Light-induced reversible manipulation of neurotransmitter release

光诱导神经递质释放的可逆操纵

• 批准号: 8177107
• 负责人: Ege T Kavalali
• 金额: $ 23.78万
• 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/8177107
• 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; 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. 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.

描述（由申请人提供）：在本申请中，我们建议采用细菌紫红素，一种从盐酸盐分子的光激活的质子泵，以操纵突触囊泡中的pH梯度。神经递质填充的突触囊泡对插入式pH值高度敏感，该pH由固有的囊泡质子泵（液泡ATPase（V-ATPase））调节。最近的研究，包括来自我们小组的工作，表明小分子抑制剂（例如，bafilymycin）对V-ATPase的抑制会导致突触释放的突触反应和阻断神经递质释放的快速使用依赖性消失。在该项目中，我们将通过使用针对神经元中突触囊泡的细菌近淡葡萄蛋白来利用突触囊泡重新填充过程的这种严格的pH依赖性，以在光诱导且快速可逆的时尚中，在膜兴奋性中没有全球变化，以在光诱导的和快速可逆的方式中模仿V-ATPase抑制剂的效果。此外，将细菌紫红素靶向其他分泌细胞器，例如在关键上依赖于内部pH值的溶酶体，可以使其适当的手术成为研究其在神经元功能中的作用的强大工具。我们建议在三个阶段开发该项目：首先，我们的目标是在功能构象中选择性地靶向突触囊泡。其次，我们将优化培养的海马神经元中囊泡细菌蛋白的光诱导的质子泵活性。最后，我们将使用果蝇中的特定神经元启动子在体内表达优化的细菌蛋白构建体，以对果蝇行为进行光诱导的操纵。综上所述，此处提出的研究在体外和完整大脑中的信息处理中桥接突触功能研究具有巨大的潜力。这种方法将使突触输入急性操纵到大脑的特定区域，以测试它们如何影响功能和行为输出。 公共卫生相关性：在本应用中，我们建议靶向细菌淡蛋白，这是一种来自盐杆菌的光激活的质子泵，特别是通过工程融合蛋白来以可逆和光引起的时尚来损害突触传播，专门用于突触囊泡。该项目的成功完成将产生重要的工具，可以使突触特定地操纵神经元电路。