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`
`0022-3565/02/3011-333–345$7.00
`THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
`Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics
`JPET 301:333–345, 2002
`
`Vol. 301, No. 1
`4642/974299
`Printed in U.S.A.
`
`4-(2-Chloro-4-methoxy-5-methylphenyl)-N-[(1S)-2-cyclopropyl-
`1-(3-fluoro-4-methylphenyl)ethyl]5-methyl-N-(2-propynyl)-1,
`3-thiazol-2-amine Hydrochloride (SSR125543A), a Potent
`and Selective Corticotrophin-Releasing Factor1 Receptor
`Antagonist. II. Characterization in Rodent Models of
`Stress-Related Disorders
`
`GUY GRIEBEL, JACQUES SIMIAND, R ´EGIS STEINBERG, MIREILLE JUNG, DANIELLE GULLY, PIERRE ROGER,
`MICHEL GESLIN, BERNARD SCATTON, JEAN-PIERRE MAFFRAND, and PHILIPPE SOUBRI ´E
`Central Nervous System Research Department, Sanofi-Synthelabo, Bagneux, France (G.G., B.S., P.R.); Exploratory Research Department,
`Sanofi-Synthelabo, Toulouse, France (D.G., M.G., J.S.); and Discovery Research Division, Sanofi-Synthelabo, Montpellier, France (R.S., M.J.,
`P.S., J.P.M.)
`Received October 11, 2001; accepted December 31, 2001
`
`This article is available online at http://jpet.aspetjournals.org
`
`ABSTRACT
`The present study investigated the effects of the novel corticotro-
`phin-releasing factor (CRF)1 receptor antagonist 4-(2-chloro-
`4-methoxy-5-methylphenyl)-N-[(1S)-2-cyclopropyl-1-(3-fluoro-4-
`methylphenyl)ethyl]5-methyl-N-(2-propynyl)-1,3-thiazol-2-amine
`hydrochloride (SSR125543A) in a variety of rodent models of
`anxiety, including conflict procedures (punished drinking and
`four-plate), exploration models (elevated plus-maze and light/
`dark), a fear/anxiety defense test battery, and several proce-
`dures based on stress-induced changes in physiological
`(isolation-induced hyperthermia and tail pinch-induced cortical
`norepinephrine release) or behavioral (social defeat-induced anx-
`iety, maternal separation-induced vocalization) parameters. More-
`over, the effects of SSR125543A were investigated in acute
`(forced swimming) and chronic (chronic mild stress; CMS) models
`of depression. SSR125543A and the CRF1 receptor antagonist
`antalarmin displayed limited efficacy in exploration-based anxiety
`models. In contrast, both compounds produced clear-cut anxio-
`
`lytic-like activity in models involving inescapable stress, including
`the conflict procedures, the social defeat-induced anxiety para-
`digm and the defense test battery (3–30 mg/kg i.p. or p.o.). These
`effects paralleled those of the anxiolytic diazepam. In addition,
`SSR125543A and antalarmin antagonized stress-induced hyper-
`thermia, distress vocalization, and cortical norepinephrine release.
`In the forced swimming test, 30 mg/kg p.o. SSR125543A and 3 to
`30 mg/kg p.o. antalarmin produced clear antidepressant-like
`effects. These latter results were strengthened by the findings
`from the CMS, which showed that repeated administration of
`10 mg/kg i.p. SSR125543A for 30 days improved the degrada-
`tion of the physical state, the reduction of body weight gain,
`and anxiety produced by stress. Together, these data indicate
`that SSR125543A shows good activity in acute and chronic
`tests of unavoidable stress exposure, suggesting that it may
`have a potential in the treatment of depression and some forms
`of anxiety disorders.
`
`Corticotropin-releasing factor (CRF) has been identified as
`a neuropeptide that plays a central role in the coordination of
`neuroendocrine, autonomic, and behavioral responses to
`stress (Vale et al., 1981). It is the main regulator of basal and
`stress-induced release of the adrenocorticotropic hormone
`(ACTH) (Stout et al., 1995). Two CRF receptor subtypes,
`CRF1 and CRF2, with distinct anatomical localization and
`pharmacology have been identified (Chalmers et al., 1996).
`CRF1 receptor expression is most abundant in neocortical,
`cerebellar, and limbic structures, whereas CRF2 receptor
`
`expression is generally prominent in subcortical structures.
`This anatomical information provided a basis for functional
`hypotheses related to CRF receptor subtypes and suggested
`that CRF may contribute significantly both to behavioral
`responses to stress and emotional behavior itself. This idea
`has been substantiated by numerous studies showing that
`i.c.v. application of CRF in rodents produces behavioral ef-
`fects similar to those observed when animals are exposed to
`stress (for review, see Griebel, 1999). Studies using CRF
`transgenic mouse lines overexpressing CRF or knockout mice
`
`ABBREVIATIONS: CRF, corticotropin-releasing factor; ACTH, adrenocorticotropic hormone; MDTB, mouse defense test battery; CMS, chronic
`mild stress; NE, norepinephrine ANOVA, analysis of variance; CP-154,526, butyl-[2,5-dimethyl-7-(2,4,6-trimethylphenyl)-7H-pyrrolo[2,3-d]pyrimi-
`din-4-yl]-ethylamine; R121919, 3-[6-(dimethylamino)-4-methyl-pyrid-3-yl]-2,5-dimethyl-N,N-dipropyl-pyrazolo[2,3-a]pyrimidin-7-amine.
`
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`
`Griebel et al.
`
`lacking the CRF1 receptor further emphasized the involve-
`ment of CRF in stress-related behaviors, because the former
`exhibited a behavioral state resembling that produced by
`anxiety, whereas the latter showed reduced emotionality (for
`review, see Contarino et al., 1999). On a clinical level, a large
`body of evidence points to increased cerebrospinal fluid CRF
`concentrations in drug-free patients with major depression
`and post-traumatic stress disorder compared with healthy
`controls (for review, see Kasckow et al., 2001). In this context,
`it was hypothesized that CRF receptor antagonists may rep-
`resent novel agents for the treatment of stress-related disor-
`ders (Chalmers et al., 1996; Holsboer, 1999).
`Several classes of nonpeptide antagonists of CRF1 recep-
`tors have been identified (for review, see Gutman et al.,
`2000). A few of them have been studied extensively in exper-
`imental models of stress. For example, peripheral adminis-
`tration of the pyrrolopyrimidine derivatives antalarmin, CP-
`154,526, and R121919 was reported to reduce the effects of
`acute and repeated stressors on behavior in rodents, and
`some activity was found in classical models used to screen
`anxiolytics and antidepressants (for reviews, see Griebel,
`1999; Gutman et al., 2000). However, negative results were
`also reported with some of these procedures. It was suggested
`that CRF1 antagonists may produce positive effects only
`when the endogenous tone of CRF is high, thereby pointing to
`a crucial importance of baseline levels of stress when inves-
`tigating the behavioral actions of these compounds. Recently,
`the results of the first open-label study examining the effects
`of R121919 in 20 patients with major depression was pub-
`lished (Zobel et al., 2000). The compound was well tolerated
`by the patients and did not significantly affect ACTH or
`cortisol levels at baseline or after a CRF challenge. More
`importantly, significant reductions in depression and anxiety
`scores were observed after 30-day treatment with the com-
`pound. Although this small open-label study does not provide
`unequivocal proof, it brings further evidence that a selective
`CRF1 receptor antagonist may represent a promising alter-
`native to agents currently used for the treatment of anxiety
`and depressive disorders.
`In the present article, we report on the psychopharmacolog-
`ical profile of 4-(2-chloro-4-methoxy-5-methylphenyl)-N-[(1S)-2-
`cyclopropyl-1-(3-fluoro-4-methylphenyl)ethyl]5-methyl-N-(2-
`propynyl)-1,3-thiazol-2-amine hydrochloride (SSR125543A), a
`2-aminothiazole derivative, which displays high affinities for
`both native and recombinant human CRF1 receptors (Ki ⫽ 1
`and 2 nM, respectively), and 1000-fold selectivity for CRF1
`compared with CRF2 receptor (Gully et al., 2002). SSR125543A
`is a potent antagonist at the CRF1 receptor as shown by its
`ability to inhibit CRF-induced cyclic AMP synthesis in human
`retinoblastoma Y 79 cells (pA2 ⫽ 8.92) and ACTH secretion in
`mouse pituitary tumor AtT-20 cells (pA2 ⫽ 9.63). After in vivo
`experiments in rats, the compound was found to inhibit CRF-
`and restraint-stress induced ACTH secretion when given orally.
`In the present series of experiments, the behavioral effects of
`SSR125543A were examined using a variety of rodent models of
`anxiety, including conflict procedures (punished drinking and
`four-plate), exploration models (elevated plus-maze and light/
`dark), a fear/anxiety defense test battery, and several test pro-
`cedures based on stress-induced changes in physiological (iso-
`lation-induced hyperthermia and tail pinch-induced cortical
`norepinephrine release) or behavioral (social defeat-induced
`anxiety, maternal separation-induced distress vocalizations)
`
`parameters. Moreover, the effects of SSR125543A were inves-
`tigated in acute (forced swimming) and chronic (chronic mild
`stress; CMS) models used for the characterization of antide-
`pressants. In this latter experiment, the compound was given
`repeatedly for 30 days. Comparative data for the CRF1 receptor
`antagonist antalarmin and the anxiolytic and antidepressant
`diazepam and fluoxetine, respectively, obtained under the same
`experimental conditions, are also provided. Finally, possible
`unwanted effects of increasing doses of SSR125543A were ex-
`amined using motor activity and memory tests.
`
`Materials and Methods
`
`Ethics
`All experimental procedures described herein were approved by
`the Animal Care and Use Committee of Sanofi-Synthelabo Recher-
`che and fully comply with French legislation on research involving
`animal subjects.
`
`Animals
`Male Sprague-Dawley and Wistar rats (Iffa Credo, L’Arbresle, and
`Charles River, Saint-Aubin-le`s-Elbeuf, France), weighing 180 to
`330 g at the time of testing, were used in the punished drinking,
`elevated plus-maze, stress-induced hyperthermia, and forced swim-
`ming tests, and in the microdialysis experiment. They were housed
`in groups of three to eight. Different strains of rats were used to
`optimize conditions. For example, preliminary data from our labora-
`tory have shown that, unlike Sprague-Dawley rats, Wistar rats from
`our laboratory animal supplier are poor responders in the elevated
`plus-maze (i.e., they display weak avoidance responses of the open
`aversive arms), making it difficult to use them for the screening of
`anxiolytics. Male CD1 (social defeat stress-induced anxiety, horizon-
`tal wire test, rotarod, passive avoidance test, and actimeter), OF1
`(mouse defense test battery; MDTB), NMRI (four-plate test), and
`BALB/c (light/dark test, chronic mild stress procedure) mice weigh-
`ing 17 to 32 g were supplied by Charles River, Iffa Credo, or Janvier
`(Le Genest, France). CD1, OF1, and NMRI mice were housed in
`groups of 20, those used in the MDTB and in the chronic mild stress
`procedure were housed singly, and BALB/c mice used in the light/
`dark test were housed in groups of six. Female guinea pigs (Dunkin-
`Hartley) with four 5-day-old pups were obtained from Harlan (Horst,
`The Netherlands). Each mother was housed individually with her
`litter provided with sawdust. Moreover, male Long Evans rats (400 –
`500 g) (Iffa-Credo) were used as threat stimulus in the MDTB. All
`animals were maintained under standard laboratory conditions (21–
`23°C; 40 – 60%relative humidity) and kept on a 12-h light/dark cycle
`with light onset at 6:00 AM.
`
`Compounds
`Compounds were prepared as solutions or suspensions in physio-
`logical saline or distilled water containing 0.1% Tween 80 (anta-
`larmin, diazepam, and fluoxetine) or 5% dimethyl sulfoxide and 5%
`Cremophor EL (SSR125543A). The
`compounds used were
`SSR125543A, antalarmin, diazepam, and fluoxetine (synthesized by
`Sanofi-Synthelabo, Bagneux and Toulouse, France). Compounds ad-
`ministered i.p. and p.o. were given in a constant volume of 5 ml/kg
`(rats), 2 ml/kg (guinea pig pups), or 20 (mice) ml/kg. The i.p. route
`was used in some experiments because an oral administration would
`have increased further the level of stress [i.e., distress vocalizations
`in guinea pig pups, stress-induced norepinephrine (NE) release in
`rats, and chronic mild stress in mice] or because it would have
`interfered with the procedure (i.e., water deprivation in the punished
`drinking test in rats).
`
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`Psychopharmacological Profile of SSR125543A
`
`335
`
`smalltube(1h)
`Confinementina
`
`(30h)
`dampsawdust
`Housinginmild
`
`light/darkcycle
`
`Inversionofthe
`
`light/darkcycle
`
`Inversionofthe
`
`Accesstoanempty
`
`bottle(2h)
`
`(30h)
`dampsawdust
`Housinginmild
`
`light/darkcycle
`
`Inversionofthe
`
`light/darkcycle
`
`Inversionofthe
`
`light/darkcycle
`
`Inversionofthe
`
`Waterdeprivation
`smalltube(1h)
`Confinementina
`
`(30min)
`waterat32°C
`Forcedwashin
`
`(16h)
`
`light/darkcycle
`
`Inversionofthe
`
`Pairedhousing
`
`(10min)
`restriction
`withfood
`
`smalltube(1h)
`Confinementina
`
`(10h)
`dampsawdust
`Pairedhousingin
`
`Waterandfood
`
`(16h)
`deprivation
`
`smalltube(1h)
`Confinementina
`
`smalltube(1h)
`Confinementina
`
`deprivation(16h)
`
`smalltube(1h)
`Confinementina
`
`Waterandfood
`
`Foodrestriction
`
`(3h)
`
`smalltube(1h)
`Confinementina
`
`(10h)
`dampsawdust
`Pairedhousingin
`
`(30min)
`waterat32°C
`Forcedwashin
`
`smalltube(1h)
`Confinementina
`
`smalltube(1h)
`Confinementina
`
`smalltube(1h)
`
`Confinementin
`
`light/darkcycle
`
`Inversionofthe
`
`Accesstoanempty
`
`(30h)
`dampsawdust
`Housinginmild
`
`bottle(2h)
`
`Waterdeprivation
`
`(16h)
`
`smalltube(1h)
`Confinementina
`
`Dark(2h)
`
`(10h)
`dampsawdust
`Pairedhousingin
`smalltube(1h)
`Confinementina
`
`smalltube(1h)
`Confinementina
`
`Foodrestriction
`
`(3h)
`
`Waterandfood
`
`(16h)
`deprivation
`
`smalltube(1h)
`Confinementina
`smalltube(1h)
`Confinementina
`
`smalltube(1h)
`Confinementina
`
`(30min)
`waterat32°C
`Forcedwashin
`
`6:00PM
`
`2:00PM
`
`9:00AM
`
`Week3
`
`6:00PM
`
`2:00PM
`
`9:00AM
`
`Week2
`
`6:00PM
`
`2:00PM
`
`9:00AM
`
`Week1
`
`Day7
`
`Day6
`
`Day5
`
`Day4
`
`Day3
`
`Day2
`
`Day1
`
`StressExposure
`
`Startof
`
`Chronicmildstressschedule
`TABLE1
`
`3
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`
`and two enclosed arms (50 ⫻ 10 ⫻ 50 cm), arranged so that the arms
`of the same type were opposite each other, connected by an open
`central area (10 ⫻ 10 cm). To prevent rats falling off, a rim of
`Plexiglas (0.5 cm in height) surrounded the perimeter of the open
`arms. The illumination in the experimental room consisted of one red
`neon tube fixed on the ceiling, so that experiments were performed
`under dim light conditions. The light intensity on the central plat-
`form was 10 lux. At the beginning of the experiment, rats were
`placed in the center of the maze, facing one of the enclosed arms, and
`observed for 4 min. The apparatus was equipped with infrared
`beams and sensors capable of measuring time spent in open arms,
`number of open-arm entries, and number of closed-arm entries (de-
`fined as entry of all four limbs into an arm of the maze). In addition,
`rats were observed via video-link by an observer located in an adja-
`cent room. This allowed the recording of a more ethologically orien-
`tated measure: 1) attempt: attempt at entry into open arms followed
`by avoidance responses. This includes stretched attend posture (the
`rat stretches forward and retracts to original position); and 2) head-
`dipping: protruding the head over the ledge of an open arm and down
`toward the floor (this response can occur while the animal’s body is
`in the closed arms, central square, or on open arms). The results
`were expressed as mean ratio of time spent in open arms to total time
`spent in both open and closed arms, mean ratio of entries into open
`arms to total entries into both open and closed arms, mean total
`number of both closed and open arm entries, mean total number of
`attempts, and mean total number of head-dips. Data were analyzed
`with one-way ANOVA. Subsequent comparisons between treatment
`groups and control were carried out using Dunnett’s t test. Experi-
`ments were performed 60 min after p.o. administration of
`SSR125543A, antalarmin, or diazepam.
`Stress-Induced Hyperthermia in Rats. The basal rectal tem-
`perature of grouped rats (5–7/cage) was measured with a telether-
`mometer (DM 852; ELLAB Instruments, Copenhagen, Denmark).
`Animals were then removed from their home cage, and placed indi-
`vidually in a small transparent plastic cage (25 ⫻ 15 ⫻ 27 cm).
`Temperature was measured again three times at 15-min intervals.
`Isolation yielded an enhanced body temperature that putatively
`reflects a stress-induced anxiogenic response. SSR125543A, diaze-
`pam, or antalarmin were administered p.o. or i.p. 60-min before
`basal temperature measurement. Data were analyzed by two-way
`ANOVA (time ⫻ treatment) with repeated measures, followed by
`Dunnett’s t test.
`Stress-Induced Cortical Norepinephrine Release in Rats.
`Cortical extracellular NE levels were increased after a 15-min tail
`pinch in awake rats (Funk and Stewart, 1996). They were measured
`in 30-␮l dialysate samples by a high-performance liquid chromatog-
`raphy system with coulometric detection as previously described
`(Marco et al., 1998), except for the mobile phase, which contained 7%
`
`336
`
`Griebel et al.
`
`Fig. 1. Effects of diazepam (f), antalarmin (_), and SSR125543A (^) in
`the punished drinking conflict test in rats. Data represent mean ⫾ S.E.M.
`多, P ⬍ 0.05 (Dunnett’s t test). n ⫽ 14 to 20.
`
`Anxiolytic-Like Activity of SSR125543A
`Punished Drinking Test in Rats. The procedure was a modifi-
`cation of the technique described by Vogel et al. (1971). At the
`beginning of the experiment, rats, deprived of water but not of food
`for 48 h before testing, were placed in cages (32 ⫻ 25 ⫻ 30 cm) with
`a stainless steel grid floor. Each cage was placed in sound-attenuated
`boxes that were well ventilated and contained a drinking tube con-
`nected to an external 50-ml burette filled with tap water. Trials were
`started only after the animal’s tongue entered in contact with the
`drinking tube for the first time. An electric shock (0.6 mA/500 ms)
`was delivered to the tongue after every 20 licks. The number of
`shocks was recorded automatically during a 5-min period. Data were
`analyzed with one-way analysis of variance (ANOVA). Subsequent
`comparisons between treatment groups and control were carried out
`using Dunnett’s t test. Experiments were performed 30 min (anta-
`larmin and diazepam) or 60 min (SSR125543A) after i.p. injection of
`the compounds. A dose of 2.5 mg/kg i.p. morphine was tested as
`negative control because this test involves the application of painful
`stimuli.
`Elevated Plus-Maze Test in Rats. The test apparatus is based
`on that described by Pellow et al. (1985). All parts of the apparatus
`were made of dark polyvinylplastic with a black rubber floor. The
`maze was elevated to a height of 50 cm with two open (50 ⫻ 10 cm)
`
`TABLE 2
`Effects of SSR125543A, antalarmin, and diazepam in the elevated plus-maze test in rats
`Data represent mean ⫾ S.E.M. n ⫽ 7 to 14.
`
`Compound
`
`Dose
`
`Time Open Arms
`
`Entries Open Arms
`
`Head-Dips
`
`Attempts
`
`Total Arm Entries
`
`SSR125543A
`
`Antalarmin
`
`Diazepam
`
`mg/kg p.o.
`0
`3
`10
`30
`0
`3
`10
`30
`0
`1
`3
`10
`
`%
`10.5 ⫾ 2.5
`13.3 ⫾ 3.5
`25.3 ⫾ 4.5
`17.2 ⫾ 3.7
`13.4 ⫾ 1.6
`14.6 ⫾ 5.6
`24.1 ⫾ 3.9
`30.9 ⫾ 5.2*
`12.4 ⫾ 2.0
`16.5 ⫾ 4.8
`32.9 ⫾ 6.3*
`49.1 ⫾ 7.2*
`
`* P ⬍ 0.05 (Dunnett’s t test).
`
`%
`22.1 ⫾ 4.3
`23.2 ⫾ 5.3
`44.7 ⫾ 6.0
`33.7 ⫾ 6.2
`32.7 ⫾ 2.9
`27.3 ⫾ 8.1
`43.3 ⫾ 5.9
`47.3 ⫾ 3.1
`30.1 ⫾ 3.1
`30.8 ⫾ 7.5
`45.4 ⫾ 7.5
`63.0 ⫾ 6.0*
`
`14.9 ⫾ 2.1
`19.9 ⫾ 3.2
`26.1 ⫾ 2.7
`19.2 ⫾ 2.6
`12.1 ⫾ 2.2
`12.9 ⫾ 2.9
`29.4 ⫾ 4.3*
`26.3 ⫾ 3.1*
`17.9 ⫾ 3.0
`16.6 ⫾ 3.5
`26.4 ⫾ 3.9
`43.4 ⫾ 5.5*
`
`7.4 ⫾ 0.6
`6.2 ⫾ 0.5*
`4.9 ⫾ 0.4*
`5.4 ⫾ 0.4*
`8.0 ⫾ 0.5
`7.9 ⫾ 0.6
`6.3 ⫾ 1.1
`5.9 ⫾ 1.0
`7.5 ⫾ 0.6
`7.5 ⫾ 0.8
`4.1 ⫾ 0.6*
`1.4 ⫾ 0.3*
`
`10.7 ⫾ 1.0
`11.1 ⫾ 0.9
`12.1 ⫾ 0.6
`12.4 ⫾ 1.0
`11.7 ⫾ 1.4
`11.9 ⫾ 1.0
`14.3 ⫾ 0.6
`12.3 ⫾ 1.1
`11.4 ⫾ 0.8
`13.3 ⫾ 1.5
`14.1 ⫾ 1.1
`12.6 ⫾ 1.6
`
`4
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`Psychopharmacological Profile of SSR125543A
`
`337
`
`a neon tube fixed on the ceiling provided the room illumination so
`that the light intensity in the center of the illuminated box was 1000
`lux. An opaque plastic tunnel (5 ⫻ 7 ⫻ 10 cm) separated the dark box
`from the illuminated one. At the beginning of the experiment, a
`mouse was placed in the illuminated box, facing the tunnel. Record-
`ing started when the animal entered the tunnel for the first time.
`The apparatus was equipped with infrared beams and sensors capa-
`ble of recording the following parameters during a 4-min period: 1)
`time spent by mice in the lit box; 2) attempt at entry into the lit box
`followed by avoidance responses (this includes stretched attend pos-
`ture; the mouse stretches forward and retracts to original position);
`3) total number of tunnel crossings; and 4) activity in the lit box.
`Data were analyzed with one-way ANOVA followed by Dunnett’s t
`test analysis. Experiments were performed 30 min (antalarmin and
`diazepam) or 60 (SSR125543A and diazepam) min after p.o. admin-
`istration of the compounds.
`Four-Plate Test in Mice. The test apparatus is based on that
`described by Boissier et al. (1968). The apparatus consisted of a cage
`with a floor composed of four rectangular metal plates connected to
`a device that can generate electric shocks (1 mA, 0.2 s). After a 15-s
`latency period, the animal is subjected to an electric shock every time
`it went from one plate to another. The number of punished crossings
`is recorded during a 1-min test period. Experiments were carried out
`60 min after p.o. administration of SSR125543A, antalarmin, or
`diazepam. In a second experiment, the duration of the anxiolytic-like
`action of 3 mg/kg p.o. SSR125543A was investigated. Mice were
`administered with the compound and placed in the apparatus 1, 2, 4,
`or 6 h later. Each animal was tested once. In a third experiment,
`possible development of tolerance to the anxiolytic-like activity was
`investigated after repeated administration of SSR12543. The com-
`pound was given orally at doses of 3 or 10 mg/kg, once daily for eight
`consecutive days. Data were analyzed by one-way ANOVA followed
`by Dunnett’s t test.
`Mouse Defense Test Battery. The test was conducted in an oval
`runway as described previously (Griebel et al., 1997). Procedure: 1)
`Pretest: 3-min familiarization period. Sixty minutes after p.o. admin-
`istration of SSR125543A, antalarmin, or diazepam, subjects were
`placed into the runway for a 3-min familiarization period, in which
`line crossings were recorded. 2) The rat avoidance test. Immediately
`after the 3-min familiarization period, the experimenter introduced a
`hand-held dead rat (killed by CO2 inhalation just before the begin-
`ning of the experiment) five times at one end of the runway and
`brought up to the subject at a speed of approximately 0.5 m/s.
`Approach was terminated when contact with the subject was made
`or the subject ran away from the approaching rat. If the subject fled,
`avoidance distance (the distance from the rat to the subject at the
`point of flight) was recorded. 3) Chase/flight test. The rat was then
`brought up to the subject at a speed of approximately 2 m/s. A
`constant distance of 2 m separated the rat and the subject when the
`former was introduced in the runway. The following parameters
`were recorded: number of stops (pause in movement), orientations
`(subject stops then orients the head toward the rat) and chase speed
`(measured when the subject is running straight). The rat was re-
`moved after the chase was completed. 4) Straight alley. By the
`closing of two doors (60 cm distant from each other), the runway was
`then converted to a straight alley in which the subject was con-
`strained. The rat was introduced in one end of the straight alley.
`During 30 s, the number of approaches/withdrawals (subject must
`move more than 20 cm forward from the closed door then return to it)
`and immobility time were recorded. After this session, it was re-
`moved from the straight alley area. 5) Forced contact. Finally, the
`experimenter brought the rat up to contact the subject in the straight
`alley. Approaches were directed quickly (within 1 s) to the subject’s
`head. For each such contact, upright postures and bites by the
`subjects were noted. 6) Post-test: Contextual defense. Immediately
`after the forced contact test, the rat was removed and the doors were
`opened. Escape attempts were recorded during a 3-min session. Data
`were analyzed by one-way ANOVA, followed by Dunnett’s t test.
`
`Fig. 2. Effects of SSR125543A, antalarmin, and diazepam on isolation
`stress-induced hyperthermia in rats. Data represent mean ⫾ S.E.M. 多,
`P ⬍ 0.05 (Dunnett’s t test). n ⫽ 7 to 17.
`
`acetonitryl as organic agent. The analytical system consisted of an
`electrochemical detector ESA Coulochem II equipped with a model
`5014 analytical cell (ESA, Chelmsford, MA). The NE levels in frac-
`tional samples were converted to a percentage of the mean value of
`the 45-min baseline measurements before treatment. Time course
`effects of tail pinch on NE levels were analyzed by ANOVA with
`repeated measures,
`followed by Dunnett’s
`test analysis.
`t
`SSR125543A and antalarmin were administered i.p. 30 min before
`tail pinch. Compound antagonism was evaluated during the tail
`pinch sampling collection. Statistical analysis was carried out using
`ANOVA followed by Dunnett’s t test.
`Light/Dark Test in Mice. The test apparatus is based on that
`described by Misslin et al. (1989). It consisted of two polyvinylchlo-
`ride boxes (20 ⫻ 20 ⫻ 14 cm) covered with Plexiglas. One of these
`boxes was darkened. A desk lamp placed 20 cm above the lit box and
`
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`Fig. 3. A, effect of 15 min tail pinch on prefrontal cortex NE release. The changes in NE levels are expressed as a percentage of the mean value of the
`three basal samples collected at ⫺60, ⫺45, and ⫺30 min before the tail pinch. Each data point represents mean ⫾ S.E.M of five to six animals, P ⬍
`0.01 compared with control group by ANOVA with repeated measures followed by Dunnett’s t test. B, reversal of tail pinch-induced NE release by
`
`SSR125543A and antalarmin given i.p. 180 min ((cid:0)) and 30 min (^) before pinch, respectively. 䡺, vehicle. Each data point represents mean ⫾ S.E.M
`
`of NE release during tail pinch from five to seven animals. 多, P ⬍ 0.05, 多多 P ⬍ 0.01 compared with respective control group by ANOVA followed by
`Dunnett’s t test.
`
`Social Defeat Stress-Induced Anxiety in Elevated Plus-
`Maze in Mice. Social defeat was used as stressor before exposure to
`the elevated plus-maze. This stressor has been shown to produce
`significant anxiogenic-like activity without any physical signs of
`distress. The procedure was a modification of the technique de-
`scribed by Miczek (1979). A naı¨ve mouse was placed in the cage of a
`resident male aggressor, which was selected for high levels of ag-
`gression (Simiand et al., 1993). Social agonistic offensive and defen-
`sive behaviors were interrupted by the experimenter and the in-
`truder removed from the area when it displayed a submissive
`posture after being attacked. Thereafter, the intruder was returned
`to the resident cage for 60 min and placed in a cylindric wire mesh
`enclosure to avoid physical contact or injury. At the end of the
`interaction period, the intruder mouse was placed onto the central
`platform of the elevated plus-maze during a 5-min period (Lister,
`1987). To increase slightly open arm exploration, a rim (1 cm in
`height) surrounded the perimeter of these arms. Time spent in open
`arms was recorded. The results were expressed as mean ratio of time
`
`spent in open arms to total time spent in both open and closed arms.
`The compounds were administered i.p. or p.o. 15 min (antalarmin),
`30 min (diazepam), or 60 min (SSR125543A) before social defeat.
`Data were analyzed by a single factor ANOVA or with the nonpara-
`metric Kruskal-Wallis test. Subsequent comparisons between treat-
`ment groups and control were carried out using Dunnett’s t test
`procedure or the nonparametric Mann-Whitney U test with ␣adjust-
`ment of Holm, respectively.
`Maternal Separation-Induced Distress Vocalizations in
`Guinea Pig Pups. The procedure was adapted from that described
`by Molewijk et al. (1996). Briefly, from day 9 of age, pups entered at
`the most, three pretest sessions (with 2-day intervals) consisting of 5
`min of isolation in a sound-attenuated cage equipped with white
`noise and white illumination, and the duration of their vocalizations
`were recorded by the experimenter. Immediately after the 5-min
`isolation, the subjects were returned to their mothers and litter-
`mates. Pups emitting vocalization during at least 120 s entered
`subsequent compound experimentation. Each pup was tested with
`
`TABLE 3
`Effects of SSR125543A, antalarmin, and diazepam in the light/dark test in mice
`Data represent mean ⫾ S.E.M. n ⫽ 12 to 14.
`
`Compound
`
`SSR125543A
`
`Diazepam
`Antalarmin
`
`Diazepam
`
`Dose
`
`mg/kg i.p.
`0
`1
`3
`10
`30
`2.5
`0
`1
`3
`10
`30
`3
`
`* P ⬍ 0.05 (Dunnett’s t test).
`a Number of beams crossed in the lit box.
`
`Time in
`Lit Box
`
`s
`20.1 ⫾ 9.2
`2.6 ⫾ 1.9
`4.0 ⫾ 2.7
`3.4 ⫾ 3.2
`14.9 ⫾ 6.7
`83.6 ⫾ 14.7*
`16.6 ⫾ 10.0
`10.7 ⫾ 6.8
`10.3 ⫾ 7.8
`14.3 ⫾ 6.8
`25.5 ⫾ 10.8
`125.8 ⫾ 22.9*
`
`Activity in
`Lit Boxa
`
`4.6 ⫾ 2.3
`0.1 ⫾ 0.1
`0.9 ⫾ 0.7
`0.3 ⫾ 0.3
`3.5 ⫾ 1.9
`27.4 ⫾ 5.4*
`6.6 ⫾ 4.0
`2.2 ⫾ 2.2
`3.0 ⫾ 2.0
`2.8 ⫾ 1.8
`5.5 ⫾ 2.2
`27.8 ⫾ 7.5*
`
`Tunnel
`Crossings
`
`5.1 ⫾ 1.7
`1.6 ⫾ 0.3
`2.4 ⫾ 0.9
`1.6 ⫾ 0.4
`5.1 ⫾ 1.4
`15.4 ⫾ 2.1*
`3.4 ⫾ 1.2
`2.3 ⫾ 0.7
`3.4 ⫾ 1.5
`4.4 ⫾ 1.8
`4.9 ⫾ 1.6
`6.8 ⫾ 1.8
`
`Attempts
`
`20.2 ⫾ 2.6
`20.4 ⫾ 2.9
`20.3 ⫾ 1.7
`22.8 ⫾ 2.4
`20.7 ⫾ 1.8
`11.8 ⫾ 2.2*
`19.5 ⫾ 1.9
`20.2 ⫾ 1.4
`23.2 ⫾ 3.9
`21.9 ⫾ 2.4
`18.0 ⫾ 2.5
`5.8 ⫾ 1.3*
`
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`Psychopharmacological Profile of SSR125543A
`
`339
`
`were analyzed by a two-way ANOVA (treatment ⫻ week) with re-
`peated measures followed by Newman-Keuls post hoc test. At the
`end of the 10-week CMS period, mice were tested in the elevated
`plus-maze and the light/dark tests (for details, see above) to assess
`the impact of CMS on anxiety levels. These behaviors were evaluated
`because individuals with a major depressive episode frequently
`present with symptoms of anxiety (DSM-IV, 1994). In the elevated
`plus-maze test, anxiety was evaluated by measuring the number of
`mice that entered the open arms, whereas in the light/dark test, it
`was assessed by measuring activity in the illuminated box. In the
`former, results were expressed as total number of mice that entered
`the open arms and analyzed by a chi square independence (␹2) test,
`whereas in the latter, data were analyzed by a Student’s t test. The
`administration of 10 mg/kg SSR125543A started 4 weeks after the
`beginning of the CMS. Animals were treated i.p. once a day until all
`experiments were completed (30 days).
`
`Evaluation of Potential Side Effects of SSR125543A
`Effects on Spontaneous Locomotor Activity in Mice: Ac-
`timeter. Testing was conducted in square, clear Plexiglas boxes
`(22 ⫻ 27 ⫻ 10 cm) equipped with infrared beams and sensors and
`placed in sound-attenuated cupboards. Horizontal locomotor activity
`was quantified as total number of beams crossed during a 20-min
`period. Sixty minutes after p.o. administration of vehicle or various
`doses of SSR125543A, subjects were placed individually in the center
`of the apparatus. ED50 values were calculated by probit analysis.
`Effects on Muscle Tone in Mice: Horizontal Wire Test. It
`consisted of individually taking mice by the tail and allowing them to
`grasp a horizontally strung wire (20 cm above the bench level, 2 mm
`in diameter, 15 cm in length) with their forepaws. Inability to grasp
`the wire with the forepaws or inability to actively grasp the wire
`within 5 s with at least one hindpaw is measured. ED50 values were
`calculated by probit analysis. Experiments were performed 60 or 120
`min after oral administration of SSR125543A.
`Effects on Motor Coordination in Mice: Rotarod Test. The
`apparatus consisted of a plastic cylinder (4 cm in diameter) turning
`at 4 turns/min. Sixty or 120 min after oral administration of
`SSR125543A, mice were placed on the turning rotarod. The occur-
`rence of fall from the rotarod was noted during the 2-min period that
`followed. ED50 values were calculated by probit analysis.
`Effects on Learning and Memory in Mice: Passive Avoid-
`ance Test. The apparatus consisted of a black and white two-
`compartment box separated by a “guillotine” door. The white com-
`partment was small (10 ⫻ 10 ⫻ 12 cm), lit by a 100-W bulb and with
`plastic floor. The black compartment was large (22.5 ⫻ 16 ⫻ 12 cm)
`with a stainless grid floor connected to a constant current shock
`generator. Sixty minutes after oral treatment with SSR125543A,
`mice were placed in the small light compartment. Entrance into the
`dark box usually occurred within 30 s and was punished by an
`electri