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 responses  Currents, voltages transmitter release  Rod and cone response differences

Pigment catches a photon Decrease [cG] closes cation Channels, Reducing depolarizing Inward current Hyperpolarizing the cell Reducing the amount of Transmitter released

RPE Cells Photoreceptors Müller Cells

Salamander Rod and Cone cell Synaptic region Ellipsoid Outer Segments

Outer segment –Rods - discs separate –Cones discs joined to plasma membrane Inner segment –Mitochondria for energy- uses O 2 Synapse (ribbon) –Pedicle vs. spherule

Light is the ligand that triggers activation of the enzyme.

Ca 2+

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 light that changes membrane voltage which in turn drives neurotransmitter release

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

Two cell types; two functions

Rod & Cone OS differ physically

Rodsvs.Cones Physical 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?

Rodsvs.Cones Biochemical 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: I 1/2 = 217 photonµm -2 S f = pA-photon - 1 µm 2 ttpeak = 98 msec T i = 93 msec A look at rod responses: the #’s

Chipmunk Cones: I 1/2 = 7,130 ± 1300 photon- µm -2 S f =4.6 E-4 pA-photon -1 µm 2 ttpeak = 51± 3 msec T i = 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/G  5/R9AP

Increasing RGS9/G  5/R9AP proteins 25 fold alters the rod response.

Calcium Feedback Light closes the outer segment cation channel reducing influx of Ca 2+, 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 Ca 2+ Calmodulin is a calcium binding protein that interacts with the cGMP channel to modulate cGMP binding affinity. Recoverin is modulated by Ca 2+ 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, G  5 & 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 Ca 2+, 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

Primate & pig cone responses can display bandpass characteristics CONE PHYSIOLOGY can predict VISUAL BEHAVIOR Human Cone

Background light induces an undershoot Dark I b = 6.08 log photons/µm 2 S

Human flicker sensitivity shows a transition from low-pass to band-pass filtering with background lights.

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

Dark Light Channels Open Large current High Energy Demand High transmitter output Channels CLOSE less current Less Energy Demand LOWER transmitter output