Figures & data
Figure 1. Deficiency in BRAP homologous protein expression caused mild anxiety-like behaviors in male mice.
(A) The expression of BRAP protein in postmortem adult human brain sections by immunohistochemistry with anti-BRAP antibody. The tissue sections were prepared from paraffined brain tissues from four cases that passed away due to sudden cardiac death with different underlying conditions. The immunostaining of the brain section of each case were indicated by numbers 1–4. Case 1: male, 28 years old, ruptured aneurysm of the right renal artery; Case 2: male, 50 years old, arrhythmia caused by pathological upregulation of fibrosis within the sinoatrial node; Case 3: male, 67 years old, pulmonary embolism; Case 4: male, 29 years old, acute left main coronary artery occlusion; (B) The expression of BRAP homologous protein in mice detected by western blot with anti-BRAP antibody. The protein extract of hippocampus tissues of three wild type mice, three heterozygous mice and three knockout mice were loaded in each well. The expression of tubulin from the same sample was used as loading controls. (C) Total numbers of entries to the open arms in the elevated plus-maze test. (D) The time spent on the open arms in the elevated plus-maze test. (E) Total distance traveled in the different regions of the open field in the OFT. (F) The distance traveled in central area in the OFT. (G) The time spent on the central area in the OFT. Two-way ANOVA was performed followed by Tukey’s multiple comparisons. Data are presented as mean ± SD. *p <.05, **p <.01, ***p <.001, n = 8.
Figure 2. The analysis of behavioral tests after exposure to chronic unpredictable mild stress (CUMS). (A) Body weight changes throughout the CUMS procedure. (B) Immobility time of bc004004+/+ and bc004004−/− mice in the forced swimming test with treatment of CUMS. (C) Analysis of sucrose preference tests during the CUMS procedure. (D) Immobility time of bc004004+/+ and bc004004−/− mice in the tail suspension test after 21-day CUMS exposure. (E) Total distance traveled in the different regions in the open field test after 21-day CUMS exposure. Two-way ANOVA was performed followed by Tukey’s multiple comparisons. (F) The expression of BRAP homologous protein in mice detected by western blot with anti-BRAP antibody. The protein extracts of hippocampus tissues of the mice was used in western blot after 21-day of CUMS treatment. The expression of β-actin from the same sample was used as loading controls. Data are presented as mean ± SD. *p <.05, **p <.01, ***p <.001,****p <.0001, n = 8.
Figure 3. Novel object recognition (NOR) test in mice exposed to 21-day of CUMS treatment.
(A) The total time of exploring objects including old and new objects. (B) Recognition index for different groups in the NOR. Recognition index is the ratio of the exploring time of new objects and the total exploring time. Two-way ANOVA was performed followed by Tukey’s multiple comparisons. Data are presented as mean ± SD.*p < .05, n = 8.
Figure 4. Analysis of dendritic morphology of the hippocampal neurons after 21-day of CUMS exposure.
(A) A representative dendritic tree of a Golgi-Cox stained hippocampal neuron (upper panel) and a reconstructed hippocampus neuron for Sholl analysis (lower panel). (B) Dendritic length of hippocampus neurons in different groups in CA1 field. (C) Dendritic length of hippocampus neurons in different groups in CA3 field. (D) The amount of dendritic branches in CA1 field of mice in different groups. (E) The amount of dendritic branches in CA3 field of mice in different groups. Two-way ANOVA was performed followed by Tukey’s multiple comparisons for each distance in (D) and (E). Data are presented as mean ± SEM. The statistical significance of interaction between CUMS and genotype, main effects of CUMS and genotype for the distances from 61 μm to 161 μm in (D) are indicated with asterisks: ∗∗interaction, ∗CUMS and ∗genotype (at 61 μm), ns for interaction, ∗∗∗CUMS and ns for genotype (at 71 μm), ns for interaction, CUMS and genotype (at 81 μm), ∗∗∗∗interaction, ns for CUMS and genotype (at 91 μm), ∗∗∗interaction, ns for CUMS and genotype (at 101 μm), ∗∗interaction, ns for CUMS and genotype (at 111 μm), ∗interaction, ns for CUMS, ∗genotype (at 121 μm), ∗interaction, ns for CUMS and genotype (at 131 μm), ns for interaction, CUMS and genotype (at 141 μm), ∗∗∗interaction, ns for CUMS and genotype (at 151 μm), ns for interaction and CUMS, ∗genotype (at 161 μm). The statistical significance of interaction between CUMS and genotype, main effects of CUMS and genotype for the distances from 31 μm to 61 μm in (E) are indicated with asterisks: ns for interaction, ∗CUMS and ∗∗genotype (at 31 μm), ∗interaction, ns for CUMS and ∗∗genotype (at 41 μm), ns for interaction and CUMS, ∗∗genotype (at 51 μm), ns for interaction and genotype, ∗∗∗CUMS (at 61 μm). The statistical tests for interaction, main effects of CUMS and genotype at all the other distances in both (D) and (E) revealed non-significance. ns: not significant, *p <.05, **p <.01, ***p <.001, ****p <.0001, n = 6.
Figure 5. The amount and morphology of different dendritic spines on hippocampal CA1 and CA3 neurons.
(A) Representative images of dendritic spines from hippocampal neurons. (B) The total dendritic spine number on hippocampal CA1 neurons, the effect of genotype: F(1, 5) = 20.06, p =.0065. (C) The total dendritic spine number on hippocampal CA3 neurons, the effect of CUMS: F(1, 5) = 7.358, p =.0421. (D) Proportions of each type of dendritic spines or protrusions (“mushroom,” “stubby,” “thin” spines and “filopodia-like” protrusions) on hippocampal CA1 neurons. (E) Proportions of each type of dendritic spines or protrusions (“mushroom,” “stubby,” “thin” spines and “filopodia-like” protrusions) on hippocampal CA3 neurons. Two-way ANOVA was performed followed by Tukey’s multiple comparisons. Data are presented as mean ± SD. ns: not significant, *p <.05, **p <.01, ***p <.001, ****p <.0001, n = 6.
Figure 6. The expression of some proteins involved in synapse plasticity of the hippocampus.
(A) Immunohistochemistry analysis of endogenous synaptophysin, PSD-95 and GluN2A in tissue sections from hippocampus after 21-day of CUMS treatment (100× and 400×). (B), (C), and (D) Quantification of areas stained by anti-synaptophysin, PSD-95 and GluN2A antibodies in tissue sections from hippocampus in immunohistochemistry analysis using ImageJ software, respectively. (E) Western blot analysis of synaptophysin and PSD-95 in protein extracts of brain tissue samples (5μg of total protein), respectively. (F) and (G) Quantification of expression levels of synaptophysin and PSD-95 as normalized by β-actin expression in western blot analysis. (H) Western blot analysis of GluN2A in brain tissue samples (5 μg of total protein). (I) Quantification of GluN2A expression in western blot analysis as normalized by β-actin expression. Two-way ANOVA with Tukey’s multiple comparisons test. *p < .05, **p < .01, ***p < .001, ****p < .0001, n = 6.
Figure 7. The impact of CUMS treatment on the expression of BDNF and TrkB in the hippocampal region of mice.
(A) Immunofluorescence labeling of endogenous BDNF in hippocampus tissue sections (400×). (B) Immunohistochemistry analysis of endogenous TrkB in hippocampus tissue sections (400×). (C) Quantification of immunofluorescence labeling of BDNF in the hippocampus by ImageJ software. (D) Quantification of positive areas stained by anti-Trk-B using ImageJ software. The interaction between CUMS and genotype: F(1, 4) = 21.28, p = .0099; the effect of CUMS: F(1, 4) =9.712, p = .0356; the effect of genotype: F(1, 4) = 58.86, p = 0016. (E) Western blot analysis of BDNF in total protein extracts of hippocampus tissue samples (3 μg of total protein). (F) and (G) Quantification of 35 kDa and 28 kDa of BDNF precursor expression in western blot analysis as normalized by β-actin expression, respectively. (H) Western blot analysis of TrkB in total protein extracts of hippocampus tissue samples (5 μg of total protein). (I) Quantification of Trk-B precursor expression in western blot analysis as normalized by β-actin expression. Two-way ANOVA with Tukey’s multiple comparisons test. *p < .05, **p < .01, ***p < .001, ****p < .0001, n = 6.
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