| 00:02 | And when we spoke about the action , this is our second lecture in |
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| 00:06 | action potential and we spoke about the potential. In the first lecture, |
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| 00:10 | already understood that it has the rising , the overshoot, the falling |
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| 00:14 | And the undershoot, we also understood there are these equilibrium, the town |
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| 00:20 | or ionic, the town charts for ion. And if there is an |
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| 00:25 | membrane potential and mills which we abbreviate VM. OK. So recall that |
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| 00:32 | equilibrium potentials are calculated using nurse And the Goldman equation is used to |
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| 00:41 | the overall membrane potential of von. the differences are is that equilibrium potential |
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| 00:49 | calculated for each ionic species. And membrane potential takes into consideration the permeability |
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| 00:58 | p for each ion and calculates membrane based at least on sodium and |
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| 01:05 | So it takes into consideration more than ionic species. We also spoke about |
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| 01:11 | period here that would be absolute refractory once the membrane potential crosses the threshold |
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| 01:18 | action potential generation and it's going through all or none event, the action |
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| 01:24 | event nothing can be elicited from this . In in in uh in the |
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| 01:32 | that no other action potential, no depolarization can be produced during this |
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| 01:39 | which we call the absolute refraction However, once it crosses back through |
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| 01:45 | threshold for action potentials in this time , you could have uh the ability |
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| 01:52 | the cells if they receive a strong input to generate an action potential. |
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| 01:56 | that's why this this period here following crossing of the threshold onwards, is |
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| 02:01 | to as relative refractory period. So happens is we talked about that once |
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| 02:10 | membrane potential reaches the threshold value. the membrane has to depolarize the threshold |
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| 02:18 | of minus 45 millivolts or so, order for voltage gated sodium channels to |
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| 02:23 | . So the first thing that happens depolarization, voltage gated sodium channels open |
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| 02:31 | , more depolarization, more sodium fluxing more sodium channels open and the membrane |
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| 02:39 | is being driven to the equilibrium potential sodium. However, there are two |
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| 02:46 | that there are two reasons why the in potential doesn't reach the equilibrium potential |
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| 02:52 | sodium. First of all, it's concept of the driving force that we |
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| 02:58 | . The driving force is the difference the number and potential, which is |
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| 03:03 | white line at any given moment along white line and equilibrium potential for specific |
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| 03:11 | . So each ion will have its driving force at any given time along |
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| 03:17 | changes of the number and potential OK. So for example, the |
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| 03:23 | force or sodium ion at this When sodium channels, voltage gated sodium |
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| 03:34 | just open up the driving force or is huge. So you can see |
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| 03:43 | this arrow here which would indicate the of the driving force for sodium and |
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| 03:50 | stopped at the equilibrium for sodium from white line of the membrane potential, |
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| 03:55 | a big driving force. But once number and potential reaches a depolarized value |
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| 04:05 | at the peak of the action Now, what you see is that |
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| 04:09 | driving force for sodium has reduced So that's one reason why the membrane |
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| 04:18 | doesn't reach the equilibrium potential for The second reason is the kinetics of |
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| 04:23 | voltage gated sodium channels that you will about is that voltage gated sodium |
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| 04:28 | Despite the fact that they're going through we call the positive feedback loop, |
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| 04:34 | depolarization, more sodium influx, more open, more sodium influx, more |
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| 04:38 | , more sodium influx, more channels , more depolarization. Despite this loop |
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| 04:43 | the opening of the channels, those close very quickly and as they close |
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| 04:49 | quickly, they're no longer open. , remember channels have to be permeable |
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| 04:55 | an ion in order to have And if the channels are closed, |
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| 04:59 | no more sodium conductance. Even in presence of the small driving force, |
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| 05:04 | still no conductance for sodium because the are now closed at the same time |
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| 05:10 | the member and potential. Once it's the peak of the action potential, |
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| 05:16 | can see that at the peak of action potential, the membrane potential has |
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| 05:20 | huge driving force for potassium. So a big difference between VM at the |
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| 05:28 | of the action potential and EK for . And that's this huge driving |
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| 05:34 | And so during the falling phase, is e flux and potassium is leaving |
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| 05:40 | south and it's driving the membrane potential its own equilibrium potential value. It |
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| 05:47 | succeeds to do that. And it has contributed to the fact that potassium |
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| 05:52 | have different kinetics, they remain open as opposed to sodium channels. So |
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| 05:58 | more hyper polarization and also remember the case of the leak potassium channels that |
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| 06:05 | dominate the potential sort of closer to rusting number of potential? Mm. |
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| 06:12 | is it clear what the driving force driving force again, is the difference |
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| 06:17 | VM which is membrane potential and E VM and E potassium VM and E |
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| 06:24 | . Each one of these ions during given moment as this membrane potential fluctuates |
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| 06:30 | have increased or reduced driving forces depending where the number and potential is at |
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| 06:36 | moment. Do we need to know like V and calcium channels open or |
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| 06:46 | so do you need to know about and calcium channels? Actually, when |
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| 06:50 | talk about action potential, we will on voltage gated sodium and potassium |
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| 06:55 | When we talk about synaptic transmission, will shift our focus to uh calcium |
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| 07:02 | preside and also posy. And when talk about inhibitory neurotransmission called Gaba or |
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| 07:10 | ergic transmission, we will talk about chloride does but chloride influencing in general |
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| 07:16 | cause hyper polarization. For the most . It's a very good question because |
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| 07:22 | said there are four ionic species. how come you're just talking about sodium |
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| 07:25 | potassium? Because if you do these of Goldman Equation and you compare it |
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| 07:30 | electrophysiological recordings, you can pretty much rely on sodium and potassium perme abilities |
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| 07:37 | these channels as the key uh uh essentially of where the number of potential |
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| 07:44 | be and the main actors in the potential. So that's why we, |
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| 07:48 | , we leave this uh on the for a minute. Very good |
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| 07:54 | OK. So we record action potentials oscilloscope. We've already talked about how |
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| 07:59 | were developed in the 19 fifties fast to pick up action potentials. So |
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| 08:04 | array oscilloscopes and if you pluck an inside the cell, you will see |
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| 08:09 | significant what looks like about 100 millivolt , the action potential, 100 millivolts |
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| 08:14 | amplitude and again about one to few in duration. However, there are |
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| 08:20 | methods of recording neuronal activity even from neurons. So when we talk about |
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| 08:26 | , we said that these wires get in the person's brain and we said |
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| 08:31 | not the same way as targeting cells the electrodes inside the cells. So |
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| 08:37 | wires are left outside neurons, maybe touching them, but they're not in |
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| 08:43 | neurons. And this is called extracellular . And then extracellular recordings are outside |
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| 08:48 | south. If you're located close enough the axon initial segment, that's where |
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| 08:54 | actual potential gets produced. You have ability to pick up this electrical activity |
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| 09:00 | through the extracellular recording, which will an inverted shape as compared to the |
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| 09:05 | reporting. And it will also be , very small in amplitude. And |
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| 09:10 | we're talking about 100 micro volts potentially amplitude versus 100 millivolts. So it's |
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| 09:17 | to the minus three. It's a story altogether which tells you that these |
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| 09:22 | recordings are not reliable at picking up potentials from single cells unless it's really |
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| 09:29 | an experimental setting. And very likely these extracellular electrodes do a lot of |
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| 09:35 | is that they pick up action potentials several nearby cells or they essentially can |
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| 09:41 | up what is called a compound activity compound action potential produced by several cells |
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| 09:48 | the area from that electrode. And what Neuralink is recording. It's |
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| 09:52 | It said we picked up a promising activity recordings with the neuralink implant and |
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| 09:58 | recording essentially extracellular activity from neuronal approximating activity probably from tens hundreds if |
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| 10:06 | thousands of cells. OK. But are different recordings. This again, |
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| 10:11 | type of intracellular recording and extracellular recordings mostly experimental, except when there is |
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| 10:17 | surgery neurosurgery. And the surgeon quite will utilize the help of a neurophysiologist |
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| 10:24 | the operating room. And the neurophysiology help a surgeon determine which parts of |
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| 10:30 | brain could be spared from surgery, parts of the brain show abnormal activity |
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| 10:35 | further corroborate what they're seeing in the room with what already they have done |
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| 10:40 | that the F MRI cat scans test uh all of these things. And |
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| 10:48 | , when the neurosurgeon has a person's , their task is to be as |
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| 10:53 | as possible. That means to remove little of the brain that is responsible |
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| 10:58 | really important functions in order to help individual and to do that, you |
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| 11:02 | want to sample electrical activity from the typically extracellularly with the help of |
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| 11:08 | In order to help the neurosurgeon to that troubled area that needs to be |
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| 11:14 | in patients. That's of course, cases when medications do not work or |
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| 11:20 | you have um uh malignant growth that's in the brain action potential patterns. |
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| 11:27 | you inject this current, if you the cell, this is the injected |
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| 11:31 | . So notice that when you inject current, what we call artificially injected |
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| 11:36 | because otherwise, biologically, it's the that create this current and action potentials |
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| 11:41 | neural transmission. But we can also this positive current and record a |
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| 11:47 | And typically, during the older you had to use two electrodes, |
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| 11:52 | that would stimulate another one that would . And in modern electro physiology, |
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| 11:56 | circuits inside these electrodes are very, fast. And so we would use |
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| 11:59 | a single electrode to both stimulate and the recording activity because it's done, |
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| 12:05 | done at such high frequence. But , anything that comes from the |
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| 12:10 | I said in biology, when you a flat line, it's not |
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| 12:15 | Uh but in instrumentation, you can many flat lines. So when you |
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| 12:20 | an artificial uh artificially injected current, like a switch on and off. |
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| 12:26 | the cell doesn't necessarily respond immediately with same. This is what we call |
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| 12:32 | wave like pulse. This electrical this is a square wave like pulse |
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| 12:38 | notice that the cell doesn't necessarily respond a square wave. It has a |
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| 12:43 | shape that's due to the resistance and properties of the cell membrane, but |
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| 12:48 | also responds obviously to the strongest stimuli the frequency of action potentials. So |
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| 12:56 | you have a weak stimulus, you depolarize the plasma membrane. Again, |
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| 13:01 | can see that these are the square that are produced by instrumentation by the |
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| 13:07 | that inject these currents. These are square waves and these are the rounded |
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| 13:13 | here because it takes time to build the charge across plasma membrane due to |
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| 13:19 | resistive incapacitated properties of the plasma And then when the stimulus stops, |
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| 13:25 | takes time and it's fast. So can be charged and recharged very quickly |
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| 13:31 | a matter of milliseconds. But it's instant like you would see in the |
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| 13:35 | wave instrumentation input. So if you a stronger stimulus, a stronger |
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| 13:43 | then there's a possibility that the membrane the cell will reach the threshold for |
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| 13:50 | potentials and will produce these five action . And if you increase the stimulus |
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| 13:57 | further inject more positive current into a like this, then what you can |
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| 14:03 | the response of the cell is a frequency of action potentials. And this |
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| 14:09 | one of the basic rudimentary codes in brain is that the strength of the |
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| 14:16 | , weak versus strong stimulus is in reflected in the frequency of the action |
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| 14:23 | that the cells produce the stronger the . The more likely that cell is |
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| 14:29 | to respond, the more likely it's to produce more action potentials during that |
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| 14:34 | . So high frequency reflects the magnitude the depolarizing current. And that magnitude |
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| 14:41 | equivalent to the strength of the weak stimulus and then not just |
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| 14:46 | it could be obviously biological electrical not just by instrumentation versus a strong |
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| 14:52 | stimulation. So you can have these , injected currents in different cells. |
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| 14:59 | we already spoke about that, that can present the exact same stimulus, |
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| 15:03 | same duration, the same amplitude and will see that the cells respond in |
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| 15:10 | different patterns. So I keep referring these patterns of dialects of actions. |
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| 15:16 | this is the code too because the of action for chars means the frequency |
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| 15:21 | neurotransmitter release, that means frequency of post synaptic response from the cell. |
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| 15:29 | you have a variety of these different . And as we discussed, most |
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| 15:34 | the diversity in these dialects in the patterns of the action potentials comes from |
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| 15:42 | inhibitory interneurons. There's a local circuit cells that will produce a diversity of |
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| 15:49 | different dialects as opposed to the projection cells that will be fairly uniform in |
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| 15:56 | functional output. But the projection cells they will communicate that output to other |
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| 16:02 | . But that output can now be by the surrounding inhibitory cells that have |
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| 16:08 | of these complex ways of talking to other and also talking to the parameter |
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| 16:12 | and telling how parameter cell is going talk to the adjacent networks or project |
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| 16:17 | information to the adjacent networks. One the coolest uh developments uh in recent |
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| 16:25 | is the fact that we have voltage channels that we talked about. We |
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| 16:30 | talk about mechanically gated channels, but are also light sensitive channels or channels |
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| 16:38 | can be responsive to light and manipulated light. And so they are these |
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| 16:45 | channels that are called channel or adoption Haller adoption and they were discovered in |
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| 16:55 | . And the interesting thing is when adoption two is activated with blue |
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| 17:02 | it will allow the influx of When Hale adoption is activated with yellow |
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| 17:11 | , it will allow the influx of . So what sodium does to the |
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| 17:17 | ? When sodium comes inside the the positive charge is going to cause |
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| 17:23 | . But when chloride channels are activated chloride comes inside the cells, it |
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| 17:29 | cause hyper polarization. So this is neat because uh although we don't talk |
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| 17:37 | chloride in relation to action potentials, is one more unique way in which |
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| 17:42 | flux of ions, the depolarizations and polarization can be studied and controls and |
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| 17:49 | in animals. So what you can is the experiments are done in |
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| 17:59 | but you can isolate these channels and general like frog side system that is |
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| 18:05 | here, it's really good for studying activity. So if you want to |
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| 18:10 | sodium channels versus potassium channels or you a new unusual sub type of the |
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| 18:17 | , nobody reported you can over express in these systems in these frog eggs |
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| 18:23 | that are very large of one millimeter diameter. And you can do electrophysiological |
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| 18:30 | . So you can use these simple . And at first of course, |
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| 18:34 | you isolate it from nature, these sensitive channels, you want to put |
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| 18:38 | in more primitive systems, you want express it. For example, in |
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| 18:42 | frog boo sides and then shine the on these eggs and actually record the |
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| 18:49 | from the channels. So again, you have a target like a channel |
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| 18:54 | you want to understand what it is , you'll say, well, why |
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| 18:57 | you just, uh instead of you've discovered this channel and while you're |
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| 19:02 | animals like rodents, why do you to go to the frog eggs to |
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| 19:07 | that channel? Uh That's because a of times you can do easy manipulations |
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| 19:15 | these systems and you can have a amount of the expression of these channels |
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| 19:21 | understand once you stimulate these channels, exactly the response is from these |
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| 19:28 | Now, you have sort of a a a very strong response from the |
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| 19:33 | that you can manipulate easily. you can take that knowledge and this |
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| 19:37 | the kinetics of this channel. you can take that knowledge and apply |
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| 19:42 | knowledge and study those channels and those and high water species uh all the |
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| 19:48 | up until humans. And in what is illustrated here is really cool |
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| 19:54 | that this is a, a mouse has a a fiber optic cable |
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| 20:01 | And remember we talked about genetic manipulations mice, we talked about transgenic |
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| 20:08 | we talked about expressing genes. So would use those techniques, you would |
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| 20:13 | a transgenic mouse, essentially, you introduce a new channel into that mouse |
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| 20:20 | that channel is going to be light . And you can introduce the depolarizing |
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| 20:26 | and the hyper polarizing channel. And really neat because you can now control |
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| 20:31 | behavior of this mouse through the light . And depending on where it is |
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| 20:38 | in the mouse's brain, you can the mouse's motor activity. If you |
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| 20:46 | depolarization and sodium with blue light, moss is going to be more |
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| 20:52 | And then through that same cable, turn on the yellow light and that |
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| 20:58 | the hyper polarizing channels. And that starts inhibiting and slowing down or eliminating |
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| 21:06 | movements, certain motor functions by, an animal. So there are multiple |
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| 21:14 | in which this technology is being But ii, I imagine if you |
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| 21:20 | eventually, of course, you cannot introduce a new gene into humans. |
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| 21:27 | if there was some technology that allowed not necessarily genetic expression, but some |
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| 21:33 | that allowed you to stimulate neurons or inhibit neurons with light and that you |
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| 21:41 | apply it through the skull because the , you can actually make them very |
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| 21:48 | , almost translucent. They will allow certain amount of light to come |
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| 21:52 | So instead of implanting electrodes to stimulate inhibit certain parts of the brain, |
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| 21:57 | would have these optic devices, so speak, that are attached to, |
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| 22:02 | your skull, that that would be that's uh potentially gonna come into the |
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| 22:08 | . But we have to find a way of having light sensitive channels in |
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| 22:13 | humans, potentially or uh imagine a in which you could express light sensitive |
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| 22:21 | only in the tumor. And by that tumor in viva in a whole |
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| 22:26 | , a whole brain to certain you would be killing that tumor. |
|
| 22:32 | that's there's a lot of different, not just electro physiology. It's |
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| 22:36 | you can use this functionality and in in general, not just with depolarization |
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| 22:41 | polarization, but potentially in pathologies as . OK. Let's uh remind ourselves |
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| 22:49 | we calculate equilibrium potentials using nurse we calculate membrane potential. Beyond using |
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| 22:56 | Goldman equation which has the permeability We know that if we want to |
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| 23:03 | a current for potassium, potassium current equal to conductance of potassium times G |
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| 23:10 | delta V or in this case, difference between the membrane potential VM and |
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| 23:17 | equilibrium potential, the driving force. you can also rewrite that the current |
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| 23:22 | potassium is conductance times the driving force that ion. And this is for |
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| 23:29 | ion. This is an example of ion, but you can rewrite it |
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| 23:33 | sodium fluoride. Uh and, and . So let's look at this situation |
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| 23:40 | where we have all of the channels the membrane, potassium and sodium |
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| 23:47 | they're in the membrane, but they're . And so if you put in |
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| 23:51 | an electrode across plasma membrane, it record zero millivolts because all the channels |
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| 23:57 | closed. And there's no flux. . So there is a reversal potentials |
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| 24:03 | minus 80 reversal or equilibrium potential minus . I use it uh interchangeably with |
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| 24:10 | reversal potential because the currents actually reverse that value and flow in the opposite |
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| 24:15 | . And you'll see that shortly, current is zero because there's no current |
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| 24:21 | , the channels are all closed. . So the conductance is zero and |
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| 24:26 | current is zero is driving 40 at . Millivolts is driving force for potassium |
|
| 24:43 | . What do you have to subtract zero at zero? Millivolts is the |
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| 24:58 | force for potassium zero. Your time up for the question. Next question |
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| 25:05 | zero. Millivolt is driving force for zero. No, right. I |
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| 25:14 | one person shaking their head. That's . No and no. Why? |
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| 25:22 | the driving force? What is the force? OK. All right. |
|
| 25:36 | start this lecture over. So the force is the difference between the memory |
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| 25:47 | . What is the mene potential? do you calculate it? All |
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| 25:52 | What is the potential? How do calculate it there? OK. So |
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| 25:57 | this value is zero, what is value for potassium include your? |
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| 26:07 | Let's go to the next slide. this is gonna be all in the |
|
| 26:13 | . So each has its own You put top that. What was |
|
| 26:21 | ? Zam is positive? 62. now wake up. So if GM |
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| 26:30 | zero and E for potassium is minus is the driving 40. No, |
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| 26:41 | VM is zero and equilibrium potential for is positive 62 driving for zero, |
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| 26:52 | VM is zero and equilibrium potential for is zero. Is there any driving |
|
| 26:58 | ? No? OK, good. we got to this point now let's |
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| 27:02 | this through. So we have driving here despite the fact that the membrane |
|
| 27:09 | VM is at zero, this is you read this figure VM is at |
|
| 27:15 | , right? But equilibrium potential for is minus 80. Equilibrium potential for |
|
| 27:20 | is positive 60 the channels are So there's no conductance. The driving |
|
| 27:28 | is huge for potassium 80. But 80 times zero of conductance gives a |
|
| 27:38 | of carbon. There's no carbon Now we open up potassium channels, |
|
| 27:44 | starts moving out of the cell. you can see that there is a |
|
| 27:49 | in the volt meter right at this . The volt meter is still measuring |
|
| 27:55 | value. There is flux of there's conductance of potassium, there's a |
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| 28:05 | driving force for potassium zero with And therefore there is a dominant potassium |
|
| 28:13 | here that can be recorded. once the membrane potential goes all the |
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| 28:22 | and hyper polarizes to minus 80 we now have a situation where the |
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| 28:28 | are open and there is conductance of going in and out. But the |
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| 28:36 | is zero. How is that That's because the driving force of minus |
|
| 28:43 | the driving force VM minus 80 minus 80 is equals zero. So this |
|
| 28:52 | you how if you have driving it doesn't mean you have a |
|
| 28:58 | you can have a huge driving But if the channels are closed, |
|
| 29:01 | no conductance, there's no current vice . You may have open channels with |
|
| 29:07 | lot of current flexing uh conductors going it, but the current is still |
|
| 29:14 | because there's no driving force. it's a zero mo and that tells |
|
| 29:19 | that there is no net flux, exact same amount of of potassium leaving |
|
| 29:26 | it is coming in. OK. keep that concept of the driving forces |
|
| 29:31 | mind. That's why I was drawing right here on the slide. |
|
| 29:37 | This is the driving force sticks right . The blue one and the yellow |
|
| 29:42 | to review this concept because I will a couple of questions regarding the driving |
|
| 29:47 | and how it's intertwined with the action and numbering potential. So at the |
|
| 29:54 | phase of the action potential, the is dominated by sodium before you have |
|
| 30:00 | depolarization. So a lot of questions I get or sometimes I get these |
|
| 30:06 | that say where does depolarization come If you have to reach the threshold |
|
| 30:11 | , where does this depolarization come You're telling us that at this threshold |
|
| 30:16 | of minus 45 millivolts voltage gated sodium will open up. So what is |
|
| 30:22 | this other depolarization for to reach. , it's actually synaptic inputs. So |
|
| 30:28 | the it's the stimulus of the synaptic . Now, these cells feel strong |
|
| 30:33 | depolarization, they depolarize and they start sodium uh at the resting membrane potential |
|
| 30:42 | dominated by potassium rising phase, dominated sodium following phase. It switches |
|
| 30:49 | you have the highest conductance for potassium the following phase and at the resting |
|
| 30:53 | potential because of the leak channels, leak channels, potassium conductance is are |
|
| 30:59 | dominating. Mhm. So a rising is sodium going in falling phase potassium |
|
| 31:09 | . Yeah. Does the current values we saw earlier indicate what direction of |
|
| 31:15 | moving in or is it just a which moving in and and yes and |
|
| 31:21 | no, because there would be positive negative uh uh p their values. |
|
| 31:28 | but uh you, you have to what eye on it is in order |
|
| 31:31 | know the direction of this fox positive versus negative um with concentration on |
|
| 31:40 | side of the battery, where is plus N versus the minus end because |
|
| 31:44 | one has their own battery. So days, we record action potentials and |
|
| 31:51 | a lot of electro physiology using this of patch clamp recordings. And this |
|
| 31:57 | an example of which we can bring electrode to the patch of the membrane |
|
| 32:02 | we can excise that patch of the and we can actually have multiple channels |
|
| 32:07 | sometimes pick up single channel activity. this is something that you apply a |
|
| 32:13 | across the uh uh channel here across whole electrode. And that voltage makes |
|
| 32:19 | channel uh ions move across and you pick up activity here from a single |
|
| 32:25 | . And I spoke that nothing in looks square wave like and it's actually |
|
| 32:29 | exactly square waved still, but the channel and channel recordings look a little |
|
| 32:35 | squarish compared to the overall number and responses that you saw that were more |
|
| 32:42 | . OK. And so the technique is really important in understanding how you |
|
| 32:51 | only record the currents but also manipulate currents and how you can use this |
|
| 32:57 | climb technique in understanding the reversals and flux of ions and also isolate individual |
|
| 33:05 | . This this technique is very important order for us to understand the subsequent |
|
| 33:10 | that we're going to talk about. . And there's a lot of it |
|
| 33:13 | we'll cover in the next couple of . So first of all, voltage |
|
| 33:17 | is a technique when you look at diagram, you're like, oh |
|
| 33:23 | Yeah, it's not that difficult. , this giant squid axon remember that |
|
| 33:30 | have a reference electrode which is your . It just says the outside of |
|
| 33:34 | uh axon and the solution is Then you have a electrode that goes |
|
| 33:42 | the Saxon mhm. This is what call internal electrode for measuring membrane |
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| 33:51 | And it's measuring the membrane potential is difference between the outside of the cell |
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| 33:56 | the inside of the cell. And is connected to voltage clamp amplifiers. |
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| 34:03 | now we're taking the measurement from this here, let's say minus 60 millivolts |
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| 34:08 | 65 millivolts address. And we're putting measurement that VM measured VM into voltage |
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| 34:16 | amplifier which compares membrane potential to the command potential. What is the command |
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| 34:25 | ? So we did not want to from South passively. If you stick |
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| 34:30 | electrode and you pass the current, can get a certain response and then |
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| 34:35 | South will come back and to its membrane potential that it likes to sit |
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| 34:42 | , let's say I the 65. that's not good enough for me. |
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| 34:46 | an experimentalist, I heard that there these equilibrium potentials for potassium minus 80 |
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| 34:52 | nernst that calculated them. I heard there is equilibrium potential for sodium of |
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| 34:57 | 62. How do I demonstrate that if it exists theoretically? And we |
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| 35:05 | it. Don't I need to have wet proof the recording of what is |
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| 35:11 | on the paper. Sure. But do that, we need a voltage |
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| 35:15 | and to do that, you have be able to command a lock membrane |
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| 35:21 | at your desired values of experimental. don't want to study membranes at minus |
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| 35:27 | . I want to study membranes at millivolts and to do that. You |
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| 35:31 | to clamp the potential, lock it at zero millivolts and that's what voltage |
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| 35:38 | allows you to do. So you the command potential here at the recording |
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| 35:45 | showing minus 60. You said the potential at minus 50. Listen. |
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| 35:51 | time when membrane is different from the potential, the clamp amplifier injects current |
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| 35:58 | the axon through the second electrode right . This brown electrode, this feedback |
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| 36:05 | causes the membrane potential to become same the command potential. So I said |
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| 36:11 | at minus 50. I'm commanding you stay at minus 50. The cell |
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| 36:15 | to minus 60 to say no, back to minus 50 I hold it |
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| 36:20 | or clamp it there. That's what clamp stands for. Literally voltage clamp |
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| 36:27 | . What you're doing, you're clamping voltage at a certain value minus 60 |
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| 36:34 | , positive 40 positive 60. And have the ability now to measure the |
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| 36:41 | and the differences that are fluxing the that is flowing back into the axon |
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| 36:46 | thus across its membrane can be measured . And this voltage clow technique is |
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| 36:52 | important in order for us to understand currents at different values along that number |
|
| 36:59 | potential scale that we've been looking at minus 80 all the way to positive |
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| 37:05 | for calcium reversals. This is exactly technique that Hoskin and Huxley used. |
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| 37:12 | in 1963 they won the Nobel Prize Physiology and Medicine for their work on |
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| 37:18 | action potential and came up with a and Huxley model of the action |
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| 37:22 | mathematical model of the action potentials. And also we're doing experiments and they |
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| 37:30 | using the voltage clamp. Yeah. remember that during the rising phase, |
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| 37:36 | the rising phase, we have an of sodium during the falling phase, |
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| 37:42 | have the outward current, which is efflux of potassium. How do we |
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| 37:47 | that we have to actually isolate these ? And so you use the voltage |
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| 37:53 | and you set the number and potential minus 26. So you depolarize it |
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| 37:58 | . And what happens is this deflection , this early transient downward deflection. |
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| 38:05 | is inward sodium current. And it that if you depolarize the plasma |
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| 38:10 | you'll have inward sodium current that later taken over by this late and sustained |
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| 38:17 | current that is outward, it's moving . So you depolarize and clamp the |
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| 38:24 | . Now with your voltage clamp at millivolts at zero millivolts, you see |
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| 38:30 | stronger influx because more depolarization, more coming in, more depolarization. So |
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| 38:36 | is more depolarization, more sodium coming . But it's showing that sodium comes |
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| 38:41 | here transiently. And at the same as you increase the sodium current immediately |
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| 38:47 | following the sodium current, you have prolonged and sustained and also larger. |
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| 38:53 | potassium current and that's because you now a greater driving force for potassium at |
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| 38:59 | millivolts. And if you depolarize further positive 26 look what happens to the |
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| 39:05 | current, it has decreased. So was larger at zero millivolts compared to |
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| 39:11 | 26. That means that the driving for sodium iron has reduced and the |
|
| 39:17 | channels are also closing and you still the sustained outward potassium current. What |
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| 39:25 | with positive 52? Let's pretend this positive 62. And that's why I |
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| 39:29 | you that different textbooks and different figures use different equilibrium potentials. But we |
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| 39:35 | talking about the equilibrium potential for sodium in our exams and in our playbook |
|
| 39:42 | everywhere in our slides is at 62 . So let's just pretend they set |
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| 39:46 | at 62 instead of 52. When said it at 62. What happens |
|
| 39:52 | inward cars? Why not exactly at qilib potential for son? But how |
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| 40:05 | there's this huge outward cur because this driving force for potassium? You're correct |
|
| 40:12 | potassium channels are now open. Now notice what happens when you go |
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| 40:17 | positive 65 value. So you cross threshold, you cross the equilibrium potential |
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| 40:22 | 62 positive for sodium. You cross . Now you're at 65 you see |
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| 40:28 | little blip here, this little blip shows you that the current right here |
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| 40:34 | of moving inward. Now, sodium starts moving outward and that's why we |
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| 40:39 | it reversal potential. Also the current reverses its direction after it crosses the |
|
| 40:46 | dodge and you still have this massive depolarization. So they proposed that there |
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| 40:53 | existence of sodium gates in the external . They studied the action potential. |
|
| 40:59 | described this early and transient inward current followed by the late uh persistent or |
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| 41:06 | potassium current and action potential. And voltage clamp, they were capable of |
|
| 41:15 | . This is the NNN word sodium . Each one of these lines in |
|
| 41:22 | , each one of these lines is single sodium channel opening. That means |
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| 41:28 | when there is depolarization, sodium channels up very quickly, but they don't |
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| 41:35 | at the same time, they all within about millisecond of time. So |
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| 41:39 | do open very quickly but not exactly the same time and they also close |
|
| 41:45 | quickly. So despite the fact that still depolarization here, there's something that |
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| 41:52 | these channels very quickly and you'll understand that something is in the next |
|
| 41:57 | if you take the combined activity, activity from all of these individual sodium |
|
| 42:03 | and you sum across them, then have the sodium current here. That's |
|
| 42:08 | sump through all of the channels. showing that there is activation during depolarization |
|
| 42:14 | during the rising phase. But there also closure of these channels right as |
|
| 42:20 | membrane potential reaches the very peak of action potential. Now, so these |
|
| 42:28 | , these channels are uh referred to fast opening, fast activating, but |
|
| 42:35 | fast and activating for sodium channels. . If you look at the |
|
| 42:41 | we are looking at the same time here. So we're looking at this |
|
| 42:45 | phase of the action potential as the starts depolarizing and the more it |
|
| 42:52 | as you can see at the very of the membrane potential, most of |
|
| 42:57 | potassium channels are now open. So depolarization, these channels are voltage |
|
| 43:04 | but sodium channels are gated very quickly closed very quickly. And potassium |
|
| 43:10 | it takes some time to open. they're referred to as delayed rectifier channels |
|
| 43:16 | they're delayed in opening and they're open and they're called rectifier because potassium conduct |
|
| 43:24 | starts to rectify or to reset the potential. Sodium hiked it up to |
|
| 43:31 | 40 potassium tries to rectify it and it back to resting membrane potential |
|
| 43:39 | So if you were to again sum all of the open potassium channels, |
|
| 43:44 | is the response that you would You would see a delayed activation of |
|
| 43:50 | channel and then prolonged, sustained activation it is rectifying the number and potential |
|
| 43:56 | resting. Now, if you were sum them across the inward versus |
|
| 44:04 | all the red and all of the activity, again, you will see |
|
| 44:08 | sodium is dominating with its influx, rising phase of the action potential and |
|
| 44:15 | outward currents. Again, this is current versus inward current, potassium, |
|
| 44:21 | currents or e flux is dominating the phase of the action potential. So |
|
| 44:27 | look at these sodium channels and why so special. This is the structure |
|
| 44:33 | voltage gated sodium channel. Each one these channels has four membrane sub |
|
| 44:41 | Each one of these subunits, 1234 six trans numbering segments. That's |
|
| 44:53 | between five and six. You have poor loop that Roderick mckinnon, we |
|
| 44:58 | about potassium channel had that very nice full hair thin lobe. And that's |
|
| 45:03 | Roderick mckinnon described also that serves to with the selectivity for this channel to |
|
| 45:10 | for sodium versus potassium. In this , sodium. That interesting thing and |
|
| 45:16 | exist between the four loop between S and the six S four has a |
|
| 45:23 | of philosophy. The trans number 87 four has a lot of positively charged |
|
| 45:31 | acid residues. Number of these are of amino acids, some of them |
|
| 45:36 | have the negative or the positive And so this one has a lot |
|
| 45:42 | positively charged amino acids within that S trans numbering segment. So these four |
|
| 45:50 | units have to come together and each of these subunits will have the poor |
|
| 45:57 | . So you will have, if have four subunits, you will have |
|
| 46:02 | loops coming into the inner lumen of channel, essentially serving as a filter |
|
| 46:08 | electrical seeing ionic seeing uh component to channel. This S four that is |
|
| 46:16 | with all of these positively uh charged acid residues is the vol of sensor |
|
| 46:22 | it's the gate, it's the the opening gate or voltage gated sodium |
|
| 46:28 | , voltage gated sodium channels at resting potential of minus 65 are closed. |
|
| 46:35 | if you depolarize the membrane, they open so closer to the threshold of |
|
| 46:40 | potential minus 45 minus 40 that gate is closed will open. And how |
|
| 46:46 | that happen? And how does this four play into it? So what |
|
| 46:53 | is the following when the channel four closed and the gates are closed. |
|
| 47:00 | positive sensor is drawn toward the inside the plasma number. The inside of |
|
| 47:09 | plasma membrane remember is negatively charged. there's a lot of minus minus minus |
|
| 47:15 | negative charge around here. And the charged S four is literally attracted to |
|
| 47:22 | negative charge and it's staying closer toward inside of the plasma membrane when there |
|
| 47:30 | depolarization, that means positive charge comes the cell. And once positive charge |
|
| 47:38 | into the South, it starts repelling S four voltage sensor. So negative |
|
| 47:46 | attracting and keeping it here. Once becomes depolarized, it starts repelling. |
|
| 47:51 | chart starts repelling the voltage sensor that sensor literally slides up within the |
|
| 48:01 | changes the confirmation of the channel and the opening of the channel. |
|
| 48:08 | So it's a, it's a physical and the confirmational change that you're seeing |
|
| 48:13 | the channel. And that's because of charge on the voltage sensor and the |
|
| 48:20 | uh attraction by negative numbering versus repulsion positively charged number. So again, |
|
| 48:29 | you look at this diagram, you have sodium channels that they open |
|
| 48:36 | very little delay. Once you have depolarization, they're very fast opening, |
|
| 48:41 | fast acting compared to potassium. they still open only for one |
|
| 48:47 | So now that we understand that this and voltage sensor slides and opens the |
|
| 48:53 | . Why does it close immediately? does it close within a millisecond? |
|
| 48:57 | has a second gate for. So can see here, depolarization in the |
|
| 49:03 | . But this channel is open and , open and closed, open and |
|
| 49:09 | . And this stimulus is sustained but more sodium channels are open. So |
|
| 49:13 | they opened and closed, now what is that you have to hyperpolarize the |
|
| 49:19 | or bring the membrane from minus 40 down to minus 65 millivolts at rest |
|
| 49:25 | then you can repeat the cycle of the channels again. Yeah. So |
|
| 49:30 | cannot open the channel again unless you the plasma membrane. And that's because |
|
| 49:39 | gated sodium channels have two gates. the way it works is that in |
|
| 49:44 | situation, it's closed, the gates closed. Nothing is fluxing. We |
|
| 49:51 | the membrane here, voltage sensor slides the numbers in previous slides. So |
|
| 49:57 | you depolarize the membrane, this s s four portion right here is going |
|
| 50:03 | slide up within the channel, it up within the channel and boom, |
|
| 50:10 | opens the channel and sodium was fluxing . But just as this sodium starts |
|
| 50:18 | in and the opening of this gate called activation gate, the activation gate |
|
| 50:24 | open and it changed the confirmation of channel. One little later, there's |
|
| 50:30 | second gate that we depict here as ball and chain. And as you |
|
| 50:34 | these arms of confirmational change for activation inactivation gate swings and plugs up the |
|
| 50:43 | channel closes it, it's called So in this position number three, |
|
| 50:50 | is corresponding to the traces above Number three, the channels are |
|
| 50:58 | And in order for this channel to closed and open again, we have |
|
| 51:04 | remove this ball and chamber. And only way this ball and Shane is |
|
| 51:10 | to leave is if we hyperpolarize the , we bring down the membrane potential |
|
| 51:16 | to minus 65 millivolt value here right to minus 65 millivolt value. What |
|
| 51:23 | is that now voltage sensor slides down it's sliding down, it's called de |
|
| 51:32 | it. It's de inactivate, it out an activation gate and allows for |
|
| 51:38 | activation gate to close and once it's , it's gonna wait for the next |
|
| 51:45 | . So activation gets open and once open, the ball and chain is |
|
| 51:50 | swing, it's gonna plug it up then it's gonna be there unless you |
|
| 51:57 | and the sensor slides into its original , deactivates and closes the channel. |
|
| 52:04 | there's two gates that are controlling And that's the reason why during the |
|
| 52:08 | potential as we talked about during the potential during the rising phase, sodium |
|
| 52:16 | not reach the equilibrium potential for One thing that we talked about is |
|
| 52:21 | size of the driving force as it . The other thing is simple fact |
|
| 52:26 | these channels close, they're open Once you open them, they |
|
| 52:31 | And the only way that you can them, you have to deactivate |
|
| 52:34 | close them and hyperpolarize the cell. . So this is a different absolute |
|
| 52:43 | period. Remember we talked about the relative versus absolute refractory period. So |
|
| 52:53 | the absolute refractory period, channels are , that's why you cannot, you |
|
| 52:57 | have them open again with more depolarization you have to hyperpolarize in order to |
|
| 53:03 | the confirmation of the channel for it open again. OK. So voltage |
|
| 53:11 | sodium channels, we already introduced a bit uh about epilepsy, um voltage |
|
| 53:21 | sodium channels and mutations in particular in gated sodium channels or voltage gated potassium |
|
| 53:33 | , calcium channels. If those mutations to neurological disorders, we refer to |
|
| 53:39 | as channelopathy. So it's channel It's a mutation that could be inherited |
|
| 53:48 | that leads to channel pathology. So things mutations in NAV NAV stands for |
|
| 54:00 | gated sodium channel and A for sodium for voltage gated. And this is |
|
| 54:07 | we're talking about. These channels are by voltage. When there is a |
|
| 54:13 | in voltage depolarization, it opens the when there's a change in involved it |
|
| 54:22 | , it flows the gates, there's chemical binding to these channels. The |
|
| 54:30 | in one direction will open the gate that direction will close the gate, |
|
| 54:35 | voltage gated channels in this case. . So N ad and it can |
|
| 54:44 | to a number of apple seeds. example is general generalized epilepsy with febrile |
|
| 54:53 | that we can abbreviate as guests generalized with febrile seizures. It's a severe |
|
| 55:02 | of childhood epilepsy. And this is voltage gated sodium channel that we looked |
|
| 55:10 | . This is the diagram of the of the voltage gated sodium channels. |
|
| 55:15 | the sequences of amina assets that we from these building blocks and everywhere you're |
|
| 55:22 | a green dot That means that a anywhere you see a green dot And |
|
| 55:29 | many sites on this channel. Any along the green areas of this channel |
|
| 55:37 | cause gaps. Let's talk a little about what this word means. Generalized |
|
| 55:47 | , generalized epilepsy typically versus non generalized focal epilepsy and generalized epilepsy and individual |
|
| 55:57 | consciousness during seizure. There's loss of . What is a febrile seizure or |
|
| 56:07 | seizure, pls, febrile seizures is good not taking things. Febrile seizures |
|
| 56:13 | hypothermia or heat induced seizures and the common type of seizures. And they're |
|
| 56:21 | common in little Children, little kids infants when your child has an infection |
|
| 56:30 | the temperature goes up above 100 You start worrying, you give them |
|
| 56:36 | medication to knock down their temperature. doesn't goes up to 100 and four |
|
| 56:42 | . You call a nurse say my is 100 and four degrees. I'll |
|
| 56:47 | go immediately rush them to emergency room hospital healthcare facility because our brains do |
|
| 56:56 | stay at 100 and four degrees comfortably a long time. As we overheat |
|
| 57:02 | our brains overheat, we experience hyperthermia we can evoke seizures that are |
|
| 57:09 | evoke seizures that are called febrile And many Children will have a febrile |
|
| 57:16 | and they'll get rushed to the hospital often and then their temperature gets knocked |
|
| 57:20 | and the infection is over and they and they'll never have another febrile seizure |
|
| 57:25 | in their lives. And they're not the left thing. And even if |
|
| 57:28 | years later, they have another infection their temperature spikes to 104 degrees, |
|
| 57:32 | kind of bring it down and they another febrile seizure again. It still |
|
| 57:37 | mean a person has epilepsy. So have to have repeated seizures and typically |
|
| 57:43 | are not provoked by anything that you , except for certain types of seizures |
|
| 57:47 | have very uh uh specific triggers such flash of strobe lights or certain sounds |
|
| 57:54 | odio GIC or sound devo seizures. febrile seizure by itself does not constitute |
|
| 58:01 | . Having single seizure on its own not qualify and give you a diagnosis |
|
| 58:06 | epilepsy. There has to be repeated uh and it has to be a |
|
| 58:13 | that's typically derived medical history observation, well as doing eeg recordings that we'll |
|
| 58:20 | about later in this course, electrons follow grounds where you record electrical activity |
|
| 58:25 | the caps that are placed on the . So, generalized epilepsy is a |
|
| 58:33 | form of epilepsy. This is a disorder. It's genetic because you have |
|
| 58:38 | mutation that leads to channelopathy as uh genes that are associated, it's called |
|
| 58:46 | one A gene. So the genes have a different name from what they |
|
| 58:50 | for, which is NAV a voltage sodium channel febrile seizure component. |
|
| 58:56 | what does that mean for individuals that this mutation? Why is the febrile |
|
| 59:02 | ? Plus? And that's because individuals have mutations in this channel, their |
|
| 59:08 | temperature doesn't have to go up to and 40 °F. Their body temperature |
|
| 59:13 | fluctuate by just two degrees outside of regular physiologic temperature, 37 to 3839 |
|
| 59:21 | and they are very likely to experience seizure. So they don't really need |
|
| 59:26 | hypothermia conditions anymore. Their seizures are easily triggered and sometimes even by ambient |
|
| 59:34 | temperatures, we have uh thermal control our bodies. Right. So we |
|
| 59:37 | sweating when it's hot or we start shivering when it's cold to keep up |
|
| 59:42 | body temperature always within a certain physiological of 36.6 °C to 37.6. And |
|
| 59:50 | just fluctuates a little bit during the . When you get sick, it |
|
| 59:54 | have an infection on the virus. . By the way, you call |
|
| 59:58 | nurse and they say the child is and four °F, right? Um |
|
| 60:06 | do I do? Um three hours from the hospital, put them in |
|
| 60:09 | eyes box. That will be actually directions the nurses will give you try |
|
| 60:14 | . You tried this, put them the eyes back. You need to |
|
| 60:18 | a child from overheating and having It's not good if you know, |
|
| 60:22 | didn't say you would, you have . So it's OK to have febrile |
|
| 60:25 | . It's not, it's not a thing. It still takes the body |
|
| 60:29 | the brain to recover quite a bit a febrile seizure. So you want |
|
| 60:32 | do everything possible to try to knock the temperature, including a a cold |
|
| 60:37 | , an ice bath and that sometimes in extreme cases get placed in |
|
| 60:42 | There's another uh uh thing that's written here. It stands for sme I |
|
| 60:49 | this SME I says with severe my epilepsy of infancy, I don't have |
|
| 60:56 | pen, but I'll write it out lecture. SME I severe my chronic |
|
| 61:01 | of infancy and you see it comes red P and that means that if |
|
| 61:07 | have a mutation anywhere where there is red dot You're very likely to develop |
|
| 61:12 | different form of epilepsy with a severe chronic epilepsy of infancy. Some of |
|
| 61:17 | may have heard it as a Dra . Also, it's another name for |
|
| 61:21 | I is Dra syndrome. Uh So where you have green dots, generalized |
|
| 61:30 | , febrile seizures mutations where you have dots, severe micronic epilepsy of |
|
| 61:36 | If you have mutations along where there those blue dots, it's another form |
|
| 61:41 | epilepsy and orange ones you have another of. So it's a, it's |
|
| 61:46 | important channel, this voltage gated channel multiple sites on it that can lead |
|
| 61:52 | severe neurological disorder. And there mostly genetic channelopathy childhood. The the |
|
| 62:00 | childhood by by that, I mean the occurrence of seizures and emergence of |
|
| 62:06 | drive syndrome is during the early the first two years of life |
|
| 62:15 | All right. So next lecture, will come back and we will talk |
|
| 62:21 | neurotoxins and voltage gated sodium channels. cool story of toshi and nara |
|
| 62:27 | But |
|
