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. 2023 Feb 28;26(3):106296. doi: 10.1016/j.isci.2023.106296

Figure 1.

Figure 1

Loss of NCLX-dependent neuronal mCa2+ efflux promotes cognitive decline

(A) Schematic of NCLX-nKO mice (NCLXfl/fl x Camk2a-Cre) mutant mouse strategy.

(B and C) (B) NCLX mRNA expression in tissue isolated from the frontal cortex of NCLX-nKO and age-matched controls (Camk2a-Cre). mRNA expression corrected to the housekeeping gene Rps13; expressed as fold change versus control, n = 3 for both groups. All data presented as mean ± SEM; ∗∗∗p<0.001; two-tailed, unpaired t-test(C). Western blots for NCLX expression in tissue isolated from the cortex of 2-month-old NCLXfl/fl x Camk2a-Cre mice compared to age-matched control Camk2a-Cre mice. VDAC, voltage-dependent anion channel, served as mitochondrial loading controls.

(D) NCLX protein expression expressed as fold-change versus Camk2a-Cre con. corrected to a mitochondrial loading control VDAC in brain cortex of 2-month-old mice. All data presented as mean ± SEM; ∗∗p<0.01; two-way ANOVA with Sidak’s multiple comparisons test.

(E and F) Y-maze spontaneous alternation test. (E) Percentage of spontaneous alternation. (F) Total number of arm entries.

(G–I) Fear-conditioning test. (G) Freezing responses in the training phase. (H) Contextual recall freezing responses, (I). Cued recall freezing responses. n = individual dots shown for each group in all graphs. All data presented as mean ± SEM. Data for percentage alternations, contextual and cued recall freezing response was analyzed using Prism (GraphPad) two-way ANOVA multiple comparison testing for an age effect with Dunnett’s post-hoc test for comparison to age 6 months and comparison of genotype across all ages using a Bonferroni’s multiple comparisons test, ∗∗p < 0.01. All comparisons were non-significant except those denoted. To ensure equivalent motor activity and behavior in the Y-maze and equivalent training for fear-conditioning behavioral testing data for Y-maze number of entries and freezing during training was analyzed using two-way ANOVA testing for comparison of genotype across all ages using a Bonferroni’s multiple comparisons test. No statistical differences were noted.