Am J Physiol Regulatory Integrative Comp Physiol
281: R839–R845, 2001.
Conditions that affect sleep alter the expression
of molecules associated with synaptic plasticity
P. TAISHI, C. SANCHEZ, Y. WANG, J. FANG, J. W. HARDING, AND J. M. KRUEGER
Department of Veterinary and Comparative Anatomy, Pharmacology, and Physiology, College
of Veterinary Medicine, Washington State University, Pullman, Washington 99164-6520
Received 16 June 2000; accepted in final form 26 April 2001
rat; rapid eye movement sleep; ambient temperature; cortex;
hippocampus
serves a synaptic function
was first proposed by Moruzzi (35). Although Moruzzi
identified the level at which sleep could serve a function, it was presented within the context of a “local
homeostasis” concept postulating that, during wakefulness, there is an accumulation of “wear and tear” in
used neurons and that sleep serves to reverse these
effects. In light of modern knowledge that neural use
enhances synaptic efficacy (for review see Ref. 39),
recent theories of sleep function have emphasized synaptic plasticity (10, 12, 23, 32). Although experimental
support for these theories is limited, many of the molecules well characterized for their participation in
sleep regulation (for review see Refs. 24 and 25) are
also posited to play a role in synaptic plasticity. The list
THE HYPOTHESIS THAT SLEEP
Address for reprint requests and other correspondence: J. M.
Krueger, Dept. of Veterinary and Comparative Anatomy, Pharmacology, and Physiology, College of Veterinary Medicine, PO Box
646520, Washington State University, Pullman, WA 99164-6520
(E-mail: [email protected]).
http://www.ajpregu.org
includes interleukin-1 (40, 49), tumor necrosis factor
(1), nitric oxide (5, 18), adenosine (9), prostaglandins
(3), nerve growth factor (29, 30), and nuclear factor-␬B
(39).
There is a relatively large literature, prompted by
the Hebbian learning hypothesis (17), demonstrating
changes in molecular markers of synaptic plasticity
induced by long-term memory tasks (for review see Ref.
2). There is yet another literature, although somewhat
smaller, demonstrating the influence of sleep on memory (42, 43). There is, however, little information concerning the influence of sleep loss or conditions that
induce excess sleep on expression of molecules previously linked to synaptic plasticity. We investigated,
therefore, the changes in mRNA associated with sleep
deprivation and excess sleep of four molecules tightly
linked to synaptic plasticity (for review see Ref. 2):
activity-regulated cytoskeleton-associated protein (Arc),
matrix metalloproteinase-9 (MMP-9), tissue plasminogen
activator (tPA), and brain-derived neurotrophic factor
(BDNF). BDNF is also directly linked to sleep regulation;
injection of exogenous BDNF induces non-rapid eye
movement sleep (NREMS) and rapid eye movement sleep
(REMS) (27), and BDNF in the cerebral cortex increases
during sleep deprivation (7, 38). We report here that
sleep loss and a mild increase in ambient temperature
(Tamb), a condition that enhances sleep in rats, affect the
expression of Arc, MMP-9, tPA, and BDNF mRNAs.
METHODS
Sleep deprivation. Adult male Sprague-Dawley rats (320–
350 g) were housed separately in a sound-attenuated environmental chamber (Hotpack 35-260) on a 12:12-h light-dark
cycle (lights on at 0800) at 25°C. Rats were acclimated to the
housing conditions for 10 days before they were killed. Four
groups of rats (n ⫽ 8 in each group) were used. Group I rats
were controls and were killed at 1600. Group II rats were
deprived of all sleep by gentle handling as previously described (48) from 0800 to 1600. Samples comprising groups I
and II were processed together. Group III rats were control
rats for group IV and were killed at 1800. Group IV rats were
sleep deprived for 8 h, allowed to recover for 2 h, and then
killed at 1800. Rats in groups III and IV were processed
separately from other groups. Rats were decapitated, and the
The costs of publication of this article were defrayed in part by the
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0363-6119/01 $5.00 Copyright © 2001 the American Physiological Society
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Taishi, P., C. Sanchez, Y. Wang, J. Fang, J. W. Harding, and J. M. Krueger. Conditions that affect sleep alter
the expression of molecules associated with synaptic plasticity. Am J Physiol Regulatory Integrative Comp Physiol 281:
R839–R845, 2001.—Many theories propose that sleep serves
a purpose in synaptic plasticity. We tested the hypothesis,
therefore, that manipulation of sleep would affect the expression of molecules known to be involved in synaptic plasticity.
mRNA expression of four molecules [brain-derived neurotrophic factor (BDNF), activity-regulated cytoskeleton-associated protein (Arc), matrix metalloproteinase-9 (MMP-9), and
tissue plasminogen activator (tPA)] was determined after 8 h
of sleep deprivation and after 6 h of a mild increase in
ambient temperature, a condition that enhances sleep in
rats. After sleep deprivation, BDNF, Arc, and tPA mRNAs in
the cerebral cortex increased while MMP-9 mRNA levels
decreased. Conversely, after enhanced ambient temperature,
BDNF, Arc, and tPA mRNAs decreased while MMP-9 mRNA
increased. In the hippocampus, sleep deprivation did not
significantly affect BDNF and tPA expression, although Arc
mRNA increased and MMP-9 mRNA decreased. Brain temperature enhancement decreased Arc mRNA levels in the
hippocampus but did not affect BDNF, MMP-9, or tPA in this
area. Results are consistent with the notion that sleep plays
a role in synaptic plasticity.
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Table 1. Sense and antisense primers for Arc, BDNF,
tPA, MMP-9, and CycA
Gene
Arc
BDNF
tPA
MMP-9
CycA
Sequence (5⬘–3⬘)
GAG TAG TCC TCC CTC AGC CGC
CTC CGT ACG TGC TCC AGC ATC
GGC TGC GCC CAT GAA AGA A
GCG CCG AAC CCT CAT AGA CAT
ACG GGA CAC GGA AGA AAC GG
CCC TCG AAG CAG GTT GCT CTG
CGT GGC CTA CGT GAC CTA TG
GGA TAG CTC GGT GGT GTC CT
CTT TGC AGA CGC CGC TGT CTC
ACG CTC CAT GGC TTC CAC AAT
Length of PCR
Product, bp
336
339
408
592
474
Arc, activity-regulated cytoskeleton-associated protein; BDNF,
brain-derived neurotrophic factor; tPA, tissue plasminogen activator; MMP-9, matrix metalloproteinase-9; CycA, cyclophilin A.
target gene) were loaded on a 3.5% polyacrylamide gel. Total
optical density of autoradiograms was measured and quantified using the Phosphoimage Processing and Analysis System (Packard Bioscience, Meriden, CT). The cycle numbers
were determined to fall within the linear ranges of amplification for each molecule (Fig. 1).
Real-time PCR for Arc and MMP-9. Real-time PCR was
performed on an iCycler iQ Multi-Color Detection System
(Bio-Rad Laboratories, Hercules, CA) using iCycler IQ 96well plates (Bio-Rad Laboratories). The PCR mixture (25 ␮l)
contained 5 ␮l of the diluted cDNA (same concentration as
above), 12.5 ␮l of 2⫻ platinum quantitative PCR Super
Mix-UDG (GIBCO BRL, Rockville, MD; 60 U/ml platinum
Taq DNA polymerase, 40 mM Tris 䡠 HCl, pH 8.4, 100 mM KCl,
6 mM MgCl2, 400 ␮M dGTP, 400 ␮M dATP, 400 ␮M dCTP,
800 ␮M dUTP, 40 U/ml UDG), 2.5 ␮l (1:10,000 dilution) of
SYBR green (Molecular Probes, Eugene, OR), and 0.5 pmol of
each primer (Table 1). The PCRs were carried out at the
initial steps at 50°C for 3 min and 95°C for 2 min followed by
40 cycles as follows: denaturation for 45 s at 94°C and 45 s at
annealing temperature (61°C for Arc, 58°C for MMP-9, and
58°C for CycA) and extension for 60 s at 72°C. Gene expression was calculated using a comparative threshold cycle (Ct)
method (User Bulletin 2 ABI PRISM 7700 sequence detection
system, PE Applied Biosystems). At the end of each cycle, the
fluorescence emitted by SYBR green was measured. All the
reactions of the samples were performed in duplicate, and
each Ct value was an average of the values obtained from
each reaction. The ⌬Ct values were determined by subtracting the average CycA Ct value from control Ct values and the
Arc or MMP-9 Ct values, i.e., 2 ⫺ [⌬Ct ⫺ ⌬Ct(control mean)],
for each set of data. Student’s t-tests were used to compare
control values with those obtained for Arc or MMP-9.
RNase protection assay for BDNF. BDNF mRNA and L32
mRNA levels were estimated by using the RiboQuant multiprobe RNase protection assay (RPA) system (Pharmingen,
San Diego, CA) according to the manufacturer’s instructions.
Briefly, the radiolabeled antisense RNA probe set was synthesized in a final 20-␮l reaction mixture, which contained 40
U of RNasin, 20 U of T7 polymerase, 100 ␮Ci of [␣-32P]UTP
(3,000 Ci/mmol; Amersham, Piscataway, NJ), 2.75 nmol each
of GTP, ATP, and CTP, 61 pmol of UTP, 200 nmol of dithiothreitol, and 1⫻ transcription buffer and the RPA templates
(Pharmingen). The probe template set generated a 315-nt
antisense probe that protects 286 nt in the rat BDNF mRNA
(GenBank accession no. D10938, nt 2138–2423) and a 141-nt
antisense probe that protects 112 nt in the rat L32 mRNA
(GenBank accession no. X06483, nt 201–312). After incubation at 37°C for 1 h, the reaction was treated with DNase I (2
U; Promega) for 30 min at 37°C, and the probe was purified
with phenol-chloroform and chloroform and precipitated with
ethanol. The target RNA (15 ␮g) was dissolved in 8 ␮l of
hybridization buffer, to which 2 ␮l (3 ⫻ 105 cpm/␮l) of
32
P-labeled probe were added. All samples, including a degradation control (10 ␮g of yeast tRNA) and Pharmingen
control RNA (2 ␮g), were overlaid with mineral oil, heated to
90°C, and then immediately reduced to 56°C and incubated
for 12–16 h. The samples were then digested by a mixture
(100 ␮l) of RNase A (80 ng/␮l) and RNase T1 (250 U/␮l) at
30°C for 45 min. Then 18 ␮l of a solution of proteinase K (10
mg/ml), yeast tRNA (2 mg/ml), and 1⫻ proteinase K buffer
were added, and the sample was incubated at 37°C for 15
min. The RNA was extracted and precipitated as described
above, air-dried and dissolved in 1⫻ loading buffer, denatured at 90°C for 3 min, and then resolved on a 5% acrylamide-8 M urea sequencing gel. Gels were dried under vacuum for 2 h at 80°C. Radioactivity was quantified with a
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cortex and hippocampus were rapidly dissected and quickly
frozen in liquid N2. The time to remove, dissect, and freeze
the tissue samples was ⬍5 min.
Enhanced brain temperature. Adult male Sprague-Dawley
rats (300–350 g) were used and housed separately as described above. Two groups of rats (n ⫽ 8 in each group) were
used. Group V rats were control rats for group VI rats, and
they were housed at 25°C; they were killed at 1400. Group VI
rats were acclimated to 25°C housing conditions as were
group V rats, but at 0800 their ambient temperature (Tamb)
was increased to 28°C for the next 6 h. This mild increase in
Tamb induces increases in NREMS and REMS (36, 47). Group
VI rats were then killed at 1400. Rats in groups V and VI
were killed, and brains were removed and dissected as described above.
RNA extraction. Total RNA was extracted by the method
developed by Chomczynski and Sacchi (6) using RNA
STAT-60 according to the manufacturer’s protocol (TelTest
B, Friendswood, TX). The RNA was dissolved in sterile water
and checked using formaldehyde-containing agarose electrophoresis, and then the RNA concentration was determined
by absorbance at 260 nm.
RT-PCR. The first-strand cDNA synthesis procedure was
described in detail previously (4). Briefly, aliquots (2 ␮g/4 ␮l)
of each dilution of RNA solutions and 0.5 ␮g of oligo(dT)12–18
were incubated at 70°C for 10 min. After the aliquot was
chilled on ice, 200 U of Superscript II RNase H reverse
transcriptase (RT; GIBCO BRL, Grand Island, NY), dNTPs,
and buffer were added to a final volume of 20 ␮l and incubated at 42°C for 70 min. The mixture was heated at 95°C for
10 min, and then the cDNA was cooled to room temperature
and stored at ⫺20°C until further analysis.
Five microliters of each dilution of cDNA (10 ng of total
RNA) were amplified by PCR using primers designed to
correspond with the rat cDNA sequences for Arc, tPA,
MMP-9 (20), BDNF, and cyclophilin A (CycA; Table 1) (14).
PCRs were conducted in a total volume of 25 ␮l containing 1
U of Taq DNA polymerase (Promega, Madison, WI), 1.75 mM
MgCl2, 0.2 mM dNTP, 4 ␮Ci of [␣-32P]dCTP (3,000 Ci/mmol;
NEN, Boston, MA), 1⫻ reaction buffer, and each gene-specific primer at 0.2 ␮M. PCR was carried out in a Stratagene
Hot Top Assembly for the RoboCycler temperature cycler as
follows: 94°C for 3 min followed by 23 cycles at 94°C for 1
min, 61°C for 1 min, and 72°C for 1.5 min for amplification of
Arc mRNA and 23 cycles at 63°C for tPA; 22 cycles at 58°C for
BDNF; 24 cycles at 58°C for MMP-9 mRNA; and 17 cycles at
61°C for CycA mRNA. PCR products (5 ␮l of CycA and 5 ␮l of
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SLEEP AND SYNAPTIC ACTIVITY
Fig. 1. A: linear ranges of amplification for brain-derived neurotrophic factor (BDNF, ), activity-regulated
cytoskeleton-associated protein (Arc, ■), matrix metalloproteinase-9 (MMP-9, Œ), and tissue plasminogen activator (tPA, F). B: linear ranges of amplification for
cyclophilin A (CycA). Aliquots (10 ␮l) were taken from
each diluted cortical or hippocampal cDNA sample and
pooled to provide templates for experimental determination of linear ranges of PCR amplification for Arc,
BDNF, tPA, MMP-9, and CycA cDNA. Each PCR was
conducted in 25 ␮l, prepared as a master mix, with all
reaction components as described in METHODS.
Sleep deprivation also enhanced the levels of Arc
mRNA in the cortex whether determined by RT-PCR or
real-time PCR. In contrast to the BDNF results, Arc
mRNA increased in the hippocampus after sleep deprivation (compare group I with group II in Table 2). After
2 h of recovery, Arc mRNA levels in the cortex and
hippocampus were similar to corresponding control
values (compare group III with group IV in Table 1).
The effect of sleep loss on MMP-9 mRNA levels was
in the opposite direction of that described for BDNF
and Arc mRNAs. Thus, after 8 h of sleep deprivation,
MMP-9 levels decreased ⬃35% in the cortex and hippocampus with RT-PCR or real-time PCR (compare
group I with group II in Table 2). After 2 h of recovery
following 8 h of sleep deprivation, MMP-9 mRNA levels
were not significantly different from corresponding
control values (compare group III with group IV in
Table 2).
tPA mRNA increased in the cortex after 8 h of sleep
deprivation (groups I and II, Table 2). tPA mRNA was
Table 2. Changes in brain of mRNA levels
determined by RT-PCR or real-time PCR
after sleep deprivation
Group
BDNF
Arc
MMP-9
tPA
1.04 ⫾ 0.10
(1.00 ⫾ 0.13)
0.68 ⫾ 0.06*
(0.64 ⫾ 0.6*)
0.51 ⫾ 0.14
0.67 ⫾ 0.3
0.50 ⫾ 0.04
Cortex
RESULTS
Effects of sleep deprivation. After 8 h of sleep deprivation, BDNF mRNA determined by RT-PCR increased by ⬃120% in the cortex (compare group I with
group II in Table 2, Fig. 2). In contrast, sleep deprivation did not affect BDNF mRNA levels in the hippocampus (Table 2). With the use of a different method
of analysis, i.e., RPA, similar results were obtained.
Thus cortical levels of BDNF mRNA determined by
RPA increased and hippocampal levels were not affected after sleep deprivation (Table 3, Fig. 3). After 2 h
of recovery following 8 h of sleep deprivation, BDNF
mRNA levels, determined by RT-PCR, were indistinguishable from corresponding control values in the
cortex and hippocampus (compare group III with group
IV in Table 2).
I
0.57 ⫾ 0.06
II
1.27 ⫾ 0.18*
III
IV
0.62 ⫾ 0.08
0.86 ⫾ 0.12
I
0.56 ⫾ 0.03
II
0.61 ⫾ 0.04
III
IV
0.64 ⫾ 0.09
0.91 ⫾ 0.11
0.36 ⫾ 0.05
(1.33 ⫾ 0.42)
1.06 ⫾ 0.13*
(3.12 ⫾ 0.61*)
0.51 ⫾ 0.14
0.51 ⫾ 0.07
0.87 ⫾ 0.06*
0.49 ⫾ 0.09
0.68 ⫾ 0.07
Hippocampus
0.45 ⫾ 0.05
(1.32 ⫾ 0.31)
0.99 ⫾ 0.19*
(3.00 ⫾ 0.70*)
0.74 ⫾ 0.08
0.92 ⫾ 0.10*
1.33 ⫾ 0.19
(1.14 ⫾ 0.01)
1.00 ⫾ 0.06*
(0.77 ⫾ 0.10*)
0.77 ⫾ 0.06
0.69 ⫾ 0.04
0.63 ⫾ 0.07
0.96 ⫾ 0.13
1.90 ⫾ 0.28
2.10 ⫾ 0.14
Values are means ⫾ SE of ratios of the radioactivity in the PCR
product of interest to that in the band of the housekeeping control
band; values in parentheses are means ⫾ SE determined by realtime RT-PCR. * Significant difference from corresponding control,
P ⬍ 0.05.
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Cyclone PhosphorImager and OptiQuant analysis software
(Packard Bioscience). Additional reagents were supplied by
the RPA kit.
cDNA analysis. PCR products were excised and purified
from agarose gels with use of a commercial gel extraction kit
(QIAEX II, Santa Clarita, CA) following recommended protocols. Ten microliters (containing 1–1.5 ␮g of cDNA) of each
gel-purified cDNA (Arc, CycA, BDNF, tPA, and MMP-9) were
then subjected to restriction digestion with BanI and BanII
(BioLabs, Beverly, MA) and SamI, SacI, and BamHI (Promega), respectively. All restriction digests included 20 U of
enzyme in a total of 50 ␮l of reaction volume and were
incubated for 2 h at 37°C. Ten microliters of each reaction
were then loaded on 1.5% agarose gel containing ethidium
bromide (0.5 ␮g/ml). Gels were run at 100 V for 60 min, and
bands were photographed under ultraviolet light using a
charge-coupled device camera (Gel Doc 1000 fluorescent gel
documentation system, Bio-Rad Laboratories). cDNA fragments obtained from each enzymatic digestion were of the
correct size on the basis of the predicted amplified sequence
(data not shown).
Statistical analysis. Using results from phosphorimaging
data, we determined the ratio of the density of the target
RNA, relative to that of the internal standard, expressed as
mean ⫾ SE. Data were analyzed using one-way analysis of
variance followed by multiple comparison testing by the
Student-Newman-Keuls method. If tests for normality failed,
the data were analyzed using Kruskal-Wallis analysis of
variance on ranks. Differences were considered significant if
probability values for t or q were less than ␣ ⫽ 0.05.
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Fig. 2. Gel electrophoresis of RT-PCR-amplified rat
cortex BDNF and CycA mRNA. PCR products were
loaded on 3.5% acrylamide gel and visualized with a
PhosphorImager. C, control; SD, 8 h of sleep deprivation; T, increased ambient temperature.
DISCUSSION
The results presented here are consistent with previous related work. Thus BDNF mRNA levels were
previously reported to increase after sleep deprivation
(7, 8, 38). Furthermore, RT-PCR-derived data were
confirmed by RPA- or real-time PCR-derived data. The
decreases in MMP-9 mRNA after sleep deprivation are
similar to decreases in MMP-9 enzymatic activity
(J. W. Harding and J. Wright, unpublished observations) after 8 h of sleep deprivation following learning
trials in a Morris water maze. Finally, Cerelli and
Tononi (7, 8) also reported that Arc mRNA and protein
levels are higher after sleep deprivation than after
sleep. The present results extend these previous findings by providing a systematic comparison of the expression of mRNA of four synaptic plasticity-associated
molecules after sleep deprivation and after 2 h of recovery following sleep deprivation. We also determined
what happens to the expression of these molecules
after an increase in Tamb, a condition that enhances
sleep (36).
Sleep deprivation is used often in sleep research to
investigate the homeostatic regulation of sleep; after
sleep deprivation, sleep rebound occurs. This rebound
sleep is thought to result from the enhanced production
during sleep deprivation of sleep-promoting and sleepregulatory substances. For instance, the transfer of
cerebrospinal fluid from sleep-deprived to non-sleepdeprived animals induces increases in sleep in the
recipient (37). As indicated in the introduction, many of
the well-characterized sleep-regulatory substances are
also involved in synaptic plasticity. BDNF may be one
of these sleep-regulatory substances, although evidence for its role in sleep regulation is limited to the
findings that if it is injected, it induces sleep (27) and
its levels in the cortex increase during sleep deprivation (7, 38). However, there is extensive evidence for its
role in neurite growth and synaptic plasticity (for review see Ref. 34). Whether the other substances measured in this study, Arc, MMP-9, or tPA, have any role
in sleep regulation has not been investigated. However, given the extensive evidence for their involvement in synaptic plasticity (for review see Ref. 2), that
their levels are affected by sleep loss and excess sleep
could be indicative of their involvement in sleep function.
Many studies in several species have confirmed the
finding that mild increases in Tamb are associated with
increases in NREMS and REMS (36). REMS is particularly sensitive to Tamb; maximum duration of REMS
occurs at thermoneutral Tamb (47). Electroencephalographic (EEG) and electromyographic recordings from
our animals would have been necessary to determine
the amount of sleep they had before death. However,
we chose not to implant EEG and electromyographic
electrodes, because wounds induce long-term upregulation of cytokines, which, in turn can affect expression
Table 3. Levels of BDNF mRNA determined by
RNase protection assay in cortex and hippocampus
after 8 h of sleep deprivation
Group
BDNF
Cortex
0.25 ⫾ 0.01
0.48 ⫾ 0.05*
I
II
Hippocampus
0.23 ⫾ 0.02
0.25 ⫾ 0.03
I
II
Values are means ⫾ SE as described in Table 2 footnote. * Significant difference from group I, P ⬍ 0.05.
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higher after sleep deprivation in the hippocampus, but
this effect was not significant. tPA mRNA levels after
2 h of recovery following 8 h of sleep deprivation were
similar in controls and experimental animals in the
cortex and hippocampus.
Effects of mild increases in Tamb. After 6 h at 28°C,
BDNF mRNA levels decreased significantly in the cortex (compare group V with group VI in Table 4; Fig. 2,
right). Although BDNF mRNA levels were less in the
hippocampus after 6 h at 28°C, they did not reach
statistical significance. Arc mRNA levels determined
by RT-PCR or real-time PCR also significantly decreased after 6 h at 28°C in the cortex. In contrast to
BDNF mRNA, the mild increase in Tamb also resulted
in a significant decrease in Arc mRNA levels in the
hippocampus (Table 4). MMP-9 levels were significantly increased in the cortex after treatment at 28°C
Tamb whether determined by RT-PCR or real-time
PCR. In the hippocampus, MMP-9 values increased
almost threefold as determined by RT-PCR (not significant) and about twofold as determined by real-time
PCR (significant). tPA mRNA levels, on the other hand,
significantly decreased in the cortex after 6 h of treatment at 28°C Tamb. tPA mRNA levels in the hippocampus were not affected by the Tamb treatment.
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Fig. 3. RNase protection assay of BDNF mRNA. BDNF mRNA levels
in the cerebral cortex (CT) increased after 8 h of sleep deprivation
(SD) compared with controls (C). BDNF mRNA levels in the hippocampus (HC) were not affected by sleep deprivation. Samples were
prepared and visualized as described in METHODS; 1 sample per
experimental group is shown. A total of 8 samples from 8 rats were
analyzed (see Table 3 for results).
of some of the molecules we measured, e.g., MMP-9
(50). Furthermore, the presence of EEG electrodes on
the surface of the dura could lead to inflammation; the
cytokine-chemokine cascade associated with inflammatory responses would likely affect expression of a
host of molecules, including those we measured. Finally, the observation that mild increases in Tamb enhance sleep is one of the most reliable, reproducible
results in sleep research. The advantage of using Tamb
Table 4. Changes in cerebral cortex and hippocampus
of mRNA levels determined by RT-PCR or real-time
PCR after mild increases in ambient temperature
Group
BDNF
Arc
V
0.46 ⫾ 0.05
VI
0.17 ⫾ 0.01*
0.51 ⫾ 0.06
(1.16 ⫾ 0.25)
0.20 ⫾ 0.03*
(0.57 ⫾ 0.07*)
V
0.89 ⫾ 0.10
VI
0.75 ⫾ 0.05
MMP-9
tPA
0.63 ⫾ 0.04
(1.02 ⫾ 0.07)
0.76 ⫾ 0.003*
(1.31 ⫾ 0.11*)
0.43 ⫾ 0.03
Cortex
0.21 ⫾ 0.02*
Hippocampus
0.91 ⫾ 0.11
(1.09 ⫾ 0.12)
0.42 ⫾ 0.03*
(0.73 ⫾ 0.05*)
1.02 ⫾ 0.10
(1.19 ⫾ 0.21)
2.94 ⫾ 1.4
(2.64 ⫾ 0.61*)
0.72 ⫾ 0.05
0.57 ⫾ 0.05
Values are means ⫾ SE as described in Table 2 footnote; values in
parentheses are means ⫾ SE as determined by real-time RT-PCR.
* Significant difference from group V, P ⬍ 0.05.
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to manipulate sleep is that it does not involve handling
the animals, nor is it a stressor.
A fundamental hypothesis forming the basis for Hebbian learning is that synaptic activation enhances the
efficacy of transmission in those synapses during subsequent activation. The enhanced efficacy is thought to
result from molecules, the upregulation and release of
which are dependent on synaptic activation (for review
see Refs. 16 and 28). Arc is an immediate-early gene
(30). Arc protein interacts with cytoskeletal protein
and likely modifies structural protein. There is strong
evidence that Arc mRNA and protein are tightly coupled to the activity state of neurons (28, 30). Within 30
min of neural activation, Arc mRNA is found throughout the dendrites of granule cell neurons (16, 28, 30,
44). Translation of Arc mRNA in specific activated
dendrite spines is likely targeted via the assembly of
the translational complex, which is induced by activation of metabotropic glutamate receptors (45). This
represents a mechanism by which the initiation of
Hebbian learning by waking activities can be targeted
to the specific circuits activated. Furthermore, caffeine,
a well-known inhibitor of sleep, induces Arc expression
via a step involving the adenosine A1 receptor (11).
That prolonged wakefulness (sleep loss) and excess
sleep alter Arc mRNA expression may reflect a disturbance of this mechanism.
A well-recognized action of tPA is to catalyze the
cleavage of plasminogen to the active protease plasmin. Plasmin is an important activator of latent
MMPs. As such, we anticipated that increased tPA
mRNA would translate into increased MMP-9 mRNA.
That it did not is likely due to the multiple roles of tPA.
Furthermore, we measured MMP-9 mRNA, not protein
activity; tPA and plasmin may not affect MMP-9
mRNA expression. Nonetheless, tPA is recognized as a
participant in neural remodeling (15), and our results
indicate that its mRNA is affected by sleep loss and
excess sleep.
The extracellular matrix is of central importance to
nervous tissue structure and function. The composition
of the extracellular matrix is in a dynamic flux that
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Perspectives
There is a growing body of evidence suggesting that
sleep is not a uniform process of the entire brain but is,
instead, a local property of highly interconnected neurons and that the local state shifts are dependent on
prior neural activity, rather than duration of wakefulness (23). Initially predicted from theoretical considerations (23, 25), it was subsequently shown that patterns of brain activity during sleep are dependent on
prior wakefulness activity. For example, Kattler et al.
(21) demonstrated that mechanical stimulation of one
hand enhances EEG slow-wave activity [a measure of
the depth of sleep (37)] in the contralateral somatosensory cortex during subsequent sleep. Drummond et al.
(13), using functional magnetic resonance imaging,
reached the conclusion that the localized effects of
sleep deprivation are dependent, in part, on the specific
cognitive task performed in prior wakefulness. These
and other data suggest that sleep is targeted and,
similar to synaptic efficacy, dependent on prior synaptic activation. Our data are consistent with this notion
to the extent that manipulations that affect sleep, sleep
deprivation and mild increases in Tamb, affected expression of molecules associated with synaptic plasticity.
This work was supported in part by National Institutes of Health
Grants NS-25378, NS-31453, and HD-36520 and by a grant from the
Sleep Medicine Education and Research Foundation, a foundation of
the American Academy of Sleep Medicine.
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