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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 Presentation
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Lecture # 16
Circadian rhythm and melanopsin3/28/13
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
• Calculate your eye’s photoreceptor acceptance angle and your resolvable spatial frequency – put on board
• Does it depend on the color combination?
Human retinal mosaic
Human retinal mosaic
For next week – HW8Short (3 paragraphs) wiki page
Chronobiology• How do organisms
sense time of day?• Why do they need to
do this?
Circadian rhythm• Many organisms follow the 24 hr light cycle of the
sun Circa = about + diem =day
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?
Measuring activity in mice
Mice are nocturnal. Active at night and not during day
During their active time, they will run on a wheel
Wheel motion detector
Monitor when and how much activity mouse has
Monitor mouse activity - running on the wheel
Day Night
Lights on at 10 am and off at 10 pm
Wheel running activity through the 24 hr day
LightOn Off
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.
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.
Jet lag aside
If fly east to west
9 pm becomes 6 pm - darkness takes longer to come
Mouse wheel running
If day shifts later, your body adjusts almost immediately
Easy to reset your clock to a later / longer time
If fly west to east
6 pm becomes 9 pm - darkness comes sooner than you expect
Mouse wheel running
It takes your body a while to adjust to your clock getting shifted (shortened)
How would mice detect light?
?????
Non-mammals• Circadian detection occurs in the pineal
organ• Pineal is on top of brain where it can easily
receive light
Mammals
• Pineal is buried in mammalian brain
• No obvious way to detect light
Circadian rhythm• Human clock involves hypothalamus
SCN is master controller of circadian clock - A few ganglion cells in eye project to SCN
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
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
Mice which lack rods and cones still have circadian rhythm!?!?!?
Light
Rodless / coneless mice studied in early 1990’sFoster et al. 1991
The search #1. Find the light sensitive cells
Science 295:1070 2002
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
Co+2 blocks rods and cones
+ drugs to block glutamate receptors
Isolated cell
Unlabeled
Inject current
Retinal neurons
Photoreceptive retinal ganglion cell
Special tract to hypothalamus and SCN
Search #2: Find the visual pigmentMouse pupillary response
Nature Neuroscience 4: 621
Mouse pupillary response
Pupil will constrict in response to light
Mouse pupil can constrict a lot!
Time course of pupil size responding to
bright light is same in WT and
rodless/coneless mice
Lucas et al 2001
Max pupil size
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
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
Action spectra
Light intensity needed to make 50% constrict = sensitivity
Measure at different wavelengths
Plot sensitivity
Log irradiance (photons/cm2 s)
Action spectra
Wild type
Rodless-coneless
Mouse visual pigmentsCone: 360 and 508 nm; rod 498 nm
Pupil response for rodless/coneless mice has different wavelength peak than rod/cone
opsins
The missing pigment
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
Berson’s retinal ganglion cells which were light sensitive
Photoreceptors
Horizontal cells
Bipolar cells
Amacrine cells
Ganglion cells
Retinal ganglion cell response matches that of pigment causing pupil response
Sensitivity has shape of pigment with λmax = 484 nm
What is the visual pigment??
Frog and fish melanophores respond to light – get smaller if light brighter
Melanophores contain an opsin = melanopsin
7 transmembrane regions
Melanophores contain an opsin= melanopsin
Melanopsin is closest to insect opsins
Melanopsin has broad expression in frogs
Melanophores in skinGives them light response
Magnocellular preoptic nucleus
SCN
Melanopsin is present in retina
RGC containing melanopsin
Hattar et al 2002 Science 295:1065
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
Melanopsin RGCs project to SCN just below hypothalamus
Label with tau-lacZ
Transgene - if introduce melanopsin into a neuron it becomes light sensitive
Cell depolarizes like an invertebrate photoreceptor and opposite to vertebrate photoreceptor
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
Mouse melanopsin
Why should circadian clock have peak sensitivity at 480 nm?
Solar spectrum
Loew and McFarland 1990
At dawn and dusk, the solar spectrum peaks at 460-480 nm both on land and in water.
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
Light response is much (100x) slower than rods and cones
Likely enables averaging of light levels over some reasonable time
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
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
Action spectra
Wild type
Rodless-coneless
Both melanopsin containing RGCs and rods and cones contribute to pupillary response
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