retina 3 ; photoreceptor physiology

Question 1

what does isomerization of visual pigments following the absorption of a photon result in an electrophysiological response in the photoreceptors rely on ?

Answer 1

this relies on the technique of recording from photoreceptors by sticking micro electrodes into them and recording any potential difference in membrane

Question 2

how where the early studies of photoreceptor physiology?

Answer 2

early studies were unsatisfactory as, although cells could be recorded they could not be identified

Question 3

how did cell identification start?

Answer 3

dye injection into recorded cells eventually allowed identification.
it showed that recordings were being made from horizontal cells although researches thought they were photoreceptors

Question 4

what can cells be injected by ?

Answer 4

cells can be injected with a variety of substances such as HRP or fluorescing dyes

Question 5

how were early recordings from photoreceptors made?

Answer 5

• early recordings were made from the photoreceptors of carp, mudpuppies, turtles and geckos as they had large photoreceptors so easier to penetrate with electrode
• these recordings showed that vertebrate photoreceptors like most of the neurons are negatively charged
• both rods and cones had resting potentials of -10 to -40 mV

Question 6

what is different about about vertebrate photoreceptors compared to other photoreceptors?

Answer 6

• with most receptors you would expect when you stimulate them they would depolarize
• vertebrate photoreceptors both cones and rods show a graded hyperpolarisation to light
• bigger the light=bigger hyperpolarisation

Question 7

what is the difference between rods and cones when exposed to light?

Answer 7

• the responses of cones are faster than those of rods
• rods are more sensitive than cones
• for any given light levels, rods give a larger response

Question 8

what is photoreceptor hyperpolarisation accompanied by ?

Answer 8

• photoreceptor hyper-polarisation in response to light is accompanied by an increase in membrane resistance of photoreceptor
• this means that light closes ionic channels thus causing hyper- polarisation

Question 9

what is an experiment that shows that ionic channels close in response to light ( membrane resistance increases ) ?

Answer 9

Ohm’s law ( V=IR )
- inject a ( electrode into photoreceptor )constant current (I) and record the voltage (V) from photoreceptor in both light and dark

• in the light, when the cell hyperpolarises, voltage goes up
• voltage is much bigger in light than dark with the same current injected
• therefore in light resistance has gone up
• if I is constant and V increases, it follow R must also have increased in the light
• in light ionic channels shut that results in hyper polarisation

Question 10

what is another experiment that shows that ionic channels close in response to light?

Answer 10

• slices of rat retina were kept alive in a perfusion chamber and 3 electrodes were positioned along the outer segment
• a current was recorded running along the outer segment in the dark
• in darkness there was a current running around the outer segment called dark current
• when you switch light on the dark current would stop
• this is in line about what we know about ionic channels
• in darkness ionic channels are open and dark current flow and when you switch light membrane resistance increases and ionic channels are shut are dark current stops

Question 11

how can dark current be recorded now ?

Answer 11

dark current can be recorded with suction electrode

brighter light = more dark current is reduced
brighter light = less current

Question 12

what is difference dark and light ?

Answer 12

in the dark:

• ionic channels are open
• photoreceptors are negatively charged
• a current runs along the outer segment

in the light:

• ionic channels shut
• photoreceptors hyperpolarise
• the dark current ceases

Question 13

what is the resting composition of a neuron ?

Answer 13

• inside neuron ( K+ and A-) negative

- outside neuron ( NA+ and Cl- ) positive

Question 14

how does photoreceptor become more negative?

Answer 14

in darkness some ion is flowing into outer segment and when you shut the ionic channel with light the photoreceptor becomes more negative

Question 15

what ion is flowing into photoreceptor in darkness and in light it stops flowing in because its ionic channels are shut and photoreceptor becomes more negative?

Answer 15

sodium
chemical force- goes from area of high concentration to low concentration
electrical force - ion attracted to opposite polarity
in darkness sodium flows into photoreceptor when ion channels are open
in light channel is shut sodium is no longer coming in to outer segment and becomes more negative

Question 16

what is the point of illumination?

Answer 16

to shut sodium channel in outer segment resulting in hyperpolarisation

Question 17

what happens to rods in darkness?

Answer 17

• sodium channel is open in darkness
• sodium flow into outer segment and potassium is flowing out the inner segment causing dark current running around inner and outer segment
• 10% of dark current is due to calcium coming in

Question 18

how much does calcium account for in cones?

Answer 18

• in cones 1/4 of dark current is due to calcium

Question 19

what happens to rods in light?

Answer 19

in light sodium channels close and sodium can no longer enter outer segment of the receptor

Question 20

where is visual pigment located?

Answer 20

visual pigment live in membrane of rod outer segment discs

Question 21

where are sodium channels located?

Answer 21

sodium channels which are opening and shutting are in the plasma membrane of outer segment

Question 22

where is visual pigment isomerisation happening ?

Answer 22

visual pigment isomerisation is happening in the disc but ion channels are in plasma membrane

Question 23

what does a secondary messenger do ?

Answer 23

a secondary messenger ( carrier ) is required to takes visual pigment isomerization in rod outer segment membrane to the ion channels in plasma membrane

Question 24

when can hyperpolarisation induced by light be recorded?

Answer 24

• the hyperpolarisation induced by light can be recorded before Wade’s cycle is complete
• this means that one of the intermediate stages must be the trigger for electrophysiological events

Question 25

what is the intermediate that triggers electrophysiological events?

Answer 25

activated form of rhodopsin is metarhodopsin

Question 26

what is the second messenger ?

Answer 26

cGMP

Question 27

what is the function of cGMP?

Answer 27

cGMP hold sodium channels open in the dark

in light cGMP level falls , sodium channels shut and the cell hyperpolarises

Question 28

how does light cause cGMP levels to fall ?

Answer 28

via an enzyme cascade

• a photon of light isomerises the visual pigment resulting in the formation of the activated rhodopsin
• causes wade cycle which causes formation of various intermediates including metarhodopsin - which is the activated form of rhodopsin
• the activated rhodopsin in turn activates a G protein ( transducin- T)
• the activated transducin then activates phosphodiesterase molecule ( PDE) which is an enzyme that hydrolyses cGMP which hold cation channel to the inactive 5’ GMP
• since this cannot hold open sodium channels they close

Question 29

how is transduction cascade terminated ?

Answer 29

The cascade is terminated by a complex series of events such as the phosphorylation of rhodopsin by rhodopsin kinase. This changes the shape of the opsin so that ‘arrestin’ can block the site where Rh* normally interacts with transducin

Question 30

what is advantage of enzyme cascade ?

Answer 30

The advantage of such a cascade is that each step amplifies the response, so that ultimately one photon can close several hundred cation channels

• one Rh can activate many T
• one T can activate many PDE
• each link chain activates more component

Question 31

what is reason why rods are more sensitive than cones ?

Answer 31

• the transduction cascade has a higher gain in rods

one photon shuts more sodium channels in rods

Question 32

what is role of calcium ?

Answer 32

• calcium has an important role to play in light adaptation
• in response to bright light you get hyperpolarisation - if light stays on hyperpolarisation decays and cell becomes photoreceptor and becomes less negative this is due to calcium

Question 33

what is function of calcium in the dark ?

Answer 33

In the dark calcium enters the cell and inhibits an enzyme, guanylate cyclase, which is responsible for the formation of cGMP (from GTP). which holds cation channel

Thus in the dark not much cGMP is made via this route.

Question 34

what happens to calcium when light ?

Answer 34

• Light closes cation channels and the cell hyperpolarises as GMP levels fall due to transduction cascade.
• However, calcium is no longer entering the cell and therefore guanylate cyclase is no longer inhibited. cGMP is therefore manufactured, cations channels open once more, and the hyperpolarisation lessens as sodium begins to flow back in.

Question 35

when do neurons release neurotransmitter ?

Answer 35

Neurons release neurotransmitter when they are relatively depolarised and cease to do so when hyperpolarised. Photoreceptors are no exception

Question 36

when do photoreceptors release their neurotransmitter ?

Answer 36

rods and cones release their neurotranmitter (glutamate) in the dark, and light serves to lessen this release

Question 37

summary of photoreceptor response to light ?

Answer 37

Resting potential of ca –40mV in dark
Non-specific cation channels are held open by cGMP
Sodium (and calcium) enters the outer segment
A dark current flows in outer and inner segment
And glutamate is released from synaptic terminal

Light activates rhodopsin
This in turn activates transducin
Which activates phosphodiesterase.
This reduces levels of cGMP which holds cation channel
Cation channels shut
The photoreceptor hyperpolarises because positive ions no longer flowing in outer segment
Glutamate release in diminished
(in time cGMP levels rise as due to the absence of calcium & the cell relatively depolarises again)