Erik D. Herzog

Vasoactive intestinal polypeptide mediates circadian rhythmicity and synchrony in mammalian clock neurons

The mammalian suprachiasmatic nucleus (SCN) is a master circadian pacemaker. It is not known which SCN neurons are autonomous pacemakers or how they synchronize their daily firing rhythms to coordinate circadian behavior. Vasoactive intestinal polypeptide (VIP) and the VIP receptor VPAC2 (encoded by the gene Vipr2) may mediate rhythms in individual SCN neurons, synchrony between neurons, or both. We found that Vip−/− and Vipr2−/− mice showed two daily bouts of activity in a skeleton photoperiod and multiple circadian periods in constant darkness. Loss of VIP or VPAC2 also abolished circadian firing rhythms in approximately half of all SCN neurons and disrupted synchrony between rhythmic neurons. Critically, daily application of a VPAC2 agonist restored rhythmicity and synchrony to VIP−/− SCN neurons, but not to Vipr2−/− neurons. We conclude that VIP coordinates daily rhythms in the SCN and behavior by synchronizing a small population of pacemaking neurons and maintaining rhythmicity in a larger subset of neurons.

A circadian clock in macrophages controls inflammatory immune responses.

Time of day-dependent variations of immune system parameters are ubiquitous phenomena in immunology. The circadian clock has been attributed with coordinating these variations on multiple levels; however, their molecular basis is little understood. Here, we systematically investigated the link between the circadian clock and rhythmic immune functions. We show that spleen, lymph nodes, and peritoneal macrophages of mice contain intrinsic circadian clockworks that operate autonomously even ex vivo. These clocks regulate circadian rhythms in inflammatory innate immune functions: Isolated spleen cells stimulated with bacterial endotoxin at different circadian times display circadian rhythms in TNF-α and IL-6 secretion. Interestingly, we found that these rhythms are not driven by systemic glucocorticoid variations nor are they due to the detected circadian fluctuation in the cellular constitution of the spleen. Rather, a local circadian clock operative in splenic macrophages likely governs these oscillations as indicated by endotoxin stimulation experiments in rhythmic primary cell cultures. On the molecular level, we show that >8% of the macrophage transcriptome oscillates in a circadian fashion, including many important regulators for pathogen recognition and cytokine secretion. As such, understanding the cross-talk between the circadian clock and the immune system provides insights into the timing mechanism of physiological and pathophysiological immune functions.

Clock controls circadian period in isolated suprachiasmatic nucleus neurons

The suprachiasmatic nucleus (SCN) is the master circadian pacemaker in mammals, and one molecular regulator of circadian rhythms is the Clock gene. Here we studied the discharge patterns of SCN neurons isolated from Clock mutant mice. Long-term, multielectrode recordings showed that heterozygous Clock mutant neurons have lengthened periods and that homozygous Clock neurons are arrhythmic, paralleling the effects on locomotor activity in the animal. In addition, cells in dispersals expressed a wider range of periods and phase relationships than cells in explants. These results suggest that the Clock gene is required for circadian rhythmicity in individual SCN cells and that a mechanism within the SCN synchronizes neurons and restricts the range of expressed circadian periods.

Sirt1 Extends Life Span and Delays Aging in Mice through the Regulation of Nk2 Homeobox 1 in the DMH and LH

The mammalian Sir2 ortholog Sirt1 plays an important role in metabolic regulation. However, the role of Sirt1 in the regulation of aging and longevity is still controversial. Here we demonstrate that brain-specific Sirt1-overexpressing (BRASTO) transgenic mice show significant life span extension in both males and females, and aged BRASTO mice exhibit phenotypes consistent with a delay in aging. These phenotypes are mediated by enhanced neural activity specifically in the dorsomedial and lateral hypothalamic nuclei (DMH and LH, respectively), through increased orexin type 2 receptor (Ox2r) expression. We identified Nk2 homeobox 1 (Nkx2-1) as a partner of Sirt1 that upregulates Ox2r transcription and colocalizes with Sirt1 in the DMH and LH. DMH/LH-specific knockdown of Sirt1, Nkx2-1, or Ox2r and DMH-specific Sirt1 overexpression further support the role of Sirt1/Nkx2-1/Ox2r-mediated signaling for longevity-associated phenotypes. Our findings indicate the importance of DMH/LH-predominant Sirt1 activity in the regulation of aging and longevity in mammals.

Clock genes, oscillators, and cellular networks in the suprachiasmatic nuclei.

The mammalian SCN contains a biological clock that drives remarkably precise circadian rhythms in vivo and in vitro. Recent advances have revealed molecular and cellular mechanisms required for the generation of these daily rhythms and their synchronization between SCN neurons and to the environmental light cycle. This review of the evidence for a cell-autonomous circadian pacemaker within specialized neurons of the SCN focuses on 6 genes implicated within the pace making mechanism, an additional 4 genes implicated in pathways from the pacemaker, and the intercellular and intracellular mechanisms that synchronize SCN neurons to each other and to solar time.

Come Together, Right… Now: Synchronization of Rhythms in a Mammalian Circadian Clock

In mammals, the suprachiasmatic nuclei (SCN) of the hypothalamus act as a dominant circadian pacemaker, coordinating rhythms throughout the body and regulating daily and seasonal changes in physiology and behavior. This review focuses on the mechanisms that mediate synchronization of circadian rhythms between SCN neurons. Understanding how these neurons communicate as a network of circadian oscillators has begun to shed light on the adaptability and dysfunction of the brains master clock.

Temporal Precision in the Mammalian Circadian System: A Reliable Clock from Less Reliable Neurons

The mammalian SCN contains a biological clock that drives remarkably precise circadian rhythms in vivo and in vitro. This study asks whether the cycle-to-cycle variability of behavioral rhythms in mice can be attributed to precision of individual circadian pacemakers within the SCN or their interactions. The authors measured the standard deviation of the cycle-to-cycle period from 7-day recordings of running wheel activity, Period1 gene expression in cultured SCN explants, and firing rate patterns of dispersed SCN neurons. Period variability of the intact tissue and animal was lower than single neurons. The median variability of running wheel and Period1 rhythms was less than 40 min per cycle compared to 2.1 h in firing rate rhythms of dispersed SCN neurons. The most precise SCN neuron, with a period deviation of 1.1 h, was 10 times noisier than the most accurate SCN explant (0.1 h) or mouse (0.1 h) but comparable to the least stable explant (2.1 h) and mouse (1.1 h). This variability correlated with intrinsic period in mice and SCN explants but not with single cells. Precision was unrelated to the amplitude of rhythms and did not change significantly with age up to 1 year after birth. Analysis of the serial correlation of cycle-to-cycle period revealed that approximately half of this variability is attributable to noise outside the pacemaker. These results indicate that cell-cell interactions within the SCN reduce pacemaker noise to determine the precision of circadian rhythms in the tissue and in behavior.

Neurons and networks in daily rhythms

Biological pacemakers dictate our daily schedules in physiology and behaviour. The molecules, cells and networks that underlie these circadian rhythms can now be monitored using long-term cellular imaging and electrophysiological tools, and initial studies have already suggested a theme — circadian clocks may be crucial for widespread changes in brain activity and plasticity. These daily changes can modify the amount or activity of available genes, transcripts, proteins, ions and other biologically active molecules, ultimately determining cellular properties such as excitability and connectivity. Recently discovered circadian molecules and cells provide preliminary insights into a network that adapts to predictable daily and seasonal changes while remaining robust in the face of other perturbations.

SIRT1 Promotes the Central Adaptive Response to Diet Restriction through Activation of the Dorsomedial and Lateral Nuclei of the Hypothalamus

Diet restriction retards aging and extends lifespan by triggering adaptive mechanisms that alter behavioral, physiological, and biochemical responses in mammals. Little is known about the molecular pathways evoking the corresponding central response. One factor that mediates the effects of diet restriction is the mammalian nicotinamide adenine dinucleotide (NAD)-dependent deacetylase SIRT1. Here we demonstrate that diet restriction significantly increases SIRT1 protein levels and induces neural activation in the dorsomedial and lateral hypothalamic nuclei. Increasing SIRT1 in the brain of transgenic (BRASTO) mice enhances neural activity specifically in these hypothalamic nuclei, maintains a higher range of body temperature, and promotes physical activity in response to different diet-restricting paradigms. These responses are all abrogated in Sirt1-deficient mice. SIRT1 upregulates expression of the orexin type 2 receptor specifically in these hypothalamic nuclei in response to diet-restricting conditions, augmenting response to ghrelin, a gut hormone whose levels increase in these conditions. Our results suggest that in the hypothalamus, SIRT1 functions as a key mediator of the central response to low nutritional availability, providing insight into the role of the hypothalamus in the regulation of metabolism and aging in mammals.

Intrinsic, nondeterministic circadian rhythm generation in identified mammalian neurons

Circadian rhythms are modeled as reliable and self-sustained oscillations generated by single cells. The mammalian suprachiasmatic nucleus (SCN) keeps near 24-h time in vivo and in vitro, but the identity of the individual cellular pacemakers is unknown. We tested the hypothesis that circadian cycling is intrinsic to a unique class of SCN neurons by measuring firing rate or Period2 gene expression in single neurons. We found that fully isolated SCN neurons can sustain circadian cycling for at least 1 week. Plating SCN neurons at <100 cells/mm2 eliminated synaptic inputs and revealed circadian neurons that contained arginine vasopressin (AVP) or vasoactive intestinal polypeptide (VIP) or neither. Surprisingly, arrhythmic neurons (nearly 80% of recorded neurons) also expressed these neuropeptides. Furthermore, neurons were observed to lose or gain circadian rhythmicity in these dispersed cell cultures, both spontaneously and in response to forskolin stimulation. In SCN explants treated with tetrodotoxin to block spike-dependent signaling, neurons gained or lost circadian cycling over many days. The rate of PERIOD2 protein accumulation on the previous cycle reliably predicted the spontaneous onset of arrhythmicity. We conclude that individual SCN neurons can generate circadian oscillations; however, there is no evidence for a specialized or anatomically localized class of cell-autonomous pacemakers. Instead, these results indicate that AVP, VIP, and other SCN neurons are intrinsic but unstable circadian oscillators that rely on network interactions to stabilize their otherwise noisy cycling.