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Special Research Seminar

Transcript of Special Research Seminar Video

Karl Berg: 00:03 Okay, well thank you very much for coming. I am Karl Berg hosting Dr. Guerrero… is visiting from Venezuela and...Dr. Guerrero is the associate professor at the University Central in Venezuela where she is also the chief of the Department of Physiology in the Faculty of Medicine...the School of Medicine. She received her bachelor's in Biology from the University Central of Venezuela in 1985, and in 1999 she completed her Ph.D. in Neuroscience at the University of Louis Pasteur in France and she's widely published in Endocrinology, especially in fish and published in just as an example: General and Comparative Endocrinology, Histochemical Cyto Chemistry and Biological signal receptors. So we are very happy to have Dr. Guerrero visiting with us. She's also collaborating with us on some work were beginning the hormonal underpinnings of a social learning processes in birds. So without further ado, thank you very much.

Dr. Guerrero: 01:34 Yes, thanks to all of you for your interest. Yes, well, we are a new group a colleagues group of science students. Some of them already go. Dr. Poleo is a colleague that works in another University in the central part of the country which...who collaborates to have facilities for fish. Because we are in Caracas, in Caracas in the Faculty of Medicine we have no facilities to have fish and to have and to do fish "in vivo" experiments. So, this for has been developed in the last nine/ten years, we began to work with this specie where nobody before was working. Then we had to do everything about this specie.

So, this is a catfish, a local catfish, and is interesting for its meat, that we can eat, and it is a very nice meat. So this is the second reason because we choose this specie, we would like to reproduce it to have it for protein, for human consumption. So, let me began to share some basic information about Melatonin and about our, our system.

Melatonin is an indoleamine synthesized from tryptophan. Four enzymatic steps to have Melatonin, ok? But Melatonin is synthesized only during the night, ok? Why? How it happens? Because Melatonin is... the Melatonin synthesis is controlled by a so called circadian clock. In mammals, is barely known all the structure of this system but is very similar in most of vertebrates, ok? In mammals, it is a neurostructures ubicated in the hypothalamus, suprachiasmatic nucleus of the hypothalamus. So this is a genetic system that controls the enzymatic transcription. In this case, the third enzyme in this Serotonin N-acetyltransferase (NAT), which is controlled, full controlled by this system, circadian clock. During the night, the circadian clock, opens the pathway to this synthesis and then by a nervous multisynaptic way, Melatonin is, excuse me, is transcripted and translated. So activity increases one hundred times, ok? This is the great, this varies during night hours. So this is increased in the activity of N-Acetylserotonin and the synthesis of nocturnal Melatonin. The fourth enzyme, Hydroxyindole-O-methyltransferase, changes, increases the activity also controlled by the clock (this is in mammals) but is a little, really the control is the third enzyme. You have a...?

Question I : 05:41 Yes, so you sort pointing at, as it transpires, the transpictural control is govern by dark, correct?

Dr. Guerrero: 05:47 Yes.

Question I: 05:48 that means that half of gains must been pretty short, in other words, by the time of daylight comes, gain some is gone.

Dr. Guerrero: 05:55 Yes, the activity of the enzyme, of the half-life, is very short. It means that when that comes all the activity is gone.

So, this is very important. We want to look if it happens like that in our model specie but our interest is, if it happens…what have to do this kind of control mechanism in a fish, in a specie that is tropical and photoperiodic, it means the time of day relatively to night are almost the same.

The second topic is the control of reproductive function. Reproductive function in most of superior vertebrates and fish is a neuronal, the brain especially the Hypothalamus through this synthesis of GnRH as hormones in mammals and, as neuropeptides in fish, can modify or stimulate the Hypophysis to synthesize two gonadotropins, GTH1 and GTH2. These hormones go to the…by and endocrine way, go to the…arrive to the general circulation and stimulate the gonads, the synthesis of sexual hormones, and with an auto-clean regulation in these glands, the synthesis of the steroids increases but at the same time the growth, the development, and the maturation of the gonads also.

So, Oogenesis and Vitellogenesis occurs and gametes are mature, spawning/espermiation is the last step to have the event of reproduction. But in animals it seems there is also the influence of other parameters, as for example: ambience parameters. Temperature, light, rainfall, photoperiod, light through photoperiod. So this environment parameters are integrated by sensor assistance of the reproductive axis and modify or trigger the changes for finally reproduction comes. One of the ways…this occurs through the light and photoperiod is when light is perceived by photoreceptor sensorial cells, specialized cells that we find in the eyes, in the retina or the pineal. There are also other structures in fish able to have photo sensorial abilities. But these are the main ones.

Light arrives to modify some of the function of the axis. The first compound within, acting in this way is Melatonin. In fact, there is evidence, about the presence of Melatonin receptors in fish, in many areas of the reproductive axis. So we ask: what is the role of Melatonin in reproduction, in controlling reproduction? To work with this we choose this specie: Oxydoras sifontesi which is a catfish. The local name is Bagre Sierra Negra, the bad translation is “black saw catfish”. Because it has a modified plax here, like a saw. This is a Doradidae, a Doradidae catfish, where the big catfish belongs. This catfish could have 1 meter or even more and some kilograms of weight. In the first year they arrive to sexual maturity. So, it is easy to have it until that moment in the laboratory, in the fish aquapuncture laboratory.

Question II 11:50 And, what is their lifespan? How long they live?

Dr. Guerrero 11:53 Years.

Ok, this specie the Orinoco basin river. Is an extensive area of the Venezuela flood planes that have a particular logic site. We have two climatic periods, very important: the dry period or dry season, and the rainy season. Here we have a graphic about precipitation, rainfall and temperature rate. When rain comes, the river spinning to increase its flow, and one or and a half month after, the river flows out. This is a time of the temperature, is minimal. This is an important moment for all the community, all the species in having in this region. Birds, insects, fish. Everybody in that region changes its status reproductive, or perform reproduction, the principal activity of reproduction that is spawning talking about fish, in that time. This is a parameter very important. Many of the parameters of the water change at this moment. The conductivity of the water, the light that arrive to the bottom of the river. Everything changes because the quantity of water. So, we know that this is not easy, to know exactly which parameter is the most important.

So, in fish we have we have the gonadal cycle: the preparatory and pre-spawning, when the Oogenesis begins and Vitellogenesis develops. The gonads begin to grow, the increase of weight of gonads, until they are already mature so the spawning occurs when rivers flow out, when the rainy season is really advanced. So it occurs, in average in May/ Jun. The dry season begins in November and continues until March, maybe April. So, in this time the preparatory phase and pre-spawning develop. After the spawning, the weight and size of gonads reduces and the fish goes to a sexual quiescence or sexual pause until the next season.

So, we begin our study by characterizing morphological aspects or features of the pineal organ. We want to know how Melatonin is created, how the structure, and things like that. We use: Light microscopy with fixative dyes, classical histological dyes and Transmission electron microscopy for ultrathin sections. We use fish that were kept in aerated tanks under natural temperature and photoperiod. Here you can see the lateral view of the fish brain, which is interesting is the very big cerebellum that is over, directly, dorsally, and anterior. Here the encephallum, optic tectum, and hypothalamus, and brain stack. The pineal is a long structure with a long stalk and a prominent gallbladder (vesícula), the pineal vesícula. It is no easy to view and to arrive to this structure.

These are the cuts that we have to do to obtain a piece of skull, a piece that contains the cranial bone and also the pineal. This piece that we take from the center of the skull, cranial skull, we cut along to have the dorsal portion. This is an internal view of the dorsal portion of the skull. Here is the cranial lobe, and here you see this dark line, is the pineal window. Is a place in fish where…the skull is really really fine even there is no bone only the membrane that allows light to travel. And the pineal is just on the pineal window. We can see by transillumination with the skull over a light under a microscope. We can see the pineal dissected

Speaker 2: 21:18 And these cells are also able to see the size. So this is a very specialized itself with a poor photo receptor poor. And also it can release Melatonin to the, of both. It also can make, uh, some synaptic contact to other neurons and other cells. So we look for this kind of infrastructure to have an approximation to the possibility for the receptionists. Finally, after many micro graph, we have this image where we find our formation very similar to a photoreceptor outer segments or segment and say I'm here and here also, this was apparently not in that part. That was the but this. This is true too. It's apparently a that that had the same strict. So we, we, we thought that is a photo receptor or sale maybe now we have to know about the feasibility of the secretion of Melatonin. So then the question, where does the separation have seen it that they be art or an annual variation?

Speaker 2: 23:06 If yes, are these melatonin rhythm's relate to sign books and are then controlled by environmental conditions. So this is a question we wanted to, to evaluate with stories about the plasma, the plasma profile of Melatonin concentration. Okay. It means the changes of illness, depression in 20. Okay. No, we decide to repeat this profile into validation. That alone, the maturation cycle many times four or five times in a year in each of this phases. And also on the three different illumination condition, it means keeping a group of fish and adapted to terminate illumination. Constant light or darkness continue, or we work with a plasma samples from collabing of tendon and the requests that are managed by a aside from embarrassment.

Speaker 2: 24:47 So this is the profile, the classic profile of the 24 hours. Melatonin secretion. The day laborers are very low. We are talking about four, five peak around 4 million during the night. Increase to around 20, sometimes more. But this is the the average compared to other species of fish for sample ceremonies. This a lot to my concentration is slow. We can find in there 800 I program, something like that. It's very, very low. I think it is because fish is not very well to her for these changes. Okay. But if it is permission and then a yellow bar intensity with Alex to we showed that the quantity of light was wasn't enough because a forger for they they take in inhibition of. Of a synthesis of NSF is a most. Have more than 5,000 have looser lumination low doesn't work. So in every apparently is like, like we know about other fishes, so we repeat into different moments in July, it means it's Bonnie period in April with postponing ring, I'm presupposing in April. So the difference in concentration was significant and it was very interesting because we know that Melatonin could have inhibitory a participation in the development of governance. Okay, in mouse and all in some fishes. So this make us to think about the possibility that in responding system when gonads are growing fast, militarily, eh, simulation is low to a lead role goes and after spawning. So it increased or reduce, disappear the sexual function.

Speaker 2: 28:04 And I saw also in humans it happens. One of the changes mentioned it when girls, when girls are in poverty just before that they becomes a sexual in nature. One of the things can happen to occur is the reduction in Melatonin secretion in the secret. So it is possible that like in the same way, if you contribute to the development of our maturity of have another structure for example. So we repeat this initials in the other periods, but it's, it's, uh, ask. The difference is during the night and at midnight, around midnight for time and for me to have only two points midday. Okay. No difference and midnight. Okay. And we check for the other periods and we here show the difference between midday, midday and midnight. So the pattern repeats July and October, which are press phoning and has center. We have a difference March and April, it's reduce and then July next year already increased.

Speaker 2: 29:49 So that was very interesting way to, to find that they have not only address the four hour profile variation of anatomy, but also this, this pattern keeps an ama profile. So the daily variation give place to an annual evaluation. So we can say that in this fish happen, it happens in seasonal animals in. But um, for example, Mama's a civilian and northern European Hansen who's a activity of revolution is only in the spring and summer. And then changes or in civilian house, the increase in Melatonin in, in the winter is very impressive. And the cornice and always representing structures are really, really reduce. So, uh, in tropical fish, is that the. That's surprised us. We can have also a profile we decide to, to, to compare this Angela a lot tonight, a profile with other, uh, variation Norway, for example, in gonna Index, that is the ratio between the weight of the governor to the body weight in percentage. So this is familiar in pink and black militant two years by two years we have an opposite comportment, that behavior of a systematic index with a municipalization in May in females also.

Speaker 2: 32:16 And we also look for sex hormones and, and a secret. And we found the same study. Oil in female fish was also very, very low windmill. Military was hard. This was a, an test the same. So these inverse relationship with sex hormones increase our, our convinced that something happened in between the Sitcom petition to modify the illumination. So we'd have three experience. Normalizes means like the mission and these two are 24 hours, but all of these animals were at that, the back condition and condition three days before the date and the time we crossed that line. And the blue is there are day in the dance. We know that darkness on team. So with constant, like no changes, no changes in the last appeal. Well with Boston, yes, we have one point significant the difference, they interpretation of these results in tow the rhythms couldn't means that that is a system may maybe a cabin feel responsible for the month of this freedom not to. Okay. Because this is genetic makeup that repeated cycles of 24 hours. Okay. But if he's able to synchronize with the extent of the pigs don't eat, permit us to, even if we are out of the light senior container, the function of certain functions with the same. A pity that ECB at the same alice. Okay, sorry.

Speaker 1: 35:03 So if you keep the fish for four days into dark, doesn't admit of Tony Devils high during those four days, keep deficient of dark for four consecutive Tony, then 12 hours as a state hie or does it

Speaker 2: 35:24 in darkness? Yes, it does. It does with outside data. And here is outside that. Okay. He was in the group was not clear from the don't because uh, the grave, our senior, nice. The outside, it's nice outside, but inside the time it is dark every time. And he is like, okay, sorry. Thank you for the piece. So when, when, when says Carolyn rhythms or they use condoms continuum in absence of lighter senior Istation, the completion is that they make like a flow is working to maintain this retina. Both. We don't know where to find this connector. No, after that I can show you this result because it's very long. But then we test the sexting the pineal glands and have a uh, in a to chamber with lighting conditions, different like new condition. And apparently we don't have a lot to mckinnon's of the rhythm. We have inhibition of the senior by life, but a week after many days was he loves he to the conclusion that if it has a psychotic, I'm not sure if it's apparently it's not inside that they find yourself. Maybe the brain.

Speaker 2: 37:39 We have to look a then we want to to look for some police stations, not seeing what damage of Melatonin receptors, a gnrh in the hypothalamus, in the brain and solely and the rain and the hypothesis we performed. This will bring these two techniques or two rather figure from sectors in sections of the brain and the chemistry of the marriage of brain sections. This was a rather graphic with the gang was Yolo County, 125 west and antibody rises with that genetic sequence is a segment that works with most of. So our first to arrive to the, to, to interpret the results we need first to perform a brain brainy, run into mediation. Then we did the colorations are alone, the brain, and then the chemistry. This is the anterior part of the brain and graphic the same.

Speaker 2: 39:31 So we found some, some very important because we know our generate sets in this, in this, uh, nuclear. And we look for a senior living of binding of Melatonin. That means receptive presence. So we found a new clinic. Okay, that's important. And, and this venture of the here in this mentor, they will look and important, very important area. That area where the principal neurons that synthesize. Now we found here the posterial carrier new. Okay. And here some labeling. So, uh, it is, it means that in the same area, no many neurons and have maybe Eh, Eh receptor formula, then hypo times the same around the third ventricle, like it's important for generations this synthesis and we found ways around your base bands with the hightest. So a finally from three important nuclear for generous since this is we found in all three, look at extension of the system, not in the same state because we are not able to do that was in the same, eh.

Speaker 2: 41:47 Then we look also by binding the hype, the hype of hypophysitis and donor and they hyperphysics clearly show the anterior part, the set of lately and generational of course. And that in the governance was not easy in general with a high density here, specific banking, but it was not clear at all. So then, um, we, that collaboration of Dr Tran's, University of Paris, we have to subsidize a preceptor of military. And Wednesday he finally, every fund that test this, this and ovaries express the military in the second and the Beatrice was even more interesting because only one of these two receptors are expressed. They may one a which is very important for women and not men, once he says to say that is not present in fish.

Speaker 2: 43:24 Okay, so with all this relative results we try to contribute to ideas. We can say that electron in a 24 hour work knife, the knife or Orlando increase senior station maybe to the hypothalamus to the bonus and during the summer and during the night, no of Melatonin because we can't plasma so we we think that an activity the day Melatonin doesn't control dysfunction but more important for us is the fact that and this vicious press assistant but it's not for the burial but associated to the big external evidence that this is the racism when may actually increase and this is the period where representation is low. Gonads reducing weight and then try to reduce and we have also changes hormonal changes. So that's all. Thank you. Open. This is the proceeds of the data where we work in collaboration with Dr. Paramount, polio and we have the inside when when we need to control. Can you look at transcripts, change or no change? No change. Actually what we're going from tree to tree nuts enough to justify the one who's changing that many, many times is it means that the bottleneck transport. So if this increasing activity, so no problem with other incentives to a the.

Speaker 1: 47:43 There was one, one of these enzymes and you told me the beginning when I asked you that that one is follows the day night cycle nat. Exactly. Net. So what controls that? That's the million dollar question.

Speaker 2: 48:04 Yes.

Speaker 1: 48:07 Yeah, but this can be something like love and hate. Come on this per se

Speaker 2: 48:16 from that three or four. The new rule is that the rice, the somebody else there we can get you is that so with the cell receptors debate. This is off the transcription to grip to the MPC people and simply go and then grab and there is inside also a small niche that is very interesting for seasonality in Mama's when when next winter they make the interest in land mechanism out or reduce. So there is an other gene, the gene who's activate later also not other than any single issue, but later added the the transcription factor produce. I love this

Speaker 1: 49:56 and if it's bacteria redundancies in that complicated, then I do a full audit. Understand localized, localized, the receptor Melatonin in the brain. How did you feed your whole identity? Is receptive just before lunch didn't

Speaker 2: 50:29 exactly. Reset is a very. It's not. We cannot be specific with the graphic because it's simply binding. Highest specificity because the concentration, because we know that a receptor of Melatonin at a high affinity receptors that the concentration in nanograms is enough to buy so that our other structures, because Melatonin is it is, is complicated. Monotony comes from the aromatic amino acid, it keeps the advocate is to traverse the member. Melatonin could traverse the member a platinum member and bind to other things inside the sale. Okay, but in mammals and this filing is with low affinity and also have other functions different to and a system the function of the many, many things. People say that Miller Melatonin's able to to work, but the concentration are always had very low concentration. Only these receptors that a, a g protein receptors hydrophilic. So we use a concentration of Melatonin for labeling. Very, very low. So in this way, we think that this is the receptive than we are, but we can't know if there is one a and one scene or one. This was a pen when my student went to to laboratory of Dr. Com the receptor and then perform the pcs and we have some results that are not yet published.

Speaker 2: 52:44 The half life is short somehow or something maybe I didn't picture for the dark, the landscaping, the darkness or in in, in this experience, this three days, three days in constant, and you begin to some for the better behave, behavioral changes, etc. Me, what other behavioral change? So many. We can't. We don't. We don't pay attention to Me. We were focusing. No way. We can't open the close. Then it's related to activity. So the the in the dark or light activity. Physical activity is. We haven't ever looked for this, but there are some words were in mouse. Even wear a for example. hamster has a activity pattern that this clearly sick. During the night they increased activity and in the eh, people have ripped in, in animals without pineal gland that has been financially to reproduce by institutions, continue to control it during the night of melatonin, so it has to be with a many, if not all the caring functions, the evidence once like a locomotive, this locomotor activity, maybe reproduction a pc to install it, but there are many physiologic permitted, even humans that have activity.

Speaker 2: 55:05 They have. I was thinking, for example, on things that I stress but also a continuous light or darkness by be masking something and these animals might be a. There are other other things that maybe happened. Days is three days. Who has the time we chose today, well after leukemia, and then not very long to have a, a rick change in physiological comportment behavior, eh, but uh, it's enough to disappear the rhythm. If it was no money but they took. So we are not sure, but we chose this time three days because of this, to, to have a time to, to, to look if there, if there is a secret then club. So three, four, five days before the darkness or continuous learning rhythms continuing. But there are eh works with, with a selfish need, efficient copy. What are the rhythms container during 10, 12 days. You with the. For the case. Because when we work and with the instructor, the reason was not present, but yes. Not with the pioneer. Second. Excuse me. Thank you.

Speaker 3: 56:59 If that's it. Thank you. Thank you everybody for coming today. Yeah.