Post on 27-Aug-2018
JPET #58206 1
Anxiolytic-like Effects of Escitalopram, Citalopram and R-citalopram
in Maternally Separated Mouse Pups
Eric W Fish, Sara Faccidomo, Sandeep Gupta, Klaus A Miczek
Department of Psychology (EWF, SF, KAM)
Department of Psychiatry, Pharmacology, and Neuroscience (KAM)
Tufts University
Medford and Boston, Massachusetts
Department of Pharmacology (SG)
Forest Laboratories
Jersey City, New Jersey
Copyright 2003 by the American Society for Pharmacology and Experimental Therapeutics.
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Running Title: JPET #58206
Correspondence: Klaus A. Miczek
Tufts University
530 Boston Ave. (Bacon Hall)
Medford, MA 02155
e-mail klaus.miczek@tufts.edu
Number of:
Text Pages = 17
Tables = 4
Figures = 3
References = 43; MAXIMUM 40
Words: 3880
Abstract: 251; MAXIMUM 250
Introduction: 765; MAXIMUM 750
Discussion: 1432; MAXIMUM 1500
Non-standard abbreviations:
USVs (ultrasonic vocalizations)
Recommended Section: Behavioral Pharmacology
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ABSTRACT
The S-enantiomer of citalopram, escitalopram, is a selective serotonin reuptake inhibitor
(SSRI) that appears to be responsible for citalopram’s antidepressant and anxiolytic
effects. Clinically, escitalopram is reported to have fewer adverse side effects than do
other SSRIs. This study compared escitalopram to other antidepressants in a pre-clinical
procedure predicting anxiolytic-like effects of drugs. CFW mouse pups (7-days old)
were separated from the dam and maintained at a temperature of 34 °C. Forty five
minutes after administering citalopram (0.56-10mg/kg), escitalopram (0.0056–3 mg/kg),
R-citalopram (1-10mg/kg), paroxetine (0.3-3 mg/kg), fluoxetine (1-30 mg/kg), or
venlafaxine (3-56 mg/kg) subcutaneously, the pups were placed individually on a 19.5 °C
surface for four minutes. Ultrasonic vocalizations (USVs) (30-80 kHz), grid crossing,
rolling (i.e. the pup turned on one side or its back), and colonic temperature were
recorded. All the drugs reduced USV emission; escitalopram was the most potent (ED50
0.05mg/kg), followed by paroxetine (0.17 mg/kg), citalopram (1.2 mg/kg), fluoxetine
(4.3 mg/kg), R-citalopram (6 mg/kg) and venlafaxine (7 mg/kg). The doses that
decreased USVs differed from those that increased motor activity. Increased grid
crossing occurred after low doses of paroxetine (0.03, 0.1 mg/kg) and fluoxetine (1
mg/kg), but only after the highest doses of the citalopram enantiomers and venlafaxine
(0.3, 10, 56 mg/kg, respectively). Except for escitalopram and venlafaxine, high doses of
the treatments increased rolling. R-citalopram caused a 10-fold rightward shift in
escitalopram’s dose-effect curve, suggesting that R-citalopram inhibits escitalopram’s
anxiolytic-like effects. These data support clinical findings that escitalopram is a potent,
well-tolerated SSRI with anxiolytic-like effects.
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Selective serotonin reuptake inhibitors (SSRIs) were initially introduced as anti-
depressants and their potential as anxiolytics has been observed in the treatment of social
phobia, post-traumatic stress disorder (PTSD), and generalized anxiety disorder (GAD)
(e.g., Nutt et al., 1999). While each of the SSRIs increases extracellular serotonin (5-HT)
in the brain by blocking the 5-HT transporter (SERT), they differ substantially in terms of
their selectivity (Nutt et al., 1999; Owens et al., 2001; Sánchez et al., 2003). Actions at
other binding sites, such as the muscarinic, histamine, adrenergic, and 5-HT2 receptors,
may contribute to some of the side effects commonly associated with SSRI treatment.
Though in general, these side effects are better tolerated than those from tricyclics and
benzodiazepines, they are a limitation in the use of SSRIs.
Citalopram and its S-enantiomer, escitalopram (Lexapro® or Cipralex®), are
highly selective inhibitors of the SERT with low or no binding to 144 other receptors
(Owens et al., 2001; Sánchez et al., 2003). Compared to citalopram and its R-enantiomer,
R-citalopram, escitalopram is at least 6-fold less potent in binding to the histamine 1 (H1)
receptor (Owens et al., 2001; Sánchez et al., 2003). Escitalopram and citalopram have
similar affinities for the sigma (σ1) receptors, which are about 2-fold less than those of
R-citalopram. Escitalopram is believed to confer the pharmacological effects of
citalopram (Hyttel et al., 1992; Sánchez et al., 2003) and its effects can be inhibited by
co-administration of R-citalopram (Sánchez, 2003a; Mørk et al., 2003). In clinical
reports, escitalopram has a faster onset of antidepressant effects compared to its racemate
citalopram and has a low incidence of side effects (Montgomery et al., 2001; Burke et al.,
2002; Wade et al., 2002; Waugh and Goa, 2003).
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Preclinical research on the anxiolytic-like effects of SSRIs is variable; some
studies find no effect of SSRIs, while others find decreases or increases in anxiety-like
behaviors. A viable hypothesis for these SSRI effects depends on whether the particular
experimental procedure measures conditioned or unconditioned behaviors particularly if
the SSRI is given acutely (Miczek et al., 1995; Griebel, 1995). One procedure that has
consistently detected an anxiolytic-like effect of acutely administered SSRIs relies on the
vocal responses of young rodents when they are briefly separated from their nest and
their dam. Separated rodent pups emit ultrasonic vocalizations (USVs) in a frequency
range between 30-80kHz that may serve as signals for the dam to initiate retrieval
(Noirot, 1972; Brunelli et al., 1994). The species-generality and unconditioned nature of
vocal reactions distinguish them from many other preclinical measures of anxiety-like
behaviors (Panksepp et al., 1980; Miczek et al., 1995; Sánchez, 2003b).
Among the neurotransmitter systems that influence separation calling, GABA and
5-HT are particularly noteworthy. Anti-anxiety treatments such as benzodiazepines and
5-HT1A agonists decrease calling, whereas benzodiazepine inverse agonists and
pentylenetetrazol can increase calling (Gardner, 1985; Insel et al., 1986; Mos and Olivier,
1989; Winslow and Insel, 1991; Nastiti et al., 1991; Molewijk et al., 1996; Vivian et al.,
1997; Fish et al., 2000). Of the SSRIs, citalopram, fluoxetine, fluvoxamine, and
paroxetine have been reported to reduce distress USVs (Mos and Olivier, 1989; Winslow
and Insel, 1990; Olivier et al., 1998). Similarly, these SSRIs, as well as escitalopram,
reduce the shock-induced USVs of adult rats (Sánchez and Meier, 1997; Schreiber et al.,
1998; Sánchez, 2003b; Sánchez et al., 2003). Unlike the procedure in adult rats, the
separation test can further be used to concurrently assess behavioral specificity, the
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degree to which drug effects on different behaviors can be dissociated. For example,
GABAA positive modulators reduce calling but their effects are often accompanied by
sedation, stimulation, and/or increased motor incoordination (Vivian et al., 1997; Fish et
al., 2000; Rowlett et al., 2001). 5-HT1A receptor agonists reduce both motor activity and
calling at a similar dose-range, while 5-HT1B receptor agonists reduce calling but
stimulate motor activity (Fish et al., 2000). While the doses of citalopram, fluvoxamine,
and fluoxetine that reduce separation distress USVs, have not been reported to affect
motor activity, a high dose of fluoxetine (20mg/kg) was reported to impair the negative
geotaxis response in maternally separated rat pups (Mos and Olivier, 1989)
The objective of the current study was to compare the anxiolytic-like and
locomotor effects of escitalopram to those of other SSRIs and the
serotonergic/noradrenergic reuptake inhibitor, venlafaxine in maternally separated 7-day
old mouse pups. Pups of this age were selected because they emit the most separation
USVs (Noirot, 1972; Fish et al., 2000; Branchi et al., 2001). To further explore the
mechanism through which escitalopram and citalopram reduce separation USVs, the
interaction between escitalopram, R-citalopram, and the H1 receptor antagonist
pyrilamine were investigated.
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Materials and Methods
Animals. CFW mice (N=774, n/treatment included in Table 1 and Table 3), from litters
of 8-12 pups, were seven days old, weighed 3.5-5.0 g and lived with both parents
(Charles River Breeding Labs, Wilmington Ma) in clear polycarbonate cages (28 x 17 x
14cm). Pine bedding covered the cage floor; food (Purina rodent chow, St. Louis, MO)
and water were freely available through a wire lid. The mouse vivarium was 21 + 1°C
with 30-40% humidity, and illuminated for 12 hours (lights on at 730). The “Guide for
the Care and Use of Laboratory Animals”
(http://www.nap.edu/readingroom/books/labrats/) was followed while caring for the mice
and all procedures were approved by Tufts University’s Institutional Care and Use
Committee (IACUC).
Apparatus and Measurements. All behaviors were measured in a sound-attenuated
chamber (49.5 x 38 x 34 cm) in a separate procedure room. The chamber had a one-way
vision window (19 x 16.5 cm) for observation, was lit by red light (10W), and held a
water bath that maintained the temperature of a square aluminum testing surface (23 x 23
cm) at 19.5 + 0.5°C. Two cm squares divided the testing surface into grids used to
measure motor activity. Equipment similar to that previously described (Vivian et al.,
1997; Fish et al., 2000; Rowlett et al., 2001) detected 30-80 kHz sounds. A high
frequency condenser microphone (Brüel and Kjær Model 4135; Nærum, Denmark)
joined to a preamplifier (Brüel and Kjær Model 2633; Nærum, Denmark) was suspended
5 cm from the testing surface. Sounds were amplified (Brüel and Kjær Model 2610;
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Nærum, Denmark) filtered (Krohn-Hite Model 3362; Avon, Ma) to produce a flat range
between 30-80 kHz and then visualized on an oscilloscope (Goldstar 059020A, Cerritos,
CA) that was connected to an analog-to-digital converter GWI-AMP (GW Instruments;
Somerville, MA). The signal was further amplified before it was connected to a
computer (Macintosh II) that ran customized signal detection software. The software
counted an ultrasound if it was 30-80 kHz, lasted longer than 10ms, and was separated
from the previous sound by at least 20 ms.. Body temperature was measured using a
lubricated thermo probe (o.d. 0.7 mm; YSI 555 N034; Yellow Springs Instruments,
Yellow Springs, IN) attached to a tele-thermometer (YSI 2100; Yellow Springs
Instruments, Yellow Springs, IN).
Procedure. The test sessions were conducted between 0730 and 1930 hours and no
differences in baseline USV rates were observed across different times of the day (Fish et
al., 2000). An entire litter of pups and some bedding were removed from the home cage,
transported to the procedure room and maintained in an incubator (11.5 x 14 x 5 cm) at
34+ 1.0°C. Twenty minutes later, the pups were weighed, individually placed in the
testing chamber for 30 seconds to screen for the emission of USVs, and marked for
identification. The pups that vocalized more than 6 times (ca. 80%) were injected with
either one dose of the drug or vehicle. After the injection, the lubricated thermo-probe
was inserted about 7mm into the rectum and held in place until the temperature
measurement stabilized (ca. 3sec). The pups were returned to the incubator for a specific
interval (see drugs section) and a second rectal temperature was taken immediately before
a 4 minute separation test. The signal detection software automatically counted USVs,
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while an experimenter, who was blind to the dose of the drug treatment, manually
counted the number of grid crossings and rolls. A grid crossing was counted when half
of the pup’s body crossed into the next grid and a roll was counted when the pup’s dorsal
surface contacted the testing surface. After the test session, the pups were euthanized by
CO2 inhalation.
Drugs. Citalopram hydrobromide (+) (1-[3-(Dimethylamino)propyl]-1-(4-fluorophenyl)-
1,3-dihydroisobenzofurn-5-carbonitrilmonohydrobromide), R-citalopram oxalate,
escitalopram oxalate (i.e. S-citalopram), paroxetine hydrochloride ((-)-trans-5[4-p-
fluorophenyl-3-piperidylmethoxy)-1,3-benzodioxole), fluoxetine hydrochloride ((+)N-
methyl-γ-[4-(trifluoromethyl)phenoxy]-benzenepropanamine), and venlafaxine (1-2-
[dimethylamino]-1-[4-methoxyphenyl]ethyl cyclohexanol) were provided by Forest
Laboratories (Jersey City, NJ). Pyrilamine maleate (mepyramine) (N-[4-Methoxy-
phenyl]methyl-N’,N’-dimethyl-N-[2-pyridinyl]-1,2-ethanediamine) was purchased from
Sigma Biochemicals (St. Louis, MO). All drugs were dissolved in 0.9% saline and
injected subcutaneously in a volume of 0.1ml/10g body weight. In the interaction
studies, drugs and/or saline were administered in two separate injections. To prevent
leakage, a small amount of glue was applied to the injection site. The separation test was
conducted 45 minutes after drug administration.
Statistics. USV and grid crossing data (transformed into percent of vehicle), rolls and
body temperatures (raw values) were analyzed using one-way between-subjects analysis
of variance (ANOVA). F-values with p<0.05, were followed by post-hoc Dunnett’s tests
to determine which individual doses were significantly different from the vehicle
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treatment. A two-way between-subjects ANOVA, followed by post-hoc Tukey’s
multiple comparison tests analyzed the interaction between escitalopram and pyrilamine.
ED50s (i.e. doses that reduced the total USVs to 50% of the vehicle mean, were estimated
by first order regression of those doses that were between ca. 20 and 80% of the vehicle
mean. r2 values for the linear regression ranged from 0.627 for fluoxetine to 0.996 for R-
citalopram. Non-overlapping 95% confidence intervals (CI) were considered statistically
significant.
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Results
Dose-Effect Studies
USVs. All of the treatments, escitalopram (F(7,92)=15.0;p<0.001; Fig 1. Table 1),
citalopram (F(5,71)=14.5;p<0.001; Fig 1. Table 1) R-Citalopram (F(3,55)=4.8;p=0.005; Fig
1. Table 1), paroxetine (F(6,72)=10.1;p<0.001; Fig 2. Table 1) fluoxetine
(F(4,86)=8.9;p<0.001; Fig 2. Table 1) and venlafaxine (F(4,58)=10.2;p<0.001; Fig 2. Table
1) dose-dependently reduced the number of separation USVs. Pups treated with the 0.3-
0.56 mg/kg doses of escitalopram, the 1-10 mg/kg doses of citalopram, the 3-10 mg/kg
doses of R-citalopram, 0.1-3 mg/kg doses of paroxetine, the 3-30 mg/kg doses of
fluoxetine, or the 3-56 mg/kg doses of venlafaxine vocalized significantly less than did
the pups treated with vehicle. The histamine H1 receptor antagonist pyrilamine
(F(5,42)=4.8;p=0.001; Table 1) also dose-dependently reduced separation USV. Pups
treated with the 3-30 mg/kg doses of pyrilamine vocalized significantly less than did the
pups treated with vehicle (p<0.05). The ED50s are shown in Table 4.
Insert Figures 1. and 2., Tables 1 and 4 about here
Grid Crossing. With the exception of citalopram, the treatments also dose-dependently
increased grid crossing: escitalopram (F(7,92)=3.1;p=0.006; Table 2); R-Citalopram
(F(3,55)=3.7;p=0.017; Table 2); paroxetine (F(6,72)=4.2;p=0.001; Table 2); fluoxetine
(F(4,76)=3.9;p=0.006; Table 2); venlafaxine (F(4,58)=2.6;p=0.04; Table 2). Pups treated
with the 0.3 and 0.56 mg/kg doses of escitalopram, the 10 mg/kg dose of R-citalopram,
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the 0.03 and 0.1 mg/kg doses of paroxetine, the 1 and 3 mg/kg doses of fluoxetine, or the
56 mg/kg dose of venlafaxine crossed significantly more grids than did the pups treated
with vehicle. Pyrilamine (F(5,42)=3.8;p=0.006; Table 2) also increased grid crossings,
significantly at the 10 mg/kg dose.
Insert Table 2 about here
Rolling. With the exception of escitalopram and venlafaxine, the treatments, citalopram
(F(5,72)=9.3;p<0.001; Table 2), R-Citalopram (F(3,55)=6.1;p=0.001; Table 2), paroxetine,
(F(6,72)=2.6;p=0.02; Table 2) fluoxetine (F(4,76)=2.8;p=0.03; Table 2) dose-dependently
increased rolling. Pups treated with the 1-10 mg/kg doses of citalopram, the 10 mg/kg
dose of R-citalopram, the 3 mg/kg dose of paroxetine, the 30 mg/kg dose of fluoxetine
rolled significantly more than did the pups administered vehicle. Pyrilamine did not
significantly affect rolling.
Body Temperature. None of the drugs, at the doses tested, significantly affected body
temperature measured immediately before the separation test (data not shown).
Interaction Studies
USVs. Escitalopram’s dose-dependent reduction of separation USVs was confirmed
when it was co-administered with either vehicle or pyrilamine (1 or 10 mg/kg)
(F(3,127)=12.0;p<0.001; Table 3). Pups treated with the 0.1 and 0.3 mg/kg doses of
escitalopram vocalized significantly less than did pups treated with vehicle alone.
Pyrilamine did not alter the effects of escitalopram on separation USVs. R-citalopram
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altered the effects of escitalopram (F(3,115)=4.6;p=0.005; Fig 3. Table 3), preventing the
reduction of USVs by the 0.1 and 0.3 mg/kg doses of escitalopram. In the presence of R-
citalopram, higher doses (i.e. 1 and 3 mg/kg) of escitalopram were required to reduce
USVs (F(2,45)=16.2;p<0.001). R-citalopram shifted escitalopram’s ED50 to the right from
0.05 (0.02,0.09) to 0.61 (0.1, 1.7) mg/kg.
Insert Figure 3 and Table 3 about here
Grid Crossing. Both escitalopram (F(3,115)=7.1;p<0.001; Table 4) and pyrilamine
(F(2,127)=3.4;p=0.04; Table 3) each significantly increased grid crossings at the 0.1 and 0.3
mg/kg and 1 mg/kg doses respectively. There was no interaction between the effects of
escitalopram and those of pyrilamine, or R-citalopram. When given with R-citalopram,
escitalopram (F(2,45)=4.3;p=0.02; Table 3) significantly increased grid crossing at the 1
mg/kg dose.
Rolling. Neither escitalopram nor pyrilamine significantly affected rolling. However,
there was a trend for an interaction between escitalopram and pyrilamine
(F(6,127)=1.9;p=0.086; Table 3) but not R-citalopram. This was probably due to a decrease
in rolling when the 0.1 mg/kg escitalopram dose was given with the 10 mg/kg pyrilamine
dose. When given with R-citalopram, the higher doses (1 and 3 mg/kg) of escitalopram
(F(2,45)=10.9;p<0.001; Table 3) increased rolling.
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Discussion
Several SSRIs and the selective noradrenergic reuptake inhibitor (SNRI) venlafaxine,
reduced the maternal separation-induced USVs of mice and differed in terms of their
potency and behavioral specificity. These results confirm earlier studies measuring
USVs in neonatal rats after treatment with clinically effective anxiolytic drugs (Gardner,
1985; Mos and Olivier, 1989; Winslow and Insel, 1991; Olivier et al., 1998). The
novelty of the present study is the comparison between the enantiomers of citalopram and
the potent reduction of USVs by its S-enantiomer, escitalopram. The rightward shift in
escitalopram’s dose-effect curve by co-administration of R-citalopram suggests that R-
citalopram inhibits some of the effects of escitalopram and that escitalopram is the active
component of citalopram.
Escitalopram was more potent than citalopram and R-citalopram at reducing
separation USVs, a result that is similar to those from behavioral and in vitro binding
studies (Hyttel et al., 1992; Owens et al., 2001; Burke et al., 2002; Sánchez et al., 2003).
However, the magnitude of the potency difference, about 20 to 125 fold more than
citalopram and R-citalopram, is larger than that predicted from the above studies. Based
on SERT affinity, escitalopram should be about 2 fold more potent than citalopram and
clinically, escitalopram appears to be at least 2-fold more potent than citalopram (Burke
et al., 2002). Development of the 5-HT transporter system could have contributed to
these discrepancies because the number and distribution of transporters substantially
change with age (e.g., Lebrand et al., 1998).
In addition to reducing USVs, all of the drugs altered motor behavior to varying
degrees, as measured by grid crossing and rolling. Escitalopram and venlafaxine were
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the most behaviorally selective in reducing pup USVs (Table 4), although escitalopram
was considerably more potent than venlafaxine (ca. 140 fold). Motor stimulation
occurred only at the highest doses of escitalopram (0.3 and 0.56 mg/kg) and venlafaxine
(56 mg/kg) which were respectively, about 30- and 19-fold greater than the minimally
effective doses (MED) for reducing USVs (Table 4). Unlike the other treatments,
escitalopram and venlafaxine did not increase rolling. Only when very high doses (1 and
3 mg/kg) of escitalopram were given in combination with R-citalopram, did escitalopram
mimic the effects of citalopram and increase rolling. The increased rolling after these
treatments is relatively modest, about half of what was observed after benzodiazepine
treatment (Rowlett et al., 2001). While the lack of increased rolling is consistent with the
clinical observations that escitalopram is well-tolerated (Montgomery et al., 2001; Burke
et al., 2002; Wade et al., 2002; Waugh and Goa, 2003), the clinical relavance of motor
stimulation observed with high doses of escitalopram and venlafaxine, remains to be
determined.
Motor stimulation has also been observed after treatment with 5-HT1B and 5-
HT2A, but not 5-HT1A, receptor agonists in mouse pups (Fish et al., 2000; Fish and
Miczek, unpublished data). Interestingly, the citalopram enantiomers and venlafaxine
showed different dose-effect relationships on motor stimulation than did paroxetine and
fluoxetine. Lower doses of paroxetine and fluoxetine were stimulating and higher doses
were not. In contrast, only the high doses of citalopram enantiomers increased grid
crossing. Similar dose-effect relationships between citalopram, paroxetine, and
fluoxetine have also been observed in adult mice placed in a novel open-field (Brocco et
al., 2002). The different dose-effect curves for the citalopram enantiomers, paroxetine
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and fluoxetine may be because of their actions at muscarinic M1 and 5-HT2C receptors,
respectively (Owens et al., 2001).
One hypothesis for the differences in potency between escitalopram and racemic
citalopram is that R-citalopram inhibits the effects of escitalopram. Co-administration of
R-citalopram reduced escitalopram’s elevation of cortical extracellular 5-HT and
reduction of shock-induced USVs (Sánchez, 2003a; Mørk et al., 2003). The results of the
interaction between escitalopram and R-citalopram in the present study are consistent
with this hypothesis. When given together, R-citalopram caused a 10 fold parallel shift to
the right in the USV reducing effects of escitalopram. There are several mechanisms
through which R-citalopram might inhibit the effects of escitalopram. One possibility is
that R-citalopram’s binding to the H1 receptor alters the effects of escitalopram. To test
this hypothesis, the H1 receptor antagonist pyrilamine was tested alone and in
combination with escitalopram. Pyrilamine (mepyramine) was chosen because it was the
ligand for the H1 receptor in escitalopram binding studies (Owens et al., 2001; Sánchez et
al., 2003). Alone, pyrilamine modestly suppressed calling, as was observed in adult rats
(Sánchez 2003b), and its effects were quite variable. When administered with
escitalopram, there was no evidence for a shift in escitalopram’s dose-effect curve,
indicating that H1 antagonism is an unlikely mechanism for R-citalopram’s inhibition of
escitalopram’s effects. Another hypothesis is that during the interaction between
escitalopram and R-citalopram, escitalopram is metabolized faster leaving higher levels
of the less effective R-citalopram to displace escitalopram from the transporter. However,
levels of escitalopram are not affected by treatment with R-citalopram in the rat brain
(Mørk et al., 2003). A third possibility is that R-citalopram’s inhibition of escitalopram’s
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effects is due to non-competitive binding to a different site on the transporter protein that
conformationally inhibits escitalopram’s binding to the transporter. Future research into
the combined effects of the single enantiomers and determining whether R-citalopram
also inhibits the effects of other SSRIs will help elucidate the mechanisms through which
R-citalopram inhibits the effects of escitalopram.
The acute anxiolytic-like effects of the SSRIs on neonatal vocalizations (Mos and
Olivier, 1989; Winslow and Insel, 1990; Molewijk et al., 1996; Olivier et al., 1998) differ
from their acute effects in humans. In humans, the anxiolytic effects of SSRIs emerge
only after chronic treatment and there are some reports that they may initially increase
anxiety (for review, Nutt et al., 1999). In preclinical studies, the acute effects of SSRIs
can be detected in several procedures that are used for characterizing anxiolytic drugs but
the nature of these effects varies markedly with the experimental procedure (for review,
Griebel, 1995; Borsini et al., 2002). Consistent anxiolytic-like effects of SSRIs occur on
measures of footshock-induced USVs (Sánchez and Meier, 1997; Schreiber et al., 1998;
Sánchez et al., 2003a), burying behavior (Njung’e and Handley 1991), conditioned
freezing (Hashimoto et al., 1996), and increased movement across the electrified grids of
the four plate test (Hascoet et al., 2000). Anxiogenic-like effects of SSRIs have been
observed on light-dark exploratory behavior (Sánchez and Meier, 1997), novelty-
suppressed feeding (Bodnoff et al., 1989), the mouse defensive battery (Griebel, 1995),
social interaction test (File et al., 1999) and the elevated plus maze (File et al., 1999).
Interpreting these contradictory results is difficult and it is clear that novel procedures for
assessing anxiety-like behaviors in animals must be developed. Though escitalopram
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inhibits both neonatal and adult vocalizations and enhances exploratory behavior in light
and dark box, it will be important to extend this assessment to several other procedures.
Procedures measuring vocalizations appear to be particularly sensitive to the acute
anxiolytic-like effects of SSRIs but there are potentially confounding variables to
consider (Winslow and Insel, 1991). Young, developing animals may be differentially
sensitive to drug treatments than are adults because of differences in number of receptors
as well as pharmacokinetics (e.g., Lebrand et al., 1998; Gow et al., 2001). Manipulations
of the 5-HT system in young animals could alter USVs by interfering with its role in
ongoing neural development (e.g., Azmitia, 2001). There is also some evidence that
certain types of vocalizations in rat pups are influenced by variables such as respiration,
thermoregulation, and heart rate (Sokoloff and Blumberg, 1997), particularly after
extended separation from the dam. Different neural microcircuits appear to subserve
different kinds of vocalizations (Jürgens and Pratt, 1979; Covington and Miczek, 2003).
Determining if these circuits are indeed the targets for the currently studied SSRIs could
address whether the present results are significantly influenced by pharmacokinetic,
developmental, or physiological variables.
Escitalopram is currently the most selective SSRI for inhibiting the SERT and its
effects in mouse pups are consistent with its clinical efficacy in treating anxiety.
Although the maternal separation procedure appears to be particularly sensitive to detect
the anxiolytic-like effects of SSRIs, it is important to examine escitalopram in other
animal models of anxiety to assess the extent to which these results are species and age
dependent. As SSRIs emerge as the pharmacotherapy of choice for anxiety disorders,
pre-clinical researchers must continue to develop externally valid procedures for
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assessing their effects in laboratory animals. Future studies may also consider the
whether differences in potency and behavioral selectivity of the SSRIs can be explained
by differential activation of particular pre-, and or post-synaptic 5-HT receptors that
mediate the USV reducing effects of escitalopram and other SSRIs. Furthermore, it will
be interesting to determine the molecular mechanisms underlying R-citalopram's
inhibitory effects on escitalopram's activity.
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Acknowledgements
The authors thank Mr. J Thomas Sopko, Dr Walter Tornatzky, and Daniel Herrewijn for
their technical assistance.
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Footnotes
This research was supported by a grant from Forest Laboratories.
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Legends for Figures
Figure 1. shows the mean number vocalizations/4-min, expressed as percent of vehicle,
by 7-day old mouse pups treated with escitalopram (triangles), citalopram (diamonds) or
R-citalopram (upside down triangles). Vertical lines represent + 1 SEM. Data points
falling between ca. 20-80% of the vehicle are fit with a first order regression line.
Asterisks denote values that are significantly different from vehicle (p<0.05). Non-
transformed values are expressed in Table 1.
Figure 2. shows the mean number vocalizations/4-min, expressed as percent of vehicle,
by 7-day old mouse pups treated with paroxetine (circles), fluoxetine (squares) or
venlafaxine (hexagons). Vertical lines represent + 1 SEM. Data points falling between
ca. 20-80% of the vehicle are fit with a first order regression line. Asterisks denote
values that are significantly different from vehicle (p<0.05). Non-transformed values are
expressed in Table 1.
Figure 3. shows the mean number of vocalizations/4-min, by 7-day old mouse pups
concurrently treated with escitalopram and saline (open triangles, open bar) or
escitalopram and R-citalopram (1.0 mg/kg) (filled triangles, filled bar). Vertical lines
represent + 1 SEM. Data points falling between ca. 20-80% of the vehicle are fit with a
first order regression line. Asterisks denote values that are significantly different from
vehicle (p<0.05). Non-transformed values are expressed in Table 3.
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Table 1. Effects of SSRIs, Venlafaxine and Pyrilamine on Separation Vocalizations
Dose (mg/kg) V 0.056 0.01 0.017 0.03 0.056 0.1 0.3 0.56 1.0 1.7 3 10 30 56
Vocalizations
Escitalopram 323+17 395+46 226+26 261+28 154+27 145+15 55+16 1.3+0.3
Citalopram 306+20 251+36 148+32 113+37 66+18 21+4.5
R-Citalopram 305+32 239+36 180+42 130+28
Paroxetine 331+42 332+29 310+72 142+39 106+33 51+17 16+2
Fluoxetine 368+36 260+39 202+49 86+21 148+33
Venlafaxine 378+23 238+48 179+43 88+27 30+14
Pyrilamine 398+32 314+27 292+61 210+42 193+55 154+45
Mice/treatment
Escitalopram 19 13 15 16 15 11 8 3
Citalopram 15 13 13 14 12 10
R-Citalopram 16 16 13 14
Paroxetine 16 10 9 14 14 10 6
Fluoxetine 16 16 16 17 16
Venlafaxine 11 16 15 12 9
Pyrilamine 11 6 4 8 10 9
The data for vocalizations are expressed as Mean + SEM. Emboldened values are significantly different from Vehicle (p<0.05)
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Table 2. Effects of SSRIs, Venlafaxine, and Pyrilamine on Motor Activity
Dose (mg/kg) V 0.056 0.01 0.017 0.03 0.056 0.1 0.3 0.56 1.0 1.7 3 10 30 56
Grid Crossings
S-Citalopram 33+2.2 38+3.0 39+2.8 36+3.1 40+3.0 42+6.5 49+6.7 64+6.4
S,R-Citalopram 32+5.1 39+3.3 39+4.0 41+6.4 42+5.6 53+7.9
R-Citalopram 26+3.3 39+4.1 39+5.4 47+6.1
Paroxetine 38+4.1 78+9.6 62+9.0 67+6.8 57+5.5 53+6.5 45+5.2
Fluoxetine 28+2.5 56+11 52+5.6 42+4.2 33+3.2
Venlafaxine 27+2.5 36+3.2 44+4.0 46+7.6 50+9.3
Pyrilamine 36+3.7 27+5.2 27+3.8 42+8.0 60+8.2 57+8.9
Rolls
S-Citalopram 3.6+0.7 2.3+1.1 2.4+0.8 3.4+1.0 2.4+0.6 4.5+1.0 6.3+1.8 6.3+ 1.9
S,R-Citalopram 2.9+0.6 4.4+0.9 8.0+1.4 9.1+1.1 11+2.3 14+1.9
R-Citalopram 1.7+0.3 2.6+0.5 1.6+0.4 4.6+ 0.8
Paroxetine 4.1+1.2 5.1+1.4 4.0+1.3 5.0+1.3 5.7+1.4 7.8+2.0 12.7+3.1
Fluoxetine 2.7+0.7 4.7+1.0 3.2+1.0 4.7+1.2 8.7+1.8
Venlafaxine 1.7+0.5 2.8+0.8 2.7+0.7 4.0+0.7 4.1+1.3
Pyrilamine 3.4+1.0 3.8+1.5 3.0+1.6 5.4+1.2 7.2+2.1 5.2+1.7
The data are expressed as Mean + SEM. Emboldened values are significantly different from Vehicle (p<0.05)
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Table 3. The interaction between Escitalopram, R-citalopram and Pyrilamine on Separation Vocalizations and Motor Activity
Escitalopram Dose (mg/kg) V 0.01 0.1 0.3 1 3
Vocalizations
+ Vehicle 352+29 275+36 138+38 54+11
+ R-Citalopram 1.0 285+37 350+39 294+44 213+41 148+34 75+29
+ Pyrilamine 1.0 309+73 247+58 155+46 85+26
+ Pyrilamine 10.0 191+76 175+60 110+46 122+44
Grid Crossings
+ Vehicle 30+3.1 31+3.2 42+3.7 49+6.1
+ R-Citalopram 1.0 31+3.8 37+5.1 48+4.0 47+7.4 43+4.6 41+5.9
+ Pyrilamine 1.0 52+14 50+7.3 49+9.6 48+9.1
+ Pyrilamine 10.0 45+7.7 37+4.8 49+8.3 52+15
Rolls
+ Vehicle 3.5+0.5 3.1+0.6 3.6+0.8 3.5+1.1
+ R-Citalopram 1.0 2.6+0.6 3.5+0.6 3.7+1.0 3.0+1.0 7.5+1.3 10.3+1.8
+ Pyrilamine 1.0 1.0+0.4 2.6+0.9 3.1+1.3 1.9+0.7
+ Pyrilamine 10.0 3.6+1.7 3.7+1.2 0.9+0.4 6.9+2.6
Mice/treatment
+ Vehicle 24 15 15 14
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+ R-Citalopram 1.0 14 13 10 12 15 7
+ Pyrilamine 1.0 7 8 8 8
+ Pyrilamine 10.0 7 9 8 8
The data for vocalizations, grid crossing, and rolling are expressed as Mean + SEM. Emboldened values are significantly different from Vehicle
(p<0.05).
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Table 4. ED50s for USVs, Minimally Effective Doses (MED) for USVs and Motor Activity, and Selectivity Ratios
Dependent Variables USV (ED50 95%CI) USV (MED) Grid Crossing (MED) Rolling (MED) Selectivity Ratio (USV:Motor)
Drug
Escitalopram 0.05 (0.02, 0.1) 0.01 0.3 ND 1:30
Paroxetine 0.17 (0.03, 0.4) .1 0.03 3 1:0.3
Citalopram 1.2 (0.6, 2.0) 1 ND 1 1:1
Fluoxetine 4.3 (0.4, 18.9) 3 1 30 1:0.3
R-Citalopram 6.0 (1.2, 19.1) 3 10 10 1:3
Venlafaxine 7.0 (1.1, 18.9) 3 56 ND 1:19
Selectivity ratio compares the MED for USVs to the lowest MED for either grid crossing or rolling. ND (not determined) means that no dose of the drug
significantly affected the behavior.
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