Progress in Neuro-Psychopharmacology & Biological Psychiatry 33 (2009) 323–329

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Progress in Neuro-Psychopharmacology & BiologicalPsychiatry

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The involvement of serotonergic system in the antidepressant effect of zinc in theforced swim test

Bernadeta Szewczyk a, Ewa Poleszak c, Piotr Wlaź d, Andrzej Wróbel e, Eliza Blicharska f, Agnieszka Cichy g,Małgorzata Dybała g, Agata Siwek g, Lucyna Pomierny-Chamioło g, Anna Piotrowska g, Piotr Brański b,Andrzej Pilc b,h, Gabriel Nowak a,g,⁎a Laboratory of Trace Elements Neurobiology, Institute of Pharmacology, Polish Academy of Sciences, Smętna 12, PL 31-343 Kraków, Polandb Department of Neurobiology, Institute of Pharmacology, Polish Academy of Sciences, Smętna 12, PL 31-343 Kraków, Polandc Department of Pharmacology and Pharmacodynamics, Medical University of Lublin, Staszica 4, PL 20-081 Lublin, Polandd Department of Animal Physiology, Institute of Biology, Maria Curie-Skłodowska University, Akademicka 19, PL 20-033 Lublin, Polande Second Department of Gynecology, Medical University of Lublin, Jaczewskiego 8, PL 20-090 Lublin, Polandf Department of Analytical Chemistry, Medical University of Lublin, Staszica 6, PL 20-081 Lublin, Polandg Laboratory of Pharmacobiology, Department of Cytobiology and Histochemistry, Collegium Medicum, Jagiellonian University, Medyczna 9, PL 30-688 Kraków, Polandh Institute of Public Health, Collegium Medicum, Jagiellonian University, Grzegórzecka 20, PL 31-531 Krakow, Poland

Abbreviations: i.p., intraperitoneally; CIT, citalopram;RIT, ritanserin; WAY, WAY 1006335; pCPA, p-chlorophenaspartate; BDNF, brain-derived neurotrophic factor; FST⁎ Corresponding author. Laboratory of Trace Eleme

Pharmacology, Polish Academy of Sciences, Smętna 12,E-mail address: nowak@if-pan.krakow.pl (G. Nowak

0278-5846/$ – see front matter © 2008 Elsevier Inc. Aldoi:10.1016/j.pnpbp.2008.12.011

a b s t r a c t

a r t i c l e i n f o

Article history:

Recent preclinical data ind Received 9 September 2008Received in revised form 30 November 2008Accepted 15 December 2008Available online 25 December 2008

Keywords:AntidepressantsFSTpCPARitanserinWAY 100635Zinc

icated the antidepressant-like activity of zinc in different tests and models ofdepression. The present study investigates the involvement of the serotonergic system in zinc activity in theforced swim test (FST) in mice and rats. The combined treatment of sub-effective doses of zinc(hydroaspartate, 2.5 mg Zn/kg) and citalopram (15 mg/kg), fluoxetine (5 mg/kg) but not with reboxetine(2.5 mg/kg) significantly reduces the immobility time in the FST in mice. These treatments had no influenceon the spontaneous locomotor activity. Moreover, while the antidepressant-like effect of zinc (5 mg/kg) inthe FST was significantly blocked by pretreatment with inhibitor of serotonin synthesis, p-chlorophenyla-lanine (pCPA, 3×200 mg/kg), 5HT-2A/C receptor antagonist, ritanserin (4 mg/kg) or 5HT-1A receptorantagonist, WAY 1006335 (0.1 mg/kg), the zinc-induced reduction in the locomotor activity was not affectedby these serotonin modulator agents. These results indicate the specific involvement of the serotonergicsystem in antidepressant but not the motion behavior of zinc in mice. Also, an increase in the swimming butnot climbing parameter of the rat FST observed following zinc administration (2.5 and 5 mg Zn/kg) indicatesthe serotonin pathway participation. This present data indicates that the antidepressant-like activity of zincobserved in the FST involves interaction with the serotonergic system.

© 2008 Elsevier Inc. All rights reserved.

1. Introduction

Zinc is a trace element essential for normal brain function (Takeda,2000). Zinc is present in specific regions of the brain including thehippocampus, amygdala and cortex (Frederickson et al., 2005). Zincseems to modulate neuronal excitability (Smart et al., 1994;Frederickson et al., 2005) and is also thought to play an importantrole in synaptic plasticity (Li et al., 2001). Zinc can also function as asignaling molecule modulating protein function (Haase and Maret,

FX, fluoxetine; RB, reboxetine;ylalanine; NMDA, N-methyl-D-, forced swim test.nts Neurobiology, Institute ofPL 31-343 Kraków, Poland.).

l rights reserved.

2005; Krezel et al., 2007). Dietary zinc deprivation influences zinchomeostasis in the brain and leads to behavioral disturbances, such asanorexia, dysphoria, depression, aggression, impaired learning andcognitive function (Takeda, 2000; Takeda et al., 2008; Tassabehji et al.,2008) and some neurological disorders (Takeda, 2000; Mathie et al.,2006). Recent data indicated that zinc is implicated in the pathophy-siology of depression and the mechanism of action by antidepressantdrugs. Preclinical studies showed the antidepressant-like activity ofzinc in tests and models of depression. Zinc was active in the forcedswim test and tail suspension test (Kroczka et al., 2000, 2001; Nowaket al., 2003a,b; Rosa et al., 2003) and enhanced the antidepressantactivity of classical antidepressants (imipramine and citalopram) inthe forced swim test (Kroczka et al., 2001; Szewczyk et al., 2002; Rosaet al., 2003). Zinc was also active in several models of depression suchas: olfactory bulbectomy (Nowak et al., 2003b); chronic unpredictablestress (Cieslik et al., 2007) and chronic mild stress (Sowa-Kućma et al.,2008). Moreover, several clinical studies indicated that major

Fig. 1. The effects of joint administration of citalopram (CIT) and zinc (Zn) on the totalduration of immobility in the FST in mice. Zn and CIT were administered 0.5 h and 1 hbefore the test, respectively. The values represent mean±SEM (n=9–11 per group).⁎pb0.01 vs control vehicle-treated group (CTR). #pb0.01 vs Zn treated group.

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depression is accompanied by a lower serum zinc level (McLoughlinand Hodge, 1990; Maes et al., 1994, 1999; Nowak et al., 1999) which isnormalized after successful antidepressant therapy (McLoughlin andHodge, 1990; Maes et al., 1997; Schlegel-Zawadzka et al., 2000). Studyperformed in pregnant women showed that lower serum zincconcentration is correlated with a severity of antepartum andpostpartum depressive symptoms (Wojcik et al., 2006). Also,preliminary studies suggests that zinc supplementation may enhanceantidepressant therapy in patients with unipolar depression (Nowaket al., 2003a; Siwek et al., in review).

While both preclinical and clinical data strongly implicated theinvolvement of zinc in the pathophysiology and therapy of depression,themechanisms involved in the antidepressant activity of zinc are stillunder investigation.

There is a large body of evidence showing that zinc modulates thepost-synaptic glutamate receptors, which may be one of the possiblemechanisms involved in the antidepressant activity of zinc (Harrisonand Gibbons, 1994; Smart et al., 1994, see Szewczyk et al., 2008).Moreover, recently, the involvement of the adenosine receptors in theantidepressant-like effects of zinc in the forced swim test wasdemonstrated (Lobato et al., 2008). Since it has already beendemonstrated that imipramine and citalopram enhanced the zincantidepressant effect in the forced swim test (Kroczka et al., 2001;Rosa et al., 2003; Szewczyk et al., 2002) and there is some datasuggesting that zinc can directly modulate the function of theserotonin system (Garcia-Colunga et al., 2005), our present studydemonstrated the interaction between the serotonergic system (usingantidepressants and other serotonin modulating agents) and zinc inthe forced swim test.

2. Materials and methods

2.1. Animals

All procedures were conducted according to the National Institutesof Health Animal care and Use Committee guidelines and wereapproved by the Ethical Committee of theMedical University of Lublinand Collegium Medicum, Jagiellonian University, Kraków. The experi-ments were carried out on male Albino Swiss mice (25–30 g) or maleWistar rats (230–250 g). The animals were kept on a normal day–night cycle with free access to food and water. Each experimentalgroup consisted of 8–10 mice and 9–10 rats.

2.2. Drug administration

2.2.1. MiceZinc hydroaspartate — Zn (2.5 or 5 mg Zn/kg, Farmapol, Poznań,

Poland) alone or in combinationwith antidepressants was administratedintraperitoneally (i.p.) 0.5 h before the test. Citalopram (15 mg/kg, CIT,Lundbeck); fluoxetine (5 mg/kg, FX, Sigma); reboxetine (2.5 mg/kg, RB,Sigma) were dissolved in 0.9% NaCl, ritanserin (4 mg/kg, RIT, Tocris, UK)andWAY1006335 (0.1mg/kg,WAY, synthesizedbyDr. J. Boksa, Institute ofPharmacology, PAS, Kraków, PL) were dissolved in DMSO/0.9% NaCl(1:250) and administered i.p.1 h before the test. Control animals receivedthe appropriate vehicle. All solutions were administered at a volume of10 ml/kg.

2.2.2. RatsZinc hydroaspartate— Zn and fluoxetine (FX) were administered i.p.

three times: 24, 5 and 1 h before the test. Zn and FX were dissolved in0.9% NaCl and administered at a volume of 2 ml/kg. Control animalsreceived 0.9% NaCl.

The doses of zinc, antidepressants, serotonin antagonists andtreatment schedules were chosen on the basis of previous studies(Kroczka et al., 2001; Poleszak 2007; Redrobe et al., 1996; Redrobe andBourin, 1997; Szewczyk et al., 2002).

2.3. Forced swim test

2.3.1. MiceThe studieswere carried out according to themethod of Porsolt et al.

(1977). Mice were placed individually in glass cylinders (height 25 cm,diameter 10 cm) containing 10 cm of water, maintained at 25 °C. Theanimals were left in the cylinder for 6 min. After the first 2 min the totalduration of immobility was measured during the following 4 min test.The mouse was judged to be immobile when it remained floatingpassively in the water.

2.3.2. RatsThe studies were carried out according to the method of Porsolt

et al. (1978). The rats were placed in glass cylinders (height 40 cm,diameter 20 cm) containing 15 cm of water, maintained at 25 °C. Twoswim sessions were conducted: an initial 15 min pretest followed 24 hlater by 5 min test. Following both sessions rats were removed fromcylinders and returned to their home cages. Behavioral scoring wasperformed according to Detke et al. (1995) and during the 5 min testsession three different behaviors were rated : 1) immobility — ratswere judged to be immobile when it remained floating passively inthe water; 2) swimming — rats were judged to be swimming if theyweremaking active swimmingmotions, more than necessary to solelymaintain their head abovewater; 3) climbing— rats were judged to beclimbing when they were making active movements in and out of thewater with their forepaws, usually directed against the walls.

2.4. Serotonergic depletion

In order to investigate the possible contribution of the serotonergicsystem to the effect of zinc in the FST, mice were pretreated with p-chlorophenylalanine (pCPA, Sigma). pCPA is known to reduce theconcentration of brain serotonin by inhibiting its biosynthesis (KoeandWeissman, 1966; O'Leary et al., 2007). In the present experimentsthe mice were injected i.p. either with saline (control group) or withpCPA. pCPAwas administered at the dose of 200mg/kg once daily for 3consecutive days. On the fourth day (24 h after the last pCPAadministration), themice received saline or zinc hydroaspartate (5 mgZn/kg) 30 min before the test.

2.5. Locomotor activity

Locomotor activity was measured with photoresistor actometers(circular cages, diameter 25 cm, two light beams). Mice were placedindividually in an actometer for 10 min. Activity was measured at 5 min

Fig. 2. The effects of joint administration of fluoxetine (FX) and zinc (Zn) on the totalduration of immobility in the FST in mice. Zn and FX were administered 0.5 h and 1 hbefore the test, respectively. The values represent mean±SEM (n=9–10 per group).⁎pb0.01 vs control vehicle-treated group (CTR). #pb0.001 vs Zn treated group.

Fig. 4. The effects of pre-treatment with pCPA (200 mg/kg; i.p. once a day per 3 consecutivedays) on the Zn-induced reduction in immobility time in the FST inmice. The values representmean±SEM (n=8–10 per group). ⁎pb0.01 vs control vehicle-treated group (CTR).

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intervals to characterize the dynamics of the changes. The number ofcrossed light beamsby the animalswas recordedas the locomotoractivity.

2.6. Statistical analysis

The obtained data was evaluated by the one- or two-way analysisof variance (ANOVA) followed by the Bonferroni test: pb0.05 wasconsidered significant.

3. Results

3.1. Effect of combined administration of Zn and CIT in the FST

The effect of the combined administration of Zn and CITon the totalduration of the immobility time in mice is shown in Fig. 1. Zn (2.5 mgZn/kg) injected in combination with CIT (15 mg/kg) significantlyreduced (by 26.6%) the immobility time in the FST in mice (Fig. 1). CIT(15 mg/kg) and zinc (2.5 mg/Zn/kg) given alone had no effect on theimmobility time (Fig. 1). Two-way ANOVA demonstrated significanteffect of CIT [F(1,36)=13.30, p=0.0008], no effect of Zn [F(1,36)=2.28,p=0.13.95] and no interaction [F(1,36)=1.72, p=0.1982].

3.2. Effect of combined administration of Zn and FX in the FST

The effect of the combined administration of Zn and FX on the totalduration of the immobility time in mice is shown in Fig. 2. Zn (2.5 mg/

Fig. 3. The effects of joint administration of reboxetine (RB) and zinc (Zn) on the totalduration of immobility in the FST in mice. Zn and RB were administered 0.5 h and 1 hbefore the test, respectively. The values represent mean±SEM (n=9–10 per group).

Zn/kg) combined and injected with FX (5mg/kg) significantly reduced(22.4%) the immobility time in the FST in mice (Fig. 2). FX (5 mg/kg)and zinc (2.5 mg/Zn/kg) given alone had no effect on the immobilitytime (Fig. 2).

Two-wayANOVAdemonstrated significant effect of FX [F(1,34)=29.78,p=0.0001], significant effect of Zn [F(1,34)=12.36, p=0.0013] andsignificant interaction [F(1,34)=8.58, p=0.006].

3.3. Effect of combined administration of Zn and RB in the FST

The effect of the combined administration of Zn and RB on the totalduration of the immobility time in mice is shown in Fig. 3. Zn (2.5 mg/Zn/kg) or RB (2.5 mg/kg) injected alone or in combination had noeffect on the immobility time in the FST in mice (Fig. 3). Two-wayANOVA demonstrated significant effect of RB [F(1,34) =4.80,p=0.0355], no effect of Zn [F(1,34)=3.38, p=0.0746] and no interaction[F(1,34)=0.73, p=0.3995].

3.4. Effect of pre-treatment with pCPA on the antidepressant-like activityof Zn in the FST

The effects of Zn on the total duration of the immobility time andeffect of pre-treatment with pCPA on the effect produced in FST inmice are shown in Fig. 4. Zn treatment (5 mg/Zn/kg) significantlyreduced (by 21.2%) the immobility time in FST in mice (Fig. 4).Administration of pCPA (200 mg/kg i.p. once a day for 3 consecutivedays) had no effect on the immobility time, although it partially (by15%) antagonized the effect elicited by zinc in this test (Fig. 4). Two-way ANOVA demonstrated no effect of pCPA [F(1,32) = 0.82,p=0.3727], significant effect of Zn [F(1,32)=12.02, p=0.0015] andsignificant interaction [F(1,32)=5.33, p=0.0275].

3.5. Effect of combined administration of Zn and RIT or WAY in the FST

The effects of Zn on the total duration of the immobility time andthe effect of pre-treatment with RIT or WAY on the effect produced inthe FST in mice are shown in Fig. 5. Zn treatment (5 mg/Zn/kg)significantly reduced (by 20.8%) the immobility time in the FST inmice(Fig. 5). Administration of RIT or WAY had no effect on the immobilitytime, although it completely antagonized the effect elicited by zinc inthis test (by 17.8% and 16.6%, respectively, Fig. 5). Two-way ANOVAdemonstrated no effect of co-treatment [F(2,49)=1.07, p=0.3522],significant effect of Zn [F(1,49)=10.57, p=0.0021] and significantinteraction [F(2,49)=10.80, p=0.0001].

Fig. 5. The effects of joint pre-treatment with ritanserin (RIT) or WAY 100635 (WAY) and zinc (Zn) on the total duration of immobility in the FST in mice. Zn was administered 0.5 hbefore the test and RIT and WAY were administered 1 h before the test. The values represent mean±SEM (n=6–13 per group). ⁎pb0.001 vs control vehicle-treated group (CTR);#pb0.001 vs Zn treated groups.

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3.6. Effect of combined administration of Zn and antidepressants onlocomotor activity in mice

The effect of the combined administration of Zn and antidepres-sants on spontaneous locomotor activity in mice is shown in Table 1.Zn or antidepressants (CIT, FX, and RB) administered either alone orcombined together had no effects on the locomotor activity in mice(Table 1). Two-way ANOVA demonstrated for: (A): significant effect ofCIT [F(1,34)=5.31, p=0.0274], no effect of Zn [F(1.34)=0.64, p=0.4285]and no interaction [F(1,34)=0.58, p=0.4521] (5 min) and significanteffect of CIT [F(1,34)=7.83, p=0.0084], no effect of Zn [F(1.34)=1.68,p=0.2041] and no interaction [F(1,34)=0.34, p=0.56381] (10 min)];(B): no effect of FX [F(1,36)=0.00, p=0.9533], no effect of Zn [F(1.36)=0.01, p=0.9223] and no interaction [F(1,36)=0.37, p=0.5465] (5 min)and no effect of FX [F(1,36)=1.22, p=0.2760], no effect of Zn [F(1.36)=0.00, p=0.9923] and no interaction [F(1,36) =0.51, p=0.4814]

Table 1Effect of zinc and antidepressants on locomotor activity in mice

Treatment Dose (mg/kg) Activity counts

5 min 10 min

AControl – 122.9±11.7 190.6±21.0Zinc 2.5 122.2±10.6 169.1±14.3CIT 15 175.5±23.1 292.7±36.5Zinc+CIT Zn 2.5+CIT 15 148.7±20.6 236.0±45.1

BControl – 125.9±13.4 201.9±20.2Zinc 2.5 119.4±13.9 187.1±26.7FX 5 117.4±11.9 164.6±13.1Zinc+FX Zn 2.5+FX 5 126.4±11.5 179.0±19.9

CControl – 115.4±7.4 181.8±8.8Zinc 2.5 109.7±10.7 155.6±20.3RB 2.5 135.9±11.1 194.5±16.8Zinc+RB Zn 2.5+RB 2.5 101.9±9.3 176.8±23.3

Zinc hydroaspartate (Zn) was administered i.p. 0.5 h before the test. Citalopram (CIT);fluoxetine (FX) and reboxetine (RB) were administered 1 h before the test. The valuesrepresent mean±SEM (n=8–10 mice per group).

(10 min)]; (C): no effect of RB [F(1,35)=0.43, p=0.5160], no effect ofZn [F(1,35)=0.0478, p=0.9223] and no interaction [F(1,35)=2.147,p=0.1526] (5 min) and no effect of RB [F(1,35)=0.83, p=0.3689], noeffect of Zn [F(1,35)=1.41, p=0.2432] and no interaction [F(1,35)=0.04,p=0.8368] (10 min).

3.7. Effect of Zn and pre-treatment with pCPA, RIT or WAY on thelocomotor activity in mice

The effect of Zn and pre-treatment with pCPA, RIT or WAY on thelocomotor activity inmice is shown inTable 2. Zn treatment (5mg/Zn/kg)reduced (by 32.9%) the locomotor activity in mice (Table 2A). Pre-treatmentwith pCPAhadno influence on the Zn-induced reduction of thelocomotor activity in mice (Table 2A). Two-way ANOVA showedsignificant effect of Zn F(1,30)=26.66, pb0.0001, no significant effect ofpCPA F(1,30)=0.69, p=0.4115 and no interaction F(1,30)=0.11, p=0.7454(5min); significanteffectof ZnF(1,30)=16.46,p=0.0003andnosignificanteffect of pCPA F(1,30)=0.09, p=0.7725 and no interaction F(1,30)=0.18,p=0.6758 (10 min). Also, neither RIT nor WAY pre-treatment influencedZn-induced locomotor alterations (Table 2B). One-way ANOVA revealed F

Table 2Effect of zinc and serotonin modulators on locomotor activity in mice

Treatment Dose (mg/kg) Activity counts

5 min 10 min

AControl – 136.9±8.5 204.8±18.0Zinc 5 91.9±8.1* 126.9±10.7pCPA 200 132.2±10.6 207.6±28.5Zinc+pCPA Zn 5+pCPA 200 81.1±8.5**,## 111.6±18.1*,#

BControl – 147.3±15.5 206.4±23.7Zinc+RIT Zn 5+RIT 4 94.2±9.4* 120.4±9.3*Zinc+WAY Zn 5+WAY 0.1 84.3±10.8* 134.3±24.2

Zinc hydroaspartate (Zn) was administered i.p. 0.5 h before the test. Ritanserin (RIT) andWAY 100635 (WAY) were administered 1 h before the test. p-Chlorophenylalanine(pCPA) was administered once daily for 3 days, and the experiment was performed onthe fourth day. The values represent mean±SEM (n=6–11 mice per group).*pb0.05, **pb0.01 vs control; #pb0.05, ##pb0.01 vs pCPA treated group (Bonferroni test).

Fig. 6. The effect of fluoxetine (A) or zinc (B) administration on immobility, swimming and climbing parameters in the FST in rats. Fluoxetine (FX, 20mg/kg) or zinc (Zn)was administeredthree times: 24, 5 and 1 h before the test. The values represent mean±SEM (n=9–10 per group). (A) ⁎pb0.0001, #pb0.0001 vs control vehicle-treated group (CTR). (B) ⁎pb0.05, #pb0.05,##pb0.01 vs control vehicle-treated group (CTR) (Bonferroni test).

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(2,20)=6.0270, p=0.0089 (5 min) and F(2,20)=0.0252, p=4.447 (10 min).RITorWAYadministered alone did not alter the locomotor activity inmice(data not shown, performed in another experiment).

3.8. Effect of administration of Zn and FX on immobility, swimming andclimbing in the FST in rats

The effect of administration of Zn and FX on immobility, swimmingand climbing in the FST in rats is shown in Fig. 6. FX in a dose of 20mg/kg: reduced (by 49.1%) immobility, increased (by 100.6%) swimmingand had no influence on the climbing time (Fig. 6A). Student two-tailed t-test: immobility t(18)=6.239; swimming t(18)=6.429; climb-ing t(18)=0.02314.

Zinc dose-dependently, reduced (5 mg Zn/kg by 42.4%) immobilityand increased (2.5 mg Zn/kg by 72% and 5 mg Zn/kg by 80.4%)swimming time in the FST (Fig. 6B). None of the administered dosesinfluenced the climbing behavior of rats in the FST (Fig. 6B). ANOVA:immobility F(3,35)=4.447, p=0.0095; swimming F(3,35)=5.295,p=0.041; climbing F(3,35)=1.013, p=0.3984.

4. Discussion

Accumulated evidence, from animal models and human studies,supports the notion that alterations in NMDA receptors maycontribute to the pathophysiology and therapy of depression (seePittenger et al., 2007). As such, compounds that reduce the NMDAreceptor function exhibit an antidepressant-like activity in animaltests and models of depression (see Pittenger et al., 2007). Zinc is apotent antagonist of the NMDA receptor complex (Harrison andGibbons, 1994; Smart et al., 1994) and, therefore, similar to the NMDAreceptor antagonists, may induce antidepressant action via thisreceptor complex (see Szewczyk et al., 2008). Furthermore, chronictreatment with zinc enhances the BDNF gene and protein expressionand reduces the affinity of glycine to the glycine/NMDA receptors(Franco et al., 2007; Nowak et al., 2004; Sowa-Kućma et al., 2008, ourunpublished data). Moreover, antidepressant-like action of zinc in theforced swim test is altered by NMDA/NO pathway modulatory agents

and is antagonized by NMDA receptor agonists' co-treatment (Rosaet al., 2003; Poleszak et al., 2008, our unpublished data). Thus, one ofthe mechanisms involved in the antidepressant-like activity of zincseems to be the NMDA/glutamate pathway.

The other mechanism possibly involved in the antidepressant-likeactivity of zincmayalso be the serotonergic system. In thepresent study,we demonstrate the effect of zinc on the action of antidepressants withdifferent pharmacological profiles: citalopram and fluoxetine — selec-tive serotonin reuptake inhibitors (Hyttel,1994; Hyttel,1977; Pawlowskiet al., 1985; Pozzi et al., 1999) and reboxetine — a potent, selective, andspecific noradrenergic reuptake inhibitor (Versiani et al., 1999). Zincadministered jointlywith reboxetine (both in subeffective doses) didnotchange the behavior of animals in the FST. However, an additive effectwasobservedwhen zincwasgiven jointlywith specific serotoninuptakeinhibitors: citalopram or fluoxetine. Previously, such an additive effectfrom the combined treatment of zinc with imipramine (serotonin/noradrenaline uptake inhibitor) or citalopram in the FST was demon-strated (Kroczka et al., 2001; Rosa et al., 2003; Szewczyk et al., 2002).Therefore, this data suggests that the antidepressant-like effect of zincseems to be mediated through an interaction with the serotonergicrather than the noradrenergic system. This may be a specificphenomenon for the FST, since the recent study of Cunha et al. (2008)showed the synergistic effects of zinc with not only “serotonergic” butalso with “noradrenergic” and “dopaminergic” profile antidepressantsin the mouse tail suspension test.

Recently, Garcia-Colunga et al. (2005) reported that zinc enhancesthe 5-HT uptake in the adult rat corpus callosum, cingulate cortex andraphe nucleus. This data contradicts the effect observed in our presentexperiments. Moreover, no in vitro effect of zinc (at 10−4 M) on [3H]Citalopram binding to the 5-HT transporter in the rat cortexwas found(our unpublished data), suggesting a lack of zinc influence on the 5HTuptake. As such, zinc may indirectly release 5HT, which in turnactivates 5HT receptors as a part of the mechanism of antidepressantactivity. In fact, the NMDA receptor antagonists release/increase theconcentration of serotonin and increase the turnover of thistransmitter in the brain (Löscher et al., 1993; Martin et al., 1988; Yanet al., 1997).

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In order to further confirm a possible contribution of the serotonintransmission in the antidepressant effect of zinc, the animals werepre-treated with pCPA, an inhibitor of serotonin synthesis. Thedepletion of serotonin by pCPA did not alter the baseline activity inthe FST but completely reduced the antidepressant-like action causedby zinc. The ability to eliminate the antidepressant-like effect of zincby 5-HT depletion further supports the notion that the behavioraleffects of zinc in the FST require the serotonin system to be intact.

Additionally,wedemonstrated the involvementof 5HT1A and5HT2A/Creceptors in zinc antidepressant-like action in the FST by using selectiveantagonists. Since zinc-induced reduction in locomotor activity was notaffected by all serotonin modulator agents (pCPA, ritanserin, WAY100635), this clearly demonstrates a specific serotonin dependence ofzinc antidepressant but not locomotor activity.

Animal behavior in the FST in rats was evaluated for specificinvolvement of serotonergic or noradrenergic pathways in the anti-depressant activity (Detke et al.,1995). Enhancementof the serotonergicsystem reduces immobility and enhances swimming time, while thenoradrenergic system is connected with decreasing immobility andenhancement of climbing behavior (Detke et al., 1995). In the presentstudy, zinc, similar to fluoxetine, besides reducing the immobility time,dose dependently increases the swimming time but not the climbing.Thus, this increase in swimming yet not climbing parameter indicatesthe serotonin rather than the adrenergic pathway's contribution to theantidepressant-like action of zinc in the FST.

The serotonergic hypothesis of depression (Głowinski and Axelrod,1964; Schildkraut, 1965) connected depressive symptoms with areduced serotonin concentration in the brain synapse and, on the otherhand, the mechanism of antidepressant action with the enhancementof concentration of this neurotransmitter. This hypothesis is con-tinuously modifying (e.g. see Cryan and Leonard, 2000 for review),implicating the role of newly recognized serotonin receptors in thisconcept. The significance of 5HT1A receptor-mediated signaling inantidepressant-like activity in the FST was demonstrated (Detke et al.,1995; Abert and Lemonde, 2004). However, a blockade instead of theenhancement of 5HT2A/C receptors seems to have an antidepressanteffect in rodent screening tests (Deakin, 1988; Redrobe and Bourin,1997) and in clinical studies (Celada et al., 2004). Zinc antidepressant-like activity in the FST is dependent on serotonergic transmission via5HT1A and 5HT2A/C, thus meaning that zinc demonstrates the 5HT1Aprofile similar to the established antidepressants, while the impor-tance of 5HT2A/C receptors in this activity remains to be recognized.

5. Conclusion

The results described in this paper indicate that the antidepres-sant-like action of zinc in the FST involves serotonergic neurotrans-mission, particularly the 5HT1A and 5HT2A/C receptors. The synergisticeffect between zinc and antidepressants with “serotonergic” profilesmay be used as a way to enhance the antidepressant effect of thesedrugs in depressed patients (Nowak et al., 2003a).

Acknowledgements

The authors thank “Lundbeck” and “Farmapol” for the generousgift of drugs. This study was partially supported by the Ministry ofEducation and Science grant no. 2 P05A 0178 29 (2005–2008) andFunds for Statutory Activity of the Institute of Pharmacology, PolishAcademy of Sciences, Collegium Medicum, Jagiellonian University,Kraków, Medical University of Lublin and Maria Curie-SkłodowskaUniversity, Lublin, Poland.

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