Membrane Excitability: Action Potential PDF

Physiology \#3 -- 01.10.21 **MEMBRANE EXCITABILITY: ACTION POTENTIAL** The resting membrane potential is the constant membrane potential recorded when a cell is electrically at rest and it cannot be considered as an electrical signal. However, neurons and muscle cells can undergo transient, rapid...

Physiology \#3 -- 01.10.21 **MEMBRANE EXCITABILITY: ACTION POTENTIAL** The resting membrane potential is the constant membrane potential recorded when a cell is electrically at rest and it cannot be considered as an electrical signal. However, neurons and muscle cells can undergo transient, rapid fluctuations in their membrane potentials producing electrical signals. These electrical signals are not a metaphysical entity but changes in the membrane potential of the excitable cells within a precise time course. 1. **Variation of membrane potential** The passive proprieties of the membrane are triggered into an active response of the membrane (alphabet). At one point, when responding to specific stimulations, the cells act autonomously comparatively to the active stimuli, as a trigger. The active responses are later organized by the neuron that translates the incoming messages in outgoing messages. Once responses are generated and organized, they are transmitted along the membrane. 2. **Depolarization and hyperpolarization** The resting membrane potential highlights the difference in potential across the membrane and its value is -70mV. This means that the membrane is polarized since there is an excess of negative charges inside and an excess of positive charges outside. This graph shows that if something happens at the membrane, measuring the change in potential by using electrodes, it will go from -70mV to -40mV: -40mV means that the membrane is still polarized but the magnitude of polarization is reduced, so it is called depolarization. Depolarizing currents drive the membrane potential towards 0mV. If the membrane potential goes towards more negative values, it is called hyperpolarization. When, from a depolarized state, the membrane comes back to its resting membrane potential it is called repolarization. When the membrane potential goes to a positive value it is called reverse polarization, meaning that the inside of a cell becomes more positive relative to the outside. 3. **The passive response of plasma membranes** *A big axon that is extracted as it can be excited and easily analyzed due to the tube shape; a recording setup with intracellular and extracellular electrodes; a voltmeter to measure the membrane potential across the membrane. The stimulating electrode injects current into the membrane.* *This setup allows us to see if the membrane potential changes when a current is injected into it.* By convention, the direction of the stimulating current is determined by the direction of the positive charges flux. As we are referring to ions and the main role is played by positively charged ions, if a current that injects positive charges is used, it is an inward current; whereas, if a current that is extracting positive charges is used, it is an outward current. Therefore, the direction of the current is the direction of the positive charge (this is not normal since normally we measure the flow of electrons). Inward current causes depolarization while outward current causes hyperpolarization. This is a square pulse stimulation because from 0mA it goes to the desired intensity very fast, then the amount of current is kept stable and then it is removed immediately. This type of stimulation is the best way to see the membrane's reaction to changes in current intensity. The pink graph shows the changes in membrane potential in response to the stimulation. A plus current is injected into the membrane: at the very beginning the membrane doesn't react but then the membrane depolarizes. Then the current is kept stable, so the membrane potential also remains stable but when the current is removed suddenly the membrane starts repolarizing. At the end, it comes back to the resting membrane potential. This is an inward current since it injects positive charges into the membrane and causes depolarization. The membrane doesn't react perfectly in sync with the stimulus and this is visible at depolarization (part 1) and repolarization (part 3) in the graph. There is a delay showing that the cell takes a while to reach a stable condition when the current is injected and when it is removed. The membrane response is locked with the stimulus, however the time course between the two is not exactly superimposable. This is a passive response since the current is injected from outside and the membrane doesn't do anything by itself. This response clearly depends on the stimulus: the higher is the stimulus intensity, the higher will be the resulting graded potential. The relationship between the stimulation intensity and the amplitude of the variation in membrane potential is a linear correlation and it is the OHM's law. An electrical equivalent model (RC circuit in parallel) which mimics the membrane's behaviour: the membrane lipids are the capacitor, the battery is the pump and the ion channels are the resistors. When the circuit is closed, the current flows through where the resistance is the lowest. The current divides at the two arms but at the very beginning most of the current will flow through the arm with the capacitor as the resistance is low since it is empty. However, the more current passes through the capacitor, the more charged the capacitor will be and this increases its resistance. Thus, as time goes on more current will pass through the other arm. When a current is injected into the membrane, at the beginning the membrane is charged so the current doesn't flow into the ion channels: there is no change in membrane potential. Then, when it starts flowing through the ion channels, the change in membrane potential occurs: the capacitor has to be completely charged in order to have a change in membrane potential. When the current is removed, it stops going through the channels but the capacitor needs time to lose the accumulated charge so it takes a while for it to come back to the resting potential. The charges do not stay stuck in one place in the ICF after they enter, but they flow from the relatively more positive depolarized region towards the more negative regions. Once they are there, they move out through ion channels into the ECF and then they are attracted by the negatively charged regions and are distributed along the field in decreasing amounts as the distance from the cell increases due to increased resistance. Once this happens across a membrane and the ions distribute along the field, the electrical field outside the cell is also changed: what happens to the membrane also influences the electrical field outside the membrane. The passive graded potential spreads along the axon as the charges spread but the amount of current flowing through two areas of the membrane depends on the difference in potential between the two points and the resistance of the material. Some current will spread along the axoplasm while the rest will move across the membrane. If the resistance of the axoplasm is low compared to the membrane it would be easier for the current to move along the axon and vice versa. This circuit depicts plasma membrane resistance in parallel and axoplasmic resistance in series. When the current moves out charges are lost and the membrane potential at that site changes. When 100 charges are injected into the axon, if the axoplasm resistance is high and the membrane resistance is low, 70% will go out and 30% will move along the axon. Out of that 30% in the next patch more charges will be lost because the membrane conductance is higher and axoplasm conductance is low: in a short distance all current will be lost. The membrane potential decreases as the distance from the stimulating site increases. This means that the current dissipates and that the membrane resistance was low compared to the axoplasm resistance and a lot of current was lost in a small distance. The space constant is the distance needed to reduce the amplitude of the graded potential at the site of stimulation to 30% of its initial value. It is the square root of the membrane resistance divided by axoplasm resistance. The higher is the membrane resistance the higher will be the space constant. The response when the membrane is stimulated by an inward or outward current is a response locked to the stimulation and its amplitude depends on the stimulus intensity: these are called graded potentials. They are local responses as they are evident at the site of stimulation but are lost as the distance from the stimulation site increases. Graded potentials require a triggering event as the membrane cannot cause it by itself so it is a passive response. The duration of the graded potential varies depending on how long the triggering event keeps the gated channels open (it starts when the triggering event is on and ends when the triggering event is off). Graded potentials spread out over short distances but with dissipation so this is called decremental conduction. In vivo, the ions are the current flowing through the membrane and the triggering event is something able to open or close ion channels. In excitable cells the resting membrane potential is due to the leak channels and the graded potentials are due to gated channels. The gated channels can be controlled by ligands, mechanical displacement, voltage or an internal messenger and when one of these mechanisms causes a change in the state of a given population of channels, it will result in an additional conductance. If Na or Ca channels open in response to a triggering event, depolarization will occur; if K or Cl channels open hyperpolarization will occur. 4. **Action potential** An action potential is an active response of the membrane. With the lowest intensity square pulse stimulation, at the start, depolarization occurs and as soon as the stimulation stops, repolarization occurs. With the medium intense stimulation the same event occurs. However, with the strongest stimulation, the cell responds as usual at the start, but when it reaches a certain value of membrane potential, the cell starts a very fast and sharp depolarisation: the membrane potential reaches zero and goes to a more positive value of +50mV; here the stimulus is off but the membrane potential goes up irrespective to the stimulus. At a certain point, it stops going up and starts repolarizing quickly and the membrane potential goes beyond the resting membrane potential and hyperpolarizes. Then, the cell slowly repolarizes again. This whole process is called action potential. An action potential is a variation of membrane potential that occurs after being triggered; however, after reaching a certain value the membrane doesn't need the stimulation to increase its potential: during an action potential, the cell will behave in the same way no matter how many times it is triggered. The distinguishing features of an action potential are: - A certain value of membrane potential, the threshold value, needs to be reached in order to trigger an action potential: when the cell is stimulated, it leads to a graded potential and if the membrane potential reaches the threshold value, an action potential will occur. - It is an all or nothing response: wither it occurs or it doesn't, there are no small or partial action potentials. - Once triggered, its time course doesn't depend on the stimulus duration - Its amplitude is greater compared to graded potentials. - Its time course is the same: the hyperpolarization phase can last longer or shorter but the first part of the action potential always occurs within the same time course. - It has an absolute and relative refractoriness. - There is no decremental conduction: once an action potential is triggered at one point, the same action potential with the same amplitude will occur at any point, no matter how far away it is from the starting point. This is important because action potentials must be conducted over large distances in our bodies without dissipation.  The black graph shows the changes in membrane potential. The first part is called the spike and it is the constant part of an action potential, its time course cannot be reduced but the hyperpolarization part can be shortened. Once the threshold is reached, the Na conductance increases very sharply (pink straight line) indicating that the Na channels are opening very fast. Then suddenly the Na conductance decreases and this time course is in parallel with the first phase of the action potential. When the membrane potential goes up, Na conductance goes up and when it decreases Na conductance also decreases: the first part of the action potential is due to a Na current. When Na enters, it tries to drive the membrane potential towards its equilibrium potential of +70mV but it fails because the Na channels close. The Na channels are voltage gated channels, since a threshold value needs to be reached in order to trigger this variation in conductance. When the membrane potential reaches the threshold value, the Na channels change their conformation and open; these channels are more likely to open when the cell is depolarized: the first Na channels open once the threshold value is reached and the entry of Na results in further depolarization and, since the Na channels are more likely to open during depolarization, more and more Na channels open. This involves a positive feedback loop. Then, the Na channels close and, with a certain delay, the voltage gated K channels open. The K channels have the same threshold value as Na channels but their time course is slower: the peak of conductance of K occurs when the cell is repolarizing and is just near the resting membrane potential. Infact, when the K channels are open while Na channels are all closed, since there are no more Na ions to counterbalance the K ion movement hyperpolarization occurs. When the K conductance moves to 0, afterhyperpolarization ends and the resting membrane potential is reached. So, Na is responsible for the hyperpolarization and repolarization of the action potential while K is responsible mainly for the afterhyperpolarization. Neither of the ions are able to reach their equilibrium potential. 5. **The voltage gated Na channels** The voltage gated Na channels have 2 gates: an activation gate and an inactivation gate. Both gates are able to close the channel but they behave oppositely in response to voltage. When the cell is at rest the activation gate is closed and the inactivation gate is open. Above threshold the activation gate opens and the inactivation gate also remains open but since the inactivation gate wants the cell to remain depolarized, it wants to close the pore. Therefore, when the cell starts depolarizing, the activation gate starts opening while the inactivation gate starts closing. Within a time interval both gates allow the entry of Na but soon the inactivation gate closes the pore resulting in a drop in potential. The Na channels opens gradually and the higher is depolarization the higher is the probability of the channels opening: it is not an on off situation but a gradual increase and decrease in conductance. The first channels to open will be the first channels to close: this leads to a stereotyped response. The duration of the action potential is always the same because, given that the two gates are equally sensitive to voltage but they perform opposite actions (one opens while the other closes), there is only a fixed amount of time during which the Na can enter. The positive feedback loop causes a huge response in a short time and could result in a huge depolarization but, as the inactivation gate is sensitive to the same voltage with the opposite effect, there won't be a problem. When the threshold is reached at b, the activation gate opens and when the value becomes more positive there is a complete opening of the activation gate, while the inactivation gate starts closing. At d there is the peak because the inactivation gate is just about to close. After the inactivation gate closes at e, the membrane potential needs to reach values below the threshold, in order to open the inactivation gate again. Until repolarization has occurred from d to f, another action potential cannot occur even if a huge amount of current is supplied because it needs to wait until the inactivation gate opens: this is the absolute refractoriness. 6. **Voltage Clamp Experiments: Ionic Currents underlying the action potential** **In** the spike, the first part of the action potential, can be seen that **dominant conductance increases the conductance of sodium**. **So, the current responsible for the depolarization, also called overshoot, is a sodium current**. After that, the cell repolarizes until it returns back to the resting  potential and goes down in hyperpolarization. Then, repolarization starts and in the first part of this phase a drop of conductance of sodium can be seen; simultaneously, we have a slow increase in the conductance of potassium. When the cell is trying to go back to resting membrane potential, it goes down to threshold, towards the negative values. Considering the conductance is negative in value, the potassium is dominant. So, potassium rise the membrane towards its equilibrium potential. After membrane comes back to the 'resting' membrane potential, afterhyperpolarization occurs. 7. **Gates and States of the channels** **The gate and the conductance of the sodium are opened thanks to the gate channel. The gate channel is composed by two gates: the activation gate and the inactivation gate.** **The behaviour of the membrane potential can be explained through the behaviour of these channels: at the value -70 mV, when membrane potential is at rest, there is excess of positive charges outside and excess of negative charges inside, and the activation gate is closed, blocking the channel pore. This prevents the sodium ions to pass through the channel and thus the inactivation gate is opened.** **Now, a depolarizing current is injected into the membrane and causes the depolarization of the membrane: if the depolarizing current succeeds and reaches the threshold, the activation gate is opened, leading the way for sodium to enter.** **Above the threshold, when the membrane gets more depolarized, the probability of the opening the activation gates is higher.** **As activation gates open, there is a progressive opening of the sodium channels. As a consequence, a fast, but gradual increase of conductance is observed. This is defined as the positive feedback loop: the more channels are opened, the more the sodium enters, which results with more depolarization. Consequently, more depolarization results in more sodium channels opening.** **The positive feedback loop is maintained by the inactivation mechanism. The action of inactivation gate during depolarization takes some time, because it requires for the cell to be very depolarized.** **When depolarization has a gradual trend, the activation gate starts opening and the inactivation gate is about to close. As depolarization increases and cell becomes very depolarized, inactivation gate starts closing, and cell loses depolarization. The inactivation gate remains closed until the membrane potential goes below threshold, and reaches to more negative values.** **e.g., assuming that the threshold is -50 mV, during the values below -49 mV, channels start opening again. When the cell is going up the threshold, the activation gate is opened; while, when the cell is going down the threshold, the inactivation gate is opened.** **This means that below threshold, the activation gate will be closed immediately and inactivation gate leaves the pore.** **When cell is depolarized, the sodium channel goes from closed to open conformation. The more the cell is depolarized, the more likely it is for the channel to be in an open conformation. When the channel has an open conformation, the cell repolarizes and the cell goes down below the threshold. When values more negative than threshold are reached, the sodium channel returns to the initial configuration.** **So, this is a cycle that, given the properties of the sodium channel, determines the time course of the spike, which is always the same. As the time course of the spikes are always the same, action potential can be stereotyped.** **The second consequence is that when the inactivation gate is closed, neither giving currents nor stimulating the cell will be able to elicit another action potential, because the more the depolarization increases, the more the inactivation gate remains closed. So, the only way to elicit another action potential is to open it.** **In the first phase, the inactivation gate is opened and the activation gate is closed. Some cells which got depolarized will reach the threshold, resulting with the inactivation gate remaining opened and activation gate starting opening. Then, depolarization is established with a high probability of lots of activation gates opening.** **Once the inactivation gate realizes the depolarization of the cell, it starts to close but it takes quite a while. So, there is only a certain time where both the activation gate and the inactivation gate are opened and this is the only window of time in which sodium can enter.** **But, as soon as +30 mV are reached, the inactivation gate starts closing and membrane drops down towards more negative values. When values below the threshold are reached, the inactivation gate starts to open again, while the activation gate closes. At this point, it is not possible to have another action potential, because the pore is blocked by the inactivation gate. This window of time, in which inducing another action potential is prevented, is called absolute refractoriness. \"Absolute\" means that if the inactivation gate is closing the pore, sodium cannot enter and, consequently, the cell can\'t generate another action potential, because it depends on sodium entry.** **The only condition that opens both the inactivation gate and the activation gate, so the only condition to have another action potential, is to wait to be below threshold. Once below the treshold, cell can be pushed towards threshold again and only when this is reached, another action potential can be generated.** **This is a positive feedback loop: in fact, the more depolarization increases, the higher is the probability to have an activation gate opened, which leads to sodium voltage-gated channels opened and, consequently, to inward current opened.** **Closure of the inactivation gate causes Na+ flow through the channel to stop, which in turn causes the membrane potential to stop rising. So, during repolarization, no more sodium can enter the cell. When the membrane potential passes -55 mV again, the activation gate closes. After that, the inactivation gate re-opens, making the channel ready to start the whole process over again.** **So, both activation and deactivation gates are activated by the treshold, but in opposite ways. The inactivation gate is oppositely sensitive to the potential.** 8. **Refractoriness** The following pic represents the **time course of action potential: the first part is the spike, the second part is afterhyperpolarization, then there is the absolute refractoriness and, finally, the relative refractoriness.** **When the value is below -55 mV (threshold), the activation gate is opened. So, if a current is given and the membrane is again driven above the threshold, another action potential takes place due to availability of the channels. This means that during the afterhyperpolarization, if current is given, another action potential can be elicited.** **When neuron is at afterhyperpolarization, the distance from the threshold is higher with respect to the resting situation, because it is more negative. So, to reach the threshold more current will be needed.** **But as a principle, during afterhyperpolarization, there can't be another action potential because the inactivation gate is closed, so in order to open the activation gate the threshold has to be reached.** **In summary, refractoriness is the property of excitable tissues that determines how closely together two action potentials can occur. This property depends on both Na+ and K+ conductance.** **There can be two types of refractoriness: absolute and relative.** **During absolute refractoriness, if currents are injected, a new spike cannot be elicited, no matter the intensity of the current. This is due to the presence and organization of the sodium channels, and it's fundamental for the action potential to be discrete.** **On the other side, during relative refractoriness, a new action potential can be elicited but starting from a disadvantageous situation due to the flow of potassium and to the hyperpolarization period. The current has to be increased to have a new spike.**  9. **The importance of the ions in the interstitial fluids** **In the graph, concentration of potassium is being increased during the resting membrane potential. This means that both the gradient, the Nernst equation and equilibrium potential will change; as a result of this, membrane potential changes spontaneously.** **In a cell that is excitable, if cell is depolarized, the threshold can be reached without any intention. If threshold is reached, an action potential is elicited to make the cell more excitable and more keen to trigger an action potential.** **Action potentials are meaningful signals, so they have consequences. They either communicate with others or induce activation of devices, for example, muscles. So, the action potential is not random, it is wanted when there is a precise sequence of events. If action potentials were random some cells would randomly get excited, causing problems (for example, in the heart which is full of excitable cells). This is why blood assessments for ions are very important.** 10. **Excitable cells show different action potentials** **Excitable cells show different action potentials.** **e.g., the time scales and time courses are very different in these 3 different cells: motor neuron, fibre of skeletal muscle and myocardial muscle cell. The spike of the motor neuron lasts 2 ms, the rapid phase of skeletal muscle fibre is 5 ms, and the action potential of myocardial muscle lasts 200 ms, which is very long.** 11. **Myelinated and unmyelinated axons** **Action potential is conducted without decremental conduction, without dissipation.** **The reason of graded potential being dissipated during conduction is due the current which enters and distributes along two possible ways: the first is the axoplasm and the second is the membrane (channels of the membrane). Dissipation occurs when current is lost through the membrane. So, the lower is the resistance of the axon of the membrane and the higher is the resistance of the axoplasm, the lower will be the distance at which the potential is lost. This is because current is lost through the membrane, and this is unavoidable. In fact, sooner or later, no variation in potential is observed if you record far from the stimulating site (because current is lost on the way).** **On the other side, action potential has almost no dissipation and this is due to its very smart way of transmitting. On the membrane, that is in charge of conductance of the transmission of action potential, there are lots of voltage gated ion channels.** **Initially, in the case of the pic above, there was a huge sodium inward current that resulted in the action potential being generated in a patch of axon, having a huge positive value. The current of the action potential flows and distributes in the neighbourhood, and near the neighbourhood there are voltage gated sodium channels. The patch of membrane which is near the one that has been stimulated and has generated the action potential, is brought to threshold. Then, another action potential is generated, and the inward current goes in the neighbourhood to generate another action potential. So, there is continuous regeneration of the action potential.** **The difference with the graded potentials is that, in the stimulating site the graded potentials have channels that mediate the variation in potential. So, the current that is responsible for variation in potential (that we call the graded potential) is concentrated in the stimulated site and is not spread out around in the rest of the membrane. So, basically graded potential is a specific outflow of the current, and there is no regeneration of the graded potential.** **The case of action potential is different as it can be seen in zone 2 (the zone that has been excited and that generates the action potential), where the inward current of sodium is applied. There is the overshoot, and then there are the sodium charges that go in the neighbourhood. So, the charges will depolarize the patch adjacent to the patch that was stimulated. If there were no channels, there would be a dissipation. If there are voltage gated channels and the membrane is brought to threshold, then they will react. Therefore, they will develop another action potential, which will lead into the depolarization of the other patch, which will generate another and another action potential and so the potential is transmitted.** **Starting from the axon hillock, membrane potential is recorded with 11 electrodes placed in series, along the axon. These are recording the membrane potential in position 1,2,3,4,5 until 11 is reached. There is a long distance from 1 to 11, and since the action potential is regenerated in all the fractures of the membrane, it takes time to reach the 11^th^ one. The membrane of an axon is equipped full with voltage gated sodium channels for sodium and potassium. This is why there is conductance without decremental.** **In the neuron, there are small regions of the axon called the nodes of Ranvier where membrane is uncovered, and there are regions which are myelinated containing layers of membrane of Schwan cells.** **During the first action potential, current wants to go in both directions. But, the myelination results with the current bumping into the wall due to the high resistance to conductance, so current only flows through the axoplasm where the resistance is lower, in only one direction.** **Then, the current reaches the node of Ranvier which has lots of voltage gated sodium ion channels, so there is low resistance-high conductance as there is no "ceiling"-like myelin. Therefore, there will be huge inward sodium current (depolarization) flowing into at the position of the rode of Ranvier, into the axoplasm, allowing the action potential to be generated, as the graded potential can reach the threshold. The node of Ranvier is the only position where action potential can be regenerated in the myelinated axons, resulting in the velocity of conductance to be higher when compared with unmyelinated axons. In an axon that is unmyelinated, the action potential should be regenerated at each patch of membrane, and so velocity of conduction is lower.** **The other factor that can affect velocity of conduction is the diameter of axon. The larger the diameter, the lower the resistance of axoplasm, so velocity is higher. So big axons are faster than small axons.** **Conduction in myelinated axon is called saltatory conduction (because action potential is generated only at the nodes of Ranvier); in this way, the propagation is faster and more efficient. In the case of a pathological condition such as multiple sclerosis, in which there is corrupted myelination, results with no signalling due to no sodium channels, and the ability to conduct the action potential is lost. So, the action potential which is considered as a command, doesn't reach the target (the effector), and consequently action will not take place.** **This graph allows to see effect of both myelination and diameter. It can be observed that even when the size of axons are the same, there is a significant difference in velocity of conduction of the cat and the squid, due to myelination. This means that if an unmyelinated squid axon has a big size, the velocity of conduction of squid is higher than a myelinated cat axon with a small diameter. So it shouldn't be generalised that all myelinated axons have higher velocity of conduction than all unmyelinated axons, diameter size is a factor as well.** 12. **Sensory neurons and receptor potential** **An action potential in a neuron is generated only in the axon hillock or in the case of sensory neurons, at the level of sensory terminal. The action potential is the final output of the neuron, something happens on the dendrites and in the soma that eventually results in the generation of action potential, that is the way of transmitting the command. This is a digital signal.** **Axon hillock is also called the trigger zone because it is the place where the action potential is originated. The end of the axon, ends in the target, the target is another neuron (in the picture above).** **The sensory neuron is exactly the same, but the job of sensory neuron is to detect the stimulus, convert the stimulus into electrical signal and to convey the signal into the brain.** **In sensory neuron, the terminal of the axon is specialized in the sensory function, it is a transducer.** **A transducer is equipped with channels and it is able to convert a specific type of energy such as thermal, into electrical signals which are action potentials that are going to travel backwards (to the CNS).** **In the picture, that is a skin receptor, the stimulus is the increase of pressure on the receptor. This will lead the sodium ion channels to open either directly, or via second messengers, resulting in depolarization, which is a graded potential. If this stimulus is high and if threshold is reached, the action potential is generated.** **The change in amplitude of the membrane potential is called receptor potential. Receptor potential is a specific way of referring to graded potential. If amplitude is high enough and reaches the threshold, the action potential is established.** **The stimulus (the mechanical stimulation of the tip of the finger) induces the receptor potential in the final portion of the membrane. The current entering in the tip of the axon travels until it reaches the first node where there are voltage gated sodium channels, and they trigger the action potential.** **Apart from one unique situation in the retina, the variation in the potential always leads to depolarization (will be covered later).** 13. **Receptor potential, sensory transduction and frequency of discharge**  *In this image a Pacinian receptor, skin receptor, is shown. The stimulus (1) is a mechanical displacement (pressure is applied, it states and then it is removed) and its timing course triggers the receptor potential, which follows the stimulus. So it is a graded potential. If stimulus increases, also graded potential increases.* The brain is not full of electrodes able to stimulate it, so it has two different ways to stimulate itself: the first is the receptor potential in sensory neurons, that corresponds to the graded potential. This potential would be transmitted with dissipation, but as soon as it reaches the first node of Ranvier or if it is a non-myelinated axon, where the membrane expresses the voltage-gated channels, depolarization happens and the action potential is triggered. Then it is transmitted without incremental transmission and it is directed towards the soma of the neuron. Sensory transduction is the conversion of all types of energies into receptor potential (so, into the electrical language) and it is necessary because the nervous system has receptors only for this type of energy (the other types are ignored). The stimulus alters the conductance and the receptor's permeability, generates current and if the current is enough to bring the membrane above threshold, the action potential is generated and it travels from the periphery to the soma (physiological direction of conductance in sensory neurons). The frequency of action potential can be modulated depending on the intensity of the stimulus: for example, if there is a digital signal, only the number of action potential, the frequencies and their occurrence in time can be modulated. In fact, "graded" potential means that it can vary in amplitude, proportional to the intensity of stimulation (e.g pressure). In the picture, a pseudounipolar sensory neuron is shown. These neurons are located in the dorsal root ganglia and present a single axon that divides into two branches: one goes towards the periphery and one enters the CNS (in this case the spinal cord). The branch that goes towards the periphery is specialized as a receptor at the end: when the stimulus interacts with the receptor, the graded potential and then the action potential are generated and the action potential is transmitted along the axon towards the soma (but it actually does not reach the soma because the neuron is pseudounipolar, therefore it goes directly towards the brain). In the image, two types of stimuli are applied (green line) and the second stimulus is more intensive than the first. Then the action potential is measured both at the level of the sensory receptor (receptor potential-orange line) and at the level of the axon (red line): at the level of the sensory receptor the magnitude of the receptor potential increases when the intensity of the stimulus increases, while at the level of the axon it is the frequency of the action potential that increases. So, the intensity is coded by variating the graded potential: the higher will be the amplitude of the graded potential, the higher will be the frequency of discharge of the axon. Consequently, the intensity is coded by the frequency of the action potential: this is called "current to frequency transduction/coding". During the relative refractoriness, other action potentials can be elicited through giving more energy. In the picture, an axon is represented (the scheme below the diagram of the action potential). The first stimulus is applied and the graded potential is generated. Then a stimulus with X intensity is applied and X is exactly the current needed to reach the threshold starting from -70 mV (it is the minimum intensity that reaches the threshold). Then, after the triggering of the action potential, the stimulus is not stopped, on the contrary, it continues to be applied, but another action potential will be generated only when it will go back to the resting potential (-70 mV). The frequency, that can be measured, is the minimum frequency of discharge of this axon and so, it is represented by the time needed to generate another action potential. This is crucial, because the duration of the after-hyperpolarization is not the same in all the neurons, it can be shorter or longer, and this means that not all the neurons discharge with the same range of frequency. But, the increase or the decrease of this frequency depends on the current that is applied: in fact, if the current applied is high, it allow the neuron to reach the threshold even if it is in hyperpolarization. So, the higher is the current applied, the more the minimum frequency of discharge can be increased (in the picture, the frequency is increased and there is the generation of 4 action potential instead of 2. This continues until the stimulus is stopped). The amplitude of the graded potential is a measure of the strength of the depolarizing currents. Then, variation in potential is what is measured and what happens is ions running reaching or not the threshold. This explains why if a receptor is stimulated by two different stimuli with different intensity, the first thing to vary is the amplitude of the graded potential, then it is transmitted towards the first portion of the axon, which has voltage-gated channels, and if the threshold is reached, action potential is triggered. *In the image, there are 3 diagrams on the top and 3 on the bottom, that represent the transduction of the action potential (Left: near the receptor; Right: middle of the axon). Threshold is reached and on the top there is the generation of 4 action potential, while on the bottom the frequency of the action potential is increased because current is higher. Also the afterhyperpolarization is shown. The curved line is lower in the central diagram and it is not represented in the diagram on the right because of dissipation of the graded potential (while action potential is not lost).* 14. **Adaptation** Adaptation means that a neuron in presence of a stimulus, that remains constant for a while, adapts (in general, decreases the reaction). In the picture, the currents of sodium and potassium are shown; they trigger the action potential and then there is afterhyperpolarization. The current is higher at the beginning and then decreases, because conductance is decreased and this determines the end of the afterhyperpolarization. Rheobase is the intensity of the stimulating current minimal to reach the threshold, applied for an infinite time (so, the minimum intensity to reach the threshold). At the rheobase there is no adaptation, because the neuron discharges at the minimal frequency. But, when the intensity of stimulation is increased, also frequency of discharge is increased at the beginning (from 80 ms passes to 31 ms and then to 52 ms). In picture B, adaptation is shown: frequency slow down from 31 ms to 52 ms, that is then maintained, so the first intervals of discharge are very high, but then the neuron set to a lower frequency, if the stimulus does not change. The current to frequency coding is maintained, so the steady state of discharge is always regulated in terms of relationship with the stimulating current. What changes are the first intervals, where the neuron reacts to the new intensity with an higher frequency and then adapts; this adaptation, which is a reduction of the discharge with respect to the first interval of stimulation, is due to the fact that during the action potential some other ions, such as calcium, enter the neuron and open and additional population of potassium channels (so, other channels for potassium, that are not only those voltage-gated, are introduced). Consequently, this increases the power of hyperpolarization, which becomes higher, the higher is the frequency of the first spike. Then the neuron adapts and remains in a regular discharge. In fact, in the image, it is shown that when more action potentials are generated in a short period of time it happens that, after the spike, there is an additional K current (I~K1~) together with the normal one (I~K~), which induces a stronger hyperpolarization and therefore more time is needed to get back to normal resting membrane potentials. The population of potassium channels increases in number depending on the frequency of the first discharges. Adaptation is a general rule in the brain, all neurons adapt. In the image, the first intervals are shorter than the others, then the steady state situation is regular and related to the intensity; so, in order to establish the intensity to frequency, the steady state, and not the first intervals, has to be measured. But the intensity is not the only important thing, also the time course matters. In this case, there are two types of receptors and the stimulus, its duration and intensity are the same. At the centre, the receptor potential is represented, while on the bottom there is the discharge of the axon. The tonic (or slow adapting) receptors, after the generation of the graded potential, adapts very slowly (at first, the frequency is high, while then it decreases and stabilizes); on the other side, the phasic (or fast adapting) receptors seem to react only when the stimulus changes (one graded potential corresponding to the onset and one to the offset, and the same for the frequency of discharge). The adaptation of the phasic receptors is faster not only for the property of the membrane, but also for the coupling with accessory structures; this structures around the receptor prevent the receptor to generate a receptor potential when the stimulus is not changing (extreme adaptation). e.g., the Meissner's receptors of the skin are axon terminals enveloped by an onion shaped collagen structure with fluid inside: when the threshold is applied, before ending up onto the axon, goes into the structure around it. The viscosity of this structure affects the threshold on the axon, such as a filter, allowing the stimulation only in the phasic time of the stimulus. So, the slow adapting receptors are the only that allow to measure the intensity, because to measure it is needed a receptor that causes an action potential also during the steady state of the stimulus. In the image, are shown mechanical stimuli applied to the skin; the measure of the intensity is in µm because of the indentation of the skin, that represents the second measure of the pressure applied. So, fast adapting receptors modulates the frequency of discharge varying the time course of the stimulus: in fact, on the right, it can be seen that the velocity changes and decreases, but the intensity is the same. So, fast receptors are good in coding transient phases (when the stimulus is changing), but they are not good when the stimulus is stable, because they adapt immediately. Summary: Among neurons, there are the sensory neurons, important for the places where the action potential originates. The action potential can originate only in two places: at the axon hillock or at the terminal. In the sensory world, it originates at the terminal, that is stimulated by a type of energy, which induces a current directly or by a second messenger. The current induces the graded potential (receptor potential), which can lead to the action potential. The intensity of the stimulus is due to the current to frequency coding (higher amplitude of the membrane receptor potential, higher frequency of discharge). This is true for all the types of sensory neurons, but they are also equipped in differently in the membrane and around the membrane of the sensory portion. These structures confers to the receptor proper different properties, and there can be tonic or slow adapting receptors (very good in coding intensity, they like to continuously monitor what is not changing) and phasic or fast adapting receptors (able to measure the velocity and the acceleration of the stimulus onset and offset). 15. **The clinical point: Multiple sclerosis (MS)** It is a pathophysiologic condition in which nerve fibers in various locations throughout the nervous system loose their myelin. Loss of myelin slows transmission of impulses in the affected neurons. A scar known as a sclerosis (meaning "hard") at the multiple sites of myelin interferes with and eventually can block the propagation of action potentials in the damaged axons.