Summary

在小鼠犁鼻急性组织切片定义细胞群的深入生理学分析

Published: September 10, 2016
doi:

Summary

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.

Abstract

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.

Introduction

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> 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。

Protocol

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.溶液的制备制备细胞外溶液S 1:4-(2-</…

Representative Results

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?…

Discussion

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?…

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

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.

Materials

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

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Ackels, T., Drose, D. R., Spehr, M. In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ. J. Vis. Exp. (115), e54517, doi:10.3791/54517 (2016).

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