1. Horacio de la Iglesia Department of Biology horaciod@u.washington.edu

2. Organization of living matter Spatial organization has been an key concept from the earliest studies of biology, and particularly physiology. Temporal organization is more subtle and usually overlooked, but equally important. The fitness of an organism depends on its physical structure and its relationship with the environment. The time domain of this physical structure, and of the relationship with the environment, is as important as the structural domain. Time as niche variable.

4. Sleep wake cycle in a human being Days Wake Sleep Days Wake Sleep The persistence of rhythms in the absence of environmental cycles is taken as evidence of an innate self-sustained timekeeping biological mechanism, that is a Biological Clock

5. Environmental Cycle (e.g.. light-dark cycle) Clock (suprachiasmatic nucleus) Photoreceptors (retina) Input pathway s Overt circadian rhythms (e.g. sleep-wake cycle) III VI IX I Output pathways Components of a Generic Biological Timing System

6. Virtually all known hormones show a daily pattern of secretion

7. LD cycle Period = 24 h Free running in constant conditions (DD or LL) Period circa-24 h 0 24 Clock time Days Analysis of circadian rhythms locomotor activity in nocturnal rodents 024 48

8. Environmental Cycle (e.g.. light-dark cycle) Clock (suprachiasmatic nucleus) Photoreceptors (retina) Input pathway s Overt circadian rhythms (e.g. sleep-wake cycle) III VI IX I Output pathways Components of a Generic Biological Timing System

9. Fitzgerald and Zucker (1976) PNAS 73: 2923-2927 The same circadian oscillator appears to control locomotor activity and estrous

10. Looking for the Circadian Clock  Lesions of suspected site should abolish rhythmicity  Transplants of suspected site should restore circadian rhythmicity in lesioned animals.  Intrinsic properties of the suspected clock should be rhythmic: in vivo and in vitro  Identification of molecular and cellular mechanisms that generate rhythmicity  Mutations of specific genes should affect circadian rhythms  There should be a pathway communicating the synchronizer with the clock

11. Lehman (1987) J. Neurosci. 7:1626-38

12. The SCN is necessary and sufficient for expression of locomotor activity circadian rhythmicity Ralph & Lehman (1991) Trends Neurosci. 14:362

13. 024 ? Lesions of the SCN abolish all endocrine circadian rhythms

14. SCN lesions abolish the rhythm of corticosterone release Moore (1972) Brain Research 42: 201

15. Everett and Sawer (1950) Endocrinol. 47:198 The release of hormones is initiated in the brain: Daily gating of the LH surge as an example

16. The release of hormones is initiated in the brain: Daily gating of the LH surge as an example

17. % of women showing an LH surge, as determined by urinary LH Edwards (1981) Nature 293:253-256

18. SCN transplants that restore locomotor activity rhythms do not restore endocrine rhythms Meyer-Bernstein et al. (1999) Endocrinol 140: 207-218

19. Identification of molecular components of the clock The first mammalian clock mutant is found It is a semi-dominant mutation Heterozygous mice in constant darkness have a longer period than wildtypes Homozygous mice in constant darkness have initially a very long period and then become arrhythmic Vitaterna et al. (1994) Science 264:719-725

20. Time mRNA or Protein level Inhibition Delay (~24 h) - Transcription Clock gene 2 + mRNA Translation Protein (negative element) mRNA Transcription Clock gene 1 Translation Protein (positive element) Transcription and translation feedback loops constitute the core clock mechanism

21. Per1 expression Light Dark This unparalleled localization of function within the SCN makes the circadian system unique for the study of the neural and molecular bases of behavior and physiology

22. De la Iglesia et al. (2000) Science 290:799-801 Splitting in the hamster

23. haPer1 RNA level in situ (SCN) Locomotor activity UNSPLIT HAMSTER ~24 hours Left SCN Right SCN Time LR

24. haPer1 RNA level Left SCN Right SCN Locomotor activity SPLIT HAMSTER ~24 hours Time in situ (SCN)

25. Dual Circadian Oscillators in the SCN The ensemble of oscillators is reflected in overt behavior Mechanism and “sticking” force? Clock outputs?

26. Split female hamsters present a circa-12-h LH surge. Swann and Turek (1985) The HPG Axis

27. The SCN could have input the neuroendocrine hypothalamus

28. SCN efferents project to LHRH cells and these projections are believed to mediate the release of LHRH that triggers the LH surge. The projections are predominantly ipsilateral.

30. de la Iglesia et al. (2003) J. Neurosci., 23:7412-7412

31. .................................... Neural and diffusible outputs of the clock. de la Iglesia and Schwartz Endocrinology (2006) 147:1148-1153

32. SCN lesions abolish the LH surge Intracerebroventricular vasopressin induces a surge in the SCN-lesioned animals van der Beek et al. (1999) Neuroscience 93: 659-666

33. Endocrinology (2012) 153: 2839–2850

34. Smarr et al.

35. Synchronization of peripheral clocks Input pathways III VI IX I Other entraining inputs Overt physiological and behavioral rhythms (activity, feeding, core body temperature, sleep- wake) Kidney Liver Skeletal muscle SCN

36. GnRH cells and the pituitary show autonomous oscillations ov clock gene expression Chappell et al. (2003) J. Neurosci. 23: 11202-11213 Yoo et al. (2003) PNAS 101-5339-5346

37. Sellix et al. (2003) Current Biol. 20:R266-R267

38. GnRH neurons (slave oscillators?) Median eminence Anterior pituitary (Peripheral clock?) GnRH LH Ovary (Peripheral clock?) E2E2 SCN neurons ER  Portal system ? ER  ? AVP VIP AVP de la Iglesia and Schwartz Endocrinology (2006) 147:1148-1153 The HPG Axis

39. Miller et al.

41. The HPA Axis

42. Ishida et al. (2005) Cell Metabolism 2:297-307 Light induces expression of clock genes in the adrenal

43. Light induction of corticosterone release is ACTH-independent

44. Light induction of Per2 expression and of corticosterone release depends on a n intact SCN

45. SCN control of glucocorticoid release

46. Melatonin tracks night length in humans Pineal Melatonin

47. Czeisler (1995) N.E.J.Medicine Light acutely inhibits melatonin production When humans are given light during the dark phase or in complete darkness when melatonin is high, melatonin levels in the blood quickly drop.

48. Czeisler (1995) N.E.J.Medicine When humans are given light during the dark phase or in complete darkness when melatonin is high, melatonin levels in the blood quickly drop. This is even observed in some blind patients. Light acutely inhibits melatonin production

49. Melatonin and artificial light in humans

50. Melatonin is a night time signal

51. Humans are seasonal breeders

52. The reproductive axis a prominent output of the circanual system

53. Entrainment and photoperiod measurement Photoperiodic measurement in most species is not based on an hourglass mechanism But on a circadian oscillation of photoinducibility

54. Clearly cannot be explained by hourglass mechanism How is it explained?

55. Resonance cycles show an entrainment-based photoperiod measurement

56. T cycles show an entrainment-based photoperiod measurement

57. Gestational photoperiod measurement Schematic design: Pregnancy After birth 12:12 Small testislarge testis