Knockout of the circadian gene, Per2, disrupts corticosterone secretion and results in depressive-like behaviors and deficits in startle responses.

BACKGROUND: The Period Circadian Regulator 2 (Per2) gene is important for the modulation of circadian rhythms that influence biological processes. Circadian control of the hypothalamus-pituitary-adrenal (HPA) axis is critical for regulation of hormones involved in the stress response. Dysregulation of the HPA axis is associated with neuropsychiatric disorders. Therefore, it is important to understand how disruption of the circadian rhythm alters the HPA axis. One way to address this question is to delete a gene involved in regulating a central circadian gene such as Per2 in an animal model and to determine how this deletion may affect the HPA axis and behaviors that are altered when the HPA axis is dysregulated. To study this, corticosterone (CORT) levels were measured through the transition from light (inactive phase) to dark (active phase). Additionally, CORT levels as well as pituitary and adrenal mRNA expression were measured following a mild restraint stress. Mice were tested for depressive-like behaviors (forced swim test (FST)), acoustic startle response (ASR), and pre-pulse inhibition (PPI). RESULTS: The present results showed that Per2 knockout impacted CORT levels, mRNA expression, depressive-like behaviors, ASR and PPI. Unlike wild-type (WT) mice, Per2 knockout (Per2) mice showed no diurnal rise in CORT levels at the onset of the dark cycle. Per2-/- mice had enhanced CORT levels and adrenal melanocortin receptor 2 (Mc2R) mRNA expression following restraint. There were no changes in expression of any other pituitary or adrenal gene. In the FST, Per2-/- mice spent more time floating (less time struggling) than WT mice, suggesting increased depressive-like behaviors. Per2-/- mice had deficits in ASR and PPI startle responses compared to WT mice. CONCLUSIONS: In summary, these findings showed that disruption of the circadian system via Per2 gene deletion dysregulated the HPA stress axis and is subsequently correlated with increased depressive-like behaviors and deficits in startle response.

Direct and indirect inhibition of the circadian clock protein Per1: effects on ENaC and blood pressure.

Circadian rhythms govern physiological functions and are important for overall health. The molecular circadian clock comprises several transcription factors that mediate circadian control of physiological function, in part, by regulating gene expression in a tissue-specific manner. These connections are well established, but the underlying mechanisms are incompletely understood. The overall goal of this study was to examine the connection among the circadian clock protein Period 1 (Per1), epithelial Na+ channel (ENaC), and blood pressure (BP) using a multipronged approach. Using global Per1 knockout mice on a 129/sv background in combination with a high-salt diet plus mineralocorticoid treatment, we demonstrated that loss of Per1 in this setting is associated with protection from hypertension. Next, we used the ENaC inhibitor benzamil to demonstrate a role for ENaC in BP regulation and urinary Na+ excretion in 129/sv mice. We targeted Per1 indirectly using pharmacological inhibition of Per1 nuclear entry in vivo to demonstrate altered expression of known Per1 target genes as well as a BP-lowering effect in 129/sv mice. Finally, we directly inhibited Per1 via genetic knockdown in amphibian distal nephron cells to demonstrate, for the first time, that reduced Per1 expression is associated with decreased ENaC activity at the single channel level.

Female C57BL/6J mice lacking the circadian clock protein PER1 are protected from nondipping hypertension.

The circadian clock is integral to the maintenance of daily rhythms of many physiological outputs, including blood pressure. Our laboratory has previously demonstrated the importance of the clock protein period 1 (PER1) in blood pressure regulation in male mice. Briefly, a high-salt diet (HS; 4% NaCl) plus injection with the long-acting mineralocorticoid deoxycorticosterone pivalate (DOCP) resulted in nondipping hypertension [<10% difference between night and day blood pressure (BP) in Per1-knockout (KO) mice but not in wild-type (WT) mice]. To date, there have been no studies that have examined the effect of a core circadian gene KO on BP rhythms in female mice. The goal of the present study was to determine whether female Per1-KO mice develop nondipping hypertension in response to HS/DOCP treatment. For the first time, we demonstrate that loss of the circadian clock protein PER1 in female mice does not significantly change mean arterial pressure (MAP) or the BP rhythm relative to female C57BL/6 WT control mice. Both WT and Per1-KO female mice experienced a significant increase in MAP in response to HS/DOCP. Importantly, however, both genotypes maintained a >10% dip in BP on HS/DOCP. This effect is distinct from the nondipping hypertension seen in male Per1-KO mice, demonstrating that the female sex appears to be protective against PER1-mediated nondipping hypertension in response to HS/DOCP. Together, these data suggest that PER1 acts in a sex-dependent manner in the regulation of cardiovascular rhythms.

The circadian gene, Per2, influences methamphetamine sensitization and reward through the dopaminergic system in the striatum of mice.

Drug addiction is a chronic and relapsing brain disorder, influenced by complex interactions between endogenous and exogenous factors. Per2, a circadian gene, plays a role in drug addiction. Previous studies using Per2-knockout mice have shown a role for Per2 in cocaine, morphine and alcohol addiction. In the present study, we investigated the role of Per2 in methamphetamine (METH) addiction using Per2-overexpression and knockout mice. We observed locomotor sensitization responses to METH administration, and rewarding effects using a conditioned place preference test. In addition, we measured expression levels of dopamine and dopamine-related genes (monoamine oxidase A, DA receptor 1, DA receptor 2, DA active transporter, tyrosine hydroxylase and cAMP response element-binding protein 1) in the striatum of the mice after repeated METH treatments, using qRT-PCR. Per2-overexpressed mice showed decreased locomotor sensitization and rewarding effects of METH compared to the wildtype mice, whereas the opposite was observed in Per2 knockout mice. Both types of transgenic mice showed altered expression levels of dopamine-related genes after repeated METH administration. Specifically, we observed lower dopamine levels in Per2-overexpressed mice and higher levels in Per2-knockout mice. Taken together, Per2 expression levels may influence the addictive effects of METH through the dopaminergic system in the striatum of mice.

Circadian control of oscillations in mitochondrial rate-limiting enzymes and nutrient utilization by PERIOD proteins.

Mitochondria are major suppliers of cellular energy through nutrients oxidation. Little is known about the mechanisms that enable mitochondria to cope with changes in nutrient supply and energy demand that naturally occur throughout the day. To address this question, we applied MS-based quantitative proteomics on isolated mitochondria from mice killed throughout the day and identified extensive oscillations in the mitochondrial proteome. Remarkably, the majority of cycling mitochondrial proteins peaked during the early light phase. We found that rate-limiting mitochondrial enzymes that process lipids and carbohydrates accumulate in a diurnal manner and are dependent on the clock proteins PER1/2. In this conjuncture, we uncovered daily oscillations in mitochondrial respiration that peak during different times of the day in response to different nutrients. Notably, the diurnal regulation of mitochondrial respiration was blunted in mice lacking PER1/2 or on a high-fat diet. We propose that PERIOD proteins optimize mitochondrial metabolism to daily changes in energy supply/demand and thereby, serve as a rheostat for mitochondrial nutrient utilization.

Loss of the clock protein PER2 shortens the erythrocyte life span in mice.

Cell proliferation and release from the bone marrow have been demonstrated to be controlled by circadian rhythms in both humans and mice. However, it is unclear whether local circadian clocks in the bone marrow influence physiological functions and life span of erythrocytes. Here, we report that loss of the clock gene Per2 significantly decreased erythrocyte life span. Mice deficient in Per2 were more susceptible to acute stresses in the erythrocytes, becoming severely anemic upon phenylhydrazine, osmotic, and H2O2 challenges. 1H NMR-based metabolomics analysis revealed that the Per2 depletion causes significant changes in metabolic profiles of erythrocytes, including increased lactate and decreased ATP levels compared with wild-type mice. The lower ATP levels were associated with hyperfunction of Na+/K+-ATPase activity in Per2-null erythrocytes, and inhibition of Na+/K+-ATPase activity by ouabain efficiently rescued ATP levels. Per2-null mice displayed increased levels of Na+/K+-ATPase α1 (ATP1A1) in the erythrocyte membrane, and transfection of Per2 cDNA into the erythroleukemic cell line TF-1 inhibited Atp1a1 expression. Furthermore, we observed that PER2 regulates Atp1a1 transcription through interacting with trans-acting transcription factor 1 (SP1). Our findings reveal that Per2 function in the bone marrow is required for the regulation of life span in circulating erythrocytes.

Renal Na-handling defect associated with PER1-dependent nondipping hypertension in male mice.

Many physiological functions have a circadian rhythm, including blood pressure (BP). BP is highest during the active phase, whereas during the rest period, BP dips 10-20%. Patients that do not experience this dip at night are termed "nondippers." Nondipping hypertension is associated with increased risk of cardiovascular disease. The mechanisms underlying nondipping hypertension are not understood. Without the circadian clock gene Per1, C57BL/6J mice develop nondipping hypertension on a high-salt diet plus mineralocorticoid treatment (HS/DOCP). Our laboratory has shown that PER1 regulates expression of several genes related to sodium (Na) transport in the kidney, including epithelial Na channel (ENaC) and Na chloride cotransporter (NCC). Urinary Na excretion also demonstrates a circadian pattern with a peak during active periods. We hypothesized that PER1 contributes to circadian regulation of BP via a renal Na-handling-dependent mechanism. Na-handling genes from the distal nephron were inappropriately regulated in KO mice on HS/DOCP. Additionally, the night/day ratio of Na urinary excretion by Per1 KO mice is decreased compared with WT (4 × vs. 7×, P < 0.001, n = 6 per group). Distal nephron-specific Per1 KO mice also show an inappropriate increase in expression of Na transporter genes αENaC and NCC. These results support the hypothesis that PER1 mediates control of circadian BP rhythms via the regulation of distal nephron Na transport genes. These findings have implications for the understanding of the etiology of nondipping hypertension and the subsequent development of novel therapies for this dangerous pathophysiological condition.

Auriculasin from Flemingia philippinensis roots shows good therapeutic indexes on hyperactive behavior in zebrafish.

Previously, period1b-/- zebrafish mutants were used to establish an attention deficit hyperactivity disorder (ADHD) model, in which hyperactive behavior was found to be a typical characteristic of ADHD due to down-regulated dopamine levels. Here, we used five prenylated isoflavones from Flemingia philippinensis roots to study their therapeutic effects on hyperactivity behavior in period1b-/- zebrafish. Results of locomotor activity assay showed that auriculasin, one of the prenylated isoflavones, significantly reduced the hyperactivity behavior in period1b-/- zebrafish. Hormone measurement results showed that auriculasin increased melatonin and dopamine content. Results of quantitative real-time polymerase chain reaction showed that auriculasin down-regulated the expression of mao but up-regulated the expression of th and per1b. Thus, auriculasin demonstrated a potential biological effect on dopamine activity to inhibit hyperactivity behavior in the ADHD zebrafish model by regulating circadian clock gene per1b.

Regulation of circadian behaviour and metabolism by REV-ERB-α and REV-ERB-ß.

The circadian clock acts at the genomic level to coordinate internal behavioural and physiological rhythms via the CLOCK-BMAL1 transcriptional heterodimer. Although the nuclear receptors REV-ERB-α and REV-ERB-ß have been proposed to form an accessory feedback loop that contributes to clock function, their precise roles and importance remain unresolved. To establish their regulatory potential, we determined the genome-wide cis-acting targets (cistromes) of both REV-ERB isoforms in murine liver, which revealed shared recognition at over 50% of their total DNA binding sites and extensive overlap with the master circadian regulator BMAL1. Although REV-ERB-α has been shown to regulate Bmal1 expression directly, our cistromic analysis reveals a more profound connection between BMAL1 and the REV-ERB-α and REV-ERB-ß genomic regulatory circuits than was previously suspected. Genes within the intersection of the BMAL1, REV-ERB-α and REV-ERB-ß cistromes are highly enriched for both clock and metabolic functions. As predicted by the cistromic analysis, dual depletion of Rev-erb-α and Rev-erb-ß function by creating double-knockout mice profoundly disrupted circadian expression of core circadian clock and lipid homeostatic gene networks. As a result, double-knockout mice show markedly altered circadian wheel-running behaviour and deregulated lipid metabolism. These data now unite REV-ERB-α and REV-ERB-ß with PER, CRY and other components of the principal feedback loop that drives circadian expression and indicate a more integral mechanism for the coordination of circadian rhythm and metabolism.

Transcriptional regulation of NHE3 and SGLT1 by the circadian clock protein Per1 in proximal tubule cells.

We have previously demonstrated that the circadian clock protein period (Per)1 coordinately regulates multiple genes involved in Na(+) reabsorption in renal collecting duct cells. Consistent with these results, Per1 knockout mice exhibit dramatically lower blood pressure than wild-type mice. The proximal tubule is responsible for a majority of Na(+) reabsorption. Previous work has demonstrated that expression of Na(+)/H(+) exchanger 3 (NHE3) oscillates with a circadian pattern and Na(+)-glucose cotransporter (SGLT)1 has been demonstrated to be a circadian target in the colon, but whether these target genes are regulated by Per1 has not been investigated in the kidney. The goal of the present study was to determine if Per1 regulates the expression of NHE3, SGLT1, and SGLT2 in the kidney. Pharmacological blockade of nuclear Per1 entry resulted in decreased mRNA expression of SGLT1 and NHE3 but not SGLT2 in the renal cortex of mice. Per1 small interfering RNA and pharmacological blockade of Per1 nuclear entry in human proximal tubule HK-2 cells yielded the same results. Examination of heterogeneous nuclear RNA suggested that the effects of Per1 on NHE3 and SGLT1 expression occurred at the level of transcription. Per1 and the circadian protein CLOCK were detected at promoters of NHE3 and SGLT1. Importantly, both membrane and intracellular protein levels of NHE3 and SGLT1 were decreased after blockade of nuclear Per1 entry. This effect was associated with reduced activity of Na(+)-K(+)-ATPase. These data demonstrate a role for Per1 in the transcriptional regulation of NHE3 and SGLT1 in the kidney.

Period 2 is essential to maintain early endothelial progenitor cell function in vitro and angiogenesis after myocardial infarction in mice.

Cellular therapeutic neovascularization has been successfully performed in clinical trials for patients with ischaemia diseases. Despite the vast knowledge of cardiovascular disease and circadian biology, the role of the circadian clock in regulating angiogenesis in myocardial infarction (MI) remains poorly understood. In this study, we aimed to investigate the role and underlying mechanisms of Period 2 (Per2) in endothelial progenitor cell (EPC) function. Flow cytometry revealed lower circulating EPC proportion in per2(-/-) than in wild-type (WT) mice. PER2 was abundantly expressed in early EPCs in mice. In vitro, EPCs from per2(-/-) mice showed impaired proliferation, migration, tube formation and adhesion. Western blot analysis demonstrated inhibited PI3k/Akt/FoxO signalling and reduced C-X-C chemokine receptor type 4 (CXCR4) protein level in EPCs of per2(-/-) mice. The impaired proliferation was blocked by activated PI3K/Akt/FoxO signalling. Direct interaction of CXCR4 and PER2 was detected in WT EPCs. To further study the effect of per2 on in vivo EPC survival and angiogenesis, we injected saline or DiI-labelled WT or per2(-/-) EPC intramyocardially into mice with induced MI. Per2(-/-) reduced the retention of transplanted EPCs in the myocardium, which was associated with significantly reduced DiI expression in the myocardium of MI mice. Decreased angiogenesis in the myocardium of per2(-/-) EPC-treated mice coincided with decreased LV function and increased infarct size in the myocardium. Per2 may be a key factor in maintaining EPC function in vitro and in therapeutic angiogenesis in vivo.

Tissue-intrinsic dysfunction of circadian clock confers transplant arteriosclerosis.

The suprachiasmatic nucleus of the brain is the circadian center, relaying rhythmic environmental and behavioral information to peripheral tissues to control circadian physiology. As such, central clock dysfunction can alter systemic homeostasis to consequently impair peripheral physiology in a manner that is secondary to circadian malfunction. To determine the impact of circadian clock function in organ transplantation and dissect the influence of intrinsic tissue clocks versus extrinsic clocks, we implemented a blood vessel grafting approach to surgically assemble a chimeric mouse that was part wild-type (WT) and part circadian clock mutant. Arterial isografts from donor WT mice that had been anastamosed to common carotid arteries of recipient WT mice (WT:WT) exhibited no pathology in this syngeneic transplant strategy. Similarly, when WT grafts were anastamosed to mice with disrupted circadian clocks, the structural features of the WT grafts immersed in the milieu of circadian malfunction were normal and absent of lesions, comparable to WT:WT grafts. In contrast, aortic grafts from Bmal1 knockout (KO) or Period-2,3 double-KO mice transplanted into littermate control WT mice developed robust arteriosclerotic disease. These lesions observed in donor grafts of Bmal1-KO were associated with up-regulation in T-cell receptors, macrophages, and infiltrating cells in the vascular grafts, but were independent of hemodynamics and B and T cell-mediated immunity. These data demonstrate the significance of intrinsic tissue clocks as an autonomous influence in experimental models of arteriosclerotic disease, which may have implications with regard to the influence of circadian clock function in organ transplantation.

A role for the circadian clock protein Per1 in the regulation of aldosterone levels and renal Na+ retention.

The circadian clock plays an important role in the regulation of physiological processes, including renal function and blood pressure. We have previously shown that the circadian protein period (Per)1 regulates the expression of multiple Na(+) transport genes in the collecting duct, including the α-subunit of the renal epithelial Na(+) channel. Consistent with this finding, Per1 knockout mice exhibit dramatically lower blood pressure than wild-type mice. We have also recently demonstrated the potential opposing actions of cryptochrome (Cry)2 on Per1 target genes. Recent work by others has demonstrated that Cry1/2 regulates aldosterone production through increased expression of the adrenal gland-specific rate-limiting enzyme 3ß-dehydrogenase isomerase (3ß-HSD). Therefore, we tested the hypothesis that Per1 plays a role in the regulation of aldosterone levels and renal Na(+) retention. Using RNA silencing and pharmacological blockade of Per1 nuclear entry in the NCI-H295R human adrenal cell line, we showed that Per1 regulates 3ß-HSD expression in vitro. These results were confirmed in vivo: mice with reduced levels of Per1 had decreased levels of plasma aldosterone and decreased mRNA expression of 3ß-HSD. We postulated that mice with reduced Per1 would have a renal Na(+)-retaining defect. Indeed, metabolic cage experiments demonstrated that Per1 heterozygotes excreted more urinary Na(+) compared with wild-type mice. Taken together, these data support the hypothesis that Per1 regulates aldosterone levels and that Per1 plays an integral role in the regulation of Na(+) retention.

Opposing actions of Per1 and Cry2 in the regulation of Per1 target gene expression in the liver and kidney.

Mounting evidence suggests that the circadian clock plays an integral role in the regulation of many physiological processes including blood pressure, renal function, and metabolism. The canonical molecular clock functions via activation of circadian target genes by Clock/Bmal1 and repression of Clock/Bmal1 activity by Per1-3 and Cry1/2. However, we have previously shown that Per1 activates genes important for renal sodium reabsorption, which contradicts the canonical role of Per1 as a repressor. Moreover, Per1 knockout (KO) mice exhibit a lowered blood pressure and heavier body weight phenotype similar to Clock KO mice, and opposite that of Cry1/2 KO mice. Recent work has highlighted the potential role of Per1 in repression of Cry2. Therefore, we postulated that Per1 potentially activates target genes through a Cry2-Clock/Bmal1-dependent mechanism, in which Per1 antagonizes Cry2, preventing its repression of Clock/Bmal1. This hypothesis was tested in vitro and in vivo. The Per1 target genes αENaC and Fxyd5 were identified as Clock targets in mpkCCDc14 cells, a model of the renal cortical collecting duct. We identified PPARα and DEC1 as novel Per1 targets in the mouse hepatocyte cell line, AML12, and in the liver in vivo. Per1 knockdown resulted in upregulation of Cry2 in vitro, and this result was confirmed in vivo in mice with reduced expression of Per1. Importantly, siRNA-mediated knockdown of Cry2 and Per1 demonstrated opposing actions for Cry2 and Per1 on Per1 target genes, supporting the potential Cry2-Clock/Bmal1-dependent mechanism underlying Per1 action in the liver and kidney.

Loss of circadian clock accelerates aging in neurodegeneration-prone mutants.

Circadian clocks generate rhythms in molecular, cellular, physiological, and behavioral processes. Recent studies suggest that disruption of the clock mechanism accelerates organismal senescence and age-related pathologies in mammals. Impaired circadian rhythms are observed in many neurological diseases; however, it is not clear whether loss of rhythms is the cause or result of neurodegeneration, or both. To address this important question, we examined the effects of circadian disruption in Drosophila melanogaster mutants that display clock-unrelated neurodegenerative phenotypes. We combined a null mutation in the clock gene period (per(01)) that abolishes circadian rhythms, with a hypomorphic mutation in the carbonyl reductase gene sniffer (sni(1)), which displays oxidative stress induced neurodegeneration. We report that disruption of circadian rhythms in sni(1) mutants significantly reduces their lifespan compared to single mutants. Shortened lifespan in double mutants was coupled with accelerated neuronal degeneration evidenced by vacuolization in the adult brain. In addition, per(01)sni(1) flies showed drastically impaired vertical mobility and increased accumulation of carbonylated proteins compared to age-matched single mutant flies. Loss of per function does not affect sni mRNA expression, suggesting that these genes act via independent pathways producing additive effects. Finally, we show that per(01) mutation accelerates the onset of brain pathologies when combined with neurodegeneration-prone mutation in another gene, swiss cheese (sws(1)), which does not operate through the oxidative stress pathway. Taken together, our data suggest that the period gene may be causally involved in neuroprotective pathways in aging Drosophila.

Temporal expression profiles of ceramide and ceramide-related genes in wild-type and mPer1/mPer2 double knockout mice.

Most living organisms exhibit circadian rhythms in physiology and behavior. These oscillations are generated by an endogenous circadian clock and control many biological processes. Ceramide has attracted attention as a signal mediator in diverse cell processes including cell death and differentiation. The relationships between ceramide expression levels and the circadian clock have not previously been investigated. To determine if there are circadian variations in the content of ceramide, we measured ceramide concentrations in the livers of wild-type (WT) and mPer1/mPer2 double knockout (DKO) mice. The ceramide concentration in WT mice was dramatically increased at Zeitgeber Time 9 (ZT9; 9 h after lights-on time) and ZT21 but no rhythmicity in ceramide expression was seen in DKO mice. Because ceramide can be generated by the hydrolysis of sphingomyelin via sphingomyelinase (SMase), or by ceramide synthase (CerS)-mediated synthesis, we assayed the expression patterns of ceramide-related genes using real-time PCR. CerS2 expression levels showed a biphasic pattern of expression in WT mice but no rhythmicity in DKO mice. While the neutral SMase (nSMase) and acidic SMase (aSMase) mRNA in WT mice were expressed in a circadian manner, the correlation between the expression levels of these SMases with times of day was weak in DKO mice. Collectively, our findings suggest that both SMases and CerS2 mRNA expression are regulated by the presence of mPer1/mPer2 circadian clock genes in vivo, and imply that ceramide may play a vital role in circadian rhythms and physiology.

Photic entrainment of period mutant mice is predicted from their phase response curves.

A fundamental property of circadian clocks is that they entrain to environmental cues. The circadian genes, Period1 and Period2, are involved in entrainment of the mammalian circadian system. To investigate the roles of the Period genes in photic entrainment, we constructed phase response curves (PRC) to light pulses for C57BL/6J wild-type, Per1(-/-), Per2(-/-), and Per3(-/-) mice and tested whether the PRCs accurately predict entrainment to non-24 light-dark cycles (T-cycles) and constant light (LL). The PRCs of wild-type and Per3(-/-) mice are similar in shape and amplitude and have relatively large delay zones and small advance zones, resulting in successful entrainment to 26 h T-cycles (T26), but not T21, with similar phase angles. Per1(-/-) mice have a high-amplitude PRC, resulting in entrainment to a broad range of T-cycles. Per2(-/-) mice also entrain to a wide range of T-cycles because the advance portion of their PRC is larger than wild types. Period aftereffects following entrainment to T-cycles were similar among all genotypes. We found that the ratio of the advance portion to the delay portion of the PRC accurately predicts the lengthening of the period of the activity rhythm in LL. Wild-type, Per1(-/-), and Per3(-/-) mice had larger delay zones than advance zones and lengthened (>24 h) periods in LL, whereas Per2(-/-) mice had delay and advance zones that were equal in size and no period lengthening in LL. Together, these results demonstrate that PRCs are powerful tools for predicting and understanding photic entrainment of circadian mutant mice.

Matrix metalloproteinase 2 and 9 dysfunction underlie vascular stiffness in circadian clock mutant mice.

OBJECTIVE: To determine if elasticity in blood vessels is compromised in circadian clock-mutant mice (Bmal1-knockout [KO] and Per-triple KO) and if matrix metalloproteinases (MMPs) might confer these changes in compliance. METHODS AND RESULTS: High-resolution ultrasonography in vivo revealed impaired remodeling and increased pulse-wave velocity in the arteries of Bmal1-KO and Per-triple KO mice. In addition, compliance of remodeled arteries and naïve pressurized arterioles ex vivo from Bmal1-KO and Per-triple KO mice was reduced, consistent with stiffening of the vascular bed. The observed vascular stiffness was coincident with dysregulation of MMP-2 and MMP-9 in Bmal1-KO mice. Furthermore, inhibition of MMPs improved indexes of pathological remodeling in wild-type mice, but the effect was abolished in Bmal1-KO mice. CONCLUSIONS: Circadian clock dysfunction contributes to hardening of arteries, which may involve impaired control of the extracellular matrix composition.

Endogenous rhythms in Period1 mutant suprachiasmatic nuclei in vitro do not represent circadian behavior.

The mammalian circadian pacemaker in the suprachiasmatic nuclei (SCN) controls daily rhythms of behavior and physiology. Lesions of the SCN cause arrhythmicity of locomotor activity, and transplants of fetal SCN tissue restore rhythmic behavior that is consistent with the periodicity of the donor's genotype, suggesting that the SCN determines the period of the circadian behavioral rhythm. While several studies have demonstrated that the circadian characteristics of in vitro SCN rhythms represent circadian behavior, others have shown that the periods of explanted SCN are not always congruent with locomotor activity. We find that the aberrant rhythms of ex vivo SCN lacking functional Period1 (Per1(-/-)) do not represent the behavioral rhythms of the mutant animals. Surprisingly, in C57BL/6J Per1(-/-) mice, the real-time circadian gene promoter activity rhythm is weak or absent in adult SCN slices in vitro even though the free-running wheel-running activity rhythm is indistinguishable from wild-type (Per1(+/+)) mice. While some neurons in Per1(-/-) SCN explants exhibit robust circadian rhythms, others have irregular and/or low-amplitude rhythms. Together, these data suggest that either a small population of rhythmic neurons in the Per1(-/-) SCN is sufficient to control wheel-running activity or that in vivo physiological factors can compensate for the aberrant endogenous rhythms of Per1(-/-) SCN.

Altered laryngeal morphology in Period1 deficient mice.

BACKGROUND: Ultrasonic vocalizations (USV) of mice are produced in and emitted by the larynx. However, which anatomical elements of the mouse larynx are involved and to which aspects of USV they contribute is not clear. Frequency and amplitude parameters of mice, deficient in the clock gene Period1 (mPer1-/- mice) are distinguishably different compared to C3H wildtype (WT) controls. Because structural differences in the larynx may be a reason for the different USV observed, we analyzed laryngeal anatomy of mPer1-/- mice and WT control animals using micro-computed-tomography and stereology. RESULTS: In mPer1-/- mice, we found laryngeal cartilages to be normally arranged, and the thyroid, arytenoid and epiglottal cartilages were similar in diameter and volume measurements, compared to WT mice. However, in the cricoid cartilage, a significant difference in the dorso-ventral diameter and volume was evident. CONCLUSION: Our findings imply that laryngeal morphology is affected by inactivation of the clock gene Period1 in mice, which may contribute to their abnormal USV.