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Independent Circadian Oscillations of Period1 in Specific Brain Areas In Vivo and In Vitro

Performed in collaboration with Erik Herzog's Lab.
Behavioral and physiological circadian rhythms in mammals are controlled by a master pacemaker in the hypothalamic suprachiasmatic nuclei (SCN). Recently, circadian oscillations of hormone secretion, clock gene expression, and electrical activity have been demonstrated in explants of other brain regions. This suggests that some extra-SCN brain regions contain a functional, SCN-independent circadian clock, but in vivo evidence for intrinsic pacemaking is still lacking. We developed a novel method to image bioluminescence in vivo from the main olfactory bulbs (OB) of intact and SCN-lesioned (SCNX) Period1::luciferase rats for 2 d in constant darkness. The OBs expressed circadian rhythms in situ with a reliable twofold increase from day to night, similar to the phase and amplitude of ex vivo rhythms. In vivo cycling persisted for at least 1 month in the absence of the SCN. To assess indirectly in vivo rhythmicity of other brain areas, we measured the phase-dependence of their in vitro rhythms on the time of surgery. Surgery reliably reset the phase of the pineal gland and vascular organ of the lamina terminalis (VOLT) harvested from SCNX rats but had little effect on the phase of the OB. We deduce that the SCN and OB contain self-sustained circadian oscillators, whereas the pineal gland and VOLT are weak oscillators that require input from the SCN to show coordinated circadian rhythms. We conclude that the mammalian brain comprises a diverse set of SCN-dependent and SCN-independent circadian oscillators.
Figure 1
Representative Per1::luc bioluminescence images from the OB transgenic rats. Light from the bilateral OB was imaged for 1 min at 4 h intervals through a glass window in the skull in an intact rat, 4 h after luciferin was delivered to the OB. Light emission peaked daily around CT 13-17 in the rat. The intact rat was enucleated 2 d before the creation of the OB window, so all times are reported relative to the light/dark cycle to which the animal was entrained previously. CT 0 denotes the daily onset of the previous light phase (subjective dawn), and CT 12 denotes subjective dusk. Pseudocolored images were superimposed over a photograph of the anesthetized rats taken immediately before the bioluminescence integration.
Figure 2
Average normalized photon flux emitted from the OB of intact rats was circadian over 2 d in constant darkness. Bioluminescence (mean ± SEM) of intact rats previously entrained to a LD cycle (A) or a DL cycle (B) peaked daily at about CT 15 in both groups. The peak-to-trough ratio for both groups averaged ~2:1. Gray and black bars denote the previous light and dark times, respectively. Values were normalized to the peak emission for each rat over the 48 h of imaging. Data from all OB (rhythmic and arrhythmic) were included in each condition.
Reference:	Abraham U, Prior JL, Granados-Fuentes D, Piwnica-Worms DR, and Herzog ED. Independent circadian oscillations of Period1 in specific brain areas in vivo and in vitro. J Neurosci 2005; 25(31): 8620-26.	PubMed Link

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