The purpose of the research proposed in this grant is to generate tools that will allow endogenous synaptic proteins to be visualized in neurons in vivo. Previously we have generated recombinant antibodies known as FingRs (Fibronectin intrabodies generated with mRNA display) that can be expressed in living neurons where they label endogenous target proteins noninvasively and with high fidelity. Furthermore, a transcriptional regulation system controls the expression levels of FingRs so that they are expressed at precisely the same level as their endogenous counterparts, insuring low background. In this grant we will generate transgenic mice that express fluorescent protein-fused FingRs that recognize synaptic proteins. Synaptic proteins in individual neurons in the brains of these mice can be visualized in vivo in real time. In turn, this will allow events at the molecular level to be correlated with events at the cellular, circuit and whole animal level. In particular, by mapping the locations and amounts of synaptic proteins in neurons, it will be possible to monitor the strength of both synaptic inputs and outputs in living neurons. The ability to monitor synaptic strength will be very useful for studying diseases associated with aberrant synaptic connectivity including schizophrenia, mental retardation and autism.
Previously we have generated recombinant antibodies that allow endogenous Gephyrin and PSD95 to be visualized in living cells. The research proposed in this grant will extend our previous results to allow synaptic proteins to be visualized in intac animals.