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）： 大脑通过改变突触活动（SA）等方式恶化或减轻自身损伤的严重程度，这可能涉及炎症介质（IM），因为它们对损伤既有促炎作用又有抗炎作用，对SA也有相反的作用。这种不同的净效应可能涉及脑组织中 IM 的产生、表达和后续效应的差异模式和刺激依赖性，而脑组织具有区域和细胞异质性。然而，由于缺乏适当的工具来同时测量来自已识别细胞和功能性脑组织内的间隙空间 (ISS) 的多个候选分子，因此之前不可能确认这一概念。我们建议通过正式结合两种新的研究工具来消除这一空白，这些工具用于测量来自啮齿动物海马器官培养物 (HOTC) 和年龄匹配的整体动物对照的已识别细胞 (和 ISS) 中 IM 蛋白的变化。将使用 Bio-Plex 蛋白质阵列系统和来自徕卡激光解剖显微镜的特定细胞类型样品对目标 IM 进行多次同步测量。我们的总体目标是建立详细的方案，用于同时测量来自已识别细胞和 ISS 的多种蛋白质，然后通过癫痫发作时看到的示例性 SA 变化来说明其使用的生物学效用。该项目将通过 3 个重要步骤来重点关注，以更清楚地说明其潜在影响。首先，我们将通过癫痫发作来改变 SA，因为这种对大脑的刻板扰动会引起 IM 的变化。其次，虽然许多 IM 会随着癫痫发作而发生变化，但我们将检查白细胞介素-1 (IL-1)，一种原型 IV。第三，将检查 6 个关键的 IL-1 家族 IM，因为了解它们的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种受体配体（IL-1α、IL-1β和IL-1受体拮抗剂（Ra））、2种受体亚型（IL-1RI和IL-1RII）和辅助蛋白（IL-1AcP）组成。虽然 IL-1α 和 IL-1β 水平升高会加剧癫痫发作，但可溶形式的 IL-1 Ra (slL-1Ra) 可起到抗惊厥作用。我们的具体目标是： 1：开发并确认同时测量已识别细胞中的 IL-1 家族 IM 以及体内 HOTC 和海马的 ISS 的方案。图2：确定体内海马和HOTC中癫痫发作引起的IL-1 IM变化的时空和细胞特异性模式。 3：确定兴奋性调节（即来自后期长时程增强 (L-LTP) 和 IL-1 IM 稳态的外源性改变）如何改变 HOTC 中的 IL-1 IM 表达、癫痫易感性和损伤，以及这些变化是否相互依赖。准确确定 IL-1 家族 IM 在已识别细胞中同时发生变化的地点、时间和程度与 R21 程序一致，并且应能加快破译 IL-1 家族 IM 如何对癫痫发作和 SA 产生双重影响。