 And hello everyone and welcome to another virtual vision web seminar. We are still very happy to see you all with us every week live. And we're going to have to continue in this format for a couple more months. So as usual, I would like to remind you to take a look at the world by the website where you will find not only the all upcoming and previous talk, but also what seminars from different neuroscience fields topic. So just take a look and we'll find sure topics that will fit your study. So today we're very happy to receive Karen Dedek from the University of Denver. Karen attended a PhD from the very same university under the supervision of Thomas chance and she continues there through her academic career. And her main interest resides on the analysis of fraternal signals processing on the similar network and be available. Currently she's working on three major topics, the functional and molecular analysis of electrical synapses. The study of secretory and functional role of fraternal internal rooms. The secretory of light dependent magnetic reception in a bird retina. And I guess that we all hope to hear a little more about this last topic today. So have a category. Hello Karen. Sorry. How are you doing today? I'm fine. Thanks Maxime. Very welcome. The stage is yours if you want to start sharing a screen. Okay. Thank you. Hi everybody. Just start sharing. So I hope everybody can see my screen now. It's perfect. And the pointer. Actually, thank you very much Maxime for the nice introduction. I have to kind of disappoint you a bit because it will be about bird retina, but not so much about magneto reception. I will allude a bit to it. But today it's more about the bird or about the retina itself, I have to say. So yeah, it's really an honor to speak in this series and I'm very happy that you tuned in to hear more about the bird retina. And in the following 45 minutes or so I will hope to convince you that the bird retina is really a fascinating topic to study. And I will start out with giving you some reasons why I think this is the case. And then I will cover briefly the structure of the bird retina and oivian photoreceptors before I show you our recent results on bipolar cells of the bird retina of the chicken. And then I will discuss transcription factor expression in the ganglion salea of the buzzard retina and the pigeon retina. So let me just start out by why I think the bird retina is interesting. Of course, the chicken retina has been a nice model for retinal development and also for myopia, but much less is known about the different cell types and their connections. Although one has to say that the birds are really highly visual animals. For example, the pigeon is a valuable model for visual attention and object categorization. And everybody knows, of course, about the superb vision of raptors. But not only the raptors have a superb vision, but also these little pacerines that fly through bushes have a superb vision because they are catching flying insects with their tiny beaks. And so these are really highly visual animals. And that makes them, of course, an ideal model to study the retina. The enormous importance of vision for these animals is also reflected in their enormous eyes. They cover large parts of the cranial volume that's shown here for the pacerine. You see the two eyes that occupy large part of the cranium here. And I once brought you a plot from a recent review from Tom Barton's lab where they plotted eye weight against brain weight. And also from that, you can appreciate that the chicken has almost a similar weight in eyes and in brain. Once again, kind of strengthening the point that these animals are really highly visual. We are also very visual, but our eyes make out much less of the cranial volume as you all know. When we compare the visual capabilities of birds, we can take a look at visual acuity. And there is also very high in many bird species. So visual acuity here would be more or less spatial acuity. So the capability to resolve fine spatial gratings or fine details. And many raptors are way better than humans at these high spatial frequencies and many bird species here are better than the typical mammalian. Species like the cat or the mouse. So these animals, these birds have really a superb vision, not only in spatial resolution, but also often in temporal resolution. So vision is a lot faster for birds than for mammalian species. The high visual acuity here is a function of the fovea. So many bird species possess retinal fovea and some diurnal raptors even possess two foveas like the buzzard here. These are images from the Gahafa camp with whom I studied the buzzard and the pigeon retina recently. And here you can see the two different fovea of the buzzard with the deep central one where the fovea lacks the ganglion cells in the center. And the shallow temporal fovea where it's more like a dent in the retinal surface and where there are still ganglion cells. But of course having a fovea retina makes a nice link to the mammalian species that possess fovea and these are the primates. So there's also from Zilke here an image from the macaque fovea with some ganglion cells still interspersed in the fovea pit. So these birds are highly visual, they have very superb vision, often superior to mammalian species and they even possess fovea. So I think they are very good comparative model to take a look at. And also many birds, the birds have very different lifestyles and foraging behaviors, some feed on seeds on the ground and some catch their prey in the air. And this also reflected, it seems in the retina structure and that's of course also very interesting to look at the different adaptations to these ecological needs. And finally, a little bit related to these ecological needs, there's another reason to study the bird retina and that is magnetoreception. So you might know that nine migratory birds are able to use the earth magnetic field to orient and one of these examples here is the European Robin and these animals are able to travel between their breeding and wintering grounds and cover distances of hundreds of kilometers and they do that at night on their own. And they use the earth magnetic field for orientation, namely the magnetic inclination angles that vary systematically from the equator towards the poles. And this so-called inclination compass was shown to be light dependent, wavelength dependent and also seems to involve green areas that process visual information. Therefore, we believe that it really starts in the retina and that's why it's also interesting to take a look at these birds and you will see in the talk that once in a while the retina of European Robin appears as a comparison to the other models that we use. So let's take a look now at the structure of the bird retina. Of course, it lines the back of the eye. The bird retina does not contain any blood vessels, but instead it developed a structure that's called pectin. It's a comb-like structure here that protrudes into the vitreous and that serves to provide nutrients to the retina. It's part of the coroid and also to maintain the pH here in the vitreous body. And the retina itself of course has the typical layout of a vertebrate retina with three nuclear layers here in the outer nuclear layer, inner nuclear and ganglion cell layer and two plexiform layers, the outer and the inner plexiform layer. And as usual, you all know that the photoreceptors convert the signal, the light signal into an electrical signal, pass it on to bipolar cells. From there, the signal goes to ganglion cells which form the optic nerve with the axons. And on the way through the retina, the signal is modulated by two classes of interneurons, the horizontal cells and the amacrine cells in the outer and inner retina, respectively. When we compare this retinal structure between mouse and chicken here, I just put two slides here, or slices here into the middle that I put to the same scale. Then you see that the bird retina is much thicker than the mouse retina. It contains many more interneurons, also many more photoreceptors and also usually two rows of ganglion cells at least. But we will see later that raptors also have five or four or five rows of ganglion cells stacked on top of each other. So there are many, many more cells inside these bird retinas. Also in terms of photoreceptors, the bird retina is more complex, sorry. It contains six types of photoreceptors, that is, one is the rods, and we have four types of single cones, and it also developed one type of double cones. The single cones come in different varieties. Some species contain UV sensitive cone, whereas other species contain a violet light sensitive single cone. And the others are common to all bird species. They are blue, green, and red cones. And in contrast to fish, the double cones of the birds contain the same option that is the long wavelength option. So these are kind of red cones. Like reptiles or some red tiles, the bird retinas contain or the photoreceptors contain oil droplets at the end of their inner segments. And these come in different colors. So that's transparent cyan, yellow, red, and pale oil droplets. And here I tried to identify them in this transmission image that An took some years ago. And here you can clearly see the yellow oil droplets, the red oil droplets of the chicken retina, a transparent one. The pale ones from the double cones, there are also rather large oil droplets, and maybe this is a small one here as a cyan colored oil droplet of a blue cone. These oil droplets serve to kind of long path filter the light. And so this shown you about this absorptance plot, just for the single cones. You see the cyan, the yellow, and the red type and absorptance plotted against wavelengths. And you can see that these oil droplets let pass or wavelengths that are on the right hand side of these curves. And when one compares the relative sensitivity of the absence. In, yeah, when you measure it with all droplets or without, then you can clearly see that the oil droplets kind of shift the wavelength sensitivity to longer wavelength. Yeah, that's these solid lines now with the oil droplets and the peaks you get also smaller and more narrow. And so that's the function of the oil droplets really do sharpen the peaks of these photoreceptor pigments. The photoreceptors of the bird, they are try stratified more or less or not that's wrong. And so the outer plexiform layer is try stratified it's not the individual photoreceptor that is try stratified but the layers in the outer plexiform layer. They are quite well defined. So here I show you I'm standing there did from chicken retina labeled for kalbindin and PSD 95. And kalbindin labels the double cones of the chicken retina and PSD 95 labels the outlines of the bird photoreceptors. And what we can see is that the double cones and here tiny one the rods that they stratify in the most distal part of the outer plexiform layer. The red and green cones that we cannot distinguish here stratify in the middle and the blue and violet cones that we also cannot tell apart stratify in the third and most proximal layer of the outer plexiform layer. As also shown here in the whole mount view. Again here we take a look on the double cones that stratify in the most distal part. So here you see the primary the principal member of the double cone and the accessory member of the double cone. Here are small profiles for the rod photoreceptor so this can be nicely discerned in these whole mounts and in the middle we have the green and the red cones that we cannot distinguish. And in the most proximal part we have the violet and blue cones that we cannot distinguish, but you can also see that they are lost a lot less common. They have the lowest number in the bird wing. These photoreceptors also have quite a number of telodendria that's shown here in this photoreceptor from the chicken retina that I injected and this can also be seen here in this image from the where we also see a lot of these telodendria protruding from the different photoreceptors. And when I added connection 36 labeling to them you can also see that connection 36 is expressed yet the crossing points of these telodendria or the junctions of these telodendria and so connection 36 seems to also in the bird couple the photoreceptors amongst each other. And it will be interesting to see what is the dependence of the coupling and maybe also how are these gap junctions regulated and what might they, what might their function be. Let me now come to the bipolar salt and their connections to photoreceptors. This is a work that was mostly done by ania grunter from our lab and it was a collaboration together with Silke alpha come Stefan years and Kevin Brickman and Henry mo Hudson here from Oldenburg and Silke alpha come Stefan years and Kevin Brickman of course from the Caesar Institute in bond and the question that we wanted to answer was so if we want to really understand the signal processing in the bird retina then of course we need to know in how many different parallel channels the visual signal is split in the outer plexiform layer and so how many bipolar cell channels do we really have that send the signal from the photoreceptors to the ganglion cells. And when we started out. What was available were some catalogs from the chicken and pigeon retinas or the pigeon catalog was provided by Mariani. And this one is from constant Kassada and both were from the late 1980s. We did some Golgi impregnations and found 11 different types of bipolar cells in the chicken retina and could define them. Major specification based on the morphological appearance of the cells. So what we did was that we use the serial electron microscopy stack to reconstruct bipolar cells and photosceptors. The one week old chicken retina and the part that we looked at was a dorsal peripheral part of the retina. The z-stack had a rather limited the stack sorry the stack had a rather limited the dimension. So it was rather thin but still it allowed us to or Anja Grinta to reconstruct many bipolar cells from the stack and also photosceptors. In total Anja reconstructed 146 different bipolar cells and 74 bipolar cells were complete. And so meaning that their dendrites and axon terminals were completely within the stack and 72 bipolar cells were present only partially. And then we group the cells into 15 different bipolar cell types based on morphological criteria. And so for example we looked at the Soma position at the dendritic stratification here. And at the stratification of the axon terminal system and you can see as in fish also the turtle the cells are multi stratified and not mono stratified like in the mouse right for example. We sorted the cells according to their to the length of their axon and so found these 15 different types that stratify in the inner plexiform layer. And to really look at the stratification you also had to decide how many layers we assigned to the inner plexiform layer. And there was eight in in this case that was based on a study published by Chen and I to where they injected individual ganglion cells and made a ganglion classification of the chicken retina and so we adopted this equidistant layering for the inner plexiform layer. So in total we found 15 different cells. Some cells were more common others were a little bit less represented in our stack. And then in addition we looked at the photoreceptor connectivity and I already explained to you that the photoreceptors of the bird have this nice stratification pattern with the double cones and the rods and the more distal parts of the outer plexiform layer and the violet and blue in the most proximal part and the green and red cones in between and this. And you could also find in the reconstructions of the photoreceptors. So here we take a look at the rods from the bottom view at the principal member and the accessory member of the double cones and the green and red cones that we had lumped together and the blue and the violet cones that we had to also put into one group because we could not distinguish between the two types, but we could at least distinguish between green and red versus blue and violet based on the stratification level. And this also shown here where we again see the rods and also the double cones stratify in the first layer, the green and red cones in the middle and the blue and violet in the most proximal part. In total, Anja reconstructed around about 700 photoreceptors and rods, double cones and single cones had about equal shares at this retinal position. That also meant that 50% of all cones were in fact double cones. When we now add this photoreceptor connectivity to the bipolar site classification, we get something like this. And here, I would just like to draw your attention to the connections to the rods. So most cells really contacted rods and when they contacted rods, they also always contacted other photoreceptors, so we did not find a cell that only contacted rods in the avian retinal. Many cells that contacted rods also contacted either the principal member or the accessory member of the double cones. So they were very often contacted indicating that they are probably more important for luminance detection and that they do not provide an additional color channel or something like that. The only more specific contacts and single cone contacts were found in type 9 and 10 bipolar cells type 9 contacted the green and all the red cones, you cannot tell, and the type 10 contacted the blue or the violet cones, or the blue and violet cones. Most likely, because we have these many cone types that we cannot tell apart, most likely we are also missing some bipolar cells that might have even longer dendrites than these ones here. And then the dendrites will run out of the volume and we might just have missed these types. And so we think it's likely that there are additional wavelength specific bipolar cells that target single cones specifically and that these might be missing from this list here. And I also looked at the response polarity or at least the putative response polarity because we cannot resolve, of course we cannot resolve that with electron microscopy so she looked at the types of contact and of course you know that in on bipolar cells. They get excited by light increments and form usually ribbon synapses and of bipolar cells, they get excited by light decrements and form basal contacts. This, because we found so many basal contacts in the avian retina it seems to be a little bit less clear. And this distinction. And was also a reason why I decided to divide the basal contacts into three further groups. The red associated contact that means, should have explained this maybe that the ribbon synapse is shown here with the ribbon and two horizontal silent rights and one invaginating bipolar cell contact so that would be a ribbon contact here, and the basal contacts are here at the base of the circle of the cone. And they were divided now into ones that are very close to the ribbon, and others that are not tried associated and then might be further away and with these middle and marginal non fried associated contacts. And when one does this one can try at least to get a handle at which type is an onset and what type is an officer. Let's just take a look at two example bipolar cells. So here's the type seven bipolar cell. We had four examples here in our data set. And these cells make basal contacts with rods and the principle member of the double cones and also with green and or red cones, but none of these basal contacts were really ribbon associated or try it associated. And that's why we think that this is a putative officer contrast we have here the type two cell. And it made many ribbon synapses to the rods and the principle member of the double cone, but also some basal contacts but these were, at least for the principle member mostly tried associated so that's why I believe we would assume that this is an office. I'm sorry that this is an onset. And I also looked at this a little bit further and use GNB three as a marker for on bipolar cells that was based on the publication rich yet in 2010 and GNB three labeling here completely. And it coincides with the PKC labeling that was used here to label individual bipolar cells. And GMB three also divided the inner plexiform layer here into these kind of half small less. And the GNB three labeled cells also expressed GNB three and they are therefore most likely on cells, but they could went a step further and looked only at the inner plexiform layer now and at this division here. So the green we are again see the GNB three. And when she looked at the synaptic ribbons that do not coincide now with the GMB three labeling and these synaptic ribbons must belong to off bipolar cells. And that is indeed what she found that in this lower half of the inner plexiform layer we also find synaptic ribbons that most likely belong to off bipolar cells. And conversely, we also find GMB three labeled CDB P two positive terminals here in the kind of off sub laminar of this inner plexiform layer. So this very, very clear division is definitely not true for the avian retina so off bipolar cells can also stratify in the more proximal half of the inner plexiform layer and on bipolar cells So this seems to be a little bit. Yeah, maybe a bit difficult to really assign the responsible polarity just based on on the stratification layering so I think we need to learn a little bit more about that. In summary I've shown you that we have 15 different types of bipolar cells identified from this serum stack. Several types might be missing because we had these long dendrites running out of the volume. And, yeah, for the response polarity we definitely need of course in the end the physiology to to solve that puzzle. And why we have been working on that. Yamagata at us or Yamagata in the Saints lab reported a chicken salad last where they found 22 different bipolar cell types based on transcriptomics, and they claim that there's putatively one on off type 10 on types and 11 off bipolar cell types. So, it might be that we still missing seven different types. And it's definitely still a little bit open, which type is on from these cell types and which is off for these ones we can be pretty sure this might be an onset and this year might be enough. So what's next. So we are trying now to correlate the classification with the bipolar cell markers and also try to look at different species to see how conserved the morphologies are and also the connectivities are. And he's just one example more or less how we do this we also use I led one as a marker for on bipolar cells that's also based on a publication from Richie et al in 2010. And also co label with GMB three as I said earlier, that also a specific for on bipolar cells. I led does not only label on bipolar cells that would be these cells here but it also labels horizontal cells here in the outer retina putative starburst our green cells, also in the ganglion salea and also ganglion cells. When you compare these two retinas here the pigeon with a chicken, one can nicely see that they look very similar. Also the lifestyle might be rather similar because both animals pack or both species pack kind of seeds from the ground. When we take a look at the European Robin retina, then we can see that it kind of has a much higher cell density. We see many more rows of ganglion cells we see the horizontal cells labeled and the putative starburst immigrants that's labeled, but we also see a whole bunch of on bipolar cells here labeled. And that's also true for the GMB three stain. So this clearly looks a little bit different between the two species. But yeah we see the enormous identity here of the Robin. And of course I used markers for bipolar cells that that I knew from the mouse retina. One example is shown here cause any lean, you might know it was type four bipolar cells in the mouse. And unfortunately the marker is not as clear as for the mouse retina because it labels several different types of bipolar cells. In this case here it seems to label one on type that is also positive for GMB three, but also some of cells that are negative here for GMB three. And when I compare now this to the European Robin. At least the markers seem to do some similar things so the overall image looks quite similar. And also I can find cells that are causing in positive here and also GMB three positive. Now also can find cells that are causing in positive but GMB three negative so they are offsets. Some markers does seem to label more than one cell type that's also true here for HCN one. Now it's combined here with eyelid one. Here the reddish ones are the horizontal cells, but then you see that Asian one is also labeling quite a bunch of bipolar cells on bipolar cells. And of course one would wish for some more specific markers and one that we found is EGFR that labels a single class of bipolar cells here in the Robin retina and this class is an on cell because it coincides here nicely with the eyelid one. Interestingly enough I bought this as a horizontal cell marker based on the Yamagata paper. It was mentioned there as a marker for horizontal cells but somehow it turned out to be a beautiful bipolar cell marker so I'm still happy to have ordered it. And yeah this is just intended as a kind of an outlook what we are going to do now with these classifications so we are trying to find markers and trying to correlate that a bit. And trying to kind of also compare the different bipolar cell types across the different bird species and see how consistent are the morphologies and also connectivities and yeah what we can learn from that. With that, I would like to come now to the last part of the talk and that is on transcription factor expression in the ganglion cell layer. This is again a collaboration with the Gava come from the Tsaisa Institute in Bonn and we looked here at the pigeon and buzzard ganglion cell layer. And you might know that the ganglion cell layer of the vertebrate retina does not only contain the ganglion cells but it also contains the displaced amacrine cells. And especially if one is interested in cell densities and that's of course an important distinction to make. Also the inner nuclear layer of the retina does not only contain the amacrine cells or the proximal half, but also displaced ganglion cells which I did not depict here, but you will see that we can also identify them in the bird retina. So we compared the buzzard retina and the pigeon retina and wanted to know whether we can use transcription factor expression to kind of tease apart a little bit the cell types. There was base on many, many studies from the mammalian retina that use this approach. And for example, amacrine cells in the mouse retina can be labeled with AP2 and on-off direction selective ganglion cells were shown to express ZP1 and ZP2 to other transcription factors which regulate gene expression in the retina. And so we use the different, different markers. Here I show the buzzard retina that all buzzard staining were done by Zilke here. And we label for FoxB1 and FoxB2 which looks rather similar in the bird labelling amacrine cells and displaced amacrine cells and ganglion cells. We have already seen and now you see here that it looks like the robin retina. So I really think that the European robin has a rapture like retina in terms of cell density and so on and the thickness of the ganglion cell layer because also here in this rapture we see at least five rows of ganglion cells in the ganglion cell layer. ZP1 and ZP2 were ganglion cell markers only that was also expected. And one surprise we had when we used AP2 it was supposed to label all amacrine cells no matter where they, where the Zoma resides, but we only found AP2 in the inner nuclear layer but not in the displaced ganglion cell layer in the buzzard. There was different in the pigeon here you see the errors are pointing to some displaced amacrine cells that were labeled with AP2 in the pigeon retina. The rest of the staining I won't go through them all here were similar to the buzzard. But ZP2 did not work for the pigeon so we labeled for CDBB2 and found that it does not only label the ribbon synapses but that it can also be used to visualize displaced amacrine cells here in the ganglion cell layer and also the amacrine cells in the inner nuclear layer. We then took a deeper look at the buzzard retina to see whether really AP2 is indeed missing out on the displaced amacrine cells. So that's shown here. I think I labeled the buzzard retina for AP2 and chat so the Julian attitude transfer race that's a marker for putative starburst amacrine cells in the bird retina as always and we have here the off starburst cells and the on starburst cells. And you see that the on starburst cells do not express AP2 in the buzzard retina. That's in stark contrast to the pigeon retina here I did a whole mountain staining with AP2 and chat. And you see an almost perfect overlap of the two label links. And only once in a while we see an AP2 labeling without a chat. These are displaced amacrine cells that are not putative starburst amacrine cells. And it does not recognize displaced amacrine cells in the buzzard. So we needed another marker if you want to really look at cell densities and tell apart the ganglion cells from the displaced amacrine cells. And, yeah, as I said we found that in CDB between. And it's just the confirmation that you did. So labeling with CDB between will indeed label here the putative starburst amacrine cells now, no matter whether you look at the central retina or at the peripheral retina of the buzzard. And now we can distinguish between the two types of cells the ganglion cells and displaced amacrine cells, and we can now count the cells. We did that along the temporal nasal access. And that should be shown here, and label the retinas for ZB1, FoxB2, DARP and CDB2 and then count it. And maybe we just focus on this plot here, where we plotted the number of retina ganglion cells and the displaced amacrine cells against the retina position here along this temporal nasal axis. And at zero we see the central fovea and here the temporal fovea and here the ganglion cell densities can be really, really high after 50,000 cells per square millimeter. And then they drop down rather sharply. Here they rise once more to the temporal fovea and then drop down also quite sharply. They even drop below the number of displaced amacrine cells at the very edges here. That's a bit different in the pigeon. We also looked there. There the retina ganglion cells are always more numerous even at the edges than the displaced amacrine cells. And here in the central fovea we find peak densities of 35,000 cells per square millimeter. And this is very close to the values that Pauli and Bingley got when they looked at the pigeon retina and counted the axons of the optic nerve. Another study by Kirubin, they claimed that there are more than 100,000 cells in the pigeon retina, but we believe now that this is an overestimate due to the method and that this might be closer to the truth here. And from this rather high level ganglion cells cell density drops down rather symmetrically to both edges. So this is a kind of a summary table. Let me just point out here that CDVP2 was really useful as a marker for these displaced amacrine cells. And that's set B1, set B2, labeled ganglion cells only, and Foxp1 and 2 labeled basically very similar sets of cells in the avian retina. It's very different from the mouse retina where they can be used to tease apart the so-called F type ganglion cells, but that does not seem to be the case in the bird retina. Or at least in these two species I have to say. To see how consistent these markers are working, I also labeled the European robin retina with just Foxp2 and set B1 in this case here and you can already see that with these two markers you can discern at least five different cell types, cell types with larger somata or nuclei I have to say, positive here only for set B1, here only for Foxp2 and here for both markers. And then we have cells with smaller nuclei, only positive for set B1 and positive for both markers and there are probably also Foxp2 only small somata like this one here. And you can see, for example, that this cell type might form a nice mosaic here across the retina. So it's really these combinations of markers might really be useful to tease apart cell types and might come in especially useful once we have something like a physiology from the avian retina and then can combine that with these markers. But of course the markers have some limits and some downsides and that's very obvious from this image here. They just give you the nuclei and you might be able to estimate the rough size of the cells but you don't know anything about their morphology and stratification. And that's why we need some other markers and looked around and found neurofilament. And then the pigeon retina and refilament 200 labels here, the axons, of course, but also large ganglion cells in the ganglion cell layer, and in addition, displaced ganglion cells in the inner nuclear layer. And these cells really have enormous somata. And in this case it seems like they are stratifying here along the two chat bands. Unfortunately, I don't have enough examples to be really sure that these cells might represent the on-off direction selective cells. In addition, the refilament in the pigeon also labels a class of amacrine cells that can also be discerned here as a soma and also here we can see the dendrites stratifying between the two chat bands. And these cells form a nice mosaic that we are focusing on here now. So these are these amacrine cells and they seem to be really one type that is completely covered with this neurofilament staining. When we look more into the inner nuclear layer we can really clearly discern these displaced ganglion cells with their huge, huge somata. And most of them are really islet positive. So that's a combination now here with islet one. These cells are positive for islet one whereas this one is negative for islet one. So there are at least probably two different types of displaced ganglion cells in the pigeon inner nuclear layer. And with this I would like to summarize this last part. So we have found that indeed transcription factors can be used to define cell types also in the avian ganglion cell layer. And it seems like CTPP2 is a more suitable marker to capture all amacrine cells. And one needs to take care if one really uses AP2 as a marker. And we found that neurofilament 200 is a suitable marker for large ganglion cells including the displaced ganglion cells. Clearly we need more markers and of course we need physiology and that applies both to the bipolar cells and to the ganglion cells here. But I think we have built here a nice foundation. So once we have the physiology we can of course also use this classification and also use the markers to verify the cell types. And that's why I think this is really helpful to have although it might seem rather descriptive at this stage. Before I conclude let me just briefly mention this cell atlas provided by Yamagata and the same lab once more. It contains this interesting table here and really describes again that the chicken retina has an enormous number of different cell types in it. When you compare it to the macaque retina you see that this is way way more and seems to be way more complex. One reason might be that the macaque has kind of transferred some of the processing stuff into its larger brain whereas the birds might process more information already in the retina. And that might relate to this higher number of cell types and higher number of bipolar cell channels for example that extract features and higher numbers of macaque and ganglion cells. And then I think it will be very interesting to look at different species. One of the things I want to tell you that my feeling is that this songbirds here, the Prasarines and especially this European Robin has more like a raptor retina with a high cell densities and the general layer outwears the pigeon and the chicken that forage on the ground seem to be also more similar. So I think if one relates this really with a behavior and the habitat of the animals one can really learn interesting things here. And with that I would like to conclude now and thank the people who have been involved in this work and once more I would like to highlight Anja Günther here did all the reconstructions from the photoreceptor since bipolar cells. This was a collaboration of our lab headed here by Henrik Mauritzen and Silke Afakam, Kevin Brinkman, Stefan Yesen, sorry at the CISA Institute in Bonn. Silke actually I have to thank twice because she also did the transcription factor study with me. I think I learned a lot when we look together at these retinas. And in this study, Lasso Albert, Vaishnavi Balaji and Pavel Neymich were also involved. And finally I also like to thank the technicians here, Bettina Kiewitz, Leonid Pfeiffer and Jessica Schmidt and Pranav Siddh for providing me with some slices for connection in the 16th. And with that, I would like finally to thank you for listening in and Maxime for inviting me and setting this all up and thank you very much and I'm very happy to take your questions. Thank you a lot Karen. That was a very interesting overviews over. That was quite interesting. Before we move for questions, I would like to remain audience that if they want to join us on this room, they can do that now, because we're going to cut down the transmission just after the questions. So the link is available in the chat. If you want to join us, please do that now. Karen, I would like to start with a couple of comments that were received at the beginning of your talk. Those kind of comments kind of highlight, I can say, it emphasize the fact that in off field we tend to focus on mammal retina. I think I will just read a comment from Mala Feller in Mala. Okay, I've never heard of all droplets. And that is crazy. Are there already all droplets? Are there an organals? Are they maintained? Why do they have different filter inputs? Maybe do you want to have a quick overview about all droplets for bird retinas? I can at least try. I'm of course also not an expert in all droplets. But yeah, I think it's interesting that they contain these all droplets. They contain these keratin noids, difficult word. And they thought to work as really long pass filters and a kind of chop off the shorter wavelength to really sharpen the absorption spectra and help probably to distinguish the different colors, I'd say. They might also act as little focal lenses and might focus the light a little bit better on the outer segments. We have seen in the literature now quite a number of suggestions what might be in the photoreceptor to kind of focus the light onto the outer segments. So all droplets are definitely one option to do that. And I think they are also present beautifully in the turtle retina. And so they seem to be a specific adaptation. I think it's mostly for color vision. That's what I'd say. I mean, they are lacking in rods, I think, but they are present in double cones, it seems, but they can have different colors there. So different species have different colors of double cone oil droplets, which is also interesting, I think it might point to different functions of the of the oil droplets in the double cones in different species. Does that answer the question? I mean, yes and no, hi Karen, that was beautiful talk. It's more like how, like, how, like, what are they? Are they organelle? Are they replenished? You know, what, like, what physically are they? So they are really, I think, they can be fractionated or not. So I think it's really kind of an oilish substance. And it's basically, I don't know, all the chemical. Like it's a micron across. Yeah, it's a micron across and it is. Yeah, it contains these carotenoids and yeah, these long. Does it have a membrane? No, I don't think there's a membrane around it. No, I think it's really an oily structure within the photoreceptor. I think if you press on the whole mound a little bit too hard and you have mounted the photoreceptor sites up, you can kind of squeeze them so that they kind of disperse. Yeah, like you put with an oil droplets really, so I don't think there's any membrane around it. So I don't think we have seen that. So the message is do investigate out of the mum already. Have a quick question and actually I've noticed that too. On one of your IPL stratification schematic tables. So you have eight different subliminite. So you show us 15 different type of Apollo cells, but none of them contacted the eggs layer. So is it here for a purpose? Are you expecting something to connect to read? Is it just for words? So we used, as I said, the genetic suggestion to divide the strata and we were surprised ourselves that we did not find a cell that's stratifying in the eighth layer. But I would presume there is a cell that stratifies there. But that might be one of the cells that we are kind of missing. It's clear that this classification does miss some types that have large, probably large dendrites and might also have large dendrites in the IPL, on terminals in the IPL. So that would be an option, right? So that we are missing out on cells that are so-called white field bipolar cells. And they might go in there. But in the Janet Naito paper where they looked at the ganglion cells and backtraced them by trace injections into the thalamus and the tectum of the bird, they had ganglion cells stratifying there. So I would presume there must be a bipolar cell stratifying there because we don't see anything like the primary rod pathway or something. On the very simple topic, I have a comment from Tom Baden who said that this littering is quite fish-like. I understand what you mean by that, but since Tommy with us, do you want to elaborate on this? Didn't expect to be called upon. Yeah, so what I meant, so you showed this one overview where you sort of guessed the on and the off blobs in the IPL. And it sort of gets this on-off thing quite clearly, but quite a lot of off right at the bottom again. That's sort of the main trend I saw and then a handful of ons in the off layer. And that is something that we've seen functionally for baby zebrafish. But I don't think it's a feature of any mammal that I've come across. So I'm wondering, do you have any thoughts on that? Yeah, I think we see clearly the difference to the mammalian bipolar cells, right? Because they are monostratified. No, they are not all monostratified, right? The guinea pig, does it have multistratified bipolar cells? But are they properly multi-ordered? Was it the Type 6 or the Type 7 in the mouse, which is sort of a little bit... Yeah, that also has these little add-ons, right? It's really hard to tell. But I think here it's definitely more obvious with the multi-stratification. But I agree, it looks like we have more off terminals interspersed in this on layer than the other way around. But I'm desperate to see physiology for the bird bipolar cells. I'm hoping you are doing that to really see whether this is true or not and what does this mean and so on. Yeah, we're still struggling on guinea pig cells. It's supposedly here. It's supposedly here. It might be difficult enough, yes. Also, yeah, we are just trying to find maybe markers that give us a clue about that. So, like the EGFR, that's a very beautiful marker. Now it labels an on-cell. But of course, it would be cool to have a marker for an off-cell that goes deep down and then we could be sure. Because it's lacking eyelid one and it's lacking GNB3 and then we could be sure that it's true that we have these off-stratification there in the on sub-laminar of the IPR. So I think that will be interesting to see whether we can find that. If I would move to the next question and to the last question, actually, just to remind all the answers for that, just to join us after we close the chat. I just want to let you know also, Karin, that you have a lot of very nice comment about your talk and especially about your images. Thank you very much. That's nice. I have one last question from Marla. Maybe Marla, you want to ask it yourself since you're here. Yeah, and I guess maybe since Tom, the fish retina expert is also here or a fish retina expert, which is, you know, I have had this theory, you know, based on really nothing that Mueller cells in the mouse retina, they have a lot of elaborations in the IPL. I thought that would be a diffusion barrier that kind of separates on and off. And this is one of the ways that kind of segregated the channels of on and off. But you clearly have many violations of that. And so I'm just wondering, I guess, what are the Miller cells look like? Like, do they have these kind of stratifications that might define different regions of the IPL like they do in the mouse? And have people looked at their kind of lateral processes coming up the stocks and how they're organized? I think I'm not. Mueller cells is really, you know, you inject them and you think shit, damn, a Mueller cell injected. Let's go ahead and you never really look at that. They are very elaborate in the outer nuclear layer. They are very broad trees there so you can easily hit them when you're aiming at folder receptors. I'm not so sure about this main part that goes through the INL. I think it also has these protrusions that we know from the mouse retina, but I never really paid close attention whether it might just fall in place with a GNB3 border that we see or something. But it would be interesting to look at. So we have a marker, I think, for Mueller cells and the bird retina. So we could try to take a look at that whether it really would fit. Because I wonder if the channels, the on and off channels are more mixed. I think they, I would guess they are more mixed than we know that from the mammalian. So I will give away my moderator rights now. So if you want to follow up on the discussion, I'd like to thank our audience to be there with us every week. So see you all next week. And we will host your ever see you soon. Thank you Maxime. Bye bye everybody.