| 00:02 | This is uh lecture eight of We're ending talking about the optical |
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| 00:08 | And when we are talking about experimental , it allows us to get to |
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| 00:15 | scales to the resolution that is a cell resolution, even subcellular resolution, |
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| 00:23 | channel resolution manipulating single channels or tracking channel activity. Uh So from all |
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| 00:30 | way from subcellular to macroscopic levels and in between, that's what we can |
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| 00:36 | experimentally. And we talked about voltage dye imaging. If you recall and |
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| 00:43 | said that voltage sensitive dye imaging is you have to actually apply these molecules |
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| 00:52 | these molecules, these little dye squiggly will incorporate themselves into the plasma |
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| 00:58 | And as there is a flux of through these ionic channels, so as |
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| 01:03 | is depolarization or hyper polarization, the potential will track exactly with delta F |
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| 01:12 | F which we are measuring essentially uh . So it's going to be linearly |
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| 01:20 | with the changes in the membrane voltage sensitive dyes. And it does |
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| 01:25 | because as the membrane potential changes, squiggly warms will change their confirmation and |
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| 01:32 | they will change their reflective properties. instead of with with fluorescent signal. |
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| 01:37 | of let's say being blue signal, would change into processing, reflecting a |
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| 01:43 | wavelength and we would process it as red signal for active cells. So |
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| 01:49 | that extent, we watched this uh , we talked about brainwaves. And |
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| 01:55 | reason why it's important to understand that that we image once it is basically |
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| 02:06 | , it it travels, the activity through the brain tissue and spreads in |
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| 02:12 | like fashion and it spreads through specifically neuronal networks and the spread of that |
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| 02:21 | is actively sculpted by inhibition. So talked about how Gabba A and Gabba |
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| 02:26 | will try to quench the EPSB. we said there's EPSB and a lot |
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| 02:31 | times inhibition will control the amplitude and duration of that EPSB. So inhibition |
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| 02:39 | is local circuit neurons, that's what talked about throughout the course, they |
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| 02:43 | their local circuit. Neurons will try shape how much of that excitatory wave |
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| 02:49 | through the interconnected circuit. So if lose a uh uh inhibition, you |
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| 02:55 | these massive sustained uh waves of activity in this case, traveling and staying |
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| 03:03 | activating synchronizing to very high level neuronal in this case in hippocampus. So |
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| 03:15 | you go to your folder again, have another article here that I already |
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| 03:23 | and that is the DEVI or JVI technologies. Now, these are, |
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| 03:31 | are different, these are also imaging . OK. But what is different |
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| 03:38 | JV is that JV is a technique uses fluorescence and that fluorescence is tagged |
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| 03:51 | the change in fluorescence is measured in specific side voltage sensitive domain. So |
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| 04:00 | not voltage sensitive dye but voltage sensitive I, voltage sensitive dye imaging is |
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| 04:08 | VSD I. In this case, SDS, voltage sensitive domain. Remember |
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| 04:13 | have different domains on these channels. of those domains will bind liens, |
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| 04:19 | antagonist, allosteric negative positive allosteric We talked about that to review, |
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| 04:26 | example, when we talked about glutamate channel, and we looked at the |
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| 04:32 | of the glutamate channel and we talked what are the different binding domains, |
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| 04:37 | are the different channel domains and where substances would bind? How would you |
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| 04:42 | these different uh parts of the Uh We also mentioned that about Gaba |
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| 04:51 | for example, gaba bonds, you , is it on the inside cytoplasmic |
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| 04:56 | cytoplasmic domain? So there's voltage sensitive . So we can genetically now express |
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| 05:03 | fluorescent protein and that protein is gonna tagged to specific channels. So what |
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| 05:12 | get is if you were, for , to tag all of the |
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| 05:25 | if you were to tag all of cells, and this is what you |
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| 05:29 | see. It would have like the sea of fluorescence and your individual neurons |
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| 05:33 | be buried in, in noise and blow. And then this is an |
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| 05:40 | in B where you are expressing the j only a small fraction of cell |
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| 05:49 | . And so in this case, compares it to almost a Gogi |
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| 05:53 | What it does is the spar stake which goi if you remember, only |
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| 05:58 | number of neurons take up goi stain reveal an entire morphology. So that's |
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| 06:04 | you would get if you had a subset of neurons expressing them. And |
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| 06:09 | is also really good. Now, seed gets even better, it is |
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| 06:16 | targeting a specific channel that is OK. So it's confining basically the |
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| 06:28 | only to the SOMA. So the is coming from the SOMA of these |
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| 06:34 | . And that's important why? Because depolarizes produces action potentials. The processes |
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| 06:41 | get depolarized but they may also get hyper polarized. So if you image |
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| 06:47 | the entire process of the cell, may average into something that is not |
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| 06:54 | representative of the output of that what is happening at the initial |
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| 06:59 | So that's something that's important to know different ways of expressing these all the |
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| 07:04 | to the subcellular location. And that's you cannot do with just applying voltage |
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| 07:10 | dye that incorporates itself throughout and gets up by all of the cells |
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| 07:14 | So via voltage sensitive dye is more a initial stain that gets picked up |
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| 07:19 | all of the cells. And j are more specific, you can drive |
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| 07:24 | to be expressed with specific promoters specific of cells and even sometimes locations if |
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| 07:30 | really good. OK. So, there's the figure that we've discussed |
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| 07:35 | So this is where you will find figure and the difference in. Uh |
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| 07:39 | , and like I said, in the sensitive dye imaging and give |
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| 07:46 | OK. So that information is there , we're always talking about this is |
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| 07:55 | another figure. We're also talking about you can do these experiments and you |
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| 08:00 | do these experiments if animal is, example, attached and is staying in |
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| 08:07 | sort of a stereotaxic and you can the activity on the macroscopic mess copic |
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| 08:15 | maybe even a single cell level. you have one of these expressions of |
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| 08:20 | and very limited populations of cells, can also have these minos copes, |
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| 08:28 | animal would have mounted on their It's like a little minos cope and |
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| 08:34 | you can potentially have like an optical that penetrates deep into the brain. |
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| 08:41 | whenever you're imaging in this situation, still unless you're using some sort of |
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| 08:46 | three photon uh imaging technique that gets deep within the tissue. If you're |
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| 08:52 | like one photon imaging or just fluorescent microscopy imaging, you will be picking |
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| 08:57 | activity from the surface of the But if you insert an optical |
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| 09:03 | you can now pick up activity from deep areas of the brain because this |
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| 09:08 | where hippocampus is in mice. It's below the cortex. So you have |
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| 09:14 | go through the whole cortex if you to monitor activity of hippocampus and |
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| 09:19 | And if you want to compare that , which you observe in vitro in |
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| 09:24 | hippocampus slides, so that's, that's amazing uh array of capabilities and techniques |
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| 09:34 | each have their own advantage and each have their own disadvantage. We won't |
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| 09:39 | time to go through that individual. , what's really interesting is that we've |
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| 09:44 | already how imaging the brain activity or activity is related to metabolic turnover or |
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| 09:51 | active is the consumption of oxygen And that is all related to an |
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| 09:58 | blood flow because that is what is to supply those active sensors of the |
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| 10:03 | . And there's another imaging technique which also have a paper in your folder |
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| 10:08 | intrinsic optical imaging probably can expect a on each one of these techniques or |
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| 10:15 | them somehow. But intrinsic optical imaging this case, does not require any |
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| 10:21 | does not require expression, genetic uh application of any chemical on the |
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| 10:29 | . And that's significant because what it you to do, it allows you |
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| 10:33 | see a change in reflect us on surface again of the cortex. Why |
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| 10:38 | there change? Because active neurons also as active neurons swell the reflective properties |
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| 10:47 | and this shows the stride cortex and primary visual cortex revealing what we call |
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| 10:53 | dominance columns using this intrins optical imaging . Now why this is significant because |
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| 11:01 | can actually do this in the clinical . This is not just an experimental |
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| 11:07 | , you can do this in the setting. And if a person, |
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| 11:10 | example, has a seizure from an that is being targeted by surgery, |
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| 11:16 | you open up their skull, these uh activities and seizure waves can be |
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| 11:23 | enough where you would actually see these spots on the surface of the of |
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| 11:29 | cortex. If it is, that's it's occurring. And if you have |
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| 11:33 | window onto it, so that's an of this tech thing. There is |
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| 11:37 | die and you can read how it to to the blood flow in this |
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| 11:42 | . And also in the paper that attached, but you can image the |
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| 11:47 | optical signal, you can also image blood flow and it's a very good |
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| 11:51 | with increases in blood flow. These . Now, uh obviously, if |
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| 11:57 | wanna go to more of a cellular level, voltage sensitive diag you get |
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| 12:04 | are gonna be better. So for example, each neuronal population which |
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| 12:09 | in primary visual cortex, each color represents a neuronal population which performs a |
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| 12:16 | different function. In this case, processes slightly different orientation of the |
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| 12:21 | So all of the cells that are yellow will process orientation of the stimulus |
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| 12:26 | this direction. All of the cells green will process them for measurement of |
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| 12:30 | in this direction to the best So then you have to go to |
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| 12:36 | cellular level techniques. Uh And the SD I was used in the |
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| 12:43 | boulder sensitive dye imaging. But GISS well can be used to refine the |
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| 12:50 | in the somatosensory cortex of rodents. have this barrel cortex. Each barrel |
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| 12:58 | the somatosensory cortex processes activity from a whisker in the whisker pad. So |
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| 13:05 | are five rows of whiskers on rod whisker pad. And there are five |
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| 13:09 | of barrels in the primary somatosensory cortex this animal. And there's exact number |
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| 13:17 | whiskers in each row, an exact of barrels representing each whisker from each |
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| 13:23 | of these rows. It's very precise . And this is also another very |
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| 13:31 | system in which you can stimulate a whisker such as row C whisker number |
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| 13:37 | and record activity in the barrel of two on the opposite side. In |
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| 13:43 | primary SOMA of sensor cortex. It's nice system because now you can place |
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| 13:47 | optic fiber or have a mount on head of the camera stimulate the |
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| 13:53 | And if you have an animal expressing dye or have the dye applied on |
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| 14:00 | tissue by stimulating the whisker, you reveal the map or see two stimulation |
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| 14:06 | some out of sensory cortex. And can see over about 30 milliseconds, |
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| 14:10 | spreads and activates much broader areas of brain wiggle wiggle Whisker E two. |
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| 14:16 | you have a small map that grows a larger map and travels as a |
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| 14:22 | wave and involves all these much larger of the brain. And in this |
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| 14:28 | , we already talked about with the agonists and antagonists. So we talked |
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| 14:33 | CNQX or A and N MD A blocked by A P five for A |
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| 14:40 | . So in this experiment on the surface, very localized where you saw |
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| 14:45 | activation of C two, you now blockers for A and MD A CD |
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| 14:51 | and A PD. You can see no map, there may be a |
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| 14:55 | bit of a surrounding residual activity of sort with no clear Whisker C two |
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| 15:02 | . So you inactivated C two and spread of that activity from C |
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| 15:06 | But if you wiggle wiggle Whisker E , you still have the same map |
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| 15:12 | because nothing has been blocked in this . And therefore the communication from this |
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| 15:18 | is also not being blocked through the of the brain. So you have |
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| 15:22 | spread of this way. OK. right. So this actually ends our |
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| 15:33 | on neuronal imaging. And I'm and |
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