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Browsing by Author "Karsten, Carley"

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    Chronic stress shifts the phase of adrenal clock gene rhythms
    (2012-04-18) Karsten, Carley
    The hypothalamic-pituitary-adrenal (HPA) axis is characterized by a robust circadian rhythm in adrenal secretion of glucocorticoids (GC), driven in part by adrenal clock genes. The GC rhythm serves to synchronize other peripheral clocks and to maintain homeostasis. Mechanisms for entrainment of the adrenal clock remain unclear. Since stress activates the HPA axis, it is possible that entrainment of the adrenal clock and, concomitantly, glucocorticoid rhythms would be susceptible to stress. The present experiment aimed to test the hypothesis that chronic stress can alter adrenal circadian rhythms. The model of chronic subordinate stress used, consisting of daily exposure to a dominant mouse, has shown a robust metabolic phenotype in previous studies. Per2::Luc mice were used to assess rhythmic expression of the clock gene Per2. Per2::Luc mice express a luciferase reporter gene driven by the Per2 promoter, so that light output measured in tissue explants reflects PER2 expression.
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    A novel in vivo approach to study circadian rhythmicity of glucocorticoids using adeno-associated virus
    (2011-09-01) Karsten, Carley; Hupalo, Sofiya; Yoder, J. Marina; Engeland, William C.
    The hypothalamic-pituitary-adrenal (HPA) axis regulates glucocorticoid release in a circadian rhythm as well as in response to stress. The adrenal gland is made up of two distinct tissues: the cortex, which produces glucocorticoids, and the medulla. The cortex responds to hormonal cues while the medulla receives input from the splanchnic nerve. Interestingly, cutting the splanchnic nerve has been shown to affect cortical function, indicating a possible interaction between the two tissues. However, the precise mechanism of this interaction remains unknown. An experimental approach that genetically manipulates adrenal activity could potentially delineate the functional relationship between medulla and cortex. One option is to infect adrenal tissue with recombinant adeno-associated virus (AAV) capable of silencing a gene of interest in a specific tissue type. This is a novel approach in the adrenal gland, so we must first develop a protocol that will optimize methodological variables such as AAV serotype, surgical procedure, injection volume, and infection time-course. We tested various combinations of these variables and used GFP-tagged AAV to assess the degree of infection in rats. Thus far, we have determined that the serotype AAV8 yields the greatest degree of infection, and AAV2 and AAV5 seem to selectively infect the cortex. In addition, the concentration of virus injected is more important than the volume, and the injection method can have a large impact. Stress tests revealed that injection of AAV-GFP does not appear to alter adrenal function. AAVs thus appear to be promising tools in neuroendocrine studies, as they provide a means for genetically modifying the adrenal gland. Future experiments will use this new technique to analyze the functional relationship between adrenal cortex and medulla.