Molecular processes underlying hippocampal LTP

海马 LTP 的分子过程

• 批准号: 7021161
• 负责人: YASUNORI HAYASHI
• 金额: $ 28.7万
• 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/7021161
• 关键词: actins; bioimaging /biomedical imaging; calmodulin dependent protein kinase; confocal scanning microscopy; conformation; enzyme activity; enzyme structure; fluorescence resonance energy transfer; genetic manipulation; genetically modified animals; green fluorescent proteins; hippocampus; laboratory mouse; laboratory rat; long term potentiation; neural plasticity; neuroimaging; neurons; optics; polymerization; synapses; technology /technique development; tissue /cell culture

DESCRIPTION (provided by applicant): 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 announcement "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 synaptic 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 that it follows LTP and LTD respectively. 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 plasticity with spatial resolution at the single synapse level on a sub-second time scale. This technique, in combination with technologies currently under development in other laboratories, such as in vivo two-photon imaging 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. It may be possible to have monkeys perform a task and observe synaptic plasticity in the brain during learning of the task.

描述（由申请人提供）：已建议突触可塑性是学习和记忆的蜂窝塑料。 但是，在给定的神经元网络中，在给定的内存范式上实际经历突触可塑性实际在视觉上监测突触实际上是不可能的。 该项目提案是针对计划公告的回应，“开发了绘制功能性神经元电路和突触变化的新型遗传方法”，描述了一种可视化生物动物神经元突触可塑性的技术的开发。 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。 我们将首先开发一种基于FRET的构建体，用于光学测量CAMKII活性和肌动蛋白聚合/去聚合平衡，这将允许对完整神经元中CAMKII激活的非破坏性光学检测。 由于在诱导NMDA受体介导的突触可塑性之后，CAMKII的酶活性会增强，并且这种活性是维持突触增强的，因此我们希望CAMKII的激活将是突触可变性的良好指标。 相反，在我们发表的一项工作中，我们发现肌动蛋白聚合/解聚平衡可以用FRET检测到分别遵循LTP和LTD。 为了在突触结构上检测FRET，我们将利用两光子显微镜。 然后，我们将通过表达神经元中的构建体并通过高速离子摩托刺激单个突触或局部电刺激或与结构可塑性结合结合来测试策略的可行性。 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。 将在这种结构中诱导突触可塑性（例如感觉剥夺）的范式测试，以查看突触可塑性是否由FRET反映。 总而言之，我们的技术将提供一个独特的系统，用于检测NMDAR介导的突触可塑性，并在单一时间尺度上以单个突触水平的空间分辨率进行空间分辨率。 该技术结合当前正在开发的其他实验室中的技术，例如自由移动的动物中的体内两光子成像和深层结构的两光子显微镜成像，将使用继电器镜头，可提供一种用于监视突触的多功能系统现有实验系统无法实现的可塑性。 将来，在病毒或转基因引入我们的记者构造之后，该技术可以应用于高级哺乳动物，例如猕猴。 在学习任务时，可能会让猴子执行任务并观察大脑中的突触可塑性。