| 00:02 | This is lecture 11 of Neuroscience. we use the acetylcholine as a sort |
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| 00:06 | a canonical example, not only at neuromuscular junction because it's easy to |
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| 00:11 | Remember, there's only one subtype of P Cylco receptor, neuromuscular junction and |
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| 00:17 | nicotinic. So that's why it's simple understand it when we come to the |
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| 00:20 | . Now, there are two types receptors on the CNS neurons. And |
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| 00:24 | nicotinic which is ionotropic allows for the of ions. It's a receptor |
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| 00:29 | And that one is muscarinic which is and it activates g protein complex that |
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| 00:35 | attached to that uh protein in the . And so we talked about how |
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| 00:41 | code will get released. We also about how a lot of molecules will |
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| 00:44 | to the reso and then they will with time. So they're reversible. |
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| 00:48 | this case, agonous cylco will open receptor channel or activate uh muscarinic |
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| 00:55 | So the agonist and a lot of will get broken down in the synaptic |
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| 01:01 | . So, acetylcholine gets broken down acetyl pins and then cline gets transported |
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| 01:08 | this presynaptic cline transporter back into the terminal where with the help of |
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| 01:15 | it gets synthesized into acetyl codeine and into the vesicles again. And you |
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| 01:21 | see this is a common theme. gets released. Glutamate doesn't get broken |
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| 01:26 | in the synapse, but it gets back. Presyn ically, Gaba gets |
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| 01:31 | . Gaba gets transported back pre And you'll also see that GLIA has |
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| 01:36 | important role to play there in regulating amount of glutamate Gaba. Then we |
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| 01:43 | acetylcholine and the light off uh botulinum , botulinum toxin will target this protein |
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| 01:51 | complex effusion and therefore will inhibit acetylcholine release. We also discussed other toxins |
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| 01:59 | can affect the all acetylcholine signaling from synaptic, from presynaptic release to poynt |
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| 02:09 | from the spiders and the snakes. then we talked about nerve gasses, |
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| 02:16 | gasses that would act uh as acetose as well as acetyl cholinesterase inhibitors with |
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| 02:25 | that serve as medications for Alzheimer's disease slow down the progression of Alzheimer's |
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| 02:33 | Uh Then we mentioned that there are receptors and we'll talk with a lot |
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| 02:39 | possy receptors. But there are also receptors in the, in, in |
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| 02:44 | instances, there are auto receptors, means that if it is a gaba |
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| 02:49 | synapse, you'll have posy the Gaba , but you will also have auto |
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| 02:56 | on that same external terminal that will binding to gaba and influencing vesicular |
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| 03:03 | So it's sort of like a feedback that's pretty common and inhibitor an excitatory |
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| 03:10 | . And in this influx of presynaptic influx of calcium is necessary for |
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| 03:17 | release. So, if you block of calcium, there is no protein |
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| 03:22 | complex fusion between vesicle and the number there is no vesicular release. So |
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| 03:28 | important mechanism. Adenosine actually blocks release glutamate by blocking influx of capsules. |
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| 03:35 | remember we mentioned some interesting neurotransmitters A and the core of it adenosine amongst |
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| 03:42 | of those interesting neurotransmitters. And so through its own receptor will target the |
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| 03:49 | calcium channel voltage gated calcium channel. there's multiple ways by which you can |
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| 03:55 | the same receptor or the same channel the membrane and achieve the same effect |
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| 04:02 | gaba release. For example, when talk about neuromodulatory or metabotropic functions, |
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| 04:09 | are talking about activation of G protein can either influence a receptor uh which |
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| 04:14 | be a channel also ion channel nearby a secondary messenger cascade downstream. We |
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| 04:23 | understand that there's a lot of excitatory inputs. There are a lot of |
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| 04:28 | synaptic inputs and neurons within milliseconds have integrate that information and decide whether they're |
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| 04:34 | enough to generate their own action potential pass that information on to the interconnected |
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| 04:41 | and networks. So there are strategies which neurons basically assure and neuronal communication |
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| 04:50 | that despite the fact that single synapse is a really small epsp in excitatory |
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| 04:57 | . For example, there's strategies and circuits and neural networks. There are |
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| 05:03 | by which that depolarization can be double increased 1020 times to assure that in |
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| 05:11 | instances. And in particular, when is strong enough stimulus or repetitive stimulus |
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| 05:17 | the cell responds to it. And of those strategies is spatial summation. |
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| 05:23 | of the excitatory synapsis will be projecting these distal apical dendrites. And this |
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| 05:30 | cell here and these excitatory inputs will to overcome a lot of inhibitory inputs |
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| 05:35 | will be quite commonly targeting what we the paras somatic regions, the regions |
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| 05:40 | the SOMA and just around the Therefore, having really strong impact because |
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| 05:46 | that the signal and all of these inhibitory inputs will eventually get integrated here |
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| 05:52 | Saloma. And the action potential is get produced here in the axon |
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| 05:59 | So that means that this region still to receive enough depolarization from these ical |
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| 06:06 | and has to overcome any hyper polarization are generated by the inhibitory inputs around |
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| 06:14 | SOMA. And so one of these is spatial summation where you'll have multiple |
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| 06:20 | . And we talked about how neurons have tens of thousands of inputs, |
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| 06:24 | of thousands in some rare instances. you'll have spatial summation where a single |
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| 06:29 | will receive multiple synapses from other neurons another network. And all of these |
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| 06:35 | be activated at the same time and will be summed across space and you |
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| 06:39 | get really large depolarization, another way repetitive signaling from the same cell. |
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| 06:47 | in this case, instead of summing space, you're summing over time or |
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| 06:52 | summation. And when we look you'll have this nice summation and an |
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| 06:57 | in signal driving that number and potential and higher towards the threshold for the |
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| 07:04 | potential. So this is what you in this example with the spatial |
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| 07:24 | But notice that this is a spatial . OK. This is an example |
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| 07:30 | the middle right here, spatial But with the temporal summation, you'll |
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| 07:34 | one epsp and there's going to be delay between these action potentials right |
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| 07:42 | Therefore, this is going to start polarizing until it depolarizes again, starts |
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| 07:51 | polarizing until it depolarizes again and produces trace number two. So you can |
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| 08:01 | that in this case, it's not effective in depolarizing. The south doesn't |
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| 08:08 | a steep of the slope. Number . As number one, the steeper |
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| 08:13 | slope, the steeper is the The higher the chance you're gonna reach |
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| 08:18 | threshold for the action potential. So we talked about the action potential, |
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| 08:26 | said that the action potential once it generated uh this axon initial segment |
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| 08:37 | it will get reproduced at each node Ranvier until it reaches external terminal and |
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| 08:46 | reproduced there in the same amplitude. that's not the case with dendrites. |
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| 08:52 | are not insulated. They don't have Lindros wrapped around them, they don't |
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| 08:58 | myelin segments around them. So this special for axons. That means that |
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| 09:04 | way that this signal gets preserved in Axon and gets insulated and protected by |
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| 09:11 | myelin right here that does not exist dendrite. Therefore, dendrites are leaky |
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| 09:20 | , instead of being insulated cables, leaky cables. And by that, |
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| 09:25 | means that if you produce a depolarization you can hear, imagine an electrode |
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| 09:31 | you can imagine a very strong synaptic onto this dendrite. And if you |
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| 09:37 | a recording electrode right next to that strong input, very strong stimulus, |
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| 09:42 | record a very large depolarization in the and potential. But if you place |
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| 09:48 | electrode just a small distance away, say five micrometers away from this first |
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| 09:53 | , 10 micrometers away. You'll notice the same depolarization now is much |
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| 09:59 | And that's because of the lack of insulation, only a portion of that |
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| 10:04 | that gets generated at the source then its terminal destination along the way it's |
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| 10:16 | . And that decay from 100% value the site of injection. So this |
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| 10:23 | 100%. This is the depolarization of site of injection over distance. Depends |
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| 10:32 | how long is this distance here is by LAMBDA over distance. This signal |
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| 10:42 | into its 37% value from 100% to . And this is referred to as |
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| 10:54 | constant or dendritic length constant where VO 100% at the source right here. |
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| 11:04 | LAMBDA is the length constant. some neurons will have longer length, |
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| 11:11 | , longer length constants means that the , if it's longer Lambda, that |
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| 11:17 | that the signal will travel further before reaches its decay. So this is |
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| 11:24 | example of short, well, I'm OK, from 100% to 37%. |
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| 11:35 | this is an example of long lamb here, this one. And so |
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| 11:42 | can see that this distance in the and the second uh iteration here it's |
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| 11:53 | longer. So this this dendritic length is much longer and different cells with |
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| 12:02 | dendrites will have different length constants. obviously, the longer is the length |
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| 12:10 | . The higher is the probability that signal will travel longer distance. |
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| 12:15 | the higher probability that neuron will still depolarized with the salmon axon to produce |
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| 12:21 | action potential. So, in dendrites are very elaborate structures that contribute |
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| 12:28 | more complex integrated properties. Why? it's not just one straight cable, |
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| 12:34 | branching, there's uh complex rules. this is a simple way of kind |
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| 12:40 | trying to understand um the communication between and integration of that signal. Many |
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| 12:47 | will have voltage gated sodium calcium and channels. They can act as amplifiers |
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| 12:52 | pho synaptic potentials, meaning that neuron we talked about has a strategy. |
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| 12:59 | it will place a lot of voltage sodium and potassium channels and nodes of |
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| 13:04 | axon initial lock. So you will a lot of NAV and KVS here |
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| 13:11 | here and here, here, you see a lot of calcium vs because |
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| 13:17 | have influx of calcium to cause neurotransmitter . And that the dendrites, if |
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| 13:24 | want to make sure that that signal the dendrite gets transported into the |
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| 13:31 | maybe you are actually gonna try to the densities of certain channels distally. |
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| 13:41 | that that signal, it gets amplified gets still transmitted down the the dirty |
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| 13:48 | . So dendritic sodium channels and some may carry electrical signals in the opposite |
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| 13:53 | from SOMA outward along dendrites. Remember that's called the back propagating action |
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| 14:00 | OK. So you have, you signals again, that will act as |
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| 14:06 | carrying information from distal dendrites into the . And this back propagating action |
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| 14:13 | small depolarization will be summing and contributing interacting with these inputs that are coming |
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| 14:21 | from the pre synoptic neurons. you have this epsp that's large by |
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| 14:29 | time it uh reaches the neuron, epsb, by the time it reaches |
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| 14:36 | SOMA, it's much smaller in And what happens if along the way |
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| 14:42 | this depolarization is traveling, trying to the SOMA, what if along the |
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| 14:48 | there's an inhibitory synapse and it is located closer to the SOMA. |
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| 14:54 | it has a really strong impact and gets activated and all of a sudden |
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| 15:00 | you're recording the signal at the level the SOMA, there's nothing. So |
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| 15:06 | means that this excitatory input was completely by this inhibitory input. And for |
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| 15:15 | Selma, the SOMA is like nothing , right? No, actually two |
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| 15:21 | happened in excitatory input and in inventory , the SOMA integrates and summ eights |
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| 15:27 | and says, OK, it's nothing have no depolarization because excitatory input got |
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| 15:34 | and it also got shunted out because you open up certain channels, you |
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| 15:39 | up and you have a condition there signal, sanatory signal becomes weaker, |
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| 15:46 | gets shunted out and you have no , no depolarization in the SOMA, |
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| 15:52 | depolarization in the AX on here. you have to have either a lot |
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| 15:56 | these inputs that overcome inhibitory inputs or inputs, active at certain times and |
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| 16:04 | inputs active at certain times so that is an interplay a long time and |
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| 16:09 | long space. Um But all of things uh are pretty complicated. |
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| 16:15 | this is just a simple example where we put it in the reality, |
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| 16:19 | looking at thousands of synapses uh being along different branches of these dendrites, |
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| 16:26 | their own length constants in the way different summations. Uh And sometimes you |
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| 16:32 | track similarities in these length constants and properties that cause different subtypes of |
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| 16:37 | So it will be similar and the subtypes of cells when we say modulation |
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| 16:44 | modulatory effects. And even the lecture the uh diffuse modulatory systems is because |
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| 16:51 | from acetylcholine, which can happen, CNS and nicotinic uh acetylcholine receptor, |
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| 16:57 | is ionotropic. All of the other that we're gonna be talking about, |
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| 17:02 | means they are acting in metabotropic And that's why a lot of times |
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| 17:08 | to as modulatory, they're slower in either membrane physiology or cellular communication on |
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| 17:17 | . And this is an example of beta receptor come back and talk about |
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| 17:23 | . This is one of the examples which can have activation of G protein |
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| 17:28 | will then activate it in a cyc . Convert a GP into cyclic K |
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| 17:34 | . That cyclic K MP can interact protein kinase. Kinas will phosphorylation |
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| 17:40 | That means they will contribute po four of kina phosphorylation that add po four |
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| 17:46 | the molecule is called phosphatases. They dephosphorylation or take the po four |
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| 17:52 | And you can see that it is to the potassium channel. And so |
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| 17:56 | of burn kin sand pho correlation opens this channel. So in the |
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| 18:03 | you can change the membrane potential. you can imagine that this process is |
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| 18:08 | to be much slower than just a binding to the channel and opening to |
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| 18:13 | receptor and opening the channel. So process is slower. It's also modulatory |
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| 18:19 | if you correlate the channel, it have a longer effect. A lot |
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| 18:23 | times than a quickly opening of the closing of the channel. Also, |
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| 18:29 | you influence the secondary messenger cascades inside south, this could have an impact |
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| 18:36 | even on the transcription factors and uh all the way um at the level |
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| 18:41 | the nucleus. Ok. So what we have? We have chemical synaptic |
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| 18:47 | , which we stressed a lot. we also talked about electrical synaptic |
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| 18:52 | right? The gap junctions and the of the gap junctions is rich diversity |
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| 18:58 | these synaptic transmission interactions on dendrites and and ss. And it allows for |
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| 19:08 | uh calculations in the brain and complex . A lot of what we're talking |
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| 19:14 | in synaptic transmission. When we talk neuropharmacology and already we discussed Botox and |
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| 19:21 | discussed Botox for Beauty Botox for migraines approved treatment. By the way, |
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| 19:27 | not endorsing Botox. And when I you those commercials, I have no |
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| 19:31 | with any of these companies. They're examples of something that you can see |
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| 19:36 | day on, on television, for . So um it explained drug |
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| 19:45 | So for example, how Alzheimer's medication , it blocks cholinesterase, right? |
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| 19:51 | a cholinesterase inhibitor explains how it We already started talking about how defective |
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| 19:58 | transmission is associated with specific neurological So, Alzheimer's acetylcholine dopamine Parkinson's disease |
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| 20:10 | it's really key to understanding your old of not only communication but also learning |
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| 20:16 | memory and why is because the stronger the communication between the Synopsis. The |
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| 20:24 | become larger, they have more they have stronger communication. They change |
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| 20:30 | . The structure changes, the presynaptic zones increase postsynaptic densities, increase the |
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| 20:36 | spines increase. It's all a process plasticity and this is the same process |
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| 20:42 | a cellular basis for learning and So when you're learning new concepts, |
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| 20:48 | lot of times it's associated learning or memory that you're using. So you're |
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| 20:55 | at something you're listening to something you're something down that involves 345, maybe |
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| 21:01 | more different tasks. And that's really best way to recall that information |
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| 21:06 | to learn that information. And as doing that, you're reshaping your |
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| 21:11 | if you're changing the synaptic transmission and changing plasticity in these synopsis as |
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| 21:20 | All right. So this leads us the last slide and we now venture |
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| 21:26 | into more, not venture back but into more neural transmission. And in |
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| 21:32 | case, just a reminder that amino neurotransmitters will be broadly expressed by billions |
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| 21:41 | neurons in the vortex of vertically with spinal cord. And when we talk |
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| 21:47 | amines, those are confined, there's limited number of cells in the specific |
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| 21:54 | that are responsible for producing all of given amine such as acetyl or dopamine |
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| 22:01 | the entire brain for the entire And then peptides. The extent of |
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| 22:09 | five discussion is remember that peptides and can be co expressed, they can |
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| 22:15 | core released. But there are different and the peptides are stored in these |
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| 22:21 | four vesicles or secret Granules. They dense four. Because if you look |
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| 22:25 | them through electron microscope, you cannot through them, they look very |
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| 22:32 | Ok. So three criteria for neurotransmitter and storage and presynaptic neuron released by |
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| 22:40 | optic neuron. It has to have postsynaptic effect. When you isolate that |
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| 22:49 | and apply it on the postsynaptic it should have the same release as |
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| 22:54 | the fact is if you stimulate the neuron. So it's uh called mimicry |
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| 22:59 | . Mimicry. On pre synaptic you have synthesizing enzymes. You have |
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| 23:09 | classical transporters. You have reuptake transporters reuptake the molecules degradated enzymes in the |
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| 23:16 | cleft or in the cell transmitter gated channels that are located by synical. |
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| 23:23 | are ionotropic G protein coupled receptors that linked to G proteins and G protein |
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| 23:29 | ion channel activation. These are all that are also activating secondary messenger |
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| 23:37 | Uh And so each chemical will have own synthesis, release machinery degradation reuptake |
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| 23:47 | par synoptic effect and also cellular effect the level of the cellular secondary |
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| 23:54 | Par synoptic CNS contains a diverse mixture synopsis that use different neurotransmitters. And |
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| 24:03 | often when we study neurotransmission, we brain slices. So brain slice is |
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| 24:10 | model to study neurotransmission, the same slice, it can be a uh |
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| 24:15 | a fish brain slice. It can a rodent brain slice. And these |
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| 24:23 | are in vitro experiments, right? vitro is something is in the |
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| 24:28 | In this case, the slice is the dish, it's in the on |
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| 24:31 | microscope, it's being folded. It's a part of the brain because it |
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| 24:35 | very similar environment that it would have the brain. But it's in vitro |
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| 24:41 | in the dish in divo, it's the whole animal. And so with |
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| 24:48 | a lot of uh electro physiology and electric physiology, we've been able to |
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| 24:54 | synopsis, stimulate certain circuits and certain . Collective measure release chemicals collect and |
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| 25:03 | data on what physi physiological changes. also don't forget the new methods that |
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| 25:13 | mentioned in the first section already. optogenetics, how we can control channels |
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| 25:20 | depolarization and flux of ions through light channels, channel adoption and hallow adoption |
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| 25:28 | we discussed that would be selected for cion or anion. So if we |
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| 25:36 | to visualize neurotransmitter release, we have have methods to visualize it. The |
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| 25:43 | most common methods is immuno chemistry and , immuno chemistry. You're using antibodies |
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| 25:53 | have been tagged with a visible marker these antibodies are specific to a neurotransmitter |
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| 26:00 | . And the way that you generate antibodies is you, for example, |
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| 26:05 | have a candidate neurotransmitter from a rodent you isolated. You inject it into |
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| 26:14 | rabbit. Rabbit is going to have immune reaction to this neurotransmitter. It's |
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| 26:20 | an invader. So it's going to antibodies and will have an immune |
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| 26:26 | So you will collect and draw its with the antibodies and then you will |
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| 26:32 | and purify these antibodies. And there's whole slew of antibodies that can bind |
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| 26:39 | different molecules and different proteins and they be polyclonal, monoclonal, many different |
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| 26:46 | . But in the end, you to take that antibody that you isolated |
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| 26:50 | tag it with visible marker. And you have the brain slice that I |
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| 26:54 | discussed in vitro. And let's say took the brain slice of the hippocampus |
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| 26:59 | you want to know what cells in hippocampus are excited for yourself that it's |
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| 27:06 | L. So you have L A that has a marker. Then you |
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| 27:13 | these antibodies on the entire slides, tissue and that slice will contain many |
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| 27:21 | cells here for simplification. For simplified , you only have two cells in |
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| 27:25 | slide. So you zoomed in on piece of the slice that has two |
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| 27:30 | . So what happens is you apply antibodies immunities to chemistry. You use |
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| 27:34 | whole procedure that takes typically about a and a half, two days, |
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| 27:40 | three days, you can use multiple , you can tag multiple antibodies, |
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| 27:47 | one with a different visible marker in fluorescence colors and one will be glowing |
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| 27:55 | , another one will be glowing red blue. So you can have multiple |
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| 28:02 | that you can apply. But in case, you apply one and you |
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| 28:06 | to know which one of these cells glutamine and you apply a little bit |
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| 28:12 | uh detergent typical it's triton X and detergent does what it actually punches little |
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| 28:21 | in the membranes of the cells and for the antibodies to come inside the |
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| 28:27 | . So then you'll say, then all of the cells will have |
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| 28:29 | antibody come in. So how are gonna know which one is really staining |
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| 28:34 | it? Then you're gonna do a of washes. So you're then literally |
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| 28:40 | change the fluid and the little tray contains your slides and you're gonna put |
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| 28:46 | on the shaker and the shaker is slosh it around for six hours and |
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| 28:51 | gonna come back later that evening and the fluids and put fresh fluid and |
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| 28:57 | gonna shake it overnight again. And the cell doesn't have the neurotransmitter, |
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| 29:04 | antibody that penetrated into that cell, just was, it will not stick |
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| 29:09 | there. There's nothing for it to to it, to stick to. |
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| 29:13 | the cells that in fact contain the of interest. And this is not |
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| 29:17 | the neurotransmitter. So you can uh uh uh proteins inside the cells, |
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| 29:26 | , uh the transmembrane proteins. in this case, only the cells |
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| 29:30 | will have that neurotransmitter candidate of interest still show a visible signal. The |
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| 29:38 | in the way will get trapped there they're bound up to, to a |
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| 29:42 | of interest. And here's an example you can, like I said, |
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| 29:47 | different colors and each color here could representing a different neurotransmitter chemical and use |
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| 29:54 | imagination. So maybe red is all , maybe green is gaba, maybe |
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| 30:00 | is microglia, maybe. So you , you know, do neuronal populations |
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| 30:06 | also glial cell populations. So we using this multiple antibodies. You can |
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| 30:12 | antibodies. When we talked about cell markers, we said this barometer cell |
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| 30:17 | CCK positive. How do we know we didn't mean that the chemistry or |
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| 30:22 | the hybridization and we know the number cells, you know, we applied |
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| 30:27 | for cholecystokinin and show that there are or cholecystokinin positive in pseudo hybridization. |
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| 30:33 | localize synthesis of protein or peptides to cell. You essentially detecting messenger RN |
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| 30:41 | . And what you have is you a strand of messenger RN A. |
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| 30:45 | we live in the post genomic era we can uh have these complementary sequences |
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| 30:53 | nucleic acids that you design. You it online and get it sent to |
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| 31:00 | lab. You will see sometimes advertisements some buildings, how much it costs |
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| 31:06 | so many kilo basis of something certain . But you produce a sequence that |
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| 31:12 | know will target MRN A will be to the messenger RN A and you |
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| 31:18 | what uh gene, this codes for synthetic uh the, the sequence that |
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| 31:24 | produce. And it's again like a sophisticated Velcro. In this case, |
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| 31:30 | have messenger names as complementary acids, acids that have to come and, |
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| 31:35 | bind together with the radioactively labeled probe . And once you have that radioactively |
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| 31:41 | probe, you have the sequence of RN A, it's the same principle |
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| 31:46 | it's using radioactivity. Um same principle the sense that you will apply it |
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| 31:53 | over the tissue, but only the will contain a molecule of interest will |
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| 31:57 | it up. Finally, uh it's lot of uh work to do this |
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| 32:07 | of work to define where molecules are . And you know, as the |
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| 32:11 | and pseudo hybridization, it shows you location and the types of the cells |
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| 32:16 | will will be expressing certain molecules. that's not all you cannot stop there |
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| 32:23 | you also want to know how these neurotransmitters affect different functional properties or signaling |
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| 32:30 | different neurons. And so the qualifying molecule evokes same response as neurotransmitter. |
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| 32:40 | what it means is that if I a glutamate axon and it caused depolarization |
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| 32:47 | this psyop cell, I should be to release glute on the same part |
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| 32:54 | the dendrite here from my electrode instead the synapse and I should be able |
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| 33:01 | mimic exactly and therefore record exact a similar response. Uh synaptic in the |
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| 33:09 | by applying glutamate. So there is is a bit of an issue here |
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| 33:16 | , with this with this kind of setup. And where is this happening |
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| 33:22 | ? So this is happening in vitro the brain lives. Most of them |
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| 33:27 | the greatest minds, brain lies skip light to stimulating synapse. And that |
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| 33:36 | is very specific, right? We that it has very specific probably |
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| 33:41 | the dendrite dendritic spine, maybe just single dendritic spine that it targets poop |
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| 33:48 | stimulation here. And when you release in the pipette, you have a |
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| 33:58 | amount of dialysis and diffusion. So is your presynaptic terminal here and it's |
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| 34:14 | this postsynaptic gid spine and there's other and spines and I can't draw that |
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| 34:21 | . But just in this example is selma, this is our axon. |
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| 34:30 | . And so this is very specialized right here. This is where a |
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| 34:36 | will have release will happen. So you stimulate the cell here, this |
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| 34:41 | what it's going to target a single spine, for example. Um |
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| 34:50 | what if you have this electrode First of all, the tip of |
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| 34:56 | electrode, it's gonna be fairly And from that electra you're going to |
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| 35:06 | glutamate the slice. Remember it's fitting the solution, it's being bathed with |
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| 35:15 | supers spinal fluid. So what happens this fluid? Well, it's going |
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| 35:25 | diffuse everywhere there's gonna be a higher of this fluid here. And there's |
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| 35:30 | be less of that active ingredient, there's going to be some sort of |
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| 35:34 | larger area because of the just dilution the fluids and diffusion. So you |
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| 35:41 | get as much of the spatial specificity you're doing this kind of experiment. |
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| 35:51 | in the last 10 years, there a new technique that was developed and |
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| 35:59 | technique is called caged neurotransmitters. So literally put glutamate molecules inside the |
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| 36:12 | Now, this is a dendrite, dendritic spine. This is just another |
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| 36:17 | here. OK. And these glutamate are everywhere but guess what? They're |
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| 36:33 | to the postsynaptic receptors because they're inside cage. So they're caged, |
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| 36:40 | they're chemically caged and you can make , you can uncage them into a |
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| 36:49 | . Now, you can then use beams or laser and point that laser |
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| 36:58 | very specific and fine location and release glutamate in just this one area around |
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| 37:06 | synapse and the glutamate that is located other synapses will stay caged. So |
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| 37:14 | is what we call uncaging neurotransmitters. this is a microscope setup that allows |
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| 37:23 | to do that and it allows you do that in four dimensions. So |
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| 37:29 | have the space dimension, which means you can find the las where they're |
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| 37:35 | , very fast. The lasers these are 10 per seconds. So the |
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| 37:41 | , very, very fast nanoseconds, say it and very confined spa. |
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| 37:50 | within nanoseconds, you can activate 10 would say in very specific areas. |
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| 37:58 | you have one dimension is this space second dimension, which is time. |
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| 38:06 | over time you can activate it very or you can go boom, this |
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| 38:10 | activated two milliseconds later, boom, milliseconds later boom in this area. |
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| 38:17 | is really cool because now you can we talked about, well, there's |
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| 38:21 | exciting thing to be here and there's inhibitory input, this gets canceled. |
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| 38:25 | I said it's really complicated because you so many different inputs all along these |
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| 38:30 | trees, right? This starts addressing question of complexity. What happens if |
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| 38:37 | activate 10 synopsis excited or inhibitory in small class of the den? What |
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| 38:42 | to the whole cell is you're recording in that cell. So you have |
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| 38:49 | and time, which is the third is that you can do it in |
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| 38:54 | dimensions. So lasers can penetrate deeper the tissue. Light microscope will allow |
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| 38:59 | to visualize typically 100 micrometers of the . You know, if you don't |
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| 39:05 | uh any enhanced fluorescence or anything. you're just looking through the tissue with |
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| 39:11 | microscope, it will have a penetration about 100 micrometers. You can only |
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| 39:17 | 100 micrometers when you have called focal . When you have lasers, you |
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| 39:23 | actually penetrate deeper into the tissue. now the depth in space becomes your |
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| 39:31 | diameter. So you have XYZ and . So you have three dimensions in |
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| 39:43 | and you have time and you have fast control of engaging these neurotransmitters. |
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| 39:49 | would be glutamate, it will be in very specific locations so long with |
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| 39:54 | , right, stimulating single synapse. let's talk about amino acid ne transmitters |
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| 40:04 | the rest of our time. We Bison Gava glutamate. You, you |
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| 40:10 | see glutamate is just decarboxylate version. is glutamate here that has Carboxyl group |
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| 40:18 | , I'm sorry, Gaba is just decarboxylate version of glutamate. And we'll |
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| 40:24 | about how Java molecules or I can this, I guess. So for |
|
| 40:41 | students, there is going to be article in your folders for four dimensional |
|
| 40:49 | and a couple of your quiz questions gonna come from that article. I'll |
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| 40:54 | you again over the next lecture and graduate students where to find it. |
|
| 41:01 | uh I'll tell you how much you know about that article. It's pretty |
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| 41:05 | uh and uh pretty extensive how much it you should know to be able |
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| 41:10 | answer those two or so questions. have graduate students for their quiz. |
|
| 41:16 | not for the 4315 section. we have this neuron and this neuron |
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| 41:27 | Gava, this neuron when it produces , it's inhibitory neuron. This neuron |
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| 41:41 | has to synthesize Gava as we talked , right? And this neuron that |
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| 41:49 | Gava actually synthesizes it from glutamate and does it through an NZ it is |
|
| 42:02 | Gad. Ok. So why is so inhibitory if the cell has glutamate |
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| 42:18 | the cell has gamma, why is cell inhibited? Because it only releases |
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| 42:27 | ? Oh, only has the release for Gaba and only has Gaba transporters |
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| 42:32 | those vesicles. It has only Uyama it needs glutamate to decarboxylate. So |
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| 42:43 | have the Banic acid decarboxylase to remove coo H and turn it into |
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| 42:50 | And Gabor neurons are a major source synaptic inhibition in the CNS. The |
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| 42:56 | source source of synaptic. In in the spinal cord was glycine. |
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| 43:01 | glycerin and or spinal cord into neurons also uh not only synthesize and release |
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| 43:08 | but also Gaba. But in the Gaba is dominant and glycine will serve |
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| 43:14 | a code factor to an MD. receptor activation is kind of interesting, |
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| 43:21 | slightly different function, almost excitatory function the CNS versus the spinal cord. |
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| 43:29 | . All right. So now we this neuron here and this neuron releases |
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| 43:37 | . So this neuron is excitatory, ? Therefore, it's gonna have to |
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| 43:45 | machinery for glutamate and also machinery for . OK. Gaba will get uploaded |
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| 43:59 | the vesicles here here in the excited synapse. Glutamate gets uploaded into the |
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| 44:08 | and those vesicles fuse and cause neurotransmitter ? Ok. So, where does |
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| 44:21 | come from? How does, how Gaba get back in once Gaba gets |
|
| 44:27 | ? How does it get back in the pre synoptic terminal. It has |
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| 44:34 | . So it will have gabber transporters it will take those ga molecules and |
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| 44:41 | will transport them here and those ga will have transporters and they will get |
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| 44:47 | into the vesicles. So you have neuronal gabba transporter, we reuptake |
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| 44:55 | then you have transported, reload it that support. What about glutamate glutamate |
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| 45:05 | glutamate gets released? Same thing you transporters, glutamate that will bring glutamate |
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| 45:15 | and that glutamate will get uploaded into vessel. This is better than the |
|
| 45:28 | , aren't you? If you take now? Ok. You know what |
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| 45:43 | sides do? Exercise will take glu and has its own transporters for glutamate |
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| 45:55 | will slurp it up and then it convert glutamate into glutamine and we'll release |
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| 46:06 | and that glutamine will go into neurons we'll get synthesized into glutamate and upload |
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| 46:14 | here. It's already taking back Um The uh gate. Why does |
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| 46:21 | need to break it down again and it back in? I don't know |
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| 46:25 | how it's been made. And so has specific transporters for glutamate, but |
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| 46:33 | not the end of the storm. what about Gaba? Let's make this |
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| 46:40 | a little bit funky looking. It has transporters for. Yeah, but |
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| 46:53 | happens to Gaba? Yeah, gets into glutamine. Glutamine gets converted, |
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| 47:07 | given to excitatory cells. It's converted glutamate, glutamine, ostracized. |
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| 47:20 | Glutamine to inhibitory cells and they like and then they de carboxyl it and |
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| 47:32 | they make gabba and then they release . So there's gabba transporters and Gaba |
|
| 47:40 | , there's glutamate transporters and glutamate But Astros will have both. They |
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| 47:45 | glutamate transporters and suction it up. that's why it's tripartite synapse. It |
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| 47:51 | gabba transporters will suck it up, put it through its own cycle of |
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| 47:56 | cycle. And again, there's no like why didn't it just give Gaba |
|
| 48:02 | in some other form? But that the, the, it just does |
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| 48:07 | through its own cycle and then gives so that there will be glutamine transporters |
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| 48:13 | there's gonna be glutamine transporters here to for it to come in. |
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| 48:20 | glutamine becomes the basis for glutamate and cells and they have the transporters for |
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| 48:27 | . Therefore, release machinery for glutamate becomes glutamate and then with gap converted |
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| 48:33 | Gaba and this cell has the transporters the machinery for Gaba. Therefore, |
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| 48:38 | is going to be released from these . So if you uh maybe we'll |
|
| 48:45 | to your question. But let's say if I wanted to apply uh glutamate |
|
| 48:52 | , does that mean that inhibitor and cells would stand for it? |
|
| 48:56 | So if you wanna stain for inhibitory , you have to stand for |
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| 49:00 | they have to stand for the synthesizing or for, in this case, |
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| 49:05 | decarboxylate enzyme is that your question or was wondering if the inhibitory cells take |
|
| 49:13 | glutamate or just glutamine, just And it's a different transporter from the |
|
| 49:20 | . One, there is a complicated that I'm actually gonna upload. That |
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| 49:27 | of represents what I talked about. is actually a simplified version of the |
|
| 49:32 | . So yeah, inside itself was that it, it goes away. |
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| 49:44 | that's why we have the transport of it back in to convert it |
|
| 49:47 | to or it brings glutamine and it , yeah, it makes Gluta maids |
|
| 49:57 | then with Gad makes Gaba. So glutamine is really the the the |
|
| 50:07 | , it's really precursor to both. an immediate precursor to glutamate and down |
|
| 50:12 | stream is a precursor to GA oh mean if let's say the gad is |
|
| 50:26 | ? Well, that's an interesting So if you would be like loading |
|
| 50:29 | up producing glutamate, but you would be able to uh upload it into |
|
| 50:34 | probably because that vesicular transporter will be . So it may be sitting there |
|
| 50:41 | accumulating, not doing much. And probably a neurological disorder at that |
|
| 50:47 | Yeah, this this this is constantly . The more glutamate released, the |
|
| 50:53 | glia has to suck it up and mediate it. Guess what happens if |
|
| 50:58 | impair glutamate transporter here in glia. , there's too much glutamate. |
|
| 51:03 | neurons become hyper excitable. So some the epilepsies and hyper excitability in the |
|
| 51:10 | could be glued to due to glutamate , dysfunctions, migraines could be due |
|
| 51:15 | glutamate transport dysfunctions because there's increased levels glutamate. Then. So like I |
|
| 51:23 | , if you can draw this do is your attempt, I'll post up |
|
| 51:28 | diagram later this week, glutamate will uh ionotropic and metabotropic receptors. Uh |
|
| 51:38 | see that some of these isotropic receptors sensitive to uh chemicals and others are |
|
| 51:45 | to chemicals and voltage. The regulated of large currents. The three subtypes |
|
| 51:53 | glutamate on the topic receptors is ample MD A and Kate receptors. A |
|
| 51:59 | of times Kate and Apple will be together and called non an MD A |
|
| 52:05 | an MD A. And that's because the kinetics of the properties of these |
|
| 52:09 | that are different than MD A versus and MD A receptors. So they |
|
| 52:14 | have their respective agonist and antagonist agonist apple and then the agonous is nm |
|
| 52:24 | the antagonist is CAQX and A P . And that will come up in |
|
| 52:29 | section and also will come up in uh third section of the course when |
|
| 52:33 | talk about glutamate transmission and the barrel . So when you have the release |
|
| 52:43 | glutamate, originally, I only told part of the story, I said |
|
| 52:48 | it will bind to the receptor and will conduct sodium inside the cells. |
|
| 52:54 | these two colors, the blue color amper receptor and the beige color is |
|
| 53:03 | MD A receptor. And when it that when glutamate is released, glutamate |
|
| 53:10 | bind into PI and an MD A . And EPSB is a reflection of |
|
| 53:19 | fluxes and opening of both of these . A and an Indian notice that |
|
| 53:27 | the MD A receptors are going to for influx of sodium but also influx |
|
| 53:33 | calcium is more calcium analy in the . And in fact, the MB |
|
| 53:37 | receptors are pretty significant source of calcium inside the cells. That's important because |
|
| 53:45 | the cell, calcium plays many different to influence the synaptic transmission also serves |
|
| 53:51 | a secondary messenger. It may not as much to changes in number and |
|
| 53:57 | depolarization versus hyper polarization. But to uh cellular physiology, influx of calcium |
|
| 54:05 | very important only certain ample receptors. , it's shown that ample receptors allow |
|
| 54:11 | influx of sodium and then e flux potassium only some ample receptors will allow |
|
| 54:18 | calcium to flux in through ample But all an MD receptors will be |
|
| 54:23 | significant source of calcium. You can that these receptor channels conduct ions inside |
|
| 54:30 | outside. So inside sodium and calcium rush in and then potassium will rush |
|
| 54:37 | and therefore the depolarizing phase of this . Remember these are graded potentials, |
|
| 54:43 | is not the action potential, but is the rising or depolarizing slope of |
|
| 54:48 | EPSP is going to be reflected by of mostly sodium through ampon and MD |
|
| 54:55 | receptors. And the rep polarizing portion this EPSP is the subsequent elu of |
|
| 55:05 | from inside of the cell to outside the cell causing this repolarization. Similarly |
|
| 55:11 | how it was acting in the action in the sense of rep polarizing the |
|
| 55:15 | back the resting membrane potential. how are they different? They're different |
|
| 55:22 | , in the following manner. First all, when E TSB gets |
|
| 55:30 | we'll talk about two components of the . This is what we call the |
|
| 55:36 | component of the Epsp. And this delayed component of sp this is delayed |
|
| 55:45 | , including the depolarization here. And is the early component which typically has |
|
| 55:51 | do with the rising depolarization here. interesting thing is as soon as glutamate |
|
| 55:59 | released, it will bind to ample and open ample receptors. So, |
|
| 56:05 | receptors are primarily responsible for the initial the early phase of this PPSB. |
|
| 56:19 | don't an MD A receptors open immediately like ample receptors. Glutamate has been |
|
| 56:25 | , glutamate bound to ample and an A receptor except that an MD A |
|
| 56:31 | is plugged up with magnesium. It a couple of binding sites for magnesium |
|
| 56:38 | are literally blocks channel. The magnesium sitting like a plug inside this |
|
| 56:45 | And when L A bonds to that channel, it's not enough to cause |
|
| 56:52 | of the confirmational change on the MD to open it. So what has |
|
| 56:58 | happen is that we have to, we call alleviate the magnesium or remove |
|
| 57:04 | magnesium block and to remove the magnesium , it has to be, as |
|
| 57:09 | shown up the top depolarization from close the R number potential minus 65 to |
|
| 57:16 | higher depolarizing value minus 55 minus 50 40. That is necessary for the |
|
| 57:25 | to be kicked out of an MD receptor channel. So that's the reason |
|
| 57:33 | an MD A receptors are responsible for late phase of the Epsp. They |
|
| 57:42 | voltage dependent and wide. So you to have glutamate release, we said |
|
| 57:48 | the glutamate release. And you have have this depolarization, visual decolonization which |
|
| 57:54 | voltage. So they are li and depending where does this initial decolonization come |
|
| 58:00 | ? And the receptors being opened as as glutamate gets released, ample receptors |
|
| 58:07 | open. They cause this initial depolarization , the rise and that opens up |
|
| 58:15 | channel kicks out the block with depolarization causes this late phase of Epsp through |
|
| 58:23 | MD A receptor. Uh So that's reason why it's also referred to as |
|
| 58:31 | Detective. It's coincidentally detecting pre synaptic release and post synaptic depolarization. It |
|
| 58:43 | very important in synaptic plasticity because it the ability essentially to know if the |
|
| 58:50 | neuron is active and the postsynaptic neuron active and it's like it's a little |
|
| 58:56 | of its own that is watching what's on with empire and other channels and |
|
| 59:01 | on the membrane. Uh neurological So misspelled here, but it is |
|
| 59:09 | in synaptic plasticity. So it's very the disc coincident detector function, the |
|
| 59:14 | to bind the pre synoptic versus cross activity. It's very important for synaptic |
|
| 59:21 | , which is the basis of learning memory. The basis of changing the |
|
| 59:25 | of the synopsis, their shapes and numbers and impairments in an MD receptor |
|
| 59:31 | associated with several neurological disorders. So plays a significant function not only in |
|
| 59:38 | and memory, but in general uh h and impairments in an MD receptor |
|
| 59:46 | are associated with several neurological disorders. , you guys remember voltage cl |
|
| 59:55 | voltage clamp. What voltage clamp It allows you to clamp the potential |
|
| 60:00 | the desired value minus 60 minus 30 , 3060. And you're using voltage |
|
| 60:07 | because you want to isolate certain You're using voltage plan because you want |
|
| 60:12 | see for example, when we study potentials where the equilibrium potential is, |
|
| 60:18 | fact experimentally not theoretically with the NS , but experimentally where is the equilibrium |
|
| 60:25 | for potassium? Where is the equilibrium for sodium? And we saw that |
|
| 60:30 | and Huxley used voltage clamp to demonstrate exactly that to isolate these currents and |
|
| 60:38 | how they get weaker inward currents and they reverse in the opposite direction past |
|
| 60:43 | equilibrium potential value. So now we to use voltage plan and understand in |
|
| 60:51 | case, we have flux of sodium calcium and potassium. We want to |
|
| 60:58 | , first of all, where is , in this case, it's not |
|
| 61:02 | potential because equilibrium potential is for a ion. In this case, it's |
|
| 61:08 | reversal potential. Lium potential is used with the reversal potential because they said |
|
| 61:15 | once it reaches that value, the direction reverses that's for just one single |
|
| 61:22 | . Uh In this case, it's reversal potential because it's no longer one |
|
| 61:27 | ion, one single ion specific It's now sodium potassium and calcium. |
|
| 61:34 | now you want to know where the are inward currents and where the currents |
|
| 61:39 | are outward currents through this receptor use voltage clamp and this is normal |
|
| 61:47 | concentration of magnesium outside the south about millimolar. And if you release |
|
| 61:55 | so this is in the presence of glutamate has been released. You're recording |
|
| 62:00 | MD A receptor currents at minus There's no an MD A receptor currents |
|
| 62:06 | the presence of glutamate at minus 30 is some opening of an MD A |
|
| 62:13 | . It reverses at zero millivolts. equi equilibrium potential for potassium minus 80 |
|
| 62:24 | potential for sodium positive 62 lubri potential calcium positive 123. What's the reversal |
|
| 62:34 | for the zero? It falls somewhere obviously, I'm not saying that you |
|
| 62:41 | add the cou potentials and, and this is how you're gonna have |
|
| 62:46 | reversal potential for uh an MD A play into that as you can |
|
| 62:52 | So the reversal is driven both by and potassium here. Now it reverses |
|
| 62:58 | more positive potentials plus 30 plus It will conduct a lot through an |
|
| 63:03 | A receptor. You can repeat the experiment. The question is, does |
|
| 63:09 | really block an MD A receptor? , I have magnesium, I released |
|
| 63:14 | and I can't see any activation of 60 but now I removed magnesium. |
|
| 63:22 | in that in vitro slice preparation, slice instead of putting 1.2 millimolar of |
|
| 63:29 | , I put 0.0 millimolar of magnesium the solution is zero magnesium and now |
|
| 63:38 | the absence of magnesium and you still glutamate of minus 60 that an MD |
|
| 63:43 | receptor is open. So this experiment again, it still reverses at |
|
| 63:49 | But it proves that if you remove in the presence of glutamate, an |
|
| 63:55 | A receptor is going to open. definitively prove these experiments at magnesium using |
|
| 64:01 | plant and glutamate, that magnesium is blocking this an MD A receptor. |
|
| 64:08 | later was confirmed also with the binding for magnesium that are present on an |
|
| 64:13 | A receptor. OK. This is an MD, a glutamate will bind |
|
| 64:20 | and things will flux an MD A has glutamate binding site and has glycine |
|
| 64:29 | site. Remember we spoke about glycine a major inhibitor, neurotransmitter in the |
|
| 64:36 | cord. But here it is referred as code factor of the NBA |
|
| 64:44 | So, when glu A B, MD A receptor, can it activate |
|
| 64:49 | MD A receptor partially. But if binds in the presence of lying with |
|
| 64:58 | presence of this cofactor, it will the robust activation of MD A |
|
| 65:04 | So there has to be certain levels glycine uh present in the next. |
|
| 65:13 | in the synapsis, basically lying as cofactor in order to properly open an |
|
| 65:20 | A receptor. And today, we're of time, but when we come |
|
| 65:25 | , we'll talk about how an MD also has different binding site of |
|
| 65:31 | glutamate zinc and other |
|
