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Distinct serotonergic pathways to the amygdala underlie separate behavioral features of anxiety

Nature Neuroscience volume 25, pages 1651–1663 (2022)Cite this article

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Abstract

Anxiety-like behaviors in mice include social avoidance and avoidance of bright spaces. Whether these features are distinctly regulated is unclear. We demonstrate that in mice, social and anxiogenic stimuli, respectively, increase and decrease serotonin (5-HT) levels in basal amygdala (BA). In dorsal raphe nucleus (DRN), 5-HT∩vGluT3 neurons projecting to BA parvalbumin (DRN5-HT∩vGluT3-BAPV) and pyramidal (DRN5-HT∩vGluT3-BAPyr) neurons have distinct intrinsic properties and gene expression and respond to anxiogenic and social stimuli, respectively. Activation of DRN5-HT∩vGluT3→BAPV inhibits 5-HT release via GABAB receptors on serotonergic terminals in BA, inducing social avoidance and avoidance of bright spaces. Activation of DRN5-HT∩vGluT3→BA neurons inhibits two subsets of BAPyr neurons via 5-HT1A receptors (HTR1A) and 5-HT1B receptors (HTR1B). Pharmacological inhibition of HTR1A and HTR1B in BA induces avoidance of bright spaces and social avoidance, respectively. These findings highlight the functional significance of heterogenic inputs from DRN to BA subpopulations in the regulation of separate anxiety-related behaviors.

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Fig. 1: Dynamics of extracellular 5-HT levels in BA and neuronal activity in DRN5-HT neurons under social and anxiogenic stimuli.
Fig. 2: DRN5-HT→BA activity regulates anxiety-related behaviors.
Fig. 3: Intrinsic electrophysiological properties of DRN-BAPyr and DRN-BAPV neurons.
Fig. 4: DRNvGluT3∩5-HT→BAPyr and DRNvGluT3∩5-HT→BAPV pathways differentially regulate social and anxiety-related behaviors.
Fig. 5: DRNvGluT3∩5-HT→BAPV inhibits 5-HT release via GABAB receptors on serotonergic terminals.
Fig. 6: GABAB receptors inhibit 5-HT release in BA.
Fig. 7: DRNvGluT3∩5-HT→BAPyr inhibits a subset of BAPyr neurons via HTR1A.
Fig. 8: HTR1A and HTR1B in BA regulate avoidance of bright spaces and social avoidance/impairments, respectively.

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Data availability

RNA sequencing raw data have been deposited in the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/) with the access number GSE214895. The data that support this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank S. M. Duan (Zhejiang University, China), H. L. Hu (Zhejiang University, China), Z. M. Lu (Zhejiang University, China), T. M. Gao (Southern Medical University, China), Y. H. Cui (Zhejiang University, China), G. P. Feng (Massachusetts Institute of Technology, USA) and all members of X.-M. Li’s Lab for discussions and suggestions on experimental design and manuscript preparation. We thank T. F. Yuan (Shanghai Jiao Tong University, China) for providing the FSCV technology. We thank Z. J. Huang (Cold Spring Harbor Laboratory, USA), H. Miao (Fudan University, China), X. H. Zhang (Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, China), Y. Rao (Capital Medical University, China) and M. M. Luo (National Institute of Biological Sciences, Beijing, China) for providing transgenic mouse lines. We thank the Chuanqi Research and Development Centre of Zhejiang University and the Core Facilities of the Zhejiang University Institute of Neuroscience for technical support. This study was funded by the major programme of the National Natural Science Foundation of China (82090031 and 82090030), Science and Technology Innovation 2030—major project of Brain Science and Brain-Like Research (2021ZD0202702 and 2021ZD0202700), General Programme of the NSFC (31871070), Key-Area Research and Development Program of Guangdong Province (2018B030334001 and 2019B030335001), Key R&D Program of Zhejiang Province (2020C03009), the Fundamental Research Funds for the Central Universities (2021FZZX001-37), CAMS Innovation Fund for Medical Sciences (2019-I2M-5-057) and Innovative and Entrepreneur Team of Zhejiang for the year 2020 biomarker-driven basic and translational research on major brain diseases (2020R01001) to X.-M.L. and Young Scientist Program of the NSFC (82001454) to H.H.

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Authors and Affiliations

  1. Department of Neurobiology and Department of Neurology of Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China

    Xiao-Dan Yu, Yi Zhu, Huiqian Huang, Hsin-Yi Lai & Xiao-Ming Li

  2. NHC and CAMS Key Laboratory of Medical Neurobiology, MOE Frontier Center of Brain Science and Brain-machine Integration, School of Brain Science and Brain Medicine, Zhejiang University, Hangzhou, China

    Xiao-Dan Yu, Yi Zhu, Qi-Xin Sun, Di Zheng, Shi-Ze Xie, Chen-Jie Shen, Jia-Yu Fu & Xiao-Ming Li

  3. Department of Neurology of the Second Affiliated Hospital, Interdisciplinary Institute of Neuroscience and Technology, Key Laboratory of Medical Neurobiology of Zhejiang Province, Zhejiang University School of Medicine, Hangzhou, China

    Xiao-Dan Yu & Hsin-Yi Lai

  4. State Key Laboratory of Membrane Biology, Peking University School of Life Sciences, Beijing, China

    Fei Deng, Jinxia Wan & Yulong Li

  5. Shanghai Key Laboratory of Psychotic Disorders, Shanghai Mental Health Center, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Wankun Gong

  6. College of Biomedical Engineering and Instrument Science, Key Laboratory for Biomedical Engineering of Ministry of Education, Zhejiang University, Hangzhou, China

    Hsin-Yi Lai

  7. Affiliated Mental Health Center & Hangzhou Seventh People’s Hospital, Zhejiang University School of Medicine, Hangzhou, China

    Hsin-Yi Lai

  8. The MOE Key Laboratory of Biosystems Homeostasis & Protection, Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China

    Jin Jin

  9. Center for Brain Science and Brain-Inspired Intelligence, Research Units for Emotion and Emotion Disorders, Chinese Academy of Medical Sciences/Guangdong–Hong Kong–Macao Greater Bay Area Center for Brain Science and Brain-Inspired Intelligence, Guangzhou, China

    Xiao-Ming Li

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Contributions

X.-D.Y. and X.-M.L. conceptualized and designed the experiment. X.-D.Y. performed the experiments and data analyses, including fiber photometry, FSCV, optogenetic, RNA-seq and ex vivo electrophysiological, behavioral, and immunohistochemical experiments. Y.Z., J.-Y.F. and D.Z. helped with the RNA scope analyses. F.D. and J.-X.W. helped with the GRAB5-HT2h signal recordings. Q.-X.S. helped with data analysis and stereotaxic surgeries. C.-J.S. and S.-Z.X. helped with stereotaxic surgeries. W.-K.G. helped with the FSCV recordings. H.Q.H., H.-Y.L., J.J. and Y.-L.L. helped with data analysis. The manuscript was written by X.-D.Y. and X.-M.L. X.-M.L. supervised all phases of the project.

Corresponding author

Correspondence to Xiao-Ming Li.

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Extended data

Extended Data Fig. 1 High Ambient Illumination Induces Anxiety-related Behaviours.

Representative traces and statistics (mean ± s.e.m) of time in open arm (F(2, 57) = 11.13, P < 0.0001; 20–300 Lux, t(57) = 3.563, **P = 0.0015; 20–600 Lux, t(57) = 4.459, ***P = 0.0001; 300–600 Lux, t(57) = 0.8959, P = 0.3741) of EPM (a), time in centre (F(2, 57) = 4.716, P = 0.0127; 20–300 Lux, t(57) = 2.760, *P = 0.0231; 20–600 Lux, t(57) = 2.547, *P = 0.0270; 300–600 Lux, t(57) = 0.2125, P = 0.8324) and total distance (F(2, 57) = 0.6638, P = 0.5188) in OFT (b), latency to feed (F(2, 57) = 22.45, P < 0.0001; 20–300 Lux, t(57) = 0.1996, P = 0.8425; 20–600 Lux, t(57) = 5.900, ****P < 0.0001; 300–600 Lux, t(57) = 5.701, ****P < 0.0001) and total food consumed (F(2, 57) = 0.1877, P = 0.8294) in NSF (c), and interaction time (F(2, 57) = 26.33, P < 0.0001; 20–300 Lux, t(57) = 3.247, **P = 0.002; 20–600 Lux, t(57) = 7.244, ****P < 0.0001; 300–600 Lux, t(57) = 0.3997, ***P = 0.0004) in social interaction test (d) (two-sided RM one-way ANOVA followed by Holm–Sidak correction), sniffing time (F(5, 114) = 18.46, P < 0.0001; S1, 20–300 Lux, t(228) = 4.591, ****P < 0.0001, 20–600 Lux, t(228) = 8.918, ****P < 0.0001, 300–600 Lux, t(228) = 4.327, ***P = 0.0002; S2, 20–300 Lux, t(228) = 1.347, P = 0.5467, 20–600 Lux, t(228) = 5.765, ****P < 0.0001, 300–600 Lux, t(228) =4.419, ***P = 0.0001) and social index (F(2, 57) = 1.358, P = 0.2653; F(2, 57) illumination = 25.31, P < 0.0001; sociability, 20–300 Lux, t(114) = 2.632, **P = 0.0097; 20–600 Lux, t(114) = 5.568, ****P < 0.0001; 300–600 Lux, t(114) = 2.936, **P = 0.0080; social novelty, 20–300 Lux, t(114) = 0.4221, P = 0.6738; 20–600 Lux, t(114) = 3.393, ****P < 0.0001; 300–600 Lux, t(114) = 2.971, **P = 0.0072) in three-chamber test (e; two-sided RM two-way ANOVA followed by Holm–Sidak correction) (n = 20 mice).

Source data

Extended Data Fig. 2 Dynamics of Extracellular 5-HT Level in BA of Female and Male Mice under Social and Anxiogenic Stimuli.

(a, b) Average traces (a, mean ± 95% CI; dash line: start of social interaction) and statistics (b, GFP, n = 5 mice; GRAB5-HT2h, n = 6 mice; two-sided RM two-way ANOVA followed by Holm–Sidak correction, F(1, 9) = 62.69, P < 0.0001; GRAB5-HT2h Baseline versus GRAB5-HT2h Social, ****P < 0.0001; GFP Social versus GRAB5-HT2h Social, ****P < 0.0001) of GFP and GRAB5-HT2h fluorescence under social stimulus in female mice. (c, d) Average traces (c, mean ± 95% CI; dash line: start of ~300 Lux stimulus) and statistics (d, GFP, n = 5 mice; GRAB5-HT2h, n = 6 mice; two-sided RM two-way ANOVA followed by Holm–Sidak correction, F(1, 9) = 23.98, P = 0.0009; GRAB5-HT2h 20 versus 300 Lux, ****P < 0.0001; 300 Lux, GFP versus GRAB5-HT2h, ****P < 0.0001) of GFP and GRAB5-HT2h fluorescence under ~300 Lux stimulus in female mice. (e, f) Average traces (e, mean ± 95% CI; dash line: start of social interaction) and statistics (f, mean ± SEM; n = 6 female and 7 male mice; two-sided unpaired t test; t(11) = 0.2916, P = 0.7760) of GRAB5-HT2h fluorescence in male and female mice under social stimulus. (g, h) Average traces (g, mean ± 95% CI; dash line: start of ~300 Lux stimulus) and statistics (h, mean ± SEM; n = 6 female and 6 male mice; two-sided unpaired t test; t(10) = 0.5323, P = 0.6062) of GRAB5-HT2h fluorescence in male and female mice under ~300 Lux stimulus.

Extended Data Fig. 3 FSCV Recordings of the Extracellular 5-HT Dynamics in BA.

(a-d) Representative pseudo colour plots (a), voltammograms (b), traces (c) and statistics (d, n = 9 events from 3 mice; two-sided paired t test; t(8) = 7.512, ****P < 0.0001) of changes of 5-HT under social stimulus, triangles in a represent time point of voltammograms in b (e-g) Representative pseudo colour plots (e) and voltammograms (f), traces (g) and statistics (h, n = 13 events from 3 mice; two-sided paired t test; t(12) = 4.495, ***P = 0.0007) of changes in 5-HT under ~300 Lux stimulus, triangles in e represent time point of voltammograms in f (i) Voltage applied to carbon fiber electrodes during FSCV recording of 5-HT. (j-m) Schematic of virus injection (j, l) and 5-HT cyclic voltammograms (k, m, background subtracted) after opto-stimulation of DRN5-HT neurons.

Extended Data Fig. 4 Differential Expressions of Genes in DRN-BAPyr and DRN-BAPV Neurons.

(a-c) FPKM of genes related to synthesis and secretion of neurotransmitters (a), ion channels (b) and neurotransmitter release (c) in DRN-BAPyr and DRN-BAPV neurons.

Extended Data Fig. 5 New Environment Exposure Induces Anxiety-like Behaviours and Increases cFos Expression in DRN-BAPV but not DRN-BAPyr Neurons.

(a) Schematics of new environment pre-exposure. (b, c) Representative traces and statistics (mean ± SEM; n = 13 mice; two-sided unpaired t test) of time in open arm (t(24) = 4.616, ***P = 0.0001) in EPM (b) and time in centre (t(24) = 2.303, *P = 0.0302) and total distance (t(24) = 0.1184, P = 0.9067) in OFT (c) of home cage (HC) or non-home cage (NHC) pre-exposure mice. (d, e) Representative images (d) and statistics (e, mean ± SEM; n = 5, 6, 6, and 6 mice; two-sided RM one-way ANOVA, F(3, 19) = 67.69, P < 0.0001; HC, vGluT2 versus PV, t(19) = 4.052, **P = 0.0016; PV, NC versus NHC, t(19) = 8.32, ****P < 0.0001) of cFos expression in RV-dsRed-positive neurons of PV-ires-cre and vGluT2-ires-cre mice in DRN exposed to home cage (HC) or non-home cage (NHC), arrows represent co-expression of cFos and dsRed, triangles represent non-cFos expression in dsRed+ neurons, scale bar: 50 μm.

Extended Data Fig. 6 BAPV Neurons are Activated under Anxiogenic but not Social Stimulus.

Schematics of anxiogenic and social stimuli (a, top), representative images (a, bottom) and statistics (b, mean ± SEM; n = 6, 8, 8 and 8 mice; F(3, 26) = 10.84, P < 0.0001; two-sided RM one-way ANOVA; Non-social, 20 versus 300 Lux, t(26) = 3.543, **P = 0.0046; Social, 20 Lux versus 300 Lux, t(26) = 4.468, ***P = 0.0008) of cFos expression in BAPV neurons, arrows: co-expression of cFos and PV, triangles: non-cFos expression in BAPV neurons, scale bar: 50 μm.

Extended Data Fig. 7 Social Stimulus Decrease cFos Expression in BAPyr neurons.

Schematics of anxiogenic and social stimuli (a, top), representative images (a, bottom) and statistics (b, mean ± SEM; n = 8 mice; F(3, 28) = 23.54, P < 0.0001; two-sided RM one-way ANOVA; 20 Lux, Non-social versus Social, t(28) = 3.389, **P = 0.0063; 300 Lux, Non-social versus Social, t(28) = 5.33, ****P < 0.0001; Non-social, 20 versus 300 Lux, t(28) 4.89, ***P = 0.0001; Social, 20 versus 300 Lux, t(28) = 2.949, *P = 0.0127) of cFos expression in BAPyr neurons, arrows: co-expression of cFos and PV, triangles: non-cFos expression in BAPyr neurons, scale bar: 50 μm.

Extended Data Fig. 8 DRN5-HT Neurons Inhibit a Subset of BAPyr Neurons via HTR1A but not GABA.

(a) Schematic of virus injection and electrophysiological recordings in BAPyr neurons under 5-ms light pulse stimulation of serotonergic terminals from DRN. (b) Representative trace of BAPyr neurons using a high chloride internal solution under 5-ms light pulse in the presence of TTX + 4AP. (c) Schematic of virus injection and electrophysiological recordings in BAPyr neurons under 55-Hz light train stimulation of serotonergic terminals from DRN. (d) Proportion of neurons with (responsive) or without (non-responsive) slow oIPSCs in BAPyr neurons during 55-Hz light train. (e) Diagrams of distribution of responsive (solid circles) and non-responsive (hollow circles) BAPyr neurons. (f) Representative traces (left, shadow: opto-activation) and statistics (right; n = 9 neurons from 3 mice; two-sided RM one-way ANOVA; F(8,16) = 27.17, P < 0.0001; ACSF versus CGP54626, t(8) = 1.132, P = 0.2904; CGP54626 versus WAY100635, t(8) = 5.815, **P = 0.0012; CGP54626 versus WAY100635, t(8) = 5.495, **P = 0.0012) of oIPSCs before and after application of CGP54626 (GABAB receptor antagonist, 1 μM) and WAY100635. (g) Representative traces (left, shadow: opto-activation) statistics (right; n = 6 neurons from 4 mice; two-sided paired t test; t(5) = 1.772, P = 0.1366) of oIPSCs before and after application of NAS-181 (1 μM).

Extended Data Fig. 9 Activation of Serotonergic Terminals in BA Modulates Glutamate Transmission to HTR1A− Pyr Neurons, but not PV or HTR1A+ Neurons, via HTR1B.

(a-c) Schematic (a), representative traces (b) and statistics (c, mean ± SEM and individual data; n = 21 neurons from 4 mice; two-sided RM one-way ANOVA, F(20, 180) = 9.516, P < 0.0001) of the amplitude of oEPSCs in BAPV neurons that showed glutamatergic responses (R-BAPV neurons) in response to opto-stimulation. (d-f) Schematic (d), representative traces (e), cumulative frequency curves of inter-spike-interval (f, outside), and statistics (f, inside) of frequency (f, left; t(16) = 0.9465, P = 0.3580) and amplitude (f, right; t(16) = 0.6886, P = 0.5009) of sEPSCs in BAPV neurons that did not show glutamatergic responses (NR-BAPV neurons) before (light off) and after (light on) opto-stimulation (n = 17 neurons from 4 mice, two-sided paired t test). (g-i) Schematic (g), representative traces (h), cumulative frequency curves of inter-spike-interval (i, outside), and statistics (i, inside) of frequency (i, left; t(11) = 0.2341, NS P = 0.8192) and amplitude (i, right; t(11) = 1.166, NS P = 0.8192) of sEPSCs in HTR1A+ BAPyr neurons before (light off) and after (light on) opto-stimulation (n = 12 neurons from 4 mice, two-sided paired t test). (j-l) Schematic (j), representative traces (k), cumulative frequency curves of inter-spike-interval (l, outside), and statistics (l, inside) of frequency (t(29) = 5.419, ****P < 0.0001) and amplitude (t(29) = 0.3166, P = 0.7538) of sEPSCs in BA HTR1A− neurons before and after opto-stimulation (n = 30 neurons from 4 mice; two-sided paired t test). (m, n) Representative traces (m), cumulative frequency curves of inter-spike-interval (n, outside), and statistics (n, inside) of frequency (t(28) = 0.9280; P = 0.3612) and amplitude (t(28) = 0.6915; P = 0.4950) of sEPSCs in BA HTR1A− neurons before and after opto-train stimulation with the presence of NAS-181 (1 μM) (n = 29 neurons from 4 mice, two-sided paired t test). Shadows in f, i, l, k: opto-activation.

Source data

Extended Data Fig. 10 Pharmacological Inhibiting HTR1A in BA Induced Avoidance of Bright Spaces but not Social Avoidance, without Affecting Locomotion.

(a) Schematic paradigm of cannula implantation and drug administration. (b-f) Statistics (mean ± SEM; n = 13 mice, two-sided unpaired t test) of time in open arm (t(24) = 4.324; ***P = 0.0002) in EPM (b), time in centre (t(24) = 3.718; **P = 0.0011) in OFT (c), latency to feed (t(24) = 3.411; **P = 0.0023) in NSF (d) and interaction time (t(24) = 1.483; P = 0.1512) in social interaction test (e), sniffing time (F(3, 48) = 13.78, P < 0.0001; E versus S1, ACSF, t(48) = 11.39, ****P < 0.0001, WAY100635, t(48) = 9.254, ****P < 0.0001; S1 versus S2, ACSF, t(48) = 4.218, ****P = 0.0001, WAY100635, t(48) = 0.703, ****P < 0.0001; ACSF versus WAY100635, S1, t(96) = 2.551, *P = 0.0245, S2, t(96) = 0.3671, P = 0.7144), social index (F(1, 24)pharmacology = 0.0945, P = 0.7611; ACSF versus WAY100635, sociability, t(48) = 0.9231, P = 0.36.6, social novelty, t(48) = 1.347, P = 0.3347) in three-chamber test (f; n = 13 mice; two-sided RM two-way ANOVA) after administration of ACSF or WAY100635 in BA bilaterally in C57 mice. (g) Schematic paradigm of cannula implantation and drug administration. (h-i) Statistics (mean ± SEM) of total distance (F(2, 36) = 1.659; P = 0.2045) in OFT (h) and total food consumed (F(2, 36) = 0.196; P > 0.8229) in NSF (i) after administration of ACSF, WAY100635 and NAS-181 in BA bilaterally in C57 mice (n = 13 mice, two-sided RM one-way ANOVA).

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Supplementary information

Supplementary Information

Supplementary Figs. 1–13 and Supplementary Table 1.

Reporting Summary

Supplementary Data 1

Statistical data for supplementary figures.

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Source Data Fig. 2

Statistical results of sniffing time (s) in three-chamber test.

Source Data Fig. 4

Statistical results of sniffing time (s) in three-chamber test.

Source Data Fig. 5

Statistical results of vGAT fluorescence intensity.

Source Data Fig. 8

Statistical results of sniffing time (s) in three-chamber test.

Source Data Extended Data Fig. 1

Statistical results of sniffing time (s) in three-chamber test.

Source Data Extended Data Fig. 9

Statistical results of sniffing time (s) in three-chamber test.

Source Data Extended Data Fig. 10

Statistical results of sniffing time (s) in three-chamber test.

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Yu, XD., Zhu, Y., Sun, QX. et al. Distinct serotonergic pathways to the amygdala underlie separate behavioral features of anxiety. Nat Neurosci 25, 1651–1663 (2022). https://doi.org/10.1038/s41593-022-01200-8

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