| 1 ObjectiveDrug addiction has important psychological and social causes and consequences. It has been recognized to be a neurological disease involving the development of complex behaviors such as drug tolerance, dependence and craving for the drugs that are characteristic of an addictive state. The molecular, cellular and physiological mechanisms that mediate the transition from occasional controlled drug use to the loss of control that, in part, defines addiction are not known. However, it is widely thought that changes in gene expression in the central nervous system (indexed by levels of mRNA) play a critical role. A primary mechanism by which drugs of abuse affect gene expression is through changing the concentration of transcription factors in the nuclei of cells. Several evidences suggest that changes in the expression and function of gene transcription factors in the nucleus accumbens, such as the cAMP response element binding protein (CREB), plays an important role in the mechanism of drug addiction. On the other hand, Learning and memory and drug addiction are modulated by the same neurotrophic factors, share certain intracellular signaling cascades, and depend on activation of the transcription factor CREB.CREB, a ubiquitously expressed transcription product, which is regulated by several intracellular signaling pathways. CREB dimers bind to specific sequences of DNA (called CREs or cAMP-response elements) in the regulatory regions of target genes. The transcriptional activity of these dimers is stimulated upon phosphorylation of CREB at Ser133 by any of several protein kinases. CREB represents a site of convergence at which diverse signaling pathways, and the external stimuli thatactivate those pathways, produce plasticity at the level of altered gene expression.However, little is known about the role of CREB during different phases of opioid addiction. It may be an important molecular basis of craving and relapse. Whether the action of morphine on CREB in hippocampus, prefrontal cortex (PFC), and the nucleus accumbens (NAc) in the process of addiction being associated is unknown.Place conditioning is commonly used to measure the rewarding or incentive properties of drugs, especially psychological dependence. At the same time, CPP model can be used as an effective tool to investigate the mechanisms underlying drug-induced reinstatement of drug seeking after extinction. In the present study, we plan to investigate the changes of CREB in hippocampus, PFC and NAc using the morphine induced conditioned place preference (CPP) paradigm in rats, and in order to elucidate the effect of morphine induced CREB during the progress of drug addiction, and use CREB as the entry point to testify the function of each brain area, to elucidate the mechanism of morphine addiction memory.2 Materials and methods Animals:Male SD rats weighing 180-220g were supplied by the Zhejiang Medical Science Research Institute. All animals were allowed to habituate to the colony room for 2 weeks upon arrival, and housed in plastic cages with food and water and maintained on a 12h light-dark cycle (light on at 7:00pm). Room temperature was maintained at22±2℃.Chemical reagents:Morphine hydrochloric (Shenyang Pharmaceutical LTD, China); Anti-CREB monoclonal antibody (Sigma); Anti-rabbit-IgG (Rockland); Nuclear and Cytoplasmic Extraction Reagents (PIERCE) Apparatus:The CPP apparatus were made with a modification with references to Schildein and others. Briefly, it consists four identical PVC shuttle boxes (60×30×30cm), each divided into two chambers (30×30×30cm) of equal size by a separator, one white with smooth floor and other black with wire mesh floor. A video camera was placed over the boxes and linked with a computer system. The rats' behavior was recorded by the video camera and analyzed by RatTrack software. The whole experiment was conducted under dim illumination (15-20Lux) and stable noise (30-40dB).Procedures: including acquisition of CPP (consisted of three phases: pre-exposure, conditioning phase, and CPP test.)... |
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