Live Imaging of Endogenous PSD-95 Using ENABLED: A Conditional Strategy to Fluorescently Label Endogenous Proteins

J. Neurosci. 2014 Dec 10; 34 (50) 16698-16712. doi: https://doi.org/10.1523/JNEUROSCI.3888-14.2014

Dale A. Fortin, Shane E. Tillo, Guang Yang, Jong-Cheol Rah, Joshua B. Melander, Suxia Bai, Omar Soler-Cedeño, Maozhen Qin, Boris V. Zemelman, Caiying Guo, Tianyi Mao* and Haining Zhong*
*Co-senior authorship

Abstract
Stoichiometric labeling of endogenous synaptic proteins for high-contrast live-cell imaging in brain tissue remains challenging. Here, we describe a conditional mouse genetic strategy termed endogenous labeling via exon duplication (ENABLED), which can be used to fluorescently label endogenous proteins with near ideal properties in all neurons, a sparse subset of neurons, or specific neuronal subtypes. We used this method to label the postsynaptic density protein PSD-95 with mVenus without overexpression side effects. We demonstrated that mVenus-tagged PSD-95 is functionally equivalent to wild-type PSD-95 and that PSD-95 is present in nearly all dendritic spines in CA1 neurons. Within spines, while PSD-95 exhibited low mobility under basal conditions, its levels could be regulated by chronic changes in neuronal activity. Notably, labeled PSD-95 also allowed us to visualize and unambiguously examine otherwise-unidentifiable excitatory shaft synapses in aspiny neurons, such as parvalbumin-positive interneurons and dopaminergic neurons. Our results demonstrate that the ENABLED strategy provides a valuable new approach to study the dynamics of endogenous synaptic proteins in vivo.