大鼠海马和杏仁体记忆相关受体和基因的研究

【作者】 张雪寒；

【导师】 李葆明；

【作者基本信息】 复旦大学 ， 神经生物学， 2005， 博士

【摘要】 NMDA受体在突触可塑性中发挥重要作用，长时程增强(LTP)被认为是学习记忆的突触机制。在许多脑区，如杏仁体和海马，LTP的诱导需要NMDA受体的激活。尤其是在海马CA1区，NMDA受体的NR2A亚基参与LTP的诱导，而NR2B亚基参与LTD的诱导。然而，NR2A和NR2B在学习记忆中的作用，知之甚少。条件性恐惧是研究学习记忆非常好的行为模型。杏仁体基底外侧核(BLA)是产生条件性恐惧，及发生与其相关的突触可塑性的关键位点。本工作中，我们分别研究了BLA和CA1区NR2A和NR2B在条件性恐惧记忆中的作用。1)我们在BLA局部注射NR2A或NR2B选择性拮抗剂，及使用基因递送技术抑制BLA的NR2A和NR2B功能。结果显示，BLA的NR2A和NR2B均参与恐惧记忆的获得，但不参与恐惧记忆的表达。2)我们在CA1区局部注射NR2A或NR2B选择性拮抗剂，观察对海马依赖的场景恐惧记忆的影响。结果显示，CA1区NR2A参与场景恐惧记忆的获得，而NR2B不参与；NR2A和NR2B均参与场景恐惧记忆的表达。学习记忆是脑的基本功能，许多基因与这一功能相关。我们利用行为学方法，筛选到新基因hippyragranin(HGN)和已知在外周系统具有抗增殖作用的基因Tob。Tob(Transducer of ErbB2)是一种细胞周期的抑制性调控因子，在外周组织发挥抑制细胞增殖的作用，而在中枢神经系统的功能不明确。使用行为学模型筛选的方法，我们发现Tob在脑内有表达，并且可能参与学习记忆过程。行为训练可以导致脑内Tob蛋白表达量的一过性升高，而且这一表达升高发生在长时记忆形成之前。在大鼠海马CA1区注射Tob蛋白的反义核酸下调Tob蛋白的表达，大鼠在Morris水迷宫行为任务中的空间学习和记忆及场景恐惧行为任务中的长时程记忆均受到伤害。另外，在CA1区局部注射Tob反义核酸，会抑制海马长时程增强。因此，这些结果表明，细胞周期的抑制性调控因子Tob是一种参与海马学习和记忆的多功能蛋白。记忆的形成、保持和读取是一个动态过程，反映的是新记忆形成和旧记忆被压抑(或清除)两个方面结合的一个结果。关于新记忆的形成已了解很多，而对参与记忆压抑(或清除)功能的分子成分和分子过程了解很少。我们鉴定出一个新的蛋白——HGN，它在海马有表达，海马的去神经支配可以降低其表达。用HGN的反义核酸下调大鼠海马CA1区HGN的表达，增强大鼠在Morris水迷宫行为任务中的学习和记忆能力，同时也能增强突触传递的长时程增强(LTP)。这些结果表明，HGN可能参与了记忆的抑制性调控。杏仁体、海马等脑区广泛接受来自蓝斑的去甲肾上腺素能投射。已知，杏仁体β受体参与情绪唤起经验相关的记忆巩固。在离体海马脑片上，激活海马CA1区β肾上腺素能受体(β受体)易化突触传递的长时程增强(LTP)。提示，CA1区β受体在海马依赖的学习和记忆中可能发挥重要作用。然而，CA1区β受体是否参与海马依赖性的学习记忆，是否参与在体LTP的诱导，尚无实验证据。这里，我们考察了CA1区β受体对在体突触可塑性的调控作用，以及对Morris水迷宫空间学习和记忆巩固的影响。在体电生理实验中，激活CA1区β受体后，正常情况下对突触传递效能仅有微小调控作用的10Hz的θ节律刺激(每串150个脉冲，1串)可显著地诱导出LTP；阻断CA1区β受体，正常情况下能显著诱导LTP的5Hz的θ节律刺激(每串150个脉冲，3串)，此时不能诱导出LTP。在Morris水迷宫实验中，训练前阻断CA1区β受体，大鼠的学习速度显著慢于对照组；训练后5分钟阻断CA1区β受体，损害48小时的空间记忆，而训练后6小时阻断CA1区β受体，对48小时空间记忆没有影响。以上结果显示，CA1区β受体参与对在体LTP的调控，参与海马依赖的空间学习和记忆。更多还原

【Abstract】 NMDA receptor plays a vital role in synaptic plasticity. Long-term potentiation (LTP), known as a synaptic mechanism of learning and memory, requires the activation of NMDA receptor in many regions, such as amygdala and hippocampus. In particular, it is suggested that NR2A subtype is involved in LTP induction, whereas NR2B in LTD induction in CA1 region. However, the roles of NR2A and NR2B subunits in memory are not well understood. Here, we investigated the effects of NR2A and NR2B subunits in amygdala and hippocampus on fear memory, respectively.1) We investigated the effects on fear memory of intra-BLA infusion of selective antagonist to NR2A or NR2B, or inhibiting the functions of NR2A or NR2B in BLA with gene delivery technology. Our results demonstrate that, both NR2A and NR2B in BLA are involved in acquisition of fear memory, but not in expression of the memory.2) We investigated the effects on fear memory of intra-CA1 of infusion of elective antagonist to NR2A or NR2B, a hippocampus-dependent task. Our data demonstrate that NR2A in area CA1 is required for both acquisition and expression of contextual fear memory, whereas NR2B is not important for acquisition, but is required for expression, of the memory.Tob (transducer of ErbB2) is a negative cell cycle regulator with anti-proliferative activity in the periphery. Using a behavioral screening paradigm to look for novel gene functions in the brain, we identified Tob as a brain-expressed protein involved in learning and memory. Behavioral training of fear-conditioning triggered a transient elevation of Tob protein, which preceded the formation of long-term memory. Functional perturbation of Tob by intra-CA1 infusion of antisense oligonucleotides in rats impaired spatial learning and memory in the Morris water maze and long-term memory for contextual fear conditioning, two behavioral paradigms that require the hippocampus. Furthermore, long-term potentiation was suppressed by Tob antisense infusion into the CA1 region. Together, these results indicate that the negative cell cycle regulator Tob is a multifunctional protein involved in hippocampus-dependent learning and memory.Memory formation, maintenance and retrieval are a dynamic process, reflecting a combined outcome of new memory formation on one hand, and older memory suppression/clearance on the other. Although much knowledge has been gained regarding new memory formation, less is known about the molecular components and processes that serve the function of memory suppression/clearance. Here we report the identification of a novel protein, termed hippyragranin (HGN), that is expressed in the rat hippocampus and its expression is reduced by hippocampal denervation. Inhibition of HGN by antisense oligonucleotide in area CA1 results in enhanced performance in Morris water maze, as well as elevated long-term potentiation (LTP). These results suggest that HGN is involved in negative memory regulator.Previous studies showed that β-adrenoceptors in amygdala are involved in the memory consolidation for emotional arousing experience and the activation of β-adrenoceptors in hippocampal CA1 region facilitates in vitro long-term potentiation (LTP). It is unclear if hippocampus-dependent learning is subjected to β-adrenergic regulation. Here, we studied roles of β-adrenoceptors in area CA1 in spatial learning and memory consolidation, and in synaptic plasticity in vivo.In Morris water maze experiment, pretraining blockade of β-adrenoceptors in area CA1, impaired spatial learning; 5-min, but not 6-h postraining blockade impaired 48-h spatial memory in rats, In electrophysiological experiment, the theta-pulse stimulation (10 Hz, 150 pulses/train, 1 train), a frequency that weakly modifies synaptic strength, induced a robust LTP when p-adrenoceptors in area CA1 were activated. By contrast, the theta-pulse stimulation (5 Hz, 150 pulses/train, 3 train), a frequency that vstrongly modifies synaptic strength, induced a significantly smaller LTP when β-adrenoceptors in area CA1 were blocked.Our results demonstrate that β-adrenoceptors in area CA1 are involved in regulating learning and memory in Morris water maze, and in vivo long-term potentioation.更多还原