海马CA1神经元对联合型恐惧记忆信息的兴奋性反应及沉默型突触去沉默化机制

发布时间：2018-02-07 14:20

本文关键词： 海马联合型学习沉默型突触记忆获取突触可塑性　出处：《华东师范大学》2016年博士论文　论文类型：学位论文

【摘要】：海马是学习与记忆的重要脑区,在记忆的获取和巩固中起到关键作用。目前的观点普遍认为,活动依赖性的神经可塑性是海马参与记忆活动的重要机制之一。过去数十年,人们对海马神经元的活动依赖性可塑性及其机制进行了系统研究,鉴定了一系列参与可塑性诱导及表达的分子和细胞机制,如膜上的NMDA受体、钙通道,胞内以CaMKII、PKC为代表的激酶系统,以及CREB等参与基因表达的分子在神经可塑性中的作用。除此之外,人们也观察到学习后海马神经元发生的一些可塑性变化,如神经元的膜兴奋性及树突形态的变化。然而,人们对于学习诱导的、与记忆编码直接相关的神经可塑性及机制知之甚少,了解这一问题对于理解海马的记忆功能至关重要。本论文中,我们主要在麻醉的成年大、小鼠上开展全细胞膜片钳记录,观察联合型学习前后海马CA1锥体细胞对感觉信息的处理及记忆编码的突触机制。我们发现：动物未进行学习时,仅少量(-30%)CA1锥体细胞能够对实验所选用的闪光刺激这一感觉信息产生明显的兴奋性或抑制性膜电位反应(兴奋性和抑制性反应的幅度分别为1.8±0.1 mV和-1.2±0.1 mV),其中部分兴奋性和抑制性反应的细胞能够对闪光刺激产生动作电位反应(比例分别为总细胞数的～9%和～7%)；进一步在清醒小鼠上获取的少量在体膜片钳记录数据也观察到一致的现象,即仅少量CA1细胞能对该闪光刺激产生反应。有趣的是,当以该闪光刺激作为联合型恐惧学习的条件刺激、训练动物建立联合型恐惧记忆之后,我们在麻醉动物上开展同样的电生理记录的实验显示：CA1锥体细胞对所记忆闪光刺激的反应比例在学习后的几天内得到大幅提高,学习当天为～70%,学习一天后该比例高达100%,这一比例在学习三天之后下降到～30%这一基础水平。并且,学习后三天内所出现的反应主要是兴奋性反应,且能有效诱发神经元动作电位发放。进一步的分析发现,学习后三天内CA1锥体细胞对闪光刺激的总体反应幅度、反应潜伏期与未学习组相比没有显著改变。此外,在双膜片钳记录的数据中,我们发现联合型学习一天后,CA1锥体细胞间自发活动的同步性水平也显著上升,而后(学习五天后的观察)、该同步性程度降为未学习水平。我们进一步对学习在CA1锥体细胞所诱发的兴奋性反应的机制进行了研究。课题组已有研究发现,在未学习时,对闪光刺激不发生反应的CA1细胞具备能潜在反应的沉默型突触(一类突触后只有NMDA受体而不含AMPA受体的谷氨酸能突触),这些神经元能在细胞去极化的情况下对闪光刺激产生兴奋性反应。因此,我们猜测该类沉默型突触的去沉默化可能是学习诱导的CA1兴奋性反应的原因。为验证这一猜想,我们首先采用CA1特异性NMDA受体亚基基因敲除小鼠进行研究,结果发现敲除鼠无法在训练后获取对闪光刺激的联合型记忆。与此对应,训练不能在CA1细胞诱发出兴奋性反应。这一结果说明,我们在正常动物观察到的、学习诱导的CA1兴奋性反应依赖于CA1自身的NMDA受体活动,也即源于CA1内部的突触可塑性修饰。因此,我们在未学习动物所观察到的、CA1神经元中与闪光刺激密切相关的沉默型突触可推测为主要的突触修饰位点,即该突触发生了去沉默化的突触增强修饰、导致兴奋性反应在大量的CA1细胞中出现。我们接下来检验学习后短期内CA1细胞是否仍存在沉默型突触介导的对闪光刺激的反应。结果显示,学习一天后,CA1锥体细胞对闪光刺激的反应潜伏期和时间特性在膜电位被去极化钳制或接近静息电位钳制两种条件下非常类似。此外,学习前后CA1锥体细胞在去极化钳制条件下对闪光刺激的平均反应强度类似。这些数据表明学习后CA1锥体细胞对闪光刺激的反应不包含沉默型突触的潜在活动,进一步说明学习前的相关沉默型突触发生了去沉默化。本论文的研究总结如下：联合型恐惧学习后,大量CA1锥体细胞能编码所记忆的感觉信息并产生兴奋性反应,在这些细胞中、沉默型突触的去沉默化机制很可能是海马记忆存储的一个重要突触机制。
[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.

【学位授予单位】：华东师范大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R338

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