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. 2017 Dec 23;21(4):382–392. doi: 10.1093/ijnp/pyx118

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© The Author(s) 2017. Published by Oxford University Press on behalf of CINP.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 3. — ERβ is necessary for cocaine conditioned place preference (CPP) in female mice. (A, B) Top panels show the location of the short hairpin RNA (shRNA) targeting ERα (shEsr1, A) and ERβ (shEsr2, B). The horizontal line represents the reference sequence transcript, with the nucleotide position indicated by a number. The open box shows the position of the protein coding sequence in the transcript, and the black vertical line shows the location of the 19 nucleotide targeting sequence in the shRNA. The bottom graphs show the relative mRNA expression of Esr1 (A) and Esr2 (B) by quantitative real-time PCR in Neuro-2a cells transfected with lentiviral plasmids expressing each of the shRNAs. Asterisks indicate a significant difference in Esr1 (A) and Esr2 (B) expression in cells transfected with shEsr1 or shEsr2, respectively, compared with the control shRNA (shScr, ***P < .001 or ****P < .0001 by Student’s t test, n = 3). (C) The left panel is a drawing of a coronal section of the mouse brain at 1.7 mm anterior to bregma, with the location of the cannulas used to infuse the lentivirus bilaterally into the nucleus accumbens (Acb) indicated by vertical lines. Adapted from the Mouse Brain Atlas (Franklin and Paxinos, 3^rd ed.). The right panel is a representative image of a coronal section of lentivirus infection in the Acb as measured by expression of green fluorescent protein, indicated by dark brown staining. (D) Timeline and design of the CPP experiment. (E) Graph of the time spent on the cocaine-paired side (in seconds) before conditioning (Test 1) and after conditioning (Test 2) with 5 mg/kg cocaine of mice expressing shEsr1, shEsr2, or shScr in the Acb. Data are presented as the means ±SEM. Significant main effect by two-way RM ANOVA (****P < .0001, n = 10). ^#Indicates significant difference between shScr and shEsr2 on Test 2 (P < .01); Significant difference between Test 1 and Test 2 within each treatment group (**P < .01 and ***P < .001) by posthoc multiple comparisons test. (F) Preference scores (Test 2-Test 1, in seconds) of mice expressing shScr, shEsr1, and shEsr2 in the Acb. A 1-way ANOVA showed a trend toward an effect of shRNA (P = .067, n = 10) with posthoc multiple comparisons test indicating a significant difference between mice expressing shEsr2 compared with shScr (P < .05). (G) Time on cocaine-paired side before conditioning (Test 1) and after conditioning (Test 2) in male mice expressing shScr or shEsr2 in the Acb (n = 8). There was a significant effect of time (****P < .0001), but no significant effect of shRNA. (H) Mean preference scores of male mice expressing shScr or shEsr2 in the Acb after cocaine CPP. aca, anterior commissure; AcbC, nucleus accumbens core; AcbSh, nucleus accumbens shell; fmi, forceps minor of the corpus callosum; LV, lateral ventricle; CPu, caudate putamen. Scale bar, 100 μm.