Name: in-depth physiological analysis of defined cell populations in acute tissue slices of the mouse vomeronasal organ
Uploaded: 2016-09-10T14:00:00
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-</...

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。

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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 ...

The overall goal of this procedure is to perform single cell electro-physiological analysis of a defined population of sensory neurons in acute tissue slices from the mouse vomeronasal organ. This method can help elucidate key questions in the field of chemosensation, such as the mechanisms of see-no chemical detection. The main advantage of this technique is that by recording from sensory neurons in acute tissue slices, these cells can be investigated in their native environment.To begin this procedure, after sacrificing the animal, remove its lower jaw, by inserting a pair of large surgical scissors through the mouth cavity, and cut the mandible bones and muscle of each side separately. Next, remove the skin of the upper jaw and around the tip of the nose with medium forceps, to gain better access to the incisors. Then use the bone scissors to cut away the largest part of the incisors at a 45 degree angle in the rostral direction to ease the VNO capsule removal from the nasal cavity.Do not cut the root of the tooth to prevent damage to the tip of the VNO capsule. After that, grab the rigid upper palate at its rostral part with medium forceps, and carefully peel back in one piece at a flat angle. Repeatedly rinse the naval cavity with ice cold solution S2.Then use micro spring scissors to cut the bone effusion between the tip of the vomer bone and the jaw.Insert the scissor tips with the curved part pointing away from the VNO. And carefully cut the bone in small steps on both the left and right side, lateral to the VNO capsule. To remove the VNO capsule, cut through the vomer bone at the caudal part, and carefully lift the vomer bone out of the nasal cavity using medium forceps.Immediately transfer the VNO to a small petri dish under a stereo microscope on an ice gel pack, where the remaining steps of the dissection should be performed. Next, rinse the VNO in a small amount of ice cold S2, to prevent the tissue from drying out. Then, separate the cartilaginous capsules that contain the VNO soft tissues by grabbing the back of the vomer bone with medium forceps.For that, position the capsule for a dorsal view so that a split between both VNOs becomes visible. Use the tip of fine forceps to separate both cartilage VNO capsules from the central bone, while keeping the vomer bone pinned down at the rear part. Make sure the medial cartilage wall that was previously attached to the vomer bone is removed.When removing cartilage around the VNO use forceps only at the rim of the cartilage capsule. And be very careful not to damage the delicate sensory epithelium. To remove remaining cartilage, turn the VNO with its curved lateral side to the bottom of the dish and securely pin down the cartilage on one side using forceps.Then carefully move the second fine forceps from the back side at a very flat angle between cartilage and VNO to loosen the connection between tissue and cartilage. To avoid damaging the sensory epithelium slowly peel the VNO away from the cartilage by holding it at its caudal tip. Once the VNO is levered from the capsule, remove all the remaining small cartilage parts as any remaining pieces of cartilage will detach the tissue from the surrounding agarose during the slicing process.Next, place a small drop of ice cold S2 on the first dissected VNO to prevent tissue damage. To embed the VNOs, fill both small petri dishes to the rim with melted S3.After that, immerse each VNO in agarose and move it horizontally back and forth several times to remove the film of extracellular solution as well as any air bubbles from its surface. Position the VNO vertically, with the rostral tip facing the bottom of the dish.Subsequently, place both the dishes on a gel ice pack, and wait until agarose has solidified. Do not change VNO orientation once the agarose has started solidifying, as this will detach the tissue from the surrounding agarose. In this procedure, use a small spatula to remove the agarose block from the small dish into the lid of a large petri dish, and flip the agarose upside down, leaving the rostral tip of the VNO facing upwards.Next, cut the block into a pyramidal shape using a surgical scalpel, and take care not to damage the embedded tissue. Then, use super glue to fix the pyramid shaped block to the center of the vibratome specimen plate. And wait about one minute for the glue to dry completely.Slice the specimen into 150 to 200 micrometer thick sections. Briefly inspect the slice morphology under the dissecting microscope. Then transfer all the slices to the oxygenating chamber until use.In this step, transfer a VNO slice to the imaging chamber and fix the slice position using stainless steel anchor wired with point one milometer thick synthetic fibers. Do not cover the tissue slice with any of the synthetic fiber threads. Next, transfer the imaging chamber to the recording setup and continuously superfuse the slice with oxygenated S2 at room temperature.Adjust the suction capillary to the surface of the solution to create a constant exchange of bath solution. Then adjust the eight in one multi barrel profusion pencil above and close to the non sensory part of the VNO slice that contains the blood vessel. Afterward connect the reference electrode and bath solution using the L shaped agar bridge filled with 150 millimolar potassium chloride.Subsequently fill the patch pipette with pipette solution S4.Mount the pipette over the silver chloride coated electrode connected to the head stage without scraping off the coating and attach firmly. Then apply slight positive pressure to the patch pipette before entering the bath. Monitor the pipette and make sure that it is between four megaohms and 10 megaohms.Visualize the VNO slice with a CCD camera using infrared optimized DIC, and identify FPR-rs3 i-Venus expressing cells or similarly labeled neurons using florescence illumination and an appropriate filter cube. Approach the cell body using hand wheels for maximum sensitivity. Due to the positive pressure a small dent on the cell soma membrane becomes visible once the pipette tip is close proximity.Then release the positive pressure and apply slight negative pressure to suck in the cell membrane in order to gain a high resistance seal of one to 20 gigaohms. Subsequently apply short and gentle suction to disrupt the cell membrane and establish the whole cell configuration. In this figure, a confocal image of a coronal VNO tissue slice shows the distribution of fluorescent FPR-rs3 Tau Venus positive neurons in the vomeronasal sensory epithelium.The FPR-rs3 Tau Venus positive neurons exhibit a single apical dendrite ending in a knob like structure at the luminal border. And shown here are the whole cell patch clamp recordings performed from the VSN soma. Here are the representative traces from wholesale patch clamp recordings of a TTX sensitive fast activating sodium current, and FPR-rs3 positive VSNs.Voltage step recordings under control conditions reveal a voltage dependent fast and transient inward current. TTX treatment at one micromolar strongly diminishes the current. And the digitally subtracted trace shown here reveals the TTX sensitive voltage gated sodium current.The representative current clamp traces show depolarization and hyper-polarization and trains of action potentials generated upon step-wise positive and negative current injection. Once mastered, the whole dissection and tissue slicing process can be done in approximately one hour for both VNOs. Following this procedure, other methods such as extra-cellular loose patch recordings or calcium imaging can be performed in order to increase throughput when analyzing larger populations of neurons.After watching this video, you should have a good understanding of how to make acute tissue slices from the mouth vomeronasal organ, and how to perform single cell electro-physiological recordings from vomeronasal sensory neurons.

Research

JoVE Journal

Neuroscience

Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation

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

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 ...

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

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 ...

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

钙成像的急性下区片的制备

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

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

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

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

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

Functional Analysis of the Larval Feeding Circuit in Drosophila

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

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

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

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

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

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 ...