Sexual dimorphism in seizure control is KCC2 mediated

描述（由申请人提供）：本申请是为Aristea S. Galanopoulou博士申请指导临床科学家发展奖（K08），由Solomon L. Mosh博士赞助。Galanopoulou博士是医学博士（雅典医学院）和博士（麦吉尔大学），曾在阿尔伯特·爱因斯坦医学院接受神经病学和临床神经生理学培训，目前是该学院的助理教授。在麦吉尔大学，Galanopoulou博士接受了分子生物学和体外基因传递方法的培训。在阿尔伯特·爱因斯坦医学院，她使用了与癫痫研究有关的体内实验方法。Galanopoulou博士的长期目标是采用新的基于分子生物学的方法来开发有效的癫痫治疗方法。网状黑质（SNR）在癫痫发作控制中起着至关重要的作用。神经内输注muscimol（一种GABAA受体激动剂）对癫痫发作具有性别、年龄和区域特异性作用。本研究将研究氯化钾共转运体KCC2在GABAA反应性雄性幼鼠SNR神经元性别分化中的作用，并确定其在癫痫发作控制中的功能。KCC2开关海马GABAA受体从去极化到超极化。Galanopoulou博士的研究结果表明，SNR中KCC2的水平可能决定GABAA受体激活是否为促惊厥，并与性别特异性膜反应和雌二醇信号传导模式相关。这些数据提出了通过过度表达KCC2和改变男性SNR的性表型来加速开发有效的癫痫发作控制系统的可能性。Galanopoulou博士将测试在PN21雄性大鼠的信噪比中过量表达KCC2是否会消除：1)肌肉醇诱导的去极化和钙升高，2)雌二醇介导的磷酸化CREB下调，3)肌肉醇诱导的促惊厥信噪比反应，以及4)将识别由KCC2调控的其他基因，这些基因抑制睾丸激素组织的肌肉醇敏感的促惊厥信噪比效应。这项研究将包括使用腺相关病毒、立体定向手术、免疫化学、原位杂交、癫痫诱导、革兰西丁穿孔膜片钳、fura-2AM成像、共聚焦显微镜、立体细胞计数、DNA微阵列和统计学进行体内基因传递。阿尔伯特·爱因斯坦医学院将提供必要的指导、实验室空间、设备和培训机会，以实现这些目标。

DESCRIPTION (provided by applicant): This application is for a Mentored Clinical Scientist Development Award (K08) for Dr. Aristea S. Galanopoulou, sponsored by Dr. Solomon L. Mosh. Dr Galanopoulou is an MD (Medical School of Athens) PhD (McGill University) trained in Neurology and Clinical Neurophysiology at Albert Einstein College of Medicine, where she is currently Assistant Professor. At McGill, Dr. Galanopoulou was trained in Molecular Biology and in vitro gene delivery methods. At Albert Einstein College of Medicine she has used in vivo experimental methods pertaining to seizure research. Dr. Galanopoulou's long- term goals are to employ novel molecular biology based approaches to develop effective treatments for epilepsy. The substantia nigra pars reticulata (SNR) has crucial role in seizure control. Intranigral infusions of muscimol, a GABAA receptor agonist, have sex, age and region specific effects on seizures. This proposal will study the role of the potassium-chloride co-transporter KCC2 in the sexual differentiation of GABAA responsive male infantile rat SNR neurons, which determine its function in seizure control. KCC2 switches hippocampal GABAA receptors from depolarizing to hyperpolarizing. Dr. Galanopoulou's findings suggest that the level of KCC2 in the SNR may determine whether GABAA receptor activation is pro-convulsant or not and correlate with sex-specific membrane responses and patterns of estradiol signaling. These data raise the possibility of accelerating the development of efficient control systems for seizures, by over expressing KCC2 and altering the sexual phenotype of male SNR. Dr. Galanopoulou will test whether in vivo over expression of KCC2 in the SNR of PN21 male rats abolishes: 1) the muscimol-induced depolarization and calcium rise, 2) the estradiol-mediated down regulation of phosphorylated CREB, 3) the muscimol-induced pro-convulsant SNR responses, and 4) will identify other genes regulated by KCC2, which suppress the testosterone-organized muscimol-sensitive proconvulsant effects of male rat SNR. This research will entail in vivo gene delivery using adeno-associated viruses, stereotactic surgery, immunochemistry, in situ hybridization, seizure induction, gramicidin perforated patch clamp, fura-2AM imaging, confocal microscopy, stereological cell counting, DNA microarrays and statistics. Albert Einstein College of Medicine will offer the necessary mentoring, laboratory space, equipment and training opportunities to achieve these goals.