Acute in vivo proximity labeling for membrane targeted proteomics in neuronal circuits

A major goal within molecular systems neuroscience is to bridge the study of neuronal circuit function with changes in protein expression and localization in awake behaving animals. However, there are limited tools for capturing changes in subcellularly-defined proteomes within neuronal circuits during activity-gated timescales in vivo. Here, we engineered targeted versions of the proximity labeling enzyme TurboID, to tag proteins at the neuronal membrane during a user-delivered biotin injection. We optimized a labeling strategy that enables a one-to-two-hour labeling window and tagged proteins in medial prefrontal cortex (mPFC) cell bodies and corresponding axons in two downstream projections. We performed proteomics to identify proteins enriched in the distinct mPFC axonal projections, and discovered proteins enriched following an acute cocaine injection in mPFC cell bodies. These advancements enable the detection of proteins at the subcellular level within short labeling windows, allowing identification of stimulus-specific proteomes in behaving mice.