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Module: Biological Foundations of Mental Health Week 3 Synaptic transmission & neurotransmitter systems Topic 1 Action potentials and synaptic transmission - part 1 of 5 Dr Philip Holland Department of Basic and Clinical Neuroscience Lecture transcript Slide 4 I’m Phil Holland. I’m a lecturer here a...

Module: Biological Foundations of Mental Health Week 3 Synaptic transmission & neurotransmitter systems Topic 1 Action potentials and synaptic transmission - part 1 of 5 Dr Philip Holland Department of Basic and Clinical Neuroscience Lecture transcript Slide 4 I’m Phil Holland. I’m a lecturer here at King’s College London, and I’m primarily interested in headache disorders including migraine, for example. And what we do in our laboratory is we use electrophysiological techniques to record the electrical impulses between nerves and how they communicate with each other. Today, I’m just going to talk over how the normal neurons set up their resting membrane potential and how then they generate and transmit these potentials. Slide 5 All cells, including neurons, have this membrane surrounding them, and that’s this phospholipid bilayer. This is a hydrophobic layer, so it allows the separation of aqueous ions between the extracellular space and the intracellular space, allowing us to set up these ionic gradients that we’re going to discuss. Now, of course, we need to move ions across these membranes, and for this purpose, we have proteins in the form of pumps, such as the sodium-potassium ATPase on the left, and ion channels, such as the sodium channels and the potassium channels here in grey and purple. Now, these ion channels can be leak channels. That is, they’re open, and they allow ions to passively flux up and down their concentration gradients. Or they can be gated in that they are closed at the resting condition and can respond to an external stimuli, be this electricity in the form of a voltagegated channel or a neurotransmitter, for example, in the form of a ligand-gated channel, causing this gate to open and allowing these channels to flux ions across the membrane. Slide 6 Now, it’s important to state that most membranes and neurons have a higher concentration of potassium leak channels, and this is important for setting up the resting membrane potential, as we’ll see. In this, ratio is 3 to 1, but this is just representative. Inside the cell, we have these large, organic anions here in red, and these are negatively charged ions on large proteins. These are locked within the cell, so they can’t cross the membrane. And as you can see, this puts a negative charge inside the cell in the intracellular space. This has the effect of drawing positively charged sodium and potassium ions, sodium in the blue and potassium in the green here, towards the extracellular space and repelling slightly negatively charged ions such as chloride ions Week 3 © King’s College London 2019 1. here, as can be seen. And that sets up a net positive charge along the extracellular space. And as we mentioned, there are more potassium leak channels in the membrane. In response to this electrostatic charge, this want for the positive charged ions to be attracted towards the negative ions inside the cell, more potassium will enter the cell, making a higher concentration of potassium inside the cell. Now of course, some sodium will also enter the cell, but because there are less sodium leak channels, this is relatively fewer than the potassium. And we also have a low concentration of chloride ions within the cell, setting up this ionic gradient across the membrane. Slide 7 We have relatively more potassium within the cell, as can be seen here in the green, and relatively fewer on the extracellular space. Again, there’s a higher concentration of sodium on the extracellular space compared to the intracellular space. These sodium-potassium ATP pumps, they act to help maintain this concentration gradient. Of course, I should point out as well that we have a higher concentration of chloride ions outside the cell as opposed to inside the cell. Obviously, these ionic gradients are established, and the sodium-potassium pump acts to pump ions against these gradients, so it’s an energy-dependent mechanism that helps to maintain the high concentration of potassium inside the cell and the lower concentration of potassium outside the cell. In order to do this, the pump needs energy. This is an energy-dependent mechanism, so it uses adenosine triphosphate and changes this, obviously, to adenosine diphosphate and an organic phosphate molecule. And this energy allows the channel to collect three sodium channels from the intracellular space, as you just saw, and actively pump them to the extracellular space. In turn, two potassium ions are gathered from the extracellular space and pumped to the intracellular space. This has two mechanisms. This, first of all increases the concentration of sodium in the extracellular space and increases the concentration of potassium in the intracellular space. But also, as three positively charged ions were pumped out of the cell and only two positively charged ions were pumped into the cell, this helps to maintain the net negativity of the intracellular space compared to the extracellular space. Slide 8 This is what we can see here. At the resting membrane potential, we have this mix of ions in the outside, largely sodium and chloride and fewer potassium, and on the inside, we have a greater concentration of potassium and fewer sodium and chloride ions. This sets up a gradient across the membrane, and these ions are under two forces. First of all, they’re under the force of the electrostatic force, and that is the charge component. The want for the positive ions to go towards the negative ions, for example, through the leak channels, as we discussed. But they’re also under the force of diffusion. That is that they want to move along their concentration gradients from an area of high concentration to an area of low concentration. So for example, under these conditions for diffusion, potassium would want to leave the cell and go to the extracellular space. It’s important for the action potential, that we’ll come to a later section, to see how these electrostatic forces influence the different ions. And for sodium, as you can see here, both the Transcripts by 3Playmedia Week 3 © King’s College London 2019 2. electrostatic force in red and the force of diffusion want to drive sodium into the cell, so sodium is very potentiated and very ready to drive into the cell should these voltage-gated sodium channels open. Potassium, on the other hand, has divergent forces. The charge component, the electrostatic component in red, as we discussed, brings potassium into the cell, but the force of diffusion wants to take potassium out of the cell. And for chloride, these are reversed where the charge component wants to repel chloride from the cell, and the force of diffusion wants to attract chloride into the cell. We have a point called the equilibrium potential, and that is the point for any ion where this net flux across the membrane would be zero, and that would be because the force of the electrostatically charged component and the force of the diffusion would be equal to each other. So under resting conditions, these ions would not move across the membrane potential. Slide 9 This is just an example of the relative concentrations of these ions, and what you can see is again highlighting that chloride and sodium are highest outside the cell, and potassium and organic anions here, the big A with the negative, are higher inside the cell. And what the resting membrane potential is, if we were to record, the electrode here on the right hand, side between the extracellular space and the intracellular space, what we’re recording is the fact that the intracellular space, due to these ionic gradients, is relatively more negative to the extracellular space. In this case, by around minus 60 to minus 70 millivolts. Transcripts by 3Playmedia Week 3 © King’s College London 2019 3. Module: Biological Foundations of Mental Health Week 3 Synaptic transmission & neurotransmitter systems Topic 1 Action potentials and synaptic transmission - part 2 of 5 Dr Philip Holland Department of Basic and Clinical Neuroscience Lecture transcript Slide 3 So in the last section, we discussed how the neurons are able to set up their resting membrane potential. Now in this section, now that they have established a resting membrane potential, we’re going to look at how they can integrate signals from, for example, a presynaptic neuron, on the left here, and how that would integrate the response to the postsynaptic neuron. Now of course, here we’re going to discuss a one-to-one relationship. But it’s important to realise that these neurons can receive multiple inputs somewhere in the region of up to 400 presynaptic inputs to a neuron. So you can see the diversity of the response here. Now, this signal, if we think about the anatomy of a neuron, is largely integrated via the dendrites and the cell body, as you can see here. So these signals are responding by the dendrites and the cell body, and then they travel through the cell body, making their way towards the axon initial segment, which we’ll discuss in relation to the generation of an action potential. Slide 4 So how is this signal transmitted? What is this signal? Well, it’s known as a graded potential, and if you remember from the previous talk, we spoke about how these ion channels in the membrane can flux specific ions, in this case, sodium, as you can see here on the left. Now, this presynaptic neuron has caused the opening of a ligand-gated channel on the postsynaptic cell. When this is opened, sodium has fluxed in, causing a positive change. And it’s this change in potential of the membrane around the ion channel that’s known as our graded potential. And this can be both positive or negative. So for example, potassium and sodium ions are positively charged. So they will depolarise the postsynaptic cell, and that is, they will move it towards the triggering threshold for an action potential. Whereas chloride ions, being negatively charged, if they were fluxed, would move the resting membrane potential away from the triggering threshold and would result in an inhibition of the likelihood of firing. Now, these graded potentials are best described by the analogy of dropping a stone into water, and you see there’s a diffusion of the wave in all directions. Now that’s what happens with this charge. It diffuses in all directions. But as it does so, it rapidly decays. So therefore, one or two inputs to a cell will rapidly decay and not have an effect on the cell itself. We need the summation of different effects to get over this quickly diminishing response that we see in graded potentials. Transcripts by 3Playmedia Week 3 © King’s College London 2019 1 Slide 5 Now, to actually result in the triggering of an action potential, as we mentioned, the presynaptic neuron here on the left has to respond. This will result, for example, in neurotransmitter release. This neurotransmitter release will activate a graded potential on the postsynaptic neuron, and the arrows here denote the size of this graded potential. As this graded potential moves through the cell body, it diminishes rapidly, as we mentioned, this electrotonic transmission. It reaches then, at the start of the axon, this region called the axon initial segment, here denoted as AIS, and as it reaches that axon initial segment, there’s a threshold, in this case, minus 55 millivolts. Now, this threshold is the triggering point above which the all or nothing event of an action potential will be initiated. If this graded potential, such as on the left here, reaches the axon initial segment and it’s below this threshold, then no action potential is generated. The graded potential decays, and the cell returns to its resting membrane state. However, if this graded potential is just above or succeeds the, in this case, minus 55 millivolt triggering potential, then we get this rapid flux of sodium ions in this very robust depolarisation and hyperpolarisation phase that we will discuss in the next lecture, known as the action potential. Slide 6 Now, as I mentioned, any single response is likely, or won’t be enough, to trigger this response, so there are a couple of ways by which the postsynaptic neuron can integrate signals from multiple responses. The first one is known as spatial summation, and that is where, in this case here, if a single channel was to respond, that graded potential may not be above the threshold and would not reach the AIS and trigger an action potential. However, if, for example three inputs-- I’m only using three in this case, but we mentioned before, there can be up to 400-- three inputs were to fire simultaneously, their responses would be summed in the cell body. So now in the red here, you can see we have a much larger graded potential, which can travel to the axon initial segment above the threshold for triggering, and generate the action potential. So this is spatial summation. This is based on the localisation of inputs around the dendrites and the cell body. Slide 7 The other method is temporal summation, and this is based on the timing of triggering, either by a single presynaptic input or by multiple inputs. And in this case, if a single action potential was to fire and it wasn’t appropriate, that wouldn’t happen. And then a second one fires, and you can see that both of these graded potentials don’t pass the threshold, so no action potential is generated. Slide 8 However, if the first and second graded potentials and postsynaptic neuron were to fire quickly, causing this graded potential in the cell body, these responses would be summed. As you can see on the right, they would sum on top of each other and potentially allow the triggering of an action potential. So by this mechanism, both the spatial localisation of inputs firing and the temporal, that is the speed, of the firing, and the speed in relation to one another, can significantly impact the action potential propagation. Slide 9 Now, the postsynaptic cell responds in a multitude of ways. We have what we call excitatory postsynaptic potentials, and these are those potential changes that happen in response to the Transcripts by 3Playmedia Week 3 © King’s College London 2019 2 positively charged ions, such as sodium and potassium. They will move the membrane potential towards the triggering threshold for an action potential - and here it would be depicted here in green. But we also have inhibitory postsynaptic potentials, and if you remember, I mentioned chloride ions. Because these are negatively charged, if chloride ions flux into the postsynaptic cell, they will move the resting membrane potential towards a hyperpolarised state, that as, more negative, and further away from this triggering threshold, which we had at minus 55 millivolts. So if the green neuron here was to fire, and we record, using a glass electrode down here, the response of the postsynaptic cell, we see this excitatory postsynaptic potential in green. But if the red neuron is to fire, we see this inhibitory postsynaptic potential. So the resting membrane potential is moved away from the triggering threshold, and as you can see in both cases, this decays relatively quickly. Now, the postsynaptic cell, as we mentioned, has somewhere in the region of about 400 inputs. So you could imagine that the response is dependent upon the integration of these multitude of signals, both positive and negative. In this case, for example, if the green and the red neuron, fluxing, causing the opening of channels fluxing in sodium and chloride ions, for example, were to fire, what the end result for the postsynaptic neuron, of course, is a balance of both this positive and negative charge. So, we can fine-tune the membrane potential and the likelihood for triggering of the postsynaptic neuron. Transcripts by 3Playmedia Week 3 © King’s College London 2019 3 Module: Biological Foundations of Mental Health Week 3 Synaptic transmission & neurotransmitter systems Topic 1 Action potentials and synaptic transmission - part 3 of 5 Dr Philip Holland Department of Basic and Clinical Neuroscience Lecture transcript Slide 3 In the last section, we discussed how these neurons can integrate information from their presynaptic partners or from the surrounding, and we spoke about how this can be very divergent with up to 400 inputs. Now after the signal is transmitted through the dendrites and the cell body to the axon initial segment, this is where it can then trigger an action potential, and that’s what we’re going to focus on today. We’re going to focus on how that action potential generation works here at the axon initial segment, this highly specialised component in the start of the axon. Slide 4 To do this, we’re really going to focus on voltage-gated sodium channels and voltage-gated potassium channels. You see this mechanism here by which we have the usual ionic gradient that you’re now becoming familiar with, whereby sodium is at much higher concentration outside the cell than inside the cell. Remember that sodium is the light blue. Therefore, if you remember back to the electrostatic and diffusion force section, sodium wants to enter the cell because it wants to be drawn towards the negatively charged intracellular space, but also it wants to enter the cell because it wants to go to the area of lower concentration within the cell. In response to an electrical stimulus, which opens this voltage-dependent gate, sodium will rapidly flux into the cell, making the intracellular space more positive with respect to the extracellular space. Slide 5 In the context of the voltage-gated potassium channel, which we now see here in the purple, the opposite is true, where potassium is higher concentrated inside the cell and lower concentrated outside the cell. If you remember that the electrostatic force, the charge component, is pulling potassium into the cell via the leak channels, but the diffusion forces want potassium to leave the cell because it’s a lower concentration outside the cell. In this circumstance, the same electrical stimulus triggering the voltage-gated potassium channel will cause potassium to leave the cell and move to the extracellular space, rendering the intracellular space more negative, and this is the hyperpolarising response. So when we talk about Week 3 © King’s College London 2019 1. hyperpolarisation, we’re talking about the inside of the cell becoming more negative. When we talk about depolarising, we’re talking about the inside of the cell becoming more positive with respect to the extracellular space. Slide 6 If you remember what we spoke about in terms of graded potential is that this incoming signal causes a localised graded potential that decays electrotonically as it traverses across the cell body. If this reaches this specialised axon initial segment at too low a threshold, we do not see a response. However, if this response is significant and above the threshold, minus 55 millivolts in this case, we see this significant depolarisation phase. So this positive phase, followed by a hyperpolarising phase, the negative phase. What we’re going to focus on now is how these channels respond to allow these phases to happen. The axon initial segment is uniquely designed to do this. It has a very high concentration of, for example, voltage-gated sodium channels, voltage-gated potassium channels, and chloride channels. So it makes it uniquely responsive to changes, and are uniquely excitable compared to other regions of the axon to trigger these action potentials, and this is very important in that context. Slide 7 The action potential-- this is a classic graph of an action potential-- is divided into a number of phases, which we’ll talk through now. First of all, we have the resting membrane potential. We’ve discussed this. We know what’s happening here. This is the polarisation of the cell that’s set up via the balance of the sodium-potassium ATPase pumps and the leak channels present on the membrane. Then in response to a depolarising stimuli, so an electrical stimuli or a graded potential, as we’ve discussed, some of these sodium channels in the postsynaptic cell might open. Now if this stimuli is large enough to pass the triggering threshold-- that’s the main 55 millivolts in the previous section-then these voltage-gated sodium channels at the axon initial segment start to open, and sodium rushes into the cell. So we get this positive depolarisation phase. This rapid entry of sodium further depolarises the intracellular space of the neuron, opening more sodium channels. So we get this very rapid flux of sodium into the cell here, in this case. The sodium channels - and we’ll come back to this - have what’s called an inactivation phase. About half a millisecond after they open, they become inactive - and we’ll come back to the mechanism for that - so they can no longer flux sodium. Slide 8 At the same time, the voltage-gated potassium channels begin to open. Now we discussed that they cause potassium to leave the cell and the cell to become more negative, but they also respond much slower than the sodium channels. They open more slowly, and they close more slowly. So as they open now, and the sodium channels are inactive, we can see here that potassium starts to leave the cell. So we’re now starting to hyperpolarise the cell. It’s becoming more negative, moving towards its resting membrane potential. Potassium continues to leave the cell, because even after the cell has been hyperpolarised back to the resting membrane potential, these channels are slower to respond, so they close more slowly. So we get this after hyperpolarisation, this overshoot of negativity, and the response to this. Then these channels close again, and in response to, potassium starts to now leak back into the cell via the leak channels, if you remember back to the leak channels. So the potassium will slowly start Week 3 © King’s College London 2019 2. to leak back into the cell, rendering it more positive and moving towards the resting membrane potential. Of course, this is helped by the sodium-potassium ATPase pump, and then we have resurrection of the normal, resting membrane potential out here. So the axon is now back at its resting state and ready to fire again. Slide 9 There are two important phases with response to these changes, and we call these the refractory period. These are the points at which the axon of the neuron either can’t fire or will find it more difficult to fire subsequent action potentials. The absolute refractory period is that point at which the sodium channels are inactivated so they can’t flux sodium. This lasts until the resting membrane potential has been restored, and we’ll come back to that. But safe to say that at this point, no action potential can be triggered in that neuron. This has two effects. It allows the neuron to control its excitability, but it also prevents back propagation. If you imagine that we only want this action potential moving in the one direction, towards the terminal field of the neuron, this helps to prevent that signal from travelling back towards the cell body. The relative refractory period is the period during this after polarisation overshoot where the potassium channels, because they are slow to close, render the membrane potential lower, so more negative, than the resting membrane potential. During this point, an action potential can be triggered, but because the membrane potential is below the resting membrane potential, it will require a greater input to do so. So this is the relative refractory period, whereby the same stimulus that end just in action potential in the previous section of the resting membrane potential may now not be sufficient, and we would need a greater stimulus. Slide 10 We just want to focus now on these different stages here. I mentioned the inactivation state of the sodium channel. It’s important to know that channels have three main states that we’re going to discuss for the purpose of these talks. They are the closed state, which is when the gate is closed and they can’t flux ions. They have the open phase in which the gate is open, and they can flux ions across the membrane. But sodium, for example here, has this inactive state and this refractory period whereby, in this case, a ball and chain mechanism is taken up into the pore and physically blocks the pore. This is a charge-dependent mechanism. Although the gate is open, as you can see - the black line here in the middle - no sodium can flux into this cell, into this membrane, so the cell can’t fire an action potential. Not all channels have the three states: voltage-gated sodium channels have all three of these states, but voltage-gated potassium channels don’t have an inactivation phase, and that’s important for their hyperpolarisation. Slide 11 It’s worthwhile now to just look in more detail at how the voltage-gated sodium channel moves between these three states across the actual action potential. In number one, number two and number three, we have the channel and how it’s responding, and in number four and number five, we have the trace of the action potential that you saw in the previous couple of slides. In number one here, at the resting condition, the gate is closed. No sodium can flux across the ion, and the neuron is at its resting membrane potential, at rest. Number two, we have this incoming electrical stimuli. This causes voltage-dependent activation of the gate, and as you see now, this starts to allow sodium to flux into the cell down towards number three. This sodium causes a depolarisation of the cell, moving it from a more negative to a more positive Week 3 © King’s College London 2019 3. component. And if this component is above the minus 55 millivolt triggering threshold, then we get rapid opening of many more sodium channels, and we get this large depolarisation phase classically seen in the action potential. As mentioned here now in number four, about half a millisecond after this activation state, because of the change in potential - if you look, the intracellular space has become more positive because of this sodium - this charge-dependent mechanism causes this ball and chain mechanism to be taken up into the pore of the ion channel, and it physically blocks the ion channel, preventing sodium flux, so the cell can’t become more depolarised. For example, equilibrium potential - if you remember back to the resting membrane potential - for sodium is somewhere in the region of 50 millivolts. But you can see here in this example, because it’s now blocking sodium influx, we’re only getting to about 30 millivolts on the depolarisation phase here. As the potassium channels now begin to open in the background and we have this hyperpolarisation phase - you can see that on the right here, number five - and as the resting membrane potential is restored, the normal charge spread across the membrane, where it’s more negative in intracellular space, is recovered, and the ball and chain mechanism simply is removed from the ion channel. At this stage, the gate closes again, so the channel is back in its resting condition ready to fire the next action potential. It’s this ball and chain mechanism that’s considered important, or is important, for the absolute refractory period, this important point when a neuron cannot fire another action potential until it has restored its normal mechanism and its normal gate in the closed state. Week 3 © King’s College London 2019 4. Module: Biological Foundations of Mental Health Week 3 Synaptic transmission & neurotransmitter systems Topic 1 Action potentials and synaptic transmission - part 4 of 5 Dr Philip Holland Department of Basic and Clinical Neuroscience Lecture transcript Slide 3 In this section, we’re now going to look at how the action potential is conducted along the axon. So we started now with understanding how the signal is integrated the dendrites and the cell body, how this then has triggered the action potential at the axon initial segment. And now we’re going to look at how both myelinated and unmyelinated fibres transmit this action potential along the length of their axon to the terminal field, where it can then have an effect on the postsynaptic neuron in this case or, for example, on muscle tissue to engage movement. Slide 4 Now, if you think back to the basic principles what we spoke about, here is the axon initial segment, the dotted circle at the start of the axon, and we have the normal distribution, whereby the inside of the cell is more negative with respect to the extracellular space here, denoted as a positive response. And the incoming graded potential, if you remember we spoke about this component-this rapidly decaying electrotonic signal. It reaches the axon initial segment, and if it’s above the threshold, which, again, we will suggest is minus 55 millivolts in this case. Slide 5 This triggers the opening of the voltage gated sodium channels, and sodium rapidly enters the neuron, depolarising the intracellular space. Therefore, the intracellular space now becomes more positive around this area of the membrane, and the extracellular space more negative. If you remember, we also said that the axon initial segment is uniquely designed for that. It has a very high concentration of voltage gated sodium channels, for example, and voltage gated potassium channels. So it’s uniquely excitable, in the context of the axon, to trigger these responses. So if you imagine it’s got a high concentration of voltage gated sodium channels, so as they trigger and open, they will further open more sodium channels and really cause a rapid influx or rapid depolarising phase. Slide 6 Now, as the sodium influxes into the cell, this will further depolarise the membrane in front of it. So we start to get the spread of the depolarisation along the membrane because it’s opening more voltage gated-dependent channels along the membrane. Week 3 © King’s College London 2019 1. Slide 7 Now, this will continue to spread. This depolarisation will spread along the axon, as you can see here. So we’re now starting to move electrotonically along the axon, the signal is progressing. And behind that, if you remember, now, the slower voltage gated potassium channels will now begin to open. There’s a half a millisecond delay in their response. They will begin to open, and potassium will begin to leave the cell, making-- because potassium now wants to get away from this positively charged environment and wants to flow down its concentration gradient. Now, at the same point, if you remember, the sodium channels-- this is when they become inactive. So because they’re inactivated now, behind this potential change, as seen in number four, the charge can only move along in that one direction towards a terminal field and can’t propagate back towards the cell body. And this is important because in certain pain states, this can become abnormal, causing an action potential to travel in both directions. Slide 8 Now, in the context of an unmyelinated fibre, if we look at that and how that conveys, this is a relatively slow process. So we have this influx of sodium, which then depolarises the membrane, moves this charge across, further opening more voltage gated sodium channels and causing further influx of sodium as we move along the axon. Behind this, the potassium is now starting to leave and the sodium channels are inactivated. So we’re starting to get a reversal of the membrane potential back to its resting state, that is, more positive on the outside and more negative on the inside. And this continues on the entire length of the axon. Now, it’s a relatively slow process because sequential voltage dependent channels have to respond along the entire length of the axon. So this is a slightly slower process causing this electrotonic spread each time the cycle has to refresh itself along entire length the axon. Slide 9 Now, in myelinated fibres, this is different. If you think, now-- and think that the myelinated fibres are surrounded by myelin generated by Schwann cells in the peripheral nervous system, or oligodendrocytes in the central nervous system. And this myelin is an insulating fatty layer. Much like the rubber cable around the wire in your house, it insulates that cable and prevents the current, or the conductance, the charge, from leaking across into the environment. For example, if you had a bare wire, you might get a shock from that. That’s what this is here to do. It’s here to prevent that. Now, in a myelinated fibre, the conduction is classically known as saltatory conduction, and that’s where the potential appears to jump from one node to the other node. And these nodes of Ranvier here are these little bare, uninsulated sections between the different oligodendrocytes in this case, because we’re talking about a central nervous system neuron, that are exposed to the extracellular space. And much like the axon initial segment, which we previously spoke about, these are very specialised compartments that have a high density of voltage gated sodium channels in the node and surrounding the node are high density, for example, voltage gated potassium channels. So again, they’re uniquely excitable compared to the insulated membrane along which the oligodendrocytes are ensheathing the axon. Now, in this case, the incoming signal causes influx of sodium at the axon initial segment. That causes then a depolarisation, so a more positive charge within the neuron. And this positive charge spreads electrotonically along the axon to the next node of Ranvier-- to the next bare component-- where it can then flux ions across the membrane. Week 3 © King’s College London 2019 2. This then happens again at this node of Ranvier. The sodium channels are triggered, sodium fluxes into the cell, and this is propagated along to the next node of Ranvier. And the same potassium mechanism is happening behind this, where at each node of Ranvier sequentially along the axon, potassium is then leaving the cell, reestablishing the resting membrane potential of the cell. And, of course, the sodium potassium ATPase pumps, you remember, are very important here as well to reestablish these membrane potentials. Now, by this mechanism, myelin, in this case, can really increase the speed of propagation because we don’t have to have sequential activation of ion channels across the entire length of the axon. We get this apparent jumping in the saltatory conduction from one node to the next. And you can really rapidly increase the conduction velocity-- the speed at which we can transmit these signals. And if you think of the brain, it’s very important because in some axons, for example, if you’ve got a motor neuron in your leg travelling up to the CNS, of course, or a higher spinal neuron, this can really be over a distance of metres. So we can really see quite a long process, where we have to get signals to the target zone very rapidly. And this is where a problem can arise, because myelination is such a specialised event, and it can really help with propagation. But in the case of disorders such as multiple sclerosis, demyelinating diseases, where we get a breakdown of this insulating sheath, the new signal, which is now conducting, can pass to this first node of Ranvier here from the axon initial segment. But if the myelin is disrupted, this charge can now leak out across the membrane potential and can now dissipate across the membrane because the membrane resistance is decreased. And in doing so, the ongoing charge that’s passed along the axon is significantly reduced. And if this potential is no longer strong enough to reach the adjacent node of Ranvier-- much like a graded potential decaying back in the cell body, if you remember that section-- then the action potential will be lost, and we will lose the ability to fire an action potential. And that is what happens in, for example, multiple sclerosis or diseases such as Guillain-Barre syndrome, where we get total loss or breakdown of the insulating myelin sheath and a loss of signal. Week 3 © King’s College London 2019 3. Module: Biological Foundations of Mental Health Week 3 Synaptic transmission & neurotransmitter systems Topic 1 Action potentials and synaptic transmission - part 5 of 5 Dr Philip Holland Department of Basic and Clinical Neuroscience Lecture transcript Slide 3 We’ve seen in our previous sections now how the dendrites in the cell body aim to create the incoming signals, how the action potential is generated at the axon initial segment, and how this is transmitted along the length of the axon. And in this section, we’re going to focus now on how this signal, then, is transmitted between axon to axon. We’re going to focus on neurotransmitter release, so chemical synapses in this section, but it’s important to note that electrical synapses also exist, and you can read further in those if you wish. Slide 4 We’re going to focus now on this very small region here between the two neurons, known as the synaptic cleft, and to do this, we have a presynaptic zone here in blue in the top. In the membrane, we have voltage-gated channels, but in this case, now, we’re focusing on voltage-gated calcium channels. We should mention, much like sodium, calcium has a much higher concentration in the extracellular space than the intracellular space, so calcium wants to come into the cell. We also have mitochondria here in yellow, because of course, there’s an energy-dependent component to it here. We have vesicles, the circles, and within these vesicles, we have neurotransmitters, the little dots, and these are stored here in the presynaptic terminal, waiting for them to be triggered and release to happen across the synaptic cleft. In response to an incoming action potential that invades the terminal field, the voltage-gated calcium channels are now opened. They trigger. Calcium can flood into the cell, and via a calcium-dependent mechanism, the vesicles are now moved to the membranes, so this process called exocytosis. They’re moved toward the extracellular membrane of the neuron where they fuse with that membrane, and there are a number of proteins involved in this process, such as SNAP25 and SNARE, and you can read up those further if you wish. This allows the membrane to fuse with the extracellular space and allows the neurotransmitter to be released into the synaptic cleft. This neurotransmitter will then diffuse across this very small gap where it can act as a ligand to trigger the ligand-gated ion channel, for example, on the postsynaptic cell. So if this was a neurotransmitter that was triggering a ligand-gated sodium channel, for example, it would open the channel, allowing sodium to flood into the postsynaptic cell. If you think back to a previous section, this would induce an excitatory postsynaptic potential, so it would excite the cell. But equally, this neurotransmitter release could cause chloride influx because there’s a chloride ion channel, and it could be inhibitory. So this is this integration component that we spoke about previously. Week 3 © King’s College London 2019 1. Slide 5 The neurotransmitter, of course, has been released, and it’s having its effect on the postsynaptic cell, but there has to be a mechanism for controlling that and what happens here. This mechanism really is a stimulus-dependent system, so the postsynaptic neuron doesn’t just want to know that my presynaptic partner fired. He wants to know what’s the relative intensity of the signal. That’s important for how the signal was transmitted. Experimentally, if we record-- so these arrows are recording electrodes, shall we say, and we’re recording the graded potential that’s coming into the cell body. We’re recording what’s happening at the axon initial segment triggering zone, and we’re going to record the action potentials from this axon. In the first example, what we can see is that the graded potential, in this case, is minus 40 millivolts, so we’ll all agree that’s above the minus is 55 millivolts threshold that we’ve been using here. That allows the triggering of an action potential here at the axon initial segment, and we can record these action potentials along the axon. These will invade the terminal field and result in the release of neurotransmitter from the presynaptic neuron based on the mechanisms we’ve just discussed. Now in the second scenario, a larger graded potential, which reaches the triggering zone in the axon initial segment with a higher threshold will induce more in action potentials as seen here by multiple action potentials in the top graph. This will invade the presynaptic terminal. Because it’s voltagedependent, or stimulus-dependent calcium channels, more of these calcium channels will be opened. Therefore, more calcium will flow into the cell. We will get more exocytosis of neurotransmitter, and therefore more neurotransmitter into the synaptic cleft to signal to the postsynaptic neuron. So we have the stimulus-dependence component where the postsynaptic neuron can sense both the activation, but also the level of activation, of its presynaptic partner. Slide 6 Now of course, once these neurotransmitters are released, we have to have a mechanism by which we can control this. We can’t just have them acting continuously. There are a number of mechanisms by which the neurotransmitters can be removed. The primary mechanism is actually reuptake into the presynaptic cell. So the presynaptic terminal will reuptake the neurotransmitters and recycle them back into vesicles to use again. But they can also be taken up by support glial cells, for example, astrocytes here on the grey on the left hand side. This is a very energy efficient mechanism by which they can recycle these neurotransmitters. Alternatively, on the postsynaptic membrane, there are mechanisms by which these neurotransmitters can be degraded. They can be broken down, and then, of course, the products are taken away into the bloodstream and then expelled. The final mechanism is just the act of simple diffusion. These neurotransmitters will diffuse away from the synaptic cleft and can be taken off into the bloodstream, and taken away from the region. But as I mentioned, the presynaptic reuptake of these neurotransmitters is the primary mechanism and the most effective mechanism. In the context of mood disorders, this is actually a very important target. So for example, the antidepressant selective serotonin reuptake inhibitors, they actually act to prevent this reuptake of the serotonin. So if you imagine, now, the serotonin is released. It’s having its effect in the postsynaptic cell. These drugs now block its reuptake, so they potentiate the effect of your normal release. They don’t have an artificial effect and increase the level of serotonin, but what they do is they potentiate your own endogenous, internal response, and that’s the mechanism by which they have their antidepressant effects. So this scenario here where we can potentiate or change the effect of the presynaptic talking to the postsynaptic neuron is very important in the context of mood disorders. Week 3 © King’s College London 2019 2. Slide 7 Just by way of review, I think it’s important to look at this in animated perspective. What we have here is we have, again, this incoming action potential, which is triggering a voltage change at the presynaptic terminal. This is opening the voltage-gated calcium channels, and calcium - positively charged calcium - is flooding into the cell. This triggers vesicle like cytosis, so these vesicles move towards the cell membrane. They then fuse with the membrane and release the neurotransmitter across the synapse. This activates, as you saw there, the local postsynaptic ion channels and allows, in this case, sodium flux into the postsynaptic dendrite. What you see here, now, is this excitatory postsynaptic potential because sodium has a depolarising effect. But equally, this could have been hyperpolarising effect had the ion channel on the surface of the postsynaptic neuron been fluxing chloride ions, for example. Week 3 © King’s College London 2019 3. Module: Biological Foundations of Mental Health Week 3 Synaptic transmission and neurotransmitter systems Topic 2: Neurotransmitters, receptors and pathways - Part 1 of 4 Dr Jon Robbins Reader in Neuroscience, Wolfson Centre for Age Related Diseases, King’s College London Lecture transcript Slide 3 Slide 4: Hello, my name’s Jon Robbins. I’m a neuroscientist at King’s College London. This Subtopic 2 is going to be on neurotransmitter systems. And you will have learned already that the neurons interact with each other by releasing neurotransmitters. You will have already heard about the synapse, in Subtopic 1, where this occurs. I have simplified this system into something I call the 2S, 3R, 2D system. The first ‘S’ stands for ‘synthesis’, the second – ‘storage’. The first ‘R’ is ‘release’, the second – ‘receptors’, the third – ‘re-uptake’. For ‘D’ – ‘D’ stands for ‘degradation’ and the final ‘D’ stands for ‘drugs’ that are targeted on the system and ‘diseases’ that involve it. Slide 5: The synapse, as you already know, is made up of two sections – the presynaptic terminal and the postsynaptic region. In between is the synaptic cleft. Synthesis occurs in the presynaptic terminal, along with storage, and reuptake, and degradation. The neurotransmitter is released into the synaptic cleft and can work on a number of receptors which can be found both postsynaptically and presynaptically. Slide 6: The first neurotransmitter I’m going to talk about is a very important one – it’s called ‘glutamate’ or ‘glutamic acid’. It’s an amino acid widely distributed in the central nervous system and it occurs at about 70 per cent of all synapses. There’s very little glutamate in the peripheral nervous system. Indeed, glutamate is the most important excitatory neurotransmitter in the central nervous system. Please note that this is a transcript. It is not a learning object. Please refer to topics for visuals and full lecture content. Week 3 @ King’s College London 2019 1. Slide 7: The first ‘S’ in my system is ‘synthesis’. So, this is how the neurotransmitter is manufactured, as required in the neuron. In fact, for glutamate, the synthesis occurs in two sorts of cells – on the left, in glial cells, and on the right, in a neuron. In glial cells, oxoglutarate is converted into glutamate by GABA transaminase. And on the right, in neurons, glutamine is turned into glutamate by glutaminase. Slide 8: The manufactured glutamate is stored in organelles called vesicles. The method by which glutamate gets into these vesicles is by a special protein, called a ‘transporter’. And this is particularly called a vesicular glutamate transporter. There are at least three types of vesicular glutamate transporter known and they all have this function of pumping glutamate into the vesicle. To get the glutamate in, hydrogen ions are pumped out. And this allows a concentration of glutamate in the vesicle to reach quite high concentrations – up to 20 millimolar. The high level of hydrogen ions found in vesicles that make them acidic and is used to pump in the glutamate is produced by a proton pump, which converts the energy of ATP into the higher concentration of hydrogen ions in the vesicle, which can then be exchanged for neurotransmitter. So, that is our storage part. Slide 9: Neurotransmitters, as you already know, are released by the nerve terminal at the axon terminal bouton. And these are released in a calcium dependent process. Calcium is required to both move and fuse the vesicles with the membrane to allow the neurotransmitter into the synaptic cleft. Slide 10: Once in the synaptic cleft, the neurotransmitter – in this case, glutamate – can act on the receptors. Glutamate has two major families of receptors – one family called ‘ionotropic glutamate receptor’, or ‘iGluR’s, and these are ion channels activated by glutamate. Pharmacologically, they could be subdivided into NMDA, AMPA, and kainate types. They’re all cation channels. And, mostly, they allow in sodium and out a little bit of potassium. However, the NMDA type cation channel also allows in significant quantities of calcium ions, which will be important later on in the module. Conversely, there’s another group of receptors that glutamate can act on and that’s the ‘metabotropic glutamate receptors’, or ‘mGluR’s. These are G-protein coupled receptors in the class ‘C’. Again, these can be subdivided into ‘Group One’, ‘Group Two’ and ‘Group Three’. Group One contains the mGluR ‘one’ and ‘five’. And these couple to particular G-proteins called ‘Gq’ and ‘G11’. Group Two include metabotropic glutamate receptors ‘two’ and ‘three’. And these couple to different G-proteins, ‘Go’ and ‘Gi’. And then the final group – Group Three – include the metabotropic glutamate receptor ‘four’ and numbers ‘six’ to ‘eight’. And these, again, all couple to the G-proteins, ‘Go’ and ‘Gi’. Slide 11: Once the neurotransmitter is released and acted on its receptors, then it can be re-uptaken back either into the neuron – as it shows on the left of this slide – or, indeed, back into glia – in this case, astrocytes, on the right hand of the slide. As we know, glutamate released into the synaptic cleft, can then diffuse. There are special transporters – proteins – that specifically take up glutamate back into the neuron. And they’re known as excitatory amino acid transporters. And they can return the glutamate back into the presynaptic terminal of the neuron, where it can be repackaged into vesicles and reused. Conversely, it can be taken up by the glial cells – in this case, astrocytes – and, here, it’s converted into glutamine by glutamine synthase. The glutamine can then be transported out of the astrocyte and into the Please note that this is a transcript. It is not a learning object. Please refer to topics for visuals and full lecture content. Week 3 @ King’s College London 2019 2. neuron by the glutamine transporter, ‘GlnT’. And that can then be synthesised back into glutamate by glutaminase. So, you can see there’s quite a complex process of removing glutamate from the synaptic cleft and recycling it. Slide 12: So, the first ‘D’ is ‘degradation’. Glutamate is quickly removed from synaptic cleft by the excitatory amino acid transporters and recycled. In astrocytes, it’s converted to glutamine by glutamine synthase. And the glutamine is transferred back to the neuron, where it’s converted back to glutamate by glutaminase. And again, this can be reused. Slide 13: The final ‘D’ covers two areas for this particular neurotransmitter. It indicates, firstly, the ‘drugs’ that act at this synapse. And these are examples for glutamate, shown on this slide. The receptors at which these drugs react are the NMDA receptor and the classic example of a drug that does this is ‘ketamine’. This is a dissociative anaesthetic and a channel blocker at this receptor. ‘Memantine’ is a competitive antagonist at this receptor. And, furthermore, another recently discovered and approved drug that acts on AMPA receptors is ‘perampanel’. And that’s a competitive antagonist. So, you can see- you can map the drugs that act on the synapse to this system. Slide 14: The second subset ‘D’ here is the ‘disease’. And, first of all, we know that some diseases are caused by the recreational uses of drugs – drug addiction and dependency. And that’s what I’ve put under recreational drugs, in the top left part of this slide. Some famous drugs, such as PCP and ketamine, are used as recreational drugs that act on the glutamate system in the brain. There are also some diseases particularly associated with a glutamatergic system. And that is epilepsy, because the control of the excitability of the brain is partly under the control of the glutamate system. In terms of function, glutamate is critical to pretty much all CNS functions. Slide 15: Once we have gone through all the particulars for that neurotransmitter, we can produce something called a ‘fact sheet’. And this is shown on the slide now. Notice the neurotransmitter is glutamate – so that’s what we’ve just done. And down on the left-hand side, I’ve given the individual letters – ‘S’, ‘S’, ‘R’, ‘R’, ‘R’ and ‘D’. And against these, you can put in the specific enzymes, ion channels, receptors etc that are associated with each part for this neurotransmitter. And on the right-hand side, you can indicate any drugs that you know of that act at these particular places. And you can subset the drugs, if you like, as I’ve done here. I’ve indicated, in green, drugs that are clinically used today. So, for glutamate, the first ‘S’ is glutaminase. And that’s one of the important enzymes that make it. The second ‘S’ – ‘storage’ – we know the storage is vesicular. The first ‘R’ is calcium dependent release at the terminal, so that’s release. And then the receptors we know about for glutamate are split into two major families – ionotropic, which include NMDA, AMPA and kainate, and then the eight subtypes of metabotropic glutamate receptors, which are G-protein coupled. Reuptake is by the excitatory amino acid transporter – EAAT. And degradation is by glutamine synthase. Please note that this is a transcript. It is not a learning object. Please refer to topics for visuals and full lecture content. Week 3 @ King’s College London 2019 3. On the right, I’ve indicated two drugs which are clinically useful – ketamine is used, as I said, as a dissociative anaesthetic and perampanel, which is used in some forms of CNS disorders. Please note that this is a transcript. It is not a learning object. Please refer to topics for visuals and full lecture content. Week 3 @ King’s College London 2019 4. Module: Biological Foundations of Mental Health Week 3 Synaptic transmission and neurotransmitter systems Topic 2: Neurotransmitters, receptors and pathways - Part 2 of 4 Dr Jon Robbins Reader in Neuroscience, Wolfson Centre for Age Related Diseases, King’s College London Slide 4: The next neurotransmitter I’m going to talk about is another important one. It’s called ‘gamma aminobutyric acid’. That’s rather a mouthful, so we’ll usually call it ‘GABA’. Again, it’s an amino acid. It’s widely distributed in the central nervous system and it’s at about 30 per cent of the synapses in the brain. There’s very little in the peripheral nervous system and this is probably the most important inhibitory neurotransmitter in the central nervous system. Slide 5: GABA is synthesised from glutamate – which is the first ‘S’ – by an enzyme called ‘glutamic acid decarboxylase’. So, its synthesis is relatively straightforward. Slide 6: It is stored, like glutamate, in vesicles, but the transporter that moves it into these vesicles is called ‘vesicular GABA transporter’, or ‘vGABAT’. Again, the vesicles also have a proton pump that fill them up with hydrogen ions which they use to exchange for the GABA neurotransmitter. And this is a common occurrence for vesicles. So, that’s the second ‘S’ – storage. Slide 7: The first ‘R’ is release. And, again, this will be a common occurrence. Again, it’s a calcium dependent vesicular release for GABA and this mainly occurs at the axon end terminal bouton. Slide 8: Receptors, once again, can be subdivided into two major classes. The ionotropic receptors, which are called the ‘GABA-A’ receptor, which is actually, in this case, an ion channel for chloride ions rather than the sodium and calcium. So, it allows negative ions into the cells. The metabotropic glutamate receptors associated with GABA are the ‘GABA-B’ receptors and they are coupled to the G-proteins, ‘Gi’ and ‘Go’. Please note that this is a transcript. It is not a learning object. Please refer to topics for visuals and full lecture content. Week 3 @ King’s College London 2019 1. Slide 9: Like glutamate, GABA is released into the synaptic cleft. Once it’s done its job of binding to the receptors, it can then be transported back, once again, into neurons and into glial cells – particularly astrocytes. The transporter protein that moves it into neurons is called the ‘GAT1’ or the ‘Neuronal GABA Transporter’. And the one that transports it into glia is appropriately named the ‘Glial GABA transporter’, ‘GAT3’. Slide 10: Degradation occurs by an important enzyme called ‘GABA transaminase’. And this occurs mostly in glial cells, such as astrocytes. a-Oxoglutarate is converted to glutamate and, at the same time, GABA is converted to an inactive compound called ‘succinic semialdehyde’. Slide 11: Finally, we talk about subset ‘D’, the last one, which includes drugs. And, here, I’ve shown drugs under the terms of receptors. And some famous drugs – that are used not clinically, necessarily – that act on the GABA A receptor are ‘muscimol’, which is an agonist and activates the receptor, ‘bicuculine’, which is a competitive antagonist, and ‘picrotoxin’, which is a GABA receptor channel blocker. Some of the clinically useful drugs that act on the GABA A receptor are the benzodiazepines, ethanol and many general anaesthetics – as these are all positive allosteric modulators of the GABA A receptor. For the GABA B receptor, ‘baclofen’ is an agonist and ‘saclofen’ is a competitive antagonist. Good examples of drugs that interfere with GABA re-uptake is ‘tiagabine’, which blocks GAT or the GABA transporter. And another drug, which is ‘vigabatrin’, blocks the important enzyme, GABA transaminase. So, there’s a number of places where you can interfere with the GABA synaptic transmission. Slide 12: The last ‘D’, here, is disease. I’ve indicated some recreational drugs – of which barbiturates are a good example – that act on GABA A receptors. The diseases you might be expected to find associated with GABA are epilepsy, anxiety, and insomnia. GABA has a major function in the central nervous system particularly associated with inhibitory actions of the brain. Slide 13: I’ll now move onto my fact sheet for GABA. Again, it’s in the same style as I’ve shown you before. Down the left are the six letters and what I’ve identified is the important aspect for each of these for GABA. So, the synthetic enzyme is ‘GAD’, the storage is ‘vesicular’, the release is ‘calcium dependent at the terminal’, and the receptors can be subdivided into ‘ionotropic GABA A receptors’ and ‘metabotropic GABA B receptors’. Reuptake is by GABA transporter and degradation is by the enzyme ‘GABA transaminase’. On the right, clinically used drugs indicated in green are ‘muscimol’ and ‘bicuculline’ – for the GABA A receptor – and the clinically used drugs that act here are benzodiazepines and anaesthetics. For the GABA B receptor, ‘baclofen’ is used clinically and ‘saclofen’ is a synthetic compound that is used for investigation action of receptors. As I previously mentioned, ‘tiagabine’ blocks the GABA transporter and ‘vigabatrine’ blocks the enzyme that breaks down GABA. So, these drugs are used clinically. Please note that this is a transcript. It is not a learning object. Please refer to topics for visuals and full lecture content. Week 3 @ King’s College London 2019 2. Module: Biological Foundations of Mental Health Week 3 Synaptic transmission and neurotransmitter systems Topic 2: Neurotransmitters, receptors and pathways - Part 3 of 4 Dr Jon Robbins Reader in Neuroscience, Wolfson Centre for Age Related Diseases, King’s College London Slide 4: Now we come on to my third example neurotransmitter – ‘dopamine’. This is a monoamine. And you can see it’s located in specific areas in the central nervous system. It has a more restricted distribution. For example, important pathways – the nigrostriatal pathway is dopaminergic and this one is particularly associated with Parkinson’s disease; the mesocortical pathway is also dopaminergic and that is associated with schizophrenia. So, you can see the distribution of cells that release dopamine are much more restricted than you see for glutamate and for GABA. Slide 5: My first S for this neurotransmitter is synthesis again. And this is a three step process where tyrosine is taken in by the diet and the rate limiting enzyme that converts this to DOPA is tyrosine hydroxylase. DOPA is converted to dopamine by dopamine decarboxylase. Slide 6: Storage, as before, occurs in vesicles. And these vesicles are very acidic, as before, because they also have the proton pump. And there are two types of vesicular monoamine transporters, called ‘VMAT1’ and ‘VMAT2’. And these can be cell type specific. Some dopaminergic neurons have one and, some, two. So, you can see this is a consistent event occurring commonly with many neurotransmitters. Slide 7: Dopamine is released. And, here, there’s some variation. It is calcium dependent, as normal. It occurs at the end terminal, as normal. But you also get a release that’s called ‘en passant’. And that’s where you have small release sites that occur all the way down the axon, as shown on this slide. And these varicosities can release dopamine all the way down, as the axon travels through tissue, as well as dopamine being released at the terminal end, as normal. Please note that this is a transcript. It is not a learning object. Please refer to topics for visuals and full lecture content. Week 3 @ King’s College London 2019 1. Slide 8: Dopamine receptors all of one type. They’re all G-protein coupled – or ‘metabotropic’ – class type. There are no ligand-gated ion channels for dopamine. They can be, again, subdivided into ‘D1-like’ and ‘D2-like’. The ‘D1-like’ family are ‘D1’ and ‘D5’. And they’re coupled to their G-protein, ‘Gs’. Whereas the ‘D2-like’ family, include ‘D2’, ‘D3’, and ‘D4’. And they’re coupled to G-proteins – ‘Gi’ and ‘Go’. Slide 9: Like before, once released, dopamine can be re-uptaken back into the neuron. And that’s by something called ‘DAT’ – ‘dopamine active transporter’. And that’s co-transported with one chloride ion and two sodium ions. Slide 10: The degradative pathway, the first D, is complicated. Dopamine can be degradated through a lot of steps to reach the common, final product, which is usually homovanillic acid. As you can see from this slide, you can go straight down-- where dopamine is first converted by monoamine oxidase-- and then an intermediate is converted into dihydroxyphenylacetic acid by catechol-Omethyltransferase. Conversely, dopamine can be converted by catechol-O-methyltransferase into three 3-methoxydopamine and then, by MAO, down to homovanillic acid. So there are a number of biochemical pathways that can lead to the breakdown of dopamine. Slide 11: For our drugs and disease section, an important drug, ‘levodopa’, used to treat Parkinson’s disease is a precursor for dopamine and, therefore, increases the amount of dopamine in peoples’ brains who have lost a component of it. A number of important drugs work on the storage of dopamine. And these work by blocking the vesicular transporter. And these drugs are ‘reserpine’ and ‘methamphetamine’. Drugs that interfere with release – ‘amantadine’ is a good example. And then some of the drugs that work on the receptors – full agonists – include dopamine itself, a compound called ‘apomorphine’ and ‘bromocriptine’. Competitive antagonists which are clinically used – ‘haloperidol’ and ‘chlorpromazine’ are also available. Reuptake of dopamine – a number of drugs work here – cocaine, for one, ‘bupropion’ and ‘methylphenidate’ – which is also called ‘Ritalin’ – work at this point. And then there are a number of important drugs that interfere with the degradation of dopamine – the monoamine oxidase inhibitors, ‘phenelzine’ and ‘selegiline’, and the COMT inhibitors, ‘entacapone’ and ‘tolcapone’. Slide 12: Then we can talk a bit about the recreational drugs that interfere with the dopaminergic system and there are some very famous, or infamous, ones here – cocaine, amphetamines and a rather unusual compound called ‘bromocriptine’, which can be found in the fungal contamination of grain and adds an important historical contribution to some of the psychosis that occurred when abnormal fungal were growing on the wheat used to make bread in the Middle Ages. The famous disease association with dopamine is Parkinson’s disease, but also schizophrenia – as you’ll find out later in the modules – also hormonal disturbances and drug dependence all involve the dopamine neurotransmitter system. What’s its role in the brain? Well, it’s possibly involved with reward systems, it’s Please note that this is a transcript. It is not a learning object. Please refer to topics for visuals and full lecture content. Week 3 @ King’s College London 2019 2. certainly involved in motor control and it possibly has an important role in thought processes and definitely in pituitary control of hormones. Slide 13: The fact sheet, as before – we’ve got our six letters down the left-hand side. And, for dopamine, the synthetic enzyme is ‘tyrosine hydroxylase’. The storage is by ‘vesicular process’. Again, we’ve got ‘calcium dependent terminal’ and, this time, ‘en passant’ release. Dopamine receptors, all Gprotein coupled receptors of five subtypes. The re-uptake protein is called ‘DAT’. And there are a number of enzymes involved in its degradation – ‘monoamine oxidase’ and ‘catechol-O-methyltransferase’. Many more drugs of clinical use are identified here – ‘L-DOPA’, which affects syntheses, ‘amantadine’, which can cause neurotransmitter release. And then a number of drugs, such as ‘apomorphine’, ‘haloperidol’ and ‘chlorpromazine’ – some of which are used for schizophrenia treatment. I’ve already indicated that ‘methylphenidate’ is used for some mental problems. And then there are some compounds that are used as adjuncts with L-DOPA and can be used to help treat Parkinson’s disease. Please note that this is a transcript. It is not a learning object. Please refer to topics for visuals and full lecture content. Week 3 @ King’s College London 2019 3. Module: Biological Foundations of Mental Health Week 3 Synaptic transmission and neurotransmitter systems Topic 2: Neurotransmitters, receptors and pathways - Part 4 of 4 Dr Jon Robbins Reader in Neuroscience, Wolfson Centre for Age Related Diseases, King’s College London Slide 4: My last neurotransmitter today is ‘5-HT’ or ‘5-Hydroxytryptamine’. It’s also called ‘serotonin’ and it’s another monoamine or, indeed, it’s, more specifically, an indolamine. Like dopamine, it has a very restricted distribution. As you can see, there’s one major nucleus that contains the cell bodies of these neurons, which is the ‘raphe’. And these project almost to everywhere in the brain. So, the cell bodies are all located in one part of the brain, but the projections go all over the brain. It’s also found in quite high concentrations in the enteric nervous system, but we won’t be discussing that now. Slide 5: As with dopamine, there are three steps to its synthesis. You’ve got ‘tryptophan’ taken into the diet. ‘Tryptophan hydroxylase’ is the rate limiting enzyme which converts it to 5-Hydroxytryptophan. ‘DOPA decarboxylase’ then finally converts it to the 5-HT, which is the active neurotransmitter. Slide 6: The second ‘S’ – storage. Again, a very similar picture to the one we saw before. We’ve got the same transporters – ‘VMAT1’ and ‘VMAT2’ – which transport 5-HT into the vesicles, again requiring hydrogen ions to be pumped out in exchange. Slide 7: The release is calcium dependent, mainly on the axon terminal bouton but, interestingly, it can be co-released with other neuropeptides, such as ‘somatostatin’ or ‘substance P’. Slide 8: Receptors fall into the two classifications, as before. You have the ligand-gated ionotropic receptor, which is the ‘5-HT3’ receptor, and that’s the only example of a 5-HT receptor that is a ligand-gated ion channel. This is a mixed cation channel, so it allows sodium and calcium into the cell and a little bit of potassium out. Please note that this is a transcript. It is not a learning object. Please refer to topics for visuals and full lecture content. Week 3 @ King’s College London 2019 1. The big list of G-protein coupled receptors, which are shown here, indicate a list of at least six families – ‘1’, ‘2’, ‘4’, ‘5’, ‘6’ and ‘7’. And these are subdivided by what G-proteins that they couple to, as we saw before. So, for example, the ‘5-HT1’ receptor family are mainly coupled to the G-proteins ‘Gi’ and ‘Go’ and, more than likely, found on the presynaptic nerve terminal. Whereas, the ‘5-HT2’ family couples to a different set of G-proteins – ‘Gq’ and ‘G11’ – which are usually postsynaptic. I will point out that the ‘5-HT5B’ receptor, although found active in animals, is a pseudogene in humans – so it does not actually get expressed. Slide 9: Reuptake – again, as before, diffusion of the 5-HT away from the synaptic cleft leads to its re-uptake by a particular protein called the ‘serotonin transporter’ or ‘SERT’, for short. Again, chloride and two sodium ions are co-transported with it to get it back into the presynaptic terminal. Slide 10: Degradation – the first ‘D’. 5-HT is converted by monoamine oxidase into ‘5-hydroxyindolealdehyde’. And then the second enzyme, ‘aldehyde hydrogenase’, converts it into ‘5-HIAA’, which is the common metabolite that monitors 5-HT. Slide 11: Then we come to drugs and disease. Important drugs – ‘L-tryptophan’ is an important precursor for the synthesis of 5-HT. It’s often used as a drug in depression. An example of a drug that works on the receptors for 5-HT is ‘sumatriptan’, which is used for migraine treatment. There are also competitive antagonists, such as ‘ondansetron’ and ‘ketanserin’. Reuptake of 5-HT can be blocked very specifically by ‘citalopram’, which is a serotonin selective re-uptake inhibitor used for depression, as is ‘imipramine’ and the monoamine oxidase inhibitor, ‘phenelzine’. You’ll hear more about these drugs as the module and course goes on. Slide 12: 5-HT has quite a large list of recreational drugs: amphetamines and its derivatives – particularly MDMA (or ecstasy, as it’s commonly known); LSD, a very famous one, works on this system and so does ‘mescaline’ and ‘psilocybin’. And psilocybin comes from magic mushrooms. Important diseases associated with 5-HT are depression, anxiety and hallucinations. As you can see, a number of the recreational drugs can produce those effects. So, therefore, 5-HT is probably important in mood, the sleep/wake cycle and appetite. Slide 13: Now we go to the fact sheet for 5-HT or serotonin. Again, down the left-hand side, as before, are the six letters. And I fill in, here, the particular important bits, which include ‘tryptophan hydroxylase’ as the synthetic enzyme, ‘vesicular’ storage, ‘calcium dependent’ release at the ‘terminal’. And then we have examples of the receptors that it works on, the re-uptake system ‘serotonin transporter’, and the important enzymes involved in its degradation – ‘monoamine oxidase’ and ‘COMT’. On the right-hand side, we see a list of drugs which ‘L-tryptophan’ is used clinically, so is ‘ondansetron’ and ‘sumatriptan’. And, again, we can identify which drugs are clinically used by colouring them green, whereas the others are useful compounds for studying the 5-HT system and maybe not used in humans. Slide 16: So, I’ve just given you four important neurotransmitters in the brain, but they’re only four of the 30 you may come across. I’ve given you a list, here, of others that are important and you are likely to come across in this Please note that this is a transcript. It is not a learning object. Please refer to topics for visuals and full lecture content. Week 3 @ King’s College London 2019 2. course, including ‘acetylcholine’, ‘ATP’ – ‘adenosine triphosphate’ – ‘bradykinin’, ‘glycine’, ‘histamine’, a whole range of neuropeptides – a couple of which I’ve already mentioned – ‘nitric oxide’ and ‘noradrenaline’. What I would suggest, as you go through the course, is make your own fact sheets as you find out about these neurotransmitters. And this will really help you build up your knowledge and get you a good understanding of the important roles of these molecules in the central nervous system. Please note that this is a transcript. It is not a learning object. Please refer to topics for visuals and full lecture content. Week 3 @ King’s College London 2019 3. Module: Biological Foundations of Mental Health Week 3 Synaptic transmission & neurotransmitter systems Topic 3 Neurotransmission defects and mental health: Focus on schizophrenia - part 1 of 3 Dr Anthony Vernon Lecturer in Neurobiology of Psychiatric and Neurodevelopmental disorders Lecture transcript Slide 4 Well hello, and welcome to this subtopic, which focuses on schizophrenia as an example of how neurotransmission deficits can cause mental health disorders. And the aim this subtopic is to give you an appreciation that defects in neurotransmission are associated with several mental health problems. You will do this mainly through looking at how impairment of dopamine signalling in the brain is implicated in schizophrenia. And you will also consider glutamate as a factor in schizophrenia. Slide 5 In order to fully understand this subtopic, you first need to be familiar with the fundamentals of neurotransmission. Neurotransmission is a fundamental brain process by which information encoded in the form of an action potential is communicated from one neuron to another within a given anatomical pathway and ultimately a neuronal network. Electrical information is received at the pre-synaptic neuronal terminal, and this is converted to chemical information through electrically stimulated neurotransmitter release which is driven by calcium influx into the pre-synaptic terminal. The released neurotransmitter then diffuses across the synaptic cleft and binds to an effector on the pre-synaptic membrane. This can either be a membrane-bound receptor protein or an enzyme and so on and so forth. The receptor becomes activated, which will activate second messenger pathways, for example, ionic flux, and depolarisation of the post-synaptic membrane. This converts the chemical information encoded by the neurotransmitter back into electrical information in the shape of action potentials, and in this way, information is propagated from one nerve cell to another. Slide 6 So what happens when neurotransmission goes wrong? Neurotransmitters, as we have seen, are essential for the transfer of electrical information between neurons within a functional brain network. So it may be said that neurotransmitters modulates the flow and rate of information transfer within a network, effectively gating synaptic plasticity. As a consequence, this process is subject to very tight regulation at several levels. In terms of neurotransmitter release, it’s controlled from the presynaptic terminal by autoreceptors. Neurotransmitter sites of action are subject to regulation-- for example, post-synaptic membrane receptors-- the number can be increased or decreased on the membrane. The neurotransmitter itself may be degraded either in the synaptic cleft by an enzyme-- an example of which would be acetylcholine, by uptake into the pre-synaptic terminal-- for example, through a Transcripts by 3Playmedia Week 3 © King’s College London 2017 1. transporter, or into surrounding glial cells. And finally, neurotransmitter synthesis and storage can be dynamically regulated by enzymes in the pre-synaptic terminal. And these multiple levels of regulation essentially ensure the correct fidelity of synaptic signalling. Ergo, when this equilibrium is altered, the final consequence is a disruption of the normal patterns of synaptic signalling. These will reverberate through neuronal networks, which ultimately manifests as a behavioural consequence. Slide 7 We’ll now move on to look at specific psychiatric disorder, schizophrenia, and how this is associated with neurotransmitters and deficits in neurotransmission. But to do that, first we need some basic facts about schizophrenia. So what is schizophrenia? Schizophrenia is a severe psychiatric disorder characterised by major disturbances in thought, emotion, and behaviour. How common is it? It is relatively common. Schizophrenia affects approximately 1% of the UK population. When does it begin? The onset of schizophrenia is typically in late adolescence or in early adulthood. How is it diagnosed? There is no diagnostic pathology for schizophrenia and diagnosis is currently based on clusters of symptoms. These are described as positive, negative, and cognitive, and we’ll look at these in more detail in a moment. How does schizophrenia relate to other psychiatric disorders? In common with other psychiatric disorders such as bipolar disorder or major depression, schizophrenia patients display cognitive impairments, but in contrast, schizophrenia is characterised by psychotic episodes consisting of both positive and negative symptoms. We will now consider these symptoms in more detail. Slide 8 The symptoms of schizophrenia can be grouped into three classes as you’ve just heard-- positive, negative, cognitive. Positive symptoms are described as additional features that are not ordinarily present. These include delusions, hallucinations that maybe auditory or visual, and thought disorder. Delusions occur and 90% of patients and represent an idiosyncratic belief or impression which is maintained despite being contradicted by reality or rational argument-- for example, I’m being watched by an alien force. Hallucinations are generally auditory-- for example, hearing voices-- and occur in 70% of patients. Patients may feel as though these voices come from the outside and they often think they’re being criticised by them. Hallucinations may also, however, been visual or related to smell, taste, or touch. Thought disorder may show up as disordered speech, including rapid changes of subject, the use of invented words, or in an appropriate emotional response to other people in a particular situation. Negative symptoms in contrast refer to a loss or reduction in a normal function. Examples include-alogia, the function of being reduced speech; affective flattening, which means a lack of emotional facial expression; avolition, meaning a diminished ability to begin and sustain an activity which is related to motivation; anhedonia, meaning you no longer find pleasure in something you used to enjoy; and asociality, meaning social withdrawal. Cognitive symptoms refer to specific impairments in certain cognitive domains and affect the patient’s general quality of life and ability to hold down a job. These include working memory, spatial memory, the ability to pay attention, and executive functions which may be defined as planning and decision making. The combination of these symptoms make it difficult for patients to interact with Transcripts by 3Playmedia Week 3 © King’s College London 2017 2. other people and may severely affect their work depending on the severity of each domain. Slide 9 Schizophrenia itself can take several courses over a patient’s lifetime. The graphs on the slide show possible life courses following a diagnosis of schizophrenia. The x-axis represents time and the y-axis symptom severity. Patients may fall into one of at least four broad categories. Number one, Group 1-- a single episode of psychosis which recovers with no lasting impairment, which corresponds to about 20% of the total number of schizophrenia patients. Group 2-- show repeated episodes of psychosis-- also referred to as relapse-remit-- with no lasting impairment, accounting for approximately 35% of patients. Group 3-- show repeated episodes of psychosis without full recovery to pre-symptomatic levels of functioning. The proportion is about 8%. In Group 4, the most serious, show repeated episodes of psychosis which increase in severity and are associated with no recovery to pre-symptomatic levels. This is about 35% of all cases on average. Slide 10 What then are the causes of schizophrenia? Epidemiological studies clearly highlight the combination of environmental factors, but there is also evidence from genetic studies that suggest genetic risk is a serious component of schizophrenia risk. In reality, it is the interaction between these environmental factors and genetic factors that determine the clinical outcome in terms of symptom severity, longterm outcome, and the life course that we just heard about. Some examples of environmental factors include obstetric complications; pre-term birth; hypoxia; exposure to infection or inflammation, either in utero or in early post-natal life; exposure to social stress, particularly during adolescence-- particularly childhood trauma is a common risk factor; and drug use, particularly addictive drugs such as cannabis, particularly during vulnerable periods of brain development have been associated with an increased risk of psychosis in the adulthood. On the genetic side, schizophrenia is clearly highly heritable, but the genetics are complex and they break down into rare variants that have large effect and are highly penetrant. And examples of this include the DISC1 gene and deletions of the gene known as neurexin-1, although there are others. More common are variants of small effect, which together interact. And this is often referred to as the polygenic score, meaning the number of these small mutations that you have in your genome. And together, as we said at the beginning, it is the interaction of these environmental factors and the genetic risk factors that define the clinical outcome. Transcripts by 3Playmedia Week 3 © King’s College London 2017 3. Module: Biological Foundations of Mental Health Week 3 Synaptic transmission & neurotransmitter systems Topic 3 Neurotransmission defects and mental health: Focus on schizophrenia - part 2 of 3 Dr Anthony Vernon Lecturer in Neurobiology of Psychiatric and Neurodevelopmental disorders Lecture transcript Slide 3 Having learned something about the basic clinical features of schizophrenia, we will now go on to consider how deficits in neurotransmission underpin the symptoms of this disease. In this section, we will focus on the role of dopamine, considering the evidence for the dopamine hypothesis of schizophrenia. To do this, we first need to know something about dopamine neurochemistry. Slide 4 On the slide is a cartoon of a dopamine-releasing neuron, illustrating the uptake, synthesis, storage, release, and re-uptake of dopamine. This basic information is necessary to understand some of the evidence that we will discuss in later slides for the role of dopamine in schizophrenia. Dopamine itself is synthesised from the amino acid tyrosine, which enters the neuron by active transport. In the cytoplasm of a dopaminergic neuron, defined as a neuron that primarily synthesises and releases dopamine, tyrosine is first converted to dihydroxyphenylalanine, or DOPA, by an enzyme called tyrosine hydroxylase. This is the rate-limiting step for dopamine synthesis and is a useful marker of how much dopamine a cell is producing, and by proxy, releasing. And this will be important later on. DOPA is converted to dopamine by L-amino acid decarboxylase also known as DOPA decarboxylase, and actively transported into synaptic vesicles through vesicular monoamine transporter 2. Following release, dopamine binds to postsynaptic dopamine receptors, which are divided into D1 and D2 subtypes. And please refer to earlier lectures by John Robbins. Dopamine can also bind to presynaptic auto receptors that inhibit further dopamine release. Dopamine in the synaptic cleft is inactivated by active transport back into the presynaptic terminal by the dopamine transporter, or DAT, where it is degraded or stored again in vesicles. Degradation of dopamine occurs presynaptically via an enzyme known as monoamine oxidase, but a small percentage may also be degraded postsynaptically by Catechol-O-Methyl Transferase, or COMT. Slide 5 Now we have some idea of how dopamine is made in a dopaminergic neuron, we next need to explore the neuronal pathways in the brain that utilise dopamine as a neurotransmitter. Dopamine is used by dopaminergic neurons in three primary pathways in the human brain, as shown on the slide. Transcripts by 3Playmedia Week 3 © King’s College London 2017 1. These are the nigrostriatal pathway, which is critical for the control of movement, and projects from the substantia nigra to the striatum. The mesolimbic and mesocortical pathways, which project from the ventral tegmental area to the nucleus accumbens, amygdala, hippocampus, to the mesolimbic pathway, and the prefrontal cortex, the mesocortical pathway. And this pathway involved in both limbic and cognitive functions, such as memory, motivation and emotional response, reward and desire, and addiction. The third pathway is the tuberoinfundibular pathway, which projects from the A8 dopaminergic nucleus via the hypothalamus to the pituitary gland. This is involved in hormonal regulation and secretion of the hormone prolactin. The mesolimbic and mesocortical pathways, as you see from their function and their topographical projections, are, therefore, well placed to contribute to the symptoms of schizophrenia. So how does this occur? Slide 6 To understand this, we now must introduce the dopamine hypothesis of schizophrenia. The basic premise of the dopamine hypothesis is that an increase in dopaminergic neurotransmission in the mesolimbic pathway leads to abnormally high levels of dopamine in the nucleus accumbens and the striatum, which are thought to underlie the positive symptoms of schizophrenia, as we’ve already heard about, including hallucinations. As you can see on the slide, in panel A, we can see the mesolimbic pathway in a normal individual projecting from the VTA to the nucleus accumbens. In panel B, this is what is happening in the schizophrenic patient. Now we can see that this projection is overactive and there is a much higher amount of dopamine in the nucleus accumbens as compared to the normal control. In contrast, a decrease in dopamine transmission-- shown in the second image-- in the mesocortical pathway leads to lower levels than normal of dopamine in the prefrontal cortex, and this is thought to explain the negative and cognitive symptoms of schizophrenia. So again, in the diagram, in panel A, we can see the situation in the normal brain where there is a normal equilibrium of dopamine neurotransmission from the VTA to the prefrontal cortex. And in panel B, we see that this pathway is reduced and there is less dopamine in these areas, leading to negative and cognitive symptoms. Slide 7 If we accept the dopamine hypothesis, we have to first understand the evidence underlying it. What is the evidence for the dopamine hypothesis? Primarily, this comes from clinical observations and experiments with dopamine-releasing drugs combined with Positron Emission Tomography, or PET, a neuroimaging method used commonly in humans, but is also possible to do in animals. Clinical observations in the 1950s, doctors treating schizophrenia patients serendipitously observed that certain drugs, such as chlorpromazine, the target of which was unknown at the time, decreased the positive symptoms of schizophrenia. This suggested that understanding how chlorpromazine had this action could provide insights into the neurobiology of schizophrenia. These drugs were thereforafter referred to as anti-psychotic drugs. In 1963, Carlsson and Lindquist subsequently showed that anti-psychotic drugs increased the amount of dopamine metabolites in the cerebral spinal fluid of schizophrenia patients. They hypothesised that this may be something to do with the brain compensating for the blockade of a dopamine receptor in the brain, although at this stage, dopamine receptors have not yet been identified. Subsequently, in the late 1980s, early 1990s, and into the 2000s, experiments in healthy people using PET who were given amphetamine showed that when amphetamine is given, dopamine release is Transcripts by 3Playmedia Week 3 © King’s College London 2017 2. stimulated, and these patients displayed positive symptoms of schizophrenia, including hallucinations. More importantly, when schizophrenia patients were given amphetamine, their symptoms became much worse. Clear evidence that increasing dopamine neurotransmission induces schizophrenia-like symptoms in otherwise healthy people and increases the severity of symptoms in patients already with a diagnosis of schizophrenia. Taken together, these clinical observations and experimental medicine studies with amphetamine were the first key pieces of evidence that led to the development of the dopamine hypothesis. Slide 8 Although it is clear that changes in the amount of dopamine could be related to the positive symptoms of schizophrenia, it was unclear where this might happen in the brain. So to understand this, we can use PET, as we discussed in the previous slide, to look at how much dopamine is present in specific parts of the brain. PET is a technique that allows the visualisation of specific proteins, such as an enzyme or neurotransmitter receptor with very high sensitivity, by combining a specific molecule that binds to these proteins with a radiolabel. Figure A shows the cartoon of the dopamine neuron you saw earlier. To visualise dopamine production in the brain, we can use a radiolabeled analogue of dopamine, 18 fluorodopa, to visualise dopamine synthesis and storage pathways in a living person, because the synapse treats this as if it were normal dopamine. 18 fluorodopa is taken up into the presynaptic terminals, where it is metabolised by DOPA decarboxylase This can provide a proxy measure of the rate of dopamine synthesis, otherwise referred to as dopamine synthesis capacity, which we would expect to be higher if there is an increased rate of dopamine release in the patient we’re studying. An example of the image this generates is shown in figure B. The areas of high signal intensity, red to green, are the caudate and putamen, which contain the highest density of dopaminergic terminals. Patients with schizophrenia have been repeatedly imaged using 18 fluorodopa PET and compared to healthy controls who do not have schizophrenia, or any other psychiatric disorder. These experiments show that schizophrenia patients, the red dots in the graph where each patient is represented by a dot, have a higher uptake value given by the rate constant KI on the y-axis of 18 F-DOPA in the striatum compared to the healthy controls indicated by the blue dots. This increase in dopamine synthesis capacity correlates positively with the severity of patient positive symptoms. Several studies greater than 50 to date have replicated these findings in different groups of schizophrenia patients around the world, and this provides the most robust evidence of dopamine dysfunction in schizophrenia localised to the mesolimbic pathway. Slide 9 Further evidence for the dopamine hypothesis comes from studies of anti-psychotic drugs and their binding to dopamine D2 receptors. As we’ve already heard, the finding that drugs like chlorpromazine block the positive symptoms of schizophrenia were serendipitous, but led to the coining of the term anti-psychotic drugs. The role of dopamine in schizophrenia was therefore strengthened by the identification of dopamine receptors in the brain, and the subsequent finding that all anti-psychotic drugs bind the dopamine D2 receptor. Indeed, the efficacy of anti-psychotics, measured as a daily dose required for the treatment of positive symptoms of schizophrenia, is closely correlated to the potency or affinity with which a Transcripts by 3Playmedia Week 3 © King’s College London 2017 3. particular anti-psychotic binds to the dopamine D2 receptor, as shown in figure A. An exception to this, however, is clozapine, which has a low affinity for the D2 receptor, but is one of the most effective anti-psychotic drugs. The reason for this is currently unclear, but clozapine and other anti-psychotics do bind to a number of other neurotransmitter receptors in the brain. This tells us that changes in dopamine neurotransmission might not be the whole story behind the symptoms of schizophrenia, which we will revisit later in this topic. Slide 10 Nevertheless, subsequent PET studies found that a specific percentage of dopamine D2 receptors must be blocked to achieve a good clinical response, defined as a reduction of positive symptoms. These studies suggest that 60% to 80% of dopamine D2 receptors must be blocked for the maximum therapeutic effect. This is shown in figure B. Here on the x-axis is the percentage of occupied or blocked D2 receptors following anti-psychotic dosing. On the y-axis is the clinical improvement in positive symptoms from none to significant improvement. It can be seen clearly that as the percentage of dopamine D2 receptors blocked increases to the critical window, more patients, indicated by the green dots, show recovery of their symptoms. We should note, however, that once the threshold of 80% dopamine D2 blockade is crossed, patients’ positive symptoms may improve, but they begin to suffer side effects, as shown by the red dots. These are described as extrapyramidal symptoms and include dyskinesia and other movement disorders, such as akathisia. These reflect the action of anti-psychotics on dopamine D2 receptors in other dopamine pathways, such as the nigrostriatal pathway, which are responsible for the control movement. This represents an elegant description of a drug therapeutic window, defined as the range of doses in which positive effects are seen without adverse side effects. As we can see, for anti-psychotics, this window is quite narrow, suggesting care must be taken in dosing. Slide 11 If the dopamine hypothesis is correct, we might ask where the excess dopamine activity comes from. It might be that the patient produces too much dopamine, doesn’t metabolise excess dopamine quickly enough, or has D2 receptors that have been modified so they respond differently to dopamine binding, principally being more sensitive to dopamine. So how could this come about? The stress diathesis model was developed to explain this excess of dopamine in the schizophrenic brain. It suggests that an individual inherits several genes that encode for abnormal proteins, leading defective dopamine function in the mesolimbic pathway, rendering the pathway hyperactive and leading to the positive symptoms. So what is the evidence for this model? The dopamine D2 receptor is one of the more significant hits in large scale studies of the genetics of schizophrenia. This genetic risk is paired with environmental stresses which further modify dopamine release. For example, stress during adolescence. Perhaps together, these can be enough to create the symptoms leading to a diagnosis of schizophrenia. However, we must also consider what evidence does not support the dopamine hypothesis model, and here, it is important to realise that a significant proportion of schizophrenia patients do not respond to anti-psychotic drugs, and their positive symptoms do not improve. This might suggest that dopamine hyperactivity is only one of the causes of the onset of schizophrenia. But is there any evidence for this? Transcripts by 3Playmedia Week 3 © King’s College London 2017 4. Slide 12 Reading slide (no audio) Slide 13 Here, we consider this possibility by looking at levels of dopamine and response to anti-psychotic treatment to understand whether there may be subtypes of schizophrenia. As we have just discussed, in about 30% of cases, the positive symptoms of schizophrenia patients do not improve following treatment with anti-psychotic drugs. If this continues, despite switching drugs or changing the dose, these patients may be described as treatment-resistant. This is a very difficult clinical problem, as essentially, there are no effective treatments for these individuals. One explanation for treatment resistance could be that the patients do not have the same abnormalities of dopamine neurotransmission as those who respond conventionally to anti-psychotic drugs. But is there any evidence to support this? Using 18 fluoro PET scans, as we discussed earlier, recent studies have revealed that this may be the case. The graph in A on the slide shows that people who respond to treatment have an increased capacity to produce dopamine. In other words, a higher dopamine synthesis capacity in the striatum, as shown by the red dots. And these are referred to as treatment responders. In contrast, people who are described as resistant to anti-psychotic treatment do not show an elevation in this dopamine synthesis capacity and are indistinguishable from healthy controls. Studies in the same individuals using a different neuroimaging technique called Magnetic Resonance Spectroscopy have found that patients who respond to anti-psychotics have normal levels of glutamate in the frontal cortex, whereas patients who are resistant have higher amounts of cortical glutamate as compared to healthy controls and treatment responders. These data confirm that treatment-resistant patients may not have an abnormality in dopamine synthesis capacity, but may have defective glutamate neurotransmission instead. This suggests that other neurotransmitters, particularly glutamate, are important for schizophrenia symptoms, not just dopamine. Clearly, there are also implications for the treatment of schizophrenia, and it is critical to be able to identify individuals who will or will not respond to anti-psychotic medication early such that other alternative drugs may be tried, including glutamatergic drugs that are currently in development. Slide 14 The dopamine and glutamate evidence that you’ve looked at is the first biological in vivo data that demonstrates that there may be at least two subtypes of schizophrenia-- one based on dopamine and one that does not seem to involve the dopamine system. Although these data require replication in a larger cohort, they confirm what has been long suspected, that the symptoms of schizophrenia cannot solely be explained by the dopamine hypothesis. Clinical evidence would support this, suggesting ant