Abstract
Innate fear has a critical role in survival of animals. Unlike conditioned fear, the neuronal circuitry underlying innate fear is largely unknown. We found that the laterodorsal tegmentum (LDT) and lateral habenula (LHb) are specifically activated by the mouse predator odorant trimethylthiazoline (TMT). Using optogenetics to selectively stimulate GABAergic neurons in the LDT immediately produced fear-like responses (freezing, accelerated heart rate and increased serum corticosterone), whereas prolonged stimulation caused anxiety-like behaviors. Notably, although selective stimulation of parvalbumin (PV)-positive interneurons similarly induced fear-like responses, stimulation of somatostatin-positive interneurons or inhibition of PV neurons in the LDT suppressed TMT-induced fear-like responses without affecting conditioned fear. Finally, activation of LHb glutamatergic inputs to LDT interneurons was sufficient to generate fear-like responses. Thus, the LHb-LDT pathway is important for regulating olfactory cue–induced innate fear. Our results provide a potential target for therapeutic intervention for anxiety disorder.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Change history
28 April 2016
In the version of this article initially published, the word "University" was missing from the affiliation of authors Hongbin Yang, Junhua Yang, Wang Xi, Sijia Hao, Liya Zhu, Huifang Lou, Yanqin Yu, Shumin Duan and Hao Wang. The correct affiliation should be "Department of Neurobiology, Key Laboratory of Medical Neurobiology (Ministry of Health of China), Key Laboratory of Neurobiology of Zhejiang Province, Zhejiang University School of Medicine, Hangzhou, China." The error has been corrected in the HTML and PDF versions of the article.
References
Blanchard, D.C. & Blanchard, R.J. Ethoexperimental approaches to the biology of emotion. Annu. Rev. Psychol. 39, 43–68 (1988).
Ohman, A. & Mineka, S. Fears, phobias, and preparedness: toward an evolved module of fear and fear learning. Psychol. Rev. 108, 483–522 (2001).
Battaglia, M. & Ogliari, A. Anxiety and panic: from human studies to animal research and back. Neurosci. Biobehav. Rev. 29, 169–179 (2005).
Knapska, E. et al. Functional anatomy of neural circuits regulating fear and extinction. Proc. Natl. Acad. Sci. USA 109, 17093–17098 (2012).
Sotres-Bayon, F., Sierra-Mercado, D., Pardilla-Delgado, E. & Quirk, G.J. Gating of fear in prelimbic cortex by hippocampal and amygdala inputs. Neuron 76, 804–812 (2012).
Maren, S., Phan, K.L. & Liberzon, I. The contextual brain: implications for fear conditioning, extinction and psychopathology. Nat. Rev. Neurosci. 14, 417–428 (2013).
Rosen, J.B. The neurobiology of conditioned and unconditioned fear: a neurobehavioral system analysis of the amygdala. Behav. Cogn. Neurosci. Rev. 3, 23–41 (2004).
Wallace, K.J. & Rosen, J.B. Neurotoxic lesions of the lateral nucleus of the amygdala decrease conditioned fear but not unconditioned fear of a predator odor: comparison with electrolytic lesions. J. Neurosci. 21, 3619–3627 (2001).
Root, C.M., Denny, C.A., Hen, R. & Axel, R. The participation of cortical amygdala in innate, odour-driven behaviour. Nature 515, 269–273 (2014).
Dielenberg, R.A., Hunt, G.E. & McGregor, I.S. “When a rat smells a cat”: the distribution of Fos immunoreactivity in rat brain following exposure to a predatory odor. Neuroscience 104, 1085–1097 (2001).
Wang, L., Chen, I.Z. & Lin, D. Collateral pathways from the ventromedial hypothalamus mediate defensive behaviors. Neuron 85, 1344–1358 (2015).
Lee, H. et al. Scalable control of mounting and attack by Esr1+ neurons in the ventromedial hypothalamus. Nature 509, 627–632 (2014).
Lin, D. et al. Functional identification of an aggression locus in the mouse hypothalamus. Nature 470, 221–226 (2011).
Gross, C.T. & Canteras, N.S. The many paths to fear. Nat. Rev. Neurosci. 13, 651–658 (2012).
Fendt, M., Endres, T., Lowry, C.A., Apfelbach, R. & McGregor, I.S. TMT-induced autonomic and behavioral changes and the neural basis of its processing. Neurosci. Biobehav. Rev. 29, 1145–1156 (2005).
Tovote, P. et al. Heart rate dynamics and behavioral responses during acute emotional challenge in corticotropin-releasing factor receptor 1-deficient and corticotropin-releasing factor-overexpressing mice. Neuroscience 134, 1113–1122 (2005).
Ekman, P., Levenson, R.W. & Friesen, W.V. Autonomic nervous system activity distinguishes among emotions. Science 221, 1208–1210 (1983).
Zhao, S. et al. Cell type–specific channelrhodopsin-2 transgenic mice for optogenetic dissection of neural circuitry function. Nat. Methods 8, 745–752 (2011).
Takahashi, L.K., Nakashima, B.R., Hong, H. & Watanabe, K. The smell of danger: a behavioral and neural analysis of predator odor-induced fear. Neurosci. Biobehav. Rev. 29, 1157–1167 (2005).
Wallace, K.J. & Rosen, J.B. Predator odor as an unconditioned fear stimulus in rats: elicitation of freezing by trimethylthiazoline, a component of fox feces. Behav. Neurosci. 114, 912–922 (2000).
Nestler, E.J. et al. Neurobiology of depression. Neuron 34, 13–25 (2002).
Wang, H.L. & Morales, M. Pedunculopontine and laterodorsal tegmental nuclei contain distinct populations of cholinergic, glutamatergic and GABAergic neurons in the rat. Eur. J. Neurosci. 29, 340–358 (2009).
Chow, B.Y. et al. High-performance genetically targetable optical neural silencing by light-driven proton pumps. Nature 463, 98–102 (2010).
Wickersham, I.R. et al. Monosynaptic restriction of transsynaptic tracing from single, genetically targeted neurons. Neuron 53, 639–647 (2007).
Likhtik, E., Stujenske, J.M., Topiwala, M.A., Harris, A.Z. & Gordon, J.A. Prefrontal entrainment of amygdala activity signals safety in learned fear and innate anxiety. Nat. Neurosci. 17, 106–113 (2014).
Sharma, A., Rale, A., Utturwar, K., Ghose, A. & Subhedar, N. Identification of the CART neuropeptide circuitry processing TMT-induced predator stress. Psychoneuroendocrinology 50, 194–208 (2014).
Janitzky, K. et al. Behavioral effects and pattern of brain c-fos mRNA induced by 2,5-dihydro-2,4,5-trimethylthiazoline, a component of fox feces odor in GAD67-GFP knock-in C57BL/6 mice. Behav. Brain Res. 202, 218–224 (2009).
Kessler, M.S. et al. fMRI fingerprint of unconditioned fear-like behavior in rats exposed to trimethylthiazoline. Eur. Neuropsychopharmacol. 22, 222–230 (2012).
Wei, P. et al. Processing of visually evoked innate fear by a non-canonical thalamic pathway. Nat. Commun. 6, 6756 (2015).
Yamaguchi, T., Danjo, T., Pastan, I., Hikida, T. & Nakanishi, S. Distinct roles of segregated transmission of the septo-habenular pathway in anxiety and fear. Neuron 78, 537–544 (2013).
Pobbe, R.L. & Zangrossi, H. Jr. Involvement of the lateral habenula in the regulation of generalized anxiety- and panic-related defensive responses in rats. Life Sci. 82, 1256–1261 (2008).
Sokolowski, K. et al. Specification of select hypothalamic circuits and innate behaviors by the embryonic patterning gene dbx1. Neuron 86, 403–416 (2015).
Pérez-Gómez, A. et al. Innate predator odor aversion driven by parallel olfactory subsystems that converge in the ventromedial hypothalamus. Curr. Biol. 25, 1340–1346 (2015).
Lenard, L.G. & Beer, B. 6-Hydroxydopamine and avoidance: possible role of response suppression. Pharmacol. Biochem. Behav. 3, 873–878 (1975).
Jhou, T.C., Fields, H.L., Baxter, M.G., Saper, C.B. & Holland, P.C. The rostromedial tegmental nucleus (RMTg), a GABAergic afferent to midbrain dopamine neurons, encodes aversive stimuli and inhibits motor responses. Neuron 61, 786–800 (2009).
Kunwar, P.S. et al. Ventromedial hypothalamic neurons control a defensive emotion state. eLife 4, e06633 (2015).
Cornwall, J., Cooper, J.D. & Phillipson, O.T. Afferent and efferent connections of the laterodorsal tegmental nucleus in the rat. Brain Res. Bull. 25, 271–284 (1990).
Berntson, G.G., Sarter, M. & Cacioppo, J.T. Anxiety and cardiovascular reactivity: the basal forebrain cholinergic link. Behav. Brain Res. 94, 225–248 (1998).
Palkovits, M. Interconnections between the neuroendocrine hypothalamus and the central autonomic system. Geoffrey Harris Memorial Lecture, Kitakyushu, Japan, October 1998. Front. Neuroendocrinol. 20, 270–295 (1999).
Lammel, S. et al. Input-specific control of reward and aversion in the ventral tegmental area. Nature 491, 212–217 (2012).
Wolff, S.B. et al. Amygdala interneuron subtypes control fear learning through disinhibition. Nature 509, 453–458 (2014).
Balaban, C.D. & Thayer, J.F. Neurological bases for balance-anxiety links. J. Anxiety Disord. 15, 53–79 (2001).
Carter, M.E. et al. Tuning arousal with optogenetic modulation of locus coeruleus neurons. Nat. Neurosci. 13, 1526–1533 (2010).
Li, K. et al. βCaMKII in lateral habenula mediates core symptoms of depression. Science 341, 1016–1020 (2013).
Li, B. et al. Synaptic potentiation onto habenula neurons in the learned helplessness model of depression. Nature 470, 535–539 (2011).
Boucetta, S., Cissé, Y., Mainville, L., Morales, M. & Jones, B.E. Discharge profiles across the sleep-waking cycle of identified cholinergic, GABAergic, and glutamatergic neurons in the pontomesencephalic tegmentum of the rat. J. Neurosci. 34, 4708–4727 (2014).
Benca, R.M., Obermeyer, W.H., Thisted, R.A. & Gillin, J.C. Sleep and psychiatric disorders. A meta-analysis. Arch. Gen. Psychiatry 49, 651–668, discussion 669–670 (1992).
Goldstein, A.N. & Walker, M.P. The role of sleep in emotional brain function. Annu. Rev. Clin. Psychol. 10, 679–708 (2014).
Acknowledgements
We thank G. Feng (Massachusetts Institute of Technology) for providing the VGAT-ChR2 (H134R)-eYFP mice and X. Zhang (Beijing Normal University) for providing the PV-Cre and SOM-Cre mice. We also thank I.C. Bruce for critically reading the paper. This work was supported by grants from the Major State Basic Research Program of China (2011CB504400, 2013CB945600 and 2015AA020515), the National Natural Science Foundation of China (31190060, 31471022, 31490590, 91232000, 91132307, 81221003 and 31471308), the National Key Technology R&D Program of the Ministry of Science and Technology of China (2012BAI01B08), the Program for Introducing Talents in Disciplines to Universities, the Zhejiang Provincial Natural Science Foundation of China (Y2110057) and Fundamental Research Funds for the Central Universities (2014FZA7007).
Author information
Authors and Affiliations
Contributions
H.Y., S.D. and H.W. designed the project, and H.Y. and J.Y. performed virus or drug injections, optogenetic behavior, electrophysiology experiments, and collected and analyzed the data. W.X. and S.H. helped to collect the data. H.Y. and S.H. performed immunohistochemistry and quantitatively analyzed the imaging data. W.X. designed the micro drive optrode system and performed freely moving recording. X.H. generated rabies and pseudo-rabies virus and F.X. supervised retrograde virus labeling experiments. B.L., H.L., L.Z., H.Y., Y.Y., S.D. and H.W. interpreted the results and commented on the manuscript. H.W. and S.D. wrote the manuscript. H.W. supervised all aspects of the project.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Mapping c-fos expression pattern in mouse brain provoked by TMT
Representative images of the c-fos expression pattern in the olfactory bulb (OB) (a), lateral hypothalamic area (LH) (b), and dorsomedial part of the ventromedial hypothalamic nucleus (VMHDM) (c) induced by 10-min exposure to saline (left), mercaptoethanol (ME) (middle), and TMT (right). Scale bar, 50 μm. (d) Average c-fos-positive cells per 250 μm2 imaging area; paraventricular thalamic nucleus (pv); lateral habenula (LHb); basolateral amygdala (Bla); central amygdala (Ce); ventral tegmental area (VTA); dorsolateral periaqueductal gray (dlPAG); ventrolateral periaqueductal gray (vlPAG); laterodorsal tegmental nucleus (LDT); locus coeruleus (LC). Samples from 5 mice, *P < 0.05, **P < 0.01, ***P < 0.001; error bars, SEM).
Supplementary Figure 2 Expression and function of ChR2 channels in GABAergic LDT neurons in VGAT-ChR2 (H134R)-eYFP (VGAT) mice
(a) Images of the LDT in coronal brain slices showing that GFP-expressing neurons were also immunopositive for anti-GAD67 staining. Scale bar, 50 μm. (b) Current-clamp (left) and voltage-clamp (right) recordings in slices showing that VGAT-positive neurons in the LDT responded to blue light; slices from 2 mice (1.18 mW/mm2, 20 Hz).
Supplementary Figure 3 Simultaneous cortical EEG and neck EMG recordings reveal that selective activation of GABAergic transmission in the LDT is unlikely to induce seizures or non-REM sleep
(a) Quantification of EEG frequency (0-50 Hz) bands with or without blue light stimulation (20 ms, 20 Hz) in the LDT of VGAT mice. (b) Quantification of EEG cumulative power (frequency from 50 to 300 Hz). (c) Enlarged figure of cortical EEG recording of boxes 1 and 2 in D. (d) Example of cortical EEG recording and neck EMG recordings. Blue lines indicate application of blue light (15 sec, 20 ms, 20 Hz). Recordings from 4 mice, *P < 0.05, **P < 0.01, ***P < 0.001; error bars, SEM.
Supplementary Figure 4 Anxiety-like responses induced by prolonged optogenetic stimulation of GABAergic transmission in the LDT are long-lasting
(a) Summary graphs for time spent and distance moved on the open arms in the elevated plus maze 24 h after photostimulation, n = 11 for WT and VGAT. (b) Summary graphs of time spent in and entries into lit compartment in the dark-light box test at 48 h before, and 30 min and 24 h after photostimulation, WT: n = 12, VGAT: n = 13. (*p < 0.05, **p < 0.01, ***p < 0.001; error bars, SEM).
Supplementary Figure 5 Optogenetic targeting of PV-ChR2 neurons
(a) Location of optical fibers within the right LDT in PV-ChR2 and control mice based on histological verification of 50-μm sections. (b) Serial sections showing localized ChR2-mCherry expression in the LDT (scale bar, 200 μm). (c) Example of optical fiber placement in the LDT. (d) Representative images from a brain slice showing that the ChR2-mCherry-expressing neurons were immunopositive for PV (right panel, scale bar, 50 μm) and the percentile of PV+ neurons that was transfected with virus (left panel, n = 6 slices from 4 mice).
Supplementary Figure 6 Optogenetic targeting of PV-Arch neurons
(a) Location of optical fibers within the right LDT of PV-Arch and control mice based on histological verification of 50-μm sections. (b) Serial sections showing that the expression of ChR2-mCherry is localized in the LDT and PV+ neurons are also projection neurons (scale bar, 200 μm). (c) Example of optical fiber placement in the LDT. (d) Representative images from a brain slice showed that Arch-GFP-expressing neurons were immunopositive for PV (Scale bar, 50 μm; n = 3 slices from 2 mice). Data were collected 4 - 6 weeks after injection of AAV-CAG-Dio-eArch3.0-eGFP(AAV 2/9) virus into the LDT of PV-Cre mice.
Supplementary Figure 7 Fear conditioned learning test in different groups
Inhibition of PV+ GABAergic neurons (1, p = 0.247; 2, p = 0.764; 3, p = 0.786; PV-GFP: n = 5, PV-Arch: n = 8) or activation of SOM+ GABAergic neurons (1, p = 0.972; 2, p = 0.927; 3, p = 0.863; SOM-cre: n = 10, SOM-ChR2: n = 10) did not affect fear-conditioned learning.
Supplementary Figure 8 Excitation of SOM+ GABAergic neurons in the LDT failed to induce fear behavior
(a) Upper left panel, sagittal slice containing the LDT from an SOM-ChR2-GFP mouse (obtained by mating SOM-Cre with Ai32 mice), Scale bar, 250 μm. Upper right panel, example of inward currents recorded from an LDT neuron expressing SOM-ChR2-GFP in response to blue light stimulation (1.18 mW/mm2, 10 Hz). Lower panels, example confocal images showing that SOM-ChR2-expressing neurons were immunopositive for anti-SOM and anti-GAD65/67, Scale bar, 50 μm. (b) The percentage of freezing time induced by activation of SOM+ GABAergic neurons with blue laser in the LDT, p = 0.274, n = 10. (c) Left, one sample of real time place preference location plots when stimulated SOM+ GABAergic neurons in the LDT. Right, activation of SOM+ GABAergic neurons in the LDT mice spent time on the stimulation side and un-stimulation side of the 10 min session, p = 0.18, n = 6.
Supplementary Figure 9 Mapping pre-synaptic inputs of LDT PV+ interneurons
(a) Coronal serial sections of trans-synaptically labeled cells in the LDT of a PV-cre mouse; yellow, starter cells; Scale bar, 100 μm. (b) Coronal representation of retrograde monosynaptic labeling of input neurons to PV+ interneurons in the LDT.
Supplementary Figure 10 Mapping pre-synaptic inputs of LDT SOM + interneurons
(a) Coronal serial sections of trans-synaptically labeled cells in the LDT of a SOM-cre mouse; yellow, starter cells; Scale bar, 100 μm. (b) Coronal representation of retrograde monosynaptic labeling of input neurons on to SOM+ interneurons in the LDT.
Supplementary Figure 11 SOM+ interneurons received local monosynaptic inputs from PV+ interneurons in the LDT
Example images of the LDT from SOM-cre mice showing that SOM+ GABAergic neurons (green) received local monosynaptic inputs from PV+ interneurons (blue). Yellow arrows, starter neurons (infected by both TVA-eGFP and rabies virus); white arrows, retrogradely-labeled PV neurons (transfected with rabies virus only). Scale bar, 50 μm.
Supplementary Figure 12 CamkIIa-ChR2 virus injected locations in the LHb
Coronal serial sections of AAV-CamkIIα-hChR2 (H134R)-mCherry (AAV 2/9) injections into the bilateral LHb. Scale bar, 200 μm.
Supplementary Figure 13 LDT unit recording in mice stimulated by optogenetic activation of terminals from the LHb or the predator odor TMT
(a, b) The same unit in the LDT showed rapid excitation by optogenetic stimulation of terminals from the LHb (a) and the predator odor TMT (b). (c, g) Schematic diagrams of recorded neurons receiving presumed monosynaptic (c) or polysynaptic (g) inputs from the LHb. (e, f) Sample traces showing the same units in the LDT phasically inhibited by activation of terminals from the LHb (e) and the predator odor (f). (d and h), Firing frequency of LDT neurons before, during, and after TMT stimulation, n = 3 and 18 cells. (*p < 0.05, **p < 0.01, ***p < 0.001)
Supplementary Figure 14 Activation of LH excitatory inputs in the LDT induces stress-like complex behaviors
(a) Coronal serial sections of AAV-CamkIIα-hChR2(H134R)-mCherry (AAV 2/9, CamkIIα-hChR2) injected into the LH. (b) Distribution of CamKIIa-ChR2-positive terminals and optical fiber placement in the LDT. (c) Schematic diagram showing injection of CamkIIα-hChR2 virus to the LH and light stimulation of their projection terminals in the LDT to induce behavioral responses. (d) Probability of freezing behavior during 15 s with light stimulation (15 ms, 20 Hz) in the LDT (p = 0.28), (e) Standing number (p = 0.264), (f) total time of standing (p = 0.001) and (g) scratching number (p = 0.047) during 30 s with light stimulation (15 ms, 20 Hz) in the LDT. Control: n = 8; ChR2: n = 11. (*p < 0.05, **p < 0.01, ***p < 0.001)
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–15 (PDF 2834 kb)
Optogenetic stimulation of LDT GABAergic interneurons in VGAT-ChR2(H134R)-eYFP mice induces immediate freezing-like behavior.
The duration of 15 s with the frequency of 20 Hz pulsed 470 nm laser is indicated by the appearance of blue "laser on" text. (MP4 4511 kb)
Photoactivation of GABAergic interneurons in the LDT failed to stop the movement of mice in the modified forced-swimming test.
The duration of light stimulation is indicated by the appearance of blue "laser on" text. (MP4 2864 kb)
Selective photoactivation of PV-positive GABAergic interneurons is sufficient to produce freezing-like behavior.
The duration of pulsed 470 nm laser is indicated by the appearance of blue "laser on" text. (MP4 8733 kb)
Yellow light-induced selective inhibition of PV-positive GABAergic interneurons transfected with archaerhodopsin suppresses the TMT-induced fear response.
A continuous 590 nm laser is given as indicated by the appearance of yellow "laser on" text. (MP4 21967 kb)
Photoactivation of SOM-positive GABAergic interneurons inhibits the TMT-induced fear response.
The duration of light stimulation is indicated by the appearance of blue "laser on" text. (MP4 6417 kb)
Photoactivation of glutamatergic terminals from the LHb to the LDT is sufficient to induce freezing-like behavior.
CamkIIa-ChR2 virus is injected into bilateral LHb 4 weeks prior to experiments and 20 Hz pulsed 470 nm laser stimulation is applied in the LDT, as indicated by the appearance of blue "laser on" text. (MP4 2963 kb)
Photoactivation of excitatory terminals in the LDT from the LH efferent pathway induced attention-like behaviors.
During the light stimulation as indicated by the appearance of blue "laser on" text, mice expresses standing and stereotyped scratching behaviors. (MP4 5029 kb)
Source data
Rights and permissions
About this article
Cite this article
Yang, H., Yang, J., Xi, W. et al. Laterodorsal tegmentum interneuron subtypes oppositely regulate olfactory cue-induced innate fear. Nat Neurosci 19, 283–289 (2016). https://doi.org/10.1038/nn.4208
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nn.4208
This article is cited by
-
Detection of neuronal defensive discharge information transmission and characteristics in periaqueductal gray double-subregions using PtNP/PEDOT:PSS modified microelectrode arrays
Microsystems & Nanoengineering (2023)
-
Male and female mice display consistent lifelong ability to address potential life-threatening cues using different post-threat coping strategies
BMC Biology (2022)
-
Reward and aversion encoding in the lateral habenula for innate and learned behaviours
Translational Psychiatry (2022)
-
A non-canonical GABAergic pathway to the VTA promotes unconditioned freezing
Molecular Psychiatry (2022)
-
Stereological estimations and neurochemical characterization of neurons expressing GABAA and GABAB receptors in the rat pedunculopontine and laterodorsal tegmental nuclei
Brain Structure and Function (2022)
