这项工作得到了NIH-NICHD向MS的校内资助。我们感谢NIH-NIMH仪器核心设施的G.Dold和T. Talbot，协助设计了促销系统。

注意: Manduca释放的细粉，可能会过敏，因此建议使用实验室安全手套和面罩。 解剖Manduca sexta以揭示上颌神经和经济特区<ol><li> 根据以下特点选择适当的性别蛾:羽化后三天具有一般健康的外观（翅膀应完全伸展，长鼻和天线应完好无损）。 <ol><li>将飞蛾单独放在塑料杯中进行运输。 </li></ol></li><li> 将飞蛾插入聚丙烯管。 注意:我们建议在通风橱中执行此步骤，以防止飞蛾的粉状鳞屑扩散。 <ol><li>管子应该比蛾体长一点（ 图1A ）。管可以通过切割15mL聚丙烯管制成。 </li> &lt;li&gt;推动蛾直到头部暴露出来，然后将绷紧的纸巾插入管的另一端，以帮助保持蛾不动（ 图1A ）。 </li></ol></li><li> 通过在其上吹入加压空气从头颅的头部（腹侧和背面）移开头发。 <ol><li>可以通过将注射器（1mL与针头，约1.4mm的ID，尖锐尖端移除）连接到加压空气源来制造空气喷射。 注意:取下头发后，可以在通风橱外进行以下所有步骤。 </li></ol></li><li> 一旦大部分头发被去除，将管子放入一个保持室，使头部的背部朝上， 如图1B所示。 <ol><li>在培养皿上，使用建模粘土建立约7厘米长，2.5厘米高的三角形底座（ 图1B ）。 </li></ol></li></ol> 图1 : Manduca解剖室的制备。 （ A ）蛾被夹在管中。头部在一端露出，而另一端用棉纸堵塞。 （ B ）显示了由培养皿和模型粘土制成的解剖室。头部的背部面朝上。 <a href="//ecsource.jove.com/files/ftp_upload/55868/55868fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <ol start="5"><li> 保护天线和长鼻子。 <ol><li>通过用剃刀刀片将移液管尖端（黄色尖端，1-200μL）切割成3片约0.5cm长的3个小管来制备。内径应足够大，以便天线和长鼻适配。 </li><li>准备一个钩子弯曲线（22 AWG，约7厘米长）。延长蛾的长鼻，然后将其插入一根小管中，用线钩将其插入，直到管到达长鼻的近端。 </li><li>用坚固的蜡染蜡固定小管（使用例如牙科电动蜡器来熔化和引导蜡）到飞蛾的头部胶囊的背部（ 图2A左侧）。 </li><li>将每个天线放入管中，并用蜡蜡固定管， 如图2A （左）所示。小心避免用热蜡损坏天线。 </li></ol></li><li> 通过切割覆盖大脑前表面的肌肉（ 即颊侧压缩肌）来稳定大脑抵抗运动。 <ol><li>在解剖显微镜下，使用微型解剖剪刀或从剃须刀芯片制成的迷你型号胶粘到牙签上以打开头部胶囊在正好在长鼻子下面的小切口（ 图2A ，左上部插图）。 </li><li>使用微型剪刀剪切颊侧压缩肌肉（见图25 ）。由于肌肉不容易看到，建议进行以下行为测试以确认其已被切割。 </li><li>在蒸馏水中稀释蔗糖以达到1M溶液。 </li><li>使用移液管将大约200μL的蔗糖溶液送至长鼻的2/3远端，观察长鼻5分钟。如果肌肉正确切割，昆虫不应该能够延伸或移动长鼻子。 </li><li>涂一层熔化的蜡染蜡以密封头胶囊中的开口。 </li></ol></li><li>将昆虫翻转过来，头盖的腹侧现在正在朝上。 </li><li> 在解剖过程中和之后灌注盐水，在头部使用的腹侧附近建立蜡杯g电蜡器:在夹层显微镜下，通过沿着头部的前部施加第一排蜡，开始建立杯子，向后移动。将吸管和天线管保持在杯内（ 图2A右侧）。 <ol><li>继续向外向上建立杯子，直到达到眼睛的水平。 </li></ol></li><li>使用镊子取两个唇唇中的一个，然后将其放在侧面，并通过添加更多的熔融蜡将其固定在蜡杯中。与其他上颌骨相同（ 图2A右图）。 </li><li> 密封蜡杯和管中的任何开口。 注意:在此步骤中，使用环氧树脂，因为它有助于轻松地密封蜡和管中的间隙，并避免任何与天线和长鼻相关的热相关损害。 <ol><li>使用牙签将塑料混合盘中的二元环氧树脂混合。保留混合盘。 </li><li>用牙签涂薄环氧树脂层到杯子的外部和内部（ 图2B ）。 </li><li>在含有长鼻子和天线的管中填充环氧树脂的空白空间，并用足够的环氧树脂填充颈部周围聚丙烯管的一部分，以将其牢固地固定（ 图2B ）。 </li><li>将环氧树脂干燥约20分钟。要检查这一点，测试塑料混合盘中剩余的环氧树脂，以确保它是固体，不再粘稠。 </li><li>用曼杜拉生理盐水填充蜡杯28 ，等待几分钟，确保没有泄漏。如果泄漏可见，请尝试用更多的热蜡和/或环氧树脂来密封。 </li></ol></li></ol><img alt="图2" class="xfigimg" src="/files/ftp_upload/55868/55868fig2v2.jpg" /> 图2 :准备人/ em&gt;为解剖。 （ A ）天线和长鼻被保护在由移液管尖端切割成长度约0.5厘米的三片的小管内。 （左图，大图）管子被固定在头部背部附近的熔融蜡染蜡。 （左图，插图）为了去除颊侧压缩机肌肉，通过在大图像中由虚线所示的小切口打开头部胶囊的背侧。 （右图）在解剖过程和记录实验期间，围绕头部腹侧构建蜡杯作为灌注盐水的储存器。 （ B ）环氧树脂用于密封蜡杯的底部与头部的背部（左图）之间的间隙，以及蜡和包含天线和长鼻的管，包括管的开口（右面板）。 <a href="//ecsource.jove.com/files/ftp_upload/55868/55868fig2v2large.jpg" target="_blank">请点击这里t</a>o查看这个数字的较大版本。 <ol start="11"><li>在夹层显微镜下，使用微分解剪刀切断阴唇。 </li><li>使用微分解剪刀或迷你斧头通过四个切口打开头部胶囊的腹侧:沿着从每个眼睛后面到前面的角质层两个切口，一个沿着头部背面的角质层切割，并且沿着头尖附近的头部前角切开一个角质层（ 图3A ）。 </li><li>使用微分解镊子轻轻拉开角质层以暴露大脑。 </li><li>通过使用两个尖锐的微型解剖镊子缓慢地小心地去除脂肪组织和覆盖经济特区的气管。避免对大脑或神经造成任何损伤（如上颌神经，视神经，子宫颈结缔组织）。通过频繁更换盐水溶液冲洗大脑，并经常调整光线，以提高可见度。注意:这可能是di的最关键部分ssection;很容易通过拉扯或压碎上颌神经而无意中损伤。 注意:此时，SEZ和上颌神经应清晰可见（ 图3B- 3C ）。然而，脑和上颌神经被薄的鞘覆盖。 </li><li>为了去除护套，用溶解在盐水中的10％（w / v）胶原酶/分散液代替蜡杯中的盐水，并将其放置在适当位置5分钟。然后，用盐水冲洗脑数次后，用超细微解剖镊子通过上颌神经从SEZ向上拉鞘，轻轻取出鞘。 </li><li>开始用新鲜的盐水灌注蜡杯。将管子从盐水滴入线放入蜡杯中，并用熔化的蜡将其固定在那里。 </li></ol><img alt="图3" class="xfigimg" src="/files/ftp_upload/55868/55868fig3.jpg" /> 图ure 3 : 人体解剖手术。 （ A ）去掉唇唇，通过点划线所示的四个切口打开头部胶囊。 （ B ）打开头部胶囊并去除脂肪组织和气管后，放大图像，允许上颌神经（MxNs）和亚食管区域的可视化（SEZ，一个术语，指的是大脑下面的区域食管）。上颌神经从长鼻中的味觉受体神经元（GRN）携带轴突到经济特区。 （ C ） 人体大脑的示意图。 <a href="//ecsource.jove.com/files/ftp_upload/55868/55868fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> 丰富的运送系统<ol><li>味觉传递系统由四个主要元素组成:水源，滋味歧管，颜色传感器和含有长鼻子，均连接到刚性塑料管（ 图4A- 4B ）。要构建该系统，请按照图4B所示的步骤进行。使用长约6厘米（ID为0.3厘米）的刚性塑料管。 </li><li>使用烙铁，在要放置长鼻子的管中形成一个小孔（ 图4A- 4B ）。 </li><li>为确保不同动物的长颈鹿总是放置在相同的位置，将塑料筛网材料（网孔，大约0.1厘米的孔尺寸）插入管中（ 图4A- 4B ）。用剃刀刀片或旋转工具将刚性管切成长约5毫米的长鼻孔。将网格放在两根管子之间。固定网眼，并通过在管的外侧涂上环氧树脂来重新连接管。将环氧树脂干燥约20分钟。 </li><li>为了促进交付使用加压灌注系统与所需的许多滋味管相连接（见下文）。使用烙铁在网格上方约1厘米处的刚性管中形成一个小孔。将灌注系统（ID 0.86 mm）的输出管插入刚性管，并通过施加环氧树脂将其固定在那里（ 图4A -4B ）。 </li><li>使用蠕动泵输送恒定流量（约40 mL / min）的蒸馏水。将其与硅胶管连接到刚性管（ 图4A -4C ）。将刚性管的另一侧连接到另一个硅管，以将输出引导到大型废物容器中（ 图4A -4C ）。 </li><li>为了提供滋味，将压缩空气注入灌注系统的歧管。这可以通过例如使用由定时电压脉冲输入控制的气动泵（10psi）来实现，以将压缩空气注入到清液中<html> d管含有所需的促味剂。 1s脉冲喷出大约0.5mL的促味剂。 </li></ol> 3.促进剂的制备和监测<ol><li>将蒸馏水稀释至所需浓度。请注意，滋味剂将被水流进一步稀释。我们测量达到长鼻的最终浓度为初始浓度的77％（参见参考文献10,29 ）。 </li><li>向每种促味剂溶液中加入人造食物染料，以确定味觉者通过长鼻子的精确时间。我们发现以0.05％w / v的绿色染料（见材料清单 ）不会激活Manduca GRNs。 </li><li> 使用颜色传感器（见表1）来检测促味剂溶液中的食物着色。使用烙铁在网孔附近和灌注系统输出管下方0.5厘米处（<html> s ="xfig"&gt;图4A-4B）。将传感器连接到刚性管，并将其固定在外部施加环氧树脂。 <ol><li>将颜色传感器电压输出与生理信号一起记录为模拟信号（见第4节）。可以使用连接到传感器的直流放大器来放大彩色传感器电压信号（ 图4A ）。 </li></ol></li><li> 确保颜色传感器正在通过传递滋味剂并记录传感器的电压输出。染料引发的信号应超过噪声水平。 <ol><li>调整恒定的水流量，使信号尽可能接近正方形。 注意:按顺序递送多种滋味剂时，请注意，新尝试的第一次试验将包含新的和以前的滋味剂的混合物。因此，我们没有分析这些第一次试验。 </li></ol></li></ol>"&gt; <img alt="图4" class="xfigimg" src="/files/ftp_upload/55868/55868fig4.jpg" /> 图4 :促销递送系统。 （ A ）用于递送和监测促进器的定时脉冲到动物的长鼻的装置的示意图。系统的主要组件用红色数字表示。通过与硅胶管连接的蠕动泵将整个长鼻子维持恒定的蒸汽水流到要放置长鼻的刚性塑料管（1）（6）。使用加压的16通道灌注系统输送含有少量无味染料的滋养物。含有滋味剂的储存器连接到附接到刚性管的歧管（2），位于要放置长鼻的孔的上方（5）。来自气动泵的压缩空气被注入到灌注系统中，以便通过定制软件进行定时控制，快速地提供促味剂。一个颜色传感器（3）用于监测促销剂。传感器连接到与吸鼻管相邻的刚性管，并且在促进剂灌注系统的出口下方连接。彩色传感器电压信号由直流放大器放大。与放大器相邻的插图上的红色迹线说明了使用定制数据采集软件记录的彩色信号;信号的快速增加和减少分别反映了到达和消化的味道。将飞蛾的长鼻子放置在位于颜色传感器正下方的孔（5）中。网孔（4）放置在孔的上方，以确保不同动物的概念总是位于同一位置。刚性管的另一侧连接到另一个硅管，以将输出引导到废物容器（7）中。 （ B ）显示连接到刚性塑料管的促味剂输送系统的主要元素的图像，其由红色数字表示，如图A所示:水源（6），滋味歧管输出管（2），颜色传感器（3），网孔（4）和要插入的长鼻孔（5）全部连接到刚性塑料管（1） 。 （ C ）显示了Manduca准备工作的安置。将飞蛾的长鼻子的远端t2 / 3引入刚性塑料管（1）的孔（5）中。如插图所示，将长鼻子用牙蜡和环氧树脂固定在适当位置。输入到刚性管的水和其输出到废物容器的水分别由数字6和7表示。数字2表示滋味歧管输出管道。 <a href="//ecsource.jove.com/files/ftp_upload/55868/55868fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> 细胞外Tetrode记录监测GRNs诱导的诱发活性<ol><li>将蛾准备放在立体显微镜下的平台上隔离表（ 图4C ）。 </li><li>将飞蛾的长鼻三分之二的长鼻子放入促销递送系统的刚性管中（ 图2B ）。要做到这一点，延长蛾的长鼻，然后用梳force钳轻轻的将其插入硬管的孔中。用软牙蜡密封长鼻，然后用一层环氧树脂密封，以避免泄漏（ 图4C ，入口）。 </li><li>将盐水灌注线连接到头部胶囊，并将灌注流量设定为约0.04L / h的恒定速率。 </li><li>将接地电极（ 即氯化银线）浸入盐水浴中（ 图5A ）。 </li><li>使用按照30中描述的制造程序制造的定制扭绞线四极（建议使用4根电极线实现良好隔离的单元并适合神经）。在实验之前，按照30中描述的步骤对电极进行电镀。 </li><li>使用手动显微操纵器将四极杆放置在上颌神经附近。推进四极直到它开始进入神经（ 图5 ）。 </li><li>放置四极杆后等待至少10分钟，使其在记录前稳定在神经内。 </li><li>放大信号（3000x），并使用设置在0.3-6 kHz之间的带通滤波器。使用数据采集软件以40 kHz采样率采集信号。 </li><li>完成实验后将蛾准备放入冷冻室。 </li></ol><img alt="图5" class="xfigimg" src="/files/ftp_upload/55868/55868fig5.jpg" /> 图5 :GRNs的Tetrode记录。 （ A ，大图像）使用微操纵器p将扭绞线四极插入上颌神经（MxN）以记录GRN的活动。如图所示，将氯化银接地电极置于盐水浴中。 （ A ，插图）显示上颌神经中四面体的大脑的放大图像。还指出了食管下段（SEZ）。 （ B ）以A为单位的Manduca大脑示意图<a href="//ecsource.jove.com/files/ftp_upload/55868/55868fig5large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

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作者没有什么可以披露的。

这里描述的方法允许来自相对简单的动物，人类性别的体内记录，以在促进递送之前，期间和之后的长时间（超过2小时）表征多个随机选择的GRN的活性。这些方法还允许用精确的时间控制快速，顺序递送多种滋味刺激，这些优点可用于研究基于滋味表现的神经机制。该协议已经被用于研究当通过与单神经元连接的中间神经元10同时监测GRN时，它们在传播给突触后靶向神经元（ 例如，在经济特区）中时，GRNs对滋味物的反应如何变换。此外，这些方法可以适应实验者的需求，允许执行复杂的范例来研究味觉编码的基本方面。 开始时我们的研究，我们有时必须解决的一个技术问题是无法用四极线检测上颌神经的尖峰信号。可能的原因是多样的，因为解剖方案是具有挑战性的，一些做法是必要的，以获得良好的准备。首先，在飞蛾解剖期间，上颌神经容易损伤，特别是在去除围绕神经组织的鞘时。第二，如果护套没有完全去除，四极线可能无法进入神经。在这两种情况下，开始新的准备工作通常是解决这些问题的最简单的方法。第三，四极线可能有问题。这可以通过测量在1kHz时应为〜270kΩ的电线的阻抗来检查。如果阻抗值高于〜300kΩ，请用金电镀电线以达到所需的阻抗（参见参考文献30 ）。第四，一件设备可能被误连接或行为不端。 另一个可能的问题是记录尖峰信号，但是神经元似乎对这些促味剂没有反应。这可能是因为记录的神经元对递送的一组滋味不敏感。同样重要的是要记住，除了GRNs的轴突之外，上颌神经还带有机械感觉纤维。因此，可以从机械感觉神经元记录，而不是或除了GRN之外。然而，味觉递送系统被设计为在整个实验中提供恒定的机械输入，使得不太可能通过对其递送的机械部件的响应来混淆对促味剂的反应。响应某些但不是其他美味的神经元，或以不同的方式对不同的美味食物进行反应，可以明确地分类为GRNs。我们建议使用新鲜稀释的滋味剂，以避免由于化合物降解或蒸发引起的滋味浓度或成分的变化的溶剂。我们还建议定期清洁系统，以避免管道污染和/或障碍物。 另一个可能的技术问题是不利的信噪比。这个问题通常可以通过重新洗涤或调整浴接地电极的位置来解决。其他解决方案可能需要屏蔽并最小化设备中每个电气连接的长度。 最后，重要的是要注意，使用四极记录获得的数据的正确分析需要仔细的尖峰分选。我们发现全自动化方法一般不足。我们建议在分析四极数据10,29,31,32,33之前熟悉穗分类文献。 我们解剖专业的替代品可以使用tocol。在这里，我们通过蛾头的腹侧部分描述了一个解剖，提供进入上颌神经和经济特区，但也可以通过背侧解剖来访问这些结构。我们发现背面制剂由于其位置较深而不适合从这些味觉结构进行记录，但是这种制剂确实具有使得能够从高级结构记录的优点，例如蘑菇体，已经与多个相关联的区域感知整合，关联学习和记忆处理34 。我们专注于使用四极电极从上颌神经记录，但是，正如我们所示，标准的细胞内尖锐电极也可以用于此目的。此外，两种技术可以组合以执行来自多个脑区域10的同时记录。神经科学文献提供了许多例子已经被证明是用于揭示感觉处理的基本原理（例如嗅觉编码）的有力工具，其适用于昆虫和脊椎动物35,36,37,38,39 。我们希望我们的方法将引导关于味觉编码的基本新见解。

味觉和嗅觉系统在环境中产生化学物质的内部表征，分别引起口味和气味的看法。这些化学感觉对于引发许多对生物存活至关重要的行为至关重要，从寻找伴侣和膳食到避免捕食者和毒素。当环境化学物质与感官受体细胞质膜中的受体相互作用时，该过程开始;这些细胞直接或通过与神经元的相互作用，将关于化学物质的身份和浓度的信息转化为电信号。然后将这些信号传输到高阶神经元和其他脑结构。随着这些步骤的进行，原始信号总是发生变化，促进生物体检测，区分，分类，比较和存储感官信息的能力，并选择适当的动作。了解胸罩如何在转化关于环境化学品的信息以最佳地执行各种任务是神经科学的一个基本问题。 风味编码被认为是相对简单的:广泛观点认为，引起味道的每种化学分子（"促味剂"）自然属于大约五种基本味道之一（ 即甜，苦，酸） ，咸和鲜） 1 。在这种"基本品味"观点中，味觉系统的工作是确定哪些基本口味是存在的。此外，神经系统中基本味觉表现的神经机制尚不清楚，并被认为是由"标记线" 2,3,4,5,6或"跨纤维图案" 7 </sup&gt; ， 8代码。在标记的线代码中，每个感觉细胞及其每个神经追随者对单一味道质量进行响应，共同形成了专门用于该味道的中枢神经系统中较高处理中心的直接和独立的通道。相比之下，在跨纤维图案代码中，每个感觉细胞可以响应多种味道品质，使得关于滋味剂的信息由感觉神经元群体的总体反应表示。味觉信息是通过基本口味，通过标签线还是通过其他机制来表达，目前尚不清楚，是近期调查的焦点3,8,9,10,11,12 。我们自己最近的工作表明，味觉系统使用时空人口代码来产生个人口味的代表，而不是基本的口味类别10 。 在这里，我们提供3种新工具来协助味觉编码的研究。首先，我们建议使用hawkmoth Manduca sexta作为一种相对简单的模型生物，适合电生理学研究的味道，并描述一种解剖程序。第二，我们建议使用细胞外"tetrodes"来记录个体GRN的活动。第三，我们建议一种新的设备，用于交付和监测精确定时的动物脉搏。这些工具根据我们实验室和其他人用来研究嗅觉系统的技术进行了改编。 昆虫如果蝇果蝇 ， 美洲蝗虫蝗虫以及蛾蛾属性，已经为数十年提供了强大的资源来了解关于神经的基本原理vous系统，包括感觉编码（ 例如，嗅觉13 ）。在哺乳动物中，味觉受体是通过复杂的第二信使途径1,4通过神经元通信的特殊细胞。它在昆虫中更简单:它们的味觉受体是神经元。此外，靠近周边的哺乳动物味道相对复杂，具有多个平行的神经路线，重要的组成部分具有挑战性，容纳在小骨骼结构15中 。昆虫味道似乎更简单。在昆虫中，GRN被包含在称为感觉器的专门结构中，位于天线，口器，翼和腿16,17中 。 GRN直接投放到食管区（SEZ），这个结构的作用被认为主要是味觉17 ，其中包含二次味觉神经元10 。从那里，信息传播到身体来驱动反射，并且到更高的脑区被整合，存储，并最终推动行为选择16 。 有必要表征外部味道反应，以了解品味信息如何在整个神经系统中从点到点传播和转化。直接监测昆虫GRNs神经活动的最常用的方法是尖端记录技术12,18,19,20,21,22,23。这包括将电极直接放置在感​​官上，其中许多是相对容易进入的。滋味剂包含在电极内，允许一个激活和分开测量GRNs在感觉中的神经元反应。但是，因为滋味剂被包含在电极中，所以不可能在促进剂被递送之前或之后除去之前测量GRN活性，或者不更换电极20来交换促味剂。另一种方法，即"侧壁"记录技术，也被用来记录GRNs的活动。这里，将记录电极插入感官敏感剂24的基底中，并且通过感觉尖端上的分离的玻璃毛细管递送促味剂。两种技术都限制从GRN到特定感官的记录。在这里，我们建议一种新技术:从随机选择的来自不同感官的GRN轴突记录，同时分别将味觉提供给长鼻子。轴突记录是通过将锋利的玻璃电极或细胞外电极束（tetrodes）放入神经中实现的，该神经带有轴突长颈鹿的GRNs经济特区10 。在Manduca ，这些轴突穿过已知是纯粹传入的上颌神经，允许明确记录感觉反应25 。这种从轴突记录的方法允许在两个以上的时间内稳定测量GRN反应之前，之后和之后的一系列促销发表。 在这里，我们描述了一种将上颌神经与SEZ一起暴露的解剖程序，这可以允许在SEZ 10中同时记录多个GRN和神经元的反应。我们还描述了使用定制的4通道扭绞线四极杆对GRN的细胞外记录的使用，当与尖峰分选方法组合时，可以同时分析多个（在我们手中，最多六个）GRN。我们进一步比较用tetrodes制作的记录与用细胞内的细胞制成的记录电极。最后，我们描述一种用于提供滋味刺激的新设备。改良了许多研究人员在嗅觉研究中长期使用的设备，以提供嗅觉研究中的气味，我们的新仪器提供了研究气味的优势:改进了以前的多通道传送系统，如Stürckow及其同事开发的（参见参考文献26,27 ），我们的设备实现了精确在提供该定时的电压读数的同时控制促味剂递送的定时;并且它允许快速，顺序地递送多个滋补刺激10 。该设备以恒定的清洁水流将吸鼻涕浸入其中可以输送滋味的受控脉冲。每个滋味脉冲通过长鼻子，然后洗去。滋味剂含有少量无味的食物着色，允许颜色传感器以准确的时间监测滋味卵的通过呃长鼻

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">Dissection and specimen preparation</td>
		</tr>
		<tr>
			<td>Polypropylene tube, 15 ml -Falcon</td>
			<td>Fisher Scientific</td>
			<td>14-959-53A</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle, Short bevel, 19G x 1-1/2&#34;&#160;</td>
			<td>&#160;MONOJET</td>
			<td>888200144</td>
			<td>For aplying air to remove the hair from the moth.</td>
		</tr>
		<tr>
			<td>Modeling Clay-Van Aken Plastalina&#160;</td>
			<td>DickBlick</td>
			<td>33268</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish-100 x 15 mm&#160;</td>
			<td>VWR International</td>
			<td>89000-304</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tip (1-200 &#181;L)</td>
			<td>USA Scientific</td>
			<td>1111-0806</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>Techni Edge</td>
			<td>TE05-071</td>
			<td></td>
		</tr>
		<tr>
			<td>22 AWG standard hookup wire</td>
			<td>AlphaWire</td>
			<td>1551</td>
			<td>For inserting the proboscis into the pippete tip.</td>
		</tr>
		<tr>
			<td>Batik wax</td>
			<td>Jacquard</td>
			<td>7946000</td>
			<td></td>
		</tr>
		<tr>
			<td>Electric waxer</td>
			<td>Almore International</td>
			<td>66000</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo Myscroscope</td>
			<td>Leica</td>
			<td>MZ75</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #1 forceps (coarse)&#160;</td>
			<td>World Precision Instruments</td>
			<td>500335</td>
			<td>For removing fat and non nervous tissue.</td>
		</tr>
		<tr>
			<td>Dumont #5 titanium forceps (fine)&#160;</td>
			<td>World Precision Instruments</td>
			<td>14096</td>
			<td>For removing fat and non nervous tissue.</td>
		</tr>
		<tr>
			<td>Dumont #5SF forceps (super-fine)</td>
			<td>World Precision Instruments</td>
			<td>500085</td>
			<td>For desheathing the nervious tissue.</td>
		</tr>
		<tr>
			<td>Vannas scissors (fine)</td>
			<td>World Precision Instruments</td>
			<td>500086</td>
			<td>For removing the cuticle.</td>
		</tr>
		<tr>
			<td>Collagenase/Dispase</td>
			<td>Sigma-Aldrich</td>
			<td>11097113001</td>
			<td></td>
		</tr>
		<tr>
			<td>Epoxy</td>
			<td>Permatex</td>
			<td>84101</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">Saline perfusion system</td>
		</tr>
		<tr>
			<td>Extension set with rate flow regulator</td>
			<td>&#160;B Braun Medical Inc.</td>
			<td>V5200</td>
			<td></td>
		</tr>
		<tr>
			<td>IV administration set with Y injection site</td>
			<td>&#160;B Braun Medical Inc.</td>
			<td>V1402</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">Tastant delivery system</td>
		</tr>
		<tr>
			<td>White Translucent Nylon Tubing OD 1/4&#34;, ID 1/8&#34;</td>
			<td>Small Parts Inc.</td>
			<td>B001JJT4SA</td>
			<td>Rigid tube that connects the four main elements of the system.</td>
		</tr>
		<tr>
			<td>Soldering iron</td>
			<td>Circuit Specialists</td>
			<td>ZD200BK</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotary tool-Dremel</td>
			<td>Dremel</td>
			<td>4200</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene mesh, hole size (hole size 0.1 x 0.13 cm)&#160;</td>
			<td>Industrial Netting</td>
			<td>XN5170</td>
			<td>For ensuring that the probosises of different animals are placed in the same location.</td>
		</tr>
		<tr>
			<td>Pressurized 16-Channel perfusion system&#160;</td>
			<td>Bioscience Tools</td>
			<td>PS-16H</td>
			<td>For tastant delivery. This system includes pinch valves, tubing, manifold, solution cylinders, valve controler and fitting accesories.</td>
		</tr>
		<tr>
			<td>Polypropylene tubing, ID 0.034&#34;, ID 0.050&#34;</td>
			<td>Becton, Dickinson &#38; Co</td>
			<td>427421</td>
			<td>&#160;Output tube from the perfusion system.</td>
		</tr>
		<tr>
			<td>Pneumatic PicoPump</td>
			<td>World Precision Instruments</td>
			<td>SYS-PV820</td>
			<td>For controlling the output channel of the perfusion system.&#160;</td>
		</tr>
		<tr>
			<td>Data acquisition software system, LabVIEW PCI-MIO-16E-4 DAQ card</td>
			<td>National Instruments</td>
			<td>LabVIEW 2011</td>
			<td>To control the pico pump for tastant delivery and to record the signals from the color sensor .</td>
		</tr>
		<tr>
			<td>Compulab 3 Manostat peristaltic pump</td>
			<td>Sigma</td>
			<td>P1366</td>
			<td>For pumping water.</td>
		</tr>
		<tr>
			<td>Silicone tubing, ID 1/16&#34; OD 1/8&#34;</td>
			<td>Cole-Parmer</td>
			<td>WU-95802-02</td>
			<td>To connect the water source to the peristaltic pump tubing, and the outlet tube of the pump to the rigid tube of the delivery system.</td>
		</tr>
		<tr>
			<td>Color sensor-digital fiber optic sensor</td>
			<td>Keyence</td>
			<td>&#160;FS-V31M</td>
			<td>For monitoring tastant delivery.&#160;</td>
		</tr>
		<tr>
			<td>Color sensor-reflective fiber unit</td>
			<td>Keyence</td>
			<td>FU35-FZ</td>
			<td>To connect the color sensor device.</td>
		</tr>
		<tr>
			<td>Dental periphery Wax</td>
			<td>Henry-Schein Dental</td>
			<td>6652151</td>
			<td>To secure the proboscis into the rigid tube.</td>
		</tr>
		<tr>
			<td>Two 3.7 L containers</td>
			<td></td>
			<td></td>
			<td>To provide water to the system, and to recollect the water waste.</td>
		</tr>
		<tr>
			<td>Fast green FCF&#160;</td>
			<td>Sigma</td>
			<td>F7258</td>
			<td></td>
		</tr>
		<tr>
			<td>Dressing forceps 25.5 cm</td>
			<td>WPI</td>
			<td>500364</td>
			<td>To introduce moths proboscis into the proboscis hole from the rigid tube.</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">Electrophysiology Equipment</td>
		</tr>
		<tr>
			<td>D.C. amplifier</td>
			<td>Brown-Lee</td>
			<td>440</td>
			<td></td>
		</tr>
		<tr>
			<td>Lamp</td>
			<td>Schott</td>
			<td>Schott Fostec Light Source DCR 2</td>
			<td></td>
		</tr>
		<tr>
			<td>Manual micromanipulator</td>
			<td>Leica</td>
			<td>micromanipulator</td>
			<td>To precicely insert the tetrodes into the animals brain. The manipulator has to allow fine and coarse movements in x, y and z axis.</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Leica</td>
			<td>MZ75</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibration-isolation table (MICRO-g lab table)</td>
			<td>TMC</td>
			<td>63-541</td>
			<td></td>
		</tr>
		<tr>
			<td>Oscilloscope</td>
			<td>Tektronix&#160;</td>
			<td>TDS2014</td>
			<td></td>
		</tr>
		<tr>
			<td>16-channle pre-amplifier and amplifier</td>
			<td>16 Channel MA-800 Amplifier System&#160;</td>
			<td>B.E.S 2013</td>
			<td></td>
		</tr>
		<tr>
			<td>Computer</td>
			<td>Dell</td>
			<td>optiplex 780</td>
			<td>The following are the minimum recommended requirements. RAM: 3.32GHz, 3GB. Processor: Intel Core&#160;2 Duo. Graphic card: integrated Intel GMA X4500.&#160;</td>
		</tr>
		<tr>
			<td>Data acquisition software system, LabVIEW PCI-MIO-16E-4 DAQ card</td>
			<td>National Instruments</td>
			<td>LabVIEW 2011</td>
			<td>To control the pico pump for tastant delivery and to record the signals from the color sensor and electrode .</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">Tastants</td>
		</tr>
		<tr>
			<td>KAc</td>
			<td>Sigma-Aldrich</td>
			<td>P5708</td>
			<td></td>
		</tr>
		<tr>
			<td>LiCl</td>
			<td>Sigma-Aldrich</td>
			<td>L9650</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>73575</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>84097</td>
			<td></td>
		</tr>
	</tbody>
</table>

new methods to study gustatory coding

味觉让动物能够检测环境中的化学物质，从而产生对生存至关重要的行为。当味觉受体神经元（GRN）检测到滋养分子时，它们将关于促进剂的身份和浓度的信息作为电活动的模式编码，然后传播到脑中的跟随神经元。这些模式构成了味觉的内在表征，然后允许动物选择动作并形成记忆。使用相对简单的动物模型一直是研究感官编码中基本原理的有力工具。在这里，我们提出了三种新的方法来研究使用飞蛾Manduca sexta的味觉编码。首先，我们提出了一个解剖程序，用于暴露上颌神经和亚食管区（SEZ），从而记录来自其轴突的GRN的活动。其次，我们描述了使用细胞外电极记录多个GRN的活动电线直接进入上颌神经。第三，我们提出了一个新的系统，以高时间精度传送和监测不同口味的脉冲。这些方法允许在递送前，之前和之后直接从GRNs 体内表征神经元反应。我们提供从多个GRN记录的电压迹线的示例，并提供了如何将尖峰分类技术应用于数据以识别个体神经元的响应的示例。最后，为了验证我们的记录方法，我们将从GRNs获得的细胞外记录与四极杆比较，用锐利的玻璃电极获得的细胞内记录。

可以使用细胞外四极电极，在促味剂递送之前，期间和之后记录GRNs的活性（ 图6A和6C ）。 图6A （中图和下图 ）显示了通过放置在上颌神经中的四根电线中每一根记录的滤波（300-6,000Hz）电压迹线，其中可以观察到反映动作电位（箭头）的不同幅度的信号。在实验开始后2秒送出1秒钟的滋味（1M蔗糖）脉冲;通过颜色传感器监测刺激的开始和偏移（ 图6A ，上图 ）。在四个通道的每个通道中可以观察到诱人的尖峰（ 图6A ，中间图 ）。当它们响应某些味觉而不是其他味道时，GRN可被识别并与机械传感器区分开（这里没有显示） 为了识别和隔离四极记录中的单个神经元响应，我们使用基于Pouzat 31和Kleinfeld的32,33方法的一组定制函数进行离线尖峰分类（这些方法在这些引文中描述:10,29）。 图6B示出了应用于图6A所示的数据的尖峰分类的示例，其中发现了三个井分离的单元。 图6C的光栅图描绘了图6B中三个分离单元对顺序递送的六种不同的促味剂（1M蔗糖，麦芽糖和NaCl; 100mM咖啡因和10mM小檗碱和叶片）的反应（4次试验s /味觉）。 如图6C所示，记录的GRN具有不同的基线活动水平，从无声（GRN1）到低或中等（GRN 2和3）。在开胃后，GRNs显示出不同的活性模式，并表现出与味觉不同的选择性。例如，GRN 1仅响应于蔗糖，而GRN 2仅在刺激开始时才响应麦芽糖和NaCl，具有峰值和峰值峰值。此外，一些反应被锁定在刺激的时间（ 例如 GRN 1对蔗糖的反应），而其他反应比刺激持续时间（ 例如，对小檗碱的GRN 3反应）或含有兴奋性和抑制性组分两者（ 例如 图7 GRN 2对NaCl和蔗糖的反应）。有关GRN的各种敏感性和活动模式的更多信息，请参见参考文献10 。 图7 ）。在连续5次蔗糖呈现试验中，在GRA 1中细胞内记录了面板A的绿色盒子中的电压迹线，并且相同的反应在图B中显示为光栅图（注意，这种类型的响应与用四极杆和记录有锐细胞内电极的GRN 2显示出更广泛的应答模式。 <img alt="图6" class="xfigimg" src="/files/ftp_upload/55868/55868fig6v2.jpg"/&gt; 图6: 具有细胞外Tetrodes的上颌神经记录的代表性结果。 （ A ）显示了上颌神经中四条电线中每一条记录的过滤（300-6,000 Hz）电压迹线（中间图）。在红色阴影指示的时间段内在中间面板中施加1秒钟的滋味脉冲。通过颜色传感器监测刺激的开始和偏移，如上图上的红色电压迹线所示，并且在中间面板上由红色阴影区域表示。虚线水平线表示+50（上），0（中）和-50（底）μV。示出了与垂直虚线内的区域对应的原始电压迹线的放大（底图）。尖峰的示例由箭头指示。 （ B ）应用于面板A所示数据的尖峰分类的例子。记录在四个细胞外线（1-4）ar中的每一个的波形e用三个不同的GRN（单元1-3）来识别有助于记录的信号。显示了三个单位的个别事件（彩色细线）和平均值（粗黑色线）。必须考虑许多统计标准以使用尖峰分类方法可靠地识别独立单位（参见参考文献10,29 ）。 （ C ）表示三种分离单位对六种不同的滋味剂的顺序的反应的光栅图（显示4种试验/味觉）。滋味递送时间（1秒）由红色阴影区域表示。滋味剂的浓度为1M（蔗糖，麦芽糖，NaCl），100mM（咖啡因）和10mM（小檗碱和小叶）。 <a href="//ecsource.jove.com/files/ftp_upload/55868/55868fig6v2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图7" c<html> lass ="xfigimg"src ="/ files / ftp_upload / 55868 / 55868fig7.jpg"/&gt; 图7 :GRN轴突的细胞内记录。 （ A ，绿色盒子）从具有尖锐玻璃细胞内电极（电阻为80-120MΩ）的GRN记录的电压迹线放置在上颌神经中，由1M蔗糖（Suc）连续5次刺激引起。 （ B ）两个GRN的响应的光栅图，包括面板A（绿色盒子面板）中所示的响应，以尖锐玻璃细胞内电极记录的顺序递送的两种不同的滋味剂（100mM灰色字体颜色; 1M黑色字体颜色）放置到上颌神经（7个试验/味觉和3个试验/水）。两个面板中的红色阴影区域表示促销交货时间。刺激的开始和偏移由颜色传感器监测，如每个面板底部的红色电压迹线所示。es / ftp_upload / 55868 / 55868fig7large.jpg"target ="_ blank"&gt;请点击此处查看此图的较大版本。

Watch this Scientific Journal Video about New Methods to Study Gustatory Coding at JoVE.com

研究味觉编码的新方法|神经科学 |视频

New Methods to Study Gustatory Coding

我们提出三种新的方法来研究味觉编码。我们使用简单的动物Manduca sexta（ Manduca ）描述了一种解剖方案，使用细胞外tetrodes来记录多种味觉受体神经元的活性，以及​​用于递送和监测精确定时的滋味脉冲的系统。

研究风味编码的新方法

The sense of taste allows animals to detect chemicals in the environment, giving rise to behaviors critical for survival. When Gustatory Receptor Neurons ...

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9.3K 次观看.  National Institutes of Health (NIH). 我们提出三种新的方法来研究味觉编码。我们使用简单的动物Manduca sexta（ Manduca ）描述了一种解剖方案，使用细胞外tetrodes来记录多种味觉受体神经元的活性，以及​​用于递送和监测精确定时的滋味脉冲的系统。 

视频: New Methods to Study Gustatory Coding

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

激光捕获显微切割技术的果蝇外周神经元

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

科研

神经科学

How to Create and Use Binocular Rivalry

Each of our eyes normally sees a slightly different image of the world around us. The brain can combine these two images into a single coherent representation. However, when the eyes are presented with images that are sufficiently different from each other, an interesting thing happens: Rather than fusing the two images into a combined conscious percept, what transpires is a pattern of perceptual alternations where one image dominates awareness while the other is suppressed; dominance alternates between the two images, typically every few seconds. This perceptual phenomenon is known as binocular rivalry. Binocular rivalry is considered useful for studying perceptual selection and awareness in both human and animal models, because unchanging visual input to each eye leads to alternations in visual awareness and perception. To create a binocular rivalry stimulus, all that is necessary is to present each eye with a different image at the same perceived location. There are several ways of doing this, but newcomers to the field are often unsure which method would best suit their specific needs. The purpose of this article is to describe a number of inexpensive and straightforward ways to create and use binocular rivalry. We detail methods that do not require expensive specialized equipment and describe each method's advantages and disadvantages. The methods described include the use of red-blue goggles, mirror stereoscopes and prism goggles.

Binocular rivalry occurs when the eyes are presented with different images at the same location: one image dominates while the other is suppressed, and dominance alternates periodically. Rivalry is useful for investigating perceptual selection and visual awareness. Here we describe several easy methods for creating and using binocular rivalry stimuli.

如何创建和使用双眼竞争

Each of our eyes normally sees a slightly different image of the world around us. The brain can combine these two images into a single coherent ...

Study Glial Cell Heterogeneity Influence on Axon Growth Using a New Coculture Method

In the central nervous system of all mammals, severed axons after injury are unable to regenerate to their original targets and functional recovery is very poor 1. The failure of axon regeneration is a combined result of several factors including the hostile glial cell environment, inhibitory myelin related molecules and decreased intrinsic neuron regenerative capacity 2. Astrocytes are the most predominant glial cell type in central nervous system and play important role in axon functions under physiology and pathology conditions 3. Contrast to the homologous oligodendrocytes, astrocytes are a heterogeneous cell population composed by different astrocyte subpopulations with diverse morphologies and gene expression 4. The functional significance of this heterogeneity, such as their influences on axon growth, is largely unknown.
To study the glial cell, especially the function of astrocyte heterogeneity in neuron behavior, we established a new method by co-culturing high purified dorsal root ganglia neurons with glial cells obtained from the rat cortex. By this technique, we were able to directly compare neuron adhesion and axon growth on different astrocytes subpopulations under the same condition.
In this report, we give the detailed protocol of this method for astrocytes isolation and culture, dorsal root ganglia neurons isolation and purification, and the co-culture of DRG neurons with astrocytes. This method could also be extended to other brain regions to study cellular or regional specific interaction between neurons and glial cells.

In this protocol, we described a new method to study the influence of glial cell heterogeneity on axon growth with an in vitro co-culture system. Rat cortical glial cells were cultured to confluence and cocultured with highly purified rat dorsal root ganglia neurons. Different glial cell influence on neurons adhesion and axon growth was compared directly in the same culture. This method provides a new way to directly study the glial cell heterogeneity influence on neuron adhesion and axon growth.

研究胶质细胞轴突的生长异质性的影响，使用一个新的共培养法

In the central nervous system of all mammals, severed axons after injury are unable to regenerate to their original targets and functional recovery is ...

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the first born neurons remain closer to the ventricle while the last born neurons migrate past the first born neurons towards the surface of the brain1. In addition to neuronal migration2, a key process for normal cortical function is the regulation of neuronal morphogenesis3. While neuronal morphogenesis can be studied in vitro in primary cultures, there is much to be learned from how these processes are regulated in tissue environments.
We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex4,6. A pSilencer modified vector is used which contains both a U6 promoter that drives the double stranded hairpin RNA and a separate expression cassette that encodes GFP protein driven by a CMV promoter7-9. Our approach allows for the rapid assessment of defects in neurite outgrowth upon specific knockdown of candidate genes and has been successfully used in a screen for regulators of neurite outgrowth8. Because only a subset of cells will express the RNAi constructs, the organotypic slices allow for a mosaic analysis of the potential phenotypes. Moreover, because this analysis is done in a near approximation of the in vivo environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown cortical function. Finally, in comparison with in vivo electroporation technology, the success of ex vivo electroporation experiments is not dependant upon proficient surgery skill development and can be performed with a shorter training time and skill.

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.

神经元形态的研究方法:<em&gt;体外</em&gt; RNAi技术在胚胎小鼠大脑皮质的电穿孔

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

Measuring the Subjective Value of Risky and Ambiguous Options using Experimental Economics and Functional MRI Methods

Most of the choices we make have uncertain consequences. In some cases the probabilities for different possible outcomes are precisely known, a condition termed "risky". In other cases when probabilities cannot be estimated, this is a condition described as "ambiguous". While most people are averse to both risk and ambiguity1,2, the degree of those aversions vary substantially across individuals, such that the subjective value of the same risky or ambiguous option can be very different for different individuals. We combine functional MRI (fMRI) with an experimental economics-based method3 to assess the neural representation of the subjective values of risky and ambiguous options4. This technique can be now used to study these neural representations in different populations, such as different age groups and different patient populations.
In our experiment, subjects make consequential choices between two alternatives while their neural activation is tracked using fMRI. On each trial subjects choose between lotteries that vary in their monetary amount and in either the probability of winning that amount or the ambiguity level associated with winning. Our parametric design allows us to use each individual's choice behavior to estimate their attitudes towards risk and ambiguity, and thus to estimate the subjective values that each option held for them. Another important feature of the design is that the outcome of the chosen lottery is not revealed during the experiment, so that no learning can take place, and thus the ambiguous options remain ambiguous and risk attitudes are stable. Instead, at the end of the scanning session one or few trials are randomly selected and played for real money. Since subjects do not know beforehand which trials will be selected, they must treat each and every trial as if it and it alone was the one trial on which they will be paid. This design ensures that we can estimate the true subjective value of each option to each subject. We then look for areas in the brain whose activation is correlated with the subjective value of risky options and for areas whose activation is correlated with the subjective value of ambiguous options.

Using functional MRI and behavioral methods to determine the neural representation of the subjective value of risky and ambiguous options in the human brain.

，测量主观价值风险和不明确的选项，使用实验经济学和功能磁共振成像的方法

Most of the choices we make have uncertain consequences. In some cases the probabilities for different possible outcomes are precisely known, a condition ...

Methods to Assay Drosophila Behavior

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases1. We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials2-4. The rapid iterative negative geotaxis (RING) assay5 has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously using large number of animals, with the high-throughput approach making it more amenable for screening experiments.

Methods to Assay Drosophila Behavior

Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.

对检测方法果蝇行为

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular ...

A Caenorhabditis elegans Model System for Amylopathy Study

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of Alzheimer's disease (AD) and is found in Lewy body dementia, inclusion body myositis and cerebral amyloid angiopathy 1-4. Amylopathies progressively develop with time. For this reason simple organisms with short lifespans may help to elucidate molecular aspects of these conditions. Here, we describe experimental protocols to study A&beta;-mediated neurodegeneration using the worm Caenorhabditis elegans. Thus, we construct transgenic worms by injecting DNA encoding human A&beta;42 into the syncytial gonads of adult hermaphrodites. Transformant lines are stabilized by a mutagenesis-induced integration. Nematodes are age synchronized by collecting and seeding their eggs. The function of neurons expressing A&beta;42 is tested in opportune behavioral assays (chemotaxis assays). Primary neuronal cultures obtained from embryos are used to complement behavioral data and to test the neuroprotective effects of anti-apoptotic compounds.

A Caenorhabditis elegans Model System for Amylopathy Study

We describe methods to study aspects of amylopathies in the worm C. elegans. We show how to construct worms expressing human A&beta;42 in neurons and how to test their function in behavioral assays. We further show how to obtain primary neuronal cultures that can be used for pharmacological testing.

一<em&gt;线虫</em&gt;研究Amylopathy模型系统

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of ...

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees (Apis mellifera)

This video demonstrates novel techniques of RNA interference (RNAi) which downregulate two genes simultaneously in honey bees using double-stranded RNA (dsRNA) injections. It also presents a protocol of proboscis extension response (PER) assay for measuring gustatory perception.	
	RNAi-mediated gene knockdown is an effective technique downregulating target gene expression. This technique is usually used for single gene manipulation, but it has limitations to detect interactions and joint effects between genes. In the first part of this video, we present two strategies to simultaneously knock down two genes (called double gene knockdown). We show both strategies are able to effectively suppress two genes, vitellogenin (vg) and ultraspiracle (usp), which are in a regulatory feedback loop. This double gene knockdown approach can be used to dissect interrelationships between genes and can be readily applied in different insect species.	
	The second part of this video is a demonstration of proboscis extension response (PER) assay in honey bees after the treatment of double gene knockdown. The PER assay is a standard test for measuring gustatory perception in honey bees, which is a key predictor for how fast a honey bee's behavioral maturation is. Greater gustatory perception of nest bees indicates increased behavioral development which is often associated with an earlier age at onset of foraging and foraging specialization in pollen. In addition, PER assay can be applied to identify metabolic states of satiation or hunger in honey bees. Finally, PER assay combined with pairing different odor stimuli for conditioning the bees is also widely used for learning and memory studies in honey bees.

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees Apis mellifera

In this protocol, we describe two strategies that simultaneously suppress two genes (double gene knockdown) in honey bees. Then we present how to use the proboscis extension response (PER) assay to study the effect of double gene knockdown on honey bee gustatory perception.

RNAi介导的双基因敲除和味觉感知测量蜜蜂（<em&gt;蜜蜂</em&gt;）

This video demonstrates novel techniques of RNA  interference (RNAi) which downregulate two genes simultaneously in honey bees  using double-stranded RNA ...

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with increasing connective tissue and decreased neuronal viability that occur with age. Here, we describe a methodology for the dissociation of healthy hypothalamic neurons from adult-aged mice. The ability to study neurons from adult-aged mice allows the use of disease models that manifest at a later age and might be more developmentally accurate for certain studies. Fluorescence imaging of dissociated neurons can be used to study the activity of a population of neurons, as opposed to using electrophysiology to study a single neuron. This is particularly useful when studying a heterogeneous neuronal population in which the desired neuronal type is rare such as for hypothalamic glucose sensing neurons. We utilized membrane potential dye imaging of adult ventromedial hypothalamic neurons to study their responses to changes in extracellular glucose. Glucose sensing neurons are believed to play a role in central regulation of energy balance. The ability to study glucose sensing in adult rodents is particularly useful since the predominance of diseases related to dysfunctional energy balance (e.g.&#160;obesity) increase with age.

The activity of single neurons from adult-aged mice can be studied by dissociating neurons from specific brain regions and using fluorescent membrane potential dye imaging. By testing responses to changes in glucose, this technique can be used to study the glucose sensitivity of adult ventromedial hypothalamic neurons.

膜下丘脑腹内侧神经元的电位染料成像从成年小鼠为研究葡萄糖传感

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with ...

A Method to Make a Craniotomy on the Ventral Skull of Neonate Rodents

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain in adulthood and during development. Although most in vivo approaches use a craniotomy to study brain regions located on the dorsal side, brainstem regions such as the pons, located on the ventral side remain relatively understudied. The main goal of this protocol is to facilitate access to ventral brainstem structures so that they can be studied in vivo using electrophysiological and imaging methods. This approach allows study of structural changes in long-range axons, patterns of electrical activity in single and ensembles of cells, and changes in blood brain barrier permeability in neonate animals. Although this protocol has been used mostly to study the auditory brainstem in neonate rats, it can easily be adapted for studies in other rodent species such as neonate mice, adult rodents and other brainstem regions.

A surgical method is described to expose the ventral skull in neonate rats. Using this approach it is possible to open a craniotomy to perform acute electrophysiology and two-photon microscopy experiments in the brainstem of anesthetized pups.

方法以使对新生儿鼠害的腹头骨开颅手术

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain ...