我们感谢林美安批判性阅读的手稿和其他成员讨论这项工作的整个过程中我们的实验室。这项工作是由由山田科学基金会和英国皇家学会的短暂访问奖学金和欧盟居里夫人国际来话团契GRR KK，和BBSRC资助计划（BB/H002278/1）和威康信托设备资助（073228/Z/03/Z，）啊

1。收集上演幼虫<ol><li>将15名女性和15名男性成年苍蝇在一个圆柱形有机玻璃笼产卵在培养皿的琼脂和葡萄汁中添加一小勺酵母膏3小时，在25°C更改板每日3-4次，弃去第一板（ 即 O / N蛋裁员）。也抛弃板的第一天。从第二天开始，保持板的鸡蛋在25°C。经过7天的收集鸡蛋，开始一个新成立的母公司苍蝇。 </li><li>约24小时后，幼虫从卵中孵化。通过挂接幼虫减薄的画笔或用钳子，35幼虫转移在从葡萄琼脂一个瓶标飞食品（10毫升）（ 图1A）。 </li><li>保持小瓶幼虫3天（96小时产蛋后，AEL）在25°C。 </li></ol> 2。解剖幼虫腹神经索文化<ol><li>清洁是nch不和手，用70％的乙醇。浸泡4染色块，2双钳子和用70％乙醇的针，并让空气干燥。 </li><li>与以下介质:一个与蒸馏水（DW）的清洁和准备4染色块池清洁的幼虫;用2ml的盾构桑M3昆虫培养基与1％青霉素和链霉素，蜕皮激素 - 自由（M3 PS中的第二个介质）解剖的幼虫;用2 M3 PS介质池解剖的腹侧神经线（VNCs）的毫升第三和第四个2毫升M3 PS中等刺VNCs。所有试剂必须预先温热至室温。 </li><li>加水的小瓶含96小时AEL幼虫。然后，通过使用刮刀，轻轻摊开食物与幼虫从小瓶湿纸巾。 10个幼虫转移到染色块DW洗掉食物。更换DW 6倍。然后更换M3 PS介质。 </li><li>幼虫之一转移到染色块M3的PS培养基中含有2毫升。 </li><li>在解剖显微镜下，二谨慎使用标准方法12 ssect幼体的大脑，但尽量减少组织损伤，要特别注意通过以下方式进行。将幼虫背侧，并保持三分之一的背侧从前端有2对产钳（每手）。虽然前部分，扯远了后端的镊子，和撕裂的表皮。脑和VNC应该是可见的前半部分的后边缘。小心地分离出的大脑和VNC复杂的消除脂肪的身体，肠道和表皮。当VNCs被损坏太多，VNCs会退化甚至完全或部分没有刺向伤害。考虑到以​​下几点: </li></ol><ul><li>不要拉或撕裂胸VNC的成虫盘和外周神经，因为这将损害VNC。为了降低末梢神经，用两对钳放置在靠近对方持有的外周神经，然后拉的神​​经与对牛肝菌。成虫盘和口部可以留到VNC。 </li><li>留下的环腺连接到大脑和淋巴结。 </li><li>切点最接近的大脑，在那里没有太多的食物中包含的肠道。 </li></ul><ol start="6"><li>通过使用P20 PIPETMAN的用针尖切断和扩大一点，转移VNC到染色块含有新鲜M3 PS中。 VNC的，任意的移液管转移之前向上和向下只用于剥离的介质，以防止的VNC粘到尖端。 </li></ol> 3。刺伤害的果蝇幼虫VNC 本节将介绍如何执行刺向受伤的VNCs，和准备刺，VNCs的时间推移分析和免疫组织化学。 （对于时间流逝分析）第5和第6条（免疫组化）后捅死文化中描述的条件和时间。 <ol><li>汇集足够的VNCs后（4-5时移和超过24染色），转让一个VNC到一个干净的染色块含M3 PS的。 </li><li>东方的VNC，使背侧的神经纤维网是在解剖显微镜下（ 图1B）。这是最自然的方向VNCs在于当连接到视叶。 </li><li>短截手动使用钨针的解剖显微镜下，从背侧几乎在一个合适的角度，并旨在用于腹部的VNC的一半（ 图1B）。 </li></ol>要特别注意以下几点: <ul><li>刺一次。 </li><li>直到针的刺击击中的玻璃底部。但是，要小心，不要损坏针尖。 </li><li>该干的持针器不能接触介质中刺向。 </li><li>解剖显微镜下是不可见的伤口。 </li></ul>注:刺伤害无关变性该过程可以刺向病变导致细胞变性内的VNC不同的。变性的查询结果中的神经纤维的孔中，并偶尔孔已被观察到在皮层非刺伤标本。标本变性影响广泛的神经纤维或变性的影响也的VNC表面必须被丢弃。变性可确定如下: <ul><li>粗糙的表面，特别是在胸部，在22小时的刺伤的横向/腹侧区域。在极端的情况下，VNC看起来突出。 </li><li>孔或液泡在胸的神经纤维，它可以是作为背景信号与荧光免疫染色的孔中可见。 </li></ul> 4。维护针和镊子<ul><li>定期检查是否足够锋利的针尖。可以是弯曲的或钝的针尖后，使用它的几个实验。在这种情况下，锐化使用阿肯色州石头或同等的前端。 </li><li>镊子必须的技巧满足完美。被损坏的提示使用。在这种情况下，调整使用阿肯色州结石或同等的前端。 </li></ul> 5。文化和时间的推移记录刺伤VNCs 若要在客厅的VNC，一个GFP蛋白陷阱线标记所有轴突- G9 13 -可以使用可视化的轴突的神经纤维，并可视化所有的神经胶质细胞（除中线神经胶质细胞）胶质驱动repoGAL4可用于表达UASdsRed的S197Y 14记者。通过杂交G9苍蝇UASdsRedS197Y; repoGAL4的苍蝇，VNCs的后代幼虫得到与绿色轴突和红色的神经胶质细胞，可被记录在活组织。 <ol><li>经过池4-5 VNCs，转移到一个干净的染色块含2毫升的M3 PS，并刺中腹部一半的VNC 1 VNC在第3节。 </li><li>聚-L-赖氨酸涂层3.5毫米玻璃底培养皿中含有1毫升的M3转移刺的VNCPS。下来将在VNCs的背侧。轻轻推VNC使用的钳子，让平坦的一面VNC坚持的菜。 </li><li>轻轻地添加1毫升的M3 PS 15％FBS的渲染的胎牛血清（FBS）的终浓度为7.5％。 </li><li>使用激光扫描共聚焦显微镜采集图像。扫描VNC，收集了Z-节系列在其整个厚度。我们使用了徕卡SP2倒置共聚焦显微镜与环境室的温度控制。任何等同的共焦显微镜的工作，但是它可能需要优化的设置进行扫描。时间的推移共聚焦显微镜的设置如下:环境箱温度:25°C; 20X物镜4倍变焦;扫描模式XYZT，512×512像素的分辨率，Z = 1微米的步骤和1小时或2 - 小时一班。 </li></ol><ul><li>徕卡SP2共聚焦显微镜扫描的文件大小的限制。通过这些设置，8-9时间点进行扫描。同其他共聚焦显微镜，它应该是能够获取图像堆栈更长的时间点， 即超过高达24小时的跨度。 </li></ul> 6。文化和刺伤和固定VNCs的免疫组化染色<ol><li>准备装有500μl的M3 PS 7.5％FBS，每孔24孔培养板。 </li><li>重复解剖的超过24 VNCs，24孔组织培养皿。 </li><li>用刀尖切断使用P20 PIPETMAN，，从池12个非刺VNCs转移到培养皿中，每孔1 VNC使用。 </li><li>刺在游泳池休息的VNCs在第3节中描述。转移到每一个每一个刺VNC。 </li><li>根据实验所需的时间将24孔板中，在25℃培养箱中培养。细胞的完整性是不变的至少24小时。 </li><li>培养后，转移12 VNCs在250μl的4％多聚甲醛的PEM在1.5ml管中通过使用的P20 PIPETMAN与前端切断。然后，fixati已经被仔细地用移液管，照顾损坏VNCs的。少量的固定液足以覆盖样品应该保持在每个管中。随后，添加新鲜固定液，轻轻地搅拌50分钟，在室温下。 </li><li>用0.3％的Triton X PBS漂洗2次，0.3％的Triton X PBS在室温下10分钟，然后洗2次。 </li><li>通过用10％正常山羊血清0.3％的Triton X PBS中孵育1小时，于RT座VNCs。可将样本存储为至少1个月，在4℃下。 </li><li>超过20小时一抗孵育VNCs，在4°C。 </li><li> 0.3％的Triton X PBS漂洗2次，然后洗3次，每次10分钟，0.3％的Triton X PBS在室温下。 </li><li>用的二抗孵育VNCs超过16小时，在4℃下</li><li>漂洗2次，然后用0.3％的Triton X PBS洗3次，每次10分钟，使用0.3％的Triton X PBS在室温RT（在黑暗中，如果使用荧光二抗）。 </li><li>更换PBS含50％甘油:50％PBS的至少一个小时。 </li><li>然后替换:用80％的甘油20％的至少一个小时的PBS。 </li><li>一个VNC安装在大提琴磁带（约0.06毫米）的2层在显微镜载玻片上一个窗口。调整的方向，然后放置一个盖玻片（18×18毫米），在窗口中。两个VNCs可以被安装在使用这种方法的滑动。 </li></ol>使用的各种不同的细胞反应的初级抗体，损伤，可以分析（ 例如，细胞数和细胞形状的变化）。 7。分析数据使用ImageJ和一系列的插件如果VNC没有刺向无关变性的，必须计算在样品分析病变的大小无关。作为刺向是手工完成，病灶大小不同，每个时间。因此，它是重要的统计学分析刺伤，尽可能的效果。 <ol><li>伤口大小测量。伤口的大小是15的维修的一个指标。，刺向病变是可见的，没有GFP表达。病变区域，可以使用可自由查​​看ImageJ软件测量经过数据，如下所示: <ol><li>使用ImageJ，打开一摞共聚焦图像从"文件"菜单，选择"导入"，从这里选择"图像序列"。这将打开的单个图像的集合到一个堆栈。 </li><li>改变"栈"到的"Hyperstack"的，要"图像"菜单，选择"Hyperstack"，然后选择"堆叠到Hyperstack"。 </li><li>使用"图像"菜单中的"属性"中设置的体素的大小。在获得的数据使用徕卡SP2共聚焦显微镜，体素的大小可以从扫描软件的方式获得，并从元数据的文本文件与图像一起保存。我们的设定，在第3节，像素尺寸为XY = 0.366211微米，Z = 0.99709微米。 </li><li>通过检查所有切片在一个时间点，画出的多边形选择工具，在工具栏上的病变区域的最大轮廓。这是一个感兴趣区域（ROI）。 </li><li>新增的投资回报率ROI经理"分析"菜单，向下滚动并选择"工具"，然后"ROI经理"，然后单击"添加"。 </li><li>在所有的时间点重复此步骤。 </li><li>点击"测量"按钮，的ROI经理，获得每个区域的面积大小。 </li></ol></li><li> VNC运动在时间的推移中记录的更正。 "Stackreg"和"Turboreg"插件16（ImageJ的网站<a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a> ）:这些插件可以纠正小样本的变动在时间的推移。所有的时间点，并具有同等代表性的光学部分通过建立一个堆栈应用的插件。它有助于观察病变随时间变化的。这些方法和使用说明书上提供的研究小组，开发这些插件的详细信息。 （ <a href="http://bigwww.epfl.ch/thevenaz/stackreg/" target="_blank">http://bigwww.epfl.c</a>的H / thevenaz / stackreg /）。 </li><li>神经胶质细胞的自动计数。 "DeadEasy神经胶质细胞"的插件（ <a href="http://www.biosciences-labs.bham.ac.uk/hidalgo/Software.html" target="_blank">www.biosciences-labs.bham.ac.uk/hidalgo/Software.html</a> ）:这是算在的REPO阳 ​​性神经胶质细胞的数量的果蝇幼虫VNC和检查胶质细胞的变化刺伤害17号所造成的。该插件准确，误差为0.1％的假阳性和假阴性4.3％17。要使用此插件，第一个标签的所有神经胶质细胞（除中线）标本中利用反回购抗体的免疫荧光的。然后，安装该插件在ImageJ的共聚焦图像，打开一个堆栈和运行插件。神经胶质细胞计数，自动在30秒左右。 </li><li>自动计数的细胞凋亡。 "DeadEasy半胱天冬酶幼虫"插件（ <a href="http://www.biosciences-labs.bham.ac.uk/hidalgo/Software.html" target="_blank">www.bioscienc</a>es-labs.bham.ac.uk/hidalgo/Software.html）18:这是算与反裂Caspase3的标记细胞的数量，和一个新版本的改编的幼虫VNCs的。刺损伤导致程序性细胞死亡增加11。要使用此插件，第一个标签中凋亡细胞的标本采用免疫荧光与防切割Caspase-3的抗体。然后，安装该插件在ImageJ的共聚焦图像，打开一个堆栈和运行插件。凋亡细胞计数，自动在30秒左右。 </li></ol> 8。试剂<ol><li>培养基:盾牌"和"桑M3昆虫培养基，7.5％胎牛血清，1％青霉素和1％链霉素。 </li><li> 0.01％聚-L-赖氨酸在无菌DW。 </li><li>固色剂:4％甲醛在质子交换膜，超纯水（0.1 M管，2毫米1毫米EGTA， 硫酸镁 ）解决方案。 </li><li>免疫染色:10％正常山羊血清，0.3％的Triton X PBS封闭溶液。 </li><li>反谷氨酰胺合成酶2抗体:1:250解堵。 </li><li>抗GFP抗体:1:1,000在封闭溶液。 </li><li>反回购抗体:1:250解堵。 </li><li>的反ELAV抗体:1:250解堵。 </li><li>反被裂解的半胱天冬酶抗体用封闭液1:1000。 </li><li>第二抗体:1:250解堵。 </li></ol>

<ol>
	<li>Fang, Y., Soares, L., Teng, X., Geary, M., Bonini, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+Drosophila+model+of+nerve+injury+reveals+an+essential+role+of+Nmnat+in+maintaining+axonal+integrity.">A novel Drosophila model of nerve injury reveals an essential role of Nmnat in maintaining axonal integrity.</a> Curr. Biol. 22, 590-595 (2012).</li><li>Xiong, X., 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=Protein+turnover+of+the+Wallenda/DLK+kinase+regulates+a+retrograde+response+to+axonal+injury.">Protein turnover of the Wallenda/DLK kinase regulates a retrograde response to axonal injury.</a> J. Cell Biol. 191, 211-223 (2010).</li><li>Leyssen, 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=Amyloid+precursor+protein+promotes+post-developmental+neurite+arborization+in+the+Drosophila+brain.">Amyloid precursor protein promotes post-developmental neurite arborization in the Drosophila brain.</a> EMBO J. 24, 2944-2955 (2005).</li><li>Kato, K., Awasaki, T., Ito, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+programmed+cell+death+induces+glial+cell+division+in+the+adult+Drosophila+brain.">Neuronal programmed cell death induces glial cell division in the adult Drosophila brain.</a> Development. 136, 51-59 (2009).</li><li>Ayaz, D., 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=Axonal+injury+and+regeneration+in+the+adult+brain+of+Drosophila.">Axonal injury and regeneration in the adult brain of Drosophila.</a> J. Neurosci. 28, 6010-6021 (2008).</li><li>Rohrbough, J., O'Dowd, D. K., Baines, R. A., Broadie, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+bases+of+behavioral+plasticity:+establishing+and+modifying+synaptic+circuits+in+the+Drosophila+genetic+system.">Cellular bases of behavioral plasticity: establishing and modifying synaptic circuits in the Drosophila genetic system.</a> J. Neurobiol. 54, 254-271 (2003).</li><li>Babcock, D. T., Landry, C., Galko, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+signaling+mediates+UV-induced+nociceptive+sensitization+in+Drosophila+larvae.">Cytokine signaling mediates UV-induced nociceptive sensitization in Drosophila larvae.</a> Curr. Biol. 19, 799-806 (2009).</li><li>Gomez-Marin, A., Louis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Active+sensation+during+orientation+behavior+in+the+Drosophila+larva:+more+sense+than+luck.">Active sensation during orientation behavior in the Drosophila larva: more sense than luck.</a> Curr. Opin. Neurobiol. 22, 208-215 (2012).</li><li>Schleyer, 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=A+behavior-based+circuit+model+of+how+outcome+expectations+organize+learned+behavior+in+larval+Drosophila.">A behavior-based circuit model of how outcome expectations organize learned behavior in larval Drosophila.</a> Learn Mem. 18, 639-653 (2011).</li><li>Brown, H. L., Cherbas, L., Cherbas, P., Truman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+time-lapse+imaging+and+dominant+negative+receptors+to+dissect+the+steroid+receptor+control+of+neuronal+remodeling+in+Drosophila.">Use of time-lapse imaging and dominant negative receptors to dissect the steroid receptor control of neuronal remodeling in Drosophila.</a> Development. 133, 275-285 (2006).</li><li>Kato, K., Forero, M. G., Fenton, J. C., Hidalgo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+glial+regenerative+response+to+central+nervous+system+injury+is+enabled+by+pros-notch+and+pros-NFkappaB+feedback.">The glial regenerative response to central nervous system injury is enabled by pros-notch and pros-NFkappaB feedback.</a> PLoS Biol. 9, e1001133 (2011).</li><li>Sullivan, W., Ashburner, M., Hawley, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila protocols. , (2000).</li><li>Morin, X., Daneman, R., Zavortink, M., Chia, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protein+trap+strategy+to+detect+GFP-tagged+proteins+expressed+from+their+endogenous+loci+in+Drosophila.">A protein trap strategy to detect GFP-tagged proteins expressed from their endogenous loci in Drosophila.</a> Proc. Natl. Acad. Sci. U.S.A. 98, 15050-15055 (2001).</li><li>Verkhusha, V. V., 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=An+enhanced+mutant+of+red+fluorescent+protein+DsRed+for+double+labeling+and+developmental+timer+of+neural+fiber+bundle+formation.">An enhanced mutant of red fluorescent protein DsRed for double labeling and developmental timer of neural fiber bundle formation.</a> J. Biol. Chem. 276, 29621-29624 (2001).</li><li>Kato, K., Forero, M. G., Fenton, J. C., Hidalgo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+glial+regenerative+response+to+central+nervous+system+injury+is+enabled+by+Pros-Notch+and+Pros-NFkB+feedback.">The glial regenerative response to central nervous system injury is enabled by Pros-Notch and Pros-NFkB feedback.</a> PLoS Biol. 9, e1001133 (2011).</li><li>Th&#233;venaz, P., Ruttimann, U. E., Unser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pyramid+approach+to+subpixel+registration+based+on+intensity.">A pyramid approach to subpixel registration based on intensity.</a> IEEE Transactions on Image Processing: A Publication of the IEEE Signal Processing Society. 7, 27-41 (1998).</li><li>Forero, M. G., Kato, K., Hidalgo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automatic+cell+counting+in+vivo+in+the+larval+nervous+system+of+Drosophila.">Automatic cell counting in vivo in the larval nervous system of Drosophila.</a> J. Microsc. 46, 202-212 (2012).</li><li>Forero, M. G., Pennack, J. A., Learte, A. R., Hidalgo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DeadEasy+caspase:+automatic+counting+of+apoptotic+cells+in+Drosophila.">DeadEasy caspase: automatic counting of apoptotic cells in Drosophila.</a> PLoS One. 4, e5441 (2009).</li><li>Lai, S. L., Lee, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+mosaic+with+dual+binary+transcriptional+systems+in+Drosophila.">Genetic mosaic with dual binary transcriptional systems in Drosophila.</a> Nat. Neurosci. 9, 703-709 (2006).</li><li>Potter, C. J., Tasic, B., Russler, E. V., Liang, L., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Q+system:+a+repressible+binary+system+for+transgene+expression,+lineage+tracing,+and+mosaic+analysis.">The Q system: a repressible binary system for transgene expression, lineage tracing, and mosaic analysis.</a> Cell. 141, 536-548 (2010).</li><li>Nicolai, L. 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=Genetically+encoded+dendritic+marker+sheds+light+on+neuronal+connectivity+in+Drosophila.">Genetically encoded dendritic marker sheds light on neuronal connectivity in Drosophila.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 20553-20558 (2010).</li><li>Tian, L., 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=Imaging+neural+activity+in+worms,+flies+and+mice+with+improved+GCaMP+calcium+indicators.">Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators.</a> Nat. Methods. 6, 875-881 (2009).</li></ol>

没有利益冲突的声明。

我们已经建立了一个协议刺伤的果蝇幼虫中枢神经系统损伤调查的细胞损伤，修复和再生。幼虫VNCs解剖和刺伤后，他们拍摄时间推移显微镜或荧光染色固定可视化神经胶质细胞和神经元，细胞凋亡或细胞分裂。随着时间的推移的病变的进展可以被测量。此方法的陪同下特意开发的软件的伤害，并在维修过程中的细胞数量变化对定量和统计分析。 我们被刺前96小时的机场快线的幼虫（和这些固定或进一步发展），发展阶段过渡到蛹和成虫。在96小时，VNCs有足够大的刺伤，它们是在中间的第三龄期的，并且从而不进行化蛹尚未;及神经系统已经是全功能的类似的广告ULT。这可能是可以刺略晚于幼虫和精确的计时一定要选择适合所研究的问题。然而，这取决于处理上的问题，但它通常将是必要文化的VNCs的一些时间来观察细胞的反应损伤。一段时间后，120小时AEL化蛹开始，一个时期的中枢神经系统改造，因此最好避免。因此是相当有限的时间窗口加文化中的幼虫捅死。使用幼虫仍然有很大的技术优势，使用成人同时，同样到了成年，人的神经系统已经是全功能的，分析对损伤的反应是相当容易和更快的幼虫。 当比较的大小和形态的头脑和VNCs在一盘VNCs在同等时间点，而无需在培养皿中培养后，解剖解剖和培养，发展是慢文化在体内 。在其他方面，发展进行通常在文化，组织的完整性是保留的，细胞是活的多个响应。伤口扩张，神经纤维的修复和神经胶质增生发生在22小时后捅死。这表明细胞对损伤的反应发生在文化和最有可能的时间超过一天，不需维护的外植体。如果更长的长期培养需要的话，协议可能需要进一步优化，如使用培养板插入物5。 重要的是，来识别品质好的样品从堕落的。优化这一协议需要一些技巧，不可避免地的变性会发生在一些标本。 VNC能够获得"菜花"的出现，反映了组织的完整性（ 图5B）击穿。变性也空泡的VNC独立的承认刺伤，它可以是完整的，非刺的标本（ 图5C </strONG&gt;）。这些样品必须被丢弃。变性是最有可能造成粗糙的解剖，它可以撕裂的神经和神经胶质细胞表面的保护层。因此，非常小心，必须采取轻轻地解剖。其他影响因素包括培养液中，必须保持清洁，并用抗生素治疗，坚持严格的清洗和时间安排，在协议中表示。最后，针与针支架，大小和锐度的针的长度是非常重要的。针座污染的培养基，并一个钝针可能会导致大的损伤，无法自我修复，会导致退化。重要的是要经常保持针尖锐。 在这个协议中，我们利用了的蛋白质陷阱GAL4-UAS系统采用传统的胶质过程的可视化可视化的神经纤维在平行线的苍蝇。这些工具也可以结合其他的二进制表达系统，如勒xA的19和Q-系统20中 ，分别独立的GAL4。这将使为实例轴突和胶质流程之间的相互作用的分析，在对损伤的反应。它与其他遗传工具，如树突21或钙离子内流22记者通过进一步结合，这种方法提供了一个很好的机会，分析背后的损伤反应在中枢神经系统的神经胶质细胞，神经元轴突和树突的细胞生物学。最后，这种方法可以结合标准的遗传学，突变和基因的过度表达，来测试基因功能的损伤和再生的反应。 该协议已经成功地领导发现了一个基因网络的基础胶质细胞的再生中枢神经系统损伤11。由于基因功能的进化保守性，解开这些细胞的活动和功能基因在果实蝇是有可能提供重要的见解了解马mmalian中枢神经系统损伤和再生。

在动物中的再生揭示细胞感受到有机体生长时是有序的和完整的，并且是一个生物体的结构完整性如何实现和维持。了解这些神秘的能力的细胞是生物学中的极大兴趣。促进再生医疗神经科学的主要目标之一。在人类中，损坏的中枢神经系统（CNS）不会再生。脊髓损伤的啮齿类动物模型，了解细胞如何响应受伤。然而，伦理问题，成本高，生命周期慢的动物制约进展。 果蝇， 果蝇是一种广泛使用的模式生物发育生物学和神经科学。由于其强大的遗传工具和生命周期短，循环导致果蝇基因功能的发现和了解人类与疾病相关的基因网络。有大量的证据进化CON从果蝇到人类基因功能的守恒。 近年来，在果蝇神经系统损伤已经建立了几个范例。一些损害周围神经沿机翼或外周神经切断后，包括感觉和运动神经元轴突2。然而，在许多方面不同的外周和中枢神经系统，这是众所周知的，在许多动物外周神经系统，而中枢神经系统不能再生。因此，了解中枢神经系统的再生，直接伤害到中枢神经系统是比较合适的。用针的刺伤害已成功地应用于果蝇成人的大脑调查的3,4受伤的。使用另一种方法，阿亚兹等。切断中枢神经系统轴突在培养成人的大脑一个压电电源microdissector，并分析了它们的再生4天5。这些后来的实验集合U的优势ps的是，他们对大脑的重点，这无疑是极大的兴趣。它的缺点是，大脑是比VNC，其中仅涉及电机和传感控制复杂得多。成人的大脑也耗费更多的时间。幼虫准备在4天的实验，而大约需要10天，成虫羽化，然后额外的5天为他们的成熟。成人的大脑也更难以处理，因为它是包裹在厚厚的角质层，这是难以去除的。 VNC具有进一步吸引脊椎动物的脊髓功能等效。 被广泛使用6-9神经科学作为一种模式生物果蝇的幼虫。虽然严格来说是处于发展阶段的幼虫，它也可以被认为是一个全功能的动物。幼虫的运动，包括嗅觉，味觉和痛，以及学习和记忆的多种感官。因此，幼虫VNC瓦特选择的中枢神经系统损伤的理想模型。 解剖和培养幼虫和蛹VNCs可以在盘中，长达24小时10仍然保持细胞的完整性。这表明该损伤可施加到VNC，然后可以被记录在时间推移在此期间的时间，或培养并固定在任何期望的时间点在此期间之内。 在这里，我们提出了一个实验范式的中枢神经系统损伤的果蝇幼虫VNC。 VNC是第一次解剖的幼虫，，手工钨针，刺向损伤。 VNC是放在玻璃底的盖玻片，和时间的推移激光扫描共聚焦显微镜拍摄的。或者，VNCs可以为所希望的一段时间内进行培养，和损伤的细胞的影响，可以使用免疫染色和共聚焦显微镜的固定标本分析。伤口区域可以被测量，和定量分析细胞怒江人數（细胞增殖和细胞死亡），可以进行与有意开发软件。可以很容易地处理大样本的大小，在统计学上验证的结果。这些方法已成功地结合强大的果蝇的遗传学发现了一个基因网络的基础胶质细胞的再生反应中枢神经系统损伤11。

<table border="1"><tbody><tr><td></td><td></td><td></td><td> 设备 </td></tr><tr><td>染色块</td><td>布鲁内尔显微镜</td><td></td><td></td></tr><tr><td>镊子第5号</td><td> 例如，精细的科学工具</td><td> 例如 11251-20 </td><td></td></tr><tr><td>钨针杆直径0.5毫米;针尖大小，1微米长，2英寸</td><td> Roboz手术器械</td><td> RS-6065 </td><td></td></tr><tr><td>持针器</td><td> Roboz手术器械</td><td> RS-6060 </td><td></td></tr><tr><td>阿肯色州石头:杜蒙钳修理工具包</td><td>精细科学工具</td><td> 29000-00 </td><td></td></tr><tr><td> 35毫米培养皿中，有27毫米的玻璃底座</td><td>磐</td><td> 3930-035 </td><td></td></tr><tr><td>徕卡SP2-AOBS共聚焦倒置显微镜与环境试验室</td><td>徕卡</td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> 试剂 </td></tr><tr><td>盾牌"和"桑M3昆虫培养基（蜕皮激素免费） </td><td>西格玛</td><td> S3652-500毫升</td><td></td></tr><tr><td>青霉素和链霉素</td><td> Invitrogen公司</td><td> 15070-063 </td><td></td></tr><tr><td>磷酸盐缓冲盐水（PBS） </td><td>查看12 </td><td></td><td></td></tr><tr><td> FBS </td><td>西格玛</td><td> F7524 </td><td></td></tr><tr><td>聚-L-赖氨酸</td><td>西格玛</td><td> P1399-25MG </td><td></td></tr><tr><td>甲醛，10％，甲醇免费，自由，超纯水</td><td> Polysciences的</td><td> 04018-1 </td><td></tD&gt; </tr><tr><td>鼠抗-谷氨酰胺合成酶抗体</td><td>密理博</td><td> MAB302 </td><td></td></tr><tr><td>兔抗GFP抗体</td><td> Life技术</td><td> A11122 </td><td></td></tr><tr><td>小鼠抗REPO抗体</td><td>发展研究杂交银行</td><td> 8D12 </td><td></td></tr><tr><td>的大鼠抗ELAV抗体的</td><td>发展研究杂交银行</td><td> 7E8A10 </td><td></td></tr><tr><td>兔抗活性半胱天冬酶3的抗体</td><td> Abcam公司</td><td> ab13847 </td><td></td></tr><tr><td>正常山羊血清</td><td>矢量实验室</td><td> S-1000 </td><td></td></tr><tr><td>抗兔的Alexa Fluor 488 </td><td> Life技术</td><td> A11034 </td><td></td></tr><tr><td> ANTI-MOUSE的Alexa Fluor 647 </td><tD&gt; Life技术</td><td> A21236 </td><td></td></tr><tr><td>灭鼠的Alexa Fluor 647 </td><td> Life技术</td><td> A21247 </td><td></td></tr></tbody></table>

an injury paradigm to investigate central nervous system repair in drosophila

已开发的实验方法，调查的细胞反应，中枢神经系统（CNS）损伤使用果蝇果蝇 。了解修复和再生的动物在生物学中是一个关键问题。人类中枢神经系统受损，无法再生，并了解如何推​​动再生医疗神经科学的主要目标之一。 果蝇强大的遗传工具包可以用来解决这个问题的中枢神经再生。 至CNS腹神经索（VNC，相当于脊椎动物脊髓）的病变施加手动用钨针。随后，VNC可以被使用激光扫描共聚焦显微镜拍摄的时间推移到24小时，顺应时代发展的病变随着时间的推移。另外，它可以进行培养，然后用免疫荧光可视化神经元和神经胶质细胞，激光共聚焦显微镜进行固定和染色。使用适当的标记，查的NGES在细胞形态和细胞状态造成的伤害可以可视化。 ImageJ的和故意的插件，定量和统计分析，可以进行测量伤口大小的变化，随着时间的推移和损伤的细胞增殖和细胞死亡的影响。这些方法可以让大样本量分析。他们可以结合强大的果蝇遗传学调查中枢神经系统的再生和修复的分子机制。

在这里，我们将展示如何执行刺向受伤的的果蝇幼虫VNC和分析细胞对损伤时间的推移和免疫共聚焦荧光显微镜。 对于时间延时数据，病变是可视化作为GFP阴性的区域内的神经纤维的标本轴承G9的轴索的标记（ 图2）。不久刺伤后，小GFP阴性区，看起来像孔或空泡，开始出现（ 图2A）。这种GFP阴性地区一般放大至大约6至8小时后，刺中（ 图2A）。随后，GFP阴性的区域缩小，甚至可能会消失（ 图2B）。刺伤后的22小时，由伤口占据的面积一般是小于刺伤后（ 图2A，B）是在6至8小时的最大面积。同样的红色荧光蛋白阴性的胶质过程最初也相应增加，但SHRINKS刺伤后的22小时。有趣的是，经常红色荧光蛋白阳性神经胶质过程填充GFP阴性的孔中的神经纤维网之前，他们的消失（ 图2C）。 在固定标本的免疫组化，精细胶质过程及其对损伤的反应，可以显示时间推移图像分辨率比。这是可以做到的，例如，使用repoGAL4胶质驱动器的膜系留记者（ 如 UAS-mCD8GFP），在果蝇中的可视化所有的神经胶质细胞（除中线的神经胶质细胞）诱导表达。这也可以与其他神经胶质标记物，如抗-GS2，该标记神经纤维网相关的神经胶质细胞（ 图3A，B）的组合。在这里，我们表明，虽然腹神经索是从背侧刺，刺中受伤导致凹痕腹部（ 图3，箭头）。刺会出现更严重的影响的神经纤维和神经胶质细胞神经​​纤维相关日的表面和皮层胶质细胞（ 图3B）。神经胶质细胞的过程显得杂乱无章的神经纤维，而他们仍然保持其在皮层组织网一样。 GS2-阳性的细胞碎片，这是不同的从正常细胞，碎片的损伤的结果是更小的，而不是连接到一个细胞体。这揭示损坏神经纤维相关的神经胶质细胞（ 图3B）。退化的神经纤维网相关的神经胶质细胞也观察到了由电子显微镜15。反裂半胱天冬酶3 +细胞凋亡染色观察神经纤维相关的神经胶质细胞15，刺向受伤后发生凋亡。也显示神经纤维相关的神经胶质细胞吞噬神经元的碎片15，和GS2 +信号也可能揭示吞噬凋亡的神经元，神经胶质的过程。劈开 - 半胱天冬酶+细胞凋亡，也观察到在皮质，至少其中的一些对应于ELAV +神经元（ <str翁&gt;图3C）。 使用固定样本和免疫组化染色的细胞反应，也使定量分析的损伤，如增殖和凋亡的影响。为此，我们特意开发了两个ImageJ的插件，DeadEasy的的半胱天冬酶幼虫和DeadEasy的神经胶质细胞，神经胶质细胞和凋亡细胞的数量自动计数，分别。他们已经验证工作幼虫VNCs，是非常准确的。通过使用这些，能够观察在通过刺损伤引起的，通过22小时（ 图4A）15 REPO阳 ​​性神经胶质细胞的数量增加。也有一个由6小时后刺向（ 图4B）15抗切割的Caspase-3的凋亡细胞的数量增加。这种果蝇的中枢神经系统损伤的神经胶质细胞增殖和凋亡的增加是在脊椎动物的中枢神经系统损伤反应的影子。 这亲的一个重要方面tocol是VNC夹层的质量。告诉在夹层的VNCs是否已被损坏或没有在这个过程中的时间，这是很难。重要的是要特别注意执行温和的解剖。然而，将不可避免地产生品质不良样品，至关重要的是，这些都是在稍后阶段识别并丢弃。在我们的手中，VNC变性似乎是无关的损伤，而造成的粗糙的解剖。我们并没有观察到一个临界值大小在受伤的伤口，以及我们分析所有受伤的样本，是不是退化。当VNCs保持其完整性，VNC面就显得不那么光滑，有光泽，无孔观察到的神经纤维（ 图5A）。可以降解的VNCs的确认24小时后解剖从表面粗糙的VNC腹侧和外侧的区域（ 图5B）。刺伤伤害无关的神经纤维的降解也可以发生，这是公认的神经纤维Ĥoles的免疫染色的标本中的背景信号。这些退化的迹象的样品必须被丢弃（ 图5C）。在我们的手中，解剖和损伤的完整和刺伤控制（YW）苍蝇的成功率都在70％左​​右。该速率将随的技能的人进行的实验，具有基因型。 <img alt="图1" src="/files/ftp_upload/50306/50306fig1.jpg" /> 图1。示意图幼虫分期和刺痛的伤害。（A）在第0天，苍蝇产卵在葡萄汁培养皿中3小时。在第1天，阴影线的第一龄幼虫（L1）收集并放置在含有标准的酵母的食品，另外3天，保留了它们的小瓶中。第4天，幼虫从食品瓶收集，和用于刺中实验。（B）侧面观幼虫的中枢神经系统。用针从背侧的腹神经索的腹部被刺中。前部是在左侧，后方右侧。 <img alt="图2" src="/files/ftp_upload/50306/50306fig2.jpg" /> 图2。时间推移恶化的VNC病变。激光共聚焦显微镜在现场VNCs GFP标记的轴突和红色荧光蛋白标记的神经胶质细胞。的基因型:UASDsRed / +; G9 / +; repoGAL4 / +（A）的病变是公认的缺乏轴突的神经纤维的荧光。刺伤后，GFP阴性会出现小洞，随后在规模和数量增加。 （B）的的5倍光学部分从刺向受伤后各时间点的预测。轴突神经纤维病变缩小，从9小时后捅死。这些图像是单一的光学部分，从不同的时间点，刺伤后受伤。要确定每个样本中的同等职位，在同一个片数从EA堆栈中的通道时间点的时间推移数据被选择了。通过比较可视化的模式与G9和repoGAL4 UASmCD8GFP在该地区不会受到刺伤，切片相当于在Z轴的位置进行了验证。中的虚线（A，B）表示的边缘的伤口。（C）的高倍率的伤口上的神经纤维。 GFP-阴性孔充满了红色荧光蛋白阳性胶质细胞的过程，随后消失（箭头）。横向视图。前了。 <img alt="图3" src="/files/ftp_upload/50306/50306fig3.jpg" /> 图3。 VNC病变细胞特性的膜系留的GFP记者为所有神经胶质细胞（除正中神经胶质细胞）染色，抗-GFP和抗-GS2神经胶质细胞，神经纤维相关标记。VNCs轴承。基因型:+ / +; UASmCD8GFP / +; repoGAL4 / +。（A）VNCs的从多尔被刺伤SAL的一面，但是这会导致腹部凹陷（箭头）。矢状单一的光学部分，背和前左（A，B）刺伤害，更严重的神经纤维相关的影响比皮质和面神经胶质细胞的神经胶质细胞。 GS2-阳性细胞碎片中的损伤的结果（箭头B 中）（B）的单一的光学部分的水平视图，前起来。箭头指向GS2 +细胞碎片（C）的细胞凋亡的标记的抗切割的Caspase-3的和示出，后损伤部分的死亡细胞在皮质的神经元的神经元标记物的抗ELAV Colocalisation。 NP，神经纤维CX，皮质。 <img alt="图4" src="/files/ftp_upload/50306/50306fig4.jpg" /> 图4。 （A）使用"DeadEasy幼虫的神经胶质细胞"的例子来计算REPO积极的神经胶质细胞和细胞凋亡DeadEasy自动计数。神经胶质细胞。左:VNC腹半染色，反REPO抗体;中间:DeadEasy幼虫的神经胶质细胞的细胞识别的插件输出;右:合并。右侧上的数字是REPO-阳性细胞中的共焦光学部分在大约30秒的整个堆栈自动计数的数目。数增加刺伤VNCs的神经胶质细胞（B）的实施例的使用&#39;DeadEasy CASPASE幼虫&#39;。左:预测的5倍光学聚焦与反裂caspase 3的抗体染色;中间的输出，显示细胞识别的插件，右:合并。在右边的数字半胱天冬酶阳性细胞自动计数在大约30秒的光学共焦部分在整个堆栈的数量。凋亡细胞的数量增加在刺VNC。 <img alt="图5" src="/files/ftp_upload/50306/50306fig5.jpg" /> 图5。例子degenerat离子（A）健康的VNC。有没有退化的迹象。（B）侧的胸部刺标本的退化（箭头）。星号表示病变的部位。（C）孔在胸神经纤维（白箭头）和皮质（橙色箭头），非刺的VNC。受损的神经纤维和丰富的空泡化的标本必须被丢弃。这里VNCs染色的神经纤维相关的胶质细胞标志物抗黑檀木。横向看，前起来。

Watch this Scientific Journal Video about An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila at JoVE.com

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

利用果蝇幼虫腹神经索，调查中枢神经系统的再生和修复受伤的范例。刺伤使用激光扫描共聚焦显微镜时间推移和固定的标本，有目的地开发的软件和遗传学与定量分析相结合，其次是中枢神经系统的再生和修复的分子机制。

调查中枢神经系统修复中受伤范式<em&gt;果蝇</em

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. ...

an-injury-paradigm-to-investigate-central-nervous-system-repair

Research

JoVE Journal

Neuroscience

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

13.2K 次观看.  University of Birmingham. 利用果蝇幼虫腹神经索，调查中枢神经系统的再生和修复受伤的范例。刺伤使用激光扫描共聚焦显微镜时间推移和固定的标本，有目的地开发的软件和遗传学与定量分析相结合，其次是中枢神经系统的再生和修复的分子机制。

视频: An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

在中央小鼠中枢神经系统的干细胞移植的多模态成像

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

科研

神经科学

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing therapeutic approaches. Lentiviral vectors are attractive tools in transduction of neurons and other cell types in CNS as they transduce both dividing and non-dividing cells, support sustained expression of transgenes, and have relatively large packaging capacity and low toxicity 1-3. Lentiviral vectors have been successfully used in transducing many neural cell types in vitro 4-6 and in animals 7-10.
Great efforts have been made to develop lentiviral vectors with improved biosafety and efficiency for gene delivery. The current third generation replication-defective and self-inactivating (SIN) lentiviral vectors are depicted in Figure 1. The required elements for vector packaging are split into four plasmids. In the lentiviral transfer plasmid, the U3 region in the 5' long terminal repeat (LTR) is replaced with a strong promoter from another virus. This modification allows the transcription of the vector sequence independent of HIV-1 Tat protein that is normally required for HIV gene expression 11. The packaging signal (&Psi;) is essential for encapsidation and the Rev-responsive element (RRE) is required for producing high titer vectors. The central polypurine tract (cPPT) is important for nuclear import of the vector DNA, a feature required for transducing non-dividing cells 12. In the 3' LTR, the cis-regulatory sequences are completely removed from the U3 region. This deletion is copied to 5' LTR after reverse transcription, resulting in transcriptional inactivation of both LTRs. Plasmid pMDLg/pRRE contains HIV-1 gag/pol genes, which provide structural proteins and reverse transcriptase. pRSV-Rev encodes Rev which binds to the RRE for efficient RNA export from the nucleus. pCMV-G encodes the vesicular stomatitis virus glycoprotein (VSV-G) that replaces HIV-1 Env. VSV-G expands the tropism of the vectors and allows concentration via ultracentrifugation 13. All the genes encoding the accessory proteins, including Vif, Vpr, Vpu, and Nef are excluded in the packaging system. The production and manipulation of lentiviral vectors should be carried out according to NIH guidelines for research involving recombinant DNA (<a href="http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf" target="_blank">http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf</a>). An approval from individual Institutional Biological and Chemical Safety Committee may be required before using lentiviral vectors. Lentiviral vectors are commonly produced by cotransfection of 293T cells with lentiviral transfer plasmid and the helper plasmids encoding the proteins required for vector packaging. Many lentiviral transfer plasmids and helper plasmids can be obtained from Addgene, a non-profit plasmid repository (<a href="http://www.addgene.org/" target="_blank">http://www.addgene.org/</a>). Some stable packaging cell lines have been developed, but these systems provide less flexibility and their packaging efficiency generally declines over time 14, 15. Commercially available transfection kits may support high efficiency of transfection 16, but they can be very expensive for large scale vector preparations. Calcium phosphate precipitation methods provide highly efficient transfection of 293T cells and thus provide a reliable and cost effective approach for lentiviral vector production.
In this protocol, we produce lentiviral vectors by cotransfection of 293T cells with four plasmids based on the calcium phosphate precipitation principle, followed by purification and concentration with ultracentrifugation through a 20% sucrose cushion. The vector titers are determined by fluorescence- activated cell sorting (FACS) analysis or by real time qPCR. The production and titration of lentiviral vectors in this protocol can be finished with 9 days. We provide an example of transducing these vectors into murine neocortical cultures containing both neurons and astrocytes. We demonstrate that lentiviral vectors support high efficiency of transduction and cell type-specific gene expression in primary cultured cells from CNS.

In this protocol we describe production, purification and titration of lentiviral vectors. We provide an example of lentiviral vector-mediated gene delivery in primary cultured neurons and astrocytes. Our methods may also apply to other cell types in vitro and in vivo.

从中枢神经系统转导细胞的慢病毒载体生产

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

标记的单细胞在中枢神经系统<em&gt;果蝇</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

啮齿动物的中枢神经系统药物直接脑室交货

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based multicolor labeling is challenging, especially when utilized for assessing interrelations between target proteins in the tissue with a high fat content such as the central nervous system (CNS).
Our protocol represents a refinement of the standard immunolabeling technique particularly adjusted for detection of both structural and soluble proteins in the rat CNS and peripheral lymph nodes (LN) affected by neuroinflammation. Nonetheless, with or without further modifications, our protocol could likely be used for detection of other related protein targets, even in other organs and species than here presented.

We here present an optimized, detailed protocol for double immunostaining in formalin-fixed, paraffin-embedded rat central nervous system (CNS) and peripheral lymph node (LN) tissue sections.

免疫组化分析大鼠中枢神经系统和周围淋巴结组织切片

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are tightly regulated, affecting the final dynamics of protein abundance. Various regulatory mechanisms exist at the translation step, rendering mRNA levels alone an unreliable indicator of gene expression. In addition, local regulation of mRNA translation has been particularly implicated in neuronal functions, shifting &#39;translatomics&#39; to the focus of attention in neurobiology. The presented method can be used to bridge transcriptomics and proteomics.
Here we describe essential modifications to the technique of polyribosome fractionation, which interrogates the translatome based on the association of actively translated mRNAs to multiple ribosomes and their differential sedimentation in sucrose gradients. Traditionally, working with in vivo samples, particularly of the central nervous system (CNS), has proven challenging due to the restricted amounts of material and the presence of fatty tissue components. In order to address this, the described protocol is specifically optimized for use with minimal amount of CNS material, as demonstrated by the use of single mouse spinal cord and brain. Briefly, CNS tissues are extracted and translating ribosomes are immobilized on mRNAs with cycloheximide. Myelin flotation is then performed to remove lipid rich components. Fractionation is performed on a sucrose gradient where mRNAs are separated according to their ribosomal loading. Isolated fractions are suitable for a range of downstream assays, including new genome wide assay technologies.

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

This protocol illustrates essential modifications of polyribosome fractionation in order to study the translatome of in vivo CNS samples. It allows global assessment of translation and transcription regulation through the isolation and comparison of total RNA to ribosome bound RNA fractions.

在中枢神经系统Translatome由多核糖体分馏体内拷问

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

在啮齿动物中枢神经系统的体内光遗传学刺激

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique non-linear deformations, dramatically hampering one's ability to evaluate cellular morphology, distribution and connectivity in the central nervous system (CNS). However, optical clearing techniques are changing the way tissues are visualized. These approaches permit one to probe deeply into intact organ preparations, providing tremendous insight into the structural organization of tissues in health and disease. Techniques such as Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) achieve this goal by providing a matrix that binds important biomolecules while permitting light-scattering lipids to freely diffuse out. Lipid removal, followed by refractive index matching, renders the tissue transparent and readily imaged in 3 dimensions (3D). Nevertheless, the electrophoretic tissue clearing (ETC) used in the original CLARITY protocol can be challenging to implement successfully and the use of a proprietary refraction index matching solution makes it expensive to use the technique routinely. This report demonstrates the implementation of a simple and inexpensive optical clearing protocol that combines passive CLARITY for improved tissue integrity and 2,2&#8242;-thiodiethanol (TDE), a previously described refractive index matching solution.

Optical clearing techniques are revolutionizing the way tissues are visualized. In this report we describe modifications of the original Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) protocol that yields more consistent and less expensive results.

鼠标中枢神经系统的光透明使用被动CLARITY

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique ...

Anatomically Inspired Three-dimensional Micro-tissue Engineered Neural Networks for Nervous System Reconstruction, Modulation, and Modeling

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory environment and the limited capacity for neurogenesis. We are developing&#160;a strategy to simultaneously address&#160;neuronal and axonal pathway loss within the damaged CNS. This manuscript presents the fabrication protocol for micro-tissue engineered neural networks (micro-TENNs), implantable constructs consisting of neurons and aligned axonal tracts spanning the extracellular matrix (ECM) lumen of a preformed hydrogel cylinder hundreds of microns in diameter that may extend centimeters in length. Neuronal aggregates are delimited to the extremes of the three-dimensional encasement and are spanned by axonal projections. Micro-TENNs are uniquely poised as a strategy for CNS reconstruction, emulating aspects of brain connectome cytoarchitecture and potentially providing means for network replacement. The neuronal aggregates may synapse with host tissue to form new functional relays to restore and/or modulate missing or damaged circuitry. These constructs may also act as pro-regenerative&#160;&#34;living scaffolds&#34; capable of exploiting developmental mechanisms for cell migration and axonal pathfinding, providing synergistic structural and soluble cues based on the state of regeneration. Micro-TENNs are fabricated by pouring liquid hydrogel into a cylindrical mold containing a longitudinally centered needle. Once the hydrogel has gelled, the needle is removed, leaving a hollow micro-column. An ECM solution is added to the lumen to provide an environment suitable for neuronal adhesion and axonal outgrowth. Dissociated neurons are mechanically aggregated for precise seeding within one or both ends of the micro-column. This methodology reliably produces self-contained miniature constructs with long-projecting axonal tracts that may recapitulate features of brain neuroanatomy. Synaptic immunolabeling and genetically encoded calcium indicators suggest that micro-TENNs possess extensive synaptic distribution and intrinsic electrical activity. Consequently, micro-TENNs represent a promising strategy for targeted neurosurgical reconstruction of brain pathways and may also be applied as biofidelic models to study neurobiological phenomena in vitro.

This manuscript details the fabrication of micro-tissue engineered neural networks: three-dimensional micron-sized constructs comprised of long aligned axonal tracts spanning aggregated neuronal population(s) encased in a tubular hydrogel. These living scaffolds can serve as functional relays to reconstruct or modulate neural circuitry or as biofidelic test-beds mimicking gray-white matter neuroanatomy.

解剖学启发的三维微组织工程神经网络用于神经系统重建，调制和建模

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory ...

Quantifying Acute Changes in Renal Sympathetic Nerve Activity in Response to Central Nervous System Manipulations in Anesthetized Rats

Renal sympathetic nerve activity (RSNA) and mean arterial pressure are important parameters in cardiovascular and autonomic research; however, there are limited resources directing scientists in the techniques for measuring and analyzing these variables. This protocol describes the methods for measuring RSNA and mean arterial pressure in anesthetized rats. The protocol also includes the approaches for accessing the brain during RSNA recordings for central nervous system (CNS) manipulations. The RSNA recording technique is compatible with pharmacologic, optogenetic, or electrical stimulation of the CNS. The approach is useful when an investigator will measure short-term (min to h) autonomic responses in non-survival experiments to correlate anatomically with CNS nuclei. The approach is not intended to be used to obtain chronic (survival) recordings of RSNA in rats. Discharges in RSNA, averaged rectified RSNA, and mean arterial pressure can be quantified and analyzed further using parametric statistical tests. Methods for obtaining venous access, recording mean arterial pressure telemetrically, and brain fixation for future histological analysis are also described in the article.

Methods for measuring sympathetic and cardiovascular responses to central nervous system (CNS) manipulations are important for advancing neuroscience. This protocol was developed to assist scientists with measuring and quantifying acute changes in renal sympathetic nerve activity (RSNA) in anesthetized rats (non-survival).

麻醉大鼠中枢神经系统操作对肾交感神经活动的急性变化量化

Renal sympathetic nerve activity (RSNA) and mean arterial pressure are important parameters in cardiovascular and autonomic research; however, there are ...

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Subtitles

调查中枢神经系统修复中受伤范式