Photo-entrainment: Photo-entrainment: Physiology and BehaviorPhysiology and Behavior

Alex HarrymanAlex Harryman

Focal Article: Melanopsin and rod-cone photoreceptive systems account for Focal Article: Melanopsin and rod-cone photoreceptive systems account for all major accessory visual functions in miceall major accessory visual functions in mice

Hattar et al.Hattar et al.20032003

Natural RhythmsNatural Rhythms

Ultradian rhythmsUltradian rhythmsExamples: Heartbeat, somite deposition during Examples: Heartbeat, somite deposition during

vertebrate embryogenesis, respiratory oscillations in vertebrate embryogenesis, respiratory oscillations in yeastyeast

Infradian rhythmsInfradian rhythmsExamples: Female estrus cycles, mating cycles, Examples: Female estrus cycles, mating cycles,

emergence of cicadasemergence of cicadas Circadian rhythmsCircadian rhythms

– From Latin From Latin circa diemcirca diem, meaning “about a day”, meaning “about a day”– 24 hour cycle, reset by light cues24 hour cycle, reset by light cues

Internal ClocksInternal Clocks

Animals possess internal clocks, which Animals possess internal clocks, which regulate hormonal control, body regulate hormonal control, body temperature, and sleep patternstemperature, and sleep patterns

Clocks under genetic control, but can be Clocks under genetic control, but can be influenced by external cues.influenced by external cues.

Light cues result in photo-entrainmentLight cues result in photo-entrainment

Components of Circadian ClockComponents of Circadian Clock

Central pacemakerCentral pacemaker with intrinsic rhythm (genetic) with intrinsic rhythm (genetic)– Regulated by number of genes and transcription Regulated by number of genes and transcription

factorsfactors

Input pathwayInput pathway to pacemaker (physiological) to pacemaker (physiological)– Exogenous cues (zeitgebers) reset pacemaker Exogenous cues (zeitgebers) reset pacemaker

Output pathwayOutput pathway to effector systems (behavioral) to effector systems (behavioral)– Synchronized response to cuesSynchronized response to cues

This paper…This paper…

Overall Goal:Overall Goal: To determine the relationship To determine the relationship between the physiological basis for photo-between the physiological basis for photo-entrainment via light transduction and the entrainment via light transduction and the behavioral response of mammals.behavioral response of mammals.

Specific Hypothesis:Specific Hypothesis: Melanopsin is the Melanopsin is the photopigment responsible for phototransduction photopigment responsible for phototransduction to the circadian pacemaker, and, as such, to the circadian pacemaker, and, as such, mammals without melanopsin (mammals without melanopsin (Opn4Opn4--) cannot ) cannot photo-entrain.photo-entrain.

Part I: Physiology of Light DetectionPart I: Physiology of Light Detection

Question:Question: Where are the photoreceptors Where are the photoreceptors responsible for photo-entrainment located in responsible for photo-entrainment located in mammals?mammals?

Experiment:Experiment: Enucleated mice. Enucleated mice.

Result:Result: Mice could no longer photo-entrain. Mice could no longer photo-entrain.

Conclusion:Conclusion: Mammalian circadian photoreceptors Mammalian circadian photoreceptors located in the retina.located in the retina.

Rods and ConesRods and Cones

What Happens When We Assume?What Happens When We Assume?

Question:Question: Are rods and cones the PRCs responsible for Are rods and cones the PRCs responsible for sending light signals to the brain?sending light signals to the brain?

Experiment:Experiment: Investigated ability of mice lacking rods and Investigated ability of mice lacking rods and cones to photo-entrain.cones to photo-entrain.

Results:Results: Mice could photo-entrain normally. Mice could photo-entrain normally.

Conclusion:Conclusion: An independent photoreceptor system exists An independent photoreceptor system exists and is responsible for photo-entrainment.and is responsible for photo-entrainment.

A New Candidate: MelanopsinA New Candidate: Melanopsin

Melanopsin isolated from Melanopsin isolated from melanophores of melanophores of Xenopus Xenopus laevislaevis

Melanopsin found in a Melanopsin found in a subset of retinal ganglion subset of retinal ganglion cells (RGCs)cells (RGCs)

Homology to non-vertebrate Homology to non-vertebrate opsinsopsins

Structure of rhodopsin (found in rods and cones)Structure of rhodopsin (found in rods and cones)

Structure of melanopsin (found in melanophores)Structure of melanopsin (found in melanophores)

Further EvidenceFurther Evidence

Melanopsin-containing RGCs are Melanopsin-containing RGCs are intrinsically photosensitive to intrinsically photosensitive to light with maximal absorption at light with maximal absorption at 480 nm480 nm

Melanopsin-containing RGCs Melanopsin-containing RGCs project to the SCN and other project to the SCN and other regions of the brain responsible regions of the brain responsible for photo-entrainmentfor photo-entrainment

PACAP, a neurotransmitter PACAP, a neurotransmitter thought to have a role in photo-thought to have a role in photo-entrainment, is found exclusively entrainment, is found exclusively in melanopsin-containing RGCsin melanopsin-containing RGCs

(From Berson, 2003)(From Berson, 2003)

(From Hattar et al., 2003)(From Hattar et al., 2003)

Icing on the CakeIcing on the Cake

Melanopsin knockout Melanopsin knockout mice expressed a mice expressed a reduced pupillary reduced pupillary response to light.response to light.

(From Lucas et. al, 2003)(From Lucas et. al, 2003)

Phototransduction ModelPhototransduction Model

Photoreceptor cells (PRCs)Photoreceptor cells (PRCs)– Responsible for detecting light Responsible for detecting light

input and relaying signals to input and relaying signals to brainbrain

Retinohypothalamic Pathway Retinohypothalamic Pathway (RHT)(RHT)– Pathway from retina to SCNPathway from retina to SCN

Suprachiasmatic Nucleus Suprachiasmatic Nucleus (SCN)(SCN)– Portion of brain responsible Portion of brain responsible

for photo-entrainment in for photo-entrainment in mammalsmammals

(From S.M. Reppert & D.R. Weaver, Nature 2002)

Part II: Behavioral ResponsePart II: Behavioral Response

One of best indicators of an animal’s One of best indicators of an animal’s ability to photo-entrain is its behavioral ability to photo-entrain is its behavioral responseresponse– Wheel running activity in rodents usedWheel running activity in rodents used– Rodents will synchronize their activity to Rodents will synchronize their activity to

light-dark cycleslight-dark cycles

““Actograms”Actograms”

(From Biemans, 2003)(From Biemans, 2003)

Link Between Physiology and Link Between Physiology and BehaviorBehavior

Determine that melanopsin was necessary Determine that melanopsin was necessary for phototransduction of light signals to for phototransduction of light signals to SCNSCN

Prove that melanopsin is the only Prove that melanopsin is the only independent non-visual photoreceptor independent non-visual photoreceptor system in mammalian retinasystem in mammalian retina

““Triple Knockout” MiceTriple Knockout” Mice

Deleted genes in rod and cone signaling Deleted genes in rod and cone signaling pathwayspathways

- Rods and cones intact, can receive light, but not transmit signal- Rods and cones intact, can receive light, but not transmit signal

Replaced melanopsin gene with tau-LacZ Replaced melanopsin gene with tau-LacZ constructconstruct– RGCs with reporter gene determined by X-gal labelingRGCs with reporter gene determined by X-gal labeling

Result: No Photo-entrainmentResult: No Photo-entrainment

(From Hattar et al., 2003)(From Hattar et al., 2003)

Summary of Behavioral Response StudiesSummary of Behavioral Response Studies

(From Panda et al., 2003)(From Panda et al., 2003)

ConclusionsConclusions

Rod-cone and melanopsin systems are the Rod-cone and melanopsin systems are the only light detecting systems in mammalian only light detecting systems in mammalian eyeeye– Demonstrated by pupillary light reflexDemonstrated by pupillary light reflex

Presence of melanopsin is essential for Presence of melanopsin is essential for photo-entrainmentphoto-entrainment– Full range of photoreception with rods and Full range of photoreception with rods and

cones presentcones present

Unanswered QuestionsUnanswered Questions

In vivo, melanopsin exhibits a maximum In vivo, melanopsin exhibits a maximum light absorbance at 484 nmlight absorbance at 484 nm

In vitro, melanopsin exhibits a blue shift In vitro, melanopsin exhibits a blue shift in absorbance, and action spectra peaks at in absorbance, and action spectra peaks at 420 nm420 nm

Unusual chromophore?Unusual chromophore?

Unstable during purification?Unstable during purification?

So, why do I want to put a light So, why do I want to put a light behind my knee???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

shiftedshifted

MechanismMechanism Irradiation of blood will cause heme photopigments to release Irradiation of blood will cause heme photopigments to release NO, which is known to be necessary in SCN for phase shiftsNO, which is known to be necessary in SCN for phase shifts

ProblemProblem 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 responseand trigger response

SO, TURN OFF THE LIGHT!SO, TURN OFF THE LIGHT!

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