The work was funded by the University of Zurich. The authors would like to thank Estrela Neto and Dr. Meriem Lamghari for helping in establishing the co-culture conditions.

所有的小鼠根据瑞士动物福利法和符合州兽医办公室，苏黎世的规定维护和处理。 1.准备材料解剖，文化传媒，微流体装置<ol><li>高压釜显微解剖镊子和剪刀（121℃，灭菌时间20分钟），并把它们存储在无菌容器中。 </li><li>通过将它们在1M HCl中24小时，在定轨摇床上，在37℃消毒盖玻片（24毫米×24毫米）。用无菌，蒸馏水用乙醇99％冲洗三次，2 O和三次。干燥然后将盖玻片，在37℃或在无菌流罩。最后，高压灭菌或暴露的盖玻片于UV光（30分钟）以完成杀菌。盖玻片然后可被储存在70％乙醇。 </li><li>使用无菌镊子小心地从包中取出的AXIS轴突隔离设备，并将其放置在无菌培养皿。 </LI&gt; <li>使用无菌活检穿孔（直径：仅1mm）中创建的培养腔室对应每采样1孔进行培养（ 图1）。 注意：不要冲太靠近微槽，因为他们可能会被施加的压力而损坏。 </li><li>通过佛光普照他们70％乙醇消毒AXIS轴突隔离设备。干那么Axon公司AXIS分离设备和盖玻片完全在无菌流罩。等待至少3小时的，然后再继续。 注：不完全的干燥，将导致在微流体器件的装配不良。 </li><li>放置每个盖玻片到35毫米的培养皿或成孔的6孔平板内。 </li><li>将AXIS轴突隔离装置上盖玻片，并轻轻按下但坚定地用钳子用弯曲的两端，以使隔离装置和玻璃盖玻片之间的完全粘连。 </li><li>在每个培养室，吸移管150微升聚-D-赖氨酸（0.1mg / ml的无菌蒸馏水的H 2O）。放置在真空下，微流体装置进行5分钟，以便从培养腔室除去所有的空气。 </li><li>如果空气仍然可以在腔室，再吸移管内看到了聚-D-赖氨酸溶液进入腔室。 </li><li>孵育用聚-D-赖氨酸O / N的装置，在37℃。 </li><li>用无菌蒸馏水H 2 O洗室三个时间</li><li>填用150微升的层粘连蛋白的工作溶液（Sigma-Aldrich公司，5微克/毫升，在PBS或无血清培养基）腔室，并孵育​​O / N在37℃。 </li><li>制备50ml培养基的三叉神经节培养物37，组成如下：48毫升Neurobasal培养基，加入1ml的B-27，100U / ml青霉素/链霉素，2mM的L-谷氨酰胺，5纳克/毫升神经生长因子（NGF） ，下午12时25阿糖胞苷。 </li><li>制备50ml培养基的牙胚培养37，组成如下：40毫升的DMEM-F12，将10毫升胎牛血清（FBS，终浓度：20％），100U / ml青霉素/链霉素，2mM的L-谷氨酰胺，150微克/毫升抗坏血酸。 </li></ol> 2.小​​鼠胚胎生成和解剖<ol><li>根据阴道塞确定胚胎的年龄（阴道塞：0.5的发展，E0.5胚胎天），并通过形态学标准确认。对于这个协议，我们一般使用E14.5-E17.5小鼠胚胎。 </li><li>清洁清扫面积和70％乙醇的立体镜。 </li><li>通过颈椎脱位牺牲孕妈妈。鼠标与第一和第二手指的颈部阻挡到电网，和拉带尾决定。 </li><li>解剖周围皮肤下腹部，并用剪刀打开腹部。定位子宫：怀孕期间的这种后期，子宫大量填充腹腔。 </li><li>解剖出子宫，并装在填充用PBS在冰上的管中。当在冰上，可将组织放置几个小时。根据机构指引抛弃母亲的尸体</li> &lt;LI&gt;解剖出胚胎从子宫并从他们胚外组织释放他们。放置在PBS中的胚胎在冰上。 </li><li>用剪刀斩首的胚胎，并使用显微解剖剪（ 图2A）从头部的其余部分分开的下颚。除去恰恰下颌，而不损坏三叉神经节;因为后者被定位在靠近下颌，其意外损坏是可能的。保留下颚和头部在冷PBS中的其余部分，在冰上。 </li><li>解剖TG，搭头，并将其放置到一个夹层玻璃培养皿，以前充满冷PBS。使用镊子，消除皮肤和颅骨。然后取出端脑和小脑通过将端脑和电梯下面镊子;端脑和小脑将翻转起来，把裸露的头骨底部。 </li><li>本地化的三叉神经节（在图2B中示出）。使用镊子TG的从三叉神经分开。消除使用解剖针为刀三叉预测的残余。将解剖的TG在培养皿中填充有冷PBS，并保持他们在冰上。 </li><li>解剖胚胎的牙齿，把下颚，以前从头骨分离，到夹层玻璃培养皿，充满冷PBS。用解剖针的刀，去除舌头和周围的下颚皮肤。通过沿颚的中线切割分开的左和右半爪。的牙胚很容易看到， 如图1C所示 。采用分离解剖针牙胚和去除多余的非牙齿组织。将解剖牙胚在培养皿中填充有冷PBS，并保持他们在冰上。 </li></ol> 3.微流控共培养<ol><li>解剖后，从微流体装置去除粘连。填写室与200微升respecti的已经媒体。 </li><li>用钳子，转移轻轻解剖TG和牙胚成通过冲压（ 图1D）形成的孔。确保牙胚不浮动，他们下沉，直到他们联系盖玻片。 </li><li>培养在37℃培养箱的样品中，5％的CO 2。 </li><li>改变培养基每48小时。不要空室完全，也不要直接将吸取文化室。的腔室将导致内室空气气泡的形成完全排空;直接移液到腔室会导致轴突损伤。为了避免这些问题，取出指向孔的外侧的吸移管的介质;类似地，吸液管上位于相对于腔室的孔中的侧面的新鲜培养基中。 </li><li>在培养期间，共培养物可以很容易地通过时间推移显微镜成像。共培养物可以保持超过10天。 </li><li>培养后，牙周D，吹打150微升PBS为每室一井，并让PBS流过腔三次洗室。 </li><li>除去PBS中并通过吸取150微升多聚甲醛的4％（在PBS中）在每室一个阱固定的样本;在室温下孵育15分钟。 </li><li>在3.6中所述，用PBS洗两次室。 </li><li>进行进一步的分析。 </li></ol>

<ol>
	<li>Pagella, P., Jim&#233;nez-Rojo, L., Mitsiadis, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+innervation+in+developing+and+regenerating+orofacial+tissues.">Roles of innervation in developing and regenerating orofacial tissues.</a> Cellular and molecular life sciences : CMLS. 71, 2241-2251 (2014).</li><li>Kumar, A., Brockes, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+dependence+in+tissue,+organ,+and+appendage+regeneration.">Nerve dependence in tissue, organ, and appendage regeneration.</a> Trends in neurosciences. 35 (11), 691-699 (2012).</li><li>Brownell, I., Guevara, E., Bai, C. B., Loomis, C. A., Joyner, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve-derived+sonic+hedgehog+defines+a+niche+for+hair+follicle+stem+cells+capable+of+becoming+epidermal+stem+cells.">Nerve-derived sonic hedgehog defines a niche for hair follicle stem cells capable of becoming epidermal stem cells.</a> Cell stem cell. 8 (5), 552-565 (2011).</li><li>Katayama, Y., Battista, 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=Signals+from+the+sympathetic+nervous+system+regulate+hematopoietic+stem+cell+egress+from+bone+marrow.">Signals from the sympathetic nervous system regulate hematopoietic stem cell egress from bone marrow.</a> Cell. 124 (2), 407-421 (2006).</li><li>Fitch, S. R., Kimber, G. 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=Signaling+from+the+sympathetic+nervous+system+regulates+hematopoietic+stem+cell+emergence+during+embryogenesis.">Signaling from the sympathetic nervous system regulates hematopoietic stem cell emergence during embryogenesis.</a> Cell stem cell. 11 (4), 554-566 (2012).</li><li>Knox, S. M., Lombaert, I. M. a., Reed, X., Vitale-Cross, L., Gutkind, J. S., Hoffman, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parasympathetic+innervation+maintains+epithelial+progenitor+cells+during+salivary+organogenesis.">Parasympathetic innervation maintains epithelial progenitor cells during salivary organogenesis.</a> Science(New York, N.Y.). 329 (5999), 1645-1647 (2010).</li><li>Knox, S. M., Lombaert, I. M. 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=Parasympathetic+stimulation+improves+epithelial+organ+regeneration.">Parasympathetic stimulation improves epithelial organ regeneration.</a> Nature communications. 4, 1494 (2013).</li><li>Oakley, B., Witt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+sensory+receptors+on+the+tongue.">Building sensory receptors on the tongue.</a> Journal of neurocytology. 33 (6), 631-646 (2004).</li><li>Oakley, B., Brandemihl, A., Cooper, D., Lau, D., Lawton, A., Zhang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+morphogenesis+of+mouse+vallate+gustatory+epithelium+and+taste+buds+requires+BDNF-dependent+taste+neurons.+Brain+research.">The morphogenesis of mouse vallate gustatory epithelium and taste buds requires BDNF-dependent taste neurons. Brain research.</a> Developmental brain research. 105 (1), 85-96 (1998).</li><li>Sun, H., Oakley, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+anterior+gustatory+epithelia+in+the+palate+and+tongue+requires+epidermal+growth+factor+receptor.">Development of anterior gustatory epithelia in the palate and tongue requires epidermal growth factor receptor.</a> Developmental biology. 242 (1), 31-43 (2002).</li><li>Mistretta, C. M., Goosens, K. a., Farinas, I., Reichardt, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+in+size,+number,+and+morphology+of+gustatory+papillae+and+taste+buds+in+BDNF+null+mutant+mice+demonstrate+neural+dependence+of+developing+taste+organs.">Alterations in size, number, and morphology of gustatory papillae and taste buds in BDNF null mutant mice demonstrate neural dependence of developing taste organs.</a> The Journal of comparative neurology. 409 (1), 13-24 (1999).</li><li>Mitsiadis, T. a., Graf, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+fate+determination+during+tooth+development+and+regeneration.">Cell fate determination during tooth development and regeneration.</a> Birth defects research. Part C, Embryo today reviews. 87 (3), 199-211 (2009).</li><li>Mohamed, S. S., Atkinson, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+histological+study+of+the+innervation+of+developing+mouse+teeth.">A histological study of the innervation of developing mouse teeth.</a> Journal of anatomy. 136 (Pt 4), 735-749 (1983).</li><li>Luukko, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+localization+of+nerve+fibres+during+development+of+embryonic+rat+molar+using+peripherin+and+protein+gene+product+9.5+antibodies.">Immunohistochemical localization of nerve fibres during development of embryonic rat molar using peripherin and protein gene product 9.5 antibodies.</a> Archives of oral biology. 42 (3), 189-195 (1997).</li><li>Johnsen, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innervation+of+teeth:+qualitative,+quantitative,+and+developmental+assessment.">Innervation of teeth: qualitative, quantitative, and developmental assessment.</a> Journal of dental research. 64, 555-563 (1985).</li><li>Mitsiadis, T. a., Dicou, E., Joffre, A., Magloire, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+localization+of+nerve+growth+factor+(NGF)+and+NGF+receptor+(NGF-R)+in+the+developing+first+molar+tooth+of+the+rat.">Immunohistochemical localization of nerve growth factor (NGF) and NGF receptor (NGF-R) in the developing first molar tooth of the rat.</a> Differentiation; research in biological diversity. 49 (1), 47-61 (1992).</li><li>Mitsiadis, T. a., Luukko, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotrophins+in+odontogenesis.">Neurotrophins in odontogenesis.</a> The International journal of developmental biology. 39 (1), 0214-6282 (1995).</li><li>Moe, K., Sijaona, A., Shrestha, A., Kettunen, P., Taniguchi, M., Luukko, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Semaphorin+3A+controls+timing+and+patterning+of+the+dental+pulp+innervation.">Semaphorin 3A controls timing and patterning of the dental pulp innervation.</a> Differentiation; research in biological diversity. 84 (5), 371-379 (2012).</li><li>Kettunen, P., L&#248;es, 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=Coordination+of+trigeminal+axon+navigation+and+patterning+with+tooth+organ+formation:+epithelial-mesenchymal+interactions+and+epithelial+Wnt4+and+Tgfbeta1+regulate+semaphorin+3a+expression+in+the+dental+mesenchyme.">Coordination of trigeminal axon navigation and patterning with tooth organ formation: epithelial-mesenchymal interactions and epithelial Wnt4 and Tgfbeta1 regulate semaphorin 3a expression in the dental mesenchyme.</a> Development (Cambridge, England). 132 (2), 323-334 (2005).</li><li>Tuisku, F., Hildebrand, C. <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+neural+influence+on+tooth+germ+generation+in+a+polyphyodont+species.">Evidence for a neural influence on tooth germ generation in a polyphyodont species.</a> Developmental biology. 165, 1-9 (1994).</li><li>Zhao, H., Feng, 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=Secretion+of+shh+by+a+neurovascular+bundle+niche+supports+mesenchymal+stem+cell+homeostasis+in+the+adult+mouse+incisor.">Secretion of shh by a neurovascular bundle niche supports mesenchymal stem cell homeostasis in the adult mouse incisor.</a> Cell stem cell. 14 (2), 160-173 (2014).</li><li>Kettunen, P., Kvinnsland, H., Luukko, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+rudimentary+diastema+tooth+primordia+are+devoid+of+peripheral+nerve+fibers.">Mouse rudimentary diastema tooth primordia are devoid of peripheral nerve fibers.</a> Anatomy and embryology. 205 (3), 187-191 (2002).</li><li>Lumsend, A., Buchanan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+experimental+study+of+timing+and+topography+of+early+tooth+development+in+the+mouse+embryo.">An experimental study of timing and topography of early tooth development in the mouse embryo.</a> Archives of oral biology. , 301-311 (1986).</li><li>Kollar, E., Lumsend, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tooth+morphogenesis:+the+role+of+the+innervation+during+induction+and+pattern+formation.">Tooth morphogenesis: the role of the innervation during induction and pattern formation.</a> Journal de Biologia Buccale. 7 (1), 49-60 (1979).</li><li>Luukko, K., Kettunen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordination+of+tooth+morphogenesis+and+neuronal+development+through+tissue+interactions:+lessons+from+mouse+models.">Coordination of tooth morphogenesis and neuronal development through tissue interactions: lessons from mouse models.</a> Experimental cell research. 325 (2), 72-77 (2014).</li><li>Lillesaar, C., Eriksson, C., Johansson, C. S., Fried, K., Hildebrand, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tooth+pulp+tissue+promotes+neurite+outgrowth+from+rat+trigeminal+ganglia+in+vitro.">Tooth pulp tissue promotes neurite outgrowth from rat trigeminal ganglia in vitro.</a> Journal of neurocytology. 28 (8), 663-670 (1999).</li><li>Lumsend, A., Davies, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotropic+effect+of+specific+target+epithelium+in+the+developing+mammalian+nervous+system.">Chemotropic effect of specific target epithelium in the developing mammalian nervous system.</a> Nature. 323 (9), 538-539 (1986).</li><li>Lillesaar, C., Fried, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurites+from+trigeminal+ganglion+explants+grown+in+vitro+are+repelled+or+attracted+by+tooth-related+tissues+depending+on+developmental+stage.">Neurites from trigeminal ganglion explants grown in vitro are repelled or attracted by tooth-related tissues depending on developmental stage.</a> Neuroscience. 125 (1), 149-161 (2004).</li><li>Lillesaar, C., Eriksson, C., Fried, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+tooth+pulp+cells+elicit+neurite+growth+from+trigeminal+neurones+and+express+mRNAs+for+neurotrophic+factors+in+vitro.">Rat tooth pulp cells elicit neurite growth from trigeminal neurones and express mRNAs for neurotrophic factors in vitro.</a> Neuroscience letters. 308 (3), 161-164 (2001).</li><li>Petrinovic, M. M., Duncan, C. 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=Neuronal+Nogo-A+regulates+neurite+fasciculation,+branching+and+extension+in+the+developing+nervous+system.">Neuronal Nogo-A regulates neurite fasciculation, branching and extension in the developing nervous system.</a> Development(Cambridge, England). 137 (15), 2539-2550 (2010).</li><li>Otsu, K., Fujiwara, N., Harada, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odontogenesis.">Odontogenesis.</a> Methods in Molecular Biology. 887, (2012).</li><li>Mitsiadis, T. a., Drouin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+of+the+Pitx1+genomic+locus+affects+mandibular+tooth+morphogenesis+and+expression+of+the+Barx1+and+Tbx1+genes.">Deletion of the Pitx1 genomic locus affects mandibular tooth morphogenesis and expression of the Barx1 and Tbx1 genes.</a> Developmental biology. 313 (2), 887-896 (2008).</li><li>Park, J. W., Vahidi, B., Taylor, A. M., Rhee, S. W., Jeon, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+culture+platform+for+neuroscience+research.">Microfluidic culture platform for neuroscience research.</a> Nature protocols. 1 (4), 2128-2136 (2006).</li><li>Hosmane, S., Tegenge, M. 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=Toll/interleukin-1+receptor+domain-containing+adapter+inducing+interferon-&#946;+mediates+microglial+phagocytosis+of+degenerating+axons.">Toll/interleukin-1 receptor domain-containing adapter inducing interferon-&#946; mediates microglial phagocytosis of degenerating axons.</a> The Journal of neuroscience the official journal of the Society for Neuroscience. 32 (22), 7745-7757 (2012).</li><li>Delamarche, E., Tonna, N., Lovchik, R. D., Bianco, F., Matteoli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacology+on+microfluidics:+multimodal+analysis+for+studying+celll-cell+interaction.">Pharmacology on microfluidics: multimodal analysis for studying celll-cell interaction.</a> Current opinion in pharmacology. 13 (5), 821-828 (2013).</li><li>Neto, E., Alves, C. 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=Sensory+neurons+and+osteoblasts:+close+partners+in+a+microfluidic+environment.">Sensory neurons and osteoblasts: close partners in a microfluidic environment.</a> Integrative Biology. , (2014).</li><li>Pagella, P., Neto, E., Jim&#233;nez-Rojo, L., Lamghari, M., Mitsiadis, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics+co-culture+systems+for+studying+tooth+innervation.">Microfluidics co-culture systems for studying tooth innervation.</a> Frontiers in physiology. 5 (August), (2014).</li><li>Connor, R., Tessier-Lavigne, M. <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+maxillary+factor,+a+maxillary+process-derived+chemoattractant+for+developing+trigeminal+sensory+axons.">Identification of maxillary factor, a maxillary process-derived chemoattractant for developing trigeminal sensory axons.</a> Neuron. 24, 165-178 (1999).</li></ol>

The authors declare that they have no competing financial interests.

上一页牙神经支配的体外研究都是基于传统的联合培养三叉神经节和牙齿的组织或细胞26,28,29的。这些研究进行主要调查这些细胞或组织上的感觉轴突38上的吸引力的影响。虽然引进外地显著的进步，几个技术问题被提出。牙胚开始培养后37几天就变质。基于这些观察，生长的神经元和牙齿在相同的培养条件影响涉及这两个组织之间的串扰的分子的任何最终分?...

支配起着器官和组织1,2的发育，稳态和再生的关键作用。此外，支配参与干细胞增殖，动员和分化3的调节- 5。实际上，实现了在颜面部复杂的组织最近的研究已经表明，唾液的发育和再生腺体6,7-期间副交感神经是必要的上皮的祖细胞的功能。类似地，已经证明，神经支配是必要的味蕾8的开发和维护- 11。因此，为了分析如牙神经支配在其他重要的口面部的器官和组织的发展却忽视的作用是很重要的。 尽管成年齿的丰富的神经支配的，相反于主体，开发板的所有其它器官和组织平齿开始在最早产后阶段进行支配。牙齿作为发展的口腔外胚层和中颅神经嵴源性间充质顺序和相互交流的结果。这些相互作用产生的上皮衍生成釉细胞和间充质来源的牙本质细胞，它们负责牙釉质和牙本质，分别为12的形成。从颈上神经节三叉神经节和交感神经感觉神经支配牙齿的成人13 - 15。在胚胎发生期间，神经纤维从向显影牙胚三叉神经节项目发出并逐步包围他们，但他们不渗透到牙乳头间质13。神经纤维进入牙髓间充质在与成牙本质细胞分化和牙本质基质沉积16关联更先进的发展阶段。牙髓的神经支配是并发症eted不久后在口腔中13牙齿萌出。先前的研究已经表明，各种脑信号和神经营养蛋白牙胚16期间参与神经支配的调节- 19。早先的研究已经清楚地表明，是支配牙齿形成鱼20的先决条件。最近的研究显示，在小鼠门齿该牙齿间充质干细胞的体内平衡是由感觉神经经由音猬（SHH）21的分泌调节。然而，神经支配在齿起始，发展和再生中的作用仍然是在哺乳动物中22高度争议- 24。 体内研究的大量已经在多种动物模型13,25,26的发展和修复过程提供了有关牙齿组织的神经支配的模式的重要信息。然而，大多数这些APPRO疼痛不是最佳突出神经纤维和靶器官和组织之间的相互作用的分子基础。共培养物构成一个有价值的方法进行调查，并操纵一个受控和隔离的环境26的神经纤维和牙齿之间的相互作用- 29。与此同时，共培养受到各种技术调整。例如，神经和特定牙组织（ 例如，牙髓，牙囊，牙上皮）通常需要不同的培养基中，以保证组织存活的时间30长时间- 32。 在过去的几十年中，使用相同的培养基中常规共培养物已进行很短的时间（ 例如 ，2天），以调查对感觉神经纤维27显影口腔和牙齿组织的吸引力或排斥力的影响- 29。然而，延长培养期必须调查神经支配牙齿形态发生和细胞分化的影响，并研究神经纤维靶器官内分枝的动态。因此，非连续的共培养将是更适合于神经，牙齿组织相互作用的研究执行。 微流体系统允许共培养的神经元和不同类型的细胞在它们的合适的培养基。在这些器件中，牙组织和神经元被分离在不同的隔室，同时允许从神经细胞体通过微通道朝向含有它们的靶组织33中的隔室的轴突的生长。微流体装置已经被用于研究癌症和新生血管35的神经元和胶质细胞34,35之间的相互作用，以及细胞与细胞的相互作用。此外，这些系统已经被用于研究DORS之间的相互作用人根神经节和成骨细胞36。 我们最近证明，三叉神经节（TG）和牙齿能够存活很长一段时间时，共培养在微流体装置37。此外，我们已经表明，来自不同发育阶段的牙齿在这些体外条件对三叉神经支配同一斥力或引力作用，它们显示出在体内 37保持。该协议提供了有关一个简单，功能强大且灵活的方式，共同培养神经节/神经与靶组织，并研究在一个可控和隔离的环境互动等特定分子的角色的信息。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>AXIS Axon Isolation Devices</td>
			<td>Millipore</td>
			<td>AX15010-TC</td>
			<td>Microchannels of different lenght are available</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma Aldrich</td>
			<td>L2020</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco</td>
			<td>17504</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse beta-NGF</td>
			<td>R&#38;D Systems</td>
			<td>1156-NG-100</td>
			<td>Human and Rat beta-NGF (R&#38;D Systems) are equivalent</td>
		</tr>
		<tr>
			<td>DMEM-F12</td>
			<td>Gibco</td>
			<td>11320-033</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of developing tooth germ innervation using microfluidic co-culture devices

支配起着器官和组织的发育，稳态和再生的关键作用。然而，这些现象背后的机制尚未很好地理解爱好。特别是，在神经支配牙齿发育和再生的作用被忽略。 几个在体内的研究已经在多种动物模型的发展和修复过程提供了有关牙齿组织的神经支配的模式的重要信息。然而，大多数这些方法不是最优的，突出的神经纤维和靶器官和组织之间的相互作用的分子基础。 共培养物构成一个有价值的方法进行调查，并操纵一个受控和隔离的环境神经纤维和齿之间的相互作用。在过去的几十年中，使用相同的培养基中常规共培养物已进行很短的时间（ 例如 ，2天）研究制定对感觉神经纤维口腔和牙齿组织的吸引力和排斥力的影响。但是，延长培养期需要调查支配牙齿形态发生和细胞分化的影响。 微流体系统允许共培养的神经元和不同类型的细胞在它们的合适的培养基。我们最近证明，三叉神经节（TG）和牙齿能够存活的很长一段时间时，共培养在微流体装置，并且它们在这些条件下保持它们显示在体内同一神经支配模式。 在此基础上，我们将描述如何分离和共培养显影三叉神经节和牙胚在微流体的共培养system.This协议描述了一个简单而灵活的方式来共培养神经节/神经和靶组织，并研究的作用在对照这种相互作用的特定分子olled和孤立的环境。

这些结果表明，分离的三叉神经节用的微流体装置的一个隔室生长，此外，所分离的牙胚的发展是持续的时间在微流体装置的另一个室长。不同的培养基中使用了两个室，两个室之间的微槽允许扩展从朝显影牙胚三叉神经节轴突的。 图3表示神经丝通过免疫荧光37可视化，在鼠标的共培养胚胎三叉神经节和小鼠胚胎门齿中所描述的微流体的共培养系统。鼠标门牙发展体内</em...

Watch this Scientific Journal Video about Analysis of Developing Tooth Germ Innervation Using Microfluidic Co-culture Devices at JoVE.com

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

Co-cultures represent a valuable method to study the interactions between nerves and target tissues and organs. Microfluidic systems allow co-culturing ganglia and whole developing organs or tissues in different culture media, thus representing a valuable tool for the in vitro study of the crosstalk between neurons and their targets.

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 ...

analysis-developing-tooth-germ-innervation-using-microfluidic-co

Research

JoVE Journal

Neuroscience

8.2K 次观看.  University of Zurich. Co-cultures represent a valuable method to study the interactions between nerves and target tissues and organs. Microfluidic systems allow co-culturing ganglia and whole developing organs or tissues in different culture media, thus representing a valuable tool for the in vitro study of the crosstalk between neurons and their targets.

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

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

二层共培养小学的大鼠皮层神经元和神经胶质

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

科研

神经科学

A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

Human primary muscle cells cultured aneurally in monolayer rarely contract spontaneously because, in the absence of a nerve component, cell differentiation is limited and motor neuron stimulation is missing1. These limitations hamper the in vitro study of many neuromuscular diseases in cultured muscle cells. Importantly, the experimental constraints of monolayered, cultured muscle cells can be overcome by functional innervation of myofibers with spinal cord explants in co-cultures.
Here, we show the different steps required to achieve an efficient, proper innervation of human primary muscle cells, leading to complete differentiation and fiber contraction according to the method developed by Askanas2. To do so, muscle cells are co-cultured with spinal cord explants of rat embryos at ED 13.5, with the dorsal root ganglia still attached to the spinal cord slices. After a few days, the muscle fibers start to contract and eventually become cross-striated through innervation by functional neurites projecting from the spinal cord explants that connecting to the muscle cells. This structure can be maintained for many months, simply by regular exchange of the culture medium.
The applications of this invaluable tool are numerous, as it represents a functional model for multidisciplinary analyses of human muscle development and innervation. In fact, a complete de novo neuromuscular junction installation occurs in a culture dish, allowing an easy measurement of many parameters at each step, in a fundamental and physiological context.
Just to cite a few examples, genomic and/or proteomic studies can be performed directly on the co-cultures. Furthermore, pre- and post-synaptic effects can be specifically and separately assessed at the neuromuscular junction, because both components come from different species, rat and human, respectively. The nerve-muscle co-culture can also be performed with human muscle cells isolated from patients suffering from muscle or neuromuscular diseases3, and thus can be used as a screening tool for candidate drugs. Finally, no special equipment but a regular BSL2 facility is needed to reproduce a functional motor unit in a culture dish. This method thus is valuable for both the muscle as well as the neuromuscular research communities for physiological and mechanistic studies of neuromuscular function, in a normal and disease context.

Cultured muscle cells are an inadequate model to recapitulate innervated muscle in vivo. A functional motor unit can be reproduced in vitro by innervation of differentiated human primary muscle cells using rat embryo spinal cord explants. This article describes how co-cultures of spinal cord explants and muscle cells are established.

一个在培养皿中的功能电机组：脊髓植和肌肉细胞共培养

Human primary muscle cells cultured  aneurally in monolayer rarely contract spontaneously because, in the absence of  a nerve component, cell ...

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture System

Proper neuron to glia interaction is critical to physiological function of the central nervous system (CNS). This bidirectional communication is sophisticatedly mediated by specific signaling pathways between neuron and glia1,2 . Identification and characterization of these signaling pathways is essential to the understanding of how neuron to glia interaction shapes CNS physiology. Previously, neuron and glia mixed cultures have been widely utilized for testing and characterizing signaling pathways between neuron and glia. What we have learned from these preparations and other in vivo tools, however, has suggested that mutual signaling between neuron and glia often occurred in specific compartments within neurons (i.e., axon, dendrite, or soma)3. This makes it important to develop a new culture system that allows separation of neuronal compartments and specifically examines the interaction between glia and neuronal axons/dendrites. In addition, the conventional mixed culture system is not capable of differentiating the soluble factors and direct membrane contact signals between neuron and glia. Furthermore, the large quantity of neurons and glial cells in the conventional co-culture system lacks the resolution necessary to observe the interaction between a single axon and a glial cell.
	In this study, we describe a novel axon and glia co-culture system with the use of a microfluidic culture platform (MCP). In this co-culture system, neurons and glial cells are cultured in two separate chambers that are connected through multiple central channels. In this microfluidic culture platform, only neuronal processes (especially axons) can enter the glial side through the central channels. In combination with powerful fluorescent protein labeling, this system allows direct examination of signaling pathways between axonal/dendritic and glial interactions, such as axon-mediated transcriptional regulation in glia, glia-mediated receptor trafficking in neuronal terminals, and glia-mediated axon growth. The narrow diameter of the chamber also significantly prohibits the flow of the neuron-enriched medium into the glial chamber, facilitating probing of the direct membrane-protein interaction between axons/dendrites and glial surfaces.

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System

This study describes the procedures of setting up a novel neuronal axon and (astro)glia co-culture platform. In this co-culture system, manipulation of direct interaction between a single axon (and single glial cell) becomes feasible, allowing mechanistic analysis of the mutual neuron to glial signaling.

神经元对神经胶质细胞在微流控文化互动平台（MCP）的神经元轴突和神经胶质细胞共培养系统的影像学分析

Proper  neuron to glia interaction is critical to physiological function of the central  nervous system (CNS). This bidirectional communication is ...

Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and morphological levels. Compound muscle action potential (CMAP) recording following phrenic nerve stimulation can be used to quantitatively assess functional diaphragm innervation by PhMNs of the cervical spinal cord in vivo in anesthetized rats and mice. Because CMAPs represent simultaneous recording of all myofibers of the whole hemi-diaphragm, it is useful to also examine the phenotypes of individual motor axons and myofibers at the diaphragm NMJ in order to track disease- and therapy-relevant morphological changes such as partial and complete denervation, regenerative sprouting and reinnervation. This can be accomplished via whole-mount immunohistochemistry (IHC) of the diaphragm, followed by detailed morphological assessment of individual NMJs throughout the muscle. Combining CMAPs and NMJ analysis provides a powerful approach for quantitatively studying diaphragmatic innervation in rodent models of CNS and PNS disease.

Compound muscle action potential recording quantitatively assesses functional diaphragm innervation by phrenic motor neurons. Whole-mount diaphragm immunohistochemistry assesses morphological innervation at individual neuromuscular junctions. The goal of this protocol is to demonstrate how these two powerful methodologies can be used in various rodent models of spinal cord disease.

隔膜的神经支配膈运动神经元的功能和形态评估

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and ...

High-resolution Volume Imaging of Neurons by the Use of Fluorescence eXclusion Method and Dedicated Microfluidic Devices

Volume is an important parameter regarding physiological and pathological characteristics of neurons at different time scales. Neurons are quite unique cells regarding their extended ramified morphologies and consequently raise several methodological challenges for volume measurement. In the particular case of in vitro neuronal growth, the chosen methodology should include sub-micrometric axial resolution combined with full-field observation on time scales from minutes to hours or days. Unlike other methods like cell shape reconstruction using confocal imaging, electrically-based measurements or Atomic Force Microscopy, the recently developed Fluorescence eXclusion method (FXm) has the potential to fulfill these challenges. However, although being simple in its principle, implementation of a high-resolution FXm for neurons requires multiple adjustments and a dedicated methodology. We present here a method based on the combination of fluorescence exclusion, low-roughness multi-compartments microfluidic devices, and finally micropatterning to achieve in vitro measurements of local neuronal volume. The high resolution provided by the device allowed us to measure the local volume of neuronal processes (neurites) and the volume of some specific structures involved in neuronal growth, such as growth cones (GCs).

Volume is an important parameter regarding physiological and pathological characteristics of cells. We describe a fluorescent exclusion method allowing full-field measurement of in vitro neuronal volume with sub-micrometric axial resolution required for the analysis of neurites and dynamic structures implied in neuronal growth.

利用荧光排斥法和专用微流控器件对神经元进行高分辨率体积成像

Volume is an important parameter regarding physiological and pathological characteristics of neurons at different time scales. Neurons are quite unique ...

Data Acquisition and Analysis In Brainstem Evoked Response Audiometry In Mice

Brainstem evoked response audiometry (BERA) is of central relevance in the clinical neurophysiology. As other evoked potential (EP) techniques, such as visually evoked potentials (VEPs) or somatosensory evoked potentials (SEPs), the auditory evoked potentials (AEPs) are triggered by the repetitive presentation of identical stimuli, the electroencephalographic (EEG) response of which is subsequently averaged resulting in distinct positive (p) and negative (n) deflections. In humans, both the amplitude and the latency of individual peaks can be used to characterize alterations in synchronization and conduction velocity in the underlying neuronal circuitries. Importantly, AEPs are also applied in basic and preclinical science to identify and characterize the auditory function in pharmacological and genetic animal models. Even more, animal models in combination with pharmacological testing are utilized to investigate for potential benefits in the treatment of sensorineural hearing loss (e.g., age- or noise-induced hearing deficits). Here we provide a detailed and integrative description of how to record auditory brainstem-evoked responses (ABRs) in mice using click and tone-burst application. A specific focus of this protocol is on pre-experimental animal housing, anesthesia, ABR recording, ABR filtering processes, automated wavelet-based amplitude growth function analysis, and latency detection.

Brainstem evoked response audiometry is an important tool in clinical neurophysiology. Nowadays, brainstem evoked response audiometry is also applied in the basic science and preclinical studies involving both pharmacological and genetic animal models. Here we provide a detailed description of how auditory brainstem responses can be successfully recorded and analyzed in mice.

脑干中的数据采集与分析 在小鼠中引发响应音频测量

Brainstem evoked response audiometry (BERA) is of central relevance in the clinical neurophysiology. As other evoked potential (EP) techniques, such as ...

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have identified a special population of vascular cells, the periventricular endothelial cells, that play a critical role in the migration and distribution of forebrain GABAergic interneurons during embryonic development. This, coupled with their cell-autonomous functions, alludes to novel roles of periventricular endothelial cells in the pathology of neuropsychiatric disorders like schizophrenia, epilepsy, and autism. Here, we have described three different in vitro assays that collectively evaluate the functions of periventricular endothelial cells and their interaction with GABAergic interneurons. Use of these assays, particularly in a human context, will allow us to identify the link between periventricular endothelial cells and brain disorders. These assays are simple, low cost, and reproducible, and can be easily adapted to any adherent cell type.

We present three simple in vitro assays-the long-distance migration assay, the co-culture migration assay, and chemo-attraction assay-that collectively evaluate the functions of human stem cell derived periventricular endothelial cells and their interaction with GABAergic interneurons.

人类干细胞衍生内皮细胞和GABAergic神经元的迁移、化学吸引和共培养测定

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have ...

Generation of Oligodendrocytes and Oligodendrocyte-Conditioned Medium for Co-Culture Experiments

In the central nervous system, oligodendrocytes are well-known for their role in axon myelination, that accelerates the propagation of action potentials through saltatory conduction. Moreover, an increasing number of reports suggest that oligodendrocytes interact with neurons beyond myelination, notably through the secretion of soluble factors. Here, we present a detailed protocol allowing purification of oligodendroglial lineage cells from glial cell cultures also containing astrocytes and microglial cells. The method relies on overnight shaking at 37 &#176;C, which allows selective detachment of the overlying oligodendroglial cells and microglial cells, and the elimination of microglia by differential adhesion. We then describe the culture of oligodendrocytes and production of oligodendrocyte-conditioned medium (OCM). We also provide the kinetics of OCM treatment or oligodendrocytes addition to purified hippocampal neurons in co-culture experiments, studying oligodendrocyte-neuron interactions.

Herein, we display an efficient method for the purification of oligodendrocytes and production of oligodendrocyte-conditioned medium that can be used for co-culture experiments.

共培养实验的奥里戈丁细胞和奥里戈丁酸基质的生成

In the central nervous system, oligodendrocytes are well-known for their role in axon myelination, that accelerates the propagation of action potentials ...

Axonal Transport of Organelles in Motor Neuron Cultures using Microfluidic Chambers System

Motor neurons (MNs) are highly polarized cells with very long axons. Axonal transport is a crucial mechanism for MN health, contributing to neuronal growth, development, and survival. We describe a detailed method for the use of microfluidic chambers (MFCs) for tracking axonal transport of fluorescently labeled organelles in MN axons. This method is rapid, relatively inexpensive, and allows for the monitoring of intracellular cues in space and time. We describe a step by step protocol for: 1) Fabrication of polydimethylsiloxane (PDMS) MFCs; 2) Plating of ventral spinal cord explants and MN dissociated culture in MFCs; 3) Labeling of mitochondria and acidic compartments followed by live confocal imagining; 4) Manual and semiautomated axonal transport analysis. Lastly, we demonstrate a difference in the transport of mitochondria and acidic compartments of HB9::GFP ventral spinal cord explant axons as a proof of the system validity. Altogether, this protocol provides an efficient tool for studying the axonal transport of various axonal components, as well as a simplified manual for MFC usage to help discover spatial experimental possibilities.

Axonal transport is a crucial mechanism for motor neuron health. In this protocol we provide a detailed method for tracking the axonal transport of acidic compartments and mitochondria in motor neuron axons using microfluidic chambers.

使用微流体腔系统在运动神经元培养物中有机细胞的斧头迁移

Motor neurons (MNs) are highly polarized cells with very long axons. Axonal transport is a crucial mechanism for MN health, contributing to neuronal ...

Visualizing the Calcitonin Gene-Related Peptide Immunoreactive Innervation of the Rat Cranial Dura Mater with Immunofluorescence and Neural Tracing

The aim of this study was to examine the distribution and origin of the calcitonin gene-related peptide (CGRP)-immunoreactive sensory nerve fibers of the cranial dura mater using immunofluorescence, three-dimensional (3D) reconstruction and retrograde tracing technique. Here, the nerve fibers and blood vessels were stained using immunofluorescence and histochemistry techniques with CGRP and fluorescent phalloidin, respectively. The spatial correlation of dural CGRP-immuoreactive nerve fibers and blood vessels were demonstrated by 3D reconstruction. Meanwhile, the origin of the CGRP-immunoreactive nerve fibers were detected by neural tracing technique with fluorogold (FG) from the area around middle meningeal artery (MMA) in the cranial dura mater to the trigeminal ganglion (TG) and cervical (C) dorsal root ganglia (DRGs). In addition, the chemical characteristics of FG-labeled neurons in the TG and DRGs were also examined together with CGRP using double immunofluorescences. Taking advantage of the transparent whole-mount sample and 3D reconstruction, it was shown that CGRP-immunoreactive nerve fibers and phalloidin-labeled arterioles run together or separately forming a dural neurovascular network in a 3D view, while the FG-labeled neurons were found in the ophthalmic, maxillary, and mandibular branches of TG, as well as the C2-3 DRGs ipsilateral to the side of tracer application in which some of FG-labeled neurons presented with CGRP-immunoreactive expression. With these approaches, we demonstrated the distributional characteristics of CGRP-immunoreactive nerve fibers around the blood vessels in the cranial dura mater, as well as the origin of these nerve fibers from TG and DRGs. From the perspective of methodology, it may provide a valuable reference for understanding the complicated neurovascular structure of the cranial dura mater under the physiological or pathological condition.

Here we present a protocol to visualize spatial correlation of calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers and blood vessels in the cranial dura mater using immunofluorescence and fluorescent histochemistry with CGRP and phalloidin, respectively. In addition, the origin of these nerve fibers was retrograde traced with a fluorescent neural tracer.

可视化与钙西通宁基因相关的肽免疫活性内活性与免疫发光和神经追踪

The aim of this study was to examine the distribution and origin of the calcitonin gene-related peptide (CGRP)-immunoreactive sensory nerve fibers of the ...