Project Summary The dynamic adaptability of the mammalian brain to environmental changes is remarkable, as it is the complexity of the networks of neurons underlying the operations that allow for such adaptations. Although we have some understanding of the anatomical and functional basis of this, we are still lacking a detailed picture of how the modulation of neuronal activity works. What is the timing and locations of these neuromodulator release and relationship with excitatory/inhibitory circuits? How does the neuromodulators circuitry accomplish the regulation of firing and synaptic properties of targeted neurons? Filling these gaps in knowledge would advance our understanding of all aspects of neuromodulator biology and allow discovery of new therapeutic strategies. To help close this gap, we have used creative approaches to the development of genetically encoded to directly report behaviorally triggered and modulated neuromodulator release including serotonin (5-HT), dopamine (DA) and norepinephrine (NE). We have disseminated these indicators to the neuroscience community and spurred major discoveries of novel mechanisms regulating neuromodulator release underlying motivation and addiction. Build on this initial success, we propose to further expand the effectiveness of this toolbox of NM sensors to enable imaging sparse release at depth and subcellular resolution. Our specific goals are to (1) improve the sensitivity of our current sensors to enable robust imaging of sparse neuromodulator release, push their spatial resolution to the subcellular level and increase linearity of response at lower concentrations; (2) expand their spectral range to red/far-red to enhance imaging depth, SNR and in vivo multiplex measurement and manipulation of multiple circuit components using two or three distinct colors, and (3) characterize the possible interference of current sensors with endogenous signaling and systematically validate emerging sensors with a wide-ranging microscopy approaches in vivo. Our strategy relies on a dynamic collaboration between the sensor design team and end users to obtain continuous feedback to implement efficient improvements to the sensors. It is our goal to rapidly disseminate a wide range of well-characterized, highly sensitive indicators for the neuroscience community to be employed to study behaving mice, fish, flies and worms, to enrich our knowledge on the functional roles of neuromodulators in the brain circuitry.