We are indebted to E. Karplus, from Sciences Wares, USA, for his precious and useful help and advices. We thank E. Carbognin, A. Avet-Rochex, M. Murmu, P. Pavot, D. Minocci, G. Vinatier, and J. Stinnakre for their contribution to the development and improvement of this technique. We also thank B. Pfeiffer and G. Rubin, Janelia Farm, H.H.M.I., Ashburn, USA, for the pJFRC65-13XLexAop2-IVS-G5A-BP line. This work was supported by the Chateaubriand Fellowship, French Embassy, Washington, USA to A. Lark, by the National Science Foundation (IOS 1352882) to T. Kitamoto, and by the French ANRs (ANR-05-NEUR-009 Drosaequorin, 2005, and FlyBrainImaging, 2011), the Conseil R&#233;gional Ile-De France (NeRF and DIM), the IFR-144, the Physique-Chimie-Biologie Interface Program of the CNRS (2009), and by the CNRS, France to JR Martin.

1.样品的制备<ol><li>溶液的制备和设置<ol><li>保持在标准的食品中，在24℃的所有果蝇线。后与他们保持低密度小瓶生成标准化的尺寸和重量的苍蝇。 <ol><li>添加10处女女性，10名男性在小瓶中，让它们交配，每2天他们转移到一个新的小瓶。然后用10天后，当苍蝇开始E关闭，每天收获苍蝇。保持苍蝇的年龄的准确记录，并让他们在良好的状态。 </li><li>在第3天，分开女性的男性和保持女性20苍蝇每小瓶。记录苍蝇在4或5天之久。 </li></ol></li><li>制备林格氏溶液具有以下浓度:130毫摩尔NaCl，5mM的氯化钾，2mM的MgCl 2的，2mM的氯化钙 ，5mM的HEPES，和36毫蔗糖和调节的溶液，以精确地7.3的pH值。准备25μ灌注液，M尼古丁和100毫米氯化钾的林格氏液。 </li><li>准备1000微升的枪头:用刀片形状尖斜面（约35从尖到底°），除去过量的碱以外，从尖端1个半厘米。 </li><li>孵育期间准备的样品箱用于储存（23厘米×17厘米×8.5厘米）。放置两个海绵（12厘米×8厘米×3厘米）水饱和的底部[防止样品干燥]的小齿条装置下面放置样品它们的制备完成。 </li><li>制备苍蝇样品，使它们包含在UAS-GFP-水母发光蛋白中的至少1个拷贝和一个副本的Gal4线的驱动的GFP-水母发光蛋白的表达。 OK107 Gal4的线（线驱动，主要表现在蘑菇体）是越过UAS-GFP-水母制定工作。 注:盒子里面必须是防水，外面必须进入箱，以防止孵化过程中腔肠降解阻挡光线。 </li></ol></li><li>预习样品aration <ol><li>冰麻醉果蝇将其传输到玻璃小瓶中，并被移动到冷冻培养皿在枪头定位前保持在冰上2分钟。在100毫升的Petri解剖显微镜下冰盘准备在稍微潮湿的滤纸吸头。 </li><li>轻轻地用钳子的地方飞枪头内。 </li><li>用毛刷轻轻推和对准fly使磁头是完全past尖的边缘和背侧区域被部分期间tip整形（ 图2B）去部分露出。 </li><li>结合牙胶的两个组成部分的一个小的（大约3-4微升）的部分。仔细涂胶各地的正面和背面头部和颈部下至和周围的枪头边缘 - 避免头顶。让胶水干燥2分钟。 </li><li>地方枪头胶合贯通孔飞在录音室（图2D）和根TLY按到位，以固定。 </li><li>结合的硅胶（约3-4微升）的两个组成部分。上腔的平侧沿边缘所在的室和枪头，满足防止林格氏液泄漏应用胶水。让胶水干燥2分钟。 </li></ol></li><li>解剖<ol><li>贴上胶带在灌注通道，短边并延伸到腔室的后面。 </li><li>放置室上解剖块飞下显微镜接通荧光灯。吸取1毫升林格氏溶液进入室。 </li><li>用细手术刀去除角质层。建立从后脑勺到触角区域平行的切口。然后，沿着眼边切割然后做一个垂直的切口上方的天线连接以前的切口。使在头的后部切口平行于最终。使用精尖钳去除角质层（ 图2C）。 </li><li>使用精尖镊子轻轻抓住了ð清除暴露呼吸组织，直到大脑和[荧光]蘑菇体是清晰可见。 </li><li>使用超锋利的钳小心地把握和捏神经上皮组织覆盖大脑允许腔肠的渗透。 注:或者木瓜蛋白酶可用于透化神经上皮9。 </li><li>用移液管，洗出两次，用林格氏液以除去从解剖和地方样品任何碎片中的暗箱。 </li><li>吸取1毫升含有5μM苄腔肠进入室林格氏溶液。关闭对话框，并允许孵育腔肠素在室温下至少2小时的。 </li></ol></li></ol> 2.影像<ol><li>建立<ol><li>开始录制系统:打开显微镜，电脑，相机和排水系统，并设置室内环境控制到25℃。 </li><li>打开测量和计算机自动化管理​​器程序。点击 ＆＃8220;设备和接口"，然后双击"NI运动器械"，最后右击"PCI-7334"，然后选择"初始化设备"。 </li><li>打开光子成像。创建新的文件夹并命名第一个录音文件。相机必须达到-80℃之前，系统将发挥作用。 </li><li>设置灌注系统 - 添加氯化钾，尼古丁和林格氏液水库和调整，以便在各溶液放电以2毫升/分。与前开始实验林格氏液清洗。 </li></ol></li><li>准备样品<ol><li>将影像装入块盘上安装显微镜下，在放大5倍，并通过穿刺插入输液管进入通道。 </li><li>设置显微镜荧光模式，然后中心将蘑菇体（MBS）成为关注焦点。调整为20X和重新中心和重点。 </li><li>在污水池的位置排水装置，调整排水分流的高度，使针尖只是ABOVË池，并运行30秒林格氏液，以保证足够的排水。 </li><li>下拉盾由轻封锁设备。在光子成像器使用自动化系统进行细微调整的焦点，并采取的MB的参考荧光图像。 </li></ol></li><li>记录<ol><li>调整光子速度到期望的记录速度（从50毫秒到1秒或更多）。选择光子模式和开放式快门。记录基因型，性别，年龄，和样本数在日志条目。通过点击ROI箱，握住控件拖动调整位置的投资回报率在箱子花萼和细胞体（CCB）和中间叶。 </li><li>为约5至10分钟，记录基线（如需要）。 </li><li>申请尼古丁1分钟。注意:开始/停止记录。用5分钟林格氏液洗涤。 </li><li>等待5分钟，恢复和准备氯化钾日志条目和定时器。 </li><li>申请氯化钾1分钟。注意:开始/停止记录。用1分钟林格氏液清洗。 </li><li>开关荧光模式，采取最后的荧光图像，并关闭林格氏液。 </li><li>按"停止"。对话框将要求关闭系统。选择"否"继续录制。 </li><li>开盾，移动排水系统，切换镜头客观的5倍，去除灌注管。从舞台上删除采样/录音盘，通过漂洗和用水擦拭镜头的两倍擦去20X镜头。 </li><li>按白色播放按钮在程序窗口的顶部以开始下一个记录。 </li></ol></li></ol> 3.分析和视频创作<ol><li>提取光子值<ol><li>打开光子分析程序 （例如，光子浏览器）和开放的第一个样品的电子表格文件。 </li><li>选择显示控制选项卡。通过点击区域，同时保持控制选择的投资回报率。调整感兴趣的区域的大小，方向和形状以包含GFP照射CCB及内侧裂片。 </li><li>添加除人的投资回报率通过点击"定义的投资回报率"，并单击并拖动屏幕上创建新的投资回报率。 </li><li>选择"动画"选项卡播放光子的视频。根据需要调整"秒宽度"和"秒的步骤"。 ROI放置在必要时重新调整。 </li><li>选择绿色荧光，尼古丁响应和氯化钾反应的代表性屏幕截图。作物的截图，粘贴图像转化为以后的分析和比较的演示幻灯片。 </li><li>同时调整"秒宽度"和"秒步骤"来记录速度（秒）。通过移动灰色标记顶部面板上的支架区域的刺激开始前，只是反应结​​束后选择的分析长度。 </li><li>选择"暂停美景，一边玩"按"&lt;&lt;快退"，按"PLAY&gt;"。选择标签"计数率表"，并根据需要和线条的颜色相匹配的投资回报率的色彩调节装置。 </li><li>当分析完成按"出口计数率数据"。结合录音建行和每一方的利益内侧瓣区域选择屏幕截图。作物的截图，粘贴图像转换成具有响应图像幻灯片。这张幻灯片将在最后的分析中供以后使用。 </li></ol></li><li>实例分析:一种方法详见提取光子数据的分析<ol><li>在"计数速率出口"的文件夹，打开电子表格文件。 </li><li>重新格式化的单元格的第一标记的列的时间，以显示在小时和分钟的时间。 </li><li>参考样本对应的幻灯片。删除所有值除列的时间和两个有代表性的投资回报。 </li><li>确定尼古丁的刺激作用开始时间 - 在主日志记录文件中获得文件夹 - 删除启动或高亮值之前的附加数据。 </li><li>查找为建行和中间叶和标志/高亮最高/峰值。 </li><li>确定响应开始和结束时间为建行和中间叶从创建使用时间点估计相应的响应绘制幻灯片，并选择由变化值接近这些点的实际值。强调这些点之间的区域，无论是建行和中间叶。另外，使用宏9。 </li><li>合并一个实验组的所有样本在一个新的电子表格。 </li><li>通过确定行值的峰值对齐是每个CCB峰值。减去从最高行值较低的值来确定大概的行值，其中样本数据必须被重新复制到。验证所有峰值对齐。 </li><li>复制片两次，删除或者内侧叶列或建行的列创建仅建行或内侧叶值的页面。 </li><li>卸下所有建行的反应已经结束后的数据。创建四个行标总光子，响应长度（持续时间），峰值振幅和响应延迟。复制和再粘贴的原稿如下。 </li><li>使用SUM函数响应过程中确定总的光子。使用COUNT函数来确定响应的长度。复制链接和最高值单元格来确定振幅峰值。使用COUNT函数 - 从尼古丁到响应的起始处的灌注的开始选择 - 判断响应延迟。 </li><li>复制值，通过使用链接功能和乘法（除了峰值）通过记录速度以秒为单位。 </li><li>酒吧/框图可能为计算的参数，如总光子，峰值振幅和响应长度来创建，如果需要的话（例如: 图5B，C，D）。 </li><li>在原始数据光子反应后的柱中，对于使用平均函数每个时间点平均的原始数据点。如果与列确定平均（SEM）的标准误差所需的后续，对于每个在时间点平均光子值。 </li><li>使用平均柱来构造的平均响应PROFILE [图]为建行样本组。为此，选择该列中的时间指定为x轴的值，并且平均光子值作为y轴的列和最后选择的散点图图形创作工具。 </li><li>与来自内侧裂片的数据重复此分析过程。 </li></ol></li><li>创建图像的视频<ol><li>打开光子观众。 </li><li>选择显示控制选项卡。点击的投资回报率，同时保持控制选择，删除和/或最小化。 </li><li>修改"秒宽度"（累积时间），以根据需要确定每个帧的内容。 </li><li>修改"秒步骤"来定义每个帧的重叠。 </li><li>选择"导出美景，一边玩"按"&lt;&lt; REW"，然后"PLAY&gt;"。对于视频图像将被导出到"查看产品出口"的文件夹中的一系列.png文件。 </li></ol></li><li>使用虚拟配音，使视频:一种方法用于视频Çreation详细<ol><li>创建在包含图像的视图出口夹名为"resultats"新文件夹。 </li><li>把脚本（renommer.VBS）为图像的文件夹。 </li><li>双击renommer.VBS运行。 </li><li>图像将自动数字重命名，并复制到"resultats"文件夹中。 </li><li>打开VirtualDub.exe。 </li><li>选择开放式的视频文件 - 首先选择图像（随后的图像会自动加载）。 </li><li>选择视频 - 根据需要选择帧速率调整。 </li><li>选择视频 - 选择压缩选择的Cinepak编解码器通过半径。 </li><li>在文件选择保存为AVI视频会自动创建。 </li></ol></li></ol>

<ol>
	<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> J Neurogenet. 22 (3), 285-307 (2008).</li><li>Shimomura, O., Johnson, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peroxidized+coelenterazine,+the+active+group+in+the+photoprotein+aequorin.">Peroxidized coelenterazine, the active group in the photoprotein aequorin.</a> Proc Natl Acad Sci USA. 75 (6), 2611-2615 (1978).</li><li>Shimomura, O., Johnson, F. H., Saiga, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Further+data+on+the+bioluminescent+protein,+aequorin.">Further data on the bioluminescent protein, aequorin.</a> J. Cell. Comp. Physiol. 62 (1), 1-8 (1963).</li><li>Baubet, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+green+fluorescent+protein-aequorin+as+bioluminescent+Ca2++reporters+at+the+single-cell+level.">Chimeric green fluorescent protein-aequorin as bioluminescent Ca2+ reporters at the single-cell level.</a> Proc Natl Acad Sci USA. 97 (13), 7260-7265 (2000).</li><li>Cabrero, P., Richmond, L., Nitabach, M., Davies, S. A., Dow, J. A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+biogenic+amine+and+a+neuropeptide+act+identically:+tyramine+signals+through+calcium+in+Drosophila+tubule+stellate+cells.">A biogenic amine and a neuropeptide act identically: tyramine signals through calcium in Drosophila tubule stellate cells.</a> Proc. Biol. Sci. 280 (1757), 20122943 (2013).</li><li>Leclerc, C., Webb, S. E., Daguzan, C., Moreau, M., Miller, A. 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+patterns+of+calcium+transients+during+neural+induction+in+Xenopus+laevis+embryos.">Imaging patterns of calcium transients during neural induction in Xenopus laevis embryos.</a> J. Cell Sci. 113 (19), 3519-3529 (2000).</li><li>Martin, J. -. R., Rogers, K. L., Chagneau, C., Br&#251;let, 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+Ca2++signalling+in+the+brain+of+Drosophila.">In vivo bioluminescence imaging of Ca2+ signalling in the brain of Drosophila.</a> PLoS One. 2 (3), e275 (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> J. Exp. Biol. 213 (24), 4163-4173 (2010).</li><li>Murmu, M. S., Stinnakre, J., R&#233;al, E., 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=Calcium-stores+mediate+adaptation+in+axon+terminals+of+Olfactory+Receptor+Neurons+in+Drosophila.">Calcium-stores mediate adaptation in axon terminals of Olfactory Receptor Neurons in Drosophila.</a> BMC Neurosci. 12, 105 (2011).</li><li>Pavot, P., Carbognin, E., 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=PKA+and+cAMP/CNG+channels+independently+regulate+the+cholinergic+Ca2+-response+of+Drosophila+mushroom+body+neurons.">PKA and cAMP/CNG channels independently regulate the cholinergic Ca2+-response of Drosophila mushroom body neurons.</a> eNeuro. 2 (2), 0054-0068 (2015).</li><li>Minocci, D., Carbognin, E., Murmu, M. S., 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+functional+calcium+imaging+of+induced+or+spontaneous+activity+in+the+fly+brain+using+a+GFP-apoaequorin-based+bioluminescent+approach.">In vivo functional calcium imaging of induced or spontaneous activity in the fly brain using a GFP-apoaequorin-based bioluminescent approach.</a> BBA-Mol Cell Res. 1833 (7), 1632-1640 (2013).</li><li>Lima, S. Q., Miesenb&#246;ck, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remote+control+of+behavior+through+genetically+targeted+photostimulation+of+neurons.">Remote control of behavior through genetically targeted photostimulation of neurons.</a> Cell. 121 (1), 141-152 (2005).</li><li>Shimomura, O., Musicki, B., Kishi, Y., Inouye, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-emitting+properties+of+recombinant+semisynthetic+aequorins+and+recombinant+fluorescein-conjugated+aequorin+for+measuring+cellular+calcium.">Light-emitting properties of recombinant semisynthetic aequorins and recombinant fluorescein-conjugated aequorin for measuring cellular calcium.</a> Cell Calcium. 14 (5), 373-378 (1993).</li><li>Berridge, M. J., Bootman, M. D., Roderick, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signalling:+dynamics,+homeostasis+and+remodelling.">Calcium signalling: dynamics, homeostasis and remodelling.</a> Nat Rev Mol Cell Bio. 4 (7), 517-529 (2003).</li><li>Fiala, A., Spall, T. <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+calcium+imaging+of+brain+activity+in+Drosophila+by+transgenic+cameleon+expression.">In vivo calcium imaging of brain activity in Drosophila by transgenic cameleon expression.</a> Sci STKE. 2003 (174), PL6 (2003).</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 (2), 271-282 (2003).</li><li>Wilson, R. I., Turner, G. C., 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=Transformation+of+Olfactory+Representations+in+the+Drosophila+Antennal+Lobe.">Transformation of Olfactory Representations in the Drosophila Antennal Lobe.</a> Science. 303 (5656), 366-370 (2004).</li></ol>

The authors have nothing to disclose.

这里介绍的最近开发的生物发光型绿色荧光蛋白水母发光蛋白的方法允许在 钙 -activity在不同的神经元体内功能记录功能，如果需要的话，以及在其他类型的细胞，如肾星状细胞的报道中卡夫雷罗等。 2013年5。 修改后，故障排除，附加部件及关键步骤 果蝇畜牧 GFP-水母发光蛋白转基因（pG5A）4插入到pUAST矢量创建三个独立转化体线 6。所有行进行了测试的生物发光，并都能够向体内 的 Ca 2+ -activity 6响应。然而最近的实验中已经指出，插在第三染色体上的GA2线产生更强大的信号比GA1线插入Øn中的第二染色体。在这里，代表结果通过使用UAS-GA2越过了MB专用线OK107获得。此外，最近B.法伊弗已产生了GFP的水母发光蛋白构建体20XUAS调控元件（20X-G5A-2）的控制下放置并该构建已在我们的实验室得到验证。简单地说，它提供了非常强烈的信号，比我们的标准GA2，因此这应该是更有效和有用的，以进一步监控的 Ca 2+ -activity在脑的深层结构，如椭球体更强。此外，它也可以是用于成像小批量或神经元的轴突终端（突触联系）是有用的。 准备成像 虽然飞必须在定位和胶合被冰麻醉，它最大限度地减少了时间上的冰是重要的。蝇应该被移动到冷冻培养皿之前被转移到玻璃小瓶中，并保持在冰上2分钟在枪头定位。我们已经注意到，麻醉的较长的时间内降低了样品的生存力，并且可以衰减响应。理想情况下，我们保持冰麻醉时间低于5-10分钟。 编带和粘合的关键步骤。关键的是要确保飞行头代替解剖和成像以及防止林格氏溶液进入吸管尖和/或从记录室的泄漏。牙科胶（和在较小程度上的硅胶）将变得粘稠并随后开始只要两个部件被组合干燥。因此，这是关键的应用胶水迅速，以避免弱粘结和胶结块。在一些情况下，我们已经注意到，替代品种胶带和胶，当与林格氏液接触，可能释放出污染物质进入林格氏溶液可影响飞及其响应，特别是因此对于实验的专用于t他研究嗅觉系统中，使用不同的气味。因此，要小心这里如果替代材料（见材料，我们用具体品种）。 几种不同形式腔肠素的已开发，以优化其活性，如光发射的速度， 钙离子-affinity（灵敏度），或发光12的强度。例如，本机腔肠素是更加稳定，从而更合适的长期成像为几个小时，而苄基腔肠素（也称为H-腔肠素）导致更快和更高的光发射用更短的半衰期。有关不同形式的腔肠的其他信息，请参阅材料清单的网址。 成像 生物发光信号可与电子倍增CCD照相机进行监测（EM-CCD，冷却至-80℃）装配到显微镜。此设置必须bË容纳在一个紧密的暗箱，以避免任何不想要的（环境）光污染。在这个手稿中描述的设置使用20X浸没物镜允许的视图的400×400微米（512×512象素）的字段。为了改善信噪比，数据被收购与0.100秒的积分时间（10赫兹），和2×2像素合并使用（1个像素= 1.2×1.2微米）。以获取和存储数据，每个检测到的光子被分配的x，y坐标和一个时间点。 500和600纳米的波长之间，在EM-CCD照相机（见材料清单精确描述）具有96％的量子效率。 一种用于体内脑功能成像的最关键的参数之一是时间分辨率。截至目前，大多数基于荧光的钙 -imaging技术例如GCaMP和卡默莱昂的提供约4赫兹（250毫秒/帧）的时间分辨率。最近，随着基于生物发光的绿色荧光蛋白水母发光蛋白，我们已经能够RAISE中的采集时间长达100毫秒/帧与EMCCD相机和甚至高达120帧/秒（8.3毫秒/帧）与电子倍增ICDD 照相机 10。在这个时间分辨率接近于的电生理技术（在约1毫秒的范围内）。而高时间分辨率是既丰富和关键专用于突触传递，并刺激诱 ​​导活性的研究，推广速度慢而必要的钙离子-kinetics也发生在神经元内，例如从细胞内钙离子-stores的钙释放（综述:贝里奇等，2003年13，）。对于这后一种类型，一个较慢的时间分辨率还是可以接受的，而相反地，他们通常需要待检测来自传感器的较好的 Ca 2+ -affinity。这一要求已取得用GFP-水母发光蛋白，以检测细胞内Ca 2+ -stores的释放在嗅觉RECEPT的轴突终端ORS神经元7,8。可替代地，根据所需的实验条件下，速度较慢的采集时间也可以使用。例如，在长O / N的记录，我们已经使用了一个2秒的积分时间，由于大量的收集的数据点（Minocci 等人，2013年）。简言之，用生物发光一个实际特征是，时间分辨率可以容易地根据所需的实验条件调整。 替代室（蝇设置）已开发，以促进实验的目标。例如，对于基于天然的刺激气味的研究中，天线必须保持无，因此像一个由A.菲亚拉14开发的方法是最适合的（我们用于Murmu 等，2010类似的方法）。简要地说，在这种方法中，苍蝇在一个minutien销在颈部，其胶合到含小孔的塑料盖玻片系留（胶合）。创建一个密封周围飞散的头部的顶部。此后，一滴振铃的沉积在头上，头壳被解剖。以这样的方式，所述天线保持自由，并且可以检测的气味。类似地，对于长期记录，如O / N或甚至几天10飞需要的，以保持为自由的约束尽可能的，在某些情况下馈送（例如:用浸在5％的葡萄糖的毛细管纸解）。在这些情况下菲亚拉的做法也是比在蓝色吸管尖（潜水法）该方法更适合。 所有药物的应用程序可以通过外部的6路多阀重力灌注系统进行控制。流可以用为2毫升/分钟的流量每个记录会话之前，校准体积灌注调节器来控制。排水装置提取液以2ml / min，从录音室经由蠕动泵的速率，允许连续流动。 在某些条件下，由于药物和/或药物使用的量的昂贵成本，一个想仅应用于小批量化合物。因此，有必要在浴中，而不是通过灌注系统直接应用它。在这种情况下，快门必须在此过程中关闭，以避免光线污染。 分析方法 每秒计数或计数每秒每坐标都被用于我们的小组进行分析。也许是最适合的，而每秒计数整个结构明亮的响应每秒计数和每个坐标（代表响应对于投资回报率的大小正常化）是最适合和准确细胞的小种群。两者可以在分析软件容易地选择。 一种使用生物发光的方法的主要优点是，获取和存储的数据（X，Y，吨的参数的方式每次发射的光子）。实际上，虽然使用了标准模式作为获得完整的图像（通常以TIFF格式）的任何其它成像技术，在该系统中，我们通过存储仅在已激活的受光像素坐标获取只有相关和显著信号的时间预固定的持续时间（其对应于采集时间或时间分辨率）。因此，数据的存储是真正的"光"相比，存储一个完整图像，因此，可以更容易地分析数据。进行数据分析，我们使用了相关软件（光子查看器），根据需要，它允许的积累时间设定。然后信号的图像的显示可以根据需要在一个目标令人信服显示数据进行调整。与此同时结果量化相同的分辨率的时间比他们的采集时间。 意义和生物发光成像的生物应用 </strong&gt;的如上述生物发光钙成像提到提供到荧光钙成像技术三个主要优点:（1）脑的更深区域可以被成像，（2）成像可连续较长时间如几个小时的O / N和甚至在一些发生例长达两天，以及（3）最近，具有较高的时间分辨率，多达120帧/秒（8.3毫秒）。的生物发光方法的主要限制是由于它不符合共聚焦照明，这因而不允许我们确定激活结构或神经元的精确深度（z计划）。 生物发光成像，脑深部区域的成像的第一个优点，先前在2007年由椭球体（EB），位于大脑的中间深层结构，内高钾诱发钙反应之前没有的成像证实电或功能报告6 </suP&gt;。同样，最近的例子是自发的钙 -activity在EB的以下应用印防己毒素（中GABA A -受体阻断剂）的证明GABA能环神经元的EB确实是抑制性神经元的记录（描述:尿布等，规定提交）。 第二个好处，时间更长，连续时间记录，已开发从我们实验室的一些研究。刺激气味已被用来检查脑神经细胞的作用在以往的研究11,15,16。使用更长期的成像，但是，我们的实验室已能嗅觉受体神经元对气味中显示动力学以前未改变。第一项研究中通过Murmu 等人（2010）显示，虽然短的刺激气味（1-3秒）表现出类似的动力学和幅度适度差异，较长的刺激气味（5秒）造成更大的幅度，以及一个改变的动力学/结构茜这归因于细胞内钙库的响应。随后的研究显示，虽然对1秒气味脉冲每隔5分钟连续5应用没有改变它们的钙反应，增加至5秒导致了响应的渐进衰减刺激的长度，这意味着一个适应的过程。此外，这种调整的现象似乎是由先前发现的钙库8的GABA能调控启动。重要的是该数据也证明了，在本实验条件下，将GFP-水母发光蛋白的钙 -sensor可以检测和跟踪从细胞内Ca 2+ -stores释放的钙。从我们的实验室另外一个独特的研究中使用生物发光成像O / N在蘑菇体10记录在大脑中的神经元的整和神经胶质人口基础（自然）的活动，以及活动。这些记录揭示蚂蚁惊人的自发活动ennal mechanosensory和运动中心（AMMC）和触角叶。此外，发生在整个晚上在神经胶质细胞的意外自治活动。在蘑菇体的研究发现这两个点状活动和两个特征峰，一短一长，其发病率变化与10岁 。 在最近的研究在我们的实验室，我们用生物发光成像检查牵连的内存辅助信号分子的操纵的影响，以及它们如何影响蘑菇体9中的钙反应。事实上，虽然傻瓜和大头菜被认定超过30年前，被称为来调节cAMP水平，对细胞和生理机制，特别是在钙 -响应其在体内的作用仍然知之甚少。随着GFP-水母发光蛋白的方法，我们已经能够同时录制两个萼（树突状structurES）和小区体，以及轴突突起在波瓣的水平，并因此使我们能够关联的水平和在这种高度复杂的结构的不同区室的响应的动力学。 最近的应用中，我们正在开发模拟物通过人为地激活神经元群体突触前到感兴趣的结构的自然反应。要做到这一点，我们表达P2X 2受体 ，其响应ATP配体门控阳离子通道，在突触前神经元，表达绿色荧光蛋白水母发光蛋白在突触后神经元群。这两种元素成不同的神经元群体的偏析需要两个分开的表达系统。为此一个LexA的构建含有绿色荧光蛋白水母发光蛋白已经创建。在我们这个系统，我们已经表达P2X 2使用GAL4-GH146与UAS-P2X 2中的蘑菇体的期票和GFP-水母发光蛋白的一个子集，初步实验使用的LexA MB247线结合的LexA-13X-GFP-水母发光蛋白。我们已经成功地记录钙应答蘑菇体以1毫摩尔ATP刺激的P2X 2受体在期票（图6）。 最后，该生物发光的GFP-水母发光蛋白的方法已被证明是在记录诱导的Ca 2+ -activity，类似于其它的 Ca 2+ -indicators刺激是有用的。但更重要的是，它也允许大脑内的检测自发的Ca 2+ -activity。这种方法开辟了长期实验和全脑成像，可以增加我们的大脑内的生理现象和活动模式深度了解未来的可能性。

基本构图和大脑及其离散结构中的活性作用长期以来一直是神经科学领域内的紧张的学习领域。也许有人用来解决这个问题的最早的成功做法是在电活动变化的直接生理测量 - 但是替代方法，允许电压，pH或钙离子浓度的变化（作为活动代理）的实时成像带来了额外的功能，研究神经元活动1。成像技术携带的优点各种各样如降低侵袭和全脑结构内监控活性的能力。此外，在动物听话遗传学，包括果蝇 ，遗传编码的钙指标，如卡默莱昂，GCaMPs和其他发展1已使研究人员能够监测单个神经元，神经元群体和全脑结构。而更传统的荧光钙成像方法使用GCaMPs（GFP融合至钙调蛋白）或FRET（CFP和YFP融合至钙调蛋白）已被证明是提供良好的空间和时间分辨率有用的技术，光激发的基础过程滋生于它的实验一些限制应用程序。由于光激发，自体荧光，光漂白，并且光毒性的性质都不可避免地产生，这进而限制了可以被成像的结构的深度，记录的持续时间，以及记录的实时连续性。换句话说，记录必须经常进行间歇地避免这些不希望的副作用。 因为这些挑战的，​​已经开发了钙成像的其他替换的方法。其中最有希望的方法之一是生物发光成像，这依赖于通过后的钙结合的酶反应产生的光。 Bioluminescen牛逼成像不需要光激发，因此不会遇到同样的困难，传统的钙成像。所述生物发光性报告水母发光蛋白-从水母中分离，同样水母从中GFP也isolated-经由它的三个的EF手结构结合钙和发生构象变化，从而导致其辅因子腔肠素的氧化和蓝光的释放（λ = 469纳米）2,3。水母发光蛋白具有钙（K D =10μM），这使得它适合用于监测的钙瞬变，因为它产生非常少的背景噪声和不与细胞内钙离子的缓冲系统 4干扰非常低的亲和力。虽然水母发光蛋白以前已用于监测神经诱导的过程在爪蟾卵5产生的光太暗用于神经元活动的实时成像。为了努力应对这一挑战，菲利普登记及＃251;让我们和他的同事在巴斯德研究所创建的基因构建融合GFP和水母发光蛋白基因，水母4中模拟天然状态。此融合基因产物经由水母发光蛋白的三个的EF手结构，其经历构象变化，导致了腔肠素，其中以非辐射到GFP传送能量的氧化再次结合钙。这种能量转移的结果，而不是蓝色的光从水母发光蛋白从GFP绿光（λ= 509纳米）的释放。 GFP和水母发光蛋白的融合也赋予更高的蛋白质稳定性帮助融合蛋白产生光比单独4水母发光蛋白明亮的19至65倍。使用大脑内生物发光方法扩展了无论是在连续记录的持续时间和结构的可记录大脑内的数表示在当前的实验应用钙成像的。概括地说在以前的工作中就意味着全球性和更大的各种大脑的区域化"活性"可以被监视（通过钙瞬变的代理）。更重要的是活性可为更长的被监测，在实时和连续的基础上，这很容易适合于追求基底函数的大脑中的完整的理解。 在过去的几年里，我们的实验室和其他人已开发转基因果蝇表达二进制表达系统的控制下的绿色荧光蛋白水母发光蛋白（GAL4 / UAS-GFP-水母发光蛋白）5,6。我们使用GFP的水母发光蛋白的生物发光通过自然刺激如香味7,8，以及调制的 Ca 2+ -activity在以下乙酰胆碱受体的刺激通过直接烟碱应用9蘑菇体研究了细胞成分产生的记录活动。此外，我们还使用GFP的水母发光蛋白，以解决了一些一直无法被预先处理实验问题，如长期成像自发钙瞬变和更深的大脑结构10的可视化。最近，我们已经使用了GAL4 / UAS系统表达的哺乳动物的ATP受体P2X 2 11的阳离子通道，在投射神经元（PNS）和使用的LexA的系统表达的GFP-水母发光蛋白在蘑菇体（MB247-LexA的， 13XLexAop2-IVS-G5A-BP）（礼节性B.法伊弗，珍妮莉娅法姆，美国）。这使我们通过应用ATP，从而激活蘑菇体（期票的下游靶标），以激活期票，模仿更自然的条件下，如响应一个刺激气味。总体来说，这一技术结合P2X 2和GFP-水母扩大实验的可能性。从广义上它提供了机会，以更好地理解活动模式在大脑中，开始对不同的刺激和干扰如何改变活动的基础图案的新研究。

<table>
	<tbody>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Merck</td>
			<td>1023911000</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>7365-45-9</td>
			<td></td>
		</tr>
		<tr>
			<td>KCL&#160;</td>
			<td>prolabo</td>
			<td>26 764.298</td>
			<td>normapur</td>
		</tr>
		<tr>
			<td>MgCl</td>
			<td>prolabo</td>
			<td>25 108.295</td>
			<td>normapur</td>
		</tr>
		<tr>
			<td>sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S0389</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotine</td>
			<td>Sigma-Aldrich</td>
			<td>N3876</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Sigma-Aldrich</td>
			<td>A2383</td>
			<td>Adenosine 5&#8242;-triphosphate disodium salt hydrate</td>
		</tr>
		<tr>
			<td>benzyl-coelenterazine</td>
			<td>Prolume, nanolight&#160;</td>
			<td>Cat #301 Coelenterazine h</td>
			<td>http://www.prolume.com/</td>
		</tr>
		<tr>
			<td>dental glue</td>
			<td>3M ESPE&#8482;</td>
			<td>46954</td>
			<td>Protemp&#8482; IV</td>
		</tr>
		<tr>
			<td>silicon glue</td>
			<td>3M ESPE&#8482;</td>
			<td>36958</td>
			<td>Express&#8482; 2 Regular Body Quick</td>
		</tr>
		<tr>
			<td>tape</td>
			<td>3M Scotch</td>
			<td>122s</td>
			<td>3M Scotch Magic Tape</td>
		</tr>
		<tr>
			<td>pipette tips</td>
			<td>Corning Incorporated</td>
			<td>4868</td>
			<td>100-1000&#181;L Univesal Pipette Tip</td>
		</tr>
		<tr>
			<td>chamber and holder (stage)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>hand made</td>
		</tr>
		<tr>
			<td>incubation box</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>hand made</td>
		</tr>
		<tr>
			<td>forceps (No 5)</td>
			<td>Fine Science Tools</td>
			<td>11252-00</td>
			<td>Micro-dissecting forceps</td>
		</tr>
		<tr>
			<td>curved forceps</td>
			<td>Sigma-Aldrich</td>
			<td>F4142</td>
			<td>Micro-dissecting forceps</td>
		</tr>
		<tr>
			<td>glue applicator&#160;</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>hand made&#160;</td>
		</tr>
		<tr>
			<td>knife</td>
			<td>Fine Science Tools</td>
			<td>10316-14</td>
			<td>5mm Depth 15&#176; stab</td>
		</tr>
		<tr>
			<td>EM-CCD camera&#160;</td>
			<td>Andor</td>
			<td>DU-897E-CS0-#BV</td>
			<td>iXon (cooled to -80&#176;C)</td>
		</tr>
		<tr>
			<td>Microscope&#160;</td>
			<td>Nikon</td>
			<td>N/A</td>
			<td>Eclipse-E800</td>
		</tr>
		<tr>
			<td>immersion objective lens</td>
			<td>Nikon</td>
			<td>N/A</td>
			<td>20x Fluor .5w</td>
		</tr>
		<tr>
			<td>dissection microscope with fluorescence</td>
			<td>Leica MZ Fl III</td>
			<td>N/A</td>
			<td>leica dissection scope and florescent lamp</td>
		</tr>
		<tr>
			<td>tight dark box</td>
			<td>Science Wares</td>
			<td>N/A</td>
			<td>Custom built by Science Wares</td>
		</tr>
		<tr>
			<td>peristaltic pump&#160;</td>
			<td>Gilson</td>
			<td>N/A</td>
			<td>Minipuls 2</td>
		</tr>
		<tr>
			<td>tubing</td>
			<td>Fisher Scientific</td>
			<td>depends on thickness</td>
			<td>Tygon R3603, St-Gobain</td>
		</tr>
		<tr>
			<td>perfusion system</td>
			<td>Warner</td>
			<td>64-0135 &#160;(VC-66CS)</td>
			<td>Perfusion valve control system, complete, with pinch valves, 6 channel</td>
		</tr>
		<tr>
			<td>perfusion regulators</td>
			<td>Leventon</td>
			<td>201108</td>
			<td>Dosi-flow 3</td>
		</tr>
		<tr>
			<td>measurement and automation explorer</td>
			<td>Sciences Wares &#38; National Instruments</td>
			<td>N/A</td>
			<td>software http://sine.ni.com/nips/cds/view/p/lang/en/nid/1380</td>
		</tr>
		<tr>
			<td>photon imager&#160;</td>
			<td>Science Wares &#38; National Instruments</td>
			<td>N/A</td>
			<td>software http://sine.ni.com/nips/cds/view/p/lang/en/nid/1380</td>
		</tr>
		<tr>
			<td>photon viewer</td>
			<td>Science Wares &#38; National Instuments</td>
			<td>N/A</td>
			<td>software http://sine.ni.com/nips/cds/view/p/lang/en/nid/1380</td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>Microsoft</td>
			<td>N/A</td>
			<td>software https://www.microsoft.com</td>
		</tr>
		<tr>
			<td>virtual dub</td>
			<td>open access&#160;</td>
			<td>N/A</td>
			<td>software http://virtualdub.sourceforge.net/</td>
		</tr>
		<tr>
			<td>UAS-GA2</td>
			<td>Martin et al., 2007, Ref. 1&#160;</td>
			<td>N/A</td>
			<td>&#160;No stocks {currently} publicly available&#160;</td>
		</tr>
		<tr>
			<td>pJFRC65-13XLexAop2-IVS-G5A-BP&#160;&#160;</td>
			<td>B. Pfeiffer, Janelia Farms, Ashburn USA.&#160;</td>
			<td>(STOCK #1117340)</td>
			<td>pfeifferb@janelia.hhmi.org</td>
		</tr>
		<tr>
			<td>P2X2</td>
			<td>Lima and Misenboeck, Ref. 11&#160;</td>
			<td>N/A</td>
			<td>&#160;No stocks {currently} publicly available&#160;</td>
		</tr>
		<tr>
			<td>OK107</td>
			<td>Bloomington stock center</td>
			<td>854</td>
			<td>http://flystocks.bio.indiana.edu/</td>
		</tr>
	</tbody>
</table>

in vivo functional brain imaging approach based on bioluminescent calcium indicator gfp-aequorin

功能体内成像已经成为一个强大的方法来研究功能和脑细胞和利益结构的生理机能。近来已经开发的 Ca 2+使用生物发光性报告的GFP-水母发光蛋白（GA）-imaging的一种新方法。这种新的技术依赖于的绿色荧光蛋白和水母发光蛋白的基因融合，产生分子能够结合钙和 - 与另外的辅助因子腔肠中 - 发光明亮的光线可以通过光子收集器进行监控。已经产生对小鼠和果蝇携带GFP的水母发光蛋白基因的转基因品系，在 果蝇中，绿色荧光蛋白水母发光蛋白基因已被放置在GAL4 / UAS二进制表达系统允许大脑内针对性表达和成像的控制之下。这个方法后来被证明是能够检测两个向内的 Ca 2+ -transients和Ca 2+从内存储-released。最重要的是它允许在连续记录，成像在大脑内的更深的深度更大的持续时间，并记录在高时间分辨率（高达8.3毫秒）。在这里，我们提出了利用生物发光成像记录和分析钙蘑菇体中2+ -activity，结构中心在飞大脑学习和记忆的基本方法。

将GFP对水母发光蛋白的融合可以让我们通过GFP的激发形象化我们之前生物发光成像的兴趣区域在显微镜上（图3A，4A，6A，6E）荧光模式。一个刺激的蘑菇体的最简单的方法是通过激活离子型乙酰胆碱受体。虽然乙酰胆碱是该受体的内源性配体，我们已发现，尼古丁产生在蘑菇体更具有可重复性的响应。这部分是因为它们特异性表达的烟碱乙酰胆碱受体，从而通过使用尼古丁我们能够避免在大脑别处所表示的毒蕈碱性乙酰胆碱受体的活化。此外，尼古丁是比乙酰更稳定，且因此允许更好和更可靠的刺激控制。同时与花萼，细胞体（CCB）和内侧叶片（ML）的快速激活典型的响应开始（参见图4一种用于标记的图像）。一般建行响应持续时间更长，表现出比所述叶片的更大的生物发光响应中所示的响应的连续面板（ 图3C-H）。 图4B示出记录在100毫秒的时间分辨率的光子10秒的积累（10赫兹）到1分钟灌注25μM尼古丁响应的高峰期。继10分钟启动1分钟灌注100毫米氯化钾的第二个刺激（ 图4C，E），它可以让我们确认我们的样本的可行性的清除期和恢复期。定量的反应可以通过在分析过程中的光子观众选择的ROI进行分析。在运行分析在光子观察者既产生图形这些值显示在选定的ROI随时间（图4D 和 E）发射的光子的数目以及可导出电子表格。 图4D是examp乐光子观众绘制的投资回报率2 [绿色]发射的光子（右CCB）和投资回报率4 [蓝]（右内侧叶）所产生的图形。类似的数据在图4E所示的氯化钾响应。导出的数据可以被用来重建该响应（图5A）的曲线以及提取有关各种参数，如持续时间，峰值幅度和总的反应（图5B-D）的附加 ​​数据。最近使用我们设计引发更自然的应答的方法的GAL4-UAS和LexA的系统。简言之，ATP门控P2X 2阳离子通道在投射神经元表达（PNS）和GFP-水母发光蛋白（GA）被表示在蘑菇体-期票的目标;这使我们能够选择性地激发期票虽然应用ATP的，其可以反过来，产生响应于蘑菇体（图6）。 <img alt="图1"SRC ="/文件/ ftp_upload / 53705 / 53705fig1.jpg"/&gt; 生物发光记者（GFP-水母发光蛋白）绿色荧光蛋白。（ 一）示意图和水母发光蛋白融合基因图1.特性与上游激活序列。 （ 二）由水母发光蛋白响应高水平钙离子的蓝光发射模型。为应对高水平钙的研究GFP-水母的绿色发光（C）型号2+。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53705/53705fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53705/53705fig2.jpg" /> 图1实验设置。（A）飞放置在枪头。 （B）的适当的胶合技术的稳定的头部并密封蝇体和吸管尖之间的区域的图。 （C）剥离开头壳示意图。 （ 四）1毫升室上支承块插入了输液管。总腔尺寸:长7.4厘米，宽2.7厘米，高0.55公分。内室尺寸:长:1.7厘米，宽:1.4厘米，高度:0.35厘米，并在腔室孔的半径:0.1厘米。 （E）查看苍蝇头显示在MBS GFP阳性信号:低光线昏暗荧光去除角质层后，来自OK107驱动的绿色荧光蛋白水母发光蛋白。 （F）显微镜光子相机和灌注系统。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53705/53705fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53705/53705fig3.jpg" /> 图3至25微米的尼古丁蘑菇体代表的响应。（A）的荧光图像在蘑菇体的绿色荧光蛋白水母发光蛋白表达GA2 / +; OK107 / +控制苍蝇的大脑（比例尺= 50微米）。 （二）在蘑菇体峰生物发光响应。 （C）的前刺激。 （四）开始反应。 （E）的峰值响应。 （六）继续建行回应。 （G）下降建设银行响应。 （H）末日的回响（BH:比例尺= 33微米）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53705/53705fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53705/53705fig4.jpg" /> 采样记录图4。代表分析 （ 一）GA2的蘑菇体的荧光图像/ +; OK107 / +控制四个投资回报率的选择在建行和中间叶（规模BA飞R = 50微米）。 （B）中，以25μM的尼古丁的生物发光响应的10秒的积累-记录在100毫秒光子反应。 （℃）至100毫米氯化钾生物发光响应结果为100毫秒的10秒的积累。 （D）分别为图的记录数在内部投资回报率的2和4尼古丁的反应发出的光子覆盖正确的建行和中间叶。记录的人数在内部投资回报率的2和4涵盖右建行和叶内侧分别氯化钾反应产生的光子（E）图。在D和E，左边的Y轴对应于整个视场范围内的光子的总数，而右边的Y轴对应的投资回报率中的光子。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53705/53705fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5"SRC ="/文件/ ftp_upload / 53705 / 53705fig5.jpg"/&gt; 图5.代表性的参数分析样本录音中的GA2 / +的蘑菇体的烟碱激活生物发光响应代表Excel的分析; OK107 / +控制飞行。 （ 一）光子反应随着时间的推移在建行和中间叶。在建行和中间叶反应（B）持续时间。 （ 三）建行和中间叶内的反应过程中的最高光子值（最大振幅）。 （ 四）在建行和整个响应内侧叶总光子。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53705/53705fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/53705/53705fig6.jpg" /> 刺激的PN THR后蘑菇体图6.两个不同的反应ough ATP和P2X 2。示例1飞表达P2X 2个PN和GFP-水母发光蛋白的蘑菇体（GH146 / +; P2X 2 / MB247-LexA的，13XLexAop2-IVS-G5A-BP（广告）（A）蘑菇体与投资回报率（规模荧光图像。酒吧= 50微米）的下列激活P2X 2个PN 500μMATP刺激，观察主要是在内侧叶蘑菇体的峰值响应。（二）生物发光图像。（C）的峰值氯化钾反应生物发光的形象。（D ）时间当然光子发射的ROI区域内，在整个反应样本2飞表达P2X 2个PN和GFP-水母发光蛋白的蘑菇体（GH146 / +; P2X 2 / MB247-LexA的，13XLexAop2-IVS-G5A-BP（EH ）。（E）与投资回报率（比例尺= 50微米）荧光图像。（F）在蘑菇体的诱导反应承接生物发光图像期票由P2X 2 ctivation刺激1mm厚的ATP，两叶及建行。 （G）的峰值氯化钾反应生物发光图像。 ROI区域内的光子发射，在整个反应（H）的时间过程。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53705/53705fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin at JoVE.com

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

在基于生物发光钙指标GFP的水母体内脑功能成像方法

在这里，我们提出了一个新颖的钙利用生物发光记者-imaging方法。这种方法使用的稠构建GFP-水母发光蛋白，其结合的 Ca 2+并发光，省去了光激发。显著此方法允许长时间连续成像，获得脑深部的结构和高时间分辨率。

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new ...

in-vivo-functional-brain-imaging-approach-based-on-bioluminescent

Nanyang Technological University

Research

JoVE Journal

Neuroscience

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

12.2K 次观看.  UMR-9197, CNRS/Université Paris Sud. 在这里，我们提出了一个新颖的钙利用生物发光记者-imaging方法。这种方法使用的稠构建GFP-水母发光蛋白，其结合的 Ca 2+并发光，省去了光激发。显著此方法允许长时间连续成像，获得脑深部的结构和高时间分辨率。 

视频: In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Functional Calcium Imaging in Developing Cortical Networks

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in intact spinal cord, brainstem, retina, cortex and dissociated neuronal culture preparations. During periods of spontaneous activity, neurons depolarize to fire single or bursts of action potentials, activating many ion channels. Depolarization activates voltage-gated calcium channels on dendrites and spines that mediate calcium influx. Highly synchronized electrical activity has been measured from local neuronal networks using field electrodes. This technique enables high temporal sampling rates but lower spatial resolution due to integrated read-out of multiple neurons at one electrode. Single cell resolution of neuronal activity is possible using patch-clamp electrophysiology on single neurons to measure firing activity. However, the ability to measure from a network is limited to the number of neurons patched simultaneously, and typically is only one or two neurons. The use of calcium-dependent fluorescent indicator dyes has enabled the measurement of synchronized activity across a network of cells. This technique gives both high spatial resolution and sufficient temporal sampling to record spontaneous activity of the developing network.
A key feature of newly-forming cortical and hippocampal networks during pre- and early postnatal development is spontaneous, synchronized neuronal activity (Katz &amp; Shatz, 1996; Khaziphov &amp; Luhmann, 2006). This correlated network activity is believed to be essential for the generation of functional circuits in the developing nervous system (Spitzer, 2006). In both primate and rodent brain, early electrical and calcium network waves are observed pre- and postnatally in vivo and in vitro (Adelsberger et al., 2005; Garaschuk et al., 2000; Lamblin et al., 1999). These early activity patterns, which are known to control several developmental processes including neuronal differentiation, synaptogenesis and plasticity (Rakic &amp; Komuro, 1995; Spitzer et al., 2004) are of critical importance for the correct development and maturation of the cortical circuitry.
In this JoVE video, we demonstrate the methods used to image spontaneous activity in developing cortical networks. Calcium-sensitive indicators, such as Fura 2-AM ester diffuse across the cell membrane where intracellular esterase activity cleaves the AM esters to leave the cell-impermeant form of indicator dye. The impermeant form of indicator has carboxylic acid groups which are able to then detect and bind calcium ions intracellularly.. The fluorescence of the calcium-sensitive dye is transiently altered upon binding to calcium. Single or multi-photon imaging techniques are used to measure the change in photons being emitted from the dye, and thus indicate an alteration in intracellular calcium. Furthermore, these calcium-dependent indicators can be combined with other fluorescent markers to investigate cell types within the active network.

Spontaneous activity of developing neuronal networks can be measured using AM-ester forms of calcium-sensitive indicator dyes. Changes in intracellular calcium, indicating neuronal activation, are detected as transient changes in indicator fluorescence with one- or two-photon imaging. This protocol can be adapted for a range of developmentally-dependent neuronal networks in vitro.

在开发皮层网络功能钙成像

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in ...

科研

神经科学

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 ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

在体内双光子成像的经验依赖的分子在大脑皮质神经元的变化

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

C.在体内神经元钙成像技术线虫

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

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

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

脑切片神经元树突的双光子钙成像

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

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 Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

幼虫斑马鱼外系头发细胞的活体钙成像

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...

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 ...

&#181;Tongue: A Microfluidics-Based Functional Imaging Platform for the Tongue In Vivo

Intravital fluorescence microscopy is a tool used widely to study multicellular dynamics in a live animal. However, it has not been successfully used in the taste sensory organ. By integrating microfluidics into the intravital tongue imaging window, the &#181;Tongue provides reliable functional images of taste cells in vivo under controlled exposure to multiple tastants. In this paper, a detailed step-by-step procedure to utilize the &#181;Tongue system is presented. There are five subsections: preparing of tastant solutions, setting up of a microfluidic module, sample mounting, acquiring functional image data, and data analysis. Some tips and techniques to solve the practical issues that may arise when using the &#181;Tongue are also presented.

 &#181;Tongue: A Microfluidics-Based Functional Imaging Platform for the Tongue In Vivo

The article introduces the &#181;Tongue (microfluidics-on-a-tongue) device for functional taste cell imaging in vivo by integrating microfluidics into an intravital imaging window on the tongue.

μTongue:一个基于微流体的功能成像平台，用于在Viva的舌头

Intravital fluorescence microscopy is a tool used widely to study multicellular dynamics in a live animal. However, it has not been successfully used in ...