Name: purification of the dendritic filopodia-rich fraction
Uploaded: 2019-05-02T13:30:00
Description: Watch this Scientific Journal Video about Purification of the Dendritic Filopodia-rich Fraction at JoVE.com

我们感谢小冈和松野幸男为海马神经元的低密度培养,Masayoshi Mishina为TLCN缺乏小鼠,三井三子和MomokoShiozaki的技术援助,以及Yoshihara实验室的成员进行有益的讨论.这项工作得到了JSPS KAKENHI格兰特Nos的支持。JP20700307、JP22700354 和 JP24500392 和 MEXT KAKENHI 授予号。JP23123525 到 YF 和 JP20022046、JP18H04683 和 JP18H05146 到 YY。

这里描述的所有方法都已获得RIKEN Wako机构动物护理和使用委员会的批准。
1. 希马坎帕神经元文化
<ol><li>培养媒介的准备
	<ol>
<li>制备200x维生素混合物。使用磁性搅拌器在500 mL的超纯水中溶解100毫克D-泛酸血酸、100毫克氯丁酸、100毫克叶酸、180毫克i-ininitol、100mg烟酰胺、100毫克皮里多什HCl和100毫克二胺HCl。溶液未完全溶解。小心地混合,在50 mL管中等分,储存在-20°C...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+a+role+of+dendritic+filopodia+in+synaptogenesis+and+spine+formation.">Evidence for a role of dendritic filopodia in synaptogenesis and spine formation.</a> Neuron. 17 (1), 91-102 (1996).</li><li>Lohmann, C., Bonhoeffer, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+local+calcium+signaling+in+rapid+synaptic+partner+selection+by+dendritic+filopodia.">A role for local calcium signaling in rapid synaptic partner selection by dendritic filopodia.</a> Neuron. 59 (2), 253-260 (2008).</li><li>Yoshihara, Y., De Roo, M., Muller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+spine+formation+and+stabilization.">Dendritic spine formation and stabilization.</a> Current Opinion in Neurobiology. 19 (2), 146-153 (2009).</li><li>Bayes, 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=Comparative+study+of+human+and+mouse+postsynaptic+proteomes+finds+high+compositional+conservation+and+abundance+differences+for+key+synaptic+proteins.">Comparative study of human and mouse postsynaptic proteomes finds high compositional conservation and abundance differences for key synaptic proteins.</a> PLoS One. 7 (10), e46683 (2012).</li><li>Bayes, 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=Characterization+of+the+proteome,+diseases+and+evolution+of+the+human+postsynaptic+density.">Characterization of the proteome, diseases and evolution of the human postsynaptic density.</a> Nature Neuroscience. 14 (1), 19-21 (2011).</li><li>Furutani, 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=Interaction+between+telencephalin+and+ERM+family+proteins+mediates+dendritic+filopodia+formation.">Interaction between telencephalin and ERM family proteins mediates dendritic filopodia formation.</a> Journal of Neuroscience. 27 (33), 8866-8876 (2007).</li><li>Mao, Y. 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=Filopodia+Conduct+Target+Selection+in+Cortical+Neurons+Using+Differences+in+Signal+Kinetics+of+a+Single+Kinase.">Filopodia Conduct Target Selection in Cortical Neurons Using Differences in Signal Kinetics of a Single Kinase.</a> Neuron. 98 (4), 767-782 (2018).</li><li>Matsuno, 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=Telencephalin+slows+spine+maturation.">Telencephalin slows spine maturation.</a> Journal of Neuroscience. 26 (6), 1776-1786 (2006).</li><li>Raemaekers, 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=ARF6-mediated+endosomal+transport+of+Telencephalin+affects+dendritic+filopodia-to-spine+maturation.">ARF6-mediated endosomal transport of Telencephalin affects dendritic filopodia-to-spine maturation.</a> The EMBO Journal. 31 (15), 3252-3269 (2012).</li><li>Mori, K., Fujita, S. C., Watanabe, Y., Obata, K., Hayaishi, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telencephalon-specific+antigen+identified+by+monoclonal+antibody.">Telencephalon-specific antigen identified by monoclonal antibody.</a> Proceedings of the National Academy of Sciences of the United States of America. 84 (11), 3921-3925 (1987).</li><li>Yoshihara, Y., Mori, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telencephalin:+a+neuronal+area+code+molecule?.">Telencephalin: a neuronal area code molecule?.</a> Neuroscience Research. 21 (2), 119-124 (1994).</li><li>Annaert, W. G., 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=Interaction+with+telencephalin+and+the+amyloid+precursor+protein+predicts+a+ring+structure+for+presenilins.">Interaction with telencephalin and the amyloid precursor protein predicts a ring structure for presenilins.</a> Neuron. 32 (4), 579-589 (2001).</li><li>Furutani, 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=Vitronectin+induces+phosphorylation+of+ezrin/radixin/moesin+actin-binding+proteins+through+binding+to+its+novel+neuronal+receptor+telencephalin.">Vitronectin induces phosphorylation of ezrin/radixin/moesin actin-binding proteins through binding to its novel neuronal receptor telencephalin.</a> Journal of Biological Chemistry. 287 (46), 39041-39049 (2012).</li><li>Nyman-Huttunen, H., Tian, L., Ning, L., Gahmberg, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=alpha-Actinin-dependent+cytoskeletal+anchorage+is+important+for+ICAM-5-mediated+neuritic+outgrowth.">alpha-Actinin-dependent cytoskeletal anchorage is important for ICAM-5-mediated neuritic outgrowth.</a> Journal of Cell Biology. 119 (Pt 15), 3057-3066 (2006).</li><li>Esselens, C., 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=Presenilin+1+mediates+the+turnover+of+telencephalin+in+hippocampal+neurons+via+an+autophagic+degradative+pathway.">Presenilin 1 mediates the turnover of telencephalin in hippocampal neurons via an autophagic degradative pathway.</a> Journal of Cell Biology. 166 (7), 1041-1054 (2004).</li><li>Furutani, Y., Yoshihara, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+Analysis+of+Dendritic+Filopodia-Rich+Fraction+Isolated+by+Telencephalin+and+Vitronectin+Interaction.">Proteomic Analysis of Dendritic Filopodia-Rich Fraction Isolated by Telencephalin and Vitronectin Interaction.</a> Frontiers in Synaptic Neuroscience. 10, 27 (2018).</li><li>Lu, Z., Piechowicz, M., Qiu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simplified+Method+for+Ultra-Low+Density,+Long-Term+Primary+Hippocampal+Neuron+Culture.">A Simplified Method for Ultra-Low Density, Long-Term Primary Hippocampal Neuron Culture.</a> Journal of Visualized Experiments. (109), (2016).</li><li>Okabe, S., Miwa, A., Okado, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+splicing+of+the+C-terminal+domain+regulates+cell+surface+expression+of+the+NMDA+receptor+NR1+subunit.">Alternative splicing of the C-terminal domain regulates cell surface expression of the NMDA receptor NR1 subunit.</a> The Journal of Neuroscience. 19 (18), 7781-7792 (1999).</li><li>Okabe, S., Vicario-Abejon, C., Segal, M., McKay, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+and+synaptogenesis+of+hippocampal+neurons+without+NMDA+receptor+function+in+culture.">Survival and synaptogenesis of hippocampal neurons without NMDA receptor function in culture.</a> European Journal of Neuroscience. 10 (6), 2192-2198 (1998).</li></ol>

利用细胞粘附分子TLCN与细胞外基质蛋白体外核酸之间的亲和力,开发了一种树突状纤维素富度分体的纯化方法。与PSD分数相比,有可能从富含树突子的丝虫片段中识别作用于未成熟突触的突触蛋白。因此,富含树突的纤维素的成分与PSD分数的成分相差74%。与PSD分数不同,我们使用培养的海马神经元主动形成噬菌体杯,细胞需要存活。为了形成方位细胞杯,我们使用了TLCN和体外核子细胞之间的相互作用。TL...

丹德炎纤维素被认为是脊柱的前体。在树突状纤维中的Actin细丝调节其延伸和缩回1,2,3。与斧子接触后,选定的树突状纤维开始成熟到脊柱,并形成突触形成4,5。脊柱的成分是由对鼻后密度分数6、7的综合分析确定的,而树突状纤维素的成分在很大程度上仍不为人所知。已经表明,端脑素(TLCN),ERM,SynGAP,Ras,PI3激酶,阿克特,mTOR,马球状激酶2,CaMKII,Syndecan-2,帕拉lemin-1,ARF6和EphB调节树突状纤维素形成5,8,9 ,10,11,虽然尚未开发一种方法,用于综合分析存在于树突状的纤维素中的分子。
TLCN(ICAM-5)是由最神经部分的尖刺神经元,即端脑12中专门表达的。TLCN在其细胞外区域有9个类似Ig的域,一个跨膜区域,一个细胞质尾13。TLCN与其细胞外区域的体外核酸素(VN)和LFA-1一元结结合,在其跨膜区域与前塞尼林结合,在其细胞质区5、8、14、15中与磷-ERM和β-actinin结合 ,16.TLCN通过磷-ERM结合到脊柱和树突状轴8、16的树突状纤维素和β-actinin的尖端。
研究表明,TLCN的过度表达增强了树突状纤维化的形成,并诱导脊柱回归到纤维化10。与TLCN细胞质区结合的ezrin的构成活性形式,增强树突状纤维化形成8。因此,TLCN通过行动素结合蛋白调节树突状纤维化形成。埃塞伦斯等人证明,微珠诱导TLCN在培养神经元上积累17。研究表明,在VN涂层微珠周围的神经元树突上形成方状的杯子结构以TLCN依赖性的方式形成15。树突状纤维素的成分与咽喉杯的成分相似。收集树突状纤维素是很困难的,但使用磁性微珠收集方位细胞杯相对容易。因此,我们开发了一种净化方丹石杯的方法,而不是树突状的纤维化18。在这里,我们描述了树突状富足部分的纯化方法。

<table>
	<tbody>
		<tr>
			<td>1 M HEPES</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>1.7 ml Low Binding MCT</td>
			<td>Sorenson BioScience</td>
			<td>39640T</td>
			<td></td>
		</tr>
		<tr>
			<td>200 mM L-Glutamine</td>
			<td>Gibco</td>
			<td>2530149</td>
			<td></td>
		</tr>
		<tr>
			<td>35-mm plastic cell culture dishes</td>
			<td>Corning</td>
			<td>430165</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-actin</td>
			<td>Sigma-Aldrich</td>
			<td>A-5060</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-alpha-Actinin</td>
			<td>Sigma-Aldrich</td>
			<td>A-5044</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-alpha-tubulin</td>
			<td>Sigma-Aldrich</td>
			<td>T-9026</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Ezrin</td>
			<td>Sigma-Aldrich</td>
			<td>clone3C12, SAB4200806</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Galphaq</td>
			<td>Santacruz</td>
			<td>sc-393</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-MAP2</td>
			<td>Chemicon</td>
			<td>clone AP20, MAB3418</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Moesin</td>
			<td>Sigma-Aldrich</td>
			<td>clone 38/87, M7060</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PLCbeta1</td>
			<td>Santacuz</td>
			<td>sc-5291</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PSD95</td>
			<td>MA2</td>
			<td>ABR</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Spectrin beta</td>
			<td>Chemicon</td>
			<td>MAB1622</td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco</td>
			<td>0080085SA</td>
			<td></td>
		</tr>
		<tr>
			<td>BCA protein assay kit</td>
			<td>Thermo</td>
			<td>23227</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>Merck</td>
			<td>1.08122.0005</td>
			<td></td>
		</tr>
		<tr>
			<td>calcium chrolide, hydrous</td>
			<td>Wako</td>
			<td>038-19735</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>Falcon</td>
			<td>353085</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer</td>
			<td>Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Choline chloride</td>
			<td>Sigma-Aldrich</td>
			<td>C7527</td>
			<td></td>
		</tr>
		<tr>
			<td>Complete EDTA free protease inhibitor cocktail</td>
			<td>Roche</td>
			<td>11873580001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytosine beta-D-arabinofuranoside</td>
			<td>Sigma-Aldrich</td>
			<td>C-6645</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase-I</td>
			<td>Sigma-Aldrich</td>
			<td>DN-25</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Pantothenic acid hemicalcium salt</td>
			<td>Sigma-Aldrich</td>
			<td>P5155</td>
			<td></td>
		</tr>
		<tr>
			<td>DynaMag-2 Magnet</td>
			<td>Thermo</td>
			<td>12321D</td>
			<td></td>
		</tr>
		<tr>
			<td>ECL Prime Western Blotting Detection Reagent</td>
			<td>GE</td>
			<td>RPN2232</td>
			<td></td>
		</tr>
		<tr>
			<td>e-PAGEL 5-20% SDS-PAGE gradient gel</td>
			<td>ATTO</td>
			<td>E-T520L</td>
			<td></td>
		</tr>
		<tr>
			<td>Folic acid</td>
			<td>Sigma-Aldrich</td>
			<td>F8758</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Gibco</td>
			<td>14175095</td>
			<td></td>
		</tr>
		<tr>
			<td>HRP-conjugated anti-rabbit IgG</td>
			<td>Jackson ImmunoResearch</td>
			<td>111-035-144</td>
			<td></td>
		</tr>
		<tr>
			<td>i-Inositol</td>
			<td>Sigma-Aldrich</td>
			<td>I7508</td>
			<td></td>
		</tr>
		<tr>
			<td>LAS-1000 mini</td>
			<td>Fuji Film</td>
			<td>LAS-1000 mini</td>
			<td>For detection of luminescence from WB membrane</td>
		</tr>
		<tr>
			<td>Magnetic polystyrene microbeads</td>
			<td>Sperotech</td>
			<td>PM-20-10</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM amino acid solution</td>
			<td>Gibco</td>
			<td>11130-051</td>
			<td>30 mM L-Arginine hydrochloride, 5 mM L-Cystine, 10 mM L-Histidine hydrochloride-H2O, 20 mM L-Isoleucine, 20 mM L-Leucine, 19.8 mM L-Lysine hydrochloride, 5.1 mM L-Methionine, 10 mM L-Phenylalanine, 20 mM L-Threonine, 2.5 mM L-Tryptophan, 10 mM L-Tyrosine, and 20 mM L-Valine</td>
		</tr>
		<tr>
			<td>Mini-slab size electrophoresis system</td>
			<td>ATTO</td>
			<td>AE-6530</td>
			<td></td>
		</tr>
		<tr>
			<td>Niacinamide</td>
			<td>Sigma-Aldrich</td>
			<td>N0636</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilin / Streptomycin</td>
			<td>Gibco</td>
			<td>15070063</td>
			<td></td>
		</tr>
		<tr>
			<td>PhosSTOP phosphatase inhibitor cocktail</td>
			<td>Roche</td>
			<td>4906845001</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine hydrobromide</td>
			<td>Nacali</td>
			<td>28360-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyridoxal HCl</td>
			<td>Sigma-Aldrich</td>
			<td>P6155</td>
			<td></td>
		</tr>
		<tr>
			<td>Riboflavin</td>
			<td>Sigma-Aldrich</td>
			<td>R9504</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver Stain 2 Kit wako</td>
			<td>Wako</td>
			<td>291-5031</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiamine HCl</td>
			<td>Sigma-Aldrich</td>
			<td>T1270</td>
			<td></td>
		</tr>
		<tr>
			<td>Trans-Blot SD Semi-Dry Transfer Cell</td>
			<td>Bio-rad</td>
			<td>1703940JA</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra pure water</td>
			<td>MilliQ</td>
			<td></td>
			<td>For production of ultra pure water</td>
		</tr>
	</tbody>
</table>

purification of the dendritic filopodia-rich fraction

丹德林丝洛波迪亚是薄和长突起基于作用素灯丝,他们延伸和缩回,仿佛寻找目标斧突。当树突状纤维化与目标斧突建立接触时,它们开始成熟成脊柱,导致突触的形成。端脑素 (TLCN) 在树突状纤维化中大量局部化,并逐渐被排除在脊柱之外。在培养的海马神经元中TLCN的过度表达诱导树突状的纤维化形成。我们表明,端脑蛋白与细胞外基质分子,体外核酸强结合。Vitronectin涂层微珠诱导神经元树突形成方位细胞杯。在方细胞杯中,TLCN、TLCN结合蛋白,如磷酸化Ezrin/Radixin/Moesin(磷-ERM)和F-actin被积累,这表明方位细胞杯的成分与树突状西多迪亚的成分相似。因此,我们开发了一种净化方丹花杯的方法,而不是树突状的纤维化。磁聚苯乙烯珠被涂覆体内蛋白,在海马神经元的培养基中大量存在,诱导神经元树突形成方位体。孵育24小时后,用洗涤剂温和溶解咽喉杯,并用磁铁分离器收集。洗涤珠子后,通过银染色和西方印迹对结合蛋白进行洗脱和分析。在结合部分中,TLCN和actin大量存在。此外,从该分数中识别的许多蛋白质被局部化为树突状纤维化;因此,我们命名结合分数作为树突状的菲洛波迪亚丰富的分数。本文介绍了树突状纤维素富分馏的纯化方法。

在培养的海马神经元中,TLCN被大量地本地化为树突状的纤维化、轴和索马,并与F-actin共同本地化(图1A,B)。当聚苯乙烯微珠被添加到培养的海马神经元中时,珠子被自动涂覆从培养基中的胎儿牛血清(FBS)中提取的体外内蛋白(VN);它们主要与树突结合,它们诱导了方位细胞杯的形成(图1B-E)。噬菌体杯基于树突上?...

Watch this Scientific Journal Video about Purification of the Dendritic Filopodia-rich Fraction at JoVE.com

Purification of the Dendritic Filopodia-rich Fraction

在此协议中,我们介绍了一种利用树突状纤维蛋白之间特定且强烈的亲和力,从培养的海马神经元上的方皮杯状突起结构中纯化树突状纤维素的富足部分的方法。粘附分子,TLCN,和细胞外基质分子,体外内丁。

富德瑞迪蒂菲洛波迪亚成分的纯化

Dendritic filopodia are thin and long protrusions based on the actin filament, and they extend and retract as if searching for a target axon. When the ...

与PSD分数相比，可以识别对树突纤维蛋白丰富的分数周围未成熟的突触作用的突触蛋白。我们用活神经元来诱导噬菌蛋白的帽形成。该技术的主要优点是，在人工起源中的活性蛋白质可以识别从树突纤维蛋白丰富的分数。首先， 通过溶解100毫克维生素B5、100毫克胆碱、100毫克叶酸、180毫克I-inositol、100毫克维生素B3、100毫克维生素B6盐酸盐和100毫克盐酸硫胺，在500毫升超纯水中溶解200毫克维生素B6和100毫克盐酸硫胺。小心地将等分液混合在 50 ml 管中，并在 20 摄氏度下储存。要准备核黄素溶液，使用磁力搅拌器在500毫克超纯水中溶解100毫克核黄素。小心地将等分液混合在 50 ml 管中，并在 20 摄氏度下储存。使用磁力搅拌器在50ml超纯水中中溶解7.35克氯化钙。对于最低基本中制剂，使用磁力搅拌机溶解400毫克氯化钾6、800毫克氯化钠、2 200毫克碳酸氢钠、158毫克磷酸钠单基二水合物、7000毫克D-葡萄糖、200毫克硫酸镁和950毫升超纯水。接下来，使用一个1ml移液器，在磁搅拌器上不断搅拌，以滴滴的方式将1.8ml的1摩尔二氯化钙加入到 MEM中。然后加入盐酸，将 MEM 的 pH 调整为 pH 7.25。然后，在 MEM 中加入 5 ml 200 倍维生素混合物和 200 微升核黄素溶液。使用超纯水将溶液的体积调整为 1000 ml。使用 0.22 微米过滤系统过滤溶液，并将其储存在 4 摄氏度。准备10倍DNase-1库存溶液溶解100毫克Dnase-1在12.5毫升的HSSS。通过 0.22 微米过滤器过滤，在 1.5 ml 管中加入等分，并在 20 摄氏度下储存管。对于 Ara-C 库存溶液制备，在 8.93 ml 的超纯水中溶解 25 毫克 Ara-C。通过 0.22 微米过滤器过滤，在 1.5ml 管中等分，并在 20 摄氏度下储存。要准备电镀介质， 混合 1 ml MEM 氨基酸溶液、750 微升 1 摩尔 HEPES、1 ml B27、125 微升 200 毫摩尔谷氨酰胺、250 微升青霉素链霉素、2.5 ml 胎儿牛血清和 44.375 ml MEM 在 50 ml 管中。为了准备停止介质，在 50 ml 管中混合 1 ml MEM 氨基酸溶液、750 微升 1 摩尔 HEPES、5 ml FBS 和 43.25 ml MEM。准备HPSS，辅以15毫摩尔 HEPES，混合49.25毫升HPSS，和750微升1摩尔HPES。对于聚-l-lysine涂层盘的制备，在25摄氏度下，用每毫升聚-l-lysine氢溴化物涂覆35毫米塑料细胞培养盘，一天。接下来，用2毫升超纯水洗三次，并在25摄氏度下用1.5毫升的停止中，直到使用。在HPSS中，在含有500微升的混合物中孵育解剖的海马，每含有2.5%的三辛和10倍Dnase-1，辅以15毫摩尔海佩斯、pH7.2，在37摄氏度下，每3分钟搅拌15分钟。然后，将海马转移到10毫升的停止中等和孵育在4摄氏度5分钟，以灭活的尝试性。接下来，在4摄氏度的10毫升新鲜停止介质中孵育解剖的海马，5分钟。将海马移入10毫升新鲜停止介质，并在4摄氏度下孵育5分钟。然后，将海马移动到停止介质的 900 微升中，在 15 ml 管中将 100 微升 10X Dnase-1 移入 100 微升中。使用1ml移液器通过移液20次将海马分离成孤立的神经元。加入9ml电镀介质，通过70微米细胞过滤器过滤到50毫升的管子中。使用血细胞计计算细胞数量，并在电镀介质中每毫升调整为 3.5 倍至第四个细胞。接下来，从聚-l-利辛涂层的盘式中吸出停止介质。板2毫升每盘细胞在涂层的盘子和孵育下5%二氧化碳在37摄氏度60-64小时。孵育后，在神经元中加入2微升的阿拉-C股票溶液，慢慢摇动盘子。将培养皿放在加湿的盒子里，在 37 摄氏度下不改变 5%二氧化碳以下的培养基。通过首先将每道菜中300万个磁性聚苯乙烯微珠添加到20个含有培养神经元的盘子中，在体外13天后净化树突纤维素丰富的分数。一天后，用三次洗涤1mlPBS中的神经元，去除中联微珠和未绑定的微珠。取出PBS后，每盘解解缓冲液用500微升来解化神经元。接下来，用细胞刮刀收集酸盐，将落酸转移到10个低蛋白结合微管中。将管子放在磁铁分离器上，等待一分钟。收集上一液，并用作银染色和西方印迹分析的未绑定分数。接下来，将珠子转移到新的低蛋白结合微管中，并在磁性分离器上设置一分钟。完全去除上流液，并添加 500 微升的解液缓冲液。使用涡流混合器清洗珠子 15 秒钟。重复清洗珠子10次，取出上一除。通过加入50微升1X SDS样品缓冲液，将管子在98摄氏度下煮沸5分钟，将蛋白质分解到珠子上。以 860xG 3000 RPM 离心管 10 秒钟，将管放在磁性分离器上 1 分钟。收集上一液，并使用它作为绑定分数。通过 BCA 蛋白质测定测量未绑定和绑定蛋白质分数的浓度。可视化具有布罗莫苯蓝色蛋白素溶液，并调整浓度为每微升5纳米的SDS页面。使用 5-20% 梯度凝胶按 SDS 页面分离绑定和未绑定分数。银色污渍凝胶。最后，根据手稿，使用适当的原抗体和二级抗体的西方印迹。在培养的海马神经元中，TLCN被大量局部化到树突状纤维素、轴和索马，并与F-actin共同本地化。编码的珠子主要与树突结合，通过神经元树突的TLCN和F-actin的积累，诱导噬菌体的形成。Thorecense 图像显示， 噬菌体杯的形成在很大程度上取决于 Tlcn 在树突中的存在。噬菌体杯只在野生型海马神经元上形成，而不是在TLCN的抗性海马神经元上形成。SDS页面结果显示，未绑定和绑定分数的蛋白质带模式几乎相同，但50和70千达顿（绑定分数）的强度低于未绑定分数中的强度。然而，从TLCN抗性培养海马神经元编写的未绑定和边界分数之间，带强度没有明显差异。西方对未绑定和绑定分数的印迹分析表明，TLCN和VN主要在绑定分数中检测。Actin、 Ezrin 、 G alpha q 、 PLC beta 1 、 MAP-2 和 Spectrin， 在绑定和未绑定分数中均检测到。在未绑定分数中检测到莫辛、PSD-95、阿尔法-阿皮宁和阿尔法-图布林。如果微珠的涂层蛋白发生变化，可采用此程序识别测定相互作用。这种技术可以识别对人造物质的蛋白质。因此，我们相信机制人工起源可以量化。我们相信，与精神障碍相关的新蛋白质可以从富含树突的纤维蛋白分馏中识别出来。并可以扩展到治疗。

Research

JoVE Journal

Neuroscience

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

在培养的中枢神经系统的神经元树突棘形态分析

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These ...

科研

神经科学

Inducing Dendritic Growth in Cultured Sympathetic Neurons

诱导培养的交感神经元的树突状增长

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

浦肯野细胞树突形态的器官切片培养分析

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

谷氨酸和GABA快速微离子导入：一个有用​​的工具，调查突触整合

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

树突分支中的海马区对小鼠的齿状回评

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

Purification of Mouse Brain Vessels

对小鼠脑血管净化

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and ...

Analyzing Dendritic Morphology in Columns and Layers

分析树突状形态的列和图层

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and ...

Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method

使用高尔基Cox方法评估老年小鼠海马树突状复杂性

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching ...

Real-time Iontophoresis with Tetramethylammonium to Quantify Volume Fraction and Tortuosity of Brain Extracellular Space

用四甲基铵进行实时离子电渗疗法，以量化体细胞分数和脑细胞外空间的变性

This review describes the basic concepts and protocol to perform the real-time iontophoresis (RTI) method, the gold-standard to explore and quantify the ...

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

嗜德维人神经元树突状树突状复杂度的定量分析

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. ...