Probing neural circuit motifs in zebrafish using holographic optogenetics

Two-photon Computer Generated Holography (CGH) based optogenetics allows activation of arbitrarily selected neurons simultaneously in 3D with cellular precision. We integrate two-photon CGH with a single-photon, dual channel light sheet microscope to record whole-brain Ca2+ activity at up to 400 frames per second, for concurrent activation and imaging. We use transgenic zebrafish larvae (5-8 days post fertilization) pan-neuronally expressing an excitatory opsin (HuC:ReaChR) and a Ca2+ indicator (HuC:GCaMP8s). This opsin-indicator combination enables flexible, simultaneous read/write access to distributed brain circuits. The initial goal is to exploit the cellular-level spatial resolution and millisecond-level temporal precision of the instrument to probe the temporal dynamics and functional connectivity of circuit motifs in a single focal plane of a live, tethered zebrafish. We are particularly interested in the iterative use of activity measurements to select targets for stimulation, in order to investigate excitatory circuit motifs (e.g., feedforward, convergent/divergent, or recurrent excitation) within or across brain regions. The overarching goal is to establish a flexible and scalable approach to deciphering functional circuit connectivity in a live animal.