Addictive drugs change synaptic properties, both at the molecular level and in terms of remodeling circuits. We will develop a novel luminescence-based methodology for monitoring synaptic activity as a new tool for functional neuroscience that can be applied to the science of drug abuse. Optogenetic methods for stimulating neural activity are revolutionizing neurobiological research in vitro and in vivo. Brief exposure to lightof cells expressing channelrhodopsin-2 (ChR2) can elicit excitatory cation fluxes (or inhibitory ion fluxes with the bacteriorhodopsin bR). To date, the impact of optogenetic stimulation has usually been monitored by electrophysiological methods that are accurate and well characterized, but are difficult and expensive to implement in freely behaving animals in vivo and/or in multiple neurons simultaneously. Optogenetic stimulation would optimally be partnered with less invasive optical methods to monitor activity among many cells. Unfortunately, the currently preferred methods for optically measuring synaptic activity are based on fluorescence methods that are poorly matched with ChR2/bR because the fluorescence excitation needed to monitor synaptic activity will trigger ChR2 and/or bR. Our new luminescence methodology will avoid the drawbacks of electrophysiology and fluorescence excitation (esp. reporter stimulation, photo bleaching & tissue auto fluorescence), and will therefore optimally partner with optogenetic methods for in vivo stimulation. Luminescence is an alternate optical technology that avoids problems associated with fluorescence. This project will develop novel luminescence probes for synaptic activity that are genetically encodable and targeted to specific cellular loci that are involved in neural activity. This will be accomplished by using Bioluminescence Resonance Energy Transfer (BRET) between a luciferase and the Venus fluorophore that is modulated by pH or Ca++ so that the spectrum of luminescent emission changes when synapses are activated optogenetically, thereby avoiding the problems associated with fluorescence excitation. These luminescence reporters of synaptic activity will be characterized in a well-studied hippocampal primary neuron culture preparation in vitro in conjunction with optogenetic stimulation before and after treatment with endocannabinoids. Finally, a viral vector encoding these reporters will be used to monitor neural activity of cortex after optogenetic stimulation in freely behaving rats in vivo. This project is appropriate for the Cutting-Edge Basic Research Award (CEBRA) R21 mechanism of the NIDA because it proposes to develop new technologies that will advance drug abuse and related neurobiological research.