Award details

Relating structure to function: Development of dendritic arborisations underlying orientation selectivity in the vertebrate visual system

Reference	BB/R000972/1
Principal Investigator / Supervisor	Professor Robert Hindges
Co-Investigators / Co-Supervisors
Institution	King's College London
Department	Developmental Neurobiology
Funding type	Research
Value (£)	571,829
Status	Completed
Type	Research Grant
Start date	02/01/2018
End date	31/05/2021
Duration	41 months

Abstract

The proposed project will use life imaging in zebrafish to investigate how the vertebrate retina shapes and aligns dendritic morphology of amacrine cells that underlie the generation of orientation selectivity in order to discover neuronal strategies to process visual information. To analyse the structure of type II/III amacrine cells in the retina through development, we will label these cells mosaically by injecting UAS-reporter plasmids (for example UAS:GFP-CAAX) into one-cell stage embryos of our established tenm3-BAC:Gal4 line driver line. Cell morphology and positioning within the retina will be determined by confocal and two-photon microscopy, as we have done in other projects before. To analyse synapse formation in developing cells we will use DNA constructs to express fluorescently tagged presynaptic markers and membrane tagged fluorescent proteins (for determining cell morphology). To analyse how light responses, neuronal activity and molecular cues influence the shaping amacrine cells, we will either dark-rear fish larvae, influence neuronal activity by expressing tetanus toxin light-chain fused to EGFP (TeNT-Lc:EGFP), the inward-rectifier potassium ion channel Kir2.1 or channelrhodopsin-YFP (UAS-ChR2-EYFP). In addition we will use existing mutant lines, tenm3KO and lakritz, to investigate the effect of synaptic adhesion molecules and the presence of synaptic targets on amacrine cell development, respectively. Analysis of dendritic morphology and orientation will be done as described above. To analyse how the dendrites of amacrine cells align with the dendritic arborisations of the postsynaptic retinal ganglion cells we will produce volume stacks of the larval zebrafish retina using Serial Blockface EM and 3D reconstructions. This will be done in our Core Facility at KCL. Finally, GFP-insertions into the tenm3 locus to label all cells in the orientation selective circuit will be based on our expertise in genome editing technology.

Summary

Structure and function in the nervous system are closely related, but it is not clear how structure-function relationships develop. One example where the structural diversity of cells is clearly evident is the vertebrate retina. It consists of over 70 different types of neurons with the function to transform light information into electrochemical signals and send them to the brain. Interestingly, visual information is pre-processed already in the retina and split into different channels, such as motion, colour or edges. This is done through the formation of distinct networks between different retinal cells, for example amacrine cells and retinal ganglion cells. Amacrine cells are the most diverse (and least studied) class of neurons in the retina, with many distinct shapes. Although some progress has been made in uncovering factors that influence shape, complexity and connectivity of neurons, our knowledge about the exact mechanisms is limited and many processes are still unclear. This project will use two specific types of amacrine cells in the retina -previously identified by us- that have a striking correspondence between morphology and function as a model to study how such neuronal structure-function relationships in the brain are generated. These cells form many processes or arbours (called dendrites) that are in the shape of elongated structures, similar to elliptic areas. We found that these ellipses are arranged radially in the eye, meaning along the axis from the middle of the retina to the outside. Our results demonstrated that these cells are crucial for detection of oriented visual stimuli, a key element in visual information. However, it is not clear a) what are the developmental steps to form these elliptic cellular structures, b) what influences the generation of this cellular shape and c) how this shape is related to their connected cells in the retina. Using the zebrafish model system, we will follow the structural development of these specificamacrine cells and identify the individual steps until they are functionally mature. To do so, we will use genetic tools already available in our laboratory to label these cells specifically and image them in live animals under the microscope. In a second set of experiments, we will investigate how different parameters will influence the development of our amacrine cells. For this we will alter activity in these neurons (for example keep the fish in the dark to abolish visual input), change the expression of certain genes in them or alter the cellular composition of the retina and then follow the structural development as before. This will answer some important questions about the mechanisms to control cellular structure. Finally, to identify which other cells connect to these specific amacrine cells and map their points of contact (synapses), we will use a state-of-the-art technology based on electron microscopy to create very detailed 3D models of the cellular structures in the retina. This will allow us to identify how the particular amacrine structure is arranged in relation to the structure of other cells and thus the underlying mechanisms for their specific function. Our work will be an important step forward towards better understanding how structure-function relationships develop in the nervous system.

Impact Summary

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 and increasing visibility of our research locally as well as nationally/internationally. - Business/Industry: 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. - Schools: Similar to the points mentioned for the general public, our research system is particularly well suited for presentation in schools. This is because the field of vision is intuitive and pupils can directly relate to it. We aim to work with students that are normally less engaged with science or from local schools identified as having low progression to Higher Education. The reason behind this is that we aim to give more opportunities to students from such schools. We will provide interactions with pupils and explain 'vision' and the science behind it as outlined in our Pathways to Impact document. Our goal here is it to create opportunities to hear about science in a special program outside the curriculum, break the student's possible reservation to science and science-related issues through our activities, increase their interest and understanding how the eye works and possibly change their opinion about how scientific research is carried out. This may also impact on their future choices to enter a scientific discipline as their profession in the future. - Visitors to Ophthalmology Clinics: People with eye-related health issues are likely to have a strong interest in how the eye works. Unfortunately, most of the knowledge on this issue is not very detailed. In recent years, the therapeutic strategies in order to restore vision have been getting more advanced, including the use of stem cells or gene therapy. However, it is sometimes very difficult for lay people to grasp the details of such findings. We therefore plan to interact with this group of people and inform them about the different cells of the eye, what kind of information is "seen" by the eye and how does our brain process this information. This should lead to a better understanding and possibly an easier dialogue with the health professionals regarding vision. - General Public: It is important that the general public is informed and given an opportunity to appreciate current research that is conducted. We aim to increase the general understanding how vision works and how the eye is built. Most of our sensory information is taken up through vision. We therefore imagine that the general public has a natural interest in our research advances in this field.

Committee	Research Committee A (Animal disease, health and welfare)
Research Topics	Neuroscience and Behaviour
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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