Keeping time in the lamina terminalis: Novel oscillator properties of forebrain sensory circumventricular organs.

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.

Neuropeptide signaling differentially affects phase maintenance and rhythm generation in SCN and extra-SCN circadian oscillators.

Circadian rhythms in physiology and behavior are coordinated by the brain's dominant circadian pacemaker located in the suprachiasmatic nuclei (SCN) of the hypothalamus. Vasoactive intestinal polypeptide (VIP) and its receptor, VPAC(2), play important roles in the functioning of the SCN pacemaker. Mice lacking VPAC(2) receptors (Vipr2(-/-)) express disrupted behavioral and metabolic rhythms and show altered SCN neuronal activity and clock gene expression. Within the brain, the SCN is not the only site containing endogenous circadian oscillators, nor is it the only site of VPAC(2) receptor expression; both VPAC(2) receptors and rhythmic clock gene/protein expression have been noted in the arcuate (Arc) and dorsomedial (DMH) nuclei of the mediobasal hypothalamus, and in the pituitary gland. The functional role of VPAC(2) receptors in rhythm generation and maintenance in these tissues is, however, unknown. We used wild type (WT) and Vipr2(-/-) mice expressing a luciferase reporter (PER2::LUC) to investigate whether circadian rhythms in the clock gene protein PER2 in these extra-SCN tissues were compromised by the absence of the VPAC(2) receptor. Vipr2(-/-) SCN cultures expressed significantly lower amplitude PER2::LUC oscillations than WT SCN. Surprisingly, in Vipr2(-/-) Arc/ME/PT complex (Arc, median eminence and pars tuberalis), DMH and pituitary, the period, amplitude and rate of damping of rhythms were not significantly different to WT. Intriguingly, while we found WT SCN and Arc/ME/PT tissues to maintain a consistent circadian phase when cultured, the phase of corresponding Vipr2(-/-) cultures was reset by cull/culture procedure. These data demonstrate that while the main rhythm parameters of extra-SCN circadian oscillations are maintained in Vipr2(-/-) mice, the ability of these oscillators to resist phase shifts is compromised. These deficiencies may contribute towards the aberrant behavior and metabolism associated with Vipr2(-/-) animals. Further, our data indicate a link between circadian rhythm strength and the ability of tissues to resist circadian phase resetting.

A riot of rhythms: neuronal and glial circadian oscillators in the mediobasal hypothalamus.

BACKGROUND: 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. 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. RESULTS: Here 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. CONCLUSION: Our 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.