A 3 photon holographic imaging and optogenetics of neurons and neural circuits

One of major challenges in neuroscience is to understanding how the cerebral cortex works. The cortex, a 1.5 mm thick sheet of tissue, is the largest part of the brain in mammals and responsible for all higher brain functions. Cortical circuits are made with multitudes of cell types, intermixed in the same territories, connected in selective patterns and implementing different functions. This creates a phenomenal methodological problem: how to record and manipulate the activity of cortical neurons in three dimensions in an awake behaving animals or in human patients. Solving the challenge will constitute a revolution in neuroscience, and enable, not just the potential understanding of how the cortex works, but also lead to major insights into biological inspired computations that could revolutionize the computer industry and generate an artificial intelligence based on biological algorithms. The strategic and economic importance of solving the challenge or comprehensive measuring and manipulating brain activity led to the US BRAIN Initiative, a 12 year, large scale project that currently involve more than 500 laboratories around the country. In fact, our MURI (Contract # W911NF-12-1-0594) ÒImaging how a neuron computesÓ), inspired both the US BRAIN Initiative [1] [2] and the International Brain Initiative [3]. In this contract, 11 laboratories in 4 universities have developed critical technology to measure the structure and function of nervous systems. It is fair to say that this MURI has carried out some of the most transformational work in developing new methods for neuroscience in the last decade. At the same time, the dream experiment to measure the activity of every neuron in a cortical column, and selectively manipulate them at will to test their functional role in behavior, remains unmet. This MURI team now proposes to build a microscope to carry out this dream experiment by harnessing two recently developed methods: three-photon imaging and holographic optogenetics. Three-photon excitation uses deep infrared lasers to optically penetrate living tissue. With it, one can optically access the entire depth of a cortical column from the surface in living animals and image neurons with single cell resolution. Holographic microscopy enables building arbitrary light patterns in 3D to perform volumetric imaging and to optogenetically activate neurons in three dimensions. Combining both methods will enable simultaneous imaging and manipulation of cortical circuits in vivo in 3D. We request funds to purchase a laser system to build this novel three-photon holographic microscope. Our strategy is feasible, according to our calculations, and should enable us to image up to 10,000 neurons simultaneous at 30Hz thorough the cortex while activating sets of neurons with single cell resolution, in a living mouse, during behavior. This work could open the door for systematic all-optical study of the cortex and become a pioneering method in brain science. While the MURI team will use this system to study some critical questions in neuroscience, our proposed microscope could also become a new standard for all-optical imaging and manipulation of complete neural circuits. The microscope will thus serve as a resource for the entire neuroscience community and a key node for training and dissemination of this technology.