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Neuropharmacology 57 (2009) 227–234

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Neuropharmacology

journal homepage: www.elsevier .com/locate/neuropharm

The group III mGlu receptor agonist ACPT-I exerts anxiolytic-like but notantidepressant-like effects, mediated by the serotonergic and GABA-ergic systems

K. Stachowicz a, A. K1odzinska a, A. Palucha-Poniewiera a, S. Schann c, P. Neuville c, A. Pilc a,b,*

a Institute of Pharmacology, Polish Academy of Sciences, Smetna 12, 31-343 Krakow, Polandb Collegium Medicum, Jagiellonian University, Krakow, Polandc Domain Therapeutics, SA, BIOPARC, Boulevard Sebastien Brant, 67400 Illkirch, France

a r t i c l e i n f o

Article history:Received 29 November 2008Received in revised form5 June 2009Accepted 8 June 2009

Keywords:AnxietyDepressionACPT-IGroup mGlu III receptorsmGlu4 receptorsmGlu8 receptorsGABASerotoninStress-induced hyperthermiaVogel testForced swim testTail suspension testElevated plus-maze testMiceRatsImipramineDiazepam

* Corresponding author at: Institute of PharmaSciences, Smetna 12, 31-343 Krakow, Poland. Tel.: þ6374500.

E-mail address: nfpilc@cyf-kr.edu.pl (A. Pilc).

0028-3908/$ – see front matter � 2009 Elsevier Ltd.doi:10.1016/j.neuropharm.2009.06.005

a b s t r a c t

Our earlier studies have demonstrated that (1S,3R,4S)-1-aminocyclo-pentane-1,3,4-tricarboxylic acidACPT-I, a group III mGlu receptor agonist, produced anxiolytic-like and antidepressant-like actions aftercentral administration. Here we describe the anxiolytic-like effects of ACPT-I after intraperitonealadministration in the stress-induced hyperthermia (SIH), elevated plus-maze (PMT) tests in mice and inthe Vogel test in rats. However, the compound did not produce antidepressant-like effects in the tailsuspension test (TST) or in the forced swim test (FST) in mice. The potential anxiolytic effect of ACPT-I(20 mg/kg) in the SIH test was inhibited by the benzodiazepine receptor antagonist flumazenil (given i.p.,10 mg/kg), and by a 5-HT1A receptor antagonist N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridynyl) cyclohexane-carboxamide (WAY100635) (0.1 mg/kg s.c.). At the same time, ritanserin (0.5 mg/kg i.p.), the 5-HT2A/C receptor antagonist, did not change the anxiolytic-like effects of ACPT-I. The resultsof these studies indicate that the GABA-ergic and serotonergic systems are involved in the potentialanxiolytic action of ACPT-I.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

The metabotropic glutamate receptors constitute a novel class ofreceptorsunderinvestigationasanewtargetformultipletherapeuticindications, including anxiety and depression (Palucha and Pilc,2002, 2007). mGlu receptors are classified intothree groups based ontheir amino acid sequence homology and intracellular coupling (Pinand Duvoisin, 1995). Group I mGlu receptors (mGlu1 and mGlu5)activate the phosphatidylinositol hydrolysis/Ca2þ signal trans-duction pathway, while group II (mGlu2 and mGlu3) and group III(mGlu4,mGlu6,mGlu7andmGlu8)receptorsinhibitadenylylcyclase

cology, Polish Academy of48 12 6623284; fax: þ48 12

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(Conn and Pin,1997). Stimulation of presynaptic group II and III mGlureceptors also leads to the inhibition of cAMP accumulation in thebrain (Schoepp et al.,1992) and to inhibition of neuronal excitability(Schoepp, 2001). Group II and III mGlu receptors are mainly localizedpresynaptically (Schoepp, 2001) and serve as autoreceptors at brainstructures (Gereau and Conn, 1995; Manzoni and Bockaert, 1995;Ugolini and Bordi, 1995; Cartmell and Schoepp, 2000). It has beenshown that activation of presynaptic mGlu receptors located at glu-tamatergic nerve terminals causes a decrease in glutamate release,therefore inhibiting glutamatergic excitatory transmission (Glaumand Miller, 1994; Cartmell and Schoepp, 2000). Hence, agentsstimulating presynaptic autoreceptors (including group II and IIImGlu receptor agonists) can act as functional antagonists of theglutamatergic system (Lovinger and McCool, 1995; Manzoni andBockaert,1995). Group III mGlu receptors are located in brain regionsrelated to mood regulation (Kinoshita et al., 1998).

K. Stachowicz et al. / Neuropharmacology 57 (2009) 227–234228

(1S,3R,4S)-1-aminocyclo-pentane-1,3,4-tricarboxylic acid (ACPT-I), a selective group III mGlu receptor agonist, was first described in1997 (Acher et al., 1997) and shows preferential affinity for mGlu4,mGlu6 and mGlu8 receptors (Goudet et al., 2008). It was found thatapplication of ACPT-I to the rat hippocampus induced significantanxiolytic-like action in the Vogel test (Tatarczynska et al., 2001,2002; Pa1ucha et al., 2004), and this anxiolytic-like effect wasreversed in a dose-dependent manner by a group III mGlu antago-nist CPPG (Pa1ucha et al., 2004). Moreover, icv injection of ACPT-Iresults in antidepressant-like effects (Pa1ucha et al., 2004). Sincerecent data indicate that ACPT-I penetrates into the brain afterperipheral administration (Palucha-Poniewiera et al., 2008), thepresent study was designed to investigate the possible anxiolytic-and antidepressant-like effects of this compound after i.p. admin-istration. The mechanism of action of ACPT-I after i.p. administrationwas also investigated. ACPT-I produced anxiolytic-like but notantidepressant-like effects. The anxiolytic-like effects of ACPT-Iwere inhibited by the benzodiazepine receptor antagonist fluma-zenil, and by a 5-HT1A receptor antagonist WAY100635. The resultsof our studies indicated that overall stimulation of mGlu4 andmGlu8 receptors evokes anxiolysis in rats and that GABA-ergic andserotonergic systems are involved in the potential anxiolytic actionof ACPT-I.

2. Materials and methods

2.1. Animals and housing

Male Wistar rats (200–250 g) were used to assess anti-anxiety effects in theconflict drinking test, male C57BL/6J mice (23–25 g) were used in the tail suspensiontest and in the locomotor activity test, and male Albino Swiss mice (23–25 g) wereused in the stress-induced hyperthermia test and elevated plus-maze or forcedswim tests. The animals were housed under standard laboratory conditions oflighting (light phase 6:00–18:00) and temperature (19–21 �C), with food and waterfreely available. All subjects were experimentally naive and used only once in eachtest. All the compounds used in behavioral studies were administered intraperito-neally (i.p.), with an exception of WAY100635, which was used in an aqueoussolution and administered subcutaneously (s.c.). In rats, the injections were given ina volume of 4 ml/kg, and in mice in a volume of 10 ml/kg. Experiments were per-formed during the light phase (10.00–14.00 h) by an observer unaware of thetreatment group. All procedures were conducted according to the guidelines of theNational Institutes of Health Animal Care and Use Committee and were approved bythe Ethics Committee of the Institute of Pharmacology, Polish Academy of Sciencesin Krakow.

2.2. Drugs

(1S,3R,4S)-1-aminocyclo-pentane-1,3,4-tricarboxylic acid (ACPT-I) (Tocris CooksonLtd, Bristol, UK), was dissolved in aqueous solution with 1.5 equivalent NaOH. In orderto obtain a dose of 10 mg/kg/ml, pH 7.0, the following procedure was used: 20 mgACPT-I was added to 1 ml of 0.276 M NaOH and 1 ml 1.8% NaCl. Lower doses of ACPT-Iwere obtained by dissolving the compound in proportionally lower concentrations ofNaOH. Flumazenil (Hoffman-La Roche, Basel), ritanserin (Research Biochemicals Inc.,Natick MA) and diazepam (Polfa, Poznan, Poland) were suspended in a 1% aqueoussolution of Tween 80 and administered intraperitoneally (i.p.). Clobazam (Urbanyl�)(Sanofi-Synthelabo), dispersed in 0.2% hydroxypropylmethylcellulose (HPMC) wasdissolved in physiological saline. N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridinyl)-cyclohexane-carboxamide trihydrochloride (WAY100635, synthesized byDr. J. Boksa, Institute of Pharmacology, Polish Academy of Sciences, Krakow, Poland)was used in an aqueous solution and administered subcutaneously (s.c.).

2.3. Conflict drinking test (Vogel test)

A modification of the method of Vogel et al. (1971) was used. On the first day ofthe experiment, the rats were adapted for 10 min to the test chamber, consisting ofa plexiglass box (27 � 27� 50 cm3), equipped with a grid floor of stainless steel barsand a drinking bottle containing tap water. After the adaptation period, the animalswere deprived of water for 24 h, and then they were placed in the test chamber foranother l0-min adaptation period during which they had a free access to thedrinking bottle. Afterwards, they were allowed a 30-min free-drinking session intheir home cage. On the next day, after another 24-h water deprivation, the ratswere placed again in the test chamber, and were allowed to drink for 30 s; imme-diately afterwards contacts to the drinking spout were punished with an electricshock (0.5 mA). The impulses, delivered as a square wave pulse between the grid

floor and the spout of the drinking bottle lasted for l s and were released every 2 s.The number of shocks accepted throughout a 5-min experimental session wasrecorded.

2.4. Modified stress-induced hyperthermia (SIH) in singly housed mice

Each experimental group consisted of ten animals in a modified stress-inducedhyperthermia (SIH) test. The animals were housed individually in a 26 � 21 �14 cmmacrolon cage, 24 h before testing. The procedure for the modified stress-inducedhyperthermia was adapted from Van der Heyden et al. (1997). For this assay, thebody temperature was measured for each mouse at t ¼ 0 min (T1) and t ¼ þ15 min(T2). Albino Swiss mice were placed into a new cage immediately following T1, withthe difference in temperature (T2 � T1) used as the measure of stress-inducedhyperthermia. The pilot studies of Spooren et al. (2002) demonstrated that a T2 � T1

interval of 15 min is optimal for the assay of SIH. A comparison between T1 invehicle-treated mice and those administered with a test-compound was used todetermine whether the agent affects body temperature alone. Rectal temperaturewas measured to the nearest 0.1 �C by an Ellab thermometer. The lubricatedthermistor probe (2 mm diameter) was inserted 20 mm into the rectum. The mousewas held at the base of the tail during this determination and the thermistor probewas left in place until a constant reading was obtained (�15 s).

2.5. Elevated plus-maze test in mice

The method, was carried out according to the method of Handley and Mithani(1984). Mice avoid open spaces (the open arms of an elevated plus-maze). Anxio-lytics increase exploratory activity in the open arms. The maze consists of 4 arms ofequal length and width (14 � 5 cm) arranged in the form of a plus sign (þ). Twoopposite arms are enclosed by 12 cm high walls (closed arms). The maze is raised56 cm above the floor. A mouse is placed in the center of the plus-maze and left toexplore for 5 min. The number of entries into the open and closed arms and the timespent in the open arms is recorded.

2.6. Tail suspension test

Immobility was induced by tail suspension according to the procedure of Steruet al. (1985). C57BL/6J mice were hung individually on a plastic string 75 cm abovethe table top with an adhesive tape placed roughly 1 cm from the tip of the tail. Theimmobility duration was recorded for 6 min. Mice were considered immobile onlywhen they hung passively and completely motionless.

2.7. Forced swim test in mice

The experiment was carried out according to the method of Porsolt et al. (1977).Briefly, the mice were individually placed in a glass cylinder (25 cm high, 10 cm indiameter) containing 6 cm of water maintained at 23–25 �C, and were left thereinfor 6 min. A mouse was regarded as immobile when it remained floating on water,making only small movements to keep its head above it. The total duration ofimmobility was measured by an experimenter during the final 4 min of a 6-min testsession, after a 2-min habituation period.

2.8. Shock threshold and free-drinking tests

To control the possibility of drug-induced changes in the perception ofa stimulus or in the thirst drive, which might have contributed to the activity inthe conflict drinking test, stimulus threshold measurements and a free-drinkingexperiment were also carried out. In both cases, the rats were treated before theexperiment in the same manner as that described in the conflict drinking test,including two 24-h water deprivation periods separated by 30 min of wateravailability. In the shock threshold test, the rats were placed in the box indi-vidually, and electric shocks were delivered through the grid floor. The shockthreshold was determined stepwise at 15 s shock free intervals by increasingmanually the current (0.1, 0.2, 0.3, 0.4, 0.5 mA). The shock lasted for l s and wasdelivered through the grid-floor until a rat showed an avoidance reaction (jumpor jerk) to the electric stimulus.

In the free-drinking test, each animal was allowed to drink from the water spout.Licking was not punished. The total amount of water (ml), consumed in 5 min, wasrecorded for each rat.

2.9. Locomotor activity

The spontaneous locomotor activity of C57BL/6J mice was measured in photo-resistor actometers (circular cages, 25 cm in diameter, 15 cm high, two light sources,two photoresistors), where the animals were placed individually 1 h after injectionof ACPT-I solution. The number of crossings of light beams was measured within10 min of experimental session. First measurement was performed 6 min afterplacing animals into actometers.

Fig. 1. The anxiolytic-like effect of ACPT-I in the Vogel test in Wistar rats. ACPT-I ordiazepam was injected 60 min before the test. Values represent the number of shocksaccepted (mean � S.E.M.) during the 5-min test, N ¼ 7–8 rats per group, ***P < 0.001versus vehicle-treated rats.

Fig. 2. Effect of ACPT-I (Fig. 2A) or diazepam (Fig. 2B) on SIH in singly housed AlbinoSwiss mice. Mice were pretreated with ACPT-I, diazepam or vehicle 60 min before thetest. Bars represent means � S.E.M. of N ¼ 10 mice. **P < 0.01, ***P < 0.001 versusvehicle-treated mice.

K. Stachowicz et al. / Neuropharmacology 57 (2009) 227–234 229

2.10. Statistical analysis

The data are presented as the mean � S.E.M. The significance of the differencesbetween means was evaluated by a one-way ANOVA, followed by Dunnett’sMultiple Comparison Test, with P < 0.05 being considered significantly different.Total SIH in mice injected with ACPT-I was analyzed by a two-way ANOVA followedby Bonferroni’s post hoc test. GraphPad Prism version 4.00 for Windows 2000(GraphPad Software, San Diego CA, USA) was used for data analysis.

3. Results

3.1. Effect of ACPT-I in Vogel’s tests

In rats, peripheral injections of ACPT-I at doses of 12.5, 25 and50 mg/kg, i.p., induced a dose-dependent increase in the numberof shocks accepted during the experimental session in the conflictdrinking test [F(4,42) ¼ 25.35, P < 0.0001], which reached signifi-cance for the two higher doses. These results suggest a potentanxiolytic-like activity of the compound. The dose of 50 mg/kg ofACPT-I increased the number of shocks accepted by about 600%,showing an efficacy similar to that of diazepam (10 mg/kg, i.p.,a dose which is routinely used in our experiments as a positivecontrol) (Fig. 1). In control experiments, neither the thresholdcurrent nor the water intake was changed by ACPT-I compared tothe vehicle treatment (Table 1). Hence, the possibility that the anti-conflict effect of that compound was caused by the reducedperception of the stimulus or by an increased thirst drive may beexcluded.

3.2. Effect of ACPT-I in the stress-induced hyperthermia test

ACPT-I, in doses of 10, 20 and 30 mg/kg, significantly reducedstress-induced hyperthermia [F(3,36)¼ 14.75, P< 0.0001] (Fig. 2 A),diazepam was more efficacious in that respect (Fig. 2B). The

Table 1Effect of ACPT-I on the shock threshold and amount of water consumed by water-deprived rats.

Compounds Dose (mg/kg) N Shock threshold (mA) Water consumption (ml)

Vehicle – 6 0.4 � 0.03 8.5 � 0.5ACPT-I 25.0 6 0.4 � 0.02 9.2 � 0.

50.0 6 0.4 � 0.02 8.6 � 0.9 nsF(2,15) ¼ 0.000, ns F(2,15) ¼ 0.2958, ns

ACPT-I was administered i.p. 60 min before the test. N ¼ number of rats per group.The values shown are the means � SEM.

anxiolytic-like effect of ACPT-I (20 mg/kg) was significantlyattenuated by flumazenil (10 mg/kg). Moreover, there was significantinteraction between the effects of ACPT-I and flumazenil[F(1,36)¼ 20.25, P< 0.0001], indicating an involvement of the GABA-ergic system in the anxiolytic-like response (Fig. 3). Furthermore, theanxiolytic-like effect of ACPT-I (20 mg/kg) was significantly attenu-ated by WAY100635 (0.1 mg/kg) [F(1,36)¼ 6.25, P¼ 0.0171] (Fig. 4A),and interaction between the effects of ACPT-I and WAY100635 wasalso significant [F(1,36) ¼ 12.25, P ¼ 0.0013]. Ritanserin (0.5 mg/kg)did not change the potential anxiolytic action of ACPT-I[F(1,35)¼ 2.24, P¼ 0.1434] (Fig. 4B). The mean basal temperature of:the control mice was 35.3 � 0.1 �C; ACPT-I (20 mg/kg) was

Fig. 3. Effect of flumazenil on the anxiolytic-like activity of ACPT-I in the SIH in mice.Mice were pretreated with ACPT-I and flumazenil 60 and 30 min before the test,respectively. Bars represent means � S.E.M. of N ¼ 10 mice. ***P < 0.001 versusvehicle-treated mice, ###P < 0.001 versus ACPT-I-treated mice.

Fig. 4. Effect of WAY100635 (Fig. 4A) or ritanserin (Fig. 4B) on the anxiolytic-likeactivity of ACPT-I in the SIH in mice. Mice were pretreated with ACPT-I or ritanserin,60 min and with WAY100635, 45 min before the test. Bars represent means � S.E.M. ofN ¼ 10 mice. ***P < 0.001 versus vehicle-treated mice, ###P < 0.001 versus ACPT-I-treated mice.

Fig. 5. Effect of treatment with imipramine or ACPT-I in the tail suspension test. ACPT-Ior imipramine was injected 60 min before the test. Values represent the immobilitytime (mean � S.E.M.) during 6-min test, N ¼ 10–9 mice per group, *P < 0.05 versusvehicle-treated mice.

K. Stachowicz et al. / Neuropharmacology 57 (2009) 227–234230

35.4 � 0.1�; Flumazenil was 35.2 � 0.1�; Ritanserin was 34.1 � 0.1�

and WAY100635 was 35.3 � 0.1�.

3.3. Effect of ACPT-I in the elevated plus-maze test

ACPT-I at a dose of 50 mg/kg, injected 30 min before the testsignificantly increased the number of open arms entries andamount of time spend in an open arms, without any influence onthe number of closed arm entries (Table 2), however it was lessefficacious than clobazam 8 mg/kg, used as positive control.

3.4. Effects of ACPT-I in the tail suspension test

ACPT-I used in doses of 25 and 50 mg/kg i.p. did not change thebehavior of mice in the tail suspension test; in contrast, imipramine(10 mg/kg), significantly decreased immobility time [F(3,30)¼ 5.890,P < 0.003], (Fig. 5).

3.5. Effects of ACPT-I in the forced swim test

Similar results were obtained in the forced swim test. ACPT-I (25and 50 mg/kg i.p.) was inactive, while imipramine (10 mg/kg),significantly decreased immobility time in mice [F(3,35) ¼ 21.21,P < 0.001],(Fig. 6). ACPT-I, administered i.p., at a dose of 10 or

Table 2Effects of ACPT-I in the elevated plus-maze test in mice.

Compound Dose(mg/kg)

N % of time in open arms % of open arm entries

Vehivcle – 10 22.2 � 4.9 2.5 � 0.6ACPT-I 12.5 10 23.6 � 5.4, P ¼ 0.8487, ns 2.7 � 0.5, P ¼ 0.7886, nsACPT-I 25 10 26.3 � 5.7, P ¼ 0.5923, ns 3.0 � 0.5, P ¼ 0.5045, nsACPT-I 50 10 54.4 � 11.4*, P ¼ 0.0181 6.1 � 1.3*, P ¼ 0.0213Clobazam 8 10 124.6 � 13.8***, P ¼ 0.0001 12.3 � 2.0***, P ¼ 0.0002

ACPT-I and clobazam was injected i.p. 30 min before the test. N ¼ number of rats pergroup. Values are means � S.E.M. Symbols indicate significance of differences inStudent’s t test, ns ¼ not significant.

30 mg/kg did not change the locomotor activity of mice within the6-min [F(2,23) ¼ 3.17, P ¼ 0.0626, ns], or 10-min [F(2,23) ¼ 2.941,P ¼ 0.0748, ns] experimental session (Fig. 7A,B).

4. Discussion

4.1. The anxiolytic-like effects of ACPT-I after peripheraladministration

The results of the current investigation show that ACPT-I,a micromolar agonist of mGlu4, mGlu6 and mGlu8 receptors(Goudet et al., 2008), induces anxiolytic-like but not antidepres-sant-like effect after i.p. administration. It was previously described,that following i.p. administration of 30 mg/kg ACPT-I brain levels of2–3 mM were reached (Palucha-Poniewiera et al., 2008). MGlu4 andmGlu8 receptors may therefore be responsible for ACPT-I anxio-lytic-like effects observed in this work, but not mGlu6 receptorswhich expression is limited to retina (Nakajima et al., 1993). Theanxiolytic-like effects of ACPT-I were previously reported afterintrahippocampal injections in rats (Tatarczynska et al., 2002;Pa1ucha et al., 2004). This report is therefore a validation of ACPT-I’sanxiolytic potential and is further proof that this amino-acid

Fig. 6. Effect of treatment with imipramine or ACPT-I in Porsolt’s test. ACPT-I orimipramine was injected 60 min before the test. Values represent the immobility time(mean � S.E.M.) during 6-min test, N ¼ 10–9 rats per group, *P < 0.01 versus vehicle-treated mice.

Fig. 7. Effects of ACPT-I, 10 or 30 mg/kg on the locomotor activity of mice. The valuesrepresent the number of light beams crossed in the actometers within 6 min (Fig. 7A)or 10 min (Fig. 7B). ACPT-I was administered i.p., 60 min before the test, 8 rats pergroup were used. Presented values are the means � S.E.M.

K. Stachowicz et al. / Neuropharmacology 57 (2009) 227–234 231

derivative is able to cross the BBB and that it produces not onlyantipsychotic-like effects after peripheral administration (Palucha-Poniewiera et al., 2008), but also anxiolytic effects.

The anxiolytic-like effects of the first selective mGlu7 receptoragonist, N,N0-dibenzyhydryl-ethane-1,2-diamine dihydrochloride(AMN082), were also recently described (Stachowicz et al., 2008).ACPT-I is an efficacious agonist at mGlu7 receptors, however itspotency for that receptor subtype lies in the millimolar range(Goudet et al., 2008), thus it is not likely that its anxiolytic-likeactivity is connected with the stimulation of mGlu7 receptors.

Whether mGlu4, mGlu8 or both receptors are involved inthe anxiolytic-like effects of ACPT-I, remains an open question. Onthe one hand, the mGlu8 receptor agonist ((S)-3,4-dicarboxy-phenylglycine) [(S)-3,4-DCPG] was able to produce anxiolytic-likeeffects in the fear-potentiated startle paradigm in rats after centraladministration (Schmid and Fendt, 2006) and in the stress-inducedhyperthermia test in mice after systemic administration (Rorick-Kehn et al., 2005). Moreover, the mGlu8 receptor knockout miceexhibit an anxiety phenotype (Linden et al., 2002; Duvoisin et al.,2005; Robbins et al., 2007). On the other hand, the selective mGlu8receptor agonist, RS-PPG ((RS)-4-phosphonophenylglycine), at a doseof up to 50 nmol/rat, did not change the number of shocks received inthe Vogel test after intrahippocampal injections (Pa1ucha et al.,2004). Also, (S)-3,4-DCPG did not produce anxiolytic-like effects afterintrahippocampal or intra-amygdalar injections in the Vogel test inrats, (Stachowicz et al., 2005). More data with the use of the moreselective mGlu8 receptor agents are necessary to clarify the problem.

As far as mGlu4 receptors are concerned, (�)-N-phenyl-7-(hydroxyimino) cyclopropa[b]chromen-1a- carboxamide (PHCCC),

a positive allosteric modulator of mGlu4 receptors (Maj et al., 2003;Marino et al., 2003), was shown to be anxiolytic after administra-tion into the basolateral amygdala (Stachowicz et al., 2004). Ifthe stimulation of mGlu8 or mGlu4 receptors, or both, brings aboutanxiolytic-like activity requires further studies using brain pene-trant selective mGlu4 or mGlu8 receptor agonists, antagonists,positive or negative allosteric modulators.

Concerning the possible mechanism of anxiolytic-like action ofACPT-I several possibilities exist:

1. Group III mGlu receptors are predominantly localized presyn-aptically on the glutamatergic terminals, and provide negativefeedback to modulate glutamate release (see Introduction andFig. 8[1]). The mechanism responsible for the anxiolytic action ofgroup III mGlu receptor agonists may resemble that reported forstimulation of presynaptic mGlu2/3 receptors (Schoepp, 2001).Group II mGlu receptor agonists, LY354740 and LY544344, havebeen proven to be effective for anxiety disorders (panic disorderas well as generalized anxiety disorder) (Schoepp et al., 2003;Danysz, 2005; Dunayevich et al., 2008). Thus, the decrease inglutamatergic neurotransmission might be responsible for theanxiolysis resulting from stimulation of either group II or groupIII mGlu receptors. The reduction of stimulatory glutamatergicneurotransmission via the activation of group III mGlu receptorsmay produce consequences similar to those that occur after theenhancement of GABA-ergic inhibitory neurotransmission bybenzodiazepines, which are the most commonly used anxiolyticdrugs (Rickels and Rynn, 2002).

2. Several studies have also shown that some mGlu receptors arepresent on non-glutamatergic neurons, such as GABA-ergicterminals, where they suppress GABA release when they areactivated (Schoepp, 2001; Vizi and Kiss, 1998). For example, therelease of GABA in the hippocampus is regulated by presynapticmGlu heteroreceptors (Janaky et al., 1994) including group IIImGlu receptors, stimulation of which suppressed GABA release(Schrader and Tasker, 1997). In our study, the benzodiazepinereceptor antagonist flumazenil inhibited the action of ACPT-I,indicating that the GABA-ergic system was involved in themodulation of the anxiolysis induced by this compound, con-firming the earlier data obtained after central administration ofACPT-I (Stachowicz et al., 2006). Inhibition of an interneuronalnetwork consisting of subsequent inhibitory GABA-ergicneurons (Freund and Buzsaki, 1996) may lead, via a disinhibitionprocess, to increased GABA-ergic neurotransmission and toanxiolysis mediated by a group III mGlu receptor agonist-ACPT-I.In that case group III mGlu receptors should function as heter-oreceptors for GABA-ergic interneuron, but not for GABA-ergicprojection neuron (see Fig. 8[2]), similar scheme was proposedto explain anxiolytic effects of LY354740, an agonist of group IImGlu receptors (Swanson et al., 2005), which share presynapticlocalization with group III mGlu receptors. The anxiolytic-likeeffects of many classes of drugs, both typical, like diazepam, andothers, including serotonergic (Fernandez-Guasti and Lopez-Rubalcava, 1998) or noradrenergic (Soderpalm et al., 1995)ligands, as well as the NMDA receptor antagonists (Przegalinskiet al., 2000), are blocked by flumazenil. This indicates thatthe GABA-ergic system is involved in the modulation of theanxiolysis mediated by the above-mentioned substances.

3. The central serotonin (5-HT) system is involved in various brainfunctions, as well as in the modulation of anxiety (see Millan,2003). Pharmacological agents that target components of the5-HT system have been successful in treating anxiety disorders.Selective serotonin reuptake inhibitors (SSRIs) are employed inthe treatment of a broad spectrum of anxiety disorders (Kentet al., 1998). The results of preclinical studies of subchronic and

Fig. 8. The possible mechanism of anxiolytic-like effects of ACPT-I. ACPT-I can evoke anxiolytic responses decreasing glutamatergic transmission by inhibition of glutamate releasevia stimulation of presynaptic group III mGlu receptors (1) or by an increase in GABA-ergic transmission. The increase in GABA-ergic transmission can occur due to inhibition ofGABA release from the GABA-ergic interneuron neuron via presynaptic group III mGlu heteroreceptors leading to disinhibition of the GABA-ergic projection neuron (2). Theinhibition of GABA release from the GABA-ergic interneuron can lead to disinhibition of serotonergic transmission (3) and via 5-HT1A receptors to an enhancement of the activity ofthe GABA-ergic projection neuron.

K. Stachowicz et al. / Neuropharmacology 57 (2009) 227–234232

chronic administration of SSRIs suggest that the outcome isa net increase in 5-HT neurotransmission (Blier and de Mon-tigny, 1999). Various 5-HT receptors exist in the central nervoussystem, among which the 5-HT1A receptors are the ones thathave been best characterized. 5-HT1A receptors are localizedpresynaptically on serotonergic neurons, where they play a roleof autoreceptors and postsynaptically mainly on non-seroto-nergic neurons. Postsynaptic 5-HT1A receptors exist mainly inlimbic structures, including hippocampus and they play a role inmood disorders. Systemic administration of partial agonistsand agonists of 5-HT1A receptors produces anxiolytic actions (fordiscussion see Millan, 2003). WAY100635, the selective 5-HT1A

receptor antagonist (Fletcher et al.,1993), was inactive in the SIHtest when given alone (confirming the data of Przegalinski et al.,1995), however, it inhibited the anxiolytic-like effect of ACPT-I,while ritanserin, the 5-HT2 receptor antagonist (Hoyer et al.,1994) was inactive in that respect, indicating the involvement of5-HT1A but not 5-HT2 receptors in the anxiolytic-like action.

While the interactions between group II mGlu receptors and 5-HT2

receptors have been described in a number of studies (e.g. Aghajanianand Marek, 2000; K1odzinska et al., 2002) and recently, a complexof 5-HT2/mGlu2 receptors has been identified and implicated inpsychosis (Gonzalez-Maeso et al., 2008), nothing is known aboutinteractions between mGlu and 5-HT1A receptors. If a complexes ofgroup III mGlu (mGlu4,8) and 5-HT1A exists and is involved inanxiolysis, obviously blockade of any component of the complexwould led to inhibition of its function, that however is to be studied.

Furthermore it is known that a number of GABA-positiveneurons are associated with serotonin receptors including 5-HT1A

ones (Peruzzi and Dut, 2004) and serotonin potentiates the inhibi-tory effects of GABA via serotonin 5-HT1A receptors (Kerr andBishop, 1992). If GABA-ergic interneuron is inhibited by ACPT-I,the serotonergic transmission to GABA-ergic projection neuron isenhanced via 5-HT1A postsynaptic receptors, leading to increased

GABA-ergic neurotransmission and to anxiolysis (see Fig. 8[3]). Ourdata strongly indicate the existence of a complex interactionsbetween the serotonergic (via 5-HT1A receptors) GABA-ergic andglutamatergic (via group III mGlu receptors) systems on the level ofthe anxiolytic-like action of group III mGlu receptor agonists. Suchinteractions were described for the effects of MTEP (Stachowiczet al., 2007a), the negative allosteric modulator of mGlu5 receptorsand to the central effects of group III mGlu receptor antagonistCPPG, which given into the amygdale produced the anxiolytic-likeeffects (Stachowicz et al., 2007b). The anxiolytic effects of bothsubstances were however blocked by ritenserine, but not byWAY100653 (Stachowicz et al., 2007a,b).

4.2. Lack of antidepressant-like effects of ACPT-I

ACPT-I did not change the behavior of mice in the tail suspensionand the forced swim tests; in contrast, imipramine significantlydecreased the immobility times. The fact that ACPT-I failed to changethe locomotor activity of rats indicated that its effect on locomotoractivity did not contribute to the lack of its effectiveness in these tests.It should be noted, however, that ACPT-I demonstrated antidepres-sant-like effects in a forced swim despair test after injections into ratbrain ventricles (Pa1ucha et al., 2004; K1ak et al., 2007). It is clear thatdifferences in species used, the routes of drug administration, andpenetration into different brain structures may account for thisdiscrepancy. Moreover as ACPT-I targets two brain mGlu subtypes(mainly mGlu4 and mGlu8) at the tested doses, further studies withmore selective ligands are needed to clarify this discrepancy betweencentral and peripheral modes of administration.

4.3. Summary

The results of this study show that ACPT-I produced anxiolytic-like effects as measured using the SIH and the elevated plus-maze

K. Stachowicz et al. / Neuropharmacology 57 (2009) 227–234 233

tests in mice and in the Vogel test in rats. Moreover, the potentialanxiolytic effect of ACPT-I was inhibited by the benzodiazepinereceptor antagonist flumazenil and by the 5-HT1A receptor antag-onist WAY100635. At the same time, the 5-HT2A/C receptorantagonist ritanserin did not change the anxiolytic-like effect ofACPT-I. The results of our studies indicate that the GABA-ergic andserotonergic systems are involved in the potential anxiolytic actionof ACPT-I. Moreover the compound did not produce antidepres-sant-like effects, therefore it can be inferred that the orthostericligands of group II and group III mGlu receptors produce anxiolytic-like and antipsychotic actions, but not antidepressant-like effectsafter peripheral administration at the preclinical level.

Acknowledgments

This study was supported in part by Funds for Statutory Activityof the Institute of Pharmacology Polish Academy of Sciences and inpart by Polpharma Foundation for Pharmacy and Medicine Grantno. 34/4/2006. K. Stachowicz was supported by grant from L’Orealfor Woman and Science.

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