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Orexin signaling in GABAergic lateral habenula neurons modulates aggressive behavior in male mice

Nature Neuroscience volume 23, pages 638–650 (2020)Cite this article

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Abstract

Heightened aggression is characteristic of multiple neuropsychiatric disorders and can have various negative effects on patients, their families and the public. Recent studies in humans and animals have implicated brain reward circuits in aggression and suggest that, in subsets of aggressive individuals, domination of subordinate social targets is reinforcing. In this study, we showed that, in male mice, orexin neurons in the lateral hypothalamus activated a small population of glutamic acid decarboxylase 2 (GAD2)-expressing neurons in the lateral habenula (LHb) via orexin receptor 2 (OxR2) and that activation of these GAD2 neurons promoted male–male aggression and conditioned place preference for aggression-paired contexts. Moreover, LHb GAD2 neurons were inhibitory within the LHb and dampened the activity of the LHb as a whole. These results suggest that the orexin system is important for the regulation of inter-male aggressive behavior and provide the first functional evidence of a local inhibitory circuit within the LHb.

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Fig. 1: Aggressive behaviors are associated with decreased LHb activity.
Fig. 2: Aggressive behaviors are associated with increased expression of Fos mRNA in GAD2 LHb neurons.
Fig. 3: Aggressive behaviors are associated with increased GAD2 LHb neuron activity.
Fig. 4: GAD2 LHb neurons are locally inhibitory and regulate aggressive behaviors in AGGs.
Fig. 5: Characterization of an LHb orexin circuit.
Fig. 6: Optogenetic manipulation of orexin inputs to the LHb modulates aggressive behavior in AGGs.
Fig. 7: Knockdown of OxR2 in GAD2 LHb modulates aggressive behavior in AGGs.

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Data availability

The data that support these findings are available from the corresponding author upon reasonable request.

Code availability

MATLAB code used to analyze photometry data is available from the corresponding author upon reasonable request.

References

  1. Hill, A. P. et al. Aggressive behavior problems in children with autism spectrum disorders: prevalence and correlates in a large clinical sample. Res. Autism Spectr. Disord. 8, 1121–1133 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  2. Cha, J. et al. Neural correlates of aggression in medication-naive children with ADHD: multivariate analysis of morphometry and tractography. Neuropsychopharmacology 40, 1717–1725 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  3. Anderson, N. E. & Kiehl, K. A. Psychopathy and aggression: when paralimbic dysfunction leads to violence. Curr. Top. Behav. Neurosci. 17, 369–393 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  4. Miles, S. R., Menefee, D. S., Wanner, J., Teten Tharp, A. & Kent, T. A. The relationship between emotion dysregulation and impulsive aggression in veterans with posttraumatic stress disorder symptoms. J. Interpers. Violence 31, 1795–1816 (2016).

    Article  PubMed  Google Scholar 

  5. May, M. E. Aggression as positive reinforcement in people with intellectual disabilities. Res. Dev. Disabil. 32, 2214–2224 (2011).

    Article  PubMed  Google Scholar 

  6. Moran, J. K., Weierstall, R. & Elbert, T. Differences in brain circuitry for appetitive and reactive aggression as revealed by realistic auditory scripts. Front. Behav. Neurosci. 8, 425 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  7. Nell, V. Cruelty’s rewards: the gratifications of perpetrators and spectators. Behav. Brain Sci. 29, 211–224; discussion 224–257 (2006).

  8. Falkner, A. L., Grosenick, L., Davidson, T. J., Deisseroth, K. & Lin, D. Hypothalamic control of male aggression-seeking behavior. Nat. Neurosci. 19, 596–604 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Golden, S. A. et al. Basal forebrain projections to the lateral habenula modulate aggression reward. Nature 534, 688–692 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Flanigan, M., Aleyasin, H., Takahashi, A., Golden, S. A. & Russo, S. J. An emerging role for the lateral habenula in aggressive behavior. Pharmacol. Biochem. Behav. 162, 79–86 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Matsumoto, M. & Hikosaka, O. Lateral habenula as a source of negative reward signals in dopamine neurons. Nature 447, 1111–1115 (2007).

    Article  CAS  PubMed  Google Scholar 

  12. Baker, P. M. et al. The lateral habenula circuitry: reward processing and cognitive control. J. Neurosci. 36, 11482–11488 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Lawson, R. P. et al. Disrupted habenula function in major depression. Mol. Psychiatry 22, 202–208 (2017).

    Article  CAS  PubMed  Google Scholar 

  14. Ranft, K. et al. Evidence for structural abnormalities of the human habenular complex in affective disorders but not in schizophrenia. Psychol. Med. 40, 557–567 (2010).

    Article  CAS  PubMed  Google Scholar 

  15. Chou, M. Y. et al. Social conflict resolution regulated by two dorsal habenular subregions in zebrafish. Science 352, 87–90 (2016).

    Article  CAS  PubMed  Google Scholar 

  16. Brinschwitz, K. et al. Glutamatergic axons from the lateral habenula mainly terminate on GABAergic neurons of the ventral midbrain. Neuroscience 168, 463–476 (2010).

    Article  CAS  PubMed  Google Scholar 

  17. Hashikawa, Y., et al. Transcriptional and spatial resolution of cell types in the mammalian habenula. Preprint at bioRxiv https://doi.org/10.1101/772376 (2019).

  18. Zhang, L. et al. A GABAergic cell type in the lateral habenula links hypothalamic homeostatic and midbrain motivation circuits with sex steroid signaling. Transl. Psychiatry 8, 50 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  19. Peyron, C. et al. Neurons containing hypocretin (orexin) project to multiple neuronal systems. J. Neurosci. 18, 9996–10015 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Tsunematsu, T., et al. Acute optogenetic silencing of orexin/hypocretin neurons induces slow-wave sleep in mice. J. Neurosci. 31, 10529–10539 (2011).

  21. Blouin, A. M. et al. Human hypocretin and melanin concentrating hormone levels are linked to emotion and social interaction. Nat. Commun. 4, 1547 (2013).

    Article  PubMed  CAS  Google Scholar 

  22. Harris, G. C., Wimmer, M. & Aston-Jones, G. A role for lateral hypothalamic orexin neurons in reward seeking. Nature 437, 556–559 (2005).

    Article  CAS  PubMed  Google Scholar 

  23. Muschamp, J. W. et al. Hypocretin (orexin) facilitates reward by attenuating the antireward effects of its cotransmitter dynorphin in ventral tegmental area. Proc. Natl Acad. Sci. USA 111, E1648–E1655 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Quarta, D. & Smolders, I. Rewarding, reinforcing and incentive salient events involve orexigenic hypothalamic neuropeptides regulating mesolimbic dopaminergic neurotransmission. Eur. J. Pharm. Sci. 57, 2–10 (2014).

    Article  CAS  PubMed  Google Scholar 

  25. Richardson, K. A. & Aston-Jones, G. Lateral hypothalamic orexin/hypocretin neurons that project to VTA are differentially activated with morphine preference. J. Neurosci. 32, 3809–3817 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Tung, L. W. et al. Orexins contribute to restraint stress-induced cocaine relapse by endocannabinoid-mediated disinhibition of dopaminergic neurons. Nat. Commun. 7, 12199 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Schwartzer, J. J., Ricci, L. A. & Melloni, R. H. Jr. Prior fighting experience increases aggression in Syrian hamsters: implications for a role of dopamine in the winner effect. Aggress. Behav. 39, 290–300 (2013).

    Article  PubMed  Google Scholar 

  28. Trusel, M. et al. Punishment-predictive cues guide avoidance through potentiation of hypothalamus-to-habenula synapses. Neuron 102, 120–127.e4 (2019).

    Article  CAS  PubMed  Google Scholar 

  29. Lazaridis, I., et al. A hypothalamus-habenula circuit controls aversion. Mol. Psychiatry 24, 1351–1368 (2019).

  30. Takahashi, A. et al. Glutamate input in the dorsal raphe nucleus as a determinant of escalated aggression in male mice. J. Neurosci. 35, 6452–6463 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Yu, Q. et al. Dopamine and serotonin signaling during two sensitive developmental periods differentially impact adult aggressive and affective behaviors in mice. Mol. Psychiatry 19, 688–698 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Wersinger, S. R., Ginns, E. I., O’Carroll, A. M., Lolait, S. J. & Young Iii, W. S. Vasopressin V1b receptor knockout reduces aggressive behavior in male mice. Mol. Psychiatry 7, 975 (2002).

    Article  CAS  PubMed  Google Scholar 

  33. Ogawa, S. et al. Abolition of male sexual behaviors in mice lacking estrogen receptors α and β (αβERKO). Proc. Natl Acad. Sci. USA 97, 14737–14741 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Takahashi, A., Nagayasu, K., Nishitani, N., Kaneko, S. & Koide, T. Control of intermale aggression by medial prefrontal cortex activation in the mouse. PLoS ONE 9, e94657 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  35. Wong, L. C. et al. Effective modulation of male aggression through lateral septum to medial hypothalamus projection. Curr. Biol. 26, 593–604 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Gunaydin, L. A. et al. Natural neural projection dynamics underlying social behavior. Cell 157, 1535–1551 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Proulx, C. D., Hikosaka, O. & Malinow, R. Reward processing by the lateral habenula in normal and depressive behaviors. Nat. Neurosci. 17, 1146–1152 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Gentile, T. A. et al. Suvorexant, an orexin/hypocretin receptor antagonist, attenuates motivational and hedonic properties of cocaine. Addict. Biol. 23, 247–255 (2018).

    Article  CAS  PubMed  Google Scholar 

  39. Sartor, G. C. & Aston-Jones, G. S. A septal–hypothalamic pathway drives orexin neurons, which is necessary for conditioned cocaine preference. J. Neurosci. 32, 4623–4631 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Shoblock, J. R. et al. Selective blockade of the orexin-2 receptor attenuates ethanol self-administration, place preference, and reinstatement. Psychopharmacology 215, 191–203 (2011).

    Article  CAS  PubMed  Google Scholar 

  41. Gonzalez, J. A., Iordanidou, P., Strom, M., Adamantidis, A. & Burdakov, D. Awake dynamics and brain-wide direct inputs of hypothalamic MCH and orexin networks. Nat. Commun. 7, 11395 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Muschamp, J. W., Dominguez, J. M., Sato, S. M., Shen, R. Y. & Hull, E. M. A role for hypocretin (orexin) in male sexual behavior. J. Neurosci. 27, 2837–2845 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Stamatakis, A. M. et al. Lateral hypothalamic area glutamatergic neurons and their projections to the lateral habenula regulate feeding and reward. J. Neurosci. 36, 302–311 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Lin, D. et al. Functional identification of an aggression locus in the mouse hypothalamus. Nature 470, 221–226 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Chemelli, R. M. et al. Narcolepsy in orexin knockout mice: molecular genetics of sleep regulation. Cell 98, 437–451 (1999).

    Article  CAS  PubMed  Google Scholar 

  46. Herring, W. J. et al. Suvorexant in patients with insomnia: results from two 3-month randomized controlled clinical trials. Biol. Psychiatry 79, 136–148 (2016).

    Article  CAS  PubMed  Google Scholar 

  47. Malherbe, P. et al. Biochemical and behavioural characterization of EMPA, a novel high-affinity, selective antagonist for the OX(2) receptor. Br. J. Pharmacol. 156, 1326–1341 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Beig, M. I., Dampney, B. W. & Carrive, P. Both Ox1r and Ox2r orexin receptors contribute to the cardiovascular and locomotor components of the novelty stress response in the rat. Neuropharmacology 89, 146–156 (2015).

    Article  CAS  PubMed  Google Scholar 

  49. Todd, W. D. et al. A hypothalamic circuit for the circadian control of aggression. Nat. Neurosci. 21, 717–724 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Mendoza, J. Circadian neurons in the lateral habenula: clocking motivated behaviors. Pharmacol. Biochem. Behav. 162, 55–61 (2017).

    Article  CAS  PubMed  Google Scholar 

  51. Golden, S. A. et al. Persistent conditioned place preference to aggression experience in adult male sexually-experienced CD-1 mice. Genes Brain Behav. 16, 44–55 (2017).

    Article  CAS  PubMed  Google Scholar 

  52. Couppis, M. H. & Kennedy, C. H. The rewarding effect of aggression is reduced by nucleus accumbens dopamine receptor antagonism in mice. Psychopharmacology 197, 449–456 (2008).

    Article  CAS  PubMed  Google Scholar 

  53. Stagkourakis, S. et al. A neural network for intermale aggression to establish social hierarchy. Nat. Neurosci. 21, 834–842 (2018).

    Article  CAS  PubMed  Google Scholar 

  54. Golden, S. A., Covington, H. E. 3rd, Berton, O. & Russo, S. J. A standardized protocol for repeated social defeat stress in mice. Nat. Protoc. 6, 1183–1191 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Krishnan, V. et al. Molecular adaptations underlying susceptibility and resistance to social defeat in brain reward regions. Cell 131, 391–404 (2007).

    Article  CAS  PubMed  Google Scholar 

  56. Arendt, D. H. et al. Anxiolytic function of the orexin 2/hypocretin A receptor in the basolateral amygdala. Psychoneuroendocrinology 40, 17–26 (2014).

    Article  CAS  PubMed  Google Scholar 

  57. Whiddon, B. B. & Palmiter, R. D. Ablation of neurons expressing melanin-concentrating hormone (MCH) in adult mice improves glucose tolerance independent of MCH signaling. J. Neurosci. 33, 2009–2016 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Daviaud, N., Friedel, R. H. & Zou, H. Vascularization and engraftment of transplanted human cerebral organoids in mouse cortex. eNeuro 5, ENEURO.0219-18.2018 (2018).

  59. Arruda-Carvalho, M. & Clem, R. L. Pathway-selective adjustment of prefrontal-amygdala transmission during fear encoding. J. Neurosci. 34, 15601–15609 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  60. Petreanu, L., Mao, T., Sternson, S. M. & Svoboda, K. The subcellular organization of neocortical excitatory connections. Nature 457, 1142–1145 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Schöne, C. et al. Coreleased orexin and glutamate evoke nonredundant spike outputs and computations in histamine neurons. Cell Rep. 7, 697–704 (2014).

  62. Calipari, E. S. et al. In vivo imaging identifies temporal signature of D1 and D2 medium spiny neurons in cocaine reward. Proc. Natl Acad. Sci. USA 113, 2726–2731 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Stagkourakis, S. et al. A neural network for intermale aggression to establish social hierarchy. Nat. Neurosci. 21, 834–842 (2018).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

The authors would like to thank S. Feng and C. Ferrer for their assistance with histology, N. Tzvaras for his assistance with microscopy and Virovek Inc. for cloning and packaging of AAV viruses. This work was supported by National Institutes of Health grants R01 MH114882-01 (to S.J.R.), R01 MH090264-06 (to S.J.R.), P50 MH096890 (to S.J.R.), P50 AT008661 (to S.J.R.), F31 MH111108-01A1 (to M.E.F.), T32 MH096678 (to M.E.F.), T32 MH087004 (to M.E.F.) and R01 MH51399 (to E.J.N.).

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Authors and Affiliations

  1. Nash Family Department of Neuroscience, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Meghan E. Flanigan, Hossein Aleyasin, Long Li, C. Joseph Burnett, Kenny L. Chan, Katherine B. LeClair, Elizabeth K. Lucas, Bridget Matikainen-Ankney, Romain Durand-de Cuttoli, Aki Takahashi, Caroline Menard, Madeline L. Pfau, Sylvain Bouchard, Erin S. Calipari, Eric J. Nestler, George W. Huntley, Roger L. Clem & Scott J. Russo

  2. Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Meghan E. Flanigan, Hossein Aleyasin, Long Li, C. Joseph Burnett, Kenny L. Chan, Katherine B. LeClair, Elizabeth K. Lucas, Bridget Matikainen-Ankney, Romain Durand-de Cuttoli, Aki Takahashi, Caroline Menard, Madeline L. Pfau, Sylvain Bouchard, Erin S. Calipari, Eric J. Nestler, George W. Huntley, Roger L. Clem & Scott J. Russo

  3. Department of Molecular Biomedical Sciences, North Carolina State University, Raleigh, NC, USA

    Elizabeth K. Lucas

  4. Laboratory of Behavioral Neuroendocrinology, University of Tsukuba, Tsukuba, Ibaraki, Japan

    Aki Takahashi

  5. Department of Psychiatry and Neuroscience, Faculty of Medicine and CERVO Brain Research Center, Université Laval, Ville de Québec, QC, Canada

    Caroline Menard

  6. Department of Biological Structure, University of Washington, Seattle, WA, USA

    Sam A. Golden

  7. Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN, USA

    Erin S. Calipari

  8. Department of Psychiatry, Yale University, New Haven, CT, USA

    Ralph J. DiLeone

  9. Department of Neuroscience II, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan

    Akihiro Yamanaka

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  1. Meghan E. Flanigan
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Contributions

Stereotaxic surgeries were performed by M.E.F., H.A., M.L.P., S.A.G. and A.T. IHC analysis and ISH were performed by M.E.F., C.M. and K.B.L. Microscopy was performed by M.E.F. Molecular cloning of miR-OxR2 constructs was performed by H.A. qPCR was performed by K.L.C and M.E.F. Fiber photometry data collection was performed by M.E.F. and C.J.B., and fiber photometry analysis was performed by M.E.F., C.J.B., E.S.C., E.J.N. and S.B. OxR2 miRNA design was performed by R.J.D. Orexin-Cre mice were made by A.Y. Behavioral experiments were performed by M.E.F., H.A., L.L, C.J.B. and K.B.L. Electrophysiology experiments were performed by R.D.C, R.L.C., E.K.L., G.W.H. and B.M.A. Results were analyzed and interpreted by M.E.F. and S.J.R. The manuscript was written by M.E.F. and S.J.R. and edited by all authors.

Corresponding author

Correspondence to Scott J. Russo.

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Competing interests

S.J.R. and M.E.F. have a patent pending (US Patent Application 62/11,233) for the use of OxR2 antagonists to treat aggression.

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Peer review information Nature Neuroscience thanks Stephen Mahler and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 LHb non-conditional fiber photometry supporting data.

a, AGG average LHb activity was reduced following a bite on day 1 of RI (two-tailed paired t-test, n = 5 biologically independent mice, 3–5 bites per mouse, t(4)=3.763, p = 0.0197). b, AGG average LHb activity was increased following a withdrawal from aggression on day 1 of RI (two-tained paired t-test, n = 5 biologically mice, 3–5 withdrawals per mouse, t(4)=3.229, p = 0.03). c, AGG average LHb activity did not differ before and after random time points during RI on day 3 (two-tailed paired t-test, n = 5 biologically independent mice, t(4)=0.6545, p = 0.5485). d, NON average LHb activity was not significantly increased following intruder approach on day 1 of RI (two-tailed paired t-test, n = 5 biologically independent mice, 3-5 approaches per mouse, t(4)=2.25, p = 0.087). e, NON average LHb activity was not significantly reduced following withdrawal from social interactions on day 1 of RI (two-tailed paired t-test, n = 5 biologically independent mice, 3-5 withdrawals per mouse, t(4)=2.353, p = 0.078). f, NON average LHb activity did not differ before and after random time points during RI on day 3 (two-tailed paired t-test, n = 5 biologically independent mice, 5 time points per mouse, t(4)=0.553, p = 0.6221). g, AGGs used for fiber photometry experiments displayed significantly higher aggression CPP scores than NONs (two-tailed student’s t-test, n = 4 biologically independent mice per group, t(6)=5.591, p = 0.0014). h, LHb peaks in the intruder paired context during the CPP preference test were negatively correlated with CPP score (two-tailed student’s t-test, n = 8 biologically independent mice, Pearson correlation coefficient = -0.94, R2 = 0.88, p = 0.0005). *p < 0.05, **p < 0.01. All data are expressed as mean ± SEM.

Extended Data Fig. 2 LHB GAD2 neuron fiber phohometry supporting data.

a, AGG LHb GAD2 neuron activity was increased following bites on day 1 of RI (two-tailed paired t-test, n = 5 biologically independent mice, 2-5 bites per mouse, t(4)=4.008, p = 0.016). b, AGG LHb GAD2 neuron activity was reduced following a withdrawal from an aggressive encounter on day 1 of RI (two-tailed paired t-test, n = 5 biologically independent mice, 3-5 withdrawals per mouse, t(4)=3.982, p = 0.0164). c, AGG LHb GAD2 neuron activity did not differ before and after random times points on day 3 of RI (two-tailed paired t-test, n = 5 biologically independent mice, 5 time points per mouse, t(4)=0.493, p = 0.6475). d, NON LHb GAD2 neuron activity was not different before and after an approach on day 1 of RI (two-tailed paired t-test, n = 5 biologically independent mice, 3-5 approaches per mouse, t(4)=1.843, p = 0.1406). e, NON LHb GAD2 neuron activity was not different before and after a withdrawal from a non-aggressive social interaction on day 1 of RI (two-tailed paired t-test, n = 5 biologically independent mice, 3-5 withdrawals per mouse, t(4)-1.633, p = 0.1777). f, NON LHb GAD2 neuron activity did not differ before and after random time points on day 3 of RI (two-tailed paired t-test, n = 5 biologically independent mice, t(4)=1.721, p = 0.1634). g, GAD2-cre AGGs used for fiber photometry experiments displayed significantly higher aggression CPP scores than GAD2-cre NONs (two-tailed paired t-test, n = 5 biologically independent mice, t(4)=2.885, p = 0.0448). h, LHb GAD2 neurons peaks in the intruder paired context during the CPP preference test were positively correlated with CPP score (two-tailed student’s t-test,A n = 10 biologically independent mice, Pearson correlation coefficient = 0.761 m, R2 = 0.586, p = 0.0161). *p < 0.05, **p < 0.01. *p < 0.05. All data are expressed as mean ± SEM.

Extended Data Fig. 3 Anterograde tracing of LHB GAD2 neuron projections.

a, Schematic of surgical manipulations for anterograde tracing of GAD2 LHb neurons. b, Representative image of viral infection in GAD2 LHb neurons. Experimental images were obtained from 3 biologically independent mice, three slices per mouse, with similar results obtained. c, Representative image of the interpeduncular nucleus (IPN) and ventral tegmental area (VTA) in mice expressing eGFP in GAD2 LHb neurons. Experimental images were obtained from 3 biologically independent mice, three slices per mouse, with similar results obtained. d, Representative image of the rostromedial tegmental nucleus (RMTg) in mice expressing eGFP in GAD2 LHb neurons. Experimental images were obtained from 3 biologically independent mice, three slices per mouse, with similar results obtained. e, Representative image of the RMTg and anterior dorsal and median raphe nuclei (DRN and MRN) in mice expressing eGFP in GAD2 LHb neurons. Experimental images were obtained from 3 biologically independent mice, three slices per mouse, with similar results obtained. f, Schematic of surgical manipulations for non-conditional anterograde tracing of LHb neurons. g, Representative image of viral infection in LHb neurons. Experimental images were obtained from 3 biologically independent mice, three slices per mouse, with similar results obtained. h, Representative image of the interpeduncular nucleus (IPN) and ventral tegmental area (VTA) in mice expressing eGFP in LHb neurons. Experimental images were obtained from 3 biologically independent mice, three slices per mouse, with similar results obtained. i, Representative image of the rostromedial tegmental nucleus (RMTg) in mice expressing eGFP in LHb neurons. Experimental images were obtained from 3 biologically independent mice, three slices per mouse, with similar results obtained. j, Representative image of the RMTg and anterior dorsal and median raphe nuclei (DRN and MRN) in mice expressing eGFP in LHb neurons. Experimental images were obtained from 3 biologically independent mice, three slices per mouse, with similar results obtained. Scale bars= 500 μm.

Extended Data Fig. 4 RI behavior in NONs during optogenetic stimulation of OxR2 over-expression in LHb GAD2 neurons.

a, ChR2-mediated stimulation of GAD2 LHb neurons in NONs did not affect attack latency during RI (two-tailed paired t-test, n = 7 biologically independent mice, t(6)=1.0, p = 0.3559). b, ChR2-mediated stimulation of GAD2 LHb neurons in NONs did not affect attack duration during RI (two-tailed paired t-test, n = 7 biologically independent mice, t(6)=1.0, p = 0.3559). c, ChR2-mediated stimulation of orexin terminals in the LHb did not affect attack latency during RI (two-tailed paired t-test, n = 6 biologically independent mice, t(5)=1.0, p = 0.3632). d, ChR2-mediated stimulation of orexin terminals in the LHb did not affect attack duration during RI (two-tailed paired t-test, n = 5 biologically independent mice, t(5)=1.0, p = 0.3632). e, Over-expression of OxR2 in GAD2 LHb neurons in NONs did not affect attack latency during RI (two-tailed student’s t-test, n = 8 biologically independent GFP mice and n = 11 biologically independent OxR2-OE mice, t(17)=0.8338, p = 0.4160). f, Over-expression of OxR2 in GAD2 LHb neurons in NONs did not affect attack duration during RI (two-tailed student’s t-test, n = 8 biologically independent GFP mice and n = 11 biologically independent OxR2-OE mice, t(17)=0.9951. All data are expressed as mean ± SEM.

Extended Data Fig. 5 Histology and 3D rendering of LHb GAD2 neurons and orexin axons.

a, Immunohistochemistry for orexin-A (red), DAPI (blue), and eGFP (green) in a GAD2-Cre mouse injected with AAV-DIO-eGFP, scale bar = 300 μm. Experimental images were taken from 3 biologically independent mice, 3 slices per mouse, with similar results obtained. b, Immunohistochemistry for orexin-A (red), DAPI (blue), and GFP (green) in a GAD2-Cre mouse injected with AAV-DIO-eGFP, scale bar = 10 μm. Experimental images were taken from 3 biologically independent mice, 3 slices per mouse, with similar results obtained. c, 3D rendering of image in b, color of GAD2 neuron coincides with estimated distance from orexin-A axon according to key in lower right corner, scale bar = 5 μm. Experimental images were taken from 3 biologically independent mice, 3 slices per mouse, with similar results obtained.

Extended Data Fig. 6 Attack latencies for AGGs and NONs used in qPCR and ISH experiments.

a, Attack latency for one day of RI in mice used for LHb qPCR, n = 9 biologically independent NON mice, n = 7 biologically independent AGG mice. b, Average attack latency for three days of RI in mice used for LHb qPCR, n = 7 biologically independent NON mice, n = 8 biologially independent AGG mice. c, Average attack latency for three days of RI in mice used for LHb OxR2 ISH, n = 6 biologically independent NON mice, n = 6 biologically independent AGG mice. d, Representative images from OxR2 ISH in AGG and NON LHb vGlut2 neurons following RI, accompanies Fig. 5j, scale bar = 20 μm. Notably, OxR2 expression was barely detectable in these neurons in AGGs or NONs, which is in line with our findings showing low OxR2 expression in vGlut2 neurons in Fig. 5b, c. Experimental images were taken from 12 biologically independent mice, 2 slices per mouse, with similar results obtained. All data are expressed as mean ± SEM.

Extended Data Fig. 7 Effects of systemic antagonism of OxR2 with EMPA on aggression and LHb activity.

a, Experimental scheme for OxR2 systemic antagonism RI experiment. b, RI test attack latency in animals treated with EMPA and vehicle (two-tailed paired t-test, n = 11 biologically independent mice per group, t(10)=0.3215, p = 0.758). c, RI test attack duration in animals treated with EMPA and vehicle (two-tailed paired t-test, n = 11 biologically independent mice per group, t(10)=2.888, p = 0.016).d, Experimental scheme for OxR2 systemic antagonism aggression CPP and locomotion experiments. e, Aggression CPP for animals treated with EMPA and vehicle (two-tailed student’s t-test, n = 12 biologically independent vehicle mice and n = 11 biologically independent EMPA mice, t(21)=2.885, p = 0.0086). f, Locomotor activity in the open field for animals treated with EMPA and vehicle (two-tailed student’s t-test, n = 12 biologically independent vehicle mice and n = 11 biologically independent EMPA mice, t(21)=0.1301, p = 0.8991). g, Anxiety-related behavior in the open field for animals treated with EMPA or vehicle (two-tailed student’s t-test, n = 11 biologically independent mice per group, t(21)=1.134, p = 0.2695) h, Representative fiber photometry traces in an animal treated with vehicle and EMPA. i, LHb GCaMP peaks during RI during vehicle and EMPA treatment (two-tailed paired t-test, n = 5 biologically independent mice, t(4)=2.946, p = 0.0421). *p < 0.05, **p < 0.01. All data are expressed as mean ± SEM.

Extended Data Fig. 8 LHb orexin-ChR2 experiments supporting data.

a, >90% of neurons infected with AAV1-DIO-YFP were positive for orexin-A protein as determined by immunohistochemistry, n = 3 biologically independent mice, 3 slices per mouse. b, Surgical manipulations for ChR2-mediated activation of orexin terminals in the LHb with concurrent knockdown of LHb OxR2. c, Optogenetic stimulation of orexin terminals in the LHb reduced attack latency in mice treated with the miR-scrambed virus, but not the miR-OxR2 virus (two-tailed paired t-test, miR-scrambled: n = 11 biologially independent mice, t(10)=2.424, p = 0.0358; miR-OxR2: n = 9 biologically independent mice, t(8)=0.5281, p = 0.6117). d, Optogenetic stimulation of orexin terminals in the LHb increased attack duration in mice treated with the miR-scrambled virus, but not the miR-OxR2 virus (two-tailed paired t-test, miR-scrambled: n = 11 biologically independent mice, t(10)=2.260, p = 0.0474; miR-OxR2: n = 9 biologically independent mice, t(8)=0.8493, p = 0.4204). *p < 0.05. All data are expressed as mean ± SEM.

Extended Data Fig. 9 In-vitro and in-vivo validation of AAV-DIO-miR-OxR2 virus.

a, N2A cells treated with miR-OxR2 construct selectively reduced OxR2 expression compared to cells treated with miR-scrambled construct, but did not reduce expression of related transcripts (two-tailed student’s t-test, n = 3 biologically independent plates per group, 3 replicates per plate; OxR2: t(4)=2.402, p = 0.0482; OxR1: t(4)=0.2123, p = 0.8423; Avpr2: t(4)=0.3686, p = 0.7311; Htr3a: t(4)=1.309, p = 0.2607; Drd4: t(4)=0.1925, p = 0.8567; Nmur: t(4)=1.672, p = 0.1699; Drd1: t(4)=0.9239, p = 0.4078; Mchr1: t(4)=1.467, p = 0.2163; Glpr1: t(4)=1.785, p = 0.1488). b, GAD2-Cre mice injected with AAV-DIO-miR-OxR2 displayed reduced expression of OxR2 compared to mice injected with AAV-DIO-miR-scrambled as determined by ISH (two-tailed student’s t-test, n = 3 mice, 2 slices per mouse, t(4)=18.44, p = 0.0001). c, Representative image of GFP expression localized to GAD2 LHb neurons in GAD2-Cre mice injected with AAV-DIO-miR-OxR2, scale bar = 25 μm. Experimental images were obtained from 6 biologically independent mice, 2 slices per mouse, with similar results obtained. d, Representative images of OxR2 expression in GAD2 LHb neurons infected with AAV-DIO-miR-OxR2 or AAV-DIO-miR-scrambled, scale bar, 20 μm. *p < 0.05, ***p < 0.001. All data are expressed as mean ± SEM.

Extended Data Fig. 10 In-vitro and in-vivo validation of AAV-DIO-OxR2 over-expression virus.

a, N2A cells treated with OxR2 over-expression construct selectively increased OxR2 expression compared to controls (two-tailed student’s t-test, n = 3 biologically independent plates per group, 3 replicates per plate, OxR2: t(4)=3.939, p = 0.0171; OxR1: t(4)=0.1238, p = 0.9075). b, GAD2-Cre mice injected with AAV-DIO-OxR2 displayed increased expression of OxR2 compared to mice injected with AAV-DIO-GFP as determined by ISH (two-tailed student’s t-test, n = 3 biologically independent mice, 2 slices per mouse, t(4)=4.417, p = 0.0069) (left). Representative image of GFP expression localized to GAD2 LHb neurons in GAD2-Cre mice injected with AAV-DIO-OxR2, scale bar = 25 μm (right). Experimental images were obtained from 7 biologically independent mice, 2 slices per mouse, with similar results obtained. c, Representative images of OxR2 expression in GAD2 LHb neurons infected with AAV-DIO-OxR2 or AAV-DIO-GFP, scale bar = 25 μm. *p < 0.05, **p < 0.01. Experimental images were taken from 7 biologically independent mice, 2 slices per mouse, with similar results obtained. All data are expressed as mean ± SEM.

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Flanigan, M.E., Aleyasin, H., Li, L. et al. Orexin signaling in GABAergic lateral habenula neurons modulates aggressive behavior in male mice. Nat Neurosci 23, 638–650 (2020). https://doi.org/10.1038/s41593-020-0617-7

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