 screen now? Not yet, not yet. I'll tell you. So hello everybody. Welcome to our new seminar. So today, give me a sec. Today I have the pleasure of introducing our speaker, which is Dr. Will Greens. So thanks Will for accepting our invitation to the Sussex NeuroTalk. It is a pleasure for us to have a young investigator like you. And today Will will talk us about an exciting new research line he's developing at the lab of Professor Jeffrey Dimon. And the title of this talk is sorry, let me find it. He will be talking about neurovascular interactions in the retina, in the mammalian retina. So I will give a brief introduction to the Dr. Will Greens. He did, sorry, he did his PhD at the laboratory of Professor Jeffrey Dimon. Sorry, I missed something. I missed the document. Sorry. He did his PhD under the supervision of Professor Jeffrey Dimon at the University of Maryland. And his thesis was called Dendritic Integration and Reciprocal Innovation in the retina. And then he moved to the Howard Hughes Medical Institute as a postdoctoral fellow. And in the University of Washington in the Department of Physiology and Biophysics, he did her postdoctoral research under the supervision of Professor Fred Rick. So again, thanks a lot, Will, for accepting our invitation. And it is a pleasure for us to share with you our new discoveries in the field. Awesome. Well, thank you very much, both Jose and Tom Baden for the invitation and for just doing a wonderful job organizing this worldwide neuroseries with the vision focus. This is just fabulous. So let me share my screen here. And my research career actually started in medium energy physics, believe it or not. And as I accepted a graduate position at the NIH to work in the field of retinal neuroscience, I was very excited to learn about the biological light detectors and the bioelectric circuits within the eye that convey visual information onto the brain. And in the end, after spending a good number of years in the field, I came to appreciate a lot more than just that about the retina itself. Okay, the retina is really sort of a window into the brain. For those of you not everyday familiar with the retina, of course, the visual images are coming through the cornea. The lens is focusing those images onto the retinal surface. You can see a sort of a blow-up of the retinal tissue on the right hand side of the screen here. Retina is a multi-layered structure with the photoreceptors on the back and several sets of synapses and neurons that ultimately process this information and convert it into spike trainings which are kicked out the optic nerve. And I've been incredibly interested in the synaptic integration and adiatic integration properties of these neurons and how they contribute to visual processing. But today I'm going to talk about another system that's also present in the eye that makes that's also similar to other brain regions. And that is the retina of humans and mice and other mammals is also vascular. And this vascular tissue supports retinal function. And today I'm going to talk about interactions between these two systems. And much of what I'm going to be showing you today is both unpublished data and also quite preliminary in some places. But I think that's sort of the fun of these types of lecture series is to really to tell people about what's going on now, not something that's already published. And so with that, now one of the godfathers, the early godfathers of retinal anatomy was Ramoni Cajal. And Ramoni Cajal was an incredible artist. And what he did is he documented through his drawings the retinas of numerous animals in the kingdom. And here in the center is one of these drawings of the retina with the photoreceptors at the top of the screen. You can see there's multiple sets, multiple layers involving some some neurons sandwiched into the middle that are conveying information from the outer retina to the inner retina. You can see the ganglion cells at the bottom of your screen there is the axons of those ganglion cells that form the optic nerve. He also identified some of the support cells that are present in these tissues. You can see an astrocyte at the very bottom of your screen down here. And this is one of these muller glia cells in the retina. Now Ramoni Cajal used the Golgi technique which allowed him to label the dendrites of individual neurons. And so what this allowed him to do is really look under the surface of these structures to see what the morphology of the different neurons. And of course looking under the surface revealed a much greater diversity in the morphology of cell types that we could appreciate from the gross retinal structure. At this point we know most of the neurons that are present in the eye you can think about these as the retinal hardware. So this represents the the neuronal types found in the retina of of mouse. So at the top of your screen you can see three types of photoreceptors those are UV and green cones. And then the longest one there is a rod photoreceptor. So there's really only three types of photoreceptors in the mouse retina. And they have dichromatic vision. Now in the first layer of the retina the horizontal cells provide feedback to those photoreceptors that come into varieties. As we continue to march deeper into the retina we see that there are roughly 12 types subtypes of bipolar cells. And we know that each of these bipolar types relay slightly different information about the visual world from the outer retina to the inner retina. And it's at this layer you can already see a high degree of parallelization and diversification of the visual signals. Now those bipolar cells make synaptic contacts on the amicron cells and ganglion cells in the inner parts of the retina. And you can see that the diversity of cell types at these later stages is even greater than it is in the outer retina. The ganglion cells ultimately transmit features of the visual world onto the brain through the optic nerve. And we know that each of these ganglion cell types and their associated circuitries are involved in extracting those particular features from the visual world. And one of the populations that has been most interesting to me throughout my career is this group of inner neurons called the amicron cells. And they actually are the most morphologically diverse cell type in the mouse retina. And these neurons, these inner neurons also release a very large array of neurotransmitters and neuromodulators that include GABA, glycine, pseocoline, dopamine, and glutamine. And while these amicrons are thought to be involved in complex processing, more than 70% of these cell types have never been studied function. And really, the majority of the work so now has been focused on four subtypes. I'll point those out. This is the A2 and the A17 amicron cells. These are both involved in scotopic vision or night vision. And the other two cell types for which quite a bit has been worked out is really the starburst amicron cells, which are involved in direction selectivity. And also the V-glute 3 amicron cell, which is involved in separating out local motion from global motion. And of course, one of the things that has allowed us, that is allowing us to dig deeper into these unknown amicron types is the genetic accessibility we get from the mouse retina. And of course, all the beautiful previous anatomical work. Now, when you're trying to study a new cell type, you really have to throw the kitchen sink. And some of the techniques we've used are viral synaptic tracing, paired recordings, optogenetics, functional calcium imaging, neuropharmacology, serial electron microscopy, and computational modeling. And we use these different techniques to get at these questions. How are these amicrons wired up? What computation do they support? And what molecular and morphological specializations contribute to these computations? And for the majority, the first 15 years of my career, I've really been focused on these questions. But some of the recent work has kicked us in a new direction. And to ask another question, which is, do amicron cells participate in neurovascular coupling pathways? And neurovascular coupling was a brand new topic for me. And so I'm going to walk you through the vasculature of the mouse retina. And similar to other brain areas, the retina is highly vascular. It's supplied, the blood is supplied by this vascular, these vessels. It supplies oxygen, glucose and nutrient and removes waste. There are three major types of vessels, the arteries and arterioles, the capillaries and the veins. And I'll point out that the organization of these vessels is different in each individual's eye. And this unique organization of these vessels is what allows security companies to use images of the retina as a biometric security feature. Now it's also important to note that blood flow is modulated locally within neural tissues. And this is called hyperremia, where there's intense neural activity and that intense neural activity triggers for the local vessels to open up and allow more blood flow to the region. And this change in blood flow is actually so tightly connected to changes in neural activity that it's actually used as a proxy for neural activity in the bold fMRI imaging technique. So changes in the vascular are very tightly associated with changes in neural activity. And of course, if we want to understand the interface between the retina and the blood and the blood, we need to look at what's commonly referred to as the blood-brain barrier. And this is the textbook description of a blood-brain barrier. So you're here on the right side of your screen. And the thelial cells make up the vessel walls, and they're held together by tight junctions. Now surrounding these vessels are parasites, which are contractile cells, and they're doing most of the mechanical work for opening and closing the vessels. And surrounding the vessels and the parasites are astrocytes, which are these glial support cells. And it's been shown that the astrocytes are really communicating with the parasites to control the vessel diameter. And of course, there's also a few cracks in between the astrocytes where neurons actually have direct access to the blood. Now this blood-brain barrier definitely controls what comes in and out of the retina or the blood. And this also limits our ability to provide pharmaceutical manipulations for diseases in particular brain regions. So this is a really important feature at the interface between blood and brain. And we wanted to look at this in the mouse retina. Okay. So on the left-hand side of the screen, you're actually looking on face at the mouse retina. These are the photoreceptors and the outer retina, all the little dots. And I've labeled the blood vessels with the sulfa-rotamine 101, and that's going to show up here in red. So this is just a Z-stack on the left. We're focusing up through the retina. You can see the blood vessels in small red blood cells that are present inside those vessels. And the main point here is that the mouse retina, just like the human retina, is a trivascular network that consists of deep, intermediate, and superficial layers. And the superficial layer here corresponds to the ganglion spill layer. And again, these are the nerve fibers that make up your optic nerve. And if we take this vasculature, the Z-stack that we just collected, this volumetric stack, and we could collapse that in one one axis, you can see this full Z projection up here. It really looks like a tangled mess. But if we instead then break the layers apart, we can see that the vessels in each layer very nicely fill out the retinal space and look quite a bit like little roadways with cul-de-sacs and traffic surface. Now the retina, as with the brain, of course, it's using the oxygen, that's being supplied. And from some beautiful work by you et al in 1994, they used one of these oxygen tension probes, which they inserted into the rat retina and slowly advanced this probe through the retinal layers while measuring oxygen tension of the tissue. Now oxygen tension really reflects the balance between oxygen consumption of that layer of the tissue and also supply of oxygen from the blood source. And so some of the numbers from the study are superimposed on top of the retina here. So the top layer, these are the ganglion cell again with the superficial blood vessels, the inner retina with these intermediate capillary levels, and the deep layers of the capillaries. And what you see is that the oxygen consumption in the middle of the retina is highest. And this means that this part of the retina is also highly subject to some sort of ischemic damage that would reduce oxygen flow to the tissue. And it's been sown that when oxygen is occluded from the central retina or retinal artery that you get this dramatic reduction in oxygen in the intermediate and the superficial layers of the retina. Now, within on the time scale of minutes, this eliminates the retinal response as seen through electroretinograms. And if the oxygen is reperfused through the tissue or the blood is reperfused the tissue, the light responses come back. However, if the retina experiences oxygen deprivation for a longer period of time, the neurons begin to die and can no longer transmit information from the retina onto the brain. Here is some, so the retina also exhibits hyperremia and that is changes in blood flow in response to changes in neural activity. And I'm showing you the superficial blood vessels from a paper, beautiful paper by Cornfield and Newman. Eric Newman is a research professor at the University of Minnesota has done just a tremendous amount of beautiful work on retinal hyperremia. These are the blood vessel layers, different blood vessels in the superficial layer. So we have an arterial first-order arterial, which branches out into a second-order arterial, which branches again into a third-order arterial here in black or capillaries. You can see these thin vessels are studded with the green parasites. That's a particular feature of the capillaries. And at the bottom of screen here is a vein that's carrying blood back back towards the heart and out of the retina. And what Cornfield showed here is that when they delivered a three hertz flickering light to the retina for 15 seconds, they see an increase in the diameter of all major vessel types of the superficial layer. And in hyperremia, this same effect is really a very local effect, as I'm going to show you here. So here I've used Sulphur Rotamine 101 to label the blood vessels in gray. You're looking at the ganglion cells on the retinal surface. So this is the superficial layer. In the top left hand point of the screen is where the blood is actually flowing into this fork in the road and is splitting to go towards the right of your screen or towards the bottom of your screen. And what I'm doing here is just using again a three hertz flicker, 150 micron spot of light that's centered on this yellow circle on one of the two branches. And at this other branch, there is no visual stimulation. And the point here is to show you that this hyperremia is a very local effect. So it's changing in response, the blood flow is changing in response to local activity. And so let's get rid of the retinal visualization. Now we can look at just the blood vessels here, and we can quantify the diameter at any location within these vessels. And we can make measurements. This guy here is around seven microns wide. This guy here is just over 10 microns. And we can now deliver the visual stimuli. And you can watch the vessels change in diameter. In particular, you'll see in the in the yellow section here, you'll see this vessel expand by 35%. But a neighboring leg coming from the same arterial is is relatively unmodulated. So this hyperremia is a very local effect. In cornfield and Newman, they also look more carefully at the different layers within the retina itself. And they found that when they compare changes in capillary diameter, the superficial and deep layers to the intermediate layer, we see the strongest modulation. And I'll remind you that this is the this is also the part of the retina, which experiences the lowest oxygen. And so modulation, these vessels are going to be particularly important for providing nutrients and oxygen to those inner retinal layers in an activity dependent manner. So the retinal inner or the blood-brain interface that I showed you earlier is typically thought to be encased in astrocytes. And as Paul shows here, the astrocytes are present in the retina, but they're really restricted to the ganglion cell layer. And these are actually the astrocytes contacting vessels in the ganglion cell layer in the mouse. Okay, so you can see these beautiful astrocytes. But what about the other two layers of vessels, the intermediate and deep layers? There's no astrocytes there. And so we really have turned our attention to these muller glia stuff, which span the length of the retina and have the ability to come in contact with all three layers of the vascular network. Now, Cajal beautifully drew out the morphology of muller glia in many different species. And a few of those are shown at the bottom of the screen, frog, carp, lizard, chicken, and cow. And while these muller cells have many functions in our present and abascular retina, I'm going to talk a little about their potential role in modulating and controlling neurobascular coupling in both mouse and potentially in humans. So here is a muller glia that I've injected with the green fluorophore, Alexa 48, and we just reconstructed one of these muller glia. You can appreciate its long morphology that it spans the entire retina. There's a very complex morphology. Again, we're labeling the blood vessels also here in yellow with the sulfa rotamine 101. So you can see it spans the trilameter network. If we just look more carefully at the contact between the muller cells and the blood vessels here in the bottom right hand corner, we're looking at the muller enfee wrapping the capillaries and the ganglion cell layer. And we can see that as we go into the intermediate and deep layers, even though this muller cell is not exactly directly adjacent to one of these vessels, it reaches out fine processes to make contact to each one of the vessels that is nearby. And so this again is consistent with the idea that these muller cells might be communicating with these blood vessels. Now muller cells have many textbook functions in the retina and many of these are true for other brain regions as well. They're involved in glycogen breakdown to help fuel aerobic metabolism for neurons. They mop up neural waste from the extracellular space. They protect neurons from excited toxicity when too much excitatory neurotransmitters are released. They control ionic homeostasis, in particular they regulate potassium concentrations near the end feet and the ganglion cell layer. They contribute to the generation of the electro-retinogram in particular the B wave and they synthesize retinic acid from retinol. And lastly it's even been shown that these long thin muller cells can act as light guides and reduce optical scatter as light is transmitted from the inner parts of the retina to the outer parts of the retina where they interact with the photoreceptors. But Eric Newman has shown of course another function for these muller cells and he finds that when he looks very carefully at muller cells and the calcium signals in these muller cells and especially when they make contact with vessels at the intermediate layer that activity in the muller cell is correlated with activity in the vessel diameter. So you can see here he's expressing a genetically coded calcium sensor in the muller cells in green and he's imaging the blood vessels which he's labeled again with the sulfurotomy. He sees these changes in calcium signal in the muller cells which correlates with an increase in vessel diameter. Okay and he's also shown that this happens in vivo and it's largely dependent on interstate or calcium stores within the muller cells themselves. Eric has also shown that these muller cells are interacting with the vessels through the arachnodonic acid metabolite pathways and I won't really be talking about those further today but I will be digging more into these muller cells. So if a muller cell is involved in hyperenia not only should it be in a position to control vessel diameter but it should also be in a position to sense neural activity. And here Eric has labeled muller cells with the calcium sensitive dye and you can see spontaneous activity that happens throughout these optical recordings. He then delivers a flickering light to the retina where he sees an increase in some of these calcium sparks within the muller cells and if we look at the average here we can see that these mullers are responding quite nicely with increases in calcium in response to visual stimulation. From a mechanistic standpoint Eric showed that the majority of these light evoked calcium signals were blocked by an ATP receptor antagonist Suraman. So this really indicates that the muller cells are sensing the release of ATP from neurons and that the release of ATP is driving calcium signals in the mullers which is playing a role in controlling vessel diameter at the intermediate capillary layer. Now since the time of Ramonica Hall our anatomical approaches have gotten a little better as well and one of the approaches that we'll be talking about today is serial block face electron microscopy. Now electron microscopy is an old approach to studying neural circuitry and it provides very high resolution images with nanometer resolution and the advancement in this technique came when people just learned that they can in fact fix tissue within one of these resin blocks. They could image get the ultra structure from the surface of this block and then they could go in and shave off make a very thin shave off the top of that block and re-image the surface and this process occurs over and over and over for several weeks which allows you to collect ultra structural data from a small block of retina and one of the most important papers at least in the retina where this was originally shown was from this Helmstetter et al paper and this really allowed them to look in a much more unbiased manner at all the cell types of a present in the retina and in this case we decided we wanted to use this technique to learn more about the fine detail of connections between the Mueller cells in the blood vessels and the Mueller cells in the neurons and the work that I'm going to show you has all been done by added sadness who is a very talented post back in the lab. I'll also say he's applying for graduate school this year and so if you're on admission committees I highly encourage you to give his application a look he's a very talented guy and a lot of fun to work with and so added really developed an eye for these Mueller cells and began reconstructing their complex morphology at the ultra structural layer level. Here are some blood vessels that have been skeletonized in red this is the intermediate capillary layer the bottom of your screen is the deep layer and these are three examples of Mueller cells reconstructed all the fine processes in the middle of the retina and he's as I mentioned we focused on two aspects one of those aspects being the relationship between the mules and the blood vessels and I should point out if we look here where these Mueller cells come in close contact with these blood vessels that they reach out and form these little hands that seem to surround these vessels here okay and these vessels are not drawn to scale they actually fill out the space and you can see that more clearly here with an on-face image of the vessel here in red the capillary vessel in red this is one of these electron micrographs so again it's just the dark areas or electron vents areas and he can identify the vessel he can identify the parasite here in light blue and he can also identify the Mueller processes and he can trace those out and color coat those and here is the the 3d reconstruction of the vessel you can see that the Mueller cells form a really a nearly complete in sheathment of the vessels and we were really struck by this feature and you can see if you remove the vessel these sheets form long stretches that appear very tunnel like in nature now at it what he did I thought this was really cool but he color coded processes from different Mueller cells or different Mueller processes different colors and so this kind of gives you an a greater appreciation that the sheet is actually made up of little tiny processes from many different Mueller cells so these Mueller cells really combined to form this continuous sheath and it's of course possible that this sheath like structure is artifactual but as we look over larger and larger volumes again we see that the sheath forms a very intense coverage of the vessel in fact it's a 98 percent coverage and this was even higher than we would expect very very interesting and nearly complete and these Mueller cells are really on top of these electrically coupled parasites here and really seem to be almost shielding them from other things that are happening in retina as I mentioned it's possible that these sheets are artifactual in some way and we know that this electron microscopy technique leads to a collapse of the extracellular space so membranes between neurons and membranes between glia cells will all be touching one another when when when fixed in this sort of the classical electron microscopy manner but so we decided to also look in a in a block created by Kevin Brigman where they were able to maintain the sort of the natural occurring extracellular space in healthy tissue and you can see in these electron graphs now there's spaces between the neurons and the various processes in the tissue we can see these Mueller cells here in purple and in the capillaries here in red and when added reconstructs larger volumes of these he sees that the Mueller sheets are still quite continuous and that the cracks between individual processes are very minimal and this includes contact between processes of different Mueller cells so this really indicates that there are probably some proteins possibly gap junctions that are holding these these sheets together and in particular different pieces from different Mueller cells and this is something we want to look at more carefully with Jackie Minehart at the University of Maryland the vessels that I've shown you so far really come from this intermediate layer and that's where we know the Mueller cells are the primary primary glia cell but we also wanted to look at these connectors which which which span the interplexiform layer and connect the vessels and the superficial in the intermediate we thought well if neural signals are activating blood vessels this is a great side of interaction but what I had it found when he looked very carefully at the Mueller cells surrounding these these little connector vessels he finds a near complete in sheathen suggesting that they they might even be even that much more insulated from neural activity in the inner retina so we then wanted to look at activity in these neural sheets and to do that we're going to use this this calcium loading method that was originally shown by Eric Newman in the in the late 1990s but the short of it here this is the mouse retina on the left hand side of your screen we're again labeling the the blood vessels here in red with sulfur rhodomy and what Eric really showed is you can use these these am calcium dyes which you can wash on the retina for 30 minutes and it'll be taken up by the astrocytes in the glial cell infi but you wind up seeing really as negatives here in the ganglion cell layer we've also included DAPI which is a nuclear stain so that's labeling all the ganglion cells and the hammering cells of this layer and as I can say you can see that that the that these are really a mutually exclusive label between the ganglion cells and the calcium dyes and this really just shows that the the calcium dyes are not loading into the ganglion cells but instead preferentially loaded into the glia and this is a really cool technique and we can activate these glia network several different ways one of the ways is just to simply puff ATP onto the very surface of the retina and I'm showing that here there's a list of lists it's calcium waves in the ganglion cell layer which spread out across the vessels and along the surface if we look deeper into the retina we can see a very clear labeling of these Mueller stocks these images on the right come from the middle of the interplexed form layer so this approach is really doing a great job of low to late labeling Mueller glia cells and allowing us to image their activity while also monitoring blood vessels um again other ways that we can elicit these calcium signals in Mueller cells one of these happens to be physical injury to the retina so we're literally just poking the retina at the beginning of this video in the bottom right hand corner of your screen that's eliciting a calcium wave it's propagating out from the site of interaction we can see when these calcium waves shown in green reach one of these vessels it causes an increase in the vessel diameter and so changes in calcium in these networks can propagate and they can cause increases in vessel diameter and most of the talk has been focused on natural methods for activating glial networks and vessels and so here actually light about calcium signals observed at the intermediate capillary level and we can see when we deliver again one of these three hertz flickering stimuli and that that spot of light is denoted by the dotted yellow line it activates calcium signals in this glia network which spread out when those calcium signals reach a vessel it causes an increase in the vessel diameter and if we look at other portions of the vessel where calcium waves do not elicit a response we also see a lack of a change in the capillary vessel and so you know this is really just to show you we have a very powerful technique where we image calcium signal in these networks and we can activate Mueller cell activity in several different ways now I want to take you of course back to our sheath observations and we wanted to see if these these sheets are functional our our block face volume also only included vessels in the intermediate level and not at the deeper layers so this this functional imaging probe gave an opportunity to look at vessels and other layers and different types of vessels as well and here I'm just puffing atb on the surface while carefully looking at the areas surrounding these vessels we're looking at intermediate level vessels on the left you can see the Mueller cells are activated in green they form these beautiful sheets to surround those vessels here is one of the connectors in the middle of the IPL you can see when it's activated you see these beautiful rings and we can look at the deepest layers which actually contain arterials and capillaries and we see beautiful Mueller cell activation along with strong sheath like activity along all major vessel types and so now we have a method for looking at at Mueller sheets under natural sort of healthy conditions and we're starting to think about the disease models where this might actually change and one of the first models we're looking at with some help from one of our graduate students IE Lee is a mouse model of retinal degeneration now and retinal degeneration the photoreceptors begin to die first and as the photoreceptors begin to die the retina begins to change and one of the big features the commonly known features this this gliosis so these change in these Mueller cell morphology and sure enough as the photoreceptors die these Mueller cells change from this nice clean columnar organization they swell and they extend processes in different directions and as the disease progresses these glial cells really take over large parts of the tissue and this happens as synapses and other features are really beginning to degrade within the disease and so this is just some preliminary data from an already 10 this is a mouse model of retinal degeneration with a slightly prolonged degeneration cycle here I'm looking at P 31 or postnatal day 31 so these tissue are these these mice still have some visual function so they're really a late stage one and so we wanted to see what are the Mueller cells and the achievements really look like under these conditions well it turns out that the achievement of the the capillaries at the intermediate level and also the inner retina look largely like they do in the healthy animal but when we go to the outer retina where we know the photoreceptors are dying off we see something quite different okay so we see sort of this blobbing calcium activation and we don't really see the clean sheets that we store under saw under normal healthy conditions and just to remind you on the left here is our calcium transients in the deep layer of a healthy mouse on the right is calcium signals in the deep layer of this already 10 so we can see that the sheets although they seem to be largely intact in the inner retina are really falling apart and seem to be abandoning the vessels of the deepest layer and of course this is just one time point throughout the degeneration cycle and we want to span this out to look at more ages to see at what point she's in that in the intermediate layer begin to break apart if in fact they do and so my part one conclusions I've shown you that Mueller wrapped capillaries and arterials throughout the retina that they really combined to form continual and sheath minute capillaries and this ranks somewhere around 98 percent coverage and that these Mueller cells may contribute to the retinas blood-brain barrier at the intermediate and deep vessel layers I've also shown you some data from us and from other groups that Mueller glitter participate in hyperemia at the capillary level and I've also shown you some preliminary data that these and sheath minutes are disrupted in the already 10 model now as far as future directions we want to look at other disease we know that for example in retinal diet retinal diet diet sorry diabetic retinopathy that increases in blood glucose levels leads to down regulation of gap junctions between Mueller cells and gap junctions between parasites and so we want to see how that affects these vessel sheets we're also working with some scientists at the University of Wisconsin to collect a serial blockface um um a serial blockface microscopy data from the primate retina and this will allow us to see if in fact these Mueller sheets are present in primates and humans as well and lastly at it is looking at the the cracks in these sheets we see uh you know it's not 100 coverage of all the vessels you know we want to see what what's really present in those cracks are they neurons are the neurons of a specific type are those neurons interacting directly with the parasites blood vessels where they simply acquiring nutrients from the vessels themselves so I'd like to move on to the second part of my talk which talks about potential role for amicron cells in neuro vascular coupling in the retina as I stated this is a very diverse cell population and they use a broad array of neurotransmitters some excitatory some inhibitory and they also use extensive gap junction networks to communicate with other neurons but one of the interesting findings early on from retinal work was that when when we used antibodies against machinery involving GABA-ergic or glycinergic transmission these are both in herb-pivotory neurotransmitters that these antibodies labeled amicrons in a mutually exclusive fashion so that amicrons were labeled either for GABA or for glycine but not both they also noted in these studies that roughly 15 percent of amicron cells didn't seem to express GABA or glycine and these cells have been named non GABA ergic non glycinergic amicron cells and interestingly a very similar percentage of amicron cells also do not express these markers in the primate and so this suggests that a similar percentage of primate amicron cells also have some neurotransmitters that we we know virtually nothing about okay and one of the amicrons which we have studied in extensive detail is this starburst amicron cell and this is a GABA ergic interneuron and some recent work by Bortier Sagdelaev in New York has really implicated these starburst amicron cells in neurovascular coupling and so on the left hand side of your screen is one of those starburst amicron cells they were named based on their beautiful symmetric morphology those starburst amicron cells released GABA and acetylcholine onto the direction selected ganglion cells and it's these actions that are involved in the in the encoding of directed motion but what these guys found is whenever they activate starburst amicron cells in this case in this panel see here by expressing white activated channels or general adopts in the starburst cells they could see a relaxation of arterials and capillaries in the retina so when they activate these starburst amicron cells and I'll call these sacs for short they saw an increase in arterial diameter and an increase in capillary diameter and when they just bath applied acetylcholine receptor antagonist agonists sorry they also saw a similar effect a relaxation of vessels both arterials and capillaries so this really suggested that a single population of amicron cells could be involved in neurovascular coupling and they hypothesized that the sacs are releasing acetylcholine which is in acting directly on the parasites or on the endothelial cells and it has been shown that these two cell types do express acetylcholine receptors however this our work from the first half of the talk and some previous work from the same lab has really shown that that Mueller cells seem to in sheet these vessels even as they traverse the inter plexiform layer and here on the right hand side is data from this Ivanova 2014 paper where they labeled the starburst cell in green the blood vessels here in red and they've also labeled Mueller cells here in white so you can say that the Mueller cell actually creates these fine sheets or processes that really seem to separate the starburst amicron cells from the vessels itself so it's quite possible that these that acetylcholine is not acting directly on the blood vessel walls of the parasite but is instead interacting through Mueller cells and treating them as an intermediate in this pathway well this led me to to look for saccabote calcium signals in Mueller cells so again I'm using the same flow for glial loading protocol that Eric Newman developed in late 90s and you can see all these Mueller cell stocks here in yellow in the middle of the inter plexiform layer and now I brought an electrode in the retina and made a whole cell patch for clamp reporting from only a single sac here and the other thing I want to mention is the sacs are in very high density so anywhere we look within the retinal tissue there are dendrites from roughly 70 overlapping sacs that are covering that area so while I'm recording from only one sac there are many sacs covering every inch of the retinal surface okay so we decided just to just to cause activity in a single sac to see if we could evoke calcium signals in the Mueller glial and that's what I'm showing you here this top trace here this is the voltage command that we're delivering through the patch electrode to this starburst amicron cell here in purple and then we're monitoring calcium and all these different Mueller stocks some of which I pointed out with the yellow circles here or the white circles here these are the individual delta f over f responses of the change in calcium signal coming from each one of those spots and below here is the average response at all these spots so we can see that when we activate a starburst amicron cell that we are able to activate these Mueller's and that the Mueller's seem to be in general integrating this calcium signal over the duration of the sac stimulation and so this is this is really up to the date on where we are on this project but of course the next stop is to use neuro pharmacology to separate out these possible pathways so the the updated model that I'm showing here is that these sacs or these starburst amicron cells are actually co-releasing acetylcholine and ATP and it's been shown that ATP is co-released at cholinergic synapses I also showed you earlier the ATP is a very effective activator of Mueller glia and so that one or both of these neurotransmitters could be activating Mueller cells which is then changing their processing of these arachnodonic acid pathways which is altering the parasites and causing a dilation of the capillaries and these pathways are indeed modulated by nitric oxide so this is another level of modulation on top of these pathways and Eric Newman has shown that when nitric oxide is high that all of the vessels in response to the light exhibit constrictions and that when nitric oxide levels are very low that all the vessels exhibit dilation so nitric oxide seems to shift the balance of these pathways from dilation and constricting but what we really believe again is that the amicron cells are working through the Mueller's as an intermediate and ultimately the neuropharmacology is going to help us pick apart these two pathways so what about other amicron cells well as I said 70 percent of the amicron cells we know virtually nothing about their function and in an attempt to dig deeper into this unknown set we use the serial blockface data sets and just simply rank the amicron cells found in that data set by density so how many cell bodies for a particular amicron cell type is found within the given area of retinal space and when we sort those amicron cells by their density we can see the largest density is of the A2 amicron cell and within this top 10 are also the starburst amicron cells and the MAC and I'll tell you a little more about why we are calling this amicron cell the MAC is the second most densely expressed amicron cell in the mouse retinal and this is the morphology these cells found in this blockface data set now we know really nothing about the neurotransmers that this cell expresses and some data I'm going to show you actually puts it into this NGNG category but luckily due to the unique morphology of this cell we were able to scour the Genset bank which has a whole array of transgenic mouse lines and we were able to find a particular line that seemed to label an amicron cell with morphology that looks similar to this MAC and sure enough this this mouse line does fluorescently label the MAC here is one of these MACs shown here this is a DAC2 GFP line you can see it has this single process that comes down into the interplexiform layer with this bushy arbor and even though we are we are studying this cell in mouse there is a very similar amicron cell found in the primate retina and this is observed from some of the earliest work in the early 40s so this amicron cell does seem to be present in both mouse and primate we know virtually nothing about it we did use some of these classic antibody labeling for mechanisms involved in inhibitory transmitters GABA and glycine and we find that it doesn't seem to label for either of these either of these markers which really does put it in this mystery category of non GABA hergic non glycine hergic and in this in our in our work we go on to look at more detail of what neurotransmitter these cells are actually using but I want to point your attention to another finding from this work and that is when at it very carefully looked at these at the ultra structure of these MACs and their association with the Mueller cells and this is one of these Mueller cells in red and these are the dendrites of a MAC here in teal what they found was that these Mueller cells really wrap the MAC processes and in some cases they even form complete in sheathments okay and here is just one of these MACs and you can see that it has a total of six in sheathments and these sheathments are coming from two different Mueller cells four of the in sheathments are coming from one Mueller cell in red and two of the in sheathments are coming from another Mueller cell in yellow and this seems to be a feature that's fairly specific to the MAC now of course we want to look at how the MAC talks to these Mueller cells and our first approach was just to simply inject small tracer molecules into one of these MACs and to look at the diffusion of these molecules into any coupled cells and this is one of the classic approaches for to look for gap junction between pairs of neurons or pairs of glial cells and what what we found here is when we injected one of these MACs with neurobiotin in this case that neurobiotin we found diffuse into approximately two neighboring Mueller glia cells and this is indicative of direct coupling between the two cell types we also included another molecule lucifer yellow which does not pass readily through gap junctions and this tracer molecule was really restricted to the MAC itself so again this really supported the notion that there is direct coupling between the amicron cell and the Mueller glia and this is what led us to name that this cell the Mueller glia coupled amicron from these tracer coupling experiments we found Mueller cell labeling in all 14 of our injections and an immediate of two Mueller cells per injected MAC which is completely consistent with our electron microscopy reconstruction we found some inconsistent labeling of wide field amicron cells but this is something we did not explore further but if you'd like to learn more about this new amicron cell including information about mouse lines that allow targeting of these cells along with some antibodies that label this cells I'd encourage you to look at this paper we published earlier this year in the Journal of Neuroscience and so from the second part of the talk I'd just like to conclude I've been talking about amicron cells and their potential role in neurovascular coupling I started by telling you about the sac the starburst amicron cell and evidence that can I can actually drive vasodilation of capillaries I've also shown you some preliminary data that sacs do elicit calcium signals and Mueller glia stocks in the inter plexiform later and at the moment it's currently unclear if the sacs act directly on the blood vessels and parasites or if the Mueller glia are acting as an intermediate and that's one of the key questions that we're going to be focused on moving forward I've also told you a little bit about a newly discovered amicron cell the MAC which seems to be heavily in sheath by Mueller glia and this is an unusual feature and in fact neuron glia coupling has to my knowledge never been described in the retina in the mammalian retina before now and I've also shown you that there's dye coupling between these MACs and Mueller cells that suggest that these two cell types are actually electrically coupled and in the future direction we want to ask if these these sacs are really using as I said the Mueller cells as an intermediate and does the activation of these Mueller cells depend on acetylcholine or ATP or both we're also looking at the possibility that MACs activate Mueller cells at these at these local and sheathments or vice versa and and do these MACs actually participate in the hypervenia function and we'd also like to look at what nitric oxide sources are modulating the dilation constriction balance and we know that there are at least three sources of nitric oxide in the retina neuronal nitric oxide inducible nitric oxide and an endothelial nitric oxide and so we really want to see which of these sources if not multiple of those sources are contributing to regulate these functions and I'll also highlight that there is an amocrine cell that indeed releases nitric oxide so it may be another amocrine cell that's involved in this process and in final conclusions we've really I've really found that the retina is this beautiful compact system for studying neurovascular coupling in her right hypervenia and it really represents a mini brain in my in my mind that allows us a much more compact system to study more complex phenomenon the evidence that we show suggests a mule glia and parasites both part of the retinal blood interface contribute to dilation and constriction of vessels that Mueller and sheathman is near complete and is altered in disease and the amocrine cells likely play a role in hypervenia and use Miller glia as an intermediate and with that I'd just like to say thank you to those involved in the work I've shown data from the individuals here in red and we also have a long list of collaborators that have contributed to these projects and I'd also like to to point out Rinalini Hoon and Ron I've sent out the University of Washington that are working with us to acquire this serial blockface data from the primary and I thank you for your attention okay well thank you very much really nice talk really impressive the amount of data and new findings so as a host I would like to ask a very you know for me a question that relates a bit with my field that is a circadian reasons I would like to ask if if if you know if these interactions between amocrine cells and Mueller cells are modulated by circadian mechanisms or what do you think the the short answer I mean this is a great question Jose and it's not something we've looked at yet we we definitely do think that circadian rhythms could play a role in the coupling and the hyperemia response we don't have any data to show that we certainly find that at the as we've begun looking at some of the cracks of the vessels at the intermediate layer we do see some M1 retinal ganglion cells so there do seem to be some melanopsin containing ganglion cells which are involved in some circadian rhythm behaviors that seem to be present at these cracks so it's possible that they you know could be signaling information directly to the blood but they could also be picking up circadian cues from the bull and in particular you know dopamine nitric oxide other things should certainly be playing a role in these sheath units great question okay so I'll go to the live chat so could I ask if you could stop to to show your screen so we can okay all right thank you very much so from the from the audience from professor Marla Feller she asked if do all the lateral processes that you show in the first part of your talk go to the basals and if a subset of lateral processes are if a subset of lateral processes anything there's this thing about them sorry I didn't catch the last part about that so I will repeat the question so if prof Marla Feller asked if do all the lateral processes go to the basals that's the first part of the question and then if a subset of lateral processes anything else distinct about them yeah yeah yeah well as added showed and Marla has shown as well there are ton of fine processes that extend from the Mueller cells within the interplexa form layer and when there's a vessel nearby these processes do seem to reach out and make contact with the vessels so if there's a vessel nearby it's likely to reach out and grab it but many of these processes are not making contact with vessel and and instead these guys seem to be surrounding synapses and also in she things some of the amicron cells one of the example I showed today is really the Mac so you know the Mueller's are you know creating substantial interactions with neurons in the interplexa form layer and substantial interactions with the vessels whenever one is sort of nearby or available okay thanks so in the same way continuing with that question Marla also asked if do other lateral processes localize to synapses or some other not neural structures yeah and that's kind of what I was showing in the latter part of the talk that that some of these processes just completely wrap this Mac cell for example and when we first looked at the electron microscopy we expected to see synapses you know but groups of vesicles maybe electron dense membrane regions that would be indicative of chemical synapses but much to our surprise we did not find chemical synapses at these points of contacts between the Mac and the Mueller cells and so at first they were a real mystery when we started in trace injecting the tracer molecules that's when we begin to get some evidence for direct coupling between the two cell types and we think that that is probably happening at the in sheathments but we our data sets don't have sufficient resolution to resolve the gap junctions themselves so you know in short some of those processes are definitely associated with neurons in some cases they're you know completely wrapping those neurons but there aren't always clear chemical synapses at these sites in action okay so um from David Berson uh hey Will beautiful work regarding the suck influence on vesicles do Mueller cell express cholinergic receptors ah yeah so this is uh this is a great question and Marla has published some work on this recently that shows in development uh Mueller cells definitely express uh cholinergic receptors although it would appear from their data and others that these cholinergic receptors are really down regulated in adult so it doesn't mean that that the cholinergic receptors are entirely gone but they aren't necessarily expressed as high levels as they are during development so uh you know there's evidence that those mechanisms are probably there um but ultimately we are going to test this signaling pathway directly and really try to get to the bottom of this question is it pure allergic activation of Mueller cells cholinergic activation of Mueller cells or is there really a direct interaction with the parasites in the vessels okay all right so i'm gonna go to the chat if there is any other question um um so from Evelyn Sernagore any idea what's happened during neonatal cholinergic waves uh yeah again this is something that Marla Feller uh can speak to much more directly than i than i can certainly those uh retina waves are activating the Mueller cells quite strongly um so i imagine they are playing you know some roles in the in the development of the the circuits and maybe the establishment of the vasculature that's something that that we really haven't begun to think about yet so i really don't have any data to talk about today but i really would point you to some of uh Marla Feller's work recent work on the topic yeah we've certainly been too focused on uh neuronal development and i think you're right this is going to be a really rich area to look at vascular development so thanks for all the plugs well so okay so can i we got one more question from the chat so Maria Kosan amazing talk and beautiful work i noticed that the basal diameters appeared much smaller in the rd10 disease model compared to the healthy retina is this related to neurodegeneration in the retina it's a great question um you know it's uh i haven't extensively looked through the different vessels at the outer retina you know this is just a couple days of preliminary data uh it's exactly the type of thing that we can quantify with this approach you know in that deep layer there are arterials and capillaries whereas the intermediate levels there's really just capillaries but in the disease certainly those sheaths seem to be largely breaking apart in that outer retina and some of the vessels we're finding are are indeed smaller this will be a great opportunity to look if you know when hyperemia really begins to sort of die out in these degenerating models we can see when these sheaths begin to sort of break apart at what ages and of course you know there may you may be able to find some way to intervene with the the degradation of these of these in sheath vents and changes in the vascular we certainly know that in disease there are a lot of associated changes with vasculature so it's quite possible that those vessels are are different up there than they are under normal healthy conditions it's a good question okay i i just want to say that this as i this is really all a new direction for me so i really appreciate everybody's questions and feedback and and this is really helping to to fuel some of our future directions decide which way to go with some of these new findings so i just want to thank again everybody for their questions and field please please contact me with any additional questions and thoughts you have on the topic okay all right so thank you one okay i think we are done with the with the question so the post if you want to continue discussing some of these ideas please join to the Zoom discussion we are already with with Jeffrey and adit so yes we will stay online for a few minutes so feel free to to join us okay thanks everybody for attending to our seminar and okay see you next time thanks oh nice nice talk thanks a lot so it's really exciting a completely new new focus so i'm just curious about why why you decided to to explore more in detail the vascular activity uh i don't know is what what was the main motivation yeah yeah uh you know i've been an amocrine cell enthusiast for a little while now now that we're off the youtube i've been an amocrine enthusiast for for quite a while now and i i you know was looking for new amocrine so i started studying that the mac and looking at the mac and we found these really interesting specializations and association with the Mueller cells and we were really struck by this and it led us to look at the ultra structural Mueller cells it led us to look at the interactions with the blood vessels and so adit really took off on the Mueller ultra structure and we just went to both ends of the cell you know what are the how do things look like at the interface with the neurons and what do things look like at the interface with the blood vessels and uh so it's really the science and the discovery of those in sheathments that fueled a lot of these other questions and ultimately we're we're really excited about it and i think it opened up the possibility that there's really a combination of amocrine cells that are really working in concert to influence and possibly even control hyperemia so um i think it is an exciting kind of set of questions and new direction uh i'm really you know new to this field uh there's a couple people that have done some work on the neuron glia interaction side of things but it it definitely seems like there's a lot of nice open questions in here you know that we have some of the tools we need to to begin asking some of those questions so we'll just one point will you are still on youtube this is a live stream so no yeah i we've got i just really conspiracy talk and stuff and don't say anything bad about lian oh hi lian hi lian all right so yes i i i will i will cut the the stream now so that's um so yes so i will end up the stream so we can continue by soon thanks everybody thanks jeff