Live cell imaging of immediate-early gene expression, calcium and mitochondria in a mammalian biological clock.

摘要

This study investigated the role of mitochondrial Ca 2+ in the hypothalamic suprachiasmatic nucleus (SCN), the mammalian central pacemaker, by imaging immediate-early gene expression, cytosolic and mitochondrial Ca2+, and mitochondrial depolarizations in brain slices and dispersed cell cultures. To image the dynamics of gene expression in individual SCN cells, an imaging system was developed to measure firefly luciferase bioluminescence in live cells and tissues from transgenic mice containing the immediate-early gene 1 promoter/enhancer of the human cytomegalovirus upstream from the luciferase gene. High expression persisted in the cytosol of dispersed cells and in the dorsal SCN of brain slices. Expression increased in response to agents acting through the transgene's Ca2+/cAMP response element. In contrast, slices imaged after culture at room temperature displayed expression in ependymal cells of the third ventricle and capillaries, indicating possible regulation through stress responses.; Mitochondria in SCN brain slices containing were identified using the mitochondrial potential dye R123 and responded with increased fluorescence to an anoxic treatment. R123 was validated as effective for labeling mitochondria in SCN cells.; To investigate patterns of Ca2+ signaling, hypothalamic cell cultures from neonatal mice containing the SCN were imaged using fluorescent Ca+2-indicators. A variety of cell responses to glutamate and elevated K+ were observed repeatedly and were cell-type dependent. Astrocytes commonly displayed Ca+2 waves while neuron-like cells gave single responses.; SCN cell cultures are typically derived from neonatal cells, whereas in vitro brain slice preparations are primarily from adult animals. Therefore, a technique was developed to maintain young adult SCN neurons in culture to enable more direct comparisons with neurons in brain slices. Responses of neuron-like cells to glutamate and elevated K+ were imaged. Finally, to determine whether these cells show evidence of a circadian rhythm in the mitochondria, they were loaded with the mitochondrial Ca2+-sensitive dye rhod-2, and cell responses during the projected day and the projected night were evaluated. A significant difference between peak percent change in fluorescence due to glutamate was observed, with a higher peak during the day. No difference was detected between when responses were induced with high K+.