我们感谢来自俄克拉荷马大学（BZ）和美国国家科学基金会赠款（IOS - 1025556，BZ和RAB）的一个内部资金，用于支持成绩单，RAB的，XP中，氧化铝，MLW，中科院，而这项研究。美国国立卫生研究院资助（RO1 NS060878，以BZ）部分支持的RAB。 RAB的承认为支持这项研究的初期阶段的美国国立卫生研究院（T32 NS07292）印度国家遥感局授予布兰代斯大学的培训资助，以及美国国立卫生研究院资助（RO1 GM21473）授予杰弗里馆，布兰代斯。我们感谢他为这个项目的持续鼓励杰弗里厅。我们感谢为我们提供与浴缸P&gt; Gal80&gt;应变克里斯汀斯科特博士。

1。 果蝇株 <ol><li> Gal4的驱动程序： GMR - GAL4，这表示在Gal4的感光细胞。 </li><li>无人机线 无人机MJD78Q（或UAS - polyQ），从而导致神经变性Gal4的表达细胞。 UAS - GFP，它表达GFP Gal4的表达细胞。 </li><li> FLP线 EY - FLP，这表示在光感受器和FLP的大脑区域的子集。 ET - FLPx2线，其中包含两个副本的FLP（FLP - IRES - FLP）的增强子陷阱线。我们已经生成〜1000，ET - FLP线（波姆等，2010），其表达模式有待特点。在这里，我们描述的幼虫和成人飞的中枢神经系统（CNS）的表达模式的选择，ET - FLPx2的表征。 </li><li> FLP记者 YW，肌动蛋白P&gt; CD2&gt; Gal4的; UAS - GFP（Pignoni Zipursky，1997年），该报告ET - FLPx2的表达模式，通过肌动蛋白 P&gt; Gal4的FLP -&gt; CD2介导的recombinational切除后的UAS - GFP。 </li><li>两个互补Gal80转换工具 浴缸P&gt; Gal80&gt;（Gordon与斯科特，2009），倒装的结构，组成表示在一个强大的微管蛋白启动子驱动的所有组织Gal80。 FLP -介导的重组后，&gt; Gal80翻转和Gal80表达被终止，只有在ET - FLPx2表达组织。 浴缸P &lt;停止&gt; Gal80（波姆等，2010），构造，不正常条件下表达Gal80翻转。 Gal80表达是由微管蛋白启动子仅在打开ET - FLPx2表达组织在FLP翻转“&gt;”停止“和在Gal80翻转。 </li><li>为测试中的复眼FLP活动的苍蝇 GMR - GAL4，UAS - polyQ;浴缸P &lt;停止&gt; Gal80（波姆等，2010），其中已变质和depigmented眼睛。在FLP表达的感光细胞，polyQ表达关闭以下Gal80倒装。 GMR - GAL4，UAS - polyQ;（波姆等，2010）浴缸P&gt; Gal80&gt;，其中有正常的眼睛。在FLP表达的感光细胞，polyQ表达打开以下Gal80倒装。 </li></ol> 2。试剂磷酸盐缓冲液（PBS，1X），pH值7.4。 4％多聚甲醛（PFA），在PBS稀释16％的煤灰，EM级（猫＃15710，电子显微镜学，哈特菲尔德，PA） PBT，1XPBS含0.1％TRITON - X 100（Sigma公司） 反Elav（单克隆抗体，9F8A9;发育研究杂交瘤细胞银行，美国爱荷华大学），污渍在果蝇中所有的神经元。 反回购（单克隆抗体8D12;发育研究杂交瘤细胞银行，美国爱荷华大学）的污渍，在果蝇的神经胶质细胞。 Alexa的594鸡和鼠二抗（Invitrogen公司） 3。测绘表达的增强子陷阱FLPx2线模式（*关键步骤） <ol><li>从YW收集处女女性，肌动蛋白P&gt; CD2&gt; Gal4的UAS - GFP的线和跨男性个人ET - FLPx2线。前者作为一个组织，ET - FLP表达GFP报告。 </li><li> *徘徊3 路龄F1幼虫解剖，保留的中枢神经系统（即脑和腹神经索，VNC）和体壁，固定在4％的煤灰为1小时，冰，用冰冷的PBS 3X其次与PBT的3倍洗涤。 解剖 <ol><li>删除徘徊3 路龄F1幼虫，它们放入一个干净的Sylgard菜（35毫米× 10毫米培养皿中充满了Sylgard 2克-混合根据生产指令） 。 <ol><li>取出食物残渣与画笔</li></ol></li><li>填写冰冷1XPBS置于冰上菜麻醉幼虫</li><li>幼虫口钩握住一双镊子和抢附近的延伸与另一钳对前气门和角质层剥离角质层对后部。中枢神经系统会附着到口，沿挂钩唾液腺，肠道，和成虫光盘。 </li><li>删除多余的组织和周围的中枢神经系统的成虫光盘。 </li><li>一个PBS填充以及耐热盘转移到中枢神经系统。 <ol><li>可以用一个硅的P200的枪头，以避免干燥的组织。 </li></ol></li><li> 200微升4％的煤灰冰1小时修复。 <ol><li>确保完全淹没在煤灰中的中枢神经系统。 <ol><li>如果中枢神经系统的花车，这通常表明，中枢神经系统不正确地从周围组织清理。 </li></ol></li></ol></li><li>与冰冷的PBS洗3X 3X清洗与PBT。 </li></ol></li><li> *解剖成人F1果蝇中枢神经系统。 <ol><li>麻醉与CO 2的苍蝇或放置在冰上10分钟的空药瓶内所载的苍蝇。 </li><li>放置成蝇到95％的乙醇填补冰玻璃盘30秒。 </li><li>转移到一个1XPBS填补玻璃盘上的冰成蝇。 <ol><li> 3X用冰冷的1X PBS清洗，以去除残留的乙醇</li></ol></li><li>放置到一个35mm Sylga飞路菜充满了冰冷的1X PBS </li><li>将腹面朝上，插入一个小虫子笔（minutiens引脚0.1毫米），通过腹部中途。 <ol><li>使用一双镊子保持基地的腿和另一双钳取出腿。 </li></ol></li><li>删除头角质层<ol><li>虽然抓住一双镊子的长鼻，放在眼睛下方和剥离角质层对飞头背中线另一回事。 </li><li>抓住另一只眼睛，消除对方的其余角质层。 </li><li>使用锋利的钳和/或削尖的钨针，取出气管周围组织的脑<ol><li>气管清洗不当，会导致自体荧光强，阻碍GFP的表达，并能使大脑浮在固定</li></ol></li></ol></li><li> VNC的夹层<ol><li>取出胸角质层<ol><li>胸部后开始，每方3 路 preepisternum钳（即抓基地的后腿）。 </li><li>轻轻分开的角质层远离腹侧中线线，重复，直到你到达基地的脖子。 </li><li>轻轻地传播胸角质层的两半，露出了VNC </li></ol></li><li>接下来，从胸部取出腹部。虽然使用一双镊子举行第二回合基地的胸部，用另一双钳抢到了第一腹sternite附近的腹部，并拉出腹部。 </li><li>若要从周围的胸组织的VNC。握住一双镊子，另一双钳轻柔撕离大多数胸胴体肱骨面积。 </li><li>在这一点上应该有一个连接周边的颈椎结缔组织（颈部）组织环。使用两个镊子撕除环。 </li><li>删除任何多余的胸椎周围组织的VNC </li></ol></li><li>大脑和VNC转移到9以及耐热菜含有冰1X PBS <ol><li>一个硅的P200的吸头可以用来避免干燥的组织</li></ol></li></ol></li><li> *固定在4％的煤灰1小时的成人中枢神经系统在冰上，用冰冷的PBS和与PBT的3X 3X。 </li><li>成人中枢神经系统准备或幼虫中枢神经系统和体壁孵育4 ° C过夜无论是神经元（反Elav，1:10）或神经胶质细胞（anti-Repo. 1:10）的抗体。清洗准备与PBT 5倍。 </li><li>育与成人或幼虫准备与Alexa的594对鼠二抗（1:100）鸡在室温下2小时，用PBS洗的准备。 </li><li>山幻灯片上的幼虫或成年中枢神经系统。为了维护中枢​​神经系统的的自然形状，作为一个小型桥的幻灯片使用一个O形塑料环（加固标签清除，艾利丹尼森，办公用品，布雷亚，CA）暂停盖玻片。 </li><li> GFP和Elav（或回购）的表达模式，荧光显微镜下检查的准备。 </li></ol> 4。在光感受器在镇压Gal4的两个互补Gal80转换工具的应用<ol><li>翻转通过浴缸Gal80 P&gt; Gal80&gt;（ 图5A）。 <ol><li>从GMR - GAL4 UAS - polyQ收集男性;浴缸P&gt; Gal80&gt;行。请注意，这些果蝇复眼的正常。 </li><li> EY - FLP与处女交配。 </li><li>收集F1的苍蝇和检查眼表型。 </li></ol></li><li>在通过浴缸翻转Gal80 P &lt;停止&gt; Gal80（ 图5B）。 <ol><li>收集GMR - GAL4 UAS - polyQ;浴缸P &lt;停止&gt; Gal80雄蝇和检查眼表型。注意的退化和这些果蝇的depigmented眼睛相比，野生型菌株（广州S） </li><li> EY - FLP与处女交配; </li><li>收集F1的苍蝇和检查眼表型。 </li></ol></li><li>选择ET - FLPx2表情纹，在感光细胞</li></ol>步骤4.1和4.2，可反复使用ET - FLPx2线，表达FLP在感光细胞的一个子集。另外，无论是GMR - GAL4 UAS - polyQ浴缸P&gt;停止&gt; Gal80苍蝇的GMR - GAL4 UAS - polyQ;浴缸P&gt; Gal80&gt;苍蝇可以越过个人ET - FLPx2线为线，以屏幕表达工党在感光细胞。有价值的，ET - FLPx2线是指那些仅表达FLP的一个子集或单一的感光细胞（ 图6） 。 5。代表性的成果一个特定的ET - FLPx2线的表达模式，可以很容易地检查和记录在F1幼虫或成蝇从YW，肌动蛋白P之间的交叉派生&gt; CD2&gt; Gal4的UAS - GFP的处女和ET - FLPx2男性（图1） 。通常情况下，三至五年重复的F1动物解剖和检查，以确定的FLP的表达模式（波姆等，2010）的可重复性。 <st荣&gt;图2显示了在ET - FLPx2幼虫和成人CNS线173A FLP表达模式。的FLP模式可以使用一个神经元或神经胶质的标记（图3＆4）进一步分析。 在图7所示的结果说明Gal80倒装的成效和翻转式系统交互联系的镇压Gal4的下列FLP -介导的重组。浴缸P &lt;停止&gt; Gal80构造不会出现有任何Gal80漏水的表达（统一的光感受器变性及色素脱失的），但它是100％，有效地翻转Gal80 eyFLP帮助压制GMR GAL4和眼睛恢复正常露面（ 图7A）。相反，浴缸P&gt; Gal80&gt;完全压制GMR - Gal4的前eyFLP引进。以下Gal80翻转，GMR - Gal4的驱动器的UAS - polyQ表达，导致变质和depigmented眼睛（图 7B） 。在这个例子中，我们用一个泛photorecetor FLP地图感光“电路”，但没有审查的行为的后果。类似的方法使用行为有关Gal4的线，我们映射CCAP - Gal4的电路的一部分调节成蝇翼扩张（波姆等，2010）。我们相信，FINGR方法将帮助我们了解行为的神经基础的宝贵。 <img src="/files/ftp_upload/2649/2649fig1.jpg" alt="图1"/> 图1。检查的FLP表达模式的ET - FLPx2线（蓝色椭圆形） 的表达模式的ET - FLPx2线可以穿过的ET - FLPx2男（左上）与肌动蛋白 P&gt; CD2&gt; Gal4的决定;无人机GFP的处女（右上）。无处不在的肌动蛋白P&gt;女在父母的Gal4的表达受到阻碍的首次登记税（灰色三角形）两侧的CD2的序列插入。在F1代中，工党将消费CD2的序列，允许肌动蛋白 P&gt; Gal4的驱动UAS - GFP的表达。 <img src="/files/ftp_upload/2649/2649fig2.jpg" alt="图2"/> 图2。 ET - FLPx2线的幼虫和成人的中枢神经系统的表达模式。这些图像描绘了从交配YW，肌动蛋白 P产生的后代解剖中枢神经系统（脑和腹神经索，VNC）&gt; CD2&gt; Gal4的UAS - GFP的家长ET - FLPx2 173A线。 FLP重组&gt; CD2&gt;卡带，激活肌动蛋白P&gt; GAL4表达（随之而来的UAS - GFP的表达） 。因此，GFP模式报告FLP表达细胞。 A组幼虫FLP表达模式， 而 B组线173A成人的表达模式。 <img src="/files/ftp_upload/2649/2649fig3.jpg" alt="图3"/> 图3。表征类型的细胞表达ET - FLPx2线＃688A flippase。一个，一个一个固定的F1 3 路龄幼虫从ET - FLPx2线＃688A和GFP Flippase报告线之间的交叉与反回购染色的复合形象。 屋宇署 ，高放大倍率（蔡司计划复消色差透镜63X石油）的观点从一个VNC后从3龄幼虫。 <img src="/files/ftp_upload/2649/2649fig4.jpg" alt="图4"/> 图4。类型的细胞内ET - FLPx2线的蘑菇体的表征＃150。交流，反回购染色固定F1 3龄幼虫的蘑菇体（63X油浸）一个高放大倍率的形象。 <img src="/files/ftp_upload/2649/2649fig5.jpg" alt="图5"/> 图5。插图Gal80翻转和翻转式的策略 。 答 ：在翻转式的策略，微管蛋白启动子驱动Gal80（浴缸P&gt; Gal80&gt;）是组成性激活，从而导致在GMR - GAL4的镇压和野生型眼表型（红眼）。引进FLP（紫色），这将翻转Gal80出（中，红色），并允许GMR - Gal4的驱动器的UAS - polyQ表达这种镇压被删除。这种感光细胞变性的结果，B（眼睛中的白点所示）。在以前的战略互惠中，微管蛋白启动子驱动Gal80表达中断两侧的“一站式”密码子的首次登记税（灰色三角形）。在Gal80的情况下，GMR - GAL4 UAS - polyQ结合，导致光感受器的神经元退化（眼睛中的白点所示）。跨越的GMR - GAL4 UAS - polyQ;浴缸P&gt;停止&gt; GAL80处女雌表达FLP的感光细胞，FLP（紫色）的男性，将促进在汽车首次登记税网站的重组，归纳的“停止”密码子（中，红色），从而使Gal80表达和GMR - Gal4的镇压。这个储备在感光细胞的神经退行性疾病（由缺乏的W所示海特点在眼）。 <img src="/files/ftp_upload/2649/2649fig6.jpg" alt="图6"/> 图6。在翻转出使用ET - FLPx2线＃688A Gal80 FINGR方法的应用程序从一个ET - FLPx2线之间的交叉＃688A处女GMR - GAL4 UAS - polyQ F1 子代 ;。tubP&gt; GAL80男性。眼小眼的中间缺乏色素的代表FLP表达＃688A线。在这方面Gal80 GMR - GAL4抑制被解除，使Gal4的驱动UAS - polyQ。 <img src="/files/ftp_upload/2649/2649fig7.jpg" alt="图7"/> 图7。或向外翻转Gal80相交的光感受器Gal4的应用程序的FINGR方法。答 ： 浴缸P &lt;停止&gt; Gal80翻转系统限制在FLP依赖性GAL4表达。 GMR - GAL4 UAS - polyQ;浴缸P&gt;停止&gt; GAL80苍蝇，感光细胞退化和depigmented（左飞）。 Gal80 eyFLP引进，是在感光细胞表达，导致GMR - GAL4，这反过来又关闭的表达polyQ，并产生与正常眼睛的苍蝇（右飞 ） 镇压。浴缸P&gt; Gal80&gt;翻转相反的方式工作的制度浴缸P &lt;停止&gt; Gal80倒装系统。浴缸P&gt; Gal80&gt;组成压抑GAL4和抑制polyQ表达（左飞，与正常的眼睛） 。 Gal80 eyFLP引进，是翻转，从感光细胞，减轻抑制GMR - GAL4表达polyQ转向。这导致苍蝇与变质，depigmented眼睛（右飞）。

<ol>
	<li>Bohm, R. A., Welch, W. P., Goodnight, L. K., Cox, L. W., Henry, L. G., Gunter, T. C., Bao, H., Zhang, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetic+mosaic+approach+for+neural+circuit+mapping+in+Drosophila.">A genetic mosaic approach for neural circuit mapping in Drosophila.</a> Proc Natl Acad Sci U S A. 107, 16378-16383 (2010).</li><li>Brand, A. H., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes.">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118, 401-415 (1993).</li><li>Bruno, B., Holbro, N., Reichert, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polycomb+group+genes+are+required+for+neural+stem+cell+survival+in+postembryonic+neurogenesis+of+Drosophila.">Polycomb group genes are required for neural stem cell survival in postembryonic neurogenesis of Drosophila.</a> Development. 134, 1091-1099 (2007).</li><li>Duffy, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GAL4+system+in+Drosophila:+a+fly+geneticist's+Swiss+army+knife.">GAL4 system in Drosophila: a fly geneticist's Swiss army knife.</a> Genesis. , 34-341 (2002).</li><li>Gordon, M. D., Scott, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+control+in+a+Drosophila+taste+circuit.">Motor control in a Drosophila taste circuit.</a> Neuron. 61, 373-384 (2009).</li><li>Lee, T., 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=Mosaic+analysis+with+a+repressible+cell+marker+for+studies+of+gene+function+in+neuronal+morphogenesis.">Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis.</a> Neuron. 22, 451-461 (1999).</li><li>McGuire, S. E., Le, P. T., Osborn, A. J., Matsumoto, K., Davis, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+rescue+of+memory+dysfunction+in+Drosophila.">Spatiotemporal rescue of memory dysfunction in Drosophila.</a> Science. 302, 1765-1768 (2003).</li><li>Osterwalder, T., Yoon, K. S., White, B. H., Keshishian, 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+conditional+tissue+specific+transgene+expression+system+using+inducible+GAL4.">A conditional tissue specific transgene expression system using inducible GAL4.</a> Proc Natl Acad Sci USA. 98, 12596-12601 (2001).</li><li>Pignoni, F., Zipursky, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+Drosophila+eye+development+by+decapentaplegic.">Induction of Drosophila eye development by decapentaplegic.</a> Development. 124, 271-278 (1997).</li><li>Roman, G., Endo, K., Zong, L., Davis, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P[Switch],+a+system+for+spatial+and+temporal+control+of+gene+expression+in+Drosophila+melanogaster.">P[Switch], a system for spatial and temporal control of gene expression in Drosophila melanogaster.</a> Proc Natl Acad Sci U S A. 98, 12602-12607 (2001).</li><li>Xu, T., Rubin, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+genetic+mosaics+in+developing+and+adult+Drosophila+tissues.">Analysis of genetic mosaics in developing and adult Drosophila tissues.</a> Development. 117, 1223-1237 (1993).</li></ol>

Gal4/UAS系统已被广泛和成功地用于各种生物研究Drosophilists（Perrimon和品牌，1993年，达菲，2002年）。迄今为止，飞行社会产生了数以千计的启动子驱动和增强子陷阱Gal4的线。一个FINGR方法的主要优势在于，它是与Gal4/UAS系统兼容，从而有可能显着扩大在果蝇研究的Gal4的电源。 FINGR方法的一个重要的应用是微调，通过Gal4/Gal80交叉口的基本行为的神经回路。这些行为包括但不限于，学习和记忆，求偶，睡觉，和昼夜节律。 FINGR方法也可以用于地图，很容易在模拟人类神经退行性疾病如阿尔茨海默氏症，肌萎缩性脊髓侧索硬化症（ALS），和帕金森氏病神经退行性疾病的关键神经元。同样，神经细胞或神经回路调解可卡因，酒精和其他药物成瘾的关键灶可能映射使用FINGR方法。应该指出，有条件的Gal4s（奥斯特瓦德等人，2001年，罗马等，2001）可能是为了争取时间控制的神经回路和行为与FINGR方法。通过使用一个温度敏感的Gal80（Gal80 TS，麦奎尔等，2003）修改的Gal80转换工具，可以产生浴缸P&gt;停止&gt; Gal80 TS 或浴缸P&gt; Gal80 TS&gt;，加入进一步颞电路操纵的灵活性。除了神经系统，FINGR方法同样适用于限制Gal4的细胞亚群的表达，无论是左翼或腿。 ET - FLPx2线将首次登记税/ FLP基于克隆分析（徐鲁宾，1993年），包括MARCM（李洛，1999）感兴趣的所有果蝇生物学家的一个宝贵的资源。还预计一些ET - FLPx2行可能取代heatshock - FLP，生产重现性克隆和反复的形态和行为分析。增强子陷阱Gal4的线一样，大多数的ET - FLPx2线是不可能的，特别要表达的细胞在人的利益。大多数行会表达利益以及其他细胞或组织细胞和FLP。然而，缺乏“组织特异性GAL4和ET - FLPx2不会FINGR方法的一个主要关注，只要其重叠的区域是一个特定的实验需要。 FINGR的设计变成精致的两个“广义”的系统。 在这些协议中说明的方法，而标准和应重现。要求人的注意力主要警示步骤固定液的选择和使用。其他定影液，比煤灰，应避免，因为他们将淬火GFP信号。我们也注意到，解剖盘，应该不会有PFA固定使用，以避免工党表达不可靠绿色荧光蛋白报告的可能性。

mapping and application of enhancer-trap flippase expression in larval and adult drosophila cns

GAL4 / UAS二进制方法是强大的基因和果蝇的神经回路操纵。然而，对于大多数的神经生物学研究，Gal4的表达是很少的组织特异性，足以让行为读数电路精确相关。为了克服这一主要障碍，我们最近开发的FINGR方法，以达到更严格的Gal4的利益的组织中的表达。 FINGR方法有三个组成部分：1）传统Gal4/UAS系统; 2）FLP / FRT -介导Gal80转换工具; 3）增强子陷阱FLP（ET - FLP）。 GAL4是用来定义主利益的神经电路。果与Gal4的一个UAS的效应，如无人机MJD78Q或UAS石TS，调节神经元的活动，又表现在飞行行为的改变。 UAS - GFP，涉及具体行为的神经回路，如额外的无人机，记者可以同时映射形态分析。 Gal4的广泛表达线，Gal4的表达，可以限制使用两个互补Gal80转换工具： 浴缸P&gt; Gal80&gt;（“翻转”）和浴缸P &lt;停止&gt; Gal80（“翻转”） 。最后，调查人员可以把Gal80上或关闭，分别与具体组织ET - FLP的帮助。在翻盖模式，Gal80压制Gal4的表达无论GAL4和ET - FLP相交。在翻转式的模式，Gal80将缓解Gal4的镇压GAL4和FLP重叠细胞。这两种方法都使GAL4定义电路中的细胞数量的限制，但在一个反模式。 FINGR方法，是与广大Gal4的线在飞社区和高度传统的克隆分析和神经回路映射多功能集合兼容。在这个协议中，我们展示了FLP表达模式的选择，ET - FLPx2线中的感光细胞FINGR方法的有效性的映射。 FINGR方法的原则也应该适用于其他遗传模型Gal4/UAS，Gal80，FLP /首次登记税是用来生物。

Watch this Scientific Journal Video about Mapping and Application of Enhancer-trap Flippase Expression in Larval and Adult Drosophila CNS at JoVE.com

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

我们描述一个Flippase引起的交互联系Gal80/Gal4镇压（FINGR）方法，允许组织特异性FLP确定Gal80表达模式。无论GAL4和FLP重叠，Gal4的表达是开启（Gal80翻转出）或关闭（Gal80翻转）。 FINGR方法是多才多艺的克隆分析和神经回路映射。

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 ...

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Research

JoVE Journal

Neuroscience

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

16.4K 次观看.  University of Oklahoma - Norman. 我们描述一个Flippase引起的交互联系Gal80/Gal4镇压（FINGR）方法，允许组织特异性FLP确定Gal80表达模式。无论GAL4和FLP重叠，Gal4的表达是开启（Gal80翻转出）或关闭（Gal80翻转）。 FINGR方法是多才多艺的克隆分析和神经回路映射。

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

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of its convenient genetics and fully sequenced genome. Additionally, the larval nervous system is an ideal model system to study mechanisms of axonal transport as the larval segmental nerves contain bundles of axons with their cell bodies located within the brain and their nerve terminals ending along the length of the body. Here we describe the procedure for visualization of synaptic vesicle proteins within larval segmental nerves. If done correctly, all components of the nervous system, along with associated tissues such as muscles and NMJs, remain intact, undamaged, and ready to be visualized. 3rd instar larvae carrying various mutations are dissected, fixed, incubated with synaptic vesicle antibodies, visualized and compared to wild type larvae. This procedure can be adapted for several different synaptic or neuronal antibodies and changes in the distribution of a variety of proteins can be easily observed within larval segmental nerves.

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster larvae provide an ideal model system to investigate the mechanisms of axonal transport within larval segmental nerves. Using this procedure, 3rd instar larvae carrying various mutations can be compared to wild type larvae.

3幼虫节段性神经的可视化 RD龄果蝇幼虫的筹备工作

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of ...

科研

神经科学

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

In vivo electroporation is a powerful method for delivering DNA expression plasmids, RNAi reagents, and morpholino anti-sense oligonucleotides to specific regions of developing embryos, including those of C. elegans, chick, Xenopus, zebrafish, and mouse 1. In zebrafish, in vivo electroporation has been shown to have excellent spatial and temporal resolution for the delivery of these reagents 2-7. The temporal resolution of this method is important because it allows for incorporation of these reagents at specific stages in development. Furthermore, because expression from electroporated vectors occurs within 6 hours 7, this method is more timely than transgenic approaches. While the spatial resolution can be extremely precise when targeting a single cell 2, 6, it is often preferable to incorporate reagents into a specific cell population within a tissue or structure. When targeting multiple cells, in vivo electroporation is efficient for delivery to a specific region of the embryo; however, particularly within the developing nervous system, it is difficult to target specific cell types solely through spatially discrete electroporation. Alternatively, enhancer trap transgenic lines offer excellent cell type-specific expression of transgenes 8. Here we describe an approach that combines transgenic Gal4-based enhancer trap lines 8 with spatially discrete in vivo electroporation 7, 9 to specifically target developing neurons of the zebrafish olfactory bulb. The Et(zic4:Gal4TA4,UAS:mCherry)hzm5 (formerly GA80_9) enhancer trap line previously described 8, displays targeted transgenic expression of mCherry mediated by a zebrafish optimized Gal4 (KalTA4) transcriptional activator in multiple regions of the developing brain including hindbrain, cerebellum, forebrain, and the olfactory bulb. To target GFP expression specifically to the olfactory bulb, a plasmid with the coding sequence of GFP under control of multiple Gal4 binding sites (UAS) was electroporated into the anterior end of the forebrain at 24-28 hours post-fertilization (hpf). Although this method incorporates plasmid DNA into multiple regions of the forebrain, GFP expression is only induced in cells transgenically expressing the KalTA4 transcription factor. Thus, by using the GA080_9 transgenic line, this approach led to GFP expression exclusively in the developing olfactory bulb. GFP expressing cells targeted through this approach showed typical axonal projections, as previously described for mitral cells of the olfactory bulb 10. This method could also be used for targeted delivery of other reagents including short-hairpin RNA interference expression plasmids, which would provide a method for spatially and temporally discrete loss-of-function analysis.

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

The temporal and spatial resolution of genetic manipulations determines the spectrum of biological phenomena that they can perturb. Here we use temporally and spatially discrete in vivo electroporation, combined with transgenic lines of zebrafish, to induce expression of a GFP transgene specifically in neurons of the developing olfactory bulb.

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 ...

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including lens-secreting cone cells, pigment cells, a bristle cell and eight photoreceptors (PRs) R1-R8 2. The PRs have specialized microvillar structures, the rhabdomeres, which contain light-sensitive pigments, the Rhodopsins (Rhs). The rhabdomeres of six PRs (R1-R6) form a trapezoid and contain Rh1 3 4. The rhabdomeres of R7 and R8 are positioned in tandem in the center of the trapezoid and share the same path of light. R7 and R8 PRs stochastically express different combinations of Rhs in two main subtypes5: In the 'p' subtype, Rh3 in pR7s is coupled with Rh5 in pR8s, whereas in the 'y' subtype, Rh4 in yR7s is associated with Rh6 in yR8s 6 7 8.
Early specification of PRs and development of ommatidia begins in the larval eye-antennal imaginal disc, a monolayer of epithelial cells. A wave of differentiation sweeps across the disc9 and initiates the assembly of undifferentiated cells into ommatidia10-11. The 'founder cell' R8 is specified first and recruits R1-6 and then R7 12-14. Subsequently, during pupal development, PR differentiation leads to extensive morphological changes 15, including rhabdomere formation, synaptogenesis and eventually rh expression.
In this protocol, we describe methods for retinal dissections and immunohistochemistry at three defined periods of retina development, which can be applied to address a variety of questions concerning retinal formation and developmental pathways. Here, we use these methods to visualize the stepwise PR differentiation at the single-cell level in whole mount larval, midpupal and adult retinas (Figure 1).

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The Drosophila retina is a crystal-like lattice composed of a small number of cell types that are generated in a stereotyped manner 1. Its amenability to sophisticated genetic analysis allows the study of complex developmental programs. This protocol describes dissections and immunohistochemistry of retinas at three discrete developmental stages, with a focus on photoreceptor differentiation.

幼虫，蛹和成人的解剖和免疫组化<em&gt;果蝇</em&gt;视网膜

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including ...

Drosophila Adult Olfactory Shock Learning

Drosophila have been used in classical conditioning experiments for over 40 years, thus greatly facilitating our understanding of memory, including the elucidation of the molecular mechanisms involved in cognitive diseases1-7. Learning and memory can be assayed in larvae to study the effect of neurodevelopmental genes8-10 and in flies to measure the contribution of adult plasticity genes1-7. Furthermore, the short lifespan of Drosophila facilitates the analysis of genes mediating age-related memory impairment5,11-13. The availability of many inducible promoters that subdivide the Drosophila nervous system makes it possible to determine when and where a gene of interest is required for normal memory as well as relay of different aspects of the reinforcement signal3,4,14,16.
Studying memory in adult Drosophila allows for a detailed analysis of the behavior and circuitry involved and a measurement of long-term memory15-17. The length of the adult stage accommodates longer-term genetic, behavioral, dietary and pharmacological manipulations of memory, in addition to determining the effect of aging and neurodegenerative disease on memory3-6,11-13,15-21.
Classical conditioning is induced by the simultaneous presentation of a neutral odor cue (conditioned stimulus, CS+) and a reinforcement stimulus, e.g., an electric shock or sucrose, (unconditioned stimulus, US), that become associated with one another by the animal1,16. A second conditioned stimulus (CS-) is subsequently presented without the US. During the testing phase, Drosophila are simultaneously presented with CS+ and CS- odors. After the Drosophila are provided time to choose between the odors, the distribution of the animals is recorded. This procedure allows associative aversive or appetitive conditioning to be reliably measured without a bias introduced by the innate preference for either of the conditioned stimuli. Various control experiments are also performed to test whether all genotypes respond normally to odor and reinforcement alone.

Drosophila Adult Olfactory Shock Learning

The method to measure adult Drosophila associative memory is described. The assay is based on the ability of the fly to associate an odor presented with a negative reinforcer (electric shock) and then recall this information at a later time, allowing memory to be measured.

果蝇嗅觉成人学习的冲击

Drosophila have been used in classical conditioning experiments for over 40 years, thus greatly facilitating our understanding of memory, including the ...

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 ...

Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal models, including monkeys, ferrets, bats, rodents, and cats. In order to draw suitable parallels between human and animal models of auditory function, it is important to establish a bridge between human functional imaging studies and animal electrophysiological studies. Functional magnetic resonance imaging (fMRI) is an established, minimally invasive method of measuring broad patterns of hemodynamic activity across different regions of the cerebral cortex. This technique is widely used to probe sensory function in the human brain, is a useful tool in linking studies of auditory processing in both humans and animals and has been successfully used to investigate auditory function in monkeys and rodents. The following protocol describes an experimental procedure for investigating auditory function in anesthetized adult cats by measuring stimulus-evoked hemodynamic changes in auditory cortex using fMRI. This method facilitates comparison of the hemodynamic responses across different models of auditory function thus leading to a better understanding of species-independent features of the mammalian auditory cortex.

Functional studies of the auditory system in mammals have traditionally been conducted using spatially-focused techniques such as electrophysiological recordings. The following protocol describes a method of visualizing large-scale patterns of evoked hemodynamic activity in the cat auditory cortex using functional magnetic resonance imaging.

听觉皮层在成年猫用高场磁共振成像功能成像

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal ...

Functional Analysis of the Larval Feeding Circuit in Drosophila

The serotonergic feeding circuit in Drosophila melanogaster larvae can be used to investigate neuronal substrates of critical importance during the development of the circuit. Using the functional output of the circuit, feeding, changes in the neuronal architecture of the stomatogastric system can be visualized. Feeding behavior can be recorded by observing the rate of retraction of the mouth hooks, which receive innervation from the brain. Locomotor behavior is used as a physiological control for feeding, since larvae use their mouth hooks to traverse across an agar substrate. Changes in feeding behavior can be correlated with the axonal architecture of the neurites innervating the gut. Using immunohistochemistry it is possible to visualize and quantitate these changes. Improper handling of the larvae during behavior paradigms can alter data as they are very sensitive to manipulations. Proper imaging of the neurite architecture innervating the gut is critical for precise quantitation of number and size of varicosities as well as the extent of branch nodes. Analysis of most circuits allow only for visualization of neurite architecture or behavioral effects; however, this model allows one to correlate the functional output of the circuit with the impairments in neuronal architecture.

Functional Analysis of the Larval Feeding Circuit in Drosophila

The feeding circuit in Drosophila melanogaster larvae serves a simple yet powerful model that allows changes in feeding rate to be correlated with alterations in the stomatogastric neural circuitry. This circuit is composed of central serotonergic neurons that send projections to the mouth hooks as well as the foregut.

在幼虫馈电电路的功能分析果蝇</i

The serotonergic  feeding circuit in Drosophila  melanogaster larvae can be used to investigate neuronal substrates of  critical importance during the ...

Live Imaging of Drosophila Larval Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.

Live Imaging of Drosophila Larval Neuroblasts

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

的实时成像<em&gt;果蝇</em&gt;幼虫神经母细胞

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective

Drosophila melanogaster is a powerful experimental model system for studying the function of the nervous system. Gene mutations that cause dysfunction of the nervous system often produce viable larvae and adults that have locomotion defective phenotypes that are difficult to adequately describe with text or completely represent with a single photographic image. Current modes of scientific publishing, however, support the submission of digital video media as supplemental material to accompany a manuscript. Here we describe a simple and widely accessible microscopy technique for acquiring high-quality digital video of both Drosophila larval and adult phenotypes from a lateral perspective. Video of larval and adult locomotion from a side-view is advantageous because it allows the observation and analysis of subtle distinctions and variations in aberrant locomotive behaviors. We have successfully used the technique to visualize and quantify aberrant crawling behaviors in third instar larvae, in addition to adult mutant phenotypes and behaviors including grooming.

Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective

Here we describe a simple and widely accessible microscopy technique to acquire high-quality digital video of Drosophila adult and larval mutant phenotypes from a lateral perspective.

收购高品质的数字视频<em&gt;果蝇</em&gt;从横向角度看幼虫及成虫行为

Drosophila melanogaster is a powerful experimental model system for studying the function of the nervous system. Gene mutations that cause dysfunction of ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

用线性琼脂糖渠道研究<em&gt;果蝇</em&gt;幼虫爬行行为

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...