Chronic corticosterone injections induce a decrease of ATP levels and sustained activation of AMP-activated protein kinase in hippocampal tissues of male mice

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

Chronic corticosterone injections induce a decrease of ATP levels and sustained activation of AMP-activated protein kinase in hippocampal tissues of male mice Yunan Zhao, Jia Shen, Hui Su, Bonan Li, Dongming Xing, Lijun Du⁎ Laboratory of Pharmaceutical Sciences, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China

A R T I C LE I N FO

AB S T R A C T

Article history:

Chronic corticosterone injections induce hippocampus tissue damage and depression-like

Accepted 9 November 2007

behavior in rodent animals, the cause of which is not known. Nevertheless, increasing

Available online 26 November 2007

evidence shows that adenylate kinase (AK) and AMP-activated protein kinase (AMPK) play a very important role in intracellular energy metabolism and are especially critical for

Keywords:

neurons which are known to have very small energy reserves and narrow margin of safety

Corticosterone

between the energy that can be generated and the energy required for maximum activity.

Depression

Abnormalities of AK or AMPK system have detrimental effects on neurons or brain function

Adenylate kinase

especially at times of increased activity. In this study, we investigated the effects of chronic

AMP-activated protein kinase

corticosterone exposure on energy metabolism, as well as AK and AMPK in hippocampal

Adenine nucleotide

tissues in male C57BL/6N mice. Our results show that chronic corticosterone injection

Mouse

induced depression-like behavior in male mice, significantly decreased the energy levels and caused a sustained increase of AMP:ATP ratio in hippocampal tissues. Interestingly, chronic corticosterone injections did not produce obvious effects on AK1 protein and mRNA levels, but caused a sustained activation of AMPK. The results indicate that sustained AMPK activation might be a mechanism by which chronic corticosterone treatment causes depression-like behavior in male mice. © 2007 Elsevier B.V. All rights reserved.

1.

Introduction

Corticosterone is a principal glucocorticoid synthesized in the rodent adrenal cortex and secreted in response to stress. There is increasing evidence that long-term exposure to high corticosterone levels produces detrimental effects on hippocampal neurons (Sapolsky et al., 1985; Woolley et al., 1990; Jacobson and Sapolsky, 1991; Mizoguchi et al., 1992; López et al., 1998; Duman et al., 1999; Karten et al., 1999), impairs hippocampal long-term potentiation (LTP) (Kim et al., 1996; Xu et al., 1997; Martin et al., 2000) and decreases spatial learning abilities of C57BL/6J mice (Grootendorst et al., 2002).

Recently, abnormalities of energy metabolism have been suggested to explain the corticosterone-induced hippocampus damages. For example, glucocorticoids could accelerate ATP loss, following metabolic insults in cultured hippocampal neurons (Lawrence and Sapolsky, 1994). In vivo, chronic corticosterone treatment induced a decrease of mitochondrial volume fraction in hippocampal area CA3 of rats (Anderson, 2004) and resulted in drastic impairment of ATP synthesis rates in the brain mitochondria of rats, as reflected by lowering of ADP phosphorylation rates (Katyare et al., 2003). As we know, central nervous system (CNS) cells need large amounts of energy. The great majority of energy used by CNS cells is for

⁎ Corresponding author. Fax: +86 10 62773630. E-mail address: [email protected] (L. Du). 0006-8993/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2007.11.027

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processes that subserve physiological functioning, some of which are even sensitive to small reductions in ATP (Hibbard et al., 1987). Consequently, abnormalities of energy metabolism may produce detrimental effects on neuro-physiological functions. It is well known that energy metabolism involves mainly energy synthesis and energy metabolism. The latter mainly consists of adenylate kinase and an AMP-activated protein kinase system. Adenylate kinase (AK, EC 2.7.4.3) is an evolutionary conserved family of enzymes that catalyzes the reversible reaction of ATP + AMP = 2 ADP (Russell et al., 1974). The reaction catalyzed by AK prevents the marked increase in the ATP/ADP ratio that would otherwise occur at the site of ∼ P generation, and the marked decrease in the ratio at the site of the ATPase. AMP-activated protein kinase (AMPK) is a member of a larger metabolite-sensing protein-kinase family. It is a αβγ heterotrimer. AMPK activity depends on phosphorylation by an upstream kinase on Thr172 in the activation loop of the α subunit, and both phosphorylation and dephosphorylation are sensitive to AMP (Hawley et al., 1996). The available evidence indicates that AMPK plays a critical role in the regulation of cellular processes which are controlled by alterations in the energy state of cells and tissues. Activation of AMPK leads to phosphorylation of serine and threonine residues in key enzymes controlling cholesterol and fatty acid synthesis and results in their inhibition and ATP conservation. On the other hand, it favors fatty acid oxidation and stimulates glycolysis by increasing glucose transport and activating 6-phosphofructo-2 kinase, thereby favoring ATP production (Carling, 2004). Nowadays, increasing evidence shows that energy regulation also plays a very important role in the organization of energy metabolism and maintenance of energy homeostasis within neurons, due to the fact that the brain has very small energy reserves, and the margin of safety between the energy that can be generated and the energy required for maximum activity is also small (Ames, 2000). Based on above rationale, here we test the hypothesis that reduced energy levels and energy deregulation might be responsible for corticosteroneinduced behavioral abnormalities.

2.

149

cantly increased immobile time of mice at week 3 (P < 0.05) and week 5 (P < 0.05), but not at week 1 (Fig. 1a). One-week corticosterone treatment even tended to decrease immobile time of mice. In the tail suspension test, two-way ANOVA also showed significant main effects of treatment (F1,84 = 5.454; P < 0.05) and time (F2,84 = 3.901; P < 0.05) with a significant interaction between these factors (F2,84 = 4.929; P < 0.05). Multiple comparison tests further revealed that the immobile time of mice treated with corticosterone decreased significantly at week 1 (P < 0.05), but increased significantly at week 3 ( P < 0.05) and week 5 (P < 0.05) (Fig. 1b).

2.2. Chronic corticosterone injections induce a decrease of ATP levels and an increase of AMP levels in hippocampal tissues In order to determine whether chronic corticosterone injections produce an effect on energy metabolism, we first

Results

2.1. Chronic corticosterone injections induce depression-like behavior After chronic corticosterone treatments, depression-like behavior of mice were assessed by using forced swimming tests and tail suspension tests at weeks 1, 3 and 5, respectively. We found that 3-week and 5-week corticosterone injections significantly increased depression-like behavior of mice in both tests. However, the 1-week corticosterone injection was not found to increase but to decrease depression-like behavior. The statistical details of these observations are given below: In the forced swimming test, there was a significant effect of time (F2,84 = 5.473; P < 0.01) and treatment on immobile time (F1,84 = 3.921; P < 0.05), as well as a significant interaction between treatment and time (F2,84 = 8.638; P < 0.001). Multiple comparison tests further showed that corticosterone signifi-

Fig. 1 – The results of the forced swim test and tail suspension test for the mice in each group (n = 15): the mean (±S.D.) of time spent immobile during the last 4 min of the tail suspension test is shown in panel (a), the mean (±S.D.) of time spent immobile during the last 4 min of the swimming test is shown in panel (b). Data were analyzed by using two-way analysis of variance (ANOVA) with treatment and time as factors. *P < 0.05, for the corticosterone vs. control mice, Tukey's Honestly Significant Difference test.

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Table 1 – Effects of chronic corticosterone injections on ATP, ADP and AMP levels in hippocampal tissues of mice ATP (nmol/mg hippocampus tissue)

Week 1 Week 3 Week 5

ADP (nmol/mg hippocampus tissue)

Control

Corticosterone

Control

1.52 ± 0.11 1.62 ± 0.12 1.57 ± 0.12

1.35 ± 0.08⁎⁎ 1.43 ± 0.08⁎ 1.48 ± 0.15

0.38 ± 0.05 0.36 ± 0.04 0.39 ± 0.06

Corticosterone 0.44 ± 0.06⁎ 0.41 ± 0.05 0.31 ± 0.03⁎

AMP (nmol/mg hippocampus tissue) Control

Corticosterone

0.041 ± 0.011 0.036 ± 0.024 0.045 ± 0.013

0.083 ± 0.025⁎⁎ 0.091 ± 0.033⁎⁎ 0.075 ± 0.024⁎⁎

Data were expressed as mean ± S.D. (n = 5). ATP, ADP and AMP values were determined by using ion-pairing HPLC–UV analysis. Statistical significance was determined by two-way analysis of variance (ANOVA) with treatment and time as factors. ⁎P < 0.05, ⁎⁎P < 0.01, for the corticosterone vs. control mice, Tukey's Honestly Significant Difference test.

measured the levels of adenine nucleotides in hippocampal tissues of mice through using ion-pairing HPLC–UV analysis technique at weeks 1, 3 and 5, respectively. We found that corticosterone injections could induce a decrease of ATP levels and an increase of AMP levels throughout the treatment. In terms of ADP levels, however, corticosterone firstly caused an increase at week 1, and lastly caused a decrease at week 5. The statistical details of these observations are given below: Two-way ANOVA showed a significant effect of time (F2,24 = 16.291; P < 0.001) and treatment (F1,24 = 28.94; P < 0.001) on ATP levels in hippocampal tissues, as well as a significant interaction between time and treatment (F2,24 = 16.291; P < 0.001). Multiple comparison tests further showed that significant differences between control and corticosteronetreated groups were observed at week 1 (P < 0.01) and week 3 (P < 0.05) with a minor decrease of 11.2% and 11.7%, respectively (Table 1). On ADP levels, two-way ANOVA revealed that the interaction between time and treatment was of statistical significance (F2,24 = 3.988; P < 0.05), as was the main effect of time (F2,24 = 48.4; P < 0.001). The main effect of treatment, however, was not statistically significant (F1,24 = 0.127; P = 0.730). Multiple comparison tests further indicated that the 1-week corticosterone injection caused a marked increase of ADP levels (P < 0.05), whereas the 5-week corticosterone injection induced a significant decrease (P < 0.05) (Table 1). On AMP levels, two-way ANOVA showed a significant effect of time (F2,24 = 4.443; P < 0.01) and treatment (F1,24 = 20.088; P < 0.001). There was, however, no interaction between time and treatment (F2,24 = 0.118; P = 0.890). Multiple comparison tests further showed that significant differences between control and corticosterone-treated groups were observed at any time, with a strong increase of 102% (P < 0.01), 152% (P < 0.01) and 67% (P < 0.01) at weeks 1, 3 and 5, respectively (Table 1).

2.3. Chronic corticosterone injections have no effect on AK1 protein and mRNA levels In order to determine whether chronic corticosterone injections produce an impact on energy regulation, we first analyzed AK1 by using Western blot and RT-PCR techniques. We found that chronic corticosterone injections did not produce obvious effects on AK1 protein and mRNA levels in hippocampus tissues. The statistical details of these observations are given below: Two-way ANOVA showed a significant main effect of time (F2,24 = 13.875; P < 0.001) on AK1 expression in hippo-

campus tissues. However, there was no effect of treatment (F1,24 = 0.408; P < 0.529) and no interaction between time and treatment (F2,24 = 1.676; P = 0.208). Multiple comparison tests further showed that there was only a tendency to decrease for AK1 mRNA levels after 5-week corticosterone treatment (P = 0.143) (Fig. 2).

Fig. 2 – The mean (±S.D.) of AK1 expression in the hippocampal tissues of mice in each group (n = 5): AK1 transcription was studied by using RT-PCR (a). Symbol ‘C’ means the control group. Symbol ‘D’ means chronic corticosterone-treated group. PCR products of β-actin and AK1 were separated on 1.5% agarose gels, visualized by ethidium bromide staining and analyzed using a FR-200A Electrophoresis Image Analysis System. The values of AK1 (b) PCR products were normalized against the amount of PCR product for β-actin obtained from the same RT sample. Data were analyzed by using two-way analysis of variance (ANOVA) with treatment and time as factors. There were no statistically significant differences between the corticosterone and control mice, Tukey's Honestly Significant Difference test, P > 0.05.

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Table 2 – Effects of chronic corticosterone injections on AMP:ATP ratio in hippocampal tissues of mice AMP/ATP

Week 1 Week 3 Week 5

Control

Corticosterone

0.027 ± 0.009 0.022 ± 0.005 0.028 ± 0.008

0.061 ± 0.017⁎⁎ 0.064 ± 0.021⁎⁎ 0.051 ± 0.016⁎⁎

Data were expressed as mean ± S.D. (n = 5). Statistical significance was determined by two-way analysis of variance (ANOVA) with treatment and time as factors. **P < 0.01, for the corticosterone vs. control mice, Tukey's Honestly Significant Difference test.

Two-way ANOVA also showed that there was no effect of time (F 2,24 = 0.307; P = 0.738) and treatment (F 1,24 = 0.194; P = 0.664) on AK1 levels in hippocampus tissues, or of the interaction between time and treatment (F2,24 = 0.811; P = 0.456) (Fig. 3b).

2.4. Chronic corticosterone injections induce a sustained increase of AMP:ATP ratio and phospho-AMPK-α levels in hippocampal tissues

Fig. 3 – The mean (± S.D.) of AK1 and phospho-AMPK-α levels in the hippocampal tissues of mice in each group (n = 5): AK1 and phospho-AMPK-α levels were determined by Western blot analysis using anti-AK1, anti-phospho-AMPK-α, anti-AMPK-α and anti-β-actin antibodies (a). Symbol ‘C’ means the control group. Symbol ‘D’ means chronic corticosterone-treated group. Protein samples were analyzed by 12% SDS-polyacrylamide gel electrophoresis. Then, the gel was transferred to Immuno-Blot PVDF membranes (0.22 μm) at a different voltage, respectively. The protein bands were quantified using a FR-200A Electrophoresis Image Analysis System. The values of phospho-AMPK-α levels (b) and AK1 (c) were normalized against the amount of AMPK-α and β-actin obtained for the same sample, respectively. Data were analyzed by using two-way analysis of variance (ANOVA) with treatment and time as factors. **P < 0.01, for the corticosterone vs. control mice, Tukey's Honestly Significant Difference test.

AMP:ATP ratio is believed to serve as a bridge between energy synthesis and energy regulation. A rise in AMP:ATP ratio can activate AMPK by phosphorylation of AMPK. In our study, AMP:ATP ratio was found to increase throughout the treatment, which made us speculate that chronic corticosterone injections might induce a sustained activation of AMPK. After we found a sustained increase of phospho-AMPK-α levels in hippocampal tissues of mice throughout the treatment, this speculation was confirmed. The statistical details of these observations are given below: Two-way ANOVA revealed a significant effect of treatment (F1,24 = 43.214; P < 0.001) and time (F2,24 = 4.23; P < 0.05) on AMP: ATP ratio with no significant interaction between two factors (F2,24 = 1.124; P = 0. 275). Multiple comparison tests further showed that significant differences between control and corticosterone-treated groups were observed at any time, with an increase of 126% (P < 0.01), 191% (P < 0.01) and 82 (P < 0.01) at weeks 1, 3 and 5, respectively (Table 2). On phospho-AMPK-α levels, two-way ANOVA revealed a significant effect of treatment (F1,24 = 63.578; P < 0.001) and time (F2,24 = 4.32; P < 0.05) with no significant interaction between two factors (F2,24 = 1.565; P = 0.230). Multiple comparison tests further showed that significant differences between control and corticosterone-treated groups were observed at any time, with an increase of 46.7% (P < 0.01), 72.2% (P < 0.01) and 78.4% (P < 0.01) at weeks 1, 3 and 5, respectively (Fig. 3c).

3.

Discussion

It is well established that high levels of corticosterone plays a very important role in the development of depression. In clinic, chronic cortisol injection can often induce behavioral and mental abnormalities. In rodent animal models, repeated corticosterone injections are also found to induce depression-

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like behavior (Kalynchuk et al., 2004). In Stone and Quartermain's study (1999), 6-week corticosterone injections at a varying dosage ranging from 10 to 50 mg/kg, all significantly induced depressive behavior in male mice in the nest-leaving test. Results form Dr. Kalynchuk's lab (Gregus et al., 2005; Johnson et al., 2006) showed that 3-week corticosterone injections increased depression-like behavior in male rats in a dose-dependent manner (10, 20 and 40 mg/kg) in the forcedswim test. In our study, we also found that both 3-week and 5-week corticosterone injections (20 mg/kg) increased depression-like behavior in male mice both in the forced-swim test and tail-suspension test, which further supports that longterm exposure to high corticosterone levels induces depression-like behavior in rodent animals. Recently, several mechanisms have been suggested to explain the corticosterone-induced behavioral abnormalities such as malfunction of glucocorticoid receptor (GR) (Belanoff et al., 2001), activation of mitogen-activated protein kinase (MAPK) signaling pathway (Yang et al., 2004; Revest et al., 2005), decreased brain-derived neurotrophic factor (BDNF) (Jacobsen and Mork, 2006; Schule et al., 2006) and excitotoxicity involving the activation of the N-methyl-D-aspartate NMDA subtype of glutamate receptor (Venero and Borrell, 1999; Danilczuk et al., 2005). Additionally, abnormalities of energy synthesis were also reported (Katyare et al., 2003; Coburn-Litvak et al., 2004). In our study, after daily corticosterone injections, we also observed the similar phenomenon through measuring the levels of ATP in hippocampus tissues, and found that ATP levels in hippocampal tissue decreased by 11.2% and 11.7% at weeks 1 and 3, respectively. As we know, large amounts of energy are required to maintain the activities of CNS cells. In Ames's review (2000) on CNS energy metabolism, the CNS energy is reported to be used for basic vegetative processes and for processes that underlie specialized physiological functions. The very speculative estimates of the relative demands on the energy were listed as follows: vegetative metabolism (5–15%), gated Na+ influx through plasma membranes (40–50%), Ca2+ influx from organelles (3–7%), processing of neurotransmitters (10–20%), intracellular signaling systems (20–30%), axonal and dendritic transport and other (20–30%). Based on above speculative estimates, once there appears to be a reduced energy level in CNS, neurotransmitter system and action potentials may be the first to be severely affected, which suggests that reduced energy levels in hippocampal tissues, caused by chronic corticosterone injections, might be responsible for depressionlike behavior. However, we also found that there was an obvious decrease of ATP levels in hippocampus tissues without depression-like behavior after 1-week corticosterone injections, which suggested that there was no direct correlation between depression-like behavior and ATP levels. The reason may be that it takes time for abnormalities of action potentials and neurotransmitter system to appear in animal behavior. Another reason may be due to the decreased ATP levels. Unlike the rapid and strong decrease (Marton et al., 1997; Ohgoh et al., 2000), slight decrease of ATP levels needs to take a longer time to produce detrimental action (Yun et al., 2000). The involved mechanisms may be associated with the durative increase of AMP:ATP ratio. It is well known that increase of AMP:ATP ratio

can activate AMP-activated protein kinase (AMPK). Therefore, it is anticipated that the durative increase of AMP:ATP ratio causes sustained activation of AMPK, as reflected by a durative and average 70% increase of phospho-AMPK-α levels in our studies. AMPK is highly expressed in brain (Turnley et al., 1999; Culmsee et al., 2001) and activated following ATP depletion, a rise of AMP levels or, more accurately, a rise in the AMP:ATP ratio within the cell. Once activated, AMPK increases cellular ATP supply through stimulating glycolysis. On the other hand, it suppresses the key enzymes involved in ATP-consuming anabolic pathways to maintain ATP level homeostasis (Hardie and Carling, 1997; Hardie et al., 2003), such as inhibiting ATPgated Cl− channel (Hallows et al., 2000) and phosphorylating GABAB receptor to cause increased GABA-dependent inhibition of presynaptic Ca2+ channels (Hardie and Frenguelli, 2007). In Dagon et al.'s (2005) study, they found that modest activation induced neurogenesis and improved cognition of animals, but that augmented AMPK activation reduced cognition and increased neural apoptosis and mortality. Results from Gorospe's lab showed that AMPK activation decreased cytoplasmic HuR (a shuttling RNA-binding protein), and consequently decreased the binding of HuR to target transcripts and diminished the expression and half-lives of such HuR target mRNA (Wang et al., 2002, 2003). Additionally, investigators found that sustained AMPK activation by either AMPK activator or active AMPK mutant, induced apoptosis ( Jung et al., 2004; Kefas et al., 2004; Dagon et al., 2006). For example, Meisse et al. (2002) found that sustained activation of AMPK induced apoptosis in liver cells through activating c-Jun N-terminal kinase. These studies demonstrated that overactivation or sustained activation of AMPK was detrimental to the neurons. Thus, sustained AMPK activation found in our study could be applied to explain why mice demonstrated depression-like behavior until 3-week corticosterone injections, although ATP levels decreased from the outset. If so, sustained AMPK activation might be another mechanism implied in chronic corticosterone-induced depression. In this paper, we also investigated whether adenylate kinase (AK) was involved in coticosterone-induced behavioral abnormalities. In vertebrates, three isozymes (AK1, AK2 and AK3) were characterized. AKl is present in the cytosol of skeletal muscle, brain and erythrocyte. AK2, which is undetectable in brain (Noma et al., 1998), is found in the intermembrane space of mitochondria of liver, kidney, spleen and heart. AK3, correctly called GTP:AMP phosphotransferase exists in the mitochondrial matrix of liver and heart (Matsuura et al., 1989; Inouye et al., 1999). It is now clear that the AK system plays an important role in buffering the ATP/ADP ratio and in energy transfer (Zeleznikar et al., 1990). AK catalyzes the reversible reaction ATP + AMP = 2 ADP. The principal effects of AK depend on the enzyme being present both at the site of energy generation and at the site of energy consumption, with diffusion of AMP from the site of consumption to the site of generation. Consequently, it prevents the marked increase in the ATP:ADP ratio at the site of energy generation and the marked decrease in the ratio at the site of the ATPase, which doubles the efficiency of the diffusion of ATP in the transfer of energy and maintains efficient intracellular energy flow (Clegg, 1984;

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Dzeja and Terzic, 2003). Thus, reduced activities of AK give rise to relatively decreased energy levels at the site of energy consumption, which may produce detrimental effects on cell or tissue functions especially at times of increased activity (Bianchi et al., 1999; Janssen et al., 2000). In our study, however, we found that chronic corticosterone injections did not produce obvious effects on AK1 protein and mRNA levels in hippocampus tissues, indicating that depression-like behavior induced by corticosterone might have no relation with AK1 within 5 weeks. Taken together, the present study emphasizes effects of chronic corticosterone injections on intracellular energy level and energy regulation in the hippocampal tissues. The results show that chronic corticosterone injections induce depression-like behavior in male mice, obviously decrease the energy levels and cause a sustained increase of AMP:ATP ratio in hippocampus tissues. In terms of energy regulation, chronic corticosterone injections do not produce obvious effects on AK1 activity, but cause a sustained activation of AMPK. Our studies suggest that sustained AMPK activation induced by reduced energy level might be one of the reasons why chronic corticosterone treatment causes depression-like behavior in male mice.

4.

Experimental procedures

4.1.

Animals and corticosterone administration

Male C57BL/6N mice (16–20 g, Vital River Laboratory Animal Technology, Beijing, China) were housed in a 12-h light/dark cycle, with lights turning off at 18:00 h, a constant temperature of 25 ± 1 °C and free access to food and tap water. Animals were treated according to the Guidelines on Accommodation and Care of Animals formulated by the Chinese Convention for the protection of vertebrate animals used for experimental and other scientific purposes. Mice were randomly assigned to six experimental groups (n = 15/group). Three groups were injected subcutaneously with corticosterone (20 mg/kg, which was suspended in physiological saline containing 0.1% DMSO and 0.1% Tween-80) in a volume of 5 ml/kg, at random times during the light phase. The other three groups were injected only with vehicle. Mice were injected daily with corticosterone or vehicle for 1, 3 or 5 weeks, after which they were tested for depression-like behaviors and then sacrificed to collect hippocampal tissues for neurochemical measurements. Five animals per group were randomly chosen for adenine nucleotides analysis, and the rest were used for Western blot (5 animals) and RT-PCR analysis (5 animals), respectively.

4.2. Forced swimming test (FST) and tail suspension test (TST) FST was similar to that described by Porsolt et al. (1977). Briefly, mice were individually placed in 10 cm of ambient temperature water (25 ± 1 °C) in 2000 mL glass beakers and were allowed to swim for 5 min, and the durations of immobility were recorded during the last 4 min of the test. Duration of immobility is defined as the absence of active, escape-oriented behaviors, such as swimming, jumping, rearing, sniffing or diving.

153

TST was similar to that described by Steru et al. (1985). After FST, mice were allowed to have a rest for 24 h, and then suspended on the edge of a shelf 58 cm above a tabletop by adhesive tape, placed approximately 1 cm from the tip of the tail. They were allowed to hang for 6 min, and the duration of immobility was recorded during the last 4 min of the test. Mice were considered immobile only when they hung passively and completely motionless.

4.3. Determination of ATP, ADP and AMP in hippocampal tissues of mice The protocol for determination of ATP, ADP and AMP was modified from Di Pierro et al. (1995). Animal heads were irradiated with a focused microwave (TMW-640, Toshiba, Tokyo, Japan) to quickly stop postmortem metabolism of purines. For mice, the microwave used was 3 kW for 1.5 s, and brains were quickly removed. Hippocampi were dissected out, and immediately homogenized (4 °C) with 0.5 ml of 0.2 M perchloric acid containing 0.05 M glutathione as antioxidant. After ultrasonic extraction for 10 min at 4 °C, the homogenate was centrifuged (4 °C) at 12,000×g for 15 min. The resultant supernatant was neutralized with a 4 M NaOH solution, and 20 μl of the supernatant was used for the ion-pairing HPLC–UV analysis on a HPLC apparatus consisting of a model 515 liquid pump, and a model 996 spectrophotometric detector (Waters Corp., Milford, MA, USA). A Supelcosil LC-18 column was used with a column oven heated to 25 °C. The mobile phase consisted of 0.1 M phosphate buffer (containing 1 mM tetrabutylammonium hydroxide solution)-acetonitrile (97.5:2.5, v/v; pH 6.0). The flow rate was 1 ml/min and the detector was set at 254 nm. The concentration of ATP, ADP and AMP in the supernatant was calculated from standard curves. Calibrations were linear from 0 to 40 μM for ATP, ADP and AMP, respectively. The standard curves for ATP, ADP and AMP were y = 35,699x, y = 30,811x and y = 33,596x, respectively [ y = peak area; x = concentration (μM)].

4.4. Reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was isolated by trizol (Bio Basic Inc., CA) extraction according to the manufacturer's instructions. Total RNA (2 μg per sample) was reverse-transcribed into first-strand cDNA using the MMLV First-Strand Synthesis System kit (Bio Basic Inc., CA) and Oligo-dT12–18. PCR reactions were carried out by using the Ready-to-Use PCR kit (Bio Basic Inc., CA). The oligonucleotide primers were designed based on GeneBank® sequence using PCR primer designing software Primer 5.0 to ensure specific and efficient amplification of target sequences. The primers synthesized by Sangon Biotechnology (Sangon, Beijing, China) and parameters for PCR amplification of AK1 and β-actin were listed in Table 3. PCR products were separated on 1.5% agarose gels, visualized by ethidium bromide (Sigma) staining, and analyzed using a FR-200A Electrophoresis Image Analysis System (Furi, Shanghai, China). Quantity of PCR product was measured by intensity of the band. The values of the AK1 PCR product were normalized against the amount of PCR product for β-actin obtained for the same RT sample.

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Table 3 – The primers and parameters for PCR amplification of AK1 and β-actin cDNA

Primer

AK1 Up: 5′-TAT GGC (mouse) TAC ACC CAC CTG TCT ACT G-3′ Down: 5′-CCG CTT CTT GAT GGT CTC CTC GT-3′ β-actin Up: 5′-GCC CAT (mouse) CTA CGA GGG CTA T-3′ Down: 5′-GCT GGA AGG TGG ACA GTG AG-3′

4.5.

Size of PCR product amplification (base pairs) 354 bp

95 °C (45 s), 58 °C (30 s), 72 °C (1 min), 25 cycles, Mg2+ (2 mM)

570 bp

95 °C (45 s), 56 °C (30 s), 72 °C (1 min), 25 cycles, Mg2+ (1.5 mM)

Western blot analysis

Animal heads irradiated with a focused microwave (3 kW for 1.5 s) were dissected and the hippocampal tissues were immediately homogenized (4 °C ) with 0.5 ml of RIPA buffer [50 mM Tris–HCl, 0.1% SDS, 1% NP-40 (Sigma), 1 mM EDTA, 150 mM NaCl, 1 mM PMSF (Sigma), 1 mM NaF, 1 mM Na3VO4, 1 μg/ml aprotinin (Sigma), 1 μg/ml leupeptin (Sigma), pH 7.5]. Aliquots of the clarified homogenized liquid containing 75 μg protein were denatured in a sample buffer [1% SDS, 1% dithiothreitol (Sigma), 10 mM Tris–HCl, 10% glycerol, 1 mM EDTA, pH 8.0] at 95 °C for 5 min. The samples were then analyzed by 12% SDS-polyacrylamide gel electrophoresis and transferred to Immuno-Blot PVDF membranes (Bio-Rad). The membrane was blocked in a buffer (PBS containing 5% non-fat dried milk and 0.2% Tween-20) for 1 h at 37 °C. Then, the membrane was in turn incubated with primary antibody, biotin-tagged secondary antibody and peroxidase-tagged streptavidin. Finally, the membrane was developed in 10 ml of freshly prepared substrate solution [0.03% H2O2, 6 mg/ml 4-chloro-1-naphthol (Sigma) and 2 mg/ml 3,3′-diaminobenzidine tetrahydro-chloride (Sigma) in PBS]. The protein bands were quantified using a FR-200A Electrophoresis Image Analysis System. The values of AK1 and phospho-AMPK-α levels were normalized against the amount of β-actin and AMPK-α obtained for the same sample, respectively. The anti-AK1 and anti-β-actin polyclonal antibodies were purchased from Bioss Biotechnology (Bioss, Beijing, China). The anti-phospho-AMPK-α (Thr172) and antiAMPK-α monoclonal antibodies were purchased from Cell Signaling Technology (Cell Signaling, Beverly, MA).

4.6.

Statistical analysis

Data were expressed as mean ± SD for the indicated number of experiments and analyzed using the statistical package for social sciences (SPSS) computer program version 10.1. Statistical significance was determined by two-way analysis of variance (ANOVA) with treatment and time as independent, between-subjects factors. In case of significant interactions, Tukey's Honestly Significant Differences test of multiple comparisons was performed. The significance level was set at P ≤ 0.05 for all statistical comparisons.

Acknowledgments The study was supported by National Natural Science Foundation of China (30572340, 90713043), the Fund for Doctoral Station of the Ministry of Education, China (20060003072) and Projects of Science Research for the 11th Five-year Plan of Ministry of Science and Technology of China (2006BAI08B0309). The authors would also like to acknowledge the invaluable help and advice from Dr. Jiming Kong and Leilanie Hall (Department of Human Anatomy and Cell Science, University of Manitoba, Winnipeg, Manitoba, Canada).

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