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Sleep deprivation effects on growth factor expression in neonatal rats: A

potential role for BDNF in the mediation of delta power

Ilana S Hairston1,2, Christelle Peyron3, Daniel P. Denning2, Norman F. Ruby2, Judith

Flores2, Robert M. Sapolsky2, H. Craig Heller2, Bruce F. O’Hara2,4

1 To whom correspondence should be addressed

Ilana Hairston2 Department of biological sciences, Stanford University

371 Serra Mall

Stanford, CA 94305-5020

Tel: +1-650-7235882; Fax: +1-650-7255356

Ilana.Hairston@stanford.edu

3 CNRS UMR5167

Faculté de Médecine RTH Laënnec

7, rue Guillaume Paradin

69 372 LYON cedex 08, France

4 Department of Biology, University of Kentucky

101 Morgan Bldg

Lexington, KY 40506-0225

Running header: Sleep deprivation in neonatal rats

FINAL ACCEPTED VERSION

Copyright (c) 2003 by the American Physiological Society.

Articles in PresS. J Neurophysiol (December 10, 2003). 10.1152/jn.00894.2003

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Abstract:

The sleeping brain differs from the waking brain in its electrophysiological and

molecular properties, including the expression of growth factors and immediate early

genes (IEG). Sleep architecture and homeostatic regulation of sleep in neonates is distinct

from that of adults. Hence, the present study addressed the question whether the unique

homeostatic response to sleep deprivation in neonates is reflected in mRNA expression of

the IEG cFos, brain-derived nerve growth factor (BDNF), and basic fibroblast growth

factor (FGF2) in the cortex. As sleep deprivation is stressful to developing rats, we also

investigated whether the increased levels of corticosterone would affect the expression of

growth factors in the hippocampus, known to be sensitive to glucocorticoid levels. At

postnatal days 16, 20, and 24 rats were subjected to either sleep deprivation, maternal

separation without sleep deprivation, sleep deprivation with two hours recovery sleep, or

no intervention. mRNA expression was quantified in the cortex and hippocampus. cFos

was increased after sleep deprivation, and was similar to control level after two hours

recovery sleep irrespective of age or brain region. BDNF was increased by sleep

deprivation in the cortex at P20 and P24, and only at P24 in the hippocampus. FGF2

increased during recovery sleep at all ages in both brain regions. We conclude that

cortical BDNF expression reflects the onset of adult sleep-homeostatic response, while

the profile of expression of both growth factors suggests a trophic effect of mild sleep

deprivation.

Key words: cFos; FGF2; corticosterone; growth factors; hippocampus; cortex

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Introduction

Neonatal sleep patterns differ from those of adults (reviewed in Davis et al. 1999).

In adult rats, sleep deprivation of up to 12 hrs causes an increase in the proportion of the

1-4 Hz frequency range (delta power) in the EEG during slow-wave sleep (SWS),

whereby the level of increase is reliably predicted by the duration of preceding wake

(Franken et al. 1991; Tobler and Borbély 1990). Conversely, neonatal rats do not show

this correlation between delta power (DP) and wake history prior to post-natal day 20

(P20) but do display a compensatory increase in the amount of SWS (Frank et al. 1998).

This suggests that the neonate’s sleep regulatory system detects sleep loss but uses a

different compensatory strategy.

In adults, the expression of a number of genes in the cortex, such as immediate

early genes (IEG), and growth factors, is higher during wake than during SWS (Cirelli

2002; O'Hara et al. 1993; Pompeiano et al. 1994; Terao et al. 2003). This study

investigated whether the distinct homeostatic response to sleep deprivation observed in

neonatal rats is reflected in the pattern of gene expression. Brain derived growth factor

(BDNF), basic fibroblast growth factor (FGF2), their receptors (TrkB and FGF2r,

respectively), and cFos were measured after sleep deprivation and recovery sleep at P16,

P20, and P24.

FGF2 mRNA increases with elevated corticosterone (CORT) levels in the

hippocampus (Chao and McEwen 1994; Hansson et al. 2000; Molteni et al. 2001).

Increased CORT levels are associated with a transient (3 hrs) increase (Marmigere et al.

2003) and long-term (>6 hrs) decrease of BDNF in the hippocampus (Nibuya et al. 1999;

Ueyama et al. 1997). Conversely, sleep deprivation and selective paradoxical sleep (PS)

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deprivation have been shown to have no effect on BDNF expression in the hippocampus

(Sei et al. 2000; Taishi et al. 2001). Consequently, as sleep deprivation has been

previously shown to induce a stress response in neonatal rats (Hairston et al. 2001), we

expected FGF2 expression in the hippocampus to increase, and BDNF expression to

either increase or remain unchanged.

We found that the onset of the sleep deprivation induced-increase in DP coincided

with the onset of sleep deprivation induced-increase in BDNF in the cortex, i.e., P20.

FGF2 expression was increased during recovery sleep irrespective of age and brain

region, consistent with the elevated levels of CORT.

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Materials and methods:

Animals: A total of 107 animals were used in this study. Long-Evans rats were bred in

the lab and maintained on a 12:12 light/dark cycle, at an ambient temperature of 22°C.

Food and water were available ad libitum. Day of birth was designated postnatal day 0

(P0), and litter sizes were culled to 8-12 pups. Surgery, recording, and sleep deprivation

procedures were performed as previously described (Hairston et al. 2001). Briefly, at P9,

six male and female pups per litter were anaesthetized with methoxyflurane inhalant

(Metofane, Mallinckrodt Veterinary, Australia). Four EEG electrodes (no. 000 stainless

steel screws) were fastened bilaterally in frontal and parietal bones. Three EMG

electrodes (stainless steel wire) were inserted into the nuchal muscles. All electrodes

were attached to a seven-pin electric socket that was fastened to the skull with dental

acrylic and the incision was sutured. After surgery pups were left to regain consciousness

on a heating pad for approximately 1 h and then returned to their litter.

EEG/EMG recording: EEG signals were recorded from either left or right hemispheres

(fronto-parietal derivation) on a Grass 7 polygraph with one channel for differential EEG

and one for differential EMG, per animal. EEG potentials were filtered at 0.3 and 35 Hz

(1/2 max, 6 dB/octave), digitized at 100 Hz and stored in 10 sec epochs on a personal

computer. The EMG signal was full-wave rectified and integrated for each epoch. The

EEG signal was Fourier transformed and vigilance states were scored using a modified

scoring algorithm (Frank and Heller 1997a). Epochs with high power in 1-4 Hz range

(delta power) and low EMG signal were scored as slow-wave sleep (SWS), epochs with

low delta power and low EMG signal were scored as paradoxical sleep (PS), epochs with

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low delta power and high EMG signal were scored as wake. To determine the effects of

sleep deprivation the amount of SWS was calculated as percent of total recording time

following sleep deprivation at all ages. Vigilance state amounts were compared to time-

matched values obtained from no-SD animals of the same age. DP within SWS was

calculated as the proportion of the mean DP in SWS during the dark phase, preceding the

sleep deprivation.

Radioimmunoassay: The radioimmunoassay was based on a procedure developed by

Jacobson et al. (1993). Briefly, triplicate samples of plasma (10 ml) were heat-denatured

at 80°C. [3H]corticosterone (Sigma, USA) and CORT antiserum (Endocrine Sciences,

CA) were added to the samples that were incubated overnight. [3H]CORT was separated

from non-radiolabeled CORT using Dextran T70 (Amersham Pharmacia Biotech AB,

Sweden) coated charcoal, and quantified in a liquid scintillation counter (Beckmen model

LS 3801, CA). Competition binding was assessed against a standard curve of CORT

(Sigma, USA) ranging from 0.01ng/ml to 5ng/ml. A standard curve was produced by a

non-linear least squares formula and best-fit analysis based on the F distribution. To

maintain between-assay reliability, only assays in which slope coefficients were within

10% of each other’s error were used.

cDNA probes: We measured the mRNA expression of BDNF, TrkB, FGF2, FGF2r, cFos

and b-actin using the following cDNA probes: 1127 bp rat BDNF cDNA (Rosenthal et al.

1991); full length (~5kb) rat TrkB cDNA (Middlemas et al. 1991) courtesy of Dr E.

Shooter); 465 bp mouse FGF2 cDNA (ATCC Cat. # 63348); 2.5 kb mouse FGF2r cDNA

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(ATCC Cat. # 63344); full length (~2 kb) rat cFos cDNA (Curran et al. 1987); and 800

bp human b-actin (Hanukoglu et al. 1983). To extract inserts, plasmids were digested for

2 hrs at 37ºC with Hind III and NarI for BDNF, HindIII and XbaI for TrkB, EcoRI and

XbaI for FGF2, HindIII and EcoRI for FGF2r, EcoRI for cFos, and PstI for b-actin (Life

technologies, GIBCO BRL). Inserts were separated from vectors by electrophoresis on

1% Low Melting Point agarose gel (Life technologies, GIBCO BRL). The insert bands

were cut from the gel, dissolved in 3 volumes of distilled water and stored at -20°C until

use. 32P-radiolabeled random primed cDNA probes were made with an activity of 5-10 x

106 CPM/mL.

RNA extraction and Northern blotting: A total of 13 northern blots were generated.

Twelve blots contained total RNA from animals of the same age group, and sacrificed at

the same time point, and included sleep-deprived (SD), recorded but not sleep deprived

(no-SD), and litter mate controls (controls). Corresponding sets for cortical and

hippocampal tissue were made for each time point and age. An additional blot contained

cortical tissue samples from no-SD animals from all three age groups, sacrificed at the

end of the sleep deprivation.

Total RNA was extracted from whole cortical and hippocampal samples using the

TRIZOL protocol (Life technologies, GIBCO BRL). Eight mg total RNA from each

sample (one animal/sample) were fractionated by electrophoresis (100mV) on 1.2%

formaldehyde/agarose gel, transferred to positively charged nylon membranes

(Hybond-N+, Amersham Pharmacia Biotech) and cross linked to the membrane by UV

irradiation.

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Following pre-hybridization, membranes were hybridized overnight at 42°C in 5x SSC,

50% formamide, 50mM sodium phosphate pH 6.8, 1% SDS, 1mM EDTA, 2.5x

Denhardt's, 200mg/ml herring sperm DNA, and a radiolabeled random prime probe

(minimum 7 X 106/mL). Membranes were washed in lX SSC for 10 min at room

temperature and then twice in 0.4X SSC, 0.2% SDS for 25 min at 55°C. Membranes were

then wrapped in a plastic sheet and exposed on BIOMAX MS film (Kodak). Films were

developed and the images were digitized.

For quantification, background densitometry measures were subtracted for each

animal/lane, and values were normalized by regression analysis. The optical density

(O.D.) values for each gene of interest were divided by the values derived from the b-

actin probe.

Experimental procedure: To habituate animals to handling during sleep-deprivation, all

pups were handled for 10 min in their home cages, without separation from dam or litter,

at least twice on the two days prior to the experiment. In preparation for EEG recording,

animals were lightly anaesthetized and flexible recording cables were connected to their

head implants that were connected to slip-ring commutators. To minimize stress due to

separation from the dam, we employed a method developed by Frank et al. (1997a), and

based on van Oers et al. (1998), which enables separation of rat pups from their mother

and litter for a significant period of time without inducing significant changes in CORT

levels (Hairston et al. 2001). Briefly, P16 animals received enriched milk formula via a

cannula inserted through their cheek. An automatic pump (Harvard Apparatus, Inc.)

delivered 53ml every 45 min. throughout the experiment. In addition, P16 animals had

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their anal-genital region gently stimulated with a Q-Tip to induce elimination reflexes.

The purpose of these interventions was to mimic maternal care. P20 and P24 animals

were fed rat chow and water. Pups were placed in 12.5 x 12.5 x 12.5 cm acrylic

incubators containing bedding from their home cage. An age-appropriate thermoneutral

environment was maintained by placing the incubators in an acrylic water bath heated to

30, 27 or 24°C for P16, P20, and P24 animals, respectively.

Figure 1 depicts the experimental groups. EEG recording started at lights-off the evening

prior to deprivation. At lights-on the next day, animals were sleep deprived by gentle

handling for 90, 140, or 180 min for P16, P20, or P24 animals, respectively. Animals in

each age group were either sleep-deprived (SD), recorded but not sleep-deprived (no-

SD), and litter-mate controls (controls). At the end of the sleep deprivation, pups were

either sacrificed immediately (no-REC) or allowed to sleep undisturbed for 2 hrs (REC)

(Figure 1). Animals were sacrificed by decapitation within five minutes of the end of the

experiment, trunk blood was collected for RIA, brains were removed, dissected and

rapidly frozen on dry ice.

Statistical analysis: Sleep measures were anlyzed using a three-way ANOVA with age

(P16, P20, P24), treatment (SD, no-SD), and proportion in each state (i.e., wake, SWS,

or PS) in 30 min bins as a repeated measures factor. Plasma CORT levels were analyzed

using a three-way ANOVA with factors age, sleep condition (REC, no-REC), and

treatment (SD, no-SD, controls). mRNA expression was determined from the northern

blots wherein for each animal the O.D. of the gene of interest was divided with the O.D.

for b-actin. Due to differences in exposure and probe potency, no comparisons were

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made across RNA probes or blots. Comparisons for each gene, within single blots, were

done using the c2 median test, and the standard residual formula was used to determine

the source of the effect for each comparison.

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Results:

Sleep measures: During the dark phase, a significant decrease in time spent asleep, was

associated with age, coupled with an equivalent increase in the amount of time spent

awake. In addition there were large age differences in the overall amounts of PS and

wake, but not in SWS. These effects were most pronounced during the latter part of the

dark phase (see figure 2). A two-way ANOVA with age as main factor and proportion

time spent in each state within 30-min bins as a repeated measures factor was performed

on the seven hours of recording from midnight to lights on. A significant decrease in time

spent in PS (F(7,33)=13.6, p<.0001), a main effect of age (F(2,39)=30.1, p<.001) and an

interaction of age by time (F(14,64)=2.9, p=.02), were found. An equivalent increase in

the amount of time spent awake (F(7,33)=22.9, p<.0001), a main effect of age

(F(2,39)=24.4, p<.001) and an interaction of age by time (F(14,64)=3.2, p<0.001) were

found. For SWS a main effect of time-bins (F(7,33)=14.0, p<.001) and an interaction

with age (F(14,64)=2.6, P=.005) were observed.

The amount of sleep during the sleep deprivation period was previously reported

(Hairston et al. 2001). From lights on to the end of the sleep deprivation, no-SD animals

slept 55-84% of recording time, with the highest amounts of sleep found in P20 animals.

At all ages, sleep deprivation reduced total sleep time by 80-85% compared to no-SD

animals. Recovery animals expressed an increase in the amount of SWS subsequent to

the sleep deprivation (fig. 3). A three way ANOVA with age and sleep deprivation as

main factors, and percent of SWS (in 30 min bins) as repeated measures factor, yielded a

main effect of sleep deprivation (F(1,45)=12.5, p=0.003), an effect of time (F(3,45)=3.6,

p=0.021) and a two-way interaction of sleep-deprivation by time (F(3,45)=3.2, p=0.033).

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A similar analysis for the amounts of PS yielded a main effect of time (F(3,45)=6.9,

p<0.001) and an interaction of sleep-deprivation by time (F(3,45)=3.2, p=0.032) (data not

shown).

There was an age-dependent effect on DP during recovery SWS sleep. A three

way ANOVA with age and sleep deprivation as main factors, and DP (in 30 min bins) as

repeated measures factor, yielded a main effect of sleep deprivation (F(1,45)=44.7,

p<0.001), a main effect of age (F(2,45)=15.4, p=0.008), and an interaction of sleep-

deprivation with age (F(2,45)=8.0, p=0.028). This interaction was due to a lack of

difference between SD and no-SD animals at P16 (contrast analysis – t(1,14)=2.15,

p=0.65), small increase in delta power at P20 in the SD group (contrast analysis –

t(1,14)=3.0, p=0.104), and a large increase at P24 (contrast analysis – t(1,14)=9.7,

p=0.008).

Effects on Plasma CORT: A summary of the effects of sleep deprivation age and

recovery sleep is shown in table 1. The changes in CORT levels in no-REC animals were

previously published (Hairston et al. 2001). Briefly, CORT levels were significantly

higher in SD animals at all ages compared with age-matched controls (F(2,64)=43.2,

p<0.0001). A significant age-dependent increase in basal levels of CORT (F(2,64)=6.6,

p=0.002) was observed. Similar analysis of the plasma collected after 2 hrs recovery

sleep (REC) yielded no effect of sleep deprivation; however the age dependent increase

was sustained (F(2,32)=16.8, p<0.0001). P16 and P20, but not P24 no-SD animals had

higher CORT levels than littermate controls (Dunnett’s t(2)=2.31, p=0.05; t(2)=2.26,

p=0.05, P16 and P20, respectively).

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Effects on gene expression: Expression of all mRNA’s was detectable at all ages in both

brain regions. Sleep deprivation had no effect on the expression of b-actin that was used

as a control probe. The BDNF probe yielded two transcripts of 4.0 and 1.6 kb. While

some variation could be observed between the two, separate analysis of each band

yielded similar results. We thus averaged the O.D. of both bands and used this value for

further analyses.

Sleep deprivation effects on gene expression in cortex (fig 4) – cFos expression was

increased in SD animals at all ages (P16: c2=7.6, p=0.02, P20: c2=6.8, p=0.03, P24:

c2=6.4, p=0.04). BDNF expression was significantly increased in SD animals at P20

(c2=9.9, p=0.03) and P24 (c2=6.2, p=0.05). FGF2 expression was elevated in P16 SD and

no-SD groups compared with controls (c2=7.0, p=0.03). There were no detectable

changes in TrkB and FGF2r expression at any age (data not shown).

Sleep recovery effects on gene expression in cortex – TrkB levels were elevated at P24

(c2=6.2, p=0.04). FGF2 expression was increased at all age groups (P16 - c2=6.2, p=0.04;

P20 - c2=8.5, p=0.01, P24 - c2=10.2, p=0.01). There were no detectable changes in cFos,

BDNF and FGF2r expression at any age (see table 2 for all p values).

Sleep deprivation effects on gene expression in hippocampus (fig. 5)– cFos expression

was increased in SD animals at all age groups (P16: c2=6.0, p=0.05, P20: c2=7.4, p=0.03,

P24: c2=8.9, p=0.01). BDNF levels were increased at P24 (c2=9.1, p=0.01). TrkB levels

were increased in SD animals at P20 (c2=6.5, p=0.04) and at P24 (c2=5.8, p=0.05) (data

not shown). FGF2 was significantly increased in SD and no-SD animals compared with

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control at P16 (c2=9.0, p=0.01). There were no detectable changes in FGF2r at any age

(data not shown).

Sleep recovery effects on gene expression in hippocampus – cFos values increased in SD

animals at P16 (c2=9.0, p<0.01). BDNF expression was increased at P24 (c2=8.5,

p=0.01), and close to significantly at P20 (c2=4.8, p=0.08). FGF2 values were increased

in both SD and no-SD animals at P16 (c2=8.0, p=0.02), but only in SD animals at P20

(c2=6.2, p=0.05) and P24 (c2=6.2, p=0.04). There were no detectable changes in TrkB

and FGF2r expression at any age (see table 2 for all p values).

Age-dependent effects on the expression of BDNF in the cortex (fig 6) - To eliminate the

possibility that constitutively high levels of BDNF expression at the younger age may

explain the lack of change in BDNF after sleep deprivation in P16 animals, a blot

containing cortical sample of no-SD animals from all three age groups was probed for

BDNF and b-actin. There were no detectable changes in BDNF expression between the

age groups (c2=3.5, p=0.17).

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Discussion

We assessed changes in gene expression following sleep deprivation in neonatal

rats. The main findings were [a] an age- and brain region-independent increase of cFos

after sleep deprivation; [b] an age-dependent increase in BDNF expression in the cortex

and hippocampus after sleep deprivation, which correlated with the age when increased

DP after sleep deprivation was observed; and [c] an age- and brain –region- independent

increase in FGF2 expression during recovery sleep.

The age dependent effects on sleep states and response to sleep deprivation were

largely consistent with previous reports (Frank and Heller 1997b, 1997a; Frank et al.

1998). Following sleep deprivation, there were no changes in DP at P16, a small increase

at P20, and a large increase at P24. Notably, Frank and Heller (1998) did not show an

increase in DP at P20. This divergence from previous published findings is probably due

to natural variance in maturation rate between cohorts.

In this study, the sleep deprivation duration was tittered to the different ages,

based on the assumption that the ability to endure sleep deprivation increases with age.

This assumption is substantiated by our previously reported observation that the

amplitude of CORT increase, due to sleep deprivation, decreased with age (Hairston et al.

2001). Within two hours of recovery sleep, CORT levels were similar to no-SD animals,

indicating that the stress response duration was similar across age groups. The conditions

used for recording sleep in neonatal rats, given the right parameters (i.e., increased

ambient temperature and stimulated elimination reflexes), caused a small increase in

CORT levels in P16 and P20 no-SD animals, suggesting that maternal separation may

have induced a mild stress response in younger animals. As CORT increases FGF2

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expression (Chao and McEwen 1994), this observation may explain the mild increases in

FGF2 in P16 recorded animals compared with littermate controls.

The sleep deprivation effects on cFos expression were consistent with the effects

in adults. cFos levels were increased independent of age and brain region, despite varying

duration of sleep deprivation and different sleep patterns between age groups. In adult

rats, cFos is highly expressed both during sleep deprivation and during spontaneous wake

(Pompeiano et al. 1994) suggesting that its expression reflects the arousal state and not

the stress of sleep deprivation

Sleep deprivation effects on the two growth factors and their receptors were

markedly different. In the cortex, BDNF expression was increased in SD P20 and P24 but

not P16, while TrkB increases were observed in P24 SD animals after recovery sleep

only. In the hippocampus, P24 SD animals demonstrated increased BDNF, while TrkB

expression was increased in SD animals at P20 and P24. FGF2 expression was increased

after recovery sleep, independent of age and brain region, while FGF2r levels were

unaffected by sleep deprivation.

The time delay between mRNA synthesis and BDNF protein translation is

unknown. However, Nanda and Mack (2000) demonstrated elevated levels of BDNF

protein, >100% of control, in the cortex 1 hr after whisker stimulation, reaching peak

values within four hrs. If a similar time course of transcription-translation occurs post

sleep deprivation it is possible that the correlation between the developmental emergence

of wake induced-expression of BDNF with the emergence of wake-dependent DP -

indicates a functional relationship between BDNF and DP augmentation.

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The age groups in this study represent a developmental period characterized by

increased synaptic connectivity of the noradrenergic and cholinergic systems to

neocortical regions. Activity in both the noreadrenergic (NA) and cholinergic systems is

associated with wake maintenance and arousal quality (Berridge and Waterhouse 2003;

Sarter and Bruno 1999). NA projections to the cortex are present and innervating the

appropriate cortical layers by P3 (Foote and Morrison 1987; Levitt and Moore 1979).

However, during the third week of postnatal development (i.e., P15-21) an increase in

noradrenaline and tyrosine hydroxylase content in the neocortex occurs, reaching adult

levels at the beginning of week four (Foote and Morrison 1987; Levitt and Moore 1979;

Loizou 1971). Similar to the NA system, cortical cholinergic afferents increase in density

throughout weeks three to six (Coyle and Yamamura 1976).

Cirelli and co-workers have shown that BDNF and TrkB transcription in the

cortex is mediated by ascending NA fibers from the locus coeruleus (Cirelli 2002; Cirelli

and Tononi 2000a; Tononi and Cirelli 2001). Additionally, BDNF is synthesized by

excitatory neurons located in projection fields of basal forebrain cholinergic neurons

(Thoenen et al. 1991), suggesting a cholinergic role in the expression of BDNF.

Mitzoguchi et al. (2002) have recently demonstrated that BDNF caused sustained

increases of intracellular Ca2+ and Ca2+-activated potassium currents in cortical neurons,

both of which are believed to mediate cellular mechanisms underlying slow-wave

oscillations observed during SWS (Bazhenov et al. 2002; Massimini and Amzica 2001).

The potential role for BDNF in activity-dependent increase of DP is further indicated by

the observations that dark rearing reduces DP from visual cortex derivation (Miyamoto et

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al. 2003), as well as locally attenuates BDNF expression in the same region (Tropea et

al. 2001).

Given the developmental changes in neuromodulator excitatory input to the

cortex, the involvement of BDNF in intracellular Ca2+ and Ca2+-activated potassium

currents, and the relationship between sensory input DP development and BDNF

expression, we propose the following sequence of events: Neuromodulator activity

during wake increases BDNF expression in the adult cortex; BDNF, in turn, facilitates

inhibitory signaling in cortical neurons via its effects on intracellular Ca2+; increased

inhibition of cortical neurons results in the augmented power in delta oscillations. At P16,

BDNF signaling is attenuated, thus slow-wave activity during SWS would not reflect

wake history. Notably, Kushikata et al. (1999) reported that intracerebroventricular

(i.c.v.) administration of BDNF did not increase DP, while an increase in SWS amount

was observed. Combined with our findings, this may suggest that BDNF is necessary but

not sufficient to mediate an increase in DP.

Sleep deprivation resulted in elevated levels of CORT, especially in younger

animals. Repeated and prolonged increases in CORT levels are associated with reduced

levels of hippocampal BDNF (Chao and McEwen 1994; Hansson et al. 2003; Nibuya et

al. 1999), although BDNF expression can be increased after a short duration of stress

(i.e., 3 hrs, (Marmigere et al. 2003), and by excitatory cholinergic (da Penha Berzaghi et

al. 1993; Thoenen et al. 1991), or glutamatergic activity (Thoenen et al. 1991). Thus,

BDNF expression in the hippocampus can be increased under conditions associated with

either decrease or increase in CORT levels.

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In this study, BDNF increased in the hippocampus only of SD P24 animals, and

remained elevated after recovery sleep. As the hippocampus is anatomically mature by

P16, and pruning constitutes the main developmental event henceforth (Gaarskjaer 1985),

it is unlikely that our results can be explained by an age dependent-increase of excitatory

input. Moreover, our findings are inconsistent with previous studies in adults in which

BDNF mRNA or protein levels in the hippocampus were unaffected by total sleep

deprivation (Taishi et al. 2001) or selective paradoxical sleep deprivation (Sei et al.

2000). On the other hand, it is possible that our findings reflect the time course of BDNF

transcription after stress. In a recent study by Marmigère et al. (2003), BDNF mRNA

levels were measured after immobilization stress of varying duration. These authors

showed that BDNF levels increased within three hours and then proceeded to decline, to

below control levels, after five hours of immobilization stress. Thus, after three hours of

sleep deprivation, our P24 animals would be exactly at the peak of the stress induced

BDNF increase.

FGF2 expression increased in both brain regions only during recovery sleep.

FGF2 expression is mediated by intracellular Ca2+, cAMP, b-adrenergic receptor

activation, and activation of cholinergic nicotinic receptors (Belluardo 1999; Belluardo et

al. 1999; Follesa and Mocchetti 1993; Riva et al. 1996; Riva et al. 1997; Roceri et al.

2000) suggesting that its transcription is activity dependent. The time course observed for

b-adrenergic induced transcription of FGF2 was 4-5 hrs in adult animals (Follesa and

Mocchetti 1993; Hansson et al. 2003). Hence, we conclude that the observed increase in

FGF2, during recovery sleep, was in response to the stimulation of the sleep deprivation.

It is unlikely that FGF2 plays a role in inducing compensatory sleep patterns, as it does

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not seem to correlate with age and it has been previously shown that FGF2 administration

had no effect on any sleep parameter (Knefati et al. 1995).

In conclusion, this study addressed the changes in gene expression after sleep

deprivation in neonatal rats. It has been established that marked changes in sleep

architecture, homeostatic, and circadian regulation of sleep occur throughout

development. While the mechanism underlying DP increase after prolonged wake is

unknown, it has been proposed to reflect experience- or use-dependent processes (Meerlo

et al. 1997; Vyazovskiy et al. 2000), and not only the time spent awake. Our findings

suggest BDNF signaling as a candidate for facilitating DP increase after prolonged wake

or enriched experience.

We also demonstrated that sleep deprivation increased FGF2 expression during

subsequent recovery sleep. FGF2 stimulates proliferation and neuronal fate (Benharroch

and Birnbaum 1990; Palmer et al. 1999), and is increased after exposure to wheel-

running (Gomez-Pinilla et al. 1997) and enrichment (Schneider et al. 2001). Thus, the

consequence of mild sleep deprivation may have long-term trophic effects. This contrasts

with reduced proliferation observed after 96 hrs of sleep deprivation by Guzman-Marin et

al. (2003) in adult rats, but agrees with the neuroprotective and synaptogenic role of

BDNF increases after short periods of sleep deprivation.

Although it remains to be determined whether sleep is necessary for the down-

stream action of growth-related proteins, our observations contribute to growing evidence

implicating waking experience in sleep quality. Early experiences such as prenatal stress,

postnatal handling, and enriched environment have long lasting effects on sleep patterns

and stress management (Dugovic et al. 1999; Meaney et al. 1991; Mirmiran et al. 1982),

JN-00894-2003.R2

21

and have long-term implications for the quality of aging (Fuxe et al. 1996; Johansson et

al. 1999; Mohammed 1993). It is possible that changes in sleep quality are a necessary

component of the cellular processes underlying these neuroprotective effects.

~ ~ ~

Acknowledgements and funding: The authors thank Dr. Eric M Shooter for generously

providing us with the plasmid containing TrkB, Dr. N. Sobel and Dr P. Franken for their

critical remarks. This work was funded by NIH grants MCHHD #HD37315; NIMH, and

F31-MH65168-01.

JN-00894-2003.R2

22

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Legends of figures and tables:

Figure 1:

The experimental design. Depicts the sleep deprivation protocol where all SD and no-SD

animals were recorded from lights off the night prior to the experiment. At lights on,

sleep deprivation by gentle handling was performed on SD animals, while no-SD animals

were not touched accept for brief stimulation of elimination reflexes at P16. Animals

were sacrificed (sac.) either at the end of the sleep deprivation (SD) or after 2 hrs

recovery sleep (REC.). An additional group of animals (cont.) were in the same room but

kept with their mother and littermates and sacrificed at equivalent time points.

Figure 2:

The distribution of arousal states at the different ages during the dark phase, starting at

midnight. Percent of 30 min bins spent in each state (PS, SWS, wake) were calculated for

each animal during the dark phase before the sleep deprivation, and averaged across age

groups. Panels a-c depict the mean and S.E.M. of the percent in each state within each

age group. There were age-dependent changes in the amounts of wake and PS with no

change in SWS expressed as a main effect of age in PS and wake (F(2,39)=30.1, p<.001,

F(2,39)=24.4, p<.001, respectively), and an interaction of the age factor with all three

sleep states (PS: F(14,64)=2.9, p=.02; SWS: F(14,64)=2.6, P=.005; wake: F(14,64)=3.2,

p<0.001) . In addition, the distribution of states within the dark phase revealed the

emergence of diurnal regulation. At P16 the three states are evenly distributed while in

the older animals an increase in the percent time spent awake increases across the dark

phase.

JN-00894-2003.R2

32

Figure 3:

Percent time spent in SWS and delta power (DP) within SWS expressed in equivalent 30

min time bins during recovery sleep (i.e., starting from the end of the sleep deprivation).

The percent of 30 min bins spent in SWS (filled bars – no-SD; white bars - SD) were

calculated for each animal and averaged across age group. Superimposed are the 1-4 Hz

integrated values (filled triangles – no-SD; white circles - SD), corrected to the mean

value derived from the baseline night recording. There were no age dependent differences

in the amount of SWS during recovery. A three way ANOVA with age and sleep

deprivation as main factors, and percent of SWS (in 30 min bins) as repeated measures

factor, yielded a main effect of sleep deprivation (F(1,45)=12.5, p=0.003), with no effect

of age. There were age dependent differences in DP, wherein a similar analysis for DP

yielded a main effect of sleep deprivation (F(1,45)=44.7, p<0.001), a main effect of age

(F(2,45)=15.4, p=0.008), and an interaction of sleep-deprivation with age (F(2,45)=8.0,

p=0.028).

Figure 4:

Age- and sleep deprivation-dependent effects on gene expression in the cortex after sleep

deprivation. a-c, depict the mean and S.E.M. for each mRNA at each age. The values for

BDNF are the average of the two bands. “*” denotes that the source of the effect is due to

the values of the marked group, as determined by the standard residual formula applied to

the c2 test. d-f, are the corresponding northern blots probed for of cFos (2.1 kb), FGF2

(6.0 kb) and BDNF (4.0 kb, upper; 1.6 kb, lower), and b-actin (1.9 kb). The letter “C”

JN-00894-2003.R2

33

represents control animals, “N” for no-SD, and “S” for SD. [a,d] At P16, SD increased

cFos expression (c2=7.6, p=0.02), FGF2 was increased in both SD and no-SD animals

(c2=7.0, p=0.03), with no change in BDNF. [b,e] At P20, cFos and BDNF were increased

in SD animals (c2=6.8, p=0.03; c2=9.9, p=0.03, respectively). [c,f] At P24, cFos and

BDNF showed significant effects of sleep deprivation (c2=6.4, p=0.04; c2=6.2, p=0.05,

respectively).

Figure 5:

Age- and sleep deprivation-dependent effects on gene expression in the hippocampus

after sleep deprivation. a-c, depict the mean and S.E.M. for each mRNA at each age. The

values for BDNF are the average of the two bands. “*” denotes that the source of the

effect is due to the values of the marked group, as determined by the standard residual

formula applied to the c2 test. d-f are the corresponding northern blots probed for of cFos

(2.1 kb), FGF2 (6.0 kb) and BDNF (4.0 kb, upper; 1.6 kb, lower), and b-actin (1.9 kb).

The letter “C” represents control animals, “N” for no-SD, and “S” for SD. [a,d] At P16,

SD increased cFos expression (c2=6.0, p=0.05), FGF2 was increased in both SD and no-

SD animals (c2=9.0, p=0.01), with no change in BDNF. [b,e] At P20, cFos was increased

in SD animals (c2=7.4, p=0.03). [c,f] At P24, cFos and BDNF showed significant effects

of sleep deprivation (c2=8.9, p=0.01; c2=9.1, p=0.01, respectively).

Figure 6:

Age-dependent effects on the expression of BDNF in the cortex of no-SD animals,

indicating that age did not affect the level of BDNF expression. [a] Mean and S.E.M. for

JN-00894-2003.R2

34

each age group. The values for BDNF are the average of the two bands. [b] Northern blot

probed for of BDNF (4.0 kb, upper; 1.6 kb, lower), and b-actin (1.9 kb). No age

dependent differences in cortical BDNF mRNA were found.

Table 1:

Mean in mg/dL and S.E.M. of CORT derived from the RIA. “*” denotes a significant

difference between the group and control, “=” denotes difference from no-SD animals.

There was an age dependent increase in the amount of CORT (F(2,64)=6.6, p=0.002),

and at all ages SD animals had high levels of CORT compared to no-SD and controls

(F(2,64)=43.2, p<0.0001). After 2 hrs recovery sleep CORT levels were similar to no-SD

and control sibling sacrificed at the same time point. ). In addition, at p16 and P20, but

not at P24, no-SD animals had significantly higher CORT levels than their littermate

controls (Dunnett’s t(2)=2.31, p=0.05; t(2)=2.26, p=0.05, respectively).

Table 2:

Gene expression after 2 hours recovery sleep. Means, S.E.M. and the results of the c2

median test for all mRNAs shown as the ratio to b-actin expression. “=” denotes a

significant difference between the SD and/or no-SD and control animals.

JN-00894-2003.R2

35

Table 1

Effects of sleep deprivation and recovery sleep on plasma CORT levels

P16 P20 P24

no-REC REC no-REC REC no-REC REC

Control 2.5 ± 1.3

n=8

2.0 ± 1.6

n=5

5.8 ± 1.4

n=8

4.4 ± 3.8

n=5

9.9 ± 5.8

n=6

13.9 ± 7.9

n=6

no-SD 5.6 ± 2.3*

n=7

2.3 ± 2.7

n=4

9.1 ± 4.5*

n=6

6.4 ± 5.8*

n=6

9.3 ± 4.3

n=6

9.9 ± 5.2

n=5

SD 15.3 ± 3.6*=

n=6

1.0 ± 1.3

n=5

15.0 ± 1.5*=

n=6

2.2 ± 1.3

n=5

15.6 ± 3.8*=

n=5

13.7 ± 7.6

n=7

Table 2

36

Gene expression after 2 hours recovery sleep

Cortex Hippocampus

P16 P20 P24 P16 P20 P24BDNF

SD 0.382±0.07 0.877±0.05 0.766±0.04 0.762±0.08 0.612±0.05 1.372±0.06=

No-SD 0.346±0.10 0.808±0.05 0.631±0.05 0.722±0.08 0.563±0.06 1.205±0.15

RE

C

Control 0.329±0.13 0.795±0.02 0.602±0.05 0.763±0.06 0.453±0.11 1.146±0.10

c2=1.3,p=0.5 c2=5.6,p=0.06 c2=4.5,p=0.1 c2=0.7,p=0.7 c2=4.8,p=0.08 c2=8.5,p<0.01

TrkB

SD 0.770±0.07 0.785±0.07 0.929±0.04= 1.721±0.24 0.852±0.13 1.620±0.17

No-SD 0.754±0.03 0.806±0.09 0.807±0.05 1.775±0.44 0.904±0.08 1.306±0.12

RE

C

Control 0.694±0.06 0.795±0.09 0.805±0.05 1.636±0.41 0.880±0.22 1.371±0.10

c2=4.8,p=0.09 c2=1.6,p=0.46 c2=6.2,p=0.04 c2=0.1,p=0.9 c2=0.5,p=1.5 c2=3.5,p=0.2

FGF2

SD 0.585±0.26= 0.746±0.09= 0.920±0.07= 0.759±0.09= 0.433±0.04= 1.310±0.02=

No-SD 0.520±0.27 0.747±0.11= 0.720±0.06 0.693±0.06 0.301±0.07 1.088±0.13

RE

C

Control 0.525±0.12 0.525±0.06 0.792±0.06 0.588±0.06 0.382±0.10 1.096±0.08

c2=6.2,p=0.04 c2=8.5,p=0.01 c2=10.2,p<0.01 c2=8.0,p=0.02 c2=6.2,p=0.05 c2=6.2,p=0.04

FGF2r

SD 0.520±0.10 0.483±0.04 0.328±0.03 0.247±0.14 0.629±0.05 0.709±0.14

No-SD 0.747±0.11 0.519±0.04 0.321±0.04 0.288±0.08 0.713±0.15 0.797±0.14

RE

C

Control 0.525±0.06 0.509±0.04 0.350±0.04 0.285±0.11 0.748±0.18 0.659±0.17

c2=2.1,p=0.4 c2=1.3,p=0.5 c2=0.1,p=1.0 c2=0.8,p=0.7 c2=1.9,p=0.4 c2=1.3,p=0.5

cFos

SD 0.516±0.19 0.678±0.17 0.581±0.07 0.695±0.05= 0.977±0.04 0.878±0.07

No-SD 0.567±0.14 0.625±0.07 0.605±0.09 0.569±0.06 0.899±0.10 0.658±0.06

RE

C

Control 0.494±0.24 0.653±0.21 0.658±0.09 0.557±0.06 0.945±0.13 0.663±0.07

c2=0.4,p=0.8 c2=1.3,p=0.5 c2=1.5,p=0.5 c2=9.0,p<0.01 c2=2.5,p=0.3 c2=4.7,p=0.1