The work was in part funded by UK Medical Research Council project grant G0601253 to G.S.B. and R.W.B.

鼠标验尸组织收获必须使用认可的道德的方法。在英国，颈椎错位是成年小鼠（英国动物上市附表1的方法（科学程序）法案，1986年，欧盟指令63分之2010/ EU）政府批准的方法。这是立法阿伯丁的动物福利和伦理审查委员会的大学谁审查和批准了以下协议中使用的所有程序在本地执行。 注意:这些方法在银行等报道研究中使用7。 1.准备耳朵电<ol><li>解剖前，制备标准生理盐水，如Liley的（1956）中，用90％ 的 O 2/5％CO 2饱和的。 Liley的盐水（MM）是由: 碳酸氢钠 （12），氯化钾（4），KH 2 PO 4（1），氯化钠（138.8），MgCl 2的（1）， 氯化钙 （2）和葡萄糖（11）。 </li><li>人道安乐死成年小鼠不破坏靠近耳朵的头骨。这里，我们使用的C57 / BL6J和MF1小鼠，但可使用任何标准的实验室小鼠品系。理想的是，使用是&lt;25 g只有电记录是与苯乙烯基染料标记进行组合，如在较大的小鼠标记是不太可靠的小鼠中，经常被仅在限制外切地区发现。 </li><li>大剪刀或骨剪取出头。 </li><li>放置头部背侧在充气Liley在宽有底解剖盘（50厘米，通常方便）在一个立体声解剖显微镜的阶段。定期（约10分钟）水闸或用浸入盐水毒气头。 </li><li>小心切开和肌肤的外耳（耳廓）与弹簧弓形剪刀和＃3镊子的基座分开，露出外耳道（EAM）的软骨。确定三叉神经的分支（下颌分裂，MDV）和耳大神经，因为他们出现˚FROM通过软骨基地下颌关节和项目的裂头骨支配凹（前）和凸（后）耳廓皮肤方面，分别为。 </li><li>附近的头骨，发现其中两个神经分支出口在颌骨和乳突之间的凹槽。轻轻拉耳廓从头骨最大限度地远离它们的长度和削减接近头骨尽可能的神经。 </li><li>移除头部与弹簧弓形剪刀耳廓，注意避免分割神经的远端树桩，并在该被除去耳朵的基最小密毛皮的量。 </li><li>转移到耳廓灵活的硅橡胶（PDMS）-lined菜充满了气液固三相Liley的液体。通过将其在其最窄（最前）点开EAM。拼合耳廓，前皮肤（凹）朝下，脚到PDMS仔细用6-8〜规则间隔很细（〜直径0.2mm）昆虫针的边缘。 </li><li>雷姆完全奥雅纳耳廓的后方面的皮肤和部分地与＃3镊子除去由钝器解剖耳廓软骨，确保前皮肤保持不变。 </li><li>用细昆虫针的角度把握以＃3钳，轻轻地延长了MDV的皮肤和软骨EAM之间出现向后的分支（通常为2）。这些引脚与最好的可能的昆虫针的PDMS，刺穿结缔组织相邻的切端，而不是神经干自己。 </li><li>删除其中大部分在周围的结缔组织，小心避免与过量拉或受损。 </li></ol> 2.电生理记录设置<ol><li>针脚耳廓皮肤与周围的边缘罚款昆虫针一个录音室的PDMS内衬基地（〜6％至8耳），放置清洗神经在耳朵附近的两个电极吸基地 - 一个用于记录（以走神经）和OT她一副无所谓的电极（提供中性信号，差分放大器，请参阅7）。 </li><li>对于记录电极，仔细匹配孔和开口的两个神经的组合厚度的内直径，所以它们适合作为紧贴尽可能和一样大的长度尽可能。 </li><li>绘制神经成通过温和抽吸从附着有硅橡胶管的另一端2毫升注射器的电极。确保神经都直了，不折叠或弯了腰。 </li><li>通过使用更强的吸力吸取结缔组织或脂肪组织，以形成一个插头，其紧紧地密封在神经周围的孔发展高电阻/阻抗配合。 </li><li>吸满盐水的其他（无所谓）电极（如有必要，也可以通过毛细作用填写）。 </li><li>放置相同记录导线（银或铂）到记录电极的内孔以接触盐水和神经（录音）或电极的收窄，火抛光结束（无所谓）。每个电极分别焊接到两芯屏蔽电缆的不同的内核。放置浴（接地）电极（银/氯化银沉淀）到浴，并将其研磨成连接到记录电极两芯电缆的屏幕。 </li><li>从两个电极饲料的电活动成差分放大器，滤波器（带过去了0.2-2千赫），并查看示波器屏幕上的独立的通道。检查通道A（记录）和通道B（无所谓）电气噪声水平看起来相似。在此阶段，可能无法看到信道A中的正常的自发动作电位之前的两个电极是平衡的。 </li><li>通过增加记录（通道A）或漠不关心（通道B）电极的阻抗纠正背景噪音电极之间的差异。通过乳晕吸吮（脂肪）结缔组织进一步陷入要么电极做到这一点，和/或在50毫升注射器施加更大的抽吸力。 <ol><li>一旦以这种方式"均衡"，切换回差（AB）记录和查找在跟踪自发动作电位（APS），或当在耳廓的保证金抚摸头发。如果没有活动（扣球）看到，重新检查神经的记录电极的紧密配合，并在冷漠电极乳晕结缔组织 - 通常是更严格的（高电阻）密封，并在两个更加平等的阻抗电极，就更好了。通过实践，这将实现良好的质量（&gt; 2:1）信号:信噪比。 </li></ol></li><li>记录通过计算机上运行的实验室接口和电软件的电图。尖峰的音频输出是非常有用的，并且可以通过一个音频放大器和相关音频扬声器馈送neurogram来实现。调整门槛只是基线噪音（因没有白-n的上述鉴定瓦兹&#39;嘶嘶声&#39;）。 </li><li>为了增加药物或荧光苯乙烯基染料获得了披结局，小心剥离附近的耳廓缘子真皮脂肪层，在打开的约5毫米×5mm的窗口露出真皮和卵泡基（ 图1A，B ）。 </li><li> （在刚生产出来的窗口层面如适用）引脚背部脂肪，清除耳部皮肤的约1毫米的折叠在领先幅度，而使皮肤并列层之间的明确生理盐水填充空白。轻轻抚摩一起用大头针或接地细镊子折叠的边缘突起，不沾皮肤毛发，来定位音频输出示波器和鲜明的最大输出AP的"持久性有机污染物"区域"点击"和。 </li></ol> 3.录音刺激诱发的动作电位<ol><li>连接到Cerami研究火抛光10厘米硼硅玻璃微电极 - 机械刺激探头位置Ç压电致动器 - 所以运动是与皮肤褶皱平行。将尖端皮肤约0.5-1毫米倍，所以倒是头发而不是外观。慢慢手动移动探头尖端偏转的头发，并观察/倾听扣球确认有效的刺激。 </li><li>使用该软件可带动1-3毛机械刺激（ 如 3秒在5 Hz的正弦波，每10秒排量探讨200-500微米），并记录在神经刺激诱 ​​发反应。 </li><li>在10秒的间隔给几个相同的机械性刺激列车。优化探针位置重复性，再根据实验方案（从10秒至30秒如降重复率）降低的刺激频率。 </li><li>使用该软件使用简单的阈值设定为〜2倍的高频噪声幅度和最大AP的〜25％以鉴别AP样活性例如 。 </li><li>算上接入点跨越的门槛为&#39;事件&#39;，量化生产的AP的频率和特点。 </li></ol> 4.录音刺激诱发射击与N-（3- triethylammoniumpropyl）-4-（4-（二丁基氨基）苯乙烯基）吡啶鎓二溴化物标记组合<ol><li>为了检验苯乙烯基染料对刺激诱发传入射击和末端标记的影响，增加选择的苯乙烯染料的适当的浓度，浴液，并继续与电气录音。 </li><li>所需的曝光时间（通常至少30分钟）后，准备用于观看毛囊的制备。通过去除固定销展开皮瓣，并露出真皮清区揭示末端标记。 </li><li>洗去外部染料与不含染料的生理盐水，使3个完整盐水变化。 </li><li>孵育毒气无染料生理盐水10-15分钟，最后变化，让最顽固染料的污染，从曝光/外部膜滤。 </li><li>去除残留在用多价螯合剂（sulfobutylatedβ-环糊精，1毫米，5分钟）在盐水膜非内化染料。 </li><li>录音室转移到直立落射荧光显微镜的舞台，并与台/滑动运动机构从事室。 </li><li>照亮适当的苯乙烯基染料的激发光的制备。使用10X 25X或荧光显微镜物镜观察卵泡标签（ 图1）。 </li></ol>

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The authors have nothing to disclose.

在这里，我们已经开发出一种相对简单的制剂，其可迅速解剖，具有低的毛囊密度，并允许少量毛囊的相对选择性机械刺激。这是电生理记录和活细胞荧光成像，包括反应染料应用可视化的机械刺激毛囊， 即成像卵泡定义的电生理反应容易获得。虽然我们还没有这样做，这个系统似乎也易于成子划分为单独的单元（单感觉轴突）记录感觉神经，并使用有针对性的GFP表达的可视化的感官终端结束的形态。 我们已经使用了耳朵皮肤制剂，调查内化和荧光膜苯乙烯pridinium染料7的释放的特点，一个技术最初开发研究局部vesic乐膜回收突触端子8。在突触，成像也很容易在确定终端8,9的反应同时电生理记录相结合。正是在这些早期研究中，我们首先注意到的染料也受到mechanosensory结局10内化。对于培养和耳蜗毛细胞感觉神经元，备受苯乙烯吡啶染料标记似乎涉及通过mechanosensory通道，然后他们阻止11,12染料。染料给它贴上标签细胞内膜，而这个标签是不可逆的。然而，在毛细胞中未机械刺激13,14和在原位完全分化初级感觉神经末梢，如在肌肉主轴15 IA结局，并且在这里7的披针形的末梢，苯乙烯基染料标记似乎反映膜的内吞作用，因为标签是可逆的，并不会阻止mechanosensory RESponses 7,15,16。而在这些神经末梢通道渗透一些染料的内化，不能完全排除，这是从染料孵育期间的持续点火和标记的可逆性清楚，大多数在原位分化终端标记的是由内化与回收囊泡膜。因此，这个简单的技术容易地用于一系列的在离体组织mechanosensory终端功能的组合电和光监视。 与大多数实用技术，重现需要重复和实践。一些关键点值得特别注意现在将描述。在整个解剖和记录会话，通过确保制剂被不断地用盐水，95％ 的 O 2/5％CO 2的完全饱和的灌流最大化组织的生存力和存活。确保毛囊在此过程中不移位，WHICH将刺激感官的结局射击。既可以使用一个连续的，层流灌注系统，或仔细气泡气体通过与细管的器官浴中从制剂的距离，或仔细地刷新溶液每20-30分钟，保持盐水表面下方的制备在任何时候。吸记录电极通过修改通常用于细胞内记录尖锐电极硼硅制成吸管。一是认真撅着尖顶与＃3钳给予相应的内部直径非常简短（&lt;1秒）接触本生灯火焰，以适应神经和火抛光（见2.4和2.5）。要获得记录时良好的信噪比，至关​​重要的是，在这两个电极上的电阻抗（电阻）既最大化和相等。通过注意在两个电极下做到这一点。对于记录电极，确保内部直径是滑动配合的神经，并且网元的最大长度RVE被吸入到记录电极。尝试使用周围神经的结缔组织，有效地密封电极尖端。可替代地，或另外地，画出一个适当大小的锥形件的脂肪组织的窄端在沿着神经。然后，通过施加强大的吸力为约1分钟与连接到管道50毫升注射器堵塞电极尖端。为一个良好的密封尖，施加强大的吸力将简单地加强插头的有效性，并且不会吸引更多的液体或神经。为了避免神经损伤，但是，需要确保结缔组织是从压缩缓冲神经对周边物质和EAM软骨。冷漠的电极应密切模仿记录电极的电阻/阻抗越好。这是通过仔细火抛光尖端小的孔尽可能不实际密封它帮助。如果需要进一步的阻力，再插上与adipo冷漠电极末端本身结缔组织，上述用于记录电极所描述。 该电瓷给出了机械位移精致控制，在空间和时间。然而，照顾电气连接 - 高温消灭他们，所以不要用太热的焊料。使用负载金属的环氧树脂胶，或使用由供应商推荐的专家推入式插座。这都将牢牢把握它，并建立电气连接。玻璃刺激探针附加到与标准环氧树脂的电瓷。火抛光的一个标准的10厘米终点X用于制备贴片或尖锐电极，用于电生理记录，以减少组织损伤的风险1.5毫米直径硼硅玻璃毛细管。如果需要单一毛囊的刺激，火抛光尖端适合单发，且探头开放开口内单发的位置。这给出了一个精致的发型控制。对于苯乙烯DYË标签，它是从年轻的动物组织通常更均匀。这是不完全清楚为什么，但是这可能反映了年轻的组织较少的机械损伤，更有效的深层组织去除。在去除泡沫层外形酷似发泡聚苯乙烯覆毛囊碱基彻底。但是，避免被过于剧烈，因为这样的风险去除神经丛层和相关披针形端子。如果有毛囊运动很少或没有电响应，和苯乙烯基染料的应用导致皮脂腺（黄/白色）的主要标记，与毛干，而不是披针形端部的基部的独特的自发荧光（橙色/黄色），过分热情的间隙破坏了下面的组织。最后，使用在成像之前的染料螯合剂极大地提高了最终图像的图像的对比度和质量。 这种技术可以在一个范围内进一步研究是有用的。这些共ULD包括，例如，筛选负责拉伸诱发反应的mechanosensory频道（），通过温育与药理学配体选择性的候选信道的准备或筛选用这样的通道基因缺失小鼠系。后者可以与在终端形态的任何改变的荧光评估由于鼠标线， 例如与NPY2R联GFP表达17遗传操作相结合。最后一个例子可以是通过检查SLV周转的调节剂的作用研究突触样小泡在这些披针形端子的作用（SLVS）7（钙，镁，latrotoxin，谷氨酸受体的配体）上拉伸诱发反应和苯乙烯基染料吸收/发布。因此，这种新技术开辟了一个范围的mechansensory神经科学研究可能感兴趣的途径。

感觉轴突的披针形端子支配毛囊哺乳动物形成围绕毛发轴上皮栅栏。他们的目的是检测它们包围的毛发的机械位移。他们是迅速，慢慢适应了主要生产活动的短脉冲响应毛发运动神经末梢的混合。活动停止很快时运动停止，即使在持续位移的存在。在这里，我们描述了结构相关研究这种小鼠耳廓模型的发展和披针形端子功能。耳廓有研究这些神经末梢许多有利的功能。首先，耳廓是皮肤的基本上两层并列背到背，很少有其他组织之间的访问的卵泡和终端干涉。皮肤非常薄，很容易解剖由于极少量的坚韧结缔组织。该神经支配是方便和识别。虽然头发folliclES都存在，他们是相对比较稀疏分布，促进卵泡的个人或小团体机械的刺激。薄底层真皮层提供良好的可访问性与药理学药物和染料的神经末梢。这使得它们特别适合于用荧光显微镜成像研究。成像可以是在活的终端，或固定和进一步的组织学处理之后。 mechanosensory神经元的响应支配毛囊历来在啮齿动物研究触须1,2，以及在较小程度上，在分离的皮肤制剂3,4。这些都告诉我们很多关于mechanosensory生理学周围毛干中神经末梢的一般原则。该vibrissal准备允许在单个毛囊的运动控制精致。然而，它可以是很难破译，由于其复杂性的输出，如vibrissal卵泡含有至少8个不同类型的解剖学不同mechanosensory结局5和这些形态类型到特定电生理反应的匹配仍然是有争议的问题。小鼠皮肤/隐神经制备一般都是在其脱毛的状态来研究触觉和疼痛反应。毛囊在这种制剂中的神经支配是较不复杂的，但毛囊的密度，再加上如此接近6三种不同卵泡类型（后卫，锥/ auchene和锯齿形的毛发）的存在下，是指研究的特异性应答单个毛囊或单一类型的结局再次挑战。此外，该制剂涉及复杂的解剖。最后，在这两个vibrissal等皮肤制剂，它是很难想象涉及而体外制剂仍然活着的结局。因此，组织切片中表达GFP的鼠标线甚至要求。 Alternatively，它需要进一步的组织学/免疫处理，例如定影和/或抗体孵育免疫荧光。 因此，我们开发了耳廓的准备，并用它来从毛囊传入受限人群进行电气录音和显示膜自行车在这些披针形的结局，由苯乙烯吡啶染料的吸收证明发生。最后，我们发现，在染料不具有机械灵敏度干扰，表明它不阻挡机械传导通道。简单的刺激和协议分析的结果所示。

<table border="0" cellpadding="0" cellspacing="0" width="1435">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PDMS - Sylgard 184</td>
			<td style="width:111px;">Dow Corning</td>
			<td style="width:119px;"></td>
			<td style="width:989px;">Flexible, inert, translucent solid silicone polymer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">No. 3 Dumont forceps</td>
			<td>Fine Science Tools</td>
			<td>11231-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Austerlitz Insect pins</td>
			<td>Fine Science Tools</td>
			<td>26002-10</td>
			<td>Very fine pins to attach pinna preparation securely to the PDMS with minimal damage.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AC Differential Preamplifier</td>
			<td>Digitimer</td>
			<td>Neurolog NL104A</td>
			<td>Amplifying the size of the incoming afferent electroneurogram. Differential recording minimises the extraneous electrical noise and baseline drift.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">High/Low-pass Filter</td>
			<td>Digitimer</td>
			<td>Neurolog NL125</td>
			<td>Signal conditioning, by reducing extraneous electrical noise to ensure best signal to noise ratio.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spike Trigger</td>
			<td>Digitimer</td>
			<td>Neurolog NL201</td>
			<td>Sets the event detector threshold and displays it on the oscilloscope. This shows the action potential detection efficacy.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Audio Amplifier &#38; speakers</td>
			<td>Digitimer</td>
			<td>Neurolog NL120S</td>
			<td>Useful audio monitoring for the presencec of electrical firing of the sensory endings while adjusting the mechanical stimulation preparation down the microscope&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oscilloscope</td>
			<td>Digitimer</td>
			<td>PM3380A</td>
			<td>We use this old model but any standard oscilloscope will suffice.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Piezo electroceramic&#160; wafer</td>
			<td>Morgan Electroceramics, Southampton UK</td>
			<td>PZT507</td>
			<td>Electrophysiology/computer interface</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Piezo electroceramic&#160; powersupply</td>
			<td>Home made</td>
			<td></td>
			<td>0-200V DC output to drive the ceramic wafer displacement, with variable electronic control of output via recording/stimulation software and computer interface. We use Spike2 software and 1401micro computer interface.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Electrophysiology Software</td>
			<td>Cambridge Electronic Design (CED)</td>
			<td>Spike2 v7</td>
			<td>Electrophysiology recording, stimulation and data analysis software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laboratory interface</td>
			<td>Cambridge Electronic Design (CED)</td>
			<td>1401 micro</td>
			<td>Electrophysiology interface, between the amplifier/filters and the computer. It inputs the electroneurogram and also drives the electroceramic movement.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FM1-43/Synaptogreen C4</td>
			<td>Biotium/Cambridge Bioscience</td>
			<td>BT70020</td>
			<td>Fluorescent membrane probe that reversibly partitions into the outer leaflet of cell membranes. Used predominantly for monitoring vesicle membrane endo-/exocytosis.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Advasep 7</td>
			<td>Biotium/Cambridge Bioscience</td>
			<td>BT70029</td>
			<td>A sulfonated b-cyclodextrin derivative that chelates FM1-43 (&#38; other styryl pyridinium dyes) out of the exposed membranes, leaving internalised dye to be seen more clearly by lowering the background labelling/fluorescence.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Retiga Exi Fast 1394</td>
			<td>Qimaging</td>
			<td></td>
			<td>Monochrome, cooled CCD camera - basic model</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Volocity 3D Image Analysis Software</td>
			<td>Perkin Elmer</td>
			<td>Volocity 6.3</td>
			<td>Image capture and analysis software.</td>
		</tr>
	</tbody>
</table>

combined recording of mechanically stimulated afferent output and nerve terminal labelling in mouse hair follicle lanceolate endings

一种新颖的解剖和记录技术是用于通过在小鼠耳廓的毛发机械位移诱发监测传入击发说明。该技术是非常划算的，并在大多数电生理实验室常见的，否则容易采购的材料很容易进行。解剖简单，快速，与由专有软件控制的通用电子陶瓷晶片提供的机械位移。同样的软件还可以记录和分析了电图输出。诱发的神经活动的记录是通过连接到火抛光标准玻璃微电极商业差分放大器。温馨提示，给出了改进的准备，刺激和拍摄条件，优化刻录品质的质量。该系统适合于测定的毛囊的栅栏神经末梢的披针形端子的电性能和光学性能，以及从它们的药理学和/或遗传操作的结果。电记录用机械刺激和标签与苯乙烯基吡啶鎓活体染料相结合的一个实例。

电生理学记录装置，与皮下脂肪组织去除以暴露毛囊，示于图1A。该露出区域的一部分被示出在图1B中明照明更高放大倍数。在这方面并回到本身折叠耳廓缘露出表皮表面。这个位置上的褶皱的边缘的毛发在水平方向突出，在用于用钝探针（未示出）。机械刺激一个理想的情况。图1C示出染料的曝光和再返回缘至展开位置后，受刺激卵泡在该位置处已利用与苯乙烯基吡啶鎓染料。从制剂Electroneurograms通常显示正在进行AP活性甚至在没有玻璃纤维探针施加运动，包括最大大小的AP的（ 图2A <str翁&gt; I-III）。活动发生在约10-20脉冲/秒（"尖峰"的迹线）的速率，示出没有结构或张力的迹象，作为刺激周期之间的间隔的自相关是很平坦的。期间5赫兹，毛发干100微米的机械刺激，整体输出通常上升到20-50脉冲/秒，或4-10每正弦周期。然后循环直方图结构揭示了响应的强夹带（所谓的"相位锁定"）（ 图2BI-ⅲ））。这是相当重复的，尽管在该反应被锁定的波形的相位在该点的运动取决于它们的位置， 即在振动探头接触到毛发。一个简单的阈值检测（水平线跨越neurogram痕迹A（I-III））已被用于该分析。然而，记录软件的功能往往可以在一个更复杂的方式来鉴别穗的大小和形状的特征。然后这可通过这些特征隔离特定传入纤维的响应，以允许进行伪单细胞记录分析。 <img alt="图1" src="/files/ftp_upload/53854/53854fig1.jpg" /> 图1:电生理记录安排与N-（3-triethylammoniumpropyl）-4-（4-（二丁基）苯乙烯基）吡啶二溴化物毛囊传入披针形的结局标签 （A）耳廓准备设立展示耳廓的电生理实验取向，神经的位置，在记录电极和卵泡神经支配的脂肪组织取出后，在暴露的区域。 （ 二 ）一些毛囊在清除覆脂肪组织下至真皮的面积亮场照明可见。黑暗中，通常bilobed，形状是皮脂腺。 （ <st荣&gt; C）的B中示出标以苯乙烯基染料N-（3- triethylammoniumpropyl）-4-（4-（二丁基氨基）苯乙烯基）吡啶鎓溴化物和通过荧光显微镜机械刺激后成像2披针末梢盒装区域）的放大图。 7，经许可使用。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53854/53854fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53854/53854fig2.jpg" /> 图2:（A）从鼠耳廓录制的刺激诱 ​​发的活动。 （AI-III）的例子，来自3个不同的实验，从连续neurograms从标准Liley的生理盐水羽片采取的活动。每个记录表示5秒部记录开始后约5分钟，包括作为正弦位移的3秒期间毛干的商场数量。机械刺激重复每30秒在整个连续记录，这通常持续1-2小时。自定义脚本文件标志着越过由水平光标（尖峰）设定的阈值，以及每个正弦循环（周期）的发病个体的事件。 （AIV）正弦位移指令信号。 （ 二 ）分析刺激诱 ​​发的扣球活动。在3°箱（ 碧-ⅲ）循环直方图分别（ 艾-ⅲ）从neurogram样品构造，示出了由玻璃探针推定披末稍对机械刺激的由毛干的5赫兹的正弦位移的响应。注意标记相位锁定，在周期的不同阶段，这取决于相对于机械探针的起始位置的位置时。 （ 起搏 ）归探头位移正弦曲线;实际幅度大约为1001;:M <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53854/53854fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Combined Recording of Mechanically Stimulated Afferent Output and Nerve Terminal Labelling in Mouse Hair Follicle Lanceolate Endings at JoVE.com

Combined Recording of Mechanically Stimulated Afferent Output and Nerve Terminal Labelling in Mouse Hair Follicle Lanceolate Endings

A simple and novel technique for recording afferent discharge due to mechanical stimulation of lanceolate terminals of palisade endings innervating mouse ear skin hair follicles is presented.

机械刺激传入输出和神经末梢标签联合录制在小鼠毛囊披针形结局

A novel dissection and recording technique is described for monitoring afferent firing evoked by mechanical displacement of hairs in the mouse pinna. The ...

combined-recording-mechanically-stimulated-afferent-output-nerve

Nanyang Technological University

Research

JoVE Journal

Neuroscience

7.4K 次观看.  University of Aberdeen. A simple and novel technique for recording afferent discharge due to mechanical stimulation of lanceolate terminals of palisade endings innervating mouse ear skin hair follicles is presented.

视频: Combined Recording of Mechanically Stimulated Afferent Output and Nerve Terminal Labelling in Mouse Hair Follicle Lanceolate Endings

Postsynaptic Recordings at Afferent Dendrites Contacting Cochlear Inner Hair Cells: Monitoring Multivesicular Release at a Ribbon Synapse

The afferent synapse between the inner hair cell (IHC) and the auditory nerve fiber provides an electrophysiologically accessible site for recording the postsynaptic activity of a single ribbon synapse 1-4. Ribbon synapses of sensory cells release neurotransmitter continuously, the rate of which is modulated in response to graded changes in IHC membrane potential 5. Ribbon synapses have been shown to operate by multivesicular release, where multiple vesicles can be released simultaneously to evoke excitatory postsynaptic currents (EPSCs) of varying amplitudes 1, 4, 6-11. Neither the role of the presynaptic ribbon, nor the mechanism underlying multivesicular release is currently well understood.
The IHC is innervated by 10-20 auditory nerve fibers, and every fiber contacts the IHC with a unmyelinated single ending to form a single ribbon synapse. The small size of the afferent boutons contacting IHCs (approximately 1 &mu;m in diameter) enables recordings with exceptional temporal resolution to be made. Furthermore, the technique can be adapted to record from both pre- and postsynaptic cells simultaneously, allowing the transfer function at the synapse to be studied directly 2. This method therefore provides a means by which fundamental aspects of neurotransmission can be studied, from multivesicular release to the elusive function of the ribbon in sensory cells.

Whole-cell patch-clamp recordings from auditory nerve fiber dendrites at the inner hair cell ribbon synapse in the mammalian cochlea.

与耳蜗内毛细胞传入树突:多泡发布监测功能区突触的突触后录音

The afferent synapse between the inner hair cell (IHC) and the auditory nerve fiber provides an electrophysiologically accessible site for recording the ...

科研

神经科学

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their close-to-surface positioning facilitates microscopic imaging of skin nerve endings in living intact animal. Using multiphoton microscopy, it is possible to obtain fine images overcoming the problem of strong light scattering of the skin tissue. Reporter transgenic mice that express EYFP under the control of Thy-1 promoter in neurons (including periphery sensory neurons) are well suited for the longitudinal studies of individual nerve endings over extended periods of time up to several months or even life-long. Furthermore, using the same femtosecond laser as for the imaging, it is possible to produce highly selective lesions of nerve fibers for the studies of the nerve fiber restructuring. Here, we present a simple and reliable protocol for longitudinal multiphoton in vivo imaging and laser-based microsurgery on mouse skin nerve endings.

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

The protocol for dynamic longitudinal imaging and selective laser lesion of nerve endings in reporter transgenic mice is presented.&#160;

在皮肤中的单神经末梢体内双光子显微镜

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their ...

Flat Mount Imaging of Mouse Skin and Its Application to the Analysis of Hair Follicle Patterning and Sensory Axon Morphology

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel cell clusters), sebaceous glands, nerves and nerve endings, and capillaries. The spatial arrangement of these structures is tightly controlled on a microscopic scale - as seen, for example, in the orderly arrangement of cell types within a single hair follicle - and on a macroscopic scale - as seen by the nearly identical orientations of thousands of hair follicles within a local region of skin. Visualizing these structures without physically sectioning the skin is possible because of the 2-dimensional geometry of this organ. In this protocol, we show that mouse skin can be dissected, fixed, permeabilized, stained, and clarified as an intact two dimensional object, a flat mount. The protocol allows for easy visualization of skin structures in their entirety through the full thickness of large areas of skin by optical sectioning and reconstruction. Images of these structures can also be integrated with information about position and orientation relative to the body axes.

Mammalian skin contains a diverse array of structures - such as hair follicles and nerve endings - that exhibit distinctive patterns of spatial organization. Analyzing skin as a flat mount takes advantage of the 2-dimensional geometry of this tissue to produce full-thickness high-resolution images of skin structures.

小鼠皮肤及其毛囊图案和感觉轴突形态分析中的应用平山成像

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel ...

Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to monitor the disease course in the follow-up period. Changes in the VEP parameters closely correlate with pathological damage in the optic nerve. This protocol provides a detailed description about the rodent model of optic nerve microinjection, in which a partial demyelination lesion is produced in the optic nerve. VEP recording techniques are also discussed. Using skull implanted electrodes, we are able to acquire reproducible intra-session and between-session VEP traces. VEPs can be recorded on individual animals over a period of time to assess the functional changes in the optic nerve longitudinally. The optic nerve demyelination model, in conjunction with the VEP recording protocol, provides a tool to investigate the disease processes associated with demyelination and remyelination, and can potentially be employed to evaluate the effects of new remyelinating drugs or neuroprotective therapies.

Focal demyelination is induced in the optic nerve using lysolecithin microinjection. Visual evoked potentials are recorded via skull electrodes implanted over the visual cortex to examine the signal conduction along the visual pathway in vivo. This protocol details the surgical procedures underlying electrode implantation and optic nerve microinjection.

在实验视神经脱髓鞘模型大鼠视觉诱发电位记录

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to ...

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles in the skin of the mouse pinna. The preparation is simple and relatively fast, reliably yielding extensive regions of multiple labeled units of living nerve terminals to study uptake and release of styryl pyridinium dyes extensively used in studies of vesicle recycling. Subdividing the preparations before labeling allows test vs. control comparisons in the same ear from a single individual. Helpful tips are given for improving the quality of the preparation, the labeling and the imaging parameters. This new system is suitable for assaying pharmacologically and mechanically-induced uptake and release of these vital dyes in lanceolate terminals in both wild-type and genetically modified animals. Examples of modulatory influences on labeling intensity are given.

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

Here we describe a novel preparation for imaging live lanceolate sensory terminals of palisade endings that innervate mouse ear skin hair follicles during staining and destaining with styryl pyridinium dyes.

在毛囊披针形结局生活神经末端标记的光学监测<em&gt;离体</em&gt;鼠耳皮肤

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles ...

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 ...

In Vitro Recording of Mesenteric Afferent Nerve Activity in Mouse Jejunal and Colonic Segments

Afferent nerves not only convey information concerning normal physiology, but also signal disturbed homeostasis and pathophysiological processes of the different organ systems from the periphery towards the central nervous system. As such, the increased activity or &#39;sensitization&#39; of mesenteric afferent nerves has been allocated an important role in the pathophysiology of visceral hypersensitivity and abdominal pain syndromes.
Mesenteric afferent nerve activity can be measured in vitro in an isolated intestinal segment that is mounted in a purpose-built organ bath and from which the splanchnic nerve is isolated, allowing researchers to directly assess nerve activity adjacent to the gastrointestinal segment. Activity can be recorded at baseline in standardized conditions, during distension of the segment or following the addition of pharmacological compounds delivered intraluminally or serosally. This technique allows the researcher to easily study the effect of drugs targeting the peripheral nervous system in control specimens; besides, it provides crucial information on how neuronal activity is altered during disease. It should be noted however that measuring afferent neuronal firing activity only constitutes one relay station in the complex neuronal signaling cascade, and researchers should bear in mind not to overlook neuronal activity at other levels (e.g., dorsal root ganglia, spinal cord or central nervous system) in order to fully elucidate the complex neuronal physiology in health and disease.
Commonly used applications include the study of neuronal activity in response to the administration of lipopolysaccharide, and the study of afferent nerve activity in animal models of irritable bowel syndrome. In a more translational approach, the isolated mouse intestinal segment can be exposed to colonic supernatants from IBS patients. Furthermore, a modification of this technique has been recently shown to be applicable in human colonic specimens.

In Vitro Recording of Mesenteric Afferent Nerve Activity in Mouse Jejunal and Colonic Segments

Mesenteric afferent nerves convey information from the gastrointestinal tract towards the brain regarding normal homeostasis as well as pathophysiology. Gastrointestinal afferent nerve activity can be assessed by mounting isolated intestinal segments with attached afferent nerves into an organ bath, isolating the nerve, and assessing basal as well as stimulated activity.

在鼠标空肠和结肠段肠系膜传入神经活性的体外记录

Afferent nerves not only convey information concerning normal physiology, but also signal disturbed homeostasis and pathophysiological processes of the ...

Loading a Calcium Dye into Frog Nerve Endings Through the Nerve Stump: Calcium Transient Registration in the Frog Neuromuscular Junction

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using calcium-sensitive fluorescent dyes that change their emission intensity or wavelength depending on the concentration of free calcium in the cell. There are several methods used to stain cells with calcium dyes. Most common are the processes of loading the dyes through a micropipette or pre-incubating with the acetoxymethyl ester forms of the dyes. However, these methods are not quite applicable to neuromuscular junctions (NMJs) due to methodological issues that arise. In this article, we present a method for loading a calcium-sensitive dye through the frog nerve stump of the frog nerve into the nerve endings. Since entry of external calcium into nerve terminals and the subsequent binding to the calcium dye occur within the millisecond time-scale, it is necessary to use a fast imaging system to record these interactions. Here, we describe a protocol for recording the calcium transient with a fast CCD camera.

Here, we describe a method for loading a calcium-sensitive dye through the frog nerve stump into the nerve endings. We also present a protocol for the recording and analysis of fast calcium transients in the peripheral nerve endings.

通过神经树桩将钙染料加载到青蛙神经末梢:青蛙神经肌肉接头中的钙瞬态登记

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using ...

Auditory Brainstem Response and Outer Hair Cell Whole-cell Patch Clamp Recording in Postnatal Rats

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to amplify the weak vibration of low-level sound signal. The morphology and electrophysiological property of outer hair cells (OHCs) develop in early postnatal ages. The maturation of outer hair cell may contribute to the development of the auditory system. However, the process of OHCs development is not well studied. This is partly because of the difficulty to measure their function by an electrophysiological approach. With the purpose of developing a simple method to address the above issue, here we describe a step-by-step protocol to study the function of OHCs in acutely dissociated cochlea from postnatal rats. With this method, we can evaluate the cochlear response to pure tone stimuli and examine the expression level and function of the motor protein prestin in OHCs. This method can also be used to investigate the inner hair cells (IHCs).

This protocol describes methods to record the auditory brainstem response from postnatal rat pups. To examine the functional development of outer hair cells, the experimental procedure of whole-cell patch clamp recording in isolated outer hair cells is described step-by-step.

产后大鼠听觉脑干反应及外毛细胞全细胞贴片钳记录

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to ...

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.