这项工作是通过美国国立卫生研究院R15区授予约翰霍恩（镍氢电池; R15MH083221）资助。

1。转基因胚胎<ol><li>这里我们使用了斑马鱼转基因株系，通过转座子介导的增强陷阱的方法8。转基因插入包括编码序列KalTA4，一个优化版本的GAL4转录激活斑马鱼，和红色荧光蛋白，mCherry。增强陷阱的方法产量线的转基因斑马鱼，这种转基因盒表达内源性增强子序列的控制之下。因此，根据特定的增强子序列的身份，KalTA4和mCherry表达是本地化到发育中的大脑的特定区域。 ET（zic4：Gal4TA4，无人机：mCherry）hzm5线，我们在这里使用，表达本地化的几个地点，脑，后脑和具有特别重要的意义，在这里，嗅球8（见图1。） 。鉴于这种表达方式，和本地化的基因组内的录像带，转基因预测zic4 发起人 8的控制之下（ <a href="http://www.helmholtz-muenchen.de/en/idg/groups/neuroimaging/lines_distel/main">见</a>也http://www.helmholtz-muenchen.de/en/idg/groups/ <a href="http://www.helmholtz-muenchen.de/en/idg/groups/neuroimaging/lines_distel/main">影像学/ lines_distel /主</a> ）。 </li><li>成人ET（zic4：Gal4TA4，无人机：mCherry）hzm5鱼杂合子转基因插入通常与WT（AB）鱼交配，导致约50％的转基因后代。 （对于转基因后代的比例更高，两个杂合子可以交配。） </li><li>为了阻止色素形成和促进荧光成像，100μM的N -苯基硫脲嘧啶（PTU）在6-8 HPF开始对待转基因胚胎。 </li></ol> 2。安装胚胎<ol><li> 24-28 HPF，转移胚胎蛋水，无机动部队。确定使用荧光显微镜的表达mCherry的转基因胚胎。取出用细镊子的转基因胚胎绒毛膜膜。传输中解脱出来，从胚胎绒毛膜培养皿中含有电加0.02％的缓冲tricaine [电穿孔缓冲：180毫米氯化钠，氯化钾5毫米，1.8毫米氯化钙 2，5毫米的HEPES，pH值7.2] 4 。允许2分钟的麻醉效果，然后再继续安装步骤。 </li><li>微波解决方案，然后放置到一个34-37 ° C培养箱解决方案，准备一个0.5％的低融点琼脂糖电缓冲区加上tricaine解决方案。一旦琼脂糖溶液的温度平衡，将大幅下降（约500μL）的琼脂糖溶液60毫米培养皿的中心。 6-8麻醉胚胎的琼脂糖下降。 </li><li>使用镊子（或减少microloader吸管提示罚款结束）的罚款，位置的胚胎，它们都面临着同一方向，并在一个垂直的行对齐。巩固琼脂糖和陷阱的胚胎中的位置，放到一个大培养皿中，用2-3毫米的冰盘。 </li><li>一旦琼脂糖凝固后，洪水电缓冲区加上tricaine菜，以确保该解决方案完全覆盖凝固的琼脂糖下降。 </li></ol> 3。显微注射DNA的表达质粒<ol><li>准备使用的MIDI或Maxi准备质粒提取试剂盒（如Qiagen公司）的绿色荧光蛋白表达质粒。挂起到终浓度为0.5μg/μL无菌水加0.03％酚红质粒。对于嗅球针对实验在这里，我们使用多个Gal4的结合位点（14无人机串联序列和基底鱼发起人，E1B）控制下的EGFP编码序列的表达质粒。将使用这种质粒，transgenically表达的KalTA4转录因子的细胞能够表达绿色荧光蛋白，因此，调解有针对性的表达绿色荧光蛋白。 注：在这里，我们使用的DNA注射，应针对发育中的大脑的其他地区适用的可视化酚红，只要该地区可以从心室​​访问。针对其他类型的细胞，需要荧光可视化，肾上腺素或Quantam点染料可能被用于可视化荧光灯照明下注射。 </li><li>显微注射移液器可以采用水平或垂直的吸管拉马。热拉强度设置会有所不同拉马，加热元件的类型和玻璃毛细管的类型。 使用一个萨特的P - 30垂直拉拔器，使用了980的热定型和拉力为960，生产薄壁硼硅毛细管玻璃细丝长，逐渐变细，尖锐的移液器[萨特仪器＃BF100 - 78 - 10]。这些设置拉Microinjetion移液器是密封的，注射前必须打破。 </li><li>使用microloader枪头，加1-2微升DNA质粒溶液注射针。 </li><li>山和争取到的高压喷射系统移液器支架装入吸管。 </li><li>回破用细镊子装入吸管，直到压力脉冲产生膨化释放红色的DNA溶液。另外，小技巧，可以迅速穿透水下，折叠实验室Kimwipe被打破。 注：因为注射移液器最初密封，必须手动回破，针尖大小和注射量是可变的。理想的注射移液器需要3-5注射脉冲完全填补24 HPF胚胎（100毫秒脉冲注入40 PSI）的前脑脑室发展。 </li><li>质粒DNA溶液注入到发育中的大脑脑室这种DNA是迫使相邻，和闰成，注定要形成嗅球的大脑区域。嗅球是来自细胞在脑脑室前壁。因此，通过插入脑室注射针等移液器指向是对发育中的大脑前结束，高压喷射力量的DNA和相邻的准嗅球。注入的DNA，直到红色染料是清晰可见脑脑室前结束。因为DNA立即开始弥漫，稀释的DNA，重要的是进行电步骤尽快注射后（例如，10秒内）。 </li></ol> 4。电穿孔<ol><li>使用自建草铂（草技术＃E2）皮下电极的电极进行电穿孔。电极连接到一个手持式探头，并最终铂电极之间的距离应为1毫米（在建筑的细节见 9） 。方波电脉冲产生的电极连接到一个草SD - 9刺激器（草技术，型号SD - 9）。 </li><li>设置SD - 9刺激产生70伏单，5毫秒电脉冲。放置电极，正电极定位毗邻的胚胎前结束，而负电极后的胚胎头。手动启动〜7-8使用的SD - 9刺激的单一模式开关的电脉冲，每个脉冲分离〜1秒。适当的电压将取决于对胚胎的年龄和所使用的电压源。年轻的胚胎需要较低的电压，以避免胚胎的损害，而年纪大的胚胎需要更高的电压 ，启动电7。 </li><li>允许胚胎，至少10分钟后，电恢复之前，使他们从琼脂糖。 </li><li>用细镊子，免费琼脂糖胚胎轻轻跟踪胚胎的轮廓，同时避免与胚胎本身的实际接触。 </li><li>一旦释放，返回的胚胎蛋水（与机动部队如果需要的话）。保持在28 ° C，直到他们将GFP表达成像。 </li></ol> 5。成像安装胚胎<ol><li>胚胎转移，从鸡蛋的水，加上警察机动部队蛋水加0.02％tricaine。允许几分钟的麻醉效果，然后再继续安装步骤。 </li><li>微波解决方案，然后放置到一个34-37 ° C培养箱解决方案，准备一个0.5％的低融点琼脂糖鸡蛋水加tricaine解决方案。一旦琼脂糖溶液的温度平衡，将大幅下降（约300μL）的琼脂糖溶液到35毫米的培养皿的中心。添加一个麻醉胚胎的琼脂糖下降。 </li><li>使用镊子（或减少microloader吸管提示罚款结束）的罚款，位置的胚胎，他们是正直的，使它能够可视化的​​背视图，发育中的大脑与一个堂堂正正的显微镜（倒置的范围将需要不同的胚胎几何和不同的菜，可以从下面的可视化 ;看到12）。琼脂糖巩固放置到一个大培养皿中，用2-3毫米的冰菜。用细镊子将有可能在凝固过程中需要重新定位，以确保胚胎不落在其副作用。 </li><li>一旦琼脂糖凝固，洪水盘鸡蛋水加tricaine确保该解决方案完全覆盖凝固的琼脂糖下降。 </li></ol> 6。代表性的成果： 虽然GFP的表达，可以观察到早6个小时后，电，在这里，我们显示了从胚胎电后第4天，充分发展的目标细胞的突起结构图像。的ET（zic4：Gal4TA4，无人机：mCherry）hzm5转基因增强子陷阱线显示mCherry组成性表达（和KalTA4）在后脑，小脑，前脑，胚胎嗅球，在5天受精后（图1A和1C）。胚胎有一个UAS - GFP表达质粒针对性电（如上所述），显示绿色荧光蛋白的表达，是限制发展嗅球细胞（图1B，1D及1E）注册成立。只有细胞transgenically表达的KalTA4转录因子能够激活这个UAS - GFP的质粒表达。这个团到非转基因胚胎的质粒不会导致任何可察觉的GFP表达。因此，它是有针对性的UAS - GFP和本地化的KalTA4驱动程序，导致发展的嗅球细胞中GFP的独家表达的转基因表达电的联合行动。表达GFP的细胞显示出典型的轴突预测嗅球僧帽细胞（图1B和1D）10。为了形象化轴突的预测，在图1B和1D的图像被暴露在胞体的区域过度曝光高层次。一个暴露的较低水平（图1E）显示，表达GFP的细胞体确实本地化嗅球（比较图1C及1E;灰线，标志着嗅球边境）。 <img src="/files/ftp_upload/2964/2964fig1.jpg" alt="图1"/> 图1。针对性的5 DPF的斑马鱼胚胎的嗅球GFP的表达。 ET（zic4：Gal4TA4，无人机：mCherry）hzm5转基因增强子陷阱线，显示构mCherry表达在后脑，小脑，前脑和嗅球（A和 C，mCherry荧光显示为红色）。这mCherry表达介导的转基因Gal4的表达。胚胎已与UAS - GFP的表达载体，电穿孔28 HPF，显示绿色荧光蛋白的表达有针对性的在受精后5天的嗅球（B，D，和 E，绿色荧光绿色显示相同的胚胎，并在A C）。一个曝光的图片的底部（e）显示，GFP的表达是有限的嗅球细胞机构。灰线表示嗅球的边界。明场（灰度），绿色荧光（绿色），和红色荧光（红色）图像都是用Olympus BX60荧光显微镜。

<ol>
	<li>Swartz, M., Eberhart, J., Mastick, G., Krull, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sparking+new+frontiers:+using+in+vivo+electroporation+for+genetic+manipulations.">Sparking new frontiers: using in vivo electroporation for genetic manipulations.</a> Dev Biol. 233, 13-21 (2001).</li><li>Bhatt, D. H., Otto, S. J., Depoister, B., Fetcho, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+AMP-induced+repair+of+zebrafish+spinal+circuits.">Cyclic AMP-induced repair of zebrafish spinal circuits.</a> Science. 305, 254-258 (2004).</li><li>Teh, C., Parinov, S., Korzh, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+ways+to+admire+zebrafish:+progress+in+functional+genomics+research+methodology.">New ways to admire zebrafish: progress in functional genomics research methodology.</a> Biotechniques. 38, 897-906 (2005).</li><li>Cerda, G. A., Thomas, J. E., Allende, M. L., Karlstrom, R. O., Palma, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+of+DNA,+RNA,+and+morpholinos+into+zebrafish+embryos.">Electroporation of DNA, RNA, and morpholinos into zebrafish embryos.</a> Methods. 39, 207-211 (2006).</li><li>Hendricks, M., Jesuthasan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation-based+methods+for+in+vivo,+whole+mount+and+primary+culture+analysis+of+zebrafish+brain+development.">Electroporation-based methods for in vivo, whole mount and primary culture analysis of zebrafish brain development.</a> Neural Development. 15, 2-6 (2007).</li><li>Tawk, M., Bianco, I. H., Clarke, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+electroporation+in+zebrafish+embryos+and+larvae.">Focal electroporation in zebrafish embryos and larvae.</a> Methods Mol Biol. 546, 145-151 (2009).</li><li>Kera, S. A., Agerwala, S. M., Horne, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+temporal+resolution+of+in+vivo+electroporation+in+zebrafish:+a+method+for+time-resolved+loss-of-function.">The temporal resolution of in vivo electroporation in zebrafish: a method for time-resolved loss-of-function.</a> Zebrafish. 7, 97-108 (2010).</li><li>Distel, M., Wullimann, M. F., K&#246;ster, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+Gal4+genetics+for+permanent+gene+expression+mapping+in+zebrafish.">Optimized Gal4 genetics for permanent gene expression mapping in zebrafish.</a> Proc Natl Acad Sci U S A. 106, 13365-13370 (2009).</li><li>Hoegler, K. J., Horne, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+the+zebrafish+optic+tectum+using+in+vivo+electroporation.">Targeting the zebrafish optic tectum using in vivo electroporation.</a> Cold Spring Harb. Protoc. , (2010).</li><li>Nobuhiko, M., Kozo, M., Tatsuya, T., Shin-ichi, H., Hitoshi, O., Yoshihiro, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+the+olfactory+bulb+to+higher+brain+centers:+genetic+visualization+of+secondary+olfactory+pathways+in+zebrafish.">From the olfactory bulb to higher brain centers: genetic visualization of secondary olfactory pathways in zebrafish.</a> J. Neurosci. 29, 4756-4766 (2009).</li><li>Davison, J. M., Akitake, C. M., Goll, M. G., Rhee, J. M., Gosse, N., Baier, H., Halpern, M. E., Leach, S. D., Parsons, 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=Transactivation+from+Gal4-VP16+transgenic+insertions+for+tissue-specific+cell+labeling+and+ablation+in+zebrafish.">Transactivation from Gal4-VP16 transgenic insertions for tissue-specific cell labeling and ablation in zebrafish.</a> Developmental Biology. , 304-811 (2007).</li><li>Distel, M., Kö, s. t. e. r., W, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=in+vivo+time-lapse+imaging+of+zebrafish+embryonic+development.">in vivo time-lapse imaging of zebrafish embryonic development.</a> CSH Protocols. , (2007).</li><li>Emelyanov, A., Parinov, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mifepristone-inducible+LexPR+system+to+drive+and+control+gene+expression+transgenic+zebrafish.">Mifepristone-inducible LexPR system to drive and control gene expression transgenic zebrafish.</a> Developmental Biology. 320, 113-121 (2008).</li><li>Knopf, F., Schnabel, K., Haase, C., Pfeifer, K., Anastassiasdis, K., Weidinger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dually+inducible+TetON+systems+for+tissue-specific+conditional+gene+expression+in+zebrafish.">Dually inducible TetON systems for tissue-specific conditional gene expression in zebrafish.</a> PNAS. , 107-10 (1993).</li><li>Zhu, P., Narita, Y., Bundschuh, S. T., Fajardo, O., Sch&#228;rer, Y. P., Chattopadhyaya, B., Bouldoires, E. A., Stepien, A. E., Deisseroth, K., Arber, S., Sprengel, R., Rijli, F. M., Friedrich, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+Dissection+of+Neuronal+Circuits+in+Zebrafish+using+Viral+Gene+Transfer+and+the+Tet+System.">Optogenetic Dissection of Neuronal Circuits in Zebrafish using Viral Gene Transfer and the Tet System.</a> Front Neural Circuits. 3, 21-21 (2009).</li></ol>

没有利益冲突的声明。

在这里，我们描述了在斑马鱼体内电穿孔法利用增强剂陷阱Gal4的转基因株系的目标电穿孔转基因表达的细胞特定的人口在发展中国家的嗅球。这种方法结合在体内电穿孔7增强剂陷阱转基因株系8介导的细胞类型的具体表达了良好的时间分辨率。虽然我们在这里所描述的嗅球的目标， 在体内电穿孔可用于其他地区的神经系统的发育2-7目标，并增强子陷阱线与Gal4的针对性体现在许多不同的特定细胞类型或组织8，11。电穿孔技术，当然，这也应该适合其他combinatioral LexA或春节系统13，14，15，如遗传方式。电穿孔的一个主要优点是，有没有需要给出一个合适的Gal4的行提供的额外的转基因株系。这样可以节省6个月内。 允许在任何神经系统发展的特定阶段的兴趣 7成神经细胞及其前体掺入的寡核苷酸在体内电。时间分辨率是亏损的功能分析特别有利，因为它可以规避与目标基因在早期发育阶段的基本职能的问题。我们在这里描述的方法也可以用来纳入丧失功能的试剂，包括RNA干扰寡核苷酸或构造和吗啉反义寡核苷酸4，7 。如显性负蛋白表达质粒或短发夹RNA干扰质粒质粒驱动的试剂也可以放在一个GAL4 UAS的控制下，允许精确的细胞类型特异性表达GFP的表达，我们已经表明这里。

试剂： <ul><li>琼脂糖，低融点（Sigma公司，A9414） </li><li>质粒的MIDI或Maxi制备的试剂盒（如Qiagen公司，＃12643] </li><li>酚红（Sigma公司，P3532） </li></ul>设备： <ul><li>毛细管玻璃长丝; ID = 0.78毫米，外径为1.0毫米（萨特仪器，＃BF100 - 78 - 10） </li><li>草SD - 9刺激（草技术，型号SD - 9） </li><li>草铂皮下电极，直针（草技术，＃E2） </li><li>微型装载机枪头（Eppendorf公司，＃X22703R） </li><li>移液器拉马（如萨特，模型P - 30） </li><li>移液器拉马（如萨特，模型P - 30） </li></ul>

targeting olfactory bulb neurons using combined in vivo electroporation and gal4-based enhancer trap zebrafish lines

在体内电穿孔是一种提供质粒DNA的表达，RNAi试剂，和吗啉反义寡核苷酸对胚胎发育的特定区域， 包括 C语言的强大方法线虫 ，鸡， 爪蟾 ，斑马鱼和小鼠1。在斑马鱼体内电穿孔已被证明有良好的空间和时间分辨率为提供这些试剂 2-7 。这种方法的时间分辨率是重要的，因为它允许这些试剂纳入发展的特定阶段。此外，因为电穿孔载体的表达发生在6小时内 7比转基因的方法，这种方法更及时。虽然空间分辨率可以非常精确的针对单个细胞2，6时，往往是最好将其纳入一个特定的细胞群，在一个组织或结构的试剂。当针对多个细胞， 在体内电穿孔是有效的传递到一个特定区域的胚胎;然而，尤其是在神经系统发育，它是很难的目标，仅仅通过特定的细胞类型空间上离散的电。另外，增强陷阱的转基因株系提供优良的转基因 8细胞类型的具体体现。在这里，我们描述了一个方法，结合体内电穿孔7，9 GAL4基于转基因的增强子陷阱线与空间上离散的专门针对发展中国家的斑马鱼嗅球的神经元。 ET（zic4：Gal4TA4，无人机：mCherry）hzm5（原GA80_9）增强子陷阱线前面所述8，显示斑马鱼的目标mCherry介导的转基因表达在发育中的大脑的多个区域，包括后脑优化Gal4的（KalTA4）转录激活因子，小脑，前脑和嗅球。要明确目标GFP表达到嗅球，与绿色荧光蛋白基因的编码序列多个Gal4的结合位点（UAS）的控制下的质粒电穿孔在受精后24-28小时（HPF）的前脑的前结束。虽然这种方法采用质粒DNA进入前脑的多个地区，GFP的表达诱导transgenically表达的KalTA4转录因子的细胞。因此，通过使用的GA080_9转基因株系，这种做法导致GFP的表达只在发展中国家的嗅球。 GFP的表达，通过这种方法有针对性的细胞呈典型的神经轴突的预测，10嗅球僧帽细胞。这种方法也可用于有针对性地提供其他试剂，包括短发夹RNA干扰表达质粒，这将提供空间和时间上离散的损失函数分析的方法。

Watch this Scientific Journal Video about Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines at JoVE.com

Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines

基因操作的时间和空间分辨率确定频谱的生物现象，他们可以扰乱。在这里，我们使用的时间和空间的离散<em&gt;在体内</em&gt;电结合，与斑马鱼的转基因株系，诱使一个绿色荧光蛋白基因的表达在发展中的嗅球神经元。

针对嗅球神经元采用组合在体内电穿孔和GAL4基于增强陷阱斑马鱼线

In vivo electroporation is a powerful method for delivering DNA expression plasmids, RNAi reagents, and morpholino anti-sense oligonucleotides to specific ...

targeting-olfactory-bulb-neurons-using-combined-vivo-electroporation

Research

JoVE Journal

Neuroscience

Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines

12.0K 次观看.  Pace University. 基因操作的时间和空间分辨率确定频谱的生物现象，他们可以扰乱。在这里，我们使用的时间和空间的离散在体内电结合，与斑马鱼的转基因株系，诱使一个绿色荧光蛋白基因的表达在发展中的嗅球神经元。

视频: Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines

Mapping and Application of Enhancer-trap Flippase Expression in Larval and Adult Drosophila CNS

The Gal4/ UAS binary method is powerful for gene and neural circuitry manipulation in Drosophila. For most neurobiological studies, however, Gal4 expression is rarely tissue-specific enough to allow for precise correlation of the circuit with behavioral readouts. To overcome this major hurdle, we recently developed the FINGR method to achieve a more restrictive Gal4 expression in the tissue of interest. The FINGR method has three components: 1) the traditional Gal4/UAS system; 2) a set of FLP/FRT-mediated Gal80 converting tools; and 3) enhancer-trap FLP (ET-FLP). Gal4 is used to define the primary neural circuitry of interest. Paring the Gal4 with a UAS-effector, such as UAS-MJD78Q or UAS-Shits, regulates the neuronal activity, which is in turn manifested by alterations in the fly behavior. With an additional UAS-reporter such as UAS-GFP, the neural circuit involved in the specific behavior can be simultaneously mapped for morphological analysis. For Gal4 lines with broad expression, Gal4 expression can be restricted by using two complementary Gal80-converting tools: tubP&gt;Gal80&gt; ('flip out') and tubP&gt;stop&gt;Gal80 ('flip in'). Finally, investigators can turn Gal80 on or off, respectively, with the help of tissue-specific ET-FLP. In the flip-in mode, Gal80 will repress Gal4 expression wherever Gal4 and ET-FLP intersect. In the flip-out mode, Gal80 will relieve Gal4 repression in cells in which Gal4 and FLP overlap. Both approaches enable the restriction of the number of cells in the Gal4-defined circuitry, but in an inverse pattern. The FINGR method is compatible with the vast collection of Gal4 lines in the fly community and highly versatile for traditional clonal analysis and for neural circuit mapping. In this protocol, we demonstrate the mapping of FLP expression patterns in select ET-FLPx2 lines and the effectiveness of the FINGR method in photoreceptor cells. The principle of the FINGR method should also be applicable to other genetic model organisms in which Gal4/UAS, Gal80, and FLP/FRT are used.

Mapping and Application of Enhancer-trap Flippase Expression in Larval and Adult Drosophila CNS

We describe a Flippase-induced intersectional Gal80/Gal4 repression (FINGR) method, allowing tissue-specific FLP to determine Gal80 expression patterns. Wherever Gal4 and FLP overlap, Gal4 expression is turned on (Gal80 flipped out) or off (Gal80 flipped in). The FINGR method is versatile for clonal analysis and neural circuit mapping.

幼虫和成人测绘和增强子陷阱Flippase表达中的应用果蝇中枢神经系统

The Gal4/ UAS binary method is powerful for gene and neural circuitry manipulation in Drosophila. For most neurobiological studies, however, Gal4 ...

科研

神经科学

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

小鼠视网膜神经节细胞的转染在体内电穿孔

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the olfactory bulb (OB). The molecular mechanisms of OSN axon growth and targeting are not well understood. Manipulating gene expression and subsequent 
visualizing of single OSN axons and their terminal arbor morphology have thus far been challenging. To study gene function at the single cell level within a specified 
time frame, we developed a lentiviral based technique to manipulate gene expression in OSNs in vivo. Lentiviral particles are delivered to OSNs by microinjection 
into the olfactory epithelium (OE). Expression cassettes are then permanently integrated into the genome of transduced OSNs. Green fluorescent protein expression 
identifies infected OSNs and outlines their entire morphology, including the axon terminal arbor. Due to the short turnaround time between microinjection and 
reporter detection, gene function studies can be focused within a very narrow period of development. With this method, we have detected GFP expression within as 
few as three days and as long as three months following injection. We have achieved both over-expression and shRNA mediated knock-down by lentiviral microinjection. 
This method provides detailed morphologies of OSN cell bodies and axons at the single cell level in vivo, and thus allows characterization of candidate gene function 
during olfactory development.

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

We present a lentiviral technique for genetic manipulation and visualization of single olfactory sensory neuron axon and its terminal arborization in vivo.

嗅觉感官神经元的慢病毒介导的遗传操作和可视化在体内</em

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the ...

Imaging Odor-Evoked Activities in the Mouse Olfactory Bulb using Optical Reflectance and Autofluorescence Signals

In the brain, sensory stimulation activates distributed populations of neurons among functional modules which participate to the coding of the stimulus. Functional optical imaging techniques are advantageous to visualize the activation of these modules in sensory cortices with high spatial resolution. In this context, endogenous optical signals that arise from molecular mechanisms linked to neuroenergetics are valuable sources of contrast to record spatial maps of sensory stimuli over wide fields in the rodent brain.
Here, we present two techniques based on changes of endogenous optical properties of the brain tissue during activation. First the intrinsic optical signals (IOS) are produced by a local alteration in red light reflectance due to: (i) absorption by changes in blood oxygenation level and blood volume (ii) photon scattering. The use of in vivo IOS to record spatial maps started in the mid 1980's with the observation of optical maps of whisker barrels in the rat and the orientation columns in the cat visual cortex1. IOS imaging of the surface of the rodent main olfactory bulb (OB) in response to odorants was later demonstrated by Larry Katz's group2. The second approach relies on flavoprotein autofluorescence signals (FAS) due to changes in the redox state of these mitochondrial metabolic intermediates. More precisely, the technique is based on the green fluorescence due to oxidized state of flavoproteins when the tissue is excited with blue light. Although such signals were probably among the first fluorescent molecules recorded for the study of brain activity by the pioneer studies of Britton Chances and colleagues3, it was not until recently that they have been used for mapping of brain activation in vivo. FAS imaging was first applied to the somatosensory cortex in rodents in response to hindpaw stimulation by Katsuei Shibuki's group4.
The olfactory system is of central importance for the survival of the vast majority of living species because it allows efficient detection and identification of chemical substances in the environment (food, predators). The OB is the first relay of olfactory information processing in the brain. It receives afferent projections from the olfactory primary sensory neurons that detect volatile odorant molecules. Each sensory neuron expresses only one type of odorant receptor and neurons carrying the same type of receptor send their nerve processes to the same well-defined microregions of &tilde;100&mu;m3 constituted of discrete neuropil, the olfactory glomerulus (Fig. 1). In the last decade, IOS imaging has fostered the functional exploration of the OB5, 6, 7 which has become one of the most studied sensory structures. The mapping of OB activity with FAS imaging has not been performed yet.
Here, we show the successive steps of an efficient protocol for IOS and FAS imaging to map odor-evoked activities in the mouse OB.

This article presents the protocols of intrinsic optical signals and flavoproteins autofluorescence signals imaging to map odor-evoked activities at the surface of the olfactory bulb in mice.

在小鼠嗅球成像气味诱发的活动，利用光学反射和自体荧光信号

In the brain, sensory stimulation activates distributed populations of neurons among functional modules which participate to the coding of the stimulus. ...

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons4,5. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

慢性成人出生的嗅觉神经元的选择性病毒转导在体内 Optogenetic刺激

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

使用超声波血脑屏障破坏和锰增强MRI的脑功能成像

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (&#60;1 mm3) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and kairomones.

完整的犁鼻器和副嗅球的体外制剂

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disorders

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium carbonate. Animal studies as well as human genetic studies indicate that lithium affects molecular targets that are involved in neuronal growth, survival and maturation, and notably molecules involved in Wnt signaling. Given the ethical challenge to obtaining brain biopsies for investigating dynamic molecular changes associated with lithium-response in the central nervous system (CNS), one may consider the use of neurons obtained from olfactory tissues to achieve this goal.The olfactory epithelium contains olfactory receptor neurons at different stages of development and glial-like supporting cells. This provides a unique opportunity to study dynamic changes in the CNS of patients with neuropsychiatric diseases, using olfactory tissue safely obtained from nasal biopsies. To overcome the drawback posed by substantial contamination of biopsied olfactory tissue with non-neuronal cells, a novel approach to obtain enriched neuronal cell populations was developed by combining nasal biopsies with laser-capture microdissection. In this study, a system for investigating treatment-associated dynamic molecular changes in neuronal tissue was developed and validated, using a small pilot sample of BD patients recruited for the study of the molecular mechanisms of lithium treatment response.

In this study, a novel platform to investigate intraneuronal molecular signatures of treatment response in bipolar disorder (BD) was developed and validated. Olfactory epithelium from BD patients was obtained through nasal biopsies. Then laser-capture microdissection was combined with Real Time RT PCR to investigate the molecular signature of lithium response in BD.

通过鼻腔活检获得联合激光捕获显微切割的嗅觉神经元：一种可能的方法来研究治疗反应的精神障碍

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium ...

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. However, there is piling evidence that olfaction is not solely limited to chemical sensitivity, but also includes temperature sensitivity. Premetamorphic Xenopus laevis are translucent animals, with protruding nasal cavities deprived of the cribriform plate separating the nose and the olfactory bulb. These characteristics make them well suited for studying olfaction, and particularly thermosensitivity. The present article describes the complete procedure for measuring temperature responses in the olfactory bulb of X. laevis larvae. Firstly, the electroporation of olfactory receptor neurons (ORNs) is performed with spectrally distinct dyes loaded into the nasal cavities in order to stain their axon terminals in the bulbar neuropil. The differential staining between left and right receptor neurons serves to identify the &#947;-glomerulus as the only structure innervated by contralateral presynaptic afferents. Secondly, the electroporation is combined with focal bolus loading in the olfactory bulb in order to stain mitral cells and their dendrites. The 3D brain volume is then scanned under line-illumination microscopy for the acquisition of fast calcium imaging data while small temperature drops are induced at the olfactory epithelium. Lastly, the post-acquisition analysis allows the morphological reconstruction of the thermosensitive network comprising the &#947;-glomerulus and its innervating mitral cells, based on specific temperature-induced Ca2+ traces. Using chemical odorants as stimuli in addition to temperature jumps enables the comparison between thermosensitive and chemosensitive networks in the olfactory bulb.

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Here we describe a protocol for measuring and analyzing temperature responses in the olfactory bulb of Xenopus laevis. Olfactory receptor neurons and mitral cells are differentially stained, after which calcium changes are recorded, reflecting a sensitivity of some neural networks in the bulb to temperature drops induced at the nose.

通过的嗅球钙监控录像的变化温度引起的神经元电活动<em&gt;非洲爪蟾</em

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. ...

Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory Sensory Neuron (OSN) is a bipolar cell with a single dendrite and a single unbranched axon. An OSN expresses only one Olfactory Receptor (OR) gene, OSNs expressing a given type of OR converge their axons to a few sets of invariant glomeruli in the Olfactory Bulb (OB). A remarkable feature of OSN projection is that the expressed ORs play instructive roles in axonal projection. ORs regulate the expression of multiple axon-sorting molecules and generate the combinatorial molecular code of axon-sorting molecules at the OSN axon termini. Thus, to understand the molecular mechanisms of OR-specific axon guidance mechanisms, it is vital to characterize their expression profiles at the OSN axon termini within the same glomerulus. The aim of this article was to introduce methods for collecting as many glomeruli as possible on a single OB section and for performing immunostaining using multiple antibodies. This would allow the comparison and analysis of the expression patterns of axon-sorting molecules without staining variation between OB sections.

Olfactory sensory neurons express a wide variety of axon-sorting molecules to establish proper neural circuitry. This protocol describes an immunohistochemical staining method to visualize combinatorial expressions of axon-sorting molecules at the axon termini of olfactory sensory neurons.

嗅球的四次免疫染色用于嗅觉感觉轴突分子识别码的可视化

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory ...