Employing subcellular calcium to control membrane voltage

 描述（由申请人提供）：本提案的目的是评估神经元活动的活动依赖性反馈控制的全分子方法的可行性。我们提出产生钙敏感的发光分子（生物发光酶，荧光素酶），其驱动光敏光遗传学元件（通道或离子泵，视蛋白）以在单细胞水平控制膜电压，用于正反馈和负反馈控制。通过调节钙敏感性和分子位置，可以使光产生特定于大事件，如爆发，或对单个尖峰或单通道活动敏感。通过将这些新的钙蛋白酶与视蛋白偶联，钙在其来源处的高度特异性感测将触发视蛋白激活以增强或抑制神经元活性，从而允许反馈控制中的高度时间和空间精度。目标将通过追求三个目标来实现：1）开发具有显著改善的速度，亮度和灵敏度范围的钙传感分裂荧光素酶; 2）将这些新分子靶向亚细胞结构域，以实现高度特异性的生物学结果; 3）将这些新的创新与光遗传学读数联系起来。我们的策略是非侵入性的，并且它可以应用于行为动物或人类中神经活动的大规模操纵，其中神经元活动的非侵入性快速反馈控制可以用于调节大脑中的临床相关活动。我们的实验还处于早期阶段，需要进行原理可行性研究的证明，但它们有可能导致一种新的策略，仅在神经元异常期间调节活动。 发射，如衰减失控活动或放大局部波动。这些可行性实验产生的分子工具本身就非常有价值，实现神经活动调节神经元自我控制的目标将是变革性的。

 DESCRIPTION (provided by applicant): The goal of this proposal is to assess the feasibility of an all-molecular method for activity-dependent feedback control of neuronal activity. We propose to generate calcium sensitive light emitting molecules (bioluminescent enzymes, luciferases) that drive light sensing optogenetic elements (channels or ion pumps, opsins) to control membrane voltage at the level of single cells for positive and negative feedback control. By adjusting calcium sensitivity and molecule location, light production can be made specific to large events such as bursts, or sensitive to individual spikes or single channel activity. By coupling these new luciferases to opsins, highly specific sensing of calcium at its source will trigger opsin activation for augmenting or suppressing neuronal activity, allowing a high degree of temporal and spatial precision in feedback control. Goals will be achieved by pursuing three aims: 1) Developing a calcium sensing split luciferase with significantly improved speed, brightness and range of sensitivity; 2) Targeting these new molecules to subcellular domains to enable highly specific biological outcomes; 3) Linking these new innovations to optogenetic readouts. Our strategy is non- invasive and it could be applied to large-scale manipulation of neural activity in behaving animals or in humans, where non-invasive, rapid feedback control of neuronal activity could be used to regulate clinically relevant activity in the brain. Our experiments are early stage, require proof of principle feasibility studies, but they have the potential to lead to a novel strategy to regulate activity only during periods of abnormal neuronal firing, such as attenuating runaway activity or amplifying local fluctuations. The molecular tools generated towards these feasibility experiments will be highly valuable in their own right, and achieving the goal of neural activity regulated self-control of neurons will be transformative.