Periodic Reporting for period 1 - ViCoZe (Molecular mechanisms underlying elemental and configural learning in zebrafish)
Período documentado: 2021-10-01 hasta 2023-09-30
The ability to learn and remember is essential for all species, including humans, as it is needed at almost every stage of life and for every task. Although research has made significant progress in understanding learning and memory mechanisms in different species (humans, rodents and insects), there is still much that is unknown about other interesting species. This is the case of the zebrafish, a teleost fish that everyone has probably come across in a pet shop, without realising that it is also a research animal model that is still widely used to study different topics in neuroscience and for translational research. In this project, we studied learning and memory in adult zebrafish by developing new, complex protocols adapted to the species and by studying the underlying brain mechanisms and signalling pathways. The results obtained in this project will allow progress to be made in several areas of research. In fact, this vertebrate has a very high genetic homology with humans, making it possible to study the genetic mechanisms underlying human diseases, such as neurodegenerative diseases, in which memory impairment is one of the main symptoms. However, this is still a young model and before it can be used effectively in this type of translational research, it is essential to understand the more fundamental mechanisms underlying the different behaviours. For this reason, the results of this project will contribute to the future use of zebrafish, including the use as a model for studying neurodegenerative, neurodevelopmental or neuropsychiatric diseases. Also, this teleost has a relatively simple and evolutionarily conserved brain, and each of our results will therefore allow us to understand whether learning and memory mechanisms are conserved in vertebrate evolution. Despite the very fundamental nature of this project, any results will have a significant impact on society by 1) allowing a better understanding of the cognition of a new species used in research 2) contributing directly or indirectly to the medical field 3) providing answers about the evolution of vertebrates.
In this project we used several approaches to achieve our goals. This started with a behavioural part where 12 semi-automated home tanks were installed in our facility to test the fish without the stress induced by the handling by an experimenter. The fish were tested for different forms of spatial learning, from the simplest (food was released in one position when coloured light is on) to the most complex (food was released in different positions depending on the colour of the light). This experiment allowed us to identify some of the cognitive limitations of zebrafish and opened up new avenues for designing new experiments that are more in line with their natural behaviour. In parallel, another complex protocol was designed to explore the ability of zebrafish to learn contextual fear. The animal received an electric shock when placed in a green light environment. After conditioning, the fish reduce their movement at the simple presentation of the green light. These different behavioural protocols were combined with different methods to study brain mechanisms. For example, ultra-high performance liquid chromatography (UHPLC) was used to measure the concentration of monoamines in the brain, allowing the involvement of the dopaminergic and serotoninergic systems in the treatment of food and electrical reinforcement to be identified. Finally, in order to study more specifically the brain areas activated during the different protocols, the brains were examined using immunohistochemistry by labelling pERK, an extracellular signal-regulated kinase, the phosphorylation of which indicates neuronal activation. All results obtained in this project will be the subject of peer-reviewed publications and have been or will be presented at scientific conferences.
We predict that findings from this project will have a substantial impact both on the field of learning and memory in zebrafish and other teleosts, as well as on the neuroscience field as a whole. Learning and memory have so far not been well-studied in adult zebrafish. Therefore, every result obtained in this project is valuable and provides new insights into how learning and memory work in this species. The behavioural part of the project has identified some cognitive limitations in adult zebrafish, raising new questions about whether these limitations are due to the protocol or to the animal's brain structures or neuronal signalling. This is not trivial and opens a new avenue for the zebrafish community to explore this question, or simply use these results to explain why zebrafish did not learn in their own projects. UHPLC and immunohistochemistry for tracking neuronal activity are two methods that have become well-used in neuroscience but have not been used routinely in adult zebrafish. This project provides new protocols and standards that will allow the zebrafish community to pursue their various lines of research using these new approaches. Overall, the project provides results that enrich our knowledge of learning and memory mechanisms in zebrafish and possibly other teleosts, including new protocols and set-ups that will be fully accessible in future publications and therefore replicable by the entire zebrafish community, and the opening of new research questions that will feed the next research projects in the field of zebrafish learning and memory. In addition to fundamental research, all the methods and results presented in this project can have a significant impact in the applied sector, for example for future drug development in diseases affecting the monoaminergic system, such as Parkinson’s disease. Although this sector has mainly used rodents so far, the development of appropriate methods in zebrafish is an important requirement for a shift to use this model system in the future.