Abstract
IntroductionMost organisms display circadian rhythms in physiology and behaviour. In mammals, these rhythms are orchestrated by the suprachiasmatic nucleus (SCN). Recently, several metabolites have emerged as important regulators of circadian timekeeping. Metabolomics approaches have aided in identifying some key metabolites in circadian processes in peripheral tissue, but methods to routinely measure metabolites in small brain areas are currently lacking.ObjectiveThe aim of the study was to establish a reliable method for metabolite quantifications in the central circadian clock and relate them to different states of neuronal excitability.MethodsWe developed a method to collect and process small brain tissue samples (0.2 mm3), suitable for liquid chromatography–mass spectrometry. Metabolites were analysed in the SCN and one of its main hypothalamic targets, the paraventricular nucleus (PVN). Tissue samples were taken at peak (midday) and trough (midnight) of the endogenous rhythm in SCN electrical activity. Additionally, neuronal activity was altered pharmacologically.ResultsWe found a minor effect of day/night fluctuations in electrical activity or silencing activity during the day. In contrast, increasing electrical activity during the night significantly upregulated many metabolites in SCN and PVN.ConclusionOur method has shown to produce reliable and physiologically relevant metabolite data from small brain samples. Inducing electrical activity at night mimics the effect of a light pulses in the SCN, producing phase shifts of the circadian rhythm. The upregulation of metabolites could have a functional role in this process, since they are not solely products of physiological states, they are significant parts of cellular signalling pathways.
Highlights
Most organisms display circadian rhythms in physiology and behaviour
We show that exposure of suprachiasmatic nucleus (SCN) and paraventricular nucleus (PVN) tissue to high extracellular K + levels at night results in upregulation of many of the measured metabolites, and in particular of TCA cycle intermediates
In the SCN there is a strong circadian rhythm in neuronal activity, which peaks at midday and is lowest at midnight (Inouye and Kawamura 1979; Sato and Kawamura 1984)
Summary
Most organisms display circadian rhythms in physiology and behaviour. In mammals, these rhythms are orchestrated by the suprachiasmatic nucleus (SCN). In the absence of external cues like light or temperature cycles, the neuronal network of the SCN sustains an autonomous circadian rhythm in electrical activity and neurotransmitter release (Gillette and Reppert 1987; Nishino et al 1976). These signals from the SCN serve as a temporal reference for the rest of the body and orchestrate circadian rhythms throughout the brain and body, including sleep-wake cycles Disturbances in circadian rhythms have been found to be associated with many diseases, including metabolic syndrome (Rudic et al 2004; Turek et al 2005) and neurodegenerative diseases (Ju et al 2013; Kondratova and Kondratov 2012; Musiek et al 2015; Wulff et al 2010)
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