1. Circadian Biology Background, quantitative analysis, and present research

2. Circadian rhythms “circa” = approximately “dies” = day Rhythms with an approximately 24-hr cycle length that are endogenous in origin

3. First demonstartion Jean Jacques d’Ortous de Mairan 1729 Leaf movements of a heliotrope plant Persistence of circadian rhythms in absence of external cues

4. Zeitgeber = “time giver” Signals from the environment that keep non-24 hour clocks in synch with the 24 hour day

5. Retinal hypothalamic tract Direct input from the retinal photoreceptors to the hypothalamus Involved in circadian system

6. Where is this clock? SCN Anterior hypothalamus Above optic chiasm Optic chiasm

7. Activity records

8. Circadian System Light/dark photoreceptor pacemaker output system rhythm inputs

9. Entrainment

10. Phase response curves

11. Different quantitative analyses

12. Circadian pacemakers are limit cycle oscillators Oscillators have a standard waveform that they return to after a perturbation Limit cycle models give better appreciation for reactions of circadian pacemakers to stimuli

13. Limit cycle

14. Simple oscillator: 2 state variables Oscillating components are called “state variables” Graphically portray changes in state variable in “phase space” – an abstract space whose coordinates describe the state of the system

15. Example: frictionless pendulum 2 state variables: 1. Position [x(t)] 2. Rate of change ofposition [x’(t)] Differs from limit cycle oscillators in 2 ways: 1. Limit cycle osc. do not damp 2. State variables return to same trajectory

16. Fourier analysis of circadian amplitude Frequency (cycles/day) Wild-typeClock/+ Clock/Clock

17. Principle components analysis

18. Cluster analysis

19. 3-Dimensional Visualization

20. Davis lab research (Fred Davis PhD)

21. TGF-alpha…the story begins SCN transplant studies in lesioned animals Encapsulated graph No axonal projections Allowed for diffusion of secreted factors Locomotor activity rhythms still persist “locomotor activating/inhibiting factors”

22. ….and continues…. A screen was preformed, using previously documented SCN factors Constant infusion of each factor took place over a period of 2-3 weeks TGF-alpha acted as expected for a locomotor inhibitory factor Completely blocked running wheel activity for the duration of the infusion Activity came back with its expected phase and period

23. Activity records for hamsters infused with different secretory factors: reversible inhibition of locomotor activity by TGF

24. Vehicle TGF- 

25. The data: in constant darkness, measured EEG, EMG, body movement, and body temperature aCSF TGF-alpha

26. Behavior Analysis Pre - Rx- Post - Vehicle TGF-  CT 6.5-8.5CT 11-12CT 6.5-8.5CT 11-12

27. TGF-  Effects on Feeding InfusionPost-Infusion -20 -10 0 10 20 CSF (n=5) TGF  (n=4) Average Change in Body Weight (grams) 7 Day Intervals 0 20 40 60 80 100 CSF (n=5) TGF  (n=4) Average Food Consumption (grams) Effects of TGF-  on Body WeightEffects of TGF-  on Feeding 7 Day Intervals InfusionPost-Infusion

28. Acute injections What effect would an acute injection have versus slow infusion? How do we test this? Any results yet?

29. Testing methods Cannulation into the third ventricle: steriotaxic surgery Obtain good activity records Injections of TGF-alpha at time of activity onset Control injections of vehicle/CSF Obtain activity results

30. Channel 8: hamster #2 CSF (2/3) TGF (2/7)

31. Channel 11: hamster #1 CSF (2/3) TGF (2/7)

32. In the process….

33. Recent Data CSF TGF-alpha

35. In summary… The study of circadian rhythms is a very broad and open area It is a multi-disciplinary science, can study anything from whole animal behavior to in situ hybridization. As shown, there are numerous opportunities for quantitative analysis

36. Sources Czeisler, C.A. (figures) Davis, F. (PI) Johnson, C. (figures) Kramer, A. (TGF-alpha, and figures) Low-Zeddies, S. (statistic figures) Snodgrass-Belt, P. (unpublished data)