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.

Alle dyr prosedyrer var i overensstemmelse med lokale og EU-lovgivning om vern av dyr som brukes til eksperimentelle formål (direktiv 86/609 / EØF) og med anbefalinger fremmet av Federation of European Laboratory Animal Science Associations (FELASA). Begge C57BL / 6 mus og fpr-RS3-i-Venus mus ble huset i grupper på begge kjønn ved værelsestemperatur på en 12 timers lys / mørke syklus med mat og vann tilgjengelig ad libitum. For eksperimenter unge voksne (6-20 uker) av begge kjønn ble brukt. Ingen åpenb...

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...

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&gt;. 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.

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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.

For å få innsikt i biofysiske og fysiologiske egenskaper av definerte cellepopulasjoner, utfører vi akutte koronale vev skiver av musen VNO (Figur 1-2). Etter disseksjon, kan skiver holdes i iskald oksygenert ekstracellulær løsning (S 2) i flere timer. Ved opptak oppsett, en konstant utveksling med friskt oksygenrikt oppløsning (figur 2D) sikrer vev levedyktighet gjennom hele eksperimentet. Vi her ansette en transgen mus...

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.

Dyptgående Fysiologisk Analyse av Definerte cellepopulasjoner i Akutt vevssnitt av musen Vomeronasal Organ

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

Imaging Pheromone Sensing i en mus vomeronasal Akutt Tissue Slice Forberedelse

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. ...

Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor

Imaging neuronal Responses i Slice Produkter av vomeronasal Organ uttrykker en Genetisk Encoded Kalsium Sensor

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

Time-lapse Imaging av Neuroblast Migrasjon i Akutt Skiver av Adult Mouse forebrain

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

Utarbeidelse av Akutt subventricular sone Slices for kalsium Imaging

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

Dual Elektrofysiologiske Opptak av Synaptically-fremkalt Astroglial og Neuronal Responses i akutt hippocampus Slices

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

In vivo Postnatal Electroporation og Time-lapse Imaging av neuroblast Migration in Mouse Akutt Brain Slices

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

Funksjonell analyse av larve Fôring Circuit i Drosophila</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

Ex vivo tilbe Intakt Vomeronasal orgel og tilbehør luktelappen

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

Slice It Hot: Akutt Adult Brain Slicing i Fysiologisk Temperatur

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

Utarbeidelse av akutt hjernen skiver med en optimalisert N-Methyl-D-glucamine beskyttende gjenopprettingsmetode

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...