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標題: | 血清張力素在大腦皮質底板神經細胞早期發育過程中所扮演的角色 The roles of serotonin in developing subplate neurons |
作者: | Chun-Chieh Liao 廖俊傑 |
指導教授: | 李立仁(Li-Jen Lee) |
關鍵字: | 細胞膜特性,突觸傳遞,樹突,發育,抗憂鬱藥物, Membrane properties,Synaptic transmission,Dendrite,Development,Antidepressant, |
出版年 : | 2011 |
學位: | 博士 |
摘要: | 血清張力素 (serotonin, 5-HT)在神經系統發育過程中扮演著重要的角色,若腦中5-HT系統異常,將會使大腦發育異常,甚至導致終生的行為及心理缺陷;然而懷孕及產後婦女常服用的SSRI(selective serotonin reuptake inhibitor) 類抗憂鬱藥物,卻會使得神經系統內的5-HT系統失衡,引發後續的負面影響。本研究旨在探討SSRI類抗憂鬱藥物暴露,是如何影響大腦皮質發育,是否會影響大腦皮質發育中最早出現的一群細胞-底板神經細胞(subplate neuron,SPn);若會影響,是否在自然狀況下5-HT就會作用在底板神經細胞上,因而5-HT失衡導致更多後續發育上的問題。
底板神經細胞(subplate neuron, SPn)在體感覺皮質區(somatosensory cortex)發育過程中,扮演視丘及皮質層間的視丘-皮質徑(thalamocortical afferents, TCAs)連接形成的中繼轉接站,而當視丘和皮質第四層神經細胞間的TCAs連結形成之後,SPn跟視丘間的連結隨即消弭;然而,SPn在出生後的成熟過程及削減過程,卻仍未有完整的研究報告。因此,在我第一部份實驗,旨在探討SPn在發育早期活動力降低的機制;因TCAs發育及投射到大腦皮質的時間大約在出生(P0表示出生當天)到出生後兩周內完成,因此我們檢測出生後兩周內大白鼠SPn的發育特性變化。研究結果發現,SPn由單一動作電位(single spike)轉為規律型連續(regular spiking)動作電位,而細胞膜特性則有漸趨成熟的連續趨勢且興奮性降低;而發育過程中TCAs投射到SPn的突觸傳遞paired-pulse ratio增加、MK-801阻斷速率減緩,則代表突觸前神經傳導物的釋放機率降低。在形態方面,SPn的細胞本體的大小隨發育增加且形狀變得較為扁平,樹突的總長度、分節點及分節片段數目在出生後第一週內隨發育增長,到出生後第十天達到最大值,隨後卻反而減少,代表有形態上的修剪。因此,由第一部份的發現我們認為,在TCAs建立過程當中,視丘和大腦皮質SPns間的功能性連結減弱及SPn本身的活動力降低,乃歸因於SPn興奮性降低、突觸傳遞減少及樹突複雜度下降這三方面所致。 在TCAs發育及SPns的成熟過程中,縫核(raphe nuclei)的5-HT生成細胞也同時投射到大腦皮質,影響大腦皮質的發育,因此5-HT在神經系統發育過程中扮演著重要的角色;然而,罹有憂鬱症懷孕或產後的婦女經常會服用SSRI(selective serotonin reuptake inhibitor) 類抗憂鬱藥物,會擾亂腦內5-HT系統的平衡,而她們所懷的胎兒或哺乳的嬰兒也會暴露在該種藥物的影響之下。因此,第二部分的實驗將探討,是否在大腦發育關鍵期暴露到SSRI,會影響幼體腦部發育,特別是較為敏感的SPn呢?因此我們把新生小鼠從P0到P4,相當於人類第三懷孕期,皮下注射百憂解prozac (fluoxetine,一種SSRI) ,然後觀察SPn的生理及形態特性是否受到影響;運用全細胞膜片箝制(whole-cell patch clamp)技術,我們測試SPn的細胞膜物理特性及動作電位特性是否受到影響。在fluoxetine處理過的小鼠,要誘導SPn產生動作電位的最小電流rheobase增加、動作電位寬度減小,代表其興奮性降低;在形態方面,早期fluoxetine處理會影響SPn樹突在正常發育過程中的重塑過程,包括樹突產生分岔、生長及修剪都發生異常。總言之,第二部份的結果顯示早期SSRI抗憂鬱藥物的暴露,會造成發育中的SPn構造及功能的異常,這可能是影響往後腦功能及正常行為的原因之一。 既然早期SSRI抗憂鬱藥物的暴露,會對發育中的造成SPn構造及功能的異常,是否代表5-HT在自然狀況下就會影響SPn的興奮性呢? 5-HT在神經發育過程中會隨同TCAs投射到大腦皮質,且5-HT回收蛋白(5-HT transporter)在發育過程中曾暫時性地表現在TCAs上,意謂早期大腦皮質神經細胞胞外有一定量的5-HT,且影響發育中的TCAs及SPns。因此,第三部分實驗我們以新生大白鼠的體感覺皮質區腦片為材料,以全細胞膜片箝制技術記錄觀察,驗證是否5-HT會對SPn的生理特性,有立即性的影響。在不同濃度的5-HT實驗中,SPn的內在生理特性並不受影響,然而,以人工刺激方式誘導產生的興奮性突觸電流卻會隨著5-HT濃度增加而呈現濃度依賴的抑制效果:5-HT濃度越高,抑制效果越明顯;然而我們以人工施加AMPA在SPn誘導產生的興奮性電流卻不受影響,因此,5-HT的抑制效果應該不是作用在突觸後的SPn上;此外,5-HT會使TCAs-SPn突觸連結的paired-pulse ratio增加及MK-801的阻斷速率減緩,代表5-HT對該突觸的抑制效果是作用在突觸前的位置上。接著我們利用5-HT受體的促效劑(agonist)及拮抗劑(antagonist)來驗證是哪一種受體引起的抑制作用,結果5-HT1B的受體促效劑CP93129具有類似的抑制效果,且它的拮抗劑SB224289則可以阻斷該抑制效果的發生。因此我們可以確認,5-HT對於新生小鼠體感覺皮質區,視丘皮質徑跟SPn間的興奮性突觸連結抑制效果,是藉由活化突觸前的5-HT1B受體所產生的。 因此,從這三個實驗我們可以推論,SPn在出生後早期仍未完全成熟且有形態上的重塑,而5-HT在此時期會以活化突觸前5-HT1B受體的方式,抑制視丘皮質神經路徑跟SPn間的興奮性突觸連結;但若在此發育關鍵期暴露到SSRI類抗憂鬱藥物,會因5-HT濃度失衡而使SPn在形態及生理上發生異常,因而使SPn相關的發育過程甚至是成年後的神經系統及心理狀態受到影響。 Serotonin (5-HT) plays important roles during neural development. Perturbation of the 5-HT system during early period might end up with life-long defects, such as altered neuronal morphology, serotonin circuitry, and behaviors. Seeing that the 5-HT fibers are present in the subplate and subplate neurons (SPns) play a pivotal role in the developing cortex, yet the roles of 5-HT on the developing SPn is totally unknown. I would like to investigate the developmental history of SPns and elucidate the role of 5-HT on SPn in somatosensory cortex of neonatal rats. Firstly, we studied the structural and functional changes of SPns in developing rat barrel cortex. Second, we verified whether neonatal antidepressant exposure, which disrupts the 5-HT system, has effects on developing SPns. Third, we investigated the effects of exceeding 5-HT level on the physiological properties of SPn and thalamocortical synaptic transmission upon these neurons. SPns are the earliest cells in the neocortex and are critical for the establishment of brain circuitry. SPns could serve as a transient relay station between the thalamus and cortical plate and assists the formation of thalamocortical projection. While the thalamus-layer IV connection is formed, the thalamic activation of subplate is diminished. In the first part of study, we aimed to explore the mechanism which may attribute to the decline of subplate activity. To resolve this issue, the developmental changes of SPns in rat somatosensory cortex were examined during the first two postnatal weeks which covers the stages prior and subsequent to the establishment of thalamocortical connection. During development, the excitability of SPns decreased as revealed by increasing rheobase and rightwardly shifted frequency-current curves. Besides, increasing paired-pulse ratio and slowing MK-801 blocking rate were noted during development, implying the reduction of presynaptic transmitter release. Morphologically, the total length, branching nodes and segments of dendrites increased significantly during the first week. However, after peaking around day 10, these values decreased, implying a pruning process. Thus, the reduction of neuronal excitability, synaptic transmission and dendritic complexity may attribute to the decline of functional connectivity between thalamus and subplate and reduction of subplate activity while the thalamocortical pathway is established. In the second part, we treated the neonatal rat pups with fluoxetine (Flx), a kind of selective serotonin reuptake inhibitor (SSRI)-type antidepressant, from the day of birth to postnatal day 4 and observed the physiological and morphological features of SPns. In SPns of Flx-treated rats, the rheobase for generating an AP was increased and the width of APs was reduced, especially in the falling phase. In the morphological aspect, the dendritic remodeling of SPns including dendritic branching, elongation and pruning were affected by early Flx treatment. Together, early SSRI exposure has teratogenic effects on the structure and function of developing SPns and these changes may lead to undesired brain activity and distorted behaviors later in life. Seeing that early SSRI exposure affected the normal development of SPns, we studied the effects of 5-HT on the electrophysiological properties of neonatal SPns in the third part. 5-HT did not affect the intrinsic properties of SPns. However, thalamus-evoked excitatory postsynaptic currents (EPSCs) to SPn was significantly suppressed by 5-HT in a dose-dependent manner. Because 5-HT did not affect AMPA-induced EPSC, postsynaptic effect of 5-HT on reducing EPSCs is unlikely. Increased paired-pulse ratio and decreased MK-801 blocking rate indicated the presence of presynaptic 5-HT receptor-mediated suppressive effect in the thalamus-subplate synapses. To elucidate the type of 5-HT receptor involved in this process, 5-HT receptor agonists and antagonists were tested. Our results showed that 5-HT1B receptor agonist CP93129 mimicked the effect of 5-HT and only 5-HT1B receptor antagonist SB224289 prevented 5-HT-mediated synaptic suppression. Taken together, our data demonstrated the presynaptic 5-HT1B receptor-mediated suppressive effect on the excitatory synapses between TCAs and SPns in the somatosensory cortex of neonatal rats. As a whole, the present works demonstrate that during early postnatal days SPns are not fully mature and are vulnerable to neonatal antidepressant exposure. Since 5-HT plays roles in adjusting the excitation-inhibition balance of SPns by 5-HT1B mechanisms and neonatal antidepressant exposure disrupts homeostasis of the 5-HT system, maturation of SPns and consequential cortical development are interfered. Usage of these drugs in pregnant women should be concerned. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/65795 |
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顯示於系所單位: | 解剖學暨細胞生物學科所 |
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