This projects aims to uncover the biological mechanisms of PTSD, a psychiatric disorder that affects over 16 million people in the US and is characterized by debilitating cognitive symptoms such as anxiety, depression, and mood instability in response to a traumatic event. To evaluate epigenetic signatures of fear memory formation in the small subset of neurons that are activated by stimuli, we plan to use TRAP (targeted recombination in active populations). TRAP uses a modified Cre-loxP recombination system to permanently label neurons with GFP when they are activated. Initiation of labelling is regulated by controlling when the drug 4-hydroxytamoxifen is injected into transgenic TRAP mice.

Experiment 1 involves fear conditioning TRAP mice, followed by INTACT to isolate nuclei from extracted brain tissue, and finally fluorescence imaging to calculate percentage GFP+ neuronal nuclei. 

To track global proteomic changes, we are combining TRAP with TurboID, a biotin ligase that robustly labels nearby proteins with biotin. Experiment 2 tests the involves transduction of neuron-like N2As with Turbo or TurboER (a form of TurboID localized to the ER) and Western blot analysis of proteome biotinylation.