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. 2017 Aug 25;7:9452. doi: 10.1038/s41598-017-10147-x

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Subsynaptic distribution of GPR37 in the mouse striatum. (A) Electron micrographs showing immunoparticles for GPR37 in the striatum of GPR37+/+ mice using the pre-embedding immunogold technique. GPR37 immunoparticles were abundant on the extrasynaptic plasma membrane (arrows) of dendritic spines (s) of striatal neurons contacted by axon terminals (at). Few immunoparticles were observed at intracellular sites (crossed arrows) in dendritic spines (s) (upper panel). Immunoparticles for GPR37 were also localized to the extrasynaptic plasma membrane (arrowheads) of axon terminals (at) establishing asymmetrical synapses with spines (s). In dendritic shafts (Den), immunoparticles for GPR37 were mainly found at the plasma membrane (arrows) (lower panel). Scale bars: 200 nm. (B) Bar graphs showing the percentage of GPR37 immunoparticles at post- and presynaptic compartments. The data are expressed as mean ± SEM of three independent experiments (***P < 0.001; Student’s t-test). (C) Representative immunoblots showing GPR37 and A_2AR immunoreactivity in striatal synaptic fractions. Striatal synaptosomes (Total) were subcellularly fractionated (see Materials and Methods section) into extrasynaptic (Extra), presynaptic active zone (Pre) and postsynaptic density (Post) fractions, which were analyzed by SDS-PAGE (20 μg of protein/lane) and immunoblotted using rabbit anti-GPR37-N, goat anti-A_2AR, rabbit anti-synaptophysin, mouse anti-PSD-95, mouse anti-SNAP-25 and rabbit anti-α-actinin antibodies. The primary antibodies were detected using a horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG, HRP-conjugated goat anti-mouse IgG, HRP-conjugated rabbit anti-goat IgG and chemiluminescence detection (see Materials and Methods). (D) Relative quantification of GPR37 enrichment in striatal presynaptic and postsynaptic fractions. The intensities of the immunoreactive bands on the immunoblotted membranes corresponding to extrasynaptic, presynaptic (Pre) and postsynaptic (Post) fractions were measured by densitometric scanning. Values were first normalized to the loading control (i.e. α-actinin) and then to the amount of GPR37 or A_2AR in the total fraction. Data are expressed as mean ± SEM of three independent experiments (*P < 0.05, Student’s t-test).