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. 2017 Jun 29;7:4379. doi: 10.1038/s41598-017-04728-z

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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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FLAG-tagged receptors reveal specificity of limited D2 receptor internalization. (A) Representative images of live neurons at baseline, following 10 minutes of agonist bath application (quinpirole 10 µM, dopamine 100 µM), and following 30 minutes of agonist-free wash. Slices were incubated in anti-FLAG M1-AF594 to label FLAG-tagged D2 receptors in the SN and LC prior to imaging. (B) FLAG D2 receptors in SN dopamine neurons show limited internalization following agonist exposure as measured by the change in the ratio of intracellular to membrane fluorescence (ΔF_r/F_r), only significant following the 30-min wash (not specified on graph). FLAG-D2 receptors in the LC show a significantly larger increase in the intracellular fluorescence relative to the membrane fluorescence (ΔF_r/F_r) when compared with FLAG-D2 receptors in SN neurons (n = 5–7, two-way ANOVA followed by Bonferroni). (C) The ΔF_r/F_r following 10 minutes of agonist exposure was not significantly different between GFP-D2 and FLAG-D2 receptors for either quinpirole or dopamine (n = 5–7, two-way ANOVA followed by Bonferroni). (D) Representative images of live neurons at baseline, following 10 minutes of agonist bath application ([Met]⁵enkephalin ME, 30 µM), and following 30 minutes of agonist-free wash. Slices were incubated in anti-FLAG M1-AF594 to label FLAG-tagged µ-opioid receptors (MORs) in the SN and LC prior to imaging. (F) In both the SN and LC, FLAG-MORs show a sharp increase in ΔF_r/F_r following agonist exposure. There was a slight decrease in ΔF_r/F_r following the 30-minute wash period. (F) FLAG-D2 receptors in the LC and FLAG-MORs in both the SN and LC show significantly larger ΔF_r/F_r following agonist application than FLAG-D2 receptors in the SN (n = 6–14, two-way ANOVA followed by Bonferroni). n.s. denotes not significant, *p < 0.05, **p < 0.01, ***p < 0.01.