Bipolar-associated miR-499-5p controls neuroplasticity by downregulating the Cav1.2 subunit CACNB2 (2022)
Figure 4

Figure 4: miR-499-5p represses CACNB2 expression in the rat hippocampus in vivo and impairs short-term recognition memory in Cacna1c+/- rats.

A) Schematic illustration of the hSyn-eGFP-scramble control (AAV-Control) and hSyn-eGFP-chimeric miR-499-5p hairpin (AAV-miR-499), and timeline of virus infection experiments.

B) miR-499-5p qPCR analysis from total RNA isolated from the right hippocampus of wild type rats injected with either AAV-control or AAV-miR-499. Data are represented as box plot with whiskers and data points (+: mean, line: median; whiskers: minimum and maximum values). (****p<0.0001, Unpaired two-sample t-Test; n=18 rats in the AAV-Control group, n=16 rats in the AAV-miR-499 group).

C) miR-499-5p qPCR analysis from total RNA isolated from the right hippocampus of Cacna1c +/- rats injected with either AAV-control or AAV-miR-499. Data are represented as box plot with whiskers and data points (+: mean, line: median; whiskers: minimum and maximum values). (****p<0.0001, Unpaired two-sample t-Test; n=15 rats in the AAV-Control group, n=16 rats in the AAV-miR-499 group).

D) Representative Western blot images of CACNB2 (upper panel) and GAPDH (lower panel) protein expression levels in the hippocampus of WT rats and Cacna1c +/- rats injected with the AAV-Control or AAV-miR-499 hairpin viruses. GAPDH was used as a loading control

E) Quantification of CACNB2 Western blots using hippocampal protein lysate from wild type rats injected with either AAV-control or AAV-miR-499. Data are represented as box plot with whiskers and data points (+: mean, line: median; whiskers: minimum and maximum values). (*p=0.0103, Unpaired two-sample t-Test; n=7 rats in the AAV-Control group, n=6 rats in the AAV-miR-499 group).

F) Quantification of CACNB2 Western blots using hippocampal protein lysate from Cacna1c +/- rats injected with either AAV-control or AAV-miR-499. Data are represented as box plot with whiskers and data points (+: mean, line: median; whiskers: minimum and maximum values). (**p=0.0044, Unpaired two-sample t-Test; n=8 rats in the AAV-Control group, n=7 rats in the AAV-miR-499 group).

G) Novel object recognition task. Percentage of time WT or Cacna1c+/- rats injected with the indicated AAV explored either the familiar or novel object. Data are represented as box plot with whiskers and data points (+: mean, line: median; whiskers: minimum and maximum values). (WT_Control: n=18 (8 males, 10 females); WT_miR-499: n=16 (7 males, 9 females); Cacna1c+/-_Control: n=15 (5 males, 10 females); Cacna1c+/-_miR-499: n=16 (10 males, 6 females). Paired t-test - novel vs familiar object exploration percentage: WT_Control: T17 = 2.383, p= 0.029*; WT_miR-499: T15 = 2.734, p= 0.015*; Cacna1c+/-_Control: T14 = 2.849, p= 0.013*; Cacna1c+/-_miR-499: T15 = 0.960, p= 0.352).

H) Elevated plus maze test. Time spent in open arms did not differ between genotypes and treatment groups. Data are represented as box plot with whiskers and data points (+: mean, line: median; whiskers: minimum and maximum values). (One-way ANOVA; WT_Control: n=19 (9 males, 10 females); WT_miR-499: n=16 (7 males, 9 females); Cacna1c+/-_Control: n=17 (7 males, 10 females); Cacna1c+/-_miR-499: n=16 (10 males, 6 females). Main effect Genotype: F1, 64= 0.044, p= 0.834; main effect Treatment: F1, 64= 1.171, p= 0.283; Interaction Genotype x Treatment: F1, 64= 0.076, p= 0.784).