Summary/Abstract, Project 5 The goal of this project is to comprehensively examine, at single-neuron resolution, the flow of information through the brain during a decision-making task. Leveraging the expertise, knowledge, and data gained from International Brain Laboratory members who have designed and studied this task, the proposed approach combines novel, advanced techniques for high- throughput, high-resolution physiological recordings and anatomical tracing to determine the substrates of inter-regional communication. In light of the extensive functional and structural diversity of neurons in the brain (for example, neurons in the auditory cortex can project to at least 11 different targets, forming 264 distinct projection patterns), the proposed experiments test the central hypothesis that specific privileged subpopulations mediate the flow of information between regions in a state-dependent manner. Such subpopulations will be identified based on transcriptionally defined cell subtypes (Aim 1) and projection types or motifs (Aim 2). In vivo two- photon calcium imaging will be combined with in vitro profiling of transcriptional and anatomical identity via the novel barcoded anatomy resolved by sequencing (BARseq) technique. BARseq is used to identify the projections and/or genetic markers of thousands or tens of thousands of neurons simultaneously in a single animal. Because the two techniques are both based on imaging, the in vivo data will be registered with in vitro data to support structure-function, state- dependent insights at the single-neuron level. The regions and circuits targeted in these experiments will be informed by the results of Projects 1 and 2. In turn, the results generated here in Project 5 will inform the modeling work proposed in Project 3.