Summary, Project 2 (Produce the cell type-specific thalamocortical projectome) Frontal cortex displays rich patterns of neural activity, which can be decomposed into 'activity modes' corresponding to specific aspects of behavior (see Overall), such as the persistent activity correlated with short- term memory, and rapidly cycling activity causing voluntary movements. Frontal cortex is strongly coupled to the thalamus, the central hub of the forebrain. Subcortical information flows through the thalamus to the cortex. Most of thalamus is non-sensory (‘higher-order’), with input from cerebellum, multiple parts of the midbrain, and hippocampus, and outputs to most cortical areas (a detailed map of inputs is part of Project 1). This project aims to uncover the thalamocortical (TC) cell types. These are excitatory neurons that receive input from subcortical areas outside of the thalamus and project to the neocortex. Understanding TC types is critical because distinct TC types likely correspond to specialized thalamocortical channels for transmission of information from sub- cortex to cortex. We currently lack even a rudimentary conceptual framework for the function of non-primary- sensory thalamus, in part because our knowledge of subcortex-thalamus-cortex circuits is at a nascent stage. A limited set of morphological reconstructions have shown that the TC neurons are diverse across and within thalamic nuclei defined by cytoarchitecture. Our preliminary data suggest that different control signals arise in different subcortical areas, with distinct effects on cortical activity modes. The input-output rules at the level of individual thalamocortical (TC) cells constrain the possible control strategies. Are subcortical control signals routed through independent TC types or even different thalamic nuclei (‘labeled lines’)? Or do multiple subcortical inputs converge at the level of TC cells, with individual TC types transmitting a mixture of control signals? To help address these questions we will establish a census of TC types across the higher-order thalamus, including neurons projecting to anterior lateral motor cortex (ALM) and medial prefrontal cortex (mPFC). We will use new methods that combine morphological reconstructions of entire TC neurons with transcriptomics for the same cells. We refer to cells defined in this manner as morpho-transcriptomic (m-t) TC types. We will further densely map cell types defined by transcriptomics, so-called t-types, across the entire thalamus. By linking morphology, transcriptomics and location at the single neuron level, these data will provide the foundation for genetic access of specific TC types (strategies for genetic access will be developed in the Molecular Science Core). Together, this information will create knowledge and tools for cell type-specific analysis of multi-regional circuits (Projects 3, 4) with thalamus in the middle.

Narrative, Project 2 (Produce the cell-type-specific thalamocortical projectome) We will uncover the diversity of thalamocortical (TC) neurons using detailed reconstructions of single cells and measuring their gene expression. We will then map molecular TC types across the thalamus using cutting-edge multiplexed spatial transcriptomics methods. These multi-modal datasets will provide the foundation for genetic access to specific TC types and provide the structural blueprint to understanding how subcortical areas influence cortical activity via the thalamus.