DYNAMICS OF SIGNAL TRANSDUCTION IN NEURONS

描述(申请人提供)：这项提案首先旨在改进、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、遗传靶向性和更长的波长。从藻胆蛋白中设计的新型远红荧光蛋白有望成为细胞周期状态、钙离子和蛋白酶活性的体内指示剂。另一种测量和控制神经元活性的新方法是设计一种人工转录因子，由高[Ca~(2+)]和光照同时激活，大大改进了c-fos等内源性活性报告。基因编码的“快照报告器”将在触发光照精确定义的时间捕捉一大群神经元的活动模式，然后驱动效应基因的表达来标记这些细胞，并允许选择性激发、抑制或消融来测试它们的功能重要性。可由红光激活的嵌合通道视紫质允许通过完整的颅骨对深层神经元进行光遗传兴奋，但其峰值波长应进一步增加，其对蓝光的残余敏感性应受到抑制。光遗传对突触释放的抑制将通过更有效的单线态氧产生蛋白来改善，并引入单线态氧感应GFP来绘制抑制的空间范围。这两种光遗传工具都将用于解剖恐惧条件反射中的杏仁核回路。这种产生单线态氧气的蛋白质现在可以分裂成两个互补的片段，只有在嵌合伙伴将它们结合在一起后，这两个片段才会变得具有光活性。这种互补系统可能允许蛋白质之间的相互作用，并捕捉到激酶和蛋白水解酶的活性，用于随后的电子显微镜观察。由于纳米粒子的开发，可以将小分子药物穿过血脑屏障运送出去，因此，一种标记在药理学定义的时间段内制造的蛋白质的基因编码标签可能会适用于完整大脑中蛋白质的图像合成和降解。这种纳米颗粒还可以帮助临床药物输送到大脑。这种技术将被用来测试一个新的假设，即非常长期的记忆，如恐惧条件反射，是以神经周围神经网络(PNN)中的空洞模式存储的，PNN是一种包裹成熟神经元并限制突触形成的专门细胞外基质。PNN和突触的三维缠绕将用连续切片电子显微镜成像。PNN和突触内成分的寿命将通过脉冲追逐15N标记和人类身体大脑中的14C含量进行比较。基因编码的指示物和抗新表位抗体应该可以提高体内蛋白酶活性的空间和时间分辨率 局部侵蚀PNN。包括基因敲除、更好的药理抑制剂和快照报告在内的新技术应该能够更精确地抑制或增强PNN侵蚀，以与行为后果进行比较。PNN组分和蛋白酶的生物合成将被成像。

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.