Multi-region two-photon calcium imaging in freely moving animals

Complex behaviors emerge from the coordinated activity of neural circuits distributed across widely separated brain regions. Existing optical approaches for monitoring neural activity lack the combined capability to record from anatomically distributed regions with high spatiotemporal resolution in freely moving animals. Here we introduce MINI2P-II, a 1.1-g 2P miniscope that achieves large-scale calcium imaging with high spatial resolution, deep imaging depth and dual-color imaging capability. Together with a dual-scope imaging system and a novel implantable periscope lens assembly, MINI2P-II enables simultaneous 2P imaging in widely separated or anatomically adjacent brain regions. In mice, we demonstrate inter-hemisphere coordination in bilateral motor cortex during climbing, egocentric boundary coding in simultaneous retrosplenial and posterior parietal cortex recordings, and in zebra finches, we showed large-scale behavior-dependent activity in both juvenile and adult individuals. MINI2P-II opens the door to multi-region 2P imaging across species and developmental stages in freely moving animals.