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. 2018 Jun 14;23(9):1851–1867. doi: 10.1038/s41380-018-0100-y

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© The Author(s) 2018

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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Fig. 3 — Diazepam-dependent loss of GABAergic synapses is prevented by inhibition of calcineurin or GABA_AR activity. a Immunolabelling of γ2-containing clusters (red) and VGAT-positive presynaptic GABArgic terminals (cyan) along MAP2-positive dendrites (20 µm; blue) following 72 h treatment with control (DMSO) or diazepam (D; 1 μM), in the absence or presence of cyclosporine A (CyA; 1 μM), and corresponding graphs showing b size (median/line-IQRs; mean/dot ± s.d. whiskers; Mann–Whitney test, *p < 0.05), and c number of synaptic γ₂ clusters (mean ± s.e.m.; ANOVA/Bonferonni post-hoc test; *p < 0.05), from n = 71 control dendrites & n = 85, n = 64, n = 73 dendrites of diazepam-, diazepam/cyclosporine A and cyclosporine A-treated cells, respectively, of a total of n = 19, n = 19, n = 18, n = 19 neurons in each group collected from two independent experiments. d Decrease in number of GABAergic terminals (mean ± s.e.m.; ANOVA/Bonferonni post-hoc test; *p < 0.05) contacting n = 71 control-treated & n = 85, n = 64, n = 73 diazepam-, diazepam/cyclosporine A- or cyclosporine A-treated dendrites for 72 h, respectively, from a total of n = 19, n = 19, n = 18, n = 19 neurons in each group, collected from two independent experiments. e Immunolabelling of γ2-containing clusters (red) and VGAT-positive presynaptic GABArgic terminals (cyan) along MAP2-positive dendrites (20 µm; blue) following 72 h treatment with control (DMSO) or diazepam (D; 1 μM), in the absence or presence of bicuculline (Bic; 50 μM), and corresponding graphs showing f size (median/line-IQRs; mean/dot ± s.d. whiskers; Mann–Whitney test, *p < 0.05), and g number of synaptic γ₂ clusters (mean ± s.e.m.; ANOVA/Bonferonni post-hoc test; *p < 0.05), from n = 60 control dendrites & n = 63, n = 61, n = 65 dendrites of diazepam-, diazepam/bicuculline and bicuculline-treated cells, respectively, of a total of n = 16, n = 19, n = 18, n = 20 neurons in each group collected from two independent experiments. h Decrease in number of GABAergic terminals (mean ± s.e.m.; ANOVA/Bonferonni post-hoc test; *p < 0.05), contacting n = 60 control-treated & n = 63, n = 61, n = 65 diazepam-, diazepam/bicuculine- or bicuculline-treated dendrites for 72 h, respectively, from a total of n = 16, n = 19, n = 18, n = 20 neurons in each group, collected from two independent experiments. Scale bars = 20 μm (a, e-upper row) and = 5 μm (a, h-lower row)