Name: in-depth physiological analysis of defined cell populations in acute tissue slices of the mouse vomeronasal organ
Uploaded: 2016-09-10T14:00:00.000+00:00
Duration: 10 min 11 s
Description: Watch this Scientific Journal Video about In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ at JoVE.com

We thank Ivan Rodriguez and Benoit von der Weid for generating the FPR-rs3-i-venus mouse line, their constructive criticism and fruitful discussions. This work was funded by grants of the Volkswagen Foundation (I/83533), the Deutsche Forschungsgemeinschaft (SP724/6-1) and by the Excellence Initiative of the German federal and state governments. MS is a Lichtenberg Professor of the Volkswagen Foundation.

所有的动物程序符合与用于实验目的动物保护（86/609 / EEC），并建议提出的欧洲实验动物科学协会联合会（FELASA）本地和欧盟的法规。既C57BL / 6小鼠和FPR-RS3-I-金星小鼠在室温下容纳在两种性别的群体与食物和水可随意获得一个12小时的光/暗周期。对于实验中使用两种性别的青少年（6-20周）。无明显的性别差异相关。 1.溶液的制备<ol><li>制备细胞外溶液S 1:4-（2-羟基-乙基）哌嗪-1-乙磺酸（HEPES）缓冲的含有（以mM计）145 NaCl的，5氯化钾，1的CaCl 2，1 MgCl 2的，10 HEPES胞外溶液; pH值= 7.3（用NaOH调节）;渗透压= 300毫渗量（以葡萄糖调整）。 </li><li>准备extracellu拉尔溶液S 2:卡波金含氧（95％O 2，5％的CO 2）含有（以mM计）125 NaCl的，25的NaHCO 3，5的KCl，1的CaCl 2，1用MgSO 4细胞外溶液中，5 BES; pH值= 7.3;渗透压= 300毫渗量（以葡萄糖调整）。 </li><li>用于组织包埋制备4％低胶凝温度的琼脂糖溶液（S 3）:4％低胶凝温度的琼脂糖。溶解2克有磁力搅拌器琼脂糖低胶凝在50ml S1的在一个100ml玻璃实验室瓶在一起。熔化琼脂糖，把瓶子在水浴（有盖未盖紧）在70℃下约20分钟或直至溶液变，同时搅拌透明。冷却并在42℃下保持熔融的琼脂糖在第二水浴中，直到进一步使用。 </li><li>制备细胞内溶液S 4:含有（以mM计）143的KCl，2 KOH，1 EGTA，0.3 氯化钙 （游离Ca 2+ = 110纳米），10 HEPES，2 MgATP移液器溶液，1 NaGTP;pH值= 7.1（用KOH调整）;渗透压= 290毫渗量。 </li><li>修改/调整S1的组合物，S 2和S 4，根据各个实验设计（ 例如 ，药理学阻断剂以分离某些类型的电压激活电流的）。 </li></ol> 2.准备工作区<ol><li>填充S 2至少30分钟之前夹层，置于冰上的温度和pH值调节和连续含氧化合物溶液充氧片储存室。 </li><li>在录制与S 2的设置填补切片灌流水库连续充氧。 </li><li>此前夹层，填充S 2 vibratome室，安排室周围的碎冰块。或者，从充氧腔进入vibratome腔室转移备用冰冷的含氧的解决方案，并保持连续充氧。 </li><li>安排手术工具和消耗品。 </li><li>将冰凝胶包下解剖显微镜下，用纸巾盖住，以防止VNO组织冻结在盘子的底部。 </li><li>通过在70％乙醇和蒸馏水简要地漂洗和安装到vibratome干净的刀片。替换为每个切片会话。 </li></ol> 3. VNO解剖和嵌入<ol><li>通过短暂暴露牺牲动物为CO 2和使用锋利的手术剪刀杀头。注意:如从牺牲动物到把VNO胶囊在冰上时间是非常关键的，应尽量减少到小于2分钟。为了保持组织活力，在不到30分钟，同时嵌入完全剖分VNOS琼脂糖。 </li><li>除去大手术剪下颌。通过口腔进入并分别切断各边的下颌骨的骨骼和肌肉。 </li><li>放置头部的其余部分在大陪替氏培养皿倒置。 </li><li>拉远上颚和周围介质镊子鼻子获得对更好的访问的前端的皮肤门牙。 </li><li>使用骨剪刀剪去门牙的最大部分在延髓方向（ 图1A）一〜45°角。这将缓解去除鼻腔VNO胶囊。 注意:不要切到牙根，以防止所述VNO胶囊的尖端损伤。 </li><li>抓住在其与介质钳子喙部刚性上腭和在一平面的角度（ 图1B）小心剥离背部在一块。 注意:用冰冷的S 2反复冲洗。 </li><li>使用微弹簧剪刀剪犁骨和下颚尖之间的骨性融合。插入有指向从所述VNO向外远离并小心切骨在左侧和右侧横向于VNO胶囊两个小步骤的前端的弯曲部的剪刀的提示。 </li><li>要删除VNO胶囊，使用微弹簧剪刀在尾椎部位通过犁骨切，小心地提起犁骨出了n个荒漠和半荒漠腔采用中等镊子。立即在其中夹层的其余步骤将执行一个冰凝胶袋所述VNO传送到一个​​小的培养皿立体显微镜下。 </li><li>冲洗所述VNO在冰冷的S 2的少量，以防止组织干燥。 </li><li>分离，通过抓取犁骨的中等钳后面包含VNO软组织软骨胶囊。这样既VNOS之间的分裂变得可见（ 图1D i）该胶囊为背视图的位置。 </li><li>使用细镊子的顶端，同时保持犁骨在后部部分牵制从中央骨分开两个软骨VNO胶囊。 注:仅在软骨胶囊的边沿使用镊子，并非常小心，不要通过刺穿软骨的微妙感觉上皮很容易损坏。 </li><li>一旦两个VNOS分离，开始除去软骨包封冷杉ŤVNO。 </li><li>抓住一个细镊子胶囊的顶部边缘和分裂出来将先前连接到犁骨（内侧）的软骨墙。 </li><li>以除去剩余软骨转动VNO其弯曲侧侧到培养皿的底部，并安全地使用镊子牵制软骨一侧。小心移动从背面侧的第二细镊子在软骨和VNO之间一个非常平坦角度松开组织和软骨之间的连接。 </li><li>通过举办它在它的尾部尖，以避免损坏感觉上皮慢慢剥离VNO由软骨了。 </li><li>一旦VNO从胶囊杠杆，确保消除所有剩余的小部分软骨软骨的任何剩余部分将在切片过程琼脂糖周围分离组织。 </li><li>将冰冷的S 2上的第一剖分VNO小降，以防止组织损伤。在侧面WIL大血管升可见这表明机构仍然完好无损，并解剖（ 图2A II）的过程中未严重受损。在情况下，血管损坏了，但它仍然是值得与只要整体形态不明确受损切片VNO进行。 </li><li>立即解剖第二VNO。 </li><li>要嵌入VNOS，请在两个小培养皿融化的第3轮辋（42℃储存在水浴见1.3）。 </li><li>慢着用细镊子更广泛的尾端的VNO以避免感官上皮损伤。 </li><li>沉浸所述VNO在琼脂糖和它水平地来回移动数次以除去细胞外溶液的膜，以及从它的表面上的任何气泡。 </li><li>在VNO与尾椎尖端对着盘底部垂直位置。而是直接镊子捏VNO的，通过移动紧密proximi镊子尖调整方向TY到VNO。 注:嵌入在导向是至关重要的，因为它在实验过程中决定切面和获取的感觉神经元。 </li><li>放置在凝胶冰包菜肴，等到琼脂糖凝固。 注意:不要更改VNO方向一旦琼脂糖已经开始凝固，因为这会从周围的琼脂糖分离组织。 </li></ol> 4.冠状VNO组织切片<ol><li>用小铲从小碟成一个大培养皿的盖子去掉琼脂糖块，翻转琼脂糖倒挂离开VNO的朝上的尾尖端。 </li><li>切割块到使用外科手术刀（3-4毫米在尖端，在底部8-10毫米）一个金字塔形。注意不要损坏包埋组织。 </li><li>使用超级胶水金字塔形块固定到vibratome样品板的中心，并且等待约1分钟为胶完全干燥。 </li><li>检体板传送到切片Çhamber并准备第二VNO，因此。保持板与第二样本在4℃下直到使用。 </li><li>使用振动切片机具有以下设置:厚度:150-200微米;速度:3.5金= 0.15毫米/秒;频率:7.5 AU = 75赫兹。转移切片补氧室，直到使用后解剖显微镜下检查短暂形态切片。片可以保持几个小时。 </li></ol> 5.单细胞的电生理记录<ol><li>对录音，使用配有浸水的目标，Dodt或红外线微分干涉对比（IR-DIC），和落射荧光以及一个冷却的CCD相机直立固定阶段显微镜。对于数据采集，利用膜片钳放大器，头部阶段，AD / DA接口板和PC（包括录音软件）。 </li><li>准备10-20补丁移液器（4-7MΩ）股票。从硼硅玻璃毛细管拉吸管（1.50 mm外径/0.86毫米ID）全光照GA微量拉马和火抛光用microforge。 注:修补相当小VSNS时火焰抛光的枪头是至关重要的。这将有助于防止在为了得到高电阻密封施加负压时破裂细胞膜。抛光移液器开场后应为约1微米。 </li><li>保持移液器在吸存储罐，以防止损坏和灰尘堆积，直到使用。 </li><li>通过填充液水库，并根据实验设计油管准备灌注系统。 注意:从管完全，因为它们会强烈地电生理记录干扰除去气泡。 </li><li>调整压力达到〜3个毫升/分钟流动。 注意:高的压力将导致电生理记录的组织切片的运动，以及终止。 </li><li>转移VNO切片成像室，并使用0.1毫米厚的SYNT有线不锈钢锚固定切片位置hetic纤维（ 图2B - C）。 注意:不要用合成纤维的一个线程，而是周围的切片琼脂糖覆盖组织切片。 </li><li>转移成像室通过一个浴应用在室温下记录的设置和不断superfuse片含氧S 2。 </li><li>调节吸入毛细管到溶液的表面上创造的浴溶液恒定交换缓慢吸入。调整8合1多管"灌注铅笔"上方，靠近到包含血管（ 图2C，3A）50所述VNO切片的非感觉的一部分。这将是有益的安排灌注铅笔和记录吸管是从面向相反方向的切片。 </li><li>连接参考电极，并使用L形琼脂桥（填充用150mM KCl）中浴溶液。 </li><li>装满吸液S 4补丁吸管。 </li><li>安装在连接到头部阶段氯化银涂覆膜片电极移液器没有刮掉涂层和附加牢固。 </li><li>应用微正压（约1ml在10毫升的塑料注射器），以补丁吸管再入浴。 </li><li>降低吸移管成使用微操纵器足够远的浴，以便能够浸入目标没有击中它（ 图2D）。 </li><li>监视使用连接到头部阶段电软件吸管电阻（R 点子 ，4-7兆欧之间）。 注:若R是PIP &lt;4MΩ玻璃针尖断裂。若R 点子 &gt; 10兆欧，尖端是最有可能堵塞和吸移管必须更换。 </li><li>可视化VNO切片用红外线优化微分干涉对比（DIC）的CCD摄像头，并使用荧光照明识别FPR-RS3-I-金星表达细胞（或类似标记的神经元）和适当的过滤立方体。 </li><li>聚焦和取决于实验设计目标的荧光或非荧光的细胞。 </li><li>接近细胞体，使用最高灵敏度手轮。由于正压，一旦枪头非常接近在细胞胞体膜的小凹痕变得可见。 </li><li>释放正压和施加轻微的负压力在细胞膜吸，以获得一个高电阻密封（1-20GΩ）。应用短期和温和的吸力破坏细胞膜，并建立全细胞配置。 </li><li>实验过程中监控接入阻力不断。 注意:只有包括神经元表现出小而稳定的接入电阻（R 输入 ≤3％;变化&lt;在实验过程中20％）为分析。 </li></ol>

The authors have nothing to disclose.

该VNO是化学感受结构检测信息素。迄今为止，大多数犁鼻受体保持为仅少数受体 - 配体对已确定要deorphanized。其中，V1rb2被描述由男性泌尿信息素的2-庚酮30可以具体地激活，V2rp5于由男性具体信息素ESP1 57以及V2r1b和V2rf2由MHC肽激活SYFPEITHI激活48和SEIDLILGY 58分别。理解受体 - 配体的关系及信号转导的先决条件是在天然环境中定义VSN种群的生物物理特性的知识。无源和有源膜性能，电压门控离子电导和动作电位放电模式定义的手段和程度受体神经元响应化学感受刺激。在急性组织切片电生理记录，特别是，谁勒细胞的膜片钳记录提供一种优良的方法非常详细分析这些特性。 在组织切片成功的，可靠的生理录音中关键的一步是准备本身。为了最大限度地提高时间跨度进行实验，组织必须切片和解剖后立即转移到冰冷的含氧的解决方案。随后，切片可以保持每个实验日细胞的大量的几个小时，允许分析。这需要一些实践以减少时间解剖，在不到30分钟嵌入一个动物的两个VNOS。在这之后，一个有经验的试验者来获得几个完整的组织片，用活细胞的成功率远高于75％。进行膜片钳记录是在比较的 Ca 2+成像的低通量的实验方法。然而，它允许活性对具有高得多的时间的单细胞水平的更详细的和通用的分析解析度。在全细胞构造中，细胞内的介质通过吸移管溶液透析。而吸管的解决方案可能不会反映确切的胞浆成分，它提供了超过两个细胞内外离子条件精确控制实验者。为结果的可靠解释，接入电阻和漏电流的连续监测是至关重要的。有时，显著增加或访问性强的变化或漏电流导致难以解释的结果，因此要求这些录音被丢弃。此外，需要注意的是某些药物化合物结合不可逆转地渲染，有必要记录每个更换后片是很重要的。 在细胞连接配置记录阻止胞内组分的透析和不影响细胞的膜电位。因此，这种技术提供了筛选的较少侵入性和相对快速的方法。然而，这种Appro公司ACH一般限于从细胞膜的外侧的超阈动作电位驱动的容性电流的记录。松补丁记录已被证明是适合于分析较大的感觉神经元的数量在VNO片38,54扣球行为。 这里使用的转基因模型提供了可靠的工具来识别和分析表达VSNS FPR-RS3的人口。此外，这里描述的技术可以扩展到无论其基因型的其他行。尽管调查一定义的细胞类型，利用转基因小鼠的巨大优势限制实验到可用线路。然而，最近，有针对性的基因组编辑已成为与CRISPR / Cas9技术59更快，更经济实惠。总之，有针对性的急性犁鼻组织切片的膜片钳记录提供一种通用的方法来表征限定populat的生物物理和生理特性感觉神经元的离子。

大多数动物在很大程度上依赖于它们的化学感官与周围环境相互作用。嗅觉效力于寻找和评估的食物，躲避捕食者和定位合适的交配伙伴的重要作用。在大多数哺乳动物，嗅觉系统包括至少四个解剖学和功能上不同的外周子系统组成:主嗅觉上皮1,2，所述Grueneberg神经节3,4，马塞拉5,6的间隔器官和犁鼻器。该VNO包括配件嗅觉系统（AOS），它在检测传达的身份，性别，社会地位和性状态7-10信息化学信号主要作用的外周感觉结构。所述VNO位于鼻中隔右腭以上的基。在小鼠实验中，它是一个双边盲结束管封闭在一个软骨胶囊11-13。该机构由两部分组成的月牙形内侧感觉上皮的lium该怀有VSNS和侧面的非感觉部分。两个上皮之间在于，其经由窄犁鼻管14连接至鼻 ​​腔粘液填充管腔。在非感觉组织的有大的横向血管提供了一种血管泵送机构，以促进通过负压15,16相对较大，大多非易失性分子如肽或小蛋白质到VNO管腔的进入。该VNO的结构部件是在出生和器官在青春期17前不久达到成人的大小。然而，啮齿动物AOS是否已在少年的功能仍然是受争论18-20。 VSNS由其上皮地点和受体的表达，他们的类型都区分。 VSNS显示与无髓鞘的轴突和凸出朝向腔和在微绒毛树突旋钮结束单个顶端树突双极形态。 VSN斧附件束状形成犁鼻神经留下的后背成尾端软骨胶囊，沿隔上升，通过筛板和项目的嗅球（AOB）21,22。犁鼻感觉上皮由两层组成:根尖层位于更靠近腔侧和港口都V1R-和所有，但一种类型的FPR-RS-表达神经元。这些神经元共表达的G蛋白α亚单位ģαi2和项目的AOB 23-25 ​​的前部。感觉神经元位于更基底层明示或V2Rs FPR-RS1一起摹αo和发送它们的轴突的AOB 26-28后区。 犁鼻神经元是由相当小的化学信息素29-33（V1Rs）或蛋白质化合物34-38（V2Rs），该被分泌到各种体液，如尿，唾液和泪液可能激活37,39-41 </sup&gt; 原位实验已经表明VSNS也由甲酰化肽和各种抗微生物/炎症联化合物25,42激活。此外，异源表达的FPR-RS蛋白与免疫系统中表达的FPR共享激动剂光谱，说明作为检测器，用于在同种或变质食品来源25疾病中的潜在作用（见附图43）。 从根本上理解受体 - 配体的关系和下游信号通路在特定人群VSN是他们的基本生物物理特性在本地环境的详细评测。在过去，细胞信号的分析，大大从转基因动物，通过共表达的荧光标记蛋白30,44-49标记定义的神经元群体中受益。在这个协议中，（FPR-RS3-I-金星）用来与一个荧光标记物一起表达FPR-RS3的转基因小鼠品系。这种方法举例说明如何使用这样的遗传修饰的小鼠品系进行急性冠状VNO组织切片使用单个神经元的膜片钳记录的光学可识别的细胞群的电生理分析。气压驱动的多管灌注系统的感官刺激和药物制剂可录制时快速，可逆和联络神经元的刺激或抑​​制作用。在片制剂全细胞记录允许固有特性，电压激活电导在细胞的天然环境中的详细分析，以及动作电位放电图形。

<table><tbody><tr><td>&lt;strong&gt;Chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Agarose (low-gelling temperature)</td><td>PeqLab</td><td>35-2030</td><td></td></tr><tr><td>ATP (Mg-ATP)</td><td>Sigma-Aldrich</td><td>A9187</td><td></td></tr><tr><td>Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES)</td><td>Sigma-Aldrich</td><td>B9879</td><td></td></tr><tr><td>Calcium chloride</td><td>Sigma-Aldrich</td><td>C1016</td><td></td></tr><tr><td>Ethylene glycol tetraacetic acid (EGTA)</td><td>Sigma-Aldrich</td><td>E3889</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G8270</td><td></td></tr><tr><td>GTP (Na-GTP)</td><td>Sigma-Aldrich</td><td>51120</td><td></td></tr><tr><td>(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma-Aldrich</td><td>M8266</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma-Aldrich</td><td>P9333</td><td></td></tr><tr><td>Potassium hydroxide</td><td>Sigma-Aldrich</td><td>03564</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>S7653</td><td></td></tr><tr><td>Sodium hydrogen carbonate</td><td>Sigma-Aldrich</td><td>S5761</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Sigma-Aldrich</td><td>S8045</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;Surgical tools and consumables&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Large Petri dish, 90 mm</td><td>VWR</td><td></td><td>decapitation, dissection of VNO capsule</td></tr><tr><td>Small Petri dish, 35 mm</td><td>VWR</td><td></td><td>lid for VNO dissection, dish for embedding in agarose</td></tr><tr><td>Sharp large surgical scissor</td><td>Fine Science Tools</td><td></td><td>decapitation, removal of lower jaw</td></tr><tr><td>Strong bone scissors</td><td>Fine Science Tools</td><td></td><td>cutting incisors</td></tr><tr><td>Medium forceps, Dumont tweezers #2</td><td>Fine Science Tools</td><td></td><td>removing skin and palate</td></tr><tr><td>Micro spring scissors, 8.5 cm, curved, 7 mm blades&amp;nbsp;</td><td>Fine Science Tools</td><td></td><td>cutting out VNO&amp;nbsp;</td></tr><tr><td>Two pairs of fine forceps, Dumont tweezers #5</td><td>Fine Science Tools</td><td></td><td>dissecting VNO out of cartilaginous capsule</td></tr><tr><td>Small stainless steel spatula</td><td>Fine Science Tools</td><td></td><td>handling agarose block and tissue slices</td></tr><tr><td>Surgical scalpel</td><td></td><td></td><td>cutting agarose block into pyramidal shape</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;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Amplifier</td><td>HEKA Elektronik</td><td>EPC-10</td><td></td></tr><tr><td>Borosilicate glass capillaries (1.50 mm OD/0.86 mm ID)</td><td>Science Products</td><td></td><td></td></tr><tr><td>CCD-camera</td><td>Leica Microsystems</td><td>DFC360FX</td><td></td></tr><tr><td>Filter cube, excitation: BP 450-490, suppression: LP 515</td><td>Leica Microsystems</td><td>I3</td><td></td></tr><tr><td>Fluorescence lamp</td><td>Leica Microsystems</td><td>EL6000</td><td></td></tr><tr><td>Hot plate magnetic stirrer</td><td>Snijders</td><td>34532</td><td></td></tr><tr><td>Microforge&amp;nbsp;</td><td>Narishige</td><td>MF-830</td><td></td></tr><tr><td>Micromanipulator Device&amp;nbsp;</td><td>Luigs &amp;amp; Neumann</td><td>SM-5</td><td></td></tr><tr><td>Micropipette puller, vertical two-step</td><td>Narishige</td><td>PC-10&amp;nbsp;</td><td></td></tr><tr><td>Microscope</td><td>Leica Microsystems</td><td>CSM DM 6000 SP5</td><td></td></tr><tr><td>Noise eliminator 50/60 Hz (HumBug)</td><td>Quest Scientific</td><td></td><td></td></tr><tr><td>Objective&amp;nbsp;</td><td>Leica Microsystems</td><td>HCX APO L20x/1.00 W</td><td></td></tr><tr><td>Oscilloscope</td><td>Tektronik</td><td>TDS 1001B</td><td></td></tr><tr><td>Osmometer&amp;nbsp;</td><td>Gonotec</td><td>Osmomat 030</td><td></td></tr><tr><td>Perfusion system 8-in-1</td><td>AutoMate Scientific</td><td></td><td></td></tr><tr><td>pH Meter five easy</td><td>Mettler Toledo</td><td></td><td></td></tr><tr><td>Pipette storage jar</td><td>World Precision Instruments</td><td>e212</td><td></td></tr><tr><td>Recording chamber&amp;nbsp;</td><td>Luigs &amp;amp; Neumann</td><td>Slice mini chamber</td><td></td></tr><tr><td>Razor blades</td><td>Wilkinson Sword GmbH</td><td>Wilkinson Sword Classic</td><td></td></tr><tr><td>Oxygenating slice storage chamber; alternative commercial chambers are: &lt;em&gt;e.g.&lt;/em&gt;, BSK1 Brain Slice Keeper (Digitimer) or the Pre-chamber (BSC-PC; Warner Instruments)</td><td>custom-made</td><td></td><td></td></tr><tr><td>Stereo microscope</td><td>Leica Microsystems</td><td>S4E</td><td></td></tr><tr><td>Trigger interface&amp;nbsp;</td><td>HEKA Elektronik</td><td>TIB-14 S</td><td></td></tr><tr><td>Vibratome&amp;nbsp;</td><td>Leica Microsystems</td><td>VT 1000 S</td><td></td></tr><tr><td>Water bath&amp;nbsp;</td><td>Memmert</td><td>WNB 45</td><td></td></tr></tbody></table>

in-depth physiological analysis of defined cell populations in acute tissue slices of the mouse vomeronasal organ

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory neurons (VSNs) express a specific type of G protein-coupled receptor (GPCR) from at least three different chemoreceptor gene families allowing sensitive and specific detection of chemosensory cues. These families comprise the V1r and V2r gene families as well as the formyl peptide receptor (FPR)-related sequence (Fpr-rs) family of putative chemoreceptor genes. In order to understand the physiology of vomeronasal receptor-ligand interactions and downstream signaling, it is essential to identify the biophysical properties inherent to each specific class of VSNs.
The physiological approach described here allows identification and in-depth analysis of a defined population of sensory neurons using a transgenic mouse line (Fpr-rs3-i-Venus). The use of this protocol, however, is not restricted to this specific line and thus can easily be extended to other genetically modified lines or wild type animals.

深入了解所定义的细胞群的生物物理和生理性能，我们执行鼠标VNO（ 图1 - 2）的急性冠状组织切片。解剖后，切片可为几个小时保持在冰冷的含氧胞外溶液（S 2）。在录音设置，用新鲜的氧合液不断地交流（ 图2D），确保整个实验过程中组织活力。我们在这里使用的转基因小鼠模型（FPR-RS3-I-金星）。 VSNS在该菌株共表达的荧光标记蛋白与FPR-RS3，所述FPR-RS基因家族（ 图3）使一个限定的感觉神经元的人口的光学识别的原型成员。电生理学记录提供装置，用于在单细胞水平的深入分析。例如，在电压钳进行电压 - 门控电流分析模式（ 图4A - B）。分离出特定类型的离子电流，切片灌流具有药理剂如TTX以抑制电压门控Na +电流（ 图4A）或硝苯地平阻断电压门控L-型Ca 2+电流（ 图4B）。此外，我们经常在电流钳构造进行全细胞记录，分析动作电位放电图形（ 图4C）。除了 ​​全细胞记录，细胞贴附&#39;松密封&#39;录音提供防止细胞内成分（图5A）的透析用较少侵入性方法。录制动作电位驱动对短期刺激申请容性电流提供了一个敏感和有效的方式筛选感官配体（ 例如，主要尿蛋白; MUPS）能激活细胞定义51（ 图5B）的人口。 ="jove_content"FO:保together.within页="1"&gt; <img alt="图1" src="/files/ftp_upload/54517/54517fig1.jpg" /> 图1:VNO的解剖 。鼠标头（A）的侧视图以说明在该门齿被切割的位置和角度。 （B）描绘了最佳点抢和剥离背面的上腭（UP）腹面观。 （C）腹面观到软骨胶囊（CC），在去除下颌，门牙和腭后怀着VNO和犁骨（VB）。解剖VNO胶囊（D）背视图描绘了两个VNOS（D i）的双边本地化。的侧视图示出了软骨的上背侧其中两个VNOS需要分开（D ii）该轮辋。比例尺= 1毫米（A - D）。517 / 54517fig1large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/54517/54517fig2.jpg" /> 图 2: 组织准备，录音室和显微镜的舞台上VNO示意性侧视图来说明过程，并在上皮细胞（A I）的非感官部分大血管（BV）的位置。虚线表示的冠状切片层。在去皮出了CC的VNO横向视图显示血管（A II）。 （B）琼脂糖包埋固定到解决方案充满录音室采用0.1毫米厚的合成纤维（SF）螺纹连接一个不锈钢锚底部冠状VNO组织切片。方框区域描绘周围的组织切片的琼脂糖。 （C）表示琼脂糖（银）的位置和方向的示意图 - 嵌入式位于两个纤维之间冠状VNO切片。 （D）放置在显微镜舞台上录音室的概述。该室装有浴应用（BA）为恒定灌流与含氧S 2，灌注铅笔（PP）的申请感官刺激或药剂，连接到放大器头阶段，参比电极的记录吸管（RP）（RE ）和吸入毛细管（SC），以保持溶液在腔室中的恒定交换。比例尺= 1毫米（AII）。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54517/54517fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54517/54517fig3.jpg" /> 图3: 冠状VNO做卷烟150微米急性冠状VNO组织切片UE片。（A）共聚焦图像（最大投影）显示荧光FPR-RS3头金星+在犁鼻感觉上皮神经元（绿色）的分布。血管（BV），内腔（L），感觉上皮（SE）。 （B）FPR-RS3头金星+神经元表现在管腔边境旋钮状结构结束了单顶树突。全细胞膜片钳记录是从VSN索玛，膜片吸管（PP）进行。 （C）与狭长的树突（D）的前端的树突旋钮（K）的一个单一的VSN的形态学，细胞胞体（S）和轴突（A）中而使体细胞在基底侧。比例尺为50μm（A），10微米（B），5微米（C）。这个数字已经从52修改。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54517/54517fig3large.jpg" target="_blank">请点击此处</a>查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/54517/54517fig4.jpg" /> 图4: 隔离电压门控钠 离子 和钙 离子 电流，以及尖峰放电 （A）从FPR-RS3 + VSNS一个TTX敏感的快速激活的Na +电流全细胞膜片钳记录代表的痕迹。控制的条件下（A I）电压步录音（胞外溶液S 1;细胞内溶液S 4）揭示了一个电压依赖快速和短暂的内向电流。 （A II）TTX处理（1μM）强烈减少的电流。数字减去跟踪（控制TTX（A iii）条 ）REVEALS的TTX敏感的电压门控钠离子电流。从控制和FPR-RS3 +分离出神经元TTX敏感的Na +电流的电流-电压关系（A IV）。 （B）代表钙电流曲线孤立药理学（10μM硝苯地平）。 （C）的代表性的电流钳痕迹表示除冰剂/超极化和后逐步阳性（C i）和负（℃ⅲ）电流注入产生（回弹）动作电位列车。注意在0 pA的电流注入（C II）测量的自发活动。 （℃ⅳ）控制和FPR-RS3 +神经元作为注入电流（I 注入 ）的函数的发射频率。在射速的逐渐增加是控制类似和FPR-RS3 + VSNS。注意，VSNS是于电流注射非常敏感，即使在低皮安范围53-56。注意使f -I曲线已经"背景校正"使用自发的扣球频率在0 pA的电流注入。数据是平均值±SEM。这个数字已经从52修改。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54517/54517fig4large.jpg" target="_blank">请点击此处查看本图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54517/54517fig5.jpg" /> 图5: 从基础VSNS外"松补丁"录音急性VNO组织切片描绘感觉上皮的基底层录制吸管的（A）IR-DIC图像。 （B）一种基础VSN在单元连接配置的代表性原始记录（＆＃39;松封&#39;）钉鞋介绍与合并重组表达的主要尿蛋白（rMUPs刺激（3秒））和升高钾浓度（50毫米，1秒的短暂瞬变脉冲响应），分别为（B I） 。在（B I）中所示的记录的较高放大倍数示出在响应于刺激（B II）的尖峰脉冲串。红条表示的刺激，跨刺激间隔= 30秒，连续拍摄，中断&lt;1秒（切痕//）的时间。比例尺= 5微米（A）。面板（A）已经从38参考修改。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54517/54517fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ at JoVE.com

In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ

Here, we describe a physiological approach that allows identification and in-depth analysis of a defined population of sensory neurons in acute coronal tissue slices of the mouse vomeronasal organ using whole-cell patch-clamp recordings.

在小鼠犁鼻急性组织切片定义细胞群的深入生理学分析

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory ...

in-depth-physiological-analysis-defined-cell-populations-acute-tissue

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

7.6K 次观看.  RWTH Aachen University. Here, we describe a physiological approach that allows identification and in-depth analysis of a defined population of sensory neurons in acute coronal tissue slices of the mouse vomeronasal organ using whole-cell patch-clamp recordings. 

视频: In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ

Multielectrode Array Recordings of the Vomeronasal Epithelium

Understanding neural circuits requires methods to record from many neurons simultaneously. For in vitro studies, one currently available technology is planar multielectrode array (MEA) recording. Here we document the use of MEAs to study the mouse vomeronasal organ (VNO), which plays an essential role in the detection of pheromones and social cues via a diverse population of sensory neurons expressing hundreds of types of receptors. Combining MEA recording with a robotic liquid handler to deliver chemical stimuli, the sensory responses of a large and diverse population of neurons can be recorded. The preparation allows us to remove the intact neuroepithelium of the VNO from the mouse and stimulate with a battery of chemicals or potential ligands while monitoring the electrical activity of the neurons for several hours. Therefore, this technique serves as a useful method for assessing ligand activity as well as exploring the properties of receptor neurons. We present the techniques needed to prepare the vomeronasal epithelium, MEA recording, and chemical stimulation.

Multielectrode array (MEA) recordings provide a method for studying the electrical activity of large populations of neurons. Here, we present the details of a MEA preparation to record from the mouse vomeronasal epithelium while simultaneously stimulating the tissue.

多电极阵列的犁鼻上皮录音

Understanding neural circuits requires methods to record from many neurons simultaneously. For in vitro studies, one currently available technology is ...

科研

神经科学

Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. Pheromones are volatile or non-volatile short-lived molecules2 secreted and/or contained in biological fluids3,4, such as urine, a liquid known to be a main source of pheromones3. Pheromonal communication is implicated in a variety of key animal modalities such as kin interactions5,6, hierarchical organisations3 and sexual interactions7,8 and are consequently directly correlated with the survival of a given species9,10,11. In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO)10,12, a paired structure located at the base of the nasal cavity, and enclosed in a cartilaginous capsule. Each VNO has a tubular shape with a lumen13,14 allowing the contact with the external chemical world. The sensory neuroepithelium is principally composed of vomeronasal bipolar sensory neurons (VSNs)15. Each VSN extends a single dendrite to the lumen ending in a large dendritic knob bearing up to 100 microvilli implicated in chemical detection16. Numerous subpopulations of VSNs are present. They are differentiated by the chemoreceptor they express and thus possibly by the ligand(s) they recognize17,18. Two main vomeronasal receptor families, V1Rs and V2Rs19,20,21,22, are composed respectively by 24023 and 12024 members and are expressed in separate layers of the neuroepithelium. Olfactory receptors (ORs)25 and formyl peptide receptors (FPRs)26,27 are also expressed in VSNs.
Whether or not these neuronal subpopulations use the same downstream signalling pathway for sensing pheromones is unknown. Despite a major role played by a calcium-permeable channel (TRPC2) present in the microvilli of mature neurons28 TRPC2 independent transduction channels have been suggested6,29. Due to the high number of neuronal subpopulations and the peculiar morphology of the organ, pharmacological and physiological investigations of the signalling elements present in the VNO are complex.
Here, we present an acute tissue slice preparation of the mouse VNO for performing calcium imaging investigations. This physiological approach allows observations, in the natural environment of a living tissue, of general or individual subpopulations of VSNs previously loaded with Fura-2AM, a calcium dye. This method is also convenient for studying any GFP-tagged pheromone receptor and is adaptable for the use of other fluorescent calcium probes. As an example, we use here a VG mouse line30, in which the translation of the pheromone V1rb2 receptor is linked to the expression of GFP by a polycistronic strategy.

In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO). Here, an acute tissue slice preparation of VNO for performing calcium imaging is described. This physiological approach allows observations of subpopulations and/or individual neurons in a living tissue and is convenient for receptor-ligand identification.

成像遥感信息素在小鼠犁鼻急性组织切片的制备

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. ...

Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within the same species 1,2. These intraspecies signals, the pheromones, as well as signals from some predators 3, activate the vomeronasal sensory neurons (VSNs) with high levels of specificity and sensitivity 4. At least three distinct families of G-protein coupled receptors, V1R, V2R and FPR 5-14, are expressed in VNO neurons to mediate the detection of the chemosensory cues. To understand how pheromone information is encoded by the VNO, it is critical to analyze the response profiles of individual VSNs to various stimuli and identify the specific receptors that mediate these responses.
The neuroepithelia of VNO are enclosed in a pair of vomer bones. The semi-blind tubular structure of VNO has one open end (the vomeronasal duct) connecting to the nasal cavity. VSNs extend their dendrites to the lumen part of the VNO, where the pheromone cues are in contact with the receptors expressed at the dendritic knobs. The cell bodies of the VSNs form pseudo-stratified layers with V1R and V2R expressed in the apical and basal layers respectively 6-8. Several techniques have been utilized to monitor responses of VSNs to sensory stimuli 4,12,15-19. Among these techniques, acute slice preparation offers several advantages. First, compared to dissociated VSNs 3,17, slice preparations maintain the neurons in their native morphology and the dendrites of the cells stay relatively intact. Second, the cell bodies of the VSNs are easily accessible in coronal slice of the VNO to allow electrophysiology studies and imaging experiments as compared to whole epithelium and whole-mount preparations 12,20. Third, this method can be combined with molecular cloning techniques to allow receptor identification.
Sensory stimulation elicits strong Ca2+ influx in VSNs that is indicative of receptor activation 4,21. We thus develop transgenic mice that express G-CaMP2 in the olfactory sensory neurons, including the VSNs 15,22. The sensitivity and the genetic nature of the probe greatly facilitate Ca2+ imaging experiments. This method has eliminated the dye loading process used in previous studies 4,21. We also employ a ligand delivery system that enables application of various stimuli to the VNO slices. The combination of the two techniques allows us to monitor multiple neurons simultaneously in response to large numbers of stimuli. Finally, we have established a semi-automated analysis pipeline to assist image processing.

The vomeronasal organ (VNO) detects intraspecies chemical signals that convey social and reproductive information. We have performed Ca2+ imaging experiments using transgenic mice expressing G-CaMP2 in VNO tissue. This approach allows us to analyze the complicated response patterns of the vomeronasal neurons to large numbers of pheromone stimuli.

在犁鼻器官的切片准备工作表示一个基因编码的钙传感器成像神经元的反应

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within ...

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

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

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

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

完整的犁鼻器和副嗅球的体外制剂

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

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

鼠标嗅觉感官神经元的完整神经上皮穿孔膜片钳记录:神经元表达鉴定的气味受体功能分析

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

An Effective Manual Deboning Method To Prepare Intact Mouse Nasal Tissue With Preserved Anatomical Organization

The mammalian nose is a multi-functional organ with intricate internal structures. The nasal cavity is lined with various epithelia such as olfactory, respiratory, and squamous epithelia which differ markedly in anatomical locations, morphology, and functions. In adult mice, the nose is covered with various skull bones, limiting experimental access to internal structures, especially those in the posterior such as the main olfactory epithelium (MOE). Here we describe an effective method for obtaining almost the entire and intact nasal tissues with preserved anatomical organization. Using surgical tools under a dissecting microscope, we sequentially remove the skull bones surrounding the nasal tissue. This procedure can be performed on both paraformaldehyde-fixed and freshly dissected, skinned mouse heads. The entire deboning procedure takes about 20-30 min, which is significantly shorter than the experimental time required for conventional chemical-based decalcification. In addition, we present an easy method to remove air bubbles trapped between turbinates, which is critical for obtaining intact thin horizontal or coronal or sagittal sections from the nasal tissue preparation. Nasal tissue prepared using our method can be used for whole mount observation of the entire epithelia, as well as morphological, immunocytochemical, RNA in situ hybridization, and physiological studies, especially in studies where region-specific examination and comparison are of interest.

有效的人工剔骨方法编写完整的鼠标鼻组织与保存的解剖组织

The mammalian nose is a multi-functional organ with intricate internal structures.  The nasal cavity is lined with various epithelia such as olfactory, ...

生物学

Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and in situ Hybridization

Animals rely on chemical communication to convey and perceive relevant environmental information, ranging from assessment of food quality to detection of available mating partners or threats. In mice, this task is executed primarily by the olfactory system and its underlying subsystems, including the main and accessory olfactory systems. Both have peripheral organs populated by sensory neurons expressing G-protein coupled receptors able to bind chemical cues that reach the nasal cavity. Even though the molecular characteristics of these receptors is well understood, little is known about their cognate specific ligands. The method described here combines in situ hybridization detection of olfactory or vomeronasal receptors with immunodetection of phosphorylated ribosomal protein S6 (pS6) - a marker of neuronal activation. This protocol was devised to identify neurons activated after a single event of exposure to purified or complex chemical stimuli detected by the olfactory organs. Importantly, this technique allows the investigation of neurons triggered in biologically relevant contexts. Ideally, this method should be used to probe the molecular biology of the olfactory system and to study olfactory behaviors.

Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and in situ Hybridization

This protocol combines in situ hybridization with immunofluorescence to identify olfactory and vomeronasal receptor genes expressed in olfactory sensory neurons after activation by chemical stimuli in the mouse.

Animals rely on chemical communication to convey and perceive relevant environmental information, ranging from assessment of food quality to detection of ...

Establishing a Whole-Cell Patch Clamp for Electrophysiological Recording from Vomeronasal Sensory Neurons

Source: Ackels, T., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/54517">In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ.</a> J. Vis. Exp. (2016)
The protocol video describes establishing a whole-cell patch clamp for electrophysiological recording from the sensory neurons of the vomeronasal organ (VNO) in mice. A coronal VNO slice with fluorescently labeled sensory neurons is placed in a recording setup. A labeled neuron is patched with a recording pipette in a whole-cell configuration for electrophysiological recording in response to chemical stimuli.

建立用于犁鼻感觉神经元电生理记录的全细胞膜片钳

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Solution Preparation

	Prepare ...

JoVE Encyclopedia of Experiments

Neurophysiology

Electrophysiology Techniques

Preparation of Mouse Vomeronasal Organ for Vibratome Sectioning

Source: Ackels, T., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/54517">In-depth physiological analysis of defined cell populations in acute tissue slices of the mouse vomeronasal organ.</a> J. Vis. Exp. (2016)
This video outlines the isolation of a mouse vomeronasal organ (VNO) and tissue preparation for vibratome sectioning. Sections are used to characterize the biophysical and physiological attributes of a specific group of sensory neurons.

用于振动切片机切片的小鼠犁鼻器官的制备

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
Both ...

Neuronal Culture Techniques

Primary Neuronal Culture

Perforated Patch-Clamp Recording of Olfactory Sensory Neurons in Mice

For this protocol, have mice available between 2 and 4 weeks old. In this presentation, mice expressing OR/IRES/tauGFP are used.
By using OR/IRES/tauGFP mice, all neurons expressing the OR of interest will be labeled with GFP. This method can be used for OR expressed in all zones. However, dissections and recordings will be easier for OR expressed in the dorsal zone.
Begin by anesthetizing and then decapitating a mouse.
The outcome of this protocol depends on the quality of the dissection. These dissection steps must be as short as possible and be precise.
To begin the surgery, use dissecting scissors to make a longitudinal cut along the dorsum. Pull skin apart to remove it. Then, cut along the lower jaw and make a coronal cut parallel to the teeth. Thus, remove the upper incisor teeth. Now, make a coronal cut around the head, behind the eyes. Discard the posterior head, and transfer the anterior portion to ice-cold Ringer&#39;s.
Now, under a dissection microscope, isolate the septum within 5 to 10 minutes. Start with a longitudinal cut along the ventral side. Then, cut the dorsal bones longitudinally along the dorsal lateral side of the nasal cavity.
Next, remove most of the bones and the palate. Transfer the isolated septum and attached epithelium to a container of oxygenated Ringer&#39;s at room temperature. Within five minutes, make the final preparations before recording.
The solution must not be too warm, or the preparation will look damaged. To access the neuroepithelium for recording, first, peel the epithelium from the underlying septum with forceps. Then, using micro-Vannas scissors, cut the anterior end of the septum to release the epithelium.
Now, carefully remove the vomeronasal organ. Cut it off where it is attached dorsally. The isolated epithelium can now be moved to the recording chamber, mucus side up. Once in place, use a harp to flatten the tissue. Then, transfer the chamber to an upright microscope. Visualize the neuroepithelium under a 40x water immersion objective while perfusing it continually with fresh Ringer&#39;s at room temperature.
To begin, search for the cells of interest, which, in this case, express EGFP. So 480-nanometers light is used to scan for cells that emit light in the 530 to 550-nanometer range. The targeted dendritic knobs and OSNs, when reviewed at 80 to 160x under bright field, should be clearly distinguishable from the supporting cells.
Now, load the electrode with the nystatin solution. Tap out any bubbles that form, and attach the electrode to the holder. Now, apply some positive pressure. The expected resistance is between 15 and 20 megaohms. Then, bring the electrode to the cell while maintaining the positive pressure.
When the resistance increases to about 40 megaohms, release the pressure and apply a slight negative pressure. If a gigaseal is made, the membrane potential should be clamped at about negative 75 millivolts.
Now that the cell is open, proceed with the experiment. Apply stimulation protocols and perfuse with pharmacological treatments as needed. To test with a single odorant, record 200 to 500 milliseconds of spontaneous activity. Apply the odorant for 500 milliseconds, and then, measure responses for up to 10 seconds.

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