Abstract
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.
Highlights
[Background] To adapt to recurring time-of-day dependent changes in their environment, many organisms have developed an endogenous circadian clock system that regulates 24-h rhythms of behavioral and physiological processes (Sharma, 2003)
A master circadian pacemaker resides in the hypothalamic suprachiasmatic nucleus (SCN)
Appetite, and metabolism are regulated by cellular circadian clocks residing in hypothalamic neurons (Cedernaes et al, 2019)
Summary
To generate E15-16 embryos, schedule the mating day of the adult mice 15-16 d before dissection. We use 2-8-month-old adult C57BL/6J mice for mating. 2. On the morning, confirm successful mating by vaginal plug check as described previously (Behringer et al, 2016). 3. Confirm pregnancy by palpitation or visually before dissection
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