Okay, so let me show you an example of a voltage clamp. Okay, we are, this is now will be a step of voltage. Remember we looked before at a step of current injected to a cell but in this case we're talking about voltage clamp. Of the Hodgkin Huxley technique. Actually, it was developed by other people, the voltage clamp, but Hodgkin used it with Huxley for the squid giant axon. So here is a voltage clamp, you start from rest, you clamp the membrane, you jump and you decide that now, the difference between the inside and the outside, is not minus 60 but it's let's say, minus 50 here. This is fixed. You cannot change it anymore. So the difference between the inside and the outside is now minus 50 rather than minus 60. And you recall that the current, that you need to inject in order to fix this voltage, and you see the capacitative current, because when you jump the voltage as we know from RC circuits, there is a capacitative current. Just because there is a change from this voltage to this voltage, DV, DT, there is a DV, there is a voltage change from here to here. So there is a capacitative current, and then you fix the voltage so there is no capacitative current any more because capacitivity of the current is, is sensitive to the change in voltage. But there is no change in voltage anymore, here it's fixed, but you get current through the resistance. The resistive current. As we knew before, form an RC circuit. So this behaves basically like an, a passive RC circuit, passive. There is a capacitance, and there is a resistive current this is already what we know. Nothing special here, this is a subthreshold regime. With the subthreshold regime, what happens when we depolarize the cell further, to the superthreshold regime. Something very interesting happens. So here I take, I start from minus 60, but in this case the voltage clamp jumps from minus 60 to zero. So this is a depolarization of 60 millivolts. And then I fix the voltage clamp, so now there is a voltage clamp to plus 60 millivolts relative to rest. Look what happens to the membrane current. I'm now showing you the membrane current. This was a surprise. This was amazing. This is really a revelation because suddenly you see something new. First you see there capacitative current. As before. Just because you change the voltage from here to here, this is a capacitative current, dvdt. It's a big dv, dt so your big, bit IC, capacitative current. But then, when you hold the voltage, when you hold the voltage at plus 60 millivolts relative to rest, you see a strange phenomena. You see that very early on, there is an inward current, inward current from the outside into the axon. This is an inward current, that is activated inward current, you continue to hold the voltage. This inward currents, current becomes smaller and smaller and smaller and smaller and smaller. Then it reverses and becomes an outward current from the inside of the axon outward. So, you see that during this Suprathreshold, voltage clamp, you get first and early inward current, activated inward current and then it reverses, it inactivates and it becomes an outward current so its a biphasic current. Although you hold the voltage fixed, there is no voltage change between the two sides of the membrane, its the same voltage. You get an inward current and an outward current. This was a new phenomenon nobody knew, that the underlying current in the squid giant membrane,during a voltage change, a voltage clamp, consists of two basic phases. The inward fast inward, activation of the current and then inactivation or reversing of the current and then becoming an outward current. This was new. What they also found, that you can block these two currents separately. They found that if you use a very potent drug a very dangerous drug, that you can find in certain fishes. Fish, the tetrodotoxin, the TTX, if you put on top of the axon, in the dish, if you put a very very low concentration of this TTX, You can get rid of one of those currents. So this is what I showed you before during voltage clamp inward and outward current, when I place TTX on the axon, on the squid axon, the first phase, this phase, the inward phase, disappears and you are left only with a outward phase of the current. So, here viewing voltage clamp, you see only outward current. This is in the presence of TTX, so the first, early first inactivating current, activating inactivating current disappear, and you are left with only one type of current. So using pharmac, pharmacology, using pharmacology agents, or drugs, using drugs, you start to separate two currents. The early current seems to be different. Then the late current. You can stay with the late current and get rid of the early current. They also found another drug, the tetraethilammonium or the TEA. This drug, when you place on top of the axon and you do a voltage plan, you find something opposite. So, here is the voltage clamp like here, and suddenly you see that the late phase, the outward phase disappears and you are left only with a inward phase. So they came to the conclusion, Hodgkin Huxley, using this separation technique, using drugs. That this response to voltage clamp, this current response, this membrane current, the inward and the outward, are probably two different currents, they are not the same current, they behave differently. They can be blocked separately. One with TTX, another one with TEA. And they found using other techniques. Changing the concentration of sodium and of potassium so they played with the concentration, both inside the axon because its so big they can change the internal concentration, they can change the external concentration of both sodium and of potassium ions. They found, very, very clearly, that the inward current is a sodium current that flows from the outside to the inside. So this is an inward current the Na, the sodium, the sodium current flows from outside, inside early on, during the voltage clamps. So, very early on. Just after your voltage clamp, some ion channels for sodium apparently open. And these sodiums enables the flow, can flow from outside into the cell. This is this part. Interestingly, you can see that this sodium flow is activated, so there are some opening of channels. Opening of pores that enables the sodium to flow inside but if you continue the voltage clamp, you'll see that this inward sodium ends, inactivates. So, this current has two phases upon it, the activation phase and the inactivation phase of the inward, first inward current. We call it fast inactivating sodium current. This is the first one. And later on with time, there is a second phase, which is an outward phase. This is the blue current here, and they show that this is consists of potassium ions. So potassium at this, at this time the potassium flows from inside, outside. This is the outward current. So the spike basically we shall see, because this is just one voltage and the spike goes all over the voltages. Because it is going up and down. But for this voltage, you can see inward sodium current fast, and inactivating and later potassium current that appears later, and seems not to be inactivated. You can see that you can continue, you can continue the voltage clamp, and the, and the, and the outward current does not inactivate. This is an inactivating current, activating, inactivating, and this outward current is not inactivated. It's activated and remains open as long as there is voltage clamp. So what is the circuit summary of what I just said. This is the drawing by Hodgkin Huxley from 1952, D, the fourth important paper for this year. They summarize what I just showed you, with an electrical circuit of the axonal membrane. We already saw circuits for the passive membrane, we saw circuits for the synaptic membrane in the dendrite. Now I show you a circuit for the axonal membrane. And here is the circuit. There is a capacitance. CM, that you already know. There is a leak or passive channels, as always there are all these channels that we mentioned, that are passive static, they don't depend on anything. This is what we call the R, or R leak. The passive channels. So this is what we saw before, the C and the R. But here are the Hodgkin Huxley added two conductances. The sodium conductance, with a positive battery inside so whenever this conductance, which is voltage dependent, and we call it excitable, because it is a voltage dependent. And when I clamp the membrane supra-threshold, I get current flow, sodium current flow from outside to the inside. Through this opening of this resistance or this conductance. The sodium conductance with the associative battery. And the reason now the conductance, the potassium conductance. So this is the potassium conductance, with a negative battery looking inside. So whenever you open this conductance, potassium actually flows from the inside, outside like it is here. Eventually it goes from here, outside. So this is the inward current and this is outward current. This is the leak current, this is the capacitative current and basically this equation comes, this, this circuit ends by saying that the total current in the membrane, is the capacitative current plus the voltage dependent excitable sodium current. The voltage dependant later potassium conductance and the passive are leak conductance. This is a very unique circuit, because it exists mostly in axons, this voltage dependence, this is what is unique about axons. They have a strong special channel, special ion channels that depends on voltage, and whenever there is enough depolarization, these channels come into play, they are open. This channel open fast, but it inactivates fast, so it opens early on, and then it closes, inactivates. And this potassium channel opens a little bit later and when it opens, there is a current flow from the inside to the outside. This is the classical conceptual jump of Hodgkin Huxley in their voyage in their journey to understand the action potential drawing this circuit. But this is not enough. They need to do more.
