Name: whole mount labeling of cilia in the main olfactory system of mice
Uploaded: 2014-12-27T14:00:00.000+00:00
Duration: 8 min 42 s
Description: Watch this Scientific Journal Video about Whole Mount Labeling of Cilia in the Main Olfactory System of Mice at JoVE.com

This work was funded by the Deutsche Forschungsgemeinschaft DFG (Exc257, SFB958).

NOT: Tüm hayvan prosedürleri hayvanların herhangi bir gereksiz acı kaçınarak Alman Hayvan Bakım yasalarına uygun olarak Charité veya Üniversite Kliniği Jena ele alındı. 1. hazırlanması Çözümler ve Diseksiyon İşyeri <ol><li> Çözümler NOT: epitel diseksiyonu başlamadan önce aşağıdaki çözümleri hazırlayın. <ol><li> Diseksiyon işlemi için Çözümler: <ol><li> 140 mM NaCI, 5 mM KCI, 10 mM HEPES, 2 mM CaCI2, 1 mM MgCI2 ve 10 mM glukoz konsantrasyonları ile Ringer çözeltisi (pH 7.4) içinde hazırlayın. </li><li> - / - 2.68 mM KCI, 1.47 mM KH 2 PO 4, 136 mM NaCI, 8.1 mM Na-2 HPO 4 konsantrasyonlar çözeltisi (pH 7.4) içinde bir PBS hazırlayın. </li><li> - / -, 0.2 mM CaCI2,% 4 sukroz ve% 4 paraformaldehid 1x PBS konsantrasyonları ile bir tespit çözeltisi (pH 7.2) ile hazırlayın. Sabitleştirici çözelti içinde sukroz kriyo artırırkesit ve pick-up cryosections arasında. -20 ° C&#39;de saklayın. </li></ol></li><li> Boyama işlemi için çözümler <ol><li> 1x PBS konsantrasyonları ile PBS + / + çözeltisi hazırlayın - / -, 0.48 mM MgCl2, 0.9 mM CaCl2. </li><li> 1x PBS + / + ve% 0.1 Triton X-100 konsantrasyonları ile, PBST + / + çözelti hazırlayın. </li><li> 1x PBST ile + / + ve% 1 jelatin konsantrasyonları ile bir blokaj çözeltisi hazırlayın. </li></ol></li></ol></li><li> Işyeri <ol><li> Diseksiyon işyeri için, parlak aydınlatma ile bir diseksiyon mikroskobu, yanı sıra sıvı-bloker kalem kullanın. </li><li> Ringer çözeltisi, ve yapışkan cam lamlar ile doldurulmuş bir petri kabı, hazırlanır. </li><li> Aşağıdaki cerrahi aletler elde: cerrahi makas keskin ve bir künt ucu, düz uç şekli ile yaylı makas, ince kavisli ucu şekli ve bir jilet ile iki forseps bir çift (olan <strong&gt; Şekil 1A). </li></ol></li></ol> Burun Septumu 2. Hazırlık <ol><li> Gıda ve su ve uygun gündüz / gece döngüsü düzenli erişimi onaylı kafeslerde ev hayvanları. </li><li> Arka ayak refleksleri test ederek% 100 izofluran ve monitör analjezi ile ıslatılmış bir kapalı hazne içeren gazlı bez anestezi gerçekleştirin. Servikal dislokasyon burun boşlukları kan neden olabildiğinden, doğrudan anestezi uygulanmış farelerin başını kesmek. </li><li> Tüm kafatası ve burun kemiği açığa çıkarmak için deri kaldırmak ve iyice bir kağıt havlu kullanarak kalan kan ve doku silin. Alt çene ve ön dişleri çıkarın. </li><li> Maksilla (yanal) den septum (medial) bir tarafını ayırmak için sütür hattına 1-2 mm mesafe paralel bilateral burun dorsal kemik İnsizyon. Tek bir kesim ile burun bölün. Kemik kalıntıları ve konka hala septum takılı ise, dikkatlice çıkarın tamamen septum maruzdokunmadan. </li><li> Septum ve burun kemiği arasında, septum dorsal kenarı boyunca bir forseps ince kavisli ucu kaydırarak dorsal nazal kemiği çıkarın. , Kemik üzerinde hafif basınç uygulayın yukarı itin ve çıkarın. </li><li> Her bir oyuğun iki septum dokulara erişime bir sağlamak için dikkatli bir şekilde baş diğer tarafında maksilla çıkarın. Büyük hayvanlar (&gt; 28 P) hazırlanırken, koku ampuller kapsayan frontal kemik ucu çıkarın. </li><li> Onun hafif sarı renk (Şekil 1B) tarafından koku epiteli belirleyin. sınırındaki solunum epiteli beyaz ve mikroskop altında görülebilir hareketli kirpikler hareketini gösterir. İnce bahar makasla ile koku ve solunum epiteli arasındaki sınır boyunca kesin. </li><li> Kesim uzatın ve vomer kemik ventral bağlantıdan septum ayırın. Sonra septum ve Os etmoidit lamina cribrosa arasındaki sınır boyunca kesilmişör. septum artık tamamen kafasına bütün bağlantıları izole edilir. Şekil 1C gösterilen kesme konumlarını kullanın. </li></ol> Immün için Koku Epitelinin 3. İzolasyonu <ol><li> Ringer çözeltisi ile doldurulmuş bir petri yapışkan bir cam slayt yerleştirin. Camın bir yarım Ringer çözeltisi (Şekil 1A) &#39;de yer alan şekilde, bir petri kenarına yerleştirin. </li><li> Dikkatle petri koku epiteli ile etmoid kemik aktarmak için bir forseps kullanın. Forseps ipuçları ile kapma olmadan kaldırın. </li><li> Bir forseps ile etmoid kemiğin dik plaka tut ve dikkatli septum bir taraftan epitel kaldırmak için ikinci bir birini kullanın. Epitel kalkmasına dik plaka ve epiteli arasındaki kavisli ucu kaydırın. </li><li> Epitel Ringer soluti çevirir durumunda, siliyer tarafını belirlemek her zaman emin olunüzerine. Epitel çevirir ise, sonradan siliyer tarafı belirlemek pek mümkün değildir. Çoğunlukla, epitel içindeki silier yüzeyi ile rulo. </li><li> Şekil 1C gösterilen kesme pozisyonları ile kayıtlı doku şeklini karşılaştırın. Sınırda bir pozisyonda epitel tut ve cam slayt üzerine çekin. Doku lezyonları önlemek için forseps ile siliyer tarafına dokunmayın. </li><li> Septum çevirin ve diğer taraftan epitel ile işlemi tekrarlayın. </li><li> Bir kağıt havlu ile koku epiteli iki adet etrafında cam slayt kurulayın ve bir sıvı-bloker kalemle doku çevreler. </li><li> Oda sıcaklığında 10 dakika boyunca 150 ul sabitleyici çözeltisi ile doku sabitleyin. Kirpikler stabilite ve bütünlüğü ile ilgili olarak, test edilen antikorların çeşitli kısa sabitleme kez kullanmak. Bu 10 dakika kirpikler içinde sabit, ama muhtemelen bütün epitel dokusu vardır. Bu durumda, hemen s sonra mikroskop ile görselprosedürü yükümlüsünüzdür. Ancak, sabitleyici katı bireysel antikorlar için değişiklik gösterebilir. </li></ol> 4. Boyama Protokolü NOT: koku alma duyu nöronlarının siliyer yapıları korumak için çok dikkatli doku tutun. Pipetleme çözüm çıkarın. Mekanik kuvvetler ince kirpikler bozabilir gibi, epiteli üzerine doğrudan herhangi bir çözüm düşürmeyin. Başka türlü belirtilmediği sürece, oda sıcaklığında bütün adımları. <ol><li> Sabitleştirici çözüm çıkarın ve PBS + / + 2 kez yıkayın. </li><li> Bloke etme çözeltisi ile en az 1 saat süre ile epitel inkübe edin. </li><li> Herhangi bir çökelti kaldırmak için tam hızda 5 dakika boyunca (bu çalışmada kullanılan antikorlar için 200 1) ve santrifüj çözeltisi bloke antikor eritilmesi ile primer antikor çözeltisi hazırlayın. </li><li> 4 ° CO / K bir nemli oda içinde antikor çözeltisi ile epitel inkübe edin. </li><li> PBS + / + için 5 dakika ile yıkayın epitel en az 3 kez. </li><li>Tam hızda 5 dakika boyunca ve santrifüj: çözeltisi (1: 500) bloke antikor eritilmesi ile ikincil antikor çözeltisi hazırlayın. </li><li> Bir karanlık oda içinde 1 saat süre ile ikinci antikor çözeltisi ile epitel inkübe edin. </li><li> PBS + / + için 5 dakika ile yıkayın epitel en az 3 kez. </li><li> Bir jilet veya kağıt havlu ile sıvı-bloker çıkarın. 5 saniye süreyle damıtılmış su ile epitel yıkayın. </li><li> Antifade montaj reaktif doku korur. NOT: gün içinde hızlı bir şekilde arka plan flüoresan artar; en geç 2 gün içerisinde montaj ortamda gömme sonra mikroskobik analiz nedenle tavsiye edilir. Özel bakım Doğru odak düzlemi ararken ciddi hazırlık zarar verebilir, çünkü doku sıkmak için değil alınmalıdır. Nedeniyle out-of-odak floresan, konfokal mikroskobu ile daha fazla incelenmesi tavsiye edilir. </li></ol>

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The authors have nothing to disclose.

The en face preparation technique described in this protocol provides a powerful tool for the detailed analysis of the olfactory system. So far, most studies characterizing the localization of signaling proteins use immunostainings of cryosections. They present a good overview of the olfactory epithelium, and protein expression in distinct cell types or regions can be easily identified. However, expression in olfactory cilia is sometimes hard to detect. Even if ciliary localization is obvious, cryosections offer...

burun boşluğunda koku epiteli fare 6-10.000.000 bipolar koku duyu nöronları 1 içerir. Her koku nöron ifade 1.200 koku reseptör gen birini seçer. Koku tespiti daha sonra 4 olf koku spesifik G protein Gα üzerinden adenilat siklaz tip III (ACIII) 3 aktive bir koku reseptörü 2, bağlanma odorant başlar. siklik adenozin monofosfat (cAMP) &#39;de elde edilen artış açan bir nükleotid kapılı seçici olmayan katyon kanalı Ca2 + ve Na +, ve daha sonra, Ca girişine yol, (CNG) 2 + akışı, bir Ca + 2 aktif Cl açılmasına neden olan siklik - Kanal 5,6. dışarı doğru Cı Elde - akışı, yüksek hücre içi Cl kolaylaştırılır -, Na + / K + / Cl ile olası emme -, - ortak taşıyıcı NKCC1, konsantrasyon sabit Cl muhafazaCI - / HCO3 - değiştirici SLC4A1 ve belki ek henüz tespit edilmesi taşıyıcıları 6-8. Bipolar koku nöronlar tek dalsız koku ampul doğrudan proje akson ve epitel yüzeyine uzanan ve özel bölmesi, dendritik topuzu gibi biten bir dendrit vardır. Kadar um 50-60 kadar bir uzunluğa ulaşabilir, bu topuz, 10-30 kirpikler itibaren, epitel yüzeyi 9 kaplayan mukus içine sızmak. Kanonik sinyal iletimi akışının Proteinler ağırlıklı bu kirpikler zarında lokalize edilmektedir. epitel artan duyusal yüzey Koku moleküllerini tespit etme yeteneğine güçlendirir. Nedeniyle duyusal nöronların yoğunluğunun, komşu dendritik kolları uzanan kirpikler içe. Epitelyumunun yüzeyi üzerine, koku reseptörlerinin farklı ifade eden farklı nöron gelen kirpikler rastgele karışımı içinde bu karışmasının sonucu. algılama ve hücreduyusal nöronların bir alt kümesi, sadece mevcut siliyer proteinlerin nitlerin ayırma cryosections de zordur. Halinde kriyoseksiyonlar kirpikler ortalama uzunluğundan daha tipik olarak daha ince olduğu için ek olarak, kirpikler boyunca bu proteinlerin kesin yeri, hemen hemen mümkün değildir. Koku nöronlarda kadar uncharacterized membran proteinlerinin siliyer lokalizasyonu soruşturma etkinleştirmek için, biz kirpikler protein lokalizasyonu detaylı analizini sağlayan bir tr yüz hazırlama tekniklerini optimize. Kısaca, fare kurban ve baş orta hatta yakın bölünür. Kıkırdakçıklar burun ve frontal kemik septum maruz bırakmak için çıkarılır. astar epitel koku kısmı ile septum burun boşluğuna tüm bağlantıları keserek gevşetilir. Ringer çözeltisi ile doldurulmuş bir petri kabına septum konulduktan sonra, epitel kaplanmış bir cam slayt aktarılır und soyulur. Kısa bir fixat ardındantaşıma kırılgan doku hasarı önlemek için mümkün olduğunca nazik ise iyon adım, immün işlemleri yapılabilir. Biz klasik cryosections ve tarif tr yüz hazırlık koku kirpikler iki farklı membran proteinlerinin boyama karşılaştırarak elde çözünürlük göstermektedir.

<table><tbody><tr><td>Spring scissors</td><td></td><td></td><td>straight tip, multiple suppliers</td></tr><tr><td>Surgical scissors</td><td></td><td></td><td>sharp and blunt end, multiple suppliers</td></tr><tr><td>Fine forceps</td><td></td><td></td><td>curved tips, Dumont #7, multiple suppliers</td></tr><tr><td>Razor blade</td><td></td><td></td><td>extra thin, multiple suppliers</td></tr><tr><td>Binocular with illumination</td><td></td><td></td><td>multiple suppliers, Stemi 2000-C, Zeiss</td></tr><tr><td>Petri dish</td><td></td><td></td><td>multiple suppliers</td></tr><tr><td>Liquid-blocker pen</td><td>Science Services</td><td>N71310</td><td></td></tr><tr><td>Polysine coated slides</td><td>Thermo Scientific</td><td>J2800AMNZ</td><td></td></tr><tr><td>Confocal microscope</td><td>Leica Microsystems</td><td>TCS SPE</td><td></td></tr><tr><td>primary antibody Goat anti-Kirrel2</td><td>R&amp;amp;D Systems</td><td>AF2930</td><td>1:200</td></tr><tr><td>primary antibody Rabbit anti-mOR-EG</td><td>Baumgart et al., 2014</td><td></td><td>1:200</td></tr><tr><td>secondary antibodies</td><td>Life Technologies</td><td>A21206, A11057</td><td>1:500</td></tr><tr><td>Mounting medium, ProLong Gold antifade reagent</td><td>Life Technologies</td><td>P36930</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>441244</td><td>toxic, work under fume hood</td></tr></tbody></table>

whole mount labeling of cilia in the main olfactory system of mice

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single dendrite to the surface of the epithelium, ending in a structure called dendritic knob. Cilia emanate from this knob into the mucus covering the epithelial surface. The proteins of the olfactory signal transduction cascade are mainly localized in the ciliary membrane, being in direct contact with volatile substances in the environment. For a detailed understanding of olfactory signal transduction, one important aspect is the exact morphological analysis of signaling protein distribution. Using light microscopical approaches in conventional cryosections, protein localization in olfactory cilia is difficult to determine due to the density of ciliary structures. To overcome this problem, we optimized an approach for whole mount labeling of cilia, leading to improved visualization of their morphology and the distribution of signaling proteins. We demonstrate the power of this approach by comparing whole mount and conventional cryosection labeling of Kirrel2. This axon-guidance adhesion molecule is known to localize in a subset of sensory neurons and their axons in an activity-dependent manner. Whole mount cilia labeling revealed an additional and novel picture of the localization of this protein.

Yüz hazırlıkları tr Koku epitel olan yerelleştirme cryosections analizinden sonra belirsizdir proteinlerin detaylı soruşturma izin, duyusal nöronların kirpikler proteinlerin yerelleştirme incelemek için kullanılabilir. Bu sorun, Irre benzeri protein 2 (Kirrel2) olarak Kin için boyama durumunda örnek olarak gösterilebilir. (Aynı zamanda Neph3 olarak da adlandırılır) Kirrel2 bir homofilik yapışma proteini olarak zar proteinleri ve fonksiyonların immünoglobulin (Ig) süper ailesinin...

Watch this Scientific Journal Video about Whole Mount Labeling of Cilia in the Main Olfactory System of Mice at JoVE.com

Whole Mount Labeling of Cilia in the Main Olfactory System of Mice

Cilia of olfactory sensory neurons contain proteins of the signal transduction cascade, but a detailed spatial analysis of their distribution is difficult in cryosections. We describe here an optimized approach for whole mount labeling and en face visualization of ciliary proteins.

Fareler Ana Koku Sistemde Cilia Whole Dağı Etiketleme

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single ...

whole-mount-labeling-of-cilia-in-the-main-olfactory-system-of-mice

Research

JoVE Journal

Neuroscience

11.1K Görüntüleme.  Universitaetsklinikum Jena. Cilia of olfactory sensory neurons contain proteins of the signal transduction cascade, but a detailed spatial analysis of their distribution is difficult in cryosections. We describe here an optimized approach for whole mount labeling and en face visualization of ciliary proteins.

Video: Whole Mount Labeling of Cilia in the Main Olfactory System of Mice

Simple and Computer-assisted Olfactory Testing for Mice

Olfaction is highly conserved among species and is required for reproduction and survival.
In humans, olfaction is also one of the senses that is affected with aging and is a strong predictor of neurodegenerative diseases. Thus, olfaction testing is used as a non-invasive diagnostic method to detect neurological deficits early on. In order to understand the mechanisms underlying olfactory network susceptibility, olfactory research in rodents has gained momentum in the past decade.
Here, we present a very simple, time efficient and reproducible olfactory testing method of innate odor perception and sensitivity in mice without the need of any prior food or water restriction. The tests are performed in a familiar environment to the mice, require only the scents and a 2 min session of odorant exposure. The analysis is performed, post-hoc, using computer-assisted commands on ImageJ and can be, therefore, carried out from start to end by one researcher.
This protocol does not require any special hardware or setup and is indicated for any laboratory interested in testing olfactory perception and sensitivity.

We present a simple and unbiased olfactory test in mice. With this protocol olfactory discrimination, preference, avoidance and sensitivity to a novel odor as compared to water can be assessed in single behavioral sessions. This method is indicated for a single experimenter and analysis is based on computer-assisted video processing.

Basit ve Bilgisayar destekli Fareler için Koku Testi

Olfaction is highly conserved among species and is required for reproduction and survival.
In humans, olfaction is also one of the senses that is affected ...

Davranış

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.

In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO). Here, an acute tissue slice preparation of VNO for performing calcium imaging is described. This physiological approach allows observations of subpopulations and/or individual neurons in a living tissue and is convenient for receptor-ligand identification.

Görüntüleme Feromon Fare Vomeronasal Akut Doku Dilim Hazırlama içinde Algılama

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. ...

Nörobilim

Imaging Odor-Evoked Activities in the Mouse Olfactory Bulb using Optical Reflectance and Autofluorescence Signals

In the brain, sensory stimulation activates distributed populations of neurons among functional modules which participate to the coding of the stimulus. Functional optical imaging techniques are advantageous to visualize the activation of these modules in sensory cortices with high spatial resolution. In this context, endogenous optical signals that arise from molecular mechanisms linked to neuroenergetics are valuable sources of contrast to record spatial maps of sensory stimuli over wide fields in the rodent brain.
Here, we present two techniques based on changes of endogenous optical properties of the brain tissue during activation. First the intrinsic optical signals (IOS) are produced by a local alteration in red light reflectance due to: (i) absorption by changes in blood oxygenation level and blood volume (ii) photon scattering. The use of in vivo IOS to record spatial maps started in the mid 1980's with the observation of optical maps of whisker barrels in the rat and the orientation columns in the cat visual cortex1. IOS imaging of the surface of the rodent main olfactory bulb (OB) in response to odorants was later demonstrated by Larry Katz's group2. The second approach relies on flavoprotein autofluorescence signals (FAS) due to changes in the redox state of these mitochondrial metabolic intermediates. More precisely, the technique is based on the green fluorescence due to oxidized state of flavoproteins when the tissue is excited with blue light. Although such signals were probably among the first fluorescent molecules recorded for the study of brain activity by the pioneer studies of Britton Chances and colleagues3, it was not until recently that they have been used for mapping of brain activation in vivo. FAS imaging was first applied to the somatosensory cortex in rodents in response to hindpaw stimulation by Katsuei Shibuki's group4.
The olfactory system is of central importance for the survival of the vast majority of living species because it allows efficient detection and identification of chemical substances in the environment (food, predators). The OB is the first relay of olfactory information processing in the brain. It receives afferent projections from the olfactory primary sensory neurons that detect volatile odorant molecules. Each sensory neuron expresses only one type of odorant receptor and neurons carrying the same type of receptor send their nerve processes to the same well-defined microregions of &tilde;100&mu;m3 constituted of discrete neuropil, the olfactory glomerulus (Fig. 1). In the last decade, IOS imaging has fostered the functional exploration of the OB5, 6, 7 which has become one of the most studied sensory structures. The mapping of OB activity with FAS imaging has not been performed yet.
Here, we show the successive steps of an efficient protocol for IOS and FAS imaging to map odor-evoked activities in the mouse OB.

This article presents the protocols of intrinsic optical signals and flavoproteins autofluorescence signals imaging to map odor-evoked activities at the surface of the olfactory bulb in mice.

Optik Yansıtma ve Otofloresans Sinyalleri kullanarak, Fare Koku Ampul Görüntüleme Koku-Uyarılmış Etkinlikler

In the brain, sensory stimulation activates distributed populations of neurons among functional modules which participate to the coding of the stimulus. ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

In Koku Reseptör Nöronlar Tüm Dağı ile immün<em&gt; Drosophila</em&gt; Anten

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

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.

Bozulmamış neuroepithelium Fare Koku Duyu Nöronlar Delikli Patch-kelepçe Kayıt: Bir Belirlenen Odorant Reseptör ifade Nöronlar Fonksiyonel Analiz

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

Tüm Dağı Diseksiyon ve Yetişkin Fare Kohlea immünofloresan

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory Sensory Neuron (OSN) is a bipolar cell with a single dendrite and a single unbranched axon. An OSN expresses only one Olfactory Receptor (OR) gene, OSNs expressing a given type of OR converge their axons to a few sets of invariant glomeruli in the Olfactory Bulb (OB). A remarkable feature of OSN projection is that the expressed ORs play instructive roles in axonal projection. ORs regulate the expression of multiple axon-sorting molecules and generate the combinatorial molecular code of axon-sorting molecules at the OSN axon termini. Thus, to understand the molecular mechanisms of OR-specific axon guidance mechanisms, it is vital to characterize their expression profiles at the OSN axon termini within the same glomerulus. The aim of this article was to introduce methods for collecting as many glomeruli as possible on a single OB section and for performing immunostaining using multiple antibodies. This would allow the comparison and analysis of the expression patterns of axon-sorting molecules without staining variation between OB sections.

Olfactory sensory neurons express a wide variety of axon-sorting molecules to establish proper neural circuitry. This protocol describes an immunohistochemical staining method to visualize combinatorial expressions of axon-sorting molecules at the axon termini of olfactory sensory neurons.

Koku Meşfatının Olfaktör Duyusal Akson Moleküler Kimlik Kodlarının Görselleştirilmesi İçin Dörtlü İmmün Sulanması

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory ...

An Objective and Reproducible Test of Olfactory Learning and Discrimination in Mice

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and parenting. Importantly, mice can be trained to associate novel odors with specific behavioral responses to provide insight into olfactory circuit function. This protocol details the procedure for training mice on a Go/No-Go operant learning task. In this approach, mice are trained on hundreds of automated trials daily for 2&#8211;4 weeks and can then be tested on novel Go/No-Go odor pairs to assess olfactory discrimination, or be used for studies on how odor learning alters the structure or function of the olfactory circuit. Additionally, the mouse olfactory bulb (OB) features ongoing integration of adult-born neurons. Interestingly, olfactory learning increases both the survival and synaptic connections of these adult-born neurons. Therefore, this protocol can be combined with other biochemical, electrophysiological, and imaging techniques to study learning and activity-dependent factors that mediate neuronal survival and plasticity.

Here, we train mice on an associative learning task to test odor discrimination. This protocol also allows for studies on learning-induced structural changes in the brain.

Koku öğrenme ve ayrımcılık farelerde bir objektif ve tekrarlanabilir testi

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and ...

An Effective Manual Deboning Method To Prepare Intact Mouse Nasal Tissue With Preserved Anatomical Organization

The mammalian nose is a multi-functional organ with intricate internal structures. The nasal cavity is lined with various epithelia such as olfactory, respiratory, and squamous epithelia which differ markedly in anatomical locations, morphology, and functions. In adult mice, the nose is covered with various skull bones, limiting experimental access to internal structures, especially those in the posterior such as the main olfactory epithelium (MOE). Here we describe an effective method for obtaining almost the entire and intact nasal tissues with preserved anatomical organization. Using surgical tools under a dissecting microscope, we sequentially remove the skull bones surrounding the nasal tissue. This procedure can be performed on both paraformaldehyde-fixed and freshly dissected, skinned mouse heads. The entire deboning procedure takes about 20-30 min, which is significantly shorter than the experimental time required for conventional chemical-based decalcification. In addition, we present an easy method to remove air bubbles trapped between turbinates, which is critical for obtaining intact thin horizontal or coronal or sagittal sections from the nasal tissue preparation. Nasal tissue prepared using our method can be used for whole mount observation of the entire epithelia, as well as morphological, immunocytochemical, RNA in situ hybridization, and physiological studies, especially in studies where region-specific examination and comparison are of interest.

Korunan Anatomik Örgütü ile bozulmamış Fare Burun Doku hazırlamak için etkili bir Manuel kemik sıyırma Yöntemi

The mammalian nose is a multi-functional organ with intricate internal structures.  The nasal cavity is lined with various epithelia such as olfactory, ...

Biyoloji

Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and in situ Hybridization

Animals rely on chemical communication to convey and perceive relevant environmental information, ranging from assessment of food quality to detection of available mating partners or threats. In mice, this task is executed primarily by the olfactory system and its underlying subsystems, including the main and accessory olfactory systems. Both have peripheral organs populated by sensory neurons expressing G-protein coupled receptors able to bind chemical cues that reach the nasal cavity. Even though the molecular characteristics of these receptors is well understood, little is known about their cognate specific ligands. The method described here combines in situ hybridization detection of olfactory or vomeronasal receptors with immunodetection of phosphorylated ribosomal protein S6 (pS6) - a marker of neuronal activation. This protocol was devised to identify neurons activated after a single event of exposure to purified or complex chemical stimuli detected by the olfactory organs. Importantly, this technique allows the investigation of neurons triggered in biologically relevant contexts. Ideally, this method should be used to probe the molecular biology of the olfactory system and to study olfactory behaviors.

Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and in situ Hybridization

This protocol combines in situ hybridization with immunofluorescence to identify olfactory and vomeronasal receptor genes expressed in olfactory sensory neurons after activation by chemical stimuli in the mouse.

Animals rely on chemical communication to convey and perceive relevant environmental information, ranging from assessment of food quality to detection of ...

Fareler Ana Koku Sistemde Cilia Whole Dağı Etiketleme

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Basit ve Bilgisayar destekli Fareler için Koku Testi

Görüntüleme Feromon Fare Vomeronasal Akut Doku Dilim Hazırlama içinde Algılama

Optik Yansıtma ve Otofloresans Sinyalleri kullanarak, Fare Koku Ampul Görüntüleme Koku-Uyarılmış Etkinlikler

In Koku Reseptör Nöronlar Tüm Dağı ile immün

Bozulmamış neuroepithelium Fare Koku Duyu Nöronlar Delikli Patch-kelepçe Kayıt: Bir Belirlenen Odorant Reseptör ifade Nöronlar Fonksiyonel Analiz

Tüm Dağı Diseksiyon ve Yetişkin Fare Kohlea immünofloresan

Koku Meşfatının Olfaktör Duyusal Akson Moleküler Kimlik Kodlarının Görselleştirilmesi İçin Dörtlü İmmün Sulanması

Koku öğrenme ve ayrımcılık farelerde bir objektif ve tekrarlanabilir testi

Korunan Anatomik Örgütü ile bozulmamış Fare Burun Doku hazırlamak için etkili bir Manuel kemik sıyırma Yöntemi

Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and in situ Hybridization