Date Published: July 28, 2014
Publisher: Public Library of Science
Author(s): Valerie L. Harbour, Yuval Weigl, Barry Robinson, Shimon Amir, Paul A. Bartell.
We performed a high temporal resolution analysis of the transcript level of two core clock genes, Period2 (Per2) and Bmal1, and a clock output gene, Dbp, in the suprachiasmatic nucleus (SCN), the master circadian clock, and in two forebrain regions, the lateral part of the central nucleus of the amygdala (CEAl), and dentate gyrus (DG), in rats. These regions, as we have shown previously, exhibit opposite rhythms in expression of the core clock protein, PERIOD2 (PER2). We found that the expression of Per2, Bmal1 and Dbp follow a diurnal rhythm in all three regions but the phase and amplitude of the rhythms of each gene vary across regions, revealing important regional differences in temporal dynamics underlying local daily rhythm generation in the mammalian forebrain. These findings underscore the complex temporal organization of subordinate circadian oscillators in the forebrain and raise interesting questions about the functional connection of these oscillators with the master SCN clock.
Circadian rhythms help organisms adapt to their cyclic environment and are important to health in humans. These rhythms are driven by a master clock located in the suprachiasmatic nucleus (SCN) and by subordinate clocks distributed throughout the rest of the brain and body . At the cellular level, the circadian clock is based on transcriptional and posttranscriptional feedback loops driven by protein products of a small set of core clock genes , . Using immunohistochemistry, we have previously identified significant daily rhythms in expression of the circadian clock protein, PER2 in multiple forebrain structures including different subregions of the amygdala, hippocampus and cortex in rats . We found, surprisingly, that the PER2 rhythms in the forebrain fall into several different phase clusters all distinct from the phase of the PER2 rhythm of the SCN. Particularly intriguing was the finding that of the different forebrain regions studied, the lateral part of central nucleus of the amygdala (CEAl) and the oval nucleus of the bed nucleus of the stria terminalis (BNSTov), which together form the central extended amygdala, exhibited daily PER2 rhythms that, uniquely, were in antiphase with the rhythms in most other structures, and in close phase with the rhythm of the SCN –. While the functional significance of these divergent PER2 rhythms is yet to be determined, these results lend support to the idea that circadian rhythms in the forebrain are attended by region specific subordinate oscillators driven by differently phased oscillations of clock genes.
We analyzed the expression of the mRNA of Per2, Bmal1 and Dbp in the CEAl, DG and SCN in brain sections from 74 rats housed under a 12 h∶12 h LD cycle and perfused at 30-min intervals around the clock. Below we first describe the rhythms of expression of each of the three genes in each region to determine whether the antiphase oscillations in PER2 expression seen previously are mirrored by antiphase oscillations in expression of the clock genes. Next, we compare the rhythms of each gene, in each of the three regions, to highlight phase differences in the expression of any one clock gene across regions. Lastly, we contrast the data on the expression of Per2 in the SCN, CEAl and DG with data for PER2 reported previously in the same rats to highlight regional differences in the phase relationship between Per2 and PER2 expression.
Our analysis reveals region-specific differences in the temporal architecture of Per2 and Bmal1 mRNA expression in the SCN, CEAl and DG. Specifically, we found that the daily Per2 and Bmal1 mRNA rhythms in the CEAl are in phase with the Per2 and Bmal1 rhythms of the SCN, and in virtual antiphase with the daily rhythms in the DG. These findings show that the antiphase rhythms in PER2 expression seen in these regions are attended by similar antiphase rhythms in the expression of Per2 as well as Bmal1 whose protein, BMAL1, plays a key role in the regulation of Per2 transcription .