我们感谢安德烈莫兰连同实验动物服务设施（LASF）的成员，他们对畜牧业和技术服务的优秀支持在斯托瓦斯研究所。这项工作是支持，从斯托瓦斯研究所和国立卫生研究院（NIDCD 008003）资金，哭了。内容完全是作者的责任，并不一定代表美国国立耳聋与其他交流障碍研究所或国立卫生研究院的官方意见。美国正在申请专利的斯托瓦斯研究所，哭​​和LM的tetO - G CaMP2小鼠。

1。溶液的配制<ol><li>根据下表，准备R1的10X，10X的R2和10X R3的解决方案。 R1 <table border="1"><tbody><tr><td>化学品</td><td>兆瓦（克/摩尔） </td><td>毫米（1X） </td><td> 10倍的股票（G / L） </td></tr><tr><td>氯化钠</td><td> 58.44 </td><td> 125 </td><td> 73.05 </td></tr><tr><td>氯化钾</td><td> 74.55 </td><td> 2.5 </td><td> 1.86 </td></tr><tr><td> MgCl 2的 </td><td> 1中号股票</td><td> 1 </td><td> 10毫升</td></tr><tr><td>氯化钙2 · 2H 2 O </td><td> 147.02 </td><td> 2 </td><td> 2.94 </td></tr><tr><td>的NaH 2 PO 4 · H 2 O </td><td> 137.99 </td><td> 1.25 </td><td> 1.72 </td></tr></tbody></table> R2的 <table border="1"><tbody><tr><td>化学</td><td>兆瓦（克/摩尔） </td><td>毫米（1X） </td><td> 10倍的股票（G / L） </td&gt; </tr><tr><td>碳酸氢钠3 </td><td> 84.01 </td><td> 25 </td><td> 21 </td></tr></tbody></table> R3 <table border="1"><tbody><tr><td>化学品</td><td>兆瓦（克/摩尔） </td><td>毫米（1X） </td><td> 10倍的股票（G / L） </td></tr><tr><td>氯化钠</td><td> 58.44 </td><td> 125 </td><td> 73.05 </td></tr><tr><td>氯化钾</td><td> 74.55 </td><td> 2.5 </td><td> 1.86 </td></tr><tr><td> MgCl 2的 </td><td> 1中号股票</td><td> 2 </td><td> 20毫升</td></tr><tr><td>氯化钙2 · 2H 2 O </td><td> 147.02 </td><td> 2 </td><td> 2.94 </td></tr><tr><td> HEPES </td><td> 1中号股票</td><td> 5 </td><td> 50毫升</td></tr></tbody></table></li><li>实验当天混合双蒸水（DDW）和广告10X R1和10X R2的100毫升100毫升1升鼠标人工脑脊髓液（mACSF）ð 1.8克葡萄糖（最后10毫米）。这种制备方法可以防止在高pH值的碳酸钙沉淀。 mACSF渗透压约为310-315 mOsm / L。如果有必要的葡萄糖或水调整渗透压。充气前至少30分钟，溶液的pH值调整到7.2-7.4 Carboxygen气体（95％的氧气和5％的二氧化碳）的解决方案。 </li><li>实验当天，1公升的林格氏液混合10X R2和DDW 10X R3的100毫升100毫升，并添加葡萄糖1.8克（10毫米）。林格氏液渗透压和pH值应该是类似的mACSF。曝气Carboxygen气体的解决方案。 </li><li>准备mACSF或林格氏液在4％的低熔点琼脂糖（LMA）。分装在Eppendorf管熔化琼脂糖和储存于4 ° C，直到使用。 LMA，这是纯化的多糖组成，流体保持在37 ° C，并迅速巩固了在温度低于25 ° C。这些特性使其理想嵌入VNO和活组织生理。如琼脂，其​​他不纯的材料，不应该选择活组织实验未知的组件可能会干扰神经元的反应。 </li><li>准备林格氏液中的信息素刺激。对于鼠标尿，1:100稀释会产生强大的反应。 </li></ol> 2。 VNO片的制备<ol><li>牺牲的动物之前，把一个加热块两管喉罩和设定温度&gt; 60 ° C至融化琼脂糖。一旦凝胶液化，管转移到37℃的热块。 </li><li>杀头一个G - CaMP2鼠标下面的CO 2安乐死。用剪刀剪的下颌骨骨去除下颌。剥落的山脊上腭组织暴露鼻腔（ 图1A）。分隔两个前门牙之间插入一个手术刀片的颚骨。小心取出颚骨暴露VNO。在这一点上，可以举起整个VNO从NASAL腔尾骨（ 图1B）控股。转让的VNO冷含氧mACSF解决方案。 </li><li>根据清扫范围，使用提示＃5镊子轻轻滑动沿鼻中隔骨（ 图1C）壁分开的两个VNOS。剥离的犁骨包绕VNO组织。轻轻抬起整个VNO组织从骨腔。在此步骤中，应采取特别小心，不损伤神经上皮。小骨组织表面上留下的碎片，必须完全删除之前嵌入。组织上留下的碎片可能会陷入切割刀片，拉的琼脂糖块组织。 </li><li>使用镊子举行的VNO组织后，轻轻地浸入到融化的琼脂糖（ 图1D） 。在冰上迅速冷却管，巩固琼脂糖。嵌入和冷却过程中应采取少于2分钟。 </li><li> VF300组织切片机（Precisionary INSTRuments，格林维尔，NC）是用来使部分。切琼脂糖块形状和胶水组织持有人（图1E和F）的VNO。插入金属圆柱体组织持有人，并填写余下额外LMA室。一旦LMA冰巩固，立即进行切片。供应切片室冷含氧mACSF，并开始在180-200微米厚度每片切割。调整的推进速度和振动频率，使该组织是在切片和VNO不属于琼脂糖压缩。收集和转移切片切片mACSF孵化室（ 图2A）。片是可行的含氧mACSF在室温下6-8小时。 图2B和C DIC和双光子图像显示一个G - CaMP2 VNO片，分别。 </li></ol> 3。成像商会成立<ol><li> VNO片放置在中间的灌注室（锡斯基尤，资助通行证），并与片切片锚（华纳仪器，哈姆登，CT）（ 图3A）。锚线程应该只压片的喉罩的一部分，但不是VNO组织。含氧mACSF交付灌注腔通过进气口〜100μL/秒，并通过出油口的吸针排出的液体是通过提供了一个新鲜mACSF 的连续流（图 3A和B）。 </li><li>填写林格氏液30毫升的注射器和钳注射泵（新时代的热泵系统，明代，纽约州）。 300-600μL/ min的设置泵的转速，提供一个连续流片（ 图3C）林格氏液。 </li><li>林格到HPLC注射液循环（Chromtech，苹果谷，MN）（ 图3D）连接插座。交付量的精确控制，可以选择不同的循环大小。我们使用在O 20μL循环UR实验。注射闭环控制两个流路线（ 图3E）。在"负载"的位置，样品可以装入样品环与汉密尔顿精密注射器。退出多余的样品通过排污口的样品循环。林格氏液注射泵注入绕过样品循环和直接出线，这是连接到灌注头（ 图3A和E）。在"注水"的位置，流经样品循环泵解决方案，并推入插座 （图3E）的刺激。成像实验之前，应气泡赶出灌注液，以确保畅通。其他商业或自定义的喷射系统，也可以刺激交付实施。 </li><li>调整灌注下的5倍或10倍镜头提示，使针尖远离VNO片约1毫米。 </li><li>当一个新的灌注系统设置，最好是测量样品的延迟时间和durATION与荧光染料。 0.1％罗丹明6G染料装入样品循环和开关阀注入后5秒的图像采集开始的位置（图3F） 。检测到荧光信号是10-30秒之间。荧光信号的平滑曲线还表明，有一点倾斜镜头和有足够的灌注含氧mACSF达到切片下产生的湍流。 </li></ol> 4。时间的推移成像<ol><li>我们用一个时间的推移成像10X或20X水浸渍镜头蔡司AxioSkope FS2显微镜。标准GFP带通滤波器（450 - 490nm处）是用于G - CaMP2信号。啶图像是由CCD相机（蔡司人力资源管理）收购与1X1或2x2分级根据的G - CaMP2的表达水平。 </li><li>设置采集速度每秒1张。调整光的强度，以尽量减少漂白的G - CaMP2信号和光损伤的细胞。对于每个前实验中，我们通常获得60帧图像堆栈（约60-70秒）。切换阀注入的位置，一个实验，在所有试验中获得一致的时间延迟在一个特定的时间点（ 例如，在图3F 5秒）。 </li><li>执行测试运行使用林格氏液的刺激。重新调整灌注设置，如果进样期间推出运动神器。 </li><li>执行积极的控制林格氏液在1:100稀释的小鼠尿液运行。典型的最大价值的G - CaMP2响应鼠标尿ΔF/ F 20-40％左右。如果需要，可以确认提供林格10毫米氯化钾刺激在实验结束时的片片的可行性。 </li><li>林格氏液洗至少三次加载后一个刺激汉密尔顿注射器。洗与林格氏液至少有三个不同的刺激之间的时间样本循环。这些步骤之间differe防止交叉污染NT样本。 </li><li>等待4-10分钟VSNS恢复，然后再应用下一个刺激。 </li></ol> 5。数据分析<ol><li>执行图像从一个切片获得的图像登记。我们AxioVision（卡尔蔡司，北美）使用自定义编写的VBA脚本来自动完成这一过程。同样的实验范围内的所有图像帧注册对付共同的选择与弹性登记（ 图 4A）的参照系。弹性又称非线性登记，注册，是一类强调目标图像的改造非硬性参考图像的图像配准技术。在这里，我们用从AxioVision实施。 这个参照系是任意选择的图像堆栈。 </li><li>执行图像减法来确定响应细胞。我们使用ImageJ v1.42定制编写的宏（ <a href="http://rsb.info.nih.gov/ij/">http://rsb.info.nih.gov/ij/</a> ，国立卫生研究院马里兰州贝塞斯达）自动完成这一过程。每个堆栈产生一个最小的投影图像。回应细胞后出现的最小投影是从原料栈（ 图4B）减去。 </li><li>减去堆栈确定感兴趣区域（ROI），并取得使用ImageJ多测量插件的ROI坐标。所有堆栈为一个实验处理，并保存所有的投资回报率的坐标在投资回报率的主列表（ 图4C） 。 </li><li>使用ROI主列表来衡量，从自定义编写的宏观和多测量插件ImageJ（图 4D）的原始图像栈的细胞反应。积响应曲线，并在Matlab中的热图（MathWorks公司，Natic​​k郡，马）。 </li></ol> 6。代表性的成果使用从四个独立的小鼠收集的尿液样本的VNO成像实验的一个例子是如图 5所示。切片响应尿液刺激与不同的patterns激活。约80细胞识别至少一个尿液的刺激，他们的反应ΔF/ F值绘制热图（ 图5A）。绘制在图5B显示尿液刺激的时间当然激活细胞1，2和3的反应痕迹。单元1显示了两个女的尿液样本，但不是男性样本，而细胞2显示激活相反的模式和响应都只有男性的样本。单元3被激活个人的男性和女性样本。细胞1和2显示了典型反应性的具体线索，尿液中的15。 <img alt="图1" src="/files/ftp_upload/3404/3404fig1.jpg" /> 图1 VNO清扫过程示意图。A. VNO在鼠标头部的解剖位置。头一个G - CaMP2鼠标已解剖，并奠定了软吨倒挂从腭揭露VNO删除的问题。入口荧光图像显示了相同的图片。 neuroepithelia的VNO是明亮的绿色。B.犁骨（上）和相同的VNO（底部）的荧光图像，是在封闭的隔离VNO 侧视图 。C.一个VNO和解剖冠状的过程。一个VNO是分开的隔，然后可以拆下来摆脱的神经上皮和犁骨 D. VNO是嵌入到LMA。大肠杆菌嵌入式块粘切片组织持有。组织持有人是在180-200每片推铁桶琼脂糖块切片微米先进。切割刀片的定位密切的铁桶。F. VF300组织切片机系统的侧视图。 BV的血管。东北，神经上皮。 <img alt="图2" src="/files/ftp_upload/3404/3404fig2.jpg" /> <st荣&gt;图2 VNO片。VNO片含氧mACSF保持在室温。B.一个VNO片DIC的图片。C.一个光子的VNO 2的G - CaMP2鼠标的形象。比例尺，50微米。 <img alt="图3" src="/files/ftp_upload/3404/3404fig3.jpg" /> 图3插图灌注系统设置。 答:典型的灌注腔入口和出口。 VNO片被定位在室中心和按用纸巾锚。中间的线程将被删除组织的锚，使VNO组织未按 。灌注腔放在显微镜下阶段下浸渍目标。 mACSF入口，吸风口和灌注头表示，单管注射泵提供通过灌注尖端林格氏液的连续流。D.高效液相色谱法仁济ction闭环系统是通过刺激方便样品装载和注射。E.示意图插图装载和注射位置的方向。林格氏液，绿色。刺激的样本，洋红色。箭头指示流向。F.延迟和洗掉灌注系统的时间测量。罗丹明6G荧光染料装入样品循环和阀切换到5秒，这是一个箭头表示的注射位置。荧光信号的变化是在10-30秒之间，减少事后检测。 <img alt="图4" src="/files/ftp_upload/3404/3404fig4.jpg" /> 图4图像处理流水线。A.一个切片图像帧注册对一个参照系。B.从原始帧堆栈的最小投影减法。后加减响应细胞变得突出。 三 ř机会性感染选择编译成主列表和它们的坐标。D.响应使用的投资回报率从原材料栈主列表细胞测量。 <img alt="图5" src="/files/ftp_upload/3404/3404fig5.jpg" /> 图5代表的G - CaMP2 VNO成像实验与个别男性和女性的尿液刺激了切片答 :ΔF/ F响应热图。收集尿液样本是从个体雄性和雌性小鼠。 X轴，响应从切片确定的细胞。 Y轴，实验中所用的尿液样本。彩条表示ΔF/ F值。B. A中箭头标记的3个有代表性的细胞响应时间当然表明刺激应用程序的时间。 F - FVB，女性FVB的F - CBA，CBA的女性，M - FVB，雄性FVB，M - BL6，雄性C57BL / 6。 特定的试剂和设备表: <table border="1"><tbody><tr><td> 名称试剂</td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>灌注腔</td><td>锡斯基尤</td><td> PC - H </td><td>水平</td></tr><tr><td>切片按住向下</td><td>华纳仪器</td><td> SHD - 22CL </td><td></td></tr><tr><td>灌注端口持有人</td><td> ALA的科学，公司</td><td> MPIOH - S </td><td></td></tr><tr><td>注射泵</td><td>新时代的泵系统，公司</td><td> NE - 300 </td><td></td></tr><tr><td>低压注射阀</td><td> Chromtech </td><td> V - 451 </td><td></td></tr><tr><td> PEEK管</td><td> Chromtech </td><td> 1531 </td><td> 1 / 16"OD，0.25毫米ID </td></tr><tr><td>聚醚醚酮样品回路</td><td> Chromtech </td><td> 1803 </td><td> 20微升</td></tr><tr><td>汉密尔顿注射器</td><td> Chromtech </td><td> 80630 </td><td>加入100μL </td></tr></tbody></table>

<ol>
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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formyl+peptide+receptors+are+candidate+chemosensory+receptors+in+the+vomeronasal+organ.">Formyl peptide receptors are candidate chemosensory receptors in the vomeronasal organ.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 9842-9847 (2009).</li><li>Riviere, S., Challet, L., Fluegge, D., Spehr, M., Rodriguez, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formyl+peptide+receptor-like+proteins+are+a+novel+family+of+vomeronasal+chemosensors.">Formyl peptide receptor-like proteins are a novel family of vomeronasal chemosensors.</a> Nature. 459, 574-577 (2009).</li><li>Yang, H., Shi, P., Zhang, Y. P., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Composition+and+evolution+of+the+V2r+vomeronasal+receptor+gene+repertoire+in+mice+and+rats.">Composition and evolution of the V2r vomeronasal receptor gene repertoire in mice and rats.</a> Genomics. 86, 306-315 (2005).</li><li>Rodriguez, I., Punta, D. e. l., Rothman, K., Ishii, A., T, ., Mombaerts, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+new+and+isolated+families+within+the+mouse+superfamily+of+V1r+vomeronasal+receptors.">Multiple new and isolated families within the mouse superfamily of V1r vomeronasal receptors.</a> Nat. Neurosci. 5, 134-140 (2002).</li><li>He, J., Ma, L., Kim, S., Nakai, J., Yu, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encoding+gender+and+individual+information+in+the+mouse+vomeronasal+organ.">Encoding gender and individual information in the mouse vomeronasal organ.</a> Science. 320, 535-538 (2008).</li><li>Holy, T. E., Dulac, C., Meister, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+of+vomeronasal+neurons+to+natural+stimuli.">Responses of vomeronasal neurons to natural stimuli.</a> Science. 289, 1569-1572 (2000).</li><li>Chamero, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+protein+pheromones+that+promote+aggressive+behaviour.">Identification of protein pheromones that promote aggressive behaviour.</a> Nature. 450, 899-902 (2007).</li><li>Leinders-Zufall, T., Ishii, T., Mombaerts, P., Zufall, F., Boehm, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+requirements+for+the+activation+of+vomeronasal+sensory+neurons+by+MHC+peptides.">Structural requirements for the activation of vomeronasal sensory neurons by MHC peptides.</a> Nat. Neurosci. 12, 1551-1558 (2009).</li><li>Kimoto, H., Haga, S., Sato, K., Touhara, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex-specific+peptides+from+exocrine+glands+stimulate+mouse+vomeronasal+sensory+neurons.">Sex-specific peptides from exocrine glands stimulate mouse vomeronasal sensory neurons.</a> Nature. 437, 898-901 (2005).</li><li>Holekamp, T. F., Turaga, D., Holy, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+three-dimensional+fluorescence+imaging+of+activity+in+neural+populations+by+objective-coupled+planar+illumination+microscopy.">Fast three-dimensional fluorescence imaging of activity in neural populations by objective-coupled planar illumination microscopy.</a> Neuron. 57, 661-672 (2008).</li><li>Leinders-Zufall, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MHC+class+I+peptides+as+chemosensory+signals+in+the+vomeronasal+organ.">MHC class I peptides as chemosensory signals in the vomeronasal organ.</a> Science. 306, 1033-1037 (2004).</li><li>He, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+signals+conveyed+by+pheromone+concentrations+to+the+mouse+vomeronasal+organ.">Distinct signals conveyed by pheromone concentrations to the mouse vomeronasal organ.</a> J. Neurosci. 30, 7473-7483 (2010).</li><li>Haga, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+male+mouse+pheromone+ESP1+enhances+female+sexual+receptive+behaviour+through+a+specific+vomeronasal+receptor.">The male mouse pheromone ESP1 enhances female sexual receptive behaviour through a specific vomeronasal receptor.</a> Nature. 466, 118-122 (2010).</li><li>Boschat, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pheromone+detection+mediated+by+a+V1r+vomeronasal+receptor.">Pheromone detection mediated by a V1r vomeronasal receptor.</a> Nat. Neurosci. 5, 1261-1262 (2002).</li><li>Hendel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+changes+of+genetic+calcium+indicators+and+OGB-1+correlated+with+neural+activity+and+calcium+in+vivo+and+in.">Fluorescence changes of genetic calcium indicators and OGB-1 correlated with neural activity and calcium in vivo and in.</a> 28, 7399-7411 (2008).</li><li>Pologruto, T. A., Yasuda, R., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+neural+activity+and+[Ca2+]+with+genetically+encoded+Ca2++indicators.">Monitoring neural activity and [Ca2+] with genetically encoded Ca2+ indicators.</a> J. Neurosci. 24, 9572-9579 (2004).</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), (2007).</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></ol>

没有利益冲突的声明。

大多数的犁鼻器受体（VRS）保持作为，因为他们发现由杜拉克和Axel 5的孤儿受体。这些化学感应受体和他们在调解动物行为中的作用的信息素配体不能很好地理解。到现在为止，只有一对配体/受体，ESP1肽及其受体，Vmn2r116（V2Rp5），已确定并传达特定的社会信息，19,23。另一个受体，V1rb2，已被证明回应2 -庚酮，这大概是从雌性小鼠24尿液丰富。然而，此配体所传达的具体信息尚不清楚。此处我们所开发的的方法，使犁鼻器神经元的反应，以大量的刺激和激活模式的分析表征。通过使用这种方法，我们发现，细胞显示男性或女性尿液中的具体反应和细胞有可能是专门运作承认具体性信息素线索15。这些细胞表达的自愿退休尚未确定。这里描述的影像处理方法，不仅可以使尿液中的信息素配体的筛选，也便于识别的特异性受体，应对信息素线索。 钙传感器的G - CAMP的家人一直在各种动物和它们的信号已被证明与钙瞬变 25-28以及关联。使用基因编码的传感器提供了敏感的神经元的反应的读数，并允许在有针对性的组织和细胞类型的传感器的具体体现。基因编码的传感器也有可能使慢性成像在活的动物。传统上，合成钙染料已被用于监测钙的反应。染料的加载程序都不可避免地侵入并经常组织造成损害。负载不均衡和有限的染料渗透在TISSUE也显著影响钙信号的检测。 在我们的实验使用的G - CaMP2小鼠中，我们发现的反应幅度看齐，或者比片制剂，钙染料加载方法更好。 VNO片可以保持在一个小时的健康状况，从而允许在一个实验中，刺激大量的分析后切片。然而，基因编码的传感器通常含有蛋白质域，可能与细胞成分相互作用，从而改变反应模式，甚至影响有机体的正常发展。一个一定要谨慎，当一个新的传感器系统介绍。仔细检查有针对性的组织是必需的。在我们以前的研究中，我们已经研究了其与生俱来的行为，犁鼻器神经元的电生理特性和嗅感觉神经元的投影模式的G - CaMP2小鼠。这些方面都没有受到影响，在G -CaMP2小鼠。然而，利用转基因动物，动物维修和基因分型招致的额外费用。此外，实验是有限生成的可用行。这些设计实验时，需要考虑的因素。

imaging neuronal responses in slice preparations of vomeronasal organ expressing a genetically encoded calcium sensor

犁鼻器（VNO）检测，化学感应信号在同一物种的1,2个人的社会，性和生殖状态信息。这些种内的信号，信息素，以及来自一些食肉动物3的信号，激活高水平的特异性和灵敏度 4犁鼻感觉神经元（VSNS） 。 G蛋白偶联受体V1R，V2R和FPR 5-14，至少有三个不同的家庭表示在犁鼻神经元的调解检测的化学感应线索。为了了解信息素的信息是如何编码的VNO，它是分析个人VSNS响应各种刺激，并确定这些反应的特异性受体介导的关键。 VNO neuroepithelia括在一对犁骨头。 VNO半盲管状结构有一个开口端（犁鼻管）连接到鼻腔。 VSNS延长树突的犁鼻器信息素线索接触表达的受体在树突状旋钮管腔部分。 VSNS的细胞体的形式与V1R和V2R伪分层表示层分别6-8在根尖和基底层。几项技术已被用于监察VSNS 4,12,15-19感官刺激的反应。急性切片准备在这些技术中，有几个优点。首先，3,17分离VSNS相比，切片的筹备工作保持在其原生形态和细胞树突的神经元保持相对完好。二，VSNS胞体很容易在冠状片的VNO访问，允许电生理研究和成像实验相比，整个上皮细胞和整个安装准备工作 12,20 。第三，这种方法可以结合分子克隆技术，让受体识别。感官刺激会引起强烈的Ca 2 + VSNS涌入，表明受体激活 4,21 。因此，我们开发的转基因小鼠的嗅感觉神经元，其中包括VSNS 15,22表达的G - CaMP2。探针的灵敏度和遗传性，极大地促进钙离子成像实验。这种方法消除了染料4,21以前的研究中使用的加载过程。我们还采用了配体传递系统，可应用各种刺激的VNO片。这两种技术的结合使我们能够监测多个神经元，同时响应大量的刺激。最后，我们已经建立了一个半自动化分析的管道，以协助图像处理。

Watch this Scientific Journal Video about Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor at JoVE.com

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

犁鼻器（VNO）检测种内的化学信号，传达社会和生殖信息。我们已经进行钙离子成像实验，利用转基因小鼠犁鼻组织中表达的G - CaMP2。这种方法可以让我们来分析复杂的犁鼻器神经元的反应模式，以大量的信息素的刺激。

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-neuronal-responses-slice-preparations-vomeronasal-organ

Nanyang Technological University

Research

JoVE Journal

Neuroscience

10.7K 次观看.  Stowers Institute for Medical Research. 犁鼻器（VNO）检测种内的化学信号，传达社会和生殖信息。我们已经进行钙离子成像实验，利用转基因小鼠犁鼻组织中表达的G - CaMP2。这种方法可以让我们来分析复杂的犁鼻器神经元的反应模式，以大量的信息素的刺激。

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

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

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory bulb1-5. Olfactory information in the external world is transmitted to the antennal lobe by olfactory sensory neurons (OSNs), which segregate to distinct regions of neuropil called glomeruli according to the specific olfactory receptor they express. Here, OSN axons synapse with both local interneurons (LNs), whose processes can innervate many different glomeruli, and projection neurons (PNs), which convey olfactory information to higher olfactory brain regions.
Optical imaging of the activity of OSNs, LNs and PNs in the antennal lobe - traditionally using synthetic calcium indicators (e.g. calcium green, FURA-2) or voltage-sensitive dyes (e.g. RH414) - has long been an important technique to understand how olfactory stimuli are represented as spatial and temporal patterns of glomerular activity in many species of insects6-10. Development of genetically-encoded neural activity reporters, such as the fluorescent calcium indicators G-CaMP11,12 and Cameleon13, the bioluminescent calcium indicator GFP-aequorin14,15, or a reporter of synaptic transmission, synapto-pHluorin16 has made the olfactory system of the fruitfly, Drosophila melanogaster, particularly accessible to neurophysiological imaging, complementing its comprehensively-described molecular, electrophysiological and neuroanatomical properties2,4,17. These reporters can be selectively expressed via binary transcriptional control systems (e.g. GAL4/UAS18, LexA/LexAop19,20, Q system21) in defined populations of neurons within the olfactory circuitry to dissect with high spatial and temporal resolution how odor-evoked neural activity is represented, modulated and transformed22-24.
Here we describe the preparation and analysis methods to measure odor-evoked responses in the Drosophila antennal lobe using G-CaMP25-27. The animal preparation is minimally invasive and can be adapted to imaging using wide-field fluorescence, confocal and two-photon microscopes.

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

We describe an established technique to measure and analyze odor-evoked calcium responses in the antennal lobe of living Drosophila melanogaster.

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 ...

科研

神经科学

Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. Pheromones are volatile or non-volatile short-lived molecules2 secreted and/or contained in biological fluids3,4, such as urine, a liquid known to be a main source of pheromones3. Pheromonal communication is implicated in a variety of key animal modalities such as kin interactions5,6, hierarchical organisations3 and sexual interactions7,8 and are consequently directly correlated with the survival of a given species9,10,11. In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO)10,12, a paired structure located at the base of the nasal cavity, and enclosed in a cartilaginous capsule. Each VNO has a tubular shape with a lumen13,14 allowing the contact with the external chemical world. The sensory neuroepithelium is principally composed of vomeronasal bipolar sensory neurons (VSNs)15. Each VSN extends a single dendrite to the lumen ending in a large dendritic knob bearing up to 100 microvilli implicated in chemical detection16. Numerous subpopulations of VSNs are present. They are differentiated by the chemoreceptor they express and thus possibly by the ligand(s) they recognize17,18. Two main vomeronasal receptor families, V1Rs and V2Rs19,20,21,22, are composed respectively by 24023 and 12024 members and are expressed in separate layers of the neuroepithelium. Olfactory receptors (ORs)25 and formyl peptide receptors (FPRs)26,27 are also expressed in VSNs.
Whether or not these neuronal subpopulations use the same downstream signalling pathway for sensing pheromones is unknown. Despite a major role played by a calcium-permeable channel (TRPC2) present in the microvilli of mature neurons28 TRPC2 independent transduction channels have been suggested6,29. Due to the high number of neuronal subpopulations and the peculiar morphology of the organ, pharmacological and physiological investigations of the signalling elements present in the VNO are complex.
Here, we present an acute tissue slice preparation of the mouse VNO for performing calcium imaging investigations. This physiological approach allows observations, in the natural environment of a living tissue, of general or individual subpopulations of VSNs previously loaded with Fura-2AM, a calcium dye. This method is also convenient for studying any GFP-tagged pheromone receptor and is adaptable for the use of other fluorescent calcium probes. As an example, we use here a VG mouse line30, in which the translation of the pheromone V1rb2 receptor is linked to the expression of GFP by a polycistronic strategy.

In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO). Here, an acute tissue slice preparation of VNO for performing calcium imaging is described. This physiological approach allows observations of subpopulations and/or individual neurons in a living tissue and is convenient for receptor-ligand identification.

成像遥感信息素在小鼠犁鼻急性组织切片的制备

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. ...

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 ...

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, ...

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

比率钙显像对单个神经元的行为线虫线虫

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...

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 ...

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a crucial component of intracellular signaling. The visualization of subcellular Ca2+ dynamics in vivo,&#160;where neurons can be studied in their native, intact circuitry, has proven technically challenging in complex nervous systems. The transparency and relatively simple nervous system of the nematode Caenorhabditis elegans enable the cell-specific expression and in vivo visualization of fluorescent tags and indicators. Among these are fluorescent indicators that have been modified for use in the cytoplasm as well as various subcellular compartments, such as the mitochondria. This protocol enables non-ratiometric Ca2+ imaging in vivo with a subcellular resolution that permits the analysis of Ca2+ dynamics down to the level of individual dendritic spines and mitochondria. Here, two available genetically encoded indicators with different Ca2+ affinities are used to demonstrate the use of this protocol for measuring relative Ca2+ levels within the cytoplasm or mitochondrial matrix in a single pair of excitatory interneurons (AVA). Together with the genetic manipulations and longitudinal observations possible in C. elegans, this imaging protocol may be useful for answering questions regarding how Ca2+ handling regulates neuronal function and plasticity.

Subcellular Imaging of Neuronal Calcium Handling In Vivo

The current methods describe a non-ratiometric approach for high-resolution, sub-compartmental calcium imaging in vivo in Caenorhabditis elegans using readily available genetically encoded calcium indicators.

体内神经元钙处理的亚细胞成像

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a ...

Calcium Imaging in Neurons

Calcium ions play an integral role in neuron function: They act as intracellular signals that can elicit responses such as altered gene expression and neurotransmitter release from synaptic vesicles. Within the cell, calcium concentration is highly dynamic due to the presence of pumps that selectively transport these ions in response to a variety of signals. Calcium imaging takes advantage of intracellular calcium flux to directly visualize calcium signaling in living neurons.This video begins with an overview of the key reagents used for this technique, known as calcium indicator dyes. The discussion includes an introduction to the commonly used dye Fura-2 and some basic principles behind how both ratiometric and non-ratiometric calcium indicators work. Next, a typical calcium imaging experiment is presented, from preparing the cells and dye to capturing and analyzing the fluorescent images. Finally, several experimental applications of calcium imaging are provided, such as the study of neuronal network activity and sensory processing.

Calcium Imaging in Neurons Using Fura-2

神经元钙成像

Calcium ions play an integral role in neuron function: They act as intracellular signals that can elicit responses such as altered gene expression and ...

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Drosophila In Vivo Calcium Imaging: A Method for Functional Imaging of Neuronal Activity

Source: Hancock, C. E., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/60288/" target="_blank">In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster</a>. J. Vis. Exp. (2019).
This video describes in vivo calcium imaging in Drosophila neurons using GCaMP. The featured protocol clip shows how to record changes in GCaMP fluorescence from mushroom body neurons during an olfactory conditioning experiment.

体内果蝇钙成像:一种神经元活动功能成像的方法

Source: Hancock, C. E., et al. In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster. J. Vis. Exp. (2019).
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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

摘要

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此视频中的章节

在气味诱发反应的钙成像果蝇触角叶

成像遥感信息素在小鼠犁鼻急性组织切片的制备

在GFP标记的下丘脑神经元的小鼠脑片成像钙反应

C.在体内神经元钙成像技术线虫

比率钙显像对单个神经元的行为线虫线虫

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

在体内小鼠劣质橄榄中的钙成像

体内神经元钙处理的亚细胞成像

神经元钙成像

体内果蝇钙成像:一种神经元活动功能成像的方法

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

摘要

探索更多视频

此视频中的章节

在气味诱发反应的钙成像果蝇触角叶

成像遥感信息素在小鼠犁鼻急性组织切片的制备

在GFP标记的下丘脑神经元的小鼠脑片成像钙反应

C.在体内神经元钙成像技术线虫

比率钙显像对单个神经元的行为线虫线虫

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

在体内 小鼠劣质橄榄中的钙成像

体内神经元钙处理的亚细胞成像

神经元钙成像

体内果蝇钙成像:一种神经元活动功能成像的方法

在体内小鼠劣质橄榄中的钙成像