Glutamatergic plumes – a novel mechanism of excitability in the brain after TBI.

项目摘要/摘要 创伤性脑损伤是造成军人和平民死亡的主要原因。同时， 脑外伤幸存者的大脑网络超兴奋性长期增加的风险更大。尽管全球 脑外伤的负担，很少有动物研究集中在突触和 网络级别。我们的目标是分析轻度脑损伤后相关兴奋性的潜在机制。 和严重的脑损伤。最近，我们观察到非典型性谷氨酸释放事件的发生率增加， 被称为谷氨酸能羽毛，在脑损伤后48小时。增加羽流的频率可以促进传播 去极化(SD)启动。SD是颅脑损伤后可检测到的一种兴奋性现象，并与 组织受损，预后不佳。因此，羽状物的流行表明，在 TBI。我们将研究大脑中这种新形式的异常谷氨酸信号，包括其后果 羽流对脑损伤后兴奋性的影响。我们将同时使用活体全细胞记录和双光子 显微镜，以及操纵羽流机制的基因工具。我们将对男性和女性进行检查 作为雌性的小鼠，在脑外伤后经历了更糟糕的兴奋性相关并发症。在目标1中，我们将确定 受控皮质撞击(CCI)和轻度TBI模型中羽流的来源，以及这些机制是如何改变的 在雌性小鼠身上。根据我们最近的数据，星形胶质细胞重新摄取谷氨酸的失败促进了羽毛的形成。因此，我们 假设脑外伤后谷氨酸羽流与星形细胞清除机制有关。为了测试这一点 假设，我们将在体内从基因上去除/增强谷氨酸清除的关键星形胶质细胞介体。这些 实验将建立脑损伤后环境中谷氨酸功能障碍(羽毛)的精确机制。 在目标2中，我们将确定羽毛是否影响脑损伤后突触的可塑性和网络动力学。 我们的初步数据显示，在脑外伤后的自发神经元活动过程中，钙负荷增加。基于 目前的文献表明，谷氨酸功能障碍，如细胞外谷氨酸的增加，可以推动钙内流 在天真的大脑里。因为细胞内钙升高是长时程增强(LTP)的重要特征 诱导，我们假设在脑损伤中的羽毛驱动短暂但强烈的突触后钙升高 有助于LTP，从而增加网络兴奋性。这一点很重要，因为 突触的可塑性与许多神经疾病有关。值得注意的是，我们将询问羽流是否以及如何诱导 通过对脑外伤后树枝状钙瞬变进行双光子成像，研究树枝状组织的可塑性。此外， 我们将研究羽流(和星形细胞机制)和SD相关的机制联系 颅脑损伤后的钙负荷。我们假设羽流为NMDA的激活提供了必要的刺激 受体，从而导致钙在脑损伤后的SD期间激增。因此，羽流可能会加剧损害。 颅脑损伤后神经细胞钙升高和SD共同作用。我们的发现将导致新的和有针对性的 创伤后兴奋性的机制，包括潜在的药物靶点。

Project Summary/Abstract Traumatic brain injury (TBI) is a major cause of mortality in both military and civilian populations. Meanwhile, TBI survivors are at greater risk for long-term increases in brain network hyper-excitability. Despite the global burden of TBI, there have been very few animal studies focused on mechanisms of excitability at synaptic and network levels. Our goal is to dissect the potential mechanisms underlying TBI-associated excitability after mild and severe brain injury. Recently, we observed an increased incidence of noncanonical glutamate release events, known as glutamatergic plumes, 48 hours after TBI. Increased frequency of plumes can facilitate spreading depolarization (SD) initiation. SD is an excitable phenomenon detected after TBI and is correlated with increased tissue damage and poor outcome. Thus, the prevalence of plumes suggests that the network is dysfunctional after TBI. We will examine this novel form of aberrant glutamate signaling in the brain, including the consequences of plumes on post-TBI excitability. We will employ simultaneous in vivo whole-cell recording and two-photon microscopy, alongside genetic tools to manipulate mechanisms of plumes. We will examine both male and female mice as females experience worse excitability-related complications post-TBI. In Aim 1, we will determine the source of plumes in controlled cortical impact (CCI) and mild TBI models, and how those mechanisms are altered in female mice. Based on our recent data, glutamate reuptake failure by astrocytes facilitates plumes. Thus, we hypothesize that astrocytic clearance mechanisms are responsible for glutamate plumes after TBI. To test this hypothesis, we will genetically ablate/enhance key astrocyte mediators of glutamate clearance in vivo. These experiments will establish a precise mechanism of glutamate dysfunction (plumes) in the post-TBI environment. In Aim 2, we will determine whether plumes can influence synaptic plasticity and network dynamics after TBI. Our pilot data shows calcium loading is enhanced during spontaneous neuronal activity after TBI. Based on current literature, glutamate dysfunction, such as increases in extracellular glutamate, can drive calcium influx in the naïve brain. Since elevation in intracellular calcium is an important feature of long-term potentiation (LTP) induction, we hypothesize that plumes in TBI drive brief but strong postsynaptic calcium elevations contributing to LTP and thus to an increase in network excitability. This is important since aberrant changes in synaptic plasticity are implicated in many neurological disorders. Notably, we will ask if and how plumes induce plasticity in dendrites by performing two-photon imaging of dendritic calcium transients after TBI. Furthermore, we will examine the mechanistic connection between plumes (and astrocytic mechanisms) and SD-associated calcium load after TBI. We hypothesize that plumes provide the stimulus necessary for the activation of NMDA receptors, thereby causing calcium to surge during SD after TBI. Thus, plumes may enhance the damage caused by both SD and neuronal calcium elevation after TBI. Our findings will result in novel and targeted mechanisms of post-traumatic excitability, including potential drug targets.