Abstract
Clock genes are expressed throughout the body, although how they oscillate in unrestrained animals is not known. Here, we show an in vivo imaging technique that enables long-term simultaneous imaging of multiple tissues. We use dual-focal 3D tracking and signal-intensity calibration to follow gene expression in a target area. We measure circadian rhythms of clock genes in the olfactory bulb, right and left ears and cortices, and the skin. In addition, the kinetic relationship between gene expression and physiological responses to experimental cues is monitored. Under stable conditions gene expression is in phase in all tissues. In response to a long-duration light pulse, the olfactory bulb shifts faster than other tissues. In Cry1−/− Cry2−/− arrhythmic mice circadian oscillation is absent in all tissues. Thus, our system successfully tracks circadian rhythms in clock genes in multiple tissues in unrestrained mice.
Highlights
Clock genes are expressed throughout the body, how they oscillate in unrestrained animals is not known
Clock gene expression is not limited to the suprachiasmatic nucleus (SCN), with expression observed in a variety of tissues[1,2,3,4,5]
The results indicate that the olfactory bulb (OB) shifts faster than other tissues in response to a long-duration light pulse
Summary
Clock genes are expressed throughout the body, how they oscillate in unrestrained animals is not known. Our system successfully tracks circadian rhythms in clock genes in multiple tissues in unrestrained mice. Monitoring of clock gene expression in vivo has been attempted using a bioluminescent reporter, either with an optical fibre in the SCN17, or using a photon detector located outside the body. In the latter case, bioluminescence emitted from tissuespecific reporters was collected efficiently using a conical wall that channels photons toward a photomultiplier tube (PMT), and the emitting tissue was identified using a tissue-specific reporter[16]. We show a method to quantitatively monitor clock gene expression simultaneously in multiple regions of freely moving mice over long durations. To overcome the technical challenge of intensity changes with time-of-day and distance from the recording apparatus, we developed a dual-focal 3D tracing (DuFT) technology and a signal-intensity calibration technique (SICT) and combined these two systems into a software application for analysing gene expression, which we call ‘Mouse Tracker’
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