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. 2019 Apr 24;13:164. doi: 10.3389/fncel.2019.00164

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Copyright © 2019 Lenoir, Varangot, Lebouvier, Galli, Hommet and Vivien.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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tPA is present in pre- and post-synaptic elements. (A) Timeline of the experiments. Representative z-stack confocal images of cortical neurons (at DIV 20) co-transfected with pCMV_tPA-HaloTag® and pCMV_GFP-homer-1C plasmids followed by immunocytochemistry raised against synapsin. tPA-HaloTag® (TMR ligand, in red) is present at synapses revealed by the presence of GFP-homer-1C (post-synaptic protein, green) and synapsin (pre-synaptic protein, blue). Scale Bar: 5 or 50 μm (whole neuron). (B) Representative diagrams showing the percentages of synapses containing or not tPA-HaloTag®. TMR staining (i.e., tPA) is present in 32% of all synapses (yellow frame: 1) and 68% of synapses are negative for tPA (white frame: 2), N = 21 neurons from N = 6 independent cultures and p < 0.0001. (C) Timeline of the experiments. Cortical neurons at DIV 20 were transfected with pCMV_tPA-HaloTag® plasmid, ICC raised against synaspin and homer-1C were performed. Representative z-stack confocal images and diagrams show the percentages of tPA located either pre or post-synaptically. Scale Bar: 5 μm. (D) 55% of all synaptic tPA is post-synaptic (green frame in confocal image; 3) and 45% of all synaptic tPA is pre-synaptic (blue frame in confocal image; 4). N = 10 neurons from N = 2 independent cultures and p = 0.025. (E) IMARIS reconstructions from confocal images C of pre-synaptic tPA (blue frame; 4) and post-synaptic tPA (green frame; 3). (F) Representative 3D-confocal images of co-transfected cortical neurons (at 3D-confocal images of co-transfected cortical neurons (at DIV20) with pCMV_tPA-HaloTag® (in red) and pCMV_GFP-homer-1-C (in green) plasmids followed by immunocytochemistry raised against synapsin (in blue). IMARIS reconstruction from confocal images. Scale Bar: 2 μm.