Abstract
Fear-related disorders (for example, phobias and anxiety) cause a substantial public health problem. To date, studies of the neural basis of fear have mostly focused on the amygdala. Here we identify a molecularly defined amygdala-independent tetra-synaptic pathway for olfaction-evoked innate fear and anxiety in male mice. This pathway starts with inputs from the olfactory bulb mitral and tufted cells to pyramidal neurons in the dorsal peduncular cortex that in turn connect to cholecystokinin-expressing (Cck+) neurons in the superior part of lateral parabrachial nucleus, which project to tachykinin 1-expressing (Tac1+) neurons in the parasubthalamic nucleus. Notably, the identified pathway is specifically involved in odor-driven innate fear. Selective activation of this pathway induces innate fear, while its inhibition suppresses odor-driven innate fear. In addition, the pathway is both necessary and sufficient for stress-induced anxiety-like behaviors. These findings reveal a forebrain-to-hindbrain neural substrate for sensory-triggered fear and anxiety that bypasses the amygdala.
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Source data are provided with this paper.
Code availability
The MATLAB script of the single-cell Ca2+ signal data analysis is available as Supplementary Code 1.
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Acknowledgements
We thank the Core Facilities of Zhejiang University Institute of Neuroscience for technical assistance, the Chuanqi Research and Development Centre of Zhejiang University and the Nanjing Brain Observatory for miniaturized two-photon microscope and data processing services. We also thank the Integration Platform for Clinical and Scientific Research on Psychiatric Disorders of the Affiliated Mental Health Center/Hangzhou Seventh People’s Hospital, Zhejiang University School of Medicine. This work was supported by the STI 2030 Major Projects (no. 2021ZD0202700 to X.-M.L.), the National Natural Science Foundation of China (nos. 82090030, 82090031 and 82288101 to X.-M.L.; 82001186 to H.W.; no. 31900723 to Q.W.; no. 32071022 to S.C.), the Non-profit Central Research Institute Fund of the Chinese Academy of Medical Sciences (no. 2023-PT310-01), the National Natural Science Foundation of China-Guangdong Joint Fund (no. U20A6005 to H.L.) and the Projects for Hangzhou Medical Disciplines of Excellence & Key Project for Hangzhou Medical Disciplines.
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H.W. and X.-M.L. initiated and designed the research and wrote the manuscript. H.W., Q.W., L.C. and X.F. performed all the experiments and analyzed the results. P.D. helped analyze the microendoscopic Ca2+ imaging results. L.T. and L.L. assisted with the histology and behavior experiments. H.L., S.C. and H.H. helped collect the fiber photometry data and interpret the results. P.C. contributed to the discussion of the results. X.-M.L. supervised the entire project.
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Extended data
Extended Data Fig. 1 Whole brain Fos expression, activities of DP exposed to different odors, glomerular tracing from serial coronal sections of MOB, and DP-projection mitral cells in MOB.
a, Typical images of taCasp3 induced cell apoptosis in BLA and CeA, CoA and MeA. b, Average escape speed curves (before and after exiting area 1 within 1 s, dotted lines indicate the point when mice exit from area 1). c, Typical image of Fos expression in OB, amygdala, and parabrachial nucleus. d, Quantification of Fos expression throughout the brain. e, Relative fold change of Fos expression throughout the brain. f, Typical image of Fos expressing neurons stained with CamkII, and quantification of co-expression ratio. g, Typical images of Fos expression in DP of mice exposed to different odors. h, Typical image of retrograde virus mixed with CTB injection in DP. i & j, For each coronal section, labeled glomeruli (in red) and unlabeled glomeruli (in white, identified by DAPI staining in blue) were individually traced in Adobe Illustrator. k, Example of glomerular tracing from serial coronal sections of MOB. l, Typical examples of trans-synaptically labelled mitral cells. Scale bar, 50 μm. m, Typical image of retrograde labeled neurons in MOB co-expressed with tdT and Slc17a7, and quantification of co-expression ratio. n, Typical image of retrograde labeled neurons in MOB co-expressed with tdT and Pcdh21, and quantification of co-expression ratio. o, Schematic and typical image for retrograde virus mixed with CTB injection in PrL and IL, and no tdT-labeled neurons were observed in MOB. Scale bar, 200 μm; Zoom, 50 μm. For (d) and (e), n = 5 brain slices from 3 mice for each group; For (f), (m), and (n), n = 6 brain slices from 3 mice. All statistical tests are two-tailed. Unpaired t test for (d). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; N.S., not significant. Data are presented as mean ± s.e.m. See Extended Source Data Fig. 1 for more detailed statistics.
Extended Data Fig. 2 Virus functional verification, photostimulation of CoA in flight-to-shelter assay, and activation of DP induced autonomic system changes.
a & b, Typical images of taCasp3-induced apoptosis in DP (a) and CoA (b), and summarized data for number of neurons. Scale bar, 200 μm. c, Average escape speed curves (before and after exiting area 1 within 1 s, dotted line indicates the point when mice exit from area 1). d, Schematic of virus injection and typical images of ChR2-EYFP expression in DP and CoA. Scale bar, 200 μm. e & f, Representative traces of inward currents, action potential firing (e), and quantification of firing rate (f) evoked by optogenetic stimulation in acute brain slices containing DP. g-j, Representative trajectory chart (g), escape speed (h), escape percentage (i), and exploration time in areas A and C of CoA activation (j). k & l, Summarized data of normalized pupil size (k) and heart rate (l) of DP activation. For (a) and (b), n = 6 brain slices from 3 mice for each group; For (f), n = 3 patched neurons; For (h)-(l), n = 8 mice per group. All statistical tests are two-tailed. Unpaired t test for (a), (b), (i), and (j); A repeated two-way ANOVA measures with bonferroni post hoc test for (h); A repeated one-way ANOVA measures with bonferroni post hoc test for (k) and (l). **p < 0.01; ***p < 0.001; ****p < 0.0001; N.S., not significant. Data are presented as mean ± s.e.m. See Extended Source Data Fig. 2 for more detailed statistics.
Extended Data Fig. 3 Virus functional verification, and comparation of opto-silencing of MOB→DP and MOB→CoA circuit for innate fear.
a, Optogenetic inhibition of MOB → OP / MOB→CoA pathway and representative images of NpHR-EYFP expression in MOB. b, Representative traces of optogenetic inhibitory effects of NpHR-expressing virus. c, Axonal terminals and fiber tracks in DP and CoA. d, Schematic of optogenetic inhibition of open field test. e & f, Average speed of opto-silencing of MOB → DP (e) and MOB→CoA (f) pathway for open field test. g, Schematic of optogenetic inhibition of TMT test. h, Representative trajectory chart of Control and MOB → DP pathway opto-silencing mice exposed to TMT. i-n, Average escape speed curves (i, dotted lines indicate the point when mice exit from area 1), escape speed (j), escape percentage (k), time in area 1 (l), aversion index (m), and freezing time percentage (n). o, Representative trajectory chart of Control and MOB→CoA pathway opto-silencing mice exposed to TMT. p-u, Average escape speed curves (p, dotted lines indicate the point when mice exit from area 1), escape speed (q), escape percentage (r), time in area 1 (s), aversion index (t), and freezing time percentage (u). v & w, Retro-AAV virus injection (v), representative images of EGFP and mCherry expression in MOB, and co-expression ratio quantification (w). Some merged yellow dotted substances were spontaneous tissue fluorescence. x, Representative trajectory chart of CoA and MeA lesion with laser OFF mice for day 1. y, Average escape speed curves (dotted lines indicate the point when mice exit from area 1). For all the representative images (a), (c), and (w), Scale bar, 200 μm; Zoom, 50 μm. For (e), (f), (j)-(n), and (q)-(u), n = 12 mice per group; For (w), n = 12 brain slices from 3 mice. All statistical tests are two-tailed. A repeated two-way ANOVA measures with bonferroni post hoc test for (e), (f), (j)-(n), and (q)-(u). *p < 0.05; ***p < 0.001; ****p < 0.0001; N.S., not significant. Data are presented as mean ± s.e.m. See Extended Source Data Fig. 3 for more detailed statistics.
Extended Data Fig. 4 Detailed features of MOBSlc17a7+→DPCamk2a+→anterior PBNslCck+→PSThTac1+ pathway.
a, Typical image of axonal terminals in posterior PBNsl and quantification of fluorescence intensity. b, Typical images of retrograde virus mixed with CTB injection in anterior and posterior PBNsl. c, Typical image of tdT-labeled neurons in DP from posterior PBNsl. d, Typical image of tdT co-labeled with Cck in posterior PBNsl, and percentage quantification. e, Typical image of starter cells in posterior PBNsl. f, DsRed-expressing neurons in DP from posterior PBNsl and quantification of labeled cell number. g, Schematic of axonal terminal staining. h, Typical images of axonal terminals from DP overlapping with Cck, Tac1, and CGRP in anterior and posterior PBNsl. i, Schematic of crossing Cck-Cre and Rosa26-tdTomato reporter mice, typical image of tdT-labeled neurons stained with Cck, and co-expression ratio quantification. j-n, Typical images of tdT-labeled neurons stained with Slc17a6 (j), Gad2 (k), CGRP (l), Tac1 (m), and CaMKII (n), and co-expression ratio quantification. o-q, Representative traces and summarized data for light-evoked EPSCs recorded in anterior PBNslCck+ neurons (o), DP (p), and PSTh (q). r-t, Typical images of tdT-labeled neurons stained with CaMKII (r), Gad2 (s), and Crf (t), and quantification of co-expression ratio. u, Schematic of axonal terminal staining and typical image of axonal terminals from anterior PBNsl overlapping with Tac1 in PSTh. v, Typical image of EYFP-labeled neurons stained with Cck and co-expression ratio quantification. w, Representative traces and summarized data for light-evoked EPSCs recorded in PSTh. For all the typical images, Scale bar, 100 μm. All statistical tests are two-tailed. For (a), (d), (f), (i)-(n), (t), and (v), n = 6 brain slices from 3 mice; (o)-(q), n = 12 patched neurons from 3 mice; For (r) and (s), n = 8 brain slices from 3 mice; For (w), n = 11 patched neurons from 3 mice. Unpaired t test for (a) and (f); A repeated one-way ANOVA measures with bonferroni post hoc test for (o)-(q), and (w). **p < 0.01; ***p < 0.001; ****p < 0.0001. Data are presented as mean ± s.e.m. See Extended Source Data Fig. 4 for more detailed statistics.
Extended Data Fig. 5 DP→anterior PBNsl circuit mediates odor-driven innate fear but not visually and auditorily evoked innate fear.
a, Typical GCaMP6s fluorescence and fiber track in DP. Scale bar, 100 μm. b, Average calcium signals and heatmap in mice exposed to peanut oil and low concentration of TMT. c, Multicurve presentation and area under the curve (AUC) of calcium activity in DP. d & e, Representative traces of mice receiving two repeated TMT stimuli (d) and peak delta F/F (e). f, Schematic of olfactory-deprived test and summarized data for latency to find food. g-k, Representative trajectory chart (g), escape percentage (h), time in area 1 (i), aversion index (j), and freezing time percentage (k). l, Average calcium signals of control and olfactory-deprived mice exposed to TMT, peak delta F/F, and AUC of calcium activity. m, Schematic of looming test and average calcium signals of mice under escape, frozen, and unresponsive conditions. n, Peak delta F/F and AUC of calcium activity for looming test. o, Schematic of auditorily evoked innate fear and average calcium signals of mice under escape, frozen, and unresponsive conditions. p, Peak delta F/F and AUC of calcium activity for auditorily evoked innate fear. q, Average speed of opto-silencing of DP→PBNsl pathway for open field test. r, Representative trajectory chart of DP→PBNsl pathway for day 1 laser OFF tests. s, Average escape speed curves (dotted lines indicate the point when mice exit from area 1). t, Typical images of pupil, average curves and summarized data of normalized pupil size. u, Average curves and summarized data of normalized heart rate. All statistical tests are two-tailed. For (c), (l), (n), and (p), n = 10 trials from 5 mice; For (e), n = 7 mice from 14 trials; For (f) and (h)-(k), n = 10 mice per group; For (q), n = 12 mice per group; For (t), n = 13 mice per group; For (u), n = 12 mice per group. One-way ANOVA with bonferroni post hoc test for (c), (n), and (p). Paired t test for (e); Unpaired t test for (f), and (h)-(l); A repeated two-way ANOVA measures with bonferroni post hoc test for (q); A repeated one-way ANOVA measures with bonferroni post hoc test for (t) and (u). *p < 0.05; **p < 0.01; ****p < 0.0001; N.S., not significant. Data are presented as mean ± s.e.m. See Extended Source Data Fig. 5 for more detailed statistics.
Extended Data Fig. 6 Anterior rather than posterior PBNslCck+ neurons contribute to odor-driven innate fear but not in visually and auditorily evoked innate fear.
a, Typical images of tdT-labeled neurons co-expressed with Fos and co-labeled percentage in anterior PBNsl. b, Typical images of Fos expression in posterior PBNsl and quantification of Fos number. c, Typical image of Fos co-labeled with tdT in posterior PBNsl, and percentage quantification. d, Schematic of virus injection and fiber implant, typical image of anterior PBNslCck+ neurons expressing GCaMP6s, and representative traces of calcium signals. e-h, Schematic of virus injection (e), average calcium signals / heatmap (f), multicurve presentation (g), peak delta F/F and AUC of calcium activity of anterior PBNslCck+ neurons (h) in mice exposed to TMT and water and DP-lesioned mice with TMT treatment. i, Schematic of virus injection, typical image of posterior PBNslCck+ neurons expressing GCaMP6s, and average calcium signals of TMT stimuli. j & l, Average calcium signals of mice under escape, frozen, and unresponsive conditions for looming (j) and auditorily (l) evoked innate fear. k & m, Peak delta F/F and AUC of calcium activity for looming (k) and auditorily (m) evoked innate fear. n, Schematic of virus injection, typical image of anterior PBNslGad2+ neurons expressing GCaMP6s, and average calcium signals. o, Heatmap of calcium transients of natural fox urine, male mice urine and peanut oil stimulations. p, Percentage of neurons excited (red), inhibited (blue), or with no response to odor (gray). q, Anterior to posterior brain slices from mouse following GCaMP6s virus injection into anterior PBNsl. r & s, Overlay of GCaMP6s virus expression (green), fibers (blue) and lens (yellow) positions. For all the typical images, Scale bar, 100 μm; Zoom, 50 μm. All statistical tests are two-tailed. For (a)-(c), n = 5 brain slices from 3 mice per group; (h), (k), and (m), n = 10 trials from 5 mice. Unpaired t test for (a) and (b); One-way ANOVA with bonferroni post hoc test for (h), (k), and (m). *p < 0.05; **p < 0.01; ****p < 0.0001; N.S., not significant. Data are presented as mean ± s.e.m. See Extended Source Data Fig. 6 for more detailed statistics.
Extended Data Fig. 7 Effects of anterior PBNslCck+ neuron ablation on TMT-induced innate fear and virus targeting.
a, Average speed of opto-silencing of PBNslCck+ neurons for open field test. b, Representative trajectory chart of PBNslCck+ neurons for day 1 laser OFF tests. c, Average escape speed curves (dotted line indicates the point when mice exit from area 1). d, Schematic of virus injection, typical images of taCasp3-induced apoptosis, and number of anterior PBNslCck+ neurons. Scale bar, 100 μm. e–k, Representative trajectory chart (e), average escape speed curves (f), escape speed (g), escape percentage (h), time in area 1 (i), aversion index (j), and freezing time percentage (k). l, Typical images of pupil. m, Average curves and summarized data of normalized pupil size. n, Average curves and summarized data of normalized heart rate. o & p, Anterior to posterior brain slices from mouse following NpHR-EYFP (o) or ChR2-EYFP (p) virus injection into anterior PBNsl and overlay of virus expression. Scale bar, 200 μm. All statistical tests are two-tailed. For (a), n = 12 mice per group; (d), n = 6 brain slices from 3 mice per group; For (g)-(k), n = 11 mice per group; For (m), n = 10 mice for EYFP group / n = 13 mice for ChR2 group; For (n), n = 10 mice for EYFP group / n = 12 mice for ChR2 group. A repeated two-way ANOVA measures with bonferroni post hoc test for (a); Unpaired t test for (d), and (g)-(k). Repeated one-way ANOVA measures with bonferroni post hoc test for (m) and (n). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; N.S., not significant. Data are presented as mean ± s.e.m. See Extended Source Data Fig. 7 for more detailed statistics.
Extended Data Fig. 8 Anterior PBNslCck+→PSTh circuit mediates odor-driven innate fear and PSTh acts as a main downstream target of anterior PBNslCck+ neurons.
a, CTB injection and typical image of CTB targeting in PSTh. Scale bar, 100 μm. b, Typical images of Fos, tdT and CTB co-labeled in anterior PBNsl, and co-expression percentage. Scale bar, 50 μm. c & d, Multicurve presentation (c) and AUC of calcium activity of PSTh-projecting anterior PBNslCck+ neurons (d). e, Representative image of axonal terminal in PSTh with fiber track. Scale bar, 100 μm. f, Average speed of opto-silencing of PBNslCck+→PSTh pathway for open field test. g, Representative trajectory chart of PBNslCck+→PSTh pathway for day 1 laser OFF tests. h, Average escape speed curves (dotted line indicates the point when mice exit from area 1). i, Typical images of pupil. j. Average curves, and summarized data of normalized pupil size. k, Average curves and summarized data of normalized heart rate. l & m, Virus injection (l), typical images of taCasp3-induced apoptosis, and number of neurons in PSTh (m). Scale bar, 100 μm. n-q, Representative trajectory chart (n), escape speed (o), escape percentage (p), and time in areas A and C (q). All statistical tests are two-tailed. For (b) and (m), n = 6 brain slices from 3 mice per group; For (d), n = 10 trials from 5 mice; For (f), n = 12 mice per group; For (j), n = 10 mice for EYFP group / n = 13 mice for ChR2 group; For (k), n = 10 mice for EYFP group / n = 12 mice for ChR2 group; For (o)-(q), n = 8 mice per group. Unpaired t test for (b), (d), (m), (p), and (q); A repeated two-way ANOVA measures with bonferroni post hoc test for (f) and (o); A repeated one-way ANOVA measures with bonferroni post hoc test for (j) and (k). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; N.S., not significant. Data are presented as mean ± s.e.m. See Extended Source Data Fig. 8 for more detailed statistics.
Extended Data Fig. 9 Activities of PSThTac1+ neurons in odor-driven innate fear, virus targeting in PSTh, and downstream target mapping.
a, Typical images of TMT exposure-induced Fos expression in PSTh and quantification of Fos number. Scale bar, 100 μm; Zoom, 50 μm. b, Typical images of tdT-labeled neurons co-expressed with Fos and co-labeled percentage in PSTh. Scale bar, 100 μm; Zoom, 50 μm. c & d, Multicurve presentation (c) and AUC of calcium activity of PSThTac1+ neurons (d). e, Average speed of opto-silencing of PSThTac1+ neurons for open field test. f, Representative trajectory chart of PSThTac1+ neurons for day 1 laser OFF tests. g, Average escape speed curves (dotted lines indicate the point when mice exit from area 1). h, Typical images of pupil. i, Average curves and summarized data of normalized pupil size. j, Average curves and summarized data of normalized heart rate. k & l, Anterior to posterior brain slices from mouse following NpHR-EYFP (k) or ChR2-EYFP (l) virus injection into PSTh and overlay of virus expression. Scale bar, 200 μm. m, EYFP expression in several downstream structures from PSThTac1+ neurons. Scale bar, 100 μm. All statistical tests are two-tailed. For (a), n = 7 brain slices from 3 mice per group; For (b), n = 5 brain slices from 3 mice per group; For (d), n = 10 trials from 5 mice; For (e), n = 12 mice per group; For (i), n = 10 mice for EYFP group / n = 12 mice for ChR2 group; For (j), n = 10 mice for EYFP group / n = 9 mice for ChR2 group. Unpaired t test for (a), (b), and (d); A repeated two-way ANOVA measures with bonferroni post hoc test for (e); A repeated one-way ANOVA measures with bonferroni post hoc test for (i) and (j). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; N.S., not significant. Data are presented as mean ± s.e.m. See Extended Source Data Fig. 9 for more detailed statistics.
Extended Data Fig. 10 Effects of ablation of specific amygdala nuclei on DP→anterior PBNsl→PSTh circuit-triggered innate fear and pain threshold.
a, Average speed of opto-silencing of DP→anterior PBNsl→PSTh pathway for open field test. b, Representative trajectory chart of DP→anterior PBNsl→PSTh pathway for day 1 laser OFF tests. c, Average escape speed curves (dotted lines indicate the point when mice exit from area 1). d, Schematic of virus injection and strategy for optogenetic manipulation of DP→anterior PBNsl→PSTh triple pathway. Symbol “&” means “and”. e, Typical images of pupil. f & g, Average curves (f) and summarized data of normalized pupil size (g). h & i, Average curves (h) and summarized data of normalized heart rate (i) amygdala-ablated mice when triple pathway activated. j & k, Anterior to posterior brain slices from mouse following Cre/Flp-dependent virus expressing NpHR-EYFP (j) or ChR2-EYFP (k) injection into anterior PBNsl and overlay of virus expression. Scale bar, 200 μm. l, Schematic of pain threshold tests including von Frey and Hargreaves test. m & n, Effects of activation of triple pathway on mechanical (m) and thermal (n) thresholds. All statistical tests are two-tailed. For (a), (m), and (n), n = 12 mice per group; For (g), n = 10 mice for EYFP group / 12 mice for ChR2 group and ChR2 + lesion group; For (i), n = 10 mice for EYFP group / 12 mice for ChR2 group and ChR2 + lesion group. A repeated two-way ANOVA measures with bonferroni post hoc test for (a), (m), and (n); A repeated one-way ANOVA measures with bonferroni post hoc test for (g) and (i). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; N.S., not significant. Data are presented as mean ± s.e.m. See Extended Source Data Fig. 10 for more detailed statistics.
Supplementary information
Supplementary Information
Supplementary Tables 1 and 2.
Supplementary Video 1
Behavioral differences of control, DP-lesioned and CoA-lesioned mice exposed to the TMT stimulus.
Supplementary Video 2
Activation of the pyramidal neurons in DP-elicited escape behavior and the CoA activation-induced aversion effect.
Supplementary Video 3
Activation of the pyramidal neurons in DP rapidly enlarged pupil size.
Supplementary Video 4
Axonal terminals from the MOB overlapped with apical dendrites of pyramidal neurons in DP projecting to PBNslCck+ neurons.
Supplementary Video 5
In vivo fiber photometry recordings of the anterior PBNsl-projecting DP neuronal activity for the TMT stimulus.
Supplementary Video 6
In vivo fiber photometry recordings of the anterior PBNslCck+ neuronal activity for the TMT stimulus.
Supplementary Video 7
Microendoscopic Ca2+ imaging of the anterior PBNslCck+ neuronal activity for the TMT stimulus.
Supplementary Video 8
In vivo fiber photometry recordings of PSThTac1+ neuronal activity for the TMT stimulus.
Supplementary Video 9
Activation of the DP→anterior PBNsl→PSTh pathway induced a decrease in the number of active nose pokes paired with sucrose.
Supplementary Code 1
Single-cell Ca2+ signal code.
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Wang, H., Wang, Q., Cui, L. et al. A molecularly defined amygdala-independent tetra-synaptic forebrain-to-hindbrain pathway for odor-driven innate fear and anxiety. Nat Neurosci 27, 514–526 (2024). https://doi.org/10.1038/s41593-023-01562-7
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DOI: https://doi.org/10.1038/s41593-023-01562-7
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