BRAIN project (Plenz): Readout and Control of Spatiotemporal Neuronal Codes of Behavior

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During the fifth year of this U19 grant, we made substantial progress in quantifying the performance of our in vivo 2-photon imaging (2PI) in combination with spatial light modulation for single cell stimulation in the awake animal. 1. Progress in co-expression of Genetically Encoded Calcium Indicators (GECIs) and opsins Co-expression of the GECI GCaMP7s and a fluorescent complementary opsin for stimulation is required for an all-optical approach to record neuronal activity and stimulates neurons using laser beams. Unfortunately, often individual cell express either the GECI or the opsin, but not both. Careful titration studies of various GECI/opsin combinations (GCaMP7s/ChrimsonR) allowed us to arrive at reasonable co-expression rates allowing us to proceed with out experimental goals. In addition, we substantially improved the co-expression of the improved GECI GCamP8 with ChrimsonR using a bicistronic vector in a transgenic mouse line. 2. Improved method for routine calibration of holographic stimulation efficacy Holographic stimulation requires the precise identification and calibration of the laser point spread function. Even small drifts in this function over the course of the experiment renders stimulation results unreliable. We combined our holographic stimulation control with a galvo-galvo routing control to synchronize our 2-photon imaging and holographic stimulation laser beams. This allows us to routine benchmark our stimulation parameters before every experiment ensuring proper stationary holographic stimulation conditions.