NEUROPHYSIOLOGIE CELLULAIRE CONSTANCE HAMMOND Chapter 3 Neurotransmitter release In chapter 3, we proose 3 videos: Observation Calcium Channel, Ca 2+ Unitary and Total Currents Ca 2+ and Neurotransmitter Release CHAPTER 3 INTRO (1:30) This third chapter has to do with the calcium current released by the arrival of the action potential to the axonal terminals. This calcium current enables the entry of calcium ions into the axonal terminals and neurotransmitter release by synaptic vesicles. What are the objectives of this chapter? At the end of the chapter, you will know about a calcium current through a voltage-gated calcium channel. You will understand the various states of a voltagegated calcium channel: closed, open, and sometimes inactivated. You will understand how intracellular calcium ions initiate synaptic vesicle exocytosis and neurotransmitter release and you will understand calcium ion clearance. Can you explain to us in simple terms this first phase of synaptic transmission? Information is transmitted between neurons in chemical form. What we are going to understand in this chapter is how axonal terminals translate the arrival of an action potential which is an electric signal into neurotransmitter release, a chemical signal. Action potential is like an electric control of a factory door which, once open, lets out employees. Except, this electric control does not work very well, and neurotransmitters are not released. What learning tools are provided in this Chapter? In addition to the course videos, supplements, quiz, practical exercises, there is a video of an experiment staged at INMED. CH. 3-1: OBSERVATION (6:18) In this Chapter 3, we are going to study how action potential arriving at the axonal terminals initiates neurotransmitter release. Let us begin by observing synaptic transmission. Let us suppose we have two connected neurons: a pre-synaptic neuron in orange and a post-synaptic neuron in blue. Since is is located after the synapse it is called pre-synaptic, and before the synapse pre-synaptic. Both the neurons are recorded in whole-cell configuration and in current-clamp mode to record changes in potential. If we stimulate the pre-synaptic neuron, we are going to record an action potential in this pre-synaptic neuron which which initiates a very small-amplitude change in potential in the post-synaptic neuron which we refer to as excitatory post-synaptic potential because it depolarizes. If we add TTX to the extracellular environment in which the neurons are submerged, we observe that the stimulation does not create a pre-synaptic action potential< which is normal, because the voltage-gated sodium channels are blocked by TTX. And we observe that there is no post-synaptic response either which is logical because there is no pre-synaptic action potential. If we submerge the experiment now in a medium that is completely free of calcium ions, 0 calcium, it does not affect the pre-synaptic action potential at all in response to stimulation but, on the other hand, there is no post-synaptic response altogether. Do calcium ions play a special part in synaptic transmission? And if so, what do they act upon? To understand what calcium ions act upon, we will work with the giant synapse of a squid. In this giant synapse, we see here the end of
the axonal terminal. This is the pre-synaptic element. It makes contact with a post-synaptic element that we see here in yellow and black, and the synapse is located under the presynaptic element. We can fill this pre-synaptic element with a calcium ion marker called aequorin. Why would we? The thing is that aequorin binds calcium ions and in this bound form becomes aequorin-calcium. Now, this molecule fluoresces differently depending on whether it is bound or free. So, using aequorin fluorescence, we can find out whether calcium ions have entered the pre-synaptic terminal. Here in the image, we record what is going on with the fluorescence, and we see here the pre-synaptic element which fluoresces without doing anything. Now we stimulate the pre-synaptic element here to induce action potentials. And we have this recording, little b. To understand what transpired between the two, we pull these two images and we see here the calcium that entered during the stimulation. We distinguish little calcium domains which we refer to as microdomains. We can see well that calcium enters the pre-synaptic terminals and enters in a non-uniform manner which suggests that there are calcium channels in this pre-synaptic element which enable calcium ions to enter. Based on the previous experiment, we can hypothesize that action potentials arriving at the presynaptic terminals open calcium channels, and calcium enters through these channels in the pre-synaptic terminal. If that is the case, what type of channel are they? What are calcium currents? What are their characteristics? Can we explain these microdomains that we have seen in the pre-synaptic terminal? To understand the types of channels and to study them, we must be able to record axonal terminals using the patch clamp method. Now, that is very difficult because they are a very small diameter, about 1 micrometer, and frequently, that is exactly the diameter of a recording pipette. But we could work with giant synapses. We just saw the giant synapse of a squid. There is also the calyx of Held which is found in the mammalian auditory central nervous system and is a giant synapse, or we could work with a neuromuscular junction which is a synapse between motor neurons and muscle cells and a giant synapse as well. We could also record the calcium channels in a soma because some calcium channels can be found in the axonal terminals of a soma. One, two, three, four types of calcium channels are described in various preparations and referred to as L, N, P/Q, and T and which are also referred to as Cav for "voltage-dependent". Which types of calcium channels are present in axonal terminals? If we study the neuromuscular junction which is easy to study because it is a giant synapse, we see that these are essentially N-type channels, and then, in other synapses, P/Q-type channels. We will confine ourselves to studying N-type channels which are most frequently to be found in axonal terminals and which enable calcium ions to enter these terminals. So, we arrive at this hypothesis that in inward voltagedependent calcium current (because it is initiated by sodium action potential via N-type highthreshold calcium channels) is responsible for a temporary increase in calcium that we have seen in the giant synapse of a squid. We arrive at the following hypothesis: an inward voltagedependent calcium current comes from N-type high-threshold channels. What does this mean, more specifically? That calcium ions enter through voltage-dependent calcium channels because these open in response to a high-threshold action potential which means that the potential should be really depolarizing for them to open, and that is what happens during an action potential. They would be responsible for a temporary increase in intracellular calcium ion concentration which we saw in the giant synapse of a squid. MOOC Neurophysiologie cellulaire Ch3 Cours 2/6
CH. 3-2: CALCIUM CHANNEL AND CALCIUM CURRENT (6:08) N-type calcium channels that are also called Cav2.2 have the following form. The are reminiscent of sodium channels, i. e. it is a single protein with 1, 2, 3, 4 similar transmembranal domains. In each transmembranal domain, we see Transmembranal Segments 1, 2, 3, 4, 5, 6, then there is the P-loop which is formed when the protein folds back on itself (here, we see it folded on itself in the form of a pseudo-tetramer). This P-loop is located in the aqueous pore and Transmembranal Segment 4 which is charged and gives this protein its voltage sensitivity. And calcium ions pass through this aqueous pore into the protein when it is folded in three dimensions. To study the unit N-type calcium current we choose the cell-attached configuration here to have very few channels under the pipette and in voltage-clamp mode to study the current. Since we are in cell-attached mode, there is extracellular fluid in the pipette. We put 110 mm of barium in this extracellular fluid instead of calcium. Why do we put barium to measure unit currents? Because calcium channels are very permeable to barium ions and this produces a unit current of greater amplitude than that produced by calcium ions. We apply blockers to channels other than the N type. And now, we record the unit current. Since we are in voltage-clamp, we apply a membrane potential of -80 mv. At this potential, there are no calcium channels that open because these are voltage-gated calcium channels. Over the course of 200 ms, we depolarize to +20 mv and we see that during this depolarization channels open with an inward negative current. And thereafter, they re-close, then open, then re-close, then open. It is a weak current of 0.5 pa. In another experiment, we see that even though there is this test, there is no response up to +20 mv. There again, there is not response. There again, there is not response. And in all other cases, there are responses that are variable. Let look at the i-v curve now which represents unit current as a function of voltage. There, we were at +20 mv, that means, for instance, about 0.5 pa here. If we now produced test potentials that are more hyperpolarized than +20, the current's amplitude would increase, therefore. This current of greater amplitude is logical because, let me remind you, that calcium ion equilibrium current is about +50 or +60 mv. So, the further we are from the reversal potential which is about here, the further we are from this reversal potential, the stronger the current. And then, toward -50, we no longer have anything because the channels are closed below -50. What are the characteristics of total N-type calcium current? To understand what they are, we record in whole-cell configuration to record several N-type calcium channels. In voltage-clamp mode to record current, we put 2 mm of calcium on the outside, and also add sodium and potassium voltage-gated channel blockers not to record these and blockers for other non-n-type calcium channels. Membrane potential is maintained at -80 mv, and we apply a depolarizing voltage up to +20 mv. We see an inward current in response to this potential step which drops off gradually during this potential step. Thus, there is a descending phase, a peak, and a gradual weakening of this current despite the fact that the potential is still +20 mv. This indicates that, in fact, the channels inactivate. The descending phase corresponds to the gradual opening of the various N-type calcium channels which opening happens rapidly. And here, we would need to see that we have, in fact, a number of current steps which are summed up to produce this smooth curve. If we repeat the experiment by applying many depolarizing steps, we record a small current at -30 mv. And then, at 0 mv, a stronger current. And at +20 mv, we are at peak current. The current-potential plot shows us that at -30 mv, we have a weak current and that this current gradually increases up to a peak which occurs towards +20 - +30 mv, and then falls off. Then why do we have a MOOC Neurophysiologie cellulaire Ch3 Cours 3/6
bell-shaped I-V curve although for the unit current, we gad a straight line? Because for a unit current, the channel either opens or does not open. Here, we see the behavior of multiple channels, of very many channels found in the membrane. The thing is that some channels open at the beginning, depolarize the membrane, and cause the channels that are not yet open to open under the influence of this depolarization. Therefore, this is like the sodium channels: they open one after another until all are open that can. And then, the current falls off because we approach the reversal potential. Normally, the current should be much stronger because here, we are very far from the reversal potential. But the fact that the channels do not open all at the same time in response to voltage causes the current to increase gradually from not much to a peak, and then falls off. CH. 3-3: CALCIUM AND NEUROTRANSMITTER RELEASE (10:12) We know now that calcium ions enter axonal terminals in response to the arrival of an action potential thanks N-type calcium channels that open that are high activation threshold channels. So, they are opened by a strong depolarization like at the time of an action potential. Calcium ions enter a pre-synaptic terminal and cause neurotransmitter release. What are the connections between calcium ions and and neurotransmitter release? Since the time delay between calcium ion entry and neurotransmitter release is extremely short, on the order of hundreds of milliseconds, it suggests that there is a physical connection between calcium channels and the vesicles containing the neurotransmitter, that somehow everything was ready for neurotransmitter release. This diagram shows an axonal terminal, which we call a pre-synaptic element, with the pre-synaptic membrane. This terminal contains synaptic vesicles which contain the neurotransmitter. Here is the synaptic cleft and across it, the postsynaptic neuron's post-synaptic membrane. We now know that there are calcium channels located very close to vesicles attached to the membrane. Sometimes we also called them docked. Let us see in this diagram how synaptic vesicles are docked to the pre-synaptic membrane and where are the calcium channels. What we see in this diagram is the presynaptic membrane which is here and here. And here, we see a vesicle with its membrane all around it. So, a vesicle and a calcium channel here. There is a physical connection to the presynaptic in the form of syntaxin, an integrated protein of the pre-synaptic membrane. There are also connections between syntaxin and proteins integrated into the vesicle or pre-synaptic membrane. So, they are called v-snares for vesicles and t-snares for targets. This entire complex here makes the vesicles attach to the pre-synaptic membrane and accounts for the fact that the calcium entrance is not far at all from the vesicle. When calcium ions enter the intracellular environment of an axonal terminal, they attach to a protein that is a calcium sensor and is called synaptotagmin. And this causes a complete change in the system configuration when the vesicle membrane fuses to the pre-synaptic membrane. When calcium ions are entering a pre-synaptic terminal because calcium channels have opened, there is a local increase in the intracellular calcium ion concentration. This cases the vesicles to fuse with the pre-synaptic membrane. This is a phenomenon known as exocytosis. The two membranes merge at a certain point in time and create a whole referred to as a fusion pore which neurotransmitter molecules use to exit. All these stages, between vesicle docking and exocytosis, involve a large number of proteins. These are complex phases which are not as yet fully understood because they involve a change in protein conformations. Afterwards, these vesicles are recycled through endocytosis, then refilled with neurotransmitters which enables MOOC Neurophysiologie cellulaire Ch3 Cours 4/6
them to participate in another neurotransmitter release. To summarize, an action potential arrives at an axonal terminal. This action potential which is going to depolarize the membrane all the way to +20 mv, or thereabouts, causes voltage-dependent high-threshold calcium channels to open. Entering calcium ions provoke exocytosis and the release of neurotransmitter that are going to attach themselves to the various receptor sites on the postsynaptic membrane. It should be noted that even when all the conditions are there, i. e. when there are several pre-synaptic action potentials, neurotransmitter release does not always occur. This is a relatively inefficient process, such that only one action potential in two to twenty will cause calcium channels to open, neurotransmitters to release, and a post-synaptic response to fire. Now, if there is a post-synaptic response, how does this response, or neurotransmitter release, stop? Once calcium ions are inside, what happens to stop vesicle fusion and exocytosis? Multiple processes are required for the axonal terminals to return to a quiescent state. As far as the membrane is concerned, it is going to repolarize, primarily because there is an inactivation of the spike sodium channels as we saw in Chapter 2. There is also an inactivation of the N-type calcium channels as we saw when we were studying the N- type total current. We saw that it was a current that inactivated fairly rapidly. And then, there is also an activation of calcium-activated potassium channels. We are not going to look at these channels right now, they are explained in a supplement. And what is their role? To transport potassium out to help repolarize the membrane, and since they are calciumactivated, as soon as calcium ions enter a pre-synaptic terminal, the intracellular part of these channels activates, the channels open, and let potassium ions out. This results in a rapid repolarization of the pre-synaptic terminal; therefore, calcium ions do not enter any more because calcium channels close. They close because the membrane is repolarized. But those calcium ions that are already inside? What are they doing? They are ejected from the presynaptic region through multiple processes: pumps that use energy in the form of ATP and that either put calcium in reserve, in an endoplasmic reticulum, for example, or outside; transporters which transport, or exchange calcium ions for sodium ones and also eject calcium to the outside; binding to proteins in the intracellular environment, such as parvalbumin, calbindin, that are able to bind calcium ions and therefore, make it disappear in its ion form which is the form in which it is active. All this makes the increase in intracellular calcium temporary, very brief which means that neurotransmitter release is also brief. Whenever an action potential arrives at an axonal terminal, the depolarization phase of this action potential enables the opening of calcium channels which essentially belong to the N or the P/Q type and which are high-threshold and open in response to strong depolarizations, such as an action potential. As a result, calcium ions enter axonal terminals and increase intracellular calcium concentrations but this increase is very local and temporary. It is temporary because calcium channels inactivate and there are also mechanisms that will eject calcium ions toward the extracellular environment thus reducing local calcium ion concentrations. Whenever the calcium that has been transported inside reaches an intracellular concentration of 0.5 to 40 micromolars, there is synaptic vesicle exocytosis, or a neurotransmitter release. Effectively, these calcium ions bind to calcium sensor proteins, such as synaptotagmin, and this changes the configuration of the vesicular and the pre-synaptic membranes and these two membranes fuse. This exocytosis occurs with a short delay of about 200 microseconds because all the calcium channels, calcium sensor proteins, and docked vesicles are very close and linked to each other. Repolarization of the axonal terminal which is important in enabling another MOOC Neurophysiologie cellulaire Ch3 Cours 5/6
synaptic transmission occurs thanks to potassium current. These are currents either of an action potential and activated by depolarization that we saw in Chapter 2 or those that we will see in a supplement that are sensitive to intracellular concentrations of calcium ions which means that these channels open whenever calcium ions are transported into axonal terminals. And while these channels are open, potassium ions exit cause the membrane to depolarize. We now know that an electrical signal, such as an action potential is created by ion currents in the form of ions, such as sodium and potassium ions, transported across the membrane. This electrical signal created at the initial axonal segment and traveling along the axon arrives at the axonal terminals. At the axonal terminals, it depolarizes the the membrane of the axonal terminals, open calcium channels, and causes calcium ions to enter. This inward transport of calcium ions is detected by calcium sensor proteins and results in the exocytosis of synaptic vesicles containing a neurotransmitter. This way, the neurotransmitter is released into the synaptic cleft. In the following courses, we will see how this neurotransmitter is going to recreate an electrical signal that is called post-synaptic potential. So, we have an electrical signal, an action potential, in a pre-synaptic neuron. An action potential causes a calcium ion current which also depolarizes the membrane (sometimes, we talk about a sodium-calcium action potential because calcium ions are involved) and causes the release of a chemical molecule. Now, we are going to see how a chemical can recreate an electrical signal. MOOC Neurophysiologie cellulaire Ch3 Cours 6/6