All-optical interrogation of millimeter-scale networks and application to developing ferret cortex

Perception and behavior require coordinated activity of thousands of neurons operating in networks that span millimeters of brain area. In vivo calcium imaging approaches have proven exceptionally powerful for examining the structure of these networks at large scales, and optogenetics can allow for causal manipulations of large populations of neurons. However, realizing the full potential of these techniques requires the ability to simultaneously measure and manipulate distinct circuit elements on the scale of millimeters. We describe an opto-macroscope, an artifact-free, all-optical system capable of delivering patterned optogenetic stimulation with high spatial and temporal resolution across millimeters of brain while simultaneously imaging functional neural activity. We find that this approach provides direct manipulation of cortical regions ranging from hundreds of microns to several millimeters in area, allowing for the perturbation of individual brain areas or networks of functional domains. Using this system we find that spatially complex endogenous networks in the developing ferret visual cortex can be readily reactivated by precisely designed patterned optogenetic stimuli. Our opto-macroscope extends current all-optical optogenetic approaches which operate on a cellular scale with multiphoton stimulation, and are poorly suited to investigate the millimeter-scale of many functional networks. It also builds upon other mesoscopic optogenetic techniques that lack simultaneous optical readouts of neural activity. The large-scale all-optical capabilities of our system make it a powerful new tool for investigating the contribution of cortical domains and brain areas to the functional neural networks that underlie perception and behavior.