Massively scalable 3D electrophysiology and two-photon imaging in freely-moving animals

总结 揭示神经回路是如何编码和实现行为体验的， 神经科学解码和剖析跨此类电路的信号流的机制需要 记录毫秒电动力学的能力，同时映射细胞的空间组织， 亚细胞电路图案，在清醒的行为动物。在自然行为中，动物主动获得感官 信息，并使用这些信息来指导正在进行的行动。在这 上下文，不受约束的运动相关信号可以让感觉系统有效地预测自我， 产生的运动，并提取有关环境的附加信息，从而形成一个稳定的内部 代表外部世界。然而，大多数记录是在头部固定的动物中进行的 这对与运动相关的信号如何塑造正在进行的感觉和记忆施加了严格的限制 在大脑中处理。进行高密度电生理和伴随的双光子钙成像 由于技术限制，目前尚不可行，因此在两个固定头中分别进行 和自由移动的制剂。非常需要能够将高分辨率联合收割机 整个脑组织体积的电记录和自由行为的双光子钙成像 动物在该提案中，我们介绍了一种新的范例，用于跨3D体积的高密度电生理学， 能够同时进行双光子钙成像。我们的创新称为NET-2 P，包括 3D纳米电极的可变高度集成到头戴式微型双光子 显微镜纳米电极阵列集成了定制设计的CMOS电子器件， 重3.5克纳米电极阵列的透明和柔性性质允许容易的双光子测量。 同时便于在大的皮质切片上进行快速电标测。我们建议使用头部- 安装的装置，以1）在触觉处理下测定皮层行波， 通过两个p成像的细胞尺度集合图和2）解开皮层集合和行波， 在学习任务后出现增强睡眠期间的记忆巩固。在初步实验中， 在头部固定清醒的动物被动胡须触摸下，我们使用平面透明电极阵列 记录和传统的双光子钙成像，并发现微观行波后， 触须接触，接触后100 ms的晚期反射波，稀疏但稳定的细胞系综结构 支持波的传播我们假设自发的行波，包括晚期的反射波， 刺激后数百毫秒出现的成分，携带与运动相关的头部方向， 意志控制信号，这将增强自由移动的小鼠的行波动力学。通过组合 在自然睡眠期间的电生理学和基于成像的整体映射，我们将分析尖峰和 跨皮质层的突触变化加强并实现了强大的功能性细胞活动景观。

SUMMARY Revealing how neural circuits encode and enable behavioral experiences is a fundamental problem in neuroscience. Decoding and dissecting the mechanisms of signal flow across such circuits necessitates the ability to record millisecond electrical dynamics and simultaneously map the spatial organization of cellular and sub-cellular circuit motifs, in awake behaving animals. During natural behavior, animals actively acquire sensory information as they move through the environment and use this information to guide ongoing actions. In this context, unconstrained movement-related signals could allow sensory systems to efficiently predict self- generated motion and extract additional information about the environment, thereby forming a stable internal representation of the external world. However, a majority of recordings are performed in head-fixed animals which imposes severe restrictions on how movement related signals shape ongoing sensory and memory processing in the brain. Performing high-density electrophysiology and concomitant two-photon calcium imaging is -at present- not feasible due to technical limitations and are therefore performed separately in both head fixed and freely moving preparations. There is a great need for technology platforms that can combine high-resolution electrical recordings across entire volumes of brain tissue and two-photon calcium imaging in freely behaving animals. In this proposal we introduce a new paradigm for high-density electrophysiology across 3D volume with capabilities to simultaneously perform two-photon calcium imaging. Our innovation termed NET-2P, comprises of 3D Nanoelectrodes of variable height integrated onto the baseplate of a head-mounted mini two-photon microscope. The Nanoelectrode array is integrated with custom-designed CMOS electronics and will in total weigh 3.5 grams. The transparent and flexible nature of the nanoelectrode array allows for easy two-photon access whilst facilitating rapid electrical mapping across large cortical sections. We propose to use the head- mounted setup to 1) assay cortical travelling waves under tactile processing whilst mapping the underlying cellular scale ensemble map via two-p imaging and 2) unravel how cortical ensembles and travelling waves that emerge after a learning task enhance memory consolidation during sleep. In preliminary experiments performed in head-fixed awake animals under passive whisker touch, we used planar transparent electrode array recordings and conventional two-photon calcium imaging, and discovered microscopic travelling waves upon whisker touch, a late reverberatory wave 100ms post touch, and sparse yet stable cellular ensemble structure that supports wave propagation. We hypothesize that spontaneous travelling waves, including late reverberatory components that emerge hundreds of milliseconds post stimulus, carry movement related, head-direction, and volitional control signals, which will enhance travelling wave dynamics in freely-moving mice. By combining electrophysiology and imaging-based ensemble mapping during natural sleep we will assay how spiking and synaptic changes across cortical layers strengthen and enable robust functional cellular activity landscapes.