So, in 1952, it's a magical year because in this year you have actually six papers. We are talking, we will talk about most before papers by Hodgkin-Huxley and also Bernard Katz, also a Nobel Laureate, so you see here three Nobel Laureates. Bernard Katz got the Nobel Prize for, for the synoptic aspect, not for the Hodgkin Huxley for the action potential. But this is a, a very basic paper where they showed that the action potential is an all or none phenomena. This was already knew, known before but here they show it again with this technique of axial resistance, axial electrode into the, into the squid giant axon. So, here I show you the all or none properties of the spike. So, you can see that these numbers is the magnitude of the depolarization current injected to the squid axon. This is a small current, no current, so this is the resting potential. This is a little bit stronger depolarizing current. So, you get as you know from membranes, when you inject depolarizing current, you get depolarization and then the, the voltage decays again to rest. We already learned about this. A little stronger depolarization. There is a voltage perturbation, there is depolarizing voltage, but it decays back to rest. So, if the depolarization current, the depolarizing current is not strong enough, you get a perturbation. The membrane potential moves in the depolarization direction, but then it decays back to rest. But if the current is strong enough, it's a supra-threshold current. There is a threshold here, voltage threshold. And if you cross this voltage threshold, for example here, you inject brief depolarizing current supra-threshold, supra-threshold. You suddenly see that the voltage, instead of going down, it goes up. And boom, it generates this new phenomena, the spike, the action potential. Goes up and then goes down as we just discussed. If you inject a stronger current, in this case here, you get an action potential that looks very similar to this one. Earlier, if you inject even a stronger current, you'll get an action potential that looks similar. So, the stronger current you get, you inject, you get this spike. The spike has the same shape. That's why we call it all or none. Because below threshold, for sub-threshold injections of depolarizing current, you don't get a spike. We can call it zero, none. If you inject enough current, supra-threshold current, you get a spike. This spike, this spike, or this spike. It's the same spike. It's a all or none. This is the all, the one, this is the zero, the none. So, there is a threshold, a voltage threshold of about ten millivolts. Above rest. So, you have to shift the membrane potential to about 10 millivolts above the rest, from minus 70 to minus 60, or from minus 60 to minus 50. You depolarize the membrane and then you get in the axon, boom. And all or none phenomena, the spike. With a very particular shape going up and going down, overshooting and undershooting the spike. So, this is a very interesting phenomena, that Hodgkin-Huxley started to analyse. So, what are the underlying currents in the membrane? This is really the big question of Hodkin-Huxley. What makes the membrane of the axon excitable? What makes the membrane of the axon enabled to after reaching a certain voltage, to generate this all or none phenomenon. What, what, what, what are the specific properties in, in the axon? Why doesn't it occur mostly, most of the time in the dendrites? What is unique about the axonal membrane that enables current to flow such that you will get this blow, this boost, this all or none action potential? So, in order to do this, Hodgkin-Huxley developed an, two techniques actually. One that is called the Space Clamp. And one that is called the voltage clip. So, I want now to discuss two techniques because these techniques, it is the technique that made the whole difference. Without these techniques, Hodgkin-Huxley would not be able to really understand the spike, in a most fundamental way. So what is this technique? So, space clamp is very simple. The space clamp technique means, that you take an axial or long axon and, and makes it electrically isopotential. What does it mean? It means that when you put, you, you, place inside the axon, an axial, an axial resistance, low resistance inside the axon, all the points along the axon become isopotential. There is no voltage drop between, let's say, this point, this point and this point. You made the axon, although it's low. Electrically, because of this resistivity that is low, actually there is no voltage draw between this point and this point and this point and this point. So, on the inside of the axon becomes isopotential. Whenever there is a spike here immediately there is a spike there, isopotential. So, the whole membrane becomes isopotential. This is not the usual case. The usual case is that when the ax, the spike starts here it propagates so there is a big spike here and no spike there at the end. But this, they got rid of, by making this axial resistance. Okay, so this is called space clamp, because you clamp the space. The space becomes shrinked electrically. There are no different points in space. The space, because of this axial resistivity, low, high conductance, inside the axon, the space becomes shrinked. And this point and this point are essentially electrically the same point. So, this is the space clamp, this is simple, making the axon as effectively isopotential. But the most sophisticated sys-, method is the voltage clamp, Vc we shall call it, the voltage clamp. The voltage clamp, I'm not going to go into the electrical aspect, but the idea is that you want to clamp the voltage. You want to set the voltage, to fix the voltage between the inside and the outside of the, of the membrane of the axon. So, you don't want the membrane of the axon to behave independently, as it wants to behave, and generate a spike. You want to fix the voltage, to clamp the voltage between the inside and the outside. So, that you will set preset. Let's say that they want the voltage difference between the inside and outside to be 20 millivolts. I can decide and set the voltage to be 20 millivolts and don't allow it anymore to move from there, or 40 millivolts or 50 millivolts above the rest. I want to preset and beside and fixed the voltage between the two sides. Why? Because I don't want the action potential to interfere. I want to see what happens to the membrane at this fixed voltage difference. So, the voltage clamps, clamp technique which is a fast feedback system enables the experimenter to dictate the desired voltage difference between the inside and the outside of the membrane. This electronic feedback system, injects current, exactly to counterbalance the excitable, the voltage dependent current that the membrane wants to generate. I will show you that this membrane generates current that is voltage dependent. For any given voltage change in the membrane, there is a new current generated inside or outside between the two sides of the membrane. And you want to hold this voltage. So, you need to inject a counter, a countercurrent to counterbalance the, the, the natural current that goes through the membrane. So, this is the voltage clamp system. You feel, using this feedback system, you feel what is the current that the membrane wants to start to generate in order to blow an action potential. Then you inject through the system a counter current, exactly the same current but in the reverse direction. So, you fix the voltage and the voltage does not change. This is the basic, the basis of voltage clamp. And because now you can fix the voltage between the two sides of the membrane, you can ask the question, what is the current that flows between the two sides of the axonal membrane for this particular voltage? If you fix another voltage, let's say you move to plus 30 millivolts above rest, what is the current that flows in the membrane for this fixed voltage? So, of course this is a very artificial system because of course the axon does not have voltage clamp in the regular sense and because of this, it blows this independent, completely independent spike. But you want to control the spike, so to speak. You want to control the voltage, fix the voltage, and then measure the current that underlies the volt, the, the, spike. Later on, they will go back to the full spike. But now, we are trying to understand what did they learn from both the space clamp, which does not allow the spike to propagate. But mostly from the voltage clamp, which enables to record direct currents through the membrane because you inject exactly the same current in the opposite direction. So, you can really measure what are the currents that flow through the membrane during voltage clamp. So, this is what I'm going to show you next, the current that flows through the membrane for a given voltage clamp. So let's look at it.
