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. 2018 May 28;11:177. doi: 10.3389/fnmol.2018.00177

FIGURE 2.

FIGURE 2

EEN1 expression in the hippocampal CA1 region of adult mice is required for long-term spatial and contextual fear memory. (A) LacZ staining in the sagittal brain section of 10-week-old EEN1 KO-first (EEN1-/-) mice. Right panel is magnification of the hippocampus. Scale bars, 1 mm in the left panel and 100 μm in the right panel. (B) Immunofluorescence staining of EEN1 in hippocampal CA1, CA2, CA3, and DG regions of 10-week-old EEN1+/+ and EEN1-/- mouse brains. Scale bar, 20 μm. (C) AAV virus was stereotaxically injected into the CA1 regions of EEN1fl/fl mice to express GFP alone or Cre and GFP. Shown are GFP signal and DAPI labeling of nuclei 21 days after viral injection. Scale bar, 1 mm. (D) Immunofluorescence staining of EEN1 in brain slices 21 days after injection of AAV virus into the CA1 region of EEN1fl/fl mice. Lower panels are magnification of the boxed areas. Scale bar, 100 μm. (E–L) The Morris water maze test. Shown are escape latency or traveled distance before escaping to the platform in the visible-platform training (E and F), escape latency, and traveled distance before escaping to the platform in the invisible-platform training (G and H), number of crossing with the 1.5× platform area and the swim trace 5 days after training in probe test (I and J), the swim trace and recall ability following training once again 1 month after training (K and L). Data represent mean ± SEM (11 GFP, 13 Cre), ∗p < 0.05, ∗∗p < 0.01. (M and N) Decrease in freezing behavior 72 h after contextual fear training in the Cre virus-injected group. Data represent mean ± SEM (9 GFP, 12 Cre), ∗∗p < 0.01.