PROJECT SUMMARYThe functions and connectivity of neurons are established and manifested by their constituentproteins. Monitoring the organization of individual proteins in specific neuronal subtypes in livebrain tissue may therefore provide important readout of cellular and circuit properties underlyingbrain function. However, it remains challenging to visualize endogenous synaptic proteins inindividual neurons in live tissue. Most studies rely on the overexpression of fluorescently taggedproteins of interest. However, protein overexpression can alter protein stoichiometry, trafficking,subcellular localization, and cell signaling, ultimately affecting cellular and circuit functions.Although `knock-in' strategies can in principle bypass protein overexpression, these strategiesare rarely employed because typical knock-in approaches result in global expression of thelabeled protein in all cells where the target protein is normally expressed. Global expressionresults in high fluorescence background and a lack of contrast in tissues, making high-resolutionimaging difficult.To solve the above problems, we recently developed a novel genetic strategy calledendogenous labeling via exon duplication (ENABLED). We have used this method to label thecritical postsynaptic marker protein PSD-95 with the yellow fluorescent protein mVenus in allneurons, in a sparse subset of neurons, or in specific neuronal subtypes. Unlike theconventional approach to visualizing PSD-95, which involves PSD-95 overexpression, ourstrategy does not result in altered neuronal functions, and, for the first time, allows for themonitoring of PSD-95 at endogenous levels in individual neurons in living mice. Despite theseadvantages, the ENABLED strategy can be further optimized to broaden its applicability andenhance its sensitivity. Furthermore, to comprehensively examine neuronal functions andconnectivity, multiple types of synaptic markers will need to be labeled. Here, we request fundsto optimize the ENABLED strategy and use it to label the excitatory and inhibitory postsynapticmarkers, PSD-95 and gephyrin, respectively, with 5-fold stronger signal in mice. We will alsogenerate ENABLED mice in which PSD-95 can be labeled using different colors for dual-coloranalyses with other proteins. We will make the reagents available to the neurosciencecommunity to provide fellow researchers with an unprecedented ability to monitor synapticconnectivity and plasticity under physiological conditions in brain slices and in behaving animals.
|Effective start/end date||7/1/16 → 6/30/18|
- National Institutes of Health: $192,500.00