Abstract
BackgroundIn mammals, the synchronized activity of cell autonomous clocks in the suprachiasmatic nuclei (SCN) enables this structure to function as the master circadian clock, coordinating daily rhythms in physiology and behavior. However, the dominance of this clock has been challenged by the observations that metabolic duress can over-ride SCN controlled rhythms, and that clock genes are expressed in many brain areas, including those implicated in the regulation of appetite and feeding. The recent development of mice in which clock gene/protein activity is reported by bioluminescent constructs (luciferase or luc) now enables us to track molecular oscillations in numerous tissues ex vivo. Consequently we determined both clock activities and responsiveness to metabolic perturbations of cells and tissues within the mediobasal hypothalamus (MBH), a site pivotal for optimal internal homeostatic regulation.ResultsHere we demonstrate endogenous circadian rhythms of PER2::LUC expression in discrete subdivisions of the arcuate (Arc) and dorsomedial nuclei (DMH). Rhythms resolved to single cells did not maintain long-term synchrony with one-another, leading to a damping of oscillations at both cell and tissue levels. Complementary electrophysiology recordings revealed rhythms in neuronal activity in the Arc and DMH. Further, PER2::LUC rhythms were detected in the ependymal layer of the third ventricle and in the median eminence/pars tuberalis (ME/PT). A high-fat diet had no effect on the molecular oscillations in the MBH, whereas food deprivation resulted in an altered phase in the ME/PT.ConclusionOur results provide the first single cell resolution of endogenous circadian rhythms in clock gene expression in any intact tissue outside the SCN, reveal the cellular basis for tissue level damping in extra-SCN oscillators and demonstrate that an oscillator in the ME/PT is responsive to changes in metabolism.
Highlights
In mammals, the synchronized activity of cell autonomous clocks in the suprachiasmatic nuclei (SCN) enables this structure to function as the master circadian clock, coordinating daily rhythms in physiology and behavior
To examine the endogenous circadian rhythmicity of multiple hypothalamic nuclei and their intra-tissue organization, we assessed clock gene expression in acute slice cultures of mediobasal hypothalamus (MBH) and SCN prepared from adult Paradigms Adult male mPer2Luc knockin mice (PER2)::LUC mice and neuronal activity in vitro using longitudinal electrophysiological recordings
The MBH expresses circadian rhythms in PER2::LUC bioluminescence and neural activity Whole MBH slice cultures were imaged in real time with an EM-CCD camera-equipped microscopy system
Summary
The synchronized activity of cell autonomous clocks in the suprachiasmatic nuclei (SCN) enables this structure to function as the master circadian clock, coordinating daily rhythms in physiology and behavior. The recent development of mice in which clock gene/protein activity is reported by bioluminescent constructs (luciferase or luc) enables us to track molecular oscillations in numerous tissues ex vivo We determined both clock activities and responsiveness to metabolic perturbations of cells and tissues within the mediobasal hypothalamus (MBH), a site pivotal for optimal internal homeostatic regulation. The coordinated daily regulation of cycles in rest and activity, food intake and metabolism are crucial for the optimal health of an individual [1,2,3,4] In mammals this daily, or circadian, timekeeping is customarily attributed to the intrinsic activities of autonomous cellular clocks within the suprachiasmatic nuclei (SCN) of the hypothalamus [5,6]. Many peripheral cells and tissues rhythmically express clock genes, the phases of which are coordinated throughout the organism by the SCN, which is itself entrained by environmental timing cues [12,13,14].
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