Sex differences in life-course social disadvantage and all-cause mortality: evidence from a longitudinal study from 1996 to 2023.

The neural computations that support sensation, cognition and behavior depend on the precise coordination of neural activity patterns within and across widely separated brain areas. Recent advances in optogenetics and three-dimensional (3D) light patterning have enabled investigators to causally probe how the activity of specific neural ensembles impacts computation and drives behavior, but only in very small, circumscribed regions of the brain (less than 1–2 mm). These techniques rely on phase modulation of the optical wavefront with a spatial light modulator (SLM), allowing the user to selectively target and photo-stimulate neural ensembles of interest. Despite the power of these new read–write optogenetic approaches, the inability to apply them to distributed brain networks has prevented investigators from causally probing the logic and principles of inter-areal communication that are central to brain function. A new technology that could overcome this technical barrier would allow neuroscientists to address key outstanding questions for the first time, which could have profound importance for understanding brain function in health, disease and neural development. The recent introduction of mesoscale two-photon (2p) microscopes, which can sample neural activity with near-micron precision across up to 25 mm of brain tissue, has vastly increased the ability of investigators to acquire physiological data on distributed neural populations in behaving animals. Although these 2p mesoscopes can monitor cellular activity, they have no ability for spatially targeted perturbations, preventing the user from probing causal relationships between the activity of specific neural ensembles in connected brain areas and behavior. Therefore, if it were possible to develop a 2p mesoscope that can not only measure but also manipulate neural activity with cellular-scale resolution, neuroscientists could use such a system to address longstanding mysteries of long-range neural communication in the brain. In practice, such a system would enable targeted perturbations of selected individual neurons or groups of neurons while simultaneously recording thousands of cells across cortical areas. However, there are multiple substantial technical challenges to achieving high-resolution holographic optogenetics in a 2p mesoscope. Existing commercial 2p mesoscopes were not designed for the integration of holographic systems, requiring a large redesign of the mesoscope build. A particularly outstanding challenge in achieving high-resolution patterned photostimulation across a mesoscopic field-of-view (FOV) is the physical limits imposed by SLM designs, which ultimately constrain the accessible photostimulation FOV. We first designed and optimized a flexible 3D holographic system fully integrated onto a 2p random-access mesoscope (2p-RAM) that enables near-single-cell resolution holographic photostimulation of neural ensembles in the brain across a millimetric FOV. We tested and validated the optical capabilities of this new read–write platform to measure and recreate highly specific patterns of activity in the brain. In particular, we showed that we can decode the identity of the specific photostimulus purely from the modulation of activity of postsynaptic neurons in downstream areas, and that we can holographically recreate and transmit visual-like information across areas in mouse visual cortex. Next, we expanded the photostimulation FOV by an order of magnitude by integrating a random-access scan module into the 2p holographic path. We further showed how one can use this system to probe functional interactions between distant brain regions. Finally, we demonstrated, for the first time to our knowledge, near-simultaneous photostimulation of specific neural ensembles across distinct remote cortical areas. Together, these data illustrate how our 2p holographic mesoscope enables the user to probe causality at both the local and the inter-areal level with near-single-cell resolution.