The Nanoneedle Net: A flexible and transparent 3D nanoelectrode array for mapping intracellular dendritic dynamics at the cortical surface

项目摘要 哺乳动物大脑中的神经元具有称为树突的复杂树状结构。这些树突状 具有独特形态特征的分支划分并形成突触输入，最终 传播到索马，形成动作电位输出（AP）- 个脑袋揭示树突如何将复杂的突触输入转化为AP输出，塑造群体水平的大脑 活动，并驱动感官输入的基础，我们的神经回路机制和大脑的理解 功能特别感兴趣的是桶皮质中第5层锥体神经元的远端树突，其 躯体输出将感觉运动信息传递到无数的大脑区域。这些深层金字塔 神经元将它们的树突垂直延伸到皮层的最表层， 来自不同大脑区域的广泛输入，并表现出无数的再生反馈事件（例如：NMDA， 钙，钠尖峰），这被认为是控制的整体输入-输出-增益的基础， neuron.然而，树突活动的时空动态，以及 体内树突和体细胞的获得仍然是未知的。这些树突的尺寸很小（直径约2 μm）， 它们的位置（距离表面<100 µm）使传统的全细胞电生理学变得不可行， 目前的黄金标准;而提出的替代方法，如钙成像，缺乏时间 分辨率来报告作为神经计算基础的快速亚阈值膜动力学。 在这里，我们的目标是绘制清醒时体感皮层远端顶端树突的电动力学， 行为小鼠使用灵活和透明的垂直纳米电极平台称为'纳米针网'。 电极帽将包括256个通道，128个平面电极和128个垂直针。每根针都将 高度为40-60 µm，尖端直径约为100 nm，并涂有脂质，以便于无缝渗透到膜中。 我们定制的阵列（电极间距为20 µm）一旦放置在大脑表面， 针穿透深度<60 µm，并与远端树枝状分支形成紧密的电密封。读数将为 通过高度多路复用的低噪声定制CMOS放大器实现。以证实两者的起源 平面表面记录和纳米针树突状记录在体内，我们将结合联合收割机传统的内部和 细胞外真实电生理学，双光子钙成像，光遗传学，使用 模板匹配和触须接触。 最终，这个平台将不仅使我们能够建立远端树突形状的计算规则， 皮层输出在积极的感觉，但提供了一个通用的方法来探测树突整合在生活中 个脑袋

Project Summary Neurons in the mammalian brain possess elaborate tree-like structures termed dendrites. These dendritic branches with distinctive morphological features compartmentalize and shape synaptic inputs, which eventually propagate to the soma to form the action-potential output (AP) – the unit currency of information transfer in the brain. Unravelling how dendrites transform complex synaptic inputs into AP output, shape population-level brain activity, and drive sensory input fundamental to our understanding of neural circuit mechanisms and brain function. Of particular interest are the distal dendrites of layer 5 pyramidal neurons in the barrel cortex, whose somatic output transfers sensorimotor information to a myriad of brain regions. These deep-layer pyramidal neurons extend their dendrites vertically into the most superficial layer of the cortex where they integrate extensive inputs from diverse brain regions, and exhibit a myriad of regenerative feedback events (E.g.: NMDA, Calcium, Sodium spikes), which are believed to be fundamental to controlling the overall input-output-gain of the neuron. However, the spatio-temporal dynamics of dendritic activity, and the overall relationship between dendritic and somatic gain in vivo remains unknown. The small size of these dendrites (~2 µm in diameter) and their location (<100 µm from the surface) has rendered conventional whole-cell electrophysiology infeasible – the current gold-standard; while the proposed alternative approaches, such as calcium imaging, lack temporal resolution to report fast sub-threshold membrane dynamics that underlie neural computation. Here, we aim to map the electrical dynamics of distal apical dendrites in the somatosensory cortex of awake behaving mice using a flexible and transparent vertical nanoelectrode platform termed ‘The Nanoneedle Net’. The Net will comprise of 256 channels with 128 planar electrodes and 128 vertical needles. Each needle will be 40-60 µm in height, ~100 nm in tip diameter, and lipid-coated to facilitate seamless penetration into a membrane. Our custom fabricated array (electrode pitch of 20 µm) once placed on the surface of the brain will allow the needles to penetrate <60 µm deep and form a tight electrical seal with distal dendritic branches. Readout will be accomplished through heavily multiplexed low noise custom CMOS amplifiers. To corroborate the origins of both planar surface recordings and nanoneedle dendritic recordings in vivo, we will combine conventional intra- and extracellular ground-truth electrophysiology, two-photon calcium imaging, optogenetics, spike sorting using template matching, and whisker touch. Ultimately, this platform will not only allow us to establish the computational rules by which distal dendrites shape cortical output during active sensation, but provide a universal method to probe dendritic integration in the living brain.