The Pharmacogenomics Journal (2006), 1–7
& 2006 Nature Publishing Group All rights reserved 1470-269X/06 $30.00
Hippocampal upregulation of the
cyclooxygenase-2 gene following neonatal
clomipramine treatment (a model of depression)
P Cassano, A Hidalgo, V Burgos,
S Adris and P Argibay
Unit of Brain Research, Instituto de Ciencias
Básicas y Medicina Experimental del Hospital
Italiano de Buenos Aires, Buenos Aires, Argentina
Correspondence:
Dr P Cassano, Unit of Brain Research, Instituto
de Ciencias Básicas y Medicina Experimental
del Hospital Italiano de Buenos Aires, Potosı́
4240, 8th floor, (C1199ACL) Buenos Aires,
Argentina.
E-mail: paola.cassano@hospitalitaliano.org.ar
Received 12 August 2005; revised 27 January
2006; accepted 16 February 2006
www.nature.com/tpj
ORIGINAL ARTICLE
Although a putative role has been attributed to inflammation in the
pathogenesis of depressive disorders, the relationship of prostaglandins,
known mediators of inflammation, and depression has not been elucidated.
Clomipramine is an antidepressive drug with a pro-depressive paradoxical
effect in adult rats when administrated neonatally. Using this effect as a
model of depression, we investigated the differential expression of the
cyclooxygenase (COX-2) gene in rat brains. Rats injected neonatally with
clomipramine showed depressive-like symptoms in adulthood, as well as
decreased levels of the brain-derived neurotrophic factor (BDNF) and a
quantitative differential expression of the COX-2 gene (Real Time PCR) and
protein (immunohistochemistry) in the hippocampus. As evidenced, the
relationship between a key enzyme in the prostaglandin synthesis and
biological and behavioral depression-like changes opens an interesting line of
investigation regarding the molecular bases of depression and its potential
treatment through immunomodulatory drugs.
The Pharmacogenomics Journal advance online publication, 28 March 2006;
doi:10.1038/sj.tpj.6500385
Keywords: COX-2; clomipramine; depressive disorders
Introduction
Clinical depression, known as major depression, is one of the most prevalent
mental disorders nowadays. It is one of the most disabling illnesses in the world,1
affecting approximately 17% of United States population.2 Since many families
have a predisposition to depression, a biological vulnerability to depression could
be inherited. External events such as a serious loss, chronic illnesses, difficult
relationships, financial problems, or any other stressor, often seem to initiate an
episode of depression. Very often, a combination of genetic, psychological, and
environmental factors are involved in the onset of a depressive disorder.3
The most acceptable theory to explain depression has been the ‘monoamine
theory’, which proposes a loss or deficits in one or more monoaminergic
neurotransmitters.4 On the other hand, there is great evidence that inflamma-
tion has much to do with depression, and that many pro-inflammatory
molecules and cytokines5 could participate in pro-depression mechanisms.
Moreover, many antidepressants could attenuate the brain expression or actions
of pro-inflammatory cytokines.6,7 Cytokines are believed to bind to glial cells,
which produce cytokines and other mediators such as prostaglandins, particu-
larly prostaglandin E2 (PGE2).8 Regarding prostaglandins, cyclooxygenase-2
 Clomipramine-induced depression and COX-2 expression
P Cassano et al
2
(COX-2), a rate limiting enzyme for prostanoids synthesis, is
induced during inflammation and participates in inflamma-
tion-mediated cytotoxicity9 and neuronal death.10 COX-2,
also modulates inflammation, glutamate-mediated cytotoxi-
city,11 and synaptic plasticity.12,13 However, in spite of the
close relationship between inflammation and prostaglan-
dins and on the other hand inflammation and depression,
the role of COX-2 and prostaglandins was clearly described
not in the clinic, neither in animal models of depression.
In order to elucidate the putative changes in the
expression of the enzyme COX-2, we chose a pharmacolo-
gical model of depression consisting of the neonatal
administration of clomipramine in rats.14 In previous
studies, this model resembled the behavioral and physiolo-
gical abnormalities found in human depression.15
Results
Behavioral tests
All test performed showed either a statistical difference or a
statistical tendency between treated and control animals.
1. Morris water maze test: treated rats spent more time
(Po0.05, ANOVA test) in the water until they found the
platform. This showed that it was more difficult for
treated animals than for controls to learn the location of
the platform.
2. Elevated plus maze: treated animals also showed a
significant difference (Po0.05, ANOVA test) at this test,
more time spent in open arms reflecting less anxiety like
behavior. It is accepted that anxious animals spend more
time in closed arms as a measure of behavior associated
with anxiety and fear.
3. Porsolt forced swim test: treated animals showed beha-
vioral differences during the steps of the test; there were
no differences in immobility (31.8715 vs 39.75717 s,
P ¼ 0.09, ANOVA test), although differences in the
behavior were observed; during the step of mobility,
Figure 1 Assessment of rat weight at the beginning of the treatment (PN8), and after the treatment (PN85). Adult treated animals showed less
body weight than controls. At the beginning of the treatment there were no differences in the body weight. White bar, control animals; gray bar,
treated animals. *Po0.01 (unpaired ‘t’-test).
The Pharmacogenomics Journal
there were no differences between the groups (98.4715
vs 98.2710.4 s); however, during the evaluation of
climbing, there was a clear statistical difference between
the groups (71.4735 vs 47.8719 s, Po0.01, ANOVA test).
4. Sucrose consumption: this test was designed as an
indirect assessment of anhedonia. Treated animals con-
sumed lower levels of sucrose solution (Po0.05, ‘t’-test).
5. Body weight: the weight of clomipramine-treated and
control animals, equal at the beginning of the experi-
ment and during the initial phase of the experiment
(PN ¼ 8–15: P40.05, ‘t’-test), showed significant differ-
ences at the end of the experiment (PN ¼ 85: P40.001,
‘t’-test) (Figure 1), reflecting lower food consumption.
Qualitative and quantitative analysis of behavior
In the treated animals, qualitative behavior such as social
isolation, indifference towards novelty, and lower solid food
consumption (Po0.05, ‘t’-test) were observed. In addition,
quantification of sexual behavior indicated a tendency
toward loss of sexual interest in treated animals (zero
mating), as compared to controls (66% mating; Po0.05,
‘Z’-test). Assessment was carried out four times daily, by
animal facility technicians trained in experimental psychol-
ogy and ethology. Also, the behavior of animals showed
ethological alterations. The cumulative time that treated
rats actively engaged in social behavior was decreased
(approximately 75%) as compared to control animals. In
addition, the socially impaired rats had an increase in the
relative amount of no contact interactions compared with
controls. Also, a decreased locomotor activity of rats was
observed. The analysis of behavior was carried out by animal
facility personnel four times a day for periods of 30 min.
Brain-derived neurotrophic factor quantification
Densitometry band analysis showed a significant (Po0.05,
‘t’-test), decrease in the brain-derived neurotrophic factor
(BDNF) bands of treated animals compared to control
animals (treated 0.85470.071; control 0.97970.071) (Figure

2). This result indicates that BDNF levels were reduced in
treated animals, which is thought to be involved in
depressive disorders as previously described.
Cyclooxygenase expression
Quantitative COX-2 gene expression on different brain
regions was determined by Real Time RT-PCR. The hippo-
campus of treated animals showed significant differences
compared with controls (Figure 3). Cyclooxygenase levels
were normalized using GPS1 and actin genes. Samples from
the neocortex showed a tendency without significant levels
of expression differences.
To confirm that the increase in COX-2 gene expression
was in concordance with that of protein localization, brains
were cut into 4-mm-thick slices and processed for immuhis-
tochemistry. In concordance with molecular biology results,
significant differences in the hippocampus of treated
animals were found (Z ¼ 5.29, Po0.05). Additionally, treated
animal cerebral cortices at the level of the entorhinal cortex
showed a significant number of COX-2 positive cells,
compared to control animals (Z ¼ 9.67, Po0.05) (Figure 4).
Discussion
The pharmacological animal model of depression used here,
previously described in the literature,14–23 was validated by
behavioral tests and by assessing BDNF, a molecule related to
depression.24,25 We studied the expression of the COX-2
gene and the localization of its protein, finding an increase
in COX-2 levels, which was consistent with previous reports
of activation of the immune system in depression disorders.
The easy modulation of COX-2 expression by COX-2
inhibitors makes it an interesting point of study to diminish
brain inflammation processes occurring in depressive peo-
ple. We have preliminary results that shown that anti-
Figure 2 Western blot of brain-derived neurotrophic factor (BDNF)
quantified as optical density of the western blot bands results. BDNF
levels were reduced in treated animals compare to control animals.
White bar, control animals; gray bar, treated animals. Po0.05 (unpaired
‘t’-test).
Clomipramine-induced depression and COX-2 expression
P Cassano et al
3
COX-2 treatment could reverse the behavioral changes
observed in clomipramine treated animals (data not shown).
There is evidence of a decreased serotonin level in adult
animals treated with clomipramine during the neonatal
period.16,26 In addition, there is evidence of behavioral and
monoaminergic mechanism alterations in adult animals
neonatally treated with clomipramine.19
On the other hand there are results that demonstrated
that the depression-like state induced by neonatal exposure
to clomipramine are reverted during the adulthood by
antidepressants drug which enhance the levels of sero-
tonin.27
In the present work, treated animals showed statistical
differences in the performance of behavioral tests. The
Morris water maze is a useful tool to assess the spatial
memory ability of rodents.28 Memory impairment and
depression were closely associated in several animal models
and cognitive processes, such as learning and memory,
which are affected in depression.29 Thus, we used this
classical test to validate our model. Our results indicate that
neonatal chronic exposure to clomipramine induces the
learning deficits and retarded locomotor effects associated
with depression. Recently, the impact of depression on
learning and memory has been directly studied in a novel
animal model of depressive behavior.30
Figure 3 Assessment of COX-2 gene expression by Real Time PCR.
Results are expressed as relative increment (in percentage) over the unit
(treated/control). There is an augmentation of COX-2 expression in
treated animals in hippocampus and entorhinal cortex. Gray bars
represent the mean7s.d. of three simultaneous experiments. (a)
Relative COX-2 mRNA differential expression (b) Relative COX-2 cDNA
quantification. *Po0.01. Analysis of relative gene expression was
performed using the 2DDCt method.
The Pharmacogenomics Journal
 Clomipramine-induced depression and COX-2 expression
P Cassano et al
4
Figure 4 COX-2 protein expression assessed through immunohisto-
chemistry analysis with a polyclonal COX-2 antibody. COX-2 protein
levels were higher in treated rats than in controls. This result is consistent
with those obtained at molecular level expression. (a) Hippocampus; (b)
entorhinal cortex. Results are expressed as the mean7s.d. of cells
counted in six consecutive fields of 100 cells. *Po0.01. (c) Micro-
photograph showing the entorhinal cortex with COX-2 positive cells
(arrows). White bar corresponds to 18 mm. (NiKON eclipse) (coolpix
4500, NIKON, Japan). Analysis was carreid out using the image analyzer
(CAS systems, Raritan, NJ, USA).
The elevated plus maze is widely used to evaluate anxiety-
like behavior in animals. The test is based on unconditioned
responses of animals to a potentially dangerous environ-
The Pharmacogenomics Journal
ment. A combination of maze height, luminosity, and open
space is assumed to induce fear or anxiety like behavior, the
degree of which is assessed by measuring the amount of time
the subject spends in various areas of the maze. Changes in
the percentage of time spent on the open arms indicate
changes in behaviors associated with anxiety. Our results
also validate the model. On the other hand, we have
observed that the neonatal treatment with clomipramine
makes little differences in the first step of a modified version
of Porsolt’s forced swim test. However, the animals showed a
low tolerance to stress in the climbing step of the test. This
behavioral despair could be associated with a depressive-like
state and was recently associated in the same model in mice
with deficit in the noradrenergic system and the modulation
of depression.31
Anhedonia is an important feature associated with the
depressive state. In the present work, we have observed at
least three related behaviors associated with an anhedonia-
like state. Treated animals consumed significantly lower
volumes of sucrose, showed less interest for food consump-
tion and also showed less interest for mating. These results
are an indirect assessment of loss of interest or of pleasure
for daily activities in the treated animals. Although it is hard
to extrapolate these results to clinical depression, we must
recognize that anhedonia32 as evaluated in this kind of
models, could be associated with a depressive like behavior.
The same could be said for the loss of interest in feeding.
In addition to the above behavioral results, we found
biological alterations related with that of depression.
Western blot for the BDNF showed different levels of
expression in the treated animals. Brain-derived neuro-
trophic factor is a member of the structurally and function-
ally homologous neurotrophin family. It is the most widely
distributed trophic factor in the brain and participates in
neuronal growth, maintenance, and use-dependent plasti-
city mechanisms, such as long-term potentiation and
learning. There are several lines of evidence supporting a
role for BDNF in the treatment of depression. Recently, it
was suggested that the BDNF gene could be a susceptibility
gene related in humans with the major depressive disorder25
If, as presumed in previous works, depression could be
related to inflammatory mechanisms,5,6,7,33 it could be of
great interest to study early unspecific inflammation
mediators, such as prostaglandins through its inducible
enzyme, COX-2, which is induced early during acute
inflammation. Functional and structural brain alterations
and unspecific immune responses (among them, COX-2
expression) have already been studied in relation with
neurological entities.34 In this work, we observed an
upregulation of the COX-2 gene in the brain of treated
animals, which coexisted with significant behavioral
changes in the adult life of the rats. Another point of
interest is the anatomical region in which the changes were
demonstrated. Our molecular and histological results
showed a significantly elevated expression of the COX-2
gene and also the protein itself. In addition, despite the fact
that we could not demonstrate a significant neocortex
expression of the COX-2 gene in the treated animals at the

molecular level, it was evident that the protein was
significantly more expressed by immunohistochemistry
than in the control group. The hippocampal expression
differences could be related to structural and biochemical
alterations present in the hippocampus of depressed
patients and in models of depression. Imaging studies have
reported volumetric alterations of the hippocampus. Addi-
tionally, functional alterations have been shown by positron
emission tomography with 18Faltanserin (A2 receptor ligand
analog).35 Moreover, those alterations have been related to
memory and learning alterations present in major depres-
sion patients, and which correlates to the results obtained
from the behavior tests carried out on the depression model
animals. Finally, our results regarding the elevated presence
of the COX-2 protein in the entorhinal cortex may indicate
the existence of a hippocampal-related mechanism. This
region of the brain is related to learning and memory and it
is connected with the hippocampus.
Regarding the potential importance of COX-2 with
inflammatory mechanisms, stress and depressive disorders,
there is some newly interesting evidence. Current biblio-
graphy suggests some association between anti COX-2
inhibitors and the treatment of psychiatric disorders.36 On
the other hand, an interesting work recently presented an
association between COX-2 inhibition and enhancement in
glucocorticoid receptor function in vitro. The relation with
depressive disorders rests in the putative alteration in that
receptor in this kind of affective disorders.37
Finally, to clarify our findings associated to depressive
illness, we should mention that COX-2 differential expres-
sion is almost limited to the hippocampus and entorhinal
cortex, which are the major areas involved in depressive
disorders.38–41
In conclusion, the model here presented resembled many
of the features of the depressive disorder as previously
described. The model also showed an upregulation of the
COX-2 gene during the adult life of the animals. This could
prove the participation of pro-inflammatory molecules,
such as prostaglandins, in the mechanisms associated with
the depressive disorder. The next step could be the
modulation of prostaglandin synthesis through the many
specific drugs currently available.
Materials and methods
Animals and treatment
Experimental procedures on the animals were performed in
compliance with international standards for humane hand-
ling, care and treatment.
Offspring from Wistar rats mated at our institutional
animal facility were born on day 21 of pregnancy (con-
sidered as postnatal day (PN ¼ 0). Rats (n ¼ 48), matched in
sex, age and weigh (P ¼ 0.771, ‘t’-test), were randomly
assigned to one of two groups: (A) Treated (n ¼ 24), were
injected subcutaneously between postnatal days 2 and 14,
twice daily with 15 mg/kg clomipramine (Anafranil R,
Laboratorio Novartis, Buenos Aires, Argentina), (B) Control
Clomipramine-induced depression and COX-2 expression
P Cassano et al
5
(n ¼ 24), were injected subcutaneously between postnatal
days 2 and 14, twice daily with saline. The dose was adjusted
according to previous bibliography.16 Clomipramine treated
animals and controls were weighed daily and food and water
consumption were controlled. On the other hand, the
behavior of the animals was observed in detail by qualified
personnel to complement the objective validation of the
model. As quantification was difficult, qualitative ethologi-
cal observations, mainly related with sociability, were
carried out on a daily basis.42
To validate the model of depression, we considered three
criteria:
1. Objective behavioral tests.
2. A biological change associated with depression.
3. Subjective analysis of daily behavior by qualified person-
nel, as well as daily record of alimentary and sexual
behavior.
1. Behavioral tests: To validate the model of depression,
clomipramine treated and control rats at the age of 90 days
(PN ¼ 90) were subject to a set of behavioral tests previously
used to evaluate depression and anxiety like symptoms and
learning and memory abilities. Tests were performed in a
randomized sequence. Animals were exposed to the elevated
plus-maze test of anxiety;43,44 the Morris water maze to
assess spatial learning and memory;45,46 the sucrose con-
sumption test to evaluate depression-induced anhedo-
nia,32,47 and the Porsolt forced swim test to evaluate
anxiety and depression.48
2. Changes in BDNF: BDNF is a member of the structurally
and functionally homologous neurotrophin family. There
are several lines of evidence supporting a role for BDNF in
the mechanism and treatment of depression.24 In this work,
BDNF protein levels were quantified by Western Blot
technique.
3. Subjective analysis of daily behavior: The behavior of the
treated and control animals were observed daily during
treatment and until they were killed. A careful record of the
alimentary behavior (food and water consumption), social
behavior (isolation, aggressiveness, passiveness), and sexual
behavior (sexual mating) was conducted.
After behavioral tests, treated and control animals were
divided in two groups for molecular analysis and immuno-
histochemistry. Brains were extracted after decapitation
under anesthesia. Hippocampus and entorhinal cortex were
obtained by trained people according to bibliography,49 and
RNA extracted. In another equivalent group, animals were
anesthetized, and perfused through the heart with 4%
paraformaldehide to observe the potential anatomical
localization of COX-2 by immunohistochemistry.
Molecular biology techniques
Real time PCR. Reverse transcription was performed on 10 mg
of total RNA (previously treated with DNasa to eliminate
any DNA contaminate) using 1 mg oligo(dT)15 (Promega,
Madison, WI, USA) and 400 U of Superscript II RNaseH
Reverse Transcriptase (Invitrogen, Buenos Aires, Argentina)
following the manufacturer’s instructions. Then RNA was
The Pharmacogenomics Journal
 Clomipramine-induced depression and COX-2 expression
P Cassano et al
6
degraded using 1 ml RNaseH (Promega). Amplification was
performed using QuantiTect SYBR Green PCR and
QuantiTect SYBR Green RT-PCR kits (in this case 250 ng of
total RNA were used as starting sample) (Qiagen, Valencia,
CA, USA) in an iCycler IQ Real-Time Detection System
(Bio-Rad, Hercules, CA, USA). The protocol used was the
following: 5 min at 941C, 40 cycles of 45 s at 941C, 30 s at the
respective melting temperature (53 or 591C) and 15 s at
721C. In all cases, similar results were obtained when PCR
were performed using RNA or cDNA as sample.
Beta-actin and G protein pathway suppressor 1 (GPS1)
were used as housekeeping genes. The primers were
designed using Primer Premier 5 (Biosoft International, Palo
Alto, CA, USA).
The COX-2 primers were designed according to gene bank
S67722 sequence and were: Forward, 50 -ACTACGCCGAGAT
TCCTGAC-30 , Reverse 50 -CAATGTTCCAGACTCCCTTG-30 .
The beta-actin primers were: Forward 50 -AGCCATGTACG
TAGCCATCCA-30 , Reverse 50 -TCTCCGGAGTCCATCACA
ATG-30 . The GPS1 primers were: Forward 50 - CCTCAG
CAATGCCCTCA-30 , Reverse 50 - CCGCTGCTCAGCAATCT-30 .
Melting curves of all samples were always performed as a
control of specificity.
Analysis of relative gene expression was performed using
the 2DDCt method.50 We compared COX-2 expression to
GPS1 and actin average.
Western blot technique
Hippocampus samples were homogenized in RIPA buffer
(50 mM Tris-HCl pH ¼ 7.4, 150 mM NaCl, 0,5% deoxycholic
acid, 1% Nonidet P-40, 0,1% SDS), containing proteinase
inhibitors (10 ml/ml Protease inhibitor cocktail, Sigma
P8340). After homogenization, the samples were centrifuged
at 10 000 g at 41C for 10 min to remove unbroken cells and
nuclei. To detect BDNF, proteins in the supernatant were
separated by sodium dodecyl sulfate-poliacrylamide gel
electrophoresis (SDS-PAGE) on 8% acrylamide gels as
previously described.51 Proteins were transferred to nitro-
cellulose membranes, blocked in 5% dried milk in TBS buffer
for 1 h, incubated with anti-BDNF antibody (Santa Cruz
Biotechnology, sc-20981, 1: 200) and antiactin antibody
(Sigma A5316, 1:1000) for 1 h at room temperature.
Membranes were washed five times in TBS and incubated
with HRP-conjugated anti-rabbit IgG (Dako code no P 0160,
1/2000) and HRP-conjugated anti-mousse IgG (Dako code
no P0447, 1:1000) for 1 h at room temperature. Immunor-
eactive bands were visualized using the enhanced chemilu-
minescence method (SuperSignal West Pico
Chemiluminescent Substrate, Prod. no 34080). Hepatocyte
homogenate was used as actin positive control and BDNF
negative control. Bands were analyzed using ImageScion
Software.
Immunohistochemistry
Brains were removed, fixed for 24 h in 4% paraformaldehide,
embedded in paraffin and cut in coronal sections of 4 mm
thick. After removing the paraffin, sections were washed in
phosphate-buffer saline (PBS) at pH 7.2. Antigen retrieval
The Pharmacogenomics Journal
was performed with citrate buffer (pH 6.7) in high-power
microwave oven with the following protocol: 15 min at
921C, 15 min at room temperature, 15 min at 921C and 1 h at
room temperature.
Incubation was carried out with goat serum (Protein
Block, BioGenex, San Ramon, USA) for 2 h followed by an
overnight incubation with polyclonal COX-2 antibody
(Cayman Chemical, Ann Arbor, MI, USA, dilution 1/200).
Samples were washed for 15 min in PBS and incubated with
the secondary antibody Multilink ((Laboratorio Biogenex
dilution 1/20) for 2 h. After a 15-min wash in PBS, a final
incubation was done in a Fluorescein-Streptavidin complex
(Vector Lab., Burlangame, USA, dilution 1/50) for 2 h and
visualized with a fluorescence microscope and photo-
graphed using a Nikon Optiphot microscope.
Coronal sections were evaluated according to a rat brain
atlas based on fixed stereotaxic coordinates (The Rat Brain in
Stereotaxic Coordinates – The New Coronal Set by George
Paxinos, Charles Watson, Academic Press; 5th edition,
November 10, 2004). To quantify the results, analysis was
done using an image analyzer (CAS systems, Raritan, NJ,
USA). Quantification was made by triplicate, counting three
random fields and the statistical difference was calculated
using the ‘Z’ proportion test, a ¼ 0.05.
Biostatistical analysis
To assess quantitative differences among treated animals or
controls regarding different variables, a software package
was utilized.52
All molecular biology and immunohistochemistry results
were controlled with adequate positive and negative con-
trols.
Duality of interest
By the present, we declare that we have no duality or conflict of
interest. The present work was financed entirely by the Hospital
Italiano of Buenos Aires, a ‘non profit’ organization.
References
1 Murray CJ, Lopez AD. Alternative projections of mortality and disability
by cause 1990–2020: Global Burden of Disease Study. Lancet 1997;
349: 1498–1504.
2 Kessler RC, McGonagle KA, Zhao S, Nelson CB, Hughes M, Eshleman S
et al. Lifetime and 12-month prevalence of DSM-III-R psychiatric
disorders in the United States. Results from the National Comorbidity
Survey. Arch Gen Psychiatry 1994; 51: 8–19.
3 Caspi A, Sugden K, Moffitt T, Taylor A, Craig I, Harrington H et al.
Influence of life stress on depression: moderation by a polymorphism in
the 5-HT gene. Science 2003; 301: 386–389.
4 Maes M, Meltzer HY. Psycopharmacology. In: Bloom FE, Kupfer DJ
(eds). The Fourth Generation of Progress. Raven Press: New York, 1995 pp
933–944.
5 Connor TJ, Leonor BE. Depression, stress and immunological activation:
the role of cytokines in depressive desorders. Life Sci 1998; 26: 583–606.
6 Leonard BE. The immune system, depression and the action of
antidepressants. Prog Neuropsychopharmacol Biol Psychiatry 2001; 25:
767–780.
7 Hopkins SJ, Rothwell NJ. Cytokines and the nervous system I: expression
and recognition. Trends Neurosci 1995; 18: 83–88.

8 Komaki G, Arimura A, Koves K. Effect of intravenous injection of IL-1
beta on PGE2 levels in several brain areas as determined by
microdialysis. Am J Physiol 1992; 262: 246–251.
9 Nogawa S, Zhang F, Ross ME, Iadecola C. Cyclooxygenase-2 expression
in neurons contributes to ischemic brain damage. J Neurosci 1997; 17:
2746–2755.
10 Kim EJ, Lee JE, Kwon KJ, Lee SH, Moon CH, Baik EJ. Differential roles of
cyclooxygenase isoforms after kainic acid-induced prostaglandin E2
production and neurodegenertation in cortical and hippocampal cell
cultures. Brain Res 2001; 908: 1–9.
11 Pepicelli O, Fedele E, Bonanno G, Raiteri M, Amone-Cat M, Grecco A et
al. In vivo activation of N-methyl-D-aspartate receptors in the rat
hippocampus increases prostaglandin E2 extracellular levels and trigges
lipid peroxidation through cyclooxygenase-mediated mechanisms. J
Neurochem 2002; 81: 1028–1034.
12 Chen C, Jeffery M, Bazan N. Cyclooxygenase-2 regulates prostaglandin
E2 signaling in Hippocampal Long-Term Synaptic Plasticity. J Neuro-
physiol 2002; 87: 2851–2857.
13 Shaw KN, Commins S, O’Mara SM. Deficits in spatial learning and
synaptic plasticity induced by the rapid and competitive broad-
spectrum cyclooxygenase inhibitor ibuprofen are reversed by increasing
endogenous brain-derived neurtrophic factor. Eur J Neurosci 2003; 17:
2438–2446.
14 Vogel G, Neill D, Hagler M, Kors D. A new animal model of endogenous
depression: a summary of present findings. Neurosci Biobehav Rev 1990;
14: 85–91.
15 Vogel G, Hartley P, Neill D, Hagler M, Kors D. Animal depression model
by neonatal clomipramine: reduction of shock induced aggression.
Pharmacol Biochem Behav 1988; 31: 103–106.
16 Feenstra MG, van Galen H, Te Riele PJ, Botterblom MH, Mirmiran M.
Decreased hypothalamic serotonin levels in adult rats treated neonatally
with clomipramine. Pharmacol Biochem Behav 1996; 55: 647–652.
17 Mavanji V, Meti B, Datta S. Sleep-wake effects of meta-chlorophenyl
piperazine and mianserin in the behaviorally depressed rat. Eur J
Pharmacol 2002; 455: 35–41.
18 Neill D, Vogel G, Hagler M, Kors D, Hennessey A. Diminished sexual
activity in a new animal model of endogenous depression. Neurosci
Biobehav Rev 1190; 14: 73–76.
19 Andersen SL, Dumont NL, Teicher MH. Differences in behavior and
monoamine laterality following neonatal clomipramine treatment. Dev
Psychobiol 2002; 41: 50–57.
20 Prathiba J, Kumar KB, Karanth KS. Hyperactivity of hypothalamic
pituitary axis in neonatal clomipramine model of depression. J Neural
Trans 1998; 105: 1335–1339.
21 Bonilla-Jaime H, Retana-Marquez S, Vazquez-Palacios G, Velazquez-
Moctezuma J. Plasma levels of corticosterone and testosterone after
sexual activity in male rats treated neonatally with clomipramine. Behav
Pharmacol 2003; 14: 357–362.
22 Bonilla-Jaime H, Retana-Marquez S, Velazquez-Moctezuma J. Pharma-
cological features of masculine sexual behavior in an animal model of
depression. Pharmacol Biochem Behav 1998; 60: 39–45.
23 Mavanji V, Datta S. Clomipramine treatment in neonatal rats alters the
brain acetylcholinesterase activity in adulthood. Neurosci Lett 2002;
330: 119–121.
24 Angelucci F, Aloe L, Iannitelli A, Gruber SH, Mathe AA. Effect of chronic
olanzapine treatment on nerve growth factor and brain-derived
neurotrophic factor in the rat brain. Eur Neuropsychopharmacol 2005;
15: 311–317.
25 Schumacher J, Jamra RA, Becker T, Ohlraun S, Klopp N, Binder EB et al.
Evidence for a relationship between genetic variants at the brain-
derived neurotrophic factor (BDNF) locus and major depression. Biol
Psychiatry 2005; 58: 307–314.
26 Hansen HH, Mikkelsen JD. Long-term effect on serotonin transporter
mRNA expression of chronic neonatal exposure to a serotonin reuptake
inhibitor. Eur J Pharmacol 1998; 352: 307–315.
27 Vazquez-Palacios G, Bonilla-Jaime H, Velazquez-Moctezuma J. Anti-
depressant effects of nicotine and fluoxetine in an animal model of
depression induced by neonatal treatment with clomipramine. Prog
Neuropsychopharmacol Biol Psychiatry 2005; 29: 39–46.
28 McNamara RK, Skelton RW. The neuropharmacological and neuro-
chemical basis of place learning in the Morris water maze. Brain Res
Brain Res Rev 1993; 18: 33–49.
Clomipramine-induced depression and COX-2 expression
P Cassano et al
7
29 Sun MK, Alon DL. Induced depressive behavior impairs learning and
memory in rats. Neurosience 2004; 129: 129–139.
30 Sun MK, Alkon DL. Related Induced depressive behavior
impairs learning and memory in rats. Neuroscience 2004; 129:
129–139.
31 Schramm NL, McDonald MP, Limbird LE. The alpha (2a)-adrenergic
receptor plays a protective role in mouse behavioral models of
depression and anxiety. J Neurosci 200; 21: 4875–4882.
32 Brennan K, Roberts DCS, Anisman H. Individual differences in sucrose
consumption in the rat: motivational and neurochemical correlates of
hedonia. Psychopharmacology 2001; 157: 269–276.
33 Licinio J, Wong ML. The role of inflammatory mediators in the biology
of major depression: central nervous system cytokines modulate the
biological substrate of depressive symptoms, regulate stress-responsive
systems, and contribute to neurotoxicity and neuroprotection. Mol
Psychiatry 1999; 4: 317–327.
34 Turrin NP, Rivest S. Innate immune reaction in response to seizures:
implications for the neuropathology associated with epilepsy. Neurobiol
Dis 2004; 16: 321–334.
35 Sheline YI, Mittler BL, Mintun MA. The hippocampus and depression.
Eur Psychiatry 2002; 17: 300–305.
36 Muller N, Riedel M, Schwarz MJ. Psychotropic effects of COX-2
inhibitors-a possible new approach for the treatment of psychiatric
disorders. Pharmacopsychiatry 2004; 37: 266–269.
37 Hu F, Wang X, Pace TW, Wu H, Miller AH. Inhibition of COX-2 by
celecoxib enhances glucocorticoid receptor function. Mol Psychiatry
2005; 10: 426–428.
38 Malberg JE, Duman RS. Cell proliferation in adult hippocampus is
decreased by inescapable stress: reversal by fluoxetine treatment.
Neuropsychopharmacology 2003; 28: 1562–1571.
39 Popoli M, Gennarelli M, Racagni G. Modulation of synaptic plasticity by
stress and antidepressants. Bipolar Disord 2002; 4: 166–182.
40 Malberg JE. Implications of adult hippocampal neurogenesis in
antidepressant action. J Psychiatry Neurosci 2004; 29: 196–205.
41 Fuchs E, Czeh B, Kole MH, Michaelis T, Lucassen PJ. Alterations of
neuroplasticity in depression: the hippocampus and beyond. Eur
Neuropsychopharmacol 2004; 14: S481–S490.
42 Lee PR, Brady DL, Shapiro RA, Dorsa DM, Koenig JI. Social interaction
deficits caused by chronic phencyclidine administration are reversed by
oxytocin. Neuropsychopharmacology 2005; 30: 1883–1894.
43 Pellow S, Chopin P, File SE, Briley M. Validation of open: closed arm
entries in an elevated mlus-maze as a measure of anxiety in the rat.
Neurosci Methods 1985; 14: 149–167.
44 Andreani R, Bacellar LF. The relationship between anxiety and
depression in animal models: a study using the forced swimming test
and elevated plus-maze. Braz J Med Biol Res 1999; 32: 1121–1126.
45 Day LB, Schalert T. Anticholinergic effects on acquisition of
place learning in the Morris water task: spatial mapping deficit
or inability to inhibit nonplace strategies? Behav neurosci 1998; 110:
998–1005.
46 Moser MB, Trommald M, Andersen P. An increase in dendritic spine
density on hippocampal CA1 pyramidal cells followin spatial learning in
adult rats suggests the formation of new synapses. Proc Natl Acad Sci
1994; 91: 12673–12675.
47 Arnone M, Maruani J, Chaperon F, Thiebot M, Poncelte M, Soubrie P et
al. Selective inhibition of sucrose and ethanol intake by SR 141416, an
antagonist of central cannabinoid (CB1) receptors. Psychopharmacology
(Berlin) 1997; 132: 104–106.
48 Hansen HH, Sanchez C, Meier E. Neonatal administration
of the selective serotonin reuptake inhibitor Lu 10-134-C
increases forced swimming-induced immobility in adult rats: a putative
animal model of depression? J Pharmacol Exp Ther 1997; 283:
1333–1341.
49 Meredith G, Arbuthnott G. IBRO Handbook Series: Methods in
Neurosciences. Wiley: England, 1993 Volume 16: Morphological Inves-
tigation of Single Neurons in vitro. Wiley.
50 Livak KJ, Schmittgen TD. Analysis of relative gene expression data using
real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.
Methods 2001; 25: 402–408.
51 Ausubel F. Current Protocols in Molecular Biology. Wiley: United States of
America, 1998.
52 Primer of Biostatistics, 5th edn, Stanton Glantz: Mc Graw-Hill, 2002.
The Pharmacogenomics Journal