Alun T.L. Hughes

Aberrant Gating of Photic Input to the Suprachiasmatic Circadian Pacemaker of Mice Lacking the VPAC2 Receptor

VIP acting via the VPAC2 receptor is implicated as a key signaling pathway in the maintenance and resetting of the hypothalamic suprachiasmatic nuclei (SCN) circadian pacemaker; circadian rhythms in SCN clock gene expression and wheel-running behavior are abolished in mice lacking the VPAC2 receptor (Vipr2–/–). Here, using immunohistochemical detection of pERK (phosphorylated extracellular signal-regulated kinases 1/2) and c-FOS, we tested whether the gating of photic input to the SCN is maintained in these apparently arrhythmic Vipr2–/– mice. Under light/dark and constant darkness, spontaneous expression of pERK and c-FOS in the wild-type mouse SCN was significantly elevated during subjective day compared with subjective night; no diurnal or circadian variation in pERK or c-FOS was detected in the SCN of Vipr2–/– mice. In constant darkness, light pulses given during the subjective night but not the subjective day significantly increased expression of pERK and c-FOS in the wild-type SCN. In contrast, light pulses given during both subjective day and subjective night robustly increased expression of pERK and c-FOS in the Vipr2–/– mouse SCN. Although photic stimuli activate intracellular pathways within the SCN of Vipr2–/– mice, they do not engage the core clock mechanisms. The absence of photic gating, together with the general lack of overt rhythms in circadian output, strongly suggests that the SCN circadian pacemaker is completely dysfunctional in the Vipr2–/– mouse.

The Circadian Clock in Murine Chondrocytes Regulates Genes Controlling Key Aspects of Cartilage Homeostasis

ObjectiveTo characterize the circadian clock in murine cartilage tissue and identify tissue-specific clock target genes, and to investigate whether the circadian clock changes during aging or during cartilage degeneration using an experimental mouse model of osteoarthritis (OA). MethodsCartilage explants were obtained from aged and young adult mice after transduction with the circadian clock fusion protein reporter PER2::luc, and real-time bioluminescence recordings were used to characterize the properties of the clock. Time-series microarrays were performed on mouse cartilage tissue to identify genes expressed in a circadian manner. Rhythmic genes were confirmed by quantitative reverse transcription–polymerase chain reaction using mouse tissue, primary chondrocytes, and a human chondrocyte cell line. Experimental OA was induced in mice by destabilization of the medial meniscus (DMM), and articular cartilage samples were microdissected and subjected to microarray analysis. ResultsMouse cartilage tissue and a human chondrocyte cell line were found to contain intrinsic molecular circadian clocks. The cartilage clock could be reset by temperature signals, while the circadian period was temperature compensated. PER2::luc bioluminescence demonstrated that circadian oscillations were significantly lower in amplitude in cartilage from aged mice. Time-series microarray analyses of the mouse tissue identified the first circadian transcriptome in cartilage, revealing that 615 genes (∼3.9% of the expressed genes) displayed a circadian pattern of expression. This included genes involved in cartilage homeostasis and survival, as well as genes with potential importance in the pathogenesis of OA. Several clock genes were disrupted in the early stages of cartilage degeneration in the DMM mouse model of OA. ConclusionThese results reveal an autonomous circadian clock in chondrocytes that can be implicated in key aspects of cartilage biology and pathology. Consequently, circadian disruption (e.g., during aging) may compromise tissue homeostasis and increase susceptibility to joint damage or disease.

Acute Suppressive and Long-Term Phase Modulation Actions of Orexin on the Mammalian Circadian Clock

Circadian and homeostatic neural circuits organize the temporal architecture of physiology and behavior, but knowledge of their interactions is imperfect. For example, neurons containing the neuropeptide orexin homeostatically control arousal and appetitive states, while neurons in the suprachiasmatic nuclei (SCN) function as the brains master circadian clock. The SCN regulates orexin neurons so that they are much more active during the circadian night than the circadian day, but it is unclear whether the orexin neurons reciprocally regulate the SCN clock. Here we show both orexinergic innervation and expression of genes encoding orexin receptors (OX1 and OX2) in the mouse SCN, with OX1 being upregulated at dusk. Remarkably, we find through in vitro physiological recordings that orexin predominantly suppresses mouse SCN Period1 (Per1)-EGFP-expressing clock cells. The mechanisms underpinning these suppressions vary across the circadian cycle, from presynaptic modulation of inhibitory GABAergic signaling during the day to directly activating leak K+ currents at night. Orexin also augments the SCN clock-resetting effects of neuropeptide Y (NPY), another neurochemical correlate of arousal, and potentiates NPYs inhibition of SCN Per1-EGFP cells. These results build on emerging literature that challenge the widely held view that orexin signaling is exclusively excitatory and suggest new mechanisms for avoiding conflicts between circadian clock signals and homeostatic cues in the brain.

Circadian oscillators in the epithalamus

The habenula complex is implicated in a range of cognitive, emotional and reproductive behaviors, and recently this epithalamic structure was suggested to be a component of the brains circadian system. Circadian timekeeping is driven in cells by the cyclical activity of core clock genes and proteins such as per2/PER2. There are currently no reports of rhythmic clock gene/protein expression in the habenula and therefore the question of whether this structure has an intrinsic molecular clock remains unresolved. Here, using videomicroscopy imaging and photon-counting of a PER2::luciferase (LUC) fusion protein together with multiunit electrophysiological recordings, we tested the endogenous circadian properties of the mouse habenula in vitro. We show that a circadian oscillator is localized primarily to the medial portion of the lateral habenula. Rhythms in PER2:: LUC bioluminescence here are visualized in single cells and oscillations continue in the presence of the sodium channel blocker, tetrodotoxin, indicating that individual cells have intrinsic timekeeping properties. Ependymal cells lining the dorsal third ventricle also express circadian oscillations of PER2. These findings establish that neurons and non-neuronal cells in the epithalamus express rhythms in cellular and molecular activities, indicating a role for circadian oscillators in the temporal regulation of habenula controlled processes and behavior.

Live imaging of altered period1 expression in the suprachiasmatic nuclei of Vipr2-/- mice.

Vasoactive intestinal polypeptide and its receptor, VPAC2, play important roles in the functioning of the brain’s circadian clock in the suprachiasmatic nuclei (SCN). Mice lacking VPAC2 receptors (Vipr2−/−) show altered circadian rhythms in locomotor behavior, neuronal firing rate, and clock gene expression, however, the nature of molecular oscillations in individual cells is unclear. Here, we used real‐time confocal imaging of a destabilized green fluorescent protein (GFP) reporter to track the expression of the core clock gene Per1 in live SCN‐containing brain slices from wild‐type (WT) and Vipr2−/− mice. Rhythms in Per1‐driven GFP were detected in WT and Vipr2−/− cells, though a significantly lower number and proportion of cells in Vipr2−/− slices expressed detectable rhythms. Further, Vipr2−/− cells expressed significantly lower amplitude oscillations than WT cells. Within each slice, the phases of WT cells were synchronized whereas cells in Vipr2−/− slices were poorly synchronized. Most GFP‐expressing cells, from both genotypes, expressed neither vasopressin nor vasoactive intestinal polypeptide. Pharmacological blockade of VPAC2 receptors in WT SCN slices partially mimicked the Vipr2−/− phenotype. These data demonstrate that intercellular communication via the VPAC2 receptor is important for SCN neurons to sustain robust, synchronous oscillations in clock gene expression.

Feedback actions of locomotor activity to the circadian clock.

The phase of the mammalian circadian system can be entrained to a range of environmental stimuli, or zeitgebers, including food availability and light. Further, locomotor activity can act as an entraining signal and represents a mechanism for an endogenous behavior to feedback and influence subsequent circadian function. This process involves a number of nuclei distributed across the brain stem, thalamus, and hypothalamus and ultimately alters SCN electrical and molecular function to induce phase shifts in the master circadian pacemaker. Locomotor activity feedback to the circadian system is effective across both nocturnal and diurnal species, including humans, and has recently been shown to improve circadian function in a mouse model with a weakened circadian system. This raises the possibility that exercise may be useful as a noninvasive treatment in cases of human circadian dysfunction including aging, shift work, transmeridian travel, and the blind.

Spatio-temporal distribution of microglia/macrophages during regeneration in the cerebellum of adult teleost fish, Apteronotus leptorhynchus: a quantitative analysis.

In contrast to mammals, adult teleost fish exhibit an enormous capacity to replace damaged neurons with newly generated ones after injuries in the central nervous system. In the present study, the role of microglia/macrophages, identified by tomato lectin binding, was examined in this process of neuronal regeneration in the corpus cerebelli of the teleost fish Apteronotus leptorhynchus. In the intact corpus cerebelli, or after short survival times following application of a mechanical lesion to this cerebellar subdivision, microglia/macrophages were virtually absent. Conversely, approximately 3 days after application of the lesion, the areal density of microglia/macrophages started to increase at and near the lesion site in the ipsilateral hemisphere, as well as in the contralateral hemisphere, and reached maximum levels at approximately 10 days post lesion. The density remained elevated until it reached background levels approximately one month after the injury. By comparing the time course of the appearance of microglia/macrophages with that of other regenerative events occurring within the first few weeks of wound healing in this model system, we hypothesize that one possible function of microglia/macrophages might be to remove debris of cells that have undergone apoptotic cell death at the lesion site.

Rhythm-promoting actions of exercise in mice with deficient neuropeptide signaling.

Daily exercise promotes physical health as well as improvements in mental and neural functions. Studies in intact wild-type (WT) rodents have revealed that the brain’s suprachiasmatic nuclei (SCN), site of the main circadian pacemaker, are also responsive to scheduled wheel running. It is unclear, however, if and how animals with a dysfunctional circadian pacemaker respond to exercise. Here, we tested whether scheduled voluntary exercise (SVE) in a running wheel for 6 hours per day could promote neural and behavioral rhythmicity in animals whose circadian competence is compromised through genetically targeted loss of vasoactive intestinal polypeptide (VIP-/- mice) or its VPAC2 receptor (Vipr2-/- mice). We report that in constant dark (DD), rhythmic VIP-/- and Vipr2-/- mice show weak free-running rhythms with a period of <23 hours and all wild-type mice are strongly rhythmic with approximately 23.5-hour periodicity. VIP-/- and Vipr2-/- mice rapidly (<7 days) synchronize to daily SVE, while WT mice take much longer (>35 days). Following 21 to 50 days of SVE, WT mice show small changes in their rhythms, and most Vipr2-/- mice now sustain robust near 24-hour behavioral rhythms, whereas very few VIP-/- mice do. This study demonstrates that scheduled daily exercise can markedly improve circadian rhythms in behavioral activity and raises the possibility that this noninvasive approach may be useful as an intervention in clinical etiologies in which there are dysfunctions of circadian time keeping.

Behavioral responses of Vipr2-/- mice to light

Vasoactive intestinal polypeptide and its receptor, VPAC2 , play important roles in the functioning of the dominant circadian pacemaker, located in the hypothalamic suprachiasmatic nuclei (SCN). Mice lacking VPAC 2 receptors (Vipr2–/– ) show altered circadian rhythms and impaired synchronization to environmental lighting cues. However, light can increase phosphoprotein and immediate early gene expression in the Vipr2–/– SCN demonstrating that the circadian clock is readily responsive to light in these mice. It is not clear whether these neurochemical responses to light can be transduced to behavioral changes as seen in wild-type (WT) animals. In this study we investigated the diurnal and circadian wheel-running profile of WT (C57BL/6J) and Vipr2–/– mice under a 12-h light:12-h complete darkness (LD) lighting schedule and in constant darkness (DD) and used 1-h light pulses to shift the activity of mice in DD. Unlike WT mice, Vipr2–/– mice show grossly altered locomotor patterns making the analysis of behavioral responses to light problematic. However, analyses of both the onset and the offset of locomotor activity reveal that in a subset of these mice, light can reset the offset of behavioral rhythms during the subjective night. This suggests that the SCN clock of Vipr2–/– mice and the rhythms it generates are responsive to photic stimulation and that these responses can be integrated to whole animal behavioral changes.

Tau haplotype frequency in frontotemporal lobar degeneration and amyotrophic lateral sclerosis

It has been reported that the H1 haplotype of the tau gene, located on chromosome 17q21, is associated with progressive supranuclear palsy. Recently, it has also been claimed that the H1 haplotype could also be a risk factor for frontotemporal dementia. However, these claims are variable and the involvement of the apolipoprotein E gene as well as the H1 haplotype has been suggested. In light of this we assessed the frequency of tau gene haplotypes in 113 cases of frontotemporal lobar degeneration and 168 control samples. We found a positive association between the H1 haplotype and frontotemporal dementia, but not with any of the other disease groupings (P = 0.029, odds ratio 1.81). We did not observe any affect on age at onset and tau haplotype or apolipoprotein E alleles, nor were any deviation from control frequencies of apolipoprotein E alleles observed. These data are consistent with the hypothesis that the tau gene, or nearby gene on the H1 haplotype, is a risk factor for frontotemporal dementia.