Protocol for minimizing off-target neuronal activation during optical stimulation in vivo

In vivo optogenetic stimulation is a powerful approach for dissecting neuronal circuit function, yet it can inadvertently lead to activation of endogenous retinal opsins, producing off-target responses that propagate to the cortex and therefore confound experimental interpretation. Here, we present a protocol for optical device implantation in the mouse brain that minimizes light leakage from the implant and skull. We also describe in vivo electrophysiological techniques to assay off-target neuronal activity and ensure its amelioration. For complete details on the use and execution of this protocol, please refer to Weiler et al.¹.