| 00:02 | Welcome back. Today is thursday, wish it was, but it's |
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| 00:11 | Tuesday February seven And we have the potential. We already started talking about |
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| 00:17 | action potential a little bit last we will continue talking about that |
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| 00:22 | And then the next lecture also we continue talking about the action potential and |
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| 00:27 | concept of the back propagating action potential back propagating spike after that, which |
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| 00:35 | basically a week from today. So these two lectures a week from today |
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| 00:40 | Tuesday, we will have our midterm review online, so we will not |
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| 00:47 | a class meeting on Tuesday next We'll have it life online over the |
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| 00:54 | and I will provide you with the link uh the day before or two |
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| 01:00 | before via the email that will go your um h e mails. |
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| 01:05 | if you have any questions on the that we discussed on any material, |
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| 01:12 | there's something you don't understand, look up before the review session, maybe |
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| 01:20 | the video on the video points. if you still have those questions come |
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| 01:26 | the review session prepared and you can those questions. What I typically do |
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| 01:32 | I cover each section In about 20 minutes, some of the more |
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| 01:40 | concepts of slides, it doesn't mean is the only thing they're going to |
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| 01:43 | on the exam, but I think maybe these are important ones that everybody |
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| 01:47 | know period And at that point you ask questions and you can raise your |
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| 01:54 | and you can type them on the and I will check periodically every 15 |
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| 01:59 | or so as we go through the and the review will also be about |
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| 02:05 | hour and 10 minutes an hour and minutes depending how we proceed. So |
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| 02:11 | so everybody's on the same page it's seven Tuesday. It's our Lecture seven |
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| 02:21 | . And if you look at your materials, what we have here is |
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| 02:27 | have some of the information that is going to relate back to what we |
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| 02:32 | the biophysical number and properties and the of the membrane equivalent circuit as well |
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| 02:43 | the concept of the I. Curves that we will be reviewing uh |
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| 02:51 | in in this lecture we'll see if get to today or in the following |
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| 02:58 | . So from the very beginning what said is that whenever you see the |
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| 03:03 | wave like boxes, right? The boxes. This is electronics. So |
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| 03:11 | the left on the diagram which you're would be what electronics produce. And |
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| 03:18 | do we talk about electronics? Because talking about recording membrane potentials. We're |
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| 03:23 | about recording action potentials. Okay and do that we need electrophysiology. We |
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| 03:34 | instrumentation and so we need electrodes and . Electronic and the electrodes. They |
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| 03:42 | very fast immediate circuits that turn on off sort of like an example would |
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| 03:48 | on much lower scales. A light . You can turn on the light |
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| 03:52 | and turn off the light switch. these would be current injections or current |
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| 03:59 | that we can produce with electrodes and this direction, their outward and in |
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| 04:04 | direction, their inward. So the as illustrated here on what we call |
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| 04:11 | ivy graph here. I stands for in Nottingham person V or membrane |
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| 04:20 | B M is in mila vaults So what we have also here is |
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| 04:27 | current by convention. So positive nana parent values is outward current, negative |
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| 04:37 | and pear. Or any ampere values this configuration by convention are inward |
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| 04:46 | So, first of all, when produce something like this on the left |
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| 04:51 | the electronics with instrumentation and you stimulate cell or in other words, you |
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| 04:58 | or pass the current to conduct the from the electrode into the cell. |
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| 05:03 | cell doesn't respond in the same square like fashion as you're seeing in a |
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| 05:11 | on the left. And that is the cell membrane has certain properties that |
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| 05:20 | that I'm talking about, the equivalent a light switch that you can turn |
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| 05:24 | or off the electronics. Turn on switch. But you can imagine the |
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| 05:30 | instead of turning on, it's sort like the dimmer that gets brighter when |
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| 05:35 | turn off, it's the dimmer that slowly back to the dark life |
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| 05:42 | So these are responses of the south it's only to the resistive and capacitive |
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| 05:51 | of the membrane. The resistance or in depends on the resting channel density |
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| 06:01 | the membrane surface area. So it how many channels you have on the |
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| 06:08 | . And also what's the surface area the membrane with the input resistance is |
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| 06:18 | . M. Which is the membrane . Because remember current cannot flow through |
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| 06:25 | membranes. The membrane is completely resistant current. But if you have channels |
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| 06:32 | those channels are opening up that means are flowing. But there's still |
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| 06:39 | That means that on all of the immediately cross. We said that they |
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| 06:43 | fast. But there is resistance And we talked about how the resistance |
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| 06:48 | neurons is very high values and tens hundreds of megatons. We talked about |
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| 06:55 | relative scales of voltage current resistance and . Which is the universe of |
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| 07:02 | Right so here you have the fact the input resistance is Iran divided by |
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| 07:10 | pi a square. Where a. the radius of hysterical neurons. So |
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| 07:19 | means that the larger this neuron The smaller the input resistance, the |
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| 07:34 | the neuron because A. Is the of hysterical neuron. So that means |
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| 07:39 | small cells will have very high input . Because they're a values are gonna |
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| 07:46 | smaller. Pie will stay the same both configurations. Resistance and capacities. |
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| 07:52 | other way to view what's happening at membrane is changing voltages equal ir equal |
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| 08:00 | or the input resistance into the zone when we're talking about capacitance. What |
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| 08:07 | capacitance? Capacitance is the ability or to store charge. So capacitance or |
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| 08:16 | in voltage can also be viewed as change in charge which is Q. |
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| 08:24 | over a capacitor of capacities. So change voltage charge has to be added |
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| 08:30 | removed from capacitor. And in this we're talking about the plasma membrane which |
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| 08:36 | a capacitor which is storing a lot charge has the capability of storing and |
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| 08:44 | now so that I see here and general we're not gonna pay that much |
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| 08:53 | about the capacity of current flows. the capacities the input capacitance is capacities |
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| 09:02 | the memory. And in this time this case times for pi a square |
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| 09:10 | a again as the radios. So the input resistance, if there's a |
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| 09:16 | south and the input resistance is high a value is small. If the |
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| 09:23 | is small and the a value is , the capacities is going to be |
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| 09:29 | . So that one is R. over four pi a squared. And |
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| 09:35 | is C N. Times four pi squared. Now in your lecture, |
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| 09:42 | you have is this description of what call membrane equivalent circuits. But what |
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| 09:49 | that mean? That means that a can be viewed as a circuit as |
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| 09:55 | electrical circuit. In other words you build it in the physics lab or |
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| 10:01 | chemistry lab you can build a circuit each channel can be viewed as a |
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| 10:10 | . So this is a symbol for . Yeah, this is the symbol |
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| 10:19 | resistance r sometimes and most of the the resistors are variable because each channel |
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| 10:29 | it's closed, if it has very resistance, if it's open it has |
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| 10:34 | resistance then it is partially open. has somewhere in between. These are |
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| 10:39 | resistors. It's also the same symbol conductors. The variable and doctors are |
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| 10:48 | resistance is not # nine, it's . That's a symbol. Each one |
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| 10:55 | the channels has electro motive force. . That that's the electrochemical, the |
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| 11:03 | of charge and the electrical gradients electro forces. Which is in a way |
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| 11:08 | channel also has its own battery. the symbol for battery is that it |
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| 11:14 | one plate that's longer here, that's and another one that's negative. And |
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| 11:20 | the representation of the membrane equivalent Or in this case of a potassium |
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| 11:29 | equivalent circuit. Yeah. And each of these channels will have their own |
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| 11:37 | sodium representation of their own variable conductor sodium and the battery conductor potassium the |
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| 11:46 | conductor for florida. And the And the battery of course, is |
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| 11:50 | to depend because the drive from the is going to depend. We will |
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| 11:55 | about the driving force already started discussing the driving forces and we'll come back |
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| 12:00 | talk about it some more electrochemical driving . What we discussed is the difference |
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| 12:09 | between the number of potential and the potential of a given, remember |
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| 12:18 | Oh so we'll come back to But this is important that you understand |
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| 12:25 | vehicles Ir or the difference here is driving force between the M. And |
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| 12:29 | . K. Because I are we then have the conductance equals one over |
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| 12:36 | . I. Is equal B over . Therefore I is equal conductance times |
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| 12:43 | or V R minus C. So the current is really conducting the |
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| 12:48 | force for that comeback. Now the symbol the will add later here that |
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| 12:58 | already discussed is a capacitor and this the symbol for capacitor and electronics, |
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| 13:16 | and capacitor. So current for potassium I. K. For potassium which |
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| 13:28 | the conductance. Right conductance stands vis like a vm minus minus conductance of |
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| 13:37 | . Times basically the driving force of stands and driving force. So the |
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| 13:45 | current generated by chemical gradient and potential . The total conductance depends on the |
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| 13:53 | of the channels that are open. you can actually calculate conductance to the |
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| 13:57 | channel. But that patch of the may have 100,000 channels. And you |
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| 14:02 | calculate the total conductors which will then the total conductors of potassium will be |
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| 14:08 | of the potassium channels and the number potassium channels times are given conductance value |
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| 14:14 | a single potassium channel. So you measure conductance values the potassium single potassium |
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| 14:21 | value And you can also estimate or the overall conductance through all of the |
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| 14:28 | channels. You can also calculate the through all of the other channels at |
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| 14:33 | same time, all of the ions the same time. This is a |
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| 14:41 | equivalent circuit representation where you have the cellular side, you have the conductors |
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| 14:46 | resistors, you have the batteries notice the battery sides and science are inverted |
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| 14:52 | because it depends on the separation of for that specific ion. And here |
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| 14:59 | have for example, active flux is sodium current is flexing okay through the |
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| 15:08 | . Okay, flexing through the conductor through the channel and it's going from |
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| 15:14 | to inside, potassium will be going inside to outside of the cell. |
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| 15:21 | this representation here you have another symbol stands for N A K pump, |
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| 15:28 | an A K A T P A . And then the capacitor and there |
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| 15:33 | certain qualities for capacitor to be a capacitor. Overall, the membrane capacitor |
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| 15:41 | leaky. So the capacitor is slightly . Remember we said that it's not |
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| 15:48 | holding the charge because it's leaking potassium potassium is most permissible addressing number of |
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| 15:55 | . So it's a leaking capacitor but the same time it's a good capacitor |
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| 16:01 | to be a good capacitor you have have a lot of surface area. |
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| 16:05 | we talked about the it depends directly the size of the radius of the |
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| 16:12 | . So the more of the surface , the more charge you can |
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| 16:15 | the two plates of the capacitor has be close to each other because if |
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| 16:21 | too far apart in space, uh charge that has to go from one |
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| 16:28 | of the member into the other is take longer time. So the closer |
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| 16:32 | are the better it is. And are the two places. The possible |
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| 16:36 | bi layer capacitor has to charge up . So when you when you stimulate |
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| 16:44 | the cell this should not take tens the milliseconds. This should take a |
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| 16:50 | milliseconds. Once the electronic light switch on, the dimmer should kick in |
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| 16:56 | a few milliseconds. Okay, the charge should load up on one side |
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| 17:01 | that aside when you stop the stimulus you turn off the switch electronics. |
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| 17:09 | the cell membrane is like a It's gonna also slowly decrease and re |
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| 17:16 | to its value before it was So these steps here are showing increasingly |
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| 17:24 | current inputs and this is measuring a in voltage. Right? So this |
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| 17:31 | current here I again and this is . So it's showing that for the |
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| 17:37 | amount of outward current or inward This cell has a linear current voltage |
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| 17:46 | or linear I. D. So it doesn't matter if you stimulate |
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| 17:50 | cell with the outward or with the current for the same amount of change |
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| 17:56 | nana in pairs for the same amount change in nana am pairs in the |
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| 18:02 | direction. Half nanogram per one nana or the outward direction. Half banana |
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| 18:08 | , one nana empire. You'll always the same change in voltage. |
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| 18:14 | so this is what gives this linear and this is I. V. |
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| 18:20 | or response from the plasma membrane. you can draw yourselves this number and |
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| 18:27 | circuits uh and make sure that you these symbols that you can recognize. |
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| 18:34 | symbols label these symbols and recognize the circuit where the currents are moving ions |
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| 18:40 | in their respective correct directions. sodium potassium outside pumps are working against concentration |
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| 18:49 | and the higher the capacitor which is the charge. You have to memorize |
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| 19:04 | part. Yes, there's only Yes. Yeah. It's very important |
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| 19:14 | this is actually the basis for many in in physics or any electronic |
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| 19:19 | capacitor, current, generator, This is what you have in your |
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| 19:29 | . All right, Where are you three different ones? This is a |
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| 19:38 | . This is a resistor. You about formulas? Well this is different |
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| 19:51 | the symbols. The symbols you have know was if you know vehicles that |
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| 19:58 | and jeez equal one over r you everything else. If you know the |
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| 20:02 | force Vm minus C. K. know, again it's only three things |
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| 20:06 | have to know and you have to what's the difference between ernst equation and |
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| 20:11 | equation. You can actually outline yourself formulas that you need to know from |
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| 20:18 | because we very clearly discussed the first Goldman equation. Okay. And then |
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| 20:24 | are these are the symbols and then information you have to remember and memorize |
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| 20:30 | this is something that is just a of the science that we're learning. |
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| 20:36 | know some of the basics of the like the threshold fractured potential, you're |
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| 20:39 | know it's -45 million holes and you're well I guess you will because it |
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| 20:45 | be on the test so you I'm sorry. No I really mentioned |
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| 20:57 | I think maybe after class last time for the learns and the Goldman equations |
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| 21:03 | don't have to know the calculations you . Don't have to do the |
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| 21:08 | You have to understand the variables and have to know the relative concentrations of |
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| 21:12 | four dominant ionic species. No and think that I use this diagram a |
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| 21:19 | of times to do this section that teaching the action for. Don |
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| 21:23 | And what I would do if I prepared for the task is I would |
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| 21:30 | this I just printed on the page have it on a power point or |
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| 21:35 | . And I will write out everything I'm seeing here, what is |
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| 21:40 | K. Write it out and write the value minus 90 is already there |
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| 21:48 | out that E. A. Is . Ionic which is a collaborative potential |
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| 21:56 | you have D. M. What it? It's number of potential right |
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| 22:01 | it's number of potential be down and calculate membrane potential. You can write |
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| 22:11 | I would use global equation calculate K. Nerds, nerds, nerds |
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| 22:20 | beyond Goldman then you say what's the between and its variables that are different |
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| 22:28 | the ability, viable. And also Goldman equation you're incorporating more than one |
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| 22:33 | . So for Goldman you can say this and red passion and sodium and |
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| 22:39 | your Goldman. This is how we plus P. Value which is |
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| 22:43 | So I think that that's what I for students to to use when you're |
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| 22:50 | know thinking what should I remember? lot of it is remembering things but |
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| 22:55 | lot of it is really understanding the concept. Uh And there's some basic |
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| 23:02 | formulas that I think are very Can label everything for example in the |
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| 23:08 | . Um What is that? What you put under a teepee energy, |
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| 23:13 | else against concentration? Great what Slow. Okay so you can use |
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| 23:19 | to make this as your major study for the action potential. Um when |
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| 23:26 | talk about these diagrams here I'm gonna about driving forces again or this yellow |
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| 23:32 | , 80 threshold, this action potential value. And so let's uh come |
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| 23:41 | to this in a second. But this will this will help you prepare |
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| 23:46 | for this slide you can just label . Capacitor resistor battery. Well pump |
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| 23:54 | already labeled here. sodium from outside potassium from inside out. It's it's |
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| 24:00 | think it's fairly simple. So now other important thing when we are looking |
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| 24:08 | these equations and we're talking about uh equation versus Goldman equation which is also |
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| 24:17 | , Hodgkin and Cats equation. You not need to calculate the VM. |
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| 24:23 | again, for example, I have that addressing member of potential, the |
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| 24:27 | dominant ion that's crossing the membrane is . So at resting number and potential |
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| 24:36 | permeability for potassium is one. The for sodium is 0.04 probability for |
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| 24:44 | 0.5. So that means that potassium way more dominant. Way more |
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| 24:53 | But then what happens and sometimes it's like 20 times more permissible at rest |
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| 24:58 | it is to sodium lines. And is how we would plug in. |
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| 25:03 | example, three values. So you plug in potassium sodium and chloride. |
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| 25:13 | is the same nonce equation VM. this case, in the equation we're |
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| 25:21 | equilibrium were given ion the ionic is . T. We have Z. |
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| 25:26 | hear we're getting rid of Z. we're adding a negative chloride also. |
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| 25:35 | our T Zia and then you have natural of permeability. PK variable times |
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| 25:48 | concentration of potassium outside versus inside PK outside inside florida outside versus inside And |
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| 25:55 | ratios they never staying the same. permeability ratio. So if the wrestling |
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| 26:01 | and potential is potassium that's leaking through leaking capacitor because it's just the way |
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| 26:07 | neurons are built. They just have open leaking potassium channels. Or potassium |
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| 26:13 | really dictating the membrane potential address because has the highest permeability to potassium during |
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| 26:21 | rising phase of the action potential permeability sodium becomes 20 times higher. How |
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| 26:28 | that happen? Because sodium channels open there's certain things that need to happen |
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| 26:34 | order for sodium channels to open. talk about it uh the the kinetics |
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| 26:41 | the sodium channels. So while addressed have potassium dominating during the rising phase |
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| 26:49 | the sodium that is dominating and during following phases potassium dominating again. So |
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| 26:59 | we have to first of all understand to to to study action potentials. |
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| 27:07 | I'm gonna come back and refer to diagram again. Remember that the cell |
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| 27:14 | is fluctuating around this RMP resting number potential value and this neuron is getting |
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| 27:22 | inputs as it's getting excitatory inputs. d polarizes but this neuron is also |
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| 27:28 | inhibitory input so it gets inhibitory inputs hyper polarizes and maybe it doesn't produce |
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| 27:34 | action potential for a while depending on state of the neuron and the stimulus |
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| 27:39 | coming into that neuron. You may this minus 65 mil of old membrane |
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| 27:49 | fluctuating up D. Polarizing hyper polarizing D. Polarizing only if it reaches |
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| 27:57 | threshold value for the action potential which actually potential threshold value here in |
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| 28:05 | Only if enough of the excitatory stimulus this value will then this neuron is |
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| 28:14 | to produce the action potential. So are all of the greatest synaptic |
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| 28:21 | Excitatory and inhibitory that are coming into south. And if they're strong enough |
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| 28:26 | for synaptic potentials it will drive the of potential, do these default values |
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| 28:33 | that it's reaches the threshold value. will produce an action potential which is |
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| 28:39 | or not. So if you reach value will always produce action but you |
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| 28:45 | go on for a while without having cell produce an action potential. Just |
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| 28:49 | what kind of inputs the cell is and which inputs are winning the inhibitor |
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| 28:55 | are winning. The cell is not to fire an action potential. So |
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| 28:59 | the rising phase which you have is influx. And if you recall if |
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| 29:04 | recall we calculated the equilibrium potential or E. Ionic for sodium positive 62 |
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| 29:14 | is positive 55 value. I'm not be too picky on the exam to |
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| 29:18 | you exact value but definitely no 55 60 positive D. N. |
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| 29:25 | And then what's happening here is that the cell is a resting number in |
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| 29:30 | . Remember that? The driving force is here in this green line |
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| 29:34 | the driving forces Vm mindless E Okay, okay, so driving force |
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| 29:44 | equal Ir Which is V. Or driving force V is equal. I |
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| 29:49 | the driving force is equal Ir So the resting membrane potential is that these |
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| 29:54 | polarized values, there's a huge difference VM Miller vault value here of let's |
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| 30:01 | minus 67 minus 70. Right, is the number of potential which is |
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| 30:07 | equation which is a combination of multiple . There's a big difference between number |
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| 30:12 | potential and equilibrium potential for sodium That number and potential. There is not |
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| 30:19 | of a driving force for potassium because potential for potassium is -90. It's |
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| 30:26 | so it happens in nature that the contain these leaky potassium channels. And |
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| 30:31 | why there's a lot of potassium that leaking out. But it's not because |
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| 30:36 | has a very large driving force pushing out. Uh huh. So as |
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| 30:44 | enough deep polarization and you open up channels, then sodium goes through what |
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| 30:51 | call the positive feedback loop sodium comes . There's more deep polarization, positive |
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| 30:57 | coming inside to sell more deep more positive charge. More deep |
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| 31:02 | So, it's a positive feedback And sodium, what it's doing is |
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| 31:08 | the overall number of potential to its equilibrium potential value of positive 55 but |
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| 31:15 | fails to reach that value because the this number and potential gets to the |
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| 31:24 | value for sodium. The smaller is driving force for sodium. So that's |
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| 31:31 | reason why sodium driving this number and never reaches the equilibrium potential for sodium |
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| 31:38 | the driving force that the more D it gets, the smaller this driving |
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| 31:44 | is getting for sodium And the more polarized that the potentials, let's say |
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| 31:50 | at positive 20. The driving force sodium here is small. Which island |
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| 31:56 | a really big driving force here, because equilibrium potential for potassium is |
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| 32:05 | The other reason why the number of never reaches the equilibrium potential for sodium |
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| 32:10 | because of the certain kinetics and the of voltage gated sodium channels. And |
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| 32:17 | not only the driving force that produces sodium, but it's also once these |
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| 32:22 | open, they also close up very . So they're very transient in their |
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| 32:28 | , in their conductance. They're fast transient so they open and close. |
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| 32:32 | for those two reasons, the number potential never reaches the equilibrium potential for |
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| 32:38 | . Although during the rising phase of action potential sodium is a dominating. |
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| 32:44 | now, once you have a lot driving force for potassium from these positive |
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| 32:50 | , there's a big difference here with potential for potassium, the number of |
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| 32:55 | is being driven down to where the potential for potassium, but it almost |
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| 33:02 | it, but it doesn't quite because more hyper polarized it gets, the |
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| 33:09 | the driving force gets for potassium. also we start encountering sodium N. |
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| 33:17 | . K pumps that are helping and this charge separation across plasma member into |
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| 33:25 | resting membrane potential values. So during rising phase of the action potential, |
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| 33:32 | cell is in the absolute refractory That means no action potential can be |
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| 33:38 | here. So if you stimulate it sell more and more really strong |
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| 33:43 | it's in the middle of action potential non event that cannot have another action |
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| 33:48 | on top of this action potential. you cannot absolutely cannot produce another action |
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| 33:54 | in this red phase here. But the number eight starts re polarizing and |
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| 34:00 | growing closer to the resting membrane the most effective way to generate the |
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| 34:07 | spike is to have the number of relax again to the wrestling number of |
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| 34:12 | values and stimulate it somewhere here. if you needed to produce another action |
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| 34:18 | in this falling phase of the action at the very end of it, |
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| 34:21 | would enter a relative refractory period during a strong enough stimulus may generate a |
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| 34:28 | action potential and then it would be at a higher frequency. And for |
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| 34:35 | you would need a stronger stimulus to that. Yes. Uh It depends |
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| 34:46 | selves have different uh inter spike We call or periods in between action |
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| 34:55 | and as you saw themselves can produce spikes and others only produced seven a |
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| 35:01 | . And that has to do with kinetics and the dynamics of the |
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| 35:05 | gated channels of the express and also I. V. Curves that these |
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| 35:09 | have. But this is what you're at basically anywhere between two spikes a |
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| 35:15 | to 600 spikes a second depending on subtype. And this relative refractory period |
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| 35:23 | gonna be different duration and different cell . So in some cells you can |
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| 35:29 | them right before they even relax the number of potentials. Others you have |
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| 35:34 | wait a couple of milliseconds so there's differences you know? Okay so we'll |
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| 35:42 | back and continue talking about these diagrams it's always nice to see how this |
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| 35:48 | started. So we're gonna watch a cool video of some of the original |
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| 35:54 | and it will remind you of some the concepts that we discussed and how |
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| 36:01 | got to this point and what was historically to study action potentials. The |
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| 36:12 | pods, body plans and habits are very different from those of humans that |
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| 36:16 | might almost be aliens from another So perhaps it's not surprising that it |
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| 36:22 | a long time for scientists to discover there are fundamental similarities between the nervous |
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| 36:27 | of pods and vertebrates yet it was recognition of a useful difference in their |
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| 36:37 | system which enabled scientists to undertake research has led to a growing understanding of |
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| 36:42 | mechanisms controlling our own nervous system. breakthrough concerned the nerves that control the |
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| 36:50 | of the mantle muscles used in jet . As this archive film shows by |
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| 36:58 | contracting its mental muscles. Even a sized squid can inject a huge amount |
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| 37:03 | water with great force. Mhm. the mid 19 thirties, the british |
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| 37:13 | Professor Jay Z. Young was engaged a study of the squid's anatomy. |
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| 37:19 | observed an array of large tubular each as much as a millimeter in |
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| 37:24 | in the squid's mantle. As these were never filled with blood, they |
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| 37:29 | not have been blood vessels from their to surrounding nerve fibers. Young thought |
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| 37:35 | must be single neurons, giant They're transmitted nerve impulses from the concentration |
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| 37:41 | nervous tissue called a ganglion to the muscles. Using electrodes, he stimulated |
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| 37:52 | surrounding nerve fibers and found that he only produce large muscle contractions in the |
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| 37:58 | when the large tubular structures remained So these were indeed giant axons. |
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| 38:14 | quickly appreciated the significance of Young's finding here at last was an axon, |
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| 38:19 | and robust enough to investigate with the available at the time and one that |
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| 38:24 | for several hours when isolated from the . The intracellular contents of the giant |
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| 38:35 | could be removed and analyzed, leading the discovery that sodium ions were more |
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| 38:40 | outside the nerve cell and potassium ions concentrated inside by refilling the empty axons |
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| 38:49 | solutions of precisely known chemical composition experimenters able to unravel the mechanisms of iron |
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| 38:55 | across the membrane. The giant axons large enough and robust enough for fine |
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| 39:06 | to be inserted through the cell membrane into the axa plasm. In these |
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| 39:17 | techniques, a fine glass tube was inserted into the axon and secured with |
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| 39:44 | . Then the tube was used to a fine wire electrode from which the |
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| 39:49 | between the inside and the outside could measured. But the formation of the |
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| 39:55 | Impulse was far too rapid for detailed with any of the electrical measuring devices |
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| 40:00 | the late 1930s, It wasn't until 1950s following the wartime improvement of electronic |
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| 40:08 | such as the cathode ray Oscilloscope. major progress was made. Scientists found |
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| 40:16 | the nerve impulse was transmitted as a wave of electrical potential and that this |
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| 40:22 | or nothing action potential was generated mainly transient movements of sodium and potassium ions |
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| 40:29 | the nerve membrane. Research on the giant axon unravel the mechanisms of the |
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| 40:37 | and propagation of the nerve action This understanding led directly to the development |
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| 40:43 | drugs that block action potential formation and act as local anesthetics now used routinely |
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| 40:50 | painkillers in dentistry and minor surgery. you you have a link to this |
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| 41:02 | your class lecture material. So you're to uh watch it as much as |
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| 41:09 | want to. Uh I think that says a lot and kind of a |
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| 41:14 | us back to a little bit of timeline that we keep mentioning throughout the |
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| 41:20 | . So only 19 fifties, you those fastest telescopes that start picking up |
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| 41:28 | . And I started doing my PhD 1996. And when I went to |
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| 41:38 | lab, there was still a little of a trepidation when we wanted to |
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| 41:44 | an action potential, it was still 40 years later, someone exciting, |
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| 41:51 | unknown and difficult to to to capture . It involves quite a bit of |
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| 41:59 | and modern day electrophysiology setup is typically quarter million to half a million and |
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| 42:07 | of microscopy and sophisticated manipulators for the and such. So it takes it |
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| 42:13 | a lot to even get to that . Even in modern day electrophysiology and |
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| 42:18 | lot of it also depends on So when you're working with marine animals |
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| 42:24 | survive in high sailing environment that, know, can live for a long |
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| 42:29 | and colder temperatures or metabolism is not same. The great models. And |
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| 42:36 | is a great model. So giant axon. Again, it's not |
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| 42:42 | giant squid. The squid wasn't this squid swallowing a ship like medieval |
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| 42:48 | but it was pretty sizable squid, the accent is one millimeter in |
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| 42:55 | And that's why it was a great model to start analyzing what's inside |
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| 43:01 | axon? What's outside the axon how we squeeze stuff out of the |
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| 43:06 | How do we inject the dye? then remember we talked about fast and |
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| 43:10 | ectoplasmic transport. So you can study ectoplasmic transport with these kind of |
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| 43:16 | Just looking at how fast the dye get transported from one end of the |
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| 43:23 | to the other and measuring the distance the time, inserting electrodes. And |
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| 43:29 | that time when professor young was doing experiments, they said that he saw |
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| 43:35 | he would stimulate the nerve and would a contraction. But he was not |
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| 43:42 | to record those action potentials for another , 20 years until Hodgkin and Huxley |
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| 43:50 | the two main scientists that recorded and and studied, analyzed and modeled action |
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| 43:59 | . And another very important technician that to come about and it came about |
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| 44:05 | uh another 2030 years later. It's voltage climb technique. And this is |
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| 44:12 | all demonstration of the voltage plan. this is what you would envision is |
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| 44:17 | similar to squid giant axon here. is the piece of axon right here |
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| 44:27 | you have a electrode here. And that electorate is green electorate is |
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| 44:35 | the current. So it's going to recording the membrane potential is going to |
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| 44:39 | action potentials is going to record positive polarization and negative hyper polarization fluctuations. |
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| 44:47 | , so why are we talking about quan? Because it's important to understand |
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| 44:53 | , what we just talked about with potentials for different ions. We did |
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| 45:01 | how we did it by doing So once you have squeezed out the |
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| 45:12 | on internal content and you know the of sodium and potassium outside versus inside |
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| 45:18 | other ions you can use that formula calculate equilibrium potentials. Right? But |
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| 45:27 | is theoretical. That is based on You sunk an electrode that says |
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| 45:39 | So you know the you know equilibrium you know. Um But well don't |
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| 45:49 | know you haven't demonstrated experimentally is their potential for sodium or potassium that we |
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| 45:58 | spend all this half an hour talking this is learns equation. But does |
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| 46:03 | exist or is it just an equation just a mathematical formula that matches |
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| 46:08 | So in order to understand individual ionic to isolate those individual and on occurrence |
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| 46:20 | to study their equilibrium potentials. We to invent this other technique called the |
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| 46:26 | clown. And without it we wouldn't an experimental demonstration of equilibrium potentials and |
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| 46:35 | lot of things that we know in . The voltage clamp is done that |
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| 46:40 | have this axon you have a recording . So one internal electrode measures VR |
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| 46:46 | potential and it is connected to a clamp amplifier. It also has a |
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| 46:53 | electrode. So if it's measuring -65 is a resting membrane potential. It's |
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| 46:59 | it as a reference to something. in this case it's measuring as a |
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| 47:04 | to neutral outside electrode which is zero neutral. So you're recording this information |
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| 47:13 | , voltage clamp amplifier compares member and to the desired command potential. So |
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| 47:22 | a second. We just talked about if I take an electrode and I |
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| 47:26 | it inside the cell is -65. if something stimulates the cell is going |
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| 47:30 | de polarize, I'm gonna record action . But I also want to control |
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| 47:36 | I'm injecting inside the cell. I to control the member and potential. |
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| 47:41 | want to command number of potential. this command potential is also a lot |
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| 47:47 | times referred to as clamp potential. voltage clamp voltage command potential. And |
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| 47:55 | do you want to command it? I want to set the number of |
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| 47:59 | at the equilibrium potential for sodium and 55. Positive 60. And see |
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| 48:04 | net flux of sodium. I want do that experimental. It's not enough |
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| 48:09 | me to calculate it. The nurse . So to do that, I'm |
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| 48:13 | say I'm gonna command whatever potential I in this dynamic range that the cell |
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| 48:18 | sustain. From -80 -60 -40 I would go to positive 60. |
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| 48:28 | gonna command it. I'm not just use the electorate as an antenna listening |
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| 48:34 | the membrane and tracing the membrane. actually gonna tell the number of potential |
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| 48:41 | have a certain potential. So I'm it when VM. So you're measuring |
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| 48:47 | here number. So, my command says I want to keep the membrane |
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| 48:53 | minus 40 sitting at minus 40. then the cell gets some inputs and |
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| 49:01 | goes to minus 60. So now VM is different from the command potential |
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| 49:07 | my my command is minus 40 but cell wants to go to minus 60 |
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| 49:12 | it's different. The client amplifying this will inject current into the axon to |
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| 49:17 | second elector of this brown electrode. this feedback arrangement causes the number of |
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| 49:25 | to become the same as the command . So, I told you to |
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| 49:30 | here at minus 40 you want to 60. I'm gonna make up the |
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| 49:35 | . I'm going to inject the 20 to bring it back to minus |
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| 49:40 | Everything that blocks us through all of synaptic currents and inputs that are coming |
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| 49:46 | . The current that is flowing back the axon and across its membrane is |
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| 49:52 | here. So, all of the that are different from my command potential |
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| 49:57 | be measured here in modern day voltage . Which essentially, you can see |
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| 50:06 | clamping with your clamping is your clamping number and you're clamping the number. |
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| 50:12 | so that you can isolate individual ionic is and study them, measure |
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| 50:19 | Block them, see where they have political potentials. It's a negative feedback |
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| 50:27 | because you can liken it to an feedback like you have in the uh |
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| 50:35 | control air conditioner system. So you it 72 Temperature goes up to |
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| 50:44 | The A. C. Kicks in brings it down to 72 negative feedback |
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| 50:50 | . This is the same way you set minus sport goes to mind the |
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| 50:54 | and you kick in the voltage clamp currents and you make up for the |
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| 50:59 | . That's a negative feedback system. voltage climb has way more complex circuits |
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| 51:05 | illustrated here and it actually only requires singular intracellular electric. So when in |
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| 51:14 | original experiments, you needed to have electorates want to record the current, |
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| 51:20 | to pass the current. The circuits so fast that the same electorate can |
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| 51:25 | and pass the current at extremely fast . That it's only one electorate that's |
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| 51:31 | a question. So to distinguish between plus influx and K. Plus |
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| 51:38 | would you do it simply in a of time? Like, okay, |
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| 51:41 | we're going to expect sodium influx. wouldn't set the reading for that. |
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| 51:44 | how would you distinguish into in terms acquiring like the correct reading. |
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| 51:50 | Uh you would actually, so if you didn't know anything and it |
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| 51:55 | like you were Hodgkin and Huxley, would just change membrane potentials probably by |
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| 52:02 | million volt at the commanding potential and would use different blockers for sodium channels |
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| 52:08 | potassium channels until you finally saw a that was replicable. And it made |
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| 52:15 | . You know. So I think that's that's how you do it. |
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| 52:19 | maybe this will also answer your Uh what the voltage clamp will actually |
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| 52:26 | or how it can be used. so Hodgkin and Huxley, Both of |
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| 52:32 | , they won a noble Friday because already in medicine in 1963 for their |
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| 52:38 | on the action. And what they is the use of all this |
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| 52:45 | And on top this red trace here the electronics. It's a square wave |
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| 52:51 | . It's a switch. It's a . Its command potential. I'm commanding |
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| 52:56 | voltage to go to negative 26. commanding the voltage to go to zero |
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| 53:02 | 26. 52 positive 65 commanding this . What I'm gonna do is as |
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| 53:12 | voltage client the potential, I'm gonna inward currents and outward currents. Remember |
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| 53:22 | convention the positive values, nana emperors outward currents. And these are negative |
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| 53:28 | values a million pair in this And they are inward currents. Uh |
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| 53:41 | what's what's happening here is you're gradually this number in potential And you're climbing |
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| 53:51 | -26. And what you're seeing, seeing the short inward current and green |
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| 53:58 | here that is followed by a much and slower outward current. That's |
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| 54:04 | And this is blue zone here. then you d polarize the membrane even |
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| 54:11 | . You de polarize and client the potential at zero mila balls. And |
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| 54:15 | seeing a much stronger inward current but still transient. You're also seeing much |
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| 54:22 | outward current because the inward current here the sodium ions coming inside influx and |
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| 54:31 | current was potassium ions leaving. So we de polarize more, there's more |
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| 54:39 | current. Remember it's positive feedback with sodium, more deep polarization, more |
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| 54:43 | channels open more deep polarization, more current. But as you de polarize |
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| 54:47 | number and potential of these positive you're also driving the potassium current outward |
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| 54:55 | that follows essentially. This is the potential is broken down into the inward |
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| 55:01 | and outward current. Notice what happens positive 26 inward current starts decreasing Because |
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| 55:09 | is coming closer to the equilibrium potential sodium positive 55 and this case is |
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| 55:16 | 52. That's why I said different . Will have a slightly different |
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| 55:20 | positive 52, positive 55, positive noble value. But it's all basically |
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| 55:26 | the same ballpark, depending on different and recordings. What happens is 52 |
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| 55:35 | , which is the equilibrium potential for . What happens to the inward |
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| 55:42 | It's gone why? Because of the potential for sodium, There is no |
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| 55:47 | current flux for sodium. So there's inward current flux ng but you still |
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| 55:52 | very large outward current. Guess what if you cross over to the other |
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| 55:57 | . You see this little blip This little blip is actually sodium current |
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| 56:03 | reversed. So equilibrium potential value of on the other side, the current |
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| 56:09 | starts flowing in the opposite direction. a lot of times this equilibrium potential |
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| 56:15 | also referred to as reversal potential value the current reverse from outward to inward |
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| 56:21 | to outward direction at the equilibrium potential beyond those values the same for potassium |
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| 56:28 | is leaving the cell. But if drive the number and below equilibrium potential |
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| 56:32 | potassium 100 million volts negative the potassium going to reverse its direction also. |
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| 56:41 | now what you have is you have early component which is the inward component |
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| 56:47 | the late component and only using the clamp where we able to tease out |
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| 56:54 | only the individual inward versus outward sodium potassium but also the dynamics of the |
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| 57:02 | the duration of these different cars. you can see that during the influx |
|
| 57:08 | phase of sodium you have multiple sodium open. They open and close very |
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| 57:15 | . So each one of these traces and red uh color under the curve |
|
| 57:22 | the opening of a sodium channel, single sodium channel. And you have |
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| 57:26 | sodium channels. You can see that all of them open at the same |
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| 57:31 | once they open, they also close quickly. So they're only open for |
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| 57:36 | millisecond or so. And that's the why the membrane potential doesn't reach the |
|
| 57:42 | potential for sodium during the d polarizing and the action potential. And if |
|
| 57:48 | some across these different channels that are you get this kind of a smooth |
|
| 57:53 | current responds here. That's the sum individual channels that are conducting sodium and |
|
| 58:00 | the outward side this is during the phase. You see this dash line |
|
| 58:06 | during the rising phase of the action during the rising phase of the action |
|
| 58:11 | that sodium channels that are opening. you look during the rising phase of |
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| 58:15 | action potential, these two dash there is barely any potassium channels open |
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| 58:21 | only at the very peak of the potential, you have the opening of |
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| 58:26 | potassium channels. Each one of these is individual potassium channel opening. You |
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| 58:32 | see very clearly. The difference is sodium channels of fast activating potassium channel |
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| 58:39 | is delayed during the action potential. channels are fast and activating or their |
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| 58:47 | is transient and the activity of potassium through potassium channels is prolonged. We |
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| 58:54 | call it sustained. So transient versus or transient versus prolonged. So the |
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| 59:01 | component of the action potential is the sodium currents. And the late component |
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| 59:09 | is the falling phase of the action is the potassium, the flocks is |
|
| 59:15 | from inside of the cell going into outside of the cell And this is |
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| 59:22 | clamp and without voltage clamp, we be able to demonstrate this experimentally. |
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| 59:27 | wouldn't be able to confirm that the potential value for sodium that we calculated |
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| 59:33 | positive 50 to a positive 55 is noted experimentally. And this was very |
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| 59:41 | technique. So this technique now allows to isolate individual currents and with the |
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| 59:46 | of different blockers and chemicals, we do really good job at isolating |
|
| 59:53 | very specific single currents of interest such sodium potassium and even more selective to |
|
| 60:00 | subtypes of sodium or potassium channels. . So yeah, um so this |
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| 60:18 | different channel, like, like so like from the area that like |
|
| 60:38 | each one conducts a different right? there's a variability in each. So |
|
| 60:57 | amplitude is not going to change, duration is different. So, if |
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| 61:03 | interested in total conductance, you're not interested in duration. You're interested in |
|
| 61:09 | . Once the channel is open, amplitude of the conductance is going to |
|
| 61:13 | the same. So if you have , it's multiplied on certain value of |
|
| 61:18 | . If you have 20 channels is towards certain value of conductance. |
|
| 61:22 | there is variability in biology and not channels will conduct the exact same number |
|
| 61:28 | arms. Not all channels will still the exact amount of time, but |
|
| 61:34 | the uh these advance there is thousands channels that are activated and it averages |
|
| 61:43 | to come out that the action potentials the same and it's reproducible in the |
|
| 61:49 | cell in the same amplitude over and and over and over with slight |
|
| 61:54 | Again, One action potential, maybe million balls. The next 1, |
|
| 62:00 | . The next 187. The next , 85 again, so there's gonna |
|
| 62:04 | slight variations. So um and this exactly the reason why you don't rely |
|
| 62:14 | on the calculations, but you actually the experiments and you can see that |
|
| 62:20 | substantial variability and you would just report with some sort of a standard |
|
| 62:24 | You know, there's an average time opening of sodium channel plus mine or |
|
| 62:30 | . Okay, But the amplitude is to be the same through these |
|
| 62:36 | supposed to be the same. So look at why sodium channels gloves and |
|
| 62:41 | because of the specific structure and sodium . Remember these channels that are participating |
|
| 62:48 | the action potential are gated by that means that voltage is going to |
|
| 62:53 | and close these channels. sodium channel four subunits. 1, 2, |
|
| 63:01 | , 4. Each subunit has six membrane segments. S 123456. The |
|
| 63:13 | loop robert Mckinnon's pore loop. The filter here, It's between five and |
|
| 63:21 | . That's five and S six As is a very interesting segment in this |
|
| 63:28 | . It has a lot of charge charge. So these are the amino |
|
| 63:34 | residues with positive charge and S four for voltage sensor portion of the |
|
| 63:43 | That means this is the portion of channel that is going to be sensitive |
|
| 63:47 | voltage. This is the portion of channel. Here is another illustration of |
|
| 63:52 | S. Four that is going to reacting to the changes in the |
|
| 63:58 | So this is the gating voltage The channel has gates, the gates |
|
| 64:04 | closed and the gates need to be and the gates are going to be |
|
| 64:10 | by voltage. And the way it's to happen is as there is this |
|
| 64:18 | charged amino acid residues they're actually attracted the negatively charged internal environment of the |
|
| 64:31 | memory. So you have this channel the gates closed. Has to gays |
|
| 64:43 | it has a voltage sensor is positively and you know that the inside of |
|
| 64:50 | num brain is negatively charged. So positively charged voltage sensor is attracted to |
|
| 64:57 | negative build up and is repelled by positive charge on the outside. But |
|
| 65:06 | the membrane starts d polarizing, if is a little bit of deep |
|
| 65:12 | this sensor we'll start getting repelled by charge that there's a positive charge. |
|
| 65:20 | up. This positive charge in the sensor is going to be repelled physically |
|
| 65:27 | it's going to move inside the channel the movement of this channel. So |
|
| 65:34 | by the charge voltage moving. This sensor will cause a confirmation a little |
|
| 65:41 | in the protein channel that caused the of the channel. Mhm. So |
|
| 65:50 | is voltage sensor. These are voltage channels. Yeah, there has to |
|
| 66:02 | deep polarization that's taking place optically in for that cell to generate an action |
|
| 66:10 | . And these deep polarization would come the synaptic inputs. And if there |
|
| 66:15 | enough of this deep polarization through synaptic inputs, then the enough positive charge |
|
| 66:22 | build up will start pushing the sensor . And as the sensor literally moves |
|
| 66:29 | being repelled through the state dimensional The two gates, the two sodium |
|
| 66:35 | open owned so the input, the positive charge build up would come from |
|
| 66:43 | positive synaptic inputs. And only if reaches that value of the threshold is |
|
| 66:51 | you will see enough positive charge and it's going to be very good enough |
|
| 66:55 | for it to to cause a conformational . So this is this is well |
|
| 67:08 | to this value right here. And remember that this you can have |
|
| 67:14 | polarization and you can have hyper These are synaptic inputs. And then |
|
| 67:21 | happens is they activate at this level synaptic inputs. If they're strong enough |
|
| 67:29 | they activate the voltage sensor and voltage sodium channels. Yeah. And that's |
|
| 67:34 | you have all our non response of action potential. What happens is that |
|
| 67:48 | a special kinetics of this channel and is fast activating and it opens |
|
| 67:58 | So it's fast opening. You can it actually has two gates, its |
|
| 68:05 | gate and this adam what we call activation gate, that's one of the |
|
| 68:10 | in which we think these two gates . If the there is enough deep |
|
| 68:16 | , the voltage sensor will slide upwards will open both of the gates. |
|
| 68:21 | as it opens, voted against this and chain swings outwards. And as |
|
| 68:28 | molten ascension keeps moving up through the , this causes another confirmation will change |
|
| 68:35 | this again, ball to come and the channel and now it's going to |
|
| 68:42 | inactivated. And in order for you go and to close the open |
|
| 68:47 | you actually have to hyper polarize the of potential. And with hyper |
|
| 68:54 | what's going to happen is the voltage is going to slide back down and |
|
| 69:04 | inactivation gate is going to move out the activation gates are going to |
|
| 69:11 | So basically there's enough voltage. This sensor is gonna pick it up cause |
|
| 69:17 | change opened both gates, but as as it opens both gates, one |
|
| 69:21 | them is opportunistic. And with this change of case says, I'm going |
|
| 69:25 | close it up right away and that's the sodium channels are open for a |
|
| 69:32 | short period of time transient because they inactivated. And that's another reason why |
|
| 69:40 | membrane potential doesn't reach equilibrium potential for is because of the inactivation not only |
|
| 69:46 | driving force decreasing, but the inactivation closed. So when it's hyper |
|
| 70:06 | that's that's what accounts for that blip we see with the potassium out flux |
|
| 70:12 | like one of the graphs that showed the N. A. Like this |
|
| 70:21 | . Yeah, this one here. a little different. It's only if |
|
| 70:28 | want to really positive potentials of Yeah. Yeah, but your I |
|
| 70:34 | it's really good good questions and good of thinking. So uh So I |
|
| 70:42 | that there's more information, there's more plots action potentials and back propagating |
|
| 70:50 | So we're doing pretty well actually on that we're going to cover for this |
|
| 70:55 | . But because we're out of time when I come back on first |
|
| 71:00 | I will go over one more The action potential dynamics, the kinetics |
|
| 71:07 | incapacity channel. If you're a little confused about anything, don't hesitate. |
|
| 71:12 | welcome questions. I may have missed question in the back. I apologize |
|
| 71:17 | you don't I'm keeping its next session the great um Yeah, and then |
|
| 71:24 | have our review in a week from . So we'll be able to cover |
|
| 71:28 | of the material we're supposed to cover this exam. Have a good |
|
| 71:36 | |
|
