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复杂的树状结构，称为树突。这些树突 具有独特形态特征的分支划分和塑造突触输入，最终 传播到体细胞以形成动作电位输出(AP)--信息传输的单位货币 大脑。揭开树突如何将复杂的突触输入转化为AP输出，塑造人群水平的大脑 活动，并驱动感觉输入，这是我们理解神经回路机制和大脑的基础 功能。特别令人感兴趣的是桶状皮质中第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大脑的无数区域。这些深层次的金字塔 神经元将树突垂直延伸到皮质最浅的一层，在那里它们整合在一起 来自不同大脑区域的广泛输入，并且表现出无数的再生反馈事件(例如：NMDA， 钙，钠尖峰)，这被认为是控制总体投入-产出-收益的基础 神经元。然而，树突活动的时空动力学，以及两者之间的总体关系 树突状细胞和体细胞在体内的获得仍不清楚。这些树枝晶的尺寸很小(直径约2微米)和 它们的位置(离表面100微米)使得传统的全细胞电生理学不可行。 目前的黄金标准；而拟议的替代方法，如钙成像，缺乏临床期 报告作为神经计算基础的快速亚阈值膜动力学的解决方案。 在这里，我们的目标是定位觉醒患者躯体感觉皮层远端心尖树突的电动力学。 使用一种灵活透明的垂直纳米电极平台的小鼠，这种平台被称为“纳米针网”。 该网络将由256个通道、128个平面电极和128个垂直针组成。每一根针都会 高40-60微米，尖端直径约100纳米，并有脂质涂层，有助于无缝渗透到膜中。 我们定制的阵列(电极间距为20微米)一旦放置在大脑表面，将允许 针头可穿透&lt；60微米深，并与远端树枝形成紧密的电密封。读数将为 通过大量多路复用的低噪声定制CMOS放大器来实现。以证实这两种生物的起源 平面表面记录和体内纳米针树突状记录，我们将结合传统的内和 细胞外真实电生理学，双光子钙成像，光遗传学，棘波分选使用 模板匹配和胡须触摸。 最终，这个平台将不仅允许我们建立远端树枝晶形成的计算规则 活跃感觉时的皮质输出，但提供了一种通用的方法来探索活体中的树突整合 大脑。

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.