Optical detection of the molecular processes underlying hippocampal LTP

海马 LTP 分子过程的光学检测

• 批准号: 7380015
• 负责人: YASUNORI HAYASHI
• 金额: $ 27.48万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7380015
• 关键词: Actins; Aging; Animals; Biological Models; Brain; Brain region; Cerebrovascular Circulation; Chemosensitization; Chromosome Pairing; Depth; Detection; Development; Disruption; Electric Stimulation; Equilibrium; Event; Fluorescence Resonance Energy Transfer; Future; Generations; Genes; Genetic; Goals; Hippocampus (Brain); Image; In Vitro; Individual; Invasive; Laboratories; Laser Scanning Microscopy; Learning; Life; Localized; Macaca; Mammals; Maps; Measures; Mediating; Memory; Methods; Microscope; Molecular; Monitor; Monkeys; N-Methyl-D-Aspartate Receptors; Neurons; Optics; Process; Publishing; Range; Reporter; Resolution; Sensory Deprivation; Series; Speed; Structure; Synapses; Synaptic plasticity; System; Techniques; Technology; Testing; Time; Transgenic Animals; Transgenic Mice; Transgenic Organisms; Validation; Viral; Work; Writing; barrel cortex; base; calmodulin-dependent protein kinase II; depolymerization; in vivo; lens; millisecond; neural circuit; novel; polymerization; programs; response; two-photon

Synaptic plasticity has been suggested to be a cellular counterpart for learning and memory. However, it is practically impossible to visually monitor synapses actually undergoing synaptic plasticity at a given memory paradigm in a given neuronal network. This project proposal, written in response to the program announce- ment "Developing Novel Genetic Methods For Mapping Functional Neuronal Circuits and Synaptic Change", describes the development of a technology for visualizing synapses that are undergoing synaptic plasticity in neurons in living animal. This goal will be accomplished by a combination of two technologies: fluorescence resonance energy transfer (FRET) and two-photon laser scanning microscopy. We will first develop a FRET-based construct for optically measuring the CaMKII activity and actin polymerization/depolymerization equilibrium, which will allow non-destructive optical detection of CaMKII activation in intact neurons. Since the enzymatic activity of CaMKII is constitutively enhanced after the induction of NMDA receptor-mediated syn- aptic plasticity and this activity is required for maintaining synaptic potentiation, we expect that the activation of CaMKII will be a good indicator of synaptic plasticity. In contrast, in a work which we published, we found actin polymerization/depolymerization equilibrium can be detected with FRET and it follow LTP and LTD respec- tively. To detect FRET at the synaptic structure, we will take advantage of a two-photon microscope. We will then test the feasibility of our strategy by expressing the construct in neurons and induce synaptic plasticity by either high-speed ionophoretic stimulation of individual synapses or local electrical stimulation or combined with detection of structural plasticity. Finally, we will generate a transgenic animal expressing this construct. The barrel cortex of the resultant animal will be observed with the two-photon microscope. Paradigms known to induce synaptic plasticity in this structure such as sensory deprivation will be tested to see whether synaptic plasticity is reflected by FRET. In summary, our technology will provide a unique system for detecting NMDAR-mediated synaptic plas- ticity with spatial resolution at the single synapse level on a sub-second time scale. This technique, in com- bination with technologies currently under development in other laboratories, such as in vivo two-photon im- aging in freely moving animals and deep-structure two-photon microscope imaging with a relay lens, will pro- vide a versatile system for monitoring synaptic plasticity that cannot be achieved with existing experimental systems. In the future, this technique could be applied to higher mammals, such as macaque monkeys, after viral or transgenic introduction of our reporter construct. Itmay be possible to have monkeys perform a task and observe synaptic plasticity in the brain during learning of the task.

已建议突触可塑性是学习和记忆的细胞。但是，是 几乎不可能在给定的内存下实际经历突触可塑性的视觉监视突触 给定神经元网络中的范式。该项目提案，响​​应该计划宣布 - “开发新的遗传方法来绘制功能性神经元电路和突触变化”，”， 描述了一种可视化正在经历突触可塑性的突触的技术的开发 活动物中的神经元。该目标将通过两种技术的组合来实现：荧光 共振能量转移（FRET）和两光子激光扫描显微镜。我们将首先发展一个 基于FRET的构造，用于光学测量CAMKII活性和肌动蛋白聚合/解聚化 平衡将允许对完整神经元中CaMKII激活的非破坏性光学检测。自从 NMDA受体介导的合成 - 维持突触增强需要的助理可塑性和此活动是必需的，我们希望激活的激活 CAMKII将是突触可塑性的良好指标。相反，在我们发表的作品中，我们发现了肌动蛋白 聚合/解聚平衡可以用FRET检测到LTP和LTD revescec- 蒂。为了在突触结构上检测FRET，我们将利用两光子显微镜。我们将 然后通过表达神经元中的构建体并诱导突触可塑性来测试我们策略的可行性 高速离子噬刺激单个突触或局部电刺激或合并 检测结构可塑性。最后，我们将产生一种表达这种结构的转基因动物。 两光子显微镜将观察到所得动物的枪管皮层。已知的范式 在这种结构中诱导突触可塑性，例如感觉剥夺，以查看突触是否是否 塑性反映了货物。 总而言之，我们的技术将提供一个独特的系统，用于检测NMDAR介导的突触插件 - 在单秒时尺度上在单个突触水平上具有空间分辨率的特性。这种技术， 与目前正在开发的其他实验室中开发的技术相结合，例如体内两光量 自由移动的动物和深层结构的两光子显微镜成像的老化，将衰老 视频使用现有实验性的多功能系统，用于监视突触可塑性 系统。将来，这种技术可以应用于高等哺乳动物，例如猕猴等高级哺乳动物 病毒或转基因引入我们的记者结构。可以让猴子执行任务 并在学习任务时观察大脑中的突触可塑性。