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The claustrum has been hypothesized to participate in high-order brain functions, but experimental studies to demonstrate these functions are currently lacking. Neural activity recording of the claustrum in freely-behaving animals allows for correlating claustral activities with specific behaviors. However, previously utilized methods for studying the claustrum make it difficult to monitor neural activity patterns of freely-behaving animals in real time. Here we applied fiber photometry to monitor Ca 2+ activity in the claustrum of freely-behaving mice. Using this method, we were able to achieve Ca 2+ activity recording in both anesthetized and freely-behaving mice. We found that the dynamics of Ca 2+ activity depended on anesthesia levels. As compared to the use of genetically encoded Ca 2+ indicators that requires a few weeks of virus-dependent expression, we used a synthetic fluorescent Ca 2+ -sensitive dye, Oregon green 488 BAPTA-1, that allows for rapidly screening neural activity of interest within a few hours that relates to certain behaviors. In this way, we found the correlation between Ca 2+ activity and specific behaviors, such as approaching an object. Our work offers an effective method for recording neural activity in the claustrum and thus for rapidly screening any behavioral relevance of the claustrum in freely-behaving mice.

Ca<sup>2+</sup>-based neural activity recording for rapidly screening behavioral correlates of the claustrum in freely behaving mice