1. Do Per1 and Per2 respond differentially to photoperiod in individual SCN neurons? Jocelyn Rice MCB186 Final Project December 13, 2006

2. SHORT DAY: contracted LONG DAY: expanded 24 6 12 18 24 In the rodent SCN, circadian oscillations reflect prior photoperiod per1 and per2 –Hamster: Nuesslein- Hildesheim et al (2000), Tournier et al (2003), de la Iglesia et al (2004) –Rat: Sumova et al (2002) –Mouse: Steinlechner et al (2002) Neural activity –Hamster: Mrugala et al (2000) –Rat: Schaap et al (2003)

3. Morning (M) and Evening (E) Oscillators? The hypothesis: –Two distinct but coupled oscillators One anchored to dawn (M), other to dusk (E) –Respond differently to light cues –M = per1/cry1 and E = per2/cry2? (Daan et al 2001) The evidence: –Bimodal activity rhythm in horizontal hamster SCN slices (Jagota et al 2000) “M” locks onto dawn and “E” locks onto dusk Can be phase-shifted independently by glutamate –“Notched” activity peaks with long photoperiod in hamster (Mrugala et al 2000) And…

4. per1 and per2: two different oscillators? Increasing photoperiod… –Broadens both peaks –Advances both rhythms –Increases phase difference One linked to dawn, other to dusk (M & E)? A way to measure photoperiod? Within individual cells, or a network of cells? H.O. de la Iglesia et al. Molecular Brain Research 127 (2004) 121–127.

5. Use transgenic mice to monitor expression of per1 and per2 simultaneously per2-RFP (need to make) per1-GFP (existing) per1 oscillation X per2 oscillation ???

6. Measure per1 and per2 expression in SCN slices from transgenic mice Entrain mice to a short or long photoperiod for four weeks, then sacrifice and prepare SCN slices –Group 1 - Short Day (LD 8:16) –Group 2 - Long Day (LD 16:8) Excite: use excitation wavelengths for GFP and RFP Detect: using time-lapse fluorescence microscopy Visualize: superimpose GFP and RFP images for each slice Mostly per1 Some per2 Equal per1 and per2 Mostly per2 Some per1

7. Quantifying expression patterns GFP (per1) and RFP (per2): fluorescence vs. time across a full circadian cycle Whole SCN Regions Individual cells Questions: Do the rhythms expand with long photoperiod and contract with short photoperiod? Does the phase difference change with photoperiod? Is there a difference between individual cell effects and whole SCN effects?

8. Scenario 1: photoperiodic effects generated in individual SCN cells When fluorescence is measured in individual neurons, some of them show this pattern. The same pattern is seen on the whole- SCN scale. Short-day mice Long-day mice per1 per2

9. Scenario 1: photoperiodic effects generated in individual SCN cells Single-cell photoperiodic effects could also result from changing phase difference alone. Peak broadening might appear only on the scale of the whole SCN. Short-day mice Long-day mice per1 per2

10. Short-day mice Long-day mice per1 per2 Scenario 2: photoperiodic effects generated by cell networks These patterns are absent from individual neurons –Waveforms and/or phase difference may be unresponsive to photoperiod, or may respond in unpredictable ways –M & E characteristics may be housed in different groups of neurons But patterns re- emerge when cell groups, regions, or whole SCN are considered

11. Scenario 2: photoperiodic effects generated by cell networks Time Number of neurons at per expression peak Short Day Long Day Maybe individual neurons change their phase relationships in response to changing photoperiod Phase spread for LP, synchrony for SP Broadened peaks with LP = combined effects of many neurons with different phases Evidence… –per1 in mice (Quintero et al 2003) –Neuronal activity in rats (Schaap et al 2003)