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&#160;</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)&#160;</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&#160;</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&#160;</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&#160;</td>
			<td>Wild Heerbrugg, Switzerland</td>
			<td>Wild M3</td>
			<td>Equipped with high-intensity illuminator (MI-150; Dolan-Jenner Inc.)&#160;</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

Universidad de Cartagena UDEC

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

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

器官型培养的神经元雪崩多电极阵列记录

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

科研

神经科学

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 ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

小脑颗粒神经元的遗传操作<em&gt;体外</em&gt;和<em&gt;在体内</em&gt;为研究神经元形态和迁移

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

诱导星形胶质细胞受体的可塑性由神经元的放电频率操作

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

新生小鼠脑侧脑室注射病毒的持续和广泛的神经元传导

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

Closed-loop Neuro-robotic Experiments to Test Computational Properties of Neuronal Networks

Information coding in the Central Nervous System (CNS) remains unexplored. There is mounting evidence that, even at a very low level, the representation of a given stimulus might be dependent on context and history. If this is actually the case, bi-directional interactions between the brain (or if need be a reduced model of it) and sensory-motor system can shed a light on how encoding and decoding of information is performed. Here an experimental system is introduced and described in which the activity of a neuronal element (i.e., a network of neurons extracted from embryonic mammalian hippocampi) is given context and used to control the movement of an artificial agent, while environmental information is fed back to the culture as a sequence of electrical stimuli. This architecture allows a quick selection of diverse encoding, decoding, and learning algorithms to test different hypotheses on the computational properties of neuronal networks.

In this paper, an experimental framework to perform closed-loop experiments is presented, in which information processing (i.e., coding and decoding) and learning of neuronal assemblies are studied during the continuous interaction with a robotic body.

闭环神经机器人实验来检验神经网络的计算性能

Information coding in the Central Nervous System (CNS) remains unexplored. There is mounting evidence that, even at a very low level, the representation ...

Conditional Genetic Transsynaptic Tracing in the Embryonic Mouse Brain

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits during embryonic maturation of the brain however is technically challenging because most transsynaptic tracing methods developed to date depend on stereotaxic tracer injection. To overcome this problem, we developed a binary genetic strategy for conditional genetic transsynaptic tracing in the mouse brain. Towards this end we generated two complementary knock-in mouse strains to selectively express the bidirectional transsynaptic tracer barley lectin (BL) and the retrograde transsynaptic tracer Tetanus Toxin fragment C from the ROSA26 locus after Cre-mediated recombination. Cell-specific tracer production in these mice is genetically encoded and does not depend on mechanical tracer injection. Therefore our experimental approach is suitable to study neural circuit formation in the embryonic murine brain. Furthermore, because tracer transfer across synapses depends on synaptic activity, these mouse strains can be used to analyze the communication between genetically defined neuronal populations during brain development at a single cell resolution. Here we provide a detailed protocol for transsynaptic tracing in mouse embryos using the novel recombinant ROSA26 alleles. We have utilized this experimental technique in order to delineate the neural circuitry underlying maturation of the reproductive axis in the developing female mouse brain.

Capitalizing on a binary genetic strategy we provide a detailed protocol for neural circuit tracing in mice that express complementary transsynaptic tracers after Cre-mediated recombination. Because cell-specific tracer production is genetically encoded, our experimental approach is suitable to study the formation and maturation of neural circuitry during murine embryonic brain development at a single cell resolution.

有条件的遗传跨突触标记在胚胎小鼠脑

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits ...

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a closed-loop event-triggered manner. Although many studies are focusing on the possible benefits and side-effects of TES in healthy and pathologic brains, there are still many fundamental open questions regarding the mechanism of action of the stimulation. Therefore, there is a clear need for a robust and reproducible method to test the acute and the chronic effects of TES in rodents. TES can be combined with regular behavioral, electrophysiological, and imaging techniques to investigate neuronal networks in vivo. The implantation of transcranial stimulation electrodes does not impose extra constraints on the experimental design while it offers a versatile, flexible tool to manipulate brain activity. Here we provide a detailed, step-by-step protocol to fabricate and implant transcranial stimulation electrodes to influence brain activity in a temporally constrained manner for months.

This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving rats.

大鼠慢性经颅电刺激及 Intracortical 记录

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

脑切片神经元树突的双光子钙成像

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively thin to properly visualize and access neurons for patch-clamping or imaging, and in vitro examination of brain circuitry is limited to only what is physically present in the acute slice. To maintain the benefits of in vitro slice experimentation while preserving a larger portion of presynaptic nuclei, we developed a novel slice preparation. This &#8220;wedge slice&#8221; was designed for patch-clamp electrophysiology recordings to characterize the diverse monaural, sound-driven inputs to medial olivocochlear (MOC) neurons in the brainstem. These neurons receive their primary afferent excitatory and inhibitory inputs from neurons activated by stimuli in the contralateral ear and corresponding cochlear nucleus (CN). An asymmetrical brain slice was designed which is thickest in the rostro-caudal domain at the lateral edge of one hemisphere and then thins towards the lateral edge of the opposite hemisphere. This slice contains, on the thick side, the auditory nerve root conveying information about auditory stimuli to the brain, the intrinsic CN circuitry, and both the disynaptic excitatory and trisynaptic inhibitory afferent pathways that converge on contralateral MOC neurons. Recording is performed from MOC neurons on the thin side of the slice, where they are visualized using DIC optics for typical patch-clamp experiments. Direct stimulation of the auditory nerve is performed as it enters the auditory brainstem, allowing for intrinsic CN circuit activity and synaptic plasticity to occur at synapses upstream of MOC neurons. With this technique, one can mimic in vivo circuit activation as closely as possible within the slice. This wedge slice preparation is applicable to other brain circuits where circuit analyses would benefit from preservation of upstream connectivity and long-range inputs, in combination with the technical advantages of in vitro slice physiology.

Integration of diverse synaptic inputs to neurons is best measured in a preparation that preserves all pre-synaptic nuclei for natural timing and circuit plasticity, but brain slices typically sever many connections. We developed a modified brain slice to mimic in vivo circuit activity while maintaining in vitro experimentation capability.

体外楔形切片准备模拟在Vivvo神经元电路连接

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively ...

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

器官型培养的神经元雪崩多电极阵列记录

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

小脑颗粒神经元的遗传操作

诱导星形胶质细胞受体的可塑性由神经元的放电频率操作

新生小鼠脑侧脑室注射病毒的持续和广泛的神经元传导

闭环神经机器人实验来检验神经网络的计算性能

有条件的遗传跨突触标记在胚胎小鼠脑

大鼠慢性经颅电刺激及 Intracortical 记录

脑切片神经元树突的双光子钙成像

体外楔形切片准备模拟在Vivvo神经元电路连接

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

器官型培养的神经元雪崩多电极阵列记录

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

小脑颗粒神经元的遗传操作

诱导星形胶质细胞受体的可塑性由神经元的放电频率操作

新生小鼠脑侧脑室注射病毒的持续和广泛的神经元传导

闭环神经机器人实验来检验神经网络的计算性能

有条件的遗传跨突触标记在胚胎小鼠脑

大鼠慢性经颅电刺激及 Intracortical 记录

脑切片神经元树突的双光子钙成像

体外楔形切片准备模拟在Vivvo神经元电路连接

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