PROJECT SUMMARY (30-lines) Movement depends on patterned spinal cord motor output necessary for coordinated muscle actions. This in turn, depends on spinal interneuron microcircuits that modulate motoneuron recruitment and firing. The recent explosion of genetically-defined interneurons has revolutionized the study of spinal motor circuits promising a more comprehensive and complete dissection of these important networks. The work started by devising genetic strategies to identify, label, study and modify activity of large groups of interneurons defined by their derivation from specific progenitor domains. This is the approach mostly taken in previous years. Much new information was obtained, but the biggest surprise was the large heterogeneity within each domain-lineage of spinal interneurons. It is thus now necessary to study the different subclasses of interneurons within each domain. This can only be accomplished with new animal models to target more restricted subgroups using intersectional genetics in which combinatorial expression of more than one gene is necessary for conditionally expressing fluorescent proteins or activity modulators to study them. This grant focuses on the V1 group. These are inhibitory interneurons with ipsilateral connections in the spinal cord and that have important roles shaping motoneuron activity. Over the years we and others have reported a variety of functions for V1 interneurons, including control of motoneuron firing by recurrent and reciprocal inhibition, being all Renshaw cells and some, but not all, Ia inhibitory interneurons (IaINs) derived from V1s. They also contribute to regulate flexion-extension alternation during movement and locomotion speed, providing respectively out-of-phase and in-phase inhibition during rhythmic locomotion. This functional diversity is parallel by the existence of many different genetically-defined subpopulations (by some estimates more than 50) organized into four major V1 clades. It is now important to analyze whether each subset is integrated in distinct microcircuits and responsible for specialized functions. We chose to focus in the next five years on the largest V1clade defined by the transcription factor Foxp2. Data gathered during the previous grant suggest this group is composed of at least three major subtypes and includes some cells with synaptology typical of IaINs. We have tested various intersectional strategies to study anatomically (using various viral approaches) the connectivity of Foxp2-V1s with different motor pools and primary afferents, and the postnatal development of these connections (Aim1). Then, we will analyze whether Foxp2-V1s are the only sources of V1-IaINs and how reciprocal inhibition is affected by genetically silencing them (Aim 2). Finally, we will reversibly silence these cells in the neonate and adult during locomotor stepping and use chronic EMG recordings and kinematics to analyze their role in muscle activation selection during rhythmi...