NBL 355 Module 9

In the last three modules of this course, I'll be talking about synaptic transmission. As a brief introduction, I wanted to talk about the discovery of synaptic transmission. And on the electrical side, this really began in the 1660s when Jan Swammerdam developed the frog neuromuscular preparation. At this time in the early 1700s, the hypothesis about the function of the nervous system really centered around the idea of animal spirits. And this concept originated with the Ancient Greeks and was advocated by Galen. In the latter part of his life, in the early part of the 1700s, Isaac Newton was actually the first person who began to think about the electrical nature of nerve signals. And he introduced the concept that electric bodies operate to greater distances and all sensation is excited, and the membranes of animal bodies move at the command of the will, namely, by the vibrations of this spirit, mutually propagated along the solid filaments of the nerves. So he really essentially described the electric and elastic spirit. Later in the 1700s, Luigi Galvani really was the pioneer who discovered the action potential, in fact. And he was an Italian doctor and professor of anatomy. And what he did was he initially began to use the preparation that Swammerdam had discovered, and this was the frog neuromuscular junction to try to understand how electricity might be involved. And in 1791, he published the commentary on the effects of electricity on muscular motion. And in these experiment, in these treaties, he described the experiments that supported the involvement of electricity in the nervous system. He further went on to hypothesize that animal electricity was produced by the brain and distributed by the nerves to the muscles. So he really was the grandfather of electrical signaling in the nervous system. Throughout the 1800s, many scientists were exploring electricity and using electricity with these types of neuromuscular preparations to investigate the electrical activity that is propagated within nerves. And in 1800s, the first action potential was stimulated using electricity, and actually the rate of propagation of the action potential was first determined. At the same time in the 1600s and the 1700s, the microscopist were really the groups who were contributing to the understanding and identification of neurons. So back in the mid to late 1600s through to the early 1700s, we had the development of the first microscopes and the use of those by Van Leewenhoek, and Odierna, and Hook, and they used these different types of lenses and objectives then to be able to visualize cells for the first time. Then in 1830s and 1840s, these anatomists and microscopist then, they were the ones who first described the cells of the nervous system. So the investigators and scientists including Valentin and Ehrenberg and Purkinje, Remak, they all were examining nervous tissue and describing the different cells that are present within the nervous system. So they noted that some of the cells had very long fiber-like processes, and even began to distinguish the myelinated fibers. Purkinje and Remak actually proposed that there are connections between the processes and also that there was a nerve cell body that they thought would be important. This microscopy actually came about because in the 1940s and 1950s, tissue stains were initially developed, and because in the absence of any staining, preserved tissue is essentially transparent and it's very difficult to see and visualize the different parts of cells. In the 1850s, additional new stains were developed, including stains like carmine red and chromic acid and the anilin dyes, and they were used by Corti and von Gerlach, Hannover, and Deiters, and they provided the first very clear images of nerve cells. So these are some of the drawings that they produced by using the specific types of dyes in order to visualize the various processes and the size and important parts of the cell body. Interestingly, so we have the cells which are called the Purkinje cells, which were first described by Purkinje, and you remember these are one of the major projection neurons that are found within the cerebellum. So in general, it was the largest neurons that were described first because these were the easiest to visualize. At the late part of the 1800s, then we have these two scientists who made a major contribution to the understanding of cells of the nervous system, so what the neurons were, and they also began to discuss some of the ideas about how synaptic transmission might occur. So Camillo Golgi was an Italian scientist. And in the mid to late 1800s, he developed a new stain which was based on using silver nitrate and silver chromate, and the stain was called the Golgi stain. And he used this to describe stain and then describe many different cells within the body. And he actually discovered what is called the Golgi complex because this was a prominent intracellular organelle that was very highly stained using the Golgi stain. We now know that the Golgi complex is involved in the movement of cargoes from the endoplasmic reticulum, through to the plasma membrane, so it's involved in membrane trafficking, it leads to the modification of specific proteins, and also the sorting of proteins into specific vesicles. Now, we have this investigator who was Santiago Ramony Cajal, and he was a Spanish neuroanatomist. And what he did was to use the Golgi stain to visualize cells in the nervous system. And so with this particular Golgi stains then, he was able to stain many different types of tissues. Now, he investigated the brains and the spinal cords and the peripheral nervous systems much more than Golgi did, so he was really a dedicated neuroanatomist, and he was also a brilliant artist as well, and he produced many beautiful and detailed drawings about what the neurons and other cells look like within the brain and the spinal cord. Based on his drawings and what he observed, Cajal proposed that the brain is composed of individual cells that are separate cells, but that they're contiguous, so they're right next to each other. And based on the junctions that he was able to see between the processes, he hypothesized that cells communicate at these specific areas, which were later called synapses. In contrast, Golgi thought that the cells in the brain were physically connected to each other and formed what he called a continuous reticulum or network. And he developed what was called the reticular theory. And the idea was that the cells were somehow physically connected to each other, and that molecules would be able to just move from one process of one cell directly to the process of another cell. So this isn't such a crazy idea because we know that in muscle cells, muscle cells are multinucleated cells. >> Groups of cells will begin to divide and then they'll lose their plasma membranes during development. And so this was a stretch of the idea that the whole brain might be one large multi nucleated cell in which the processes would be able to directly send information from one cell to another cell. But Cajal thought that was different at this time, the cell theory really had a great hold. And so he proposed again that all of the cells in the brain were single cells and somehow they would communicate at these specific junctions between them. Now with the microscopy that was available to Cajal at the time, he was not actually able to observe the synaptic cleft, but he could see things that were sort of reminiscent of the post synaptic density. And he thought that there was clearly a junction between cells that would show that the cells are separate, but somehow that communication would take place there. So he developed what is called the neuron doctrine today. And the whole idea that the cells communicate at synapse was called the neuron doctrine at the early part of the 20th century. And it's neat that both Golgi and Cajal shared the Nobel Prize in 1906 for their contributions to the understanding of the cells and these junctions in the nervous system. Now this shows one of the drawings by Cajal, and you can see that this is a drawing of cells that are found in the neocortex. This Golgi stain really stains the neurons and the neuronal processes very well. And so you can see predominantly the neurons here, the glial cells are in between the neurons, especially the astrocytes and maybe a few oligodendrocytes. And you can see the level of detail and what beautiful drawings that he was able to produce. So that we now call this region where neurons communicate with each other it's now called the synapse. This idea of there being a synapse was developed in the late 1800s by Dr. Charles Sherrington, actually by another investigator, Dr. Foster, at the suggestion of a classical scholar who was Arthur Verrall. The word synapse comes from the Greek synapsis, which means conjunction. And together to fasten together this conjunction. And this was just a decade or so after the actual word for the cells that are involved in signaling within the nervous system as neurons was proposed by von Waldeyer. And the concept of the axon was proposed by Kolliker and dendrites by Wilhelm His. These were a group of many neuroanatomists then, who were interested in using the observation of this tissue to try to understand what its function is. And today we have neuro anatomists still who are able to use microscopy and many sophisticated types of microscopy like immunofluorescence to be able to study the cells of the nervous system. So going back to the electrical signaling and the investigation in terms of the electrical properties of neurons then, at the beginning of the 20th century, then, there emerged big controversy about what the basis of synaptic transmission or neurotransmission was. And we had these two groups who were called the soups and the sparks. The soups were really the pharmacologist who thought that the cells of the nervous system communicated by chemical signaling. And the sparks were the electrophysiologists who thought that the neurons communicated by electrical signaling. And this debate about the basis of communication between neurons really ranged from the latter part, the 1800s all the way through until about the 1930s or the 1940s, when the first synaptic transmitters were identified and the mechanism of synaptic transmission was worked out. Of course, now we know that synaptic transmission at the majority of synapses, involves both electrical and chemical signaling. So this was not a mutually exclusive type of debate, though the investigators didn't realize this at the time. So now we have a characterization of the synapse. And the synapse is identified as the region of a neuron or the region between a neuron and its target cell, where synaptic transmission or what we call neurotransmission takes place. So going back to our first lecture, we talked about how neurons can form synapses and communicate with many different types of cells. This depicts the majority of synaptic transmission within the body. So this is the communication between one neuron and its target neuron. This is how the majority of signaling occurs within the central nervous system. In addition, neurons can communicate with muscle cells. They can communicate with either skeletal muscle, smooth muscle, or cardiac muscle. This is going to be a very different type of synaptic junction because the target cell will be very different. And of course the target cell has a specific function and that is in order to induce either muscle contraction or muscle relaxation. In the third type, we have a neuron which can communicate with a gland. And there are many examples of the junction between a neuron and a gland. For example, the synaptic communication that controls the adrenal gland or the lachrymal gland or the pancreas, for example. Because these target cells can be very different and their function of the target cell is very different, these different types of synapses are very different with respect to their neurotransmitters, their morphology, and of course, with the function, the functional responses that are produced in the postsynaptic cell. Going back to galvani this preparation, the neuromuscular junction, has turned out to be the best characterized of all the synapses within the body. Mainly because it is a very large synapse and it's very accessible, And it's quite a simple synapse. Its main function is to induce contraction of the muscle cell. So as we go through talking about synaptic transmission, we'll talk about the different neurotransmitters, acetylcholine, at the neuro muscular junction. And then in the last modules, we'll be talking about synaptic transmission between neurons in what we call central synapses. These synapses are much smaller, they can be much more complex. And of course, the function of the target neuron is to integrate all of the incoming synaptic activity and then produce an action potential. So it's much more complex with respect to the integration of synaptic responses. >> So we'll begin by talking about the two different types of synaptic transmission that can occur between a neuron and its target cell. And I should mention that the presynaptic part, so the sending region in synaptic transmission, is always an axon. It's always a presynaptic axon. So that is something that is constant even though the posynaptic cell and the posynaptic responses can be very different. So when we talk about synaptic transmission, we'll always depict a presynaptic axon as the place where the information will be flowing from. So the two main types of synaptic transmission occur at what we call an electrical synapse and a chemical synapse. And these are very different in terms of their mechanisms of synaptic transmission. At an electrical synapse, there is a very close juxtaposition between the plasma membrane of the presynaptic axon and the postsynaptic cell. And these membranes actually come in physical contact with each other because they form what are called gap junctions. And these are very large channel or pore like complexes, and half of the protein of this complex is contributed by the presynaptic axon, and half is contributed by the postsynaptic cell. These gap junctions allow for the movement of small molecules, including ions, directly from the presynaptic axon to the postsynaptic neuron. And so there are no chemicals involved except for the molecules that are moving through the gap junctions and there is no change in the electrical activity and there's no use of neurotransmitters at this type of synapse. Chemical synapses on the other hand are the synapses that use neurotransmitters and they have neurotransmitter receptors. So at a chemical synapse, there is the release of neurotransmitter into the synaptic cleft, the neurotransmitter binds to specific receptors that are found on the postsynaptic membrane, and its these receptors that produce the responses in the postsynaptic cell. Now, in terms of the numbers, there are a much greater number of chemical synapses than there are electrical synapses. And electrical synapses, though somewhat well characterized in invertebrates, haven't really been as well characterized in the mammalian nervous system. And so we actually don't know that much about the role and the function of electrical synapses within the mammalian brain. However, they are present and I'd like to talk about them for a few minutes, and this will be the only time that I'll really be discussing electrical synapses within this course and then I'll spend the rest of the time talking about the much more abundant chemical synapses. And this is the typical type of chemical transmission that you would have been introduced to in your previous foundation courses. So the electrical synapse. In an electrical synapse, what you can see is that if we measure a presynaptic action potential, this will produce a postsynaptic response that occurs very rapidly after the presynaptic action potential. So there is almost no delay between the presynaptic response, which is the action potential, and the postsynaptic response in this case, which is a large depolarization of the membrane. So the delay is very short, often on the order of only a tenth to two tenths of a millisecond. Now, because of the presence of these gap junction channels, small molecules such as ions can move directly from the presynaptic to the postsynaptic neuron. So you remember that the movement of ions is current. And so this means that the current will flow directly from the pre to the postsynaptic neuron. And these channels are called connexons, and each connexon is composed of six connexins that are located on the presynaptic membrane and six connexin proteins that are on the postsynaptic membrane. And these connexon gap junction channels are fairly non selective, and they will allow ions and other small molecules to move directly across the channel down their electro chemical gradient into the postsynaptic neuron. So ionic current will flow through the connexons then, and so if there is a depolarization of the presynaptic neuron because of the sodium current that is present underlying the action potential, which is followed by the potassium current, you'll see exactly the same currents in the postsynaptic neuron. So you have an initial sodium current that would be followed by a potassium current. So these electrical synapses are very fast, and this is much faster than chemical synaptic transmission because it involves just this direct flow of current. So electrical synapses have some very specific properties, and you can see here this depicts the sodium ions that would be moving into the postsynaptic neuron. And then these channels don't have any directionality to them and so potassium ions could be moving in the other direction as the action potential currents are moving through this channel. So as I mentioned, these channels actually form, they allow for these membranes to come essentially to become one. So this is the synaptic cleft in the typical gap between the pre and postsynaptic neuron. And this synaptic cleft is usually on the order of about 20 nanometers in diameter. But at the region where the connexons are forming, these gap junctions then will prevent there being a cleft here. So there's actually no space between the pre and the postsynaptic neuron. So as I mentioned, these synapses are thought to be pretty rare in the mammalian nervous system. So only a few or a small percentage of all the total synapses involve gap junctions. They're very different because of the distance between the pre and the postsynaptic neuron, because these channels form, they will then allow for these membranes to be very close to each other. And this type of communication is very fast. It is extremely rapid because it doesn't involve the release of anything, it just involves the movement of current across the gap junction channel. And interestingly, these electrical synapses are typically bidirectional. And that means that small molecules can move also from the postsynaptic cell to the presynaptic neuron. However, this type of synaptic transmission is very limited. And the reason for this is because it can only occur at the places where the presynaptic and postsynaptic neuron are actually right next to each other. And we're going to be talking about a type of transmission which is called volume transmission in which in chemical synaptic transmission, the chemical can actually diffuse quite a distance around a neuron and can affect target cells that are not as close to the presynaptic axon. The other limitation is there's no gain. So whatever response is produced in the presynaptic cell if it's a depolarization, will be exactly the same type of response but smaller because as you have the movement of current, that current can also leak out into the synaptic cleft and so the current will be lost just as it is in any type of graded signaling. So these are not very efficient synapses and they're also not adaptable. So there's no way to boost the signal and there's no way to change the type of response that we're going to see in chemical synaptic transmission, how the sine of a response can actually be changed by the presynaptic neuron receptors that are present there. But these synapses are very fast and they're important. They have been shown to be present in the hypothalamus and among mammalian brain hormone secreting neurons, they've also been demonstrated in the hippocampal interneurons and this thought to allow for the synchronization of neurons. And they're also present within the retina. So even though they haven't been that well characterized yet, they're believed to be very important in the mammalian nervous system as well, of course, in invertebrates where they're used in specific escape responses. >> Chemical synaptic transmission is very different, and this represents the great majority of synapses that are present in the mammalian nervous system. And in chemical synaptic transmission, you have the communication between a presynaptic axon and its postsynaptic target, even though as I mentioned, these targets can be very different. In chemical synaptic transmission you have the rapid release and diffusion of a neurotransmitter from the presynaptic axon into the synaptic cleft, then that neurotransmitter will bind to a receptor, and this results in a response in the target cell. Now these postsynaptic responses can be either what we call excitatory responses involving depolarization of the membrane potential, they can be inhibitory responses that lead to hyperpolarization of the membrane potential and in some cases, the responses don't involve any change in the membrane potential, but rather lead to the production of second messengers and other signaling cascades within the postsynaptic neuron. And depending on the type of receptor and the type of neurotransmitter, transmission in chemical transmission can be either what we call fast and direct where there is a direct effect on the membrane potential, or it can be slow and indirect, where there are slower and more long lasting changes within the membrane potential. And this depends on both the neurotransmitter that is being released at a chemical synapse, as well as the type of receptor that is detecting and responding to that neurotransmitter. So as I mentioned the chemical synaptic transmission represents the majority of synapses in both the central and peripheral nervous systems, and even though it is slower and the time delay between the presynaptic action potential and the postsynaptic response is on the order of 0.3-1 millisecond. And the reason for this is because it takes time to release the neurotransmitter for that neurotransmitter to bind to diffuse across the cleft, to bind to its postsynaptic receptors, and for those postsynaptic receptors to induce their specific functions. So if we were to look at the time course of the presynaptic and the postsynaptic response, you can see here that there is a time delay in this case on the order of about a half to six tenths of a millisecond between the peak of the presynaptic response and the peak of the postsynaptic response. Now, in chemical synaptic transmission the presynaptic signal is always an action potential because we've just learned about the action potential conduction down to the presynaptic region, and this is the only way, this is the only type of electrical signal that will be present in the presynaptic neuron so it's always going to be excitatory, but depending on the neurotransmitter and the receptors as I mentioned, the postsynaptic responses can be either excitatory inhibitory or modulatory. The advantage to this type of synaptic transmission is that it is much more plastic, so it can be adapted and it can be modulated so depending on what neurotransmitter is released and the types of receptors, then those responses can be modified and modulated. And because of the biochemical components that are involved there are many places for modulation of synaptic transmission, both at the presynaptic and on the postsynaptic side. So an interesting finding that has really just been demonstrated in the last decade or so, is that both chemical and electrical synapses are present in the mammalian CNS, and we don't really know exactly what the configuration of this combination of electrical and chemical synapses is, but here are some ideas about how they might be integrated. So here is a typical type of chemical synapse where we have the release of neurotransmitter and the presence of receptors on the postsynaptic membrane and here's our typical electrical synapses, where these membranes between the pre and postsynaptic neurons are very close to each other and they're going to allow for the formation of the gap junction channels which will allow the movement of current and other small molecules between the cells. So one idea is that they might both be present using the same presynaptic axon, but there might be two different postsynaptic cells, and these two different postsynaptic neurons might be using the two different types. Another idea is that an individual synapse so this would represent two distinct synapses from which a single presynaptic axon would be communicating with two different cells. In a mixed synapse, the idea would be that both of these types of synaptic transmission might occur between the same pre and postsynaptic neuron, and so you would have one region of the synapse which would employ chemical synaptic transmission, and the other part which would employ the electrical synapse. Another idea is that you could have a postsynaptic region which could receive two inputs from two different types of axons that would use different synaptic transmission so here is a region of the postsynaptic neuron receiving input from an axon that uses a chemical synapse and a different axon in another region that would be using the electrical synapse as its mechanism to communicate. And then this is the idea that for this has actually been demonstrated within lower vertebrate brits so in fish where this type of synapse has actually been identified using immunofluorescence microscopy, where at exactly the same synapse we have the presence of both gap junctions and some receptors which indicate that chemical synaptic transmission would be taking place. Of course these ideas are not mutually exclusive and it might be that some neurons in the nervous system use these two types of signaling at different target cells, or within the same target cells so I think that these different hypotheses are actually plausible. And it might be that these have different functions so one idea is that, in this direct electrical synaptic transmission might be involved in producing some type of current that would be very rapid and initially produced and then in chemical synaptic transmission, you might have an additional current which would be produced, which could either augment or offset this type of response. So in the central nervous system then we have talked about how synapses can be excitatory or inhibitory and so I wanted to spend a few minutes to talk about the different types of chemical synapses which are present within the central nervous system. And just to go back and remind you, an individual neuron can receive many hundreds to many thousands of different synaptic inputs. So even though we often draw a neuron like this where it's got a single synaptic input, the reality is that most neurons receive thousands of inputs and these inputs can be either excitatory or inhibitory or modulatory. And this is an example of this, so here we have a presynaptic region, which is called a presynaptic bouton, bouton for the French word for button and here we have this presynaptic axon that is communicating with two dendritic spines that are very close to each other, and so you can see this particular spine here then is going to be engaged in synaptic transmission on this part and this spine will receive the chemical neurotransmitter release on this part. So even though we often talk about synaptic transmission as being between a presynaptic axon and a dendritic spine, or a dendritic target it's much more complicated than this and the neuron will be integrating the responses, both EPSPs and IPSPs, as well as modulatory responses from these many thousands of inputs, all during the lifetime of the neuron when it is signaling. >> We talk about synaptic transmission and this type of synaptic transmission that we have typically introduced is actually what is now called wiring transmission, or point to point transmission. And this is the canonical type of synaptic transmission where you have a presynaptic axon and it releases neurotransmitter, and the receptors are found on the postsynaptic cell that are just right across from the presynaptic neuron. And so you have this very specific and direct type of chemical transmission. But it turns out that there is actually an additional type of neurotransmission, and this is called volume neurotransmission. In this type of transmission, the neurotransmitter is released into the cleft, but there's no barrier to its diffusion outside into the extra cellular fluid. And so in volume transmission, a presynaptic axon can actually lead to responses in a number of neurons or a number of dendritic spines that would be found in an area that would be around the presynaptic axon. So the neurotransmitter would work then in a volume around where it is being released to produce probably the coordinated response in a number of target regions. So now together, we often talk about synaptic transmission as neurotransmission because it involves both this point to point synaptic transmission and also the volume transmission. One of the ideas about the function of the astrocytes at the synapse is that it helps to compartmentalize this type of transmission. So here we have an Astrocyte. The Astrocyte would be producing some extra cellular proteins in this area and together, the Astrocyte and these proteins would form a barrier to the diffusion of the neurotransmitter from outside of the cleft. In addition, there is the presence of the extracellular matrix. So these are extracellular proteins and if they are found around this snaps too, they can also function as a barrier to the diffusion of the neurotransmitter. So it's thought that these barriers then are essential for the wiring transmission and to make sure that this is going to be compartmentalized. And there will be specificity such that only this region, in this case a postsynaptic spine, will be able to respond to the neurotransmitter. For volume transmission, the idea would be that there's a lack of either an astrocyte or the lack of extracellular matrix which will then remove the barrier and allow for these neurotransmitters to diffuse around into the extracellular fluid and be able to work on many targets that would be within the vicinity of the release site. So in addition to its function in taking up neurotransmitters and possibly releasing gleo transmitters. We talked about one of the function for the astrocyte in the tripartite synapse. And additional function then might just be to act as a physical barrier and to determine the specificity of the response and determine whether or not a particular region will be participating in only point to point wiring transmission or whether this presynaptic axon will be able to communicate with a variety of postsynaptic areas. Functionally we can talk about the different types of synapses that are present in the central nervous system and I've already introduced this idea. There are three major types of synapses, which are called inhibitory synapses, excitatory synapses, and then the third type, which is not depicted here, is the modulatory synapse. And these functional types of synapses are named based on the response that is being produced in the postsynaptic cell. Again, the presynaptic communicating region is always the presynaptic axon. And the response of the presynaptic region is almost exclusively using the action potential. So the action potential we think about as a large excitatory response followed by a repolarization of the membrane potential. But depending on the neurotransmitter and type of receptor, the response in the postsynaptic cell can be either inhibitory, excitatory, or modulatory. This depicts one of the observations in neuro anatomy, and that is that the spine synapses. So the synapses that occur between a dendritic spine are predominantly excitatory synapses, and they predominantly also use glutamate as their neurotransmitter. So about 70% of all of the synapses that are found within the central nervous system are located on dendritic spines. They use glutamate and they produce excitatory responses and this is a cartoon of an inhibitory synapse, which you can see is present on the dendritic shaft. And these are predominantly inhibitory, as shown here, though they can also be many excitatory synapses on the dendritic shaft as well. Here is this uses Gaba, Gaba is the main inhibitory neurotransmitter used in the central nervous system. And this produces an inhibitory response on the dendritic shaft region. So here this depicts the four morphological types of CNS synapses, so where a presynaptic axon can form a synapse and communicate with a postsynaptic neuron. I've already talked about these two. This is a spine synapse between an axon, presynaptic axon, and dendritic spine. This is a shaft synapse between a presynaptic axon and a dendritic shaft. And again, these are mainly excitatory. The shaft synapses can be either excitatory inhibitory or in some cases modulatory. Now the other place that there are a number of synapses on a neuron is on the cellsoma. And again, these synapses can be either excitatory or inhibitory but in general, there are more inhibitory synapses that are present on the cellsoma then there are excitatory synapses. And though it's rare, there is also an additional type of synapse, and this is called an axoaxonic synapse, where a presynaptic axon will actually make a synapse with the presynaptic region of another axon. And of course, if this is a myelinated axon, this would be the first place along the axon where the myelin would actually end and where this synapse could occur. But these are important because these will regulate the action potentials as they arrive at the presynaptic region. I wanted to introduce an additional morphological type of synapse, which has really only been demonstrated and characterized in the last decade. And it has not really made its way into the majority of neuroscience textbooks yet. And this is called en passant synapse. In en passant synapse, we have a presynaptic axon and what you can see is that this presynaptic axon has got lots of synaptic vesicles, and it is making a synapse with this postsynaptic dendritic spine region here. But what distinguishes the en passant synapse is that this axon does not end at this place. This axon will continue on and it will form a downstream synapse with another dendritic spine where there's a pre synaptic region. And it is now been speculated that there a great majority of the synapses that occur between a presynaptic axon and a postsynaptic dendritic spine are actually this type of en passant synapse. I will refer to this type of synapse as what I will call a terminal synapse, because the axon you can see here would be terminating at this region of the dendritic spine. >> We have these two major types of presynaptic morphologies then, where we have a terminal synapse between an axon and its target. We have an en passant synapse. I'm going to be talking about the number of neurons that are present within the central nervous system and the calculation as to the number of synapses that are present. And when we do this calculation, it is very useful to know that a single axon can make thousands of synapses with postsynaptic targets. And we're going to do this calculation in the next lecture, and it really helps us to do some bookkeeping so that all of the presynaptic and posyaptic regions will be able to add up with each other. So this is a micrograph showing an axon that is forming en passant synapses, And you can see what distinguishes this axon is the presence of the presynaptic synaptic vesicles, which are present in these presynaptic regions. So this is really the proof that this is forming functional synapses with many target spines and will continue on downstream. So you can imagine in terms of the time course, you would have an action potential which would be propagating along this axon, where it will induce synaptic transmission at this presynaptic region. And then a tenth or so of a millisecond later, it will induce a synaptic response in this postsynaptic dendritic spine. So that's a really interesting concept when we think about how information is integrated within a neuron where you would have a depolarization, for example, of a dendritic spine, which would then be initially produced in this region of the dendrite, and then would be moving along the dendrite where that information would then be integrated. I'd like to mention something else that I talked about when I talked about development of the nervous system. And that is even though we usually depict the synaptic cleft as being this large gap between the pre and the postsynaptic neuron, that's on the order of about 20-40 nanometers in diameter. Two things. First of all, the synaptic vesicle diameter is about 40 nanometer. Usually the synaptic cleft is drawn much larger than it actually is. The cleft is about the diameter of a single synaptic vesicle. So the pre and the postsynaptic neuron are much closer than they're usually depicted in the drawings. The other thing is that there are cell adhesion molecules which span the membrane from the presynaptic to the postsynaptic cell. And these are large cell Hees molecules which have a very large extracellular domain. So as you can imagine, they would need many amino acids to be able to span this distance. And some of these cell adhesion molecules form what we call homotypic interactions. So for example, the N-cadherins or the NCAMs, which stand for the neuronal cell adhesion molecules will bind to exactly the same molecule on the opposite membrane. Some of these cell adhesion molecules involve heterotypic interactions. So here, for example, you have the neurexin protein from the presynaptic side which binds to the neuroligin protein on the postsynaptic side. And these interactions involve the types of non covalent protein interactions that we talked about that help to form the secondary and tertiary and quaternary types of structures. These are reversible types of bonds, but there are hundreds of these different bonds which will then help to form and stabilize this interaction. Even though we often depict the pre and the postsynaptic neuron as really not having any type of communication with each other, except during synaptic transmission, through these transmembrane proteins then, these call adhesion molecules can actually lead to the communication at all times, even before and after synaptic transmission. You can see here that these cell adhesion molecules have got intracellular domains, and these intracellular domains combine to proteins that we call scaffolding proteins, which help to build a complex scaffold of proteins at, for example, the postsynaptic density region. They also combine to cytoskeletal proteins, so actin binding proteins, and regulatory proteins that regulate the actin cytoskeleton. And it can also bind signaling proteins and probably lead to the activation or inhibition of specific signaling cascades. Now, one of the reasons that the cell adhesion molecules have received a lot of attention in the last decade or so is because mutations in these two proteins, the neurexins and the neuroligins were identified as the first genetic mutations that are present in autism individuals. So through genetic analyses, a number actually up to about 75 different genes have been implicated as contributing to autism and autism spectrum disorders. And these mutations in these two proteins were the first to be identified. So we've talked about the astrocyte then and we talked about the astrocyte when we talked about the function of glial cells. About how astrocytes can take up neurotransmitters and they can also potentially release molecules into the synaptic cleft, and this can contribute to synaptic transmission. And then now I've also mentioned another potential function for the astrocyte, and that is to determine whether synaptic transmission will be point to point or volume transmission. So this is just a review slide to show the importance of the astrocyte in this region here. And because we have a presynaptic neuron, a postsynaptic neuron, and now an astrocyte, this is how the tripartite synapse got its name. And I will end with this topic in this lecture, and that is the idea that synapses are not fixed during development, and they are not fixed in the adult brain either. So this shows that during development there is a peak of synaptogenesis, which occurs between birth and about four to five years of age. But then we have the loss of synapses through synaptic pruning and the process of synaptic elimination. And this is thought to be an activity dependent process in which the synapses are initially produced in an overabundance, but only the active synapses will be retained and strengthened. The synapses that don't receive any activity are the synapses which are thought to be eliminated. And these processes of synaptogenesis and synaptic elimination are thought to occur throughout our adult life in specific regions within the brain. Here we have a process of long term potentiation in which a dendritic spine is becoming larger. There are more receptors here. And this synapse would be producing a larger response. And so we have synaptic plasticity, which leads to both an increase or a decrease in specific synapses. This type of synaptic plasticity is also thought to be extremely important not only during development but in the adult brain because it's thought that this type of plasticity is the mechanism underlying learning and memory for specific types of memory systems.

In this lecture, I'll continue to talk about synaptic transmission. Synaptic transmission can be divided into specific stages. And in the next two lectures, I'll be talking about these specific stages which are depicted in this figure. First, synaptic vesicles, which contain neurotransmitters, have to be filled with those neurotransmitters by specific mechanisms following their synthesis. Then the action potential travels down the axon to the presynaptic region, and this will depolarize the presynaptic membrane. And following that, the neurotransmitter is released, it diffuses across the cleft, binds to receptors, and produces changes in the postsynaptic membrane or which lead to changes in the membrane potential or the production of second messengers. Before I start talking about this specific mechanisms, I wanted to discuss how the first neurotransmitter was discovered. So it turns out that acetylcholine, and the structure acetylcholine as shown here, was the first neurotransmitter to be identified. And it was first identified by Dr. Otto Loewi in Germany in experiments that he published in the early 1920s. And in his experiment, he actually used an isolated system to demonstrate that a chemical was involved in mediating synaptic transmission. And together with Henry Dale, he shared the Nobel Prize in Physiology or Medicine in 1936. So what Dr. Otto Loewi's experiment was that he had originally determined that when he stimulated the vagus nerve, which is one of the parasympathetic nerves, this leads to a decrease in the heart rate as well as the force of contraction. And this is a depiction of the vagus nerve. It originates within the brain stem within the medulla oblongata, it extends out of the brain stem, and it will innervate specific regions of the heart. Now importantly, the heart is also innervated by a sympathetic autonomic neuron, and it originates within the spinal cord. But what Dr. Loewi did was to remove the heart from an animal together with the vagus nerve intact. And using this experiment, what he did was, with this isolated preparation, he could stimulate the vagus nerve and this would then lead to a decrease in the contraction force and the contraction rate that he could measure within this isolated preparation. So the experiment was that he isolated an additional heart and put it in a chamber which was connected to the original heart chamber. And so the solution from this initial chamber could flow to the second chamber. And what he noticed was when he stimulated the vagus nerve and measured a decrease in the force of contraction in the innervated heart, that a short time after that, if he measured the contraction in this naive heart that was connected to the perfuse, he saw a similar but slightly delayed type of response. And he initially called, this perfuse substance, he called it vagusstoff. And this was then later identified as acetylcholine. So the idea was that something was released, some diffusible molecule was released from the vagus nerve, and when it was applied to this naive denervated heart, it would produce the same type of response in the second heart. And a few years later, he together with other investigators, purified the perfused and discovered that the molecule was acetylcholine. Now, what's interesting about this discovery is, first of all, it was discovered in the peripheral nervous system. So when we actually think about the total number of synapses that are in the nervous system altogether this peripheral nervous system type of synapse actually represent just a small fraction of the total. The other thing is that we're going to be talking about the mechanism of acetylcholine action. And it turns out that this mechanism involves a type of receptor, which is called the muscarinic acetylcholine receptor which actually uses a second messenger pathway in order to produce its effects. So we're going to be talking about the two different types of neurotransmitter receptors, which are the ionotropic and metabotropic receptors. And this was actually a type of metabotropic receptor which mediates the slower type of synaptic transmission. Now there are about a dozen or so neurotransmitters that have been identified. And back in the 1960s and '70s the game was on to try to identify novel neurotransmitters. And at that time, neuroscientists came up with a set of rules that had to be followed or had to be met in order for a particular substance to really be considered a neurotransmitter. Of course, it has to be present. It has to be synthesized and packaged and released from the presynaptic neuron from that axon. And it would work, it would act by binding to and activating postsynaptic receptors and producing a biological effect. In addition, it was thought that after that chemical would be released, it would need to be removed from the synaptic cleft. Because it was already known that synaptic transmission was transient. And so the idea was that there needed to be some a removal mechanism. And then in experimental preparations, it needed to be demonstrated that if one applied the chemical to the postsynaptic cell, it would be able to produce the same type of response in the postsynaptic neuron as the endogenous neurotransmitter would. And following these types of experiments then there were two major categories of neurotransmitters that were discovered and defined. And we now refer to these as the conventional neurotransmitters and the unconventional neurotransmitters. The conventional neurotransmitters meet all of the criteria of these type of historical neurotransmitters, whereas the unconventional neurotransmitters actually do not. So within the conventional neurotransmitters, there are two major categories. And these are the small molecule neurotransmitters and the neuropeptide neurotransmitters. So first, the small molecule neurotransmitters have a small molecular weight that's similar to acetylcholine in terms of its molecular mass. These neurotransmitters are synthesized in the presynaptic terminus, so that at the end of that axon, within the terminus itself, or within the pre synaptic region. Then these small molecule neurotransmitters will be pumped into and stored into the specific type of vesicles, which are the synaptic vesicles. Those are those 40 nanometer vesicles. So they're smaller than a secretory vesicle typically, and they butt off from the recycling endosome that is found within the presynaptic neuron. Small molecule neurotransmitters can produce either very rapid effects or slower effects on their target cells. So they can lead to direct changes in the membrane potential, or they can have neuromodulatory effects through the production of second messengers. And in general, one of the rules is that most neurons synthesize only one specific type of small molecule neurotransmitter. So if you have for example the vagus nerve, which would be coming from those motor neurons within the brain stem. Those neurons would only be synthesizing acetylcholine. However, more recently it's actually been discovered that there are quite a few exceptions to this rule. So there are some neurons, for example, in the spinal cord, which are thought to synthesize both GABA and glycine, which are inhibitory neurotransmitters. And some other places in the nervous system where there might actually be too small molecule neurotransmitters which are synthesized and released. These small molecule neurotransmitters are produced by enzymes which are found in the presynaptic region. And as soon as they're synthesized, then they will get transported within the synaptic vesicles. Neuropeptides are also neurotransmitters, but they're quite different both with respect to their molecular structure, as well as how they are released. As peptides, and peptides, you remember, are small proteins. And secreted proteins are synthesized within the endoplasmic reticulum, they get trafficked through the Golgi and transported into specific vesicles. And for most neurons, this machinery for the synthesis of proteins and peptides is located within the cell body. So neuropeptides are synthesized within the cell soma, and they are transported in vesicles down to the presynaptic terminus by fast axonal transport. They are packaged into secretory vesicles. And in some cells, these secretory vesicles are referred to as dense core granules because they have a dense inner region as viewed by electron microscopy. Because they have a lot of proteins, a lot of these peptides inside the granules which pick up the EM stain. The effects of neuropeptides are almost always neuromodulatory. So they don't usually produce fast or slow, excitatory, or inhibitory postsynaptic potentials, but they work through the production of second messengers. And these second messengers can change the activities of a lot of enzymes and functions within the posnaptic neuron. In general, neuropeptides are released together within the same pre synaptic region with small molecule neurotransmitters. Together, they're actually released, of course, from separate vesicles, but an individual neuron usually will synthesize and release one type of small molecule neurotransmitter, and one type of neuropeptide. And we often refer to a specific type of neuron as a subtype, depending on what neuropeptide it will produce. So we can see here that these types of conventional neurotransmitters are packaged into vesicles, and they are usually released from the presynaptic terminus. Unconventional neurotransmitters, on the other hand, are very different. These types of neurotransmitters are hydrophobic molecules, and there are two types. One group are derived from fatty acids and these include what are called the endocannabinoids, and these are lipid molecules which are released in response to synaptic transmission. The other category are the small molecule gases, like nitric oxide, and carbon monoxide, and hydrogen sulfide. As small molecule gases, these are also hydrophobic molecules and therefore, they can diffuse directly across the plasma membrane. So they are not stored within vesicles. These also are usually neuromodulatory, they can be synthesized by either the presynaptic or the posynaptic neuron. They are not stored in vesicles because they are hydrophobic, and they can cross the membrane very easily. And in many cases, these are produced in response to synaptic transmission that involves a small molecule or neuropeptide neurotransmitter. So as we talk about synaptic transmission, we're going to talk about these unconventional neurotransmitters because they have this hydrophobic nature. In some cases, it's thought that they might actually function as what are called retrograde signals in synaptic transmission. So they may be synthesized by the postsynaptic neuron, and because they can diffuse across the membrane, they can diffuse back to the presynaptic neuron and modulate the presynaptic neurons activity. So in the next few lectures, we'll be talking and focusing on the conventional neurotransmitters. And we can distinguish these from the unconventional neurotransmitters, again, because they are stored in vesicles. For the small molecule neurotransmitters, they are stored in synaptic vesicles, whereas the neuropeptide transmitters are stored in secretory vesicles. So one of the differences between these types of vesicles is that synaptic vesicles are smaller. They're about 40-50 nanometers in diameter, whereas the secretory vesicles are larger, on the order of about 100-200 nanometers. In addition, if you use electron microscopy to visualize these peptide, or small molecule containing vesicles, then you can see that they can be distinguished by how they look in electron microscopy. The synaptic vesicles are usually clear so they don't have a dense core. And the reason for this is because the synaptic vesicles do not contain any proteins, or peptides in the lumen of the vesicles. In contrast, the dense core vesicles contain lots of peptides, because that's what's going to be released from these vesicles, and so they pick up this particular stain, and they have a dense core to them. So they differ. These two types of vesicles both function as neurotransmitters but they differ with respect to their synthesis, their packaging within the vesicles as well as their localization, and what is required for them to be released. In general, the synaptic vesicles are usually found very close to the region of the neuron that is right next to the region of the axon that is juxtaposed to the postsynaptic cell. Whereas these secretory vesicles containing neuropeptides are usually localized much further away from this particular presynaptic zone. And because of their differences in localization, they have different requirements in order for them to release their neurotransmitter into the synaptic cleft or in the case of the neuropeptides into the extracellular fluid. So we'll begin to talk about the different types of small molecule neurotransmitters and neuropeptides. And we'll discuss where these neurotransmitters are synthesized, where they're released, where their mechanisms are. So it turns out that there are three major categories of small molecule neurotransmitters. We have the amines, so these are small molecule transmitters that are distinguished by their amino groups. There are the amino acids, and these are neurotransmitters which are either directly the L amino acids like glutamate and glycine which are synthesized and also used for protein synthesis. And then the third category are the purines. Now we can see within the amine category there is acetylcholine and it gets its own specific category because there are no other neurotransmitters that are similar in structure or in function. The other category within the amines are what we call the biogenic amines, and another name for these, is the monoamines. And within this category, there are three subcategories which are the catecholamines, including dopamine, norepinephrine, and epinephrine. The indoleamine, which is serotonin, and the imidazoleamine which is histamine. Interestingly, the biogenic amine neurons are localized. The neurons which synthesize and release these neurotransmitters, their cell bodies are localized in some very discrete nuclei that are found within either the brain stem or within the hypothalamus. For acetylcholine, there are cholinergic neurons that are localized both in the CNS as well as the PNS. And for the amino acids and the purines, these are typically used predominantly within the central nervous system. The other category of conventional neurotransmitters are the neuropeptides. And this shows an example of some of the neuropeptides. So neuropeptides are often synthesized as larger precursors. So you can see here's an example of a larger precursor. And these larger precursors will undergo a specific cleavage by proteases. And the proteases will break this precursor. They will break the bonds and produce smaller molecular weight peptides from the larger precursor. So this is the structure of a peptide neurotransmitter that has 1, 2, 3, 4, 5 amino acids in it and this is just a typical size. Neuropeptides can range from about three amino acids up to about 36 amino acids in length. And the smallest ones are usually produced from larger precursors. And then of course, for the larger ones, they don't need to undergo any different type of proteolysis. Now, over the course of the last 30 or 40 years, many different neuropeptides have been discovered. And one way that neuropeptides can be categorized is where they are synthesized or by what their function is. So we have, for example, the brain gut peptides, the pituitary peptides, the hypothalamic releasing peptides. So these are all named by their specific place where the neurons are that synthesize and release them. And then we have another category, which is called the opioid peptides. And these are the endogenous peptides that work at the opioid receptors and activate the similar types of responses as the opioid drugs do. Now we'll begin first to talk about the small molecule neuropeptides, and then we'll talk about the neuropeptides. But I wanted to compare and contrast just where they are synthesized and where they are localized again. So small molecule neurotransmitters are synthesized at the pre-synaptic region. And usually it is soluble enzymes that are going to synthesize these neurotransmitters. Once the enzymes will use the substrate to produce these small molecule neurotransmitters which are of course, small soluble molecules, these will be transported into synaptic vesicles, which have just been generated by the early endosome. And then these synaptic vesicles will be localized at a specific region which is called the active zone. And the active zone in the presynaptic region is a zone or an area where the neurotransmitter will be released. And so these vesicles will then be clustered in the presynaptic region. We'll talk about the mechanisms of neurotransmitter release later on in this slide. But first, these vehicles are just localized and docked within the presynaptic region. All of the membranes and the transmembrane proteins that are involved in the synaptic vesicle have to be shipped down by fast exonal transport to the presynaptic area. In contrast, the enzymes which are soluble enzymes which are involved in the synthesis of neurotransmitter, actually just depend on slow axonal transport in order to make their way from the cell body where they're synthesized down to the presynaptic terminus. In contrast, neuropeptide transmitters are synthesized within the cell soma, and here is the endoplasmic reticulum, which will be synthesizing these neuropeptides. And they'll be inserted into the ER lumen co-translationally. They get packaged into vesicles, and those vesicles fuse with the Golgi apparatus, and then those vesicles will move through the Golgi, and eventually, they will be sorted into secretory vesicles at the trans Golgi network. In many cases, the neuropeptides are synthesized as a larger precursor. And they get packaged together with the proteases that will cleave them. And so as the vesicle moves along the microtubules by fast axonal transport, the neuropeptides will undergo maturation, which involves the cleavage from the large precursor into the final smaller neuropeptide that has matured. These vesicles, usually the neuropeptides are usually localized in these regions that are much further away from the active zone. So in many cases, they'll be localized on the side of the presynaptic region or in this region here. And in some cases, they will be released into the synaptic cleft, but in other cases, they'll actually be released into the extracellular fluid that is found in this region, outside of the synaptic cleft. Because they become localized much further away from the active zone, they require much greater presynaptic activity in order to get those neuropeptides released. And as I mentioned, most neurons release at least one small molecule neurotransmitter and one neuropeptide transmitter. So this is a single presynaptic axon terminus, and it is forming a synapse with a single postsynaptic target. And we have the transport of the enzymes which will be producing the small molecule neurotransmitters. And then the small molecule neurotransmitters will get packaged. They'll get transported into the synaptic vesicles and they will then release the neurotransmitters into the cleft. So as this vesicles fuse, then they'll release the neurotransmitter, and it will then be localized in this specific region where it will interact with its receptors. For the neuropeptides, which are localized in the secretory vesicles here called dense core vesicles. You can see that they will end up being much further away from the actual synaptic region. They'll be much further away, and these neuropeptides will then be released into an area that surrounds the synapse in the extracellular fluid. And therefore, it's thought that the neuropeptides can then work both on synaptic receptors as well as receptors that are localized in what's called the extra synaptic region, and also potentially on nearby neurons. So neuropeptides traditionally work in what we call volume transmission. And some of the small molecule neurotransmitters in this case depicted here will be working in point to point or wiring synaptic transmission. So I want to make an important distinction before we talk about the mechanisms of synaptic transmission. And that is that we already talked about how neurons receive many hundreds to thousands of different synaptic inputs. So they're going to receive neurotransmitters at the postsynaptic side from many different types of neurons. However, when we talk about the name of a specific neuron, or the type of a specific neuron, we name that not for all of the synaptic inputs that it receives, but rather for the neurotransmitter that that particular neuron will synthesize and release from its axon. So this, for example, could be a glutamatergic neuron. This particular neuron is synthesizing and releasing glutamate from its presynaptic terminus but you can see here that it is receiving both excitatory glutamatergic, as well as inhibitory GABAergic, and probably lots of modulatory inputs as well. And as I mentioned before, the postsynaptic neuron and synaptic transmission can become very complex. So this is a depiction of a dendritic spine, and you can see here, this is a presynaptic glutamate, a neuron, a glutamatergic neuron which will release glutamate at this dendritic spine. But you see here, this is a neuron which has an axon, this is coming from a dopaminergic neuron and you can see on this part of the dendritic spine, there will be dopamine released, and there'll be dopamine receptors here, which are going to be able to integrate with the glutamate response to produce the overall response within this dendritic spine. And then it becomes even more complicated, because this would be an axon from a neuron that synthesizes acetylcholine, so this would be from a cholinergic neuron. And you can see that it would be releasing acetylcholine by volume transmission into this entire area here and would be able to work on receptors for acetylcholine that would be present on this particular axon or this dendritic spine, or this dopaminergic axon. And so the mechanisms of synaptic transmission integration can become exceedingly complex as we begin to build these types of circuits in terms of how the brain really functions. So one thing that is similar about all of the small molecule neurotransmitters is that they're synthesized by enzymes and they're transported into synaptic vesicles at the presynaptic terminus. And so here we have the presence of our synaptic vesicles and these would be the synaptic vesicles that would be specific for the different types of neurotransmitters. But what you can see here is that each one of these mechanisms for the transport of the neurotransmitter into the synaptic vesicle is very similar. So small molecule neurotransmitters are transported into synaptic vesicles by a specific type of transporter that's called the vesicular Neurotransmitter transporter. And I've already introduced this when I was talking about secondary active transporters. These transporters use a gradient, which is the proton gradient that is established first by the proton ATPAs, which is in the membrane of the synaptic vesicle. And then they use that in an antiport mechanism to transport that specific small molecule neurotransmitter into the lumen of the vesicle. And we think about just trying to estimate the concentration of neurotransmitters. It turns out that there are approximately 3,000-9,000 neurotransmitter molecules within a single vesicle. So the concentration of neurotransmitter is actually quite high within the inside of these vesicles. And neurophysiologists often talk about synaptic transmission as being quantal, and when we go back and think about this quantal release of neurotransmitter, we can consider the amount of neurotransmitter that's present within a single synaptic vehicle, we'll refer to that as a quantum of neurotransmitter. Because it's a discrete unit and it's packaged within this discrete unit. So now I'll start talking about the mechanisms of synaptic transmission. So keeping track on our figure of the numbered steps in synaptic transmission, I've just gone through Step 1, which is the synthesis and packaging of neurotransmitters into their specific vehicles. The second stage is that the action potential arrives within the presynaptic area. And we've talked all about action potential generation and conduction and the action potential will be conducted in this myelinated axon in one direction, from the cell all the way down to this presynaptic region here. Now, this slide also depicts the two different types of morphological types of axons and presynaptic termini that they can develop. This would be considered what we call a terminal synapse. So in this type of synaptic connection, then we would have an axon which would end, it would terminate when it made a synapse on the postsynaptic target. But as I've mentioned, there are also these synapses which are called en passant synapses and they contribute a substantial number to the total number of synapses that are present within the central nervous system. So in these synapses, we have an axon, which is depicted here, and it will form a synapse so this will be the presynaptic region. But then the axon will continue on, where it will be able to make a synapse with the downstream, another downstream target. And there can be many thousands of synapses that are produced by a single axon. In both cases, what will happen is that this depolarization will reach the presynaptic region, either here at the terminus or here at this presynaptic area here. Because action potential, we know is a large depolarization of the membrane potential, followed by a repolarization and what happens is this depolarization of the membrane potential will activate a specific type channel, which is a voltage gated calcium channel that is localized within this presynaptic region. Through the activation of these voltage gated calcium channels, this will lead to the influx of calcium, which will then stimulate and activate the fusion of the synaptic vesicles and the release of neurotransmitter into the synaptic cleft. So here is our depolarization and repolarization then and that is going to occur within the presynaptic region that membrane, and having the voltage sensors there, which are the voltage gated calcium channels, is going to allow for the conversion of the electrical change, the membrane potential, into an increase in calcium levels at the presynaptic neuron. Now, in the next lecture, I'll be talking about calcium. Calcium is the major chemical that first converts that electrical signal into a stimulation of the neurotransmitter release.

In this lecture, I'll be talking about the mechanisms of synaptic transmission at the presynaptic side. As I mentioned before, the different steps of chemical synaptic transmission have been identified. And the first step is that the synaptic vesicles are filled with neurotransmitter. And we talked about this in the last lecture, and I'll discuss this further when we talk about the individual neurotransmitter systems. Then the action potential travels down the axon and arrives at the presynaptic membrane. And what this does is to depolarize the presynaptic membrane. Then in the next steps, the neurotransmitter is released, diffuses across the cleft, binds to receptors, and induces changes in the postsynaptic membrane potential, or leads to the production of second messengers. So today we'll be talking about the steps which involve the release of neurotransmitter, how that depolarization is then converted into an increase in neurotransmitter levels in the synapse. So the action potential arrives at the presynaptic terminus. And we often talk about this function as being the invasion or the depolarization of the presynaptic terminus. And for a synapse, which is a terminal synapse then, this will lead to a final depolarization of the membrane in the axon. For en passant synapses, when you have an axon that makes a synapse and then continues on, then the action potential will depolarize the presynaptic region, but then the action potential will continue on because of the presence of voltage gated potassium and sodium channels, and the action potential then will be conducted along the axon, and this will depolarize the presynaptic region of each of the synapses that are found along the length of that axon. And you remember that the action potential is a large depolarization of the membrane potential from the resting membrane potential all the way up to the peak of the action potential, which is anywhere between +20 and +40 millivolts. This is a very large depolarization. About a 100 millivolts change in the membrane potential, it's very short lived on the order of about a millisecond or so to the peak, and then the repolarization phase, which involves bringing the membrane potential back to the resting membrane potential and hyperpolarizing it even beyond, such that the total action potential is about 3-4 milliseconds. So this is a transient depolarization of the membrane potential at the presynaptic region. Before I start talking about synaptic transmission at the presynaptic side, I need to introduce some characteristics of the calcium ion. The calcium ion is a divalent cation and it's a metal ion. And unlike the other ions that we talked about, sodium and potassium and chloride, calcium ions also have an additional function, and that is that they act as messenger molecules. Now, the concentration of the calcium ion is manipulated. It's affected by changing its concentration within the cell and in different compartments. Unlike other messenger molecules, it is not synthesized or degraded, and therefore, its concentration has to be changed by changing its localization within the cell. Once calcium levels increase within the cell, it can bind to specific calcium binding proteins, and when it binds to those proteins, it induces conformational changes that lead to a change in the function of those proteins. Now, unlike the other ions that we talked about, sodium and potassium and chloride, which have gradients on the order of about 10 times to 20 times between the outside and the inside, or the inside and the outside, the concentration gradient for calcium is much greater. The concentration of extracellular calcium is on the order of about 1-2 millimolar, but on the inside of the cell, the concentration is on the order of about 50-100 nanomolar. And what this means is that there is an approximate 10,000 fold gradient in the calcium concentration between the outside and the inside. And one of the reasons that this has arisen during evolution, it's thought, is because when the calcium concentrations are fairly high within the cell for a sustained period of time, this is a signal for apoptosis. And so it's thought that in order for cells to maintain their cell survival and prevent apoptosis then, they have evolved some sophisticated mechanisms in order to keep the intracellular calcium concentration at an extremely low level. These mechanisms include both primary and secondary active transporters, as well as calcium binding proteins. At the level of the plasma membrane, there are two active transporters which essentially pump calcium out of the cell in order to remove the calcium from the inside to the outside and keep the calcium intracellular concentration very low. One of these is a calcium ATPase or a calcium pump. And like the sodium potassium ATPase, it uses the energy that's provided when ATP is hydrolyzed in order to pump calcium up its concentration gradient outside of the cell. So this is a type of primary active transporter. In addition, there is a secondary active transporter, which is the sodium calcium exchanger, which exchanges a molecule of sodium for a molecule of calcium. So this type of secondary active transporter uses the energy that's built into the sodium gradient, so sodium flows down its concentration gradient and at the same time it exchanges calcium which is pumping calcium outside of the cell. Now interestingly, there are also intracellular mechanisms that help to regulate the calcium concentration as well. The first one is found in the endoplasmic reticulum. And this is another calcium ATPase, or calcium pump. The primary active transporter that is a different gene product than the plasma membrane transporter, but it has very similar function and structure. And basically what it does is to pump calcium from the cytoplasm inside the lumen of the endoplasmic reticulum and it uses ATP hydrolysis to do that movement of the ion. There is a calcium transporter which is found in mitochondria, which is called the calcium uniporter. It's a secondary active transporter that transports protons inside the mitochondria and brings calcium with it. This pool of calcium is thought to be a fairly stable pool. It's not a dynamic pool, whereas the endoplasmic reticulum calcium pool is thought to be a dynamic calcium pool. And the reason for this is that there are calcium channels that are localized within the endoplasmic reticulum membrane, and when these gated calcium channels are opened, they allow calcium to flow from the inside of the ER into the cytoplasm and can increase the cytoplasmic calcium concentration through this intracellular store. In addition, there are calcium binding proteins that are found in the cytoplasm which help to buffer the calcium mechanisms. So through all of these different mechanisms then the intracellular calcium concentration is kept at this extremely low level on the order of about 10^-7 molar. >> We now know that calcium is required for synaptic transmission. And in a series of experiments, it was demonstrated that calcium is essential for synaptic transmission. However, it is not involved in the action potential in the presynaptic neuron. So in order to do this type of experiment, investigators depolarized the presynaptic axon. And then were able to measure a response, a depolarization, in the postsynaptic cell. If this experiment is done in the presence of a specific inhibitor, which is cadmium, and it turns out that cadmium blocks the calcium channels which are present in the presynaptic neuron. Then what that does is to block the postsynaptic response in response to a presynaptic depolarization. Now what this cadmium does is it blocks the calcium current that is activated by the depolarization in the presynaptic neuron. So here you can see that there is a presynaptic calcium current that occurs in response to depolarization and this presence of cadmium blocks this. So this demonstrates that calcium is required for the production of a postsynaptic response that is produced by a presynaptic depolarization and here this mimics the action potential. Now in this similar type of experiment, this shows an endogenous action potential that is being produced. And then the investigators could measure a postsynaptic depolarization of the membrane that would be produced in response to this presynaptic action potential. If a calcium buffer such as EGTA is present within the presynaptic neuron, you can see that what this does is to block the posynaptic depolarization that is produced in response to the presynaptic action potential. But what you can also see here is that the presynaptic action potential is not affected by the presence of the calcium buffer at all. Which demonstrates that while calcium is required for synaptic transmission, it is not required for the presynaptic action potential. And then they went on and did another nice experiment to determine whether calcium alone is sufficient to induce synaptic transmission. So in this experiment, they injected calcium into the presynaptic axon and then measured the postsynaptic response. And you can see here, the injection of calcium on its own, even without a presynaptic action potential, is able to produce a postsynaptic response. So from these studies, then, it was concluded that calcium is necessary for synaptic transmission, and it's also sufficient in the presynaptic side for synaptic transmission as well, but it is not required for the action potential. Now, the way that the calcium levels are increased in the presynaptic neuron is by activation of a voltage gated calcium channel. So we've learned about voltage gated sodium and voltage gated potassium channels. And the voltage gated calcium channels are very similar in their structure to the voltage gated sodium channels. And they work by a very similar type of mechanism as all the voltage gated channels that we've talked about. It turns out that there are 12 transmembrane spanning domains, and these are repeated four times, and within each one of these repeats is this region which is called segment 4, the S4 region. And just like the voltage gated sodium and potassium channels, this S4 region is the voltage sensor region. What happens is when the membrane potential depolarizes within the presynaptic neuron, where these voltage gated calcium channels are localized. This leads to a movement of the voltage sensor, and then that conformational change is transduced into the opening of the voltage gated calcium channels. So while the voltage gated sodium and potassium channels are required to both produce and to propagate or conduct the action potential it's the voltage gated calcium channel that then senses the depolarization that is produced by the action potential. And this leads to an opening of the voltage gated calcium channels and an influx of calcium into the presynaptic neuron. Now, because we typically depict the presynaptic neuron as being in this region and the post neuron as being underneath it, then I've just flipped around this channel so that we can depict it as it would be working in the presynaptic region. So here is the voltage gated calcium channel, and you can see that it has this large Alpha sub unit here, which contains these 12 transmembrane spanning domains. Each one is a repeat, and each one of these repeats has got the S4 region. Interestingly, in addition, just like the voltage gated sodium channels, there are also auxiliary subunits for the voltage gated calcium channel. And it's thought that these subunits are very important for localizing the voltage gated channels to a specific regions within the posnaptic terminus or the posnaptic region. Now this is a dendrogram that depicts the different voltage calcium channels that have been identified. And you can see that there are several families of voltage gated channels. There is a family called the L-type, which stands for the large type, the P/Q, and the N-type of channels which are related in terms of their structure, and then are the T-type of channels. Each one of these channels is a different gene. It's a different type of protein, and it has differential expression in neurons and muscle cells. And for the presynaptic neurotransmitter release. It turns out that it's the N-type and the P/Q-types of channels that are localized in the presynaptic region. Their dedicated function is to be involved in the release of neurotransmitter. These other types of voltage gated calcium channels, the L and the T-type are found in muscle cells. They're also found in the dendrites of neurons. And they have slightly different channel properties and activities and they have different functions. For example, the L-type channels that are found in skeletal muscle are involved in what's called excitation contraction coupling. So they basically are involved in transducing the muscle action potential into the release of calcium and muscle contraction. And then there are the T-type channels which are involved in regulating pacemaker activity within the heart cardiac muscle. >> What happens is that we have the action potential and it travels down the axon to the pre synaptic region. And the voltage gated calcium channels are localized at a particular region, which is called the active zone. Now at the active zone in the presynaptic region, this is the region where the synaptic vesicles are going to fuse with the presynaptic membrane and release their neurotransmitter into the cleft. These voltage gated calcium channels are closed at the resting membrane potential, and then they are activated and open in response to the depolarization that is produced by the action potential when it arrives at the presynaptic terminus. The calcium will then flow down its electrochemical gradient, this calcium will then stimulate the release of neurotransmitter into the synaptic cleft. These voltage gated calcium channels are very interesting because while they have a voltage sensor region, and they also have an overall structure that is very similar to the voltage gated sodium channels, they do not in general inactivate. They're much more similar to the voltage gated potassium channels in this particular feature. As I mentioned, they are localized to specific regions which are very close to where the synaptic vesicles are located, and so there is a very local increase in calcium concentration that is produced when these voltage gated calcium channels will open. The next step then, so we have an increase in the calcium concentration in the presynaptic neuron. And the next step is to induce the fusion of the synaptic vesicles with the presynaptic membrane. This is a cartoon depiction of a typical synaptic vesicle, and you can see that it contains a number of transmembrane proteins and also some peripheral associated proteins as well. Now, two of these proteins are critical for filling these synaptic vesicles with neurotransmitters. This is the proton ATPase, and this is a primary active transporter that pumps protons inside the lumen of the synaptic vesicle. Then we have the vesicular Neurotransmitter transporter, and its job is to use the proton gradient that is established by the proton ATPase to exchange that for a neurotransmitter molecule and fill the neurotransmitter inside the lumen of the synaptic vesicle, so that the concentration will be very high inside the vesicle. Now the other important proteins that are localized in the synaptic vesicle are this protein here, which is called synaptotagmin. This is the protein that is the sensor for calcium. It will sense the rise in the cytoplasmic calcium concentration that's mediated by the influx through the voltage gated calcium channels, and then it will induce a conformational change within the synaptotagmin protein. Then the other two vesicles, the other two proteins that are important are this protein here which is called the V-SNARE. This protein is also named VAMP. This is the protein that is involved in actually producing the mechanism of fusion of the synaptic vesicle with the presynaptic terminus. In addition, there's this protein which is called synaptophysin and there's SV2. And it's thought that these are important in docking and localizing the synaptic vesicles to specific regions, and they have other regulatory functions as well. In addition, this protein synapsin is involved in helping to bind and tether synaptic vesicles to the actin cytoskeleton that is found within the presynaptic terminus. Synaptic vesicles are initially generated by the early endosome, which is also called the synaptic endosome. It turns out that there are three distinct pools of synaptic vesicles that are produced. This pool here is called the resting or the reserve pool. This pool is associated with the actin cytoskeleton and is sitting here further back, further away from the active zone. Then there's this pool called the recycling pool. The recycling pool can be very rapidly recruited to a region close to the active zone if the synaptic vesicle pool that's found here becomes used up or depleted. This pool is called the readily releasable pool. Many of these vesicles are located very close to the plasma membrane, and when the calcium comes into the presynaptic terminus, it's these vesicles which will undergo exocytosis and release their neurotransmitter into the synaptic cleft. Now these pools are all bio chemically and metabolically connected to each other, so as soon as a synaptic vesicle is released, the proteins within that synaptic vesicle can be taken back up and they can be refilled into the recycling pool, or this vesicle can go back to the early endosome. This ensures then that there's always a specific number of synaptic vesicles that are found in this readily releasable pool, and so the neuron is always ready and is going to be able to respond to a presynaptic action potential. What calcium does when it flows across the channel and increases the calcium concentration in the presynaptic cytoplasm, is this calcium stimulates exocytosis of the synaptic vesicle. Here we have a high magnification cartoon of a synaptic vesicle just showing a portion of the synaptic vesicle, and this is where the synaptotagmin protein would be localized. The synaptotagmin is the calcium sensor. It binds calcium ions, this is a non covalent binding, so it's a reversible binding, but it has what we call a high affinity for calcium. When the calcium comes in, when the calcium flows across the membrane through the activation of the voltage gated calcium channels, those calcium ions will bind to synaptotagmin. What this does is to induce a conformational change in the synaptotagmin protein. Now it turns out that the synaptotagmin is also associated with this protein, which is the V-SNARE or the VAMP. The VAMP protein, the vesicle SNARE protein forms a large complex with similar types of proteins that are found on the presynaptic plasma membrane of the axon. It forms this large complex that includes the V-SNARE. It also contains the T-SNARE, which is this blue protein called syntaxin. And then there are also several cytoplasmic proteins, including this protein, which is called SNAP25, which enters into this complex. This whole complex that includes the V-SNAREs, and the T-SNAREs is called the SNARE complex. This SNARE complex, what it does in the absence of calcium is to bring this vesicle very close to the presynaptic plasma membrane, but it actually inhibits and prevents the fusion of these vesicles. But then when a calcium ions flow in, bind to synaptotagmin, the synaptotagmin communicates with the SNARE complex, and it's going to bring this vesicle very close to the plasma membrane, and this will induce exocytosis, the fusion of the vesicle and exocytosis of the contents which are inside, and these are the neurotransmitter molecules. >> So here is a depiction of what happens during these events. So first we have the docking of the vesicle. So we have the v-SNARE and the t-SNARE molecules which are going to come together, and they will form the SNARE complex, and this is going to lead to the docking of the vesicle at the presynaptic membrane. Then through a series of reactions, this vesicle is said to be primed. It depends on energy, where we have the formation of the SNARE complex between the vesicle SNAREs and the target SNAREs, the t-SNAREs in the presynaptic membrane. When the calcium concentration increases in the cytoplasm in this active zone region because of the influx through the voltage-gated calcium channels then, that's going to lead to this conformational change in the synaptotagmin protein, which is then communicated to the SNARE complex. These proteins will further change their conformation, bringing this vesicle so close to the plasma membrane that the membrane, the vesicle membrane and the presynaptic plasma membranes will actually fuse with each other, and you remember this is exocytosis, and so now the neurotransmitter will be released into the synaptic cleft. So it's through these conformational protein-protein interactions then, that are going to pull this vesicle so close to the plasma membrane, that this vesicle will undergo exocytosis and release its contents into the synaptic cleft. The neurotransmitter being now in the aqueous, extracellular fluid of the synaptic cleft will be able to diffuse all around. Importantly, it can diffuse to the postsynaptic neuron, where it will then bind to and activate specific neurotransmitter receptor proteins that are found in the postsynaptic membrane. So it turns out that there are different types of synaptic vesicle that can be characterized by how quickly they occur, and also whether or not there's just partial fusion or full fusion with the presynaptic plasma membrane. The first type is called kiss-and-run. This is a very fast type of fusion. It's ultra fast. And what happens is, it is so fast that it just will fuse with the presynaptic membrane, but then before all of these proteins and lipids can actually become incorporated into the presynaptic membrane, this vesicle will undergo endocytosis and basically will leave the synaptic vesicle intact, because it's got the neurotransmitter transporter. These vehicles can then be refilled with the neurotransmitter, and can be reduct and reused very rapidly. There's also ultrafast endocytosis and clathrin-mediated endocytosis. So in this particular situation, this is also an ultra-fast type of mechanism that's similar to the kiss-and-run. It's just that the endocytosis occurs in a slightly different mechanism. And even though it is very rapid, you can see that as soon as the membrane has fused, this synaptic vesicle proteins can diffuse within the membrane, and so some of these synaptic vesicle proteins will become part of the presynaptic membrane. But this is also very rapid and you can see that there's still lots of synaptic vesicle proteins there. And so these vesicles can be rapidly recycled. For the other type, the slow type, there's much more complete mixing of the synaptic vesicle proteins with the presynaptic membrane. And this has to go through a much slower type of endocytosis. This vesicle, because it's going to have lots of presynaptic proteins as well as synaptic vesicle proteins in it, it has to go back to the early endosome and be sorted into synaptic vesicles at that point. So the critical feature of the voltage-gated calcium channels is that it detects the presynaptic action potential. And then through the mechanism of calcium-dependent exocytosis, it is going to convert that presynaptic electrical signal into a release of neurotransmitter at the cleft. So in this mechanism, the presynaptic action potential, which is an electrical signal, is first converted into an increase in the calcium concentration. And calcium being a chemical, then produces a chemical signal in the presynaptic region. Then this calcium chemical signal is converted into neurotransmitter release, and the neurotransmitter being a chemical will produce a chemical signal. So we can see here that there is an interconversion of information. Electrical information is converted into chemical information by the presynaptic machinery. Now, these neurotransmitters, one of the mechanisms whereby they act is by binding to specific ligand-gated channels, which can produce a postsynaptic change in the membrane potential. So we're going to see in the next few lectures, we're going to be talking about how the electrical signals can be produced in the postsynaptic neuron. And interestingly, it is this voltage-gated calcium channel that is the detector and the protein that is going to then convert this electrical signal into a chemical signal for synaptic transmission. We've talked about the neural code, and we've talked about how there is something about the frequency, or the timing of action potentials which encodes information. Now, because the voltage-gated calcium channels are the proteins which are going to be activated by the presynaptic action potentials then, it turns out that this is the protein that decodes the information that is encoded in the action potential frequency, or trains of action potentials into the calcium signal and the release of neurotransmitter at the cleft. So if we look here, this would be the neurotransmitter release or the amount of calcium that is present in the presynaptic neuron. And you can see that if we have a frequency of action potentials at this particular rate for this number of action potentials within this train, we get a response either in the calcium signal, or in the amount of neurotransmitter released. That looks something like this. As we increase the frequency of action potentials, and if we increase the total number, so the duration of the train of action potentials, you can see that that is transduced into a larger calcium signal and a larger amount of neurotransmitter that is released. So the release of the neurotransmitter then depends on the presynaptic calcium signal that is present within the presynaptic axon. And again, the voltage-gated calcium channel decodes the information that is encoded within the frequency and timing of action potentials in the presynaptic neuron. >> This is an experiment showing the increase in calcium concentration either in a terminal synapse or an en passant synapse. You can see using a calcium sensitive die that shows an increase in the pseudocolored a micro graph here where the increase in calcium is shown by either changes in the fluorescence as reflected by a change in the color from green, yellow to red. You can see that during the action potential, there is an increase in calcium concentration in the pre synaptic region within this en passant synapse. Now, the posnaptic part of this synapse is not shown here, so this is just the presynaptic change in calcium concentration. What you can also see are there are some little hot spots here where the calcium concentration is particularly high, and these are called calcium micro domains and it turns out these are the regions where the voltage gated calcium channels are localized at the active zone. This are the regions where the calcium concentration is going to be highest because the calcium is flowing into the presynaptic neuron within these regions. This is a motor neuron. You can see how much larger the axon is at this motor end plate compared with this neuron to neuron en passant synapse. But again, you can also see that there are specific hot spots with a micro domains of calcium are particularly highly localized. Now getting back to the function of the voltage gated calcium channels, the majority of calcium channels do not inactivate. This is a really important function because they will be able then to direct transduce the number of action potentials, so the frequency of action potentials directly into the level of calcium that is being produced in the presynaptic neuron. If the voltage gated calcium channel is inactivated then as soon as that first action potential would arrive, if there was another action potential that arrived very rapidly, then those voltage gated channels would remain inactivated and closed. Would have to wait, like the sodium channels do, for the resting membrane potential to be reached in order to reopen again. This is a key feature in that they do not inactivate, so that they can faithfully transduce the frequency and timing of action potentials in the presynaptic neuron into the amount of calcium that is present in the presynaptic neuron and then the amount of neurotransmitter that is released into the synaptic cleft. The voltage dependence of the voltage gated calcium channels is slightly greater than it is for the voltage gated sodium and potassium channels. It's around -40 to -45 millivolts. That's threshold for the activation of the voltage gated channels. They require a slightly greater depolarization of the membrane potential to be opened compared with the voltage gated sodium and potassium channels. But you remember the action potential is this, very large depolarization, 100 millivolt depolarization of the membrane potential. Which is always going to be great enough to activate the voltage gated channels that are found in the presynaptic neuron. The calcium influx through the voltage gated channels then will induce the synaptic vesicle exocytosis. The neurotransmitter will then be released into the synaptic cleft. In the next few lectures, we're going to be talking about the receptors that are present on the posynaptic membrane, and how they are involved in detecting and transducing the neurotransmitter into specific responses that are found in the postsynaptic cell. An interesting feature of neurotransmitters is that the majority of neurotransmitters are removed by specific mechanisms from the synaptic cleft. One mechanism is neurotransmitter degradation. Neurotransmitters can be degraded and for the synaptic transmission using acetylcholine. This is the mechanism for removal of acetylcholine from the cleft. It is the only neurotransmitter system for which there is a dedicated and specific degradative enzyme which removes the neurotransmitter and that's acetylcholine. The majority of other neurotransmitters are removed by what are which are called plasma membrane neurotransmitter transporters. These are a type of secondary active transporter that is found on the presynaptic plasma membrane, the postsynaptic plasma membrane, and importantly, also on the plasma membrane of nearby astrocytes that are forming the tripartite synapse. These transporters will pump the neurotransmitter across the membrane, back into the cytoplasm, removing it from the synaptic cleft. Now, all neurotransmitters also diffuse out of the cleft, depending on whether or not there is an astrocyte there or whether or not there is extracellular matrix. That's another mechanism for diffusion as well. In contrast to the small molecule neurotransmitters, the neuropeptide neurotransmitters are released at regions that are much further away from the active zones and when we talk about neuropeptides, we'll be talking about their requirements for release. They're actually released into the extracellular fluid at the extra synaptic sites, which are much further away from the synapse. In addition, the neuropeptides do not have specific uptake or degradative mechanisms. Their levels within the synapse or in the extracellular fluid are usually regulated just by diffusion. Now, in addition to the removal of the neurotransmitter, there is also recycling of the synaptic vesicle proteins. After the fusion of neurotransmitter, especially in the fusion which involves the full fusion, the membrane is recycled. It's initially retrieved by endocytosis. If the endocytosis, if this is a very rapid, if this is the ultra fast fusion, these vesicles will endocytose before the synaptic vesicle proteins can diffuse within the membrane. Those vesicles can be rapidly recycled by filling them with neurotransmitter, and then they will go back into the reserve pool. If the vesicle has undergone full fusion, it will probably need to go back to the synaptic endosome, where it buds off a new synaptic vesicle which will be filled by neurotransmitter. But this is a very important process because if there was not the endocytosis process to balance the exocytosis of the synaptic vesicles, then the presynaptic membrane would just expand and become larger and larger. This endocytic process is very important for the recycling of the membranes, and the synaptic vesicles and is critical for the process so that a presynaptic neuron can be ready and will have synaptic vesicles which are localized, docked and primed and ready to fuse in response to the presynaptic calcium signal. Now, in the next few lectures, we're going to be talking about the postsynaptic responses, the receptors, and their different functions, and we'll also be talking about the different neurotransmitter systems.

Camillo Golgi proposed the reticular theory, that cells in the nervous system form a continuous network of interconnected processes, and electrical signals can flow unimpeded through this continuous network. In contrast, the neuron doctrine is the concept that the nervous system is composed of individual cells (each with its own processes) that communicate at the gaps between them, later called synapses. It was proposed by Santigo Ramon y Cajal, Foster and others. Cajal was correct in describing chemical synapses; Golgi was somewhat correct in describing electrical synapses. These concepts are important because it raised questions about the mechanisms whereby neurons could communicate at synapses, which led to the discoveries of chemical synaptic transmission, neurotransmitters, receptors, membrane potentials, electrical signaling via the action potential, and GAP junctions.

At an electrical synapse, ions (and small molecules) from the pre-synaptic region flow directly to the post-synaptic terminus via gap junction channels known as connexons. A change in membrane potential in the pre-synaptic neuron leads directly to a change in membrane potential in the post- synaptic neuron by the flow of current through the connexon. Electrical synapses are thought to be much less abundant than chemical synapses in the mammalian CNS. An advantage of an electrical synapse is that it is very fast. A limitation is that communication can only occur only at the specific regions of the membranes that are physically connected (forming the gap junction), and there is no adaptability. In electrical synapses, the pre-synaptic and post-synaptic neurons always have the same response, and there is no mechanism to boost the signal (there is no gain). The term “electrical synapse” is confusing because at chemical synapses, the response often involves an electrical response in the target cell.

At a chemical synapse, the electrical signal from the presynaptic neuron (the AP) is converted to chemical information through the release of neurotransmitters into the synaptic cleft. The neurotransmitter binds to receptors on the target cell leading to a response in the target. There is often an interconversion of electrical and chemical information. Chemical synapses make up the majority of synapses in the CNS and PNS. An advantage is that this type of synapse is very adaptable. The postsynaptic response can be excitatory, inhibitory, or modulatory. Transmission can be fast and brief or slow and long-lasting. One presynaptic neuron can communicate with numerous postsynaptic target regions (in volume transmission). A limitation is that it involves multiple biochemical steps that results in a brief time delay, so transmission at a chemical synapse is slower than at an electrical synapse.

Golgi’s reticular theory was the idea that neuronal processes (axons and dendrites) form a continuous reticular network, and electrical signals can flow directly through this continuous network. At an electrical synapse, the depolarization of the presynaptic membrane is transmitted directly to the postsynaptic neuron by current flow via GAP junction channels. Consequently, the pre-synaptic and post-synaptic neurons are in an electrical continuum with each other (similar to what Golgi proposed, though only a few synapses function this way). In Cajal’s neuron doctrine, the idea is that neurons are separate cells that communicate at the spaces between them, the synapse, which would be similar to what occurs at chemical synapses where neurotransmitter is released and binds to postsynaptic receptors. This is how the majority of synapses in the mammalian CNS function.

Some synaptic junctions appear along the length of an axon as it extends—these are called en passant ("in passing") synapses and can be in the hundreds or even the thousands along one axon. Other synapses appear as terminals at the ends of axonal branches. Swellings termed axonal varicosities or axonal boutons are typically the sites where synapses occur. Boutons form as terminal bulbs at the end of an axon, and/or along the length of individual axons as boutons en passant. Many en passant synapses are thought to occur between an axon and multiple dendritic spines located along a dendrite and/or many dendrites. Spine synapses are usually glutamate/excitatory synapses. This may be important to coordinate synaptic transmission between one axon and multiple spines along a dendrite, to produce a large response in the target dendrite

In the CNS, neurons communicate with other neurons. In the PNS, neurons communicate with other neurons, skeletal muscles, cardiac (heart) muscles, smooth muscles, and glands

Wiring transmission is point-to-point neurotransmission that occurs at a synapse between a presynaptic neuron and its postsynaptic target. Neurotransmitter is released from synaptic vesicles at the active zone. Communication is localized to the synapse, and is usually fairly short lived (on the order of msecs to secs) and localized since the neurotransmitter is rapidly removed from the synapse. Wiring transmission often involves electrical responses in the postsynaptic cell, in the form of EPSPs and IPSPs. Nearby astrocytes or extracellular matrix (ECM) proteins could help to enclose the synapse and prevent the NT from diffusing to nearby regions. Volume transmission is not localized to the synapse. Neurotransmitters are released into the synapse where they can spill out, or be released outside the synapse, and diffuse into the extracellular fluid. In volume transmission, NTs can act on receptors found at the synapse, outside the synapse (perisynaptic or extrasynaptic receptors) and on receptors in the cell body and neighboring neurons. Communication can occur in a volume around the synapse, and is often longer lived (secs to mins) and not localized since the NT diffuses a long distance and is only slowly removed. Volume transmission often involves receptors that mediate neuromodulation

The three functional types of synapses are excitatory, inhibitory and modulatory/neuromodulatory. The four types of anatomical synapses (defined by the target) are axo-dendritic (on dendritic spines or dendritic shafts), axo-somatic (on the cell body), and axo-axonic (on another axon). Two additional types of synapses (defined by the axon) are terminal synapses (where the presynaptic axon ends where it forms a synapse on its target) and en passant synapses (where the axon forms a synapse by a bouton that blebs out from the axon, but does not end there but rather it continues on to form additional synapses).

Many cell adhesion molecules are localized at synaptic sites in neuronal axons and dendrites. These molecules bridge presynaptic and postsynaptic specializations but do far more than simply provide a mechanical link between cells... Synaptic adhesion proteins participate in the formation, maturation, function and plasticity of synaptic connections. Together with conventional synaptic transmission mechanisms, these molecules are an important element in the trans-cellular communication mediated by synapses.

Following synaptogenesis, in early infancy and childhood, synapses can be strengthened, weakened or removed, in an activity dependent manner in a process called synaptic refinement (the removal of synapses is often called synapse elimination). Microglia are proposed to function in the removal of synapses. Similar types of activity dependent plasticity have been documented in the adult brain, in the hippocampus and cerebral cortex, where long term potentiation (LTP) and long term depression (LTD) are proposed to be mechanisms involved in learning and memory

Acetylcholine was the first neurotransmitter discovered by Dr. Otto Loewi in the 1920s in the PNS in the synapse between the vagus nerve and the heart. Loewi was one of the “soups” a group of scientists who proposed that communication between neurons occurs by chemicals. The criticism of this idea was the use of chemical signaling would be much too slow for the fast communication that occurs in the brain. The other group, the “sparks” proposed that communication occurred by electrical communication, but one criticism of that idea is that since there are synaptic gaps between neurons (the synaptic cleft), the mechanism for electrical communication was not apparent or obvious. Both were correct. The action potential is an electrical signal used within neurons (to send a signal from the cell body to the presynaptic terminus). At chemical synapses (the majority of synapses), the action potential is converted into release of neurotransmitter at the synapse. Then in many synapses the chemical signal is converted into an electrical signal in the postsynaptic membrane. Postsynaptic potentials can summate to trigger an action potential.

The two types of conventional NTs are small molecule NTs and neuropeptide NTs. Small molecule NTs have a low molecular weight (between 75-500 daltons). They are synthesized in the presynaptic region and packaged/transported into synaptic vesicles by vesicular NT transporters. Neuropeptide NTs are synthesized in the cell soma by the RER and packaged at the trans Golgi network (TGN) into secretory vesicles (sometimes called dense core vesicles/granules), and transported to the presynaptic terminus by fast axonal transport. Many neuropeptides are synthesized as larger precursors and undergo proteolytic cleavage during maturation as the vesicles are transported by fast axonal transport from the cell body to the presynaptic region

A. Amines: acetylcholine and the monoamines: dopamine, norepinephrine, epinephrine, serotonin, and histamine

B. Amino Acids: glutamate, aspartate, GABA, and glycine

C. Purines: ATP and adenosine One thing that is surprising about this list is that with all the variety of basic survival, behavioral, motor, sensory and cognitive functions that the nervous system is involved in and controls, there are actually so few neurotransmitters used. For many of these neurotransmitters there are different types of receptors and receptor subunit genes, which may help provide the diversity required for so many different types of functions of the brain

Synaptic vesicles are generated by budding off from the early endosome (EE) found at the presynaptic area/terminus. This specific EE is often called a synaptic endosome. Small molecule NTs are synthesized at the presynaptic region and transported into synaptic vesicles by NT transporters. NT transporters are a type of secondary active transporter that use the proton (H+) gradient established by the H+ ATPase, to transport NTs inside synaptic vesicles, up their concentration gradient. A single synaptic vesicle contains from 3,000 to 8,000 molecules of NT, depending on the NT and the synapse

Neurons are often named for the small molecule NT they synthesize and release. For example: cholinergic, glutamatergic, GABAergic, glycinergic, dopaminergic, noradrenergic (norepinephrine releasing neurons), adrenergic (epinephrine releasing neurons), serotonergic, and histaminergic neurons. Most neurons release one small molecule NT and one neuropeptide NT from the same presynaptic terminus. Hence some neurons, such as GABAergic inhibitory interneurons located in the neocortex and other areas, are identified by the neuropeptide they co-release. For example, somatostatin or VIP expressing GABAergic interneurons. Neurons receive thousands of inputs, and those inputs can be from all the different types of neurons, for example, GABAergic, glutamatergic, dopaminergic etc, so a neuron will express lots and lots of different types of NT receptors, which are specific for the NT released at those synapses

Ca2+ is a divalent cation. The Ca2+ gradient is a 10,000:1 concentration ratio between the outside and the inside. Intracellular Ca2+ is kept low by a Na+-dependent Ca2+ exchanger (secondary active transport) and a Ca2+ pump, also called the Ca2+ ATPase (primary active transporter), both located on the plasma membrane. The intracellular Ca2+ pool is the Ca2+ stored within the endoplasmic reticulum (ER) of a cell. There is a Ca2+ ATPase on the ER membrane, which pumps Ca2+ inside the ER. (It is called the SERCA pump for sarcoplasmic/endoplasmic reticulum Ca2+ ATPase, also expressed in skeletal muscle). There are also Ca2+ binding proteins in the cytosol, and mitochondria also take up Ca2+. Neurons need to keep their intracellular Ca2+ levels low because Ca2+ acts as a secondary messenger, involved in NT and NP release, and regulates gene expression. In addition, an excess of Ca2+ would lead to apoptosis, a type of cell death.

The action potential (AP) requires activation of voltage gated (VG) Na+ and VG K+ channels. The AP is conducted along the length of the axon and causes depolarization of the presynaptic membrane and this depolarization causes activation of VG Ca2+ channels. Blocking VG Ca2+ channels, or buffering/removing Ca2+ in the presynaptic neuron blocks the postsynaptic response (but not the AP). Hence Ca2+ is required for synaptic transmission. An increase in presynaptic intracellular Ca2+ is the signal that is required to trigger exocytosis of synaptic vesicles and NT release. In fact, Ca2+ is likely to be the only signal that is required for this. Injection of Ca2+ into the presynaptic neuron (in the absence of an AP) is sufficient to induce synaptic vesicle fusion/exocytosis and NT release. That said, presynaptic Ca2+ does also have a number of other effects in the presynaptic neuron, including the trafficking/mobilization of synaptic vesicles among the three pools of synaptic vesicles

A voltage gated Ca2+ channel is composed of 24 transmembrane-spanning domains (including four voltage-sensing S4 regions). The voltage sensor (S4) domains contain positively charged amino acids along one side. The VG Ca2+ channel is generally similar to the VG Na+ channel but lacks the inactivation segment/gate. The VG Ca2+ channels are activated by depolarization, but they have a slightly higher threshold than the VG Na+ channel, requiring a depolarization to about -45 to -40 mV to be activated (compared with -55 mV for the VG Na+ channels). When activated, the VG Ca2+ channel changes conformation and opens, allowing Ca2+ to move through the channel as Ca2+ flows down its electrochemical gradient. VG Ca2+ channels are localized at the presynaptic plasma membrane at the active zone, very close to where the docked synaptic vesicles are located. The localization to the active zone is one of the functions of the non-channel auxiliary α2, β, γ and δ subunits. It is important the VG Ca2+ channels do not inactivate so they can accurately translate the frequency, pattern and number of presynaptic action potentials into the concentration of Ca2+ present at the presynaptic terminus, and thus, the amount and duration of NT released.

NTs are released from synaptic vesicles after Ca2+ flows into the presynaptic terminus through the VG Ca2+ channels. The active zone is the region where the VG Ca2+ channels are localized and this is right next to where the docked and primed synaptic vesicles are located. Depolarization of the presynaptic terminus by the AP causes opening of VG Ca2+ channels and presynaptic Ca2+ levels to increase, and the Ca2+ causes fusion of the synaptic vesicles (exocytosis) and the release of NT. The Ca2+ sensor is located on the synaptic vesicle membrane and is called synaptotagmin. (Synaptotagmin has multiple transmembrane spanning domains and the Ca2+ binding domain faces the cytoplasm.) V- SNARE proteins (synaptobrevin/VAMP) are also found on the synaptic vesicle membrane and interact with the t-SNAREs (syntaxin and SNAP25) on the presynaptic plasma membrane and cytoplasm to form the SNARE complex. When synaptotagmin binds to Ca2+, it regulates the SNARE complex and this induces fusion of the vesicle with the presynaptic membrane (exocytosis) and release of contents of the vesicle, the NT, into the synaptic cleft

After exocytosis, the membrane is retrieved by endocytosis. After partial fusion (kiss and run), the synaptic vesicle undergoes very rapidly endocytosis before the synaptic vesicle transmembrane proteins and lipids can mix with the presynaptic plasma membrane. Thus after partial fusion, the endocytosed synaptic vesicle can be immediately refilled with NT. In the case of full fusion, following exocytosis, the synaptic vesicle transmembrane proteins and lipids have time to diffuse and comingle with the presynaptic plasma membrane proteins and lipids, and when an endocytic vesicle is pinched off from the presynaptic plasma membrane it will contain synaptic vesicle proteins and presynaptic plasma membrane proteins. Those endocytic vesicles need to traffic to and fuse with the synaptic/early endosome, and new synaptic vesicles are produced by budding off, and are then refilled with NT by the vesicular NT transporters. Membrane recycling involves the recycling of membrane proteins and lipids following exocytosis and endocytosis.

All those mechanisms that keep intracellular Ca2+ levels low are present in the presynaptic region/terminus. Therefore, after Ca2+ flows into the presynaptic neuron through VG Ca2+channels, that Ca2+ will be rapidly pumped or exchanged out, bound to cytoplasmic Ca2+ binding proteins and sequestered into ER compartments and mitochondria. This means that the increase in presynaptic Ca2+ is transient and localized to the area around the active zone. Since the docked synaptic vesicles are close to the active zone, only those docked vesicles will fuse and this produces a small amount of NT release. However, if there is a train of APs, then the VG Ca2+ channels will open again and again, the Ca2+ increase will be much longer lasting and the Ca2+ will diffuse further into the presynaptic neuron. This will lead to the fusion of more synaptic vesicles and more NT will be released into the synaptic cleft.

When the AP arrives at the presynaptic active zone, the large depolarization of the membrane potential activates voltage gated Ca2+ channels. The Ca2+ flows into the presynaptic neuron (This is the Ca2+ signal, a chemical signal.) The Ca2+ activates synaptotagmin and the SNARE complex, causing the fusion of synaptic vesicles, exocytosis and release of NT into the cleft. (This is the NT signal, a chemical signal at the cleft.) The AP involves repolarization of the membrane potential back to the resting membrane potential, which leads to closing of the VG Ca2+ channels, and termination of synaptic vesicle fusion.

The frequency, pattern and number of action potentials determines the magnitude, area and duration of the presynaptic Ca2+ signal. The location of the VG Ca2+ channels determines the initial location of the Ca2+ signal. The presynaptic Ca2+ signal then determines the amount and location of NT release. Information is encoded in the AP frequency/pattern/duration (the neural code). Since the VG Ca2+ channels are activated-opened by the APs, they decode the information that was encoded by the APs, and produce the presynaptic Ca2+ signal and NT release that reflects the AP code

Many of the synaptic vesicles, which contain small molecule NTs, are localized very close to the presynaptic active zones where the voltage gated Ca2+ channels are localized and where the synaptic vesicles will fuse. The release of small molecule NTs from synaptic vesicles requires only low frequency action potentials, which is sufficient to produce small, local increases in Ca2+ near the active zone. In contrast, neuropeptide NTs, which are packaged into secretory vesicles, are localized much further away from the presynaptic active zone. Hence, to induce the release of neuropeptide NTs, high frequency action potentials are required. High frequency action potentials will activate the voltage gated Ca2+ channels repeatedly, which is required to produce a much larger increase in presynaptic Ca2+, so that enough Ca2+ will diffuse to induce fusion of the neuropeptide NT vesicles

All NTs can diffuse from where they are released, so diffusion is one mechanism that leads to a decrease in NT levels. The presence of astrocytes and extracellular matrix around the synapse can affect the rate of diffusion of NS out of the synapse. Acetylcholine is the one NT that is specifically degraded at the cleft. The other NTs (amino acids and monoamines) are removed by uptake into neurons and astrocytes by NT transporters located on the plasma membrane. It’s not clear how purines are recycled. Neuropeptide NTs can also be degraded by nonselective proteases in the cleft. NT removal is important because we want neuronal responses to be fairly short lived and specific. For example, control of muscle contraction needs to be precise, and often short lived. When contraction is no longer needed, the muscle must be able to relax, which requires removal of the NT. In sensory systems, once the stimulus is gone, the transmission from the sensory neuron transmission needs to be stopped, which occurs by uptake of the NT. Then for all the integration and computation that the CNS controls, the signals and codes must be precisely controlled.

Luigi Galvani and the Discovery of Action Potential

• Who: Italian doctor and professor of anatomy.

• Discovery: Action potential using frog neuromuscular junction.

• Publication: Commentary on electricity's effects on muscular motion in 1791.

• Hypothesis: Animal electricity from brain to muscles.

• Significance: Pioneer of electrical signaling in the nervous system.