Regulation of extraysynaptic GABA-A receptors in health and disease

本项目主要研究创伤性脑损伤引起的细胞和分子变化。 (TBI)，特别是影响海马区GABA能信号的那些。不适应的变化 细胞和分子特性有助于脑外伤的认知和神经精神后遗症，以及 癫痫发作。脑外伤后导致功能障碍的慢性细胞和分子改变，以及 由创伤性脑损伤引发的导致这些慢性改变的近端因素尚不完全清楚。 小胶质细胞是大脑的常驻免疫细胞，在健康的大脑中起着有益的作用，但在 对伤害的反应他们的行为可能是不适应的、伤害性的，并放大了脑损伤的后果。 因此，小胶质细胞及其炎症信号是触发病理生理的候选因素。 颅脑损伤后细胞和分子的变化。先前的工作已经确立了下调对 突触外GABA受体是脑损伤在海马区的一贯特征。建议进行的实验 这里将检验这样的假设：i)小胶质细胞激活的减弱将限制脑损伤引起的变化 在突触外GABA受体的表达和功能以及II)小胶质细胞激活的减弱 减少脑外伤(癫痫)后自发性癫痫的发生率。 结合使用膜片钳电生理学和免疫组织化学技术， 突触外GABA受体(GABAA和GABAB)在海马区的表达和功能 神经元将在严重脑损伤(受控皮质撞击，CCI)模型中进行评估。评估角色 脑外伤后这些分子变化中的小胶质细胞的变化，这是一种强大的药理学工具，显著地 将使用耗竭的脑小胶质细胞(CSF1-R抑制剂PLX5622)。PLX5622治疗的动物将被 暴露于CCI，允许评估TBI后功能性分子改变是否 依赖于小胶质细胞的激活。具体地说，拟议的实验将确定在 脑创伤后突触外突触GABA受体依赖于小胶质细胞的激活。作为这一目标的一部分，其他 将检查受脑损伤影响的区域(丘脑、小脑)的CCI相关变化。 突触外GABA受体功能。 脑损伤的细胞效应和结果也将被调查，以评估它们对 小胶质细胞激活。在CCI前用PLX5622耗尽小胶质细胞的方法，时间 脑外伤后神经元死亡的过程和程度(对于所有神经元和特定的神经元间亚型) 将确定为对照和PLX5622处理的动物。苔藓纤维发芽( DGGC轴突对损伤的病理生理树枝反应)是发生的另一种细胞变化 作为脑损伤的结果，苔藓纤维发芽将被量化，并在两者之间进行比较 组。这里建议研究的细胞和分子变化都被认为是有助于 癫痫的发生和发作，但不管这些特定的细胞/分子变化， 对CCI后癫痫发生的显著小胶质细胞依赖效应的认识是最重要的 重要性。视频-脑电监测将用于确定自发性癫痫的发生率 对照组和PLX5622组动物CCI后的癫痫(即癫痫)和发作特征。这些数据 将提供证据支持(或驳斥)小胶质细胞在CCI后癫痫发生中的重要作用。 需要这些洞察力来为未来的调查和治疗方法提供信息。

This project is focused on the cellular and molecular changes caused by traumatic brain injury (TBI), specifically those affecting GABAergic signaling in the hippocampus. Maladaptive changes in cellular and molecular properties contribute to the cognitive and neuropsychiatric sequelae of TBI, and epileptic seizures. The chronic cellular and molecular alterations causing dysfunction after TBI, and the proximal factors triggered by TBI that induce these chronic changes, are incompletely understood. Microglia, the resident immune cells of the brain, serve beneficial functions in the healthy brain but in response to injury their actions can be maladaptive, injurious, and magnify the consequences of TBI. As such, microglia and their inflammatory signaling are candidate factors to trigger pathophysiological cellular and molecular changes after TBI. Prior work has established that downregulation of extrasynaptic GABA receptors is a consistent feature of TBI in the hippocampus. Experiments proposed here will test the hypotheses that: i) attenuation of microglia activation will limit TBI-induced changes in extrasynaptic GABA receptor expression and function and ii) attenuation of microglia activation will decrease the incidence of spontaneous seizures after TBI (epileptogenesis). Using a combination of patch-clamp electrophysiology and immunohistochemical techniques, the expression and function of extrasynaptic GABA receptors (both GABAA and GABAB) in hippocampal neurons will be assessed in a model of severe TBI (controlled cortical impact, CCI). To assess the role of microglia in these molecular alterations after TBI, a powerful pharmacological tool that dramatically depletes brain microglia will be used (PLX5622, a CSF1-R inhibitor). PLX5622-treated animals will be exposed to CCI, allowing the assessment of whether functional molecular alterations after TBI are dependent on microglia activation. Specifically, the proposed experiments will determine if changes in extasynaptic GABA receptors after TBI are dependent on microglia activation. As part of this aim, other areas affected by TBI (thalamus, cerebellum) will be examined for CCI-associated changes in extrasynaptic GABA receptor function. The cellular effects of TBI and outcomes will also be investigated to assess their dependence on microglia activation. Using the approach of depleting microglia with PLX5622 prior to CCI, the time course and magnitude of neuronal death after TBI (for all neurons and specific interneuronal subtypes) will be determined for control and PLX5622-treated animals. Mossy fiber sprouting (the pathophysiological arborizing response of DGGC axons to injury) is another cellular change that occurs as a consequence of TBI; mossy fiber sprouting will be quantified and compared between the two groups. The cellular and molecular changes proposed for study here are all believed to contribute to epileptogenesis and seizures, but irrespective of these specific cellular/molecular changes the recognition of significant microglia-dependent effects on epileptogenesis after CCI is of greatest importance. Video-EEG monitoring will be used to determine the incidence of spontaneous seizures (i.e. epilepsy) and seizure characteristics after CCI in control and PLX5622-treated animals. These data will provide evidence to support (or refute) a significant role of microglia in epileptogenesis after CCI. These insights are needed to inform areas of future investigation and therapeutic approaches.