J Huazhong Univ Sci Technol［Med Sci］ 34(6):838-844,2014 DOI 10.1007/s11596-014-1362-5 J Huazhong Univ Sci Technol［Med Sci］ 34(6):2014

838

Glutamate Transporter 1-mediated Antidepressant-like Effect in a Rat Model of Chronic Unpredictable Stress* Jian-xin CHEN (陈建新)1, Li-hua YAO (姚丽华)1, Bi-bo XU (徐碧波)2, Kun QIAN (钱 坤)3, Hui-ling WANG (王惠玲)1, Zhong-chun LIU (刘忠纯)1, Xiao-ping WANG (王晓萍)1, Gao-hua WANG (王高华)1# 1 Department of Psychiatry, Renmin Hospital, Wuhan University, Wuhan 430060, China 2 Department of Psychology, Faculty of Education, Hubei University, Wuhan 430062, China 3 Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China © Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2014

Summary: In recent years, more attention has been paid to the role of the glutamate transporter 1 (GLT-1, EAAT2) in major depressive disorder (MDD). However, experimental data on brain GLT-1 levels are, to some extent, inconsistent in human postmortem and animal studies. These discrepancies imply that the role of GLT-1 in the pathophysiology of MDD and the action of antidepressants remain obscure. This work was designed to study the impact of chronic unpredictable stress (CUS) for 2 sessions per day for 35 days and four weeks of fluoxetine (FLX) on depressive-like behaviors in rats, as well as the concomitant expression of the GLT-1 protein in the hippocampus. Behavioral changes were assessed by the sucrose preference and open field tests. GLT-1 levels were detected by immunohistochemistry and Western blot analysis. Our study demonstrated that the animals exposed to CUS showed depressive-like behaviors and exhibited a significant decrease in GLT-1 expression in the hippocampus. Chronic FLX treatment reversed the behavioral deficits and the CUS-induced decrease in GLT-1 levels. Taken together, our results support the reduction of GLT-1 in human postmortem studies in MDD and suggest that GLT-1 may be involved in the antidepressant activity of FLX. Our studies further support the notion that GLT-1 is an attractive candidate molecule associated with the fundamental processes of MDD and may be a potential, and novel pharmacological target for the treatment of MDD. Key words: chronic unpredictable stress; glutamate transporter 1; glutamate; fluoxetine; hippocampus 

Major depressive disorder (MDD), also known as major depression (MD), is the most common and chronic recurring psychiatric illness, and it affects a rising percentage of the world’s population[1]. To date, it has been recognized that monoamine deficits are not solely sufficient to explain the pathophysiology of MDD and the action mechanism of antidepressants[2, 3]. Moreover, increasing evidence indicates an important role for the glutamatergic system in fundamental processes related to the occurrence and treatment of MDD[4, 5]. The transporter EAAT2 (rodent nomenclature glutamate transporter 1, GLT-1), which is a predominantly astroglial glutamate transporter in the hippocampus and the prefrontal cortex (PFC)[6], is responsible for the majority of extracellular glutamate uptake[7]. Data from patients with MDD[8] and animal models of depression[9–11] have revealed alterations in the expression levels of GLT-1. However, experimental data on brain GLT-1 levels are, to some extent, inconsistent in human postmortem tissue[8] and animal studies[9, 10]. These discrepancies imply that the role of GLT-1 in the pathophysiology of MDD and the action mechanism of Jian-xin CHEN, E-mail: [email protected] # Corresponding author, E-mail: [email protected] * This project was supported by the Key Technology Research of Major Mental Illness Prevention and Treatment for the Barriers to the Recognition and Prevention of Depression and Anxiety in the General Hospital, China (No. 2012BAI01B05).

antidepressants remain obscure. The relationship between GLT-1 and MDD requires further study. Fluoxetine (FLX), a selective serotonin (5-HT) reuptake inhibitor, is one of the most commonly prescribed antidepressants. However, the mechanisms that underlie its antidepressant action remain unclear. A recent study demonstrated that FLX treatment induced the expression of EAAT2 (GLT-1) in the normal rat hippocampus and cortex[12]. However, this approach is in contrast to clinical practice in which treatment is restricted to the sufferers of MDD. Therefore, further exploration of the relationship between FLX and GLT-1 through animal models of depression is necessary. Based on the previous GLT-1 findings, we hypothesized that chronic unpredictable stress (CUS) for 2 sessions per day for 35 days would lead to a decreased expression of GLT-1 within the rat hippocampus, which could be reversed by four weeks of FLX treatment. The results may contribute to an improved understanding of the neurobiology of MDD, as well as an improved treatment of MDD. 1 MATERIALS AND METHODS 1.1 Animals Sixty male Sprague-Dawley (SD) rats used in this experiment were obtained from the Center of Experimental Animals of Wuhan University (China). All rats were housed in plastic cages in groups of four or five and

J Huazhong Univ Sci Technol［Med Sci］ 34(6):2014

were maintained under standard laboratory conditions (12/12-h light/dark cycle: lights on at 8:00 p.m., 22±2°C, food and water ad libitum). The experimental procedures were in accordance with the Chinese Health Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee of Wuhan University. All efforts were undertaken to minimize the number of rats and their suffering. The animals weighed between 250 and 300 g prior to the first stress exposure. After one week of habituation, the rats were randomly separated into three groups: the control+saline (control+S; n=20), CUS+saline (CUS+S; n=20), and CUS+FLX (n=20) groups. The sucrose preference and open field tests were conducted after CUS and FLX treatment. The rats were housed in groups during the experimental period, but were stressed, injected and underwent behavioral assessments in individual housing. All rats were decapitated immediately after the behavioral experiment. Ten rats in each group were used for detection of the GLT-1 expression by immunohistochemistry. An additional 10 rats in each group were used for GLT-1 Western blotting analysis. The details of the experimental procedure are depicted in fig. 1.

Fig. 1 Details of the experimental procedure Rats in the control+S, CUS+S and CUS+FLX groups were tested (sucrose preference test and open field test) at 36th and 65th day and killed within 12 h of the last test.

1.2 CUS The CUS model was established using the procedures described previously[13, 14] with slight modifications. The rats in the CUS+S group and CUS+FLX group were subjected to various stressors for 5 weeks (2 stressors per day). Ten stressors were used in the experiment: immobilization for 2 h (in a 25 cm×8 cm cylindrical plastic rodent restrainer), nip trail for 1 min, swimming in 4°C cold water for 5 min, swimming in 45°C warm water for 5 min, cage tilting (45°) for 24 h, damp sawdust (200 mL water in a cage) for 24 h, shaking for 15 min (120 r/min rocking bed), food deprivation for 24 h, water deprivation for 24 h, and alterations of the light-dark cycle. The rats were exposed to the stressors individually. The same stressor was not administered to any individual for two consecutive stages. The rats in the control+S group were not subjected to stressors. 1.3 Sucrose Preference Test All rats were trained to consume a palatable weak sucrose solution (1%), which was used to assess anhedonia. The training courses included an initial 48 h of sucrose solution exposure without any other food or water available and an additional 1 h per day of sucrose solution exposure for 5 consecutive days. Before each test, the rats were deprived of water for 12 h (8:00 p.m. to

839 8:00 a.m.). The rats were allowed to drink a bottle of 1% sucrose solution and water for 1 h (8:00 a.m. to 9:00 a.m.). The total consumption of the sucrose solution and water was measured by comparing the bottle weight before and after the test. The sucrose preference was calculated by the following formula: the sucrose preference (%)=total sucrose solution consumption/(total water+total sucrose solution consumption)×100%. 1.4 Open Field Test Each animal was placed at the center of a dimly illuminated rectangular cage (120 cm×90 cm×35 cm) and observed for 10 min continuously by a camera[14], which was used to assess spontaneous activity. The total traveled distance, moved velocity, and frequencies of rearing (standing upright on the hind legs while the forepaws are free) were automatically calculated with a video tracking system (Ethovision 3.0; Noldus, Netherlands). The cage was cleaned thoroughly before the next test. 1.5 FLX Treatment FLX (Shanghai JinHuan, China) was dissolved in saline immediately prior to administration. The rats received a daily intraperitoneal injection of FLX (10 mg/kg)[15–18] or an equal volume of saline vehicle for 4 weeks. 1.6 Immunohistochemistry Analysis The rats were euthanized by decapitation, and the hippocampus was dissected, embedded in paraffin, and then cut into 5-µm thick sections. The sections were first treated with 3% H2O2 in methanol for 30 min and then in the microwave for 15 min in 0.01 mmol/L citrate buffer (pH 7.2). After a preincubation in 10% normal goat serum for 30 min, the sections were incubated with rabbit polyclonal anti-GLT-1 antibody (1:200, ab41621, Abcam, USA) over night at 4°C. After washing with phosphate-buffered saline (PBS), they were subsequently incubated for 60 min at 37°C with the secondary antibody biotinylated goat anti-rabbit IgG (1:100, sc-3840, Santa Cruz Biotechnology, Santa Cruz, USA). Finally, the sections were washed three times in 0.1 mol/L PBS and stained with DAB for approximately 10 min. Images of the positively stained GLT-1 in the CA1, CA3, and DG regions of the hippocampus were captured at 200× magnification by an Olympus BX50 microscope (Olympus, Japan). A computer-assisted image analysis system (JEDA801D, Jieda Science and Technology Company Limited, China) was used for quantitative analysis of the integrated absorbance (A) for immunohistochemical staining of GLT-1. Ten sections were randomly selected for the quantification per rat. 1.7 Western Blot Analysis For determination of the GLT-1 protein levels, the protein extracts were obtained from the hippocampus. Briefly, the samples for Western blot analysis were removed from the –80°C freezer and immediately homogenized at 4°C with 0.5 mL of lysis buffer [0.01 mol/L Tris-HCl buffer (pH 7.6) containing 0.25 mol/L sucrose, 0.1 mol/L NaCl, 1 mmol/L EDTA, and 1 mmol/L phenylmethylsulfonyl fluoride]. The homogenates were centrifuged at 12 000 r/min for 5 min at 4°C, and the supernatant was analyzed. Protein concentrations were measured through the BCA protein assay. Aliquots of the clarified homogenized liquid, which contained 50 µg of protein, were denatured in a sample buffer [0.1 mol/L Tris-HCl buffer (pH 6.8) containing 0.2 mol/L

840 DTT, 4% SDS, 20% glycerol, and 0.1% bromophenol blue]. The samples were then analyzed by 10% SDSpolyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore). The primary antibodies included rabbit polyclonal anti-GLT-1 (1:300, ab41621, Abcam, USA) and rabbit polyclonal anti-GAPDH (1:300, ab9485, Abcam, USA). The secondary antibody included horseradish peroxidase conjugated goat anti-rabbit IgG (1:5000, sc-2004, Santa Cruz Biotechnology, Santa Cruz, USA). The membranes were detected with SuperSignal West Pico Substrate (Thermo Scientific) and exposed to X-ray film. The images were later scanned, and the densitometric analysis was performed using a video imaging CMIASWIN system (Bio-Rad, USA). The changes in the relative content of GLT-1 expression were represented with the ratio of the densitometric value of the band of GLT-1 to that of GAPDH (GLT-1/GAPDH×100%). 1.8 Statistical Analysis Data analysis was performed using SPSS (Statistical Package for the Social Sciences) version 17 software. The results are expressed as ±s. Data were analyzed by

J Huazhong Univ Sci Technol［Med Sci］ 34(6):2014

one-way ANOVA followed by the Bonferroni test for post hoc multiple range comparisons. The statistically significant difference was set as P