Antidepressant-like activity of zinc: further behavioral and molecular evidence
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Zinc exhibits antidepressant-like activity in preclinical tests (the forced swim test and tail suspension test) and in olfactory bulbectomy and chronic unpredictable stress; two models of depression. Zinc also enhances the treatment of depression in humans. In the present study we evaluated the antidepressant activity of zinc in another model of depression—chronic mild stress (CMS) and the effect of zinc treatment on BDNF protein and the mRNA level. In CMS zinc hydroaspartate (10 mg/kg) exhibited a rapid (after 1 week of treatment) antidepressant-like effect. Chronic treatment with zinc induced a 17–39% increase in the BDNF mRNA and protein level in the hippocampus. These data indicate a rapidly acting antidepressant-like activity of zinc in CMS and the involvement of zinc in the regulation of BDNF.
KeywordsZinc chronic mild stress BDNF depression
Zinc is a trace element involved in a wide range of biological processes. Zinc deprivation influences brain zinc homeostasis and leads to alterations in behavior, learning and mental function (Takeda 2000). Several studies indicated the involvement of zinc in the pathophysiology of depression and the mechanism of action of antidepressant drugs. Clinical data demonstrated a lower serum zinc concentration in patients with depression (Maes et al. 1994; McLoughlin and Hodge 1990). Moreover zinc exhibits antidepressant-like activity both in preclinical tests (the forced swim test and tail suspension test) and some models (olfactory bulbectomy and chronic unpredictable stress) of depression (Kroczka et al. 2000, 2001; Nowak et al. 2003b; Rosa et al. 2003; Cieślik et al. 2007; for review, Nowak et al. 2005).
Preclinical screening tests and animal models of depression are employed to detect the potential antidepressant activity of newly tested compounds. Out of several procedures used in such studies, the chronic mild stress (CMS) model of depression appears to be the most extensively validated and investigated. In this model, rats or mice are exposed sequentially to a variety of mild stressors which change every few hours over a period of weeks or months. Among other behavioral, biochemical, and physiological impairments, this procedure causes a substantial and long-lasting decrease in responsiveness to rewarding stimuli, which can be effectively reversed by chronic treatment with various classes of antidepressants and the repeated application of an electroconvulsive shock. Conversely, non-antidepressant drugs, such as chlordiazepoxide, haloperidol, chlorprothixene, amphetamine, and morphine have been found to be ineffective in the CMS model (e.g., see Papp et al. 1996; Willner 2005). One of the aims of our present study was to investigate the antidepressant-like activity of zinc in the CMS model of depression.
There are dozens of drugs used in the treatment of depression, although the precise mechanisms underlying the therapeutic efficacy of these drugs remains unclear. Recent hypothesis linked the adaptation of intracellular proteins and genes as a factor contributing to the efficacy of antidepressant drugs (Duman et al. 1997). Several reports suggest that alteration in the brain-derived neurotrophic factor (BDNF) gene expression plays a key role in the pathophysiology and therapy of affective disorders (depression). In general, the brain (hippocampal and/or cortical) BDNF expression is reduced in the psychopathology of stress or depression, while antidepressant treatment increases this neurotrophic factor (Chen et al. 2001; Castren 2004). Furthermore, BDNF infusions into the midbrain (Siuciak et al. 1997) and hippocampus (Shirayama et al. 2002) produce antidepressant effects in rodents. Several clinical studies have demonstrated decreased blood levels of BDNF in depressed patients (Shimizu et al. 2003; Karege et al. 2005). Thus, measurement of the BDNF expression may be a suitable indicator in the discovery efforts for new antidepressants. We demonstrated previously (Nowak et al. 2004) that chronic treatment of zinc induced an increase in the cortical but not hippocampal BDNF mRNA level in rats, so the second aim of this paper is to evaluate the time course of the influence of the dose of zinc active in CMS on the BDNF mRNA and protein level in the rat brain.
The experiments were performed on male Wistar rats (230–270 g). Except as described below (chronic mild stress), the animals were housed with food and water freely available, and were maintained on a 12-h light–dark cycle and in constant temperature (22 ± 2°C) and humidity (55 ± 5%) conditions. Experiments were carried out between 9:00 a.m. and 2 p.m. Each experimental group consisted of 6–10 animals. All injections were given intraperitoneal (i.p.) at a volume of 2 ml/kg. All experimental procedures were approved by the Animal Care and Use Committee at the Institute of Pharmacology, Polish Academy of Sciences in Krakow.
CMS model of depression
Male Wistar rats (our own breeding stock) were brought into the laboratory 2 months prior to the start of the experiment. The animals were first trained to consume a 1% sucrose solution; training consisted of eleven 1-h baseline tests in which sucrose was presented in the home cage, following 14 h periods of food and water deprivation; the sucrose intake was measured by weighing bottles at the end of the test. Subsequently, sucrose consumption was monitored at weekly intervals throughout the experiment. On the basis of their sucrose intakes in the final baseline test, the animals were divided into two matched groups. One group of animals was subjected to the CMS procedure for a period of 8 consecutive weeks. Each week of the stress regime consisted of: two periods of food or water deprivation, two periods of 45° cage tilt, two periods of intermittent illumination (lights on and off every 2 h), two periods of soiled cage (250 ml water in sawdust bedding), one period of paired housing, two periods of low intensity stroboscopic illumination (150 flashes/min), and three periods of no stress. All stressors were 10–14 h in duration and were applied individually and continuously. Control animals were housed in separate rooms and had no contact with the stressed animals. They were deprived of food and water for the 14 h preceding each sucrose test, but otherwise food and water were freely available in the home cage. On the basis of their sucrose intake following the initial 2 weeks of the stress conditions, both stressed and control animals were each divided further into matched subgroups (n = 8), and for the subsequent 5 weeks they received daily i.p. injections of the vehicle solution (saline, 2 ml/kg), zinc hydroaspartate (5, 10 and 20 mg/kg) or imipramine (10 mg/kg) as the reference treatment. The drugs were administered at 10:00 a.m. and the weekly sucrose tests were carried out 24 h following the last drug injection. Stress was continued throughout the period of treatment.
Northern blot analysis of BDNF mRNA
Male Wistar rats were treated once a day with zinc hydroaspartate at a dose of 10 mg/kg (~1.8 mg Zn/kg) or saline for 1, 7, 14 or 35 days. Twenty-four hours after the last treatment, the animals were killed and their brains were removed. Dissected brain structures (the frontal cortices and hippocampi) were frozen on dry ice and stored at −80°C.
The procedure for the determination of the BDNF mRNA levels was performed according to Legutko et al. (2006). The total RNA was extracted using TRIzol Reagent (Life Technologies) following the manufacturer’s protocol. Northern blot analysis was performed with 10 μg of the total RNA, separated on 1% of denaturing agarose–formaldehyde gel, transferred subsequently to a nylon membrane (Nytran, Schleicher and Schuell) and immobilized by ultraviolet (UV) radiation. A probe for the rat BDNF was generated by polymerase chain reaction (PCR) from cDNA, using primers: 5′-ACTCTGGAGAGCGTGAATGG-3′ and 5′-CAGCCTTCCTTCGTGTAACC-3′. The 470 bp product was cloned into pCRII TA cloning vector. The cloned insert was isolated by restriction with EcoRI and radio-labeled with α-[P32]dCTP by random-priming. This probe was purified with Prime-It RmT (Stratagene, La Jolla, CA). Hybridization was performed in Church’s buffer at 65°C overnight. Hybridized filters were washed for 30 min in 2X saline–sodium citrate (SSC) buffer/0.1% sodium dodecyl sulfate (SDS) at room temperature and 30 min in 0.1xSSC/0.1% SDS at 55°C and exposed. Following exposure, the filters were stripped (washed three times in 0.1xSSC/0.1% SDS at 100°C for 10 min), and re-hybridized with β-actin cDNA probe (Clontech) to normalize the RNA loading. Northern blots were analyzed quantitatively with a PhosphorImager (Image Gauge 4.0, Fuji).
Western blot analysis of the BDNF protein
Male Wistar rats were treated once a day with zinc hydroaspartate at a dose of 10 mg/kg (1.8 mg Zn/kg) or with saline for 7 or 14 days. Twenty-four hours after the last treatment, the animals were killed and their brains were removed. Dissected brain structures (the frontal cortices and hippocampi) were frozen on dry ice and stored at −80°C.
The tissue was homogenized in ice in 2% SDS, denaturated for 10 min at 95°C, and centrifuged for 5 min at 10,000 rpm at 4°C. Protein concentration in the supernatant was determined using bicinchoninic acid (Pierce). The samples containing 20 μg of protein were fractionated by 12.5% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Invitrogen, Paisley, UK). Non-specific binding was blocked for 30 min at 4°C with 10% nonfat dry milk in Tris-buffered saline with 1% Tween 20 (TBS-T). Then membranes were incubated overnight at 4°C with polyclonal rabbit anti-BDNF antibodies (1:500 dilution, Santa Cruz Biotechnology, Santa Cruz, CA). The membranes were then washed with TBS-T and incubated for 30 min at room temperature with a goat horseradish peroxidase-conjugated anti-rabbit IgG (1:25,000 dilutions, Upstate/Millipore, Lake Placid, NY). Antibody detection was performed with an enhanced chemiluminescence reaction (ECL Western blotting detection, Amersham Life Science). The BDNF signal was visualized and quantified with the FUJI-LAS 1000 system and Fuji Image Gauge v 4.0 software. To confirm equal loading of the samples on the gel, the blots were incubated for 30 min with mouse anti-β-actin antibody (1:1,000 dilution, Sigma) and then processed as described above. The density of each BDNF protein band was normalized to the density of the β-actin band.
Data was evaluated using GraphPad Prism software (ver. 4.0, San Diego, CA).
All results are presented as means ± SEM. P < 0.05 was considered as statistically significant. Northern blot and Western blot results are presented as a percentage of the control BDNF/actin ratio and were evaluated using Student’s t-test. All CMS results obtained in this study were analyzed by multiple analyses of variance with two between-subject factors (stress/control, drug treatments) and successive sucrose tests as within-subject factor. The Fisher’s LSD test was used for the post-hoc comparisons of means.
Effect of imipramine or zinc administration on CMS-induced alterations in sucrose drinking
As shown in Fig. 1a, 5 weeks of treatment with imipramine had no significant effect on the sucrose intake in control animals [F(1,14) = 0.159; NS] and led to a gradual increase in sucrose consumption in stressed animals, resulting in a significant treatment effect [F(1,14) = 14.484; P < 0.001 and treatment × weeks interaction [F(5,70) = 10.656; P < 0.001]. As compared to week 0 scores, the increases in sucrose intake in stressed animals reached statistical significance after 3 weeks of treatment (P < 0.01), and this effect was maintained during a further 2 weeks of treatment (see Fig. 1).
As shown in Fig. 1b, 5 weeks of treatment with zinc had no significant effects in control animals [treatment effect: F(3,28) = 0.854; NS] but in stressed groups this compound caused a significant effect [F(3,28) = 4.584; P < 0.01 and treatment × weeks interaction: F(15,140) = 2.718; P < 0.05]. Zinc appears to have an inverse dose-dependent effect in the CMS model. Thus, only the dose of 10 mg/kg fully reversed the decrease of sucrose drinking in stressed animals; the two other doses (5 and 20 mg/kg) were ineffective against the CMS-induced deficit in sucrose consumption. As compared to the values at week 0, the increase of sucrose intake in stressed animals receiving 10 mg/kg of zinc already reached significance after the first week of treatment (P < 0.05). This effect was maintained and further enhanced thereafter, and at week 5 the amount of sucrose solution drunk by these animals was comparable to that of vehicle-treated controls (P = 0.503) and significantly higher than that of vehicle-treated stressed animals (P < 0.01).
Effect of zinc treatment on BDNF mRNA level
Effect of zinc treatment on BDNF protein level
A growing body of evidence indicates an antidepressant activity of zinc and a significant role of this ion in the mechanism(s) of action of antidepressant drugs. Recently the antidepressant-like activity of zinc was demonstrated in a rodent FST (Kroczka et al. 2000, 2001; Nowak et al. 2003b; Rosa et al. 2003), tail suspension test (TST) (Rosa et al. 2003), and in animal models of depression, such as olfactory bulbectomy (OB) or chronic unpredictable stress (Nowak et al. 2003b; Cieslik et al. 2007). Moreover, joint administration of sub-effective doses of zinc and antidepressants (imipramine or citalopram) enhanced the effects of these antidepressants in the FST or under conditions of chronic unpredictable stress (Kroczka et al. 2001; Szewczyk et al. 2002; Cieślik et al. 2007). The present data, demonstrating the activity of zinc in a very reliable model of depression, chronic mild stress, further supports the notion of the antidepressant properties of zinc. Furthermore, the effect of zinc in this model is evident early, after 1 week of treatment. By comparison, Sánchez et al. showed that in CMS significant effects of classic antidepressants were achieved from week 1 for escitalopram, from week 2 for citalopram, from week 3 for fluoxetine and from week 4 for imipramine (Sánchez et al. 2003). The rapid effect of zinc observed in CMS might imply the rapid therapeutic action of zinc also in human depression or may suggest that zinc supplementation can accelerate the effect of classic antidepressants, but these hypotheses need to be further evaluated. Our preliminary clinical study demonstrated the benefit of zinc supplementation in antidepressant therapy in major depression (Nowak et al. 2003a). Efficacy of antidepressant therapy and the status of patients in this study were evaluated by using the Hamilton Depression Rating Scale (HDRS) and Beck Depression Inventory (BDI). Zinc supplementation significantly reduced scores in both measures after 6- and 12-weeks supplementation when compared with placebo treatment. This preliminary study is the first demonstration of the benefit of zinc supplementation in antidepressant therapy in human and strongly supports the effects of zinc observed in all preclinical studies.
Preclinical and clinical studies put forward a neurotrophic hypothesis of the pathophysiology and therapy of depression in which BDNF plays a central role (Nibuya et al. 1995). This is based on the regulation of BDNF activity by antidepressants (Nibuya et al. 1995; reviewed in Duman and Monteggia 2006) and antidepressant-like effects of BDNF in behavioral models of depression (Siuciak et al. 1997; Shirayama et al. 2002). The data on transgenic mice with eliminated or reduced BDNF activity in the brain demonstrated that these animals are insensitive to antidepressants in behavioral tests (Saarelainen et al. 2003). Recent data published by Adachi et al. suggest that the loss of the hippocampal BDNF is not sufficient to mediate depression-like behavior. However, selective loss of BDNF in dentate gyrus (DG) of the hippocampus seems to be necessary for mediating the therapeutic effect of antidepressants (Adachi et al. 2008). Thus, these data indicated that agents which elevate BDNF or increase the activity of the BDNF pathway may be useful in treating depression. Although there is no clear relationship between the dose and time-of-treatment in the effect on BDNF expression, generally it is accepted that antidepressants induce an increase in the expression and function of BDNF in the hippocampal and/or cortical regions. In our recent paper (Nowak et al. 2004), we demonstrated that 2 weeks of zinc hydroaspartate (65 mg/kg; ~11 mg/kg of zinc) administration increases BDNF mRNA in the rat cortex but not in the hippocampus. These data are consistent with the data published by Franco et al., who also found an increase in BDNF expression in the cerebral cortex but not in the hippocampus, after chronic treatment with zinc (Franco et al. 2008). In both studies, rather high doses of zinc (~11 and ~ 15 mg/kg, respectively) were used. Our present study demonstrated that even 1 week of zinc hydroaspartate (10 mg/kg; ~1.8 mg/kg of zinc) treatment is sufficient to induce a 40% increase in hippocampal BDNF mRNA. This effect is present also after following 2 and 5 weeks of treatment. Thus, a lower dose of zinc than previously examined increases hippocampal BDNF after 1 week of treatment, whereas 5 weeks of zinc treatment is required for a result of an increase in BDNF mRNA in the cortex. This may indicate that a mechanism regulating the BDNF expression is more sensitive to zinc in the hippocampus than in the cortex. Examination of the effect of zinc on BDNF protein revealed alterations similar to those for mRNA. An increase in the BDNF protein level in the hippocampus after 1 and 2 weeks of zinc treatment and no changes in the cortex are parallel to the mRNA profiles. These observations indicated that zinc not only increases BDNF transcript expression, but also protein synthesis.
Several reports suggest that clinically effective antidepressants (affecting monoamine transmitter re-uptake or metabolism) may inhibit the function of the NMDA receptor by increasing BDNF activity (Duman et al. 1997a,b; Skolnick et al. 2001). Zinc is an antagonist of the NMDA receptor complex (Harrison and Gibbsons 1994), so one of the potential mechanisms of the antidepressant activity of zinc might be related to its direct antagonism of the NMDA receptor. The relationship between antagonism at the NMDA receptor complex and BDNF activity is not clear and published data are divergent. Different authors report increases, no changes or decreases in the BDNF transcript and/or protein expression following treatment with NMDA antagonists (Castren et al. 1993; Marvanova et al. 2001; Semba et al. 2006; Toyomoto et al. 2005). Another issue to consider is the general relationship between the BDNF and NMDA receptor. BDNF increases the activity of the NMDA receptor under physiological conditions (Caldeira et al. 2007; Glazner et al. 2000; Xu et al. 2006), although in “non-physiological” situations (stress-, drug-, pathology-induced hyper-stimulation) this relationship may be altered (Brandoli et al. 1998). As proposed by Sandoval et al., BDNF may enhance the NMDA receptor activity via TrkB receptors, while it inhibits this activity by stimulation of the p75NTR receptors (Sandoval et al. 2007). Additionally, chronic treatment with BDNF down regulates the TrkB receptors (Knusel et al. 1997), which may reduce NMDA transmission. Such mechanism is conceivable for cases of chronic treatment with antidepressants; when increases in BDNF expression would be accompanied by a reduction in NMDA receptor function (Skolnick et al. 2001). Thus, zinc like most antidepressants, increases expression of BDNF while inhibiting the activity of the NMDA receptor pathways (our unpublished data).
The present data demonstrates the quick acting antidepressant activity of zinc in a CMS model, as well as the involvement of zinc in the regulation of BDNF expression, which may reflect an effect on the NMDA/glutamate pathway. These findings support the possibility of the use of zinc as a potential antidepressant agent.
This study was supported by MNiSW grant no. 2 P05A 0178 29 (2005–2008) and funds for statutory activity of the Institute of Pharmacology PAS and Collegium Medicum Jagiellonian University, Kraków. The authors thank Farmapol Sp. z o.o. Poznań, Poland for the generous gift of zinc hydroaspartate.
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