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. 2018 Sep 13;8:13752. doi: 10.1038/s41598-018-30589-1

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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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Nbea knockdown in the dorsal hippocampal CA1 region resulted in impairments in extinction after contextual fear retrieval. (A) Validation of shNbea efficiency in cultured hippocampal neurons. Left, Western blot image of NBEA expression from AAV-scrambled or AAV-shNbea-treated cultured hippocampal neurons. Right, Quantification of NBEA expression. Scrambled (100 ± 6.321, n = 4) vs shNbea (32.14 ± 7.286, n = 4), ***p = 0.0004, t-test, two-tailed. (B) Upper, Representative image of AAV virus injection into the CA1 of the dorsal hippocampus. Middle, Immunohistochemistry for NBEA and NeuN in AAV-scrambled-injected CA1. Lower, Immunohistochemistry for NBEA and NeuN in AAV-shNbea-injected CA1. (C) Locomotor activity in the open field test. There was no significant difference in the locomotor activity between AAV-scrambled and AAV-shNbea-injected mice. F(5, 125) = 0.692, p = 0.6304, two-way ANOVA, post hoc Sidak’s multiple comparison test. Scale bars indicate 50 μm. (D) Anxiety-like behavior in the open field test. There was no significant difference in time spent in the center of the open field for the first 5 min of the test between the AAV-scrambled and AAV-shNbea-injected mice. Scrambled (18.05 ± 3.96, n = 14) and shNbea (19.94 ± 4.404, n = 13), p = 0.7519, t-test, two-tailed. (E) Schematic representation of the experimental protocol. (F) Fear conditioning. There was no significant difference in the freezing level during fear conditioning between the AAV-scrambled and AAV-shNbea-injected mice. F(4, 100) = 0.8908, p = 0.4725, two-way ANOVA, post hoc Sidak’s multiple comparison test. (G) Fear retrieval. F(1, 25) = 8.984, **p = 0.0061, two-way ANOVA with post hoc Sidak’s multiple comparisons test. There was no significant difference in the freezing level during retrieval 1 (RT1) between AAV-scrambled and AAV-shNbea-injected mice. Scrambled (72.93 ± 3.599, n = 14) and shNbea (69.17 ± 2.744, n = 13), p = 0.2945. However, there was a significant difference in the freezing level during retrieval 2 (RT2) between AAV-scrambled and AAV-shNbea-injected mice. Scrambled (53 ± 2.733, n = 14) and shNbea (77.75 ± 2.603, n = 13), ***p = 0. 0.0003.