Generation of Mouse Primary Hypothalamic Neuronal Cultures for Circadian Bioluminescence Assays.

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, e.g., 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.