論文使用權限 Thesis access permission:自定論文開放時間 user define
開放時間 Available:
校內 Campus: 已公開 available
校外 Off-campus: 已公開 available
論文名稱 Title |
古柯鹼影響KCC2在大鼠腦區表現之研究 Effect of cocaine exposure on K+-Cl- cotransporter 2 expression in rat |
||
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 |
89 |
|
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 |
2011-10-26 |
繳交日期 Date of Submission |
2011-12-26 |
關鍵字 Keywords |
古柯鹼、藥物成癮、第二型鉀氯離子轉運蛋白、γ-丁氨基酪酸、年齡專一性 cocaine, drug addiction, K+-Cl- cotransporter, GABA, age-dependent |
||
統計 Statistics |
本論文已被瀏覽 5662 次,被下載 267 次 The thesis/dissertation has been browsed 5662 times, has been downloaded 267 times. |
中文摘要 |
在懷孕期間吸食古柯鹼 (prenatal cocaine)對胎兒的腦神經發育會產生長期負面影響,例如運動功能障礙或學習能力、記憶力低落等問題。在成熟的腦神經系統中γ-amino-butyric acid (GABA)是主要的抑制性神經傳遞物質,但在初生時期的未成熟神經細胞中,GABA扮演的卻是興奮性神經傳遞物質。這興奮性和抑制性之間的改變與隨時間相對表現的第二型鉀氯轉運蛋白(K+-Cl- cotransporter 2, KCC2) 有密切關聯,且自大鼠出生開始GABA從興奮性到出生後三十天已完全轉換成抑制性神經傳遞物。因此在本論文中我們想了解懷孕期間吸食古柯鹼是否會對胎兒腦部神經系統中KCC2的表現產生影響,我們選擇分別與認知功能有關的前額葉皮層 (prefrontal cortex)、與形成記憶有關的海馬迴 (hippocampus)、報償迴路中最重要腹側被蓋區(ventral tegmental area, VTA)和伏隔核 (nucleus accumbens, NAc)做為研究對象。實驗天數從大鼠出生後 postnatal day (PND) 第八天到第三十天並以偶數天數進行取樣,對照組 (control) 跟實驗組 (prenatal cocaine) 皆使用西方點墨法 (western blot) 觀察KCC2轉運蛋白之表現量並繪製成圖表。試驗結果顯示在前額葉皮層 (prefrontal cortex)的組別皆有增加的趨勢,但在KCC2的表現量是實驗組 (prenatal CA)低於對照組 (control )。此外,我們亦觀察正常出生後的大鼠重複給予古柯鹼在不同年齡(PND 24 及 PND 32)對KCC2的表現影響,以及古柯鹼對行為敏感化 (locomotor activity) 的影響,結果顯示PND24的大鼠在行為敏感化較易上癮,且其KCC2的表現量低於PND32大鼠。故進一步分成A組 (PND 22∼27 ) 和B組 (PND 29∼34) 探討持續投予古柯鹼後第一天和第五天的大鼠於藥物成癮(drug addiction )後對KCC2表現的影響,結果顯示A組抑制KCC2表現量比B組明顯,可說明B組的KCC2表現高於A組。故B組中GABA有明顯的抑制作用,且在行為模式相對跑比較慢,所以藥物成癮方面A組比較容易導致成癮。最後藉由腦切片浸泡藥物了解可能的訊息傳遞路徑,在腦切片結果得知古柯鹼會抑制KCC2的表現量,隨之再給SCH23390(dopamine D1-receptor 抑制劑)或eticlopride(dopamine D2-receptor 抑制劑)發現兩者都能降低對KCC2表現的抑制作用,另外在prenatal cocaine組的腦切片結果發現以eticlopride 處理會增加KCC2的表現,但以SCH23390反而會抑制了KCC2的表現,顯示D1與D2 receptor 在Cocaine 影響KCC2 的表現均扮演一定的角色! |
Abstract |
Cocaine (CA) exposure during pregnancy causes long-lasting negative effects on fetal brain development and eventually results in motor dysfunction or changes in learning and memory performance. γ-amino-butyric acid (GABA) is the primary inhibitory neurotransmitter in the adult brain and undergo a switch from excitatory to inhibitory during early postnatal period. The excitatory/inhibitory switch is resulted in the relative temporal expression of K+-Cl- cotransporter 2 (KCC2). GABA is the neurotransmitter in the rat was born from excitement to inhibition and until the growth of thirty days have completely inhibitory. Here we test the effect of CA prenatal exposure on the expression of KCC2 in prefrontal cortex (recognition), hippocampus (memory), VTA (reward) and nucleus accumbens (reward). Protein expression profile of control or prenatal CA treated groups were evaluated by western blot in 2 days interval from postnatal day (PND) 8 to 30. The expression of KCC2 was time-dependently enhanced from PND 8 and reaches its maximal expression around PND 18 in prenatal CA exposure groups. The time-dependent profile of KCC2 expression in prefrontal cortex and NAc was significantly delayed in prenatal CA exposure group. We then correlate the KCC2 expression and the cocaine sensitivity by locomotor activity assay. We found group A shows a higher sensitivity to cocaine than group B in control rats. Surprisingly, group A of prenatal cocaine reduce the sensitive to cocaine to a similar extend like group B in control rats, suggesting prenatal exposure of cocaine might enhance the KCC2 expression. Furthermore, age range of A group (PND 22~27) and B group (PND 29~34) to repeated cocaine exposure resulted in up-regulation of KCC2 expression in B group earlier than A group. We found that the KCC2 expressions of repeated cocaine exposure in B group were higher than A group. In other words, in the B group, the inhibitory effect of GABA was significant and the locomotor activity was relatively slow. Therefore, the A group was more easy be cocaine addiction than B group. We next explore the signaling mechanism underlying cocaine exposure-induced KCC2 expression inhibition. Brain slices were incubated with cocaine with or without dopamine receptor antagonists and KCC2 expression was evaluated by western blot. Either SCH23390 (dopamine D1-receptor inhibitor) or eticlopride (dopamine D2-receptor inhibitor) significantly hamper the inhibition of KCC2 expression by cocaine in normal slices. However, only D2 antagonist eticlopride but not SCH23390 is effective reverse cocaine-induced KCC2 expression inhibition. Overall, results from our current studies provide a further insight into the molecular mechanism of cocaine-induced synaptic modification. |
目次 Table of Contents |
目 錄 縮寫表……………………………………………………………. 1 中文摘要…………………………………………………………. 2 Abstract……………………………………………………………4 緒論………………………………………………………………. 6 實驗目的…………………………………………………………. 16 材料與方法………………………………………………………. 17 I. 實驗動物購製……………………………………………... 17 II. SD rat 腦組織切片製備…………………………….…......17 III. 取樣時程…………………………………………....…….19 IV. 西方點墨法…………………………………….……........20 V. 運動活性分析……………………………....…….……….24 VI. 實驗數據統計與分析……………………………….…....26 實驗結果…………………………………………………………. 27 討論………………………………………………………………. 45 參考文獻…………………………………………………………. 53 圖表………………………………………………………………. 59 |
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. Al-Tikriti MS, Roth RH, Kessler RM, Innis RB (1992) Autoradiographic localization of dopamine D1 and D2 receptors in rat cerebral cortex following unilateral neurotoxic lesions. Brain Res 575: 39-46. Bakshi K, Gennaro S, Chan CY, Kosciuk M, Liu J, et al. (2009) Prenatal cocaine reduces AMPA receptor synaptic expression through hyperphosphorylation of the synaptic anchoring protein GRIP. J Neurosci 29: 6308-6319. 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. Beaulieu JM, Sotnikova TD, Marion S, Lefkowitz RJ, Gainetdinov RR, et al. (2005) An Akt/beta-arrestin 2/PP2A signaling complex mediates dopaminergic neurotransmission and behavior. Cell 122: 261-273. Ben-Ari Y (2002) Excitatory actions of gaba during development: the nature of the nurture. Nat Rev Neurosci 3: 728-739. Ben-Ari Y, Cherubini E, Corradetti R, Gaiarsa JL (1989) Giant synaptic potentials in immature rat CA3 hippocampal neurones. J Physiol 416: 303-325. Berninger B, Marty S, Zafra F, da Penha Berzaghi M, Thoenen H, et al. (1995) GABAergic stimulation switches from enhancing to repressing BDNF expression in rat hippocampal neurons during maturation in vitro. Development 121: 2327-2335. Bray JG, Mynlieff M (2009) Influx of calcium through L-type calcium channels in early postnatal regulation of chloride transporters in the rat hippocampus. Dev Neurobiol 69: 885-896; erratum 897-912. Broersen LM, Heinsbroek RP, de Bruin JP, Olivier B (1996) Effects of local application of dopaminergic drugs into the medial prefrontal cortex of rats on latent inhibition. Biol Psychiatry 40: 1083-1090. Caillard O, Ben-Ari Y, Gaiarsa JL (1999) Long-term potentiation of GABAergic synaptic transmission in neonatal rat hippocampus. J Physiol 518 ( Pt 1): 109-119. Carlezon WA, Jr., Duman RS, Nestler EJ (2005) The many faces of CREB. Trends Neurosci 28: 436-445. 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. Chen G, Trombley PQ, van den Pol AN (1996) Excitatory actions of GABA in developing rat hypothalamic neurones. J Physiol 494 ( Pt 2): 451-464. Cherubini E, Gaiarsa JL, Ben-Ari Y (1991) GABA: an excitatory transmitter in early postnatal life. Trends Neurosci 14: 515-519. Cherubini E, Rovira C, Gaiarsa JL, Corradetti R, Ben Ari Y (1990) GABA mediated excitation in immature rat CA3 hippocampal neurons. Int J Dev Neurosci 8: 481-490. Cohen BI (2002) The significance of ammonia/gamma-aminobutyric acid (GABA) ratio for normality and liver disorders. Med Hypotheses 59: 757-758. Connor JA, Tseng HY, Hockberger PE (1987) Depolarization- and transmitter-induced changes in intracellular Ca2+ of rat cerebellar granule cells in explant cultures. J Neurosci 7: 1384-1400. Cordero-Erausquin M, Coull JAM, Boudreau D, Rolland M, De Koninck Y (2005) Differential maturation of GABA action and anion reversal potential in spinal lamina I neurons: Impact of chloride extrusion capacity (vol 19, 9613, 2005). Journal of Neuroscience 25: 10574-10575. Crandall JE, Hackett HE, Tobet SA, Kosofsky BE, Bhide PG (2004) Cocaine exposure decreases GABA neuron migration from the ganglionic eminence to the cerebral cortex in embryonic mice. Cereb Cortex 14: 665-675. Delaney-Black V, Covington C, Ostrea E, Jr., Romero A, Baker D, et al. (1996) Prenatal cocaine and neonatal outcome: evaluation of dose-response relationship. Pediatrics 98: 735-740. Di Chiara G, Bassareo V, Fenu S, De Luca MA, Spina L, et al. (2004) Dopamine and drug addiction: the nucleus accumbens shell connection. Neuropharmacology 47 Suppl 1: 227-241. Emamian ES, Hall D, Birnbaum MJ, Karayiorgou M, Gogos JA (2004) Convergent evidence for impaired AKT1-GSK3beta signaling in schizophrenia. Nat Genet 36: 131-137. Friedman E, Yadin E, Wang HY (1996) Effect of prenatal cocaine on dopamine receptor-G protein coupling in mesocortical regions of the rabbit brain. Neuroscience 70: 739-747. 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. Ganguly K, Schinder AF, Wong ST, Poo M (2001) GABA itself promotes the developmental switch of neuronal GABAergic responses from excitation to inhibition. Cell 105: 521-532. Ikeda Y, Nishiyama N, Saito H, Katsuki H (1997) GABAA receptor stimulation promotes survival of embryonic rat striatal neurons in culture. Brain Res Dev Brain Res 98: 253-258. Jarolimek W, Lewen A, Misgeld U (1999) A furosemide-sensitive K+-Cl- cotransporter counteracts intracellular Cl- accumulation and depletion in cultured rat midbrain neurons. J Neurosci 19: 4695-4704. Jeffery KJ (2003) The neurobiology of spatial behaviour. Oxford: Oxford University Press. xxxi, 316 p. p. Jones LB, Stanwood GD, Reinoso BS, Washington RA, Wang HY, et al. (2000) In utero cocaine-induced dysfunction of dopamine D1 receptor signaling and abnormal differentiation of cerebral cortical neurons. J Neurosci 20: 4606-4614. Jones S, Bonci A (2005) Synaptic plasticity and drug addiction. Curr Opin Pharmacol 5: 20-25. Kakazu Y, Akaike N, Komiyama S, Nabekura J (1999) Regulation of intracellular chloride by cotransporters in developing lateral superior olive neurons. J Neurosci 19: 2843-2851. Kalivas PW (2007) Cocaine and amphetamine-like psychostimulants: neurocircuitry and glutamate neuroplasticity. Dialogues Clin Neurosci 9: 389-397. Kalivas PW, Pierce RC, Cornish J, Sorg BA (1998) A role for sensitization in craving and relapse in cocaine addiction. Journal of Psychopharmacology 12: 49-53. Kalivas PW, Stewart J (1991) Dopamine transmission in the initiation and expression of drug- and stress-induced sensitization of motor activity. Brain Res Brain Res Rev 16: 223-244. Keller RW, Jr., Snyder-Keller A (2000) Prenatal cocaine exposure. Ann N Y Acad Sci 909: 217-232. Khazipov R, Leinekugel X, Khalilov I, Gaiarsa JL, Ben-Ari Y (1997) Synchronization of GABAergic interneuronal network in CA3 subfield of neonatal rat hippocampal slices. J Physiol 498 ( Pt 3): 763-772. Kirsch J, Betz H (1998) Glycine-receptor activation is required for receptor clustering in spinal neurons. Nature 392: 717-720. Kneussel M, Betz H (2000) Clustering of inhibitory neurotransmitter receptors at developing postsynaptic sites: the membrane activation model. Trends Neurosci 23: 429-435. Koya E, Uejima JL, Wihbey KA, Bossert JM, Hope BT, et al. (2009) Role of ventral medial prefrontal cortex in incubation of cocaine craving. Neuropharmacology 56: 177-185. Le AD, Funk D, Harding S, Juzytsch W, Li Z, et al. (2008) Intra-median raphe nucleus (MRN) infusions of muscimol, a GABA-A receptor agonist, reinstate alcohol seeking in rats: role of impulsivity and reward. Psychopharmacology (Berl) 195: 605-615. Leinekugel X, Tseeb V, Ben-Ari Y, Bregestovski P (1995) Synaptic GABAA activation induces Ca2+ rise in pyramidal cells and interneurons from rat neonatal hippocampal slices. J Physiol 487 ( Pt 2): 319-329. Little JZ, Teyler TJ (1996) Prenatal cocaine exposure leads to enhanced long-term potentiation in region CA1 of hippocampus. Brain Res Dev Brain Res 92: 117-119. Liu J, Jordan LM (2005) Stimulation of the parapyramidal region of the neonatal rat brain stem produces locomotor-like activity involving spinal 5-HT7 and 5-HT2A receptors. J Neurophysiol 94: 1392-1404. Liu QS, Pu L, Poo MM (2005) Repeated cocaine exposure in vivo facilitates LTP induction in midbrain dopamine neurons. Nature 437: 1027-1031. LoTurco JJ, Owens DF, Heath MJ, Davis MB, Kriegstein AR (1995) GABA and glutamate depolarize cortical progenitor cells and inhibit DNA synthesis. Neuron 15: 1287-1298. 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 J, Karadsheh M, Delpire E (1999) Developmental regulation of the neuronal-specific isoform of K-Cl cotransporter KCC2 in postnatal rat brains. J Neurobiol 39: 558-568. Luhmann HJ, Prince DA (1991) Postnatal maturation of the GABAergic system in rat neocortex. J Neurophysiol 65: 247-263. 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. Mangiavacchi S, Wolf ME (2004) D1 dopamine receptor stimulation increases the rate of AMPA receptor insertion onto the surface of cultured nucleus accumbens neurons through a pathway dependent on protein kinase A. J Neurochem 88: 1261-1271. Marty S, Berninger B, Carroll P, Thoenen H (1996) GABAergic stimulation regulates the phenotype of hippocampal interneurons through the regulation of brain-derived neurotrophic factor. Neuron 16: 565-570. Mueller AL, Taube JS, Schwartzkroin PA (1984) Development of hyperpolarizing inhibitory postsynaptic potentials and hyperpolarizing response to gamma-aminobutyric acid in rabbit hippocampus studied in vitro. J Neurosci 4: 860-867. Nestler EJ (2005) The neurobiology of cocaine addiction. Sci Pract Perspect 3: 4-10. Obrietan K, van den Pol AN (1995) GABA neurotransmission in the hypothalamus: developmental reversal from Ca2+ elevating to depressing. J Neurosci 15: 5065-5077. Owens DF, Boyce LH, Davis MB, Kriegstein AR (1996) Excitatory GABA responses in embryonic and neonatal cortical slices demonstrated by gramicidin perforated-patch recordings and calcium imaging. J Neurosci 16: 6414-6423. Payne JA, Rivera C, Voipio J, Kaila K (2003) Cation-chloride co-transporters in neuronal communication, development and trauma. Trends Neurosci 26: 199-206. Pistis M, Perra S, Pillolla G, Melis M, Muntoni AL, et al. (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. Plotkin MD, Snyder EY, Hebert SC, Delpire E (1997) Expression of the Na-K-2Cl cotransporter is developmentally regulated in postnatal rat brains: a possible mechanism underlying GABA's excitatory role in immature brain. J Neurobiol 33: 781-795. Reichling DB, Kyrozis A, Wang J, MacDermott AB (1994) Mechanisms of GABA and glycine depolarization-induced calcium transients in rat dorsal horn neurons. J Physiol 476: 411-421. Rivera C, Li H, Thomas-Crusells J, Lahtinen H, Viitanen T, et al. (2002) BDNF-induced TrkB activation down-regulates the K+-Cl- cotransporter KCC2 and impairs neuronal Cl- extrusion. J Cell Biol 159: 747-752. Robinson TE, Berridge KC (1993) The neural basis of drug craving: an incentive-sensitization theory of addiction. Brain Res Brain Res Rev 18: 247-291. Robinson TE, Berridge KC (2003) Addiction. Annu Rev Psychol 54: 25-53. Salisbury AL, Lester BM, Seifer R, Lagasse L, Bauer CR, et al. (2007) Prenatal cocaine use and maternal depression: effects on infant neurobehavior. Neurotoxicol Teratol 29: 331-340. Shankaran S, Lester BM, Das A, Bauer CR, Bada HS, et al. (2007) Impact of maternal substance use during pregnancy on childhood outcome. Semin Fetal Neonatal Med 12: 143-150. Steketee JD, Kalivas PW (2011) Drug wanting: behavioral sensitization and relapse to drug-seeking behavior. Pharmacol Rev 63: 348-365. Svenningsson P, Fienberg AA, Allen PB, Moine CL, Lindskog M, et al. (2000) Dopamine D(1) receptor-induced gene transcription is modulated by DARPP-32. J Neurochem 75: 248-257. Thompson BL, Levitt P, Stanwood GD (2005) Prenatal cocaine exposure specifically alters spontaneous alternation behavior. Behav Brain Res 164: 107-116. Uslaner J, Badiani A, Day HE, Watson SJ, Akil H, et al. (2001) Environmental context modulates the ability of cocaine and amphetamine to induce c-fos mRNA expression in the neocortex, caudate nucleus, and nucleus accumbens. Brain Res 920: 106-116. Vanderschuren LJ, Kalivas PW (2000) Alterations in dopaminergic and glutamatergic transmission in the induction and expression of behavioral sensitization: a critical review of preclinical studies. Psychopharmacology (Berl) 151: 99-120. Vezina P (1996) D1 dopamine receptor activation is necessary for the induction of sensitization by amphetamine in the ventral tegmental area. J Neurosci 16: 2411-2420. Vu TQ, Payne JA, Copenhagen DR (2000) Localization and developmental expression patterns of the neuronal K-Cl cotransporter (KCC2) in the rat retina. J Neurosci 20: 1414-1423. Walters CL, Kuo YC, Blendy JA (2003) Differential distribution of CREB in the mesolimbic dopamine reward pathway. Journal of Neurochemistry 87: 1237-1244. Wang HY, Runyan S, Yadin E, Friedman E (1995) Prenatal exposure to cocaine selectively reduces D1 dopamine receptor-mediated activation of striatal Gs proteins. J Pharmacol Exp Ther 273: 492-498. Wang J, Reichling DB, Kyrozis A, MacDermott AB (1994) Developmental loss of GABA- and glycine-induced depolarization and Ca2+ transients in embryonic rat dorsal horn neurons in culture. Eur J Neurosci 6: 1275-1280. Yuste R, Katz LC (1991) Control of postsynaptic Ca2+ influx in developing neocortex by excitatory and inhibitory neurotransmitters. Neuron 6: 333-344. Zhen X, Torres C, Wang HY, Friedman E (2001) Prenatal exposure to cocaine disrupts D1A dopamine receptor function via selective inhibition of protein phosphatase 1 pathway in rabbit frontal cortex. J Neurosci 21: 9160-9167. Zuckerman B, Frank DA, Hingson R, Amaro H, Levenson SM, et al. (1989) Effects of maternal marijuana and cocaine use on fetal growth. N Engl J Med 320: 762-768. |
電子全文 Fulltext |
本電子全文僅授權使用者為學術研究之目的,進行個人非營利性質之檢索、閱讀、列印。請遵守中華民國著作權法之相關規定,切勿任意重製、散佈、改作、轉貼、播送,以免觸法。 論文使用權限 Thesis access permission:自定論文開放時間 user define 開放時間 Available: 校內 Campus: 已公開 available 校外 Off-campus: 已公開 available |
紙本論文 Printed copies |
紙本論文的公開資訊在102學年度以後相對較為完整。如果需要查詢101學年度以前的紙本論文公開資訊,請聯繫圖資處紙本論文服務櫃台。如有不便之處敬請見諒。 開放時間 available 已公開 available |
QR Code |