CSB346 Lecture 7 Notes

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University of Toronto St. George
Cell and Systems Biology
John Peever

CSB346 Lecture 7 Notes – Peripheral and Central Oxygen Sensing (March 5, 2013) Slide 1 – Peripheral and central oxygen sensing - Central chemoreceptors are the brain cells and the brain regions that are somehow able to taste CO2 levels and then turn that into a meaningful respiratory signal. However, O2 sensing and how that affects breathing is complicated. It is complicated because the signal, unlike that for CO2 detection, can vary immensely across developmental age, depending on how one has been experiencing levels of hypoxia (e.g., low levels of O2). It is a complicated system. It changes in young animals, with age, during pathologies, etc. It is also complicated because a large component of O2 sensing is in the peripheral nervous system (e.g., carotid bodies). Slide 2 – Chemical regulation of breathing - We are going to focus on this branch of chemical regulation of breathing. In terms of peripheral chemoreceptors, it’s not possible to talk about how they work unless we consider both CO2 and O2, which is another complicated factor in explaining the system. There are two types of central chemoreceptors. - There is substantial evidence that the brain is able to measure and respond to O2 levels. However, human brains don’t seem to notice hypoxia as much as other mammalian brains. - In terms of PO2, there are two separate mechanisms that are both quite powerful. One is responded to by the peripheral chemoreceptors. This is the one that most of you will read about in textbooks. There is evidence that parts of the brain can also listen to and respond to low levels and high levels of O2. Therefore, we have to think of there being two factors. Typically with CO2, we focus on a classic central chemoreceptor that we discussed in the last lecture. - Today, we will look at how are low levels of O2 or changing levels in O2 sensed by the central nervous system, as well as the peripheral nervous system, and how do those changing values in O2 impact breathing? - When CO2 goes up, ventilation goes up. When O2 goes down, ventilation typically responds by increasing, but this response is plastic and can change based on whether you are a child, infant, older person, or someone who spent their whole life living at high altitude. Slide 3 – Regulation of breathing by hypoxia - O2 returns from the lung circulation and is going to the brain, in which it has to go past the carotid arteries. The carotid bodies are sitting at the bifurcation of the internal and external carotid arteries. The carotid body is one of the most vascularised structures in the body. For its size and volume of tissue, it has more blood vessels than most parts of the brain. This is the first clue that the carotid body must be doing something important and looking at what is in the blood if it is so vascularised. Since so much information is passing through it, it must be listening very carefully to some message in the blood. The information from the CBs is then sent by afferents into the NTS (e.g., part of the brainstem that is dorsal to the VRG). This information going from the CBs to the CNS, in particular the NTS, is the first step in detecting how the brain recognizes that levels of O2 have changed. CBs are tasting or sampling O2 levels in the blood, and relaying the information back to the CNS to the NTS, which then communicate with various parts of the VRG (e.g., where breathing is initiated and modulated). The goal is to make sure that enough O2 is reaching the brain and the working tissues. - How do changes in O2 levels affect breathing? This is not straightforward. It is not a linear response. As O2 levels decrease, ventilation doesn’t linearly increase in response to that. The response can be modified by how much CO2 is in the blood, by developmental stages of the CNS or the PNS, or by how long you have been exposed to a various level of O2 (e.g., if you live at high altitude, your brain adapts to this low level of O2, which can affect how O2 affects breathing). - Where is O2 sensed? What are the mechanisms that do it? The CBs are a key player. - How do cells or nervous system cells sense O2? If O2 levels change, what is it that the cells recognize? We are going to only talk about cells in the CBs. Unlike the central chemoreceptors where there are cells in various parts of the brain, we are going to talk about cells that are confined to the CBs. Slide 4 – Regulation of breathing by hypoxia - When O2 levels decrease, this is called a hypoxic stimulus. When ventilation changes in response to hypoxia, this is called the hypoxic ventilatory response (HVR). This is similar to the hypercapnic ventilatory response, but we don’t talk about this as much as the HVR. - The HVR is complicated. o Neonates and adults respond to hypoxia differently.  Given the same low level of O2, they respond completely different. o The HVR can be a time-dependent phenomenon.  Unlike CO2 levels where CO2 levels go up, breathing stays up the whole time. The brain gets lazy with O2 levels. When O2 levels go down, breathing will initially go up but then it will often fall off and go down. o The HVR is explicitly sensitive to different patterns of O2.  Most people tend to think that if you have five minutes of hypoxia, then breathing is affected in exactly the same way over the five minutes.  In real life, ducks that sit in ponds have to constantly go under the water to find their food. They become repeatedly hypoxic when their heads are in the water (e.g., they are not breathing). Animals have these intermittent periods of hypoxia, which is quite natural for them. Unfortunately in people, we experience intermittent hypoxia, but it is a pathological scenario for us. Again, because it is a repeated stimulus and not a constant one, our brains don’t deal with it in a simple straightforward way. We adapt to repeated periods of hypoxia. The HVR is different for sustained periods of hypoxia (e.g., constant long periods) than it is for intermittent periods of hypoxia (e.g., short periods). - Some of these different patterns could be mediated the fact that we have both central (e.g., sensors in the brain) and peripheral (e.g., CBs). We have a range of responses to hypoxia. Slide 5 – The HVR in newborn mammals - This is the first example of the confusing nature of the HVR. o Here is one example of one type of HVR. - This is tidal volume, frequency, and overall levels of ventilation in a 1 day-old rat and an 8 day- old rat. At the arrow, the rat goes from breathing room air (e.g., 21% O2) and the rat becomes hypoxic because we take half of the O2 out of the air (e.g., 12% O2). This is a serious stimulus, but not life threatening. o Look at what happens in a 1 day-old rat.  The overall levels of ventilation in room air vary over time. In room air, over time, ventilation varies between 10-18 ml/min. Breathing seems unstable.  At the arrow, when we drop the inspired O2 level from 21% to 12%, ventilation goes up. This is called phase 1 of the HVR (e.g., when O2 levels fall, breathing goes up). The increase in ventilation is somewhat attributable to both an increase in how many times per minute the rat was breathing in and more loosely, the volume of each breathe. Remember that ventilation is the product of frequency times volume.  The rat is still experiencing 12% hypoxia, but ventilation starts falling. This is called the second phase of the HVR. This is a normal response. o Look at what happens in an 8 day-old rat.  Look at how consolidated things are. The dots are much more tightly organized. Overall, the response itself is much more collected or consistent.  The rat is breathing room air, and ventilation is stable. For the first few minutes before we drop O2 levels to make the rat hypoxic, ventilation is really stable.  When the rat becomes hypoxic, ventilation goes up steeply. Then ventilation falls off at a consistent fashion.  The same thing is obvious in tidal volume. The tidal volume goes up when hypoxia is experienced, and then it falls off.  Frequency is a little bit more unpredictable. - This is what it looks like for a sustained period of hypoxia. This is about 2.5 hours of hypoxia. This is a fairly consistent response. o This doesn’t look like anything that would happen in humans during hypoxia. For babies, they experience this kind of HVR. For adults, the magnitude of ventilation increases and stays elevated. - We think it is a protective mechanism. Why continue ventilating heavily when O2 levels aren’t going up? You can’t get more O2 by breathing faster. This is called a biphasic response. It is primarily experienced in infant animals. The more immature you are, the more you have this up and down response. The more mature an animal is, the less dramatic this is. o This is only one form of HVR. o Another common HVR is that they stop breathing all together. If there is no O2, then why am I wasting my time breathing? They are using energy to move the respiratory muscles. They would stop breathing to conserve energy. There is a more complicated strategy involved in this. - This is in neonatal mammals. Metabolism is massively suppressed. If you were measuring metabolic rate, metabolic rate falls off at the same rate that respiration does. Ventilation falls off because the brain shuts off body metabolism. The animals enter into a state of suspended animation. o The mature CNS cannot do this. The brain tries to keep working and it doesn’t reduce its metabolic rate. It uses all the O2 that is stored in your body and it starves to death. Slide 6 – Sensitization of the HVR - The HVR is also mutable, or it can change based on previous experiences or the types of experiences. - This is showing you how it doesn’t remain constant over time. It can either become more sensitive or less sensitive to a stimulus depending on how it sees hypoxia. - This is 8 consecutive days where you alter between hypoxia at 10.5% O2 and room air. It is going to outline how across an 8 day period, an animal doesn’t respond to the same stimulus with a stable response (e.g., it changes). - This is breathing rate or frequency, volume, and overall levels of ventilation. Rats are active during the night, and are quiet during the day. Breathing goes up during the dark phase when the rats are active, and goes down during the light phase when they are sleeping and quiet. When you are active, you breathe more. When you are quiet, you breathe less. o The same thing happens for ventilation and overall levels of volume. o This is showing you that breathing is tracking behavioural state (e.g., awake or asleep). - Look at what happens when you release the animals into a constant hypoxic condition. o Notice that the breathing rate immediately goes up. They become hypoxic and breathing has gone up above normal levels. Across the 8 day period, it continues to elevate. The stimulus didn’t get any worse, but the overall breathing rate continued to increase. If you have a constant stimulus, why does the response change when the stimulus isn’t changing? The level of hypoxia the rats experience isn’t getting worse across time, but breathing frequency was elevated across time.  This is one type of example of how the respiratory response to hypoxia becomes sensitized (e.g., more sensitive). This is called sensitization.  The respiratory system can take the same stimulus and listen to it differently. This is an example of how the HVR is not a constant phenomenon. It can continuously change and adapt. Slide 7 – Desensitization of the HVR - This is an example of desensitization. For people born at high altitudes, their brain has adapted some mechanism where it doesn’t seem to care so much about O2 levels. - These three graphs show how changes from normal levels of arterial O2 affect ventilation. o The control group are people who are born at sea level (e.g., exposed to 21% O2).  As arterial O2 becomes hypoxic, ventilation goes up in a predictable way. This is in adults. The HVR is very sensitive in people who don’t normally experience hypoxia. o There are people living at high altitudes. They live there but they weren’t born there.  This is the response to the same stimulus. They seem they start to ignore it. This makes sense. What is the point of noticing a stimulus if you are constantly living with it? o There are people who are native to high altitudes. They were born there, live there, and still live there.  They have no HVR. Their breathing doesn’t change in response to hypoxia. This is an example of desensitization to the HVR. The HVR is absent in people who are living at chronic levels of hypoxia. Their breathing system has desensitized to this stimulus. - Hypoxia doesn’t just do one thing. Slide 8 – Peripheral versus central hypoxia - Neurons are highly sensitive to levels of O2. The brain listens to O2 levels. The CBs are not the only thing that is sensing O2. O2 levels have a profound effect on the way the brain works. Cells that don’t have anything to do with O2 sensing are smart. They have to modify their behaviour in response to low levels of O2 because they want to conserve their levels of ATP. Channel arrest is one of the ways that cells can reduce their activity in response to low levels of O2. - What are parts of the brain that are sensitive to changes in O2 that actually might change breathing? Lots of cells and neurons in the brain notice that O2 levels have changed, but they are not involved in breathing. Their job is to recognize that that O2 levels have fallen, but their job is not to then send that information to the respiratory center. For example, if you are subjected to high altitude, you feel lightheaded because the low levels of O2 affects how the brain cells are trying to conserve energy. - Are there any particular brain areas that detect low levels of O2 that might have a role in affecting breathing? Some of the examples are the thalamus, hypothalamus, pons, and medulla. These are some of the areas that respond to low levels of O2 and may affect respiration. I am going to focus on the medulla because the medulla is where the VRG is sitting, so it must be extra important in respiratory control. Slide 9 – Hypoxia-induced C-fos expression - C-fos is a tool that can be used to determine if cells are responding to a particular stimulus. A cell is said to make c-fos if it is activated by some stimulus. In this context, does hypoxia turn a particular cell on? You will find various cells in the brain that have turned on or expressed c-fos, so those cells in those brain areas has responded to hypoxia. - These are some of the areas in the medulla. There are areas that show lots of c-fox expression when a rat is exposed to hypoxia. These are likely brain areas that are involved in controlling respiration when an animal is experiencing hypoxia. - These are the number of cells in the structure during room air compared to hypoxia. o The NTS is a critical brain structure that listens to afferent input from the CBs. Cells in the NTS are activated during hypoxia because the CB is listening to the hypoxic stimulus, sending the information to the NTS, and the NTS cells are activated. This makes good empirical sense. o The VLM (e.g., ventrolateral medulla) is the area that houses the VRG. These cells tend to be more sensitive to hypoxia. o The raphé has a doubling of the number of cells during hypoxia, indicating that the raphé area of the brain is listening to hypoxia. o The LC is sitting in the pons. They also seem to increase the number of cells that are c- fox positive. - These are all potential candidates for being parts of the brain that we know (1) if you stimulate those areas, it affects breathing and (2) if you stimulate those areas with hypoxia, the number of cells in that area that appear to become active increases. These candidate areas are areas that might be involved in turning on during hypoxia and hence, affecting the HVR. - This is a loose piece of evidence, but it is supportive of the idea that these brain areas could be responsible for mediating the HVR. Slide 10 – Central hypoxia in vitro - Some of the responses to hypoxia are also switching off the brain. - This is a piece of the medulla in the brainstem that has the respiratory network that has been plucked out of a baby rat and put into a dish of aCSF. We record the phrenic nerve that goes to the diaphragm. We can record the hypoglossal nerve that goes to the tongue. Both of those have a respiratory rhythm. You can record the phrenic activity in the piece of tissue. o When it becomes hypoxic, breathing shuts off. - A o The lines are representing inspiration. The tissues don’t have the peripheral chemoreceptors (e.g., the CBs). You make the tissue hypoxic, it stops firing. Breathing is being reduced. - B o In a different preparation or animal, when you make the tissue hypoxic, breathing shuts off. - C o It is breathing rapidly in and out, but when you make it hypoxic, breathing becomes slow. - This is an example of how just the CNS can also not deal with this anymore when there is too much hypoxia around. There are cells in the medulla that are responsible for detecting hypoxia. o One idea is that some of these cells could be listening to that stimulus. The idea is that in an immature brain, hypoxia can inhibit brain function and not excite it. - There are no peripheral chemoreceptors here. This is all about the brain. The brain is doing all of the O2 sensing. You made the brain hypoxic, and it responded by reducing breathing in a predictable and coordinated way. If you remove the hypoxia, then breathing returns to normal. o The idea that just the CNS itself listened to changes in O2 and affected breathing accordingly. The structures that are able to detect O2 must be sitting in the CNS because there is no PNS intact. - If you expose a piece of tissue from a neonatal animal to hypoxia, breathing goes down in response to hypoxia. o This shows you that the CNS can respond to hypoxia. Breathing is going down in a predictable and organized manner. The low level of O2 hasn’t prevented the brain from doing its job. Breathing goes down in an organized fashion. The respiratory network hasn’t been messed up by this stimulus. It is unable to continue responding. This means that if ventilation is going down, then you can think of it as being inhibited by O2. The inhibitory network means that there is a group of cells that are inhibited by O2. - Where could this be going on? Slide 11 – The pre-BotC responds to hypoxia - She just made the pre-BotC of an adult cat hypoxic by injecting NaCN. It kills you because it prevents cells from using O2. When you give a cell NaCN, it really becomes hypoxic because the cell can’t use O2 anymore and by definition the cell then becomes hypoxic. - They microinjected NaCN into the pre-BotC because it is important for generating breathing. o She prevents the pre-BotC from using O2 by making it chemically hypoxic. Breathing changes as soon as she injects NaCN. It becomes much more rapid and deeper. - The point is that manipulating the ability of those cells to use O2 massively affects breathing. o The pre-BotC is able to sense O2. o Others might argue that the pre-BotC couldn’t work well anymore, which is why the breathing was changing. She argued that if you kill the pre-BotC so that it couldn’t function anymore, then it wouldn’t have created a stable breathing and breathing would have shut off.  The breathing after NaCN injection is repeatedly stable. Then the cat returns to normal breathing.  She hasn’t killed the pre-BotC. She demonstrated that the pre-BotC is sensitive to changes in O2 levels. Slide 12 – Mechanisms for sensing hypoxia - What is the biophysics of ion channels? How do ion channels detect and sense changing levels of O2? How do the CBs respond to low levels of O2? Why the CBs? What is the evidence that the CBs are doing the majority of O2 sensing? How do cells in the CBs detect O2? Slide 13 – Central O2-sensing mechanisms - There are two hypotheses. o A cell can detect O2 through some property of its membrane. O2 levels drop, the membrane of the cell (e.g., protein or receptor on the cells) detects that the O2 levels have changed. This is called the membrane hypothesis. It proposes that some ion channel or some protein is directly responsible for sensing O2, and that those changes affect the excitability of that cell. The cell can then say that O2 levels have dropped. This is pertaining to cells in the CBs.  I am going to focus on the membrane hypothesis. o The other hypothesis is complicated and confusing. The data for it is contradictory. This gets into mitochondrial function in how the energy powerhouse of the cell could be detecting changes in O2, which in turn affect the O2 transport, which ultimately affects cell excitability.  You do not have to know about the metabolism hypothesis. Slide 14 – K+ channels and O2-sensing - How could a cell notice that O2 levels have changed? - Remember that K+ channels may be the smallest thing in the brain that notices how CO2 levels change. K+ channels are vital sensors on the outside of a cell. They can sense a lot of different things such as O2 and pH. They are extremely useful at regulating how a cell is excitable or quiet. If you open a K+ channel, K+ flows out of the cell, and the cell hyperpolarizes (e.g., less excitable). - O2 sensing could affect these K+ channels. o This is a neocortical cell. This is not a respiratory cell, which is not the point. The point is how could a cell be affected by O2 levels? Not only are respiratory cells impacted by O2, but cells that keep you alert and active are too. o This is an example of how a non-respiratory cell is affected by O2 levels. This is a model of how O2 could affect the cells that are doing the actual sensing of changes in O2 levels so that respiration is changed. o This is in vitro (e.g., sitting in a dish). This is when the cell is exposed to normal levels of O2 and to extremely low levels of O2.  Look at the line. This is when the K+ channel is opened and when it is closed. As the line goes up, K+ channels are closed. When O2 levels fall, the K+ channels are constantly closed. What does this mean for the cell? This means the cell is becoming more excitable.  This is just looking at when a K+ channel is open and when a K+ channel is closed. The K+ channel is sitting on a neocortical neuron.  When the cell is sitting at normal levels of O2, the channel is open a lot, so the cell is not excitable. When the cell is exposed to a highly hypoxic environment, the K+ channels close, and makes the cells extremely excitable. o This is an example of how O2 could be sensed by a brain cell. I am not saying that cortical neurons are the cells that are detecting O2 and changing breathing. I am using this as an example of how O2 could affect a cell.  Does this similar mechanism exist in the CB? Slide 15 – Ca2+ channels and O2-sensing - The other option is that Ca2+ could be a sensor for O2 levels. - This slide is showing you that you could affect the excitability of a cell by affecting a Ca2+ channel, which is sensitive to O2. o This is control levels during hypoxia.  During control levels, at various levels of hyperpolarization o the cell, you have a particular magnitude of response. From -40 mV to 0 mV, you can see the measured change in Ca2+ flux through a particular channel. o The change in Ca2+ flux becomes smooth when the cell is exposed to hypoxia.  There is a change in the flow of Ca2+ through this channel compared to high levels of O2 (e.g., control). - These voltage-gated Ca2+ channels are primary means of Ca2+ influx. It helps make the cell more excitable. Activation of these channels is regulated by membrane potential. As you change the membrane potential, the cells open in a predictable voltage-dependent way. This is mediated mostly by K+ channel activity. o The change in Ca2+ activity in response to changes in voltage is tied into a K+ channel. This suggests that Ca2+ channels may also be directly sensitive to changes in O2. o The activation of a specific type of Ca2+ (e.g., L-type) in pre-BotC cells have been shown to be very responsive to O2 levels. o This implies hypoxia can excite a pre-BotC cell.  C  These were reported from a pre-BotC cell from a slice. This shows that these channels are very sensitive to hypoxia. o The point is that we know have a respiratory neuron that generates breathing. The channel on the pre-BotC cell is sensitive to hypoxia. This means that you make the cell more excitable in response to more hypoxia. - Now we have two mechanisms: K+ channel and Ca2+ channel. They that both seem to be sensitive to O2. The complicated factor is that the K+ channel and the Ca2+ channel have an interplay, but this implies that there might be more than one way of doing the same thing. - This is the evidence that we have two types of channels that are both sensitive to O2. These are coming from the CNS proper. One is from a neocortical neuron. One is from a pre-BotC neuron. - There are two types of brain cells that are sensitive to changes in O2 because O2 affects the way a K+ channel works and the way a Ca2+ channel works. Because O2 affects those channels, it affects the excitability of the cells. o How does a cell notice that O2 levels are changing? It could be noticing a change in the Ca2+ channel or K+ channel. They are somehow abl
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