Dyptgående Fysiologisk Analyse av Definerte cellepopulasjoner i Akutt vevssnitt av musen Vomeronasal Organ
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
De fleste dyr avhengige av deres kjemiske sanser for å samhandle med sine omgivelser. Luktesansen spiller en avgjørende rolle for å finne og vurdere mat, unngå rovdyr og finne egnede parring samarbeidspartnere. I de fleste pattedyr, luktsystemet består av minst fire anatomisk og funksjonelt distinkte perifere delsystemer: hoved olfaktoriske epitel 1,2, den Grueneberg ganglion 3,4, septal organ for Masera 5,6 og vomeronasal organ. VNO omfatter perifer sensorisk strukturen av tilbehøret luktsystemet (AOS), som spiller en viktig rolle i å oppdage kjemiske signaler som formidler informasjon om identitet, kjønn, sosial rang og seksuell tilstand 7-10. VNO ligger ved foten av neseskilleveggen rett over ganen. I mus, er det en bilateral blind-ending rør innelukket i en kapsel brusk 11-13. Orgelet består av både en halvmåneformet medial sensoriske epitheLium som havner på VSNs og av en ikke-sensorisk del på den laterale side. Mellom begge epithelia ligger noen slimfylte lumen som er koblet til nesehulen via den smale vomeronasal kanalen 14. En stor lateral blodåre i den ikke-sensorisk vev tilveiebringer en vaskulær pumpemekanisme for å lette innføring av forholdsvis store, hovedsakelig ikke-flyktige molekyler slik som peptider eller små proteiner inn i VNO lumen ved undertrykk 15,16. De strukturelle komponenter i VNO er til stede ved fødselen og orgelet når voksen størrelse kort tid før puberteten 17. Men om den gnager AOS er allerede funksjonelle i yngel er fortsatt gjenstand for debatt 18-20.
VSNs utmerker seg ved både sin epithelial plassering og type reseptor de uttrykker. VSNs viser en bipolar morfologi med en unmyelinated axon og en enkel apikal dendrite som stikker mot lumen og ender i en microvillous dendrittiske knott. VSN øksons fasciculate å danne Vomeronasale nerve som forlater brusk kapsel til dorso-caudal slutten, stiger langs septum, passerer cribriform plate og prosjekter til tilbehøret luktelappen (AOB) 21,22. Den Vomeronasale sensorisk epitel består av to lag: den apikale lag ligger nærmere luminal side og havner både V1R- og alle, men en type FPR-rs-uttrykke nevroner. Disse nevronene coexpress G-protein α-subenheten G αi2 og prosjekt til fremre del av AOB 23-25. Sensoriske nevroner lokalisert i de mer basale lag ekspress V2Rs eller FPR-RS1 sammen G αo og sender sine aksoner til bakre region av AOB 26-28.
Vomeronasal nevroner er sannsynlig aktiveres ved ganske små semiochemicals 29-33 (V1Rs) eller proteinforbindelser 34-38 (V2Rs) som skilles ut i ulike kroppsvæsker som urin, spytt og tårevæske 37,39-41 </sup>. In situ forsøk har vist at VSNs er også aktiveres av formylert peptider og ulike antimikrobielle / betennelse bundet forbindelser 25,42. Videre, heterologt uttrykt FPR-rs-proteiner dele agonist-spektra med FPRs uttrykt i immunsystemet, noe som indikerer en potensiell rolle som detektorer for sykdom i conspecifics eller fordervet mat kilder 25 (se referanse 43).
Grunnleggende for å forstå reseptor-ligand relasjoner og nedstrøms signalkaskader i bestemte VSN populasjoner er en detaljert evaluering av deres grunnleggende biofysiske egenskapene i en innfødt miljø. I det siste, har analysen av cellesignale sterkt nytte av genmodifiserte dyr som markerer en definert populasjon av nevroner ved coexpressing en fluoriserende fargestoff protein 30,44-49. I denne protokollen, en transgen muselinje som uttrykker FPR-RS3 sammen med en fluoriserende fargestoff (FPR-RS3-i-Venus) brukes.Denne tilnærmingen eksemplifiserer hvordan å ansette en slik genmodifisert mus belastning å utføre elektrofysiologisk analyse av en optisk identifiserbar cellepopulasjon ved hjelp av enkelt nervecellen patch-clamp opptak i akutte koronale VNO vev skiver. En lufttrykket drevet multi-fat perfusjon system for sensoriske stimuli og farmakologiske midler gir rask, reversible og focal neuronal stimulering eller hemming under opptak. Hel-celle opptak i skive forberedelsene gi rom for en detaljert analyse av iboende egenskaper, spenningsaktiverte conductances, samt handlings potensielle utslippsmønstre i cellens opprinnelige miljø.
Protocol
Representative Results
Discussion
VNO er en chemosensory struktur som oppdager semiochemicals. Til dags dato, forblir de fleste vomeronasal reseptorer som skal deorphanized som bare få reseptor-ligand-par er blitt identifisert. Blant disse V1rb2 ble beskrevet å være spesielt aktiveres av mannlig urin feromon 2-heptanon 30, V2rp5 for å bli aktivert av den mannlige spesifikk feromon ESP1 57 samt V2r1b og V2rf2 å bli aktivert av MHC peptidene SYFPEITHI 48 og SEIDLILGY 58, henholdsvis. En forutsetning f…
Disclosures
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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