Perforeret Patch-clamp Registrering af Mouse Olfactory sensoriske neuroner i Intakt neuroepithelium: Funktionel analyse af neuroner udtrykker en identificeret odorantreceptor
Summary
Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.
Abstract
Analysere de fysiologiske reaktioner olfaktoriske sensoriske neuroner (OSN), når stimuleres med specifikke ligander er afgørende at forstå grundlaget for olfaktoriske drevne adfærd og deres modulation. Disse kodning egenskaber er stærkt afhængige den oprindelige samspil mellem lugtmolekyler og olfaktoriske receptor (OR) udtrykt i OSN. Identiteten, specificitet og ligand spektrum af udtrykt eller er kritiske. Sandsynligheden for at finde den ligand af OR udtrykkes i en OSN valgt tilfældigt inden epitelet er meget lav. For at løse denne udfordring, denne protokol bruger genetisk mærkede mus, der udtrykker det fluorescerende protein GFP under kontrol af promotoren af definerede yderste periferi. OSN er placeret i en stram og organiseret epitel foring næsehulen, med omkringliggende celler påvirke deres modning og funktion. Her beskriver vi en fremgangsmåde til isolering af et intakt lugteepitel og registrere gennem patch-clamp optagelser egenskaber OSN expressing definerede lugtstofreceptorer. Protokollen gør det muligt at karakterisere OSN membranegenskaber samtidig holde indflydelsen af tilstødende væv. Analyse af patch-clamp resultater giver en præcis kvantificering af ligand / ELLER interaktioner, transduktionsveje og farmakologi, OSN 'kodning egenskaber og deres modulation på membranen niveau.
Introduction
Olfaktoriske sensoriske neuroner (OSN) repræsenterer det første skridt i olfaktoriske perception. Beliggende i den olfaktoriske epitel foring næsehulen hos gnavere, de omdanne kemiske oplysninger af lugtstoffer i aktionspotentialer sendes gennem deres axon til hjernen. For bedre at forstå de olfaktoriske kodning mekanismer, er det nødvendigt at karakterisere transduktion og membran egenskaber OSN. Indtil for nylig blev de fleste af de teknikker, der anvendes til at karakterisere egenskaber pattedyr OSN udført på dissocieret OSN 1-4. Dissociationsprocessen anvender forskellige mekaniske og kemiske (dvs. enzymer) processer at befri OSN fra deres omgivelser. Disse processer inducere et lavt antal tilgængelige celler til optagelser. Dette lave antal kan være endnu mere kritisk i tilfælde af GFP mærkede celler. Dissociation fjerner også de lokale celle-til-celle-interaktioner mellem OSN og andre celler i det olfaktoriske epitel, der kan forbedre survival og modulering af OSN egenskaber. For at omgå dissociation procedure blev en intakt præparat udviklet 5.
Hver OSN udtrykker én olfaktoriske receptor (OR) udvalgt fra en stor multigenfamilie 6. Der er ~ 1.000 yderste periferi udtrykt i de vigtigste olfaktoriske epitel i musen. På grund af det store antal OR i vildtype dyr, at chancerne optage OSN udtrykker det samme eller er meget lave. For at overvinde disse begrænsninger er tilgængelige gen målrettede mus, hvor alle OSN udtrykker en identificeret eller er mærket med et fluorescerende protein 7-9. Disse mærkede OSN blev anvendt til at gøre funktionel analyse i dissocierede præparater 7,10,11 med ulemperne nævnt tidligere. En intakt epitel forberedelse 5 af genetisk mærkede mus omgår derfor disse spørgsmål. Det gør det muligt at overvåge aktiviteten af OSN udtrykker præcist definerede yderste periferi i et miljø så tæt på i vivo som muligt. Desuden patch-clamp optagelser af OSN tillader også præcis analyse af ejendomme membran, transduktion pathway farmakologi, ligand / ELLER interaktioner. Alle disse emner kan næppe analyseres under anvendelse ekstracellulære optagelser. Vi brugte denne teknik til at overvåge svarene fra OSN udtrykker de lugtstofreceptorer SR1 og MOR23 12,13. Muligheden for teknikken blev bekræftet af andre grupper på MOR23 udtrykker OSN 14 samt andre yderste periferi udtrykker neuroner 15,16. Overvågningen af en defineret population af OSN kan føre til en analyse af deres egenskaber i mange forskellige sammenhænge, såsom udvikling 14, aldring 17, lugtstof-induceret plasticitet 18, og den rolle, variationer i lugtstof receptorens sekvens i lugt kodning 15. Denne protokol giver således et effektivt værktøj til at overvåge de funktionelle egenskaber af definerede OSN på membranen niveau.
Protocol
Representative Results
Discussion
Evnen af denne protokol korrekt overvåge egenskaber sunde OSN afhænger i høj grad af kvaliteten af præparatet. Derfor dissektion trin er kritisk. Først er det vigtigt at være opmærksom på kvaliteten (pH, osmolaritet), iltning og temperatur (iskold, men ikke frosne) i dissektion medium. For det andet skal manipulation af epitel med dissekere værktøjer være så begrænset som muligt for at undgå skader. Endelig er det afgørende at opnå en sammensætning så flad som muligt for at få adgang til d…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Authors would like to thank Peter Mombaerts for the generous gift of OR-GFP mice; Anne Lefranc and the CSGA animal facility for excellent animal care. Funding was provided by CNRS through an ATIP and ATIP Plus grants, by Conseil Régional de Bourgogne (FABER and PARI grants), by Université de Bourgogne (BQR program).
Materials
| heavy equipment | |||
| vibration table with Faraday cage | TMC | 63-500 SERIES | required : isolates the recording system from vibrations induced by the environment (movements of experimenter, vibrations of equipment such as fans for cooling computers, etc); can also be purchased with a Faraday cage, or equipped by a custom made Faraday cage; this cage is recommended to avoid electric noise from the environment |
| optics | |||
| microscope | Olympus | BX51WI | upright microcope equipped with epifluorescence; fixed or moving stage depending on the user's preference |
| objectives | Olympus | LUMPLFL40XW | at least 2 objectives required: a 4x or 10x for coarse approach to the cell; and a 40x immersion long distance example Olympus LUMPLFL40XW/IR/0,8/WD:3.3 MM |
| magnifier | Olympus | U-TVCAC | ABSOLUTELY REQUIRED: placed in the light path between the objective and the camera; allows to magnify the image on the screen in order to reach precisely the knob with the recording electrode |
| camera | Olympus | DP72 | a good camera is required to see the neurons in fluorescence as well as in bright field; the controlling software is simple and allows to take pictures and do live camera image to monitor the approach of the electrode to the cell. An ultrasensitive camera is not necessary |
| filters | Olympus/Chroma | depending on the fluorescent protein used in the mice; example for GFP: excitation : BP460-490: emission: HQ530/50m | |
| recording electrodes /system | |||
| amplifier | HEKA | EPC10 USB | monitors the currents flowing through the recording electrode and also controls the puffing by sending a TTL signal to the spritzer; the EPC10 setup is controled by computer |
| software | HEKA | Patchmaster | controls the amplifier during the experiment |
| micromanipulator | Sutter | MP225 | precision micromanipulator, allows precise movements down to 1/1Oth of a micrometer; this model is very stable; avoid hydraulic manipulators that may drift |
| electrode puller | sutter | P97 | with a FT345-B wide trough filament; to prepare recording pipets of about 2µm diameter with a long tip to reach the cells; the resistance should be 15 to 20Mohm with perforated patch clamp solution |
| glass | sutter | BF120-69-10 | in our recording conditions, this glass is ideal for recording pipets |
| recording chamber | warner instruments | RC-26G | a chamber is needed to set the preparation under the microscope. To maintain the preparation in the center of the chamber, a net/anchor should be used. |
| stimulation | |||
| glass | WPI | TW100F-4 | attached in groups of 7, these pipettes are used to prepare prepulled stimulating pipettes |
| multibarrel puller | MDI | PMP-107-Z | by association of pull and twist, this puller allows us to prepare puffing electrodes with 7 barrels |
| precision pressure injector | Toohey Company | P/N T25-1-900 Single Channel | this precision pressure injector controls the pressure ejected in the multibarrel puller; it is controlled manually or by the amplifier by a 5V TTLs |
| micromanipulator | Narishige | YOU-1 | a coarse manipulator is enough to bring the puffing electrode close to the recording site |
| tubings | N/A | tygon tubing to bring the pressure from the puffer to the puffing pipette | |
| solutions/perfusion/chemicals | |||
| vacuum pump | gardner denver | 300 series | a vibrating membrane pump is more quiet and efficient than other types of pumps |
| perfusion system | N/A | N/A | gravity perfusion system with polyethlylen tubing to bring in and out the external solution from the recording chamber |
| nystatin | Sigma-Aldrich | N3503 | mandatory to perpare internal solution for perforated patch clamp |
| DIMETHYL SULFOXIDE | Sigma-Aldrich | D5879 | used to disolve nystatin for internal solution for perforated patch |
| Sodium chloride | Sigma-Aldrich | S9625 | extracellular solution |
| Potassium chloride | Sigma-Aldrich | P4504 | intracellular/extracellular solution |
| Calcium chloride di hydrate | Sigma-Aldrich | C7902 | extracellular solution |
| Sodium phosphate monobasic monohydrate (NaH2PO4) | Sigma-Aldrich | S9638 | extracellular solution |
| Magnesium sulfate heptahydrate (MgSO4 7H2O) | Sigma-Aldrich | 63140 | extracellular solution |
| Glucose | Sigma-Aldrich | G8270 | extracellular solution |
| Sodium bicarbonate | Sigma-Aldrich | S6297 | extracellular solution |
| EGTA (Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid) | Sigma-Aldrich | E3889 | internal solution |
| Potassium hydroxyde | Sigma-Aldrich | P1767 | internal solution |
| MethylSulfoxide | Sigma-Aldrich | 47,135-6 | intracellular solution |
| Hepes-Na | Sigma-Aldrich | H7006 | intracellular solution |
| Sucrose | Sigma-Aldrich | S0389 | intracellular solution |
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