Ethanol sensitivity--native /recombinant NMDA receptors

描述(由申请人提供)： 这项申请是对目前资助的K02奖的竞争性更新，重点是提高PI研究乙醇对大脑离子通道的作用的能力。PI已经对酒精对NMDA受体的影响进行了研究，目前由NIAAA通过R01资助，以继续这项工作。PI利用目前资助的K02奖励期，获得了双电极电压钳、全细胞膜片钳电生理学和分子生物学方面的培训。他使用这些技术对NMDA受体中酒精作用的分子部位做出了重要发现。利用野生型和突变型NMDA受体转染卵母细胞和HEK293细胞的实验结果使人们更好地理解了亚单位组装、细胞内信号传递过程和跨膜结构域如何调节这种受体对酒精的敏感性。在这项应用中，PI将通过学习技术来扩展这些发现的相关性，这些技术用于在脑神经元中表达具有改变的乙醇敏感性的突变NMDA受体亚单位，并评估分子生物学、电生理学和动物行为的特定领域。具体地说，PI将：1)学习生成和使用基于病毒的基因载体将具有改变的酒精敏感性的NMDA受体亚单位输送到培养和体内培养的神经元中所需的技术；2)学习用于产生表达具有改变的酒精敏感性的突变的NMDA受体亚单位的转基因动物的方法；3)学习从表达突变的具有酒精敏感性的NMDA受体的动物脑片分离的离子通道电流的方法；以及4)学习评估酒精在表达具有改变的酒精敏感性的突变的NMDA受体的动物中的神经行为行为所需的技术。这项培训将在一系列会议期间进行，由具有技术专长的各种顾问进行监督。这项培训计划的成功完成将大大提高PI的研究专业知识和实现其研究目标的能力。

DESCRIPTION (provided by applicant): This application is a competitive renewal of a currently funded K02 award and is focused on advancing the PI's ability to investigate the actions of ethanol on brain ion channels. The PI has conducted research into the effects of alcohol of the NMDA receptor and is currently funded by NIAAA through an R01 to continue this work. The PI has utilized the currently funded K02 award period to obtain training in two-electrode voltage-clamp, whole-cell patch-clamp eletrophysiology and molecular biology. He has used these techniques to make important discoveries with regard to the molecular sites of alcohol action of the NMDA receptor. Results from experiments utilizing transfection of oocytes and HEK293 cells with wild-type and mutant NMDA receptors has led to a better understanding of how subunit assembly, intracellular signaling processes and transmembrane domains modulate the sensitivity of this receptor to alcohol. In this application, the PI will extend the relevance of these findings by learning techniques used to express mutant NMDA receptor subunits with altered ethanol sensitivity in brain neurons and to assess specific areas of molecular biology, electrophysiology, and animal behavior. Specifically, the PI will: 1) learn techniquesneeded to generate and use viral-based gene vectors to deliver NMDA receptor subunits with altered ethanol sensitivity into neurons grown in culture and in vivo; 2) learn methods used to generate genetically modified animals that express mutant NMDA receptor subunits with altered ethanol sensitivity; 3) learn methods to prepare and record ion channel currents from brain slices isolated from animals expressing mutant NMDA receptors with altered ethanol sensitivity and; 4) learn techniques needed to assess the neurobehavioral actions of alcohol in animals expressing mutant NMDA receptors with altered ethanol sensitivity. This training will be obtained during a series of sessions supervised by a variety of consultants with demonstrated expertise in the techniques. The successful completion of this training plan will substantially enhance the PI's research expertise and his ability to fulfill the goals of his research.