Our development of an imaging technique based on two-photon fluorescence lifetime imaging microscopy (2pFLIM) and extensively optimized biosensors has allowed us to monitor signaling activity in single synaptic compartments in brain slices. Using this technique, we have recently succeeded in measuring the activity of signaling proteins important for LTP and structural LTP (sLTP) in single dendritic spines. The new results provided many insights into the spatiotemporal regulation of biochemical signaling during LTP and sLTP.

Besides monitoring each element of the signaling cascade, tools to manipulate signaling activity in a spatially and temporally controlled manner will be extremely useful to understand the information flow in the signaling network. Previously several photo-activatable proteins for activating small GTPase proteins and gene transcription have been developed. However, it is not easy to scale up these strategies to many more proteins. We have been developing photo-inducible inhibitors using a scalable design based on the light oxygen voltage domain 2 (LOV2)-Jα helix domain of Phototropin1. LOV2-Jα changes its conformation from a closed form to an open form upon blue light absorption. We fuse a pseudo-substrate inhibitor peptide to LOV2-Jα so that LOV2-Jα disables the action of the inhibitor due to steric hindrance when it is in the closed conformation, and upon light absorption, the inhibitor peptide action is switched on by the conformational change of LOV2-Jα and reversed spontaneously. Using this strategy, we developed a photo-activatable kinase inhibitor for CaMKII, a kinase important for learning and memory. This tool allows us to measure the timing.

Imaging endogenous proteins with high specificity, resolution, and contrast is critical for defining their subcellular localization, especially in the brain, due to its extremely complicated morphology. However, a scalable and high-throughput approach to imaging endogenous proteins has not been established. Toward this goal, we have developed a new method termed Single-cell Labeling of ENdogenous proteins by endonuclease-mediated homology-Directed Repair (SLENDR), which allows in vivo genome editing in the mammalian brain for single-cell labeling of endogenous proteins by inserting a tag sequence to a gene of interest through CRISPR-Cas9-mediated homology-directed repair (HDR). SLENDR has allowed us to rapidly determine the subcellular localization and dynamics of endogenous proteins with the resolution of micro- to nanometers in various cell types in widespread brain regions. Therefore, SLENDR provides a novel platform for comprehensive, large-scale analyses.

Imaging neuronal activity using genetically-encoded Ca2+ indicators with two-photon microscopy has been invaluable for the analysis of functional circuits and their plasticity with high spatiotemporal resolution in vivo. However, the behaviors and dynamics of each neuron critically depend on their intracellular biochemical states, and thus it is important to analyze the underlying biochemical signaling that regulates neuronal plasticity. To elucidate the missing connection between the biochemical states and circuit plasticity, the goal of this project is to simultaneously image neural circuit activity and signaling dynamics in individual synapses and neurons comprising the network. Thus, our objective is to combine 2-photon Ca2+ imaging with 2-photon fluorescence lifetime imaging microscopy (2pFLIM) of ongoing intracellular signaling and to implement the system in vivo.