 So, good afternoon, everybody. Welcome to our Sasex neuro vision seminar for today. I would like to introduce myself first. My name is Jose Moya Diaz. I'm working as a postdoctoral researcher at the laboratory of Professor Leo Laganado. And from this term, I will be part of the committee, which is organizing the Sasex vision talks. And today I have the pleasure of introducing our next speaker, Professor Miha Riblin from the Waysman Institute in Israel. So, yes, today I would like to give a very brief introduction about Professor Riblin before starting. And, well, Professor Riblin did her baccalaureate studies in mathematics and computer science at the Ebreu University of Jerusalem in Israel. Then in the year 2002, she started her PhD in the interdisciplinary center for neural computation at the Ebreu University of Jerusalem in Israel under the supervision of Professor Hagai Bergman and Professor Jifat Pruth. And the title of her thesis was synchronous oscillations in the basal ganglia cortical networks. Do they generate tremor and other symptoms of Parkinson's disease? After finishing her PhD, she moved to the States for developing her postdoctoral research in the Department of Molecular and Cell Biology at the University of Berkeley under the supervision of Professor Marla Feller. Then she moved back to Israel in the year 2013. And actually she's an assistant professor at the Neurobiology Department at the Waysman Institute of Science in Israel. So, going deep in terms of the description of Professor Riblin's lab and interest, I have to say that Dr. Michael Michal Riblin studies visual processing in the retina and their dynamic nature. During her postdoc in Marla Feller's lab, she found that the retinal code is flexible, showing that neurons in the direction selective circuit qualitatively change their light response with stimulus history. In her lab, Dr. Riblin uses this phenomenon to decipher new mechanisms for direction selectivity and other retinal circuits. Her lab also found that light responses of retinal neurons can change with their location within the retina. Recently, she became interested in how neurons in the brain can affect visual processing in the retina. So, today, she will be presenting her talk, which is titled Top-down Modulation of the Retinal Code via Histamineurgic Neurons in the Hypothalamus. Thanks, Michal, for accepting our invitation. It's a pleasure to have you with us today. Thank you very much, Jose, for the introduction and for the invitation to speak. Do you see my screen? Yeah, yeah. All right, cool. So, yeah, I'm going to talk about top-down modulations of retinal code via histamineurgic neurons in the hypothalamus. As you can see, this is a drawing of a retina by Ramoni Kachal. Although it is quite common to start a retina talk with one of Kachal's drawings, I have a special reason to bring you this drawing as I'll show you later. And from Kachal's work, I would like to show you paintings of my daughter, which emphasize the differences in the visual scene that arrive at a retina at different times of the day. And indeed, the visual image as well as our behavioral requirement constantly change with the environment and with time. In my lab, we are interested to see how the retina deals with these changes. So, the retina contains more than 80 different subtypes of neurons which form very specific connections between them. Its architecture results in multiple pathways conveyed by multiple subtypes of retinal ganglion cells, the output neurons of the retina. Each subtype encodes a specific modality in the visual field, such as edges, motion, or color. This modality is thought to be fixed and hardwired. However, when others find that retinal neurons can in fact change the modality they encode. Let me give you two examples. Most retinal ganglion cells belong to the on pathway responding to light onset or to the off pathway responding to light offset. However, polarity preference may change with illumination level. Here's the peristimulus time histogram recorded from our retinal ganglion cell in response to two second light offset illustrated here by the dark bar. This reveals an off response of the cell. When overall ambient light level is reduced, the cell continues to respond to light offset but starts encoding light onset as well. Another example comes from direction selective ganglion cells. Direction selective ganglion cell encode the direction of motion in the visual field. The polar plot here represents the number of spikes evoked by the cell in response to grading driftings at 12 different directions. The traces are example of the spiking activity and the arrow represents the preferred direction of the cell. The opposite direction is called the null direction. During my postdoc in Mara Feller's lab, I found that following a short repetitive visual stimulation in the time scale of minutes, a given direction selective ganglion cell can reverse its directional preference as you see in this example. So we know now that there is dynamic computing in the retina as a function of the visual input and if you're interested in more details, we have a review with Will Graves and Fred Rickey on this. And now we're asking, can the brain affect visual processing in the retina? If that is true, it might suggest that there is also dynamic computing in the retina as a function of the behavioral state of the animal. So the outline of my talk today would be the following. I will start to briefly show you only some of our findings on the direction selective circuit. The reason would be because I want to introduce you with a new simulation modeling of the retina, which I hope that some of you might find useful. And then we will move on to the main part of my talk and see whether the brain signals onto the retina and can change the processing there. Okay, so direction selectivity first. We all know that motion is an important modality and it is already encoded by the retina, as I showed you. Why is it important? Well, we see motion all the time. Here we see Lea Ante in the lab moving. On the right, you see what maybe Lea sees while she is moving in the corridors of the lab. And then we see that she sleeps and falls and everything involves motion. So motion is important. And here I showed you my funny video. Okay, so what underlies direction selectivity in the retina? The key inputs onto direction selective ganglion cells, the key cells that immediate direction selectivity in the retina are starburst amachrine cells. Starburst amachrine cells form asymmetric inhibitory connections onto direction selective ganglion cell. And on top of this, they also show their own directional preference. So in fact, each process of the starburst amachrine cell would prefer motion outward from cell soma or what we call centrifugal motion. So the centrifugal preference of starburst amachrine cells together with asymmetric inhibition onto the direction selective ganglion cell mediates direction selectivity in the retina. So what exactly mediates the reversal of directional preference in direction selective ganglion cells following the repetitive visual stimulation? To answer this, we recorded from starburst amachrine cells before and after repetitive stimulation. And we specifically looked at their centrifugal preference to see how it changes with the repetitive visual stimulation. And to measure their centrifugal preference, we presented the starburst amachrine cells either with centrifugal motion of expanding ring or centripetal motion of collapsing rings. And as expected, we find that a starburst amachrine cell reveals centrifugal preference. You can see that the voltage amplitudes are a larger in response to the centrifugal motion here in blue than in response to the centripetal motion in red. Following the repetitive visual stimulation, we find that the starburst amachrine cells lose their directional or their centrifugal preference. And you can see that the response amplitude is now similar in both centrifugal and centripetal motion. When we have a careful look on the traces, you can see that they are also shifted in time. So the repetitive visual stimulation does not only abolishes the centrifugal preference in terms of amplitude. It also does shift in the timing of the response. And because this study is already published, I will not get into the details, but basically we find that these changes both in the amplitude and the timing are reflected in the inhibitory inputs onto direction-selective ganglion cells and mediate the reversal. But what we'll learn from here is that really the centrifugal preference of starburst amachrine cells is crucial for direction-selectivity. And if we lose it, we might lose direction-selectivity and even reverse it. So we wondered what mediates the centrifugal preference in starburst amachrine cells? Now, we are suddenly not the first to do it. Many, many others and some of them are listed here, search for the mechanism underlying the centrifugal preference of starburst amachrine cells and there are suggested intrinsic mechanism as well as exact excitatory inputs or inhibitory inputs. Now, we focused on the excitatory and inhibitory inputs and wanted to see how they act together to mediate the centrifugal preference. So for that, we build a simulation in which we control the precise excitation onto the starburst amachrine cells as well as adding the inhibitory network between starburst amachrine cells. Now, these findings which the study was conducted by Elisai Ezratsoor, Oren Amsalem, Lea Anchry, together with British Patil and in collaboration with Edan Segev, and you can now find it in Bioarchive. I will not get into the results of what we found, but I would like to tell you that in order to find these results, we built the retinal stimulation modeling environment, what we call a RSME, which allows you to build neural networks with detailed morphology, biophysical constraint. You can define precise connectivity patterns between your neurons and it also allows you to implement new visual inputs or existing ones. And if you're interested in the work or in using this simulation, I will refer you to the Bioarchive paper and you can see the GitHub and use it for your own purposes. I hope you will find it useful. And from here, I would move to the main subject of the talk and this is trying to see if the brain can affect the retina at all. This project was led by Rebecca Warwick and Serena Richitelli, postdocs in the lab, together with Alina Hoykamp and Hadaria Akhov, Ph.D. students. And here we go back to Ramonik Haas drawing. Now, if you look at Ramonik Haas drawing, then you can see the photoreceptors here and the arrows that point on the direction of the information flow from the photoreceptors to the bipolar cell to retinal ganglion cells and from them to the brain. But if you look carefully, you can also observe some axons in the opposite direction, actually suggesting that these axons come from the optic disc from the brain to innervate the retinal neurons. Now, these axons, called retinal pedal axons, were found already by Ramonik Haas, as you can see, 130 years ago, and they were primarily found in the Ayn avian species. However, there were some reports that they also exist in mammalian species. And later on, using immunohistochemistry, it was shown that at least in the mouse rat in guinea pig and primates, these or part of these retinal pedal axons are histamine energy. Now, you might wonder because we used to know histamine more in relation to our immune system and all these allergies that some of us has, but histamine can also act as a neuromodulator in the brain. Okay, so if it is coming from histamine, and if these retinal pedal axons are histamine energy, it means that we can actually maybe identify where they are coming from. And the reason is that the sole source for histamine in the brain is coming from neurons in the tuberomarmillary nucleus or the TMN, which lays in the posterior hypothalamus. And here you can see in red the tuberomarmillary nucleus, it's a tiny nucleus, but it's there, and it contains the histamine energy neurons. And it was shown that these histamine energy neurons project all over the brain and maybe also to the retina. And it was also shown that these histamine energy neurons have wake on sleep off kind of activity. So basically, when we are asleep, the firing rate is low. And when we are attentive waking, you can see that their activity goes up. So overall, it might suggest that histamine coming from the brain might change our processing in the retina as a function of the arousal state. And our goal in this study were to identify the histamine energy retinal pedal axons in the mouth and to reveal how histamine coming from the brain shapes the retinal code. Okay, so to identify the histamine energy retinal pedal axons, we injected to the TMN of wild type mice, a labular virus of titty tomato or m cherry. We waited for four weeks, and then we looked at the retina and tried to find for the retinal pedal axons. First, you can see here a cross a coronal section from an injected mouse. You can see the virus here in red in the TMN and much over. You can also see in a green HDC. HDC stands for histidine decarboxylase. This is the enzyme that actually synthesizes a histamine. So basically, it is found in histamine energy neurons. And we can see that indeed the virus transfected the histamine energy neurons. And then if we move on to the retina, then you can see very beautifully that there were just few axons that depart from the optic disc, but they branch extensively to cover the dorsal part of our retina. So it looks kind of cool because we kind of found the retinal pedal axons. However, you can see that on top of them we see from time to time a ganglion cells or a label sometimes with their dendrites. And we think that maybe these retinal ganglion cells send their axons close by to the injection site and take up the virus which is being retrograde label taking path. And we also are not sure that these retina pedal axons are indeed histamineergic because as you can see, although histamineergic neurons took the virus, there were some non histamineergic neurons that were also affected by the virus. So in order to make sure and be more specific, we decided to move to other mouse lines, the HDC CreLine. Either from the Jackson laboratory, these mice were generated in Bill Wisden lab or from the Ginsat project. And now we inject a predependent virus with TD tomato to the TMM. And here in the Sagittal view, you can see the specificity of the expression. The virus is now expressed in HDC positive neurons. And now we're looking for the axons to see whether they arrive at the retina. So first, if you look at the axon, if you look at the axons from the optic chiasma along the optic nerve, you can see a few axons that they make it all the way to the retina. From time to time, we saw axons that seem to be ending their terminals in the middle of the optic nerve. We do not know if this is real or maybe some limitation of our imaging techniques. But in any event, when we look at the retina, we do see a few axons that emerge from the optic disc. And here you see this nice hardship that they covered the dorsal part of the retina, and again, an extensively branched to cover large portions of the retina. These axons tend to travel in the ganglion cell layer. And then at a certain point, they go deeper in the tissue and innervate also the inner plexiform layer. So basically, the histamineurgic retinal petal axons are not specific to one specific layer in the retina. And they're also like to mention that a histamine acts in a paracrine fashion. So basically, it can spread more than just its exact location. All right. So at that point, we were very happy that we found the retinal petal axons because it's in Zoom and you cannot celebrate it. I will show you the celebration. What you see here is the Selenah and Adab celebrating the findings of the histamineurgic retinal petal axons. Cool. So we managed to identify them. And then we were still somewhat worried, the fact that we got a non-uniform coverage and that the axons were tuned and more dense in the dorsal retina. We didn't know if this is real or maybe just some limitation of our approach. But then when you think of it, the mouse retina is not really uniform. And so in our recent review by our labs, we here, for example, we look at the density of different retinal ganglion cells. Here it is color coded. And you can see that many types of retinal ganglion cells do not show a uniform density. And they usually tend to vary along the dorsal ventral axis. Some of them also change the size of their dendritic tree in the different locations, showing us that indeed topographic variations in the mouse retina exist. On top of that, Rebecca Warwick in the lab did some targeted recordings from off transient alpha retinal ganglion cells. And what she found that in response to a dark spot, the transient of alpha cells change the response duration as a function of their location in the retina. And basically, you can see here the duration of the response of the function of the ventral dorsal axis, where you can see that neurons in the ventral axis tend to be more transient, whereas neurons that are closer to the dorsal part of the retina tend to be more sustained. So the retina of the mouse is not really uniform. We can even see some functional differences in portion of the ganglion cells. And so why does this happen? Well, if we think of it, then the mouse is a very small animal that lives close to the ground and views the environment from close to the ground. So basically it means that the dorsal retina is mostly responsible for encoding the ground. And what it does is many encoding for raging and hunting for food, while the ventral retina primarily sees the sky and may be more in the part of the predator detection. Now, the fact that the histamineurgic axons nonuniformly innovate the retina and are denser in the dorsal retina. First, maybe in the ventral retina, we don't really need to change the encoding, because whenever there is a predator, you want to know that there is a predator regardless of your arousal state, maybe. And here it might suggest that the histamineurgic axons act in order to tune the retinal code for a foraging and a orientation in the environment. But this is, of course, merely a suggestion. And we can discuss it later. But overall, it seems reasonable to us that the nonuniform coverage of the retinal pedal axons is real. And it is possible that histamine affects mainly one portion of the retina. And then we're moving to the main question, which is, can and how histamine affect the activity of the neurons in the retina? So in order to answer this question, we performed two photon calcium imaging from the ganglion cell layer of Taiwan g-comp 6F mice, which expressed g-comp in a subset of the neurons in the ganglion cell layer. Although there are also maybe displaced amocrine cells there for simplicity, I will refer to all the neurons as retinal ganglion cells or RTCs. So now we're looking at retinal ganglion cells, calcium transient, this is spontaneous activity. And then we add histamine to the solution. And you can see that some neurons do not react to histamine application, whereas other neurons increase their calcium transients in response to the histamine. Now, we know that histamine in the CNS acts on G-protein coupled receptors, and specifically in the mammalian retina, three types of histamine receptors were found, one and two both thought to be more excitatory and three thought to be more inhibitory. And so in order to make sure that this effect is really made by the histamine receptors, we blocked all receptors pharmacologically, and then we apply histamine, and you see that now in the presence of the blockers, the neurons do not increase their calcium transients with histamine. This is a summary plot showing you the percentage of neurons that actually increased their activity in the two photon imaging in five micromolar histamine, in 20 micromolar histamine, about a quarter of the cells increased their activity, whereas in the presence of the blockers, the cells did not increase activity. So that is cool. This is a calcium. We also wanted to verify this in the spiking activity of the neurons. So for that, we moved to multi-electrode array recording. And again, we're only looking at spontaneous activity. We are adding a histamine to the aim solution. And you can see that some cells increased their firing rate when histamine is added. You can see the distribution of the difference in firing rate when we added histamine. It is here in orange. In blue, you can see the distribution in a control experiment where we did not really add histamine. And indeed, I think you can appreciate that many of the retinal ganglion cells increased their firing rate. And about more than 40% of the neurons tended to increase their firing rate when we added histamine to the solution. And then we wondered, oh, by the way, these results of an increase in firing rate are in line with previous results that were obtained in the rat retina, which also showed a large increase in the spontaneous activity of retinal ganglion cells when histamine is added. We then wondered whether these neurons that actually increase their firing rate with histamine, whether they belong to a specific subtype of retinal ganglion cells. So we looked at the large responses to a full field flash. This is, again, only the neurons that reacted to histamine. And you can see that those neurons actually come from different populations. We see on retinal ganglion cells, on off cells, as well as off retinal ganglion cells. So this is not specific to a specific population. And so histamine increases the spontaneous activity. We next obviously wanted to see whether it also changes the light responses of retinal ganglion cells. And what you see here is two photon costume imaging, again, of the ganglion cells. We present the retina with a two-second bright spot, UV spot. And we repeat these experiments in three conditions, in the precondition. And then when we add histamine, this would be the orange curve. And then after washing out the histamine, you can see in this example cell, cell number five, that some neurons were not affected by histamine application. Other cells were affected by histamine application, either increasing the responses or decreasing the responses. And you can see that other cells even more dramatically change. So this sustained on cell, for example, lost its response when histamine was added. And this cell that used to be an off cell now gained an on response while one histamine is in the solution. And this was recovered in the in the washout. So I would like to emphasize that what we count here for the retinal ganglion cells is only cells that showed a stable activity. And by stable, we compare the responses in the pre and the washout condition. And overall, if we look at the percentage of neurons that change, then in a control experiment where we do not really add histamine, we just repeat the same protocol three times, a 3% of the neurons change, while when we add histamine, more than a quarter of the neurons change. And the colors here present the on cells, off cells and non responsive cells. These are the cells that actually changed. And these a strong red ourselves that they were not responsive, but but gained a response. So we know by the two vote on calcium imaging that retinal ganglion cells can change their life responses. But we do not know exactly how to differentiate between these cells and to tell which cells change and which not. In order to answer this, we moved on and conducted patch clamp recordings. And and we specifically targeted on alpha retinal ganglion cells, we identified them by the large cell bodies. And then once we touched on the cell, we clustered the alpha cells based on on sustained off a transient and so on. And this is example from an unsustainable for it no ganglion cell. You see when when we add histamine, nothing really much changes in the spiking activity. We also recorded the responses to a bright spots of different sizes. And as you can appreciate by the responses of the cells to the content in the control solution, after histamine was added, it seems like the unsustainable right now ganglion cells don't change much when histamine is in the solution, at least not to this specific stimuli. Here's a population looking at the maximum firing rate versus diameter of the spot for control condition and with histamine. And this is looking at the duration of the response of the function of diameter. And we see that histamine doesn't change these cells, at least not significantly. The same was true for off sustained alpha retinal ganglion cells, which also kept their responses when applying histamine to the solution. However, some cells did change. And if we look here at the off transient alpha retinal ganglion cell, you can see that application of histamine increased its continuous activity. And this increase was maintained. As you can see in the live responses here, the basal firing rate when histamine was added is increased. But this was not the only change. We can also see that the response duration got shortened in the presence of histamine. And we also noted that there was a reduction in the overall spiking activity. And this is quantified here. Again, you can see recordings from 12 off transient alpha cells, their maximum firing rate in control solution is in black. And in the presence of histamine is in orange, you can appreciate that the firing rate was reduced as well as the response duration. We also wanted to target, as I told you, we work a lot on direction selective ganglion cells in the lab. So we targeted these cells, this was done using a transgenic mouse line that specifically labels posterior preferring direction selective ganglion cells. And what we found is that histamine increases their spontaneous firing rate. And it also changes their on off responses to the spot stimuli. And then we wondered, since the spot responses, I mean, usually the direction selective ganglion cells are not so great in responding to a stationary stimuli. So we moved on and we wanted to see whether these cells change their directional tuning when we apply histamine to the solution. And so here you see recordings from posterior preferring direction selective ganglion cell in response to either drifting gratings or moving bar in control conditions. You can see the tuning here is marked to the right posterior direction. And then when we add histamine, you can see that the cells do not change their directional preference. However, it does seem that the directional tuning gets broader with the when histamine is added. And we can we can measure this using the normalized pictorial summation, which is a measure of how sharp the tuning is, the higher the value, the sharper the tuning. And you can see that with histamine application, indeed, the tuning gets broader both in response to the gratings and also in response to the bars. All right. So histamine now does a lot. We see it changes the activity in the spontaneous activity is also changing transient of alpha cells, a posterior preferring direction selective cells. But then we were wondering whether it also affects other direction selective ganglion cells. And in order to answer this question, we moved on and used the multi electrode array and presented the retina with drifting gratings and in all direction and measured the directional tunings of direction selective ganglion cells. So basically we identify direction selective ganglion cells based on their directional tuning. And again here we only stick to those cells that they were stable. So we have a precondition, histamine condition and washout. And we only take these cells that based on their firing rate, the firing the firing rate did not dramatically increase or decrease between the pre and the washout conditions. So those cells, we are now including them in our analysis. And you can see that in control conditions were actually we did not add any histamine just repeated the measurements three times. You can see that we got 40 direction selective ganglion cells and five percent of them changed their directional tuning when we did not really add histamine, but in the second control. And if you look at the experiments that did involve histamine, then you can see that's more than a third of the direction selective ganglion cells changed their directional tuning with histamine. And in general you can see that they reduced their normalized vectorial summation, meaning that their tuning became broader when we added histamine and there was a recovery once we washed the histamine out. And these are just two examples from a control condition where no histamine was added. And these examples come from a histamine experiment where you can nicely see the broadening of the tuning of the direction selective ganglion cells, which then recover when histamine is washed out. But are there really different direction selective ganglion cells? You can see that only a third of the direction selective ganglion cells responded to histamine or changed their tuning with histamine. When we look at their directional preference, we see that indeed those cells that were affected come from different populations because they show a different direction, different preferred directions. And if we overall look at the activity of all the neurons that change and just look at their firing rate, then you can see that this is the firing rate in the precondition. And what histamine tends to do is to increase the firing rate in all directions. So it looks like histamine changes spontaneous activity of retinal ganglion cells, some of them, as well as changes the light responses. And specifically we found that it changes the light responses of transient of alpha ganglion cells and posterior preferring direction selective ganglion cells as well as other direction selective ganglion cells. And now we're asking whether we can, instead of just pouring histamine and add it to the solution, whether we can really activate the histamineurgic pitino pedal axons and cause some similar effects in the retina. And so what we're doing now is we're going back to the HDC reline and we now inject a virus to the TMA. And again, this is now an excitatory dread. And after the expression of the dread in the axons, we expect that once applying CNO to the solution, this would activate the excitatory dread and we will see this will release histamine and we will see some effects on the retinal ganglion cells. And that would tell us that not only adding histamine to the solution does it, but really these retino pedal axons can also do it. And then you see the selective expressions of the virus in HDC positive neurons. And here you can see the axon in the retina. This is after recording from the retina on the multi electrode array. And what we're doing now, so we are looking at the spontaneous activity of the retinal ganglion cells in the precondition and after we added CNO. Now we know that CNO might have some off-target effects and so we do the experiments in two populations. First on injected mice that really have the excitatory dread and then on injected mice and where we can actually just see how CNO spontaneously affects the firing rate. And this is what you see here. So this is the firing rate before adding CNO and this is the firing rate after adding the CNO. This is of the control experiment where the non-injected retinas. And you can see that there were some variations of the firing rate of these neurons. But if you look at the retinas where we added CNO in the injected retinas then you can see that the sub-population of the neurons either most strongly increased or decreased their firing activity once we added CNO. And overall about a little more than 10% of the retinal ganglion cells that we reported changed their spontaneous activity when CNO was added in the injected mice. So it looks like the retinal beta axons do something on this spontaneous activity but then we wondered well we specifically focused on the direction selective ganglion cells and we wondered whether the activation of the retinal beta axons by CNO can cause the same change in the tuning of the direction selective ganglion cells just by activating these axons. And so now what we're doing is again we put the retina was on the mirror already and we present it with drifting gratings to identify direction selective ganglion cells. And we do it again in control conditions. This is on the non-injected retinas. So this might look at the off-targets effects of CNO. And we do it on the injected mouse retinas. And what you see here is in the control experiments about 9% of the neurons change their tuning with a CNO application but a quarter of the neurons change their tuning with CNO application in the injected mice. And here you can see some examples from control solution and from the injected mouse retina where you can see nicely the broadening of the tuning curve when CNO is added which is trying to simulate the histamine that we add to the solution. And here again you can see the firing rate of the neurons in the precondition and this is after adding the CNO and you can appreciate that overall the increase in all directions is similar to what we found for the histamine for the histamine condition suggesting that indeed the CNO activation in the injected mice caused a similar effect like the histamine. So it could be that the retinopada system is active and vivid in the mouse retina. Can we see the same effect in human? So obviously human is much more complicated. We cannot. I mean we won't take their retinas and put them on the MEA but what we can do is to test their vision. And here specifically what I'm showing you is our test of the peripheral vision of humans and the test is done as follows basically the subject looks at the center of the screen and you have 60 different points here and every time at a different point is presented with a small spot illuminated at the different light intensities and the subject needs to report whether whenever it sees or detects the spot and it is done for all the spots here and basically this is a way to measure the visual sensitivity of the subject. So we did it in the periphery visual field so this is between 30 and 60 degrees of our vision and we repeated these tests twice. So once the subject got antihistamine H1 blocker and in the other it got a placebo and the two tests were separated by at least two weeks and it was a single blind test so the subject did not know what they got whether the placebo or the fennel steel the H1 blocker and these experiments were carried by a Nua Gilad ophthalmologist in a Kaplan hospital and we did this on eight subjects. Okay so I can tell you that overall when we started to look at the results what we found is no real significant difference in the peripheral visual field with and without the antihistamine drug. However we then remembered that the mouse retina is non-uniformly innervated by the retinopatal axons and then we thought well maybe we do not really expect all these regions to have the same the same trend by the antihistamine and so when we looked at the specific areas in the periphery visual field and here they are color-coded by the change in sensitivity so basically red would mean increase in sensitivity with the H1 blockers and blue would mean decrease in sensitivity with the antihistamine and you can see that indeed the visual field is non-uniformly affected by the antihistamineergic drug and so if we look at this area and we define them then you could see that at least in the superior visual field this is now the visual sensitivity in the control when they took the placebo or the antihistamine H1 blocker you can see that overall antihistamine in fact increases the sensitivity in the superior visual field for these subjects each doc here represents one eye of the subjects. Now this is interesting it is a bit maybe counterintuitive because we might expect that histamine when we are aroused and attend would actually increase our sensitivity but it looks that the sensitivity increases when we block H1 receptors but I would like to mention that in the primate retina recording from a baboon retinal Gandion cells showed that histamine application to the retina actually decreases their flash sensitivity so this might be in line with our results. I would like to summarize what I showed you today so I started by revealing the mouse histamineergic retinal pedale axons which asymmetrically innervate the retina and originate in the TMM. We saw that histamine changes both the spontaneous activity and the live responses of some retinal ganglion cells and we identified some of these neurons like the transient of alpha cell and the direction selective ganglion cell and then we showed that the chemogenetic activation of the histamineergic retinal pedale axons can modulate also the spontaneous activity of retinal ganglion cells and the tuning of the direction selective ganglion cells and finally we saw that in human there is some evidence that maybe already at the retina histamine or its blockage may change the sensitivity in the visual field. Overall if we take into account histamine changes its activity and the level of histamine is changes with the arousal state of the animal it might mean that the retinal code also changes and is tuned by the arousal state of the animal and I think this resonates quite nicely with two recent studies from Sylvia Schroder and Leang Leang from Matteo Corandini and Mark Anderman's lab who looked at the terminals of retinal ganglion cells either in the super coliculus or the LGN and they showed that the live responses and sometimes also the spontaneous activity of these terminals may also change with the arousal state of the animal and there they measured the arousal state via locomotion and pupil size and of course there can be some effects that happen locally in the thalamus or in the superior coliculus but maybe together with our finding it suggests that some of the changes originate already at the retina via the retinopatal axons. I would like to emphasize again those who did the work Rebekah Warwick, Serena Richitelli, Alina Hoykamp and Hadaria Akov, Noah Gilad was the medical doctor who carried the experiments in humans, thank the funding agencies and of course thank you for listening. Oops, there you go. Okay thanks a lot, Mihaal, nice talk, really nice talk. So we have some some questions. Anna Blasitz asks if do you have a sense of what the range of concentrations of histamines are released from the hypothalamus into the retina? Yes, that's a good question. So basically when we measure, we don't but when other measure the concentrations of a histamine in the retina it was found to be parallel to the concentrations of histamine in other brain regions and I can tell you the number but I don't think it will tell you a lot but we also did some a test in our histamine using the MEA looking at the concentration dependent effect and we find that when we start from 2 micromolar we all already start to see changes in the activity which is probably what already exists in the retina when histamine is being released there and we also see that the effects are a concentration dependent so when you add more histamine the effects are even stronger. Okay, so Leon is asking if do you think that the actions of histamines on RGCs are direct and if histamine acts into amaprincelles? Yeah, that's a great, great, great question. So to be honest we think both. So first we see that the retina petal axons run in the ganglion cell layer as I showed you and where am I? Well this was a long time ago. Okay, yeah it runs in the ganglion cell layer but it also gets into the inner plexiform layer and as I said because it acts in a parkland fashion it can be more spread than this and when we did some intercellular recordings from direction selective ganglion cells with histamine it looked like the spike shape changed and we also know that histamine receptors are expressed in retinal ganglion in direction selective ganglion cells for example so we do think that there are actions probably via age two receptors on the direction selective ganglion cells but probably there are also effects on amaprincelles and we know that dopamine amaprincelles for example express age one receptors and it was shown that other amaprincelles may be changing their activity via age two or age three receptors so I would tend to think that this is a combination both on the retinal ganglion cell and the amaprincelles and maybe also bipolar cells. Okay so Tim Gollish is asking yeah is telling us that is it known which retinal neurons have histamine receptors and are the histamine effects on calcium and firing rate restricted to the dorsal retina? I didn't get the the second part of what you asked. Sorry so Tim is asking if is it known which retinal neurons have histamine receptors that's the first part of the question and the second part of the question is if are the histamine effects on calcium and firing rate restricted to the dorsal retina? Oh yeah I understand yes so basically if we look at a data based on single cell RNA sequencing then it looks like the receptors are kind of all over so we know that the age three is based on this data existing all in all types of retinal neurons age two is more in the amaprincelles and ganglion cells and here you see age one receptor and because of the asymmetric innovation we too were interested to see whether the effect are different but when we same for the receptors at least age one receptors we can see them all over the retina so they were not exclusively demonstrated in the dorsal retina and also when we apply histamine and look at the live responses we see that both the dorsal and the ventral retina can change with histamine application but what we think happens is that because the retinal petal axons are asymmetric physiologically usually the ventral retina is less affected. Okay well we have a lot of questions well George is actually curious about if what do you know if you know anything about the nasotemporal distribution of posterior preferring dorsal selective ganglion cells if i understood well about the nasotemporal distribution of the posterior preferring direction selective ganglion cells if you see any any nasotemporal particular pattern of distributions of the direction selective ganglion cells the posterior preferring one i don't when i recorded from these posterior preferring neurons i usually divided my retina to dorsal and ventral and then i did observe some differences in the size and in the density of the posterior preferring neurons but i never looked at the nasotemporal maybe andi uberman looked at this i need to check i don't remember oh no worries so sylvia sylvia Schroder is asking how does baseline firing and visual response change with histamine within the same RGCs so they tend to increase uh many of the retinal ganglion cells that we look at tend to increase their firing rate when histamine is added to the solution okay yes am i answering this so i repeat the question but i think how does baseline firing and visual response change with histamine within the same RGCs i think yes yeah okay you made the point and anablasit is asking why do you think that turning off the direction selective ganglion cells gets more growth when the histamine is high which will be during the awake time yeah that's a very good question so it's again i mean just like the human data that shows that histamine that is supposed to be released when we're attentive actually reduces uh the sensitivity it is also price striking for us that the direction selective ganglion cells broaden their tuning with histamine because it seems as if you know the cells are less tuned and less tuned and so one thing that we're doing now is we thought that maybe looking at the single cells and understanding what each one of them does separately will not give us the answer to the overall picture of histamine so we're trying to work now in collaboration with Adzschneidmann and Jonny Mazel to really look at how maybe the population code changes when histamine is added and i hope that soon i will be able to say something smarter about why this exactly happens at least at the joint activity level of the retinal ganglion cells okay and i think this is the last question at the moment uh Anton Nicolae uh he said i probably missed this but do you do cno and histamine experiments for the same cell ah no we didn't we didn't i want to say that these the cno experiments were so difficult that i i admire Hadar and Serena for managing to do it we did not do it although it's a great suggestion uh we did not okay all right okay okay so if there is not any question thanks a lot Michal for the great talk and just for letting people know we already posted the link for the Zoom meeting if people wants to join and discuss more in detail about the data and project you can join now so okay thanks everybody for attending to our sus exhibition series and see you next week thank you very much Jose no worries so we will keep the the the the live stream open so people can access to the link if they want to join so micah in the first part of your talk you um you're showing us the differences between uh uh the adaptive effects uh you start video uh you're showing in the video you don't have to Verena and Lea we can hear you i went and i muted them i hope they don't mind oh god Marla Marla when you came on there i thought it was Andy Warhol i'll take that as a compliment i don't know Anna was that a compliment i can't tell i have no idea okay all right well Lea and you don't look anything like Andy Warhol no no no it was a beautiful talk that was great all right i'll let Leanne talk yes so this this really powerful adaptive effect uh when you looked at the centripetal versus centrifugal stimulus yeah um did i kind of see right that the phase shift was much larger for the centripetal effect than the centrifugal after adaptation so yeah the phase shift after adaptation just relative to the stimulus not relative to each other necessarily yeah um wow i need to have a second look at this so basically what i can tell you let me see here um i don't know if you can really say that we can i can send you the images and then we can discuss but basically what what we found is that uh since say uh the um the responses arise at the center of mouse of the of the receptive field we saw that the receptive field actually changes being stronger in the center at the beginning and then the surround takes over after the repetitive visual stimulation and therefore the responses shifting time because they they are now activated by the center of mouse of mass of the receptive field and uh that's what we have there and maybe there is a side but but both of them change both of them are changing yes okay so it's primarily you think a kind of um depressing adaptation in the excitatory bipolar cell input probably towards the center of the receptive field so basically what we think is that the i i think it's kind of changing the center surround organization starting already at the bipolar cell level yes and uh the hypothesis is that it is mediated by changing interactions between roads and cones basically we believe that the roads are saturated with the strong repetitive visual stimulation and therefore horizontal cells transfer negative input via them and that's what starts all the changes uh in the polarity and in the center of surround organization okay you can really i'll send you down yes yes the support of the chat yes sorry i have another question here in the internal zoom uh by maria cosine uh she's asking if uh what are your thoughts uh on the effect of other neuro modulators on the retina such such as serotonin or endopamine wow yeah i i must admit that i'm very curious about this specifically serotonin i think when we look at the old um papers and not also not the very old one looking at the retina pedal axon so some of them are thought to be histamine energetic other were suggested to be serotonergic and i think that they certainly can do this very interesting interplay between the histamine and serotonin that we know might might be there all over not only in the retina um the reason we started with histamine is because we know that there is evidence that there are no histamine energy neurons in the retina and therefore when you apply histamine it's easier to study the effect and i'm afraid that dopamine for sure and also serotonin might have some uh and some origins also in the retina and then this might be a little bit more complicated to study but i believe in them uh personally we started very slightly to have a look at it but say we did not get very very deep in there well i have a question you know i've been working on in circadian retina and dopamine and we know that the dopaminergic levels vary across daytime what do you think about histamine are they under donor control in the in some of you know synaptic activity or using the yeah it's a good question me and Serena talked a lot about this um so in the mouse we usually know that uh histamine what from from uh most of the studies looked at histamine as a function of attention and not as a function of the circadian rhythm but one study did look at histamine release and hgc expression with the circadian rhythm and it did also show that histamine changes with the circadian rhythm being higher well during night for mice you would expect it to be higher uh during the day for uh for us and then it's really it's it's really difficult to separate between you know the circadian rhythm and the arousal state and it seems reasonable that you know they act in the same way both of them are pushing in the same direction but uh still i mean i would assume that if you would uh take a person at night and do a test that really requires its attention then i believe that histamine will still be produced there so i guess it's um playing ground between the two although i would love to hear the thoughts of any of you if you have something better to say i would love to know okay uh yeah i i mean you know we've been just working with fish michael and i i i don't know if there's any evidence in fish that histamine is um i didn't read anything about fish it it it's certainly not the transmitter released by the uh you know centrifugal fibers in fish that uh that they seem to be primarily releasing a kind of ephomerephomide a kind of peptide yeah but i i guess actually that was one of my questions whether um all whether in in mice and perhaps other mammals all the centrifugal fibers are histamine allergic whether there are any other types types of yeah yeah there are i think there are yes you know variety okay yeah yes i have a question yeah hello i have a question to you selvia i think you know so much about the arousal changes there do you think it's histamine whatever you recorded or at least partly well hard to say so probably not for two reasons okay so i think most of my receptor fields were were not looking down right they were either in the upper or kind of in the uh horizon i guess or yeah so i'm not sure the histamine would would be there i think in the center at night still there for us okay but the second one we saw mostly and i think also liam um we mostly saw depression of the responses whereas when i understood you correctly the responses mostly go up right i don't know so i think you two reported on some increases in this fortanous activity right am i wrong uh so i