摘要
阿片依赖是一种慢性复发性脑病,以神经生理功能紊乱及病态行为如强制性的药物使用及持久的心理渴求为特征。目前阿片依赖发生的详细机制尚未阐明。但大量研究表明,机体长期接触阿片类物质产生的神经元适应性改变是阿片依赖发生的生物学基础。这些代偿性适应不仅发生在阿片受体作用系统本身,还涉及到许多非阿片受体作用系统,如兴奋性氨基酸受体系统、多巴胺受体系统、乙酰胆碱受体系统、咪唑啉受体系统等。
磷脂酰乙醇胺结合蛋白(phosphatidylethanolamine binding protein,PEBP)是本课题组在前期蛋白质组学研究中发现的吗啡依赖大鼠脑区差异表达蛋白。该蛋白不仅是海马胆碱能神经刺激肽(hippocampal cholinergic neurostimulating peptide,HCNP)的前体蛋白,还具有与疏水性配体(如膜磷脂、阿片受体、核苷酸等)结合,参与不同信号转导途径(Raf-1/MEK/ERK、G蛋白偶联受体)等多种功能。因此,本研究目的为观察该蛋白是否在阿片依赖过程中发挥作用并探讨其可能机制。
我们首先建立大鼠吗啡依赖模型,以确证PEBP在吗啡依赖大鼠不同脑区的差异表达。蛋白免疫印迹实验结果显示,相对于生理盐水组,该蛋白表达量在吗啡依赖大鼠的海马、伏隔核有显著性上调(分别为1.6倍、1.22倍,P < 0.01,与生理盐水组相比),在内侧前额叶皮层有显著性下调(0.65倍,P < 0.05,与生理盐水组相比);而在纹状体、小脑与脑干等脑区无统计学显著性改变。该实验结果与蛋白质组结果(海马、前额皮层、纹状体均有上调)并不完全一致,但提示PEBP蛋白表达量在吗啡依赖大鼠具有脑区差异表达。
蛋白质组与蛋白免疫印迹实验结果均显示PEBP蛋白表达在吗啡依赖大鼠海马区具有显著上调。海马是哺乳动物脑边缘系统的主要组成部分,可接受多途径的传入信息,与吗啡精神依赖与躯体依赖过程均密切相关。为了进一步研究PEBP在吗啡依赖病理过程的调节,我们建立了吗啡依赖与自然戒断大鼠模型,观察大鼠海马区PEBP蛋白表达在不同时程的变化。PEBP蛋白C端抗体(针对152-164位氨基酸)免疫印迹实验结果显示PEBP具有随时程逐渐变化的趋势:与盐水对照组大鼠相比,在吗啡依赖4 d、8 d海马PEBP表达量均有显著性上调(分别为2倍与2.7倍,P < 0.01,与生理盐水组相比);而自然戒断3 d PEBP蛋白下调至正常水平,在自然戒断7 d、14 d蛋白表达量再次显著上调且逐渐增加(分别为2倍与2.4倍,P < 0.05,与生理盐水组相比),至21 d天蛋白表达量下降,但与对照组相比仍有显著性增加(1.8倍,P < 0.05,与生理盐水组相比);该上调趋势持续至自然戒断28 d。
在上述吗啡依赖与自然戒断模型中采用戒断症状评分法观察30 min内各组大鼠的躯体戒断症状以及体重变化。结果发现,在自然戒断前9 d,自然戒断大鼠症状评分均显著高于生理盐水对照组大鼠;其中自然戒断第2、3 d分数较高,然后逐渐降低。自然戒断大鼠体重在戒断第1 d开始下降,至第2 d降至最低点,第3 d开始回升,第6 d恢复至戒断前体重水平。生理盐水对照组大鼠体重在相同时程则持续增加。该部分结果提示,大鼠体重与戒断症状有一定的相关性;PEBP表达量在自然戒断症状较重时下调至最低,随着大鼠自然戒断症状逐渐减轻而逐渐上调,呈一定负相关性。
为了进一步确定PEBP与阿片躯体依赖病理过程的关系,我们采用反义核酸干涉技术研究下调PEBP后对吗啡躯体依赖戒断症状表达的影响。首先应用Sfold、Mfold以及RNAstructure等软件设计七条针对PEBP编码序列(coding sequence,CDS)的反义寡核苷酸(antisense oligodeoxynuclecotide,ASODN);然后以PC12细胞做为筛选模型,根据RT-PCR与蛋白免疫印迹实验确定具有生物学活性的ASODN(能下调PEBP基因与蛋白表达量)。得到有生物学活性的ASODN后,通过在大鼠海马区连续微量注射PEBP-ASODN来观察PEBP蛋白表达量下调对吗啡依赖大鼠催促戒断行为的影响。行为学实验结果显示,与吗啡依赖组大鼠相比,PEBP-ASODN微量注射的慢性吗啡处理组大鼠戒断症状评分显著增加(13.67分,P < 0.05,与吗啡处理组相比),其中某些症状显著加重(湿狗样抖动,激惹、流涎等);而PEBP-正义寡核苷酸(sense oligodeoxynuclecotide,SODN)微量注射对慢性吗啡处理大鼠的戒断症状表达并无显著影响(9.67分,P>0.05,与吗啡处理组相比)。实验结果提示,PEBP确实参与了阿片依赖病理过程,海马区PEBP蛋白表达量下调可促进吗啡躯体依赖大鼠某些戒断症状显著加重。
由PEBP蛋白氨基端第2-12位氨基酸解离下的海马胆碱能神经刺激肽(HCNP)在中枢胆碱能系统的神经发育中具有重要作用,可通过促进胆碱乙酰转移酶活性增强中枢海马胆碱能系统功能。而有研究显示,海马胆碱能系统与阿片躯体依赖也密切相关。因此,我们在上述实验结果的基础上,继续在大鼠吗啡依赖与自然戒断模型中观察中枢海马胆碱能系统活性的变化,研究PEBP是否通过该系统参与吗啡躯体依赖过程。分别采用PEBP C端抗体(针对PEBP 152-164位氨基酸)与N端抗体(针对PEBP 2-12位氨基酸)进行蛋白免疫印迹实验,间接推测吗啡依赖与自然戒断不同时程大鼠海马区HCNP的表达量变化。实验结果显示,大鼠海马HCNP表达量在吗啡依赖与自然戒断不同时程具有与PEBP相同的变化趋势。采用紫外分光光度法测定大鼠海马区胆碱乙酰转移酶(choline acetyl transferase,ChAT)活性的变化。实验结果显示,与生理盐水对照组相比,吗啡处理组大鼠第4 d ChAT活性即显著增高(138 nmol/h.mg pro,生理盐水对照组为118 nmol/h.mg pro,P < 0.01);第8 d吗啡处理组与催促戒断组大鼠ChAT活性均有显著升高(分别为149 nmol/h.mg pro与147 nmol/h.mg pro,生理盐水对照组为126 nmol/h.mg pro,P < 0.05);而在自然戒断3 d自然戒断大鼠ChAT活性下降至生理盐水对照组水平(分别为97 nmol/h.mg pro与101 nmol/h.mg pro,P > 0.05);随后自然戒断组大鼠ChAT活性再次逐渐增高,在自然戒断第14d显著高于生理盐水对照组水平(150 nmol/h.mg pro与115 nmol/h.mg pro,P < 0.01));该增高趋势持续至自然戒断第21 d,ChAT活性恢复至正常水平。该实验结果提示,吗啡依赖与自然戒断不同时程大鼠海马区HCNP含量、CHAT活性与PEBP表达呈现较为一致的变化趋势,提示PEBP可能通过海马胆碱能系统参与阿片躯体依赖过程。
综上所述,本研究在国内外首次发现PEBP在吗啡依赖与自然戒断病理过程中具有脑区与时程特异性的差异表达,大鼠海马区PEBP表达量下调可促进纳络酮催促戒断症状的发生,PEBP可能通过调节海马胆碱能系统功能参与阿片躯体依赖过程。
Drug addiction is a chronic, relapsing disorder in brain in which compulsive drug-seeking and drug-taking behavior persists despite serious negative consequences. Prolonged or repeated use of addictive substances, such as opioids, induces adaptive changes in the central nervous system that lead to tolerance, physical dependence, sensitization, craving and relapsing. Although the exact mechanisms are still unknown, researches in recent years have shown that these adaptations not only happened in the opioid receptor system itsself, but also in other non-opioid systems such as excitatory amino acids receptor , dopamine receptor, acetylcholine receptor and imidazoline receptor systems.
Proteomic analysis of our previous study revealed that the expression of phosphatidylethanolamine binding protein (PEBP) changed in some brain regions of morphine dependent rats. Many researches have shown that PEBP played a variety of biological roles. It is the precursor protein of hippocampal cholinergic neurostimulating peptide (HCNP), which may play an important role in the septal cholinergic development of the hippocampus. Also PEBP is associated with an increasing number of diseases through its involvement in several cell signaling cascades (Raf-1/MEK/ERK, GPCRs). Thus the aim of this study is to find out whether PEBP is involved in the pathogeny of opioid dependence and to investigate the possible mechanisms.
The first step in this study was to identify the result of proteomics. Results of Western blot showed that among six brain regions studied, PEBP was significantly up-regulated by chronic morphine in hippocampus and nucleus accumbens (60%, 22% increase vs. saline group respectively), while down-regulated in medial prefrontal cortex (35% decrease vs. saline group). On the other hand, there were no significant changes of PEBP expression in striatum, brain stem and cerebellum. Although these results were not completely consistent with proteomic analysis, we confirmed that PEBP was regulated by chronic morphine in special brain regions.
According to the results of proteomics and Western blot, the expression of PEBP was up-regulated significantly by chronic morphine in hippocampus. To learn more about the regulation of PEBP in opioid dependence, the changes of PEBP expression in hippocampus during different time courses were observed by Western blot in morphine dependent and natural withdrawal rat models. Results showed that PEBP was up-regulated 4 d and 8 d after morphine injection compared with untreated rats. While 3 d after the final morphine injection (3 d of natural withdrawal), the expression of PEBP decreased to the control level and then up-regulated again 7 d after natural withdrawal. This up-regulation of PEBP remained until 28 d after natural withdrawal. However, the expression of PEBP showed no change during the same time courses of saline-treated rats. Meanwhile, we observed withdrawal symptoms with the same animal models to investigate the possible behavioral significance of this regulation. Compared with saline-treated control group, morphine dependent rats displayed significant withdrawal symptoms in the early 9 days of natural withdrawal period. The highest score was obtained on the second day. Also the weight of the natural withdrawal rats decreased from the first day and reached the lowest level on the second day. These results suggested PEBP might have negative correlation with the expression of morphine withdrawal syndroma.
Since above results indicated the expression of hippocampal PEBP altered during morphine dependence and withdrawal, antisense oligonucleotide strategy was used to study whether down-regulation of PEBP in hippocampus influence the development and/or expression of physical dependence. Softwares such as Sfold, Mfold and RNA structure were used to design ASODN targeting the coding sequence (CDS) of PEBP. Then the effective ASODN was screened by RT-PCR and Western Blot in PC12 cells. Finally precipitated withdrawal behavior of morphine dependent rats was observed after down-regulation of PEBP by continued microinjecting of ASODN in hippocampus. It was showed several (wet dog shakes, irritability and sialorrhea) but not all withdrawal symptoms were markedly enhanced in PEBP-down-regulation rats. The overall withdrawal score was significantly increased in ASODN-treated morphine dependent rats, while this did not happen in SODN-treated group.Then results of Western blot identified down-regulation of expression of PEBP in hippocampus of ASODN-treated morphine dependent rats compared with NS-treated group and morphine-treated group.
HCNP is an 11 amino acid peptide which is released from N-terminal of PEBP. It plays an important role in the septal cholinergic development of the hippocampus by enhancing the activity of ChAT. Previous studies have found that the hippocampal cholinergic system was involved in the development and expression of opioid physical dependence. Thus we continued to observe activity changes of the hippocampal cholinergic system during morphine dependence and natural withdrawal to see if PEBP was involved in opioid dependence through the hippocampal cholinergic system. Using different antibodies of PEBP, we observed the level of HCNP by Western blot. Results showed that the changes of HCNP level in hippocampus of morphine dependent and withdrawal rats had a similar trend to the changes of PEBP. Then ChAT activity was measured to observe if this enzyme activity changed with the level of HCNP. We found ChAT activity in hippocampus varied with the level of HCNP: it was increased significantly 4 d and 8 d after morphine administration, then decreased to the control level after 3 d and increased again after 14 d which continued until 21 d of natural withdrawal. This time-dependent changes in HCNP level and ChAT activity were similar to those in PEBP expression, suggesting PEBP regulated morphine dependence and withdrawal through the hippocampal cholinergic system.
In conclusion, in the present study we found hippocampal PEBP participated in morphine dependence at first time. The expression of PEBP in hippocampus had different changes during morphine dependence and natural withdrawal. Down-regulation of PEBP in hippocampus enhanced the expression of precipitated withdrawal symptoms, and the modulation to hippocampal cholinergic system functions mediated the involvement of PEBP in morphine dependence.
引文
[1] 罗旭.禁毒战争的遏制战略—访国家禁毒委员会副主任、公安部副部长张新枫.光明日报,2007-06-25
[2] 孙晓素,吕博雄.吸毒者艾滋病毒感染率升至 7.5%.南方日报,2007-12-03
[3] Leshner AI. Addiction is a brain disease, and it matters. Science, 1997,278: 45-47.
[4] 李树春, 李博, 程德纲等. 中医药治疗阿片类物质依赖的研究进展. 北京中医药大学学报,2005,28(01): 84-88
[5] Nestler EJ, Aghajanian GK. Molecular and cellular basis of addiction. Science,1997,278(5335): 58-63.
[6] Williams JT, Christie MJ, Manzoni O. Cellular and synaptic adaptations mediating opioid dependence. Physiol Rev,2001,81(1):299-343.
[7] Taylor DA, Fleming WW. Unifying perspectives of the mechanisms underlying the development of tolerance and physical dependence to opioids. J Pharmacol Exp Th,2001,297(1):11-18
[8] Heidbreder CA, Hagan JJ. Novel pharmacotherapeutic approaches for the treatment of drug addiction and craving. Curr Opin Pharmacol,2005,5: 107-118
[9] 秦晓辉,米卫东,张宏. 椎管内非阿片类药物镇痛研究进展. 国外医学麻醉学与复苏分册, 2005,26(03): 167-170.
[10] 李锦. 阿片功能调节剂. 中国药理学会通讯.,2002,19: 14-15
[11] Li J, Zhang XM, Wu N, He JT, Liu BY, Wang HX, Su RB. Comparative Proteomic Analysis of Hippocampus in Morphine-dependent Rats. Mol Cell Proteomics,2004,3.10: S178
[12] Bernier I, Jolles P. Purification and characterisation of a basic 23 kDa cytosolic protein from bovine brain. Biochim .Biophi Acta,1984,790: 174-181
[13] Ojika K,Mitake S,Tohdoh N, et al. Hippocampal cholinergic neurostimulating peptides(HCNP).Progress in Neurobiology,2000,60: 37-83
[14] Keller ET, Fu Zheng, Brennan M.The role of Raf kinase inhibitor protein (RKIP) in health and disease. Bioch Pharm,2004,68: 1049-1053
[15] Yeung K, Janosch P, McFerran N, et al.Mechanism of suppression of the Raf/Mek/extracellular signal-regulated kinase pathway by the raf kinase inhibitor protein. Mol Cell Biol.2000, 20: 3079-3085
[16] Lorenz K, Lohse MJ, Quitterer U. Protein kinase C switches the Raf kinase inhibitor from Raf-1 to GRK-2. Nature ,2003,426: 574-579
[17] Keller ET, Fu Z, Brennan M. The role of Raf kinase inhibitor protein (RKIP) in health and disease. Biochem Pharmacol,2004,68: 1049-1053
[18] Kroslak T, Koch T, Kahl E, et al. Human phosphatidylethanolamine-binding protein facilitiesheterotrimeric G protein-dependent signaling. J Biol Chem,2001,276: 39772-39778
[19] Ojika K, Katada E, Tohdoh N, et al.Demonstration of deacetylated hippocampal cholinergic neurostimulating peptide and its precursor protein in rat tissues. Brain Research,1995,701: 19-27
[20] Tohdoh N, Tojo S, Agui H, et al. Sequence homology of rat and human HCNP precursor proteins,bovine phosphatidylethanolamine-binding protein and rat 23-kDa protein associated with the opioid-binding protein. Mol Brain Res,1995,(30): 381-384
[21] Banfield MJ, Barker JJ, Perry ACF, et al. Function from structure? The crystal structure of human phosphatidylethanolamine-binding protein suggests a role in membrane signal transduction.Structure, 1998,6: 1245-1254
[22] Maki M, Matsukawa N, Yuasa H, et al,Decreased expression of hippocampal chiolinergic neurostimulating peptide precursor protein mRNA in the hippocampus in Alzheimer disease. J Neuropathol Exp Neurol,2002,61(2): 176-85
[23] George AJ, Holsinger RM, Mclean CA, et al.Decreased phosphatidylethanolamine binding protein expression correlated with Abeta accumulation in the Tg2576 mouse model of Alzheimer’s disease. Neurobiol Aging,2006,27(4): 614-623
[24] Tuttle KR, Anderson PW. A novel potential therapy for diabetic nephropathy and vascular complications:protein kinase C beta inhibition. Am J Kidney Dis, 2003,42(3): 456-65
[25] Mitake S, Ojika K, Katada E, et al. Distribution of hippocamal cholinergic neurostimulating peptide(HCNP) immunoreactivity in the central nervous system of the rat. Brain Research,1996,706: 57-70
[26] Trujillo KA, Akil H. Inhibition of morphine tolerance and dependence by the NMDA receptor antagonist MK-801.science,1991,251(4989): 85-87
[27] Fan GH, Wang LZ, Qin HC, et al.Inhibition of calcium/calmodulin-dependent protein kinase Ⅱin rat hippocampus attenuates morphine tolerance and dependence.Mol Pharmacol,1999,56(1): 39-45
[28] Salmanzadeh F, Fathollahi Y, Semnanian S, et al.Long-term potentiation as an electrophysiological assay for morphine dependence and withdrawal in rats:an in vitro study.J Neurosci Meth,2003,124(2): 189-96
[29] Harriosn JM, Allen RG, Pellegrino MJ, et al. Chronic morphine treatment alters endogenous opioid control of hippocampal mossy fiber synaptic transmission. J. Neurophysiol.2002,87(5),2464-2470.
[30] Zhang LC, Buccafusco JJ. Adaptive changes in M1 muscarinic receptors localized to specific rostral brain region during and after morphine withdrawal. Neuropharmocology,2000,39: 1720-31
[31] Carní J, Farré M. Mechanisms of Disease, Drug Addiction. N Engl J Med,2003,349: 975-86
[32] Ziegler A, Luedke GH, Fabbro D, et al.Induction of apoptosis in small-cell lung cancer cellsby an antisense oligonucleotide targeting the bcl-2 coding sequence.J Natl Cancer Inst,1997,87: 1027-36
[33] Mologni L, Nielsen PE, Gambacorti-Passerini C. In vitro transcriptional and translational block of the bcl-gene operated by peptide nucleic acid.Biochem and Biophy Res Commu,1999,264: 537-43
[34] Robinson ESJ, Nutt DJ, Jackson HC, et al.The uptake of a fluorescently labelled antisense oligonucleotide in vitro and in vivo. J Neurosci Meth,2005,147: 48-54
[35] Yin JB, Ma YX, Yin Q, et al. Involvement of over-expressed BMP4 in pentylenetetrazol kindling-induced cell proliferation in the dentate gyrus of adult rats.Biochem and Biophy Res Commu,2007,355:54-60
[36] Countryman R, Kaban NL, Colonbo PJ, et al. Hippocampal c-fos is necessary for long-term memory of a socially transmitted food preference. Neurobiology of Learning and Memory,2005,84:175-83
[37] Buccafusco J, Zhang LC, Shuster LC, et al. Prevention of precipitated withdrawal symptoms by activating central cholinergic systems during a dependence-producing schedule of morphine in rats. Brain Research,2000,853:76-83
[38] McDonough JL, Neverova I, Van-Eyk JE, et al. Proteomic analysis of human biopsy samples by single-two dimensional electrophoresis: Coomassie, silver, mass spectrometry, and Western blotting. Proteomics,2002,2:978-987
[39] Corthals GL, Wasinger VC, Hochstrasser DF, et al. The dynamic range of protein expression: a challenge for proteomic research.Electrophoresis,2000,21(6):1104-15
[40] Wasinger V, Pollack JD, Humphery-Smith I. The proteome of Mycoplasma genitalium.Chaps-soluble component. Eur J Biochem,2000,267(6):1571-82
[41] Vetulani J. Drug addiction.Part Ⅱ .Neurobiology of addiction.Pol J Pharmacol, 2001,53:303-17
[42] Nakagawa T, Kaneko S. Neuropsychotoxicity of abused drugs: molecular and neural mechanisms of neuropsychotoxicity induced by methamphetamine,3,4 -methylenedioxymethamphetamine(ecstasy), and 5-methoxy-N,N-diisopropyltryptamine(foxy). J Pharmacol Sci,2008,106(1):2-8
[43] Muris P, Schnidt H, Lanbrichs R, et al. Protective and vulnerability factors of depression in normal adolescerts.Behav Res Ther,2001,39(5):555-65
[44] Zarrindast MR, Lashgari R, Rezayof A, et al. NMDA receptors of dorsal hippocampus are involved in the acquisition,but not in the expression of morphine-induced place preference.Euro J Pharma,2007,568:192-98
[45] Neisewander JL, Baker DA, Fuchs RA, et al. Fos protein expression and cocaine-seekingbehavior in rats after exposure to a cocaine self-administration environment.J Neurosci,2000,20 (2):798-805
[46] Countryman R, Kaban NL, Colonbo PJ, et al. Hippocampal c-fos is necessary for long-term memory of a socially transmitted food preference. Neurobiology of Learning and Memory,2005,84:175-83
[47] Weiss B, Davidkova G, Zhang S. Antisense strategies in neurobiology. Neurochem Int,1997,31(3): 321-48
[48] James DT. Applications of antisense and siRNAs during preclinical drug development. DDT,2002,7(17):912-17
[49] Senn C, Hangartner C,Moes Suzette, et al. Central administration of small interfering RNAs in rats: A comparison with antisense oligonucleotides. Euro J Pharma,2005,522:30-7
[50] Andronescu M., Condon A., Hoos H.H., et al. Efficient parameter estimation for RNA secondary structure prediction.Bioinformatics,2007,23(13):i19-i28
[51] Kurreck J, Wyszko E, Gllen C, et al. Design of antisense oligonucleotides stabilized by locked nucleic acids.Nucleic Acids Res,2002,30(9):1911-18
[52] Sczakiel G, Homann M, Rittner K, et al.Computer-aided search for effective antisense RNA target sequences of the human immuno-deficiency virus type 1. Antisense Res Dev,1993,3:45-52
[53] Schroeder SJ, Turner DH. Factors affecting the thermodynamic atsbility of small asymmetric internal loops in RNA. Biochemistry,2000,39:9257-74
[54] Tang L, Shukla PK, Wang ZJ. Disruption of acute opioid dependence by antisense oligodeoxynucleotides targeting neutogranin. Brain Research,2007,1143:78-82
[55] 周文华,杨国栋.吗啡依赖与毒蕈碱型乙酰胆碱受体的适应.中国药物滥用防治杂志,2005,11(2):83-6
[56] Ojika K, Mitake S, Kamiya T, et al. Two different molecules,NGF and free-HCNP,stimulate cholinergic activity in septal nuclei in vitro in a different manner. Dev Brain Res,1994,79:1-9
[57] Ojika K, Katada E, Tohdoh N, et al. Demonstration of deacetylated hippocampal cholinergic neurostimulatinf peptide and its precursor protein in rat tissues. Brain Res,1995,701:19-27
[58] Shiba K, Ogawa K, Kinuya S, et al. A simple and rapid radiochemical choline acetyltransferase (ChAT) assay screening test. J Neuro Meth,2006,157:98-102
[59] Siegal D,Erickson J,Varoqui H,et al. Brain vesicular acetylcholine transporter in human users of drugs of abuse. Synapse,2004,52(4):223-32
[60] Eisenach JC. Muscarinic-mediated analgesia.Life science,1999,64:549-54
[61] Crossland J, Ahmed KZ. Brain acetylcholine during morphine withdrawal. Neurochem Res,1984,9:351-366
[62] Buccafusco JJ. Inhibition of the morphine withdrawal syndrome by a novel muscarinicantagonist (4-DAMP).Life Sci,1991,48:749-56
[63] Sharif SI, Kadi AO. The role of cholinergic systems in the expression of morphine withdrawal. Neurosci Res,1996,25:155-60
[64] Shiba K, Ogawa K, Kinuya S, et al. A simple and rapid radiochemical choline acetyltransferase (ChAT) assay screening test. J Neuro Meth,2006,157:98-102
[65] Siegal D, Erickson J, Varoqui H, et al. Brain vesicular acetylcholine transporter in human users of drugs of abuse. Synapse,2004,52(4):223-32
[66] Iwase T, Ojika K, Matsukawa N, et al. Muscarinic cholinergic and glutamatergic reciprocal regulation of expression of hippocampal cholinergic neurostimulating peptide precursor protein gene in rat hippocampus.Neuroscience,2001,102(2):341-52
[67] Ojika K, Tsugu Y, Mitake S, et al. NMDA receptor activation enhances the release of a cholinergic differentiation peptide(HCNP) from hippocampal neurons in vitro. Devl Brain Res,1998,106:173-80
[68] 王晓菲,吴宁,苏瑞斌,等.胍丁胺对吗啡长期处理引起的 NMDA 受体蛋白表达改变的影响.中国药物依赖杂志,2006,15(4):267-72
[69] Markram H, Segal M. Acetylcholine potentiates responses to N-methyl-D-aspartate in the rat hippocampus.Neurosci Lett,1990,113:62-5
[70] Bliss TV, Collingridge GL. A synaptic model of memory:long-term potentiation in the hippocampus.Nature,1993,361(6407):31-9
[71] Saal D, Malenka RC. The role of synaptic plasticity in addiction. Clin Neurosci Res,2005,5:141-46
[72] Nicoll RA. The septo-hippocampal projection: a model cholinergic pathway. Trends Neurosci,1985,8:533-36
[73] Imperato A, Obinu MC, Casu MA, et al. Chronic morphine increases hippocampal acetylcholine release: possible relevance in drug dependence. Eur J Pharmacol,1996,302(1~3):21-26
[74] Albin MS, Orr MD, Bunegin L, et al. Tetrahydroaminoacridine antagonism to narcotic addiction, Exp Neurol,1975,46:644-48
[75] Zhang LC, Buccafusco JJ. Prevention of morphine-induced muscarinic(M2) adaption suppresses the expression of withdrawal symptoms. Brain Res,1998,803:114-121
[76] Robbins TW, Everitt BJ. Drug addiction: bad habits add up. Nature,1999,398:567-70
[77] Valle BS, Coadou G, Labbe H, et al. Peptides corresponding to the N-and C-terminal parts of PEBP are well-structured in solution:new insights into their possible interaction with partners in vivo. J Pept Res,2003,61(2):47-57
[78] Wang HY, Frienman E, Olmstead MC, et al. Ultra-low-dose naloxone suppresses opioidtolerance.dependence and associated changes in mu opioid receptor-G protein coupling and Gβν signaling. Neuroscience,2005,13:247-61
