Midbrain circuits for defensive behaviour

Abstract

Survival in threatening situations depends on the selection and rapid execution of an appropriate active or passive defensive response, yet the underlying brain circuitry is not understood. Here we use circuit-based optogenetic, in vivo and in vitro electrophysiological, and neuroanatomical tracing methods to define midbrain periaqueductal grey circuits for specific defensive behaviours. We identify an inhibitory pathway from the central nucleus of the amygdala to the ventrolateral periaqueductal grey that produces freezing by disinhibition of ventrolateral periaqueductal grey excitatory outputs to pre-motor targets in the magnocellular nucleus of the medulla. In addition, we provide evidence for anatomical and functional interaction of this freezing pathway with long-range and local circuits mediating flight. Our data define the neuronal circuitry underlying the execution of freezing, an evolutionarily conserved defensive behaviour, which is expressed by many species including fish, rodents and primates. In humans, dysregulation of this ‘survival circuit’ has been implicated in anxiety-related disorders.

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Figure 1: Glutamatergic vlPAG neurons drive defensive responses.
Figure 2: A disinhibitory pathway from CEA to vlPAG.
Figure 3: GABAergic vlPAG neurons control freezing.
Figure 4: Glutamatergic output to the Mc drives freezing.
Figure 5: Interactions between PAG freezing and flight pathways.

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Acknowledgements

We thank C. Müller, J. Lüdke, K. Bylund, J. Alonso, T. Lu, P. Argast and P. Buchmann for technical assistance, J. J. Letzkus for input on the manuscript, and all members of the Lüthi and Arber laboratories for discussions and other help with the project. We thank L. Gelman and S. Bourke for help with microscopy, and M. Stadler for statistical advice. We are grateful to G. Keller for providing viruses for optogenetics, Z. J. Huang for initially providing the Gad2-ires-Cre mouse line and L. Xiao and P. Scheiffele for the anti-rabiesG antibody. This work was supported by the Novartis Research Foundation, by the National Center of Competences in Research: ‘SYNAPSY — The Synaptic Bases of Mental Diseases’ (financed by the Swiss National Science Foundation) as well as by a Swiss National Science Foundation Core Grant, and a European Research Council Advanced Grant to A.L. Support for S.A. and M.S.E. was provided by a European Research Council Advanced Grant, the Swiss National Science Foundation and the Kanton Basel-Stadt. P.T. and J.P.F. were supported by NARSAD Young Investigator Grants by the Brain and Behavior Foundation. M.S.E. was also supported by a long-term post-doctoral fellowship of the Human Frontier Science Program and a Synapsis Foundation Grant. F.C. and C.H. were supported by grants from the European Research Council (ERC) under the European Union’s Seventh Framework Program (FP7/2007-2013)/ERC grant agreement number 281168 and the Fondation pour la Recherche Médicale.

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Authors

Contributions

P.T. conceived, designed, performed and analysed most of the experiments and wrote the manuscript. M.S.E. conceived, designed, performed and analysed neuroanatomical tracing experiments. P.B. performed and analysed in vitro slice recordings. F.C. and C.H. performed single-unit recordings in the PAG. S.B.E.W. established optogenetic methodology. J.P.F. performed experiments. M.M. designed and tested viruses for optogenetics. C.R., L.F. and K.D. produced viruses for optogenetics. S.A. designed viruses for tracing and optogenetics. A.L. conceived the project and wrote the manuscript. All authors contributed to the experimental design and commented on the manuscript.

Corresponding authors

Correspondence to Philip Tovote or Andreas Lüthi.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1

a, Expression of ChR2 throughout vlPAG in consecutive coronal brain sections51. b, c, Fibre placements in Vglut2-ires-Cre mice of experimental and control groups. d, Supplementary Video stills with superimposed representative movements tracks during the snake open-field test for unconditioned defensive responses. e, Colour-coded plot for a mouse’s motion before, during and after light-mediated inhibition of vlPAG glutamatergic neurons expressing Arch during a snake open-field test session. f, Freezing responses plotted against mouse–snake distance during ‘light on’ and ‘light off’ periods showing no linear correlation. g, Effects of light-mediated inhibition of vlPAG glutamatergic neurons on anxiety-like behaviour in the open field test with no snake present. Inhibition of vlPAG glutamatergic cells resulted in enhanced track length (n = 6 mice, paired two-tailed Student’s t-test) and more frequent visits to the centre of the open field (n = 6 mice, paired two-tailed Student’s t-test). h, Example of an entire snake open-field test session, with track length, freezing episodes and mouse–snake distance. Values are means ± s.e.m.

Extended Data Figure 2

a, In vitro slice recordings of functional connectivity between CEA and GABAergic or glutamatergic neurons in vlPAG. b, Targeting of fluorescently labelled GAD1+ neurons for whole-cell patch clamp recordings (triangle indicates patch pipette, scale bar, 20 μm). c, Example traces of three GAD1+ cells (from three slices of three mice) showing eIPSCs upon light stimulation (10 ms duration) of afferents from the CEA (upper traces), and blockage of eIPSCs by PTX (lower traces). d, Targeting of fluorescently labelled vGluT2+ neurons for whole-cell patch clamp recordings (triangle indicates patch pipette; scale bar, 20 μm). e, Example traces of three light non-responsive vGluT2+ neurons (from the same three slices as in c). f, Quantification of eIPSCs amplitudes. g, Analysis of specificity of AAV-mediated expression of TVA and rabiesG (scale bar, 10 μm). h, Analysis of specificity of TVA-dependent EnvA-ΔG–mCherry–rabies infection (scale bar, 20 μm). i, k, Lack of EnvA-ΔG–mCherry–rabies infection after injection into wild-type mice (scale bar, 100 μm). j, k, Leakiness analysis of the combined AAV and EnvA-ΔG–mCherry–rabies tracing system in wild-type mice (scale bar, 200 μm). Values are means ± s.e.m.

Extended Data Figure 3

a, Electrode placements for the recordings of unidentified single units. b, Placements of optrodes for the recordings of identified neurons. c, Raster-frequency plot of an optically identified vlPAG vGluT2+ neuron. d, A glutamatergic neuron exhibiting marked optical activation during constant illumination for 20 s. e, Identified vGluT2+ neurons (n = 6) showed both increased and decreased activity during freezing. f, g, Fibre placements in Gad2-ires-Cre mice expressing inhibitory or excitatory optical actuators. h, i, Activation of vlPAG GAD2+ neurons resulted in reduced conditioned contextual freezing (n = 12 ChR2, n = 7 control, paired two-tailed Student’s t-test) and lower innate freezing levels during the snake open-field test (n = 10 ChR2, n = 8 control, two-tailed Wilcoxon signed-rank test). j, Optical activation of vlPAG GAD2+ neurons had no effect on freezing in naive mice (n = 8 ChR2, n = 8 control, two-tailed Wilcoxon signed-rank test). Box–whisker plots indicate median, interquartile range, and 5th–95th percentiles of the distribution. *P < 0.05.

Extended Data Figure 4

a, Projection pattern of glutamatergic vlPAG axonal inputs to the rostral medulla. Terminals of vGluT2+ vlPAG projection neurons were labelled by AAV-mediated expression of GFP fused to presynaptic marker synaptophysin (top; scale bar, 400 μm), and ChR2–mCherry expression was visualized using immunohistochemistry (bottom). b, Concomitant AAV-mediated expression of Syn–GFP in vlPAG neurons (left panel, scale bar, 200 μm) labelled presynaptic terminals within Mc (right panel, scale bar, 100 μm). c, High-resolution image of a retrogradely traced Mc pre-motor neuron (rabies–mCherry) and SynGFP+ vlPAG inputs (top), and visualization of identified glutamatergic synaptic contacts (bottom; scale bar, 10 μm). d, e, Density of vGluT2+ vlPAG synaptic inputs to pre-motor neurons in Mc (n = 8 cells from three mice), and quantification of their distribution between the dendritic or somatic compartment. f, Intersectional EnvA-ΔG–mCherry–rabies tracing approach to identify local GABAergic inputs to vlPAG-to-Mc-projecting glutamatergic cells. g, TVA-, rabiesG- and EnvA-ΔG–mCherry–rabies triple-positive cells were identified as starter cells (left panels), while GAD1 and EnvA-ΔG–mCherry–rabies double positive cells indicated presynaptic GABAergic neurons (right panels; scale bar, 20 μm). h, Quantification of GAD1+ or GAD1 presynaptic cells (n = 2 mice; dark grey, rabies+/GAD1+; light grey, rabies+/GAD1). i, Example picture of glutamatergic vlPAG neurons retrogradely traced from the Mc, expressing ChR2 in presence of Cre and Flp recombinase (scale bar, 200 μm). j, Analysis of viral efficacy and leakiness. Overlaps of ChR delivered by AAV-CreONFlpON-ChR2, HSV-delivered Cre-dependent Flp–mCherry and Cre-dependent β-Gal were quantified in Vglut2::LacZ reporter mice. k, Fibre placement in Vglut2-ires-Cre mice expressing ChR2 in glutamatergic vlPAG-to-Mc projection neurons. l, Example of an entire session of light activation of glutamatergic vlPAG-to-Mc projection neurons, with the mouse’s motion, cumulative track length and light-induced freezing bouts. Values are means ± s.e.m.

Extended Data Figure 5

a, Expression of ChR2 throughout PAG in consecutive coronal brain sections (left), and fibre placements in Vglut2-ires-Cre mice of experimental and control groups (right). b, Light-evoked effect on freezing behaviour induced by activation of different glutamatergic subpopulations of PAG neurons in naive animals (n = 12 PAG, n = 10 vlPAG, n = 7 vlPAG-to-Mc, Kruskal–Wallis test, P < 0.001, Dunn’s multiple comparison post-hoc test). c, EnvA-ΔG–mCherry–rabies-mediated, Cre-dependent monosynaptic retrograde tracing of inputs to GAD2+ and vGluT2+ neurons in the vlPAG and dl/lPAG. d, Rabies-mediated labelling of presynaptic neurons within PMD (left panels) and CEA (right panels) of Gad2-ires-Cre and Vglut2-ires-Cre mice (scale bar, 100 μm). e, Statistical analysis reveals differential input to vGluT2+ (n = 3 mice for each vlPAG and dl/lPAG) and GAD2+ (n = 4 mice) neurons within vlPAG or dl/lPAG. While CEA preferentially targets GAD2+ neurons of the vlPAG (1 × 3 ANOVA, F(2,6) = 21.67, P < 0.01, Tukey’s post-hoc test), vGluT2+ neurons of the dl/lPAG receive stronger inputs from PMD (1 × 3 ANOVA, F(2,7) = 287, P < 0.0001, Tukey’s post-hoc test). f, Cell-type-specific monosynaptic rabies tracing of VMH and LH inputs to vGluT2+ neurons in the dl/lPAG, vlPAG and vlPAG GAD2+ neurons (scale bars in overview, 200 μm; in zoom-in, 25 μm). g, Quantification of starter cells in the PAG and presynaptic cells in CEA and hypothalamic subregions. Boxes indicate median and 25th–75th percentiles of the distribution. **P < 0.01; ***P < 0.001.

Extended Data Figure 6 Schematic representation of the freezing pathway.

PN, projection neuron; IN, interneuron; MN, motor neuron.

Supplementary information

Supplementary Information

This file contains a Supplementary Discussion, Statistics Table and Supplementary References. (PDF 236 kb)

Freezing behavior evoked by activation of glutamatergic vlPAG neurons.

Freezing behavior evoked by activation of glutamatergic vlPAG neurons. (MP4 5535 kb)

Inhibition of glutamatergic vlPAG neurons diminishes conditioned cued freezing

Inhibition of glutamatergic vlPAG neurons diminishes conditioned cued freezing. (MP4 11318 kb)

Selective activation of glutamatergic vlPAG neurons projecting to Mc evokes freezing behavior

Selective activation of glutamatergic vlPAG neurons projecting to Mc evokes freezing behavior. (MP4 6429 kb)

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Tovote, P., Esposito, M., Botta, P. et al. Midbrain circuits for defensive behaviour. Nature 534, 206–212 (2016). https://doi.org/10.1038/nature17996

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