Genetically Encoded Phototriggers of Neuronal Activity

描述(由申请人提供)： 能够将光刺激转化为电信号的异源蛋白质可以用来控制完整组织或生物体中可兴奋细胞的功能。这些可选择性寻址的去极化电流源在遗传上受限于细胞靶标群体，可用于为神经回路提供分布式输入，阐明功能性突触连接，探测电路和系统的响应特性，并揭示分布式神经表征中携带的行为相关信息。为了实现基因定位的光刺激，我们在通常缺乏配体门控离子通道的神经元中表达这些通道，并通过用光可移除阻断基团(CAGE)进行化学修饰使这些通道的电导门控激动剂具有生物惰性。因此，钥匙和锁机制确保了光刺激的时间控制和细胞类型特异性：动作电位的启动需要释放自由激动剂(钥匙)的光脉冲，以及已被遗传编程表达同源配体门控离子通道(锁)的靶神经元。该项目的目标是促进具有快速动力学的高选择性光触发器的开发(特定目标1和2)，创建模块化的哺乳动物表达系统(特定目标3和4)，并启动对哺乳动物大脑中功能神经电路的光学分析，重点是皮质和海马区抑制性(GABA能)中间神经元网络(特定目标5)。GABA能回路对信息的处理、存储和检索具有基本的重要性，乙醇和苯二氮卓类药物的作用以及中间神经元参与精神分裂症和阿尔茨海默病等疾病的作用说明了这一点。

DESCRIPTION (provided by applicant): Heterologous proteins capable of transducing optical stimuli into electrical signals can be used to control the function of excitable cells in intact tissues or organisms. Restricted genetically to circumscribed populations of cellular targets, these selectively addressable sources of depolarizing current can be used to supply distributed inputs to neural circuits, elucidate functional synaptic connections, probe the response characteristics of circuits and systems, and unveil behaviorally relevant information carried in distributed neural representations. To achieve genetically localized photostimulation, we express ligand-gated ion channels in neurons that normally lack them, and render the agonists that gate the conductances of these channels biologically inert by chemical modification with photoremovable blocking groups ('cages'). A key-and-lock mechanism thus ensures temporal control and cell-type specificity of photostimulation: the initiation of an action potential requires a light pulse that liberates free agonist (the 'key'), and a target neuron that has been genetically programmed to express the cognate ligand-gated ion channel (the 'lock'). The Objective of this project is to advance the development of highly selective phototriggers with fast kinetics (Specific Aims 1 and 2), create modular mammalian expression systems (Specific Aims 3 and 4), and initiate optical analyses of functional neural circuits in the mammalian brain, with an emphasis on networks of inhibitory (GABAergic) interneurons in the cortex and hippocampus (Specific Aim 5). GABAergic circuits are of basic importance to the processing, storage, and retrieval of information, as illustrated by the effects of agents such as ethanol and benzodiazepines, and by the involvement of interneurons in diseases such as schizophrenia and Alzheimer's disease.