Stephen Liberles Transcript
SPEAKER 0Thank you Anna! And, uh, hello. It is my pleasure to introduce Steve Liberles, who's visiting us today from the Department of Cell Biology at Harvard Medical School. and he is an HHMI Investigator Uh, Steve is a phenomenal scientist. Any of you who have taken my neurogenetics classes have at least had one of his papers that we did. I won't go into details because they're very memorable papers I'm sure anyone in the class could attest to that, but um so Steve was an undergraduate at Harvard, And, then a graduate student at Harvard with Stew Schreiber, where he did sort of chemical biology kind of stuff. He Then became a post doc with Linda Buck when she was a at Harvard Med school And then when she moved out to the Hutch in Seattle, Steve moved with her. During that time, he actually made some really important contributions to olfactory research, discovering some new families, perceptors, which were really interesting, he's not going to talk about them today But he discovered the tars Tracy being perceptors, which is really important because, for example, they are found in things like bobcat urine and are what Trigger innate avoidance responses. So a lot of that Steve's work also discovered some important roles. Potentially important roles for, uh, receptors. These FDR receptors that recognize formulated proteins. And we're using the immune system. We found rules for those in the Barbara nasal organ. Yeah, since, uh, coming to Harvard, he's made of ton really important contributions, which I'm not going to use this time to describe, but really interesting discoveries in terms of taste, mechanic sensation, um, smell etcetera, looking not only at a function of these receptors but the revolution. And it's a really interesting work over the last eight years, or so he has moved into the vagus nerve, which is something that physiologist knew in the early 20th centuries were really important for things like controlling breathing and heart rate and all kinds of really important on functions. But basically lain dormant has not really a model to look into, uh, with cellular and molecular studies And I think Steve's work led the away in bringing this really important organ system signaling system. This was signaling system back on understanding how it works. So that's what Steve's gonna talk about today. So thank you very much for joining us. Yeah, we're about to do that
SPEAKER 1
Thank you. Very much for that kind introduction. I'm not gonna be a Zen like and sit on the table Um, yes, and my My lab studies sensory systems to gain access to neural circuits that control physiology and behavior. And we study a number of sensory systems in the lab and I'll talk about our work on the biggest nerve today. So our external senses enable us to perceive the world around us. And discoveries of sensory receptors that detect odors tastes light in touch. I think he's really landmark achievements in neuroscience, less well understood are internal senses within the body that survey the state of internal organs and the vagus nerve is a major body brain connection that controls basic functions of the respiratory, cardiovascular and digestive systems. And it displaces strikingly beautiful anatomy. So it's a two way information highway. They're both sensory neurons that take information from the body to the brain and the motor neuron to take information the other direction. Today, I'm gonna focus on the sensory neurons whose soma residing ganglia at the base of the skull and from there send projections throughout the body to the heart, the lung the stomach, the intestine. And these were very long neurons that interface both with internal organs and the brain and the ascending Axons cross through the jugular frame and innovative region of the brain stem, where they activate neural circuits that orchestrate multi organ physiological responses. Now vagal sensory neurons detective variety of stimuli in the airways, they detect stretch of the lungs with every breath irritants that make you cough in the cardiovascular system that detects changes in blood pressure and altered respiratory gas levels associated with hypoxia and hypercapnia And in the digestive system they detect stretch of the stomach as occurs during a meal, nutrients that make you feel full and perhaps toxins that make you feel sick. And I would argue that when we began this work, how any of these senses were detected at a molecular level was unknown. And moreover, the number of sensations mediated by the vagus nerve has not previously been quantified, and I'll show data that some senses have been hidden to the field so far. So the vagus nerv has really fascinated people for over 100 years, but these fundamental problems were unsolved, so we set out to use molecular and genetic approaches to deconstruct the system uh, we began with the hypothesis. That's a heterogeneous structure containing different sensory neuron types, each of which might target particular internal organ detective specific stimulus and, in response, selectively control autonomic physiology. So We began by charting the diversity of vagal sensory neurons and then adapting genetic approaches so that we can map, image and control different neural types. And our long term goal is really to understand mechanism, including the identification of internal organ sensory receptors. Now we've used a variety of approaches to chart cellular diversity in the vagus nerve and unpublished data. Sarah Prescott from the lab has turned a single cell RNA sequencing. Like everyone these days, so she has harvested vagal ganglia from wild type mice and then she can enrich for neuron. She can make a single cell suspension and enrich for neurons. And then, using a 10 X Genomics platform, she can isolate individual neurons and micro phonetics derived Droplets containing, uh, gel beads with bar coded primers. For reverse transcription. She could make a single cell C DNA and then on next generation sequencing platform, attain single cell transcriptomes and then has a bioinformatics pipeline to analyze the data. And to make a long story short, she's done this now on over 50,000 cells from vagal ganglia. I should note that in the mouse, the vagal ganglia actually fused the biggest nervous, the 10th cranial nerve and the gangly refused with the ninth cranial nerve, the glasal fringial ganglia. So we have these data sets together, and what we see is a staggering diversity of cell types. So a conservative estimate we think we think there are 37 at least 37 molecularly distinct neuron types. So for comparison in the visual system, you think of rods, cones and maybe melan opsin producing cells and the taste system, you think of maybe five taste modalities here. There's just a tremendous diversity, and I think it's exciting to think, you know, this neuron type might innovate the heart and control heart rate. This one might innovate the lungs and control breathing. And so our approach to really understand what these different neuron types are doing is been a genetic one. We built various Ires-cre in mice so we can access different neural populations, and highway two of these here one is marked by a pure and urgent receptor. P2ry1 one another by an orphan receptor. GPR 65 these are each about 10 to 15% of vagal sensory neurons and have different functions. So I'll talk a lot about the P2ry1 neurons today. These enervate the airways and control breathing while the GPR 65 neurons innovate intestinal ville and stomach mucosa and they function as chemo sensors in the G I track. Now from Sarah's data set, we realized that there were many neuron types that we had no idea where they were projecting in the body what they were detecting, what they were controlling. These are, genetically uncharted neurons. And so we affectionately called this the dark matter of the vagus nerve um and so. Sarah has really, uh, taken a tour de force approach To getting as many different cree lines as possible, making some, in some cases obtaining them in other cases. So now she has sort of an expanded genetic toolkit to access various vagal sensory neuron types, and we think of the 37 populations we can now target about 33 of these either individually or at least in small clusters and so This is enormous mouse cost. Um, spend more on mice than people these days, but I just wanna highlight one other tool in the toolbox. So vagal sensory neurons are glutamatergic while motor neurons are Coenergic So we obtained a vglut2-ires-cre line from Brad. Vogel's Lab, which hits what we think. is essentially all vagal sensory neurons without hitting motor fibers of passage. Okay, so with these tools in hand, I wanna walk today through three different reflexes that are mediated by the vagus nerve. First, I want to talk about sensory neurons in the airways that control breathing. So there is a diversity of vagal sensory neurons that enervate the airways that not not only those that control breathing but also neurons involved in airway defense such as neurons, that evoke cough. There are other neurons that may interface with the immune system, giving the sensation of a sore throat. There are other neurons that enervate vocal cords. It might be important for speech appropiaception Today I'm gonna focus on a particular class of airway intervening sensory neuron that detects airway stretch, so mechanic receptors in the airways that mediate a classic respiratory reflux that was first discovered 150 years ago by Herring and Brewer, who observed that mechanical distension of the airways revoked reflective inhibition of breathing or apnea. And we can visualize this herring brewer reflects. This is an experiment from Ray Chang in the lab, where he's recording airway pressure through the tracheal cannula and then administers, uh, stepwise increases in airway pressure through a second cannula. And what you see here is a series of spikes. Each of these spikes is the change in pressure that accompanies a breath and as the increased airway pressure. There's a reduction and ultimately a termination in breathing. And this is the classic herring brewer reflux And the question is, how is force in the airway sensed at a molecular level? So our strategy to solving this problem was to first identify the neuron types that might be relevant for airway stretch sensation. And the approach that Ray Chang in the lab used was an opto genetics one. So you wanted to find neurons that when you activated them, evoked a similar apnea or an inhibition and breathing. And so what Ray does, is he expresses Channel Robson, a white activated ion channel in somewhere all vagal sensory neurons. And then in an anesthetized mouse, you can just shine light on the vagus nerve trunk and in mice containing channel adoption under the v-glut2 driver. So all sensory neurons when he shines light on the trunk, you can record these robust, light developed action potential, so the technique works nicely. And when Ray activates all vagal sensory neurons using opted genetic approaches, you can powerful control autonomic physiology with light. You could impact several major physiological systems with these robust, light induced drops and breathing and heart rate and gastric pressure, for example, and we wondered whether or not we can associate these different physiological functions of the vagus nerve and begin to assign them to different neuron types. And so, just coming back to the two Cre lines I showed earlier when Ray activates just vagal p2ry1 neurons, he's activating a couple 100 neurons in the entire mouse, and the animals stopped breathing. They're trapped in a state of exhalation and while you observe these powerful effects on breathing over 10 second trial, there's no effect on heart rate or on gastric pressure. Now when he does the same experiment, but he's using GPR 65 Channel reduction line. Now he sees an orthogonal phenotype. There's no effect on breathing or heart rate, but he gets a reduction in both the tonic and phase components of gastric pressure. And these findings indicate that individual sensory neurons can engage neural circuits that control these whole body physiological systems with high selectivity. I think it indicates a fine tune division of labor among different sensory neuron populations for controlling autonomic physiology. Now, for the purposes of this section of the talk, I wanna focus on neurons impact breathing. So this is work initiated by Ray in the lab and carried out, extended by Sarah with her additional Cre lines. And so we look for neurons that when we opti genetically activated, then we inhibited respiration. We actually found several different classes of apnea promoting neurons in the vagus nerve. I want to highlight, two of them. One is marked by this p2ry1 in this p2ry1 cre mice another in this paz2 cre mouse and come back to this in a little bit. We also found many neurons that did not impact breathing. and We found one neuron thes Kalbe, one neurons that actually call it the hyperventilation phenotype. I'm not gonna talk about these neuron today, but these neurons are they enervate the corraded body and the aortic bodies we think these are neurons that are involved in hypoxia sensing So there looks like there are multiple types of apnea promoting neurons which of these might be airway stretch sensors? And so, to address this, we turn to in vivo imaging in vagal ganglia. So this is an experiment from Erica Williams and M. D. PH D. Student in the lab who expresses G camps in vagal ganglia. So We consider that many of the cellular responses in vagal sensory neurons were not so autonomous they would be dependent on the architecture of peripheral tissue. So we wanted to do these experiments. Is imaging experiments in vivo. So in an anesthetized mouse, you surgically exposes the vagal ganglia. She has to cut the connections proximal to the brain to, lift the ganglia out of the mouse for stable image ing, but she leaves the connections to the periphery intact. And then she could make various perturbations in the periphery and record responses in real time. At the level of ganglia and the first experiment ill show you. She simply puts an electrical stimulator on the nerve trunk and gives pulses of electricity. And this is what she sees. This is vagal ganglia, G camp fluorescence. She gives pulses of electricity with each pulse she can see neurons firing She can image about 150 neurons per an image ing session. She's single neuron resolution, and she can also genetically tag the responding neurons with Cree lines in orthogonal four for us, So electrical stimulation is to control. Erica uses to intern around viability at the end of every image ing session, and then she moved to other physiological stimuli so she can stretch the stomach by inflating a surgically implanted gastric balloon. And this activates about 15% of vagal sensory neurons she can profuse or inject liquid diet through this, uh, small intestine. The very proximal small intestine deactivates other sensory neurons. Or she could extend the airways by forcing air through trichial cannula and this activates other sensory neurons. And she can apply these stimuli in tandem in the same mouse and see that neurons can have selective response properties. And moreover, neurons with different response characteristics are intermingled in the ganglion with a salt and pepper organization. Now, for the next few slides, I want to talk about this particular sensation, the sensation of Airway Stretch. Erica observes a rare cohort about 80 neurons per ganglion that respond to airway stretch These neurons respond similarly whether she stretches the airwaves with ambient air with oxygen or with the inert gas nitrogen consistent with mechanical response. Now these lung mechanic receptors air active every time the animal inhales during title breathing, and we can observe this by in vivo imaging. So here is a G camp fluorescence trace of a representative neuron responding over time. Uh, gray bars are the sights of airway stretch sensation, and you can see this neuron responds repeatedly, Uh, airwave stretch. This is the electrical stimulation paradigm at the end of the image ing session. And if you look at the baseline activity of these neurons, so I should say up front that these are slam dunk stimuli that really are probably super physiological, invoke really robust responses. If you look at the baseline activity, these neurons. You can see that these neurons are rhythmically active and the oscillations are perfectly and trained to the respiratory cycle. And we don't see these oscillations in neurons that don't respond to airway Stretch. So it's not, for example, a movement. Artifacts breathing. So these neurons are active with each inhalation responding to increases in airway volume. How are they sensing this at a molecular level? Well, our first coup came when Rui Chang in the lab observed that the ion channels Piezo1 and Piezo2 were expressed in subsets of vagal sensory neuron. So, for example, about 25 to 30% vagal sensory neurons express Piezo2 so as background. The Piezo's were identified by Ardem P at scripts there ion channels that are intrinsically gated by force. In the absence of auxiliary factors, they're important for our senses of touch and for limb propio exception knowing where our limbs are in space. And so we wondered, based on expression here, whether Piezos might contribute to internal organ salute, internal organ mechanic sensation as well. And so we reached out to our Ardem and began what's really been a fantastic collaboration. So I want to highlight just mentioned that there were two different types of neurons that inhibited breathing that I showed. One was marked in P2ry1 cre mice and other in the Piezo2 cre mice, and we wondered whether either of these might encompass the airway stretch sensors. I should note that about 50% of the p2ry1 neurons contain Piezo2, and vice versa. So there are many cells that express both in our initial model. Going into this was that maybe the airwaves stretch sensors were simply encompassed. As a yellow neuron by both of these Cree lines I'm gonna show you is actually that there are two different apnea promoting neurons one green and one red cell. And the reason we think that is based on this experiment. So this is an in vivo imaging experiment where we mark different cell populations with Cre during the in vivo imaging session. This is a little bit of a complicated slide, so walk me through it. First, there's some genetic gymnastics, So Erica took different cre lines, crossed in with the Td tape tomato reporter and then introduced a global G camp allele so she could introduce a measure, responses and pre positive and pre negative expert neurons in the same experiment. What you're looking at the responses of a couple 100 neurons over time. Each row is the response of one neuron on the responses. Are color coded the in magenta Are the Cre expressing cells and some of the Cree negative cells are shown on the bottom and the airway stretch stimuli are shown. A green bar and what Erica observed was that the airway stretch responses predominantly occurred in Piezo2 positive. But p2ry1 negative neurons. So this really indicated that there were two separate populations of neurons that impacted breathing that we could see here. These neurons are the airway stretch neurons that express largely express Piezo2 and inhibit breathing thes are a different population of neurons that are marked in the P2ry1. neurons, as I've shown by opted genetic experiments, also inhibit breathing. And I'm gonna come back to these neurons at the end of the talk. But we think these are doing so obvious question is Piezo 2 essential for airway stretch sensation so as background. Um, Ardem observed that knock out of Piezo2 is lethal to mice this is a Piezo2 knockout pup global Piezo2 knock out up on the right these mice have first off this. Mice had severe appropriate septal defects. It can't even write itself. It also displays frequent gasping and die soon after birth, due the respiratory distress and upon histology there cyanosis. And there's decreased oxygen saturation in the blood and the airways have not fully developed in these mice. so ardem was interested in tracking the cell types that are relevant for this respiratory lethality. So he crossed he, generated a fox Piezo2 to allele and crossed it with many different cre lines and one of the Cree lines that across the two is a Phox2b Cre shown here. Phox2BCre drives expression in the notice ganglion, which is the largest of the vagal ganglia as well as the inferior Petroleo ganglia. And these mice actually survive to adulthood, so they weren't relevant for ardems respiratory lethality. But then we were very excited because now we could query the role of Piezo2 in any notice ganglion mediated sensory process. So he sent us these mice and Ray began testing them. To a panel of different internal organ stimuli, and Ray observed that airway stretch responses were completely lost in these mice. So this is, Ah, whole nerve electrophysiology experiment where Ray is recording responses to airway stretch. And he's sort of a nice graded response to airway stretch, as well as a response to inter peritoneal serotonin, which activates other vagal sensory neurons and in the piezo2 cell specific knockout responses to airway stretcher, completely lost, even at very high magnitudes of airway distension. So piezo2 is absolutely essential for airway stretch sensation by the vagus nerve. What does this mean physiologically Well, these animals have, um, increased title volume during passive breathing, and they've also lost this classic respiratory reflexes. Herring Brewer reflexes, just to remind you thes are breast that you're visualizing here. And as you increase area pressure, there's a reduction and ending a breathing if you do the same exact experiment. But now on the piezo2 knock out what you see is that these mice continued to breathe normally in a manner that's independent of airway volume, so they're insensitive to airway volume and their breathing dynamics. Um and they lost his classical reflex. Okay, so that's sort of where we stand on Airway stretch sensation. There are many interesting questions I think to follow up on, including the architecture of the peripheral terminals, that sense airway stretch. That's a work in progress. Uh, now I'm gonna turn into a second sense of the vagus nerve. And that's the sense of barrier reception or blood pressure sensation so There are many neurons that innovate the cardiovascular system of the vagus nerve, including neurons. enervate the great arteries. And These include mechanic receptors that sends blood pressure. Also, chemo receptors that detect changes in blood gas levels associate with epoxy and hyper campania. They're also sensory neuron to innovate the heart itself and these air poorly studied. They include cardiac mechanic receptors and cardiac chemo receptors that mediate a variety of different reflexes. Um, I want to focus today on Baro reception. So, uh, this is the barareception reflux is A classic reflux where changes in blood pressure, for example, during postural changes or movement caused a real time moment by moment, changing cardiac output to ensure real time stabilization of blood flow to the brain and body and the way they're several hot spots in the cardiovascular system where blood pressure is sensed. Um, the vagus nerve since has a branch of the vagys nerve called the aortic depressed enervates right at the aortic arch, the largest blood vessel in the body. Um, the corroted, um sorry. The glas al fring al nerve, Which again mouse the Gaza Frenzel nerve and vagus nerve is actually fused, which is convenient for us because we couldn't manipulate all the baro receptors simultaneously that will show the glass of fringe deal Nerve sends a fiber to the carotid Sinus, which is also another hot spot for Baro reception. So these bars receptor Akron's innovate regions of the blood vessel wall that are unusually elastic. What happens is that with each heart beat the blood pressure pulse that emanates radially stretches the elastic vessel wall, and it's this radial stretch that's picked up by sensory neuron. So these are essentially stretch receptors as well, uh, the Afrins then transmit the information up to the brain stem where simultaneously engages parasympathetic motor neurons that inhibit sympathetic motor neurons, ultimately decreasing heart rate and blood pressure. So the question that remained open is how is blood pressure? Sensed a molecular and architectural level. So again, we used a similar approach where we search for vagel, certain sensory neurons that impacted heart rate and blood pressure in a way that mimics the baroreceptor reflex. And we came across way observed that again piezo2 neurons and I promise you piezo 2 neurons do not do everything. Show reflects at the end where there's another reflects independent of piezo2 But in this sensory modality, Piezo2 neurons again opted Genetic activation of them caused a decrease in heart rate and blood pressure that was reminiscent of the barrier receptor reflects. And we didn't observe this in any other Cree line that we used and i Show a couple examples here. This is M C four R neurons, which I'll show also innovate the aorta and GPR, 65 neurons that do not. So our piezo2 neurons essential for the bearer receptor reflects. To address this, we use a genetic approach to a blade piezo 2 neurons and viggle ganglia. So mouse cells are normally resistant to the theory of toxins, so we used the diphtheria toxin guided approach that could be rendered sensitive by expression of the human diphtheria toxin receptor d T r. And there's a Cree dependent DTR allele that exists in mice. So we we generated mice that express d tr under control the piezo2 through Cre and these mice We then bilaterally injected theory toxin into vagel ganglia, and this results in very efficient ablation. piezo2 neurons over 95% of piezo two neurons in vagel on Gaza friend Jill Ganglia are bladed, while other piezo2 neurons in the body are spared after a careful titratation of DT and also other Cree expressing cell are crea negative cells in vagel ganglia are also spared by this injection, so it's efficient and it's selective. I should note that if we do this on all vagal sensory neurons, it's lethal to mice on. We've tracked some neuron types that are relevant for survival, but piezo2 neurons are dispensable. They actually can remove the Piezo2 neurons and they survive the procedure so that we can examine the integrity of the barrier receptor reflux. After doing this and we can see that the barrier receptor reflux is severely compromised. So this is we evoke the barrow receptor reflex. Pharmacologically we inject fennel ephron, which is Veysel constrictor intravenously. Uh, this result in an increase in blood pressure and this increase in blood pressure secondarily triggers the decrease in heart rate through the barrier receptor reflex. And if we blade the piezo2 neurons, we actually get a more dramatic and exaggerated increase in blood pressure. And despite this exaggerated increase, we see a severely attenuated change in heart rate. So the bearer in the normal barrier receptor reflexes lost um and If we blade other neurons, we have no effect on the barrier. Receptor reflects so piezo2 to itself is essential for the bearer receptor reflects again is piezo2 protein essential? Um, there we got a little bit of a surprise that stumped us for a little while. So knock out of piezo2 actually had no effect. These phoz2bcre. piezo2 knockouts had no effect and Wei Zang in Ardems Lab observed that as part of this collaboration, that knock out of both Piezo one and piezo two was essential for eliminating the barriers after reflex. So here you've seen wild type mice here is the federal after an injection induced increase in blood pressure and the heart rate response and wild type mice. And if you knock out both Piezo one and Piezo2 you eliminate this reflex so taken together with the ablation data, there multiple explanations for why you might need both Piezo one and Piezo two. But a parsimonious interpretation eyes that perhaps these receptors are functioning together in the same Afrin cooperating to mediate blood pressure sensation. We don't know that for sure, but that's our leading model. And whether not they form mixed head rumors or whether or not they're operating peril, I think it's still an open question. So there's more study that needs to be done about the molecular level to understand how these proteins are functioning. So as a first step way next wanted to look at the architecture of the barrier receptor terminals. What do they look like in the aorta? And so we turned to a technique that was previously developed in the lab by Dave Strudwick to map both the peripheral and central projections of vagal sensory neurons. And what he does, or what they did is he injects Cree dependent dental associate ID viruses, A V s directly into vagal ganglia and these a V s and code for us and proteins that enable us to visualize the fibers. And in the first set of experiments, he used Vigo to cre mice So again, all sensory neurons injects with creative tomato virus, and you can see the beautiful vagal fibers in the heart and the lung in the stomach or intestine. And you can also look at the patterning of different types of vagal fibers in brain stem. I'm now focus on the aorta. So this is the aortic arch, the largest blood vessel in the body on I love this fiber. Here, this is the erotic depressed, a nerve that comes down and interviews right at the peak of the aortic arch and then bye for Cates, with both dorsal and ventral branches ratifying and sort of a satellite pattern across the aortic arch. And we observed a few different types of terminals in the aorta. They've only been described before in one study, but we see the same terminals is that study from 20 or 30 years ago from Ed Foxes Lab and we look for cre lines that might mark these different terminal More apologies. And we found three different types of cells that innovated the aorta. One was marked by piezo2. And I'll come back to these things terminal morphology and a little bit. We see another cell type mark in by M. C. Four R cre on a third cell type marked by CALB1 one neuron. So these neurons actually contact rare glioma cells in the aorta, and they also integrate created bodies and they show the hyperventilation phenotype from these mice. So we think these are the chemo, the vascular chemo receptors. But these two different terminal types, um, have been unknown function. So M c four. Our neurons form what were previously described this flower spray terminals based on their characteristic morphology. These were actually the major terminals in the aorta, and they're largely confined to the saddle region. So if you use a non specific tracing approach like dye I, you're looking mostly at these terminals. The piezo2 neurons formed a different terminal type called enna endings, which are long linear branches that emanate from the principal fiber tract, and these again are the bearer receptors. So in early studies with proposed that maybe these were different types of bearer sector terminals, but there have not been tools available for selected manipulation of each of these terminal More apologies. Now that we can eliminate the piezo 2 nuerons neurons, we eliminate this terminal morphology while preserving this terminal mythology, and we eliminate the barrier receptor reflects the barrier receptors form these ending endings. The functions of these flowers, great terminals are mysterious. So these are not mediating known arterial chemo receptor, arterial mechanic, receptor, um, reflexes. Presumably they're sensing something that has been hidden to the field were very interested in what these neurons are doing. The piezo2 neurons adopt a distinct macroscopic architectures so they don't innovate the saddle region. There fibers actually extend beyond the saddle region and form. What we describe is aortic clause that look like they're grasping the entire aorta, the dorsal and ventral branches. reconverged near the arterial ligament at the base of the aorta. In some cases, the proximity of these, um, dorsal and ventral branches give the appearance of an aortic ring. And, the terminal sort of dance off these off the claws laterally and based on some preliminary data. We think the piezo2 protein is localized to these ending endings. And right now we're trying to get some electron microscopy of these different terminal endings to understand how forced the sense of the structural level. But we don't have the answer at this point. Um okay, so that's that's what I wanted to say about Barry receptors. And now I'm gonna turn, uh, to the 3rd 3rd. Reflects and that is, uh, back to the Airways again, actually. So we're talking about neurons in the upper airways that are important for airway protection. So, um, through to a hiccup of evolution, there is, uh, a danger zone in the upper airways, where the dirty gastrointestinal tract shown in red in the pristine respiratory tract shown in Blue Cross. And every time you eat a meal, you run the risk of material from your gastrointestinal tract infiltrating the airways. So there's a dense network of sensory neurons that guards guards at this for angel crossing to protect the airways and evoke a constellation of reflexes. So first, you have occlusion reflexes, protective reflexes. So every time you swallow, there is an airway protected motor program that ensues. So there's a brief apnea transient apnea that prevents aspiration of material into the lungs. The vocal chords will adopt. They will close, and that provides a physical barrier to prevent material from entering the trachea. And the wearing itself will actually close. The window vestibule will shut the EPA goddess, which is sort of the lid of the respiratory system, if you will, that descends on the larynx. So all those provided physical barrier to help prevent material from entering the wear rings every time you swallow. If material does enter the larynx there, then secondary clearance of X reflexes that evoked, for example, cough. People think about cough mice off. There's also what's called an exploratory reflux, which is, or NGO cough that has slightly different kid Matics from a cough and swallowing is also evoked by airway defense so fringy will swallow or secondary swallow. It's similar to a coffin that expunge is material from the larynx. Ah, cough sends it out of your mouth, while a secondary swallow will send it down into your gastrointestinal tract. But that also helps clear out the lyrics and these airway defense reflexes are really important. So dysfunction of these reflexes can cause severe clinical problems on these air, common in elderly patients as well, and a concurrent stroke after stroke or nerve injury on conclude choking, um, dysplasia or difficulty swallowing or aspiration pneumonia, which is actually a leading cause of death in elderly patients and what you're looking at here, this is a barium swallow and actually a healthy individual who has temporary anesthesia on a particular nerve branch of the vagus nerve, the superior of Orangeville nerve and what you'll see your to swallowing events. The first swallowing event occurs normally down The esophagus and the second swelling events. The material gets caught up in the larynx. And then there's a NATPE ihr ation event where material penetrates the trickiest. That's the normal event. Here's the aspiration. Event material gets caught up and goes down, uh, wearing. So that's what the sensory neurons air there to guard against. And so there's a tremendous diversity of Vega LaFrentz in the lyrics that are very poorly studied. so there's a branch of the vagus nerve called The Superior lariningeal nerv this is the branch. That was, uh, anesthetizing the prior study that innovates the wearing from the vocal folds up to the epicodus as well as the coddle pharynx. There's also a second branch called The Recurrent Laryngeal Nerve. This is the poster child against intelligent design. Uh, so this neuron goes all the way down the neck underneath the aortic arch in comes back up the throat. So in giraffe, for example, and giraffes, the cell bodies and innovation point of pretty close. It still takes this long and winding trajectory so not intelligently designed. But, uh, so the were current ones you under, uh, innovates, the trachea, the upper lungs, the trachea and some innovation of the larynx as well. And so there is a tremendous diversity of terminal, morphologic in the larynx, um, that have been really it's a congested hotspot for innovation. And over the decades of research in this field, people have used different creative descriptors to describe all these terminals. Ivy like terminals had their form Terminals Corp, muscular terminals, chandelier terminals, free endings. And it's so hard to know if different people are describing the same terminals, different adjectives or whether not individual terminals might be come together with the same name. And i So we set out to genetic approaches, which we hope will provide some sort of consistency across study and also allow us to link the terminal morphology to the function, the response properties and ultimately to identify receptors in the media, it sensory functions of the larynx. And so we set out to use genetic approaches in our entry into the system is really again through an optic genetic approach. So I showed before then, when we activate all vagal sensory neurons that we evoked changes in breathing and heart rate and gastric function. When we change the anesthetic, we actually see a different FINA types. Now we moved from Isil for into urethane, and now we see that when we do that opted genetic activation of all vagal sensory neurons evokes repetitive swallowing. The mice will continue to swallow for the duration that you activate vagal sensory neurons and they'll stop when you turn the light off. So I want you to focus your attention here. This is the hyoid bone which will elevate with each swallow. So if you here's the wear rings and the hyoid bone. If you you'll see this elevating, it'll be obvious when the white is turned on from these experiments. So here's the mouse at rest. Nothing interesting is happening and then the white turns on. You'll see that the mouse will just swallow repetitive. Where's the swallow? There's a swallow. There is a swallow. This mouse will swallow six times while the white is turned on. And then when the light is turned off, it'll return to baseline. Okay, so, uh, swallowing is actually complex motor action that you can divide into several phases. There's the oral phases swallowing, which includes which is thought to be volitional so does not occur under anesthesia and involves tongue movements that we did not observe. The secondary phases. Following is a fringe ALS swallow which again is place Portland Airway Defense Onda Third piece of swallowing is Asafa Gill, which involves esophageal Paracelsus, which we also do not observe with consistency. So we think we're observing for Angel swallows. We measured several physiological parameters. So when we measured tracheal pressure received a brief apnea that occurs with um each each opted genetically evoked swallow. We see an increase in, uh PMG activity of the diagnostic muscle which sits above the hyoid bone, which is a signature of swallow a spike inferential pressure. Now, in addition to seeing these features of swallow, we also see another reflex. When we often genetically activate all vagal sensory neurons, we see expert Torrey reflexes, thes air again. The orange all costs what they're sometimes nickname their costs. Um, it's been debated whether mice Cough Cough has is associated with a prior deep inspiration before violent expiration and an expert Torrey reflex. You sort of lack this prior inspiration but have this forceful explanation, and we see these as well in mice when we apologetically activate the sensory neurons. And this shows what a representative trace looks like with when we're looking at these parameters over time so this particular mouse will swallow five times and we'll display to expert Torrey reflexes. And so next we one of the using our collection of pre lines, asked which cell types might be relevant for swallowing an expatory reflexes on what Sarah observed was when she activated these p2ry1 neurons under one anesthetic state. These neurons caused this inhibition of breathing. As we change the anesthesia, we see that these neurons now evoke swallowing with transient apnea and That's consistent, I should say when electrical stimulation experiments of the S L N have shown these sort of anesthetic dependent responses. Uh, To consistent with this When we look at many other sensory neuron types of the Vagus, we don't see similar levels of swallowing. We see some when we activate these M p Y. One. Our neurons and way also don't see it with the chat neurons, which are the motor neurons. We don't see what the piezo2 neurons finally phenotype. No piezo2 role and and I should say these air sensory neurons that we transect the vagus nerve and then stimulate above and below the transaction site. We put the light fiber above the transaction site we evoke to swallow, consistent with this being a role for sensory neurons. Furthermore, when we moved the light to different nerve branches, we see this effect when we moved the light on the superior, or NGO nerve that integrates the larynx. So again, these were NGO sensory neurons that are relevant for the pheno type. Looking at our map of cell types p2ry1 neurons hit what we think are four distinct clusters within the it was in the map with high levels, and you could detect lower levels of P2ry1 by and five other clusters. So nine different clusters to consider eight of these clusters, are hit by Cree lines that do not drive swallowing and the 1 cluster that we think is relevant is this NP 19 neuron cluster. This is a high expressing P2ry1 neuron a small cluster about 50 sensory neurons that we think is driving a constellation of airway defense reflexes that include apnea vocal cord up. I'm showing that yet, yeah, expert Terry reflexes inferential swallowing. And we want to look at another hallmark of airway defense, and that is vocal cord deduction. And so again, every time you swallow, the vocal chords will close to provide a physical barrier that prevents entry of material into the trachea on. But you'll see, I'll show a video here. We're measuring the dynamics of the vocal chords, using a miniaturized endoscope in the mouse and what you'll see in during normal breathing cycles. You see the vocal chords, um, expanding, abducting during each inhalation and then partially adopting during each X elation And then when there's a challenge, the vocal chords will completely adopt and that way see the same thing when we activate all vagal sensory neurons and speak with two drivers. So here's the vocal cords they're opening and closing. With each breath we turn the light on. We now get a complete deduction event to the vocal chords completely closed, and then we turn the light back. When we turn off, the normal dynamics come back. Hope that hope that's clear and when we do the same experiment with the p2ry1 neurons. We see a slightly different phenotype, so we don't see these complete deduction events. Instead, what we see are these transient episodes of complete blotted closure that occur every time the animal swallows. So again, it's part of this airway protective program that's occurring when we ah pathetically about swallowing and when we actually piezo2 neurons, for example, we don't see any episodes of complete, clotted closure we see under this anesthetic condition, Um, change in, um, in the extent of Gothic abduction. So the point of this is that the vagal p2ry1 nueron seem to evoke this coordinated motor program for airway protection. What are the stimuli that are relevant? so first. What we wanted to do is defined, uh, the Lawrenceville stimuli that evoke swallowing. So, Ben humans, the graduate student lab developed a preparation where you could profuse stimuli through the laryxn And, he did this attack very slow flow rate. So when uses PBS and constantly flows about 100 micro liters per minute, there is no reflexes swallowing due to For such of the PBS. This is similar to the mucus that would normally be present in the throat. And if he switches from one PBS solution to a second PBS solution, he also does not evoke swallowing from the changes in flow that accompany switching stimulus solutions. If he switches to water, to acid or to high salt, he starts seeing reflective swallowing. When the stimulator refused to the larynx, uh, many others, he also sees it. If you in sorts of mechanical probe up the larynx you'll see reflective swallowing. However, other stimuli did not evoke swallowing, including sweet stimuli like saccharin and sucrose, umami stimuli, MST and Alan. Some bitter stimuli trip. One trip the one agonist. So the Warren Geul stimuli that evoked swallowing, or at least partially distinct from classical taste modalities. These are occurring through Lauren Geul sensation. Not, for example, through aural sensation. Because we can cut the superior Lauren Geul nerve and eliminate responses to the water, salt and acid response and diminished responses to the mechanical probe. Mechanical pro probably extends into Caudal Ferenc, so we're getting some activation of Glasgow fringy LaFrance with the stimulus is So what P2ry1 neurons do. So we turned again to this ablation approach, using diphtheria toxin, where we can a blade to p2ry1 neurons they survive the procedure as well, and what we observe is after blading the p2ry1 neurons responses. The high salt mechanical force are intact, but responses to water and acid are lost in divination. so, p2ry1 neurons, seem to mediate specific modalities of sensation in the larynx So how do they sense the stimuli? Water sensation has been a big mystery in the throat, and it's been debated, for example, whether water is detected primarily by sensory neurons or by upstream sentinel cells that communicate with neurons. So we thought, by tracing the anatomy of these P2ry1 neurons in the larynx, we could gain some insight. So we turned back to this anatomical approach, Um, that they've developed a Sarah now took the p2ry1 cre mice and injected vagal ganglia with pre dependent reporters. She first did this with many Cree lines, and I'll just say that there's a tremendous diversity of neuronal of morphology is in the larynx, Um, that she could see with different cre lines. So trip a one Cree or there are multiple subtypes of trip A one cree, actually different form different free endings in different regions of the larynx. Kalbe one neurons this'll different. Calvi, one neuron from the crowded body. These from chandelier terminals in the larynx. We observed ghabra one neurons in Moscow on different two different piezo2 neurons, one near the coastal glens and another in Lauren Ziegel muscle. I'll say, with the exception of some of the trip, a one neurons that are detecting environmental irritants from this venue or it and others, I would say we don't understand what any of these neurons are really doing in the lyrics, but those air subjects for future study the p2ry1 neurons form two distinct terminal morphology. So this is an open book preparation of the larynx So we filet it by cutting, uh, the EPA goddess here, and what we see is there's dense innovation of the war in jail, surface of the epiglottis as well as nearly written noid. So the epithelium over the vocal folds. And if you zoom in, what you can see is that these p2ry1 neurons seem to form core puzzles that about 40 40 microns in diameter. And these core puzzles directly opposed Lawrenceville taste buds. so There these think of taste buds. You possibly think of taste buds in your mouth on your tongue, mostly and in your palate. But about 10% of taste buds are found in the larynx where their function has been pretty mysterious. And so these words your taste buds air directly innovated by these vagal p2ry1 neurons, and that raised the possibility that the sensations mediated by p2ry1 neurons is involved. Primarily primary detection by taste buds. And taste cells that communicate with these neurons. So we wanted to test this by either performing Gaiman's function or positive function experiments in epithelial cells directly. So first we wanted to opera genetically stimulate epithelial cells, uh So we used the carrot and eat promoter, and I should note that character Nate promoter drives expression nicely to taste buds. But there are some other epithelial cells that are hit by the carrot Nate promoters. So let's put that out there. But it does not hit vagal sensory neurons. All right, So when we shine light on the epithelium of the different regions of the throat, when you move the light into the larynx, we can evoke repetitive swallowing. When we drive channel adoption in epithelial cells. If we move the light to the vagal ganglia again, we don't evoke swallowing. So this is just the activation of epithelial cells is sufficient to trigger this neural arc resulting in swallowing reflex. Now. Now, we wanted to disrupt epithelial cell to neuron communication and see if we could disrupt, um, sensations that evokes swallow. And so we relied on work from Tom Fingers Lab, who showed that taste transaction in the mouth So bitter, sweet, salty, sour umami taste all relied on I a tropic 80 p receptors and knock out mice lacking P two x two p two x three Lose all these taste sensations and it turns out that thes np 19 neurons robustly expressed both p two x two and p two x three. So we tested whether or not these knockout mice might have deficits in Lawrenceville sensations, we took them and tested them in our internal profusion essay, and what we observed is the p two x two p two x three. Double knockout mice have normal responses to high salt mechanical force but have diminished responses, responses acid and have lost responses. To water. And so our current model, which we're doing more experiments to tease this apart further, is that upstream epithelial cells, taste cells or another epithelial cell type is first sensing water. Uh, then this releases atp, which activates on a tropic receptors on vagal sensory neurons. In particular, the N P 90 neurons that are marked in the p2ry1 cre mice This takes information up to the brain, evoking a constellation of airway defense reflexes, including friends you'll swallow and associated apnea and vocal cord reduction, as well as expert Torrey reflexes. Now it's interesting to note that, um, cough and humans chronic cough can be treated by inhibitors of, in some cases by inhibitors via tropic ATP receptors, the mechanism by which people thought this work their proposed that this work is that a T P is providing a damage signal that somehow sensitizing Lauren Geul neurons.