Undergraduate Honors Theses

Thesis Defended

Fall 2015

Document Type

Thesis

Type of Thesis

Departmental Honors

Department

Integrative Physiology

First Advisor

Kenneth P. Wright, Jr.

Second Advisor

Robert L. Spencer

Third Advisor

David Sherwood

Abstract

Circadian rhythms are maintained through the self-regulatory, oscillatory molecular clock, which includes the core clock genes Per1, Per2, and Bmal1. Disruptions to clock gene expression have been associated with numerous mood and behavior disorders including major depression, anxiety disorders, and bipolar disorders (1-2). The molecular clock has been well-characterized in the suprachiasmatic nucleus (SCN), the master clock of the body (3-7). Many peripheral tissues and extra-SCN brain regions have also been shown to have circadian rhythms in these core clock genes, but considering that the SCN has few direct projections to these brain and body regions, the question remains how the SCN communicates to extra-SCN molecular clocks (4-7). Glucocorticoids (CORT) are a potential candidate by which the SCN signals to other brain and body regions as glucocorticoid receptors (GR) are found ubiquitously throughout the brain and body, with the notable exception of the SCN. Furthermore, CORT is released in a diurnal manner, with peak plasma levels occurring immediately upon the animal’s active phase. Interestingly, there is a glucocorticoid response element (GRE) in the promoter regions of the Per1 and Per2 genes, which may be a mechanism by which CORT can induce Per1 and Per2 expression, and thereby entrain the molecular clock (8). We compared clock gene expression in mice that had a conditional forebrain glucocorticoid receptor knockout (FBGRKO) to GR floxed mice to determine the necessity of GRs in diurnal core clock gene expression. FBGRKO (C57BL/6 pure strain of the T29-1 founder line containing Cre+ recombinase transgene) mice have been previously well-characterized to have disruptions in GR expression in the forebrain including the hippocampus, cortex, and nucleus accumbens, while the central nucleus of the amygdala (CEA) had a 50% deletion and the paraventricular nucleus (PVN) was not affected (9-11). Mice were sacrificed under basal conditions in the light phase (zeitgeber time (ZT) 1.5) or dark phase (ZT13). In situ hybridization was used to measure mRNA. Our results show there is a time of day difference for Per1, Per2, and Bmal1 clock genes in the SCN and for Per1 and Bmal1 in the PVN. The subregions of the prefrontal cortex (anterior cingulate, prelimbic, infralimbic, ventral orbital) and insula show a time of day difference only for Bmal1. Only Bmal1 showed a time of day difference in the subregions of the hippocampus (CA3, supra dentate gyrus, infra dentate gyrus) and amygdala (central, basolateral, medial), while Per1 was significant only in medial amygdala, and Per2 only in CA3. CA1 of the hippocampus did not show time of day differences for any clock gene investigated. There were no genotype differences for all brain regions examined. These results are expected in the SCN and PVN, as hypothalamic GRs would not be affected by the FBGRKO. The lack of evident FBGRKO effect in the HPC, AMY, PFC, and insula may be due to the fact that the sacrifice times (1.5 and 13 hours after lights on) are not at the diurnal peaks and troughs of these clock genes and therefore there may not be enough temporal resolution to see slight shifts in the diurnal rhythmic expression. It is also possible that GRs are not necessary for diurnal clock gene expression in these tissues or that CORT modulates the diurnal rhythm of Per1 and Bmal1 in the forebrain regions through a series of non-GR mediated neuronal projections.

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