Regulation of extraysynaptic GABA-A receptors in health and disease

本项目的重点是创伤性脑损伤引起的细胞和分子变化 (TBI)特别是影响海马体中GABA能信号传导的那些。适应不良的变化 细胞和分子特性导致TBI的认知和神经精神后遗症， 癫痫发作TBI后引起功能障碍的慢性细胞和分子改变， 由TBI触发的诱发这些慢性变化的近端因素尚未完全了解。 小胶质细胞，大脑的常驻免疫细胞，在健康的大脑中发挥有益的功能，但在 他们的行为可能是适应不良的、有害的，并放大了TBI的后果。 因此，小胶质细胞和它们的炎症信号传导是触发病理生理学变化的候选因素。 TBI后的细胞和分子变化。先前的工作已经确定，下调 突触外GABA受体是海马中TBI的一致特征。拟议的实验 这里将测试以下假设：i）小胶质细胞激活的减弱将限制TBI诱导的变化 在突触外GABA受体表达和功能中，以及ii）小胶质细胞活化的减弱将 降低TBI后自发性癫痫发作的发生率（癫痫发生）。 结合膜片钳电生理学和免疫组织化学技术， 海马突触外GABA受体（GABAA和GABAB）的表达和功能 将在严重TBI（受控皮质撞击，CCI）模型中评估神经元。评估角色 小胶质细胞在TBI后的这些分子改变中的作用，这是一种强大的药理学工具， 将使用消耗脑小胶质细胞的细胞因子（PLX 5622，CSF 1-R抑制剂）。PLX 5622给药动物将 暴露于CCI，允许评估TBI后的功能性分子改变是否 依赖于小胶质细胞的激活。具体来说，拟议的实验将确定是否改变 TBI后突触外GABA受体依赖于小胶质细胞活化。作为这一目标的一部分，其他 将检查受TBI影响的区域（丘脑、小脑）的CCI相关变化， 突触外GABA受体功能。 还将研究TBI的细胞效应和结果，以评估其对以下因素的依赖性： 小胶质细胞激活。在CCI之前使用PLX 5622消耗小胶质细胞的方法， TBI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） 将确定对照组和PLX 5622给药组动物的平均存活时间。苔藓纤维发芽（ DGGC轴突对损伤的病理生理学分支反应）是另一种细胞变化， 作为TBI的结果;苔藓纤维发芽将被量化并在两者之间进行比较 组这里提出的研究的细胞和分子变化都被认为有助于 癫痫发生和癫痫发作，但不管这些特定的细胞/分子变化， 认识到CCI后癫痫发生的显著小胶质细胞依赖性作用是最重要的， 重要性视频脑电图监测将用于确定自发性癫痫发作的发生率 (i.e.癫痫）和CCI后的癫痫发作特征。这些数据 将提供证据支持（或反驳）小胶质细胞在CCI后癫痫发生中的重要作用。 需要这些见解来为未来的研究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.