- Email: [email protected]
Zinc deficiency induces depression-like symptoms in adult rats
Physiology & Behavior 95 (2008) 365–369
Contents lists available at ScienceDirect
Physiology & Behavior j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / p h b
Zinc deficiency induces depression-like symptoms in adult rats Nadine M. Tassabehji a, Rikki S. Corniola b, Almamoun Alshingiti a, Cathy W. Levenson a,b,⁎ a b
Department of Nutrition, Food & Exercise Sciences, Florida State University, Tallahassee, Florida 32306-4340, United States Program in Neuroscience, Florida State University, Tallahassee, Florida 32306-4340, United States
A R T I C L E
I N F O
Article history: Received 15 November 2007 Received in revised form 29 May 2008 Accepted 27 June 2008 Keywords: Zinc Depression Anxiety Anhedonia Anorexia Fluoxetine
A B S T R A C T There is mounting evidence suggesting a link between serum zinc levels and clinical depression. Not only is serum zinc negatively correlated with the severity of symptoms, but zinc levels appear to be lowest in patients who do not respond to antidepressant drug therapy. It is not known if reduced zinc levels are contributing to depression, or the result of dietary or other factors associated with major depression. Thus, we designed this study to test the hypothesis that dietary zinc deficiency would induce depression-like behaviors in rats. Two-month-old male rats were fed zinc adequate (ZA, 30 ppm), deficient (ZD, 1 ppm), or supplemented (ZS, 180 ppm) diets for 3 weeks. Consistent with the development of depression, ZD rats displayed anorexia (p b 0.001), anhedonia (reduced saccharin:water intake, p b 0.001), and increased anxietylike behaviors in a light–dark box test (p b 0.05). Furthermore, the antidepressant drug fluoxetine (10 mg/kg body wt) reduced behavioral despair, as measured by the forced swim test, in rats fed the ZA and ZS rats (p b 0.05), but was ineffective in ZD rats. Together these studies suggest that zinc deficiency leads to the development of depression-like behaviors that may be refractory to antidepressant treatment. © 2008 Elsevier Inc. All rights reserved.
1. Introduction The World Health Organization (WHO) estimates that over 120 million people world-wide suffer from depression and has predicted that by 2020 unipolar major depression will be the second leading cause of disease or injury after ischemic heart disease. Unfortunately, current drug therapies for depression are not ideal. While there have been recent advances in the development and use of pharmacologic agents, many patients do not respond to these treatments. Others experience only modest improvements while suffering side effects that can include insomnia, weight gain, and sexual dysfunction [1]. Unfortunately, the ability to design more effective drugs has been hampered by our lack of understanding of the causes and mechanisms of depression. Over the last two decades there has been mounting evidence suggesting a link between zinc deficiency and clinical depression. One of the first reports to suggest this link was a case study that identified low serum zinc levels in a patient with treatment-resistant depression. Subsequent work showed that patients with major depression had serum zinc levels that were 88–84% of controls (p b 0.0001) and that there was a relationship between serum zinc levels and the severity of the symptoms of depression [2–6]. For example, in a study conducted in 80 subjects, patients with minor depression had serum zinc levels that were approximately 93% of control, while those with major depression ⁎ Corresponding author. Program in Neuroscience, Florida State University, 237 Biomedical Research Facility, Tallahassee, FL 32306-4340, United States. Tel.: +1 850 644 4122; fax: +1 850 644 0989. E-mail address: [email protected] (C.W. Levenson). 0031-9384/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2008.06.017
had serum zinc levels that were 88% of control (p b 0.001) [3]. Furthermore, two separate studies have shown a significant negative correlation between the Hamilton Depression Rating Scale (HDRS) score and serum zinc levels in depressed individuals [3,6]. It is also interesting to note that treatment-resistant patients appear to have lower serum zinc levels than those who respond to pharmacological intervention (p b 0.005) [2]. Dietary zinc deficiency has been identified throughout the developing world including countries such as Ethiopia [7], Bangladesh [8], Iran [9], Indonesia [10], Peru [11] and India [12]. However, it has also been reported in Europe [13] and the United States, with zinc deficiency being reported in all age groups including the elderly [14], adolescents [15], and pre-school children [16]. There is also evidence that specific populations, such as athletes, may be at risk for mild to moderate zinc deficiency [17]. Despite the prevalence of zinc deficiency, the link between a dietary deficiency of this essential micronutrient and the development of depression is merely correlational, and it is unknown that zinc deficiency is actually contributing to the development of depression or if it results from the behaviors (including changes in diet) that accompany the onset of depressive symptoms. Thus, the work described here was designed to test the hypothesis that dietary zinc deficiency contributes to the development of depression-like symptoms in laboratory rats. 2. Materials and methods 2.1. Animal care Two-month-old male Sprague–Dawley rats (Charles Rivers Laboratories, Wilmington, MA) were individually housed in temperature-
366
N.M. Tassabehji et al. / Physiology & Behavior 95 (2008) 365–369
controlled rooms with a 12-hour light–dark cycle. All rats were fed a commercially prepared (Research Diets Inc., New Brunswick, NJ) zinc adequate diet (ZA, 30 ppm) for 3 days. After this baseline period, rats were fed a ZA, zinc deficient (ZD, 1 ppm), or zinc supplemented (ZS, 180 ppm) diet for a period of 3 weeks. Because zinc deficiency is known to cause anorexia, an additional group of rats was pair-fed to the ZD rats. Pair-fed (PF) rats were provided with the weighed amount of ZA food eaten by the ZD rats on the previous day. Body weight, food intake, and water intake were monitored daily. 2.2. Anhedonia The ability of zinc deficiency to induce the depression-like symptom anhedonia was tested using a standard two-bottle choice paradigm for saccharin preference [18], two-month-old individually housed male rats (n = 12) were given a choice between deionized water and a 0.05% saccharin solution. Water and saccharin intakes were measured daily for 4 days and averaged for each animal. To avoid preferences associated with bottle placement, the positions of the bottles were changed daily. After baseline preferences were established, rats with a saccharin to water intake ratio N5 were provided with the ZD diet for two weeks. At the end of the two-week dietary period, saccharin preference was again tested for 4 days. 2.3. Light–dark box Anxiety-like behaviors were measured using standard protocols for light–dark box exploratory behavior [19] in animals (n = 10) from each dietary group (ZA, ZD, ZS, PF). An additional group of ZD rats
Fig. 2. Zinc deficiency induces anxiety-like behaviors. Anxiety-like behaviors were quantified using a light–dark box by measuring (A) the number of explorations into the light and (B) time spent in the light in rats fed a zinc adequate (ZA, 30 ppm), zinc deficient (ZD, 1 ppm), zinc supplemented (ZS, 180 ppm), or pair-fed (PF) diet for 3 weeks. Bars (mean ± SD) with different letters are significantly different at p b 0.05.
(n = 5) was also treated with the antidepressant fluoxetine, administered daily in drinking water during the 3-week dietary treatment period. Water intake was monitored daily to insure the constant administration of fluoxetine at 10 mg/kg/day. Non-FLX treated rats were later used in the swim protocol. Animals were monitored for the number of entries into the light box as well as the amount of time spent in the light during a 10 min test period. Differences between dietary treatment groups were analyzed by one-way ANOVA and a Tukey's post-hoc test. To determine the effect of fluoxetine in ZA and ZD diets, entries into light box were analyzed by two-way ANOVA with a Bonferroni post-hoc test.
Fig. 1. Effect of dietary zinc on food intake and body weight. Two-month-old male rats were fed a zinc adequate (ZA, 30 ppm), zinc deficient (ZD, 1 ppm), or zinc supplemented (ZS, 180 ppm) diet for 3 weeks. Additional rats were pair-fed (PF) to each ZD rat to control for anorexia. (A) Rats fed the ZD diet had significantly reduced food intake beginning on day 10 (p b 0.05) that was maintained throughout the study (p b 0.001 at day 21). (B) Body weights were also reduced in ZD rats compared to ZA controls (p b 0.001 at day 21).
Fig. 3. Effect of fluoxetine on anxiety-like behaviors in zinc deficient rats. Adult male rats were fed zinc adequate (ZA, 30 ppm) and zinc deficient (ZD, 1 ppm) diets, with (FLX) or without (H2O) oral fluoxetine for 21 days. Anxiety-like behaviors were quantified using a light–dark box by measuring the number of explorations into the light. Bars represent mean ± SD with a significant effect of FLX (p b 0.001).
N.M. Tassabehji et al. / Physiology & Behavior 95 (2008) 365–369
367
2.4. Porsolt swim test The Porsolt forced swim test is based on the observation that antidepressant drugs increase active swimming and reduce the amount of behavioral despair (immobility). This method has been well documented as a test for antidepressant drug efficacy [20]. After three weeks on the ZA, ZS or ZD diets, rats (n = 5) were placed individually into a temperature-controlled (25 °C) cylindrical water tank measuring 25 cm in diameter and 75 cm high for a period of 15 min. At the end of this period, the animals were removed from the tank and injected i.p. with either 10 mg/kg fluoxetine or an equal volume of vehicle (0.9% saline). The following day, rats were placed in the swim tank for 5 min during which time swimming behaviors were digitally recorded and later used to quantify immobility time by investigators blinded to the treatments. Because this test is stressful, no further behavioral tests were conducted on rats after the swim test. Differences between treatment groups were analyzed by ANOVA and a Tukey's post-hoc test. 3. Results 3.1. Food intake and body weight The effect of dietary zinc on food intake over the 3-week experimental period is shown in Fig. 1A. At the beginning of the experiment food intake was similar for all groups, with a significant decline (p b 0.05) in food intake in ZD animals observed by day 10 compared to both ZA and ZS rats. Reductions continued throughout the study such that by day 21, mean body weights of ZD rats were approximately 75% of ZA rats (p b 0.001; Fig. 1B). ZD rats developed the previously characterized [21] 4-day feeding cycle. 3.2. Anxiety-like behavior Zinc deficiency resulted in a significant decrease in both the number of explorations into the light side of the light–dark box (p b 0.01, Fig. 2A) and the amount of time spent exploring the lighted compartment (p b 0.05, Fig. 2B). These decreases, which have previously been used to quantify anxiety-like behaviors in rodents [19], were not the result of decreases in food intake. Pair-fed rats consumed the same amount of food each day as the ZD rats, but showed no differences in either measure of anxiety compared to ZA controls. Three weeks of zinc supplementation significantly increased
Fig. 5. Zinc deficiency impairs fluoxetine efficacy in the forced swim test. Adult male rats were fed a zinc adequate (ZA, 30 ppm), zinc supplemented (ZS, 180 ppm), or zinc deficient (ZD, 1 ppm) diet for 21 days and then subjected to the forced swim test to examine fluoxetine efficacy. Drug efficacy (FLX) in the 5 min swim test is determined by the significant reduction in immobility time compared to saline-treated controls (SAL). Bars (mean ± SD) indicate immobility duration (seconds). ⁎Significantly different from saline-treated controls at p b 0.05.
the time spent in light (p b 0.05) compared to ZA rats, but did not change the number of explorations (Fig. 2A and B). Fig. 3 shows that fluoxetine administration eliminated the anxiety-like behaviors ZD rats. Two-way ANOVA suggested a significant main effect of fluoxetine (p b 0.001, F = 20.8) and well as an interaction between drug and diet (p b 0.05, F = 7.35) on the number of explorations. Similar effects were seen on the amount of time spent in the light with a significant interaction between drug and diet (p b 0.05, F = 6.44), but no significant independent effects of either drug or diet. 3.3. Anhedonia Eleven of 12 ZA rats preferred the saccharin solution to water with a saccharin to water intake ratio N1.0. Eight animals exhibited a clear preference for saccharin with an intake ratio of N5.0. When these 8 animals were put on the ZD diet for two weeks, the intake ration dropped for every animal (Fig. 4). The mean intake ratio was reduced from 8.2 ± 1.8 prior to the zinc deficiency to 3.6 ± 2.0 after 2 weeks of the ZD diet (p b 0.001; Fig. 4 inset). 3.4. Fluoxetine efficacy Consistent with its use as an antidepressant, treatment with the SSRI fluoxetine significantly decreased immobility time (behavioral despair) in both ZA and ZS rats (p b 0.01; Fig. 5). However, in ZD rats, fluoxetine was unable to act as an effective antidepressant with no differences between saline-treated and fluoxetine-treated animals (Fig. 5). 4. Discussion The Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) lists symptoms of major depression in humans that include significant changes in appetite and weight, anhedonia, anxiety, and reduced physical activity [22]. The data reported here suggest a causative role for zinc deficiency in the induction of these symptoms in rodents.
Fig. 4. Zinc deficiency induces the depression-like behavior of anhedonia. The average saccharin:water intake ratio of each zinc adequate (ZA, 30 ppm) rat (n = 8) was first determined to be N 5.0. After two weeks on a zinc deficient diet (ZD, 1 ppm), intake ratios were again measured and found to be reduced in every rat. Bars represent mean intake ratios for all rats over a 4-day measurement period. Inset shows mean intake ratios (mean ± SD) for all eight rats. ⁎Significantly different from ZA at p b 0.001.
4.1. Anorexia We have known for over 3 decades that zinc deficiency induces anorexia in rats [23]. Most of the work on the role of zinc in food intake has been conducted in developing animals, beginning at or just after
368
N.M. Tassabehji et al. / Physiology & Behavior 95 (2008) 365–369
weaning. In young animals, anorexia develops rapidly with significant decreases in food intake in less than a week [24]. Even in the older animals used in this study (2 months old), with higher expected zinc stores, anorexia developed in approximately 10 days. We have previously shown that zinc deficiency-induced anorexia can be corrected rapidly after re-feeding zinc. In fact, when zinc deficient animals are fed a zinc adequate diet, food intake is increased within approximately 24 h, suggesting a specific role for zinc [21]. When reduced food intake accompanies human depression, it is important to consider the possibility that zinc deficiency is the result, not the cause, of the depression-related anorexia. However, previous work has shown that zinc deficiency is not simply a function of reduced food intake, and found no relationship between serum zinc and total food intake in depressed patients [2,3]. Together these data suggest that zinc deficiency is not simply the result of reduced food intake in depressed patients, but may actually be contributing to the development of the symptoms. 4.2. Anhedonia The development of anhedonia in ZD rats, as measured by a preference for saccharin-sweetened water, further supports a role for zinc deficiency in the development of depression. Because previous work has shown that when given a choice between dietary carbohydrate, fat, and protein, ZD rats preferentially reduce carbohydrate intake [25], in this work we were careful to use the model of saccharin-based anhedonia in the two-bottle test, rather than a sucrose-based test that has also been used [26]. It should also be noted that zinc deficiency is commonly believed to reduce taste acuity. A decrease in taste acuity for saccharin would have been a confounding variable in the current work if ZD rats were unable to taste the concentration of saccharin used in this test. However, the most recent reports on the role of zinc in taste acuity suggest that zinc deficiency raises the threshold for salt detection, but not sweet tastes [27]. Thus the reduction in the saccharin:water intake ratios reported here are the result of anhedonia, not decreased taste acuity. 4.3. Anxiety As many as 85% of people who suffer from major depression have co-morbid anxiety. This may be characterized by both generalized anxiety, as well as debilitating panic attacks and panic disorder [28]. Thus, we examined ZD rats for the presence of anxiety-like behaviors and found evidence of anxiety-like behaviors in adult rats that were not evident in controls. These findings are consistent with a recent report of anxiety-like behaviors in young zinc deficient rats [29], as well as thigmotaxia in young ZD rats in earlier work [30]. While food deprivation may be expected to increase anxiety, the finding of anxiety in ZD rats is not confounded by food intake in the current work because the anxiety-like behavior exhibited by ZD rats was not seen in pair-fed rats consuming the same amount of food. 4.4. Response to fluoxetine While there have been recent advances in the development and use of SSRIs, the now preferred pharmacological treatment for depression, it has been reported that 19–34% of patients fail to respond to drug therapy, even when they are fully compliant with the treatment regimen. Another 12–15% of patients achieve only a partial response to treatment [31]. While previous work in rats has shown that fluoxetine may have anti-panic properties [32,33] when administered chronically, there is also evidence that it can be anxiogenic [34]. In the current work fluoxetine increased exploratory behavior in both ZA and ZD rats. Thus, caution is warranted when interpreting these data because it is likely that fluoxetine increases exploratory behavior rather than correcting the effects of zinc deficiency.
While effective at decreasing immobility in zinc adequate rats as measured by the Porsolt swim test, fluoxetine did not decrease immobility in zinc deficient rats, suggesting that this SSRI may be rendered less effective in zinc deficiency. It should be noted that while zinc deficient animals did not respond to fluoxetine in the swim test, they also did not show increased immobility compared to controls. This is because, as designed, the Porsolt swim test is a test of drug efficacy, rather than a test for depression. The efficacy of acute injections of antidepressant drugs in this test are used as a screening test for the effectiveness of chronic doses in humans [20]. We have also used the Porsolt swim test in zinc deficient animals treated with chronically (3 weeks) of fluoxetine and confirmed that fluoxetine administration does not reduce immobility time in zinc deficient rats. While the current data suggest that the effectiveness of fluoxetine is reduced by zinc deficiency, it should be noted that the mechanisms responsible for this are not known. While we would hypothesize central neurobiological mechanisms are at work to impair fluoxetine efficacy, it is also possible that zinc deficiency impairs fluoxetine uptake or absorption. Ours findings are supported by an earlier report showing that patients who are refractory to common pharmacological treatments for depression have lower serum zinc levels than those who respond to drug interventions [2]. The stress associated hormones epinephrine and glucocorticoids increase liver metallothionein and reduce serum zinc [35]. Thus, this could indeed be part of the mechanisms where by depressed individuals have reduced serum zinc levels. Regardless of the mechanism of this decrease, our data suggest that this decrease could not only exacerbate the depression, but could also prevent the patient from responding fully to antidepressant drug therapies. These data also raise the issue of zinc supplementation in depressed patients, particularly those who fail to respond to drug therapy. In a double-blind trial, 20 patients diagnosed with major depression using the DSM-IV criteria were given oral zinc supplementation (25 mg/day) or a placebo in addition to standard antidepressant drug therapy. Patients were assessed before treatment and at 2, 6 and 12 weeks using two different depression inventory tests. These measures of depression status showed that 6 weeks of zinc supplementation augmented antidepressant drug therapy by over 50%. This difference was not only statistically significant (pb 0.05), but was sustained through the full 12 weeks of the study [36]. The upper tolerable limit for zinc has been set at 40 mg/day. Thus, levels of zinc used in this trail are not toxic and can safely be used in most patients. Thus, in light of the current data, it may be prudent to consider zinc supplementation of up to 25 mg/day as an adjunct to antidepressant therapy. While it is unlikely that zinc would have an independent effect in depressed patients, correcting an underlying zinc deficiency in depressed patients may increase drug efficacy. Acknowledgements The authors would like to acknowledge generous support from the Susan E. Lucas Fellowship, the inaugural award of the Dr. Allen Barrett and Gail Chatt-Ellis Graduate Research Fellowship Series in Neuroscience to N.M.T and R.C.S. We would also like to thank Charles Badland, Florida State University, for his invaluable help with the photomicrographs and figures, Allison Reiter for technical support, and Dr. Mohamed Kabbaj for the valuable advice and support. References [1] Dording CM, Mischoulon D, Petersen TJ, Kornbluh R, Gordon J, Nierenberg AA, et al. The pharmacologic management of SSRI-induced side effects: a survey of psychiatrists. Ann Clin Psychiatry 2002;14:143–7. [2] Maes M, Vandoolaeghe E, Neels H, Demedts P, Wauters A, Meltzer HY, et al. Lower serum zinc in major depression is a sensitive marker of treatment resistance and of the immune/inflammatory response in that illness. Biol Psychiatry 1997;42:349–58. [3] Maes M, D'Haese PC, Scharpe S, D'Hondt P, Cosyns P, De Broe ME. Hypozincemia in depression. J Affect Disord 1994;31:135–40. [4] Nowak G. Alterations in zinc homeostasis in depression and antidepressant therapy. Pol J Pharmacol 1998;50:1–4.
N.M. Tassabehji et al. / Physiology & Behavior 95 (2008) 365–369 [5] Maes M, De Vos N, Demedts P, Wauters A, Neels H. Lower serum zinc in major depression in relation to changes in serum acute phase proteins. J Affect Disord 1999;56:189-19. [6] Nowak G, Zieba A, Dudek D, Krosniak M, Szymaczek M, Schlegel-Zawadzka M. Serum trace elements in animal models and human depression. Part I. Zinc. Hum Psychopharmacol 1999;14:83–6. [7] Hotz C, Brown KH. Identifying populations at risk of zinc deficiency: the use of supplementation trials. Nutr Rev 2001;59:80–4. [8] Fischer Walker CL, Baqui AH, Ahmed S, Zaman K, El Arifeen S, Begum N, et al. Lowdose weekly supplementation of iron and/or zinc does not affect growth among Bangladeshi infants. Eur J Clin Nutr Sept 19 2007 [Electronic publication ahead for print]. [9] Mahmoodi MR, Kimiagar SM. Prevalence of zinc deficiency in junior high school students of Tehran City. Biol Trace Elem Res 2001;81:93–103. [10] Lind T, Lonnerdal B, Stenlund H, Ismail D, Seswandhana R, Ekstrom EC. A community-based randomized controlled trial of iron and zinc supplementation in Indonesian infants: interactions between iron and zinc. Am J Clin Nutr 2003;77:883–90. [11] Richard SA, Zavaleta N, Caulfield LE, Black RE, Witzig RS, Shankar AH. Zinc and iron supplementation and malaria, diarrhea, and respiratory infections in children in the Peruvian Amazon. Am J Trop Med Hyg 2006;75:126–32. [12] Chakravarty I, Sinha RK. Prevalence of micronutrient deficiency based on results obtained from the national pilot program on control of micronutrient malnutrition. Nutr Rev 2002;60:S53–58. [13] Cruz JA. Dietary habits and nutritional status in adolescents over Europe–Southern Europe. Eur J Clin Nutr 2000;54(Suppl 1):S29–35. [14] Vaquero MP. Magnesium and trace elements in the elderly: intake, status and recommendations. J Nutr Health Aging 2002;6:147–53. [15] Brown KH, Peerson JM, Rivera J, Allen LH. Effect of supplemental zinc on the growth and serum zinc concentrations of prepubertal children: a meta-analysis of randomized controlled trials. Am J Clin Nutr 2002;75:1062–71. [16] Skinner JD, Carruth BR, Houck KS, Bounds W, Morris M, Cox DR, et al. Longitudinal study of nutrient and food intakes of white preschool children aged 24 to 60 months. J Am Diet Assoc 1999;99:1514–21. [17] Micheletti A, Rossi R, Rufini S. Zinc status in athletes: relation to diet and exercise. Sports Med 2001;31:577–82. [18] Pucilowski O, Overstreet DH, Rezvani AH, Janowsky DS. Chronic mild stressinduced anhedonia: greater effect in a genetic rat model of depression. Physiol Behav 1993;54:1215–20. [19] Cryan JF, Kelly PH, Neijt HC, Sansig G, Flor PJ, van Der Putten H. Antidepressant and anxiolytic-like effects in mice lacking the group III metabotropic glutamate receptor mGluR7. Eur J Neurosci 2003;17:2409–17.
369
[20] Porsolt RD, Bertin A, Jalfre M. Behavioral despair in mice: a primary screening test for antidepressants. Arch Int Pharmacodyn Ther 1977;229:327–36. [21] Evans SA, Overton JM, Alshingiti A, Levenson CW. Regulation of metabolic rate and substrate utilization by zinc deficiency. Metabolism 2004;53:727–32. [22] 4th ed. Washington, DC: American Psychiatric Association; 2000. p. 356. [23] Chesters JK, Quarterman J. Effects of zinc deficiency on food intake and feeding patterns of rats. Br J Nutr 1970;24:1061–9. [24] Cui L, Takagi Y, Wasa M, Iiboshi Y, Inoue M, Khan J, et al. Zinc deficiency enhances interleukin-1alpha-induced metallothionein-1 expression in rats. J Nutr 1998;128:1092–8. [25] Rains TM, Shay NF. Zinc status specifically changes preferences for carbohydrate and protein in rats selecting from separate carbohydrate-, protein-, and fatcontaining diets. J Nutr 1995;125:2874–9. [26] Slattery DA, Markou A, Cryan JF. Evaluation of reward processes in an animal model of depression. Psychopharmacology (Berl) 2007;190:555–68. [27] Stewart-Knox BJ, Simpson EE, Parr H, Rae G, Polito A, Intorre F, et al. Taste acuity in response to zinc supplementation in older Europeans. Br J Nutr 2007;26:1–8. [28] Gorman JM. Comorbid depression and anxiety spectrum disorders. Depress Anxiety 2005;4:160–8. [29] Takeda A, Tamano H, Kan F, Itoh H, Oku N. Anxiety-like behavior of young rats after 2-week zinc deprivation. Behav Brain Res 2007;177:1–6. [30] Chu Y, Mouat MF, Harris RB, Coffield JA, Grider A. Water maze performance and changes in serum corticosterone levels in zinc-deprived and pair-fed rats. Physiol Behav 2003;78:569–78. [31] Fava M, Davidson KG. Definition and epidemiology of treatment-resistant depression. Psychiatr Clin North Am 1996;19:179–200. [32] Thompson MR, Li KM, Clemens KJ, Gurtman CG, Hunt GE, Cornish JL, et al. Chronic fluoxetine treatment partly attenuates the long-term anxiety and depressive symptoms induced by MDMA (‘Ecstasy’) in rats. Neuropsychopharmacol 2004;2:694–704. [33] Poltronieri SC, Zangrossi Jr H, de Barros Viana M. Antipanic-like effect of serotonin reuptake inhibitors in the elevated T-maze. Behav Brain Res 2003;147:185–92. [34] Drapier D, Bentué-Ferrer D, Laviolle B, Millet B, Allain H, Bourin M, et al. Effects of acute fluoxetine, paroxetine and desipramine on rats tested on the elevated plusmaze. Behav Brain Res 2007;176:202–9. [35] Cousins RJ, Dunn MA, Leinart AS, Yedinak KC, DiSilvestro RA. Coordinate regulation of zinc metabolism and metallothionein gene expression in rats. Am J Physiol 1986;251:E688–94. [36] Nowak G, Siwek M, Dudek D, Zieba A, Pilc A. Effect of zinc supplementation on antidepressant therapy in unipolar depression: a preliminary placebo-controlled study. Pol J Pharmacol 2003;55:1143–7.
Contents lists available at ScienceDirect
Physiology & Behavior j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / p h b
Zinc deficiency induces depression-like symptoms in adult rats Nadine M. Tassabehji a, Rikki S. Corniola b, Almamoun Alshingiti a, Cathy W. Levenson a,b,⁎ a b
Department of Nutrition, Food & Exercise Sciences, Florida State University, Tallahassee, Florida 32306-4340, United States Program in Neuroscience, Florida State University, Tallahassee, Florida 32306-4340, United States
A R T I C L E
I N F O
Article history: Received 15 November 2007 Received in revised form 29 May 2008 Accepted 27 June 2008 Keywords: Zinc Depression Anxiety Anhedonia Anorexia Fluoxetine
A B S T R A C T There is mounting evidence suggesting a link between serum zinc levels and clinical depression. Not only is serum zinc negatively correlated with the severity of symptoms, but zinc levels appear to be lowest in patients who do not respond to antidepressant drug therapy. It is not known if reduced zinc levels are contributing to depression, or the result of dietary or other factors associated with major depression. Thus, we designed this study to test the hypothesis that dietary zinc deficiency would induce depression-like behaviors in rats. Two-month-old male rats were fed zinc adequate (ZA, 30 ppm), deficient (ZD, 1 ppm), or supplemented (ZS, 180 ppm) diets for 3 weeks. Consistent with the development of depression, ZD rats displayed anorexia (p b 0.001), anhedonia (reduced saccharin:water intake, p b 0.001), and increased anxietylike behaviors in a light–dark box test (p b 0.05). Furthermore, the antidepressant drug fluoxetine (10 mg/kg body wt) reduced behavioral despair, as measured by the forced swim test, in rats fed the ZA and ZS rats (p b 0.05), but was ineffective in ZD rats. Together these studies suggest that zinc deficiency leads to the development of depression-like behaviors that may be refractory to antidepressant treatment. © 2008 Elsevier Inc. All rights reserved.
1. Introduction The World Health Organization (WHO) estimates that over 120 million people world-wide suffer from depression and has predicted that by 2020 unipolar major depression will be the second leading cause of disease or injury after ischemic heart disease. Unfortunately, current drug therapies for depression are not ideal. While there have been recent advances in the development and use of pharmacologic agents, many patients do not respond to these treatments. Others experience only modest improvements while suffering side effects that can include insomnia, weight gain, and sexual dysfunction [1]. Unfortunately, the ability to design more effective drugs has been hampered by our lack of understanding of the causes and mechanisms of depression. Over the last two decades there has been mounting evidence suggesting a link between zinc deficiency and clinical depression. One of the first reports to suggest this link was a case study that identified low serum zinc levels in a patient with treatment-resistant depression. Subsequent work showed that patients with major depression had serum zinc levels that were 88–84% of controls (p b 0.0001) and that there was a relationship between serum zinc levels and the severity of the symptoms of depression [2–6]. For example, in a study conducted in 80 subjects, patients with minor depression had serum zinc levels that were approximately 93% of control, while those with major depression ⁎ Corresponding author. Program in Neuroscience, Florida State University, 237 Biomedical Research Facility, Tallahassee, FL 32306-4340, United States. Tel.: +1 850 644 4122; fax: +1 850 644 0989. E-mail address: [email protected] (C.W. Levenson). 0031-9384/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2008.06.017
had serum zinc levels that were 88% of control (p b 0.001) [3]. Furthermore, two separate studies have shown a significant negative correlation between the Hamilton Depression Rating Scale (HDRS) score and serum zinc levels in depressed individuals [3,6]. It is also interesting to note that treatment-resistant patients appear to have lower serum zinc levels than those who respond to pharmacological intervention (p b 0.005) [2]. Dietary zinc deficiency has been identified throughout the developing world including countries such as Ethiopia [7], Bangladesh [8], Iran [9], Indonesia [10], Peru [11] and India [12]. However, it has also been reported in Europe [13] and the United States, with zinc deficiency being reported in all age groups including the elderly [14], adolescents [15], and pre-school children [16]. There is also evidence that specific populations, such as athletes, may be at risk for mild to moderate zinc deficiency [17]. Despite the prevalence of zinc deficiency, the link between a dietary deficiency of this essential micronutrient and the development of depression is merely correlational, and it is unknown that zinc deficiency is actually contributing to the development of depression or if it results from the behaviors (including changes in diet) that accompany the onset of depressive symptoms. Thus, the work described here was designed to test the hypothesis that dietary zinc deficiency contributes to the development of depression-like symptoms in laboratory rats. 2. Materials and methods 2.1. Animal care Two-month-old male Sprague–Dawley rats (Charles Rivers Laboratories, Wilmington, MA) were individually housed in temperature-
366
N.M. Tassabehji et al. / Physiology & Behavior 95 (2008) 365–369
controlled rooms with a 12-hour light–dark cycle. All rats were fed a commercially prepared (Research Diets Inc., New Brunswick, NJ) zinc adequate diet (ZA, 30 ppm) for 3 days. After this baseline period, rats were fed a ZA, zinc deficient (ZD, 1 ppm), or zinc supplemented (ZS, 180 ppm) diet for a period of 3 weeks. Because zinc deficiency is known to cause anorexia, an additional group of rats was pair-fed to the ZD rats. Pair-fed (PF) rats were provided with the weighed amount of ZA food eaten by the ZD rats on the previous day. Body weight, food intake, and water intake were monitored daily. 2.2. Anhedonia The ability of zinc deficiency to induce the depression-like symptom anhedonia was tested using a standard two-bottle choice paradigm for saccharin preference [18], two-month-old individually housed male rats (n = 12) were given a choice between deionized water and a 0.05% saccharin solution. Water and saccharin intakes were measured daily for 4 days and averaged for each animal. To avoid preferences associated with bottle placement, the positions of the bottles were changed daily. After baseline preferences were established, rats with a saccharin to water intake ratio N5 were provided with the ZD diet for two weeks. At the end of the two-week dietary period, saccharin preference was again tested for 4 days. 2.3. Light–dark box Anxiety-like behaviors were measured using standard protocols for light–dark box exploratory behavior [19] in animals (n = 10) from each dietary group (ZA, ZD, ZS, PF). An additional group of ZD rats
Fig. 2. Zinc deficiency induces anxiety-like behaviors. Anxiety-like behaviors were quantified using a light–dark box by measuring (A) the number of explorations into the light and (B) time spent in the light in rats fed a zinc adequate (ZA, 30 ppm), zinc deficient (ZD, 1 ppm), zinc supplemented (ZS, 180 ppm), or pair-fed (PF) diet for 3 weeks. Bars (mean ± SD) with different letters are significantly different at p b 0.05.
(n = 5) was also treated with the antidepressant fluoxetine, administered daily in drinking water during the 3-week dietary treatment period. Water intake was monitored daily to insure the constant administration of fluoxetine at 10 mg/kg/day. Non-FLX treated rats were later used in the swim protocol. Animals were monitored for the number of entries into the light box as well as the amount of time spent in the light during a 10 min test period. Differences between dietary treatment groups were analyzed by one-way ANOVA and a Tukey's post-hoc test. To determine the effect of fluoxetine in ZA and ZD diets, entries into light box were analyzed by two-way ANOVA with a Bonferroni post-hoc test.
Fig. 1. Effect of dietary zinc on food intake and body weight. Two-month-old male rats were fed a zinc adequate (ZA, 30 ppm), zinc deficient (ZD, 1 ppm), or zinc supplemented (ZS, 180 ppm) diet for 3 weeks. Additional rats were pair-fed (PF) to each ZD rat to control for anorexia. (A) Rats fed the ZD diet had significantly reduced food intake beginning on day 10 (p b 0.05) that was maintained throughout the study (p b 0.001 at day 21). (B) Body weights were also reduced in ZD rats compared to ZA controls (p b 0.001 at day 21).
Fig. 3. Effect of fluoxetine on anxiety-like behaviors in zinc deficient rats. Adult male rats were fed zinc adequate (ZA, 30 ppm) and zinc deficient (ZD, 1 ppm) diets, with (FLX) or without (H2O) oral fluoxetine for 21 days. Anxiety-like behaviors were quantified using a light–dark box by measuring the number of explorations into the light. Bars represent mean ± SD with a significant effect of FLX (p b 0.001).
N.M. Tassabehji et al. / Physiology & Behavior 95 (2008) 365–369
367
2.4. Porsolt swim test The Porsolt forced swim test is based on the observation that antidepressant drugs increase active swimming and reduce the amount of behavioral despair (immobility). This method has been well documented as a test for antidepressant drug efficacy [20]. After three weeks on the ZA, ZS or ZD diets, rats (n = 5) were placed individually into a temperature-controlled (25 °C) cylindrical water tank measuring 25 cm in diameter and 75 cm high for a period of 15 min. At the end of this period, the animals were removed from the tank and injected i.p. with either 10 mg/kg fluoxetine or an equal volume of vehicle (0.9% saline). The following day, rats were placed in the swim tank for 5 min during which time swimming behaviors were digitally recorded and later used to quantify immobility time by investigators blinded to the treatments. Because this test is stressful, no further behavioral tests were conducted on rats after the swim test. Differences between treatment groups were analyzed by ANOVA and a Tukey's post-hoc test. 3. Results 3.1. Food intake and body weight The effect of dietary zinc on food intake over the 3-week experimental period is shown in Fig. 1A. At the beginning of the experiment food intake was similar for all groups, with a significant decline (p b 0.05) in food intake in ZD animals observed by day 10 compared to both ZA and ZS rats. Reductions continued throughout the study such that by day 21, mean body weights of ZD rats were approximately 75% of ZA rats (p b 0.001; Fig. 1B). ZD rats developed the previously characterized [21] 4-day feeding cycle. 3.2. Anxiety-like behavior Zinc deficiency resulted in a significant decrease in both the number of explorations into the light side of the light–dark box (p b 0.01, Fig. 2A) and the amount of time spent exploring the lighted compartment (p b 0.05, Fig. 2B). These decreases, which have previously been used to quantify anxiety-like behaviors in rodents [19], were not the result of decreases in food intake. Pair-fed rats consumed the same amount of food each day as the ZD rats, but showed no differences in either measure of anxiety compared to ZA controls. Three weeks of zinc supplementation significantly increased
Fig. 5. Zinc deficiency impairs fluoxetine efficacy in the forced swim test. Adult male rats were fed a zinc adequate (ZA, 30 ppm), zinc supplemented (ZS, 180 ppm), or zinc deficient (ZD, 1 ppm) diet for 21 days and then subjected to the forced swim test to examine fluoxetine efficacy. Drug efficacy (FLX) in the 5 min swim test is determined by the significant reduction in immobility time compared to saline-treated controls (SAL). Bars (mean ± SD) indicate immobility duration (seconds). ⁎Significantly different from saline-treated controls at p b 0.05.
the time spent in light (p b 0.05) compared to ZA rats, but did not change the number of explorations (Fig. 2A and B). Fig. 3 shows that fluoxetine administration eliminated the anxiety-like behaviors ZD rats. Two-way ANOVA suggested a significant main effect of fluoxetine (p b 0.001, F = 20.8) and well as an interaction between drug and diet (p b 0.05, F = 7.35) on the number of explorations. Similar effects were seen on the amount of time spent in the light with a significant interaction between drug and diet (p b 0.05, F = 6.44), but no significant independent effects of either drug or diet. 3.3. Anhedonia Eleven of 12 ZA rats preferred the saccharin solution to water with a saccharin to water intake ratio N1.0. Eight animals exhibited a clear preference for saccharin with an intake ratio of N5.0. When these 8 animals were put on the ZD diet for two weeks, the intake ration dropped for every animal (Fig. 4). The mean intake ratio was reduced from 8.2 ± 1.8 prior to the zinc deficiency to 3.6 ± 2.0 after 2 weeks of the ZD diet (p b 0.001; Fig. 4 inset). 3.4. Fluoxetine efficacy Consistent with its use as an antidepressant, treatment with the SSRI fluoxetine significantly decreased immobility time (behavioral despair) in both ZA and ZS rats (p b 0.01; Fig. 5). However, in ZD rats, fluoxetine was unable to act as an effective antidepressant with no differences between saline-treated and fluoxetine-treated animals (Fig. 5). 4. Discussion The Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) lists symptoms of major depression in humans that include significant changes in appetite and weight, anhedonia, anxiety, and reduced physical activity [22]. The data reported here suggest a causative role for zinc deficiency in the induction of these symptoms in rodents.
Fig. 4. Zinc deficiency induces the depression-like behavior of anhedonia. The average saccharin:water intake ratio of each zinc adequate (ZA, 30 ppm) rat (n = 8) was first determined to be N 5.0. After two weeks on a zinc deficient diet (ZD, 1 ppm), intake ratios were again measured and found to be reduced in every rat. Bars represent mean intake ratios for all rats over a 4-day measurement period. Inset shows mean intake ratios (mean ± SD) for all eight rats. ⁎Significantly different from ZA at p b 0.001.
4.1. Anorexia We have known for over 3 decades that zinc deficiency induces anorexia in rats [23]. Most of the work on the role of zinc in food intake has been conducted in developing animals, beginning at or just after
368
N.M. Tassabehji et al. / Physiology & Behavior 95 (2008) 365–369
weaning. In young animals, anorexia develops rapidly with significant decreases in food intake in less than a week [24]. Even in the older animals used in this study (2 months old), with higher expected zinc stores, anorexia developed in approximately 10 days. We have previously shown that zinc deficiency-induced anorexia can be corrected rapidly after re-feeding zinc. In fact, when zinc deficient animals are fed a zinc adequate diet, food intake is increased within approximately 24 h, suggesting a specific role for zinc [21]. When reduced food intake accompanies human depression, it is important to consider the possibility that zinc deficiency is the result, not the cause, of the depression-related anorexia. However, previous work has shown that zinc deficiency is not simply a function of reduced food intake, and found no relationship between serum zinc and total food intake in depressed patients [2,3]. Together these data suggest that zinc deficiency is not simply the result of reduced food intake in depressed patients, but may actually be contributing to the development of the symptoms. 4.2. Anhedonia The development of anhedonia in ZD rats, as measured by a preference for saccharin-sweetened water, further supports a role for zinc deficiency in the development of depression. Because previous work has shown that when given a choice between dietary carbohydrate, fat, and protein, ZD rats preferentially reduce carbohydrate intake [25], in this work we were careful to use the model of saccharin-based anhedonia in the two-bottle test, rather than a sucrose-based test that has also been used [26]. It should also be noted that zinc deficiency is commonly believed to reduce taste acuity. A decrease in taste acuity for saccharin would have been a confounding variable in the current work if ZD rats were unable to taste the concentration of saccharin used in this test. However, the most recent reports on the role of zinc in taste acuity suggest that zinc deficiency raises the threshold for salt detection, but not sweet tastes [27]. Thus the reduction in the saccharin:water intake ratios reported here are the result of anhedonia, not decreased taste acuity. 4.3. Anxiety As many as 85% of people who suffer from major depression have co-morbid anxiety. This may be characterized by both generalized anxiety, as well as debilitating panic attacks and panic disorder [28]. Thus, we examined ZD rats for the presence of anxiety-like behaviors and found evidence of anxiety-like behaviors in adult rats that were not evident in controls. These findings are consistent with a recent report of anxiety-like behaviors in young zinc deficient rats [29], as well as thigmotaxia in young ZD rats in earlier work [30]. While food deprivation may be expected to increase anxiety, the finding of anxiety in ZD rats is not confounded by food intake in the current work because the anxiety-like behavior exhibited by ZD rats was not seen in pair-fed rats consuming the same amount of food. 4.4. Response to fluoxetine While there have been recent advances in the development and use of SSRIs, the now preferred pharmacological treatment for depression, it has been reported that 19–34% of patients fail to respond to drug therapy, even when they are fully compliant with the treatment regimen. Another 12–15% of patients achieve only a partial response to treatment [31]. While previous work in rats has shown that fluoxetine may have anti-panic properties [32,33] when administered chronically, there is also evidence that it can be anxiogenic [34]. In the current work fluoxetine increased exploratory behavior in both ZA and ZD rats. Thus, caution is warranted when interpreting these data because it is likely that fluoxetine increases exploratory behavior rather than correcting the effects of zinc deficiency.
While effective at decreasing immobility in zinc adequate rats as measured by the Porsolt swim test, fluoxetine did not decrease immobility in zinc deficient rats, suggesting that this SSRI may be rendered less effective in zinc deficiency. It should be noted that while zinc deficient animals did not respond to fluoxetine in the swim test, they also did not show increased immobility compared to controls. This is because, as designed, the Porsolt swim test is a test of drug efficacy, rather than a test for depression. The efficacy of acute injections of antidepressant drugs in this test are used as a screening test for the effectiveness of chronic doses in humans [20]. We have also used the Porsolt swim test in zinc deficient animals treated with chronically (3 weeks) of fluoxetine and confirmed that fluoxetine administration does not reduce immobility time in zinc deficient rats. While the current data suggest that the effectiveness of fluoxetine is reduced by zinc deficiency, it should be noted that the mechanisms responsible for this are not known. While we would hypothesize central neurobiological mechanisms are at work to impair fluoxetine efficacy, it is also possible that zinc deficiency impairs fluoxetine uptake or absorption. Ours findings are supported by an earlier report showing that patients who are refractory to common pharmacological treatments for depression have lower serum zinc levels than those who respond to drug interventions [2]. The stress associated hormones epinephrine and glucocorticoids increase liver metallothionein and reduce serum zinc [35]. Thus, this could indeed be part of the mechanisms where by depressed individuals have reduced serum zinc levels. Regardless of the mechanism of this decrease, our data suggest that this decrease could not only exacerbate the depression, but could also prevent the patient from responding fully to antidepressant drug therapies. These data also raise the issue of zinc supplementation in depressed patients, particularly those who fail to respond to drug therapy. In a double-blind trial, 20 patients diagnosed with major depression using the DSM-IV criteria were given oral zinc supplementation (25 mg/day) or a placebo in addition to standard antidepressant drug therapy. Patients were assessed before treatment and at 2, 6 and 12 weeks using two different depression inventory tests. These measures of depression status showed that 6 weeks of zinc supplementation augmented antidepressant drug therapy by over 50%. This difference was not only statistically significant (pb 0.05), but was sustained through the full 12 weeks of the study [36]. The upper tolerable limit for zinc has been set at 40 mg/day. Thus, levels of zinc used in this trail are not toxic and can safely be used in most patients. Thus, in light of the current data, it may be prudent to consider zinc supplementation of up to 25 mg/day as an adjunct to antidepressant therapy. While it is unlikely that zinc would have an independent effect in depressed patients, correcting an underlying zinc deficiency in depressed patients may increase drug efficacy. Acknowledgements The authors would like to acknowledge generous support from the Susan E. Lucas Fellowship, the inaugural award of the Dr. Allen Barrett and Gail Chatt-Ellis Graduate Research Fellowship Series in Neuroscience to N.M.T and R.C.S. We would also like to thank Charles Badland, Florida State University, for his invaluable help with the photomicrographs and figures, Allison Reiter for technical support, and Dr. Mohamed Kabbaj for the valuable advice and support. References [1] Dording CM, Mischoulon D, Petersen TJ, Kornbluh R, Gordon J, Nierenberg AA, et al. The pharmacologic management of SSRI-induced side effects: a survey of psychiatrists. Ann Clin Psychiatry 2002;14:143–7. [2] Maes M, Vandoolaeghe E, Neels H, Demedts P, Wauters A, Meltzer HY, et al. Lower serum zinc in major depression is a sensitive marker of treatment resistance and of the immune/inflammatory response in that illness. Biol Psychiatry 1997;42:349–58. [3] Maes M, D'Haese PC, Scharpe S, D'Hondt P, Cosyns P, De Broe ME. Hypozincemia in depression. J Affect Disord 1994;31:135–40. [4] Nowak G. Alterations in zinc homeostasis in depression and antidepressant therapy. Pol J Pharmacol 1998;50:1–4.
N.M. Tassabehji et al. / Physiology & Behavior 95 (2008) 365–369 [5] Maes M, De Vos N, Demedts P, Wauters A, Neels H. Lower serum zinc in major depression in relation to changes in serum acute phase proteins. J Affect Disord 1999;56:189-19. [6] Nowak G, Zieba A, Dudek D, Krosniak M, Szymaczek M, Schlegel-Zawadzka M. Serum trace elements in animal models and human depression. Part I. Zinc. Hum Psychopharmacol 1999;14:83–6. [7] Hotz C, Brown KH. Identifying populations at risk of zinc deficiency: the use of supplementation trials. Nutr Rev 2001;59:80–4. [8] Fischer Walker CL, Baqui AH, Ahmed S, Zaman K, El Arifeen S, Begum N, et al. Lowdose weekly supplementation of iron and/or zinc does not affect growth among Bangladeshi infants. Eur J Clin Nutr Sept 19 2007 [Electronic publication ahead for print]. [9] Mahmoodi MR, Kimiagar SM. Prevalence of zinc deficiency in junior high school students of Tehran City. Biol Trace Elem Res 2001;81:93–103. [10] Lind T, Lonnerdal B, Stenlund H, Ismail D, Seswandhana R, Ekstrom EC. A community-based randomized controlled trial of iron and zinc supplementation in Indonesian infants: interactions between iron and zinc. Am J Clin Nutr 2003;77:883–90. [11] Richard SA, Zavaleta N, Caulfield LE, Black RE, Witzig RS, Shankar AH. Zinc and iron supplementation and malaria, diarrhea, and respiratory infections in children in the Peruvian Amazon. Am J Trop Med Hyg 2006;75:126–32. [12] Chakravarty I, Sinha RK. Prevalence of micronutrient deficiency based on results obtained from the national pilot program on control of micronutrient malnutrition. Nutr Rev 2002;60:S53–58. [13] Cruz JA. Dietary habits and nutritional status in adolescents over Europe–Southern Europe. Eur J Clin Nutr 2000;54(Suppl 1):S29–35. [14] Vaquero MP. Magnesium and trace elements in the elderly: intake, status and recommendations. J Nutr Health Aging 2002;6:147–53. [15] Brown KH, Peerson JM, Rivera J, Allen LH. Effect of supplemental zinc on the growth and serum zinc concentrations of prepubertal children: a meta-analysis of randomized controlled trials. Am J Clin Nutr 2002;75:1062–71. [16] Skinner JD, Carruth BR, Houck KS, Bounds W, Morris M, Cox DR, et al. Longitudinal study of nutrient and food intakes of white preschool children aged 24 to 60 months. J Am Diet Assoc 1999;99:1514–21. [17] Micheletti A, Rossi R, Rufini S. Zinc status in athletes: relation to diet and exercise. Sports Med 2001;31:577–82. [18] Pucilowski O, Overstreet DH, Rezvani AH, Janowsky DS. Chronic mild stressinduced anhedonia: greater effect in a genetic rat model of depression. Physiol Behav 1993;54:1215–20. [19] Cryan JF, Kelly PH, Neijt HC, Sansig G, Flor PJ, van Der Putten H. Antidepressant and anxiolytic-like effects in mice lacking the group III metabotropic glutamate receptor mGluR7. Eur J Neurosci 2003;17:2409–17.
369
[20] Porsolt RD, Bertin A, Jalfre M. Behavioral despair in mice: a primary screening test for antidepressants. Arch Int Pharmacodyn Ther 1977;229:327–36. [21] Evans SA, Overton JM, Alshingiti A, Levenson CW. Regulation of metabolic rate and substrate utilization by zinc deficiency. Metabolism 2004;53:727–32. [22] 4th ed. Washington, DC: American Psychiatric Association; 2000. p. 356. [23] Chesters JK, Quarterman J. Effects of zinc deficiency on food intake and feeding patterns of rats. Br J Nutr 1970;24:1061–9. [24] Cui L, Takagi Y, Wasa M, Iiboshi Y, Inoue M, Khan J, et al. Zinc deficiency enhances interleukin-1alpha-induced metallothionein-1 expression in rats. J Nutr 1998;128:1092–8. [25] Rains TM, Shay NF. Zinc status specifically changes preferences for carbohydrate and protein in rats selecting from separate carbohydrate-, protein-, and fatcontaining diets. J Nutr 1995;125:2874–9. [26] Slattery DA, Markou A, Cryan JF. Evaluation of reward processes in an animal model of depression. Psychopharmacology (Berl) 2007;190:555–68. [27] Stewart-Knox BJ, Simpson EE, Parr H, Rae G, Polito A, Intorre F, et al. Taste acuity in response to zinc supplementation in older Europeans. Br J Nutr 2007;26:1–8. [28] Gorman JM. Comorbid depression and anxiety spectrum disorders. Depress Anxiety 2005;4:160–8. [29] Takeda A, Tamano H, Kan F, Itoh H, Oku N. Anxiety-like behavior of young rats after 2-week zinc deprivation. Behav Brain Res 2007;177:1–6. [30] Chu Y, Mouat MF, Harris RB, Coffield JA, Grider A. Water maze performance and changes in serum corticosterone levels in zinc-deprived and pair-fed rats. Physiol Behav 2003;78:569–78. [31] Fava M, Davidson KG. Definition and epidemiology of treatment-resistant depression. Psychiatr Clin North Am 1996;19:179–200. [32] Thompson MR, Li KM, Clemens KJ, Gurtman CG, Hunt GE, Cornish JL, et al. Chronic fluoxetine treatment partly attenuates the long-term anxiety and depressive symptoms induced by MDMA (‘Ecstasy’) in rats. Neuropsychopharmacol 2004;2:694–704. [33] Poltronieri SC, Zangrossi Jr H, de Barros Viana M. Antipanic-like effect of serotonin reuptake inhibitors in the elevated T-maze. Behav Brain Res 2003;147:185–92. [34] Drapier D, Bentué-Ferrer D, Laviolle B, Millet B, Allain H, Bourin M, et al. Effects of acute fluoxetine, paroxetine and desipramine on rats tested on the elevated plusmaze. Behav Brain Res 2007;176:202–9. [35] Cousins RJ, Dunn MA, Leinart AS, Yedinak KC, DiSilvestro RA. Coordinate regulation of zinc metabolism and metallothionein gene expression in rats. Am J Physiol 1986;251:E688–94. [36] Nowak G, Siwek M, Dudek D, Zieba A, Pilc A. Effect of zinc supplementation on antidepressant therapy in unipolar depression: a preliminary placebo-controlled study. Pol J Pharmacol 2003;55:1143–7.