3D Scanning Two-photon Fiberscope Technology for Simultaneous Multi-region Multi-cell-type Imaging in Freely-moving Rodents

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PROJECT SUMMARY Brain activities involve neurons generating fast-propagating signals to encode and relay information within dynamic neural networks. Neuroscientists aspire to obtain access to such networks in unconstrained animal models (e.g., rodents) with high spatiotemporal resolution, which will shed light on the fundamental working mechanisms of the brain. Optical imaging, particularly multiphoton microscopy, has played a significant role in this endeavor. The past decade has seen impressive progresses, from head-restrained benchtop microscopy with virtual navigation to large FOV microscopy for neuron population imaging, three-photon microscopy for deep brain imaging, and two-photon (2P) miniscopy for in vivo imaging in freely-walking (but limited rotation) mice. Despite these exciting technological advances, tools for simultaneous, large-scale, and high-resolution imaging over multiple brain regions in freely-behaving rodents are still lacking. Successful development of such tools can accelerate the process of uncovering general principles of neural networks in a working brain under nearly natural conditions. The free-moving style for imaging would minimize the differences between experimentally controlled actions and natural spontaneous behaviors, thus allowing for precise examination of neural network functions. The capability of simultaneous imaging over two interconnected neural populations would provide a comprehensive and precise timeline of the neural circuit dynamics associated with various behaviors at both cellular and population levels. Our proposed research is motivated by the need for such imaging tools with the above-mentioned features. The main objective is to develop a 3D-scanning, ultrathin and light 2P fiberscope technology for enabling high- resolution, simultaneous imaging of dynamic neural activities over a large FOV at two brain regions in freely- moving rodents. To achieve our objective, we propose the following aims: (1) To develop a fast scanning 2P fiberscope of a large FOV (Ø500 um) using a cascaded magnification strategy while maintaining a compact probe size (Ø2.5 mm). The larger FOV will be achieved by using an innovative micro-optics design. In addition, a modular scanner head design will be implemented in the 2P fiberscope to improve the probe robustness for in vivo imaging at a high scanning frequency (e.g., ~2.8 kHz); (2) To develop a miniature (Ø2mm) tunable lens that can be integrated into our 2D scanning fiberscope for enabling depth (focus) scanning/selection over 150 um. Focus scanning allows for convenient selection of a proper layer or population of neurons. The tunable lens can create a curved refractive index profile when applied with a low-voltage (<10 V, safe) electrical drive. Compared with other tunable lenses, the tunable lens will be extremely compact and light, critical for imaging freely-moving rodents. A fiberscope integrated with a tunable lens will be developed and tested using phantoms, fluorescent tissue slides, and a mouse model in vivo. (3) To develop a dual-probe system, enabling simultaneous 2P imaging of two brain regions in freely- walking/rotating mice. The ultracompact size and lightweight of the fiberscope permit two fiberscopes to be mounted a mouse head, allowing for simultaneous imaging of two brain regions (cortex or deep brain). A novel, proactive, dual-probe optoelectrical commutator (dpOEC) will be developed for the first time to sense and compensate the torque built up in the fiberscopes, allowing the mouse to walk/rotate freely during imaging; (4) To assess the feasibility of the dual-probe 2P technology for exploring neural network dynamics in two different brain regions simultaneously during social decision making. Social behavior involves sensory, cognitive, and motor functions and thus depends on the interactions of many neurons, but until now no technology is available to record from a large population of neurons with subcellular resolution over multiple interconnected regions in freely-behaving mice. Here we choose to study the dynamic neural connectivity between the primary motor cortex (M1) and a critical sensory information routing node, periaqueductal gray (PAG). Both areas are critically involved in social behavior, but how these interconnected regions synergize to process information remains almost completely unknown. In addition to testing the performance of the 2P fiberscopy technology, this aim could also shed light on how social preference is encoded. As a control, we will monitor these regions during a locomotion (but nonsocial) activity (Rotarod running), for which the information on M1 that is independent of PAG is already available. In summary, successful completion of the proposed study will establish a new two-photon fiberscope imaging platform for the neuroscience community to enable simultaneous high-resolution imaging of neural network dynamics of different cell types over different brain regions in freely-behaving rodents. In addition, focus/depth scanning will be made possible. The fiberscope can be easily attached to and detached from the mouse head, permitting repeated use. Although beyond the scope of current proposal, the technology can also have many translational applications, including internal luminal organ imaging for diagnosis or guidance of intervention.