Name: 3d scanning technology bridging microcircuits and macroscale brain images in 3d novel embedding overlapping protocol
Uploaded: 2019-05-12T13:00:00
Description: Watch this Scientific Journal Video about 3D Scanning Technology Bridging Microcircuits and Macroscale Brain Images in 3D Novel Embedding Overlapping Protocol at JoVE.com

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。

这里描述的所有实验程序都已获得京都大学动物护理委员会的批准。
1. 动物(第1天)
<ol><li>准备雌性C57BL/6J小鼠(n = 6,3~5周)。 注:该协议适用于所有啮齿动物物种。</li>
</ol>2. MRI 设置(第 1 天)
<ol><li>使用加热器垫控制器将鼠标放入放在加热垫上调节至 37°C 的丙烯酸麻醉盒中。</li>
	<li>使用由非脱脂蒸发器、气流计和空气压缩机组成的麻醉...

<ol>
	<li>Vogelsberger, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+galaxies+reproduced+by+a+hydrodynamic+simulation.">Properties of galaxies reproduced by a hydrodynamic simulation.</a> Nature. 509 (7499), 177-182 (2014).</li><li>Zhang, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+systems+approach+identifies+genetic+nodes+and+networks+in+late-onset+Alzheimer&#8217;s+disease.">Integrated systems approach identifies genetic nodes and networks in late-onset Alzheimer&#8217;s disease.</a> Cell. 153 (3), 707-720 (2013).</li><li>Sporns, O., Chialvo, D. R., Kaiser, M., Hilgetag, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization,+development+and+function+of+complex+brain+networks.">Organization, development and function of complex brain networks.</a> Trends in Cognitive Sciences. 8 (9), 418-425 (2004).</li><li>Shimono, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-uniformity+of+cell+density+and+networks+in+the+monkey+brain.">Non-uniformity of cell density and networks in the monkey brain.</a> Scientific Reports. 3, 2541 (2013).</li><li>DeFelipe, J., Jones, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cajal on the Cerebral Cortex: An Annotated Translation of the Complete Wrings. , (1988).</li><li>Kandel, E. R., Schwartz, J. H., Jessell, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles of Neural Science. , (2013).</li><li>Pine, J., Taketani, M., Baudry, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+history+of+MEA+development.">A history of MEA development.</a> Advances in Network Electrophysiology. , 3-23 (2006).</li><li>Grienberger, C., Konnerth, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+calcium+in+neurons.">Imaging calcium in neurons.</a> Neuron. 73 (5), 862-885 (2012).</li><li>Shimono, M., Beggs, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+clusters,+hubs,+and+communities+in+the+cortical+microconnectome.">Functional clusters, hubs, and communities in the cortical microconnectome.</a> Cerebral Cortex. 25 (10), 3743-3757 (2014).</li><li>Amunts, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BigBrain:+An+Ultrahigh-Resolution+3D+Human+Brain+Model.">BigBrain: An Ultrahigh-Resolution 3D Human Brain Model.</a> Science. 340, 1472-1475 (2013).</li><li>Ali, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rigid+and+non-rigid+registration+of+polarized+light+imaging+data+for+3D+reconstruction+of+the+temporal+lobe+of+the+human+brain+at+micrometer+resolution.">Rigid and non-rigid registration of polarized light imaging data for 3D reconstruction of the temporal lobe of the human brain at micrometer resolution.</a> NeuroImage. 181, 235-251 (2018).</li><li>Kozai, T. D. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasmall+implantable+composite+microelectrodes+with+bioactive+surfaces+for+chronic+neural+interfaces.">Ultrasmall implantable composite microelectrodes with bioactive surfaces for chronic neural interfaces.</a> Nature Materials. 11, 1065-1073 (2012).</li><li>Kampasi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+color+optogenetic+control+of+neural+populations+using+low-noise,+multishank+optoelectrodes.">Dual color optogenetic control of neural populations using low-noise, multishank optoelectrodes.</a> Microsystems &amp; Nanoengineering. 4 (1), 10 (2018).</li><li>Hellwig, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+analysis+of+the+local+connectivity+between+pyramidal+neurons+in+layers+2/3+of+the+rat+visual+cortex.">A quantitative analysis of the local connectivity between pyramidal neurons in layers 2/3 of the rat visual cortex.</a> Biological Cybernetics. 82 (2), 111-121 (2000).</li><li>Bezgin, G., Reid, A. T., Schubert, D., K&#246;tter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matching+spatial+with+ontological+brain+regions+using+Java+tools+for+visualization,+database+access,+and+integrated+data+analysis.">Matching spatial with ontological brain regions using Java tools for visualization, database access, and integrated data analysis.</a> Neuroinformatics. 7 (1), 7-22 (2009).</li><li>Holmgren, C., Harkany, T., Svennenfors, B., Zilberter, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyramidal+cell+communication+within+local+networks+in+layer+2/3+of+rat+neocortex.">Pyramidal cell communication within local networks in layer 2/3 of rat neocortex.</a> The Journal of Physiology. 551 (1), 139-153 (2003).</li><li>Boucsein, C., Nawrot, M., Schnepel, P., Aertsen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+the+cortical+column:+abundance+and+physiology+of+horizontal+connections+imply+a+strong+role+for+inputs+from+the+surround.">Beyond the cortical column: abundance and physiology of horizontal connections imply a strong role for inputs from the surround.</a> Frontiers in Neuroscience. 5 (32), 1-13 (2011).</li><li>Edelsbrunner, H., Aronov, J. P. B., Basu, S., Sharir, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+reconstruction+by+wrapping+finite+point+sets+in+space.">Surface reconstruction by wrapping finite point sets in space.</a> Discrete and Computational Geometry - The Goodman-Pollack Festschrift. , 379-404 (2003).</li><li>Roland, P. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+brain+atlas:+For+high&#8208;resolution+functional+and+anatomical+mapping.">Human brain atlas: For high&#8208;resolution functional and anatomical mapping.</a> Human Brain Mapping. 1 (3), 173-184 (1994).</li><li>Johnson, G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Waxholm+space:+an+image-based+reference+for+coordinating+mouse+brain+research.">Waxholm space: an image-based reference for coordinating mouse brain research.</a> NeuroImage. 53 (2), 365-372 (2010).</li><li>Evans, A. C., Janke, A. L., Collins, D. L., Baillet, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+templates+and+atlases.">Brain templates and atlases.</a> NeuroImage. 62 (2), 911-922 (2012).</li><li>Okabe, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain/MINDS&#8211;a+new+program+for+comprehensive+analyses+of+the+brain.">Brain/MINDS&#8211;a new program for comprehensive analyses of the brain.</a> Microscopy. 64 (1), 3-4 (2015).</li><li>Shimono, M., Hatano, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+communication+dynamics+on+macro-connectome,+and+the+propagation+speed.">Efficient communication dynamics on macro-connectome, and the propagation speed.</a> Scientific Reports. 8 (1), 2510 (2018).</li></ol>

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?...

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。

<table>
	<tbody>
		<tr>
			<td>Air compressor</td>
			<td>Kimura Medical</td>
			<td>KA-100</td>
			<td>Animal preparation for MRI</td>
		</tr>
		<tr>
			<td>All-in-one fluorescence microscope</td>
			<td>KEYENCE</td>
			<td>BZ-X710</td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthesia box</td>
			<td>Bio Research Center</td>
			<td>RIC-01</td>
			<td>Animal preparation for MRI</td>
		</tr>
		<tr>
			<td>Anesthesia system</td>
			<td>ACOMA Medical Industry</td>
			<td>NS-5000A</td>
			<td>Animal preparation for MRI</td>
		</tr>
		<tr>
			<td>Anti-GAD67, clone 1G10.2</td>
			<td>Merk Millipore</td>
			<td>MAB5406</td>
			<td>For immunostaining</td>
		</tr>
		<tr>
			<td>Calcium Chrolide</td>
			<td>nacalai tesque</td>
			<td>06729-55</td>
			<td>aCSF</td>
		</tr>
		<tr>
			<td>Choline Chloride</td>
			<td>nacalai tesque</td>
			<td>08809-45</td>
			<td>aCSF</td>
		</tr>
		<tr>
			<td>curved blunt forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposal scalpel</td>
			<td>Kai</td>
			<td>10</td>
			<td></td>
		</tr>
		<tr>
			<td>D-PBS(-) without Ca and Mg, liquid(10x)</td>
			<td>nacalai tesque</td>
			<td></td>
			<td>For immunostaining</td>
		</tr>
		<tr>
			<td>D(+)-Glucose</td>
			<td>Wako</td>
			<td>049-31165</td>
			<td>aCSF</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>nacalai tesque</td>
			<td>16605-42</td>
			<td>re-secctioning</td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488</td>
			<td>Invitrogen</td>
			<td>A32723</td>
			<td>For immunostaining</td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 555</td>
			<td>Invitrogen</td>
			<td>A32732</td>
			<td>For immunostaining</td>
		</tr>
		<tr>
			<td>Heater mat</td>
			<td>Bio Research Center</td>
			<td>HM-10</td>
			<td>Animal preparation for MRI</td>
		</tr>
		<tr>
			<td>Heater mat controller</td>
			<td>Bio Research Center</td>
			<td>BWT-100A</td>
			<td>Animal preparation for MRI</td>
		</tr>
		<tr>
			<td>Heater system</td>
			<td>SA Instruments</td>
			<td>MR-compatible Small Animal Heating System</td>
			<td>Animal preparation for MRI</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>AbbVie</td>
			<td></td>
			<td>Animal preparation for MRI</td>
		</tr>
		<tr>
			<td>Isoflurane vaporizer</td>
			<td>ACOMA Medical Industry</td>
			<td>MKIIIai</td>
			<td>Animal preparation for MRI</td>
		</tr>
		<tr>
			<td>Linear Slicer</td>
			<td>DOSAKA</td>
			<td>Neo Linear Slicer MT</td>
			<td></td>
		</tr>
		<tr>
			<td>L(+)-Ascorbic Acid Sodium Salt</td>
			<td>Wako</td>
			<td>196-01252</td>
			<td>aCSF</td>
		</tr>
		<tr>
			<td>Magnesium Chrolide Hexahydrate</td>
			<td>Wako</td>
			<td>135-00165</td>
			<td>aCSF</td>
		</tr>
		<tr>
			<td>MaxOne Single-Well MEA</td>
			<td>MaxWell Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Metal Spatula</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Monitoring system</td>
			<td>SA Instruments</td>
			<td>Model 1025</td>
			<td>Animal preparation for MRI</td>
		</tr>
		<tr>
			<td>Monitoring software</td>
			<td>SA Instruments</td>
			<td>PC-SAM V.5.12</td>
			<td>Animal preparation for MRI</td>
		</tr>
		<tr>
			<td>MRI compatible cradle</td>
			<td>Bruker BioSpin</td>
			<td>T12812</td>
			<td>Animal preparation for MRI</td>
		</tr>
		<tr>
			<td>MRI coil</td>
			<td>Bruker BioSpin</td>
			<td>T9988</td>
			<td>For MRI</td>
		</tr>
		<tr>
			<td>MRI operation software</td>
			<td>Bruker BioSpin</td>
			<td>ParaVision 5.1</td>
			<td>For MRI</td>
		</tr>
		<tr>
			<td>Neo LinearSlicer MT</td>
			<td>D.S.K.</td>
			<td>NLS-MT</td>
			<td></td>
		</tr>
		<tr>
			<td>NeuN (D4G40) XP Rabbit mAb</td>
			<td>Cell Signaling</td>
			<td>24307</td>
			<td>For immunostaining</td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>Wako</td>
			<td>143-06561</td>
			<td>For immunostaining</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Wako</td>
			<td>163-03545</td>
			<td>aCSF</td>
		</tr>
		<tr>
			<td>Polyethylene Glycol Mono-p-isooctylphenyl Ether</td>
			<td>nacalai tesque</td>
			<td>12967-45</td>
			<td>For immunostaining</td>
		</tr>
		<tr>
			<td>Pressure-sensitive respiration sensor</td>
			<td>SA Instruments</td>
			<td>RS-301</td>
			<td>Animal preparation for MRI</td>
		</tr>
		<tr>
			<td>Preclinical MRI scanner</td>
			<td>Bruker BioSpin</td>
			<td>BioSpec 70/20 USR</td>
			<td>For MRI</td>
		</tr>
		<tr>
			<td>Pyruvic Acid Sodium Salt</td>
			<td>nacalai tesque</td>
			<td>29806-54</td>
			<td>aCSF</td>
		</tr>
		<tr>
			<td>SCAN in a BOX</td>
			<td>Open Technologies srl</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sieve bottle</td>
			<td>TIGERCROWN</td>
			<td>81</td>
			<td>For 3D scan</td>
		</tr>
		<tr>
			<td>SlowFade Gold Antifade Mountant</td>
			<td>Invitrogen</td>
			<td>S36937</td>
			<td>For immunostaining</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Wako</td>
			<td>191-01665</td>
			<td>aCSF</td>
		</tr>
		<tr>
			<td>Sodium Dihydrogenphosphate</td>
			<td>Wako</td>
			<td>197-09705</td>
			<td>aCSF</td>
		</tr>
		<tr>
			<td>Sodium Hydrogen Carbonate</td>
			<td>Wako</td>
			<td>191-01305</td>
			<td>aCSF</td>
		</tr>
		<tr>
			<td>Sodium Hydrogensulfite</td>
			<td>nacalai tesque</td>
			<td>31220-15</td>
			<td>For immunostaining</td>
		</tr>
		<tr>
			<td>Thermistor temperature probe</td>
			<td>SA Instruments</td>
			<td>RTP-101-B, PLTPC-300</td>
			<td>Animal preparation for MRI</td>
		</tr>
		<tr>
			<td>Tooth bar</td>
			<td>Bruker BioSpin</td>
			<td>T10146</td>
			<td>Animal preparation for MRI</td>
		</tr>
		<tr>
			<td>Winged intravenous needle</td>
			<td>TERUMO</td>
			<td>SV-23CLK</td>
			<td>For perfusion</td>
		</tr>
		<tr>
			<td>1 mol/l-Tris-HCl Buffer Solution</td>
			<td>nacalai tesque</td>
			<td>35436-01</td>
			<td>For immunostaining</td>
		</tr>
		<tr>
			<td>1 mol/l-Hydrochloric Acid</td>
			<td>nacalai tesque</td>
			<td>37314-15</td>
			<td>For pH adjustment of solution</td>
		</tr>
		<tr>
			<td>16%-Paraformaldehyde Aqueous Solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td>For immunostaining</td>
		</tr>
	</tbody>
</table>

3d scanning technology bridging microcircuits and macroscale brain images in 3d novel embedding overlapping protocol

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)。

我们评估了通过剥离 MRI 体积产生的皮质表面与从提取的大脑的 3D 扫描中获得的表面之间的距离。距离直方图的模值仅为 55 μm (图3a)。此外,当从距离等于零的点累积直方图时,累积值在 ±300 μm 时达到总样本数的 90%(图 3b)。两个表面之间的距离的最终直方图显示了一个典型峰值约50μm。如果我们从宏观的角度来解释这个值,有趣的...

Watch this Scientific Journal Video about 3D Scanning Technology Bridging Microcircuits and Macroscale Brain Images in 3D Novel Embedding Overlapping Protocol at JoVE.com

3D Scanning Technology Bridging Microcircuits and Macroscale Brain Images in 3D Novel Embedding Overlapping Protocol

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

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)。

The human brain, being a multiscale system, has both macroscopic electrical signals, globally flowing along thick white-matter fiber bundles, and ...

该协议是首次引入解剖、特别是神经解剖学研究领域的3D扫描技术，并取得了高精度的重叠性能。3D 神经嵌入重叠（或 3D 神经协议）将微尺度无菌套接插入宏尺度大脑图像，并无缝地桥接这些不同的空间尺度。我们还必须将 3D 神经协议应用于人类 MRIS 和死后大脑。重叠使我们能够识别已知的与疾病相关的 MRI 对比度模式。3D扫描系统对提取的生物生物周围的水很敏感，因此非常小心地擦水至关重要。此外，必须尽快执行该程序。从准备鼠标以及文本协议中描述的 MRI 采集和设备准备开始此过程。第二天，用手术剪刀沿着下垂缝合线切割先前安乐死小鼠的头皮。使用手术剪刀将头骨从羊角以对角方向切割到小刀锋前面的一点。然后，用弯曲的钳子轻轻地从大脑中剥下头骨。用铲子抬起大脑，用冰冷切割溶液将其转移到培养皿中。使用带受控旋转泵速度的外部气体炸弹，流空气中含有95%的氧气和5%的二氧化碳，在冰冷的切割溶液中产生小气泡。一到两分钟后，将大脑放在微纤维布上，其表面稍微被面粉筛覆盖。使用超纤维布轻轻擦拭大脑表面的液体。将大脑，其后面向上，放在样品支架上。然后，将样品支架放在自动转盘的中心。在 3D 扫描期间使房间变暗，并在转盘上执行 3D 扫描。要测试 3D 扫描是否正常工作，请单击 3D 扫描，选择两个镜头之间的角度为 22.5，起始角度为零，最终角度为 360。将大脑移动到培养皿中，在切割溶液中使大脑冒泡约10秒。用手术刀将大脑切成两个块，在日冕平面中间。然后，使用手术铲轻轻地将两个脑块移动到平坦的表面。使用即时胶水连接脑块基座的脑块。使用超细纤维布，在一到两分钟内从大脑表面轻轻小心地擦拭液体。然后，再次执行 3D 扫描。将刀片连接到振动器的刀片支架上。将覆盖脑块底座中心的黑色胶带连接到振动器的切割阶段中心。将切割溶液倒入切割阶段，并在振动器上设置切割阶段。调整切削速度和振幅。从两个脑块中制作两到五片300微米的日冕片。如果可能，将溶液保持切割阶段冒泡。优化系统的切割速度、频率和振动振幅，将速度设置为每分钟 12.7 毫米，频率设置为 87 至 88 赫兹，秋千宽度设置为 0.8 至 1.0 毫米。在切割大脑切片时，以格式记录切片坐标，包括前后坐标、半球和其他条件。使用厚塑料移液器轻轻地将脑片转移到装满预热ACSF的烧杯。在大约34摄氏度的烧杯中孵育大脑切片一小时。在此期间，对切割阶段的剩余脑块进行 3D 扫描。现在，在录制单元上设置多电子阵列或 MEA 芯片。使用两个管将芯片连接到近位泵。使用一根管子将相同的 ACSF 引导到 MEA 芯片中，使用另一根管引导 ACSF 离开 MEA 芯片。将两根针（连接在两根管子的尖端）连接到 MEA 芯片壁的顶部。在 MEA 芯片的内壁下，用提示固定其位置。将 ACSF 的流量设置为 4.1 RPM。执行 MRI 数据处理以提取文本协议中描述的皮质体积。要执行 MRI 图像处理，请下载 3D 切片器免费软件。打开使用 3D 切片器中的体积渲染和编辑器模块制作的提取大脑的 MR 图像。将模式从编辑器更改为音量渲染，然后单击目标按钮，使大脑的图像位于屏幕中心。选择 MRT-2 大脑模式，通过移动移位栏调整阈值。然后，从音量渲染移回编辑器，然后单击阈值效果按钮。应用标签41，大脑皮层。然后，通过将文件格式从 VTK 更改到表单清单上的 STL，将大脑表面数据保存为 STL 文件。在扫描序列中从 8 或 16 个不同角度拍摄的 8 或 16 张图像之间执行自动核心校正，以纠正小不匹配。为此，请单击对齐选项中包含的"全局注册"并集成图像。从不同角度重复扫描，以获得整个大脑表面。如果从不同角度扫描的图像之间的集成不成功，请单击"手动对齐"并选择一对固定图像和移动图像。通过选择不同图像中的三个或四个公共点，开始图像的手动对齐。然后，单击"确定"。优化算法是迭代最接近点算法，不包括非线性变形。通过选择所有图像并单击网格生成按钮，使所有对齐图像的网格。然后，选择小艺术对象选项，以以最高分辨率获取网格。将图像另存为 STL 二进制或 ASCII 格式。现在，打开 MRI 表面，并使用表面处理软件将其与 3D 扫描表面合并。执行手动对齐过程，如上一样。然后，再次保存这些核心已注册的曲面图像。如有必要，通过清除大脑区域周围的小噪音（尤其是在 MRI 数据的情况下）以及填充任何孔（尤其是 3D 扫描数据）来清理单个表面数据。最后，使用MATLAB等数据分析软件打开表面数据。生成并评估两个曲面之间最小距离的直方图。通过剥离 MRI 体积产生的皮质表面与从提取的大脑的 3D 扫描中获得的表面之间的距离进行了评估。距离直方图的模式值只有 55 微米。此外，当从距离等于零的点累积直方图时，累积值达到大约 300 微米的样本总数的 90%。两个表面之间距离的最终直方图显示，典型峰值约为 50 微米。从宏观角度来看，此模式值对应于几何限制，即从 MRIs 的体素大小到 100 微米。这一点间接表明，MRI 和 3D 扫描之间的重叠算法效果非常好，MRI 和 3D 扫描的噪声水平都作为低值被抑制。该协议是首次通过扫描技术向生物有机体推广。该技术最初用于纯工程需求。将这项技术应用于医学可以回答新的问题。在3D扫描之后，我们可以使用钙成像壁片克录音，在时间、空间分辨率和可记录的细胞数量方面获得互补知识。该协议提供的高精度重叠性能将使解剖空间尺度和零空间比例之间的无缝关联比以前更逼真。

Research

JoVE Journal

Neuroscience

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

神经微电路急性脑片电生理和形态特征采用配对膜片钳记录

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with ...

科研

神经科学

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

接口3D工程神经细胞微电极阵列：一种创新<em&gt;体外</em&gt;实验模型

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the ...

Utilizing 3D Printing Technology to Merge MRI with Histology: A Protocol for Brain Sectioning

利用3D打印技术来合并MRI与组织学：一个大脑切片协议

Magnetic resonance imaging (MRI) allows for the delineation between normal and abnormal tissue on a macroscopic scale, sampling an entire tissue volume ...

High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis

血脑屏障的高分辨率共聚焦成像: 成像、3D 重建和 Transcytosis 的量化

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

用反褶积显微镜观察小鼠原发性下丘脑神经元 GLUT4 蛋白的动态图像

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

活细胞 Sod1 突变蛋白聚集物高含量定量测定方法的建立

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

A Novel In Vitro Model of Blast Traumatic Brain Injury

A Novel In Vitro Model of Blast Traumatic Brain Injury

一种新的脑外伤体损伤模型

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the ...

High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue

溶剂清除脑组织狂犬病病毒感染的高分辨率3D成像

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and ...

A Method for 3D Reconstruction and Virtual Reality Analysis of Glial and Neuronal Cells

胶质细胞和神经细胞的三维重建与虚拟现实分析方法

Serial sectioning and subsequent high-resolution imaging of biological tissue using electron microscopy (EM) allow for the segmentation and reconstruction ...

Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

近红外荧光和高分辨率扫描测量啮齿类动物大脑中蛋白质的表达

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study ...