MULTIPLEXED & CELL-SPECIFIC ANALYSES OF BRAIN FUNCTION

描述（由申请人提供）： 大脑通过包括改变突触活动（SA）的方式减轻或减轻自身损伤的严重性，所述改变突触活动可能涉及炎症介质（IM），因为它们对损伤具有促炎和抗炎作用，加上对SA具有同样相反的作用。这种发散的净效应可能涉及IM在脑组织中的产生、表达和后续效应的差异模式和刺激依赖性，脑组织是区域和细胞异质的。然而，确认这一概念以前是不可能的，因为缺乏适当的工具，允许同时测量来自功能脑组织内已识别细胞和间隙（ISS）的多个候选分子。我们建议通过正式结合2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鉴定细胞（和ISS）中的IM蛋白变化。将使用Bio-Doppler蛋白质阵列系统和来自Leica激光解剖显微镜的特定细胞类型样本对靶向IM进行多次和同时测量。我们的总体目标是建立详细的协议，同时测量多种蛋白质从确定的细胞加上ISS，然后说明其使用的生物效用与示例性SA的变化与癫痫发作。该项目将通过三个重要步骤来重点关注，以更清楚地说明其潜在影响。首先，我们将通过使用癫痫发作来改变SA，因为这种刻板的大脑扰动会引起IM的变化。其次，虽然许多IM的变化与癫痫发作，我们将检查白细胞介素-1（IL-1），一个原型IV。第三，将检查6个关键的IL-1家族IM，因为理解它们的同时行为对于最终破译IL-1如何影响SA和脑功能至关重要。IL-1家族IM由3种受体配体（IL-1 α、IL-1 β和IL-1受体拮抗剂（Ra））、2种受体亚型（IL-1 RI和IL-1 RII）和一种辅助蛋白（IL-1AcP）组成。虽然IL-1 α和IL-1 β水平升高可增强癫痫发作，但可溶性形式的IL-1 Ra（sIL-1 Ra）可作为抗惊厥剂。我们的具体目标是：一曰：开发并确认用于同时测量鉴定细胞中的IL-1家族IM和体内HOTC和海马的ISS的方案。2：确定由体内海马和HOTC中的癫痫发作诱导的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，来自L-LTP和IL-1 IM稳态的外源性改变）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在识别的细胞中同时变化的位置、时间和程度与R21程序一致，并且应该加速破译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.