Functional analysis of visual circuits in the zebrafish Robo2 mutant.

The formation of functional neural circuits is critically dependent on the formation of highly specific connections between neurons. Understanding how such precise neural circuits arise is therefore a fundamentally important challenge in developmental neuroscience. The enormous complexity of the brain means that this challenge is a formidable one. However, a common feature of the brain is the arrangement of neural connections with similar functional properties into layers. This organization provides an anatomical point of reference for studying how neurons target one another during periods of circuit formation. This has led to wealth of information concerning the mechanisms that drive the structural development of layers and of the neurons that contribute to them. However, the functional significance of layer formation is poorly understood. Why do we have a layered brain, and what are the consequences for brain function if these layers don't form properly during development? Intuitively one would think that disordered layering leads to functional deficits in the brain but this may not necessarily be the case. The developing brain is highly plastic and adaptable and developing neurons may still connect with their correct partners even if they are in the wrong place. Understanding how the developing brain copes with a disordered neural landscape will give us insight into the extent of the brains intrinsic adaptability. This has direct implications for understanding how the damaged brain copes with neurological damage caused by trauma or disease. The aim of this proposal is to use the visual system of zebrafish as a model system to try and understand how neural circuits develop when the layered architecture of the brain is disrupted. In the visual system retinal ganglion cell (RGC) axons, which are the neural information cables that connect the eye to the brain, are organized into layers within the brain. However, defects in the robo2 gene in zebrafish results in a disordered arrangement of RGC axons. We aim to understand how the functional properties of the target neurons are impacted by the disordered arrangement of their RGC axonal inputs. We will use zebrafish that have fluorescent indicators of neural activity within the brain. Active neurons fluoresce brightly while silent neurons are dimly fluorescent. Thus, the neurons themselves can tell us when they are active and when they are not. Because young zebrafish are translucent we can see these fluorescent signals inside the brains of living intact larvae. We are using these fluorescent fish to characterize the response properties of neurons within visual regions of the brain when the layered organization of their RGC inputs from the retina is disrupted. Can functional visual circuits form despite the disordered arrangement of layers and the absence of Robo2? If so what mechanisms does the developing brain use to compensate for the layering defects? If, on the other hand, we find that the function of visual is disrupted we will have provide valuable new insight into the functional importance of Robo2 and layer formation in the brain.

A common feature of the brain is the arrangement of neural connections with similar functional properties into layers. This provides an assessable system for studying how neurons target one another during periods of circuit formation and has led to many studies of the mechanisms that drive the structural development of layers. The functional significance of layer formation is poorly understood however. The aim of this proposal is to use the robo2 (astray) mutant zebrafish as model to address this. Retinal ganglion cell (RGC) axons show layering defects in the tectum of astray mutants but the consequences of this for tectal function are not known. The first aim of this proposal is to use in vivo functional imaging of RGC axon terminals within the tectum of astray mutants to describe the functional development of three classes of direction selective (DS), and three classes of orientation (OS) RGC. We will also generate high resolution functional maps of these RGC axons within the tectum to provide the first detailed picture of how axons of defined functional classes are disrupted in the astray mutant. These experiments will provide a platform for the second aim of this proposal which is to ask how and whether development of DS and OS tectal neurons are affected by the disordered arrangement of their RGC inputs. The proposed experiments are designed to test the functional significance of layers in the brain. They will also provide insight into the extent of the brain's intrinsic plasticity by asking how the developing brain copes with a disordered neural landscape.

1. Academic beneficiaries: The topics addressed in this proposal will be of broad interest to those in the neuroscience field. The molecular and activity-dependent mechanisms that ensure lamina-specific targeting of neurites is an area of intense research, particularly within the visual system. The experiments outlined here will be important for placing these mainly molecular and anatomical studies in context. The Robo family of transmembrane receptors and their Slit ligands have also been studied extensively but the functional importance of Robos in the brain has received relatively little attention. Lastly we will be asking what plasticity mechanisms are engaged by the brain to cope with the disordered layering of RGC inputs. This will be of interest to those studying plasticity and repair in the brain in general. To maximise the impact of our research on the academic community all findings related to this proposal will be published in international journals and be made available on PubMed central. Additionally we will attend national and international conferences and seminars to present our findings to the scientific community.

2. Communications and engagement with the general public. The Meyer lab regularly aims to promote scientific research as a career by hosting undergraduate summer students from King's University and other UK academic institutions and by hosting one day visits from school children. Members of the lab have also taken part in the London Science Festival: All Things Bright and Small, by presenting images obtained during the course of our research, and the science behind them, to members of the general public. We are also working with four Masters students from St Martins Art College who are engaged in a communications design project that will represent our work as part of an exhibition for the non-scientific community. Lastly I have presented our research to the Judd Grammar School science club in February of this year. This will be a yearly occurrence. By bringing science to life through lab visits, talks and exhibitions we hope to generate enthusiasm and an appreciation of scientific research within the general public.


3. Contribution to the nation's health: By addressing how the brain copes with a failure to form layers we will be studying the extent of the brain's intrinsic plasticity- can functional tectal circuits develop despite spatial disorder in RGC inputs? Studies of neural plasticity are not only important for understanding how the brain develops but also for understanding functional recovery of neural circuits following neurological trauma or disease. Thus, while these experiments do don't address disease related questions directly they are necessary to understand and treat neurological disorders which, collectively, place a major burden on society.