In Vivo Analysis of Neural Circuit Formation in the Zebrafish Retinotectal System

We are trying to decipher the mechanisms that guide the precise formation of the vast numbers of connections, known as synapses between nerve cells in the brain. By investigating this problem we hope to a gain insight into the processes that lead to nervous system malformation and that limit the capacity of the adult brain for self-repair. We know from watching neurons as they form synapses in the intact brain of live zebrafish embryos that there is a surprising amount of trial and error involved- many more synapses are formed that are maintained. But how does the brain distinguish between correct and incorrect synapses? Unlike a computer, which is fully assembled before you turn it on, the brain begins to function as it develops. We are investigating the possibility that this early brain activity tests each new synapse and maintains the ones that are correct. Also, similar to the connections inside computers that are welded together, synapses between neurons must be held together by ‘cellular glue‘. By investigating the molecular nature of this glue we hope to gain insight into how synaptic connections in the brain are formed.

The vertebrate brain is organised into precise, highly stereotyped and evolutionarily conserved functional systems. The aim of my research is to investigate how such precise wiring is achieved, how synapses form between neurons, and to elucidate the cellular and molecular events that regulate synaptogenesis. I hope to address these questions using the zebrafish retinotectal projection as a model system. Retinotectal circuit formation is characterized by the extension of retinal ganglion cell (RGC) axons from the retina to their midbrain target, the optic tectum, where they elaborate an axonal arbour and establish synaptic connections with tectal cell dendrites. In vivo imaging of RGC axons and tectal cell dendrites expressing fluorescent synaptic marker proteins as they arborized in the optic tectum of live zebrafish larvae demonstrates that most new synapses form rapidly on newly extended axonal/dendritic processes. Also, axonal and dendritic branches are initiated almost exclusively at synaptic sites. Furthermore, only a small fraction of nascent synapses are maintained, and these synapses in-turn maintain the otherwise unstable axonal process on which they form. These data reveal two distinct mechanisms by which synapse formation can guide axonal and dendritic arbour growth; (1) by acting as preferential sites for the initiation of new branches, and (2) by selective stabilization of a subset of those new branches upon which further new synapses are formed.
Given that formation of a stable synapse seems to have such a profound effect on dendritic and axonal arbour form, a fundamental question is: what underlies the process of synapse formation and stabilization? The goals of the proposed research are to investigate the influence of 1) neural activity, and 2) Synaptic cell adhesion molecule (SynCAM) mediated cell-cell adhesion on synapse formation and stability, and arbour growth in the retinotectal system. I plan to address these questions in vivo using molecular genetic approaches, time-lapse microscopy of retinotectal circuit formation and electrophysiological recording of neural function. The combination of these techniques, particularly when used in developing zebrafish, will allow the study of circuit formation in vivo at a level of detail that is not easily attainable in other systems, and thus lead to a greater understanding of processes that lead to nervous system malformation and that limit regenerative processes in the adult nervous system.