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).

Ce protocole suit les directives de protection des animaux de l&#39;Université de Bourgogne et a été approuvé par le comité d&#39;éthique de l&#39;Université de Bourgogne. 1. Les animaux <ol><li> Utilisez la souris OU-IRES-tauGFP génétiquement disponibles au Laboratoire Jackson. Ces souris ont été développés dans le laboratoire du Dr Peter Mombaerts afin d&#39;analyser le ciblage et le développement des axones du système olfactif 19. Par exemple, la ligne MOR23-IRES-tauGFP, numéro de stock 006643, porte le nom officiel de la souche B6; 129P2- Olfr16 tm2Mom / MomJ; de même, la ligne SR1-IRES-tauGFP, stock number 6717 porte le nom B6 officielle; 129P2- Olfr124 tm1Mom / MomJ. </li><li> En ce qui concerne l&#39;âge des animaux: pour un meilleur résultat du protocole, utiliser des animaux entre 2 et 4 semaines d&#39;âge. Dans ce groupe d&#39;âge, la dissection est plus facile (os plus doux, plus ferme épithélium olfactif) et les boutons dendritiques sont bigger comparer aux animaux plus âgés. </li></ol> 2. Préparation des électrodes et Solutions <ol><li> Pour les pipettes de stimulation: achat prepulled stimuler pipettes. Sinon, les préparer manuellement. <ol><li> Utiliser une flamme, pliez six pipettes en verre de 1 mm à environ 1 cm de la pointe. Insérez ces six pipettes droites plus un 7 ème dans 1,5 cm gaine thermorétractable renforcer par un oeillet. </li><li> Thermorétractable tube pour maintenir les barils attachés ensemble. Fixez un tube thermorétractable additionnelle à l&#39;autre extrémité des barils. Tirez cette pipette stimulant avec un extracteur multi-canon. </li><li> Ajouter un peu de colle liquide blanc autour de l&#39;oeillet de renforcer les conseils pliés. Laisser sécher O / N. </li></ol></li><li> Préparez 1 ou 2 L de solution extracellulaire normale Ringer (en mM: NaCl 124, KCl 3, MgSO4 1,3, CaCl2 2, NaHCO 3 26, NaH 2 PO 4 1,25, le glucose 15; pH 7.6 et 305 mOsm). Conserver à 4 ° Cjusqu&#39;à utilisation. </li><li> Préparer une solution stock intracellulaire (en mM): KCl 70, 53 KOH, l&#39;acide méthane 30, EGTA 5, HEPES 10, 70 saccharose; pH 7,2 (KOH) et 310 mOsm. Conserver à 4 ° C jusqu&#39;à utilisation. Conseillé pour plusieurs semaines. </li><li> Préparer la solution intracellulaire d&#39;enregistrement avec nystatine extemporanée (à la dernière minute) avant l&#39;expérience. <ol><li> Peser 3 mg de nystatine, ajouter 50 pi de DMSO; vortex 20 sec Soniquer puis 2-3 min jusqu&#39;à entièrement diluée. </li><li> Ajouter 20 pi de solution de DMSO-nystatine dans 5 ml de solution de réserve intracellulaire. Vortex 20 sec, puis Soniquer 3 min. Gardez cette solution à 4 ° C et de protéger de la lumière directe, la nystatine est sensible à la lumière. Cette solution peut être utilisée pendant quelques heures. Remplacer tous les jours. </li><li> Une fois la préparation de l&#39;épithélium olfactif est sous le microscope, prendre un peu de cette solution dans une seringue de 1 ml avec une pointe jaune flamme allongée pour remplir les électrodes; protéger de la lumière directe. Une fois à température ambiante, la solution de nystatineest pas stable; remplacer la solution dans la seringue de 1 ml toutes les heures ou le garder dans la glace. </li></ol></li><li> Tirez électrodes d&#39;enregistrement avec un extracteur d&#39;obtenir à long cou et petit pourboire (~ 2 um) avec une résistance de 15-20 MQ avec la solution de nystatine interne. </li><li> Préparer la solution odorant à 0,5 M dans le DMSO sous une hotte; aliquote et de garder à -20 ° C jusqu&#39;à utilisation. Diluer substance odorante dans la solution de Ringer jusqu&#39;à la concentration désirée. Remplissez la pipette stimulant. </li></ol> 3. Préparation de l&#39;épithélium olfactif Remarque: souris OU-IRES-tauGFP expriment la tauGFP sous le contrôle de l&#39;OU promoteur. Chez ces souris, tous les neurones exprimant l&#39;OU d&#39;intérêt sont étiquetés avec la GFP. Ce protocole est adapté pour les RUP exprimées dans toutes les zones. Cependant, les dissections sont plus faciles et les enregistrements de RUP exprimées dans la zone dorsale. <ol><li> Anesthésier l&#39;animal par injection d&#39;un mélange de kétamine et de xylazine (150 mg / kg et 10 mg / kg DBOpoids y, respectivement). Décapitation peut être effectuée avec des ciseaux pointus pour les jeunes souris ou avec une guillotine de rongeur bien entretenu pour les souris âgées. <ol><li> Aide de la bague des ciseaux de dissection font une incision médiane longitudinale à travers la peau du dos. Retirer la peau en le tirant à part. En utilisant les ciseaux, couper les mâchoires inférieures à l&#39;articulation de la mâchoire. Retirez les incisives supérieures par une coupe coronale parallèle aux dents. </li><li> Faire une coupe coronale de la tête derrière les yeux et de ne conserver que la partie antérieure de la tête. Tremper dans une solution de Ringer glacée pour la dissection sous la portée. </li></ol></li><li> Disséquer sous le champ d&#39;application: dans la face ventrale, faire une coupe longitudinale le long de la mâchoire supérieure / les dents. Couper les os dorsale longitudinalement, suivant la partie dorso-latérale de la cavité nasale. Retirer la plupart des os et du palais; transférer le septum et l&#39;épithélium attaché à elle dans oxygéné Ringer à RT. </li><li> Pour la dissection final: Juste avant de commencer l&#39;enregistrementsession, décoller l&#39;épithélium de la cloison sous-jacent avec une pince. Détachez l&#39;épithélium avec une pince et avec deux coupes 4-5 mm de ciseaux (utilisation microvannas ciseaux) à la partie antérieure de la cloison où l&#39;adhérence est plus forte. <ol><li> Retirez délicatement l&#39;organe voméronasal par découpe le long de sa connexion dorsale à l&#39;épithélium septale. Transfert de l&#39;épithélium à une chambre d&#39;enregistrement avec la couche de mucus vers le haut; garder à plat dans la chambre avec une harpe. </li></ol></li><li> Installez chambre sous un microscope droit équipé d&#39;une optique de fluorescence et une caméra sensible. Visualisez la préparation sur l&#39;écran d&#39;ordinateur à fort grossissement 40X par un objectif immersion dans l&#39;eau (ouverture numérique de 0,8) et un 2X ou 4X grossissement supplémentaire atteint par une lentille grossissante. Perfuser continu avec oxygéné Ringer à RT (1-2 ml / min). </li></ol> 4. Enregistrement de session <ol><li> Rechercher cellule fluorescente: exciter la préparation à 480nm pour EGFP, qui émet de la lumière dans la gamme 530-550 nm; cibler un seul bouton dendritique qui est fiable visible dans fluorescence et sous champ lumineux. </li><li> Remplir l&#39;électrode avec une solution intracellulaire à la nystatine; éliminer les bulles en tapotant doucement sur l&#39;électrode. </li><li> Insérez électrode sur un support d&#39;électrode, appliquer une pression positive dans la pipette; La résistance doit être 15-20 MQ. </li><li> Apportez électrode proche de la cellule; une fois la résistance atteint ~ 40 MQ, relâcher la pression positive et appliquer une légère pression négative pour atteindre un gigaseal. </li><li> Une fois joint est atteint, réglez le potentiel de membrane à environ -75 mV; </li><li> Une fois que la cellule est ouverte, procéder à des protocoles de stimulation, les traitements pharmacologiques. Pour mesurer la réponse à une stimulation odorante unique, fiche 200-500 ms de l&#39;activité spontanée, stimuler pour 500 ms et mesurer la réponse de la cellule pour un maximum de 10 sec 13. Pour les traitements pharmacologiques, perfuser les agents pharmacologiques àla 20 concentration souhaitée. </li></ol> 5. Analyse des données <ol><li> Analyser les courants induits par la stimulation odorante comme suit: l&#39;amplitude maximale, le temps de montée (temps nécessaire pour atteindre 90% de l&#39;amplitude maximale, en ms), le courant total (aire sous la courbe à pas), le temps à 50% (temps entre le début et le décalage de la réponse à 50% de l&#39;amplitude maximale, en ms). <ol><li> En utilisant les courants de pointe de transduction (amplitude maximale atteinte) à différentes concentrations, dessiner et ajuster une courbe dose-réponse en utilisant l&#39;équation de Hill: I = Imax / (1 ​​+ (K 1/2 / C) n), où je représente le courant de crête , Imax la réponse maximale à des concentrations de saturation, K1 / 2, la concentration à laquelle la moitié de la réponse maximale a été atteinte, C la concentration de substance odorante et n le coefficient de Hill. </li></ol></li><li> Analyser les enregistrements de potentiel de membrane en pince ampèremétrique pour la dépolarisation maximale, la total potentiel suscité (aire sous la courbe dans mVsec); enregistrent l&#39;activité de dopage spontanée en 30 sec à 1 min enregistrements; fiche excitabilité travers pointes provoquées par injection de courants (5-10 Pa). </li></ol>

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The authors declare that they have no competing financial interest.

La capacité de ce protocole pour surveiller correctement les propriétés des ARS en bonne santé dépend fortement de la qualité de la préparation. Par conséquent, les étapes de dissection sont critiques. D&#39;abord, il est essentiel de prêter attention à la qualité (pH, osmolarité), l&#39;oxygénation et la température (glace-froid mais non congelé) du milieu de dissection. Deuxièmement, la manipulation de l&#39;épithélium avec des outils de dissection doit être aussi limité que possible pour éviter ...

Les neurones sensoriels olfactifs (OSN) représentent la première étape de la perception olfactive. Situé dans l&#39;épithélium olfactif tapissant la cavité nasale chez les rongeurs, ils transforment les informations chimique de substances odorantes dans des potentiels d&#39;action envoyés par leurs axones dans le cerveau. Pour mieux comprendre les mécanismes de codage olfactif, il est nécessaire de caractériser les propriétés de transduction et membranaires de ARS. Jusqu&#39;à récemment, la plupart des techniques utilisées pour caractériser les propriétés des ARS de mammifères ont été effectuées sur dissocié ARS 4.1. Le processus de dissociation utilise divers (c.-à-enzymes) des procédés mécaniques et chimiques pour libérer les ARS de leur environnement. Ces processus induisent un faible nombre de cellules disponibles pour les enregistrements. Ce faible nombre peut être encore plus critique dans le cas des cellules GFP étiqueté. La dissociation supprime également les interactions locales de cellule à cellule entre les ARS et d&#39;autres cellules de l&#39;épithélium olfactif qui peuvent améliorer SURVIVal et la modulation des propriétés de ARS. Afin de contourner la procédure de dissociation, une préparation intacte 5 a été développé. Chaque OSN exprime un récepteur olfactif (OU) choisi parmi une grande famille multigénique 6. Il ya ~ 1000 RUP exprimées dans la principale épithélium olfactif chez la souris. En raison du grand nombre d&#39;OR dans les animaux de type sauvage, les chances d&#39;enregistrer ARS exprimant le même OU sont très faibles. Pour surmonter ces limitations, les souris de gènes ciblés sont disponibles dans lequel tous les ARS exprimant une personne identifiée ou sont étiquetés avec une protéine fluorescente 7-9. Ces ARS marquées ont été utilisés pour faire l&#39;analyse fonctionnelle dans les préparations dissociées 7,10,11 avec les inconvénients mentionnés plus haut. Une préparation de l&#39;épithélium intact 5 de souris génétiquement marqués contourne donc sur ces questions. Il permet le suivi de l&#39;activité des ARS exprimant RUP précisément définies dans un environnement aussi près dans vivo que possible. En outre, les enregistrements de patch-clamp de ARS permettent également une analyse précise des propriétés de la membrane, transduction voie pharmacologie, ligand / ou des interactions. Tous ces sujets peuvent difficilement être analysées à l&#39;aide des enregistrements extracellulaires. Nous avons utilisé cette technique pour surveiller les réponses des ARS exprimant les récepteurs olfactifs SR1 et MOR23 12,13. La faisabilité de la technique a été confirmée par d&#39;autres groupes sur MOR23 exprimant ARS 14 ainsi que sur les autres RUP exprimant neurones 15,16. Le suivi d&#39;une population définie de ARS peut conduire à l&#39;analyse de leurs propriétés dans de nombreux contextes différents tels que le développement 14, 17 vieillissement, odorant induite plasticité 18, et le rôle des variations de la séquence du récepteur olfactif de l&#39;odeur de codage 15. Ce protocole fournit ainsi un outil puissant pour surveiller les propriétés fonctionnelles des ARS définies au niveau de la membrane.

<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

Analysant les réactions physiologiques des neurones sensoriels olfactifs (OSN) lorsqu&#39;elles sont stimulées par des ligands spécifiques est essentiel de comprendre la base de comportements olfactifs moteur et leur modulation. Ces propriétés de codage dépendent fortement de l&#39;interaction initiale entre les molécules d&#39;odeur et le récepteur olfactif (OU) exprimées dans les ARS. L&#39;identité, la spécificité et le ligand spectre du exprimés ou sont critiques. La probabilité de trouver le ligand de l&#39;OU exprimé dans un OSN choisi au hasard dans l&#39;épithélium est très faible. Pour relever ce défi, ce protocole utilise des souris génétiquement marquées exprimant la protéine fluorescente GFP sous le contrôle du promoteur des RUP définies. ARS sont situés dans un épithélium serré et organisé garniture de la cavité nasale, avec les cellules voisines influencer leur maturation et la fonction. Nous décrivons ici une méthode pour isoler un épithélium olfactif intact et enregistrer par patch-clamp enregistrements les propriétés des ARS expressing récepteurs olfactifs définies. Le protocole permet de caractériser les propriétés de la membrane OSN tout en maintenant l&#39;influence du tissu voisin. Analyse des résultats de patch-clamp donne une quantification précise de ligand / ou des interactions, des voies de transduction et de la pharmacologie, les propriétés de codage de ARS et leur modulation au niveau de la membrane.

Le résultat de ce protocole dépend de la qualité de la dissection. Cette dissection étapes doivent être courts (moins de 10 à 15 min) et précis (par exemple, pour éviter les dommages de l&#39;épithélium). La figure 1 illustre comment une préparation idéale ressemble à différents niveaux de grossissement. A un faible grossissement sous champ lumineux les différents types de cellules (comme les poignées de ARS, des cellules de soutien) se distinguent (figure 1A). ...

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.

Perforé patch-clamp enregistrement des souris neurones sensoriels olfactifs dans neuroépithélium Intact: Analyse fonctionnelle des neurones exprimant un récepteur odorant Identifié

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

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JoVE Journal

Neuroscience

26.6K vues.  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.

Vidéo: 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.

Réponses Analyse des souris neurones sensoriels olfactifs Utilisation de l&#39;enregistrement Electroolfactogram Air-phase

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

Recherche

Neurosciences

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.

Photolyse des composés en cage dans les cils olfactifs des neurones sensoriels

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.

Odorant induites réponses enregistrées à partir de neurones récepteurs olfactifs en utilisant la technique de la pipette d&#39;aspiration

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.

Marquage fluorescent rétrograde Permet ciblée extracellulaire seule unité d&#39;enregistrement de neurones identifiés<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.

Enregistrement de l&#39;activité électrique de neurones identifiés dans le cerveau intact des poissons transgéniques

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.

Isolation des neurones sensoriels de<em&gt; Aplysia californica</em&gt; Patch pour les enregistrements de serrage des courants glutamatergiques

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.

Intracellulaire enregistrement, le mappage des champs sensorielle, et la culture de neurones identifiés dans le Leech, 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.

Tout le mont immunomarquage des récepteurs olfactifs neurones dans le<em&gt; Drosophila</em&gt; Antenne

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.

Enregistrement Light-réponses évoquées dans les neurones post-synaptiques dans Dark-adaptés, Préparations rétine de la souris tranche en utilisant des techniques de patch clamp

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.

Préparations et protocoles pour toute cellule Patch Clamp enregistrement des neurones Tectal Xenopus laevis

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