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?...

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

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. ...

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, ...

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

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

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

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

完整的犁鼻器和副嗅球的体外制剂

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

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with enhanced [Ca2+]i signals. It has been proposed that astrocytes in turn regulate neurons and blood vessels through calcium-dependent mechanisms, such as the release of signaling molecules. However, [Ca2+]i imaging in entire astrocytes has only recently become feasible with genetically encoded calcium indicators (GECIs) such as the GCaMP series. The use of GECIs in astrocytes now provides opportunities to study astrocyte [Ca2+]i signals in detail within model microcircuits such as the striatum, which is the largest nucleus of the basal ganglia. In the present report, detailed surgical methods to express GECIs in astrocytes in vivo, and confocal imaging approaches to record [Ca2+]i signals in striatal astrocytes in situ, are described. We highlight precautions, necessary controls and tests to determine if GECI expression is selective for astrocytes and to evaluate signs of overt astrocyte reactivity. We also describe brain slice and imaging conditions in detail that permit reliable [Ca2+]i imaging in striatal astrocytes in situ. The use of these approaches revealed the entire territories of single striatal astrocytes and spontaneous [Ca2+]i signals within their somata, branches and branchlets. The further use and expansion of these approaches in the striatum will allow for the detailed study of astrocyte [Ca2+]i signals in the striatal microcircuitry.

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

The properties and functions of astrocyte intracellular Ca2+ signals in the striatum remain incompletely explored. We describe methods to express genetically encoded calcium indicators in striatal astrocytes using adeno-associated viruses of serotype 2/5 (AAV2/5), as well as procedures to reliably image Ca2+ signals within striatal astrocytes in situ.

成像细胞内Ca<sup&gt; 2+</sup&gt;从成年小鼠使用基因编码的钙指标纹状体星形胶质细胞信号

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with ...

Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. Retinal neurons in the network comprise of numerous subtypes. More than 10 subtypes of bipolar cells, ganglion cells, and amacrine cells have been identified by morphological studies. Multiple subtypes of retinal neurons are thought to encode distinct features of visual signaling, such as motion and color, and form multiple neural pathways. However, the functional roles of each neuron in visual signal processing are not fully understood. The patch clamp method is useful to address this fundamental question. Here, a protocol to record light-evoked synaptic responses in mouse retinal neurons using patch clamp recordings in dark-adapted conditions is provided. The mouse eyes are dark-adapted O/N, and retinal slice preparations are dissected in a dark room using infrared illumination and viewers. Infrared light does not activate mouse photoreceptors and thus preserves their light responsiveness. Patch clamp is used to record light-evoked responses in retinal neurons. A fluorescent dye is injected during recordings to characterize neuronal morphological subtypes. This procedure enables us to determine the physiological functions of each neuron in the mouse retina.

We will demonstrate how to prepare retinal slices from the mouse eye and record light responses in retinal neurons. The entire procedure is conducted in dark-adapted conditions.

记录光诱发神经元突触后反应在暗适应，小鼠视网膜切片准备利用膜片钳技术

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. ...

Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate goal is to image neuronal activity in vivo, one must be able to image activity of a single cell to ensure successful in vivo preparations. This procedure will describe how to image membrane potential in a single cell to provide a foundation to eventually image in vivo. Here we describe methods for imaging GEVIs consisting of a voltage-sensing domain fused to either a single fluorescent protein (FP) or two fluorescent proteins capable of F&#246;rster resonance energy transfer (FRET) in vitro. Using an image splitter enables the projection of images created by two different wavelengths onto the same charge-coupled device (CCD) camera simultaneously. The image splitter positions a second filter cube in the light path. This second filter cube consists of a dichroic and two emission filters to separate the donor and acceptor fluorescent wavelengths depending on the FPs of the GEVI. This setup enables the simultaneous recording of both the acceptor and donor fluorescent partners while the membrane potential is manipulated via whole cell patch clamp configuration. When using a GEVI consisting of a single FP, the second filter cube can be removed allowing the mirrors in the image splitter to project a single image onto the CCD camera.

A method for imaging changes in membrane potential using genetically encoded voltage indicators is described.

成像膜电位与基因编码荧光电压传感器的两种类型

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate ...

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

一个神经元蛋白在神经元使用遗传编码的非天然氨基酸的光控制

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the experimental time, and increases the number of animals that are utilized per study. This limitation specifically impacts protocols such as calcium imaging that require prolonged pre-incubation with bath-applied dyes. Exponential bacterial growth within 3 - 4 h after slicing is tightly correlated with a decrease in tissue health. This study describes a method for&#160;limiting the proliferation of bacteria in acute preparations to maintain viable neuronal tissue for prolonged periods of time (&#62;24 h) without the need for antibiotics, sterile procedures, or tissue culture media containing growth factors. By cycling the extracellular fluid through UV irradiation and keeping the tissue in a custom holding chamber at 15 - 16 &#176;C, the tissue shows no difference in electrophysiological properties, or calcium signaling through intracellular calcium dyes at &#62;24 h postdissection. These methods will not only extend experimental time for those using acute neuronal tissue, but will reduce the number of animals required to complete experimental goals, and will set a gold standard for acute neuronal tissue incubation.

Once removed from the body, neuronal tissue is greatly affected by environmental conditions, leading to eventual degradation of the tissue after 6 - 8 h. Using a unique incubation method, which closely monitors and regulates the extracellular environment of the tissue, tissue viability can be significantly extended for &#62;24 h.

对于电生理和钙成像急性神经组织的长时间培养

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

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.