Light-induced reversible manipulation of neurotransmitter release

描述(申请人提供)：在这项申请中，我们建议使用细菌视紫红质，一种来自盐生盐生杆菌的光激活质子泵，来操纵突触小泡中的pH梯度。突触内充满神经递质的囊泡对囊泡内的pH高度敏感，而囊泡内的pH受固有的囊泡质子泵--空泡ATPase(v-ATPase)调节。最近的研究，包括我们团队的工作，表明小分子抑制剂(如巴非霉素)对v-ATPase的抑制会导致突触反应的快速衰减和神经递质释放的阻断。在这个项目中，我们将利用突触小泡再充盈过程的这种严格的pH依赖性，使用针对神经元突触小泡的细菌视紫红质，以光诱导和快速可逆的方式模拟v-ATPase抑制剂的作用，而不会整体改变膜的兴奋性。此外，将细菌视紫红质靶向于其他分泌细胞器，如溶酶体，可以成为研究它们在神经元功能中的作用的有力工具(S)，这些细胞器的正常运行严重依赖于囊内的pH。我们建议这个项目分三个阶段进行：第一，我们的目标是选择性地靶向突触小泡功能构象中的细菌视紫红质。其次，我们将优化培养的海马神经元中囊泡细菌视紫红质的光诱导质子泵活性。最后，我们将使用果蝇特定的神经元启动子在体内表达优化的细菌视紫红质结构，用于光诱导操纵果蝇的行为。综上所述，本文提出的研究在体外突触功能研究和完整脑中的信息处理方面具有巨大的潜力。这种方法将允许对大脑特定区域的突触输入进行急性操作，以测试它们如何影响功能和行为输出。 公共卫生相关性：在这项应用中，我们建议靶向细菌视紫红质，一种来自盐生盐生杆菌的光激活质子泵，通过工程融合蛋白以可逆和光诱导的方式削弱突触传递，特别针对突触小泡。这个项目的成功完成将产生重要的工具，能够实现突触对神经元电路的特定操作。

DESCRIPTION (provided by applicant): In this application, we propose to employ bacteriorhodopsin, a light-activated proton pump from Halobacterium salinarium, to manipulate the pH gradient in synaptic vesicles. Synaptic vesicle filling with neurotransmitters is highly sensitive to intravesicular pH, which is regulated by an intrinsic vesicular proton pump, vacuolar ATPase (v-ATPase). Recent studies, including work from our group, suggests that inhibition of v-ATPase by small molecule inhibitors (e.g. bafilomycin) results in fast use-dependent rundown of synaptic responses and blockade of neurotransmitter release. In this project, we will exploit this strict pH-dependence of the synaptic vesicle refilling process by using bacteriorhodopsin targeted to synaptic vesicles in neurons to emulate the effect of v-ATPase inhibitors in a light-induced and rapidly reversible fashion without global changes in the membrane excitability. In addition, targeting of bacteriorhodopsin to other secretory organelles such as lysosomes that critically depend on the intravesicular pH for their proper operation can be a powerful tool to investigate their role(s) in neuronal function. We propose to develop this project in three stages: First, we aim to selectively target bacteriorhodopsin in a functional conformation to synaptic vesicles. Second, we will optimize light-induced proton pump activity of the vesicular bacteriorhodopsin in cultured hippocampal neurons. Finally, we will express optimized bacteriorhodopsin constructs in vivo using specific neuronal promoters in Drosophila for light-induced manipulation of Drosophila behavior. Taken together the research proposed here has significant potential in bridging synaptic functional studies in vitro and information processing in the intact brain. This approach will enable acute manipulation of synaptic inputs into a particular area of the brain to test how they may influence function as well as behavioral output. PUBLIC HEALTH RELEVANCE: In this application we propose to target bacteriorhodopsin, a light-activated proton pump from Halobacterium salinarium, specifically to synaptic vesicles by engineering fusion proteins to impair synaptic transmission in a reversible and light-induced fashion. Successful completion of this project will yield important tools that can enable synapse specific manipulation of neuronal circuits.