マウス鋤鼻器官の急性組織スライスで定義された細胞集団の中で、深さ生理学的解析
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
ほとんどの動物は、それらの周囲と対話するために、それらの化学的感覚に大きく依存しています。嗅覚は、適切な交配パートナーを見つけ、食品を評価する、捕食者を回避し、配置するための重要な役割を果たしています。主嗅上皮1,2、Grueneberg神経節3,4、マゼーラ5,6の中隔臓器と鋤鼻器官：ほとんどの哺乳動物では、嗅覚系は、少なくとも4つの解剖学的および機能的に異なる周辺のサブシステムから構成されています。 VNOは、ID、性別、社会的地位や性的な状態7-10についての情報を伝える化学手がかりを検出するのに重要な役割を果たしているアクセサリーの嗅覚システム（AOS）、の末梢感覚構造を含みます。 VNOは、右口蓋上記の鼻中隔の基部に位置しています。マウスでは、軟骨カプセル11-13で囲まれた二国間の盲終わる管です。臓器は、三日月形の内側の感覚epitheの両方で構成されVSNをを保有し、側面上の非感覚一部のlium。両方の上皮との間の狭い鋤鼻ダクト14を介して鼻腔に接続された粘液で満たされた内腔に位置しています。非感覚組織の大きな横方向の血管は負圧15,16を介してVNO腔にそのようなペプチドまたは小型タンパク質のような比較的大きい、主に非揮発性の分子の進入を容易にするために、血管ポンピング機構を提供します。 VNOの構造部品は、出生時に存在し、臓器は、まもなく思春期17の前に大人のサイズに達します。しかし、齧歯類のAOSはすでに少年で機能しているかどうか18-20を議論することが課題です。
VSNを、それらの上皮場所とそれらが発現する受容体のタイプの両方によって区別されます。 VSNをは無髄軸索内腔に向かって突出しており、微絨毛の樹状ノブで終わる単一の先端樹状突起を持つバイポーラ形態を示します。 VSNの斧アドオン束生の、背尾側端での軟骨カプセルを残す中隔に沿って上昇し、副嗅球（AOB）21,22に篩板やプロジェクトを渡し鋤鼻神経を形成します。鋤鼻感覚上皮は2層で構成されています：頂端層が腔側に近い位置に配置し、V1R-とFPR-RS-発現ニューロンの1タイプではなく、すべての両方を保有されています。これらのニューロンは、AOB 23-25 の前部にGタンパク質αサブユニットGのαi2とプロジェクトを共発現します。感覚ニューロンは、GαOと一緒に、より基底層特急V2RsまたはFPR-RS1に位置し、AOB 26-28の後部領域への軸索を送ります。
鋤鼻ニューロンはおそらく、尿、唾液などの各種体液中に分泌し、涙液されているという小さな情報化学物質29-33（V1Rs）またはタンパク質化合物34-38（V2Rs）によって活性化される37,39-41 </sup> その場での実験でのVSNもホルミル化ペプチドおよび種々の抗菌剤/炎症結合化合物25,42によって活性化されることが示されています。また、異種FPR-RSタンパク質が（参照43を参照）同種または腐った食物源25で病気のための検出器としての潜在的な役割を示し、免疫系において発現のFPRでアゴニストスペクトルを共有して表明しました。
特定のVSN集団における受容体 – リガンドの関係および下流のシグナル伝達カスケードを理解するための基本的には、ネイティブ環境での基本的な生物物理学的特性の詳細な評価です。過去には、細胞内シグナル伝達の解析が大幅に蛍光マーカータンパク質30,44-49を共発現することによってニューロンの定義された人口をマークする遺伝子改変動物の恩恵を受けています。このプロトコルでは、蛍光マーカー（FPR-RS3-I-ビーナス）と共にFPR-RS3を発現するトランスジェニックマウス系統が使用されます。このアプローチは、急性冠状VNOの組織切片内の単一ニューロンのパッチクランプ記録を用いて光学的に識別可能な細胞集団の電気生理学的分析を行うために、そのような遺伝的に改変されたマウス系統を使用する方法を例示します。感覚刺激と薬理学的薬剤のための空気圧駆動式マルチバレル灌流システムは、録音時に、迅速な可逆焦点神経刺激または阻害することができます。スライス標本における全細胞記録は固有の特性、電圧活性化コンダクタンス、ならびに細胞のネイティブな環境での活動電位放電パターンの詳細な分析を可能にします。
Protocol
Representative Results
Discussion
VNOは、情報化学物質を検出する化学感覚構造です。今日まで、鋤鼻受容体の大多数は、ほんの数の受容体 – リガンド対が同定されているようにdeorphanizedされていません。このうち、V1rb2は、特にMHCペプチドSYFPEITHI 48とSEIDLILGY 58によって活性化されることが、男性特有のフェロモンESP1 57と同様にV2r1bとV2rf2によって活性化されることが、男性の尿フェロモン2-ヘプタノン…
Divulgations
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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