Discovering the cell type basis of the IT projection system and its dysregulation after perinatal HIE

Abstract: Intratelencephalic (IT) excitatory cortical neurons project only within telencephalic structures – the cortex and striatum – and make only callosal, corticostriatal, and intrahemispheric connections. They exhibited massive amplification and diversification during mammalian cortical evolution and are therefore thought to

underlie the unique capabilities of human cognition. Despite their importance, little is currently known about distinct IT subtypes and their contributions to cortical organization, function, and dysfunction in disease. Upper layer and deep layer IT neurons diverge in their axonal trajectories: upper layer IT neurons make

predominantly callosal cortico-cortical, intrahemispheric, and ipsilateral corticostriatal connections, whereas deep IT neurons project fewer callosal axons and instead project more heavily to bilateral striatum. Upper layer IT neurons demonstrate high differential gene expression in autism, a syndrome with predominant cognitive

symptoms, while IT neurons with corticostriatal connections are hypothesized to contribute to motor disorders. Thus, dissecting IT subtypes is essential to understanding their unique contributions to neurological disease. One such disease that presents with varying cognitive and motor impairment is perinatal hypoxic

ischemic encephalopathy (HIE), the most common brain injury in term neonates. HIE often injures the cortex and striatum, prime targets of IT neurons. Sequelae include cognitive or motor symptoms, suggesting that HIE may differentially disrupt cortico-cortical and corticostriatal circuits mediated by distinct IT subtypes. My

preliminary transcriptomic data suggest that deep IT neurons demonstrate a greater burden of differential gene expression after HIE than upper IT neurons, particularly in gene pathways that regulate axon development. In this proposal, I utilize two novel knock-in mouse lines, Wfs1-Flp and Deptor-CreER, that label

superficial and deep IT subsets, respectively. In Aim 1, I will perform anterograde and retrograde axonal tracing to fully characterize the cortico-cortical and corticostriatal axonal projections from primary motor cortex in each mouse line. In Aim 2, I will perform the Vannucci model of HIE in Wfs1-Flp and Deptor-CreER mice to assess

changes in cortico-cortical and corticostriatal axonal projections from upper and deep layer IT neurons after HIE. Finally, in Aim 3, to assess cell-specific changes in gene expression after HIE with high spatial precision, I will utilize the cutting-edge spatial transcriptomics platform MERFISH in mouse cortex after HIE compared to control

cortex. I will amplify the power of this approach by integrating MERFISH data with my existing single nucleus RNA sequencing data from mouse cortex after HIE, providing an innovative informatics pipeline that combines the high detection power of single nucleus transcriptomics with the laminar precision of spatial transcriptomics.

Through this work, I will disentangle the cell-specific responses of IT subtypes to HIE, laying the groundwork to understand how disruptions in IT microcircuitry differentially contribute to cognitive versus motor symptoms and uncovering candidate targets for future pursuit as cell-targeted therapies to ameliorate IT dysfunction after HIE.