Fig. 6: conditional knockout of Kdm4a in DG neurons facilitates memory formation in pattern separation task.
From: Kdm4a is an activity downregulated barrier to generate engrams for memory separation
a (Left) Strategy for construction of conditional Kdm4a knockout mice. (Right) PCR identification results of Kdm4awt/fl mice. b RT-qPCR analysis of Kdm4a mRNA expression levels in the DG. Kdm4af/f, n = 3; Nes-Cre;Kdm4af/f, n = 3. Two-tailed unpaired t-test, t4 = 3.911, * p = 0.0174. Data are presented as mean ± s.e.m. c Western blot detection of Kdm4a knockout efficiency in the hippocampus of Nes-Cre;Kdm4af/f mice. Kdm4af/f, n = 4; Nes-Cre;Kdm4af/f, n = 4. Two-tailed unpaired t-test, t6 = 3.456, * p = 0.0135. Data are presented as mean ± s.e.m. d Genomic DNA PCR detection of Kdm4a knockout efficiency of Nes-Cre;Kdm4af/f mice. CX, cortex; HP, hippocampus; ST, stratum; TH, thalamus. C, control (Kdm4af/f); K, Kdm4a cKO (Nes-Cre;Kdm4af/f). LoxP: Kdm4a loxP primer; IP Ctrl: intrinsic positive control primer. Kdm4af/f, n = 3; Nes-Cre;Kdm4af/f, n = 3. Two-way ANOVA followed by Dunnett test, F1,4 = 113.3, p = 0.0004. **** p < 0.0001. Data are presented as mean ± s.e.m. e Immunofluorescence staining of Kdm4a in the Kdm4af/f versus Nes-Cre;Kdm4af/f mice. Scale bar, 500 μm. Kdm4af/f, n = 3, region = 31; Nes-Cre;Kdm4af/f, n = 3, region = 28. Two-tailed unpaired t-test, t57 = 34.13, **** p < 0.0001. Data are presented as mean ± s.e.m. f Open field test. The locomotor ability of Kdm4a cKO mice was normal. Kdm4af/f, n = 12; Nes-Cre;Kdm4af/w, n = 12; Nes-Cre;Kdm4af/f, n = 11. One-way ANOVA followed by Dunnett test, Distance moved: F2,32 = 0.02494, p = 0.9754; Time in center: F2,32 = 0.6745, p = 0.5165. Data are presented as mean ± s.e.m. g Elevated plus maze test. Neuron-specific knockout of Kdm4a did not cause increased anxiety levels in mice. Kdm4af/f, n = 10; Nes-Cre;Kdm4af/w, n = 10; Nes-Cre;Kdm4af/f, n = 9. One-way ANOVA followed by Dunnett test, Time in open arm: F2,26 = 0.4922, p = 0.6168; Time in closed arm: F2,26 = 0.2663, p = 0.7682; Percentage in open arm: F2,26 = 0.492, p = 0.6168. Data are presented as mean ± s.e.m. h There was no difference in freezing levels between Kdm4a cKO mice and control mice during the memory retrieval phase of contextual fear conditioning. Kdm4af/f, n = 12; Nes-Cre; Kdm4af/f, n = 11. Two-way ANOVA followed by Sidak test, F1,21 = 0.07286, p = 0.7898. Data are presented as mean ± s.e.m. i, The shock speed when the mice received footshock was not significantly different between Kdm4a cKO mice and control mice. Kdm4af/f, n = 12; Nes-Cre; Kdm4af/f, n = 11.Two-tailed unpaired t-test, t21 = 0.7363, p = 0.4697. Data are presented as mean ± s.e.m. j Kdm4a cKO mice and control mice were subjected to the contextual fear discrimination test. k (Left) There was no difference in freezing levels between Kdm4a cKO mice and control mice in context A. Kdm4af/f, n = 12; Nes-Cre; Kdm4af/f, n = 11.Two-way ANOVA followed by Sidak test, F1,21 = 0.9275, p = 0.3465. CFD: contextual fear discrimination. (Middle) The freezing level of Kdm4a cKO mice in context B was significantly lower than that of control mice. Two-way ANOVA followed by Sidak test, F1,21 = 11.77, ** p = 0.0025. (Right) The discrimination index of Kdm4a cKO mice in the contextual fear discrimination test was significantly higher than that of control mice. Two-way ANOVA followed by Sidak test, F1,21 = 5.497, * p = 0.029. Data are presented as mean ± s.e.m.
