MULTIPLEXED & CELL-SPECIFIC ANALYSES OF BRAIN FUNCTION

描述(由申请人提供)： 大脑通过改变突触活动(SA)来恶化或减轻自身损伤的严重程度，突触活动可能涉及炎症介质(IM)，因为它们对损伤既有促炎作用，也有抗炎作用，而且对SA有同样相反的作用。这种不同的净效应可能涉及IM在脑组织中的产生、表达和随后的影响的不同模式和刺激依赖性，这在区域和细胞上是不同的。然而，证实这一概念以前是不可能的，因为缺乏适当的工具，可以同时测量来自已识别细胞和功能脑组织中的间质间隙(ISS)的多个候选分子。我们建议通过正式耦合两个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(Hotc‘s)和年龄匹配的全动物对照的已识别细胞(和ISS)中IM蛋白的变化。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本，将对目标IM进行多次和同时测量。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的多个蛋白质，然后通过癫痫发作所见的典型SA变化来说明它们的生物效用。该项目将通过3个重要步骤进行重点阐述，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，因为大脑的这种刻板印象会导致IM的变化。其次，虽然许多IM随着癫痫发作而改变，但我们将检查IL-1，一种典型的IV。第三，我们将检查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个受体配体(IL-1α、IL-1β和IL-1受体拮抗剂(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-1α和IL-1β水平升高会增加癫痫发作，但可溶性形式的IL-1ra(SLL-1ra)起到了抗惊厥的作用。我们的具体目标是：1：建立和确认在活体内同时测量已识别细胞和来自Hotc‘s和海马体的ISS中IL-1家族IM的方案。2：确定癫痫发作诱导的IL-1 IM变化的时空和细胞特异性模式。3：确定兴奋性条件反射(即晚期长时程增强和外源性IL-1 IM稳态改变)如何改变Hotc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。准确地确定IL-1家族IM在已识别细胞中同时变化的地点、时间和程度与R21程序一致，并应能加快破译IL-1家族IM如何对癫痫发作和SA造成双重影响。

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.