Neuropharmacology and AnalgesiaChronic morphine administration induces over-expression of aldolase C with reduction of CREB phosphorylation in the mouse hippocampus
Introduction
In recent studies, alterations of the activity and expression of enzymes in cell metabolism, such as glycolysis, were detected in morphine-dependent patients and animals, indicating that a disturbance of glycometabolism may be involved in the molecular events of morphine dependence (Sharma et al., 2003, Xiang et al., 2006, Serres et al., 2005, Chen et al., 2007). Aldolase catalyzes a step of the glycolytic pathway, the aldolhydrolysis of fructose-1, 6-biphosphate into dihydroxyacetone phosphate and glycerol-3-phosphate, and the two substrates play key roles in producing ATP through the tricarboxylic acid cycle and other energy metabolism pathways. Vertebrate aldolases exist as three isozymes with different tissue distribution and kinetics: A, B, and C. Aldolase C is the brain-specific isoform of fructose-1, 6-bisphosphate aldolase (Lebherz and Rutter, 1969). Recently, several proteomics studies have reported altered expression levels of aldolase C in different brain regions of chronic morphine-administered animals (Li et al., 2006, Kim et al., 2005).
Increasing evidence demonstrates that the hippocampus is an important region associated with morphine dependence (Nestler, 2002, Morón et al., 2007). The rodent hippocampus is functionally involved in morphine-induced conditioned place preference (Rezayof et al., 2003, Zarrindast et al., 2006) and withdrawal behavior (Done et al., 1992, Lu et al., 2000, Dong et al., 2008). One recent study found downregulation of some energy metabolism enzymes, lower ATP levels, and an impaired ability to convert glucose into ATP in the mouse hippocampus following chronic morphine treatment, which indicates that an abnormality in hippocampal energy metabolism may contribute to morphine dependence (Chen et al., 2007). However, whether morphine treatment induces alterations in the expression of aldolase C in the hippocampus remains unclear.
Modulation of transcription factors such as the cAMP response element binding protein (CREB) is an important mechanism underlying the development of morphine dependence (Williams et al., 2001, Deisseroth et al., 1996). CREB is regulated through phosphorylation of Ser133, which modulates the transcription of genes containing cAMP response elements (CRE) in their promoters (Lonze and Ginty, 2002). Evidence suggests that chronic morphine administration induces changes in the expression and function of CREB in several brain regions such as the nucleus accumbens and locus coeruleus, which may contribute to withdrawal behaviors and neural adaptations associated with morphine dependence (Guitart et al., 1992, Lane-Ladd et al., 1997, Shaw-Lutchman et al., 2002). However, few studies have reported regulation of CREB phosphorylation in the hippocampus associated with morphine dependence.
In this study, we used Western blot and immunofluorescence to evaluate the effect of morphine on the regulation of aldolase C in the mouse hippocampus. In addition, we examined the expression of phosphorylated CREB (p-CREB) by Western blot and immunohistochemistry, hypothesizing that chronic morphine administration would cause alterations in CREB phosphorylation. Finally, we examined the co-expression of aldolase C and p-CREB in hippocampal neurons using laser-scanning confocal microscopy in order to study changes in aldolase C expression associated with changes in the regulation of morphine-mediated CREB phosphorylation.
Section snippets
Animals and treatment
Thirty-two male ICR mice (20–25 g; Experimental Animal Center of Peking University Health Science Center, Beijing, China) were maintained in a colony room at 24 ± 2 °C on a 12 h light/dark cycle with food and water available freely. One week after arrival, mice were randomly divided into the following 4 groups, containing 8 mice per group: a control group receiving saline (Sal), a morphine-treated group (Mor), a naloxone-precipitated withdrawal group (Mor + Nal), and a naloxone-pretreated morphine
The assessment of morphine withdrawal
We found that naloxone precipitation induced robust withdrawal jumping in morphine-dependent mice (Fig. 1A). The effect was not observed with naloxone pretreatment before morphine administration in mice (P < 0.05). To observe the effect of chronic morphine treatment on weight loss, the weight of each animal was measured before injection with morphine or saline each day. We found that mice treated with morphine showed significantly less weight gain than the control group from day 2 to day 5 (all P <
Discussion
Recently, several studies have measured the concentrations of glycolytic intermediates in rodent brains following chronic exposure to morphine using nuclear magnetic resonance spectroscopy. They found that the rate of glycolysis was increased and related metabolic enzymes were coordinately activated, which indicates that an abnormality of glycolysis may be involved in the molecular mechanism of morphine dependence (Sharma et al., 2003, Xiang et al., 2006, Serres et al., 2005). The hippocampus
Acknowledgements
We thank Prof. Qi-Hua He (The Medicine and Health Analysis Center of Peking University) for the gifts of FITC-conjugated and TRITC-conjugated antibodies and her help with laser-confocal microscopy analysis. We also thank Prof. Xiao-Yan Qiu and Prof. Jing-Rong Zhang (The Human Disease Genomics Research Center of Peking University) for their technical assistance. We are grateful to Dr. Quan Li and Dr. Xin Zhao for their helpful comments. This work was supported by a grant from the National
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