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|Title:||CIRCADIAN LOCOMOTOR ACTIVITY AND PER2 RHYTHMS IN THE LIMBIC FOREBRAIN ARE DISRUPTED BY DOUBLE-STRANDED RNA TO PER2 INFUSIONS INTO THE AREA OF THE SCN|
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|Authors/Affiliations:||1 Alex Gavrila*; 1 Barry Robinson; 2 Julia Hoy; 2 Aditi Bhargava; 1 Shimon Amir; |
1 Concordia University, Montreal, QC, Canada; 2 University of California San Francisco, CA, USA
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|Content:||Objectives: Studies in mutant mice with targeted disruption in the Period2 (Per2) gene suggest that the PER2 protein participates in the regulation of circadian behavioral rhythms. Moreover, it has been shown that direct suppression of PER2 expression in the suprachiasmatic nucleus (SCN, the master circadian clock) with antisense oligonucleotides disrupts photic resetting of the SCN clock. However, despite significant progress in the field in recent years, the effect of such suppression on behavioral rhythms is still unknown. The objective of this work was to investigate the effect of PER2 suppression in the SCN on the disruption of locomotor activity and PER2 rhythms in the limbic forebrain.|
Materials and Methods: Bilateral infusions of double-stranded RNA (dsRNA) to Per2 were performed in the area of the SCN (6 µg/side) in order to transiently suppress PER2 expression. Control rats were administered bilateral β-globin dsRNA infusions in the area of the SCN. Circadian locomotor activity was monitored throughout the course of all experiments. Experiment 1 investigated the effect of PER2 suppression in the SCN on locomotor activity in rats housed in constant darkness. In experiment 2, rats were perfused at Zeitgeber time 13 (ZT13, one hour after lights off), three, six and 12 days post-infusion and PER2-immunoreactivity was assessed. Finally, experiment 3 probed the specificity of the dsRNA to Per2 used by staining alternate coronal brain slices for PER2- and cFos-immunoreactivity in rats perfused at ZT1 and ZT13, six days post-surgery.
Results: Bilateral infusions of dsRNA into the SCN disrupted circadian wheel running activity rhythms for up to 8 days in experimental rats housed in constant darkness; whereas, control infusions into the SCN or dorsal infusions of dsRNA to Per2 had no effect. Relative to controls, PER2 suppression in the SCN was evident 12 days post-dsRNA infusion (26% suppression); however, maximal suppression was observed three days post-surgery (60% suppression). In addition, a blunted PER2 rhythm was observed in the oval nucleus of the bed nucleus of the stria terminalis (BNSTov), central nucleus of the amygdala (CEA) and dentate gyrus (DG). The specificity of this effect was validated by demonstrating no difference between cFos expression in any of the above areas in control rats and rats treated with dsRNA to Per2.
Conclusion: These results provide direct evidence that the expression of PER2 in the SCN is essential for the maintenance of circadian locomotor activity and PER2 rhythms in the limbic forebrain of rats.
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