Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

A pontomesencephalic PACAPergic pathway underlying panic-like behavioral and somatic symptoms in mice

Nature Neuroscience volume 27, pages 90–101 (2024)Cite this article

Subjects

Abstract

Panic disorder is characterized by uncontrollable fear accompanied by somatic symptoms that distinguish it from other anxiety disorders. Neural mechanisms underlying these unique symptoms are not completely understood. Here, we report that the pituitary adenylate cyclase-activating polypeptide (PACAP)-expressing neurons in the lateral parabrachial nucleus projecting to the dorsal raphe are crucial for panic-like behavioral and physiological alterations. These neurons are activated by panicogenic stimuli but inhibited in conditioned fear and anxiogenic conditions. Activating these neurons elicits strong defensive behaviors and rapid cardiorespiratory increase without creating aversive memory, whereas inhibiting them attenuates panic-associated symptoms. Chemogenetic or pharmacological inhibition of downstream PACAP receptor-expressing dorsal raphe neurons abolishes panic-like symptoms. The pontomesencephalic PACAPergic pathway is therefore a likely mediator of panicogenesis, and may be a promising therapeutic target for treating panic disorder.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Fig. 1: Activity of PACAPPBL→DR neurons increases in panicogenic conditions.
Fig. 2: Activity of PACAPPBL→DR neurons decreases during retrieval in the fear conditioning test.
Fig. 3: Activation of PACAPPBL→DR neurons produces panic-like symptoms without forming associative memory.
Fig. 4: PACAPPBL→DR axon terminal activation produces panic-like symptoms but does not create conditioned fear memory.
Fig. 5: Inhibition of PACAPPBL→DR neurons attenuates panic-like symptoms.
Fig. 6: PAC1RDR neurons receive monosynaptic input from PACAPPBL neurons, and their activation is necessary and sufficient for induction of panic-like symptoms.
Fig. 7: Infusion of PAC1R antagonist PACAP (6-38) into DR attenuates panic-like symptoms.

Similar content being viewed by others

Data availability

All data associated with this study are available in the supplementary information. Source data are provided with this paper.

References

  1. Killgore, W. D. S. et al. Cortico-limbic responses to masked affective faces across PTSD, panic disorder, and specific phobia. Depress Anxiety 31, 150–159 (2014).

    PubMed  Google Scholar 

  2. Crowe, R. R., Noyes, R., Pauls, D. L. & Slymen, D. A family study of panic disorder. Arch. Gen. Psychiatry 40, 1065–1069 (1983).

    CAS  PubMed  Google Scholar 

  3. Katon, W. Panic disorder and somatization. Review of 55 cases. Am. J. Med. 77, 101–106 (1984).

    CAS  PubMed  Google Scholar 

  4. Meuret, A. E., Kroll, J. & Ritz, T. Panic disorder comorbidity with medical conditions and treatment implications. Annu. Rev. Clin. Psychol. 13, 209–240 (2017).

    PubMed  Google Scholar 

  5. Gorman, J. M., Kent, J. M., Sullivan, G. M. & Coplan, J. D. Neuroanatomical hypothesis of panic disorder, revised. Am. J. Psychiatry 157, 493–505 (2000).

    CAS  Google Scholar 

  6. Coplan, J. D. & Lydiard, R. B. Brain circuits in panic disorder. Biol. Psychiatry 44, 1264–1276 (1998).

    CAS  PubMed  Google Scholar 

  7. Shekhar, A., Sajdyk, T. J., Gehlert, D. R. & Rainnie, D. G. The amygdala, panic disorder, and cardiovascular responses. Ann. N. Y. Acad. Sci. 985, 308–325 (2003).

    CAS  PubMed  Google Scholar 

  8. Kim, J. E., Dager, S. R. & Lyoo, I. K. The role of the amygdala in the pathophysiology of panic disorder: evidence from neuroimaging studies. Biol. Mood Anxiety Disord. 2, 20 (2012).

    PubMed  PubMed Central  Google Scholar 

  9. Yoon, S. et al. Subregional shape alterations in the amygdala in patients with panic disorder. PLoS ONE 11, e0157856 (2016).

    PubMed  PubMed Central  Google Scholar 

  10. Feinstein, J. S., Adolphs, R., Damasio, A. & Tranel, D. The human amygdala and the induction and experience of fear. Curr. Biol. 21, 34–38 (2011).

    CAS  PubMed  Google Scholar 

  11. Khalsa, S. S. et al. Panic anxiety in humans with bilateral amygdala lesions: pharmacological induction via cardiorespiratory interoceptive pathways. J. Neurosci. 36, 3559–3566 (2016).

    CAS  PubMed Central  Google Scholar 

  12. Feinstein, J. S. et al. Fear and panic in humans with bilateral amygdala damage. Nat. Neurosci. 16, 270–272 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Chiang, M. C. et al. Parabrachial complex: a hub for pain and aversion. J. Neurosci. 39, 8225–8230 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Han, S., Soleiman, M. T., Soden, M. E., Zweifel, L. S. & Palmiter, R. D. Elucidating an affective pain circuit that creates a threat memory. Cell 162, 363–374 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Davern, P. J. A role for the lateral parabrachial nucleus in cardiovascular function and fluid homeostasis. Front. Physiol. 5, 436 (2014).

    PubMed  PubMed Central  Google Scholar 

  16. Yahiro, T., Kataoka, N., Nakamura, Y. & Nakamura, K. The lateral parabrachial nucleus, but not the thalamus, mediates thermosensory pathways for behavioural thermoregulation. Sci. Rep. 7, 5031 (2017).

    PubMed  PubMed Central  Google Scholar 

  17. Liu, S. et al. Neural basis of opioid-induced respiratory depression and its rescue. Proc. Natl Acad. Sci. USA 118, e2022134118 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Bourin, M., Baker, G. B. & Bradwejn, J. Neurobiology of panic disorder. J. Psychosom. Res. 44, 163–180 (1998).

    CAS  PubMed  Google Scholar 

  19. Singewald, N. & Sharp, T. Neuroanatomical targets of anxiogenic drugs in the hindbrain as revealed by Fos immunocytochemistry. Neuroscience 98, 759–770 (2000).

    CAS  PubMed  Google Scholar 

  20. Brannan, S. et al. Neuroimaging of cerebral activations and deactivations associated with hypercapnia and hunger for air. Proc. Natl Acad. Sci. USA 98, 2029–2034 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Perna, G., Caldirola, D. & Bellodi, L. Panic disorder: from respiration to the homeostatic brain. Acta Neuropsychiatr. 16, 57–67 (2004).

    PubMed  Google Scholar 

  22. Cardoso, J. C. R., Garcia, M. G. & Power, D. M. Tracing the origins of the pituitary adenylate-cyclase activating polypeptide (PACAP). Front. Neurosci. 14, 366 (2020).

    PubMed  PubMed Central  Google Scholar 

  23. Zhang, L. et al. Behavioral role of PACAP signaling reflects its selective distribution in glutamatergic and GABAergic neuronal subpopulations. eLife 10, e61718 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Stroth, N., Holighaus, Y., Ait-Ali, D. & Eiden, L. E. PACAP: a master regulator of neuroendocrine stress circuits and the cellular stress response. Ann. N. Y. Acad. Sci. 1220, 49–59 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Ressler, K. J. et al. Post-traumatic stress disorder is associated with PACAP and the PAC1 receptor. Nature 470, 492–497 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Cho, J.-H. et al. Pituitary adenylate cyclase-activating polypeptide induces postsynaptically expressed potentiation in the intra-amygdala circuit. J. Neurosci. 32, 14165–14177 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Iurato, S. et al. DNA methylation signatures in panic disorder. Transl. Psychiatry 7, 1287 (2017).

    PubMed  PubMed Central  Google Scholar 

  28. Erhardt, A., Lucae, S., Ising, M., Holsboer, F. & Binder, E. B. Association of PACAP and PACAPR1 gene variants with unipolar depression and panic disorder. Pharmacopsychiatry 46, A89 (2013).

    Google Scholar 

  29. Evans, A. K. & Lowry, C. A. Pharmacology of the β-carboline FG-7142, a partial inverse agonist at the benzodiazepine allosteric site of the GABAA receptor: neurochemical, neurophysiological, and behavioral effects. CNS Drug Rev. 13, 475–501 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Ye, J. & Veinante, P. Cell-type specific parallel circuits in the bed nucleus of the stria terminalis and the central nucleus of the amygdala of the mouse. Brain Struct. Funct. 224, 1067–1095 (2019).

    CAS  PubMed  Google Scholar 

  31. Boucher, M. N., Aktar, M., Braas, K. M., May, V. & Hammack, S. E. Activation of lateral parabrachial nucleus (LPBn) PACAP-expressing projection neurons to the bed nucleus of the stria terminalis (BNST) enhances anxiety-like behavior. J. Mol. Neurosci. 72, 451–458 (2021).

    PubMed  PubMed Central  Google Scholar 

  32. Missig, G. et al. Parabrachial nucleus (PBn) pituitary adenylate cyclase activating polypeptide (PACAP) signaling in the amygdala: implication for the sensory and behavioral effects of pain. Neuropharmacology 86, 38–48 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Ziemann, A. E. et al. The amygdala is a chemosensor that detects carbon dioxide and acidosis to elicit fear behavior. Cell 139, 1012–1021 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Leibold, N. K. et al. CO2 exposure as translational cross-species experimental model for panic. Transl. Psychiatry 6, e885 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Perna, G. et al. Carbon dioxide/oxygen challenge test in panic disorder. Psychiatry Res. 52, 159–171 (1994).

    CAS  Google Scholar 

  36. Dorow, R., Horowski, R., Paschelke, G., Amin, M. & Braestrup, C. Severe anxiety induced by FG 7142, a β-carboline ligand for benzodiazepine receptors. Lancet 322, 98–99 (1983).

    Google Scholar 

  37. McGregor, I. S., Lee, A. M. & Westbrook, R. F. Stress-induced changes in respiratory quotient, energy expenditure and locomotor activity in rats: effects of midazolam. Psychopharmacology 116, 475–482 (1994).

    CAS  PubMed  Google Scholar 

  38. Patki, G. et al. Tempol treatment reduces anxiety-like behaviors induced by multiple anxiogenic drugs in rats. PLoS ONE 10, e0117498 (2015).

    PubMed  PubMed Central  Google Scholar 

  39. Federici, L. M., Caliman, I. F., Fitz, S. D., Shekhar, A. & Johnson, P. L. Select panicogenic drugs and stimuli induce consistent increases in tail skin flushes and decreases in core body temperature. Behav. Pharmacol. 30, 376–382 (2019).

    CAS  PubMed  Google Scholar 

  40. Salchner, P. et al. Airjet and FG-7142-induced Fos expression differs in rats selectively bred for high and low anxiety-related behavior. Neuropharmacology 50, 1048–1058 (2006).

    CAS  PubMed  Google Scholar 

  41. Johnson, P. L. et al. Orexin 1 receptors are a novel target to modulate panic responses and the panic brain network. Physiol. Behav. 107, 733–742 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Palmiter, R. D. The parabrachial nucleus: CGRP neurons function as a general alarm. Trends Neurosci. 41, 280–293 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Kang, S. J. et al. A central alarm system that gates multi-sensory innate threat cues to the amygdala. Cell Rep. 40, 111222 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Iversen, S. D. 5-HT and anxiety. Neuropharmacology 23, 1553–1560 (1984).

    CAS  PubMed  Google Scholar 

  45. Kahn, R. S., Asnis, G. M., Wetzler, S. & Praag, H. M. Neuroendocrine evidence for serotonin receptor hypersensitivity in panic disorder. Psychopharmacology 96, 360–364 (1988).

    CAS  PubMed  Google Scholar 

  46. Kahn, R. S., Wetzler, S., Praag, H. M., Asnis, G. M. & Strauman, T. Behavioral indications for serotonin receptor hypersensitivity in panic disorder. Psychiatry Res. 25, 101–104 (1988).

    CAS  Google Scholar 

  47. Ren, J. et al. Anatomically defined and functionally distinct dorsal raphe serotonin sub-systems. Cell 175, 472–487.e20 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Paul, E. D. & Lowry, C. A. Functional topography of serotonergic systems supports the Deakin/Graeff hypothesis of anxiety and affective disorders. J. Psychopharmacol. 27, 1090–1106 (2013).

    CAS  PubMed  Google Scholar 

  49. Luskin, A. T. et al. A diverse network of pericoerulear neurons control arousal states. Preprint at bioRxiv https://doi.org/10.1101/2022.06.30.498327 (2022).

  50. Borkar, C. D. et al. Sex differences in behavioral responses during a conditioned flight paradigm. Behav. Brain Res. 389, 112623 (2020).

    PubMed  Google Scholar 

  51. Dorofeikova, M. et al. Effects of footshock stress on social behavior and neuronal activation in the medial prefrontal cortex and amygdala of male and female mice. PLoS ONE 18, e0281388 (2023).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Eiden, L. E., Goosens, K. A., Jacobson, K. A., Leggio, L. & Zhang, L. Peptide-liganded G protein-coupled receptors as neurotherapeutics. ACS Pharmacol. Transl. Sci. 3, 190–202 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Spampanato, J., Polepalli, J. & Sah, P. Interneurons in the basolateral amygdala. Neuropharmacology 60, 765–773 (2011).

    CAS  PubMed  Google Scholar 

  54. Gilpin, N. W., Herman, M. A. & Roberto, M. The central amygdala as an integrative hub for anxiety and alcohol use disorders. Biol. Psychiatry 77, 859–869 (2015).

    Google Scholar 

  55. Duvarci, S., Popa, D. & Paré, D. Central amygdala activity during fear conditioning. J. Neurosci. 31, 289–294 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Fogaça, M. V. & Duman, R. S. Cortical GABAergic dysfunction in stress and depression: new insights for therapeutic interventions. Front. Cell. Neurosci. 13, 87 (2019).

    PubMed  PubMed Central  Google Scholar 

  57. Brambilla, P., Perez, J., Barale, F., Schettini, G. & Soares, J. C. GABAergic dysfunction in mood disorders. Mol. Psychiatry 8, 721–737 (2003).

    CAS  PubMed  Google Scholar 

  58. Maller, R. G. & Reiss, S. Anxiety sensitivity in 1984 and panic attacks in 1987. J. Anxiety Disord. 6, 241–247 (1992).

    Google Scholar 

  59. Goodwin, R. D. et al. Panic attack as a risk factor for severe psychopathology. Am. J. Psychiatry 161, 2207–2214 (2004).

    PubMed  Google Scholar 

  60. Coryell, W. et al. Depression and panic attacks: the significance of overlap as reflected in follow-up and family study data. Am. J. Psychiatry 145, 293–300 (1988).

    CAS  PubMed  Google Scholar 

  61. Walker, D. L., Toufexis, D. J. & Davis, M. Role of the bed nucleus of the stria terminalis versus the amygdala in fear, stress, and anxiety. Eur. J. Pharmacol. 463, 199–216 (2003).

    CAS  PubMed  Google Scholar 

  62. Lee, Y. & Davis, M. Role of the hippocampus, the bed nucleus of the stria terminalis, and the amygdala in the excitatory effect of corticotropin-releasing hormone on the acoustic startle reflex. J. Neurosci. 17, 6434–6446 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. Waddell, J., Morris, R. W. & Bouton, M. E. Effects of bed nucleus of the stria terminalis lesions on conditioned anxiety: aversive conditioning with long-duration conditional stimuli and reinstatement of extinguished fear. Behav. Neurosci. 120, 324–336 (2006).

    PubMed  Google Scholar 

  64. Taugher, R. J. et al. The bed nucleus of the stria terminalis is critical for anxiety-related behavior evoked by CO2 and acidosis. J. Neurosci. 34, 10247–10255 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  65. Kaur, S. & Saper, C. B. Neural circuitry underlying waking up to hypercapnia. Front. Neurosci. 13, 401 (2019).

    PubMed  PubMed Central  Google Scholar 

  66. Song, G. & Poon, C.-S. Lateral parabrachial nucleus mediates shortening of expiration and increase of inspiratory drive during hypercapnia. Respir. Physiol. Neurobiol. 165, 9–12 (2009).

    PubMed  Google Scholar 

  67. Breier, A., Charney, D. S. & Heninger, G. R. Agoraphobia with panic attacks: development, diagnostic stability, and course of illness. Arch. Gen. Psychiatry 43, 1029–1036 (1986).

    CAS  PubMed  Google Scholar 

  68. Kessler, R. C. et al. The epidemiology of panic attacks, panic disorder, and agoraphobia in the National Comorbidity Survey replication. Arch. Gen. Psychiatry 63, 415–424 (2006).

    PubMed  PubMed Central  Google Scholar 

  69. Chiang, M. C. et al. Divergent neural pathways emanating from the lateral parabrachial nucleus mediate distinct components of the pain response. Neuron 106, 927–939.e5 (2020).

    CAS  PubMed  Google Scholar 

  70. Sun, L. et al. Parabrachial nucleus circuit governs neuropathic pain-like behavior. Nat. Commun. 11, 5974 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  71. Deakin, J. F. & Graeff, F. G. 5-HT and mechanisms of defence. J. Psychopharmacol. 5, 305–315 (1991).

    CAS  PubMed  Google Scholar 

  72. Graeff, F. G. Serotonin, the periaqueductal gray and panic. Neurosci. Biobehav. Rev. 28, 239–259 (2004).

    CAS  PubMed  Google Scholar 

  73. Pobbe, R. L. H., Zangrossi, H., Blanchard, D. C. & Blanchard, R. J. Involvement of dorsal raphe nucleus and dorsal periaqueductal gray 5-HT receptors in the modulation of mouse defensive behaviors. Eur. Neuropsychopharmacol. 21, 306–315 (2011).

    CAS  PubMed  Google Scholar 

  74. Bago, M. & Dean, C. Sympathoinhibition from ventrolateral periaqueductal gray mediated by 5-HT1A receptors in the RVLM. Am. J. Physiol. 280, R976–R984 (2001).

    CAS  Google Scholar 

  75. Teissier, A. et al. Activity of raphé serotonergic neurons controls emotional behaviors. Cell Rep. 13, 1965–1976 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  76. Correia, P. A. et al. Transient inhibition and long-term facilitation of locomotion by phasic optogenetic activation of serotonin neurons. eLife 6, e20975 (2017).

    PubMed  PubMed Central  Google Scholar 

  77. Seo, C. et al. Intense threat switches dorsal raphe serotonin neurons to a paradoxical operational mode. Science 363, 538–542 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  78. Walsh, J. J. et al. 5-HT release in nucleus accumbens rescues social deficits in mouse autism model. Nature 560, 589–594 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  79. Cathala, A. et al. Serotonin2B receptor blockade in the rat dorsal raphe nucleus suppresses cocaine-induced hyperlocomotion through an opposite control of mesocortical and mesoaccumbens dopamine pathways. Neuropharmacology 180, 108309 (2020).

    CAS  PubMed  Google Scholar 

  80. Kusljic, S. & Van Den Buuse, M. Differential role of serotonin projections from the dorsal and median raphe nuclei in phencyclidine-induced hyperlocomotion and fos-like immunoreactivity in rats. Synapse 66, 885–892 (2012).

    CAS  PubMed  Google Scholar 

  81. Sukamoto, T., Yamamoto, T., Watanabe, S. & Ueki, S. Cardiovascular responses to centrally administered serotonin in conscious normotensive and spontaneously hypertensive rats. Eur. J. Pharmacol. 100, 173–179 (1984).

    CAS  PubMed  Google Scholar 

  82. Gradin, K., Qadri, F., Nomikos, G. G., Hillegaart, V. & Svensson, T. H. Substance P injection into the dorsal raphe increases blood pressure and serotonin release in hippocampus of conscious rats. Eur. J. Pharmacol. 218, 363–367 (1992).

    CAS  PubMed  Google Scholar 

  83. Lovick, T. A. Influence of the dorsal and median raphe nuclei on neurons in the periaqueductal gray matter: role of 5-hydroxytryptamine. Neuroscience 59, 993–1000 (1994).

    CAS  PubMed  Google Scholar 

  84. Courtney, N. A. & Ford, C. P. Mechanisms of 5-HT1A receptor-mediated transmission in dorsal raphe serotonin neurons. J. Physiol. 594, 953–965 (2016).

    CAS  PubMed  Google Scholar 

  85. Gantz, S. C., Levitt, E. S., Llamosas, N., Neve, K. A. & Williams, J. T. Depression of serotonin synaptic transmission by the dopamine precursor l-DOPA. Cell Rep. 12, 944–954 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  86. Perna, G., Schruers, K., Alciati, A. & Caldirola, D. Novel investigational therapeutics for panic disorder. Expert Opin. Investig. Drugs 24, 491–505 (2015).

    CAS  PubMed  Google Scholar 

  87. Kaur, S. et al. Role of serotonergic dorsal raphe neurons in hypercapnia-induced arousals. Nat. Commun. 11, 2769 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  88. Cho, H.-Y., Kim, M. & Han, J.-H. Specific disruption of contextual memory recall by sparse additional activity in the dentate gyrus. Neurobiol. Learn. Mem. 145, 190–198 (2017).

    PubMed  Google Scholar 

  89. Jeong, Y. et al. Synaptic plasticity-dependent competition rule influences memory formation. Nat. Commun. 12, 3915 (2021).

    CAS  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank members of the Han laboratory for critical discussion of the paper and D. O’Keefe for critical input on the manuscript. We also thank S. Park for proofreading the response letter to the reviewers. The study was funded by a National Institutes of Mental Health Biobehavioral Research Award for Innovative New Scientists (BRAINS) grant 1R01MH116203 (S.H.) and a Bridge to Independence award from the Simons Foundation Autism Research Initiative SFARI no. 388708 (S.H.).

Author information

Author notes

  1. Jong-Hyun Kim

    Present address: Center for Cognition and Sociality, Institute for Basic Science, Daejeon, Republic of Korea

  2. These authors contributed equally: Sukjae J. Kang, Jong-Hyun Kim.

Authors and Affiliations

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

    Sukjae J. Kang, Jong-Hyun Kim, Dong-Il Kim, Benjamin Z. Roberts & Sung Han

  2. Neuroscience Graduate Program, University of California San Diego, La Jolla, CA, USA

    Benjamin Z. Roberts & Sung Han

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

    Sung Han

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

    Sung Han

Authors
  1. Sukjae J. Kang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jong-Hyun Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Dong-Il Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Benjamin Z. Roberts
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sung Han
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

S.H., J.-H.K. and S.J.K. conceptualized the study. S.H., J.-H.K. and S.J.K. developed the methodology. J.-H.K. and S.J.K. performed the investigations and visualizations. S.H. acquired funding. D.-I.K. provided viruses. S.H., J.-H.K., S.J.K. and B.Z.R. wrote the manuscript.

Corresponding author

Correspondence to Sung Han.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Neuroscience thanks Kerry Ressler and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

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

Extended data

Extended Data Fig. 1 Projections from PACAPPBL neurons.

a, Schematic and histological confirmation of Cre-dependent expression of EYFP in the PBL of an Adcyap1Cre/+ mouse. Scale bars: 100 µm. b, Representative images of the output regions of PACAPPBL neurons. Scale bars: 100 µm. Injections were repeated on three mice with similar results.

Extended Data Fig. 2 Behavioral changes of the PACAPPBL→DR::GCaMP6s mice during panicogenic conditions.

a, Heat map and distance traveled before- (normal air) and after CO2 exposure. Paired two-sided t-test, P < 0.001. n = 7 mice. b, Heat map and distance traveled following FG-7142 or control injection. Repeated measure two-way ANOVA with Sidak’s multiple comparisons test. n = 7 mice. Data are presented as the mean ± SEM; see also Supplementary Table 3 for statistical details. *P < 0.05, **P < 0.01.

Source data

Extended Data Fig. 3 Calcium activity changes in PACAPPBL→DR neurons do not respond as a general multi-modal aversive circuit.

a, Average calcium trace during “Conditioning” phase of the fear conditioning test. Blue shading indicates “Tone ON” periods and orange arrows indicate when the foot shock was given. n = 3 mice. b, Average calcium trace during the looming test with corresponding AUC analysis. Pink shading indicates the 2-s looming exposure. Repeated measure one-way ANOVA with Sidak’s multiple comparisons test, F (2, 6) = 240.127, P < 0.001. n = 4 mice. Data are presented as the mean ± SEM; see also Supplementary Table 3 for statistical details. **P < 0.01.

Source data

Extended Data Fig. 4 Calcium activity changes in PACAPPBL→DR neurons during anxiogenic conditions.

a, Schematic of the elevated platform test. b, Average calcium trace during elevated platform assessment, and corresponding area under curve (AUC) analysis. Paired two-sided t-test, P = 0.0017. n = 4 mice. c, Schematic of the elevated plus maze. d, Average calcium trace during the seconds immediately preceding and following open arm entry and exit, with corresponding AUC analysis. Paired two-sided t-test, P < 0.0001. n = 4 mice. e, Calcium traces from individual animals. Pink shading indicates when mice were in the open arm. n = 4 mice. Data are presented as the mean ± SEM; see also Supplementary Table 3 for statistical details. **P < 0.01, ***P < 0.001.

Source data

Extended Data Fig. 5 Inputs to PACAPPBL→DR neurons.

a, Schematics of Cre- and Flp-dependent retrograde tracing in PBL of an Adcyap1Cre/+ mouse for identification of inputs to PACAPPBL→DR neurons. b, Histology of TVA and RVdG expression in PBL. Scale bars: 50 µm. Injections were repeated on three mice with similar results. c, Representative images of cTRIO tracing in BNST and CeA. Scale bars: 50 µm. Injections were repeated on three mice with similar results. d, Schematic of inputs to PACAPPBL→DR neurons. Repeated measure one-way ANOVA with Sidak’s multiple comparisons test, F (4, 8) = 21.871, P = 0.0073. n = 3 mice. e,f, Histological images of RVdG and VGAT expression, and graphical representation of the percentage of VGAT colocalization in BNST (e) and CeA (f). Scale bars: 50 µm. n = 3 mice. Data are presented as the mean ± SEM; see also Supplementary Table 3 for counting and statistical details.

Extended Data Fig. 6 Inhibition of PACAPPBL→DR neurons attenuates panic-like symptoms.

a, Distance traveled before (normal air) and after CO2 exposure during chemogenetic inhibition of PACAPPBL→DR neurons. Mixed-effects analysis with Sidak’s multiple comparisons test. n = 9 control mice, n = 6 hM4Di mice. b,c, Distance traveled (b, repeated measures two-way ANOVA with Sidak’s multiple comparisons test) and velocity (c, repeated measures two-way ANOVA with Sidak’s multiple comparisons test) after FG-7142 injection during chemogenetic inhibition of PACAPPBL→DR neurons. n = 9 control mice, n = 5 hM4Di mice. Data are presented as the mean ± SEM; see also Supplementary Table 3 for statistical details. *P < 0.05, **P < 0.01.

Source data

Extended Data Fig. 7 PAC1RDR neurons are monosynaptically connected with PACAPPBL neurons.

a, Single channel histological images of Fig.7g. Presynaptic PACAPPBL neurons are also VGluT2 (Slc17a6) positive. b, Single channel histological images of Fig.7h. Presynaptic PACAPPBL neurons do not overlap with VGAT (Slc32a1). c, Histological images of DR after perfusing the retrograde rabies tracing mice. TVA (green), RVdG (red). Starter cells are observable. d, Representative images of presynaptic cells (RVdG, red) in PBL. Scale bars: 100 µm. This experiment was repeated on three mice with similar results.

Extended Data Fig. 8 Inhibition of PAC1RDR neurons blocks panic-like symptoms.

a, Heat map of mouse activity before (air) and after CO2 exposure during chemogenetic inhibition of PAC1RDR neurons. n = 7 mice per group. b,c, Heat map of mouse activity (b) and velocity (c, mixed-effects analysis with Sidak’s multiple comparisons test and adjusted P values) after FG-7142 injection during chemogenetic inhibition of PAC1RDR neurons. n = 7 mice per group. Data are presented as the mean ± SEM; see also Supplementary Table 3 for statistical details. **P < 0.01, ***P < 0.001.

Source data

Extended Data Fig. 9 The majority of PAC1R (Adcyap1r1) neurons are SERT-positive.

a, Representative in situ hybridization images of PAC1R- (Adcyap1r1) and SERT-positive neurons in the DR (left). b, Graphical representation of percentages of fluorescent neurons. n = 7 mice. Scale bars: 50 µm. Data are presented as the mean ± SEM.; see also Supplementary Table 2 for counting details.

Extended Data Fig. 10 Projection targets of PAC1RDR neurons.

a, Histological images of output regions from PAC1RDR neurons expressing ChR2. Abbreviations: lateral septal nucleus (LS), BNST, CeA, paraventricular nucleus of the thalamus (PVT), nucleus of reuniens (RE), paraventricular hypothalamic nucleus (PVH), lateral hypothalamus (LHA), dorsomedial nucleus of the hypothalamus (DMH), lateral geniculate complex (LGd), subparafascicular nucleus parviceullar part (SPFp), peripeduncular nucleus (PP), substantia nigra compact part (SNc), ventral tegmental area (VTA), ifterfascicular nucleus raphe (IF), rostral linear uncleus raphe (RL), Peri-locus ceruleus (Peri-LC), LC, nucleus raphe magnus (RM), and pontine reticular nucleus (PRN). Scale bars: 100 µm. n = 5 mice.

Supplementary information

Supplementary Information

Supplementary Tables 1–3.

Reporting Summary

Supplementary Video 1

Photostimulation of PACAPPBL→DR neurons induced immediate panic-like behaviors. ChR2 was expressed in PBL of an Adcyap1Cre/+ mouse Cre- and Flp-dependently for optogenetic activation of PACAPPBL→DR neurons. The mouse immediately immobilized during the 20-Hz photostimulation.

Supplementary Video 2

Photostimulation of PACAPPBL→DR neurons in the control group has no effect. EYFP was expressed in the PBL of an Adcyap1Cre/+ mouse Cre- and Flp-dependently as a control for optogenetic activation of PACAPPBL→DR neurons. The mouse showed no behavioral change during photostimulation.

Supplementary Video 3

Optogenetic conditioning of PACAPPBL→DR neurons did not form contextual fear memory. Optogenetically conditioned PACAPPBL→DR::ChR2 mice did not show freezing behaviors during the context test.

Supplementary Video 4

Optogenetic conditioning of PACAPPBL→DR neurons did not form cued fear memory. Optogenetically conditioned PACAPPBL→DR::ChR2 mice did not show freezing behaviors during the cue test.

Supplementary Video 5

Photostimulation of PACAPPBL→DR neurons induced tail-rattling in the initial phase of stimulation. Optogenetic 20-Hz photostimulation of PACAPPBL→DR::ChR2 mice sometimes induced tail-rattling in the initial phase of the stimulation.

Supplementary Video 6

Photostimulation of PAC1RDR neurons induced immediate immobilization. Optogenetic 40-Hz photostimulation of PAC1RDR::ChR2 mice induced immediate immobilization.

Source data

Source Data Fig. 1

Statistical source data.

Source Data Fig. 2

Statistical source data.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 4

Statistical source data.

Source Data Fig. 5

Statistical source data.

Source Data Fig. 6

Statistical source data.

Source Data Fig. 7

Statistical source data.

Source Data Extended Data Fig. 2

Statistical source data.

Source Data Extended Data Fig. 3

Statistical source data.

Source Data Extended Data Fig. 4

Statistical source data.

Source Data Extended Data Fig. 6

Statistical source data.

Source Data Extended Data Fig. 8

Statistical source data.

Rights and permissions

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

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kang, S.J., Kim, JH., Kim, DI. et al. A pontomesencephalic PACAPergic pathway underlying panic-like behavioral and somatic symptoms in mice. Nat Neurosci 27, 90–101 (2024). https://doi.org/10.1038/s41593-023-01504-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41593-023-01504-3

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing