我们承认这种方法的发展，巴勃罗·绘制在图1的示意图使用固定块蓝图和丹妮拉Pelz Traversa，西尔克Sachse和Beate Eisermann。我们是感谢帕Ramdya西尔克Sachse手稿的意见和拉斐尔Rytz。 R.贝尔是由勃林格殷格翰基金会博士研究生奖学金的支持。由欧洲研究会，开始独立研究员格兰特和瑞士国家科学基金会资助在R.顿的实验室研究。

1。安装块组件<ol><li>安装的有机玻璃块（图1A）应作出的蓝图中给出确切的规格（可从 <a href="http://neuro.uni-konstanz.de/jove_mountingblock_blueprint/" target="_blank">http://neuro.uni-konstanz.de/j ove_mountingblock_blueprint /</a> ）允许易于安装和解剖苍蝇。主题通过从背面的螺丝，但不超出块前，他们的立场将在动物准备调整（见2.7）。 </li><li>使用精密播种机，在飞的圆形腔上缘的凹痕，挖下的表面，使苍蝇的身体的空间，减少块的厚度，在这一点上。 </li><li>用手术刀垂直切的铜网和接近底部的缝隙，创造一个“白领”。确保两个结果皮瓣水平，因为这将有助于飞插入。 </li><li>用牙签安装到上面飞的圆形腔块的强力胶，放置一个小水滴。将胶水和位置上的铜网，使狭缝中心块（图1A）。轻轻按下并删除任何多余的胶水。用的镊子，每侧皮瓣下添加小滴胶水，并在前面块，使狭缝指向垂直向下折叠的网格。确保电网的顶部是块级。允许完全干燥。 </li><li>组装线架：削减了一半的塑料盖玻片，删除中央的一块做一个“U”形。使用蜂蜡粘上一块导线跨越的“U”的顶端，不使线材过于绷紧。 </li><li>构建的触角盾：使孔用塑料纸孔，打孔盖玻片。修剪到0.5×0.7厘米的盖玻片的边缘。胶水的镂空的塑料盖玻片胶带，然后将通过孔接触到磁带上的乙醇下降删除的粘合剂。 </li></ol> 2。动物的制备<ol><li>相应的基因型的果蝇 - 即轴承所需的“司机”活动，记者转基因组合 - 应该是1-3周;角质层年轻的苍蝇是太软，难以削减。当实验需要，为了配合转基因表达水平直接比较不同基因型，苍蝇应该是年龄相匹配的，以避免与年龄有关的生理上的差异。如果性别并不重要，我们更倾向于使用雌蝇，因为他们有更大的元首和更容易操作。 </li><li>麻醉苍蝇，在冰上冷却。 </li><li>安装块引入一个单一的苍蝇。由右翼联合举行的苍蝇和滑动，使背，表面沿缝的铜网，它被暂停其颈部（图1B-C的）。如果有必要，旋转它，直到它低头。 </li><li>放置在前面的F罚款仙人掌脊柱ly的头，以防止它逃跑（图1B-C的）。放置在前面的长鼻仙人掌脊柱，以防止其移动。仙人掌脊椎修复使用蜂蜡块。确保头顶是块顶部的水平。 </li><li>解决苍蝇的头块与松香的小水滴（溶于乙醇）。块，放置在一个加湿盒，让约一小时的强化松香。 </li><li>使用镊子，将触角板和拖放（蜡端出来;见1.5）的持有人之间的天线和头部表皮褶皱线。固定支架与蜂蜡块前。 </li><li>使用成块建造的螺丝，轻轻向前推线架，创造一些触角板和头部之间的空间。 </li><li>在苍蝇的头顶部中央位置的孔，将触角盾（见1.6）。蜂蜡修复安装块顶部。 </li><li>使用刀片分离器，铜TA在磁带上的小孔上盾暴露背后的天线头苍蝇的角质层。孔不应超出眼睛的准备，以防止泄漏。 </li><li>双组分硅密封孔之间的磁带和角质层。从苍蝇的头顶部删除任何多余的硅。 </li><li>将一滴果蝇林格氏液的生理盐水（MgCl 2的 2毫米，130毫米氯化钠，氯化钾5毫米，2毫米氯化钙2，蔗糖36毫米，5毫米肝素钠，pH值7.3）的头。确保有没有泄漏：天线应保持完全干燥。使用蓝宝石刀片，切在角质层孔。首先，轻轻地沿着表皮褶皱后面的天线直接得分，然后切沿眼睛整个单眼的背面。最后，折叠打进边缘的一块角质层，从下部切，以避免切断触角神经。 </li><li>用细镊子小心取出腺体和气管。冲洗多次与果蝇 </em&gt;的林格氏液，头部留下了大降一些。 </li></ol> 3。刺激气味传递<ol><li>气味注射器：2毫升的塑料注射器插入到它的清洁钳和吸管（使用过滤嘴）20μL气味稀释后，在适当的溶剂所需，纤维素垫，使用枪头推到每桶垫深。插头和帽的注射器，直到需要。新鲜的气味稀释应准备至少每3个月（或更频繁的高挥发性和/或经常使用的气味），新垫应为每个录制会话之前的气味注射器准备。不要使用超过三刺激气味垫，因为气味浓度会衰减。 </li><li>定制嗅觉（图2）：主气流（1升/分钟）直接通过聚四氟乙烯管（内部直径0.4毫米）;上游，该管的出口，二次空气流（1升/分钟27厘米）的补充。两种气流所产生的泵膜，流量调节通过两个独立的流量计。空气流，可以通过在煤气洗涤瓶的水加湿，但应小心，以避免过度加湿（即超过〜60％）。二次空气流直接通过气味注射器或一个空的注射器：注射器相连的气流通过注射器针头刺入柱塞（外径1.2毫米）。三路的单位（如哈佛仪器VC6中）通过阀门控制器控制电磁阀用于切换之间的空气清新和刺激气味。 </li></ol> 4。 在体内成像<ol><li>我们在这里描述的成像使用一个堂堂正正的荧光显微镜（如直立固定舞台蔡司Axio上考官D1），配备20倍水的目标（例如蔡司W“的计划复消色差透镜”20X / 1,0 M27的迪爱生）（图2）。对于与G-CAMP（激发/发射：488/509纳米）成像，使用滤波器模块具有以下属性：450-490 nm激发过滤器分色镜（T495LP）和500-550 nm发射滤波器（色度等）。将安装块包含在显微镜下的飞。苍蝇的触角面前的嗅觉约0.5厘米的出口位置。 </li><li>降低的目标，直到它触及林格氏液对果蝇的头部的解决方案。通过观众看明场照明下，位置的准备，所以，在头壳孔为中心，其边界重点。切换到荧光灯和调整的重点，直到你可以看到标记的神经元（从基础的G-CAMP的绿色荧光明显）。 </li><li>收购了电荷耦合器件图像利益肾小球微调对焦（CCD）相机（如CoolSNAP HQ2数码相机系统）安装在显微镜的使用适当的采集软件（如Metafluor）（图3A）。关闭照明光路中的隔膜限制激发光成像领域。 </li><li>对于录制的气味诱发活动，采集速率为4赫兹设置和调整曝光时间获得您的CCD相机的动态范围内的荧光值，但不超过1000（任意单位）至少12位的动态分辨率较低。曝光时间不应超过100毫秒，如果可能的话，减​​少的G-CAMP漂白。如果你的相机的空间分辨率允许的话，你可以增加的分级（井数的芯片将进入一个数字像素分级），以减少曝光时间。我们取得的350x225像素（一个2x2的分级），准备在210x135微米的图像。这些设置是最佳捕捉肾小球气味诱导的G-CAMP反应，具有良好的空间和时间分辨率，同时限制记者漂白。 </li><li>设置测量参数。我们通常记录为10秒（即40帧），气味刺激通常开始收购期开始后的4秒（15帧）和持久的一秒钟（UNTIL 19帧）。一只苍蝇准备通常可以测试高达25-30不同气味的刺激，往往局限于记者，由于漂白的荧光信号不可逆转的下降。刺激间的时间间隔将取决于观察反应的强度和漂白率。可确定适当的刺激间的时间间隔，以避免感官适应长度约反复阳性对照气味（如果已知）中试实验的演示。列入这种气味可以让每〜10在一个给定的一系列刺激核查的持续经营能力和准备的一致响应。 </li></ol> 5。数据处理和分析数据处理使用ImageJ的（与Mac插件生物光子学， <a href="http://www.macbiophotonics.ca/">www.macbiophotonics.ca</a> ）和自定义写在Matlab和R方案如下： <ol><li>纠正抽样移动文物：将对应一个动物准备（如30测量，每40帧）使用ImageJ的） “刚体”StackReg / TurboReg模式（ <a href="http://bigwww.epfl.ch/thevenaz/stackreg">bigwww.epfl.ch / thevenaz / stackreg的</a>所有图像。这一步是必需的，以消除在收购框架内和之间的测量标神经元的位置的变化。然而，它是唯一可能消除在XY平面上的变化。如果强的重点发生变化的数据应丢弃。 </li><li>堆栈段成单个10秒（40帧）测量。 </li><li>漂白文物的正确曝光荧光灯会导致荧光衰减，在一个单一的测量10。在某些情况下，重要的是要纠正这种衰变分析数据之前。每个测量，计算出一个背景框架。这应该是几帧的平均发病前刺激（如帧6-14）。这backg除以每帧圆框获得相对荧光的变化。由G-CAMP标记的区域内所有像素平均计算每帧的平均相对荧光值。适合双指数的平均荧光曲线，给人更强的重量之前的刺激和权重为零的气味反应的时间（20帧，在我们的例子刺激后发病）的框架。减去从每个像素的相对荧光曲线的拟合曲线。乘以纠正的背景框架，重新获得原始数据帧。这种矫正，可能会导致在反应动力学的修改，如果信号不返回到基线测量结束（例如，如果抑制或非常强烈的兴奋反应诱发）（图4）。校正虽然漂白文物的是在许多情况下非常有用，时间参数的解释应谨慎，如果漂白校正。 </li><li>计算荧光的相对变化（德LTA，F / F）的每个帧每次测量（F的第I-0）/ F 0 * 100，其中F 0是背景框架（见5.3），和F 我的第i帧的荧光值测量。 </li><li>计算平均ΔF/ F在肾小球直径为10个像素，以获得活动的痕迹（图5A）（图3B）的兴趣的一个圆形区域内。如果需要，这些痕迹转化为热图使用ImageJ的（图5A）。 </li><li> ΔF/ F四个帧响应高峰期间的平均刺激前减去平均ΔF/ F四个帧，计算峰值振幅的响应。同样的程序使用说明空间响应模式（见图3B）和量化在跨苍蝇利益的特定区域响应的幅度（见图5B）。也可以量化的峰值响应，结合曲线下的面积，在时间的刺激，或averagin克帧左右的最大响应。 </li></ol> 6。代表结果我们使用了上述协议，以记录和分析丙酸诱发反应苍蝇嗅觉合作的的受体IR8A，这是活跃在几个不同的嗅觉神经元的人口29,30（表达启动子的控制下的G-CaMP1.6 28图3-5）。 1％丙酸的刺激引起的G-CAMP荧光的增加 - 这表明细胞内钙的增加 - 在嗅觉神经元支配两个肾小球，DC4〜DP1l（图3B）。我们代表从多个苍蝇在图5中的数据说明了不同的方式：行活动的痕迹，热图和酒吧情节的峰值响应。丙酸诱发反应有明显的峰值幅度在DC4〜和DP1l和时空动态。此外，虽然反应一般健壮，动物个体之间的变异性也可以被观察（看到他都肾小球图5A atmaps）。量化的峰值响应（图5B），允许简单的统计比较不同肾小球和动物之间的不同气味。然而，气味诱发的钙反应可以有非均匀的时空动态，量化与一个单一的值，其规模将无视这种复杂性。 <img alt="图1" src="/files/ftp_upload/2976/2976fig1.jpg" /> 图1。安装块示意图（a）在（二）引进后飞，和（c）在近距离顶视图（改编）从31。 <img alt="图2" src="/files/ftp_upload/2976/2976fig2.jpg" /> 图2。实验设置的示意图。 <img alt="图3" src="/files/ftp_upload/2976/2976fig3.jpg" /> （一）2.8步后（左图）和原始赌注的荧光图像的飞行准备的顶视图图3。NNAL叶标记与G-CaMP1.6（基因型：IR8A子：GAL4的;无人机G-CaMP1.6）。 DC4〜和DP1l肾小球内可区分它们的形态，大小和位置，并在标有红色和蓝色，分别。 （二）彩色编码图像丙酸（1％V / V）的响应。图像对应的ΔF/ F响应峰值计算步骤5.6解释（参见图5A）。左侧colorscale表示％ΔF/ F值。黑色圆圈表示，用量化的反应（见图5）的利息区域的位置和大小。 <img alt="图4" src="/files/ftp_upload/2976/2976fig4.jpg" /> 图4。纠正漂白的荧光记者的信号。漂白矫正效果，在步骤5.3中所述的例子。没有重大变化响应的形状，在左侧面板上的样本数据，可以得到纠正。在右侧面板中的样本数据是严重影响漂白校正，可能因为信号下降和气味刺激终止后保持低于基线。 <img alt="图4" src="/files/ftp_upload/2976/2976fig5.jpg" /> 图5（a）顶：DC4〜肾小球（左）和DP1l（右）丙酸（1％V / V）的钙离子反应的时空动态。黑色痕迹表明在该地区的利益，跨越8个动物（见图3B）的平均ΔF/ F的中位数。灰色表面之间的分布在第一和第三四分表示的范围。演讲中位数和四分位值，观察到的反应比均值和标准差的变异和分布提供了更多的信息。绿色和洋红框显示帧平均计算如图3B所示，在图5B量化的峰值响应。底部：热图显示为单独的行中的所有动物个体的响应数据，ΔF/ F值再版esented由的colorscale（右一）。水平黑边显示的时间和持续时间的刺激。 （二）跨越8个DC4〜DP1l肾小球的动物到丙酸的中位数峰值响应。错误横道之间的第一和第三四分分布范围。

<ol>
	<li>Galizia, C. G., Rossler, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+olfactory+systems+in+insects:+anatomy+and+function.">Parallel olfactory systems in insects: anatomy and function.</a> Annu. Rev. Entomol. 55, 399-420 (2010).</li><li>Vosshall, L. B., Stocker, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Architecture+of+Smell+and+Taste+in+Drosophila.">Molecular Architecture of Smell and Taste in Drosophila.</a> Annu. Rev. Neurosci. 30, 505-533 (2007).</li><li>Wilson, R. I., Mainen, Z. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+events+in+olfactory+processing.">Early events in olfactory processing.</a> Annu. Rev. Neurosci. 29, 163-201 (2006).</li><li>Masse, N. Y., Turner, G. C., Jefferis, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+information+processing+in+Drosophila.">Olfactory information processing in Drosophila.</a> Curr. Biol. 19, 700-713 (2009).</li><li>Hansson, B. S., Anton, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+and+morphology+of+the+antennal+lobe:+new+developments.">Function and morphology of the antennal lobe: new developments.</a> Annu. Rev. Entomol. 45, 203-231 (2000).</li><li>Galizia, C. G., Joerges, J., Kuttner, A., Faber, T., Menzel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+semi-in-vivo+preparation+for+optical+recording+of+the+insect+brain.">A semi-in-vivo preparation for optical recording of the insect brain.</a> J. Neurosci. Methods. 76, 61-69 (1997).</li><li>Galizia, C. G., Menzel, R., Holldobler, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+odor-evoked+glomerular+activity+patterns+in+the+antennal+lobes+of+the+ant+Camponotus+rufipes.">Optical imaging of odor-evoked glomerular activity patterns in the antennal lobes of the ant Camponotus rufipes.</a> Naturwissenschaften. 86, 533-537 (1999).</li><li>Okada, K., Kanzaki, R., Kawachi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-speed+voltage-sensitive+dye+imaging+of+an+in+vivo+insect+brain.">High-speed voltage-sensitive dye imaging of an in vivo insect brain.</a> Neurosci. Lett. 209, 197-200 (1996).</li><li>Carlsson, M. A., Knusel, P., Verschure, P. F., Hansson, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatio-temporal+Ca2++dynamics+of+moth+olfactory+projection+neurones.">Spatio-temporal Ca2+ dynamics of moth olfactory projection neurones.</a> The European journal of neuroscience. 22, 647-657 (2005).</li><li>Galizia, C. G., Vetter, R. S., Christensen, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Insect Sensory Neuroscience (Frontiers in Neuroscience). 13, 349-392 (2005).</li><li>Nakai, J., Ohkura, M., Imoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high+signal-to-noise+Ca&#40;2+&#41;+probe+composed+of+a+single+green+fluorescent+protein.">A high signal-to-noise Ca&#40;2+&#41; probe composed of a single green fluorescent protein.</a> Nat. Biotechnol. 19, 137-141 (2001).</li><li>Tian, L. <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><li>Miyawaki, A., Griesbeck, O., Heim, R., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+and+quantitative+Ca2++measurements+using+improved+cameleons.">Dynamic and quantitative Ca2+ measurements using improved cameleons.</a> Proc. Natl. Acad. Sci. U.S.A. 96, 2135-2140 (1999).</li><li>Martin, J. R., Rogers, K. L., Chagneau, C., Brulet, P. <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+bioluminescence+imaging+of+Ca+signalling+in+the+brain+of+Drosophila.">In vivo bioluminescence imaging of Ca signalling in the brain of Drosophila.</a> PLoS One. 2, 275-275 (2007).</li><li>Murmu, M. S., Stinnakre, J., Martin, 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=Presynaptic+Ca2++stores+contribute+to+odor-induced+responses+in+Drosophila+olfactory+receptor+neurons.">Presynaptic Ca2+ stores contribute to odor-induced responses in Drosophila olfactory receptor neurons.</a> The Journal of Experimental Biology. 213, 4163-4173 (2010).</li><li>Yuste, R., Miller, R. B., Holthoff, K., Zhang, S., Miesenbock, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synapto-pHluorins:+chimeras+between+pH-sensitive+mutants+of+green+fluorescent+protein+and+synaptic+vesicle+membrane+proteins+as+reporters+of+neurotransmitter+release.">Synapto-pHluorins: chimeras between pH-sensitive mutants of green fluorescent protein and synaptic vesicle membrane proteins as reporters of neurotransmitter release.</a> Methods Enzymol. 327, 522-546 (2000).</li><li>Benton, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitivity+and+specificity+in+Drosophila+pheromone+perception.">Sensitivity and specificity in Drosophila pheromone perception.</a> Trends Neurosci. 30, 512-519 (2007).</li><li>Elliott, D. A., Brand, A. 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+GAL4+system+:+a+versatile+system+for+the+expression+of+genes.">The GAL4 system : a versatile system for the expression of genes.</a> Methods Mol. Biol. 420, 79-95 (2008).</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>Yagi, R., Mayer, F., Basler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Refined+LexA+transactivators+and+their+use+in+combination+with+the+Drosophila+Gal4+system.">Refined LexA transactivators and their use in combination with the Drosophila Gal4 system.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 16166-16171 (2010).</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>Wang, J. W., Wong, A. M., Flores, J., Vosshall, L. B., Axel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+calcium+imaging+reveals+an+odor-evoked+map+of+activity+in+the+fly+brain.">Two-photon calcium imaging reveals an odor-evoked map of activity in the fly brain.</a> Cell. 112, 271-282 (2003).</li><li>Fiala, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+expressed+cameleon+in+Drosophila+melanogaster+is+used+to+visualize+olfactory+information+in+projection+neurons.">Genetically expressed cameleon in Drosophila melanogaster is used to visualize olfactory information in projection neurons.</a> Curr. Biol. 12, 1877-1884 (2002).</li><li>Ng, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+of+olfactory+information+between+three+populations+of+neurons+in+the+antennal+lobe+of+the+fly.">Transmission of olfactory information between three populations of neurons in the antennal lobe of the fly.</a> Neuron. 36, 463-474 (2002).</li><li>Sachse, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-dependent+plasticity+in+an+olfactory+circuit.">Activity-dependent plasticity in an olfactory circuit.</a> Neuron. 56, 838-850 (2007).</li><li>Pelz, D., Roeske, T., Syed, Z., de Bruyne, M., Galizia, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+receptive+range+of+an+olfactory+receptor+in+vivo+&#40;Drosophila+melanogaster+Or22a&#41;.">The molecular receptive range of an olfactory receptor in vivo &#40;Drosophila melanogaster Or22a&#41;.</a> J. Neurobiol. 66, 1544-1563 (2006).</li><li>Silbering, A. F., Galizia, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+of+odor+mixtures+in+the+Drosophila+antennal+lobe+reveals+both+global+inhibition+and+glomerulus-specific+interactions.">Processing of odor mixtures in the Drosophila antennal lobe reveals both global inhibition and glomerulus-specific interactions.</a> J. Neurosci. 27, 11966-11977 (2007).</li><li>Reiff, D. F. <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+performance+of+genetically+encoded+indicators+of+neural+activity+in+flies.">In vivo performance of genetically encoded indicators of neural activity in flies.</a> J. Neurosci. 25, 4766-4778 (2005).</li><li>Abuin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+architecture+of+olfactory+ionotropic+glutamate+receptors.">Functional architecture of olfactory ionotropic glutamate receptors.</a> Neuron. 69, 44-60 (2011).</li><li>Benton, R., Vannice, K. S., Gomez-Diaz, C., Vosshall, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variant+ionotropic+glutamate+receptors+as+chemosensory+receptors+in+Drosophila.">Variant ionotropic glutamate receptors as chemosensory receptors in Drosophila.</a> Cell. 136, 149-162 (2009).</li><li>Pelz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Functional characterization of Drosophila melanogaster Olfactory Receptor Neurons. , (2005).</li><li>Ai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acid+sensing+by+the+Drosophila+olfactory+system.">Acid sensing by the Drosophila olfactory system.</a> Nature. 468, 691-695 (2010).</li><li>Martin, 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=In+Vivo+Brain+Imaging:+Fluorescence+or+Bioluminescence,+Which+to+Choose?.">In Vivo Brain Imaging: Fluorescence or Bioluminescence, Which to Choose?.</a> Journal of Neurogenetics. , 1-23 (2008).</li><li>Jayaraman, V., Laurent, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+a+genetically+encoded+optical+sensor+of+neural+activity+using+electrophysiology+in+intact+adult+fruit+flies.">Evaluating a genetically encoded optical sensor of neural activity using electrophysiology in intact adult fruit flies.</a> Front. Neural Circuits. 1, 3-3 (2007).</li><li>Pfeiffer, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tools+for+neuroanatomy+and+neurogenetics+in+Drosophila.">Tools for neuroanatomy and neurogenetics in Drosophila.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 9715-9720 (2008).</li></ol>

作者没有透露。

这里描述的制备和分析方法可以用来分析气味诱发反应，在任何一个相应的驱动程序转基因是在触角叶（嗅觉神经元，LN和PNS）的神经元的人口。虽然我们形容它的应用程序使用荧光显微镜，光学成像该制剂同样可以进行使用共聚焦或双光子显微镜22,32。事实上，这些后者文书缓解宽视场显微镜的光散射的问题，它可以减少图像的分辨率，特别是当大量神经元表达的数字记者。通常允许这种准备至少一个半小时的录音气味诱发反应，但这个时间可以显着或长或短，取决于记录的测量值的数量和长度，曝光时间用于每个帧和刺激间的时间间隔。此外，双光子激发可以减少漂白的荧光代表orter，以及细胞的光毒性，允许在更长的时间周期22,32测量。 无论选择显微镜，头壳内苍蝇的大脑运动是最经常出现的问题时，成像在完整动物。虽然运动校正可以纠正小问题（步骤5.1），它是通常最好只记录从高度稳定的筹备工作。降低移动文物的战略包括：（我）适当限制的仙人掌脊柱飞的长鼻;（二）固定修复与二十烷（事先要加强2.6）（二十烷可以被融化在他们的舞台，苍蝇的腿~~ 37°C，使用银线，以扩大和完善尖用于此目的的烙铁）;（三）刻痕用细镊子食管（可见光之间的触角神经）。 另外的G-CaMP1.6用在这里，提供一个优化版本的G-CAMP及其他钙指标的数量，不同的信号信噪比，基线荧光和12,28,33的时空动态。精确的传感器选择应考虑到所有这些属性，类型的神经元进行分析和生物正在解决的问题。电流传感器报告的钙变化的关联与动作电位的12,34，和他们进一步改善，相当不错，再加上越来越多的特定基因驱动线35将继续加强在完整的苍蝇的神经活动的光学成像的潜力。

<table border="1"><tbody><tr><td>试剂</td><td>供应商</td><td>目录编号</td><td>评论</td></tr><tr><td>仙人掌脊柱</td><td>花园中心</td><td></td><td> 〜7毫米长薄，不太灵活</td></tr><tr><td>松香</td><td>弦乐器店</td><td></td><td></td></tr><tr><td>两个组件的硅</td><td> WPI的</td><td> KWIK-SIL </td><td></td></tr><tr><td>定制的有机玻璃安装块</td><td>蓝图从<a href="http://neuro.uni-konstanz.de/jove_mountingblock_blueprint/" target="_blank">http://neuro.uni-konstanz.de/j ove_mountingblock_blueprint /</a> </td><td></td><td></td></tr><tr><td>铜网格</td><td>雅典娜网格</td><td> G220-5 </td><td>通常用于电子显微镜的样品。 </td></tr><tr><td>螺丝</td><td>五金店</TD&gt; <td></td><td>直径2毫米</td></tr><tr><td>塑料盖玻片</td><td>普莱诺-EM </td><td> L4193 </td><td> 22 x 22毫米</td></tr><tr><td>烙铁头</td><td>康拉德电子</td><td> 830283-62 </td><td></td></tr><tr><td>稳压电源</td><td>康拉德电子</td><td> 511407-62 </td><td></td></tr><tr><td>细铁丝</td><td> isabellenhütte </td><td> ISA欧姆</td><td> 0.013毫米</td></tr><tr><td>二十烷</td><td>西格玛</td><td> 21,927-4 </td><td></td></tr><tr><td>显微镜</td><td>蔡司</td><td> axioexaminer D1的</td><td></td></tr><tr><td> CCD相机</td><td> visitron </td><td> CoolSnap总部</td><td></td></tr><tr><td> metafluor </td><td> visitron </td><td></td><td>采集软件</td></tr><tr><td>单色</td><td> visitron </td><td> visichrome </td&gt; <td></td></tr><tr><td>易碎的刀片</td><td>罚款科学工具</td><td> 10050-00 </td><td></td></tr><tr><td>持有的刀片</td><td> WPI的</td><td> 14134 </td><td></td></tr><tr><td>蓝宝石刀片</td><td> WPI的</td><td> 500314 </td><td>双刃刀片1毫米</td></tr><tr><td>膜气体泵</td><td> KNF纽伯格公司</td><td> NMP的830 Kaposi水痘样疹</td><td></td></tr><tr><td>蓝宝石刀架</td><td> WPI的</td><td> 500317 </td><td></td></tr><tr><td>三通阀</td><td>李公司</td><td> LFAA1200118H </td><td>配置é </td></tr><tr><td>流量计500毫升/分钟</td><td> analyt-MTC的</td><td> 112-08SA </td><td></td></tr><tr><td>流量计5升/分钟</td><td> analyt-MTC的</td><td> 102-08SA </td><td></td></tr><tr><td>蜂蜡</td><td> siladent技术公司</td><td> 209212 </td><td>; </td></tr><tr><td>纤维素垫</td><td> kettenbach医学</td><td> 31003 </td><td></td></tr><tr><td>镊子</td><td>普莱诺-EM </td><td> T5130 </td><td>杜蒙生物学＃5 </td></tr><tr><td>注射器</td><td>屋宇署</td><td> 300928 </td><td> 2毫升，Discardit二，2块</td></tr><tr><td>针</td><td>布劳恩Sterican </td><td> 4665120 </td><td> 1.2毫米外径</td></tr></tbody></table>

calcium imaging of odor-evoked responses in the drosophila antennal lobe

触角叶是主要在昆虫的大脑嗅觉中心，并表示脊椎动物嗅球1-5的解剖和功能上等同于。在外部世界的嗅觉信息传送到触角叶嗅感觉神经元（嗅觉神经元），其中分隔称为肾小球的神经纤维的不同地区，根据具体的嗅觉受体，他们表达。在这里，OSN的轴突突触与本地的interneurons（LNS），其过程可支配许多不同的肾小球和投射神经元（PNS），传达出较高的嗅觉脑区的嗅觉信息。 光学成像的嗅觉神经元，LNS和PNS在触角叶的活动 - 传统使用合成钙指标（如钙绿色，FURA-2）或电压敏感染料（如RH414） - 一直是重要的技术，了解如何嗅觉刺激表示为空间和时间模式在许多种昆虫6-10肾小球活动。发展基因编码的神经活动，如荧光钙指标的G-CAMP 11,12和13卡默莱昂，发光钙指标GFP的水母14,15，或突触传递的记者，记者，突触pHluorin 16取得了嗅觉系统的果蝇， 果蝇 ，特别是神经生理学成像，配合其全面描述分子，电生理及神经解剖性质2,4,17。这些记者可以通过二进制转录调控系统的神经元的特定人群（例如，Q系统21 18 GAL4/UAS，LexA蛋白/ LexAop 19,20）内的嗅觉电路剖析如何气味诱发的高时空分辨率的选择性表达神经活动的代表，调制和转换22-24 &lt;/ SUP&gt;。 这里，我们描述的编制和分析方法来衡量使用的G-CAMP 25日至27日在果蝇触角叶的气味诱发反应。动物准备是微创和可适应采用宽视场荧光，共聚焦和双光子显微镜的成像。

Watch this Scientific Journal Video about Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe at JoVE.com

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

我们描述了一个既定的测量和分析技术在触角叶的生活气味诱发的钙反应<em&gt;果蝇</em&gt;。

在气味诱发反应的钙成像果蝇触角叶

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory ...

calcium-imaging-odor-evoked-responses-drosophila-antennal

Research

JoVE Journal

Neuroscience

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

25.8K 次观看.  University of Lausanne. 我们描述了一个既定的测量和分析技术在触角叶的生活气味诱发的钙反应果蝇。

视频: Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

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. ...

科研

神经科学

Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within the same species 1,2. These intraspecies signals, the pheromones, as well as signals from some predators 3, activate the vomeronasal sensory neurons (VSNs) with high levels of specificity and sensitivity 4. At least three distinct families of G-protein coupled receptors, V1R, V2R and FPR 5-14, are expressed in VNO neurons to mediate the detection of the chemosensory cues. To understand how pheromone information is encoded by the VNO, it is critical to analyze the response profiles of individual VSNs to various stimuli and identify the specific receptors that mediate these responses.
The neuroepithelia of VNO are enclosed in a pair of vomer bones. The semi-blind tubular structure of VNO has one open end (the vomeronasal duct) connecting to the nasal cavity. VSNs extend their dendrites to the lumen part of the VNO, where the pheromone cues are in contact with the receptors expressed at the dendritic knobs. The cell bodies of the VSNs form pseudo-stratified layers with V1R and V2R expressed in the apical and basal layers respectively 6-8. Several techniques have been utilized to monitor responses of VSNs to sensory stimuli 4,12,15-19. Among these techniques, acute slice preparation offers several advantages. First, compared to dissociated VSNs 3,17, slice preparations maintain the neurons in their native morphology and the dendrites of the cells stay relatively intact. Second, the cell bodies of the VSNs are easily accessible in coronal slice of the VNO to allow electrophysiology studies and imaging experiments as compared to whole epithelium and whole-mount preparations 12,20. Third, this method can be combined with molecular cloning techniques to allow receptor identification.
Sensory stimulation elicits strong Ca2+ influx in VSNs that is indicative of receptor activation 4,21. We thus develop transgenic mice that express G-CaMP2 in the olfactory sensory neurons, including the VSNs 15,22. The sensitivity and the genetic nature of the probe greatly facilitate Ca2+ imaging experiments. This method has eliminated the dye loading process used in previous studies 4,21. We also employ a ligand delivery system that enables application of various stimuli to the VNO slices. The combination of the two techniques allows us to monitor multiple neurons simultaneously in response to large numbers of stimuli. Finally, we have established a semi-automated analysis pipeline to assist image processing.

The vomeronasal organ (VNO) detects intraspecies chemical signals that convey social and reproductive information. We have performed Ca2+ imaging experiments using transgenic mice expressing G-CaMP2 in VNO tissue. This approach allows us to analyze the complicated response patterns of the vomeronasal neurons to large numbers of pheromone stimuli.

在犁鼻器官的切片准备工作表示一个基因编码的钙传感器成像神经元的反应

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within ...

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the precise molecular steps as well as the activity of specific neurons in multi-functional nuclei of brain areas such as the hypothalamus remain. This problem includes identification of the molecular components involved in the regulation of various neurohormone signal transduction cascades. Elevations of intracellular Ca2+ play an important role in regulating the sensitivity of neurons, both at the level of signal transduction and at synaptic sites.
New tools have emerged to help identify neurons in the myriad of brain neurons by expressing green fluorescent protein (GFP) under the control of a particular promoter. To monitor both spatially and temporally stimulus-induced Ca2+ responses in GFP-tagged neurons, a non-green fluorescent Ca2+ indicator dye needs to be used. In addition, confocal microscopy is a favorite method of imaging individual neurons in tissue slices due to its ability to visualize neurons in distinct planes of depth within the tissue and to limit out-of-focus fluorescence. The ratiometric Ca2+ indicator fura-2 has been used in combination with GFP-tagged neurons1. However, the dye is excited by ultraviolet (UV) light. The cost of the laser and the limited optical penetration depth of UV light hindered its use in many laboratories. Moreover, GFP fluorescence may interfere with the fura-2 signals2. Therefore, we decided to use a red fluorescent Ca2+ indicator dye. The huge Stokes shift of fura-red permits multicolor analysis of the red fluorescence in combination with GFP using a single excitation wavelength. We had previously good results using fura-red in combination with GFP-tagged olfactory neurons3. The protocols for olfactory tissue slices seemed to work equally well in hypothalamic neurons4. Fura-red based Ca2+ imaging was also successfully combined with GFP-tagged pancreatic &beta;-cells and GFP-tagged receptors expressed in HEK cells5,6. A little quirk of fura-red is that its fluorescence intensity at 650 nm decreases once the indicator binds calcium7. Therefore, the fluorescence of resting neurons with low Ca2+ concentration has relatively high intensity. It should be noted, that other red Ca2+-indicator dyes exist or are currently being developed, that might give better or improved results in different neurons and brain areas.

In this protocol, we update recent progress in imaging Ca2+ signals of GFP-tagged neurons in brain tissue slices using a red fluorescent Ca2+ indicator dye.

在GFP标记的下丘脑神经元的小鼠脑片成像钙反应

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings (GCMR) in the Insect Antennal Lobe

All organisms inhabit a world full of sensory stimuli that determine their behavioral and physiological response to their environment. Olfaction is especially important in insects, which use their olfactory systems to respond to, and discriminate amongst, complex odor stimuli. These odors elicit behaviors that mediate processes such as reproduction and habitat selection1-3. Additionally, chemical sensing by insects mediates behaviors that are highly significant for agriculture and human health, including pollination4-6, herbivory of food crops7, and transmission of disease8,9. Identification of olfactory signals and their role in insect behavior is thus important for understanding both ecological processes and human food resources and well-being.
	To date, the identification of volatiles that drive insect behavior has been difficult and often tedious. Current techniques include gas chromatography-coupled electroantennogram recording (GC-EAG), and gas chromatography-coupled single sensillum recordings (GC-SSR)10-12. These techniques proved to be vital in the identification of bioactive compounds. We have developed a method that uses gas chromatography coupled to multi-channel electrophysiological recordings (termed 'GCMR') from neurons in the antennal lobe (AL; the insect's primary olfactory center)13,14. This state-of-the-art technique allows us to probe how odor information is represented in the insect brain. Moreover, because neural responses to odors at this level of olfactory processing are highly sensitive owing to the degree of convergence of the antenna's receptor neurons into AL neurons, AL recordings will allow the detection of active constituents of natural odors efficiently and with high sensitivity. Here we describe GCMR and give an example of its use.
	Several general steps are involved in the detection of bioactive volatiles and insect response. Volatiles first need to be collected from sources of interest (in this example we use flowers from the genus Mimulus (Phyrmaceae)) and characterized as needed using standard GC-MS techniques14-16. Insects are prepared for study using minimal dissection, after which a recording electrode is inserted into the antennal lobe and multi-channel neural recording begins. Post-processing of the neural data then reveals which particular odorants cause significant neural responses by the insect nervous system.
	Although the example we present here is specific to pollination studies, GCMR can be expanded to a wide range of study organisms and volatile sources. For instance, this method can be used in the identification of odorants attracting or repelling vector insects and crop pests. Moreover, GCMR can also be used to identify attractants for beneficial insects, such as pollinators. The technique may be expanded to non-insect subjects as well.

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings GCMR in the Insect Antennal Lobe

Olfactory cues mediate many different behaviors in insects, and are often complex mixtures comprised of tens to hundreds of volatile compounds. Using gas chromatography with multi-channel recording in the insect antennal lobe, we describe a method for the identification of bioactive compounds.

用气相色谱 - 多单元的录音（GCMR）昆虫触角叶嗅觉挥发物的鉴定

All  organisms inhabit a world full of sensory stimuli that determine their  behavioral and physiological response to their environment. Olfaction is ...

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model animal for endocrine research, Drosophila melanogaster, which has sophisticated genetic tools and genome information, is being increasingly used. This article describes a method for the calcium imaging of Drosophila brain explants. This method enables the detection of the direct signaling of a hormone to the brain. It is well known that many peptide hormones act through G-protein-coupled receptors (GPCRs), whose activation causes an increase in the intracellular Ca2+concentration. Neural activation also elevates intracellular Ca2+ levels, from both Ca2+ influx and the release of Ca2+ stored in the endoplasmic reticulum (ER). A calcium sensor, GCaMP, can monitor these Ca2+ changes. In this method, GCaMP is expressed in the neurons of interest, and the GCaMP-expressing larval brain is dissected and cultured ex vivo. The test peptide is then applied to the brain explant, and the fluorescent changes in GCaMP are detected using a spinning disc confocal microscope equipped with a CCD camera. Using this method, any water-soluble molecule can be tested, and various cellular events associated with neural activation can be imaged using the appropriate fluorescent indicators. Moreover, by modifying the imaging chamber, this method can be used to image other Drosophila organs or the organs of other animals.

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

This paper describes a protocol for ex vivo calcium imaging of the Drosophila brain. In this method, natural or synthetic compounds can be applied to the buffer to test their ability to activate particular neurons in the brain.

前体钙成像用于可视化大脑对果蝇内分泌信号的反应

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model ...

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. Learning-induced changes in synaptic transmission are often distributed across many neurons and levels of processing in the brain. Therefore, methods to visualize learning-dependent synaptic plasticity across neurons are needed. The fruit fly Drosophila melanogaster represents a particularly favorable model organism to study neuronal circuits underlying learning. The protocol presented here demonstrates a way in which the processes underlying the formation of associative olfactory memories, i.e., synaptic activity and their changes, can be monitored in vivo. Using the broad array of genetic tools available in Drosophila, it is possible to specifically express genetically encoded calcium indicators in determined cell populations and even single cells. By fixing a fly in place, and opening the head capsule, it is possible to visualize calcium dynamics in these cells whilst delivering olfactory stimuli. Additionally, we demonstrate a set-up in which the fly can be subjected, simultaneously, to electric shocks to the body. This provides a system in which flies can undergo classical olfactory conditioning - whereby a previously na&#239;ve odor is learned to be associated with electric shock punishment - at the same time as the representation of this odor (and other untrained odors) is observed in the brain via two-photon microscopy. Our lab has previously reported the generation of synaptically localized calcium sensors, which enables one to confine the fluorescent calcium signals to pre- or postsynaptic compartments. Two-photon microscopy provides a way to spatially resolve fine structures. We exemplify this by focusing on neurons integrating information from the mushroom body, a higher-order center of the insect brain. Overall, this protocol provides a method to examine the synaptic connections between neurons whose activity is modulated as a result of olfactory learning.

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Here we present a protocol with which pre- and/or postsynaptic calcium can be visualized in the context of Drosophila learning and memory. In vivo calcium imaging using synaptically localized calcium sensors is combined with a classical olfactory conditioning paradigm such that the synaptic plasticity underlying this type of associative learning may be determined.

在Vivo光学钙成像学习诱导突触可塑性在果蝇黑色素增生

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. ...

Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte function is not fully known. Pericytes have been suggested to be involved in capillary development, regulation of endothelial barrier tightness and trancytosis activity, regulation of capillary tone and to play crucial roles in certain brain pathologies.
Pericytes are challenging to investigate in the intact brain due to the difficulties in visualizing processes in the brain parenchyma, as well as the close proximity to the other cells of the NVU. The present protocol describes a method for isolation and culture of primary bovine brain capillary pericytes and their following usage in calcium imaging studies, where effects of agonists involved in brain signaling and pathologies can be investigated. Cortical capillary fragments are allowed to attach to the bottom of culture flasks and, after 6 days, endothelial cells and pericytes have grown out from the capillary fragments. The endothelial cells are removed by gentle trypsinization and pericytes are cultured for 5 additional days before passaging.
Isolated pericytes are seeded in 96-well culture plates and loaded with the calcium indicator dye (Fura-2 acetoxymethyl (AM)) to allow for measurements of intracellular calcium levels in a plate reader setup. Alternatively, pericytes are seeded on coverslips and mounted in cell chambers. Following loading with the calcium indicator (Cal-520 AM), calcium live-imaging can be performed using confocal microscopy at an excitation wavelength of 488 nm and emission wavelength of 510-520 nm.
The method described here has been used to obtain the first intracellular calcium measurements from primary brain capillary pericytes, demonstrating that pericytes are stimulated via ATP and are able to contract in vitro.

Brain capillary pericytes are essential players in the regulation of blood-brain barrier properties and blood flow. This protocol describes how brain capillary pericytes can be isolated, cultured, characterized with respect to cell type and applied for investigations of intracellular calcium signaling with fluorescent probes.

脑毛细管中培养细胞细胞钙测量和钙成像研究

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte ...

In vivo Calcium Imaging of Mouse Geniculate Ganglion Neuron Responses to Taste Stimuli

Within the last ten years, advances in genetically encoded calcium indicators (GECIs) have promoted a revolution in in vivo functional imaging. Using calcium as a proxy for neuronal activity, these techniques provide a way to monitor the responses of individual cells within large neuronal ensembles to a variety of stimuli in real time. We, and others, have applied these techniques to image the responses of individual geniculate ganglion neurons to taste stimuli applied to the tongues of live anesthetized mice. The geniculate ganglion is comprised of the cell bodies of gustatory neurons innervating the anterior tongue and palate as well as some somatosensory neurons innervating the pinna of the ear. Imaging the taste-evoked responses of individual geniculate ganglion neurons with GCaMP has provided important information about the tuning profiles of these neurons in wild-type mice as well as a way to detect peripheral taste miswiring phenotypes in genetically manipulated mice. Here we demonstrate the surgical procedure to expose the geniculate ganglion, GCaMP fluorescence image acquisition, initial steps for data analysis, and troubleshooting. This technique can be used with transgenically encoded GCaMP, or with AAV-mediated GCaMP expression, and can be modified to image particular genetic subsets of interest (i.e., Cre-mediated GCaMP expression). Overall, in vivo calcium imaging of geniculate ganglion neurons is a powerful technique for monitoring the activity of peripheral gustatory neurons and provides complementary information to more traditional whole-nerve chorda tympani recordings or taste behavior assays.

Here we present how to expose the geniculate ganglion of a live, anesthetized laboratory mouse and how to use calcium imaging to measure the responses of ensembles of these neurons to taste stimuli, allowing for multiple trials with different stimulants. This allows for in depth comparisons of which neurons respond to which tastants.

在活体钙成像小鼠致病性甘里昂神经元对味觉刺激的反应

Within the last ten years, advances in genetically encoded calcium indicators (GECIs) have promoted a revolution in in vivo functional imaging. Using ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

在体内 小鼠劣质橄榄中的钙成像

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

评估视网膜神经节细胞层中的钙活性

Calcium Imaging In Electrically Stimulated Flat-Mounted Retinas

Retinal dystrophies are a leading cause of blindness worldwide. Extensive efforts are underway to develop advanced retinal prostheses that can bypass the impaired light-sensing photoreceptor cells in the degenerated retina, aiming to partially restore vision by inducing visual percepts. One common avenue of investigation involves the design and production of implantable devices with a flexible physical structure, housing a high number of electrodes. This enables the efficient and precise generation of visual percepts. However, with each technological advancement, there arises a need for a reliable and manageable ex vivo method to verify the functionality of the device before progressing to in vivo experiments, where factors beyond the device's performance come into play. This article presents a comprehensive protocol for studying calcium activity in the retinal ganglion cell layer (GCL) following electrical stimulation. Specifically, the following steps are outlined: (1) fluorescently labeling the rat retina using genetically encoded calcium indicators, (2) capturing the fluorescence signal using an inverted fluorescence microscope while applying distinct patterns of electrical stimulation, and (3) extracting and analyzing the calcium traces from individual cells within the GCL. By following this procedure, researchers can efficiently test new stimulation protocols prior to conducting in vivo experiments.

作者聚焦：一种使用钙成像进行神经电刺激的创新方法 

Retinal prostheses have the ability to generate visual perceptions. To advance the development of new prostheses, ex vivo methods are needed to test devices before implantation. This article provides a comprehensive protocol for studying calcium activity in the retinal ganglion cell layer when subjected to electrical stimulation.

电刺激平装视网膜中的钙成像

Retinal dystrophies are a leading cause of blindness worldwide. Extensive efforts are underway to develop advanced retinal prostheses that can bypass the ...