SLEEP 2011 Abstract Supplement

A. Basic Science II. Cell and Molecular Biology and Genetics
0033
NEUROTENSIN RECEPTOR 1 (NTSR1) IS INVOLVED IN
BOTH AFFECT AND SLEEP
Fitzpatrick K 1 , Hotz Vitaterna M 1 , Olker C 1 , Millstein J 3 , Gotter AL 2 ,
Winrow CJ 2 , Renger JJ 2 , Turek FW 1
1
Neurobiology & Physiology, Northwestern University, Evanston, IL,
USA, 2 Neuroscience Department, Merck Research Laboratories, West
Point, PA, USA, 3 Statistical Genetics, Sage Bionetworks, Seattle, WA,
USA
Introduction: Neurotensin, a neuropeptide that interacts with the dopaminergic
system, has been previously associated with many psychiatric
illnesses such as anxiety, addiction, and schizophrenia. Based on results
from a large-scale sleep-wake phenotype and genotype analysis of 269
adult male mice from a [C57BL/6J X (BALB/cByJ X C57BL/6J F1)]
N2 segregating cross, we identified neurotensin receptor 1 (NTSR1) as a
candidate gene for affect and sleep through quantitative trait loci (QTL)
and expression QTL (eQTL) analyses.
Methods: 10 control C57BL/6 mice and 10 NTSR1 knockout mice were
behaviorally tested in the open field, elevated plus maze, tail suspension,
and forced swim tests. The same animals were used to record baseline
sleep, sleep deprivation, and subsequent recovery, of which 8 knockout
animals and 9 wild type controls successfully completed the protocol.
Results: NTSR1 knockouts showed increased anxious behavior as compared
to the wild types in the open field test, with significant differences
in distance traveled, percent of time spent in the center, and percent of
time spent in the corners. NTSR1 knockouts also showed an increase in
despair behavior in the tail suspension test, with significant differences
in bouts of immobility. NTSR1 knockouts also displayed a lower percentage
of sleep time spent in REM sleep in the dark phase, and larger
diurnal variation of REM minutes than the wild type controls in baseline
conditions. In the recovery period following sleep deprivation, the
NTSR1 knockout animals presented with more wake and less non-REM
rebound sleep.
Conclusion: Anxiety and depression have long been associated with
alterations in sleep. Despite a wealth of evidence for a genetic component
for depression and anxiety, the specific genes and gene networks
associated with these affective disorders remain largely unknown. Here
we present that a knockout of a candidate gene in the region of a mouse
QTL for sleep characteristics, known to have comorbidity in humans
with affective disorders, shows similar behavioral characteristics associated
with affective disorders in humans.
Support (If Any): Merck & Co., Inc.
0034
TOLL-LIKE RECEPTOR 4 IS A REGULATOR OF
MONOCYTE AND ELECTROENCEPHALOGRAPHIC
RESPONSES TO SLEEP LOSS
Wisor J, Clegern WC, Schmidt MA
Washington State University, Spokane, WA, USA
Introduction: Sleep loss triggers changes in inflammatory signaling
pathways in the brain and periphery. The mechanisms that underlie
these changes are ill-defined. The Toll-like receptor 4 (TLR4)
activates inflammatory signaling cascades in response to endogenous
and pathogen-associated ligands known to be elevated in association
with sleep loss. TLR4 is therefore a possible mediator of some of the
inflammation-related effects of sleep loss. Here we describe the baseline
electroencephalographic sleep phenotype and the biochemical and
electroencephalographic responses to sleep loss in TLR4-deficient mice.
Methods: : TLR4-deficient mice and wild type controls were subjected
to electroencephalographic and electromyographic recordings during
spontaneous sleep/wake cycles and during and after sleep deprivation
sessions of 3, 6 and 24-hr duration.
Results: Relative to wild type control mice, TLR4-deficient mice exhibited
an increase in the duration of the primary daily waking bout oc-
curring at dark onset in a light/dark cycle. The amount of time spent
in non-rapid eye movement sleep by TLR4-deficient mice was reduced
in proportion to increased wakefulness in the hours immediately after
dark onset. Subsequent to sleep deprivation, EEG measures of increased
sleep drive were attenuated in TLR4-deficient mice relative to wild
type mice. TLR4 was enriched 10-fold in brain cells positive for the
cell surface marker CD11b (cells of the monocyte lineage) relative to
CD11b-negative cells in wild type mouse brains. To assess whether this
population was affected selectively by TLR4 knockout, flow cytometry
was used to count F4/80- and CD45-positive cells in the brains of sleepdeprived
and time of day control mice. While wild type mice exhibited a
significant reduction in the number of CD11b-positive cells in the brain
after 24-hr sleep deprivation, TLR4-deficient mice did not.
Conclusion: These data demonstrate that innate immune signaling pathways
active in the monocyte lineage, including presumably microglia,
detect and mediate in part the cerebral reaction to sleep loss.
Support (If Any): These experiments were supported by a Washington
State University, Spokane Faculty Seed grant and a Washington State
University New Faculty Seed grant.
0035
GENE EXPRESSION IN SLEEP-DEPRIVED PURINE TYPE
2X7 RECEPTOR KNOCKOUT MICE
Honn KA, Davis CJ, Bohnet SG, Krueger JM
WWAMI Medical Education Program, Sleep and Performance
Research Center, Washington State University, Spokane, WA, USA
Introduction: The purine type 2X7 receptor (P2X7R) is involved in
cytokine release and sleep regulation. ATP is released during neurotransmission.
Further, P2X7R expression changes with sleep loss and
time of day. Mice lacking the P2X7R have reduced duration of NREMS
and electroencephalography (EEG) slow wave activity (SWA) during
NREMS following sleep deprivation (SD) as compared with wildtype
mice (WT). We posit that the attenuated sleep responses to sleep loss
observed in the P2X7R knockout (KO) mice is accompanied by changes
in mRNAs previously linked to sleep regulation.
Methods: Levels of mRNAs previously linked to sleep were determined
using reverse transcriptase polymerase chain reaction (PCR).
Two strains of mice were used, control C57BL6 and P2X7RKO mice.
For each strain, one group of mice (N=8) was deprived of sleep by gentle
handling during the last 6h of daylight and the control groups (N=8
each) were allowed to sleep undisturbed. All mice were sacrificed at
dark onset. The hypothalamus and the somatosensory cortex were harvested,
RNA extracted, cDNA prepared and PCR performed by standard
methods.
Results: SD enhanced brain-derived neurotrophic factor (BDNF), the
P2X4 receptor (P2X4R), and adenosine deaminase (Ada) mRNAs in
the cortices of both strains. In the hypothalamus of WT mice, BDNF,
P2X4R, and Ada mRNAs also increased, but in this tissue these mRNAs
failed to change in the P2X7RKO mice. SD enhanced tumor necrosis
factor alpha (TNF) mRNA in WT cortices but not in P2X7RKO cortices.
In contrast, in the hypothalamus, SD failed to alter TNF mRNA expression
in WT mice but decreased it in the P2X7RKO mice.
Conclusion: Differential brain expression in WT and P2X7RKO mice
of BDNF, P2X4R, Ada and TNF mRNAs may be involved in the biochemical
causal pathways that lead to the differential sleep responses to
sleep loss exhibited by these two strains of mice.
Support (If Any): NIH NS031453, NS025378
A15
SLEEP, Volume 34, Abstract Supplement, 2011