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

NOTE: Toutes les procédures d&#39;animaux ont été traités à la Clinique universitaire Charité ou Iéna en accord avec les lois de protection des animaux allemands en évitant toute souffrance indue des animaux. 1. Préparation et Solutions Dissection travail <ol><li> Solutions NOTE: Préparer les solutions suivantes avant de commencer la dissection de l&#39;épithélium. <ol><li> Solutions pour la procédure de dissection: <ol><li> Préparer la solution de Ringer (pH 7,4) avec des concentrations de NaCl 140 mM, KCl 5 mM, HEPES 10 mM, CaCl2 2 mM, MgCl2 1 mM, et 10 mM de glucose. </li><li> Préparer une PBS - / - solution (pH 7,4) avec des concentrations de 2,68 mM de KCl, 1,47 mM de KH 2 PO 4, NaCl 136 mM et 8,1 mM de Na 2 HPO 4. </li><li> Préparer une solution de fixateur (pH 7,2) avec des concentrations de 1 x PBS - / -, 0,2 mM de CaCl2, 4% de saccharose, et 4% de paraformaldehyde. Le saccharose dans la solution de fixateur améliore cryosectionnement et pick-up de cryosections. Stocker à -20 ° C. </li></ol></li><li> Solutions pour la procédure de coloration <ol><li> Préparer un + / + PBS solution avec des concentrations de 1 x PBS - / -, 0,48 mM de MgCl2, 0,9 mM de CaCl2. </li><li> Préparer une PBST + / + solution avec des concentrations de 1 x PBS + / + et 0,1% de Triton X-100. </li><li> Préparer une solution de blocage avec des concentrations de 1 x PBST + / + et 1% de gélatine. </li></ol></li></ol></li><li> Lieu de travail <ol><li> Pour le lieu de travail de dissection, utiliser un microscope de dissection avec un éclairage lumineux, ainsi que d&#39;un stylo liquide-bloquant. </li><li> Préparer une boîte de Pétri, rempli de la solution de Ringer, et des lames de verre adhésives. </li><li> Obtenir les instruments chirurgicaux suivants: une paire de ciseaux chirurgicaux avec une forte et une pointe émoussée, une paire de ciseaux à ressort avec la forme de la pointe droite, deux pinces de forme de pointe recourbée fine et une lame de rasoir ( <strong&gt; La figure 1A). </li></ol></li></ol> 2. Préparation de la cloison nasale <ol><li> animaux de maison dans des cages approuvées ayant un accès régulier à la nourriture et de l&#39;eau et le cycle jour / nuit approprié. </li><li> Effectuer l&#39;anesthésie dans un récipient contenant de la gaze imbibée fermé avec 100% d&#39;isoflurane et le moniteur analgésie en testant les réflexes du pied arrière. Depuis la dislocation cervicale peut causer le sang dans les cavités nasales, décapiter directement souris anesthésiées. </li><li> Retirer la peau pour exposer l&#39;os de l&#39;ensemble du crâne et le nez, et essuyer le sang et le tissu restant soigneusement avec une serviette en papier. Retirer la mâchoire inférieure et les dents de devant. </li><li> Inciser l&#39;os du nez dorsale bilatéralement 1-2 mm de distance parallèlement à la ligne de suture pour séparer un côté de la cloison (médiane) de la mâchoire (latéralement). Diviser le nez avec une seule coupe. Si les restes d&#39;os et les cornets sont encore attachés à la cloison, retirez-les attentivement afin d&#39;exposer complètement le septumsans le toucher. </li><li> Retirez la dorsale os nasal en faisant glisser la pointe recourbée amende de une pince le long de la face dorsale de la cloison entre la cloison et l&#39;os nasal. Appliquer une légère pression sur l&#39;os, pousser vers le haut et le retirer. </li><li> Pour permettre l&#39;accès aux deux tissus du septum, un de chaque cavité, retirez délicatement le maxillaire de l&#39;autre côté de la tête. Lors de la préparation des animaux plus âgés (P&gt; 28), retirer la pointe de l&#39;os frontal couvrant les bulbes olfactifs. </li><li> Identifier l&#39;épithélium olfactif par sa couleur légèrement jaune (figure 1B). L&#39;épithélium respiratoire bordure est blanc et montre le mouvement des cils mobiles qui peut être vu sous le microscope de dissection. Coupez le long de la frontière entre l&#39;épithélium olfactif et respiratoire avec une paire de ciseaux fins de printemps. </li><li> Prolonger la coupe et séparer la cloison de la connexion ventrale à l&#39;os du vomer. Ensuite, couper le long de la frontière entre la cloison et la lame criblée de l&#39;ethmoïde Ose. Le septum est maintenant complètement isolé de toutes les connexions à la tête. Utilisez les positions de coupe illustrés à la figure 1C. </li></ol> 3. Isolement de l&#39;épithélium olfactif pour Immunocoloration <ol><li> Passer une lame de verre adhésif dans la boîte de Pétri remplie de la solution de Ringer. Placer sur le bord de la boîte de Pétri, de sorte que la moitié de la vitre se trouve en solution (Figure 1A) de Ringer. </li><li> Utilisez une pince pour transférer attentivement l&#39;ethmoïde avec l&#39;épithélium olfactif à la boîte de Pétri. Soulevez sans saisissant avec les forceps conseils. </li><li> Prenez la lame perpendiculaire de l&#39;ethmoïde avec une pince et d&#39;utiliser un deuxième pour retirer soigneusement l&#39;épithélium d&#39;un côté de la cloison. Faites glisser la pointe courbe entre la lame perpendiculaire et l&#39;épithélium à peler l&#39;épithélium off. </li><li> Soyez toujours sûr d&#39;identifier le côté ciliaire, en cas de l&#39;épithélium des culbutes dans la soluti de Ringersur. Si l&#39;épithélium retourne, il ne est guère possible d&#39;identifier le côté ciliaire après. La plupart du temps, l&#39;épithélium se enroule avec la surface à l&#39;intérieur ciliaire. </li><li> Comparer la forme du tissu inscrits avec les positions de coupe de la Figure 1C. Prenez l&#39;épithélium à une position à la frontière et tirez sur la lame de verre. Ne touchez pas la face ciliaire avec la pince pour éviter des lésions du tissu. </li><li> Tournez le septum et répéter la procédure avec l&#39;épithélium de l&#39;autre côté. </li><li> Sécher la lame de verre autour des deux morceaux de l&#39;épithélium olfactif avec une serviette en papier et entourer le tissu avec un stylo liquide-bloquant. </li><li> Fixer le tissu avec 150 ul solution de fixateur pendant 10 min à température ambiante. En ce qui concerne la stabilité et l&#39;intégrité cils, utiliser de courts délais de fixation pour une variété d&#39;anticorps testés. Au sein de ces 10 min cils sont fixés, mais probablement pas l&#39;ensemble du tissu de l&#39;épithélium. Ainsi, immédiatement visualiser au microscope après les scontenant procédure. Cependant, les temps peuvent varier fixants pour les anticorps individuels. </li></ol> 4. Protocole de coloration REMARQUE: Manipulez le tissu très soigneusement pour préserver les structures ciliaires des neurones sensoriels olfactifs. Retirer solutions par pipetage. Ne pas tomber des solutions directement sur l&#39;épithélium, que les forces mécaniques pourraient perturber l&#39;amende cils. Effectuez toutes les étapes à la température ambiante sauf indication contraire. <ol><li> Retirer la solution de fixateur et laver deux fois avec du PBS + / +. </li><li> Incuber l&#39;épithélium pendant au moins 1 heure avec une solution de blocage. </li><li> Préparer la solution d&#39;anticorps primaire par dissolution de l&#39;anticorps dans une solution de blocage (1: 200 pour les anticorps utilisés dans cette étude) et centrifuger pendant 5 min à vitesse maximale pour enlever les précipités. </li><li> Incuber l&#39;épithélium avec une solution d&#39;anticorps dans une chambre humide à 4 ° CO / N. </li><li> Laver avec du PBS épithélium + / ​​+ pendant 5 minutes au moins trois fois. </li><li>Préparer la solution d&#39;anticorps secondaire par dissolution de l&#39;anticorps dans une solution de blocage (1: 500) et centrifuger pendant 5 min à vitesse maximale. </li><li> Incuber l&#39;épithélium avec une solution d&#39;anticorps secondaire pendant 1 h dans une chambre sombre. </li><li> Laver avec du PBS épithélium + / ​​+ pendant 5 minutes au moins trois fois. </li><li> Retirer le liquide-bloquant avec une lame de rasoir ou une serviette en papier. Laver l&#39;épithélium avec de l&#39;eau distillée pendant 5 s. </li><li> Préserver le tissu dans antifade réactif de montage. NOTE: fluorescence de fond augmente rapidement dans les jours; analyse microscopique au plus tard deux jours après l&#39;intégration en milieu de montage est donc recommandé. Une attention particulière doit être prise de ne pas presser le tissu lors de la recherche du plan focal correct, car il peut endommager gravement la préparation. En raison de la fluorescence out-of-focus, une enquête plus approfondie avec la microscopie confocale est recommandé. </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...

L&#39;épithélium olfactif de la souris dans la cavité nasale comprend 6-10000000 neurones sensoriels olfactifs bipolaires 1. Chaque neurone olfactif choisit l&#39;un des 1200 gènes de récepteurs olfactifs d&#39;expression. La détection des substances odorantes commence par se lier à un récepteur olfactif 2, qui active alors l&#39;adénylyl cyclase de type III (ACIII) 3 via la protéine G spécifique olfactif Ga olf 4 odorant. L&#39;augmentation de l&#39;adénosine monophosphate cyclique (AMPc) résultant ouvre un nucleotide-gated cyclique (CNG), un canal cationique non sélectif qui conduit à l&#39;afflux de Ca2 + et de Na + et Ca2 + influx ensuite conduit à une ouverture de Ca2 + activés Cl - 5,6 canal. Le résultant extérieur Cl - flux est facilitée par une haute Cl intracellulaire - concentration maintenue constante par prise de Cl -, probablement par la Na + / K + / Cl - cotransporteur NKCC1, leCl - / HCO 3 - échangeur SLC4A1, et peut-être d&#39;autres transporteurs encore être identifiés 6-8. Neurones olfactifs bipolaires sont simples, non ramifiés axones qui font saillie directement vers le bulbe olfactif, et une dendrite qui se étend à la surface de l&#39;épithélium et se termine comme un compartiment spécialisé, le bouton dendritique. De ce bouton, 10-30 cils, qui peut atteindre une longueur maximale de 50 à 60 um, émaner dans le mucus recouvrant la surface épithéliale neuf. Les protéines de la cascade de transduction du signal canonique sont principalement localisés dans la membrane de ces cils. La surface sensorielle accrue de l&#39;épithélium amplifie la capacité à détecter les substances odorantes. En raison de la densité des neurones sensoriels, les cils se étendant à partir de boutons dendritiques voisins se entremêlent. Ce brassage conduit à un mélange aléatoire de différentes franges de neurones qui expriment différents types de récepteurs olfactifs, sur la surface de l&#39;épithélium. La détection et la celluleparti- allocation de protéines ciliaires qui ne sont présents dans un sous-ensemble de neurones sensoriels est donc difficile dans cryosections. En outre, la localisation précise de ces protéines ainsi que les cils ne est guère possible, puisque coupes cryogéniques sont généralement plus mince que la longueur moyenne des cils. Pour activer enquête sur ciliaire localisation de protéines membranaires jusqu&#39;à présent non caractérisés dans les neurones olfactifs, nous avons optimisé une technique de préparation face en permettant l&#39;analyse détaillée de la localisation des protéines dans les cils. En bref, la souris est sacrifiée et la tête près de la ligne médiane divisée. Cornets, nasale, et des os frontaux sont retirés pour exposer le septum. Le septum avec la partie olfactive de l&#39;épithélium de revêtement est desserré en coupant toutes les connexions à la cavité nasale. Après avoir mis le septum dans une boîte de Pétri remplie de la solution de Ringer, l&#39;épithélium est décollée und transféré sur une lame de verre revêtue. Après une courte FIXATétape ions, procédures immunocoloration peut être effectuée si la manipulation est aussi douce que possible pour éviter d&#39;endommager le tissu fragile. Nous démontrons la résolution réalisable en comparant la coloration de deux protéines membranaires différentes dans les cils olfactifs dans cryosections classiques et dans la préparation de visage en décrit.

<table border="0" cellpadding="0" cellspacing="0" width="629">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:127px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Spring scissors</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">straight tip, multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Surgical scissors</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">sharp and blunt end, multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fine forceps</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">curved tips, Dumont #7, multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Razor blade</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">extra thin, multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Binocular with illumination</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">multiple suppliers, Stemi 2000-C, Zeiss</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petri dish</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">multiple suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Liquid-blocker pen</td>
			<td style="width:127px;">Science Services</td>
			<td style="width:119px;">N71310</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Polysine coated slides</td>
			<td style="width:127px;">Thermo Scientific</td>
			<td style="width:119px;">J2800AMNZ</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Confocal microscope</td>
			<td style="width:127px;">Leica Microsystems</td>
			<td style="width:119px;">TCS SPE</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">primary antibody Goat anti-Kirrel2</td>
			<td style="width:127px;">R&#38;D Systems</td>
			<td style="width:119px;">AF2930</td>
			<td style="width:167px;">1:200</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">primary antibody Rabbit anti-mOR-EG</td>
			<td style="width:127px;">Baumgart et al., 2014</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">secondary antibodies</td>
			<td style="width:127px;">Life Technologies</td>
			<td style="width:119px;">A21206, A11057</td>
			<td style="width:167px;">1:500</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Mounting medium, ProLong Gold antifade reagent</td>
			<td style="width:127px;">Life Technologies</td>
			<td style="width:119px;">P36930</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Paraformaldehyde</td>
			<td style="width:127px;">Sigma</td>
			<td align="right" style="width:119px;">441244</td>
			<td style="width:167px;">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.

Épithélium olfactif en préparations de visage peut être utilisé pour étudier la localisation des protéines dans les cils des neurones sensoriels, permettant l&#39;étude détaillée des protéines dont la localisation ne est pas claire après analyse de cryosections. Ce problème peut être illustrée par le cas de la coloration pour Kin IRRE-like protein 2 (Kirrel2). Kirrel2 (également appelé Neph3) est un membre de la superfamille des immunoglobulines (Ig) et les fonctions des protéines mem...

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.

Mont entiers étiquetage des Cilia dans le système olfactif principal de souris

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

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Neuroscience

11.0K vues.  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.

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

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudi&egrave;re et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

We describe the procedure to prepare staged Drosophila embryos for the visualization of the embryonic nervous system during embryogenesis.

La visualisation du système nerveux embryonnaire, en totalité montage Drosophile Embryons

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

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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. ...

DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord

Here we present a technique to label the trajectories of small groups of DRG neurons into the embryonic spinal cord by diffusive staining using the lipophilic tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)1. The comparison of axonal pathways of wild-type with those of mouse lines in which genes are mutated allows testing for a functional role of candidate proteins in the control of axonal branching which is an essential mechanism in the wiring of the nervous system. Axonal branching enables an individual neuron to connect with multiple targets, thereby providing the physical basis for the parallel processing of information. Ramifications at intermediate target regions of axonal growth may be distinguished from terminal arborization. Furthermore, different modes of axonal branch formation may be classified depending on whether branching results from the activities of the growth cone (splitting or delayed branching) or from the budding of collaterals from the axon shaft in a process called interstitial branching2 (Fig. 1).
The central projections of neurons from the DRG offer a useful experimental system to study both types of axonal branching: when their afferent axons reach the dorsal root entry zone (DREZ) of the spinal cord between embryonic days 10 to 13 (E10 - E13) they display a stereotyped pattern of T- or Y-shaped bifurcation. The two resulting daughter axons then proceed in rostral or caudal directions, respectively, at the dorsolateral margin of the cord and only after a waiting period collaterals sprout from these stem axons to penetrate the gray matter (interstitial branching) and project to relay neurons in specific laminae of the spinal cord where they further arborize (terminal branching)3. DiI tracings have revealed growth cones at the dorsal root entry zone of the spinal cord that appeared to be in the process of splitting suggesting that bifurcation is caused by splitting of the growth cone itself4 (Fig. 2), however, other options have been discussed as well5.
This video demonstrates first how to dissect the spinal cord of E12.5 mice leaving the DRG attached. Following fixation of the specimen tiny amounts of DiI are applied to DRG using glass needles pulled from capillary tubes. After an incubation step, the labeled spinal cord is mounted as an inverted open-book preparation to analyze individual axons using fluorescence microscopy.

The stereotyped projections of sensory afferents into the rodent spinal cord offer an easily accessible experimental system to study axonal branching through the tracing of single axons.

DII-étiquetage des neurones DRG pour l&#39;étude de branchement axonal dans une préparation Mont entiers de souris embryonnaires de la moelle épinière

Here we  present a technique to label the trajectories of small groups of DRG neurons  into the embryonic spinal cord by diffusive staining using the ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

Étiquetage des cellules isolées dans le système nerveux central des<em&gt; Drosophila melanogaster</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

Mont entiers immunofluorescence de la rétine de souris néonatal chargé d&#39;enquêter sur l&#39;angiogenèse<em&gt; In vivo</em

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat muscles can offer significant advantage over traditionally used thicker muscles, such as those from the hind limb (e.g. gastrocnemius). Thin muscles allow for comprehensive overview of the entire innervation pattern for a given muscle, which in turn permits identification of selectively vulnerable pools of motor neurons. These muscles also allow analysis of parameters such as motor unit size, axonal branching, and terminal/nodal sprouting. A common obstacle in using such muscles is gaining the technical expertise to dissect them. In this video, we detail the protocol for dissecting the transversus abdominis (TVA) muscle from young mice and performing immunofluorescence to visualize axons and neuromuscular junctions (NMJs). We demonstrate that this technique gives a complete overview of the innervation pattern of the TVA muscle&#160;and can be used to investigate NMJ pathology in a mouse model of the childhood motor neuron disease, spinal muscular atrophy.

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

In this video we demonstrate a protocol for dissection of the transversus abdominis muscle of the mouse and use immunofluorescence and microscopy to visualize neuromuscular junctions.

Dissection du muscle Transversus Abdominis pour l’analyse de jonction neuromusculaire à montage entier

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat ...

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 ...

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various neurological deficits. The current ex vivo live imaging of motile ependymal cilia technique allows for a detailed study of ciliary dynamics following several steps. These steps include: mice euthanasia with carbon dioxide according to protocols of The University of Toledo&#8217;s Institutional Animal Care and Use Committee (IACUC); craniectomy followed by brain removal and sagittal brain dissection with a vibratome or sharp blade to obtain very thin sections through the brain lateral ventricles, where the ependymal cilia can be visualized. Incubation of the brain&#8217;s slices in a customized glass-bottom plate containing Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)/High-Glucose at 37 &#176;C in the presence of 95%/5% O2/CO2 mixture is essential to keep the tissue alive during the experiment. A video of the cilia beating is then recorded using a high-resolution differential interference contrast microscope. The video is then analyzed frame by frame to calculate the ciliary beating frequency. This allows distinct classification of the ependymal cells into three categories or types based on their ciliary beating frequency and angle. Furthermore, this technique allows the use of high-speed fluorescence imaging analysis to characterize the unique intracellular calcium oscillation properties of ependymal cells as well as the effect of pharmacological agents on the calcium oscillations and the ciliary beating frequency. In addition, this technique is suitable for immunofluorescence imaging for ciliary structure and ciliary protein localization studies. This is particularly important in disease diagnosis and phenotype studies. The main limitation of the technique is attributed to the decrease in live motile cilia movement as the brain tissue starts to die.

Using high-resolution differential interference contrast (DIC) microscopy, an ex vivo observation of the beating of motile ependymal cilia located within the mouse brain ventricles is demonstrated by live-imaging. The technique allows a recording of the unique ciliary beating frequency and beating angle as well as their intracellular calcium oscillation pacing properties.

Imagerie en direct de la épendymaire Cilia dans les ventricules latéraux du cerveau de souris

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various ...

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.

Mont entiers Dissection et immunofluorescence de la cochlée de souris adultes

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 ...

HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2. Consequently, investigation of structural details of the interaction between 5-HT2A and mGlu2 affecting schizophrenia-related behaviors represents a powerful translational tool. As previously shown, the head-twitch response (HTR) in mice is elicited by hallucinogenic drugs and this behavioral response is absent in 5-HT2A knockout (KO) mice3,4. Additionally, by conditionally expressing the 5-HT2A receptor only in cortex, it was demonstrated that 5-HT2A receptor-dependent signaling pathways on cortical pyramidal neurons are sufficient to elicit head-twitch behavior in response to hallucinogenic drugs3. Finally, it has been shown that the head-twitch behavioral response induced by the hallucinogens DOI and lysergic acid diethylamide (LSD) is significantly decreased in mGlu2-KO mice5. These findings suggest that mGlu2 is at least in part necessary for the 5-HT2A receptor-dependent psychosis-like behavioral effects induced by LSD-like drugs. However, this does not provide evidence as to whether the 5-HT2A-mGlu2 receptor complex is necessary for this behavioral phenotype. To address this question, herpes simplex virus (HSV) constructs to express either mGlu2 or mGlu2&#916;TM4N (mGlu2/mGlu3 chimeric construct that does not form the 5-HT2A-mGlu2 receptor complex) in the frontal cortex of mGlu2-KO mice were used to examine whether this GPCR heteromeric complex is needed for the behavioral effects induced by LSD-like drugs6.

This article describes how to inject viral vectors into the mouse frontal cortex to test behavioral assays that require GPCR heteromeric formation.

HSV-Mediated Expression du transgène de chimériques Constructs pour étudier la fonction Behavioral des GPCR hétéromères chez la souris

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2.  ...