由美国国立卫生研究院/ NCRR科罗​​拉多CTSI授权号UL1 RR025780和落基山脉Neurlogical障碍核心中心授予美国国立卫生研究院P30NS048154部分支持NIH / NIDCD R01 DC 011582.成像实验在美国科罗拉多州安舒茨医学校区先进的光学显微镜核心的大学进行支撑。萨沙迪拉克博士索尔克研究所为我们提供了GlyT2-GFP小鼠。

1.光学基因分型乳鼠1.光学基因分型<ol><li>检查使用适当的激发波长的激光指针的各荧光标记物的表达（在这里描述的实验中为405nm）和相应的过滤器护目镜阻断激发波长不过将发射波长（450 - 700在此处描述的实验纳米） 。点激光指示器在头部或鼠标小狗脊髓的背面（ 参见图1）。避免闪耀的激光进入眼睛和皮肤的激光的冗长曝光。 </li></ol>较早的动物2.光学基因分型<ol><li>通过腹膜内注射（120毫克/公斤体重）深深麻醉小鼠（老化P14到在这里描述的实验P138）用戊巴比妥的过量。通过检查动​​物的反射确认适当的麻醉（简称捏个尖Ë耳朵或后腿之一，引起耳或腿）的回缩反射。 </li><li>注意:尽管使用戊巴比妥（120毫克/千克体重）的过量，我们指的是注射过程为"深度麻醉"，而不是安乐死，因为动物是理想情况下还活着（ 即它的心脏还在跳动），当手术和灌注开始。这确保了内部器官包括脑的更有效的灌注。 </li><li>一旦动物没有显示任何反射，小心地通过使在皮肤覆盖头部的背面的切口和切割到头骨的中部去除皮肤覆盖在头骨（图2A）。暴露揭露头骨激光光并如上所述通过过滤器护目镜观察荧光。如果一个积极的信号GFP观察，继续执行步骤2，如果不通过断头牺牲动物（秒/确保安乐死下列表格我们IACUC规定）。 </li></ol>注意:在更老的小鼠（&gt; 1个月）的荧光可以通过动物的眼睛中观察到。 2.穿心灌注和脑准备<ol><li>用冰冷的磷酸盐缓冲盐水灌注transcardially（PBS;氯化钠:137毫，氯化钾:2.7毫，KH 2 PO 4:1.76毫米， 的 Na 2 HPO 4:10毫米），使用地下30注射器针从很大程度上除去血这个动物。 <ol><li>首先，小心地打开胸廓用锋利的剪刀，然后将针头推入心脏的左心室。启动抽PBS到心脏和主动脉，并紧跟这一步打开心脏的右心房与剪刀允许血液离开人体。注意:灌注需要5 - 10分钟取决于动物的大小。 </li></ol></li><li>通过灌注放血后，杀头的动物，保护其头部引脚needles（或为30G注射器针头）通过眼窝刺入他们的准备菜，从头骨取出大脑。 <ol><li>首先，用锋利的剪刀剪下皮肤覆盖在颅骨:做一个小切口在脑后，然后沿头部的背侧中线切开，切成只是尾部的动物的眼睛两侧而在尾方向最后切到了脑后彻底清除皮肤的皮瓣。 </li><li>接下来，重复颅骨本身相同的切削模式，确保在这个过程中删除这两个耳蜗，因为这将显著简化到底切除脑干。此过程后大脑的尾半应处于完全暴露。 </li><li>在下一步骤，使用锋利刀片或手术刀贯穿全前脑削减在约45°至约2毫米喙从小脑的角度。舀出大脑中与AB的分离的前半耳鼻喉科锅铲。 </li><li>使用抹刀提升大脑的尾侧一半在其嘴侧端并通过仍连接到脑干其腹侧脑神经随后切割。作为一个最终的结果，脑包括脑干尾端一半应该自由地脱落配制的动物头的其余部分。 </li><li>翻转大脑以暴露其腹侧的除去尾半和沿冠状面只是喙切（对于顺行注射）或只是尾椎（对于逆行注射）的含有梯形体的腹侧位于"灯泡"（见步骤2.3）。 <ol><li>始终使用锋利的刀片或手术刀的切割程序，以尽量减少对组织造成过度的机械应力性细胞死亡。作为一个最终的结果，应该已经获得了脑外植体跨越长度约1厘米，并包含沿等脑区的完整的上橄榄复合体。 </li></ol></li></ol> &lt;/ LI&gt; </ol>注意:此协议演示程序示踪剂注射入位于听觉脑干梯形体。根据需要适应切割面和注射部位的位置和方向。如果兴趣谎言足够接近脑表面，使得它们可以被识别并且不切断脑部注射的荧光脑区（参见图2B），然后在冠状切割步骤可以省略。 <ol start="3"><li>确定梯形体含有梯形体（VNTB）腹侧核2腹突出的"灯泡"。 </li></ol>注:有关这些切割面的近似立体定位进一步解剖参考，请参阅寰富兰克林和Paxinos，2008年面板78，前囟〜-5.7毫米显示内侧核的梯形体（= MNTB）的标示为"Tz的"。面板69显示梯形波腹核DY（VNTB）标记为"MVPO"（medioventral periolivary核）19。 3.顺行/逆行追踪注意:在RT（25℃）下进行所有下面的步骤。 <ol><li>切割后，将脑干外植体在制备培养皿含有充氧（95％O 2，5％的CO 2）解剖溶液（氯化钠:125毫，氯化钾:2.5毫米， 氯化镁 :1mM的， 氯化钙 2:0.1毫米，葡萄糖:25mM的，将NaH 2 PO 4:1.25毫， 碳酸氢钠 :25mM的，抗坏血酸:0.4mM的，肌醇:3毫米，丙酮酸:2毫米），用30克的注射器针头固定它。确保注射区域朝上。 </li><li>从硼硅玻璃拉注射移液器，并与以下三种溶液中的一个填充它们:a）一种为1.0mg / ml的溶液霍乱毒素亚基-B的共轭到Alexa的荧光团555在PBS 12-13日 （主要用于逆行运输），B ）1％-dilution在葡聚糖四甲555（3000兆瓦，主要用于逆行运输）或c）1％-dilution的0.9％盐水中的万MW葡聚糖TRITC 555 0.9％的盐水（主要用于顺行运输）。 <ol><li>确保注射针的电阻范围为2.5至3.5兆欧姆，并且他们在3被制造 - 4拉周期。对于这里描述的实验中，通过在吸管拉马选择预编程的拉算法实现这一目标。 </li></ol></li><li>照亮使用适当波长的静态安装激光指示器大脑外植体（项目编号2 图 3A;在这里描述的实验中405nm的波长）。使用激光指针作为用于注射剂的导向，并且旨在将激光束在荧光标记的脑核或感兴趣的细胞将被注入。 </li><li>查找并通过双目显微镜观察照射目标区域，而穿着兼容的过滤器眼罩（450 - 700纳米的带通滤波器在这里描述的实验）。插入通过显微注射电极到感兴趣的区域。 </li><li>注射示踪物质。注射由2 - 10压脉冲在15psi的每个50毫秒，利用压力注射装置，定向垂直成的感兴趣区（ROI）的区域中的脑切片内。管理每个压力脉冲的10个间隔 - 15秒，以允许染料扩散。 </li><li>在四甲（TRITC）注射剂的情况下，附加地刺激电极通过电穿孔，以提高上染率与放置在感兴趣的脑区域（MNTB和VNTB在这里描述的实验）的电极。 <ol><li>使用的8伏的TTL 8脉冲50毫秒与50毫秒脉冲间间隔，通过刺激器驱动并放大到55 V用刺激隔离单元（项目在图3B中第7号和9）。使用10 - 20重复该协议，adminis的羊羔数分钟7,8。确保正确接地电极 - 接地线应接在洗澡的解决方案！ </li></ol></li><li>检查注射成功。自的荧光示踪剂具有较长的发射波长也发射在激光激发的荧光信号以更短的波长，在视觉上检查该染料吸收/分散在注射的目标区域而照射与激光​​的区域，并通过过滤器护目镜观察。 </li></ol> 4.孵化<ol><li>注射后，孵育脑干中含氧人工脑脊液（ACSF;氯化钠:125毫，氯化钾:2.5毫米， 氯化镁 :1mM的， 氯化钙 2:2毫米，葡萄糖:25毫米，的NaH 2 PO 4:1.25毫，的NaHCO 3:25毫米，抗坏血酸:0.4mM的，肌醇:3毫米，丙酮酸:2毫米，通入95％O 2 - 5％的CO 2）在室温（RT，25℃）为1 - 4小时，而染料被输送。泡沫在整个孵化期的解决方案。 </li><li>接着，转移脑干中的4％多聚甲醛（PFA）溶解在PBS（约100个​​ml的; pH为7的PFA / PBS中混合），并培育它们在4℃下，以允许后固定过夜（O / N）。 </li></ol> 5.组织切片和安装<ol><li>第二天，洗涤脑干三次在PBS（5分钟，每次洗涤步骤），包括它在4％琼脂，然后切成50 - 80微米厚的薄片上的vibratome。使用在载玻片上，和盖玻片封固剂安装片。 </li><li>任选，（以1例如蓝色尼斯尔:100稀释在AB培养基）标记有荧光尼氏切片25分钟。 <ol><li>为了制备AB介质，混合250个毫升0.2M磷酸缓冲液（PB; 51毫KH 2 PO 4，150mM的磷酸氢二钠），15毫升5 M氯化钠，15毫升10％的Triton-X，220 ml的DDH 2 O的和5g牛血清白蛋白。 </li><li> Prece德和成功由3洗涤步骤的尼斯尔标记步骤在PBS（每次10分钟）。 </li></ol></li></ol>注意:在情况下，厚的部分需要安装和分析（在这里描述的这种切片实验是100 - 厚以保持过更大的距离轴突连接500微米），该技术可以与组织清除组合。我们发现ClearT2 12是成功的20。当一个组织清除步骤包括，安装部分之前执行它。

<ol>
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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+development+of+the+calyx+of+Held+synapse.">Dynamic development of the calyx of Held synapse.</a> PNAS. 105 (14), 5603-5608 (2008).</li><li>Albrecht, O., Dondzillo, A., Mayer, F., Thompson, J. A., Klug, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitory+projections+from+the+ventral+nucleus+of+the+trapezoid+body+to+the+medial+nucleus+of+the+trapezoid+body+in+the+mouse.">Inhibitory projections from the ventral nucleus of the trapezoid body to the medial nucleus of the trapezoid body in the mouse.</a> Front. Neural Circuits. 8, 83 (2014).</li><li>Benson, D. A., Teas, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+unit+of+binaural+interaction+in+the+auditory+cortex+of+the+chinchilla.">Single unit of binaural interaction in the auditory cortex of the chinchilla.</a> Brain Res. 103, 313-338 (1976).</li><li>Koka, K., Jones, H. G., Thornton, J. L., Lupo, J. E., Tollin, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sound+pressure+transformations+by+the+head+and+pinnae+of+the+adult+Chinchilla+(Chinchilla+lanigera).">Sound pressure transformations by the head and pinnae of the adult Chinchilla (Chinchilla lanigera).</a> Hear Res. 272 (1-2), 135-147 (2011).</li><li>Ryan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hearing+sensitivity+of+the+mongolian+gerbil,+Meriones+unguiculatus.">Hearing sensitivity of the mongolian gerbil, Meriones unguiculatus.</a> J. Acoust. Soc. Am. 59 (5), 1222-1226 (1976).</li><li>Zwicker, E., Fastl, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Psychoacoustics Facts and Models. , (1990).</li><li>Chu, G., Hayakawa, H., Berg, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+for+the+efficient+transfection+of+mammalian+cells+with+DNA.">Electroporation for the efficient transfection of mammalian cells with DNA.</a> Nucleic Acids Res. 15 (3), 1311-1326 (1987).</li><li>Klenchin, V. A., Sukharev, S. I., Serov, S. M., Chernomordik, L. V., Chizmadzhev, Y. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrically+induced+DNA+uptake+by+cells+is+a+fast+process+involving+DNA+electrophoresis.">Electrically induced DNA uptake by cells is a fast process involving DNA electrophoresis.</a> Biophys J. 60 (4), 804-811 (1991).</li><li>Elias, L., Kriegstein, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+Slice+Culture+of+E18+Rat+Brains.">Organotypic Slice Culture of E18 Rat Brains.</a> J. Vis. Exp. (6), e235 (2007).</li><li>G&#228;hwiler, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+monolayer+cultures+of+nervous+tissue.">Organotypic monolayer cultures of nervous tissue.</a> J. Neurosci. Methods. 4 (4), 329-342 (1981).</li><li>Tallini, Y. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BAC+transgenic+mice+express+enhanced+green+fluorescent+protein+in+central+and+peripheral+cholinergic+neurons.">BAC transgenic mice express enhanced green fluorescent protein in central and peripheral cholinergic neurons.</a> Physiol Genomics. 27 (3), 391-397 (2006).</li></ol>

我们什么都没有透露。

体外示踪剂电穿孔的一般强度，相对于在体内跟踪研究中，是可以让研究人员的利益，因此大脑区域更好的访问，不涉及昂贵立体定向（通常电）设备。此外，所需的脑外植体存活期跨越只有几个小时（1 - 4）中的体内示踪注射的情况下，而不是数天或甚至数周（见关于使用葡聚糖胺和在其他示踪剂的详细审查体内注射由Lanciego和Wouterlood，2011 22，但也罗德里格?...

为了定义一个特定的神经元（微）电路，必须通过找出所述电路，和它们的连接图形的各个参与者启动。自从沃勒的出版约neurofiber通过损毁1种类繁多的神经解剖跟踪技术跟踪已经建立。这些技术的一些可以在固定的组织验尸2-4被应用，更为依赖于染料的活的神经元的活性的运输，如发现于1971年5-6。后者可进一步细分为两组利用活性逆行方法区分（从注入区域到给定突起的来源 ， 即所突出的所述区的神经元的胞 ​​体）和活性顺行（从注入面积给定突起的目标 ， 即轴突突起和标记的神经元）传输的轴突终端。此外，在一些情况下，TRA减量材料注入活的动物，然后注入由数天或数周（体内示踪剂注射）存活，而在其他情况下取出的大脑注射和注入人工脑脊液后孵育几个小时（体外示踪剂注射） 。 在这个协议中，我们修改了现有的体外电击技术7-8在使用霍乱毒素亚基B和四甲右旋糖酐作为跟踪物质顺行和逆行追踪实验标记神经元胞体和进程。此协议的总体目标是提供神经科学家与一个有效的工具来跟踪不同的大脑核团神经元之间的连接方式，同时采取可转基因小鼠线和基本的光学设备的优势，以便在示踪剂注射，以增加定位精度。虽然顺行和逆行追踪的方法，用霍乱毒素和葡聚糖胺和它们各自的荧光标记的偶联物是不是新的9-13，（由于是电穿孔的方法 ，例如:Haas等14），示踪注射涉及脑组织的块的组合与电穿孔在体外制备是一个比较新的发展7。使用相同类型的示踪染料的活体动物过度神经元的跟踪技术的主要优点是由于较高的效率与该电穿孔的染料被拉紧的神经元的增加的标记强度。一个附加的优点是示踪剂注射期间缩短的潜伏期（用于染料运输）和其增加的目标准确度，因为实验者具有视觉地控制喷射的目标区域。后者也意味着不需要昂贵的立体定向的设备，需要找到感兴趣的核或大脑区域。 为了阿迪tionally增加靶向准确性，我们采取了转基因小鼠系，它表达的GFP在其甘氨酸神经元15和基本的光学设备组成的405nm的波长和匹配带通滤波护目镜的手持式激光指针（450的亚群的优势- 700纳米）。因此，我们在通过其荧光信号识别注射区靶向精度和通过提供一种更细的方式，通过观测土著GFP信号和所述示踪剂的相互作用的时，控制该注射区域内的染料扩散取得显著进一步增加荧光。我们的技术还允许揭示的电路的功能以及通过识别GFP阳性抑制性神经元及其连通（或兴奋性在其它小鼠品系）填充的示踪剂。 总之，我们进一步增强了强大的神经科学工具，研究脊椎动物脑的连接组和评估吨他给定neurocircuit不同的神经解剖学特征。通过使用转基因小鼠以及便宜且广泛可用的光学设备，我们能够显著增加我们注射剂的靶向精度。此外，转基因小鼠使我们能够确定被跟踪的连接，这有助于揭示在听觉脑干的抑制微电路的功能的类型。

<table><tbody><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>S7653</td><td>All chemicals are from Sigma-Aldrich, unless noted otherwise.</td></tr><tr><td>Potassium chloride&amp;nbsp;</td><td></td><td>P9333</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td></td><td>P5655</td><td></td></tr><tr><td>Sodium phosphate di-basic</td><td></td><td>S907</td><td></td></tr><tr><td>Magnesium chloride</td><td></td><td>M2670</td><td></td></tr><tr><td>Calcium chloride</td><td></td><td>C5080</td><td></td></tr><tr><td>Glucose</td><td></td><td>G7528</td><td></td></tr><tr><td>Sodium bicarbonate</td><td></td><td>S6297</td><td></td></tr><tr><td>Ascorbic acid</td><td></td><td>A4544</td><td></td></tr><tr><td>Myo-inositol</td><td></td><td>I5125</td><td></td></tr><tr><td>Sodium pyruvate</td><td></td><td>P2256</td><td></td></tr><tr><td>Bovine serum albumine</td><td></td><td>A2153</td><td>optional, for additional (immuno)histochemistry</td></tr><tr><td>Triton-X-100</td><td></td><td>X100</td><td>optional, for additional (immuno)histochemistry</td></tr><tr><td>Poly(ethylene glycol), 8000 MW</td><td></td><td>P2139</td><td>optional, for brain clearing</td></tr><tr><td>Formamide</td><td>Fisher Scientific</td><td>F84</td><td>optional, for brain clearing</td></tr><tr><td>Choleratoxin subunit-b</td><td>Molecular Probes</td><td>C-34776 (Alexa 555)&amp;nbsp;</td><td></td></tr><tr><td>Dextrane tetramethyl-rhodamine</td><td>Molecular Probes</td><td>D-7162 (Alexa 555)</td><td></td></tr><tr><td>Fluorescent Nissl</td><td>Invitrogen</td><td>N-21479 (blue)</td><td>optional</td></tr><tr><td>Paraformaldehyde</td><td>Fisher Scientific</td><td>SF93</td><td></td></tr><tr><td>Agarose</td><td>Invitrogen</td><td>16520</td><td></td></tr><tr><td>Fluoromount-G</td><td>Southern Biotech</td><td>0100-01</td><td></td></tr><tr><td>Pentobarbi-tal</td><td>Vortech Pharmaceuticals</td><td>Fatal-Plus&amp;nbsp;</td><td></td></tr><tr><td>Borosilicate glass filaments</td><td>Harvard Apparatus</td><td>G150F-10</td><td></td></tr><tr><td>Pipette puller</td><td>Zeitz Instruments, Germany</td><td>DMZ Universal Puller</td><td></td></tr><tr><td>Perfusion setup</td><td>Custom-made</td><td></td><td></td></tr><tr><td>Laser pointer&amp;nbsp;</td><td>laserpointerpro.com, Hong Kong</td><td>HK-88007294 (405 nm)</td><td></td></tr><tr><td>Filter/safety goggles</td><td>Dragon Lasers, China</td><td>LSG09 (band-pass 450-700 nm)</td><td></td></tr><tr><td>Bionocular microscope&amp;nbsp;</td><td>Wild Heerbrugg, Switzerland</td><td>Wild M3</td><td>Equipped with high-intensity illuminator (MI-150; Dolan-Jenner Inc.)&amp;nbsp;</td></tr><tr><td>Picospritzer</td><td>Parker Instruments</td><td>Picospritzer III</td><td></td></tr><tr><td>PC with installed MC Stimulus software</td><td>Multi Channel Sys-tems, Germany (software)</td><td></td><td></td></tr><tr><td>2-channel stimulator</td><td>Multi Channel Sys-tems, Germany</td><td>STG-1002</td><td></td></tr><tr><td>Stimulation isolation unit</td><td>A.M.P.I., Israel</td><td>Iso-Flex</td><td></td></tr><tr><td>Micromanipulator</td><td>Narishige, Japan</td><td>YOU-1</td><td></td></tr><tr><td>Vibratome</td><td>Leica, Germany</td><td>VT1000S</td><td></td></tr></tbody></table>

laser-guided neuronal tracing in brain explants

我们提出结合的体外示踪剂注射方案，它使用了一系列的电气和压力脉冲的过程中，通过电穿孔，增加脑外植体与靶向激光照明和匹配滤波器护目镜染料吸收的技术。 在体外的电本身所描述的技术产生了比较良好的视觉控制目标定位大脑的某些区域。通过将其与荧光遗传标记的激光激发和它们的读出通过带传递滤波器护目镜，其中可以拿起遗传标记的细胞/细胞核和荧光示踪染料的排放量，研究者可以大幅提高注射剂的精度相结合通过找到感兴趣的区域，并更有效地控制了该染料扩散/吸收在注射区域。此外，激光照射技术允许研究给定neurocircuit通过省的功能iding约神经元投射到一特定区域的情况下的GFP表达被链接到发射机的由神经元的亚群表达的类型的类型的信息。

图1和2示出了如何将激光指针和激光护目镜可用于快速，低成本从垫料基因型GFP阳性的动物。在年轻小鼠幼仔情况下，该技术可以用于通过头骨和覆盖皮肤（图1A - D）的非侵入性地确定在动物的大脑的GFP标签。发射的荧光可以通过皮肤和小鼠幼仔头骨至少高达3日龄（图1C）可以看出。该过程示于图1，为绿色荧光蛋白（GFP...

Watch this Scientific Journal Video about Laser-guided Neuronal Tracing In Brain Explants at JoVE.com

Laser-guided Neuronal Tracing In Brain Explants

我们描述了一种技术，通过顺行或逆行示踪剂注射标记的神经元和它们的过程变成使用体外制备脑核。我们修改的I N通过利用荧光标记的小鼠突变体和基本的光学设备，以便增加贴标精度体外示踪剂电穿孔的现有方法。

激光制导神经跟踪在脑外植体

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake ...

laser-guided-neuronal-tracing-in-brain-explants

Research

JoVE Journal

Neuroscience

8.8K 次观看.  University of Colorado School of Medicine. 我们描述了一种技术，通过顺行或逆行示踪剂注射标记的神经元和它们的过程变成使用体外制备脑核。我们修改的I N通过利用荧光标记的小鼠突变体和基本的光学设备，以便增加贴标精度体外示踪剂电穿孔的现有方法。 

视频: Laser-guided Neuronal Tracing In Brain Explants

Intraventricular Transplantation of Engineered Neuronal Precursors for In Vivo Neuroarchitecture Studies

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in vivo gene control of neocortical projection neuron morphology. This method is based on (1) in vitro lentiviral engineering of neuronal precursors as "test" and "control" cells, (2) their co-transplantation into wild-type brains, and (3) paired morphometric evaluation of their neuronal derivatives. Specifically, E12.5 pallial precursors from panneuronal, genetically labeled donors, are employed for this purpose. They are engineered to take advantage of selected promoters and tetON/OFF technology, and they are free-hand transplanted into neonatal lateral ventricles. Later, upon immunofluorescence profiling of recipient brains, silhouettes of transplanted neurons are fed into NeurphologyJ open source software, their morphometric parameters are extracted, and average length and branching index are calculated. Compared to other methods, this one offers three main advantages: it permits achieving of fine control of transgene expression at affordable costs, it only requires basic surgical skills, and it provides statistically reliable results upon analysis of a limited number of animals. Because of its design, however, it is not adequate to address non cell-autonomous control of neuroarchitecture. Moreover, it should be preferably used to investigate neurite morphology control after completion of neuronal migration. In its present formulation, this method is exquisitely tuned to investigate gene control of glutamatergic neocortical neuron architecture. Taking advantage of transgenic lines expressing EGFP in other specific neural cell types, it can be re-purposed to address gene control of their architecture.

Based on in vitro lentiviral engineering of neuronal precursors, their co-transplantation into wild-type brains and paired morphometric evaluation of "test" and "control" derivatives, this method allows accurate modeling of in vivo gene control of neocortical neuron morphology in a simple and affordable way.

工程神经前体宫内移植,用于Vivo神经结构研究

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in ...

科研

遗传学

Using Laser Tweezers For Manipulating Isolated Neurons In Vitro

In this paper and video, we describe the protocols used in our laboratory to study the targeting preferences of regenerating cell processes of adult retinal neurons in vitro. Procedures for preparing retinal cell cultures start with the dissection, digestion and trituration of the retina, and end with the plating of isolated retinal cells on dishes made especially for use with laser tweezers. These dishes are divided into a cell adhesive half and a cell repellant half. The cell adhesive side is coated with a layer of Sal-1 antibodies, which provide a substrate upon which our cells grow. Other adhesive substrates could be used for other cell types. The cell repellant side is coated with a thin layer of poly-HEMA. The cells plated on the poly-HEMA side of the dish are trapped with the laser tweezers, transported and then placed adjacent to a cell on the Sal-1 side to create a pair. Formation of cell groups of any size should be possible with this technique. "Laser-tweezers-controlled micromanipulation" means that the investigator can choose which cells to move, and the desired distance between the cells can be standardized. Because the laser beam goes through transparent surfaces of the culture dish, cell selection and placement are done in an enclosed, sterile environment. Cells can be monitored by video time-lapse and used with any cell biological technique required. This technique may help investigations of cell-cell interactions.

This video describes the manipulation of cultured neurons using laser tweezers in vitro.

使用激光光镊操纵隔离神经细胞在体外

In this paper and video, we describe the protocols used in our laboratory to study the targeting preferences of regenerating cell processes of adult ...

生物学

Neural Explant Cultures from Xenopus laevis

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During axon outgrowth, the growth cone must integrate multiple sources of guidance cue information to modulate its cytoskeleton in order to propel the growth cone forward and accurately navigate to find its specific targets1. How this integration occurs at the cytoskeletal level is still emerging, and examination of cytoskeletal protein and effector dynamics within the growth cone can allow the elucidation of these mechanisms. Xenopus laevis growth cones are large enough (10-30 microns in diameter) to perform high-resolution live imaging of cytoskeletal dynamics (e.g.2-4 ) and are easy to isolate and manipulate in a lab setting compared to other vertebrates. The frog is a classic model system for developmental neurobiology studies, and important early insights into growth cone microtubule dynamics were initially found using this system5-7 . In this method8, eggs are collected and fertilized in vitro, injected with RNA encoding fluorescently tagged cytoskeletal fusion proteins or other constructs to manipulate gene expression, and then allowed to develop to the neural tube stage. Neural tubes are isolated by dissection and then are cultured, and growth cones on outgrowing neurites are imaged. In this article, we describe how to perform this method, the goal of which is to culture Xenopus laevis growth cones for subsequent high-resolution image analysis. While we provide the example of +TIP fusion protein EB1-GFP, this method can be applied to any number of proteins to elucidate their behaviors within the growth cone.

Neural Explant Cultures from Xenopus laevis

Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.

的神经植体培养<em&gt;非洲爪蟾</em

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

神经科学

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function.1,2 Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al.,3 has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types.4 We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 &deg;C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.

We describe how to use laser capture microdissection (LCM) to obtain enriched populations of hippocampal neurons or single neurons from frozen sections of the injured rat brain for subsequent gene expression analysis using quantitative real time PCR and/or whole-genome microarrays.

激光捕获显微切割的丰富种群的创伤性脑损伤后神经元或单个神经元的基因表达分析

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that ...

Laser-scanning Photostimulation of Optogenetically Targeted Forebrain Circuits

The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the interactions of these forebrain circuits has been aided recently by the development of optogenetic methods for light-mediated modulation of neuronal activity. Here, we describe a protocol for examining the functional organization of forebrain circuits&#160;in vitro using laser-scanning photostimulation of channelrhodopsin, expressed optogenetically via viral-mediated transfection. This approach also exploits the utility of cre-lox recombination in transgenic mice to target expression in specific neuronal cell types. Following transfection, neurons are physiologically recorded in slice preparations using whole-cell patch clamp to measure their evoked responses to laser-scanning photostimulation of channelrhodopsin expressing fibers. This approach enables an assessment of functional topography and synaptic properties. Morphological correlates can be obtained by imaging the neuroanatomical expression of channelrhodopsin expressing fibers using confocal microscopy of the live slice or post-fixed tissue. These methods enable functional investigations of forebrain circuits that expand upon more conventional approaches.

We describe a method for characterizing the functional topography and synaptic properties of forebrain circuits using an optogenetic approach to photostimulate neuronal populations&#160;in vitro.

Optogenetically有针对性的前脑电路的激光扫描光刺激

The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the ...

A Method to Make a Craniotomy on the Ventral Skull of Neonate Rodents

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain in adulthood and during development. Although most in vivo approaches use a craniotomy to study brain regions located on the dorsal side, brainstem regions such as the pons, located on the ventral side remain relatively understudied. The main goal of this protocol is to facilitate access to ventral brainstem structures so that they can be studied in vivo using electrophysiological and imaging methods. This approach allows study of structural changes in long-range axons, patterns of electrical activity in single and ensembles of cells, and changes in blood brain barrier permeability in neonate animals. Although this protocol has been used mostly to study the auditory brainstem in neonate rats, it can easily be adapted for studies in other rodent species such as neonate mice, adult rodents and other brainstem regions.

A surgical method is described to expose the ventral skull in neonate rats. Using this approach it is possible to open a craniotomy to perform acute electrophysiology and two-photon microscopy experiments in the brainstem of anesthetized pups.

方法以使对新生儿鼠害的腹头骨开颅手术

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain ...

Laser Nanosurgery of Cerebellar Axons In Vivo

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex5-9.
This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.

Laser Nanosurgery of Cerebellar Axons In Vivo

Two-photon imaging, coupled to laser nanodissection, are useful tools to study degenerative and regenerative processes in the central nervous system with subcellular resolution. This protocol shows how to label, image, and dissect single climbing fibers in the cerebellar cortex in vivo.

小脑轴突的激光Nanosurgery<em&gt;在体内</em

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to ...

In Vivo Intracerebral Stereotaxic Injections for Optogenetic Stimulation of Long-Range Inputs in Mouse Brain Slices

Knowledge of cell-type specific synaptic connectivity is a crucial prerequisite for understanding brain-wide neuronal circuits. The functional investigation of long-range connections requires targeted recordings of single neurons combined with the specific stimulation of identified distant inputs. This is often difficult to achieve with conventional and electrical stimulation techniques, because axons from converging upstream brain areas may intermingle in the target region. The stereotaxic targeting of a specific brain region for virus-mediated expression of light-sensitive ion channels allows selective stimulation of axons originating from that region with light. Intracerebral stereotaxic injections can be used in well-delimited structures, such as the anterior thalamic nuclei, in addition to other subcortical or cortical areas throughout the brain.
Described here is a set of techniques for precise stereotaxic injection of viral vectors expressing channelrhodopsin in the mouse brain, followed by photostimulation of axon terminals in the brain slice preparation. These protocols are simple and widely applicable. In combination with whole-cell patch clamp recording from a postsynaptically connected neuron, photostimulation of axons allows the detection of functional synaptic connections, pharmacological characterization, and evaluation of their strength. In addition, biocytin filling of the recorded neuron can be used for post-hoc morphological identification of the postsynaptic neuron.

This protocol describes a set of methods to identify the cell-type specific functional connectivity of long-range inputs from distant brain regions using optogenetic stimulations in ex vivo brain slices.

在Vivo脑内立体注射中，用于小鼠脑切片中远距离输入的光遗传学刺激

Knowledge of cell-type specific synaptic connectivity is a crucial prerequisite for understanding brain-wide neuronal circuits. The functional ...

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones -the sensory structures located at the distal tips of developing axons- execute this process. Studying the structure and dynamics of the growth cone is crucial to understanding axonal development and the interactions with the surrounding central nervous system (CNS) that enable it to form neural circuits. This is essential when devising methods to reintegrate axons into neural circuits following injury in fundamental research and pre-clinical contexts. Thus far, the general understanding of growth cone dynamics is primarily founded on studies of neurons cultured in two dimensions (2D). Although undoubtedly fundamental to the current knowledge of growth cone structural dynamics and response to stimuli, 2D studies misrepresent the physiological three-dimensional (3D) environment encountered by neuronal growth cones in intact CNS tissue. More recently, collagen gels were employed to overcome some of these limitations, enabling the investigation of neuronal development in 3D. However, both synthetic 2D and 3D environments lack signaling cues within CNS tissue, which direct the extension and pathfinding of developing axons. This protocol provides a method for studying axons and growth cones using organotypic brain slices, where developing axons encounter physiologically relevant physical and chemical cues. By combining fine-tuned in utero and ex utero electroporation to sparsely deliver fluorescent reporters along with super-resolution microscopy, this protocol presents a methodological pipeline for the visualization of axon and growth cone dynamics in situ. Furthermore, a detailed toolkit description of the analysis of long-term and live-cell imaging data is included.

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

This protocol demonstrates a straightforward and robust method to study in situ axon growth and growth cone dynamics. It describes how to prepare ex vivo physiologically relevant acute brain slices and provides a user-friendly analysis pipeline.

原位 急性 离体 胚胎脑切片培养物中轴突生长和生长锥动力学的可视化

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones ...

Intracerebral Transplantation and In Vivo Bioluminescence Tracking of Human Neural Progenitor Cells in the Mouse Brain

Cell therapy has long been an emerging treatment paradigm in experimental neurobiology. However, cell transplantation studies often rely on end-point measurements and can therefore only evaluate longitudinal changes of cell migration and survival to a limited extent. This paper provides a reliable, minimally invasive protocol to transplant and longitudinally track neural progenitor cells (NPCs) in the adult mouse brain. Before transplantation, cells are transduced with a lentiviral vector comprising a bioluminescent (firefly-luciferase) and fluorescent (green fluorescent protein [GFP]) reporter. The NPCs are transplanted into the right cortical hemisphere using stereotaxic injections in the sensorimotor cortex. Following transplantation, grafted cells were detected through the intact skull for up to five weeks (at days 0, 3, 14, 21, 35) with a resolution limit of 6,000 cells using in vivo bioluminescence imaging. Subsequently, the transplanted cells are identified in histological brain sections and further characterized with immunofluorescence. Thus, this protocol provides a valuable tool to transplant, track, quantify, and characterize cells in the mouse brain.

Intracerebral Transplantation and In Vivo Bioluminescence Tracking of Human Neural Progenitor Cells in the Mouse Brain

We describe the intraparenchymal transplantation of human neural progenitor cells transduced with a dual reporter vector expressing luciferase-green fluorescent protein (GFP) in the mouse brain. After transplantation, the luciferase signal is repeatedly measured using in vivo bioluminescence and GFP-expressing grafted cells identified in brain sections using fluorescence microscopy.

小鼠大脑中人类神经祖细胞的脑内移植和 体内 生物发光跟踪

Cell therapy has long been an emerging treatment paradigm in experimental neurobiology. However, cell transplantation studies often rely on end-point ...

激光制导神经跟踪在脑外植体

摘要

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此视频中的章节

工程神经前体宫内移植,用于Vivo神经结构研究

使用激光光镊操纵隔离神经细胞在体外

的神经植体培养

激光捕获显微切割的丰富种群的创伤性脑损伤后神经元或单个神经元的基因表达分析

Optogenetically有针对性的前脑电路的激光扫描光刺激

方法以使对新生儿鼠害的腹头骨开颅手术

小脑轴突的激光Nanosurgery

在Vivo脑内立体注射中，用于小鼠脑切片中远距离输入的光遗传学刺激

原位急性离体胚胎脑切片培养物中轴突生长和生长锥动力学的可视化

小鼠大脑中人类神经祖细胞的脑内移植和体内生物发光跟踪

激光制导神经跟踪在脑外植体

摘要

探索更多视频

此视频中的章节

工程神经前体宫内移植,用于Vivo神经结构研究

使用激光光镊操纵隔离神经细胞在体外

的神经植体培养

激光捕获显微切割的丰富种群的创伤性脑损伤后神经元或单个神经元的基因表达分析

Optogenetically有针对性的前脑电路的激光扫描光刺激

方法以使对新生儿鼠害的腹头骨开颅手术

小脑轴突的激光Nanosurgery

在Vivo脑内立体注射中，用于小鼠脑切片中远距离输入的光遗传学刺激

原位 急性 离体 胚胎脑切片培养物中轴突生长和生长锥动力学的可视化

小鼠大脑中人类神经祖细胞的脑内移植和 体内 生物发光跟踪

原位急性离体胚胎脑切片培养物中轴突生长和生长锥动力学的可视化

小鼠大脑中人类神经祖细胞的脑内移植和体内生物发光跟踪