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.

项目总结 要了解皮质环路中的信息流，需要了解解剖学上的连接性 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 现在可以用技术来研究细胞和大脑区域的连通性，但树突整合 这些输入很难观察，因为我们无法接触到细小树枝晶中的复杂电信号。 荧光电压传感器可能彻底改变我们对树突状细胞功能作用的理解 大脑皮层回路中的整合已经被认识到几十年了。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，无法使其 在具有挑战性的活体树突状成像应用中的应用。此外，许多荧光电压传感器 不适合于双光子成像，这是在单个分支的尺度上分解树枝所需的 并在完整的组织中刺入数百微米深的脊椎。新一代基因编码的“绝地”传感器 在BCM的圣皮埃尔实验室开发，克服了许多这些限制，现在已经得到验证 用于体内的双光子体细胞成像。这些验证实验表明绝地传感器具有 必要的灵敏度、光稳定性和亮度，以便能够对活体树突中的电活动进行成像。 作为对这些传感器开发的补充，树枝状双光子成像技术包括 用于提高空间分辨率的自适应光学和用于稀疏标记树突成像的贝塞尔波束整形 是由伯克利的纪万昌实验室开发的。这些技术已经用树枝状钙进行了验证 成像，但尚未与体内树突中的绝地电压传感器相结合。在这份R34提案中，我们 将执行一系列仔细的验证测量，使我们能够优化高级双光子 用于询问树突电生理的绝地电压传感器的成像。这些实验将 毫不含糊地揭示该方法具有足够的时间和空间分辨率以实现 在体内观察树突整合的关键方面。优化这些技术将具有广泛的价值 ，并将使未来的大脑电路项目能够回答关于如何 初级视觉皮层回路中的锥体神经元整合了不同来源的视觉信息 树突状乔木，以及这一整合过程是如何由神经调节剂，如乙酰胆碱 不同的大脑状态。