This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A goal of our present proposal is to establish an assay system by which circadian rhythms can be monitored using human fibroblasts with a circadian reporter and real time bioluminescence monitoring, and to test our hypothesis that clinical response to lithium can be predicted using human peripheral cells. b. Studies and Results. The goal of the first Aim is to apply in vitro circadian rhythm recording method using human primary fibroblasts. So far eleven healthy participants were recruited and six fibroblast cell lines have been established. After cultivation and expansion, the circadian clock Bmal1 promoter-luciferase gene was delivered to fibroblasts, and Bmal1-luciferase cell lines were established. We optimized culture and recording conditions. Robust and sustained circadian rhythms have now been recorded in these human fibroblast cell lines. Average period length was 24.60 ? 0.16 h (N=5). The results were in agreement with those previously published by Dr. Brown and his colleagues (Brown et al., 2005) and, therefore, we have completed the Aim 1.
In Aim 2 we plan to examine the effects of lithium on circadian rhythm parameters in fibroblasts obtained from lithium responders and non-responders. We have been optimizing lithium treatment conditions to be applied to bipolar disorder patients'cells. Since high lithium concentrations were used to test the effects of lithium on the period length in previous studies, we tested 3, 5, 7, and 10 mM lithium stimulation on the period length. At the same time, we want to test the effects of lithium at therapeutic concentrations, e.g., 1 mM. Since acute 1mM lithium stimulation did not show significant impact, we have been culturing cells in the presence of l mM lithium for one or four weeks before rhythm recording. We chose four weeks as well because the clinical lithium sensitivity is first determined four weeks after lithium therapy.
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