Recombinant Probes for Visualizing Endogenous Synaptic Proteins in Living Neurons

• DOI: 10.1016/j.neuron.2013.04.017
• Publication Date: 2013-06-19T18:46:03Z
• Authors: Garrett G. Gross; Jason A. Junge; Rudy J. Mora; Hyung-Bae Kwon; C. Anders Olson; Terry T. Takahashi; Emily R. Liman; Graham C.R. Ellis...; Aaron W. McGee; Bernardo L. Sabatini; Richard W. Roberts; Don B. Arnold
• Keywords: Gephyrin

The ability to visualize endogenous proteins in living neurons provides a powerful means to interrogate neuronal structure and function. Here we generate recombinant antibody-like proteins, termed Fibronectin intrabodies generated with mRNA display (FingRs), that bind endogenous neuronal proteins PSD-95 and Gephyrin with high affinity and that, when fused to GFP, allow excitatory and inhibitory synapses to be visualized in living neurons. Design of the FingR incorporates a transcriptional regulation system that ties FingR expression to the level of the target and reduces background fluorescence. In dissociated neurons and brain slices, FingRs generated against PSD-95 and Gephyrin did not affect the expression patterns of their endogenous target proteins or the number or strength of synapses. Together, our data indicate that PSD-95 and Gephyrin FingRs can report the localization and amount of endogenous synaptic proteins in living neurons and thus may be used to study changes in synaptic strength in vivo.