Circadian Rhythms in the Amygdala

Analysis of circadian rhythm genes in the amygdala of male and female SHR rats by RT-qPCR show a great deal of dysregulation when compared to their WKY controls. The pattern of dysregulation was markedly different than those seen in the SCN and hippocampus.

Females especially showed a large amount of dysregulation with five of eight circadian genes analyzed showing differences. Female SHR rats showed an upregulation in Bmal1 (26-fold;

p < 0.01), Npas2 (21-fold; p < 0.001), Clock (15-fold; p < 0.01), Per1 (16.5-fold; p < 0.01) and Per2 (11-fold; p < 0.001) (Figure 12A). Females had an overall trend of upregulation in the circadian panel tested. Males also showed dysregulation compared to their WKY counterparts, however a different pattern of dysregulation compared to the females was observed. They showed significant changes in two of the eight genes with Per1 being significantly upregulated (6.6-fold; p < 0.01) and Cry2 being significantly downregulated at a fold change of 507 (p < 0.001) (Figure 12B).

Figure 10. mRNA levels of circadian rhythm genes in the suprachiasmatic nucleus of 19 week old SHR rats relative to WKY control. Fold changes in mRNA levels of Bmal1, Npas2, Clock, Per1, Per2, Per3, Cry1 and Cry2 in female (A) and male (B) rats. Fold changes in gene expression were calculated by relative quantification (Ct) of RT-qPCR threshold cycles (Ct) as per Livak and Schmittgen⁸⁷ using mean Ct values of housekeeping genes GAPDH, Ywhaz and CycA. Data is expressed as mean fold change of SHR relative to the WKY group  SEM (n = 8). Statistical significance between WKY and SHR groups is shown

Bmal1

Figure 11. mRNA levels of circadian rhythm genes in the hippocampus of 19 week old SHR rats relative to WKY control. Fold changes in mRNA levels of Bmal1, Npas2, Clock, Per1, Per2, Per3, Cry1 and Cry2 in female (A) and male (B) rats. Fold changes in gene expression were calculated by relative quantification (Ct) of RT-qPCR threshold cycles (Ct) as per Livak and Schmittgen⁸⁷ using mean Ct values of housekeeping genes GAPDH, Ywhaz and CycA. Data is expressed as mean fold change of SHR relative to the WKY group

 SEM (n = 8). Statistical significance between WKY and SHR groups is shown by: * p <

Bmal1

Figure 12. mRNA levels of circadian rhythm genes in the amygdala of 19 week old SHR rats relative to WKY control. Fold changes in mRNA levels of Bmal1, Npas2, Clock, Per1, Per2, Per3, Cry1 and Cry2 in female (A) and male (B) rats. Fold changes in gene expression were calculated by relative quantification (Ct) of RT-qPCR threshold cycles (Ct) as per Livak and Schmittgen⁸⁷ using mean Ct values of housekeeping genes GAPDH, Ywhaz and CycA. Data is expressed as mean fold change of SHR relative to the WKY group  SEM (n

= 8). Statistical significance between WKY and SHR groups is shown by: * p < 0.05, ** p <

0.01, *** p < 0.001.

3.2.4 Circadian Rhythms in the PVN

Results from RT-qPCR analysis of the paraventricular nucleus of SHR brains showed significant changes in the circadian rhythm gene panel relative to their WKY controls. These changes are different from the dysregulation patterns seen in the three other brain areas above, however, the pattern in the female PVN is similar to the pattern seen in the female amygdala with regard to trends. Female offspring showed a great deal of statistically significant changes in the circadian genes analyzed with five out of eight being significantly upregulated. Females showed upregulations in Bmal1 (5-fold; p < 0.05), Clock (98-fold; p < 0.0001), Per1 (7-fold;

p < 0.01), Per2 (3.5-fold; p < 0.05) and Cry1 (5.5-fold; p < 0.05) (Figure 13A). It should be noted that expression levels of Per3 were so extremely low that its relative expression was unable to be detected in the PVN of female SHR brains. Male offspring showed statistically significant upregulations in six of eight circadian genes examined, however, these changes were different from those seen in the females. The males saw significant upregulations of Bmal1 (29-fold; p < 0.001), Npas2 (8-fold; p < 0.05), Clock (11.7-fold; p < 0.05), Per2 (15.4-fold; p < 0.001), Cry1 (9.2-(15.4-fold; p < 0.05) and Cry2 (15.2-(15.4-fold; p < 0.01) (Figure 13B).

Figure 13. mRNA levels of circadian rhythm genes in the paraventricular nucleus of 19 week old SHR rats relative to WKY control. Fold changes in mRNA levels of Bmal1, Npas2, Clock, Per1, Per2, Per3, Cry1 and Cry2 in female (A) and male (B) rats. Fold changes in gene expression were calculated by relative quantification (Ct) of RT-qPCR threshold cycles (Ct) as per Livak and Schmittgen⁸⁷ using mean Ct values of housekeeping genes GAPDH, Ywhaz and CycA. Data is expressed as mean fold change of SHR relative to the WKY group  SEM (n = 8). Statistical significance between WKY and SHR groups is shown

Bmal1

3.2.5 Circadian Rhythms in the PFC

The results from the RT-qPCR analysis of circadian rhythm genes found in the prefrontal cortex of male and female SHR animals show a dysregulated pattern when compared to their WKY controls. These dysregulation patterns were quite different from those seen in all other brain areas. Female offspring showed five statistically significant changes across the selected circadian genes. The results show upregulations in Npas2 (3.3-fold; p <

0.0001), Per1 (2.2-fold; p < 0.001), Per3 (2.4-fold; p < 0.001), Cry1 (1.5-fold; p < 0.05) and Cry2 (2.7-fold; p < 0.0001) (Figure 14A). Male offspring also showed five statistically significant changes in the circadian gene panel with similar genes that were dysregulated in females. Upregulations in Npas2 (4.1-fold; p < 0.0001), Per1 (2.3-fold; p < 0.0001), Per3 (2.4-fold; p < 0.0001), Cry2 (2.3-fold; p < 0.0001) and a downregulation in Per2 (1.3-fold; p

< 0.01) (Figure 14B) were observed in males. In this brain area the male and female dysregulation patterns were quite similar with most genes showing a similar trend in fold change.

Figure 14. mRNA levels of circadian rhythm genes in the prefrontal cortex of 19 week old SHR rats relative to WKY control. Fold changes in mRNA levels of Bmal1, Npas2, Clock, Per1, Per2, Per3, Cry1 and Cry2 in female (A) and male (B) rats. Fold changes in gene expression were calculated by relative quantification (Ct) of RT-qPCR threshold cycles (Ct) as per Livak and Schmittgen⁸⁷ using mean Ct values of housekeeping genes GAPDH, Ywhaz and CycA. Data is expressed as mean fold change of SHR relative to the WKY group

 SEM (n = 8). Statistical significance between WKY and SHR groups is shown by: * p <

0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.