June 15 Poster Session
Time-of-day specific changes in metabolic detoxification
and insecticide resistance in the malaria mosquito
Anopheles gambiae
Giles Duffield , Nathaniel Balmert , Samuel Rund , John Ghazi ,
Peng Zhou
1Biological Sciences, University of Notre Dame, Notre Dame, IN, UNITED STATES
5University 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 latenight 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.
Daily rhythms in antennal protein and olfactory sensitivity in
the malaria mosquito Anopheles gambiae
Nicolle Bonar , Samuel Rund , Matthew Champion , John Ghazi ,
Cameron Houk , Matthew Leming , Zainulabeuddin Syed ,
Giles Duffield
1Department of Biological Sciences , University of Notre Dame, Notre Dame, IN, UNITED STATES
3Department 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 bloodfeeding 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.
Light-regulated blood-feeding and flight activity behavior
and a light phase response curve for the Anopheles
gambiae malaria mosquito
Aaron Sheppard , Gary George , Erin Clark , Hannah Yu ,
Samuel Rund , Giles Duffield
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.
Analysis of Locomotor Activity Rhythms in a Population of
Free-Behaving C. elegans
Ari Winbush , Alexander van der Linden
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 nonrobustness 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.
Physical and psychological stress as potent synchronizers
of mouse peripheral circadian clocks
Yu Tahara , Takuya Shiraishi , Yosuke Kikuchi , Atsushi Haraguchi ,
Daisuke Kuriki , Shigenobu Shibata
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.
Redox oscillations in fruit flies
Utham Kashyap Valekunja , Rachel Edgar , John O'Neill , Akhilesh
B. Reddy
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,
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.
Shock O'Clock The circadian clock in endotoxic shock systemic versus local clock regulation
Veronika Lang , Achim Kramer , Bert Maier
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.
Chronic Stress Induces Physiological and Brain Region
Specific Molecular Disruptions of Circadian Amplitude in
Nicole Edgar , Andrea Gillman , Ryan Logan , Daniel Hoffman ,
Colleen McClung
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.
CCA1, a central circadian oscillator mediates ER stress
response in Arabidopsis
Andrew Melencion , In Jung Jung , Hee Jin Park , Joon–Yung Cha , Mi
Ri Kim , Min-Gab Kim , Sang Yeol Lee , Dae-Jin Yun , Woe-Yeon Kim
1Division of Applied Life Science , Gyeongsang National University, Jinju, KOREA
3Plant 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.
Assessing the Impact of Chronic Sleep Restriction and
Acute Sleep Deprivation on Performance-Associated
Regional Brain Activation Using Near-Infrared Spectroscopy
Michael Lee , Gary Strangman , Joseph Hull , Tushar Kamath ,
Shadab Rahman , Steven Lockley , jameela lokhandwala , Quan Zhang ,
Charles Czeisler , Elizabeth Klerman
1Medicine and Neurology, Brigham and Women's Hospital/Harvard Medical School, Boston, MA, UNITED
2Psychiatry, Harvard Medical School, Boston, MA, UNITED STATES
4Massachusetts Institute of Technology, Boston, UNITED STATES
5Harvard Medical School, Boston, MA, UNITED STATES
6Brigham & Women's Hospital, Boston, MA, UNITED STATES
7Chemistry, Southern Methodist University, Dallas, TX, UNITED STATES
9Division 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
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
Sleep deprivation alters hepatic metabolism and the
peripheral clock
Jessica Ferrell , Shannon Boehme , John Y.L. Chiang
1IMS, Northeast Ohio Medical University, Rootstown, OH, UNITED STATES
2Northeast 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
Hyper-sensitivity of the circadian system to light in Delayed
Sleep Phase Disorder
Sean Cain , Jessica Stanghi , Olivia McConchie , Clare Anderson
1Psychological Sciences, Monash University, Clayton, Victoria, AUSTRALIA
2Monash 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.
Inter-individual differences in night-time behavioral and
cerebral responses to high and low sleep pressure
Micheline Maire , Carolin F. Reichert , Virginie Gabel , Antoine U. Viola ,
Klaus Scheffler , Markus Klarhöfer , Werner Strobel ,
Christian Cajochen , Christina Schmidt
1Centre for Chronobiology, Psychiatric Hospital of the University of Basel, Basel, SWITZERLAND
5Max-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, , SWITZERLAND
9Center 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 interindividual 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 sleeppressure-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.
Circadian activity splitting in two sighted individuals with
non-24 hour sleep-wake disorder
Sabra Abbott , Lirong Zhu , Kathryn Reid , Phyllis Zee
1Northwestern University, Chicago, IL, UNITED STATES
2Neurology, 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
Novel PER2 alleles for familial advanced sleep phase
Christin Chong , Geetha Bhagavatula , William Hallows , Philip Kurien ,
Yong Huang , Emily Quinn , Len Pennacchio , Christopher Jones ,
Louis Ptacek , Ying-Hui Fu
1UCSF, San Francisco,
2George Washington University, Washington D.C.,
6Icahn School of Medicine at Mount Sinai, New York, NY, UNITED STATES
7Lawrence 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.
An Important Role of 5 Evening Neurons in Drosophila
Circadian Rhythms and Sleep
Fang Guo and Michael Rosbash
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 nighttime 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
A circadian lens on human population activity patterns:
Inferences from the power grid
Caitlin Crosier , Adam Stowie , Mario Amacarelli , Taylor Fredrickson ,
Spencer Keith , Kassandra Leasure , Emily Socha , Ryan Mymko ,
Brandon Boesiger , J. David Glass
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.
Chronobiology meets Big Data: Humans 'in the wild'
Dimitri Perrin , Craig Jolley , Hiroki Ueda
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.
Circadian disfuntion in chronic kidney disease
Inês 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 sleepwake 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.
The effect of light containing spatial structure on the
suprachiasmatic nucleus
Josh Mouland , Tim Brown , Rob Lucas
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.
Trypanosoma brucei accelerates the mouse circadian clock
Filipa Rijo-Ferreira , Joseph S Takahashi , Luisa M Figueiredo
1UT Southwestern / Instituto Medicina Molecular, Dallas / Lisbon, USA / PORTUGAL
2Howard 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.
The Role of Sustained GABA-A Receptor Activation within
the SCN in Light-induced Phase Shifts is Phase Dependent
Daniel Hummer , John McNeill , Tony Larkin , H. Elliott Albers
1Psychology, Morehouse College, Atlanta, GA, UNITED STATES
2Neuroscience 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 CT14.519.5, (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 (CT14.519.5: -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 receptormediated effect is phase dependent.
Supported by NIH NS078220 and NSF IOS-1022050
Effects of chronic nighttime light exposure on the daily
rhythms in locomotor activity and clock gene expression in
the SCN
Lily Yan , Greg Leach
1Psychology & Neuroscience Program, Michigan State University, East Lansing, MI, UNITED STATES
2Psychology, 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.
Suprachiasmatic clues to circadian dysfunction in the
BACHD mouse model of Huntington's disease.
Dika Kuljis , Danny Truong , Dawn Loh , Takashi Kudo ,
Christopher Colwell
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.
Temporal pattern of GABAA receptor δ subunit expression
in the suprachiasmatic nucleus of male Syrian hamsters.
James Walton , John (Mac) McNeill , Elliott Albers
1Neuroscience Institute, Georgia State University, Atlanta, GA, UNITED STATES
3Georgia 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
CRYPTOCHROME and its role in controlling circadian
rhythms using electrophysiological techniques in
Drosophila melanogaster
Marie Nugent , Ezio Rosato , Joern Steinert
1Genetics, University of Leicester, Leicester, Leicestershire, UNITED KINGDOM
2Genetics, 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 (lLNvs). 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 coexpressed 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
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.
Phase-mapping the mouse brain with a CRY1::mCherry
fluorescent reporter
Arthur Millius , Rikuhiro Yamada , Junko Yoshida , Hideki Ukai ,
Hiroki Ueda
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 lightsheet microscopy. We anticipate this new mouse reporter line will aid in understanding
the circadian spatiotemporal coupling between the SCN and different regions of the
Phosphorylation of the Cryptochrome 1 C-terminal tail
regulates circadian period length
Peng Gao , Seung-Hee Yoo , Kyung-Jong Lee , Clark Rosensweig ,
Joseph S. Takahashi , Benjamin P. Chen , Carla B. Green
1Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX,
3Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, TX, UNITED
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 DNAPK 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.
Structural and Functional Characterization of the
Interactions between Cryptochromes and Xenobiotic
Anna Kriebs , Erin Soto , Emma Henriksson , Katja Lamia
1Chemical Physiology, The Scripps Research Institute, La Jolla, CA, UNITED STATES
3The 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.
Characterising of the role of Cryptochromes in Retinal
Responses to Light
Jovi Chau-Yee Wong , Elizabeth Maywood ,
Russell Foster Stuart Peirson
1Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, Oxon, UNITED KINGDOM
2MRC-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.
Structural Characterization of Fungal Photoreceptor-Envoy
Jameela Lokhandwala , Brian Zoltowski
1Chemistry, Southern Methodist University, Dallas, TX, UNITED STATES
3Southern 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 bluelight. 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 BlueLight-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.
Differential Mechanisms of Phase Advancing versus
Delaying Light Pulses in Drosophila.
Pooja Vinayak , Emma Garren , Sina Mizaheri , Jay Hirsh
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.
Identification of a second region regulating nuclear
localization of the circadian clock protein mouse
Cryptochrome 1
Karla Marz , Pagkapol Y. Pongsawakul , Kimberley V. Sukhum ,
Monica E. Johnson
1Biology, Gustavus Adolphus College, Saint Peter, MN, UNITED STATES
2Salk Institute for Biological Studies, La Jolla, CA, USA
3Washington University in St. Louis, St. Louis, MO, UNITED STATES
4Gustavus 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 HEK293 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.
Altered cryptochrome degradation influences GABAergic
signaling and excitation of suprachiasmatic nucleus
Sven Wegner , Mino D.C. Belle , Alun T.L. Hughes , Hugh D. Piggins
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.
Magnetoreception in Drosophila melanogaster
Giorgio Fedele , Edward Green , Ezio Rosato , Charalambos Kyriacou
1University of Leicester, Leicester, Leicestershire, UNITED KINGDOM
2Genetics, 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.
Roles of C-terminal truncated Bmal1 on circadian rhythm
Noheon Park , Solmi Cheon , Hee-Dae Kim , Kyungjin Kim
1Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX, UNITED STATES
2School 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.
Structure/function interrogation of mCRY1 defines a
distributed binding interface with the CLOCK/BMAL1
Clark Rosensweig , Peng Gao , Carla Green
1Neuroscience, UT Southwestern Medical Center, Dallas, TX, UNITED STATES
2UT 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 rhythmgenerating capacity. Cry1-/-/Cry2-/- mouse embryonic fibroblasts, which are arrhythmic, were cotransfected 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. Coimmunoprecipitation 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.
The effect of interneuronal communication between clock
neurons in Drosophila
Qi Zhang , Orie Shafer
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.
Translational control of the circadian clock through the capbinding protein eIF4E
Ruifeng Cao , Andrew Liu , Shimon Amir , Nahum Sonenberg
1Department of Biochemistry, McGill University, Montréal, QC, CANADA
2Department of Biological Sciences, The University of Memphis, Memphis, TN, UNITED STATES
3Center for Studies in Behavioral Neurobiology, Concordia University, Montreal, QC, CANADA
4Department of Biochemistry and Goodman Cancer Research Center, McGill University, Montreal, QC,
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.
The NRON/KPNB1 Complex Regulates Nuclear
Translocation and Function of the Circadian Clock
YOOL LEE , Au Reum Jang , Lauren J Francey , Amita Sehgal , John
B Hogenesch
1Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA, UNITED
2Department of Neuroscience, University of Pennsylvania School of Medicine, Philadelphia, PA, UNITED
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.
Development of circadian pacemaker cells in the Drosophila
tianxin liu , GURUSWAMY MAHESH , Jerry Houl , Paul Hardin
1Biology, Texas A&M University, College Station, TX, UNITED STATES
3Department of Biology and Center for Biological Clocks Research, Texas A&M University, College Station,
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
The beginning of in vivo clock gene expression rhythmicity
in the fetal rat SCN
Pavel Houdek , Alena Sumova
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 E2122. 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, Reverbα 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
An ultradian rhythm of somite formation is modulated by
xBmal1 and xNocturnin in Xenopus laevis
Kristen Curran , Latoya Allen , Nicole Johnson , Chelsea Lope ,
Gail Willadsen , ELizabeth Campbell , Brett VonBergen ,
Devon Winfrey , Morgan Hadley , Thomas Kerndt
1Biological Sciences, University of Wisconsin Whitewater, Whitewater, WI, UNITED STATES
10BIological 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.
The circadian molecular clock regulates adult hippocampal
neurogenesis by controlling the timing of cell-cycle entry
and exit.
Pascale Bouchard-Cannon , Lucia Mendoza-Viveos , Andrew Yuen ,
Mads Kaern , Hai-Ying Mary Cheng
1Biology, University of Toronto Mississauga, Mississauga, OR, CANADA
4Cellular 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.
Hyperoxia affects neonatal lung circadian dynamics and
worsens injury
Shaon Sengupta , Guang Yang , Phyllis Dennery
1Neonatology, Children's Hospital of Phialdelphia, Philadelphia, PA, UNITED STATES
3University 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)
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.
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.
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.
Maternal effects on circadian gene expression in fetal
Krisztina Meszaros , Linda Pruess , Matthias Gondan , Eberhard Ritz ,
Franz Schaefer
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.
Dopamine and Melatonin Regulate Ocular Circadian
Kenkichi Baba , Susana Contreras-Alcantara , Gianluca Tosini
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 DLuciferin 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.
Utilizing Electroretinograms (ERG) to Analyze Circadian
Rhythms in Grompadorhina Portentosa Photoreceptor
Wil Bogue , Andrew Urdiales , Edgar Mantes , Aaron Schirmer ,
Frederick Prete
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.
Retinal Muller Cells are Circadian Clock Cells and Clock
Genes Impact Retinal Neovascularization
Douglas McMahon , Lili Xu , John Penn , Andrew Liu
1Vanderbilt University, Nashville, TN, UNITED STATES
4University 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.
Physiological and behavioural consequences of
destabilised entrainment in melanopsin knockout mice
Violetta Pilorz , Russell Foster , Stuart Peirson
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.
Retinal projections to the suprachiasmatic nucleus: from
morphology to function
DIEGO FERNANDEZ , Shih-Kuo Chen , Samer Hattar
1Biology, Johns Hopkins University, Baltimore, MD, UNITED STATES
2Institute 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.
ipRGC neurotransmitters, glutamate and PACAP, are
distinct in their contributions to non-image forming
William Keenan , Samer Hattar
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 reentrainment 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.
Involvement of 5-HT3 and 5-HT4 receptors in the regulation
of circadian clock gene expression in mouse small intestine
Natsumi Aoki , Hiroyuki Watanabe , Kazuya Okada , Kazuyuki Aoki ,
Yuko Ikeda , Atsushi Haraguchi , Yu Tahara , Shigenobu Shibata
1School of Advanced Science and Engineering, Waseda Univercity, Shinjuku-ku, Tokyo, JAPAN
2School of Advanced Science and Engineering, Waseda University, Shinjuku-ku, Tokyo, JAPAN
5School ofAdvanced Science and Engineering, Waseda University, Shinzyuku-ku, Tokyo, JAPAN
7Waseda 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, parachlorophenylalanine, 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.
Serotonergic enhancement of photic phase shifts: BMY7378
does not require the serotonergic fibers connecting the
median raphe nucleus to the suprachiasmatic nucleus
Victoria Smith , Michael Antle
1Department of Psychology, University of Calgary, Calgary, AB, CANADA
2Psychology, 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 5HT1A 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
Investigating ASIC1a as a potential link between circadian
disruption and mood disorders in mice
Jonathan Shelton , Sujin Yun , James Shoblock , Natalie Welty ,
Christine Dugovic
1Janssen R&D, San Diego, CA, UNITED STATES
5Jassen 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 antidepressant/anxiolytic/mood stabilizing properties of ASIC1a inhibitors.
A Mutation in PERIOD3 Causes Familial Advanced Sleep
Luoying Zhang , Chris Jones , Noriaki Sakai , Ying Xu , Noriko Saigoh ,
Kazumasa Saigoh , Shu-Ting Lin , Seiji Nishino , Louis Ptacek , YingHui Fu
1Neurology, University of California, San Francisco, San Francisco, CA, USA
2University of Utah, Salt Lake City,
3Stanford University, Palo Alto, CA, UNITED STATES
4University 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
Perinatal photoperiod affects the serotonergic system
Noah Green , Chad Jackson , Douglas McMahon
1Biological Sciences, Vanderbilt University, Nashville, TN, UNITED STATES
2Vanderbilt 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.
In patients with Alzheimer’s disease, correlations in motor
activity fluctuations respond to bright light therapy are
associated with mood and cognition
Kun Hu , Frank Scheer , Steven Shea , Rixt Riemersma - van der Lek ,
Eus Van Someren
1Brigham & Women's Hospital/Harvard Medical School, Boston, MA, UNITED STATES
3Oregon Institute of Occupational Health Sciences, Oregon Health & Science University, Portland, OR,
4University 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
Early wake therapy phasae-delays advanced melatonin
offset and improves mood in depressed pregnant women
Barbara Parry , Charles Meliska , Diane Sorenson , Fernando Martinez ,
Ana Lopez , Henry Orff , Richard Hauger
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 phasedelayed 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 crossover 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
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.
Inhibition of specific classes of histone deacetylases reduce
anxiety- and depression-like behaviors in ClockΔ19 mutant
Ryan Logan , Rachel Arey , Angela Ozburn , Nicole Edgar ,
Colleen McClung
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 rewardrelated 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)).
Association of Depression with Variations of Melatonin and
Cortisol Rhythms in Delayed Phase Sleep Disorder (DSPD)
Seong Jae Kim , Kathryn Reid , Sabra Abbott , Phyllis Zee
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
Circadian abnormalities in the Myshkin mouse model of
Joseph TImothy , Harshmeena Sanghani , Mino Belle , Hugh Piggine
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 sleepwake 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 neuronspecific 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 goaldirected 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.
Brain circadian clocks in a mouse model of depression
Dominc Landgraf , Christophe D. Proulx , Roberto Malinow , David
K. Welsh
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.
Daily Temporal Rhythms in Cellular Activity in the Lateral
Hugh Piggins , Kanwal Sakhi , Sven Wegner , Mino Belle ,
Michael Howarth , Tim Brown
1Faculty of Life Sciences, University of Manchester, Manchester, UNITED KINGDOM
2Faculty of Life Sciences, University of Mancheser, Manchester, UNITED KINGDOM
4Faculty of Life Science, University of Manchester, Manchester, Greater Manchester, UNITED KINGDOM
6University 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 Per1luc 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.
Cyanobacterial Clock Output Feeds Back through
Metabolism to Regulate Clock Input
Gopal K. Pattanayak , Connie Phong , Michael J. Rust
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.
Intracellular Distributions of the KaiABC Proteins During the
Cyanobacterial Circadian Cycle: A Spatiotemporal
Stefanie Hertel , Pål O. Westermark
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.
Circadian regulation of oxidative stress-induced Stress
Julio Pusterla , Juan Lescano , Victoria A. Acosta Rodríguez , Mario
E. Guido , Eduardo Garbarino-Pico
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.
Circadian regulation of actin dynamics
Ned Hoyle , John O'Neill
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
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.
Diverse Circadian Periods from Individual Cells: Stochastic
or Clonal?
Yan Li , Yongli Shan , Hung-Chung Huang , Seung-Hee Yoo ,
Joseph Takahashi
1Department of Neuroscience, The University of Texas Southwestern Medical Center, Dallas, TX, UNITED
4Department of Biochemistry and Molecular Biology, UT Health Science Center at Houston, Houston, TX,
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.
An Ultradian Rhythm in Mouse Embryonic Fibroblast (MEF)
Cell Lines
Shuzhang Yang , Seung-Hee Yoo , Yongli Shan , Yan Li ,
Joseph Takahashi
1Howard Hughes Medical Institute, Department of Neuroscience, UT Southwestern Medical Center, Dallas,
2Department of Biochemistry and Molecular Biology, UT Health Science Center at Houston, Houston, TX-
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.
Transcriptional responses during synchronization of clocks
in mouse and human cells.
Julie Baggs , Jason DeBruyne
1Neuroscience Institute, Dept. of Pharmacology & Toxicology, Morehouse School of Medicine, GA, USA
2Neuroscience Institute/Dept. of Pharmacology & Toxicology, Morehouse School of Medicine, Atlanta, GA,
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.
Genome-wide analysis of circadian clock properties in
human fibroblasts
Ludmila Gaspar , Steven Brown
1University of Zurich, Institute of Pharmacology and Toxicology, Zurich, SWITZERLAND
2Institute 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
The circadian clock in the Antarctic krill Euphausia superba
Benjamin Hunt , Özge Özkaya , Paul Seear , Ted Gaten ,
Nathaniel Davies , Eran Tauber , Charalambos Kyriacou ,
Geraint Tarling , Ezio Rosato
1Genetics, University of Leicester, Leicester, Leicestershire, UNITED KINGDOM
3Biology, 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
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.
Understanding timekeeping in an intertidal crustacean
Eurydice pulchra
Lin Zhang , David C Wilcockson , Michael H Hastings , Simon
G Webster , Charalambos P Kyriacou
1Genetics Department, Leicester University, Leicester, UNITED KINGDOM
2Institute of Biological, Environmental, and Rural Sciences, Aberystwyth University, Aberystwyth, UNITED
3Laboratory 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
First description of circadian rhythms in visual sensitivity,
predatory behavior, and locomotion in a praying mantis
Aaron Schirmer , Frederick Prete , Edgar Mantes , Wil Bogue ,
Andrew Urdiales
1Biology, Northeastern Illinois University, Chicago, IL, UNITED STATES
3Northeastern 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.
Clock Silencing in Adulthood Impairs Rhythmic Insulin
Release and Reprograms Protein Secretion Transcription
Mark Perelis , Biliana Marcheva , Wenyu Huang , Chiaki Omura ,
Yumiko Kobayashi , Yumiko Kobayashi , Grant D. Barish , Joseph Bass
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.
Sustained inhibition of Na+/K+/Cl- co-transporter 1 (NKCC1)
enhances the magnitude of light-induced phase delays of
the circadian clock.
John (Mac) McNeill , James Walton , Elliott Albers
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 freerunning 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
Constitutive activation of glycogen synthase kinase 3
induces metabolic dyssynchrony and impairment in mice
Karen Gamble , Jodi Paul , Russell Johnson , William Ratcliffe ,
Martin Young
1Psychiatry and Behavioral Neurobiology, University of Alabama at Birmingham, Birmingham, AL, UNITED
2University of Alabama at Birmingham, Birmingham, AL, UNITED STATES
4Medicine, Cardiovascular Disease Division, University of Alabama at Birmingham, Birmingham, AL,
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-ofday-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.
Influence of circadian rhythms on postprandial triglyceride
metabolism: Role of the Suprachiasmatic Nucleus
1ANATOMY, UNAM, Mexico, Distrito Federal, 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
Novel Cry Stabilizing Compounds Reinforce the Peripheral
Clock Mechanism and Lower Blood Glucose in Diabetic
Jeffrey Johnson , Travis Renner , Tod Steinfeld , Eric Mabery ,
Erik Willis , Tim Rantz , Timothy Park , Erin Riegler , Paul Humphries ,
Kerryn McCluskie
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 glucagondependent 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
Metabolic disturbances in a model of chronic jetlag
LP Casiraghi , J J Chiesa , L Marpegan , Diego Golombek
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.
Insulin-FOXO3 signaling modulates circadian rhythms via
regulation of Clock transcription
Ines Chaves , Gijsbertus van der Horst , Raymond Schellevis ,
Romana Nijman , Marian Groot Koerkamp , Frank Holstege ,
Marten Smidt , Marco Hoekman
1Genetics, Erasmus MC, Rotterdam, NETHERLANDS
3Rudolf 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 insulinFOXO3-Clock axis in the modulation of circadian rhythms in response to metabolic signals.
Bmal1 in brown adipocytes is not required for rhythmic
oscillations of core body temperature
Georgios Paschos , GUANGRUI YANG , Teresa Reyes ,
Garret FitzGerald
1University of Pennsylvania, Philadelphia, 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 adipocytespecific 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.
Human skeletal muscle clock: implications in myokine
secretion and insulino-resistance
Laurent Perrin , Svetlana Skarupelova , Hubert Vidal , Etienne Lefai ,
Charna Dibner
1Division of Endocrinology, Diabetes and Nutrition, University of Geneva, Geneva, Geneva, SWITZERLAND
5Endocrinology, Diabetes and Nutrition, Faculty of Medicine, University of Geneva, Geneva,
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.
Circadian clocks and Polyamines – a metabolic feedback
ziv zwighaft , Judith Kraut-Cohen , Moran Shalev , Rona Aviram ,
Liat Rousso Noori , Marina Golik , Asaph Aharoni , Chaim Kahana ,
Gad Asher
1Biological chemistry, Weizmann Institute of Science, Rehovot, ISRAEL
2Weizmann Institute of Science, Rehovot,
5Biological chemstry, Weizmann Institute of Science, Rehovot, ISRAEL
9Department 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.
Effects of light, food, and methamphetamine on the
circadian activity rhythm in mice
Julie Pendergast , Shin Yamazaki
1Medicine-Diabetes, Endocrinology and Metabolism, Vanderbilt University Medical Center, Nashville, TN,
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 lightdark 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.
Food-entrainable circadian oscillations of PER2:LUC in the
mouse olfactory bulb: critical role for olfactory input
Ilya Pavlovski , Cong Xu , Hanna Opiol , Mateusz Michalik ,
Jennifer Evans , Ralph Mistlberger
1Simon Fraser University, Vancouver, CANADA
5Biomedical 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)
Phase advanced locomotor activity during timed restricted
feeding persists in tissue-type plasminogen activator knock
out (tPA-/-) mice.
Jessica Murphy , Eric Mintz
1Department of Biological Sciences, Kent State University, Kent, OH, UNITED STATES
2Biological 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 tissuetype 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.
Circadian properties of food-anticipatory activity reexamined: entrainment limits and scalar timing in operant
and general activity
Christian Petersen , Danica Patton , Teresa Dattolo
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
Meal shift experiments reveal unusual properties of
circadian food anticipatory rhythms in rats and mice.
Andrea Smit , Mateusz Michalik , Danica Patton , Christian Petersen ,
Ralph Mistlberger
1Simon Fraser University, Vancouver, BC, CANADA
5Psychology, 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)
Exploring interactions between copper homeostasis and
transport and the SCN circadian clock
Yukihiro Yamada , Rebecca Prosser
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, 12841295) 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 Cuinduced 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
MicroRNAs cooperate with rhythmic transcription to shape
circadian gene expression
Ngoc-Hien Du , Bulak Arpat , Mara De Matos , David Gatfield
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.
Neurospora crassa Circadian Rhythms in Continuous
Chemostat Cultures
Allison Cockrell , Kathleen Cusick , Emily Petersen , Carissa Soto ,
Russell Pirlo , Bradley Ringeisen , Justin Biffinger
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 clockassociated 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.
Use of mouse substrains identifies a QTL for circadian
Vivek Kumar , Joseph Takahashi
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 nonsynonymous 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.
A Novel ENU-Induced Mutation in the Melanocortin-4
Receptor (MC4R) Gene in Mice Leads to Altered BodyWeight Regulation and Expression of Circadian
Marleen de Groot , Jennifer Mohawk , Vivek Kumar , Joseph Takahashi
1Neuroscience, HHMI/UTSouthwestern, Dallas, TX, UNITED STATES
2Neuroscience, UTSouthwestern, Dallas, TX, UNITED STATES
3Neuroscience, 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 wheelrunning activity under normal ad libitum feeding conditions, and that these changes can be
reversed by exposure to conditions of temporal restricted feeding.
The circadian clock controls pre-mRNA splicing through the
Lin Zhang , Yufeng Wan , Xianyun Chen , Dongni Wang , Xinyang Yu ,
Hanjie Shen , Xiaodi Liang , Wei Liu , Jinhu Guo
1Life Science School, Sun Yat-Sen University, GuangZhou, GuangDong, CHINA
9Sun 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.
Circadian gene expression patterns on the periphery
depend on mouse genotype
Rok Kosir , Jure Acimovic , Ursula Prosenc Zmrzljak , Anja Korencic ,
Martina Perse , Damjana Rozman
1Center for Functional Genomics and Bio-chips, Faculty of Medicine, University of Ljubljana, Ljubljana,
3Institute of Oncology Ljubljana, Ljubljana, SLOVENIA
5Institute 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
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
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 clockbased diseases.
Estrogen response elements in clock genes: a bioinformatic
Jessica Lensie , Eric Mintz , Chi-Hua Chiu
1Biological Sciences, Kent State University, Kent, OH, UNITED STATES
2Department 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.
Circadian clock-dependent and -independent rhythmic
proteomes implement distinct diurnal functions in mouse
Daniel Mauvoisin , Jingkui Wang , Céline Jouffe , Eva Martin ,
Florian Atger , Patrice Waridel , Manfredo Quadroni , Frédéric Gachon ,
Félix Naef
1Diabetes & Circadian Rhythms, Nestle Institute of Health Science , Lausanne, Vaud, SWITZERLAND
2Institute of Bioengineering, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne,
Lausanne, Vaud, SWITZERLAND
4Diabetes & 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 posttranslational 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.
The mouse liver displays circadian rhythms in the
phospholipid metabolism and in the activity of its
synthesizing enzymes
Lucas D. Gorné , Victoria Acosta Rodríguez , Susana J. Pasquaré ,
Gabriela A. Salvador , Norma M. Giusto Mario E. Guido
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,
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.
New insight into post-transcriptional regulation of circadian
rhythms using a system wide identification of RNA-binding
Pauline Gosselin , Alfredo Castello , Matthias Hentze , Ueli Schibler
1Molecular Biology, University of Geneva, GENEVA, SWITZERLAND
2Molecular 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 mRNAbinding 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.
Role of the circadian clock regulated ATF5 transcription
capucine BOLVIN , frederic GACHON
1circadian rhytms, NIHS, lausanne, vaud, SWITZERLAND
2circadian 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 nightday 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.
Orchestration of the rhythmic translation by the circadian
Florian Atger , Eva Martin , Fréderic Gachon
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.
Chronic phase shifting paradigms disrupt locomotor rhythm
entrainment in C57BL/6J but not BALB/cJ mice.
Tyrus Takacs , Penny Molyneux , David Bonsall , Mary Harrington ,
Todd Weber
1Biology, Rider University, Lawrenceville, NJ, UNITED STATES
2Psychology, 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 freerunning 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
Drosophila mechanosensory organs and Ionotopic
Receptors (IRs) contribute to clock synchronization by
temperature cycles and proprioceptive feedback
Chenghao Chen , Alekos Simoni , Werner Wofgang , Min Xu ,
Richard Benton , Joerg Albert , Ralf Stanewsky
1Department of Cell developmental biology, UCL, London, UNITED KINGDOM
2School of biological and chemical sciences, Queen Mary, University of London, London, UNITED
4Department of Cell developmental Biology, UCL, London, UNITED KINGDOM
5Center for Integrative Genomics, University of Lausanne, Lausanne, SWITZERLAND
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.
Genetic engineering of an S714 mutation in PER1 leads to
an advanced feeding rhythm phase in mice
Zhiwei Liu , Moli Huang , Guangsen Shi , Lijuan Xing , Xi Wu ,
Zhipeng Qu , Jie Yan , Ling Yang , Satchidananda Panda , Ying Xu
1Nanjing University, Nanjing, CHINA
2Soochow University, Soochow,
3Model Animal Research Center, Nanjing University, Nanjing, Jiangsu, CHINA
7School of Mathematical Sciences, Soochow University, Suzhou, Jiangsu, CHINA
8Center for Systems Biology, Soochow University, Suzhou, Jiangsu, CHINA
9Salk 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.
Sustained Inhibition of GABAA Receptors in the SCN is
Necessary to Inhibit Light-induced Phase Delays
Tony Larkin , Daniel Hummer , H. Elliott Albers
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
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