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A midline thalamic circuit determines reactions to visual threat

Naturevolume 557pages183189 (2018) | Download Citation

Abstract

How our internal state is merged with our visual perception of an impending threat to drive an adaptive behavioural response is not known. Mice respond to visual threats by either freezing or seeking shelter. Here we show that nuclei of the ventral midline thalamus (vMT), the xiphoid nucleus (Xi) and nucleus reuniens (Re), represent crucial hubs in the network controlling behavioural responses to visual threats. The Xi projects to the basolateral amygdala to promote saliency-reducing responses to threats, such as freezing, whereas the Re projects to the medial prefrontal cortex (Re→mPFC) to promote saliency-enhancing, even confrontational responses to threats, such as tail rattling. Activation of the Re→mPFC pathway also increases autonomic arousal in a manner that is rewarding. The vMT is therefore important for biasing how internal states are translated into opposing categories of behavioural responses to perceived threats. These findings may have implications for understanding disorders of arousal and adaptive decision-making, such as phobias, post-traumatic stress and addictions.

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Acknowledgements

This work was supported by the National Institutes of Health (NIH) National Eye Institute grant R01 EY022157 (A.D.H.), by NIH U01 NS090562 (A.D.H.) and by a National Science Foundation Graduate Research Fellowship (L.D.S.). We thank L. Giacomo and C. Mallory for advice and assistance with electrophysiology.

Reviewer information

Nature thanks D. Lin, H. Shin and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Affiliations

  1. Department of Neurobiology, Stanford University School of Medicine, Palo Alto, CA, USA

    • Lindsey D. Salay
    • , Nao Ishiko
    •  & Andrew D. Huberman
  2. Stanford Neurosciences Institute, Stanford University School of Medicine, Palo Alto, CA, USA

    • Andrew D. Huberman
  3. BioX, Stanford University School of Medicine, Palo Alto, CA, USA

    • Andrew D. Huberman
  4. Department of Ophthalmology, Stanford University School of Medicine, Palo Alto, CA, USA

    • Andrew D. Huberman

Authors

  1. Search for Lindsey D. Salay in:

  2. Search for Nao Ishiko in:

  3. Search for Andrew D. Huberman in:

Contributions

L.D.S. and A.D.H. conceived the design of the study. L.D.S. and N.I. performed the experiments and analysed the data. L.D.S. and A.D.H. wrote the paper.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Andrew D. Huberman.

Extended data figures and tables

  1. Extended Data Fig. 1 hM3Dq activates vMT, whereas hM4Di inactivates vMT.

    Refers to Figs. 1 and 2. a, Timeline of the c-Fos induction protocol. bd, Recently active c-Fos+ neurons (green) in the vMT of mice that were exposed to the looming stimulus, with XFP (b), hM4Di (c), or hM3Dq (d) injections (red) into the vMT and CNO delivered intraperitoneally. e, After CNO delivery, hM3Dq increased whereas hM4Di decreased the number of c-Fos+ cells in the vMT relative to XFP controls. Scale bars, 100 μm. Data are mean ± s.e.m. *P < 0.05, ***P < 0.001. See Supplementary Table 1 for statistical analysis and sample sizes.

  2. Extended Data Fig. 2 Control groups do not differ in their defensive responses to looming threat.

    Refers to Figs. 24. ah, Comparison of different control mice reveals no significant differences across any of the behaviours performed in response to the looming threat: tail rattling (ae), running (b, f), freezing (c, g), or hiding (d, h). Controls include: mice with no treatments; mice with AAV-XFP and CNO; mice with CAV-Cre, AAV-DIO-XFP and CNO; mice with CAV-Cre and AAV-DIO-hM3Dq but without CNO; and mice with AAV-XFP, optrode implant and sham stimulation. il, Comparison of male and female control mice (i, k) and mice with vMT activation (j, l) reveals no significant sex differences across any of the behaviours performed in response to the looming threat. Notably, both male and female mice tail-rattle in response to the looming threat. Data are mean ± s.e.m.; NS, not significant. See Supplementary Table 1 for statistical analysis and sample sizes.

  3. Extended Data Fig. 3 Activating vMT→NAc does not increase tail rattling or saliency-enhancing behaviours.

    Refers to Fig. 3. ad, Mice were injected with AAV-GFP in the vMT (n = 17 mice; a, b) and axons were observed in the NAc (c, d). Representative image of GFP+ neurons in the VMT (b) and GFP+ axons in the NAc (d). e, To activate vMT neurons that project to the NAc, CAV-Cre was injected into the NAc and Cre-dependent hM3Dq was injected into the vMT. f, Representative image of hM3Dq+ neurons in the vMT that project to the NAc. g, Activating the vMT→NAc pathway did not significantly change the behavioural responses to looming as compared to controls. Scale bars, 100 μm. Data are mean ± s.e.m.; NS, not significant. See Supplementary Table 1 for statistical analysis and sample sizes.

  4. Extended Data Fig. 4 Viral targeting and number of cells infected in the vMT.

    Refers to Figs. 3 and 4. a, To activate vMT neurons that project to the mPFC or the BLA, CAV-Cre was injected into the mPFC or the BLA and Cre-dependent hM3D was injected into the vMT. b, The average number of infected hM3d–mCherry+ vMT cells did not differ between the vMT→BLA and the vMT→mPFC pathway activation groups. ce, Locations of injections to activate the vMT→mPFC pathway. d, Relative expression of hM3D in vMT→mPFC cells does not scale with tail-rattling behaviour. f, g, Representative images of hM3dq–mCherry/Cre+ neurons (red) in the vMT that project to the mPFC. h, Mice were injected with AAV-ChR2 in the vMT to activate the vMT. ij, Representative images of ChR2–eYFP+ neurons (green) in the vMT and the fibre tracts composed of vMT axons that project to the mPFC. Data are mean ± s.e.m.; NS, not significant. See Supplementary Table 1 for statistical analysis and sample sizes.

  5. Extended Data Fig. 5 vMT activation results in saliency-enhancing behaviours performed in the open arena.

    Refers to Figs. 3 and 4. a, Percentage of mice tail-rattling in response to looming after vMT→PFC or vMT→BLA activation. All of the mice with vMT→mPFC optogenetic stimulation displayed tail-rattling behaviour. b, c, Percentage of tail-rattling (b) or running (c) events performed in the open arena as opposed to in the shelter. Mice with vMT→ mPFC optogenetic stimulation perform most tail-rattling and running events in the open. d, Optogenetic activation of the vMT results in most mice tail-rattling. e, Mice with vMT optogenetic stimulation perform most tail-rattling events in the open arena as opposed to in the shelter. f, Mice with vMT optogenetic stimulation perform most running events within the open arena as opposed to towards the shelter. Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001; NS, not significant. See Supplementary Table 1 for statistical analysis and sample sizes.

  6. Extended Data Fig. 6 vMT activation does not change locomotion, aggression or anxiety, but does result in less freezing in response to a predator odour.

    Refers to Figs. 4 and 5. a, Mice were injected with AAV-ChR2 in the vMT in order to activate the vMT during presentation of the predator-odour threat. b, Activating the vMT (n = 9 mice) decreased freezing as compared to controls with sham stimulation (n = 7 mice). c, Activating the vMT decreased avoidance as compared to controls with sham stimulation (avoidance index = (P − 50)/50, where P is the percentage of time the mouse spent on the side of the arena away from the odour). d, The visual cliff test. e, Activating the vMT (hM3D + CNO, n = 15 mice) or inactivating the vMT (hM4D + CNO, n = 9) during the visual cliff test did not change the number of times in which the mouse chose the shallow side as compared to control mice (n = 9 mice, P < 0.05). f, The RTPP test. g, Activating the vMT (ChR2, n = 7 mice) did not change the relative activity (distance covered) in the RTPP test as compared to control mice (n = 6 mice). h, Mice were tested on the resident-intruder test for aggressive behaviours. i, Activating the vMT (ChR2, n = 7 mice) did not change the average number of tail-rattling events in the resident-intruder test as compared to control mice (n = 14 mice). j, k, Activating the vMT (ChR2, n = 7 mice) did not change the percentage of mice attacking (j) or the latency to attack (k) in the resident-intruder test as compared to control mice (n = 14 mice). l, Mice were subjected to an open field test to analyse anxiety-related behaviour; representative tracing of a control mouse (AAV-GFP, left) and a mouse with vMT activation (AAV-ChR2, right) in the open field test. m, Activating the vMT (ChR2, n = 10 mice) did not change the percentage of time in the centre of the open field as compared to control mice (n = 10 mice). n, Activating the vMT (ChR2, n = 10 mice) did not change the relative activity (distance covered) in the open field test as compared to control mice (n = 10 mice). Data are mean ± s.e.m. *P < 0.05; NS, not significant. See Supplementary Table 1 for statistical analysis and sample sizes.

  7. Extended Data Fig. 7 vMT activation results in increased arousal.

    Refers to Fig. 5. a, Schematic of the experimental set-up to analyse light-driven pupillary responses in mice. Treating control mice with CNO did not change pupil size across all light levels. b, Chronic vMT activation by chemicogenetic methods (+CNO) significantly increases pupil size across all light levels as compared to the same mice without activation (−CNO). c, Chronic vMT inactivation (+CNO) did not the change pupil size across all light levels as compared to the same mice without activation (−CNO). d, Schematic of experimental set-up to analyse arousal-driven pupillary responses in mice with vMT activation, mice with vMT inactivation and control mice. The pupil was measured in constant light (100 lx) conditions in mice with and without CNO. e, After CNO delivery, mice with hM3Dq had a significant increase in relative pupil size. Mice with XFP and hM4Di did not have a significant change in pupil size after administration of CNO. f, In constant dark conditions, vMT activation significantly increased pupil size, whereas vMT inactivation did not change pupil size as compared to control mice with CNO. g, In constant dark conditions, optogentic activation of the vMT significantly increased pupil size (n = 11 mice) compared to control mice (n = 12 mice). Data are mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant. See Supplementary Table 1 for statistical analysis and sample sizes.

  8. Extended Data Fig. 8 Looming stimuli induce vMT activation in naive, but not experienced mice habituated to looms.

    Refers to Fig. 6. a, Schematic of the experimental protocol. b, c, Quantification of c-Fos+ cells in the vMT of naive or pre-exposed mice that experienced looming from above revealed a significant decrease in c-Fos+ cells in the vMT (b) and Xi (c) after looming from above in pre-exposed mice that are habituated to the looms (n = 6 mice) as compared to naive mice (n = 7 mice). Data are mean ± s.e.m. **P < 0.01; ***P < 0.001. Supplementary Table 1 for statistical analysis and sample sizes.

  9. Extended Data Fig. 9 Tail rattling and running scales with arousal.

    Refers to Figs. 4 and 5. a, b, Mice in which ChR2 had been injected into the vMT were divided into three groups based on the extent to which vMT activation increased arousal responses: low, moderate or high (n = 7, n = 9 and n = 10 mice, respectively). Mice with high arousal had significantly more tail-rattling (a) and running (b) events in response to looming, as compared to mice with low arousal (P < 0.001 and P < 0.001). c, d, Mice with high arousal spent similar amounts of time freezing (c) and hiding (d) in response to looming, as compared to mice with low arousal. ef, 100% of mice with high arousal tail-rattled (e) and ran (f) in response to looming, whereas only 33% and 20% of mice with low arousal tail-rattled and ran, respectively. gh, Similar numbers of mice with high arousal froze (g) and hid (h) in response to looming as compared to mice with low arousal. Data are mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant. See Supplementary Table 1 for statistical analysis and sample sizes.

  10. Extended Data Fig. 10 Hindbrain inputs to the vMT.

    a, Schematic illustration of ΔG-rabies-XFP injection into the vMT to map vMT inputs. b, Quantification of the relative density of projection neurons to the vMT (n = 12 mice). ce, Representative images showing the expression of ΔG-rabies-XFP in transynaptically labelled cells in the superior colliculus (c), dorsal raphe (d), periaqueductal grey (d), and median raphe (e). DRN, dorsal raphe; MRN, median raphe; PAG, periaqueductal grey; PRN, pontine reticular nucleus; SCm, superior colliculus, motor; SCs, superior colliculus, sensory. Scale bars, 100 μm.

Supplementary information

  1. Supplemental Table 1

    This table contains the statistical analysis

  2. Reporting Summary

  3. Supplemental Video 1

    Looming stimuli cause freeze behavior (refers to Figure 1) Control mice freeze in response to overhead looming stimuli. Refer to figure 1 for quantification

  4. Supplemental Video 2

    Looming stimuli cause escape behavior (refers to Figure 2) Control mice also run to the provided shelter in order to hide in response to overhead looming stimuli. Refer to figure 1 for quantification

  5. Supplemental Video 3

    vMT inactivation promotes saliency-reducing responses to looming stimuli (refers to Figure 3) Mice with vMT inactivation freeze in response to overhead looming stimuli. Refer to figure 2 for quantification

  6. Supplemental Video 4

    vMT activation promotes saliency-enhancing responses to looming stimuli (refers to Figure 4) Mice with vMT activation tail rattle within the open arena in response to overhead looming stimuli. Refer to figure 2 for quantification

  7. Supplemental Video 5

    Optogenetic activation of the vMT promotes saliency-enhancing responses to looming stimuli (refers to Figure 5) Mice with optogenetic vMT activation tail rattle and run within the open arena in response to overhead looming stimuli. Refer to figure 4 for quantification

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