Nous tenons à remercier Scott Hamilton, et des membres du laboratoire O&#39;Halloran la lecture attentive de ce manuscrit. Nous remercions également notre relecteur anonyme pour d&#39;excellentes suggestions et commentaires pertinents.

1. Construction de la GFP Tagged EGL-4 animaux exprimant <ol><li> Cloner la fusion traductionnelle GFP :: EGL-4 sous le promoteur pour le gène odr-3 (2678 pb utilisation directement en amont du codon d&#39;initiation): (p) odr-3 :: GFP :: EGL-4. L&#39;expression ODR-3 disques de promoteur dans les paires de neurones amphid: AWA, AWB, AWC, et faiblement dans l&#39;ASH. </li><li> Injecter le plasmide (p) odr-3 :: GFP :: EGL-4 de type sauvage dans (N2) animaux à 50 ng / ul avec les marqueurs de co-injection en utilisant des techniques de transformation de la lignée germinale 8: (p) ofm-1: : GFP 9 (25 ng / pl) et (p) odr-1 :: dsRED 10 (25 ng / pl). Le odr-1 promoteur dirige l&#39;expression dans l&#39;AWC et AWB amphid paires de neurones, et la ofm-1 promoteur dirige l&#39;expression dans les coelomocytes. </li><li> Soumettre les animaux transgéniques exprimant les tableaux détaillés extrachromosomiques à l&#39;étape 1.2 de l&#39;ultra-violet (UV) / triméthylpsoralène (TMP) protocole d&#39;intégration11. </li><li> Bleach synchroniser les 12 animaux et de les cultiver sur des nématodes croissance des médias (NGM) des plaques contenant OP50 E. coli à la phase de L4. Lavez-L4 animaux assiettes NGM avec M9 tampon pour éliminer OP50 E. coli. </li><li> Éliminer le tampon M9 et ajouter 50-100 ul de 30 pg / ml solution TMP travail sur le culot à vis sans fin dans le tube de 1,5 ml. Enroulez le tube de 1,5 ml dans du papier pour bloquer la lumière ambiante et agiter doucement sur un agitateur pendant 15 min dans la feuille gainé de tube de 1,5 ml. Transfert vers dans la solution TMP / ver sur une plaque non ensemencée NGM grande. </li><li> Exposer les vers sur la plaque NGM aux UV 350 pJ (X100) à l&#39;aide de 2400 Stratalinker. Ensuite, ajoutez les vers à une plaque OP50 NGM contenant et laisser incuber dans l&#39;obscurité pendant 5 heures. </li><li> Choisissez 50 vers transgéniques à environ 10 plaques ensemencées (2-5 vers chaque plaque). Transfert P0 est à chaque nouvelles plaques de 24 h pendant 3 jours. </li><li> Choisissez une 250 F1 clonalenimals (1 animal par plaque). Cloner 2-4 animaux F2 de chaque plaque F1. Vérifiez animaux F3 pour rechercher une ligne de transmission de 100% en consultant d&#39;(p) ofm-1 :: GFP modèle d&#39;expression sous une loupe binoculaire fluorescent. </li><li> Outcross la dernière ligne intégrée (s) avec les animaux de type sauvage N2 5 fois. Nous nous référerons à la dernière ligne intégrée pyIs500, et la GFP-étiqueté EGL-4 molécule GFP :: EGL-4. </li></ol> 2. La culture et l&#39;entretien des animaux d&#39;analyses de translocation nucléaire <ol><li> Cultiver les animaux pyIs500 à 25 ° C sur des plaques standard de 10 cm NGM (voir ci-dessous pour la recette) ensemencé avec OP50 E. coli 13 (figure 1). </li><li> Le jour 1, ramasser quatre à cinq L4 pyIs500 animaux de plaques cultivées à 25 ° C sur cinq différentes 10 cm OP50 E. coli ensemencées plaques et incuber à 25 ° C (c&#39;est, 4-5 L4 animaux par plaque). </li><li> Le jour 4, lavez adulte populations de pyIs500 animaux hors des plaques NGM volumineux à l&#39;aide de S-basale tampon (voir ci-dessous pour la recette). Utiliser des pipettes en verre à usage unique pour transférer les animaux, car ils adhérer à la paroi de pointes de pipettes en plastique. </li></ol> 3. À long terme Odeur induite analyses translocation nucléaire <ol><li> Laver les animaux pyIs500 3 fois dans S-basale pour éliminer les bactéries. Ne pas centrifuger les animaux entre les lavages, mais plutôt permettre aux animaux de se déposer par gravité dans un stationnaire tube de 1,5 ml. Il est essentiel de s&#39;assurer que toutes les bactéries est retiré, et que les pâtés NGM sont exempts de contamination, car les bactéries résiduelles affectera négativement le résultat du test de translocation. En outre, nous avons constaté que l&#39;autoclave de 1,5 ml microtubes produit un &quot;collant&quot; bien sur les parois intérieures du tube qui entraîne les animaux à coller aux parois, et ainsi utiliser non autoclavé tubes de 1,5 ml microtubes. </li><li> Alors que les animaux s&#39;installent entre les lavages, maquillage til adaptation solution. Ajouter 100 ml de S-basale tampon pour un cylindre de 100 ml diplômés. Ajouter adapter l&#39;odeur de la S-basale et scelle le cylindre à l&#39;aide d&#39;une bande de parafilm. Si vous effectuez l&#39;adaptation benzaldéhyde ajouter 7,5 ul de benzaldéhyde dans 100 ml de S-basale, d&#39;adaptation butanone ajouter 11 ul de 2-butaone à 100 ml de S-basale. Retourner doucement (ne pas secouer) le cylindre contenant diplôme S-basales et les odeurs 30 fois pour créer une émulsion uniforme. </li><li> Après le lavage final dans S-basale, permettre aux animaux de se déposer et retirer tout le liquide. Une étiquette tube &quot;+ ve&quot; ou &quot;adapter&quot; et l&#39;autre tube &quot;-ve&quot; ou &quot;unadapt&quot;. Puis ajouter 1 ml du mélange adaptation au microtube adaptation (+ ve) et ajouter 1 ml de S-basale de la microtube de contrôle inadapté (-ve). Essayer de maintenir le nombre d&#39;animaux entre 100 et 200 dans chaque tube. Avec un peu de pratique, il est possible d&#39;estimer le nombre approximatif d&#39;animaux dans une pastille à l&#39;oeil. </li><li> Placer un tube de 1,5 ml capuchon protecteurdans chaque tube (empêche les couvercles de popping ouvert) et placer les tubes sur un agitateur pendant 80 min. Nous avons trouvé que l&#39;exposition odeur entre 60 min et 80 min donne des résultats similaires (à la fois en raison des mesures d&#39;adaptation à long terme). Cependant, 80 min exposition fournit des résultats plus cohérents dans nos mains. Ainsi, tous nos essais d&#39;adaptation à long terme sont normalisés en utilisant une exposition odeur min 80 (figure 2). </li><li> Après 80 min, laver les animaux 3 fois en S-basale. Permettre aux animaux de se déposer par gravité entre chaque lavage. </li></ol> 4. À court terme induits par l&#39;odeur analyses de translocation nucléaire <ol><li> Dans le cas de l&#39;adaptation odeur de courte durée, utiliser le même protocole que par des essais à long terme translocation odeurs provoquées par étapes (3.1-3.4), mais au lieu d&#39;incubation des animaux à s&#39;adapter pyIs500 odeur pendant 80 minutes, utilisez une exposition de 30 min . La quantification des odeurs provoquées par des tests de translocation nucléaire décrites ci-dessous (étape 5) est le même à long terme et à court terexpositions m. </li></ol> 5. Notation de l&#39;événement odeur induite par translocation nucléaire <ol><li> Faire 2% d&#39;agarose contenant des plaquettes de 5 mM d&#39;azoture de sodium (NaN 3) en dissolvant dans l&#39;électrophorèse sur gel d&#39;agarose 2 0 dH. Placez une seule goutte d&#39;agarose fondu contenant NaN 3 sur une lame de microscope en verre. Placer immédiatement une autre lame de microscope en verre sur la première diapositive pour aplatir la goutte d&#39;agarose fondu. Laissez-le pendant 30 secondes, puis doucement démêler le microscope glisse en laissant une lame de microscope avec une semelle d&#39;agarose de ~ 1 mm. </li><li> Après le lavage final dans S-basale, permettre aux vers de terre pour s&#39;installer dans le tube de 1,5 ml et éliminer tout le liquide. Ensuite, ajoutez les animaux goutte à goutte sur les plages d&#39;agarose de plusieurs lames de microscope. Il est important de garder le nombre d&#39;animaux par lame entre 50 et 100. Trop d&#39;animaux par lame va compliquer la notation et créent souvent une lueur bleu-après disperser la lumière d&#39;excitation. L&#39;excès de liquide peutêtre méchant du tampon d&#39;agarose en utilisant un petit Kim-wipe. Placez un verre 0,5 mm lamelle sur le tampon et laisser reposer pendant 2 minutes pour permettre le NaN 3 à immobiliser les animaux. Remarque, les animaux peuvent être récupérés à partir des diapositives après avoir marqué. Les animaux se remettre de l&#39;anesthésie NaN 3 après ~ 1 h en cas de transfert d&#39;une plaque ensemencée NGM dans une goutte de tampon M9. </li><li> A ce stade, il est impératif que chaque expérimentateur élaborer une clé de suivre coulissant est l&#39;expérimental (+ ve) coulissant et qui glissent est le contrôle (-ve) coulissant, et passer les diapositives à une autre personne qui aveuglément marquer la GFP: EGL-4 modèle de localisation. Ceci permet de contrôler les biais de l&#39;expérimentateur. </li><li> Score entre 50 et 100 animaux par lame à l&#39;aide d&#39;un microscope droit fluorescent avec objectifs à fort grossissement 10X, 40X et 63X et GFP / RFP filtres. Tout d&#39;abord, localiser les animaux sous un grossissement 10X en utilisant éclairage sur fond clair. Ensuite, localisez le neurone AWC en éteignant l&#39;bright-champ d&#39;éclairage et en utilisant le filtre DP. Les (p) odr-1 :: lecteurs dsRED expression dans les neurones et AWB AWC. Les neurones ont AWC distinctifs ovales en forme de corps cellulaires par rapport aux corps cellulaires AWB qui sont plus petits et de forme circulaire (figure 2). </li><li> Une fois que le corps de la cellule AWC a été localisé, passer au filtre GFP et d&#39;enregistrer la localisation de la GFP: EGL-4 comme le nucléaire ou cytoplasmique. En utilisant un compteur permet à l&#39;expérimentateur de garder regarder dans l&#39;oculaire tout en marquant la diapositive. Note: 5.1 à 5.5 étapes sont répétées au moins 3 fois à des jours différents pour produire des données statistiquement significatives (figures 1-2). </li></ol> 6. Surveillance GFP-étiqueté EGL-4 lors de la récupération de l&#39;adaptation odeur à long terme <ol><li> Après l&#39;étape 3.5, diviser les populations d&#39;animaux pyIs500 en aliquotes multiples et notes pour la GFP nucléaire par rapport cytoplasmique :: EGL-4 dans l&#39;AWC à zéro point dans le temps (c&#39;est-à-après 80 min d&#39;exposition) et multiples points de temps ultérieurs (figure 3). </li><li> Transfert à la fois le contrôle (-ve) et expérimentales (+ ve) animaux utilisant des pipettes en verre jetables sur des plaques NGM. </li><li> Permettre aux animaux de récupérer pour différentes longueurs de temps et de réexaminer la localisation de la GFP :: EGL-4in AWC à chaque point dans le temps comme dans les étapes 5.1 à 5.5. </li></ol> 7. Matériels Plaques NGM: Pour ajouter 1 L à 1 L ddH 2 0: 21 g de Bacto-Agar, 3 g Chlorure de sodium (NaCl), 2,5 g de Bacto-peptone. Médias autoclave. Après autoclavage, refroidir la gélose jusqu&#39;à ce que la température indique 54 ° C Ajouter les solutions suivantes: 25 ml de tampon phosphate de potassium 1M, 1 ml 1 M Sulfate de magnésium (MgSO 4), 1 ml chlorure de calcium 1 M (CaCl 2); 1 Cholestérol ml dans de l&#39;éthanol (5 mg / ml). 1 M dibasique K 2 HPO 4 174,2 g / mol: 500 ml Pour: Dans 400 ml ddH O 2, dissoudre 87,1 g Dibasic K 2 HPO 4. Réglez le volume à 500 L avec ddH 2 O. Stériliser par filtration à l&#39;aide d&#39;une unité de 500 ml stérile filtre. 1 M monobasique KH 2 PO 4 136,1 g / mol: Pour 1 L: Dans 800 ml ddH O 2, dissoudre 136,1 g monobasique KH 2 PO 4. Réglez le volume à 1 L avec ddH 2 O. Stériliser par filtration à l&#39;aide d&#39;une unité de 500 ml stérile filtre. 1 M de phosphate de potassium (K 3 PO 4) pH 6,0 Tampon: Pour 1 L: Dans un 1 L Unité de filtration stérile, ajouter 868 ml de 1 M monobasique KH 2 PO 4. Laissez l&#39;appareil à filtrer à travers, puis ajouter ml 132 de 1 M dibasique K 2 HPO 4. Laissez l&#39;appareil pour filtrer tout le liquide. Retirez l&#39;unité de filtre et ajuster le pH à 6,0. Conservez-la à température ambiante. S-basale: Pour 1 litre: ajouter 50 ml 1M K 3 PO 4 tampon, pH ~ 6,0, et 20 ml de NaCl 5M aq. Remplir à 1 L avecddH 2 O. Solution autoclave. M9: Pour 1 litre: 3 g KH 2 PO 4, 6 g de Na 2 HPO 4, 5 g de NaCl, 1 ml 1 M MgSO 4, H 2 O. Remplir à 1 L avec ddH 2 0. Stériliser à l&#39;autoclave.

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Aucun conflit d&#39;intérêt déclaré.

L&#39;entrée des odeurs nucléaire induite d&#39;une GFP-étiqueté EGL-4 molécule décrite ici fournit une lecture solide moléculaire de l&#39;adaptation olfactive chez C. elegans. Les analyses des odeurs provoquées par translocation nucléaire sont simples et ne nécessitent que quelques jours de temps de préparation. L&#39;animal pyIs500 que nous avons construit pour ces essais, exprime un marqueur qui illumine le neurone AWC ainsi que l&#39;expression de la GFP-étiqueté EGL-4 protéines. Ain...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> Bacto Agar </td><td> Difco </td><td> DF0140-07-4 </td><td> Plaques NGM </td></tr><tr><td> Chlorure de sodium </td><td> Fisher Chemical </td><td> S671-10 </td><td> Plaques NGM </td></tr><tr><td> Bacto Peptone </td><td> Difco </td><td> DF0118-07-2 </td><td> Plaques NGM </td></tr><tr><td> Phosphate de potassium dibasique </td><td> Fisher Chemical </td><td> S375-500 </td><td> S-basale tampon et plaques NGM </td></tr><tr><td> Phosphate de potassium monobasique </td><td> Fisher Chemical </td><td> P285-500 </td><td> S-basale tampon et plaques NGM </td></tr><tr><td> Kimwipes - Petit </td><td> Kimberly-Clark </td><td> LS2770 </td><td></td></tr><tr><td> L&#39;éthanol à 100% </td><td> Shield Gold chimiqueCie. </td><td> 43196-115 </td><td> les odeurs de dilution pour les essais de chimiotaxie </td></tr><tr><td> Chlorure de calcium </td><td> Sigma-Aldrich </td><td> C8106-500G </td><td> Plaques NGM </td></tr><tr><td> Sulfate de Magnésium </td><td> MP Biomédical </td><td> 150136-500G </td><td> Plaques NGM </td></tr><tr><td> D&#39;azoture de sodium à 99% </td><td> Fisher Scientific </td><td> ICN10289180 </td><td> Anesthésique </td></tr><tr><td> Agarose - UltraPure </td><td> Invitrogen </td><td> 16500-500 </td><td> Coussinets d&#39;agarose </td></tr><tr><td> Benzaldéhyde </td><td> Sigma-Aldrich </td><td> B1334-100G </td><td> Odeur AWC </td></tr><tr><td> Butanone, ACS grade </td><td> Sigma-Aldrich </td><td> 360473-500ML </td><td> Odeur AWC </td></tr><tr><td> Microtubes à centrifuger - 1,5 ml de couleur </td><td> Denville </td><td> LS8147 </td><td></td></tr><tr><td> Pipette Pasteur en verre jetable 5-3/4 &quot; </td><td> Fisher Scientific </td&gt; <td> 13-678-20B </td><td></td></tr><tr><td> Stratalinker </td><td> Stratagene </td><td> Stratalinker 2400 </td><td> L&#39;intégration UV </td></tr><tr><td> Bouteille vide Filter - 500 ml </td><td> Nalgene </td><td> 09 à 740-25B </td><td></td></tr></tbody></table>

a molecular readout of long-term olfactory adaptation in c. elegans

Lors d&#39;une stimulation soutenue des neurones sensoriels les plus adaptera sa réponse en diminuant leur sensibilité au signal. La réponse d&#39;adaptation permet l&#39;attention de forme et protège également les cellules de la sur-stimulation. L&#39;adaptation dans le circuit olfactif de C. elegans a d&#39;abord été décrit par Colbert et Bargmann 1,2. Ici, les auteurs ont défini les paramètres du paradigme d&#39;adaptation olfactive, dont ils se servaient pour concevoir un crible génétique pour isoler des mutants défectueux dans leur capacité à s&#39;adapter aux odeurs volatiles captées par les cellules Wing Amphid de type C (AWC) des neurones sensoriels. Lorsque de type sauvage C. elegans animaux sont exposés à une jolie AWC-senti l&#39;odeur 3 pour 30 minutes, ils vont adapter leur réactivité à l&#39;odeur et ensuite ignorer l&#39;odeur adaptation dans un essai de chimiotaxie comportement pendant ~ 1 heure. Lorsque de type sauvage C. elegans animaux sont exposés à une jolie AWC-senti l&#39;odeur pendant ~ 1 heure ils seront alors ignorer l&#39;odeur adaptation en achemotaxis analyse comportementale pour ~ 3 h. Ces deux phases d&#39;adaptation olfactive chez C. elegans ont été décrits comme adaptation à court terme olfactif (induite après une exposition de 30 min odeur), et adaptation à long terme olfactif (induite après exposition de 60 min odeur). Des travaux ultérieurs de L&#39;Etoile et al., 4 découvert une protéine kinase G (PKG) a appelé EGL-4 qui est nécessaire à la fois pour l&#39;adaptation à court terme et à long terme dans les neurones olfactifs AWC. La EGL-4 protéine contient une séquence de localisation nucléaire qui est nécessaire à long terme des mesures d&#39;adaptation olfactifs, mais dispensable à court terme des mesures d&#39;adaptation olfactifs dans les 4 AWC. Par le marquage EGL-4 avec une protéine fluorescente verte, il était possible de visualiser la localisation de EGL-4 dans l&#39;AWC lors d&#39;une exposition prolongée odeur. L&#39;utilisation de ce entièrement fonctionnel GFP-étiqueté EGL-4 (GFP :: EGL-4) molécule que nous avons pu développer une lecture moléculaire adaptation à long terme olfactive dans les 5 AWC. L&#39;utilisation de ce mlecture olecular d&#39;adaptation olfactive, nous avons été en mesure d&#39;effectuer les deux écrans avant et arrière génétiques pour identifier les animaux mutants qui présentent des modèles défectueux localisation subcellulaire de la GFP :: EGL-4 dans l&#39;6,7 AWC. Nous décrivons ici: 1) la construction de la GFP :: EGL-4 animaux exprimant; 2) le protocole pour la culture des animaux à long terme analyses de translocation induite par l&#39;odeur nucléaires, et 3) la notation de long terme induite par l&#39;odeur Si la translocation nucléaire et la récupération (re-sensibilisation) de la GFP nucléaire :: EGL-4 Etat.

Un exemple de modèle de localisation de la GFP :: EGL-4in l&#39;AWC avant et après exposition prolongée odeur est montré dans la figure 2. Avant l&#39;exposition prolongée odeur, la GFP :: EGL-4 est localisée dans le cytosol de la AWC (figure 2B), et après 80 min d&#39;exposition odeur GFP :: EGL-4 est localisée dans le noyau de l&#39;AWC (figure 2D). Au niveau comportemental, les animaux avec cytosolique GFP :: EGL-4 dans AWC sont attirés par une source ponctu...

Watch this Scientific Journal Video about A Molecular Readout of Long-term Olfactory Adaptation in C. elegans at JoVE.com

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

Nous décrivons ici une lecture moléculaire adaptation à long terme olfactif<em&gt; Caenorhabditis elegans</em&gt;. La protéine kinase G, EGL-4, est nécessaire pour les mesures d&#39;adaptation stables dans la paire de neurone sensoriel primaire appelé AWC. Pendant de longues odeur d&#39;exposition EGL-4 translocation du cytosol vers le noyau de l&#39;AWC.

Un indicateur moléculaire de l&#39;adaptation à long terme olfactive dans<em&gt; C. elegans</em

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps ...

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JoVE Journal

Neuroscience

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

10.7K vues.  George Washington University. Nous décrivons ici une lecture moléculaire adaptation à long terme olfactif Caenorhabditis elegans. La protéine kinase G, EGL-4, est nécessaire pour les mesures d'adaptation stables dans la paire de neurone sensoriel primaire appelé AWC. Pendant de longues odeur d'exposition EGL-4 translocation du cytosol vers le noyau de l'AWC.

Vidéo: A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment through neuron-specific detection of food and chemical odors1, 2, and can associate nutritive states with chemical odors3, temperature4, and the pathogenicity of a food source5.
Here, we describe assays of C. elegans associative learning and short- and long-term associative memory. We modified an aversive olfactory learning paradigm6 to instead produce a positive response; the assay involves starving ~400 worms, then feeding the worms in the presence of the AWC neuron-sensed volatile chemoattractant butanone at a concentration that elicits a low chemotactic index (similar to Toroyama et al.7). A standard population chemotaxis assay1 tests the worms' attraction to the odorant immediately or minutes to hours after conditioning.
After conditioning, wild-type animals' chemotaxis to butanone increases ~0.6 Chemotaxis Index units, its "Learning Index". Associative learning is dependent on the presence of both food and butanone during training. Pairing food and butanone for a single conditioning period ("massed training") produces short-term associative memory that lasts ~2 hours. Multiple conditioning periods with rest periods between ("spaced training") yields long-term associative memory (&lt;40 hours), and is dependent on the cAMP Response Element Binding protein (CREB),6 a transcription factor required for long-term memory across species.8
Our protocol also includes image analysis methods for quick and accurate determination of chemotaxis indices. High-contrast images of animals on chemotaxis assay plates are captured and analyzed by worm counting software in MatLab. The software corrects for uneven background using a morphological tophat transformation.9 Otsu's method is then used to determine a threshold to separate worms from the background.10 Very small particles are removed automatically and larger non-worm regions (plate edges or agar punches) are removed by manual selection. The software then estimates the size of single worm by ignoring regions that are above a specified maximum size and taking the median size of the remaining regions. The number of worms is then estimated by dividing the total area identified as occupied by worms by the estimated size of a single worm.
We have found that learning and short- and long-term memory can be distinguished, and that these processes share similar key molecules with higher organisms.6,8 Our assays can quickly test novel candidate genes or molecules that affect learning and short- or long-term memory in C. elegans that are relevant across species.

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

Here we describe methods to test C. elegans associative learning and short- and long-term associative memory. These population assays employ the worms abilities to chemotax toward volatile odorants, and form positive associations upon pairing food with the chemoattractant butanone. Increasing the number of conditioning periods induces long-term memory.

C. elegans à l&#39;apprentissage butanone, à court terme et à long terme Tests mémoire associative

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment ...

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Neurosciences

In vivo Laser Axotomy in C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron is damaged by injury or disease, it may regenerate. Cell-intrinsic and extrinsic factors influence the ability of a neuron to regenerate and restore function. Recently, the nematode C. elegans has emerged as an excellent model organism to identify genes and signaling pathways that influence the regeneration of neurons1-6. The main way to initiate neuronal regeneration in C. elegans is laser-mediated cutting, or axotomy. During axotomy, a fluorescently-labeled neuronal process is severed using high-energy pulses. Initially, neuronal regeneration in C. elegans was examined using an amplified femtosecond laser5. However, subsequent regeneration studies have shown that a conventional pulsed laser can be used to accurately sever neurons in vivo and elicit a similar regenerative response1,3,7.
We present a protocol for performing in vivo laser axotomy in the worm using a MicroPoint pulsed laser, a turnkey system that is readily available and that has been widely used for targeted cell ablation. We describe aligning the laser, mounting the worms, cutting specific neurons, and assessing subsequent regeneration. The system provides the ability to cut large numbers of neurons in multiple worms during one experiment. Thus, laser axotomy as described herein is an efficient system for initiating and analyzing the process of regeneration.

In vivo Laser Axotomy in C. elegans

A protocol to cut neurons in C. elegans with a MicroPoint pulsed laser is presented. We describe setting up the system, immobilizing worms, and severing labeled neurons. Advantages include a relatively low-cost system and the ability to sever neuronal processes or ablate cells in vivo.

In vivo laser axotomie en C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron ...

Automated Quantification of Synaptic Fluorescence in C. elegans

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall neural circuit function. Synapse strength critically depends on the abundance of neurotransmitter receptors clustered at synaptic sites on the postsynaptic membrane. Receptor levels are established developmentally, and can be altered by receptor trafficking between surface-localized, subsynaptic, and intracellular pools, representing important mechanisms of synaptic plasticity and neuromodulation. Rigorous methods to quantify synaptically-localized neurotransmitter receptor abundance are essential to study synaptic development and plasticity. Fluorescence microscopy is an optimal approach because it preserves spatial information, distinguishing synaptic from non-synaptic pools, and discriminating among receptor populations localized to different types of synapses. The genetic model organism Caenorhabditis elegans is particularly well suited for these studies due to the small size and relative simplicity of its nervous system, its transparency, and the availability of powerful genetic techniques, allowing examination of native synapses in intact animals.
Here we present a method for quantifying fluorescently-labeled synaptic neurotransmitter receptors in C. elegans. Its key feature is the automated identification and analysis of individual synapses in three dimensions in multi-plane confocal microscope output files, tabulating position, volume, fluorescence intensity, and total fluorescence for each synapse. This approach has two principal advantages over manual analysis of z-plane projections of confocal data. First, because every plane of the confocal data set is included, no data are lost through z-plane projection, typically based on pixel intensity averages or maxima. Second, identification of synapses is automated, but can be inspected by the experimenter as the data analysis proceeds, allowing fast and accurate extraction of data from large numbers of synapses. Hundreds to thousands of synapses per sample can easily be obtained, producing large data sets to maximize statistical power. Considerations for preparing C. elegans for analysis, and performing confocal imaging to minimize variability between animals within treatment groups are also discussed. Although developed to analyze C. elegans postsynaptic receptors, this method is generally useful for any type of synaptically-localized protein, or indeed, any fluorescence signal that is localized to discrete clusters, puncta, or organelles.
The procedure is performed in three steps: 1) preparation of samples, 2) confocal imaging, and 3) image analysis. Steps 1 and 2 are specific to C. elegans, while step 3 is generally applicable to any punctate fluorescence signal in confocal micrographs.

Automated Quantification of Synaptic Fluorescence in C. elegans

The abundance of neurotransmitter receptors clustered at synapses strongly influences synaptic strength. This method quantifies fluorescently-labeled neurotransmitter receptors in three dimensions with single-synapse resolution in C. elegans, allowing hundreds of synapses to be rapidly characterized within a single sample without distortions introduced by z-plane projection.

Quantification automatisée de la fluorescence dans Synaptic<em&gt; C. elegans</em

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall ...

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

En imagerie calcique in vivo des neurones en C. elegans

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

Long-term Behavioral Tracking of Freely Swimming Weakly Electric Fish

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and social interactions can be best studied by observing unrestrained, freely behaving animals. Weakly electric fish (WEF) display readily observable exploratory and social behaviors by emitting electric organ discharge (EOD). Here, we describe three effective techniques to synchronously measure the EOD, body position, and posture of a free-swimming WEF for an extended period of time. First, we describe the construction of an experimental tank inside of an isolation chamber designed to block external sources of sensory stimuli such as light, sound, and vibration. The aquarium was partitioned to accommodate four test specimens, and automated gates remotely control the animals&#39; access to the central arena. Second, we describe a precise and reliable real-time EOD timing measurement method from freely swimming WEF. Signal distortions caused by the animal&#39;s body movements are corrected by spatial averaging and temporal processing stages. Third, we describe an underwater near-infrared imaging setup to observe unperturbed nocturnal animal behaviors. Infrared light pulses were used to synchronize the timing between the video and the physiological signal over a long recording duration. Our automated tracking software measures the animal&#39;s body position and posture reliably in an aquatic scene. In combination, these techniques enable long term observation of spontaneous behavior of freely swimming weakly electric fish in a reliable and precise manner. We believe our method can be similarly applied to the study of other aquatic animals by relating their physiological signals with exploratory or social behaviors.

We describe a set of techniques for studying spontaneous behavior of freely swimming weakly electric fish over an extended period of time, by synchronously measuring the animal&#39;s electric organ discharge timing, body position and posture both accurately and reliably in a specially designed aquarium tank inside a sensory isolation chamber.

À long terme de suivi comportemental de librement piscine faiblement poisson électrique

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and ...

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.

Microchambres agarose à long terme de l&#39;imagerie calcique<em&gt; Caenorhabditis elegans</em

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

Dissecting the neuronal and neuromuscular circuits that regulate behavior remains a major challenge in biology. The nematode Caenorhabditis elegans has proven to be an invaluable model organism in helping to tackle this challenge, from inspiring technological approaches,&#160;building the human brain connectome, to actually shedding light on the specific molecular drivers of basic functional patterns. The bulk of the behavioral studies in C. elegans have been performed on solid substrates. In liquid, animals exhibit behavioral patterns that include movement at a range of speeds in 3D, as well as partial body movements, such as a posterior curl without anterior shape change, which introduce new challenges for quantitation. The steps of a simple procedure, and use of a software that enables high-resolution analysis of C. elegans swim behavior, are presented here. The software, named CeleST, uses a specialized computer program that tracks multiple animals simultaneously and provides novel measures of C. elegans locomotion in liquid (swimming). The measures are mostly grounded in animal posture and based on mathematics used in computer vision and pattern recognition, without computational requirements for threshold cut-offs. The software tool can be used to both assess overall swimming prowess in hundreds of animals from combined small batch trials and to reveal novel phenotypes even in well-characterized genetic mutants. The preparation of specimens for analysis with CeleST is simple and low-tech, enabling wide adaptation by the scientific community. Use of the computational approach described here should therefore contribute to the greater understanding of behavior and behavioral circuits in the C. elegans model.

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

An efficient and simple methodology for computer-based analysis of nematode swimming behavior in liquid is described. The method requires little to no investment for C. elegans laboratories. The hardware used is standard, and the computer software for the behavioral analysis (CeleST) is an open source one.

Analyse automatisée de<em&gt; C. elegans</em&gt; Comportement Swim Utiliser Celest Software

Dissecting the neuronal and neuromuscular circuits that regulate behavior remains a major challenge in biology. The nematode Caenorhabditis elegans has ...

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

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

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

Immunomarquage quadruple de l&#39;ampoule olfactive pour la visualisation des codes d&#39;identité moléculaire des axes sensoriels olfactifs

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

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its completely mapped nervous system and transparent anatomy, presents an ideal model for understanding real-time neural dynamics using calcium indicators. In combination with microfluidic technologies and experimental designs, calcium-imaging studies using these indicators are performed in both free-moving and trapped animals. However, most previous studies utilizing trapping devices, such as the olfactory chip described in Chronis et al., have devices designed for use in the more common hermaphrodite, as the less common male is both morphologically and structurally dissimilar. An adapted olfactory chip was designed and fabricated for increased efficiency in male neuronal imaging with using young adult animals. A turn was incorporated into the worm loading port to rotate the animals and to allow for the separation of the individual neurons within a bilateral pair in 2D imaging. Worms are exposed to a controlled flow of odorant within the microfluidic device, as described in previous hermaphrodite studies. Calcium transients are then analyzed using the open-source software ImageJ. The procedure described herein should allow for an increased amount of male-based C. elegans calcium imaging studies, deepening our understanding of the mechanisms of sex-specific neuronal signaling.

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

The use of an adapted &#34;olfactory chip&#34; for the efficient calcium imaging of C. elegans males is described here. Studies of male exposure to glycerol and a pheromone are also shown.

En utilisant une puce olfactif microfluidique adapté pour l’imagerie de l’activité neuronale en réponse aux phéromones dans les neurones de tête mâle C. Elegans

The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its ...

Generation and Long-term Maintenance of Nerve-free Hydra

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The interstitial cells can be eliminated through two consecutive treatments with colchicine, a plant-derived toxin that kills dividing cells, thus erasing the potential for renewal of the differentiated cells that are derived from the interstitial stem cells. This allows for the generation of Hydra that lack nerve cells. A nerve-free polyp cannot open its mouth to feed, egest, or regulate osmotic pressure. Such animals, however, can survive and be cultured indefinitely in the laboratory if regularly force-fed and burped. The lack of nerve cells allows for studies of the role of the nervous system in regulating animal behavior and regeneration. Previously published protocols for nerve-free Hydra maintenance involve outdated techniques such as mouth-pipetting with hand-pulled micropipette tips to feed and clean the Hydra. Here, an improved protocol for maintenance of nerve-free Hydra is introduced. Fine-tipped forceps are used to force open the mouth and insert freshly killed Artemia. Following force-feeding, the body cavity of the animal is flushed with fresh medium using a syringe and hypodermic needle to remove undigested material, referred to here as "burping". This new method of force-feeding and burping nerve-free Hydra through the use of forceps and syringes eliminates the need for mouth-pipetting using hand-pulled micropipette tips. It thus makes the process safer and significantly more time efficient. To ensure that the nerve cells in the hypostome have been eliminated, immunohistochemistry using anti-tyrosine-tubulin is conducted.

Generation and Long-term Maintenance of Nerve-free Hydra

Through a double treatment with colchicine, a plant-derived toxin that kills dividing cells, nerve-free Hydra vulgaris can be generated. These Hydra cannot feed or egest on their own. This paper describes an improved method for long-term maintenance of nerve free Hydra vulgaris in the laboratory.

Génération et entretien à long terme sans nerve<em&gt; Hydra</em

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The ...