Chapter 6 subtitles postsynaptic integration

CELLULAR NEUROPHYSIOLOGY CONSTANCE HAMMOND Chapter 6 subtitles postsynaptic integration INTRODUCTION (1:56) This sixth and final chapter deals with the summation of presynaptic currents. Glutamate and GABA released by axon terminals have bound to their respective receptor channels and have produced inward or outward currents. Now, what is happening in the postsynaptic neuron? What are the objectives of this chapter? At the end of the chapter, you will know how to add up EPSPS and IPSPs as they propagate along dendrites and you will understand the final effect on the sodium channels located at the axon initial segment and the final effect on the presynaptic neuron activity. Can you explain in simple terms how the various postsynaptic potentials add up to influence neuronal activity? It's a little like voting for a project or against a project. If there are more votes for it, the project goes ahead. If there are more votes against it, the project does not go ahead. The project here is making an action potential. If depolarization exceeds hyperpolarization in the postsynaptic membrane, and particularly at the postsynaptic initial segment, there will be an action potential. If, on the contrary, hyperpolarization exceeds depolarization, there will be no action potential triggered. CH. 6-1 : COMPOUND PSP, PSP PROPAGATION AND SUMMATION (11:19) What do postsynaptic potentials, EPSPsand IPSPs, become once created at postsynaptic membranes? How do they propagate along dendrites? And what is their role? Here is a neuron with two afferent glutamatergic synapses, in red, and two GABAergic synapses afferent to this neuron, in blue. If now we record the activity of this neuron in whole-cell configuration and current-clamp mode to record the changes of potential, we record this compound somatic PSP. "Somatic" because we record it from the soma and "PSP" for postsynaptic potential. So this is the depolarization. Now, if we add a blocker of GABAergic synapses, it means that the blue here are blocked and we record only the effect of the red ones, we record a bigger depolarization of higher amplitude than the green one here. It means that the glutamatergic synapses depolarize the membrane and if the GABAergic synapses are active at the same time, it reduces the effect of the depolarization. Now if we do the contrary, we apply blockers of the glutamatergic transmission, APV for NMDA channels, CNQX for AMPA and kainate channels, we record a hyperpolarization of the membrane which is not big, here, and and no depolarization at all. Now the green one is in fact the sum of these two and you can see here, at the beginning, a small hyperpolarization followed by a depolarization of smaller amplitude than the orange one. If we plot all the traces at the same scale, you see here, of course, that the depolarization without GABAergic synapses is larger and the change of potential without the glutamatergic synapses, with the GABAergic synapses only, is a hyperpolarization. You see here that synapses are all along the dendritic tree and the soma here. These synapses are called axo-spinous because they're between an axon terminal and a spine. Number 2 is axo-dendritic, because it's between an axon terminal and the shaft of a dendrite, and axo-somatic for number 3, because it's between an axon terminal and the soma. Imagine that you have a lot of these types of

synapses on the dendritic branches and the soma. How do they sum along the dendritic trees and, first of all, how do they propagate? Here, we record the activity of a single synapse. The configuration is called whole- dendrite because we are attached first to a dendrite and then a small hole is performed so that we have access to the whole dendrite. This is theoretical, because it's very difficult to record the activity of a single synapse, but let's suppose we can do it. We would record this. First, in current-clamp mode, we record an excitatory postsynaptic potential. I remind you that it's called excitatory because it's depolarizing and, when it depolarizes the membrane, it has a tendency to open sodium channels at the axon initial segment. So here is an EPSP and, underlying this EPSP is a current that we can record now in voltage-clamp mode. This current is an inward current due to the entry of sodium and small exit of potassium ions. This glutamatergic current is short and inward, so there is a net entry of + charges. After this entry of + charges, how do these charges propagate so that we can record an EPSP at the soma and then at the axon initial segment? I remind you that what is very important is what happens here at the axon initial segment, because this is where the sodium channels can be activated and give rise to an action potential. How does this EPSP propagate? We now record at two sites, the dendritic site here, and a somatic site here, in whole-cell configuration and current-clamp mode to record changes of membrane potential. The synapse is on the dendrite here far from the soma. This is the origin of the EPSP. If we recorded it here, we would have an EPSP of that amplitude at time t0. Then at t1, when it's here, between the soma and the dendrite, at t2, when it's in the soma, how is the amplitude of the EPSP? Here the amplitude has decreased and here, in the soma, it has decreased a lot. You can also see that the rise time here is longer. So when the EPSP propagates, its amplitude decreases and its rise time lengthens. How does it propagate? What are the mechanisms? The + charges that entered are essentially sodium ions. These sodium ions are here in the intracellular compartment where there are a lot of other + ions, the potassium ions. They repel each other, so that that they repel, they repel, they repel each other, and they go further down like that, repelling each other. That's how they can accumulate in the soma and then in the axon initial segment. But while they repel, there are also leak potassium currents in the membrane, so that now some potassium ions exit. You remember that the electrochemical force for potassium ions is to push them out, so some of them go out. So we lose + charges, while the EPSP propagates, and at the end, of course, the amplitude is a lot decreased, because there are less + charges than at the origin of the EPSP. We say that this propagation is passive, because it's a passive repulse of + charges, and decremental, because it loses in amplitude while propagating. It should be noted that + charges propagate in two directions: they don't only propagate downstream here towards the axon initial segment, they also propagate upstream, of course, because there is no reason that there be a direction of propagation. But upstream we are not interested by it, what is interesting is what it does at the axon initial segment. We just saw how one a single EPSP propagates, now what happens when you have two here (but it could be also much more than two) glutamatergic synapses on the dendritic branches. So trace 1 alone, here, is recorded in the soma and has the amplitude here in dotted lines. Synapse 2 alone, when recorded in the soma, has this amplitude, here, in dotted lines. but when the two are active close in time, they arrive at nearly the same time in the soma. How do they sum? We see here the resultant, which is exactly the geometric sum of 1 + 2. We say that there is linear spatial summation of EPSPs. Why is this sum linear? MOOC Cellular neurophysiology Ch.6 2/6

It's because the two synapses are not on the same branch, so there is no interference with each other. They sum geometrically here in the soma. This is called spatial summation because the two synapses are located in different points of the dendrites, they are not located close to each other. They are on different branches so this is called a spatial summation. There is a different form of summation, which is called temporal: it happens when the same synapse is active several times in a row. Now let's record the effect of this synapse in the soma, whole-cell recording, current-clamp mode, to record the changes of membrane potential. The synapse is active once, twice, and three times, at very close times. It means that the second time it's active, the first EPSP is not yet back to normal, to control value, to basal value, and the second one and the third one also happens when the second one is still decaying. This summation is called nonlinear: you immediately see that the third one is not equal to three times the first one. It's less than that. Why? It's a question of driving force. The current underlying the EPSP is a cation current. It equals G (the conductance of the membrane) multiplied by the driving force. The equilibrium potential for cations is around 0 mv, so when the membrane depolarizes, it goes closer and closer to the equilibrium potential. If we put the equilibrium potential here at 0, and you have here the depolarization, when you depolarize the membrane, you get closer to this equilibrium potential, and the driving force decreases, therefore the current is decreased and the amplitude of the resultant EPSP is decreased. This is the nonlinear temporal summation of EPSPs. CH. 6-2 : IPSP PROPAGATION AND SUMMATION (2:50) Now what about IPSPs, the inhibitory postsynaptic potentials? Do they add up the same way as EPSPs? Do we also have a temporal and a spatial summation? This is what we are going to see now. Let's suppose that we record the activity of a single GABAergic synapse at its dendritic point of origin. We record a small hyperpolarization of the membrane called IPSP, for inhibitory postsynaptic potential. I remind you that it's called inhibitory because this type of hyperpolarization prevents sodium channels here at the axon initial segment to open. Underlying this small hyperpolarization is an outward current, a GABAergic outward current, and we saw that it results from an entry of negatively-charged chloride ions. These negative charges will propagate exactly the same as the positive charges that we saw before: they will propagate upstream and downstream but what is interesting, as I told you, it's downstream, because it's the effect on the axon initial segment which is important. They propagate the same way means that anions repel other anions and, like that, they go down to the soma and the axon initial segment. But meanwhile there are also channels that open and some charges go out and are lost so at the very end here, this small hyperpolarization is nearly nothing, nearly non existent, so you need a lot of GABAergic synapses, especially when they are in the dendrites, to obtain a significant hyperpolarization of the membrane here at the axon initial segment. They sum up the same way as EPSPs: you can have a linear summation when the synapses are on different dendritic branches, and you get the geometric sum of two or many IPSPs, and you can also have a nonlinear temporal summation when the same synapse here is active several times in a row. MOOC Cellular neurophysiology Ch.6 3/6

CH. 6-3 : SUMMATION OF EPSPS AND IPSPS (7:25) Let's look at the propagation and summation of EPEPs and IPSPs whenever they are produced together and give rise to a compound PSP that we saw on the first slide. We will proceed step by step. At point 1, here is a glutamatergic synapse, and we record an EPSP of a certain amplitude. At point 3, in the soma, we record, in current-clamp mode, the EPSP once it has propagated along the dendritic branch, and it's of much smaller amplitude and slower rise time. Now we have a synapse which is GABAergic at point 2. If we record it at point 1, because the propagation can go upstream, we have nearly no change of membrane potential, because it's far away. If we record quite close, here, at the somatic point 3, we get a small hyperpolarization of the membrane. Now if the two synapses are active so that the PSPs arrive at the same time at point 3, here it's in green, though the EPSP has quite a large amplitude at point 1, when it arrives at point 3, it's nearly null. So the sum here of EPSP and IPSP gives no change of membrane potential. Suppose now that the two synapses, the glutamatergic one and the GABAergic one, are on the same dendritic branch. Look at the glutamatergic one first. We record here at the point of initiation and we have an EPSP here of a certain amplitude. At point 2, this EPSP is smaller because of the decremental propagation. At point 3, it's even smaller, again because of the decremental passive propagation of the + charges. Now, if the GABAergic synapse, which is also on the same dendrite, fires at point 2, its point of initiation, we see a small hyperpolarization. Once it propagated to point 1, it's of course of much smaller amplitude, and propagated to point 3, nearly nothing, it does not exist. If the two are active nearly at the same time, we see here the corresponding recording. Let's suppose 1 here is active first, so there is no change of EPSP. It's active first alone. Then it arrives at point 2 when the GABAergic synapse is active, so the two sum and there is a large reduction of the EPSP. Now when this EPSP propagates to the soma, it's nearly nothing. So from a small EPSP, when this synapse downstream, the GABAergic synapse downstream the glutamatergic one, is active, it cancels the EPSP. This is a sort of nonlinear summation. Because of the change of membrane potential at point 2, the EPSP is much smaller. We previously studied in chapter 5 that sometimes GABAergic synapses have no effect because the membrane potential equals the equilibrium potential of chloride ions. Let's suppose this is the case here. We have the glutamatergic synapse on dendritic point 1 and it gives an EPSP, here, of a certain amplitude. Once propagated alone down to the soma, the amplitude is reduced. Suppose now that the GABAergic synapses that we put here at somatic location 2 are active alone. They have nearly no effect because the soma is exactly at the equilibrium potential of chloride ions. So no effect, no change of potential. Of course, when propagated upstream, there is absolutely no change of potential in the dendrite. Now suppose that the two types of synapses are active in this way: when the EPSP arrives at the soma, that time, the GABAergic synapses are active. We will get this recording of EPSP, of a very small EPSP. You see here it's not the linear summation of the EPSP plus the silent inhibition, no IPSP. Why? This is called the shunting effect. The shunting effect is this: when a lot of GABA-A channels open, of course the membrane resistance decreases, because there are a lot of channels open. There are a lot of ions going through so the membrane resistance decreases. We could say also that the conductance increases. With Ohm's law, if we have a change in current, let's suppose here it's a glutamatergic current, this will give a change of potential, which is here the EPSP. If the membrane resistance decreases, of course, the EPSP amplitude decreases, though the current is the MOOC Cellular neurophysiology Ch.6 4/6

same. So it means that even though here we have the same amplitude of EPSP, when it arrives at the soma where here the somatic membrane has a decreased resistance, the EPSP is much smaller. This shunting effect is always present. Why do we speak about it only with GABAergic synapses? Because of course, when you open any channel, you have a decrease in resistance, but it's more important with GABAergic synapses because they have a tendency to be grouped at the soma, here, or very near the axon initial segment where, as they are grouped, they have a much bigger effect on membrane resistance. CH. 6-4 : ROLES OF POSTSYNAPTIC POTENTIALS (7:04) What is the role of the compound PSP that results from the summation of all the IPSPs and EPSPs that occur nearly at the same time in the dendrites and the soma? What is its role, be it depolarizing, hyperpolarizing or silent? We studied, in chapter 2, that the action potential is initiated at the axon initial segment, because there, there are a lot of sodium channels and these sodium channels have the lowest threshold of activation. All the synapses here, when they are active all together, their role is to open (or not) the sodium channels here. If they open the sodium channels, it means that the sum of all the EPSPs and IPSPs reaches the threshold of the opening of sodium channels. So here on the left, there is a closed sodium channel, and if the PSP (this is the sum of EPSPs and IPSPs) is of sufficient amplitude to open these channels, sodium ions go through and an action potential is initiated. Here the channel is open. Here is the recording. We have the PSP here, and here, the action potential. When the amplitude of the PSP, which is the sum of EPSPs and IPSPs, reaches the threshold for action potential initiation, then there is an action potential. So it has to be above this threshold to immediately generate an action potential. If only a few glutamatergic synapses are active at the same time, or there is a lot of GABAergic synapses active at the same time as glutamatergic ones, the resulting PSP is not of sufficient amplitude to allow the opening of sodium channels. If you look at sodium channels in the left, they stay closed and here it's shown that the amplitude of the PSP is too small to reach the amplitude that we call the threshold of action potential. So there is no action potential. This means that, though glutamate is released, all the synaptic currents happen: propagation, summation..., there is no effect. So you see that, to get an action potential in the postsynaptic neuron, it requires a lot of glutamatergic synapses to be active at the same time, and not too many GABAergic synapses active at the same time as the excitatory ones. Following the activity of glutamatergic synapses, an inward current is generated and produces an EPSP. Following the activity of GABAergic synapses, an outward current is generated and produces an IPSP or no potential change. When both synapses are active, they produce EPSPs and IPSPs that add up in a spatial and temporal manner. If the sum is a depolarization of sufficient amplitude to open sodium channels, an action potential will be triggered at the axon initial segment. If not, there will be no action potential. Inward synaptic currents result from the entry of cations through glutamate receptor channels. They evoke postsynaptic depolarizations called EPSPs. Here, the amplitude of the EPSP, VEPSP, equals the resistance of the membrane multiplied by the current carried by the cations. Outward synaptic currents that are carried by chloride ions? Now it's an entry of chloride ions, of negatively-charged ions, through GABA-A channels. It evokes postsynaptic hyperpolarizations called IPSPs. The amplitude of an IPSP results from the resistance of the membrane multiplied by the current carried by chloride ions. Often, the equilibrium potential of chloride equals membrane MOOC Cellular neurophysiology Ch.6 5/6

potential, so the current is very weak and even null, and there is no IPSP. It's what we call silent inhibition. Once they are initiated, EPSPs and IPSPs propagate upstream but also downstream to the axon initial segment. They passively propagate along dendrites and, because they passively propagate, their amplitude decreases and their rise time lengthens, because some charges are lost during propagation, they go out of the membrane. While propagating, PSPs summate. They can summate linearly, it means that the PSP sum equals the geometric sum of EPSPs and IPSPs, when they are generated far away from each other, but they can also sum in a nonlinear manner. The nonlinear summation says that the PSP sum is inferior to the geometric sum of EPSPs and IPSPs. If these PSPs are generated at close locations on a single dendritic branch, because then they have an effect on each other, or at a single dendritic location but multiple times in a row, the resulting compound PSP will generate action potentials if its amplitude reaches the sodium channel opening threshold, or it will reduce the probability of evoking action potentials if it is hyperpolarizing or silent. GLOBAL CONCLUSION First, receptor-channels (glutamate or GABAA receptor-channels) open, because two molecules of neurotransmitter (glutamate or GABA) bind to them. Then ions cross the membrane through ion channels thanks to the electrochemical force = Vm - Eion. (Membrane potential Vm must be different from the ions' reversal potential Eion, for a current to be generated. If Vm = Eion, there is no current.) When ions cross the membrane, they create a current, and this current of + or - charges depolarizes (EPSP) or hyperpolarizes (IPSP) the postsynaptic membrane. Postsynaptic potentials (EPSPs and IPSPs) are passively propagated to the soma and axon intial segment. When the sum of post-synaptic potentials reaches the opening threshold of voltage-gated sodium channels at the axon initial segment, an action potential is created and propagates along the axon without attenuating. It arrives at axon terminals, depolarizes the membrane of axon terminals and open voltage-gated calcium channels. This gives rise to an inward calcium current, a transient and local increase in calcium concentration, which leads to the exocytosis of synaptic vesicles and the release of neurotransmitter into the synaptic cleft. This neurotransmitter (glutamate or GABA) then binds to post-synaptic receptors and thus opens channels (glutamate or GABA receptorchannels). And this takes us back to the beginning, where the summation of the various IPSPs and EPSPs will give or fail to give rise to action potentials. Overall, all of this course is a loop that closes in on itself. In the MOOC, we began by explaining the action potential and ended by talking about the summation of PSPs, but we could as well have begun by PSPs and ended by exocytosis of synaptic vesicles. MOOC Cellular neurophysiology Ch.6 6/6