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. 2018 Jun 26;12:389. doi: 10.3389/fnins.2018.00389

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Copyright © 2018 Verstraelen, Van Dyck, Verschuuren, Kashikar, Nuydens, Timmermans and De Vos.

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 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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Synapse marker quantification as morphological readout for connectivity. (A) Immunocytochemical labeling (ICC) of pre- and postsynaptic markers in primary hippocampal neurons to label all [synaptophysin (Syph)], excitatory (vGLUT, PSD-95, and AMPA-R) or inhibitory (vGAT, gephyrin) synapse compartments. Some antibodies give rise to non-specific staining in neuronal cell bodies and astrocytes (PSD-95; arrows) or in neuronal nuclei (gephyrin; arrowhead), underlining the need for thorough antibody validation. Scale bar: 20 μm. (B) Genetic labeling of synaptic markers allows for temporal follow-up. Constitutive overexpression of PSD-95-GFP and synaptophysin-mKate2 (Syph-mKate2) gives rise to overexpression artifacts such as overfilling of the neuronal soma and dendrites by PSD-95-GFP, and a high number of synaptophysin-mKate2 puncta that do not colocalize with ICC of another presynaptic protein, synapsin (Syn). Targeting of GFP toward endogenous PSD-95 using FingR technology (Gross et al., 2013) results in good colocalization of PSD-95-FingR and ICC, making this approach more attractive for synapse screening than constitutive overexpression of fusion constructs. Scale bars: 20 μm. (C) MAP2-stained neurites can be analyzed for area fraction, but also skeletonized to determine the length and number of branching points. Scale bar: 50 μm. (D) Synaptic marker spots are segmented within the boundaries of the neurite mask to calculate synaptic marker density. An overlay of the raw image and the detected spots shows accurate spot detection. Scale bar: 20 μm. (E) To avoid detection of immature synapses, extrasynaptic and a specific staining, the apparent colocalization of pre- and postsynaptic markers can be evaluated as a proxy for mature synapses. Pre- and postsynaptic images of corresponding Z-planes are considered to avoid overdetection of mature synapses from different axial positions. After segmentation, pre- and postsynapse masks are combined and analyzed for colocalization, yielding parameters such as % overlap (O) or synapse density. A mature synapse can be defined by an arbitrary cut-off, e.g., 30% overlap between post/pre or pre/post. Alternatively, an intensity-based Pearson coefficient (P) can be calculated for the pre- and postsynaptic images. This method is independent of spot segmentation. For visual representation, the ‘product of the differences from the mean’ (PDM) is shown (H, high colocalization; L, low). Scale bar: 20 μm.