Project 3. Abstract Descending control of orofacial behavior (Kleinfeld lead; Mukamel, Svoboda, Wang) This Research Project will define the connectivity and neural mechanisms of descending control by motor cortex of orofacial behavior. Orofacial movements can be rhythmic and coordinated with each other. However, individual movements can also be specifically controlled to achieve behavioral goals, such as consuming a reward at a particular location and time. We will use a behavioral assay in which mice have to make a directional tongue movement at a particular time to receive a reward. This further serves as an example of set- point control. The brainstem level controls for licking are driven by the motor neurons in the hypoglossal nucleus whose activity in turn is modulated by premotor neurons in the intermediate nucleus of the reticular formation. Our studies will explore the anatomical connectivity, i.e. ?Components? and signal flow, i.e., ?Wiring Diagrams? that converge on this region of the reticular formation. We will further refine the description of hypoglossal premotor subregions, nominally referred to here as ?hIRt?, that are relevant to licking. Our preliminary data indicates that motor cortex can direct the timing of licking bouts and direction of licking, but not the timing of individual licks. We will trace these command signals from the motor cortex through the superior colliculus and into the hIRt. We will measure neural signals in another descending pathway from the basal ganglia that converges on the superior colliculus and the hIRt. We will measure how these descending inputs are synaptically coupled to defined neuron types in the hIRt. Together this project will provide a mechanistic account of how descending signals from multiple sources are integrated at the level of premotor neurons in the brainstem, complementing our studies on projections that control the set-point of whisking (Project 2). An additional focus will set the stage for the next generation dissection of the neural circuits in the brainstem. This requires molecular profiling of specific cell classes to provide transcriptomes that will facilitate future fine-grained analyses, including genetic labeling, manipulation, and transsynaptic tracing. Specific populations of premotor neurons will be isolated by transsynaptic labeling and mRNA will be isolated using Translating Ribosome Affinity Purification (TRAP), a technology that isolates mRNAs associated with translating ribosomes and is especially favorable in the densely myelinated brainstem. This is followed by deep sequencing and yields a new method, TRAP:Seq.