Retina - Biochemistry and Physiology - Part 2 -

Question 1

Briefly discuss the inactivation mechanisms of phototransduction: role of rhodopsin kinase, arrestin, recoverin, guanylate cyclase and calcium levels.

Answer 1

R* is phosphorylated by a rhodopsin kinase (RK), partially blocking the activity of R*
♣ Recoverin
a. A calcium-binding protein
b. Regulates rhodopsin kinase (RK) and phosphorylation of rhodopsin
♣ Guanylate cyclase in photoreceptor
a. Guanylate cyclase activity is regulated by a calcium-feedback mechanism
1) Inhibited by physiologic concentrations of calcium (dark, unstimulated state of photoreceptor)
2) Activated when calcium levels drop during phototransduction (light, stimulated state)

Question 2

Discuss the mechanisms of light adaptation involving the role of calcium:
1) regulating guanylate cyclase activity and cGMP levels (most important mechanism in regulating photoreceptor response to steady light)

Answer 2

Role of calcium
(1) Guanylate cyclase
- Cytoplasmic Ca++ has a powerful negative-feedback action on synthesis of cGMP
- Modulates photoreceptor sensitivity
- Regulates activity of guanylate cyclase (guanylyl cyclase)
Cytoplasmic calcium levels fall with activation of visual pigment
°Light ultimately results in activating PDE, promoting cGMP hydrolysis and closing cGMP-gated channels.
°Ca++ (and Na+) influx stops, but Ca++ efflux continues due to the Na+/Ca++ exchanger (in outer segment plasma membrane.
°Cytosolic Ca++ levels decrease, activating guanylate cyclase
°Partial recovery in cGMP level, causing adaptation to light

Question 3

Discuss the mechanisms of light adaptation involving the role of calcium:
2) effect on recoverin.

Answer 3

(2) Recoverin – under high calcium levels recoverin binds to rhodopsin kinase. As calcium levels fall, recoverin detaches from rhodopsin kinase, and RK binds to R*.

Question 4

What is light-induced translocation of proteins; how does it affect transducin & arrestin?

Answer 4

Movements of proteins within photoreceptor occur at high (saturating) light intensities
°Continuous bright light causes majority of transducin in rods (but not cones) to move from the outer segment to inner segment. Reduces transducin activation rate
°Intense light causes translocation of arrestin in both rods and cones, moving from inner to outer segment.

Question 5

Describe the 2 phases of dark adaptation.

Answer 5

Fast phase =- cones (<10 min)
Cones have rapid recovery from light exposure compared to rods
1) Inactivation of phototransduction is much more rapid in cones than rods
2) Cones regenerate 11-cis-retinal from both RPE + Muller cells.
Slow phase = rods (20-30 min) More sensitive to a single photon than cones, but slower kinetics (shutting off phototransduction response) and rods saturate with bright light.

Question 6

How do cone responses differ from rods?

Answer 6

Cones have faster kinetics than rods. Thus, cones operate over a wide range of bright light intensities without saturating.

Question 7

What is the rate limiting step of dark adaptation?

Answer 7

Rate limited kinetics due to rate-limited delivery of 11-cis-retinal from the RPE to the opsin in the outer segments

Question 8

Electroretinography: measures

Answer 8

an extracellular potential arising from currents that flow through the retina as a result of neuronal signaling. Early receptor potential: no latency, reflects the bleaching of visual pigment within 1.5 milliseconds (e.g., rhodopsin to metarhodopsin II).

Question 9

Concept of parallel processing

Answer 9

There is simultaneous processing of visual information through independent circuits (pathways).

a. Representation of the visual scene is reduced to a limited number of specialized parallel circuits.
b. The photoreceptor signal is processed and integrated within the retina in the terms of center-surround receptive fields.
- Retinal processing can be described in the context of a receptive field.
c. Retinal neurons enhance and segregate different sensory cues of the visual stimulus

Question 10

Convergence:

Answer 10

126 million photoreceptors converge onto 1 million ganglion cells. Signals from many rod photoreceptors can converge onto several bipolar cells, then to one ganglion cell. Rods pool signals to provide high sensitivity for dark-adapted vision. The lower convergence for cones preserves higher spatial discrimination & acuity.

Question 11

Divergence:

Answer 11

a cone can transmit a signal to several bipolar cells of different types, then onto several ganglion cell classes.

Question 12

List the vertical connections for processing the photoreceptor signal; what neurotransmitter do these neurons utilize?

Answer 12

• vertical pathway: photoreceptors to bipolar/ganglion cells
• photorecptors and bipolar cells use glutamate as the neurotransmitter to encode retinal signals
• glutamate provides fast vertical channel signaling

Question 13

Which cells of the vertical pathway are multipolar neurons?

Answer 13

• ganglion cells

Question 14

Which of these cells give a graded response?

Answer 14

• photoreceptors & bipolar cells

- glutamate release varies with changes in illumination

Question 15

An action potential?

Answer 15

• ganglion cells generate action potentials, sending info to the visual cortex about how the light is distributed in space and time

Question 16

Realize that photoreceptors and bipolar cells have ribbon synapses - what is the general advantage of ribbon release?

Answer 16

• use ribbon synapses for high rates of tonic neurotransmitter release

Question 17

List the neurons responsible for lateral interactions.

Answer 17

• lateral interactions: horizontal cell connections in outer plexiform layer (OPL) & amacrine cell connections in the inner plexiform layer (IPL)
• lateral pathways emphasize differences in signals between neighboring photoreceptors

Question 18

Realize the general concept that photoreceptor glutamate has a different effect on bipolar cells depending

Answer 18

on the receptor on the bipolar dendrites.

Question 19

Realize that the type of glutamate receptor defines the type of bipolar cell:

Answer 19

(Bipolar cells are classified as OFF or ON- cells based on the light conditions when the cell is depolarized).

Question 20

OFF bipolar cells:

Answer 20

ionotropic receptors, depolarized when light is OFF

Question 21

ON bipolar cells:

Answer 21

metabotropic receptors, hyperpolarized in response to steady release of glutamate (photoreceptor resting state) and depolarized when the light is ON.

Question 22

At what level of the retina are cone signals split into on- and off- bipolar cell pathways?

Answer 22

• Cone signals are split into off and on channels at the outer plexiform layer (synapse with off and on bipolar cells)

Question 23

Describe the pathway by which rods send their signals to ganglion cells

Answer 23

rod —> rod bipolar cell (ON cell) —> all amacrine cell —> cone bipolar cell (ON and OFF pathways) —> ganglion cell

Question 24

At what level of retina do rod signals split into on- and off- pathways?

Answer 24

• INNER plexiform layer

Question 25

Describe the relationship between circadian rhythm and photo-entrainment.

Answer 25

• circadian rhythms persist when experimental animals are kept in total darkness
• this free-running period is species-specific and usually slightly different from 24 hours
• however, if circadian rhym continues on a free-running basis, within days the organism will be desynchronized from external time cues (light/dark cycles)
• a phase-resetting mechanism excists for the circadian clock, where daily light cues (irradiance, solar day) can shift the phase of a circadian rhythm, resulting in synchronization, or circadian photoentrainment

Question 26

Discuss the retinal cell and photopigment involved in entrainment.

Answer 26

overview: a subset of retinal ganglion cells, the ipRGCs, contain melanopsin and are responsible for light entrainment (synchronization) of circadian rhythms
- a small subset of retinal ganglion cells responds to light: the intrinsically photosensitive retinal ganglion cells (ipRGCs)
- contains a photopigment, melanopsin
- much less sensitive to light than rods and cones
- low photon-capture probability
- low density of melanopsin, which is found only in the plasma membrane
- detect ambient levels of light (irradiance)
- depolarize in response to light, triggering action potentials
- photoentrainment appears to be independent of rod and cone photoreceptors

Question 27

What neural pathways are involved in the regulation of circadian rhythm?

Answer 27

the melanopsin-producing ipRGCs project to the suprachiasmatic nuclei of the ventral hypothalamus, just superior to the optic chiasm

• suprachiasmatic nucleus (SCN) is the master circadian pacemaker
• ipRGCs —> SCN comprises the retinohypothalamic tract
• A multisynaptic pathways extends from the SCN to the pineal gland and functions to:
• transmit info about light and circadian time (light entrainment)
• entrain the rhythmic production and secretion of melatonin by the pineal (light suppresses melatonin secretion / inhibits sleep)

Question 28

what retinal cell detects light for the pupillary light reflex?

Answer 28

• intrinsically photosensitive retinal ganglion cells (ipRGCs)

Question 29

List the classical biochemical pathways of the photoreceptor

Answer 29

• Rate of metabolism in the retina relates to its blood supply
• RPE and photoreceptors receive blood supply from choriocapillaris and have high metabolic activity
• Inner retina receives blood supply from branches of the central retinal artery and has less metabolic demand
• Glucose metabolism
• Oxygen consumption

Question 30

B) Discuss glucose metabolism in photoreceptors.

Answer 30

• retina cells utilize glucose independent of insulin
• transport mechanisms are regulated directly by extracellular concentration of glucose rather than indirectly by insulin
• glucose transport occurs by facilitated diffusion (GLUT 1 and GLUT 3 transporter proteins)
• photoreceptors account for the most glucose consumption in the retina (>80%)
• although aerobic respiration occurs at high rates, a large amount of glucose is converted to lactate
  retina produces ATP as its energy source

Question 31

Is insulin required for the utilization of glucose by retinal cells?

Answer 31

NO

-retina cells utilize glucose independent of insulin

Question 32

Why does the photoreceptor need ATP, describe its requirement for oxygen.

Answer 32

• photoreceptors use 3-4 times more oxygen than other retinal/CNS neurons; twice as much again in dark adapted condition
• probably cells of the body with the highest rate of oxidative metabolism (mitochondria in inner segment)
• ATP is utilized to:
• maintain structure of outer segments
• maintain ionic polarization (dark current)

Question 33

What pathway generates NADPH and what is the role of NADPH in the retinoid cycle?

Answer 33

glucose utilization in Pentose Phosphate Pathway

• NADPH (from PPP) utilized for:
• visual (retinoid) cycle: conversion of all-trans-retinal —> all-trans-retinol
• maintains glutathione reduction

Question 34

F) Briefly describe the role of Muller cells in glucose metabolism.

Answer 34

• take in glucose via a glucose transporter, converting it into glycogen
• Muller cells are the only site for GLYCOGEN STORAGE in the retina
• release lactate, which can be metabolized by photoreceptors