Abstract
Tools for genetically-determined visualization of synaptic circuits and interactions are necessary to build connectomics of the vertebrate brain and to screen synaptic properties in neurological disease models. Here we develop a transgenic FingR (fibronectin intrabodies generated by mRNA display) technology for monitoring synapses in live zebrafish. We demonstrate FingR labeling of defined excitatory and inhibitory synapses, and show FingR applicability for dissecting synapse dynamics in normal and disease states. Using our system we show that chronic hypoxia, associated with neurological defects in preterm birth, affects dopaminergic neuron synapse number depending on the developmental timing of hypoxia.
Highlights
Our implementation of an inducible expression system for FingRs in transgenic zebrafish provides a novel approach for visualizing and quantifying development, dynamics, and the effects of experimental or disease manipulations on the post-synaptic proteins PSD-95 and GPHN
In control experiments we found no changes in synapse structure or in behavior in animals expressing FingRs
Our approach is built upon an inducible expression system using Gal4/UAS; and we have shown that in vivo monitoring of synapse maturation and dynamics is feasible
Summary
Injections of UAS:PSD95.FingR-GFP plasmid into embryos of the line Tg(otpb.A:Gal4), which expresses in diencephalic dopaminergic neurons[11], resulted in GFP expression in neuronal cell bodies and neurites (Fig. 1C). To test whether the FingR system affected expression of synaptic proteins or numbers of synapses, we determined the level of Synapsin expression using immunohistochemistry in defined regions of the telencephalon, or with western blot of the entire embryo, in animals with pan-neuronal expression of FingR(PSD95)-GFP.
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