Upload
kiril
View
30
Download
3
Tags:
Embed Size (px)
DESCRIPTION
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. RPE Cells Photoreceptors Müller Cells. Outer - PowerPoint PPT Presentation
Citation preview
Electrophysiology of Photoreceptors (from counting photons in starlight to the blazing sun snowy slopes)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
RPE Cells
Photoreceptors
Müller Cells
RPE Cells
Photoreceptors
Müller Cells
Brett Gerwin MSII
Salamander Rod and Cone cellSalamander Rod and Cone cell
Synaptic region
Ellipsoid
OuterSegments
Light is the ligand that triggers activation of the enzyme.
Ca2+
Rods AND ConesRods 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
Detecting A Single QuantumDetecting A Single Quantum
In rods a single photon generates an electrical signal that is sufficiently above the noise so as to be reliably detectable.
Photocurrents are graded responses to lightthat changes membrane voltage which in turn drives neurotransmitter release
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 sensitivityRod sensitivity is high at a cost of speed, slow temporal sensitivity
Toad rod recording at 20°C
Two cell types; two functionsTwo cell types; two functions
Rod & Cone OS differ physically Rod & Cone OS differ physically
Rods vs. ConesRods vs. Cones 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
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
Rods vs. Cones: Kinetic & Sensitivity DifferencesRods vs. Cones: Kinetic & Sensitivity Differences
Where do these differences arise?Where do these differences arise?
Rods vs. Cones BiochemicalRods vs. 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 response Greater Ca++ permeability
through cGMP channel CONES NEVER
SATURATE to steady light.
10x faster PDE inactivation.
Chipmunk Rod:
I1/2 = 200 photonµm -2
Sf = 0.090 pA-photon -1µm2
ttpeak = 100 msec
Ti = 90 msec
-20
-15
-10
-5
0
R (pA)
0.60.40.20.0time (s)
A look at rod responses: the #’sA look at rod responses: the #’s
-14
-12
-10
-8
-6
-4
-2
0
2
R (pA)
0.8s0.60.40.20.0
time (sec)
Chipmunk Cones:I1/2 = 7,000 photon- µm-2
Sf =0.0005 pA-photon-1µm2
ttpeak = 50 msec
Ti = 40 msec
Cone responses: the number’s
Chipmunk Cones:
I1/2 = 7,000 photon- µm-2
Sf =0.0005 pA-photon-1µm2
ttpeak = 50 msec
Ti = 40 ms
ROD/CONE Ratios
Chipmunk Rod:
I1/2 = 200 photonµm -2
Sf = 0.090 pA-photon -1µm2
ttpeak = 100 ms
Ti = 90 ms
Rod/Cones:
I1/2 : 1/35
Sf : 1/180
ttpeak : 2
Ti : 2.25
Inactivation steps control sensitivity and timingInactivation steps control sensitivity and timing
ROD and CONE transduction are different! 2x timing 50 to 100x in sensitivity
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 CascadePhototranscduction 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.
Phototranscduction CascadePhototranscduction Cascade
RGS9/G5/R9AP
Cyclic activity of enzymes in CascadeCyclic activity of enzymes in Cascade
Increasing RGS9/G5/R9AP proteins 25 fold alters the rod response.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 FeedbackCalcium Feedback
Light closes the outer segment cation channel reducing influx of Ca2+, a potent feedback signal in phototransduction.
Calcium FeedbackCalcium 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 PhototransductionShutting 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 timingInactivation 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 behaviorElectrical 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
What is adaptation?What is adaptation?
When a constant stimulus results in a variable response. Usually smaller
AdaptationMeasures of toad rod sensitivity on various backgrounds
Measures of toad rod sensitivity on various backgrounds
Toad Rod recording
Later Yau and colleagues find some adaptation in monkey rods
Later Yau and colleagues find some adaptation in monkey rods
Human rod adaptation:Follows Weber’s law
Human rod adaptation:Follows Weber’s law
Minor changes in kinetics accompany light adaptation.
Adaptation in rods may take several seconds to manifest
Adaptation in rods may take several seconds to manifest
Rod photoreceptors do adapt
-very slowly
-to only a limited degree
Cone Adaptation begins within 1 sec
Cone Adaptation begins within 1 sec
Primate cones follow Weber’s law with background light
Cone responses on backgroundCone responses on backgroundThe presence of a background light shifts the operating range of the cone: so increments and decrement can be encoded.
Cones adapt to center their operational range near 50% VmaxCones adapt to center their operational range near 50% Vmax
Background light causes the cones to shift their operating range
Background light causes the cones to shift their operating range
14
12
10
8
6
4
2
0
0.20.0s
14
12
10
8
6
4
2
0
0.20.0s
14
12
10
8
6
4
2
0
0.20.0s
Background light induces undershootBackground light induces undershoot
Dark Dark
Ib = 6.08 log photons/µm2S
Temporal shape of the response can influence behavior as well.
Human flicker sensitivity shows a transition from low-pass to band-pass filtering with background lights.