1. Photo-entrainment: Physiology and Behavior Alex Harryman Focal Article: Melanopsin and rod-cone photoreceptive systems account for all major accessory visual functions in mice Hattar et al. 2003

2. Natural Rhythms Ultradian rhythms Ultradian rhythms Examples: Heartbeat, somite deposition during vertebrate embryogenesis, respiratory oscillations in yeast Infradian rhythms Infradian rhythms Examples: Female estrus cycles, mating cycles, emergence of cicadas Circadian rhythms Circadian rhythms –From Latin circa diem, meaning “about a day” –24 hour cycle, reset by light cues

3. Internal Clocks Animals possess internal clocks, which regulate hormonal control, body temperature, and sleep patterns Animals possess internal clocks, which regulate hormonal control, body temperature, and sleep patterns Clocks under genetic control, but can be influenced by external cues.  Light cues result in photo-entrainment

4. Components of Circadian Clock Central pacemaker with intrinsic rhythm (genetic) Central pacemaker with intrinsic rhythm (genetic) –Regulated by number of genes and transcription factors Input pathway to pacemaker (physiological) Input pathway to pacemaker (physiological) –Exogenous cues (zeitgebers) reset pacemaker Output pathway to effector systems (behavioral) Output pathway to effector systems (behavioral) –Synchronized response to cues

5. This paper… Overall Goal: To determine the relationship between the physiological basis for photo- entrainment via light transduction and the behavioral response of mammals. Overall Goal: To determine the relationship between the physiological basis for photo- entrainment via light transduction and the behavioral response of mammals. Specific Hypothesis: Melanopsin is the photopigment responsible for phototransduction to the circadian pacemaker, and, as such, mammals without melanopsin (Opn4 - ) cannot photo-entrain. Specific Hypothesis: Melanopsin is the photopigment responsible for phototransduction to the circadian pacemaker, and, as such, mammals without melanopsin (Opn4 - ) cannot photo-entrain.

6. Part I: Physiology of Light Detection Question: Where are the photoreceptors responsible for photo-entrainment located in mammals? Experiment: Enucleated mice. Result: Mice could no longer photo-entrain. Conclusion: Mammalian circadian photoreceptors located in the retina.

7. Rods and Cones

8. What Happens When We Assume? Question: Are rods and cones the PRCs responsible for sending light signals to the brain? Experiment: Investigated ability of mice lacking rods and cones to photo-entrain. Results: Mice could photo-entrain normally. Conclusion: An independent photoreceptor system exists and is responsible for photo-entrainment.

9. A New Candidate: Melanopsin Melanopsin isolated from melanophores of Xenopus laevis Melanopsin isolated from melanophores of Xenopus laevis Melanopsin found in a subset of retinal ganglion cells (RGCs) Melanopsin found in a subset of retinal ganglion cells (RGCs) Homology to non-vertebrate opsins Homology to non-vertebrate opsins Structure of rhodopsin (found in rods and cones) Structure of melanopsin (found in melanophores)

10. Further Evidence Melanopsin-containing RGCs are intrinsically photosensitive to light with maximal absorption at 480 nm Melanopsin-containing RGCs are intrinsically photosensitive to light with maximal absorption at 480 nm Melanopsin-containing RGCs project to the SCN and other regions of the brain responsible for photo-entrainment Melanopsin-containing RGCs project to the SCN and other regions of the brain responsible for photo-entrainment PACAP, a neurotransmitter thought to have a role in photo- entrainment, is found exclusively in melanopsin-containing RGCs PACAP, a neurotransmitter thought to have a role in photo- entrainment, is found exclusively in melanopsin-containing RGCs (From Berson, 2003) (From Hattar et al., 2003)

11. Icing on the Cake Melanopsin knockout mice expressed a reduced pupillary response to light. Melanopsin knockout mice expressed a reduced pupillary response to light. (From Lucas et. al, 2003)

12. Phototransduction Model Photoreceptor cells (PRCs) Photoreceptor cells (PRCs) –Responsible for detecting light input and relaying signals to brain Retinohypothalamic Pathway (RHT) Retinohypothalamic Pathway (RHT) –Pathway from retina to SCN Suprachiasmatic Nucleus (SCN) Suprachiasmatic Nucleus (SCN) –Portion of brain responsible for photo-entrainment in mammals (From S.M. Reppert & D.R. Weaver, Nature 2002)

13. Part II: Behavioral Response One of best indicators of an animal’s ability to photo-entrain is its behavioral response One of best indicators of an animal’s ability to photo-entrain is its behavioral response – Wheel running activity in rodents used – Rodents will synchronize their activity to light-dark cycles light-dark cycles

14. “Actograms” (From Biemans, 2003)

15. Link Between Physiology and Behavior Determine that melanopsin was necessary for phototransduction of light signals to SCN Determine that melanopsin was necessary for phototransduction of light signals to SCN Prove that melanopsin is the only independent non-visual photoreceptor system in mammalian retina Prove that melanopsin is the only independent non-visual photoreceptor system in mammalian retina

16. “Triple Knockout” Mice Deleted genes in rod and cone signaling pathways Deleted genes in rod and cone signaling pathways - Rods and cones intact, can receive light, but not transmit signal Replaced melanopsin gene with tau-LacZ construct Replaced melanopsin gene with tau-LacZ construct – RGCs with reporter gene determined by X-gal labeling

17. Result: No Photo-entrainment (From Hattar et al., 2003)

18. Summary of Behavioral Response Studies (From Panda et al., 2003)

19. Conclusions Rod-cone and melanopsin systems are the only light detecting systems in mammalian eye Rod-cone and melanopsin systems are the only light detecting systems in mammalian eye –Demonstrated by pupillary light reflex Presence of melanopsin is essential for photo-entrainment Presence of melanopsin is essential for photo-entrainment –Full range of photoreception with rods and cones present

20. Unanswered Questions In vivo, melanopsin exhibits a maximum light absorbance at 484 nm In vivo, melanopsin exhibits a maximum light absorbance at 484 nm In vitro, melanopsin exhibits a blue shift in absorbance, and action spectra peaks at 420 nm In vitro, melanopsin exhibits a blue shift in absorbance, and action spectra peaks at 420 nm  Unusual chromophore?  Unstable during purification?

21. So, why do I want to put a light behind my knee??? Concept: Humoral phototransduction Concept: Humoral phototransduction  Circadian rhythms of body temperature and melatonin can be  Circadian rhythms of body temperature and melatonin can be shifted shifted Mechanism Mechanism  Irradiation of blood will cause heme photopigments to release NO, which is known to be necessary in SCN for phase shifts NO, which is known to be necessary in SCN for phase shifts Problem Problem  No evidence of how NO released in knee can reach the brain  No evidence of how NO released in knee can reach the brain and trigger response and trigger response

22. SO, TURN OFF THE LIGHT!

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