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

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

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

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

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.

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.

Rods and Cones

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.

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)

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)

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)

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)

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

“Actograms” (From Biemans, 2003)

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

“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

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

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

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

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?

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

SO, TURN OFF THE LIGHT!

References Focal Article Hattar S, Lucas RJ, Mrosovsky N, Thompson S, Douglas RH, Hankins MW, Lem J, Hofmann F, Foster RG, Yau KW (2003) Melanopsin and rod-cone photoreceptive systems account for all major accessory visual functions in mice. Nature 175: 1 – 5. Primary Sources Hannibal J, Hinderson P, Knudson SM, Georg B, Fahrenkrug J (2001) The photopigment melanopsin is exclusively present in pituitary adenylate cyclase-activating polypeptide-containing retinal ganglion cells of the retinohypothalamic tract. Journal of Neuroscience 21 (191): RC1 – RC7. Lucas RJ, Hattar S, Takao M, Berson DM, Foster RG, Yau KW (2003) Diminished pupillary light reflex at high irradiance in melanopsin-knockout mice. Science 299: 245 – 247. Ma’ayan S, Peirson S, Lupi, Lucas RJ, Jeffrey G, Foster RG (2003) Melanopsin retinal ganglion cells and the maintenance of circadian and papillary responses to light in aged rodless/coneless (rd/rd cl) mice. Experimental Journal of Neuroscience 17: 1793 – Panda S, Sato TK, Castrucci AM, Rollag MD, DeGrip WJ, Hogenesch JB, Provencio I, Kay SA (2002) Melanopsin (Opn4) requirement for normal light-induced circadian phase shifting. Science 298: 2213 – Provencio I, Jiang G, DeGrip WJ, Par Hayes W, Rollag M (1998) Melanopsin: an opsin in melanophores, brain, and eye. Proceedings of the National Academy of Sciences of the United States of America 95(1): 340 – 345. Provencio I, Rodriguez IR, Jiang G, Hayes WP, Moreira EF, Rollag MD (2000) A novel human opsin in the inner retina. The Journal of Neuroscience 20(2): 600 – 605. Ruby NF, Barakat MT, Heller HC (2004) Phenotypic differences in reentrainment behavior and sensitivity to nighttime light pulses in Siberian hamsters. Journal of Biological Rhythms 19(6): 530 – 541. Thapan K, Arendt J, Skene DJ (2001) An action spectrum for melantonin suppression: evidence for a novel non-rod, non-cone photoreceptor system in humans. Journal of Physiology 535(1): 261 – 267.

And even more references…… Secondary Sources Argamasa SM, Froehlich AC, McCall MA, Nevo E, Provencio I, Foster RG (1995) Argamasa SM, Froehlich AC, McCall MA, Nevo E, Provencio I, Foster RG (1995) Photopigments and circadian systems of vertebrates. Biophysical Chemistry 56: 3 – 11. Photopigments and circadian systems of vertebrates. Biophysical Chemistry 56: 3 – 11. Aton SJ, Block GD, Tei H, Yamazaki S, Herzog ED (2004) Plasticity of circadian behavior and the suprachiasmatic nucleus following exposure to non-24-hour light cycles. Journal of Biological Rhythms 19(3): 198 – 207. Aton SJ, Block GD, Tei H, Yamazaki S, Herzog ED (2004) Plasticity of circadian behavior and the suprachiasmatic nucleus following exposure to non-24-hour light cycles. Journal of Biological Rhythms 19(3): 198 – 207. Bellingham J, Foster RG (2002) Opsins and mammalian photoentrainment. Cell Tissue Bellingham J, Foster RG (2002) Opsins and mammalian photoentrainment. Cell Tissue Research 309: 57 – 71. Berson DM (2003) Strange vision: ganglion cells as circadian photoreceptors. TRENDS in Neuroscience 26(6): 314 – 320. Berson DM (2003) Strange vision: ganglion cells as circadian photoreceptors. TRENDS in Neuroscience 26(6): 314 – 320. Berson DM, Dunn FA, Takao M (2002) Phototransduction by retinal ganglion cells that set the circadian clock. Science 295: 1070 – Berson DM, Dunn FA, Takao M (2002) Phototransduction by retinal ganglion cells that set the circadian clock. Science 295: 1070 – Campbell SS, Murphy PJ (1998) Extraocular circadian phototransduction in humans. Science 279: 396 – 399. Campbell SS, Murphy PJ (1998) Extraocular circadian phototransduction in humans. Science 279: 396 – 399. Colwell CS, Michel S, Itri J, Rodriguez W, Tam J, Lelievre V, Hu Z, Waschek JA (2004) Selective deficits in the circadian light response in mice lacking PACAP. American Journal of Physiology – Regulatory, Integrative, and Comparative Physiology 287: 1194 – Colwell CS, Michel S, Itri J, Rodriguez W, Tam J, Lelievre V, Hu Z, Waschek JA (2004) Selective deficits in the circadian light response in mice lacking PACAP. American Journal of Physiology – Regulatory, Integrative, and Comparative Physiology 287: 1194 – Evans JA, Elliott JA, Gorman MR (2004) Photoperiod differentially modulates photic and nonphotic phase response curves of hamsters. American Journal of Physiology – Regulatory, Integrative, and Comparative Physiology 286: 539 – 546. Evans JA, Elliott JA, Gorman MR (2004) Photoperiod differentially modulates photic and nonphotic phase response curves of hamsters. American Journal of Physiology – Regulatory, Integrative, and Comparative Physiology 286: 539 – 546. Foster RG (1998) Shedding light on the biological clock. Neuron 20: 829 – 832. Foster RG (1998) Shedding light on the biological clock. Neuron 20: 829 – 832. Foster RG, Hankins MW (2002) Non-rod, non-cone photoreception in the vertebrates. Progress in Retinal and Eye Research 21: 507 – 527. Foster RG, Hankins MW (2002) Non-rod, non-cone photoreception in the vertebrates. Progress in Retinal and Eye Research 21: 507 – 527. Gooler JJ, Lu J, Fischer D, Saper CB (2003) A broad role for melanopsin in nonvisual photoreception. The Journal of Neuroscience 23(18): 7093 – Gooler JJ, Lu J, Fischer D, Saper CB (2003) A broad role for melanopsin in nonvisual photoreception. The Journal of Neuroscience 23(18): 7093 – Hannibal J, Fahrenkrug J (2004) Target areas innervated by PACAP- immunoreactive retinal ganglion cells. Cell Tissue Research 316: 99 – 113. Hannibal J, Fahrenkrug J (2004) Target areas innervated by PACAP- immunoreactive retinal ganglion cells. Cell Tissue Research 316: 99 – 113. Hattar S, Liao HW, Takao M, Berson DM, Yau KW (2002) Melanopsin- containing retinal ganglion cells: architecture, projections, and intrinsic photsensitivy. Science 295: 1065 – Hattar S, Liao HW, Takao M, Berson DM, Yau KW (2002) Melanopsin- containing retinal ganglion cells: architecture, projections, and intrinsic photsensitivy. Science 295: 1065 – Hirota T, Fukada Y (2004) Resetting mechanism of central and peripheral circadian clocks in mammals. Zoological Science 21: 359 – 368. Hirota T, Fukada Y (2004) Resetting mechanism of central and peripheral circadian clocks in mammals. Zoological Science 21: 359 – 368. Johnson CH (1992) Phase response curves: what can they tell us about circadian clocks? Found in: Circadian Clocks from Cell to Human (ed. Hiroshige T, Honma K) Johnson CH (1992) Phase response curves: what can they tell us about circadian clocks? Found in: Circadian Clocks from Cell to Human (ed. Hiroshige T, Honma K) Keiner, J (2001) Where vision begins – phototransduction in rods and cones. Publication data not available. Keiner, J (2001) Where vision begins – phototransduction in rods and cones. Publication data not available. Lewy AJ, Wehr TA, Goodwin FK, Newsome DA, Markey SP (1980) Light suppresses melatonin secretion in humans. Science 210(4475): 1267 – Lewy AJ, Wehr TA, Goodwin FK, Newsome DA, Markey SP (1980) Light suppresses melatonin secretion in humans. Science 210(4475): 1267 – Lucas RJ, Freedman MS, Munoz M, Garcia-Fernandez JM, Foster RG (1999) Regulation of the mammalian pineal by non-rod, non-cone ocular photoreceptors. Science 284: 505 – 507. Lucas RJ, Freedman MS, Munoz M, Garcia-Fernandez JM, Foster RG (1999) Regulation of the mammalian pineal by non-rod, non-cone ocular photoreceptors. Science 284: 505 – 507. Metz HS (2003) Light and the circadian clock. Journal of AAPOS 7(4): Editorial. Metz HS (2003) Light and the circadian clock. Journal of AAPOS 7(4): Editorial. Newman LA, Walker MT, Brown RL, Cronin TW, Robinson PR (2003) Melanopsin forms a functional short-wave photopigment. Biochemistry 42: – Newman LA, Walker MT, Brown RL, Cronin TW, Robinson PR (2003) Melanopsin forms a functional short-wave photopigment. Biochemistry 42: – Panda S, Provencio I, Tu DC, Pires SS, Rollag MD, Castrucci AM, Pletcher MT, Sato TK, Wiltshire T, Asdahazy M, Kay SA, Van Gelder RN, Hogenesch JB (2003) Melanopsin is required for non-image- forming photic responses in blind mice. Science 301: 525 – 527. Panda S, Provencio I, Tu DC, Pires SS, Rollag MD, Castrucci AM, Pletcher MT, Sato TK, Wiltshire T, Asdahazy M, Kay SA, Van Gelder RN, Hogenesch JB (2003) Melanopsin is required for non-image- forming photic responses in blind mice. Science 301: 525 – 527. Provencio I, Rollag MD, Castrucci AM (2002) Photoreceptive net in the mammalian retina. Nature 145: 493. Provencio I, Rollag MD, Castrucci AM (2002) Photoreceptive net in the mammalian retina. Nature 145: 493. Rollag MD, Berson DM, Provencio I (2003) Melanopsin, ganglion-cell photoreceptors, and mammalian photoentrainment. Journal of Biological Rhythms 18(3): 227 – 234. Rollag MD, Berson DM, Provencio I (2003) Melanopsin, ganglion-cell photoreceptors, and mammalian photoentrainment. Journal of Biological Rhythms 18(3): 227 – 234. Ruby NF, Brenna TJ, Xie X, Cao V, Franken P, Heller HC, O’Hara BF (2002) Role of melanopsin in circadian responses to light. Science 298: 2211 – Ruby NF, Brenna TJ, Xie X, Cao V, Franken P, Heller HC, O’Hara BF (2002) Role of melanopsin in circadian responses to light. Science 298: 2211 – Ruby NF, Dark J, Burns DE, Heller HC, Zucker I (2002) The suprachiasmatic nucleus is essential for circadian body temperature rhythms in hibernating ground squirrels. The Journal of Neuroscience 22(1): 357 – 364. Ruby NF, Dark J, Burns DE, Heller HC, Zucker I (2002) The suprachiasmatic nucleus is essential for circadian body temperature rhythms in hibernating ground squirrels. The Journal of Neuroscience 22(1): 357 – 364. Ruby NF, Joshi N, Heller NC (2002) Constant darkness restores entrainment to phase-delayed Siberian hamsters. American Journal of Physiology – Regulatory, Integrative, and Comparative Physiology 283: R1314 – R1320. Ruby NF, Joshi N, Heller NC (2002) Constant darkness restores entrainment to phase-delayed Siberian hamsters. American Journal of Physiology – Regulatory, Integrative, and Comparative Physiology 283: R1314 – R1320. Wee R, Castrucci AM, Provencio I, Gan L, Van Gelder RN (2002) Loss of photic entrainment and altered free-running circadian rhytms in math5-/- mice. The Journal of Neuroscience 22(23): – Wee R, Castrucci AM, Provencio I, Gan L, Van Gelder RN (2002) Loss of photic entrainment and altered free-running circadian rhytms in math5-/- mice. The Journal of Neuroscience 22(23): –