With the calcium that a chelator and remove of calcium, people have demonstrating that the transmitter release will significantly get perturbed. In fact it will completely abolish if there's no external calcium, okay? But how do you know at which step calcium works which step calcium works to trigger transmitter release, okay? In our model that we discuss that the action potential propagate to the nerve terminal to open and comes in and the transmitter release. But how do you if you applied EGTA or BAPTA. And if you apply for example, or if you make new solution without calcium. It does not have the resolution, does not have enough resolution, to tell us, when does calcium work? Okay, is calcium important for the propagation of action potential to a nerve terminal, okay? Or calcium is important for the opening of the postsynaptic receptors. That only with calcium the postsynaptic receptor can open, and then therefore has a response, okay? If you. Prediction in calcium is important in transmission release. And only important for transmission release and then your prediction will be, calcium influx will be always happening just before the transmitted get released after the action potential. Propagate to the nerve terminal. So how do you do experiment to prove that, demonstrate that? Or how do you do experiment to demonstrate at what time calcium is doing their job? So if you have ways to measure calcium. Okay, if you have a ways to measure calcium, and then we are measuring the transmitted release you can simultaneously measure calcium. And also measure transmitted release and look at that relationship, okay? To see at the what times the calcium level will goes up or goes down, and then how does it the calcium level up and down related to the transmitter release, okay? So this is again, can only be done in 1970's or 1980's, but in fact in 1965 Bernard Katz, who got the Nobel Prize, who is the personal hero for my PhD advisor, that's his big hero, Bernard Katz, a British scientist, designed an experiment actually to measure the timing of calcium in the synapsids mission. So here is what I did. It's a relative simple apparatus. What I did is that they can perfuse the neuromuscular junction with no calcium okay? And in that case if you stimulate the nerve and then if you record you insert here to recall a post-synaptic response, you find, there is no synapse transmission. There is no transmitter release. For example, here, this indicates the time you stimulate the nerve. Okay? You stimulate the nerve, you generate an artifact. Okay? Because, there's some electrical field that can be sensed by these electrons. So you know the nerve are stimulating, okay? And then the action potential will propagate through to the axon into the neuromuscular junction. And this indicates the action potential propagation that you can record. By your electrode here. Okay, only when the action potential propagate very close to electrode, you can sort of, like extracellular recording, you can sense, there's an action potential propagating here, okay? So that recording extracellularly, okay? And then in that condition, you observe there's no transmission at all there's no transmitter with this at all. This is why few of you already know because of if you remove calcium there's no transmitter release, okay? You completely remove all calcium but [INAUDIBLE] is they feel to the calcium solution in one of the electrical, and non-electrical. And then produce electrical very close to the neuromuscular junction, okay? So then they can sort of profuse in the low coding. You're only adding that calcium in a specific location at a specific time. How do you add in that calcium at a specific time. Well, this is very simple. Because calcium is positive attract. So we can use a technique call metafrasis. Okay? It's very simple because if you add some electrical positive charges in your electrode, then calcium will be repelled. And, rejected out. Okay? If they have the same charge. They will repel each other, right? So what you do is you can add a more simple circuitry, that can open and close at different time, okay? So then what you can do is, that you can open this circuitry, inject calcium before the transmitter release, okay? And then in that case you can see, well again, you stimulate and you found there is transmitter release you can record, okay? And then you can changing the time to open, or to activate, electrophoresis. Again, the electrical circuit It's much, much more precise than you manually changing the solution which takes many seconds. Okay? So they can achieve really second resolution and then they focus only what you apply kerosene. In a few milliseconds, of the action potential arrival, you can trigger transmitter release, okay? That is, if you shortly, or almost at the same time, When the action potential arrive in the nerve terminal your applied calcium when calcium is available. You can get the transmitter release, okay? So this narrowed down the action of calcium not just for the survival or propagation of action potential. In D, you can see here, even without calcium, you can still record the action potential propagate into the nerve terminal. There's just no transmitter released, okay? So this narrows the calcium's temporal role close to the action potential arrival. Okay? But does this experiment and the previous experiment demonstrate that is the calcium influx that it trigger transmitter release. Previous some student attention to be specific propose well removing the calcium outside then there's no transmitter release, okay? And [INAUDIBLE] so 1965 is from [INAUDIBLE] calcium only means to be close to [INAUDIBLE], small after of [INAUDIBLE] and is required to generate transmitter release. Do those two experiments, prove, or demonstrate, that the calcium calcium get into the cell to trigger that transmitter release? Do they prove, or do they. >> [INAUDIBLE] >> You don't think they prove. Okay, so great, I also don't think they prove that the calcium working, is get into the cell, into the, specifically, to the presynaptic cell, to trigger transmitter release. Okay? Then what is your alternative explanation. So if calcium may or may not get in to the cell. We have already know from our previous review that a calcium needs get in to the cell. But why would we our explanation that how calcium triggers transmitter release? >> Maybe the calcium attract the one tibular receptor on the [INAUDIBLE]. >> Great! The other possibility is calcium does not need to get into a cell. Calcium can work. On the receptors are working on the membrane. Biting into receptor in membrane, near terminal. And this receptor going through this signal transduction to trigger the transmitter release. The previous experiment do not eliminate this possibility. The only proof calcium is important and is walking at a small time window, but it has no specifically pointing out where does calcium at, okay. Calcium walking inside outside at least that's two possibility. So [FOREIGN] How do you design experiment to prove or disprove that calcium is working outside or inside? Need to get in fact inside the cell to trigger transmitter release. Okay great. One possibility is, again, if we have certain dye's that can emit signals upon calcium binding, okay. We can pull off dye's into the cell and then we can observe once calcium comes in. Then it's style will peacefully, hopefully reports the calcium influx okay. All right, again, so this needs to be wait until the calcium died has been generated right, without it, how do we do it? Listen to me. So, listen, listen. Don't calcium die. Then how do you do it? Great, well, if calcium, which is a charged ion, right, we have already talk about and. Use voltage clamp to record. During depolarization, the sodium current, and potassium current mediated by those ion channel. If calcium Is moving. Again, whenever you have a charged particle moving across the electrical field, you generate a current. Okay, so if you can record, specifically, the calcium current in the paths membrane. Okay, now you know how there's a calcium influx. So these are two possibilities.