CSB346 Lecture 5 Notes

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

CSB346 Lecture 5 2 - We talked about the experiment where you manipulate the pre-BötC in an anesthetized rat. - Other people wanted Paul to do the same experiments, but in awake animals. o “Scientists kill rats by stopping them from breathing while awake.” This was the single handed problem with these experiments. The idea of this experiment was a problem. - The hypothesis is that if you disrupt the pre-BötC, it should stop normal breathing (or eupnea). 3 - This is about how they achieved these goals. This explains how they can shut off pre-BötC cells temporarily to show that they are responsible for generating respiration. - The allatostatin receptor is a bug receptor, hence “bugging”. Humans do not make these receptors. Humans do not have any transmitters that can talk to allatostatin receptors. o This means that if you can push a pre-BötC to express allatostatin receptors, you can then add the transmitter that makes that receptor turn on or off, and then you can control the activity of a pre-BötC cell. - They used an AAV2, which drives the expression of AlstR in pre-BötC cells. o It is a method for inserting these receptors into neurons. o Once the cell expresses the receptor, it turns green because the virus also drives the expression of a green protein, so that you know the cell is expressing the receptor because the dye is present. o Once the AlstR is stuck into the pre-BötC cells, it allows us to manipulate the cell. When you give allatostatin, it allows you to open up the receptor.  When it opens up, K+ flows out. K+ is happily charged. There is a lot of K+ inside the cell. When the receptor opens, K+ rushes out of the cell. The cell becomes less excitable or less positive. - When you stick the AlstR in a pre-BötC cell, you can then add allatostatin to it. When you do, it opens the receptor that has been virally transduced into the neuron. K+ flows out of the cell. The cell becomes really hard to activate. It makes the cell unable or less likely to fire an action potential. o This allows quick but temporary reversal of pre-BötC cells. This allows you to activate the AlstR and turn the cells in the pre-BötC off, in which you will stop breathing. 4 - Here is the pre-BötC. The cells are green because they express AlstR. They are yellow because they have NK1R. pre-BötC are supposed to be exclusively expressing NK1R. o This is a snapshot of the pre-BötC area. Green means they have the AlstR. Yellow means they express NK1R. Chances are that you have the AlstR in the right cells in the brain (e.g., the pre-BötC cells). - Instead of doing it in intact animals, they had to do it under anesthesia. I don’t think this is the best experiment because it needs to be done under behaving conditions. - The idea is that breathing should stop. 5 - The outcome is what you would have expected. - (A) This is phrenic activity. When you add AL into the animal, breathing gets shallower or the height of the ventilations start to go down. Then you get to this point where the rat stops breathing. At that point, they have to mechanically ventilate the animal. At some point, the AL goes away from the receptor and the cells can become active. The rat starts to breathe by itself again. - (B) This is the data showing you what I just explained, but for a whole bunch of rats. This is group data showing you that the frequency drops off when you add AL. - (C) The height of each breath goes along normally andthen it goes off when you add AL. - Adding AL opens up the receptor, K+ flows out of the pre-BötC cells, and it makes them unable to fire action potentials. This silences them from doing their normal job. The outcome of that silencing is that breathing stops. So they must be really important in generating breathing. 6 - People still did not believe him because it was done under anesthesia. o They used a softer, less invasive way of doing the same thing. They used an approach called saporin, which is a neurotoxin. Saporin is bound to an antibody that recognizes an antigen. The antigen is a receptor.  You have an antibody and a saporin. The saporin kills the cell once it gets taken into the cell. 7 - You have an antibody against the NK1R. The antibody is attached to saporin. If you inject the antibody and saporin into the pre-BötC, the saporin gets internalized into the cell that expresses NK1R receptors (i.e., presumed pre-BötC cell). Through a series of processes, saporin becomes toxic and kills the cell. - They dusted the pre-BötC with saporin. It is toxic. What happens when you kill the pre-BötC cells? In the last experiment, the virus transduces a receptor onto pre-BötC cells that shuts them up temporarily but they just come back again. This is actually killing the pre-BötC cells. 8 - They called the pre-BötC the NA region (e.g., scNA = pre-BötC) because they are kind of close to each other. - (A) This is the pre-BötC area in normal control animal where they didn’t make any toxic injections. (B) This is the pre-BötC area after they add SAP. This is in a different rat. You can’t do histology in the same rat. The cells are expressing substance P. Then you add SAP, and the cells appear to be gone. The idea is that SAP killed the cells. o You inject SAP into the area. You let the rat recover for a while because it doesn’t kill them immediately. It takes time to kill the cells. - This is a histological demonstration. This is a slice of tissue after injecting SAP. This is to show that the SAP killed the cells. 9 - Does it screw up breathing? o It wasn’t a straightforward result. The result is open to some interpretation. - This is a normal rat. This is the control. - This rat had the pre-BötC on the left and right side leisioned using saporin. You still have the in and out breathing, but it looks crazy. The breathing rhythm is screwed up. They are having a lot of trouble breathing, but they are able to do it. - Here is normal room air and breathing in a control rat. - Breathing is faster and higher when you expose the rat to 5% CO2 in the control. o CO2 is extremely stimulatory to breathing. o Not much happens to this rat without the pre-BötC. This rat does not breathe any faster. It either can’t detect the CO2 or it is unable to do anything about it. Maybe it is able to respond to CO2, but it can’t do anything to change its breathing to be able to do it. - They exposed the rat to pure O2. It usually slows downbreathing. o In this rat, it almost stops breathing. - Lesions of the pre-BötC screw up normal breathing and perturbs the ability to respond to respiratory stimuli such as CO2 (e.g., hypercapnia) and O2. o Normal breathing is massively screwed up. - This is about a minute of breathing. This is what it looks like up close. In the leisioned rat, the breathing is messy. - The arrow is the rat taking a sigh, which is normal. 11 - This is where the experiment is tricky. - Saporin doesn’t act right away, unlike the allatostatin experiments where you shut the cells off quickly and turn them on very quickly again. If you poison someone slowly, that is what was happening to the pre-BötC. They died off slowly. o This means that you can’t simply take a snapshot and say that the cells are dead and this is what happened to breathing. They had to look at it across several weeks. o They are looking at the death of the pre-BötC and how it screws up breathing. - Iamp and DIA EMG is the diaphragm activity. - EEG activity is the cortical activity of the brain surface. This is what we use to determine if you are awake, in NREM (slow wave deep sleep; deep sleep), or in REM (dreaming sleep; paradoxical sleep; active sleep). They did it because they have to know what state the animal is in (e.g., awake or asleep) because breathing is carefully tied to whether you are awake or you are asleep. Your breathing goes down when you sleep. o You need to convince me that the animal is not simplyhaving a change in breathing because it is falling asleep. One cannot be convinced by this unless you measure brain activity to see that the rat is either awake or asleep. - In sleep studies, you have to know what overall generalized muscle tone is doing. Muscle tone goes down when you fall asleep. Neck EMG is an example of overall levels of muscle tone. They drop off when the animal enters into REM sleep. You use overall levels of muscle tone or EMG activity and cortical activity to gauge what state the animal is in. Then you look at how breathing changes during those states. - (A) This is before any injection. - (B) The W doesn’t look as clear as compared to the control. The control is sharp and clear breathing. The rat is having a hard time coordinating its breathing activity. o In NREM sleep, the rat is still breathing in NREM. When it enters into REM sleep, breathing almost completely stops. They cannot detect any breathing movements if you look at overall muscle activity. - (C) Much still doesn’t change. It looks uncoordinated during W. o The breathing from NREM to REM shuts off. He is not breathing. For about 15 seconds, the animal is physically not breathing. Every time thishappens, he physically wakes up to breathe. - (D) It looks crazy during W. o Now during W, he is also having periods of apnea. o They didn’t go past 10 days because the animal’s breathing became so ataxic or uncoordinated that it was no longer effective. The breathing is not being effective in causing the chest to move expand and relax. They had to terminate the experiment at 10 days because it started to occur during W as well. - This is the final reallygood proof that the pre-BötC is extremely important. As the pre-BötC dies off slowly, breathing becomes messier. When you kill the pre-BötC in an awake animal, it stops breathing effectively. Why does the rat breathe in W and NREM but not in REM? o In REM sleep, the brain looks like it is awake. The cortical activity looks identical to W. All the muscles in the body except the diaphragm, the breathing muscles, and the eyes are completely relaxed. You go into REM sleep paralysis. o When you enter into REM sleep, the muscles just relax. The diaphragm relaxes a little bit. This is why we think that as the rat enters into REM sleep, the breathing is shutting off because the pre-BötC is no longer able sending enough of a signal to the diaphragm. When the REM sleep enters, it just shuts everything off a little bit more. It is too much to generate normal breathing. 13 - This summarizes what I talked about. - The pre-BötC is important for generating breathing. - The studies in vivo (e.g., in the context of life and behaviour) showed that we had two different approaches to manipulate the pre-BötC in a behaving rat. These experiments have been done in cats, dogs, and goats. The experiments demonstrate that the pre-BötC is important in generating eupnea (e.g., normal breathing). - However, they show that the pre-BötC neurons are NOT essential for RRG. o If they were essential and you got rid of them, breathing would have stopped in an intact animal where you support to damage it. It disrupted it severely. Breathing became ataxic, but it did not stop the animal from breathing. - In their absence, other neurons must be capable of generating breathing, albeit, in an ataxic and uncoordinated way. o This is not the interpretation that the authors of the paper generated from their own data. They did not generate this. They said that the pre-BötC is required for breathing and that it is so important that breathing became uncoordinated when it was leisioned, therefore it must be critical. o It means that breathing becomes uncoordinated, but it’s not essential. I think it’s a critical part of the brain but it may not be the only part. I think the assignment that you red should point out that there is another part that is also important in breathing. I am making this general conclusion that is different from the author’s. - The location of these neurons and their biochemical profiles is not entirely clear. o I told you that they express two different types of receptors, but it is not a particularly distinguishing feature of a neuron, but it is a marker. - However, they have clues that point us in the right direction to understand where these cells are and how they might behave. - While we know that the pre-BötC plays a critical role in RRG, we still have to ask how these cells communicate with one another. o The two general ideas in the field of respiratory science are that you can have the cells in the pre-BötC generating breathing by one of two different mechanisms or models. 14 - I have only been talking about an area in the brain called the pre-BötC. The pre-BötC is where breathing happens, but how does it happen? - How do the cells talk to each other? When are they talking to each other? What are their messages? Where are they located? - The human brain has billion of neurons. We only located a general area in the brainstem called the pre-BötC that is important, but how they talk to each other is hard to figure out. - When we do unit recording studies in the VRG or the pre-BötC, there are lots of different cells. Some of them fire during inspiration. Some of them fire during expiration. Some have different patterns of activity. The different patterns and types of cells are doing something. - The idea is that there are different ways that you can model the way breathing occurs based on the firing patterns of cells. o The three models of RRG are all hypothetical. It is based on pure observation. - This is an example of how the RRG network might be working in the region of the pre-BötC. - The pacemaker model o The pacemaker neurons have an intrinsic activity. The idea is that there is an intrinsic set of activity in the cells in the pre-BötC that cause it to fire over and over again. - The network model o You need a whole bunch of cells that work well together. Some are firing during inspiration. Some are firing during expiration. That communication is allowing effective breathing to occur. It relies on the inhibition between two sets of populations. o You have to have an inhibition. You can’t breathe in forever. There is a group of cells that are telling the cells that cause you to breathe in to stop. They require inhibition (e.g., the signal to stop inspiring). GABA and glycine are two of the primary transmitters that cause cells to stop talking or become quiet (e.g., cause inhibition). - These models are based on the observations on how cells behave when you stick electrodes to record when they are active and when they are quiet. When they are active, how their activity looks and when it occurs. - Which model is right? - I believe that there’s probably a combination. The hybrid model says that maybe pacemaker and network work together. - These pictures show you an example of the pacemaker model and an example of the network model, but I am not expecting you to know anything about those pictures. 15 - People have long thought that the pre-BötC cells are pacemaker. - What is a pacemaker? It is an intrinsic set of properties in a neuron that allow it to fire without any drive. A pacemaker is doing its job without anyone telling it to do its job. It generates its own rhythm. It does this by a series of ionic movements into and out of the cell. o Look at what defines a cardiac pacemaker because it is the same idea. - Na+, K+ and Ca2+ flux into and out of the cell. This fluxcauses the cell to fire an action potential repetitively without anything causing it to occur. Pacemakers are special cells. They are not common types of cells. They are special because breathing is special. Breathing doesn’t fail because cells have these inherent, intrinsic rhythms. - How can you test whether or not you have the pre-BötC region? Is it a pacemaker model? Does it best fit a pacemaker model or a network model? o Are the cells in the pre-BötC pacemaker cells and do those pacemaker cells generate breathing? Or is it a complicated scenario whereby you have a network of different types of cells having a conversation to generate effective respiratory rhythm? 16 - The cell depolarizes, fires an action potential, hyperpolarizes, starts to build up, fires an action potential, repolarizes, and so on. - The action potential is caused by a change in the permeability of the pacemaker cell of these three primary ions. The permeability is decreasing for K+. This decrease in permeability starts to make the cell more excitable. The permeability for Na+starts to increase. o K+ typically wants to go outside of the cell. It makes the cell more negative. When the permeability decreases, K+ is not escaping so much. It keeps the positive charge in the cell. The cell starts to get more excitable. It approaches a threshold. o At the same time, the permeability for Na+ starts to go up. Na+ typically wants to move into the cell and making the cell more positive. o The permeability for Ca2+ starts to go up. Ca2+ flows into the cell by a concentration gradient. - It causes the cell to fire an action potential. The permeability then changes in the opposite direction. It is more permeable to K+. The cell quiets down
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