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Background: Circadian disruption has long been recognized as a symptom of Alzheimer’s disease (AD); however, emerging data suggests that circadian dysfunction occurs early on in disease development, potentially preceding any noticeable cognitive deficits. Objective: This study compares the onset of AD in male and female wild type (C57BL6/J), transgenic (AβPP/PS1), and knock-in (APPNL-F/NL-F) AD mouse models from the period of plaque initiation (6 months) through 12 months. Methods: Rhythmic daily activity patterns, glucose sensitivity, cognitive function (Morris water maze, MWM), and AD pathology (plaques formation) were assessed. A comparison was made across sexes. Results: Sex-dependent hyperactivity in AβPP/PS1 mice was observed. In comparison to C57BL/6J animals, 6-month-old male AβPP/PS1 demonstrated nighttime hyperactivity, as did 12-month-old females. Female AβPP/PS1 animals performed significantly worse on a MWM task than AβPP/PS1 males at 12 months and trended toward increased plaque pathology. APPNL-F/NL-F 12-month-old males performed significantly worse on the MWM task compared to 12-month-old females. Significantly greater plaque pathology occurred in AβPP/PS1 animals as compared to APPNL-F/NL-F animals. Female AβPP/PS1 animals performed significantly worse than APPNL-F/NL-F animals in spatial learning and memory tasks, though this was reversed in males. Conclusion: Taken together, this study provides novel insights into baseline sex differences, as well as characterizes baseline diurnal activity variations, in the AβPP/PS1 and APPNL-F/NL-F AD mouse models.

We have previously demonstrated hippocampal hyperglutamatergic signaling occurs prior to plaque accumulation in AβPP/PS1 mice. Here, we evaluate 2-Amino-6-(trifluoromethoxy) benzothiazole (riluzole) as an early intervention strategy for Alzheimer's disease (AD), aimed at restoring glutamate neurotransmission prior to substantial Beta amyloid (Aβ) plaque accumulation and cognitive decline. Male AβPP/PS1 mice, a model of progressive cerebral amyloidosis, were treated with riluzole from 2-6 months of age. Morris water maze, in vivo electrochemistry, and immunofluorescence were performed to assess cognition, glutamatergic neurotransmission, and pathology, respectively, at 12 months. Four months of prodromal riluzole treatment in AβPP/PS1 mice resulted in long-lasting procognitive effects and attenuated glutamatergic tone that was observed six months after discontinuing riluzole treatment. Riluzole-treated AβPP/PS1 mice had significant improvement in long-term memory compared to vehicle-treated AβPP/PS1 mice that was similar to normal aging C57BL/6J control mice. Furthermore, basal glutamate concentration and evoked-glutamate release levels, which were elevated in vehicle-treated AβPP/PS1 mice, were restored to levels observed in age-matched C57BL/6J mice in AβPP/PS1 mice receiving prodromal riluzole treatment. Aβ plaque accumulation was not altered with riluzole treatment. This study supports that interventions targeting the glutamatergic system during the early stages of AD progression have long-term effects on disease outcome, and importantly may prevent cognitive decline. Our observations provide preclinical support for targeting glutamate neurotransmission in patients at risk for developing AD. Read the Editorial Highlight for this article on page 399.

An internal circadian clock regulates timing of systemic energy homeostasis. The central clock in the hypothalamic suprachiasmatic nucleus (SCN) directs local clocks in peripheral tissues such as liver, muscle, and adipose tissue to synchronize metabolism with food intake and rest/activity cycles. Aryl hydrocarbon receptor (AhR) interacts with the molecular circadian clockworks. Activation of AhR dampens rhythmic expression of core clock genes, which may lead to metabolic dysfunction. Given the importance of appropriately-timed adipose tissue function to regulation of energy homeostasis, this study focused on mechanisms by which AhR may influence clock-controlled adipose tissue activity. We hypothesized that AhR activation in adipose tissue would impair lipolysis by dampening adipose rhythms, leading to a decreased lipolysis rate during fasting, and subsequently, altered serum glucose concentrations. Levels of clock gene and lipolysis gene transcripts in mouse mesenchymal stem cells (BMSCs) differentiated into mature adipocytes were suppressed by the AhR agonist β-napthoflavone (BNF), in an AhR dependent manner. BNF altered rhythms of core clock gene and lipolysis gene transcripts in C57bl6/J mice. BNF reduced serum free fatty acids, glycerol and liver glycogen. Chromatin immunoprecipitation indicated that BNF increased binding of AhR to E-Box elements in clock gene and lipolysis gene promoters. These data establish a link between AhR activation and impaired lipolysis, specifically by altering adipose tissue rhythmicity. In response to the decreased available energy from impaired lipolysis, the body increases glycogenolysis, thereby degrading more glycogen to provide necessary energy.

Chronically elevated basal glutamate levels are hypothesized to attenuate detection of physiological signals thereby inhibiting memory formation and retrieval, while inducing excitotoxicity-mediated neurodegeneration observed in Alzheimer’s disease (AD). However, current medication targeting the glutamatergic system, such as memantine, shows limited efficacy and is unable to decelerate disease progression, possibly because it modulates postsynaptic N-methyl-D-aspartate receptors rather than glutamate release or clearance. To determine if decreasing presynaptic glutamate release leads to long-term procognitive effects, we treated AβPP/PS1 mice with LY379268 (3.0 mg/kg; i.p.), a metabotropic glutamate receptor (mGluR)(2/3) agonist from 2–6 months of age when elevated glutamate levels are first observed but cognition is unaffected. C57BL/6J genetic background control mice and another cohort of AβPP/PS1 mice received normal saline (i.p.) as vehicle controls. After 6 months off treatment, mice receiving LY379268 did not show long-term improvement as assessed by the Morris water maze (MWM) spatial learning and memory paradigm. Following MWM, mice were isoflurane anesthetized and a glutamate selective microelectrode was used to measure in vivo basal and stimulus-evoked glutamate release and clearance independently from the dentate, CA3, and CA1 hippocampal subregions. Immunohistochemistry was used to measure hippocampal astrogliosis and plaque pathology. Similar to previous studies, we observed elevated basal glutamate, stimulus evoked glutamate release, and astrogliosis in AβPP/PS1 vehicle mice versus C57BL/6J mice. Treatment with LY379268 did not attenuate these responses nor diminish plaque pathology. The current study builds upon previous research demonstrating hyperglutamatergic hippocampal signaling in AβPP/PS1 mice; however, long-term therapeutic efficacy of LY379268 in AβPP/PS1 was not observed.

The 3T3-L1 murine pre-adipocyte line is an established cell culture model for screening Metabolism Disrupting Chemicals (MDCs). Despite a need to accurately identify MDCs for further evaluation, relatively little research has been performed to comprehensively evaluate reproducibility across laboratories, assess factors that might contribute to varying degrees of differentiation between laboratories (media additives, plastics, cell source, etc.), or to standardize protocols. As such, the goals of this study were to assess interlaboratory variability of efficacy and potency outcomes for triglyceride accumulation and pre-adipocyte proliferation using the mouse 3T3-L1 pre-adipocyte cell assay to test chemicals. Ten laboratories from five different countries participated. Each laboratory evaluated one reference chemical (rosiglitazone) and three blinded test chemicals (tributyltin chloride, pyraclostrobin, and bisphenol A) using: 1) their Laboratory-specific 3T3-L1 Cells (LC) and their Laboratory-specific differentiation Protocol (LP), 2) Shared 3T3-L1 Cells (SC) with LP, 3) LC with a Shared differentiation Protocol (SP), and 4) SC with SP. Blinded test chemical responses were analyzed by the coordinating laboratory. The magnitude and range of bioactivities reported varied considerably across laboratories and test conditions, though the presence or absence of activity for each tested chemical was more consistent. Triglyceride accumulation activity determinations for rosiglitazone ranged from 90 to 100% across test conditions, but 30–70 % for pre-adipocyte proliferation; this was 40–80 % for triglyceride accumulation induced by pyraclostrobin, 80–100 % for tributyltin, and 80–100 % for bisphenol A. Consistency was much lower for pre-adipocyte proliferation, with 30–70 % active determinations for pyraclostrobin, 30–50 % for tributyltin, and 20–40 % for bisphenol A. Greater consistency was observed for the SC/SP assessment. As such, working to develop a standardized adipogenic differentiation protocol represents the best strategy for improving consistency of adipogenic responses using the 3T3-L1 model to reproducibly identify MDCs and increase confidence in reported outcomes. Over-arching project supported by grants [R01 ES016099 to HMS; R00 ES030405 to CDK] from the National Institute of Environmental Health Sciences (NIEHS); University of Turin; European Union's Horizon 2020 research and innovation program under grant agreement GOLIATH No. 825489; Brunel University London; NIEHS (1K22ES026208 and R01ES027863); NIEHS (Z0ES102785); Spanish Institute of Health Carlos III (grant FIS-PI16/01812).

Per-Arnt-Sim (PAS) domain-containing proteins are critical to homeostatic regulatory networks that mediate responsiveness to environmental change. PAS domains are multifunctional structural motifs that allow protein-protein interactions among family members, typically forming heterodimeric transcription factors to affect the transcription of target genes. Prototypical PAS domain-dependent pathways include the circadian clock network and metabolic regulation of the xenobiotic response through the aryl hydrocarbon receptor (AhR). Both pathways are increasingly linked to health, and alteration in their function contributes to development of disease. The aryl hydrocarbon receptor (AhR) demonstrates promiscuity in ligand binding and selectivity during heterodimer formation, which allows varied combinations of protein/protein interactions with other Per-Arnt-Sim (PAS) domain containing proteins and crosstalk among signaling pathways, including the molecular clockworks. AhR and the circadian signaling pathways are highly integrated and reciprocally regulated. AhR exhibits a rhythmic expression and time-dependent sensitivity to activation by AhR agonists. Conversely, AhR influences amplitude and phase of rhythms in circadian clock genes, hormones, and behavior. Understanding the molecular interactions between AhR and the clock provides insight into physiological regulation of rhythmic processes and provides an innovative approach to development of therapeutics.

High fat diet (HFD) consumption alters the synchronized circadian timing system resulting in harmful loss, gain or shift of transcriptional oscillations. The aryl hydrocarbon receptor (AhR) shares structural homology to clock genes, containing both PAS domains and basic helix-loop helix structural motifs, allowing for interaction with components of the primary circadian feedback loop. Activation of AhR alters circadian rhythmicity, primarily through inhibition of Clock/Bmal1-mediated regulation of Per1. AhR-deficient mice are protected from diet-induced metabolic dysfunction, exhibiting enhanced insulin sensitivity and glucose tolerance. This study examined whether AhR haploinsufficiency can also protect against diet-induced alterations in rhythm. After feeding AhR+/+ and AhR+/- mice an HFD (60% fat) for 15 weeks, samples were collected every 4 hours over a 24-hour period. HFD altered the rhythm of serum glucose and the metabolic transcriptome, including hepatic nuclear receptors Rev-erbα and PPARγ in wild-type c57bl6/j mice. AhR reduction provided protection against diet-induced transcriptional oscillation changes; serum glucose and metabolic gene rhythms were protected from the disruption caused by HFD feeding. These data highlight the critical role of AhR signaling in the regulation of metabolism and provide a potential therapeutic target for diseases characterized by rhythmic desynchrony.

The prevalence of metabolic syndrome, a clustering of three or more risk factors that include abdominal obesity, increased blood pressure, and high levels of glucose, triglycerides, and high-density lipoproteins, has reached dangerous and costly levels worldwide. Increases in morbidity and mortality result from a combination of factors that promote altered glucose metabolism, insulin resistance, and metabolic dysfunction. Although diet and exercise are commonly touted as important determinants in the development of metabolic dysfunction, other environmental factors, including circadian clock disruption and activation of the aryl hydrocarbon receptor (AhR) by dietary or other environmental sources, must also be considered. AhR binds a range of ligands, which prompts protein–protein interactions with other Per-Arnt-Sim (PAS)-domain-containing proteins and subsequent transcriptional activity. This review focuses on the reciprocal crosstalk between the activated AhR and the molecular circadian clock. AhR exhibits a rhythmic expression and time-dependent sensitivity to activation by AhR agonists. Conversely, AhR activation influences the amplitude and phase of expression of circadian clock genes, hormones, and the behavioral responses of the clock system to changes in environmental illumination. Both the clock and AhR status and activation play significant and underappreciated roles in metabolic homeostasis. This review highlights the state of knowledge regarding how AhR may act together with the circadian clock to influence energy metabolism. Understanding the variety of AhR-dependent mechanisms, including its interactions with the circadian timing system that promote metabolic dysfunction, reveals new targets of interest for maintenance of healthy metabolism.

BACKGROUND/OBJECTIVES Epidemics of obesity and diabetes are escalating. High-calorie/high-fat food is a major cause for these global health issues, but molecular mechanisms underlying high-fat, diet-induced obesity are still not well understood. The aryl hydrocarbon receptor (AhR), a transcription factor that acts as a xenobiotic sensor, mediates environmental toxicant-induced obesity, insulin resistance and development of diabetes. AhR also influences lipid metabolism and diet-induced obesity. The effects of AhR deficiency on diet-induced obesity, hepatic steatosis and insulin resistance were examined. METHODS : Male wild type (WT), AhR null (AhR−/−) and AhR heterozygote (AhR+/−) mice were fed a normal chow diet (NCD, 10% kcal from fat) or a high-fat diet (HFD, 60% kcal from fat) for up to 14 weeks. Adiposity, adipose and liver morphology, insulin signaling, metabolic parameters and gene profiles were assessed. RESULTS AhR deficiency protected against HFD-induced obesity, hepatic steatosis, insulin resistance and inflammation. Moreover, AhR deficiency preserved insulin signaling in major metabolic tissues. These protective effects result from a higher energy expenditure in AhR-deficient mice compared to WT. Levels of transcript for both the thermogenic gene, uncoupling protein 1 (Ucp1), in brown adipose tissue and mitochondrial β-oxidation genes in muscle were significantly higher in AhR−/− and AhR+/− mice compared to WT. CONCLUSIONS This work documents a physiologically relevant function for AhR in regulation of body weight, hepatic fat deposition, insulin sensitivity and energy expenditure under HFD exposure, suggesting that AhR signaling may be developed as a potential therapeutic target for treatment of obesity and metabolic disorders.

Hypothalamic histaminergic tuberomammillary (TM) neurons in rats express high densities of nicotinic acetylcholine receptors (nAChRs) whose Ca 2+ permeability, kinetic and pharmacological properties are similar to those of heterologous homomeric α7 nAChRs. However, native α7 nAChR subunits can co-assemble with β or α5 nAChR subunits to form functional heteromeric α7-containing α7β or α7α5 nAChRs with kinetics and pharmacology similar to those of α7 homomers. Therefore, although TM nAChRs have been used as an ex vivo model of functional α7 homomers, the molecular makeup of TM nAChRs has not been determined and the expression of functional α7-containing heteromers in TM neurons has not been excluded. To determine the profile of TM nAChR subunit transcripts, we have conducted single-cell qRT-PCR experiments using acutely dissociated TM neurons in rats. TM neurons were found to express transcripts of only principal α3, α6 and α7 nAChR subunits. Transcripts of other known mammalian neuronal subunits (α2, α4–5, α9–10, β2–4) were not detected. In the absence of β and α5 subunits, the expression of functional α7-containing heteromers in TM neurons is highly unlikely because principal α3, α6 and α7 nAChR subunits alone are not known to form functional heteromeric nAChRs. These results support the exclusive expression of native functional α7 homomers in rat TM neurons and introduce these neurons as a unique reliable source of native functional homomeric α7 nAChRs suitable for ex vivo and in vitro pharmacological assays in developing selective α7 nAChR agents.

PAS domain–containing proteins can act as environmental sensors that capture external stimuli to allow coordination of organismal physiology with the outside world. These proteins permit diverse ligand binding and heterodimeric partnership, allowing for varied combinations of PAS-dependent protein-protein interactions and promoting crosstalk among signaling pathways. Previous studies report crosstalk between circadian clock proteins and the aryl hydrocarbon receptor (AhR). Activated AhR forms a heterodimer with the circadian clock protein Bmal1 and thereby functionally inhibits CLOCK/Bmal1 activity. If physiological activation of AhR through naturally occurring, endogenous ligands inhibits clock function, it seems plausible to hypothesize that decreased AhR expression releases AhR-induced inhibition of circadian rhythms. Because both AhR and the clock are important regulators of glucose metabolism, it follows that decreased AhR will also alter metabolic function. To test this hypothesis, rhythms of behavior, metabolic outputs, and circadian and metabolic gene expression were measured in AhR-deficient mice. Genetic depletion of AhR enhanced behavioral responses to changes in the light-dark cycle, increased rhythmic amplitude of circadian clock genes in the liver, and altered rhythms of glucose and insulin. This study provides evidence of AhR-induced inhibition that influences circadian rhythm amplitude.

The molecular circadian clock mechanism is highly conserved between mammalian and avian species. Avian circadian timing is regulated at multiple oscillatory sites, including the retina, pineal, and hypothalamic suprachiasmatic nucleus (SCN). Based on the authors' previous studies on the rat ovary, it was hypothesized that ovarian clock timing is regulated by the luteinizing hormone (LH) surge. The authors used the chicken as a model to test this hypothesis, because the timing of the endogenous LH surge is accurately predicted from the time of oviposition. Therefore, tissues can be removed before and after the LH surge, allowing one to determine the effect of LH on specific clock genes. The authors first examined the 24-h expression patterns of the avian circadian clock genes of Bmal1, Cry1, and Per2 in primary oscillatory tissues (hypothalamus and pineal) as well as peripheral tissues (liver and ovary). Second, the authors determined changes in clock gene expression after the endogenous LH surge. Clock genes were rhythmically expressed in each tissue, but LH influenced expression of these clock genes only in the ovary. The data suggest that expression of ovarian circadian clock genes may be influenced by the LH surge in vivo and directly by LH in cultured granulosa cells. LH induced rhythmic expression of Per1 and Bmal1 in arrhythmic, cultured granulosa cells. Furthermore, LH altered the phase and amplitude of clock gene rhythms in serum-shocked granulosa cells. Thus, the LH surge may be a mechanistic link for communicating circadian timing information from the central pacemaker to the ovary.

The aryl hydrocarbon receptor (AhR) is a period-aryl hydrocarbon receptor nuclear transporter-simple minded domain transcription factor that shares structural similarity with circadian clock genes and readily interacts with components of the molecular clock. Activation of AhR by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters behavioral circadian rhythms and represses the Period1 (Per1) gene in murine hematopoietic stem and progenitor cells. Per1 expression is driven by circadian locomotor activity cycles kaput-brain muscle ARNT-like (CLOCK-BMAL1)-dependent activation of Eboxes in the Per1 promoter. We hypothesized that the effects of AhR activation on the circadian clock are mediated by disruption of CLOCK-BMAL1 function and subsequent Per1 gene suppression. Effects of AhR activation on rhythmic Per1 transcripts were examined in livers of mice after treatment with the AhR agonist, TCDD; the molecular mechanisms of Per1 repression by AhR were determined in hepatoma cells using TCDD and beta-napthoflavone as AhR activators. This study reports, for the first time, that AhR activation by TCDD alters the Per1 rhythm in the mouse liver and that Per1 gene suppression depends upon the presence of AhR. Furthermore, AhR interaction with BMAL1 attenuates CLOCK-BMAL1 activity and decreases CLOCK binding at Ebox1 and Ebox3 in the Per1 promoter. Taken together, these data suggest that AhR activation represses Per1 through disrupting CLOCK-BMAL1 activity, producing dysregulation of rhythmic Per1 gene expression. These data define alteration of the Per1 rhythm as novel signaling events downstream of AhR activation. Downregulation of Per1 could contribute to metabolic disease, cancer, and other detrimental effects resulting from exposure to certain environmental pollutants.

Although it is generally accepted that the circadian clock provides a timing signal for the luteinizing hormone (LH) surge, mechanistic explanations of this phenomenon remain underexplored. It is known, for example, that circadian locomotor output cycles kaput (clock) mutant mice have severely dampened LH surges, but whether this phenotype derives from a loss of circadian rhythmicity in the suprachiasmatic nucleus (SCN) or altered circadian function in gonadotropin-releasing hormone (GnRH) neurons has not been resolved. GnRH neurons can be stimulated to cycle with a circadian period in vitro and disruption of that cycle disturbs secretion of the GnRH decapeptide. We show that both period-2 (PER2) and brain muscle Arnt-like-1 (BMAL1) proteins cycle with a circadian period in the GnRH population in vivo. PER2 and BMAL1 expression both oscillate with a 24-hour period, with PER2 peaking during the night and BMAL1 peaking during the day. The population, however, is not as homogeneous as other oscillatory tissues with only about 50% of the population sharing peak expression levels of BMAL1 at zeitgeber time 4 (ZT4) and PER2 at ZT16. Further, a light pulse that induced a phase delay in the activity rhythm of the GnRH-eGFP mice caused a similar delay in peak expression levels of BMAL1 and PER2. These studies provide direct evidence for a functional circadian clock in native GnRH neurons with a phase that closely follows that of the SCN.

Transcription factors belonging to the Per/Arnt/Sim (PAS) domain family are highly conserved and many are involved in circadian rhythm regulation. One member of this family, aryl hydrocarbon receptor (AhR), is an orphan receptor whose physiological role is unknown. Recent findings have led to the hypothesis that AhR has a role in circadian rhythm, which is the focus of the present investigation. First, time-of-day-dependent mRNA expression of AhR and its signaling target, cytochrome p4501A1 ( Cyp1a1), was determined in C57BL/6J mice by quantitative RT-PCR. Circadian expression of AhR and Cyp1a1 was observed both in the suprachiasmatic nucleus (SCN) and liver. Next, the circadian phenotype of mice lacking AhR (AhRKO) was investigated using behavioral monitoring. Intact AhRKO mice had robust circadian rhythmicity with a similar tau under constant conditions compared to wild-type mice, but a significant difference in tau was observed between genotypes in ovariectomized female mice. Time to reentrainment following 6-h advances or delays of the light/dark cycle was not significantly different between genotypes. However, mice exposed to the AhR agonist 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD; 1 µg/kg of body weight) displayed decreased phase shifts in response to light and had altered expression of Per1 and Bmal1. These results suggest that chronic activation of AhR may affect the ability of the circadian timekeeping system to adjust to alterations in environmental lighting by affecting canonical clock genes. Further studies are necessary to decipher the mechanism of how AhR agonists could disrupt light-induced phase shifts. If AhR does have a role in circadian rhythm, it may share redundant roles with other PAS domain proteins and/or the role of AhR may not be exhibited in the behavioral activity rhythm, but could be important elsewhere in the peripheral circadian system.

Gonadotropin-releasing hormone (GnRH) neurons in the hypothalamus play an important role in reproductive function. These cells originate in the nasal compartment and migrate into the basal forebrain in association with olfactory/vomeronasal nerves in embryonic life in rodents. Here, we studied the role of neuropilins and their ligands, semaphorins, in the development of the olfactory-GnRH system. We focused onNeuropilin-2knock-out (Npn-2−/−) mice, because they are known to display defasciculation of olfactory nerves and reduced fertility. We found a significant decrease in the number of GnRH neurons in the hypothalamus and a marked reduction in their gonadal size. We then observed an abnormal increase of GnRH neurons in the noses ofNpn-2−/−mice, indicating that these cells failed to migrate into the forebrain. However, because neuropilins and semaphorins are involved in events of neuronal migration in the brain, we asked whether the observed reduction in GnRH neurons was directly attributable to the action of these molecules. Using fluorescence-activated cell sorting and reverse transcription-PCR on mRNA derived from embryonic green fluorescent protein (GFP)–GnRH transgenic mice, we found expression of class 3 semaphorins and their receptors (neuropilin-1/2 and plexin-A1) in GnRH neurons. Furthermore, double-immunofluorescence experiments showed that migrating GnRH neurons, as well as associated olfactory fibers, express Npn-2 in the nasal region. We then used a line of immortalized GnRH neurons (GN11 cells) that display the same expression patterns for semaphorins and their receptors as GFP–GnRH cells and found that class 3 semaphorins and vascular endothelial growth factors modulate their migratory activity. These studies provide support for the direct involvement of neuropilins and their ligands in the establishment of the GnRH neuroendocrine system.

It is unclear whether pre-symptomatic Alzheimer’s disease (AD) causes circadian disruption or whether circadian disruption accelerates AD pathogenesis. In order to examine the sensitivity of learning and memory to circadian disruption, we altered normal lighting phases by an 8 h shortening of the dark period every 3 days (jet lag) in the APPSwDI NOS2–/– model of AD (AD-Tg) at a young age (4-5 months), when memory is not yet affected compared to non-transgenic (non-Tg) mice. Analysis of activity in 12-12 h lighting or constant darkness showed only minor differences between AD-Tg and non-Tg mice. Jet lag greatly reduced activity in both genotypes during the normal dark time. Learning on the Morris water maze was significantly impaired only in the AD-Tg mice exposed to jet lag. However, memory 3 days after training was impaired in both genotypes. Jet lag caused a decrease of glutathione (GSH) levels that tended to be more pronounced in AD-Tg than in non-Tg brains and an associated increase in NADH levels in both genotypes. Lower brain GSH levels after jet lag correlated with poor performance on the maze. These data indicate that the combination of the environmental stress of circadian disruption together with latent stress of the mutant amyloid and NOS2 knockout contributes to cognitive deficits that correlate with lower GSH levels.

A molecular device that measures time on a daily, or circadian, scale is a nearly ubiquitous feature of eukaryotic organisms. A core group of clock genes, whose coordinated function is required for this timekeeping, is expressed both in the central clock and within numerous peripheral organs. We examined expression of clock genes in the rat ovary. Transcripts for core oscillator elements (Arntl, Clock, Per1, Per2, and Cry1) were present in the ovary as indicated by quantitative real-time RT-PCR. Rhythmic expression patterns of Arntl and Per2 transcripts and protein products were out of phase with respect to the central oscillator and in complete antiphase to each other. Expression of Arntl was significantly elevated after the LH surge on the day of proestrus. Finally, hCG treatment induced cyclic expression of both Arntl and Per2 gene products in hypophysectomized, immature rats primed with eCG. Collectively, these data suggest that the core underpinnings of the transcriptional/translational feedback loop that drives circadian rhythmicity is present in the rat ovary. Furthermore, the study identifies LH as a potential regulator of circadian clock gene rhythms in the ovary.

Summary The cell cycle is another molecular timing device found within biological systems. Similar to the circadian Circadian clocks comprise a cyclic series of dynamic clock, the free-running period of mitosis is 24 hr in cellular states, characterized by the changing avail- many eukaryotic cells (Tyson et al., 2002). Cell replicaability of substrates that alter clock time when acti- tion is controlled by complex intracellular signaling pathvated. To determine whether circadian clocks, like the ways that integrate information about the extracellular cell cycle, exhibit regulation by key phosphorylation environment with intracellular cues. The events of the events, we examined endogenous kinase regulation cell cycle are predominantly controlled at the protein of timekeeping in the mammalian suprachiasmatic nu- level by a small number of heterodimeric protein kicleus (SCN). Short-term inhibition of PKG-II but not nases. These kinases, which fluctuate over the course PKG-I using antisense oligodeoxynucleotides de- of the cycle, exert control over multiple proteins that layed rhythms of electrical activity and Bmal1 mRNA. are involved in transcription, DNA replication, and cytoPhase resetting was rapid and dynamic; inhibition of kinesis by phosphorylation at specific regulatory sites, PKG-II forced repetition of the last 3.5 hr of the cycle. rendering certain proteins active while inactivating othChronic inhibition of PKG-II disrupted electrical activ- ers (Nurse, 2002; Tyson et al., 2002). ity rhythms and tonically increased Bmal1 mRNA. Knowledge of cell cycle regulation prompted our inPKG-II-like immunoreactivity was detected after coim- vestigation of critical kinases for regulating the circadian munoprecipitation with CLOCK, and CLOCK was clock. Clock elements are regulated by complex cellular phosphorylated in the presence of active PKG-II. PKG- processes that include posttranslational modifications

The suprachiasmatic nucleus (SCN) circadian clock exhibits a recurrent series of dynamic cellular states, characterized by the ability of exogenous signals to activate defined kinases that alter clock time. To explore potential relationships between kinase activation by exogenous signals and endogenous control mechanisms, we examined clock-controlled protein kinase G (PKG) regulation in the mammalian SCN. Signaling via the cGMP-PKG pathway is required for light- or glutamate (GLU)-induced phase advance in late night. Spontaneous cGMP-PKG activation occurred at the end of subjective night in free-running SCNin vitro. Phasing of the SCN rhythmin vitrowas delayed by ∼3 hr after treatment with guanylyl cyclase (GC) inhibitors, PKG inhibition, or antisense oligodeoxynucleotide (αODN) specific for PKG, but not PKA inhibitor or mismatched ODN. This sensitivity to GC-PKG inhibition was limited to the same 2 hr time window demarcated by clock-controlled activation of cGMP-PKG. Inhibition of the cGMP-PKG pathway at this time caused delays in the phasing of four endogenous rhythms: wheel-running activity, neuronal activity, cGMP, andPer1. Timing of the cGMP-PKG-necessary window in both rat and mouse depended on clock phase, established by the antecedent light/dark cycle rather than solar time. Because behavioral, neurophysiological, biochemical, and molecular rhythms showed the same temporal sensitivities and qualitative responses, we predict that clock-regulated GC-cGMP-PKG activation may provide a necessary cue as to clock state at the end of the nocturnal domain. Because sensitivity to phase advance by light-GLU-activated GC-cGMP-PKG occurs in juxtaposition, these signals may induce a premature shift to this PKG-necessary clock state.

The rotation of the earth on its axis creates the environment of a 24 h solar day, which organisms on earth have used to their evolutionary advantage by integrating this timing information into their genetic make-up in the form of a circadian clock. This intrinsic molecular clock is pivotal for maintenance of synchronized homeostasis between the individual organism and the external environment to allow coordinated rhythmic physiological and behavioral function. Aryl hydrocarbon receptor (AhR) is a master regulator of dioxin-mediated toxic effects, and is, therefore, critical in maintaining adaptive responses through regulating the expression of phase I/II drug metabolism enzymes. AhR expression is robustly rhythmic, and physiological cross-talk between AhR signaling and circadian rhythms has been established. Increasing evidence raises a compelling argument that disruption of endogenous circadian rhythms contributes to the development of disease, including sleep disorders, metabolic disorders and cancers. Similarly, exposure to environmental pollutants through air, water and food, is increasingly cited as contributory to these same problems. Thus, a better understanding of interactions between AhR signaling and the circadian clock regulatory network can provide critical new insights into environmentally regulated disease processes. This review highlights recent advances in the understanding of the reciprocal interactions between dioxin-mediated AhR signaling and the circadian clock including how these pathways relate to health and disease, with emphasis on the control of metabolic function.

We investigated a role for cAMP/protein kinase A (PKA) in light/glutamate (GLU)-stimulated state changes of the mammalian circadian clock in the suprachiasmatic nucleus (SCN). Nocturnal GLU treatment elevated [cAMP]; however, agonists of cAMP/PKA did not mimic the effects of light/GLU. Coincident activation of cAMP/PKA enhanced GLU-stimulated state changes in early night but blocked light/GLU-induced state changes in the late night, whereas inhibition of cAMP/PKA reversed these effects. These responses are distinct from those mediated by mitogen-activated protein kinase (MAPK). MAPK inhibitors attenuated both GLU-induced state changes. Although GLU inducedmPer1mRNA in both early and late night, inhibition of PKA blocked this event only in early night, suggesting that cellular mechanisms regulatingmPer1are gated by the suprachiasmatic circadian clock. These data support a diametric gating role for cAMP/PKA in light/GLU-induced SCN state changes: cAMP/PKA promotes the effects of light/GLU in early night, but opposes them in late night.

Daily “about-24-hour” cycles of physiology and behavior, referred to as circadian rhythms, are generated by molecular “clocks” that are present in virtually all cells of the body. Individual clocks are synchronized to each other and to solar time by a molecular “master clock” in the suprachiasmatic nuclei (SCN) of the anterior hypothalamus. The SCN detects environmental light and uses this time-of-day information to coordinate clocks throughout the body. Thus, the SCN directs a systemic circadian clock network to drive whole-animal regulation of 24 h rhythmicity. The importance of homeostatic regulation of systemic circadian processes to health maintenance is only beginning to be understood. Clock disruption brought on by shift work in human populations predisposes the affected people to numerous health risks, including increased risk of breast and prostate cancers, increased risk of cardiovascular disease, and increased incidence of metabolic disorders, including type 2 diabetes. This chapter describes the current state of knowledge concerning systemic regulation of the circadian clock network by exploring: (1) the molecular underpinnings of cellular clocks that use transcriptional and translational feedback networks to generate oscillations in individual cells, (2) the generation and entrainment of circadian rhythmicity by the SCN, and (3) the transmission of timekeeping information to the output network.

Anatomical studies have suggested that the germinal disc (GD) region (GDR; GD plus overlying granulosa cells) is the growth center of the avian preovulatory follicle. The objective of this study was to characterize the physiology of the GDR by comparing the functions of two morphologically distinct populations of granulosa cells. The three markers of the physiology of individual granulosa cells examined were 1) proliferation, 2) production of plasminogen activator (PA), and 3) production of progesterone. The effect of LH on each of these functions was also evaluated. Sections 8 mm in diameter were obtained from granulosa cells associated with the GD (GD granulosa cells) and from granulosa cells on the layer distal to the GD (nonGD granulosa cells) from the five largest preovulatory follicles (F5-F1, F1 designated the largest) 12-14 h (before the LH surge) or 2 h (after the LH surge) before ovulation. Proliferation was measured using [3H]thymidine incorporation. PA activity was measured using the chromogenic substrate S-2251. Progesterone was measured by RIA. Incorporation of [3H]thymidine was very high in GD and nonGD granulosa cells from F5 and F4 follicles and decreased dramatically as the follicle progressed through the hierarchy, but remained significantly higher in GD granulosa cells compared to nonGD granulosa cells at all stages of development examined (F5-F1). Exposure of follicles to LH in vivo inhibited [3H]thymidine incorporation by GD granulosa cells in all follicles except the F5. In contrast, in vivo exposure to LH had no effect on [3H]thymidine incorporation by nonGD granulosa cells. PA production by GD granulosa cells was high throughout the stages of maturation studied (F5-F1), whereas PA production by nonGD granulosa cells decreased as follicles matured from F5 to F1. Interestingly, LH stimulated PA production by F5 GD granulosa cells, had no effect on PA production by F3 GD granulosa cells, and inhibited PA production by F1 GD granulosa cells. In contrast, LH inhibited PA production by nonGD granulosa cells in F3 and F1 follicles. Progesterone production by GD granulosa cells was low in F3 and F1 follicles. Progesterone production by nonGD granulosa cells increased as the follicle matured from the F3 to F1 stage and was stimulated significantly by LH. These data indicate that physiological differences in granulosa cell function are dependent upon the location of granulosa cells relative to the GD. GD granulosa cells are less mature, proliferate more rapidly, and produce more PA than nonGD granulosa cells, which produce more progesterone and less PA. Differences in granulosa cell function may be due to the influence of the GD, providing physiological evidence that the GDR may be the growth center of the follicle.

Beta-naphthoflavone (BNF, DB06732) is an agonist of aryl hydrocarbon receptor (AhR) and a putative chemotherapeutic agent that has antitumor activity against mammary carcinomas in vivo. However, the mechanism by which BNF exerts this antitumor effect remains unclear. Thus, we explored mechanisms of BNF's antitumor effects in human breast cancer cells. This study showed that BNF suppressed cell proliferation and induced cell cycle arrest in the G0/G1 phase with downregulation of cyclin D1/D3 and CDK4 and upregulation of p21(Cip1/Waf1), leading to a senescence-like phenotype in estrogen receptor (ER)-positive MCF-7 cells, but not in ER-negative MDA-MB-231 cells. In addition, BNF inhibited PI3K/AKT signaling, and the PI3K inhibitor, LY294,002, exhibited the same inhibitory effects on cyclinD1/D3, CDK4 and the cell cycle as BNF. Interestingly, BNF activated mitogen-activated protein kinase-extracellular signal-regulated kinase (MAPK-ERK) signaling, and more notably, MEK inhibitor PD98059 significantly blocked the BNF-induced cell cycle arrest and upregulation of p21(Cip1/Waf1). Furthermore, specific ERα and AhR siRNA studies indicate that ERα is required in BNF-induced p21(Cip1/Waf1) expression, and BNF-mediated cell cycle arrest and modulation of AKT and ERK signaling is AhR-dependent. Taken together, AhR-dependent inhibition of the PI3K/AKT pathway, activation of MAPK/ERK and modulation of ERα is a novel mechanism underlying BNF-mediated antitumor effects in breast cancer, which may represent a promising strategy to be exploited in future clinical trials.

Plasminogen activator (PA) is hypothesized to be important in the remodeling of the extracellular matrix during follicular growth. The granulosa layer produces high amounts of PA in response to a stimulatory factor, produced by the theca layer, that is inhibited by LH. To determine the site and mechanism by which LH inhibits PA production, we asked 1) whether LH acts on the granulosa layer and/or the theca layer to inhibit PA production by the largest preovulatory follicle (F1), and 2) whether LH affects PA production by acting at the mRNA or protein level. Sections (10 mm in diameter) of granulosa layers obtained from the F1 follicle before (14 h before ovulation) or after (2 h before ovulation) the LH surge were incubated (24 h at 37 degrees C) in theca-conditioned medium; this medium had been prepared by incubation of 10-mm-diameter sections of theca layers, obtained before (14 h before ovulation) or after (2 h before ovulation) the LH surge, in Dulbecco's Modified Eagle's Medium for 24 h at 37 degrees C. PA production in culture medium was measured with use of the chromogenic substrate S-2251. PA production was high when granulosa layers obtained before the LH surge were incubated in theca-conditioned medium obtained before the LH surge; it was also high when granulosa layers obtained before the LH surge were incubated in theca-conditioned medium obtained after the LH surge. PA production was low when granulosa layers obtained after the LH surge were incubated in theca-conditioned medium obtained before the LH surge, and was also low when granulosa layers obtained after the LH surge were incubated in theca-conditioned medium obtained after the LH surge. Northern and Western blots and activity assays performed on granulosa layer homogenates indicated that PA mRNA, protein, and activity were high before the LH surge and low after the LH surge. Production of the stimulatory factor by the theca layer is apparently unaffected by LH. After exposure to LH, the granulosa layer is no longer capable of producing PA, even in the presence of the theca-derived stimulatory factor. We conclude that the granulosa layer is the site of mRNA and/or protein regulation of PA production by LH.

Microscopic analysis of ovarian follicles in the domestic hen has revealed differences in the cellular structure of granulosa cells that are dependent upon the location of granulosa cells relative to the germinal disc, which contains the female gamete. These differences appear as a morphological gradient, which implies variations in granulosa cell function. This observation prompted us to hypothesize that the germinal disc region (GDR) of the avian preovulatory follicle participates in the process of follicular growth by producing factors that act in a paracrine manner to stimulate proliferation of and inhibit steroidogenesis in the granulosa layer, establishing a gradient in the morphology and physiology of the granulosa layer. To test our hypothesis, we asked two questions : 1) Are physiological gradients of proliferation and steroidogenesis present within the granulosa layer of a preovulatory follicle ? 2) Does the GDR secrete factors that affect granulosa cell proliferation and/or steroidogenesis ? Incorporation of 3 H-thymidine was used as a measure of proliferation, and production of progesterone was used as a measure of steroidogenesis. In the first experiment, 8-mm-diameter sections were obtained from three morphologically distinct regions of the granulosa monolayer : 1) the GDR, 2) granulosa cells distal to the GDR (distal granulosa), and 3) granulosa cells midway between the GDR and distal granulosa cells (proximal granulosa cells). The GDR incorporated the most 3 H-thymidine and produced the least progesterone. Distal granulosa cells incorporated the least 3 H-thymidine and produced the most progesterone. Proximal granulosa cells incorporated an intermediate amount of 3 H-thymidine and produced an intermediate amount of progesterone. To answer the second question, conditioned medium was prepared from GDRs and distal granulosa cells (control) obtained from the F1 (largest preovulatory follicle) and F3 (the third-largest preovulatory follicle) follicles. Sections (8-mm in diameter) of the distal granulosa layer (F3 for 3 H-thymidine incorporation, F1 for progesterone production) were incubated in GDR-conditioned medium or granulosa cell-conditioned medium to determine whether factors secreted into the medium by the GDR and distal granulosa cells affect granulosa cell proliferation and/or steroidogenesis. Certain samples of GDR-conditioned medium and granulosa cell-conditioned medium were boiled, protease-treated or charcoal-stripped. F3 and F1 GDRs produced heat- and protease-sensitive factors that promoted proliferation and inhibited progesterone production by granulosa cells. These data indicate that diametrically opposed gradients of proliferation and steroidogenesis are present within the granulosa layer of an individual preovulatory follicle. Furthermore, the GDR produces proliferation-stimulating and steroidogenesis-inhibiting factors that may act in an autocrine or paracrine manner to influence proliferation and steroidogenesis in granulosa cells.

Light-stimulated adjustment of the circadian clock is an important adaptive physiological response that allows maintenance of behavioral synchrony with solar time. Our previous studies indicate that the aryl hydrocarbon receptor (AhR) agonist 2,3,7,8- tetrachlorodibenzo-p-dioxin attenuates light-induced phase resetting in early night. However, the mechanism of inhibition remains unclear. In this study, we showed that another potent AhR agonist-β-naphthoflavone (BNF)-significantly decreased light-induced phase shifts in wild-type (WT) mice, whereas AhR knockout mice had an enhanced response to light that was unaffected by BNF. Mechanistically, BNF blocked light induction of the Per1 transcript in suprachiasmatic nucleus and liver in WT mice, and BNF blocked forskolin (FSK)-induced Per1 transcripts in Hepa-1c1c7 (c7) cells. An E-box decoy did not affect BNF inhibition of FSK-induced Per1 transcripts in c7 cells. cAMP-response element (CRE)-dependent induction of Per1 promoter activity in response to FSK in combination with phorbol 12-tetradecanoate 13-acetate was suppressed in cells that expressed high levels of AhR (c7) compared with cells lacking functional AhR activity (c12). In addition, the inhibitory effect of BNF on FSK-induced Per1 was dependent on phosphorylation of JNK. Together, these results suggest that AhR activation inhibits light-induced phase resetting through the activation of JNK, negative regulation of CREs in the Per1 promoter, and suppression of Per1.

The stages of follicular maturation of a preovulatory follicle in the hen can be divided into an extended proliferative phase (prior to LH surge) and a brief ovulatory phase (after LH surge). Previous studies suggest involvement of plasminogen activator (PA) in both the proliferative and ovulatory phases. The goals of the present study were 1) to determine whether PA production by granulosa and theca is dependent upon interaction of the two cell layers; 2) to investigate whether the structural difference of the stigma (site of follicular rupture) and nonstigma regions of the theca layer affect PA production; 3) to determine whether there is a change in the ability of the granulosa layer and stigma or nonstigma regions of the theca layer to produce PA as the follicle makes the transition from the proliferative to the ovulatory phase; and 4) to characterize the type(s) of PA produced by the hen follicle. Equal proportions of the granulosa layer (10-mm diameter) and stigma or nonstigma regions of the theca layer (10 mg) obtained from the F1 preovulatory follicle 8 h before ovulation (before LH surge) or 2 h before ovulation (after LH surge) were incubated alone or in combination for 24 h. PA was measured in tissue homogenates and medium by use of the chromogenic substrate S-2251. The granulosa layer or stigma or nonstigma regions of the theca layer incubated alone and obtained either 8 h or 2 h before ovulation had very low amounts of PA activity in the medium and tissue homogenates.(ABSTRACT TRUNCATED AT 250 WORDS)

Progesterone conjugated to bovine serum albumin (BSA) was used as a probe to study sex differences and the effects of hormonal status on binding of progesterone to crude synaptosomal membrane preparations (P2) derived from the mediobasal hypothalamic-anterior hypothalamic-preoptic area or the corpus striatum. Binding of 125I-labeled BSA linked to progesterone at the 11 position of the steroid (P-11-BSA) was decreased by competition with unlabeled P-11-BSA or P-3-BSA (in which progesterone is bound to BSA at the 3 position). P-3-BSA displayed higher affinity than P-11-BSA. Hypothalamic and striatal preparations from adult females show high specific binding (60-80%) to the progesterone-BSA conjugate. Specific binding was reduced more than 80% 14 days after ovariectomy. Estrogen treatment (10 micrograms per rat for 4 days) of 14-day ovariectomized rats restored specific binding to levels equivalent to intact females. In contrast, adult males displayed drastically reduced or no specific binding in either tissue. No specific binding was detected after orchidectomy. Estrogen treatment of orchidectomized animals induced specific binding sites similar to those in intact females. Additionally, an affinity probe was developed by linking primary amines on the P-3-BSA conjugate to agarose activated aldehydes in an AminoLink column. A digitoxin-solubilized fraction from female rat P2 cerebellum preparations yielded a single major band after affinity purification with an estimated molecular mass of 40-50 kDa in an SDS/PAGE system after silver stain. These results show a reversible sex difference in the specific binding of progesterone to synaptosomal membrane sites in the central nervous system of male and female rats which is dependent on estrogen.

Activation of the aryl hydrocarbon receptor (AhR) by the highly toxic, prototypical ligand, 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD) or other dioxin-like compounds compromises ovarian function by altering follicle maturation and steroid synthesis. Although alteration of transcription after nuclear translocation and heterodimerization of AhR with its binding partner, aryl hydrocarbon nuclear transporter (ARNT), is often cited as a primary mechanism for mediating the toxic effects of dioxins, recent evidence indicates that crosstalk between AhR and several other signaling pathways also occurs. Like the circadian clock genes, AhR is a member of the basic helix-loop-helix, Per-ARNT-SIM (bHLH-PAS) domain family of proteins. Thus, these studies tested the hypothesis that TCDD can act to alter circadian clock regulation in the ovary. Adult female c57bl6/J mice entrained to a typical 12 h light/12 h dark cycle were exposed to a single 1 µg/kg dose of TCDD by gavage. Six days after exposure, animals were released into constant darkness and ovaries were collected every 4 h over a 24 h period. Quantitative real-time PCR and immunoblot analysis demonstrated that TCDD exposure alters expression of the canonical clock genes, Bmal1 and Per2 in the ovary. AhR transcript and protein, which displayed a circadian pattern of expression in the ovaries of control mice, were also altered after TCDD treatment. Immunohistochemistry studies revealed co-localization of AhR with BMAL1 in various ovarian cell types. Furthermore, co-immunoprecipitation demonstrated time-of-day dependent interactions of AhR with BMAL1 that were enhanced after TCDD treatment. Collectively these studies suggest that crosstalk between classical AhR signaling and the molecular circadian clockworks may be responsible for altered ovarian function after TCDD exposure.

Anesthetics are widely used in experiments investigating neurotoxicity and neuroprotection; however, these agents are known to interfere with the outcome of these experiments. The purpose of this overview is to review these effects and suggest methods for minimizing unintended consequences on experimental outcomes. Information on the neuroprotective and neurotoxic effects of isoflurane, dexmedetomidine, propofol, ketamine, barbiturates, halothane, xenon, carbon dioxide, and nitrous oxide is summarized. The pertinent cell signaling pathways of these agents are discussed. Methods of humane animal euthanasia without anesthetics are considered. Most anesthetics alter the processes of neuronal survival and death. When designing survival surgeries, sham controls subjected to anesthesia but not the surgical intervention should be compared with controls subjected to neither anesthesia nor surgery. Additional controls could include using an anesthetic with a different mechanism of action from the primary anesthetic used. Because the effects of anesthetics lessen with time after surgery, survival surgeries should include later time points until at least 7 d after the procedure. Humane methods of animal euthanasia that do not require anesthetics exist and should be used whenever appropriate.

A comprehensive understanding of neuroprotective pathways is essential to progress in the battle against numerous neurodegenerative conditions. The hypothalamic suprachiasmatic nucleus (SCN) is endogenously resistant to glutamate (Glu) excitotoxicity in vivo. This study was designed to determine whether immortalized SCN neurons (SCN2.2 cells) retain this characteristic. We first established that SCN2.2 cells retained the ability to respond to Glu. SCN2.2 cells expressed N-methyl-d-aspartate (NMDA) receptor subtypes NR1 and NR2A/2B, suggesting the presence of functional receptors. mRNA for the NMDA receptor subunits NR2A and NR2B were higher in the SCN2.2 than in the control hypothalamic neurons (GT1-7). Specific NMDA receptor antagonists (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate and d-(–)-2-amino-5-phosphonovaleric acid blocked Glu-induced activation of gene expression. SCN2.2 cells were resistant to Glu excitotoxicity compared with GT1-7 neurons as assessed with a mitochondrial function assay, cell death by trypan blue exclusion and apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labeling. SCN2.2 resistance to Glu excitoxicity was retained in the presence of the broad spectrum Glu transport inhibitor, l-trans-pyrrolidine-2,4 dicarboxylate, excluding glial Glu uptake as a major neuroprotective mechanism. Collectively, these observations demonstrate endogenous neuroprotection in SCN2.2 cells; this cell line is resistant to excitotoxicity under conditions that are toxic to other immortalized cell lines. Thus, the SCN2.2 cell line may provide insights into the molecular mechanisms that confer endogenous neuroprotection in the SCN.

Time-of-day-dependent variation in neuronal ischemia is well documented. Whether this results from changes in time-of-day variation in susceptibility or from other causative factors remains unclear. We hypothesize that hippocampal cells exhibit variation in activation of cell death predictive markers in response to ischemia induced at different times-of-day. Changes in hippocampal circadian clock gene rhythmicity may also be associated with ischemia. Transient global ischemia was induced in rats at three times of day and animals were sacrificed 24 h later. Hippocampal caspase-3, -8 and -9 transcripts and active proteins and calbindin protein were measured in the CA1 region of the hippocampus. In a second study, 24-h rhythms of circadian regulatory transcripts were determined in hippocampus after global ischemia. Caspase-3, -8 and -9 transcripts and active protein levels were increased substantially when ischemia occurred in early night (ZT14); smaller changes were observed in late night (ZT20, or day ZT6). Calbindin levels decreased following ischemia, especially at ZT14. Ischemia shifted the rhythm of the Per1 transcript; peak expression occurred 6 h earlier following ischemia. Rhythms of Cry1 and Bmal1 were not altered. Greater induction of caspases and decline of calbindin when ischemia was performed at ZT14 than at ZT20 or ZT6 support the concept of increased hippocampal susceptibility to ischemia at ZT14. Alteration of the Per1 transcript suggests a potential role for the circadian clock in this process. Notably, ZT14 represents the beginning of the rats' nocturnal period of activity, corresponding to the time when humans experience the greatest neuronal ischemic damage from stroke.

The aryl hydrocarbon receptor (AhR) mediates adverse effects of dioxins, but its physiological role remains ambiguous. The similarity between AhR and canonical circadian clock genes suggests potential involvement of AhR in regulation of circadian timing. Photoproducts of tryptophan (TRP), including 6-formylindolo [3,2-b]carbazole (FICZ), have high affinity for AhR and are postulated as endogenous ligands. Although TRP photoproducts activate AhR signaling in vitro, their effects in vivo have not been investigated in mammals. Because TRP photoproducts may act as transducers of light, we examined their effects on the circadian clock. Intraperitoneal injection of TRP photoproducts or FICZ to C57BL/6J mice dose dependently induced AhR downstream targets, cytochrome P4501A1 (CYP1A1) and cytochrome P4501B1 mRNA expression, in liver. c-fos mRNA, a commonly used marker for light responses, was also induced with FICZ, and all responses were AhR dependent. A rat-immortalized suprachiasmatic nucleus (SCN) cell line, SCN 2.2, was used to examine the direct effect of TRP photoproducts on the molecular clock. Both TRP photoproducts and FICZ-increased CYP1A1 expression and prolonged FICZ incubation altered the circadian expression of clock genes (Per1, Cry1, and Cry2) in SCN 2.2 cells. Furthermore, FICZ inhibited glutamate-induced phase shifting of the mouse SCN electrical activity rhythm. Circadian light entrainment is critical for adjustment of the endogenous rhythm to environmental light cycle. Our results reveal a potential for TRP photoproducts to modulate light-dependent regulation of circadian rhythm through triggering of AhR signaling. This may lead to further understanding of toxicity of dioxins and the role of AhR in circadian rhythmicity.

A molecular device that measures time on a daily, or circadian, scale is a nearly ubiquitous feature of eukaryotic organisms. A core group of clock genes, whose coordinated function is required for this timekeeping, is expressed both in the central clock and within numerous peripheral organs. We examined expression of clock genes in the rat ovary. Transcripts for core oscillator elements (Arntl, Clock, Per1, Per2, and Cry1) were present in the ovary as indicated by quantitative real-time RT-PCR. Rhythmic expression patterns of Arntl and Per2 transcripts and protein products were out of phase with respect to the central oscillator and in complete antiphase to each other. Expression of Arntl was significantly elevated after the LH surge on the day of proestrus. Finally, hCG treatment induced cyclic expression of both Arntl and Per2 gene products in hypophysectomized, immature rats primed with eCG. Collectively, these data suggest that the core underpinnings of the transcriptional/translational feedback loop that drives circadian rhythmicity is present in the rat ovary. Furthermore, the study identifies LH as a potential regulator of circadian clock gene rhythms in the ovary.

The recent identification of specific genes responsible for the generation of endogenous circadian rhythmicity in the suprachiasmatic nucleus presents a new level of investigation into endogenous rhythmicity and mechanisms of synchronization of this circadian clock with the environmental light?dark cycle. This article describes techniques that employ antisense and decoy oligodeoxynucleotides (ODN) to determine the roles of specific molecular substrates both in endogenous rhythmicity and in regulating the effects of light on the mammalian circadian clock. Application of antisense ODN technology has revealed a role for timeless (Tim) in the core clock mechanism and established that induction of period1 (Per1) is required for light responsiveness. Likewise, a decoy ODN designed to sequester activated CREB protein definitively demonstrated a requirement for CRE-mediated transcription in light signaling. Experiments designed with these molecular tools offer new insights on the interaction of cellular processes and signaling with the molecular clockworks.

The recent identification of specific genes responsible for the generation of endogenous circadian rhythmicity in the suprachiasmatic nucleus presents a new level of investigation into endogenous rhythmicity and mechanisms of synchronization of this circadian clock with the environmental light?dark cycle. This article describes techniques that employ antisense and decoy oligodeoxynucleotides (ODN) to determine the roles of specific molecular substrates both in endogenous rhythmicity and in regulating the effects of light on the mammalian circadian clock. Application of antisense ODN technology has revealed a role for timeless (Tim) in the core clock mechanism and established that induction of period1 (Per1) is required for light responsiveness. Likewise, a decoy ODN designed to sequester activated CREB protein definitively demonstrated a requirement for CRE-mediated transcription in light signaling. Experiments designed with these molecular tools offer new insights on the interaction of cellular processes and signaling with the molecular clockworks.

Despite a central circadian role in Drosophila for the transcriptional regulator Timeless ( dTim ), the relevance of mammalian Timeless ( mTim ) remains equivocal. Conditional knockdown of mTim protein expression in the rat suprachiasmatic nucleus (SCN) disrupted SCN neuronal activity rhythms, and altered levels of known core clock elements. Full-length mTim protein (mTIM-fl) exhibited a 24-hour oscillation, where as a truncated isoform (mTIM-s) was constitutively expressed. mTIM-fl associated with the mammalian clock Period proteins (mPERs) in oscillating SCN cells. These data suggest that mTim is required for rhythmicity and is a functional homolog of dTim on the negative-feedback arm of the mammalian molecular clockwork.

Light is a prominent stimulus that synchronizes endogenous circadian rhythmicity to environmental light/dark cycles. Nocturnal light elevates mRNA of the Period1 (Per1) gene and induces long term state changes, expressed as phase shifts of circadian rhythms. The cellular mechanism for Per1 elevation and light-induced phase advance in the suprachiasmatic nucleus (SCN), a process initiated primarily by glutamatergic neurotransmission from the retinohypothalamic tract, was examined. Glutamate (GLU)-induced phase advances in the rat SCN were blocked by antisense oligodeoxynucleotide (ODN) against Per1 and Ca(2+)/cAMP response element (CRE)-decoy ODN. CRE-decoy ODN also blocked light-induced phase advances in vivo. Furthermore, the CRE-decoy blocked GLU-induced accumulation of Per1 mRNA. Thus, Ca(2+)/cAMP response element-binding protein (CREB) and Per1 are integral components of the pathway transducing light-stimulated GLU neurotransmission into phase advance of the circadian clock.