CSB346 Lecture 6 Notes

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Cell and Systems Biology
John Peever

CSB346 Lecture 6 – Central Chemoreception (February 12, 2013) Slide 1 - We want to move away from how the rhythm is generated. - We want to think about how the rhythm is affected by stimuli that we know are critical. - We are going to talk about central chemoreceptors. Slide 2 - We talked about how the pre-BötC is responsible for generating the rhythm. - The rhythm is sent to the respiratory MNs (e.g., hypoglossal and phrenic). Those muscles generate the force that enables muscle contraction so that ventilation can occur. - Respiratory frequency multiplied by volume gives you ventilation. - How does CO2 get sensed? Slide 3 - CO2 is the most powerful stimulus for breathing. Oxygen is critical. You breathe to acquire oxygen, but you breathe because CO2 makes you breathe. - Breathing depends not only on the pre-BötC to generate it, but it also has to have input from other things that detect changes in O2 and CO2 level. It is important that you are constantly monitoring how much O2 is in the arterial blood supply going to the brain and how much CO2 is in the blood supply. If there is too much, you need to breathe more quickly to get rid of it. - A chemoreceptor is a receptor that senses the chemicals (e.g., O2, CO2, pH, H+). - O2 chemoreceptors are mainly external to the brain. CO2 is detected both by the brain and by the carotid bodies. - Central chemoreceptors are sensing CO2, pH, or H+ that are located in the brain. - CO2/pH levels are related to acid-base balance in the blood and brain. Their levels reflect adequacy of lung ventilation to tissue metabolism. o If you are running and you slow down your breathing, your working tissues are metabolising more quickly because you are exercising, producing more CO2, and if you are not exhaling the CO2, it starts to build up in the body. CO2 levels go up. pH goes down. This is because you are hypoventilating. Lung ventilation is designed to meet metabolic demands. If you are producing more CO2, it is a powerful drive to breathe more rapidly, not because you want to acquire more O2, but because you want to get rid of the CO2 that you are producing because it can change your pH balance. pH is highly regulated. The respiratory system is the rapid way by which pH is regulated. Slide 4 - These were experiments that were done in lambs. - VT stands for tidal volume. ML stands for the amount of air in each respiratory cycle. The lamb is breathing in and out. - How much of this ventilation is due to CO2 itself? o For a long time, people thought you were breathing in and out because you were trying to get O2. But CO2 is the most important signal that was driving you to breathe, not O2. - They took the blood out of the lamb and went through an extracorporeal loop. He took out the same amount of CO2 that the lamb was producing just by normal bodily metabolism. o This is when the lamb is letting O2 go into its lung so that CO2 levels are fluctuating across the respiratory cycle. o He started to switch so that the lamb’s blood was prevented from going to the lungs. o He added the O2 and took out the CO2 of the blood. It is called an extracorporeal lung. - As soon as the lamb got switched, so that its lungs are no longer in control of how much O2 is being added and CO2 is being removed, you can see that breathing becomes slower and more shallow (e.g., the volume of each breath is going down). - Then he takes control entirely of how much CO2 and O2 the lamb has in its arterial blood supply. o He kept both of them constant. He provided the amount of O2 required to keep metabolism constant. He took out CO2 so it never fluctuated. o The lamb completely stops breathing. - This demonstrates that when CO2 falls below a particular threshold, you don’t breathe. If you are provided the right amount of O2 and you have no fluctuation in CO2 (e.g., constant; he is taking CO2 out in the same rate that the lamb is producing CO2), the lamb stops breathing. o He did a whole bunch of other tests to show that it was CO2, and not O2. - CO2 is the stimulus that is pushing you to breathe rhythmically. Without that stimulus, your brain seems to say “I don’t need to breathe anymore because I have enough O2 and I don’t have CO2 building up in my venous blood supply, so why would I spend energy breathing.” - This study shows you that CO2 is an important signal for the brain to listen to. When the signal isn’t there, you didn’t seem to generate the effort to breathe. There was no need. o Metabolism drives ventilation. o When metabolism is not fluctuating (e.g., the metabolism is not reaching the), then what is the point of breathing? When metabolism goes up, you breathe more quickly. When it drops below a certain threshold as it does in these experiments, you stop breathing. o CO2 production is a powerful stimulus to cause you to breathe. Slide 5 - This is the same experiment done in humans. - They told the patient to hyperventilate. You are blowing CO2 off more rapidly than you are producing it. After they stop hyperventilating, they stopped breathing for a second. o Like in the lamb, when they got rid of the CO2, the brain doesn’t feel the need to breathe because there no CO2 build up that is signalling the brain to cause you to breathe in and out. - This experiment prompted a whole series of experiments. o Why would this happen? o Why would patients stop breathing when they hyperventilate? - They showed that the exact same hyperventilation caused almost 1 minute without breathing. o They got the patient to fall into stage I sleep. Then the machine hyperventilated the person. When they hyperventilated the patients, they stopped breathing for up to 60 seconds. - They repeated the same type of intervention at a different sleep stage, called deeper sleep. - When CO2 levels fall (e.g., 40 mmHg  33 mmHg) by hyperventilating, you stop breathing. o CO2 levels are extreme tracked by the brain. When they fall below a particular value, you seem to stop breathing. o The brain listens extremely carefully to CO2. o A drop in CO2 is signalling the brain to not listen or to not generate respiratory rhythm. - O2 level do not change during hyperventilation. o Remember in the O2 dissociation curve, Hb is 98.5% saturated. Breathing faster at sea level doesn’t do much to get more O2 into the system. Slide 6 - Increases in CO2 are extremely powerful. o The two previous experiments looked at dropping CO2 levels in the venous blood supply, in which you seemed to stop breathing. o What would happen to breathing if CO2 goes up? - Look at the slope of the line. o The X axis is how much arterial CO2 you have in mmHg. o The graph is how much ventilation is going on relative to how much CO2 is in the blood. o It goes up a little bit, breathing goes up. It goes up a little bit more, breathing goes way up. It goes a little bit more, it goes shooting to the roof. o The steeper the curve, the more powerful the stimulus is having on breathing.  A tiny change in CO2 causes a massive change in breathing.  If CO2 goes down, you stop breathing.  If CO2 goes up a little bit, you breathe incredibly heavily. Ventilation increases massively. - The rat is breathing in and out with room air. o When I give the rat 7% CO2, the breathing goes up in terms of frequency (e.g., the number of breaths per unit of time) and height (e.g., amplitude or tidal volume). o Ventilation is tracking how much CO2 the rat is producing. Slide 7 - Where are the central chemoreceptors in the brain? - The overall levels of blood flow are an index of how active certain parts of the brain are. - He used NMR. o He looked at overall levels of brain activity. o He would take a patient, give them a whiff of CO2 in hypercapnic air, and he would see some part of the brain light up. This would tell him that it is the part of the brain that is sensing CO2 changes. - The whole brain went wild. o Not surprising because one of the most immediate responses to a high amount of CO2 is panic. - Look at how bright the brainstem gets (e.g., yellow). The yellower it is, the more excited the cells appear to be in that area. The ventral portion of the brainstem seems to be quite activated. Although there was a lot of activity in the brain, there was still an enormous amount of activation in the ventral portion of the brainstem. o This is an area that we know that seems to be involved in sensing CO2, at least in cats. o This was one of the first pieces of evidence to suggest that the brainstem is also noticing changes in CO2. - How do you know that the central chemoreceptors are not in the periphery? o There is a group of people who get cancer in the carotid bodies. The carotid bodies are important in detecting CO2 and O2. Some people get cancer and have to get them surgically removed. Slide 8 - He got a collection of patients who had their carotid bodies removed. - If there are important receptors in the brain that detects CO2, either patients without carotid bodies won’t notice CO2 or something will change. - The top two traces is the response to changes in CO2 in a patient who has intact carotid bodies. VT is measuring the volume of air moving in and out of the lungs in a human. o You step CO2 way up from 40 mmHg. o In the red box, there is an increase in ventilation. It is quick, rapid, and it starts to increase. The amplitude of the inspiration goes up almost instantaneously. CO2 goes up and the brain listened right way. - In a group of people who were CB denervated, he did the same experiment. o Look at the difference in the red box. The time in the red box is wider. o When you increase CO2, you still get an increase in breathing, but it took five times longer to get there. - This idea led to the notion that peripheral chemoreceptors are listening to CO2 because when they are intact, you get a rapid response. But when they are gone, you still get an increase in breathing, so something must be detecting the increase in CO2, but it takes longer for it to happen. It takes time for blood to go from the lungs to the brain. The carotid bodies are sitting at the bifurcation of the common carotid. They are located close to the source of changes in lung CO2 levels. o The idea is that the CB detects CO2 quickly. The central chemoreceptors also detect CO2, but it takes longer for CO2 to get to them. - This is the idea that there are peripheral and central chemoreceptors. There is something detecting CO2 in CB denervated patients because they don’t have CB chemoreceptors. There is some other structure, presumably, in the brain, must be responsible for this phenomenon. Slide 9 - This shows that there are two sets of chemoreceptors. This is overall level of ventilation under resting conditions in a human. CO2 is allowed to build up in a bag that the patient is breathing in. All of a sudden, at a certain level, CO2 starts to increase. It keeps going up. Then it goes up again. - The idea in these experiments is that there are two thresholds. It goes up, and then it goes up more rapidly. There are two slopes to this line. o This slope is mediated by the peripheral chemoreceptors. o This slope is mediated by the central chemoreceptors. - This is another piece of evidence that there are two different types of responses in breathing to the same stimulus. One is that the stimulus gets more and more severe, but there are two lines that look like there are two responses. - This is not proof that there two sets of chemoreceptors. This is the hypothesis. It is hypothesized that peripheral chemoreceptors respond first, and central chemoreceptors respond second. Slide 10 - If there are central chemoreceptors, where are they? - This is evidence by the accumulation of three different scientists. They defined CO2 sensitive zones (e.g., chemosensors). They took anesthetized cats and they opened up the cat so that they could remove some of the tissue over the ventral part of the brainstem. o They thought the ventral surface of the brainstem was detecting changes in CO2. o They put some acidic drops onto the surface of the ventral medulla around the VRG. - They found that in three separate areas, when you acidified them, the cats would breathe much more deeply. - These three people called these areas the three chemosensitive zones. - This is the ventral surface of the brainstem. These are the three areas that they acidified. They are mimicking high levels of CO2. They noticed the cats would breathe more quickly. - They acidified long the ventral surface of the brainstem. The cat breathed more quickly. These must be important for central chemoreception. Slide 11 - Each of them independently discovered that there are spots on the ventral surface of the brain just ventral to the VRG. When you acidified them in a cat, the cat would breathe more quickly. We call it region M, region S, and region L. - Those are the three people who initially discovered some potential region of the central chemoreceptors. This was done in cats. - Central chemoreception is how the CNS (i.e., the brain) detects CO2 blood levels. - What parts of the brain were able to detect CO2 or pH? Three people identified three areas (M, S, and L) on the ventral surface of the brainstem. They exposed the ventral surface of the medulla. They acidified those areas. They noticed the cat would breathe more deeply or more rapidly. They decided that the ventral surface of the medulla must be critical in mediating the acid changes that are accompanied by putting acid on the surface on the brain. - This is historically the first place where we got to understand where CO2 was being sensed in the brain. Slide 12 - All the areas in orange are areas that when you acidify them, breathing changes. o Acidification is the equivalent of high levels of CO2. CO2 is combined rapidly with water, which is converted into bicarbonate and H+ ions. The H+ ions predict pH. High levels of CO2 drive up H+ ion levels. H+ ion changes pH. - The RTN, NTS, FN, and LC are all regions that when you acidify them, breathing changes rapidly. - Multiple areas are involved in CO2 detection, not just the ventral chemosensitive zones. - This is the sagittal view. The orange areas denote brain regions where breathing changes in response to changing pH or CO2 levels. This is the area that is equivalent to where the three people acidified before. The area in orange shows that breathing changes when you acidify it. RTN would be like the M. - This is half of a transverse section. These are some of the areas that are CO2 sensitive and that we think are part of network of brain centers that are involved in CO2 detection. Slide 13 - There are these areas that are CO2 sensitive. Breathing changes when you drop acid onto those areas. How are those parts of the brain detecting that? How are they doing it? Where are those neurons sitting? How do the neurons respond to CO2 changes? Slide 14 - What is being sensed? Is it CO2 or pH? - CO2 immediately combines itself with water and converts into hydrogen ion and bicarbonate. It immediately finds itself attracted to water molecules and immediately converted into hydrogen ion and bicarbonate. If CO2 is being converted immediately into hydrogen, which is an index of pH, it seems hard to imagine that the brain cells are detecting CO2 because CO2 doesn’t get to sit around on its own very much. How could CO2 be sensed by brain cells if it is rapidly converted into pH? - It is assumed that pH is the variable that brain cells are detecting, and not CO2. If you are going to study how the brain is detecting a change in CO2, you have to ask yourself, can it actually detect CO2? It is going to be hard because it is never sitting around to be detected. Even though you have CO2 being the variable that you are thinking about, if it is not able to turn into a useful signal for the brain, then it is not that useful. It looks like hydrogen ion, which is an index of pH, is the detected variable. Slide 15 - Let us assume that pH is the detected variable. How are the cells doing this? o Is it the actual membrane of the cell that is detecting the pH? Do some cells have a specialized function where their function is to sense pH in the brain? o Does CO2 and pH change affect breathing by altering the way two cells talk to each other (e.g., synaptic transmission between cells)? Breathing is generated by a network of neurons (e.g., pre-BotC or RTN). Is it possible that when pH changes, the conversation between neuron in those rhythm generating areas is sped up (e.g., breathing frequency increases or breathing depth deepens)? Synaptic transmission could be another mechanism by which the brain is detecting pH. pH changes the way two cells talk to each other. o The pH across the cell membrane is being regulated so that the excitability of a candidate group of cells is affected. - Studies in vitro performed in the LC, NTS, and the caudal raphe show that neuronal depolarization (or hyperpolarization) is produced by hypercapnia. This means that cells in those brain areas respond to changes in pH (like on slide 13). The effect has been attributed to intrinsic membrane properties, perhaps involving a potassium channel. Perhaps there is an intrinsic cell membrane property that is affected by pH, perhaps either in the caudal raphe, NTS, or LC. - The other example are the type I cells of the carotid body. They are rapidly depolarized by acidosis (e.g., change in H+ ion concentration or pH). A potassium channel could be thought to underlie that response. - How are these areas of the brain, which respond to changes in pH and hence trigger changes of ventilation, sensing the signal? Slide 16 - Is it the inside or the outside of the cell that is detecting pH? o You can have a situation whereby the outside of the cell notices that there is a change in H+ ion concentration, the cell is going to be excited or inhibited, and the cell is going to change breathing. o You can have a change across a cell membrane that is signalling the cell to recognize it. o A pH sensor on a cell has an external part of the receptor on the cell membrane. Then there are the inner workings on the inside of the membrane. This is the idea that there could be intracellular and extracellular changes in pH that are being detected. This is getting down to the cellular mechanism by which pH might actually be detected. There is evidence to support both of these ideas. - K+ channels are involved in regulating pH. There could be a K+ channel that is directly or indirectly responding to changes in the pH. - There is no evidence that CO2 itself can be sensed, but that does not mean that it is not impossible. There is good evidence that pH sensing is going on, but there is no evidence that CO2 isn’t being sensed. This doesn’t mean that it is not a feasible mechanism. Slide 17 - These are some of the possible mechanisms by which a channel or protein can sense pH. o TASK channels are involved in pH sensing. They are very sensitive to changes in pH. o K+ channels are sitting on various respiratory cells. When pH changes, they change the way the way K+ channels function. This, in turn, affects the excitability of a cell. - The idea is that something on a cell has to affect the way that cell is behaving, so that it can then tell its neighbours or where it projects to, that something about CO2 has changed. o For example, a pre-BotC cell is one of the things that are helping generate respiratory rhythm. If it possesses K+ channels, and the K+ channels are responsive to changes in pH, then it is an effective mechanism whereby that cell can then change breathing. o Another option is that pH can affect gap junctions. You don’t have to affect the way that a protein on a cell affects the membrane of a cell, but you can allow cells to talk to each other via gap junctions more easily. It is a different way of relaying communication. Gap junctions have been shown to be sensitive to pH. If you affect a gap junction in relation to pH, then it is just a way that you can send a signal throughout a network of cells that generate breathing. o I am going to focus on K+ channels because the most evidence comes from these channels and they are the most ubiquitous throughout the nervous system and the peripheral nervous system that pH profoundly influences the effect of K+ channels.  K+ channels are very powerful channels because they are really leaky, which means that they have a lot of control on how excitable a cell is. If you have a cell that is full of K+ channels, and they are all open, the bad news for the cell is that it is going to be reasonably powerless because when they are all open, K+ is running outside from the inside to the outside and taking the positive charge with it, leaving the cell unable to generate an action potential because it is hyperpolarized. o There are various pH sensitive membrane ion transport proteins. One of them is a Na+/H+ exchange protein subtype 3. It is another protein sitting on the surface of the cell that changes how the excitability of the cell is regulated and it responds to pH. - These are four candidate mechanisms by which pH can affect the excitability of a cell, and hence possibly the overall level of ventilation. o We have to look at not only the brain regions and the cells that are in them, but what are the ways that pH could affect the cells themselves. Slide 18 - This is a recording of an individual cell. This is looking at how the current that moves across the cell membrane changes in response to pH. Current moving in and out of a cell is an index of the ions that are flowing across its membrane, regulating its excitability. - Specific molecules in brainstem neurons are sensitive to CO2 and pH, and they regulate cellular excitability. Kir channels are sensitive to pH and they regulate membrane potential and hence cell excitability. - There is evidence that Kir channels detect CO2 levels. They can detect CO2 levels either by changing their response to hypercapnia or hypocapnia. They are not just responsive to high levels of CO2, but they are also responsive to low levels of CO2. A nothing response is also a type of response. This tells you that changes in CO2 (either high or low) can affect the functioning of these K+ channels. - In situ hybridization studies indiciate that Kir channels are co-expressed in brainstem cardio- respiratory nuclei. It is likely that they contribute to pH sensitivity. - A o This is a Kir channel is affected by changing intracellular pH. Remember that K+ channels open and K+ leaves the cell. At a pH of 7.4, this is the current that is moving through these Kir channels. o When you acidify the cell (7.4  7.0), there is less K+ moving through because you can see that they are all squished together. This is showing you that there is less current flowing through the cell. If less K+ going out of the cell, then more K+ is staying in. If K+ is staying in the cell, it is keeping its positive charge inside the cell, which means the cell is becoming more excitable. o When you go to a lower pH (e.g., hypercpania), you have less current, which means the cell is becoming more excitable. When you go down to pH 6.6, you can see that almost no K+ is leaving, which indicates that the Kir channels are closing or the Kir channels are restricting the flow of K+ from the inside to the outside. So they are potentially making the cell more excitable as pH decreases. o They can reverse it going back to 7.4, which looks a lot like the first 7.4. This is a reversible phenomenon. The cell isn’t dead. This is showing you that you can take the cell back. The changes in the conductance through the Kir channels haven’t damaged the cell. - You are looking at the level of current in a cell across a tiny period of time. As the lines move up with the reduction in pH, it means less current is flowing. The K+ is sitting inside the cell because it is not going through the cell. It makes the cell more excitable. You can make the current go down to almost no current and no K+ flowing out of the cell (e.g., you would be dead by now because this is a vicious pH). When you return the pH to 7.4, the cell gets restored to the way it was before. This did not kill the cell. This is showing that rectifying the Kir channel is sensitive to a change in pH. It doesn’t tell you anything about breathing. Slide 19 - This tells you something about breathing. This is the firing rate of different cells in the midline raphe. Here are slices of the brain. The tubes are showing you parts of the respiratory nuclei. The blue line, where he recorded cells from, is the raphe nucleus. The idea is that raphe cells communicate with the pre-BotC. The raphe cells are responsive to CO2. If this is true, then if I impale a raphe cell with an electrode so that I can record when it fires an action potential (e.g., when it is communicating information), then it should change its firing potential when pH changes. - A o This is a raphe cell that nothing happened to. The cell doesn’t change its firing rate when the pH changes. - B o When the %CO2 goes from 5% to 9%, the firing rate goes up. The cell in the raphe nucleus fired faster when exposed to a higher level of CO2. The cell is listening to CO2 levels. When you increase CO2 levels, the cell fires faster. When you decrease CO2 levels, it fires slower. The raphé cell must be tracking CO2 changes. - C o Not every cell does the same thing. This cell does the opposite. - This is meant to give you a real picture of what is going on. Some cells don’t give a crap about CO2. Some cells fire faster when CO2 goes up and fire slower when CO2 does down. Some cells fire faster when CO2 is lower. The point is not the direction of the change. The point is that they are noticing that pH is changing. It says that the raphé nucleus has cells that are pH/CO2 sensitive. The raphé nucleus has cells in it that respond to CO2. Slide 20 - How is synaptic transmission affected? If pH may affect synaptic transmission, you are going to have to have a cell changing the way that it’s talking when it’s responding to CO2. For example, if the raphé cell changes its firing pattern in relation to CO2, then its sy
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