Functional investigation of neural circuits in larval zebrafish

Abstract

A fundamental goal of modern neuroscience is to understand the principles by which neural circuits within the brain lead to cognition and behaviour. Technological advances in the fields of microscopy and optogenetics have very recently allowed for the activity of all cells within a functional circuit to be observed and recorded, meaning that this goal is now within reach. Due to the physical and optical properties of neural tissue, these technologies remain most useful for the analysis of circuits that span very small volumes, meaning that small animal model organisms, with brains less than approximately 1 mm across, are most amenable to these types of analyses. The larval zebrafish model has emerged as an optimal balance between small size and the high complexity of a vertebrate brain. This thesis describes the development and application of several of these technological advances to examine neural circuits underlying perception and behaviour in the larval zebrafish.Due to the relative recency of zebrafish as a model for functional neuroscience, the genetic and experimental tools required for in depth circuit analysis have not been widely available. Thirteen transgenic zebrafish lines have been developed during this thesis for expressing optogenetic proteins throughout the brain, such as GCaMP5G for imaging neuronal calcium dynamics, and ChR2(ET/TC) and eNpHR3.0 for activating and silencing neuronal activity, respectively. These biological tools were developed in combination with optical techniques for probing neural circuits. This thesis describes the design and construction of a custom selective plane illumination microscope for the rapid imaging of neuronal activity across large areas of the zebrafish brain. The use of a spatial light modulator is also described for the targeted stimulation of ChR2(ET/TC) in the cerebellum of larval zebrafish. This experiment provided preliminary data for the functional investigation of connections between cerebellar cells and the optic tectum, as well as confirming the validity of this technique for analysis of other neuronal circuits in vivo.Since learning is one of the most interesting yet ill-defined processes controlled by the brain, this thesis aimed to use the tools developed above to examine the changes across neuronal circuit activity responsible for learning in larval zebrafish, specifically those in the cerebellum responsible for motor learning. A classical P ag e 2 conditioning assay was administered to 7-day-old zebrafish by pairing a non-startling tone with an aversive tail shock stimulus. This paradigm has been extensively used in other model organisms, however failed to produce a significant learning behaviour in the experiments performed for this thesis. More recent evidence suggests that zebrafish only begin to show learning from approximately one month of age.However, the data presented here indicate that zebrafish up to six weeks of age were still unable to undergo classical conditioning to pairings of auditory and electric stimuli. A number of is...