这项工作部分得到JSPS科学研究援助赠款(C)(第22500336号、25430068号、16K07061号)(Y. Sasaki)的支持。我们感谢野中泰郎博士和木崎美子女士(横滨城市大学)提供生物素素的LRRTM2蛋白。我们还感谢小池和石井的技术援助。

本手稿中描述的实验是根据横滨城市大学机构动物护理和使用委员会概述的准则进行的。
1. 神经元球作为悬垂培养物的准备(天体外(DIV) 0-3)
注:此处描述的神经元球培养制备过程基于Sasaki组先前报告的方法,并作了一些修改9,10。我们还从Banker方法中采用了几种程序来分离文化14。
<ol><li>
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<ol>
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H., Li, P., Shen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+molecular+mechanisms+underlying+presynapse+formation.">Cellular and molecular mechanisms underlying presynapse formation.</a> Journal of Cell Biology. 203 (1), 11-22 (2013).</li><li>S&#252;dhof, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroligins+and+neurexins+link+synaptic+function+to+cognitive+disease.">Neuroligins and neurexins link synaptic function to cognitive disease.</a> Nature. 455 (7215), 903-911 (2008).</li><li>Saitsu, H., 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=CASK+aberrations+in+male+patients+with+Ohtahara+syndrome+and+cerebellar+hypoplasia.">CASK aberrations in male patients with Ohtahara syndrome and cerebellar hypoplasia.</a> Epilepsia. 53 (8), 1441-1449 (2012).</li><li>Friedman, H. V., Bresler, T., Garner, C. C., Ziv, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+of+New+Individual+Excitatory+Synapses.">Assembly of New Individual Excitatory Synapses.</a> Neuron. 27 (1), 57-69 (2000).</li><li>Bresler, T., 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=Postsynaptic+density+assembly+is+fundamentally+different+from+presynaptic+active+zone+assembly.">Postsynaptic density assembly is fundamentally different from presynaptic active zone assembly.</a> The Journal of neuroscience the official journal of the Society for Neuroscience. 24 (6), 1507-1520 (2004).</li><li>Parvin, S., Takeda, R., Sugiura, Y., Neyazaki, M., Nogi, T., Sasaki, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fragile+X+mental+retardation+protein+regulates+accumulation+of+the+active+zone+protein+Munc18-1+in+presynapses+via+local+translation+in+axons+during+synaptogenesis.">Fragile X mental retardation protein regulates accumulation of the active zone protein Munc18-1 in presynapses via local translation in axons during synaptogenesis.</a> Neuroscience Research. , (2018).</li><li>Sasaki, 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=Phosphorylation+of+Zipcode+Binding+Protein+1+Is+Required+for+Brain-Derived+Neurotrophic+Factor+Signaling+of+Local+&#946;-Actin+Synthesis+and+Growth+Cone+Turning.">Phosphorylation of Zipcode Binding Protein 1 Is Required for Brain-Derived Neurotrophic Factor Signaling of Local &#946;-Actin Synthesis and Growth Cone Turning.</a> Journal of Neuroscience. 30 (28), 9349-9358 (2010).</li><li>Sasaki, Y., Gross, C., Xing, L., Goshima, Y., Bassell, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+axon-enriched+microRNAs+localized+to+growth+cones+of+cortical+neurons.">Identification of axon-enriched microRNAs localized to growth cones of cortical neurons.</a> Developmental neurobiology. 74 (3), 397-406 (2014).</li><li>Linhoff, M. W., 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=An+Unbiased+Expression+Screen+for+Synaptogenic+Proteins+Identifies+the+LRRTM+Protein+Family+as+Synaptic+Organizers.">An Unbiased Expression Screen for Synaptogenic Proteins Identifies the LRRTM Protein Family as Synaptic Organizers.</a> Neuron. 61 (5), 734-749 (2009).</li><li>de Wit, J., 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=LRRTM2+Interacts+with+Neurexin1+and+Regulates+Excitatory+Synapse+Formation.">LRRTM2 Interacts with Neurexin1 and Regulates Excitatory Synapse Formation.</a> Neuron. 64 (6), 799-806 (2009).</li><li>Ko, J., Fuccillo, M. V., Malenka, R. C., S&#252;dhof, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LRRTM2+Functions+as+a+Neurexin+Ligand+in+Promoting+Excitatory+Synapse+Formation.">LRRTM2 Functions as a Neurexin Ligand in Promoting Excitatory Synapse Formation.</a> Neuron. 64 (6), 791-798 (2009).</li><li>Kaech, S., Banker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+hippocampal+neurons.">Culturing hippocampal neurons.</a> Nature protocols. 1 (5), 2406-2415 (2006).</li><li>Hirai, H., 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=Structural+basis+for+ligand+capture+and+release+by+the+endocytic+receptor+ApoER2.">Structural basis for ligand capture and release by the endocytic receptor ApoER2.</a> EMBO reports. , (2017).</li><li>Yasui, N., 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=Functional+Importance+of+Covalent+Homodimer+of+Reelin+Protein+Linked+via+Its+Central+Region.">Functional Importance of Covalent Homodimer of Reelin Protein Linked via Its Central Region.</a> Journal of Biological Chemistry. 286 (40), 35247-35256 (2011).</li><li>Bassell, G. J., Warren, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fragile+X+Syndrome:+Loss+of+Local+mRNA+Regulation+Alters+Synaptic+Development+and+Function.">Fragile X Syndrome: Loss of Local mRNA Regulation Alters Synaptic Development and Function.</a> Neuron. 60 (2), 201-214 (2008).</li><li>Darnell, J. C., Klann, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+translation+of+translational+control+by+FMRP:+therapeutic+targets+for+FXS.">The translation of translational control by FMRP: therapeutic targets for FXS.</a> Nat Neurosci. 16 (11), 1530-1536 (2013).</li><li>Richter, J. D., Bassell, G. J., Klann, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysregulation+and+restoration+of+translational+homeostasis+in+fragile+X+syndrome.">Dysregulation and restoration of translational homeostasis in fragile X syndrome.</a> Nature Reviews Neuroscience. 16 (10), 595-605 (2015).</li><li>Lucido, A. L., 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=Rapid+Assembly+of+Functional+Presynaptic+Boutons+Triggered+by+Adhesive+Contacts.">Rapid Assembly of Functional Presynaptic Boutons Triggered by Adhesive Contacts.</a> Journal of Neuroscience. 29 (40), 12449-12466 (2009).</li><li>Taylor, A. M., Wu, J., Tai, H. C., Schuman, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+Translation+of+&#946;-Catenin+Regulates+Synaptic+Vesicle+Dynamics.">Axonal Translation of &#946;-Catenin Regulates Synaptic Vesicle Dynamics.</a> Journal of Neuroscience. 33 (13), 5584-5589 (2013).</li><li>Batista, A. F. R., Mart&#237;nez, J. C., Hengst, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intra-axonal+Synthesis+of+SNAP25+Is+Required+for+the+Formation+of+Presynaptic+Terminals.">Intra-axonal Synthesis of SNAP25 Is Required for the Formation of Presynaptic Terminals.</a> Cell reports. 20 (13), 3085-3098 (2017).</li></ol>

我们开发了利用神经元球培养法检测用LRRTM2-珠刺激的先增前形成的方法。目前,大多数前突生形成测定包括多-D-流源(PDL)涂层珠和分离培养/微流体室20,21,22。该方法的优点之一是LRRTM2珠子。当LRRTM2与神经素相互作用,形成兴奋的先阴突子,特别是11,12,13,PDL-珠诱导兴奋和抑制预突生非<...

突触形成是神经回路1、2、3开发过程中的关键步骤之一。由突触前和突触后组成的专门隔间的形成是一个复杂和多步骤的过程,涉及适当识别斧子和树突,形成活动区和后突密度,以及正确对齐电位通道和神经递质受体1,2。在每个过程中,许多种类的突触蛋白通过从细胞体中运输突触蛋白和/或通过隔间中的局部翻译,在适当的时间积累到这些专用隔间。这些突触蛋白被认为是以有组织的方式排列,以形成功能性突触。一些突触蛋白的机能导致神经疾病4,5。然而,目前还不清楚突触蛋白是如何在适当的时间积累。
为了研究突触蛋白如何以有组织的方式积累,有必要按时间顺序检查突触蛋白的积累。一些报告演示了实时成像,以观察神经元分离培养的突触形成6,7。然而,它是费时的神经元,刚刚开始在显微镜下突触形成。为了有效地观察突触蛋白的积累,突触的形成必须在研究人员想要诱导形成的时候开始。第二个挑战是区分突触蛋白的积累,由于从细胞体或突触中的局部翻译。为此,翻译水平是必要的,在不允许从细胞体运输突触蛋白的条件下进行测量。
我们利用神经元球培养物与珠子的结合,开发出了新型的先发性形成测定,以诱导前突子形成8。神经元球培养是开发来检查斧状表型,由于形成围绕细胞体的斧板9,10。我们使用磁珠与富含亮氨酸的重复跨膜神经元2(LRRTM2)结合,这是一个前突触组织者诱导兴奋前突触(图1A)11,12,13。通过使用 LRRTM2 磁珠,在应用磁珠时开始预突子形成。这意味着,前突子的形成开始于神经元球的千个斧子在同一时间,因此它允许有效地检查突触蛋白积累的精确时间过程。此外,神经元球培养很容易通过去除细胞体来阻止从体中传递突触蛋白(图1B)8。我们已经证实,没有细胞体的斧子可以存活,并在去除细胞体后至少4小时健康。因此,此方案适用于研究突触蛋白的衍生位置(细胞体或斧子),以及突触蛋白如何以有组织的方式积累。

<table>
	<tbody>
		<tr>
			<td>Antibody diluent</td>
			<td>DAKO</td>
			<td>S2022</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 AffiniPure Donkey Anti-Mouse IgG (H+L)</td>
			<td>Jackson ImmunoResearch</td>
			<td>715-585-151</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 AffiniPure Donkey Anti-Rabbit IgG (H+L)</td>
			<td>Jackson ImmunoResearch</td>
			<td>711-545-152</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse anti-Munc18-1</td>
			<td>BD Biosciences</td>
			<td>610336</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement (50X), serum free</td>
			<td>Thermo Fisher Scientific</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Alubumin (BSA)</td>
			<td>Nacalai Tesque</td>
			<td>01863-48</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell-Culture Treated Multidishes (4 well dish)</td>
			<td>Nunc</td>
			<td>176740</td>
			<td></td>
		</tr>
		<tr>
			<td>Complete EDTA-free</td>
			<td>Roche</td>
			<td>11873580001</td>
			<td></td>
		</tr>
		<tr>
			<td>cooled CCD camera</td>
			<td>Andor Technology</td>
			<td>iXON3</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip</td>
			<td>Matsunami</td>
			<td>C015001</td>
			<td>Size: 15 mm, Thickness: 0.13-0.17 mm</td>
		</tr>
		<tr>
			<td>Cytosine &#946;-D-arabinofuranoside (AraC)</td>
			<td>Sigma-Aldrich</td>
			<td>C1768</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#39;,6-Diamidino-2-phenylindole Dihydrochloride (DAPI)</td>
			<td>Nacalai Tesque</td>
			<td>11034-56</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyribonuclease 1 (DNase I)</td>
			<td>Wako pure chemicals</td>
			<td>047-26771</td>
			<td></td>
		</tr>
		<tr>
			<td>Expi293 Expression System</td>
			<td>Thermo Fisher Scientific</td>
			<td>A14635</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>Sigma-Aldrich</td>
			<td>H1270</td>
			<td></td>
		</tr>
		<tr>
			<td>image acquisition software</td>
			<td>Nikon</td>
			<td>NIS-element AR</td>
			<td></td>
		</tr>
		<tr>
			<td>Image analysis software</td>
			<td>NIH</td>
			<td>Image J</td>
			<td>https://imagej.nih.gov/ij/</td>
		</tr>
		<tr>
			<td>Inverted fluorecent microscope</td>
			<td>Nikon</td>
			<td>Eclipse Ti-E</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal media</td>
			<td>Thermo Fisher Scientific</td>
			<td>#21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum (NGS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>#143-06561</td>
			<td></td>
		</tr>
		<tr>
			<td>N-propyl gallate</td>
			<td>Nacalai Tesque</td>
			<td>29303-92</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Nacalai Tesque</td>
			<td>26126-25</td>
			<td></td>
		</tr>
		<tr>
			<td> 
			Paraplast Plus</td>
			<td>Sigma-Aldrich</td>
			<td>P3558</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine Hydrobromide (MW &#62; 300,000)</td>
			<td>Nacalai Tesque</td>
			<td>28359-54</td>
			<td></td>
		</tr>
		<tr>
			<td>poly (vinyl alcohol)</td>
			<td>Sigma</td>
			<td>P8136</td>
			<td></td>
		</tr>
		<tr>
			<td>Prepacked Disposable PD-10 Columns</td>
			<td>GE healthcare</td>
			<td>17085101</td>
			<td></td>
		</tr>
		<tr>
			<td>rabbit anti-vesicular glutamate transporter 1</td>
			<td>Synaptic Systems</td>
			<td>135-302</td>
			<td></td>
		</tr>
		<tr>
			<td>SCAT 20X-N (neutral non-phosphorous detergent)</td>
			<td>Nacalai Tesque</td>
			<td>41506-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin-coated magnetic particles</td>
			<td>Spherotech Inc</td>
			<td>SVM-40-10</td>
			<td>diameter: 4-5 &#181;m</td>
		</tr>
		<tr>
			<td>TritonX-100</td>
			<td>Nacalai Tesque</td>
			<td>35501-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Nacalai Tesque</td>
			<td>18172-94</td>
			<td></td>
		</tr>
	</tbody>
</table>

presynapse formation assay using presynapse organizer beads and &ldquo;neuron ball&rdquo; culture

在神经元发育过程中,突触形成是建立神经回路的重要一步。要形成突触,突触蛋白必须通过从细胞体的传输和/或未成熟的突触中的局部翻译以适当的顺序提供。然而,它不完全了解突触蛋白如何积累在突触以适当的顺序。本文提出了一种利用神经元球培养与珠子结合来诱导前突触形成的新方法。神经元细胞聚集的神经元球提供远离细胞体和树突的斧状片,因此可以通过避免细胞体的压倒性信号来检测前突子的弱荧光信号。作为珠子来触发前突动的形成,我们使用珠子与富含亮氨酸的重复跨膜神经元2(LRRTM2)结合,这是一个兴奋的先发前的组织者。使用这种方法,我们证明了囊性谷氨酸输送剂1(vGlut1),一种突触性囊泡蛋白,在预突触中积累得比Munc18-1(一种活性区蛋白)快。Munc18-1 在切除细胞体后,在先行中积累翻译依赖性。这一发现表明Munc18-1通过局部平移在斧子中积累,而不是从细胞体传输。总之,该方法适用于分析突触前蛋白的积累和突触蛋白的来源。由于神经元球培养简单,无需使用特殊仪器,该方法可适用于其他实验平台。

在这里,我们展示了在神经元球培养的斧板的LRRTM2诱导前突触中预突触蛋白的积累代表性的结果。作为前突触蛋白,我们分析了兴奋性突触性囊泡蛋白vGlut1和活性区蛋白Munc18-1。我们还研究了前突子中vGlut1和Munc18-1积累的时间过程,并利用去突子去除细胞体和蛋白质合成抑制剂的突厥前Munc18-1来源得到结果。最近,我们研究了脆弱X智力迟钝蛋白(FMRP)在预突子8中Munc18-1的积累作用。FMRP是?...

Watch this Scientific Journal Video about Presynapse Formation Assay Using Presynapse Organizer Beads and Ã¢â‚¬Å“Neuron BallÃ¢â‚¬Â Culture at JoVE.com

Presynapse Formation Assay Using Presynapse Organizer Beads and ÃƒÂ¢Ã¢â€šÂ¬Ã…â€œNeuron BallÃƒÂ¢Ã¢â€šÂ¬Ã‚Â Culture

预突生形成是一个动态过程,包括以适当的顺序积累突触蛋白。在这种方法中,前突子形成由与"神经元球"培养的正交片上与前突子组织者蛋白结合的珠子触发,因此在先发形成期间很容易分析突触蛋白的积累。

使用预突子形成测定使用预突子组织者珠和"神经元球"文化

During neuronal development, synapse formation is an important step to establish neural circuits. To form synapses, synaptic proteins must be supplied in ...

该协议适用于研究前突触蛋白的来源：细胞体或轴突。以及突触形成期间，前突触蛋白如何以有组织的方式在预酶中积累。这种技术可以同时诱导数千个预收缩，并且不使用任何特殊装置来维持培养中的轴子，从而能够有效地分析轴子中许多预轴的形成。和研究生霍纳米一起演示这个程序的将是我实验室的本科生里石井。从文本协议中描述的鼠标安乐死开始此过程。解剖腹部以获得E16胚胎。在细尖钳的帮助下，小心地从胚胎中去除大脑，并将其转移到含有四毫升 HEPES 缓冲盐溶液或 HBSS 的 60 毫米细胞培养皿中。取出毛丝，切开嗅觉球。使用立体显微镜下钳子的细小头从每个脑半球分离皮质，并转移到另一个含有新鲜 HBSS 的 60 毫米培养皿中。每个单独的神经元球培养使用至少三到五个胚胎。用微分弹簧剪刀在层流细胞培养罩中将皮质切成小块。现在，将切碎的皮质转移到15毫升的管子里。在37摄氏度的水浴中，在4毫升0.125%的三辛辛中尝试4.5分钟。通过无菌转移移液器将细胞聚集转移到含有 10 毫升 HBSS 的新 15 毫升管中。在37摄氏度下孵育5分钟，然后再重复这一步。然后将细胞聚集转移到一个新的15毫升管，其中包含两毫升的NGB培养基、0.01%的DNase I和10%的马血清。使用火抛光的精细玻璃巴斯德移液器反复上下移液三到五次，使三辛化皮质发生三到五次。为了准备神经元球，使用NGB介质将细胞悬浮液中的细胞密度调整到每毫升一百万个细胞。在培养皿的底部加入七毫升 PBS。培养皮质神经元作为10微升悬挂滴，每滴含有10，000个细胞，在10厘米培养皿的上盖内。在37摄氏度下将盘子放在孵化器中三天，在加湿条件下使用5%的CO2，以便形成神经元球。在 60 毫米的盘式中，使用每毫升 15 微克 PLL 溶液在玻酸盐缓冲液中将聚 L-Lysine 或 PLL 涂到石蜡珠玻璃盖上。在37摄氏度的二氧化碳培养箱中至少保留一小时。使用 PBS 洗涤四次后，将 PLL 涂层盖滑移至包含 350 微升 NGB 介质的四井板中，每个井中装有三微摩尔 AraC。AraC 被添加到介质中以杀死分裂细胞。在 CO2 培养箱中孵育含有 PLL 涂层盖滑的四孔板至少 20 分钟，以确保介质的温度达到 37 摄氏度，然后再转移神经元球。在 DIV 3 时，当神经元球形成得非常好时，将它们转移到 PLL 涂层盖滑上。在四井板内，每井添加五个神经元球。在DIV 11中，将20微升从悬浮物涂层磁性颗粒转移到微离心管。将珠子固定到与镉永磁体相连的手工设备上，并在 1.5 毫升微离心管中用 100 微升 PBS-MCBC 洗涤三次。从珠子上完全去除 PBS-MCBC 后，向洗涤的珠子中加入预定体积的 LRRTM2 库存。在四摄氏度下用旋转器孵育混合物一至两个小时。孵育后，用100微升PBS-MCBC洗涤珠子两次。然后，用100微升的NGB介质清洗珠子。将 LRRTM2 珠子重新用于 50 微升的 NGB 介质中，用于神经元球培养。现在，在立体显微镜下用剃须刀刀片以 45 度角切割黄色尖端的端部。将黄色尖端放在神经元球的细胞体区域，然后通过吸气去除细胞体。在神经元球培养上应用 LRRTM2 和控制珠子。然后，使用铁氧体磁铁将珠子淹没到神经元球培养的板块底部一分钟，开始突触前形成。此过程可确保同时接触所有珠子。修复和染色神经元在神经元球培养后，前突触形成与珠子，如文本协议中所述。使用 60X 油浸透镜，使用冷却的 CCD 摄像机在倒置荧光显微镜下捕捉差分干扰对比度和免疫荧光图像。测量轴突中预突触中的免疫荧光强度。使用珠子上感兴趣区域的免疫荧光强度。减去离珠区域强度除以极光强度，沿珠子20微米。减去背景强度。这种比率强度提供蛋白质积累指数。要量化使用LRRTM2珠子诱导的先酶的累积水平，请始终选择与细胞体分开的两个视场或更多的区域。为了进行准确的测量，请根据盖玻片选择五个不同的斧头字段。这里展示的是LRRTM2珠子在DIV 11诱导Munc18-1在神经元球轴子预结合中神经元球培养中的应用。神经元球轴上的珠子在相位显微镜图像中可见。免疫荧光图像观察了前突触蛋白的积累。即使在被移除的细胞体轴子中，在珠子下观察到Munc18-1的积累，类似于具有细胞体的神经元球的轴子。当通过高放大倍率目标透镜分析轴膜表的外围区域时，vGlut1和Munc18-1清楚地积聚在带细胞体和没有细胞体的珠子下的轴子预声中。时间过程实验表明，在30分钟内，vGlut1在预分析中积累显著增加。另一方面，Munc18-1的积累在两小时后开始显著增加，并在四小时后到达高原。这些数据表明，突触囊泡蛋白vGlut1在预酶中积累得早于活性区蛋白Munc18-1。Fmr1-KO神经元预发的Munc18-1积累比野生神经元增加1.5倍，表明FMRP参与Munc18-1积累。蛋白质合成抑制剂，非异霉素，抑制Munc18-1在轴星中显著积累，有或没有细胞体。这表明积累是蛋白质合成依赖。使用火抛光的精细玻璃巴斯德移液器移液，是重要的一步。实验者准备了具有两到三个不同直径的火抛光移液器，并选择了直径合适的移液器。使用此过程，通过实时成像测量由 LRRTM2 珠子诱导的预囊突触释放。这个额外的方法将回答轴突中的蛋白质合成是否参与突触释放的调节。使用这种方法，研究人员检查前突触蛋白如何以有组织的方式在预酶酶中积累，以及前突触蛋白的来源位置。

Research

JoVE Journal

Neuroscience

Presynapse Formation Assay Using Presynapse Organizer Beads and &ldquo;Neuron Ball&rdquo; Culture

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

成年鼠神经干细胞的分离和扩大使用神经干含量

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

科研

神经科学

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

建立小鼠胚胎神经干细胞培养神经球含量

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Preparation and Culture of Chicken Auditory Brainstem Slices

鸡脑干切片的制备及文化

The chicken auditory brainstem is a well-established model system that has been widely used to study the anatomy and physiology of auditory processing at ...

Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay

人类胶质母细胞瘤肿瘤细胞的分离和扩大使用的神经球含量

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in ...

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

彗星试验测定DNA损伤的海马神经元

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been ...

Isolation and Culture of Mouse Cortical Astrocytes

小鼠脑皮层星形胶质细胞的分离和培养

Astrocytes  are an abundant cell type in the mammalian brain, yet much remains to be  learned about their molecular and functional characteristics. In ...

Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors

Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors

在共同的文化整合模式中GABA能神经元的刺和HEK293细胞抑制性突触形成稳定表达GABA<sub&gt;一个</sub&gt;受体

Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA ...

Analysis of Developing Tooth Germ Innervation Using Microfluidic Co-culture Devices

微流控采用共培养开发设备牙胚支配分析

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena ...

Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay

实时 cAMP 检测气味受体激活的活细胞测量

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. ...

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

生活成像的脑皮层细胞使用二维培养系统

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...