An expanded toolbox for genetic access to molecularly defined cortical intratelencephalic neuron subtypes

Among the multiple cortical glutamatergic neuron classes, intratelencephalic (IT) neurons represent the largest class and project within the cortex and striatum. IT neurons have been amplified and diversified throughout mammalian evolution and are thought to underlie increasingly complex mammalian cognition. However, despite their diversity and significance in cortical processes, few existing tools connect the molecular signatures to the projection patterns of distinct IT subtypes, limiting our ability to understand their organization and function at a subtype resolution. Here, we illustrate six novel gene knock-in mouse lines based on IT-specific markers, labeling subpopulations with unique cortico-cortical and corticostriatal projection patterns. These include two broad subpopulations with upper-layer (Cux1-Flp) versus lower-layer (Kcnh5-CreER) expression biases, two narrow upper-layer subpopulations with contrasting callosal (Wfs1-Flp) and local (Igfn1-Flp) projection patterns, and two narrow lower-layer subpopulations with contrasting bilateral (Deptor-CreER) versus ipsilateral-predominant (Ptprm-CreER) corticostriatal projection patterns. Upper-layer Wfs1 ⁺ and lower-layer Ptprm ⁺ IT neurons also project to perirhinal/ectorhinal cortex with densities that vary across cortical areas. Finally, we use in vivo calcium imaging in mice performing a reach-withdraw-drink task to demonstrate subtype-specific temporal dynamics in upper-layer Wfs1 ⁺ versus lower-layer Deptor ⁺ neurons. Our work expands the toolbox of transgenic mice to access molecularly defined IT neuron subpopulations.