Fig. 6: Adaptive optogenetic photostimulation target selection during functional calcium imaging in zebrafish.

a improv pipeline for targeting direction-selective neurons for photostimulation. The ‘Adaptive Targeting’ actor provides the target locations to the ‘Photostimulation’ actor to control the next photostimulation event. b improv orchestrates a phase of visual stimulation to characterize neurons in real time, automatically switching after reaching characterization criteria to a closed-loop photostimulation phase. c Example visual tuning curves of neurons chosen for photostimulation, normalized to their maximum response. The ‘Adaptive Targeting’ actor selects a direction-selective neuron with rsChRmine expression for photostimulation. d Average fluorescence across all neurons showing stimulus-locked responses to visual stimuli (colored bars) followed by a photostimulation phase (gray lines). e Pretectum (Pt) and hindbrain (Hb) in a zebrafish expressing nuclear-targeted GCaMP6s (greyscale). Photostimulating a target neuron (red dot) results in evoked activity in responsive neurons (orange dots). Color intensity encodes the response magnitude, and lines denote the top 10 strongest responders. f Fluorescence responses to photostimulation of the red neuron in (e) (top trace; Target), and three simultaneously recorded putatively responsive neurons (bottom): (1) responsive, upregulated; (2) non-responsive; and (3) consistently responding. Extracted fluorescence traces are obtained from ‘Caiman Online’. g Example neurons’ visual and photostimulation tuning curves. The visual tuning curve is calculated as the average response during a given angular direction of the stimulus (45°, 90°, …). The photostimulation tuning curve is calculated as the average response during photostimulation of other neurons whose tuning peaks were located at a given direction, such as the 45°, forward-right tuned neuron in c). The left plot shows a neuron that is less activated by photostimulation but with similar directional tuning, and the two left neurons show stronger universal activation from stimulated neurons. h Analysis of all neurons across fish (N = 3; ~1300 neurons) responsive to visual stimuli (red dot) and photostimulation events (black dot), compared to their overall responsiveness. Most neurons responded to visual and optogenetic stimulation, but some neurons were only discovered as responsive during photostimulation. Diagonal trends on either side indicate neurons preferring either visual or photostimulation.