Just a word about the timing of all of these events. So chemical synapses are slower than electrical synapses, but chemical synapses are still quite fast. And it really is remarkable how quickly these activities can unfold. for example, the process of forming a synaptic vesicle and filling it with neurotransmitter, docking it, having it ready to go in an active zone. it might take place in less than a minute. The actual fusion events, where the membrane fuses, causing a release of neurotransmitter, can happen in about a millisecond or less. And then, finally, the recycling of that membrane back into an endosomal compartment can take place in less than a minute. So, very quickly, this process can unfold at every synaptic terminal in our brain. Now, let's consider the critical role of Calcium in the fusion of presynaptic vesicles with the presynaptic terminal membrane. So, I'd like for us to, again, to consider some evidence that supports the important role of Calcium in the process of vesicles fusing with the presynaptic terminal. This data come, once again, from our favorite animal model for these kinds of studies, the squid. Where there are some really huge synapses that allow experiments to be done, like can't be done in nearly any other known system. And what's being done here is a presynaptic neuron is being stuck with microelectrodes that allow for clamping the voltage or for injecting current. And there's a postsynaptic process, postsynaptic neuron for which the postsynaptic membrane potential is going to be recorded. And so, what's being demonstrated here in these data is the important role of Calcium. So, if the presynaptic terminal is depolarized, which is what we see here there's an inward current, and that inward current is carried by Calcium. So basically, Calcium is rushing in to the presynaptic terminal as voltage-gated calcium channels open. That leads to an action potential in the postsynaptic neuron, okay? Now, this presynaptic terminal is large enough that the scientists can actually infuse a compound that blocks Calcium channels. And when that's done, the same kind of depolarization of the presynaptic membrane fails to result in the opening of these voltage-gated Calcium channels. So, there's pretty much no change and no action potential. So, without Calcium in the presynaptic terminal, we do not record an action potential in the postsynaptic process. Likewise, a series of experiments were done that further this theme, which illustrate that Calcium is both necessary and sufficient to trigger vesicle fusion and the release of neurotransmitter. Now using the same preparation, it's possible to inject Calcium directly into the presynaptic terminal. And when you do that, you can measure a depolarization of the postsynaptic neuron. So, this suggest that infusion of Calcium to the presynaptic terminal leads to some vesicle fusion events, the release of neurotransmitter, the opening of cation selective channels in the postsynaptic membrane, and the depolarization of that cell. Here, again, is further evidence for the importance of Calcium in the controlled experiment. The presynaptic membrane is depolarized such that an action potential is generated. About a millisecond later, an action potential is recorded in the postsynaptic cell. But if a Calcium chelator is injected into the presynaptic cell, this is a compound that is going to prevent a sudden rise of intercellular Calcium. So, so now we're not blocking the Calcium channel, we're just sucking up the Calcium that enters the presynaptic terminal. So, even though we have a nice robust action potential In the presynaptic neuron, there's only a very modest sub-threshold depolarization seen in the postsynaptic cell. So, this experiment suggests that a sudden rise of Calcium in the presynaptic terminal was critical for the release of neurotransmitter and the generation of an action potential in the postsynaptic cell, Okay? Well, so far, we haven't really discussed how all of this comes about. And to begin that discussion, we need to consider some of the molecules that are affiliated with the synaptic vesicle membrane, as well as the membrane at the presynaptic terminal. So, here's a rather fanciful look at a synaptic vesicle. And what we see is an artist rendition, now a couple of years old but still just conveys the point of the complexity from a molecular perspective of the surface of the vesicular membrane. And this complexity reflects the precise means by which the presynaptic terminal regulates the trafficking of synaptic vesicles and controls their fusion with the presynaptic terminal. So, I show you this just to make the point that there are dozens of molecules in the vesicular membrane that are important for regulating the activity at a chemical synapse. One of these molecules is particularly important, and I will highlight that for you. It's a protein called Synaptotagmin. And here, it is illustrated here. So, Synaptotagmin appears to be the molecule in the vesicular membrane that binds to Calcium. So, as Calcium rushes into that presynaptic terminal, Synaptotagmin seems to be the Calcium sensor that interacts with Calcium. And eventually, sets into motion the events that quickly unfold leading to the fusion of the membrane in the pre synaptic terminal. The way that this happens is through the activation of the set of proteins, some of which are found in the synaptic vesicle membrane. Others are found in the membrane of the presynaptic plasma membrane. Together, these proteins form something called the SNARE Complex. So, SNARE is actually an acronym. you don't need to be concerned with knowing that acronym but it's helpful to use this acronym because it reminds you as to the function of this complex of proteins. So, the SNARE complex is involved in snaring the vesicle membrane to the cytoplasmic face of the presynaptic terminal. This is, in fact, the docking process. So as these membranes approach one another there is an interaction among these proteins. Basically, these proteins, such as Synaptobrevin SNAP-25, and some others, they associate with one another and perhaps form some sort of helical interaction that allows the synaptic vesicle to be docked to the presynaptic terminal. Now, notice the proximity of the SNARE complex to Synaptotagmin. Synaptotagmin, again, is the molecule that binds Calcium ions as Calcium enters this presynaptic terminal with the arrival of depolarization. So, let's sort of step through that process and see how this works. Initially, we have proteins affiliated with the vesicle membrane, we have Synaptobrevin, SNAP-25 and Synaptotagmin. But, not yet do we have sig, significant depolarization arriving. So, the SNARE complex begins to dock a vesicle membrane. And this happens in a Calcium independent fashion. This allows a vesicle to be docked, even without an inrush yet of Calcium into the presynaptic terminal. So, when the SNARE complex forms, we can say that these membranes have been pulled together, and this vesicle is now docked. So, this is a picture of what a docked vesicle looks like. The SNARE complex is formed, this vesicle is primed and ready for action with the arrival of a wave of depolarization. So, once that happens voltage-gated Calcium channels open, Calcium rushes into the presynaptic terminal. And now, Calcium binds to Synaptotagmin. And the interaction of Calcium in Synaptotagmin leads to a series of confirmational changes in the SNARE complex that tugs these two membranes together. And as that happens, then the membranes will fuse and neurotransmitter now is free to diffuse out of the synaptic vesicle through what's called Fusion Pore that forms as the vesicle membrane in the presynaptic terminal unite. Well, now that the neurotransmitter is diffused out of the synaptic vesicle what's left is the retrieval of the membrane from the synaptic vesicle. And this involves a variety of proteins some of which are illustrated here in this slide. I would highlight just a couple of them for you. one that's very important is this protein called Clathrin. so Clathrin coats the vesicle membrane and with interactions with other proteins such as this Dynamin protein. we have the retrieval of those membranes, and here's a picture of what this looks like. So, the Clathrin molecule has an interesting geometrical shape. We see it up here to the upper left. It forms a structure called a Triskelion, and that Triskelion then can associate with other molecules and essentially form this buckyball-like ring around the membrane. And so, as these Clathrin, Triskelion begin to form, then the vesicle membrane is pulled back away from the presynaptic terminal. at some point in here, Dynamin gets involved and appears to nip these vesicle membranes, allowing for a completely coated vesicle membrane to now be retrieved. And via interactions with Actin filaments and a variety of other proteins this vesicle membrane is retrieved. The Clathrin coat is removed, and the vesicle is now ready for repackaging with neural transmitter without loss of all of those specialized proteins that are associated with this vesicle. Like Synaptotagmin in the SNARE complex that are important for the function of this membrane as it's recycled and repackaged with more neurotransmitter. Now, I'd like to conclude this tutorial by thinking about the activity of a toxin that's found in nature, from the Clostridium family of bacteria. And it's over the last couple of decades or so, has been used relatively widely in clinical practice. and also, unfortunately been somewhat abused perhaps, in the cosmetic industry. And, of course, what I am speaking of is Botulinum Toxin, otherwise known as Botox. So, Botox can be used in the therapeutic setting to reduce muscle tension, to reduce muscle spasticity perhaps to quiet a twitching muscle. these are all very good and appropriate uses of botox. botox can also be used cosmetically to try to reduce muscular activity that might give rise to wrinkles or furrows in the skin. And for a variety of reasons, botox has become a fairly widely used drug in our time. So, how does it work? Well, the way botox works is by cleaving the SNARE complex. So, botox is a Protease. And it is a Protease that disrupts the SNARE complex in one of several sites depending upon exactly what formulation of Botulinum Toxin we're speaking of. one of the most common and widely used forms of botox is, is BoTX-A, and we now know exactly where. The cleavage site for BoTX-A is within the SNARE complex. And a variety of other botox formulations have their own cleavage sites on one member or another of the SNARE complex. So, you can imagine why this works so well. So, if the SNARE complex is cleaved, then no matter how much Calcium rushes into the presynaptic terminal, that vesicle is very unlikely to fuse on it's own. The SNARE complex is what facilitates bringing together these membranes and twisting and turning, resulting in the fusion of the vesicle membrane with the presynaptic terminal. Without the SNARE complex to pull those membranes together, we're very unlikely in having a fusion event. Therefore, we're very unlikely to release a neurotransmitter at that synaptic terminal. So, botox is a very powerful molecular tool that can be used to silence a chemical synapse. Okay? Well, we've come to the end of the tutorial. And I would encourage you to view one of the animations that will review these processes that are involved in synaptic transmission. It's animation 5.1. You can follow the hyperlink at the end of the handout or navigate there via our website. Next time, we'll talk about neurotransmitters, and after that, the receptors for neurotransmitters. So, I'll see you then.
