Chronic valproate normalizes behavior in mice overexpressing calcineurin
Claire J. Herzoga, Stéphanie Miota, Isabelle M. Mansuyb, Bruno Girosa, Eleni T. Tzavaraa,⁎
aINSERM U-513, 8 rue de Général Sarrail, 94000, Créteil, France
bBrain Research Institute, University of Zürich and Swiss Federal Institute of Technology, Zürich, Switzerland
Received 16 July 2007; received in revised form 12 October 2007; accepted 18 October 2007
Available online 25 October 2007
Calcineurin (PP2B) is a Ca2+-dependent protein phosphatase enriched in the brain that takes part in intracellular signaling pathways regulating
synaptic plasticity and complex brain functions. We report here that when these pathways are activated by transgenic expression of calcineurin,
locomotor activity of mice in response to novelty is increased, as well as the behavioral and molecular responses of the psychostimulant cocaine.
We also observed that the anxious-like behavior is altered. These behavioral changes are indicative of a generally increased behavioral
responsiveness and could be normalized by chronic treatment with the mood stabilizer valproate. These results provide proof of concept that
calcineurin-dependent dephosphorylation plays an important role in behavioral reactivity and in the effects of mood regulators. Mice
overexpressing calcineurin represent a novel tool to study affective responses related to psychiatric disorders.
© 2007 Elsevier B.V. All rights reserved.
Keywords: Calcineurin; Cocaine; Valproate; Behavioral reactivity; Affective disorders; Mood stabilizer
There is an increasing shift from single neurotransmitter-
based theories towards global network approaches, advocating
an important role of signal-transduction pathways in multifac-
eted patterns of behavior and in the pathophysiology of psy-
chiatric disorders. The underlying hypothesis is that psychiatric
disorders have too complex a semiology to be attributed to a
single neurotransmitter system dysregulation. Signaling path-
ways, on the other hand, broadly affect neuronal networks by
dynamically regulating the activity of multiple neurotransmitter
systems, as well as processes such as synaptic activity, receptor
desensitization, cell survival, neuroplasticity, and cellular resi-
lience. Minor variations in ubiquitous regulators of signaling
pathways can affect complex functions (Charney and Manji,
2004) and the same second messenger system can be implicated
in seemingly unrelated brain functions. For example calcium-
stimulated-adenylyl-cyclase-dependent signal-transduction has
been involved in drug dependence (Valverde et al., 1996;
Tzavara et al., 2002), in circadian rhythms (Tzavara et al., 1996)
and in learning and memory (Xia and Storm, 2005); all of which
can be affected during the life-time of an individual suffering
from schizophrenia or mood disorder. It has been suggested that
category per se but with more specific aspects of the phenotype,
such as affective symptoms and cognitive effects, which cross
traditional psychiatric diagnostic boundaries (Craddock et al.,
A central feature in signal-transduction is the dynamic bal-
ance between protein kinases and protein phosphatases that is
essential for synaptic plasticity. Recent evidence shows that the
expression and activity of various kinases/phosphatases or their
downstream targets are modified (i) following psychoactive
drug administration (Ron and Jurd, 2005), (ii) in animal models
of psychiatric disorders (Angelucci et al., 2005) and (iii) in post-
mortem samples from psychiatric patients (Albert et al., 2002).
For instance, the protein kinase DARPP-32 (cAMP-regulated-
phosphoprotein-of-Mr32,000) mediates effects of common
psychotomimetics (Svenningsson et al., 2003) as well as
antidepressants (Svenningsson et al., 2002). In the balance of
kinase/phosphatase activity, the phosphatase calcineurin acts as
a major negative regulator of protein phosphorylation. Interest-
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European Journal of Pharmacology 580 (2008) 153–160
⁎Corresponding author. Tel.: +33 1 49813572; fax: +33 1 49813685.
E-mail address: firstname.lastname@example.org (E.T. Tzavara).
0014-2999/$ - see front matter © 2007 Elsevier B.V. All rights reserved.
ingly, mice in which calcineurin activity is increased experi-
mentally by expression of an active form in the mouse forebrain
(CN98 mice), show impaired synaptic plasticity (Winder et al.,
1998). Using this transgenic mouse model, we recently showed
that calcineurin is involved in the mechanisms of action of
antidepressants (Crozatier et al., 2007).
Here we have assessed affective-like responses in CN98
mice. Mice were subjected to an open field (actimeter), elevated
plus-maze and zero-maze test for the assessment of their be-
havioral reactions to novelty and stress. Since psychostimulant
states in humans, we also assessed the effects of acute cocaine
administration on locomotor activity in CN98 mice. In parallel
we have measured c-fos expression upon acute cocaine in-
jection. The expression of this immediate early gene is a marker
of neuronal activation and is used widely to evaluate neuronal
Steiner and Gerfen, 1993; Svenningsson et al., 2003). CN98
mice showed enhanced locomotor activity in response to nov-
elty, reduced anxiety-like behavior on the elevated plus- and
zero-mazes, and increased sensitivity to the locomotor stimulant
actions of cocaine, accompanied by elevated c-fos expression in
the cortex, striatum, and nucleus accumbens after acute cocaine
injection. The increased behavioral reactivity that we observed
in CN98 mice as compared to their wild type (WT) littermates,
was reminiscent of the altered behaviors observed in animal
models of emotional lability, mania or bipolar disorder (Gessa
et al., 1995). Therefore, we evaluated weather the anticonvul-
sant/mood regulator valproate, a drug widely used in the clinical
treatment of patients, was able to reverse the altered behavioral
pattern of the CN98 mice.
2. Materials and methods
The CN98 transgenic mice that express a constitutively
active form of calcineurin Aα subunit (Winder et al., 1998)
were used in this study and their WT littermates were used as
controls. This transgene is driven by the CAMKII (Ca2+/
calmodulin-dependent-protein-kinase) promoter, that restricts
its expression to the forebrain and throughout the hippocampus
and dentate gyrus (Winder et al., 1998). I. Mansuy provided
heterozygous couples and a colony was established in our
laboratory (Crozatier et al., 2007); breeding was maintained on
a C57/Bl6 background in standard laboratory conditions (12-
h light/dark cycle with light on at 07:30; room temperature 21±
1 °C, food and water ad libitum). All experiments were carried
out in accordance with the European Communities Council
Directive (86/809/EEC) and approved by the local ethical
committee. Behavioral tests were performed by an experimenter
blinded to genotype [wild type (WT) or transgenic (CN98)
mice] and treatment. Except when effects of habituation were
assessed, each mouse was tested only once, and for each
experiment, and the numbers of animals utilized were 8–12 per
experimental group, for both behavioral and in situ hybridiza-
Drugs, purchased from Sigma (L'Isle d'Abeau Chesnes,
France), were dissolved in 0.9%NaCl and injected intraperito-
neally (10 ml/kg). Valproate (200 mg/kg; i.p.) was administered
chronically, once daily for 21 days. Cocaine (5, 15, 30 or 45 mg/
kg; i.p.) was injected acutely and locomotor activity was regis-
tered immediately after.
2.3. Behavioral tests
2.3.1. Locomotor activity
connected by an interface to a computer (Imetronic, Bordeaux,
France) and located in a sound-attenuated experimental room.
Ambulations (horizontal locomotion) were defined as the num-
ber of photocell beams (located 15 mm above the floor) broken
per unit of time.
a. Locomotor activity in response to novelty: Reactivity to
novelty was examined by placing naive mice, never exposed
to the apparatus, in the box and measuring horizontal loco-
motor activity for 30 min. The same mice were used the
following day to measure locomotor activity under habitu-
b. Locomotor activity in habituated animals: The aforemen-
tioned mice were placed in the apparatus for 20 min (habit-
uation period) and then locomotor activity was measured for
c. Locomotor activity in response to cocaine: Another group of
mice was used for pharmacological experiments. These mice
were habituated to the apparatus for 30 min the day before
the experiment. The day of the experiment mice were placed
in the apparatus, cocaine or drugs were injected after a
20 min habituation period and locomotor activity measured
for an additional 30 min period.
2.3.2. Elevated plus-maze test
The maze (Pellow and File, 1986; Linden et al., 2004)
consisted of two open arms (37.5×5×0.3 cm) and two closed
arms (37.5×5×15 cm) linked by a central platform (5×5 cm)
and positioned 63 cm above the ground.
On the test day, animals were transferred to the testing room
in their home cages and allowed to acclimate for at least 1 h
prior to testing. A test session began by placing a mouse on the
central platform facing the open arm to allow free exploration of
the maze for 5 min. An arm entry was recorded when all four
legs on the mouse were on the arm. The total amount of entries
(in closed and open arms) was used to determine the locomotor
activity of each tested mouse. After each test session the maze
was cleaned before placing the next mouse.
2.3.3. Zero-maze test
The maze (Shepherd et al., 1994) consisted of a 7 cm wide
ring (external diameter 47 cm) divided into four equal parts of
alternating closed and open sections and positioned 50 cm
above the ground. On the test day, animals were transferred to
154C.J. Herzog et al. / European Journal of Pharmacology 580 (2008) 153–160
the testing room in their home cages and allowed to acclimate
for at least 1 h prior to testing. Mice were individually placed in
one of the closed compartments and their ambulatory behaviors
were recorded with a with a video-track system. The time spent
in the open compartment during the 5 min of the test session
was measured. The total time each mouse was engaged in
locomotor activity (in the closed and open compartments) was
calculated as in index of locomotor activity. After each test
session the maze was cleaned before placing the next mouse.
Each mouse was tested in the plus-maze or the open maze
only once. Mice tested in the plus-maze (or the zero-maze) were
either naïve to all manipulation or were previously subjected to
a single actimeter session. With completely naïve mice or mice
already subjected to a single actimeter session we observed
identical results in the elevated plus-maze and zero-maze.
2.4. In situ hybridization
Fifteen minutes after cocaine injection (15 mg/kg), mice were
sacrificed by decapitation and brains quickly removed and
frozen. Coronal sections were taken at −20 °C and mounted on
glass slides. c-fos specific antisense oligonucleotides (5′-GTT
GAC AGG AGA GCC CAT GCT GGA GAA GGA GTC GGC
TGG GGA ATG -3′) were labeled with [35S]dATP, using term-
inal transferase (Amersham Biosciences), to a specific activity of
5×10−8dpm/μg. Sections were covered with 100 μl of a solu-
tion containing 50% hybridization solution (Amersham Bios-
ciences), 40% deionized formamide (Merck Eurolab, Strasbourg,
dithiothreitol, and 3–5×10−5dpm of each labeled oligonucleo-
tide. The samples were incubated overnight at 42 °C, washed in
saline sodium citrate, followed by PBS, and exposed to X-ray
films (Biomax; Eastman Kodak, Rochester, NY) for 21 days.
2.5. Data analysis
Data represent mean±S.E.M. of the indicated number of
Fig. 1. Locomotor activity in mice overexpressing calcineurin. (A) Locomotor
activity in response to novelty is increased in CN98 mice. Horizontal locomotor
activity(ambulations)for WT(grey bars)andCN98(white bars) miceplaced for
the first time in an actimeter. n=8–10 animals per group. *Pb0.05 for WT
versus CN98 animals. (B) No difference in locomotor activity in habituated
CN98 and WT mice. Horizontal locomotor activity (ambulations) for WT (grey
bars) and CN98 (white bars) mice habituated to the actimeter. n=8–10 animals
per group. *Pb0.05 for WT versus CN98 animals.
activity (i.e. time active in all compartments) (D) for WT (grey bars) and CN98 (white bars) mice. n=8–12 animals per group. *Pb0.05 for WT versus CN98 animals.
155C.J. Herzog et al. / European Journal of Pharmacology 580 (2008) 153–160
way ANOVA, followed by Duncan's post-hoc test when appro-
priate. All analyses were run with the Statistica Software.
We evaluated locomotion in CN98 upon exposure to novelty.
When mice were introduced in a novel environment (first ex-
posure to the actimeter cage) we observed a higher exploratory
activity in CN98 than WT mice (F(1,15)=5.37, Pb0.05; one-
way ANOVA, Fig. 1A). This was however no longer observed
upon subsequent exposure to the actimeter (F(1,15)=0.95; n.s.;
Fig. 1B), indicating a selective hyperlocomotor effect of novelty
in CN98 and not a general increase in locomotor activity.
Behavioral inhibition and anxiety-like behaviors were
investigated in the elevated plus-maze and zero-maze tests
(Ognibene et al., 2005; Olausson et al., 2001; Zorner et al.,
2003). In the plus-maze, CN98 mice showed reduced anxiety
and a behavioral disinhibition expressed as an increase in the
time spent in open arms compared to WT mice (F(1,22)=4.56
Pb0.05, one-way ANOVA, Fig. 2A). Total entries (i.e. the sum
of entries in the open and closed arms) were not different bet-
ween CN98 and WT mice (F(1,22)=0.7 n.s., one-way ANOVA,
Fig. 2B). Similarly in the zero-maze, CN98 mice spent sig-
nificantly more time in the bright (open) compartment than did
WT mice (F(1,7)=6.22, Pb0.05, one-way ANOVA, Fig. 2C)
and engaged in more exploratory activity in this compartment
Fig. 3. Response to cocaine is increased in CN98 mice. (A) Cocaine-induced hyperactivity is increased in CN98 mice. Horizontal locomotor activity (ambulations)
measured in an actimeter after an acute injection of vehicle or 5, 15, 30 or 45-mg/kg cocaine in WT (grey bars) and CN98 (white bars) mice. n=8–12 animals per
group. *Pb0.05 for animals treated with cocaine versus animals of the same genotype treated with vehicle,#Pb0.05 for WT versus CN98 animals under the same
treatment. (B) C-fos expression in response to cocaine is increased in CN98 mice. Expression of the immediate early gene c-fos in striatum, nucleus accumbens, and
frontal cortex after administration of vehicle or 15 mg/kg cocaine in WT (grey bars) or CN98 (white bars) mice. n=8–12 animals per group. *Pb0.05 for animals
treated with cocaine versus animals of the same genotype treated with vehicle,#Pb0.05 for WT versus CN98 animals under the same treatment.
156C.J. Herzog et al. / European Journal of Pharmacology 580 (2008) 153–160
(F(1,7)=7.18, Pb0.05, not shown). Total time engaged in
locomotor activity (i.e. the sum of activity in all compartments)
was not different between CN98 and WT mice (F(1,7)=1.7,
n.s., one-way ANOVA, Fig. 2D). It should be noted that unlike
the actimeter experiment, locomotor activity, as reflected by
total arm entries, in the plus-maze and zero-maze did not differ
between CN98 and WT mice. However, as seen in the literature
when testing genetically modified mice or when testing
pharmacological effects of compounds in anxiety and locomo-
tion tests, total arm entries in the plus-maze do not always
coincide with conventional measures of locomotion (ambula-
tions) in activity tests. Total arm entries in the plus-maze
sometimes fail to picture changes in locomotor activity that are
observed with an open field or an actimeter (see for instance
Kuzmin et al., 2006; Ferguson et al., 2004).
When injected with cocaine (5, 15, 30 or 45 mg/kg), CN98
mice habituated to the actimeter exhibited a significantly higher
hyperlocomotion than WT mice (effect of treatment F(4,61)=
15.09, Pb0.001 and genotype F(1,61)=5.19, Pb0.05, two-
way ANOVA). At the 45 mg/kg dose appearance of massive
stereotypic movements parasitized locomotor activity and in-
terfered with horizontal locomotion measurements. When we
analyzed the effects of cocaine in CN98 and WT mice we
show a significant interaction between genotype and treatment
(F(3,53)=2.81; Pb0.05, two-way ANOVA). Subsequent post-
hoc analysis shows that while in WT mice cocaine had no effect
on locomotion at 5 mg/kg, it did increase activity by 100% in
CN98 mice. At 15 and 30 mg/kg, cocaine increased locomotion
in both groups, but this increase was more pronounced in CN98
than WT animals (Fig. 3A).
In another set of CN98 and WT mice, c-fos mRNA
expression was examined in the striatum, nucleus accumbens,
and frontal cortex, major targets of dopaminergic inputs after a
single cocaine injection (15 mg/kg). Interestingly, both the
pattern and extent of c-fos increase were different in CN98 as
compared to WT mice. In WT mice, c-fos mRNA expression
was increased only in the striatum and nucleus accumbens
(47%±11 and 44%±10 respectively). In CN98 mice, it was
increased in the striatum, nucleus accumbens, but also in the
frontal cortex; the increase was significantly higher in all struc-
tures (striatum 159%±8, F(1,71)=37 Pb0.001, nucleus
accumbens, 83%±8, F(1,77)=4.3 Pb0.05, frontal cortex
81%±13, F(1,54)=10.91 Pb0.01, two-way ANOVA for geno-
type and treatment) (Fig. 3B).
Finally, we investigated whether the mood stabilizer val-
proate, a drug widely used in the clinical treatment of patients,
could reverse behavioral hyperactivity observed (i) upon first
exposure to an actimeter, (ii) after a single injection of cocaine
or (iii) after exposure to the elevated plus-maze in CN98 mice.
Chronic valproate treatment prevented the increase in locomo-
tor activity induced by novelty (Fig. 4A; genotype × chronic
treatment (valproate or vehicle) interaction F(1,46)=4.4, two-
way ANOVA). Chronic valproate also prevented cocaine-in-
duced hyperlocomotion in CN98 mice (significant genotype ×
chronic treatment (valproate or vehicle) × acute treatment (co-
caine or saline) interaction F(1,74)=4.57, Pb0.05, three-way
ANOVA). Chronic vehicle treatment had no such effect and
cocaine-induced hyperlocomotion was still markedly enhanced
in CN98 animals when compared to WT (Pb0.001) (Fig. 4B).
In the elevated plus-maze, chronic valproate treatment normal-
ized the time CN98 mice spent in open arms (genotype ×
chronic treatment (valproate or vehicle) interaction F(1,35)=5
Pb0.05, two-way ANOVA) (Fig. 4C). In addition, valproate
treated CN98 mice made fewer entries in the open arms of
the elevated plus-maze as compared to vehicle treated CN98
mice (11±1 entries for vehicle treated versus 6±1 for valproate
treated CN98 mice).
Fig. 4. The mood regulator valproate normalizes behavior in CN98 mice.
(A) Horizontal locomotion (ambulations) upon a first exposure to the actimeter,
for WT (grey bars) and CN98 mice (white bars) pre-treated chronically with
vehicle (v) or valproate (val). n=8–12 animals per group. *Pb0.05 for CN98
versus WT animals;#Pb0.05 for animals treated chronically with valproate
versus animals of the same genotype treated with vehicle, (B) locomotor activity
in response to an acute cocaine injection (15 mg/kg), for WT (grey bars) and
CN98 mice (white bars) pre-treated chronically with vehicle (v) or valproate
(val). n=8–12 animals per group.+Pb0.05 for animals treated acutely with
cocaine versus animals of the same genotype and pretreatment treated acutely
animals of the same genotype and acute treatment that were treated chronically
with vehicle, (C) total time spent in the open arms of the plus-maze, for WT
(grey bars) and CN98 mice (white bars) pre-treated chronically with vehicle (v)
or valproate (val). n=8–12 animals per group. *Pb0.05 for CN98 versus WT
animals;#Pb0.05 for animals treated chronically with valproate versus animals
of the same genotype treated with vehicle.
#Pb0.05 for animals treated chronically with valproate versus
157C.J. Herzog et al. / European Journal of Pharmacology 580 (2008) 153–160
The aim of this study was to assess affective-like responses
in CN98 mice, assess their behavioral and c-fos response to an
acute cocaine challenge, and evaluate whether chronic
valproate treatment can reverse the altered behaviors of
CN98 mice. CN98 mice displayed a stronger locomotor
response to novelty and spent more time in the open arms of
the elevated plus-maze and zero-maze than WT mice did.
They also expressed increased locomotor response to cocaine
injection, accompanied by an altered magnitude and pattern of
c-fos mRNA expression in response to cocaine. We also show
that chronic valproate treatment attenuated the hyperrespon-
siveness of CN98 mice to novelty and cocaine injection, and
reduced the time spent in open arms of the plus-maze. Thus,
towards a comprehensive assessment of behavioral reactivity
and disinhibition, we suggest that CN98 mice display a
context dependent hypersensitivity that is normalized by the
mood regulator valproate.
Increased activity upon exposure to novelty, and reduced
anxiety-like behaviors could be relevant to behavioral
disinhibition, namely reduced neophobia, increased risk-taking
behavior and augmented agitation in response to contextual
changes. Such a behavioral pattern has been described in
putative genetic models of affective disorders (Roybal et al.,
2007; Rozeboom et al., 2007; White et al., 2007). In CN98
mice acute administration of cocaine had more pronounced
stimulatory effects on c-fos expression in the dorsal and ventral
striatum, as well as in the cortex. In this later region cocaine
induced c-fos expression only in CN98 mice. The behavioral
response to cocaine was also increased in these mice. The
parallel increases in c-fos expression and locomotor activity
that we observed are in agreement with previous studies
having established c-fos as a reliable marker of neuronal
activation in response to psychostimulants (Svenningsson et
al., 2003). Psychostimulant induced hyperlocomotion in
rodents is thought to reflect manic state in humans, and
enhanced biochemical and behavioral responsiveness to
psychostimulants is at the basis of pharmacological models
of mania (Lenox et al., 2002; Machado-Vieira et al., 2004).
In our study, the behavioral supersensitivity of the CN98
mice was reversed by chronic treatment with valproate, an anti-
regulators, (lithium or the anticonvulsants valproate and car-
bamazepine) are first line treatments for affective disorders. In
animal models of bipolar disorder, mood regulators reduce
psychostimulant or novelty induced hyperlocomotion (Arban
et al., 2005;Shaldubina et al., 2002). Interestingly, it has been
proposed that mood regulators could act by resetting intracel-
lular kinase/phosphatase equilibrium (Du et al., 2004). The fact
that valproate reduced cocaine hypersensitivity in CN98 and
normalized their emotional reactivity to novelty further impli-
action of mood regulators and in the pathophysiology of af-
Our results confirm and extend previous studies suggest-
ing that calcineurin dysfunction could be involved in
molecular networks associated with response to stress,
memory of aversive experiences (Lin et al., 2003),
antidepressant mechanism of action (Crozatier et al., 2007)
and psychotic behavior (Miyakawa et al., 2003). It was shown
that mice invalidated for calcineurin show increased basal
locomotor activity. Our results may seem surprising in the light
of this study and of previous reports showing that PKA
activation (which is antagonistic to calcineurin activation) me-
diates behavioral and biochemical effects of psychostimulants
(Svenningsson et al., 2003). Phosphorylation/dephosphoryla-
tion dependent cascades show bidirectional plasticity (Bhalla et
al., 2002). For instance it is well known that calcineurin
dephosphorylates downstream targets of PKA and therefore
calcineurin overexpression is predicted to inhibit cyclic AMP/
PKA signaling. However the recent discovery of calcineurin
activated adenylyl cyclase in the hippocampus (Chan et al.,
2005) establishes a putative mechanism by which calcineurin
overexpression may increase cyclic AMP signaling. Because of
such bidirectional plasticity signal-transduction-cascades were
proposed as key mediators in affective disorders, in which both
positive and negative affective responses can coexist (Manji et
al., 2003). It is also important to note that the calcineurin
knockout mice utilized by Miyakawa and collaborators
(Miyakawa et al., 2003; Zeng et al., 2001) are forebrain specific
knockout of the CNB1 subunit of calcineurin, which is the
regulatory subunit of calcineurin in the brain. In our model, the
CN98 mouse over-express CNAa, the catalytic subunit of cal-
cineurin. Even if the regulatory and catalytic calcineurin subunits
by Miyakawa and collaborators and the CN98 mice that we use
here cannot totally be considered as mirror models. Finally it
should be noted that such apparently contradictory results were
memory paradigms. Thus, in CN98 mice, the expression of an
plasticity, learning and memory (Mansuy et al., 1998a,b: Winder
enhances synaptic plasticity and cognitive functions in the mouse
(Malleret et al., 2001). However, full inhibition by pharmacolog-
In conclusion, congruent with recent studies that relate cal-
cineurin and its inter-player DARPP-32 to psychosis in animal
models (Miyakawa et al., 2003) and in humans (Gerber et al.,
2003), our results suggest that genetic variations of calcineurin
could predispose to a higher responsiveness to stimuli and could
be related to the greater vulnerability to environmental situa-
tions that more easily trigger a pathological emotional state in
patients that suffer from affective disorders. This study provides
proof that manipulation of calcineurin, a candidate signal-trans-
duction protein for affective disorders, results in relevant be-
havioral changes, which are corrected by the mood regulator
valproate. CN98 mice might be helpful for the investigation of
neurobiological substrates underlying the vulnerability to af-
fective disorders and the identification of novel therapeutic
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