Successful application of two-photon imaging with genetic tools in awake macaque monkeys will enable fundamental advances in our understanding of higher cognitive function at the level of molecular and neuronal circuits. Here we report techniques for long-term two-photon imaging in awake macaque monkeys. Using genetically encoded indicators including GCaMP5 and GCaMP6s delivered by AAV2/1 into the visual cortex, we demonstrate that high-quality two-photon imaging of large neuronal populations can be achieved and maintained in awake monkeys for months. Simultaneous intracellular recording and two-photon calcium imaging confirm that fluorescence activity is linearly proportional to neuronal spiking activity across a wide range of firing rates (10 Hz to 150 Hz). By providing two-photon imaging access to cortical neuronal populations at single-cell or single dendritic spine resolution in awake monkeys, the techniques reported can help bridge the use of modern genetic and molecular tools and the study of higher cognitive function.
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