Progress in Neuro-Psychopharmacology & Biological Psychiatry 56 (2015) 254–263

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Progress in Neuro-Psychopharmacology & BiologicalPsychiatry

Zinc deficiency in rats is associated with up-regulation of hippocampalNMDA receptor

Urszula Doboszewska a, Magdalena Sowa-Kućma a, Katarzyna Młyniec b, Bartłomiej Pochwat a,Malgorzata Hołuj c, Beata Ostachowicz d, Andrzej Pilc a,e, Gabriel Nowak a,b, Bernadeta Szewczyk a,⁎a Department of Neurobiology, Institute of Pharmacology, Polish Academy of Sciences, Smętna 12, PL 31-343 Kraków, Polandb Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9, PL 30-688 Kraków, Polandc Department of Behavioral Neuroscience & Drug Development, Institute of Pharmacology, Polish Academy of Sciences, Smętna 12, PL 31-343 Kraków, Polandd Faculty of Physics and Applied Computer Sciences, AGH University of Science and Technology, Mickiewicza 30, PL 30-059 Kraków, Polande Faculty of Health Sciences, Jagiellonian University Medical College, Michałowskiego 20, PL 31-126 Kraków, Poland

Abbreviations: CaMK, Ca2+ calmodulin-dependent prstress; CREB, cyclic AMP response element binding protestress;BDNF,brain-derivedneurotrophic factor; FST, forcedMDD,major depressive disorder; p-CREB, phosphorylatedpartate receptor; PFC, prefrontal cortex; p-TrkB, phosphodensity; PSD-95, postsynaptic density protein 95; TrkB, troceptor; TXRF, Total Reflection X-Ray Fluorescence; ZnA, zin⁎ Corresponding author. Tel.: +48 126623362; fax: +4

E-mail address: szewczyk@if-pan.krakow.pl (B. Szewc

http://dx.doi.org/10.1016/j.pnpbp.2014.09.0130278-5846/© 2014 Elsevier Inc. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 1 July 2014Received in revised form 31 August 2014Accepted 19 September 2014Available online 5 October 2014

Keywords:Forced swim testNMDA receptorSocial interaction testSucrose intake testZinc deficiency

Rationale: Data indicated that zinc deficiency may contribute to the development of depression; howeverchanges induced by zinc deficiency are not fully described.Objectives: In the present paper we tested whether the dietary zinc restriction in rats causes alterations inN-methyl-D-aspartate receptor (NMDAR) subunits in brain regions that are relevant to depression.Methods: Male Sprague Dawley rats were fed a zinc adequate diet (ZnA, 50 mg Zn/kg) or a zinc deficient diet(ZnD, 3mg Zn/kg) for 4 or 6 weeks. Then, the behavior of the rats was examined in the forced swim test, sucroseintake test and social interaction test. Western blot assays were used to study the alterations in NMDAR subunitsGluN2A and GluN2B and proteins associated with NMDAR signaling in the hippocampus (Hp) and prefrontalcortex (PFC).Results: Following 4 or 6 weeks of zinc restriction, behavioral despair, anhedonia and a reduction of social behav-ior occurred in rats with concomitant increased expression of GluN2A and GluN2B and decreased expression of

the PSD-95, p-CREB and BDNF protein levels in theHp. The up-regulation of GluN2Aproteinwas also found in thePFC, but only after prolonged (6 weeks) zinc deprivation.Conclusions: The procedure of zinc restriction in rats causes behavioral changes that share some similarities to thepathophysiology of depression. Obtained data indicated that depressive-like behavior induced by zinc deficiencyis associated with the changes in NMDAR signaling pathway.

© 2014 Elsevier Inc. All rights reserved.

1. Introduction

Depression is a chronic debilitating disorder with an increasingprevalence (WHO, 2008). It has a neurobiological basis and is associatedwith functional and structural brain abnormalities, e.g., neuroimagingstudies have established the relevance of the prefrontal cortex (PFC)and the hippocampus (Hp) to major depression (Kupfer et al., 2012;Palazidou, 2012). The first line-medications for major depressive disor-der (MDD) are based on the monoamine hypothesis of depressionand antidepressant action (Coppen, 1967; Schildkraut, 1965), have a

otein kinase; CMS, chronicmildin; CUS, chronic unpredictableswimstress;Hp,hippocampus;CREB; NMDAR, N-methyl-D-as-rylated TrkB; PSD, postsynapticpomyosine-related kinase B re-c adequate; ZnD, zinc deficient.8 126374500.zyk).

delayed onset of action, and fail to produce a response in a large percentof patients (Trivedi et al., 2006). In contrast, the infusion ofsubanaesthetic doses of the N-methyl-D-aspartate receptor (NMDAR)antagonist ketamine exerts rapid antidepressant effects in patientswith treatment resistant MDD (Aan Het Rot et al., 2012; Berman et al.,2000; Zarate et al., 2006). These observations suggest that strategies be-yond the monoaminergic systemmight be beneficial for improving thetreatment outcome in depressed patients. Exploring glutamatergic tar-gets, including NMDAR, may be a promising approach to the discoveryof novel antidepressants (Pilc et al., 2013). Furthermore, magnetic reso-nance spectroscopy studies have shown altered levels of glutamate inthe central nervous system of depressed patients (Grimm et al., 2012).These data strongly support the hypothesis that the glutamatergic sys-tem and NMDAR play an important role in the pathophysiology andtreatment of depression (Skolnick et al., 2009).

The NMDAR functions as a heteromeric complex composed of foursubunits surrounding a central cation-selective pore. Three major sub-types of NMDAR subunits have been described: GluN1, GluN2A–D andGluN3A–B (Cull-Candy et al., 2001). The most widely expressed

Fig. 1. Experimental paradigm. Ratswere fed the zinc adequate diet (ZnA, 50mg Zn/kg) orthe zinc deficient diet (ZnD, 3 mg Zn/kg) for 4 or 6 weeks and were further used for thebehavioral tests or biochemical analysis.

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NMDAR is composed of two glycine binding GluN1 subunits and twoglutamate-binding GluN2 subunits (GluN2B or GluN2A or a mixture ofthe two). A growing body of evidence has shown that distinct NMDARfunctions probably depend on the subunit composition (Köhr, 2006).Stimulation of NMDAR results in the Ca2+ influx and induction ofdiverse intracellular signaling pathways including: Ca2+ calmodulin-dependent protein kinase (CaMK); cyclic AMP response element bind-ing protein (CREB), brain-derived neurotrophic factor (BDNF) and itsreceptor, tropomyosin-related kinase B (TrkB) (Duman and Voleti,2012).

The best characterized effect of zinc is the inhibition of NMDAR andtwo different mechanisms of action for zinc were identified: a voltage-independent, non-competitive (allosteric) inhibition, responsible for re-ducing channel-opening frequency, and voltage-dependent inhibition,representing an open channel blocking effect of zinc (Christine andChoi, 1990; Paoletti et al., 2009). The comparison of GluN1/GluN2Aand GluN1/GluN2B receptors revealed that the voltage-dependent inhi-bition is similar in both types of receptors but the voltage-independentzinc inhibition is highly subunit-specific, with an affinity ranging fromlow nM for GluN1/GluN2A receptors to 1 μM for GluN1/GluN2B recep-tors and ≥10 μM for GluN1/GluN2C and GluN1/GluN2D receptors(Chen et al., 1997; Paoletti et al., 1997). However, the maximal effectof zinc is smaller at receptors containing GluN2A than at this with theGluN2B subunit because zinc exerts only a partial inhibition of theGluN1/GluN2A receptors yet fully inhibits the GluN1/GluN2B receptors(Williams, 1996).

Developing evidence suggests that modifiable lifestyle behaviors arerisk factors for mental disorders. Within these behaviors, diet quality isthemost recent area of attention in the lifestyle-mental health researchfield (Jacka et al., 2012b). The intake of zinc is among the dietary factorsthat may be associated with a risk for depression (Szewczyk et al.,2011). Recently, some large, epidemiological studies have suggestedthat a low dietary zinc intake is associated with depression (Jackaet al., 2012a; Maserejian et al., 2012; Vashum et al., 2014). The meta-analysis study published recently by Swardfager et al., 2013 showedclearly that MDD is associated with a lower concentration of zinc inperipheral blood (Swardfager et al., 2013). Moreover, evidence pointstoward potential benefits of zinc supplementation in depressed patients(Lai et al., 2012). Zinc supplementation was found to be beneficial as anadjunct therapy (Nowak et al., 2003a; Ranjbar et al., 2013; Siwek et al.,2009) or as a stand-alone intervention (Solati et al., 2014) for depression.

Data from animal studies further suggest that an inadequate dietaryzinc intake may contribute to the development of depression. Studiesperformed in rodents indicate a causative role of zinc deficiency in theinduction of depressive-like symptoms (Mlyniec and Nowak, 2012;Mlyniec et al., 2012; Tamano et al., 2009; Tassabehji et al., 2008;Whittle et al., 2009) or anhedonia (Cope et al., 2011; Tassabehji et al.,2008); however the mechanisms induced by zinc deficiency are notyet fully described. A number of preclinical tests and models of depres-sion showed antidepressant activity of zinc (Kroczka et al., 2000, 2001;Nowak et al., 2003b; Rosa et al., 2003; Cunha et al., 2008; Cieslik et al.,2007; Sowa-Kucma et al., 2008) and NMDAR was found to participatein this activity (Szewczyk et al., 2010).

Considering the involvement of the NMDAR in the pathophysiologyof depression and the mechanism of action of zinc we investigatedwhether the time-course restriction of zinc causes alterations inNMDAR subunits and proteins involved in NMDAR regulating signalingpathway in the brain regions relevant to depression, i.e., the PFC and theHp.

2. Methods

2.1. Animals and diet

All procedures were conducted according to the National Institutesof Health Animal Care and Use Committee guidelines and were

approved by the Ethics Committee of the Institute of Pharmacology,Krakow. The experiments were carried out on male Sprague Dawleyrats (Charles River Laboratories, Germany) at the age of 4 weeks andhabituated to the laboratory conditions for 1 week prior to use. Duringthe habituation phase, the rats were fed a standard diet with 35 mgZn/kg. Following the habituation phase, the animals were divided intogroups; each group consisted of 8–10 rats that were fed a zinc adequatediet (ZnA) of 50 mg Zn/kg or a zinc deficient diet (ZnD) of 3 mg Zn/kgfor 4 or 6 weeks (Fig. 1). All of the diets were purchased from AltrominGmbH (Lage, Germany). The animals were housed 5 per cage (with theexception of the rats used in the sucrose consumption or social interac-tion tests, whichwere housed individually) in a controlled environment(temperature 22 ± 2 °C, 12 h light/dark cycle, 40–50% humidity) withfree access to food and water.

2.2. Forced swim test (FST)

Following 4 or 6 weeks of the ZnA or ZnD diet, the forced swim test(FST) was performed, as published previously (Szewczyk et al., 2009).The rats were placed in glass cylinders (height 40 cm, diameter20 cm) containing 15 cm of water that was maintained at 23–25 °C.Two swim sessions were conducted: an initial 15 min pretest wasfollowed 24 h later by a 5 min test. Following both sessions, the ratswere removed from the cylinders and returned to their home cages.Behavioral scoring was performed during the 5 min test session.

2.3. Locomotor activity

Twenty four hours after the FST, the locomotor activity of rats wasmeasured. Locomotor activitywas recorded individually for each animalin Opto-Varimex cages (Columbus Instruments, USA) linked on-line to acompatible IBM-PC. The behavior of the rats was analyzed using Auto-Track software (Columbus Instruments, Columbus, USA). Each cage(43 cm× 44 cm× 25 cm)was equippedwith a 15 × 15 array of infraredemitters located 3 cm from the floor surface. The number of light beamsinterrupted by an animal was recorded at 5 min intervals during the15 min test and was presented as the distance traveled in cm.

2.4. Sucrose consumption test

Individually housed rats were fed the ZnA or the ZnD diet for 4 or6 weeks. Following 4 or 6 weeks of the ZnA or the ZnD diet, the sucroseconsumption test was performed, as published by Luo et al. (2013). Atest session was preceded by a training session. Both, the training andthe test sessions were conducted in home cages equipped with two

Fig. 2. FST following a 4-week (A) or a 6-week (B) zinc restriction (*p b 0.05, ** p b 0.01, *** p b 0.0001 vs. ZnA by Student's t-test; values expressed as the mean ± SEM). Spontaneouslocomotor activity following a 4-week (C) or a 6-week (D) zinc deficiency (by the Two-way Repeated Measures ANOVA; values expressed as the mean ± SEM).

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bottles, one bottle contained 1% sucrose solution and the other onecontained water. In the training session, the animals were trained toconsume a 1% sucrose solution for 48 h. To prevent the possible effectsof side preference, the positions of the bottles were switched every12 h. Following the training session, sterile water was provided for 6 hand for the next 18 h the food andwaterwerewithheld. The test sessionwas performed 24 h after the training session and lasted 1 h. The ratswere given choice between water and 1% sucrose solution. The sucroseintake of the rats wasmeasured byweighing pre-weighed bottles at theend of the tests.

2.5. Social interaction test

Following 4 or 6 weeks of the ZnA or ZnD diet, the social interactiontest was performed. The experiments were conducted in the open fieldarena (length × width × height: 57 × 67 × 30 cm) made of blackPlexiglas. The arena was dimly illuminated with an indirect light of18 Lux. The behavior of the rats was recorded by two cameras placedabove the arena and connected to the NoldusMPEG recorder 2.1. Videoswere analyzed off-line by the Noldus The Observer® XT, version 10.5.

Fig. 3. The effects of the zinc deficiency on the consumption of 1% sucrose solution following a 4values expressed as the mean ± SEM).

Rats were individually housed for 5 days prior to the procedure. Onthe fifth day of social isolation, the rats were transferred to the experi-mental room and individually adapted to the open field arena for7 min. Afterward, the rats were handled, weighed and half were dyedwith a gentian violet (2% methylrosanilinium chloride) on the rearpart of the body. On the test day (the sixth day of social isolation), twounfamiliar rats of matched body weight (±20 g), one white and onedyed, were placed in the open field arena and their behavior wasrecorded for 10 min. Social interaction time was measured for each ratseparately and expressed as a summed score per each pair of rats. Thefollowing active social behaviors were scored: sniffing (rat sniffs theconspecific's parts of the body, including the anogenital region), socialgrooming (rat licks and chews the fur of the conspecific), following(rat moves toward and follows the other rat), mounting (rat stands onthe conspecific's back) and climbing (rat climbs over the conspecific'sback).

2.6. Total Reflection X-ray Fluorescence (TXRF)

Serum zinc level was measured by Total Reflection X-ray Fluores-cence (TXRF) method, as described previously (Opoka et al., 2010). As

-week (A) or a 6-week (B) zinc restriction. (*p b 0.05, **p b 0.01 vs. ZnA by Student's t-test,

Fig. 4. The effects of the zinc deficiency on the total duration of active social behavior following a 4-week (A) or a 6-week (B) zinc restriction (*p b 0.01, **p b 0.001 vs. ZnA by Student's t-test, values expressed as the mean ± SEM).

Table 1The effect of the diet on a serum zinc concentration following 4 or 6 weeks of zincrestriction.(* p b 0.0001 vs. ZnA by Student's t-test; values expressed as the mean ± SEM).

ZnA ZnD

4 weeks 1.391 ± 0.038 0.5010 ± 0.032*6 weeks 1.524 ± 0.054 0.6246 ± 0.041*

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an internal standard, selenium standard was added, so that the finalconcentration was 5 mg/l. From the resulting solutions, 5 μl was pipet-ted on reflectorsmadeof clean glass, used for TXRF analysis. Concentrat-ed nitric acid, suprapur quality additionally cleaned by subboilingdistillation procedure, was purchased from Merck (Darmstadt,Germany). Selenium standard, 1000 mg/l selenium in nitric acid, waspurchased from Fluka, Sigma Aldrich (Saint Louis, Missouri, UnitedStates). NANOHUNTER TXRF Spectrometer from Rigaku (Japan) wasused. Mo X-ray tube (50 kV, 0.8mA)was applied. Each sample wasmea-sured three times.

2.7. Western blotting

We tested the levels of the following proteins: NMDAR GluN2Aand GluN2B subunits, postsynaptic density protein 95 (PSD-95), thetranscription factor cyclic AMP response-element binding protein(CREB), brain-derived neurotrophic factor (BDNF) and its receptor,high-affinity tropomyosine-related kinase B receptor (TrkB).

Following4 or 6 weeks of the ZnAor ZnDdiet, the ratswere killed byrapid decapitation; their brainswere rapidly dissected and immersed incooled (2–8 °C) 0.9% sodium chloride solution. Complete hippocampi(dorsal and ventral) and prefrontal cortices were dissected on a coldplate, immediately frozen on dry ice and stored at−80 °C until analysis.Hippocampi and prefrontal cortices were homogenized in 2% sodiumdodecyl-sulfate solution, denatured at 95 °C for 10min and centrifugedat 10,000 RPM at 4 °C for 5 min. The total protein concentration wasquantified in the supernatant using a Pierce BCA Protein Assay Kit(Pierce Biotechnology, Inc., Rockford, IL, USA). The samples containing50 μg of protein were resolved on a 12.5% SDS-polyacrylamide gel(for further detection of CREB, BDNF or TrkB protein) or a 7.5%SDS-polyacrylamide gel (for further detection of GluN2A, Glu2B orPSD-95 protein) and were transferred on a nitrocellulose membrane(Invitrogen, Paisley, UK). The membranes were blocked for 60 minand incubated with rabbit polyclonal antibodies targeting phosphorylat-ed CREB at Ser 133 (p-CREB, Ser 133, 1:200, Santa Cruz Biotechnology,Dallas, Texas), BDNF (N-20, 1:200, Santa Cruz Biotechnology, Dallas,Texas), GluN2A (1:200, Santa Cruz Biotechnology, Dallas, Texas),GluN2B (1:200, Santa Cruz Biotechnology, Dallas, Texas), or tyrosine-phosphorylated TrkB (p-TrkB, Y-515, 1:500, Abcam, Cambridge, UK), orwith the mouse monoclonal antibody against PSD-95 (1:200, Pierce Bio-technology, Inc., Rockford, IL, USA), or the mouse monoclonal antibodyagainst β-actin (1:1000, Sigma, Germany) at 2–8 °C overnight. The nextday, the membranes were incubated for 30 min with horseradishperoxidase-linked secondary antibodies (BM ChemiluminescenceWB kit (Mouse/Rabbit), Roche Diagnostic, Mannheim, Germany) underconstant shaking at room temperature. The secondary antibodies weredetected using a BM Chemiluminescence WB kit (Mouse/Rabbit)(RocheDiagnostic,Mannheim, Germany). The protein bandswere visual-izedwith the Fuji-LAS 4000 System. The density of each protein bandwasanalyzed using imaging software (Fuji Image Gauge v 4.0) and was

normalized by the optical density of the corresponding β-actin band(Fig. 6 and 8).

2.8. Statistics

Data were analyzed using a Student's t-test or two-way repeatedmeasures ANOVA followed by a Bonferroni post-hoc test. All resultsare presented as the mean ± SEM. p b 0.05 was considered statisticallysignificant with 95% confidence (Prism, GraphPad, San Diego, CA).

3. Results

3.1. Effects of zinc deprivation on the behavior of rats in the FST, sucrose in-take test and social interaction test.

3.1.1. Forced swim testA4-week zinc restriction significantly increased the immobility time

[t(19) = 5.120, p b 0.0001] and decreased swimming [t(19) = 5.237,p b 0.0001] and climbing [t(19) = 2.727, p b 0.05] parameters in theFST (Fig. 2A). A 6-week depletion of zinc significantly increased the im-mobility time [t(15)= 3.764, p b 0,01], although it did not influence theswimming [t(15)= 1.617, p= 0.1266] or the climbing [t(15)= 1.632,p = 0.1235] parameters in the FST (Fig. 2B).

No difference was found in the spontaneous locomotor activity(measured at the first time point of 5 min, which equals to the FST as-sessment period) of rats fed with the ZnD diet for either 4 weeks(Fig. 2C) or 6 weeks (Fig. 2D) when compared to animals fed with theZnA diet. Two-way ANOVA demonstrated no interaction [F(2,24) = 2.97,p = 0.0706], no effect of the diet [F(1,12) = 0.54, p = 0.4770], but asignificant effect of time (between different time point measurements,taken at 5, 10 or 15 min) [F(2,24) = 30.57, p b 0.0001] on locomotoractivity following a 4-week zinc restriction (Fig. 2C). Two-way ANOVAshowed no interaction [F(2,28) = 2.22, p = 0.1277] and no effect ofthe diet [F(1,14) = 0.06, p = 0.8094], but a significant effect of time(between different time point measurements, taken at 5, 10 or 15 min)[F(2,28) = 27.04, p b 0.0001] on the locomotor activity following a6-week zinc depletion (Fig. 2D).

3.1.2. Sucrose intake testBoth the 4-week and the 6-week zinc deprivation induced a reduc-

tion in consumption of 1% sucrose solution [t(16) = 2.966, p b 0.01

Fig. 5. The effects of the zinc deficiency on the expression of proteins in the Hp (left) and PFC (right) following 4 weeks of zinc restriction: GluN2A (5A, B), GluN2B (5C, D), PSD-95 (5E,F),p-CREB (5G, H), BDNF (5I, J) and p-TrkB (5K, L). The results (mean± SEM) are presented as theGLUN2A, GluN2B, PSD-95, p-CREB, BDNF or p-TrkB/β-actin ratio (% of control) (*p b 0.05 vs.ZnA by Student's t-test, values expressed as the mean ± SEM).

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following 4 weeks (Fig. 3A); t(14) = 2.342, p b 0.05 following 6 weeks(Fig. 3B)].

3.1.3. Social interaction testBoth the 4-week and the 6-week zinc deprivation induced a reduc-

tion of social behavior [t(12) = 4.364, p b 0.001 following 4 weeks(Fig. 4A); t(6) = 5.169, p b 0.01 following 6 weeks (Fig. 4B).

3.2. Effect of dietary zinc deprivation on the serum zinc level.

The 4- and the 6-week zinc restriction loweredmarkedly and signif-icantly the serum zinc levels [t(12) = 15.68, p b 0.0001 following4 weeks; t(12) = 11.32, p b 0.0001 following 6 weeks] (Table 1).

3.3. Effects of zinc deprivation on the alterations in NMDAR subunits.

3.3.1. Time-course changes in NMDAR subunit (GluN2A, GluN2B), PSD-95,p-CREB, BDNF and p-TrB in rat Hp

4 weeks of zinc restriction significantly increased the expression ofGluN2A [by 54%, t(16) = 2.180, p b 0.05] (Fig. 5A) and GluN2B [by84%, t(19) = 3.588, p b 0.01] (Fig. 5C) proteins in the rat Hp. Further-more, the levels of both proteins were significantly increased after6 weeks of the zinc regimen. The level of GluN2A protein was increasedby 79% [t(11) = 2.216, p b 0.05] (Fig. 7A), whereas the level of GluN2Bprotein was increased by 77% [t(13) = 2.554, p b 0.05] (Fig. 7C) in theHpof the zinc deficient group following a 6-week depletion of zinc com-pared to the control group. However, the level of PSD-95 protein in theHp of the zinc deficient rats was decreased by 48% after the 4-weekzinc restriction [t(20) = 2.249, p b 0.05] (Fig. 5E) and by 57% after the6-week zinc deprivation, [t(12) = 1.363, p = 0.1980] (Fig. 7E). In addi-tion, the levels of p-CREB and BDNF proteins were significantly lower inthe Hp of the zinc deficient animals following both the 4-week and the6-week zinc restrictions. After 4 weeks of zinc deprivation, the level ofp-CREB protein was decreased by 91% [t(8) = 2.380, p b 0.05] (Fig. 5G),the level of BDNF protein was decreased by 65% [t(11) = 2.481,p b 0.05] (Fig. 5I), and the level of p-TrkB protein was decreased by 31%[t(20) = 2.106, p b 0.05] (Fig. 5K) in the Hp of the zinc deficient rats.After 6 weeks of zinc depletion, the level of p-CREBproteinwas decreasedby 43% [t(12) = 2.477, p b 0.05] (Fig. 7G) and the level of BDNF proteinwas decreased by 55% [t(12) = 2.539, p b 0.05] (Fig. 7I) in the Hp ofthe zinc deficient group. However, a significant increase [by 84%,t(14) = 2.294, p b 0.05] (Fig. 7K) in p-TrkB protein was observed in theHp of the zinc deficient rats following a 6-week restriction of zinc com-pared to the control animals.

Fig. 6. Representative blots of GluN2A (177 kDa), GluN2B (178 kDa), PSD-95 (105 kDa),p-TrkB (92 kDa), BDNF (14 kDa), p-CREB (43 kDa) and β-actin (42 kDa) in the Hp (left)and PFC (right) after 4 weeks of zinc deficiency.

3.3.2. Time-course changes in NMDAR subunit (GluN2A, GluN2B), PSD-95,p-CREB, BDNF and p-TrB in rat PFC

A 4-week zinc restriction did not significantly affect the GluN2A[t(18) = 0.9965, p = 0.3322] (Fig. 5B) or the GluN2B [t(20) = 0.8446,p= 0.4083] (Fig. 5D) protein levels; however, the zinc restriction did sig-nificantly increase the level of PSD-95 [by 52%, t(14) = 2.579, p b 0.05](Fig. 5F) protein in the PFC. At 4 weeks, no statistically significantdifferences in the levels of p-CREB [t(16) = 0.5247, p = 0.6070](Fig. 5H), BDNF [t(16) = 0.3212, p = 0.7522] (Fig. 5J) or p-TrkB[t(20) = 0.09296, p = 0.9269)] (Fig. 5L) proteins between the zincdeficient and control rats were observed in the PFC (Fig. 6).

A 6-week deprivation of zinc resulted in a significant increase inGluN2A [by 200%, t(12) = 2.294, p b 0.05] (Fig. 7B) protein levels inthe PFC of the zinc deficient group compared to the control group,whereas GluN2B [t(14) = 0.04675, p = 0.9634] (Fig. 7D) and PSD-95[t(14) = 0.1548, p = 0.8792] (Fig. 7F) protein levels remained un-changed. The elevated level of GluN2A protein in the PFC following a6-week depletion of zinc was accompanied by lower levels of p-CREBand BDNF proteins. The level of p-CREB protein was decreased by 22%[t(14) = 2.155, p b 0.05] (Fig. 7H), and the level of BDNF protein wasdecreased by 53% [t(11) = 2.393, p b 0.05] (Fig. 7J) in the PFC ofthe zinc deficient rats following a 6-week restriction of zinc comparedto the control animals. Moreover, a significant increase [by 153%,t(12) = 2.463, p b 0.05] (Fig. 7L) in p-TrkB protein was observed inthe PFC of the zinc deficient rats.

4. Discussion

To induce depressive-like behavior we subjected rats to 4 and6 weeks of zinc deficiency, based on the previous data indicating zincdeficiency as an animal model of depression (Mlyniec et al., 2013,2014; Whittle et al., 2009). In our hands the 4- and 6-week zinc restric-tion diets increased immobility time in the FST, without alterations inlocomotor activity. To further confirm the depressive-like behaviorof rats following 4 or 6 weeks of zinc restriction we performed thesucrose intake test and social interaction test. We observed anhedonia(measured as a decrease in sucrose intake) and a reduction of social be-havior in rats after 4 and 6 weeks of zinc deficiency. We also measuredthe serum zinc concentration and we found that both 4 and 6 weeks ofzinc deficiency significantly decrease the serum zinc level in rats. Thesebehavioral andbiochemical results are consistentwith the recently pub-lished data suggesting that zinc deficiencymight induce depressive-likebehavior both inmice and rats and further indicate zinc deficiency as anexperimental model of depression.

Pre-clinical and clinical data have indicated a link between thealtered composition of NMDAR subunits and the pathophysiology of de-pression. These alterations have been observed in experimental modelsof depression based on stress exposure, e.g., the increased hippocampallevel of GluN2A protein was demonstrated in the chronic mild stress(CMS) (Calabrese et al., 2012; Pochwat et al., 2014). Moreover, socialstress paradigm induced an increase in hippocampal GluN2B mRNAand protein levels in stressed mice (Sterlemann et al., 2010), alterna-tively, in the study by Quan et al. (2011) chronic unpredictable stress(CUS) induced a reduction in the expression of GluN2B receptor in thePFC of stressed rats (Quan et al., 2011). In post-mortem tissues, a signif-icant reduction in GluN2A and GluN2B proteins was found in the PFC ofdepressed patients (Feyissa et al., 2009). Recently, a significantly elevat-ed amount of GluN2A protein and reduced amount of GluN2B proteinwere observed the in the Hp of suicide victims (Sowa-Kucma et al.,2013). The present study demonstrates, for the first time the up-regulation of GluN2A and GluN2B proteins in the Hp following the 4-week and the 6-week zinc deficiency. Furthermore, the up-regulationof GluN2A protein occurred in the PFC, but only after a longer (a 6-week) period of zinc deprivation. It seems that the hippocampalNMDAR is more susceptible to zinc restriction than the NMDAR in thePFC, perhaps due to the fact that in the brain, the highest zinc

Fig. 7. The effects of the zinc deficiency on the expression of proteins in the Hp (left) and PFC (right) following 6 weeks of zinc restriction: GluN2A (7A, B), GluN2B (7C, D), PSD-95 (7E, F),p-CREB (7G, H), BDNF (7I, J) and p-TrkB (7K, L). The results (mean± SEM) are presented as theGLUN2A, GluN2B, PSD-95, p-CREB, BDNF or p-TrkB/β-actin ratio (% of control) (*p b 0.05 vs.ZnA by Student's t-test, values expressed as the mean ± SEM).

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Fig. 8. Representative blots of GluN2A (177 kDa), GluN2B (178 kDa), PSD-95 (105 kDa),p-TrkB (92 kDa), BDNF (14 kDa), p-CREB (43 kDa) and β-actin (42 kDa) in the Hp (left)and PFC (right) after 6 weeks of zinc deficiency.

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concentrations are present in the Hp (Frederickson et al., 2000). Sincethe biochemical measures were done in different animals than thoseused for the behavioral tests, the abnormalities observed in our studyare due to zinc deficiency but not due to the stress evoked bybehavioral tests.

Studies indicated that administration of antidepressants leads tomodulation of NMDAR function through the activation of BDNF(Skolnick, 1999). It has been also demonstrated that long termexposureof cerebellar granule cell neurons to BDNF reduces mRNA and proteinlevels of NMDAR-2A and -2C (Brandoli et al., 1998). Therefore, one ofthe possible mechanisms that may mediate the increased expressionof GluN2A protein after dietary zinc deprivation might be the reductionof BDNF.

Our present study showing reduced expression of p-CREB (Ser 133),BDNF and p-TrkB (Y-515) proteins in the Hp, but not in the PFC, of ratsfollowing a 4-week zinc deficiencymight suggest that theHp, the regionof the brain that plays a critical role in neurogenesis (Eriksson et al.,1998), is more susceptible to changes in the CREB/BDNF pathwayelicited by zinc deficiency than the PFC. This observation is consistentwith previous study on the zinc deficiency model in mice (Mlyniecet al., 2014) and further suggests that the changes in CREB/BDNF/TrkBprotein levels are dynamic in response to zinc deprivation and vary inregions of the brain over the course of time (the 6-week zinc depletioninfluences the CREB/BDNF/TrkB pathway not only in the Hp but also thePFC). Moreover, when comparing mouse (Mlyniec et al., 2014) and ratmodels, the changes in the CREB/BDNF pathway after 6 weeks of zincrestriction are more widespread in the rats, affecting both the Hp andthe PFC.

Interestingly, in our rat model, after an initial hippocampal p-TrkBdown-regulation (4 weeks of zinc deficiency), an up-regulation ofp-TrkB occurs both in the Hp and the PFC (6 weeks of zinc deficiency).Decreased expression of hippocampal TrkB was also observed in thezinc deficiency model in mice, (Mlyniec et al., 2014). TrkB expressionin the Hp was decreased in the postmortem brain of suicide subjects(Dwivedi et al., 2003). On the other hand it was demonstrated that aTrkB antagonist showed antidepressant-like properties in the FST andtail-suspension test (TST) (Cazorla et al., 2011). Moreover, repeated im-mobilization stress and unpredictable stress increased levels of TrkBmRNA (Nibuya et al., 1999). Indeed, increasing evidence suggests thata prolonged increased TrkB activation is observed in many pathologicalconditions, including psychiatric disorders (Boulle et al., 2012),with ourrat model reflecting these changes.

In the present study we also examined the effect of zinc deficiencyon the expression of PSD-95 protein in rat brain. PSD-95 interacts

with the cytoplasmic tail of GluN2A and GluN2B proteins, anchoringNMDAR in the postsynaptic density (PSD) (Kornau et al., 1995; Shengand Kim. 2011). A microarray and electron microscopic stereologystudy showed a significant down-regulation of synaptic protein genesin MDD (Kang et al., 2012). The down-regulation of PSD-95 protein inthe PFC has been implicated in MDD (Feyissa et al., 2009) and wasrecently found in the Hp of suicide victims (Sowa-Kucma et al., 2013).In contrast, the administration of the NMDAR antagonist ketamine in-creased the levels of synaptic proteins, including PSD-95 (Li et al.,2010). PSD-95 has a crucial role in maintaining the integrity of thePSD (Chen et al., 2011). The decreased level of PSD-95 in the Hp furthermimics the neurobiological changes associated with depression; more-over, it suggests a disrupted integrity of the hippocampal PSD in the zincdeficiency model in rats. GluN2A and GluN2B subunits can be locatedeither synaptically or extrasynaptically (Thomas et al., 2006). Theincreased levels of GluN2A and GluN2B proteins with a concomitantreduction in PSD-95 may suggest that the synaptic NMDARs are de-creased, while the extrasynaptic NMDARs are increased. The activationof extrasynaptic NMDARs was found to be neurotoxic and attenuatedCREB signaling (Hardingham and Bading, 2010; Hardingham et al.,2002). Hence, the decreased pCREB level might result from the activa-tion of extrasynaptic NMDARs. However, the research on the differentialrole of synaptic and extrasynaptic NMDARs was conducted in culturedneurons (Hardingham and Bading, 2010; Hardingham et al., 2002)and may not be applicable to the in vivo network. Furthermore, thetotal amount of GluN2A and GluN2B proteins did not differ betweenthe wild-type and mutant mice lacking PSD-95 (Migaud et al., 1998).This indicates that postsynaptic NMDAR clustering does not solelydepend on the PSD-95 (Migaud et al., 1998). Therefore, despite a de-creased level of PSD-95, an increase in GluN2A and GluN2B proteinsmay have occurred in the Hp following zinc deprivation.

5. Conclusions

Our data indicate that zinc deficiency is associated with alterationsof NMDAR (different alterations of the subunits in different brainregions). Considering the importance of NMDAR hyperactivity to thepathophysiology of depression and presented data, zinc deficiencycauses neurochemical alterations that share some similarities withthat observed in depression. Behavioral data indicating that zinc deficien-cy induces anhedonia and impairs social behavior in rats increases valueof experimentally induced zinc deficiency as a model of depression.

Acknowledgments

The study was partially supported by the Statutory Activity of theInstitute of Pharmacology PAS and Jagiellonian University MedicalCollege in Krakow, grant POIG.01.01.02-12-004/09-00, and financialsupport from the project Interdisciplinary PhD Studies “Molecular sci-ences for medicine” (co-financed by the European Social Fund withinthe Human Capital Operational Programme, POKL.04.01.01-00-056/10). All authors declare that there are no conflicts of interest.

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