Let there be light.

And then what. . . .

Defining a receptor: FILTER(S) TRANSDUCER–ENCODER• Filters external (what reaches the eye) and internal

(what reaches the cells) – eg. Age-related lens yellowing, macular pigment, oil

droplets in birds and reptile cone photoreceptors.

• Energy specificity (direction of temperature change, wavelength).– Visual transducer = visual pigment

• Encoder ( spike trains vs. graded potentials )

Defining a receptor: FILTER(S) TRANSDUCER–ENCODER• What information is carried by the cells:

– Intensity (how much stimulus is there?)– Temporal (when did the stimulus arrive?)

• What information is not carried by individual cells:– Modality (fine touch vs. pain vs. light are not encoded

by the spike train).

• Where does the signal originate? (Spatial info. not carried by individual sensors, but by the array of sensing elements, and central wiring)

Electrophysiology of Photoreceptors (from counting photons in

starlight to the blazing sun snowy slopes)

Phototransduction Cascade quick review

Single Cell responsesCurrents, voltages transmitter release

Rod and cone response differences

Pigment catches a photon

Decrease [cG] closes cationChannels,

Reducing depolarizing Inward current

Hyperpolarizing the cell

Reducing the amount ofTransmitter released

RPE Cells

Photoreceptors

Müller Cells

Salamander Rod and Cone cell

Synaptic region

Ellipsoid

OuterSegments

• Outer segment– Rods - discs separate

– Cones discs joined to plasma membrane

• Inner segment – Mitochondria for

energy- uses O2

• Synapse (ribbon)– Pedicle vs. spherule

Light is the ligand that triggers activation of the enzyme.

Ca2+

Rods AND Cones• Circulating current between the OS and IS in the dark partially

depolarizes the cells.

• Light triggers HYPERPOLARIZATION and decreased transmitter release. Glutamate is the neurotransmitter.

• Biochemical cascade initiated by absorption of one photon by chromophore (11-cis retinal).

• Activated opsin acts as an enzyme. Rhodopsin and cone opsins are the classical G-protein couple receptor (GPCR).

• Opsin activates transducin, which activates phosphodiesterase (PDE). Activated PDE destroys cGMP

• cGMP is the 2nd messenger that keeps cation channels open

Dynamic balance of [cG] determines membrane potential

Single cell recording

Detecting A Single Quantum

Photocurrents are graded responses to lightthat changes membrane voltage which in turn drives

neurotransmitter release

-1

0

r (pA)

1.00.50.0

time (s)

Rod sensitivity is high at a cost of speed, slow temporal sensitivity

Two cell types; two functions

Rod & Cone OS differ physically

Rods vs. ConesPhysical

• Rod shaped OS• Separate discs

– Slower pigment regeneration (renewal)

• Synaptic ending is small round spherule with few ribbons

• Connect only to On-type, rod bipolars

• One pigment type– No color vision

• Cone shaped OS• Fused discs, continuous

with extracellular space• Pedicle shaped synaptic

terminal with More ribbon synapses (20)

• Connects to many types of BOTH on & off Bipolar cells

• Two or three types of visual pigments– Color discrimination

Rods

Cones

Rods are slow to respond,Very sensitive, and

Adapt only over a small range

Cones respond quickly,

biphasically, are rather

insensitive, and adapt over a very

large range of light intensities

Rods vs. Cones: Kinetic & Sensitivity Differences

Where do these differences arise?

Rods vs. ConesBiochemical

• Very stable visual pigment

• Greater biochem gain• Slower responses• Lower Ca++

permeability through cGMP channel

• Saturation– Limited operating range

• Less stable visual pigment• Lower sensitivity(gain)• Faster temporal resp.• Greater Ca++ permeability

through cGMP channel• CONES NEVER

SATURATE to steady light.• 10x greater RGS9 content

(leads to faster PDE inactivation).

Chipmunk Rod:

I1/2 = 217 photonµm -2

Sf = 0.087 pA-photon -1µm2

ttpeak = 98 msec

Ti = 93 msec

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0

R (pA)

0.60.40.20.0time (s)

A look at rod responses: the #’s

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-8

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-2

0

2

R (pA)

0.8s0.60.40.20.0

time (sec)

Chipmunk Cones:I1/2 = 7,130 ± 1300 photon- µm-2

Sf =4.6 E-4 pA-photon-1µm2

ttpeak = 51± 3 msec

Ti = 39± 6 msec

(n=23) undershoot 0 of 2 S cones 15 of 21 M cones (31%)

Cone responses: the number’s

Inactivation steps control sensitivity and timing

• ROD and CONE transduction are different!

• Although the specific details of the differences is not yet known. . . .– Kinases for phosphorylation of R* differ

– The cGMP gated channels are different

– GCAP proteins that are Ca++ sensitive feedback signal are different

– Inactivation of PDE* by RGS9 are probably different (Cones have 10x rod levels of RGS9)

Phototranscduction Cascade

– Photon of light generates R*• Stage 1: R* collides with G protein (both on the membrane

disk) (500 to 800 fold amplification)– G-GDP : R* promotes exchange of GTP for GDP

– G protein splits to become active G-GTP and the G

• Stage 2: G-GTP collides with and attaches to the enzyme PDE () complex dislodging an inhibitory unit (PDE) Gain factor 1.– The G-GTP- PDE complex greatly enhances PDE activity

• Stage 3: activated PDE hydrolyzes cGMP -> 5’ GMP – Gain factor 6-50

• TOTAL GAIN about 5000 cGMP destroyed, 1,000,000 Na/Ca ions excluded from outer segment of rod photoreceptor outer segment.

Cyclic activity of enzymes

RGS9/G5/R9AP

Increasing RGS9/G5/R9AP proteins 25 fold alters the rod response.

1.0

0.5

0.0

Fractional response

1.51.00.50.0

Control eGFP

Calcium Feedback

Light closes the outer segment cation channel reducing influx of Ca2+, a potent feedback signal in phototransduction.

Calcium Feedback

• Guanylate cyclase replaces the cGMP to reopen the channel to repolarize the membrane back to resting levels.– Cyclase activity is cubically dependent on Ca2+

• Calmodulin is a calcium binding protein that interacts with the cGMP channel to modulate cGMP binding affinity.

• Recoverin is modulated by Ca2+ and is part of the rhodopsin recovery pathway.

Shutting off Phototransduction

• The size of the signal (the gain) depends on how long the cascade remains active.

• Each step of the cascade must be reversed to shut off the signal (Enzymes inactivated)– SPEED vs. SENSITIVITY

• The inactivation of PDE* depends on a complex of 3 proteins: RGS9, G5 & R9AP– Rod vs. Cone gain may depend on PDE*

inactivation rate and RGS9 amounts.

Inactivation steps control sensitivity and timing• ROD and CONE transduction are different!• Although the specific details of the differences is

not yet known. . . .– Kinases for phosphorylation of R* differ (GRK1 & GRK7)

– Inactivation of PDE* by RGS9 are probably different (Cones have 10x rod levels of RGS9)

– Cone channel admits more Ca2+, providing a faster feedback signal to

• Guanylate cyclase (replenishes cGMP to open channels, GCAP)• Recoverin (inhibits Kinase that shuts off R*)• Through Calmodulin acting on channel itself (increase K1/2)

Electrical responses can shape visual behavior

• Simple - if the photoreceptors can’t see it, How can the visual system?– At threshold the rods are counting photons at

the rate of 1/85 minutes!!!• Summation at the bipolar cells

• Temporal shape of the response can influence behavior as well. . . Next slide

10

5

0

R(pA)

0.40.20.0 sec

P347L Pig cone responses

10-4

10-3

10-2

pA2/Hz

12 3 4 5 6 7 8 9

102 3 4 5Hz

Human P347L Pig

Primate & pig cone responses can display bandpass characteristics

CONE PHYSIOLOGY can predict VISUAL BEHAVIOR

Human

Cone

14

12

10

8

6

4

2

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0.20.0s

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12

10

8

6

4

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0.20.0s

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Background light induces an undershoot

Dark Dark

Ib = 6.08 log photons/µm2S

Human flicker sensitivity shows a transition from low-pass to band-pass

filtering with background lights.

Spectral sensitivityColor vision depends on the presence of at least two photopigments (two cone types).

-6

-5

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0

Log S

700600500400Wavelength (nm)

Dark LightChannels Open

Large current

High Energy Demand

High transmitteroutput

Channels CLOSE

less current

Less Energy Demand

LOWER transmitter output