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Serotonin engages an anxiety and fear-promoting circuit in the extended amygdala

Nature volume 537, pages 97–101 (2016)Cite this article

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Abstract

Serotonin (also known as 5-hydroxytryptamine (5-HT)) is a neurotransmitter that has an essential role in the regulation of emotion. However, the precise circuits have not yet been defined through which aversive states are orchestrated by 5-HT. Here we show that 5-HT from the dorsal raphe nucleus (5-HTDRN) enhances fear and anxiety and activates a subpopulation of corticotropin-releasing factor (CRF) neurons in the bed nucleus of the stria terminalis (CRFBNST) in mice. Specifically, 5-HTDRN projections to the BNST, via actions at 5-HT2C receptors (5-HT2CRs), engage a CRFBNST inhibitory microcircuit that silences anxiolytic BNST outputs to the ventral tegmental area and lateral hypothalamus. Furthermore, we demonstrate that this CRFBNST inhibitory circuit underlies aversive behaviour following acute exposure to selective serotonin reuptake inhibitors (SSRIs). This early aversive effect is mediated via the corticotrophin-releasing factor type 1 receptor (CRF1R, also known as CRHR1), given that CRF1R antagonism is sufficient to prevent acute SSRI-induced enhancements in aversive learning. These results reveal an essential 5-HTDRN→CRFBNST circuit governing fear and anxiety, and provide a potential mechanistic explanation for the clinical observation of early adverse events to SSRI treatment in some patients with anxiety disorders1,2.

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Figure 1: Optogenetic identification of a 5-HTDRN→BNST projection that elicits anxiety and fear-related behaviour.
Figure 2: Serotonin activates a local population of CRFBNST neurons that inhibits outputs to the midbrain.
Figure 3: Acute fluoxetine elicits aversive behaviour by engaging inhibitory CRF circuits in the BNST.

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Acknowledgements

We acknowledge B. Roth for providing DREADD viral constructs and SertCre mice, and B. Lowell for providing CrfCre mice. We also thank A. Lopez, D. Perron, and A. Kendra for technical assistance with stereotaxic surgeries on mice, B. Geenen for technical assistance with immunohistochemistry and E. Dankoski for technical assistance with the FSCV. This work was supported by NIH grants AA019454, AA011605 (T.L.K.), the Wellcome Trust (098012) and the Biotechnology and Biological Sciences Research Council grant (BB/K001418/1) (L.K.H.) and by NIH grant K01AA023555 and the Alcohol Beverage Medical Research Fund (Z.A.M.). C.A.M. was supported by a postdoctoral NIAAA F32 fellowship (AA021319-02). C.M.M. is supported by a predoctoral NIAAA F31 fellowship (F31AA023440).

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Author notes

  1. Catherine A. Marcinkiewcz and Christopher M. Mazzone: These authors contributed equally to this work.

Authors and Affiliations

  1. Bowles Center for Alcohol Studies, University of North Carolina at Chapel Hill, Chapel Hill, 27599, North Carolina, USA

    Catherine A. Marcinkiewcz, Christopher M. Mazzone, J. Andrew Hardaway, Jeffrey F. DiBerto, Cayce E. Dorrier, Gregory J. Tipton, Todd E. Thiele, Zoe A. McElligott & Thomas L. Kash

  2. Curriculum in Neurobiology, School of Medicine, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, 27599, North Carolina, USA

    Christopher M. Mazzone & Thomas L. Kash

  3. Rowett Institute of Nutrition and Health, University of Aberdeen, Aberdeen, AB25 2ZD, UK

    Giuseppe D’Agostino, Claudia Cristiano & Lora K. Heisler

  4. National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, 20852-9411, Maryland, USA

    Lindsay R. Halladay & Andrew Holmes

  5. Department of Psychology & Neuroscience, College of Arts and Sciences, University of North Carolina at Chapel Hill, Chapel Hill, 27599, North Carolina, USA

    Montserrat Navarro, Nathan Burnham, Todd E. Thiele & Thomas L. Kash

  6. Department of Bioengineering, Stanford University, Stanford, 94305, California, USA

    Charu Ramakrishnan & Karl Deisseroth

  7. Hayward Genetics Center, Tulane University, New Orleans, 70112, Louisiana, USA

    Tamas Kozicz

  8. Department of Anatomy, Radboud University Nijmegen Medical Center, Nijmegen, 6500HB, The Netherlands

    Tamas Kozicz

  9. Department of Psychiatry, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, 27599, North Carolina, USA

    Zoe A. McElligott

  10. Department of Pharmacology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, 27599, North Carolina, USA

    Thomas L. Kash

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  1. Catherine A. Marcinkiewcz
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Contributions

C.A.M., C.M.M., G.D., Z.A.M., L.K.H. and T.L.K. designed the experiments. A.H. and J.F.D. performed triple label fos/tryptophan hydroxylase/Fluor-Gold staining and image analysis. L.R.H. performed electrode placement surgeries and in vivo recordings during fear acquisition and recall. C.A.M. performed stereotaxic surgeries for evoked 5-HT electrophysiology and optogenetic behavioural experiments. Z.A.M. performed slice FSCV experiments and C.A.M. performed evoked 5-HT electrophysiology experiments. C.A.M. performed stereotaxic surgeries, behavioural and data analysis for 5-HTDRN→BNST optogenetic experiments. C.A.M. performed all slice electrophysiology experiments and C.M.M. and C.A.M. performed stereotaxic surgeries for these experiments (retrograde tracers, ChR2 infusions, and hM3D and hM4D infusions), C.M.M. performed stereotaxic surgeries for chemogenetic manipulations in CRFBNST neurons that were used in fluoxetine fear conditioning experiments and C.A.M. performed behavioural and data analysis. C.E.D. performed surgeries for electrophysiological recordings and data analysis for fear conditioning experiments. M.N. and J.F.D. performed surgeries for chemogenetic manipulations in CRFBNST neurons that were used in fluoxetine anxiety (EZM) assays and N.B. and C.A.M. performed behavioural and data analysis. C.M.M. and J.F.D. performed stereotaxic surgeries for HSVCre::hM3DBNST behavioural manipulations and C.A.M. performed behavioural and data analysis. C.M.M. also performed imaging and analysis for optogenetic experiments, chemogenetic, and Intrsect experiments. C.R. and K.D. designed Intrsect viral constructs. G.D. and C.C. performed surgeries, behavioural and data analysis for Htr2cCre::hM3DBNST experiments. C.A.M., C.M.M. and T.L.K. wrote the manuscript with input from Z.A.M., L.R.H., J.F.D., J.A.H., G.D., T.E.T., A.H., L.K.H. and T.K.

Corresponding author

Correspondence to Thomas L. Kash.

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Competing interests

The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks A. Sahay and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 In vivo recordings in BNST neurons during fear conditioning reveal opposite patterns of activation during acquisition and recall.

a, b, Representative neuronal firing rate (a) and population Z score of the firing rate (b) for BNST neurons (n = 45 cells from 7 mice) 30 s before conditioned stimulus (tone), during the conditioned stimulus (CS), and 30 s after the unconditioned stimulus. c, Percentage time spent freezing during fear acquisition, cued fear recall and contextual fear recall. d, Electrode placements for BNST recordings. e, Raw firing rates during freezing (blue) versus movement (red) epochs were averaged across all putative principal neurons (firing rate < 10 Hz). Acquisition: cells in BNST exhibited greater average firing rates during freezing epochs compared to movement epochs during CS3 (t44 = 2.88, P < 0.01, Student’s unpaired two-tailed t-test), CS4 (t44 = 3.14, P < 0.01, Student’s unpaired two-tailed t-test), and CS5 (t44 = 4.4, P < 0.001, Student’s unpaired two-tailed t-test) (n = 45 cells from 7 mice). CS recall: average firing rates during freezing epochs decreased over CS presentations such that firing during block 5 was significantly less than block 1 (t41 = 3.44, P = 0.001, Student’s unpaired two-tailed t-test). Freezing firing rates during block 5 were also significantly less than movement epochs during block 5 (t41 = 4.03, P < 0.001, Student’s unpaired two-tailed t-test) (n = 42 cells from 7 mice). CX test: average firing rate was significantly greater during movement versus freezing epochs during minute 1 (t44 = 4.83, P < 0.001, Student’s unpaired two-tailed t-test), minute 2 (t44 = 3.17, P < 0.01, Student’s unpaired two-tailed t-test), and minute 5 (t44 = 4.36, P < 0.001, Student’s unpaired two-tailed t-test) (n = 45 cells from 7 mice). f, Freezing-related changes in firing rates during the CS were determined by measuring the ratio of average firing rates during freezing versus movement epochs for each session. Acquisition: activity during freezing epochs increased significantly relative to movement epochs during CS4 (t45 = 3.26, P < 0.01, Student’s unpaired two-tailed t-test) and CS5 (t45 = 2.17, P < 0.05, Student’s unpaired two-tailed t-test) (n = 46 cells from 7 mice). CS recall: freezing significantly suppressed activity relative to movement epochs during the last two CS presentations (t47 = 5.29, P = <0.001, Student’s unpaired two-tailed t-test) (n = 48 cells from 7 mice). CX test: freezing significantly suppressed activity during minutes 1 (t44 = 6.06, P < 0.001, Student’s unpaired two-tailed t-test), minute 2 (t44 = 2.92, P < 0.01, Student’s unpaired two-tailed t-test), and minute 5 (t44 = 3.55, P = .001, Student’s unpaired two-tailed t-test) (n = 45 cells from 7 mice). g, Plots showing correlation between freezing behaviour and firing rate of BNST neurons across sessions and for all sessions. Data are mean ± s.e.m. *P < 0.05 **P < 0.01; ***P < 0.001. Scale bar, 100 μm.

Extended Data Figure 2 Effects of optogenetic stimulation of 5-HT inputs to the BNST on feeding, anxiety and locomotion.

a–c, SertCre::ChR2DRN→BNST mice exhibited reduced probability (t15 = 2.67, P < 0.05, Student’s unpaired two-tailed t-test, n = 8 control, n = 9 ChR2) and latency (t15 = 1.003, P > 0.05, Student’s unpaired two-tailed t-test, n = 8 control, n = 9 ChR2) to enter the open arms of the EPM without exhibiting locomotor deficits. d, e, Photostimulation of 5-HTDRN→BNST terminals had no effect on locomotor activity in the open field (d) (n = 9 control, n = 11 ChR2) or home cage feeding (e) (n = 4 control, n = 6 ChR2). Data are mean ± s.e.m. *P < 0.05.

Extended Data Figure 3 Chemogenetic activation of 5-HT2CR-expressing neurons in the BNST increases anxiety-like behaviour.

a, Confocal images of coronal BNST slices obtained from Htr2cCre mice following double fluorescence in situ hybridization for 5-HT2CR and Cre. Yellow arrows indicate cells in which there is co-localization, red arrows indicate cells in which only Cre is expressed and green arrows indicate cells in which only 5-HT2CR is expressed. b, Pie chart representing the distribution of genetic markers in BNST neurons. c, Experimental configuration in Htr2ccre::hM3DqBNST mice. d, Coronal images showing c-fos induction in 5-HT2CR expressing neurons in the BNST of Htr2cCre::hM3DqBNST or Htr2cCre::mCherryBNST mice following CNO injection. e, Bath application of CNO depolarized 5-HT2CR-expressing neurons expressing hM3Dq in slice (n = 3 cells from 3 mice). f, Chemogenetic stimulation of 5-HT2CR expressing neurons in BNST increased latency to feed in the NSF (t11 = 2.591, P < 0.05, Student’s unpaired two-tailed t-test, n = 6; mCherry, n = 7 hM3Dq). g, Chemogenetic activation of 5-HT2CR-expressing BNST neurons had no effect on home cage feeding (n = 5 mCherry, n = 6 hM3Dq). h, Confocal images from Htr2cCre::mCherryBNST mice showing mCherry expression in 5-HT2CR-expressing soma in the BNST and fibres in the LH and VTA. Data are mean ± s.e.m. *P < 0.05. Scale bar, 100 μm.

Extended Data Figure 4 Electrophysiological characterization of 5-HT responses and 5-HT receptor expression in CRFBNST neurons.

a, A pie chart showing the distribution of CRFBNST neurons that were depolarized, hyperpolarized, or had no response to 5-HT (n = 8 cells from 4 mice). b, Coronal images of the BNST showing co-localization of 5-HT2CRs with CRF mRNA using double fluorescence in situ hybridization. c, d, Histograms showing the percentage of 5-HT2C neurons that express CRF and the percentage of CRF neurons that express 5-HT2CRs in the BNST (n = 3 slices from 3 mice). e, Recording configuration in CRFBNST neurons. f, Slice electrophysiology in BNST of Crf reporter mice showing depolarization of all (VTA-projecting and non-projecting) CRF neurons following bath application of the 5-HT2 receptor agonist mCPP (n = 12 cells from 6 mice) and blockade of this response by the 5-HT2C receptor antagonist RS-102221 (n = 5 cells from 3 mice). g, Change in membrane potential induced by mCPP (t12 = 2.18, P < 0.05, one-sample t-test, n = 13 cells from 6 mice) is blocked by a 5-HT2CR antagonist (n = 5 cells from 3 mice). h, mCPP selectively depolarizes non-VTA-projecting CRFBNST neurons (n = 5 cells from 2 mice non-VTA-projecting CRF, n = 5 cells from 4 mice VTA-projecting CRF). Data are mean ± s.e.m. *P < 0.05.

Extended Data Figure 5 5-HT activates inhibitory microcircuits in the BNST that modulate outputs to the LH.

a, Recording configuration in CRF reporter mice infused with retrograde tracer beads in the LH. b, Average traces of 5-HT induced depolarization in LH projecting versus non-projecting neurons. c, Histograms showing 5-HT induced depolarization in non-LH projecting BNST neurons (t4 = 4.425, P < 0.05, one-sample t-test, n = 5 cells from 3 mice) and hyperpolarization in LH-projecting neurons (t5 = 2.789, P < 0.05, one-sample t-test, n = 6 cells from 3 mice). d, Confocal image of retrogradely CTB-labelled VTA (red) and LH (green) outputs in a CRF-L10a reporter (blue). e, f, Pie charts depicting the percentage of LH-projecting only, VTA-projecting only, collateralizing, and CTB-negative (unlabelled) CRF in neurons in the dorsal and ventral aspects of the BNST (n = 6 hemispheres from 3 mice). g, Experimental schematic depicting viral infusions into the BNST and retrograde tracer bead infusions into the LH of CrfCre::ChR2BNST mice. h, Recording configuration in CrfCre::ChR2BNST mice with LH tracer beads. i, Representative trace of light evoked IPSCs in LH-projecting neurons (n = 7 cells from 4 mice) and blockade of this light evoked response by GABAzine (n = 2 cells from 2 mice). j, Recording configuration in VTA-projecting neurons in the BNST of C57BL/6 mice. k, l, 5-HT has no effect on miniature IPSC frequency or amplitude in BNST→VTA projecting neurons (n = 7 from 4 mice). m, n, 5-HT has no effect on sIPSC frequency or amplitude in the presence of the 5-HT2CR antagonist RS-102221 (n = 5 cells from 4 mice). o, Recording configuration in LH projecting neurons in the BNST of C57BL/6 mice. p, Representative traces showing an increase in sIPSC frequency in the presence of 5-HT for 6 cells from 3 mice. q, r, 5-HT increases sIPSC frequency but not amplitude in BNST→LH projecting neurons (F11,55 = 11.65, P < 0.01, repeated measures one-way ANOVA, n = 6 cells from 3 mice). s, t, 5-HT has no effect on miniature IPSC frequency or amplitude (n = 5 cells from 3 mice). u, v, 5-HT has no effect on sIPSC frequency or amplitude in the presence of RS-102221 (n = 6 cells from 4 mice). Data are mean ± s.e.m. *P < 0.05.

Extended Data Figure 6 5-HT does not alter GABAergic transmission in CRF neurons nor does it directly excite non-CRF VTA-projecting neurons in the BNST.

a, Recording configuration in CRFBNST neurons in a CRF reporter. b, c, 5-HT has no effect on sIPSC frequency or amplitude in the total population of CRF neurons (n = 5 cells from 3 mice). d, Recording configuration in non-CRF, VTA-projecting neurons in the BNST and average trace of 5-HT effect on membrane potential in non-CRF, VTA-projecting neurons in the presence of tetrodotoxin. e, Histogram summarizing 5-HT effects on membrane potential in local and VTA-projecting CRF neurons and local CRF neurons in the presence of the 5-HT2C receptor antagonist RS-102221 (same data shown in Fig. 2b) juxtaposed with the lack of effect of 5-HT on membrane potential in non-CRF, VTA-projecting neurons (t4 = 0.9381, ns, one-sample t-test, n = 5 cells from 3 mice). Data are mean ± s.e.m. **P < 0.01; ***P < 0.001.

Extended Data Figure 7 The 5-HT2 agonist mCPP increases GABAergic but not glutamatergic transmission in the BNST.

a, b, mCPP increases sIPSC frequency (F15,30 = 1.863, P < 0.001, Repeated measures one-way ANOVA, n = 3 cells from 3 mice) but not amplitude in the BNST of C57BL/6 mice. c, d, mCPP has no effect on spontaneous excitatory postsynaptic current (sEPSC) frequency or amplitude in the BNST of C57BL/6 mice (n = 5 cells from 3 mice). Data are mean ± s.e.m. *P < 0.05.

Extended Data Figure 8 Optogenetic and intrsectional characterization of 5-HT-CRF circuits in the BNST and outputs to the midbrain.

a, Experimental design and recording configuration from SertCre::ChR2DRN→BNST mouse with retrograde tracer beads in the VTA. b, Representative traces for 5 cells from 3 mice depicting the increase in sIPSCs in VTA-projecting neurons in the BNST following light-evoked 5-HT release. c, Histogram summarizing the effect of light evoked 5-HT release on sIPSC frequency in VTA-projecting neurons (t4 = 4.890, P < 0.01, one-sample t-test, n = 5 cells from 3 mice). d, Experimental configuration in CrfCre::Intrsect-ChR2BNST mice. e, Representative images from 4 CrfCre::HSV-LSL1-mCherry-flpoVTA/LH mice and 4 CrfCre::HSV-LSL1-mCherryVTA/LH mice injected with Intrsect-ChR2-eYFP in the BNST. f, Cell counts of eYFP+ neurons from HSV-LSL1-flpo and HSV-LSL1-mCherry injected CrfCre::Intrsect-ChR2BNST mice indicating the number of non-projecting CRF neurons compared to the total CRF population in the dorsal (top panel; t14 = 1.959, ns, Student’s unpaired two-tailed t-test, n = 4 mice, 8 hemispheres per group) and ventral aspects of the BNST (bottom panel; t7 = 2.431, P < 0.05, Student’s unpaired Welch’s corrected two-tailed t-test, n = 4 mice, 8 hemispheres per group). g, Recording configuration and light-evoked IPSC showing local GABA release from non-projecting CRF neurons in the BNST. h, Sterotaxic injection of ChR2 in CrfCre mouse. i, j, Light evoked IPSCs in the VTA and LH indicating that CRF projections to these regions are GABAergic. Data are mean ± s.e.m. *P < 0.05; **P < 0.01.

Extended Data Figure 9 Pharmacological blockade of CRF1 receptors reduces fluoxetine-induced aversive behaviour and 5-HT enhancement of GABAergic transmission in the BNST.

a, Experimental schedule of injections and behaviour. b, CRF1R antagonist does not modify fear acquisition but reduces fluoxetine enhancement of cued fear recall (F1,20 = 13.70, P < 0.01, two-way ANOVA, n = 6 per group). c, Recording configuration in BNST neurons that project to the LH in C57BL/6 mice. d, Bath application of a CRF1R antagonist blocks the 5-HT induced increase in sIPSC frequency in LH-projecting neurons in the BNST (F10,30 = 0.2213, ns, Repeated measures one-way ANOVA, n = 4 cells from 2 mice). e, There was a reduction in sIPSC amplitude during 5-HT bath application and CRF1R blockade (F10,30 = 2.941, P < 0.05, Repeated measures one-way ANOVA, n = 4 cells from 2 mice). Data are mean ± s.e.m. **P < 0.01.

Extended Data Figure 10 Model of a serotonin-sensitive inhibitory microcircuit in the BNST that modulates anxiety and aversive learning.

Serotonin inputs to the BNST activate 5-HT2CRs expressed in non-projecting ‘local’ CRF neurons. These local CRF neurons promote anxiety and fear by inhibiting anxiolytic outputs to the VTA and LH that are putatively GABAergic. Another discrete subset of CRF neurons, which are inhibited by 5-HT, send direct, inhibitory projections to the VTA and LH. These CRFBNST output neurons are GABAergic and putatively anxiolytic and stress buffering. Blue dashed lines indicate hypothesized additional synapses between CRFBNST neurons. Dashed red line indicates a putatively GABAergic synapse.

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Marcinkiewcz, C., Mazzone, C., D’Agostino, G. et al. Serotonin engages an anxiety and fear-promoting circuit in the extended amygdala. Nature 537, 97–101 (2016). https://doi.org/10.1038/nature19318

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