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>
	<li>Fiala, J. C., Feinberg, M., Popov, V., Harris, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptogenesis+via+dendritic+filopodia+in+developing+hippocampal+area+CA1.">Synaptogenesis via dendritic filopodia in developing hippocampal area CA1.</a> Journal of Neuroscience. 18 (21), 8900-8911 (1998).</li><li>Portera-Cailliau, C., Pan, D. T., Yuste, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-regulated+dynamic+behavior+of+early+dendritic+protrusions:+evidence+for+different+types+of+dendritic+filopodia.">Activity-regulated dynamic behavior of early dendritic protrusions: evidence for different types of dendritic filopodia.</a> Journal of Neuroscience. 23 (18), 7129-7142 (2003).</li><li>Ziv, N. E., Smith, S. 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。

purification-of-the-dendritic-filopodia-rich-fraction

Research

JoVE Journal

Neuroscience

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These structures are rich in actin and have been shown to be highly dynamic. In response to classical Hebbian plasticity 
as well as neuromodulatory signals, dendritic spines can change shape and number, which is thought to be critical for the refinement of neural 
circuits and the processing and storage of information within the brain. Within dendritic spines, a complex network of proteins link extracellular 
signals with the actin cyctoskeleton allowing for control of dendritic spine morphology and number. Neuropathological studies have demonstrated that 
a number of disease states, ranging from schizophrenia to autism spectrum disorders, display abnormal dendritic spine morphology or numbers. 
Moreover, recent genetic studies have identified mutations in numerous genes that encode synaptic proteins, leading to suggestions that these 
proteins may contribute to aberrant spine plasticity that, in part, underlie the pathophysiology of these disorders. In order to study the potential 
role of these proteins in controlling dendritic spine morphologies/number, the use of cultured cortical neurons offers several advantages. Firstly, 
this system allows for high-resolution imaging of dendritic spines in fixed cells as well as time-lapse imaging of live cells. Secondly, this in 
vitro system allows for easy manipulation of protein function by expression of mutant proteins, knockdown by shRNA constructs, or pharmacological 
treatments. These techniques allow researchers to begin to dissect the role of disease-associated proteins and to predict how mutations of these 
proteins may function in vivo.

Numerous recent studies have identified mutations in synaptic proteins associated with brain pathologies. Primary cultured cortical neurons offer great flexibility in examining the effects of these disease-associated proteins on dendritic spine morphology and motility.

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

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 4-6. Altered patterns of dendritic growth and plasticity are associated with impaired neurobehavioral function in experimental models 7, and are thought to contribute to clinical symptoms observed in both neurodevelopmental disorders 8-10 and neurodegenerative diseases 11-13. Such observations underscore the functional importance of precisely regulating dendritic morphology, and suggest that identifying mechanisms that control dendritic growth will not only advance understanding of how neuronal connectivity is regulated during normal development, but may also provide insight on novel therapeutic strategies for diverse neurological diseases.
Mechanistic studies of dendritic growth would be greatly facilitated by the availability of a model system that allows neurons to be experimentally switched from a state in which they do not extend dendrites to one in which they elaborate a dendritic arbor comparable to that of their in vivo counterparts. Primary cultures of sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rodents provide such a model. When cultured in defined medium in the absence of serum and ganglionic glial cells, sympathetic neurons extend a single process which is axonal, and this unipolar state persists for weeks to months in culture 14,15. However, the addition of either bone morphogenetic protein-7 (BMP-7) 16,17 or Matrigel 18 to the culture medium triggers these neurons to extend multiple processes that meet the morphologic, biochemical and functional criteria for dendrites. Sympathetic neurons dissociated from the SCG of perinatal rodents and grown under defined conditions are a homogenous population of neurons 19 that respond uniformly to the dendrite-promoting activity of Matrigel, BMP-7 and other BMPs of the decapentaplegic (dpp) and 60A subfamilies 17,18,20,21. Importantly, Matrigel- and BMP-induced dendrite formation occurs in the absence of changes in cell survival or axonal growth 17,18.
Here, we describe how to set up dissociated cultures of sympathetic neurons derived from the SCG of perinatal rats so that they are responsive to the selective dendrite-promoting activity of Matrigel or BMPs.

We describe a protocol for using bone morphogenetic protein-7 (BMP-7) or Matrigel to selectively induce dendritic growth in primary sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rats.

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

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

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

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

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

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity and extent of dendritic arborization correlates well with the synaptic plasticity in these cells. A reliable method for assessment of dendritic arborization is needed to characterize the deleterious effects of neurological disorders on these structures and to determine the effects of therapeutic interventions. However, quantification of these structures has proven to be a formidable task given their complex and dynamic nature. Fortunately, sophisticated imaging techniques can be paired with conventional staining methods to assess the state of dendritic arborization, providing a more reliable and expeditious means of assessment. Below is an example of how these imaging techniques were paired with staining methods to characterize the dendritic arborization in wild type mice. These complementary imaging methods can be used to qualitatively and quantitatively assess dendritic arborization that span a rather wide area within the hippocampal region.

We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

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

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 immune cells between the brain and the blood. Moreover, the huge neuronal metabolic demand requires a moment-to-moment regulation of blood flow. Notably, abnormalities of these regulations are etiological hallmarks of most brain pathologies; including glioblastoma, stroke, edema, epilepsy, degenerative diseases (ex: Parkinson&#8217;s disease, Alzheimer&#8217;s disease), brain tumors, as well as inflammatory conditions such as multiple sclerosis, meningitis and sepsis-induced brain dysfunctions. Thus, understanding the signaling events modulating the cerebrovascular physiology is a major challenge. Much insight into the cellular and molecular properties of the various cell types that compose the cerebrovascular system can be gained from primary culture or cell sorting from freshly dissociated brain tissue. However, properties such as cell polarity, morphology and intercellular relationships are not maintained in such preparations. The protocol that we describe here is designed to purify brain vessel fragments, whilst maintaining structural integrity. We show that isolated vessels consist of endothelial cells sealed by tight junctions that are surrounded by a continuous basal lamina. Pericytes, smooth muscle cells as well as the perivascular astrocyte endfeet membranes remain attached to the endothelial layer. Finally, we describe how to perform immunostaining experiments on purified brain vessels.

We describe a protocol allowing the purification of the mouse brain's vascular compartment. Isolated brain vessels include endothelial cells linked by tight junctions and surrounded by a continuous basal lamina, pericytes, vascular smooth muscle cells, as well as perivascular astroglial membranes.

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

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 columns to facilitate brain wiring during development and information processing in developed animals. Postsynaptic neurons elaborate dendrites in type-specific patterns in specific layers to synapse with appropriate presynaptic terminals. The fly medulla neuropil is composed of 10 layers and about 750 columns; each column is innervated by dendrites of over 38 types of medulla neurons, which match with the axonal terminals of some 7 types of afferents in a type-specific fashion. This report details the procedures to image and analyze dendrites of medulla neurons. The workflow includes three sections: (i) the dual-view imaging section combines two confocal image stacks collected at orthogonal orientations into a high-resolution 3D image of dendrites; (ii) the dendrite tracing and registration section traces dendritic arbors in 3D and registers dendritic traces to the reference column array; (iii) the dendritic analysis section analyzes dendritic patterns with respect to columns and layers, including layer-specific termination and planar projection direction of dendritic arbors, and derives estimates of dendritic branching and termination frequencies. The protocols utilize custom plugins built on the open-source MIPAV (Medical Imaging Processing, Analysis, and Visualization) platform and custom toolboxes in the matrix laboratory language. Together, these protocols provide a complete workflow to analyze the dendritic routing of Drosophila medulla neurons in layers and columns, to identify cell types, and to determine defects in mutants.

Here, we show how to analyze dendritic routing of Drosophila medulla neurons in columns and layers. The workflow includes a dual-view imaging technique to improve the image quality and computational tools for tracing, registering dendritic arbors to the reference column array and for analyzing the dendritic structures in 3D space.

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

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching variations of the neuronal dendrites within the hippocampus are implicated in cognition and memory formation. There are several approaches to Golgi staining, all of which have been useful for determining the morphological characteristics of dendritic arbors and produce a clear background. The present Golgi-Cox method, (a slight variation of the protocol that is provided with a commercial Golgi staining kit), was designed to assess how a relatively low dose of the chemotherapeutic drug 5-flurouracil (5-Fu) would affect dendritic morphology, the number of spines, and the complexity of arborization within the hippocampus. The 5-Fu significantly modulated the dendritic complexity and decreased the spine density throughout the hippocampus in a region-specific manner. The data presented show that the Golgi staining method effectively stained the mature neurons in the CA1, the CA3, and the dentate gyrus (DG) of the hippocampus. This protocol reports the details for each step so that other researchers can reliably stain tissue throughout the brain with high quality results and minimal troubleshooting.

Here we present a Golgi-Cox protocol in extensive detail. This reliable tissue stain method allows for a high-quality assessment of the cytoarchitecture in the hippocampus, and throughout the entire brain, with minimal troubleshooting.

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

This review describes the basic concepts and protocol to perform the real-time iontophoresis (RTI) method, the gold-standard to explore and quantify the extracellular space (ECS) of the living brain. The ECS surrounds all brain cells and contains both interstitial fluid and extracellular matrix. The transport of many substances required for brain activity, including neurotransmitters, hormones, and nutrients, occurs by diffusion through the ECS. Changes in the volume and geometry of this space occur during normal brain processes, like sleep, and pathological conditions, like ischemia. However, the structure and regulation of brain ECS, particularly in diseased states, remains largely unexplored. The RTI method measures two physical parameters of living brain: volume fraction and tortuosity. Volume fraction is the proportion of tissue volume occupied by ECS. Tortuosity is a measure of the relative hindrance a substance encounters when diffusing through a brain region as compared to a medium with no obstructions. In RTI, an inert molecule is pulsed from a source microelectrode into the brain ECS. As molecules diffuse away from this source, the changing concentration of the ion is measured over time using an ion-selective microelectrode positioned roughly 100 &#181;m away. From the resulting diffusion curve, both volume fraction and tortuosity can be calculated. This technique has been used in brain slices from multiple species (including humans) and in vivo to study acute and chronic changes to ECS. Unlike other methods, RTI can be used to examine both reversible and irreversible changes to the brain ECS in real time.

This protocol describes real-time iontophoresis, a method that measures physical parameters of the extracellular space (ECS) of living brains. The diffusion of an inert molecule released into the ECS is used to calculate the ECS volume fraction and tortuosity. It is ideal for studying acute reversible changes to brain ECS.

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

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. Drosophila larval neuronal dendritic arborization (da) is an ideal model for studying morphogenesis of neural dendrites and gene function in the development of nervous system. There are four classes of da neurons. Class IV is the most complex with a branching pattern that covers almost the entire area of the larval body wall. We have previously characterized the effect of silencing the Drosophila ortholog of SOX5 on class IV neuronal dendritic arborization complexity (NDAC) using four parameters: the length of dendrites, the surface area of dendrite coverage, the total number of branches, and the branching structure. This protocol presents the workflow of NDAC quantitative analysis, consisting of larval dissection, confocal microscopy, and image analysis procedures using ImageJ software. Further insight into da neuronal development and its underlying mechanisms will improve the understanding of neuronal function and provide clues about the fundamental causes of neurological and neurodevelopmental disorders.

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

This protocol focuses on quantitative analysis of neuronal dendritic arborization complexity (NDAC) in Drosophila, which can be used for studies of dendritic morphogenesis.

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. ...