Multichannel fiber photometry for mapping axonal terminal activity in a restricted brain region in freely moving mice

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, [Formula: see text]-wide brain region in freely moving mice. This device consists of four bundled multimode fibers, each with a [Formula: see text] core diameter and a scientific complementary metal-oxide semiconductor camera to simultaneously acquire fluorescence. We achieved a sampling rate of [Formula: see text] and sufficient sensitivity to acquire data from axonal terminals. To avoid interference with neighboring channels, the recording depth of each channel was restricted to [Formula: see text]. Furthermore, the small-core-diameter fibers did not restrict mouse locomotion. Using the [Formula: see text] indicator GCaMP5G, we validated the system by recording [Formula: see text] 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 [Formula: see text] 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.