How does the calcium control these transmitter readings. Well, in 1967, Dutch Rahamimoff already characterized the action of the calcium in newer muscular junction by simply profuse different concentrations of calcium ions and natural amount of transmitter release. As you can see in this average result, the calcium level increases gradually you have a much larger increase of the transmitter release. Nowadays we can also use the optical mass telemetry. This is unpublished results from our lab. We can put the pH sensitive frozen protein inside of the cylinder vessicle. Okay? By fusing with some special vessicle proteins. And because net vesicle are acidic, when you arr triggering a transmitter release, then this is will fill with puss membrane and reach in the neutral environment. And this pH sensitive frozen protein will increase that [INAUDIBLE]. Okay, so you can see in this case we could use this optical mass to measure the transmitter release and the relationship with the calcium concentration applying outside of the cell. For example, in the regular 2 [INAUDIBLE] molar calcium concentration. When your stimulus cell, you can see some optical response. The force increase, reflecting the exocytosis. And subsequently, this signal goes down reflecting that this protein can internalize. And the signal get [INAUDIBLE] once the [INAUDIBLE] that vesicle become acidic. Okay. So you can do this experiment just by changing the external calcium concentration. For example, you can do that again, you can see the same response. But if you chelate external calcium, and the stimulation, while you're there's no transmitter release. And then you can out by increase the calcium concentrated gradually, and then, you can see the transmitter release also increase. And then you can change it back to two calcium. So why when do those experiment we want to change it back to this two minimal calcium. We already tested two minimal calcium at the beginning and then we changing our different concentrations calcium. Why do we want to do that to change it back to two minimal calcium? To demonstrate, this in fact, is reversible because you are stimulating the cell over and over again. Maybe the cell, after certain profusion and stimulation, they are becoming unhealthy, so abnormal response. Then, that we see that it's two minimal calcium. The response will be different than at the beginning. But they are not, so you can demonstrate that even after this different trials of different calcium concentration. If you are turning back, you're getting a same calcium concentration, right, induced response. So, this is a totally calcium dependent reversible response. So this make it very clear that this response most likely will be physiological is reversible. It's not due to the cell, that there will be damage along this perfusion. Okay. And those by this optical or more conventionally more than 40 years ago by this physiology. Then we can get this calcium dependent post [INAUDIBLE] response curve. Okay. And if you look at that, interestingly it's not a linear relationship. That is, when you gradually increase calcium, you have a linear increase of transmitter [INAUDIBLE]. Rather If you look at this curve, the curve is a power function, okay. So this is the experiments that are based on the optical response and an actual [INAUDIBLE] response is also a power relationship. Okay. The reason why the curve looks slightly different is because of the plotting, the skill is different than this linear scale. Okay. But many other experiments, both in neuromuscular junction from [INAUDIBLE] from our central nervous system. People reached a consensus that calcium dependent transmitter release is a power function of n equal to three or four. That is if you look at this curve with the small amount of calcium increase, actually you have a much larger non-linear increase of transmitter release. Let's just look at it more closely, for example in this curve we are changing the external calcium concentration from point two to point three in the middle okay? You can see that the amount transmitter release increased from almost zero to maybe 0.05 here okay? But so that is how you increase point one millimole of calcium. But if you increase another point where minimal, 2.4 minimal, my observe is that the amount of response will be reaching to 1.5, okay. So again, that's a non-linear, much larger increase of the post synaptic response. So small calcium alteration or increase can translate into a much larger post-synaptic, much larger transmitter release. This is a unique feature of transmitter release, where neurons using this calcium to amplify the synaptic transmission, okay? Again, this holds true for many other synapse. And if we look at the exact timing of transmitter readings with calcium. Then you can see that indeed the chemicals in that transmission is very very rapid. Okay, again this is the first experiment done in in 1980s, early 1980s. Here is a pair of recordings from a prisoner terminal and the post synaptic neurons. Okay you can see here this curve indicates in the. Is the true mean of the action potential. So usually the presynaptic action potential comes here, and then you immediately get to the postsynaptic response. You can see that's the delay. The time scale here is one millisecond, so the arrival of the action potential recall in the presynaptic terminal. And the post [INAUDIBLE] response is at one millisecond time scale [INAUDIBLE]. Okay. But if you look more closely, because we know that the actual potential, the function is to open at the water [INAUDIBLE] calcium channels. Okay so, under that condition, using this action potential like depolarization, and if you can manage to record the associated calcium which is showing here in this, this is more inset. Euler found, even more interestingly, that the calcium and transmitter release are more closely associated. First, the calcium influx is following the depolarization of action potential. More specifically, you can look right here, actually the peak of the calcium response It's at the hyper polarization phase. It's not at the depolarizing phase. Rather that at the going down, decay phase of the action potential. And this calcium influx Immediately trigger the postsynaptic response by releasing transmitter. Why the calcium current pick happening at the decaying phase of the action potential? Well, the reason being that the calcium channel, they are sensitive. He actually requires the writing face of the wattage to open the calcium channel okay so a lot of delays in the chemical synapse transmission. At least about 0.5 milliseconds if you look at the transcript. The delays, this 0.5 millisecond delay, is due to the delay of opening of voltage gated [INAUDIBLE] calcium channel, okay? It needs to be open. And then, even after you open, why the peak is after this decay phase? Well, because if you still remember equation all the calcium reverse potential. When you have membrane potential. The driving force for the calcium influx actually is not so big. The reward potential for calcium is probably very positive, plus 50 or plus 60. So at the peak of action potential, actually even though the calcium channel may be already opening, there's not too much of a driving force for calcium and iron to go through that channel. Well, at the decaying phase, the drops but the calcium channels are still open. Okay? And then you have a larger driving force, so what you get is this peak of the calcium current. So nature decides the principles of this action potential and [INAUDIBLE] channel to precisely control the timing of a count of influx which was subsequently to trigger transmitter rays. So, this is a very neat feature and, after 20 years or 15 years, people start to delay. In the [INAUDIBLE] central brain, and then they found similarly like [INAUDIBLE] the calcium signal and the transmitter radius following the similar principle, okay. So this is unique feature of both the calcium Intensity of the concentration dependent, a non-linear curve, and then the
