作者想感谢以下为这项工作提供资金:大方的启动资金从生物医学工程系，美国华盛顿大学，麦中心系统神经科学经费，办公室美国海军研究资助（批准编号:N000141210089）到BR

1。气味准备工作和实施<ol><li>稀释在矿物油中的气味的解决方案（体积），以达到所需的浓度水平。存储一个20毫升的混合物，矿物油和的加臭剂，在60毫升的玻璃瓶。成的橡胶止动件（表19），一个从底部和其他从顶部插入两个注射器针头，以提供一个入口和一个出口管线。该橡胶塞密封的玻璃瓶中，并附加一个自定义设计的活性炭过滤器的入口线（ 图1A）。 </li><li>的碳过滤器是由使用两个6毫升注射器。切一半，丢弃的注射器的柱塞端。与棉和活性炭填充每个剩余的片前，将它们连接在一起使用热收缩管。 </li><li>出口管线连接的气味瓶（使用聚油管，ID0.86 ​​毫米）到塑料管（的Nalgene FEP套管，ID5.8毫米）提供一个恒定的气流通过天线（ 图1B </st荣&gt;）。 </li><li>直接的碳 - 过滤，除湿后的空气（载气流量为0.75升/分钟），使用的塑料管内放置几个厘米蝗虫天线朝向蝗虫。 </li><li>对于刺激气味，取代上述的气味在瓶中溶液的静态顶空恒定的体积（0.1升/分钟）。这是通过使用一个小型的泵（WPI，PV-820）注入等量的除湿后的空气进入瓶子。从气味瓶的蒸气被引导通过的气流管（ 图1B）上的出口管线。 </li><li>取出输送气味的气体，通过放置一个真空漏斗〜10厘米背后的蝗虫天线。 </li></ol> 2。准备拍摄单感器的蝗虫天线<ol><li>选择完全成熟的翅膀，不论男女，一个年轻的成年蝗虫，，但交配前阶段从拥挤的殖民地。为了抑制蝗虫，第一次截肢的腿。密封组织的粘合剂（Vetbond，3M）截肢网站。安全吨他蝗虫一个自定义的设计室搭着一小块电工胶带围绕其胸部（ 图2A）。 </li><li>在解剖显微镜下，使天线放置在蜡中的平台（ 图2A），一个浅的凹槽。将天线放置入槽中，并使其稳定在两端的天线（ 图2B;一个electrowaxer是用于熔化的蜡）使用蜡染蜡。 </li><li>接地电极（氯化银线）插入到胸部（〜1厘米的距离，从头部）。使用蜡染蜡到两个密封切口部位和持有的接地线（ 图2A）。 </li></ol> 3。单感器监测记录到的气味诱发反应的嗅觉受体神经元（ORNs） <ol><li>稳定的蝗虫天线放置在立体显微镜下（徕卡M205C）的振动隔离表（TMC）（ 图3A）。请确保记录感器的基础是明确的光年可见（ 图3B）。 </li><li>使用微管拆卸器（萨特P-1000），制作玻璃电极（阻抗3-10MΩ时，充满了蝗虫盐水，齿顶圆直径1-3微米），使用高硼硅玻璃毛细管（外径1.2毫米，ID0.69毫米）。 </li><li>玻璃电极放置到一个微量吸移管的保持器，其被安装到机动的微操纵器（萨特MP-285）。轻轻插入电极插入底座的感受器（ 图3B）。需要注意的是每个感器可以包含3-50 ORNs在蝗虫10。 </li><li>放大的信号（10000倍），使用一个AC放大器（草P-55）。过滤器0.3 -之间的信号为10.0 kHz和15 kHz的采样率（ 图3D）数据采集系统（LabVIEW软件，PCI-MIO-16E-4数据采集卡，美国国家仪器公司）收购。 </li></ol> 4。蝗虫触角叶和蘑菇体的录音解剖程序<ol><li>关注的restraini的纳克第2.1节中描述的步骤。 图4A和 B所示的蝗虫在定制设计室的位置。 </li><li>灌注生理盐水后，解剖过程中，建立一个蜡杯左右的蝗虫头。的蜡杯应该从正上方的口部，并超出围绕的两个天线之间的区域，如在图4C中所示的化合物的眼延伸。 </li><li>为了让天线通过的蜡杯，使用塑料（聚乙烯）管材（5毫米长，ID0.86​​毫米），双方的小隧道。确保塑料管材，可通过滑动的蜡杯。实现，后者是通过使用紧密环绕的塑料管的橡胶垫圈，但被连接到的蜡杯形（ 图4C）。 </li><li>使用环氧树脂，附加的基础上的触角的塑料管的底端。此步骤可确保天线即使在周围的地方举行角质层除去。 </li><li>保持从这时开始用生理盐水填充蜡杯。首先通过除去之间的两个天线（与前后轴对齐的矩形的长边）的中心的矩形区域。接着，在天线（ 图4D）的基础上，而不会干扰的化合物的眼睛和角质层去除角质层在相邻区域。 </li><li>用细镊子，轻轻地取下空气囊和大脑周围的脂肪体。在此步骤结束时，应该清楚地看到蝗虫大脑（ 图4D）。请注意，这个过程中的的嗅觉信息（浅黄色色素沉着）的大脑区域位于两个天线之间。 </li><li>在蝗虫肠道下方的大脑和身体长度的沿运行。为了防止潜在的不稳定准备的肠道运动，轻轻一拉肠，并把它用细剪刀。做一个小切口在腹部，以上的直肠，取出肠道的通过拉后肠粗钳。为了防止盐水泄漏，配合前腹部的切口部位的缝合线。 </li><li>使用涂有一层薄薄的蜡，以提升大脑和稳定11 如图4D所示期间电生理薄的金属丝制成的一个小平台。 </li><li>昆虫的大脑覆盖由一个薄的绝缘鞘，实验之前需要被移除。若要desheath大脑，轻轻地散布少量的一种酶（0.3-0.4毫克蛋白酶，Sigma Aldrich公司）的表面上使用细钳子9的大脑。 〜5-10秒的酶应用后，用生理盐水彻底冲洗大脑。使用超精细镊子轻轻捏，拉套，随后撕毁它打开了记录位置（AL和MB所示的图4E，F）。 </li></ol> 5。多单元录音和触角叶蕈状体<ol><li>放置的蝗虫制剂（ 图5A），在立体显微镜下对隔振表下的悬臂支架暂停。 </li><li>整个实验过程中保持恒定的盐水灌注率（约0.04升/小时）。使用氯化银线作为接地电极浸没在盐水充注蜡杯。 </li><li> PN录音，使用一个16通道的，硅探头（NeuroNexus技术，项目，中央与特区关系的TET-3MM-150-150-121-A16， 图5B）。每次实验前，电极电镀金实现在200〜300kΩ范围的阻抗。使用图7中所示的电路用于电镀。 </li><li>定位电极靠近触角叶的表面，并轻轻地插入到使用手动微操纵器（WPI，M3301R）（ 图5D）的组织。 </li><li>在〜10μm的步骤推进电极。等待2-3分钟，在每一个步骤，并评估收购D信号质量。在一个理想的录制现场，将拿起细胞外信号由多个记录通道，将有一个高的信号噪声比（SNR&gt; 3-5倍，噪音SDS）。 </li><li>对于KC录音，使用一个定制的双绞线四极管（ 图5C，一步一步的制作过程，在第6）。所讨论的，在步骤5.3，电镀这些电极。 KC胞体被限制的表面层的MB 8放置的MB的表面上（ 图5E）的四极管。 </li><li> PN和KC录音可以同时从同一蝗虫制备，如在图5A中示意性地示出。 </li><li>至少等待15分钟后，发现记录的位置，让稳定的电极。 </li><li>收购在15千赫之间0.3-6千赫，过滤器，所有细胞外信号放大（10000次）使用的是16通道AC放大器（生物电子商店，加州理工学院，帕萨迪纳，CA）（图6A，B）。 </li></ol> 6。 KC录音程序，使绞丝电极<ol><li>要设计一个多单元的电极为KC录音，使用绝缘镍铬丝（RO800，0.0005"丝）8。 </li><li>如果需要8个电极，然后包裹的导线周围的一块长10-15厘米的硬纸板4倍。纸板的端部可以用塑料管覆盖，以防止导线从切割边缘。虽然包装，确保有一点松弛，但保证线未拉紧。小心处理线，因为它打破容易。 </li><li>一束导线合在一起在纸板的顶部，并用一条胶带（时间带，T-534-RP）保持在一起。切的股线在其另一端。删除钢绞线和组线切端，以及使用另一块的磁带。 </li><li>使用滴度板振动筛（赛默飞世尔科技，型号4625Q），风绞合在一起（〜72转/分3分钟），以形成一个绞合线。录音股的底部可以夹在滴定板转子和顶部可以夹在一个繁荣的立场。卷绕时，导线应具有稍有懈怠，但不绷紧。 </li><li>熔体的绝缘层，用热枪（韦勒6966C）坚持个人绞合在一起，形成一个单一的线。 3-4缓慢通行证（3-4秒）的线的长度上应该是足够的，以加热并熔化的绞合在一起。然后松开线的底部，并允许它放松。修剪线附近的两端取出磁带。 </li><li>一端插入的导线通过一个5-6厘米长，玻璃毛细管（外径1.0毫米，编号0.58毫米）。取笑除了双绞线的一端，用细镊子，非常简要用火焰烧毁年底，除去包衣。这一步必须做仔细的电线暴露在火焰中的时间过长将导致股熔化和纠结。 </li><li>轻轻分开8股的火烧端一个第二焊每股分别采用8引脚IC插座。外套的IC插座的顶部与环氧持有的股线和玻璃毛细管到位。还放置一小滴的环氧树脂，在其另一端的毛细管按住丝的地方（ 图5C）。 </li><li>最后，切割导线的前端在一个45度的角度与硬质合金剪刀从毛细管尖端约0.5厘米。 </li></ol>

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antennal+Lobe+Processing+Correlates+to+Moth+Olfactory+Behavior.">Antennal Lobe Processing Correlates to Moth Olfactory Behavior.</a> Journal of Neuroscience. 32, 5772-5782 (2012).</li><li>Silbering, A. F., Bell, R., Galizia, C. G., Benton, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+Imaging+of+Odor-evoked+Responses+in+the+Drosophila+Antennal+Lobe.">Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe.</a> J. Vis. Exp. (61), e2976 (2012).</li><li>Skiri, H. T., Galizia, C. G., Mustaparta, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Representation+of+Primary+Plant+Odorants+in+the+Antennal+Lobe+of+the+Moth+Heliothis+virescens+Using+Calcium+Imaging.">Representation of Primary Plant Odorants in the Antennal Lobe of the Moth Heliothis virescens Using Calcium Imaging.</a> Chemical Senses. 29, 253-267 (2004).</li></ol>

没有利益冲突的声明。

大多数感官刺激，唤起组合分布在歌舞团的神经元的反应。因此，同时监控多神经元活动是有必要了解如何刺激特定的信息在大脑中的神经回路表示和处理。在这里，我们已经证明细胞外多单位记录技术的气味诱发反应的特点前三加工中心以及昆虫嗅觉通路。我们注意到，这里所介绍的技术已嗅觉编码的以前的研究中使用的一些，并成为一个标准的实践在此字段中3,6,13-17。相结合的技术?...

<table border="1"><tbody><tr><td> 名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td></td><td></td><td></td><td> 电设备 </td></tr><tr><td> AC放大器</td><td> GRASS </td><td>型号P55 </td><td>单感器录音的</td></tr><tr><td>音频显示器（型号3300） </td><td> AM系统</td><td> 940000 </td><td></td></tr><tr><td>定做16个信道，前置放大器和放大器</td><td>卡尔。技术。生物电子商店</td><td></td><td> AL和MB录音</td></tr><tr><td>数据采集​​单元</td><td>美国国家仪器公司</td><td> BNC-2090 </td><td></td></tr><tr><td>光纤灯</td><td> WPI </td><td> SI-72-8 </td><td> &lt;/ TD&gt; </tr><tr><td>光源115 V </td><td> WPI </td><td> NOVA </td><td></td></tr><tr><td>手动显微操纵器</td><td> WPI </td><td> M3301R </td><td>蝗虫大脑录音的</td></tr><tr><td> Stereomicroscope1繁荣的立场</td><td>徕卡</td><td> M80 </td><td>蝗虫大脑录音的</td></tr><tr><td> Stereomicroscope2 </td><td>徕卡</td><td> M205C </td><td>单感器录音的</td></tr><tr><td>振动隔离表</td><td> TMC </td><td> 63-500系列</td><td></td></tr><tr><td>电动微操作机器人</td><td>萨特仪器</td><td> MP285 / T </td><td></td></tr><tr><td>示波器</td><td>泰克公司</td><td> TD2014B </td><td></td></tr><tr><td></td><td></td><td></td><td> 电极/建筑工具 &lt;/ TD&gt; </tr><tr><td> 16通道的电极</td><td> NeuroNexus </td><td>中央与特区关系的TET-3MM-150-121 </td><td>触角叶的录音</td></tr><tr><td>高硼硅毛细管长丝，ID0.69毫米</td><td>萨特仪器</td><td> BF120-69-10 </td><td>用于制造玻璃电极</td></tr><tr><td>微管拉马</td><td>萨特仪器</td><td> P-1000 </td><td></td></tr><tr><td>函数信号发生器</td><td>万用表仓库</td><td> SG1639A </td><td>镀金电极</td></tr><tr><td>镀金溶液（非氰化物） </td><td> SIFCO产业</td><td> NC SPS 5355 </td><td></td></tr><tr><td>阻抗测试仪</td><td>比克电子有限公司</td><td> IMP-2 </td><td>镀金电极</td></tr><tr><td>开关旋转</td><td> ELECTROSWITCH </td><td> C7D0123N </td><td>金PLating电极</td></tr><tr><td>脉冲隔离器</td><td> WPI </td><td> A365 </td><td>镀金电极</td></tr><tr><td> Q系列电极持有人</td><td>华纳仪器</td><td> 64-1091 </td><td></td></tr><tr><td>银线直径0.010" </td><td> AM系统</td><td> 782500 </td><td>接地电极</td></tr><tr><td> 8引脚DIP IC插座</td><td> DIGIKEY </td><td> ED90032-ND </td><td></td></tr><tr><td>高硼硅毛细管长丝，ID0.58毫米</td><td>华纳仪器</td><td> 64-0787 </td><td>双绞线四极管建设</td></tr><tr><td>热风枪</td><td>韦勒</td><td> 6966C </td><td></td></tr><tr><td> Rediohm-800线</td><td>康泰尔精密技术</td><td> PF002005 </td><td></td></tr><tr><td>滴度板振动筛</td><td>热科学的</td><td> 4625Q </td><td>扭线</td></tr><tr><td>硬质合金剪刀，4.5" </td><td>生物医学研究INSTR </td><td> 25-1000 </td><td>为削减扭曲四极线</td></tr><tr><td>细点镊子</td><td> HECO </td><td> 91-EF5-SA </td><td>除了戏弄四极管电线</td></tr><tr><td></td><td></td><td></td><td> 气味交货 </td></tr><tr><td> 6毫升注射器</td><td>肯德尔</td><td> 1180600777 </td><td>定制设计的活性炭过滤器</td></tr><tr><td>布朗气味瓶</td><td>费舍尔</td><td> 08-912-165 </td><td></td></tr><tr><td>木炭</td><td> <a href="http://BuyActivatedCharcoal.com" target="_blank">BuyActivatedCharcoal.com</a> </td><td> GAC-48C </td><td></td></tr><tr><td>干燥剂</td><td>燥石膏</td><td> 23005 </td><td></td></tr><tr><td>燥石膏气体干燥罐</td><td>菲舍尔科学</td><td> 09-204 </td><td></td></tr><tr><td>热缩管</td><td> 3M </td><td> EPS-200 </td><td>气味过滤器准备</td></tr><tr><td>注射针铝轮毂，计19 </td><td>肯德尔</td><td> 8881-200136 </td><td>用于提供入口和出口管线的气味瓶</td></tr><tr><td>矿物油</td><td> Mallinckrodt公司化学品</td><td> 6357-04 </td><td>气味稀释</td></tr><tr><td> NALGENE塑料管材，890 FEP </td><td> Thermo Scientific的</td><td> 8050-0310 </td><td>载波气体输送</td></tr><tr><td>气动picopump </td><td> WPI </td><td>系统pv820 </td><td>气味交付</td></tr><tr><td>聚乙烯气管内径0.86毫米</td><td> Intramedic </td><td> 427421 </td><td>气味瓶出口CONNECTI组件和生理盐水丰富的管</td></tr><tr><td>瓶塞</td><td>实验室纯</td><td> 97041 </td><td>密封气味瓶</td></tr><tr><td>时间的录像带</td><td> PDC </td><td> T-534-RP </td><td></td></tr><tr><td>管的luer </td><td> Cole-Parmer公司</td><td> 30600-66 </td><td></td></tr><tr><td>真空集热管</td><td>麦克马斯特 - 卡尔</td><td> 5488K66 </td><td></td></tr><tr><td></td><td></td><td></td><td> 准备工作/夹层 </td></tr><tr><td> 100×15毫米陪替氏培养皿</td><td> VWR国际</td><td> 89000-304 </td><td></td></tr><tr><td> 18 AWG铜绞线</td><td> LAPP KABEL </td><td> 4510013 </td><td>电线绝缘层被用作橡胶垫片</td></tr><tr><td> 22 AWG绞合联播线</td><td> AlphaWire </td><td> 1551 </td><td>大脑平台</td></tr><tr><td>蜡染蜡</td><td>提花</td><td> 7946000 </td><td></td></tr><tr><td>牙齿周边蜡， </td><td>亨利·施恩牙科</td><td> 6652151 </td><td></td></tr><tr><td> Electrowaxer </td><td> Almore国际</td><td> 66000 </td><td></td></tr><tr><td>环氧树脂，5分钟</td><td> Permatex </td><td> 84101 </td><td></td></tr><tr><td>注射针铝轮毂</td><td>肯德尔</td><td> 8881-200136 </td><td></td></tr><tr><td>从灰色链霉菌蛋白酶</td><td> Sigma-Aldrich公司</td><td> P5147 </td><td> ，为desheathing蝗虫大脑</td></tr><tr><td>缝合线的非无菌</td><td>费舍尔</td><td> NC9087024 </td><td>绑在腹部肠切除后</td></tr><tr><td> Vetbond </td><td> 3M </td><td> 1469SB </td><td>密封是putation网站</td></tr><tr><td>杜蒙＃1钳（粗） </td><td> WPI </td><td> 500335 </td><td></td></tr><tr><td>杜蒙＃5级钛金属钳（精） </td><td> WPI </td><td> 14096 </td><td></td></tr><tr><td>杜蒙5SF钳（超精细） </td><td> WPI </td><td> 500085 </td><td> desheathing蝗虫大脑</td></tr><tr><td> 10厘米解剖剪刀</td><td> WPI </td><td> 14393 </td><td>为消除腿和翅膀</td></tr><tr><td> Vannas剪刀（精） </td><td> WPI </td><td> 500086 </td><td>去除角质层，切割前肠， </td></tr><tr><td></td><td></td><td></td><td> 盐水绮靡 </td></tr><tr><td>扩展率流量调节器</td><td>摩尔医疗</td><td> 69136 </td><td>调节盐水流量</td></tr><tr><td>静脉注射设置Ÿ注射部位</td><td>摩尔医疗</td><td> 73190 </td><td>调节盐水流量</td></tr></tbody></table>

multi-unit recording methods to characterize neural activity in the locust (schistocerca americana) olfactory circuits

嗅觉检测和解释的许多生物的生存是至关重要的。值得注意的是，跨门类的物种都有着惊人的相似嗅觉系统在进化的时间1，化学传感，生物方法进行了优化。在昆虫的嗅觉系统，增味剂，转嗅觉受体神经元（ORN）的天线，这将化学刺激的动作电位的列车。从ORNs的感官输入，然后转发至触角叶（AL;类似于脊椎动物的嗅球结构）。在AL，神经表征气味采取的形式分布在歌舞团的主要神经元（PNS，也被称为投射神经元）2,3的时空放电模式。随后处理的AL输出由肯扬细胞（角质形成）在下游蘑菇体（MB），相关联的结构与嗅觉记忆和学习4,5。她的E，我们提出了电生理记录技术，在这些嗅觉电路的监控气味诱发的神经反应。 首先，我们提出一个单一的感受器记录的方法来研究人口的ORNs 6,7气味诱发反应。我们讨论使用生理盐水填充尖锐的玻璃吸管作为电极细胞外监控ORN反应。接下来，我们提出了一种方法来使用商业16通道的电极细胞外PN反应。类似的方法使用一个定制的8路双绞线四极管展示了肯扬细胞记录8。我们提供详细的实验装置，目前这些技术代表记录的痕迹。

在图3D中示出一个单一的ORN两种不同醇的气味诱发反应。根据记录位置（放置的电极）的多单元录音感器类型，可以实现的。 图6A中所示的原料外从AL记录波形。可以观察到的动作电位或尖峰的幅度变化，来自不同的PN，在此电压跟踪。虽然蝗虫触角叶投射神经元有兴奋和抑制神经元，唯一的PN生成钠尖峰，可以检测到细胞外3。这一观察表明?...

Watch this Scientific Journal Video about Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits at JoVE.com

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

我们证明细胞外的多单元的记录技术中的前三个阶段的无脊椎动物的嗅觉路径表征气味诱发反应的变化。这些技术可以很容易地适应以及其他神经系统检查合奏活动。

多单位记录方法来描述蝗虫的神经活动（<em&gt; Schistocerca美洲</em&gt;）嗅觉电路

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

multi-unit-recording-methods-to-characterize-neural-activity-locust

Research

JoVE Journal

Neuroscience

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

26.9K 次观看.  Washington University in St. Louis. 我们证明细胞外的多单元的记录技术中的前三个阶段的无脊椎动物的嗅觉路径表征气味诱发反应的变化。这些技术可以很容易地适应以及其他神经系统检查合奏活动。

视频: Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also propagation of suprathreshold spiking activity involves populations of neurons. Empirical studies addressing cortical function directly thus require recordings from populations of neurons with high resolution. Here we describe an optical method and a deconvolution algorithm to record neural activity from up to 100 neurons with single-cell and single-spike resolution. This method relies on detection of the transient increases in intracellular somatic calcium concentration associated with suprathreshold electrical spikes (action potentials) in cortical neurons. High temporal resolution of the optical recordings is achieved by a fast random-access scanning technique using acousto-optical deflectors (AODs)1. Two-photon excitation of the calcium-sensitive dye results in high spatial resolution in opaque brain tissue2. Reconstruction of spikes from the fluorescence calcium recordings is achieved by a maximum-likelihood method. Simultaneous electrophysiological and optical recordings indicate that our method reliably detects spikes (&gt;97% spike detection efficiency), has a low rate of false positive spike detection (&lt; 0.003 spikes/sec), and a high temporal precision (about 3 msec) 3. This optical method of spike detection can be used to record neural activity in vitro and in anesthetized animals in vivo3,4.

Understanding the function of the vertebrate central nervous system requires recordings from many neurons because cortical function arises on the level of populations of neurons. Here we describe an optical method to record suprathreshold neural activity with single-cell and single-spike resolution, dithered random-access scanning. This method records somatic fluorescence calcium signals from up to 100 neurons with high temporal resolution. A maximum-likelihood algorithm deconvolves the underlying suprathreshold neural activity from the somatic fluorescence calcium signals. This method reliably detects spikes with high detection efficiency and a low rate of false positives and can be used to study neural populations in vitro and in vivo.

阈上的神经活动的单细胞和单穗的分辨率的光学的方法记录

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also ...

科研

神经科学

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

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

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

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

用气相色谱 - 多单元的录音（GCMR）昆虫触角叶嗅觉挥发物的鉴定

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

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 ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

运行轮活动在啮齿类动物的昼夜节律的记录和分析

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the underlying neuronal components in the circuits1-6. Application of optogenetics7,8 in the larval neural circuit allows us to manipulate neuronal activity in spatially and temporally patterned ways9-13. Typically, specimens are broadly illuminated with a mercury lamp or LED, so specificity of the target neurons is controlled by binary gene expression systems such as the Gal4-UAS system14,15. In this work, to improve the spatial resolution to "sub-genetic resolution", we locally illuminated a subset of neurons in the ventral nerve cord using lasers implemented in a conventional confocal microscope. While monitoring the motion of the body wall of the semi-intact larvae, we interactively activated or inhibited neural activity with channelrhodopsin16,17 or halorhodopsin18-20, respectively. By spatially and temporally restricted illumination of the neural tissue, we can manipulate the activity of specific neurons in the circuit at a specific phase of behavior. This method is useful for studying the relationship between the activities of a local neural assembly in the ventral nerve cord and the spatiotemporal pattern of motor output.

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Here we describe a protocol for optogenetic manipulation of motoneuronal activity while monitoring changes in motor output (muscle contraction) in semi-intact Drosophila larvae using lasers within a conventional confocal microscope. This technique enables researchers to achieve local perturbation of neural activity in a few neuromeres to elucidate the dynamics of motor circuits.

光遗传学的扰动激光照射半完好的神经活动<em&gt;果蝇</em&gt;幼虫运动

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the ...

Neural Activity Propagation in an Unfolded Hippocampal Preparation with a Penetrating Micro-electrode Array

This protocol describes a method for preparing a new in vitro flat hippocampus preparation combined with a micro-machined array to map neural activity in the hippocampus. The transverse hippocampal slice preparation is the most common tissue preparation to study hippocampus electrophysiology. A longitudinal hippocampal slice was also developed in order to investigate longitudinal connections in the hippocampus. The intact mouse hippocampus can also be maintained in vitro because its thickness allows adequate oxygen diffusion. However, these three preparations do not provide direct access to neural propagation since some of the tissue is either missing or folded. The unfolded intact hippocampus provides both transverse and longitudinal connections in a flat configuration for direct access to the tissue to analyze the full extent of signal propagation in the hippocampus in vitro. In order to effectively monitor the neural activity from the cell layer, a custom made penetrating micro-electrode array (PMEA) was fabricated and applied to the unfolded hippocampus. The PMEA with 64 electrodes of 200 &#181;m in height could record neural activity deep inside the mouse hippocampus. The unique combination of an unfolded hippocampal preparation and the PMEA provides a new in-vitro tool to study the speed and direction of propagation of neural activity in the two-dimensional CA1-CA3 regions of the hippocampus with a high signal to noise ratio.

We have developed an in vitro unfolded hippocampus which preserves CA1-CA3 array of neurons. Combined with the penetrating micro-electrode array, neural activity can be monitored in both the longitudinal and transverse orientations. This method provides advantages over hippocampal slice preparations as the propagation in the entire hippocampus can be recorded simultaneously.

神经活动的传播的展开海马准备具有穿透微电极阵列

This protocol describes a method for preparing a new in vitro flat hippocampus preparation combined with a micro-machined array to map neural activity in ...

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and awake animals. Therefore, methods allowing the manipulation of synaptic systems in awake animals are required in order to determine the contribution of synaptic inputs to neuronal processing unaffected by anesthetics. Here, we present methodology for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. By combining these procedures, we performed microiontophoretic injections of gabazine to selectively block GABAA receptors in neurons of the inferior colliculus of head-restrained mice. Gabazine successfully modified neural response properties such as the frequency response area and stimulus-specific adaptation. Thus, we demonstrate that our methods are suitable for recording single-unit activity and for dissecting the role of specific neurotransmitter receptors in auditory processing.
The main limitation of the described procedure is the relatively short recording time (~3 hr), which is determined by the level of habituation of the animal to the recording sessions. On the other hand, multiple recording sessions can be performed in the same animal. The advantage of this technique over other experimental procedures used to manipulate the level of neurotransmission or neuromodulation (such as systemic injections or the use of optogenetic models), is that the drug effect is confined to the local synaptic inputs to the target neuron. In addition, the custom-manufacture of electrodes allows adjustment of specific parameters according to the neural structure and type of neuron of interest (such as the tip resistance for improving the signal-to-noise ratio of the recordings).

We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest.

神经元电活动外记录与神经活性物质在清醒小鼠微电泳应用相结合

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and ...

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. However, there is piling evidence that olfaction is not solely limited to chemical sensitivity, but also includes temperature sensitivity. Premetamorphic Xenopus laevis are translucent animals, with protruding nasal cavities deprived of the cribriform plate separating the nose and the olfactory bulb. These characteristics make them well suited for studying olfaction, and particularly thermosensitivity. The present article describes the complete procedure for measuring temperature responses in the olfactory bulb of X. laevis larvae. Firstly, the electroporation of olfactory receptor neurons (ORNs) is performed with spectrally distinct dyes loaded into the nasal cavities in order to stain their axon terminals in the bulbar neuropil. The differential staining between left and right receptor neurons serves to identify the &#947;-glomerulus as the only structure innervated by contralateral presynaptic afferents. Secondly, the electroporation is combined with focal bolus loading in the olfactory bulb in order to stain mitral cells and their dendrites. The 3D brain volume is then scanned under line-illumination microscopy for the acquisition of fast calcium imaging data while small temperature drops are induced at the olfactory epithelium. Lastly, the post-acquisition analysis allows the morphological reconstruction of the thermosensitive network comprising the &#947;-glomerulus and its innervating mitral cells, based on specific temperature-induced Ca2+ traces. Using chemical odorants as stimuli in addition to temperature jumps enables the comparison between thermosensitive and chemosensitive networks in the olfactory bulb.

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Here we describe a protocol for measuring and analyzing temperature responses in the olfactory bulb of Xenopus laevis. Olfactory receptor neurons and mitral cells are differentially stained, after which calcium changes are recorded, reflecting a sensitivity of some neural networks in the bulb to temperature drops induced at the nose.

通过的嗅球钙监控录像的变化温度引起的神经元电活动<em&gt;非洲爪蟾</em

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

时间依赖性增加对微电极阵列刺激神经元细胞培养物的网络响应

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory Sensory Neuron (OSN) is a bipolar cell with a single dendrite and a single unbranched axon. An OSN expresses only one Olfactory Receptor (OR) gene, OSNs expressing a given type of OR converge their axons to a few sets of invariant glomeruli in the Olfactory Bulb (OB). A remarkable feature of OSN projection is that the expressed ORs play instructive roles in axonal projection. ORs regulate the expression of multiple axon-sorting molecules and generate the combinatorial molecular code of axon-sorting molecules at the OSN axon termini. Thus, to understand the molecular mechanisms of OR-specific axon guidance mechanisms, it is vital to characterize their expression profiles at the OSN axon termini within the same glomerulus. The aim of this article was to introduce methods for collecting as many glomeruli as possible on a single OB section and for performing immunostaining using multiple antibodies. This would allow the comparison and analysis of the expression patterns of axon-sorting molecules without staining variation between OB sections.

Olfactory sensory neurons express a wide variety of axon-sorting molecules to establish proper neural circuitry. This protocol describes an immunohistochemical staining method to visualize combinatorial expressions of axon-sorting molecules at the axon termini of olfactory sensory neurons.

嗅球的四次免疫染色用于嗅觉感觉轴突分子识别码的可视化

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory ...

多单位记录方法来描述蝗虫的神经活动（

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