Structural and functional analysis of zebrafish visual circuits specified by teneurin-3

The brain is built through connections that are formed during development. These connections have to be very precise in order to allow the brain to fulfil the functions important for our behaviour. Moreover, they also have to be specific so that nerve cells for particular functions are able to communicate with each other. It is therefore not surprising that one of the major challenges in Neuroscience is to understand the mechanisms that lead to such specificity: How does one cell recognise its appropriate partner and form a connection with it? What happens when these connections are not formed correctly? In recent years, several studies, mostly done in cell culture experiments, have identified a number of molecules localised on the surface of cells that play a role in this cell recognition process. However, this list of proteins is far from complete to fully explain the diversity of connections found in the brain. Moreover, what is urgently needed is a robust system where the formation and function of such connections can be studied in the living organism. Our model described in this proposal, the visual system in zebrafish, fulfils this requirement and is excellently suited for such studies. Different neurons in the visual system are responsible to transmit various aspects of visual information to the brain, for example colour, light intensity, or the direction of a movement. Functionally related neurons connect to each other at specific places in the eye and the brain. In a previous published study, we have found that the deletion of a particular protein at the surface of neurons found in the eye and the brain leads to defects in both, cell connectivity and a specific visual function involving motion. This suggests that 1) this protein is important for the appropriate connectivity between certain neurons and 2) these cells and their connections are responsible for transmitting a specific visual information. However, there are still a lot of unknowns to fully understand this process. For example: In which neurons is the candidate protein present and what is their function? How do these cells form the connections with each other during development? Are all cells containing this protein connected to each other and transmit the same functional property or are they diverse? Do other, related proteins have a similar function?
With this project we will address these questions in a clear and defined work plan. Our experiments are based on techniques that we have successfully applied before. In a first step, we will use genetic methods to label all the cells, which are positive for our gene, so that we can analyse how these cells look like and with which other cells they connect. We will then characterise the functional properties of these cells. Our previous data suggests that this visual function has to do with a specific motion of objects that are seen by the eye, but it is possible that we will uncover additional functions as well. We will analyse what happens to nerve connections when we delete the gene. Importantly, we already have generated the genetic tools needed for these experiments in our preliminary work presented in this application. Once we have uncovered the function of these cells containing our protein, we will determine if its presence is needed on both, the information-sending and information-receiving neuron. This will give important insights on the mechanism of action for these proteins. Finally, we will assess if other proteins that are related to our candidate fulfil similar functions in specifying connections and for functionally related neurons.
Our work described in this project impacts on the general understanding of building connections in the brain. Such knowledge is important not only for our comprehension on brain development, but also because it can help us to develop strategies for a functional recovery after brain damage through trauma or disease.

Our experiments are based on genetically altered zebrafish that we will use for structural analysis of neural circuits and population or single cell functional imaging of visual responses. Most of the tools described here are already present in our lab, including the tenm3-BAC line and both the tenm3 and tenm4 mutant lines that stem form other independent projects. The work described in our different Objectives can be generally categorised in the following approaches:

Structural analysis: We will label tenm3 positive cells in the retina and the tectum by using our existing tenm3-BAC-Gal4 line in combination with reporter lines, including UAS-Citrine or -RFP. The Gal4 expression pattern will be verified independently with newly generated tenm3 antibodies. Cell morphology and stratification of their neurites will be assessed in retina and tectum by confocal and two-photon microscopy, as we have done before. Analyses will be done also in the background of our tenm3 mutant line to assess consequences of gene deletion. To analyse synapse formation between tenm3-positive retinal cells, we will inject DNA constructs to express pre- and postynaptic markers under the control of the tenm3-BAC-Gal4 line. Co-localisation of fluorescent puncta will be assessed by confocal/two-photon microscopy.

Functional analysis: In vivo functional imaging will be a core technique for the project outlined here. We will use the tenm3-BAC-Gal4 driver line in combination with our genetically encoded reporter lines for neural activity, UAS-SyGCaMP3 or UAS-GCaMP5. Light and dark drifting bars will be presented in 12 different directions to immobilised larvae (using a ViSaGe MKII Stimulus generator) while we record visually evoked SyGCaMP3 or GCaMP5 responses using confocal microscopy. Tenm3 and tenm4 mutant lines will be imaged in the genetic backgound of a Isl2b-Gal4;UAS-GCaMP3 transgenic line. Data will be analysed in a voxel-wise strategy, as done previously.

We identify the following categories that will benefit from the project presented here:

- Academia: Our work is mostly related to Neuroscience, however also impacts on other disciplines, including Developmental Biology and Bioinformatics. As outlined in the Academic Beneficiaries section, this impact can be recognised on several levels: Generating new knowledge in multiple disciplines, training highly skilled researchers, increasing visibility of our research, and thus at the same time of our Institute and University.

- Public Sector: We will create new knowledge to understand the functioning of our nervous system. This will directly influence the quality of state-of-the-art teaching that will be conducted at Universities. In a first step this impacts only on the local University where our staff is directly involved in teaching, but upon dissemination of our findings, it will also influence the general content of teaching in other institutions. An example is our previous work that is now featured in basic neurobiology text books. This increase in education quality will, on a longer term, benefit also the economy because better students will enter the work force in the future.

- Business/Industry: Our work will make use of new technologies and approaches to analyse complex data to explain the functional responses in the nervous system. In addition, we work towards discovering possible strategies to build a functional circuit in the brain. These themes are potentially interesting for the Business and Industry sectors that are working in related fields. Complex data analysis is becoming ever more important in different applications and the appropriate algorithms for our work will impact the development of others as well. Our results about generating neural connections are important to develop novel therapeutic strategies to treat brain damage after trauma or disease. It therefore directly impacts on industrial involvement in these endeavours.
In addition, we will enhance the work force, primarily here in the UK, but possibly also internationally. This will depend on the suitable candidates we will find for the project. With this project, we will educate and form highly skilled researcher and technical personnel, ready to enter the workforce, not only in academia, but also in the Industry sector, and therefore contributing to the UK economy.

- General Public: It is important that the general public is informed and given an opportunity to appreciate current research that is conducted. Because our project focuses on a sensory system, vision, it is well-suited to be understood by the general public. This work therefore will increase general awareness of scientific advancements, through public lectures and interactions with the public.
Because our basic work will enhance knowledge on the formation of neural circuits, it will also impact on our understanding of age-related conditions, where circuits can undergo degeneration. On a long term, this will influence existing and make possible novel strategies to reduce degenerative processes, therefore impact on the well being of the general public.

- Schools: Similar to the points mentioned for the general public, our research system is particularly well suited for presentation in schools. This is because vision is intuitive and pupils can directly relate to it. We will be able to provide interactions with pupils and explain neuroscience. This will impact not only on their general understanding if science but also possibly on their future choices to enter a scientific discipline as their profession in the future.