Vestibular neurons link motion sickness, behavioural thermoregulation and metabolic balance in mice

Longlong Tu; Xing Fang; Yongjie Yang; Meng Yu; Hailan Liu; Hesong Liu; Na Yin; Jonathan C Bean; Kristine M Conde; Mengjie Wang; Yongxiang Li; Olivia Z Ginnard; Qingzhuo Liu; Yuhan Shi; Junying Han; Yi Zhu; Makoto Fukuda; Qingchun Tong; Benjamin Arenkiel; Mingshan Xue; Yang He; Chunmei Wang; Yong Xu

doi:10.1038/s42255-025-01234-9

. Author manuscript; available in PMC: 2026 Jul 17.

Published in final edited form as: Nat Metab. 2025 Mar 21;7(4):742–758. doi: 10.1038/s42255-025-01234-9

Vestibular neurons link motion sickness, behavioural thermoregulation and metabolic balance in mice

Longlong Tu ^1,⁷, Xing Fang ¹, Yongjie Yang ¹, Meng Yu ¹, Hailan Liu ¹, Hesong Liu ¹, Na Yin ¹, Jonathan C Bean ¹, Kristine M Conde ¹, Mengjie Wang ¹, Yongxiang Li ¹, Olivia Z Ginnard ¹, Qingzhuo Liu ¹, Yuhan Shi ¹, Junying Han ¹, Yi Zhu ¹, Makoto Fukuda ¹, Qingchun Tong ², Benjamin Arenkiel ³, Mingshan Xue ^3,^4,⁵, Yang He ¹, Chunmei Wang ¹, Yong Xu ^1,^6,⁷

PMCID: PMC13374115 NIHMSID: NIHMS2175782 PMID: 40119169

Abstract

Motion sickness is associated with thermoregulation and metabolic control, but the underlying neural circuitry remains largely unknown. Here we show that neurons in the medial vestibular nuclei parvocellular part (MVePC) mediate the hypothermic responses induced by motion. Reactivation of motion-sensitive MVePC neurons recapitulates motion sickness in mice. We show that motion-activated neurons in the MVePC are glutamatergic (MVePC^Glu), and that optogenetic stimulation of MVePC^Glu neurons mimics motion-induced hypothermia by signalling to the lateral parabrachial nucleus (LPBN). Acute inhibition of MVePC-LPBN circuitry abrogates motion-induced hypothermia. Finally, we show that chronic inhibition of MVePC^Glu neurons prevents diet-induced obesity and improves glucose homeostasis without suppressing food intake. Overall, these findings highlight MVePC^Glu neurons as a potential target for motion-sickness treatment and obesity control.

Forty percent of people in the United States are living with obesity and another 30% are overweight, according to the US Centers for Disease Control and Prevention. These individuals are at higher risk for many chronic diseases, including metabolic syndrome, cardiovascular diseases, cancers and depression. Obesity and the associated metabolic syndrome are also risk factors for poor COVID-19 outcomes¹. Effective treatments to reduce obesity and improve metabolic health are urgently needed. Unfortunately, current therapies aimed at suppressing appetite are not suitable for some patients with obesity owing to serious adverse effects and the compensatory decrease in energy expenditure^2,3. Thus, alternative strategies are needed to improve body weight and metabolic health.

Emerging evidence suggests a link between metabolic balance and motion sickness, which affects approximately one-third of the population. This condition arises from sensory conflicts commonly experienced during water or air travel and virtual movements in video games^4,5. Symptoms of motion sickness in humans, including facial pallor, nausea and vomiting, have been well-documented and investigated⁶. Notably, motion sickness leads to changes in thermoregulation, as evidenced by a reduction in body temperature observed in mouse, shrews, rats and humans^7–12. In addition, individuals with motion sickness often seek cooler environments to lower their body temperature¹³. Notably, research suggests a potential link between obesity and impairments in balance and sensory processing, which contribute to motion sickness¹⁴. Moreover, excess adipose tissue in obese animals might affect thermogenesis and heat dissipation, key mechanisms contributing to motion-induced hypothermia⁶. Interestingly, many genetic variants associated with motion sickness are implicated in the regulation of glucose tolerance, insulin sensitivity and body-weight balance¹⁵. However, the exact relationship between motion sickness, metabolic regulation and the underlying mechanisms remains unclear.

The functional vestibular organs in the inner ear seem to be essential for typical motion sickness to occur. For instance, dogs exhibit resistance to motion sickness following bilateral labyrinthectomy¹⁶, as do humans with bilateral vestibular deficiency^17,18. Another study has revealed that bilateral labyrinthectomy can prevent motion-induced hypothermia in rats¹⁹. Our previous studies have shown that mice lacking the α9 cholinoreceptor subunit, predominantly expressed in vestibular hair cells, exhibit attenuated hypothermic responses during provocative motion⁷. Nonetheless, the specific brain region(s), neuronal populations and associated circuits that govern key aspects of motion sickness remain largely unidentified.

The Suncus murinus (house musk shrew), an insectivore, is used extensively in motion-sickness studies because its emetic reflex is similar to that of humans^9,10,20,21. However, experimental tools are limited in this species given that its genome sequence is unknown^9,22–24. Although mice lack the emetic reflex and cannot vomit, as a model they offer an abundance of molecular and genetic tools, as well as relevant behavioural assays, to elucidate the neural substrates of motion sickness. Here, we establish a mouse model of motion sickness, which exhibits prominent hypothermia during provocation, accompanied by associated perturbed thermoregulatory and other physiological adaptations typical of what is seen in people experiencing the condition. Using this model, we found that motion activates MVePC^Glu neurons. We further demonstrated that the activation of these neurons is both required and sufficient to mediate motion-induced thermal adaptations. Finally, we examined the metabolic effects in mice with genetically manipulated MVePC^Glu neurons, identifying a motion-sensitive brain region as a potential target for metabolic interventions.

Results

Establishment of a mouse motion-sickness model

To assess motion-induced hypothermia, mice were implanted with a G2 E-Mitter telemetric probe in the abdominal cavity to monitor core body temperature and locomotor activity before being subjected to motion (1 Hz, 4-cm horizontal displacement) for 30 min (Fig. 1a). Before motion exposure, body temperature did not differ between the sham and motion groups (Fig. 1b). However, mice in the motion group experienced a rapid, substantial decrease in body temperature compared with the sham group (sham versus motion, −0.25 versus −3.97 °C in male mice; sham versus motion, −0.29 versus −4.47 °C in female mice), but their locomotor activity was not affected (Fig. 1b–d). Of note, nocturnal mice exhibit minimal basal locomotor activity during the day, when all studies were performed. The motion-induced decrease in body temperature was partially influenced by ambient room temperature (Supplementary Fig. 1a,b); this could be due to alterations in basal thermogenesis and other homeostatic responses when mice are exposed to 4 °C or thermoneutral ambient temperature. Nonetheless, we performed all subsequent studies at room temperature. To evaluate thermoregulatory behaviour in response to motion, another cohort of male and female mice was placed in a thermal gradient box immediately after the motion stimulus concluded (Fig. 1e). The sham mice preferred an ambient temperature of ~30 °C, which is within their thermoneutral zone²⁵ (Fig. 1f,g). Of note, mice in the motion group preferred ~26.0 °C, indicative of cold-seeking behaviour (Fig. 1f,g). Furthermore, both sated and hungry mice showed reduced appetite after the provocation period (Fig. 1h,i). In addition, motion also induced conditioned taste aversion in both male and female mice, as shown in a two-bottle assay (Fig. 1j,k). In another experiment, mice were placed in an open field arena following the motion stimulus. Provocation elicited a reduction in total distance travelled, velocity and time spent in the central zone in both male and female mice compared with those in the sham group (Fig. 1l–o). To identify neurons activated by motion stimuli in the brain, we performed unbiased brain-wide mapping and stained for the immediate early gene product c-Fos. This approach revealed c-Fos-positive cells in the nucleus tractus solitarius (NTS), MVePC, LPBN, locus coeruleus (LC), central nucleus of the amygdala (CeA), paraventricular nucleus of the hypothalamus (PVH) and anterior part of bed nucleus of the stria terminalis (αBNST) (Fig. 1p–s and Extended Data Fig. 1a–c). The raphe pallidus nucleus (RPa) and the medial part of preoptic area of the hypothalamus (MPA) are known for their roles in whole-body thermoregulation^26,27. However, compared with the sham group, mice exposed to motion did not display a significant increase in c-Fos expression in the RPa or MPA (Extended Data Fig. 1a–c).

Fig. 1 | — a, Illustration of the mouse motion-sickness model in the laboratory. b–d, Motion (1 Hz, 4-cm horizontal displacement, 30 min) induced hypothermia (b,c), but did not affect locomotor activity (d). n = 6 mice per group; two-sided Student’s t-test was used for statistical analysis in c in the same sex. e–g, Schematic of the thermal gradient box (e), distribution of the time mice spent in each temperature zone (f) and preferred ambient temperature (g) after motion. n = 10 male and 8 female mice per group in f and g; two-sided Student’s t-test was used for statistical analysis in g in the same sex. h,i, Motion decreased food intake in both sated (h) and hungry (i) mice. n = 6 mice; two-way analysis of variance (ANOVA) followed by Bonferroni post hoc test was used for statistical analysis in the same sex. j,k, Schematic for conditioned taste aversion assay paired with motion sickness (j), and motion caused aversion in both male and female mice (k). n = 8 mice per group; two-sided Student’s t-test was used for statistical analysis in k in the same sex. j was created using https://BioRender.com. l–o, Distance travelled (l), velocity (m) and central-zone duration (n) were reduced; corresponding heat maps in an open field test following motion are shown (o). n = 10 for male/sham, 6 for male/motion, 9 for female/sham and 7 for female/motion; two-sided Student’s t-test was used for statistical analysis within the same sex. p–s, Representative photomicrographs illustrating c-Fos immunostaining (p) and the number of c-Fos⁺ cells induced by motion in the MVePC (q), LPBN (r) and PVH (s). n = 8 for male/sham, 9 for male/motion, 7 for female/sham and 8 for female/motion; two-sided Student’s t-test was used for statistical analysis within the same sex. LPBD, lateral parabrachial nucleus, dorsal part; LPBC, lateral parabrachial nucleus, central part; LPBE, lateral parabrachial nucleus, external part; scp, superior cerebellar peduncle. t–v, The effect of scopolamine on body temperature (t,u) and locomotor activity (v) in the presence or absence of motion. n = 6 mice per group; two-sided Student’s t-test was used for statistical analysis in u within the same sex. w,x, Effect of scopolamine on preferred ambient temperature in the presence or absence of motion in female mice. n = 8 mice per group; one-way ANOVA followed by Bonferroni post hoc test was used for statistical analysis in x. Data are expressed as mean ± s.e.m.

Although some studies have reported sex-linked differences in motion sickness in humans (for example, nausea) and animals (for example, emesis in shrews and defecations in rats), a consensus has not yet been reached^28–30. Our statistical analysis between male and female mouse data (Fig. 1a–s) revealed no significant differences between sexes.

Scopolamine, a muscarinic acetylcholine receptor antagonist, has been used for decades to treat motion sickness in humans, despite its reported side effects³¹. Furthermore, treatment with scopolamine has proven effective against motion sickness in rats and shrews^32,33. Thus, we used scopolamine (10 mg kg⁻¹ body weight, intraperitoneal (i.p.)) to validate whether we had developed a bona fide mouse model of motion sickness. Although scopolamine did not affect basal body temperature or locomotor activity, it significantly reversed motion-induced hypothermia, as well as cold-seeking behaviour (Fig. 1t–x). However, scopolamine did not abolish motion-induced anorexia; rather, it suppressed food intake on its own (Extended Data Fig. 2a). We also found that scopolamine reversed the motion-induced drop in locomotor activity, but it induced anxiety-like behaviour in the open field test (Extended Data Fig. 2b–e). Overall, we developed a mouse model of motion sickness by assessing gradual hypothermia along with cold-seeking behaviour, which we validated with the anti-motion-sickness drug scopolamine.

Activity-dependent genetic labelling of MVePC neurons

Because we found that male mice gain body weight faster than females, and this rapid accumulation of adiposity might affect thermogenesis and heat loss–key mechanisms of motion-induced hypothermia^7,34–we used female mice for subsequent experiments, unless stated otherwise.

To genetically label motion-specific neurons in the brain, we crossed TRAP2 mice, which express tamoxifen-inducible CreER recombinase under the Fos promoter, with a conditional tdTomato reporter line, resulting in TRAP2/Rosa26-LSL-tdTomato mice³⁵. Female mice harbouring these alleles were exposed to motion 1 h after receiving an injection of 4-hydroxytamoxifen (4-OHT, 50 mg kg⁻¹ body weight, i.p.), which enabled CreER in motion-specific neurons to enter the nucleus and activate tdTomato expression in neurons exhibiting elevated activity³⁵. Two weeks after provocation, we observed a large number of tdTomato-labelled neurons in the NTS, MVePC, LPBN, LC, PVH, CeA and BNST (Extended Data Fig. 3a,b). Immunohistochemical staining for c-Fos after the second round of provocation confirmed that most of ‘TRAPed’ neurons labelled by tdTomato in female TRAP2/Rosa26-LSL-tdTomato mice were selectively activated by motion (Extended Data Fig. 3a,b).

We next investigated which brain region(s) mediate the hypothermic responses induced by motion. Because MVePC, LPBN and PVH have been reported to engage in thermoregulation^36–40, we stereotaxically injected a Cre-dependent adeno-associated virus (AAV) vector carrying hM3Dq-mCherry into these areas bilaterally. After a 2-week recovery period, the mice received a single dose of 4-OHT and were then subjected to sham or motion conditions 1 h later (Fig. 2a). This protocol TRAPed motion-activated neurons in the MVePC, LPBN or PVH using targeted expression of hM3Dq-mCherry; to reactivate these neurons, a single i.p. injection of clozapine-N-oxide (CNO; 3 mg kg⁻¹ body weight) was given 2 weeks later. Reactivation of PVH neurons showed a trend towards increased body temperature (Extended Data Fig. 4a,b) without altered locomotor activity (Extended Data Fig. 4c,d). Moreover, activation of these PVH neurons led to a reduction in food intake, without influencing preferred ambient temperature or locomotor activity, as shown in the open field test (Extended Data Fig. 4e–k). We confirmed the expression of hM3Dq-mCherry by observing an increase in c-Fos-positive cells in the PVH after CNO injection (Extended Data Fig. 4l,m). Reactivation of LPBN neurons resulted in decreased body temperature, anorexia and locomotor activity in the open field test, without affecting preferred ambient temperature (Extended Data Fig. 5a–k). However, the reduction in body temperature was not as pronounced as the motion-induced reduction of body temperature (LPBN versus motion, −1.00 versus −4.47 °C, P < 0.001, t-test) (Fig. 1c and Extended Data Fig. 5b). We then validated hM3Dq-mCherry expression by observing increased c-Fos-positive cells in the LPBN after CNO injection (Extended Data Fig. 5l,m). Of note, reactivation of MVePC neurons induced an even lower body temperature than did motion (MVePC versus motion, −6.07 versus −4.47 °C) but did not influence locomotor activity (Fig. 2b–e). Moreover, when motion-specific MVePC neurons were reactivated, the mice displayed cold-seeking behaviour in the thermal gradient box (Fig. 2f,g), and anorexia and reduced locomotor activity in an open field test (Fig. 2h–l). The expression of hM3Dq-mCherry in the MVePC was validated by an increase of c-Fos-positive cells (Fig. 2m,n). Overall, these results suggest that reactivation of motion-specific MVePC neurons can recapitulate motion-induced physiological responses in mice.

Given that reactivation of motion-specific MVePC neurons mimics provocation-induced hypothermia (Fig. 2b,c), and that scopolamine can largely abolish motion-induced hypothermia (Fig. 1t,u), we thus hypothesized that motion-specific MVePC neurons are the direct targets of scopolamine. To test this, we stereotaxically injected a Cre-dependent AAV vector harbouring hM3Dq-mCherry into the MVePC in female TRAP2 mice that were then subjected to the TRAPed protocol (see above). We then conducted whole-cell electrophysiological recordings in acute brain slices to assess the activity of motion-specific hM3Dq-mCherry-expressing neurons. As expected, CNO treatment significantly increased the firing frequency and membrane potential of hM3Dq-mCherry-expressing neurons in the MVePC (Extended Data Fig. 6a–d). However, pretreatment with scopolamine (10 μM) largely abolished CNO-induced electrical responses (Extended Data Fig. 6b–d). Furthermore, the effect of scopolamine can be reversed, because CNO continued to increase firing frequency and membrane potential after washout (Extended Data Fig. 6e–g). Of note, scopolamine did not impact the basal activity of recorded MVePC neurons, consistent with a previous report⁴¹. This suggests that the side effects (for example, increased anxiety and anorexia) of scopolamine could stem from other brain regions in which the drug disrupts the normal function of the cholinergic system³¹. Consistent with this, the unaffected basal activity of recorded neurons rules out any non-specific effects of scopolamine on hM3Dq itself. Furthermore, scopolamine can attenuate motion-induced c-Fos-expressing cells in the MVePC (Extended Data Fig. 6h,i). Together, these data provide evidence that MVePC neurons represent the direct target of the anti-motion-sickness efficacy of scopolamine.

Because activation of motion-specific MVePC neurons can induce hypothermia, we investigated whether motion-specific MVePC neurons can be activated by warm temperatures. To this end, we used slice electrophysiology to interrogate the electrical responses of motion-specific MVePC neurons (labelled by tdTomato in female TRAP2/Rosa26-LSL-tdTomato mice) to different ambient temperatures. Current clamp recordings of neurons exhibiting spontaneous action potentials and firing frequencies at different temperatures (33 °C, 36 °C and 39 °C) revealed that all motion-specific MVePC neurons (11 out of 11) are warm-sensitive (Fig. 2p–r). As negative controls, eight tdTomato-negative neurons in the MVePC were recorded, of which five were inhibited by increased ambient temperature, one did not respond and two showed slightly increased firing frequencies and elevated membrane potentials (Extended Data Fig. 7a–d). For motion-specific MVePC neurons, we conducted the same electrophysiological recordings while administering a cocktail of synaptic blockers (tetrodotoxin (TTX), 6,7-dinitroquinoxaline-2,3-dione (DNQX), D-2-amino-5-phosphonopentanoic acid (D-AP5) and bicuculline). We found that these cells remained activated by high temperature, showing an increased membrane potential; this result suggests that their warm sensitivity is intrinsic and independent of synaptic inputs (Extended Data Fig. 7e,f). By contrast, under the same experimental conditions, the membrane potential of tdTomato-negative neurons in the MVePC was not changed (Extended Data Fig. 7g,h).

Given that AMP-activated protein kinase (AMPK) is a canonical regulator of thermogenesis⁴², we investigated its role in the warm-sensitive properties of MVePC neurons. Pretreatment with the AMPK blocker Compound C (10 μM) eliminated high-temperature-induced increase in firing frequency and membrane potential (Extended Data Fig. 8a–c). To further investigate which temperature-sensitive transient receptor potential (TRP) channels might mediate the warm-sensing properties of MVePC neurons, we manually collected 12 tdTomato-positive and 12 tdTomato-negative neurons in the MVePC region in female TRAP2/Rosa26-LSL-tdTomato mice and then performed single-cell quantitative reverse transcription PCR (RT–qPCR). Eleven potential genes encoding temperature-sensitive channels were analysed–Trpm2, Trpm3, Trpm4, Trpm5, Trpm8, Trpv1, Trpv2, Trpv3, Trpv4, Trpa1 and Trpc5 (refs. 43,44). We found that Trpm5, Trpm8, Trpv1 and Trpa1 are not expressed in these neurons, and expression levels of Trpv3 and Trpv4 are low. The results further revealed that the expression levels of Trpm2, Trpm3 and Trpv2 are significantly higher in tdTomato-positive than in tdTomato-negative neurons (Fig. 2t); notably, the ion channels encoded by these three genes have been reported to specifically respond to high temperatures^45–47.

To determine the molecular characteristics of motion-specific MVePC neurons, we performed a triple RNAscope against glutamatergic (labelled with Vglut2), GABAergic (labelled with Gad1) and motion-specific MVePC (labelled with tdTomato) neurons in the MVePC region after performing the TRAPed protocol in female TRAP2/Rosa26-LSL-tdTomato mice. We found that 87.8% of motion-activated MVePC neurons were glutamatergic, and 12.2% of them were GABAergic (Fig. 2u,v). Furthermore, rabies retrograde tracing revealed that motion-specific MVePC neurons receive synaptic inputs from rostral NTS neurons (Supplementary Fig. 2a–c).

Activating MVePC neurons mimics motion-induced hypothermia

The AAV expression of humanized ChR2-H134R fused to enhanced yellow fluorescent protein (eYFP) driven by the CaMKIIa promoter has been widely used for the optogenetic activation of glutamatergic neurons⁴⁸. Using this method, we stereotaxically injected AAV-CaMKIIa-hChR2-H134R-eFYP into the MVePC of wild-type (WT) female mice, to selectively express eYFP-tagged ChR2 in the glutamatergic neurons of the MVePC (MVePC^Glu) (Fig. 3a and Extended Data Fig. 9a). During the same surgery, we implanted an optic fibre 0.4 mm above the MVePC for optogenetic stimulation, along with a G2 E-Mitter telemetric probe in the abdominal cavity to measure core body temperature and locomotor activity. Although delivery of yellow light (589 nm, 20 Hz, as control) had no effect, blue-light delivery (473 nm) at various frequencies (2, 5, 10 and 20 Hz) reduced body temperature in a frequency-dependent manner (Fig. 3b,c). Meanwhile, compared with the yellow-light control, blue-light stimulation also decreased locomotor activity (Fig. 3d,e). Notably, mice displayed elevated basal locomotor activity after the implanted fibres were tethered to the optical cable during baseline, despite the experiments occurring during daytime hours when mice are generally inactive. Intriguingly, optogenetic stimulation of MVePC^Glu neurons caused a negative valence in the real-time place preference test, possibly reflecting the discomfort associated with motion sickness (Extended Data Fig. 9b–d). These results indicate that activation of MVePC^Glu neurons can replicate the motion-induced hypothermic response. Because 12.2% of motion-specific MVePC neurons are GABAergic, we next set out to determine the role of MVePC GABAergic (MVePC^Vgat) neurons in regulating body temperature. To this end, we stereotaxically injected AAV-Ef1-fDIO-hChR2-H134R-eFYP into the MVePC of female Vgat-flpo mice, and implanted an optic fibre 0.4 mm above the MVePC, enabling optogenetic stimulation. We also implanted a G2 E-Mitter telemetric probe to monitor core body temperature and locomotor activity (Fig. 3f and Extended Data Fig. 9e). As before, yellow-light delivery (589 nm, 20 Hz, as control) did not affect core body temperature (Fig. 3g,h). Although blue-light delivery (473 nm) at 20 Hz significantly reduced body temperature, this decrease was less pronounced than that caused by motion (Figs. 1b,c and 3g,h; −2.50 versus −4.47 °C, P < 0.01, t-test). Of note, blue-light delivery at other frequencies (that is, 10, 5 and 2 Hz) did not affect body temperature (Fig. 3g,h). Moreover, stimulation of MVePC^Vgat neurons had no impact on locomotor activity (Fig. 3i,j) and did not induce a negative valence (Extended Data Fig. 9f–h). Taken together, these data suggest that MVePC^Glu neurons play a dominant role in motion-induced hypothermia, whereas the effects of MVePC^Vgat neurons are modest.

Fig. 3 | — a, Schematic of injection of pAAV9-CaMKIIa-hChR2-H134R-eYFP into the MVePC in female WT mice. b–e, Stimulation of MVePC^Glu neurons induced hypothermia (b,c) and decreased locomotor activity (d,e) in a frequency-dependent manner. n = 6 mice; one-way ANOVA followed by Bonferroni post hoc test was used for statistical analysis in c and e. f, Schematic of injection of AVV-fDIO-ChR2 into the MVePC in female Vgat-flpo mice. g–j, Optogenetic stimulation of MVePC^Vgat neurons reduced body temperature (g,h), but did not influence locomotor activity (i,j). n = 6 mice; one-way ANOVA followed by Bonferroni post hoc test was ussed for statistical analysis in h. k, Representative images of expression of hChR2-eYFP with injection of pAAV9-CamKIIa-hChR2-H134R-eYFP in the MVePC, and dense terminals and fibres were observed in the LPBN and PVH in three mice. Pr, prepositus nucleus; CG, central gray; LVe, lateral vestibular nucleus; 3V, third ventricle. l, Schematic of the injection of pAAV-CaMKIIa-hChR2-H134R-eYFP into the MVePC, and implantation of an optical fibre over the PVH in female WT mice. m–p, Stimulation of terminals of MVePC^Glu neurons in the PVH did not affect body temperature (m,n) or locomotor activity (o,p). n = 5 mice. q, Schematic diagram of injection of pAAV-CaMKIIa-hChR2-H134R-eYFP into the MVePC and implantation of an optical fibre over the LPBN in WT female mice. r–u, Stimulation of terminals of MVePC^Glu neurons in the LPBN induced hypothermia (r,s) and decreased locomotor activity (t,u) in a frequency-dependent manner. n = 6 mice; one-way ANOVA followed by Bonferroni post hoc test was used for statistical analysis in s and u. v, Representative confocal image showing that ChR2-labelled MVePC^Gluoriginated fibres are in close proximity to glutamatergic neurons in the LPBN in three mice. w,x, Schematic for electrophysiological recordings of LPBN neurons in response to photostimulation of ChR2-labelled MVePC^Glu-originated fibres (w); photostimulation of ChR2-labelled MVePC^Glu-originated fibres induced activation of LPBN neurons (x). n = 8 neurons from 2 mice; two-tail paired t-test was used for statistical analysis. y,z, Light-evoked EPSCs in LPBN neurons in the presence or absence of 4-AP, TTX, DNQX and D-AP5 (y). Quantification of EPSCs of LPBN neurons in the presence or absence of different blockers (z). n = 9 neurons from 2 mice; two-tail paired t-test was used for statistical analysis in z. 4-AP, 4-aminopyridine. Data are expressed as mean ± s.e.m.

To investigate the downstream target(s) mediating the functions of MVePC^Glu neurons, we stereotaxically injected AAV-CaMKIIa-hChR2-H134R-eYFP into the MVePC of WT female mice. As expected, we observed abundant eYFP-labelled neuron cell bodies and fibres in the MVePC, confirming the successful infection of MVePC^Glu neurons (Fig. 3k). In addition, we detected abundant eYFP-labelled fibres and terminals in the LPBN and PVH, confirming that MVePC^Glu neurons project to these two brain regions (Fig. 3k). Thus, we generated another cohort of female mice with MVePC^Glu neurons expressing hChR2-H134R-eYFP, as depicted in Fig. 3a. During the same surgery, we implanted an optic fibre above the PVH for optogenetic stimulation of the MVePC^Glu→PVH circuit (Fig. 3l) and a G2 E-Mitter telemetric probe in the abdominal cavity to monitor core body temperature and locomotor activity. Blue-light delivery (473 nm) at frequencies of 2, 5, 10 and 20 Hz did not affect body temperature or locomotor activity (Fig. 3m–p), indicating that the MVePC^Glu→PVH circuit is not involved in thermoregulation. In another female cohort with MVePC^Glu neurons expressing hChR2-H134R-eYFP, we implanted an optic fibre above the LPBN to facilitate optogenetic stimulation of the MVePC^Glu→LPBN circuit (Fig. 3q and Extended Data Fig. 9i) and a G2 E-Mitter telemetric probe in the abdominal cavity. Yellow-light delivery (589 nm, 20 Hz) did not have an effect; however, blue-light delivery (473 nm) at various frequencies (2, 5, 10 and 20 Hz) reduced body temperature in a frequency-dependent manner (Fig. 3r,s). Furthermore, blue light at 20 Hz also decreased locomotor activity (Fig. 3t,u). Similarly, optogenetic stimulation of eYFP-labelled fibres and terminals in the LPBN resulted in negative valence in the real-time place preference test (Extended Data Fig. 9j–l). These results indicate that optogenetic stimulation of MVePC^Glu neurons activates downstream LPBN neurons, leading to reductions in body temperature and locomotor activity, along with a negative valence.

To further investigate the synaptic connection between MVePC^Glu and LPBN neurons, we stereotaxically injected AAV-CaMKIIa-hChR2-H134R-eYFP into the MVePC of WT female mice. Our observations revealed that the terminals of MVePC^Glu neurons are in close proximity to the soma of glutamatergic neurons in the LPBN, suggesting that MVePC^Glu neurons establish synapses with glutamatergic neurons in the LPBN (Fig. 3v). To further explore the synaptic connectivity, we prepared brain slices containing both the MVePC and LPBN for ChR2-assisted circuit mapping of LPBN neurons⁴⁹ (Fig. 3w). In current clamp mode, we found that blue-light delivery at 20 Hz induced an increase in firing frequency of LPBN neurons (Fig. 3x). In voltage clamp mode, we detected blue-light-evoked excitatory postsynaptic currents (EPSCs) in these neurons (Fig. 3y,z). These light-evoked EPSCs remained intact despite the presence of 100 μM 4-AP and 1 μM TTX, indicative of monosynaptic connectivity. To evaluate the role of NMDA and/or AMPA receptors, we repeated the brain-slice recording with 20 μM DNQX alone and then with 20 μM DNQX followed by 50 μM D-AP5. DNQX alone partially reduced the EPSC, and the addition of D-AP5 further diminished the EPSC, ultimately abolishing it (Fig. 3y,z). Thus, these data indicate that MVePC^Glu neurons provide monosynaptic glutamatergic inputs to LPBN neurons.

Inhibiting MVePC→LPBN circuit attenuates motion sickness

Given that the activation of the MVePC→LPBN circuit mimics motion-induced hypothermia, we next investigated whether inhibiting this circuit would attenuate it. First, we tested whether inhibition of MVePC^Glu neurons could eliminate motion-induced hypothermia. To do this, we stereotaxically injected AAV2-CaMKIIa-hM4Di(Gi)-mCherry bilaterally into the MVePC of WT female mice (Fig. 4a–d). Control WT female mice received the same stereotaxic injections with AAV2-CaMKIIa-mCherry (Fig. 4a–d). During the same surgery, we implanted a G2 E-Mitter telemetric probe in the abdominal cavity to monitor core body temperature and locomotor activity. Four weeks after the surgery, we administered a single i.p. injection of CNO (3 mg kg⁻¹ body weight) 20 min before exposure to motion to inhibit hM4Di-expressing neurons in the MVePC. As previously reported, provocation consistently reduced core body temperature in the mCherry group, whereas inhibition of MVePC^Glu neurons significantly ameliorated the motion-induced drop in body temperature without affecting locomotor activity (Fig. 4e–g). Moreover, inhibition of MVePC^Glu neurons did not affect food intake (Fig. 4h) but did lessen the motion-induced reduction in locomotor activity as well as cold-seeking behaviour (Fig. 4i–m). Next, we asked whether inhibition of the MVePC→LPBN neural circuit could reduce motion sickness. To this end, we stereotaxically injected retro-DIO-hM4Di-mCherry bilaterally into the LPBN, and AAV-Cre-GFP bilaterally into the MVePC, such that LPBN-projecting MVePC neurons expressed hM4Di-mCherry (Fig. 4n–q). As controls, WT female mice received the same injections of retro-DIO-mCherry in the LPBN, and AAV-Cre-green fluorescent protein (GFP) in the MVePC (Fig. 4n–q). During the same surgeries, we implanted a G2 E-Mitter telemetric probe in the abdominal cavity to monitor temperature and locomotor activity. Four weeks after surgery, a single i.p. injection of CNO (3 mg kg⁻¹ body weight) was administered 20 min before the motion stimulus was applied, to inhibit LPBN-projecting MVePC neurons. Consistently, motion induced hypothermia in controls without affecting locomotor activity (Fig. 4r–t). Notably, inhibiting this circuit ameliorated motion-induced decreases in body temperature (Fig. 4r,s). Additionally, inhibition of this circuit also rescued motion-induced cold-seeking behaviour, but not anorexia or locomotor activity (Fig. 4u–z). Overall, these experiments revealed that inhibition of the MVePC→LPBN circuit could abrogate motion-induced hypothermia along with cold-seeking behaviour.

Fig. 4 | — a, Schematic of injection of pAAV2-CaMKIIa-hM4D(Gi)-mCherry into the MVePC, and representative images from six mice of hM4D(Gi)-mCherry expression in the MVePC. b–d, Typical action potential trace (b), firing frequency (c) and resting membrane potential (d) of MVePC neurons expressing hM4Di in response to 10 μM CNO. n = 6 neurons; a two-tailed paired t-test was used for statistical analysis in c and d. e–i, Inhibition of MVePC^Glu neurons, in the presence or absence of motion, affects body temperature (e,f), locomotor activity (g), food intake (h) and preferred ambient temperature (i). n = 6 mice per group; for statistical analysis, a two-way ANOVA followed by Bonferroni test was used in h, and a one-way ANOVA followed by Bonferroni test was used in f and i. j–m, Inhibition of MVePC^Glu neurons, in the presence or absence of motion, affects distance travelled (j), velocity (k) and time spent in the central zone (l); corresponding heat maps (m) in an open field test are shown. n = 6 mice per group; one-way ANOVA followed by Bonferroni test was used for statistical analysis. n, Schematic of injection of AAV-Cre-GFP into the MVePC and retroAAV-DIO-hM4Di into the LPBN, and representative images from six mice of expression of hM4D(Gi)-mCherry in the MVePC. o–q, Typical action potential trace (o), firing frequency (p) and resting membrane potential (q) of MVePC neurons expressing hM4DI in response to 10 μM CNO. n = 5 neurons; two-sided paired t-test was used for statistical analysis in p and q. r–v, Inhibition of the MVePC^Glu→LPBN neural circuit, in the presence or absence of motion, affects body temperature (r,s), locomotor activity (t), food intake (u) and preferred ambient temperature (v). n = 6 mice; two-way ANOVA followed by Bonferroni test was used for statistical analysis in u, and one-way ANOVA followed by Bonferroni test was used for statistical analysis in s and v. w–z, Inhibition of the MVePC^Glu→LPBN neural circuit, in the presence or absence of motion, affects distance travelled (w), velocity (x) and time spent in the central zone (y); corresponding heat maps (z) in an open field test are shown. n = 6 mice per group; one-way ANOVA followed by Bonferroni test was used for statistical analysis. Data are expressed as mean ± s.e.m.

Functional identification of the MVePC→BAT axis

Reduced thermogenesis is a key mechanism underlying motion-induced hypothermia^7,8. Given the significance of brown adipose tissue (BAT) in energy homeostasis, we next investigated the role of BAT thermogenesis in hypothermia during motion sickness. To this end, we implanted an IPTT-300 probe underneath the skin and above the interscapular BAT in female mice to measure dynamic temperature changes of BAT. Notably, before the onset of provocation, there was no difference in BAT temperature between the sham and motion groups (Extended Data Fig. 10a). However, motion caused a robust drop in BAT temperature compared with the change in the sham group (Extended Data Fig. 10a,b; −0.22 versus −2.77 °C). These findings indicate that reduced BAT thermogenesis indeed contributes to motion-induced hypothermia. Although we have demonstrated the dominant role of MVePC^Glu neurons in motion-induced hypothermia, it still remains unclear whether MVePC^Glu neurons affect BAT temperature. To investigate the potential connectivity between MVePC^Glu neurons and BAT, we injected AAV-CaMKIIa-hChR2-H134R-eFYP into the MVePC, followed by the retro grade polysynaptic tracer PRV-RFP into the BAT of WT female mice (Extended Data Fig. 10c). We identified a large number of PRV-infected neurons in the RPa and PVH, but not the LPBN (Extended Data Fig. 10d), consistent with previous reports^50,51. Abundant PRV-infected cells were observed in the MVePC (Extended Data Fig. 10d); nevertheless, it remains unknown why LPBN neurons receive dense projections from the MVePC yet show no connectivity with the BAT through this method.

Next, we investigated whether manipulating MVePC^Glu neurons would impact BAT thermogenesis. To do this, we stereotaxically injected AAV-CaMKIIa-hChR2-H134R-eFYP into the MVePC of WT female mice, and implanted an optic fibre 0.4 mm above the MVePC for optogenetic stimulation, as illustrated in Fig. 3a. During the same surgery, we also implanted an IPTT-300 probe to measure BAT temperature (Extended Data Fig. 10e). Notably, activation of MVePC^Glu neurons through blue light (20 Hz) resulted in a dramatic drop in BAT temperature compared with that induced by yellow light (−0.18 versus −3.32 °C; Extended Data Fig. 10f,g). Furthermore, we interrogated the effect of inhibition of MVePC^Glu neurons on BAT thermogenesis. To this end, we stereotaxically injected AAV2-CaMKIIa-hM4Di(Gi)-mCherry into the MVePC of WT female mice, as shown in Fig. 4a, and implanted an IPTT-300 probe to measure BAT temperature in the same mice (Extended Data Fig. 10h). Notably, the inhibition of MVePC^Glu neurons through CNO injection significantly increased BAT temperature (0.20 versus 1.00 °C; Extended Data Fig. 10i,j). Collectively, our findings suggest that the MVePC→BAT axis contributes to hypothermia during motion sickness.

Inhibition of MVePC^Glu neurons prevents diet-induced obesity

Our previous model demonstrated that acute inhibition of MVePC^Glu neurons through CNO injection led to a slight increase in core body temperature and locomotor activity in the absence of motion (Supplementary Fig. 3a–d). Moreover, inhibition of these neurons promoted BsAT thermogenesis (Extended Data Fig. 10h–j). These results suggest that energy expenditure in mice is increased when MVePC^Glu neurons are inhibited. We next investigated whether this inhibition could offer metabolic benefits over the long term. To address this possibility, we stereotaxically injected AAV-CaMKII-Cre and AAV-EF1-Kir2.1-P2A-dTOMATO into the MVePC to chronically inhibit MVePC^Glu neurons in WT female mice (Fig. 5a–d). Control WT female mice received the same stereotaxic injections of AAV-CaMKII-Cre and AAV-DIO-mCherry (Fig. 5a–d). After surgery, all mice were placed on regular chow for 4 weeks, followed by a 9-week high-fat diet (HFD). Notably, body-weight gain during the HFD period was significantly lower in mice with chronic inhibition of MVePC^Glu neurons than in controls (Fig. 5e,f). There were no differences in caloric intake between groups during both the 4-week regular-chow period and 9-week HFD period (Fig. 5g). Fat mass, not lean mass, accounted for the body-weight differences between these two groups (Fig. 5h,i). In accordance, adipocytes were smaller in Kir2.1-treated mice than in control mice (Supplementary Fig. 4a). In addition, mice with chronic inhibition of MVePC^Glu neurons also had a higher body temperature during the dark cycle, and a trend was observed only during the day (Fig. 5j,k). Moreover, these mice exhibited higher BAT norepinephrine and epinephrine levels (Fig. 5l,m), suggesting increased sympathetic tone towards BAT thermogenesis. Furthermore, chronic inhibition of MVePC^Glu neurons resulted in a higher expression of UCP-1 in the BAT (Fig. 5n,o). In line with their reduced body weight and adiposity, mice with chronic inhibition of MVePC^Glu neurons displayed an improved glucose tolerance and increased insulin sensitivity compared with the control mice (Fig. 5p,q). To explore the mechanisms underlying the observed leaner body weight in Kir2.1-treated mice, which had a comparable caloric intake to the control mice, we introduced all the mice into the metabolic cages. As expected, chronic inhibition of MVePC^Glu neurons led to a significant increase in energy expenditure, particularly during the dark cycle (Fig. 5r,s), when analysed using analysis of covariance with body weight as a covariate. This was accompanied by robust increases in locomotor activity during the dark cycle (Fig. 5t,u). Both groups of mice consistently consumed comparable amounts of food in the metabolic cages (Fig. 5v). In addition to using metabolic cages to measure energy expenditure and locomotor activity (quantified as breaks per beam), we used another approach, the HomeCageScan, to measure locomotor activity. We therefore introduced the mice to the HomeCageScan, which can monitor a variety of spontaneous behaviours, including locomotor activity. We found that Kir2.1-treated mice travelled 416.8 m the light cycle (total 12 h) and 2,289.6 m during the dark cycle (total 12 h) cycle, whereas control mice covered only 51.7 m and 113.8 m during the same periods, respectively (Fig. 5w,x). Further, Kir2.1-treated mice and controls spent comparable amounts of time eating, but Kir2.1-treated mice exhibited a significant reduction in the time spent drinking, sleeping, grooming and sniffing during the dark cycle (Fig. 5y,z and Supplementary Fig. 4b–i). Taken together, these results highlight that chronic inhibition of MVePC^Glu neurons prevents diet-induced obesity through increased energy expenditure in female mice.

Fig. 5 | — a, Schematic of injection of pENN-AAV-CamKIIa-Cre and AAVEF1a-DIO-Kir2.1-P2A-dTOMATO into the MVePC, and representative images of expression of dTOMATO in the MVePC from seven mice. b–d, Representative traces of action potential of a control MVePC^Glu neuron and a Kir2.1-labelled MVePC^Glu neuron (b), and quantification of firing frequency and membrane potential (c,d). n = 6 neurons; two-sided Student’s t-test was used for statistical analysis. e–g, Body weight (e), body-weight gain (f) and caloric intake (g) in control and Kir2.1-teated mice. n = 7 mice per group; a two-way ANOVA followed by Bonferroni test was used for statistical analysis. The arrow indicates the time at which mice were introduced into metabolic cages. h,i, Fat (h) and lean (i) mass. n = 7 mice; two-sided Student’s t-test was used for statistical analysis. j–m, Body temperature in the light (j) and dark (k), and norepinephrine (l) and epinephrine (m) levels in the BAT. n = 7 mice; two-sided Student’s t-test was used for statistical analysis in k–m. n,o, UCP-1 expression in the BAT and representative immunostaining images. n = 7 mice; two-sided Student’s t-test was used for statistical analysis in n. p,q, Glucose levels and the area under the curve (AUC) during the glucose tolerance test (p) and insulin sensitivity test (q). n = 7 mice per group; two-way ANOVA followed by Bonferroni test was used for statistical analysis in the individual time-points analysis, and two-sided Student’s t-test was used for AUC analysis. r,s, Energy expenditure (r), energy expenditure analysed using analysis of covariance (ANCOVA) using body weight as a covariate and predicted energy expenditure using body weight of a 30-g mouse (s). n = 7 mice per group; two-sided Student’s t-test was used for statistical analysis in **s. t**,u, Locomotor activity measured in the metabolic cages over a 72-h period (t), and averaged locomotor activity for the light, dark and full day (u). n = 7 mice; two-way ANOVA followed by Bonferroni test was used for statistical analysis in u. v, Cumulative food intake during the 72-h period in the metabolic cages. w,x, Distance travelled, measured by HomeCageScan (w), and average distance travelled during the light and dark periods (x). n = 7 mice per group; two-way ANOVA followed by Bonferroni test was used for statistical analysis in x. y,z, Eating duration measured by HomeCageScan over a 48-h period (y), and averaged eating duration during the light and dark periods (z). n = 7 mice. Data are expressed as mean ± s.e.m.

Discussion

In this work, we explored the neural substrates underlying motion-induced thermal adaptations, which led to an interesting observation: chemogenetic inhibition of MVePC^Glu neurons resulted in a 0.5–1 °C increase in body temperature, along with elevated locomotor activity, that lasted approximately 3 h. We also observed increased BAT thermogenesis when MVePC^Glu neurons were inhibited. These physiological alterations suggest that chronic inhibition of these neurons could result in higher energy expenditure in mice. This has led us to investigate the potential metabolic benefits of chronic inhibition of MVePC^Glu neurons. As expected, mice with chronic inhibition of these cells experienced less weight gain and exhibited better glucose tolerance and enhanced insulin sensitivity without changes in feeding behaviour. The underlying mechanism affecting this is likely linked to the drastic increase in locomotor activity during the dark cycle, along with enhanced sympathetic tone towards BAT thermogenesis. These results highlight the underappreciated function of the vestibular system in metabolic balance, and further raise the possibility that a better understanding of the neural basis for thermoregulation during motion sickness could provide unconventional targets for obesity treatment.

It has been estimated that 5–10% of the general population is highly susceptible to motion sickness, and others are moderately susceptible⁵². The most frequently reported symptom of motion sickness in people is nausea. Indeed, for ethical reasons, clinical studies of the condition usually stop before vomiting has occurred^53,54. The only unequivocal and objective sign of motion sickness in animals is vomiting⁵⁵. However, common laboratory animals (for example, mice and rats) cannot vomit. Assessing motion sickness in laboratory animals is even more challenging given that subjective indices (for example, latency to the nausea onset and nausea rating) are unavailable⁵⁶. The objective assessment of motion sickness in animals mainly relies on behavioural indices with a relatively low temporal resolution (for example, pica) or requires training paradigms (for example, conditioned taste aversion)⁶.

In the present study, we profiled thermoregulatory behaviour in mice after provocation and progressive hypothermia during provocation. Notably, mice demonstrated a thermoregulatory response that led them to actively seek out colder temperatures after experiencing motion sickness. This behaviour seems paradoxical given that body temperature fell during and after the provocation. The underlying reasons are unknown, but it could be related to a perturbed body-temperature set point or distorted sensation of ambient temperature due to provocation. In retrospective motion-sickness studies in humans, to the best of our knowledge, one clinical report has documented similar phenomena in people with motion sickness¹³. In addition to thermoregulatory adaptations, we also observed a reduction in locomotor activity and anorexia after provocation, consistent with previous rat studies^6,57. However, we acknowledge that the symptoms of motion sickness, or those that can be used to evaluate motion sickness, could extend beyond the definitions in the present work. For instance, increased sweating and cutaneous vasodilation, which leads to heat loss associated with reduced BAT thermogenesis, occurs during episodes of motion sickness^6,7.

Also, sex differences are occasionally reported in people’s susceptibility to motion sickness. Some studies have reported that women experience more nausea^28,58, but this was not observed in other clinical studies⁵⁹. The underlying reasons are unclear, but sex differences could be drivsen by individual susceptibility (for example, age, genetic factors or past experience) to some extent, or even could vary depending on the type of motion stimulation. Likewise, there is no consensus in animal studies (for example, in shrews) regarding sex differences associated with motion sickness^30,60. In this work, we examined thermoregulatory behaviours (that is, gradual hypothermia and cold-seeking behaviour) as indicators of motion sickness in mice. We tested both male and female cohorts and found comparable alterations of thermoregulatory behaviours induced by motion in both sexes.

Our findings on c-Fos activity patterns evoked by motion stimuli in mice are consistent with previous work in rats and shrews^10,29. The MVePC, LPBN and PVH have been associated with thermoregulation^36–40. We therefore examined which of these three brain regions is responsible for motion-induced hypothermic responses. To do this, we used activity-dependent genetic labelling along with excitatory chemogenetics to reactivate motion-specific neurons in each brain region. Our results indicate that only the reactivation of MVePC neurons fully reproduces motion-induced hypothermia, cold-seeking behaviour, anorexia and reduced locomotor activity. This was surprising because there were fewer neurons activated by motion in the MVePC than in the LPBN and PVH. Also noteworthy is that MVePC neurons can modulate BAT thermogenesis. More specifically, activation of MVePC neurons decreased BAT temperature, whereas inhibition of MVePC neurons increased BAT temperature. These results could help to explain how activation of MVePC neurons could recapitulate motion-induced hypothermia, and inhibition of these neurons can largely abrogate it. However, it is unclear how MVePC neurons modulate BAT temperature, although we discovered a connection between BAT and MVePC neurons using PRV-mediated retrograde polysynaptic viral tracing. It should be noted that adult humans do not possess BAT pads in the same way as adult mice⁶¹. Thus, our findings regarding the BAT innervation and BAT thermogenesis in mice during motion sickness might not fully recapitulate the mechanisms in humans.

Slice electrophysiology studies revealed that all motion-specific MVePC neurons are activated in response to warm temperatures, which to our knowledge has never been reported in this group of neurons before. However, caution is needed in interpreting these results. Are these neurons really activated by increased temperature in the MVePC during the initial phase of provocation? Unfortunately, to our knowledge, there is no study monitoring the dynamic changes of temperature in the MVePC during provocation. We used an infrared camera to monitor the surface temperature of the head during provocation in mice, and the temperature actually fell in the same manner as the core body temperature, although the extent was smaller⁷. Flavia and colleagues reported that a noticeable fall in hypothalamic temperature was observed within 5 min after onset of motion in rats⁶². Hypothalamic temperature does not necessarily refer to the temperature of the MVePC. Given that motion-specific MVePC neurons are warm-activated, does this mean motion-induced hypothermia would, in turn, inhibit these motion-activated MVePC neurons per se? Perhaps this is why we did not detect as many c-Fos-positive cells as might be expected in the MVePC, including those induced by motion and excitatory chemogenetics. These open questions require further mechanistic studies.

Our findings indicating that MVePC neurons are involved in thermoregulation during motion sickness are consistent with a previous report, which demonstrated that Vglut2 neurons in the medial vestibular complex mediate gravitational-stress-induced hypothermic responses in mice³⁶. Here, we further demonstrated that activation of MVePC^Glu neurons, but not MVePC^Vgat neurons, causes a negative valence that might represent the debilitating sensations of motion sickness. Furthermore, we showed that MVePC^Glu neurons, which are involved in hypothermia and negative valence, exert their effects through downstream targets in the LPBN, not the PVH. Chemogenetic inhibition of MVePC^Glu neurons can almost fully abolish motion-induced hypothermia, as well as cold-seeking behaviour. Moreover, inhibition of the MVePC→LPBN circuit largely alleviates provocation-induced hypothermic responses and reverses cold-seeking behaviour. Recently, Machuca-Márquez and colleagues have demonstrated that cholecystokinin-expressing vestibular neurons drive rotation-induced autonomic changes through projection to Calca-expressing neurons in the LPBN⁶³. This also confirms the functional roles of vestibular neurons in motion sickness induced by different paradigms, such as hypergravitation, rotation and horizontal or orbital shaking^7,63,64.

Since the 1950s, anticholinergics (for example, scopolamine) have been the most common prescription for preventing and treating motion sickness in humans. Nonetheless, the mechanisms underlying how these drugs prevent and treat motion sickness are unclear. Do these medications target the motion-activated MVePC neurons or other brain regions (for example, NTS or PVH) in which neurons are also activated by motion? Considering the wide distribution of muscarinic receptors (that is, targets of scopolamine) in the brain⁶⁵, it is of essential importance to identify the specific brain region and neurons that are responsible for anti-motion-sickness efficacy versus associated side effects. Here, we revealed that motion-activated MVePC neurons are the targets of scopolamine in reversing motion-induced hypothermia. This finding is noteworthy because it is now possible to separate the anti-motion-sickness efficacy from the side effects of scopolamine (for example, increased anxiety-like behaviour and reduced food intake). The side effects might stem from targeted neurons in other brain regions, and its anti-motion-sickness effects are linked to MVePC neurons. By pinpointing the specific brain region involved and associated neural circuits (that is, MVePC→LPBN), researchers could develop more specific treatments that are more effective while minimizing side effects in future.

In summary, we established a mouse motion sickness model in the laboratory through assessments of gradual hypothermia and cold-seeking behaviour, validating it with the anti-motion-sickness drug scopolamine. All motion-specific MVePC neurons are warm-activated, and most are glutamatergic. Notably, we reveal that MVePC^Glu neurons play a dominant role in mimicking motion sickness through downstream targets in the LPBN, and that inhibition of the MVePC→LPBN neural circuit alleviates motion sickness. Last, chronic inhibition of MVePC^Glu neurons prevents diet-induced obesity through increased energy expenditure in mice. Together, these findings could help to provide potential targets to treat motion sickness in humans, and highlight the underappreciated function of the vestibular system in obesity control.

Methods

Mice

All animal care and procedures were approved by the Baylor College of Medicine Institutional Animal Care and Use Committees. Mice were housed in a temperature-controlled environment at 22–24 °C with 40–65% humidity with a 12-h light, 12-h dark cycle. The mice were fed regular chow (Pico Lab, LabDiet, no. 5V5R) or a HFD (60% kcal fat; Research Diets, no. D12492), as specified in each study. Water was provided ad libitum.

The following mouse strains were used: Rosa26-LSL-tdTomato mice (no. 007905), Vgat-flpo (no. 029591) and TRAP2 mice (Fos2A-iCreER; no. 03331), all from the Jackson Laboratory, and 57BL/6J mice from the Baylor College of Medicine mouse facility.

Immunohistochemistry

To map motion-induced c-Fos expression throughout the brain, male and female mice (8 weeks of age) were perfused 1 h after exposure to a motion stimulus. To minimize environmental stress and other non-specific stimuli affecting c-Fos expression, we brought all mice to the procedure room every day for three consecutive days. Frozen tissues were sectioned at 25 μm in the coronal plane using a freezing microtome and incubated at room temperature for 1 h in 0.03% H₂O₂. The free-floating sections were blocked with 1.5% normal goat serum containing 0.3% Triton X-100 in PBS (Vectastain Elite ABC kit, Vector Laboratories) for 2 h. After three washes with PBS, sections were then incubated with rabbit anti-c-Fos antibody (1:5,000, no. 226003, Synaptic Systems) for 48 h at 4 °C. The sections were subsequently washed and incubated with secondary goat-anti-rabbit antibody (1:200; Vector Laboratories) for 2 h, followed by Vectastain avidin–biotin complex reagent for 1 h (1:100; Vectastain Elite ABC kit, Vector Laboratories). c-Fos expression was visualized using a commercially available peroxidase substrate (Vector VIP kit, Vector Laboratories). For c-Fos immunostaining, the sections were incubated with an Alexa-Fluor-488-conjugated donkey anti-rabbit antibody (1:500, no. A21206, Invitrogen) for 2 h at room temperature after incubation with an antibody to c-Fos (1:1,000). Following the same procedures as for c-Fos immunostaining, anti-glutamate antibody (1:200, G6642, Sigma-Aldrich) was used to stain glutamatergic neurons in the LPBN.

TRAP induction

4-hydroxytamoxifen (4-OHT) was dissolved in pure ethanol at a concentration of 20 mg ml⁻¹. The resulting solution was mixed with oil (4 parts sunflower seed oil and 1 part castor oil) at a concentration of 10 mg ml⁻¹ by shaking at room temperature for 15 min; then, the ethanol was evaporated by vacuum centrifugation (3,000 r.p.m., 20 min). The final 10 mg ml⁻¹ 4-OHT solution was i.p. injected at a dose of 50 mg kg body⁻¹ weight⁶⁶. For the motion-TRAP protocol, singly housed mice were acclimated to the procedure room for three consecutive days and subjected to handling to mimic catching and injection daily. On the fourth day, mice were introduced to the procedure room at 9:00. 4-OHT was injected at 12:30, followed by provocative motion between 13:30 and 14:00. Mice were left undisturbed in the procedure room until 18:00.

RNAscope

Two weeks after exposure to provocative motion and 4-OHT injection, female TRAP2 mice were anaesthetized and perfused with saline, followed by 10% formalin. Their brains were removed and post-fixed in 10% formalin for 16 h at 4 °C and cryoprotected in 30% sucrose for 48 h. Brains were frozen and sectioned at 14 μm using a cryostat and washed in diethylpyrocarbonate-treated PBS for 10 min. Sections were mounted on charged slides, dried for 0.5 h at room temperature and stored at −80 °C. On the day of the RNAscope assay, the slides were thawed and rinsed two times in PBS and baked in an oven for 30 min at 60 °C. After that, slides were post-fixed in 10% formalin for 15 min at 4 °C. The slides were then gradually dehydrated in ethanol (50%, 70% and 100%, 5 min each) and underwent target retrieval for 5 min at 100 °C. After incubation in protease III (322337, Advanced Cell Diagnostics) for 30 min at 40 °C, the slides were rinsed in distilled water and incubated in mouse RNAscope probes for tdTomato (317041-C3, Advanced Cell Diagnostics), Mm-Slc32a1 (319191-C2, Advanced Cell Diagnostics) and Mm-Slc17a6 (319171-C1, Advanced Cell Diagnostics) for 2 h at 40 °C. Sections were then processed using RNAscope Fluorescent Multiplex Detection Reagents (320851, Advanced Cell Diagnostics), according to the manufacturer’s instructions. The slides were cover-slipped and analysed using a Leica DM5500 fluorescence microscope with OptiGrid structured illumination.

Chemogenetics

To reactivate motion-specific MVePC, LPBN and PVH neurons, female TRAP2 mice received stereotaxic injection of a Cre-dependent AAV vector carrying hM3Dq-mCherry (no. 44361-AAV8, Addgene) into the MVePC (the injection coordinates were anterior–posterior (AP), −6.12 mm; medial–lateral (ML), ±0.80 mm; dorsal–ventral (DV), +4.50 mm), LPBN (AP, −5.30 mm; ML, ±1.50 mm; DV, +3.70 mm) or PVH (AP, −0.8 mm; ML, ±0.20 mm; DV, +4.85 mm). All mice underwent intra-abdominal implantation of a G2 E-Mitter telemetric probe for real-time monitoring of core body temperature and locomotor activity using a G2 receiver underneath the cage⁶⁷. After a 2-week recovery period, these mice received a single dose of 4-OHT (50 mg kg⁻¹ body weight, i.p.), and were exposed to the same provocative motion 1 h later. This protocol TRAPed the motion-activated MVePC, LPBN or PVH neurons with targeted expression of hM3Dq-mCherry. A single i.p. CNO (3 mg kg⁻¹ body weight; no. 16882, Cayman) injection was used to reactivate TRAPed motion-activated MVePC, LPBN or PVH neurons.

AAV vectors with the CaMKIIa promoter have been widely used to drive gene expression in glutamatergic neurons⁶⁸. To inhibit MVePC^Glu neurons, pAAV2-CaMKIIa-hM4D(Gi)-mCherry (no. 50447-AAV2, Addgene) or control virus pAAV-CaMKIIa-mCherry (no. 114469-AAV2, Addgene) was therefore injected bilaterally in the MVePC (AP, −6.12 mm; ML, ±0.80 mm; DV, +4.50 mm) in female WT mice. Meanwhile, all mice received intra-abdominal implantation of G2 E-Mitter telemetric probe to allow for real-time monitoring of core body temperature and locomotor activity using a G2 receiver underneath the cage⁶⁷. All mice were allowed to recover for 4 weeks after surgery. To investigate whether inhibition of MVePC^Glu neurons attenuates motion-induced hypothermia, a single i.p. injection of CNO was administered 20 min before provocative motion. Likewise, to examine whether inhibition of MVe^Glu neurons perturbs thermoregulatory behaviour, mice were placed in the thermal gradient test box (Bio-TGT2, Bioseb) for an ambient-temperature preference test immediately after provocative motion.

To determine whether inhibition of the MVePC→LPBN neural circuit can ameliorate motion-induced hypothermia, retrograde pAAV-hsyn-DIO-hM4D(Gi)-mCherry (no. 44362, Addgene) or retrograde pAAV-hsyn-DIO-mCherry (no. 50459, Addgene) was injected bilaterally into the LPBN (AP, −5.30 mm; ML, ±1.50 mm; DV, +3.70 mm), and rAAV8-hsyn-GFP-Cre (no. AV5053D, UNC) was injected bilaterally in the MVePC in female WT mice. We also implanted the G2 E-Mitter telemetric probe in the abdominal cavity to measure core body temperature. A single i.p. injection of CNO was administered 20 min before provocative motion.

Rabies retrograde tracing

To uncover the upstream synaptic inputs to motion-specific MVePC neurons, female TRAP2 mice received a stereotaxic injection of 200 nl of AAV2-EF1a-FLEX-GTB (Viral Vector Core, Salk Institute for Biological Studies) into the MVePC. Two weeks later, mice were subjected to the motion-TRAP protocol. One week after the mice underwent the motion-TRAP protocol, 200 nl of EnVA-G-deleted Rabies-GFP GTB (Viral Vector Core, Salk Institute for Biological Studies) was injected into the MVePC. One week later, the mice were perfused, and their brains were coronally sectioned. Images were captured using a Leica DM5500 fluorescence microscope with OptiGrid structured illumination configuration.

Optogenetic studies

We stereotaxically injected AAV-CaMKIIa-hChR2-H134R-eFYP (no. 26969, Addgene) into the MVePC of female WT mice to express ChR2-eYFP in glutamatergic neurons. During the surgery, we also implanted an optic fibre (RWD) 0.4 mm above the MVePC, enabling optogenetic stimulation of MVePC^Glu neurons, and also implanted the G2 E-Mitter telemetric probe in the abdominal cavity to measure core body temperature. Mice were allowed at least 4 weeks for recovery before experimental stimulation. Blue light (473 nm, 10 ms per pulse; MGL-FN-589, CNI LASER) served for experimental stimulation, and yellow light (595 nm, 10 ms per pulse, 20 Hz; MGL-FN-589, CNI LASER) was used as a control.

To stimulate the terminals of MVePC^Glu neurons in the LPBN, we stereotaxically injected AAV-CaMKIIa-hChR2-H134R-eFYP (no. 26969, Addgene) into the MVePC of female WT mice. We also implanted an optic fibre (RWD) 0.4 mm above the LPBN to facilitate optogenetic stimulation of MVePC^Glu neuron terminals, and implanted the G2 E-Mitter telemetric probe in the abdominal cavity to measure core body temperature. Blue light (473 nm, 10 ms per pulse; MGL-FN-589, CNI LASER) was used for experimental stimulation, and yellow light (595 nm, 10 ms per pulse, 20 Hz; MGL-FN-589, CNI LASER) served as the control.

Electrophysiology

Mice were deeply anaesthetized with isoflurane and transcardially perfused with a modified ice-cold sucrose-based cutting solution (pH 7.3) containing 10 mM NaCl, 25 mM NaHCO₃, 195 mM sucrose, 5 mM glucose, 2.5 mM KCl, 1.25 mM NaH₂PO₄, 2 mM sodium pyruvate, 0.5 mM CaCl₂ and 7 mM MgCl₂, bubbled continuously with 95% O₂ and 5% CO₂. The mice were then decapitated, and entire brains were removed and immediately submerged in the cutting solution. Coronal brain slices (220 mm) containing the MVePC or LPBN were cut with a Microm HM 650 V vibratome (Thermo Fisher Scientific) in oxygenated cutting solution. Slices were then incubated in oxygenated artificial cerebrospinal fluid (aCSF; 126 mM NaCl, 2.5 mM KCl, 2.4 mM CaCl₂, 1.2 mM NaH₂PO₄, 1.2 mM MgCl₂, 11.1 mM glucose and 21.4 mM NaHCO₃, balanced with 95% O₂/5% CO₂, pH 7.4) to recover for ~25 min at 32 °C and subsequently for 1 h at room temperature before recording. Slices were transferred to a recording chamber and allowed to equilibrate for at least 10 min before recording. The slices were superfused at 32 °C in oxygenated aCSF at a flow rate of 1.8–2 ml min⁻¹. eYFP-, tdTomato- or mCherry-labelled neurons were visualized using epifluorescence and IR-DIC imaging on an upright microscope (Eclipse FN-1, Nikon) equipped with a movable stage (MP-285, Sutter Instrument). Patch pipettes with resistances of 3–5 MΩ were filled with intracellular solution (pH 7.3) containing 128 mM potassium gluconate, 10 mM KCl, 10 mM HEPES, 0.1 mM EGTA, 2 mM MgCl₂, 0.05 mM Na-GTP and 4 mM Mg-ATP. Recordings were made using a MultiClamp 700B amplifier (Axon Instrument), sampled using Digidata 1440A and analysed offline with pClamp 10.3 software (Axon Instruments). Series resistance was monitored throughout the recording, remaining generally <10 MΩ without compensation. The liquid junction potential was +12.5 mV and was corrected after the experiment. Data were excluded if the series resistance increased dramatically during the experiment or if there was no overshoot in action potentials. Currents were amplified, filtered at 1 kHz and digitized at 20 kHz. Current clamp was engaged to test neural firing frequency and resting membrane potential. To test the warmth sensitivity of motion-specific MVePC neurons, the temperature recording chamber was periodically changed to characterize the thermosensitivity of each recorded neuron. Neuron firing activity was monitored for at least 3 min at every temperature set point (33 °C, 36 °C and 39 °C). Compound C (10 μM) was used to investigate whether AMPK is involved in the warmth sensitivity of MVePC neurons. For the light-evoked EPSC recordings, the internal recording solution contained: 125 mM CsCH₃SO₃; 10 mM CsCl; 5 mM NaCl; 2 mM MgCl₂; 1 mM EGTA; 10 mM HEPES; 5 mM (Mg)ATP; 0.3 mM (Na)GTP (adjusted to pH 7.3 with NaOH). mEPSCs in the LPBN neurons were measured in voltage clamp mode with a holding potential of −60 mV in the presence of 1 μM TTX and 50 μM bicuculline. 4-AP and TTX were used to confirm whether the evoked eEPSC currents were monosynaptic. In some recordings, fluorescent-guided whole-cell patch–clamp recordings were performed in Kir2.1-dTomato-expressing MVePC neurons. The baseline neuronal firing frequency and resting membrane potential were compared in MVePC neurons expressing Kir2.1-dTomato. CNO (Cayman, no. 16882, 10 μM, 5 s puff) was used to test neuronal firing frequency and resting membrane potential in hM3Dq-mCherry-expressing neurons before and after acute treatment. To characterize scopolamine’s effect on CNO-induced neuronal activation, brain slices were pretreated with scopolamine (10 μM) for 5 min before CNO was administered.

Single neuron RT–qPCR

The method has been described in our previous publication⁶⁹. In brief, after the TRAPed protocol, whole brains of female TRAP2/Rosa26-LSL-tdTomato mice were removed and immediately submerged in ice-cold sucrose-based cutting solution (adjusted to pH 7.3) containing (in mM) 10 NaCl, 25 NaHCO₃, 195 sucrose, 5 glucose, 2.5 KCl, 1.25 NaH₂PO₄, 2 sodium pyruvate, 0.5 CaCl₂ and 7 MgCl₂, bubbled continuously with 95% O₂ and 5% CO₂, as before. The slices containing the MVePC region (250 μm) were cut with a Microm HM 650 V vibratome (Thermo Fisher Scientific). The slices were recovered for 1 h at 34 °C and then maintained at room temperature in aCSF (pH 7.3) containing 126 mM NaCl, 2.5 mM KCl, 2.4 mM CaCl₂, 1.2 mM NaH₂PO₄, 1.2 mM MgCl₂, 11.1 mM glucose and 21.4 mM NaHCO₃ saturated with 95% O₂ and 5% CO₂ before recording. Slices were transferred to a recording chamber, and the MVePC region was identified and excised using a fluorescence-equipped dissecting microscope. Single tdTomato-positive and tdTomato-negative neurons in the MVePC region were visualized using epifluorescence and IR-DIC imaging on an upright microscope equipped with a moveable stage (MP-285, Sutter Instrument). Single neurons were manually collected using a pipette for RNA extraction and reverse transcription using the Ambion Single-Cell-to-CT Kit (Ambion, Life Technologies), according to the manufacturer’s instructions. In brief, 10 μl single-cell lysis solution containing DNase I was added to each sample, and the entire cell contents underwent cDNA synthesis (25 °C for 10 min, 42 °C for 60 min and 85 °C for 5 min). Preamplification on the cDNA samples was conducted using a primers mix for the genes listed in Supplementary Table 1, followed by real-time PCR on a CFX384 Real-Time System (Bio-Rad).

Glucose tolerance test and insulin tolerance test

For the glucose tolerance test, after an overnight fast, mice received an i.p. injection of 1.5 g kg⁻¹ body weight d-glucose (G8270, Sigma) at 9:00. Blood glucose was measured from tail blood using a glucometer (OneTouch Ultra) at the specified time points, as indicated in figures (0, 15, 30, 60 and 120 min). For the insulin tolerance test, after a 2-h fast in the morning, mice received an i.p. injection of insulin (1.0 U kg⁻¹ body weight) at 11:00. Blood glucose was measured at times 0, 15, 30, 60 and 120 min after injection.

Food intake, body weight and energy expenditure

Female C57BL/6J mice received stereotaxic injections of AAV-CamMKII-Cre (no. 105558-AAV5, Addgene) and a Cre-dependent AAV-EF1a-DIO-Kir2.1-P2A-dTOMATO⁷⁰ into the MVePC bilaterally. In these mice, MVePC^Glu neurons selectively expressed Kir2.1 (an inward-rectifier potassium channel), which results in the constant inhibition of these neurons. As controls, female WT mice received injections of AAV-CamMKII-Cre and AAV-DIO-mCherry (no. 50459-AAV8, Addgene) into the MVePC. Food intake and body weight were measured every week after surgery. Quantitative magnetic resonance was used to determine body composition. Energy expenditure was measured in temperature-controlled (23 °C) cabinets containing 16 TSE PhenoMaster metabolic cages. Mice were acclimated to the metabolic cages for 3 days before data collection. Data collected from days 4–7 were used for analyses, and energy expenditure was analysed using the online CalR tool^71,72.

Temperature preference test

Immediately after experiencing the motion stimulus, mice were placed in a thermal gradient test box (Bio-TGT2, Bioseb) with one side set at 10 °C and the other side set at 55 °C. Mice were allowed to habituate for 30 min and were then recorded for 30 min. The thermal gradient box was divided into 20 calibrated zones, each with a corresponding temperature. The preferred temperature was defined as the temperature of the zone in which the mouse spent the longest time. The time spent in temperature zones was recorded and analysed using Thermal Gradient Test software (Bioseb).

Behavioural assays

All tests were performed in a dedicated sound-proof behavioural facility. Mice were acclimated to the procedure room 2 h before the start of each test and remained there throughout the test. The conditioned place preference apparatus comprised two identical conditioning chambers (chambers 1 and 2) that were connected by an opening (12.5 cm) in the centre. Each chamber was 30 × 30 × 25 cm (length × width × height) and had white plexiglass walls and white plexiglass floors. Blue light (473 nm, 10 ms per pulse, 10 Hz) was shone whenever the mouse entered chamber 2 and ceased when it entered chamber 1. Yellow light (595 nm, 10 ms per pulse, 10 Hz) was used as a control in a different trial. Each trial lasted 10 min.

The open field test was conducted in a clear plexiglass open field arena (40 × 40 × 30 cm). For the open field test after provocative motion, mice were introduced into the arena immediately after motion stimulus and were recorded for 10 min. In other conditions, 20 min after CNO injection, mice were introduced to the arena, followed by a 10-min recording. Ethovision XT 14 was used to record and analyse all behavioural assays.

The conditioned-taste-aversion assay followed a 6-day protocol involving daily 1-h introductions to a test arena containing two water bottles⁷³. Water was restricted in the home cage, but mice were given ad libitum water access from 9:00 to 10:00. In the first 3 days, both water bottles contained unflavoured water. On day four, both water bottles were filled with either grape- or cherry-flavoured water (grape or cherry Kool-Aid) sweetened with 0.2% saccharin, with each flavour presented in half of the trials. Immediately after a 1-h introduction on the conditioning day, the mice were subjected to motion. On day five, both water bottles contained unflavoured water. On day six, one water bottle contained cherry-flavoured water, and the other contained grape-flavoured water. Consumption from each water bottle was measured manually.

HomeCageScan

To evaluate the spontaneous behaviour of control and Kir2.1-treated mice, we placed mice into the HomeCageScan (CleverSys) to record their behaviours for 2 days. Behaviours were analysed by the HomeCageScan software.

Statistical analysis

No statistical methods were used to predetermine sample sizes, but our sample sizes are similar to those reported in a previous publication⁷⁴. Data collection was randomized and conducted in a blinded manner. For most physiological measurements (for example, body temperature), 6–8 mice per group were included. The data are presented as mean ± s.e.m. unless otherwise stated. Data distribution was assumed to be normal but this was not formally tested. Methods of statistical analyses were chosen on the basis of the design of each experiment and are indicated in the figure legends, and P < 0.05 was considered to be statistically significant.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Extended Data

Extended Data Fig. 3 | — (a) Schematic timeline of TRAP protocol coupled with c-fos immnuostaining. (b) In the left panel, tdTomato-labeled neurons refer to ‘TRAP’ed motion-specific neurons, and c-fos positive cells indicate neurons were activated after mice being subjected to a second-round of motion. Percentage of colocalization between tdTomato-labeled neurons and c-fos positive cells in each specific brain region was shown in the right panel. n = 3 mice. Data are expressed as mean ± standard errors of the mean. Arrows point to co-localized cells between tdTomato and c-fos positive cells. (Relative to Fig. 2).

Extended Data Fig. 4 | — (**a–d**) Re-activation of motion-specific PVH neurons did not affect body temperature (a-b) or locomotor activity (c-d). n = 6 for control, and n = 7 for TRAP mice. (**e–g**) Re-activation of motion-specific PVH neurons inhibited food intake in fasting mice (e), but did not affect preferred ambient temperature (f-g). n = 6 for control, and n = 7 for TRAP mice, two-way ANOVA followed by Bonferroni post hoc test for (e). (**h–k**) Distance traveled (h), velocity (i), central zone duration (j), and representative heat-maps (k) in an open field test after motion-specific PVH neurons were re-activated via CNO injection. n = 6 for control, and n = 7 for TRAP mice. (**l, m**) Expression of hM3Dq-mCherry in the PVH that was validated by c-fos immunostaining after CNO injection, and quantification of c-fos cells (m). n = 6 for control, and n = 7 for TRAP mice, two-sided student t text for (m). Data are expressed as mean ± standard errors of the mean. (Relative to Fig. 2).

Extended Data Fig. 5 | — (**a–d**) Re-activation of motion-specific LPBN neurons slightly decreased body temperature (a-b), but did not influence locomotor activity (c-d). n = 6 mice, one-way ANOVA followed by Bonferroni post hoc test for (b). (**e–g**) Re-activation of motion-specific LPBN neurons inhibited food intake in fasting mice (e), but did not affect preferred ambient temperature (f-g). n = 6 mice, two-way ANOVA followed by Bonferroni post hoc test for (e). (**h–k**) Distance traveled (h), velocity (i), central zone duration (j), and representative heat-maps (k) in an open field test after motion-specific LPBN neurons were re-activated via CNO injection. n = 6 mice, one-way ANOVA followed by Bonferroni post hoc test for (h) and (i). (**l, m**) Expression of hM3Dq-mCherry in the LPBN that was validated by c-fos immunostaining after CNO injection, and quantification of c-fos cells (m). n = 6 mice, two-sided student t text for (m). Data are expressed as mean ± standard errors of the mean. (Relative to Fig. 2).

Extended Data Fig. 6 | — (**a–d**) Experimental illustration of a recorded motion-specific MVePC neuron that was labeled by hM3Dq-mCherry (a), scopolamine attenuated CNO-induced increase in firing frequency and membrane potential (b-c), and raw traces of a recorded hM3Dq-mCherry-expressing neuron among different treatments (d). n = 8 neurons from 2 mice, two-tail paired t test for (b) and (c). (**e–g**) After washout of scopolamine, CNO induced increase in firing frequency and membrane potential of hM3Dq-mCherry-expressing neurons (e-f), and raw traces of a recorded hM3Dq-mCherry-expressing neuron in response to CNO after scopolamine washout (g). n = 6 neurons from 2 mice, two-tail paired t test for (e) and (f). (**h, i**) Representative photomicrographs illustrating c-fos immunostaining and number of c-fos cells in the MVePC following scopolamine treatment against motion in female mice. n = 6 mice in each group, one-way ANOVA followed by Bonferroni post hoc test. Data are expressed as mean ± standard errors of the mean. (Relative to Fig. 2).

Extended Data Fig. 7 | — (a) Experimental illustration of a recorded non-motion-specific MVePC neuron from 2 female TRAP2/Rosa26-LSL-tdTomato mice. (**b–d**) Firing frequency and membrane potential of non-motion-specific MVePC neurons during different ambient recording temperatures (b-c), and representative raw traces of one recorded non-motion-specific MVePC neuron (d). n = 7 neurons from 2 mice. (**e, f**) Membrane potential of motion-specific MVePC neurons during different ambient recording temperatures in the presence of a cocktail of synaptic blockers (TTX, CNQX, D-AP5 and bicuculline), and representative raw trace of one recorded neuron in this condition. n = 13 neurons from 2 mice. One-way ANOVA followed by Bonferroni tests for (e). (**g, h**) Membrane potential of non-motion-specific MVePC neurons during different ambient recording temperatures in the presence of a cocktail blockers of synaptic blockers (TTX, CNQX, D-AP5 and bicuculline), and representative raw trace of one recorded neuron in this condition. n = 6 neurons from 2 mice. Data are expressed as mean ± standard errors of the mean. (Relative to Fig. 2).

Extended Data Fig. 8 | — (**a, b**) Firing frequency and membrane potential of motion-specific MVePC neurons exposed to different ambient temperatures in the presence of compound C (10 μM). n = 8 neurons from 2 mice. (c) Representative raw traces of one recorded neuron. Data are expressed as mean ± standard errors of the mean. (Relative to Fig. 2).

Extended Data Fig. 9 | — (**a–d**) Representative image showing fiber position and hChR2 (H134R)-EFYP expression in the MVePC^Glu neurons (a), and time spent, velocity and distance travelled in each respective chamber that was coupled with MVePC^Glu stimulation during the real-time place preference test (b-d). n = 7 mice, two-tail paired t test for (b). (**e–h**) Representative image showing fiber position and hChR2 (H134R)-EFYP expression in the MVePC^Vgat neurons (e), and time spent, velocity and distance travelled in each respective chamber that was coupled with MVePC^Vgat stimulation during the real-time place preference test (f-h). n = 7 mice, two-tail paired t test. (**i–l**) Representative image showing fiber position and terminals of hChR2 (H134R)-EFYP expression in the LPBN (i), and time spent, velocity and distance travelled in each respective chamber that was coupled with stimulation of terminals of MVePC^Glu in the LPBN during the real-time place preference test (j-l). n = 6 mice, two-tail paired t test for (j). Data are expressed as mean ± standard errors of the mean. (Relative to Fig. 3).

Extended Data Fig. 10 | — (**a, b**) Motion decreased BAT temperature in female mice. n = 6 mice, two-way ANOVA followed by Bonferroni tests for (a), and two-sided student t test for (b). (**c, d**) Experimental illustration of PRV-mediated retrograde polysynaptic tracing in BAT (c), and representative images showing RFP⁺ neurons in MVePC, RPa, PVH, but not LPBN (d) from 3 mice. (**e–g**) Activation of MVePC^Glu neurons via blue light delivery decreased BAT temperature. n = 6 mice, two-way ANOVA followed by Bonferroni tests for (e), and two-tail paired t test for (g). (**h–j**) Inhibition of MVePC^Glu neurons via CNO injection increased BAT temperature. n = 6 mice, two-way ANOVA followed by Bonferroni tests for (i), and student t test for (j). Data are expressed as mean ± standard errors of the mean. (Relative to Fig. 4).

Supplementary Material

supplementary

NIHMS2175782-supplement-supplementary.pdf^{(811.8KB, pdf)}

Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s42255-025-01234-9.

Acknowledgements

The investigators are supported by grants from the NIH (P01DK113954, R01DK115761, R01DK117281, R01DK125480 and R01DK120858 to Y.X.; R01DK104901 and R01DK12665 to M.F.; R01MH117089 to M.X.); USDA/CRIS (51000–064–01S to Y.X.; 51000–064–02S to M.F.; 3092–51000–062–04(B)S to C.W.; 1F32DK13868501A1 to X.F.); the McKnight Foundation to M.X.; and an American Heart Association Postdoctoral Fellowship (2020AHA000POST000204188 to L.T.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the paper.

Footnotes

Competing interests

The authors declare no competing interests.

Additional information

Extended data is available for this paper at https://doi.org/10.1038/s42255–025-01234–9.

Peer review information Nature Metabolism thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editors: Jean Nakhle and Ashley Castellanos-Jankiewicz, in collaboration with the Nature Metabolism team.

Data availability

All data generated or analysed during this study are included in this published article. No third-party materials were included in this paper. Further information and requests for resources and reagents should be directed to and will be fulfilled by Y.X. (yongx@bcm.edu). Source data are provided with this paper.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

supplementary

NIHMS2175782-supplement-supplementary.pdf^{(811.8KB, pdf)}

Data Availability Statement

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PERMALINK

Vestibular neurons link motion sickness, behavioural thermoregulation and metabolic balance in mice

Longlong Tu

Xing Fang

Yongjie Yang

Meng Yu

Hailan Liu

Hesong Liu

Na Yin

Jonathan C Bean

Kristine M Conde

Mengjie Wang

Yongxiang Li

Olivia Z Ginnard

Qingzhuo Liu

Yuhan Shi

Junying Han

Yi Zhu

Makoto Fukuda

Qingchun Tong

Benjamin Arenkiel

Mingshan Xue

Yang He

Chunmei Wang

Yong Xu

Abstract

Results

Establishment of a mouse motion-sickness model

Fig. 1 |. Establishment of a mouse motion-sickness model in the laboratory.

Activity-dependent genetic labelling of MVePC neurons

Fig. 2 |. Reactivation of motion-specific MVePC neurons recapitulates motion sickness in female mice.

Activating MVePC neurons mimics motion-induced hypothermia

Fig. 3 |. Optogenetic stimulation of the MVePCGlu→LPBN neural circuit recapitulates motion sickness in female mice.

Inhibiting MVePC→LPBN circuit attenuates motion sickness

Fig. 4 |. Inhibition of the MVePCGlu→LPBN neural circuit alleviates motion sickness in female mice.

Functional identification of the MVePC→BAT axis

Inhibition of MVePCGlu neurons prevents diet-induced obesity

Fig. 5 |. Chronic inhibition of MVePCGlu neurons prevented diet-induced obesity in female mice.

Discussion

Methods

Mice

Immunohistochemistry

TRAP induction

RNAscope

Chemogenetics

Rabies retrograde tracing

Optogenetic studies

Electrophysiology

Single neuron RT–qPCR

Glucose tolerance test and insulin tolerance test

Food intake, body weight and energy expenditure

Temperature preference test

Behavioural assays

HomeCageScan

Statistical analysis

Reporting summary

Extended Data

Extended Data Fig. 1 |. Motion elicits c-fos expression in the brain.

Extended Data Fig. 2 |. Validation of the mouse motion sickness model with the anti-motion sickness drug scopolamine.

Extended Data Fig. 3 |. Validation of ‘TRAP’ed motion-specific neurons via c-fos immunostaining.

Extended Data Fig. 4 |. Re-activation of motion-specific PVH neurons.

Extended Data Fig. 5 |. Re-activation of motion-specific LPBN neurons.

Extended Data Fig. 6 |. Effect of scopolamine on electrical responses of motionspecific MVePC neurons.

Extended Data Fig. 7 |. Electrical responses of MVePC neurons exposed to different ambient temperatures.

Extended Data Fig. 8 |. Compound C abolishes excitation of MVePC neurons elicited by high ambient temperature.

Extended Data Fig. 9 |. Validation of hChR2 expression and real-time place preference test.

Extended Data Fig. 10 |. MVePCGlu neurons modulates BAT thermogenesis in female mice.

Supplementary Material

Acknowledgements

Footnotes

Data availability

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

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Fig. 3 |. Optogenetic stimulation of the MVePC^Glu→LPBN neural circuit recapitulates motion sickness in female mice.

Fig. 4 |. Inhibition of the MVePC^Glu→LPBN neural circuit alleviates motion sickness in female mice.

Inhibition of MVePC^Glu neurons prevents diet-induced obesity

Fig. 5 |. Chronic inhibition of MVePC^Glu neurons prevented diet-induced obesity in female mice.

Extended Data Fig. 10 |. MVePC^Glu neurons modulates BAT thermogenesis in female mice.