3D 扫描技术在 3D 新颖嵌入重叠协议中桥接微电路和宏观大脑图像
Summary
本文介绍了一种利用三维扫描技术将两个空间尺度连接起来的实验方案:由MRI在>100 μm处成像的全脑解剖结构的宏观空间尺度,以及使用神经元分布的微观空间尺度免疫组织化学染色和多电极阵列系统等方法(±10μm)。
Abstract
人脑是一个多尺度系统,它同时具有宏观电信号,沿着厚厚的白质纤维束在全球流动,以及微小的神经元尖峰,沿着斧头和树突传播。两种尺度都补充了人类认知和行为功能的不同方面。在宏观层面,MRI是目前的标准成像技术,其中最小的空间分辨率,体素大小,是0.1×1毫米3。此外,在微观层面上,以前的生理学研究知道这种体素内的神经元结构不均匀。本研究通过将生物科学研究与3D扫描技术的进步相连接,为将微观数据准确嵌入宏观图谱而开发一种强有力的方法。由于3D扫描技术一直主要用于工程和工业设计,它第一次被重新用于将微连接体嵌入整个大脑,同时保留活脑细胞中的天然尖峰。为了实现这一目标,首先,我们构建了一个扫描协议,从生物生物中获取准确的3D图像,由于潮湿和反射表面,对图像本身具有挑战性。其次,我们训练保持速度,以防止活脑组织的退化,这是保持更好的条件和记录更多的自然神经元尖峰从脑组织的活动神经元的关键因素。两个皮质表面图像,从两个不同的成像模块(即 MRI 和 3D 扫描仪表面图像)独立提取,令人惊讶的是,作为直方图的模值,其距离误差仅为 50 μm。这种精度在尺度上可与细胞间距离的微观分辨率相媲美;此外,它是稳定的不同单独的小鼠。这种新协议,3D新嵌入重叠(3D-NEO)协议,桥接由该集成协议派生的宏观和微观水平,并加速新的科学发现,以研究综合连接架构(即,微连接)。
Introduction
不同物理和生物组织的非均匀多尺度架构通常见1,2。大脑也是一个非常不统一和多尺度的网络组织3,4。各种认知功能被编码在这样的网络组织中,在亚毫秒时间分辨率中保存神经元群的电尖模式的时间变化。从历史上看,神经元之间的复杂网络被结构上详细观察使用染色技术由圣地亚哥拉蒙和卡哈尔从150多年前5。为了观察活动神经元的群体行为,研究人员开发了各种记录技术6,7,8,而最近这些技术的重大发展使我们能够记录同时来自大量神经元的电活动。此外,通过这些功能活动,科学家已经成功地重建了大量神经元之间的因果相互作用网络,并宣告了其复杂相互作用的拓扑结构”微连接体”9.对大脑的宏观观察也允许将整个大脑视为一个网络组织,因为许多大脑区域都通过多个纤维束连接。将微连接体嵌入到全球大脑图谱中,在目前的技术进步中仍有明显的局限性,这就是为什么这种嵌入协议如此重要的原因。但是,嵌入协议的开发面临着许多挑战。例如,为了观察纯隔离的大脑区域中活的局部神经元回路的活动,需要为体外记录制作脑切片。此外,从脑切片进行体外录音仍然是一个重要的选择,至少有两个原因。首先,从比±1.5毫米深的大脑区域和高时间分辨率(<1 ms)同时观察许多活个体神经元的活动仍然不容易。其次,当我们希望了解局部神经元回路的内部结构时,我们需要停止来自外部大脑区域的所有输入,以消除混淆因素。为了确定所生成脑切片的方向和位置,还需要使用坐标集成这些生成的脑切片的空间位置。然而,有一些系统可靠的方法,使大脑切片有组织的方式10,11。这里引入了一种新的共注册协议,使用3D扫描技术进行神经科学研究,以提供一体化协议。该协议用于协调微尺度和宏尺度,并通过提取的大脑的 3D 扫描表面,将数据嵌入到宏观 MRI 空间上,并将多电极阵列 (MEA) 微数据12、13和染色数据嵌入到宏观 MRI 空间上,非侵入性记录的大脑。令人惊讶的是,这显示距离误差仅为 ±50 μm,作为直方图的模式值。因此,所有六个小鼠的 MRI 表面与扫描的 3D 表面之间最小距离的模值接近 50 μm,这是检查个体之间的共性时的适当数字。典型的切片宽度记录的峰值活动约为 300 μm。
Protocol
Representative Results
Discussion
我们开发了一种称为3D-NEO协议的新协议,通过比以前更精确地重叠两个大脑表面来桥接宏观和微观空间尺度。最初,在创建该协议时有两个挑战,这使得两个大脑表面图像的准确重叠和记录来自生物体的健康神经元活动成为可能。首先,有必要在从头骨中提取切出切出后,在不伤害大脑有机体的情况下,有效地擦拭被提取的大脑周围的切割溶液(协议步骤6.2)。其次,由于干燥和时间延迟的第一和第三个条件?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
M.S.感谢医学院医学院医学信息工程课程全体教职员工的支持,并感谢高村正彦教授、野本教授和多里斯·扎基安教授的帮助。评论。这项研究得到了挑战性探索研究援助资助和教育部(教育、文化、体育、科学和技术部)对M.S.的优秀青年研究人员领导计划的支持。这项工作的MRI实验在日本京都大学医学院小动物MRI系医学研究支持中心进行。
Materials
| Air compressor | Kimura Medical | KA-100 | Animal preparation for MRI |
| All-in-one fluorescence microscope | KEYENCE | BZ-X710 | |
| Anesthesia box | Bio Research Center | RIC-01 | Animal preparation for MRI |
| Anesthesia system | ACOMA Medical Industry | NS-5000A | Animal preparation for MRI |
| Anti-GAD67, clone 1G10.2 | Merk Millipore | MAB5406 | For immunostaining |
| Calcium Chrolide | nacalai tesque | 06729-55 | aCSF |
| Choline Chloride | nacalai tesque | 08809-45 | aCSF |
| curved blunt forceps | |||
| Disposal scalpel | Kai | 10 | |
| D-PBS(-) without Ca and Mg, liquid(10x) | nacalai tesque | For immunostaining | |
| D(+)-Glucose | Wako | 049-31165 | aCSF |
| Gelatin | nacalai tesque | 16605-42 | re-secctioning |
| Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 | Invitrogen | A32723 | For immunostaining |
| Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 555 | Invitrogen | A32732 | For immunostaining |
| Heater mat | Bio Research Center | HM-10 | Animal preparation for MRI |
| Heater mat controller | Bio Research Center | BWT-100A | Animal preparation for MRI |
| Heater system | SA Instruments | MR-compatible Small Animal Heating System | Animal preparation for MRI |
| Isoflurane | AbbVie | Animal preparation for MRI | |
| Isoflurane vaporizer | ACOMA Medical Industry | MKIIIai | Animal preparation for MRI |
| Linear Slicer | DOSAKA | Neo Linear Slicer MT | |
| L(+)-Ascorbic Acid Sodium Salt | Wako | 196-01252 | aCSF |
| Magnesium Chrolide Hexahydrate | Wako | 135-00165 | aCSF |
| MaxOne Single-Well MEA | MaxWell Biosystems | ||
| Metal Spatula | |||
| Monitoring system | SA Instruments | Model 1025 | Animal preparation for MRI |
| Monitoring software | SA Instruments | PC-SAM V.5.12 | Animal preparation for MRI |
| MRI compatible cradle | Bruker BioSpin | T12812 | Animal preparation for MRI |
| MRI coil | Bruker BioSpin | T9988 | For MRI |
| MRI operation software | Bruker BioSpin | ParaVision 5.1 | For MRI |
| Neo LinearSlicer MT | D.S.K. | NLS-MT | |
| NeuN (D4G40) XP Rabbit mAb | Cell Signaling | 24307 | For immunostaining |
| Normal Goat Serum | Wako | 143-06561 | For immunostaining |
| Potassium Chloride | Wako | 163-03545 | aCSF |
| Polyethylene Glycol Mono-p-isooctylphenyl Ether | nacalai tesque | 12967-45 | For immunostaining |
| Pressure-sensitive respiration sensor | SA Instruments | RS-301 | Animal preparation for MRI |
| Preclinical MRI scanner | Bruker BioSpin | BioSpec 70/20 USR | For MRI |
| Pyruvic Acid Sodium Salt | nacalai tesque | 29806-54 | aCSF |
| SCAN in a BOX | Open Technologies srl | ||
| scissors | |||
| Sieve bottle | TIGERCROWN | 81 | For 3D scan |
| SlowFade Gold Antifade Mountant | Invitrogen | S36937 | For immunostaining |
| Sodium Chloride | Wako | 191-01665 | aCSF |
| Sodium Dihydrogenphosphate | Wako | 197-09705 | aCSF |
| Sodium Hydrogen Carbonate | Wako | 191-01305 | aCSF |
| Sodium Hydrogensulfite | nacalai tesque | 31220-15 | For immunostaining |
| Thermistor temperature probe | SA Instruments | RTP-101-B, PLTPC-300 | Animal preparation for MRI |
| Tooth bar | Bruker BioSpin | T10146 | Animal preparation for MRI |
| Winged intravenous needle | TERUMO | SV-23CLK | For perfusion |
| 1 mol/l-Tris-HCl Buffer Solution | nacalai tesque | 35436-01 | For immunostaining |
| 1 mol/l-Hydrochloric Acid | nacalai tesque | 37314-15 | For pH adjustment of solution |
| 16%-Paraformaldehyde Aqueous Solution | Electron Microscopy Sciences | 15710 | For immunostaining |
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