2P-NucTag: On-demand phototagging for molecular analysis of functionally identified cortical neurons

Neural circuits are characterized by genetically and functionally diverse cell types. A mechanistic understanding of circuit function is predicated on linking the genetic and physiological properties of individual neurons. However, it remains highly challenging to map the molecular properties onto functionally heterogeneous neuronal subtypes in mammalian cortical circuits in vivo. Here, we introduce a high-throughput two-photon nuclear phototagging (2P-NucTag) approach for on-demand and stable labeling of single neurons via a photoactivatable red fluorescent protein following in vivo functional characterization in behaving mice. Using this optimized function-forward pipeline to selectively label and transcriptionally profile previously inaccessible "place" and "silent" cells in the hippocampus of behaving mice, we identify unexpected differences in gene expression. Furthermore, we demonstrate multiple downstream experimental directions that 2P-NucTag enables, including ex vivo slice electrophysiology and histology. Thus, 2P-NucTag opens a new way to uncover the molecular principles that govern the functional organization of neural circuits.