healthmedicinet

Animals

Female and male Sprague–Dawley rats were obtained from Chongqing Medical University
Animal Care Centre, and they mated in the laboratory colony of Children’s Hospital
of Chongqing Medical University. The offspring of these rats were used in the present
study. Pregnant females were housed individually in plastic cages in the temperature-controlled
(21 °C) colony room on a 12/12 h light/dark cycle (8:00 a.m.–8:00 p.m.), and were
free to food and water. The pregnant rats were divided into 4 groups: normal rearing
(control), sleep deprivation at early pregnancy stage (ESD, gestational days 1–7),
sleep deprivation at middle pregnancy stage (MSD, gestational days 8–14) and sleep
deprivation at late pregnancy stage (LSD, gestational days 15–21). Within 24 h of
birth, all litters were culled to 10 pups with a goal of balancing the number of males
and females equally. Young adult rats from postnatal day (PND) 42–56 were used for
behavioral and electrophysiological experiments. All experimental protocols were approved
by Chongqing Medical University Animal Care Committee.

Sleep deprivation

The sleep deprivation was performed by gentle handling for 6 h per day (12:00–18:00)
as previously described 10], 20]. Briefly, pregnant rats were kept awake by gentle tapping or rattling of the cage
and, if necessary, by gently being touched with a soft brush if behavioral signs of
sleep were observed, such as closed eyes and immobility. Food and water were available
ad libitum throughout the sleep deprivation period.

Reagents and antibodies

All drugs were purchased from Sigma-Aldrich. Mouse Anti-BrdU monoclonal antibody was
purchased from Sigma-Aldrich. Rabbit anti-NeuN monoclonal antibody was purchased from
Millipore. Rabbit anti-GFAP monoclonal was purchased from Abcam. Complete protease
inhibitor cocktail tablets and phosphatase inhibitor cocktail tablets were purchased
from Roche Applied Science.

Morris water maze

Spatial learning and memory were examined with the Morris water maze using programs
similar to those described previously 21], 22]. Briefly, rats were trained in a circular fiberglass pool (180-cm diameter) over
4 trials per day for 6 consecutive days to find a hidden platform. During each trial,
the rats that cannot find the hidden platform within 60 s were guided to the platform
where they remained for 20 s. A probe test was performed 24 h after the last learning
trial. All trials were recorded and analyzed by using an Any-maze tracking system
(Stoelting, USA).

Elevated plus maze test

The plus maze apparatus consisted of two opposite open arms and two opposite closed
arms (20-cm-tall walls on the closed arms) arranged at right angles. At the beginning
of the test, rats were put in the center of the apparatus, which is elevated 1 m above
the floor. The number of entries and the time spent in each arm were recorded for
10 min by ANY-maze video tracking system.

Novelty-suppressed feeding test

Novelty-suppressed feeding test was performed as described previously 23]. In brief, rats were deprived of food for 48 h prior to the test. During test, a
single pellet of food was placed on a white filter paper located in the center of
the arena (60?×?60 cm), and rat was placed in a corner of the arena to explore the
arena for 12 min. The latency to begin eating food and the amount of food consumption
were recorded. The rats were immediately returned to their home cages after test,
where food consumption was monitored for another 30 min.

Forced swimming test

Rats were forced to swim in a cylinder filled with water (temperature 24–25 °C; 20 cm
in diameter, 40 cm in height) for 10 min. The latency to immobility and total immobility
time were recorded and analyzed by using ANY-maze video tracking system.

Immunohistochemistry

To label newborn cells, rats were subjected to BrdU injection (100 mg/kg, i.p.) at
age of 2 weeks or 6 weeks, and were sacrificed 4 weeks or 24 h after the last BrdU
injection, respectively. The animals were deeply anesthetized and transcardially perfused
with 4 % paraformaldehyde in 100 mM phosphate buffer, pH 7.4. Immunohistochemistry
was performed on 30-?m coronal sections as previously described 23], 24]. Every sixth slice with the same reference position was stained. Positive cells were
quantitated using a 40× objective (Leica). Obtaining numbers were multiplied by 6
to determine the estimated total number of positive cells per dentate gyrus (DG) of
rat.

Electrophysiology in vivo

Rats were deeply anesthetized with sodium pentobarbital at a dose of 60 mg/kg (i.p.)
and then mounted to a stereotaxic frame (Stoelting Co.). The core temperature was
monitored and kept at 36.5 °C?±?0.5 °C. Stimulating and recording electrodes (a pair
of 100 ?m outer diameter Teflon-coated wires; A-M Systems Inc.) were located at the
Schaffer collaterals of dorsal hippocampus and ipsilateral striatum radiatum of hippocampal
CA1 area, respectively. Final positions of the electrodes were determined when an
optimal response of field excitatory post-synaptic potential (fEPSP) was obtained.
Baseline responses were recorded at 0.033 Hz for 30 min, with an intensity that evoked
half of maximum amplitude. Once the stable baseline was obtained, a HFS consisted
of 100 pulses at 100 Hz was delivered to induce LTP.

Electrophysiology in vitro

Rats were deeply anesthetized with 25 % urethane (1.5 g/kg, i.p.) and transcardially
perfused with NMDG artificial cerebral spinal fluid (in mM: NMDG 93, HCl 93, KCl 2.5,
NaH
₂
PO
₄
1.2, CaCl
₂
0.5, MgSO
₄
10, NaHCO
₃
30, HEPES 20, Na-ascorbate 5.0, Na-pyruvate 3.0, Thiourea 2.0, NAC 12, and D-glucose
25, pH?=?7.3.) prior to decapitation. The brain was rapidly dissected and placed in
ice-cold NMDG ACSF. Hippocampal slices (400 ?m) were coronally sectioned with a vibratome
(VT1200S, Leica Microsystems, Bannockburn, IL) and then were incubated in HEPES ACSF
(in mM: NaCl 92, KCl 2.5, NaH
₂
PO
₄
1.2, CaCl
₂
0.5, MgSO
₄
10, NaHCO
₃
30, HEPES 20, Na-ascorbate 5.0, Na-pyruvate 3.0, Thiourea 2.0, NAC 12, and 25 D-glucose,
pH?=?7.3.) for 1 h at 30 °C.

Miniature excitatory postsynaptic currents (mEPSCs) of hippocampal CA1 pyramidal neurons
were recorded with pipette filled with internal solution (in mM: Cs-methanesulfonate
130, MgCl
₂
2.0, EGTA 0.5, HEPES 10, QX-314 5.0, K
₂
ATP 5.0, and Na
₂
GTP 0.3, pH?=?7.3), resistance of which was 3–5 M?. Bicuculline methiodide (10 ?M)
and TTX (1 ?M) were added in ACSF (in mM: NaCl 120, KCl 2.5, NaH
₂
PO
₄
1.25, CaCl
₂
2.0, MgSO
₄
2.0, NaHCO
₃
26, glucose 10, pH?=?7.3) to block GABA receptors and Na
⁺
channels respectively. Data acquisition (filtered at 3 kHz and digitized at 10 kHz)
was performed with PatchMaster v2.73 (HEKA Electronic, Lambrecht/Pfalz, Germany) with
holding potential at ?70 mV. Mini Analysis Program 6.0.3 (Synaptosoft Inc., Decatur,
GA) was used to automatically detected mEPSCs.

Statistical analysis

All data are presented as mean?±?SEM. Spatial learning data were analyzed by a two-way
ANOVA, with treatment (group) as the between-subjects factor and learning day as the
within-subjects factor. All the other data were analyzed by a one-way ANOVA, with
treatment (group) as the between-subjects factor. Significance level was set at p??0.05.