Authors would like to thank Peter Mombaerts for the generous gift of OR-GFP mice; Anne Lefranc and the CSGA animal facility for excellent animal care. Funding was provided by CNRS through an ATIP and ATIP Plus grants, by Conseil R&#233;gional de Bourgogne (FABER and PARI grants), by Universit&#233; de Bourgogne (BQR program).

Bu protokol Université de Bourgogne hayvan bakım kuralları takip ve Université de Bourgogne etik kurul tarafından onaylandı. 1. Hayvanlar <ol><li> Jackson Laboratuvarı&#39;nda mevcut genetik olarak OR-IRES-tauGFP fareler kullanın. Bu fareler koku alma sisteminin 19 akson hedefleme ve gelişimini analiz etmek için Dr. Peter Mombaerts &#39;laboratuvarda geliştirilmiştir. Örneğin, MOR23-IRES-tauGFP hattı, stok numarası 006643, resmi gerilme adı B6 taşır; 129P2- Olfr16 tm2Mom / MomJ; Benzer şekilde, SR1-IRES-tauGFP hattı, stok numarası 6717 resmi adı B6 taşır; 129P2- Olfr124 tm1Mom / MomJ. </li><li> Hayvanların yaşı ile ilgili olarak: protokolünün daha iyi bir sonuç için, yaş 2 ve 4 hafta arasında hayvanları kullanın. Bu yaş grubunda, diseksiyon daha kolay (yumuşak kemikler, sıkı koku epiteli) ve dendritik topuzlar bi olmasıdırgger yaşlı hayvanlarda karşılaştırın. </li></ol> Elektrotlar ve Çözümleri 2. Hazırlık <ol><li> Uyarıcı pipetler için: Satın alma pipetler uyarıcı prepulled. Aksi takdirde, elle onları hazırlamak. <ol><li> Bir alevi kullanarak, ucundan yaklaşık 1 cm altı 1 mm cam pipetler bükün. Bir delikten tarafından güçlendirmek 1,5 cm ısıyla daralan boru bu altı pipetler artı düz 7 inci takın. </li><li> Isı birlikte ekli varil korumak için boru küçültmek. Varil diğer ucuna ek bir ısı shrink boru takın. Çok varil çektirmesi ile bu uyarıcı pipet çekin. </li><li> Bükülmüş ipuçları güçlendirmek için deliğinden etrafında bazı beyaz sıvı tutkal ekleyin. Kuru O / N edelim. </li></ol></li><li> Normal Ringer hücre dışı solüsyon, 1 veya 2 L hazırlayın (mM olarak: NaCl 124, KCI 3, MgSO 4 1.3, CaCl2 2, NaHCO 3 26 NaH 2 PO 4 1.25, glükoz 15; pH 7.6, 305 mOsm). 4 ° C&#39;de tutunkullanılıncaya kadar. </li><li> (MM olarak) hücre içi bir stok çözelti hazırlayın: KCI 70, KOH 53, metansülfonik asit, 30 EGTA 5, HEPES 10, sukroz 70; pH 7.2 (KOH) ve 310 mOsm. Kullanılana kadar 4 ° C &#39;de muhafaza edin. Birkaç hafta için iyi. </li><li> Deneyden önce (son dakikada) ekstemporane nystatin ile hücre içi kayıt çözeltisi hazırlayın. <ol><li> DMSO 50 ul ekleyin, nistatin 3 mg tartılır; tamamen seyreltilmiş kadar vorteks 20 sn sonra 2-3 dakika sonikasyon. </li><li> Hücre içi stok çözeltisi, 5 ml DMSO-nistatin çözeltisi 20 ul ekle. Vortex 20 sn, daha sonra 3 dakika sonikasyon. 4 ° C &#39;de, bu çözüm tutun ve doğrudan ışıktan korumak, nistatin ışığa duyarlıdır. Bu çözelti, birkaç saat süre ile kullanılabilir. Her gün değiştirin. </li><li> Koku epiteli hazırlık mikroskop altında sonra, elektrotlar doldurmak için bir alev uzatılmış sarı ucu ile 1 ml şırınga içinde bu çözümün bir kısmını almak; Doğrudan ışıktan korumak. Oda sıcaklığında, nistatin çözelti bir kezsabit değildir; her saat şırınga veya buz tutmak ml, 1 çözüm değiştirin. </li></ol></li><li> İç nystatin çözeltisi ile 15-20 M? Bir direnç ile uzun boyun ve küçük bir ipucu (~ 2 mikron) elde etmek için bir çektirmenin ile kayıt elektrotları çekin. </li><li> Davlumbaz altında DMSO içinde 0.5 M de odorant çözeltisi hazırlayın; eş-payı ve kullanılana kadar -20 ° C&#39;de saklayın. Istenen konsantrasyona kadar, Ringer çözeltisi içinde Deodorantını seyreltin. Uyarıcı pipet doldurun. </li></ol> Koku Epitelinin 3. hazırlanması Not: TD-IRES-tauGFP fareler ya da promotörün kontrolü altında tauGFP ifade eder. Bu farelerde, ilgi OR ifade tüm nöronlar GFP ile etiketlenmiş. Bu protokol tüm bölgeler ifade ameliyathanelerinde için uyarlanmıştır. Ancak, disseksiyonları ve kayıtlar dorsal bölgesinde ifade ameliyathanelerinde için daha kolaydır. <ol><li> Ketamin ve ksilazin (150 mg / kg ve 10 mg / kg Yönetim Kurulu karışımı enjekte edilerek hayvan anestezisiy ağırlık). Dekapitasyon genç farelerde ya da yaşlı fareler için uygun bir şekilde muhafaza kemirgen giyotinle birlikte keskin bir makasla gerçekleştirilebilir. <ol><li> Kullanımı halka diseksiyon makas dorsal deri yoluyla uzunlamasına bir medial kesi yapmak. Bunu dışında çekerek cildi çıkartın. Makas kullanarak, çene eklemi alt çene kesti. Dişlere koronal kesim paralel olarak üst ön dişleri çıkarın. </li><li> Gözlerinin arkasında başın bir koronal kesim olun ve başın sadece ön kısmını tutun. Kapsamında diseksiyon için buz Ringer çözeltisi içinde bunu batırın. </li></ol></li><li> Kapsamında ayır: ventral tarafında, üst çene / diş boyunca uzunlamasına kesim yapmak. Burun boşluğunun dorso-yan tarafında takip, uzunlamasına sırt kemikleri kesin. En kemikleri ve damak kaldır; oda sıcaklığında oksijenli Ringer buna bağlı septum ve epitel aktarın. </li><li> Son diseksiyon için: Sağ kayda başlamadan önceoturum forseps ile altta yatan septum gelen epitel uzak soyun. Forseps ile ve yapışma güçlü septum ön kısmında iki 4-5 mm makas darbeleri (kullanım microvannas makas) ile epitel ayırın. <ol><li> Dikkatle septal epiteline dorsal bağlantısı boyunca dışarı keserek vomeronasal organı çıkarın. Yukarı bakacak şekilde mukus tabakası ile bir kayıt odasına epitel aktarın; Bir harp ile odasında düz tutun. </li></ol></li><li> Floresan optik ve hassas bir kamera ile donatılmış dik bir mikroskop altında odasını takın. 40X su daldırma hedefi (sayısal açıklık 0.8) ve bir büyüteç ile elde ekstra bir 2X veya 4X büyütme ile yüksek büyütmede bilgisayar ekranında hazırlık gözünüzde canlandırın. Oda sıcaklığında oksijenli Ringer (1-2 ml / dakika) sürekli serpmek. </li></ol> 4. Kayıt Oturumu <ol><li> Floresan hücreye Ara: 480 de hazırlık heyecanlandırmak530-550 nm aralığında ışık yayar EGFP nm; floresan ve parlak bir alan altında güvenilir görünür tek dendritik topuzu hedef. </li><li> Nystatin ile hücre içi çözelti ile elektrot doldurun; hafifçe elektrot üzerinde dokunarak kabarcıkları. </li><li> Elektrot tutucu elektrot yerleştirin pipet pozitif basınç uygulayın; Direnç 15-20 M? Olmalıdır. </li><li> Hücreye yakın elektrot getirin; Direnç ~ 40 M? ulaştığında, pozitif basınç serbest bırakın ve bir gigaseal ulaşmak için hafif negatif basınç uygulayın. </li><li> Conta ulaşıldığında, yaklaşık -75 mV zar potansiyeli ayarlanır; </li><li> Hücre açıldıktan sonra, stimülasyon protokolleri, farmakolojik tedaviler ile devam edin. Tek bir odorant stimülasyon, kayıt spontan aktivite 200-500 milisaniye kadar yanıtı ölçmek için, 500 milisaniye boyunca canlandırmayı ve 10 saniye 13 hücre yanıtı ölçmek. Farmakolojik tedaviler için de farmakolojik ajanlar serpmekİstenen bir konsantrasyon, 20. </li></ol> 5. Veri Analizi <ol><li> Olarak takip Odorant uyarılmasıyla ortaya akımları analiz: (msn maksimum genlik% 90 ulaşmak için gerekli zaman,) maksimum genlik, yükselme zamanı, toplam akım (PAS eğrinin altında kalan alan), zaman% 50 (başlangıcı ve msn&#39;de, azami amplitüd% 50 karşılık mahsup arasındaki süre). <ol><li> Çizip Hill denklemi kullanarak bir doz yanıt eğrisi ayarı, farklı konsantrasyonda (maksimum amplitüd ulaştı) tepe iletim akımları kullanılarak: I = Imax / (1 ​​+ (K 02/01 / C) n-i) tepe akımını temsil eder, , maksimum cevabın yarısı erişildiği K1 / 2 konsantrasyonu, C odorant konsantrasyonu ve n Hill katsayısı doyurarak konsantrasyonlarda maksimum cevap IMAX. </li></ol></li><li> Maksimum depolarizasyon için geçerli kelepçe tota membran potansiyeli kayıtları analizl potansiyeli (mVsec eğrinin altında kalan alan) ortaya çıkardı; 30 sn 1 dk kayıtları üzerinde rekor spontan spike aktivitesi; akımlar (5-10 pA) enjeksiyonu ile ortaya sivri aracılığıyla kayıt heyecanlanma. </li></ol>

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The authors declare that they have no competing financial interest.

doğru, sağlıklı OSNs özelliklerini izlemek için bu protokolün yeteneği hazırlık kalitesine büyük ölçüde bağlıdır. Bu nedenle, diseksiyon adımlar önemlidir. Önce kalite (pH, osmolarite), oksijenasyon ve sıcaklık (buz-soğuk ama dondurulmuş değil) diseksiyon orta dikkat etmek önemlidir. İkincisi, diseksiyon araçları ile epitel manipülasyon olarak zarar görmemesi için mümkün olduğunca sınırlı olmalıdır. Son olarak, mümkün olduğunca büyük OSN popülasyonu erişmek amacıyla, müm...

Koku alma duyu nöronları (OSN) koku algı ilk adımı temsil etmektedir. Kemirgenlerde burun boşluğunu çevreleyen koku epiteli bulunan beyinle kendi akson yoluyla gönderilen aksiyon potansiyelleri içine koku kimyasal bilgi dönüşümü. Daha iyi koku kodlama mekanizmaları anlamak için, OSNs iletimi ve membran özelliklerini karakterize etmek gereklidir. Yakın zamana kadar, memeli OSNs özelliklerini karakterize etmek için kullanılan teknikler çok ayrışmış OSNs 1-4 üzerinde gerçekleştirilmiştir. ayrışma süreci çevrelerinden OSNs boşaltmak için çeşitli mekanik ve kimyasal (yani enzimler) süreçleri kullanır. Bu işlemler, kayıtlar için uygun bir hücre, düşük uyarmaktadır. Bu az sayıda GFP etiketli hücrelerin durumunda daha da kritik öneme sahip olabilir. Aynşması da OSNs ve Surviv artırabilir koku epitel başka hücreler arasındaki yerel hücre-hücre etkileşimleri ortadan kaldırırEl ve OSNs &#39;özelliklerinin modülasyonu. Ayrışma işlemini atlamak için, sağlam bir preparat 5 geliştirilmiştir. Her OSN büyük bir çoklu gen ailesi 6 arasından seçildiği (OR), bir koku reseptörü ifade eder. Fare ana koku epiteli olarak ifade 1,000 OR ~ vardır. Nedeniyle vahşi tip hayvanlarda OR çok sayıda, büyük ihtimalle aynı ifade OSNs kayıt YA çok düşük etmek. Bu sınırlamaları aşmak için, gen hedeflenen fareler mevcut olduğu bir tespit VEYA floresan proteini 7-9 ile etiketlenmiş ifade tüm OSNs. Bu etiketli OSNs ayrışmış hazırlıkları Daha önce de belirttiğimiz sakıncaları ile 7,10,11 fonksiyonel analiz yapmak için kullanıldı. Genetik etiketli farelerden alınan sağlam bir epitel hazırlık 5 Dolayısıyla bu sorunları circumvents. Vi yakın olarak bir ortamda tam olarak tanımlanmış ORs ifade OSNs aktivitesi izlenmesine olanak sağlarmümkün olduğunca vo. Ayrıca, OSNs patch-kelepçe kayıtları da zar özellikleri, iletim yolu farmakoloji, ligand / VEYA etkileşimlerinin hassas analiz sağlar. Bütün bu konular pek dışı kayıtları kullanılarak analiz edilebilir. Biz koku reseptörü SR1 ve MOR23 12,13 ifade OSNs yanıtları izlemek için bu tekniği kullanılır. teknik fizibilite nöronlar 15,16 ifade OSNs 14 eksprese eden MOR23 üzerinde diğer gruplar yanı sıra, diğer ORs ile teyit edilmiştir. OSNs tanımlanmış bir nüfus izleme 17 yaşlanma, böyle bir gelişme 14 gibi pek çok farklı bağlamlarda özelliklerinin analizi yol açabilir, odorant indüklenen plastisite 18 ve 15 kodlama koku odorant reseptörün dizilimindeki rolü. Bu protokol, böylece zar düzeyinde tanımlanan OSNs fonksiyonel özelliklerini izlemek için güçlü bir araç sağlar.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td colspan="4">heavy equipment</td>
		</tr>
		<tr>
			<td>vibration table with Faraday cage</td>
			<td>TMC</td>
			<td>63-500 SERIES</td>
			<td>required : isolates the recording system from vibrations induced by the environment (movements of experimenter, vibrations of equipment such as fans for cooling computers, etc); can also be purchased with a Faraday cage, or equipped by a custom made Faraday cage; this cage is recommended to avoid electric noise from the environment</td>
		</tr>
		<tr>
			<td colspan="4">optics</td>
		</tr>
		<tr>
			<td>microscope</td>
			<td>Olympus</td>
			<td>BX51WI</td>
			<td>upright microcope equipped with epifluorescence; fixed or moving stage depending on the user&#39;s preference</td>
		</tr>
		<tr>
			<td>objectives</td>
			<td>Olympus</td>
			<td>LUMPLFL40XW</td>
			<td>at least 2 objectives required: a 4x or 10x for coarse approach to the cell; and a 40x immersion long distance example Olympus LUMPLFL40XW/IR/0,8/WD:3.3 MM</td>
		</tr>
		<tr>
			<td>magnifier</td>
			<td>Olympus</td>
			<td>U-TVCAC</td>
			<td>ABSOLUTELY REQUIRED: placed in the light path between the objective and the camera; allows to magnify the image on the screen in order to reach precisely the knob with the recording electrode</td>
		</tr>
		<tr>
			<td>camera</td>
			<td>Olympus</td>
			<td>DP72</td>
			<td>a good camera is required to see the neurons in fluorescence as well as in bright field; the controlling software is simple and allows to take pictures and do live camera image to monitor the approach of the electrode to the cell. An ultrasensitive camera is not necessary</td>
		</tr>
		<tr>
			<td>filters</td>
			<td>Olympus/Chroma</td>
			<td></td>
			<td>depending on the fluorescent protein used in the mice; example for GFP: excitation : BP460-490: emission: HQ530/50m</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">recording electrodes /system</td>
		</tr>
		<tr>
			<td>amplifier</td>
			<td>HEKA</td>
			<td>EPC10 USB</td>
			<td>monitors the currents flowing through the recording electrode and also controls the puffing by sending a TTL signal to the spritzer; the EPC10 setup is controled by computer</td>
		</tr>
		<tr>
			<td>software</td>
			<td>HEKA</td>
			<td>Patchmaster</td>
			<td>controls the amplifier during the experiment</td>
		</tr>
		<tr>
			<td>micromanipulator</td>
			<td>Sutter</td>
			<td>MP225</td>
			<td>precision micromanipulator, allows precise movements down to 1/1Oth of a micrometer; this model is very stable; avoid hydraulic manipulators that may drift</td>
		</tr>
		<tr>
			<td>electrode puller</td>
			<td>sutter</td>
			<td>P97</td>
			<td>with a FT345-B wide trough filament;&#160; to prepare recording pipets of about 2&#181;m diameter with a long tip to reach the cells; the resistance should be 15 to 20Mohm with perforated patch clamp solution</td>
		</tr>
		<tr>
			<td>glass</td>
			<td>sutter</td>
			<td>BF120-69-10</td>
			<td>in our recording conditions, this glass is ideal for recording pipets</td>
		</tr>
		<tr>
			<td>recording chamber</td>
			<td>warner instruments</td>
			<td>RC-26G</td>
			<td>a chamber is needed to set the preparation under the microscope. To maintain the preparation in the center of the chamber, a net/anchor should be used.</td>
		</tr>
		<tr>
			<td colspan="4">stimulation</td>
		</tr>
		<tr>
			<td>glass</td>
			<td>WPI</td>
			<td>TW100F-4</td>
			<td>attached in groups of 7, these pipettes are used to prepare prepulled stimulating pipettes</td>
		</tr>
		<tr>
			<td>multibarrel puller</td>
			<td>MDI</td>
			<td>PMP-107-Z</td>
			<td>by association of pull and twist, this puller allows us to prepare puffing electrodes with 7 barrels</td>
		</tr>
		<tr>
			<td>precision pressure injector&#160;</td>
			<td>Toohey Company</td>
			<td>P/N T25-1-900 Single Channel&#160;&#160;&#160;</td>
			<td>this precision pressure injector&#160; controls the pressure ejected in the multibarrel puller; it is controlled manually or by the amplifier by a 5V&#160; TTLs</td>
		</tr>
		<tr>
			<td>micromanipulator</td>
			<td>Narishige</td>
			<td>YOU-1</td>
			<td>a coarse manipulator is enough to bring the puffing electrode close to the recording site</td>
		</tr>
		<tr>
			<td>tubings</td>
			<td>N/A</td>
			<td></td>
			<td>tygon tubing to bring the pressure from the puffer to the puffing pipette</td>
		</tr>
		<tr>
			<td colspan="4">solutions/perfusion/chemicals</td>
		</tr>
		<tr>
			<td>vacuum pump</td>
			<td>gardner denver</td>
			<td>300 series</td>
			<td>a vibrating membrane pump is more quiet and efficient than other types of pumps</td>
		</tr>
		<tr>
			<td>perfusion system</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>gravity perfusion system with polyethlylen tubing to bring in and out the external solution from the recording chamber</td>
		</tr>
		<tr>
			<td>nystatin</td>
			<td>Sigma-Aldrich</td>
			<td>N3503</td>
			<td>mandatory to perpare internal solution for perforated patch clamp</td>
		</tr>
		<tr>
			<td>DIMETHYL SULFOXIDE</td>
			<td>Sigma-Aldrich</td>
			<td>D5879</td>
			<td>used to disolve nystatin for internal solution for perforated patch&#160;</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S9625</td>
			<td>extracellular solution</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P4504</td>
			<td>intracellular/extracellular solution</td>
		</tr>
		<tr>
			<td>Calcium chloride di hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C7902</td>
			<td>extracellular solution</td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic monohydrate (NaH2PO4)</td>
			<td>Sigma-Aldrich</td>
			<td>S9638</td>
			<td>extracellular solution</td>
		</tr>
		<tr>
			<td>Magnesium sulfate heptahydrate (MgSO4 7H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>63140</td>
			<td>extracellular solution</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270</td>
			<td>extracellular solution</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S6297</td>
			<td>extracellular solution</td>
		</tr>
		<tr>
			<td>EGTA (Ethylene glycol-bis(2-aminoethylether)-N,N,N&#8242;,N&#8242;-tetraacetic acid)</td>
			<td>Sigma-Aldrich</td>
			<td>E3889</td>
			<td>internal solution</td>
		</tr>
		<tr>
			<td>Potassium hydroxyde</td>
			<td>Sigma-Aldrich</td>
			<td>P1767</td>
			<td>internal solution</td>
		</tr>
		<tr>
			<td>MethylSulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>47,135-6</td>
			<td>intracellular solution</td>
		</tr>
		<tr>
			<td>Hepes-Na</td>
			<td>Sigma-Aldrich</td>
			<td>H7006</td>
			<td>intracellular solution</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S0389</td>
			<td>intracellular solution</td>
		</tr>
	</tbody>
</table>

perforated patch-clamp recording of mouse olfactory sensory neurons in intact neuroepithelium: functional analysis of neurons expressing an identified odorant receptor

Özel bağ ile uyarıldıkları zaman koku duyu nöronlarının fizyolojik tepkilerini (OSN) analiz koku tahrikli davranışları ve modülasyon temelini anlamak önemlidir. Bu kodlama özellikleri koku molekülleri ve koku reseptörü (OR) arasındaki ilk etkileşim ağır bağımlı OSNs olarak ifade edilmiştir. ifade YA kritik kimliği, özgünlüğü ve ligand spektrumu. OR ligandı bulmak için olasılık epitel içinde rastgele seçilen bir OSN çok düşüktür dile getirdi. Bu sorunu çözmek için, bu protokol, genetik olarak tanımlanmış ORs promoterinin kontrolü altında floresan protein GFP ifade isim levhası fareler kullanır. OSNs komşu hücrelerin olgunlaşması ve fonksiyonu etkileyen ile burun boşluğuna astar sıkı ve organize epitel yer almaktadır. Burada OSNs e özelliklerini sağlam bir koku epiteli izole ve patch-kelepçe kayıtları ile kaydetmek için bir yöntem tariftanımlanan koku reseptörü xpressing. protokol komşu dokuya etkisi tutarken tek OSN membran özelliklerini karakterize sağlar. Patch-kelepçe sonuçlarının analizi ligand / VEYA etkileşimleri, iletim yollarının ve farmakoloji, OSNs &#39;kodlama özellikleri ve membran düzeyde modülasyon kesin miktarının verir.

Bu protokolün sonuç diseksiyon kalitesine bağlıdır. Bu diseksiyon adımlar kısa (en az 10 dk 15) ve (yani, epitel zarar görmemesi için) kesin olmalıdır. Şekil 1 ideal hazırlık farklı büyütme seviyelerinde nasıl göründüğünü göstermektedir. Parlak saha altında düşük büyütme (bu tür hücreleri desteklemek OSNs tokmakları gibi), farklı hücre tipleri ayırt (Şekil 1A) &#39;dir. En yüksek büyütme düzeyinde, genellikle 80X 160x, aydınlık alanda...

Watch this Scientific Journal Video about Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant

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

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 ...

perforated-patch-clamp-recording-mouse-olfactory-sensory-neurons

Research

JoVE Journal

Neuroscience

26.6K Görüntüleme.  Université de Bourgogne. 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.

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

Analyzing Responses of Mouse Olfactory Sensory Neurons Using the Air-phase Electroolfactogram Recording

Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and other social interactions. The electroolfactogram (EOG) recording is an informative, easy to conduct, and reliable method to assay olfactory function at the level of the olfactory epithelium. Since the 1956 description of the EOG by Ottoson in frogs1, the EOG recording has been applied in many vertebrates including salamanders, rabbits, rats, mice, and humans (reviewed by Scott and Scott-Johnson, 2002, ref. 2). The recent advances in genetic modification in mice have rekindled interest in recording the EOG for physiological characterization of olfactory function in knock-out and knock-in mice. EOG recordings have been successfully applied to demonstrate the central role of olfactory signal transduction components3-8, and more recently to characterize the contribution of certain regulatory mechanisms to OSN responses9-12.
Odorant detection occurs at the surface of the olfactory epithelium on the cilia of OSNs, where a signal transduction cascade leads to opening of ion channels, generating a current that flows into the cilia and depolarizes the membrane13. The EOG is the negative potential recorded extracellularly at the surface of the olfactory epithelium upon odorant stimulation, resulting from a summation of the potential changes caused by individual responsive OSNs in the recording field2. Comparison of the amplitude and kinetics of the EOG thus provide valuable information about how genetic modification and other experimental manipulations influence the molecular signaling underlying the OSN response to odor. 
Here we describe an air-phase EOG recording on a preparation of mouse olfactory turbinates. Briefly, after sacrificing the mouse, the olfactory turbinates are exposed by bisecting the head along the midline and removing the septum. The turbinate preparation is then placed in the recording setup, and a recording electrode is placed at the surface of the olfactory epithelium on one of the medial turbinates. A reference electrode is electrically connected to the tissue through a buffer solution. A continuous stream of humidified air is blown over the surface of the epithelium to keep it moist. The vapor of odorant solutions is puffed into the stream of humidified air to stimulate the epithelium. Responses are recorded and digitized for further analysis.

The electroolfactogram (EOG) recording is an informative, easy-to-conduct, and reliable way of assessing olfactory function at the level of the olfactory epithelium. This protocol describes a recording setup, mouse tissue preparation, data collection, and basic data analysis.

Hava-faz Electroolfactogram Kayıt Fare Koku Duyu Nöronlar analizi Yanıtları

Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and ...

Nörobilim

Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory  Neurons

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. Caged compounds are molecules made physiologically inactive by a chemical cage that can be broken by a flash of ultraviolet light. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged compounds for the study of olfactory transduction in dissociated mouse olfactory sensory neurons. The process of olfactory transduction (Figure 1) takes place in the cilia of olfactory sensory neurons, where odorant binding to receptors leads to the increase of cAMP that opens cyclic nucleotide-gated (CNG) channels2. Ca entry through CNG channels activates Ca-activated Cl channels. We show how to dissociate neurons from the mouse olfactory epithelium3 and how to activate CNG channels or Ca-activated Cl channels by photolysis of caged cAMP4 or caged Ca5. We use a flash lamp6,7 to apply ultraviolet flashes to the ciliary region to uncage cAMP or Ca while patch-clamp recordings are taken to measure the current in the whole-cell voltage-clamp configuration8-11.

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged cAMP or caged Ca for the study of olfactory transduction in dissociated mouse olfactory sensory neurons.

Koku Duyu Nöronlar Cilia Kafesli Bileşiklerin Flash Fotoliz

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. ...

Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex behaviors such as food choice, predator, conspecific and mate recognition and other socially relevant cues. Olfactory receptor neurons (ORNs) are located in the dorsal part of the nasal cavity embedded in the olfactory epithelium. These bipolar neurons send an axon to the olfactory bulb (see Fig. 1, Reisert &amp; Zhao1, originally published in the Journal of General Physiology) and extend a single dendrite to the epithelial border from where cilia radiate into the mucus that covers the olfactory epithelium. The cilia contain the signal transduction machinery that ultimately leads to excitatory current influx through the ciliary transduction channels, a cyclic nucleotide-gated (CNG) channel and a Ca2+-activated Cl- channel (Fig. 1). The ensuing depolarization triggers action potential generation at the cell body2-4.
In this video we describe the use of the "suction pipette technique" to record odorant-induced responses from ORNs. This method was originally developed to record from rod photoreceptors5 and a variant of this method can be found at jove.com modified to record from mouse cone photoreceptors6. The suction pipette technique was later adapted to also record from ORNs7,8. Briefly, following dissociation of the olfactory epithelium and cell isolation, the entire cell body of an ORN is sucked into the tip of a recording pipette. The dendrite and the cilia remain exposed to the bath solution and thus accessible to solution changes to enable e.g. odorant or pharmacological blocker application. In this configuration, no access to the intracellular environment is gained (no whole-cell voltage clamp) and the intracellular voltage remains free to vary. This allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body9. The difference in kinetics between these two signals allows them to be separated using different filter settings. This technique can be used on any wild type or knockout mouse or to record selectively from ORNs that also express GFP to label specific subsets of ORNs, e.g. expressing a given odorant receptor or ion channel.

Olfactory receptor neurons (ORNs) convert odor signals first into a receptor current that in turn triggers action potentials that are conveyed to second order neurons in the olfactory bulb. Here we describe the suction pipette technique to record simultaneously the odorant-induced receptor current and action potentials from mouse ORNs.

Emme Pipet Tekniği kullanılarak Olfaktör Reseptör Nöronlar Kaydedilmiş odorant bağlı Cevapları

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

Retrograd Floresan Etiketleme Belirlenen Nöronlar gelen Hedefli Hücre dışı Tek ünitesi Kayıt izin verir<em&gt; In vivo</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be performed in an intact brain preparation where the neural circuitry of the CNS remains intact. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with green fluorescent protein for identification in the intact brain. Fish have multiple populations of GnRH neurons, and their functions are dependent on their location in the brain and the GnRH gene that they express1 . We have focused our demonstration on GnRH3 neurons located in the terminal nerves (TN) associated with the olfactory bulbs using the intact brain of transgenic medaka fish (Figure 1B and C). Studies suggest that medaka TN-GnRH3 neurons are neuromodulatory, acting as a transmitter of information from the external environment to the central nervous system; they do not play a direct role in regulating pituitary-gonadal functions, as do the well-known hypothalamic GnRH1 neurons2, 3 .The tonic pattern of spontaneous action potential firing of TN-GnRH3 neurons is an intrinsic property4-6, the frequency of which is modulated by visual cues from conspecifics2 and the neuropeptide kisspeptin 15. In this video, we use a stable line of transgenic medaka in which TN-GnRH3 neurons express a transgene containing the promoter region of Gnrh3 linked to enhanced green fluorescent protein7 to show you how to identify neurons and monitor their electrical activity in the whole brain preparation6.

In this video, we will demonstrate how to record electrical activity from identified single neurons in a whole brain preparation, which preserves complex neural circuits. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with a fluorescent protein for identification in the intact brain preparation.

Transgenik Balık Sağlam Beyin Tespit Edilen nöronlar Elektrik Aktivitesi Kayıt

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be ...

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

Duyusal nöronlar izolasyonu<em&gt; Aplysia californica</em&gt; Glutamaterjik Akımlar Patch Kelepçe Kayıtlar için

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of neurophysiology, neuroethology, and developmental biology. The goal of this report is to consolidate experimental techniques from the leech system into a single article that will be of use to physiologists with expertise in other nervous system preparations, or to biology students with little or no electrophysiology experience. We demonstrate how to dissect the leech for recording intracellularly from identified neural circuits in the ganglion. Next we show how individual cells of known function can be removed from the ganglion to be cultured in a Petri dish, and how to record from those neurons in culture. Then we demonstrate how to prepare a patch of innervated skin to be used for mapping sensory or motor fields. These leech preparations are still widely used to address basic electrical properties of neural networks, behavior, synaptogenesis, and development. They are also an appropriate training module for neuroscience or physiology teaching laboratories.

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

This article describes three nervous system preparations using leeches: intracellular recording from neurons in ventral ganglia, culturing neurons from ventral ganglia, and recording from a patch of innervated skin to map sensory fields.

Hücre içi Kaydı, Duyu Alan Haritalama ve Sülük de Kültüre Belirlenen Nöronlar Hirudo medicinalis</i

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of ...

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 ...

Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. Retinal neurons in the network comprise of numerous subtypes. More than 10 subtypes of bipolar cells, ganglion cells, and amacrine cells have been identified by morphological studies. Multiple subtypes of retinal neurons are thought to encode distinct features of visual signaling, such as motion and color, and form multiple neural pathways. However, the functional roles of each neuron in visual signal processing are not fully understood. The patch clamp method is useful to address this fundamental question. Here, a protocol to record light-evoked synaptic responses in mouse retinal neurons using patch clamp recordings in dark-adapted conditions is provided. The mouse eyes are dark-adapted O/N, and retinal slice preparations are dissected in a dark room using infrared illumination and viewers. Infrared light does not activate mouse photoreceptors and thus preserves their light responsiveness. Patch clamp is used to record light-evoked responses in retinal neurons. A fluorescent dye is injected during recordings to characterize neuronal morphological subtypes. This procedure enables us to determine the physiological functions of each neuron in the mouse retina.

We will demonstrate how to prepare retinal slices from the mouse eye and record light responses in retinal neurons. The entire procedure is conducted in dark-adapted conditions.

Kayıt Patch Kelepçe Teknikleri Kullanarak Koyu adapte Fare Retina Dilim Hazırlıkları Nöronlar içinde postsinaptik Yanıtları Işık-uyarılmış

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. ...

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic tectum, is a popular model to study how neural circuits self-assemble. The ability to carry out whole cell patch clamp recordings from tectal neurons and to record RGC-evoked responses, either in vivo or using a whole brain preparation, has generated a large body of high-resolution data about the mechanisms underlying normal, and abnormal, circuit formation and function. Here we describe how to perform the in vivo preparation, the original whole brain preparation, and a more recently developed horizontal brain slice preparation for obtaining whole cell patch clamp recordings from tectal neurons. Each preparation has unique experimental advantages. The in vivo preparation enables the recording of the direct response of tectal neurons to visual stimuli projected onto the eye. The whole brain preparation allows for the RGC axons to be activated in a highly controlled manner, and the horizontal brain slice preparation allows recording from across all layers of the tectum.

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

In this paper, we discuss three brain preparations used for whole cell patch clamp recording to study the retinotectal circuit of Xenopus laevis tadpoles. Each preparation, with its own specific advantages, contributes to the experimental tractability of the Xenopus tadpole as a model to study neural circuit function.

Ürünleri ve tüm hücre yama kelepçe kayıt Xenopus laevis Tectal nöronlar için iletişim kuralları

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic ...