We are recording the transmitter because we are seeing the postsynaptic receptor gated by the transmitter. They are open. Once they are open, they will allowed ions, positive ions get into a cell. And that's the reason we see this increase in the membrane potential, because there's more positive ions getting into a cell while you record is the membrane potential increase. But as I just mentioned this, you cannot distinguish what is a transmitter increase, postsynaptic sensitivity increase. And what he described, what the student described answering my question is while you can measure in the transmitter release, then the question is how? How are you going to measure transmitter release? You can no longer use this postsynaptic cell as a reference, right? Because then you are going to be different, then they are going to be difficult to measure it, to distinguish the possibility. Okay, so one way, design an independent sensor that is different than the postsynaptic receptors but can be sensitive to the amount of transmitter released, right? So there are a number of ways. How do you do it where you can sense the glutamate release, right? If you are using optical methods, then you can design some sensor that can sense glutamate. How do you do it? You can use some glutamate binding domains and then they're coupled with some fluorescent protein, and hoping that this sensor will be sensitive to glutamate. Well, indeed, people have already developed that and displayed it on surface and they can sense glutamate. So that will be one way. The other way to sense a transmitter release is you can have a way to directly measure the exocytosis in a presynaptic cell. For example, if the presynaptic cell, they have the release of transmitters, then they will have increase of the membrane area because of the vesicle will fuse with the plasma membrane, right? So if you have a neat way to measure how much the membrane area increase, for example, by measuring the capacitance. Then we say in this case it would detect indeed there is a much larger membrane area increase, comparing with our LTP, okay? You have your control, right? So he says, at least this correlate if we assume the transmitter is correlated with the vesicle membrane and that will be the increase of the transmitter release. And there's other ways, for example, you can also, Put some artificial transmitter, for example, isofluorescence, if you think, into the vesicles. And if we get release, you'll see that isofluorescence will decrease. Again, then you can compare before the plasticity and after the plasticity, if there's change or not. And we can also try that, because of the presynaptic terminal have this vesicle, and this vesicle is acidic, has a lot of proton inside. And then you can put some fluorescent protein that is pH sensitive. And if the vesicle gets released, then these proton acidic environment is going to be changed into neutral environment. And if you put the pH-sensitive indicators there, then the pH will be different. So you indirectly measure the pH sensor. But because you strategically put a pH sensor only inside the lumen side, so you can use that as an indirect way to measure exocytosis and transmitter release. So at least the way to do that is to have an independent way to measure the transmitter release. You can also measure from another source. For example, in this case you can now measure the postsynaptic receptors because you want to directly study whether this effect is here or here. You want to distinguish whether it's pre or post. But what you can do is, and people have done that, if there's another adjacent cell, for example, a glial cell. It turns out that some of the glial cell will sense the release of glutamate, to open up the special transporter on the glial cells, to sort of regenerate more glutamate in the future. So then you can record this glial cell. You are not recording this postsynaptic neuron. You are recording this glial cell. And you say, you can do the control experiment. The glial cell can sense different amount of glutamate release leaking out from the synaptic cleft. And then you are using the plasticity protocol to induce it and then recall a glial cell as your independent way to measure the amount of transmitter released. Okay, all of those things can demonstrate a transmitter release is auto or not. Then here's another question is very excited to answer. How do you do the experiment to probe whether the postsynaptic sensitivity is auto or not? Previously you are just measuring independent weight, the transmitter release is changing or not. But how do you design an experiment to directly test that postsynaptic sensitivity is changing or not? And a difficulty to really add a glutamate. But see we have a ways to use uncaging, right? We can use the cage glutamate. We can just use in the sense, UV light. Because if we directly inject glutamate, it will trigger a lot of side effects, the desensitization, diffusion. But you can use an uncaging glutamate, and there you can directly measure the sensitivity changing or not.
