JoVE Journal > Neuroscience
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
Automatic Translation
Neuroscience

Dybdegående Fysiologisk Analyse af Definerede Cell populationer i Akut Tissue Skiver af Mouse vomeronasale organ

Published: September 10, 2016
doi:
10.3791/54517
, ,
1Department of Chemosensation, Institute for Biology II,RWTH Aachen University, 2Mill Hill Laboratory,The Francis Crick Institute

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 er stærkt afhængige deres kemiske sanser til at interagere med deres omgivelser. Lugtesansen spiller en afgørende rolle for at finde og vurdere mad, undgå rovdyr og finde egnede parring partnere. I de fleste pattedyr, den olfaktoriske system består af mindst fire anatomisk og funktionelt forskellige perifere delsystemer: den vigtigste lugteepitel 1,2, den Grueneberg ganglion 3,4, den septale organ Masera 5,6 og vomeronasale organ. Den VNO omfatter perifer sensorisk struktur af tilbehøret olfaktoriske system, (AOS), som spiller en stor rolle i at afsløre kemiske signaler, der formidler oplysninger om identitet, køn, social rang og seksuel tilstand 7-10. VNO er ​​placeret i bunden af ​​næseskillevæggen ret ovenfor ganen. Hos mus er det en bilateral blind-ending rør indesluttet i en bruskagtig kapsel 11-13. Orglet består af både en halvmåneformet medial sensorisk epitheTrifolium, som huser de VSNs og er af ikke-sensorisk del på den laterale side. Mellem begge epiteler ligger en mucus-fyldte hulrum, som er forbundet til næsehulen via den smalle vomeronasal kanalen 14. En stor lateral blodkar i den ikke-sensoriske væv tilvejebringer en vaskulær pumpemekanisme for at lette optagelse af relativt store, for det meste ikke-flygtige molekyler, såsom peptider eller små proteiner i VNO lumen gennem undertryk 15,16. De strukturelle komponenter i VNO er til stede ved fødslen, og orglet når voksenstørrelse kort før puberteten 17. Men om gnaver AOS er allerede funktionel i unge er stadig genstand for debat 18-20.

VSNs udmærker sig ved både deres epitel placering og typen af ​​receptor de udtrykker. VSNs viser en bipolær morfologi med en ikke-myelinerede axon og en enkelt apikal dendritceller, der rager mod lumen og ender i en microvillous dendritisk knop. VSN økseons fasciculate at danne vomeronasal nerve, der forlader bruskspidserne kapsel på dorso-caudale ende, stiger langs septum, passerer cribriform plade og projekter til tilbehøret lugtekolben (AOB) 21,22. Den vomeronasale sensoriske epithel består af to lag: den apikale lag er placeret tættere på den luminale side og havne både V1R- og alle undtagen én type FPR-RS-udtrykkende neuroner. Disse neuroner co-udtrykke G-protein α-subunit G αi2 og projekt til den forreste del af AOB 23-25. Sensoriske neuroner beliggende i de mere basale lag udtrykkelige V2Rs eller FPR-RS1 sammen G αo og sende deres axoner til den bageste region af AOB 26-28.

Vomeronasal neuroner sandsynligvis aktiveres af temmelig små semiochemicals 29-33 (V1Rs) eller proteinholdige forbindelser 34-38 (V2Rs), der udskilles i forskellige kropsvæsker såsom urin, spyt og tårevæske 37,39-41 </sup>. In situ eksperimenter har vist, at VSNs også aktiveres af formyleret peptider og forskellige antimikrobielle / inflammation forbundne forbindelser 25,42. Desuden udtrykte heterologt FPR-rs proteiner deler agonist spektre med FPRs udtrykt i immunsystemet, hvilket indikerer en potentiel rolle som detektorer for sygdom i artsfæller eller forkælede fødekilder 25 (se referere 43).

Grundlæggende for forståelsen receptor-ligand relationer og downstream signalering kaskader i bestemte VSN befolkningsgrupper er en detaljeret evaluering af deres grundlæggende biofysiske karakteristika i en indfødt miljø. I fortiden, har analysen af cellulære signalering stærkt nydt godt af genmodificerede dyr, der markerer en defineret population af neuroner ved at co-udtrykke en fluorescerende markør protein 30,44-49. I denne protokol, en transgen mus linje, der udtrykker FPR-RS3 sammen med en fluorescerende markør (FPR-RS3-i-Venus) anvendes.Denne tilgang eksemplificerer, hvordan at ansætte sådan en genetisk modificeret musestamme til at udføre elektrofysiologiske analyse af et optisk identificerbare cellepopulation anvendes enkelt neuron patch-clamp optagelser i akutte koronale VNO væv skiver. Et lufttryk drevet multi-tønde perfusion for sensoriske stimuli og farmakologiske agenter giver hurtig, reversibel og fokal neuronal stimulation eller hæmning under optagelserne. Whole-celle optagelser i slice præparater mulighed for en detaljeret analyse af iboende egenskaber, spænding-aktiverede konduktanser, samt action potentielle udledning mønstre i cellens oprindelige miljø.

Protocol

Alle procedurer dyr var i overensstemmelse med lokale og EU-lovgivning om beskyttelse af dyr, der anvendes til forsøg (direktiv 86/609 / EØF) og med henstillinger fra Sammenslutningen af ​​Europæiske Laboratorium Animal Science Associations (FELASA). Begge C57BL / 6-mus og Fpr-RS3-i-Venus-mus blev holdt i grupper af begge køn ved stuetemperatur på en 12 timers lys / mørke-cyklus med foder og vand tilgængeligt ad libitum. Til forsøg unge voksne (6-20 uger) af begge køn blev anvendt. Ingen åbenlyse …

Representative Results

For at få indsigt i de biofysiske og fysiologiske egenskaber definerede cellepopulationer, vi udfører akutte koronale vævssnit med musen VNO (figur 1 – 2). Efter dissektion, kan skiverne holdes i iskold iltet ekstracellulær opløsning (S 2) til flere timer. Ved opsætningen optagelse, en konstant udveksling med frisk iltet løsning (figur 2D) sikrer levedygtighed væv i hele forsøget. Vi her anvender en transgen musemodel…

Discussion

Den VNO er ​​en chemosensory struktur, der registrerer semiochemicals. Til dato er de fleste af vomeronasal receptorer mangler at blive deorphanized som kun få receptor-ligand-par er blevet identificeret. Blandt disse blev V1rb2 beskrevet for specifikt aktiveres af den mandlige urin feromon 2-heptanon 30, der V2rp5 aktiveres af den mandlige specifikke feromon ESP1 57 samt V2r1b og V2rf2 at blive aktiveret af MHC-peptider SYFPEITHI 48 og SEIDLILGY 58, henholdsvis. En forud…

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
DOWNLOAD MATERIALS LIST

References

  1. Firestein, S. How the olfactory system makes sense of scents. Nature. 413 (6852), 211-218 (2001).
  2. Mombaerts, P. Genes and ligands for odorant, vomeronasal and taste receptors. Nat. Rev. Neurosci. 5 (4), 263-278 (2004).
  3. Fuss, S. H., Omura, M., Mombaerts, P. The Grueneberg ganglion of the mouse projects axons to glomeruli in the olfactory bulb. Eur. J. Neurosci. 22 (10), 2649-2654 (2005).
  4. Roppolo, D., Ribaud, V., Jungo, V. P., Lüscher, C., Rodriguez, I. Projection of the Grüneberg ganglion to the mouse olfactory bulb. Eur. J. Neurosci. 23 (11), 2887-2894 (2006).
  5. Adams, D. R. Fine structure of the vomeronasal and septal olfactory epithelia and of glandular structures. Microsc. Res. Tech. 23 (1), 86-97 (1992).
  6. Ma, M., et al. Olfactory signal transduction in the mouse septal organ. J. Neurosci. 23 (1), 317-324 (2003).
  7. Dulac, C., Torello, A. T. Molecular detection of pheromone signals in mammals: from genes to behaviour. Nat. Rev. Neurosci. 4 (7), 551-562 (2003).
  8. Luo, M., Katz, L. C. Encoding pheromonal signals in the mammalian vomeronasal system. Curr. Opin. Neurobiol. 14 (4), 428-434 (2004).
  9. Brennan, P. A., Kendrick, K. M. Mammalian social odours: attraction and individual recognition. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 361 (1476), 2061-2078 (2006).
  10. Tirindelli, R., Dibattista, M., Pifferi, S., Menini, A. From Pheromones to Behavior. Physiol. Rev. 89, 921-956 (2009).
  11. Jacobson, L., Trotier, D., Doving, K. B. Anatomical description of a new organ in the nose of domesticated animals by Ludvig Jacobson (1813). Chem. Senses. 23 (6), 743-754 (1998).
  12. Keverne, E. B. The Vomeronasal Organ. Science. 286 (5440), 716-720 (1999).
  13. Breer, H., Fleischer, J., Strotmann, J. The sense of smell: multiple olfactory subsystems. Cell. Mol. Life Sci. C. 63 (13), 1465-1475 (2006).
  14. Liberles, S. D. Mammalian pheromones. Annu. Rev. Physiol. 76, 151-175 (2014).
  15. Meredith, M., O’Connell, R. J. Efferent control of stimulus access to the hamster vomeronasal organ. J. Physiol. 286, 301-316 (1979).
  16. Pankevich, D., Baum, M. J., Cherry, J. A. Removal of the superior cervical ganglia fails to block Fos induction in the accessory olfactory system of male mice after exposure to female odors. Neurosci. Lett. 345 (1), 13-16 (2003).
  17. Giacobini, P., Benedetto, A., Tirindelli, R., Fasolo, A. Proliferation and migration of receptor neurons in the vomeronasal organ of the adult mouse. Brain Res. Dev. Brain Res. 123 (1), 33-40 (2000).
  18. Coppola, D. M., O’Connell, R. J. Stimulus access to olfactory and vomeronasal receptors in utero. Neurosci. Lett. 106 (3), 241-248 (1989).
  19. Hovis, K. R., et al. Activity Regulates Functional Connectivity from the Vomeronasal Organ to the Accessory Olfactory Bulb. J. Neurosci. 32 (23), 7907-7916 (2012).
  20. Mucignat-Caretta, C. The rodent accessory olfactory system. J. Comp. Physiol. A Neuroethol. Sensory, Neural, Behav. Physiol. 196 (10), 767-777 (2010).
  21. Jia, C., Halpern, M. Subclasses of vomeronasal receptor neurons: differential expression of G proteins (Giα2 and G(αo)) and segregated projections to the accessory olfactory bulb. Brain Res. 719 (1-2), 117-128 (1996).
  22. Del Punta, K., Puche, C. A., Adams, N. C., Rodriguez, I., Mombaerts, P. A divergent pattern of sensory axonal projections is rendered convergent by second-order neurons in the accessory olfactory bulb. Neuron. 35 (6), 1057-1066 (2002).
  23. Belluscio, L., Koentges, G., Axel, R., Dulac, C. A map of pheromone receptor activation in the mammalian brain. Cell. 97 (2), 209-220 (1999).
  24. Rodriguez, I., Feinstein, P., Mombaerts, P. Variable patterns of axonal projections of sensory neurons in the mouse vomeronasal system. Cell. 97 (2), 199-208 (1999).
  25. Rivière, S., Challet, L., Fluegge, D., Spehr, M., Rodriguez, I. Formyl peptide receptor-like proteins are a novel family of vomeronasal chemosensors. Nature. 459 (7246), 574-577 (2009).
  26. Martini, S., Silvotti, L., Shirazi, A., Ryba, N. J. P., Tirindelli, R. Co-expression of putative pheromone receptors in the sensory neurons of the vomeronasal organ. J. Neurosci. 21 (3), 843-848 (2001).
  27. Matsuoka, M., et al. Immunocytochemical study of Gi2alpha and Goalpha on the epithelium surface of the rat vomeronasal organ. Chem. Senses. 26 (2), 161-166 (2001).
  28. Dulac, C., Torello, A. T. Molecular detection of pheromone signals in mammals: from genes to behaviour. Nat. Rev. Neurosci. 4 (7), 551-562 (2003).
  29. Leinders-Zufall, T., et al. Ultrasensitive pheromone detection by mammalian vomeronasal neurons. Nature. 405 (6788), 792-796 (2000).
  30. Boschat, C., et al. Pheromone detection mediated by a V1r vomeronasal receptor. Nat. Neurosci. 5 (12), 1261-1262 (2002).
  31. Novotny, M. V. Pheromones, binding proteins and receptor responses in rodents. Biochem. Soc. Trans. 31, 117-122 (2003).
  32. Nodari, F., et al. Sulfated steroids as natural ligands of mouse pheromone-sensing neurons. J. Neurosci. 28 (25), 6407-6418 (2008).
  33. Isogai, Y., et al. Molecular organization of vomeronasal chemoreception. Nature. 478 (7368), 241-245 (2011).
  34. Leinders-Zufall, T., et al. MHC class I peptides as chemosensory signals in the vomeronasal organ. Science. 306 (5698), 1033-1037 (2004).
  35. Chamero, P., et al. Identification of protein pheromones that promote aggressive behaviour. Nature. 450 (7171), 899-902 (2007).
  36. Kimoto, H., Haga, S., Sato, K., Touhara, K. Sex-specific peptides from exocrine glands stimulate mouse vomeronasal sensory neurons. Nature. 437 (7060), 898-901 (2005).
  37. Ferrero, D. M., et al. A juvenile mouse pheromone inhibits sexual behaviour through the vomeronasal system. Nature. 502 (7471), 368-371 (2013).
  38. Kaur, A. W., et al. Murine pheromone proteins constitute a context-dependent combinatorial code governing multiple social behaviors. Cell. 157 (3), 676-688 (2014).
  39. Ben-Shaul, Y., Katz, L. C., Mooney, R., Dulac, C. In vivo vomeronasal stimulation reveals sensory encoding of conspecific and allospecific cues by the mouse accessory olfactory bulb. PNAS. 107 (11), 5172-5177 (2010).
  40. Kimoto, H., et al. Sex- and strain-specific expression and vomeronasal activity of mouse ESP family peptides. Curr. Biol. 17 (21), 1879-1884 (2007).
  41. Spehr, M., et al. Parallel processing of social signals by the mammalian main and accessory olfactory systems. Cell. Mol. Life Sci. C. 63 (13), 1476-1484 (2006).
  42. Chamero, P., et al. G protein G{alpha}o is essential for vomeronasal function and aggressive behavior in mice. PNAS. , (2011).
  43. Bufe, B., Schumann, T., Zufall, F. Formyl peptide receptors from immune and vomeronasal system exhibit distinct agonist properties. J. Biol. Chem. 287 (40), 33644-33655 (2012).
  44. Bozza, T., Feinstein, P., Zheng, C., Mombaerts, P. Odorant receptor expression defines functional units in the mouse olfactory system. J. Neurosci. 22 (8), 3033-3043 (2002).
  45. Grosmaitre, X., Vassalli, A., Mombaerts, P., Shepherd, G. M., Ma, M. Odorant responses of olfactory sensory neurons expressing the odorant receptor MOR23: a patch clamp analysis in gene-targeted mice. PNAS. 103 (6), 1970-1975 (2006).
  46. Oka, Y., et al. Odorant receptor map in the mouse olfactory bulb: in vivo sensitivity and specificity of receptor-defined glomeruli. Neuron. 52 (5), 857-869 (2006).
  47. Ukhanov, K., Leinders-Zufall, T., Zufall, F. Patch-clamp analysis of gene-targeted vomeronasal neurons expressing a defined V1r or V2r receptor: ionic mechanisms underlying persistent firing. J. Neurophysiol. 98 (4), 2357-2369 (2007).
  48. Leinders-Zufall, T., Ishii, T., Mombaerts, P., Zufall, F., Boehm, T. Structural requirements for the activation of vomeronasal sensory neurons by MHC peptides. Nat. Neurosci. 12 (12), 1551-1558 (2009).
  49. Pacifico, R., Dewan, A., Cawley, D., Guo, C., Bozza, T. An olfactory subsystem that mediates high-sensitivity detection of volatile amines. Cell Rep. 2 (1), 76-88 (2012).
  50. Veitinger, S., et al. Purinergic signalling mobilizes mitochondrial Ca2+ in mouse Sertoli cells. J. Physiol. 589 (Pt 21), 5033-5055 (2011).
  51. Kaur, A. W., et al. Murine pheromone proteins constitute a context-dependent combinatorial code governing multiple social behaviors. Cell. 157 (3), 676-688 (2014).
  52. Ackels, T., von der Weid, B., Rodriguez, I., Spehr, M. Physiological characterization of formyl peptide receptor expressing cells in the mouse vomeronasal organ. Front. Neuroanat. 8, 1-13 (2014).
  53. Liman, E. R., Corey, D. P. Electrophysiological characterization of chemosensory neurons from the mouse vomeronasal organ. J. Neurosci. 16 (15), 4625-4637 (1996).
  54. Cichy, A., et al. Extracellular pH Regulates Excitability of Vomeronasal Sensory Neurons. J. Neurosci. 35 (9), 4025-4039 (2015).
  55. Shimazaki, R., et al. Electrophysiological properties and modeling of murine vomeronasal sensory neurons in acute slice preparations. Chem. Senses. 31 (5), 425-435 (2006).
  56. Hagendorf, S., Fluegge, D., Engelhardt, C., Spehr, M. Homeostatic control of sensory output in basal vomeronasal neurons: activity-dependent expression of ether-à-go-go-related gene potassium channels. J. Neurosci. 29 (1), 206-221 (2009).
  57. Haga, S., et al. The male mouse pheromone ESP1 enhances female sexual receptive behaviour through a specific vomeronasal receptor. Nature. 466 (7302), 118-122 (2010).
  58. Leinders-Zufall, T., et al. A family of nonclassical class I MHC genes contributes to ultrasensitive chemodetection by mouse vomeronasal sensory neurons. J. Neurosci. 34 (15), 5121-5133 (2014).
  59. Jinek, M., et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 337, 816-821 (2012).

Tags

Keyword Extraction:Physiological AnalysisCell PopulationsMouse Vomeronasal OrganChemosensationSensory NeuronsNasal CavityVomer BoneVNO CapsuleCartilaginous Capsules
check_url/54517?article_type=t

Play Video
PDF
DOI
DOWNLOAD MATERIALS LIST
Cite This Article
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).
Download Citation
Reprints and Permissions

View Video

✖

To prove you're not a robot, please enter the text in the image below

CAPTCHA Image
Play CAPTCHA Audio
Refresh Image