Anxiolytic-Like Effects of Escitalopram, Citalopram, and R- Citalopram in Maternally Separated Mouse Pups
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0022-3565/04/3082-474 –480$20.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 308, No. 2
Copyright © 2004 by The American Society for Pharmacology and Experimental Therapeutics 58206/1120858
JPET 308:474–480, 2004 Printed in U.S.A.
Anxiolytic-Like Effects of Escitalopram, Citalopram, and R-
Citalopram in Maternally Separated Mouse Pups
Eric W. Fish, Sara Faccidomo, Sandeep Gupta, and Klaus A. Miczek
Department of Psychology (E.W.F., S.F., K.A.M.) and Department of Psychiatry, Pharmacology, and Neuroscience (K.A.M.),
Tufts University, Medford and Boston, Massachusetts; and Department of Pharmacology and Toxicology (S.G.), Forest
Research Institute, Jersey City, New Jersey
Received August 6, 2003; accepted October 16, 2003
Downloaded from jpet.aspetjournals.org at ASPET Journals on February 4, 2015
ABSTRACT
The S-enantiomer of citalopram, escitalopram, is a selective the drugs reduced USV emission; escitalopram was the most
serotonin reuptake inhibitor (SSRI) that appears to be respon- potent (ED50 0.05 mg/kg), followed by paroxetine (0.17 mg/kg),
sible for citalopram’s antidepressant and anxiolytic effects. citalopram (1.2 mg/kg), fluoxetine (4.3 mg/kg), R-citalopram (6
Clinically, escitalopram is reported to have fewer adverse side mg/kg), and venlafaxine (7 mg/kg). The doses that decreased
effects than do other SSRIs. This study compared escitalopram USVs differed from those that increased motor activity. In-
to other antidepressants in a preclinical procedure predicting creased grid crossing occurred after low doses of paroxetine
anxiolytic-like effects of drugs. Carworth Farms Webster (CFW) (0.03 or 0.1 mg/kg) and fluoxetine (1 mg/kg), but only after the
mouse pups (7 days old) were separated from the dam and highest doses of the citalopram enantiomers and venlafaxine
maintained at a temperature of 34°C. Forty-five minutes after (0.3, 10, and 56 mg/kg, respectively). Except for escitalopram
administering citalopram (0.56 –10 mg/kg), escitalopram and venlafaxine, high doses of the treatments increased rolling.
(0.0056 –3 mg/kg), R-citalopram (1–10 mg/kg), paroxetine R-Citalopram caused a 10-fold rightward shift in escitalopram’s
(0.3–3 mg/kg), fluoxetine (1–30 mg/kg), or venlafaxine (3–56 dose-effect curve, suggesting that R-citalopram inhibits escita-
mg/kg) subcutaneously, the pups were placed individually on a lopram’s anxiolytic-like effects. These data support clinical
19.5°C surface for 4 min. Ultrasonic vocalizations (USVs) findings that escitalopram is a potent, well tolerated SSRI with
(30 – 80 kHz), grid crossing, rolling (i.e., the pup turned on one anxiolytic-like effects.
side or its back), and colonic temperature were recorded. All
Selective serotonin reuptake inhibitors (SSRIs) were ini- hibitors of the SERT with low or no binding to 144 other
tially introduced as antidepressants, and their potential as receptors (Owens et al., 2001; Sánchez et al., 2003). Com-
anxiolytics has been observed in the treatment of social pho- pared with citalopram and its R-enantiomer, R-citalopram,
bia, post-traumatic stress disorder, and generalized anxiety escitalopram is at least 6-fold less potent in binding to the
disorder (e.g., Nutt et al., 1999). Although each of the SSRIs histamine 1 (H1) receptor (Owens et al., 2001; Sánchez et al.,
increases extracellular serotonin (5-hydroxytryptamine, 2003). Escitalopram and citalopram have similar affinities
5-HT) in the brain by blocking the 5-HT transporter SERT, for the (1) receptors, which are about 2-fold less than those
they differ substantially in terms of their selectivity (Nutt et of R-citalopram. Escitalopram is believed to confer the phar-
al., 1999; Owens et al., 2001; Sánchez et al., 2003). Actions at macological effects of citalopram (Hyttel et al., 1992; Sánchez
other binding sites, such as the muscarinic, histamine, ad- et al., 2003), and its effects can be inhibited by coadministra-
renergic, and 5-HT2 receptors, may contribute to some of the tion of R-citalopram (Mørk et al., 2003; Sánchez, 2003a). In
side effects commonly associated with SSRI treatment. Al- clinical reports, escitalopram has a faster onset of antide-
though in general, these side effects are better tolerated than pressant effects compared with its racemate citalopram and
those from tricyclics and benzodiazepines, they are a limita- has a low incidence of side effects (Montgomery et al., 2001;
tion in the use of SSRIs. Citalopram and its S-enantiomer, Burke et al., 2002; Wade et al., 2002; Waugh and Goa, 2003).
escitalopram (Lexapro or Cipralex), are highly selective in- Preclinical research on the anxiolytic-like effects of SSRIs
is variable; some studies find no effect of SSRIs, whereas
This research was supported by a grant from Forest Laboratories. others find decreases or increases in anxiety-like behaviors.
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org. A viable hypothesis for these SSRI effects depends on
DOI: 10.1124/jpet.103.058206. whether the experimental procedure measures conditioned
ABBREVIATIONS: SSRI, selective serotonin reuptake inhibitor; 5-HT, 5-hydroxytryptamine (serotonin); SERT, serotonin transporter; USV,
ultrasonic vocalization; CFW, Carworth Farms Webster; MED, minimally effective dose.
474Anxiolytic-Like Effects of Citalopram Enantiomers in Mice 475
or unconditioned behaviors, particularly when the SSRI is
30 ⴞ 14
given acutely (Griebel, 1995; Miczek et al., 1995). One pro-
56
9
cedure that has consistently detected an anxiolytic-like effect
of acutely administered SSRIs relies on the vocal responses of
148 ⴞ 33
88 ⴞ 27
154 ⴞ 45
young rodents when they are briefly separated from their
16
12
9
30
nest and their dam. Separated rodent pups emit ultrasonic
vocalizations (USVs) in a frequency range between 30 and 80
kHz that may serve as signals for the dam to initiate re-
21 ⴞ 4.5
130 ⴞ 28
86 ⴞ 21
179 ⴞ 43
193 ⴞ 55
trieval (Noirot, 1972; Brunelli et al., 1994). The species gen-
10
14
17
15
10
10
erality and unconditioned nature of vocal reactions distin-
guish them from many other preclinical measures of anxiety-
like behaviors (Panksepp et al., 1980; Miczek et al., 1995;
66 ⴞ 18
180 ⴞ 42
202 ⴞ 49
238 ⴞ 48
210 ⴞ 42
16 ⴞ 2
Sánchez, 2003b).
12
13
16
16
6
8
3
Among the neurotransmitter systems that influence sepa-
ration calling, GABA and 5-HT are particularly noteworthy.
Antianxiety treatments such as benzodiazepines and 5-HT1A
113 ⴞ 37
agonists decrease calling, whereas benzodiazepine inverse
1.7
14
agonists and pentylenetetrazol can increase calling (Gard-
Downloaded from jpet.aspetjournals.org at ASPET Journals on February 4, 2015
ner, 1985; Insel et al., 1986; Mos and Olivier, 1989; Nastiti et
148 ⴞ 32
51 ⴞ 17
239 ⫾ 36
260 ⫾ 39
292 ⫾ 61
al., 1991; Winslow and Insel, 1991; Molewijk et al., 1996;
1.0
13
16
10
16
4
Vivian et al., 1997; Fish et al., 2000). Of the SSRIs, citalo-
pram, fluoxetine, fluvoxamine, and paroxetine have been
reported to reduce distress USVs (Mos and Olivier, 1989;
1.3 ⴞ 0.3
251 ⫾ 36
Winslow and Insel, 1990; Olivier et al., 1998). Similarly,
0.56
13
3
these SSRIs, as well as escitalopram, reduce the shock-in-
duced USVs of adult rats (Sánchez and Meier, 1997; Schre-
Dose (mg/kg)
iber et al., 1998; Sánchez, 2003b; Sánchez et al., 2003). Un-
55 ⴞ 16
106 ⴞ 33
314 ⫾ 27
like the procedure in adult rats, the separation test can
0.3
14
8
6
The data for vocalizations are expressed as mean ⫾ S.E.M. Bold values are significantly different from vehicle (p ⬍ 0.05).
further be used to concurrently assess behavioral specificity,
the degree to which drug effects on different behaviors can be
dissociated. For example, GABAA-positive modulators reduce
145 ⴞ 15
142 ⴞ 39
calling, but their effects are often accompanied by sedation,
0.1
11
14
stimulation, and/or increased motor incoordination (Vivian et
al., 1997; Fish et al., 2000; Rowlett et al., 2001). 5-HT1A 310 ⫾ 72
receptor agonists reduce both motor activity and calling at a
0.056
similar dose range, whereas 5-HT1B receptor agonists reduce
9
calling but stimulate motor activity (Fish et al., 2000). Al-
though the doses of citalopram, fluvoxamine, and fluoxetine
154 ⴞ 27
332 ⫾ 29
that reduce separation distress USVs have not been reported
Effects of SSRIs, venlafaxine, and pyrilamine on separation vocalizations
0.03
15
10
to affect motor activity, a high dose of fluoxetine (20 mg/kg)
was reported to impair the negative geotaxis response in
maternally separated rat pups (Mos and Olivier, 1989).
261 ⫾ 28
0.017
The objective of the current study was to compare the
16
anxiolytic-like and locomotor effects of escitalopram to those
of other SSRIs and the serotonergic/noradrenergic reuptake
226 ⴞ 26
inhibitor, venlafaxine, in maternally separated 7-day-old
0.01
mouse pups. Pups of this age were selected because they emit
15
the most separation USVs (Noirot, 1972; Fish et al., 2000;
Branchi et al., 2001). To further explore the mechanism
395 ⫾ 46
through which escitalopram and citalopram reduce separa-
0.056
13
tion USVs, the interaction among escitalopram, R-citalo-
pram, and the H1 receptor antagonist pyrilamine was inves-
tigated.
323 ⫾ 17
306 ⫾ 20
305 ⫾ 32
331 ⫾ 42
368 ⫾ 36
378 ⫾ 23
398 ⫾ 32
Vehicle
19
15
16
16
16
11
11
Materials and Methods
Animals. Carworth Farms Webster (CFW) mice (n ⫽ 774, number
Mice/Treatment
R-Citalopram
R-Citalopram
Escitalopram
Escitalopram
Venlafaxine
Venlafaxine
Citalopram
Citalopram
Vocalizations
Pyrilamine
Pyrilamine
per treatment included in Table 1 and Table 3), from litters of 8 to 12
Paroxetine
Paroxetine
Fluoxetine
Fluoxetine
pups, were 7 days old, weighed 3.5 to 5.0 g, and lived with both
TABLE 1
parents (Charles River Laboratories Inc., Wilmington, Ma) in clear
polycarbonate cages (28 ⫻ 17 ⫻ 14 cm). Pine bedding covered the
cage floor; food (Purina rodent chow; Purina, St. Louis, MO) and476 Fish et al.
water were freely available through a wire lid. The mouse vivarium Statistics. USV and grid crossing data (transformed into percent-
was 21 ⫾ 1°C with 30 to 40% humidity, and was illuminated for 12 h age of vehicle), rolls, and body temperatures (raw values) were ana-
(lights on at 7:30 AM). The “Guide for the Care and Use of Labora- lyzed using one-way between-subjects analysis of variance. F-values
tory Animals” (http://www.nap.edu/readingroom/books/labrats/) was with p ⬍ 0.05 were followed by post-hoc Dunnett’s tests to determine
followed while caring for the mice and all procedures were approved which individual doses were significantly different from the vehicle
by Tufts University’s Institutional Care and Use Committee. treatment. A two-way between-subjects analysis of variance, fol-
Apparatus and Measurements. All behaviors were measured in lowed by post-hoc Tukey’s multiple comparison tests, analyzed the
a sound-attenuated chamber (49.5 ⫻ 38 ⫻ 34 cm) in a separate interaction between escitalopram and pyrilamine. ED50 values (i.e.,
procedure room. The chamber had a one-way vision window (19 ⫻ doses that reduced the total USVs to 50% of the vehicle mean) were
16.5 cm) for observation, was lit by red light (10 W), and held a water estimated by first-order regression of those doses that were between ca.
bath that maintained the temperature of a square aluminum testing 20 and 80% of the vehicle mean. r2 values for the linear regression
surface (23 ⫻ 23 cm) at 19.5 ⫾ 0.5°C. Two-centimeter squares di- ranged from 0.627 for fluoxetine to 0.996 for R-citalopram. Nonoverlap-
vided the testing surface into grids used to measure motor activity. ping 95% confidence intervals were considered statistically significant.
Equipment similar to that previously described (Vivian et al., 1997;
Fish et al., 2000; Rowlett et al., 2001) detected 30- to 80-kHz sounds. Results
A high-frequency condenser microphone (Brüel and Kjær model
4135; Brüel and Kjær, Nærum, Denmark) joined to a preamplifier Dose-Effect Studies
(Brüel and Kjær model 2633) was suspended 5 cm from the testing USVs. All of the treatments, escitalopram (F(7,92) ⫽ 15.0;
surface. Sounds were amplified (Brüel and Kjær model 2610), fil-
p ⬍ 0.001; Fig. 1; Table 1), citalopram (F(5,71) ⫽ 14.5; p ⬍
tered (Krohn-Hite model 3362; Krohn-Hite Corporation, Brockton,
Downloaded from jpet.aspetjournals.org at ASPET Journals on February 4, 2015
0.001; Fig. 1; Table 1), R-citalopram (F(3,55) ⫽ 4.8; p ⫽ 0.005;
MA) to produce a flat range between 30 and 80 kHz, and then
visualized on an oscilloscope (Goldstar 059020A; Goldstar, Cerritos, Fig. 1; Table 1), paroxetine (F(6,72) ⫽ 10.1; p ⬍ 0.001; Fig. 2;
CA) that was connected to an analog-to-digital converter GWI-AMP Table 1) fluoxetine (F(4,86) ⫽ 8.9; p ⬍ 0.001; Fig. 2; Table 1),
(GW Instruments; Somerville, MA). The signal was further amplified and venlafaxine (F(4,58) ⫽ 10.2; p ⬍ 0.001; Fig. 2; Table 1)
before it was connected to a computer (Macintosh II) that ran cus- dose dependently reduced the number of separation USVs.
tomized signal detection software. The software counted an ultra- Pups treated with the 0.3 to 0.56 mg/kg doses of escitalo-
sound if it was 30 to 80 kHz, lasted longer than 10 ms, and was pram, the 1 to 10 mg/kg doses of citalopram, the 3 to 10 mg/kg
separated from the previous sound by at least 20 ms. Body temper- doses of R-citalopram, the 0.1 to 3 mg/kg doses of paroxetine,
ature was measured using a lubricated thermo probe (o.d. 0.7 mm; the 3 to 30 mg/kg doses of fluoxetine, or the 3 to 56 mg/kg
YSI 555 N034; YSI Inc., Yellow Springs, IN) attached to a tele-
doses of venlafaxine vocalized significantly less than did the
thermometer (YSI 2100; YSI Inc.).
pups treated with vehicle. The histamine H1 receptor antag-
Procedure. The test sessions were conducted between 7:30 AM
and 7:30 PM, and no differences in baseline USV rates were observed onist pyrilamine (F(5,42) ⫽ 4.8; p ⫽ 0.001; Table 1) also dose
across different times of the day (Fish et al., 2000). An entire litter of dependently reduced separation USV. Pups treated with the
pups and some bedding were removed from the home cage, trans- 3 to 30 mg/kg doses of pyrilamine vocalized significantly less
ported to the procedure room, and maintained in an incubator than did the pups treated with vehicle (p ⬍ 0.05). The ED50
(11.5 ⫻ 14 ⫻ 5 cm) at 34 ⫾ 1.0°C. Twenty minutes later, the pups values are shown in Table 4.
were weighed, individually placed in the testing chamber for 30 s to Grid Crossing. With the exception of citalopram, the
screen for the emission of USVs, and marked for identification. The treatments also dose dependently increased grid crossing:
pups that vocalized more than six times (ca. 80%) were injected with escitalopram (F(7,92) ⫽ 3.1; p ⫽ 0.006; Table 2), R-citalopram
either one dose of the drug or vehicle. After the injection, the lubri-
(F(3,55) ⫽ 3.7; p ⫽ 0.017; Table 2), paroxetine (F(6,72) ⫽ 4.2;
cated thermo-probe was inserted about 7 mm into the rectum and
p ⫽ 0.001; Table 2), fluoxetine (F(4,76) ⫽ 3.9; p ⫽ 0.006; Table
held in place until the temperature measurement stabilized (ca. 3 s).
The pups were returned to the incubator for a specific interval (see 2), and venlafaxine (F(4,58) ⫽ 2.6; p ⫽ 0.04; Table 2). Pups
“Drugs” section) and a second rectal temperature was taken imme-
diately before a 4-min separation test. The signal detection software
automatically counted USVs, 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)pro-
pyl]-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-5-carbonitrilmono-
hydrobromide)], 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-␥-[4-(trifluoromethyl)phenoxy]-benzenepropanamine), and ven-
lafaxine (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-Aldrich
(St. Louis, MO). All drugs were dissolved in 0.9% saline and injected Fig. 1. The mean number of vocalizations per 4 min, expressed as per-
subcutaneously in a volume of 0.1 ml/10 g body weight. In the interac- centage of vehicle, by 7-day old mouse pups treated with escitalopram
(triangles), citalopram (diamonds), or R-citalopram (inverted triangles).
tion studies, drugs and/or saline were administered in two separate
Vertical lines represent ⫾1 S.E.M. Data points falling between ca. 20 and
injections. To prevent leakage, a small amount of glue was applied to 80% of the vehicle are fit with a first-order regression line. Asterisks
the injection site. The separation test was conducted 45 min after drug denote values that are significantly different from vehicle (p ⬍ 0.05).
administration. Nontransformed values are expressed in Table 1.Anxiolytic-Like Effects of Citalopram Enantiomers in Mice 477
50 ⴞ 9.3
4.1 ⫾ 1.3
56
8.7 ⴞ 1.8
33 ⫾ 3.2
46 ⫾ 7.6
57 ⫾ 8.9
4.0 ⫾ 0.7
5.2 ⫾ 1.7
30
47 ⴞ 6.1
60 ⴞ 8.2
53 ⫾ 7.9
42 ⫾ 4.2
44 ⫾ 4.0
4.7 ⫾ 1.2
2.7 ⫾ 0.7
7.2 ⫾ 2.1
10
52 ⴞ 5.6
14 ⴞ 1.9
4.6 ⴞ 0.8
12.7 ⴞ 3.1
42 ⫾ 5.6
39 ⫾ 5.4
45 ⫾ 5.2
36 ⫾ 3.2
42 ⫾ 8.0
3.2 ⫾ 1.0
2.8 ⫾ 0.8
5.4 ⫾ 1.2
3
11 ⴞ 2.3
41 ⫾ 6.4
1.6 ⫾ 0.4
Fig. 2. The mean number vocalizations per 4 min, expressed as percent-
1.7
age of vehicle, by 7-day-old mouse pups treated with paroxetine (circles),
fluoxetine (squares), or venlafaxine (hexagons). Vertical lines represent
Downloaded from jpet.aspetjournals.org at ASPET Journals on February 4, 2015
⫾1 S.E.M. Data points falling between ca. 20 and 80% of the vehicle are
fit with a first-order regression line. Asterisks denote values that are
9.1 ⴞ 1.1
39 ⫾ 4.0
39 ⫾ 4.1
53 ⫾ 6.5
27 ⫾ 3.8
7.8 ⫾ 2.0
4.7 ⫾ 1.0
3.0 ⫾ 1.6
significantly different from vehicle (p ⬍ 0.05). Nontransformed values are
56 ⴞ 11
1.0
expressed in Table 1.
treated with the 0.3 and 0.56 mg/kg doses of escitalopram,
the 10 mg/kg dose of R-citalopram, the 0.03 and 0.1 mg/kg
64 ⴞ 6.4
8.0 ⴞ 1.4
39 ⫾ 3.3
6.3 ⫾ 1.9
2.6 ⫾ 0.5
doses of paroxetine, the 1 and 3 mg/kg doses of fluoxetine, or
0.56
the 56 mg/kg dose of venlafaxine crossed significantly more
Dose (mg/kg)
grids than did the pups treated with vehicle. Pyrilamine
(F(5,42) ⫽ 3.8; p ⫽ 0.006; Table 2) also increased grid cross-
49 ⴞ 6.7
57 ⫾ 5.5
27 ⫾ 5.2
6.3 ⫾ 1.8
4.4 ⫾ 0.9
5.7 ⫾ 1.4
3.8 ⫾ 1.5
ings, significantly at the 10 mg/kg dose.
0.3
Rolling. With the exception of escitalopram and venlafax-
ine, 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
67 ⴞ 6.8
42 ⫾ 6.5
4.5 ⫾ 1.0
5.0 ⫾ 1.3
0.1
(F(6,72) ⫽ 2.6; p ⫽ 0.02; Table 2), and fluoxetine (F(4,76) ⫽ 2.8;
The data are expressed as mean ⫾ S.E.M. Bold values are significantly different from vehicle (p ⬍ 0.05).
p ⫽ 0.03; Table 2) dose dependently increased rolling. Pups
treated with the 1 to 10 mg/kg doses of citalopram, the 10
62 ⫾ 9.0
4.0 ⫾ 1.3
mg/kg dose of R-citalopram, the 3 mg/kg dose of paroxetine,
0.056
or the 30 mg/kg dose of fluoxetine rolled significantly more
than did the pups administered vehicle. Pyrilamine did not
significantly affect rolling.
78 ⴞ 9.6
40 ⫾ 3.0
2.4 ⫾ 0.6
5.1 ⫾ 1.4
Body Temperature. None of the drugs, at the doses
0.03
tested, significantly affected body temperature measured im-
mediately before the separation test (data not shown).
Effects of SSRIs, venlafaxine, and pyrilamine on motor activity
36 ⫾ 3.1
3.4 ⫾ 1.0
0.017
Interaction Studies
USVs. Escitalopram’s dose-dependent reduction of separa-
tion USVs was confirmed when it was coadministered with
39 ⫾ 2.8
2.4 ⫾ 0.8
either vehicle or pyrilamine (1 or 10 mg/kg) (F(3,127) ⫽ 12.0;
0.01
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
38 ⫾ 3.0
2.3 ⫾ 1.1
0.056
effects of escitalopram on separation USVs. R-citalopram
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-
33 ⫾ 2.2
32 ⫾ 5.1
26 ⫾ 3.3
38 ⫾ 4.1
28 ⫾ 2.5
27 ⫾ 2.5
36 ⫾ 3.7
3.6 ⫾ 0.7
2.9 ⫾ 0.6
1.7 ⫾ 0.3
4.1 ⫾ 1.2
2.7 ⫾ 0.7
1.7 ⫾ 0.5
3.4 ⫾ 1.0
Vehicle
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
R-Citalopram
R-Citalopram
Escitalopram
Escitalopram
0.05 (0.02, 0.09) to 0.61 (0.1, 1.7) mg/kg.
Grid Crossings
Venlafaxine
Venlafaxine
Citalopram
Citalopram
Pyrilamine
Pyrilamine
Paroxetine
Paroxetine
Fluoxetine
Fluoxetine
Grid Crossing. Escitalopram (F(3,115) ⫽ 7.1; p ⬍ 0.001;
TABLE 2
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
Rolls
mg/kg and 1 mg/kg doses, respectively. There was no inter-478 Fish et al.
TABLE 3
The interaction between escitalopram, R-citalopram, and pyrilamine on separation vocalizations and motor activity
The data for vocalizations, grid crossing, and rolling are expressed as mean ⫾ S.E.M. Bold values are significantly different from vehicle (p ⬍ 0.05).
Escitalopram Dose (mg/kg)
Vehicle 0.01 0.1 0.3 1 3
Vocalizations
⫹ Vehicle 352 ⫾ 29 275 ⫾ 36 138 ⴞ 38 54 ⴞ 11
⫹ R-Citalopram, 1.0 mg/kg 285 ⫾ 37 350 ⫾ 39 294 ⫾ 44 213 ⫾ 41 148 ⴞ 34 75 ⴞ 29
⫹ Pyrilamine, 1.0 mg/kg 309 ⫾ 73 247 ⫾ 58 155 ⴞ 46 85 ⴞ 26
⫹ Pyrilamine, 10.0 mg/kg 191 ⫾ 76 175 ⫾ 60 110 ⴞ 46 122 ⴞ 44
Grid Crossings
⫹ Vehicle 30 ⫾ 3.1 31 ⫾ 3.2 42 ⴞ 3.7 49 ⴞ 6.1
⫹ R-Citalopram, 1.0 mg/kg 31 ⫾ 3.8 37 ⫾ 5.1 48 ⫾ 4.0 47 ⫾ 7.4 43 ⴞ 4.6 41 ⫾ 5.9
⫹ Pyrilamine, 1.0 mg/kg 52 ⴞ 14 50 ⫾ 7.3 49 ⫾ 9.6 48 ⫾ 9.1
⫹ Pyrilamine, 10.0 mg/kg 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 mg/kg 2.6 ⫾ 0.6 3.5 ⫾ 0.6 3.7 ⫾ 1.0 3.0 ⫾ 1.0 7.5 ⴞ 1.3 10.3 ⴞ 1.8
⫹ Pyrilamine, 1.0 mg/kg 1.0 ⫾ 0.4 2.6 ⫾ 0.9 3.1 ⫾ 1.3 1.9 ⫾ 0.7
⫹ Pyrilamine, 10.0 mg/kg 3.6 ⫾ 1.7 3.7 ⫾ 1.2 0.9 ⫾ 0.4 6.9 ⫾ 2.6
Mice/Treatment
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⫹ Vehicle 24 15 15 14
⫹ R-Citalopram, 1.0 mg/kg 14 13 10 12 15 7
⫹ Pyrilamine 1.0, mg/kg 7 8 8 8
⫹ Pyrilamine, 10.0 mg/kg 7 9 8 8
tion-induced USVs of mice and differed in terms of their
potency and behavioral specificity. These results confirm ear-
lier 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 reduc-
tion of USVs by its S-enantiomer, escitalopram. The right-
ward shift in escitalopram’s dose-effect curve by coadminis-
tration 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;
Fig. 3. The mean number of vocalizations per 4 min, by 7-day-old mouse Sánchez et al., 2003). However, the magnitude of the potency
pups concurrently treated with escitalopram and saline (open triangles, difference, about 20- to 125-fold more than citalopram and
open bar) or escitalopram and R-citalopram (1.0 mg/kg) (filled triangles,
filled bar). Vertical lines represent ⫾1 S.E.M. Data points falling between R-citalopram, is larger than that predicted from the above
ca. 20 and 80% of the vehicle are fit with a first-order regression line. studies. Based on SERT affinity, escitalopram should be
Asterisks denote values that are significantly different from vehicle (p ⬍ about 2-fold more potent than citalopram, and clinically,
0.05). Nontransformed values are expressed in Table 3.
escitalopram appears to be at least 2-fold more potent than
action between the effects of escitalopram and those of pyril- citalopram (Burke et al., 2002). Development of the 5-HT
amine or R-citalopram. When given with R-citalopram, es- transporter system could have contributed to these discrep-
citalopram (F(2,45) ⫽ 4.3; p ⫽ 0.02; Table 3) significantly ancies because the number and distribution of transporters
increased grid crossing at the 1 mg/kg dose. substantially change with age (e.g., Lebrand et al., 1998).
Rolling. Neither escitalopram nor pyrilamine signifi- In addition to reducing USVs, all of the drugs altered
cantly affected rolling. However, there was a trend for an motor behavior to varying degrees, as measured by grid
interaction between escitalopram and pyrilamine (F(6,127) ⫽ crossing and rolling. Escitalopram and venlafaxine were the
1.9; p ⫽ 0.086; Table 3) but not R-citalopram. This was most behaviorally selective in reducing pup USVs (Table 4),
probably due to a decrease in rolling when the 0.1 mg/kg although escitalopram was considerably more potent than
escitalopram dose was given with the 10 mg/kg pyrilamine venlafaxine (ca. 140-fold). Motor stimulation occurred only at
dose. When given with R-citalopram, the higher doses (1 and the highest doses of escitalopram (0.3 and 0.56 mg/kg) and
3 mg/kg) of escitalopram (F(2,45) ⫽ 10.9; p ⬍ 0.001; Table 3) venlafaxine (56 mg/kg), which were, respectively, about 30-
increased rolling. and 19-fold greater than the minimally effective doses
(MEDs) for reducing USVs (Table 4). Unlike the other treat-
Discussion ments, escitalopram and venlafaxine did not increase rolling.
Several SSRIs and the mixed noradrenergic-serotonergic Only when very high doses (1 and 3 mg/kg) of escitalopram
reuptake inhibitor venlafaxine reduced the maternal separa- were given in combination with R-citalopram did escitalo-Anxiolytic-Like Effects of Citalopram Enantiomers in Mice 479
TABLE 4
ED50 values for USVs, MEDs for USVs and motor behavior, and selectivity ratios
Selectivity ratio compares the MED for USVs to the lowest MED for either grid crossing or rolling. N.D. (not determined) means that no dose of the drug significantly affected
the behavior.
Dependent Variables
Drug
USV (ED50, 95% CI) USV (MED) Grid Crossing (MED) Rolling (MED) Selectivity Ratio (USV/Motor)
Escitalopram 0.05 (0.02, 0.1) 0.01 0.3 N.D. 1:30
Paroxetine 0.17 (0.03, 0.4) 0.1 0.03 3 1:0.3
Citalopram 1.2 (0.6, 2.0) 1 N.D. 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 N.D. 1:19
CI, confidence interval.
pram mimic the effects of citalopram and increase rolling. R-citalopram’s inhibition of escitalopram’s effects. Another
The increased rolling after these treatments is relatively hypothesis is that during the interaction between escitalo-
modest, about half of what was observed after benzodiaz- pram and R-citalopram, escitalopram is metabolized faster,
epine treatment (Rowlett et al., 2001). Although the lack of leaving higher levels of the less effective R-citalopram to
increased rolling is consistent with the clinical observations displace escitalopram from the transporter. However, levels
Downloaded from jpet.aspetjournals.org at ASPET Journals on February 4, 2015
that escitalopram is well tolerated (Montgomery et al., 2001; of escitalopram are not affected by treatment with R-citalo-
Burke et al., 2002; Wade et al., 2002; Waugh and Goa, 2003), pram in the rat brain (Mørk et al., 2003). A third possibility
the clinical relevance of motor stimulation observed with is that R-citalopram’s inhibition of escitalopram’s effects is
high doses of escitalopram and venlafaxine remains to be due to noncompetitive binding to a different site on the trans-
determined. porter protein that conformationally inhibits escitalopram’s
Motor stimulation has also been observed after treatment binding to the transporter. Future research into the com-
with 5-HT1B and 5-HT2A, but not 5-HT1A, receptor agonists bined effects of the single enantiomers and determining
in mouse pups (Fish et al., 2000; E. W. Fish and K. A. Miczek, whether R-citalopram also inhibits the effects of other SSRIs
unpublished data). Interestingly, the citalopram enanti- will help elucidate the mechanisms through which R-citalo-
omers and venlafaxine showed dose-effect relationships on pram inhibits the effects of escitalopram.
motor stimulation different from those of paroxetine and The acute anxiolytic-like effects of the SSRIs on neonatal
fluoxetine. Lower doses of paroxetine and fluoxetine were vocalizations (Mos and Olivier, 1989; Winslow and Insel,
stimulating and higher doses were not. In contrast, only the 1990; Molewijk et al., 1996; Olivier et al., 1998) differ from
high doses of citalopram enantiomers increased grid cross- their acute effects in humans. In humans, the anxiolytic
ing. Similar dose-effect relationships between citalopram, effects of SSRIs emerge only after chronic treatment, and
paroxetine, and fluoxetine have also been observed in adult there are some reports that they may initially increase anx-
mice placed in a novel open-field (Brocco et al., 2002). The iety (for review, see Nutt et al., 1999). In preclinical studies,
different dose-effect curves for the citalopram enantiomers, the acute effects of SSRIs can be detected in several proce-
paroxetine and fluoxetine, may be because of their actions at dures that are used for characterizing anxiolytic drugs, but
muscarinic M1 and 5-HT2C receptors, respectively (Owens et the nature of these effects varies markedly with the experi-
al., 2001). mental procedure (for review, see Griebel, 1995; Borsini et
One hypothesis for the differences in potency between es- al., 2002). Consistent anxiolytic-like effects of SSRIs occur on
citalopram and racemic citalopram is that R-citalopram in- measures of footshock-induced USVs (Sánchez and Meier,
hibits the effects of escitalopram. Coadministration of R- 1997; Schreiber et al., 1998; Sánchez et al., 2003), burying
citalopram reduced escitalopram’s elevation of cortical behavior (Njung’e and Handley 1991), conditioned freezing
extracellular 5-HT and reduction of shock-induced USVs (Hashimoto et al., 1996), and increased movement across the
(Sánchez, 2003a; Mørk et al., 2003). The results of the inter- electrified grids of the four plate test (Hascoet et al., 2000).
action between escitalopram and R-citalopram in the present Anxiogenic-like effects of SSRIs have been observed on light-
study are consistent with this hypothesis. When given to- dark exploratory behavior (Sánchez and Meier, 1997), novel-
gether, R-citalopram caused a 10-fold parallel shift to the ty-suppressed feeding (Bodnoff et al., 1989), the mouse de-
right in the USV-reducing effects of escitalopram. There are fensive battery (Griebel, 1995), the social interaction test
several mechanisms through which R-citalopram might in- (File et al., 1999), and the elevated plus maze (File et al.,
hibit the effects of escitalopram. One possibility is that R- 1999). Interpreting these contradictory results is difficult,
citalopram’s binding to the H1 receptor alters the effects of and it is clear that novel procedures for assessing anxiety-
escitalopram. To test this hypothesis, the H1 receptor antag- like behaviors in animals must be developed. Although es-
onist pyrilamine was tested alone and in combination with citalopram inhibits both neonatal and adult vocalizations
escitalopram. Pyrilamine (mepyramine) was chosen because and enhances exploratory behavior in the light and dark box,
it was the ligand for the H1 receptor in escitalopram binding it will be important to extend this assessment to several
studies (Owens et al., 2001; Sánchez et al., 2003). Alone, other procedures.
pyrilamine modestly suppressed calling, as was observed in Procedures measuring vocalizations appear to be particu-
adult rats (Sánchez, 2003b), and its effects were quite vari- larly sensitive to the acute anxiolytic-like effects of SSRIs,
able. When administered with escitalopram, there was no but there are potentially confounding variables to consider
evidence for a shift in escitalopram’s dose-effect curve, indi- (Winslow and Insel, 1991). Young, developing animals may
cating that H1 antagonism is an unlikely mechanism for be differentially sensitive to drug treatments than are adults480 Fish et al.
because of differences in the number of receptors as well as Hascoet M, Bourin M, Colombel MC, Fiocco AJ, and Baker GB (2000) Anxiolytic-like
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157–160.
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