WIDE FIELD-OF-VIEW MULTI-PLANE TWO-PHOTON MICROSCOPY ENABLING CORTICAL NETWORK ANALYSIS AND SINGLE-CELL OPTOGENETICS

Brain computation emerges from the network activity of intricately interconnected neuronal populations spanning multiple brain regions. To elucidate the computational principles of the brain, it is essential to record neural activity across multiple cortical regions and layers while causally intervening in network dynamics by modulating key neurons involved in information encoding. However, microscope technologies capable of simultaneously achieving large-scale recording and precise single-cell manipulation have remained limited. Here, we developed a wide-field two-photon microscope that enables multi-plane imaging combined with three-dimensional single-cell optogenetics. Using this system, we performed simultaneous wide-field Ca²⁺ imaging from cortical layer 2 and layer 5. We further evaluated the stability of neuronal responses to photoactivation and confirmed the spatial specificity of optogenetic stimulation at single-cell resolution. Applying this newly developed microscope, we estimated the structure of neural networks spanning layer 2 and layer 5 across multiple brain regions in mice during tactile stimulation. Our results revealed distinct spatial response properties to tactile stimuli between layer 2 and layer 5. In addition, network analyses were conducted to characterize network features, including node degree, hub neurons, and the spatial distribution of functional connections. As a next step, we aim to combine optogenetic manipulation of key network nodes—such as hub neurons and highly responsive cells—with tactile stimulation to investigate how cortical networks achieve stable tactile information representation at single-cell resolution.