Sexual dimorphism in seizure control is KCC2 mediated

描述(由申请者提供)：本申请是为授予Aristea S.Galanopoulou博士的临床科学家发展奖(K08)，由所罗门·L·莫什博士赞助。Galanopoulou博士是雅典医学院的医学博士(McGill大学)，在阿尔伯特·爱因斯坦医学院接受神经学和临床神经生理学方面的培训，目前是该学院的助理教授。在麦吉尔，格兰诺普鲁博士接受了分子生物学和体外基因传递方法方面的培训。在阿尔伯特·爱因斯坦医学院，她使用了与癫痫研究有关的活体实验方法。Galanopoulou博士的长期目标是使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。黑质网状部(SNR)在癫痫控制中起重要作用。黑质内注入一种GABAA受体激动剂麝香酚对癫痫发作有性别、年龄和区域特异性的影响。本研究将研究KCC2在对GABAA反应的雄性幼鼠SNR神经元的性别分化中的作用，以确定其在癫痫控制中的功能。KCC2将海马区GABAA受体从去极化转换为超极化。Galanopoulou博士的发现表明，SNR中KCC2的水平可能决定GABAA受体的激活是否有利于惊厥，并与性别特异性的膜反应和雌二醇信号模式相关。这些数据增加了通过过度表达KCC2和改变男性SNR的性别表型来加速开发有效的癫痫控制系统的可能性。Galanopoulou博士将在体内测试KCC2在PN21雄性大鼠的SNR中的过度表达是否可以消除：1)蝇草醇诱导的去极化和钙离子升高，2)雌二醇介导的磷酸化CREB下调，3)蝇草醇诱导的促惊厥反应，4)KCC2调节的其他基因，这些基因可以抑制雄性大鼠SNR由睾酮组织的对蝇草酚敏感的促惊厥作用。这项研究将包括用腺相关病毒、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·爱因斯坦医学院将为实现这些目标提供必要的指导、实验室空间、设备和培训机会。

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.