lecture # 16
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Lecture # 16. Circadian rhythm and melanopsin 3/28/13. Measuring human eye resolution . Pick two of the four patterns in the hall – have each person in your group walk away from the pattern until you can’t see the stripes any more – measure distance to wall - PowerPoint PPT PresentationTRANSCRIPT
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Lecture # 16
Circadian rhythm and melanopsin3/28/13
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Measuring human eye resolution
• Pick two of the four patterns in the hall – have each person in your group walk away from the pattern until you can’t see the stripes any more – measure distance to wall
• Calculate your eye’s photoreceptor acceptance angle and your resolvable spatial frequency – put on board
• Does it depend on the color combination?
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Human retinal mosaic
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Human retinal mosaic
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For next week – HW8Short (3 paragraphs) wiki page
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Chronobiology• How do organisms
sense time of day?• Why do they need to
do this?
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Circadian rhythm• Many organisms follow the 24 hr light cycle of the
sun Circa = about + diem =day
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Light• Cycle needs to be entrained
Without light, cycle free runs
• Loss of light at end of the day signals cycle
• Why might cycle be useful?
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Measuring activity in mice
Mice are nocturnal. Active at night and not during day
During their active time, they will run on a wheel
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Wheel motion detector
Monitor when and how much activity mouse has
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Monitor mouse activity - running on the wheel
Day Night
Lights on at 10 am and off at 10 pm
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Wheel running activity through the 24 hr day
LightOn Off
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Mouse wheel running – multiple days
If you shift the light / dark boundary, it takes the mice a few days to shift back.
They shift forward almost instantly
If you remove the light, they still follow the 24 hour cycle.
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Mouse wheel running
If you shift the light / dark boundary, it takes the mice a few days to shift back.
They shift forward almost instantly
If you remove the light, they still follow the 24 hour cycle.
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Jet lag aside
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If fly east to west
9 pm becomes 6 pm - darkness takes longer to come
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Mouse wheel running
If day shifts later, your body adjusts almost immediately
Easy to reset your clock to a later / longer time
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If fly west to east
6 pm becomes 9 pm - darkness comes sooner than you expect
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Mouse wheel running
It takes your body a while to adjust to your clock getting shifted (shortened)
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How would mice detect light?
?????
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Non-mammals• Circadian detection occurs in the pineal
organ• Pineal is on top of brain where it can easily
receive light
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Mammals
• Pineal is buried in mammalian brain
• No obvious way to detect light
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Circadian rhythm• Human clock involves hypothalamus
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SCN is master controller of circadian clock - A few ganglion cells in eye project to SCN
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Setting the clock
• Need eyes to set clock• Just a few of retinal ganglion cells project to
SCNSCN keeps master clock
• The clock is set or photoentrained at twilightBiological clock is set to local timeZeitgeber = time giver
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Light detection
• For 150 years people thought only rods and cones detected light in the vertebrate eye
• Earliest eyes didn’t form imagesStill sensitive to light
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Mice which lack rods and cones still have circadian rhythm!?!?!?
Light
Rodless / coneless mice studied in early 1990’sFoster et al. 1991
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The search #1. Find the light sensitive cells
Science 295:1070 2002
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Berson et al 2002
Inject dye into SCN in brainRetrograde labeling of ganglion cells in retina
Measure light response of the labeled ganglion cells
SCN = superchiasmatic nucleus
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Co+2 blocks rods and cones
+ drugs to block glutamate receptors
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Isolated cell
Unlabeled
Inject current
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Retinal neurons
Photoreceptive retinal ganglion cell
Special tract to hypothalamus and SCN
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Search #2: Find the visual pigmentMouse pupillary response
Nature Neuroscience 4: 621
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Mouse pupillary response
Pupil will constrict in response to light
Mouse pupil can constrict a lot!
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Time course of pupil size responding to
bright light is same in WT and
rodless/coneless mice
Lucas et al 2001
Max pupil size
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Record action spectra
• The pupil contracts in proportion to the amount it is stimulated
• The stimulation is based on the amount of light it absorbs
• Response should mirror pigment absorbance properties
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Light response• More light shine on eye,
more pupil constricts
• Plot % constriction vs light intensity
• Find light intensity needed to give 50% responsePigment absorption will be inversely proportional to this light intensity
Light intensity
Pupi
l con
stric
tion
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Action spectra
Light intensity needed to make 50% constrict = sensitivity
Measure at different wavelengths
Plot sensitivity
Log irradiance (photons/cm2 s)
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Action spectra
Wild type
Rodless-coneless
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Mouse visual pigmentsCone: 360 and 508 nm; rod 498 nm
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Pupil response for rodless/coneless mice has different wavelength peak than rod/cone
opsins
The missing pigment
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Missing visual pigment
• There must be a visual pigment with peak sensitivity at 480 nmIt must be in the retinaIt is not in rods or cones
• Controls multiple effectsCircadian entrainmentPupillary responseMelatonin suppression
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Berson’s retinal ganglion cells which were light sensitive
Photoreceptors
Horizontal cells
Bipolar cells
Amacrine cells
Ganglion cells
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Retinal ganglion cell response matches that of pigment causing pupil response
Sensitivity has shape of pigment with λmax = 484 nm
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What is the visual pigment??
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Frog and fish melanophores respond to light – get smaller if light brighter
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Melanophores contain an opsin = melanopsin
7 transmembrane regions
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Melanophores contain an opsin= melanopsin
Melanopsin is closest to insect opsins
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Melanopsin has broad expression in frogs
Melanophores in skinGives them light response
Magnocellular preoptic nucleus
SCN
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Melanopsin is present in retina
RGC containing melanopsin
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Hattar et al 2002 Science 295:1065
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Look for melanopsin in mouse retina: found in retinal gangion cells
Find a few RGCs which label with melanopsinIn rat, 2300 cellsIn mouse, 800 cells
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Melanopsin RGCs project to SCN just below hypothalamus
Label with tau-lacZ
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Transgene - if introduce melanopsin into a neuron it becomes light sensitive
Cell depolarizes like an invertebrate photoreceptor and opposite to vertebrate photoreceptor
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Mice lacking rods, cones and melanopsin have no clock
So melanopsin is key to photoentrainment of circadian rhythm
Also key to pupil response - mouse mutants lack this too
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Mouse melanopsin
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Why should circadian clock have peak sensitivity at 480 nm?
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Solar spectrum
Loew and McFarland 1990
At dawn and dusk, the solar spectrum peaks at 460-480 nm both on land and in water.
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In melanopsin expression systems, if add drug to block Gt – there is no effect.
If block Gq then no light response.
If block PLC, no light response
Gt
Gq
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Light response is much (100x) slower than rods and cones
Likely enables averaging of light levels over some reasonable time
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Rule out other possibilities
• Cryptochromes are light sensitive moleculesImportant in plantsAre expressed in inner retinaUse flavin instead of 11-cis retinal as chromophore
• Mouse KO for 11-cis has no circadian clockCryptochromes may be important for clock function but not clock entrainment
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Melanopsin pathway
• Shares inputs with rod / cone pathway-Loss of rod / cones decreases pupil response-Loss of melanopsin decreases (but doesn’t totally lose) circadian response-If lose both, then no circadian response
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Action spectra
Wild type
Rodless-coneless
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Both melanopsin containing RGCs and rods and cones contribute to pupillary response
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Disease implications
• May be diseases where melanopsin pathway is defective which might lead to sleep disorders
• Loss of eyes impacts both sight and circadian rhythm