A top-down slow breathing circuit that alleviates negative affect in mice | Nature Neuroscience

Nature Neuroscience volume 27, pages 2455–2465 (2024)Cite this article

9950 Accesses

2 Citations

830 Altmetric

Metrics details

Although breathing is primarily automatic, its modulation by behavior and emotions suggests cortical inputs to brainstem respiratory networks, which hitherto have received little characterization. Here we identify in mice a top-down breathing pathway from dorsal anterior cingulate cortex (dACC) neurons to pontine reticular nucleus GABAergic inhibitory neurons (PnCGABA), which then project to the ventrolateral medulla (VLM). dACC→PnC activity correlates with slow breathing cycles and volitional orofacial behaviors and is influenced by anxiogenic conditions. Optogenetic stimulation of the dACC→PnCGABA→VLM circuit simultaneously slows breathing and suppresses anxiety-like behaviors, whereas optogenetic inhibition increases both breathing rate and anxiety-like behaviors. These findings suggest that the dACC→PnCGABA→VLM circuit has a crucial role in coordinating slow breathing and reducing negative affect. Our study elucidates a circuit basis for top-down control of breathing, which can influence emotional states.

This is a preview of subscription content, access via your institution

Access Nature and 54 other Nature Portfolio journals

Get Nature+, our best-value online-access subscription

$29.99 / 30 days

cancel any time

Subscribe to this journal

Receive 12 print issues and online access

$209.00 per year

only $17.42 per issue

Buy this article

Prices may be subject to local taxes which are calculated during checkout

All data are available from the corresponding author upon request. All data associated with statistical analyses are available as source data. The source data are also available at figshare repository (https://doi.org/10.6084/m9.figshare.26888749 (ref. 53)). Source data are provided with this paper.

Feldman, J. L., Mitchell, G. S. & Nattie, E. E. Breathing: rhythmicity, plasticity, chemosensitivity. Annu. Rev. Neurosci. 26, 239–266 (2003).

Article CAS PubMed PubMed Central Google Scholar

Del Negro, C. A., Funk, G. D. & Feldman, J. L. Breathing matters. Nat. Rev. Neurosci. 19, 351–367 (2018).

Article PubMed PubMed Central Google Scholar

Dick, T. E. et al. Facts and challenges in respiratory neurobiology. Respir. Physiol. Neurobiol. 258, 104–107 (2018).

Article CAS PubMed Google Scholar

Pagliardini, S., Funk, G. D. & Dickson, C. T. Breathing and brain state: urethane anesthesia as a model for natural sleep. Respir. Physiol. Neurobiol. 188, 324–332 (2013).

Article CAS PubMed Google Scholar

Arthurs, J. W., Bowen, A. J., Palmiter, R. D. & Baertsch, N. A. Parabrachial tachykinin1-expressing neurons involved in state-dependent breathing control. Nat. Commun. 14, 963 (2023).

Article CAS PubMed PubMed Central Google Scholar

Park, H.-D. et al. Breathing is coupled with voluntary action and the cortical readiness potential. Nat. Commun. 11, 289 (2020).

Article CAS PubMed PubMed Central Google Scholar

Betka, S., Adler, D., Similowski, T. & Blanke, O. Breathing control, brain, and bodily self-consciousness: toward immersive digiceuticals to alleviate respiratory suffering. Biol. Psychol. 171, 108329 (2022).

Article PubMed Google Scholar

Moore, J. D. et al. Hierarchy of orofacial rhythms revealed through whisking and breathing. Nature 497, 205–210 (2013).

Article CAS PubMed PubMed Central Google Scholar

Hegland, K. W., Bolser, D. C. & Davenport, P. W. Volitional control of reflex cough. J. Appl. Physiol. 113, 39–46 (2012).

Article CAS PubMed PubMed Central Google Scholar

Watanabe, Y., Abe, S., Ishikawa, T., Yamada, Y. & Yamane, G. Y. Cortical regulation during the early stage of initiation of voluntary swallowing in humans. Dysphagia 19, 100–108 (2004).

Article PubMed Google Scholar

Martin-Harris, B. Coordination of respiration and swallowing. GI Motility online https://www.nature.com/gimo/contents/pt1/full/gimo10.html (2006).

Homma, I. & Masaoka, Y. Breathing rhythms and emotions. Exp. Physiol. 93, 1011–1021 (2008).

Article PubMed Google Scholar

Holt, P. E. & Andrews, G. Hyperventilation and anxiety in panic disorder, social phobia, GAD and normal controls. Behav. Res. Ther. 27, 453–460 (1989).

Article CAS PubMed Google Scholar

Leander, M. et al. Impact of anxiety and depression on respiratory symptoms. Respir. Med. 108, 1594–1600 (2014).

Article PubMed Google Scholar

Masaoka, Y., Jack, S., Warburton, C. J. & Homma, I. Breathing patterns associated with trait anxiety and breathlessness in humans. Jpn. J. Physiol. 54, 465–470 (2004).

Article PubMed Google Scholar

Bondarenko, E., Hodgson, D. & Nalivaiko, E. Prelimbic prefrontal cortex mediates respiratory responses to mild and potent prolonged, but not brief, stressors. Respir. Physiol. Neurobiol. 204, 21–27 (2014).

Article CAS PubMed Google Scholar

Bondarenko, E. & Nalivaiko, E. Pharmacological inhibition of infralimbic prefrontal cortex abolishes sniffing behavior and respiratory response to stress. FASEB J. 30, 1261 (2016).

Article Google Scholar

Liu, S. et al. Divergent brainstem opioidergic pathways that coordinate breathing with pain and emotions. Neuron 110, 857–873 (2022).

Article CAS PubMed Google Scholar

Valenza, M. C. et al. Effectiveness of controlled breathing techniques on anxiety and depression in hospitalized patients with COPD: a randomized clinical Trial. Respir. Care 59, 209–215 (2014).

Article PubMed Google Scholar

Strigo, I. A. & Craig, A. D. Interoception, homeostatic emotions and sympathovagal balance. Philos. Trans. R. Soc. Lond. B 371, 20160010 (2016).

Article Google Scholar

Balban, M. Y. et al. Brief structured respiration practices enhance mood and reduce physiological arousal. Cell Rep. Med. 4, 100895 (2023).

Article PubMed PubMed Central Google Scholar

Herrero, J. L., Khuvis, S., Yeagle, E., Cerf, M. & Mehta, A. D. Breathing above the brain stem: volitional control and attentional modulation in humans. J. Neurophysiol. 119, 145–159 (2018).

Article PubMed Google Scholar

Mckay, L. C., Evans, K. C., Frackowiak, R. S. & Corfield, D. R. Neural correlates of voluntary breathing in humans. J. Appl. Physiol. 95, 1170–1178 (2003).

Article CAS PubMed Google Scholar

Tremoureux, L. et al. Does the supplementary motor area keep patients with Ondine’s curse syndrome breathing while awake? PLoS ONE 9, e84534 (2014).

Article PubMed PubMed Central Google Scholar

Macey, K. E. et al. Inspiratory loading elicits aberrant fMRI signal changes in obstructive sleep apnea. Respir. Physiol. Neurobiol. 151, 44–60 (2006).

Article PubMed Google Scholar

Davenport, P. W. & Vovk, A. Cortical and subcortical central neural pathways in respiratory sensations. Respir. Physiol. Neurobiol. 167, 72–86 (2009).

Article PubMed Google Scholar

Liotti, M. et al. Brain responses associated with consciousness of breathlessness (air hunger). Proc. Natl Acad. Sci. USA 98, 2035–2040 (2001).

Article CAS PubMed PubMed Central Google Scholar

Harper, R. M. et al. Hypercapnic exposure in congenital central hypoventilation syndrome reveals CNS respiratory control mechanisms. J. Neurophysiol. 93, 1647–1658 (2005).

Article CAS PubMed Google Scholar

Evans, K. C. et al. BOLD fMRI identifies limbic, paralimbic, and cerebellar activation during air hunger. J. Neurophysiol. 88, 1500–1511 (2002).

Article PubMed Google Scholar

Kelly, B. N., Huckabee, M. L., Jones, R. D. & Carroll, G. J. The influence of volition on breathing-swallowing coordination in healthy adults. Behav. Neurosci. 121, 1174–1179 (2007).

Article PubMed Google Scholar

Jürgens, U. Neural pathways underlying vocal control. Neurosci. Biobehav. Rev. 26, 235–258 (2002).

Article PubMed Google Scholar

Biskamp, J., Bartos, M. & Sauer, J.-F. Organization of prefrontal network activity by respiration-related oscillations. Sci. Rep. 7, 45508 (2017).

Article CAS PubMed PubMed Central Google Scholar

Karalis, N. & Sirota, A. Breathing coordinates cortico-hippocampal dynamics in mice during offline states. Nat. Commun. 13, 467 (2022).

Article CAS PubMed PubMed Central Google Scholar

Baertsch, N. A., Baertsch, H. C. & Ramirez, J. M. The interdependence of excitation and inhibition for the control of dynamic breathing rhythms. Nat. Commun. 9, 843 (2018).

Article PubMed PubMed Central Google Scholar

Hirrlinger, J. et al. GABA-glycine cotransmitting neurons in the ventrolateral medulla: development and functional relevance for breathing. Front. Cell. Neurosci. 13, 517 (2019).

Article CAS PubMed PubMed Central Google Scholar

Sherman, D., Worrell, J. W., Cui, Y. & Feldman, J. L. Optogenetic perturbation of preBötzinger complex inhibitory neurons modulates respiratory pattern. Nat. Neurosci. 18, 408–414 (2015).

Article CAS PubMed PubMed Central Google Scholar

Hülsmann, S., Hagos, L., Eulenburg, V. & Hirrlinger, J. Inspiratory off-switch mediated by optogenetic activation of inhibitory neurons in the preBötzinger complex in vivo. Int. J. Mol. Sci. 22, 2019 (2021).

Article PubMed PubMed Central Google Scholar

Yang, C. F., Kim, E. J., Callaway, E. M. & Feldman, J. L. Monosynaptic projections to excitatory and inhibitory preBötzinger complex neurons. Front. Neuroanat. 14, 58 (2020).

Article CAS PubMed PubMed Central Google Scholar

Zingg, B. et al. AAV-mediated anterograde transsynaptic tagging: mapping corticocollicular input-defined neural pathways for defense behaviors. Neuron 93, 33–47 (2017).

Article CAS PubMed Google Scholar

Zingg, B., Peng, B., Huang, J., Tao, H. W. & Zhang, L. I. Synaptic specificity and application of anterograde transsynaptic AAV for probing neural circuitry. J. Neurosci. 40, 3250–3267 (2020).

Article CAS PubMed PubMed Central Google Scholar

Scott, G., Westberg, K.-G., Vrentzos, N., Kolta, A. & Lund, J. Effect of lidocaine and NMDA injections into the medial pontobulbar reticular formation on mastication evoked by cortical stimulation in anaesthetized rabbits. Eur. J. Neurosci. 17, 2156–2162 (2003).

Article CAS PubMed Google Scholar

McAfee, S. S. et al. Minimally invasive highly precise monitoring of respiratory rhythm in the mouse using an epithelial temperature probe. J. Neurosci. Methods 263, 89–94 (2016).

Article PubMed PubMed Central Google Scholar

Liu, S. & Han, S. Simultaneous recording of breathing and neural activity in awake behaving mice. STAR Protoc. 3, 101412 (2022).

Article PubMed PubMed Central Google Scholar

Huckabee, M. L., Deecke, L., Cannito, M. P., Gould, H. J. & Mayr, W. Cortical control mechanisms in volitional swallowing: the Bereitschaftspotential. Brain Topogr. 16, 3–17 (2003).

Article PubMed Google Scholar

Le Gal, J. P. et al. Modulation of respiratory network activity by forelimb and hindlimb locomotor generators. Eur. J. Neurosci. 52, 3181–3195 (2020).

Article PubMed Google Scholar

Fanning, J. et al. Relationships between respiratory sinus arrhythmia and stress in college students. J. Behav. Med. 43, 308–317 (2020).

Article CAS PubMed Google Scholar

Manuel, J. et al. Deciphering the neural signature of human cardiovascular regulation. eLife 9, e55316 (2020).

Article CAS PubMed PubMed Central Google Scholar

Menuet, C. et al. PreBötzinger complex neurons drive respiratory modulation of blood pressure and heart rate. eLife 9, e57288 (2020).

Article CAS PubMed PubMed Central Google Scholar

Molle, L. & Coste, A. The respiratory modulation of interoception. J. Neurophysiol. 127, 896–899 (2022).

Article PubMed Google Scholar

Klein, A. S., Dolensek, N., Weiand, C. & Gogolla, N. Fear balance is maintained by bodily feedback to the insular cortex in mice. Science 374, 1010–1015 (2021).

Article CAS PubMed Google Scholar

Liu, Y. et al. Molecular regulation of sexual preference revealed by genetic studies of 5-HT in the brains of male mice. Nature 472, 95–99 (2011).

Article CAS PubMed PubMed Central Google Scholar

Beny-Shefer, Y. et al. Nucleus accumbens dopamine signaling regulates sexual preference for females in male mice. Cell Rep. 21, 3079–3088 (2017).

Article CAS PubMed Google Scholar

Jhang, J. et al. A top-down slow breathing circuit that alleviates negative affect. figshare https://doi.org/10.6084/m9.figshare.26888749 (2024).

Download references

We thank all members of the Han Laboratory for scientific discussions and support. This work was supported by grants from the KAVLI Institute for Brain and Mind Innovative Research Grants (IRGS 2020-1710). All members of the Han Laboratory are committed to upholding principles of equity and inclusion, ensuring that no person is discriminated against based on gender, race, age, religion, sexual orientation, veteran status or disability.

Shijia Liu

Present address: Howard Hughes Medical Institute, Department of Neurobiology, Harvard Medical School, Boston, MA, USA

Peptide Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA, USA

Jinho Jhang, Seahyung Park, Shijia Liu & Sung Han

Department of Neurobiology, Division of Biological Sciences, University of California San Diego, La Jolla, CA, USA

Shijia Liu & Sung Han

Research Development Department, The Salk Institute for Biological Studies, La Jolla, CA, USA

David D. O’Keefe

Center for Neuroscience Imaging Research, Institute for Basic Science, Suwon, Republic of Korea

Sung Han

Department of Biomedical Engineering, Sungkyunkwan University, Suwon, Republic of Korea

Sung Han

You can also search for this author in PubMed Google Scholar

S.H. and J.J. designed the study and secured funding. J.J., S.P., D.D.O. and S.H. wrote the paper. J.J. performed the experiments and analyzed the data. S.P. performed electrophysiology. S.L. helped with thermistor implantation surgeries.

Correspondence to Sung Han.

The authors declare no competing interests.

Nature Neuroscience thanks Julien Bouvier, Nadine Gogolla and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

a, Summary of the criteria used to search candidate circuits. Axonal projection images were searched through the Mouse Brain Connectivity Atlas (Allen Brain Institute). In situ hybridization (ISH) images were searched through the Mouse Brain ISH Data Atlas (Allen Brain Institute). b, Sample images showing the projections of labeled PnC neurons observed in the ventrolateral medulla (VLM). Left, the AAV injection site (PnC). Right, the observed terminals in the VLM. c, Sample image from a Vgat ISH experiment. d, Sample images showing the projections of PFC neurons observed in the PnC. Left image shows the AAV injection site (PFC, prefrontal cortex; covering the dACC and vACC) with the expression of eGFP. Middle and right (in segmentation view) images show the eGFP-labeled axon terminals in the PnC. PnC, pontine reticular nucleus; VLM, ventrolateral medulla; PFC, prefrontal cortex. Scale bars, 200 μm in b–d. These are representative data (b–d) available from the Allen Brain Atlas.

Source data

a, Representative raw breathing trace measured by inductance plethysmography experiment under anesthesia from a mouse expressing ChR2 in dACC→PnC neurons. b–f, Analyses of breathing rates and inspiratory/expiratory durations during photoactivation of dACC→PnC neurons with reduced light intensity (b–f; ChR2, N = 6; eGFP, N = 5 mice). b,c, Percent change of normalized breathing rates during the photoactivation with ~6 mW (b) and ~3 mW (c) intensity. d, Correlation between the change of breathing rate and intensity of light stimulation. e,f, Correlation between the change of inspiratory (e) or expiratory (f) duration and intensity of light stimulation. g, Photaoactivation of dACC→PnC neurons decreases breathing rates (shown in breath per minute; BPM). h, Schematic of breathing monitoring using a nasal thermistor sensor. i,j, Photoactivation of dACC→PnC neurons led to a decrease in breathing rate in awake mice (ChR2, N = 6; eGFP N = 6 mice). Average breathing rate (5-s window smoothed; i); light-induced changes (ON– OFF) in breathing rates (j). N.SP > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001. Line graphs are shown as mean ± s.e.m. Box-whisker plot is shown as median and interquartile range with minimum and maximum.

a, Schematic of CTB-555 injection into the dACC (left) and representative image showing the injection site (right). Scale bar, 500 μm. b, Known afferent regions of the dACC (claustrum and basolateral amygdala) and the PnC. No retrograde labeling occurred in the PnC (N = 4 mice). CLA, claustrum; LA, lateral nucleus of amygdala; BLA, basolateral nucleus of amygdala. Scale bar, 200 μm.

a, Raw breathing trace recorded during ChR2-mediated photoactivation of PnCGABA neurons. Scale bar, 10 s. b, Raw breathing trace recorded during eNpHR3.0-mediated photoinhibition of PnCGABA neurons. Scale bar, 10 s. c,d, Schematic of simultaneous recording of jaw movement and breathing signals using inductance plethysmography under anesthesia (c, left) or using nasal thermistor sensor in awake state (d, left). No instance of jaw opening or mastication was observed throughout the duration of photostimulation (right; c, N = 4 mice; d, N = 5 mice).

a, Surgical procedure for expressing jGCaMP7s in PnCGABA neurons, and image showing jGCaMP7s expression (right). Scale bar, 200 μm. b, Schematic showing observation of voluntary drinking behavior. c, Representative traces showing breathing cycles and PnCGABA activity during drinking behavior. d, Average PnCGABA activity during drinking behavior (N = 5 mice). Dashed line shows the moment that a water droplet was taken into oral cavity. e, Schematic of submersion experiments in which mice were removed after 1–2 s (withdrawal). f, Representative traces showing breathing cycles and PnCGABA activity during withdrawal experiments. g, Average PnCGABA neuronal activity during withdrawal experiments (N = 5 mice). h, Schematic of submersion experiments in which mice were released. i, Representative traces showing breathing cycles and PnCGABA activity during release/swim experiments. j, Average PnCGABA neuronal activity during release/swim experiments (N = 5 mice). Data are shown as mean ± s.e.m. or individual value from each subject.

Source data

a, Illustrations showing GCaMP7s injection sites and fiber placement targeting dACC area, in mice used for elevated plus maze, elevated platform and foot shock exposure experiments (N = 6 mice). b, Representative traces of dACC→PnC neuronal activity (top) and breathing rate (bottom) during the elevated plus maze exposure. c, Representative raw breathing signals observed during an exit episode. d, Analysis of the length of inspiratory and expiratory phases before and after exit (N = 6 mice). e, Breathing rates during exploration of an exposed area classified by episodes with refrained behavior (n = 43 episodes) and full exploration (n = 10 episodes). ***P < 0.001. Data are shown as mean ± s.e.m. or individual value from each subject.

Source data

a, Axon collaterals of dACC→PnC neurons observed in regions other than the PnC. Scale bar, 200 μm. b,c, Photoactivation of dACC→PnC neurons (ChR2, N = 6; eGFP, N = 7 male mice) did not change approach response to female odor (b) but reduced avoidance response to TMT (c). d–f, Photoactivation of dACC→PnC neurons during light/dark choice test (d, ChR2, N = 9 and eGFP control, N = 8 mice). Photoactivation of dACC→PnC neurons reduces ΔBPM (light–dark; e) and increases time spent in the light zone (f). g,h, Photoinhibition of dACC→PnC projections (eNpHR3.0, N = 7; eGFP control, N = 8 male mice) did not change approach response to female odor (g) or avoidance response to TMT (h). LV, lateral ventricle; CPu, caudate putamen; cc, corpus callosum; D3V, dorsal third ventricle; PVT, paraventricular thalamic nucleus; MD, mediodorsal thalamic nucleus; VMT, ventromedial thalamic nucleus; DpME, deep mesencephalic nucleus; RMC, red nucleus, magnocellular; ZI, zona incerta; LH, lateral hypothalamus; mt, mammillothalamic tract; ic, internal capsule. N.SP > 0.05, *P < 0.05, **P < 0.01. Bar graphs are shown as mean ± s.e.m. Box-whisker plots are shown as median and interquartile range with minimum and maximum.

Source data

a, Efferent projections of PnCGABA neurons. Right, quantification of projection density of eYFP-labeled axons (N = 5 mice, Vgat-ires-Cre). Scale bar, 200 μm. b, Images showing the viral injection site in the PnC, and brainstem regions receiving efferent fibers expressing ChR2-eYFP. Scale bar, 200 μm. BST, bed nucleus of stria terminalis; LHb, lateral habenula; PVT, paraventricular nucleus of thalamus; PB, parabrachial nucleus; VLM, ventrolateral medulla. Data are shown as mean ± s.e.m.

Source data

a, Inductance plethysmography experiments performed with photoactivation of PnCGABA→VLM terminals (ChR2, N = 7; eGFP, N = 7 mice), tested with different frequencies. b, Changes in breathing rates (% of baseline) induced by stimulation of PnCGABA→VLM terminals (ChR2, N = 7; eGFP, N = 7 mice). c, Inductance plethysmography experiments performed with photoactivation of PnCGABA→LHb/PVT terminals (ChR2, N = 7; eGFP, N = 8 mice), tested with different frequencies. d, Changes in breathing rates (% of baseline) induced by stimulation of PnCGABA→LHb/PVT terminals (ChR2, N = 7; eGFP, N = 8 mice). e–g, Breathing rates shown as breaths per minute (BPM) during photoactivation experiments (15-Hz stimulation; e, PnCGABA→VLM; f, PnCGABA→LHb/PVT; g, AAV1-based labeling). ***P < 0.001. Line graphs are shown as mean ± s.e.m. Box-whisker plots are shown as median and interquartile range with minimum and maximum.

Source data

Supplementary Figs. 1–5 and Table 1.

Statistical source data.

Statistical source data

Statistical source data.

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

Jhang, J., Park, S., Liu, S. et al. A top-down slow breathing circuit that alleviates negative affect in mice. Nat Neurosci 27, 2455–2465 (2024). https://doi.org/10.1038/s41593-024-01799-w

Download citation

Received: 07 October 2023

Accepted: 23 September 2024

Published: 19 November 2024

Issue Date: December 2024

DOI: https://doi.org/10.1038/s41593-024-01799-w

Anyone you share the following link with will be able to read this content:

Sorry, a shareable link is not currently available for this article.

Provided by the Springer Nature SharedIt content-sharing initiative

Nature Communications (2025)

Nature Neuroscience (2024)