Validation and Optimization of Two-Photon Dendritic Voltage Imaging in Vivo

项目摘要 要理解皮层回路中的信息流， 以及对神经元的输入进行积分以生成尖峰输出的方式。许多 现在有技术可以研究细胞和大脑区域之间的连接，但是树突状细胞的整合， 观察这些输入是具有挑战性的，因为我们无法获得精细树突中的复杂电信号。 荧光电压传感器的潜力彻底改变了我们对树突状细胞功能作用的理解。 几十年来，人们已经认识到皮层回路中的整合。然而，在实践中，荧光电压传感器 在亮度、灵敏度和光稳定性方面缺乏必要的特性， 用于体内树突状成像的挑战性应用。此外，许多荧光电压传感器 不适合双光子成像，这需要在单个分支的尺度上分辨树突 在完整的组织中有数百微米深的刺新一代基因编码的“JEDI”传感器 在圣皮埃尔实验室开发的，克服了许多这些限制，现在已经得到验证 用于体内双光子体细胞成像。这些验证实验表明，JEDI传感器具有 这需要必要的灵敏度、光稳定性和亮度，以能够对体内树突中的电活动进行成像。 作为对这些传感器的补充，树突的双光子成像技术，包括 用于提高空间分辨率的自适应光学器件和用于稀疏标记树突成像的贝塞尔光束整形 是由伯克利的Ji实验室开发的。这些技术已被验证与树突状钙 成像，但尚未与体内树突中的JEDI电压传感器组合。在这个R34提案中，我们 将进行一系列仔细的验证测量，使我们能够优化先进的双光子 用于询问树突电生理学的JEDI电压传感器的成像。这些实验将 明确揭示了这种方法在多大程度上具有足够的时间和空间分辨率， 观察体内树突整合的关键方面。优化这些技术将具有广泛的价值 适用于该领域，并将使未来的BRAIN Circuits项目能够回答有关如何 初级视觉皮层回路中的锥体神经元将不同来源的视觉信息整合到它们的 树突状乔木，以及这种整合过程是如何通过神经调质，如乙酰胆碱， 不同的大脑状态

PROJECT SUMMARY Understanding information flow in cortical circuits requires understanding both the anatomical connectivity between neurons and the way in which inputs to a neuron are integrated to generate a spiking output. Many techniques are now available to study connectivity across cells and brain areas, but the dendritic integration of these inputs is challenging to observe because we lack access to the complex electrical signals in fine dendrites. The potential for fluorescent voltage sensors to revolutionize our understanding of the functional role of dendritic integration in cortical circuits has been recognized for decades. However, in practice, fluorescent voltage sensors have lacked the necessary characteristics in terms of brightness, sensitivity, and photostability to enable their use in the challenging application of dendritic imaging in vivo. Furthermore, many fluorescent voltage sensors are unsuitable for two-photon imaging, which is required to resolve dendrites at the scale of individual branches and spines hundreds of microns deep in intact tissue. A new generation of genetically-encoded “JEDI” sensors developed in the St. Pierre lab here at BCM overcome many of these limitations, and have now been validated for two-photon somatic imaging in vivo. These validation experiments suggest that JEDI sensors have the necessary sensitivity, photostability, and brightness to enable imaging of electrical activity in dendrites in vivo. Complementing the development of these sensors, technologies for two-photon imaging of dendrites, including adaptive optics for increasing spatial resolution and Bessel beam shaping for imaging sparsely-labeled dendrites have been developed by the Ji lab at Berkeley. These techniques have been validated with dendritic calcium imaging, but have not been combined with JEDI voltage sensors in dendrites in vivo. In this R34 proposal, we will perform a careful series of validation measurements that will enable us to optimize advanced two-photon imaging of JEDI voltage sensors for interrogating dendritic electrophysiology. These experiments will unambiguously reveal the extent to which this approach has sufficient temporal and spatial resolution to enable observations of key aspects of dendritic integration in vivo. Optimizing these techniques will be widely valuable for the field, and will enable a future BRAIN Circuits project to answer fundamental questions about how pyramidal neurons in primary visual cortex circuits integrate different sources of visual information in their dendritic arbors, and how this process of integration is shaped by neuromodulators such as acetylcholine across different brain states.