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博碩士論文 etd-0801110-184540 詳細資訊
Title page for etd-0801110-184540
論文名稱
Title
古柯鹼影響 PKMζ 在 VTA 區域表現之研究
Role of Cocaine-Induced Protein Kinase Mzeta Expression in the Ventral Tegmental Area
系所名稱
Department
畢業學年期
Year, semester
語文別
Language
學位類別
Degree
頁數
Number of pages
90
研究生
Author
指導教授
Advisor
召集委員
Convenor
口試委員
Advisory Committee
口試日期
Date of Exam
2010-06-21
繳交日期
Date of Submission
2010-08-01
關鍵字
Keywords
蛋白激酶Mζ、長期增益效應、腹側盖區、神經可塑性、藥物成癮
long-term potentiation, Protein Kinase Mζ, ventral tegmental area, synaptic plasticity, drug addiction
統計
Statistics
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中文摘要
藥物成癮是長期使用成癮性藥物後造成腦內類似神經可塑性(synaptic plasticity) 的生理變化進而對藥物產生依賴性,其中腦內的中腦邊緣多巴胺系統 (mesolimbic dopamine system) 的神經迴路改變被認為與成癮發生最為相關,此迴路包含了腹側盖區 ( ventral tegmental area ;VTA )、依核 (nucleus accumbens ; NAC ) 及其他相關的邊緣系統相關區域 (limbic structure) 如海馬迴 (hippocampus) 等。神經可塑性其中的機制之一「長期增益效應 (long-term potentiation ;LTP)」是突觸間的神經迴路受到外界的刺激而使突觸間傳導強度改變的現象,為目前最常被用來解釋學習以及記憶形成的機制之一。在近來的研究中發現蛋白激酶MMζ (Protein Kinase Mζ ; PKMζ ) 可藉由增加hippocampus 中突觸後
細胞膜上AMPA (α-amino-3-hydroxyl-5-methyl-4 -isoxazole-propionate)受器的數目來增加突觸間的傳遞強度,進而在記憶形成時扮演重要角色,故在本實驗中以古柯鹼誘導成癮後,希望探討PKMζ 在中腦邊緣多巴胺系統中的VTA 以及其他相關區域中所扮演的角色及可能的
調控路徑。在本實驗中給予不同年齡層的大鼠腹腔注射古柯鹼而誘導成癮的發生後分別在不同時間點取出VTA、NAc、hippocampus 等區域的腦組織並分析其PKMζ 表現量的變化。我們發現年輕組的大鼠(Postnatal day ; P18~P30) 在接觸單一劑古柯鹼注射後的 VTA 其
PKMζ 的確會被誘導而表現,並且在第30 分鐘有顯著增加,而此PKMζ 蛋白質在 1小時以內的增是來自於對既存的 PKMζ mRNA 轉譯的調控,其中包含了 PKMζ 對自身表現正回饋的調控,以及受到MEK1 (mitogen-activated protein kinase kinase 1)、蛋白激酶A
(cAMP-dependent protein kinase ; PKA)、PI3K (Phosphatidylinositol 3-kinases)、多巴胺受器D1- 及D2- 亞型 (dopamine receptor D1- 及D2- subtype) 等的影響,並且不受 CaMK II(Calcium/calmodulindependent
protein kinase II) 的影響。同時於 VTA 的 PKMζ mRNA 表現量也在第1 小時增加,顯示具有轉錄上的調控。此外單一劑古柯鹼注射也會24 小時內於 NAc 與 hippocampus 中誘導出PKMζ 的表現。而重複連續注射古柯鹼 5 天也會在VTA 、NAc、hippocampus等區域誘導PKMζ 持續的表現。在VTA 與 NAc 中,年長組 (P45~P50)的大鼠對古柯鹼所誘導的PKMζ 表現量較年輕組的不顯著,並且年長組對古柯鹼所誘導行為影響 (locomotor sensitization) 也較年輕組
不明顯。胚胎時期即接觸到古柯鹼也會減少於出生後再度接觸到古柯鹼在 VTA 與 NAc 中所誘導出的 PKMζ 表現量,並且對古柯鹼所誘導行為影響 (locomotor sensitization) 也相近於年長組的表現。
Abstract
The mesolimbic dopamine system, including dopaminergic projections from the ventral tegmental area (VTA) to nucleus accumbens (NAc), is critically involved in the development of addiction to many drugs of abuse, including cocaine (CA). Although there is an attractive hypothesis that the modifications of mesolimbic reward circuit following repeated drug exposure are responsible for cocaine-addicted causes behaviors change, however, our understanding in the underlying molecular mechanisms at the neural circuit level is still in its infancy. It has been suggested PKMzeta, a constitutively active atypical isoform of PKC, plays a critical role in spatial memory formation and long-term synaptic potentiation in hippocampus. To define the relationship among PKMzeta, CA-induced synaptic long-term potentiation and CA addiction, we examined the regulation of PKMzeta after CA administration in Sprague-Dawley rat. We found single CA injection elicits an increase in PKMzeta protein expression in the VTA region. The increase was first observed 10 min after CA administration and lasted for 7 days, the longest sampling time point of our experimental design. The PKMzeta protein expression can also be induced in 10 minutes while incubating the acute isolated brain slice with CA, the expression within 1 hr can be eliminated at the present of Chelerythrine (PKC inhibitor) and ZIP (PKMzeta inhibitor) suggests a positive feedback loop. The PKMzeta mRNA expression can be induced within 1 hr, and Actinomycin d (transcription inhibitor) had no effect on the PKMzeta protein expression indicating CA increases PKMζ translation from preexisting PKMζ mRNA. Furthermore,real time PCR-based analysis showed resembling increase profile ofPKMζ mRNA after single CA injection, suggesting a co-upregulation of transcription and translation of PKMζ after CA administration in VTA.
Eticlopride (dopamine receptor D2-subtype antagonist) 、SCH-23390(dopamine receptor D1-subtype antagonist)、H-89 (PKA inhibitor)、
Wortmannin (PI3K inhibitor)、PD98059 (MEK1 inhibitor) decreasedcocaine-induced PKMζ expression within 1 hr in VTA. On the contrary,
KN-62 (CaMK II inhibitor) has no obvious effect on PKMζ expression.
CA challenge not only induces the PKMζ expression in the VTA region but also in the NAc and hippocampus region. The CA-induced PKMζ
expression is more obvious in elder group (>45 days in age) than younger group (18~30 days in age), similar results also showed in the locomotor
activity assay. Prenatal CA exposure decreased the postnatal CA-induced PKMζ expression and the locomotor sensitivity in younger group.
Overall, results from our current experiments have raised the possibility of PKMζ involvement in CA addiction. How CA regulates PKMζ
expression and the context dependence between PKMζ and CA-induced behavior change and synaptic long-term potentiation remains further elucidation.
目次 Table of Contents
頁數
圖表索引 1
縮寫表 2
中文摘要 3
英文摘要 5
緒論 7
實驗材料與方法
實驗動物 15
1.腦組織切片製備 15
2.西方點墨法
2-1.蛋白質萃取 17
2-2.蛋白質定量 18
2-3.硫酸十二酯鈉-聚丙烯醯胺凝膠電泳 18
2-4.蛋白質電泳轉印 19
2-5.免疫染色 19
2-6. 冷光持續曝光拍攝 20
3.即時-反轉錄聚合酶連鎖反應
3-1. RNA 萃取 21
3-2. RNA 定量及反轉錄 21
3-3.聚合酶連鎖反應 23
4.運動活性測量 24
5.實驗數據統計與分析 26
6.實驗試劑與供應商 26
7.實驗試劑的作用 27
結果 28
討論 43
參考文獻 52
圖表 58
參考文獻 References
Adriani W, Laviola G (2004) Windows of vulnerability to psychopathology and therapeutic strategy in the adolescent rodent model. Behav Pharmacol 15:341-352.
Baumeister SE, Tossmann P (2005) Association between early onset of cigarette, alcohol and cannabis use and later drug use patterns: an analysis of a survey in European metropolises. Eur Addict Res 11:92-98.
Biondi RM (2004) Phosphoinositide-dependent protein kinase 1, a sensor of protein conformation. Trends Biochem Sci 29:136-142.
Bliss TV, Lomo T (1973) Long-lasting potentiation of synaptic transmission in the dentate area of the anaesthetized rabbit following stimulation of the perforant path. J Physiol 232:331-356.
Bliss TV, Collingridge GL (1993) A synaptic model of memory: long-term potentiation in the hippocampus. Nature 361:31-39.
Borgland SL, Malenka RC, Bonci A (2004) Acute and chronic cocaine-induced potentiation of synaptic strength in the ventral tegmental area: electrophysiological and behavioral correlates in individual rats. J Neurosci 24:7482-7490.
Boudreau AC, Wolf ME (2005) Behavioral sensitization to cocaine is associated with increased AMPA receptor surface expression in the nucleus accumbens. J Neurosci 25:9144-9151.
Bustamante D, You ZB, Castel MN, Johansson S, Goiny M, Terenius L, Hokfelt T, Herrera-Marschitz M (2002) Effect of single and repeated methamphetamine treatment on neurotransmitter release in substantia nigra and neostriatum of the rat. J Neurochem 83:645-654.
Cao J, Lotfipour S, Loughlin SE, Leslie FM (2007) Adolescent maturation of cocaine-sensitive neural mechanisms. Neuropsychopharmacology 32:2279-2289.
Carr DB, Sesack SR (2000) Projections from the rat prefrontal cortex to the ventral tegmental area: target specificity in the synaptic associations with mesoaccumbens and mesocortical neurons. J Neurosci 20:3864-3873.
Cooper DC, Klipec WD, Fowler MA, Ozkan ED (2006) A role for the subiculum in the brain motivation/reward circuitry. Behav Brain Res 174:225-231.
Cornish JL, Kalivas PW (2001) Repeated cocaine administration into the rat ventral tegmental area produces behavioral sensitization to a systemic cocaine challenge. Behav Brain Res 126:205-209.
Delaney-Black V, Covington C, Ostrea E, Jr., Romero A, Baker D, Tagle MT, Nordstrom-Klee B, Silvestre MA, Angelilli ML, Hack C, Long J (1996) Prenatal cocaine and neonatal outcome: evaluation of dose-response relationship. Pediatrics 98:735-740.
Eisch AJ, Harburg GC (2006) Opiates, psychostimulants, and adult hippocampal neurogenesis: Insights for addiction and stem cell biology. Hippocampus 16:271-286.
Gabbott PL, Warner TA, Jays PR, Salway P, Busby SJ (2005) Prefrontal cortex in the rat: projections to subcortical autonomic, motor, and limbic centers. J Comp Neurol 492:145-177.
Girault JA, Valjent E, Caboche J, Herve D (2007) ERK2: a logical AND gate critical for drug-induced plasticity? Curr Opin Pharmacol 7:77-85.
Hernandez AI, Blace N, Crary JF, Serrano PA, Leitges M, Libien JM, Weinstein G, Tcherapanov A, Sacktor TC (2003) Protein kinase M zeta synthesis from a brain mRNA encoding an independent protein kinase C zeta catalytic domain. Implications for the molecular mechanism of memory. J Biol Chem 278:40305-40316.
Hyman SE, Malenka RC (2001) Addiction and the brain: the neurobiology of compulsion and its persistence. Nat Rev Neurosci 2:695-703.
Jackson A, Mead AN, Stephens DN (2000) Behavioural effects of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-receptor antagonists and their relevance to substance abuse. Pharmacol Ther 88:59-76.
Jones N, O'Neill MJ, Tricklebank M, Libri V, Williams SC (2005) Examining the neural targets of the AMPA receptor potentiator LY404187 in the rat brain using pharmacological magnetic resonance imaging. Psychopharmacology (Berl) 180:743-751.
Kalivas PW, Volkow ND (2005) The neural basis of addiction: a pathology of motivation and choice. Am J Psychiatry 162:1403-1413.
Kalivas PW, O'Brien C (2008) Drug addiction as a pathology of staged neuroplasticity. Neuropsychopharmacology 33:166-180.
Kauer JA, Malenka RC (2007) Synaptic plasticity and addiction. Nat Rev Neurosci 8:844-858.
Kelleher RJ, 3rd, Govindarajan A, Jung HY, Kang H, Tonegawa S (2004) Translational control by MAPK signaling in long-term synaptic plasticity and memory. Cell 116:467-479.
Keller RW, Jr., Snyder-Keller A (2000) Prenatal cocaine exposure. Ann N Y Acad Sci 909:217-232.
Kelly A, Lynch MA (2000) Long-term potentiation in dentate gyrus of the rat is inhibited by the phosphoinositide 3-kinase inhibitor, wortmannin. Neuropharmacology 39:643-651.
Kelly MT, Crary JF, Sacktor TC (2007) Regulation of protein kinase Mzeta synthesis by multiple kinases in long-term potentiation. J Neurosci 27:3439-3444.
Kessels HW, Malinow R (2009) Synaptic AMPA receptor plasticity and behavior. Neuron 61:340-350.
Koob G, Kreek MJ (2007) Stress, dysregulation of drug reward pathways, and the transition to drug dependence. Am J Psychiatry 164:1149-1159.
Kourrich S, Rothwell PE, Klug JR, Thomas MJ (2007) Cocaine experience controls bidirectional synaptic plasticity in the nucleus accumbens. J Neurosci 27:7921-7928.
Kuhar MJ, Ritz MC, Boja JW (1991) The dopamine hypothesis of the reinforcing properties of cocaine. Trends Neurosci 14:299-302.
Ling DS, Benardo LS, Serrano PA, Blace N, Kelly MT, Crary JF, Sacktor TC (2002) Protein kinase Mzeta is necessary and sufficient for LTP maintenance. Nat Neurosci 5:295-296.
Liu QS, Pu L, Poo MM (2005) Repeated cocaine exposure in vivo facilitates LTP induction in midbrain dopamine neurons. Nature 437:1027-1031.
Lu H, Lim B, Poo MM (2009) Cocaine exposure in utero alters synaptic plasticity in the medial prefrontal cortex of postnatal rats. J Neurosci 29:12664-12674.
Lu L, Koya E, Zhai H, Hope BT, Shaham Y (2006) Role of ERK in cocaine addiction. Trends Neurosci 29:695-703.
Malanga CJ, Pejchal M, Kosofsky BE (2007) Prenatal exposure to cocaine alters the development of conditioned place-preference to cocaine in adult mice. Pharmacol Biochem Behav 87:462-471.
Malenka RC, Bear MF (2004) LTP and LTD: an embarrassment of riches. Neuron 44:5-21.
Mendelson JH, Mello NK (1996) Management of cocaine abuse and dependence. N Engl J Med 334:965-972.
Morris RG (1989) Synaptic plasticity and learning: selective impairment of learning rats and blockade of long-term potentiation in vivo by the N-methyl-D-aspartate receptor antagonist AP5. J Neurosci 9:3040-3057.
Morrow BA, Elsworth JD, Roth RH (2005) Prenatal exposure to cocaine selectively disrupts the development of parvalbumin containing local circuit neurons in the medial prefrontal cortex of the rat. Synapse 56:1-11.
Moser MB, Moser EI (1998) Functional differentiation in the hippocampus. Hippocampus 8:608-619.
Muslimov IA, Nimmrich V, Hernandez AI, Tcherepanov A, Sacktor TC, Tiedge H (2004) Dendritic transport and localization of protein kinase Mzeta mRNA: implications for molecular memory consolidation. J Biol Chem 279:52613-52622.
O'Brien CP (2005) Anticraving medications for relapse prevention: a possible new class of psychoactive medications. Am J Psychiatry 162:1423-1431.
Palmer RH, Dekker LV, Woscholski R, Le Good JA, Gigg R, Parker PJ (1995) Activation of PRK1 by phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate. A comparison with protein kinase C isotypes. J Biol Chem 270:22412-22416.
Pastalkova E, Serrano P, Pinkhasova D, Wallace E, Fenton AA, Sacktor TC (2006) Storage of spatial information by the maintenance mechanism of LTP. Science 313:1141-1144.
Perkel DJ, Nicoll RA (1993) Evidence for all-or-none regulation of neurotransmitter release: implications for long-term potentiation. J Physiol 471:481-500.
Pierce RC, Kumaresan V (2006) The mesolimbic dopamine system: the final common pathway for the reinforcing effect of drugs of abuse? Neurosci Biobehav Rev 30:215-238.
Pistis M, Perra S, Pillolla G, Melis M, Muntoni AL, Gessa GL (2004) Adolescent exposure to cannabinoids induces long-lasting changes in the response to drugs of abuse of rat midbrain dopamine neurons. Biol Psychiatry 56:86-94.
Roberson ED, Sweatt JD (1996) Transient activation of cyclic AMP-dependent protein kinase during hippocampal long-term potentiation. J Biol Chem 271:30436-30441.
Roberson ED, English JD, Sweatt JD (1996) A biochemist's view of long-term potentiation. Learn Mem 3:1-24.
Sacktor TC (2008) PKMzeta, LTP maintenance, and the dynamic molecular biology of memory storage. Prog Brain Res 169:27-40.
Sacktor TC, Osten P, Valsamis H, Jiang X, Naik MU, Sublette E (1993) Persistent activation of the zeta isoform of protein kinase C in the maintenance of long-term potentiation. Proc Natl Acad Sci U S A 90:8342-8346.
Salisbury AL, Lester BM, Seifer R, Lagasse L, Bauer CR, Shankaran S, Bada H, Wright L, Liu J, Poole K (2007) Prenatal cocaine use and maternal depression: effects on infant neurobehavior. Neurotoxicol Teratol 29:331-340.
Sanes JR, Lichtman JW (1999) Can molecules explain long-term potentiation? Nat Neurosci 2:597-604.
Schenk S, Snow S (1994) Sensitization to cocaine's motor activating properties produced by electrical kindling of the medial prefrontal cortex but not of the hippocampus. Brain Res 659:17-22.
Shalev U, Grimm JW, Shaham Y (2002) Neurobiology of relapse to heroin and cocaine seeking: a review. Pharmacol Rev 54:1-42.
Shankaran S, Lester BM, Das A, Bauer CR, Bada HS, Lagasse L, Higgins R (2007) Impact of maternal substance use during pregnancy on childhood outcome. Semin Fetal Neonatal Med 12:143-150.
Shema R, Sacktor TC, Dudai Y (2007) Rapid erasure of long-term memory associations in the cortex by an inhibitor of PKM zeta. Science 317:951-953.
Shilatifard A (1998) Factors regulating the transcriptional elongation activity of RNA polymerase II. FASEB J 12:1437-1446.
Sonnenburg ED, Gao T, Newton AC (2001) The phosphoinositide-dependent kinase, PDK-1, phosphorylates conventional protein kinase C isozymes by a mechanism that is independent of phosphoinositide 3-kinase. J Biol Chem 276:45289-45297.
Sweatt JD (1999) Toward a molecular explanation for long-term potentiation. Learn Mem 6:399-416.
Sweatt JD, Atkins CM, Johnson J, English JD, Roberson ED, Chen SJ, Newton A, Klann E (1998) Protected-site phosphorylation of protein kinase C in hippocampal long-term potentiation. J Neurochem 71:1075-1085.
Thomas KL, Laroche S, Errington ML, Bliss TV, Hunt SP (1994) Spatial and temporal changes in signal transduction pathways during LTP. Neuron 13:737-745.
Thomas MJ, Kalivas PW, Shaham Y (2008) Neuroplasticity in the mesolimbic dopamine system and cocaine addiction. Br J Pharmacol 154:327-342.
Ungless MA, Whistler JL, Malenka RC, Bonci A (2001) Single cocaine exposure in vivo induces long-term potentiation in dopamine neurons. Nature 411:583-587.
Volkow N, Li TK (2005) The neuroscience of addiction. Nat Neurosci 8:1429-1430.
Vorel SR, Liu X, Hayes RJ, Spector JA, Gardner EL (2001) Relapse to cocaine-seeking after hippocampal theta burst stimulation. Science 292:1175-1178.
Wolf ME (1998) The role of excitatory amino acids in behavioral sensitization to psychomotor stimulants. Prog Neurobiol 54:679-720.
Yang CR, Mogenson GJ (1986) Dopamine enhances terminal excitability of hippocampal-accumbens neurons via D2 receptor: role of dopamine in presynaptic inhibition. J Neurosci 6:2470-2478.
Yao Y, Kelly MT, Sajikumar S, Serrano P, Tian D, Bergold PJ, Frey JU, Sacktor TC (2008) PKM zeta maintains late long-term potentiation by N-ethylmaleimide-sensitive factor/GluR2-dependent trafficking of postsynaptic AMPA receptors. J Neurosci 28:7820-7827.
Zuckerman B, Frank DA, Hingson R, Amaro H, Levenson SM, Kayne H, Parker S, Vinci R, Aboagye K, Fried LE, et al. (1989) Effects of maternal marijuana and cocaine use on fetal growth. N Engl J Med 320:762-768.
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