Name: imaging pheromone sensing in a mouse vomeronasal acute tissue slice preparation
Uploaded: 2011-12-06T12:00:00
Duration: 9 min 31 s
Description: Watch this Scientific Journal Video about Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation at JoVE.com

Мы хотим поблагодарить особенно Моник Nenniger Tosato за отличную техническую поддержку, а также Жан-Ив Чаттон за научные советы и платформы визуализации СИФ UNIL для микроскопа оборудования. Финансирование было предоставлено Швейцарского национального научного фонда.

<ol>
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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+detection+of+pheromone+signals+in+mammals:+from+genes+to+behaviour.">Molecular detection of pheromone signals in mammals: from genes to behaviour.</a> Nat. Rev. Neurosci. 4, 551-562 (2003).</li><li>Brennan, P. A., Zufall, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pheromonal+communication+in+vertebrates.">Pheromonal communication in vertebrates.</a> Nature. 444, 308-315 (2006).</li><li>Mondor, E. B., Roitberg, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inclusive+fitness+benefits+of+scent-marking+predators.">Inclusive fitness benefits of scent-marking predators.</a> Proc. Biol. Sci. 271, 341-343 (2004).</li><li>Holy, T. 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R., Leinders-Zufall, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pheromone+detection+by+mammalian+vomeronasal+neurons.">Pheromone detection by mammalian vomeronasal neurons.</a> Microsc. Res. Tech. 58, 251-260 (2002).</li><li>Ciges, M., Labella, T., Gayoso, M., Sanchez, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructure+of+the+organ+of+Jacobson+and+comparative+study+with+olfactory+mucosa.">Ultrastructure of the organ of Jacobson and comparative study with olfactory mucosa.</a> Acta. Otolaryngol. 83, 47-58 (1977).</li><li>Munger, S. D., Leinders-Zufall, T., Zufall, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subsystem+organization+of+the+mammalian+sense+of+smell.">Subsystem organization of the mammalian sense of smell.</a> Annu. Rev. Physiol. 71, 115-140 (2009).</li><li>Mombaerts, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+and+ligands+for+odorant,+vomeronasal+and+taste+receptors.">Genes and ligands for odorant, vomeronasal and taste receptors.</a> Nat. Rev. Neurosci. 5, 263-278 (2004).</li><li>Ryba, N. J. P., Tirindelli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+multigene+family+of+putative+pheromone+receptors.">A new multigene family of putative pheromone receptors.</a> Cell. 19, 371-379 (1997).</li><li>Matsunami, H., Buck, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multigene+family+encoding+a+diverse+array+of+putative+pheromone+receptors+in+mammals.">A multigene family encoding a diverse array of putative pheromone receptors in mammals.</a> Cell. 90, 775-784 (1997).</li><li>Herrada, G., Dulac, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+family+of+putative+pheromone+receptors+in+mammals+with+a+topographycally+organized+and+sexually+dimorphic+distribution.">A novel family of putative pheromone receptors in mammals with a topographycally organized and sexually dimorphic distribution.</a> Cell. 90, 763-773 (1997).</li><li>Dulac, C., Axel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+family+of+genes+encoding+putative+pheromone+receptors+in+mammals.">A novel family of genes encoding putative pheromone receptors in mammals.</a> Cell. 83, 195-206 (1995).</li><li>Young, J. M., Massa, H. F., Hsu, L., Trask, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extreme+variability+among+mammalian+V1R+gene+families.">Extreme variability among mammalian V1R gene families.</a> Genome. Res. 20, 10-18 (2009).</li><li>Yang, H., Shi, P., Zhang, Y. P., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Composition+and+evolution+of+the+V2r+vomeronasal+receptor+gene+repertoire+in+mice+and+rats.">Composition and evolution of the V2r vomeronasal receptor gene repertoire in mice and rats.</a> Genomics. 86, 306-315 (2005).</li><li>Levai, O., Feistel, T., Breer, H., Strotmann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cells+in+the+vomeronasal+organ+express+odorant+receptors+but+project+to+the+accessory+olfactory+bulb.">Cells in the vomeronasal organ express odorant receptors but project to the accessory olfactory bulb.</a> J. Comp. Neurol. 498, 476-490 (2006).</li><li>Riviere, S., Challet, L., Fluegge, D., Spehr, M., Rodriguez, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formyl+peptide+receptor-like+proteins+are+a+novel+family+of+vomeronasal+chemosensors.">Formyl peptide receptor-like proteins are a novel family of vomeronasal chemosensors.</a> Nature. 459, 574-577 (2009).</li><li>Liberles, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formyl+peptide+receptors+are+candidate+chemosensory+receptors+in+the+vomeronasal+organ.">Formyl peptide receptors are candidate chemosensory receptors in the vomeronasal organ.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 9842-9847 (2009).</li><li>Liman, E. R., Corey, D. P., Dulac, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRP2:+A+candidate+transduction+channel+for+mammalian+pheromone+sensory+signaling.">TRP2: A candidate transduction channel for mammalian pheromone sensory signaling.</a> Proc. Natl. Acad. Sci. U.S.A. 96, 5791-5796 (1999).</li><li>Kelliher, K. R., Spehr, M., Li, X. H., Zufall, F., Leinders-Zufall, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pheromonal+recognition+memory+induced+by+TRPC2-independent+vomeronasal+sensing.">Pheromonal recognition memory induced by TRPC2-independent vomeronasal sensing.</a> Eur. J. Neurosci. 23, 3385-3390 (2006).</li><li>Rodriguez, I., Feinstein, P., Mombaerts, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variable+patterns+of+axonal+projections+of+sensory+neurons+in+the+mouse+vomeronasal+system.">Variable patterns of axonal projections of sensory neurons in the mouse vomeronasal system.</a> Cell. 97, 199-208 (1999).</li><li>Boschat, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pheromone+detection+mediated+by+a+V1r+vomeronasal+receptor.">Pheromone detection mediated by a V1r vomeronasal receptor.</a> Nature. Neuroscience. 5, 1261-1262 (2002).</li><li>Brechbühl, J., Klaey, M., Broillet, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Grueneberg+ganglion+cells+mediate+alarm+pheromone+detection+in+mice.">Grueneberg ganglion cells mediate alarm pheromone detection in mice.</a> Science. 321, 1092-1095 (2008).</li></ol>

Нет конфликта интересов объявлены.

Метод визуализации кальция, представленные здесь позволяет записывать, в остром подготовки ломтик, феромонов ответы нейронов мыши ВНО. При таком подходе, рассечение от животного к вомероназального нейронов, с некоторой практикой, легко достижимо и дает долгоживущих подготовки ткани. Он позволяет много времени эксперименты, как фармакологические исследования или изучения феромонов кодирования. При этом физиологические техники, большой численностью населения, а также точный нейронных субпопуляций могут быть проанализированы в частности. Кроме того, этот метод может быть легко адаптирована к иммуногистохимического подходы или экспериментов, основанных на других красителей кальция. Использование этих комбинированных методик, безусловно, позволяют выявлению новых феромонов лигандами для многих хеморецепторы сирот выражается в орган мыши вомероназального.

<table border="1"><tbody><tr><td> Название реагента </td><td> Компания </td><td> Номер по каталогу </td><td> Комментарии </td></tr><tr><td> Аденозин-5&#39;-трифосфат динатрия солевой раствор (АТФ) </td><td> Sigma-Aldrich </td><td> A6559-25UMO </td><td></td></tr><tr><td> Агар </td><td> Sigma-Aldrich </td><td> A7002-100G </td><td> Предпочтительно для иммуногистохимии </td></tr><tr><td> Агар (легкоплавкие) </td><td> Sigma-Aldrich </td><td> A0701-25G </td><td> Предпочтительно для кальция изображений </td></tr><tr><td> CL-100 Биполярный регулятора температуры </td><td> Warner инструменты </td><td> W64-0352 </td><td></td></tr><tr><td> Конфокальной микроскопии </td><td> Leica Microsystems </td><td> SP5 AOBS </td><td></td></tr><tr><td> Cy5-AffiniPure антимышиного IgG </td><td> Джексон ImmunoResearch </td><td> 115-175-071 </td><td> В защищенном от света </td></tr><tr><td> ДМСО (диметилсульфоксид) </td><td> Merck </td><td> 317 275-100мл </td><td></td></tr><tr><td> Вложение пресс-форм, Пил-A-Way </td><td> Polysciences </td><td> 18646A-1 </td><td> 22 х 22 х 20 мм </td></tr><tr><td> Флуоресцентные монтажа СМИ (Vectashield с DAPI, 10 мл) </td><td> Rectolab </td><td> H-1200 </td><td></td></tr><tr><td> Фура-2 утра </td><td> Teflabs </td><td> 0103 </td><td> В защищенном от света </td></tr><tr><td> Glisseal N (вакуумной смазки, 60 г) </td><td> Борер Chemie </td><td> Glisseal N </td><td></td></tr><tr><td> Большая камера Запись ванны (RC-26G) </td><td> Warner инструменты </td><td> 64-0235 </td><td></td></tr><tr><td> MetaFluor (программное обеспечение для визуализации кальция; v.7.6.5.0) </td><td> Visitron системы </td><td></td><td></td></tr><tr><td> Моноклональные Анти-тубулина, acetylateг мышиного антитела </td><td> Sigma-Aldrich </td><td> T6793-.2ML </td><td></td></tr><tr><td> Нормальный козел сыворотки (NGS, 10 мл) </td><td> Interchim </td><td> UP379030 </td><td></td></tr><tr><td> Платформа для камер (P-1) </td><td> Warner инструменты </td><td> 64-0277 </td><td></td></tr><tr><td> Pluronic F-127, 2 г </td><td> Invitrogen </td><td> P-6867 </td><td></td></tr><tr><td> Roti Колл (дозирования бутылку, 10 г) </td><td> Карл Рот </td><td> 0258,1 </td><td></td></tr><tr><td> SC-20 Dual In-линия Решение обогреватель / охладитель </td><td> Warner инструменты </td><td> W64-0353 </td><td></td></tr><tr><td> Фрагмент якорь (SHD-26GKIT) </td><td> Warner инструменты </td><td> 64-0266 </td><td></td></tr><tr><td> Stereofluorescence микроскопом </td><td> Leica Microsystems </td><td> MZ16FA </td><td></td></tr><tr><td> Супер PAP PEN (гидрофобныеручкой) </td><td> Pelco Международного </td><td> 22309 </td><td></td></tr><tr><td> Тритон Х-100 </td><td> Fluka </td><td> 93 420-250мл </td><td></td></tr><tr><td> Вибрационный лезвие микротома VT 1200S </td><td> Leica Microsystems </td><td> 14048142066 </td><td></td></tr><tr><td> VisiChrome системы высокой скорости полихроматор </td><td> Visitron системы </td><td></td><td></td></tr><tr><td> 3D Визуализация данных (Imaris; v.6.3.1) </td><td> Bitplane Научно Software </td><td></td><td></td></tr></tbody></table>

imaging pheromone sensing in a mouse vomeronasal acute tissue slice preparation

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. Pheromones are volatile or non-volatile short-lived molecules2 secreted and/or contained in biological fluids3,4, such as urine, a liquid known to be a main source of pheromones3. Pheromonal communication is implicated in a variety of key animal modalities such as kin interactions5,6, hierarchical organisations3 and sexual interactions7,8 and are consequently directly correlated with the survival of a given species9,10,11. In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO)10,12, a paired structure located at the base of the nasal cavity, and enclosed in a cartilaginous capsule. Each VNO has a tubular shape with a lumen13,14 allowing the contact with the external chemical world. The sensory neuroepithelium is principally composed of vomeronasal bipolar sensory neurons (VSNs)15. Each VSN extends a single dendrite to the lumen ending in a large dendritic knob bearing up to 100 microvilli implicated in chemical detection16. Numerous subpopulations of VSNs are present. They are differentiated by the chemoreceptor they express and thus possibly by the ligand(s) they recognize17,18. Two main vomeronasal receptor families, V1Rs and V2Rs19,20,21,22, are composed respectively by 24023 and 12024 members and are expressed in separate layers of the neuroepithelium. Olfactory receptors (ORs)25 and formyl peptide receptors (FPRs)26,27 are also expressed in VSNs.
Whether or not these neuronal subpopulations use the same downstream signalling pathway for sensing pheromones is unknown. Despite a major role played by a calcium-permeable channel (TRPC2) present in the microvilli of mature neurons28 TRPC2 independent transduction channels have been suggested6,29. Due to the high number of neuronal subpopulations and the peculiar morphology of the organ, pharmacological and physiological investigations of the signalling elements present in the VNO are complex.
Here, we present an acute tissue slice preparation of the mouse VNO for performing calcium imaging investigations. This physiological approach allows observations, in the natural environment of a living tissue, of general or individual subpopulations of VSNs previously loaded with Fura-2AM, a calcium dye. This method is also convenient for studying any GFP-tagged pheromone receptor and is adaptable for the use of other fluorescent calcium probes. As an example, we use here a VG mouse line30, in which the translation of the pheromone V1rb2 receptor is linked to the expression of GFP by a polycistronic strategy.

Watch this Scientific Journal Video about Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation at JoVE.com

Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation

У мышей способность обнаруживать феромоны в основном при посредничестве вомероназального орган (ВНО). Здесь, острый срез подготовки ткани для проведения ВНО кальция изображений описан. Эта физиологическая подход позволяет наблюдений субпопуляций и / или отдельных нейронов в живой ткани и удобен для рецептор-лиганд идентификации.

Изображениями феромонов зондирования в мышь вомероназального острого Подготовка фрагментов тканей

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. ...

Research

JoVE Journal

Neuroscience

Multielectrode Array Recordings of the Vomeronasal Epithelium

Многоэлектродной массива Записи вомероназального Эпителий

Understanding neural circuits requires methods to record from many neurons simultaneously. For in vitro studies, one currently available technology is ...

Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

Хронический изображений Мыши зрительной коры Использование тоньше черепа-подготовка

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The ...

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Патч зажим Recordings от мыши нейронов сетчатки в темно-адаптированной фрагмент подготовка

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus

Подготовка фрагментов, органотипической культивирования тканей и люциферазы Запись часы активность генов в супрахиазматическое ядро

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the ...

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

Несколько мыши нейроанатомической Магнитно-резонансная томография

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

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

Изображений нейронов Ответы на фрагмент Препараты вомероназального орган выражения генетически закодированные Кальций датчика

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within ...

Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

Подготовка тканей и Иммуноокрашивание Мыши чувствительные нервные волокна, иннервирующие кожу и кости конечностей

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in ...

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

Подготовка Острый субвентрикулярной фрагменты зона для кальция изображений

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

Экс Vivo Препараты неповрежденной вомероназального органа и аксессуаров обонятельная луковица

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: Острая взрослом мозге нарезки в физиологической температуре

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments ...