Transcribed by Tina Park Neuroscience Lecture 18 Synapses I by Dr.

Joel Schiff

Monday, February 3, 2014

Hour 2: Synapses I [00:59:55] Dr. Joel Schiff Alright now, what we basically discussed here is the action potentials propagating along an axon. But what I never addressed is: what triggers an axon potential initially and when it comes to the end of the axon what does I do then? And in most cases, the answer to both of those questions is synapses. Because something is going to initiate the action potential at the beginning of the axon near the cell body by the axon hillock and all that you have, all these dendrites, and at the further end of the axon where the axon potential reaches that, somehow that cell in going to have to signal that this action potential has arrived. The other cell might be another neuron, muscle, gland, or any form of tissue that responds to the existence of the action potential and the axon. What happens in these cases is we have whats called a synapse. You have an axon or neuron. Heres a neuron. And you have connection to another cell, and the connection here is referred to as a synapse. Now there are some rare cases where the connection is actually electrical. It can be mediated by a gap junction, which has a connection between cytoplasm of one cell to cytoplasm of another. Im not going to spend much time on this. What you find is generally not in neurons but such other tissues such as cardiac muscles, epithelium tissues or other organs where you have multiple cells where you can find connection between one cell to another. What typically happens in something involving a neuron is this. It doesnt necessary have to be another neuron. Its another cell. Heres the synapse. So the axon that is carrying the action potential towards the synapse is referred to as presynaptic. And the cell that meets at synapse is referred to as postsynaptic. And for most synapses, when an action potential comes along it releases chemical into the space between the two cells. That chemical is referred to as the transmitter. The transmitter has some influence on the second cell the postsynaptic cell. Now what happens here is, the action potential travels along, might be myelinated or not myelinated, and it causes cell to release the transmitter. Two possibilities. One is action potential tells nerve axon ending to synthesize transmitter and release it. Or the axon ending has been synthesizing transmitter all along storing it and then what action potential does is release of transmitter molecules from wherever its being stored and thats the general case. Now, whats going here? It enlarged the nerve ending. The presynaptic nerve ending. Theres your axon. And yo u have storage compartments in presynaptic neuron known as presynaptic vesicles. And the transmitter having been synthesized is stored in vesicles, and when the action potential comes along it causes these vesicles tend to release their content of transmitter. Now how does that work? Lets start off with transmitters in vesicles. Action potential comes along. Before we do that lets not bring along the action potential. Why do you need an action potential? To release the transmitter. Theres two mechanis m of releasing transmitter. One is, heres your membrane inside, outside. Heres your vesicle. The vesicle comes, drifts towards the cell membrane, and fuses with the cell membrane. And then opening between these membranes appears so you end up with this vesicle open to the outside world and the transmitter can get released. Now, the question I mentioned first is, how does action potential cause this to happen? And the key, way, in which to understanding this is thats the wrong question. The right question to ask is if you dont have an action potential, why doesnt this happen? Now, occasionally it does happen. You sometimes get spontaneous release of transmitter. But the overall scheme of things is unusually. Not unusually rare but unusually seldom. Why doesnt this vesicle filled with transmitter bump into the cell membrane and then fuse with it then release transmitter in absence of action potential? What is this cell membrane made of? I bring you back to Dr. Kinnallys lectures. Youve got a bilayer of lipids. And on the aqueous phases of inside the cell, you have some hydrophilic attachments. It might be a sulfate group. It might be a carboxyl group; might be an amine. Might be a hydroxine thats not charged generally but hydrophilic to certain content. Amines are rare. Mostly what you have are carboxy hydroxy, which can be partially ionized especially if theyre attached to some sort of an aromatic structure, cholesterol You have phosphate sulfate hydroxy group, which are negative charges and rare amines. You have hydroxy groups which might be O-. Whole point is, molecules that make up cell membranes with the lipids also have hydrophilic groups that include negative charges and this has nothing to do with membrane potential because about same the

inside and outside of cell. So doesnt contribute to any electrical field or charges but you have fixed negative charges part of the membrane. And you have these same sort of structures on these vesicle membranes. So what happens when you have vesicle with negative charges around it? In the vicinity of a cell membrane that has negative charges over it. Or what if you have two synapses close to each other. Both of which have ... The answer is they repel. These happen constantly, which we call Brownian Movement. Trivia question: who first worked out the mechanism of Brownian Movement? It wasnt Brown. Einstein. In part of his big year in 1904 and 1905 where he came out with special theory of relativity and the photoelectric effect and got noble prize for photoelectric effect. So these vesicles repel each other as they are moving around and are also being repelled by cell membrane so they dont bump into cell membrane and dont release transmitter by exocytosis. The answer of course is they do occasionally. Thats the side comment I was eluding to because what happens is when you have these Brownian Movement of vesicles at nerve endings, what you end up with is their kinetic energies (number of vesicles, kinetic energies) depending on the temperature that have Boltamine distribution most of them have relatively little kinetic energy. They all have some except for some very rare cases. But theres a small entail. In order for vesicle thats negative charged to strike the cell membrane, which is also negativ e charge and would repel it. It has to have enough Kinetic energy to overcome the electrical repulses, coulomb forces. If thats a negative charged wall Im holding a negative charged baseball and toss it, the ball will slow down and come back away without actually touching the wall. Well if takes some imagination. But if I throw it hard it might touch the wall because the kinetic energy is enough to overcome the electrostatic repulsion of like charges. So occasionally you might get spontaneous release of vesicle full of transmitter. If you look at a neuromuscular synapse. I remember the first time I ever did look at one, was when I was a student a frog nerve muscle synapse. And you poke an electrode into the muscle fiber and you measure the electrode in the muscle fiber, and occasionally you get a little depolarization, a mV or os. Doesnt trigger action potential but these happen once a second on average and these are spontaneous release of transmitter from nerve ending because sometimes theres a vesicle thats out on this tail that has enough kinetic energy to overcome repulsion. So it does happen occasionally. Happens once or twice but not thats not a biggy. What happens when an action potential comes along this axon? Well you get an increase in permeability of Na, increase delay of permeability of K, depolarization, hyperpolarization, and then it goes back to normal. So the membrane potential goes up, down, and then it levels off. The key here is there is voltage gated depending on membrane potentials, Ca ion channels in these nerve endings, that have threshold of -40 mV. And when youre more positive of -40 mV, Ca can move across cell membrane. In which direction? In extracellular fluid, Ca concentration is something like 2.5mMol, 2.5x1-^-3. In the cytoplasm of most cells, if youre not doing anything exciting, you have Ca ion concentration of 10^-7 Mol. The key is that Ca ion concentration inside most cells is kept very low. About 10^-7molar. And this is about 25 times this. Big ratio. If you increase Ca ion permeability, Ca comes into the cell. Because Ca concentration at rest is so low, the amount of Ca coming into the cytoplasm of axon may significantly increase concentration of Ca ion in the cytoplasm. Whats that going to do? Well the key here is that Ca ion is divalent ion two plus charges. So dropped a lot of Ca++ ion concentration [plus plus, plus plus, plus plus[ and you being to neutralize the negative charges that are structurally part of cell membrane and vesicle membranes, which involves vesicle membrane to approach cell membrane. Not just crashing but anywhere where there are release sites, anchors or bars or protein that sort of sit in there. And once they get close enough, actin filaments, which you will encounter in muscle, that will grab on to the vesicle and pull it into the dock. Like ferry boats approaching to the piers. What you have here then is this docking mechanism involving the release points in cell membrane involving actin filaments that pull you into the dock. These don t come into place unless the vesicle is sufficiently close to the docking point and then they pull vesicle in and release the transmitter. So, typically whats happening here is, you get action potential to arrive, Ca permeably goes up and when it goes to -40, it goes back down. And during this period of time which is part of duration of action potential, a millisecond or two, Calcium is coming in neutralizing charges allowing vesicles to get close enough to make docking mechanism work and you end up with transmitter release. Weve gone this far, now what? The transmitter, once its outside, it binds to... Heres the postsynaptic cell and there are receptor molecules on postsynaptic cell. And the transmitters bind to receptor so what you end up with is. Transmitter + receptor, reversible binding and things happen. And thats what Ill be discussing Thursday. But here , you have the vesicle docks, opens up, release transmitters into outside world where it can reach receptors.

Then, what? Well there are two possibilities: One is you notice this docked vesicle opened to the outside world vaguely resembles capital Greek omega. These are called omega forms and they can be on electron microscopes. There are two possibilities. One is that the vesicle fuses completely with the cell membrane and then end up with what Dr. Kinnally described as a raft what used to be vesicle membrane floating around on the surface of the axon membrane. And remember the fluid mosaic model of the membrane where these little rafts of slightly different structures. Vesicle membranes can be seen floating around on the surface of the axon. Thats possibility number one. The other possibility is that the membrane conceals and the vesicle, now empty, can wander off into the cytoplasm. This fusion was always believed to be the classic mechanism. But more recently we found situations in which the vesicles release its contents then separate from this membrane, leaving an empty vesicle. This process here is referred to as Kiss and Run. Obviously named by someone who is hung up in adolescence. So now what are we doing here? Weve got an action potential bringing Ca into the cytoplasm, which leads to release of transmitter. And what the transmitter does after it binds to receptors, Ill get to T hursday. Thats the postsynaptic effects. But ill bring back that magic word homeostasis. All of these changes that weve just made has to be undone so when next action potential comes along were ready to go through same process again. Youve got to rest ore parameters. Well, what changes have we made in releasing the transmitter? Oh, let me just point to this graph here. If thats the kinetic energy distribution of the vesicles in the cell, and this is the threshold, it has to have to release transmitter to fuse with the membrane and thats why it happens very seldom. Notice you put Ca in to neutralize these charges theres a lot of threshold force you have to apply a lot less kinetic energy you have to have to slam into the cell membrane. So lets just say only this much kinetic energy is needed. As you can see, under these conditions after the action potential, a lot more transmitter vesicles are going to release their transmitter. So you dont change the overall kinetic energy profile. What you did was you moved the threshold down always so that now a lot of vesicles can be released. Again the most widely studied of synapses is the one from motor neuron to skeletal muscle in the frog. This goes back decade; almost a century. Well not quite. 70 or so years. And what was discovered is on average once the threshold is lower, you release contents of about three hundred something vesicles. Thats a lot. Ill get back to that. Thursday. That fact. So what do you have to recycle now to maintain homeostasis? You have action potential coming along. Alright, its ended. Its done you have Na -K ATPase and an axon thats removing Na and bringing in K that crossed membrane during action potential. Thats true everywhere. The next thing you did was increase the Ca concentration in the cytoplasm from 10^-7 to 10^- 6 or even 10 ^-5 molar by letting all these Ca come in while the voltage gated Ca permeability gates were opened during action potential. So we have to get that Ca out. And there are two ways of doing that. First thing is you have to lower the Ca concentration back to 10^-7 so you stop releasing transmitter. You want to bring threshold back up to what it is at rest you have to get Ca out somehow. And the important thing is as fast as possible because might be another action potential coming along. You want to lower the Ca concentration inside the axon. Now theres a fast mechanism, but it cant really handle all the Ca. And it certainly cant if youve got more than one action potential going on. Its pretty fast to lower the Ca concentration but its temporary measure. And that is Ca is bo und by smooth endoplasmic reticulum within the axon cells. Its sort of sequestered, hidden away, and bound up. If you have a pipe burst in your basement and you might want to at least pick up some water with a sponge but over the long run thats not going to help much. In the long run youve got to get that water out of the house, out of the basement. So, either two slower but more complete, mechanisms that are going on. One is a Ca ATPase in the cell membrane of the axon, which is for every ATP it splits, pumps one Ca ion out of the cell. One for one. This is called a primary Ca transporter because the movement of the Ca is linked directly to splitting of ATP. You let the Ca in; you pump it out. Youre doing work to get back to where you had been to k eep things unchanged running up and down the escalator again. The other mechanism is there is a Na-Ca exchanger. It will move one Ca out and moves 3 Na in. So remember across cell membrane theres more Na outside than inside cell so Na wants to move into the cell. So you have this counter transport this antiporter. That takes 3 Na from outside lets them into the cell and moves one Ca out. Thats essentially a secondary Ca transporter because its not directly linked to an ATPase. But what you got to keep in mind is over the long run it does rely on ATP hydrolysis. Because had you got those Na outside cell and got Na in now you got 3 Na pumped out at expense of ATP. So you let them in now you have to let them out. Youre still running up and down the escalator. Even though not directly hydrolyzing ATP in this exchanger, you still have to do work to undo that linked change. So you have to get Ca back out of the cell. Thats number one.

The second thing you did in the course of this releasing the transmit ter is youve used up some vesicles full of transmitters so what you have to do now is take these new vesicles or old ones and refill them. If you use this kiss and run mechanisms. Then youve got to take these vesicles and fill them up with transmitters. If you went this way with membrane fusion going on, you have two things to worry about. You took vesicle membrane and fused with vesicle membrane and have rafts floating around. If you had this gong on for a long time youd have increase in surface area of presynaptic neuron because adding more lipid to it and at same time running out of lipid storage. So youd have to recycle lipids fused into cell membrane. So what happens here is this: theres your cell membrane, and heres a raft of what used to be vesicle membrane and it floats around to a recycling point- where you take your newspaper, in this case formally vesicle lipids. Whats happening here is: its covered with molecules called clathrin. Clathrin coats rafts with what used to be vesicle membrane and then starts pinching in as clathrin polymerizes. So what happens is membrane pulling into the cell with clathrin here and eventually it goes through a reverse of this sequence. With the clathrin out here and eventually this pinches off of the membrane and you end up with membrane and vesicle coated with clathrin and thats referred as coated vesicle. Now youve got two types of vesicles, empty because doesnt have transmitters in them, as a result of kiss and run which are plain vesicles that dont have transmitters and youve got clathrin coated vesicles rebuilt out of rafts floating in fluid mosaic cell membrane. And both of these are taken to whats referred to as the cis side of the golgi apparatus. Golgi apparatus is an intracellular structure that packages two things into a vesicle. So it would take the transmitter wherever it is and package it into a new vesicle. The old vesicle attaches on and eventually releases a transmitter vesicle from whats referred to as the trans side of the golgi apparatus. So now youve recycled lipids to make new vesicles, loaded them with transmitter youve reduced Ca concentration in cytoplasm youve got Na pump to handle Na-K movement of action potential and now you have more or less brought things back to the way they were before when action potential cam along presynaptic axon so that when another action potential comes along presynaptic axon this whole thing can repeat. One other thing, you manage to release transmitter into space between presynaptic neuron and postsynaptic cell whatever it is. So, how do you get rid of transmitter? If you go back to the where you had been the original state had no transmitter in space between the cell so what happens to the transmitter? Now here are there are multiple stories depending on what the transmitter is. And theyre basically three types categories of transmitters. One is acehytlcholine, which is unique onto itself. Another is chatecholamines, which includes epinephrine, norepinephrine, dopamine, and few others. Plus a single indolamine called 5hydroxytriptomine, 5-HT. But its also called, the same compound, serotonine. Curiously people tend to say serotonine but when they write it they write 5-HT so thats the reason for it. And each of these is recycled in similar ways. And then theres everything else and ill pick up with this on Thursday so we can discuss it all.