Revealing neural circuits underlying zebrafish behavior using mesoscopic light field microscopy

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ABSTRACT A critical step towards understanding how neural circuits drive behavior is the ability to record the activity of all neurons in an organism while it interacts with its environment in an unconstrained manner. A promising vertebrate model system in this regard is the zebrafish larva, which performs complex visually-driven behaviors such as hunting from an early age, and due to its transparency allows large-scale neural imaging at single- neuron resolution. However so far most investigations into the neural basis of zebrafish hunting behavior have used tethered fish which are unable to move freely. While some new assays have recently begun to overcome this limitation, these still significantly perturb the fish’s natural hunting behavior by requiring both an extremely shallow water depth, and rapid cancellation of lateral movement in order to maintain the fish within the field of view. Here we will develop a new `MesoLFM’ imaging technology overcoming both of these limitations, thus permitting the neural basis of truly unconstrained hunting behavior to be investigated for the first time in zebrafish. We will achieve this by combining light field microscopy (LFM), an electrically tunable lens (ETL), and the MesoLens, a giant microscope objective with a 4x/0.47 NA objective, a 10 mm working distance and a 5 mm field of view. LFM allows single-shot capture of 3D volumes, enabling imaging at many Hz. The ETL removes the need for rapid stage or objective movements in z, enabling imaging in relatively deep water. The MesoLens allows long sequences of movements to be captured within a single field of view, enabling imaging of hunting without the need for rapid xy motion cancellation. First, we will perform optical simulations of MesoLFM in order to determine the optimal parameters for components such as the microlens array. Second, we will construct the MesoLFM according to these specifications, and calibrate it using measurements of fluorescent beads and a standard resolution target. Third, we will directly demonstrate MesoLFM imaging of the zebrafish brain. Together this work will establish a new platform for investigating the neural bases of unconstrained behavior. In a future R01 application we will use the MesoLFM to reveal how neural circuits drive natural goal-directed behavior and decision-making.