Summary, Project 3 (Impact of subcortical inputs on frontal cortex via thalamus) The goal of this project is to elucidate the functional influence of subcortical inputs on the frontal cortex via non- sensory ('higher-order') thalamus. Each part of the frontal cortex receives inputs from multiple thalamic nuclei, each of which in turn receives inputs from diverse subcortical brain regions. Our recent work has established clear motifs for how different thalamic inputs can engage specific populations of pyramidal cells and interneurons in the frontal cortex. However, we still lack even a basic understanding of how subcortical inputs are organized in the thalamus, including how they are routed at the level of thalamic nuclei and single thalamocortical (TC) cells. Here we test the hypothesis that each subcortical input either excites or inhibits a defined subset of TC cells to engage specific networks in the frontal cortex. In Aim 1, we use electrophysiology to study the organization and influence of specific subcortical inputs on the thalamus and cortex in the intact brain. This work leverages new, high-density Neuropixels 2.4 probes that allow dense sampling of TC cell activity across thalamic nuclei. These experiments will define convergence and divergence rules in the thalamus and determine how subcortical signals propagate to frontal cortex. In Aim 2, we use brain slice electrophysiology to determine the properties of subcortical connections onto identified TC cells in the thalamus. We incorporate patch-Seq analysis to assess gene expression of each recorded neuron, linking processing of subcortical inputs with TC cell type. This work builds on Projects 1 and 2 and the Molecular Science Core, providing a foundational analysis of subcortical connections to the thalamus. In Aim 3, we then use voltage imaging in slices to examine how specific subcortical-thalamic pathways engage distinct networks across the frontal cortex, focusing on medial prefrontal cortex and motor cortex. Here we use anterograde viruses to conditionally express optogenetic tools in TC cells that then engage specific networks in the frontal cortex. We also employ mFISH to assess the genetic signatures of imaged excitatory and inhibitory cells, again allowing for sub-populations to be determined. Together, our experiments will establish the organization and properties of connections from subcortical inputs to higher-order thalamus and in turn the frontal cortex. They will serve as a strong foundation for our behavioral studies in Project 4, and constrain multi-regional models of neural computation developed in Project 5.