| 00:00 | Mm. This is lecture seven of and we're discussing the action potential. |
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| 00:06 | first of all, we learned several things in the last couple of lectures |
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| 00:12 | we discussed. First of all, we spoke about the resting member and |
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| 00:17 | , we spoke about thing, a that is called Eion or equilibrium potentials |
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| 00:25 | Eion where we realize that each the calcium chloride, sodium and |
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| 00:32 | the four species that we're studying here their own equilibrium potential. And that |
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| 00:37 | calculate that equilibrium potential you used Nernst . And so you should be familiar |
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| 00:46 | what ERNST equation is and what are terms of ERNST equation as we discussed |
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| 00:55 | class? OK. OK. So is N equation that will allow you |
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| 01:09 | calculate equilibrium potential for each ion. membrane potential will calculate membrane potential. |
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| 01:18 | Goldman equation. The Goldman equation will take the same components. But instead |
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| 01:28 | e ionic, it's measurement of DM is number and potential. And it |
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| 01:36 | into this nernst equation. More than ionic species such as potassium and potassium |
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| 01:43 | such as sodium and sodium permeability and concentration on the outside versus the |
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| 01:50 | So I'm not gonna rewrite the whole , but you can look it |
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| 01:53 | So each ion has its own equilibrium with its own equilibrium potentials are listed |
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| 01:59 | with these black dashes right here. . These dash lines here are indicate |
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| 02:07 | important potentials that you should know for exam. So resting number and potential |
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| 02:12 | behind the 65 threshold for action potential 45 millivolts zero millivolts is when there |
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| 02:20 | an overshoot of the actual potential. now, the rising phase of action |
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| 02:26 | is driven by sodium influence. And talked about the fact that cell numbers |
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| 02:32 | depolarize and hyperpolarize so they can constantly their membrane potential. So, membrane |
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| 02:40 | beyond is at any given moment. we talked about if this is resting |
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| 02:45 | and potential R and P of minus it can fluctuate, it can |
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| 02:51 | it can depolarize, it can depolarize, depolarize more. But if |
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| 02:56 | reaches this threshold for action potential will generate an all or none. Even |
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| 03:01 | this point, this is the point the sodium channels, voltage gated sodium |
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| 03:06 | open. So in order for this to reach a depolarization, most of |
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| 03:12 | inputs are gonna be synaptic inputs that learn through receptor channels can depolarize the |
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| 03:18 | and plasma membranes. So when we about the membrane potential or and and |
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| 03:26 | membrane potential VM or voltage of the membrane potential measured in mets when we |
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| 03:34 | about the membrane potential, we're talking this white trace here, this is |
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| 03:39 | action potential trace, this is the potential. And we're looking at how |
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| 03:44 | number in potential is relating to the potentials and to the driving force for |
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| 03:52 | . We discussed, started discussing this of the driving force called the driving |
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| 04:00 | is the difference between VM and E . So each ion will have their |
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| 04:09 | driving force because each ion has its equilibrium potential and it's going to fluctuate |
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| 04:18 | the number and potential fluctuates. So we already talked about is that when |
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| 04:23 | reach this threshold, that's when you all of the voltage you get at |
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| 04:27 | channels. When they open, the starts depolarizing very quick, more sodium |
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| 04:34 | in more depolarization, more voltage gated channels open. So this is gated |
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| 04:40 | voltage, more voltage gated sodium channels . And the whole membrane potential is |
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| 04:48 | to equilibrium potential for sodium because this the dominant ion that's influx during the |
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| 04:54 | phase of the action potential. And lines, if you recall, I |
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| 04:58 | that if you are at the just the activation of voltage gated sodium channels |
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| 05:06 | this membrane potential level, here, a huge driving force for sodium because |
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| 05:12 | a big difference between where the white is the membrane potential versus where the |
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| 05:19 | potential for sodium is as sodium It is trying to reach the equilibrium |
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| 05:28 | for sodium. But it cannot, does not. There are two reasons |
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| 05:33 | why it does not reach the equilibrium for sodium. The first reason is |
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| 05:39 | once the membrane potential is here at peak of the action potential, the |
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| 05:45 | force for sodium now reduces to very driving force. At the same |
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| 05:52 | there's something about sodium channel kinetics that learn today is that these channels as |
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| 05:59 | as they open within millisecond or so all close, it's called inactivation. |
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| 06:05 | that's just the nature of the way build these voltage gated sodium channels is |
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| 06:09 | soon as they open, they're very at opening, they're also very fast |
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| 06:13 | closing. While the membrane potential is the peak here of the action potential |
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| 06:20 | this stage, there's a huge driving . There's difference here between membrane potential |
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| 06:26 | potassium equilibrium potential. And therefore potassium the dominant ion during the falling phase |
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| 06:33 | the action potential. Potassium is e and it's now driving the membrane potential |
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| 06:38 | its own equilibrium potential value. And almost succeeds to do that because it |
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| 06:44 | a very strong drive and it also the leak channels that are constantly open |
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| 06:48 | leaking. So it almost goes to potential for potassium but then gets restored |
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| 06:54 | its pre uh action potential membrane potential resting membrane potential with the help of |
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| 07:02 | TPNAK pumps. Ok. So there many different ways in which we can |
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| 07:10 | the action potentials in neurons. And talking about when we look at these |
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| 07:15 | , we're talking about intracellular recordings of action potentials and what you're seeing here |
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| 07:20 | if you block an electrode into the , it will immediately show minus 65 |
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| 07:27 | . But when you record an action , there is this change of about |
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| 07:31 | millivolts over about a couple of So the amplitude and the response that |
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| 07:37 | get from intracellular recordings, it's it's on the water of 100 |
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| 07:42 | However, there are other ways in we can pick up neuronal activity. |
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| 07:47 | this electrode here is actually picking up on the outside of the neurons. |
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| 07:52 | as the charge is changing on the of neurons here, this electrode right |
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| 07:59 | , extracellular electrode is going to record very small and inverted looking action |
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| 08:06 | but it's going to be only on order of about 100 micro volts milly |
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| 08:12 | to the minus three versus micro 10 the minus six really, really small |
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| 08:19 | . So if you're recording from outside the cells, you're getting really small |
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| 08:24 | . And that's what neuralink is And that's what a lot of uh |
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| 08:27 | electrode implants in the brain are They're recording electrical activity from outside of |
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| 08:33 | and not just one neuron typically synchronized . That means that activity that is |
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| 08:39 | at the same time across a collection neurons or synchronized neural network. And |
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| 08:45 | the type of the activity that these recordings are really good at picking out |
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| 08:50 | especially in vivo in human implants. experimentally, we can pick up single |
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| 08:56 | if we have these electrodes sitting right that Axion initial segment. Because this |
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| 09:03 | where the action potential gets generated, can pick up a small extracellular |
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| 09:10 | So from the very beginning, I about the fact that different cells have |
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| 09:14 | own dialects. And these dialects really representations of the patterns and the frequencies |
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| 09:21 | the action potentials. The different cells can produce. And this is just |
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| 09:26 | illustration of if you, for in the older days wanted to do |
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| 09:30 | types of recordings, you have to two electrodes. One would be a |
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| 09:34 | electrode, putting a positive current inside cell. The second one would be |
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| 09:38 | recording electrode in modern electric physiology. circuits that sample uh electrical activity extremely |
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| 09:45 | . And so we can do both and recording with just a single |
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| 09:50 | If you look at this top trace , it shows that injected current and |
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| 09:55 | looks very on like like a switch on immediate, we call this square |
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| 10:01 | pulse. And typically I told you you see a flat line in |
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| 10:05 | that's not good, but you can a lot of flat and square lines |
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| 10:08 | sinusoids in instrumentation. So when we're electrophysiological recordings and we're stimulating these |
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| 10:16 | We're using instrumentation, we're flipping a and as we flip the switch |
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| 10:23 | there's a current being passed inside the . If you look at the cellular |
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| 10:28 | , though cellular response does not necessarily like a square, it has a |
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| 10:32 | bit of the delay because you have charge up the number and it has |
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| 10:37 | incapacitated of properties. And what it you that if the stimulus is strong |
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| 10:43 | that you can inject this current into neuron, and this injection of the |
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| 10:47 | will essentially mimic a stimulus onto that and then subsequently record a response of |
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| 10:55 | pattern of electrical activity from those neurons a pattern of the action potentials. |
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| 11:01 | really small stimulations. Again, this instrumentation on top produces these square wave |
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| 11:07 | pulses. The response of the cell not look square, it takes a |
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| 11:13 | milliseconds. So it's a really good , neuronal membrane capacitors are holding a |
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| 11:19 | of charge and neuronal membrane is holding lot of charge on both sides of |
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| 11:22 | membrane, but it takes a few to charge that capacitor. So it's |
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| 11:27 | good, it's really fast, but not immediate like you would see in |
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| 11:31 | instrumentation and when you stop the it takes a few milliseconds for the |
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| 11:37 | to reshuffle itself across plasma membrane into membrane. If the stimulus here from |
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| 11:45 | from instrumentation is stronger, you will get a response where a cell can |
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| 11:50 | , let's say five action potentials. if that same cell receives a even |
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| 11:55 | stronger input or a stronger stimulus, may be a response and instead of |
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| 12:03 | , 1011, 12 action potentials. so this is something that we know |
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| 12:08 | the code in neurons. The code such that frequency of action potentials reflects |
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| 12:15 | magnitude of depolarizing current, which essentially that it reflects the magnitude of the |
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| 12:24 | or the magnitude of the input small , no response, larger stimulus, |
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| 12:30 | action potentials, large large stimulus, lot of action potentials. So that's |
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| 12:35 | of a like a code, it's a digital code uh that neurons produce |
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| 12:40 | producing these action potentials. And we that there are these dialects, I |
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| 12:47 | to them as dialects of these action . And we have a variety of |
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| 12:53 | types of cells, but in particular interneurons as we discussed them very early |
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| 12:59 | are the ones that exhibit diversity, functional diversity and morphological diversity that's much |
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| 13:07 | than the exciter current projection cells. if these neurons that can be located |
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| 13:13 | a small patch of the cortex, you'll have a variety of neurons that |
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| 13:17 | produce their own frequencies and patterns of potentials that I refer to as |
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| 13:26 | A very interesting thing that has been in the science is control of depolarization |
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| 13:33 | hyper polarization and control of channels. we were talking about channels as the |
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| 13:39 | blocks. So the channel channels will conducting the ions that are made out |
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| 13:43 | the amino acids. And we're interested studying these channels and we can depolarize |
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| 13:49 | hyperpolarize the cells with these electrodes that just saw in the previous slide. |
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| 13:54 | you can inject positive current or you inject negative current through this electrode and |
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| 14:00 | will get either depolarization with positive current hyper polarization with a negative current. |
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| 14:05 | it's strong enough, you will get action potential. So this is typically |
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| 14:09 | we're used to man stipulating the cell or changing its memory potentials and action |
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| 14:17 | is through electrophysiology. And in the 15 years, there has been a |
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| 14:23 | interesting technique that has been developed. called optogenetics and optogenetics is remember, |
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| 14:30 | talked about genetic manipulations of rodents and talked about knock in knockout uh trans |
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| 14:40 | trans genes. So here you are a uh uh introducing a foreign gene |
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| 14:47 | you're expressing these ion channels and the of your choice. What does that |
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| 14:54 | ? A system of your choice? these channels are very special, these |
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| 15:01 | , a lot of uh single cell uh they are reacting to light and |
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| 15:11 | actually have these light sensitive channels. there is a channel of adoption |
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| 15:16 | And it's a channel and single cell that in the presence of blue |
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| 15:23 | it will allow for conductance of sodium the cells. So when the blue |
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| 15:27 | is on the number and potential of cell will be polarizing. You can |
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| 15:31 | depolarize it uh all the way to threshold so that the cells will be |
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| 15:37 | action protections. And there's another channel adoption. And if you express hallow |
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| 15:46 | , hallow adoption is sensitive to yellow . So when it's exposed to yellow |
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| 15:51 | , now it's gonna let inside flora going inside the cell will cause these |
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| 15:58 | , chloride going inside the cell will these hyper polarization. We can do |
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| 16:04 | with electrodes or we can do it this really interesting and unique technique. |
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| 16:09 | yellow light on and now there's hyper and you can actually co express these |
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| 16:14 | channels in different systems, as I of your choice. So what is |
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| 16:18 | system of choice when you find some organism someplace in the salt plains of |
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| 16:26 | ? And you bring it back to lab and you realize that this organism |
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| 16:31 | light sensitive channels. You now want isolate that channel and you want to |
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| 16:38 | that channel and you want to see you can express that channel. And |
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| 16:43 | you go to the simpler systems, of the simple system is systems is |
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| 16:50 | sides. So the large frog it's really easy to actually introduce a |
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| 16:55 | gene and over express a gene of over express a channel of interest in |
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| 17:02 | . It's been done for decades because we discover a certain channel, a |
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| 17:08 | of times we try to look for channel and brain slices and the |
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| 17:13 | And so you get to the, the electrode to one of these cells |
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| 17:18 | it looks like it may have that , but it's somewhat buried in noise |
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| 17:22 | you're not sure. So you hunt the next cell and you kind of |
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| 17:26 | maybe current from that channel again, you're not certain. So what you |
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| 17:31 | do is you can take an over that channel on the simple system like |
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| 17:35 | side. So you know, you a lot of that channel electrophysiology, |
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| 17:39 | would over express a certain channel like potassium channel, for example, and |
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| 17:44 | you would produce stimulations and you would the responses of this potassium current. |
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| 17:50 | now you understand how this channel, currents that channel produces. Now you |
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| 17:57 | go into more complicated systems like let's in brain slice or in a whole |
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| 18:02 | in vivo and try to record. now you have a comparison. Now |
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| 18:07 | have really revealed a lot of the of this channel and therefore the kinetics |
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| 18:14 | this car, it's really cool because can use these primitive systems also to |
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| 18:20 | and over express channels that are light . And so the first step would |
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| 18:25 | to to test it in a simple like frog ci to understand how you |
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| 18:31 | by light, how you depolarize and and then move it into a more |
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| 18:36 | system. So the next thing is may want to do it in |
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| 18:40 | And the, the, the thing you want to do is you wanna |
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| 18:43 | one channel in rodents and you put in the slice and you do |
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| 18:48 | So you put an electrode and you the blue light and the electrode records |
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| 18:55 | and uh awesome, it's working. you have one of these express. |
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| 19:01 | , you would want to express both want to depolarize neurons and hyperpolarize |
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| 19:06 | So you can co express them, can have drivers expressing them in specific |
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| 19:12 | . So now you have the ability make the cell more excitable, depolarize |
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| 19:16 | with blue light or make the cell excitable or inhibit its activity with a |
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| 19:22 | light. So if you can show functionality in vitro with electrophysiology together, |
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| 19:29 | you can move in and start doing vivo experiments and whole animal. And |
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| 19:34 | is an illustration where there's going to an implant or there's a off the |
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| 19:40 | off the cable here that's attached to animal's head. And this animal has |
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| 19:47 | sensitive channels that it expresses. And example, you can now not only |
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| 19:54 | the excitability of depolarizing the membranes versus the membranes, you actually can control |
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| 20:01 | behavior. So if you shine the light on a certain region, let's |
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| 20:06 | a motor region that animal is gonna active. Um And if you shine |
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| 20:11 | yellow light on that same region, animal is going to slow down |
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| 20:16 | and it's, and it's not as simple as that, of |
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| 20:19 | and there as we know his brain really complex, but you can now |
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| 20:24 | expressing these light sensitive channels and specific of cells and manipulating specific subtypes of |
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| 20:31 | . And that's that's really exciting. this is another way instead of electrophysiology |
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| 20:38 | to essentially depolarize and hyperpolarize the cells light. And you know, you |
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| 20:44 | think of like, well, what the application for humans here? |
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| 20:49 | the application for humans there is that course, you cannot over express or |
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| 20:53 | a new uh gene and channel into brains. Uh But you know, |
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| 21:00 | are things like gene therapies that are , there are things that maybe there |
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| 21:06 | something that we can tag temporarily somehow those channels that, that, that |
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| 21:13 | is not genetically expressed. And the would be to control brain activity with |
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| 21:22 | . And if you have something that responsive in the brain to light. |
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| 21:27 | , of course, when you're looking humans and some sort of a therapy |
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| 21:31 | based on light, you know, have to overcome a lot of |
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| 21:34 | a lot of ethical hurdles. Uh again, a mechanism, some sort |
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| 21:42 | a cellular mechanism by which uh you manipulate neurons with light uh potentially through |
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| 21:52 | channels. Maybe there's something else that be discovered in the next few |
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| 21:59 | So when we are recording action there's two several important things uh that |
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| 22:06 | talk about. Here. Again, a review of the equilibrium potential for |
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| 22:12 | ion. Here, it's a review Goldman Equation. How you calculate the |
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| 22:17 | and potential for each ion here is law rewritten for car for potassium. |
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| 22:25 | car for potassium is equal conductance of times of driving force. Remember the |
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| 22:31 | force is VM minus an E ion this case, E for potassium. |
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| 22:36 | this current as we measure or each conducted for each ion is it depends |
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| 22:43 | the equilibrium potential for that particular Let's look at this scenario here, |
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| 22:52 | ? You have no channels that are , you have both sodium and potassium |
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| 22:59 | inserted in the membrane. And if put an electrode inside, there's no |
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| 23:04 | fluxing. So your electrode will show millivolts. Yeah. Equilibrium potential for |
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| 23:13 | is minus 80. Equilibrium potential for is 62. Let's look at the |
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| 23:19 | for potassium at zero. There's no because channels are closed. Is there |
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| 23:28 | driving force or potassium? There's a driving force for potassium because the VM |
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| 23:38 | at zero and the K is minus . So this is actually 80 |
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| 23:45 | However, the conductance is zero. , the current is zero So you |
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| 23:50 | have a huge driving force. But the channels are not open, nothing |
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| 23:54 | fluxing, the current is zero. this situation here, we open up |
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| 24:00 | channels and potassium starts e fluxing from to outside. Now, we can |
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| 24:06 | that this number and potential is now . There is conductance for potassium. |
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| 24:12 | greater than zero because potassium channels are and conducting. There is also the |
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| 24:19 | force because uh equilibrium potential for potassium minus 80. So there's still a |
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| 24:26 | difference between where this arrow is and minus 80 is going to be on |
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| 24:30 | volt meter. Therefore, there is significant current that now can be recorded |
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| 24:37 | potassium. In this situation, we're at minus 80 millivolts in the volter |
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| 24:47 | minus 80 millivolts is also equilibrium potential potassium that tells us that the driving |
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| 24:55 | is zero. And this is a where although the channels are open and |
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| 25:04 | plenty of conductance, there's no net of potassium on the outside versus |
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| 25:11 | it's equal amount of potassium crossing. the driving force of mine is 80 |
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| 25:18 | is zero. Therefore, the current zero. It's the same amount going |
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| 25:24 | both directions. Therefore, the it's zero because both directions is opposite |
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| 25:29 | , inward and outward, canceling each out. So once again, keep |
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| 25:36 | mind these concepts of equilibrium potential, equilibrium potential relates to the driving |
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| 25:44 | right, conductance and how you calculate uh the currents in the presence of |
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| 25:51 | and uh driving force where you have current or in the absence of either |
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| 25:58 | , you don't have any problems during resting membrane potential. And here it's |
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| 26:07 | minus 80 millivolts which is really close the potassium uh equilibrium potential. And |
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| 26:15 | because the membrane is leaking. And I said, this value is going |
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| 26:18 | be fluctuating minus 65 minus 70 so . But during the resting membrane |
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| 26:24 | we already know that potassium has way conductance than sodium. OK? Because |
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| 26:32 | the leak channels for potassium leak For potassium, we know that once |
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| 26:37 | number and potential crosses the threshold for potential generation, it's dominated by sodium |
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| 26:47 | . We also know that sodium is to reach the equilibrium potential. The |
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| 26:54 | and potential sodium is driving the number potential to reach the equilibrium potential for |
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| 26:59 | . But it fails because of the driving force. And because of the |
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| 27:03 | kinetics that we'll discuss in a few . At the same time, sodium |
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| 27:09 | are closing here, they're all At the same time, potassium channels |
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| 27:13 | wide open and potassium conductance is now the falling phase of the action |
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| 27:21 | And again, at resting membrane potential close to more negative values of minus |
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| 27:28 | . It's dominated by potassium again by potassium leak channels. There's a rising |
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| 27:33 | is so you're moving inside inward it's positive chart moving inside it's inward |
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| 27:40 | , positive charge, moving outside potassium outward current. OK. The movement |
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| 27:47 | the current is in the direction of positive charge movement to record these kind |
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| 27:55 | uh activities and and neurons action potentials understand why. How do we know |
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| 28:03 | that it's sodium versus potassium. We to start isolating individual channels and studying |
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| 28:09 | channels. And in this case, has shown is that you have a |
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| 28:14 | and in some instances instead of inserting pet inside of the cells, you |
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| 28:19 | actually pluck a little piece of the membrane out. And that piece will |
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| 28:24 | sodium channels for example of interest. now you can stimulate electric through the |
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| 28:32 | or you can introduce some chemicals chemically the solution of the electrode and measure |
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| 28:38 | it affects sodium channels. And so told you that nothing in nature looks |
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| 28:42 | way, but this is the This is the square away from the |
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| 28:47 | and this is a single channel opening closing for sodium. So it looks |
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| 28:52 | little bit square but it's not it's just really fast in this |
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| 28:56 | OK. But this is a single opening. So this is really neat |
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| 29:00 | you can isolate currents through a single or currents through many channels that belong |
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| 29:09 | one species like sodium or potassium. I'll tell you how it's done. |
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| 29:13 | done with the help of voltage clown this is a diagram that explains the |
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| 29:19 | clamp. What is voltage clamp? what it is. It's voltage |
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| 29:25 | voltage is voltage. It's a number voltage clamp is a clamp, it's |
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| 29:30 | something. So in this case, holding the voltage or clamping the voltage |
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| 29:35 | a desired value for me, the said it wants to be at minus |
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| 29:40 | and minus 55 and minus 70. want to be someplace else where I |
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| 29:47 | it to be. It's like why I want it to be anywhere |
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| 29:53 | Because you know, when people calculate like Nernst equation using equilibrium potential for |
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| 30:02 | , this is the calculation if you to demonstrate it. So how do |
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| 30:07 | demonstrate it? How do you demonstrate the reversal potential or equilibrium potential for |
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| 30:14 | is positive 62 if the cell lives minus 65 and then transiently goes to |
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| 30:20 | during action potential. How do how do you do that? The |
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| 30:23 | way to do that is to use voltage clamp to isolate these currents? |
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| 30:28 | the way that this is done is this is our giant axon from the |
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| 30:33 | . You have a reference electrode on ground, these green electrodes one |
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| 30:38 | And this is measuring internal electrode measuring potential and it's connected to the voltage |
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| 30:44 | amplifier. So it's measuring here, a minus 60. I wanted to |
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| 30:50 | at minus 80 all the time. I am here. I am commanding |
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| 30:57 | voltage. It's in, I'm in of that. The experimenter and I've |
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| 31:01 | this voltage clap apple part number and to the desired command potential. So |
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| 31:06 | command potential is minus 80 the solids minus 60. And because the cell |
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| 31:11 | at minus 60 my voltage clamp amplifier will notice that I set my |
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| 31:17 | The cell number is at minus When there's a difference in VM from |
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| 31:22 | command potential, the clamp will now the current into the axon through a |
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| 31:28 | electrode. We'll make sure it's not . So if it's a minus 60 |
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| 31:34 | gonna inject negative 20 millivolts of current drive it down to M and keep |
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| 31:39 | clamped with moo. So this is same as command potential. The current |
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| 31:45 | flows back into the axon and across membrane can be measured here. So |
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| 31:50 | we're, when we're measuring the these are all of the deflections that |
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| 31:54 | happening from what we have clamped it . OK. And this is what |
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| 31:58 | clamp is. Don't worry, it be more clear. So Hoskin and |
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| 32:11 | used the voltage clamp, were you membrane potential, any chosen value? |
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| 32:18 | they said, you know what, see what happens if we depolarize the |
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| 32:23 | into minus 26 to 0, positive positive 52. Let's assume actually that |
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| 32:32 | is positive 62 this particular thing. Well, yeah, this is positive |
|
| 32:45 | . I just made it. It's positive 62. There you go. |
|
| 32:51 | . So they're depolarizing the membrane. are they doing it? They're doing |
|
| 32:55 | with a voltage clamp that we just . But what they see is when |
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| 33:00 | depolarize and plant the number in a 20 at negative 26. This little |
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| 33:06 | here, it's an inward current by . The inward currents have negative uh |
|
| 33:12 | or peak value. And what's noted that there's a small inward current and |
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| 33:20 | inward current increases when you depolarize the to zero. And that inward current |
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| 33:25 | followed by a much slower outward current the other direction. And when it |
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| 33:31 | to positive 26 that inward current actually decreasing. That sodium carb starts decreasing |
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| 33:40 | the outward current is sustained and it's . And when you put it a |
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| 33:45 | 62 which is the equilibrium potential for , there's no inward cars. And |
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| 33:54 | you clamp the potential of positive 65 the other side of the equilibrium |
|
| 34:00 | you see this little blip here, now the sodium current from inward becoming |
|
| 34:07 | outward current. That's why we call reversal potential. Because if that equilibrium |
|
| 34:12 | value, if you cross it, currents start fluxing in the opposite |
|
| 34:16 | If they were going in, they're be start coming out. If they're |
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| 34:19 | out, they're gonna start coming in the positive flux of charge. So |
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| 34:26 | was noted is that this early inward closes? So Hodgkin and Huxley did |
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| 34:35 | experimentally. They created a Hutin and model of the action potentials. And |
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| 34:41 | also postulated that there are gates that regulating these voltage gated sodium channels that |
|
| 34:48 | talk about in just a couple of . And that this early component is |
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| 34:53 | component, that the late component, component is a potassium component. But |
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| 34:59 | cannot, you cannot see this without voltage plan, you cannot see the |
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| 35:05 | in inward currents decrease as the membrane is reaching value closer to equilibrium potential |
|
| 35:13 | sodium. That there is no sodium , equilibrium potential for sodium. That |
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| 35:19 | sodium current is in the opposite reverse . Once you pass the equilibrium |
|
| 35:25 | this is all experiments that could not done without voltage plant. So it's |
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| 35:31 | really important that you know theo theory and and and formulas essentially that do |
|
| 35:39 | of these measurements uh get reconfirmed with recordings in this case and using voltage |
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| 35:47 | and 1963 Hodgkin and Huxley uh collected Prize in physiology in medicine for their |
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| 35:56 | work on the action potentials and the of action potentials and the gating properties |
|
| 36:01 | the sodium channels that they described. if we look at this kind of |
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| 36:07 | composite activity across individual channels during the phase, and this is sodium |
|
| 36:14 | So as soon as the threshold is with this dashed line. These are |
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| 36:19 | sodium channels, they will open but notice they do not open all |
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| 36:23 | the same time. So there is slight delay, there's a sub millisecond |
|
| 36:28 | between opening of all of these But also as soon as they |
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| 36:32 | they also close. So independent of fact that there is significant depolarization still |
|
| 36:39 | on here, this shadow opened and and it's not going to reopen |
|
| 36:43 | And that's the second reason it's called . That's the second reason why numbering |
|
| 36:50 | doesn't reach the equilibrium potential for sodium the peak. It's because the sodium |
|
| 36:55 | they inactivate at the same time. we look what happens if you. |
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| 37:00 | this is the rising phase of the potential already toward the very end of |
|
| 37:04 | rising phase of the action potentials, channels are opening up. This is |
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| 37:11 | potassium channels. And you can see the difference that sodium channels are very |
|
| 37:18 | opening, but also closing very And potassium channels are delayed in |
|
| 37:26 | But once they open, they have sustained or prolonged activation. So if |
|
| 37:33 | were to take an average of all these sodium channels in Oakland, this |
|
| 37:38 | the average inward current during the rising of the action potential. And this |
|
| 37:44 | the rising phase that ends here, ending here. They already have activation |
|
| 37:50 | potassium currents. And these are the potassium currents calculated from the sum of |
|
| 37:57 | of the individual potassium channels that open . And if you overlap and subtract |
|
| 38:04 | versus outward, and you see where inward is dominating sodium during the early |
|
| 38:09 | and the potassium during the late phase the falling phase of the action |
|
| 38:16 | So both potassium and sodium open uh response to depolarization, that's why these |
|
| 38:26 | are called voltage gated channels. They're by voltage. The voltage is the |
|
| 38:30 | that is going to open their gates also going to close their gates, |
|
| 38:37 | gates open later than sodium gates. potassium is referred to as delayed |
|
| 38:43 | what it is trying to do it is trying to drive the number |
|
| 38:47 | potential back to its resting number and or to rectify or reset the number |
|
| 38:53 | potential to its preaction potential level to resting number and potential. So they're |
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| 39:00 | to as delayed rectify because they're delayed activation, but they're sustained or open |
|
| 39:07 | than the sodium challenge. And so are also good uh good uh points |
|
| 39:12 | know for the exam. The difference in the and not only influx of |
|
| 39:18 | during rising phase and elu during falling of potassium, but the kinetics of |
|
| 39:25 | channels and in particular the sodium because in the next couple of |
|
| 39:31 | we're going to discuss exactly how voltage these channels, how can it gate |
|
| 39:38 | channels? What does it do to the voltage of the channels open. |
|
| 39:45 | this is a structure of voltage gated channels and voltage gated sodium channels will |
|
| 39:54 | designated as N A DNA for sodium . For volt educated, each one |
|
| 40:02 | them is comprised of four subunits. four subunits come together and form a |
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| 40:13 | in between each one of the sub has six trans numbering segments in between |
|
| 40:22 | S five segment and the six S six of the sub unit, |
|
| 40:28 | is four loops also that was described Roderick mckinnon as hairpin loop. And |
|
| 40:34 | poor loop exists in all of the units coming together close into the inner |
|
| 40:40 | of the channel to act as a filter or ionic seating mechanism. If |
|
| 40:47 | will to selectively in this case, select or just sodium to only allow |
|
| 40:54 | sodium to pass through sodium voltage gated channels, sodium voltage gated potassium channels |
|
| 41:00 | potassium doesn't mean that they're gonna flux each other's channels. Although sometimes that |
|
| 41:06 | happen too. Another important feature of sub unit is the trans number in |
|
| 41:13 | four S four contains a lot of charged amino acid residues. Remember that |
|
| 41:22 | are the channels that are built from acids, they're strung together and that |
|
| 41:27 | of them will contain positive and some them will contain negative amino acid residues |
|
| 41:36 | so it happens that S four, s four region has a lot of |
|
| 41:43 | acids with a positive charge in And as the membrane is sitting at |
|
| 41:50 | minus 65 N volts, the inside the membrane is negatively charged and hyper |
|
| 42:00 | . And this positive charge with the four sub unit is very much attracted |
|
| 42:06 | the negative charge of the membrane. it's keeping the S four subunit in |
|
| 42:12 | position. And it's keeping the activation , these two arms activation gates closed |
|
| 42:21 | there is depolarization from minus 65 to 45 from minus 40. The threshold |
|
| 42:28 | action potential generation those channels will open a change in the numbering potential. |
|
| 42:36 | when there is a change in the potential, in this case, |
|
| 42:41 | these channels will open again, that depolarization will come from a stimulus from |
|
| 42:48 | input from receptor channels that we'll talk when we talk about synaptic transmission. |
|
| 42:54 | it needs to happen in order for gates to open. And the way |
|
| 42:58 | happens is that if you depolarize this , now the inside of the membrane |
|
| 43:06 | not as negatively charged. In it can turn positively more positively charged |
|
| 43:12 | it was in this resting membrane And now these positively charged voltage sensors |
|
| 43:20 | the amino acid residues in the voltage cause the physical movement and the displacement |
|
| 43:27 | the voltage sensor inside the channel, the confirmation of the channel and causing |
|
| 43:34 | opening of the activation gates. Uh address the negative charges keeping it attracting |
|
| 43:43 | positive segment here to stay in place keep the gates closed, the activation |
|
| 43:49 | closed and with accumulation of positive charge the number and with depolarization, it |
|
| 43:55 | repelling this voltage sensor. And as repels the voltage sensor opens up, |
|
| 44:03 | opens up the gates. So gating voltage poor selectivity, it's selected for |
|
| 44:11 | . So these are voltage gated sodium was called voltage gated potassium channels. |
|
| 44:16 | selected to a single. OK. now we understand how these channels |
|
| 44:22 | But I also told you that as as these channels open, they also |
|
| 44:28 | . Why? And that's because these have a second gauge. And the |
|
| 44:34 | gate here is depicted by this ball chain. And so as this trans |
|
| 44:40 | segment moves up, s four moves and opens the activation gate. That |
|
| 44:47 | confirmational change that causes the opening of gate also swings this ball into the |
|
| 44:56 | of the channel and plugs it So this is called inactivation. So |
|
| 45:01 | has two gates. It's just the of this channel is that the sensor |
|
| 45:07 | up, the gates open for a until this one comes and just plugs |
|
| 45:13 | back up and inactivates it. Now inactivated. So notice this diagram here |
|
| 45:20 | this shows a depolarization from minus 65 minus 40 mills to threshold. So |
|
| 45:28 | depolarize the number in here and immediately are voltage gated sodium channels that |
|
| 45:34 | open, open. So in number , they're open and conducting. This |
|
| 45:40 | number two, they're open and conducting number two, but immediately they close |
|
| 45:46 | they enter in this state. Number , which is inactivation state. When |
|
| 45:53 | you have this other gate blocking it notice that this is still sustained |
|
| 46:01 | You're still sitting at minus 40 but those channels are closed. So |
|
| 46:09 | needs to happen is this inactivation gate to be removed. And the only |
|
| 46:17 | to remove it is to release the potential back to minus 65 millivolts |
|
| 46:25 | Number four. In which case, inactivation gau leaves, the inactivation gate |
|
| 46:36 | . OK. And the activation gate and it, it cannot be |
|
| 46:46 | it has to be 123412341. So cycle has to repeat. And that's |
|
| 46:53 | reason why you have the absolute refractory that you cannot produce any action potentials |
|
| 47:00 | all of these channels are closed. you can add more depolarization in this |
|
| 47:05 | here, those channels will not open you regroup them, unless you reorganize |
|
| 47:11 | confirmation again to where they are closed and ready to be open. |
|
| 47:18 | you essentially have to restore the And as you hyperpolarize what's gonna happen |
|
| 47:24 | this voltage sensor, it's going to attracted to the negative charge of the |
|
| 47:29 | again and it's gonna slide down and the closure of this game. So |
|
| 47:36 | how these channels are voltage gated. opens with a little delay but really |
|
| 47:43 | . There's much greater delay in potassium opening as you saw in this diagram |
|
| 47:50 | . This is depolarization. Sodium channels up immediately and potassium channels take some |
|
| 47:56 | to open up sodium channels open up and they immediately close. Also |
|
| 48:06 | Fast activating. Compared to potassium, stay open for about one millisecond because |
|
| 48:15 | inactivate with this mechanism here. And cannot be open again immediately by depolarization |
|
| 48:25 | they get hyper polarized, repositioned. they can become functional. Again. |
|
| 48:32 | the reason why sodium channels close. the reason why the action potential doesn't |
|
| 48:37 | the equilibrium potential for sodium to open close and the driving force reduces at |
|
| 48:42 | same time. So you have closed , open inactivated state. When the |
|
| 48:50 | is removed, it's called de inactivation closure again of the channel as you |
|
| 48:56 | in position one. So let's talk voltage gated sodium channels and some of |
|
| 49:07 | neurological disorders. So it's a pretty uh complex three dimensional structure of this |
|
| 49:22 | . And you have a lot of elements, six transmembrane segments, the |
|
| 49:29 | po the voltage sensor area for sub . And there are a lot of |
|
| 49:40 | neurological genetic diseases in particular epilepsies that linked to mutations in channels. And |
|
| 49:49 | mutations and channels such as in voltage sodium channel are referred to as channelopathy |
|
| 49:56 | the mutation in N AD which stands voltage gated sodium channel. And you'll |
|
| 50:02 | in the next lecture that there are sub types of voltage gated sodium |
|
| 50:07 | And by the way, there are sub types of voltage gated sodium |
|
| 50:10 | Also in the heart, not just the brain, we're talking about the |
|
| 50:15 | , but there are different subtypes of A DS. However, if there |
|
| 50:20 | mutations in NAV S, they can in severe forms of childhood epilepsy. |
|
| 50:27 | we'll talk about a couple of those of childhood epilepsy. The first one |
|
| 50:33 | called generalized epilepsy with febrile seizures, or gaps. Plus and I want |
|
| 50:41 | to know this what is generalized Generalized epilepsy is loss of consciousness is |
|
| 50:50 | you think about somebody having generalized epilepsy generalized seizures, you are thinking similarly |
|
| 50:59 | when we spoke about 10 gauge and epilepticus. You're thinking about a person |
|
| 51:05 | collapsed, having contractions, we call clonic components. There's a generalized |
|
| 51:13 | a person who loses consciousness and it last from seconds to hours and typically |
|
| 51:19 | to be stopped within minutes not to detrimental to the brain activity because it's |
|
| 51:25 | like short circuiting the neurons just like short circuit the electrical circuits and they |
|
| 51:30 | work anymore. So, generalized epilepsy other forms of epilepsy, like uh |
|
| 51:37 | focal epilepsy that confined to one area the brain and may not result in |
|
| 51:42 | loss of consciousness. So a person have a seizure without losing consciousness. |
|
| 51:48 | person may have a twitch or lock in the right arm and it is |
|
| 51:52 | be diagnosed as epilepsy seizure. There so many different types of seizures. |
|
| 52:00 | can be evoked by strobe lights. can be evoked by strong auditor signal |
|
| 52:08 | OD audiogenic seizures. Others have no . We don't know why they come |
|
| 52:14 | . And the epilepsy as such is by neurologist is typically referred as epilepsies |
|
| 52:22 | the symptomology can be so varied from staring space for 10 seconds to uh |
|
| 52:28 | is an apon seizure. It's also seizure with a very brief loss of |
|
| 52:34 | to status epilepticus and generalized tonic clonic that last for a very, very |
|
| 52:39 | time to sometimes seizures, not having motor component at all and having instead |
|
| 52:47 | emotional component bout of aggression and but also not remembering what happened during |
|
| 52:56 | period of time. So, it's a very diverse group of neurological |
|
| 53:01 | epilepsies. And within that, you a variety of different seizures. In |
|
| 53:07 | case, it's generalized seizures with febrile , generalized epilepsy with febrile seizures. |
|
| 53:15 | is a febrile seizure? Febrile seizure hyperthermia induced seizure. It's the most |
|
| 53:22 | type of seizure. It's very common infants when they get an infection and |
|
| 53:29 | body temperature rises. And when you the nurse, if you're a parent |
|
| 53:35 | you're supervising an infant or child, say, uh, you know, |
|
| 53:41 | 100 °F 102 °F. What have done? Well, I tried to |
|
| 53:47 | off the temperature with some medications. , how's it working? It's not |
|
| 53:52 | , it's going to 100 and four . Ok. The suggestion is gonna |
|
| 53:55 | go immediately seek help emergency room, , ambulance, whatever way you can |
|
| 54:02 | the child to under clinical or medical . Uh, parents says I have |
|
| 54:09 | way this is gonna take half an for somebody to get here. Uh |
|
| 54:15 | you will hear instructions from nurses say the child in the eyes bath because |
|
| 54:20 | have to bring down the temperature. the brain cannot stay at 100 4 |
|
| 54:26 | for long periods of time without things really bad. And one of those |
|
| 54:31 | that happen that goes really bad and in infants and young Children is a |
|
| 54:35 | seizure. So they'll go up to 4 °F and they will experience that |
|
| 54:41 | seizure and it will really scare the and there might be AAA follow up |
|
| 54:47 | from emergency room with a neurologist. but having a single seizure, febrile |
|
| 54:56 | does not make you a person with to qualify as a person or patient |
|
| 55:06 | epilepsy. You have to have repeated and a lot of times not only |
|
| 55:11 | seizures but unprovoked. So we don't exactly the cause of these seizures and |
|
| 55:17 | more than one seizure that you have have within a certain period of time |
|
| 55:21 | order to essentially have diagnosis of epilepsy febrile seizures can occur in Children and |
|
| 55:28 | never have another seizure again. Sometimes may have two febrile seizures because they |
|
| 55:33 | two back to back infections at very age and they have some sort of |
|
| 55:37 | sensitivity to temporary changes, hypothermia. still doesn't qualify them as epileptic. |
|
| 55:45 | you have to monitor cases of multiple . But typically what happens then in |
|
| 55:51 | Children is everywhere. You're seeing a dot Here, there's a mutation and |
|
| 55:57 | this voltage gated sodium channel that can up causing generalized epilepsy with febrile |
|
| 56:03 | In that case, this mutation is that an individual's temperature doesn't have to |
|
| 56:09 | up to 100 and four. It fluctuate just by a couple of degrees |
|
| 56:14 | normal physiological and centigrade is 37 to . So 100 and four is about |
|
| 56:21 | °C. So it can go from centigrade 37 to 38.5. And the |
|
| 56:27 | would experience a AAA febrile seizure so their threshold for seizures that are evoked |
|
| 56:36 | this case by hypothermia, the threshold been lowered. And in fact, |
|
| 56:41 | lot of these Children with gaps plus parents, they dread hot summer months |
|
| 56:47 | although we have internal body temperature it's hot, you sweat when it's |
|
| 56:51 | , you shiver. There isn't as of the adjustment as fast with the |
|
| 56:56 | . Sometimes the ambient temperatures outside temperatures affect these Children to the degree that |
|
| 57:02 | will experience seizures. Yes, that syndrome that is noted here and that |
|
| 57:11 | important because we're still talking about the channel. That syndrome that is noted |
|
| 57:18 | stands for severe myoclonic epilepsy of infancy SME I and another name for SME |
|
| 57:43 | that you may have heard of is syndrome. It has had some national |
|
| 57:53 | because J syndrome patients are uh very to cannabis and cannabinoid medications in particular |
|
| 58:02 | or cannabidiol. And in fact, a pharmaceutical uh CBD medication that is |
|
| 58:08 | in the United States for J syndrome or SME I patients. And the |
|
| 58:14 | why I wanted to highlight that first of all, I've worked with |
|
| 58:18 | syndrome mutation and in rodents for a of years. And we found some |
|
| 58:24 | interesting things about this mutation and also to control potentially seizures using a |
|
| 58:33 | So we've done significant amount of work filed a patent with the University of |
|
| 58:38 | on manipulation or control of seizures and abnormal physiological using adenosine agonist specific chemicals |
|
| 58:47 | denison. And you'll understand what that because it's very well related to something |
|
| 58:52 | you consume absolutely every day. adenosine is a neurotransmitter in your |
|
| 58:57 | It helps you sleep, it goes at night, but adenosine receptors in |
|
| 59:01 | brain interact with caffeine. So you'll that as we talk about neurotransmission in |
|
| 59:06 | second section of the response. The why I'm highlighting Driveway Syndrome is not |
|
| 59:11 | I only uh not only because I it and I really cared about understanding |
|
| 59:16 | disease and helping the patients, these with gas flo and sme I in |
|
| 59:22 | , about 30% of the Children that these mutations. Now, if you |
|
| 59:26 | gas, it's where the green spots . If it's sme I a different |
|
| 59:30 | in a different location now can lead SME I. But 30% of these |
|
| 59:35 | are un unresponsive to pharmaceutical medications. they have to have cocktails of |
|
| 59:43 | They have to seek alternative treatments. are uh either seeking under supervision or |
|
| 59:49 | medicating with cannabis or cannabinoids, but really, really miserable for 30% of |
|
| 59:56 | Children that have this disease. It's the seizures cannot be controlled if they |
|
| 60:01 | be reduced, the number of seizures be reduced, but they still may |
|
| 60:04 | . Some of these Children may have of seizures a day and then they |
|
| 60:08 | pumped with a lot of drugs that similarly to alcohol, benzodiazepines and that |
|
| 60:15 | them act almost like drunk. So have a little child that is uh |
|
| 60:20 | around, stumbling like a like a person. This is significant, |
|
| 60:25 | So we need more therapies for these of severe disorders. Developmental disorders. |
|
| 60:31 | if the child is having so many , they cannot learn, they cannot |
|
| 60:36 | , their brains don't develop and you know, the consequences of that |
|
| 60:40 | , are, are throughout the However, over 20% of these Children |
|
| 60:46 | unexpectedly from what we call sudden unexpected in epilepsy or so, typically happens |
|
| 60:54 | night, it is linked to cardiac , but the parents don't know and |
|
| 60:59 | really knows when and if it will . And that's really tragic. And |
|
| 61:05 | these forms of epilepsy are a lot times referred as catastrophic forms of |
|
| 61:11 | They're catastrophic, not only for a that has it in their brain |
|
| 61:16 | it's catastrophic for the families that have deal with a child that has such |
|
| 61:20 | severe disorder. And in case of death or sudden expected death and |
|
| 61:25 | it's just a just a horrible outcome , for families that are already trying |
|
| 61:30 | help their child get through life with sorts of medications and preparation. |
|
| 61:37 | so understanding these diseases, understanding how affect voltage gated sodium channels, knowing |
|
| 61:42 | there are these things, channelopathy are for you guys because there's a |
|
| 61:48 | there's a need for a lot more and clinical interventions, validated novel approaches |
|
| 61:56 | could help these Children that could help Children develop normally and could prevent that |
|
| 62:03 | . That's uh that's that, that's significant percentage of these Children. |
|
| 62:10 | So we're gonna end here today because want to take attendance. Don't |
|
| 62:14 | I will tell you how this is work and when we come back, |
|
| 62:17 | gonna talk about what I call. , I didn't put it on here |
|
| 62:21 | mouthwatering tales of toxins and puffer All right. So let me stop |
|
| 62:30 | |
|
