Rapid seizure-induced changes in dendritic spines

描述(申请人提供)：癫痫影响2-5%的人，通常与长期的神经缺陷有关，如学习障碍和脑瘫。虽然癫痫发作在某些临床情况下可能直接导致神经元死亡，但在其他情况下，癫痫发作似乎不会导致神经元死亡，但仍可能对神经元结构和功能产生有害影响。虽然癫痫通过兴奋性毒性和细胞凋亡诱导的神经元死亡已被广泛研究，但癫痫非致死性神经元损伤的机制尚不清楚。最近发现，神经元的树突棘对各种生理刺激表现出快速的运动，这表明脊髓运动在学习等重要神经元功能中发挥着作用。由于癫痫发作涉及过度的神经生理活动，本研究建议的主要假设是癫痫发作引起树突棘的直接、快速变化，这种变化取决于特定的癫痫发作性质。该方案的主要目的是利用先进的细胞成像技术，如共聚焦显微镜、双光子成像和表达绿色荧光蛋白的转基因小鼠，研究癫痫发作对动物癫痫模型树突棘密度、形态和运动性的快速影响。我们将在固定的脑切片上观察红藻氨酸和戊四氮诱发的在体树突上癫痫发作的时间进程和影响。在完整的麻醉动物体内，将分析药物诱发癫痫发作期间树突状细胞结构和运动的实时变化。将在体外活体脑片中研究介导癫痫诱发的脊柱快速变化的细胞机制。首席研究员(PI)是一位在临床癫痫和基础癫痫研究方面接受过培训的学术内科科学家。尽管PI在与动物癫痫模型相关的细胞和系统生理学方面拥有丰富的经验，但由本研究事业奖(K02)支持的高分辨率细胞成像的增加维度应该会为癫痫非致命性神经元损伤的机制提供重要的见解，并在很大程度上帮助PI发展癫痫的独立研究事业。华盛顿大学拥有无与伦比的尖端技术资源和细胞成像方面的专家研究人员，是PI完成本提案目标的理想环境。

DESCRIPTION (provided by applicant): Epilepsy affects 2-5% of people and is often associated with long-term neurological deficits, such as learning disabilities and cerebral palsy. Although seizures may directly cause neuronal death in some clinical contexts, in other situations seizures do not appear to induce neuronal death, but may still have detrimental effects on neuronal structure and function. While seizure-induced neuronal death via excitotoxicity and apoptosis has been studied extensively, mechanisms of non-lethal neuronal injury from seizures are poorly understood. Recently, dendritic spines of neurons have been found to exhibit rapid motility in response to various physiological stimuli, suggesting a role of spine motility in important neuronal functions, such as learning. As seizures involve excessive neurophysiological activity, the major hypothesis of this research proposal is that seizures induce direct, rapid changes in dendritic spines, which vary depending on specific seizure properties. The main objective of this proposal is to investigate the rapid effects of seizures on dendritic spine density, morphology and motility in animal seizure models, utilizing advanced cellular imaging techniques, such as confocal microscopy, two-photon imaging, and transgenic mice expressing green-fluorescent protein. The time course and effect of kainate and pentylenetetrazole-induced in vivo seizures on dendrites will be examined in fixed brain sections. Real-time changes in dendritic structure and motility will be analyzed during pharmacologically induced electrographic seizures in intact anesthetized animals in vivo. Cellular mechanisms mediating rapid seizure-induced spine changes will be investigated in live brain slices in vitro. The principal investigator (PI) is an academic physician-scientist trained in both clinical epilepsy and basic epilepsy research. Although the PI has substantial experience in cellular and systems physiology related to animal seizure models, the added dimension of high-resolution cellular imaging supported by this Research Career Award (K02) should provide important insights into mechanisms of non-lethal neuronal injury from seizures and significantly aid the PI in developing an independent research career in epilepsy. Washington University possesses unmatched resources in cutting-edge technology and expert researchers in cellular imaging and represents an ideal environment for the PI to complete the goals of this proposal.