Metabolic and cardiovascular processes controlled by the hindbrain exhibit 24 h rhythms, but the extent to which the hindbrain possesses endogenous circadian timekeeping is unresolved. Here we provide compelling evidence that genetic, neuronal, and vascular activities of the brainstem's dorsal vagal complex are subject to intrinsic circadian control with a crucial role for the connection between its components in regulating their rhythmic properties. Robust 24 h variation in clock gene expression in vivo and neuronal firing ex vivo were observed in the area postrema (AP) and nucleus of the solitary tract (NTS), together with enhanced nocturnal responsiveness to metabolic cues. Unexpectedly, we also find functional and molecular evidence for increased penetration of blood borne molecules into the NTS at night. Our findings reveal that the hindbrain houses a local network complex of neuronal and nonneuronal autonomous circadian oscillators, with clear implications for understanding local temporal control of physiology in the brainstem.

BACKGROUND Heart rate follows a diurnal variation, and slow heart rhythms occur primarily at night.

Drinking behavior and osmotic regulatory mechanisms exhibit clear daily variation which is necessary for achieving the homeostatic osmolality. In mammals, the master clock in the brain's suprachiasmatic nuclei has long been held as the main driver of circadian (24 h) rhythms in physiology and behavior. However, rhythmic clock gene expression in other brain sites raises the possibility of local circadian control of neural activity and function. The subfornical organ (SFO) and the organum vasculosum laminae terminalis (OVLT) are two sensory circumventricular organs (sCVOs) that play key roles in the central control of thirst and water homeostasis, but the extent to which they are subject to intrinsic circadian control remains undefined.Using a combination of ex vivo bioluminescence and in vivo gene expression, we report for the first time that the SFO contains an unexpectedly robust autonomous clock with unusual spatiotemporal characteristics in core and noncore clock gene expression. Furthermore, putative single-cell oscillators in the SFO and OVLT are strongly rhythmic and require action potential-dependent communication to maintain synchrony. Our results reveal that these thirst-controlling sCVOs possess intrinsic circadian timekeeping properties and raise the possibility that these contribute to daily regulation of drinking behavior. K E Y W O R D Scircadian, circumventricular organ, fluid balance, OVLT, SFO, suprachiasmatic

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29In the human, there is a circadian rhythm in the resting heart rate and it is higher during the day in 30 preparation for physical activity. Conversely, slow heart rhythms (bradyarrhythmias) occur primarily 31 at night. Although the lower heart rate at night is widely assumed to be neural in origin (the result 32 of high vagal tone), the objective of the study was to test whether there is an intrinsic change in 33 heart rate driven by a local circadian clock. In the mouse, there was a circadian rhythm in the heart 34 rate in vivo in the conscious telemetrized animal, but there was also a circadian rhythm in the 35 intrinsic heart rate in denervated preparations: the Langendorff-perfused heart and isolated sinus 36 node. In the sinus node, experiments (qPCR and bioluminescence recordings in mice with a Per1 37 luciferase reporter) revealed functioning canonical clock genes, e.g. Bmal1 and Per1. We identified 38 a circadian rhythm in the expression of key ion channels, notably the pacemaker channel Hcn4 39 (mRNA and protein) and the corresponding ionic current (funny current, measured by whole cell 40 patch clamp in isolated sinus node cells). Block of funny current in the isolated sinus node 41 abolished the circadian rhythm in the intrinsic heart rate. Incapacitating the local clock (by cardiac-42 specific knockout of Bmal1) abolished the normal circadian rhythm of Hcn4, funny current and the 43 intrinsic heart rate. Chromatin immunoprecipitation demonstrated that Hcn4 is a transcriptional 44 target of BMAL1 establishing a pathway by which the local clock can regulate heart rate. In 45 conclusion, there is a circadian rhythm in the intrinsic heart rate as a result of a local circadian 46 clock in the sinus node that drives rhythmic expression of Hcn4. The data reveal a novel regulator 47 of heart rate and mechanistic insight into the occurrence of bradyarrhythmias at night. 483 Living things including humans are attuned to the 24 h day-night cycle and many biological 49 processes exhibit an intrinsic ~24 h (i.e. circadian) rhythm. In the human, the resting heart rate (in 50 the absence of physical activity) shows a circadian rhythm and is higher during the day when we 51 are awake 1,2 . The heart is therefore primed, anticipating the increase in demand during the awake 52 period. Conversely, bradyarrhythmias primarily occur at night 1,3 . Previously, the circadian rhythm in 53 heart rate in vivo has been attributed to the autonomic nervous system: to high sympathetic nerve 54 activity accompanying physical activity during the awake period and high vagal tone during the 55 sleep period 4 . This is partly based on heart rate variability as a surrogate measure of autonomic 56 tone 5-7 ; however, we have shown that heart rate variability cannot be used in any simple way as a 57 measure of autonomic tone 8 . Therefore, the mechanism underlying the circadian rhythm in heart 58 rate is still unknown. We asked the question whether there is a circadian rhythm in the intrinsic 59 heart rate set by the pacemaker of the he...