Long-term all-optical interrogation of cortical neurons in awake-behaving non-human primates

ABSTRACT Whereas optogenetic techniques have proven successful in their ability to manipulate neuronal populations in species ranging from insects to rodents—with high spatial and temporal fidelity—significant obstacles remain in their application to non-human primates (NHPs). Robust optogenetics-activated behavior and long-term noninvasive monitoring of target neurons, have been challenging in NHPs. Here we present a method for all-optical interrogation (AOI), integrating optical stimulation and simultaneous two-photon imaging of neuronal populations in the primary visual cortex (V1) of awake rhesus macaques. A red-shifted channel-rhodopsin transgene (C1V1) and genetically-encoded calcium indicators (GCaMP5 or GCaMP6s) were delivered by AAVs, and subsequently expressed in V1 neuronal populations for months with high stability. We achieved optogenetic stimulation using both single-photon (1P) activation of neuronal populations and two-photon (2P) activation of single-cells, while simultaneously recording 2P calcium imaging in awake monkeys. Optogenetic manipulations of V1 neuronal populations produced reliable artificial visual percepts. Together, our advances show the feasibility of precise and stable all-optical interrogation of cortical neurons in awake NHPs, which may facilitate broad applications in high-level cognition and pre-clinical testing studies.