Optical Fiber-Based Recording of Climbing Fiber Ca²⁺ Signals in Freely Behaving Mice

The olivocerebellar circuitry is important to convey both motor and non-motor information from the inferior olive (IO) to the cerebellar cortex. Several methods are currently established to observe the dynamics of the olivocerebellar circuitry, largely by recording the complex spike activity of cerebellar Purkinje cells; however, these techniques can be technically challenging to apply in vivo and are not always possible in freely behaving animals. Here, we developed a method for the direct, accessible, and robust recording of climbing fiber (CF) Ca²⁺ signals based on optical fiber photometry. We first verified the IO stereotactic coordinates and the organization of contralateral CF projections using tracing techniques and then injected Ca²⁺ indicators optimized for axonal labeling, followed by optical fiber-based recordings. We demonstrated this method by recording CF Ca²⁺ signals in lobule IV/V of the cerebellar vermis, comparing the resulting signals in freely moving mice. We found various movement-evoked CF Ca²⁺ signals, but the onset of exploratory-like behaviors, including rearing and tiptoe standing, was highly synchronous with recorded CF activity. Thus, we have successfully established a robust and accessible method to record the CF Ca²⁺ signals in freely behaving mice, which will extend the toolbox for studying cerebellar function and related disorders.