And here is another experiment demonstrating moving. That is direct injecting of calcium into the terminal. The experiment done in 1970s. Again, terminal. So this is the experiment recording in the post synaptic neurons. Well in a pre-synapse neuron, you are inserting the calcium electron and inject the amount of calcium. And then you can very small, yet detectable, post-synaptic calcium response. And people take it as evidence that calcium itself injecting into the presynaptic terminal will trigger transmitter release, okay? But if you look at the response, first you look at response to the. This is a 0.2 millivolt, and the temporal response here is 2 seconds. Okay, this is a response. If we fast backward to the response in the screen. This is the screen of terminal, and you use this action potential. And what I've found is look at the here is 10 mV. Calcium in one millisecond. That's a huge difference between this one mV response, and 2 seconds, that is 2000 millisecond response. Why? There will be such a huge difference. When you are injecting calcium Into the nerve terminal. The response seems to be very small and slow. Why this is the case? Well, in the action potential trigger condition you have this calcium channel just near the membrane. So once they open, the calcium. The source of calcium coming from the mouth of the water do calcium channel, very close to a membrane. And indeed in the studies, electro-microscopical studies, or biochemical studies people found that water is gaining calcium channel. In physical proximity with those synaptic axon. Once calcium enters a cell, reaches the synaptic axon locally. Putting it in other words, that is the calcium levels are very high in the synaptic. And the of calcium actually is much shorter. You have the calcium diffusion into a terminal for only locally you have calcium concentration. And if you have a calcium sensor that only responds to high concentration of calcium, low affinity sensor. That will trigger a transmitter release, and the further lower calcium concentration has no effect. But if we are injecting calcium Into the nerve terminal. Well, actually, doing this we have no idea of the calcium concentration and temporal profile in the nerve terminal. Inside the cell, there are a lot of calcium buffering mechanisms okay, different calcium buffers there are a lot of transporters, pumps reactively will remove calcium along the way. So you are actually not precisely controllable temporally, and spatially the calcium concentration in a nerve terminal. So the fact that this has a much smaller response most likely is because of the maybe it's too little calcium reach the nerve terminal. To slowly, okay? And the calcium dynamics is different than the calcium dynamics trigger by the wattage KG channel. And indeed if one has those calcium sensors one can do those experiment to the ready measure the calcium dynamics to see whether they are seen or not, okay? And therefore people even use that method to more precisely and rapidly control calcium. And here again, taking advantage of the chemistry, this is the so-called caged calcium. Again, it has this EDTA or EGTA like it has the four carboxic groups that can chelate calcium. So that is the building block that can specifically bind to calcium. But you also have another chemical modification here. Has this aromatic ring with some electrons donating groups, and withdrawing groups. And previously, chemists know that if you attach this aromatic ring, actually it will render this chemical compound photosensitive, especially to a UV light. So upon UV illumination, actually the bottom will break at this stage close to the attachment. So then there is in this wave. You can inject a mixture of this calcium buffer, a photosynthesis calcium buffer or what we call caged calcium. So we premix calcium with this caged group into a nerve terminal. The calcium in that case will be chelating by this full caged group. They are not free, okay? And therefor even when calcium keeps casing around the nerve terminal. This nerve terminal is a big nerve terminal in a more radiant system called kitex. Well it has a kittie like structure. Which our principle terminal references the postsynaptic. Cell body in the auditory pathway. This is called MNTB cells in the auditory pathway. And you can through Inject this caged calcium. Because calcium are caged, solely on path. And they are caged so they cannot find who their effector. [INAUDIBLE] But then, at a time you want it to be released, you can show the light, using a UV light, a brief pulse of UV light to uncage or photolyze the caged calcium. And in this case, if you break this chelator into these two groups, then these four carboxyl groups are going to break into two pieces. Then they have much lower affinity to chelate calcium, and then free calcium get released, okay. And because of the illumination and this photo uncaging speed are much, much faster in terms of millisecond or even shorter. So you can mimic chemically using this caged calcium you can mimic the rapid release or influx of calcium in enough terminal, okay. So in that case, if put some calcium die at the syntax in to enough terminal and you do that and is probably. First to use the relatively shorter, lower energy of UV line and you will say, the calcium signal will increase a little bit okay. And then you can use a more stronger energy of UV line and you can say, the [INAUDIBLE] grows up but even higher. And okay in this case you do not change the voltage or action potential in the nerve terminal. And just purely using the photochemical method, you can induce the calcium level changes. And if you have the recording electrode in the post-synapse cell Correspondingly you can record the response form this cell. Meaning that this cell, receiving the transmitter release, triggered by calcium, and then you will change its current in the wattage craft. And as you can see that here actually is a illustration of a non linear relationship between calcium and transmitter release. That is, you have about two fold increase of calcium transit in this case, but you actually have a much larger increase of transmitter release. Again, following what we described of how non-linear, a poly function of calcium in transmitter release. And because of this new, for the chemical approaches, approaches, you can more precisely control the temporal dynamics of calcium. Okay, and, there, you can achieve a EPSC like response, similarly, like triggering atom tension in an known to open calcium channels
