Light-induced reversible manipulation of neurotransmitter release

描述（由申请人提供）：在本申请中，我们建议使用细菌视紫红质，一种来自盐盐杆菌的光激活质子泵，来操纵突触囊泡中的pH梯度。神经递质填充的突触囊泡对囊内pH值高度敏感，pH值由囊泡内质子泵、液泡atp酶（v- atp酶）调节。最近的研究，包括我们小组的工作，表明用小分子抑制剂（如巴菲霉素）抑制v- atp酶会导致突触反应的快速使用依赖性下降和神经递质释放的阻断。在这个项目中，我们将利用这种严格的ph依赖性突触囊泡再填充过程，通过使用靶向神经元突触囊泡的细菌视紫红质，以光诱导和快速可逆的方式模拟v- atp酶抑制剂的作用，而不会改变膜的兴奋性。此外，细菌视紫红质靶向其他分泌细胞器，如溶酶体，它们的正常运作严重依赖于囊泡内pH值，这可能是研究它们在神经元功能中的作用的有力工具。我们计划分三个阶段开展该项目：首先，我们的目标是选择性地将具有功能构象的细菌视紫红质靶向突触囊泡。其次，我们将优化培养海马神经元中水泡性细菌视紫红质的光诱导质子泵活性。最后，我们将在果蝇体内使用特定的神经元启动子表达优化的细菌视紫红质构建物，用于光诱导果蝇行为的操纵。综上所述，本文提出的研究在连接体外突触功能研究和完整大脑中的信息处理方面具有重要的潜力。这种方法将能够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。

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.