Circadian Biology and the Neurovascular Unit.

Mammalian physiology and cellular function are subject to significant oscillations over the course of every 24-hour day. It is likely that these daily rhythms will affect function as well as mechanisms of disease in the central nervous system. In this review, we attempt to survey and synthesize emerging studies that investigate how circadian biology may influence the neurovascular unit. We examine how circadian clocks may operate in neural, glial, and vascular compartments, review how circadian mechanisms regulate cell-cell signaling, assess interactions with aging and vascular comorbidities, and finally ask whether and how circadian effects and disruptions in rhythms may influence the risk and progression of pathophysiology in cerebrovascular disease. Overcoming identified challenges and leveraging opportunities for future research might support the development of novel circadian-based treatments for stroke.

Dim light in the evening causes coordinated realignment of circadian rhythms, sleep, and short-term memory.

Light provides the primary signal for entraining circadian rhythms to the day/night cycle. In addition to rods and cones, the retina contains a small population of photosensitive retinal ganglion cells (pRGCs) expressing the photopigment melanopsin (OPN4). Concerns have been raised that exposure to dim artificial lighting in the evening (DLE) may perturb circadian rhythms and sleep patterns, and OPN4 is presumed to mediate these effects. Here, we examine the effects of 4-h, 20-lux DLE on circadian physiology and behavior in mice and the role of OPN4 in these responses. We show that 2 wk of DLE induces a phase delay of ∼2 to 3 h in mice, comparable to that reported in humans. DLE-induced phase shifts are unaffected in Opn4-/- mice, indicating that rods and cones are capable of driving these responses in the absence of melanopsin. DLE delays molecular clock rhythms in the heart, liver, adrenal gland, and dorsal hippocampus. It also reverses short-term recognition memory performance, which is associated with changes in preceding sleep history. In addition, DLE modifies patterns of hypothalamic and cortical cFos signals, a molecular correlate of recent neuronal activity. Together, our data show that DLE causes coordinated realignment of circadian rhythms, sleep patterns, and short-term memory process in mice. These effects are particularly relevant as DLE conditions-due to artificial light exposure-are experienced by the majority of the populace on a daily basis.

Recurring circadian disruption alters circadian clock sensitivity to resetting.

A single phase advance of the light:dark (LD) cycle can temporarily disrupt synchrony of neural circadian rhythms within the suprachiasmatic nucleus (SCN) and between the SCN and peripheral tissues. Compounding this, modern life can involve repeated disruptive light conditions. To model chronic disruption to the circadian system, we exposed male mice to more than a month of a 20-hr light cycle (LD10:10), which mice typically cannot entrain to. Control animals were housed under LD12:12. We measured locomotor activity and body temperature rhythms in vivo, and rhythms of PER2::LUC bioluminescence in SCN and peripheral tissues ex vivo. Unexpectedly, we discovered strong effects of the time of dissection on circadian phase of PER2::LUC bioluminescent rhythms, which varied across tissues. White adipose tissue was strongly reset by dissection, while thymus phase appeared independent of dissection timing. Prior light exposure impacted the SCN, resulting in strong resetting of SCN phase by dissection for mice housed under LD10:10, and weak phase shifts by time of dissection in SCN from control LD12:12 mice. These findings suggest that exposure to circadian disruption may desynchronize SCN neurons, increasing network sensitivity to perturbations. We propose that tissues with a weakened circadian network, such as the SCN under disruptive light conditions, or with little to no coupling, for example, some peripheral tissues, will show increased resetting effects. In particular, exposure to light at inconsistent circadian times on a recurring weekly basis disrupts circadian rhythms and alters sensitivity of the SCN neural pacemaker to dissection time.

Deconstructing circadian disruption: Assessing the contribution of reduced peripheral oscillator amplitude on obesity and glucose intolerance in mice.

Disturbing the circadian regulation of physiology by disruption of the rhythmic environment is associated with adverse health outcomes but the underlying mechanisms are unknown. Here, the response of central and peripheral circadian clocks to an advance or delay of the light-dark cycle was determined in mice. This identified transient damping of peripheral clocks as a consequence of an advanced light-dark cycle. Similar depression of peripheral rhythm amplitude was observed in mice exposed to repeated phase shifts. To assess the metabolic consequences of such peripheral amplitude depression in isolation, temporally chimeric mice lacking a functional central clock (Vgat-Cre+ Bmal1fl/fl ) were housed in the absence of environmental rhythmicity. In vivo PER2::LUC bioluminescence imaging of anesthetized and freely moving mice revealed that this resulted in a state of peripheral amplitude depression, similar in severity to that observed transiently following an advance of the light-dark cycle. Surprisingly, our mice did not show alterations in body mass or glucose tolerance in males or females on regular or high-fat diets. Overall, our results identify transient damping of peripheral rhythm amplitude as a consequence of exposure to an advanced light-dark cycle but chronic damping of peripheral clocks in isolation is insufficient to induce adverse metabolic outcomes in mice.

Evidence for Internal Desynchrony Caused by Circadian Clock Resetting.

Circadian disruption has been linked to markers for poor health outcomes in humans and animal models. What is it about circadian disruption that is problematic? One hypothesis is that phase resetting of the circadian system, which occurs in response to changes in environmental timing cues, leads to internal desynchrony within the organism. Internal desynchrony is understood as acute changes in phase relationships between biological rhythms from different cell groups, tissues, or organs within the body. Do we have strong evidence for internal desynchrony associated with or caused by circadian clock resetting? Here we review the literature, highlighting several key studies from measures of gene expression in laboratory rodents. We conclude that current evidence offers strong support for the premise that some protocols for light-induced resetting are associated with internal desynchrony. It is important to continue research to test whether internal desynchrony is necessary and/or sufficient for negative health impact of circadian disruption.

Methods for Detecting PER2:LUCIFERASE Bioluminescence Rhythms in Freely Moving Mice.

Circadian rhythms are driven by daily oscillations of gene expression. An important tool for studying cellular and tissue circadian rhythms is the use of a gene reporter, such as bioluminescence from the reporter gene luciferase controlled by a rhythmically expressed gene of interest. Here we describe methods that allow measurement of circadian bioluminescence from a freely moving mouse housed in a standard cage. Using a LumiCycle In Vivo (Actimetrics), we determined conditions that allow detection of circadian rhythms of bioluminescence from the PER2 reporter, PER2::LUC, in freely behaving mice. The LumiCycle In Vivo applies a background subtraction that corrects for effects of room temperature on photomultiplier tube (PMT) output. We tested delivery of d-luciferin via a subcutaneous minipump and in the drinking water. We demonstrate spikes in bioluminescence associated with drinking bouts. Further, we demonstrate that a synthetic luciferase substrate, CycLuc1, can support circadian rhythms of bioluminescence, even when delivered at a lower concentration than d-luciferin, and can support longer-term studies. A small difference in phase of the PER2::LUC bioluminescence rhythms, with females phase leading males, can be detected with this technique. We share our analysis scripts and suggestions for further improvements in this method. This approach will be straightforward to apply to mice with tissue-specific reporters, allowing insights into responses of specific peripheral clocks to perturbations such as environmental or pharmacological manipulations.

Cell-Type-Specific Circadian Bioluminescence Rhythms in Dbp Reporter Mice.

Circadian rhythms are endogenously generated physiological and molecular rhythms with a cycle length of about 24 h. Bioluminescent reporters have been exceptionally useful for studying circadian rhythms in numerous species. Here, we report development of a reporter mouse generated by modification of a widely expressed and highly rhythmic gene encoding D-site albumin promoter binding protein (Dbp). In this line of mice, firefly luciferase is expressed from the Dbp locus in a Cre recombinase-dependent manner, allowing assessment of bioluminescence rhythms in specific cellular populations. A mouse line in which luciferase expression was Cre-independent was also generated. The Dbp reporter alleles do not alter Dbp gene expression rhythms in liver or circadian locomotor activity rhythms. In vivo and ex vivo studies show the utility of the reporter alleles for monitoring rhythmicity. Our studies reveal cell-type-specific characteristics of rhythms among neuronal populations within the suprachiasmatic nuclei ex vivo. In vivo studies show Dbp-driven bioluminescence rhythms in the liver of Albumin-Cre;DbpKI/+ "liver reporter" mice. After a shift of the lighting schedule, locomotor activity achieved the proper phase relationship with the new lighting cycle more rapidly than hepatic bioluminescence did. As previously shown, restricting food access to the daytime altered the phase of hepatic rhythmicity. Our model allowed assessment of the rate of recovery from misalignment once animals were provided with food ad libitum. These studies confirm the previously demonstrated circadian misalignment following environmental perturbations and reveal the utility of this model for minimally invasive, longitudinal monitoring of rhythmicity from specific mouse tissues.

A Modified Golgi-Cox Procedure for use in Undergraduate Courses.

The Golgi staining method has a long history in the field of neuroscience, and remains an important teaching tool in undergraduate laboratory settings. The ability to visualize a cell with all the processes makes the method useful when teaching introductory neuroscience courses. However, the amount of time required for post-stain tissue processing makes it a difficult procedure to use when teaching laboratory classes. We detail a modified Golgi-Cox method with a short incubation period and minimal post-stain processing that produces well differentiated cells, making it ideal for use in an undergraduate laboratory.

CIRCADA: Shiny Apps for Exploration of Experimental and Synthetic Circadian Time Series with an Educational Emphasis.

Circadian rhythms are daily oscillations in physiology and behavior that can be assessed by recording body temperature, locomotor activity, or bioluminescent reporters, among other measures. These different types of data can vary greatly in waveform, noise characteristics, typical sampling rate, and length of recording. We developed 2 Shiny apps for exploration of these data, enabling visualization and analysis of circadian parameters such as period and phase. Methods include the discrete wavelet transform, sine fitting, the Lomb-Scargle periodogram, autocorrelation, and maximum entropy spectral analysis, giving a sense of how well each method works on each type of data. The apps also provide educational overviews and guidance for these methods, supporting the training of those new to this type of analysis. CIRCADA-E (Circadian App for Data Analysis-Experimental Time Series) allows users to explore a large curated experimental data set with mouse body temperature, locomotor activity, and PER2::LUC rhythms recorded from multiple tissues. CIRCADA-S (Circadian App for Data Analysis-Synthetic Time Series) generates and analyzes time series with user-specified parameters, thereby demonstrating how the accuracy of period and phase estimation depends on the type and level of noise, sampling rate, length of recording, and method. We demonstrate the potential uses of the apps through 2 in silico case studies.

Nobiletin affects circadian rhythms and oncogenic characteristics in a cell-dependent manner.

The natural product nobiletin is a small molecule, widely studied with regard to its therapeutic effects, including in cancer cell lines and tumors. Recently, nobiletin has also been shown to affect circadian rhythms via their enhancement, resulting in protection against metabolic syndrome. We hypothesized that nobiletin's anti-oncogenic effects, such as prevention of cell migration and formation of anchorage independent colonies, are correspondingly accompanied by modulation of circadian rhythms. Concurrently, we wished to determine whether the circadian and anti-oncogenic effects of nobiletin differed across cancer cell lines. In this study, we assessed nobiletin's circadian and therapeutic characteristics to ascertain whether these effects depend on cell line, which here also varied in terms of baseline circadian rhythmicity. Three cell culture models where nobiletin's effects on cell proliferation and migration have been studied previously were evaluated: U2OS (bone osteosarcoma), which possesses robust circadian rhythms; MCF7 (breast adenocarcinoma), which has weak circadian rhythms; and MDA-MB-231 (breast adenocarcinoma), which is arrhythmic. We found that circadian, migration, and proliferative effects following nobiletin treatment were subtle in the U2OS and MCF7 cells. On the other hand, changes were clear in MDA-MB-231s, where nobiletin rescued rhythmicity and substantially reduced oncogenic features, specifically two-dimensional cell motility and anchorage-independent growth. Based on these results and those previously described, we posit that the effects of nobiletin are indeed cell-type dependent, and that a positive correlation may exist between nobiletin's circadian and therapeutic effects.

Visualizing jet lag in the mouse suprachiasmatic nucleus and peripheral circadian timing system.

Circadian rhythms regulate most physiological processes. Adjustments to circadian time, called phase shifts, are necessary following international travel and on a more frequent basis for individuals who work non-traditional schedules such as rotating shifts. As the disruption that results from frequent phase shifts is deleterious to both animals and humans, we sought to better understand the kinetics of resynchronization of the mouse circadian system to one of the most disruptive phase shifts, a 6-h phase advance. Mice bearing a luciferase reporter gene for mPer2 were subjected to a 6-h advance of the light cycle and molecular rhythms in suprachiasmatic nuclei (SCN), thymus, spleen, lung and esophagus were measured periodically for 2 weeks following the shift. For the SCN, the master pacemaker in the brain, we employed high-resolution imaging of the brain slice to describe the resynchronization of rhythms in single SCN neurons during adjustment to the new light cycle. We observed significant differences in shifting kinetics among mice, among organs such as the spleen and lung, and importantly among neurons in the SCN. The phase distribution among all Period2-expressing SCN neurons widened on the day following a shift of the light cycle, which was partially due to cells in the ventral SCN exhibiting a larger initial phase shift than cells in the dorsal SCN. There was no clear delineation of ventral and dorsal regions, however, as the SCN appear to have a population of fast-shifting cells whose anatomical distribution is organized in a ventral-dorsal gradient. Full resynchronization of the SCN and peripheral timing system, as measured by a circadian reporter gene, did not occur until after 8 days in the advanced light cycle.

Dexras1 potentiates photic and suppresses nonphotic responses of the circadian clock.

Circadian rhythms of physiology and behavior are generated by biological clocks that are synchronized to the cyclic environment by photic or nonphotic cues. The interactions and integration of various entrainment pathways to the clock are poorly understood. Here, we show that the Ras-like G protein Dexras1 is a critical modulator of the responsiveness of the master clock to photic and nonphotic inputs. Genetic deletion of Dexras1 reduces photic entrainment by eliminating a pertussis-sensitive circadian response to NMDA. Mechanistically, Dexras1 couples NMDA and light input to Gi/o and ERK activation. In addition, the mutation greatly potentiates nonphotic responses to neuropeptide Y and unmasks a nonphotic response to arousal. Thus, Dexras1 modulates the responses of the master clock to photic and nonphotic stimuli in opposite directions. These results identify a signaling molecule that serves as a differential modulator of the gated photic and nonphotic input pathways to the circadian timekeeping system.

Ghrelin effects on the circadian system of mice.

The orexigenic peptide ghrelin stimulates both food intake and growth hormone release and is synthesized in the stomach and in hypothalamic areas involved in feeding control. The suprachiasmatic nuclei of the hypothalamus (SCN) control most circadian rhythms, although there is evidence that some oscillators, such as food-entrainable oscillators, can drive activity rhythms even after SCN ablation. Ghrelin levels exhibit a circadian rhythm and closely follow feeding schedules, making this peptide a putative candidate for food-related entraining signals. We examined the response of the SCN to ghrelin treatments in vitro, by means of electrophysiological and bioluminescence recordings, and in vivo, by assessing effects on the phase of locomotor activity rhythms. Ghrelin applied at circadian time 6 in vitro to cultured SCN slices induced an approximately 3 h phase advance. In addition, ghrelin phase advanced the rhythm of PER2::LUC (Period2::Luciferase) expression in cultured SCN explants from mPer2(Luc) transgenic mice. In vivo, intraperitoneal administration of ghrelin or a synthetic analog, growth hormone-releasing protein-6 (GHRP-6), to ad libitum fed animals failed to alter circadian phase. When injected after 30 h of food deprivation, GHRP-6 induced a phase advance compared with saline-injected animals. These results indicate that ghrelin may play a role in the circadian system by exerting a direct action on the SCN and that the system as a whole may become sensitive to ghrelin and other feeding-related neuropeptides under conditions of food restriction.

Circadian entrainment aftereffects in suprachiasmatic nuclei and peripheral tissues in vitro.

Circadian rhythms are endogenous 24-h rhythms. The suprachiasmatic nuclei (SCN) of the mammalian hypothalamus serve as the master circadian pacemaker, entraining peripheral organs which also demonstrate circadian rhythms. Entrainment to LD cycles of non-24 h duration (T-cycles) induces aftereffects on period that act to bring the intrinsic period closer to the entraining cycle. Both parametric effects, such as changes in endogenous period, and non-parametric effects of light, such as instantaneous phase shifts, act synergistically to accomplish entrainment of the SCN. It is not yet known if entrainment of peripheral oscillators similarly involves both parametric and non-parametric effects. In this study, mPer2(Luc) knockin mice were entrained to either long or short T-cycles, placed into constant darkness (DD) for 3 days to measure behavioral free-running period (FRP), and then PER2::LUC bioluminescence from SCN, spleen, esophagus, lung and thymus was measured in vitro. The FRP of SCN samples was negatively correlated with the FRP of behavioral rhythms, replicating prior results in mPer1-Luc mice. The FRP of the four peripheral oscillators tested did not correlate with behavioral rhythm FRP. Evidence that the SCN may entrain peripheral tissues by shifting phase relationships was observed, in that the phase of PER2::LUC in the SCN relative to peripheral tissues and also to the onset of behavioral activity varied between groups. Our study suggests that aftereffects on FRP may be an emergent property of the system that cannot be explained by the period changes in the system components. Further, we demonstrate that the phase relationship between the rhythm in PER2 in the SCN and these peripheral tissues is altered following T-cycle entrainment.

Alterations in glutamatergic signaling contribute to the decline of circadian photoentrainment in aged mice.

Robust physiological circadian rhythms form an integral part of well-being. The aging process has been found to negatively impact systems that drive circadian physiology, typically manifesting as symptoms associated with abnormal/disrupted sleeping patterns. Here, we investigated the age-related decline in light-driven circadian entrainment in male C57BL/6J mice. We compared light-driven resetting of circadian behavioral activity in young (1-2 months) and old (14-18 months) mice and explored alterations in the glutamatergic pathway at the level of the circadian pacemaker, the suprachiasmatic nucleus (SCN). Aged animals showed a significant reduction in sensitivity to behavioral phase resetting by light. We show that this change was through alterations in N-Methyl-D-aspartate (NMDA) signaling at the SCN, where NMDA, a glutamatergic agonist, was less potent in inducing clock resetting. Finally, we show that this shift in NMDA sensitivity was through the reduced SCN expression of this receptor's NR2B subunit. Only in young animals did an NR2B antagonist attenuate behavioral resetting. These results can help target treatments that aim to improve both physiological and behavioral circadian entrainment in aged populations.

Neuropeptide Y does not reset the circadian clock in NPY Y2-/- mice.

Mammalian circadian rhythms are modulated by neuropeptide Y (NPY), a peptide contained in the projection from the intergeniculate leaflet to the suprachiasmatic nuclei of the circadian pacemaker. NPY resets the circadian clock during the subjective day, mediating non-photic inputs. Previous studies using receptor-selective agonists have indicated that this action of NPY is mediated by the Y2 receptor in hamsters. The present study determined if NPY applied to the suprachiasmatic nuclei in the mid-subjective day can phase-advance the rhythm of spontaneous firing rate of Y2-/- mice. We observed that NPY did reset the rhythm of control mice but did not significantly shift the phase of this rhythm in the Y2-/- mice. These results provide strong evidence for the role of the Y2 receptor mediating neuropeptide Y subjective day phase-advance shifts in mice.

Neuropeptide Y differentially suppresses per1 and per2 mRNA induced by light in the suprachiasmatic nuclei of the golden hamster.

Neuropeptide Y (NPY), present in an input pathway to the suprachiasmatic nuclei (SCN), can block the effects of light on circadian rhythms. The authors have studied this interaction using an in vitro brain slice technique. Effects of NPY on light-induced period1 and period2 mRNA in the SCN were examined in vitro following a light pulse during early subjective night. Golden hamsters (n = 91) were housed under a 14:10 LD cycle and then moved to constant dim red light for 3 days. Hamsters were exposed to a 5-min light pulse previously shown to induce phase shifts and prepared for in vitro application of NPY. Hypothalamic slices containing the SCN were maintained in vitro for 40 min to 4 h after the light pulse, then quick-frozen. Sections were evaluated by in situ hybridization with [35S]-labeled cRNA probes for per mRNA. Rapid light induction of both per1 and per2 by 40 min and 1 h after the light pulse, respectively, was apparent, with NPY inhibition of this response significant by at least these same time points. However, although striking suppression of per2 mRNA by the NPY continued through the peak for per2 at 2 h, per1 mRNA levels rebounded quickly to equal the per1 induction peak at 1 h and mirrored the control light induction pattern for per1 thereafter. Delaying NPY to 30 min after slice preparation demonstrated that NPY is capable of suppressing peak per1 levels. These results confirm the feasibility of measuring light-induced gene expression in the SCN in vitro. A differential regulation of per1 and per2 transcription might be of critical importance for the modulation of circadian responses to light.