SLEEP 2011 Abstract Supplement

A. Basic Science II. Cell and Molecular Biology and Genetics
0027
A NOVEL TECHNIQUE TO IDENTIFY THE TARGETS OF
CIRCADIAN MODIFIERS
Anafi R 1 , Dong AC 1 , Kim J 2 , Hogenesch JB 3
1
Division of Sleep Medicine, University of Pennsylvania, Philadelphia,
PA, USA, 2 Department of Biology and Penn Genome Frontiers
Institute, University of Pennsylvania, Philiadelphia, PA, USA,
3
Department of Pharmacology and the Institute for Translational
Medicine and Therapeutics, University of Pennsylvania, Philiadelphia,
PA, USA
Introduction: A recent genome-wide RNAi screen identified hundreds
of genes that influence circadian rhythms in the U2OS model system.
Core clock genes form a highly interconnected network and most modifiers
impact the expression of many clock genes. We have developed
a strategy to distinguish directly mediated changes in clock gene expression
from changes mediated by circadian network interactions. We
demonstrate the application of this strategy in identifying likely targets
of these circadian modifiers.
Methods: Using the U2OS model system, we knocked down each core
clock gene in a dose dependent fashion. The expression of all clock
genes was measured in every sample by quantitative PCR. For each
clock gene, we collected the data from all samples in which RNAi was
not targeted against the gene in question. The pooled data was fit using
singular value regression. The resulting equation is used to predict the
expression of the specified clock gene in new samples, given the expression
of all other core clock components. PCR was then performed to
measure clock gene expression in samples transfected by the recently
identified circadian modifiers. Changes in core clock gene expression
that are well predicted by the changes in the other core clock genes are
taken to represent network effects. Poorly predicted changes are taken
to represent influences not mediated by other core clock genes. In order
to test the utility of this approach, we applied this methodology to genes
with known core clock interactions
Results: The expression of each core clock gene was found to depend
strongly on the expression of others. Singular value regression was able
to describe a significant fraction of this variance and allows predictions
of clock gene expression.
Conclusion: This methodology permits rapid, preliminary determination
of the molecular targets of circadian modifiers and will improve our
understanding of the inputs influencing the circadian network.
Support (If Any): American Sleep Medicine Foundation Physician Scientist
Training Grant
0028
THE ROLE OF A SYNAPTIC ADHESION MOLECULE IN
SLEEP REGULATION
El Helou J 1,2 , Bélanger-Nelson E 1 , Dorsaz S 4 , Curie T 4 , Franken P 4 ,
Mongrain V 1,3
1
Center for Advanced Research in Sleep Medicine, Sacre-Coeur
Hospital of Montreal, Montreal, QC, Canada, 2 Biological Sciences,
Université de Montréal, Montreal, QC, Canada, 3 Psychiatry, Université
de Montréal, Montreal, QC, Canada, 4 Center for Integrative Genomics,
University of Lausanne, Lausanne, Switzerland
Introduction: Sleep need builds up during wakefulness and decays during
sleep and this has been linked to changes in synaptic strength. Accordingly,
sleep deprivation was shown to decrease NMDA receptors
(NMDAr) functioning in different brain areas. NMDAr functioning is
regulated by synaptic adhesion molecules (SAM), and one of these was
shown to determine NMDAr synaptic localization. Here, we assessed
the effect of sleep pressure on the expression of this SAM and its role in
sleep regulation.
Methods: 1) Male mice from 3 inbred strains were submitted or not to a
6h sleep deprivation (SD) by gentle handling starting at ZT0 (Zeitgeber
time 0: lights on), and sacrificed at ZT6 for brain sampling. Total RNA
was extracted and expression levels measured by microarray or qPCR.
2) AKR/J male mice were submitted to a 6h SD at ZT0, followed by
protein extraction of brain cortical tissue and Western blot. 3) The EEG
of mutant male mice not expressing the targeted SAM was recorded during
24h of baseline, during a 6h SD starting at ZT0 and 18h of recovery.
Results: We observed that the mRNA expression of the targeted SAM
was consistently decreased by SD in all inbred strains, whereas the
SD-dependent decrease in its protein level was not significant for total
cortical protein samples. Preliminary observations indicate that mice homozygote
for the SAM mutation are much more difficult to keep awake
during SD. Also, mutant and heterozygote mice had increased non-rapid
eye movement sleep time during baseline compared to wild-type littermates.
A similar trend was observed for recovery.
Conclusion: Additional analyses are underway to assess the effect of
this SAM on the dynamics of EEG markers of sleep pressure. Our preliminary
data suggest a role for this SAM in sleep regulation.
Support (If Any): University of Lausanne, FNS (3100A0-111974),
NSERC grants (386623-2010 and 390478-2010)
0029
HIPPOCAMPAL GENE EXPRESSION DURING
PARADOXICAL SLEEP AS REVEALED BY CDNA
MICROARRAY, QPCR AND IMMUNOHISTOCHEMISTRY
Renouard L, Ogawa K, Camargo N, Abdelkarim M,
Lakhdarchaouche Y, Gay N, Scoté-Blachon C, Salvert D, Fort P,
Luppi P
CNRS UMR5167, Lyon Cedex 08, France
Introduction: Paradoxical sleep (PS), the state during which dreams
occurs is present in mammals and birds. Its function remains unknown
although several studies indicate that it might play a role in learning and
memory. It has been named PS because the EEG shows a cortical activity
similar to that of waking while the muscle tone is completely abolished.
To investigate what modifications PS induces at molecular level
in the neurons of the hippocampal formation, we profiled gene expression
by cDNA microarrays approach in the hippocampal formation of
rats displaying different quantities of PS and then localized the neurons
with a modification in gene expresson.
Methods: A first group of rats (n=12) was deprived of PS (PSD rats) by
the multiple platform method during 78h. A second group of rats was allowed
to recover from this deprivation (PSR) during 6 hours (n=12). A
third group of rats remained in their home cage during all the protocol
(PSC, n=12). All animals were sacrificed at the same time of the day. Total
mRNA was extracted from the hippocampal formation and hybridized on
Affymetrix cDNA microarrays. Quantitative PCRs were then made to validate
the results of the microarrays. Then, we used immunohistochemistry
to localize neurons that express the proteins of interest in the hippocampal
formation of another set of 3 group of rat (PSC, PSD, PSR, n=12).
Results: The expression of 83 transcripts was modified in the hippocampal
formation by the protocol. 32 of these transcripts increased their
expression level after PS recovery. The majority of them are implicated
in synaptic plasticity, learning and memory (Homer1a, Arc, c-Fos, Zif-
268, Cox2) and others are implicated in the nervous system development
(Bdnf, Nptx2, Mas1). 24 transcripts increased their expression
level after PS deprivation and are mainly implicated in metabolism
regulation. In the PSR animals, the number of Fos and Arc immunoreactive
granular cells in the dentate gyrus was 10 time higher than in the
other groups. The number of Cox2+ cells in the dentate gyrus and CA3
was 2 time greater than in the other groups. We have also verified if
theses IEG+ neurons were young cells. No double labelled cells Fos+ or
Arc+/doublecortin were observed. Morever we injected BrdU 3, 6 and
12 weeks before the protocol of privation/recovery of PS and no Fos or
Arc neurons expressed BrdU+.
Conclusion: Our results indicate that a subpopulation of the granular
dentate gyrus neurons shows a strong activation and plasticity process
during PS.
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SLEEP, Volume 34, Abstract Supplement, 2011