anxiolytic-like effects of escitalopram, citalopram and r...

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.

JPET Fast Forward. Published on October 30, 2003 as DOI:10.1124/jpet.103.058206This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on October 30, 2003 as DOI: 10.1124/jpet.103.058206

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Running Title: JPET #58206

Correspondence: Klaus A. Miczek

Tufts University

530 Boston Ave. (Bacon Hall)

Medford, MA 02155

e-mail [email protected]

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

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on October 30, 2003 as DOI: 10.1124/jpet.103.058206

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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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This article has not been copyedited and form

atted. The final version m

ay differ from this version.

JPET

Fast Forward. Published on O

ctober 30, 2003 as DO

I: 10.1124/jpet.103.058206 at ASPET Journals on August 27, 2018 jpet.aspetjournals.org Downloaded from



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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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