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\begin{document}

\title{The Crucial Role of Hippocampus Cyclooxygenase-2 Plays in Synaptic
Plasticity and Memory}



\author[1]{Hong Ni}%
\author[1]{Zhongzhao Guo}%
\author[2]{Yue Wu}%
\author[3]{Jie Wang}%
\author[1]{Zilu Zhu}%
\author[4]{Yang Yang}%
\author[1]{Deheng Wang}%
\affil[1]{Shanghai University of Traditional Chinese Medicine}%
\affil[2]{Shanghai University of Traditional Chinese Medicine Yueyang Hospital of Integrated Traditional Chinese Medicine and Western Medicine}%
\affil[3]{Shuguang Hospital}%
\affil[4]{Shanghai University of TCM}%


\vspace{-1em}



  
  \date{May 26, 2024}


\begingroup
\let\center\flushleft
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\endgroup





\selectlanguage{english}
\begin{abstract}
It is generally accepted that Cyclooxygenase-2 (COX-2) is activated to
cause inflammation. However, COX-2 is also constitutively expressed at
the postsynaptic dendrites and excitatory terminals of the cortical and
spinal cord neurons. Although some evidence suggests that COX-2 release
during neuronal signaling may be pivotal for regulating the function of
memory, the significance of constitutively expressed COX-2 in neuron is
still unclear. This research aims to discover the role of COX-2 in
memory beyond neuroinflammation and to determine whether the inhibition
of COX-2 can cause cognitive dysfunction by influencing dendritic
plasticity and its underlying mechanism. The cognitive ability was
assessed by novel object recognition task (NORT) and Morris water maze
(MWM) test. Immunofluorescence, Golgi-cox staining were used to observe
dendritic synaptic. Gamma oscillation in hippocampus CA1 was performed
by Tetrode in-vivo recording. Prostaglandins were measured by HPLC/mass
spectrometry. We observed the expressions of cyclic adenosine
monophosphate (cAMP)/ brain-derived neurotrophic factor (BDNF) pathway
proteins in hippocampus and N2a cells by Elisa and western blot. We
found COX-2 gene knockout (KO) could significantly impact the learning
and memory ability; reduce the expression of postsynaptic density
protein 95 (PSD95) in the neuron; cause synaptic disorder; influence
gamma oscillation and reduce the expression prostaglandin (PG) E2, cAMP,
phosphorylated protein kinase A (p-PKA), phosphorylated cAMP response
element binding protein (p-CREB) and BDNF in the hippocampus. It
suggested COX-2 might play a critical role in learning, regulating
synaptic plasticity and gamma oscillation in the hippocampus CA1 by
regulating COX-2/BDNF signaling pathway.%
\end{abstract}%



\sloppy


\textbf{The Crucial Role of Hippocampus Cyclooxygenase-2 Plays in
Synaptic Plasticity and Memory}

Hong Ni\textsuperscript{1\#}, Zhongzhao Guo\textsuperscript{1\#}, Yue
Wu\textsuperscript{4\#}, Jie Wang\textsuperscript{3}, Zilu
Zhu\textsuperscript{2}, Yang Yang\textsuperscript{1}, Deheng
Wang\textsuperscript{2*}

\emph{1.The Academy of Integrative Medicine, Shanghai University of
Traditional Chinese Medicine, 201203, Shanghai, China.}

\emph{2. The Academy of Basic Medical Sciences, Shanghai University of
Traditional Chinese Medicine, 201203, Shanghai, China}

\emph{3. Department of Peripheral Vascular Surgery, Shuguang Hospital
Affiliated to Shanghai University of Traditional Chinese Medicine,
201203, Shanghai, China.}

\emph{4.} \emph{Department of~Gastroenterology, Yueyang Hospital
of~Integrated Traditional Chinese and~Western Medicine, 200437,
Shanghai,~China.}

\textsuperscript{\#}These authors contributed equally to this work.

*Corresponding author.

\textbf{Running title} : Deletion of cyclooxygenase-2 impair memory

\textbf{Correspondence:}

E-mail: Dr. Wang: wangdeheng@shutcm.edu.cn;

Word counts:4748

\textbf{ABSTRACT}

It is generally accepted that Cyclooxygenase-2 (COX-2) is activated to
cause inflammation. However, COX-2 is also constitutively expressed at
the postsynaptic dendrites and excitatory terminals of the cortical and
spinal cord neurons. Although some evidence suggests that COX-2 release
during neuronal signaling may be pivotal for regulating the function of
memory, the significance of constitutively expressed COX-2 in neuron is
still unclear. This research aims to discover the role of COX-2 in
memory beyond neuroinflammation and to determine whether the inhibition
of COX-2 can cause cognitive dysfunction by influencing dendritic
plasticity and its underlying mechanism. The cognitive ability was
assessed by novel object recognition task (NORT) and Morris water maze
(MWM) test. Immunofluorescence, Golgi-cox staining were used to observe
dendritic synaptic. Gamma oscillation in hippocampus CA1 was performed
by Tetrode in-vivo recording. Prostaglandins were measured by HPLC/mass
spectrometry. We observed the expressions of cyclic adenosine
monophosphate (cAMP)/ brain-derived neurotrophic factor (BDNF) pathway
proteins in hippocampus and N2a cells by Elisa and western blot. We
found COX-2 gene knockout (KO) could significantly impact the learning
and memory ability; reduce the expression of postsynaptic density
protein 95 (PSD95) in the neuron; cause synaptic disorder; influence
gamma oscillation and reduce the expression prostaglandin (PG)
E\textsubscript{2}, cAMP, phosphorylated protein kinase A (p-PKA),
phosphorylated cAMP response element binding protein (p-CREB) and BDNF
in the hippocampus. It suggested COX-2 might play a critical role in
learning, regulating synaptic plasticity and gamma oscillation in the
hippocampus CA1 by regulating COX-2/BDNF signaling pathway.

\textbf{KEYWORDS:} COX-2; Memory; Synaptic plasticity; Gamma
oscillation; BDNF

\textbf{INTRODUCTION}

Cyclooxygenase (COX) is a prostaglandin G/H synthase (PGHS), which forms
prostaglandin (PG) H\textsubscript{2} from arachidonic acid (AA), a
rate-limiting enzyme associated with inflammatory processes.
PGH\textsubscript{2} is metabolized to physiologically active PGs, such
as PGE\textsubscript{2}, PGI\textsubscript{2}, PGD\textsubscript{2},
PGF\textsubscript{2a}, and thromboxane (TX) A\textsubscript{2}
\textsuperscript{{[}1{]}}. COX-1 and COX-2 are the isoforms of COX.
Different genes translate them with differences in tissue distribution
and biological functions \textsuperscript{{[}2{]}}.

COX-1 is constitutively expressed in most
cells\textsuperscript{{[}3{]}}. In contrast, COX-2 is an inducible
enzyme expressed due to pro-inflammatory stimuli
\textsuperscript{{[}4{]}}, associated with inflammation and injury
response\textsuperscript{{[}5{]}}. However, recent studies indicate a
greater functional significance of COX-2 \textsuperscript{{[}6-8{]}}.
Compared with COX-1, the expression of COX-2 is elevated in the
glutamatergic neurons of the hippocampus and cortical
regions\textsuperscript{{[}9{]}} but not in astrocytes and microglial
cells\textsuperscript{{[}10{]}}. Thus, COX-2 has a specific role in
neurons. Neuronal COX-2 overexpression is observed in cerebral ischemia,
seizures, brain injury, and neurodegenerative diseases, such as
Parkinson's, Alzheimer's, Huntington's,
etc.\textsuperscript{{[}10-14{]}}. These are induced by inflammatory
mediators, excitotoxic, and oxidative\textsuperscript{{[}9{]}}. However,
inhibiting COX-2 exacerbates rather than reduces inflammation-related
brain damage \textsuperscript{{[}15{]}}. This could lead to AD
progression, elevated COX-2 neuron levels in the early
stages\textsuperscript{{[}16{]}}, and decreased in the late
stages\textsuperscript{{[}17{]}}. Additionally, cell proliferation and
neuroblast differentiation inside the dentate gyrus are reduced by COX-2
deficiency because of genetic and pharmacological
inhibition\textsuperscript{{[}18-20{]}}. Although consolidating spatial
memory in the hippocampus is governed by COX-2 \textsuperscript{{[}21,
22{]}}, its role in brain function remains controversial. This is due to
the significant localization of COX-2 in dendritic spines of neurons and
modifies synapses \textsuperscript{{[}23, 24{]}}. However, the
participation mechanisms of COX and PGs in the hippocampal synaptic
plasticity remain unclear.

Therefore, the role of COX-2 was investigated in the hippocampal neuron
with wild-type (WT) and COX-2KO mice. The ability to learn and memorize
was significantly affected by COX-2 gene knockout. This knockout also
decreases the neuronal expression of PSD95, leading to synaptic changes,
and influences gamma wave oscillation through
COX-2/PGE\textsubscript{2}/cAMP/PKA/CREB/BDNF signaling.

\textbf{MATERIALS AND METHODS}

\textbf{Animals}

The C57BL/6 male mice were obtained from Nanjing University's Model
Animal Research Center, while the COX-2KO mice were kindly provided by
the Chinese Academy of Sciences. All mice were bred in the Experimental
Animal Center of Shanghai University of Traditional Chinese Medicine's
Experimental Animal Center. The following two groups were established
with fifteen mice each: WT group and COX-2KO group, both at 9 months
old. During the 12-hour light/dark cycle (lights on 7:00-19:00), all
mice were housed in an environment with a temperature of 22 \selectlanguage{ngerman}°C and a
humidity of 55\%. Water and food are available ad libitum. In accordance
with the guidelines of the National Institutes of Health for the care
and use of laboratory animals, all experimental protocols were strictly
complied with, and were approved by Shanghai University of Traditional
Chinese Medicine Animals Research Ethics Committee
(No.PZSHUTCM200821019) based on the guidelines provided by the Ministry
of Science and Technology of the People's Republic of China.

\textbf{Genotyping}

A DNA identification procedure was performed in order to evaluate the
reliability of COX-2KO transgenic mice. Approximately 4 mm was cut from
the tails of mice. Total DNA was isolated from the mouse with a DNA
extraction kit (Beijing Tiangen Biochemical Technology, China) and was
amplified by PCR. The following conditions were applied to the PCR
reaction system: initial denaturation for 4 minutes at 94 °C, followed
by 31 cycles of denaturation for 30 seconds at 94 °C, annealing for 30
seconds at 61 °C, and extension for 1 minute at 72 °C, followed by a
final extension for 5 minutes at 72 °C. A gel imaging device (Bio-Rad,
USA) is used to visualize the PCR products after they have been labeled
with SYBR Green (Sigma-Aldrich, USA), separated by electrophoresis (130V
constant voltage, continued for 30 minutes) and visualized with SYBR
Green (Sigma-Aldrich, USA). The following primers (Sangon Biotech,
China) were used: CDF141: 5'-ATC GCC TTC TTG ACG AGT TC-3'; COX2KO
probe1: 5'- ACC TCT GCG ATG CTC TTCC-3'; COX2KO probe2: 5'- ACT GGT CAA
ATC CTG TGC TC-3'.

\textbf{Morris Water Maze (MWM)}

By training mice to find a hidden platform submerged in water, a MWM
paradigm was used to evaluate the cognitive abilities of mice. As
described previously (Vorhees \& Williams, 2006), by adding edible
titanium dioxide to opaque water (35 °C) within a circular pool with a
diameter of 120 cm and wall height of 50 cm, the mice were allowed to
swim in a circular pool. An 8 cm diameter platform was hidden 1\selectlanguage{english} cm below
the surface of the water. Mice were trained to find the hidden platform
by markers around the pool within 60\selectlanguage{english} s. If the mouse failed to find the
platform within 60\selectlanguage{english} s, it was guided to the platform and requested to
stay there for 30\selectlanguage{english} s. Three trials were conducted for each mouse,
starting at different points in the afternoon each day for six
consecutive days in order to locate the platform beneath the water's
surface. A probe test was administered to the mice following a training
period of six days. For the probe test, the mice were left to swim
without the platform for 60 s. EthoVision video tracking system (Noldus
Information Technology, Netherlands) was used to analyze the escape
latency, swim speed, distance traveled, time spent within the target
quadrant, and crossing times of the platform during the entire test.

\textbf{The novel object recognition task (NORT)}

A NORT experiment was conducted to evaluate the mice's ability to
recognize new objects versus familiar objects when they were allowed to
explore freely (Dere \emph{et al.} , 2007). The mice were placed in the
testing room 30 minutes before the experiment on day one following MWM
to become accustomed to the conditions. All the mice were allowed to
acclimate themselves to an open-field arena (50 cm x 50 cm, dim light,
24 degC) and two blocks in it for 10\selectlanguage{english} min twice a day for 2 days. All mice
were allowed to explore two blocks for five minutes on the following
day, and 1 hour and 24 hours later, mice were given five minutes to
explore an unfamiliar block with a different shape and color. The
recognition indexes (\%) were calculated by the ratio of the time spent
exploring a novel object over the total amount of time spent in each
group to evaluate memory.

\textbf{Double Immunofluorescence Staining}

In order to obtain histological results, mice were anesthetized with 1\%
barbital sodium (100 mg/kg) (Aladdin, China) before being transcranially
perfused with 0.1 M phosphate-buffered saline (PBS, pH 7.4) followed by
4\% paraformaldehyde (PFA). After collecting the brains, they were fixed
in 4\% PFA at 4 degC for 24 hours and cryopreserved in 30\% sucrose until
sectioning. A sliding microtome (Leica, Germany) was used to section the
brain coronally into 35 m sections containing the hippocampal area which
is about 2 mm from the Bregma. Then the brain sections from hippocampus
were blocked for 10 min in PBS containing 10\% bovine serum albumin
(BSA) and then incubated overnight at 4 degC with the primary antibodies
NeuN (Mouse monoclonal) (1:100, Abcam, USA) and synaptophysin (Rabbit
monoclonal) (1:100, CST, USA) or PSD95 (Rabbit monoclonal) (1:100, CST,
USA). Subsequently, the sections were incubated with the secondary
antibodies {[}Alexa Fluor 555-conjugated anti-mouse (1:1000 dilution,
Thermo Fisher Scientific, USA), Alexa Fluor 488-conjugated anti-rabbit
(1:1000 dilution, Thermo Fisher Scientific, USA){]} for 2h at room
temperature. ~Each of the steps above was followed by three rinses in
PBS lasting 10 minutes each. At the end the sections were mounted by
fluorescent mounting medium (Sigma, USA). Laser-scanning confocal
microscopes (Leica TCS SP2, Germany) were used to capture the confocal
images.

\textbf{Western blot}

A radioimmunoprecipitation (RIPA) assay buffer containing a mixture of
protease and phosphatase inhibitors was used to extract the protein from
the hippocampus. The protein concentration was measured by BCA Protein
Assay kit (Thermo Fisher Scientific, USA) after brain cells had been
suspended, homogenized, sonicated, and centrifuged for 15 minutes at
14,000g and 4 degC. Afterwards, proteins were boiled for 10 minutes at 100
degC in a buffer containing glycerol, Eagle's medium, and SDS. After ten
micrograms of protein had been loaded on 12.5\% SDS-PAGE (Yeasen
Biotech, China), the separated proteins were transferred to
nitrocellulose membrane (Yeasen Biotech, China). The membrane was
blocked by 5\% BSA in the TBST buffer (10 mmol/L of Tris, pH 7.5; 100
mmol/L of NaCl; and 0.1\% Tween 20), and was incubated overnight at 4 degC
with specific primary antibodies (1:1000 dilution in 5\% nonfat milk in
the TBST buffer) of synaptophysin (CST, USA), PSD95 (CST, USA),
\selectlanguage{greek}β-\selectlanguage{english}tubulin (Proteintech, USA), PKA (Abcam, USA), p-PKA (Abcam, USA), CREB
(Proteintech, USA) , p-CREB (Proteintech, USA), BDNF (CST, USA). On the
second day, primary antibody was detected with anti-rabbi secondary
antibodies (1:5000; Invitrogen, USA) and the immunoreactive bands were
visualized with an enhanced chemiluminescence kit (Beyotime
Biotechnology, China). Then Image J software was used to quantify the
band density. A minimum of three blots were performed for each
condition.

\textbf{Golgi-Cox Staining}

After the mice have been anesthetized with 10\% chloral hydrate (400
mg/kg) (Aladdin, China), brains were removed quickly from the skull,
rinsed with 0.1M PBS. Staining of brain slices was performed according
to the manufacturer's instructions using the FD Rapid Golgi Stain kit
(FD Neuro Technologies, USA). Brains were cryosectioned coronally into
100 \selectlanguage{greek}µ\selectlanguage{english}m sections containing the hippocampal area with a cryostat
microtome (Leica, Germany). The slices were mounted on gelatin-coated
microscope slides, stained, dehydrated, and covered with Vecta Mount
Mounting Medium (Vector, USA).

\textbf{Sholl Analysis}

Sholl Analysis is a classical method to analyze neuronal complexity by
drawing a series of concentric circles with the soma as the center. This
is done by obtaining the number of intersections of dendrites that have
distances at 10mm intervals from the soma. The number of intersections
is used to reflect the complexity of the cell. We used Imaris software
(version 7.7.2) for dendritic complexity sholl analysis. The steps for
dendritic complexity sholl analysis are as follows: color
inversion--select the soma as a center--select the dendritic
end--perform semi-automatic dendritic analysis. Dendrite branching
should be flipped to 3D to match the selection in order to distinguish
real dendrite branches. When the drawing is finished, select specific
values in the statistics tab - filament No. sholl intersections
(Pillai\emph{et al.} , 2012).

\textbf{cAMP ELISA}

The level of cAMP in the hippocampus was measured by a direct
competitive ELISA kit (Abcam, USA) as directed by the manufacturer.
After weighing the frozen tissue, 0.1M HCl was added in a 5X volume.
Then the sample was homogenized on ice and centrifuged at high speed for
five minutes, collecting the supernatant. After loading the samples into
96-well plates, the samples were incubated for an hour with the cAMP
antibody. The wells were incubated for an additional hour with cAMP-
Horseradish Peroxidase (HRP) and washed three times with wash buffer
before the final step. Thereafter, HRP developer was added and incubated
for 1 hour. A 1 M HCl solution was used to stop the reaction, and the
color developed was measured using an ELx800 plate reader (BioTek, USA).

\textbf{Measurement of PGs by HPLC/mass spectrometry}

As previously reported by Yu (Yu \emph{et al.} , 2010), the amount of PG
profiles was measured in the hippocampus of mice. Dissection and
homogenization of hippocampus samples were carried out in ice-cold PBS
containing 100 micrograms of indomethacin inside stainless steel beads.
The supernatant was collected and spiked immediately with 5ng PGD2-d4
(Cayman Chemical, USA), PGE2-d4 (Cayman Chemical, USA), 6-keto
Prostaglandin F1\selectlanguage{greek}α-\selectlanguage{english}d4 (Cayman Chemical, USA), PGF2\selectlanguage{greek}α-\selectlanguage{english}d4 (Cayman Chemical,
USA), TxB2-d4 (Cayman Chemical, USA), then purified by solid phase
extraction using StrataX C18 cartridges (Phenomenex, USA). Afterwards, a
1ml acetonitrile solution was used to condition and equilibrate the
solid phase extraction cartridge. After applying the sample to the
cartridge, it was washed with 1ml of 5\% acetonitrile in water, and
dried for 15 minutes under vacuum. Next, a solution of 5\% acetonitrile
in ethyl acetate was used to elute the analyte and internal standards
from the cartridge. A gentle stream of nitrogen was used to dry the
eluate. In a solution of 200 liters of 5\% acetonitrile in water, the
resulting residue was reconstituted and centrifuged using nylon
microspin filters (Alltech Associates, USA), in the end quantified by
liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS)
analyses described previously (Song \emph{et al.} , 2007).

\textbf{RNA extraction and real-time PCR}

TRIzol (Invitrogen, USA) was used to extract total RNA from the
supernatant of the cells and the mouse hippocampal tissue. By using
NanoDrop 2000 micro-ultraviolet spectrophotometry (1011U, NanoDrop
Technologies, USA), the purity and concentration of RNA were determined.
Then the cDNA was synthesized according to the instructions provided
with the cDNA Reverse Transcription Kit (Takara, Japan). PCR was
conducted as follows: 95 \selectlanguage{ngerman}°C for 30 seconds, followed by 40 cycles of 5
seconds at 9 5°C and 34 seconds at 60 °C. The expression of the target
genes was normalized relative to that of glyceraldehyde-3-phosphate
dehydrogenase (GAPDH), and calculated using the 2\selectlanguage{english}-\selectlanguage{greek}ΔΔ\selectlanguage{english}Ct method
(Ayuk\emph{et al.} , 2016). The primer sequences are: COX-2, forward:
CAC TAC ATC CTG ACC CAC TT, reverse: ATG CTC CTG CTT GAG TAT GT; TNF-a,
forward: GAT AAA GGG ACA GCG TCA GC, reverse: CAG CCT TGT CCC TTG AAG
AGA ACC; iNOS, forward: GAT AAA GGG ACA GCG TCA GC, reverse: CCT TCG GGC
CAA AGA TCC TG; IL-1\selectlanguage{greek}β, \selectlanguage{english}forward: ATC TCG CAG CAG CAC ATC AAC, reverse:
TGT TCA TCT CGG AGC CTG TAGT; GAPDH, forward: GCC AAA TTC AAC GGC ACA
GT, reverse: AGA TGG TGA TGG GCT TCCC.

\textbf{Cell Culture}

Mouse neuroblastoma cells (N2a cells) were cultured in Dulbecco's
Modified Eagle's Medium (DMEM) supplemented with 10\% fetal bovine serum
(FBS) (Gibco, Thermo Fisher Scientific, USA) and 1\%
penicillin--streptomycin (Invitrogen, USA), and maintained in a 5\%
humidified CO\textsubscript{2} and 95\% air at 37~\selectlanguage{ngerman}°C.
1×10\textsuperscript{4} N2a cells/well were seeded onto a 6-well plate
until the cells adhered to the well. When the cell grew to 70 to 80
percent, 10\selectlanguage{greek}μ\selectlanguage{english}mol/L COX-2 selective inhibitor Celecoxib (Selleck, USA) was
added. Then the cell were incubated for 24hour until the culture medium
was collected to measure the mRNA of COX-2 by RT-qPCR. Celecoxib was
added for 24h in the cell culture medium before stimulation with
PGE\textsubscript{2} (1\selectlanguage{greek}μ\selectlanguage{english}m; Selleck, USA) for 4 hours, then the culture
medium was collected to measure the expression of proteins by western
blot.

\textbf{Tetrode electrode implantation surgery and data collecting}

We used the Self-made movable bundles of the 32-channel tetrode
electrode (Song \emph{et al.} , 2020) to target the hippocampus CA1 (AP
2 mm; ML 1.8 mm and DV 1.1 mm) of the anesthetized mice on the
stereotactic frame. The local field potentials (LFP) value refers to the
real-time potential under the sampling frequency of 1000 Hz which is
digitized by the Plexon OmniPlex Neural Data Acquisition System (Plexon,
USA), and analyzed by NeuroExplorer Version5.306 (Nex Technologies,
USA).

\textbf{Statistical analysis}

All the behavioral data were presented as means \selectlanguage{ngerman}± SEM. The rest of data
were reported as means ± SD and analyzed by Student's t test or one-way
ANOVA with GraphPad 7.0 software (San Diego, CA). A p value of
\textless{}0.05 was considered as statistically significant.

\textbf{RESULTS}

\selectlanguage{english}\textbf{Effects of COX-2 depletion on the learning and memory ability.}

Firstly, it was observed that after genotyping COX-2KO
(COX-2\textsuperscript{-/-}) mice by PCR (Fig. 1A), no COX-2 protein was
detected by Western blot (Fig. 1B).Then a comparison between WT and
COX-2KO mice revealed no differences in the body weights, body
temperatures, or basal inflammation (TNF-a, iNOS, and IL-1\selectlanguage{greek}β\selectlanguage{english}) in the
hippocampus (Fig.1C-E). Furthermore, to determine whether COX-2
contributes to learning and memory ability, we used Morris water maze
(MWM) memory test and novel object recognition task (NORT). In the MWM
test, the results showed that the escape latency of mice in each group
decreased with the increase in training times, indicating that the
training of mice was effective. Compared with WT group, the escape
latency at training day 4 and 5 were significantly shorter for COX-2
knocked out mice (Fig.1C). Furthermore, to measure spatial learning and
memory retention, we next performed probe trials on the 6th day. In the
comparison with WT mice, COX-2KO ones failed to find the target platform
with less time spending (Fig.1D, F) and fewer platform crossings
(Fig.1E) in the target quadrant. In addition, no differences were
observed between WT and COX-2KO mice in terms of swimming speed (Fig.1I)
and total distance traveled (Fig.1J).Next, we used NORT to assess the
recognition memory of different groups. As illustrated in Figure 1G, the
recognition index of COX-2KO mice were reduced compared to the WT group
at the test of both 1h and 24h. Moreover, the total distance traveled by
COX-2KO and WT mice in the NORT (Fig.1M) was also not different. These
results suggested that COX-2KO mice were impaired in learning and memory
ability.

\textbf{Effects of COX-2 depletion on the SYP and PSD95.}

Then we detected synaptophysin (SYP), PSD95 by double immunofluorescence
and Western blot. We stained PSD95 with red and SYP with green in the
hippocampus CA1 (Fig. 2A). We found there was almost no change in the
fluorescence intensity of SYP between WT and COX-2KO those two groups,
however, compared with WT group, the expression of PSD95 was almost
absent in COX-2KO samples with a faint green staining (Fig. 2B).
Consistent with the immunofluorescence result, we found COX-2 depletion
could significantly downregulate the expression of PSD95 protein but not
SYP in the hippocampus (Fig. 2C). These results indicated that COX-2KO
could influence the expression of PSD95 instead of SYP in the brain.

\textbf{Effects of COX-2 depletion on the neuronal structure.}

Because hippocampal neuronal structure is believed to be the basis of
learning and memory (Parra-Damas \emph{et al.} , 2017), we then examined
the alterations in neuronal structure in WT and COX-2KO mice hippocampus
CA1 with Golgi staining. It revealed that dendritic complexity, which
was evaluated by Sholl analysis, was stunted in response to COX-2KO
(Fig. 3A, B). At the same time the dendrite length was less after
COX-2KO (Fig. 3C). Furthermore, COX-2 depletion could not only decrease
the density of dendritic spines but also reduce the percentage of
mushroom-type spines (Fig. 3D, E, F). These data suggested that the loss
of COX-2 gene resulted in neuronal structure disturbances in COX-2KO
mice.

\textbf{Effects of COX-2 depletion on Gamma oscillation in hippocampus
CA1.}

Then we carried out local field potential (LFP) recording in the
hippocampus CA1 by implanted 32-channel tetrodes. After analyzed the
relative power of oscillations in COX-2KO and WT control mice, we
observed no significantly altered in the relative power of theta
(4--12Hz), alpha (13--15Hz), and beta (16--30Hz) oscillations (Figure 4
D). However, as illustrated in figure 4, we found a significant decrease
in the relative power of low gamma (30-60Hz), and high gamma (60-90Hz)
oscillation in COX-2KO mice, compared with WT ones (Figure 4 B, C, D).
These results suggested that the deletion of COX-2 may lead to weaker
gamma oscillation in the hippocampus CA1.

\textbf{Effects of COX-2 depletion on the expression of PGs.}

In the result of HPLC/mass spectrometry, we found the expression of
PGE\textsubscript{2} decreased significantly in COX-2KO mice compared
with WT ones (Fig. 5A). However, PGD\textsubscript{2},
PGF\textsubscript{2a}, 6-Keto-PGF\textsubscript{1a} and
TxB\textsubscript{2} these four types of prostaglandins showed no
significant difference between WT and COX-2KO group (Fig. 5B-D).

\textbf{Effects of COX-2 depletion on cAMP/PKA/CREB/BDNF signaling
pathway.}

Then we detect the expression of cAMP with Elisa, total and
phosphorylated PKA/CREB and BDNF with western blot in the hippocampus of
each group. As it illustrated in Figure 6A and B, COX-2KO mice showed
lower levels of cAMP in comparison with WT mice, which were accompanied
by a lower level of phosphorylated PKA in total PKA, phosphorylated CREB
in total CREB, as well as BDNF. The result showed that the cAMP, p-PKA,
p-CREB and BDNF can all be reduced in the hippocampus of COX-2KO mice.

\textbf{Effects of Celecoxib and PGE\textsubscript{2} on SYP/PSD95 and
cAMP/PKA/CREB/BDNF signaling in N2a cells.}

To identify the effects COX-2 and PGE\textsubscript{2} in vitro, we
treated N2a cells with COX-2 selective inhibitor Celecoxib. As shown in
RT-qPCR result, the inhibition ability of Celecoxib for COX-2 was
dose-dependent, while 10\selectlanguage{greek}μ\selectlanguage{english}m was the most efficient among 1\selectlanguage{greek}μ\selectlanguage{english}m, 5\selectlanguage{greek}μ\selectlanguage{english}m, 10\selectlanguage{greek}μ\selectlanguage{english}m
and 20\selectlanguage{greek}μ\selectlanguage{english}m these four concentration gradients (Fig. 7A). After N2a cells
were treated with 10\selectlanguage{greek}μ\selectlanguage{english}m Celecoxib for 24h, evidently, we found not only
the expression of COX-2 and PSD95 protein decreased but also cAMP and
its downstream signal pathway p-PKA/PKA, p-CREB/CREB, BDNF all
downregulated. But no change was found in the SYP expression after the
treatment of Celecoxib. While all of these cytokines significantly
increased after the treatment of PGE\textsubscript{2} (1\selectlanguage{greek}μ\selectlanguage{english}mol/L) for 4
hours (Fig. 7B, C). These results suggest that Celecoxib has the effect
of reducing PSD95 and cAMP/PKA/CREB/BDNF signaling by inhibiting the
releasing of PGE\textsubscript{2} in N2a cell lines.

\textbf{DISCUSSION}

Evidence suggests the constitutive expression of ''inducible'' COX-2 in
the brain\textsuperscript{{[}25{]}}, kidney\textsuperscript{{[}26,
27{]}}, and a few other organs\textsuperscript{{[}28{]}}, with basal
activity involvement in cellular functions. Previous studies have
demonstrated that neuronal COX-2 and its metabolites are essential in
memory and learning. For the first time, Yamagata established that
hippocampal neuronal COX-2 correlates with neural activity and memory
consolidation in rats \textsuperscript{{[}29{]}}. COX-2 is also
implicated in various cognitive functions, such as
acquiring\textsuperscript{{[}30{]}}, consolidating
\textsuperscript{{[}31{]}}, and passively avoiding
\textsuperscript{{[}32{]}} memories. Celecoxib is a selective COX-2
inhibitor and could impair the spatial memory of mice due to infusion
into the hippocampal CA1 region \textsuperscript{{[}33, 34{]}} without
any effect on COX-1 inhibition\textsuperscript{{[}21{]}}. Our data also
demonstrated the importance of COX-2 in mice's memory, consistent with
these findings.

Many studies have examined the molecular mechanisms associated with
synaptic modifications during memory development and mediated by various
proteins \textsuperscript{{[}35-38{]}}. Furthermore, COX-2 is enriched
in neuronal dendritic spines with active
synapses\textsuperscript{{[}39,40{]}}. Synapses form the structural
basis for neuronal communication and facilitate memory functions. PSD95
and SYP regulate the structure and function of
synapses\textsuperscript{{[}41{]}}. PSD95, a significantly available
scaffold protein in the postsynaptic region, helps in the signal
transmission of neurons \textsuperscript{{[}42{]}}. A complete PSD95
knockout in mice causes severe deficits in learning and memory of
spatial areas, establishing the significance of PSD95 in developing
synapses\textsuperscript{{[}43, 44{]}}. Therefore, COX-2 signaling plays
a crucial role in the postsynaptic region, regulating PSD95 expression
and contributing to memory formation.

Learning and memory are associated with activity-induced changes in
dendritic spine density, maturity, size, and
shape\textsuperscript{{[}45{]}}. New memories are directly related to
the density of dendritic spines inside the CA1 region of the hippocampal
structure \textsuperscript{{[}46-49{]}}. Dendritic spines mature into
mushroom spines to achieve spatial working
memory\textsuperscript{{[}45{]}}. Decreased spine density or maturity is
correlated with mental retardation, memory loss, and Alzheimer's
disease\textsuperscript{{[}50-52{]}}. Dendritic tree structure and
arborization provide insight into how neurons receive and transmit
synaptic inputs\textsuperscript{{[}53{]}}. The dendritic diameter in the
cerebellum of C57BL/6 male mice positively correlated with the total
postsynaptic density (PSD). Moreover, a greater diameter is associated
with a greater PSD area \textsuperscript{{[}54{]}}. Dendrite thickness
regulates postsynaptic potential transmission by affecting electrical
resistance, with faster signal propagation among thicker
dendrites\textsuperscript{{[}55, 56{]}}. Evidence indicates
COX-2/PGE\textsubscript{2} signaling alters dendrite and dendritic spine
morphology \textsuperscript{{[}57{]}}. This study supported the
importance of dendritic spine density and maturity in memory formation.
The study also significantly reduced dendritic spine density in COX-2KO
mice. Thus, OX-2 was crucial in mediating memory-related neuronal
structure in the hippocampal CA1 region.

Local population activities or the assembly of neurons across multiple
brain regions cause a rhythmic fluctuation known as neural oscillation.
It is detected using electrocorticography (ECoG), local field potentials
(LFP), magnetoencephalography (MEG), and electroencephalography (EEG)
under various frequencies. These include delta (0.5--3 Hz), theta (4--12
Hz), alpha (13--15 Hz), beta (16--30 Hz), low gamma (30--60 Hz), and
high gamma(60-120 Hz) \textsuperscript{{[}58-60{]}}. They regulate
neuronal spike timing on the microscale and coordinate macroscale data
dispersed in the cortical region to provide information concerning
temporospatial localizations \textsuperscript{{[}61{]}}. Group
coordination is necessary for effective communication between
presynaptic and postsynaptic groups \textsuperscript{{[}62{]}}.
Selective COX-2 inhibitors significantly reduced LTP induction in
hippocampal dentate granule neurons \emph{in vitro} . In contrast, COX-1
inhibitors were ineffective, reversed by PGE\textsubscript{2} treatment,
but not by other PGs \textsuperscript{{[}63{]}}. Additionally,
activating neuronal COX-2 was necessary to induce LTP in
learning\emph{in vivo} \textsuperscript{{[}21{]}}. Gamma oscillations
have fast rhythms to escape inhibition through network excitation for
the time being. This elevates the effectiveness, overall precision, and
selection of communication among various network areas
\textsuperscript{{[}64{]}}. The changing gamma oscillation power could
be a disease biomarker to investigate novel therapies for treating
cognitive impairment in diseases such as Alzheimer's
\textsuperscript{{[}65, 66{]}}. A strong correlation was observed
between the \emph{in vivo} gamma oscillation power and the cognitive
functions of the test animals\textsuperscript{{[}67{]}}. The present
study depicts that COX-2KO in mice effectively decreases gamma
oscillation power. Thus, COX-2 could affect learning and memory ability
by modulating gamma oscillations in the hippocampal CA1 area.

Our findings indicate that only PGE\textsubscript{2} expression
significantly decreased in COX-2KO mice among all the five PG types. One
possible explanation for the altered PG profile would be that COX-2
predominantly metabolizes AA to PGE\textsubscript{2}. On the other hand,
COX-1 could be responsible for other PGs \textsuperscript{{[}68{]}}.
PGE\textsubscript{2} is characterized as the lipid mediator, enabling
nervous system development. Meanwhile, various studies demonstrate that
abnormal PGE\textsubscript{2} signaling leads to neurodevelopmental
disorders \textsuperscript{{[}69, 70{]}}. The
COX-2/PGE\textsubscript{2}signaling pathway is associated with several
vital processes in developing neurons. This includes proliferation,
migration\textsuperscript{{[}71, 72{]}}, synaptogenesis
\textsuperscript{{[}73, 74{]}}, and the formation and plasticity of
dendritic spines\textsuperscript{{[}72, 75{]}}. Some rat studies have
revealed that uncontrolled elevation or reduction of
PGE\textsubscript{2} expression affects cerebellar
\textsuperscript{{[}76{]}}and hippocampal\textsuperscript{{[}77{]}}
dendritic arborization during critical periods after delivery. In
contrast, inhibiting PGE\textsubscript{2}production by locally injecting
COX-2 inhibitors can reduce the arborization of the dendritic tree and
the density of excited synapses\textsuperscript{{[}78{]}}. As depicted
in the COX-2KO mouse model\textsuperscript{{[}79-81{]}}, many developing
genes have differential expression due to the reduced
PGE\textsubscript{2} levels. This disrupts synaptic transmission
pathways, long-term nerve potentiation, navigating the neurites, and
developing the dendritic spine \textsuperscript{{[}82, 83{]}},
consistent with our results.

PKA is the primary downstream kinase of cAMP and could phosphorylate
CREB. Thus, parts of the transcription factor are recruited within the
promoter regions, including the BDNFgene \textsuperscript{{[}84{]}}. The
cAMP/PKA/CREB/BDNF signaling pathway promotes the survival of neurons,
modulates synaptic morphology, and strengthens synaptic
transmission\textsuperscript{{[}85{]}}. The pathway modulates survival,
development, cognition, and mood across epilepsy, depression, and
Alzheimer's disease animal model neurons \textsuperscript{{[}86, 87{]}}.
Our study observed lower phosphorylation expression in the COX-2KO group
of cAMP, PKA, and CREB with BDNF expression than in WT. Thus, COX-2
could participate in the synaptic plasticity modulation of hippocampal
neurons using cAMP/PKA/CREB/BDNF signaling.

Our findings revealed the COX-2 signaling pathway as a new mechanism for
regulating the hippocampal dendritogenic process and gamma oscillation
to affect learning and memory ability. The exogenous application of
PGE\textsubscript{2} effectively reversed the actions caused by the
inhibition of COX-2. COX-2 could participate in hippocampal synaptic
plasticity by regulating COX-2/PGE\textsubscript{2}/cAMP/PKA/CREB/BDNF
signaling in the postsynaptic membrane PSD95 modulation. Therefore, this
study can elevate our understanding of COX-2 physiologic function and
its role in memory formation.

\textbf{ACKNOWLEDGEMENTS}

This research was supported by the National Natural Science Foundation
of China (31271142); Shanghai Key Laboratory of Health Identification
and Assessment (13DZ2261000).

\textbf{ABBREVIATIONS}

AA: Arachidonic acid; BDNF: brain-derived neurotrophic factor; BSA:
Bovine serum albumin; cAMP: Cyclic adenosine monophosphate; COX:
Cyclooxygenase; CREB: cAMP response element binding protein; DMEM:
Dulbecco's modified Eagle's medium; ECoG: Electrocorticography; EEG:
electroencephalography; FBS: fetal bovine serum; GAPDH:
Glyceraldehyde-3-phosphate dehydrogenase; HFS: high-frequency
stimulations; HRP: Horseradish Peroxidase; KO: knockout; LC/MS/MS:
Liquid chromatography/mass spectrometry/mass spectrometry; LFP: local
field potentials; LTP: long-term potentiation; MEG:
Magnetoencephalography; PBS: Phosphate-buffered saline; PFA:
Paraformaldehyde; PG: prostaglandin; PGHS: Prostaglandin G/H synthase;
PKA: protein kinase A; PSD: Post Synaptic Density; RIPA:
Radioimmunoprecipitation; SYP: Synaptophysin; TX: Thromboxane; WT: Wild
type.

\textbf{CONFLICT OF INTEREST}

The authors declare no financial conflict of interest that might be
construed to influence the results or interpretation of the manuscript.

\textbf{AUTHOR CONTRIBUTIONS}

Deheng Wang designed the study; Hong Ni wrote the manuscript and
performed the study; Zhongzhao Guo, Yue Wu, Zilu Zhu and Jie Wang
analyzed the data; Yang Yang complete the experiment of revision, and
all authors have reviewed and approved the final version of the
manuscript.

\textbf{DATA AVAILABILITY STATEMENT}

All data analyzed and presented in this study are available from the
corresponding author on reasonable request.

\textbf{REFERENCES}

Ahmed, T. \& Gilani, A.H. (2011) A comparative study of curcuminoids to
measure their effect on inflammatory and apoptotic gene expression in an
Abeta plus ibotenic acid-infused rat model of Alzheimer's
disease.\emph{Brain Res} , \textbf{1400} , 1-18.

Aid, S., Langenbach, R. \& Bosetti, F. (2008) Neuroinflammatory response
to lipopolysaccharide is exacerbated in mice genetically deficient in
cyclooxygenase-2. \emph{J Neuroinflammation} , \textbf{5} , 17.

Ayuk, S.M., Abrahamse, H. \& Houreld, N.N. (2016) The role of
photobiomodulation on gene expression of cell adhesion molecules in
diabetic wounded fibroblasts in vitro. \emph{J Photochem Photobiol B}
,\textbf{161} , 368-374.

Beltran-Campos, V., Prado-Alcala, R.A., Leon-Jacinto, U.,
Aguilar-Vazquez, A., Quirarte, G.L., Ramirez-Amaya, V. \& Diaz-Cintra,
S. (2011) Increase of mushroom spine density in CA1 apical dendrites
produced by water maze training is prevented by ovariectomy. \emph{Brain
Res} , \textbf{1369} , 119-130.

Bird, A.D. \& Cuntz, H. (2019) Dissecting Sholl Analysis into Its
Functional Components. \emph{Cell Rep} , \textbf{27} , 3081-3096 e3085.

Bosetti, F., Langenbach, R. \& Weerasinghe, G.R. (2004) Prostaglandin E2
and microsomal prostaglandin E synthase-2 expression are decreased in
the cyclooxygenase-2-deficient mouse brain despite compensatory
induction of cyclooxygenase-1 and Ca2+-dependent phospholipase
A2.\emph{J Neurochem} , \textbf{91} , 1389-1397.

Brock, T.G., McNish, R.W. \& Peters-Golden, M. (1999) Arachidonic acid
is preferentially metabolized by cyclooxygenase-2 to prostacyclin and
prostaglandin E2. \emph{J Biol Chem} , \textbf{274} , 11660-11666.

Burks, S.R., Wright, C.L. \& McCarthy, M.M. (2007) Exploration of
prostanoid receptor subtype regulating estradiol and prostaglandin E2
induction of spinophilin in developing preoptic area
neurons.\emph{Neuroscience} , \textbf{146} , 1117-1127.

Candelario-Jalil, E. (2009) A role for cyclooxygenase-1 in
beta-amyloid-induced neuroinflammation. \emph{Aging (Albany NY)}
,\textbf{1} , 350-353.

Chen, C., Magee, J.C. \& Bazan, N.G. (2002) Cyclooxygenase-2 regulates
prostaglandin E2 signaling in hippocampal long-term synaptic
plasticity.\emph{J Neurophysiol} , \textbf{87} , 2851-2857.

Chen, R., Zhang, J., Fan, N., Teng, Z.Q., Wu, Y., Yang, H., Tang, Y.P.,
Sun, H., Song, Y. \& Chen, C. (2013) Delta9-THC-caused synaptic and
memory impairments are mediated through COX-2 signaling. \emph{Cell}
,\textbf{155} , 1154-1165.

Choi, S.H., Aid, S. \& Bosetti, F. (2009) The distinct roles of
cyclooxygenase-1 and -2 in neuroinflammation: implications for
translational research. \emph{Trends Pharmacol Sci} , \textbf{30} ,
174-181.

Clemente, S.M., Martinez-Costa, O.H., Monsalve, M. \& Samhan-Arias, A.K.
(2020) Targeting Lipid Peroxidation for Cancer
Treatment.\emph{Molecules} , \textbf{25} .

Cole, S.R. \& Voytek, B. (2017) Brain Oscillations and the Importance of
Waveform Shape. \emph{Trends Cogn Sci} , \textbf{21} , 137-149.

Cowley, T.R., Fahey, B. \& O'Mara, S.M. (2008) COX-2, but not COX-1,
activity is necessary for the induction of perforant path long-term
potentiation and spatial learning in vivo. \emph{Eur J Neurosci}
,\textbf{27} , 2999-3008.

Dean, S.L., Knutson, J.F., Krebs-Kraft, D.L. \& McCarthy, M.M. (2012a)
Prostaglandin E2 is an endogenous modulator of cerebellar development
and complex behavior during a sensitive postnatal period. \emph{Eur J
Neurosci} , \textbf{35} , 1218-1229.

Dean, S.L., Wright, C.L., Hoffman, J.F., Wang, M., Alger, B.E. \&
McCarthy, M.M. (2012b) Prostaglandin E2 stimulates estradiol synthesis
in the cerebellum postnatally with associated effects on Purkinje neuron
dendritic arbor and electrophysiological properties.\emph{Endocrinology}
, \textbf{153} , 5415-5427.

DeLarge, A.F. \& Winsauer, P.J. (2021) Effects of (9)-THC on memory in
ovariectomized and intact female rats. \emph{Horm Behav} , \textbf{127}
, 104883.

Dere, E., Huston, J.P. \& De Souza Silva, M.A. (2007) The pharmacology,
neuroanatomy and neurogenetics of one-trial object recognition in
rodents. \emph{Neurosci Biobehav Rev} , \textbf{31} , 673-704.

Elias, G.M., Funke, L., Stein, V., Grant, S.G., Bredt, D.S. \& Nicoll,
R.A. (2006) Synapse-specific and developmentally regulated targeting of
AMPA receptors by a family of MAGUK scaffolding proteins. \emph{Neuron}
,\textbf{52} , 307-320.

Fiala, J.C., Spacek, J. \& Harris, K.M. (2002) Dendritic spine
pathology: cause or consequence of neurological disorders? \emph{Brain
Res Brain Res Rev} , \textbf{39} , 29-54.

Flemming, A. (2012) Stroke: Can PSD95 inhibitors widen the therapeutic
window? \emph{Nat Rev Drug Discov} , \textbf{11} , 272.

Fries, P. (2015) Rhythms for Cognition: Communication through
Coherence.\emph{Neuron} , \textbf{88} , 220-235.

Fujimi, K., Noda, K., Sasaki, K., Wakisaka, Y., Tanizaki, Y., Iida, M.,
Kiyohara, Y., Kanba, S. \& Iwaki, T. (2007) Altered expression of COX-2
in subdivisions of the hippocampus during aging and in Alzheimer's
disease: the Hisayama Study. \emph{Dement Geriatr Cogn Disord}
,\textbf{23} , 423-431.

Green, T., Gonzalez, A.A., Mitchell, K.D. \& Navar, L.G. (2012) The
complex interplay between cyclooxygenase-2 and angiotensin II in
regulating kidney function. \emph{Curr Opin Nephrol Hypertens}
,\textbf{21} , 7-14.

Grossman, Y.S., Fillinger, C., Manganaro, A., Voren, G., Waldman, R.,
Zou, T., Janssen, W.G., Kenny, P.J. \& Dumitriu, D. (2022) Structure and
function differences in the prelimbic cortex to basolateral amygdala
circuit mediate trait vulnerability in a novel model of acute social
defeat stress in male mice. \emph{Neuropsychopharmacology} , \textbf{47}
, 788-799.

Guo, H., Cheng, Y., Wang, C., Wu, J., Zou, Z., Niu, B., Yu, H., Wang, H.
\& Xu, J. (2017) FFPM, a PDE4 inhibitor, reverses learning and memory
deficits in APP/PS1 transgenic mice via cAMP/PKA/CREB signaling and
anti-inflammatory effects. \emph{Neuropharmacology} , \textbf{116} ,
260-269.

Harland, B.C., Collings, D.A., McNaughton, N., Abraham, W.C. \&
Dalrymple-Alford, J.C. (2014) Anterior thalamic lesions reduce spine
density in both hippocampal CA1 and retrosplenial cortex, but enrichment
rescues CA1 spines only. \emph{Hippocampus} , \textbf{24} , 1232-1247.

Harris, R.C. (2008) An update on cyclooxygenase-2 expression and
metabolites in the kidney. \emph{Curr Opin Nephrol Hypertens}
,\textbf{17} , 64-69.

Ho, L., Pieroni, C., Winger, D., Purohit, D.P., Aisen, P.S. \&
Pasinetti, G.M. (1999) Regional distribution of cyclooxygenase-2 in the
hippocampal formation in Alzheimer's disease. \emph{J Neurosci Res}
,\textbf{57} , 295-303.

Holscher, C. (1995) Prostaglandins play a role in memory consolidation
in the chick. \emph{Eur J Pharmacol} , \textbf{294} , 253-259.

Honda, S., Matsumoto, M., Tajinda, K. \& Mihara, T. (2020) Enhancing
Clinical Trials Through Synergistic Gamma Power Analysis. \emph{Front
Psychiatry} , \textbf{11} , 537.

Hoozemans, J.J., Rozemuller, J.M., van Haastert, E.S., Veerhuis, R. \&
Eikelenboom, P. (2008) Cyclooxygenase-1 and -2 in the different stages
of Alzheimer's disease pathology. \emph{Curr Pharm Des} , \textbf{14} ,
1419-1427.

Hwang, I.K., Yi, S.S., Yoo, K.Y., Park, O.K., Yan, B., Kim, I.Y., Kim,
Y.N., Song, W., Moon, S.M., Won, M.H., Seong, J.K. \& Yoon, Y.S. (2010)
Effects of treadmill exercise on cyclooxygenase-2 in the hippocampus in
type 2 diabetic rats: correlation with the neuroblasts. \emph{Brain Res}
, \textbf{1341} , 84-92.

Ju, Z., Li, M., Xu, J., Howell, D.C., Li, Z. \& Chen, F.E. (2022) Recent
development on COX-2 inhibitors as promising anti-inflammatory agents:
The past 10 years. \emph{Acta Pharm Sin B} , \textbf{12} , 2790-2807.

Kaufmann, W.E., Worley, P.F., Pegg, J., Bremer, M. \& Isakson, P. (1996)
COX-2, a synaptically induced enzyme, is expressed by excitatory neurons
at postsynaptic sites in rat cerebral cortex. \emph{Proc Natl Acad Sci U
S A} , \textbf{93} , 2317-2321.

Kepka, A., Ochocinska, A., Chojnowska, S., Borzym-Kluczyk, M., Skorupa,
E., Knas, M. \& Waszkiewicz, N. (2021) Potential Role of L-Carnitine in
Autism Spectrum Disorder. \emph{J Clin Med} , \textbf{10} .

Kissoondoyal, A. \& Crawford, D.A. (2022) Prostaglandin E2 Increases
Neurite Length and the Formation of Axonal Loops, and Regulates Cone
Turning in Differentiating NE4C Cells Via PKA. \emph{Cell Mol Neurobiol}
, \textbf{42} , 1385-1397.

Leal, G., Bramham, C.R. \& Duarte, C.B. (2017) BDNF and Hippocampal
Synaptic Plasticity. \emph{Vitam Horm} , \textbf{104} , 153-195.

Li, L., Sun, R., Zenga, J., Himburg, H., Wang, L., Duan, S., Liu, J.,
Bui, D., Xie, Z., Du, T., Xie, L., Yin, T., Wong, S., Gao, S. \& Hu, M.
(2022) Comparison of Absolute Expression and Turnover Number of COX-1
and COX-2 in Human and Rodent Cells and Tissues. \emph{J Inflamm Res}
,\textbf{15} , 4435-4447.

Li, S., Quan, T., Xu, C., Huang, Q., Kang, H., Chen, Y., Li, A., Fu, L.,
Luo, Q., Gong, H. \& Zeng, S. (2019) Optimization of Traced Neuron
Skeleton Using Lasso-Based Model. \emph{Front Neuroanat} , \textbf{13} ,
18.

Li, W., Wu, S., Ahmad, M., Jiang, J., Liu, H., Nagayama, T., Rose, M.E.,
Tyurin, V.A., Tyurina, Y.Y., Borisenko, G.G., Belikova, N., Chen, J.,
Kagan, V.E. \& Graham, S.H. (2010) The cyclooxygenase site, but not the
peroxidase site of cyclooxygenase-2 is required for neurotoxicity in
hypoxic and ischemic injury. \emph{J Neurochem} , \textbf{113} ,
965-977.

Liu, R., Tan, Q. \& Luo, Q. (2018) Decreased expression level and
DNA-binding activity of specificity protein 1 via cyclooxygenase-2
inhibition antagonizes radiation resistance, cell migration and invasion
in radiation-resistant lung cancer cells. \emph{Oncol Lett} ,\textbf{16}
, 3029-3037.

Loopuijt, L.D., Silva Filho, M., Hirt, B., Vonthein, R. \& Kremers, J.
(2007) Dendritic thickness: a morphometric parameter to classify mouse
retinal ganglion cells. \emph{Braz J Med Biol Res} , \textbf{40} ,
1367-1382.

Lopez, D.E. \& Ballaz, S.J. (2020) The Role of Brain Cyclooxygenase-2
(Cox-2) Beyond Neuroinflammation: Neuronal Homeostasis in Memory and
Anxiety. \emph{Mol Neurobiol} , \textbf{57} , 5167-5176.

Lu, C.B., Jefferys, J.G., Toescu, E.C. \& Vreugdenhil, M. (2011) In
vitro hippocampal gamma oscillation power as an index of in vivo CA3
gamma oscillation strength and spatial reference memory. \emph{Neurobiol
Learn Mem} , \textbf{95} , 221-230.

Ma, J., Zhang, Z., Kang, L., Geng, D., Wang, Y., Wang, M. \& Cui, H.
(2014) Repetitive transcranial magnetic stimulation (rTMS) influences
spatial cognition and modulates hippocampal structural synaptic
plasticity in aging mice. \emph{Exp Gerontol} , \textbf{58} , 256-268.

Mahmmoud, R.R., Sase, S., Aher, Y.D., Sase, A., Groger, M., Mokhtar, M.,
Hoger, H. \& Lubec, G. (2015) Spatial and Working Memory Is Linked to
Spine Density and Mushroom Spines. \emph{PLoS One} , \textbf{10} ,
e0139739.

Mathalon, D.H. \& Sohal, V.S. (2015) Neural Oscillations and Synchrony
in Brain Dysfunction and Neuropsychiatric Disorders: It's About
Time.\emph{JAMA Psychiatry} , \textbf{72} , 840-844.

Meier, M.A., Lemercier, C.E., Kulisch, C., Kiss, B., Lendvai, B., Adham,
N. \& Gerevich, Z. (2020) The novel antipsychotic cariprazine stabilizes
gamma oscillations in rat hippocampal slices. \emph{Br J Pharmacol}
,\textbf{177} , 1622-1634.

Mirisis, A.A., Alexandrescu, A., Carew, T.J. \& Kopec, A.M. (2016) The
Contribution of Spatial and Temporal Molecular Networks in the Induction
of Long-term Memory and Its Underlying Synaptic Plasticity. \emph{AIMS
Neurosci} , \textbf{3} , 356-384.

Morton, R.S. \& Dongari-Bagtzoglou, A.I. (2001) Cyclooxygenase-2 is
upregulated in inflamed gingival tissues. \emph{J Periodontol}
,\textbf{72} , 461-469.

Nakayama, M., Uchimura, K., Zhu, R.L., Nagayama, T., Rose, M.E.,
Stetler, R.A., Isakson, P.C., Chen, J. \& Graham, S.H. (1998)
Cyclooxygenase-2 inhibition prevents delayed death of CA1 hippocampal
neurons following global ischemia. \emph{Proc Natl Acad Sci U S A}
,\textbf{95} , 10954-10959.

Nam, S.M., Kim, J.W., Yoo, D.Y., Choi, J.H., Kim, W., Jung, H.Y., Won,
M.H., Hwang, I.K., Seong, J.K. \& Yoon, Y.S. (2013) Effects of treadmill
exercise on neural stem cells, cell proliferation, and neuroblast
differentiation in the subgranular zone of the dentate gyrus in
cyclooxygenase-2 knockout mice. \emph{Neurochem Res} , \textbf{38} ,
2559-2569.

Nam, S.M., Kim, J.W., Yoo, D.Y., Choi, J.H., Kim, W., Jung, H.Y., Won,
M.H., Hwang, I.K., Seong, J.K. \& Yoon, Y.S. (2015) Comparison of
pharmacological and genetic inhibition of cyclooxygenase-2: effects on
adult neurogenesis in the hippocampal dentate gyrus. \emph{J Vet Sci}
,\textbf{16} , 245-251.

Nishiyama, J. (2019) Plasticity of dendritic spines: Molecular function
and dysfunction in neurodevelopmental disorders. \emph{Psychiatry Clin
Neurosci} , \textbf{73} , 541-550.

Niwa, K., Araki, E., Morham, S.G., Ross, M.E. \& Iadecola, C. (2000)
Cyclooxygenase-2 contributes to functional hyperemia in whisker-barrel
cortex. \emph{J Neurosci} , \textbf{20} , 763-770.

O'Banion, M.K., Winn, V.D. \& Young, D.A. (1992) cDNA cloning and
functional activity of a glucocorticoid-regulated inflammatory
cyclooxygenase. \emph{Proc Natl Acad Sci U S A} , \textbf{89} ,
4888-4892.

Parajuli, L.K., Urakubo, H., Takahashi-Nakazato, A., Ogelman, R.,
Iwasaki, H., Koike, M., Kwon, H.B., Ishii, S., Oh, W.C., Fukazawa, Y. \&
Okabe, S. (2020) Geometry and the Organizational Principle of Spine
Synapses along a Dendrite. \emph{eNeuro} , \textbf{7} .

Parra-Damas, A., Chen, M., Enriquez-Barreto, L., Ortega, L., Acosta, S.,
Perna, J.C., Fullana, M.N., Aguilera, J., Rodriguez-Alvarez, J. \&
Saura, C.A. (2017) CRTC1 Function During Memory Encoding Is Disrupted in
Neurodegeneration. \emph{Biol Psychiatry} , \textbf{81} , 111-123.

Penzes, P., Cahill, M.E., Jones, K.A., VanLeeuwen, J.E. \& Woolfrey,
K.M. (2011) Dendritic spine pathology in neuropsychiatric
disorders.\emph{Nat Neurosci} , \textbf{14} , 285-293.

Perez-Pouchoulen, M., Yu, S.J., Roby, C.R., Bonsavage, N. \& McCarthy,
M.M. (2019) Regulatory Control of Microglial Phagocytosis by Estradiol
and Prostaglandin E2 in the Developing Rat Cerebellum.\emph{Cerebellum}
, \textbf{18} , 882-895.

Pillai, A.G., de Jong, D., Kanatsou, S., Krugers, H., Knapman, A.,
Heinzmann, J.M., Holsboer, F., Landgraf, R., Joels, M. \& Touma, C.
(2012) Dendritic morphology of hippocampal and amygdalar neurons in
adolescent mice is resilient to genetic differences in stress
reactivity. \emph{PLoS One} , \textbf{7} , e38971.

Rai-Bhogal, R., Ahmad, E., Li, H. \& Crawford, D.A. (2018) Microarray
analysis of gene expression in the cyclooxygenase knockout mice - a
connection to autism spectrum disorder. \emph{Eur J Neurosci}
,\textbf{47} , 750-766.

Rall, J.M., Mach, S.A. \& Dash, P.K. (2003) Intrahippocampal infusion of
a cyclooxygenase-2 inhibitor attenuates memory acquisition in
rats.\emph{Brain Res} , \textbf{968} , 273-276.

Ramires Junior, O.V., Dos Santos, T.M., Silveira, J.S., Leite-Aguiar,
R., Coutinho-Silva, R., Savio, L.E.B. \& Wyse, A.T.S. (2022)
Rivastigmine Reverses the Decrease in Synapsin and Memory Caused by
Homocysteine: Is There Relation to Inflammation? \emph{Mol Neurobiol}
,\textbf{59} , 4517-4534.

Rossini, L., De Santis, D., Mauceri, R.R., Tesoriero, C., Bentivoglio,
M., Maderna, E., Maiorana, A., Deleo, F., de Curtis, M., Tringali, G.,
Cossu, M., Tumminelli, G., Bramerio, M., Spreafico, R., Tassi, L. \&
Garbelli, R. (2021) Dendritic pathology, spine loss and synaptic
reorganization in human cortex from epilepsy patients. \emph{Brain}
,\textbf{144} , 251-265.

Ruan, J., Liu, L., Shan, X., Xia, B. \& Fu, Q. (2019) Anti-depressant
effects of oil from fructus gardeniae via PKA-CREB-BDNF
signaling.\emph{Biosci Rep} , \textbf{39} .

Runyan, J.D., Moore, A.N. \& Dash, P.K. (2004) A role for prefrontal
cortex in memory storage for trace fear conditioning. \emph{J Neurosci}
,\textbf{24} , 1288-1295.

Sharifzadeh, M., Naghdi, N., Khosrovani, S., Ostad, S.N., Sharifzadeh,
K. \& Roghani, A. (2005) Post-training intrahippocampal infusion of the
COX-2 inhibitor celecoxib impaired spatial memory retention in
rats.\emph{Eur J Pharmacol} , \textbf{511} , 159-166.

Sharifzadeh, M., Tavasoli, M., Soodi, M., Mohammadi-Eraghi, S.,
Ghahremani, M.H. \& Roghani, A. (2006) A time course analysis of
cyclooxygenase-2 suggests a role in spatial memory retrieval in
rats.\emph{Neurosci Res} , \textbf{54} , 171-179.

Sheynikhovich, D., Otani, S., Bai, J. \& Arleo, A. (2022) Long-term
memory, synaptic plasticity and dopamine in rodent medial prefrontal
cortex: Role in executive functions. \emph{Front Behav Neurosci}
,\textbf{16} , 1068271.

Song, D., Wang, D., Yang, Q., Yan, T., Wang, Z., Yan, Y., Zhao, J., Xie,
Z., Liu, Y., Ke, Z., Qazi, T.J., Li, Y., Wu, Y., Shi, Q., Lang, Y.,
Zhang, H., Huang, T., Wang, C., Quan, Z. \& Qing, H. (2020) The
lateralization of left hippocampal CA3 during the retrieval of spatial
working memory. \emph{Nat Commun} , \textbf{11} , 2901.

Song, W.L., Lawson, J.A., Wang, M., Zou, H. \& FitzGerald, G.A. (2007)
Noninvasive assessment of the role of cyclooxygenases in cardiovascular
health: a detailed HPLC/MS/MS method. \emph{Methods Enzymol}
,\textbf{433} , 51-72.

Strauss, K.I. (2008) Antiinflammatory and neuroprotective actions of
COX2 inhibitors in the injured brain. \emph{Brain Behav Immun}
,\textbf{22} , 285-298.

Strekalova, T., Pavlov, D., Trofimov, A., Anthony, D.C., Svistunov, A.,
Proshin, A., Umriukhin, A., Lyundup, A., Lesch, K.P. \& Cespuglio, R.
(2022) Hippocampal Over-Expression of Cyclooxygenase-2 (COX-2) Is
Associated with Susceptibility to Stress-Induced Anhedonia in
Mice.\emph{Int J Mol Sci} , \textbf{23} .

Tabrizian, K., Hashemzaei, M., Nasiri, A.A., Najafi, S., Amelinia, F.,
Sanati, M., Shamshirgaran, F. \& Fanoudi, S. (2018) Interactive
involvement of hippocampal cAMP/PKA and cyclooxygenase-2 signaling
pathways in spatial learning in the Morris water maze. \emph{Folia
Neuropathol} , \textbf{56} , 58-66.

Tamiji, J. \& Crawford, D.A. (2010) The neurobiology of lipid metabolism
in autism spectrum disorders. \emph{Neurosignals} , \textbf{18} ,
98-112.

Teather, L.A., Packard, M.G. \& Bazan, N.G. (2002) Post-training
cyclooxygenase-2 (COX-2) inhibition impairs memory
consolidation.\emph{Learn Mem} , \textbf{9} , 41-47.

Thomas, N., Krishnapillai, R., Bindhu, P.R. \& Thomas, P. (2019)
Immunohistochemical expression of cyclooxygenase-2 in oral squamous cell
carcinoma. \emph{Indian J Dent Res} , \textbf{30} , 102-106.

Tiesinga, P.H., Fellous, J.M., Salinas, E., Jose, J.V. \& Sejnowski,
T.J. (2004) Inhibitory synchrony as a mechanism for attentional gain
modulation. \emph{J Physiol Paris} , \textbf{98} , 296-314.

Vorhees, C.V. \& Williams, M.T. (2006) Morris water maze: procedures for
assessing spatial and related forms of learning and memory. \emph{Nat
Protoc} , \textbf{1} , 848-858.

Wang, Y.Y., Zhao, B., Wu, M.M., Zheng, X.L., Lin, L. \& Yin, D.M. (2021)
Overexpression of neuregulin 1 in GABAergic interneurons results in
reversible cortical disinhibition. \emph{Nat Commun} , \textbf{12} ,
278.

Woolf, N.J., Milov, A.M., Schweitzer, E.S. \& Roghani, A. (2001)
Elevation of nerve growth factor and antisense knockdown of TrkA
receptor during contextual memory consolidation. \emph{J Neurosci}
,\textbf{21} , 1047-1055.

Wright, C.L. \& McCarthy, M.M. (2009) Prostaglandin E2-induced
masculinization of brain and behavior requires protein kinase A,
AMPA/kainate, and metabotropic glutamate receptor signaling. \emph{J
Neurosci} , \textbf{29} , 13274-13282.

Yagami, T., Koma, H. \& Yamamoto, Y. (2016) Pathophysiological Roles of
Cyclooxygenases and Prostaglandins in the Central Nervous
System.\emph{Mol Neurobiol} , \textbf{53} , 4754-4771.

Yamagata, K., Andreasson, K.I., Kaufmann, W.E., Barnes, C.A. \& Worley,
P.F. (1993) Expression of a mitogen-inducible cyclooxygenase in brain
neurons: regulation by synaptic activity and
glucocorticoids.\emph{Neuron} , \textbf{11} , 371-386.

Yang, H.W. (2009) {[}COX-2 regulation of prostaglandins in synaptic
signaling{]}. \emph{Sheng Li Ke Xue Jin Zhan} , \textbf{40} , 317-320.

Yasuda, R., Hayashi, Y. \& Hell, J.W. (2022) CaMKII: a central molecular
organizer of synaptic plasticity, learning and memory. \emph{Nat Rev
Neurosci} .

Yermakova, A.V. \& O'Banion, M.K. (2001) Downregulation of neuronal
cyclooxygenase-2 expression in end stage Alzheimer's
disease.\emph{Neurobiol Aging} , \textbf{22} , 823-836.

Yu, Y., Stubbe, J., Ibrahim, S., Song, W.L., Smyth, E.M., Funk, C.D. \&
FitzGerald, G.A. (2010) Cyclooxygenase-2-dependent prostacyclin
formation and blood pressure homeostasis: targeted exchange of
cyclooxygenase isoforms in mice. \emph{Circ Res} , \textbf{106} ,
337-345.

Yui, K., Imataka, G. \& Yoshihara, S. (2022) Lipid-Based Molecules on
Signaling Pathways in Autism Spectrum Disorder. \emph{Int J Mol Sci}
,\textbf{23} .

Zhang, H., Watrous, A.J., Patel, A. \& Jacobs, J. (2018) Theta and Alpha
Oscillations Are Traveling Waves in the Human Neocortex. \emph{Neuron}
,\textbf{98} , 1269-1281 e1264.

Zhong, Y., Chen, J., Li, L., Qin, Y., Wei, Y., Pan, S., Jiang, Y., Chen,
J. \& Xie, Y. (2018) PKA-CREB-BDNF signaling pathway mediates
propofol-induced long-term learning and memory impairment in hippocampus
of rats. \emph{Brain Res} , \textbf{1691} , 64-74.

Zidar, N., Odar, K., Glavac, D., Jerse, M., Zupanc, T. \& Stajer, D.
(2009) Cyclooxygenase in normal human tissues--is COX-1 really a
constitutive isoform, and COX-2 an inducible isoform? \emph{J Cell Mol
Med} , \textbf{13} , 3753-3763.

\par\null

\textbf{Figure legend}

\textbf{Figure 1.} \textbf{The genotyping of COX-2KO mice and the effect
of COX-2 depletion on the learning and memory ability in MWM and NOR
test.} \textbf{(A)} Genotyping of COX-2 by PCR. The 800 bp DNA fragments
were detected in WT (COX-2+/+) mice; 400 bp were detected in COX-2KO
homozygote (COX-2-/-) mice; both 400 and 800 bp DNA fragments could be
detected in COX-2KO heterozygote (COX-2+/-) mice. \textbf{(B)} COX-2
protein expression in hippocampus in WT and COX-2KO mice were detected
by western blot. \textbf{(C)} Body weight of WT and COX-2KO
mice.\textbf{(D)} Body temperature of WT and COX-2KO mice.
\textbf{(E)}Expression of mRNAs for TNF-a, iNOS and IL-1\selectlanguage{greek}β \selectlanguage{english}in the
hippocampus of WT and COX-2KO mice. \textbf{(F)} The escape latency in
finding a target platform during the 5-day period training trials.
\textbf{(G)} Time spent in the target quadrant on the last day without
platform.\textbf{(H)} Counts crossing the platform area on the last day
without platform. \textbf{(I)} Swimming speed of WT and COX-2KO mice on
the last day of MWM. \textbf{(J)} Total distance traveled of WT and
COX-2KO mice on the last day of MWM. \textbf{(K)} The track of probe
test.\textbf{(L)} The recognition index. \textbf{(J)} Total distance
traveled of WT and COX-2KO mice in NORT. (The bars indicate the means \selectlanguage{ngerman}±
SEM; n = 10 per group; *\selectlanguage{english}? \textless{} 0.05).

\selectlanguage{english}\textbf{Figure 2. Effects of COX-2 depletion on the SYP and PSD95.
(A)}Representative immunofluorescence photomicrographs of SYP, PSD95 and
DAPI in hippocampus CA1. \textbf{(B)} Quantification of SYP (+) and
PSD95 (+) fluorescence intensity. \textbf{(C)} The expression of SYP and
PSD95 protein in the hippocampus of WT and COX\selectlanguage{english}-2KO mice was detected by
Western Blot. (The bars indicate the means +- SD; n = 5 per group; *\selectlanguage{english}?
\textless{} 0.05; Scale bar = 100\selectlanguage{greek}μ\selectlanguage{english}m)

\textbf{Figure 3.} \textbf{Effects of COX-2 depletion on the neuronal
structure. (A)} Representative Golgi staining photomicrographs in
hippocampus CA1. Bar=20\selectlanguage{greek}μ\selectlanguage{english}m. \textbf{(B)} The Sholl analysis was performed
to evaluate the dendritic complexity. n=25 neurons from 5 mice for each
group. \textbf{(C)} Quantitative analysis of the dendrite
length.\textbf{(D)} Representative Golgi staining photomicrographs for
the dendritic spines. Bar=2\selectlanguage{greek}μ\selectlanguage{english}m. \textbf{(E)} Quantitative analysis of the
spine density. \textbf{(F)} Quantitative analysis of the percentage of
mushroom spines. (The bars indicate the means \selectlanguage{ngerman}± SD; n = 5 per group; *\selectlanguage{english}?
\textless{} 0.05)

\selectlanguage{english}\textbf{Figure 4. Effects of COX-2 depletion on Gamma oscillation in
hippocampus CA1. (A)} Representative LFP and filtered theta, alpha,
beta, low gamma and high gamma component of the signal from WT and
COX\selectlanguage{english}-2KO mice. \textbf{(B)} Representative Power spectrogram of LFP from
WT and COX\selectlanguage{english}-2KO mice. \textbf{(C)} Power spectral density of LFP from 0
to 120Hz (left) and 30 to 120Hz (right) in WT and COX\selectlanguage{english}-2KO
mice.\textbf{(D)} The power of theta oscillation (4--12Hz), alpha
oscillation (13--15Hz), beta oscillation (16--30Hz), low gamma
oscillation (30--60Hz) and high gamma oscillation (60--120Hz). (The bars
indicate the means +- SD; n = 5 per group; *\selectlanguage{english}? \textless{} 0.05)

\selectlanguage{english}\textbf{Figure 5. Effects of COX-2 depletion on the expression of PGs.
(A-E)} Results of PGE\textsubscript{2}, PGD\textsubscript{2},
PGF\textsubscript{2a}, 6-keto-PGF\textsubscript{1a} and
TxB\textsubscript{2} levels by HPLC/mass spectrometry in the hippocampus
of WT and COX-2KO mice. (The bars indicate the means +- SEM; n = 5 per
group; *\selectlanguage{english}? \textless{} 0.05)

\textbf{Figure 6.} \selectlanguage{english}\textbf{Effects of COX-2 depletion on
cAMP/PKA/CREB/BDNF signaling pathway.} \textbf{(A)} The expression of
PKA, p-PKA, CREB, p-CREB, and BDNF protein in the hippocampus of WT and
COX\selectlanguage{english}-2KO mice was detected by Western Blot. \textbf{(B)} The expression
of cAMP was detected by Elisa. (The bars indicate the means +- SD; n = 5
per group; *\selectlanguage{english}? \textless{} 0.05)

\textbf{Figure 7. Effects of Celecoxib and PGE\textsubscript{2} on
SYP/PSD95 and cAMP/PKA/CREB/BDNF signaling in N2a cells. (A)} RT-qPCR
result of COX-2 mRNA in N2a cells treated with Celecoxib in different
concentration. \textbf{(B)} ELISA result of cAMP level.
\textbf{(C)}Western blot result of COX-2, SYP, PSD95, PKA, p-PKA, CREB,
p-CREB and BDNF proteins in N2a cells treated with Celecoxib and
PGE\textsubscript{2}. (The bars indicate the means +- SD; n = 4 per
group; *\selectlanguage{english}? \textless{} 0.05)\selectlanguage{english}
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