Neuropharmacology 89 (2015) 318e324

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Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm

Insulin-like growth factor 2 mitigates depressive behavior in a rat model of chronic stress Yan-Wei Luo a, b, Yang Xu b, Wen-Yu Cao a, Xiao-Lin Zhong a, Juan Duan a, Xue-Qin Wang a, Zhao-Lan Hu a, Fang Li a, Jian-Yi Zhang a, Ming Zhou b, Ru-Ping Dai c, Chang-Qi Li a, * a b c

Department of Anatomy and Neurobiology, Xiangya School of Medicine, Central South University, Changsha, Hunan Province, 410013, China Cancer Research Institute, Central South University, Changsha, Hunan Province, 410008, China Department of Anesthesiology, The Second Xiangya Hospital of Central South University, Changsha, Hunan Province, 410011, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 16 May 2014 Received in revised form 4 October 2014 Accepted 8 October 2014 Available online 19 October 2014

Depression is a common psychiatric disorder associated with chronic stress. Insulin-like growth factor 2 (IGF2) is a growth factor that serves important roles in the brain during development and at adulthood. Here, the role of IGF2 expression in the hippocampus was investigated in a rat model of depression. A chronic restraint stress (CRS) model of depression was established in rats, exhibiting depression-like behavior as assessed with the sucrose preference test (SPT) and forced swimming test (FST), and with evaluation of the corticosterone levels. Hippocampal IGF2 levels were significantly lower in rats suffering CRS than in controls, as were levels of pERK1/2 and GluR1. Lentivirus-mediated hippocampal IGF2 overexpression alleviated depressive behavior in restrained rats, elevated the levels of pERK1/2 and GluR1 proteins, but it did not affect the expression of pGSK3b, GluR2, NMDAR1, and NMDAR2A. These results suggest the chronic restraint stress induces depressive behavior, which may be mediated by ERKdependent IGF2 signaling, pointing to an antidepressant role for this molecular pathway. © 2014 Elsevier Ltd. All rights reserved.

Keywords: IGF2 Depression Antidepressant ERK1/2 Hippocampus

1. Introduction Chronic stress plays an important role in various psychiatric disorders, such as anxiety and major depression (Hill et al., 2012). Pre-clinical and clinical studies suggest that some parts of the brain, including the anterior cingulated cortex (ACC), amygdala, and hippocampal formation are activated or inhibited by chronic stress (Maras and Baram, 2012; McEwen, 2007). This may lead to abnormal gene expression and neuroendocrine dysfunction. In this way, the hippocampus is an important region for stress response because it has many direct and indirect connections with the hypothalamicepituitary axis (Isgor et al., 2004; Jacobson and Sapolsky, 1991). The dentate gyrus is one of the main parts of the brain featured in adult neurogenesis (Kuhn et al., 1996). This is modulated by stress and related to antidepressant therapy (Dias et al., 2012; Kheirbek et al., 2012). Insulin growth factors (IGF1 and IGF2) belong to the insulin growth factor family. They can modulate the proliferation, survival, and differentiation of many types of neuronal systems, including cholinergic, dopaminergic, and serotonergic neurons (Estil et al.,

* Corresponding author. Tel.: þ86 0731 82650421; fax: þ86 0731 82650426. E-mail address: [email protected] (C.-Q. Li). http://dx.doi.org/10.1016/j.neuropharm.2014.10.011 0028-3908/© 2014 Elsevier Ltd. All rights reserved.

2012; Silva et al., 2000). IGF2/ mice have developmental deficits, as evidenced by reduced brain volume (Constancia et al., 2002). Environmental factors appear to alter IGF2 expression in the brain, whereby they affect neuronal premise and function. Specifically, IGF2 gene expression is significantly lower in the hippocampus and amygdala after chronic restraint stress in adult rats (Andrus et al., 2012; Jung et al., 2012). Chronic unpredictable stress and repeated restraint stress are the two most important animal models of depression, and both induce hypothalamicepituitaryeadrenal (HPA) axis dysfunction as indicated by alterations in corticosterone levels and changes in the immune system and other pathophysiological aspects found in depressive patients (Valvassori et al., 2013). Some researchers believe that repeated homotypic stressors may lead to habituation but this is not the case with heterotypic stressors (Armario et al., 2008; Armario and Nadal, 2013; Babb et al., 2014). However, depending on duration and intensity, chronic restraint stress may also induce depressive-like behaviors, such as loss of body weight, anhedonia, and increased immobility in the forced swimming test (Hetzel and Rosenkranz, 2014; Yoon et al., 2014). This mimics the repeated homotypic stressors associated with increased depressive symptoms in human life that occur on a daily basis, such as medical problems, work stress, and poverty (Blackmore et al., 2007; Keller

Y.-W. Luo et al. / Neuropharmacology 89 (2015) 318e324

et al., 2007). However, the role of IGF2 and the manner of modulation of chronic stress-induced behavioral changes are poorly understood. Here, this issue was explored in a rat model of depression by repetitive chronic restraint stress (CRS). The mRNA and protein levels of IGF2 and its putative upstream and downstream signaling molecules were determined in the hippocampal formation in CRS and control groups. The effects of induced in vivo IGF2 overexpression on the behavior and relevant protein expression were explored further. 2. Materials and methods 2.1. Animals Adult male SpragueeDawley rats (2 months of age) were obtained from Central South University Animal Services (Changsha, China), and housed under standard conditions (12 h light/12 h dark cycle, 22 ± 1  C, 52 ± 2% humidity). Rats were given free access to food and water. The experimental protocol was approved by the Animal Care and Use Committee of Central South University in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. 2.2. Chronic restraint stress (CRS) procedure Rats were habituated for 7 days before experiments. Rats were divided randomly into a control group (Con) and CRS group (CRS). The control group animals were handled several times daily. The CRS group rats were repeatedly placed in a plastic restrainer (350 ml cubage water bottle, Wahaha Corporation Limited, Hangzhou, China) for 10 days. The restraint stress was induced daily from 13:00 to 17:00 under 200 lux light conditions at 22  C, with rats returned to their cages afterwards. 2.3. Measurement of body weight Rats were weighed before restraint stress on day 3 and after stress treatment on day 13. 2.4. Behavioral tests 2.4.1. Sucrose preference test (SPT) Sucrose preference testing (SPT) was performed as previously described (Li et al., 2012). In order to get rats to habituate with sucrose solution, two bottles of 1% sucrose solution were placed in each cage for 24 h. The rats were deprived of water for 12 h. The SPT procedure was carried out at 13:00 to 17:00 on day 14. Consumption of sucrose solution and water was determined for 4 h. Each animal was given free access to two bottles, one with 1% sucrose solution and one with water. For each group, the consumption of sucrose solution and water was measured by weighing the bottles. The relative amount of sucrose consumed was calculated using the following formula: Sucrose consumption rate (%) ¼ sucrose consumption/(water consumption þ sucrose consumption). 2.4.2. Forced swimming test (FST) The forced swimming test was performed as previously described (Cao et al., 2014). Rats were introduced to pre-swimming in an open cylindrical container (diameter 20 cm, height 50 cm), containing 45 cm of water at 23 ± 2  C, for 15 min on the day before experimentation. The water in the chamber was changed between each rat. On the day of the experiment, rats were forced to swim for 5 min in the container, and the water was changed. The course of swimming was recorded by a video camera. Rats were considered immobile if they floated, making only those movements necessary to keep their heads above the surface of the water. The relative amount of time spent immobile was recorded by an observer blind to the experimental conditions. Statistical analysis followed. 2.5. ELISA determination of corticosterone A Rat Corticosterone ELISA Kit (CUSABIO, WuHan, China) was used to determine the corticosterone levels, according to the manufacturer's instructions. Briefly, 24 h after the end of 10-day CRS, normal control and CRS-exposed rats were decapitated and trunk blood was collected and centrifuged for 15 min at 1000 g at 4  C. The supernatants were collected and used to measure the corticosterone levels of each sample. Then 50 ml of standard or sample was added to each well; 50 ml of HRPconjugate and 50 ml of antibody were also added to each well. All the components were mixed thoroughly and then incubated for 1 h at 37  C. Then they were incubated with horseradish peroxidase-labeled anti-rabbit antibody for half an hour at room temperature. Wells were then developed with tetramethylbenzidine reagent in dark and the absorbance was measured at 450 nm.

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qPCR was performed with a Power SYBR Green PCR Master Mix on a MiniOpticon™ Real-Time PCR System (BioRad, U.S.). All qPCR reactions were performed using the same protocol (Stage 1: 95  C, 10 s; Stage 2: 95  C 5 s; Stage 3, 65  C, 10 s; repeat the stages 2 and 3 for 40 cycles) in biological and technical triplicates. Data were analyzed using the CFX Manager Software package (BioRad) and Microsoft Excel. bactin served as an internal control. The 2DDCt method and student's t-test were used for data analysis. All primer sequences used are listed in Supplemental Table 1. 2.7. Immunohistochemistry IGF2 immunohistochemistry was performed as described previously (Li et al., 2014). Rats (n ¼ 6) were anesthetized with an overdose of chloral hydrate (80 mg/ kg). The brains were fixed overnight with 4% paraformaldehyde after perfusion and then cryoprotected in 10e30% gradient sucrose in phosphate buffer (pH 7.4). Coronal sections (30 mm) of the brain were cut in a cryostat at 20  C. One set that containing eight slices of hippocampus were randomly obtained from six sets of serial slices from each rat at 2.3 mm to 3.8 mm anteroposterior to the bregma for immunostaining. Antibodies and their working concentrations are described in Supplemental Table 2. The slices were captured using a digital camera that was attached to the microscope (Nikon, H600L Light Microscope) and analyzed using Motic Images Advanced 3.2 software (Motic, XiaMen, China) (Li et al., 2008). The average optical density of the IGF2 immunoreactivity (IR) was detected in regions of CA1, CA3, and DG at the same magnification factor and light intensity. Analysis was conducted by a researcher blind to treatment conditions. Data were acquired from eight sections/brain region/animal and the data were averaged to produce a single value per subject. 2.8. Western blot The hippocampus was removed and snap-frozen in nitrogen liquid. Frozen samples were homogenized (PRO Scientific Inc., U.S.) in a cocktail lysis buffer containing protease inhibitors and phosphatase inhibitors (Roche Applied Science, Mannheim, Germany). Samples were then centrifuged at 12,000 g for 20 min at 4  C and the supernatants were used for Western blot analysis. A total of 60 mg protein from each sample was loaded in and separated by 10% Bis-Tris SDS-PAGE gel. The blotted proteins were transferred to a 0.22-mm nitrocellulose membrane. Membranes were blocked in 5% nonfat milk for 3 h at room temperature and incubated overnight with the following primary antibodies listed in Supplemental Table 2. Membranes were washed and further incubated with HRP-conjugated secondary antibodies (1:2000) for 2 h at room temperature, followed by exposure to photographic films for 30 s to 10 min. Films were scanned. Western blot bands were quantified by the mean gray value using NIH Image J 7.0. Reduced glyceraldehydephosphate dehydrogenase (GAPDH) was used as internal loading control. 2.9. Lentivirus injection and observation To investigate the function of IGF2 in depressive-like behaviors, IGF2 was overexpressed, mediated by lentivirus in the hippocampal formation. Lv-IGF2 and Lv-GFP lentiviral suspensions were purchased from GeneChem Corporation (Shanghai, China) and stored at 80  C until use. The titer of the lentiviral recombinant vectors was 2  109 titer units (TU)/ml. For this experiment, rats were divided into seven groups, including a normal control group (Con, n ¼ 5), sham-operation group (Con þ Sham, n ¼ 5), control group with normal saline injection (Con þ NS, n ¼ 6), control group with Lv-GFP injection (Con þ GFP, n ¼ 6), control group with Lv-IGF2 injection (Con þ IGF2, n ¼ 6), CRS group with Lv-GFP injection (CRS þ GFP, n ¼ 7), and CRS group with Lv-IGF2 injection (CRS þ IGF2, n ¼ 6). After the last CRS exposure, lentiviral suspension was microinjected into the hippocampus as described previously (Li et al., 2014). A total volume of 1.5 ml lentiviral suspension (diluted 10 times with enhance infected solution) was injected bilaterally into hippocampi using the following coordinates: anteroposterior 3.50 mm relative to bregma; lateral ± 1.50 mm; dorsoventral 3.5 mm from the skull. The injection was carried out over 5 min, allowing slow diffusion. Animals were allowed to survive for 3 weeks, after behavioral tests their brains were harvested for anatomic or biochemical analysis. 2.10. Statistical analysis Statistical analyses were performed using Graphpad Prism 5 (Graphpad Software, San Diego, CA, U.S.) or SPSS13.0 (IBM), and data are presented as mean ± SD. Unpaired two-tailed Student's t test or one ANOVA or two ANOVA were used for mean comparisons followed by Bonferroni post tests multiple comparison test if applicable. Differences were considered to be significant when the P value

Insulin-like growth factor 2 mitigates depressive behavior in a rat model of chronic stress.

Depression is a common psychiatric disorder associated with chronic stress. Insulin-like growth factor 2 (IGF2) is a growth factor that serves importa...
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