Name: new methods to study gustatory coding
Uploaded: 2017-06-29T15:00:00.000+00:00
Duration: 10 min 59 s
Description: Watch this Scientific Journal Video about New Methods to Study Gustatory Coding at JoVE.com

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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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><tbody><tr><td>&lt;strong&gt;Dissection and Specimen Preparation&lt;/strong&gt;</td><td></td><td></td><td></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, 19 G x 1 - 1/2&quot;&amp;nbsp;</td><td>&amp;nbsp;MONOJET</td><td>888200144</td><td>For applying air to remove the hair from the moth.</td></tr><tr><td>Modeling Clay-Van Aken Plastalina&amp;nbsp;</td><td>DickBlick</td><td>33268</td><td></td></tr><tr><td>Petri dish -100 x 15 mm&amp;nbsp;</td><td>VWR International</td><td>89000-304</td><td></td></tr><tr><td>Pipette tip (1 - 200 &amp;micro;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)&amp;nbsp;</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)&amp;nbsp;</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 nervous 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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Saline Perfusion System&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Extension set with rate flow regulator</td><td>&amp;nbsp;B Braun Medical Inc.</td><td>V5200</td><td></td></tr><tr><td>IV administration set with Y injection site</td><td>&amp;nbsp;B Braun Medical Inc.</td><td>V1402</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Tastant Delivery System&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>White Translucent Nylon Tubing OD 1/4&quot;, ID 1/8&quot;</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)&amp;nbsp;</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&amp;nbsp;</td><td>Bioscience Tools</td><td>PS-16H</td><td>For tastant delivery. This system includes pinch valves, tubing, manifold, solution cylinders, valve controller and fitting accessories.</td></tr><tr><td>Polypropylene tubing, ID 0.034&quot;, ID 0.050&quot;</td><td>Becton, Dickinson &amp;amp; Co</td><td>427421</td><td>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.&amp;nbsp;</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&quot; OD 1/8&quot;</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>&amp;nbsp;FS-V31M</td><td>For monitoring tastant delivery.&amp;nbsp;</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&amp;nbsp;</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 moth proboscis into the proboscis hole in the rigid tube.</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Electrophysiology Equipment&lt;/strong&gt;</td><td></td><td></td><td></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 precisely insert the tetrodes into the animal&#039;s 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&amp;nbsp;</td><td>TDS2014</td><td></td></tr><tr><td>16-channle pre-amplifier and amplifier</td><td>16 Channel MA-800 Amplifier System&amp;nbsp;</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: 3.32 GHz, 3GB. Processor: Intel Core&amp;nbsp;2 Duo. Graphic card: integrated Intel GMA X4500.&amp;nbsp;</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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Tastants&lt;/strong&gt;</td><td></td><td></td><td></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.bdigitaluss.remotexs.co/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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SCIENTISTS IN ACTION

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

Technique to Collect Fungiform (Taste) Papillae from Human Tongue

The sense of taste is critical for human life. It informs the body about the quality of food that will be potentially ingested and stimulates metabolic processes that prepare the alimentary canal for digestion. Steady progress is being made towards understanding the early biochemical and molecular events underlying taste transduction (for a review, Breslin and Spector, 20081). However, progress to date has largely resulted from animal models. Yet, since marked differences in receptor specificity and receptor density vary among species, human taste transduction will only be understood by using human taste tissue. Here we describe a biopsy technique to collect human fungiform papillae, visible as rounded pink anterior structures, about 0.5 mm in diameter that contain taste buds. These biopsied papillae are used for several purposes including the isolation of viable taste bud cells, in situ hybridization, immunohistochemistry and, through techniques of molecular biology, the identification of taste-specific novel proteins.

Technique to Collect Fungiform Taste Papillae from Human Tongue

Knowledge of molecular mechanisms underlying gustatory transduction has recently enjoyed significant advances, largely due to using animal models. However, the wide diversity in taste sensitivity and specificity among mammals warrants studies in human tissue. We describe a biopsy technique to collect living taste cells from the papillae on human tongue.

技术收集人舌菌状（味道）乳头

The sense of taste is critical for human life. It informs the body about the quality of food that will be potentially ingested and stimulates metabolic ...

科研

医学

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the researcher to measure gustatory responses directly and quantitatively, reflecting the sensory input that the insect nervous system receives from taste stimuli in its environment. This protocol outlines all key steps in performing this technique. The critical steps in assembling an electrophysiology rig, such as selection of necessary equipment and a suitable environment for recording, are delineated. We also describe how to prepare for recording by making appropriate reference and recording electrodes, and tastant solutions. We describe in detail the method used for preparing the insect by insertion of a glass reference electrode into the fly in order to immobilize the proboscis. We show traces of the electrical impulses fired by taste neurons in response to a sugar and a bitter compound. Aspects of the protocol are technically challenging and we include an extensive description of some common technical challenges that may be encountered, such as lack of signal or excessive noise in the system, and potential solutions. The technique has limitations, such as the inability to deliver temporally complex stimuli, observe background firing immediately prior to stimulus delivery, or use water-insoluble taste compounds conveniently. Despite these limitations, this technique (including minor variations referenced in the protocol) is a standard, broadly accepted procedure for recording Drosophila neuronal responses to taste compounds.

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

This protocol describes extracellular recording of the action potential responses fired by labellar taste neurons in Drosophila.

电生理记录从<em&gt;果蝇</em&gt; Labellar味感器

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the ...

神经科学

Taste Exam: A Brief and Validated Test

The emerging importance of taste in medicine and biomedical research, and new knowledge about its genetic underpinnings, has motivated us to supplement classic taste-testing methods in two ways. First, we explain how to do a brief assessment of the mouth, including the tongue, to ensure that taste papillae are present and to note evidence of relevant disease. Second, we draw on genetics to validate taste test data by comparing reports of perceived bitterness intensity and inborn receptor genotypes. Discordance between objective measures of genotype and subjective reports of taste experience can identify data collection errors, distracted subjects or those who have not understood or followed instructions. Our expectation is that fast and valid taste tests may persuade researchers and clinicians to assess taste regularly, making taste testing as common as testing for hearing and vision. Finally, because many tissues of the body express taste receptors, taste responses may provide a proxy for tissue sensitivity elsewhere in the body and, thereby, serve as a rapid, point-of-care test to guide diagnosis and a research tool to evaluate taste receptor protein function.

This protocol measures human taste responses and includes a brief anatomical assessment, a short taste test, and a validation method using the subject&#39;s reported sensation and taste receptor genotype.

味觉考试: 一个简短的验证测试

The emerging importance of taste in medicine and biomedical research, and new knowledge about its genetic underpinnings, has motivated us to supplement ...

Whole-Mount Staining, Visualization, and Analysis of Fungiform, Circumvallate, and Palate Taste Buds

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells communicate with nerve fibers that carry this information to the brain. Because taste-transducing cells continuously die and are replaced throughout adulthood, the taste-bud environment is both complex and dynamic, requiring detailed analyses of its cell types, their locations, and any physical relationships between them. Detailed analyses have been limited by tongue-tissue heterogeneity and density that have significantly reduced antibody permeability. These obstacles require sectioning protocols that result in splitting taste buds across sections so that measurements are only approximated, and cell relationships are lost. To overcome these challenges, the methods described herein involve collecting, imaging, and analyzing whole taste buds and individual terminal arbors from three taste regions: fungiform papillae, circumvallate papillae, and the palate. Collecting whole taste buds reduces bias and technical variability and can be used to report absolute numbers for features including taste-bud volume, total taste-bud innervation, transducing-cell counts, and the morphology of individual terminal arbors. To demonstrate the advantages of this method, this paper provides comparisons of taste bud and innervation volumes between fungiform and circumvallate taste buds using a general taste-bud marker and a label for all taste fibers. A workflow for the use of sparse-cell genetic labeling of taste neurons (with labeled subsets of taste-transducing cells) is also provided. This workflow analyzes the structures of individual taste-nerve arbors, cell type numbers, and the physical relationships between cells using image analysis software. Together, these workflows provide a novel approach for tissue preparation and analysis of both whole taste buds and the complete morphology of their innervating arbors.

This paper describes methods for tissue preparation, staining, and analysis of whole fungiform, circumvallate, and palate taste buds that consistently yield whole and intact taste buds (including the nerve fibers that innervate them) and maintain the relationships between structures within taste buds and the surrounding papilla.

真菌形态、环形和味觉味蕾的全镶嵌染色、可视化和分析

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells ...

In vivo Calcium Imaging of Mouse Geniculate Ganglion Neuron Responses to Taste Stimuli

Within the last ten years, advances in genetically encoded calcium indicators (GECIs) have promoted a revolution in in vivo functional imaging. Using calcium as a proxy for neuronal activity, these techniques provide a way to monitor the responses of individual cells within large neuronal ensembles to a variety of stimuli in real time. We, and others, have applied these techniques to image the responses of individual geniculate ganglion neurons to taste stimuli applied to the tongues of live anesthetized mice. The geniculate ganglion is comprised of the cell bodies of gustatory neurons innervating the anterior tongue and palate as well as some somatosensory neurons innervating the pinna of the ear. Imaging the taste-evoked responses of individual geniculate ganglion neurons with GCaMP has provided important information about the tuning profiles of these neurons in wild-type mice as well as a way to detect peripheral taste miswiring phenotypes in genetically manipulated mice. Here we demonstrate the surgical procedure to expose the geniculate ganglion, GCaMP fluorescence image acquisition, initial steps for data analysis, and troubleshooting. This technique can be used with transgenically encoded GCaMP, or with AAV-mediated GCaMP expression, and can be modified to image particular genetic subsets of interest (i.e., Cre-mediated GCaMP expression). Overall, in vivo calcium imaging of geniculate ganglion neurons is a powerful technique for monitoring the activity of peripheral gustatory neurons and provides complementary information to more traditional whole-nerve chorda tympani recordings or taste behavior assays.

Here we present how to expose the geniculate ganglion of a live, anesthetized laboratory mouse and how to use calcium imaging to measure the responses of ensembles of these neurons to taste stimuli, allowing for multiple trials with different stimulants. This allows for in depth comparisons of which neurons respond to which tastants.

在活体钙成像小鼠致病性甘里昂神经元对味觉刺激的反应

Within the last ten years, advances in genetically encoded calcium indicators (GECIs) have promoted a revolution in in vivo functional imaging. Using ...

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

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

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

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

μTongue:一个基于微流体的功能成像平台，用于在Viva的舌头

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

一种增强昆虫味觉神经元活性分析的新方法

Base Recording: A Technique for Analyzing Responses of Taste Neurons in Drosophila

Insects taste the external world through taste hairs, or sensilla, that have pores at their tips. When a sensillum comes into contact with a potential food source, compounds from the food source enter through the pore and activate neurons within. For over 50 years, these responses have been recorded using a technique called tip recording. However, this method has major limitations, including the inability to measure neural activity before or after stimulus contact and the requirement for tastants to be soluble in aqueous solutions. We describe here a technique that we call base recording, which overcomes these limitations. Base recording allows the measurement of taste neuron activity before, during, and after the stimulus. Thus, it allows extensive analysis of OFF responses that occur after a taste stimulus. It can be used to study hydrophobic compounds such as long-chain pheromones that have very low solubility in water. In summary, base recording offers the advantages of single-sensillum electrophysiology as a means of measuring neuronal activity - high spatial and temporal resolution, without the need for genetic tools - and overcomes key limitations of the traditional tip recording technique.

作者聚焦：化学接收的进步 – 从昆虫气味受体到非编码 RNA

A rarely used method of electrophysiological recording, base recording, allows analysis of features of taste coding that cannot be examined by conventional recording methods. Base recording also allows the analysis of taste responses to hydrophobic stimuli that cannot be studied using traditional electrophysiological methods.

基础记录：一种分析果蝇味觉神经元反应的技术

Insects taste the external world through taste hairs, or sensilla, that have pores at their tips. When a sensillum comes into contact with a potential ...

In Vivo Calcium Imaging of Taste-Induced Neural Responses in Adult Drosophila

For nearly two decades, in vivo calcium imaging has been an effective method for measuring cellular responses to taste stimuli in the fruit fly model organism, Drosophila melanogaster. A key strength of this methodology is its ability to record taste-induced neural responses in awake animals without the need for anesthesia. This approach employs binary expression systems (e.g., Gal4-UAS) to express the calcium indicator GCaMP in specific neurons of interest. This protocol describes a procedure in which flies expressing GCaMP are mounted with the labellum securely positioned, enabling fluorescence in the brain to be recorded at millisecond resolution under a confocal microscope while a solution is applied to the labellum, stimulating all labellar taste sensilla. The examples provided focus on calcium responses in primary gustatory receptor neurons of D. melanogaster. However, this approach can be adapted to record from other neurons of interest within the brain of Drosophilids or other insect species. This imaging method enables researchers to simultaneously record collective calcium responses from groups of gustatory neurons across the labellum, complementing electrophysiological tip recordings that quantify action potentials from individual neurons. The in vivo calcium imaging technique outlined here has been instrumental in uncovering molecular and cellular mechanisms of chemosensation, identifying unique temporal response patterns in primary taste neurons, investigating mechanisms of gustatory modulation, and exploring taste processing in downstream circuits.

A calcium imaging approach enables the recording of taste-induced responses in the brains of awake flies while a solution is applied to the labellum. Primary gustatory responses in Drosophila melanogaster are used as an example, but this protocol can be adapted to study downstream neurons or other species.

体内研究成年果蝇味觉诱导神经反应的钙成像

For nearly two decades, in vivo calcium imaging has been an effective method for measuring cellular responses to taste stimuli in the fruit fly model ...

研究风味编码的新方法

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此视频中的章节

技术收集人舌菌状（味道）乳头

电生理记录从

味觉考试: 一个简短的验证测试

真菌形态、环形和味觉味蕾的全镶嵌染色、可视化和分析

在活体钙成像小鼠致病性甘里昂神经元对味觉刺激的反应

μTongue:一个基于微流体的功能成像平台，用于在Viva的舌头

基础记录：一种分析果蝇味觉神经元反应的技术

基础记录：一种分析果蝇味觉神经元反应的技术

基础记录：一种分析果蝇味觉神经元反应的技术

体内研究成年果蝇味觉诱导神经反应的钙成像