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