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