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

体内双光子树突电压成像的验证和优化

• 批准号: 10658307
• 负责人: Jacob Reimer
• 金额: $ 33.9万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-17 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10658307
• 关键词: Acetylcholine; Action Potentials; Anatomy; Animals; Area; Axon; Back; Benchmarking; Brain; Calcium; Calcium Spikes; Cells; Characteristics; Complement; Complex; Contralateral; Data; Dendrites; Dendritic Spines; Detection; Development; Dissociation; Electrophysiology (science); Event; Feedback; Future; Generations; Image; Individual; Injections; Label; Measurement; Measures; Mediating; Membrane; Methods; Microscopy; Neuromodulator; Neurons; Output; Pattern; Performance; Play; Process; Property; Proxy; Pyramidal Cells; Resolution; Role; Series; Shapes; Signal Transduction; Site; Sodium; Source; Synapses; Techniques; Technology; Testing; Thalamic structure; Tissues; Trees; Validation; Vertebral column; Visual; Visual Cortex; adaptive optics; area striata; calcium indicator; experimental study; feasibility testing; hippocampal pyramidal neuron; in vivo; in vivo imaging; insight; neural; performance tests; response; sensor; temporal measurement; two-photon; visual information; voltage

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.

项目概要 了解皮质回路中的信息流需要了解解剖学连接 神经元之间的关系以及神经元的输入被整合以产生尖峰输出的方式。许多 现在可用于研究细胞和大脑区域之间的连接性，但树突整合 这些输入很难观察，因为我们无法获得精细树突中的复杂电信号。 荧光电压传感器有可能彻底改变我们对树突功能的理解 几十年来，皮质回路的整合已得到认可。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，以使其能够 用于具有挑战性的体内树突成像应用。此外，许多荧光电压传感器 不适合双光子成像，而双光子成像需要在单个分支的尺度上解析树突 并在完整的组织中形成数百微米深的刺。新一代基因编码“JEDI”传感器 BCM 的 St. Pierre 实验室开发的技术克服了许多这些限制，现已得到验证 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 必要的灵敏度、光稳定性和亮度，以实现体内树突电活动的成像。 树突双光子成像技术补充了这些传感器的发展，包括 用于提高空间分辨率的自适应光学器件和用于对稀疏标记的树突进行成像的贝塞尔光束整形 由伯克利的 Ji 实验室开发。这些技术已通过树突状钙得到验证 成像，但尚未与体内树突中的 JEDI 电压传感器结合。在这个 R34 提案中，我们 将执行一系列仔细的验证测量，这将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 电压传感器成像。这些实验将 明确揭示该方法具有足够的时间和空间分辨率的程度，以实现 体内树突整合关键方面的观察。优化这些技术将具有广泛的价值 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大脑中。 树突状乔木，以及这种整合过程是如何由神经调节剂（例如乙酰胆碱）塑造的 不同的大脑状态。