Schmidt Cosima X, Tsang Anthony H, Oster Henrik
Institute of Neurobiology, Center of Brain, Behavior and Metabolism, University of Lübeck, Lübeck, Germany.
Bio Protoc. 2021 Mar 5;11(5):e3944. doi: 10.21769/BioProtoc.3944.
An endogenous circadian clock system enables organisms to adapt to time-of-day dependent environmental changes. In consequence, most physiological processes exhibit daily rhythms of, ., energy metabolism, immune function, sleep, or hormone production. Hypothalamic circadian clocks have been identified to play a particular role in coordinating many of these processes. Primary neuronal cultures are widely used as a physiologically relevant model to study molecular events within neurons. However, as circadian rhythms include dynamic molecular changes over longer timescales that vary between individual cells, longitudinal measurement methods are essential to investigate the regulation of circadian clocks of hypothalamic neurons. Here we provide a protocol for generating primary hypothalamic neuronal cultures expressing a circadian luciferase reporter. Such reporter cells can be used to longitudinally monitor cellular circadian rhythms at high temporal resolution by performing bioluminescence measurements.
内源性昼夜节律时钟系统使生物体能够适应依赖于时间的环境变化。因此，大多数生理过程呈现出每日节律，例如能量代谢、免疫功能、睡眠或激素分泌。下丘脑昼夜节律时钟已被确定在协调其中许多过程中发挥特殊作用。原代神经元培养物被广泛用作研究神经元内分子事件的生理相关模型。然而，由于昼夜节律包括在较长时间尺度上的动态分子变化，且这些变化在单个细胞之间存在差异，因此纵向测量方法对于研究下丘脑神经元昼夜节律时钟的调节至关重要。在这里，我们提供了一种生成表达昼夜节律荧光素酶报告基因的原代下丘脑神经元培养物的方案。这种报告基因细胞可用于通过进行生物发光测量，以高时间分辨率纵向监测细胞昼夜节律。
