Optochemical Genetics
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| Anzahl der Teile | 163 | |
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| Lizenz | CC-Namensnennung - keine Bearbeitung 4.0 International: Sie dürfen das Werk in unveränderter Form zu jedem legalen Zweck nutzen, vervielfältigen, verbreiten und öffentlich zugänglich machen, sofern Sie den Namen des Autors/Rechteinhabers in der von ihm festgelegten Weise nennen. | |
| Identifikatoren | 10.5446/50407 (DOI) | |
| Herausgeber | 05jdrrw50 (ROR) | |
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00:12
NeurobiologieBiosyntheseEnhancerMolekülAlpha-1-RezeptorSyntheseölFaserplatteLegierenAktivität <Konzentration>Chemische ForschungBesprechung/Interview
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02:08
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02:44
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03:02
MolekülChemischer ProzessEisflächeSystemische Therapie <Pharmakologie>Biosynthese
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03:40
TellerseparatorChemisches Experiment
04:05
MolekülBiologisches MaterialBesprechung/Interview
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05:28
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06:19
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07:06
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07:21
RingbrennkammerChemisches Experiment
07:38
RingbrennkammerChemisches Experiment
07:51
Eukaryontische ZelleHydrophobe WechselwirkungTransdermales therapeutisches SystemBrillenglasChemisches ExperimentComputeranimation
08:05
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08:46
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09:03
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09:18
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09:32
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09:50
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10:24
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10:49
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11:47
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12:03
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12:20
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12:33
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12:51
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13:05
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14:25
Computeranimation
Transkript: Englisch(automatisch erzeugt)
00:19
Hello and welcome to my laboratory. I'm Dirk Trauner,
00:23
Professor of Chemical Genetics at the Ludwig's Maximilians Universität in Munich, Germany. Today, I'm going to tell you about our research program, which is dedicated to both natural products and neurons. However, today's feature will focus on our work on what we like to call
00:41
optochemical genetics, where we try to merge small synthetic molecules with natural receptors in neurobiology to influence neural activity with light. This requires both synthesis and neurobiology. First, the synthetic part will be introduced by my greatest student, Matthias Schoenberger, and then my resident neuroscientist, Dr.
01:03
Martin Somser, is going to take over and demonstrate to you how optochemical genetics works. My name is Matthias Schoenberger. I'm a doctoral student working with Dirk Trauner in the very exciting field of optochemical genetics, and
01:22
I will show you a little bit about the chemical part of our research project, which is the synthesis of the light-sensitive molecules that we use for our experiments. So this is one typical photochromic ligand. That is the molecule that has two functionalities, which is being a ligand in this part of the molecule. This is a glutamate,
01:45
and this part of the molecule is a light-sensitive handle that works as a photo switch. As you can see, we have this comparatively large conjugated pi system with this double bond. So this is diazo benzene, and if it's illuminated with a certain wavelength, in this case 380 nanometers,
02:04
it summarizes from the trans-azobenzene to the cis-azobenzene, which looks like this. So, as you can see, when in this case, this fennel ring pointed up, now it's pointing down,
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and the glutamate part of the molecule does not change at all. So we have changed the geometry. So what happens is that the trans-azomer is flat and stiff, and then after absorption of the light, the molecule
02:43
bends around this molecule in a certain angle, and it's not as conjugated anymore. It has a higher dipole moment, and certainly it changes the the steric room that it takes in the ligand binding pocket. As a consequence, this molecule will bind very differently to a receptor than this molecule. Also,
03:04
this molecule is much less stable, which means that it relaxes back to the trans-azomer thermally, or if we take another distinct wavelength, for example 500 nanometer, we can switch it back very fast. So this is a rapid, reversible process between these two isomers,
03:24
which is the trans and the cis-azomer of the diazobenzene. So to get our hands on these exciting new molecules, we just run through a normal multi-step organic synthesis, and before we go on with the biological evaluation, we make sure that we have a clean and characterized product. For example, here
03:40
I'm purifying a new molecule. You see the nice separation between an impurity and the product that we see right here. And then we characterize it, for example, using NMR, HPLC, mass spectroscopy, and then once identified and being cleaned, we hand over the product to our colleagues. For example, Martin Sumser, who does a biological evaluation in a different laboratory.
04:08
Hi, I'm Martin Sumser, the senior scientist of Professor Traune, and I just got the sample from Matthias in the lab, and I will evaluate now this molecule in a biological preparation.
04:27
Here a mouse brain is incubating an ice-cold artificial cerebrospinal fluid. This is necessary to keep the cells in the brain alive, and I will now prepare brain slices with which I will do the electrophysiological evaluation later on.
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To fix the brain in the chamber, we use regular superglue, which I put in the chamber, and then I will transfer the brain from the storing solution into the chamber.
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I need to remove some of the liquid before, and then I glue it onto the chamber. Like this. Refill it with the artificial cerebrospinal fluid.
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So I put now the chamber on the vibrotome, which is a custom made vibrotome. I lower the blade, and first I will cut off a rather thick piece of the brain to trim the brain.
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And later on, the following slices will be around 300 micrometer in thickness. And with these, I will perform the experiments. I slowly move the blade through the brain to cut off the top part. You see the two hemispheres on the left and the right, and the brain stem here, the whitish matter.
06:07
I'm slicing now from the top to the bottom of the brain, first through the cortex. So this slice is now 300 micrometers thick, and this is necessary to later see the cells
06:26
under the microscope. If it is thicker, then you don't see the cells anymore. If it's thinner, then the cells die earlier, but 300 microns is thickness-wise the best to carry out the experiments.
06:42
So that will be the first slice. Slices are rather fragile, as you might expect, being 300 micrometer thick. I cut through the middle to have the two hemispheres separated. And I transfer now the slices to the incubation solution, which is also ACSF,
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but at 34 degrees rather than ice cold. After 30 minutes of incubation in the ACSF, I transfer the slice, one slice, to the recording chamber to have a look at it under the microscope.
07:32
The slice is fixed with a grid, which basically pushes the slice to the bottom of the recording
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chamber. Let's have a look at the cells via the camera.
08:04
Here you see cortical neurons, and one of these I will try to patch. Now I will pull the glass electrodes, which I will use for the patch clamping. Therefore, I fix this glass electrode and start the pull-up.
08:40
So now I take one of these, fill it with the intracellular solution, which is essentially a buffer containing ATP and GTP to keep the cells happy. With the electrode holder, I will position the electrode underneath the objective, but before I enter the perfusion bath, I have to put the reference electrode into the chamber,
09:05
and then with the micromanipulator, I can move the glass electrode on a micrometer scale.
09:22
So I try to find the pipette now underneath the objective, and here we are. And now I lower the pipette, or the glass electrode, to the surface of the slice and
09:42
approach my cell, which will be this one, slowly, and put a positive pressure on the pipette. So as soon as I get close to the cell, the membrane shows a small dimple because of the intracellular solution, which I blow out from the tip of the pipette, and this
10:04
indicates that I'm close to the cell, and when I relieve the pressure, the membrane flips onto the glass electrode and makes the contact which is needed to later break into the cell and get this patch I need for the electrophysiological recording.
10:38
So now I have achieved the gigaseal on this cell, and I will try to break into the cell now.
10:46
Okay, now we are in the cell. Now you see that we have access to the cell because there is an increase in current, which is due to the fact that now the membrane is not blocking the glass electrode anymore,
11:01
but it is ruptured, so we have an electrical contact to the interior of the cell. And now if I switch to current clamp mode, we can record action potential firing by simply injecting current into the cell and thereby depolarizing the cell up to a threshold
11:25
in which the AP, the action potential, is initiated. So now there is an action potential, and we can reliably initiate action potential firing, which is now only done via current injection, but now we wash in our photo-switchable
11:49
molecule, which is diluted in artificial cerebrospinal fluid, and then we can control this action potential firing not by current injection, but simply by applying a light pulse
12:05
which switches our molecule and then activates the neuron. And the molecule is now washed in through the perfusion system into our recording chamber, and as soon as it arrives there, we can start our light pulse protocol.
12:29
So now we apply our light stimulation protocol. We switch between 500 and 380 nm, and thereby activating our molecule, which then will
12:43
bind to a glutamate receptor, which these neurons intrinsically express, and with this activation of the glutamate receptor, we can control the activity of the neuron. So let's see whether we can evoke action potential firing.
13:00
So now we are at 370 nm, switching to 500 nm, 370 nm again, and nice action potential firing, as you can see. And this is a repeatable process, looks good. So I hope we have been able to demonstrate to you optochemical genetics in action, how
13:21
small organic molecules, synthetic molecules, can be used in combination with transmembrane proteins that are genetically manipulated or not to influence neuronal activity in combination with light. And we believe that our photo switches are useful on the one hand as investigative tools that they can be used to dissect neuronal circuitry to understand how the nervous
13:44
system works, but on the other hand we also believe that they have a great potential as therapeutics. We have a serious effort going on in my laboratory and in combination with colleagues in the United States and in Germany to restore vision. In patients who have lost their natural photoreceptors, we believe that we can use
14:02
the remainder of the circuitry with optochemical genetics to restore visual responses. On the other hand, we are also interested in influencing pain sensation with light. And if you want to read more about photo switches, I invite you to take a look at our thematic issue in the Beilstern Journal of Organic Chemistry dedicated to photo switches.