A nose-to-brain axis for spoiled food odor-triggered defensive responses in male mice

Domain: neuroscience
Published: 2026-01-03
Editorial outcome: major_revision

Abstract
Rancid odors can trigger retching and aversion in humans that prevent consumption of spoiled food, and consequently metabolic dysregulation, but the underlying neural mechanisms remain unclear. Here, we show that exposure to 2-methylbutyric acid (2MBA) odor triggers such defensive responses in male mice, mediated by a nose-to-brain axis. 2MBA perception in olfactory epithelium activates neurons in olfactory bulb (OB) projecting to glutamatergic neurons in anterior piriform cortex (aPirGlu), which in turn project to either the mediodorsal thalamic nucleus (MD), which innervates respiratory muscles to drive retching-like behaviors, or project to the nucleus accumbens (NAc) to drive aversion. Artificial inhibition of the nose→OB→aPir→MDGlu→respiratory muscles circuit diminishes retching-like behaviors, but not aversion, while inhibiting the nose→OB→aPir→NAc circuit diminishes aversion, but not retching-like behaviors. These findings thus establish a nose-to-brain axis for rancid odor-triggered retching and aversion in male mice, advancing our understanding of sensory-motor integration and whole-body neural bases of defensive responses to odor.

Introduction
Olfactory perception is required for maintaining metabolic homeostasis, and has been proposed as a potential dietary intervention for treating metabolic disorders based on evidence that normal food odors could regulate lipid mobilization and insulin resistance1–3. In humans, spoiled food odors can signal warnings of health risks and trigger defensive response behaviors, such as vomiting, retching, or diarrhea to empty noxious chemicals from the gastrointestinal tract, or aversion that prevents ingestion of noxious substances4,5. Based on their effects in reducing or avoiding infection and poisoning, these odor-inducible behaviors can effectively mitigate metabolic burden on hepatic detoxification pathways and preserve renal clearance capacity6–8.

Olfactory perception begins with scent molecules binding to receptors on olfactory sensory neurons in specialized tissue of the nasal cavity known as the olfactory epithelium; the axons of these neurons converge in small spherical structures known as glomeruli in the olfactory bulb (OB)9,10. Although a topological pattern of activated glomeruli has been identified as a basis for distinguishing different smells9,10, little is known about how such topographical information from the OB is transmitted and subsequently interpreted in the brain to spoiled food odors.

Output cells of the OB relay information from individual glomeruli to olfactory cortical areas, the largest of which is the piriform cortex (Pir), via diffuse and overlapping projections, thus allowing individual Pir neurons to incorporate inputs from different combinations of glomeruli11,12. The anterior and posterior Pir (aPir and pPir) exhibit neuroanatomical differences, with the aPir receiving more extensive inputs from OB than pPir13. The aPir in turn sends projections to numerous brain regions associated with defensive responses, including the prefrontal cortex14,15. However, it remains unclear whether and how the OB-aPir pathway participates in defensive responses to spoiled food odors.

The neural mechanism underlying vomiting triggered by spoiled food odors has been difficult to resolve, as the emetic species (e.g., cat, dog, ferret) used in traditional paradigms for this research have relatively fewer experimental resources for detailed molecular and cellular investigations compared to mice16–18, such as the capacity for straightforward genetic manipulation and the broad availability of transgenic strains19. Although laboratory mice lack vomiting behavior because the smooth muscle of the esophagus is not strong enough to expel food from the gastrointestinal tract, they do exhibit retching-like behavior, which has been used in recent studies to investigate vomiting-related responses6,19.

In the present study, we found that exposure to 2-methylbutyric acid (2MBA), a rancid odor associated with spoiled food20, triggers a retching-like behavior in male mice, which could promote emptying noxious chemicals, as well as aversion behaviors that could prevent consumption of potentially harmful substances. Through a combination of viral tracing, in vivo multi-tetrode electrophysiological recordings, and microendoscopic calcium imaging, we subsequently characterized the organization of a nose→OB→anterior piriform cortex (aPir)→mediodorsal thalamic nucleus→respiratory muscles circuit that is activated by exposure to 2MBA odor and triggers retching-like behavior in mice. We also uncovered a nose→OB→aPir→accumbens nucleus circuit that is activated in parallel and mediates a conditioned aversion to 2MBA odor in mice. Beyond defining the afferent to efferent pathway of a circuit mechanism for sensory-motor integration and periphery-brain communication, the present study provides a conceptual basis for developing odor-based therapies and intranasal neuromodulation strategies aimed at maintaining metabolic homeostasis or treating metabolic disorders.

Results
2-methylbutyric acid (2MBA)-triggered retching-like behavior and aversion in male mice
Inspired by human defensive response to unpleasant odors, we established a male mouse paradigm for odor-triggered retching-like behavior. Specifically, filter papers were individually placed in the center of a box. Several unpleasant odorants, including 2-methylbutyric acid (2MBA), (+)-fenchone (FEN), hexanol (HEX), limonene (LIM)21, predator-associated odor (trimethylthiazoline, TMT)22, bacterial metabolite odor (hydrogen sulfide, H2S)23, and bacterial virulence-and antibiotic resistance-associated odor (MgrB)24 at different concentrations (diluted with ddH2O), were each pipetted onto a filter paper and left to evaporate in respective boxes. Mice were placed within the box and permitted to move freely during a 20 min odor inhalation phase, while a camera was positioned horizontally outside the box to record their behaviors (Fig. 1a). Among these treatments, only exposure to 2MBA, a rancid odor associated with spoiled food, triggered significant mouth-opening actions analogous to retching behavior in humans, while none of the other unpleasant odorants, nor the ddH2O control, triggered such behavior (Fig. 1b–d, Supplementary Figs. 1 and 2, and Supplementary Video 1). Notably, the number of mouth-opening actions increased along with 2MBA concentration (Fig. 1c,d).

Retching is physiologically characterized by activation of the diaphragm (inspiratory) and abdominal (expiratory) muscles6. To determine whether the mouth-opening actions triggered by 2MBA inhalation indeed represented retching-like behavior, we conducted electromyogram (EMG) recordings of the activities of these muscles during 2MBA exposure. The amplitude and frequency of the diaphragm and abdominal external oblique muscles activities both significantly increased during the mouth-opening actions, whereas this increase was absent during exposure to other unpleasant odorants (FEN, HEX, LIM, TMT, H2S, and MgrB) or ddH2O (Fig. 1e–g and Supplementary Figs. 1 and 2). We therefore refer to the phenotypic readout of our 2MBA paradigm as retching-like behavior.

As rancid odors have also been shown to induce psychological responses21,25, we next investigated whether 2MBA exposure triggered an aversive response in mice. For this purpose, we conducted conditioned place aversion (CPA) tests, following previously described methods (Fig. 1h)26,27. Heatmap analysis showed that mice spent less time in the 2MBA-paired chamber, resulting in a higher CPA score, compared to the ddH2O-paired group (Fig. 1i,j). These data demonstrated that 2MBA could trigger aversion in mice.

To investigate whether the observed responses were triggered specifically through the olfactory system, we employed a previously described method for inducing anosmia28 in which ZnSO4 was bilaterally administered by intranasal irrigation for 5 successive days (Supplementary Fig. 3a). Exposure to 2MBA did not lead to retching-like behavior or aversion in the anosmia model mice as it did in saline-treated sham mice (Supplementary Fig. 3b–j). These results indicated that olfactory perception of spoiled food odor could induce defensive responses (retching-like behavior and aversion) in mice.

2MBA activates glutamatergic neurons in the anterior piriform cortex (aPirGlu) to trigger retching-like behavior and aversion
The olfactory sensory neurons (OSNs) in the olfactory epithelium, responsible for perceiving odor molecules, are well-known to project to the olfactory bulb (OB)9. To identify brain regions downstream of the OB that are activated by 2MBA, we used FosCreERT2 (FosTRAP2) mice. Briefly, we injected an AAV expressing Cre-dependent FLP and a mCherry reporter (AAV2/1-DIO-FLP-mCherry) into the naris of FosTRAP2 mice; we also injected an AAV expressing a FLP-dependent EGFP (AAV-fDIO-EGFP) into the OB. Three weeks after AAV injection, mice were intraperitoneally injected with the Cre-inducing agent, 4-hydroxytamoxifen (4-OHT)29, then exposed to 2MBA using the same protocol as for the retching-like behavior paradigm (Fig. 2a and Supplementary Fig. 4a). This strategy enabled mCherry labeling of 2MBA-activated OSNs, with EGFP labeling of OB neurons innervated by 2MBA-activated OSNs. In whole brain sections, we could observe EGFP+ fibers positioned downstream of the EGFP-labeled OB neurons (Fig. 2b and Supplementary Fig. 4b). Examination of regions comprising the olfactory cortex, including the anterior piriform cortex (aPir) and posterior piriform cortex (pPir)30, revealed that EGFP+ fibers were abundant in the aPir, but very scarce in other brain regions, including the well-known downstream of OB (pPir; olfactory tubercle, OT; or cortical amygdala, CoA) (Fig. 2c and Supplementary Fig. 4c–g). We therefore focused on the potential role of the aPir in 2MBA-triggered retching-like behavior and aversion.

To investigate whether aPir neurons participated in the olfactory response to 2MBA, we crossed FosTRAP2 mice with Ai14-tdTomato mice, to enable tdTomato labeling in activated neurons expressing Fos, a marker of immediate-early neural activity, upon intraperitoneal injection of 4-OHT31,32. We then monitored neuronal activation during 2MBA inhalation, as above, and found that tdTomato expression was significantly increased in the aPir compared to that ddH2O-treated controls (Supplementary Fig. 5a, b). These results indicated that olfactory exposure to 2MBA activated aPir neurons. As aPir neurons are known to comprise both glutamatergic and GABAergic neurons33, we conducted immunofluorescence staining to determine which cell type was activated in the aPir. This analysis showed that 85.1% tdTomato signals colocalized with glutamate antibodies (Supplementary Fig. 5c, d). These results indicated that glutamatergic neurons in the aPir (aPirGlu) respond to 2MBA.

We next explored the possibility that a specific subset of aPirGlu neurons responds to 2MBA. For this purpose, we examined aPirGlu neuronal responses to several odor stimuli (2MBA, FEN, HEX, LIM) at single-neuron resolution by injecting the aPir with an AAV expressing the Cre-dependent fluorescent Ca2+ indicator, GCaMP6m (AAV-DIO-GCaMP6m), and mounting a microendoscopic gradient index (GRIN) lens immediately above the aPir in CaMK2-Cre mice (Fig. 2d). We found that 30% of aPir neurons were activated by 2MBA inhalation alone, while 33.3% of neurons were activated by simultaneous exposure to 2MBA with other odors, and 36.7% of neurons exhibited no detectable response to 2MBA (Fig. 2e–g and Supplementary Fig. 6). To examine the neural activity of these 2MBA-activated aPirGlu neurons, we performed optical fiber photometry recordings in FosTRAP2 mice. This assay revealed that the Ca2+ signal of 2MBA-activated aPir increased following inhalation of 2MBA, FEN, HEX, or LIM, but not ddH2O, and the ΔF/F value was higher after 2MBA inhalation than after exposure to other odors (Fig. 2h–k). We also found that 2MBA-TRAPed neuronal activity increased in aPir in a concentration-dependent pattern, with inhalation of high 2MBA (30%) inducing greater ΔF/F values than low concentration (15%) (Supplementary Fig. 7a–c). These results were consistent with previous studies that also showed concentration-dependent responses to odors in Pir neurons33,34. As these results collectively suggested that aPirGlu neurons selectively respond to 2MBA inhalation, we subsequently focused on these 2MBA-activated aPirGlu neurons.

We next investigated whether the 2MBA-activated aPirGlu neurons were essential for 2MBA-triggered retching-like behavior and aversion. Given that aPirGlu neuronal activity increased upon exposure to 2MBA, we selectively chemogenetically inhibited 2MBA-activated aPir neuronal activity by injecting a Cre-dependent hM4Di-mCherry virus (AAV-DIO-hM4Di-mCherry) into the aPir of FosTRAP2 mice. After 3 weeks, mice were intraperitoneally injected with 4-OHT, then exposed to 2MBA odor. Two weeks later, these mice were intraperitoneally injected with the hM4Di-induction agent, clozapine-N-oxide (CNO), and evaluated for retching-like behavior and aversion (Fig. 2l, m). We observed that retching-like behavior (i.e., number of retching-like actions) and CPA scores were both significantly decreased in mice with inhibition of 2MBA-activated aPirGlu neurons compared with mCherry expressing controls (Fig. 2n). To confirm TRAP efficiency, mice were injected with the AAV-DIO-hM4Di-mCherry virus, then treated with 4-OHT induction and 2MBA odors prior to sacrifice (Supplementary Fig. 7d). Immunofluorescent staining for c-Fos in the aPir showed that 92.35% of tdTomato+ neurons colocalized with c-Fos+ nuclei, thus confirming the high efficiency of TRAP labeling in 2MBA-responsive neurons (Supplementary Fig. 7e, f). These results indicated that an increase in the aPirGlu neuronal activity contributes to spoiled food odor-triggered retching-like behavior and aversion.

2MBA-activated aPirGlu neurons project to the mediodorsal thalamic nucleus
To identify downstream neuronal targets of aPirGlu neurons potentially involved in retching-like behavior and aversion triggered by 2MBA odor, we injected an AAV-DIO-EGFP into the aPir of FosTRAP2 mice. Three weeks after AAV injection, mice were exposed to 2MBA odor following intraperitoneal injection of 4-OHT (Fig. 3a). Two weeks later, we observed EGFP+ neurons in the aPir (Fig. 3b) and EGFP+ fibers in several brain regions, including the mediodorsal thalamic nucleus (MD), accumbens nucleus (NAc), lateral hypothalamic area (LH), and paratenial thalamic nucleus (PT) (Fig. 3c and Supplementary Fig. 8a, b).

To investigate whether these brain regions rich in EGFP+ fibers were also involved in the 2MBA-triggered responses, we monitored neuronal activity in these regions during exposure to 2MBA odor in FosTRAP2:Ai14 mice as above. We found that tdTomato expression was significantly increased in the MD (84% colocalized with a glutamate-specific antibody) and NAc (93% colocalized with a GABA-specific antibody), but not LH and PT, upon 2MBA exposure (Supplementary Fig. 8c–f). Given the relatively strong signals evoked by 2MBA, we focused on aPirGlu→MD and aPirGlu→NAc circuits in subsequent studies.

As the MD, composed of glutamatergic and GABAergic neurons, has been previously implicated in transmitting sensory information during pain and olfactory discrimination behaviors35,36, we first sought to dissect the functional connectivity of the aPir→MD circuit by anterograde monosynaptic tracing. To this end, we injected an AAV2/1-Cre-mCherry virus into the aPir and an AAV-DIO-EGFP virus into the MD (Fig. 3d). After 3 weeks, we observed EGFP+ neurons in the MD that colocalized with a glutamate-specific antibody rather than a GABA-specific antibody (Fig. 3e, f), indicating that aPir neurons projected to glutamatergic neurons in the MD (MDGlu).

Next, to characterize the detailed organization of this aPirGlu→MD pathway, we used a cell-type-specific retrograde trans-monosynaptic tracing strategy in which helper viruses (AAV-EF1α-DIO-TVA-mCherry and AAV-EF1α-DIO-RVG) were injected into the MD of CaMK2-Cre mice. After 3 weeks, we injected a rabies virus (RV) EnvA-pseudotyped RV-ΔG-EGFP into the MD (Fig. 3g). At one week post RV injection, we detected EGFP+ neurons that co-localized with glutamate-specific antibody in the aPir (Fig. 3h and Supplementary Fig. 9). These data demonstrated that aPirGlu neurons project to MDGlu neurons.

We next examined functional connections of this aPirGlu→MDGlu pathway by injecting an AAV-DIO-ChR2-mCherry into the aPir and an AAV-CaMK2-EGFP into the MD of FosTRAP2 mice (Fig. 3i). At three weeks after injection, mice were exposed to 2MBA odor following intraperitoneal injection of 4-OHT. Two weeks later, mCherry+ fibers and EGFP+ neurons could be detected in the MD (Fig. 3j). Light stimulation of ChR2-containing terminals in the MD reliably triggered excitatory postsynaptic currents (EPSCs) in MDGlu neurons, and this increase in EPSCs was eliminated upon treatment with 6,7-dinitroquinoxaline-2,3(1H,4H)-dione (DNQX), an AMPA receptor antagonist (Fig. 3k, l). These findings suggested the presence of functional connections between 2MBA-activated aPirGlu neurons and MDGlu neurons.

2MBA-activated aPirGlu neurons project to the NAc
Previous studies have shown that the NAc (95% of which comprises GABAergic neurons) is activated in odor-based discrimination tasks36,37, indicating that it participates in olfactory processing. To explore the functional connectivity of this proposed aPirGlu→NAc circuit, we first injected an anterograde monosynaptic AAV2/1-Cre-mCherry into the aPir and an AAV-DIO-EGFP virus into the NAc of C57 mice (Fig. 4a). After 3 weeks, EGFP+ neurons that co-localized with a GABA-specific antibody rather than a glutamate-specific antibody could be observed in the NAc (Fig. 4b, c). In addition, Cre-dependent helper viruses (AAV-EF1α-DIO-TVA-mCherry and AAV-EF1α-DIO-RVG) were injected into the NAc of GAD2-Cre mice, and 3 weeks later, RV-ΔG-EGFP was injected into the NAc (Fig. 4d). At one week post-RV injection, we identified EGFP+ neurons that colocalized with a glutamate-specific antibody in the aPir (Fig. 4e and Supplementary Fig. 10). These data suggested that aPirGlu neurons also project to NAcGABA neurons.

To then characterize functional connections of this aPirGlu→NAcGABA circuit, we injected AAV-DIO-ChR2-mCherry into the aPir and AAV-mDLx-EGFP into the NAc of FosTRAP2 mice (Fig. 4f). At three weeks after injection, mice were intraperitoneally injected with 4-OHT, then exposed to 2MBA odor. Two weeks later, mCherry+ fibers and EGFP+ neurons could be observed in the NAc (Fig. 4g). Light stimulation of ChR2-containing terminals in the NAc reliably triggered EPSCs in NAcGABA neurons, which were subsequently abolished by DNQX treatment (Fig. 4h). These results thus confirmed the presence of functional connections from 2MBA-activated aPirGlu neurons to NAcGABA neurons.

To determine whether MD and NAc neurons received projections from the same aPir neuron, we injected retro-AAV-EGFP into the MD and retro-AAV-mCherry into the NAc of C57 mice (Supplementary Fig. 11a). We found that 75.44% of EGFP+ neurons in the aPir were not co-localized with mCherry+ neurons (Supplementary Fig. 11b–d). This suggested that distinct aPir neurons project to the MD and NAc, indicating that these aPirGlu-innervated MD neurons and NAc neurons may mediate different behaviors induced by 2MBA. Taken together, these results showed that 2MBA-activated aPirGlu neurons project to MDGlu and NAcGABA neurons (Fig. 4i).

The nose→OB→aPir→MD circuit is essential for 2MBA-triggered retching-like behavior
To investigate the functional roles of the aPirGlu→MDGlu and aPirGlu→NAcGABA circuits in 2MBA-triggered retching-like behavior and aversion, we first conducted in vivo multi-tetrode electrophysiological recordings in the MD and NAc of freely moving mice (Fig. 5a and Supplementary Fig. 12a). Interestingly, time-locked recordings indicated that retching-like behavior triggered a significant increase in the firing rate of MD neurons, whereas no increase was observed in NAc neurons (Fig. 5b, c). Alternatively, we found that firing rate significantly increased in NAc neurons, but not MD neurons, when mice traveled from the 2MBA-unpaired chamber to the 2MBA-paired chamber in CPA post-tests (Supplementary Fig. 12b, c). Based on these firing patterns, we focused on the MD in 2MBA-triggered retching-like behavior and the NAc in 2MBA-triggered aversion.

To characterize the function of the aPirGlu→MDGlu circuit in 2MBA-triggered retching-like behavior, we used optical fiber photometry to monitor MDGlu activity in freely moving CaMK2-Cre mice injected with an AAV-DIO-GCaMP6m in the MD following 2MBA exposure (Fig. 5d). Calcium signal recordings showed that 2MBA-induced retching-like behavior was associated with an obvious, time-locked increase in MDGlu neuronal activity, while no such increase was observed upon travel from a 2MBA-unpaired chamber to a 2MBA-paired chamber in CPA post-tests (Fig. 5e, f and Supplementary Fig. 12d–f). To selectively monitor the response of aPir-innervated MD neurons at single-neuron resolution, we injected an AAV2/1-Cre into the aPir and an AAV-DIO-GCaMP6m into the MD of C57 mice carrying a GRIN lens mounted at the top of the MD (Fig. 5g). Consistent with the above results, 2MBA-triggered retching-like behavior was associated with time-locked increased calcium activity in aPir-innervated MD neurons, while no increase in calcium activity was observed during CPA post-tests (Fig. 5h, i and Supplementary Fig. 12g–i). A similar strategy was conducted in the mice exposed to TMT, H2S or MgrB odors, we found no time-locked increased calcium activity in aPir-innervated MD neurons with odor exposure or CPA tests (Supplementary Fig. 12j–q). Together, these data indicate that MD neurons were specifically activated during 2MBA-triggered retching-like behavior.

To test whether increased activity in this aPir→MD circuit was required for 2MBA-triggered retching-like behavior, we chemogenetically inhibited the circuit through injection of the bilateral aPir with AAV-DIO-hM4Di-mCherry in FosTRAP2 mice with cannulas implanted in the bilateral MD. After virus expression, CNO was administered to MD via intracranial microinjection (Supplementary Fig. 13a, b). We found that chemogenetic inhibition of the aPir→MD circuit alleviated the 2MBA-triggered retching-like behavior, but not aversion (Supplementary Fig. 13c). These results indicated that the aPir→MD circuit was essential for retching-like behavior, but not aversion, in response to 2MBA.

To assess whether activation of the aPir→MD circuit alone was sufficient to elicit retching-like behavior, we bilaterally injected AAV-DIO-ChR2-mCherry into the aPir and bilaterally implanted optical fibers in the MD of FosTRAP2 mice (Supplementary Fig. 13d, e). We found that optical activation of the aPir→MD circuit could induce retching-like behavior in mice, but not aversion in real-time place aversion (RTPA) tests (Supplementary Fig. 13f). These cumulative results suggested that the aPir→MD circuit is necessary and sufficient to induce retching-like behavior akin to a rancid odor response.

To visualize the entire proposed nose→OB→aPir→MD circuit, we injected AAV2/1-Cre into the bilateral nares, AAV-Con Fon-hM4Di-EGFP into the bilateral OB, Cre-dependent helper viruses into the bilateral aPir, and retro-Cre into the bilateral MD. After 3 weeks of virus expression, RV-Flp-DsRed was injected into the bilateral aPir (Fig. 5j). At one week post-injection with RV, we observed neurons co-labeled with EGFP+ and DsRed+ in the OB (Fig. 5k), and thus visualized the entire proposed nose→OB→aPir→MD circuit.

To investigate whether this nose-to-brain axis was essential for 2MBA odor to trigger retching-like behavior and conditioned aversion, we used the same viral tracing strategy as above and bilaterally implanted cannulas in the aPir (Fig. 5l). Upon intracranial microinjection of CNO into the aPir to inhibit the nose→OB→aPir→MD circuit, we observed that retching-like behavior, but not conditioned aversion, was significantly reduced in response to 2MBA odor compared to that in mCherry control mice (Fig. 5m–p). These results thus demonstrated that the nose→OB→aPir→MD circuit was required for 2MBA-triggered retching-like behavior, but not aversion.

The nose→OB→aPir→NAc circuit is essential for 2MBA-triggered aversion
To next investigate the role of the aPirGlu→NAcGABA circuit in mediating retching-like behavior and aversion to 2MBA odor, we conducted fiber photometry recordings of NAcGABA activity in freely moving GAD2-Cre mice expressing AAV-DIO-GCaMP6m in the NAc (Fig. 6a). After three weeks of virus expression, time-locked recordings confirmed that calcium activity significantly increased when mice traveled from an unpaired chamber to a 2MBA-paired chamber in CPA post-tests, but did not increase during retching-like behavior (Fig. 6b, c and Supplementary Fig. 14a–c). To selectively monitor the response of aPir-innervated NAc neurons at single-neuron resolution, we injected the aPir with an AAV2/1-Cre and delivered an AAV-DIO-GCaMP6m virus to the NAc of C57 mice, then mounted a GRIN lens over the NAc (Fig. 6d). In line with our above findings, calcium activity showed a significant, time-locked increase upon entry into a 2MBA-paired chamber from an unpaired chamber in CPA post-tests, while no increase was observed during retching-like behavior (Fig. 6e–g and Supplementary Fig. 14d–f). Similar strategy was conducted in the mice exposed to TMT, H2S or MgrB odors, we found time-locked increased calcium activity in aPir-innervated NAc neurons with odor exposure or entry into a TMT/H2S/ MgrB-paired chamber in CPA post-tests (Supplementary Fig. 14g–n). Together, these data indicate that NAc neurons were activated during odor-triggered aversion.

In light of this increased aPir→NAc circuit activity in conditioned aversion to 2MBA, we selectively blocked this circuit by chemogenetic inhibition via bilateral aPir injection of AAV-DIO-hM4Di-mCherry in FosTRAP2 mice and intracranial CNO administration through implanted cannulas in the bilateral NAc (Supplementary Fig. 15a, b), behavioral tests indicated that 2MBA-triggered aversion, but not retching-like behavior, was significantly decreased compared to that in mCherry controls (Supplementary Fig. 15c). These results indicated that the aPir→NAc circuit was required for aversion triggered by 2MBA odor.

To assess whether activation of the aPir→NAc circuit alone was sufficient to elicit aversion, we bilaterally injected AAV-DIO-ChR2-mCherry into the aPir and bilaterally implanted optical fibers in the NAc of FosTRAP2 mice (Supplementary Fig. 15d, e). Optical activation of the aPir→NAc circuit in the absence of 2MBA odor induced aversion to a chamber coupled with the optical activation in RTPA tests, but did not result in observable retching-like behavior (Supplementary Fig. 15f). Together, these results suggested that the aPir→NAc circuit was necessary and sufficient to aversion, but not retching-like behavior.

To visualize the entire proposed nose→OB→aPir→NAc circuit, we bilaterally injected AAV2/1-Cre virus into the nares, AAV-Con Fon-hM4Di-EGFP into the bilateral OB, Cre-dependent helper viruses in the bilateral aPir, and retro-Cre virus into the bilateral NAc. After 3 weeks, we injected RV-Flp-DsRed into the bilateral aPir (Fig. 6h) and found EGFP+ DsRed+ neurons in the OB after one week of RV expression. These results thus depicted the anatomical connections of a nose→OB→aPir→NAc circuit (Fig. 6i).

To investigate whether this nose-to-brain axis was essential for the observed responses to 2MBA, we used the same viral tracing strategy as above and bilaterally implanted cannulas in the aPir (Fig. 6j). Behavioral assays following intracranial CNO application into the aPir to inhibit the nose→OB→aPir→NAc circuit showed that inhibiting this circuit alleviated conditioned aversion to 2MBA, but did not decrease 2MBA-triggered retching-like behavior (Fig. 6k–n). These results indicated that the nose→OB→aPir→NAc circuit was required for 2MBA-triggered aversion, but not retching-like behavior.

The nose-brain-muscle axis is essential for the retching-like behavior triggered by 2MBA
Given our observation that the nose→OB→aPir→MD circuit mediates 2MBA-triggered retching-like behavior and that retching behavior involves increased activity of diaphragm and abdominal muscles, we next explored the possible basis through which the MD could modulate the activity of respiratory muscles. For this purpose, we injected a PRV-RFP virus into the abdominal external oblique muscle, and a PRV-EGFP virus into the diaphragm muscle of C57 mice (Fig. 7a). Four days later, RFP+EGFP+ co-labeled neurons were observed in several brain regions, including the PVN, raphe magnus nucleus, peritrigeminal zone, PAG, LH, and lateral reticular nucleus, as well as the ventral respiratory group (VRG), a region previously shown to contain respiratory neurons that coordinate muscle activity during retching38 (Fig. 7b and Supplementary Fig. 16). To characterize connections between the MD and VRG in greater detail, we injected an AAV-DIO-mCherry in the MD and a retro-EGFP in the VRG. Three weeks later, we could detect EGFP+ neurons in the ventrolateral periaqueductal gray (VLPAG) surrounded by mCherry+ fibers (Fig. 7c–e). These results suggested that the MD likely regulates abdominal and diaphragm muscles through an MD→VLPAG→VRG circuit.

To determine whether activation of the MD→VLPAG circuit could trigger retching-like behavior, we injected an AAV-DIO-ChR2-mCherry into the MD and implanted optical fibers in the VLPAG of CaMK2-Cre mice. A 473 nm blue light was delivered to activate terminals in the VLPAG from MDGlu neurons. We recorded retching-like behavior and EMG after optogenetic activation of the MDGlu→VLPAG pathway. We found that optogenetic activation of this pathway induced increased retching-like behaviors and increased frequency and amplitude of abdominal external oblique muscle and diaphragmatic muscle activity compared with mCherry control mice (Supplementary Fig. 17).

Taken together, these results demonstrated that distinct neural circuits participate in retching-like behavior (nose→OB→aPir→MD→VLPAG→VRG→respiratory muscles circuit) and aversion (nose→OB→aPir→NAc circuit) in response to rancid odor stimulus (Fig. 7f).

Methods
Animals
Male mice from the following strains were used in all experiments: C57BL/6J, CaMK2-Cre, GAD2-Cre, FOS2A-iCreER (FosTRAP2), and Ai14 (RCL-tdT). The mice, aged 8–10 weeks, were obtained from Charles River or Jackson Laboratories. They were housed in groups of five per cage under standard colony conditions, with ad libitum access to water and standard mouse chow. The animals were kept on a 12 h light/dark cycle (light on from 8:00 AM to 8:00 PM) at a controlled temperature of 23–25 °C and 50% humidity. Deviations from these conditions occurred only during specific procedures, such as GRIN lens or tetrode array implantation. Mice were randomly assigned to experimental groups, and all procedures were approved by the Animal Care and Use Committee at the University of Science and Technology of China (USTC).

Retching-like behavior recording
Mice were placed in a transparent box (24 cm × 12 cm × 18 cm) and allowed to move freely. A piece of filter paper was positioned at the center of the box to facilitate the uniform evaporation of odorants. We pipetted 1 mL of odorants onto the filter paper, including ddH2O, 2-methylbutyric acid (2MBA, 15% and 30%, corresponding to 1.375×10⁻³ mol and 2.75×10⁻³ mol, respectively), (+)-fenchone (FEN), hexanol (HEX), and limonene (LIM), trimethylthiazoline (TMT), NaHS (a H2S-releasing agent), MgrB peptide. To record the behavior, we positioned a high-speed camera (160 frames/s) horizontally outside the box. The behavior was recorded continuously for 20 minutes.

Electromyogram (EMG) recording
EMG was recorded following previously described protocols in mice6,53. Mice were anesthetized with isoflurane and maintained body temperature at 37 °C using a heating pad. To monitor muscle activity, EMG electrodes (A-M Systems, No. 793200) were implanted in the diaphragm and abdominal external oblique muscles, and fixed with UV glue. Subsequently, mice were head-fixed in a stereotaxic device (RWD). A ground electrode and a reference electrode were connected to the skull of mice. Connection cables of electrodes were attached firmly to the skull using tissue glue and dental cement. After surgery, the EMG signal was recorded, filtered (10–500 Hz), and digitized using a multi-channel signal acquisition apparatus (Medusa, Bio-Signal Technologies). A muscle was considered activated when the amplitude of the EMG signals increased by at least 30% above the basal level.

Conditioned position aversion (CPA) test
The CPA test was conducted using a custom-made two-chamber apparatus (50 cm × 25 cm × 25 cm) following previously reported protocols26,27. A camera was mounted directly above the apparatus to track the movement of each mouse, and the data were analyzed using EthoVision XT14 software (Noldus).

Pre-Phase (Day 1 and Day 2). Mice were gently introduced to the apparatus and allowed to explore both chambers freely for 15 min. The time spent in the preferred chamber on Day 2 served as the baseline.

Pairing-Phase (Day 3 and Day 4). For 2MBA-paired group mice, mice were confined to their preferred chamber and exposed to 2MBA for 30 min and were confined to the other chamber for 30 min paired with ddH2O. For ddH2O-paired group mice, mice were confined to their preferred chamber and exposed to ddH2O for 30 min and were confined to the other chamber for 30 min paired with ddH2O.

Post-Phase (Day 5). 24 hours after the final pairing phase, mice were allowed to freely explore both chambers of the apparatus for 15 min. The CPA score was calculated as the difference between the time spent in the preferred chamber during the post-test and the time spent in the same chamber during the pre-test: CPA score = (time in the preferred chamber paired with 2MBA or ddH2O)Post - (time in the preferred chamber paired with 2MBA or ddH2O)Pre.

Real-time place aversion (RTPA) test
The same experimental apparatus used for CPA tests was employed for RTPA tests, following previously reported protocols26,27. One day before RTPA testing, mice underwent a 15-minute pre-test. During this pre-test, they were placed at the center of the apparatus and allowed to freely explore both chambers. Mice that showed a strong preference for one side of the apparatus, spending more than 65% (>585 s) or less than 35% (<315 s) of the total exploration time on one side, were excluded from further testing. On the test day, we applied photostimulation in one randomly selected chamber (designated as the treatment-paired chamber), while the other chamber served as the no-stimulation (unpaired) chamber. Mice were placed in the center of the apparatus and allowed to explore freely for 20 min. Each entry into the treatment-paired chamber triggered photostimulation (473 nm, 5 ms pulses at 20 Hz) using a Master 8 pulse stimulator (A.M.P.I.). We recorded all stimulations and real-time travel trajectories using EthoVision XT14 software (Noldus) for subsequent analysis. RTPA score = (time in the treatment-paired chamber)Test - (time in the treatment-paired chamber)Pre.

Anosmia induced by zinc sulfate (ZnSO4) application
We induced the anosmia model by intranasally irrigating mice with ZnSO4, following a previously described protocol28. Mice were anesthetized with isoflurane, and 50 μL of 5% ZnSO4 was administered to each naris. This irrigation was repeated for 5 consecutive days. Sham mice received intranasal saline irrigation.

Immunohistochemistry
Mice were anesthetized with intraperitoneal pentobarbital sodium (20 mg/kg) and perfused with 0.9% (w/v) saline, followed by 4% (w/v) paraformaldehyde (PFA). After perfusion, the brains were extracted and fixed overnight in 4% PFA at 4 °C. The tissue was then incubated in 20% (w/v) and 30% (w/v) sucrose solutions at 4 °C until the brain tissue sank. We cut 40-μm-thick coronal slices using a cryostat (Leica CM1860) for immunofluorescence. Following three washes with phosphate-buffered saline (PBS), the slices were blocked for 1 h at room temperature with a solution containing 5% bovine serum albumin (BSA) (w/v) and 0.5% Triton X-100 in PBS. Primary antibody incubation was conducted in a solution containing 3% BSA, 3% donkey serum, and 0.3% Triton X-100 in PBS for at least 24 hours at 4 °C. We used anti-glutamate (1:500, rabbit, Sigma), anti-GABA (1:500, rabbit, Sigma), and anti-c-Fos (1:500, rabbit, Synaptic Systems) as primary antibodies. Afterward, the slices were incubated with secondary antibodies conjugated to fluorophores (1:500, Invitrogen) for 1.5 h at room temperature. Finally, the slices were stained with 4,6-diamidino-2-phenylindole (DAPI; 1:2000, Sigma) for 6 min at room temperature. Fluorescence signals were observed and imaged using an FV3000 microscope.

Stereotaxic surgeries and viral injections
Mice were anesthetized with isoflurane (3% for induction; 1.5–2% for maintenance) and head-fixed in a stereotaxic device (RWD) on a heating pad to maintain body temperature. After shaving the fur, we made a small incision in the scalp to carefully expose the skull. Once exposed, we used a syringe equipped with a pulled glass microelectrode to ensure proper alignment of the skull in all planes. A small hole was drilled at the injection site, and the virus was injected at an infusion rate of 35 nl/min through the glass microelectrode, which was connected to an infusion pump (Micro4, WPI). The pipette was left in place for 5 min post-injection to prevent backflow or spread of the virus.

For virus injection into the naris, mice were placed in a supine position on a heating blanket, with the mouth fixed. The glass microelectrode was inserted into the naris, and 1.5 μL of either rAAV-EF1α-DIO-FLP-P2A-mCherry (AAV2/1-DIO-FLP-mCherry) or rAAV2/1-hSyn-Cre-pA (AAV2/1-Cre, 5.75×10¹³ vg/ml) was injected.

For anterograde tracing of the aPir→MD or aPir→NAc projection, we injected rAAV-EF1α-Cre-P2A-mCherry (AAV2/1-Cre-mCherry, AAV2/1, 6.83×10¹² vg/ml, 240 nl) into the aPir (ML: −2.90 mm, AP: +1.20 mm, DV: −4.24 mm). Subsequently, we injected rAAV-Ef1α-DIO-EGFP-WPRE-pA (AAV-DIO-EGFP, AAV2/9, 1.95×10¹² vg/ml, 200 nl) bilaterally into the MD (ML: −0.25 mm, AP: −0.46 mm, DV: −3.00 mm) or the NAc (ML: −1 mm, AP: +1.18 mm, DV: −4.00 mm). After waiting for 3 weeks to allow for viral expression, we perfused the mice and prepared brain slices to detect signals.

For retrograde tracing of the aPir→MD or aPir→NAc projections, we first injected a helper virus containing rAAV-Ef1α-DIO-RVG-WPRE-pA (AAV-DIO-RVG, AAV2/9, 2×10¹² vg/ml) and rAAV-Ef1α-DIO-mCherry-F2A-TVA-WPRE-pA (AAV-DIO-TVA-mCherry, AAV2/9, 2×10¹² vg/ml) at a 1:1 ratio (200 nl) into the MD or NAc unilaterally. After three weeks, we injected RV-CVS-ENVA-N2C(ΔG)-EGFP (2×10⁸ IFU/ml, 200 nl) into the same location of the MD or NAc. Following a 7-day period for virus expression, we perfused the mice for subsequent experiments.

For retrograde tracing from the abdominal and diaphragm muscles, we injected 4 μL of RFP-expressing pseudorabies virus (PRV-RFP; 2.00E+09 PFU/ml) into the abdominal external oblique muscle and 4 μL of EGFP-expressing pseudorabies virus (PRV-EGFP; 2.00E+09 PFU/ml) into the diaphragm muscle. Seven days after injection, we perfused the mice to detect the PRV signal.

For retrograde tracing from the VRG, we injected AAV2/Retro-hSyn-EGFP (retro-AAV-EGFP, 5.08×10¹² vg/ml, 200 nl) into the VRG (ML: −1.2 mm, AP: −7.67 mm, DV: −4.80 mm). Three weeks after injection, we perfused the mice to detect EGFP+ signal.

In vivo optogenetic manipulations
For optogenetic manipulation, we injected the Cre-dependent virus rAAV-Ef1α-DIO-hChR2(H134R)-mCherry-WPRE-pA (AAV-DIO-ChR2-mCherry, AAV2/9, 1.63×10¹³ vg/ml, 200 nl) into the aPir of FosTRAP2 mice or into the MD of CaMK2-Cre mice. Following viral injection, we implanted optic fibers (200 μm diameter, Newdoon) into the MD, NAc or VLPAG of anesthetized mice. To secure the optical fibers, we used tissue glue and dental cement to attach them firmly to the skull. Mice were allowed at least 3 weeks to recover before starting the experiments. To activate the circuits, we connected the optical fibers to a laser generator through fiber sleeves. A 473 nm blue light (2–5 mW, 20 Hz) using a Master-8 pulse stimulator (A.M.P.I.). Control mice received the Cre-dependent virus rAAV-Ef1α-DIO-mCherry-WPRE-pA (AAV-DIO-mCherry, AAV2/9, 1.63×10¹³ vg/ml, 200 nl) and underwent the same surgical procedure.

In vivo chemogenetic manipulations
For chemogenetic experiments, we injected the Cre-dependent virus rAAV-Ef1α-DIO-hM4D(Gi)-mCherry-WPRE-pA (AAV-DIO-hM4Di-mCherry, AAV2/9, 2.25×10¹² vg/ml, 200 nl) into the aPir of FosTRAP2 mice. Control mice received an injection of rAAV-Ef1α-DIO-mCherry-WPRE-pA (AAV-DIO-mCherry, AAV2/9, 2.12×10¹² vg/ml). For chemogenetic inhibition of 2MBA-activated aPir neurons, we administered CNO intraperitoneally. To inhibit the aPir→MD and aPir→NAc circuits, we implanted cannulas (internal diameter 0.25 mm, RWD) into the MD or NAc. CNO (3 μM, 150 nL) was then microinjected through the cannulas54.

In vivo optical fiber photometer recording
To record calcium signals, we injected the AAV-DIO-GCaMP6m virus and implanted an optic fiber (200 μm diameter, Newdoon) into the aPir of FosTRAP2 mice, the MD of CaMK2-Cre mice, and the NAc of GAD2-Cre mice. Recordings were conducted three weeks after viral expression. A monofiber optic patch cord was connected to the fiber photometry system (Inper) and secured to the implanted fiber using a ceramic sleeve and black heat-shrinkable tubing. A 470 nm LED light (3 ms pulses, 40 Hz) was bandpass-filtered (470/10 nm) and delivered to the fiber tip to excite GCaMP6m fluorescence. The emitted fluorescence was captured at 40 fps using Inper Signal. A 410-nm LED light, bandpass-filtered (410/10 nm), served as the isosbestic control channel. Fluorescence changes (ΔF/F) were calculated using the formula ΔF/F (%) = [(F(duration) - F(baseline)) / F(baseline)]×100%55. GCaMP6m fluorescence intensity was recorded while mice were exposed to odor stimuli, with signals recorded 5 seconds before the odor presentation used as the baseline. Fluorescence was also measured during retching-like behavior and CPA tests, with baseline signals defined as those recorded 5 seconds prior to retching or traveling in odor-paired chambers. Heatmaps and averaged calcium traces were generated using Inper Plot software (Inper Technology, Hangzhou).

In vivo electrophysiological recordings
Mice were housed individually before the implantation of tetrode arrays. Following a 3-day adaptation period, we implanted custom-made movable tetrode arrays with screw-driven microdrives and four implantable tetrodes into the MD or NAc. After surgery, mice were given a recovery period of at least 3 days. During this time, electrodes attached to their heads were connected to cables for daily 20 min adaptation sessions. During recordings, mice were placed in behavioral boxes surrounded by copper mesh and allowed to move freely. Neuronal signals were amplified, filtered (300–5000 Hz bandwidth), and stored using Neurostudio software. Offline analyses were performed with Neuroexplorer 4 (Nex Technologies) and Offline Sorter 4 (Plexon).

In vitro electrophysiological recordings
Brain slice preparation. Mice were deeply anesthetized using isoflurane. After anesthesia, we performed intracardiac perfusion with ice-cold, oxygenated artificial cerebrospinal fluid (NMDG ACSF; osmolarity: 300-305 mOsm/kg, pH: 7.3-7.4). The NMDG ACSF consisted of the following components (in mM): 93 NMDG, 2.5 KCl, 25 glucose, 0.5 CaCl2, 5 Na-ascorbate, 20 HEPES, 3 Na-pyruvate, 1.2 NaH2PO4, 3 glutathione, 30 NaHCO3, 10 MgSO4, and 2 thiourea. We cut coronal slices (300 μm) containing the MD or NAc using a vibrating microtome (VT1200s, Leica, Germany). The slices were then transferred to HEPES ACSF solution for at least 1 hour. This HEPES ACSF solution (28 °C, osmolarity: 300-305 mOsm/kg, pH 7.3–7.4) contained (in mM): 2.5 KCl, 3 glutathione (GSH), 92 NaCl, 3 sodium pyruvate, 1.2 NaH2PO4, 2 MgSO4, 2 thiourea, 30 NaHCO3, 25 glucose, 20 HEPES, 5 Na-ascorbate, and 2 CaCl2. After incubation, we placed the brain slices in a recording chamber (Warner Instruments, USA) for whole-cell recordings. The slices were continuously perfused with standard ACSF (32 °C, osmolarity: 300–310 mOsm/kg, pH: 7.3–7.4). This ACSF contained (in mM): 3 HEPES, 10 glucose, 129 NaCl, 3 KCl, 2.4 CaCl2, 1.3 MgSO4, 1.2 KH2PO4, and 20 NaHCO3, at a flow rate of 3 ml/min.

Light-evoked response. We visualized neurons in the MD and NAc brain regions using a 40× water immersion objective on an upright microscope (BX51WI, Olympus, Japan). Patch pipettes were fabricated by horizontally pulling and fire-polishing borosilicate glass capillary tubes to achieve a tip resistance of 5–7 MΩ. These pipettes were filled with a potassium gluconate-based internal solution. An optical fiber (200 μm diameter) was positioned 0.2 mm above the brain slice and aligned with the target neurons to deliver blue laser light. To confirm the functionality of the AAV-DIO-ChR2-mCherry virus, we visualized neurons expressing ChR2-mCherry in the aPir and stimulated them with blue laser light (473 nm, 5–10 mW) at pulse widths of 15 ms and frequencies of 5 Hz or 10 Hz. Following blue laser light stimulation of ChR2-mCherry-expressing aPir neuronal fibers in MD or NAc brain slices, we recorded excitatory postsynaptic currents (EPSCs) in voltage-clamp mode at a holding potential of −70 mV.

Microendoscope imaging and data processing
For microendoscope imaging, we delivered the Cre-dependent virus rAAV-Ef1α-DIO-GCaMP6m-WPRE-pA (AAV-DIO-GCaMP6m, AAV2/9, 1.63×10¹³ vg/ml) separately into the aPir, MD, and NAc. The injection pipette was left in place for 20 min following virus delivery to ensure proper diffusion. Subsequently, we implanted microendoscopic gradient index (GRIN) lenses (6.1 mm in length, 0.5 mm in diameter; or 4.00 mm in length, 1.0 mm in diameter, Inscopix, USA) into the aPir, MD, and NAc of anesthetized mice. The GRIN lenses implanted in the MD were modified as described56–58.

After 21 days of surgical recovery, we attached an integrated miniature fluorescence microscope (Inscopix, USA) to the baseplate on the mice’s heads. To ensure adaptation, the mice underwent 15 min sessions of habituation for 3 consecutive days before the recording sessions. During data acquisition, we recorded image data at 20 Hz per session. The collected image data were processed offline using Inscopix Data Processing Software (version 1.1.6, Inscopix, USA), following previously established protocols56–58.

Statistical analysis
We performed statistical comparisons between two groups using paired or unpaired two-tailed Student’s t tests. For multiple-group comparisons, we applied one-way or two-way ANOVA followed by post hoc analyses. Statistical significance was defined as follows: *P<0.05, **P<0.01, and ***P<0.001. Results are presented as means±s.e.m. Statistical analysis and graphing were conducted using OriginPro 2023 (OriginLab, USA) and GraphPad Prism 8 (GraphPad Software, USA). Offline analysis of in vitro electrophysiological data was performed using Clampfit software (version 10.7, Axon Instruments, USA). For in vivo electrophysiological data, we used NeuroExplorer (Plexon, USA). Microendoscopic imaging data were analyzed offline using Inscopix Data Processing Software (version 1.1.6, Inscopix, USA). All images were created by the authors using Adobe Illustrator.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This study focuses on the neural mechanisms of defensive responses triggered by spoiled food odors. It discovers that exposure to 2 - methylbutyric acid (2MBA) odor in mice can trigger retching-like behavior and aversion.By using techniques such as viral tracing, in vivo electrophysiological recordings, and calcium imaging, the researchers identify two neural circuits. The nose-OB-aPir-MD circuit is essential for 2MBA-triggered retching-like behavior, while the nose-OB-aPir-NAc circuit is crucial for 2MBA-triggered aversion.The study further reveals that 2MBA activates glutamatergic neurons in the anterior piriform cortex. These activated neurons project to the mediodorsal thalamic nucleus and the nucleus accumbens, respectively, to mediate different defensive behaviors.This research defines the circuit mechanisms for sensory-motor integration and periphery-brain communication in response to spoiled food odors. It also provides a conceptual basis for developing odor-based therapies and intranasal neuromodulation strategies related to metabolic homeostasis and disorders.\nPrevious studies showed that bacterial toxins for food poisoning can induce retching-like behavior. The results of this study strongly support the existence of retching-like behavior in rodents. And it greatly expands the study of retching-like behavior from interoception to olfactory modality. This study is well designed and fully executed. The data are compelling and fully clarify the brain circuits for olfaction-induced retching and nausea.I do not have major concerns. Only several minor concerns are here:\n1) Fig.3l, the N value of this experiment is only 4. Please perform more experiments, at least in 10 cells to prove the synapse is glutamatergic. Moreover, the amplitude of EPSCs should be always positive .\n2) Fig.4h, the amplitude of EPSCs should always be positive. Please correct it.\n3) Fig.5n, the N values for animal number are 5. Please increase the N values to 7-10.\n4) Fig. 6l, please also increase the N values.", "reviewer_2": "This study investigates the neural circuit mechanisms by which the rancid odors triggers retching-like behaviors via the olfactory system. Utilizing a series of contemporary techniques, including TRAP2 labeling of activated neurons, viral tracing, microendoscopic calcium imaging, and chemogenetic/optogenetic manipulations, the authors reveal a fascinating nose-to-brain axis for olfaction-triggered defensive behaviors. These findings provide a theoretical foundation for developing odor-based anti-emetic therapies, such as managing the side effects of obesity treatment drugs. Specifically, the pathway nose-aPir→MDglu→ventrolateral periaqueductal gray (VLPAG)→ventral respiratory group (VRG)→respiratory muscles controls retching, while aPir→NAcGABA mediates aversion. The results are significant and novel, supported by well-designed experiments and clearly presented data. However, to further strengthen the findings, some concerns should be addressed.\n1. The study lacks activation experiments (only Pir-MD/NAc pathway activation of TRAP neurons was performed). It would be better to test if MDglu -VLPAG pathway activation could trigger retching-like behavior. Please provide electromyography (EMG) and time-event plots of retching-like behaviors.\n2. In fact, MOB mitral cells project to several brain regions, with dense innervation including the piriform cortex, cortical amygdala, and olfactory tubercle (Liberles, Stephen D. 2014 Annual Review of Physiology). The aPir is a key nucleus in this study. Fig2a identified the OB-aPir pathway connection using viral tracing methods. However, do the OB neurons trapped by 2MBA have other downstream projections? Please present other EGFP fiber-labeled nuclei and clarify whether these nuclei are also involved in retching-like behaviors. Additionally, the role of aPir in olfaction-triggered innate defense is insufficiently discussed in both the introduction and discussion sections.\n3. Predator odor TMT (Wang, Hao, et al. 2024 Nature Neuroscience), bacterial metabolite H2S (Koike, Kohei et al. 2021 Neuron), and MgrB peptide (Bufe, Bernd, et al. 2019 Nature Communications) can also induce innate escape or defensive avoidance behaviors in mice. These were not included as control odors to confirm the pathway's specificity to 2MBA. Did this aPir→MDglu/ NAcGABA pathways also response to TMT (Calcium data)?\n4. The miniature endoscopic calcium imaging experiments do not specify the number of mice used or the Grin lens trajectory.\n5. Figure 2h-k does not indicate the number of mice or trials. Since TRAP-labeled neurons appear to respond to different odors, is the activity of these neurons correlated with varying concentrations of 2MBA? Odorants induced behavioral responses can be concentration-dependent. 15%, 30% (vol:vol) were used in 2MBA-triggered retching-like behavior and aversion tests. Could provide molar concentration? Is these concentrations too high to apply in these behaviors according to doi: 10.1038/nature06281 ? This reference should be included in the Introduction.\n6. For the fiber photometry recordings in Figure 5d and Figure 6c, please specify the number of mice and trials.\n7. Please provide the co-localization statistics for tdTomato with Glu and GABA in MD and NAc in Extended Data Figure 6e.\n8. Figure 2m should include c-Fos co-staining to confirm TRAP efficiency."}, "author_response": "Response to Reviews\nManuscript ID: NCOMMS-25-29279A\nTitle: A nose-to-brain axis for spoiled food odor-triggered defensive responses\nWe sincerely appreciate the positive and helpful evaluations provided by the editor and\nReviewers. Guided by these thoughtful comments, we have completed an extensive set of\nadditional experiments to address their specific concerns. We have also revised the manuscript\nwhere appropriate, incorporating content germane to comments of each Reviewer. A revised\nversion of the manuscript with tracked changes (highlighted in blue) has been uploaded as a\nseparate file. The detailed changes and our point-by-point responses to each of the Editor’s or\nReviewers’ questions and comments are presented below.\nContents:\nResponse to Reviewer #1: Page 02-05\nResponse to Reviewer #2: Page 06-18\nReference: Page 19-20\nReviewer #1 (Remarks to the Author):\nThis study focuses on the neural mechanisms of defensive responses triggered by spoiled food\nodors. It discovers that exposure to 2-methylbutyric acid (2MBA) odor in mice can trigger\nretching-like behavior and aversion. By using techniques such as viral tracing, in vivo\nelectrophysiological recordings, and calcium imaging, the researchers identify two neural\ncircuits. The nose-OB-aPir-MD circuit is essential for 2MBA-triggered retching-like behavior,\nwhile the nose-OB-aPir-NAc circuit is crucial for 2MBA-triggered aversion. The study further\nreveals that 2MBA activates glutamatergic neurons in the anterior piriform cortex. These\nactivated neurons project to the mediodorsal thalamic nucleus and the nucleus accumbens,\nrespectively, to mediate different defensive behaviors. This research defines the circuit\nmechanisms for sensory-motor integration and periphery-brain communication in response to\nspoiled food odors. It also provides a conceptual basis for developing odor-based therapies and\nintranasal neuromodulation strategies related to metabolic homeostasis and disorders.\nPrevious studies showed that bacterial toxins for food poisoning can induce retching-like\nbehavior. The results of this study strongly support the existence of retching-like behavior in\nrodents. And it greatly expands the study of retching-like behavior from interoception to\nolfactory modality. This study is well designed and fully executed. The data are compelling and\nfully clarify the brain circuits for olfaction-induced retching and nausea. I do not have major\nconcerns. Only several minor concerns are here:\n1) Fig.3l, the N value of this experiment is only 4. Please perform more experiments, at least in\n10 cells to prove the synapse is glutamatergic. Moreover, the amplitude of EPSCs should be\nalways positive.\nRESPONSE: First, we would like to thank the reviewer for their positive comments regarding\nour manuscript and for their guidance towards improving our study. As the postsynaptic\nglutamatergic responses before and after light stimulation were obvious in our\nelectrophysiology recordings, we therefore presented representative data from only five cells\n(kindly note there are overlapping markers in the plot). However, we agree that greater\nstatistical rigor is necessary and, as suggested, we have replicated the experiment to increase\nthe sample size to 14 cells. To this end, we injected an AAV-DIO-ChR2-mCherry into the aPir\nand an AAV-CaMK2-EGFP into the MD of FosTRAP2 mice. After three weeks of virus expression,\nmice were injected with 4-OHT and exposed to 2MBA. Two weeks later, in vitro\n2\nelectrophysiological recordings of ChR2-expressing MDGlu neuronal terminals revealed that\nlight stimulation reliably triggered excitatory postsynaptic currents (EPSCs), and were\neliminated upon treatment with the AMPA receptor antagonist, 6,7-dinitroquinoxaline-2,3\n(1H,4H)-dione (DNQX) (Response Document Figure 1, and also see new Figure 3l). These\nfindings confirmed the presence of functional connections between 2MBA-activated aPirGlu\nneurons and MDGlu neurons.\nIn original Fig.3l, we presented EPSC amplitude as negative values to illustrate that these\ncurrents were below baseline. Based on the reviewer’s comment, we now understand that this\npresentation is inappropriate, and we have adjusted the amplitude in the figure to show positive\nvalues (Response Document Figure 1, and also see new Figure 3l).\nWe have updated these results with our new data in the revised manuscript.\nResponse Document Figure 1 | Functional connections of the aPirGlu→MDGlu circuit. a,\nSchematic for virus injection and recording configuration in acute slices of FosTRAP2 mice. b,\nRepresentative images of mCherry+ fibers in the MD surrounding EGFP+ neurons. Scale bar,\n10 µm. c, Representative traces (left) and summarized data (right) for light-evoked currents\n(blue bar) before and after treatment with AMPA receptor antagonist, DNQX (10 µM) (n = 14\ncells from 10 mice per group). Significance was assessed using two-tailed paired Student’s t-\ntests in panel c. All data are presented as the mean ± s.e.m. *** P < 0.01. ACSF, artificial\ncerebrospinal fluid; DNXQ, 6,7-dinitroquinoxaline-2,3 (1H,4H)-dione.\n2) Fig.4h, the amplitude of EPSCs should always be positive. Please correct it.\nRESPONSE: As suggested, this has been corrected in the revised manuscript.\n3) Fig.5n, the N values for animal number are 5. Please increase the N values to 7-10.\nRESPONSE: As suggested, we have performed more experiments to increase the sample size\nto 10 mice. Specifically, we injected AAV2/1-Cre into the bilateral nares, AAV-Con Fon-\nhM4Di-EGFP into the bilateral OB, Cre-dependent helper viruses into the bilateral aPir, and\nretro-Cre into the bilateral MD. After 3 weeks of virus expression, RV-Flp-DsRed was injected\ninto the bilateral aPir. At one week post injection with RV, we applied CNO to the aPir by\n3\nintracranial microinjection to inhibit the nose→OB→aPir→MD circuit. Upon treatment with\n2MBA odor, we observed that retching-like behavior, but not conditioned aversion, was\nsignificantly reduced compared to that in mCherry control mice (Response Document Figure\n2, and also see new Figures 5m-p). These findings were consistent with data in original figure\n5j-p, thus demonstrating that the nose→OB→aPir→MD circuit is required for 2MBA-triggered\nretching-like behavior, but not aversion responses.\nWe have replaced the original results with these new data in the revised manuscript.\nResponse Document Figure 2 | The nose→OB→aPir→MD circuit controls 2MBA-\ntriggered retching-like behavior. a, Schematic for chemogenetic inhibition of the\nnose→OB→aPir→MD circuit. b,c, Example raster plots (b) and counts (c) of retching-like\nbehavior in mice following inhibition of the nose→OB→aPir→MD circuit (n = 10 mice per\ngroup). d, Summarized data of electromyography (EMG) recordings of diaphragm muscle\nfollowing inhibition of the nose→OB→aPir→MD circuit (n = 10 mice per group). e,\nSummarized data of CPA tests in mice inhibition of the nose→OB→aPir→MD circuit (n = 10\nmice per group). Significance was assessed using two-tailed unpaired Student’s t-tests in panels\nc, d and e. All data are presented as means ± s.e.m. *** P < 0.001, n.s., not significant.\n4) Fig. 6l, please also increase the N values.\nRESPONSE: As suggested, we replicated this experiment to increase the sample size to 10\nmice by injecting AAV2/1-Cre into the bilateral nares, AAV-Con Fon-hM4Di-EGFP into the\nbilateral OB, Cre-dependent helper viruses into the bilateral aPir, and retro-Cre into the bilateral\nNAc of C57 mice. After 3 weeks of virus expression, RV-Flp-DsRed was injected into the\nbilateral aPir. At one week post injection with RV, we administered CNO in the aPir by\n4\nintracranial microinjection to inhibit the nose→OB→aPir→NAc circuit. Behavioral tests again\nshowed that conditioned aversion, but not retching-like behavior, was significantly reduced in\nresponse to 2MBA odor compared to that in mCherry control mice (Response Document Figure\n3, and also see new Figures 6k-n). These findings were consistent with data in original figures\n6h-n, supporting that the nose→OB→aPir→NAc circuit is required for 2MBA-triggered\naversion, but not retching-like behavior.\nThese data obtained from 10 mice per group now replace the original results in the revised\nmanuscript.\nResponse Document Figure 3 | The nose→OB→aPir→NAc circuit controls 2MBA-\ntriggered aversion. a, Schematic for chemogenetic inhibition of the nose→OB→aPir→NAc\ncircuit. b,c, Example raster plots (b) and counts (c) of retching-like behavior following\ninhibition of the nose→OB→aPir→NAc circuit (n = 10 mice per group). d, Summarized data\nof EMG recordings of diaphragm muscle with inhibition of the nose→OB→aPir→NAc circuit\n(n = 10 mice per group). e, Summarized data of CPA tests following inhibition of the\nnose→OB→aPir→NAc circuit (n = 10 mice per group). Significance was assessed using two-\ntailed unpaired Student’s t-tests in panels c, d and e. All data are presented as means ± s.e.m.\n*** P < 0.001, n.s., not significant.\nWe again thank the reviewer for their time, careful review, and expert guidance, which has\nhelped to us to improve the statistical rigor of our work and reinforce the conclusions of our\nstudy.\nThis study investigates the neural circuit mechanisms by which the rancid odors triggers\nretching-like behaviors via the olfactory system. Utilizing a series of contemporary techniques,\nincluding TRAP2 labeling of activated neurons, viral tracing, microendoscopic calcium\nimaging, and chemogenetic/optogenetic manipulations, the authors reveal a fascinating nose-\nto-brain axis for olfaction-triggered defensive behaviors. These findings provide a theoretical\nfoundation for developing odor-based anti-emetic therapies, such as managing the side effects\nof obesity treatment drugs. Specifically, the pathway nose-aPir→MDglu→ventrolateral\nperiaqueductal gray (VLPAG)→ventral respiratory group (VRG)→respiratory muscles\ncontrols retching, while aPir→NAcGABA mediates aversion. The results are significant and\nnovel, supported by well-designed experiments and clearly presented data. However, to further\nstrengthen the findings, some concerns should be addressed.\n1. The study lacks activation experiments (only Pir-MD/NAc pathway activation of TRAP\nneurons was performed). It would be better to test if MDglu -VLPAG pathway activation could\ntrigger retching-like behavior. Please provide electromyography (EMG) and time-event plots\nof retching-like behaviors.\nRESPONSE: First, we would like to thank the reviewer for their positive review of our work\nand for their expert insights, which have guided several improvements to our study. As\nsuggested, we have conducted new experiments in which we activate the MDGlu→VLPAG\npathway. Specifically, we injected AAV-DIO-ChR2-mCherry into the MD, and implanted\noptical fibers in the VLPAG of CaMK2-Cre mice. Upon delivery of 473 nm blue light (2-5 mW,\nretching-like behavior and electromyography (EMG) recordings after optogenetic activation of\nMDGlu→VLPAG pathway showed significant increases in both retching-like behaviors, as well\nas increased frequency and amplitude of abdominal external oblique muscle and diaphragmatic\nmuscle activity compared with corresponding responses in mCherry control mice (Response\nDocument Figure 4, also see new Extended Data Fig. 17). These results collectively\ndemonstrated that the MDGlu→VLPAG pathway is essential for triggering retching-like\nbehavior.\nWe have added these results to the revised manuscript, including time-event plots\nquantifying retching-like behaviors and EMG data, as requested.\nbehavior. a, Schematic for optogenetic activation of the MDGlu→VLPAG circuit in CaMK2-\nCre mice. b, Representative images of mCherry+ neurons in the MD (left) and mCherry+ fibers\nwith the optical fiber tract in the VLPAG (right). Scale bars, 100 μm. c,d, Example raster plots\n(c) and counts (d) of retching-like behavior following optogenetic activation of the\nMDGlu→VLPAG circuit. e,f, Summarized data from EMG recordings in abdominal external\noblique (Abd., e) and diaphragm (Dia., f) muscle upon optogenetic activation of the\nMDGlu→VLPAG circuit. Significance was assessed using two-tailed unpaired Student’s t-tests\nin panels d, e, and f. All data are presented as means ± s.e.m. * P < 0.05, ** P < 0.01, *** P <\n0.001.\n2. In fact, MOB mitral cells project to several brain regions, with dense innervation including\nthe piriform cortex, cortical amygdala, and olfactory tubercle (Liberles, Stephen D. 2014\nAnnual Review of Physiology). The aPir is a key nucleus in this study. Fig2a identified the OB-\naPir pathway connection using viral tracing methods. However, do the OB neurons trapped by\n2MBA have other downstream projections? Please present other EGFP fiber-labeled nuclei and\nclarify whether these nuclei are also involved in retching-like behaviors. Additionally, the role\nof aPir in olfaction-triggered innate defense is insufficiently discussed in both the introduction\nand discussion sections.\nRESPONSE: We thank the reviewer for this insightful suggestion. As the reviewer correctly\nnoted, mitral cells in the main olfactory bulb (MOB) have been shown to project to multiple\nregions, including the piriform cortex (Pir), cortical amygdala (CoA), and olfactory tubercle\n(OT).1 To address whether 2MBA-TRAPed OB neurons innervate nuclei other than the Pir, we\n7\nexamined the distribution of EGFP signals in whole brain slices of 2MBA-treated FosTRAP2\nmice following AAV2/1-DIO-FLP-mCherry injection in the naris, AAV-fDIO-EGFP injection\ninto the OB, and subsequent 4-OHT administration by i.p. injection (Response Document\nFigure 5a, and also see new Figure 2a). We found that EGFP+ fibers were abundant in the aPir,\nbut very scarce in other region, including the well-known downstream of the OB (pPir, OT or\nCoA) (Response Document Figure 5b, and also see new Extended Data Figure 4). These data\nconfirmed that 2MBA-TRAPed OB neurons predominantly project to the aPir.\nWe completely agree that our introduction does not sufficiently address the role of aPir in\nolfaction-triggered innate defense. We have therefore added the following content to the revised\nintroduction:\n“Output cells of the OB relay information from individual glomeruli to olfactory\ncortical areas, the largest of which is the piriform cortex (Pir), via diffuse and\noverlapping projections, thus allowing individual Pir neurons to incorporate inputs\nfrom different combinations of glomeruli.1,2 The anterior and posterior Pir (aPir\nand pPir) exhibit neuroanatomical differences, with the aPir receiving more\nextensive inputs from OB than pPir.3 The aPir in turn sends projections to numerous\nbrain regions associated with defensive responses, including the prefrontal\ncortex.4,5 However, it remains unclear whether and how the OB-aPir pathway\nparticipates in defensive responses to spoiled food odors.”\nAdditionally, we have added related content to the revised Discussion section, as follows:\n“Mitral cells in the OB project to multiple regions, including the Pir, CoA, and OT.1\nPrevious studies have revealed amygdala neurons encode odor valence, which\npreferentially respond to liked odors versus disliked odors.6,7 The OT has also been\nshown to contain spatially-segregated functional domains involved in distinct\nmotivations or hedonic behaviors associated with olfaction.8-10 We found that\n2MBA-TRAPed OB neurons predominantly innervate the aPir, rather than other\nbrain regions. The specificity of such neural circuits may be related to the function\nof these nuclei. Finding in our current study demonstrated that the aPir is involved\nin retching-like behavior and aversion triggered by rancid odor.\nThe aPir encodes odorant information, perception, and odor-associated\nvalues, which is then communicated to other, high-order cortical areas, such as the\n8\nprefrontal cortex, thereby supporting the establishment of odor associations with\nepisodic-like or emotional memories.4,5,11 Anatomically, the aPir is well positioned\nto transform sensory representations into value representations that may\nsubsequently lead to appropriate behavioral outputs, including defensive\nresponses.5 Our results showed that aPir projects to the MD to drive odor-triggered\nretching-like behaviors, or project to the NAc to drive odor-triggered aversion,\nindicating that aPir integrates olfactory information, and serves as a hub linking\nodor sensory processing to behaviorally outputs.”\nWe have added these new results and expanded introduction and discussion content to the\nrevised manuscript.\nResponse Document Figure 5 | Viral tracing in FosTRAP2 mice. a, Schematic for viral tracing\nin FosTRAP2 mice. b-e, A series of coronal sections showing the distributions of EGFP+ fibers\n9\nacross different brain areas. Scale bars, 100 μm (b and c) and 1 mm (d and e). OSN, olfactory\nsensory neuron; OB, olfactory bulb; aPir, anterior piriform cortex; pPir, posterior piriform\ncortex; amOT, anteromedial domain of the OT; lOT, lateral domain of the OT; CoApl,\nposterolateral cortical amygdala; CoAa, anterior cortical amygdala.\n3. Predator odor TMT (Wang, Hao, et al. 2024 Nature Neuroscience), bacterial metabolite H2S\n(Koike, Kohei et al. 2021 Neuron), and MgrB peptide (Bufe, Bernd, et al. 2019 Nature\nCommunications) can also induce innate escape or defensive avoidance behaviors in mice.\nThese were not included as control odors to confirm the pathway’s specificity to 2MBA. Did\nthis aPir→MDglu/ NAcGABA pathways also response to TMT (Calcium data)?\nRESPONSE: We appreciate the reviewer’s insightful suggestion regarding control odors and\npathway specificity. Although trimethylthiazoline (TMT), H S, and MgrB peptide are known\n2\nto trigger innate defensive avoidance in mice, these odors can also elicit responses other than\ndefensive behaviors. Specifically, TMT triggers intense fear responses,12 H S causes\n2\nrespiratory/neurological toxicity,13,14 and MgrB peptide is associated with bacterial virulence\nand antibiotic resistance, thus posing biosafety concern.15 2MBA is more closely associated\nwith the olfactory sensation of rancid food, which is frequently encountered in daily life,\nwithout reported adverse effects.16,17 Given these potential off-target effects of other stimuli, we\ntherefore employed 2MBA as the relevant stimulus for studying rancid odor-triggered defensive\nresponses in our study. However, we agree that incorporating TMT, H S and MgrB peptide as\n2\ncontrol odors should confirm the specificity of this pathway to 2MBA.\nTo address this concern, we conducted behavioral assays and microendoscopic calcium\nrecordings in mice exposed to these odors. Consistent with previous studies, we found that TMT,\nNaHS (a H S-releasing agent13), and MgrB peptide odors (concentrations according to previous\n2\nstudies12,13,15) all triggered aversion in CPA experiments, but not retching-like behaviors\n(Response Document Figure 6, and also see new Extended Data Figure 2). Moreover, none of\nthese odors led to increased amplitude or frequency in EMG recordings (Response Document\nFigure 6, and also see new Extended Data Figure 2).\nMicroendoscopic calcium recordings showed that aPir-innervated NAc neurons displayed\na significant, time-locked increase in calcium activity upon exposure to these odors, and entry\ninto a chamber paired with TMT, H S, or MgrB peptide odors compared to unpaired chamber\n2\nentries in CPA post-tests (Response Document Figure 7a-f, and also see new Extended Data\nFigure 14g-n). These results were consistent with previous studies that showed NAc\ninvolvement in aversion triggered by different stimuli.18-20 However, we detected no time-\n10\nlocked increase in calcium activity in aPir-innervated MD neurons of freely moving C57 mice\nupon exposure to TMT, H S or MgrB peptide or in CPA post-tests (Response Document Figure\n2\n7g-l and also see new Extended Data Figure 12j-q). Moreover, only 2MBA could stably trigger\nretching-like behaviors, indicating that the aPir→MDGlu pathway was specific to 2MBA-\ntriggered retching-like behavior.\nWe have added these new results to the revised manuscript.\nResponse Document Figure 6 | Absence of retching-like behaviors in response to TMT,\nH S, or MgrB peptide odors. a-l, Schematics of the experimental design for exposure to TMT\n2\n(a, trimethylthiazoline), H S (e, hydrogen sulfide), or MgrB peptide (i) odors; corresponding\n2\ncounts of mouth-opening activity (b, f, and j, respectively); representative traces and\nsummarized data of EMG amplitude and frequency of abdominal external oblique (Abd.) and\ndiaphragm (Dia.) muscle (c, g, and k, respectively); summarized data for CPA tests (d, h, and l,\nrespectively). 2% TMT, 10 μM NaHS and 200 μM MgrB were used for CPA tests. Significance\nwas assessed using two-tailed paired Student’s t-tests in panels c, g, k, and two-tailed unpaired\nStudent’s t-tests in panels d, h, l. All data are presented as the mean ± s.e.m. * P < 0.05, ** P <\n0.01, *** P < 0.001, n.s., not significant.\nNAc and MD neurons. a, Schematic for virus injection and GRIN lens implantation for in vivo\nmicroendoscope imaging of aPir-innervated NAc neurons in freely moving C57 mice. b,\nRepresentative image shows virus expression and Grin lens tract in the NAc. Scale bar, 100 μm.\nc,d, Summarized data of calcium transients (c) and averaged z-score (d) of GCaMP6m-\nexpressing NAc neurons before and during odor inhalation. e,f, Summarized data showing the\nevents (e) and averaged z-score (f) of GCaMP6m-expressing NAc neurons in CPA post-tests.\ng, Schematic for virus injection and in vivo microendoscopic calcium recording of aPir-\ninnervated MD neurons in C57 mice. h, Representative image shows virus expression and Grin\n12\nlens tract in the MD. Scale bar, 100 μm. i,j, Summarized data of calcium transients (i) and\naveraged z-score (j) of GCaMP6m-expressing MD neurons before and during odor inhalation.\nk,l, Summarized data of calcium transients (k) and averaged z-score (l) of GCaMP6m-\nexpressing MD neurons during CPA post-tests. Significance was assessed using two-tailed\npaired Student’s t-tests in panels c, d, e, f, i, j, k and l. All data are presented as the mean ±\ns.e.m. ** P < 0.01, *** P < 0.001, n.s., not significant.\n4. The miniature endoscopic calcium imaging experiments do not specify the number of mice\nused or the Grin lens trajectory.\nRESPONSE: We agree that this information is critical to assess statistical rigor. As\nrecommended, we now explicitly include the number of animals for calcium imaging\nexperiments in the corresponding figure legends. We have also added images showing Grin lens\ntrajectories to illustrate the delivery sites, where appropriate, in the revised manuscript\n(Response Document Figure 8, and also see new Figures 5g, 6d, and Extended Data Figure 6b).\nResponse Document Figure 8 | Representative images of GRIN lens implantation sites. a,\nSchematic (left) and representative image (right) for virus injection and GRIN lens implantation\nfor microendoscopic calcium imaging of aPir neurons. Scale bar, 100 μm. b, Schematic (left)\nand representative image (right) for virus injections and GRIN lens implantation of MD neurons.\nScale bar, 100 μm. c, Schematic (left) and representative image (right) for virus injections and\nGRIN lens implantation of NAc neurons. Scale bar, 100 μm.\n5. Figure 2h-k does not indicate the number of mice or trials. Since TRAP-labeled neurons\nappear to respond to different odors, is the activity of these neurons correlated with varying\nconcentrations of 2MBA? Odorants induced behavioral responses can be concentration-\ndependent. 15%, 30% (vol:vol) were used in 2MBA-triggered retching-like behavior and\naversion tests. Could provide molar concentration? Is these concentrations too high to apply in\nthese behaviors according to doi: 10.1038/nature06281? This reference should be included in\nthe Introduction.\nRESPONSE: Specific questions from this comment are addressed individually below:\nRESPONSE: In Figure 2h-k, eight FosTRAP:Ai mice were used for experiments demonstrating\nresponses to different odors among individual mice. Each row of heatmaps corresponds to one\ntrial conducted with an individual mouse. We have added the number of mice used in each\nexperiment to the Figure legends.\nQ2：Since TRAP-labeled neurons appear to respond to different odors, is the activity of these\nneurons correlated with varying concentrations of 2MBA?\nRESPONSE: To assess whether 2MBA-TRAPed neurons respond to different concentrations\nof 2MBA, we conducted new experiments wherein AAV-DIO-GCaMP6m was injected and\noptical fibers were implanted into the aPir of FosTRAP2 mice. After sufficient virus expression\nand labeling by 4-OHT injection, we performed optical fiber photometry recordings in these\nmice upon exposure to different concentrations of 2MBA. We found that the neural activity of\n2MBA-TRAPed aPir neurons increased in a concentration-dependent pattern, with 30% 2MBA\ninhalation resulting in higher ΔF/F than 15% 2MBA (Response Document Figure 9, and also\nsee new Extended Data Figures 7a-c). These findings were consistent with previous studies that\nalso showed concentration-dependent neuronal responses to odors in Pir (e.g. ethyl butyrate,\nhexanal, and lavender essential oil).21,22\nResponse Document Figure 9 | Effects of 2MBA inhalation on calcium activity in aPir of\nFosTRAP2 mice. a, Schematic for in vivo fiber photometry recordings of 2MBA-activated aPir\nneurons in FosTRAP2 mice. b,c, Average calcium activity (b) and heatmaps (c) of time-locked\nCa2+ signals in GCaMP6m-expressing aPir neurons upon exposure to varying concentrations\nof 2MBA. Dashed line indicates onset of odor exposure.\nQ3: Odorants induced behavioral responses can be concentration-dependent. 15%, 30%\n(vol:vol) were used in 2MBA-triggered retching-like behavior and aversion tests. Could\nprovide molar concentration? Is these concentrations too high to apply in these behaviors\naccording to doi: 10.1038/nature06281? This reference should be included in the Introduction.\nconcentrations. As the density of 2MBA is 0.936 g/mL, the molar mass is 102.13 g/mol. Taking\n30% as an example, the volume of 2MBA per 1 mL solution is 0.3 mL, mass = 0.3 mL ×\n0.936 g/mol = 0.2808 g; and therefore molar unit = 0.2808 g / 102.13 g/mol ≈ 2.75×10⁻³ mol.\nSimilarly, 15% 2MBA corresponds to of 1.375×10⁻³ mol. We have added molar units to define\n2MBA concentrations in the revised Methods section.\nAs the reviewer noted, these 2MBA concentrations are high compared to concentrations\napplied by Sakano et. al. in which doses of 1.7×10⁻7 ~ 1.7×10⁻4 mol were used to trigger\navoidance responses.16 Inspired by this work, we tested a wider concentration gradient, ranging\nfrom 0.938% to 30% per mL solution (8.595×10⁻5 mol ~ 2.75×10⁻3 mol，Response Document\nTable1). We found that ≥1.875% (or 1.719×10⁻4 mol) concentrations could trigger aversion in\nCPA test, which similar to that in Sakano et. al (Response Document Figure 10a-c). However,\nonly high concentrations (i.e., ≥7.5% or 6.875×10⁻4 mol) could trigger retching-like behavior\n(Response Document Figure 10d-f). As mentioned by the reviewer, odor-triggered behavioral\nresponses can be concentration-dependent, and our results demonstrated that high\nconcentrations of 2MBA can trigger retching-like behavior.\nWe have added these new results, revised the figure legends, and accordingly modified the\nMethods section to include molar units of 2MBA. Additionally, we have cited this pioneering\nwork by Sakano and colleagues in revised Introduction section.\nvol: vol mol\n0.938% 8.594×10⁻5 mol\n1.875% 1.719×10⁻4 mol\n3.75% 3.438×10⁻4 mol\n7.5% 6.875×10⁻4 mol\n15% 1.375×10⁻³ mol\n30% 2.75×10⁻³ mol\nResponse Document Table 1 | 2MBA concentration gradient tested for behavioral\nresponses with corresponding molar units.\nSchematic of experimental paradigm for rancid odor-triggered retching-like behavior using\n2MBA evaporated in an enclosed space. b,c, Example raster plots (b) and summarized data (c)\nof retching-like behaviors during exposure to 2MBA odor. d, Schematic of experimental\nparadigm for rancid odor-induced conditioned place aversion (CPA) tests in ddH O-paired\n2\ngroup and 2MBA-paired group. e,f, Summarized data for CPA tests in ddH O-paired group and\n2\n2MBA-paired group. Significance was assessed using one-way ANOVA with post hoc\ncomparison between groups in panel c, e and f. All data are presented as means ± s.e.m. * P <\n0.05, ** P < 0.01, *** P < 0.001. n.s., not significant.\n6. For the fiber photometry recordings in Figure 5d and Figure 6c, please specify the number\nof mice and trials.\nResponse: In Figure 5d and 6c, each row of heatmaps corresponds to a trial conducted in one\nmouse. As suggested, we have added the number of mice and trials to the Figure legends.\n7. Please provide the co-localization statistics for tdTomato with Glu and GABA in MD and\nNAc in Extended Data Figure 6e.\nResponse: As suggested, we conducted quantitative image analysis of tdTomato colocalization\nwith Glu or GABA antibody in the MD and NAc. We found that 84% of tdTomato⁺ neurons\ncolocalized with glutamatergic antibody in the MD, while 93% colocalized with GABAergic\nantibody in the NAc (Response Document Figure 11, and also see new Extended Data Figure\n8f). These results demonstrated that 2MBA odor primarily activates glutamatergic neurons in\nthe MD and GABAergic neurons in the NAc.\nResponse Document Figure 11 | Immunohistochemistry staining of 2MBA-TRAPed\nneurons in MD and NAc. a,b, Representative images (top) and quantitative analysis (bottom)\nof colocalization between tdTomato signals and glutamate-specific antibody in the MD (a) or\nGABA-specific antibody in the NAc (b). Scale bars, 10 μm.\n8. Figure 2m should include c-Fos co-staining to confirm TRAP efficiency.\nResponse: To address this concern, we injected AAV-DIO-hM4Di-mCherry into the aPir of\nFosTRAP2 mice. After 3 weeks of virus expression, mice were intraperitoneally injected with 4-\nOHT, then exposed to 2MBA odor. Two weeks later, these mice were exposed to 2MBA again\nfor c-Fos immunostaining in brain sections containing aPir (Response Document Figure 12a,b,\nand also see new Extended Data Figure 7d,e). Quantitative imaging analysis revealed that 92.35%\nof TRAP-labeled mCherry⁺ neurons colocalized with c-Fos⁺ neurons (Response Document\nFigure 12c, and also see new Extended Data Figure 7f). This extensive colocalization between\nTRAPed neurons and c-Fos signals supported the high efficiency of TRAP-based labeling in\nthis study.\nWe have added these new results to the revised manuscript.\nsignals in the aPir. a, Schematic for TRAP labeling and c-Fos immunostaining to capture aPir\nneurons activated by 2MBA inhalation in FosTRAP2 mice. b,c, Representative images (b) and\nquantitative analysis (c) of the 2MBA-responsive neural ensemble in the aPir. Scale bar, 10 μm.\nWe would like to again thank the reviewer for their extremely helpful guidance and supportive\ncomments that have helped to substantially improve our study.\nphysiol-021113-170334 (2014).\ndistributed coding. 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Congratulations!", "reviewer_2": "The authors have addressed all of my concerns satisfactorily. I have no further suggestions and I believe the revised manuscript is now ready for publication."}, "author_response": ""}]

Decision letter