本文關(guān)鍵詞： 海馬 聯(lián)合型學(xué)習(xí) 沉默型突觸 記憶獲取 突觸可塑性　出處：《華東師范大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：海馬是學(xué)習(xí)與記憶的重要腦區(qū),在記憶的獲取和鞏固中起到關(guān)鍵作用。目前的觀點(diǎn)普遍認(rèn)為,活動依賴性的神經(jīng)可塑性是海馬參與記憶活動的重要機(jī)制之一。過去數(shù)十年,人們對海馬神經(jīng)元的活動依賴性可塑性及其機(jī)制進(jìn)行了系統(tǒng)研究,鑒定了一系列參與可塑性誘導(dǎo)及表達(dá)的分子和細(xì)胞機(jī)制,如膜上的NMDA受體、鈣通道,胞內(nèi)以CaMKII、PKC為代表的激酶系統(tǒng),以及CREB等參與基因表達(dá)的分子在神經(jīng)可塑性中的作用。除此之外,人們也觀察到學(xué)習(xí)后海馬神經(jīng)元發(fā)生的一些可塑性變化,如神經(jīng)元的膜興奮性及樹突形態(tài)的變化。然而,人們對于學(xué)習(xí)誘導(dǎo)的、與記憶編碼直接相關(guān)的神經(jīng)可塑性及機(jī)制知之甚少,了解這一問題對于理解海馬的記憶功能至關(guān)重要。本論文中,我們主要在麻醉的成年大、小鼠上開展全細(xì)胞膜片鉗記錄,觀察聯(lián)合型學(xué)習(xí)前后海馬CA1錐體細(xì)胞對感覺信息的處理及記憶編碼的突觸機(jī)制。我們發(fā)現(xiàn)：動物未進(jìn)行學(xué)習(xí)時(shí),僅少量(-30%)CA1錐體細(xì)胞能夠?qū)?shí)驗(yàn)所選用的閃光刺激這一感覺信息產(chǎn)生明顯的興奮性或抑制性膜電位反應(yīng)(興奮性和抑制性反應(yīng)的幅度分別為1.8±0.1 mV和-1.2±0.1 mV),其中部分興奮性和抑制性反應(yīng)的細(xì)胞能夠?qū)﹂W光刺激產(chǎn)生動作電位反應(yīng)(比例分別為總細(xì)胞數(shù)的～9%和～7%)；進(jìn)一步在清醒小鼠上獲取的少量在體膜片鉗記錄數(shù)據(jù)也觀察到一致的現(xiàn)象,即僅少量CA1細(xì)胞能對該閃光刺激產(chǎn)生反應(yīng)。有趣的是,當(dāng)以該閃光刺激作為聯(lián)合型恐懼學(xué)習(xí)的條件刺激、訓(xùn)練動物建立聯(lián)合型恐懼記憶之后,我們在麻醉動物上開展同樣的電生理記錄的實(shí)驗(yàn)顯示：CA1錐體細(xì)胞對所記憶閃光刺激的反應(yīng)比例在學(xué)習(xí)后的幾天內(nèi)得到大幅提高,學(xué)習(xí)當(dāng)天為～70%,學(xué)習(xí)一天后該比例高達(dá)100%,這一比例在學(xué)習(xí)三天之后下降到～30%這一基礎(chǔ)水平。并且,學(xué)習(xí)后三天內(nèi)所出現(xiàn)的反應(yīng)主要是興奮性反應(yīng),且能有效誘發(fā)神經(jīng)元動作電位發(fā)放。進(jìn)一步的分析發(fā)現(xiàn),學(xué)習(xí)后三天內(nèi)CA1錐體細(xì)胞對閃光刺激的總體反應(yīng)幅度、反應(yīng)潛伏期與未學(xué)習(xí)組相比沒有顯著改變。此外,在雙膜片鉗記錄的數(shù)據(jù)中,我們發(fā)現(xiàn)聯(lián)合型學(xué)習(xí)一天后,CA1錐體細(xì)胞間自發(fā)活動的同步性水平也顯著上升,而后(學(xué)習(xí)五天后的觀察)、該同步性程度降為未學(xué)習(xí)水平。我們進(jìn)一步對學(xué)習(xí)在CA1錐體細(xì)胞所誘發(fā)的興奮性反應(yīng)的機(jī)制進(jìn)行了研究。課題組已有研究發(fā)現(xiàn),在未學(xué)習(xí)時(shí),對閃光刺激不發(fā)生反應(yīng)的CA1細(xì)胞具備能潛在反應(yīng)的沉默型突觸(一類突觸后只有NMDA受體而不含AMPA受體的谷氨酸能突觸),這些神經(jīng)元能在細(xì)胞去極化的情況下對閃光刺激產(chǎn)生興奮性反應(yīng)。因此,我們猜測該類沉默型突觸的去沉默化可能是學(xué)習(xí)誘導(dǎo)的CA1興奮性反應(yīng)的原因。為驗(yàn)證這一猜想,我們首先采用CA1特異性NMDA受體亞基基因敲除小鼠進(jìn)行研究,結(jié)果發(fā)現(xiàn)敲除鼠無法在訓(xùn)練后獲取對閃光刺激的聯(lián)合型記憶。與此對應(yīng),訓(xùn)練不能在CA1細(xì)胞誘發(fā)出興奮性反應(yīng)。這一結(jié)果說明,我們在正常動物觀察到的、學(xué)習(xí)誘導(dǎo)的CA1興奮性反應(yīng)依賴于CA1自身的NMDA受體活動,也即源于CA1內(nèi)部的突觸可塑性修飾。因此,我們在未學(xué)習(xí)動物所觀察到的、CA1神經(jīng)元中與閃光刺激密切相關(guān)的沉默型突觸可推測為主要的突觸修飾位點(diǎn),即該突觸發(fā)生了去沉默化的突觸增強(qiáng)修飾、導(dǎo)致興奮性反應(yīng)在大量的CA1細(xì)胞中出現(xiàn)。我們接下來檢驗(yàn)學(xué)習(xí)后短期內(nèi)CA1細(xì)胞是否仍存在沉默型突觸介導(dǎo)的對閃光刺激的反應(yīng)。結(jié)果顯示,學(xué)習(xí)一天后,CA1錐體細(xì)胞對閃光刺激的反應(yīng)潛伏期和時(shí)間特性在膜電位被去極化鉗制或接近靜息電位鉗制兩種條件下非常類似。此外,學(xué)習(xí)前后CA1錐體細(xì)胞在去極化鉗制條件下對閃光刺激的平均反應(yīng)強(qiáng)度類似。這些數(shù)據(jù)表明學(xué)習(xí)后CA1錐體細(xì)胞對閃光刺激的反應(yīng)不包含沉默型突觸的潛在活動,進(jìn)一步說明學(xué)習(xí)前的相關(guān)沉默型突觸發(fā)生了去沉默化。本論文的研究總結(jié)如下：聯(lián)合型恐懼學(xué)習(xí)后,大量CA1錐體細(xì)胞能編碼所記憶的感覺信息并產(chǎn)生興奮性反應(yīng),在這些細(xì)胞中、沉默型突觸的去沉默化機(jī)制很可能是海馬記憶存儲的一個(gè)重要突觸機(jī)制。
[Abstract]:Hippocampus is an important brain area for learning and memory, the memory of the acquisition and consolidation plays a key role. The current view is generally believed that activity dependent neural plasticity is one of important mechanisms involved in hippocampal memory activities. In the past few decades, people of activity dependent plasticity of hippocampal neurons and its mechanism of system a series of research, induction and expression of plasticity in the molecular and cellular mechanisms were identified, such as the NMDA receptor on the membrane, calcium channels, intracellular CaMKII, PKC as the representative of the kinase system and CREB molecules involved in gene expression in neural plasticity. In addition, it is also observed some plastic changes of hippocampal neurons after learning, such as changes in neuronal excitability and dendritic morphology. However, the learning induced neural plasticity, and the mechanism is directly related to memory encoding Little is known about the understanding of this issue is crucial for understanding the hippocampal memory function. In this paper, we mainly in anesthetized adult, to carry out the whole cell patch clamp mice, observe the combined learning and memory encoding before and after the treatment of hippocampal CA1 pyramidal cells of sensory information to synaptic mechanisms. We found that animal without learning when only a small amount of (-30%) CA1 pyramidal cells can used in the flash stimulation of this sensory information have obvious inhibitory or excitatory membrane potential response (excitatory and inhibitory response amplitude were 1.8 + 0.1 and -1.2 + 0.1 mV mV), the excitatory and inhibitory responses of the part cells can generate action potentials in response to photic stimulation (% of total cell number ~ 9% and ~ 7%); further obtained in awake mice on the small body in the patch clamp data were also observed in uniform The phenomenon is only a small amount of CA1 cells can respond to the flash stimulation. Interestingly, when using the flash stimulation as a joint type of fear learning stimulus conditions, after the establishment of a joint training animal type fear memory, that we carry out the same electrophysiological recording in anesthetized animal experiments on CA1 pyramidal cells on the reaction ratio the memory of flash stimulation in learning a few days after the increase, learning the day to 70%, the proportion of learning a day up to 100%, this proportion dropped to 30% in three days after learning the basic level of learning. And, within three days after the reaction is mainly excitatory the reaction, and can effectively release evoked neuronal action potentials. Further analysis found that CA1 pyramidal cells within three days of the overall response amplitude of flash stimulation after learning the latency compared with the untreated group. This study did not change significantly Outside, in the double patch recording data, we found that the combined learning after a synchronization level of CA1 pyramidal cells between spontaneous activity also increased significantly, and then (after five days of learning, the observation of the degree of synchronization) reduced to learning level. We further study on excitatory responses evoked in CA1 pyramidal cells the mechanism was studied. The research group has been found, without learning, no reaction of flash stimulated CA1 cells can silence synaptic potential reactions (glutamic acid a postsynaptic NMDA receptor only without AMPA receptors and synaptic depolarization of these neurons), can in the cell under the condition of flash stimulation induced excitatory response. Therefore, we speculate that this kind of silent synapses to silence may be the reason why CA1 excitatory responses induced by learning. In order to verify this conjecture, we first used CA1 specific NMDA receptor subunit gene knockout mice were studied. The results showed that the knockout mice cannot get the memory of the combined flash stimulation after training. Correspondingly, the training can not be in CA1 cells induced by excitatory reaction. This result suggests that we observed in the normal animal, NMDA receptor activity learning induced by CA1 the excitatory response depends on the CA1 itself, namely the modification of synaptic plasticity is derived from CA1's interior. Therefore, we have to study animal observed, CA1 neurons and synapses are closely related to the silent flash stimulation can be speculated as the major synaptic modification sites, which happened to the silent synapse modification of excitatory synaptic potentiation, resulting in response to a large number of CA1 cells. Then we test the learning in a short time after CA1 cells are still silent synapses mediated to flash stimulation reaction. The results show that learning day After the CA1 pyramidal cell response latency and time characteristics of flash stimulation in the membrane potential was clamped depolarization or near the resting potential clamp under the two conditions are very similar. In addition, after the study of CA1 pyramidal cells in the depolarizing clamp under the condition of average intensity of reaction of flash stimulation is similar. These data demonstrate the potential of learning activities after the reaction of CA1 pyramidal cells to flash stimuli do not contain silent synapses, further explain silence synapses before learning happened to silence. The research of this thesis are summarized as follows: Joint fear learning, a large number of CA1 pyramidal cells encoding memory sensory information and generate excitatory responses in these cells, silence synapses silence mechanism is likely an important mechanism of synaptic hippocampal memory storage.

【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R338

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