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?...

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 border="0" cellpadding="0" cellspacing="0" width="846">
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			<td height="21" style="height:21px;width:323px;">Chemicals</td>
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			<td style="width:116px;"></td>
			<td style="width:275px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Agarose (low-gelling temperature)</td>
			<td style="width:133px;">PeqLab</td>
			<td style="width:116px;">35-2030</td>
			<td style="width:275px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:323px;">ATP (Mg-ATP)</td>
			<td style="width:133px;">Sigma-Aldrich</td>
			<td style="width:116px;">A9187</td>
			<td style="width:275px;"></td>
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			<td height="42" style="height:42px;width:323px;">Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES)</td>
			<td style="width:133px;">Sigma-Aldrich</td>
			<td style="width:116px;">B9879</td>
			<td style="width:275px;"></td>
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			<td height="21" style="height:21px;width:323px;">Calcium chloride</td>
			<td style="width:133px;">Sigma-Aldrich</td>
			<td style="width:116px;">C1016</td>
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			<td height="21" style="height:21px;width:323px;">Ethylene glycol tetraacetic acid (EGTA)</td>
			<td style="width:133px;">Sigma-Aldrich</td>
			<td style="width:116px;">E3889</td>
			<td style="width:275px;"></td>
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			<td height="21" style="height:21px;width:323px;">Glucose</td>
			<td style="width:133px;">Sigma-Aldrich</td>
			<td style="width:116px;">G8270</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:323px;">GTP (Na-GTP)</td>
			<td style="width:133px;">Sigma-Aldrich</td>
			<td style="width:116px;">51120</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td>
			<td style="width:133px;">Sigma-Aldrich</td>
			<td style="width:116px;">H3375</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Magnesium chloride</td>
			<td style="width:133px;">Sigma-Aldrich</td>
			<td style="width:116px;">M8266</td>
			<td style="width:275px;"></td>
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			<td height="84" style="height:84px;width:323px;">Oxygenating slice storage chamber; alternative commercial chambers are e.g. BSK1 Brain Slice Keeper (Digitimer) or the Pre-chamber (BSC-PC; Warner Instruments)</td>
			<td style="width:133px;">custom-made</td>
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			<td height="21" style="height:21px;width:323px;">Stereo microscope</td>
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			<td height="21" style="height:21px;width:323px;">Water bath&#160;</td>
			<td style="width:133px;">Memmert</td>
			<td style="width:116px;">WNB 45</td>
			<td style="width:275px;"></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?...

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

Research

JoVE Journal

Neuroscience

7.5K 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

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. ...

科研

神经科学

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 ...

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals1. Though being a crucial part of the visual processing by shaping the retinal output2, most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event.
Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage5. Also, it leaves the retina responsive to visual stimuli since infrared light (&gt;850 nm) is only poorly absorbed by photopigments6.
In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.

This article depicts the recording of individual cells from fluorescently tagged neuronal populations in the intact mouse retina. By using two-photon infrared excitation transgenetically labeled cells were targeted for patch-clamp recording to study their light responses, receptive field properties, and morphology.

在完整的视网膜电生理特性表达GFP的细胞群

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack ...

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells in restricted areas of the adult mammalian brain. Newborn neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to their final destination in the olfactory bulb (OB)1. In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes2,3 using blood vessels as a structural support and a source of molecular factors required for migration4,5. In the OB, neuroblasts detach from the chains and migrate radially into the different bulbar layers where they differentiate into interneurons and integrate into the existing network1, 6.
In this manuscript we describe the procedure for monitoring cell migration in acute slices of the rodent brain. The use of acute slices allows the assessment of cell migration in the microenvironment that closely resembling to in vivo conditions and in brain regions that are difficult to access for in vivo imaging. In addition, it avoids long culturing condition as in the case of organotypic and cell cultures that may eventually alter the migration properties of the cells. Neuronal precursors in acute slices can be visualized using DIC optics or fluorescent proteins. Viral labeling of neuronal precursors in the SVZ, grafting neuroblasts from reporter mice into the SVZ of wild-type mice, and using transgenic mice that express fluorescent protein in neuroblasts are all suitable methods for visualizing neuroblasts and following their migration. The later method, however, does not allow individual cells to be tracked for long periods of time because of the high density of labeled cells. We used a wide-field fluorescent upright microscope equipped with a CCD camera to achieve a relatively rapid acquisition interval (one image every 15 or 30 sec) to reliably identify the stationary and migratory phases. A precise identification of the duration of the stationary and migratory phases is crucial for the unambiguous interpretation of results. We also performed multiple z-step acquisitions to monitor neuroblasts migration in 3D. Wide-field fluorescent imaging has been used extensively to visualize neuronal migration7-10. Here, we describe detailed protocol for labeling neuroblasts, performing real-time video-imaging of neuroblast migration in acute slices of the adult mouse forebrain, and analyzing cell migration. While the described protocol exemplified the migration of neuroblasts in the adult RMS, it can also be used to follow cell migration in embryonic and early postnatal brains.

We describe a protocol for real-time videoimaging of neuronal migration in the mouse forebrain. The migration of virally-labeled or grafted neuronal precursors was recorded in acute live slices using wide-field fluorescent imaging with a relatively rapid acquisition interval to study the different phases of cell migration, including the durations of the stationary and migration phases and the speed of migration.

急性片的成年小鼠前脑的神经细胞迁移的延时成像

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.
In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.

成神经细胞迁移的小鼠急性脑片在体内电穿孔产后和时间推移成像

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

Functional Analysis of the Larval Feeding Circuit in Drosophila

The serotonergic feeding circuit in Drosophila melanogaster larvae can be used to investigate neuronal substrates of critical importance during the development of the circuit. Using the functional output of the circuit, feeding, changes in the neuronal architecture of the stomatogastric system can be visualized. Feeding behavior can be recorded by observing the rate of retraction of the mouth hooks, which receive innervation from the brain. Locomotor behavior is used as a physiological control for feeding, since larvae use their mouth hooks to traverse across an agar substrate. Changes in feeding behavior can be correlated with the axonal architecture of the neurites innervating the gut. Using immunohistochemistry it is possible to visualize and quantitate these changes. Improper handling of the larvae during behavior paradigms can alter data as they are very sensitive to manipulations. Proper imaging of the neurite architecture innervating the gut is critical for precise quantitation of number and size of varicosities as well as the extent of branch nodes. Analysis of most circuits allow only for visualization of neurite architecture or behavioral effects; however, this model allows one to correlate the functional output of the circuit with the impairments in neuronal architecture.

Functional Analysis of the Larval Feeding Circuit in Drosophila

The feeding circuit in Drosophila melanogaster larvae serves a simple yet powerful model that allows changes in feeding rate to be correlated with alterations in the stomatogastric neural circuitry. This circuit is composed of central serotonergic neurons that send projections to the mouth hooks as well as the foregut.

在幼虫馈电电路的功能分析果蝇</i

The serotonergic  feeding circuit in Drosophila  melanogaster larvae can be used to investigate neuronal substrates of  critical importance during the ...

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 ...

Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments that largely determines the quality of results. It has been shown that omitting the cooling step during cutting procedure is beneficial in obtaining healthy slices and cells, especially when dealing with highly myelinated brain structures from mature animals. Even though the precise mechanism whereby elevated temperature supports neural health can only be speculated upon, it stands to reason that, whenever possible, the temperature in which the slicing is performed should be close to physiological conditions to prevent temperature related artifacts. Another important advantage of this method is the simplicity of the procedure and therefore the short preparation time. In the demonstrated method adult mice are used but the same procedure can be applied with younger mice as well as rats. Also, the following patch clamp experiment is performed on horizontal cerebellar slices, but the same procedure can also be used in other planes as well as other posterior areas of the brain.

In this paper we show a method for preparing acute brain slices in physiological temperature, using a conventional physiological solution without special modifications for the cutting (such as adding sucrose) and without intracardial perfusion of the animal before slice preparation.

切片热门:成人急性脑切片在生理温度

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

用优化的n-甲基 d-葡乙胺保护恢复法制备急性脑切片

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...