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. 2020 Nov 24;11:5962. doi: 10.1038/s41467-020-19780-z

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© The Author(s) 2020

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. 6 — a, b Repeated ketamine exposure (15 mg/kg) increased PRDM5 protein expression in the nucleus of DLS neurons (unpaired two-tailed t test, t(6) = 2.533, P = 0.0445). PRDM5 is observed as two bands at 66 and 70 kDa (two isoforms). c Co-immunostaining for PRDM5 (red), NeuN (green), GFAP (wathet blue), and DAPI (deep blue) in the DLS. d Ketamine (15 mg/kg) increased PRDM5 protein expression in DLS neurons (unpaired two-tailed t test, t(6) = 3.572, P = 0.0118). e, g EMSA experiments were performed with 5 μg of PRDM5 protein and biotin-labeled Mgll DNA oligos (probe1 and probe2). The shifted protein–DNA band (red rectangle; lanes 2, 3, and 6 in e; lanes 2, 3, and 7 in g was competed with excess and unlabeled DNA oligos (lane 4). Preincubation with anti-PRDM5 antibodies resulted in supershifted antibody–protein–DNA bands (blue rectangle; lanes 7, 8, and 9 in e; lanes 9 and 10 in g that migrate more slowly in the native gel. f, h Ketamine (15 mg/kg) enriched PRDM5 at the promoter of gene Mgll (site 1 and site 2; site1, unpaired two-tailed t test, t(6) = 12.87, P < 0.0001; site2, unpaired t test, t(6) = 20.32, P < 0.0001). Compared to SAL group, *P < 0.05, ***P < 0.001. i, j shRNA2 targeting Prdm5 effectively interfered with PRDM5 expression (unpaired two-tailed t test, t(10) = 3.908, P = 0.0029). Compared to SCR group, **P < 0.01. k AAV-shPrdm5 reduced ketamine-seeking behaviors (n = 21 mice/group, two-way repeated measured ANOVA, followed by Dunnett’s multiple comparisons test, virus F(3,48) = 38.24, P < 0.0001; time F(8,384) = 2.848, P = 0.0044; interaction, F(24,384) = 2.056, P = 0.0027). l AAV-shPrdm5 reduced ketamine-taking behaviors (n = 21 mice/group, two-way repeated measured ANOVA, followed by Dunnett’s multiple comparisons test, virus F(3,48) = 31.97, P < 0.0001; time F(8,384) = 2.554, P = 0.0101; interaction, F(24,384) = 1.016, P = 0.4441). m AAV-shPrdm5 restored the DLS 2-AG level in ketamine self-administration test (one-way ANOVA, followed by Dunnett’s multiple comparisons test, F(3,30) = 5.848, P = 0.0029; SCR + KET vs. SCR + SAL, P = 0.0461; SCR + KET vs. AAV-shPrdm5 + KET, P = 0.0123). n AAV-shPrdm5 could not reduce ketamine-induced hyperlocomotor activity (n = 21 mice/group, two-way repeated measured ANOVA, followed by Dunnett’s multiple comparisons test: virus F(3,80) = 7.706, P = 0.0001; time F(7,560) = 8.764, P < 0.001; interaction, F(21,560) = 2.867, P < 0.001). o AAV-shPrdm5 restored the 2-AG level in the DLS in ketamine hyperlocomotion test (one-way ANOVA, followed by Dunnett’s multiple comparisons test, F(3,28) = 5.727, P = 0.0035; SCR + KET vs. SCR + SAL, P = 0.0142; SCR + KET vs. AAV-shPrdm5 + KET, P = 0.0031). Compared to SCR + SAL group, *P < 0.05, **P < 0.01, ***P < 0.001; compared to SCR + KET group, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001. Data are shown as mean ± SEM. SAL saline, KET ketamine, SCR scrambled shRNA. Source data provided as a Source Data file.