Two-photon patterned optogenetic excitation is a powerful emerging tool for perturbing distributed neural activity with cellular precision and specificity. However, current techniques for two-photon patterned illumination in vivo target a limited number of neurons with relatively poor temporal resolution, have not been validated across brain areas, and have not yet been reported to drive mammalian behavioral responses. The Technology Core will tackle a set of technically advanced yet feasible dissemination and development steps, advancing the team?s optics hardware, software, and optogenetics capabilities. These advances will enable two-photon optogenetic stimulation to achieve robust and precise distributed neural control in behaving animals across brain areas and cell types. All three research projects will work with the Technology Core, leveraging the unique expertise of the core laboratories in advanced optical technologies that allow us to sculpt laser wavefronts to interrogate brain circuits with single-neuron resolution in different sensory regions of the rodent brain. First, the Technology Core will integrate and validate the best practices for all-optical imaging and patterned two-photon perturbation, experimental management software, and light-sensitive probes. These state-of-the- art tools will be disseminated across the team. Next, the core will advance and optimize the specificity and scale of patterned two-photon stimulation, by exploring new optical, algorithmic, and probe-targeting solutions for improving stimulation specificity and robustness, new hardware and optical designs for extending the accessible stimulation field in both lateral and axial dimensions, and closed-loop software for brain motion compensation. Finally, the core will advance and optimize the temporal precision of patterned two-photon stimulation using rate-optimized optogenetic probes, spatial light modulators (SLMs), and high pulse-energy lasers, and by accurately synchronizing the optical perturbations with behavioral events and with intrinsic electrical dynamics. By enabling the stimulation of hundreds of neurons with unprecedented (below 10 ms) temporal resolution in behaving animals across brain areas and cell types, the work of the Technology Core will enable the research objective of elucidating how the cooperative activity of large groups of neurons drives behavior. Optimization of optical systems, software systems, and probes will be a critical component of result comparisons across sensory modalities. A unique strength of our proposal is that the technical teams are involved in performing the in vivo experiments, ensuring that technical expertise is directly available during experiments and that the problem solving is driven by experimentally relevant concerns.
