Neurobiological project Rybka Project
The memory of humans and vertebrates still remains one of the main mysteries of neurobiology. Despite numerous studies, the final answer to the question “where is the memorized information stored in the brain?” Has not yet been found.
Our project is also designed to shed light on this question: the study of the molecular-biological mechanisms of long-term memory with the help of the zebrafish known as “zebrafish” using the Danio Rerio fish.
This is a popular laboratory fish, which is a standard model organism in studies on embryology, stem cells, etc. The danio genome is sequenced, which simplifies its genetic modification.
The fish has a brain rather complex to be able to learn and for having memory. So, for example, she can learn to go through simple mazes, swim for food in a certain place by the sound of a bell, and some other simple "tricks".
On the other hand, a fish is a fish, i.e. aquatic animal with external fertilization, which greatly facilitates its genetic transformation and rearing. Indeed, it is not necessary to isolate the eggs, fertilize them in vitro, and then inject a modified zygote into the uterus, as is the case in mammals.
Figure 1. Daniorerio ordinary (“wild”), above and its fry, below.
The next important quality of the fish is its body capable of transmitting light. In the “wild” forms of danios (Fig. 1), only fry are transparent, but there is a special breed called Casper, which has no pigments (Fig. 2).
Figure 2. Danio breed Casper
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Caspers are almost transparent even in adulthood, which allows them to look inward non-invasively, that is, to study what is happening inside them with a conventional camera and without surgery, in the process of their normal life activity. In the experiment, we will use the technology of luminous protein, which will highlight the processes of interest to us in fish, more on this below.
That is why we chose a zebrafish for a neurobiological project called the Rybka Project ( http://rybka.org.ua ).
Our project is aimed at studying the mechanisms of long-term memory, its consolidation (transition of short-term memory into long-term memory), as well as synaptic plasticity (see link). According to the literature [1-4], several dozen proteins participate in the process of memory formation.
Our task is to investigate the activity of some of them in real conditions, the dynamics of their synthesis in time and space (distribution by regions and fragments of the brain). For this purpose, we are going to use such genetic modifications (transfections) of the fish, in which the DNA segment encoding GFP (green fluorescen tprotein, green fluorescent protein) is linked to the promoter of the gene of interest (which encodes the protein of interest).
Thus, we make the expression of our target gene also entail the synthesis of a fluorescent protein that glows when illuminated with ultraviolet light. This makes visible brain processes involving this gene.
This technique has become quite popular in the last decade, including in experiments with fish [5]. An experiment close to ours, which is described here ( http://habrahabr.ru/sandbox/57219/ ), is also based on the luminous protein technology.
Figure 3. Zif268 binds to DNA.
For example, consider one of the "memory proteins" that appear in our project. It is called zif268 (Fig. 3) and is a metalloprotein because "Ready" protein molecule includes two zinc atoms. In addition, it is a transcription factor and is able to interact with DNA.
After the transgenic fish are grown, our task is to observe them using high-resolution cameras - such that will allow them to see their brain in detail, fixing the intensity of the glow of green protein in different parts of it.
In this case, the fish will solve various tasks for learning and memorizing such as passing simple labyrinths, developing conditional behavior (eg sailing to feed on a bell), learning unfamiliar surroundings, etc.
We will be able to see in which parts and structures of the brain a particular protein is formed, track the dynamics of its appearance in relation to the training session, the connection of various proteins with the type of images in memory, and other interesting facts.
We hope that such experiments will help to come closer to the disclosure of secrets of memory, to understand the mechanisms for the long-term storage of information in the brain, as well as to give a key to the development of more realistic models of biological neural systems. In addition to the main task of the project, other related results can also be obtained: from the study of the expression of other protein groups in vertebrates to the “production” of various variations of luminous fish for aquariums. At the moment, the project is at the initial stage, theoretical analysis, the design of transfection protocols (genetic modification), methods of recording and analyzing data are underway. It is planned to purchase fish, equipment and reagents. There is also a search for funds for the project, because at the moment he does not have any financial support. It is possible to apply for a grant or crowdfunding.
There is a successful experience of crowdfunding for the project “I am a little mouse and I want to live longer!” , Many people from the team who also participate in the Rybka Project.
We plan to publish news on the development of the project, and the results of research - when they appear. Comments and suggestions from all who are interested are welcome.
Our project is also designed to shed light on this question: the study of the molecular-biological mechanisms of long-term memory with the help of the zebrafish known as “zebrafish” using the Danio Rerio fish.
This is a popular laboratory fish, which is a standard model organism in studies on embryology, stem cells, etc. The danio genome is sequenced, which simplifies its genetic modification.
Inset about model organisms
Since the main motivator of biological research is medicine, it can be said that “the main one” is the only type of HomoSapiens, and all the others are “models” on which experiments can be performed. Therefore, such organisms are called model. Their range is extremely wide: from bacteria to chimpanzees. Among the standard model organisms present are E. coli E. coli, bread yeast, C. Elegans nematode worm, Drosophila fly, Danio fish, frog, Axolotl, mouse and rat, chicken, 2 species of macaques, chimpanzees. Each organism is good for certain types of experiments: for example, bacteria quickly evolve, generations can be replaced in minutes, axolotl is used for regeneration experiments, and higher primates - for studying cognitive abilities, occurrence of speech, intelligence, etc. Many model organisms have pure lines - bred by closely interbreeding. Such organisms are called linear, any 2 individuals from one line have almost identical genomes.
Our hero is a zebrafish , a popular aquarium fish from the family Carps, also known as the brachidanio rerio or zebrafish. In the wild, this fish lives in the Ganges basin in India and neighboring countries. Danio can grow to 6-7 centimeters, quite unpretentious in the aquarium content and easily reproduces. The zebrafish embryogenesis lasts 48 hours, after which the larva forms, the fish reaches sexual maturity at 3 months. More than 10 laboratory danios lines are known, its genome is decoded, the fish has 25 chromosomes.
Our hero is a zebrafish , a popular aquarium fish from the family Carps, also known as the brachidanio rerio or zebrafish. In the wild, this fish lives in the Ganges basin in India and neighboring countries. Danio can grow to 6-7 centimeters, quite unpretentious in the aquarium content and easily reproduces. The zebrafish embryogenesis lasts 48 hours, after which the larva forms, the fish reaches sexual maturity at 3 months. More than 10 laboratory danios lines are known, its genome is decoded, the fish has 25 chromosomes.
The fish has a brain rather complex to be able to learn and for having memory. So, for example, she can learn to go through simple mazes, swim for food in a certain place by the sound of a bell, and some other simple "tricks".
On the other hand, a fish is a fish, i.e. aquatic animal with external fertilization, which greatly facilitates its genetic transformation and rearing. Indeed, it is not necessary to isolate the eggs, fertilize them in vitro, and then inject a modified zygote into the uterus, as is the case in mammals.
Figure 1. Daniorerio ordinary (“wild”), above and its fry, below.
The next important quality of the fish is its body capable of transmitting light. In the “wild” forms of danios (Fig. 1), only fry are transparent, but there is a special breed called Casper, which has no pigments (Fig. 2).
Figure 2. Danio breed Casper
')
Caspers are almost transparent even in adulthood, which allows them to look inward non-invasively, that is, to study what is happening inside them with a conventional camera and without surgery, in the process of their normal life activity. In the experiment, we will use the technology of luminous protein, which will highlight the processes of interest to us in fish, more on this below.
That is why we chose a zebrafish for a neurobiological project called the Rybka Project ( http://rybka.org.ua ).
Our project is aimed at studying the mechanisms of long-term memory, its consolidation (transition of short-term memory into long-term memory), as well as synaptic plasticity (see link). According to the literature [1-4], several dozen proteins participate in the process of memory formation.
Our task is to investigate the activity of some of them in real conditions, the dynamics of their synthesis in time and space (distribution by regions and fragments of the brain). For this purpose, we are going to use such genetic modifications (transfections) of the fish, in which the DNA segment encoding GFP (green fluorescen tprotein, green fluorescent protein) is linked to the promoter of the gene of interest (which encodes the protein of interest).
Thus, we make the expression of our target gene also entail the synthesis of a fluorescent protein that glows when illuminated with ultraviolet light. This makes visible brain processes involving this gene.
This technique has become quite popular in the last decade, including in experiments with fish [5]. An experiment close to ours, which is described here ( http://habrahabr.ru/sandbox/57219/ ), is also based on the luminous protein technology.
Figure 3. Zif268 binds to DNA.
For example, consider one of the "memory proteins" that appear in our project. It is called zif268 (Fig. 3) and is a metalloprotein because "Ready" protein molecule includes two zinc atoms. In addition, it is a transcription factor and is able to interact with DNA.
After the transgenic fish are grown, our task is to observe them using high-resolution cameras - such that will allow them to see their brain in detail, fixing the intensity of the glow of green protein in different parts of it.
In this case, the fish will solve various tasks for learning and memorizing such as passing simple labyrinths, developing conditional behavior (eg sailing to feed on a bell), learning unfamiliar surroundings, etc.
We will be able to see in which parts and structures of the brain a particular protein is formed, track the dynamics of its appearance in relation to the training session, the connection of various proteins with the type of images in memory, and other interesting facts.
We hope that such experiments will help to come closer to the disclosure of secrets of memory, to understand the mechanisms for the long-term storage of information in the brain, as well as to give a key to the development of more realistic models of biological neural systems. In addition to the main task of the project, other related results can also be obtained: from the study of the expression of other protein groups in vertebrates to the “production” of various variations of luminous fish for aquariums. At the moment, the project is at the initial stage, theoretical analysis, the design of transfection protocols (genetic modification), methods of recording and analyzing data are underway. It is planned to purchase fish, equipment and reagents. There is also a search for funds for the project, because at the moment he does not have any financial support. It is possible to apply for a grant or crowdfunding.
There is a successful experience of crowdfunding for the project “I am a little mouse and I want to live longer!” , Many people from the team who also participate in the Rybka Project.
We plan to publish news on the development of the project, and the results of research - when they appear. Comments and suggestions from all who are interested are welcome.
Literature
- Garcia-Osta A. and Alberini CM (2009) Amyloid beta mediates memory formation. Learning and Memory. 16: 267-272.
- Alberini CM (2009) Transcription Factors in Long-Term Memory and Synaptic Plasticity. Physiol Rev. 89: 121-45.
- Chen DY, Stern SA, Garcia-Osta A, Saunier-Rebori B, Pollonini G, Bambah-Mukku D, Blitzer RD, Alberini CM. (2011) A critical role for IGF-II in memory consolidation and enhancement. Nature 469: 491-497.
- Milekic, Maria H., and Cristina M. Alberini. "Temporally graded requirement for protein synthesis following memory reactivation." Neuron 36.3 (2002): 521-525.
- Kimberly Dooley, Leonard I Zon, Zebrafish: Current Opinion in Genetics & Development, Volume 10, Issue 3, June 1, 2000, Pages 252-256, ISSN 0959-437X.
Source: https://habr.com/ru/post/224465/
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