Light-induced reversible manipulation of neurotransmitter release

光诱导神经递质释放的可逆操纵

• 批准号: 8269870
• 负责人: Ege T Kavalali
• 金额: $ 19.85万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8269870
• 关键词: Acute; Animals; Area; Bacteriorhodopsins; Behavior; Behavioral; Brain; Cells; Chemicals; Chimeric Proteins; Dependence; Drosophila genus; Electric Stimulation; Electrophysiology (science); Engineering; Fiber; Fluorescence; Global Change; Goals; Halobacterium salinarium; Hippocampus (Brain); Image; In Vitro; Knowledge; Larva; Life; Light; Lysosomes; Measures; Membrane; Membrane Potentials; Methodology; Molecular Conformation; Monitor; Neurons; Neurosciences; Neurotransmitters; Optical Methods; Optics; Organelles; Output; Population; Process; Property; Proteins; Proton Pump; Research; Role; Signal Transduction; Staging; Synapses; Synaptic Transmission; Synaptic Vesicles; System; Testing; Transgenic Organisms; Work; base; fly; in vivo; information processing; inhibitor/antagonist; neurotransmitter release; operation; optogenetics; pH gradient; promoter; research study; response; sensor; small molecule; synaptic depression; tool; tool development; trafficking; vacuolar H+-ATPase

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.

描述（由申请人提供）：在本申请中，我们提出采用细菌视紫红质（一种来自盐生盐杆菌的光激活质子泵）来操纵突触囊泡中的pH梯度。充满神经递质的突触囊泡对囊泡内pH高度敏感，而囊泡内pH受内源性囊泡质子泵-液泡ATP酶（v-ATP酶）调节。最近的研究，包括我们小组的工作，表明小分子抑制剂（如巴弗洛霉素）对v-ATP酶的抑制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值依赖性的突触囊泡再填充过程中，通过使用细菌视紫红质针对神经元中的突触囊泡，以模仿的v-ATP酶抑制剂的作用，在光诱导和快速可逆的方式，而没有全球性的变化膜兴奋性。此外，将细菌视紫红质靶向其他分泌细胞器，如溶酶体，其严格依赖于囊内pH值以进行其适当操作，这可以是研究其在神经元功能中的作用的有力工具。我们建议分三个阶段开发该项目：首先，我们的目标是选择性地将功能构象中的细菌视紫红质靶向突触囊泡。其次，我们将优化光诱导的质子泵活性的泡状菌紫质在培养的海马神经元。最后，我们将在果蝇中使用特定的神经元启动子在体内表达优化的细菌视紫红质构建体，用于光诱导操纵果蝇行为。总之，本文提出的研究在体外突触功能研究和完整大脑信息处理方面具有重要的潜力。这种方法将能够对进入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以及行为输出。