Brain Structure and Function

, Volume 224, Issue 1, pp 19–31 | Cite as

A suprachiasmatic-independent circadian clock(s) in the habenula is affected by Per gene mutations and housing light conditions in mice

  • Nora L. Salaberry
  • Hélène Hamm
  • Marie-Paule Felder-Schmittbuhl
  • Jorge MendozaEmail author
Original Article


For many years, the suprachiasmatic nucleus (SCN) was considered as the unique circadian pacemaker in the mammalian brain. Currently, it is known that other brain areas are able to oscillate in a circadian manner. However, many of them are dependent on, or synchronized by, the SCN. The Habenula (Hb), localized in the epithalamus, is a key nucleus for the regulation of monoamine activity (dopamine, serotonin) and presents circadian features; nonetheless, the clock properties of the Hb are not fully described. Here, we report, first, circadian expression of clock genes in the lateral habenula (LHb) under constant darkness (DD) condition in wild-type mice which is disturbed in double Per1−/−-Per2Brdm1 clock-mutant mice. Second, using Per2::luciferase transgenic mice, we observed a self-sustained oscillatory ability (PER2::LUCIFERASE bioluminescence rhythmicity) in the rostral and caudal part of the Hb of arrhythmic SCN-ablated animals. Finally, in Per2::luciferase mice exposed to different lighting conditions (light-dark, constant darkness or constant light), the period or amplitude of PER2 oscillations, in both the rostral and caudal Hb, were similar. However, under DD condition or from SCN-lesioned mice, these two Hb regions were out of phase, suggesting an uncoupling of two putative Hb oscillators. Altogether, these results suggest that an autonomous clock in the rostral and caudal part of the Hb requires integrity of circadian genes to tick, and light information or SCN innervation to keep synchrony. The relevance of the Hb timing might reside in the regulation of circadian functions linked to motivational (reward) and emotional (mood) processes.


Circadian Habenula Per2 luciferase Suprachiasmatic Clock genes 



We thank deeply Prof. Urs Albrecht (University of Fribourg) for providing the Per1−/−Per2Brdm1 mutant mice. Per1 and Cry2 plasmids were kindly donated by Prof. H. Okamura (Kyoto University, Japan). mClock and rRevErbα plasmids were generously provided by Prof. J. Takahashi (Northwestern University) and Dr. Hugues Dardente (University of Tours), respectively. We thank the personal of the Chronobiotron Platform (UMS 3414, Strasbourg) for animal care. Funding sources of the present study were provided by the Agence National de la Recherche (ANR-14-CE13-0002-01 ADDiCLOCK JCJC to JM and NLS PhD fellow) and the Centre National de la Recherche Scientifique (JM and MPFS).

Compliance with ethical standards

Conflict of interest

Authors declare no potential conflicts of interest.

Human or animal rights

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Ethical approval

All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.

Supplementary material

429_2018_1756_MOESM1_ESM.doc (4 mb)
Supplementary material 1 (DOC 4057 KB)


  1. Abraham D, Dallmann R, Steinlechner S et al (2006) Restoration of circadian rhythmicity in circadian clock-deficient mice in constant light. J Biol Rhythms 21:169–176. CrossRefGoogle Scholar
  2. Aizawa H, Cui W, Tanaka K, Okamoto H (2013) Hyperactivation of the habenula as a link between depression and sleep disturbance. Front Hum Neurosci 7:826. CrossRefGoogle Scholar
  3. Bae K, Jin X, Maywood ES et al (2001) Differential functions of mPer1, mPer2, and mPer3 in the SCN circadian clock. Neuron 30:525–536CrossRefGoogle Scholar
  4. Besing RC, Rogers CO, Paul JR et al (2017) GSK3 activity regulates rhythms in hippocampal clock gene expression and synaptic plasticity. Hippocampus 27:890–898. CrossRefGoogle Scholar
  5. Bianco IH, Wilson SW (2009) The habenular nuclei: a conserved asymmetric relay station in the vertebrate brain. Philos Trans R Soc Lond B Biol Sci 364:1005–1020. CrossRefGoogle Scholar
  6. Brancaccio M, Patton AP, Chesham JE et al (2017) Astrocytes control circadian timekeeping in the suprachiasmatic nucleus via glutamatergic signaling. Neuron 93:1420–1435.e5. CrossRefGoogle Scholar
  7. Brinschwitz K, Dittgen A, Madai VI et al (2010) Glutamatergic axons from the lateral habenula mainly terminate on GABAergic neurons of the ventral midbrain. Neuroscience 168:463–476. CrossRefGoogle Scholar
  8. Cheng R-K, Krishnan S, Lin Q et al (2017) Characterization of a thalamic nucleus mediating habenula responses to changes in ambient illumination. BMC Biol 15:104. CrossRefGoogle Scholar
  9. Christiansen SL, Bouzinova EV, Fahrenkrug J, Wiborg O (2016) Altered expression pattern of clock genes in a rat model of depression. Int J Neuropsychopharmacol 19:pyw061. CrossRefGoogle Scholar
  10. Christoph GR, Leonzio RJ, Wilcox KS (1986) Stimulation of the lateral habenula inhibits dopamine-containing neurons in the substantia nigra and ventral tegmental area of the rat. J Neurosci 6:613–619CrossRefGoogle Scholar
  11. Concha ML, Wilson SW (2001) Asymmetry in the epithalamus of vertebrates. J Anat 199:63–84CrossRefGoogle Scholar
  12. Crumbley C, Burris TP (2011) Direct regulation of CLOCK expression by REV-ERB. PLoS One 6:e17290. CrossRefGoogle Scholar
  13. Dreosti E, Vendrell Llopis N, Carl M et al (2014) Left-right asymmetry is required for the habenulae to respond to both visual and olfactory stimuli. Curr Biol 24:440–445. CrossRefGoogle Scholar
  14. Fernandez DC, Fogerson PM, Lazzerini Ospri L et al (2018) Light affects mood and learning through distinct retina-brain pathways. Cell. Google Scholar
  15. Gonzalez MMC, Aston-Jones G (2008) Light deprivation damages monoamine neurons and produces a depressive behavioral phenotype in rats. Proc Natl Acad Sci USA 105:4898–4903. CrossRefGoogle Scholar
  16. Gottesfeld Z (1983) Origin and distribution of noradrenergic innervation in the habenula: a neurochemical study. Brain Res 275:299–304. CrossRefGoogle Scholar
  17. Granados-Fuentes D, Prolo LM, Abraham U, Herzog ED (2004) The suprachiasmatic nucleus entrains, but does not sustain, circadian rhythmicity in the olfactory bulb. J Neurosci 24:615–619. CrossRefGoogle Scholar
  18. Guilding C, Piggins HD (2007) Challenging the omnipotence of the suprachiasmatic timekeeper: are circadian oscillators present throughout the mammalian brain? Eur J Neurosci 25:3195–3216. CrossRefGoogle Scholar
  19. Guilding C, Hughes ATL, Brown TM et al (2009) A riot of rhythms: neuronal and glial circadian oscillators in the mediobasal hypothalamus. Mol Brain 2:28. CrossRefGoogle Scholar
  20. Guilding C, Hughes ATL, Piggins HD (2010) Circadian oscillators in the epithalamus. Neuroscience 169:1630–1639. CrossRefGoogle Scholar
  21. Hamada T, Sutherland K, Ishikawa M et al (2016) In vivo imaging of clock gene expression in multiple tissues of freely moving mice. Nat Commun 7:11705. CrossRefGoogle Scholar
  22. Hattar S, Kumar M, Park A et al (2006) Central projections of melanopsin-expressing retinal ganglion cells in the mouse. J Comp Neurol 497:326–349. CrossRefGoogle Scholar
  23. Hernández VS, Vázquez-Juárez E, Márquez MM et al (2015) Extra-neurohypophyseal axonal projections from individual vasopressin-containing magnocellular neurons in rat hypothalamus. Front Neuroanat 9:130. Google Scholar
  24. Hikosaka O (2010) The habenula: from stress evasion to value-based decision-making. Nat Rev Neurosci 11:503–513. CrossRefGoogle Scholar
  25. Honma S, Ono D, Suzuki Y et al (2012) Suprachiasmatic nucleus: cellular clocks and networks. Prog Brain Res 199:129–141. CrossRefGoogle Scholar
  26. Hughes ATL, Croft CL, Samuels RE et al (2015) Constant light enhances synchrony among circadian clock cells and promotes behavioral rhythms in VPAC2-signaling deficient mice. Sci Rep 5:14044. CrossRefGoogle Scholar
  27. Jaeger C, Sandu C, Malan A et al (2015) Circadian organization of the rodent retina involves strongly coupled, layer-specific oscillators. FASEB J 29:1493–1504. CrossRefGoogle Scholar
  28. Jagota A, de la Iglesia HO, Schwartz WJ (2000) Morning and evening circadian oscillations in the suprachiasmatic nucleus in vitro. Nat Neurosci 3:372–376. CrossRefGoogle Scholar
  29. Karatsoreos IN (2014) Links between circadian rhythms and psychiatric disease. Front Behav Neurosci 8:162. CrossRefGoogle Scholar
  30. Karolczak M, Burbach GJ, Sties G et al (2004) Clock gene mRNA and protein rhythms in the pineal gland of mice. Eur J Neurosci 19:3382–3388. CrossRefGoogle Scholar
  31. Lammel S, Lim BK, Ran C et al (2012) Input-specific control of reward and aversion in the ventral tegmental area. Nature 491:212–217. CrossRefGoogle Scholar
  32. Lecourtier L, Kelly PH (2007) A conductor hidden in the orchestra? Role of the habenular complex in monoamine transmission and cognition. Neurosci Biobehav Rev 31:658–672. CrossRefGoogle Scholar
  33. Leise TL, Wang CW, Gitis PJ, Welsh DK (2012) Persistent cell-autonomous circadian oscillations in fibroblasts revealed by six-week single-cell imaging of PER2::LUC bioluminescence. PLoS One 7:e33334. CrossRefGoogle Scholar
  34. Lin Q, Jesuthasan S (2017) Masking of a circadian behavior in larval zebrafish involves the thalamo-habenula pathway. Sci Rep 7:4104. CrossRefGoogle Scholar
  35. Mathis V, Cosquer B, Avallone M et al (2015) Excitatory Transmission to the Lateral Habenula Is Critical for Encoding and Retrieval of Spatial Memory. Neuropsychopharmacology 40:2843–2851. CrossRefGoogle Scholar
  36. Matsumoto M, Hikosaka O (2007) Lateral habenula as a source of negative reward signals in dopamine neurons. Nature 447:1111–1115. CrossRefGoogle Scholar
  37. Mizumori SJY, Baker PM (2017) The Lateral Habenula and Adaptive Behaviors. Trends Neurosci 40:481–493. CrossRefGoogle Scholar
  38. Mohawk JA, Green CB, Takahashi JS (2012) Central and peripheral circadian clocks in mammals. Annu Rev Neurosci 35:445–462. CrossRefGoogle Scholar
  39. Namboodiri VMK, Rodriguez-Romaguera J, Stuber GD (2016) The habenula. Curr Biol 26:R873–R877. CrossRefGoogle Scholar
  40. Ohta H, Yamazaki S, McMahon DG (2005) Constant light desynchronizes mammalian clock neurons. Nat Neurosci 8:267–269. CrossRefGoogle Scholar
  41. Paxinos G, Franklin KBJ (2001) The mouse brain in stereotaxic coordinates, 2 edn. Academic Press, San DiegoGoogle Scholar
  42. Pittendrigh CS, Daan SA (1976) Functional analysis of circadian pacemakers in nocturnal rodents. V. Pacemaker structure: a clockfor all seasons. J Comp Physiol A 106:333–355CrossRefGoogle Scholar
  43. Poller WC, Madai VI, Bernard R et al (2013) A glutamatergic projection from the lateral hypothalamus targets VTA-projecting neurons in the lateral habenula of the rat. Brain Res 1507:45–60. CrossRefGoogle Scholar
  44. Preitner N, Damiola F, Lopez-Molina L et al (2002) The orphan nuclear receptor REV-ERBalpha controls circadian transcription within the positive limb of the mammalian circadian oscillator. Cell 110:251–260CrossRefGoogle Scholar
  45. Prolo LM, Takahashi JS, Herzog ED (2005) Circadian rhythm generation and entrainment in astrocytes. J Neurosci 25:404–408. CrossRefGoogle Scholar
  46. Qu T, Dong K, Sugioka K, Yamadori T (1996) Demonstration of direct input from the retina to the lateral habenular nucleus in the albino rat. Brain Res 709:251–258. CrossRefGoogle Scholar
  47. Refinetti R, Lissen GC, Halberg F (2007) Procedures for numerical analysis of circadian rhythms. Biol Rhythm Res 38:275–325. CrossRefGoogle Scholar
  48. Reuss S, Decker K (1997) Anterograde tracing of retinohypothalamic afferents with Fluoro-Gold. Brain Res 745:197–204. CrossRefGoogle Scholar
  49. Ripperger JA, Albrecht U (2012) The circadian clock component PERIOD2: from molecular to cerebral functions. Prog Brain Res 199:233–245. CrossRefGoogle Scholar
  50. Ruan G-X, Allen GC, Yamazaki S, McMahon DG (2008) An autonomous circadian clock in the inner mouse retina regulated by dopamine and GABA. PLoS Biol 6:e249. CrossRefGoogle Scholar
  51. Sakhi K, Belle MDC, Gossan N et al (2014a) Daily variation in the electrophysiological activity of mouse medial habenula neurones. J Physiol (Lond) 592:587–603. CrossRefGoogle Scholar
  52. Sakhi K, Wegner S, Belle MDC et al (2014b) Intrinsic and extrinsic cues regulate the daily profile of mouse lateral habenula neuronal activity. J Physiol (Lond) 592:5025–5045. CrossRefGoogle Scholar
  53. Shieh K-R (2003) Distribution of the rhythm-related genes rPERIOD1, rPERIOD2, and rCLOCK, in the rat brain. Neuroscience 118:831–843CrossRefGoogle Scholar
  54. Shuboni DD, Cramm SL, Yan L et al (2015) Acute effects of light on the brain and behavior of diurnal Arvicanthis niloticus and nocturnal Mus musculus. Physiol Behav 138:75–86. CrossRefGoogle Scholar
  55. Sutherland RJ (1982) The dorsal diencephalic conduction system: a review of the anatomy and functions of the habenular complex. Neurosci Biobehav Rev 6:1–13CrossRefGoogle Scholar
  56. Takahashi JS, Hong H-K, Ko CH, McDearmon EL (2008) The genetics of mammalian circadian order and disorder: implications for physiology and disease. Nat Rev Genet 9:764–775. CrossRefGoogle Scholar
  57. Talbi R, Klosen P, Laran-Chich M-P et al (2016) Coordinated seasonal regulation of metabolic and reproductive hypothalamic peptides in the desert jerboa. J Comp Neurol 524:3717–3728. CrossRefGoogle Scholar
  58. Tavakoli-Nezhad M, Schwartz WJ (2006) Hamsters running on time: is the lateral habenula a part of the clock? Chronobiol Int 23:217–224. CrossRefGoogle Scholar
  59. Tournier BB, Menet JS, Dardente H et al (2003) Photoperiod differentially regulates clock genes’ expression in the suprachiasmatic nucleus of Syrian hamster. Neuroscience 118:317–322. CrossRefGoogle Scholar
  60. Welsh DK, Takahashi JS, Kay SA (2010) Suprachiasmatic nucleus: cell autonomy and network properties. Annu Rev Physiol 72:551–577. CrossRefGoogle Scholar
  61. Wyse CA, Coogan AN (2010) Impact of aging on diurnal expression patterns of CLOCK and BMAL1 in the mouse brain. Brain Res 1337:21–31. CrossRefGoogle Scholar
  62. Xu H, Zhang C, Zhao H (2015) Effect of suprachiasmatic nucleus lesion on Period2 and C-fos expression in habenular nucleus. Int J Chem 7:163CrossRefGoogle Scholar
  63. Yoo S-H, Yamazaki S, Lowrey PL et al (2004) PERIOD2::LUCIFERASE real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues. Proc Natl Acad Sci USA 101:5339–5346. CrossRefGoogle Scholar
  64. Zahm DS, Root DH (2017) Review of the cytology and connections of the lateral habenula, an avatar of adaptive behaving. Pharmacol Biochem Behav 162:3–21. CrossRefGoogle Scholar
  65. Zhang C, Truong KK, Zhou Q-Y (2009) Efferent projections of prokineticin 2 expressing neurons in the mouse suprachiasmatic nucleus. PLoS One 4:e7151. CrossRefGoogle Scholar
  66. Zhang L, Hernández VS, Vázquez-Juárez E et al (2016) Thirst Is associated with suppression of habenula output and active stress coping: is there a role for a non-canonical vasopressin-glutamate pathway? Front Neural Circuits. Google Scholar
  67. Zhang L, Hernández VS, Swinny JD et al (2018) A GABAergic cell type in the lateral habenula links hypothalamic homeostatic and midbrain motivation circuits with sex steroid signaling. Transl Psychiatry 8:50. CrossRefGoogle Scholar
  68. Zhao H, Rusak B (2005) Circadian firing-rate rhythms and light responses of rat habenular nucleus neurons in vivo and in vitro. Neuroscience 132:519–528. CrossRefGoogle Scholar
  69. Zhao Z, Xu H, Liu Y et al (2015) Diurnal expression of the Per2 gene and protein in the lateral habenular nucleus. Int J Mol Sci 16:16740–16749. CrossRefGoogle Scholar
  70. Zheng B, Larkin DW, Albrecht U et al (1999) The mPer2 gene encodes a functional component of the mammalian circadian clock. Nature 400:169–173. CrossRefGoogle Scholar
  71. Zheng B, Albrecht U, Kaasik K et al (2001) Nonredundant roles of the mPer1 and mPer2 genes in the mammalian circadian clock. Cell 105:683–694CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Institute of Cellular and Integrative Neurosciences, CNRS UPR-3212StrasbourgFrance

Personalised recommendations