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Effects of neonatal dexamethasone treatment on hippocampal synaptic function.

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Effects of Neonatal Dexamethasone
Treatment on Hippocampal
Synaptic Function
Hsiao-Ju Lin, MS,1 Chiung-Chun Huang, PhD,1 and Kuei-Sen Hsu, PhD1,2
Objective: Synthetic glucocorticoid dexamethasone (DEX) is frequently used as a therapeutic agent to lessen the morbidity of
chronic lung disease in premature infants. Surprisingly, little is known about the long-term neurodevelopmental outcomes of this
therapy.
Methods: Using a schedule of tapering doses of DEX similar to that used in premature infants, we examined the consequences
of neonatal DEX treatment on hippocampal synaptic plasticity of infants and associative memory later in their lives.
Results: Neonatal DEX treatment changed the direction of synaptic plasticity, favoring low-frequency, stimulation-induced,
long-term depression and opposing the induction of long-term potentiation by high-frequency stimulation in adolescent (5week-old) rats, but these alterations disappeared in young adult (8-week-old) rats. The effects of DEX on long-term depression
and long-term potentiation were found to correlate with an increase in the autophosphorylation of Ca2⫹/calmodulin-dependent
protein kinase II and a decrease in the protein phosphatase 1 activity. Neonatal DEX treatment also disrupted memory retention
in 5-week-old (but not 8-week-old) rats subjected to passive avoidance learning tasks.
Interpretation: These results suggest that neonatal DEX treatment alters hippocampal synaptic plasticity and contextual fear
memory formation in later life, but these impairments apparently are not permanent.
Ann Neurol 2006;59:939 –951
Severe respiratory distress syndrome is common in
very-low-birth-weigh infants, resulting in various degrees of mechanical ventilation and/or oxygen dependency and subsequent onset of chronic lung disease.
Synthetic glucocorticoid dexamethasone (DEX) frequently is used as a therapeutic agent to lessen the progression of chronic lung disease in premature infants.1–3
Typically, high doses of DEX are administered for several weeks, notably during a critical period in the development of the infant brain. Therefore, concern
about the long-term effect of this therapy on brain development of the child has been growing.4,5 Although
long-term follow-up studies are scarce, a recent study
reported that school-age children who were treated
with DEX as preterm infants have impaired neuromotor skills and cognitive function,6 supporting the view
that neonatal DEX therapy adversely affects brain development. Barrington’s review7 of 8 randomized controlled studies enrolling 679 infants also showed a significant risk for neurodevelopmental impairment and
cerebral palsy after DEX treatment. These findings
raise additional questions concerning the mechanism(s)
underlying these neurodevelopmental abnormalities
and whether these alterations are long-lasting and lifelong. Because controlled prospective and mechanistic
studies in humans are limited, a plausible way to address these questions is to use an experimental animal
model that simulates the DEX treatment used clinically
during neonatal life.
During the past decade, a number of clinically relevant rodent models have been used to investigate the
long-lasting neurological outcomes of neonatal DEX
treatment. In one such model (the neonatal rat pup on
postnatal days 1–3 [P1-3], which correlates to the neurodevelopmental age of human infants when exposed
to this agent in the neonatal intensive care unit), a
3-day tapering course of DEX treatment caused a longlasting deficit in the storage of spatial memory.8 Furthermore, a 4-day tapering course of DEX treatment in
the neonatal rat pup on P3 to P6 has been reported to
change neurodevelopment and neuroendocrine function in adulthood.9,10 Although these results highlight
From the 1Department of Pharmacology, College of Medicine; and
2
Center for Gene Regulation and Signal Transduction Research,
National Cheng Kung University, Tainan, Taiwan.
Address correspondence to Dr Hsu, Department of Pharmacology,
College of Medicine, National Cheng Kung University, No. 1, University Road, Tainan City 701, Taiwan.
E-mail: [email protected]
Received Nov 11, 2005, and in revised form Apr 3, 2006. Accepted
for publication Apr 10, 2006.
Published online May 22, 2006 in Wiley InterScience
(www.interscience.wiley.com). DOI: 10.1002/ana.20885
© 2006 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
939
the risk for long-lasting behavioral and physiological alterations associated with neonatal DEX treatment, they
do not show the specific mechanisms behind these abnormalities. Furthermore, the results have varied from
study to study, in part because of the differences between studies in behavioral testing methodology and
variations in timing and dosage regimens. This motivated us to evaluate in detail the long-lasting effects of
a DEX treatment protocol (resembling the one used
clinically to treat preterm human neonates) on brain
function during early development.
This study asks two main questions: (1) Does neonatal DEX treatment permanently alter hippocampal
synaptic plasticity and associative memory in later life;
and (2) if so, what is the responsible molecular mechanism(s)? To this end, neonatal pups were treated with
tapering doses of DEX on P1 to P3, which is the age
of the rat brain comparable with that of human brain
in last trimester of pregnancy and in premature infants
treated with DEX.11,12
Materials and Methods
Animals
Pregnant Sprague–Dawley rats (250 –280gm; Central Animal
Laboratory, National Cheng Kung University, Tainan, Taiwan) were housed singly under controlled illumination (12/
12-hour light/dark cycle, “on” at 7:00 AM) and ambient temperature (24°C) and had ad libitum access to food and water.
Pups were born on day 22 or 23 of gestation. Within 6 to 8
hours after delivery (day 0), pups were removed from their
nests and six male pups were placed back with each dam.
Pups were weaned at 21 days old and remained group
housed with littermates until experimentation at 5 to 8
weeks old. All procedures were performed according to National Institutes of Health (NIH) guidelines for animal research (Guide for the Care and Use of Laboratory Animals,
NRC, 1996) and approved by the Institutional Animal Care
and Use Committee at National Cheng Kung University. All
efforts were made to minimize animal suffering and to use
only the number of animals necessary to produce reliable scientific data.
Dexamethasone Treatment Protocol
Each litter was assigned to one of two treatment groups: a
saline (SAL)-treated or DEX-treated group. All pups within
each litter were removed from their home cage and separated
from their mother for injection and body weight measurement (between 11:00 AM and 1:00 PM) for a period of 5
minutes. Pups in the DEX group received a daily intraperitoneal injection of DEX (Sigma, St. Louis, MO) between P1
and P3. DEX was given in tapering doses of 0.5mg/kg on
P1, 0.3mg/kg on P2, and 0.1mg/kg on P3. In some experiments, tapering doses of DEX were administered at P4 to P6
or P15 to P17. Animals in the vehicle group received equivalent volumes of intraperitoneal injection of sterile SAL as
the pups in the DEX group. Electrophysiological, biochemical, and behavioral experiments were performed using different groups of rats at the age of 5 or 8 weeks.
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Hippocampal Slice Preparations and Electrophysiology
Hippocampal slices (400␮m in thickness) were prepared
from 5- or 8-week-old rats using standard procedures,13,14
allowed to recover for a minimum of 1 hour, and then transferred to a submersion-type recording chamber continually
perfused at 30 to 32°C with oxygenated artificial cerebrospinal fluid solution (in mM): 117 NaCl, 4.7 KCl, 2.5 CaCl2,
1.2 MgCl2, 25 NaHCO3, 1.2 NaH2PO4, 11 glucose. Area
CA3 was removed surgically after sectioning. Extracellular
and whole-cell recordings were performed using an
Axoclamp-2B or Axopatch 200B amplifier (Axon Instruments, Union City, CA). The responses were low-pass filtered at 2kHz, digitally sampled at 5 to 10kHz, and analyzed
using pCLAMP software (version 8.0; Axon Instruments).
Postsynaptic responses were evoked in CA1 stratum radiatum by stimulation of Schaffer collateral/commissural afferents at 0.033Hz with a bipolar stimulating electrode. The
stimulation strength was set to elicit a response with an amplitude that was 30 to 40% of the maximum spike-free response. Field excitatory postsynaptic potentials (fEPSPs) were
recorded with a glass pipette filled with1M NaCl (2–3M⍀
resistance), and the initial slope was measured. Paired-pulse
facilitation (PPF) was assessed using a succession of paired
pulses separated by interpulse intervals of 20, 40, 60, 80,
100, 150, and 200 milliseconds. The LTP was induced by
high-frequency stimulation (HFS) at the test pulse intensity
(ie, two 1-second trains of 100Hz stimuli separated by an
intertrain interval of 20 seconds), and the saturated LTP was
induced by six 1-second trains of 100Hz stimuli separated by
an intertrain interval of 5 minutes. Long-term depression
(LTD) was induced by stimulation delivered at 1Hz for 15
minutes (900 pulses). The stimulation intensity during stimulation application was the same as the test pulse intensity.
Whole-cell recording of excitatory postsynaptic currents (EPSCs) was made from CA1 pyramidal cells, which were identified under a differential interference contrast microscope.
Cells were held at ⫺70mV, whereas series and input resistances were monitored throughout each experiment, as described previously.14 Patch pipettes (3–5M⍀) filled with the
following internal solution were used (in mM): 110 potassium gluconate, 30 KCl, 10 HEPES, 1 MgCl2, 0.5 EGTA, 4
Na2ATP, 0.3 Na3GTP, 7 phosphocreatine, 5 lidocaine
N-ethyl bromide quaternary [QX-314], pH 7.3, 290 to 295
milliosmoles. The amplitude of evoked EPSCs was measured. To ensure stability of the whole-cell recordings, we
initiated electrical stimulation before the cell was patched.
We waited for approximately 5 minutes in the cell-attached
configuration before break-in to wash off any residual internal solution spilled from the approaching pipette.
Passive Avoidance Training and Testing
A one-way passive avoidance learning task was selected as the
tool for behavioral assessment to measure associative memory
retention in rats from each group, as described previously.15
Each rat was placed individually into the lit compartment of
an automated passive avoidance system (Ugo Basile, Comerio, Italy) and, after entering the dark compartment, was
given a scrambled foot shock (0.5mA for 2 seconds). The
retention test was conducted 24 hours later with the rat
again being placed in the lit compartment and subjected to
the same protocol described earlier in the absence of a foot
shock. The latency (maximum, 300 seconds) of entry into
the dark chamber was scored.
Preparation of Postsynaptic Density Fractions
Isolation of postsynaptic density (PSD) fractions was performed according to Wells and colleagues’ procedure.16 In
brief, the microdissected CA1 regions were homogenized in
ice-cold Ca2⫹, Mg2⫹-free buffer (50mM HEPES, 100mM
NaCl, and 3mM KAc, pH 7.4) with RNase inhibitor (15U/
ml), and the homogenate was centrifuged at 2,000g for 1
minute. Supernatants were passed through two 100␮m nylon mesh filters, followed by a 5␮m pore filter. The filtrate
was centrifuged at 1,000g for 10 minutes, and then gently
resuspended in the same buffer at a protein concentration of
2mg/ml.
Western Blotting
For each experimental group, homogenates from at least
three slices were pooled. The microdissected subregions were
lysed in ice-cold tris(hydroxymethyl)aminomethane (Tris)HCl buffer solution (pH 7.4) containing a cocktail of protein phosphatase and proteinase inhibitors (50mM Tris-HCl,
100mM NaCl, 15mM sodium pyrophosphate, 50mM sodium fluoride, 1mM sodium orthovanadate, 5mM EGTA,
5mM EDTA, 1mM phenylmethylsulfonyl fluoride, 1␮M
microcystin-LR, 1␮M okadaic acid, 0.5% Triton X-100
[Sigma], 2mM benzamidine, 60␮g/ml aprotinin, and
60␮g/ml leupeptin) to avoid dephosphorylation and degradation of proteins, and then ground with a pellet pestle
(Kontes Glassware, Vineland, NJ). Samples were sonicated
and spun down at 15,000g at 4°C for 10 minutes. The supernatant was then assayed for total protein concentration
using Bio-Rad Bradford Protein Assay Kit (Bio-Rad, Hercules, CA). Each sample of tissue homogenate or each PSD
fraction was separated in 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel. After the transfer on nitrocellulose membranes, blots were blocked in buffer solution
containing 5% milk and 0.1% Tween 20 in phosphatebuffered saline (124mM NaCl, 4mM KCl, 10mM
Na2HPO4, and 10mM KH2PO4, pH 7.2) for 1 hour, and
then blotted for 2 hours at room temperature with monoclonal antibodies that recognize phosphorylated Ca2⫹/
calmodulin-dependent protein kinase II␣ (CaMKII␣) at the
threonine-286 site (1:2,000; Affinity Bioreagents, Golden,
CO), phosphorylated glutamate receptor 1 (GluR1) at
serine-831 or serine-845 (1:1,000; Upstate Biotechnology,
Lake Placid, NY), PP1 (1:1,000; Upstate Biotechnology), or
“striatal-enriched phosphatase” STEP (1:1,000; Upstate Biotechnology). Blots then were probed with horseradish peroxidase–conjugated secondary antibody for 1 hour and developed using the enzyme chemiluminescence immunoblotting
detection system (Amersham Biosciences, Buckinghamshire,
United Kingdom), according to manufacturer’s instructions.
The immunoblots using phosphorylation site-specific antibodies were subsequently stripped and reprobed with an antibody that recognizes CaMKII␣ (1:1,000; Affinity Bioreagents)
or GluR1 (1:2,000; Upstate Biotechnology). Phosphorylation
site-specific antibody and the phosphorylation-independent
antibody were used to determine the relative amount of
CaMKII␣ or GluR1 phosphorylation (ie, the ratio of the signals). Immunoblots were analyzed by densitometry using
Bio-profil BioLight PC software (BioLight, Danderyd, Sweden).
Protein Phosphatase Activity Assay
Hippocampal slices were treated and dissected as described
earlier. A single CA1 subregion was homogenized in 50mM
Tris-HCl (pH 7.5), 0.2mM EDTA, 0.2mM EGTA,
60␮g/ml aprotinin, and 60␮g/ml leupeptin. Phosphatase activity of 20ng protein of the CA1 homogenate was measured
with the Serine/Threonine Phosphatase Assay System (Promega, Madison, WI), according to the manufacturer’s instructions.17
Data Analysis
All data are expressed as means ⫾ standard error of the
mean. Data of body weight and passive avoidance memory
retention test were analyzed by one-way analysis of variance,
followed by Fisher’s least significance difference test. For
LTP, LTD, and PPF experiments, statistical analysis was performed using the Mann–Whitney U test. Western blot and
protein phosphatase activity data were analyzed using an unpaired Student’s t test. The number of animals used is indicated by n. p values less than 0.05 were considered to represent significant differences.
Results
Maternal Behavior
To determine whether neonatal DEX treatment influences maternal care of pups, we measured the latency
of dams to retrieve pups and to adopt a nursing posture after the DEX- or SAL-treated pups were returned
to their nests. Testing was performed after injection on
P1 to P3. There was no difference in maternal pupdirected behavior between DEX- and SAL-treated
groups (data not shown).
Body Weight
To investigate whether neonatal DEX treatment affected somatic growth in later life, we compared body
weights of rats obtained from DEX- and SAL-treated
groups at various postnatal ages. As shown in Figure
1A, neonatal DEX treatment significantly reduced
body weight gain on P3, P14, P28, and P35 ( p ⬍
0.05), but by P56, there was no weight difference between groups.
Effects of Neonatal Dexamethasone Treatment on
Glutamatergic Synaptic Transmission
We next examined whether the basal glutamatergic
synaptic transmission at the Schaffer collateral-CA1
synapses in adolescent (5-week-old) pups was altered
by neonatal DEX treatment; stimulus–response relations for extracellular fEPSP obtained from DEX- and
SAL-treated rat slices were compared. Neonatal DEX
treatment (relative to SAL treatment) led to a signifi-
Lin et al: DEX Alters Synaptic Function
941
Fig 1. Effect of dexamethasone (DEX) treatment on increase in body weight, basal synaptic transmission, and paired-pulse facilitation (PPF). (A) DEX-treated rats had significantly lower body weights compared with saline (SAL)-treated rats at early ages. Each
vertical bar represents the means ⫾ standard error of the mean for each group. (B) Input-output curve of field excitatory postsynaptic potentials (fEPSP; V/sec) versus stimulus intensity (␮A) at the Schaffer collateral-CA1 synapses of hippocampal slices from SAL(open circles, n ⫽ 5) and DEX-treated (solid circles, n ⫽ 5) rats. Inset shows overlaid traces (each an average of three responses)
evoked in a slice from a SAL- (left set of traces) and DEX-treated (right set of traces) rats. (C) The magnitude of the ␣-amino-3hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor component (EPSCAMPA) estimated by the size of the EPSC measured
5 milliseconds after the start of the EPSC at ⫺70mV and expressed relative to the magnitude of the N-methyl-D-aspartate
(NMDA) receptor–mediated component of EPSCs (EPSCNMDA) was estimated by the amplitude of the synaptic currents measured 50
milliseconds after the start of the EPSC at ⫹40mV. Neonatal DEX treatment (n ⫽ 6) significantly increased the ratio of EPSCAMPA to EPSCNMDA in the CA1 pyramidal cells when compared with SAL-treated rats (n ⫽ 6). Inset shows example EPSCs
(average of three responses) recorded at ⫺70 and ⫹40 mV in cells from SAL- and DEX-treated rats. (D) Comparison of PPF in
slices from SAL- (open circles, n ⫽ 6) and DEX-treated (solid circles, n ⫽ 6) rats. The plot summarizes facilitation of the second
fEPSP slope relative to the first one as a function of the interpulse intervals of 20 to 200 milliseconds. Inset shows example PPF
(average of three responses) obtained with interpulse interval of 40 milliseconds in slices from SAL- and DEX-treated rats. *p ⬍
0.05 versus SAL-treated group by unpaired Student’s t test.
cant leftward shift of the stimulus–response curve ( p ⬍
0.05), indicating an increase in basal glutamatergic
transmission (see Fig 1B). Because fEPSPs are a rather
coarse measure of synaptic transmission, to further examine the effects of neonatal DEX treatment on the
␣-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid
(AMPA) and N-methyl-D-aspartate (NMDA) receptor
function, we compared the synaptic response mediated
by either AMPA receptors or NMDA receptors in CA1
cells from DEX- and SAL-treated rats using whole-cell,
patch-clamp recording techniques. AMPA receptor–
mediated responses were recorded at a holding potential of ⫺70mV, and NMDA receptor–mediated responses were recorded at a holding potential of
⫹40mV. Because neonatal DEX treatment specifically
potentiated AMPA receptor–mediated synaptic currents (EPSCAMPA), but not NMDA receptor–mediated
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synaptic currents (EPSCNMDA), the ratio of EPSCAMPA/EPSCNMDA was significantly larger in slices
from DEX-treated versus SAL-treated rats (see Fig 1C).
These results suggest that postsynaptic AMPA receptor
function and/or number has been modified at the
Schaffer collateral-CA1 synapses after neonatal DEX
treatment.
To determine whether neonatal DEX treatment alters the presynaptic function, we examined the PPF, a
transient form of presynaptic plasticity in which the
second of two closely spaced stimuli elicits enhanced
transmitter release.18 As shown in Figure 1D, pairs of
presynaptic fiber stimulation pulses delivered at interpulse intervals of 20, 40, 60, 80, 150, and 200 milliseconds evoked nearly identical amounts of PPF in
slices from DEX- and SAL-treated rats. These results
suggest that the presynaptic function at the Schaffer
collateral-CA1 synapses remains normal after neonatal
DEX treatment.
Neonatal Dexamethasone Treatment Impairs LongTerm Potentiation but Enhances Long-Term
Depression Induction
To examine the effects of neonatal DEX treatment on
long-term synaptic plasticity, we analyzed LTP and
LTD in the CA1 region of the hippocampus. In slices
from SAL-treated 5-week-old rats, a protocol that normally produces a nonsaturating, short-lasting LTP (two
1-second trains of 100Hz stimuli separated by an intertrain interval of 20 seconds), induced a robust LTP,
whereas in slices from 5-week-old DEX-treated rats,
LTP was significantly reduced (50 minutes after HFS:
SAL, 156.5 ⫾ 9.2% of baseline, n ⫽ 8; DEX,
117.5 ⫾ 6.2% of baseline, n ⫽ 8; p ⬍ 0.05) (Fig 2A),
suggesting that neonatal DEX treatment impairs LTP
induction in later life. LTD was induced by applying
low-frequency stimulation (LFS) at 1Hz lasting 15
minutes (900 pulses) to the Schaffer collateral afferent
inputs. As shown in Figure 2B, slices from DEXtreated but not SAL-treated 5-week-old rats showed a
robust LTD (50 minutes after the end of LFS: 64.2 ⫾
6.7% of baseline, n ⫽ 8 vs 96.5 ⫾ 6.7% of baseline,
n ⫽ 8; p ⬍ 0.05), suggesting that neonatal DEX treatment may facilitate the induction of LTD in later life.
These results are consistent with previous results showing that DEX treatment during neonatal life alters the
inducibility of hippocampal CA1 LTP and LTD in
later life.8
In an attempt to investigate whether changes in inhibitory synaptic transmission contribute to the reduction in LTP observed in DEX-treated neonatal rats, we
blocked inhibitory synaptic transmission by including
bicuculline methochloride (10␮M) in the artificial cerebrospinal fluid solution and examined the inducibility of LTP. In these experiments, the average magnitude of LTP measured 50 minutes after HFS was
164.7 ⫾ 8.2% of baseline (n ⫽ 4) in slices from SALtreated rats compared with 123.4 ⫾ 7.9% of baseline
(n ⫽ 4) in slices from DEX-treated rats ( p ⬍ 0.05)
(data not shown), indicating that the LTP deficit observed in DEX-treated rats is not caused by the changes
in inhibitory synaptic transmission in the hippocampal
CA1 region.
The findings that neonatal DEX treatment impairs
LTP but enhances LTD induction suggest the possibility that this treatment may change synaptic properties.
To explore this possibility, we stimulated the Schaffer
collateral afferent fibers with a range of frequencies (5,
10, 20, and 50Hz) and examined the consequent
changes in the synaptic strength. Our results confirmed
the theoretical model of synaptic plasticity that Bienenstock, Cooper, and Munro19 postulated; HFS leads to
LTP, intermediate frequency stimulation produces only
a minor or no change in synaptic strength, and LFS
produces LTD. In slices from SAL-treated rats, 900
pulses of 1 and 5Hz produced no significant change in
synaptic strength. Moreover, the slices from SALtreated rats showed a significant potentiation with
stimulation of 10 or 20Hz (900 pulses). In contrast,
900 pulses of 1 and 5Hz induced a significant LTD in
slices from DEX-treated rats. The slices from DEXtreated rats also showed significant depression in synaptic strength with stimulation of 10 or 20Hz (900
pulses). Two 1-second trains of 50Hz stimuli induced
an intermediate magnitude of LTP in slices from SALtreated rats but had little effect on synaptic strength in
slices from DEX-treated rats (50 minutes after HFS:
SAL, 129.8 ⫾ 8.7% of baseline, n ⫽ 5; DEX,
108.3 ⫾ 6.7% of baseline, n ⫽ 5; p ⬍ 0.05). The
results obtained with the whole range of different conditioning frequencies are shown in Figure 2C. In these
experiments, the frequency–response function for the
slices from DEX-treated rats favors LTD at frequencies
less than 10Hz and obliterates LTP induction at frequencies greater than 50Hz.
Having established that neonatal DEX treatment alters the induction of LTP and LTD in later life, we
next asked whether these changes are permanent. To
address this issue, we compared the inducibility of LTP
and LTD in slices from young adult (8-week-old) rats
that received DEX or SAL treatment between P1 and
P3. In contrast with the differences in LTP and LTD
induction observed between the 5-week-old groups,
there were no differences between the 8-week-old
groups: both showed normal LTP (50 minutes after
HFS: 154.6 ⫾ 9.3% of baseline, n ⫽ 8 vs 156.8 ⫾
8.2% of baseline, n ⫽ 8; p ⬎ 0.05) and LTD (50
minutes after the end of LFS: 93.5 ⫾ 4.8% of baseline,
n ⫽ 8 vs 94.7 ⫾ 3.9% of baseline, n ⫽ 8; p ⬎ 0.05;
see Fig 2D vs 2E), suggesting that the effects of neonatal DEX treatment on hippocampal synaptic plasticity do not persist into adulthood.
During early postnatal development, the neonatal
rat displays a reduced hypothalamic-pituitary-adrenal
(HPA) response to many types of environmental stressors.20,21 This early period has been termed the stress
hyporesponsive period (SHRP).22 The SHRP has been
suggested to extend from P4 to P14, with glucocorticoids falling to their lowest levels on P9.23,24 Previous
studies have shown that neonatal DEX treatment could
affect the HPA response.9 This prompted us to investigate whether the timing of neonatal DEX treatment
relative to the SHRP contributes to deficits in hippocampal synaptic plasticity. To this end, we compared
the inducibility of LTP and LTD in slices from rats
treated with tapering doses of DEX before SHRP (P13), during early SHRP (P4-6), and after SHRP (P1517). Notably, slices from rats that were treated with
DEX during the early SHRP consistently displayed an
Lin et al: DEX Alters Synaptic Function
943
Fig 2. Neonatal dexamethasone (DEX) treatment impairs long-term potentiation (LTP) induction but facilitates the induction of
long-term depression (LTD) in the CA1 region of the hippocampus at 5 weeks old but not at 8 weeks old. (A) The slices from neonatal DEX-treated rats at 5 weeks old displayed a deficit in high-frequency stimulation (HFS)–induced (two 1-second trains of
100Hz stimuli separated by an intertrain interval of 20 seconds) LTP that was evident immediately after the tetanus and persisted
for at least 1 hour. Open circles denote saline (SAL)-treated rats (n ⫽ 8); solid circles denote DEX-treated rats (n ⫽ 8). (B)
The hippocampal slices obtained from DEX-treated rats (n ⫽ 8) at 5 weeks old showed a reliable LTD after a prolonged lowfrequency stimulation (LFS; 900 stimuli delivered at 1Hz), whereas slices from SAL-treated rats (n ⫽ 8) did not. (C) Frequency–
response curve in slices from SAL- (n ⫽ 8, 5, 5, 5, 8 animals, respectively, for each frequency) and DEX-treated rats (n ⫽ 8, 5,
5, 5, 8 animals, respectively, for each frequency) at 5 weeks old. The percentage change in synaptic strength from baseline in all
slices was measured at 50 minutes after stimulation at the indicated frequencies. *p ⬍ 0.05 versus SAL-treated group by unpaired
Student’s t test. (D) Summary graphs of LTP induced with an HFS (two 1-second trains of 100Hz stimuli) in slices from SAL(n ⫽ 8) and DEX-treated rats (n ⫽ 8) at 8 weeks old. There were no significant differences observed between groups in the magnitude of LTP. (E) Summary graphs of LTD induced with a prolonged LFS (900 stimuli delivered at 1Hz) in slices from SAL(n ⫽ 8) and DEX-treated rats (n ⫽ 8) at 8 weeks old. No difference in the magnitude of LTD was noted between groups. The
superimposed field excitatory postsynaptic potentials (fEPSPs; each an average of three responses) in the inset illustrate respective recordings from example experiments taken at the time indicated by number. Horizontal bar denotes the period of delivery of 1Hz
LFS. Dotted lines show level of baseline.
impaired LTP (50 minutes after HFS: SAL, 154.5 ⫾
9.3% of baseline, n ⫽ 6; DEX, 121.1 ⫾ 6.5% of baseline, n ⫽ 6; p ⬍ 0.05; Fig 3A) and enhanced LTD (50
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minutes after the end of LFS: SAL, 94.5 ⫾ 4.3% of
baseline, n ⫽ 6; DEX, 57.1 ⫾ 8.3% of baseline, n ⫽
6; p ⬍ 0.05; see Fig 3B), as was observed in rats
Fig 3. Effects of neonatal dexamethasone (DEX) treatment at different stages of the stress hyporesponsive period (SHRP) on the induction of long-term potentiation (LTP) and long-term depression (LTD) in later life. (A) Summary graphs of LTP induced with
a high-frequency stimulation (HFS; two 1-second trains of 100Hz stimuli) in slices from 5-week-old rats that received saline (SAL;
open circles, n ⫽ 6) and DEX treatment (solid circles, n ⫽ 6) during early SHRP (postnatal days 4 – 6 [P4-6]). (B) Summary
graphs of LTD induced with a prolonged LFS (900 stimuli delivered at 1Hz) in slices from 5-week-old rats that received SAL
(n ⫽ 6) and DEX treatment (n ⫽ 6) during early SHRP (P4-6). (C) Summary graphs of LTP induced with an HFS (two
1-second trains of 100Hz stimuli) in slices from 5-week-old rats that received SAL (n ⫽ 6) and DEX treatment (n ⫽ 6) after
SHRP (P15-17). (D) Summary graphs of LTD induced with a prolonged low-frequency stimulation (LFS; 900 stimuli delivered at
1Hz) in slices from 5-week-old rats that received SAL (n ⫽ 6) and DEX treatment (n ⫽ 6) after SHRP (P15-17). The superimposed field excitatory postsynaptic potentials (fEPSPs; each an average of three responses) in the inset illustrate respective recordings
from example experiments taken at the time indicated by number. Horizontal bar denotes the period of delivery of 1Hz LFS. Dotted lines show level of baseline.
treated before the SHRP (see Figs 2A, B). In contrast,
post-SHRP DEX treatment did not significantly influence the induction of LTP (50 minutes after HFS:
SAL, 156.8 ⫾ 7.9% of baseline, n ⫽ 6; DEX,
153.6 ⫾ 8.9% of baseline, n ⫽ 6; p ⬎ 0.05; see Fig
3C) and LTD (50 minutes after the end of LFS: SAL,
95.2 ⫾ 4.1% of baseline, n ⫽ 6; DEX, 96.1 ⫾ 5.7%
of baseline, n ⫽ 6; p ⬎ 0.05) in 5-week-old rats (see
Fig 3D). These results suggest that the DEX-induced
deficits in hippocampal synaptic plasticity depend on
the timing of DEX treatment relative to the SHRP.
Putative Mechanisms Underlying the Effects of
Neonatal Dexamethasone Treatment on the
Induction of Long-Term Potentiation
and Long-Term Depression
We next identified the possible mechanisms underlying
the long-lasting effects of DEX. Given that AMPA receptor function increased significantly (see Fig 1C)
without a deficit in PPF (see Fig 1D) in hippocampal
slices of neonatal DEX-treated rats, changes in LTP
and LTD induction are likely due to a deficit in the
postsynaptic machinery. Because intracellular factors
are necessary for LTP and LTD induction, we investigated the potential role of CaMKII, a major PSD protein that may serve as a regulator of synaptic
strength.25,26 Autophosphorylation of CaMKII␣ at one
site, threonine-286, allows the kinase to be active even
in the absence of Ca2⫹27 and promotes association
with PSD by binding to the NMDA receptors.28 According to this view, the levels of CaMKII␣ autophosphorylation at threonine-286 within PSD complexes
may determine the size and direction of synaptic
change caused by synaptic patterns of activity. To determine whether the DEX effect is mediated by changing the basal CaMKII activity, we used Western blotting with a phosphor-specific antibody against
CaMKII␣ phosphorylated at threonine-286 to measure
Lin et al: DEX Alters Synaptic Function
945
the level of autophosphorylated CaMKII in PSD proteins. As shown in Figure 4A, significantly more
CaMKII␣ phosphorylation was observed in slices from
5-week-old DEX-treated rats than SAL-treated rats
(levels were increased to 185.3 ⫾ 5.6% of that in the
SAL-treated group, n ⫽ 10; p ⬍ 0.05); however, this
difference was not observed in 8-week-old rats
(106.9 ⫾ 3.5% of that in the SAL-treated group, n ⫽
10; p ⬎ 0.05). Because phosphorylation at threonine286 is associated with constitutive, calciumindependent activation of CaMKII,27 these findings
suggest that neonatal DEX treatment may induce a
lasting activation of CaMKII in PSD.
To investigate whether the period of DEX treatment
relative to the SHRP influences increase in the
threonine-286 phosphorylation of CaMKII␣ in PSD
of DEX-treated rats, we compared the basal CaMKII␣
phosphorylation at threonine-286 in slices from rats
treated with tapering doses of DEX before SHRP (P13), during early SHRP (P4-6), and after SHRP (P1517). During early SHRP, the level of CaMKII␣ phosphorylation at threonine-286 was significant higher in
slices from 5-week-old DEX-treated rats compared
with SAL-treated rats. In contrast, after SHRP, no significant between-group difference in basal CaMKII␣
phosphorylation at threonine-286 was observed (see
Fig 4B).
In addition to its abundant localization in hippocampus, CaMKII␣ also is expressed in other brain
areas such as amygdala.29 Therefore, we asked whether
the increase in autophosphorylation of CaMKII␣ observed in DEX-treated rats was selectively expressed in
the hippocampal CA1 region. Thus, the basal
threonine-286 phosphorylation levels of CaMKII␣ in
the PSD proteins of other brain areas were compared
between 5-week-old DEX- and SAL-treated rats. As
shown in Figure 4C, significantly more phosphorthreonine-286 CaMKII␣ immunostaining in the PSD
fractions from amygdala and prefrontal cortex, but not
the cerebellum, was observed in DEX- compared with
SAL-treated rats. We also found that the increase in
CaMKII␣ phosphorylation at threonine-286 in the
PSD fractions of DEX-treated rats was significantly
higher in the hippocampal CA1 region than amygdala
and prefrontal cortex ( p ⬍ 0.05).
Because the phosphorylation of AMPA receptor
GluR1 subunits at serine-831 or serine-845 also contributes to bidirectional synaptic modifications,17,30 we
evaluated whether neonatal DEX treatment alters the
levels of GluR1 phosphorylation. The quantitative immunoblotting using two phosphorylation site-specific
antibodies that recognize the serine-831 and serine-845
sites of GluR1 subunits, respectively, showed GluR1
phosphorylation at both the serine-831 and serine-845
sites of PSD proteins from neonatal 5-week-old DEXtreated rats were significantly increased (serine-831:
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154.5 ⫾ 5.3%, n ⫽ 7; p ⬍ 0.05; serine-845: 136.5 ⫾
4.5%, n ⫽ 7; p ⬍ 0.05) in comparison with the SALtreated control rats (see Fig 4D). These findings indicate that neonatal DEX treatment also is coupled to
the signaling pathway that elicits an increased phosphorylation of AMPA receptor GluR1 subunits.
Because PP1 contributes to the regulation of
CaMKII autophosphorylation by dephosphorylating
the threonine-286 site, we evaluated whether the increase in CaMKII␣ phosphorylation of DEX-treated
rats was related to a decrease in PP1 activity. PP1 activity in the CA1 region of the hippocampus was compared between DEX- and SAL-treated rats. Under our
assay conditions, greater than 90% of the PP activity
could be attributed to PP1, that is, was inhibitable by
a thiophosphorylated inhibitor-I protein (data not
shown), as reported previously.17,31 Figure 4E shows
that the basal PP1 activity was significantly lower in
slices from DEX-treated rats than from SAL-treated
rats at 5 weeks old ( p ⬍ 0.05; DEX: 4.2 ⫾ 0.3nmol/
mg/min, n ⫽ 6; SAL: 6.8 ⫾ 0.2nmol/mg/min, n ⫽
5), but not at 8 weeks old ( p ⬎ 0.05; DEX: 5.9 ⫾
0.4nmol/mg/min, n ⫽ 6; SAL: 6.5 ⫾ 0.4nmol/mg/
min, n ⫽ 5). A comparison of basal PP1 activity in
slices from rats treated with DEX or SAL before, during early, and after SHRP showed that activity in the
hippocampal CA1 region was significantly lower in
5-week-old DEX-treated compared with SAL-treated
rats during early SHRP but not after SHRP (see Fig
4F).
The decreased basal PP1 activity observed in neonatal DEX-treated rats could be due to a reduction of
PP1 protein production. Western blot analysis found
significant reduction in PP1 protein in 5-week-old, but
not 8-week-old, DEX-treated rats compared with their
SAL-treated control rats (see Fig 4G). We also examined the levels of STEP, a protein tyrosine phosphatase
that is also preferentially expressed in the hippocampal
neurons and has been implicated in regulating the induction of LTP at the Schaffer collateral-CA1 synapses.32 In contrast with the effect of DEX on PP1,
neonatal DEX treatment produced no significant effect
on the STEP levels. Thus, neonatal DEX treatment selectively reduced PP1 expression, but not STEP, in the
hippocampal CA1 region. Furthermore, in the 5-weekold DEX-treated rats, the levels of PP1 protein in the
hippocampal CA1 region was significantly lower during early SHRP but not after SHRP (see Fig 4H). Together, these data suggest that the increase in the
CaMKII␣ phosphorylation at threonine-286 observed
in the neonatal DEX-treated rats is coupled to the signaling pathway that elicits decreased PP1 protein production.
Memory Retention of a Passive Avoidance Task Is
Impaired after Neonatal Dexamethasone Treatment
Finally, a one-trial passive avoidance task (a form of
contextual fear conditioning) was used to determine
whether neonatal DEX treatment leads to impaired
hippocampal-dependent associative learning and memory. The biochemical requirements of memory formation of this task in the hippocampus have been studied.33 Twenty-four hours after one training session
consisting of a single low-intensity shock, the rats were
returned to the passive avoidance chamber for memory
retention testing. No significant between-group differences in performance were noted during the training.
But on the day of testing, performance of 5-week-old
DEX-treated rats was poorer (ie, they entered the darkened chamber in which they were previously shocked
more rapidly [21.3 ⫾ 5.3 seconds; n ⫽ 8] than SALtreated rats [46.8 ⫾ 4.5 seconds; n ⫽ 8]; Fig 5A).
However, the latencies to enter the dark compartment
were similar between the two groups (47.8 ⫾ 5.2 vs
54.9 ⫾ 4.9 seconds) at 8 weeks old (see Fig 5B). We
further observed that 5-week-old DEX-treated rats
have significantly shorter step-through latencies than
the SAL-treated rats during early SHRP (25.3 ⫾ 4.3
seconds, n ⫽ 5 vs 57.2 ⫾ 4.6 seconds, n ⫽ 6; see Fig
5C) but not after SHRP (52.8 ⫾ 4.6 vs 57.2 ⫾ 4.6
seconds; see Fig 5C). These results suggest that neonatal DEX treatment may impair the formation of contextual fear memory in the adolescent, but this deficit
does not persist into adulthood.
Discussion
Although early neonatal glucocorticoid therapy has
been used successfully to prevent and lessen chronic
lung disease for the past two decades,1–3 much concern
about its long-term effects remains. Little is known
about the long-term effects of neonatal DEX treatment
on structure and functional brain development. Although neonatal DEX treatment leads to the alterations of hippocampal synaptic plasticity and the formation of contextual fear memory in later life, our
study found these effects do not persist into adulthood.
In addition, our results show that the effects of neonatal DEX treatment on LTP and LTD induction were
correlated with an increase in the autophosphorylation
of CaMKII at threonine-286 and a decrease in PP1
activity. Furthermore, the timing of DEX treatment
relative to the SHRP determined whether DEX affects
hippocampal functions.
The stimulus–response relations in the CA1 region
were steeper in slices from DEX- compared with SALtreated rats, suggesting that neonatal DEX treatment
leads to an enhancement of basal synaptic transmission.
Although this could result from an increase in presynaptic glutamate release, alteration in postsynaptic
AMPA receptor functions is a more likely explanation,
because slices from DEX-treated rats exhibited an increase in the ratio of AMPA receptor– to NMDA receptor–mediated synaptic responses in CA1 pyramidal
neurons, and changes in PPF did not occur after neonatal DEX treatment. Increased AMPA receptor–mediated synaptic transmission may be due to increased
unitary conductance,34 increased number of synaptic
AMPA receptors,35 or both. Previous studies indicated
that phosphorylation of GluR1 subunit at serine-831
site by activated CaMKII leads to an increased conductance of AMPA receptor channels.36,37 Because phosphorylation of AMPA receptor GluR1 subunit at
serine-831 was increased in DEX-treated rats, whereas
the level of GluR1 subunits was unaltered, we therefore
suggest that increased channel conductance contributes, at least in part, to the heightened AMPA receptor–
mediated synaptic transmission.
In line with previous findings,8 the frequency–
response curves of LTP and LTD for DEX-treated rats
were modified to favor LTD induction, indicating that
neonatal DEX treatment alters the thresholds for synaptic modifiability (reduces threshold for induction of
LTD and increases threshold for LTP induction).
What mechanisms might be involved? Steady-state
level of autonomous CaMKII activity plays a pivotal
role in setting the value of the LTP and LTD threshold,25,26 and in DEX-treated rats, basal CaMKII autophosphorylation increased at threonine-286. Indeed,
transgenic mice overexpressing a mutated form of
CaMKII␣ with autonomous activity display a downward shift of the frequency–response curve, favoring
LTD over LTP induction in the hippocampal CA1 region.26 How might CaMKII␣ autophosphorylation alter threshold for synaptic modification? Threonine-286
phosphorylation is sufficient to convert the CaMKII to
the high-affinity form.38 Thus, the greater level of autophosphorylated CaMKII␣ may bind a greater
amount of Ca2⫹/calmodulin, which, in turn, leads to
systematic shift in favor of LTD at all submaximal levels of Ca2⫹ influx triggered by low-frequency stimulation.26 Also, autophosphorylated CaMKII␣ may activate some molecular machinery required to support
LTP, which, in turn, blocks subsequent LTP induction.
How does neonatal DEX treatment lead to a lasting
increase in CaMKII␣ phosphorylation? Because the increase in CaMKII␣ phosphorylation observed in DEXtreated rats is correlated with a reduction of PP1 activity and protein expression, one possibility is that
neonatal DEX treatment may act through an unidentified mechanism to decrease PP1 expression, which
renders the CaMKII␣ persistently hyperphosphorylated. Indeed, in the cytoplasm, CaMKII␣ is dephosphorylated primarily by PP2A, whereas in the PSD,
dephosphorylation is almost exclusively mediated by
PP1.39 Thus, future studies should delineate whether
Lin et al: DEX Alters Synaptic Function
947
Figure 4
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Annals of Neurology
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June 2006
the decreased expression of PP1 protein in neonatal
DEX-treated rats is due to inhibition of its synthesis or
stimulation of its degradation.
These results demonstrate that the timing of treatment relative to the SHRP determines whether DEX
adversely affects hippocampal plasticity and contextual
fear memory formation. The relatively lower activation
of the HPA response to stress during the SHRP20,21
suggests that postnatal maturation of the HPA axis
helps protect neonates from an exposure to high levels
of glucocorticoids during the period of brain development. Although the cause of the SHRP-related DEX
response remains to be elucidated, it may be linked, at
least in part, to the low basal corticosterone concentration and immaturity of the HPA-negative feedback system during the SHRP.40 Consequently, DEX may profoundly affect the brain during this period. From these
results, we speculate that the effects of neonatal DEX
treatment that we observed may be mediated through
its influence on HAP axis function.
In this study, DEX treatment induced a significant
retardation of body weight gain in the rat pups that
lasted for up to 4 weeks after treatment, which is in
line with observations of others9,10; growth retardation
is also a consistent finding in DEX-treated preterm human infants.41 The decreased somatic growth observed
in the DEX-treated pups may be due to inadequate
nutritional intake during the postnatal period. Alternatively, DEX may elevate protein catabolism beyond the
capacity for an anabolic state, resulting in reduced
growth and lean body mass.42 Whether the decreased
somatic growth observed in the DEX-treated pups is
Š
associated with long-lasting deficits in hippocampal
synaptic function in later life remains to be delineated.
Another important result of our study is that, although neonatal DEX treatment changes hippocampal
synaptic plasticity and the formation of contextual fear
memory in adolescence, these effects do not persist into
adulthood. These findings are not consistent with
those of Kamphuis and colleagues,8 who reported that
a similar DEX treatment regimen caused deficits in
hippocampal synaptic plasticity and spatial learning
that persisted into adulthood. The reason for this discrepancy is unclear, but it could be attributed to that
different animal strains (Wistar rats vs Sprague–Dawley
rats in our study) were used. In fact, there is abundant
evidence that the time of onset, duration, and extent of
the endocrine response to stressful stimuli or DEX
treatment differ among rat strains.43,44 Because the
maternal care can influence defensive responses to
treatment,45 naturally occurring subtle variations in
maternal behavior may, in part, account for the variability of results. Notably, Kamphuis and colleagues8
tested only the effects of neonatal DEX treatment in
adult rats at 3 to 4 months old. Thus, their results are
not sufficient to conclude whether the alterations of
neonatal DEX treatment on hippocampal function
may persist throughout life.
In conclusion, our work supports the notion that
short-term neonatal treatment with DEX, before and
during SHRP, may lead to alterations of hippocampal
synaptic plasticity and contextual fear memory formation in later life and, more importantly, identifies specific molecular mechanisms to support this contention.
Fig 4. Ca2⫹/calmodulin-dependent protein kinase II␣ (CaMKII␣) autophosphorylation at threonine-286 and glutamate receptor 1
(GluR1) phosphorylation at both serine-831 and serine-845 are increased in hippocampal CA1 postsynaptic density (PSD) purified
from 5-week-old, but not 8-week-old, rats neonatally treated with dexamethasone (DEX). (A) Representative blot showing that
higher phosphorylation of CaMKII␣ at threonine-286 in PSD from DEX-treated rats compared with saline (SAL)-treated rats at 5
weeks old, but not 8 weeks old. Group data showing the normalization of phosphorylation of CaMKII␣ to the nonphosphorylated
form were determined in each group. (B) Representative blot showing that the level of CaMKII␣ phosphorylation at threonine-286
in hippocampal CA1 PSD from 5-week-old rats that received SAL and DEX treatment during (postnatal days 4 – 6 [P4-6]) or
after stress hyporesponsive period (SHRP; P15-17). Group data showing the normalization of phosphorylation of CaMKII␣ to the
nonphosphorylated form were determined in each group. (C) Representative blot showing that the level of CaMKII␣ phosphorylation
at threonine-286 in prefrontal cortex (PFC), amygdala (Amy), and cerebellum (Cb) PSD from DEX-treated rats compared with
SAL-treated rats at 5 weeks old. Group data showing the normalization of phosphorylation of CaMKII␣ to the nonphosphorylated
form were determined in each group. (D) Representative blot showing that higher phosphorylation of GluR1 at serine-831 and
serine-845 in hippocampal CA1 PSD from DEX-treated rats compared with SAL-treated rats at 5 weeks old, but not 8 weeks old.
Group data showing the normalization of phosphorylation of GluR1 to the nonphosphorylated form were determined in each group.
(E) Vertical bar plots representing the analysis of PP1 activity in the CA1 region of hippocampal slices from neonatally SAL- or
DEX-treated rats at 5 or 8 weeks old. Note that the basal PP1 activity is decreased in DEX-treated rats at 5 weeks old compared
with SAL-treated rats. (F) Vertical bar plots representing the analysis of PP1 activity in CA1 region of hippocampal slices from
5-week-old rats that received SAL and DEX treatment during (P4-6) or after SHRP (P15-17). (G) Representative blot showing
the level of PP1 and striatal-enriched tyrosine phosphatase (STEP) in the hippocampal CA1 homogenate from neonatally SAL- or
DEX-treated rats at 5 and 8 weeks old age. PP1 is selectively reduced in 5-week-old DEX-treated rats. Group data showing the
normalization of PP1 or STEP to ␤-actin were determined in each group. (H) Representative blot showing the level of PP1 in the
hippocampal CA1 homogenate from 5-week-old rats that received SAL and DEX treatment during (P4-6) or after SHRP (P1517). Group data showing the normalization of PP1 protein to ␤-actin were determined in each group. *p ⬍ 0.05 versus SALtreated group by unpaired Student’s t test.
Lin et al: DEX Alters Synaptic Function
949
rectly to humans, this study strengthens recent concerns that this therapy has adverse effects on the longterm development and functions of the brain in
children and may help to evaluate the risks and benefits of postnatal steroid therapy. Further long-term
follow-up studies are necessary to evaluate this issue in
humans.
This work was supported by the National Science Council of Taiwan (NSC94-2752-B-006-002-PAE, K.-S.H.).
References
Fig 5. Neonatal dexamethasone (DEX) treatment results in
impaired memory formation for one-trial passive avoidance
task, a form of contextual fear conditioning. Retention test was
performed 24 hours after training. (A) Vertical bar plots representing the latencies to enter the dark component on retention testing in 5-week-old rats that neonatally received saline
(SAL) or DEX treatment. Note that the latency is significantly
decreased in DEX- compared with SAL-treated rats. (B) The
latencies to enter the dark compartment for DEX-treated rats
was not significantly different from those of SAL-treated rats
at 8 weeks old. (C) Vertical bar plots representing the latencies to enter the dark component on retention testing in
5-week-old rats that received SAL and DEX treatment during
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*p ⬍ 0.05 versus SAL-treated group by one-way analysis of
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Although it is unclear whether these findings apply di-
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