MULTIPLEXED & CELL-SPECIFIC ANALYSES OF BRAIN FUNCTION

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• 批准号: 6606561
• 负责人: Richard P Kraig
• 金额: $ 17.63万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-02-01 至 2005-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6606561
• 关键词: biological signal transduction; brain electrical activity; brain injury; cytokine receptors; electrophysiology; epilepsy; hippocampus; inflammation; interleukin 1; interstitial; laboratory rat; laser capture microdissection; long term potentiation; neurophysiology; protein structure function; synapses; western blottings

DESCRIPTION (provided by applicant): Brain worsens or lessens the severity of injury to itself by means that include altered synaptic activity (SA) that likely involves inflammatory mediators (IM's) since they have both pro- and anti-inflammatory effects on injury plus equally opposite effects on SA. Such divergent, net effects likely involve differential patterns and stimulus dependency of the production, expression and subsequent effects of IM's in brain tissue which is regionally and cellular heterogeneous. However, confirming this notion has previously been impossible due to the absence of appropriate tools that would allow needed simultaneous measurements of multiple candidate molecules from identified cells and the interstitial space (ISS) within functioning brain tissue. We propose to remove this void by formally coupling 2 new investigative tools for measurement of IM protein changes in identified cells (and ISS) from rodent hippocampal organ cultures (HOTC's) and age-matched whole animal controls. Multiple and simultaneous measurements of targeted IM's will be made using the Bio-Plex Protein Array System and specific cell-type samples derived from a Leica laser dissection microscope. Our general goal is to establish detailed protocols for simultaneous measurement of multiple proteins from identified cells plus the ISS and then illustrate the biologic utility for their use with the exemplary SA change seen with seizures. This project will be focused via 3 important steps to more clearly illustrate its potential impact. First, we will alter SA through the use of seizures, since this stereotypic perturbation of brain induces changes in IM's. Second while many IM's change with seizures, we will examine interleukin-1 (IL-1), a prototypic IV. Third, 6 key IL-1 family IM's will be examined since understanding their simultaneous behavior is essential to eventually deciphering how IL-1 effects SA and brain function. IL-1 family IM's consist of 3 receptor ligands (IL-1alpha, IL-1beta & IL-lreceptor antagonist (Ra)), 2 receptor subtypes (IL-1RI & IL-1RII) and an accessory protein (IL-1AcP). While elevated levels of IL-1alpha and IL-1beta enhance seizures, the soluble form of IL-1 Ra (slL-1Ra) acts as an anticonvulsant. Our specific aims are: 1: Develop and confirm protocols for simultaneously measuring IL-1 family IM in identified cells and the ISS from HOTC's and hippocampus in vivo. 2: Determine the spatiotemporal and cell-specific pattern of IL-1 IM changes induced by seizures in hippocampus in vivo and in HOTC's. 3: Determine how excitability conditioning (i.e., from late-long-term potentiation (L-LTP) and exogenous alteration of IL-1 IM homeostasis) alters IL-1 IM expression, seizure susceptibility & injury in HOTC's and whether these changes are interdependent. Accurately determining the where, when and to what degree IL-1 family IM's simultaneously change in identified cells is consistent with the R21 program and should speed deciphering how IL-1 family IM's cause dual effects on seizures and SA.

描述（由申请人提供）： 大脑会通过包括改变的突触活动（SA）的方式来恶化或减轻损伤的严重程度，而突触活动（SA）可能涉及炎症介质（IM），因为它们对损伤具有促疾病和抗炎作用，以及对SA的相反作用。这种不同的净作用可能涉及差异模式和刺激依赖性，IM在脑组织和细胞异质性的脑组织中的刺激依赖性。但是，由于缺乏适当的工具，该概念以前是不可能的。我们建议通过正式耦合2种新的调查工具来消除这一空隙，以测量啮齿动物海马器官培养物（HOTC）和年龄匹配的整个动物对照组中识别细胞（和ISS）中IM蛋白质变化的方法。使用Bio-Plex蛋白阵列系统和源自Leica Laser Laser解剖显微镜的特定细胞类型样品进行靶向IM的多个和同时测量。我们的一般目标是建立详细的方案，以同时测量来自已识别细胞加上ISS的多种蛋白质，然后说明其与癫痫发作所看到的示例性SA变化一起使用的生物学实用程序。该项目将通过3个重要步骤集中精力，以更清楚地说明其潜在影响。首先，我们将通过使用癫痫发作来改变SA，因为这种刻板印象的大脑扰动会引起IM的变化。其次，尽管许多IM随着癫痫发作的变化，但我们将检查白介素-1（IL-1），原型IV。第三，将检查6个密钥IL-1家族IM，因为了解其同时行为对于最终破译IL-1如何影响SA和大脑功能至关重要。 IL-1家族IM由3个受体配体（IL-1Alpha，IL-1Beta＆il-LeReceptor拮抗剂（RA）），2个受体亚型（IL-1RI和IL-1RII）和一个辅助蛋白（IL-1ACP）组成。虽然IL-1Alpha和IL-1Beta的水平升高增强了癫痫发作，但IL-1 RA（SLL-1RA）的可溶形式充当抗惊厥药。我们的具体目的是：1：开发和确认协议，用于同时测量已识别的细胞和来自HOTC和Hippocampus的IS中的IL-1家族IM。 2：确定由海马体内和HOTC中的癫痫发作引起的IL-1 IM变化的时空和细胞特异性模式。 3：确定IL-1 IM稳态的兴奋性调节（即，从较晚的长期增强（L-LTP）和外源性改变）会改变IL-1 IM的表达，HOTC中的癫痫敏感性和损伤，以及这些变化是否相互依存。准确地确定IL-1家族在何时何地，何时何地与识别细胞的同时变化与R21程序一致，并应加快解密IL-1家族IM对癫痫发作和SA的双重影响。