DYNAMICS OF SIGNAL TRANSDUCTION IN NEURONS

神经元信号传导的动力学

• 批准号: 8782641
• 负责人: ROGER Y TSIEN
• 金额: $ 61.29万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-09-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8782641
• 关键词: 3-Dimensional; Ablation; Age; Anabolism; Antibodies; Apolipoprotein E; Appearance; Autopsy; Behavioral; Blood - brain barrier anatomy; Brain; Cadaver; Cell Cycle; Cells; Chemosensitization; Cleaved cell; Clinical; Development; Drug Delivery Systems; Dyes; Electron Microscopy; Electron Transport; Electrons; Engineering; Extinction (Psychology); Extracellular Matrix; FOS gene; Face; Family; Gelatinase A; Gelatinase B; Genes; Genetic; Goals; Health; Hour; Human; Hybrids; Image; Imagery; In Vitro; Individual; Knock-out; Knockout Mice; Label; Learning; Light; Lighting; Longevity; Maps; Mass Spectrum Analysis; Matrix Metalloproteinase Inhibitor; Measures; Methods; Microscopic; Microtomy; Molecular; Monitor; Mus; Nematoda; Nerve Degeneration; Neurons; Nuclear Weapon; Optics; Pathway interactions; Pattern; Peptide Hydrolases; Pharmaceutical Preparations; Phosphotransferases; Phycobiliproteins; Physiologic pulse; Play; Protease Inhibitor; Protein Biosynthesis; Protein Engineering; Protein Isoforms; Proteins; Recipe; Reporter; Residual state; Resolution; Role; Scanning; Signal Transduction; Singlet Oxygen; Synapses; Synaptophysin; Synaptosomes; System; Techniques; Testing; Time; Traumatic Brain Injury; Use of New Techniques; Viral; activating transcription factor; active control; addiction; base; conditioned fear; conditioning; cranium; directed evolution; dosage; forgetting; improved; in vivo; inhibitor/antagonist; long term memory; mature animal; nanoparticle; novel; optogenetics; overexpression; presynaptic; prevent; protein degradation; protein protein interaction; red fluorescent protein; research study; small molecule; spatial relationship; synaptic inhibition; synaptogenesis; temporal measurement; tool; transcription factor; uptake; vesicle-associated membrane protein; voltage

DESCRIPTION (provided by applicant): This proposal first aims to improve, develop, and test powerful new molecular techniques to monitor and manipulate dynamic signal transduction in neurons. Goals for voltage indicators based on photoinduced electron transfer include greater sensitivity, genetic targeting, and longer wavelengths. New far-red fluorescent proteins engineered from phycobiliproteins are promising building blocks for in vivo indicators of cell cycle status, Ca2+, and protease activity. A novel alternative approach to measuring and manipulating neuronal activity is to engineer an artificial transcription factor activated by simultaneous high [Ca2+] and illumination, greatly improving on endogenous activity reporters such as c-fos. The genetically encoded "snapshot reporter" will capture the pattern of activity throughout a large ensemble of neurons at a time precisely defined by the triggering illumination, then drive expression of effector genes to mark those cells and allow selective excitation, inhibition, or ablation to test their functional importance. A chimeric channelrhodopsi activatable by red light permits optogenetic excitation of deep neurons through the intact skull, but its peak wavelength should be further increased and its residual sensitivity to blue light suppressed. Optogenetic inhibition of synaptic release will be improved by a more efficient singlet-oxygen generating protein, and introducing a singlet- oxygen-sensing GFP to map the spatial extent of inhibition. Both optogenetic tools will be applied to dissect amygdalar circuits n fear conditioning. The singlet-oxygen generating protein can now be split into two complementary fragments that only become photoactive after being brought together by chimeric partners. This complementation system may allow protein-protein interactions and kinase and protease activity to be captured for subsequent visualization by electron microscopy. A genetically encoded tag that marks proteins made during a pharmacologically defined period may become applicable to image synthesis and degradation of proteins in intact brain, thanks to development of nanoparticles that deliver small molecule drugs across the blood-brain barrier. Such nanoparticles may also aid clinical drug delivery to the brain. Such techniques will be used to test a new hypothesis that very long-term memories such as fear conditioning are stored as the pattern of holes in the perineuronal net (PNN), a specialized extracellular matrix that envelops mature neurons and restricts synapse formation. The 3-D intertwining of PNN and synapses will be imaged by serial-section electron microscopy. Lifetimes of PNN vs. intrasynaptic components will be compared by pulse-chase 15N labeling in mice and 14C content in human cadaver brains. Genetically encoded indicators and anti-neoepitope antibodies should improve spatial and temporal resolution of the in vivo activity of proteases that locally erode PNN. New techniques including genetic knockouts, better pharmacological inhibitors, and the snapshot reporter should enable more precise inhibition or potentiation of PNN erosion to compare with behavioral consequences. Biosynthesis of PNN components and proteases will be imaged.

描述（由申请人提供）：该提案的第一个目的是改进，开发和测试强大的新分子技术，以监测和操纵神经元中的动态信号转导。基于光致电子转移的电压指示器的目标包括更高的灵敏度、遗传靶向和更长的波长。新的远红荧光蛋白工程藻胆蛋白是有前途的积木在体内指标的细胞周期状态，Ca 2+，和蛋白酶活性。测量和操纵神经元活动的一种新的替代方法是设计一种人工转录因子，该因子通过同时高[Ca 2 +]和照明激活，极大地改善了内源性活性报告基因（如c-fos）。基因编码的“快照报告”将在触发照明精确定义的时间捕获整个大型神经元集合的活动模式，然后驱动效应基因的表达以标记这些细胞，并允许选择性激发，抑制或消融以测试其功能重要性。一个嵌合channelrhodopsi激活的红光允许光遗传学激发深层神经元通过完整的头骨，但其峰值波长应进一步增加，其残余的敏感性蓝光抑制。突触释放的光遗传抑制将通过更有效的单线态氧产生蛋白质和引入单线态氧感测GFP以映射抑制的空间范围来改善。这两种光遗传学工具将被应用于解剖恐惧条件反射中的杏仁核回路。产生单线态氧的蛋白质现在可以分裂成两个互补的片段，只有在嵌合伴侣聚集在一起后才能变得具有光活性。这种互补系统可以允许蛋白质-蛋白质相互作用以及激酶和蛋白酶活性被捕获，用于随后通过电子显微镜进行可视化。一个基因编码的标签，标志着蛋白质在一个特定的时期可能成为适用于图像合成和蛋白质在完整的大脑降解，由于纳米粒子的发展，提供小分子药物穿过血脑屏障。这样的纳米颗粒也可以帮助临床药物递送到大脑。此类技术将用于测试一个新假设，即恐惧条件反射等非常长期的记忆以神经元周网（PNN）中的孔模式存储，神经元周网是一种专门的细胞外基质，包裹成熟神经元并限制突触形成。PNN和突触的三维缠绕将通过连续切片电子显微镜成像。将通过小鼠中的脉冲追踪15 N标记和人尸体脑中的14 C含量来比较PNN与突触内组分的寿命。遗传编码的指示剂和抗新表位抗体应改善蛋白酶的体内活性的空间和时间分辨率， 局部侵蚀PNN。新技术，包括基因敲除，更好的药理学抑制剂，和快照报告应该能够更精确地抑制或增强PNN侵蚀的行为后果进行比较。将对PNN组分和蛋白酶的生物合成进行成像。