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Transcribed by Matt Asaro Neuroscience Lecture 23- Vision by Dr.

Schiff

February 15, 2014

This hour Im going to be talking about vision, and even though I gave you a sort of general scheme of things for sensory transducers, Im beginning to wonder if I should have put vision off for a little bit alter but your sense of vision violates just about all the rules I just gave. So, your primary transducer cells are the retinal cells the rods, and cones, although, there are some other cells that are also light sensitive in the retina that we are just beginning to learn about. Life gets complicated as you know more. Basically, what Im going to start doing is dealing with rod cell, and cones are a variant on rods that differ in 3 different ways and form there we will move on a bit. You have an eye (draws on board), you have a lens, light comes in. In the back of the eye is the retina and in the retina what you have are rod cells and cone cells. And Im going to be talking about rod cells because they are sort of easier to deal with and cone cells are a little more complicated so well start with the simple thing. You have a rod cell which looks like this- you have a structure that is referred to as the outer segment. In the outer segment, there are disc like structures packed in. This is the outside and this is in. So, this is the outer segment. Its out but its surrounded by whats called the pigment layer which lines the outer surface of the visual part of the retina. These disc like structures are called saccules. They are formed from the cell membrane at the inner part of the outer segment. What happens is an invagination appears at the inner part of the outer segment, the membrane sort of dips in. Heres the membrane dipping in. This one sort of works its way across, then eventually the disc splits off, seals off at the outside edge of the cell membrane so you have this totally internal structure called the saccule. As more are made at the inner part of the outer segment, these old ones move outward and when theyre each the end of the segment (the outer most part), they break down and they are garbage. They are transported out of the rod cell, basically the rod cell closes down, forms a new membrane and leaves them down in the pigment layer. The pigment layer has phagocytic cells of various types and they eat them up . Youll find there are three functions of the pigment layer. The retina of old sacuoles (otherwise the rod cell outer segment would keep getting bigger and bigger. If youre making new saccules you have to get rid of the old ones and in case you care, this whole cycle of life span of a saccule in the rod cell outer segment is about 11 days. A narrowing part that then connects it to whats referred to as the inner segment. So heres the inner segment. The inner segment is characterized by lots of mitochondria. There are thousands. The eye is very dependent on oxidative metabolism. Then the structure goes into theres an axon which is interrupted by the cell body with the nucleus and then at the other end there is something called the synaptic body. Now, what is the status of this rod cell lets say in the dark and then well see what light does to it. (answering a students question: The invagination starts just distal to the isthmus the characteristic of the isthmus is that if you were to take an Em of the isthmus itself, youd find microtubules in the 9+2 arrangement that youd find in a cilium. So at some point in evolution this was a cilium which eventually developed all of these other properties.)

Now lets take a look at a rod cell in the dark. Just leave it in the dark. The membrane potential of this rod cell is hardly something youd expect to find something resting in the dark. Minus 25 mV or so. Its relative to what we usually find cells. This is depolarized. Why is it depolarized? The reason its depolarized is you have in the outer segment a lot of open sodium channels and when you open the sodium channels the membrane potential moves towards +60. Where youd expec tit to be for the normal distribution of ions the cell is depolarized bc you have a high PNA in the outer segment. Now the reason you have a high PNA in the outer segment is that theres a guanlyl cyclase present in the outer segment that produces cyclic GMP- It produces cyclic GMP which somehow acts on the sodium channels to open them. If you have lots of sodium leaking into the cell here, lets get running up our down escalator, you wanna keep everything as constant state so if youre letting the sodium leak in, youve got to get sodium back out of the cell because there are lots of mitochondria in the inner segment and there is lots of Sodium potassium ATPase (3 Na out) So the work that is done running up the down escalator is in the inner segment where you have a lot of mitochondria producing a lot of ATP which drives a lot of sodium, so you have this constant flux of sodium. So you have this sodium in the outer segment, presumably travels along the isthmus to the inner segment where it gets pumped out. This is all in the dark, this is a busy little cell. The other thing that happens is because your membrane potential is -25 thats north of -40, so wherever you have voltage gated calcium channels and you do out here in the synaptic body that has synaptic vesicles in it, you have a high PCA, calcium channels are kept open by high PCA in the synaptic body. So whats that gonna do? Youre releasing transmitter! So heres a cell. The transmitter by the way, appears to be glutamate. So glutamate is the transmitter being released. Okay youre sitting I the dark and unlike any other transmitter and transducer in body, without stimulus its depolarize and its releasing its transmitter, thats the opposite of what I just said for two cell transducer but this one works backwards. By the way, think of this rod cell. If youre in the retina and the outer membrane is buried in this pigment layer, which way does light come into the eye? Through the cornea through the lens. SO the light is coming this way. If you were designing an eye, wouldnt you aim the light sensitive part towards the light? But its backwards. So this is sort of unlike what you might expect but a lot what the light is doing is not what you would expect. These rod cells are incredibly sensitive- they can respond to 1 photon! So what happens is you bring in some light and it strikes one of the saccules. It goes through everything, it strikes one of the saccules in the outer segment. Then what, what do you have? On the saccule membrane, you have a protein which is called opsin, its a protein but theres more to it than that. This is a membrane bound protein on the saccules (so heres your saccules, here youre opsin- and attached to the opsin is another molecule- retinal. Found that because its found in the retina and ends in al because its an aldehyde chemically. So retinal is attached to it. What does retinal look like? Among other things, it has a poly unsaturated conjugated double bond system so if you have a conjugated double bond system and all of these substitutions are trans (remember organic chemistry) this molecule is in a straight line overall. Thats not what is attached to the opsin. Whats attached to opsin is something

called 11-cis retinal which has your conjugated double bonds until you get to carbon 11 where there is a cis substituent. Where does that lead to? Its bent. While its bent like this, the cis retinal can attach to the opsin protein at two different points (different ends). Its bent in the middle. Its anchored to the opsin. Along comes the proton, one photon rather. What does the photon do? It causes a transition from the 11-cis to all trans light. It flips that cis bond into a trans just like all the others. Now what happens here. The 11-cis flips into all trans so now this angled molecule here which was nicely firmly attached and therefore very stable (bound to opsin protein) suddenly wants to be straight. The all trans version of the molecule is a straight line. So this thing wants to straighten out it cant do that while attached to both ends. SO it lets go of both ends The opsin lets go of retinal and now you have retinal. Now you only have retinal attached at one end- straight molecule when it was in cis form, it was bound at two points to opsin so it was very stable, If you break off one end now you have this molecule sort of waving like a flag in the breeze and its sure as hell going to break off at the other end so what you have is in response to light, you go from 11 cis to trans. The trans breaks off into opsin The opsin stays bound to membrane of saccules but retinal is floating off somewhere in solution of outer segment where it might break down, it might find another opsin to attach to- cant attach stably cause its in a straight line. The breaking off- when this goes from 11 cis into trans- it goes through 5 intermediate steps before it completely lets go- not gonna require you to remember the names of the name. Somewhere in that chain of configurations of half attached rodopsin thats what this molecule with the retinal is called probably cause it comes in rods but its Rh- somewhere in the process of the separating it activates a G protein- so in this configuration- it activates a G protein. Its G protein activated- it drops its GTP and picks up GTP and its alpha subunit which detaches from beta gamma subunit and you have this activated alpha subunit with GTP. That G protein is called transducing. What transducing then does is it activates an enzyme- thats what G proteins do- the enzyme it activates is a phosphodiesterase that breaks down cyclic GMP into ordinary GMP. Where have we seen cyclic GMP? Here in the dark. Whats the cyclic GMP doing in the dark? The cyclic GMP is increasing the sodium permeability in keeping the cell depolarized. What you do is what you do when light comes when it comes in is it breaks down rhodopsin- activating the g protein transducing which activates a phosphodiesterase that breaks down the cyclic GMP so now you have lower cyclic GMP concentration and that will stop keeping sodium channels open. Lets start a parallel column here for what light does. (11-cis to trans break down of rhodopsinas this breaks down it activates transducing which activates phosphodiesterase which decreases cyclic GMP which decreases sodium permeability cause less cyclic GMP you dont keep channels open. And what will that do? If you increase permeability that will have opposite effect- moves cell more negative- the membrane potential moves to about -65 mV. The point is this -65 is more negative than -40. Where did -40 come in? Whenever you hear -40 think calcium. So you have a decrease in calcium permeability and therefore you turn off transmitter release. And your glutamate is no longer being released so if you think about this a little bitit almost makes sense to refer tot eh rod cell as the dark detector rather than light detector because in the light its not releasing transmitter- its membrane potential is

normal range about -65 in the light and its not releasing any transmitter. When you put it in the dark- it depolarizes and releases its transmitter. SO you can almost think of it as a dark receptor and basically this is what happens in the rod cell- the rod cell itself has a single absorption spectrum for light and its very sensitive to light- rods are what we see with in very dim light. And because theres only one of them- we dont see color. Lets do our housekeeping- our recycling- have to maintain our homeostasis- what are we doing here? Well we broke down a bunch of cyclic AMP but theres always present in the outer segment- a guanlyl cyclase so it will eventually make more and replace it. And that will open the sodium channels and make the cell less negative and more positive and that will restart the transmitter release and so on and so forth- the transmitter release and recycling vesicles as it takes place in any presynaptic neuron. But what else are we doing here? We broke down rhodopsin and essentially separated out the retinal- and we flipped it into an all trans state- So one thing we have to do is take the retinal and theres an enzyme present- retinal isomerase- that can take the normal all trans retinal that weve released and bend that molecule so that it becomes 11 cis. So thats one part of the recycling- but if you have the retinal part of the rhodopsin released into the cytoplasm of the outer segment and theres a constant turnover of outer segment at the distal end of outer segment youre always losing tissue- youre at some point going to be losing retinal so you have to replace that retinal. The retinal is stored , well its precursor is stored in the pigment layer. Storage of actually retinol (ol because its an alcohol) and then it has to be oxidized to an aldehyde to make reintal. Retinol you get when you consume vitamin A. the pigment layer of the retina stores tons of retinol which is the precursor of retinal which then can be made 11 cis by the retinal isomerase and then stick back onto the rhodopsin- remember I said there were 3 functions of the pigment layer- this is what is referred to as the privalged site for restoring retinol Vitamin A. When you take Vitamin A into your body, a significant part of what is taken in is stored in the retina. Somewhere a good ten years ago, some middle school science teacher told you that if you have a deficiency of Vit A you will develop night blindness. Remember that? You will lose your sensitivity to light. If you remember that, promptly forget it. Not true! Effectively, this is such a privileged storage location for vit A and its derivate that your liver would have to be totally deprived of Vit A before you start losing any from your retina layer. Youre never going to run out of Vit A bc its a lipid soluble compound because its stored there very intensively. You dont run out of VIt A what you run out of is this- the oxidation of retinol into retinal is a redox reaction and it requires one of the B vitamins (paradoxin B6) shortage of that- so that you cant convert retinol into retinal- will lead to night blindness- so eat your B vitamins- okay. Rods. Cones differ from rods in three ways. 4 ways actually. 1. Cones are skinnier than rods. So you have a rod cell, right next to it you might have a cone cell. Theyre thinner and they dont have saccules into the outer segment- what the cone cell has in its outer segment is these invaginations never break off and seal into saccules so what you get are a lot of membrane area but its all part of the cell membrane and it tapers in shape (hence cone shape) you have your isthmus, you have your inner segment. You are pumping sodium out here and its leaking in here and so on. So the

functioning is about the same. Where the cones differ is in the protein that replaces opsin. They have three different proteins that replace opsin. There are 3 different types of cones in your retina. If youre colorblind, dont step back. Most people have 3 diff proteins that replace opsin (cone opsins) and they each have a slightly different light absorption spectrum because the protein effects the frequency of light that optimally causes the cis-trans flip to occur when photon to hit it- you basically have as far as wavelength is concerned- blue is short red is long- you have cones that a sensitivity here and here and here. As opposed to rods whose sensitivity to light looks something like this. A high school science teacher in England proposed that there are 3 different kinds of cone cells each with a different light absorption spectrum and eh published the suggestion that there are blue cones, yellow cones, and red cones, sensitive to three different colors. Three years later, Herman Von Helm Holtz, very big guy in 1890s science. He was a physician by education and he also was one of the leading physicists of the time and did a lot of mathematical analysis of music- Helm Holtz independently came up with this very same theory and someone managed to point out to him that this guy young had beat him to it by a few years. Helm Holtz effectively announced that credit should go to Young. So this is referred to as Young Helm Holtz Theory or Trichromatic theory of color vision. Their maximum light sensitive is blue, yellow, and sort of yellow orange. Theres nothing fundamental about 3 by the way. There are other animals that have up to 7 different kinds of cone equivalence. There are some that dont even have retinas that have different receptors (different butterflies) but Im not going into that. But the cone sensitivity is much less than rod sensitivity so that in very dim light the cones sort of turn off and the rods are what we see with since rods cant see colors- everything sort of looks gray in the dark. We have this distribution of cones- the cones are thinner and therefore you can pack them a lot more closely together than rods. In the center of your retina- you have the fovea which is made up only of cones this is what you use for acute vision- a lot of cones packed tightly for acute vision- ratio of cones to rods shifts as you move away from center of retina and as you get out towards the edges, its virtually all rods. When light, a photon, comes into your eye, it will either hit a rod cell or a cone cell or it will hit the pigment layer. In humans, the third function of the pigment layer is to absorb light. We are not especially good nocturnal animals because anything that doesnt hit a rod or a cone effectively gets absorbed and may as well not have been there. Some animals that are nocturnal cats, deer, things you see dark in the headlights they have eyes that reflect back and thats because theyre pigment layer is reflective- advantage is if a photon misses a rod or a cone it can bounce around inside the eye from the shiny reflective pigment layer until it hits another rod or cone. The advantage to us is that if you see a photon coming from somewhere you know where it came from so we have good visual acuity. To cat or deer you dont have as clear an image but they are better at detecting any sudden change in the light dark pattern and youre much more sensitive to very low levels of light so they tend to survive. Okay, maybe Ill just stop right here.