Optical detection of the molecular processes underlying hippocampal LTP

海马 LTP 分子过程的光学检测

• 批准号: 7191766
• 负责人: YASUNORI HAYASHI
• 金额: $ 28.04万
• 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/7191766
• 关键词: Actins; 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; NIH Program Announcements; 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; response; two-photon

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）：突触可塑性已被认为是学习和记忆的细胞对应物。 然而，实际上不可能在给定的神经元网络中以给定的记忆范式直观地监视实际经历突触可塑性的突触。 该项目提案是为了响应项目公告“开发用于绘制功能神经元回路和突触变化的新型遗传方法”而编写的，描述了一种用于可视化活体动物神经元中正在经历突触可塑性的突触的技术的开发。 这一目标将通过两种技术的结合来实现：荧光共振能量转移（FRET）和双光子激光扫描显微镜。 我们将首先开发一种基于 FRET 的结构，用于光学测量 CaMKII 活性和肌动蛋白聚合/解聚平衡，这将允许对完整神经元中的 CaMKII 激活进行无损光学检测。 由于在诱导 NMDA 受体介导的突触可塑性后，CaMKII 的酶活性会持续增强，并且这种活性是维持突触增强所必需的，因此我们预计 CaMKII 的激活将是突触可塑性的良好指标。 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，并且它分别遵循 LTP 和 LTD。 为了检测突触结构处的 FRET，我们将利用双光子显微镜。 然后，我们将通过在神经元中表达该构建体并通过单个突触的高速离子电渗刺激或局部电刺激或与结构可塑性检测相结​​合来诱导突触可塑性来测试我们策略的可行性。 最后，我们将产生表达该构建体的转基因动物。 将用双光子显微镜观察所得动物的桶状皮层。 将测试已知在该结构中诱导突触可塑性的范式（例如感觉剥夺），以查看 FRET 是否反映了突触可塑性。 总之，我们的技术将提供一种独特的系统，用于检测 NMDAR 介导的突触可塑性，在亚秒时间尺度上在单个突触水平上具有空间分辨率。 该技术与其他实验室目前正在开发的技术相结合，例如自由活动动物的体内双光子成像和使用中继透镜的深层结构双光子显微镜成像，将提供一种用于监测突触的多功能系统。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报告基因构建体后，这项技术可以应用于高等哺乳动物，例如猕猴。 也许可以让猴子执行一项任务，并在学习该任务的过程中观察大脑中的突触可塑性。