And there is also not only this fast ligand-gated ion channel response. There is also relative slow synaptic response. Most likely regulated, or mediated, by the gpci, the seven transmembrane. What type of tropic receptors? So rather than neurotransmitters directly gate to this ion channel itself. Some of the neurotransmitters or most of the neurotransmitters they can also activate the gpci. Specific gpci and this gpci show the signal transduction for example you can amplify the signal by activating a second messenger. In this case, G [INAUDIBLE] can interact with the adenalyte cyclase to generate [INAUDIBLE] AMP and [INAUDIBLE] AMP, for example, one of the classical target will be activating protein kinase A. And that can lead to the phosphorylation of some specific ion channel. And depends on what ion channel type, for example in this case is a potassium channel. And the potassium channel will be either regulated to open or close. And that will lead to the excitability change of this cell, because once potassium channel open, then there will be more potassium comes in. The cell will be difficult to fire action. Potentially, it will be hyper-polarized. Okay? And this response takes many steps, okay? So, in the the receptor itself, which is open so it will be fast. But in this case it takes this G protein and cyclase and kinase in the ion channel, so you'll be slower. But it could be. Signal query amplify many times bigger, okay? For example, if I see a tiger, then I get scared, my heartbeat might change, okay? And this is, again, should some special activate the downstream's gpci and the G protein and this G protein through, so in this case, G beta gamma sub unit itself can gauge some potassium channel channel. G protein even will rectify potassium channel. And that channel will close and make the cell much easier to fire action potentials, so your heartbeat would be fast, okay? So this is a peripheral example, but in the central nervous system it's the same principle. And in the synapse that integrate both of these IPSP and EPSP, excitatory and inhibitory inpulse. And once it's sending out this output to the nerve terminals, the nerve terminals can also have special relocation. For example in a nerve terminal, there might be some special inhibitor neuron. Can release for example GABA to inhibit the transmitter release at this nerve terminal, okay. And so anatomy, this nerve terminal can also target different cell. So indeed, the nervous system is is a combination of many excitatory, inhibitory target specific different concourse. It's a combination of all those properties make the neuron network could be as complex as possible to mediate higher cognitive function. And one of the challenges is to untangle, to identify specific cells, the key cells, the key roles of certain synapse in certain behavior as I just mentioned. Okay, so this is part of the summary of how post-synaptic cells sense their transmitters. They are excitatory and inhibitory, and then can be fast and slow. Depends on different ion channel and that modulation can be at a different component, the dendrites, the spine, your cell bodies actually initial circuit ones. And the pre-synaptic terminals as I illustrated here. It's all those combinations. So the classical works illustrates this. But if we time travel back switch gears, time travels back to after our understanding. Time travels back to, for example 30 years ago. After our understanding of this basic building principle of nervous system, we still having big challenges. For example, we know, through electrophysiology recording, that the neurons in our central brain will in the synapse, they will sense glutamate as the transmitter. But it will still have no idea in more than 30 years ago or 40 years ago. We still have no idea what molecule, what protein are indeed the glutamate receptors. What genes coding for those glutamate receptors? So now what you can see. How will you solve this problem if you time travels back? Again, through our recordings we know the transmitter can release where some people who worked on that understood the reduced machinery some key protein. And for electrical recording, for muscle, for neurons, we see the post-synaptic response and we know they are sensitive to glutamate. How you identify the genes that coding for the glutamate receptors? So indeed the way a few people, actually a few labs almost simultaneously achieve the identification a glutamate receptor. And then the way they are doing so called expression cloning. Functional expression of a glutamate receptors. How do you do it? Well, this is similar as one of the students has to look at MRI, okay. So what you can do is, you can isolate the MRI that might encode the glutamate receptors, right? And then you can count it, you reverse transcribe them into cDNA and then you can clone them into a vector with this different cDNA and it has a promoter. Then what you can do is, you can separate this library for example, into ten different groups. For example, here using your medical accounting, you can elaborate with 10,000 different genes. And then you separate into ten different groups. And each group will have 1,000 gene. And, in this is a 1,000 genes. It's a mixture. DNA are small, right? And, what you can do is, you force them pressed in these cells. For example, some big cells will be oversized. [FOREIGN] Which is very easy to record. Very big. So, you can insert it naturally. Also other cells, hex cells, are also easy to record and to inspect. And you can record it. And what you can do is in the other slide for example, you separated. Say here you only have one copy of your gene. You don't know which one is the photon receptor, but you separate it into ten different groups. And many are recaught in one of the old set that is part of one of these ten groups. And glutamate, if in that group, for example number seven group here. Just happen to have this gene and it will get expressed. And then one you get a glutamate, you will see the response. Right? And then you know, a ha! Some of the glutamate receptor is within these 1000 genes. How are you going to do later to identify which one is the gene? Okay, so similarly, then you can separate, you already got this 1000 genes. Then those are the genes, those are the DNA, again one unique advantage of DNA is, once you clone it, you can amplify that by any amount. Okay? You can replicate a DNA, you have a bacteria to help you or you have other methods. So you just separate them again, free them into a hundred groups. Each group, ten group, each group has 100, right? And then for the number eight here, this 100 has a response. That probably will save you quite some time, even though we'll appreciate your diligence, right? We still want to save some time so again, similarly, if it's 100 affinity divide. If it's ten then maybe you can test individual one. Right? So this is called a functional expression counting. The advantage of this assay is that if you have a robust, functional assay, even if the affinity is low, you can test it. Okay? It doesn't require high affinity. It just add a glutamate you just say higher concentration glutamate and but the response needs to be robust. But what might be the disadvantage of these approaches? What might be the limitation of this approach if you think? >> [INAUDIBLE] >> Okay. So I got ten, so I have several response group seven, eight, nine all have low response. But then if I am the professor, I'd be great, maybe that's three different glutamate receptors. Let me just separate them individually and then I clone three different glutamate receptors. This would be an advantage rather than disadvantage, isn't it? And it turns out, it turns out there are more than four different glutamate receptors in the brain. And several different labs simultaneously doing this cloning. It turns out they clone all different isophones. And then later, they say, I'm the first, I'm the first, it turns out just they're similar homologues, they have just different isoforms. I will call a pass rather than a disadvantage. It's very sensitive, so it could even clone different isoforms. That would be cool. What would be the limitation, you think? So there would be limitations sometimes if the requirement is that if you only have one gene that it would be sufficient to mediate this response. So the possibility that it might require something on the surface, but as somebody as you just pointed out if you require additional components maybe we cut a trafficking molecule to deliver on the surface. So, we will have this total pool. It already contains two components where it's pressed. And you'll say that a glutamate response but then where's the divide ten group? In this one has an A component, here is B component and you know you require A and B together to have the response. Then you divide it into ten groups, then you say, none of them has any response, right? Then this is going to be difficult, right? It might be very useful, but it also has some limitations. Again if we have A and B then you need to think, you need to, sort of, mix. Right? You divide it, if it is only two component, right? So, you divide it by the ten group. One way to overcome this limitation is you can mix A and B, A and C and D, right? How many times are they going to mix? Probably C ten two, right? Do you agree? So study biology sometimes still need some high school math, right? And if you have three component, A, B, C, again, it needs more mixing, right? Then once you mix them, and then you can separate so then that would be much more complicated.
