Recombinant Probes for Visualizing Endogenous Synaptic Proteins in Living Neurons

•Recombinant intrabodies (FingRs) generated with mRNA display bind PSD-95 or Gephyrin•PSD-95 and Gephyrin FingRs accurately label endogenous targets in living neurons•Transcriptional control matches expression of FingRs to targets, reducing background•PSD-95 and Gephyrin FingRs do not affect neuronal morphology or function

SummaryThe 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.