| 00:00 | morning on this computer, and this Lecture seven of Neuroscience. Last |
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| 00:07 | We talked about resting membrane potential. discuss the main cast off characters chemical |
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| 00:14 | , the ions involved in setting the membrane potential, which is really unequal |
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| 00:21 | off ions. Such a sodium, chloride and calcium across the plasma membrane |
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| 00:28 | these four ions will influence the membrane the most, and we know that |
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| 00:34 | and potassium will influence the membrane potential most out of those four ions. |
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| 00:40 | we talked about is each one of ions has a certain concentration certain. |
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| 00:45 | Moeller concentration on the outside, extra solution of cellular fluid versus the inside |
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| 00:52 | the cytoplasmic fluid. So for you have five potassium mill, Imola |
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| 00:58 | and 100 million Mueller inside, so inside of the cell is dominated by |
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| 01:03 | ill, and the outside of the is dominated by sodium and chloride. |
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| 01:09 | in addition, an important thing here not only the concentrations but the ratio |
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| 01:14 | these concentrations on the outside of the versus the inside of the south. |
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| 01:18 | there's 20 more times of potassium on inside of the cell. There's 10 |
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| 01:25 | times of sodium on the outside of cell versus the inside of the |
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| 01:29 | and there is 10,000 more times of on the outside of the cell compared |
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| 01:35 | the inside of the cell. So we discussed, is that calcium has |
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| 01:39 | greatest chemical radiant disparity across plasma membrane . There's 11.5 more times on the |
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| 01:48 | on the inside of the cell, so you have to remember for the |
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| 01:54 | the concentrations off the outside and the , and the ratios of these ions |
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| 01:59 | you'll have to recognize and then equilibrium for these ions. And so we |
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| 02:05 | the concentrations of these ions, and learn how to calculate a Kulaib reum |
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| 02:11 | for a given ions such as E A, which stands for equilibrium potential |
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| 02:17 | potassium positive 56 million miles. To that value, we use the nursed |
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| 02:23 | nursed equation, which is the ion potential forgiven. One species ion equals |
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| 02:31 | are TCF log of the ion on outside over the ion concentration on the |
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| 02:37 | of the south. Where are his constant T is the temperature is disease |
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| 02:44 | . F is the Barry Days electrical log based on logarithms ionic concentration outside |
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| 02:52 | inside. And once you plug in the actual million Moeller concentrations. If |
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| 02:58 | could put here five million Moeller versus million Mueller for potassium or the ratios |
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| 03:04 | , the ratio is shown here. you simplify two on 303 rtc app |
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| 03:13 | 61.54 million volts for cat irons minus 54 no vaults for and I on |
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| 03:26 | 30.77 for a dive. Aylin Catalon two Plus. Once you do these |
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| 03:34 | , you then derive the equilibrium potential for each ion. So for potassium |
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| 03:40 | smile the stadium, the levels. here notice that these values are written |
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| 03:46 | . This diagram it says that potassium potential year is minus 102 million |
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| 03:55 | And this calculation here shows that potassium oracle liberal potential is minus 80 million |
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| 04:02 | and the point is not to trick here. But the fact of the |
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| 04:06 | is that there are differences in these Grady INTs and those differences are noticeable |
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| 04:14 | difference, uh, cellular subtypes in own little shop types and also in |
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| 04:20 | parts of the central nervous system where Aquarius and the Ionic environments and the |
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| 04:26 | vary from place to place. So will tell you how you need |
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| 04:31 | Remember these reversal potentials because I made graph for you that you can use |
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| 04:37 | full again. All of these equilibrium and you'll understand why use interchangeably reversal |
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| 04:43 | with equilibrium potential? When I talked equilibrium potentials, the other equation that |
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| 04:49 | discussed last time is the Goldman equation the Goldman equation. In this situation |
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| 04:57 | into consideration permeability. So it is much the same abbreviation from Nerds |
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| 05:05 | But now we introduce permeability and, of for equilibrium, potential forgiven |
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| 05:11 | We're calculating membrane potential in that membrane defense, mostly on potassium, sodium |
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| 05:17 | a little part to chloride. And you plug in the permeability ratio, |
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| 05:23 | is 40 times permeability for potassium and from the ability for sodium and you |
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| 05:30 | in in this case, you're plugging their million Moeller. Here you were |
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| 05:35 | in 1/20 which represents these ratios about 1/20 here. Five over 100 is |
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| 05:42 | same as 1/20 can, either pulling the mill Imola value or the ratio |
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| 05:46 | value. So 40 times more permeability potassium, one permeability for sodium their |
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| 05:54 | and you get the membrane potential address mine and 65 kilovolts. So this |
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| 06:00 | very important. That's one of the . All potential on this potential again |
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| 06:06 | derived by using the Goldman equation which is different by for calculating an |
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| 06:14 | ionic delivery. Um, potential is here. The important thing is that |
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| 06:21 | that we discussed, like Astra side important function and with this diagram on |
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| 06:27 | left shows it shows on the Y a member and potential. And then |
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| 06:32 | X axis concentration of potassium and so concentration of potassium is anywhere between 3.5 |
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| 06:38 | Mueller to five million Mueller on the of the south and at that concentration |
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| 06:45 | potential. Overall numbering potential is about 70 million balls about resting membrane |
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| 06:52 | But look what happens if you double potassium mill Imola concentration just by a |
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| 06:58 | million Moeller to 10 million Moeller. you're reaching close to minus 50 million |
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| 07:04 | if you increase it to about 12 . Moeller. You already reached the |
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| 07:08 | for action Potential Generation, which is minus 40 minus 45 million volts, |
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| 07:12 | ? Yeah, So changes in extra potassium solution. Potassium eyes using outside |
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| 07:20 | the cells. Remember the cells and permissible for potassium. And that's because |
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| 07:25 | a lot of potassium channels that So potassium is leaking out of these |
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| 07:30 | , and neurons in general are like capacitors. They're leaking potassium. They're |
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| 07:36 | . Charge essentially off the capacitor which is plasma membrane. And by |
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| 07:43 | this, it's creating ah, negative inside the cell because positive potassium minus |
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| 07:51 | . Now, if you increase the concentration on the outside of the |
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| 07:55 | now that leakage drive is reduced and you have a drive of potassium that |
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| 08:01 | not a strong for it to come the inside of the cell to the |
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| 08:05 | , and accumulation of extra cellular potassium be detrimental. Accumulation of extra Celia |
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| 08:11 | and increases can cause persistent firing and and high potassium models from high potassium |
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| 08:18 | models are often used as models with and epilepsy. And so this is |
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| 08:23 | neurological disorder, Apple FC Apple To be diagnosed with epilepsy. You |
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| 08:30 | tow, have seizure activity and seizures in very different types and forums. |
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| 08:37 | can have Ah, motor component with , Kalanick, contractions and tremors. |
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| 08:44 | you can have ah, no motor . You have very strong emotional |
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| 08:49 | It can result in the loss of or in a seizure that does not |
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| 08:55 | in the loss of consciousness and is thio smaller area of the brain. |
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| 09:00 | refer to Jesus focal seizures. The we discussed from early on would be |
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| 09:07 | in the hospital by using electrons so ground it's B e g, the |
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| 09:11 | that would be placed on top of skull and would pick up the electrical |
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| 09:16 | from underneath the skull. And so you actually increase potassium and if you |
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| 09:22 | inhibition and increase potassium so there's a more firing of the exciting terry |
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| 09:28 | This is one of the models for and seizures. Increasing potassium, which |
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| 09:33 | polarizes neurons and makes neurons fire the of Astra sizes to siphon off these |
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| 09:40 | increases in the potassium concentrations or other , through its very extensive processes through |
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| 09:46 | network and through this interconnected network through Astra sides. And this is referred |
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| 09:52 | a special buffering, essentially specially buffering concentration of potassium so that there is |
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| 09:59 | significant increase in extra cellular potassium persistent and that can affect the cells |
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| 10:05 | the South content fire and synchronize and excessive excited terry activity, which can |
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| 10:10 | Thio epileptic seizure. So this function Astra side off essentially monitoring the Ionic |
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| 10:18 | the neurotransmitter environment and the synapses is of the key functions. And distributing |
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| 10:24 | these local ionic concentration increases is very , because if it is persistent, |
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| 10:30 | it actually lead not only to but it could also lead Thio cell |
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| 10:36 | . So this is an important slide keep in mind. And I believe |
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| 10:42 | time we talked about Roderick MacKinnon, somebody remind me if we discussed this |
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| 10:48 | slide and I'm looking at the chat so I can see if you have |
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| 10:52 | questions on the syllabus you mentioned? might have homework and assignments will be |
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| 10:57 | later. Maybe we had something like . Maybe you missed it. I |
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| 11:03 | one homework assignment, which was, example, Thio. Determine which number |
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| 11:11 | Manet area corresponds. Thio broke US and to Vernon CAS area. |
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| 11:18 | this is a good exam question. I said, You guys should look |
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| 11:21 | up. I also said that it's as much homework but really keeping track |
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| 11:26 | the notes off the neurological disorders that already discussed. How often decisions need |
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| 11:31 | occur to be considered that once you tow, have mawr, then one |
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| 11:36 | for it to be considered apple saying, Um, there's very different |
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| 11:42 | of seizures and very different types of . Um, actually, it's very |
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| 11:49 | for little infants to have the seizure when the temperature goes up their called |
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| 11:56 | seizures or heat induced seizures, and if it doesn't repeat that you don't |
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| 12:02 | get diagnosed with epilepsy. So at multiple seizures you have tow confirm it |
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| 12:08 | multiple methods, and one of them be e g recording covered Yes, |
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| 12:17 | . So just remember, can it happen in adolescent seizures can happen in |
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| 12:25 | ages, but there is a higher of epilepsy and seizure activity. And |
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| 12:31 | Children, Uh, and then that increases again at an older age of |
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| 12:38 | . Plus, uh, there are forms of seizures. Some of them |
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| 12:43 | developmental forms of epilepsy and seizures to in on set within the 1st 23 |
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| 12:49 | of life. There's at the lesson of seizures that are more common that |
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| 12:54 | in during the teenage years and into early twenties. But that is really |
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| 12:59 | very complicated question. That's a very question, because epilepsy and seizures do |
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| 13:07 | . It's a zit can be a off genetic mutation, environmental changes, |
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| 13:14 | induce, seizures, visually sound induced and such. So very good questions |
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| 13:20 | , Hold on to it. Maybe talk more about epilepsy during the |
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| 13:24 | The teacher A message from this diagram Roderick MacKinnon and the fact that he |
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| 13:29 | driven by the quest to solve the of the Potassium Channel and then to |
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| 13:35 | the potassium channel. Using X ray . He was using genetic mutations and |
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| 13:42 | flies, which is a model of , because if you mutated potassium |
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| 13:47 | not just raise extra, sell your concentration shaker flies would present a epileptic |
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| 13:54 | syndromes and flies that are likable to you're seeing. Humans Gene Mutations Air |
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| 14:00 | hairpin loop is this poor loop that discovered. And this is the selectivity |
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| 14:06 | inside the channel in our loop conserved acids. This is important. You |
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| 14:11 | these mutations because you want to Of course, what the fruit |
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| 14:16 | potassium channel, how it functions and toxins, uh, can bind to |
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| 14:22 | parts of this channel using your pharmacology using electrophysiology. You can determine which |
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| 14:29 | the fact activity of these channels. the point is that a lot of |
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| 14:32 | channels air across conserved across species. so we may have a home ology |
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| 14:39 | 70 80% uh, to potassium channel and morphology that you see in fruit |
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| 14:47 | . So these discoveries that go from dish from isolated channels understanding their functions |
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| 14:55 | quite often not only a political for , but then result in aging humans |
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| 15:01 | using the science to their advantage be further discoveries for for discovery of new |
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| 15:09 | and new treatments for different disorders. action potential, we're gonna talk about |
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| 15:17 | potential will understand that from resting. goes to the rising phase and goes |
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| 15:22 | an overshoot which is above zero millet , then goes into the following phase |
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| 15:27 | goes into the undershoot, which essentially a potential that is below the resting |
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| 15:33 | and potential re Ford recovers with the of the palms back to the resting |
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| 15:38 | and potential. So I'm gonna, , recording all arrived as much |
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| 17:05 | Tonto, right? Just Yeah, , yeah. Mhm. Yeah, |
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| 20:32 | could. Yeah. Oh, okay. So isolating the squid damped |
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| 20:59 | on this is how we know the of islands inside the Exxon because it's |
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| 21:07 | big that we can actually squeeze its solution out. Um, which is |
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| 21:15 | . And some of the experiments and ionic transport. Remember, we talked |
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| 21:19 | actual plas Mick transport, slow or . Some of this ionic transport mechanisms |
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| 21:26 | described by using essentially some of these experimental data knowledge. So let me |
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| 21:36 | with you the diagram that I made helps us understand a lot about action |
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| 21:43 | . So I drew this and I'm post this, uh, online |
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| 21:55 | But this is very important that quite few things are summarized in in this |
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| 22:06 | and some of the very important things are important for us to now. |
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| 22:14 | , so remember we drew this this last lecture Did you guys do it |
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| 22:24 | lecture? Anybody? Did we go the circuit with you? So we |
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| 22:47 | about numbering equivalent circuits. We described . Each channel can serve as a |
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| 22:53 | variable conductor variable resistor that each one these channels, because of the equilibrium |
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| 22:58 | on the electro motive forces has its battery that each channel will have its |
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| 23:05 | circuit representing plasma membrane that you calculate current the strength of the current for |
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| 23:13 | which is I the equals I Okay, or I is equal |
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| 23:22 | InsWeb is the inverse of resistance times but VM here is the difference between |
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| 23:30 | and minus E k. What does mean? This is the driving |
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| 23:36 | So the current for potassium will depend the conductors for potassium and the driving |
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| 23:41 | for that given ion for that potassium and the total conductors is the sum |
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| 23:47 | all of the conducting sister individual potassium times the number of potassium channel Senator |
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| 23:53 | membrane and equally so you can have strength of the sodium current calculated like |
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| 23:58 | and other cards using the driving We talked about the fact that membrane |
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| 24:04 | a great capacitor. Uh, we about resistance and that resistance. The |
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| 24:10 | the south, the larger the The smaller is the input resistance into |
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| 24:15 | south, but the larger the surface for the membrane, the greater is |
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| 24:20 | capacitance properties of the cell. We about capacitors, good capacitors inside the |
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| 24:27 | . They accumulate the charge pretty They discharge of pretty fast. The |
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| 24:30 | plates are located close to each and we also discussed this what we |
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| 24:35 | Ivy Block. This is the voltage over a current plot showing a linear |
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| 24:41 | between voltage and current, but will back to some of the plots. |
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| 24:45 | lecture discussed, Um, some or greater detail. Finally, this was |
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| 24:49 | slide about the circuits that basically said if you have a passive circuit, |
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| 24:54 | will not have any conductors through that . In reality, if you have |
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| 25:00 | passive and active circuits, there's circus the plasma member and, to be |
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| 25:04 | correctly, should have a capacitor built . So you have. See, |
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| 25:08 | here you have the n a K , which works against concentration, |
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| 25:12 | Um And then you have the electro forces driving potassium from the inside to |
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| 25:18 | and driving sodium from the outside the of the cell. So these were |
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| 25:22 | membrane equivalent circuits. And so the thing thio now understand before we talk |
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| 25:29 | action potential is that these from inability that we discussed the dressed 40 times |
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| 25:35 | permeability to potassium over sodium. We see that chloride has small permeability that |
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| 25:45 | those permeability numbers and ratios change. the top line represents resting member and |
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| 25:50 | middle line of PK ratio To PN two p c o represents the ratio |
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| 25:57 | the ability during the action potential. , what happens is that there is |
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| 26:01 | 20 times more permeability thio potassium to ion during the action potential compared to |
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| 26:09 | potassium ion, and also noticed that for co arrive addressed or during the |
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| 26:16 | potential doesn't change that march. so it's not a very important |
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| 26:21 | but nonetheless, we can also use , Hodgkin and tax equation with the |
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| 26:26 | are TCF Natural log P, p N A. For sodium and |
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| 26:31 | fa chloride. And you would see if you plug in the chloride |
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| 26:36 | it's not going to have a significant fact from the membrane potential value. |
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| 26:41 | because it doesn't change from rest action , it doesn't have a significant effect |
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| 26:47 | the actual potential value either. I'm gonna You should whip out your |
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| 27:00 | anding course notebook or blank piece of . The other blind piece of paper |
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| 27:09 | not the one little where you drew circuits so that you can recognize |
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| 27:13 | conductors, battery capacitors. This page this notebook is gonna be dedicated. |
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| 27:22 | Action potential, The whole page, whole page. Okay, so get |
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| 27:28 | . I drew this yesterday with a . You're lucky it's already done. |
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| 27:32 | can go faster over it, but are very important things. All of |
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| 27:36 | will be on the exam. First all, the equilibrium potentials for protection |
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| 27:40 | , Mark minus 90 have a Colombian for fluoride, which is minus 70 |
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| 27:46 | volts. You have resting number of RMP, which is minus 65 million |
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| 27:52 | . You have action. Potential threshold minus 45 million balls. A sexual |
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| 27:59 | threshold value minus 40 of abnormal. zero value is where you would have |
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| 28:07 | overshoot where the cell d polarizes and zero normal value equilibrium Potential for sodium |
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| 28:14 | positive. 55 positive. 60. a break here in the scale and |
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| 28:18 | have a deliberate potential council about 120. So what did he |
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| 28:29 | Raised it. You can draw it . This is what I'm going to |
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| 28:37 | . So here is our resting membrane . This is a cell and it |
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| 28:43 | polarizes and hyper polarized. High Hyper high proposed deep polarizes, deep |
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| 28:49 | , deep polarizes, deep polarizes, polarized. Deep polarizes, hyper polarizes |
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| 28:55 | . Yeah. Never reaches the threshold action. Potential never generates an action |
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| 29:06 | . Okay, it stays sub So these fluctuations, these deep polarization |
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| 29:12 | driving the cell thio more positive These deflections upward deflections is positive imports |
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| 29:21 | represent excited terry inputs and activation of Terry synopsis and these hyper polarization They |
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| 29:28 | this downward deflections to hyper polarized the their response of the cell. The |
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| 29:35 | in the member of potential because of inhibitory inputs in inhibitory synapses. So |
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| 29:41 | no, uh, reaching the threshold , which is indicated by Yellow |
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| 29:49 | And so the membrane potential states sub in the way you can think of |
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| 29:54 | is analog and coding mode erased it . So I said, You have |
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| 30:01 | draw now there's a number of potential polarized Whoa, hyper polarized, |
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| 30:07 | polarized, deep, polarized, polarized , threshold, fraction potential. What |
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| 30:13 | all or not? Event? actual potential happens. So if it |
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| 30:20 | this value off the threshold for the potential, which is about minus 45 |
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| 30:26 | 40 mil levels, that means that is strong enough excited Terry input coming |
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| 30:31 | the cell, maybe from thousands of on the dendritic trees gets to the |
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| 30:36 | of the cell, then the accident segment by the Soma. If it's |
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| 30:40 | polarized enough, it then produces all non action potential of them. |
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| 30:49 | yeah, So if we have two these events repeating, there's several important |
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| 30:56 | that are happening. When you have polarization, you have more sodium |
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| 31:02 | more sodium influx inside the cell causes cell to dip polarized Further, more |
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| 31:06 | polarization inside the cell causes more so you have this positive feedback |
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| 31:13 | Mhm positive feedback loop. That means . Deep polarization, more sodium, |
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| 31:18 | and more deep polarization, more going in and with sodium influx. |
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| 31:24 | sodium coming inside the cell is trying do. It's trying to drive the |
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| 31:28 | member in potential, which is measured on the Y axis on the X |
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| 31:34 | is time milliseconds trying to drive this in potential. That's close to the |
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| 31:40 | liberal potential for sodium as possible, it doesn't achieve that. And you |
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| 31:45 | say, Well, if it is , positive feedback loop more so, |
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| 31:50 | more deeply ization. More so, multiple ization. What happens then? |
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| 31:54 | come sodium doesn't reach a cooler brand potassium? Well, we talked about |
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| 32:00 | important concepts driving force, the M minus. The difference between the membrane |
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| 32:09 | , which is the equilibrium potential for , is its driving force. |
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| 32:15 | there are larger the driving force, larger the car inflow, the larger |
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| 32:19 | driving force. Okay, the mawr specific ion has driven across plasma |
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| 32:28 | So when you think about resting member potential here and action potential threshold, |
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| 32:36 | this driving force it was driving for . That's awful color. This driving |
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| 32:48 | is great for sodium. It's very separation between equilibrium potential facility in versus |
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| 32:55 | membrane potential, either dressed or during threshold of action potential. But what |
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| 33:04 | is the cell membrane potential keeps rising deep polarizing. This driving force keeps |
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| 33:11 | and shrinking and shrinking and shrinking and and becoming really small. This difference |
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| 33:18 | V, M and e sodium is smaller and smaller and smaller as the |
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| 33:24 | polarizes. So unless the Selby something else happens. Now you're actually |
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| 33:34 | at how far away this member and is from equilibrium potential for potassium. |
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| 33:44 | you're all of a sudden realizing that driving force, VM mindless potassium, |
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| 33:49 | much greater to potassium, so the in the driving force, in part |
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| 33:55 | responsible for the decrease of sodium The other part is responsible for decrease |
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| 34:02 | sodium influxes, inactivation of sodium So there's gating properties of the sodium |
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| 34:08 | that will discuss later today that you understand why the sodium channel shuts down |
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| 34:15 | why there is no MAWR sodium conductors , and it doesn't restrict Librium potential |
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| 34:21 | sodium. However, what happens is starts the flock sing, so potassium |
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| 34:28 | leaving the cell, and this is for the falling face off the action |
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| 34:33 | for the re polarization phase. And you have and undershoot and potassium is |
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| 34:41 | to drive the overall VM member and to its equilibrium potential value of about |
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| 34:48 | 90 million volts over the year. it also doesn't succeed quite to do |
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| 34:55 | as you have now, decrease in driving force for potassium, a significant |
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| 35:02 | and increase for driving force for sodium this stage here and activation off sodium |
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| 35:10 | a d p. Ace pumps reshuffling thes ions across plasma membrane back |
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| 35:16 | the wrestling member and potential. And it gets deep polarized enough again and |
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| 35:22 | the threshold for action potential, it another all or none event. That's |
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| 35:28 | few milliseconds to too few milliseconds in . Uh huh. These are all |
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| 35:36 | important numbers, values, dynamics, all of this is going to be |
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| 35:42 | your test. That a thing that gonna try to do here. I'm |
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| 35:46 | try to match this up pretty I was able to do that |
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| 35:53 | I took this diagram from the Look at that. Wow. Pretty |
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| 35:58 | . All right. My action potential not that bad of an action potential |
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| 36:03 | . So you have deep polarization And during this, uh, days |
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| 36:08 | the action potential, you're in the refractory period. That means that during |
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| 36:13 | red zone on deep polarization and during re polarization, you cannot produce another |
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| 36:20 | potential. The cell is not equipped produce another action potential. The membrane |
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| 36:27 | to re polarize significantly. It's shown close close to the action potential threshold |
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| 36:35 | in order for the cell to enter cell number in potential to enter in |
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| 36:40 | relative refractory period during relatively factory Martin Blue is when the salad it |
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| 36:47 | another very strong excited Terry. Another , deep, polarizing input it can |
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| 36:53 | the relative refractory period produced another action . So the frequency of self firing |
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| 37:00 | lot of times depends on the especially on the relative refractory period. |
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| 37:06 | the relative refractory period very much depends the ions and the re shuffling off |
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| 37:12 | across different subtypes of different neuronal Okay, so this is the absolute |
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| 37:20 | period versus the relative or factory That's very important to understand why, |
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| 37:27 | we talk further about, some of channel dynamics assume recording action potential, |
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| 37:34 | we'll talk about action potential. But do we know? And how do |
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| 37:38 | understand the action potential? Whether some the things that we needed? So |
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| 37:42 | we watched this movie, Professor brilliant, anonymous, he dissected, |
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| 37:48 | was stimulating. You saw the but he needed faster equipment and that |
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| 37:54 | equipment didn't come until World War Two incorporated very fast electrical circuits. A |
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| 38:01 | of them came from Navy, in , some of the connections and still |
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| 38:05 | like connections and the lab that we for some of the electrophysiology cords and |
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| 38:12 | and oscilloscopes that we connect. So Clamp was unnecessary technique. And what |
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| 38:17 | the voltage climb? So we have understand what was mentioned in the |
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| 38:21 | That Whoa, that's sweet. John was so big that he was able |
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| 38:25 | put an electrode inside squid John Paxson outside squid giant tax on. So |
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| 38:31 | was able Thio have two electrodes and the difference across plasma membrane. You |
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| 38:38 | very fast equipment in order to pick the action potential because action potential last |
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| 38:43 | two milliseconds. So you need to very high sampling rates. And the |
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| 38:46 | rates are incredibly high in modern We're talking about 400 uh samples for |
|
| 38:56 | samples, 40,000 samples per second. what you have here is an circuit |
|
| 39:03 | explains to you how voltage Kwan So the green electric goes inside the |
|
| 39:08 | john tax on that goes into the plasma and the other green electrode is |
|
| 39:13 | reference electrode, or the or the , which is basically comparing the voltage |
|
| 39:20 | the inside of the cell with the electoral to the outside of the |
|
| 39:24 | where outside of the cell is set zero million ball voltage. One internal |
|
| 39:30 | measures voltage and is connected to the clamp amplifier. So you're measuring voltage |
|
| 39:35 | is your measuring voltage your reporting? voltage to the voltage clamp amplifier here |
|
| 39:40 | green voltage clamp amplifier compares membrane potential the desired command potential. So, |
|
| 39:48 | , what are you talking about? is the command potential command potential or |
|
| 39:54 | membrane holding or clamping membrane? It's Samos commanding for the membrane potential to |
|
| 40:01 | held at a certain mill evolve So essentially, once you report this |
|
| 40:11 | and you said you're voltage plan, command the voltage clamped Experimenter says. |
|
| 40:17 | want the member and potential to be minus 30 in any fluctuation from minus |
|
| 40:23 | . I want this current when this from minus sturdy and the VM and |
|
| 40:29 | and potential is different from my Then I want this amplifier and two |
|
| 40:35 | jack current into the acts on through Second Orange Electric. And this feedback |
|
| 40:41 | causes the member and potential to become same as command potential and the current |
|
| 40:47 | back across the acts on in order correct for the setting. In order |
|
| 40:52 | inject this current, the current flowing into the action across the membrane can |
|
| 40:57 | measured, then can be measured as current can be measured to snap. |
|
| 41:01 | current can be measured as any physiological that happened that this voltage clamp amplifier |
|
| 41:09 | thio negatively feedback and reset it back the holding or command potential that it |
|
| 41:16 | was set by the experiment. So of the num brain that is important |
|
| 41:22 | remember the reversal or ionic equilibrium We did the calculations for a |
|
| 41:29 | um, potentials, But have we them experimentally? Not until we have |
|
| 41:34 | plan, so it allows you. measure effects of changes in membrane potential |
|
| 41:42 | change the membrane potential overall and then individually on the conduct, insists and |
|
| 41:48 | what happens to currents going inside the and going outside the cell. It's |
|
| 41:53 | negative feedback system, different from positive and and put in sodium conduct us |
|
| 42:00 | we just discussed this negative feedback and more like a thermostat in the summer |
|
| 42:06 | . Too hot school air kicks in the wintertime too cold, warm air |
|
| 42:10 | in. So the same way, there is a difference between the membrane |
|
| 42:15 | VM and the command potential, this the output that will come out from |
|
| 42:21 | voltage. Clamp this V out. as it balances, any fluctuations is |
|
| 42:26 | a actual activity off currents flowing across membrane. Modern voltage plan has ah |
|
| 42:35 | more complex circuit and only requires a intracellular like. So when I showed |
|
| 42:41 | patch clamp recordings, um, we'll some of them. Even today, |
|
| 42:46 | talked about only a single electrode. that single electrode can inject and record |
|
| 42:50 | can inject record card. In other , a single electron now can serve |
|
| 42:55 | a clamping and is a recording You still need a reference electorate, |
|
| 42:59 | is a ground which is always Any electric physiological electrical recording. This |
|
| 43:06 | voltage climb. The voltage clown was to let me check the chat. |
|
| 43:17 | , see the questions in Is your law slide? There's a resistant voltage |
|
| 43:22 | elements years with typical structure that represents child alone or that childhood memory. |
|
| 43:28 | ion channel and really the member. you, you would think with the |
|
| 43:33 | they represented by the capacitor, capacitance capacitor. This is the input during |
|
| 43:38 | relative refractory period, need to be that is greater than the threshold, |
|
| 43:43 | just enough to be polar as a to the threshold. Um, depending |
|
| 43:51 | where the membrane potential, as if is very close to the threshold and |
|
| 43:56 | get strong stimulus input, you will an action potential If you arm or |
|
| 44:02 | polarized, you will need a stronger in order to regenerate that action |
|
| 44:07 | So the, uh, earlier you in the refractory period, the faster |
|
| 44:13 | can generate second action potential. I that that's what you're getting at. |
|
| 44:19 | think if the stimulus doesn't reach the cellphone too polarized UK. Ignore |
|
| 44:24 | . Okay. I think that the polarizes, but if it doesn't reach |
|
| 44:29 | threshold, it doesn't produce the action . I think this is the correct |
|
| 44:33 | of saying that, uh, what said the surge isn't quite. Answer |
|
| 44:38 | question I have. Yes, I . Can you explain briefly the purpose |
|
| 44:44 | the voltage clamping? Great. So are on this, actually, just |
|
| 44:48 | the purpose of voltage clamping. Why would you create all of these |
|
| 44:52 | ? Like I said, Look, nurse equation, you were able to |
|
| 44:58 | individual reversal potentials based on what not on electrical recordings based on the |
|
| 45:06 | radiance and based on the neurons and goldman on the nurse equation for equilibrium |
|
| 45:11 | . The EU's voltage clamp. In example, Hodgkin and Huxley, who |
|
| 45:16 | in 1963 awarded Nobel prize in physiology medicine under work on the action potential |
|
| 45:23 | pictured here and what we know about potential in the model Hodgkin and Huxley |
|
| 45:28 | of action potential is still very much play very much dominating than Europe. |
|
| 45:33 | , modern nor physiology. Voltage clamp you dip, polarize the cell membrane |
|
| 45:38 | in red line to minus 26 million . That shows you two things. |
|
| 45:43 | shows you this downward blip. This blip here on the green represents inward |
|
| 45:49 | . And it shows you that as as you do polarize the south, |
|
| 45:52 | have this downward blip inward current going and then shortly afterwards you have this |
|
| 45:58 | zone current, which is the outward . It shows you that despite the |
|
| 46:03 | that you have this prolonged be polarizing , the inward currents just is |
|
| 46:09 | It doesn't persist, but the outward persists as long as there is deep |
|
| 46:16 | . If you d polarize the cell you voltage clam, that this is |
|
| 46:20 | experiment in which you're going to be to voltage clamp. Okay, sufficiently |
|
| 46:25 | clamped the member in that zero Mila . What happens is that you observe |
|
| 46:29 | a stronger in lord card mawr sodium in more do polarization, more |
|
| 46:34 | but it's still transient, but what is zero moviles. Now there's a |
|
| 46:39 | strong outward current because they're strong driving for potassium that has been created at |
|
| 46:45 | very positive, uh, D polarizing . If you do polarize the |
|
| 46:51 | even mawr now in the positive potentials positive 26. Now you're seeing a |
|
| 46:57 | , small reduction in this inward card this blip here on the green and |
|
| 47:04 | big increase in the outward card. you do polarize the self deposited 52 |
|
| 47:09 | balls. Whoa, what happened to inward current? It's gone. |
|
| 47:15 | Because this is equilibrium potential for sodium happens of equilibrium potential. There is |
|
| 47:21 | net flow of sodium ions. They're inside and outside. There is no |
|
| 47:26 | change in the movement of the There is no more inward signal. |
|
| 47:31 | have reached an equilibrium potential for which is generating this inward signal, |
|
| 47:38 | now you have just persistent Outward which is still delayed. It doesn't |
|
| 47:44 | immediately, is compared to these to inward currents generated by by sodium but |
|
| 47:49 | potassium outward current still persists. What if you go to positive 65 if |
|
| 47:55 | 50 to a positive 55 eyes the potential value for sodium? What happens |
|
| 48:01 | positive 65. Very interesting, If you were to zoom in a |
|
| 48:06 | bit more on these images, what see is you see a little bit |
|
| 48:10 | a early blip here and you see I'm saying. What is this early |
|
| 48:14 | ? This is sodium. This is , but now, on the other |
|
| 48:19 | of its equilibrium, potential off 62 a positive 65 sodium is no |
|
| 48:26 | flowing inside the cell. The current and sodium flows to the outside of |
|
| 48:32 | cell. And that's why when I said, just off the tip of |
|
| 48:35 | tongue, it always comes out Potential reversal potential. It's the same |
|
| 48:39 | equilibrium. Potentially used these two terms , and the reason for it is |
|
| 48:45 | at this value, the current reverses sodium card instead of coming inside, |
|
| 48:51 | becomes an outward card. Of physiological. If you kept the sustained |
|
| 48:56 | polarization of positive 65 mil levels, would mean cell death, but |
|
| 49:02 | in order to clamp the voltage, clamping it. You're holding this voltage |
|
| 49:07 | of positive 52. It allows you reveal the dynamics of individuals, conducted |
|
| 49:14 | dynamics off sodium conductors, which is and transient versus the potassium conductors, |
|
| 49:20 | is late in this persistent. And thanks to these two giants that we |
|
| 49:26 | understanding off the action potential you understand on probably will know more about actual |
|
| 49:33 | You thought you'd never want to Now, if you look at the |
|
| 49:38 | of sodium during the rising phase of action potential and you look and be |
|
| 49:44 | , each one of these wiggly traces single sodium channel. And when sodium |
|
| 49:51 | opens, you have this red curve the opening of the channel. What |
|
| 49:56 | shows is that the sodium channels open on during the polarization deep polarization. |
|
| 50:02 | they open it slightly different times during rising phase of the action potential, |
|
| 50:07 | then see what you have is you the combination of the some of all |
|
| 50:11 | these individual sodium channels opening up some , a sodium current showing a very |
|
| 50:21 | but early activated transient sodium, inward sodium carb. And if you |
|
| 50:29 | on the same, this this, to dash lines represent the rising phase |
|
| 50:35 | the action potential. If you look the diagram with potassium, these two |
|
| 50:40 | lines, which still represent the rising of the actual potential, shows you |
|
| 50:44 | potassium channels, which are outward, insists and indie You have individual potassium |
|
| 50:51 | opening up. Potassium channels do not , they're opening, is delayed, |
|
| 50:57 | they're opening is sustained. The activity the current flow through individual potassium channels |
|
| 51:02 | much, much longer and represents the of the DIF organization and the cell |
|
| 51:08 | as compared to the sodium channels. they're late activated, a delayed activation |
|
| 51:14 | the persistent activated. That means that long as there is deep polarization, |
|
| 51:18 | going to be very long, awkward currents and e. Of course, |
|
| 51:22 | have the sum of all of these potassium channels opening into the overall potassium |
|
| 51:29 | that is shown in the blue curve and on the top right, and |
|
| 51:33 | have basically net trans member and current the rising phase. During these two |
|
| 51:39 | realized lines of the action potential It's by sodium in flex, but that |
|
| 51:45 | transient. You can see that the it's still deep polarized at the peak |
|
| 51:50 | it still is D polarized at the dash line, but the sodium channels |
|
| 51:56 | closed, and now potassium channels are up peaking. The opening of the |
|
| 52:01 | is, during the falling phase, peak of the falling phase of the |
|
| 52:05 | potential. And that's when you have e flexing and leaving the cell from |
|
| 52:10 | inside to the outside to re balance shark very quickly. And this is |
|
| 52:15 | again over a period of one too milliseconds in time. So sodium channel |
|
| 52:23 | understanding of the sodium channel dynamics are . But understanding the morphology is equally |
|
| 52:29 | important because that tells us something about sodium channel dynamic sodium channel. In |
|
| 52:34 | case, when we talk about actual , we're talking about voltage gated |
|
| 52:38 | channel in voltage gated potassium channel. does that mean? That means that |
|
| 52:43 | channel has gates. That's why it's . That's not delegates, its Gates |
|
| 52:51 | gates. It's gated, gaining channels and and has a voltage. It's |
|
| 52:59 | to changes a voltages so it's gated voltage. It has this voltage |
|
| 53:04 | If we look at the overall morphology the channel, we have four trans |
|
| 53:12 | sub units. We have nitrous and Oxo and on the side of plasma |
|
| 53:18 | . Each one of the sub which is noted here in Roman numeral |
|
| 53:24 | contains six trans membrane segments noted. s 123456 The hairpin poor loop. |
|
| 53:33 | selectivity Felter is located between segment five six. Segment four of this Trans |
|
| 53:40 | and in each of the sub units a lot of positively charged amino acid |
|
| 53:47 | . And these positively charged amino acid are attracted by the negatively charged inside |
|
| 53:54 | the plasma membrane. And it has and you'll notice it actually has two |
|
| 53:59 | of gates. So what happens is resting membrane potential that inside off the |
|
| 54:09 | , it gets negatively charged and it negatively charged. And this voltage |
|
| 54:14 | which isn't trans membrane six it's really dimensional collection of Amina assets that are |
|
| 54:20 | , positively polarized, attracted by this charge. And they're sitting attracted close |
|
| 54:26 | the cytoplasmic side of the plasma membrane the activation gave indicated here is closed |
|
| 54:33 | the sodium. But what happens with polarization from minus 65 to minus 40 |
|
| 54:39 | balls is that instead of this negative , you now have accumulation of positive |
|
| 54:45 | on the inside of the membrane. this positive charge starts repelling the voltage |
|
| 54:52 | . And what that means is that sensor actually changes and starts moving. |
|
| 55:00 | structure, changes its confirmation and starts upwards and away from positive charge. |
|
| 55:07 | as it induces, this confirmation will across the entire protein channel. The |
|
| 55:14 | gave the channel gates for the sodium now open. Okay, so sodium |
|
| 55:21 | opened by the change in voltage across membrane and by the confirmation will change |
|
| 55:28 | gets induced by the movement off the sensor that's located and trans membrane segment |
|
| 55:35 | off each one of the sub So sodium channel kinetics are such that |
|
| 55:42 | discussed It's fast opening, but it's fast inactivating. It's called inactivation. |
|
| 55:49 | one millisecond or so sodium channels open sodium channels closed, you can have |
|
| 55:55 | deep polarization here, shown above from 65 to minus 40 million balls but |
|
| 56:02 | doesn't matter for sodium channel. It from home one millisecond and then immediately |
|
| 56:08 | . And then for this channel to opened again. You actually have the |
|
| 56:12 | polarized plasma number in here showed on of the Blue Line. In order |
|
| 56:17 | it, Thio be ableto open And so there's a cycle here. |
|
| 56:22 | this, in fact, shows that channels has two types of gates. |
|
| 56:29 | , it has Activation Gate, which closed, and it has a second |
|
| 56:35 | hanging out down here, sort of a ball on a change, and |
|
| 56:38 | that's called Inactivation Gate. Two types gates. The activation game has shown |
|
| 56:45 | to arms closed. Inactivation gate is ball, and chain number one sodium |
|
| 56:53 | here corresponds to the traces of the opening shown above in number one. |
|
| 56:59 | channel is closed because they're just Polarization started as soon as deep polarization |
|
| 57:04 | . A number two activation gate is because the voltage sensor has moved up |
|
| 57:10 | opened up activation gate and sodium channel open and sodium ions of flowing into |
|
| 57:17 | sodium channel for about one millisecond. because of that confirmation will change that |
|
| 57:22 | generated by the sliding off the voltage up wars onto the channel That confirmation |
|
| 57:29 | change now causes the inactivation gate to and swing and close the channel |
|
| 57:35 | Plug it up. It's called so the movement of that bolted sense |
|
| 57:41 | first opens activation gates. But as changes the confirmation of the channel, |
|
| 57:46 | channel used the second gate and activation and in activates. So Number |
|
| 57:52 | you have an activation, and that takes only one millisecond. Now what |
|
| 57:58 | to happen in order for you? , open the channel again. What |
|
| 58:02 | have to do is you have to the inactivation gave. I didn't make |
|
| 58:07 | these terms, and it's called Deep . You deep inactivate you remove inactivation |
|
| 58:13 | . How do you remove the inactivation ? The only way to do it |
|
| 58:17 | by hyper polarizing the South to its number and potential. Once you do |
|
| 58:24 | , then the inactivation gave swings back this position called Dean Activation and activation |
|
| 58:33 | close shut. Now you're in for same as one. Now you're ready |
|
| 58:39 | get another D polarizing stimulus for the Channel to transit. We open and |
|
| 58:44 | , generating very fast action potential. cannot go 1 to 1. You |
|
| 58:48 | go. 1231 You have to 123 Closed again. Open inactivated. |
|
| 58:57 | activated. Closed. Okay, this the dynamics. And this is the |
|
| 59:03 | reason why sodium ions never reach equilibrium for sodium. During the rising phase |
|
| 59:10 | the action potential, the driving force number one. We discussed decreases for |
|
| 59:16 | mile. The driving force of polarized potentials increases for potassium instead. |
|
| 59:22 | second thing that happens is the channel , the inactivation of the sodium channel |
|
| 59:28 | does not allow for the member and to be driven all the way to |
|
| 59:32 | equilibrium potential for sodium this inactivation of sodium channel dynamics that come into the |
|
| 59:40 | . Therefore, you also should relate to the refractory period's that if you |
|
| 59:46 | hyper polarized the South a certain you do not Dean. Activate these |
|
| 59:51 | and close them. Therefore, you generate another action to control, and |
|
| 59:56 | why you cannot generate another action potential like absolute refractory period until you reach |
|
| 60:01 | certain level of hyper polarization and you into the relatively factory period in which |
|
| 60:08 | ion channels for sodium are now closed closing. Some of them are |
|
| 60:14 | Most of them will be closed when tell fully re polarizes, but now |
|
| 60:18 | open to generating another action potential during relatives. Refractory period. So this |
|
| 60:27 | a diagram that shows patch clamp recording you can actually patch onto the pieces |
|
| 60:32 | the plasma membrane, and you will sodium channels and you can withdraw pieces |
|
| 60:38 | the plasma membrane, and this is you can pass the current. |
|
| 60:42 | remember, the pie pads can now the current and record the current. |
|
| 60:45 | air very fast electrical pie pads, glass pipettes filled with a solution and |
|
| 60:51 | electorate sitting inside of them that's communicating of the electrical information. So you |
|
| 60:57 | to have these very fast recordings, you can withdraw black remembering, and |
|
| 61:01 | can produce the current. You polarize membrane and you can record individual channel |
|
| 61:07 | so you can actually record individual molecular opening and closing and how the ions |
|
| 61:16 | flow through that individual channel. So different techniques that we use in modern |
|
| 61:29 | on those online here. They're all referred to this patch clamp technique. |
|
| 61:35 | again goes back to the voltage Is your clamping onto something mostly of |
|
| 61:41 | the member and potential of the cell of potential? If you have this |
|
| 61:48 | than these Electra's air, actually, showed you in the lab. They're |
|
| 61:53 | underneath the microscopes or targeting these sort little cell sick collective connected to the |
|
| 61:59 | and from amplifiers. You have wires off and you actually have a suction |
|
| 62:05 | coming off from the amplifier that's connected this electrode. And as you visualize |
|
| 62:10 | bring this electrode close. Thio the . You actually suction the sound of |
|
| 62:18 | . This is why it's called Mile . So you wonder how you get |
|
| 62:21 | section through your mouth or through the . You actually pulled slightly through this |
|
| 62:27 | . What you do, you suction the plasma number. Once you have |
|
| 62:33 | electrode attached, you can perform what called cell attached recordings. So one |
|
| 62:38 | of the recording, which forms a contact between the pipe that and the |
|
| 62:42 | , and you can pick up cell recording currents from that cell Now, |
|
| 62:49 | you go by the labs electrophysiology you'll see guys sitting there. If |
|
| 62:54 | actively patching south what we call sitting these wires with this pipette wires actually |
|
| 63:02 | something like more like we call it kiss. So strong false of suction |
|
| 63:08 | actually break the plasma membrane. And you have the complete access to |
|
| 63:14 | The cytoplasm becomes continuous. Yeah, cytoplasm and the cell attacks. Recording |
|
| 63:18 | not continues, but in the whole recording the side of plasmas continuous with |
|
| 63:24 | intracellular electorate recording solution. And so of the currents passing in and out |
|
| 63:29 | the South will deal picked up by whole cell recording recording what the whole |
|
| 63:34 | is doing and as far as the passing through plasma membrane. Now, |
|
| 63:40 | you attach and you have the cell recording instead of strong suction, you |
|
| 63:44 | actually withdraw your electrode. And it's . All of this is science, |
|
| 63:52 | and lock and persistence. Remember that get to these electric physiological and feature |
|
| 63:58 | that may take you a couple hours three hours to set up the solutions |
|
| 64:02 | set up the tissues to perform a on the animal toe. Isolate brain |
|
| 64:07 | to put it under a microscope on know me the beginning, and it's |
|
| 64:14 | 12 PM it's almost one now. still have to do the experiment, |
|
| 64:19 | it takes me our to get a pash, maybe half a. |
|
| 64:24 | if I'm lucky, I may lose sell because somebody walks in the lab |
|
| 64:29 | the equipment vibrates and you lose the . So it's very, very, |
|
| 64:35 | precise work. Very dexterous work. , you're keeping track constantly of different |
|
| 64:42 | . They're happening around you, and not to discourage you, but to |
|
| 64:45 | you about electrophysiology. That's what something in your books. If you withdraw |
|
| 64:50 | plasma membrane now you have what is inside out. Recording. You are |
|
| 64:56 | the cytoplasmic domain of this channel Prodi the extra cellular environment. You think |
|
| 65:01 | it. It's always being exposed to environment. Now is all of a |
|
| 65:05 | extra sally environment. Why would you that? Because once you're inside the |
|
| 65:10 | , you don't really know how things different chemicals affect this channel. But |
|
| 65:15 | you have a controlled environment. You pretend you're the cell the side of |
|
| 65:20 | in your excess, Ellis was. can change things in a controlled |
|
| 65:24 | ions and chemicals, and observe how conducted through this channel how the currents |
|
| 65:29 | this channel are affected by it. , if you do both the kiss |
|
| 65:36 | the withdrawal of the electron, what happen if you're lucky, you may |
|
| 65:40 | on outside out recording. In this , you break the plasma membrane on |
|
| 65:45 | inside of the electrode and you withdraw so the pieces break off from the |
|
| 65:52 | membrane on the outside here of the membrane surrounding this piece of interest for |
|
| 65:59 | . What happens? The plasma member Rian eels and reforms the plasma |
|
| 66:03 | Remember that it's fossil Olympic bile So if you put these fossil Olympic |
|
| 66:08 | together, they form the walls that the mycelium. They can really Neil |
|
| 66:13 | they can reform, and in this now you have an experimental set up |
|
| 66:18 | which the extra cellular or the outside of the channel is exposed to the |
|
| 66:24 | cellular solution. So you have an out recording versus the inside out |
|
| 66:31 | It's very important. It's very important pharmacology because you may want to see |
|
| 66:36 | certain chemicals that can pass through plasma , how they affect the cytoplasmic |
|
| 66:44 | how they affect the flow of current cytoplasmic domain. Likewise, if you |
|
| 66:51 | some chemicals, you don't know if pastor plasma member or not, you |
|
| 66:56 | precisely address what effect they have on channel. If they buy on the |
|
| 67:00 | cellular domain side of this channel, that there are very different binding size |
|
| 67:05 | different chemicals and toxins and molecules on channels, because each one of these |
|
| 67:12 | it's a very complex, three dimensional structure comprised off, you know, |
|
| 67:19 | . And so it's very important for to know we're different molecules bind and |
|
| 67:24 | binding of these molecules may affect the or the current flow through these |
|
| 67:30 | It would be employing all of these to understand electrical activity across plasma membrane |
|
| 67:37 | the whole cell or across individual So this segment is one of my |
|
| 67:43 | , actually, because I talked about I called mouthwatering tails off toxins, |
|
| 67:49 | we're gonna talk about the fact that so many toxins in nature and then |
|
| 67:56 | for the most part try to avoid toxins in nature. We know to |
|
| 68:01 | out for poisonous plans for poisonous animals , uh, frogs that have poison |
|
| 68:11 | snakes that have venom for fish that stab you and so on. It's |
|
| 68:18 | toxins. Set it out in And then there's a certain contingent of |
|
| 68:23 | that are thrill seekers and constantly seek little bit of excitement from what do |
|
| 68:29 | toxins feel like? And so I'm share with the video that talks |
|
| 68:37 | you know, watching episode of Simpsons and the disclaimer and I get to |
|
| 68:41 | classes that they're so funny unless our after everybody. So if your offensive |
|
| 68:48 | should be equally offensive to everybody, like Simpsons are. And so the |
|
| 68:53 | is not to offend anyone here, we can all handle this is an |
|
| 68:58 | . But to watch this funny this funny episode should remind you half |
|
| 69:04 | the lecture today. Easy, bankable , If you just cut and probably |
|
| 69:15 | , yes, he just but in yes. Yes, it is |
|
| 69:24 | potentially fatal. But if slice properly be quite misty, I must get |
|
| 69:29 | master. Uh huh. smokes. ? Mr You are needing Indication E |
|
| 69:42 | colorfully dart master, we need your hands by skilled hands are busy. |
|
| 69:48 | do it. Uh, okay, me fill you in a little bit |
|
| 69:58 | saying that, uh, Simpson's go sushi restaurant. And he's so hungry |
|
| 70:05 | ate everything and he identifies Fukuda puffer or blow fish on the menu. |
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| 70:15 | this is where it survived. Concentrate that Yeah. Fuku testing. |
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| 70:38 | back to me. Oh, beautiful , Liberty. Oh, God, |
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| 70:47 | e j. Simpson says I shall , But we have reason to believe |
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| 70:53 | have eaten e o. Tell me . No need to panic. There's |
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| 71:00 | map to the hospital on the back the menu. I don't take me |
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| 71:07 | . Well, you, Homer, never heard of a poison pork |
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| 71:11 | Do I have agreed that I should this to you? Tony? |
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| 71:15 | I can read Mars like a Uh huh. It's good news, |
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| 71:21 | it? No, Mr Simpson, in fact, you have consumed the |
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| 71:24 | of the Blowfish and from what the has told me, it's quite |
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| 71:28 | You have 24 hours to live Well, 22. I'm sorry I |
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| 71:34 | you waiting so long, March. gonna die. I'm gonna die. |
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| 71:40 | , if there's one consolation is that feel no pain at all until sometime |
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| 71:45 | evening when your heart suddenly explodes. , a little death anxiety is |
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| 71:50 | You can expect to go through five . The first is denial. No |
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| 71:54 | . Because I'm not dying. Second angered by U E O for |
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| 71:59 | Yeah. What's up? Your You to get me out of this. |
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| 72:03 | make it worth your while. Acceptance. Well, you're going to |
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| 72:08 | jump kind, Mr Soups. And progress astounds me. I should leave |
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| 72:11 | two alone. Perhaps this pamphlet will helpful. So last week? |
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| 72:33 | So to do this, you have go through special training because food official |
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| 72:40 | fish contains a toxic. And the that prepare these very specific slices of |
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| 72:49 | they have to go through, I about seven years of training. So |
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| 72:55 | have toe remove certain parts of the . Is there a dissecting yet? |
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| 73:04 | has to be done very carefully. ? So, again, the take |
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| 73:14 | |
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