 Okay, welcome everyone to the Provost Lecture Series. So today we're here to celebrate Professor Takahashi before he leaves Oise for his beautiful retirement back in Kyoto. And so please take a seat if you can move towards the front. And so in addition, so we have several speaker phones. Speaker phones, so if you have any questions after the talk please come forward, okay? And just a very brief kind of summary. Since we started the Provost Lecture Series in November 2022, and so we have many excellent speakers and all OISE faculty members, these are they have received significant awards or reach significant milestones such as being promoted to associate professor or full professors. And so today is the number 11 for the lecture series. And again I want to thank everyone who has been very supportive of this Provost Lecture Series covering many different divisions from Office of the Provost, the CPR, and also the core facilities engineering support section. They have been helping us to also CPR to design the gifts and also 3D print the test from the frame, special picture frame. So I just want to again thank everybody who has been a strong supporters for this lecture series. And so there are also some upcoming lectures before the end of the fiscal year. So in 22 days, Yaxia will be given Peace Provost Lecture Series and also professor Satoshi Mitori and also Yoko. And so the two last lectures are pretty close to each other due to people's availability and also the chair's availability. And so we'll send out reminders to remind everyone. And without further ado, so I would like to ask Professor Tengizoya to introduce Professor Takahashi and chair Takashi. Thank you Amy, yeah, right. So it is my great pleasure to take the role of a chair for Tomoyuki's special lecture. Yeah, I was honored and surprised that Tomoyuki nominated me as the chair for his lecture. Yeah, so but, yeah. So probably one of the reason is that we are graduated from the same high school. So in Komaba, yeah. So I didn't know that when I was a high school student but he was a few years, several years senior to me. And then actually, so his classmates includes quite the important people. For example, Dr. Omi who took the role of guiding Japan through the COVID and also Mr. Kuroda who guided the Japan's economy for the last decade, right. Yeah, so and after a few decades, I started my postdoc in New York biology and at that time the patch clamp was the coolest new technology. And I don't know that at that time but this is the kind of method Tomoyuki developed together with the birth sacrament. Yeah, so and then after joining OST, we took serotonin as one of the major research target. And then I realized that Tomoyuki was already doing in very pioneering work of how the serotonin works. For example, in this case in the spinal cord. Yeah, so and then basically wherever I go, I found Tomoyuki's previous footsteps. So I'm very much impressed. And furthermore, most recently we are working on like a functional MRI of mice using our mouse MRI facility here. So at that time, the difference of the brain response with and without anesthesia was a big issue for us to get the paper accepted. And then I again realized that Tomoyuki has already been working on this problem. Yeah, so I'm very honored to have this senpai, the senior from my school leading us in many different ways. So today you will hear a lot about his research career in the last half a decade, half a century. But yeah, so most recently I received an email like this. So Tomoyuki is also like an active person in the OIST tennis team. And then we have, I get this kind of mail and when I go to tennis court, I always find that Tomoyuki always already playing, yeah. So and then without spending any more time, I would like to ask Tomoyuki to present his lecture. Thank you. Thank you very much, Kenji and Ami for nice and introduction. And so I didn't know that I left my footstep. And but I know that he's coming from the same high school, yeah. And it's very nice to know. Anyway, so today I'm going to talk about my past work. And in the initial part I'd like to play back very quickly the work before I come to OIST. And thereafter I'd like to go into a little bit more through a play back of the work I did in OIST. And but it is, I found it very difficult to squeeze 50 years into a 40 minute, to be honest. And so let's start. So when I was a student in a medical school and I was interested in the brain, how brain works like other students. And I was particularly oriented to do a basic brain science and decided to do that. And they eventually wants to contribute to the therapy of the neuronal brain diseases, if possible, that was my dream. So I knocked the door of the pharmacology laboratory which is headed by Masanori Otsuka, who has discovered GAVA as an inhibitory neurotransmitter in Harvard University in the Kufla's laboratory. And now he's a professor there. So I asked him and he proposed me a very interesting project, interesting but very challenging project which is to discover primary sensory neurotransmitters. So sensory neurotransmitter was not totally unknown. The motor transmitter was established to be acetylcholine in 1931 by 35 by Henry Dale, but no information about sensory transmitter. So if you know a little bit of anatomy in the spinal cord, there are two roots. One is a dorsal root and the other one is a ventral root. And the dorsal root is exclusively sensory fiber and ventral roots is a motor fiber which innervates to the muscle. And so the strategy or working hypothesis of Masanori was that if there is a sensory transmitter it must be in the dorsal roots but not in the ventral roots. So it's a very strong hypothesis. But, and so we then started to find a bioactive substance which is exclusively expressed in dorsal roots. So you went to the slaughterhouse and collected the dorsal roots and ventral roots and made a rough, very crude extraction and put it to the ghetto filtration column. And we found that there is indeed a bioactive peak which is just on the dorsal roots but not in the ventral roots. Do you see here on the side? And so this peak has a peptide properties can be abolished via chymotropic treatment. So we called it dorsal root peptide with unknown origin. But anyway, so it has a gut contracting bioactivity. So it's a far from the sensory transmitter by itself. But it is anyway, it is a dorsal root bioactivity. And so and then we realized that the substance peak has a similar bioactivity. Substance peak was discovered by Von Neuerer in 1931. And so this was actually the primary sequence was elicited at that moment. And it was a undecopt peptide. And then we obtained the synthetic substance peak and compare the properties dorsal root peptide in two-dimensional electrophoresis, high voltage electrophoresis and found that they're identical. So we identify the dorsal root peptide is a substance peak. And so and then as a next step, I wanted to know the sensory ending in the spinal code where it goes and where is it substance peak is concentrated. And I did a separation of the region in the dorsal in the spinal code and found it in the dorsal lateral part of the dorsal home. It was highly concentrated there. But when you did that, so when we transact, when we cut the dorsal roots and if you wait several days, then this high concentration in the dorsal lateral part of the spinal cord is disappeared. And also at the cut end of the dorsal roots and there is a huge accumulation of this substance peak activity. So it means that substance peak is synthesized in the dorsal root ganglia and it is transported to the spinal cord and mainly released as a dorsal lateral part of the spinal cord. And so this actually is okay for a candidate for a sensory transmitter, but I thought it is more likely to be a pain transmitter rather than a general sensory transmitter. But Masanori has a different opinion. And so he wants to regard it as a general sensory transmitter. And so the key question is whether it has an excitatory response to in the motor neuron. So I tried to apply it to the motor neuron in first in vivo and failed for one year because pharmacology in vivo and was extremely difficult. And there is no clear cut results obtained by that. So I changed my strategy and into the slice. And I thought I have to see the motor neuron directly to make a proper pharmacology. So to see that, you need a thin slice. So for the right trapezee transplant, so I made 130 micrometer thick slice. It's open to a millimeter. And then this is a spinal cord slice. And then I managed to see the motor neuron under the TIC condenser with 40X auto-imagine objective. And I penetrates the micro, glass microelectrode into the motor neuron and I recorded the action potential and spontaneous potential. And when I applied glutamate by all of races to the motor neuron directly, I have got various responses of the glutamate. So it has, it certainly has a glutamate receptors, but unfortunately it couldn't produce a substance-free response. And then later it was established that substance-free the pain transfer effect. And it has no effect on the motor neuron, no direct effect on the motor neuron. So my failure was not too long. And about that, but at least I had got a preparation here. So which might be extended in the future, I thought. But I quit the project here and then went to the post-doc training to the University College London in 1977 in the laboratory of Bernard Katzen-Milleri. And so where I was trained to monitor the intracellular carcin dynamics. And that was away from my previous experience, but as a post-doc, it's a good experience, I thought. So I followed that. And then we have used a cooling, which is a jellyfish luminescence protein. And which we are donated from Dr. Shimomura. And then we injected it, I injected it into the skeletal muscle of the first twitch and throat twitch muscle. And I found that the kinetics of the intracellular calcium is very different. In the first twitch muscle, the calcium dynamics is much, much faster. So it corresponds well to the first construction and through construction. And this is against the dogma that the first and through construction is made of the myosin properties difference. So but it is, anyway, it is a results. So then, anyway. So after that, I come back to Kyoto. And I come back to Japan and I joined in Kyoto in the laboratory of Moto-Ikuno Physiology. And we are in the same campus. The famous molecular biologist, Numa, was cloning a lot of function receptors. And he actually cloned acetylchone receptor and asked us to help him, help them. With measuring the current caused by acetylcholine to be sure it is functions of their cloned receptors are real functional receptors. So they have used the CDNA, CRNA, into the xenophilic sites alphabetically modulated subunits. And I have made a two volt electrode voltage clamp and applied acetylcholine. And then I had a response which was reversibly blocked by the Clare, which is antagonist of the acetylcholine receptors. And thereafter, Numa wanted to go into the depths of each individual subunit function. And so, in fact, he has found alphabet gamma delta subunit and also epsilon subunit. So, and it was a bit too much for me to work on that. And they have decided to collaborate with Bruce Suckman in getting in. So I went to getting in. And then in the laboratory of the Bruce Suckman, we have used the outside expressed with alphabet gamma delta epsilon first. And then we have seen two different type of channels. One is slow and small conductance. The other one is fast and large conductance. But when we separate, separately expressed, alphabet gamma delta and alphabet beta epsilon delta, they are clearly separated. So it means, and then when we have seen it, we have got an idea. It must be a developmental switch from gamma to epsilon because it has been reported that the receptor channel is small and slow in early stage of the development and it become fast and larger. And it, in fact, it was confirmed in a bovine muscle in the Twitter and adult muscle channels coincided with gamma and epsilon containing receptors. So these are the kind of collaboration with numerous group and I have got that. For the first time, I've got a collaboration with a molecular biogest and it was quite educational and but it was also very, very hard because that the way of thinking is very different between molecular biogest and physiogest. So we had a lot of collision many times. But it was by itself, it was quite educational too. And during the time, I realized that I'm a physiologist. So I talked to Bruce Sackman and whether he's interested in applying the Patrick Lamp technique, he devised it to the Synthesized Collaboration which I developed in 1978. And he become extremely excited and become very positively agreed with it. And so we decided to collaborate together. And in the next two years, I come back and forth between getting in and Kyoto and we established the method. But it was, it took so long time, but at the end it turned out to be very, very simple. So to do the Patrick Lamp recording from the slice neuron, you have to remove the tissue covering the surface of the neuron. And to do that, you have to blow it up. And so with post-pressure, like a grass-cutter does, you see. And so we have applied the post-pressure from pipette, big pipette, and then it removed. Now this is a standard method now. And so, and it was established in 1989. And so this method has distributed to many, many laboratory in the world because this is the first time that one can record from the real neurons in the slice with Patrick Lamp method. And the advantage of Patrick Lamp is that it is extremely stable so that you can record one hour easily. And also you can control the intracellular ionic concentration, et cetera. So it is far better than microelectrode recording I did initially. And hence, it becomes universally available. And then I have to survive. So after several years, I was appointed to chairman in Tokyo University. And I've started, decided to start recording from press and after the tunnel. It's more challenging, not just from the neuron. And so to do that, I invited Ian Forsyth from West University and who has been working at this particular synapse colleagues held. That is a giant synapse in the auditory brainstem. And it's a glutamatergic synapse. And by doing the plan post-synaptic paired Patrick Lamp recording, we are able to see the response like that. And also we can load anything we like into the press and after tunnel. So that I think is a quite useful method. And so we decided to concentrate our work to the press and after tunnel because nothing much is known about the press and after property whereas post-synaptic properties are well known. So this is a black box and a new field. So we can actually cultivate by using this method. So among many things, I most my, one of my favorite work is about the challenge to the saturation hypothesis. So it was strongly believed that post-synaptic glutamate saturate post-synaptic receptor, glutamate receptor and preceptor. And if it saturate the synaptic efficacy is determined by a receptor density or receptor sensitivity, not from there is no room for the post-synaptic contribution. So but I wanted to test it directly and the direct method we can do is to, to load glutamate at high concentration in the post-synaptic tunnel. So that if we, if the receptor saturated the high, if you load high concentration glutamate that is incorporated in the vesicle by a vesicle transporter using the ATP. And then, then it is released but then it should, if it is saturated, it doesn't work. So there should be no difference in the response size. But when we unloaded the glutamate at high concentration, in fact the single vesicular response and multiple response are both enhanced clearly. So it means that is post-synaptic receptor is far from saturation. So this is quite revolutionary at this moment. And at that moment and other synapses, there are different strategies with experience made. And now people believe that the receptors are not saturated. So, so this strategy we use in opposite way. And then we go ahead. But so that was after I retired at Tokyo University and I went to the, I moved to the Doshan University in Kyoto and in 2007 and where I worked with Tetsuhori here. And so, we first wash out the glutamate from the presynaptic site soil. Then the EPSC size declines to the close to zero. And then we, at the same time, we load a caged glutamate. Caged glutamate is glutamate but it is inactive because it is bound to caged compound. But if you apply UV light, it is cleaved so that the glutamate is freed and the glutamate concentration quickly goes up. So we used it and then the EPSC amplitude and goes back to the original size. So from this time course of the recovery, we estimate the refilling time of glutamate into the vesicle and which turn out to be 14 second at room temperature and seven second at physiological temperature. And so this is quite slow in a way. And so it means that it's a late limiting step of the vesicle reuse after recycling. So if there is a very fast endocytosis, something like sub second and millisecond endocytosis, it doesn't mean much to the total physiology because the vesicle is empty so it doesn't do any physiological contribution. So the vesicle has to wait another 10th of a minute, 10th of a second before it is fully filled with neurotransmitter. So it has a big physiological impact to measure the time course of the refilling. And another piece of work is made on the question of the distance between the synoptic vesicle and the calcium channel in the nerve terminal. And so this was made just by collaboration with Angus Silva in the University of Scotland and David DeGorio in the past two. And Rui Shigemoto in Aista and so we work together and so Nakamura headed this work. And we measure the calcium transient from the terminal, calyx of the terminal in the top, which follows action after action potentials in a confocal spots in different spots of the terminal. And we loaded EGTA into the present terminal to chelate calcium and to see how much the EPSC size is attenuated. And we made a simulation out of this and other results, including the freeze fracture identification of the gold-stained calcium channel particles by Shigemoto. And we found it and he found it is clustered in the terminal. So it's a calcium terminal clustered and calcium channel has a cluster. And so the measurement should be from the vesicle to the cluster of the calcium channel. So it's a perimeter of the calcium channel. So this perimeter vesicle distance is turned out to be a 10th of a nanometer, which we calculated and estimated from the simulation. And then it fits very well to the reality. And when this distance become longer, the response become smaller and slower so that the transmission, signal transfer becomes smaller. And in fact, it was a case in the very immature synapses, the distance is longer, and then it becomes shorter during the development. And that's other synapses like hippocampus in this coupling distance is much longer. So the response itself is very slow in hippocampus compared with the delay synapse, the KXFERD, which is extremely fast synapses. So then I, at the same time to the Doshi University, I've got an appointment in OIST. So from now it is working in OIST. So in OIST, I have got a small laboratory in Urumasete and that was with Yamashita and Oisei Buchi and worked and worked on the building also. And so Yamashita, I have been working in the Urumasete and I have been working in the Urumasete and I have been working in the Urumasete so Takayuki Yamashita introduced the capacitance measurements, which is a capacitance measurements of the pre-snap membrane. The membrane capacitance is proportional to the membrane area, as you know, if you know a bit of physics. And this, so that it is to measure the membrane, pre-snap membrane, yeah? And so in fact, one femur followed response to 12.7 vesicle. vesicle has a 50 nanometer outer diameter so you can actually calculate that. And so when vesicles are exocytosis and they are the release transmitters at the same times, many, many vesicles used at the same time. So there is a big jump in initially this exocytosis followed by a slow recovery, that is because of the endocytosis, which is occurring more randomly. And so that using this capacitance measurement, you can actually separate exocytosis and endocytosis. So this is a very useful tool for the dissecting mechanism of the pre-snapt function. So, and Eguchi used this, Yamashita used this and reported that endocytosis by itself is calcium dependent, not just exocytosis, but endocytosis is also calcium dependent. And Eguchi found that exocytosis and endocytosis are kind of balancing. So when there is a massive exocytosis, endocytosis become faster and to compensate the loss of vesicles. And so this compensation is mediated by the very sophisticated cascade, starting from glutamate to NMDA receptor, neutral nucleic acid NO, and PKG, G-kindness and low-kindness and PAP2 to the endocytosis. And so you can say that because if you use a blocker, it can be easily abolished. So the monitoring the endocytosis slowing by the, in the presence of the blockers, which was in many cases loaded directly into the north tunnel, Kaxwell, the president of the tunnel, and we have to use that. And that dimeter of after it become a full time professor in OIST 19 at 15, no, 22, sorry, 215 dimeter of has developed a very nice preparation, for culture preparation. He dissected out the presnapdic and post-snapdiculation separately and co-culture and the four new snaps which is turned out to be giant. And so this is a calyx-like terminal in culture. So if you put it in the culture from slice, the advantage is that it is transparent so that you can easily make an imaging studies. And also you can easily manipulate genetically. So this advantage we take the after the, after it. And using this preparation, the low end gear, monitored, loaded basic, basic fluorescent tag into the, into the, into the vesicle during the endocytosis. And he actually levered abundant vesicles in the nocturne and monitored dynamics of the vesicles directly in the nocturne. And Oomachi, as a postdoc, he loaded instead a few vesicles and then follow the single vesicle traffic. And during the high frequency stimulation, he managed to see the directional movement which started after stimulation. And so these are the kind of advancement. And the ZAK topic and who came from Maruyama unit and joined us and helps us for the PRT issues and everything. And but now he become interested in the proteomics and he devised, he elaborated very high resolution proteins by himself. And so I advise him to learn the synaptic vesicle fraction from Reinhardt yarn in getting in. So he visited Reinhardt yarn in getting in and learned how to separate synaptic vesicle fraction from the synaptosomal fraction. And so he did a synaptosomal and vesicle fraction proteomics and found three times more abundant protein than previously reported. So it's a lot of protein hidden because of the similarity in the structure and also the abundance is low. So he has actually picked up many or almost every less abundant, unabundant protein. And so this is a quite a nice catalog and a platform for the future study. And in fact, many of the this protein including the very rare proteins can be associated with the neuronal disease and cognitive and sensory and motor and these are all associated like this. So this is a big future for the future studies. So but we are still in the electrophysiological side and we thought it's important to characterize the synaptic dysfunction in the major brain disease and which is Parkinson's disease and other disease which is most abundant among people and which is very serious and very difficult to cure. And these two diseases have a common features and both start from the low part of the brain for instance and go up to the higher upper brain. And so the protein, both are carried by protein. In the protein, the alpha-synclin for Parkinson's disease and tau for the Alzheimer's disease and alpha-synclin is a wild type alpha-synclin monomer is soluble type alpha-synclin accumulates and then polymerized and then precipitates into the so-called levy body, LV. And then you can see by history where it is so that they found that it is in the basal ganglia which is the center of the motor control. So when the alpha-synclin precipitates into the basal ganglia, motor control is distorted. And this propagation is like prion and it's an interneuronal or trans-synaptic transfer. And in the case of Alzheimer's disease and it is tau protein which is tau is usually attached to the micro-tubes and in fact it is assembling multiple micro-tubes but when it is phosphorylated it's detached from the micro-tubes and phosphorylation is made by different factors including beta-ameloid. And the beta-ameloid usually increase early in the age say up to 50 or 60 years old and then when it comes to the plateau the tau starts to increase. And as tau increases the cognitive problem starts so it has a more direct correlation between tau and cognitive impairment. And so we thought it is important to follow up the tau as a target. So first we wanted to know what happens if you inject tau or alpha-synaptic into the characteristics of the trans-synaptic tunnel. So we did it and then we found that it works quite strongly and it actually reduced the EPSC size to 20% or less within 30 minutes in the case of tau but when it is stimulated at one hertz that when it's stimulated open to one hertz you have a very little effect. So it's a frequency dependent run down of the EPSC. And similarly the alpha-synocrine when it is loaded at open to O3 hertz stimulation it has no effect but when it is stimulated at 100 hertz the transmission from plateau to post-synaptic by action potentials decrease. So it's a fidelity of transmission is declined with time. So and then alpha-synocrine actually repairs the fidelity strongly in the presence of others in the native network. And so this is so together this is a sort of you know low pass filter effect. So it just blocks the high frequency transmission but low frequency transmission is going through. So it's a low pass filter so-called. And this low pass filter we see also in the effect of the general anesthetics isofluoran so which was found by Honey One post-doc. So in the case of the isofluoran blocks synaptic transmission and that it is more strongly at higher frequencies at 200 hertz it has a very strong effect but open to hertz it has no effect. So it has it is again it is a low pass filter. So the frequency of the transmission is very, very important. And the low frequency transmission is important for the maintain the life. And if you block it the life is lost but high frequency stimulation you can lose it but you lose a high function between such as a cognition and a motor control or the release of the important molecules like a monamine transmitter like a dopamine, silicon and noradrenaline and also peptides including substance. So this needs a high frequency stimulation to release. So the high frequency stimulation is a kind of a key point to the high function of the brain. And so these key functions are in here. So it is not surprising that if the tower accumulates if the alpha-cylinder accumulates a basal ganglia it's function is impaired. And if it accumulates the tower accumulates in the hippocampus it appears it's low role of the cognition, the learning in the memory. And it is not surprising also that when general anesthesia is applied everything is attended. Everywhere. So that is our tentative conclusion. And so the next step is to find out the target of the peptide and the pathogenic protein. And so we did a capacitance measurement and we found that it was endocytosis. It was attacked first. So the tower alpha-cylinder actually appears endocytosis first. And when endocytosis is compromised then the exocytosis gradually declines. And because of the recycling and the recycling cannot catch up the basic supply at the release sites which is more severe when the release is at high frequency. So it explains why high frequency transmissions appear so easily. So, but anyway the target, main target is endocytosis. So the next question is why is the endocytosis appeared by tower synchrine? So we have, and you have to remember that the tower is a microtubus binding protein and it actually assembles tubulin into microtubus. And also our synchrine has a similar effect. So, and in fact if you inject the tower into the karyx terminal in the green picture and you'll see that the tower loaded in that karyx and in this karyx microtubus also increases in pink color. And the important thing is that there is one another player which is called the dynamin. And the dynamin, the GTP is nomadic and GTP is of the 100 kilodalton large monomeric GTP. And this is important for the endocytosis because it cut off the membrane from the plasma membrane to the vesicle. So it's the final cutting off of the membrane. And the dynamin, surprisingly the dynamin is a microtubus binding protein which I found in the old literature. And it was discovered as a microtubus binding protein. So I thought that it must be the dynamin which sequestered, which is sequestered by the microtubus over assembled by the tower. So that was my hypothesis and we actually tested that. And in fact here in the vitro experiments made by Takei and Nupayama and so dynamin by itself is soluble most of the time but if you put the microtubus, most of them are kind of insoluble. So it's a PPD in this. Likewise, in the slice loaded with the spikes with the tower, the insoluble immobile dynamin in places. So it means that the free soluble dynamin decreases. So this is consistent. So it's a time to rescue the disease. And so we actually, the simplest way to rescue the synaptic dysfunction is to depolymerize the microtubus. So we have used the nocodazole which is known to be depolymerizer and it's nicely or perfectly this decovers and the synaptic entailment in both of the synaptic tower and the NBTSC lander and the fidelity lander. So all are actually solved by breaking out the microtubus. But we need more specific. We need to rescue because nocodazole is not going to be a good drug. So we actually try to find out the interaction side between microtubus and dynamin and which is not known. So we had to make a screening assay and we randomly made 20 peptides at the pH domain which is sort of the same as the NBTSC lander. pH domain which is sort of the pH domain which is instructing so. And we found that the number five peptide which is D-H-D-P5 we call has a risking effect. So we could rescue the in vitro binding and rescue the endocytosis in the brew and then rescue the synaptic lander. So this peptide pH-D-P5 is quite good in vitro. So we have a very small time left but we have another two slides to go. So then we wanted to load it to the NBTSC lander real animal in the brew. And so we decided to infuse it into the Alzheimer's disease model mask. And so before that we have to be sure that it goes into the brain. So we put the other cell permeable peptide sequence at the pH domain and monitor to monitor the FITC in the end tunnel. And also we made a scrambly control and then we infuse the mice through the nose. So nasal infusion. So five times, no, no, four, five days, once in one day and five days a week except for Saturday and Sunday. For four weeks. And then when we see how that cutting the tissue section we found that fortunately this peptide was found in the hippocampus. So this is because probably the noses, nasal membrane is very close to the hippocampus. That is one advantage. Another advantage that that part is not very much protected by the blood-brain barrier. So these two, we have got that dragging into the brain. These are the hippocampus region. And these white spots on the right-hand side is the other FITC of the THD design. And then we did a behavior task. And so this is a Morris water mice, so-called the famous and standard method for the learning and memory test. And so when the mice is put into the autobus, which is a target solution, and although there is a platform, mice don't know whether there is. And the mice learn how to get it with time. And it becomes faster and faster in normal cases. But in this AD, Alzheimer's disease is more than mouse. It never learns, and so it is nearly flat. Even after all these try-ups, and it's the same for the scrambled infused mice. But if you infuse this DP5, then it learns like a wild type, so it actually acquires memory, learning ability during the Pfizer infusion. And the right-hand side is a memory test. It's testing the memory retention. So in the fifth day, you remove the platform and let the mice swim in the bus, and then you follow the trajectory. And the original place of the platform is one quadrant. And so how many times the mice visit that quadrant is evaluated. And in the case of the AD mouse, it never visits there, so preferentially. So it's less than 20%, 5%, but in the case of PhDP5 applied mice, this improved and more than nearly 40% of time they are staying in that program. So they are remembering whether that there is a platform in that quadrant. So it actually recovers the memory, so learning a memory is required. So it is our interpretation. So just to follow my trajectory, I started to find out the sense of the transmitter and then made a since last preparation and the test and the excitation. And then after some training in the UCL and also in collaboration with molecular biologists, I developed a particular method in synthesize. And I actually put it to the personality kind of preparation for this health. And we introduced capacitance measurement and basically N-cytosis. And also presented to the measuring instruments, but then we tested the synaptic dysfunction mechanism in Alzheimer's disease and part of the disease. And we were able to rescue synaptic dysfunction at least in the AD mouse mode. So the final end should be to rescue the patient. But the time is over. So that is, but it's okay. It's not my job to do that. So it's a job of the pharmaceutical company for the clinician. So I'm happy to open these results to the world and to some good laboratory to advance that. So these are our members, staffs. And so thank you very much for your listening. Thank you very much, Tomoki, for your long-time contribution to OIST and then the high level of research. Yeah, okay. Yeah, and then there's a reward for this seminar. So the provost office prepared this plaque so that sentence is a dear Tomoki in admiration of your pioneering investigation into what really happens in the synapses in our brain. So enjoy more time to play tennis in Kyoto. Thank you very much.