June 15 Poster Session

  P1

Time-of-day specific changes in metabolic detoxification and insecticide resistance in the malaria mosquito

Anopheles gambiaeGiles Duffield1, Nathaniel Balmert1, Samuel Rund1, John Ghazi1,

Peng Zhou5

1Biological Sciences, University of Notre Dame, Notre Dame, IN, UNITED STATES5University of notre dame, notre dame, IN, UNITED STATES

Mosquitoes exhibit ~24 hour rhythms in physiology and behavior, regulated by the cooperative action of an endogenous circadian clock and the environmental LD cycle. Here, we characterize diel (observed under LD conditions) time-of-day changes in metabolic detoxification and resistance to insecticide challenge in Anopheles gambiae mosquitoes. A better understanding of mosquito chronobiology will yield insights into developing novel control strategies for this important disease vector. We have previously identified >2000 rhythmically expressed An. gambiae genes (Rund et al., 2011, PNAS 108:E421-E430; Rund et al., 2013, BMC Genomics 14:218). These include metabolic detoxification enzymes peaking at various times throughout the day. Especially interesting was the identification of rhythmic genes encoding enzymes capable of pyrethroid and/or DDT metabolism (CYP6M2, CYP6P3, CYP6Z1, and GSTE2). We hypothesized that these temporal changes in gene expression would confer time-of-day specific changes in metabolic detoxification and responses to insecticide challenge. An. gambiae mosquitoes (adult female Pimperena and Mali-NIH strains) were tested by gene expression analysis for diel rhythms in key genes associated with insecticidal resistance. Biochemical assays for total GST, esterase, and oxidase enzymatic activities were undertaken on time-specific mosquito head and body protein lysates. To determine for rhythmic susceptibility to insecticides by survivorship, mosquitoes were exposed to DDT or deltamethrin across the diel cycle. We report the occurrence of temporal changes in GST activity in samples extracted from the body and head with a single peak at late-night to dawn, but no rhythms were detected in oxidase or esterase activity. The Pimperena strain was found to be resistant to insecticidal challenge, and subsequent genomic analysis revealed the presence of the resistance-conferring kdr mutation. We observed diel rhythmicity in key insecticide detoxification genes in the Mali-NIH strain, with peak phases as previously reported in the Pimperena strain. The insecticide sensitive Mali-NIH strain mosquitoes exhibited a diel rhythm in survivorship to DDT exposure and a bimodal variation to deltamethrin challenge. Our results demonstrate rhythms in detoxification in An. gambiae mosquitoes; this knowledge could be incorporated into mosquito control and

experimental design strategies, and contributes to our basic understanding of mosquito biology. Supported by the Eck Institute for Global Health.

 

  P2

Daily rhythms in antennal protein and olfactory sensitivity in the malaria mosquito Anopheles gambiae

Nicolle Bonar1, Samuel Rund1, Matthew Champion3, John Ghazi1, Cameron Houk1, Matthew Leming1, Zainulabeuddin Syed1,

Giles Duffield8

1Department of Biological Sciences , University of Notre Dame, Notre Dame, IN, UNITED STATES3Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN, UNITED STATES

8Biological Sciences, University of Notre Dame, Notre Dame, IN, UNITED STATES

We recently characterized 24-hr daily rhythmic patterns of gene expression in Anopheles gambiae mosquitoes (Rund et al., 2011, PNAS 108:E421-E430; Rund et al., 2013, BMC Genomics 14:218) . These include numerous odorant binding proteins (OBPs), soluble odorant carrying proteins enriched in olfactory organs. Here we demonstrate that multiple rhythmically expressed genes including OBPs and takeout proteins, involved in regulating blood feeding behavior, have corresponding rhythmic protein levels as measured by quantitative proteomics. This includes AgamOBP1, previously shown as important to An. gambiae odorant sensing. Further, electrophysiological investigations demonstrate time-of-day specific differences in olfactory sensitivity of antennae to major host-derived odorants. The pre-dusk/dusk peaks in OBPs and takeout gene expression correspond with peak protein abundance at night, and in turn coincide with the time of increased olfactory sensitivity to odorants requiring OBPs and times of increased blood-feeding behavior. This suggests an important role for OBPs in modulating temporal changes in odorant sensitivity, enabling the olfactory system to coordinate with the circadian niche of An. gambiae. Supported by the Eck Institute for Global Health, Center for Rare and Neglected Disease, and the Indiana CTSI. 

  P3

Light-regulated blood-feeding and flight activity behavior and a light phase response curve for the Anopheles

gambiae malaria mosquito Aaron Sheppard1, Gary George1, Erin Clark1, Hannah Yu1,

Samuel Rund1, Giles Duffield1

1Biological Sciences, University of Notre Dame, Notre Dame, IN, UNITED STATES

Biting behaviors in anopheline mosquitoes are time-of-day specific, with a greater abundance of biting occurring during the dark phase of their photoperiod (Rund et al., 2013, Scientific Reports 3: 2494). We investigated whether a single light pulse administered during the early dark phase of the LD cycle would inhibit biting behavior. An. gambiae locomotion/flight activity has a distinct circadian rhythm, characterized by nocturnal activity bouts. We investigated how precisely timed light pulses delivered throughout the circadian cycle can shift the flight activity rhythm, leading to the synthesis of an An. gambiae Phase Response Curve (PRC). Mosquitoes were maintained on a 12:12 LD cycle, including 1 hr dawn/dusk simulations. To investigate biting inhibition, two incipient An. gambiae species (S and M molecular forms) were treated with white light (10 min, 150-800 lux) at the onset of dark phase of the LD cycle (ZT12; end of dusk), and the percentage taking a blood meal was recorded every 2 hr up to 8 hr. To produce an anchored PRC, S-form mosquitoes received a single 30 min pulse of white light (300 lux) at various times during the immediate 24 hr transitioning from LD to DD. The pulse significantly reduced biting tendency in the S-form mosquito for 2 hr after administration (at 0.20 hr and 2 hr), with variable responses observed at 4 hr, and no differences detected at 6 and 8 hr (one factor ANOVA, p < 0.05). Conversely, M-form mosquitoes were unresponsive to the light treatment, i.e. their biting tendency remained high and did not change (n.s.). For the PRC analysis, as seen in most other examined species, e.g. Drosophila, mouse and human, An. gambiae mosquitoes demonstrated distinct delays and advances in circadian phase when light was presented during the early and late subjective night, respectively. These data reveal a strain-specific effect of acute light treatment on biting behavior that is both immediate and sustained (up to 2 hr but not 6 hr). The An. gambaie PRC is qualitatively similar to several model insect and vertebrate organisms. At present, insecticidal treated bed-nets designed to prevent mosquito-human contact and kill mosquitoes, are heavily relied upon to prevent malaria transmission; as mosquitoes and malaria parasites are becoming increasingly resistant to insecticidal and drug treatments, respectively, there is a necessity for the ongoing development of novel and innovative control strategies. The inhibitory and phase shifting effects of light may prove to be an effective tool in assisting with these strategies.

  P4

Analysis of Locomotor Activity Rhythms in a Population of Free-Behaving C. elegans

Ari Winbush1, Alexander van der Linden1

1Biology, University of Nevada, Reno, NV, UNITED STATES

Locomotor activity is used extensively as a behavioral output to study the underpinnings of the circadian clock in different animals. Our previous work has established the nematode Caenorhabditis elegans (C. elegans) as a new attractive model to study temperature control of the circadian clock. C. elegans is a well-established system to study temperature responses; it has a well-mapped brain circuitry that senses small changes in temperature, and exhibits circadian rhythms of locomotor activity entrained by low-amplitude temperature cycles as well as light [1, 2]. To record these circadian rhythms of locomotor activity, long-term recording imaging systems have been previously developed [1, 2]. However, these imaging systems are limited to monitoring locomotion of individual adult animals in a liquid or on a standard agar medium, and individual animals often show high variability in circadian parameters, and a non-robustness of rhythms. We and others have hypothesized that a populational approach is more adequate for the study of circadian rhythmicity in C. elegans, and for screening of circadian mutants. We have developed a system to record locomotory activity rhythms of a

population of C. elegans on a standard agar plate environment. To this end, we utilized the previously developed multi-worm tracker (MWT) [3] to establish a real-time tracking system able to record locomotor activity in a small population of freely moving animals over extended periods of time. We describe the setup here and present evidence that C. elegans locomotion can be modified by light/dark and temperature cycles in a circadian fashion. In summary, our new setup will allow for long-term, robust measurement of circadian rhythmicity in C. elegans locomotor behavior. [1] Saigusa et al 2002, Current Biology; [2] Simonetta et al 2011, PLoS One; [3] Swierczek et al 2011, Nature Methods.

  P5

Physical and psychological stress as potent synchronizers of mouse peripheral circadian clocks

Yu Tahara1, Takuya Shiraishi2, Yosuke Kikuchi2, Atsushi Haraguchi2, Daisuke Kuriki2, Shigenobu Shibata6

1Laboratory of Physiology and Pharmacology, School of Advanced Science and Engineering, Waseda University, Waseda University, Shinjuku-ku, Tokyo, JAPAN

2Laboratory of Physiology and Pharmacology, School of Advanced Science and Engineering, Waseda University, Tokyo, JAPAN

6School of Advanced Scinces and Enginnering, Waseda university, Tokyo, JAPAN

We investigated the effect of restraint stress on mouse peripheral circadian clocks by the in vivo monitoring of peripheral PER2::LUC bioluminescence (Tahara et al., 2012). Restraint stress for 2 h each day at the same time strongly altered the phase of the PER2::LUC rhythm. This changed depended on the time-of-day and/or the number of days of restraint stress. Three days of restraint stress was found to induce 3–5 h of phase-advance of PER2::LUC rhythms in all recorded organs. RT-PCR analysis showed that other clock genes were also phase-shifted by the restraint stress in the same organs. Interestingly, stress exposure at the beginning of the light phase (ZT 0-2) caused disruption of the PER2::LUC rhythm in the kidney and the desynchronization of phases among three organs. We confirmed that these effects were not associated with the HPA axis or sleep deprivation. Activation of sympathetic pathways or the induction of oxidative stress can also cause similar phase-shifts of peripheral clocks. In addition, acute expression changes of many clock genes in each tissue were detected after single restraint stress, whereas the phase of PER2 protein rhythm in the SCN did not change following restraint stress. These results suggest that peripheral clocks are directly entrained by the external stress. Finally, an elevated platform stress, for 2 h, also induced phase-shifts of peripheral clocks, suggesting that not only physical but also psychological stressors might be potent synchronizers of peripheral clocks. To the best of our knowledge, our results demonstrate for the first time that peripheral clocks directly and immediately entrain to external stressors through sympathetic and/or oxidative stress pathways.

  P6

Redox oscillations in fruit fliesUtham Kashyap Valekunja1, Rachel Edgar2, John O'Neill3, Akhilesh B.

Reddy4

1Department of Clinical Biochemistry, Wellcome Trust-MRC Institute of Metabolic Science, Cambridge, Cambridgeshire, UNITED KINGDOM

2Wellcome Trust-MRC Institute of Metabolic Science, University of Cambridge, Cambridge, Cambridgeshire, UNITED KINGDOM

3MRC LMB, Cambridge, Cambs., UNITED KINGDOM

4University of Cambridge Metabolic Research Laboratories, Wellcome Trust-MRC Institute of Metabolic Science, University of Cambridge, Cambridge, Cambridgeshire, UNITED KINGDOM

Circadian oscillators are modeled by transcription/translation feedback loops, which use post-transcriptional and post-translational mechanisms to generate ~24 hour rhythms. The existence of non-transcriptional oscillators in specialized mammalian cells highlight, however, that redox oscillations are likely to be independent of transcriptional rhythms and are evolutionarily conserved from bacteria to humans, suggesting that redox oscillations may be a core feature of circadian clocks.Using a genetic and biochemical approach, we behaviourally screened Drosophila using the UAS-Gal4 system to knock down expression of a variety of redox genes in clock neuron subsets within the fly brain. In addition, we performed biochemical assays of recombinant fly peroxiredoxin proteins. We further dissected redox oxidation using whole animal lysates, and by using Schneider 2 (S2) cell lines. Although on going, our work shows that specific redox genes affect circadian behaviour, whereas many do not, highlighting a complex interplay between the transcriptional and non-transcriptional mechanisms in vivo.

  P7

Shock O'Clock The circadian clock in endotoxic shock - systemic versus local clock regulationVeronika Lang1, Achim Kramer2, Bert Maier1

1Charité Universitätsmedizin , Berlin, GERMANY

24 hour periodicity in the environment has led to the evolution of molecular circadian clocks in organisms ranging from cyanobacteria to humans. The impact of the circadian system on the regulation of immune function becomes apparent by experiments done by Francis Halberg in 1960: he observed that the mortality rate in mice challenged with the same dose of lipopolysaccharide (LPS) varied 8-fold depending on what time of day LPS was administered. One of the keyplayers in endotoxic shock is the innate immune system, as it is the first to respond to LPS. Amongst these macrophages are important initiators of the pro-inflammatory immune response. Previously, our group showed that macrophages not only have an intrinsic circadian clock, but also a circadian cytokine response upon LPS treatment in vitro and ex vivo.Until today it remains unclear (i) to what extent circadian systemic factors play a role in the outcome of endotoxic shock and (ii) how important the immune cell intrinsic clock is in the regulation of the LPS induced immune response. Using the widely utilized model of LPS induced endotoxic shock we aim to unravel the biological implications of circadian regulation of immune functions. In a first step, we confirmed the diurnal mortality observed by Halberg in the standard lab mouse strain C57Bl/6. Further, we investigated the impact of external time cues such as light-dark rhythms on time-of-day dependent mortality and susceptibility to LPS. Our results demonstrate that sensitivity to LPS and thus mortality is regulated by the circadian system rather than driven by light-dark cycles. Surprisingly,

susceptibility to LPS was increased up to 3-fold in mice kept in DD compared to LD. Finally pan clock knockout (MOP3-/-; Cry1-/- Cry2-/-) and conditional macrophage clock knockout (LysM Cre x Bmal1 flox; LysM Cre x Clock flox) mouse models will be used to dissect the role of the systemic versus local information in the circadian regulation of the pathophysiology of endotoxic shock. In summary this study will give us further insight into the role and level of circadian clock regulation in endotoxic shock.

  P8

Chronic Stress Induces Physiological and Brain Region Specific Molecular Disruptions of Circadian Amplitude in

MiceNicole Edgar1, Andrea Gillman1, Ryan  Logan1, Daniel Hoffman1,

Colleen McClung1

1Psychiatry, University of Pittsburgh, Pittsburgh, PA, UNITED STATES

Increasing evidence implicates circadian abnormalities as a component of the pathophysiology of major depressive disorder (MDD). The suprachiasmatic nucleus (SCN) of the hypothalamus coordinates rhythms throughout the brain and body. On a cellular level, rhythms are generated by a cycle of gene expression, including the PERIOD2 (PER2) protein. Here, we examined whether unpredictable chronic mild stress (UCMS): 1) elicits physiological circadian disruptions similar to MDD subjects and 2) induces circadian disruptions in MDD-associated brain regions.Activity and body temperature rhythms were recorded in wild type c57BL/6 mice (N= 7 control, 16 UCMS) before, during, and after exposure to four weeks of UCMS. A second cohort of mice carrying a fusion gene for Per2 and luciferase (N= 18-25 control, 20-25 UCMS) was exposed to UCMS and tissue explants for six MDD-associated brain regions (mPFC, CeA, BLA, NAc, VTA, SCN) were assessed for rhythmicity for one week. UCMS significantly decreased circadian amplitude of activity and body temperature in WT mice (p<0.001), similar to findings in MDD subjects. UCMS elicited a decrease in the amplitude of molecular rhythms in the SCN (p<0.01) but surprisingly, increased the amplitude of molecular rhythms in the NAC (p<0.05). Brain region-specific changes in circadian amplitude were correlated with mood-related behaviors.Chronic stress selectively alters circadian amplitude of physiological and molecular rhythms in mice and induces opposing changes in MDD-associated brain regions that are correlated with mood-related behavior. The results of this study will have implications for future investigations targeting region-specific circadian mechanisms in MDD.

  P9

CCA1, a central circadian oscillator mediates ER stress response in Arabidopsis

Andrew Melencion1, In Jung Jung1, Hee Jin Park3, Joon–Yung Cha1, Mi Ri Kim1, Min-Gab Kim6, Sang Yeol Lee1, Dae-Jin Yun1, Woe-Yeon Kim1

1Division of Applied Life Science , Gyeongsang National University, Jinju, KOREA3Plant Molecular Biology and Biotechnology Research Center , Gyeongsang National University, Jinju,

6College of Pharmacy and Research Institute of Pharmaceutical Science, Gyeongsang National University, ,

We examined the evidence regarding the role of circadian clock in modulating Unfolded Protein Response (UPR) pathway under ER stress conditions in plants. UPR is an essential means with important physiological roles in all cells and ER stress arises under both single and multiple stress conditions. The circadian clock uses environmental rhythm to fine-tune and adapt a wide range of developmental and physiological processes. Our results support the evidence that UPR pathway is controlled by circadian clock in non-stress and stress conditions. Furthermore, we found that ER chaperone gene BiP3 is under circadian control of Circadian Clock Associated 1 (CCA1) and bZIP28, an ER stress-specific transcription factor forms a transcriptional complex with CCA1 to activate UPR-associated genes under ER stress. In addition, overexpression of CCA1 exhibits tolerance to ER stress in Arabidopsis. Our results suggest that circadian clock plays a pivotal role in the UPR in plants.

  P10

Assessing the Impact of Chronic Sleep Restriction and Acute Sleep Deprivation on Performance-Associated

Regional Brain Activation Using Near-Infrared SpectroscopyMichael Lee1, Gary Strangman2, Joseph Hull1, Tushar Kamath4,

Shadab Rahman5, Steven Lockley6, jameela lokhandwala7, Quan Zhang2, Charles Czeisler9, Elizabeth Klerman10

1Medicine and Neurology, Brigham and Women's Hospital/Harvard Medical School, Boston, MA, UNITED STATES

2Psychiatry, Harvard Medical School, Boston, MA, UNITED STATES4Massachusetts Institute of Technology, Boston, UNITED STATES

5Harvard Medical School, Boston, MA, UNITED STATES6Brigham & Women's Hospital, Boston, MA, UNITED STATES

7Chemistry, Southern Methodist University, Dallas, TX, UNITED STATES9Division of Sleep and Circadian Disorders, Harvard Medical School, Boston, MA, UNITED STATES

10Div. Sleep Medicine, Brigham and Women's Hospital/Harvard Medical School, Boston, MA, UNITED STATES

Sleep deprivation alters prefrontal cortex (PFC) activity, a brain region important for cognitive performance and executive function. It remains unknown, however, if PFC activity reflects cognitive performance and whether such activity exhibits a circadian rhythm or is altered by chronic or acute sleep loss. We have employed a novel neuroimaging approach—Near-Infrared Spectroscopy (NIRS)—to quantify hemodynamic changes in oxygenated and deoxygenated blood concentration that reflect alterations in regional brain activity.We used NIRS to monitor PFC activity during a 10-min visual psychomotor vigilance task (PVT) in healthy volunteers participating in either (i) a 32-day forced desynchrony inpatient study assessing the effects of chronic sleep restriction (CSR; multiple consecutive days of insufficient sleep) or (ii) an inpatient study that included a 30-hr acute sleep deprivation (ASD; single episode of extended wake).Preliminary assessment of the averaged PFC hemodynamic response to individual PVT trials showed a 2-second lag between trial presentation before both (i) a rapid increase in oxygenated blood concentration to levels ~5-fold greater than baseline (which primarily reflects cardiac oscillation) before returning to baseline concentrations after ~8 seconds, and (ii) a rapid decrease

in deoxygenated blood to levels ~4-fold greater than baseline (which primarily reflects cardiac oscillation)  before returning to baseline concentrations. This PFC hemodynamic response to PVT trial presentations can be characterized for further analyses using metrics including amplitude and area under the curve. Analyses of the effects of circadian timing and sleep loss on PFC response are ongoing.The results may lead to identification of times of decreased vigilance in shift-working and other populations at risk of sleepiness-related accidents. Support: Lee: NSBRI PF03002*. Strangman: NSBRI SMST 02801*. Lockley: NSBRI HFP02801*. Klerman: NSBRI HFP02802*, NIH K24-HL105664, P01 AG09975, Catalyst 1UL1 TR001102-01 and R01-HL-114088. * Supported by the National Space Biomedical Research Institute through NASA NCC 9-58

  P11

Sleep deprivation alters hepatic metabolism and the peripheral clock

Jessica Ferrell1, Shannon Boehme2, John Y.L. Chiang2

1IMS, Northeast Ohio Medical University, Rootstown, OH, UNITED STATES2Northeast Ohio Medical University, Rootstown, OH,

Circadian disruption is associated with increased incidence of cancer, cardiovascular events, inflammatory diseases and symptoms of metabolic syndrome including dyslipidemia and obesity. Bile acids (BA) moderate glucose and lipid homeostasis via activation of the liver and intestinal nuclear receptors FXR and TGR5. Cyp7a1, the rate-limiting enzyme that converts cholesterol to BA, is regulated by nutrients, insulin, and BA themselves. Cyp7a1 displays a circadian rhythm in gene expression and activity and this rhythm is shifted by high fat diet and altered time of feeding, leading to de-regulated metabolism and metabolic disease. The mechanisms by which perturbed rhythms or altered diet negatively affect Cyp7a1 and ultimately glucose and lipid metabolism are unclear. We therefore utilized a sleep deprivation protocol to examine circadian rhythms of hepatic metabolism. Cohorts of female wild type mice were sleep deprived from ZT 2 – ZT 8 for 5 consecutive days using gentle stimulation techniques. Control and experimental mice were sacrificed at ZT 2, 6, 10, 14, 18, and 22 (n=5-6) and core molecular clock and key hepatic metabolism genes were examined. Under 12:12 LD, liver Clock gene expression was significantly suppressed across all times in sleep-deprived mice, while BMAL1 expression remained unchanged. Liver Per1 and Per2 expression was significantly suppressed, while Reverbα peak expression was advanced by approximately 4 hr. The well-established nighttime peak in Cyp7a1 expression was abolished by sleep deprivation, while expression of the branch-point BA enzyme Cyp8b1 was unchanged. 24 hr lipid profiling revealed significantly increased serum and liver free fatty acids and altered triglyceride rhythms in serum and liver of sleep-deprived mice. Taken together, these data indicate that acute circadian disruption may have a profound effect on hepatic metabolism via altered lipid homeostasis, thus contributing to the etiology of metabolic disease. DK58379 and DK44442 to JYLC and DK096784 to JMF

  P12

Hyper-sensitivity of the circadian system to light in Delayed Sleep Phase Disorder

Sean Cain1, Jessica  Stanghi2, Olivia McConchie2, Clare Anderson2

1Psychological Sciences, Monash University, Clayton, Victoria, AUSTRALIA2Monash University, Clayton,

Delayed Sleep Phase Disorder (DSPD) is a prevalent primary sleep disorder, characterized by a difficulty in initiating sleep at night and subsequent difficulty in waking at times required for work or school. A core feature of DSPD is a delay in the timing of circadian rhythms that control sleep and wake. The physiological basis of the disorder remains unknown. We tested the hypothesis that hyper-responsiveness of the circadian clock to the evening (delaying) light is a physiological basis of DSPD. We examined the effect of light levels typically encountered in the evening on the suppression of melatonin and on acute alertness. Four individuals with DSPD and five controls were studied under highly-controlled conditions. We assessed the effect of a 3 h light exposure of regular room light on melatonin suppression, as well as subjective and objective measures of alertness. To minimize the effects of postural changes on melatonin levels, participants maintained a controlled posture (sitting upright) throughout the in-lab protocol. Lights were dim (< 3 lux) in the five hours preceding habitual bedtime in order to measure unsuppressed melatonin levels. Three hours of regular room light exposure (200 lux at corneal level) began at habitual bedtime. Saliva samples for the measurement of melatonin were taken every 30 minutes. Suppression was calculated as percent levels relative to the sample immediately preceding light exposure. As hypothesized, light exposure resulted in greater melatonin suppression in the DSPD group compared to controls. Suppression was greater in the DSPD group at all time points, with significant differences at 60 and 180 minutes of light exposure  and trends at 30 and 90 minutes. Overall suppression in DSPD participants was ~120% of control levels (38.7% vs. 17.5% suppression). For measures of alertness, participants completed the Karolinska Sleepiness Scale (KSS; measure of subjective sleepiness) every 30 minutes and the Psychomotor Vigilance Task (PVT; reaction time test of objective alertness) every hour. The Karolinska Drowsiness Test (KDT; EEG/EOG measure of objective alertness) was completed hourly. Particpants also wore Optalert™ real-time drowsiness detection system (eye-glass frame-mounted infra-red reflectance oculography). DSPD participants displayed an enhanced alerting response to the indoor light relative to controls for all measures. These findings support the notion that the circadian systems of individuals with DSPD are hyper-responsive to the effects of evening light of a moderate level.

  P13

Inter-individual differences in night-time behavioral and cerebral responses to high and low sleep pressure

conditionsMicheline Maire1, Carolin F. Reichert1, Virginie Gabel1, Antoine U. Viola1,

Klaus Scheffler5, Markus Klarhöfer6, Werner Strobel7, Christian Cajochen8, Christina Schmidt8

1Centre for Chronobiology, Psychiatric Hospital of the University of Basel, Basel, SWITZERLAND5Max-Planck-Institut für Biologische Kybernetik, Tübingen, GERMANY

6Universität Basel, Institut für Radiologie, Basel, 7Universität Basel, Pneumologie, SWITZERLAND

8Center for Chronobiology, Psychiatric Hospital of the University of Basel, Basel, , SWITZERLAND9Center for Chronobiology, Psychiatric Hospital of the University of Basel, Basel, , SWITZERLAND

Previous reports suggest circadian- and sleep-wake-dependent impacts on cognition-related cortical and subcortical brain activity. Furthermore, there is increasing evidence that the response to a misalignment between the circadian and homeostatic systems as triggered by sleep deprivation or by scheduling sleep at the inappropriate times of the day presents large inter-individual variability. Here, we quantified task-related BOLD activity during fMRI sessions at specific time points within the 24-hour cycle in a cohort of healthy young volunteers, genotyped for a VNTR polymorphism in the hPER3 clock gene. Twenty-nine volunteers, 14 homozygous carriers of the long repeat allele (PER35/5) and 15 homozygous carriers of the short repeat allele (PER34/4) underwent a 40-h sleep deprivation (SD) and a 40-h multiple nap protocol (NP; 10 cycles of 160 min wakefulness/80 min nap). A psychomotor vigilance task was performed every four hours, whereof every 2nd session took place in a 3T MRI scanner. Saliva was collected for melatonin assays along with several questionnaires at regular time intervals, and the EEG was continuously recorded. We reveal a consistent pattern of greater vulnerability to high sleep pressure levels in PER35/5 than PER34/4 carriers on the physiological and behavioral level. In particular, we observ higher subjective sleepiness, more slow eye movements and unintentional sleep episodes, as well as more attentional lapses in PER35/5 than PER3 4/4 carriers (all ps < 0.05). So far, the fMRI analysis (n=28) during the attentional task focused on the night-time window encompassing maximal circadian sleep promotion. Results indicate a PER3- and sleep-pressure-level-dependent BOLD activity pattern in a series of attention-related cerebral brain regions (e.g. bilateral inferior frontal gyrus and anterior cingulate cortex, left inferior parietal and thalamic regions, all pcorr < 0.05). Lower activations or greater deactivations in these areas were observed in PER35/5 carriers under SD, compared to NP and/or higher activity levels in PER34/4 carriers than in PER35/5 carriers under SD. Our data confirm the detrimental effects of elevated sleep pressure, as consistently detected at the behavioral, psycho-physiological and cerebral level. They further support the existence of large inter-individual differences in these responses, especially at the circadian arousal trough occurring at the end of the biological night.

  P14

Circadian activity splitting in two sighted individuals with non-24 hour sleep-wake disorder

Sabra Abbott1, Lirong Zhu2, Kathryn  Reid2, Phyllis Zee2

1Northwestern University, Chicago, IL, UNITED STATES2Neurology, Northwestern University, Chicago, Illinois, UNITED STATES

BACKGROUND: Circadian activity splitting is a phenomenon that has been well documented in hamsters, and has also been described in a variety of other mammals, including CS mice, rats and diurnal rodents.  However, a similar phenomenon has not previously been demonstrated in humans.METHODS: Two sighted subjects who were previously clinically diagnosed with non-24 hour sleep-wake disorder underwent actigraphy monitoring for at least 2 weeks.RESULTS:  Both individuals were observed to have a period of decreased a, followed by one day of increased activity and splitting of the activity rhythm into two components. These activity periods subsequently re-fused, with the prior morning activity period occurring during the subjective evening, and the evening activity period occurring during the subjective morning.CONCLUSIONS: We describe here the first human examples of splitting in sighted individuals with N24, similar to that previously observed in rodents under constant light conditions.  Splitting has previously been thought to be due to the presence of two distinct oscillators that have dissociated.  Furthermore, in CS mice, who are prone to develop bimodal or split rhythms, the SCN is unable to properly coordinate peripheral

oscillators, resulting in difficulty with entrainment.   Sighted N24 is a poorly understood disorder in which individuals are unable to maintain entrainment to the 24 hour environment, despite having normal vision.  The etiology of this disorder remains unclear however the ability for these individuals to develop behavioral splitting could point to an underlying instability of the coupling of the two oscillators that may in turn affect entrainment ability.  Further evaluation of this phenomenon may lead to better understanding of the pathophysiology as well as potential treatment options for this disorder.

  P15

Novel PER2 alleles for familial advanced sleep phaseChristin Chong1, Geetha Bhagavatula2, William Hallows1, Philip Kurien1,

Yong Huang1, Emily Quinn6, Len Pennacchio7, Christopher Jones8, Louis Ptacek1, Ying-Hui Fu1

1UCSF, San Francisco, 2George Washington University, Washington D.C.,

6Icahn School of Medicine at Mount Sinai, New York, NY, UNITED STATES7Lawrence Berkeley National Laboratory, Berkeley, CA, UNITED STATES

8University of Utah, Salt Lake City, ,

Familial advanced sleep phase (FASP) is the first human Mendelian autosomal dominant sleep phenotype identified, which is characterized by exceptionally early sleep-wake onset and offset. Transgenic mice carrying human FASP mutations such as PER2-S662G recapitulate the circadian phase advancement of behavioral activity, and shortening of circadian period observed in FASP individuals. Further supporting the causative role of PER2, novel PER2 alleles for FASP were recently identified. These mutations provide a unique opportunity for investigating cellular substrates underlying timing of sleep-wake activity. Similar to PER2-S662G, nuclear clearance of novel FASP PER2 variants is accelerated, suggesting that nuclear transport of PER2 may calibrate the cellular clock when it is genetically perturbed. Supporting the involvement of nuclear export for phasic regulation, administration of leptomycin B in vitro results in marked phase advancement. Understanding FASP mutations may reveal novel counterbalancing mechanisms for the maintenance of circadian rhythms.

 

  P16

An Important Role of 5 Evening Neurons in Drosophila Circadian Rhythms and SleepFang Guo and Michael Rosbash1

1HHMI/Brandeis Univ, , UNITED STATES

Like mammalian sleep, Drosophila sleep is modulated by two systems, a homeostat influenced by daily experience and a circadian process controlled by the clock. The functional and anatomical overlap between these two systems is largely unknown as is the nature of the sleep homeostat. We report here that 5 LNd neurons play an important role in the circadian network and are also involved in homeostatic sleep regulation. Activation of these neurons either transiently with the dTrpA1 channel or constantly with the NachBac sodium channel not only increased the circadian morning anticipation peak and locomotor activity but also induced night-time sleep loss and affected sleep rebound. Blocking output of these 5 LNds not only caused DD arrhythmicity and reduced the evening activity peak but also increased sleep. RNA profiling and RNAi experiments indicate that these 5 activity-promoting LNds are cholinergic and use

acetylchoine to promote locomotor activity and wakefulness. The profiling and activity manipulations also suggest that these cells contribute to shaping the sexually dimorphic activity/seep pattern differences between males and females. The 5 LNds function downstream of PDF signaling, which has a surprising and to our knowledge unprecedented inhibitory effect on their activity. This result is consistent with the surprising effects of signal transduction pathway manipulations within the 5 cells. This connection “back” to PDF pacemaker neurons is complemented by a “forward” connection to activity-sleep centers in the dorsal region of the brain, further indicating that these 5 cells contribute to and bridge both the circadian and homeostatic systems.

  P17

A circadian lens on human population activity patterns: Inferences from the power grid

Caitlin Crosier1, Adam Stowie1, Mario Amacarelli1, Taylor Fredrickson1, Spencer Keith1, Kassandra Leasure1, Emily Socha1, Ryan Mymko1,

Brandon Boesiger1, J. David Glass1

1Biological Sciences, Kent State University, Stow, OH, UNITED STATES

Few, if any studies have focused on the daily rhythmic nature of modern industrialized populations. The present study utilized real-time load data from the U.S. Pacific Northwest electrical power grid as a reflection of human operative household (i.e. "homecage") activity. This approach involved ClockLab analyses of continuously streaming internet data (provided in 5 min bins) from a human subject pool of approximately 43 million residential users. Rhythm analyses reveal striking seasonal and intra-week differences in human activity patterns, largely devoid of industrial and automated load interference. While length of the diurnal activity period (alpha) is longer during the summer than winter (17.3 h vs. 16.5 h, respectively; p<0.001), a distinct, stable bimodal pattern of alpha, coinciding with morning and evening activity peaks is evident throughout the year, except during hot summer periods where a single prolonged peak is seen. As expected, significantly more activity occurs in the solar dark phase during the winter than during the summer (6.3 h vs. 2.0 h, respectively; p<001). Interestingly, throughout the year a "weekend effect" is evident, where morning activity onset occurs approximately 1 h later than during the work week, significantly shortening alpha (15.6 h vs. 16.5 h, respectively; p<0.001). This indicates a general phase-delaying response to the absence of job-related or other weekday morning arousal cues, substantiating a preference or need to sleep longer on weekends produced by the long human circadian period and/or homeostatic sleep debt. The use of grid power load as a means for human actimetry assessment thus offers new insights into the collective diurnal activity patterns of large human populations.

  P18

Chronobiology meets Big Data: Humans 'in the wild'Dimitri Perrin1, Craig Jolley1, Hiroki Ueda1

1RIKEN, Kobe, JAPAN

Human chronobiology studies are either done in laboratories or have to rely on volunteers reporting their activities using questionnaires such as the Munich ChronoType Questionnaire. Despite their success, both approaches have limitations, particularly in terms of their ability to handle very large sample sizes. Till Roenneberg recently advocated the idea of a large-scale human sleep project that would rely, in part, on data being automatically acquired from a large number of participants through ad-hoc devices. As a proof-of-concept for such an initiative, we looked at an existing Big Data repository and tried to analyze it in a chronobiology context. Even though the data was not optimized for such a study and mostly contains information on road

traffic, telecommunications and electricity usage, we were able to extract relevant information. This is an encouraging first step, just as sleep-specific apps and devices are being released.

  P19

Circadian disfuntion in chronic kidney diseaseInês Chaves and G.T.J. van der Horst

Erasmus MC, ROtterndam, The Netherlands

Circadian clocks regulate appropriate timing of physiology, behaviour and metabolism by generating rhythms with a period of approximately 24 hours. The master circadian pacemaker resides in the suprachiasmatic nuclei (SCN) in the brain, and this structure is synchronized every day by light information. The SCN communicates day-night phase information to peripheral circadian clocks that exist in almost all cells and organs, including the kidney. Our previous research has shown a disturbed circadian sleep-wake rhythm and impaired secretion of melatonin (a key hormone in the regulation of sleep-wake rhythms) in chronic kidney disease (CKD). Furthermore, a disturbed circadian blood pressure rhythm (a non-dipper profile) and an impaired circadian rhythm of urine production have been found in patients with renal failure. Thus, these clinical manifestations point to a possible disruption of the circadian system in CKD patients.The overall goal of our study is to provide molecular evidence for disturbed circadian rhythms in peripheral tissues in CKD patients. Particularly, we tested the hypothesis that the circadian deregulation in CKD patients is caused by an excess of clock-resetting compounds in the blood, resulting from renal failure, which affect circadian gene expression in peripheral tissues.

  P20

The effect of light containing spatial structure on the suprachiasmatic nucleus

Josh Mouland1, Tim Brown1, Rob Lucas1

1University of Manchester, Manchester, Lancashire, UNITED KINGDOM

The current view is that the suprachiasmatic nucleus (SCN) acts as an irradiance detector, integrating light intensity across time and space to obtain the most accurate view of time of day. However, the retinal ganglion cells upon which it relies for light information have finite receptive fields and respond to light steps with an initial transient excitation that is larger than the sustained excitation. As a result, the activity of these retinal ganglion cells, both individually and as a population, should be modulated by spatial patterns. We have explored this possibility by recording electrophysiological responses to visual stimuli of the murine SCN in vivo. Using this method we have mapped spatial receptive fields of individual SCN units; and determined their response to drifting gratings of various spatial and temporal frequencies. We have further addressed the question of whether spatially structured stimuli produce a change in time averaged firing rate when compared to irradiance matched spatially uniform stimuli. To examine the effect of non-uniform spatial stimulation on the suprachiasmatic nucleus as a whole we compared phase shifts in free-running rhythms of locomotor activity induced by spatially uniform vs patterned stimuli of matched irradiance.

  P21

Trypanosoma brucei accelerates the mouse circadian clock Filipa Rijo-Ferreira1, Joseph S Takahashi2, Luisa M Figueiredo3

1UT Southwestern / Instituto Medicina Molecular, Dallas / Lisbon, USA / PORTUGAL2Howard Hughes Medical Institute, UT Southwestern, Dallas, Texas, USA

3Instituto de Medicina Molecular, Lisboa, PORTUGAL

Sleeping sickness is a parasitic disease that, if left untreated, leads to coma and eventually death. It is caused by Trypanosoma brucei - a unicellular and extracellular eukaryotic parasite that lives in the bloodstream and interstitial spaces of several tissues. In humans, this parasite disrupts the host’s circadian rhythm, including the sleep/wake pattern, body temperature and endocrine secretion. We hypothesize that T. brucei disrupts the host circadian rhythm by interfering with clock gene expression. This circadian rhythm disruption may result from a direct interaction with the parasite and/or an indirect effect such as the immune response or metabolic conditions. We used a mouse model to better understand the exact effect of a T. brucei infection on its host circadian rhythm. First, using running wheels, we showed that T. brucei changes circadian behavior in mice. Similar to that observed in humans, the amount of activity during rest phase increased 10-20% compared to healthy animals. Second, infections in PER2::LUC reporter mice, revealed ex vivo that highly parasitized tissues have a robust circadian rhythm but with a 2hr shorter period. The same observations were reproduced in vitro when parasites were directly co-cultured with isolated fibroblasts, suggesting that parasites may have a direct effect on the host cell circadian clock. Finally we observed that gene expression of clock genes in vivo is significantly affected in peripheral tissues, especially the most highly parasitized. These results show that (i) T. brucei infection causes significant molecular and behavioral changes of the mouse circadian rhythm; (ii) this effect may be partly caused by a direct interaction with the parasite. 

  P22

The Role of Sustained GABA-A Receptor Activation within the SCN in Light-induced Phase Shifts is Phase Dependent

Daniel Hummer1, John McNeill2, Tony Larkin1, H. Elliott Albers2

1Psychology, Morehouse College, Atlanta, GA, UNITED STATES2Neuroscience Institute, Georgia State University, Atlanta, GA, UNITED STATES

Cells in the SCN exhibit a sustained response to light, including increases in cell firing and Per expression that last for several hours (Albrecht et al. (1997); Yamamoto et al. (2001); Yan et al. (2002); Kuhlman et al. (2003); Hamada et al. (2004)).  We hypothesize that the sustained activation of GABA receptors in the SCN mediates the ability of light to phase shift the circadian clock.  We have demonstrated that sustained activation of GABAA receptors in the SCN between CT14.5 and CT19.5 is necessary and sufficient to produce light-induced phase delays of the circadian clock.  In the present experiment we tested the prediction that the inhibition of GABAA receptors must occur between CT14.5 and CT19.5 in order to inhibit phase delays resulting from a LP at CT13.5.  Male Syrian hamsters were implanted with guide cannula aimed at the SCN.  After recovery, they were allowed to establish a stable free-running activity rhythm in DD.  Animals were randomly assigned to receive a 15-minute LP at CT13.5 followed by injection of the GABAA antagonist bicuculline in the following regimens: (1) hourly injections from CT14.5-19.5, (2) hourly injections from CT17.5-22.5, or (3) hourly injections from CT23.5-4.5.  The administration of the GABAA receptor antagonist at CT14.5-19.5 and CT17.5-22.5 each resulted in significantly smaller light-induced phase delays compared to vehicle-treated controls (CT14.5-19.5: -30.12±9.63 min versus controls: -61.44±7.54 min); CT17.5-22.5: -43.07±6.96 min versus controls: -70.65±13.17 min).  Light-induced phase delays exhibited by hamsters treated with the

GABAA receptor antagonist at CT23.5-4.5, however, did not differ from those exhibited by vehicle controls (CT23.5-4.5: -47.95±9.35 min; versus controls: -56.70±5.93 min).  These data are consistent with the hypothesis that the sustained activation of GABAA receptors in the SCN mediates the ability of light to phase shift the circadian clock, but that this GABAA receptor-mediated effect is phase dependent.

Supported by NIH NS078220 and NSF IOS-1022050

  P23

Effects of chronic nighttime light exposure on the daily rhythms in locomotor activity and clock gene expression in

the SCNLily Yan1, Greg Leach2

1Psychology & Neuroscience Program, Michigan State University, East Lansing, MI, UNITED STATES2Psychology, Michigan State University, East Lansing, MI, UNITED STATES

The environmental light/dark (LD) cycle is the most salient cue for entraining circadian rhythms, which are regulated by the principal circadian clock located in the suprachiasmatic nucleus (SCN). Experimental paradigms utilizing altered lighting conditions have demonstrated unique responses of the SCN, which have revealed the robustness and resilience of the SCN neural network in the face of environmental perturbation. The present study contributes to this rich body of literature, by evaluating the responses of rhythms in behavior and in the expression of clock genes in the SCN in a paradigm simulating the lighting conditions experienced by night shift workers. CD1 mice were first entrained to 12:12h LD cycle, then a 4h light exposure starting at ZT16 was introduced, which resulted in a 12:4:4:4 h LDLD condition. In cage activity was monitored with infrared motion sensors. Although the majority of the activity occurred at night, the activity bouts appeared to be more fragmented than those of the LD controls. The expression of PER1 and PER2 was analyzed in the SCN after two weeks in LDLD condition. The results revealed moderate levels of PER1 or PER2 expression across all time points examined, without detectable rhythms in the whole SCN or within the core/shell subregions. The loss of overt rhythms in the SCN is likely due to the loss of synchrony among the cellular oscillators within the nucleus, which has been shown to involve the VIP-VPAC2 pathway. VIP in the SCN was then examined and was found to be higher in the LDLD compared to the LD group. The results show that the chronic nighttime light exposure is detrimental to the time-keeping function of the SCN. This paradigm provides a unique model to further elucidate the mechanisms underlying the robustness and resilience of the SCN network.

  P24

Suprachiasmatic clues to circadian dysfunction in the BACHD mouse model of Huntington's disease.

Dika Kuljis1, Danny Truong2, Dawn  Loh2, Takashi  Kudo4, Christopher Colwell2

1Neurobiology, UCLA, Los Angeles, CA, 2Psychiatry and Biobehavioral Science, UCLA, Los Angeles, CA,

4Psychiatry and Biobehavioral Science , UCLA, LOS ANGELES, CA,

Huntington’s disease (HD) is an autosomal dominant disorder caused by an expanded trinucleotide repeat (>37) in the gene encoding huntingtin protein. Motor symptom onset typically occurs at mid-life and results in the HD diagnosis. Even before the onset of the motor symptoms,

HD gene carriers commonly exhibit altered sleep/wake rhythms, experiencing non-motor symptoms such as daytime sleepiness, delayed sleep onset and fragmentation. We believe addressing disrupted sleep is an important consideration for the treatment of HD patients and have employed the bacterial artificial chromosome (BAC) HD mouse model to mechanistically determine its etiology. Previously we have shown the BACHD mouse recapitulates important aspects of patient behavioral phenotypes, and additionally, found evidence of circadian system dysfunction. Daily rhythms in electrical activity of suprachiasmatic nuclei (SCN) neurons orchestrate biological rhythms throughout the brain and body, but this rhyth is lost in BACHD mouse. Here we test for changes to SCN neuronal rhythmic electrophysiological properties and examine likely currents in the search for the ionic mechanism underlying the loss of rhythmic electrical activity in BACHD SCN.

  P25

Temporal pattern of GABAA receptor δ subunit expression in the suprachiasmatic nucleus of male Syrian hamsters.

James Walton1, John (Mac) McNeill1, Elliott Albers3

1Neuroscience Institute, Georgia State University, Atlanta, GA, UNITED STATES3Georgia State University, Atlanta, GA, UNITED STATES

GABAA receptors (GABAARs) are pentameric assemblages generally comprised of three different proteins from 19 subunits. Although all GABAARs are ligand gated ion channels, subunit composition determines channel properties and location of the receptor on the cell membrane. In contrast to classical phasic inhibition mediated by synaptic GABAARs, GABAARs containing the δ subunit are tonically active high-affinity non-desensitizing channels found at extrasynaptic locations. GABAA δ receptors mediate neuronal activity by monitoring ambient extrasynaptic GABA concentrations and spillover from nearby synapses. Altered tonic inhibition mediated by extrasynaptic GABAARs has been implicated in multiple psychiatric disorders, including sleep disorders. Recent work has demonstrated that GABAA δ receptors also have a role in the regulation of photic input into the SCN in a circadian phase-specific manner, however whether these receptors are differentially expressed over the LD cycle in the SCN is not known. Toward this end, we collected SCN tissues via micropunch from male Syrian hamsters exposed to a 14:10 L:D cycle at ZT1, 6, 13, 17, and 19 to assess GABAA δ expression. In common with the circadian rhythm in the effects of GABAA δ receptors on photic input to the SCN, GABAA δ mRNA expression varied across the LD cycle, with levels at nadir during the dark phase. We are currently assessing GABAA δ protein across the LD cycle. Taken together, these findings indicate that there is a 24 hr rhythm in expression of the GABAA δ subunit that may contribute to the circadian pattern of responsiveness to GABA at extrasynaptic receptors in the SCN. Supported by NIH NS078220 

  P26

CRYPTOCHROME and its role in controlling circadian rhythms using electrophysiological techniques in

Drosophila melanogaster Marie Nugent1, Ezio Rosato2, Joern Steinert3

1Genetics, University of Leicester, Leicester, Leicestershire, UNITED KINGDOM2Genetics, Univesity of Leicester, Leicester, Leicestershire, UNITED KINGDOM

3MRC Toxicology Unit, Leicester, Leicesteshire, UNITED KINGDOM

This project investigates the role of CRY in the regulation of neuronal excitability in a specific subset of neurons of the Drosophila CNS called the large ventral lateral neurons (l-LNvs). Overexpression of a constitutively active form of CRY (CRY∆) is used to study the role of CRY within the

circuitry involved in circadian clock regulation.  Overexpression of a constitutively active form of CRY (CRY∆) as well as CRY knock-outs (CRY0 ) enable us to study the functional role of CRY within the circuitry involved in circadian clock regulation.  Electrophysiological characterisation of voltage gated potassium currents and synaptic activities modulated by overexpression of CRY∆ or in CRY0 flies will reveal CRY’s contributions to neuronal excitability and synaptic transmission within the network of circadian neurons. Whole cell patch clamp recordings of l-LNvs in whole brain preparations of adult Drosophila were performed (Multiclamp 700B, pClamp 10, Axon Instruments).  Female flies w1118 /UAS cry∆72.3; TIM-GAL4/ UAS GFP co-expressed CRY∆ and GFP in clock cells, including the l-LNvs.  w1118 /+; tim-GAL4/ UAS GFP females were used as controls.  Recordings were conducted during ZT10-12 to eliminate any changes in the activity seen in the l-LNvs due to a circadian effect.   Electrophysiological recordings reveal that overexpression of CRY∆ induces increased outward potassium currents in l-LNvs between holding potentials of 0 mV to 50 mV (in 10 mV increments) compared to controls (678 pA at 50mV in control vs 820 pA in CRY∆, p=0.043 with Two-Way ANOVA).  Investigations of the spontaneous action potential (AP) firing frequencies were also conducted which show a trend in increased firing with CRY∆ at various holding potentials (-30 mV, -40 mV, -50 mV and -60 mV) compared to controls (p=0.025 with Two-Way ANOVA). Analysis of the pre-synaptic currents also reveals an increase in frequency with CRY∆ at various holding potentials (-30 mV, -40 mV, -50 mV and -60 mV) compared to controls (p=0.023 with Two-Way ANOVA).Larval locomotor activities were measured as crawling distance in 10min (ANYmaze) using female larvae that were w1118; elav-GAL4/+; UAS-cry∆4.1/+ and compared to w1118; elav-GAL4/+; +/+ for control.  Data are expressed as mean ±SEM (n), * indicates p<0.05 with unpaired t-test.  Larval locomotor assays to investigate the motor effects of CRY∆ overexpression at the larval neuromuscular junction (NMJ) have shown an increase in the distance travelled by CRY∆ expressing larvae from 0.54 m (14) to 0.69 m ±0.04 (17)*. This data indicates that CRY∆ expression is able to mediate changes in activity at the NMJ as well as in CNS clock cells by modulating neuronal firing, potassium channel currents and locomotor activities.  Further experiments will reveal how CRY is modulating synaptic activity and neuronal excitability to allow firm conclusions from the electrophysiological data collected so far.

  P27

Phase-mapping the mouse brain with a CRY1::mCherry fluorescent reporter

Arthur Millius1, Rikuhiro Yamada1, Junko Yoshida1, Hideki Ukai1, Hiroki Ueda1

1Laboratory for Systems Biology, RIKEN CDB, Kobe, JAPAN

Daily biological rhythms occur in a variety of organisms from simple, single-celled eukaryotic alga to people struggling to recover from the effects of airplane travel. In mammals, the central time-keeping mechanism is located in a tiny structure of the brain

called the suprachiasmatic nucleus (SCN), but many other tissues also have clock machinery and circadian rhythms.  For example, we examined extra-SCN brain tissue by making 1 mm horizontal, coronal, and sagittal sections in week-old PER2::LUC reporter mice and found all slices in different regions of the brain maintained 24-hour luminescent oscillations with different degrees of amplitude for >7 days.  To understand the phase relationship between the SCN and the rest of the brain with higher spatial resolution, we constructed a c-terminal fusion of CRY1 with the red fluorescent protein mCherry driven by the Cry1 promoter and intronic enhancer (P(Cry1)-Cry1::mCherry). Transient transfection of P(Cry1)-Cry1::mCherry restored Cry1-/-:Cry2-/- mouse embryonic fibroblast P(Per2)-dLuc rhythms to wild-type levels with similar phase, period, and amplitude.  Dual luminescent/fluorescent imaging revealed that CRY1::mCherry accumulated in both the cytosol and nucleus, and peak expression was delayed relative to P(Per2)-dLuc in individual cells.  Currently, we are generating a CRY1::mCherry transgenic mouse and plan to image whole transparent brains from these mice using light-sheet microscopy.  We anticipate this new mouse reporter line will aid in understanding the circadian spatiotemporal coupling between the SCN and different regions of the brain.

  P28

Phosphorylation of the Cryptochrome 1 C-terminal tail regulates circadian period length

Peng Gao1, Seung-Hee  Yoo1, Kyung-Jong  Lee3, Clark  Rosensweig1, Joseph S. Takahashi1, Benjamin P.  Chen3, Carla B.  Green1

1Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX, 3Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, TX, UNITED

STATES

The Cryptochrome (CRY) proteins are critical components of the mammalian circadian clock and act to rhythmically repress the activity of the transcriptional activators CLOCK and BMAL1 at the heart of the clock mechanism. The CRY proteins are part of a large repressive complex, the components of which are not completely known. Using mass spectroscopy, we identified the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) as a CRY-interacting protein and found that loss or inhibition of this kinase results in circadian rhythms with abnormally long periods. We then identified serine 588 in the C-terminal tail of mouse CRY1 as a potential DNA-PK phosphorylation site but surprisingly found that the phosphomimetic mutation S588D also results in long period rhythms, similar to the loss of DNA-PK. Consistent with this, we found that phosphorylation of this site is increased in cells lacking DNA-PK, suggesting that DNA-PK negatively regulates the phosphorylation of this site most likely through indirect means. Furthermore, we found that phosphorylation of this site increases the stability of the CRY1 protein and prevents FBXL3-mediated degradation. The phosphorylation of this site is robustly rhythmic in mouse liver nuclei, peaking in the middle of the circadian day at a time when CRY1 levels are declining. Therefore, these data suggest a new role for the C-terminal tail of CRY1 in which phosphorylation rhythmically regulates CRY1 stability and contributes to the proper circadian period length.

  P29

Structural and Functional Characterization of the Interactions between Cryptochromes and Xenobiotic

ReceptorsAnna Kriebs1, Erin Soto1, Emma Henriksson3, Katja Lamia1

1Chemical Physiology, The Scripps Research Institute, La Jolla, CA, UNITED STATES3The Scripps Research Institute, La Jolla, CA, UNITED STATES

Circadian clocks sustain rhythmic patterns in behavior and physiology and increase organisms' fitness in the context of cyclic changes in the environment. Studies have shown that disruption of natural circadian rythms (e.g. through shift work) leads to an increased risk of developing heart disease, stroke, obesity or diabetes, underlining the importance of synchronization to the environment in humans. The molecular mechanism underlying mammalian circadian rhythms is based on a transcriptional feedback loop featuring the transcription factors CLOCK, BMAL1, CRY, and PER. This core clock directly or indirectly drives oscillating transcription of a large number of target genes. Approximately 10 % of the genome is transcribed rhythmically in various body tissues including liver. These circadian clock controlled genes include key metabolic enzymes and nuclear receptors. Interestingly, the liver xenobiotic receptors PXR and CAR show a robust interaction with circadian clock component cryptochromes (CRY1 and CRY2). Hypothesizing that these interactions alter PXR and CAR function we will characterize the interaction between CRY1 and/or CRY2 with PXR/CAR biochemically and structurally. We will determine the influence of CRY1 and/or CRY2 on PXR/CAR mediated xenobiotic metabolism and establish how CRY1 and/or CRY2 control the efficacy of drug treatments. This will present exciting new information that may help to optimize drug administration routines.

  P30

Characterising of the role of Cryptochromes in Retinal Responses to Light

Jovi Chau-Yee Wong1, Elizabeth Maywood2, Russell Foster1Stuart Peirson1

1Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, Oxon, UNITED KINGDOM2MRC-LMB, Cambridge, Cambs, UNITED KINGDOM

Mammalian Cryptochromes (CRYs) are a constituent component of the core circadian clock mechanism. Recent work has suggested that CRY may have additional physiological roles as a modulator or component of the retinal clock and/or as a putative light-dependent magnetoreceptor. Reports differ in consensus on the localisation of cryptochrome in the retina. Previous work has suggested CRY2 is expressed in cones as well as the majority of cell types in the inner nuclear layer (INL) and ganglion cell layer (GCL), while convincing CRY1 immunohistochemistry has never been reported in the retina. However, many commercially available CRY antibodies do not provide specific staining, and produce a similar signal in retinae from mice lacking CRY. Here we report a novel pattern of CRY1 and CRY2 expression in the retina using newly raised CRY antibodies that have undergone extensive validation (Anand et al., 2013, Maywood et al., 2011). Furthermore, colocalization studies were performed with CRY1/2 and retinal cell markers (TH – dopaminergic amacrine cells, ChAT – cholinergic

amacrine cells, Brn3A – retinal ganglion cells, GABA, GlyT1 – glycinergic amacrine cells, UVS – UV cones, and cone arrestin – all cones), as well as c-fos following a light pulse (light-activated cells). Colocalization data between CRY1/2 and clock proteins CLOCK and PER1, and the circadian photopigment OPN4 (melanopsin) are reported as well. These data suggest that CRY1 is the dominant form of CRY in the mammalian retina. Testing the role of CRY in the retina also requires functional assays of the retinal circadian clock. For example, cone ERG responses demonstrate a circadian rhythm in amplitude that is abolished in Cry1-/-Cry2-/- mice (Cameron et al., 2008). Here we show that wildtype mice show attenuated pupillary responses during the subjective night (ZT/CT 18) compared to the subjective day (ZT/CT 6). These differences persist under both LD and DD cycles. Furthermore, this effect is unattenuated in Opn4-/- (melanopsin knockout) mice, a model which shows attenuated diurnal variations in cone ERG responses. Here we report the circadian tuning of the pupillary light response in Cry1-/-, Cry2-/- and Cry1-/-Cry2-/- mice.

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Structural Characterization of Fungal Photoreceptor-EnvoyJameela Lokhandwala1, Brian Zoltowski1

1Chemistry, Southern Methodist University, Dallas, TX, UNITED STATES3Southern Methodist University, Dallas, TX, UNITED STATES

Organisms contain elaborate mechanisms to sense and adapt to environmental stimuli.  In particular, circadian clocks sense external cues to coordinate metabolism, growth, and sexual development to the diurnal light cycle.  In organisms such as plants, fungi and animals, these clocks consist of complex networks of feedback loops integrated into a central circadian oscillator.  We have a reasonable understanding of how the central oscillator operates, however, how multiple stimuli are integrated into the core clock to generate a coherent cellular response is poorly understood.  In the filamentous fungi Hypocrea jecorina (anamorph T. reesei) cellulase gene expression, carbon catabolite repression, sulfur metabolism and adaptive stress responses are all under control of blue-light.  A Light-Oxygen-Voltage (LOV) domain containing photoreceptor, ENVOY (ENV1) acts as a central node to integrate multiple input pathways into transcriptional machinery.  This is achieved by two primary mechanisms:  1) As a direct photoreceptor, ENV1 acts in conjunction with another LOV domain containing photoreceptor Blue-Light-Receptor-1 (BLR1) to regulate clock controlled gene transcription by the BLR1/BLR2 complex.  2) ENV1 acts in a light-dependent and light-independent mechanism to regulate cellulase gene expression, stress responses and sexual development through intersection of G-protein coupled receptor (GPCR) signaling. Using a combination of solution biophysics and structural characterization we have demonstrated that ENV1 employs a divergent mechanism to incorporate oxidative stress and blue-light signaling into regulation of gene expression.  Interestingly, although the core circadian machinery in T. reesei is conserved with N. crassa,(VVD=ENV1, Blr1=WC1, Blr2=WC2), they differ on the level of fundamental chemistry and signaling

mechanisms.  Thus, even closely related species adapt integration of environmental variables into circadian regulation.

  P32

Differential Mechanisms of Phase Advancing versus Delaying Light Pulses in Drosophila.

Pooja Vinayak1, Emma Garren1, Sina Mizaheri1, Jay Hirsh1

1Biology, University of Virginia, Charlottesville, VA,

We study the light sensitivity of light pulse induced phase shifts at limiting levels of light to determine whether phase advancing vs phase delaying light pulses proceed via similar mechanisms. Literature values for light sensitivity of light pulses that lead to advances vs delays of circadian phase in Drosophila indicate rather low light sensitivity, with no consistent differences for advances vs delays, and are highly variable between publications. Here we show that both variability and poor light sensitivity result from bright light during light/dark (LD) entrainment conditions that cause a severe and persistent loss of the circadian photoreceptor, crytochrome (CRY).  Our use of CRY-preserving dim light for LD entrainment results in high light sensitivity for both phase delaying and advancing light pulses. However, delaying pulses are >10x more light sensitive than advancing pulses, implying a distinct mechanism from advancing light pulses. We are investigating whether the differential intracellular localization of the CRY target TIM during early vs late night can account for this differential light sensitivity.  Finally, we investigate the neuronal requirements for CRY expression for phase advancing vs delaying light pulses. Surprisingly, CRY expresssion in differential subsets of circadian pacemaker neurons are required for each light response.

  P33

Identification of a second region regulating nuclear localization of the circadian clock protein mouse

Cryptochrome 1Karla Marz1, Pagkapol Y.  Pongsawakul2, Kimberley V. Sukhum3, Monica

E. Johnson4

1Biology, Gustavus Adolphus College, Saint Peter, MN, UNITED STATES2Salk Institute for Biological Studies, La Jolla, CA, USA

3Washington University in St. Louis, St. Louis, MO, UNITED STATES4Gustavus Adolphus College, Saint Peter, MN, UNITED STATES

The sequences of the C-terminal tails of vertebrate Cryptochromes (CRYs), though different in sequence, have both been shown to be necessary for CRY nuclear localization and sufficient to drive a cytoplasmic protein into the nucleus (van der Schalie et al., 2007).  mCRY1 and mCRY2 exhibit conserved sequence differences in another region, and in order to characterize this region, we generated a panel of site-directed hybrid mCRY1/2 mutants that had mCRY2 residues substituted into mCRY1. We found that these mutants were less effective than either mCRY1 or mCRY2 in repressing CLOCK/BMAL1-activated transcription in a luciferase reporter assay.  Three selected mutants from this panel exhibited decreased nuclear localization in HEK-293 cells, despite each having an intact C-terminal tail.  In addition, mutation of mCRY1 Serine71 to aspartate, a phosphomimetic mutation at a position shown to be phosphorylated by AMPK (Lamia et al., 2009), nearly abrogated mCRY1 nuclear localization.  Interestingly, the equivalent mCRY2 mutant, mCRY2(S89D), localized to the nucleus to the same degree as wild-type mCRY2.  The region altered in the mCRY1/mCRY2 hybrid mutants, which is adjacent to Serine71/Serine89 in

mCRY1/mCRY2, contains a loop/helix that is disordered in the recent crystal structure of mCRY1 (Czarna et al., 2013) but ordered in mCRY2 (Xing et al., 2003).  Because disorder in crystal structures often indicates increased mobility, it is possible that, in mCRY1 but not in mCRY2, this loop shifts in response to phosphorylation of a nearby serine to a shape that promotes retention of the protein in the cytoplasm.

  P34

Altered cryptochrome degradation influences GABAergic signaling and excitation of suprachiasmatic nucleus

neurons.Sven Wegner1, Mino D.C.  Belle1, Alun T.L. Hughes1, Hugh D. Piggins1

1Faculty of Life Sciences, University of Manchester, Manchester, UNITED KINGDOM

The mammalian suprachiasmatic nucleus (SCN) orchestrates circadian rhythms in physiology and behavior. Key components of the SCN molecular clock include the Cryptochrome (Cry1/2) genes and their protein products CRY1/2. The after-hours (Afh/Afh) mouse model carries a loss-of-function mutation in an ubiquitin ligase complex (Fbxl3), thereby delaying CRY degradation. As a result Afh/Afh mice exhibit lengthened circadian periods (~26.5h) and reduced rhythm strength in wheel-running activity. However, it is unknown how this alteration in CRY degradation influences the electrical activity of SCN neurons. Here, we made whole-cell patch-clamp recordings from SCN brain slices prepared from Afh/Afh and congenic control (+/+) mice housed under 12h light-12h dark and determined if and how the FBXl3 mutation affects SCN neuronal activity. In both genotypes, spontaneous electrical activity was higher during the day than at night. However, at night the ventral SCN cells from Afh/Afh mice are more hyperpolarized than their +/+ counterparts (~-47 mV in Afh/Afh versus ~-40mV in +/+). A similar though less pronounced genotype-related difference was also observed in night-time recordings from cells in the dorsal SCN (~-42 mV in Afh/Afh mice versus ~-38mV in +/+ mice, respectively).  Recordings from SCN slices prepared from Afh/Afh and +/+ mice housed in constant darkness also demonstrated these genotype-related differences at subjective night, indicating that they are intrinsic to the SCN and not driven by the light-dark cycle. Measurement of synaptic transmissions within the SCN at night showed that in comparison to +/+ ventral SCN cells, the GABAergic tone was elevated in corresponding Afh/Afh cells. This suggests that delayed CRY degradation prolongs night-time hyperpolarized states particularly of ventral SCN cells through increased GABAergic synaptic transmission. Such changes in electrophysiological behavior and neurotransmitter balance may also contribute to the reduced rhythm strength in behavior and SCN molecular oscillations observed in the Afh/Afh animals.

  P35

Magnetoreception in Drosophila melanogasterGiorgio Fedele1, Edward Green2, Ezio Rosato2, Charalambos Kyriacou4

1University of Leicester, Leicester, Leicestershire, UNITED KINGDOM2Genetics, University of Leicester, ,

4Genetics, UNiversity of Leicester, Leicester, UNITED KINGDOM

Many higher animals have evolved the ability to use the Earth’s magnetic field, particularly for orientation and navigation. However, the biophysical mechanism underlying magnetoreception remains elusive. One theoretical model (the radical pair mechanism - RPM) proposes that the geomagnetic field is perceived by chemical reactions involving the activated blue-light photoreceptor Cryptochrome (CRY). Recent evidence supports the RPM in Drosophila melanogaster, revealing a mechanistic link with the circadian clock. Here we reveal that exposure to a low frequency electromagnetic field (AC-EMF) or a Static Field (SF) robustly affects three behavioural phenotypes in the fruitfly, circadian rhythms, hyperactivity and negative geotaxis.  We genetically manipulate CRY both intra- and intercellularly and our results reveal some novel features of CRY-mediated magnetoreception that may indirectly support the RPM, but not in its classic form.

  P36

Roles of C-terminal truncated Bmal1 on circadian rhythmNoheon Park1, Solmi Cheon2, Hee-Dae Kim2, Kyungjin Kim2

1Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX, UNITED STATES2School of Biological Science, Seoul National University, Seoul, Seoul, KOREA SOUTH

Circadian rhythm is an endogenous biological clock which is comprised by the transcriptional /translational feedback loops of clock genes. Bmal1 is an indispensible transcription factor for consisting of circadian rhythm. Here, we report a new circadian mutant mice harboring C-terminal truncated Bmal1 (Bmal1mut). Mice carrying the Bmal1mut allele were generated from the gene-trapped embryonic stem cells. The homozygotic mutant (Bmal1mut/mut) mice immediately lost their rhythms under constant darkness condition. Interestingly, the heterozygotic (Bmal1+/mut) mice gradually lost their rhythms, whereas Bmal1+/- mice sustained the rhythms. Interestingly, Bmal1mut/mut mice sustained their fertility, whearas Bmal1-/- mice were sterile. The circadian gene expression profiles of Bmal1mut/mut mice were arrhythmic in vivo and in vitro implying that the arrythmicities were resulted from the impaired molecular clock machinery. Although there was no difference in the cellular localization and heterodimerization with CLOCK, the overexpression of Bmal1mut was unable to activate the transcription of Per1 promoter.  These results indicate that C-terminal region of Bmal1 has pivotal roles on the regulation of circadian rhythm and the Bmal1mut mice provide a new model system for evaluating the consequence of circadian rhythms.

  P37

Withdrawn

 

  P38

Structure/function interrogation of mCRY1 defines a distributed binding interface with the CLOCK/BMAL1

heterodimerClark Rosensweig1, Peng Gao2, Carla Green1

1Neuroscience, UT Southwestern Medical Center, Dallas, TX, UNITED STATES2UT Southwestern Medical Center, Dallas, TX, UNITED STATES

CRY1 is a critical part of the repressive machinery of the core molecular clock. However, there are many open questions regarding CRY1’s role in rhythm generation at the cellular level. In a screen to identify mCRY1 mutants that are weak repressors of CLOCK/BMAL1 transcriptional activity, we identified point mutations that resulted in decreased repressive activity. We used these mutants to test the hypothesis that repression by CRY1 is the key component of its rhythm-generating capacity. Cry1-/-/Cry2-/- mouse embryonic fibroblasts, which are arrhythmic, were co-transfected with a Luciferase reporter (under the control of the Per2 promoter) and mCry1 (under the control of its own promoter and intronic elements). The cells were synchronized by dexamethasone treatment and luciferase activity was monitored in a LumiCycle over the course of a week. Wild-type mCRY1 rescues rhythmic activity in these cells. However, our various point mutants have a range of effects on the cells’ rhythmic activity; several mutants failed to rescue rhythms in the cells outright, while others rescue rhythms, but with a change in period length. The half-lives of the mutants were indistinguishable from wild-type mCRY1, suggesting that decay dynamics were not a factor in either rescue capabilities or the period length changes. Co-immunoprecipitation experiments indicated that the mutants had variable binding affinities for CLOCK and BMAL1, which correlated with rescue activity. These data suggest that there is a threshold of interaction between CRY1 and the CLOCK/BMAL1 heterodimer necessary to drive rhythmic activity in cells. Moreover, the mutations are widely distributed across the mCRY1 structure and largely superficial when mapped to the existing crystal structure, which suggests a substantial binding interface between the three proteins.

  P39

Withdrawn.

  P40

The effect of interneuronal communication between clock neurons in Drosophila

Qi Zhang1, Orie Shafer1

1University of Michigan, Ann Arbor,

Daily circadian physiology and behavior are essential for living of animals. In Drosophila, the central circadian clock, consisting of about 150 clock neurons, orchestrates circadian behaviors such as feeding, mating, locomotor activity and sleep. The clock neurons express Period (Per), Timeless (Tim), Clock (Clk), Cycle (Cyc) and other circadian clock genes to form a transcriptional and translational feedback loop. Though circadian rhythms rely on this cell-autonomous molecular clock, interneuronal communication between different groups of clock neurons is critical for normal circadian rhythms. Such communication is thought to adjust the molecular clock of the LNd neurons of the fly to maintain a synchronized and robust clock neuron network. Indeed, recent evidence in mammals and insects suggests that connectivity between clock neurons can overcome some genetic loss of molecular clock function within the brain. For example, the interneuronal restores rhythmic Per2-Luc activity in the Cry null Suprachiasmatic Nucleus, (Maywood et al. 2011). However little is known about the effects of interneuronal communication on molecular circadian clocks within the clock neuron network. To address such communication, we restored the Per function in Per01 mutants specifically in the PDF expressing LNv neurons and addressed which aspects of molecular timekeeping were rescued in the LNds. We found that Tim cycling was not rescued in LNd by the restoration of molecular clock in specifically in LNv. In contrast, the cycling of PDP1 Ɛ, another circadian molecular in positive limb of feedback loop, was restored in LNd presumably through interneuronal signaling from sLNv. We concluded that only some components of circadian clock can be rescued through interneuronal communication. Since Pdp1 Ɛ is in the positive limb of feedback loop, we hypothesize that the positive limb of feedback loop in LNd can be restored through interneuronal signaling from the sLNv and the rescued positive limb in LNd is important to rhythmic locomotor output.

  P41

Translational control of the circadian clock through the cap-binding protein eIF4E

Ruifeng Cao1, Andrew Liu2, Shimon Amir3, Nahum Sonenberg4

1Department of Biochemistry, McGill University, Montréal, QC, CANADA2Department of Biological Sciences, The University of Memphis, Memphis, TN, UNITED STATES

3Center for Studies in Behavioral Neurobiology, Concordia University, Montreal, QC, CANADA4Department of Biochemistry and Goodman Cancer Research Center, McGill University, Montreal, QC,

CANADA

The mammalian circadian clock is endogenously driven by interlocking transcriptional/posttranslational feedback loops. However, little is known on how translational regulation modulates the physiology of the master circadian pacemaker in the brain, the suprachiasmatic nucleus (SCN).  mRNA translation is a highly regulated process. Control of translation predominantly occurs at the level of translation initiation, which begins with the recognition of the m7GpppN (where N is any nucleotide) structure (5’cap) by the eukaryotic translation initiation factor 4E (eIF4E). Because eIF4E is the least abundant initiation factor, 5’cap-recognition by eIF4E is rate-limiting for translation and therefore a major target for regulation. eIF4E amounts and activity are controlled at many levels including transcription, phosphorylation, and interactions with its binding partners including  the family of 4E binding proteins (4E-BPs) . 4E-BPs are phosphorylated and deactivated by the PI3K/AKT/mTOR pathway.    eIF4E is phosphorylated by MNKs as a target of the MAPK signaling pathway. Thus, eIF4E is a converging point of regulation by the mTOR and MAPK pathways. In this presentation I will discuss the roles of eIF4E-dependent translational control in the circadian clock.

I will first discuss the functions of the pivotal translational repressor, 4E-BP1 in the SCN.  The activity of 4E-BP1 is under circadian control via the mechanistic target of rapamycin (mTOR) signaling.  4E-BP1 knockout (KO) leads to accelerated circadian re-synchronization to a shift in the light/dark cycle at the behavioral and cellular level.  Remarkably, the mice lacking 4E-BP1 are less susceptible to forced clock desynchrony by constant light.  The behavioral phenotypes of KO

mice are associated with increased vasoactive intestinal peptide (VIP) signaling in the SCN, inasmuch as 4E-BP1 preferentially represses Vip mRNA translation.  These results reveal a key role of 4E-BP1-mediated translational control in synchrony and entrainment of the circadian clock. Next, I will talk about the role of eIF4E phosphorylation in the mammalian circadian clock.  eIF4E is phosphorylated at Ser209 by Mnk1/2 via the MAPK/ERK pathway. We found that eIF4E is rhythmically phosphorylated in the SCN as well as in peripheral tissues such as the liver.  eIF4E phosphorylation is regulated by light in the SCN. To study the biological functions of this event, we generated the eIF4E mutant mice (eIF4E KI), in which Ser209 is mutated to Alanine and therefore cannot be phosphorylated.  These mice show shorter circadian period and attenuated photic entrainment.  The behavioral phenotypes are consistent with decreased level of PERIOD proteins in the brain, which can be mechanistically explained by decreased Per1 and Per2 mRNA translation in the eIF4E KI mice.  Thus, eIF4E phosphorylation facilitates PER1 and PER2 protein expression at the level of mRNA translation. These results give insights on how protein synthesis is regulated in the circadian clock and provide potential pharmacological targets to manipulate the functions of the circadian clock.

  P42

The NRON/KPNB1 Complex Regulates Nuclear Translocation and Function of the Circadian Clock

YOOL LEE1, Au Reum  Jang2, Lauren J Francey1, Amita Sehgal2, John B Hogenesch1

1Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA, UNITED STATES

2Department of Neuroscience, University of Pennsylvania School of Medicine, Philadelphia, PA, UNITED STATES

Regulated nuclear translocation is a critical and evolutionarily conserved aspect of signal transduction. Here we report that components of the NRON/KPNB1 complex, first identified in both flies and mammals as key regulators of NFAT signaling, regulate nuclear translocation and function of the circadian clock. Complex members function in phosphorylation (GSK3β, CK1ε, DYRK2), regulated proteolysis (PSMD11, CUL4B), and nuclear import (KPNB1) and export (CSE1L). Depletion of most pathway components leads to period length deficits or arrhythmic clock function in human cells. We show that complex members interact with circadian clock proteins and co-localize at the nuclear membrane. Inhibition of a conserved complex member, importin β (KPNB1), traps clock factors in the cytoplasm and abolishes gene expression rhythms in human cells and behavioral rhythms in flies. In sum, these data show this complex integrates regulated phosphorylation, proteolysis, and nuclear translocation functions of the circadian clock, adding an important new layer of understanding to the circadian clock mechanism.

  P43

Development of circadian pacemaker cells in the Drosophila brain

tianxin liu1, GURUSWAMY MAHESH2, Jerry Houl3, Paul  Hardin3

1Biology, Texas A&M University, College Station, TX, UNITED STATES2BIOLOGY, TEXAS A&M UNIVERSITY, COLLEGE STATION, TX, UNITED STATES

3Department of Biology and Center for Biological Clocks Research, Texas A&M University, College Station, Texas, UNITED STATES

 The identification and analysis of clock genes in Drosophila revealed that the circadian clock is driven by a transcriptional feedback loop in which CLOCK-CYCLE (CLK-CYC) heterodimers activate transcription of their feedback repressors PERIOD(PER) and TIMELESS (TIM). Since CLK is required to intiate feedback loop function, CLK expression can be used as a primary marker for clock cells. CLK is detected in presumptive brain pacemaker neurons as early as the embryonic stage (ES) 16, and initiates rhythmic PER expression in ~16 brain pacemaker neurons during the L1 larval stage. PER marks ~16 brain pacemaker neurons throughout larval development, and late in pupal development PER expression expands to include all ~150 brain pacemaker neurons found in adults. We hypothesize that the expansion of PER positive brain neurons results from the proliferation of clock neurons in pupae. To detect CLK protein expression in brain neurons with high sensitivity, transgenic flies expressing a CLK-GFP fusion protein were generated and probed with an anti-GFP antibody. We found that CLK-GFP is expressed in ~16 brain neurons in L2 larvae, but then CLK-GFP expression rapidly expands just after the L2-L3 transition to encompass ~150 brain pacemaker neurons. Based on their location, these neurons represent the vast majority of brain pacemaker neurons found in adults. However, only ~16 of these CLK-GFP positive brain pacemaker neurons express PER, consistent with previous reports. CLK-GFP expression is only detected in cells that co-express the post-mitotic marker ELAV, demonstrating that CLK-GFP is expressed in differentiated rather than actively dividing cells. These results suggest that essentially all brain pacemaker neurons found in adults are present in L3 larvae, but only a small subset of theses neurons have a functional clock. Ongoing experiments to test whether the brain pacemaker neuron circuit is already assembled in L3 larvae and determine why most CLK-GFP expressing cells are non-functional will be presented.

 

  P44

The beginning of in vivo clock gene expression rhythmicity in the fetal rat SCN

Pavel Houdek1, Alena Sumova2

1Department of Neurohumoral Regulations, Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, CZECH REPUBLIC

2Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, CZECH REPUBLIC

The mammalian SCN and its rhythmicity develops gradually during ontogenesis. In the rat, the SCN is formed from the embryonic day (E) 14 through E17, and the gestation terminates at E21-22. Intrinsic SCN rhythmicity is already present in the late embryonic stage; day-night oscillation of metabolic activity, Avp mRNA levels and firing rate of the SCN neurons were detected already from E19, at E21 and at E22, respectively. Our previous study demonstrated an absence of circadian rhythms in Per1, Per2, Cry1, Bmal1 and Clock expression at E19, and the rhythms developed via increasing amplitude since birth up to the postnatal day 5-10 (Sladek at al., 2004). It remains to be elucidated whether the intrinsic rhythmicity during prenatal period results from a functional fetal SCN clock, or it is driven by rhythmical maternal cues. Therefore, the aim of the present study was to ascertain in detail prenatal development of clock gene expression rhythms. To achieve this, we used a more sensitive method for detection of the levels of clock gene transcripts than in the previous studies. Daily profiles of gene expression were detected in the rat SCN at E19 and E21. The fetal brains were collected in 3 h intervals during the 24 h period, the SCN were precisely dissected (using laser dissection), and expression of clock genes Per2, Rev-erbα and Bmal1, and of c-fos, Avp and Vip was detected by qRT PCR. The results demonstrated that at E19, expressions of canonical clock genes Per2 and Bmal1 were not rhythmic. However,

expressions of other studied genes followed clear circadian rhythms. At E21, Bmal1 exhibited still a very low amplitude rhythm, but Per2 was already expressed rhythmically with a high amplitude. The expressions of the other genes were also rhythmic. The data confirmed our previous finding on the absence of the Per2 and Bmal1 rhythms in the fetal SCN at E19. Moreover, the results provide evidence that in the fetal SCN, the intrinsic rhythmicity is present before the molecular clock mechanism fully develops. Therefore, the intrinsic rhythmicity in the fetal SCN is driven by maternal rather then fetal circadian system. 

 The study was supported by the Czech Science Foundation grant P303121108

  P45

An ultradian rhythm of somite formation is modulated by xBmal1 and xNocturnin in Xenopus laevis

Kristen Curran1, Latoya Allen1, Nicole Johnson1, Chelsea Lope1, Gail Willadsen1, ELizabeth  Campbell1, Brett VonBergen1,

Devon Winfrey1, Morgan Hadley1, Thomas Kerndt10

1Biological Sciences, University of Wisconsin Whitewater, Whitewater, WI, UNITED STATES10BIological Sciences, University of Wisconsin Whitewater, Whitewater, WI, UNITED STATES

We have been investigating whether xBmal1and xNocturnin play a role in somitogenesis, a cyclic developmental process with an ultradian period.    Previous work from our lab shows that circadian genes (xPeriod1, xPeriod2, xBmal1, and xNocturnin) are expressed in developing somites.  In Xenopus, a pair of somites is formed about every 40-50 minutes from anterior to posterior.  Somites eventually form the vertebrae, muscles of the back, and dermis.   We were intrigued by the co-localization of circadian genes in an embryonic tissue known to be regulated by an ultradian clock.   Cyclic expression of genes involved in Notch signaling has been implicated in the somite clock.  Disruption of Notch signaling in humans has been linked to skeletal defects in the vertebral column.  We found that both depletion (morpholino) and overexpression (mRNA) of a xBMAL1 protein (bHLH transcription factor) and xNOCTURNIN protein (deadenylase) on one side of the developing embryo led to a significant decrease in somite number with respect to the untreated side (p<0.001).  These manipulations also significantly affect expression of a somite clock component (xESR9; p<0.02).    We observed opposing effects on somite size.  Depletion of xBMAL1 or xNOCTURNIN caused a statistically significant decrease in somite area (quantified using NIH ImageJ; p<0.002), while overexpression of these proteins caused a significant dose dependent increase in somite area (p<0.001).  We speculate that xBmal1 and xNocturnin play two roles during somitogenesis.  First, they influence the timing of somitogenesis by modulating the type and amount of bHLH proteins available in each cell.  Later, xBmal1 and xNocturnin promote myogenesis.

  P46

The circadian molecular clock regulates adult hippocampal neurogenesis by controlling the timing of cell-cycle entry

and exit.Pascale Bouchard-Cannon1, Lucia Mendoza-Viveos1, Andrew Yuen1,

Mads Kaern4, Hai-Ying Mary Cheng1

1Biology, University of Toronto Mississauga, Mississauga, OR, CANADA4Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, CANADA

The subgranular zone (SGZ) of the adult hippocampus contains a pool of quiescent neural progenitor cells (QNPs) that are capable of entering the cell cycle and producing newborn neurons. The mechanisms that control the timing and extent of adult neurogenesis are not well understood. Here, we show that QNPs of the adult SGZ express molecular-clock components and proliferate in a rhythmic fashion. The clock proteins PERIOD2 and BMAL1 are critical for proper control of neurogenesis. The absence of PERIOD2 abolishes the gating of cell-cycle entrance of QNPs, whereas genetic ablation of bmal1 results in constitutively high levels of proliferation and delayed cell-cycle exit. We use mathematical model simulations to show that these observations may arise from clock-driven expression of a cell-cycle inhibitor that targets the cyclin D/Cdk4-6 complex. Our findings may have broad implications for the circadian clock in timing cell-cycle events of other stem cell populations throughout the body.

  P47

Hyperoxia affects neonatal lung circadian dynamics and worsens injury

Shaon Sengupta1, Guang Yang1, Phyllis Dennery3

1Neonatology, Children's Hospital of Phialdelphia, Philadelphia, PA, UNITED STATES3University of Pennsylvania, Philadelphia, PA, UNITED STATES

Background: Exposure to cigarette smoke (oxidative stress) has been associated with changes in lung circadian gene expression and lung function. However, the role of circadian genes, especially Rev-erbα in the developing lung in response to stressors such as hyperoxia is not known. Objective: We hypothesized that the expression of the key circadian genes, Rev-erbα, Bmal and Per1, is regulated by hyperoxia in neonatal mice and that disrupted circadian rhythm may exacerbate hyperoxic lung injury. Also, given the importance of Rev-erbα, this response to this injury maybe modulated by altering the expression of Rev-erbα in animals [by using a Rev-erbα KO and phosphomutant with stabilized Rev-erbα (SD) mice.]Design/Methods: Newborn (<12 hours old) C57Bl/6, Rev-erbα KO, Rev-erbα SD (over-expressor of Rev-erbα) mice pups were exposed to any of the following groups: >95% oxygen (O2) + CL (constant light);>95% O2 + 12hr light-dark (LD) cycles;RA (Room Air) + CL;RA + 12h LD cycle(each for 0-72 hrs).The lungs were removed at the end of exposure (day 3). Another subset was exposed to either RA or O2 for 3 days under 12hr LD cycling and samples were collected at 6am (ZT0), 10am (ZT4), 6pm (ZT12), 10pm (ZT16). Expression of circadian genes and their downstream targets (p21, cmyc, PGC1α, OGG1) and lung histology was studied.Results:In WT animals, Bmal1, per2, Rev-erbα and Rev-erbß as well as downstream targets p21 and PGC1α showed significant oscillations in normoxia. In hyperoxia, this rhythm was dampened. In the hyperoxia exposure group,

light had a differential effect on the expression of Rev-erbα and Bmal1 expression. We found significantly increased levels of anti-proliferative gene, p21 in O2+CL group of Rev-erbαKO mice, while this increase is relatively modest in Rev-erbαSD mice. Rev-erbα KO lungs had significantly decreased PCNA positivity (index of proliferation) at baseline, which was further exacerbated by hyperoxia exposure. In contrast, Rev-erb alpha SD mice had significantly incresed proliferation at baseline, which was much less reduced after hyperoxic exposure.Conclusions: In the neonatal lung, hyperoxia disrupted the rhythmic expression of clock genes. Using transgenic mice to alter the expression of key circadian gene, Rev-erbα the expression of anti-proliferative gene, p21 can be modulated. This suggests that of Rev-erbα may have a protective role in mediating hyperoxic lung injury.

  P48

Maternal effects on circadian gene expression in fetal kidneys

Krisztina  Meszaros1, Linda Pruess2, Matthias Gondan3, Eberhard Ritz4, Franz Schaefer1

1Division of Pediatric Nephrology, Center for Pediatrics and Adolescent Medicine, Heidelberg University, Heidelberg, GERMANY

2 Heidelberg University, Heidelberg, 3Department of Psychology, University of Copenhagen, , , DENMARK

4Department of Internal Medicine, Heidelberg University, Heidelberg, GERMANY

Background: The circadian rhythm of key kidney functions such as electrolyte and water excretion involving blood pressure control is driven by the clock gene network. In adults, broad adverse effects of the disturbed circadian rhythm are known, whereas potential role of the maternal circadian clock disturbances in prenatal programming of renal function has not been studied yet. We sought to explore the effect of altered light exposure and restricted feeding schedule on the development of circadian gene expression in the fetal kidney.

Methods: Pregnant rats were randomly allocated into 5 groups. The rats were fed ab libitum under 12h:12h light-dark cycle (LD), constant light (LL), constant darkness (DD), or ultradian, 6h:6h light-dark cycle (6-6LD). Another group of pregnant rats was kept under 12h:12h light-dark cycle with food access restricted to the light period (FR). Mothers (6-10 per group) with their offspring were sacrificed at 4-h intervals one day before the expected delivery. Intrarenal circadian gene expression patterns were profiled by real-time rtPCR for the canonical clock genes Rev-erbα and Per2 and the clock-controlled genes NHE3, αENaC, SGK1, and AVPR2.

Results: Kidneys of fetuses from mothers kept at LD displayed circadian expression of the investigated genes [Rev-erbα (p = 0.014), Per2 (p = 0.014), NHE3 (p < 0.001), αENaC (p < 0.001), SGK1 (p = 0.017), and AVPR2 (p = 0.003)]. Intrarenal gene expression of the fetuses from mothers exposed to LL exhibited robust circadian expression with phases similar to those observed at LD [Rev-erbα (p = 0.027), Per2 (p < 0.001), NHE3 (p < 0.001), αENaC (p < 0.001), SGK1 (p < 0.001), and AVPR2 (p = 0.022)]. In contrast, circadian fluctuations of intrarenal gene expression were completely absent in fetuses with maternal DD and 6-6LD exposure. In the animals exposed to maternal FR circadian rhythmicity persisted for Per2, was lost for Rev-erbα and showed a distinct phase shift for the clock-controlled genes.

Conclusion: Our findings show that in the developing kidney the circadian clock network becomes operational even before birth and oscillatory gene expression is affected differently by different types of light cue modification during pregnancy. In addition, the oscillatory expression of kidney specific clock-controlled genes responds to nutritional cues independently of canonical clock gene input.

  P49

Dopamine and Melatonin Regulate Ocular Circadian Rhythms

Kenkichi Baba1, Susana  Contreras-Alcantara1, Gianluca Tosini1

1Pharmacology/Toxicology, Morehouse School of Medicine, Atlanta, GA, UNITED STATES

Purpose: Previous studies have shown that the retina, the retinal pigment epithelium (RPE) and the cornea contain a circadian clock that controls the circadian rhythms in PER2::LUC bioluminescence. Additional studies have indicated that only in the retina the PER2::LUC rhythm can be phase-shifted by light, thus suggesting that other signals are used by the retina to entrain the circadian rhythms in other ocular structures. Melatonin (MLT) and dopamine (DA) are known to be the key molecules to regulated retinal circadian rhythms. In this study we investigated the role of DA and MLT to and their associated receptors in the regulation of RPE and cornea circadian rhythm.

Methods: Eyes were obtained from PER2::LUC mice, the eye-cups were dissected, and the retina, RPE-choroid and cornea were cultured at 37 oC with 199 medium containing 0.1mM D-Luciferin K salt. The bioluminescence emitted by these tissues was measured using a LumiCycle. After 3-4 days of culture, DA (100uM), MLT (100nM) and DA or MLT receptor agonists were added to the culture at different circadian times, and the recording was continued another 5 days.

Results: Administration of DA, but not MLT, phase-shifted PER2::LUC bioluminescence rhythm in the RPE-choroid. Sumanirole (D2R agonist) induced a significant phase-shift during the late night -early subjective day, whereas PD168077 (D4R agonist) did not produce a significant phase-shift of the PER2::LUC bioluminescence rhythm. Conversely, an administration of MLT, but not DA, phase-shifted PER2::LUC bioluminescence rhythm in the cornea. Our further experiment showed IIK7 (MT2 agonist) induced a significant phase-shift during night.

Conclusions: Our data indicate that DA via D2 receptors can phase-shift the circadian rhythm in PER2::LUC bioluminescence rhythm in the mouse RPE, whereas MLT via MT2 can phase-shift the circadian rhythm in PER2::LUC bioluminescence in the cornea. Thus indicating that the retina uses DA and MLT to entrain other tissues within the eye.  Our data also indicate that activation of these signaling pathways is the correct timing of circadian functions in these tissues.

  P50

Utilizing Electroretinograms (ERG) to Analyze Circadian Rhythms in Grompadorhina Portentosa Photoreceptor

Sensitivity.Wil  Bogue1, Andrew Urdiales1, Edgar Mantes1, Aaron Schirmer1,

Frederick  Prete1

1Biology, Northeastern Illinois University , Chicago, IL, UNITED STATES

 The electroretinogram (ERG) is a sub-corneal, extracellular recording reflecting the summed electrical responses of the compound eye photoreceptors in response to square wave light pulses. Previous studies using several species of cockroach have shown the ERG to be a robust technique by means of which to analyze changes in photoreceptor activity under various experimental conditions. Our analysis revealed that Grompadorhina Portentosa’s ERG is a complex waveform consisting of four distinct components: transient and sustained ON potentials elicited by light stimulus onset, and transient and sustained OFF potentials elicited by stimulus offset. ERG recordings at 15 min intervals over 96 continuous hours under constant darkness conditions revealed circadian rhythms in the sustained ON and OFF components of the ERG reflecting oscillations in photoreceptor sensitivity. Further analyses revealed rhythmic changes in the latencies to the maximum amplitudes of both the sustained ON and OFF potentials; however, the period of these oscillations does not appear to be circadian in nature. Interestingly, the circadian periods observed only emerged after the roach was adapted to the experimental set-up for 3-4 days. This first analysis of G. portentosa’s ERG provides a novel source of data regarding the cellular and physiological changes in its visual systems. We believe these changes well adapt them to their dimly lit ecological niche. Future research will examine the molecular underpinnings of these circadian oscillations in this cockroach’s visual physiology.

 

  P51

Retinal Muller Cells are Circadian Clock Cells and Clock Genes Impact Retinal Neovascularization

Douglas McMahon1, Lili Xu1, John Penn1, Andrew Liu4

1Vanderbilt University, Nashville, TN, UNITED STATES4University of Memphis, Memphis, TN,

Circadian rhythms generation is likely distributed across several cell types in the retina.  Here, we have shown that retinal Muller glia express the full complement of core circadian clock genes and exhibit circadian clock function, demonstrating circadian rhythms in bioluminescence from purified mouse Muller cells derived from PER2::LUC circadian reporter mice.  Retinal Muller cell cultures exhibit robust freerunning near 24-hour rhythms in gene expression that persist in for several days.  These rhythms are inhibited by knockout, or knockdown of the clock genes Period1 or Bmal1.  Human Muller cells transduced with lentiviral circadian gene reporters also exhibit robust rhythms.  Given that Muller cells are an important source of vascularizing signals in the retina, and Period clock genes have been shown to influence VEGF in tumor cell lines, we examined the potential impact of the Period1 and Period 2 on VEGF secretion and retinal neovascularization in a mouse model of oxygen induced retinopathy (OIR).  We found that in Per1/Per2 double knockout mice VEGF secretion was influenced rhythmically by the circadian clock, that peak VEGF levels were increased in OIR in Per1/2 double knockout retinas vs wt, and that there was an increase in retinal neovascularization in the Per1/2 double knockout retinas.  These results indicate that retina Muller glia are circadian clocks, even when isolated from other retinal cell types, and suggest that clock genes, perhaps in the Muller glia, are important regulators of  neovascularization signals. COI/Financial Disclosure None.

  P52

Physiological and behavioural consequences of destabilised entrainment in melanopsin knockout mice

Violetta  Pilorz1, Russell Foster1, Stuart Peirson1

1Department of Clinical Neurosciences , University of Oxford , Oxford , Oxford , UNITED KINGDOM

Light is the primary environmental stimulus for entraining the master circadian pacemaker of mammals to the 24hr solar cycle - a process termed photoentrainment. As such, numerous physiological and behavioural rhythms are either directly or indirectly regulated by the light/dark cycle. Melanopsin-expressing photosensitive retinal ganglion cells (pRGCs) play a key role in transmitting the light input from the retina to the suprachiasmatic nuclei (SCN). However, classical rod and cone photoreceptors are able to fully compensate in the absence of melanopsin, mediating photoentrainment via the pRGC pathway.  As such, a direct contribution of melanopsin to photoentrainment appears limited. Here we show that in the absence of melanopsin, mice show increased activity and feeding duration during the light phase. These findings suggest a change in the stability of entrainment in OPN4-/- mice, which are confirmed by differences in the phase angle of entrainment under extreme photoperiods. Differences in physiological and behavioural rhythms are also reflected by increased activity and food intake throughout the dark phase. Under light/dim-light conditions, OPN4-/- mice exhibit accelerated food anticipatory activity without changes in body mass and food consumption. The increased diurnal feeding and activity, coupled with lower body mass compared to controls provides evidence to suggest that destabilised entrainment in OPN4-/- mice results in metabolic consequences. In summary, this data provides new insight into the role of melanopsin in stabilising circadian rhythms, and as a result, energetic balance during long term entrainment.  

  P53

Retinal projections to the suprachiasmatic nucleus: from morphology to function

DIEGO FERNANDEZ1, Shih-Kuo  Chen2, Samer Hattar1

1Biology, Johns Hopkins University, Baltimore, MD, UNITED STATES2Institute of Zoology, National Taiwan University, , TAIWAN

Light detected by the mammalian eye allows conscious perception of images, detection of movement and the synchronization of circadian rhythms to the solar day. The majority of retinal ganglion cells (RGCs) project to image-forming centers in the brain. However, recently discovered RGCs that express the photopigment melanopsin (Opn4) are themselves atypical photoreceptors (intrinsically photosensitive (ip)RGCs) and project to several brain nuclei that regulate non-image forming functions, such as the suprachiasmatic nucleus (SCN). The SCN is located in the anterior hypothalamus, and is the locus of a circadian clock responsible for the temporal regulation of physiological and behavioral rhythms. In most morphological studies done so far, it has been difficult to decipher which cell types are innervated by RGCs because of the dense retinal innervation that the SCN received. Using detailed morphological and tracing analysis, we characterized the cell types in the SCN that receive synaptic inputs from the retina. Furthermore, we generated a melanopsin (Opn4) driven conditional Cre line that causes Cre-mediated excision and the expression of a tracer (Opn4creert2/+; Z/AP). Using these

animals in combination with very low dosis of Tamoxifen, we were able to label single ipRGCs in the retina and trace their axonal trajectories to the brain. We found that most cells in the SCN received retinal projections, at somal and dendritic level. Interestingly, we also observed that a single ipRGC showed a complex arborization pattern that, by sending collaterals, could innervate different brain areas.

The SCN also received projections from the intergeniculate leaflet (IGL), a brain area also involved in the control of the circadian activity that received retinal input. In base of this interesting interconnected circuit, we studied the pattern of innervation from the IGL to the SCN. We found that IGL fibers make functional contacts with most SCN neurons and also with retinal projections. Genetic ablation of ipRGCs that constitute the retinal input to the SCN, drastically affected the IGL innervation.

Together, these results shed light into the complex pattern of retinal and IGL innervation to the SCN. In base of these data, it is plausible to hypothesize that retinal terminals play a central role in the correct circuit formation within the SCN.

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ipRGC neurotransmitters, glutamate and PACAP, are distinct in their contributions to non-image forming

behaviorsWilliam Keenan1, Samer Hattar1

1Biology, Johns Hopkins University, Baltimore, MD, UNITED STATES

Intrinsically photosensitive retinal ganglion cells (ipRGCs) are required for relaying light information for subconscious visual behaviors such as circadian photoentrainment and the pupillary light reflex (PLR). However, the mechanisms by which ipRGCs mediate the fast and sustained signaling dynamics required for the PLR and photoentrainment are poorly understood. One possible way by which ipRGCs could achieve this flexibility is through the use of multiple neurotransmitters. ipRGCs are known to contain two neurotransmitters: glutamate and PACAP, and, remarkably, our data indicate that these neurotransmitters each contribute to distinct aspects of photoentrainment and the PLR. Specifically, we find glutamate is required for rapid re-entrainment to a shifting light cycle and the fast kinetics of the PLR but not required for long-term photoentrainment and PLR maintenance. These results provide insight into how a single cell type is capable of broad and dynamic regulation of behavior.

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Involvement of 5-HT3 and 5-HT4 receptors in the regulation of circadian clock gene expression in mouse small intestine

Natsumi Aoki1, Hiroyuki Watanabe2, Kazuya Okada2, Kazuyuki Aoki2, Yuko Ikeda5, Atsushi Haraguchi2, Yu Tahara7, Shigenobu Shibata8

1School of Advanced Science and Engineering, Waseda Univercity, Shinjuku-ku, Tokyo, JAPAN2School of Advanced Science and Engineering, Waseda University, Shinjuku-ku, Tokyo, JAPAN

5School ofAdvanced Science and Engineering, Waseda University, Shinzyuku-ku, Tokyo, JAPAN7Waseda University, Shinjuku-ku, Tokyo, JAPAN

8School of Advanced Scinces and Enginnering, Waseda university, Tokyo, Tokyo, JAPAN

Several lines of evidence suggest that 5-HT receptors play a critical role in the expression of clock genes in the suprachiasmatic nucleus, the main circadian oscillator in hamsters. The contributions of 5-HT receptor subtypes in the intestine, where they are expressed at high concentrations, are however not yet clarified. The 5-HT synthesis inhibitor, para-chlorophenylalanine, attenuated the daily rhythm of Per1 and Per2 gene expression in the intestine. Injection of 5-HT and agonists of the 5-HT3 and 5-HT4 receptors increased Per1/Per2 expression and decreased Bmal1 expression in a dose-dependent manner. Although treatment with antagonists of 5-HT3 and 5-HT4 alone did not affect clock gene expression, co-injection of these antagonists with 5-HT blocked the 5-HT-induced changes in clock gene expression. Increased tissue levels of 5-HT due to treatment with the antidepressants clomipramine and fluvoxamine did not affect clock gene expression. The present results suggest that the 5-HT system in the small intestine may play a critical role in regulating circadian rhythms through 5-HT3/5-HT4 receptor activation.

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Serotonergic enhancement of photic phase shifts: BMY7378 does not require the serotonergic fibers connecting the median raphe nucleus to the suprachiasmatic nucleus

Victoria Smith1, Michael Antle2

1Department of Psychology, University of Calgary, Calgary, AB, CANADA2Psychology, University of Calgary, Calgary, Alberta, CANADA

Serotonin mixed agonists/antagonists have been shown to greatly potentiate photic phase shifts, and this potentiation has been shown to require the 5-HT1A receptor. Preliminary findings from our lab suggest that this potentiation of photic phase shifts occurs as a result of receptor binding in the median raphe nucleus (MRN), but not in the suprachiasmatic nucleus (SCN). The goal of the present experiment was to examine the effects of BMY7378 on light-induced phase shifts when administered systemically following a loss of the serotonergic fibers connecting the MRN and the SCN. Adult male hamsters were exposed to either a 5,7-DHT or sham lesion to the SCN, eliminating the serotonergic fibers connecting these two structures. Housed in constant darkness, hamsters were then exposed to a systemic pretreatment of either BMY7378 (5mg/kg) or vehicle control 45 minutes prior to a 15-minute light pulse (40 lux) at CT18. Pretreatment with BMY7378 resulted in a potentiation of the photic phase shift that was comparable in magnitude following either a 5,7-DHT or sham lesion to the SCN. These results suggest that BMY7378 binding to 5-HT1A receptors in the MRN does not potentiate photic phase shifts by altering the magnitude of serotonin released by the fibers that directly connect the MRN to the SCN, indicating that the indirect route via the intergeniculate leaflet is the likely pathway pertinent to this potentiation effect.

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Investigating ASIC1a as a potential link between circadian disruption and mood disorders in mice

Jonathan Shelton1, Sujin Yun1, James Shoblock1, Natalie Welty1, Christine Dugovic5

1Janssen R&D, San Diego, CA, UNITED STATES5Jassen R&D, San Diego,

Recent clinical and animal studies have demonstrated an association between circadian disruption and mood disorders.  Therefore, pursuing genes that stabilize circadian rhythms may prove useful as a therapeutic intervention for mood disorders such as depression.  One such target, the acid sensing ion channel (ASIC1a) is localized in tissues known to regulate circadian rhythms including the SCN and whose activation has been implicated in mood disorders.  To investigate a potential link between ASIC1a and circadian rhythms in relation with mood disorders, wheel running locomotor activity was monitored in ASIC1a KO animals under both entrained and free running conditions and in parallel depressive-like behavior was assessed using the tail suspension test in these animals. 

ASIC1a KO and WT mice were placed on running wheels so that locomotor activity could be assessed under an entrained light/dark schedule and also constant darkness.  Various circadian parameters under both lighting conditions were then measured.  In a separate study to assess depressive-like behaviors, ASIC1a KO and WT mice were subjected to the tail suspension test.

During a normal light/dark schedule, ASIC1a KO mice demonstrated less anticipatory activity prior to dark onset and the activity during the first 4 hours of the dark phase was attenuated by half when compared to WT controls.  During constant darkness, wheel running activity was similarly decreased in the ASIC1a KO animals and the period was significantly longer than WT mice.  In the tail suspension test, the immobility time in the ASIC1a KO mice was significantly reduced when compared to WT controls indicative of an anti-depressant-like phenotype and thus replicating a previously published study.   

Given the current preliminary data demonstrating a potential role for ASIC1a in the regulation of circadian rhythms and its association with an anti-depressant-like phenotype, decreasing ASIC1a activity by therapeutic intervention may prove beneficial in alleviating mood disorders by altering circadian rhythms.  Future studies are planned to evaluate the potential anti-depressant/anxiolytic/mood stabilizing properties of ASIC1a inhibitors.

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A Mutation in PERIOD3 Causes Familial Advanced Sleep Phase

Luoying Zhang1, Chris Jones2, Noriaki Sakai3, Ying Xu4, Noriko Saigoh4, Kazumasa Saigoh4, Shu-Ting Lin4, Seiji Nishino3, Louis Ptacek4, Ying-

Hui Fu4

1Neurology, University of California, San Francisco, San Francisco, CA, USA2University of Utah, Salt Lake City,

3Stanford University, Palo Alto, CA, UNITED STATES4University of California, San Francisco, San Francisco,

Familial advanced sleep phase (FASP) is a human phenotype characterized by early evening sleep time and early morning awakening. Here we report a mutation in the circadian clock gene PERIOD3 (PER3) to be associated with FASP. Besides having circadian phenotypes, these FASP subjects also exhibit elevated depression index and seasonality. Transgenic flies carrying the mutant hPER3 show advanced phase and shortened period of activity rhythm compared to hPER3-WT flies, mimicking the human FASP phenotype. Transgenic mice expressing the mutant hPER3 also show altered behavioral period, accompanied by depression-like behavior. Further molecular characterizations reveal that the mutation destabilizes PER3 protein and enhances the degradation of PERIOD2 protein, which plays a critical role in timing the circadian clock. Taken together, our findings suggest that a mutation in PER3 result in phase-advanced clock and depression in human, implicating that PER3 serves as a nexus for circadian rhythm and mood regulation.

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Perinatal photoperiod affects the serotonergic systemNoah Green1, Chad  Jackson2, Douglas McMahon2

1Biological Sciences, Vanderbilt University, Nashville, TN, UNITED STATES2Vanderbilt University, Nashville,

Depression and anxiety disorders are significant problems for human health and are thought to involve alterations in brain serotonin signaling. Our laboratory has recently demonstrated that the circadian photoperiod experienced during perinatal development has enduring effects on depression and anxiety related behaviors and on serotonergic neuronal function.  Dorsal raphe neurons are acutely modulated by melatonin and mice developed on different seasonal photoperiods are presumed to have different amounts of melatonergic signaling during development due to the different duration of the dark period.  We hypothesize that perinatal light cycles exert their enduring influence on depression and anxiety behaviors in part through developmental melatonergic programming of raphe serotonergic neuronal function. We have developed C3Hf+/+ mice, on an equinox (12:12), long (16:8) or short (8:16) photoperiods to determine the effect these developmental light cycles have on the serotonergic system.  Our results demonstrate that perinatal photoperiod significantly affects serotonergic neuronal firing rate, 5-HT and its metabolite concentrations, expression of key serotonergic genes and depression and anxiety related behaviors during young adulthood.  We have also observed that the changes in serotonergic firing rate are negated in melatonin 1 receptor knockout mice suggesting melatonin is an integral player in the developmental photoperiodic programming of serotonergic neuronal physiology.  These experiments will establish a novel model for exploration of how a pervasive environmental signal, the light dark cycle, may influence the development and long-term function of brain serotonergic neurons and affective behaviors. 

 

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In patients with Alzheimer’s disease, correlations in motor activity fluctuations respond to bright light therapy are

associated with mood and cognitionKun Hu1, Frank Scheer1, Steven Shea3, Rixt  Riemersma - van der Lek4,

Eus Van Someren5

1Brigham & Women's Hospital/Harvard Medical School, Boston, MA, UNITED STATES3Oregon Institute of Occupational Health Sciences, Oregon Health & Science University, Portland, OR,

UNITED STATES4University Medical Center Groningen, Groningen, NETHERLANDS

5Netherlands Institute for Neuroscience, Amsterdam, NETHERLANDS

Fluctuations in motor activity in many species, including humans, have similar temporal correlations at different time scales, from minutes up to 24h. Those activity correlations at time scales >2h are greatly influenced by the circadian pacemaker. The source of activity correlations at time scales <2 h is unknown. Certain recent studies indicate depression reduced these correlations but other studies provided controversial results. Depression is common in patients with dementia and Alzheimer’s disease (AD), and is associated with reduced cognitive ability and quality of life. Thus, we hypothesize that AD patients with severe depression and cognitive decline have reduced short-term activity correlations. Since light and melatonin treatments can be beneficial for cognitive functions in AD patients, we further hypothesize that these treatments can help to maintain/improve activity correlations in AD. To test these hypotheses, we analyzed

activity data of 164 AD patients who underwent a double-blind, placebo-controlled, 2x2 factorial randomized trial for up to ~3.5 years: (1) 44 subjects exposed to whole-day bright light (1000 lux) every day; (2) 41 taking 2.5 mg melatonin every evening; (3) 41 receiving both light and melatonin treatments; and (4) 38 without either treatment. For each assessment, ~14 days of activity data were analyzed and correlations at time scales from 0.02-2 h were quantified by a detrended fluctuation analysis-derived exponent α, i.e., α=0.50 for white noise without correlations, and larger α (>0.5) for stronger correlations. In the present sample of AD patients, α was between 0.75 and 1.18 at baseline and decreased by ~0.03 per year on average (p<0.0001). Light treatment partially counteracted the decrease in activity correlations, increasing α by 0.018 per year (p=0.015). Melatonin treatment had no significant influence on α. After accounting for the yearly changes of cognitive and depression measures, mixed model analyses showed that smaller α was associated with lower scores in the mini–mental state examination (p=0.0053), and higher scores in the Cornell Scale for Depression in Dementia (p=0.0048), the Multi Observational Scale for Elderly Subjects withdrawn behavior subscale (p=0.00063) and the Philadelphia Geriatric Center affect rating scale (negative affect; p=0.022). These results indicate that reduced activity correlations at small time scales reflect cognitive decline and depressive symptoms in AD and that light treatment may decelerate behavioral disturbances in these patients.

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Early wake therapy phasae-delays advanced melatonin offset and improves mood in depressed pregnant women

Barbara Parry1, Charles Meliska2, Diane Sorenson2, Fernando Martinez2, Ana Lopez2, Henry Orff2, Richard Hauger2

1University of California, San Diego, La Jolla, 2UCSD, La Jolla, CA

Aims:  To test the hypothesis, based on previous data indicating that melatonin circadian rhythms were phase-advanced in pregnant depressed women (DW) and phase-delayed in postpartum DW, that early night wake therapy (EWT) that delays sleep, would benefit mood in pregnant DW more than late-night wake therapy (LWT) that advances sleep.  Methods:   In 58 women, 31 pregnant (7 DW; 24 healthy women-HW) and 27 postpartum (14 DW; 13 HW), we measured plasma melatonin every 30 minutes in dim (<50 lux)/dark conditions from 18:00-11:00 h.  In 21 DW (7 pregnant, 14 postpartum) and 37 HW (24 pregnant, 13 postpartum), mean age 28 years, we randomized in a cross-over design to EWT (sleep 03:00-07:00 h) vs. LWT (sleep 21:00-01:00 h) followed by a night of recovery sleep (RS-22:30-06:30 h).  A clinician administered mood ratings (Hamilton Depression Rating Scale-HDRS) pre- and post-treatment (after RS).   Results: At baseline, in pregnancy, DW vs. HW had earlier melatonin offset (p=.045) and lower mean total melatonin (p=0.01), peak (p=.037) and area under the curve (AUC, p=.016); postpartum DW vs. HW had higher mean morning melatonin (p=.031), somewhat higher AUC (p > .05) and shorter melatonin duration (p=.055).  Mood scores by HDRS improved with LWT in pregnant (p=.004) and postpartum (p=.014) DW, and with EWT in pregnant (p= .002) and postpartum (p=.011) DW.  LWT improved HDRS scores by 48.3% in pregnant and 46.2% in postpartum DW; EWT improved mood scores by 57.1% in pregnant and by 34.5% in postpartum DW. Melatonin onset (p=.013) and synthesis offset (p=.048) were delayed after EWT in

pregnant, but not postpartum DW (p>.05); melatonin changes after LWT were inconsistent in pregnant and postpartum DW.  Conclusion:  Both EWT and LWT reduced depressive symptoms as measured by the HDRS in pregnant and postpartum women. In pregnant DW with phase-advanced melatonin offset time, EWT, which delays sleep onset time, corrected the melatonin abnormality and improved mood.

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Inhibition of specific classes of histone deacetylases reduce anxiety- and depression-like behaviors in ClockΔ19 mutant

miceRyan Logan1, Rachel Arey2, Angela Ozburn3, Nicole Edgar3,

Colleen McClung3

1Univeresity of Pittsburgh, Pittsburgh, 2Princeton University, Princeton,

3University of Pittsburgh, Pittsburgh, PA, UNITED STATES

Emerging evidence implicates alterations of epigenetic mechanisms and circadian rhythms as putative contributors to the pathophysiology and the treatment of mood disorders. Our previous studies indicate mice carrying a mutation in Clock (ClockΔ19) display a behavioral repertoire similar to the clinical symptomology of human bipolar mania (e.g., reduced anxiety and depression). CLOCK and other circadian proteins epigenetically regulate gene transcription through multiple mechanisms suggesting links between epigenetics, circadian rhythms, and mood disorders. A commonly prescribed mood stabilizer for bipolar disorder, valproic acid, directly inhibits the activity of class I and II histone deacetylases (HDACs). HDACs are epigenetic enzymes that regulate gene transcription, such as by chromatin and post-translational modifications. Other diseases are currently being treated with HDAC inhibitors. These inhibitors have the potential to cause long-lasting and stable transcriptional changes that may contribute to their “therapeutic utility” for mood disorders. Therefore, our studies have begun to determine the “therapeutic utility” of HDAC inhibitors on bipolar disorder by examining the effects of HDAC inhibitors on anxiety- and depression-like behaviors in an animal model of bipolar mania (ClockΔ19 mice). Male and female wild-type and ClockΔ19 mice were treated with valproic acid (class I and II HDAC inhibitor), suberoylanilide hydroxamic acid (SAHA; pan-HDAC inhibitor), MS275 (class I HDAC inhibitor), or MC1568 (class IIa HDAC inhibitor) for 10-14 days then tested in the elevated plus maze, dark-light box, open-field, and forced swim tests. Following treatment, acetylation of histones, protein, and gene expression were measured in mood- and reward-related brain regions. Each of the treatments reduced anxiety- and depression-like behaviors selectively in male and female ClockΔ19 mice, except MC1568, which caused a mixed behavioral state. Valproic acid and SAHA increased global acetylation at histones H3 and H4 and had differential effects on the expression of dopamine-related genes in the ventral tegmental area. The effects of valproic acid to reduce manic-like behaviors in ClockΔ19 mice may be via class I HDAC inhibition, as treatment with MS275 caused similar effects on behavior. The present findings further support the potential of HDAC inhibitors for the treatment of mood disorders. Ongoing studies using ChIP-seq and RNA-seq, combined with viral-mediated gene knockdown, are planning to identify therapeutic targets of these inhibitors. (Support: IMHRO Johnson & Johnson (McClung) and NARSAD (Logan)).

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Association of Depression with Variations of Melatonin and Cortisol Rhythms in Delayed Phase Sleep Disorder (DSPD)

PatientsSeong Jae Kim1, Kathryn Reid1, Sabra Abbott1, Phyllis Zee1

1Neurology, Northwestern University Feinberg School of Medicine, Chicago, IL, UNITED STATES

Introduction: Depressive symptoms are commonly reported by those with delayed sleep phase disorder (DSPD), and is thought to be associated with alterations in circadian rhythm function. Abnormal alterations in phase and amplitude of melatonin and cortisol have been reported in endogenously depressed patients. Therefore, we tested the hypothesis that in DSPD, alterations in circadian timing and amplitude of cortisol and melatonin are associated with depressive symptoms. Methods: Twenty-one DSPD (32.2±11.1years; M: F=12:9) and 15 controls (31.7±14.3 years; M: F=7:8) completed an inpatient study at the Northwestern Clinical Research Unit (CRU). Participants with major psychiatric disorders were excluded. Participants maintained a regular habitual sleep schedule for 1 week prior to the CRU admission. Plasma melatonin and cortisol were collected at 30- to 60-min intervals under a modified constant routine condition (dim light < 10 lux), mood was assessed using the global affect (GA) visual analogue scales every 2 hours. Dim light melatonin onset (DLMO) defined as 2SD above baseline and nadir of cortisol were used as circadian phase markers, and second peak value was used for cortisol amplitude. Cross-correlation analyses were performed to determine time-ordered relationships between the changes of GA and cortisol and melatonin over 24hours. Results: Compared to control, DSPD patients had a significant decreased average GA scores (68.4±11.6 vs 81.5±9.3, p<0.05) and increased average cortisol amplitude (98.4±33.9 vs 73.5±26.8, p=0.058), and there were effects of time and group on mood state (F11, 319=4.116; F1,29=8.38, p<0.01) and cortisol amplitude (F11, 264 =32.94, p<0.01; F1,24 =3.87, p=0.06). DSPD patients showed significantly delayed phase in DLMO and cortisol nadir (23.8±2.0h vs 20.2±1.6h; 25.2±2.7hvs20.4±4.0h, p<0.01), and higher amplitude of the cortisol rhythm (251.5±85.3vs183.3±65.3, p=0.039). For total sample, average GA scores were negatively correlated with DLMO and timing of peak cortisol (p<0.001), and a trend of a negative correlation with cortisol amplitude (p=0.055). In addition, there was a negative correlation between GA scores and cortisol peak by 1hour lead (r=-0.29, p<0.001) and 1hour lag(r=-0.28, p<0.001) for DSPD patients, but lead& lag correlations were not observed in control subjects. Conclusion: The relationship between alterations in the timing and amplitude of circadian rhythms with depressive symptoms, supports a role of circadian dysregulation in the high prevalence of depression in DSPD. Depression in DSPD patients seems to stimulate cortisol secretion, but the elevated cortisol might have a negative effect rather than a compensatory effect on mood, suggesting abnormal cortisol endocrine response in depression.

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Circadian abnormalities in the Myshkin mouse model of mania

Joseph TImothy1, Harshmeena Sanghani1, Mino Belle1, Hugh Piggine1

1University of Manchester, Manchester,

Sleep and circadian rhythm disturbance are common pathologies observed in many neuropsychiatric disorders. For example bipolar disorder (BPD), which is characterised by oscillating episodes of mania and depression,  is often accompanied by changes in the sleep-wake cycle. Interest in the role of the circadian system in the aetiology and pathology of BPD was heightened following the observation of mania-like phenotypes in mice possessing mutations in clock genes, Clock (Clock∆19) and Fbxl3 (Afterhours).

Recently, a mouse line known as Myshkin, which carries a mutation in the neuron-specific α3 subunit of the Na+/K+-ATPase, was identified as a model of the manic phase of BPD. These animals showed hyperlocomotion, reduced anxiety, increase in goal-directed behaviours and reduced sleep. Some of these phenotypes were attenuated through treatment with either lithium or valproic acid. As major questions persist as to the role of the circadian system in affective disorders, we used this model to understand changes to the circadian physiology underlying the Myshkin phenotype.

Under constant dark the wheel-running rhythms of Myshkin animals show an array of characteristics that point towards alterations to the underlying regulation of circadian behaviour. These changes included long free-running period (~24.2hrs) extremely lengthened alpha duration (~19hrs), reduced rhythm amplitude and spontaneous alterations in tau. Myshkin animals also show unusual responses to light, including abnormal phase re-setting during the late subjective night.

Having observed overt changes in circadian behaviour, alterations in the functioning of the Myshkin SCN were investigated using whole-cell current-clamp recordings and fura-2 calcium imaging. Acute electrophysiological properties of Myshkin SCN neurons were not grossly abnormal from control +/+ animals yet over the 24-hour day, failed to show a clear rhythm in spontaneous firing rate or resting membrane potential. Responses to GABA and the glutamate receptor agonist, AMPA, showed no genotype difference, indicating that Myshkin mouse neurons retain normal synaptic responses.

These findings show that the physiology of the SCN in the Myshkin model of mania is altered resulting in a decrease in the amplitude of day-night output that potentially contributes to abnormal circadian behaviour. This establishes that the Myshkin mouse has many circadian disturbances, from the cellular to the whole-animal level, which are consistent with observations of disruption to biological timekeeping in human BPD.

 

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Brain circadian clocks in a mouse model of depressionDominc Landgraf1, Christophe D. Proulx1, Roberto Malinow1, David

K. Welsh1

1UCSD, San Diego, CA, UNITED STATES

Neuropsychiatric disorders like major depressive disorder (MDD) are associated with disrupted circadian rhythms and sequence variation in circadian clock genes. Therapies for mood disorders, including light treatment and lithium, also affect the circadian system. While circadian rhythms are mainly dependent on the master pacemaker in the hypothalamic suprachiasmatic nucleus (SCN), the SCN synchronizes circadian clocks in other brain regions and peripheral organs, which harbor less robust rhythms. Since critical reward circuits, like the dopamine and serotonin systems, are under circadian control, we hypothesize that mood disorders are associated with disturbances of circadian rhythms predominantly in non-SCN brain regions involved in mood regulation. Using the PER2::LUC reporter, we identified alterations of rhythms in mood-related brain regions in mice undergoing learned helplessness, a behavioral model of depression. We found that helplessness in mice is frequently associated with arrhythmicity in a subset of brain regions implicated in mood regulation, including the nucleus accumbens. In these brain regions, arrhythmicity arises from significantly fewer cells expressing PER2, a broader phase distribution of PER2 rhythms, or both. Based on our studies, we believe that strong circadian rhythms in the nucleus accumbens and other mood-regulating brain areas increase resilience against depression-like behavior. In future experiments, manipulating rhythms in specific brain regions in healthy mice will provide the opportunity to test the role of circadian rhythms as a causal factor in mood disorders. Since mood-regulating brain regions play similar roles in mice and humans, our study has great potential to improve the understanding and treatment of MDD.

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Daily Temporal Rhythms in Cellular Activity in the Lateral Habenula

Hugh Piggins1, Kanwal Sakhi2, Sven Wegner1, Mino Belle4, Michael Howarth1, Tim Brown6

1Faculty of Life Sciences, University of Manchester, Manchester, UNITED KINGDOM2Faculty of Life Sciences, University of Mancheser, Manchester, UNITED KINGDOM

4Faculty of Life Science, University of Manchester, Manchester, Greater Manchester, UNITED KINGDOM6University of Manchester, Manchester, Lancashire, UNITED KINGDOM

Intrinsic daily or circadian rhythms in physiology and behaviour emerge through the synchronization of brain and local tissue-specific clocks with environmental time cues. Much research has focused on the timekeeping properties of the master circadian clock in the hypothalamic suprachiasmatic nuclei (SCN) and established that SCN neurons express rhythms in clock gene expression and electrical activity, even when isolated from the rest of the brain and body as tissue slice explant. Rhythmic clock gene expression has been noted in other brain structures distal to the SCN, but little is known about the organization and expression of neuronal activity rhythms in these extraSCN sites.  One such candidate extraSCN oscillator is the lateral habenula (LHb) which is located in epithalamus, adjacent to the dorsal third ventricle. In this study, we used a coronal brain slice preparation that isolates from the LHb from the SCN, and assessed, using whole cell patch-clamp electrophysiology, whether LHb neurons express a daily rhythm in input resistance (Rinput), resting membrane potential (RMP), and spontaneous action potential firing rate (SFR).  The majority of LHb neurons recorded, showed spontaneous spiking, while a minority were in non-spiking or near quiescent states. We found that SFR varied

significantly across the projected day-night cycle, with lower spiking rate in the morning as compared to the late day and early night.  By contrast, neither Rinput nor RMP varied significantly across the projected day-night cycle.  The daily change in SFR was absent in LHb slices prepared from Cry1-/-Cry2-/- mice, which lack a molecular circadian clock.  Assessment of Per1-luc expression in LHb brain slices indicated a low amplitude rhythm in bioluminescence in slices prepared from Cry1+/+Cry2+/+ mice, whereas such rhythmic activity was absent in LHb explants from Cry1-/-Cry2-/- mice.  In summary, these investigations reveal that the LHb expresses a daily rhythm in electrical activity and clock gene expression that appears to depend on the presence of a functional molecular clock. We are now determining how environmental and other extrinsic signals influence LHb neuronal activity.

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Cyanobacterial Clock Output Feeds Back through Metabolism to Regulate Clock Input

Gopal K. Pattanayak1, Connie Phong1, Michael J.  Rust1

1Department of Molecular Genetics and Cell Biology, Institute for Genomics and Systems Biology, University of Chicago, Chicago, IL,

Circadian clocks are biological oscillators that allow an organism to anticipate daily changes in its external environment. In a simple clock found in cyanobacteria, metabolic signals that include ATP/ADP ratio and the redox state of the plastoquinone pool impinge directly on oscillator components and couple the clock to rhythmic changes in the environment. But it is unclear how these metabolic mechanisms are related to known clock input mutants, and what factors determine the metabolic response to unanticipated darkness. We find that a mutant with a desensitized clock input phenotype acts by misregulating clock output genome-wide leading to altered dark metabolism.  This metabolic effect is sufficient to recapitulate a desensitized input response in an in vitro model of the clock. We show that the circadian clock generates a rhythm in energy storage metabolism and conversely, mutations that disrupt dark metabolism make clock input hypersensitive to darkness. Our results reveal there is a feedback loop that connects clock output back to clock input through rhythmic metabolism.

  P68

Intracellular Distributions of the KaiABC Proteins During the Cyanobacterial Circadian Cycle: A Spatiotemporal

Simulation Stefanie Hertel1, Pål O. Westermark1

1Institute for Theoretical Biology, Charité - Universitätsmedizin Berlin, Berlin, Berlin, GERMANY

In the model cyanobacterium Synechococcus elongatus PCC 7942, the interactions among KaiA, KaiB and KaiC proteins generate circadian oscillations in the KaiC phosphorylation states. These rhythms are even observed in an in vitro system. During the circadian cycle, KaiAC, KaiBC and KaiABC complexes assemble and disassemble.

The standard ratio of Kai proteins in the in vitro KaiC phosphorylation system is evidently different from that in vivo. The relative amount of KaiA to KaiC in vitro is 1/3 whereas it is at least 1/20 in cyanobacterial cells. When the latter ratio is used in the in vitro system, oscillations are damped. Furthermore, fractions of KaiB and KaiC are localized in the membrane whereas KaiA is found in the cytoplasm. The three proteins have also been observed at one of the cell poles but polar localization of KaiA is dependent on the presence of KaiB and phosphorylated KaiC. Two questions arise from these findings. First, do the different intracellular distributions of the Kai proteins eventually lead to effective intracellular ratios that are close to those observed in vitro? And second, how do spatial concentration gradients of KaiA, KaiB and KaiC affect KaiABC complex formation during the circadian cycle? To address these, we reformulated an existing mathematical model of the KaiABC protein oscillator (Brettschneider et al., MSB 2010) as a reaction-diffusion system. Such a model takes into account not only biochemical reactions, but also diffusion of molecules. We analyzed the kinetics of KaiA, KaiB and KaiC considering diffusion of the three Kai proteins between membrane and cytoplasm. This spatiotemporal simulation makes it possible to understand the role of concentration gradients of KaiA, KaiB and KaiC for the functioning of the KaiABC clock.

The results of these simulations strongly point to a need for considering localization in order to understand the in vivo KaiABC clock quantitatively.

  P69

Circadian regulation of oxidative stress-induced Stress Granules

Julio Pusterla1, Juan Lescano1, Victoria A. Acosta Rodríguez1, Mario E. Guido1, Eduardo Garbarino-Pico1

1CIQUIBIC-CONICET, Dpto de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Córdoba, ARGENTINA

It is believed that the exposition to high levels of UV-radiation during the daytime was one of the strongest selective forces that led to circadian rhythms emergence. Accordingly, the stress response is circadianly modulated in numerous model systems. When are exposed to stress, many cell types form microscopically visible foci called stress granules (SG). SG are mainly formed by stalled translational initiation complexes. Since a number of SG components display circadian rhythms, we hypothesized that SG formation may be circadian regulated. To investigate that, we administrated arsenite to synchronized NIH3T3 cell cultures to generate oxidative stress and induce the formation of SG. We found temporal changes in number, signal intensity and size of arsenite-induced SG detected with an anti-eIF3 antibody by ICC. Since total eIF3 protein levels remained constant across the time, the temporal differences observed suggest a redistribution of the protein. Furthermore, we did not find any change along time in the phosphorylation of eIF2α, an event involved in SG nucleation. We next analyzed the mRNA levels of several RNA-binding proteins that are component of SG and could be involved in the

temporal changes observed. Interesting, Tia1, Brf1, hnRNPQ, and Lark transcripts presented temporal changes in their levels. Indeed, we observed important circadian changes in the expression of TIA-1, a protein capable of inducing SG nucleation. We also detected that LARK protein levels (also known as RBM4) were highly induced by oxidative stress and the magnitude of these induction was time-dependent. Our results show that SG formation exhibits circadian rhythms and that this phenomenon may be involved in the circadian regulation of stress response.

  P70

Circadian regulation of actin dynamicsNed Hoyle1, John O'Neill1

1MRC Laboratory of Molecular Biology, Cambridge, Cambridgeshire, UK

The actin cytoskeleton is a dynamic structure critical to cellular form and function.  Regulation of actin polymerisation and depolymerisation is known to directly affect cell morphology and motility.  It has recently been reported that actin polymeric state is circadian in mouse liver and spleen, but the nature of the circadian control of actin is unknown.Interplay between actin dynamics and the circadian clock is possible in multiple modes.  Firstly, the actin regulating factors Cofilin and Arp2 are expressed with a circadian profile at the mRNA level in mouse liver, suggesting direct control of actin polymerisation through well-defined actin regulation pathways.  However, actin dynamics have been shown to control gene expression, for example through the sequestration of the transcription factor MRTF, a known regulator of PER2 expression. This raises the possibility of feedback control though the actin cytoskeleton.  Furthermore, direct oxidation of actin monomers can stimulate disassembly of actin fibres.  Cellular rhythms in redox status may therefore provide a control point for the modulation of actin dynamics and secondary effects on cellular metabolism. We present data showing global rhythms in actin polymeric state in immortalised fibroblasts, indicating that actin dynamics are regulated over circadian time in a cell intrinsic manner.  Using cells from the arrhythmic Cry1-/-/Cry2-/- double homozygous null mouse strain we demonstrate that these rhythms are linked to the functionality of the canonical BMAL: CLOCK - PER: CRY transcription-translation feedback loop.  We present live cell imaging data visualising actin dynamics over circadian time and investigate the mechanistic basis of circadian actin dynamics using a combination of immuno-precipitation and mass spectrometry.  

  P71

Diverse Circadian Periods from Individual Cells: Stochastic or Clonal?

Yan Li1, Yongli Shan1, Hung-Chung Huang1, Seung-Hee Yoo4, Joseph Takahashi5

1Department of Neuroscience, The University of Texas Southwestern Medical Center, Dallas, TX, UNITED STATES

4Department of Biochemistry and Molecular Biology, UT Health Science Center at Houston, Houston, TX, UNITED STATES

5Department of Neuroscience, Howard Hughes Medical Institute, The University of Texas Southwestern Medical Center, Dallas, TX, UNITED STATES

Biolumininescence imaging of immortalized ear fibroblast cells isolated from mPer2LucSV knockin mice revealed that individual cells can oscillate robustly with diverse circadian periods (range: 21.7-27.5 hr, mean: 24.8 ± 1.1 hr SD) and independent phases. To investigate whether the diverse distribution of periods is due to stochastic or clonal factors, we made 150 clonal cell lines derived from single cells from the parent culture described above. Periods calculated from lumicycle recording of the 150 clonal cell lines exhibited broadly distributed periods and were relatively stable over generations demonstrating that period is, at least in part, clonally determined. Ten clonal cell lines were chosen for detailed analysis: 5 with short periods (< 23.5 hr) and 5 with long periods (> 26 hr). Single-cell imaging with further statistical analysis suggested that clonal cell lines with longer period exhibited more period variability. Within a clonal cell line, period length was not correlated with cell size. We also produced a second generation of single-cell derived clonal cell lines from two of the above-mentioned clonal cell lines, with short or long period, respectively. Lumicycle data of the subclones and single-cell imaging analysis of the corresponding parent clone demonstrated comparable period distribution with the consistent mean value. From these experiments we can conclude that both environmental/stochastic and heritable/clonal factors play important roles in determining circadian period heterogeneity. Further effort was made to elucidate what the heritable/clonal factors might be. Neither exonic DNA sequencing, nor whole genome sequencing revealed any causative gene mutations associated with period variability. Core clock gene expression profiles also showed no significant difference between long or short period clonal cell lines. Preliminary data suggest that epigenetic modifications may be involved in periodicity determination.

  P72

An Ultradian Rhythm in Mouse Embryonic Fibroblast (MEF) Cell Lines

Shuzhang Yang1, Seung-Hee Yoo2, Yongli Shan3, Yan Li3, Joseph Takahashi1

1Howard Hughes Medical Institute, Department of Neuroscience, UT Southwestern Medical Center, Dallas, TX, UNITED STATES

2Department of Biochemistry and Molecular Biology, UT Health Science Center at Houston, Houston, TX-Texas, UNITED STATES

3Department of Neuroscience, UT Southwestern Medical Center, Dallas, TX-Texas, UNITED STATES

Ultradian rhythms have been reported in yeast, a strain of cyanobacterium Cyanothece sp., and a chondrogenic cell line ATDC5. Here report the existence of ultradian rhythms in mouse embryonic fibroblast (MEF) cell lines. Using luciferase as reporter we detected ultradian rhythms in multiple MEF cell lines, with periods ranging from 4 to 12 hours. The ultradian rhythms develop gradually upon cell-cell contact, and are synchronized, likely through gap junctions. Inhibition of oxidative phosphorylation, but not glycolysis, suppressed ultradian cycles. Chelating extracellular calcium also blocked ultradian cycles. The ultradian cycles can be induced in other cells which do not have bona fide ultradian cycles, such as human osteoblastoma cell line U2OS and mouse fibroblast cell line NIH3T3 by mixing and co-culture with ultradian MEFs. These induced ultradian cycles required cell-cell contact with ultradian MEF cells in order to be expressed.

  P73

Transcriptional responses during synchronization of clocks in mouse and human cells. Julie Baggs1, Jason DeBruyne2

1Neuroscience Institute, Dept. of Pharmacology & Toxicology, Morehouse School of Medicine, GA, USA2Neuroscience Institute/Dept. of Pharmacology & Toxicology, Morehouse School of Medicine, Atlanta, GA,

UNITED STATES

Immortalized cell line models have been extremely useful for understanding how circadian clocks function. Human U2-OS and mouse NIH3T3 cell lines have been broadly used to explore circadian clockwork mechanisms, and thus far, those mechanisms appear to be very similar. We sought to determine if these similarities extend to processes underlying clock resetting as well by comparing the transcriptional responses occurring during synchronization. We explored the responses in U2-OS and NIH3T3 cells to three commonly used synchronization agents: dexamethasone, forskolin and 50% horse serum (serum shock).  Cultures of each cell line were harvested at dense time intervals for the first six hours to determine the acute transcriptional responses using microarrays, and at three-hour intervals thereafter to compare circadian dynamics by qPCR. Overall, we found a surprisingly large number of genes whose expression was varied> 10-fold in response to each stimulus. Transcriptional responses could be sorted into 4-8 self-organizing patterns, depending on cell line and treatment. In addition, we found that many long non-coding RNAs dynamically responded to these inputs. Overall, these data should shed some light on the signaling and resetting processes shared between mouse and human circadian machinery.

  P74

Genome-wide analysis of circadian clock properties in human fibroblasts

Ludmila Gaspar1, Steven Brown2

1University of Zurich, Institute of Pharmacology and Toxicology, Zurich, SWITZERLAND2Institute of Pharmacology and Toxicology, University of Zurich, Zurich, SWITZERLAND

Humans show large inter-individual differences in organizing their behavior within the 24-hour day, and multiple studies showed that this diurnal preference, or chronotype, is highly heritable. Until now, only a few reports about the genetic basis of human natural diurnal variation have been published. Taking advantage of genomic approaches and the conservation of circadian clocks in peripheral tissues, we employed a lentiviral circadian reporter system and real-time bioluminometry to determine different clock properties in a fully genotyped cohort of 200 human primary umbilical cord fibroblast lines, with the aim of deciphering functional genetic variants that determine natural human individual differences in circadian chronotype at a cellular level. Using this methodology, we have uncovered hundreds of polymorphisms reaching suggestive significance, including a novel SNP in a putative Per1 enhancer region, as well as two SNPs reaching stringent genome-wide significance criteria and affecting the expression of Per2.  Currently, we are validating candidates by genome-wide RNAi-based screen technologies and by looking for global enrichment of relevant alleles in extreme chronotypes cohorts. The same technology can be applied as well to examine links between the circadian clock and disease.  Beyond the core circadian clockwork, many other conserved signaling pathways influencing both circadian clock function and its effects upon human

physiology and behavior can also be examined. For example, although we saw no changes in core circadian function in fibroblasts from a cohort of patients with Bipolar Disorder, we made the further observation that the amplitude of drug-activated CREB signalling in cells from human skin biopsies correlated with bipolar disorder in affected individuals.  cAMP/CREB signalling is known to be important to circadian hormonal variation and to synaptic plasticity.  Knowing that inter-individual differences in cAMP/CREB signaling affect both the human endocrine response to light and susceptibility to bipolar disorder would make it a common neurological signaling pathway that could help explain the observed relationship between this disorder, circadian behavior, and light exposure. Consistent with this hypothesis, we also found that subjects with elevated CREB signaling in fibroblasts showed reduced suppression of melatonin levels by light in vivo, reflecting the dependence of melatonin synthesis upon adrenergic mechanisms. Given the role of the cAMP/CREB-signaling pathway in synaptic plasticity, as a part of the circadian clockwork, and in the regulation of melatonin synthesis, the relationships that we uncover could help furnish therapeutically useful endophenotypes of bipolar disorder and its treatment.

 

  P75

The circadian clock in the Antarctic krill Euphausia superbaBenjamin Hunt1, Özge Özkaya1, Paul Seear3, Ted Gaten3, Nathaniel Davies1, Eran Tauber1, Charalambos Kyriacou1,

Geraint Tarling8, Ezio Rosato1

1Genetics, University of Leicester, Leicester, Leicestershire, UNITED KINGDOM3Biology, University of Leicester, Leicester, Leicestershire, UNITED KINGDOM

8Biological Sciences Division, British Antarctic Survey, Cambridge, Cambridgeshire, UNITED KINGDOM

The Antarctic krill Euphausia superba is a keystone species of the Southern Ocean, the major link between primary production and predators at higher trophic levels. The behaviour,  development and metabolism of Euphausia is synchronised to environmental cycles ranging from the circadian to the circannual, from daily vertical migration patterns and swarming behaviour to strategies of maturation, reproduction and quiescence tracking the longer rhythms of sea ice extent and algal blooms, and there is evidence that these phenomena may be influenced by the biological clock.  Little is known, however, of the Euphausia clock at a molecular level. Here we describe the cloning and characterisation of the main molecular components of the krill clock as well as a number of regulatory components through the use of degenerate PCR and the creation of a de novo assembled transcriptome using RNA-seq. We have furthermore begun analysis of their pattern of expression via quantitative reverse-transcription PCR (Q-RT-PCR). Based on domain analysis and Q-RT-PCR results we speculate that the central circadian heterodimer may possess unique characteristics that enable Euphausia to sustain biological rhythmicity during the extreme

summer and winter polar light cycles. Phylogenetic analysis of the central components of the clock show Euphausia orthlogs near the base of vertebrate (EsCLOCK and EsBMAL1) and invertebrate (EsPERIOD and EsTIMELESS) branches. Combined with the discovery of a light-sensitive, Drosophila -like CRY1, this evidence suggests that the Euphausia clock may possess an ancient, perhaps unique design.

  P76

Understanding timekeeping in an intertidal crustacean Eurydice pulchra

Lin Zhang1, David C Wilcockson2, Michael H Hastings3, Simon G Webster4, Charalambos P Kyriacou1

1Genetics Department, Leicester University, Leicester, UNITED KINGDOM2Institute of Biological, Environmental, and Rural Sciences, Aberystwyth University, Aberystwyth, UNITED

KINGDOM3Laboratory of Molecular Biology, MRC, Cambridge, UNITED KINGDOM

4School of Biological Sciences, Bangor University, Bangor, UNITED KINGDOM

Animals that inhibit intertidal zone adjust their activities to the ebb and flow of the tides as well as to the light dark cycles. However, the molecular mechanisms underlying tidal behaviour and physiology are poorly understood. Here we demonstrate that an intertidal crustacean, Eurydice pulchra, exhibits self-sustained robust tidal swimming activities with a ~12.4 h period that is temperature compensated and entrainable by a vibration stimulus. Eurydice also shows ~24 h circadian rhythms of chromatophore-pigment dispersion, which can be entrained with light/dark cycles and a circadian molecular cycle in timeless expression. Using both environmental and molecular manipulations we were able to disrupt circadian physiological, behavioural and molecular rhythms. Tidal behaviour however just kept on ticking normally. Our results suggest that a working circadian clock in not required for Eurydice’s tidal behaviour.

Lin Zhang, Michael H. Hastings, Edward W. Green, Eran Tauber, Martin Sladek, Simon G. Webster,Charalambos P. Kyriacou, and David C. Wilcockson. (2013). Dissociation of Circadian and Circatidal Timekeeping in the Marine Crustacean Eurydice pulchra. Current Biology 23(19),1863-73.

  P77

First description of circadian rhythms in visual sensitivity, predatory behavior, and locomotion in a praying mantis

Aaron Schirmer1, Frederick Prete1, Edgar Mantes3, Wil Bogue3, Andrew Urdiales3

1Biology, Northeastern Illinois University, Chicago, IL, UNITED STATES3Northeastern Illinois University, Chicago, IL, UNITED STATES

Although circadian rhythms have been described in a variety of insect species, studies across multiple levels of analysis have only been conducted in a small number of key model systems such as Drosophila, cockroaches, and crickets.  Further, with the exception of two studies conducted over thirty years ago, no thorough analyses have been carried out on the praying mantises. We used a multilevel experimental approach to determine whether and/or to what extent circadian rhythms modulate several key physiological and behavioral parameters in the praying mantis, Hierodula patellifera.  The experiments included chronic electroretinograms (ERG) to assess compound eye sensitivity, photographic colorimetric analyses of changes in

compound eye color resulting from the migration of shielding pigments, analyses of gross locomotor activity on a modified treadmill apparatus, and assessment of the differences between responsiveness to prey-like, computer generated visual stimuli during periods of maximum vs. minimum compound eye sensitivity. Our results clearly indicate that circadian clocks modulate the target behaviors across all levels of our analysis.  Specifically, strong rhythms, which persisted in constant conditions with periods of approximately 24 hours, were discovered in optic lobe sensitivity to light, appetitive responsiveness to prey-like stimuli, and gross locomotor activity. Further, circadian clocks modulating both pigment migration and locomotor behavior strongly responded to light/dark cycles, suggesting these clocks were able to anticipate and entrain to environmental light cues.  These data indicate that circadian rhythms are present at the cellular, systems, and organismal level in the praying mantis H. patellifera.  This is the first time that such rhythms have been described in a praying mantis, and our data represent an important step forward in our understanding of the complexities of circadian rhythms in the praying mantis.

  P78

Clock Silencing in Adulthood Impairs Rhythmic Insulin Release and Reprograms Protein Secretion Transcription

NetworksMark Perelis1, Biliana Marcheva1, Wenyu Huang1, Chiaki Omura1,

Yumiko Kobayashi1, Yumiko Kobayashi1, Grant D. Barish1, Joseph Bass1

1Department of Medicine, Division of Endocrinology, Metabolism and Molecular Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL, UNITED STATES

The molecular clock is encoded by a transcription-translation feedback loop in brain and peripheral tissues that maintains physiologic constancy through transcriptional programming. Studies in liver and pancreas show that clock transcription factors vary in abundance across the light-dark cycle and generate temporal windows in the transcription of metabolic gene networks, raising the possibility that clock control of transcription may impact physiologic homeostasis. Indeed, pancreatic clock deregulation throughout development and adulthood causes hypoinsulinemic diabetes, although the cellular and genomic basis for this pathology has not been defined.  Here we show that wild-type mice display pronounced circadian oscillations of insulin release in response to both glucose and KCl and that selective clock ablation within beta cells during adulthood causes hypoinsulinemia and impaired glucose tolerance.  Beta cell failure occurs when clock function is abrogated in adulthood due to impaired nutrient-stimulated insulin exocytosis, while oxygen consumption is intact, indicating a defect downstream of metabolism.  RNA sequencing further reveals that clock transcription factors regulate a repertoire of beta cell genes involved in vesicle trafficking, docking, and exocytosis. Collectively, these findings demonstrate that the circadian clock produces rhythmic cycles of glucose-stimulated insulin secretion due to programming of islet transcription networks involved in peptide hormone trafficking and release into the circulation.

 

  P79

Sustained inhibition of Na+/K+/Cl- co-transporter 1 (NKCC1) enhances the magnitude of light-induced phase delays of

the circadian clock.John (Mac) McNeill1, James Walton1, Elliott Albers1

1Neuroscience Institute, Georgia State University, Atlanta, GA, UNITED STATES

The suprachiasmatic nucleus (SCN) generates circadian rhythmicity and receives light signals to entrain this endogenous rhythmicity to the environmental light/dark cycle. γ -Aminobutyric acid (GABA) and its associated synthesis, release, receptor, and uptake proteins are expressed in most neurons of the SCN. An abundance of evidence from our lab indicates that acute activation of GABAA receptors (GABAARs) inhibits the phase shifting effects of photic cues and promotes the phase shifting effects of non-photic cues. More recently we have shown that the sustained activation of GABAARs can mimic the phase delaying effects of light and that the sustained inhibition of GABAARs can inhibit the phase delaying effects of light. Since recent data indicate that excitatory responses in the adult SCN to GABAAR activation are enhanced across the active phase and are dependent on the chloride co-transporter, Na+/K+/Cl- co-transporter 1 (NKCC1), we determined if sustained excitatory responses to GABAAR activation are necessary for light to induce phase delays. Adult male Syrian hamsters were allowed to establish free-running activity rhythms in constant dark conditions. On test day, hamsters received a phase delaying 15-minute light pulse (LP) in the early subjective night (CT 13.5), followed by six microinjections into the SCN region (1 injection/hour, CT 14.5-19.5) of bumetanide (a Na-K-Cl cotransporter 1 inhibitor) or vehicle. Surprisingly, NKCC inhibition significantly increased the magnitude of LP-induced phase delays (bumetanide, -2.1 +/- 0.4 hrs. vehicle, -0.97 +/- 0.2 hrs.). These data suggest that excitatory GABAAR responses may inhibit the ability of light to induce phase delays.

Supported by NIH NS078220

  P80

Constitutive activation of glycogen synthase kinase 3 induces metabolic dyssynchrony and impairment in mice

Karen Gamble1, Jodi Paul2, Russell Johnson1, William Ratcliffe4, Martin Young4

1Psychiatry and Behavioral Neurobiology, University of Alabama at Birmingham, Birmingham, AL, UNITED STATES

2University of Alabama at Birmingham, Birmingham, AL, UNITED STATES4Medicine, Cardiovascular Disease Division, University of Alabama at Birmingham, Birmingham, AL,

UNITED STATES

Twenty-four hour rhythms in activity/physiology, including metabolism, are regulated by circadian clocks, which allow the body to respond appropriately to daily environmental stimuli such as food availability. The 24-h timing mechanism is maintained by transcriptional-translational feedback loops of a set of core “clock genes.” The importance of the circadian clock in metabolic regulation is underscored by reports of obesity or altered metabolism in clock gene mutant mouse models. One enzyme that may link the clock to metabolism is glycogen synthase kinase 3 (GSK3). GSK3 is an established modulator of insulin sensitivity; genetic loss of GSK3α results in increased

glucose tolerance and insulin sensitivity as well as reduced body fat. Importantly, phospho-GSK3 (an inverse marker of activity) is rhythmic in the liver, heart, and brain. Therefore, we sought to determine the metabolic consequences of constitutive GSK3 activation (i.e., non-rhythmic) by utilizing a double transgenic mouse, with serine-alanine substitutions in the inhibitory phosphorylation sites of both isoforms of GSK3 (α and β). Our data indicate that constitutively active GSK3 knock-in (GSK3-KI) mice have significantly increased body mass and adiposity (compared to wild-type controls) coupled with increased food intake during the day, insulin resistance, and dampened rhythms in respiratory exchange ratios (RER; an indication of time-of-day-dependent substrate utilization). When fed a high fat diet (HFD) for 16 weeks, both genotypes display increased reliance on lipid/fatty acid oxidation. Interestingly, the RER acrophase for GSK3-KI mice on HFD showed high inter-subject variability, indicating dyssynchrony between substrate utilization rhythms with the light-dark cycle. In summary, these data support the model that rhythmic GSK3 activity is necessary for normal daily energy balance, and that disruption of this rhythm leads to circadian misalignment and metabolic dysfunction.

This research is supported by the UAB Nutrition and Obesity Research Center.

  P81

Influence of circadian rhythms on postprandial triglyceride metabolism: Role of the Suprachiasmatic Nucleus

SOFIA MORAN-RAMOS1, NATALI N. GUERRERO-VARGAS2, CAROLINA ESCOBAR1, RUUD M. BUIJS2

1ANATOMY, UNAM, Mexico, Distrito Federal, MEXICO2CELLULAR BIOLOGY AND PHYSIOLOGY, UNAM, MEXICO, DF, MEXICO

Energy metabolism follows a diurnal pattern responding to the light/dark cycle and food availability. Several lines of evidence suggest that a mismatch between the daily rhythms of activity and food intake, or rest and fasting can lead to development of metabolic abnormalities such as cardiovascular disease. Recently postprandial lipemia was suggested as a good predictor of vascular events. To assess the effects of circadian rhythms on postprandial lipid metabolism, male Wistar rats were fasted for 24 h and gavaged with a lipid bolus during the day (ZT2) or night (ZT14). The results show a clear difference in postprandial triglyceride (TG) levels that is not associated to the rate of intestinal absorption, but rather due to a difference in TG uptake by tissues. To further investigate whether the suprachiasmatic nucleus (SCN) is responsible for these daily fluctuations in postprandial lipid uptake, SCN-lesioned rats were gavaged with the fat bolus at the different time points. The differential daily effects in postprandial TG were not observed in SCN-lesioned rats and their postprandial TG profile was similar to intact rats at ZT14. In parallel SCN-lesioned rats showed higher mean daily body core temperature compared to intact rats with no daily fluctuations. Based on the role of brown adipose tissue in thermogenesis and in TG clearance, we suggest that the SCN influence postprandial triglyceride clearance by its influence on brown adipose tissue to achieve the daily changes in body core temperature.

  P82

Novel Cry Stabilizing Compounds Reinforce the Peripheral Clock Mechanism and Lower Blood Glucose in Diabetic

Mice

Jeffrey Johnson1, Travis Renner1, Tod Steinfeld1, Eric Mabery1, Erik Willis1, Tim Rantz1, Timothy  Park1, Erin Riegler1, Paul Humphries1,

Kerryn McCluskie1

1Reset Therapeutics, South San Francisco, CA, UNITED STATES

Numerous studies in animals and humans have demonstrated a close link between circadian disruption and metabolic dysfunction. High fat feeding or hyperphagia in mice results in major alterations in the circadian pattern of expression of genes regulating both carbohydrate and lipid metabolism, and these changes contribute to metabolic disease. Phenotypic screening of small molecule compound libraries by Hirota et al. (Science 2012 337:1094-7) resulted in the identification of compounds that interact with and stabilize Cry proteins. These compounds impacted both core clock and metabolic gene transcription and were able to suppress glucagon-dependent glucose production in mouse hepatocytes. We have developed a series of Cry stabilizers with improved drug-like properties, and have used them to translate these findings to the level of the whole organism. Compound A has similar effects to the previously described Cry stabilizers in Per2::Luc and Bmal1::Luc screening assays, but it also can suppress E-box gene transcription in vitro and in multiple peripheral tissues of mice following oral dosing. This compound also leads to a phase delay in Bmal1 mRNA accumulation in liver and skeletal muscle of DIO C57Bl/6j mice. Numerous clock output genes are also affected as well as genes directing carbohydrate and lipid metabolism, and these changes prompted us to examine the effect of Compound A on glucose metabolism in DIO mice. After 7 days of QD oral dosing at 100 mg/kg, Compound A caused a substantial reduction in fasting blood glucose (FBG) and improved glucose tolerance) in an oral glucose tolerance test (OGTT) (p < 0.0001, 1-way ANOVA for both FBG and OGTT AUC). The effects of Compound A and another Cry stabilizer compound (Compound B) approached that of the PPARγ agonist rosiglitazone. However, in contrast to rosiglitazone, neither Compound A nor Compound B resulted in weight gain over the seven days of treatment. Plasma insulin was also lower in mice treated with Compound A, and calculation of HOMA-IR indicated a substantial (50%) improvement in insulin sensitivity (similar to rosiglitazone). We conclude that stabilization of Cry by Compound A works through the peripheral clock mechanism to impact carbohydrate and lipid metabolism and improve glucose metabolism in hyperglycemic mice. This represents the first demonstration that direct pharmacological manipulation of the core clock can have a positive effect on metabolic function in vivo and forms the basis of efforts to develop a new class of therapeutics for diabetes and other metabolic disorders.

 

  P83

Metabolic disturbances in a model of chronic jetlagLP Casiraghi1, J J Chiesa1, L Marpegan1, Diego Golombek1

1Universidad Nacional de Quilmes/CONICET, , ARGENTINA

Animal models of circadian disruption represent valuable tools for the understanding of the basis of shiftwork-related complications. In this work we describe metabolic disturbances appearing under a chronic jet lag (CJL) protocol that we have previously shown to produce forced desynchronization of activity rhythms in mice. In addition we tested for possible strategies to alleviate such negative effects. Mice were housed under a schedule consisting of 6 hour advances of the LD cycle every two days (ChrA), or under control LD, in three different cage conditions: a) individually (ChrA), b) in groups of 3-4 animals (G-ChrA), and c) individually in a cage equipped with a running wheel (W-ChrA). A fourth group was housed individually under a symmetrical delaying CJL schedule (ChrD). Weight and food intake were monitored for 30 days. Weight gain was significantly elevated in the ChrA group, while no difference was found in any of

the other experimental groups. No differences were found in food intake for any group. Metabolic disturbances, including altered triglycerides blood levels and adipocytes size, were found in the ChrA group at blood and tissue levels that may underlie the weight gain observations. Our CJL model appears as a convenient one to test for metabolic disturbances related to circadian disturbances.

  P84

Insulin-FOXO3 signaling modulates circadian rhythms via regulation of Clock transcription

Ines Chaves1, Gijsbertus van der Horst1, Raymond Schellevis3, Romana Nijman1, Marian Groot Koerkamp5, Frank Holstege5,

Marten Smidt7, Marco Hoekman7

1Genetics, Erasmus MC, Rotterdam, NETHERLANDS3Rudolf Magnus Institute for Neuroscience, Utrecht,

5University Medical Center Utrecht, Utrecht, 7Swammerdam Institute of Life Sciences, University of Amsterdam, Amsterdam, UNITED STATES

In mammals, light sensed by the retina is transmitted to the SCN master clock, where it is translated into regulation of clock gene transcription. The signaling pathways responsible for coupling metabolic cues to the molecular clock are still being uncovered. Here, we show that the forkhead transcription factor FOXO3 is a crucial modulator of hepatic circadian rhythmicity via direct regulation of Clock. Knockdown of FoxO3 dampens circadian amplitude, whereas this effect can be rescued by overexpression of Clock. Binding of FOXO3 to two Daf Binding Elements located in the Clock-promoter area further identifies Clock as a direct transcriptional target of FOXO3. Remarkably, FOXO3 deficient mice display a normal circadian behavior, but the liver oscillator is severely affected. Finally, we show that insulin signaling regulates transcription of Clock in a PI3K and FOXO3 dependent manner. Our data point to a key role of the insulin-FOXO3-Clock axis in the modulation of circadian rhythms in response to metabolic signals.

  P85

Bmal1 in brown adipocytes is not required for rhythmic oscillations of core body temperature

Georgios Paschos1, GUANGRUI YANG2, Teresa Reyes1, Garret FitzGerald1

1University of Pennsylvania, Philadelphia, PA, UNITED STATES2UNIVERSITY OF PENNSYLVANIA, DREXEL HILL, PA, UNITED STATES

Body temperature exhibits circadian oscillation that is conserved among mammals. The circadian clock orchestrates several homeostatic pathways to maintain body temperature in response to diurnal changes in environmental ambient temperature. However, the mechanisms under the control of the circadian clock responsible for coordinating circadian rhythms of body temperature are largely unknown. Brown adipose tissue thermogenesis produces heat used for the maintenance of body temperature and contributes to total energy expenditure. Here we show that adipocyte Bmal1, an indispensable component of the circadian clock, is not required for rhythmic oscillations of body core temperature. Global post-natal deletion of Bmal1 in 16week old mice abolished the rhythms of core body temperature. On the other hand, mice with an adipocyte-specific deletion of Bmal1 (Ad-Bmal1-/-) showed intact rhythms of core body temperature. Exposure of mice to cold during peak and trough BMAL1 activity showed no difference in body temperature drop and recovery. Consistent with this finding, deletion of Bmal1 in adipocytes had

no effect on body temperature response to cold exposure. Expression of Ucp1 in response to cold in adipocytes lacking Bmal1 was similar to adipocytes from wild-type mice. Electron microscopy of brown adipocytes from Ad-Bmal1-/- mice revealed normal mitochondria morphology. These results suggest no role of Bmal1 in brown adipocyte thermogenesis and the rhythmic oscillation of body temperature.

  P86

Human skeletal muscle clock: implications in myokine secretion and insulino-resistance

Laurent Perrin1, Svetlana Skarupelova1, Hubert Vidal3, Etienne Lefai3, Charna Dibner5

1Division of Endocrinology, Diabetes and Nutrition, University of Geneva, Geneva, Geneva, SWITZERLAND3CarMeN, INSERM, Lyon, FRANCE

5Endocrinology, Diabetes and Nutrition, Faculty of Medicine, University of Geneva, Geneva, SWITZERLAND

Circadian rhythms are functional in all organisms allowing an adaptation to the external world by controlling most of physiological processes. In mammals, the suprachiasmatic nucleus of the hypothalamus is the central pacemaker that controls peripheral clocks in organs. Mouse skeletal muscle clock has been shown to play a role in glucose metabolism regulation. We aimed at characterizing circadian rhythms in human primary skeletal muscle cells established from healthy and insulino-resistant donors. This was done by continuous recording of bioluminescent reporters Bmal1-luciferase and Per2-luciferase, introduced by lentiviral transduction, by endogenous circadian transcriptome, and by circadian myokine analysis employing perifusion system. Our experiments revealed that differentiated human primary myotubes synchronized in vitro exhibited robust circadian rhythm with an oscillation period of about 24.6 hours. The expression and secretion levels of Interleukin 6 (IL-6) were assessed in the presence and absence (by siClock transfection) of functional clock. Il6 expression and secretion profiles are circadian, and secretion is strongly down-regulated upon the clock disruption. In addition, insulin sensitivity assessed around-the-clock in synchronized myotubes by measuring Akt phosphorylation levels in response to insulin pulse might exhibit circadian profile. Finally, TNFα-induced insulin resistant myotubes might lose their functional clocks, while myotubes derived from overweight and obese donors exhibited longer oscillation period. In conclusion, we demonstrate that the human skeletal muscle possesses a high-amplitude circadian rhythm, and that this clock might plays a role in the regulation of the skeletal myotube transcriptome, myokine secretion and insulin-sensitivity.

  P87

Circadian clocks and Polyamines – a metabolic feedback loop

ziv zwighaft1, Judith Kraut-Cohen2, Moran Shalev2, Rona Aviram2, Liat Rousso Noori5, Marina Golik2, Asaph Aharoni2, Chaim Kahana2,

Gad Asher9

1Biological chemistry, Weizmann Institute of Science, Rehovot, ISRAEL2Weizmann Institute of Science, Rehovot,

5Biological chemstry, Weizmann Institute of Science, Rehovot, ISRAEL9Department of Biological Chemistry, Weizmann Institute of Science, Israel, Rehovot, , ISRAEL

The physiology and metabolism of mammals are subject to daily oscillations that are driven by an endogenous circadian clock. Concomitantly, circadian clocks are tightly coupled to cellular metabolism and respond to feeding. The molecular mechanisms through which metabolism

regulates clocks’ function are just starting to emerge. Here we show the involvement of polyamines, essential, positively charged molecules in circadian rhythmicity. Both feeding and clock-dependent mechanisms drive the daily expression of key enzymes in polyamine biosynthesis (e.g. Odc and Srm) and polyamine accumulation. Moreover, we show that BMAL1/CLOCK rhythmically bind to conserved E-box elements on the Odc gene. In turn, polyamine homeostasis is essential for circadian oscillations as excess or depletion of polyamines affects circadian oscillations. Preliminary results suggest that polyamines might modulate both the phosphorylation status and the interaction of core clock proteins. We propose a feedback loop whereby circadian clocks regulate the daily accumulation of polyamines, which are necessary for accurate circadian rhythmicity.

 

  P88

Effects of light, food, and methamphetamine on the circadian activity rhythm in mice

Julie Pendergast1, Shin Yamazaki2

1Medicine-Diabetes, Endocrinology and Metabolism, Vanderbilt University Medical Center, Nashville, TN, UNITED STATES

2Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX, UNITED STATES

The circadian rhythm of locomotor activity in mice is synchronized to environmental factors such as light and food availability. It is well-known that entrainment of the activity rhythm to the light-dark cycle is attained by the circadian pacemaker in the suprachiasmatic nucleus (SCN). Locomotor activity is also controlled by two extra-SCN oscillators; periodic food availability entrains the food-entrainable oscillator (FEO) and constant consumption of low-dose methamphetamine reveals the output of the methamphetamine-sensitive circadian oscillator (MASCO). In this study, we sought to investigate the relationship between the SCN, FEO, and MASCO by examining the combinatorial effects of light, food restriction, and/or methamphetamine on locomotor activity. To investigate coupling between the SCN and FEO, we tested whether food anticipatory activity, which is the output of the FEO, shifted coordinately with phase shifts of the light-dark cycle. We found that the phase of food anticipatory activity was phase-delayed or phase-advanced symmetrically with the respective shift of the light-dark cycle, suggesting that the FEO is strongly coupled to the SCN and the phase angle between the SCN and FEO is maintained during ad libitum feeding. To examine the effect of methamphetamine on the output of the FEO, we administered methamphetamine to mice undergoing restricted feeding and found that food-entrained activity was delayed by methamphetamine treatment. In addition, restricted feeding induced dissociation of the MASCO and SCN activity rhythms during short-term methamphetamine treatment, when these rhythms are typically integrated. In conclusion, our data suggest that the outputs of the SCN, FEO and MASCO collectively drive locomotor activity.

  P89

Food-entrainable circadian oscillations of PER2:LUC in the mouse olfactory bulb: critical role for olfactory input

Ilya Pavlovski1, Cong Xu1, Hanna Opiol1, Mateusz Michalik1, Jennifer Evans5, Ralph Mistlberger6

1Simon Fraser University, Vancouver, CANADA5Biomedical Sciences, Marquette University, Milwaukee, WI, UNITED STATES

6Psychology, Simon Fraser University, Burnaby, BC, CANADA

Daily feeding schedules entrain clock gene rhythms in brain and peripheral tissues. The location of oscillators mediating food anticipatory behavioral rhythms, and the stimuli that entrain these oscillators, remain to be clarified. We are using PERIOD2::luciferase knock-in mice to characterize food-entrainable oscillators in isolated brain regions and determine stimuli and input pathways that mediate entrainment. The olfactory bulb (OB) in PerLuc rats and mice exhibits robust circadian oscillations of bioluminescence that do not require an intact suprachiasmatic nucleus (SCN). To determine if this rhythm is food-entrainable, homozygous Per2Luc mice were fed ad-lib or for 4h/day at ZT6 (LD 12:12). Food restricted mice exhibited robust food anticipatory activity rhythms. Tissue slices were collected at ZT8-11. Bioluminescence was recorded using Lumicycle. In ad-lib fed mice, PER2::LUC bioluminescence peaked at ZT14.8 + .4 in the SCN  and at ZT2.5 + 1.1 in the OB. In food restricted mice fed at ZT6 for >30 days, bioluminescence peaked at ZT17.0 + 0.5 in the SCN  and at ZT19.9 + 1.9 in the OB. Explants collected on days 1, 2, 3, 5, and 7 of restricted feeding  revealed that resetting of the OB rhythm by daytime feeding is gradual and requires at least 7 days to reach a stable new phase. To identify mealtime associated stimuli responsible for OB clock resetting, Per2Luc mice received intra-nasal infusions of ZnSO4 to ablate olfactory receptor input. The mice were given 5 days to recover and were then placed on the ZT6 restricted feeding schedule or fed ad-lib. Behavioral anosmia was confirmed by daily food search tests.  Unexpectedly, the OB rhythm in anosmic mice fed ad-lib was inverted relative to intact mice. Restricted daytime feeding for 7 days did not shift this rhythm. These results establish the OB circadian clock as food-entrainable, and indicate that the entrained phase of this clock relative to the daily rhythm of food intake (nocturnal when fed ad-lib; diurnal  when fed at ZT6) requires olfactory input. Supported by NSERC (Canada)

 

  P90

Phase advanced locomotor activity during timed restricted feeding persists in tissue-type plasminogen activator knock

out (tPA-/-) mice.Jessica Murphy2, Eric Mintz1

1Department of Biological Sciences, Kent State University, Kent, OH, UNITED STATES2Biological Sciences, Kent State University, Akron, OH, UNITED STATES

In response to food being made available for only a few hours each day, mice display increased locomotor activity in the hours preceding food presentation. This food anticipatory activity (FAA) continues as long as the food restriction schedule is maintained, but disappears quickly once ad libitum feeding conditions are restored. To study the mechanisms underlying FAA, we placed mice in conditions alternating restricted food (RF; constant) and free food (FF: variable) cycles in multiple phases. The present study was performed with two genotypes, memory deficient tissue-type plasminogen activator knock out (tPA-/-) male mice and wild type (WT, C57BL/6J) males. tPA-/- mice are severely deficient in long-term potentiation, long-term depression, and hippocampal-based learning and memory tasks.  We have previously shown that these mice show increased FAA.  Two groups (control and experimental; n=6) of each genotype were individually maintained in 12L:12D photoperiod with FF. After entraining to LD conditions, animals had a week of restricted food (RF: ZT6-10; ZT0: lights onset). In control groups, all RF phases were followed with 3 days (constant) of FF, while in the experimental groups, these RF phases were followed with 3, 6 and 9 day FF cycles (variable), sequentially. Our results suggest that mice lose weight after the first FR bout, but not in subsequent bouts, showing adaptation to RF even with intervening FF periods.  During subsequent FR bouts, tPA-/- mice recover FAA more quickly

than WT, suggesting that this is not the result of hippocampal-dependent learning.  In addition, daytime locomotor activity persists in the FF periods in tPA-/- mice, which also show increased activity during the ZT10-12 period.  This increased activity may result from a reduced masking effect of light or from altered phase resetting of peripheral clocks during the intervening FF periods. Taken together, these data suggest that underlying mechanisms of FAA regulation may be exposed in tPA-/- mice.

  P91

Circadian properties of food-anticipatory activity re-examined: entrainment limits and scalar timing in operant

and general activityChristian  Petersen1, Danica Patton1, Teresa  Dattolo1

1Simon Fraser University, Vancouver, BC, CANADA

Rats maintained on a single daily meal provided at a fixed time of day exhibit a daily rhythm of food anticipatory activity (FAA). Early studies failed to observe FAA if the schedule of food availability deviated greatly from 24h, suggesting that the timing mechanism was a food-entrained oscillator with a circadian period and entrainment limits. Circadian limits for FAA have since been disputed (Crystal, 2001). Rats were reported to anticipate meals at long but non-circadian intervals of 14-21h. Additionally, FAA was reported to exhibit characteristics consistent with the scalar property, a hallmark of interval timing. In these studies, FAA was measured by an operant response, a method seldom used in chronobiology. To address this we re-examined circadian limits to FAA using 3 separate groups of rats maintained on 18h and 24h for ~ 1 month each. Group 1 rats (N=16) were entrained to LD 12:12 with activity recorded by motion sensors. Group 2 rats (N=20) were maintained in constant bright light to eliminate free-running rhythms, and activity was recorded by telemetry. Group 3 rats (N=7) were maintained in  LD, and activity was recorded by lever pressing (operant) and motion sensors. To test whether FAA exhibits the scalar property, Group 3 rats were then re-entrained to the 24h restricted feeding schedule, and mealtime was delay shifted once every 22 days to sample 7 different mealtimes across the LD cycle. In all groups and all individual rats, FAA was robust under 24h schedules and absent on 18h schedules, regardless of lighting condition or activity measure. Furthermore, in Group 3 rats, the duration of anticipation to each of 7 mealtimes, in both measures of activity, failed to scale with the intervals between lights-on (or off) and mealtime, violating the scalar property. These results provide no evidence that non-circadian interval timing processes contribute to the behavior of rats maintained on 18h and 24h feeding schedules, and confirm a critical role for a circadian entrainment process in both operant and non-operant food anticipatory activity. Supported by NSERC

  P92

Meal shift experiments reveal unusual properties of circadian food anticipatory rhythms in rats and mice.

Andrea Smit1, Mateusz Michalik1, Danica Patton1, Christian Petersen1, Ralph Mistlberger5

1Simon Fraser University, Vancouver, BC, CANADA5Psychology, Simon Fraser University, Burnaby, BC, CANADA

Anticipation of one or two daily meals in nocturnal rats and mice is a circadian process that has been modeled as the output of two coupled food-entrainable oscillators (FEOs) (A. Phillips et al, SRBR 2012).  In meal shift experiments, we have observed cases in which food anticipatory

activity (FAA) associated with a daytime meal appears to persist at this time of day for many days following an 8h delay of mealtime, in parallel with emergence of a second bout of FAA at the new mealtime.  A summary of observations from seven experiments is as follows.  1. Persistence of activity at a prior mealtime is most likely to be observed following delay shifts of mealtime from ZT3 to ZT11, in LD 12:12.  2. Persistence can be observed in rats and mice. 3. In some cases activity persists at the original mealtime in parallel with anticipation at the new mealtime, while in other cases, persisting activity gradually delays to merge with the new bout of anticipation. 4. Persistence varies with the measure of activity. Persistence was observed with motion sensors and running wheels. No unambiguous cases were observed when activity was measured using abdominal radiotransmitters, possibly due to high background levels of daytime movement detected by this method. No cases were observed using food-reinforced lever pressing. 5. No persistence was observed in rats that were first made arrhythmic by long-term exposure to constant light. 6. If mealtime is gradually shifted from ZT3 to ZT11 by 1h/day, FAA delays in parallel each day, but during subsequent 2 day food deprivation tests, activity reappears at the original mealtime. These results add complexity to multiple oscillator models of FAA. Observation #3 could be modeled by a dual coupled FEO model, if coupling is weak and the response zone of the PRC is close to 8h, so that the new meal falls outside of the response zone in some cases (activity persists at prior mealtime) and in the delay zone in other cases (activity delays toward the new mealtime, concurrent with induction of a second FEO at the new mealtime.) Observations #5 and 6 suggest that mealtimes might also be tagged by the phase of the SCN pacemaker. Supported by NSERC (Canada)

  P93

Exploring interactions between copper homeostasis and transport and the SCN circadian clock

Yukihiro Yamada1, Rebecca Prosser2

1Biochemistry, cellular and molecular biology, University of Tennessee, Knoxville, TN, 2Biochemistry and Cellular and Molecular Biology, Univ. Tennessee, Knoxville, TN, UNITED STATES

Glutamate (Glu) signaling coupled with TrkB signaling shifts the phase of neuronal activity rhythms in the suprachiasmatic nucleus (SCN), the mammalian master clock. Glu and TrkB signaling activate kinase pathways that result in changes in gene transcription and translation. Downstream activation of different pathways during the early night versus late night causes delays or advances, respectively, in SCN clock phase. The MAPK/ERK pathway has been implicated in both phase delays and advances, in vivo and in vitro. Previous work from our lab using acute brain slices prepared from adult, male C57Bl/J6 mice has shown that bath-application of copper (Cu) or a Cu-specific chelator, tetrathiomolybdate (TTM), causes phase shifts in SCN neuronal activity rhythms in vitro. We investigated the involvement of glutamate and TrkB receptors in Cu and TTM effects, but the intracellular mechanisms underlying the phase shifts remain unknown. Previous studies show that Cu application increases MAPK/ERK signaling while Cu chelation inhibits ERK activation. One study (Molecular and Cellular Biology (2012): 32, 1284-1295) further demonstrated that Cu binds MEK1 (a MAPK kinase), and ERK phosphorylation is dependent on Cu import by Copper transporter-1 (CTR1) in vivo. Another copper transporter, ATP7A, is important in neuronal function, and during excess Cu conditions, has been shown to efflux Cu. Here we investigate the role of MAPK/ERK signaling in Cu- and TTM-induced phase shifts and whether these Cu transporters could play a role in SCN circadian rhythms. By recording SCN neuronal activity extracellulary from coronal slices, we determined that Cu-induced phase shifts are blocked by co-application of the MEK inhibitor U0126, but U0126 does not inhibit TTM-induced phase shifts.  These results suggest that TTM-induced phase delays do not require MEK activity although U0126 attenuates Glu-induced phase delays in vitro and TTM effects during the subjective night are NMDAR-dependent. As TTM also induces daytime phase advance independent of NMDAR activation, daytime TTM experiments involving U0126 are ongoing. Furthermore, we show that both the CTR1 protein, which could be involved in MEK

activity, and ATP7A are expressed in the SCN. Future experiments will test for changes in expression of these transporters. The role of Cu in the SCN remains unclear, but the putative interaction between Cu homeostasis and circadian rhythms highlights the need for further investigation.

  P94

MicroRNAs cooperate with rhythmic transcription to shape circadian gene expression

Ngoc-Hien Du1, Bulak Arpat1, Mara De Matos1, David Gatfield1

1Center for Integrative Genomics, University of Lausanne, Lausanne, SWITZERLAND

Over the last decade, microarray analyses and high-throughput sequencing from various mammalian tissues have indicated that up to 15% of the transcriptome are under circadian control. It was generally assumed that the majority of rhythmic mRNA accumulation is generated by rhythmic transcription. However, recent studies have suggested that a considerable proportion of mRNA cycling may actually be generated by post-transcriptional mechanisms. MicroRNAs are a class of regulatory molecules that are involved in the post-transcriptional control of mRNA stability and translation. How miRNAs, in a genome-wide fashion, influence circadian gene expression is, however, yet to be unveiled.

Using a mouse model in which miRNA biogenesis can be inactivated in hepatocytes (conditional Dicer knockout mouse), we have now addressed the role of these regulatory molecules play in rhythmic gene expression in the liver. Whole transcriptome sequencing revealed that the hepatic core clock was surprisingly resilient to total miRNA loss. However, we found that miRNAs acted as important regulators of clock-controlled gene expression. Co-regulation by miRNAs, which affected up to 30% of rhythmically transcribed genes, thus led to the modulation of phases and amplitudes of mRNA abundance rhythms. By contrast, only very few transcripts were dependent on miRNAs for their rhythmic accumulation. Finally, our study highlights several miRNAs that could preferentially modulate circadian gene expression and identifies pathways in the liver that appeared particularly prone to dual regulation through miRNAs and the clock. Overall, our study provides a comprehensive analysis of miRNA activity in shaping hepatic circadian gene expression and can serve as a valuable resource for further investigations into the regulatory roles that miRNAs play in liver gene expression and physiology. 

  P95

Neurospora crassa Circadian Rhythms in Continuous Chemostat Cultures

Allison Cockrell1, Kathleen Cusick1, Emily Petersen1, Carissa Soto1, Russell Pirlo1, Bradley Ringeisen1, Justin Biffinger1

1Naval Research Laboratory, Washington, DC, UNITED STATES

   Neurospora crassa is one of the model organisms used for understanding molecular mechanisms of circadian rhythms. The rhythms in N. crassa have predominately been studied using substrate and/or batch culture growth conditions. While these experiments are necessary and undoubtedly informative, these environments are not ideally controlled. For example, the defined nutrient conditions become depleted during growth, waste products accumulate and can alter the medium pH, and the O2 concentration is not always monitored or controlled. To address these issues, we have grown N. crassa cells in continuous chemostat cultures. These growth conditions provide a continuous supply of fresh glucose medium to the cells while constantly

removing waste products. The pH, temperature, and concentration of dissolved O2 are all tightly controlled and monitored over time. 

   The goal of this project was to determine how the circadian rhythms of N. crassa changed under these controlled growth conditions. Circadian rhythms were studied by monitoring clock-associated gene transcription levels with RT-PCR and FRQ protein expression levels using luminescence. During the first ~44 hours of continuous culture the FRQ protein expression showed ~23-hour periodicity. Over time (170 hours) no defined control over circadian clock genes was observed in constant darkness. These genes were still responsive to light and dark cycling and temperature entrainment. In this experiment FRQ expression cycled with ~22-hour periods over the course of the experiment, suggesting light entrainment and confirming the loss of circadian clock control in the constant darkness experiment. By cultivating N. crassa in continuous culture chemostats we can eliminate secondary effects (i.e., nutrient deprivation, waste accumulation) and contribute to the understanding of circadian rhythm mechanisms which are clearly linked to environmental stressors.

  P96

Use of mouse substrains identifies a QTL for circadian amplitude

Vivek Kumar1, Joseph Takahashi1

1Neuroscience, UT Southwestern, Dallas, TX, USA

Forward genetic approaches have been essential in identifying genes and pathways regulating complex behaviors.  Here, we characterize circadian wheel running behavior between two closely related mouse substrains, C57BL/6J (B6J) and C57BL/6N (B6N).  Using QTL analysis we map a single causative locus regulating circadian amplitude on chromosome 11. Within the QTL interval there is only one non-synonymous mutation in cytoplasmic FMR interacting protein 2 (Cyfip2) present in the B6N strain.  Modeling and biochemical characterization reveals that the mutant CYFIP2 present in B6N strain is less stable with a shorter half-life.  We have tested the Cyfip2 conventional and conditional knockout mice for wheel running behavior and will report the findings. There are no significant QTLs for circadian period or activity between B6J and B6N making it ideal strain for mapping ENU mutants for these two traits. 

 

  P97

A Novel ENU-Induced Mutation in the Melanocortin-4 Receptor (MC4R) Gene in Mice Leads to Altered Body-

Weight Regulation and Expression of Circadian Rhythmicity.

Marleen de Groot1, Jennifer Mohawk2, Vivek Kumar3, Joseph Takahashi4

1Neuroscience, HHMI/UTSouthwestern, Dallas, TX, UNITED STATES2Neuroscience, UTSouthwestern, Dallas, TX, UNITED STATES3Neuroscience, UT Southwestern, Dallas, TX, UNITED STATES

4HHMI/UTSouthwestern, Dallas, TX, UNITED STATES

The intricate balance among energy consumption, conservation and expenditure is integral to both biological (or circadian) rhythmicity and body-weight regulation. There is increasing evidence that these two phenomena are inextricably intertwined. In order to discover novel genes and pathways involved in body-weight regulation, we set out to identify new mutant mouse lines with abnormally high or low body weight. A line of obese mice was identified as part of a large forward genetic ENU mutagenesis screen for dominant mutations. These mice carried a mutation that mapped to a locus on chromosome 18, and were found to have a single amino acid change within the melanocortin-4 receptor (MC4R) gene. This G-protein-coupled receptor is expressed in the hypothalamus and is stimulated by a-melanocyte-stimulating hormone (a-MSH). Stimulation of this receptor decreases energy consumption and leads to behaviors associated with satiety. Like humans with mutations in this gene, and other genetic mouse models, mice carrying this novel mutation show early-onset obesity on regular chow, as well as abnormal responses to high fat diet. In addition, we report that mice with this mutation show altered patterns of daily wheel-running activity under normal ad libitum feeding conditions, and that these changes can be reversed by exposure to conditions of temporal restricted feeding.

  P98

The circadian clock controls pre-mRNA splicing through the spliceosome

Lin Zhang1, Yufeng Wan1, Xianyun Chen1, Dongni Wang1, Xinyang Yu1, Hanjie Shen1, Xiaodi Liang1, Wei Liu1, Jinhu Guo9

1Life Science School, Sun Yat-Sen University, GuangZhou, GuangDong, CHINA9Sun Yat-sen University, Guangzhou, Guangdong, CHINA

Increasing evidence points to the connection between pre-mRNA splicing and the circadian clock; however, the underlying mechanism of this connection remains largely elusive. In this study, we demonstrated that the splicing of the clock core gene frequency (frq) is auto-regulated by the circadian clock, in the filamentous fungus Neurospora crassa. In strains with knocked down or knocked out for spliceosomal components and spliceosome-associated factors, including prp5, prmt5 and the U5, U4-2 and U4-1 snRNA genes, circadian rhythms were disturbed. The circadian clock directly governs the expression of a number of spliceosomal genes and the assembly of the spliceosome, which further regulates the circadian/diurnal rhythms of splicing of frq and a downstream gene NCU09649. We also showed that the exosome, a complex that is responsible for RNA turnover, regulates the alternative splicing of frq, by affecting the RNA stability or expression of a number of spliceosomal genes. Collectively, these data suggest that the circadian clock regulates the pre-mRNA alternative splicing in interconnected pathways.

  P99

Circadian gene expression patterns on the periphery depend on mouse genotype

Rok Kosir1, Jure Acimovic1, Ursula Prosenc Zmrzljak3, Anja Korencic1, Martina Perse5, Damjana Rozman1

1Center for Functional Genomics and Bio-chips, Faculty of Medicine, University of Ljubljana, Ljubljana, SLOVENIA

3Institute of Oncology Ljubljana, Ljubljana, SLOVENIA5Institute of Pathology, Faculty of Medicine, University of Ljubljana, Ljubljana, SLOVENIA

Introduction. Biological clock is an important component in body homeostasis. However, it is not clear how the genetic background of each individual influences circadian expression and

regulation. We addressed this question by investigating gene expression in liver and adrenal glands of inbreed mouse strains 129/SvPas plus C57BL/6J and pure C57BL/6J, in DD or LD conditions. Methods. 24 h gene expression profiles were fitted using various trigonometric functions to obtain the circadian amplitudes and phases. Genome variation data files were downloaded from dbSNP database for the three 129 mouse strains and compared to the reference strain C57BL/6J. Results. Robustness of circadian expression depends on genotype and tissue. In adrenal glands under LD many genes differ in circadian profiles between mouse strains. Steroidogenic genes (Cyp11a1,Cyp17a1,Cyp21a1,Cyp51) are phase-shifted between strains at least in one of the lightening conditions. The majority of steroidogenic and core clock genes are expressed at higher levels with higher amplitudes in 129/SvPas mixed strain, exceptions are Arntl and Cry1. Liver seems to maintain a more robust circadian regulation with fewer differences observed. Since the genomes of 129 and C57BL/6J mice are already sequenced we questioned whether the modified circadian expression derives from mutations in these genes or their regulatory regions. We identified 16.900 sequence variations in 193 selected genes. The majority of variations (> 97%) were discovered in intron and promoter regions.  All three 129 strains have the same variations in coding regions of Per3, Vipr2, Opnn4 and Dusp4. Nucleotide variations were also observed in intron and promoter regions of genes that showed differences in gene expression.Conclusions. Light has greater impact on circadian expression of core clock and metabolic genes in the 129/SvPas background. Together with the genotype, the light influences primarily the amplitudes of core clock genes while the amplitudes and phases are affected in metabolic genes. 86% of analyzed genes in three different 129 strains harbor genetic variations compared to the reference strain C57BL/6J. The majority of these are in intron and promoter regions that could affect gene expression and thus also the circadian changes observed in our experiment. These findings might have important implications for understanding the genetic bases of the circadian rhythm differences in human individuals and their susceptibility to develop the clock-based diseases.

  P100

Estrogen response elements in clock genes: a bioinformatic analysis.

Jessica Lensie1, Eric Mintz2, Chi-Hua  Chiu3

1Biological Sciences, Kent State University, Kent, OH, UNITED STATES2Department of Biological Sciences, Kent State University, Kent, OH, UNITED STATES

3Kent State University, Kent, Ohio, UNITED STATES

Steroid hormones influence circadian rhythms by a number of different mechanisms. In the case of estrogens, there is strong evidence of an influence on circadian clock function at both the behavioral and molecular levels, including the ability to change the length of free-running period in hamsters, alter the response of the suprachiasmatic nucleus to light, and alter the expression of clock genes including Per1, Per2 and Cry2. However, these studies have not examined direct interactions between estrogens and the clock genes. In this study, we used a bioinformatic approach to examine the presence of estrogen response elements (EREs) in the non-coding regions of genes included in the positive and negative transcription/translation feedback loops of the circadian clock. These genes were examined in mouse, rat, and human to determine the degree of sequence conservation and thus the likelihood that EREs contribute to the transcription of circadian clock genes. EREs were found in all of the clock genes through the use of Dragon ERE finder, with a wide variation between genes in number of EREs and the localization of the EREs to either the intronic regions or the flanking regions of the gene. Thus, estrogen may affect the transcription of the circadian clock genes either by enhancing or inhibiting the transcription of the gene based on the location of the ERE in the non-coding regions. Of the genes found to contain EREs, we see no partiality to either the positive or negative limb of the clock.  The total

number of EREs in intronic regions is similar between mouse and human, and a more detailed analysis of the conservation of EREs in clock genes is in progress.  This study is the first to examine several circadian clock genes for EREs in non-coding regions and the conservation of these EREs in clock genes across species.  The results suggest the potential for a large number of potential sites where estrogen receptor activity could directly influence clock gene transcription.

  P101

Circadian clock-dependent and -independent rhythmic proteomes implement distinct diurnal functions in mouse

liverDaniel Mauvoisin1, Jingkui Wang2, Céline Jouffe1, Eva Martin4,

Florian Atger4, Patrice Waridel6, Manfredo Quadroni6, Frédéric Gachon4, Félix Naef2

1Diabetes & Circadian Rhythms, Nestle Institute of Health Science , Lausanne, Vaud, SWITZERLAND2Institute of Bioengineering, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne,

Lausanne, Vaud, SWITZERLAND4Diabetes & Circadian Rhythms, Nestle Institute of Health Science, Lausanne, Vaud, SWITZERLAND

6Protein Analysis Facility, University of Lausanne, Lausanne, Vaud,

Diurnal oscillations of gene expression controlled by the circadian clock underlie rhythmic physiology across most living organisms. While such rhythms have been extensively studied at the level of transcription and mRNA accumulation, little is known on the accumulation patterns of proteins. Here, we quantified temporal profiles in the murine hepatic proteome under physiological light-dark conditions using SILAC quantitative mass spectrometry (MS). Our analysis identified over five thousand proteins of which several hundred showed robust diurnal oscillations with peak phases enriched in the morning and during the night and related to core hepatic physiological functions. Combined mathematical modeling of temporal protein and mRNA profiles indicated that proteins accumulate with reduced amplitudes and significant delays, consistent with protein half-live data. Moreover, a group comprising about half of the rhythmic proteins showed no corresponding rhythmic mRNAs, indicating significant translational or post-translational diurnal control. Such rhythms were highly enriched in secreted proteins accumulating tightly during the night. Also, these rhythms persisted in clock deficient animals, suggesting that food related entrainment signals influence rhythms in circulating plasma factors.

 

  P102

The mouse liver displays circadian rhythms in the phospholipid metabolism and in the activity of its

synthesizing enzymesLucas D. Gorné1, Victoria Acosta Rodríguez1, Susana J. Pasquaré3,

Gabriela A. Salvador3, Norma M. Giusto3Mario E. Guido1

1CIQUIBIC-CONICET, Dpto de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Córdoba, ARGENTINA

3INIBIBB-CONICET, Universidad Nacional del Sur, Bahía Blanca, Argentina, Bahía Blanca, Buenos Aires, ARGENTINA

Disruption of circadian clocks leads to severe metabolic disorders and pathologies involving lipid metabolism such as obesity and diabetes. Glycerophospholipids (GPLs) are the most abundant

class of phospholipids that constitutes cell membranes and their synthesis is a crucial process in the liver function; however, its temporal regulation remains unknown. Here we investigated whether the GPL metabolism oscillates in mouse liver fed ad libitum. We first found that the endogenous content of some GPLs (phosphatidylcholine: PC, phosphatidylinositol: PI) exhibited a clear daily rhythmicity in mouse livers collected under constant darkness. Overall, GPLs showed higher levels during the subjective day and lowest levels at CT 20 in the subjective night. In addition, the activity of GPL-synthesizing and -remodeling enzymes: phosphatidate phosphohydrolase 1 (PAP-1, /Lipin) and lysophospholipid acyltransferases (LPLATs) displayed significant variations with higher levels during the subjective day and dusk. Since PC is an essential and abundant hepatic GPL exhibiting a highly regulated process of synthesis, we evaluated whether the enzymes involved in PC synthesis exhibit a temporal regulation in both expression and activity. In the liver, PC is mainly synthesized through the Kennedy pathway with Choline Kinase (ChoK) as one of the key regulatory enzymes or through an alternative pathway catalyzed by the phosphatidylethanolamine (PE) N-methyltransferase (PEMT) converting PE into PC. In this respect, we observed that the ratio in the endogenous PC to PE content exhibited daily variations with the lowest levels during the subjective night. In addition, we found significant oscillations in ChoKα and PEMT mRNA expression with maximal levels during the subjective night (CT 16-20). Results demonstrate that the metabolism of liver GPLs from LD-synchronized mice oscillates rhythmically with a precise temporal control in the expression and activities of specific synthesizing/remodeling enzymes. Our findings provide new insights to understand the temporal regulation of biological processes implicating phospholipid metabolism.   

  P103

New insight into post-transcriptional regulation of circadian rhythms using a system wide identification of RNA-binding

proteinsPauline Gosselin1, Alfredo Castello2, Matthias Hentze3, Ueli  Schibler1

1Molecular Biology, University of Geneva, GENEVA, SWITZERLAND2Molecular biology, European Molecular Biology Laboratory (EMBL), HEIDELBERG, GERMANY

3Molecular Biology, European Molecular Biology Laboratory (EMBL), Heidelberg, GERMANY

Circadian clocks are internal molecular time-keeping systems that enable organisms to adjust their physiology and behavior to the daily environmental changes. In recent years, it has become clear that post-transcriptional mechanisms and temperature cycles play important roles in governing circadian gene expression. To examine the possible temperature-dependent impact of protein-RNA interactions on a transcriptomes-wide scale, we used the UV-crosslinking interactome capture strategy recently developed by Hentze and coworkers (1). RNA-binding proteins (RBP) associated with polyadenylated RNAs in NIH3T3 fibroblasts at two different temperatures (33 °C and 38 °C) were identified by mass spectrometry. Indeed, our laboratory previously demonstrated that simulated mouse body temperature oscillations were able to synchronize the phase of circadian gene expression in these cells. Proteins such as eIF3G or GRSF1 were found among the proteins bound to mRNAs in a temperature specific manner. Our experiments will hopefully help us to investigate changes in the composition of mRNA-binding proteins and to identify key RBPs involved in the post-transcriptional control of circadian gene expression. We are also adapting the interactome capture technology to study mRNA-protein interactions around the clock in mouse liver. Indeed, RNA polymerase II chromatin

immunoprecipitation experiments and mRNA profiling studies in this tissue have indicated that the cyclic accumulation of most circadian mRNAs is regulated by post-transcriptional rather than transcriptional mechanisms.

 (1)   1. Castello A., Horos R., Strein C., Fischer B., Eichelbaum K., Steinmetz L.M., Krijgsveld J., Hentze M.W. System-wide identification of RNA-binding proteins by interactome capture. Nat. Protoc. 2013;8:491–500.

  P104

Role of the circadian clock regulated ATF5 transcription factor

capucine BOLVIN1, frederic GACHON2

1circadian rhytms, NIHS, lausanne, vaud, SWITZERLAND2circadian rhythms, NIHS, Lausanne, Vaud, SWITZERLAND

By regulating the metabolism of fatty acids, carbohydrates and xenobiotic the mammalian circadian clock plays a fundamental role in the liver. Impairment of this rhythm leads to disorders such as metabolic syndrome. To date, clock controlled metabolism in liver is known to be regulated mainly at the transcriptional level. However, we recently show that, via a circadian clock-coordinated 12-hours period rhythmic activation of one of the unfolded protein response (UPR) pathways, the circadian clock regulates posttranslational modifications of liver enzymes. In this context, ATF5, a bZip transcription factors, attracts our attention. Indeed its mRNA is rhythmically expressed with a previously unseen 24 hours cycle peaking during night-day transition. Moreover, its translation is regulated by the UPR. UPR activation has been linked to tumour growth and to chemotherapeutics treatments resistance and ATF5 has been shown as a mediator of cell survival. Thus, we planned to characterize the potential role of ATF5 in liver metabolism after activation of the UPR. First we characterised that the ATF5 protein induction after Tunicamycin (tm) (ER stress inducer) injection in WT mice displayed a rhythmic pattern picking at the night-day transition matching the Atf5 mRNA peak. To characterize the role of ATF5 in vivo, we developed an Atf5 KO mouse and performed transcriptomic profiling of wild-type compared to KO mice after activation of the UPR. Microarrays were performed on liver RNAs and we identified differently expressed genes between Atf5 KO and WT after UPR activation. These genes were involved in the inflammatory response and cellular regeneration process. To get further insight into the inflammatory response, cellular regeneration mechanisms and the regulation of this process, we investigated responses after Tm injections on WT mice. Interestingly, mice responded differently depending on the injection time and western blotting and qPCR analysis confirmed Tm effect on inflammatory and cellular regeneration markers. Also, as expected, ATF5 protein expression was induce in WT mice still with remarkably differences depending on tm injection time.

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Orchestration of the rhythmic translation by the circadian clock

Florian Atger1, Eva Martin1, Fréderic Gachon1

1Circadian Rhythms, NIHS, Lausanne, SWITZERLAND

The transcriptional regulation of circadian genes and outputs have been imply investigated. Transcriptome analysis revealed that the expression of 5-10% of genes exhibits circadian rhythmicity at steady-state mRNA level in any given organism or tissue and led to the former paradigm that circadian output is primarily controlled at the transcriptional level. However, proteomic analysis in mouse liver revealed that half of the rhythmic proteome does not exhibit corresponding rhythmic mRNA expression, suggesting the importance of post-transcriptional (PT) regulation to generate circadian outputs. Indeed, core circadian genes and outputs exhibit PT regulation such as mRNAs half-life, IRES dependent translation and the recruitment of RBPs or microRNAs.Recently, our laboratory described how the clock coordinates ribosome biogenesis. This conclusion is supported by the rhythmic activation of molecular pathways involved in translation regulation. Indeed, mTOR, ERK and AMPK pathways are rhythmically activated, resulting in a rhythmic binding of 4E-BP1 proteins on m7GTP beads mimicking the mRNA cap structures. These results and the amount of circumstantial evidences lead us to hypothesize that the clock may be involved in a larger panel of regulation related to the translational processes. To verify this hypothesis, we are currently investigating the global rhythmic mRNA translation through ribosome profiling strategy in Wild-Type, Bmal1KO and Cry1/Cry2 double KO mice.  The rhythmic translation of ribosomal protein (RPs) is not supported by a rhythmic expression of RPs genes since the total RP mRNAs level was shown to be constant. Thus, the regulation of ribosome biogenesis should act at the PT level. RPs mRNAs possess a TOP (Terminal Oligopyrimidine Tract) in their 5’-UTR that comprises the core of their translational cis-regulatory element. However, the trans-acting factors that potentially bind the 5’-TOP and mediate the RP translation remain to be discovered. To investigate them, we studied the candidate trans-acting factors found in the literature and developed a mRNA pull down strategy coupled with mass spectrometry to identify new 5’-TOP mRNAs binding proteins.

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Chronic phase shifting paradigms disrupt locomotor rhythm entrainment in C57BL/6J but not BALB/cJ mice.

Tyrus Takacs1, Penny  Molyneux2, David Bonsall2, Mary Harrington2, Todd Weber1

1Biology, Rider University, Lawrenceville, NJ, UNITED STATES2Psychology, Smith College, Northampton, MA, UNITED STATES

BALB/cJ mice show rapid re-entrainment of wheel-running rhythms to large phase advances of the light-dark cycle, within 1-2 cycles compared to 5-7 cycles required of C57BL/6J mice.  The duration of the photophase influences the rate of re-entrainment and acute phase shifting is not different in BALB/cJ and C57BL/6J mice.  In preliminary experiments using Per2::Luc+/-  F2-hybrids of BALB/cJ & C57BL/6J mice with a BALB/cJ circadian locomotor phenotype (i.e. rapid re-entrainment of wheel-running rhythms) and slowly re-entraining C57BL/6J mice, bioluminescence rhythms in SCN explants showed similar phase angle of entrainment to the new LD cycle on day 2 following a 6-hour phase advance of the light-dark cycle.  Bioluminescence rhythms from spleen and thymus explants from “BALB-like” hybrids showed a 3-hour phase advance on day 2 following the phase shift, whereas rhythms in explants from C57BL/6J mice were unmoved.  All bioluminescence rhythms were re-entrained in samples from both groups taken on day 9 following the shift.  In a separate experiment, BALB/cJ and C57BL/6J mice were subjected to chronic phase advance or phase delay paradigms of differing frequencies and durations.  When light-dark cycles were phase-advanced by 6 hours every 4 days (6x4) or by 4 hours every 3 days (4x3), BALB/cJ mice maintained entrainment for up to 10 weeks with a typical phase relationship between onset of darkness and onset of activity, confirmed by subsequent release to DD.  Conversely, C57BL/6J mice reacted to chronic phase advances with 1-2 weeks of desynchrony followed eventually by entrainment to the LD cycle with a delayed phase angle, also confirmed by subsequent release to DD.  Both groups entrained to chronic 4x3 phase delays for 10 weeks.  C57BL/6J mice showed significantly greater aftereffects in free-running period from chronic advance and chronic delay paradigms than did BALB/cJ mice.  Results suggest that the propensity of the BALB/cJ circadian system to maintain a relatively short period, combined with a stronger coupling between SCN and peripheral tissues, may underlie accelerated re-entrainment to large phase advances of the light-dark cycle.

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Drosophila mechanosensory organs and Ionotopic Receptors (IRs) contribute to clock synchronization by

temperature cycles and proprioceptive feedbackChenghao Chen1, Alekos  Simoni2, Werner  Wofgang2, Min Xu4, Richard

Benton5, Joerg  Albert6, Ralf Stanewsky7

1Department of Cell developmental biology, UCL, London, UNITED KINGDOM2School of biological and chemical sciences, Queen Mary, University of London, London, UNITED

KINGDOM4Department of Cell developmental Biology, UCL, London, UNITED KINGDOM

5Center for Integrative Genomics, University of Lausanne, Lausanne, SWITZERLAND6UCL, London, UNITED KINGDOM

7Department of Cell Developmental Biology, UCL, London, UNITED KINGDOM

Circadian clocks are synchronized by the natural daily fluctuations of light and temperature. Previous work suggests that in Drosophila melanogaster peripheral mechanosensory organs, otherwise known to function as stretch receptors (chordotonal organs; ChO), also function as temperature sensors. The

temperature receptors mediating this synchronization have not been identified. Transient Receptor Potential (TRP) channels function as thermoreceptors in animals and we have previously shown that the Pyrexia (Pyx) TRP channel mediates temperature synchronization in the lower range (16°C:20°C Temperature Cycles; TC). Here, we isolated proteins interacting with the Nocte protein, known to play a role in temperature entrainment and ChO function. A member of the Ionotropic Receptor (IR) family, a new class of chemosensory receptors in flies, physically interacts with Nocte and is also expressed in ChO. IR loss-of-function mutants fail to synchronize their behaviour to TC in the higher range (25:27°C) and clock protein oscillations in subsets of the clock neurons are blunted and abolished in peripheral clocks. Interestingly, mechanical stimulation of the ChO using (12h:12h) cycles of vibration and silence (VS) also results in synchronization of the circadian clock requiring a functional clock and functional chordotonal organs. Our results suggest that ChO form part of the temperature-sensing or -signaling apparatus from peripheral sensory organs to the clock neurons in the brain covering different intervals of the physiological relevant TC fruit flies are normally exposed to in nature. In addition we propose that proprioceptive feedback from ChO to the brain clock may help an animal to keep its circadian clock in sync with its own, stimulus-induced activities.

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Genetic engineering of an S714 mutation in PER1 leads to an advanced feeding rhythm phase in mice

Zhiwei Liu1, Moli Huang2, Guangsen Shi3, Lijuan Xing1, Xi Wu1, Zhipeng Qu1, Jie Yan7, Ling Yang8, Satchidananda Panda9, Ying Xu3

1Nanjing University, Nanjing, CHINA2Soochow University, Soochow,

3Model Animal Research Center, Nanjing University, Nanjing, Jiangsu, CHINA7School of Mathematical Sciences, Soochow University, Suzhou, Jiangsu, CHINA

8Center for Systems Biology, Soochow University, Suzhou, Jiangsu, CHINA9Salk Institute for Biological Studies, La Jolla, CA, UNITED STATES

The circadian clock allows an organism to anticipate environmental cyclic changes, such as the light-dark cycle and food availability, and thus provides a greater adaptive advantage when compared with random processes . Although the basic function of the molecular clock is largely evolutionarily conserved, mammals employ multiple paralogous clock genes, whereas Drosophila does not. These clock components emerged and expanded in mammalian circadian systems, resulting in a high level of functional divergence in physiological functions. Mapping specific function in each paralogous gene is likely to provide general insights into the understanding of how adjustments to the clock components achieve balance between clock systems and physiological homeostasis. A particularly interesting example comes from the individuals within a single-family pedigree that carry an S662G dominant mutation in hPER2 that causes familial advanced sleep-phase syndrome (FASPS). The S662 position of the hPER2 protein is the first of five serines spaced three amino acids apart, and the S662G mutation impedes sequential phosphorylation. The SXXS motif is highly conserved in mammalian PER proteins, and t his serial phosphorylation of the PER2 protein is tightly coupled with light-dark cues. We hypothesise that this motif arose before duplications to retain the essential time-keeping function and similar selective pressure constraints on this motif. We predicted that mutational behaviour can be observed through an amino-acid change in the first serine of this motif in the hPER1 protein by applying the general principles learned from PER2 regulation, while knockout mice showed subtle phenotype owing to family gene

redundancy. Here, we generated bacterial artificial chromosome (BAC) transgenic mice carrying an S714G mutation in the hPER1 and found that Per1 is a key gene that drives behavioral rhythms in food intake and plays a critical role in physiological optimisation for feeding behaviour and energy expenditure.

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Sustained Inhibition of GABAA Receptors in the SCN is Necessary to Inhibit Light-induced Phase Delays

Tony Larkin1, Daniel Hummer1, H. Elliott Albers3

1Psychology, Morehouse College, Atlanta, GA, 3Neuroscience Institute, Georgia State University, Atlanta, GA, UNITED STATES

The mammalian circadian clock, located in the suprachiasmatic nucleus (SCN), is entrained to the environment via photic and non-photic cues.   Light exposure in the early or late night produces phase delays or phase advances, respectively.   We hypothesize that sustained activation of GABA A receptors in the SCN mediates the ability of light to phase shift the circadian pacemaker.   Consistent with this hypothesis, previous studies in our lab have demonstrated that sustained inhibition of GABA A receptors in the SCN inhibit the phase delaying effects of a light pulse given in the early night.   Specifically, 6 or 8 (but not 3) consecutive hours of GABA A antagonist (bicuculline) administration beginning at CT14.5 significantly reduced light-induced phase delays resulting from a LP at CT13.5.   The current study tests the hypothesis that more than 4 consecutive hours of GABA A receptor blockade is necessary to inhibit light-induced phase delays.   Male Syrian hamsters were implanted with a guide cannula aimed at the SCN and allowed to establish a stable free-running rhythm in DD.   Animals were randomly assigned to receive a 15-minute LP at CT13.5 followed by one of six microinjection regimens of bicuculline into the SCN: (1) 3 hourly injections beginning at CT17.5, (2) 3 hourly injections beginning at CT19.5, (3) 4 hourly injections beginning at CT14.5, (4) 4 hourly injections beginning at CT18.5, or (5) 3 hourly injections beginning at CT14.5 plus 3 hourly injections beginning at 19.5.   The magnitude of light-induced phase delays did not differ between any of the 5 experimental groups and vehicle-treated controls.   These data are consistent with the hypothesis that at least five consecutive hours of GABA A receptor inhibition is necessary to inhibit light induced phase delays.

Supported by NIH NS078220