data collectively reveal that MD-upstream APCGABA neurons critically regulate fear impact on pain and nerve injury-induced MD neural dysfunction.

To investigate the physiological role of MD-projecting APCGABA neurons, we inhibited them in naïve mice without 2MT exposure (Supplementary Fig. 14a). This inhibition induced both mechanical and thermal hypersensitivity (Supplementary Fig. 14b) and established an aversive state, as measured by conditioned place aversion (CPA) (Supplementary Fig. 14c). These results demonstrate that this pathway exerts a tonic inhibitory control over basal pain perception, in addition to its critical role in mediating fear-induced analgesia.

The APCGABA-MD circuit mediates 2MT-induced analgesia.
To deepen our understanding of how the APCGABA-MD circuit mediates fear analgesia, we first tested the necessity of APCGABA-MD projections in fear-induced analgesia. We thus injected AAV-VGAT-eNpHR-EYFP into the APC region and implanted optic fibers bilaterally over the MD, enabling a circuit-specific inhibition in freely moving mice. AAV-VGAT-EYFP was injected as the control (Fig. 7a). The accuracy of optic fiber placement was validated (Fig. 7b). Electrophysiological recordings from the eNpHR-EYFP+ APC neurons across multiple mice confirmed the viability of functional virus expression (Fig. 7c). We showed that optogenetic inhibition of APCGABA terminals within the MD greatly diminished the analgesic effects of 2MT, both in naïve mice and those with SNI (Fig. 7d-f).

Without the 2MT exposure, optogenetic inhibition of the MD-projecting APCGABA terminals in naïve mice resulted in hypersensitivity and the formation of aversive memory (Supplementary Fig. 15a-c), as observed in mice when the APCGABA neurons were chemogenetically inhibited (Supplementary Fig. 11a-c).

Next, we conducted experiments to assess the sufficiency of APCGABA-MD circuit activity in fear analgesia. To this end, AAV-VGAT-ChR2-mCherry or AAV-VGAT-mCherry was injected into the APC and optic fibers were bilaterally implanted over the MD (Fig. 7g). Current clamp recordings on ChR2-mCherry+-expressing APC neurons showed that blue light stimulation (20 Hz) reliably evoked neural firing across multiple neurons (Fig. 7h). As expected, optogenetic activation of APCGABA terminals within the MD was sufficient to produce analgesia in various pain tests (Fig. 7i-k), phenocopied the behavioral pattern in the innate fear context (Fig. 1) or the effects of APCGABA neural activation (Fig. 3i-k). Furthermore, we observed a striking dissociation in conditioned place preference: SNI mice showed a significant preference for the chamber paired with optogenetic activation of APCGABA neuron terminals in the MD (Fig. 7l), whereas naïve mice displayed no such preference (Supplementary Fig. 16). This pain-context-specific behavioral reinforcement establishes that the rewarding effect is contingent upon the relief of neuropathic pain, rather than a general rewarding effect.

In the pain test with 2MT exposure, APCGABA-MD circuit activation further enhanced the fear-evoked analgesia on chronic neuropathic pain (Supplementary Fig. 16d-g).

Collectively, we present compelling evidence demonstrating that the APCGABA-MD circuit mediates innate fear-induced analgesia and regulates pain perception.

The innate fear responses are not affected by pain.
To ensure survival, mammals need to cope with the most critical threats. We then examined how the pain affects innate fear behavior. We observed that prior exposure to a 47℃ hot plate or 10 min-pinprick stimuli pinprick has no influence on the fear reaction assessed by the proportion of freezing time, movement distance, odor zone entries, and time in the odor zone during innate fear contexts (Fig. 8a-d). Mice experiencing inflammatory pain or neuropathic pain also exhibited similar levels of 2MT-induced fear reaction when compared to their respective control groups (Fig. 8e-h). These findings suggest that the innate fear is not affected by pain.

Methods
Animals. Adult (8–10 weeks) C57BL/6J, FosTRAP2, and Rosa26-tdTomato (Ai14) male mice were used in the experiments. C57BL/6J mice were purchased from Beijing Vital River Laboratory Animal Technology. CaMKII-Cre STOCK B6.Cg-Tg(Camk2a-cre)T29-1Stl/J (IMSR_JAX:005359), VGAT-Cre STOCK B6J.129S6(FVB)Slc32a1tm2(cre)Lowl/MwarJ (IMSR_JAX:028862), and Ai14 STOCK B6;129S6-Gt(ROSA)26Sortm14(CAGtdTomato)Hze/J (IMSR_JAX:007914) mice were initially purchased from Jackson Laboratories. FosTRAP2 STOCK Fostm2.1(icre/ERT2)Luo/J (Trap2) (IMSR_JAX:030323) mice were gifted by Dr. Shenbin Liu (Fudan University, China). To generate TRAP2::Ai14 mice for activity-dependent labeling experiments, FosTRAP2 mice were crossed with Ai14 mice. All mice were housed in groups of three to five mice per cage with free access to water and food in a biorhythm of 12 h light/12 h dark. The room temperature was kept at 23±1 °C and a consistent humidity of 50 ± 5%. All animal procedures and protocols were approved by the Animal Care and Use Committee of the University of Science and Technology of China.

Stereotaxic injection. The mice were administered intraperitoneal (i.p.) injections of pentobarbital sodium at a dosage of 20 mg/kg, and were then carefully positioned and adjusted on a stereotactic device (RWD Life Technologies Co., Ltd., China). To maintain the body temperature of the mice, a heating pad was utilized. Additionally, eye ointment was applied to protect the mice's eyes from any potential light irritation. A glass micropipette was utilized for injecting the viruses which are packaged and obtained by BrainVTA (Wuhan, China) in a volume of (300-320 nl for APC and 200-220 nl for MD) at a speed of 50 nl/min. After the injection, the micropipette was left in place for 10 minutes, allowing the virus to spread. Once the mice had recovered from the anesthesia, they were returned to home cages. Behavioral tests were performed 2-3 weeks later. The coordinates were defined as anterior-posterior (AP) from bregma, mediolateral (ML) from the midline, and dorsoventral (DV) from the brain surface (in mm).

Virus injection and optical fiber implantation. To manipulate APCGABA neurons, an adeno-associated virus (AAV) carrying the inhibitory hM4Di receptor under the control of the GABAergic neuron promoter (rAAV2/9-VGAT1-hM4D(Gi)-mCherry-WPRE-pA, 5.07E+12 vg/ml, Cat. No. PT-0488) or the excitatory hM3Dq receptor (rAAV2/9-VGAT1-hM3D(Gq)-mCherry-WPRE-pA, 5.44E+12 vg/ml, Cat. No. PT-0489) or rAAV2/9-VGAT1-mCherry-WPRE-pA (5.36E+12 vg/ml, Cat. No. PT-0325) as control were injected into the APC (AP, +0.75 mm; ML, ± 3.25 mm; DV, -5.2 mm) bilaterally.

To inhibit MD-projecting APCGABA neurons, rAAV2/Retro-hSyn-CRE-WPRE-hGH-pA (5.22E+12 vg/ml, Cat. No. PT-2334) was injected into the MD (AP, -1 mm; ML, ±0.5 mm; DV, -3.3 mm) of C57 mice bilaterally, and rAAV2/9-VGAT1-DIO-hM4D(Gi)-mCherry-WPRE-hGH-pA (5.02E+12 vg/ml, Cat. No. PT-0618) or rAAV2/9-VGAT1-DIO-mCherry-WPRE-hGH-pA (5.79E+12 vg/ml, Cat. No. PT-3225) was injected into the APC bilaterally.

To optogenetically manipulate APCGABA terminals within the MD, rAAV2/9-VGAT1-eNpHR3.0-EYFP-WPRE-hGH-pA (5.00E+12 vg/ml, Cat. No. PT-0182) or rAAV2/9-VGAT1-hChR2(H134R)-mCherry-WPRE-hGH-pA (5.66E+12 vg/ml, Cat. No. PT-0643) were injected into the APC bilaterally. rAAV2/9-VGAT1-EYFP-WPRE-hGH-pA (5.33E+12 vg/ml, Cat. No. PT-0190) or rAAV2/9-VGAT1-mCherry-WPRE-pA (5.44E+12 vg/ml) was used as controls.

To verify the specificity of the VGAT-promoter driven viruses, we injected a mixture of rAAV2/9-DIO-EYFP (5.14E+12 vg/ml, Cat. No. PT-0012) and rAAV2/9-VGAT1-mCherry-WPRE-pA viruses (1:1) into the bilateral APC of VGAT-Cre mice.

Fiber photometry. To assess calcium changes, rAAV2/9-hSyn-GCaMp6m-WPRE-hGH-pA (300 nl, 5.14E+12 vg/ml, Cat. No. PT-0148), rAAV2/9-VGAT1-GCaMp6m-WPRE-hGH-pA (300 nl, 5.79E+12 vg/ml, Cat. No. PT-3317), or rAAV2/9-CaMKIIa-GCaMp6m-WPRE-hGH-pA (300 nl, 5.04E+12 vg/ml, Cat. No. PT-0111) was injected into the APC (AP, +0.75 mm; ML, -3.25 mm; DV, -5.2 mm) unilaterally in C57 mice. Additionally, rAAV2/9-DIO-GCaMP6m-WPRE-pA (300 nl, 6.21E+12 vg/ml, Cat. No. PT-0283) was injected into the APC of VGAT-Cre or CaMKII-Cre mice. Two weeks later, the optical fiber (diameter: 200 μm; N.A.: 0.37; length: 5.5 mm; Inper, China) was embedded at the APC by attaching dental cement to the skull. After a one-week recovery, Ca2+ signals of APC were recorded by the three-color single-channel fiber photometry system (ThinkerTech, China).

Mice were initially placed in a square box and allowed habituation for 30 minutes. During the fiber photometry experiments, odor stimuli—including water, anisole (Sigma, Cat. No. 296295), and 2MT (Tokyo Chemical Industry, Cat. No. M0285) were presented via air puff or cotton swab. Each odor was tested across six repeated trials with 1-minute inter-trial intervals. In each trial, mice were exposed to the odor for 1 second. For air-puff or cotton swab delivery, a Pasteur pipette or a cotton swab containing 50 μl of the odor solution was manually introduced into the chamber for 1 second. By clicking the Mark button on the software (ThinkerTech, China) at the moment when the odorant stimuli were delivered, the behavioral and fluorescent events could be time-locked. The fluorescence signal of each mouse was analyzed six times.

Neurons and terminals expressing GCaMP6m were stimulated by laser intensity at 470 nm wavelength (40 μW) to obtain calcium-dependent fluorescence signals, while motion artifacts were corrected by laser signal at 410 nm (20 μW). The obtained calcium signal was analyzed by MATLAB software and calculated (F−F0)/ F0 (average baseline fluorescence signal) to calculate the changed value of the Ca2+ signal (ΔF/F). The 5-second window around the event was analyzed, using a 2-second window before the stimulus as the baseline. The result was the averaged data from the first six Ca2+ responses (six trials) of each mouse.

Optogenetic manipulation. To optogenetically manipulate APCGABA terminals within the MD, two weeks after the bilateral virus injection into the APC, the optical fibers (diameter: 200 μm; N.A.: 0.37; length: 4.0 mm; Inper, China) were embedded at the MD by attaching dental cement to the skull bilaterally. One week later, behavioral tests were performed. In experiments, the optical fiber in the mice's heads was connected to the laser source through a sleeve line.

For optogenetic manipulation, the intensity of the blue light (473 nm, 2.5 mW, 10 ms, 20 Hz) or the yellow light (594 nm, 5 mW, 10 ms, 20 Hz) was measured and determined by the photometer and then controlled by the device (QAXK-LASER, ThinkerTech, China). On the first day, mice had their heads interfaced with the laser system but were not exposed to any light stimuli. The following day, they underwent distinct exposures to either blue or yellow light stimuli to observe differences in behavioral phenotypes. Mice in the control virus groups had the same stimulation regimen.

In detail, during the entire pain experiment, we initially exposed the neurons or circuits to light for 1 minute to enable control. Following this, we employed a stimulation regimen of 1 minute ON and 5 minutes OFF to assess mechanical and thermal sensitivity. During the ON phase, we conducted the necessary measurements. Afterward, the mice were given a 5-minute rest before we repeated the aforementioned steps. For the hot plate test, we used the 3 minutes ON and 5 minutes OFF stimulation regimen.

Chemogenetic manipulation. To manipulate the activity of specific neurons, mice expressing chemogenetic viruses were intraperitoneally injected with CNO (2 mg/kg, APExBIO Technology LLC, Cat. No. A3317) prior to the experiment. After a period of 30 minutes, during which the drug became effective, behavioral experiments were conducted. In the actual experimental procedure, mice are initially administered saline intraperitoneally on the first day to assess their behavioral phenotypes. On the following day, CNO is injected intraperitoneally into the mice to evaluate any differential behavioral phenotypes.

Innate fear detection. In experiments to test the fear, the proportion of the freezing time, the movement distance, and the behavior adjacent to the odor were chosen as indicators to detect fear. To conduct these experiments, the mice were placed in a square glass box lined with clean filter paper. The mice were allowed to acclimate to the apparatus for 30 minutes the day before the innate fear-associated experiments. On the day of the experiment, the mice were initially placed in the box to explore freely for 10 minutes. Then, either water (20 μl), anisole (20 μl), or 2MT (20 μl) was dropped onto the upper left corner of the filter paper using a pipette. The mice were then exposed to the specific odor for 20 minutes. Finally, the proportion of the freezing time and the movement distance, including the trajectory of the mice within the 20-minute period, were analyzed using software (SuperFcs system, XinRuan, Shanghai, China). For data analysis, we utilized the built-in SuperMaze animal behavior video analysis software, Version 2.0. This software allowed us to adjust the Mobility/Immobility threshold to accurately differentiate between the mobility and immobility states of the mice, as well as modify the Immobility/Freezing threshold to effectively distinguish between immobility and freezing states induced by fear.

After conducting multiple tests, we determined the optimal initial values displayed on the software to be a Mobility/Immobility threshold of 16 and an Immobility/Freezing threshold of 19. Subsequently, we proceeded with the formal experiment. In all freezing-related experiments mentioned in this article, these preset threshold values were consistently used for analysis.

Certain experiments include the quantification of metrics, including the duration of residence in the odor zone and the number of entries into that zone, all of which are processed using SMART V3.0 software.

Timeline for odor delivery. For pain testing experiments, mice were first acclimatized to the behavioral chamber. Following an acclimatization period, a continuous 2MT (or control solvent) odor was initiated by placing a filter paper saturated with the stimulus at the top of the chamber. Pain assays (von Frey, Hargreaves, etc.) began 2 minutes after the onset of odor exposure, and the odor was maintained throughout the entire testing session to ensure animals remained in a sustained state of innate fear during nociceptive measurements.

Complete Freund's adjuvant (CFA) model. To induce chronic inflammation, CFA (10 μl, Sigma, Cat. No. 8. 18752) was injected into the hind paws of the mice. After a period of seven days following the injection, the withdrawal threshold was measured using the von Frey test, while the withdrawal latency was measured using Hargreaves’ test. In order to establish a baseline for comparison, mice that received an injection of saline (0.9% NaCl) were used as control subjects.

Spared nerve injury (SNI) model. Mice were anesthetized under the intraperitoneal injection of pentobarbital sodium (20 mg/kg) and then subjected to nerve injury or sham surgery. The skin on the lateral surface of the thigh was cut, and the sciatic nerve was exposed by separating the muscle with a glass minute needle. The common peroneal nerve and tibial nerve were separated from the sural nerve and ligated with nonabsorbent 4-0 chromic gut sutures (Ethicon), and the nerve 2-4 mm distal to the ligation site was resected. In the sham operation, the sciatic nerve and its three branches were exposed, but the nerve was not ligated. Fourteen days after surgery, the withdrawal threshold and withdrawal latency were measured by the von Frey test and Hargreaves’ test, respectively.

Inflammatory pain measurement (Formalin test). Mice were placed in a square glass box and allowed to acclimate for 10 minutes. A solution of formalin (10%, Xinhongfu Biotechnology) was prepared, which was then diluted to a 1% concentration using normal saline. Subsequently, the diluted solution (50 μl) was injected subcutaneously into the hind paw of each mouse The licking behavior of the mice was then videotaped with a camera immediately after injection. The number of licking episodes in one hour was recorded. The experiment was divided into two phases. The first phase, known as the acute phase, is comprised of the initial ten minutes. The second phase, referred to as the inflammatory phase, consisted of the remaining 50 minutes.

Mechanical sensitivity measurement (von Frey test). On the day of the behavioral experiment, the mice were initially acclimated to the plexiglass chambers (6.5 x 6.5 x 6 cm) and placed on a wire mesh for one hour. The Up-Down method was employed for the experiment75. Briefly, a series of von Frey filaments (North Coast Medical Inc., USA) were used to stimulate the plantar area of the hind paw for a duration of 3 seconds. The responses of the mice to each monofilament were recorded. Each monofilament was tested five times. There was a minimum interval of 5 minutes between each measurement. The stimulus strength that produced a 50% response rate was determined and taken as the withdrawal threshold.

Thermal sensitivity measurement (Hargreaves’ test). On the day of the behavioral experiment, the mice were initially given a one-hour period to acclimate to the plexiglass chambers, which measured 6.5 x 6.5 x 6 cm, on the Hargreaves’ Apparatus (IITC Life Science Inc., CA, USA). During the experiment, a radiant heat beam emitted by the machine was focused on the hind paws of the mice. Each mouse was subjected to a total of five exposures to the radiant heat beam, and the incubation period of paw withdrawal was carefully recorded. To ensure accurate data, there was a minimum interval of 10 minutes between each experiment. In order to prevent tissue damage and minimize the potential for pain sensitization, the duration of the radiant heat beam exposure was limited to a maximum of 20 seconds.

Acute thermal pain measurement (Hot plate test). The mice were placed on a hot plate (IITC Life Science Inc., CA, USA) at a temperature of 47 °C, and both the latency (time taken to respond) and episodes of nocifensive responses (such as jumping and licking) were carefully recorded. To avoid potential tissue damage, the duration of the 47 °C hot plate test was limited to 3 minutes. The control group was exposed to a temperature of 25 °C.

Acute mechanical pain measurement (Pinprick Test). The hind paw of the mice was gently stimulated ten times with a blunt needle that wouldn’t pierce or injure the skin. There was at least a one-minute interval between the each stimulation. The number of paw withdrawal reactions was recorded during these ten experiments, and the percentage of reactions was calculated as: (number of paw lifts)/(total trials [n=10]) × 100%.

Rotarod Test. Mice were tested on an accelerated rotary rod (IITC Life Science Inc., CA, USA). The training phase spanned the first two days, during which the mice were trained to maintain balance. The starting speed at 5 rounds per minute, gradually reaching a maximum speed of 20, and the duration of each training session was 5 minutes. On the third day, the test phase began, with the starting speed set at 5 and the maximum speed set to 40. The experimental duration was 5 minutes, during which the instrument automatically recorded the time it took for the mice to drop. The experiment was repeated twice, and the average value was calculated.

Balance beam. The balance beam test was performed using a 1-meter-long beam with a 12 mm wide flat surface, positioned 50 cm above the bench and supported by two rods. A black box containing nesting material from the home cage was placed at the far end of the beam to serve as an incentive. Mice were acclimatized to the apparatus for at least two days prior to testing. A refresher training session was conducted approximately 10 minutes before the formal test. If a mouse stalled, sniffed, or failed to advance, it was gently encouraged from behind using a gloved finger. It is worth noting that overtraining could increase stalling behavior due to overfamiliarity with the task. Conversely, mice that failed to traverse the beam after two training days received additional training. Once a mouse entered the black box, it remained there for approximately 15 seconds before the next trial. On the test day, the time taken to cross the beam (s) was recorded for each animal. The apparatus was cleaned with water and 75% ethanol after each trial to remove urine and feces.

Withdrawal kinetics. Withdrawal kinetics were assessed by applying gentle mechanical stimulation to the mouse hind paw using a blunt needle that did not puncture the skin. The response was recorded with slow-motion video, and the maximum vertical distance (mm) the stimulated paw was lifted from the ground was measured. The time (s) from initial lift-off to the highest point of withdrawal was also recorded. Based on these parameters, the paw withdrawal velocity (mm/s) was calculated. Three trials were conducted per mouse, and the average value was determined.

Grip strength. Grip strength was assessed using a grip strength meter (BIOSEB, EB Instruments). Each mouse was positioned on a metal grid and allowed to stabilize. The tail was then gently and steadily pulled backward along the plane of the grid until all four limbs were completely released. The peak grip force (g) displayed on the device was recorded. Sixteen consecutive trials were performed per animal, and the mean grip strength was calculated based on the last 12 trials (trials 5–16).

Conditional place avoidance and preference (CPA/CPP) test. The experimental setup consisted of two chambers with distinct visual cues: one chamber had black walls with black ground, while the other chamber had white walls with white ground. There was also a neutral middle area between the two chambers. The mice were allowed to move freely between the chambers, and their positions were recorded using an overhead camera. On the first day of the experiment, mice were acclimated to the device for 30 minutes, allowing the mice to familiarize themselves with the chambers and the visual cues. On day 2, the mice were given a 10-minute period to freely explore the entire device. During this exploration period, the time spent by each mouse in each chamber was recorded.

On days 3, 4, and 5, the mice underwent training sessions. For the CPA test, mice were placed in the white box with no light or saline injection for a duration of 30 minutes on the morning of the training day. Four hours later, mice were placed in the black box and were exposed to either yellow light stimulation or received an injection of CNO. For the CPP test, mice were kept in the black box with no light or saline injection. Four hours later, mice were placed in the white box and were subjected to blue light stimulation or CNO injection.

On day 6, mice were once again given a 10-minute period to freely explore the entire apparatus, and the time they spent in each chamber was recorded. The changes in position preference of mice before and after training were analyzed.

Activity-dependent Fos induction by innate fear. 4-hydroxytamoxifen (4-OHT, Sigma, Cat. No. H6278) was dissolved in ethanol at a concentration of 20 mg/mL and shaken at 37 °C for 15 minutes, after which the solution was aliquoted and stored at -20 °C. Before use, 4-OHT was re-dissolved in ethanol by shaking at 37 °C for 15 minutes, followed by the addition of corn oil to obtain a final 4-OHT concentration of 10 mg/mL. Finally, the ethanol was removed by vacuum evaporation through centrifugation prior to use.

To label neurons specifically activated by innate fear odor 2MT, mice were housed individually before the odor exposure experiment. On the day of the experiment, 2MT solution (20 μl) was applied to filter papers, with H2O (20 μl) or anisole (20 μl) used for the control group. These filter papers were then placed in the cages for 1.5 hours. Subsequently, the mice received an intraperitoneal injection of 4-OHT at a dosage of 50 mg/kg in a final concentration of 10 mg/ml and were exposed to 2MT for an additional 1.5 hours. One week later, the brains of these mice were harvested for further analysis.

For the 2MT re-exposure experiment, TRAP2::Ai14 mice were acclimated to the experimental environment for two days prior to the initial exposure. On the day of the first exposure, the mice were exposed to 2MT (20 μL) for a duration of 3 hours. At 1.5 hours after the start of this exposure, 4-OHT (50 mg/kg) was administered by intraperitoneal injection. One week later, the re-exposure experiment was conducted, with the mice again being adapted to the environment for two days beforehand. During this re-exposure experiment, one group of mice received 2MT (20 μL) and another group received anisole (20 μL) for a period of 3 hours. Following the completion of the odor exposure, the brains were perfused for c-Fos staining.

Immunofluorescence and imaging. Mice were deeply anesthetized with isoflurane and perfused transcardially with 0.1 M PBS (phosphate-buffered saline) followed by PFA (4% paraformaldehyde). Dissected brain tissues were fixed in 4% PFA for an overnight period and then transferred to the sucrose (30%) at 4 °C. After the brains were dehydrated completely by sinking in sucrose, the brains were frozen in Tissue-Tek O.C.T (From SAKURA) and sectioned coronally (35 μm) with the freezing microtome (Leica CM1950, Leica Biosystems). The obtained sections were washed three times with PBS (at least five minutes each) and then incubated with blocking buffer (10% sheep serum in PBS plus 0.3% Triton X-100 in PBS) for one hour at room temperature. Brain sections were incubated with primary antibodies (Rabbit anti-GABA, 1:500, Sigma, Cat. No. A2052; Rabbit anti-c-Fos, 1:500, Synaptic Systems, Cat. No. 226 003) in the blocking buffer overnight at 4 °C. After washing three times with PBS the next day, brain sections were incubated with secondary antibodies (Goat anti-rabbit Alexa Fluor 488, 1:1000, Jackson, Cat. No. 111-545-003) for two hours in the blocking buffer. In the end, the sections were washed again three times with PBS, and then stained with DAPI, washed, mounted, and sealed after dropping anti-fluorescence quench. Immunostained brain sections were subjected to acquisition imaging using the Olympus FV3000 microscope.

Images obtained by laser confocal microscopy were analyzed using Image J or Adobe Photoshop. To quantify the cell proportion of c-Fos positive neurons or anti-GABA-positive neurons in the PC, three to five randomly selected brain sections were chosen for analysis. Dual-color fluorescence was quantified as above. The final percentage for each result was the average percentage of slices in each mouse.

Fluorescence in situ hybridization (FISH). On the first day, brain slices are washed in DEPC-PBS for 5 minutes, followed by a 5-minute wash in DEPC-PTW (0.1% Tween-20 in PBS), and then incubated in 2x SSC + 0.5% Triton X-100 for 30 minutes. This is followed by two 5-minute washes in DEPC-PTW, after which the slices are incubated in 3% hydrogen peroxide-methanol for 10 minutes to block endogenous peroxidase activity, and then washed twice for 5 minutes in DEPC-PBS. The tissues are then incubated for 10 minutes in triethanolamine-HCl buffer (pH 8.0) containing 0.25% acetic anhydride without ribonuclease to acetylate free amines, followed by three 5-minute washes in DEPC-PBS. The tissue is prehybridized in prehybridization buffer (50% formamide, 25% 2x SSC, 0.3 mg/ml yeast RNA, 0.1 mg/ml heparin, 1x Denhardt’s solution, 0.1% Tween-20, 5 mM EDTA) for 2 hours at room temperature, before hybridizing with digoxigenin-labeled antisense cRNA probes for CAMKPII (873 bp amplified by primers AGTCTCCAAGCCAACCCC and ATAGAGNCGCACACCAGGC from NM_009792.1) and GAD2 (869 bp amplified by primers TCTTTTCTCCTGGTGGCG and AAATCTTGTCCGAGGCGTTCG from NM_008078.1) for 20 hours at 65 °C in the same buffer.

On the second day, the brain slices undergo a 30-minute wash in preheated (65 °C) prehybridization buffer, followed by a 30-minute wash in a 1:1 mixture of preheated (65 °C) prehybridization buffer and TBST. Next, there are two 5-minute washes in TBST, a brief wash in a 1:1 mixture of TBST and TAE, and three 5-minute washes in TAE. Unbound probes are removed by electrophoresis at 60V for 2 hours in a 2% agarose gel, after which the tissue is incubated overnight at 4 °C for 36 hours in Anti-Digoxigenin-POD antibody (Roche, Cat. No. 11207733910), diluted 1:1000 in PBT supplemented with 10% sheep serum.

On the third day, the brain slices are washed three times in TNT buffer, each for 10 minutes, and incubated with anti-IF 488 antibody (Servicebio, Cat. No. G1231), diluted 1:1000 in TBST containing 3% H2O2, for 20 minutes at room temperature, followed by immunofluorescence staining using antibodies against Chk pAb to mCherry (1:1000, abcam, Cat. No. ab205402) and Goat pAb to Chk IgY labeled with Alexa Fluor 594 (1:500, abcam, Cat. No. ab150172).

Brain slice electrophysiology. Mice were anaesthetized with isoflurane and then perfused with ice-cold oxygenated N-Methyl-D-glucamine (NMDG) artificial cerebrospinal fluid (ACSF) (20 mL) that contained (in mM): KCl (2.5), NMDG (93), NaHCO₃ (30), NaH₂PO₄ (1.2), glucose (25), HEPES (20), thiourea (2), Na-pyruvate (3), CaCl₂ (0.5), Na-ascorbate (5), MgSO₄ (10), and glutathione (3) (pH 7.3-7.4, 300-305 mOsm). Coronal slices (300 μm thickness) were then removed to ice-cold oxygenated NMDG-ACSF and sectioned on a vibrating microtome (VT1200s, Leica). The brain slices were transferred to oxygenated NMDG-ACSF at 32 °C for incubation and recovery, followed by oxygenated N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) ACSF that contained (in mM) 92 NaCl, 2.5 KCl, 1.2 NaH2PO4, 30 NaHCO3, 20 HEPES, 25 glucose, 3 Na-pyruvate, 5 Na-ascorbate, 2 thiourea, 2 CaCl2, 2 MgSO4 and 3 GSH (PH 7.3-7.4, 300-305 mOsm) for more than 1 h at 25 °C. Whole-cell patch-clamp recordings were performed in oxygenated standard ACSF that contained (in mM) 129 NaCl, 1.2 KH2PO4, 3 KCl, 2.4 CaCl2, 3 HEPES, 1.3 MgSO4, 10 glucose, and 20 NaNHCO3 (PH 7.3-7.4, 300-310 mOsm, oxygenated with 95% O2 and 5% CO2) at 25 °C.

Patch-clamp electrophysiology data were acquired with a MultiClamp 700B amplifier (Molecular Devices) and Clampfit pClamp 10.0 software. Signals were low-pass filtered at 2 kHz and digitized at 5 kHz using Digidata 1550B (Molecular Devices). The pipette (6-8 MΩ) was pulled by a micropipette puller (P-1000, Sutter instrument) and filled with the internal solution that contained (in mM): 130 K-gluconate, 5 KCl, 4 Na2ATP, 0.5 NaGTP, 20 HEPES, 0.5 EGTA (PH 7.28, 290-300 mOsm). To record an input-output curve of neuronal excitability, 10 pA command current steps with 500 ms pulses were injected from 0 to +150 pA, and the number of action potentials (APs) was quantified for each step. To confirm the efficacy of hM4Di-mediated inhibition and hM3Dq-mediated excitation, CNO (10 μM) was bath-applied. Optical stimulation was delivered using a laser through an optical fiber 200 μm in diameter positioned 0.2 mm from the surface of the brain slice. To confirm the efficacy of ChR2-mediated activation, fluorescently labeled neurons were stimulated with a 473 nm laser using 20 Hz stimulation protocols with a pulse width of 10 ms. In similar, the functional expression of eNpHR3.0 was assessed by applying yellow (594 nm) laser light stimulation.

To examine functional inhibitory projections from the APC to the downstream regions and sIPSCs of MD neurons, recordings were conducted in the presence of CNQX (10 μM, Sigma) and patch pipettes were filled with the chloride-based internal solution that contained (in mM): 145 CsCl, 10 EGTA, 10 HEPES, 2 MgCl2, 2 CaCl2, 2 Mg-ATP and 5 QX-314. Neurons were held at −70 mV using the voltage clamp for recording inward Cs+-IPSCs. For evaluating synaptic identities, GABA-mediated IPSCs were blocked by the bath application of bicuculline (10 μM, Sigma, Cat. No. 14343). To test direct synaptic connections, both TTX (1 μM, Sigma, Cat. No. A0224) and 4-AP (500 μM, Sigma, Cat. No. 275875) were used to restore monosynaptic current.

To investigate the effect of 2MT exposure on neuronal excitability, mice were placed in a cage containing 2MT for 1 hour prior to the experiment. Subsequently, they were sacrificed for brain slicing to ensure the sustained effect of the 2MT odor.

To investigate the effect of odor exposure on neuronal excitability, mice were continuously exposed to 2MT or water for 60 minutes by applying 20 μl odorant solutions to filter paper positioned at the testing arena. Acute brain slices containing the MD thalamus were then prepared for electrophysiological recordings.

To investigate the effect of chemogenetic inhibition on neuronal excitability, mice received intraperitoneal CNO injections (30 minutes prior) to either 1-hour 2MT or water exposure, after which acute brain slices containing the MD thalamus were prepared for electrophysiological recordings.

Quantification and statistical analysis. All experiments and data analyses were performed in a double-blind manner. GraphPad Prism v.8.0.1, Olympus FV10-ASW 4.0a Viewer, ImageJ, Clampfit software v.10.0, SuperFcs system, SMART V3.0 software, and MATLAB R2017b software were used to analyze the data. Unpaired Student’s t-test (or Mann-Whitney U test), paired Student’s t-test (or Wilcoxon signed-rank test), and one-way ANOVA or two-way ANOVA test followed by Bonferroni’s test for multiple comparisons were used to determine statistical differences. Shapiro-Wilk test was used to test whether the data followed a normal distribution. When data were normally distributed, paired and unpaired t-tests were implemented. When data did not conform to a normal distribution, the Wilcoxon signed-rank test was used for paired comparisons, while the Mann-Whitney U test was used for unpaired comparisons. Results with *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 were statistically significant. All data were presented as the mean ± s.e.m except for Supplementary Fig. 6 and 12 shown as box and whisker plots (medians, quartiles (boxes), and ranges from minimum to maximum (whiskers)). Detailed analysis results are shown in Supplementary Data 1.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This study by Jia et al. examines the intersection between innate fear and pain. They start by characterizing how an innately fear-inducing odor affects pain across multiple modalities and timescales. 2MT induced analgesia in naïve animals as well as attenuated mechanical and thermal hypersensitivity in models of chronic pain. Figure 1 is quite a nice behavioral demonstration. The authors then perform experiments and reach the conclusion that GABAergic neurons in the anterior piriform cortex are activated by 2MT and that activating or inhibiting these neurons or their projections to the medial dorsal thalamus bidirectionally affect baseline and injury-induced nociception. They also find that pain does not affect fear responses, suggesting a hierarchy of survival behaviors that prioritizes predator evasion over attention to noxious stimuli. The study is interesting. However, here are concerns – particularly regarding if the statements made are substantiated by the data- that need to be addressed by the authors before publication.\nMajor comments:\n1. It is unclear that the manipulations are being done on a functionally relevant population. The cFOS in Figure 1 is quite different from the DREADD/mCherry expression for the recordings and manipulations in figure 2/3.\n2. The authors claim that inhibiting APC GABA neurons attenuates the effect of predator odor on pain and that activating APC GABA neurons recapitulates the effect of odor on pain. The data in the main and supplementary figures do not necessarily support this claim.\na. The activation of these neurons (hm3d and ChR2 experiments) should recapitulate what happens with the 2MT response. Figure 3 paints a far more complicated picture. Although, the authors state in the abstract “Conversely, activation of APCGABA neurons or the APCGABA-MD circuit recapitulates fear-induced analgesia.” This statement is not supported by figure 3 or 5.\nb. APC GABA neuron inhibition affects pain independent of odor. The authors argue that because there was no significant difference between the inhibited groups with and without odor, the effect is not due to a change in baseline sensitivity. At face value this is confusing. First, this was only true for mechanical stimuli which was acknowledged but not discussed. Second, the authors should report the ANOVA interactions in Fig. S7 rather than comparing the two CNO groups. This would show if the effect of inhibition was different with and without 2MT, which I believe is the comparison the authors are trying to make.\nc. The fact that only mechanical sensitivity seems to be further heightened by inhibition and the fact that activation produces analgesia even when predator odor is present (and presumably already activating APC neurons) makes it difficult to fully support the claim that this circuit underlies predator odor analgesia and not nociception in general. While it is important and appreciated that the authors showed the effects without odor (inhibition) and with odor (excitation), it would be helpful to explore the possibility that different manipulations might be able to better distinguish baseline nociceptive effects from odor.\ni. Could the authors try changing their stimulation and inhibition parameters in a way that would not affect baseline thresholds but would still affect odor-induced analgesia?\nd. What proportion of APC GABA neurons are activated by 2MT? Is there subtype specificity that may be confounding the results? Would manipulating 2MT TRAPed APC inhibitory neurons more specifically affect predator odor analgesia and have less of an effect on baseline nociceptive thresholds?\n3. It is inappropriate to say that APC GABA neurons ‘encode’ fear-related olfactory information. Based on the data, the strongest conclusion that can be made is that they respond (transiently) to 2MT. Furthermore, the excitatory neurons in the APC also respond, making any conclusions about encoding more difficult.\n4. There appears to be a mismatch between the neural activity recordings and the effect of this circuit on pain. How long were mice exposed to odors during the fiber photometry experiments? The neurons show clear bursts in activity, but the increase only lasts a few seconds. How could these neurons influence pain across minutes and hours? It would be beneficial to show neural responses during longer exposures that are similar to those the mice experience during behavioral tests.\na. Relatedly, there is some detail lacking in the results and methods sections on how mice were exposed to the odors during behavioral assays.\n5. Figure 5G-I is confusing as presented and the claim based on these data are not currently fully supported. The authors claim that inhibition of APC GABA   MD, “2MT failed to rescue SNI-elicited MD hyperactivity…” (line 299). At minimum, the authors should include mCherry and hM4 groups that were not exposed to 2MT to show the effect in controls and compare it to the effect after inhibition. It would also be helpful to include “SNI” and “2MT” in the labels of these panels and indicate that these data are from MD and not APC slices.\n6. While figure 5 (retro-cre) is not miswritten, there remains the caveat that these neurons could have collateral projections.\n7. Supplementary figure 9 kind of undercuts the story… a very small proportion of fos expressing neurons seem to project to the MD.\n8. As camkII neurons were also activated, it is unclear why they were not functionally tested.\nMinor\n1. Given the fact that excitatory APC neurons are activated by both aversive and neutral odors it would be interesting to know if APC excitatory neurons also project to MD.\n2. What is physiological and pathological pain – these terms are a bit novel for the abstract.\n3. Hargreave’s should be written Hargreaves or Hargreaves’\n4. The specificity of the transgene targeting using such promoter based viral vectors is always complicated – how often was specificity tested?\nIn summary, the authors convincingly show that this circuit can modulate pain responses, but more work should be done if the authors wish to make the claim that this pathway is the central mechanism/circuit for innate fear-induced analgesia.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "In this study, Jia et al. provide an extensive assessment of the role of the anterior piriform cortex in mediating the analgesic effect of innate fear. The topic is highly interesting but complex as it is not easy to conceptually link the impact of innate fear on pain sensation. To do so the authors exposed mice to the odor of predators and then proceed to a battery of pain-related tests, showing that animals previously exposed to the odor predator exhibit reduced nocifensive behavioral responses.\nUsing an extensive combination of anatomical, imaging, and functional approaches they progressively identify a GABAergic projections from the anterior proportion of the piriform cortex to the mediodorsal thalamus that may be both necessary and sufficient for this effect. This result calls for partcular attention since long-distance inhibitory projections from cortex to the MD has never been reported before in mice. The amount of experimental work is impressive and all approaches are by themselves state-of-the-art, but the meaning and interpretation of all these experiments also appear as somewhat questionable. In addition, crucial methodological details are missing as explained below.\nMethodology/behavioral paradigm\nThe exact protocol for odor exposure is not clear enough in relation with the many experiments reported. When exactly and for how long are animals exposed to the different odours? Is it acute or multiple exposures? Is it during each beahvioral assay or just before? Dpending on the answers to these questions there may be new issues to consider. Too many crucial details are lacking preventing the reader from understanding what exactly has been done.\nFor all histological controls, not enough data are provided to document the spread of viral infusion. For all interventions (DREADD and opto), general histology should be provided showing shades of spread for each included individual as is usually the norm.\nWithout these essential controls/information, the conceptual ground of the whole paper is unclear.\nGABAergic projection from cortex to thalamus\nThe authors should really comment on whether this was expected or not. Very importantly, greater care is needed to ensure that this important claim is unambiguously supported by included data.\nFor example, neurons that are positive for vgat may also transmit glutamate, how do we know if that is not the case here?\nViral spread into TRN which is mentionned in the paper is guaranteed to yield thalamic inhibition but this is not taken into consideration. The authors also reasonned that there is a higher proportion of GAD2+ proportion of cells labelled for dtomato than it is for CamKII cells (Figure 2c and 2d, the first d should be c though) but the panel c shows very few GAD2+ cells whereas CAMKII+ cells are abundant. This casts further doubt on that inhibitory CT route which would be further fraction at best of those GAD2+ cells (those projecting to the MD). All these issues add on each other, making the whole point not compelling given the importance of the claim.\nTaken together, these issues largely undermine the paper to the extent that it is unclear whether the main points could still hold if they can be adequately addressed and making any more minor comments irrelevant at this stage. The paper would also benefit from professional level proof-reading for english.", "reviewer_4": "The manuscript by Jia et al. describes a very interesting set of findings revealing a potential circuit underlying pain suppression by innate fear. Of particular interest this is a novel long-range GABAergic projection originating from Anterior Piriform Cortex (APC) and targeting MD thalamus. While I think the basic story is sound, there are some issues with statistical comparisons and the interpretation of results that need to be addressed. In addition, it is unclear how the slice results relate to the behavioral results – there are questions about how exactly manipulations were done and what were the possible mechanisms that lead to long-lasting effects of 2MT exposure / chemogenetic inhibition persisting into slice experiments.\n1. It would be useful to know what % of APC neurons activated by fear are a) GABAergic, b) project to MD, and c) are both.\n2. For 4e – what was the protocol for 2MT exposure? Specifically what was the frequency and duration of 2MT exposure, and what was the timing relative to brain slice preparation? What do the authors think is the mechanism underlying this effect?\n3. The causal demonstration of the role of this circuit is in Fig. 5 and 6. Fig. 5 shows that chemogenetically inhibiting MD-projecting GABAergic neurons in APC increases pain sensitivity in normal mice and SNI mice exposed to 2MT. Fig. 5g-i show that chemogenetic inhibition of GABAergic APC-MD neurons increases the excitability of MD neurons in 2MT-exposed SNI mice.\na. The protocol for 2MT exposure and chemogenetic inhibition are not explained. Specifically what was the frequency and duration of 2MT exposure? What was the timing of 2MT exposure relative to brain slice preparation? Was chemogenetic inhibition done preceding/during every 2MT exposure, and if not, what was the timing of chemogenetic inhibition relative to 2MT exposure?\nb. Does the effect of chemogenetic inhibition on MD neuron excitability depend on 2MT exposure, or does the same effect occur in mice which undergo chemogenetic inhibition but have not been exposed to 2MT? This seems like an important and straightforward experiment.\nc. What is the possible mechanism for this? Was there a change in input resistance suggestive of increases in spontaneous inhibitory input (potentially from GABAergic APC-MD terminals) in MD neurons? Did the authors measure spontaneous inhibitory input? This mechanism is important because it’s hard to imagine how transient inhibition of an inhibitory input would end up leading to persistent changes in the excitability of MD neurons.\n4. Fig. 6 shows that inhibition of GABAergic APC-MD terminals increases pain sensitivity in mice exposed to 2MT +/- SNI. Based on Supp. Fig 6+7, it seems that the effect is really the same regardless of whether mice have been exposed to 2MT and/or SNI – inhibiting these cells always leads to an exacerbation of pain sensitivity (albeit this effect is smaller in absence of 2MT). So it’s not just that it abolishes the effects of 2MT, but it also seems to increase pain sensitivity at baseline – this is an important point that should be noted explicitly and clearly.\n5. Based on Supp Fig. 6c, inhibiting APC-MD GABAergic neurons increases CPA. This means that inhibiting these neurons is aversive, independent of pain. Conversely increases in the activity of these neurons are probably rewarding. This is a caveat that makes the interpretation of Fig. 6i less clear – specifically, the CPP shown in Fig. 6i may not be related to an alleviation of pain but rather to independent rewarding effects of the stimulation.\n6. It’s a bit odd to use 20Hz pulse trains to activate eNpHR – its kinetics are too slow to respond to these pulse trains so it’s a bit better / simpler to just use continuous light. Related to this, was the reported light power the peak light power, i.e., each pulse reaches a peak of 5 mW, or was the reported light power of 5 mW the net / average power measured over light ON and light OFF periods?\n7. Line 209 – the authors should not report “a trend towards a statistically significant decrease.” The decrease is not statistically significant. They can say that this was a trend that did not reach statistical significance, or they can perform additional experiments to increase the n to confirm that the result does / does not reach statistical significance.\n8. Related to this, almost all of the statistical tests are performed as comparisons within a group on two days, where the author report significant changes in the experimental group but not in the control group. Really, this is not appropriate – the authors should perform statistical tests which directly compare the experimental and control groups, either using a 2-way ANOVA, or by calculating a difference score for each animal across the two days and then comparing these difference scores between the experimental and control groups to show a significant difference. I think the authors need to do this, if not in all cases, for their key findings.", "reviewer_5": "In this manuscript by Jia and colleagues, the authors investigate the neural mechanisms by which innate fear suppresses pain sensitivity in male mice, identifying a GABAergic projection from the anterior piriform cortex (APC) to the mediodorsal thalamus (MD) as a key component. Using a combination of viral tracing, electrophysiology, fiber photometry, chemogenetics, and optogenetics, the authors claim to show that innate fear—triggered by predator odor (2MT)—induces analgesia via this corticothalamic circuit.\nWhile the study is methodologically rich and addresses an important topic, several conceptual, methodological, and interpretive issues significantly dampened the excitement about the manuscript and its current impact. The manuscript overstates its mechanistic conclusions. In my opinion, the core effects observed are better interpreted as modulations of pain sensitivity, rather than evidence of a distinct analgesic state induced by innate fear.\nMajor points:\n1. The manuscript relies on the premises that the 2MT odor promotes innate fear, but the behavioral characterization of the 2MT is very poor. The only information we have is supplementary Figure 1, with an example of the trajectories and the overall quantification of freezing.\na. The methods section lacks a description of how freezing is calculated. The authors only mention that it's done via software.\nb. Regarding the methods, the authors mention six trials for each odor, but we always see only the averages of the six trials. Is there a desensitization/habituation to the odor? In addition, it's mentioned that there is a one-second exposure of the odor per trial, but there is no mention of how long the wait between trials is.\nc. Do all mice display innate fear responses to the odor? Were mice excluded?\nd. There is a critical inconsistency in the odor delivery protocols. The method section for the odor exposure to induce innate fear described for the fiber photometry are different from the one described in innate fear detection. More importantly, I could not find any information on how the odor is integrated with the pain assays in the manuscript. Is the odor delivered continuously while the pain assays (like von Frey test, Hargreave's test, etc) are ongoing? Are the mice exposed to it only once before initiating the battery of pain assays or at the beginning of each pain test? The timeline for the behavioural manipulations needs to be clarified.\nAll these points should be carefully addressed in order to understand the following figures of the manuscript and how exactly innate fear impacts pain sensitivity. This is particularly true since all the key readouts (e.g., increased thresholds, reduced nocifensive responses) may reflect motor suppression (freezing) rather than analgesia.\n2. Although not clearly stated, the figures, figure caption and main text suggest that the same mice undergo the series of pain sensitivity tests for each experiment. For example, the same group of mice undergoes all the tests in the main figure 1. This suggests the authors rely on repeated pain testing in the same mice across multiple conditions without proper counterbalancing. This could lead to several problems. Stress-Induced Analgesia (SIA) is a well-documented phenomenon where repeated exposure to stress or pain itself alters subsequent sensitivity, and this could have a significant impact on the overall results and lead to possible confounds. There is no evidence that the pain sensitivity test exposure order was controlled, counterbalanced or test-order randomized. This weakens the overall conclusions of the manuscript.\n3. The supplementary figure 6 Shows that modulation (particularly the inactivation) of APC GABA neurons regulates pain sensitivity without innate fear. This undermines the central claim that fear is required for analgesia. In response, the authors add Supplementary Figures 7 and 11, in which a comparison between the pain thresholds following neuronal inhibition with and without innate fear exposure is shown. However, these comparisons are convoluted, spread across multiple figures, and not clearly described. In detail:\na. Supplementary Figure 7 a-b - compared with supplementary figure 6 a-c with main figure 3 a-d\nb. Supplementary Figure 7 c-d - compares supplementary figure 10 with main figure 5 a-d\nc. Supplementary Figure 7 e-f - compares supplementary figure 11 a-c with main figure 6 a-d\nd. And then, Supplementary Figure 11d-g relates to the main figure 6g-l\nAll of this back and forth is very chaotic and takes the attention away from the fact that there is a modulation of pain sensitivity alone, without the need for innate fear. Moreover, on the main text, the authors claim that these supplementary controls have \"no significance differences\" (line193-195;) and \"did not show any differences in mechanical nociception (line 312-313)\" and that is not true, which is simply incorrect based on the actual data of the supplementary figure. Moreover, the results of the thermal pain are not mentioned or compared. The data suggest that APC-GABA neurons modulate pain tonically and do not require fear for their effect. The manuscript selectively emphasizes data that support the fear angle and buries conflicting data in chaotic supplements.\n4. No effort is made to dissociate freezing-induced motor suppression from true analgesic effects. Since 2MT induces freezing, reduced reactivity to pain stimuli could reflect behavioral suppression, not analgesia. The rotarod test is used to argue that circuit manipulations do not impair motor function. However, the rotarod primarily assesses gross motor coordination and balance, not fine motor output or spontaneous movement. - Paw withdrawal tests rely on quick reflex-like responses that may not be fully reflected in rotarod performance. Thus, motor slowing, freezing behavior, or motivational changes could still confound nociceptive assays.\n5. The authors fail to adequately address the possibility that observed pain suppression results from inhibited motor output (such as freezing) rather than altered pain perception. The rotarod primarily assesses gross motor coordination and balance, not fine motor output or spontaneous movement. - Paw withdrawal tests rely on quick reflex-like responses that may not be fully reflected in rotarod performance. In addition, it seems that the activation of the APC-GABA promotes freezing in naive mice and decreases locomotion. Thus, motor slowing, freezing behavior, or motivational changes could still confound nociceptive assays. There can be a change in pain sensitivity merely because the manipulations impact motion.\n6. In several key experiments (e.g., Fig. 2d: 3 mice; Fig. 2g–l: 5 mice per group), sample sizes are extremely small. The authors do not provide power analyses or variance estimates to justify these choices. How did the authors access the statistical power?\nMinor points:\n1. The introduction lacks referencing, e.g., lines 30, 32, 40, and 46.\n2. The data referenced from lines 161 to 168 is not provided. It would be relevant to include such a dataset, yet only mechanical nociception is mentioned; thermal is missing.\n3. Fig. 1b—The latency and number of licks or jumps are taken into consideration together, but there is massive variability. These are distinct behaviors and should be analyzed separately.\n4. Fig. 1c - How is the % of response for the pinprick test calculated?\n5. Fig. 2d - typo, the representative figure is panel c instead of d\n6. Fig. 2g-l, heatmaps—not all animals seem to have a modulation of the neuronal activity during the delivery of 2MT. What could be the explanation?\n7. Fig. 7 - Why, for physiological pain conditions, was only the thermal pain tested and not the mechanical?\n8. Supplementary Figure 3 b-d - anisole group missing, therefore the conclusion on lines 120-121 that \"These findings provide additional evidence for the involvement of APC neurons in processing fear-related olfactory information.\" could potentially be wrong since there is no odor that does not elicit fear responses.\n9. Supplementary Figure 3g and j - only cotton swab. Airpuff is missing, and no explanation is provided for it to be the case.\n10. Supplementary Figure 4 - The re-exposure to the arena should have been counterbalanced.\n11. Supplementary Figure 9 and 11 - Typo, the letters associated with the panels are in CAPS.\nThe manuscript's conceptual framework offers an important mechanistic insight into how innate fear modulates pain sensitivity via an olfactory-cortical-thalamic GABAergic circuit. It is conceptually valuable and could contribute strongly to understanding how fear can suppress pain and lead to potential therapeutic targets for pain management. However, the current version of this manuscript contains significant interpretive overreach and methodological limitations. The central claim is not supported by the data as presented, and several key results are confounded. With substantial revision and reframing, the core finding—that an APC-GABA-MD circuit modulates pain sensitivity—could still represent a publishable advance, but not in its current conceptual framing as a fear-induced analgesia mechanism."}, "author_response": "We sincerely appreciate the reviewer’s thoughtful evaluation and constructive suggestions.\nMajor comments:\n1. It is unclear that the manipulations are being done on a functionally relevant population. The cFOS in Figure 1 is quite different from the DREADD/mCherry expression for the recordings and manipulations in figure 2/3.\nWe appreciate the reviewer’s insightful comment regarding the difference between the cFOS patterns and the DREADD/mCherry expression. As shown in original Figure 1d, 2MT exposure activates both inhibitory and excitatory neurons, whereas in Figs. 2 and 3, the DREADD/mCherry experiments specifically target GABAergic neurons, which include a subset of Fos+ neurons. Thus, though partially overlapping, the two groups appeared to unidentical.\nTo directly address this concern, we now employed the TRAP2 strain, which enables genetic labeling and manipulation of neurons activated by a defined stimulation.1 To this end, we bilaterally injected AAV-VGAT-DIO-hM4Gi-mCherry into the APC of TRAP2 mice, followed by 2MT exposure paired with 4-OHT injection. Using this approach, we were able to specifically manipulate 2MT-activated inhibitory neurons. Inhibiting this ensemble significantly attenuated 2MT-induced fear expression and reduced 2MT-induced analgesia, but did not affect baselines thermal/mechanical nociception or locomotion (Response Fig. 1a-k; revised Fig. 4a-k). These findings establish that 2MT-responsive APCGABA neurons are necessary for innate fear-induced analgesia while being functionally distinct from basal nociceptive processing.\nFurthermore, selective activation of 2MT-trapped APC inhibitory neurons was sufficient to evoke freezing and robust analgesia in the absence of 2MT, again without affecting motor performance (Response Fig. 1m-u; revised Fig. 4m-u). These complementary loss- and gain-of-function results demonstrate that 2MT-responsive APCGABA neurons are both necessary and sufficient for innate fear-related analgesia.\nWe have incorporated these data into the revised manuscript (lines 233-246; 275-282).\nResponse Fig 1. Chemogenetic manipulation of 2MT-activated inhibitory neurons in the APC.\na, l, Schematic of the viral injection and behavioral experimental timelines. b, m, Left: image of coronal section showing hM4Di (b) or hM3Dq (m) expression in APC. Right: the diffusion areas of viral expression at coronal level of APC. Subjects are individually colored (n=7). c, n, Effect of chemogenetically inhibiting (c) or activating (n) 2MT-TRAPed APCGABA neurons on the proportion of freezing time and total moving distance (n=7 mice/group). d, o, Baselines pain sensitivity following chemogenetic inhibition (d) or activation (o) of 2MT-TRAPed APCGABA neurons (n=7 mice/group). e, f, Effect of chemogenetically inhibiting 2MT-TRAPed APCGABA neurons on basal nociception (e) or chronic neuropathic pain (f) of 2MT-exposed mice (n=7 mice/group). g-k, q-u, Effect of chemogenetically inhibiting (g-k) or activating (q-u) 2MT-TRAPed APCGABA neurons on motor performance (n=7 mice/group). p, Effect of chemogenetically activating 2MT-TRAPed APCGABA neurons on neuropathic pain (n=7 mice/group). All data are presented as mean ± SEM except for the data in (b) depicted as box and whisker plots. “ns”, no significance; *p < 0.05, **p < 0.01. Significance was assessed by two-tailed paired Student’s t-test or Wilcoxon signed-rank test in (c, d, g-k, n, q-u), and two-way ANOVA followed by Bonferroni’s multiple comparisons test in (e, f, o, p).\n2. The authors claim that inhibiting APC GABA neurons attenuates the effect of predator odor on pain and that activating APC GABA neurons recapitulates the effect of odor on pain. The data in the main and supplementary figures do not necessarily support this claim.\na. The activation of these neurons (hm3d and ChR2 experiments) should recapitulate what happens with the 2MT response. Figure 3 paints a far more complicated picture. Although, the authors state in the abstract “Conversely, activation of APCGABA neurons or the APCGABA-MD circuit recapitulates fear-induced analgesia.” This statement is not supported by figure 3 or 5.\nWe sincerely appreciate the reviewer’s thoughtful assessment of our claims regarding circuit recapitulation. We agree that our original abstract overstated the equivalence between natural 2MT responses and artificial activation paradigms. To more accurately reflect our findings, we have revised the statement to: “activation of APCGABA neurons or the APCGABA-MD circuit induces freezing responses and relieves pain, mimicking fear-induced analgesia” (lines 24-26).\nb. APC GABA neuron inhibition affects pain independent of odor. The authors argue that because there was no significant difference between the inhibited groups with and without odor, the effect is not due to a change in baselines sensitivity. At face value this is confusing. First, this was only true for mechanical stimuli which was acknowledged but not discussed. Second, the authors should report the ANOVA interactions in Fig. S7 rather than comparing the two CNO groups. This would show if the effect of inhibition was different with and without 2MT, which I believe is the comparison the authors are trying to make.\nWe appreciate the reviewer’s insightful critique regarding the interpretation of our inhibition experiments.\nThe observation that basal nociception is affected by manipulation of APCᴳᴬᴮᴬ neurons indeed highlights the complexity of the interpretation. With TRAP2-based strategy to selectively target 2MT-responsive APC inhibitory neurons as presented above, we demonstrate that 2MT-responsive APCGABA neurons are both necessary and sufficient for innate fear-induced analgesia and are functionally distinct from those regulating basal nociceptive processing.\nTo streamlines the presentation and eliminate redundancy, we removed original supplementary Figure 7 and incorporated TRAP data into the revised manuscript (Revised Fig.4, lines 233-246; 275-282).\nWe thank the reviewer again for prompting these important improvements to our study's statistical rigor and interpretation.\nc. The fact that only mechanical sensitivity seems to be further heightened by inhibition and the fact that activation produces analgesia even when predator odor is present (and presumably already activating APC neurons) makes it difficult to fully support the claim that this circuit underlies predator odor analgesia and not nociception in general. While it is important and appreciated that the authors showed the effects without odor (inhibition) and with odor (excitation), it would be helpful to explore the possibility that different manipulations might be able to better distinguish baselines nociceptive effects from odor.\ni. Could the authors try changing their stimulation and inhibition parameters in a way that would not affect baselines thresholds but would still affect odor-induced analgesia?\nWe appreciate the reviewer’s thoughtful suggestions regarding the distinction between baselines nociception and odor-specific effects. As suggested by the reviewer, we conducted optogenetic experiments testing new parameters using a reduced frequency (5 Hz). Unfortunately, we found that the 5Hz-stimulation alters the baselines pain thresholds as well (Response Fig. 3).\nResponse Fig 3. Effects of 5Hz-stimulation of APC-MD circuit on basal nociception.\na, c, Schematic of the viral injection. b, d, Effect of chemogenetically inhibiting (c) or activating (m) APCGABA neural terminals at 5Hz within the MD on basal nociception (n=7 mice/group). *p < 0.05, **p < 0.01*, ***p < 0.001. Significance was assessed by two-tailed paired Student’s t-test in (b, d).\nd. What proportion of APC GABA neurons are activated by 2MT? Is there subtype specificity that may be confounding the results? Would manipulating 2MT TRAPed APC inhibitory neurons more specifically affect predator odor analgesia and have less of an effect on baselines nociceptive thresholds?\nWe thank the reviewer for raising these important questions regarding molecular and functional specificity among APCGABA neurons. To address this, we quantified the proportion of APC GABA neurons that are activated by 2MT. Using traditional c-Fos staining, we found that ~40% of APC GABA neurons (Gad2) were Fos-positive after 2MT (Response Fig. 4, revised Fig. 12a-c).\nAs nicely suggested by the reviewer, we now specifically manipulated 2MT-TRAPed APC inhibitory neurons (Response Fig. 1; revised Fig. 4). In contrast to manipulating the entire APCGABA population, 2MT-trapped neural ensemble clearly dissociates odor-specific from general antinociception. We thank the reviewer again for this informative suggestion.\nResponse Fig 4. Quantification of proportion of APC GABA neurons that are activated by 2MT.\na, A cartoon illustrating c-Fos induction by 2MT exposure. b, c, Representative image (b) and summary data (c) for the percentage of GAD2-positive neurons (ISH, red) co-localized with c-Fos staining (blue) in the APC (n=15 sections from 5 mice). Scale bars, 500 μm and 50 μm. Data are presented as box and whisker plots (medians, quartiles (boxes), and ranges from minimum to maximum (whiskers)).\n3. It is inappropriate to say that APC GABA neurons ‘encode’ fear-related olfactory information. Based on the data, the strongest conclusion that can be made is that they respond (transiently) to 2MT. Furthermore, the excitatory neurons in the APC also respond, making any conclusions about encoding more difficult.\nWe thank the reviewer for this thoughtful comment. We agree that our original wording overstated the interpretation. We have replaced “encode” with “respond to” or “are activated by” 2MT throughout the manuscript to more accurately reflect our findings.\n4. There appears to be a mismatch between the neural activity recordings and the effect of this circuit on pain. How long were mice exposed to odors during the fiber photometry experiments? The neurons show clear bursts in activity, but the increase only lasts a few seconds. How could these neurons influence pain across minutes and hours? It would be beneficial to show neural responses during longer exposures that are similar to those the mice experience during behavioral tests.\na. Relatedly, there is some detail lacking in the results and methods sections on how mice were exposed to the odors during behavioral assays.\nWe thank the reviewer for this valuable comment and apologize for not providing sufficient methodological details in the original submission, which may have created the impression of a discrepancy between neural activity and behavioral effects.\nDuring the fiber photometry experiments, odor stimuli were delivered via air puff or cotton swab presentation. Each odor was tested across six repeated trials with 1-minute inter-trial intervals. In each trial, mice were exposed to odors for 1 second. This transient delivery paradigm was optimized to capture the rapid temporal dynamics of population activity. In contrast, during behavioral assays, odors were continuously presented via filter paper near the testing arena to mimic sustained naturalistic exposure, which can engage downstream plasticity in regions such as the MD (Figs. 5-6) and mediate longer-lasting behavioral modulation.\nIn response to the reviewer's suggestion, we conducted additional fiber photometry recordings during prolonged odor exposure (20-min duration) under conditions similar to our behavioral assays. Our data demonstrated comparable APCGABA neural dynamics during odor zone visits between the initial 5-min phase and the terminal 5-min phase (Response Fig. 5), demonstrating persistent APCGABA activity throughout the exposure period. We have incorporated the above details regarding odor exposure during behavioral testing into both the Results and Methods sections (lines 153-154; 612-619; 684-690).\nResponse Figure 5. Similar APCGABA neural activity in initial vs. terminal 5-min of 20-min odor exposure\na, Schematic of viral injection and fiber implantation in VGAT-Cre mice (left) and a diagram of fiber photometry recordings triggered by entries into the odor zone containing 2MT (right). b, c, Heatmaps (b) and AUC analysis (c) showing Ca2+ transients upon entry into the 2MT zone during the initial and terminal five-minute periods. All data are presented as mean ± s.e.m. “ns”, no significance. Significance was assessed by two-tailed unpaired Student’s t-test in (c).\n5. Figure 5G-I is confusing as presented and the claim based on these data are not currently fully supported. The authors claim that inhibition of APC GABA  MD, “2MT failed to rescue SNI-elicited MD hyperactivity…” (lines 299). At minimum, the authors should include mCherry and hM4 groups that were not exposed to 2MT to show the effect in controls and compare it to the effect after inhibition. It would also be helpful to include “SNI” and “2MT” in the labels of these panels and indicate that these data are from MD and not APC slices.\nWe thank the reviewer for the thoughtful critique of our MD thalamus electrophysiology data and agree that additional controls and clearer presentation are needed to support our conclusions.\nIn response, we performed additional patch clamp recordings from MD neurons in SNI mice expressing mCherry or hM4Di. We found that inhibition of MD-projecting APCGABA neurons did not further alter the SNI-induced MD hyperactivity, as measured by firing rate and rheobase, likely reflecting a ceiling effect of SNI-induced MD neural hyperactivity. Following 2MT treatment, MD hyperactivity was normalized in mCherry-expressing mice, but this rescue effect was abolished in hM4Di-expressing mice (Response Fig. 6, revised Fig. 6g, h).\nWe have also revised the figure presentation by adding “SNI” and “2MT” labels and explicitly indicating that the recordings were performed from MD, not APC, slices. These new data and clarifications have been incorporated into the revised manuscript (lines 363-376).\nResponse Fig 6. Inhibition of MD-projecting APC neurons attenuates 2MT-reversed MD hyperactivity in chronic pain.\na, b, Effects of the chemogenetic inhibition of MD-projecting APCGABA neurons on the firing rate (a) and rheobase (b) of MD neurons recorded from either water or 2MT-exposed SNI mice (DIO-hM4Di SNI&H O, n=18 neurons from 4 mice; DIO-mCherry SNI&H O, n=26 neurons from 4 mice; 2 2 DIO-hM4Di SNI&2MT, n=27 neurons from 3 mice; DIO-mCherry SNI&2MT, n=23 neurons from 3 mice). Significance was assessed by two-way ANOVA followed by Bonferroni’s multiple comparisons test in (a) and one-way ANOVA followed by Bonferroni’s multiple comparisons test in (b). All data are presented as mean ± s.e.m. **P < 0.01, ***P < 0.0001, not significant (ns).\n6. While figure 5 (retro-cre) is not miswritten, there remains the caveat that these neurons could have collateral projections.\nWe thank the reviewer for this insightful comment. We agree that, while the retro-Cre approach effectively targets APC→MD projection neurons, it cannot exclude the possibility that these neurons possess collateral projections to other regions. We have now acknowledged this caveat in the revised discussion (lines 511–522). To address this limitation, our optogenetic experiments specifically manipulated the APC neural terminals within the MD and recapitulated the behavioral phenotypes observed during suppression of MD-presynaptic APCGABA neurons, demonstrating the functional specificity of this pathway.\n7. Supplementary figure 9 kind of undercuts the story… a very small proportion of fos expressing neurons seem to project to the MD.\nWe thank the reviewer for this thoughtful observation. We fully acknowledge that the relatively small proportion of Fos⁺ neurons projecting to the MD thalamus, as shown in Supplementary Figure 9, warrants careful interpretation.\nMethodologically, the limited overlap may partly stem from inherent constraints of retrograde tracing, as some activated MD-projecting neurons might not be labeled. Biologically, however, the high activation rate (~78%) within this subpopulation suggests functional specificity rather than weak circuit engagement. These findings indicate that, although numerically limited, a specialized subset of MD-projecting APCGABA neurons is preferentially recruited by 2MT and exerts a functionally significant influence on fear-related analgesia. We have clarified this point in the revised manuscript (lines 495–504).\n8. As CamKII neurons were also activated, it is unclear why they were not functionally tested.\nWe appreciate this insightful question regarding the CaMKII-positive neurons in our study. We initially focused on GABAergic populations because 2MT elicited stronger and more selective calcium responses in inhibitory neurons, whereas excitatory neurons showed comparable activation to both 2MT and anisole (Fig. 2g-l). This specificity suggested a predominant role of APCGABA neurons in fear-induced analgesia.\nIn addition, when we manipulated the whole population of glutamatergic APC neurons by virally expressing DREADDs under the CaMKII promoter in wild-type mice, epileptic behavior was induced, precluding further behavioral testing (see attached supplementary video). This pro-epileptic effect aligns with previous studies.2,3 Further investigation is needed to determine whether 2MT-responsive APCGLU neurons contribute to innate fear-induced analgesia. This point has been incorporated into the revised discussion (lines 541-544).\nMinor\n1. Given the fact that excitatory APC neurons are activated by both aversive and neutral odors it would be interesting to know if APC excitatory neurons also project to MD.\nWe thank the reviewer for this insightful suggestion. Our anterograde tracing (AAV-CaMKII-mCherry injected into the APC) reveals mCherry+ terminals in the MD thalamus (Response Fig. 8).\nResponse Fig 8. Anterograde tracing data reveal glutamatergic projections from the APC to MD.\na, Schematic of the viral injection (left) and the representative image of mCherry-expressing neurons (red) co-localized with anti-GLU staining (green) in the APC. Scale bars, 250 μm and 50 μm. b, Representative images of mCherry-expressing signals in MD of C57 mice. Scale bar, 300 μm.\n2. What is physiological and pathological pain-these terms are a bit novel for the abstract.\nWe thank the reviewer for the helpful comment. We have replaced “physiological and pathological pain” with “acute and chronic pain” in the abstract.\n3. Hargreave’s should be written Hargreaves or Hargreaves’\nWe thank the reviewer for catching this typographical error. We have corrected “Hargreave’s” to “Hargreaves’” throughout the manuscript.\n4. The specificity of the transgene targeting using such promoter based viral vectors is always complicated-how often was specificity tested?\nWe thank the reviewer for raising this important point regarding the specificity of promoter-based viral targeting. To assess the accuracy of VGAT promoter–driven expression, we co-injected AAV-DIO-EYFP and AAV-VGAT-mCherry into the APC of VGAT-Cre mice. Approximately 72% of mCherry⁺ neurons were also EYFP⁺ (Response Fig. 9; revised Supplementary Fig. 6), confirming substantial but not complete specificity.\nIn addition, the specificity of VGAT promoter based viral expression was verified in revised Fig 6b showing that ~81% of mCherry-expressing neurons were GABA-positive.\nWe acknowledge that this level of specificity indicates potential off-target expression, which cannot be fully excluded. This limitation has been noted in the revised manuscript under “Limitations of the Study” (lines 540–541).\nResponse Fig 9. Verification of the specificity of VGAT-promoter-driven viral expression.\na, Schematic of the viral injection. b, Representative images show mCherry-expressing neurons (red) co-localized with EYFP-expressing neurons (green) in the APC. Scale bars, 250 μm. c, Summary data for the percentage of mCherry+ neurons that co-localize with EYFP+ neurons (n=9 sections from 3 mice). All data are presented as box and whisker plots (medians, quartiles (boxes), and ranges from minimum to maximum (whiskers)).\nIn summary, the authors convincingly show that this circuit can modulate pain responses, but more work should be done if the authors wish to make the claim that this pathway is the central mechanism/circuit for innate fear-induced analgesia.\nReviewer #2 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nWe sincerely appreciate the time and expertise that the co-reviewer has invested in evaluating our manuscript.\nReviewer #3 (Remarks to the Author):\nIn this study, Jia et al. provide an extensive assessment of the role of the anterior piriform cortex in mediating the analgesic effect of innate fear. The topic is highly interesting but complex as it is not easy to conceptually link the impact of innate fear on pain sensation. To do so the authors exposed mice to the odor of predators and then proceed to a battery of pain-related tests, showing that animals previously exposed to the odor predator exhibit reduced nocifensive behavioral responses. Using an extensive combination of anatomical, imaging, and functional approaches they progressively identify a GABAergic projections from the anterior proportion of the piriform cortex to the mediodorsal thalamus that may be both necessary and sufficient for this effect. This result calls for particular attention since long-distance inhibitory projections from cortex to the MD has never been reported before in mice. The amount of experimental work is impressive and all approaches are by themselves state-of-the-art, but the meaning and interpretation of all these experiments also appear as somewhat questionable. In addition, crucial methodological details are missing as explained below.\nRESPONSE: We sincerely appreciate the reviewer's thoughtful evaluation of our study and the important concerns. Below we provide point-by-point responses to strengthen the manuscript.\nMethodology/behavioral paradigm\nThe exact protocol for odor exposure is not clear enough in relation with the many experiments reported. When exactly and for how long are animals exposed to the different odours? Is it acute or multiple exposures? Is it during each beahvioral assay or just before? Dpending on the answers to these questions there may be new issues to consider. Too many crucial details are lacking preventing the reader from understanding what exactly has been done.\nWe thank the reviewer for raising this important point regarding the odor exposure protocol. We have now revised the Methods section to include detailed, step-by-step description of each procedure to ensure full experimental transparency:\n• Pain behavior assays: Mice were first acclimated to the behavioral chamber for 30 minutes prior to testing. After acclimation, a continuous 2MT (or control solvent) odor was introduced by placing a filter paper saturated with the stimulus at the top of the chamber. Measurements of thermal or mechanical sensitivity (Hargreaves and von Frey tests) began 2 minutes after odor onset, and the odor stimulus was maintained throughout the entire testing session. This ensured that the animals remained in a sustained state of innate fear during nociceptive assessment.\n• Odor-evoked calcium imaging: Mice were habituated for 30 minutes in a square testing box before odor presentation. Odor stimuli (water, anisole, or 2MT) were delivered either by air puff or cotton swab, with six repeated trials per odor and 1-minute inter-trial intervals. Each odor exposure lasted 1 second per trial. For delivery, a Pasteur pipette or cotton swab containing 50 μl of odor solution was briefly introduced into the chamber.\n• Electrophysiology experiments: Mice received intraperitoneal CNO (30 minutes prior) to either 1-hour 2MT or water exposure, after which acute brain slices containing the mediodorsal (MD) thalamus were prepared for electrophysiological recordings.\nWe apologize for the lack of methodological details in the original submission. The revised manuscript now adds these procedures. The revised manuscript now includes these clarifications in the Methods section to facilitate reproducibility and interpretation of our findings.\nFor all histological controls, not enough data are provided to document the spread of viral infusion. For all interventions (DREADD and opto), general histology should be provided showing shades of spread for each included individual as is usually the norm.\nWithout these essential controls/information, the conceptual ground of the whole paper is unclear.\nWe thank the reviewer for emphasizing the importance of providing comprehensive histological documentation of viral spread. In the original study, viral expression was microscopically verified for all animals included in the analyses; however, we acknowledge that not all histological sections were documented photographically, and therefore, only a portion of the corresponding images is currently available. We have now compiled and included all existing histological images from these earlier cohorts in the revised submission (Response Figure 10). While these data represent only a subset of the DREADD and optogenetic experiments, they consistently show correct targeting and confinement of viral expression within the intended regions, supporting the reliability of our injection procedures.\nTo fully address the reviewer’s recommendation, we conducted a new, independently replicated chemogenetic experiment using TRAP2 mice, for which viral expression was systematically analyzed and documented for every individual animal. The revised supplementary Fig. 4 (Response Figure 11) now provides representative micrographs and schematic reconstructions for all subjects, aligned to standard brain atlas plates to clearly illustrate the extent and reproducibility of viral spread.\nWe acknowledge the limitation that complete photographic documentation of viral spread is not available for every animal from the earlier cohorts. Nonetheless, the consistency observed across the available images and the newly generated, fully quantified dataset together demonstrate that our viral manipulations were well targeted and reproducible, reinforcing the validity of the reported findings.\nResponse Fig 10. Verification of the shades of viral spread in C57 mouse.\na, c, e, g, Schematic of the viral injection. b, d, f, h, The diffusion areas of hM4Di (b), hM3Dq (d), NpHR (f) and ChR2 (h) expression in the APC. Subjects are individually colored (n=7 mice/group).\nResponse Fig 11. Verification of the shades of viral spread for each included TRAP2 mouse.\na, d, Schematic of the viral injection. b, e, The diffusion areas of hM4Di (b) or hM3Dq (e) expression. Subjects are individually colored (n=7 mice/group). c, f, Images of coronal section showing hM4Di (c) or hM3Dq (f) expression in the APC.\nGABAergic projection from cortex to thalamus\nThe authors should really comment on whether this was expected or not. Very importantly, greater care is needed to ensure that this important claim is unambiguously supported by included data.\nWe thank the reviewer for this important point. The identification of a long-range GABAergic projection from the anterior piriform cortex (APC) to the mediodorsal (MD) thalamus was indeed unexpected, as the cortico-thalamic pathway is classically considered glutamatergic4,5. Based on the established findings regarding long-range cortical GABAergic projections (auditory cortex→lateral amygdala pathway6, and medial prefrontal cortex→to the retrosplenial cortex pathway7), our study provides evidence for the existence and functional significance of a GABAergic projection from the APC to the MD.\nSpecifically, our multidisciplinary approach combining viral tracing, optogenetics, chemogenetics and electrophysiological recordings provide the anatomical evidence of APC→MD inhibitory projection, and demonstrates crucial role of the APC→MD pathway in innate fear-induced analgesia.\nThe discovery of this APC→MD pathway aligns with the growing recognition of cortical long-range inhibitory circuits as important regulators of distant brain regions (such as amygdala and cortex)6,7. Our results extend this emerging paradigm to the olfactory system, revealing a previously uncharacterized inhibitory corticothalamic circuit that may coordinate cross-regional behavioral outputs during threat perception.\nThese points have been incorporated into the revised discussion (lines 486-487; 489-494).\nFor example, neurons that are positive for vgat may also transmit glutamate, how do we know if that is not the case here?\nWe thank the reviewer for highlighting this important point. Indeed, a subset of VGAT-positive neurons has been reported to co-release glutamate under certain conditions. While the precise incidence of such co-transmission within the specific cortical population we targeted remains to be determined, evidence from the mouse cerebral cortex indicates that co-expression of VGAT and VGLUT1 at the synaptic level is rare, approximately 3.6 ± 0.2% of VGAT⁺ puncta8. Importantly, the net physiological effect of optogenetic activation in our study was pain suppression, a response that is inconsistent with predominant glutamatergic excitation. Thus, while we cannot completely exclude the possibility that a small subset of VGAT⁺ neurons may co-release glutamate, our functional and anatomical data strongly support that the observed effect is primarily mediated through GABAergic transmission. Future intersectional strategies selectively deleting glutamatergic signaling components in this population will help clarify any potential modulatory contribution of co-released glutamate to the observed fear-induced analgesia.\nViral spread into TRN which is mentionned in the paper is guaranteed to yield thalamic inhibition but this is not taken into consideration.\nWe thank the reviewer for raising this important concern. We fully agree that off-target viral expression in the thalamic reticular nucleus (TRN) could potentially confound the interpretation of our results by introducing unintended thalamic inhibition. As now documented in the Response Fig. 12, we confirmed that in the TRAP2 mice included in the final behavioral analyses, viral expression was largely restricted to the intended cortical target regions, with no detectable spread into the TRN.\nResponse Fig 12. Verification of the shades of viral spread in the TRN for each included TRAP2 mouse.\na, c, Schematic of the viral injection. b, d, Images of coronal section showing hM4Di (b) or hM3Dq (d) expression in the TRN.\nThe authors also reasonned that there is a higher proportion of GAD2+ proportion of cells labelled for dtomato than it is for CamKII cells (Figure 2c and 2d, the first d should be c though) but the panel c shows very few GAD2+ cells whereas CAMKII+ cells are abundant. This casts further doubt on that inhibitory CT route which would be further fraction at best of those GAD2+ cells (those projecting to the MD). All these issues add on each other, making the whole point not compelling given the importance of the claim.\nWe thank the reviewer for this comment and apologize for any lack of clarity in our original manuscript. The key point is not a direct comparison between the absolute proportions of GAD2+ and CaMKII+ cells within the tdTomato-labeled population.\nIt’s true that the absolute proportion of CaMKII+ cells within the tdTomato-labeled population is higher than GAD2+ cells. Rather, the critical finding is the differential change in these proportions between experimental groups (2MT versus anisole; 2MT versus H O). As we now state more clearly in the revised manuscript, upon 2MT 2 exposure, the proportion of GAD2+ cells among the tdTomato-labeled neurons significantly increased compared to the control groups. In contrast, the proportion of CaMKII+ cells remained unchanged. This specific, divergent response underscores that the fearful stimulus selectively recruits the inhibitory neurons, thereby validating their functional relevance.\nTaken together, these issues largely undermine the paper to the extent that it is unclear whether the main points could still hold if they can be adequately addressed and making any more minor comments irrelevant at this stage. The paper would also benefit from professional level proof-reading for english.\nWe sincerely thank the reviewer for their thoughtful and constructive feedback, which has greatly strengthened the quality and clarity of our manuscript. Below, we summarize the major revisions made to address the concerns raised:\n1) Expanded methodological details on odor exposure:\nThe Methods section has been substantially revised to provide step-by-step descriptions of odor presentation across all experimental paradigms. Detailed information is now included regarding the timing, duration, and delivery method of odor stimuli for behavioral assays, calcium imaging, and electrophysiological recordings, ensuring transparency and reproducibility.\n2) Comprehensive histological validation of viral expression:\nWe performed detailed histological verification of viral expression in both the original and newly conducted experiments. All available histological images from earlier cohorts have been compiled, confirming accurate targeting of the anterior piriform APC. Additional chemogenetic experiments in TRAP2 mice were conducted, with viral expression systematically quantified and documented for each individual animal. These data show that viral expression was largely restricted to the APC, with minimal off-target spread.\nWe thank the reviewer for this stringent assessment and fully acknowledge that the initial submission lacked crucial methodological details and controls, which undermined the conceptual foundation of our study.\nHowever, we fully acknowledge several limitations of the current study. The VGAT promoter-based viral system used to target GABAergic neurons in the APC may still carry off-target effects that cannot be completely excluded. It also remains to be determined whether a direct APCGLU-MD projection contributes to fear-induced analgesia. Given the known local feedback and feedforward inhibition within the piriform cortex9,10, further studies will be required to dissect the relative contributions of excitatory–inhibitory interactions within the APC and potential disynaptic pathways involving long-range excitatory activation of MD GABAergic neurons.\nThese considerations have been explicitly incorporated into the revised Discussion.\nFinally, the entire manuscript has undergone comprehensive professional English editing to improve clarity, precision, and overall readability.\nReviewer #4 (Remarks to the Author):\nThe manuscript by Jia et al. describes a very interesting set of findings revealing a potential circuit underlying pain suppression by innate fear. Of particular interest this is a novel long-range GABAergic projection originating from Anterior Piriform Cortex (APC) and targeting MD thalamus. While I think the basic story is sound, there are some issues with statistical comparisons and the interpretation of results that need to be addressed. In addition, it is unclear how the slice results relate to the behavioral results – there are questions about how exactly manipulations were done and what were the possible mechanisms that lead to long-lasting effects of 2MT exposure / chemogenetic inhibition persisting into slice experiments.\nWe sincerely appreciate the reviewer’s positive evaluation of our work and their constructive feedback.\n1. It would be useful to know what % of APC neurons activated by fear are a) GABAergic, b) project to MD, and c) are both.\nWe thank the reviewer for this important suggestion. To quantify the neuronal populations involved, we conducted additional experiments and observed that approximately 30% of Fos+ neurons are GABAergic and ~40% of GABAergic neurons were Fos-positive (Response Fig. 13, revised Supplementary Fig. 12a-c). Retrograde tracing detected only ~6% of Fos+ neurons projecting to MD (Response Fig. 13d-f). Methodologically, the limited overlap may partly stem from inherent constraints of retrograde tracing, as some activated MD-projecting neurons might not be labeled. Because of this limitation, the triple overlapping experiment of Fos/ GABA/retro-EYFP is challenging. Clearly, this will be underestimated due to the low efficiency of retrograde labeling.\nNotably, although MD-projecting GABAergic neurons constitutes a small fraction of all 2MT-activatied neurons, ~78% of this specific subpopulation was activated by 2MT (Response Fig. 13g-i, revised Supplementary Fig. 12d-f).\nMethodologically, the limited overlap may partly stem from inherent constraints of retrograde tracing, as some activated MD-projecting neurons might not be labeled. Biologically, however, the high activation rate within this subpopulation suggests functional specificity rather than weak circuit engagement. These findings indicate that, although numerically limited, a specialized subset of MD-projecting APCGABA neurons is preferentially recruited by 2MT and exerts a functionally significant influence on fear-related analgesia. We have clarified this point in the revised manuscript (page 18, lines 495–504).\nResponse Fig. 13. MD-projecting APCGABA neurons were activated by 2MT.\na, d, Schematic of the viral injection and a cartoon illustrating c-Fos induction by 2MT exposure. b, c, Representative image (b) and summary data (c) for the percentage of c-Fos-positive neurons (blue) co-localized with GAD2 (red), and GAD2-positive neurons co-localized with c-Fos staining in the APC (n=5 mice). Scale bars, 500 μm and 50 μm. e, f, Representative image (e) and quantitative summary for the percentage of c-Fos+ neurons (blue) co-localized with EGFP (green) in the APC (f, n=3 mice). Scale bars, 250 μm and 50 μm. Data are presented as box and whisker plots (medians, quartiles (boxes), and ranges from minimum to maximum (whiskers)).\n2. For 4e – what was the protocol for 2MT exposure? Specifically what was the frequency and duration of 2MT exposure, and what was the timing relative to brain slice preparation? What do the authors think is the mechanism underlying this effect?\nWe thank the reviewer for requesting clarification regarding the 2MT exposure protocol and its underlying mechanism.\nMice were continuously exposed to 2MT for 60 minutes by applying 20 μL of odorant solution onto filter paper placed near the testing arena, after which acute brain slices containing the MD thalamus were prepared for electrophysiological recordings. These methodological details have now been added to the Methods section.\nAs elaborated in our response to Point #3c, we propose that the rescue effect of 2MT arises from enhanced synaptic inhibition onto MD neurons, as supported by our electrophysiological recordings (Response Fig. 15). This mechanism provides a plausible neural substrate linking 2MT exposure to the observed behavioral analgesia.\n3. The causal demonstration of the role of this circuit is in Fig. 5 and 6. Fig. 5 shows that chemogenetically inhibiting MD-projecting GABAergic neurons in APC increases pain sensitivity in normal mice and SNI mice exposed to 2MT. Fig. 5g-i show that chemogenetic inhibition of GABAergic APC-MD neurons increases the excitability of MD neurons in 2MT-exposed SNI mice.\na. The protocol for 2MT exposure and chemogenetic inhibition are not explained. Specifically what was the frequency and duration of 2MT exposure? What was the timing of 2MT exposure relative to brain slice preparation? Was chemogenetic inhibition done preceding/during every 2MT exposure, and if not, what was the timing of chemogenetic inhibition relative to 2MT exposure?\nWe thank the reviewer for this important question regarding the timing of 2MT exposure and chemogenetic inhibition. Mice received intraperitoneal CNO injections (30 minutes prior) to either 1-hour 2MT or water exposure, after which acute brain slices containing the MD thalamus were prepared for electrophysiological recordings. These methodological details have been added to both the Results and Methods sections of the revised manuscript.\nb. Does the effect of chemogenetic inhibition on MD neuron excitability depend on 2MT exposure, or does the same effect occur in mice which undergo chemogenetic inhibition but have not been exposed to 2MT? This seems like an important and straightforward experiment.\nWe thank the reviewer for this insightful comment regarding whether the effect of chemogenetic inhibition on MD neuron excitability depends on 2MT exposure. To address this, we performed additional patch clamp recordings from MD neurons in SNI mice expressing mCherry or hM4Di that have not been exposed to 2MT.\nIn the absence of 2MT, inhibition of MD-projecting APCGABA neurons did not alter SNI-induced hyperexcitability, as assessed by firing rate and rheobase, compared with mCherry controls. This was likely due to a ceiling effect from maximal SNI-induced MD neural hyperactivity. In contrast, while 2MT exposure effectively rescued MD hyperactivity in control (mCherry-expressing) mice, this therapeutic effect was completely abolished in inhibitory hM4Di expressing mice (Response Fig. 14, revised Fig. 6g, h). Thus, functional APC-MD GABAergic transmission is specifically required for the 2MT-mediated reversal of SNI-induced hyperactivity. We have incorporated these data into the revised manuscript (lines 363-376).\nResponse Fig 14. Inhibition of MD-projecting APC neurons attenuates 2MT-reversed MD hyperactivity in chronic pain.\na, b, Effects of the chemogenetic inhibition of MD-projecting APCGABA neurons on the firing rate (g) and rheobase (h) of MD neurons recorded from either water or 2MT-exposed SNI mice (DIO-hM4Di SNI&H O, n=18 neurons from 4 mice; DIO-mCherry SNI&H O, n=26 neurons from 4 mice; 2 2 DIO-hM4Di SNI&2MT, n=27 neurons from 3 mice; DIO-mCherry SNI&2MT, n=23 neurons from 3 mice). Significance was assessed by two-way ANOVA followed by Bonferroni’s multiple comparisons test in (a), and one-way ANOVA followed by Bonferroni’s multiple comparisons test in (b). All data are presented as mean ± s.e.m. **P < 0.01, ***P < 0.0001, not significant (ns).\nc. What is the possible mechanism for this? Was there a change in input resistance suggestive of increases in spontaneous inhibitory input (potentially from GABAergic APC-MD terminals) in MD neurons? Did the authors measure spontaneous inhibitory input? This mechanism is important because it’s hard to imagine how transient inhibition of an inhibitory input would end up leading to persistent changes in the excitability of MD neurons.\nWe thank the reviewer for these insightful mechanistic questions. To explore how 2MT exposure induces persistent changes in MD neuron excitability, we performed additional electrophysiological analyses.\nFirst, as nicely suggested by the reviewer, we compared the input resistance (R_in) of MD neurons between SNI&H O and SNI&2MT groups and found a significant 2 decrease in Rin following 2MT exposure (Response Fig. 15a; revised Fig. 5h), suggesting enhanced spontaneous inhibitory input.\nConsistent with this, recordings of spontaneous inhibitory postsynaptic currents (sIPSCs) in MD neurons in SNI mice revealed that 2MT treatment 2MT significantly increased sIPSC frequency without affecting amplitude (Response Fig. 15b; revised Fig. 5i), indicating a presynaptic enhancement of inhibitory transmission onto MD neurons. These findings provide a plausible mechanism by which 2MT exposure strengthens APCGABA→MD inhibition, leading to persistent normalization of MD excitability. The new data and interpretations have been incorporated into the revised manuscript (lines 317-326).\nResponse Fig 15. 2MT decreases input resistance while attenuating sIPSCs in MD neurons of SNI mice.\na, Summary data of input resistance (h) for MD neurons from water-exposed or 2MT-exposed SNI mice (SNI & H O, n=23 neurons from 3 mice; SNI & 2MT, n=22 neurons from 3 mice). b, 2 Representative traces and summary data of sIPSCs in MD neurons from water-exposed or 2MT-exposed SNI mice (n=18 neurons from 3 mice/group). Significance was assessed by two-tailed unpaired Student’s t-test or Mann-Whitney test in (a, b). All data are presented as mean ± s.e.m. “ns”. *P < 0.05, ***P < 0.001.\n4. Fig. 6 shows that inhibition of GABAergic APC-MD terminals increases pain sensitivity in mice exposed to 2MT +/- SNI. Based on Supp. Fig 6+7, it seems that the effect is really the same regardless of whether mice have been exposed to 2MT and/or SNI – inhibiting these cells always leads to an exacerbation of pain sensitivity (albeit this effect is smaller in absence of 2MT). So it’s not just that it abolishes the effects of 2MT, but it also seems to increase pain sensitivity at baselines – this is an important point that should be noted explicitly and clearly.\nWe sincerely thank the reviewer for this important observation and fully agree that this point should be stated explicitly. We have clarified this in the revised Discussion: “Notably, chemogenetic inhibition of the entire APC-MD GABAergic terminals not only abolished the analgesic effect of 2MT but also consistently induced pain hypersensitivity in naïve mice. Thus, this pathway plays a dual role in pain modulation: it mediates fear-induced analgesia and provides a tonic inhibitory tone that continuously gates basal nociceptive transmission, as its inhibition alone was sufficient to induce pain hypersensitivity.”\nIn addition, given that only ~40 % of APC GABA neurons were activated by 2MT (revised supplementary Fig. 12a-c), we recognized that broad manipulation of APCGABA neurons could confound odor-specific effects. To directly address this, we now employed the TRAP2 strain, which enables genetic labeling and manipulation of neurons activated by a defined stimulation.1 To this end, we bilaterally injected AAV-VGAT-DIO-hM4Gi-mCherry into the APC of TRAP2 mice, followed by 2MT exposure paired with 4-OHT injection. Using this approach, we were able to specifically manipulate 2MT-activated inhibitory neurons. Inhibiting this ensemble significantly attenuated 2MT-induced fear expression and reduced 2MT-induced analgesia, but did not affect baselines thermal/mechanical nociception or locomotion (Response Fig. 16a-k; revised Fig. 4a-k). These findings establish that 2MT-responsive APCGABA neurons are necessary for innate fear-induced analgesia while being functionally distinct from basal nociceptive processing.\nFurthermore, selective activation of 2MT-trapped APC inhibitory neurons was sufficient to evoke freezing and robust analgesia in the absence of 2MT, again without affecting motor performance (Response Fig. 16m-u; revised Fig. 4m-u). These complementary loss- and gain-of-function results demonstrate that 2MT-responsive APCGABA neurons are both necessary and sufficient for innate fear-related analgesia.\nWe have incorporated these data into the revised manuscript (lines 231-246; 275-282).\nResponse Fig 16. Chemogenetic manipulation of 2MT-activated inhibitory neurons in the APC.\na, l, Schematic of the viral injection and behavioral experimental timelines. b, m, Left: image of coronal section showing hM4Di (b) or hM3Dq (m) expression in APC. Right: the diffusion areas of viral expression at coronal level of APC. Subjects are individually colored (n=7). c, n, Effect of chemogenetically inhibiting (c) or activating (n) 2MT-TRAPed APCGABA neurons on the proportion of freezing time and total moving distance (n=7 mice/group). d, o, Baselines pain sensitivity following chemogenetic inhibition (d) or activation (o) of 2MT-TRAPed APCGABA neurons (n=7 mice/group). e, f, Effect of chemogenetically inhibiting 2MT-TRAPed APCGABA neurons on basal nociception (e) or chronic neuropathic pain (f) of 2MT-exposed mice (n=7 mice/group). g-k, q-u, Effect of chemogenetically inhibiting (g-k) or activating (q-u) 2MT-TRAPed APCGABA neurons on motor performance (n=7 mice/group). p, Effect of chemogenetically activating 2MT-TRAPed APCGABA neurons on neuropathic pain (n=7 mice/group). All data are presented as mean ± SEM except for the data in (b) depicted as box and whisker plots. “ns”, no significance; *p < 0.05, **p < 0.01. Significance was assessed by two-tailed paired Student’s t-test or Wilcoxon signed-rank test in (c, d, g-k, n, q-u), and two-way ANOVA followed by Bonferroni’s multiple comparisons test in (e, f, o, p).\n5. Based on Supp Fig. 6c, inhibiting APC-MD GABAergic neurons increases CPA. This means that inhibiting these neurons is aversive, independent of pain. Conversely increases in the activity of these neurons are probably rewarding. This is a caveat that makes the interpretation of Fig. 6i less clear – specifically, the CPP shown in Fig. 6i may not be related to an alleviation of pain but rather to independent rewarding effects of the stimulation.\nWe sincerely appreciate the reviewer's insightful critique regarding the potential confound between pain relief and reward in our APC-MD GABAergic neuron manipulations.\nFollowing the reviewer's suggestion, we conducted a new conditioned place preference (CPP) test in naïve mice without SNI following APC GABA-MD circuit activation. We found that APC-MD stimulation did not produce a rewarding effect (Response Fig. 17; revised supplementary Fig. 16). This pain-context-specific behavioral reinforcement supports our interpretation that APCGABA-MD circuit activation mediates analgesia under neuropathic conditions, rather than a general rewarding effect. We have incorporated these data into the revised manuscript (lines 410-413).\nResponse Figure 17. The APCGABA-MD circuit activation does not induce CPP in naïve mice.\na, Schematic of the viral injection. b, Experimental schematic (left) and quantification (right) of conditioned place preference (CPP) test (n=7 mice). Significance was assessed by Wilcoxon signed-rank test. All data are presented as mean ± s.e.m. not significant (ns).\n6. It’s a bit odd to use 20Hz pulse trains to activate eNpHR – its kinetics are too slow to respond to these pulse trains so it’s a bit better / simpler to just use continuous light. Related to this, was the reported light power the peak light power, i.e., each pulse reaches a peak of 5 mW, or was the reported light power of 5 mW the net / average power measured over light ON and light OFF periods?\nWe appreciate the reviewer's expertise regarding eNpHR kinetics and light delivery specifications. We agree that continuous illumination would be more appropriate for eNpHR-mediated inhibition. The initial use of 20 Hz pulse trains was intended to maintain methodological consistency with our ChR2 stimulation paradigm. It is worth noting that even with these suboptimal parameters, 20 Hz pulsed stimulation remained effective in suppressing neuronal activity (completely abolishing neural firing).\nThe reported light power of 5 mW is the average power measured over light ON and light OFF periods.\n7. Line 209 – the authors should not report “a trend towards a statistically significant decrease.” The decrease is not statistically significant. They can say that this was a trend that did not reach statistical significance, or they can perform additional experiments to increase the n to confirm that the result does / does not reach statistical significance.\n8. Related to this, almost all of the statistical tests are performed as comparisons within a group on two days, where the author report significant changes in the experimental group but not in the control group. Really, this is not appropriate – the authors should perform statistical tests which directly compare the experimental and control groups, either using a 2-way ANOVA, or by calculating a difference score for each animal across the two days and then comparing these difference scores between the experimental and control groups to show a significant difference. I think the authors need to do this, if not in all cases, for their key findings.\nWe sincerely appreciate the reviewer's expert guidance on proper comparative statistics. First, for original Fig. 3i and 3j, we have conducted additional experiments to measure thermal sensitivity. Our new data showed that “both mechanical and thermal nociception were significantly decreased”.\nSecond, we have reanalyzed our key datasets using appropriate two-way ANOVA or difference score analyses to compare the magnitude of changes between groups. These additional analyses confirmed the robustness of our main findings and are now reported in the revised manuscript.\nReviewer #5 (Remarks to the Author):\nIn this manuscript by Jia and colleagues, the authors investigate the neural mechanisms by which innate fear suppresses pain sensitivity in male mice, identifying a GABAergic projection from the anterior piriform cortex (APC) to the mediodorsal thalamus (MD) as a key component. Using a combination of viral tracing, electrophysiology, fiber photometry, chemogenetics, and optogenetics, the authors claim to show that innate fear—triggered by predator odor (2MT)—induces analgesia via this corticothalamic circuit.\nWhile the study is methodologically rich and addresses an important topic, several conceptual, methodological, and interpretive issues significantly dampened the excitement about the manuscript and its current impact. The manuscript overstates its mechanistic conclusions. In my opinion, the core effects observed are better interpreted as modulations of pain sensitivity, rather than evidence of a distinct analgesic state induced by innate fear.\nMajor points:\n1. The manuscript relies on the premises that the 2MT odor promotes innate fear, but the behavioral characterization of the 2MT is very poor. The only information we have is supplementary Figure 1, with an example of the trajectories and the overall quantification of freezing.\nWe thank the reviewer for this important critique regarding the behavioral characterization of 2MT-induced fear responses and agree that detailed behavioral validation is essential. 2MT is a well-established innate fear odor. This phenomenon has been extensively documented and replicated in multiple independent reports. In the revised manuscript, we now explicitly cite these foundational studies and clarify that our behavioral results (Supplementary Fig. 1) are consistent with this well-established literature11,12.\na. The methods section lacks a description of how freezing is calculated. The authors only mention that it's done via software.\nWe thank the reviewer for pointing out the need for additional detail regarding the freezing quantification. We have now included the following description in the Methods section:\n“The experiment was conducted using the SuperFcs Conditional Fear Conditioning System, which was provided by Shanghai XinRuan Information Technology Co., Ltd. For data analysis, we utilized the built-in SuperMaze animal behavior video analysis software, Version 2.0. This software allowed us to adjust the Mobility/Immobility threshold to accurately differentiate between the mobility and immobility states of the mice, as well as modify the Immobility/Freezing threshold to effectively distinguish between immobility and freezing states induced by fear.\nAfter conducting multiple tests, we determined the optimal initial values displayed on the software to be a Mobility/Immobility threshold of 16 and an Immobility/Freezing threshold of 19. Subsequently, we proceeded with the formal experiment. These preset parameters were applied consistently across all freezing-related experiments in this study.”\nb. Regarding the methods, the authors mention six trials for each odor, but we always see only the averages of the six trials. Is there a desensitization/habituation to the odor? In addition, it's mentioned that there is a one-second exposure of the odor per trial, but there is no mention of how long the wait between trials is.\nWe appreciate the reviewer’s thoughtful question regarding potential odor habituation. To address this, we analyzed trial-by-trial photometry data within each session in both transgenic and C57 mice. The analysis revealed stable calcium response magnitudes across all six trials of anisole or 2MT exposure (Response Fig. 18; revised Supplemental Fig. 5), indicating negligible habituation under our experimental conditions. We have incorporated these data into the revised manuscript (lines 161-165).\nTo minimize potential olfactory adaptation, a 1-min inter-trial interval was maintained between successive odor trials in our experiments. This interval was chosen based on prior literature showing that it provides sufficient recovery time for olfactory receptor cell to reset after stimulation13. We have added the detailed exposure protocol in the Methods part (lines 612-619).\nResponse Figure 18. Calcium response magnitudes across six trials of anisole or 2MT exposure.\na-d, The AUC illustrates Ca²⁺ transients in APC neurons triggered by six trials of odor exposure delivered via air puff (a, c) or cotton swab (b, d) of anisole and 2MT in transgenic mice (n=5 mice/group). e-h, The AUC illustrates Ca²⁺ transients in APC neurons triggered by six trials of odor exposure delivered via air puff (e, g) or cotton swab (f, h) of anisole and 2MT in C57 mice (AAV-VGAT-GCaMP6m: n=6 mice/group; AAV-CaMKII-GCaMP6M: n=5 mice/group). Significance was assessed by one-way ANOVA with Bonferroni post hoc analysis in (a-h). All data are presented as the mean ± s.e.m. *P < 0.05, not significant (ns).\nc. Do all mice display innate fear responses to the odor? Were mice excluded?\nWe thank the reviewer for this question. In our experiments, all mice tested exhibited robust innate fear responses to 2MT. No animals were excluded from the analysis.\nd. There is a critical inconsistency in the odor delivery protocols. The method section for the odor exposure to induce innate fear described for the fiber photometry are different from the one described in innate fear detection. More importantly, I could not find any information on how the odor is integrated with the pain assays in the manuscript. Is the odor delivered continuously while the pain assays (like von Frey test, Hargreave's test, etc) are ongoing? Are the mice exposed to it only once before initiating the battery of pain assays or at the beginning of each pain test? The timelines for the behavioral manipulations needs to be clarified.\nAs pointed by the reviewer, the protocol used to induce innate fear in the Supplementary Fig. 1 was different from that used in photometry recordings. For 2MT-induced innate fear, mice were exposed to continuous 2MT odor delivery for 20 minutes to elicit robust freezing. A separate cohort of mice received six trials of 1-second 2MT exposure, with 1-minute inter-trial intervals. This design allowed us to isolate odor-evoked neural dynamics while minimizing olfactory adaptation.\nFor pain testing experiments, mice were first acclimatized to the behavioral chamber. Following an acclimatization period, a continuous 2MT (or control solvent) odor was initiated by placing a filter paper saturated with the stimulus at the top of the chamber. Pain assays (von Frey, Hargreaves, etc.) began 2 minutes after the onset of odor exposure, and the odor was maintained throughout the entire testing session to ensure animals remained in a sustained state of innate fear during nociceptive measurements.\nWe apologize for the lack of clarity in the original submission. The revised manuscript now includes a new Methods subsection titled “Timelines for Odor delivery” (lines 684-690), detailing these procedures.\nAll these points should be carefully addressed in order to understand the following figures of the manuscript and how exactly innate fear impacts pain sensitivity. This is particularly true since all the key readouts (e.g., increased thresholds, reduced nocifensive responses) may reflect motor suppression (freezing) rather than analgesia.\nWe sincerely thank the reviewer for emphasizing the importance of clarifying how innate fear impacts pain sensitivity and for raising the possibility that reduced nocifensive responses could reflect motor suppression rather than true analgesia. We have taken several steps to address this concern comprehensively.\nFirst, we have clarified all odor delivery protocols and experimental timelines to ensure that the relationship between innate fear and pain sensitivity is clearly understood in the context of each behavioral assay (see revised Methods section, “Timelines for odor delivery”).\nSecond, to dissociate motor suppression from analgesic effects, we performed additional analyses and fine motor behavioral assays (see responses to points 4 and 5). Quantification of locomotor activity during nociceptive testing showed no significant differences in movement or freezing levels between groups when mechanical or thermal stimuli were applied, indicating that changes in pain thresholds were not confounded by motor inhibition.\nThird, our conditioned place aversion (CPA) and conditioned place preference (CPP) experiments provide direct behavioral evidence for genuine analgesia: activation of the APC-MD GABAergic circuit was reinforcing only under neuropathic pain conditions, not in naïve animals, supporting the interpretation that the observed behavioral changes reflect pain relief rather than motor suppression or reward.\n2. Although not clearly stated, the figures, figure caption and main text suggest that the same mice undergo the series of pain sensitivity tests for each experiment. For example, the same group of mice undergoes all the tests in the main figure 1. This suggests the authors rely on repeated pain testing in the same mice across multiple conditions without proper counterbalancing. This could lead to several problems. Stress-Induced Analgesia (SIA) is a well-documented phenomenon where repeated exposure to stress or pain itself alters subsequent sensitivity, and this could have a significant impact on the overall results and lead to possible confounds. There is no evidence that the pain sensitivity test exposure order was controlled, counterbalanced or test-order randomized. This weakens the overall conclusions of the manuscript.\nWe thank the reviewer for this important comment and apologize for the ambiguous description of our behavioral tests paradigms. In the revised manuscript, we have clarified that separate cohorts of mice were used for the series of pain sensitivity tests shown in Figure 1. For experiments involving neural manipulations, pain sensitivity tests, from mild, basal nociception to post-injury assessments, were conducted in the same cohort of mice to allow within-subject comparisons under consistent physiological conditions.\nAs the reviewer correctly noted, repeated pain testing could potentially alter subsequent sensitivity through stress-induced analgesia (SIA) or test-order effects. To minimize these confounds, we employed a counterbalanced testing design in revision. In naïve C57BL/6 mice, four test modalities (von Frey, Hargreaves’, hot plate, and pinprick tests) were counterbalanced across animals. In nerve-injured and CFA-injected groups, two tests (von Frey and Hargreaves’) were counterbalanced (Response Fig. 19; revised Supplementary Fig. 10). Our analyses showed that baselines pain sensitivity remained stable across all test modalities regardless of order.\nImportantly, the 2MT-induced analgesic effect was unaffected by testing sequence, confirming that test order did not influence our main conclusions.\nWe have incorporated these data into the revised manuscript (lines 208-219).\nResponse Fig 19. The sequence of pain sensitivity testing does not affect 2MT-induced analgesia.\na, f, Outlines of experimental designs. b-e, g, h, Evaluation of fear-induced analgesia across different pain test sequences, encompassing basal nociception (b), acute mechanical (c), thermal (d), chronic neuropathic (g), and inflammatory pain (f) models (n=7 mice/group). Significance was assessed by two-tailed paired Student’s t-test or Wilcoxon signed-rank test in (b, c, e-g). All data are presented as the mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ***P < 0.001, not significant (ns). Details of the statistical analyses are presented in Supplementary Table 1.\n3. The supplementary figure 6 Shows that modulation (particularly the inactivation) of APC GABA neurons regulates pain sensitivity without innate fear. This undermines the central claim that fear is required for analgesia. In response, the authors add Supplementary Figures 7 and 11, in which a comparison between the pain thresholds following neuronal inhibition with and without innate fear exposure is shown. However, these comparisons are convoluted, spread across multiple figures, and not clearly described. In detail:\na. Supplementary Figure 7 a-b - compared with supplementary figure 6 a-c with main figure 3 a-d\nb. Supplementary Figure 7 c-d - compares supplementary figure 10 with main figure 5 a-d\nc. Supplementary Figure 7 e-f - compares supplementary figure 11 a-c with main figure 6 a-d\nd. And then, Supplementary Figure 11d-g relates to the main figure 6g-l\nAll of this back and forth is very chaotic and takes the attention away from the fact that there is a modulation of pain sensitivity alone, without the need for innate fear. Moreover, on the main text, the authors claim that these supplementary controls have \"no significance differences\" (lines193-195;) and \"did not show any differences in mechanical nociception (lines 312-313)\" and that is not true, which is simply incorrect based on the actual data of the supplementary figure. Moreover, the results of the thermal pain are not mentioned or compared. The data suggest that APC-GABA neurons modulate pain tonically and do not require fear for their effect. The manuscript selectively emphasizes data that support the fear angle and buries conflicting data in chaotic supplements.\nWe sincerely appreciate the reviewer’s insightful critique regarding the interpretation of our inhibition experiments and agree that additional clarification is needed.\nThe observation that basal nociception is affected by manipulation of APCᴳᴬᴮᴬ neurons indeed highlights the complexity of the interpretation. Importantly, since only ~40% of APC GABA neurons are activated by 2MT (revised Supplementary Fig 12a-c), manipulating the whole APCGABA neuronal population could obscure odor-specific effects and confound the interpretation of fear-dependent analgesia.\nTo address this, we performed new experiments using a TRAP2-based strategy to selectively target 2MT-responsive APC inhibitory neurons. TRAP2 mice were bilaterally injected with either AAV-VGAT-DIO-hM4Di-mCherry or AAV-VGAT-DIO-mCherry into the APC, followed by 2MT exposure paired with 4-OHT injection. This approach enabled us to specifically manipulate the neuronal ensemble activated by 2MT. Chemogenetic inhibition of this 2MT-responsive ensemble attenuated both 2MT-induced fear expression and analgesia, without affecting baselines mechanical or thermal nociception or locomotor activity (Response Fig. 20a-k; revised Fig. 4a-k). Conversely, selective activation of this ensemble was sufficient to induce freezing behavior and robust analgesia even in the absence of 2MT, again without altering motor performance (Response Fig. 20m-u; revised Fig. 4m-u).\nTogether, these findings demonstrate that 2MT-responsive APCGABA neurons are both necessary and sufficient for innate fear–induced analgesia and are functionally distinct from those regulating basal nociceptive processing. We believe these experiments significantly strengthen our ability to distinguish general nociceptive effects from odor-specific modulation.\nTo streamlines the presentation and eliminate redundancy, we removed original supplementary Figure 7 and incorporated TRAP data into the revised manuscript (lines 233-246; 275-285).\nResponse Fig 20. Chemogenetic manipulation of 2MT-activated inhibitory neurons in the APC.\na, l, Schematic of the viral injection and behavioral experimental timelines. b, m, Left: image of coronal section showing hM4Di (b) or hM3Dq (m) expression in APC. Right: the diffusion areas of viral expression at coronal level of APC. Subjects are individually colored (n=7). c, n, Effect of chemogenetically inhibiting (c) or activating (n) 2MT-TRAPed APCGABA neurons on the proportion of freezing time and total moving distance (n=7 mice/group). d, o, Baselines pain sensitivity following chemogenetic inhibition (d) or activation (o) of 2MT-TRAPed APCGABA neurons (n=7 mice/group). e, f, Effect of chemogenetically inhibiting 2MT-TRAPed APCGABA neurons on basal nociception (e) or chronic neuropathic pain (f) of 2MT-exposed mice (n=7 mice/group). g-k, q-u, Effect of chemogenetically inhibiting (g-k) or activating (q-u) 2MT-TRAPed APCGABA neurons on motor performance (n=7 mice/group). p, Effect of chemogenetically activating 2MT-TRAPed APCGABA neurons on neuropathic pain (n=7 mice/group). All data are presented as mean ± SEM except for the data in (b) depicted as box and whisker plots. “ns”, no significance; *p < 0.05, **p < 0.01. Significance was assessed by two-tailed paired Student’s t-test or Wilcoxon signed-rank test in (c, d, g-k, n, q-u), and two-way ANOVA followed by Bonferroni’s multiple comparisons test in (e, f, o, p).\n4. No effort is made to dissociate freezing-induced motor suppression from true analgesic effects. Since 2MT induces freezing, reduced reactivity to pain stimuli could reflect behavioral suppression, not analgesia. The rotarod test is used to argue that circuit manipulations do not impair motor function. However, the rotarod primarily assesses gross motor coordination and balance, not fine motor output or spontaneous movement. - Paw withdrawal tests rely on quick reflex-like responses that may not be fully reflected in rotarod performance. Thus, motor slowing, freezing behavior, or motivational changes could still confound nociceptive assays.\n5. The authors fail to adequately address the possibility that observed pain suppression results from inhibited motor output (such as freezing) rather than altered pain perception. The rotarod primarily assesses gross motor coordination and balance, not fine motor output or spontaneous movement. - Paw withdrawal tests rely on quick reflex-like responses that may not be fully reflected in rotarod performance. In addition, it seems that the activation of the APC-GABA promotes freezing in naive mice and decreases locomotion. Thus, motor slowing, freezing behavior, or motivational changes could still confound nociceptive assays. There can be a change in pain sensitivity merely because the manipulations impact motion.\nWe thank the reviewer for highlighting this crucial interpretive challenge. We agree that rotarod performance alone cannot exclude subtle motor impairments in fine motor output or motivational state. To rigorously dissociate true analgesic effects from motor suppression, we conducted a comprehensive battery of behavioral assessments evaluating both fine and gross motor functions.\nFirst, to directly test whether 2MT-induced freezing impairs motor performance, we measured forelimb grip strength and found no significant difference between 2MT-exposed and water-exposed mice (Response Fig. 21a; revised Supplementary Fig. 2a). Next, high-speed paw kinematic analysis during the pinprick assay revealed comparable paw withdrawal velocities and amplitudes between groups (Response Fig. 21b; revised Supplementary Fig. 2b), indicating that 2MT exposure does not impair reflexive motor output.\nWe further assessed motor coordination and balance using the balance beam and rotarod tests. No differences were detected in traversal time (balance beam) or latency-to-fall (rotarod) between 2MT- and water-treated mice (Response Fig. 21c, d; revised Supplementary Fig. 2c, d). Together, these results demonstrate that the observed analgesic effects of 2MT are not attributable to motor impairment. We have incorporated these data into the revised manuscript (lines 97-111).\nIn experiments involving APC and APC–MD circuit manipulations, we applied the same battery of motor assessments and similarly found no evidence of motor suppression (Response Fig. 22; revised Supplementary Fig. 9), further confirming that the analgesic effects reflect altered pain perception rather than reduced motor output. We have incorporated these data into the revised manuscript (lines 203-205; 263-266; 350-352).\nImportantly, our conditioned place aversion (CPA) and conditioned place preference (CPP) assays provide independent behavioral validation of true analgesia. For instance, inhibition of the APC-MD GABAergic pathway increased aversive responses, while its activation produced preference behavior only under neuropathic pain conditions, not in naïve animals (Revised Supplementary Fig. 16). This context-dependent reinforcement pattern supports the conclusion that APC-MD circuit activity alleviates pain rather than producing general reward or locomotor effects, further confirming that the observed reductions in nocifensive behaviors represent genuine analgesic outcomes.\nResponse Fig 21. 2MT does not affect motor performance.\na, Cartoon illustrating grip strength test and quantification of the grip strength of mice exposed to H O 2 and 2MT (n=7 mice/group). b, Cartoon illustrating paw withdrawal kinetics test and quantification of the withdrawal amplitude and velocity of mice exposed to H O and 2MT (n=7 mice/group). c, Cartoon 2 illustrating balance beam test and quantification of the traversal time of mice exposed to H O and 2MT 2 (n=7 mice/group). d, Cartoon illustrating rotarod test and quantification of the latency-to-fall of mice exposed to H O and 2MT 2 (n=7 mice/group). Significance was assessed by two-tailed paired Student’s t-test or Wilcoxon signed-rank test in (a-d). All data are presented as the mean ± s.e.m. Not significant (ns).\nResponse Fig 22. Manipulation of APCGABA or MD-projecting APCGABA neurons does not affect motor performance.\na, Schematic of the viral injection. b-f, Effects of chemogenetic inhibition of APCGABA neurons on motor performance, including grip strength (b), paw withdrawal amplitude and velocity (c), traversal time on a balance beam (d), latency-to-fall from rotarod (e), and total locomotor distance in the OFT (f) (n=7 mice/group). g-k, Effects of chemogenetic activation of APCGABA neurons on motor performance (n=7 mice/group). l-p, Effects of chemogenetic inhibition of MD-projecting APCGABA neurons on motor performance (n=8 mice/group). Significance was assessed by two-tailed paired Student’s t-test or Wilcoxon signed-rank test. All data are presented as the mean ± s.e.m. Not significant (ns).\n6. In several key experiments (e.g., Fig. 2d: 3 mice; Fig. 2g–l: 5 mice per group), sample sizes are extremely small. The authors do not provide power analyses or variance estimates to justify these choices. How did the authors access the statistical power?\nWe sincerely thank the reviewer for raising this important point regarding sample sizes and statistical power. We acknowledge that several of the key experiments involve technically demanding procedures, and we have accordingly increased the number of biological replicates in the revised manuscript.\nNotably, although the number of animals per group is modest in certain assays, the statistical power is enhanced by the large number of independent observations per animal. For example, in Figure 2d (immunostaining), data from n = 5 mice represent the averaged values from five sections per animal. In Figure 2g-l (fiber photometry), each mouse contributes continuous recordings across six trials, providing multiple within-subject measures that increase analytical robustness.\nMinor points:\n1. The introduction lacks referencing, e.g., lines 30, 32, 40, and 46.\nWe have added references.\n2. The data referenced from lines 161 to 168 is not provided. It would be relevant to include such a dataset, yet only mechanical nociception is mentioned; thermal is missing.\nWe sincerely thank the reviewer for noting this omission. We have now added the full dataset, including both mechanical and thermal nociception results (Response Fig. 23; revised Supplementary Fig. 7), to the revised manuscript (lines 192-197).\nResponse Fig 23. 2MT-induced analgesia on basal nociception over two consecutive days.\na, 2MT-induced analgesia on basal mechanical nociception over two consecutive days (n=8 mice/group). b, 2MT-induced analgesia on basal thermal nociception over two Significance was assessed by two-tailed paired Student’s t-test. All data are presented as the mean ± s.e.m. Not significant (ns).\n3. Fig. 1b, The latency and number of licks or jumps are taken into consideration together, but there is massive variability. These are distinct behaviors and should be analyzed separately.\nWe thank the reviewer for this valuable suggestion. In response, we have re-analyzed the data presented in Figure 1b. The original panel has been replaced with new figures (Response Fig. 24; revised Fig. 1b) that separately display the latency and frequency of licking and jumping behaviors. This re-analysis confirmed that 2MT exposure significantly increased response latencies and reduced the number of episodes for both behaviors compared to the water control, whereas anisole produced no significant effect.\nResponse Fig 24. 2MT-induced analgesia in the hot plate test.\na, b, Latency (a) and number of (b) licking or jumping behavior of mice in the hot plate test (n=12 mice/group). Significance was assessed by one-way ANOVA with Bonferroni post hoc analysis. All data are presented as mean ± s.e.m. “ns”. *P < 0.05, **P < 0.01, ***P < 0.0001, not significant (ns).\n4. Fig. 1c - How is the % of response for the pinprick test calculated?\nWe sincerely appreciate the reviewer’s detailed question. In our paradigm, each mouse underwent ten independent pinprick trials, and the percentage of responses was calculated as the frequency of paw withdrawal reflexes across these trials: (number of paw lifts)/(total trials [n=10]) × 100%. We have added the detail in the Method section:\nEach mouse was subjected to ten consecutive pinprick trials. The percentage of responses was calculated as: (number of paw lifts)/(total trials [n=10]) × 100% (lines 744-747).\n5. Fig. 2d - typo, the representative figure is panel c instead of d\nWe thank the reviewer for catching this typographical error. We have corrected it.\n6. Fig. 2g-l, heatmaps: not all animals seem to have a modulation of the neuronal activity during the delivery of 2MT. What could be the explanation?\nWe thank the reviewer for this thoughtful comment. The variation in heatmap intensity indeed reflects expected individual differences in the absolute magnitude of neural responses to 2MT, which can arise from technical factors (e.g., differences in viral transduction efficiency, fiber placement, or signal quality).\n7. Fig. 7 - Why, for physiological pain conditions, was only the thermal pain tested and not the mechanical?\nWe sincerely appreciate the reviewer’s insightful question regarding our choice of pain modality. To address this point, we have now included mechanical pain data obtained using the pinprick assay. The results show that prior exposure to ten pinprick trials did not affect fear-related behaviors, including the proportion of freezing time, movement distance, odor-zone entries, or time spent in the odor zone during innate fear testing (Response Fig. 25, revised Fig. 8c, d). These new data confirm that the observed effects are not modality-dependent and have incorporated these data into the revised manuscript (lines 421-424).\nResponse Fig 24. The innate fear responses are not affected by mechanical pain.\na, Experimental design for innate fear test under mechanical pain. b, Innate fear reactions assessed by the proportion of freezing time, moving distance, odor zone entries, and time in the odor zone of 2MT-exposed mice following 10-trial hindpaw pinprick stimuli (n=7 mice/group). Significance was assessed by two-tailed unpaired Student’s t-test or Mann-Whitney U test. All data are presented as mean ± s.e.m. not significant (ns).\n8. Supplementary Figure 3 b-d - anisole group missing, therefore the conclusion on lines 120-121 that \"These findings provide additional evidence for the involvement of APC neurons in processing fear-related olfactory information.\" could potentially be wrong since there is no odor that does not elicit fear responses.\nWe thank the reviewer for this important insight. We agree that including a non-aversive odor control is essential to establish the specificity of APC involvement in fear-related, rather than general olfactory, processing.\nTo address this, we performed new experiments using anisole as a neutral odor control. The results show that APC neurons exhibit significantly stronger activation in response to 2MT than to anisole, confirming the fear-specific engagement of APC neuronal activity (Response Fig. 25; revised supplementary Fig. 4a-d). We have incorporated these data into the revised manuscript (lines 131-137).\nResponse Fig 25. APC neurons exhibit response to fear odor 2MT.\na, Schematic of the viral injection (left), representative image of viral expression and optic fiber implantation (right). Scale bar, 250 μm. b, Schematic of the fiber photometry recording in response to different odor delivered via airpuff or cotton swabs in vivo. c, d, Average responses (left), heatmaps (middle) and AUC during 0–5 s (right) showing Ca2+ transients in APC neurons evoked by H O, 2 anisole and 2MT, delivered via airpuff (c) or cotton swabs (d) (n=7 mice/group). Significance was assessed by one-way ANOVA with Bonferroni post hoc analysis. All data are presented as the mean ± s.e.m. *p < 0.05, not significant (ns).\n9. Supplementary Figure 3g and j - only cotton swab. Airpuff is missing, and no explanation is provided for it to be the case.\nWe sincerely appreciate the reviewer’s careful attention to methodological completeness. We have now added the missing air-puff stimulation data (Response Fig. 26; revised Supplementary Fig. 4g, k). The results show a similar activation pattern to that elicited by the cotton swab, confirming the consistency of sensory responses across different odor stimuli. We have incorporated these data into the revised manuscript (lines 166-169).\nResponse Figure 26. APCGABA exhibits a more specific response to 2MT.\na, e, Schematic of the viral injection (left), representative image of viral expression and optic fiber implantation (right). Scale bar, 250 μm. b, f, Schematic of the fiber photometry recording in response to H O and 2MT delivered via airpuff or cotton swabs in vivo. c, d, g, h, Average responses (left), 2 heatmaps (middle), and AUC during 0–5 s (right) showing Ca2+ transients evoked by airpuff (c, g) or cotton swabs (d, h) of H O, anisole and 2MT in the APCGABA neurons (AAV-VGAT-GCaMP6m, n=6 2 mice/group; AAV-CAMKII-GCaMP6m, n=5 mice/group). Significance was assessed by one-way ANOVA with Bonferroni post hoc analysis. All data are presented as the mean ± s.e.m. *p < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, not significant (ns).\n10. Supplementary Figure 4 - The re-exposure to the arena should have been counterbalanced.\nWe sincerely appreciate the reviewer’s suggestion regarding the experimental design. In the revised experiments, we implemented a counterbalanced re-exposure design to control for potential context-specific effects in the arena. The results showed that CNO injection administered prior to salines exposure did not alter 2MT-induced freezing levels (Response Fig. 27, revised Supplementary Fig. 8), confirming that the behavioral outcomes were not influenced by the order of re-exposure. We have incorporated these data into the revised manuscript (lines 199-201; 347-349).\nResponse Fig 27. Inhibition of APCGABA or MD-projecting APCGABA neurons does not affect the 2MT-induced freezing behavior.\na, d, Schematic of the viral injection and behavioral test. b, c, Effects of the chemogenetic inhibition of APCGABA neurons on the proportion of freezing time (b) and moving distance (c). Mice received salines and CNO injections in reverse orders [salines then CNO in b (n=10 mice/group); CNO then salines in c (n=7 mice/group)], allowing for within-subject comparison. e, f, Effects of the chemogenetic inhibition of MD-projecting APCGABA neurons on the proportion of freezing time (e) and moving distance (f). Mice received salines and CNO injections in reverse orders [salines then CNO in g (n=6 mice/group); CNO then salines in h (n=8 mice/group)], allowing for within-subject comparison. Significance was assessed by two-tailed paired Student’s t-test or Wilcoxon signed-rank test. All data are presented as the mean ± s.e.m. Not significant (ns).\n11. Supplementary Figure 9 and 11 - Typo, the letters associated with the panels are in CAPS.\nWe thank the reviewer for catching this typographical error. We have corrected them.\nThe manuscript's conceptual framework offers an important mechanistic insight into how innate fear modulates pain sensitivity via an olfactory-cortical-thalamic GABAergic circuit. It is conceptually valuable and could contribute strongly to understanding how fear can suppress pain and lead to potential therapeutic targets for pain management. However, the current version of this manuscript contains significant interpretive overreach and methodological limitations. The central claim is not supported by the data as presented, and several key results are confounded. With substantial revision and reframing, the core finding—that an APC-GABA-MD circuit modulates pain sensitivity—could still represent a publishable advance, but not in its current conceptual framing as a fear-induced analgesia mechanism.\nWe are deeply grateful for the reviewer's insightful comments and constructive suggestions regarding the interpretation of our results and methodological limitations. In response to these important concerns, we have conducted a series of rigorous additional experiments:\nWe included new control experiments to systematically rule out potential confounding effects of motor suppression induced by 2MT or neural manipulation. We implemented comprehensive counterbalancing to test whether order effects in pain behavioral assessments exit. Importantly, through targeted manipulation of 2MT-activated APCGABA neuronal populations, we demonstrated no significant effect on baselines nociceptive thresholds, but selective modulation of 2MT-induced pain suppression. These new data substantially strengthen our conclusion that at least a distinct subpopulation of APCGABA neurons mediates fear-related pain suppression rather than general nociceptive processing.\nIn addition, we revised the title as “A corticothalamic circuit modulates pain sensitivity and mediates innate fear-induced analgesia in male mice”.\nWe believe these changes will address the reviewer’s concerns about overinterpretation while preserving the importance of identifying this previously unknown pathway in fear-pain interactions. We thank the reviewer for their suggestions that helped us improve the manuscript.\nReferences"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have carefully revised their manuscript and adequately addressed the main concerns raised. Below are a few minor points and possible errors:\n1. The authors may want to cite (and discuss, if appropriate) new work on the intersection of pain and survival threats, including predator odor (PMID: 41062698)\n2. In Figure 6e and 6f, should all bars be yellow?\n3. Check sentence “For most animals…” line 45\n4. Check sentence “To further investigate this…” line 174, specifically the word “bench” in line 176\n5. Check line 503-504, “exerts a functionally influence”", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "I thank the authors for the important additional work performed to address some of the issues. The more detailed methodology and histology clearly add value to the paper.\nI remain mildly concerned about how the existence of the long-distance inhibitory projection from APC to MD is contextualized. To the best of my knowledge, only one previous study has reported a similar pathway (Mazo et al., Nature Communications 2022). Although it was not the central claim of that paper and has not been widely cited, it should be acknowledged given the novelty of this finding. I hope this helps the authors appreciate the potential impact of their findings in the thalamocortical field specifically. A stronger effort to properly contextualize this result is still warranted.\nGiven the present evidence, the lack of converging data in the literature, and the relatively low number of such projecting GABA neurons (along with potential methodological caveats acknowledged by the authors), I believe the authors should adopt a careful and cautious tone in claiming the significance of this connection.", "reviewer_4": "The authors have done a good job of responding to my comments.", "reviewer_5": "The revised manuscript by Jia et al. is much improved and strengthened. I appreciate the additional work that the authors have done to address my comments and those of the other reviewers. It's a substantial amount of work and that should be commendable. Overall, I'm more positive, however, one critical interpretational issue remains around locomotion/freezing confounds, and I believe one additional analysis is necessary to fully lock the interpretation.\nThe current supplementary Fig. 8 shows that activation of APC-GABA neurons increases freezing and decreases total distance. However, the manuscript (lines 263-264) still claims that \"chemogenetic activation of APC GABA neurons did not affect motor performance, which was assessed by grip strength, high-speed paw kinematics, balance beam, rotarod, and OFT tests\". This is inconsistent with the OFT result and should be corrected.\nSimilarly, in the new TRAP2 experiments (Fig. 4), the same is observed: inactivation leads to a decrease in freezing and activation leads to an increase in freezing and a decrease in the distance in the OFT. Given that these manipulations shift freezing/locomotion, any change in pain readouts could be secondary to general motor rather than pain modulation per se. The authors' added motor battery is useful but may not fully deconfound movement-dependent pain readouts.\nGrip/beam/rotarod test capacity and coordination; Hargreaves, von Frey, hot plate, and pinprick still depend on movement thresholds and can be biased by freezing/locomotion.\nI have two suggestions that should address this concern, are feasible, and will have a high impact.\n1. Speed-/freezing-matched analyses.\nRecompute the pain effects within bins of matched locomotor state (e.g., speed 0–1, 1–3, >3 cm/s; or \"freezing\" vs \"non-freezing\"). If analgesia persists in matched bins, that is strong evidence against a pure motor account.\n2. Trial-by-trial mixed models with locomotion covariates.\nFit a GLMM/LMM: pain readout ~ manipulation + instantaneous speed + (manipulation×speed) + random intercepts per mouse.\n- A significant manipulation main effect with small/NS interaction suggests locomotion does not explain it.\n- Report effect sizes and partial R²\nIn the absence of these controls, the current data cannot exclude the possibility that pain changes are a consequence of altered freezing/locomotion rather than a modulation of nociception per se.\nWith these additions—and sustained, tempered wording that this pathway modulates pain and mediates a substantial component of odor-evoked analgesia—the work will be suitable for publication."}, "author_response": "We sincerely thank the reviewer for recognizing the value added by the additional methodological and histological analyses.\nI remain mildly concerned about how the existence of the long-distance inhibitory projection from APC to MD is contextualized. To the best of my knowledge, only one previous study has reported a similar pathway (Mazo et al., Nature Communications 2022). Although it was not the central claim of that paper and has not been widely cited, it should be acknowledged given the novelty of this finding. I hope this helps the authors appreciate the potential impact of their findings in the thalamocortical field specifically. A stronger effort to properly contextualize this result is still warranted.\nGiven the present evidence, the lack of converging data in the literature, and the relatively low number of such projecting GABA neurons (along with potential methodological caveats acknowledged by the authors), I believe the authors should adopt a careful and cautious tone in claiming the significance of this connection.\nWe thank the reviewer for these critical and constructive comments. We fully agree that the identification of this specific long-range GABAergic projection requires careful contextualization within the existing literature and a cautious interpretation of its functional significance.\nTo address this point, we now include the proper citation (Mazo et al., Nature Communications, 2022), as nicely noted by the reviewer, which reported a long-range inhibitory projection originating from the APC. In addition, we have revised the Discussion (lines 493–501) to more carefully contextualize our findings:\n“In this study, we dissect a direct inhibitory pathway from the APC to the MD through neural tracing and electrophysiological recordings. This observation aligns with the growing recognition for long-range inhibitory cortical circuits, including APC→ olfactory bulb62, auditory cortex→lateral amygdala63, and medial prefrontal cortex→ retrosplenial cortex pathways64. While our findings extend this emerging framework to the APC→MD axis, the relatively sparse anatomical representation of these projecting GABAergic neurons suggests that their contribution may be context-dependent, and the broader functional significance of this pathway remains to be further defined.”\nReviewer #4 (Remarks to the Author):\nThe authors have done a good job of responding to my comments.\nWe appreciate the positive feedback and constructive input throughout the review process.\nReviewer #5 (Remarks to the Author):\nThe revised manuscript by Jia et al. is much improved and strengthened. I appreciate the additional work that the authors have done to address my comments and those of the other reviewers. It's a substantial amount of work and that should be commendable. Overall, I'm more positive, however, one critical interpretational issue remains around locomotion/freezing confounds, and I believe one additional analysis is necessary to fully lock the interpretation.\nWe sincerely thank the reviewer for the thorough and constructive review.\nThe current supplementary Fig. 8 shows that activation of APC-GABA neurons increases freezing and decreases total distance. However, the manuscript (lines 263-264) still claims that \"chemogenetic activation of APC GABA neurons did not affect motor performance, which was assessed by grip strength, high-speed paw kinematics, balance beam, rotarod, and OFT tests\". This is inconsistent with the OFT result and should be corrected.\nThank you for your careful attention to this detail. The discrepancy might arise from the different durations and primary purposes of the two behavioral assessments:\nThe statement on lines 263-264 refers to a short-duration (5-minute) OFT used to screen for gross motor deficits. In this context, we did not observe a significant difference in total distance traveled. The data in Supplementary Figure 8 employ a longer time used for assessing freezing behavior1,2. We have corrected it as:\n“Furthermore, gross or fine motor control, assessed by grip strength, high-speed paw kinematics, balance beam, rotarod tests, along with the total distance traveled in a 5-minute OFT remained unchanged following chemogenetic activation of APCGABA neurons (Supplementary Fig. 9g-k).” (line 263-267). Meanwhile, we clearly labeled time duration in different contexts for clarification.\nSimilarly, in the new TRAP2 experiments (Fig. 4), the same is observed: inactivation leads to a decrease in freezing and activation leads to an increase in freezing and a decrease in the distance in the OFT. Given that these manipulations shift freezing/locomotion, any change in pain readouts could be secondary to general motor rather than pain modulation per se. The authors' added motor battery is useful but may not fully deconfound movement-dependent pain readouts. Grip/beam/rotarod test capacity and coordination; Hargreaves, von Frey, hot plate, and pinprick still depend on movement thresholds and can be biased by freezing/locomotion.\nI have two suggestions that should address this concern, are feasible, and will have a high impact.\n1. Speed-/freezing-matched analyses.\nRecompute the pain effects within bins of matched locomotor state (e.g., speed 0–1, 1–3, >3 cm/s; or \"freezing\" vs \"non-freezing\"). If analgesia persists in matched bins, that is strong evidence against a pure motor account.\n2. Trial-by-trial mixed models with locomotion covariates.\nFit a GLMM/LMM: pain readout ~ manipulation + instantaneous speed + (manipulation×speed) + random intercepts per mouse.\n- A significant manipulation main effect with small/NS interaction suggests locomotion does not explain it.\n- Report effect sizes and partial R²\nIn the absence of these controls, the current data cannot exclude the possibility that pain changes are a consequence of altered freezing/locomotion rather than a modulation of nociception per se.\nWith these additions—and sustained, tempered wording that this pathway modulates pain and mediates a substantial component of odor-evoked analgesia—the work will be suitable for publication.\nWe thank the reviewer for this careful and insightful critique regarding the potential confounding influence of locomotor state on pain-related readouts. We agree that freezing and locomotion can, in principle, influence behavioral measurements and appreciate the thoughtful analytical suggestions.\nIn our study, standard reflexive nociceptive assays (von Frey, Hargreaves, pinprick) were performed under a widely accepted behavioral criterion: stimuli were delivered only when animals were stationary and the hindpaw was in a relaxed, weight-bearing position. Experimenters deliberately waited for periods of immobility prior to stimulus application to avoid confounding effects of voluntary movement on reflexive motor responses. Consequently, for all valid measurements, instantaneous locomotor speed at the time of testing was effectively zero by design, leaving no meaningful variance in locomotion to match across conditions or include as a covariate. For this reason, speed-matched binning and trial-by-trial mixed models incorporating locomotion are not applicable to these reflexive assays in our experimental framework.\nThat said, we fully agree that the proposed speed-matched and mixed-model approaches are highly appropriate for paradigms in which pain-related readouts are sampled continuously during free movement (e.g., measures of ongoing home-cage behavioral states, or decision-making tasks). We also acknowledge that, more broadly, for acute nociceptive responses measured using classical reflex-based assays, changes in freezing or locomotion can be difficult, and in many cases practically impossible, to fully deconfound. We have now explicitly added this limitation to the Discussion to clarify that altered motor state may influence the interpretation of certain reflexive pain measures as: “Finally, altered motor states like freezing can confound movement-dependent pain assessments. Therefore, integrating movement-independent readouts, such as facial grimace scales, will help to achieve a more comprehensive understanding of pain modulation by the APC→MD circuit.” (line 555-558).\nTo further address this concern, we also conducted Conditioned Place Aversion (CPA) and Conditioned Place Preference (CPP) assays in the manuscript, which assess the affective-motivational component of pain and do not rely on reflexive motor outputs. Importantly, during the test phase of CPA/CPP, animals freely explored the apparatus without neural manipulation, thereby eliminating acute effects on locomotion or freezing. Notably, the total distance traveled on the test day (Day 6) for CPP did not differ significantly between the hM3Dq and mCherry groups (Response Fig 1). We have now expanded the Discussion to clarify that measures of affective dimension of pain can help substantiate a modulation of nociception (line 475-479).\nResponse Fig 1. Total distance traveled during the CPP test phase.\na, Schematic of the conditioned place preference (CPP) experimental design. b, Quantification (left) and representative movement traces (right) of the total distance traveled during the CPP test phase (n = 5 mice per group). Data are presented as mean ± s.e.m. No significant difference (ns) was observed between groups.\nUsing these paradigms, APCGABA neurons or APC→MD circuit inhibition induced pain-associated aversion, whereas activation produced pain-relief-associated place preference. These results provide movement-independent evidence supporting a role of APCGABA neurons and APC→MD circuit in analgesia, particularly in the affective dimension of pain. Together, by clarifying the interpretational boundaries of reflexive nociceptive assays, our CPP/CPA results and discussion support the conclusion that APCGABA neurons or APC→MD circuit manipulation modulates pain and contributes substantially to odor-evoked analgesia.\nReference:"}, {"round": 3, "reviewer_comments": {"reviewer_5": "I commend the authors for their candid acknowledgement of the study's limitations and for the thorough, thoughtful responses to each reviewer. They have addressed all my critiques. I have no further comments. This study will be an important contribution to the field."}, "author_response": ""}]

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