Fiber photometry has been increasingly popular in neuroscience research in freely behaving animals. In combination with genetically encoded calcium indicators, it allows for real-time monitoring of neural activity in neuronal somata, dendrites, and axonal terminals. We developed a multichannel fiber photometry device to map the activity of axonal terminals in a restricted, 100 - μ m -wide brain region in freely moving mice. This device consists of four bundled multimode fibers, each with a 50 - μ m core diameter and a scientific complementary metal-oxide semiconductor camera to simultaneously acquire fluorescence. We achieved a sampling rate of 100 frames / s and sufficient sensitivity to acquire data from axonal terminals. To avoid interference with neighboring channels, the recording depth of each channel was restricted to < 250 μ m . Furthermore, the small-core-diameter fibers did not restrict mouse locomotion. Using the Ca 2 + indicator GCaMP5G, we validated the system by recording Ca 2 + signals in axonal terminals from the medial entorhinal cortex layer II to the hippocampal dentate gyrus (DG) in freely moving mice. We detected spatially separated Ca 2 + signals at four different sites in the DG. Therefore, our multichannel fiber photometry device provides a simple but powerful method to functionally map axonal terminals in spatially confined brain areas of freely moving animals.

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