Revealing cell type-specific connectivity motifs in orofacial premotor circuits

PROJECT SUMMARY Orofacial movements are the product of convergent, descending synaptic input from high-level motor centers to low-level premotor circuits in the intermediate and parvocellular reticular nuclei (IRN/PARN) of the brainstem. The IRN and PARN contain central pattern generators and complex feedback loops that, while often flexibly driven by higher-level motor centers, are also capable of organizing basic orofacial motor output independent of descending input. Yet, due in large part to a fundamental lack of knowledge about the cellular and circuit structure of the IRN and PARN, how these circuits integrate control signals from higher-level motor centers to produce goal-directed behavior remains unknown. Progress in connectomic mapping of medullary reticular nuclei like the IRN and PARN has lagged behind other brain regions due to these structures’ exceptional cellular diversity and cytoarchitectonic disorganization. Recent landmark developments in spatial and single-cell whole-brain transcriptomic atlases have now uncovered the precise transcriptomic identity and spatial arrangement of cell types within medullary reticular nuclei, enabling positive identification of cell types within these structures for the first time. By combining this information with cutting edge techniques for transsynaptic viral labeling and multiplexed fluorescence in situ hybridization (mFISH), I will seek to answer fundamental questions about cell type-specific circuit structure within the IRN and PARN. Aim 1 will design and test mFISH gene panels optimized for identifying as many cell types within the IRN and PARN as possible. Aim 2 will identify which cell types in the IRN and PARN receive direct synaptic input from motor cortex, the subtanstia nigra, the superior colliculus, and/or the fastigial nucleus of the deep cerebellar nuclei. Aim 3 will identify which cell types in the IRN and PARN project to motor neurons in the trigeminal, facial, and/or hypoglossal motor nuclei, as well as which cell types synapse locally on these premotor neurons. Results obtained in the proposed project will determine whether cell types in the IRN and PARN are organized into sequential processing layers, how many synapses such a structure may employ between descending projections and motor neurons, and whether or not this structure is multiplexed for different input sources and target motor nuclei. This foundational knowledge about IRN and PARN circuit structure will provide a rigorous conceptual framework for future anatomical and physiological studies of orofacial motor control. The work proposed here will also provide rich opportunities for training, including the development of computational tools for mining BRAIN initiative datasets, learning multistep rabies virus-mediated retrograde transsynaptic tracing, and building of computational frameworks for collecting and processing large mFISH imaging datasets.