Ce travail a été soutenu par le NIH aux subventions NS27501 DKOD. Un appui supplémentaire pour ce travail a été fourni par une subvention à l&#39;UC Irvine en faveur de DKOD l&#39;entremise du Programme Professeur HHMI.

Les préparatifs avant le jour de la culture: <ol><li> Faire une solution stérile de dissection. </li><li> Faire stériles DMEM et garder en aliquotes de 10 ml à 4 ° C pendant 2 semaines. </li><li> Faire stériles DDM2 suppléments et geler en 50 ou 100 aliquotes pendant 1 mois. </li><li> Faire ConA / laminine. </li><li> Manteau lamelles. Optionnel: Faire CNBM stériles et conserver au congélateur jusqu&#39;à quatre mois. </li></ol> Le jour de la culture I. Préparer la solution enzymatique (ES) en hotte à flux laminaire <ol><li> Mettre 5 ml de solution de dissection (DS) dans un tube à centrifuger de 15 ml. </li><li> Peser à 0,8 mg de L-cystéine dans un eppendorf de 0,6 ml et ajouter 150 ul de la DS. Pipeter haut en bas jusqu&#39;à cristaux ont disparu. </li><li> Ajouter 150 ml de DS / cystéine dans les 5 ml DS et bien mélanger. </li><li> Ajouter 50 U (unités) de la papaïne. </li><li> Ajouter 7 ml de 0,1 N Na OH et bien mélanger. Solution sera clair dans les 30 min. lorsqu&#39;il est activé. </li><li> Solution enzymatique filtre avec filtre seringue 0,2 mm dans un tube de 1,5 à vis stériles ml microcentrifugeuse bouchon Papaïne: Voulez-vous 50 U dans DS 5 ml. Chaque lot est légèrement différente, donc faut calculer combien: 37,3 mg / ml et 28,3 U / mg 37,3 x 28,3 = 1055,59 U / ml (1000 ml) 50 U = 47,4 ml </li></ol> II. Faire DDM2 des médias Le jour de la culture: ajouter les suppléments suivants pour faire DDM2 A 10 ml de DMEM ajouter les suppléments, juste avant la culture: <ol><li> 100 ul de la transferrine </li><li> 100 ul Putrescine </li><li> 100 ul Sélénium </li><li> La progestérone 100 ul </li><li> Insuline 50 pl </li><li> 10 ul 20 Hydroxyecdysone </li></ol> Remarque: Assurez assez DDM2 pour toutes les cultures prévues (chaque cerveau plaqué sur une lamelle unique dans un plat individuel de 35 mm de Pétri, 1,5 ms de média / culture) Etape III-VI peut être à une table de laboratoire non stériles fait et devrait être terminé en 45 minutes ou moins. III. Les chrysalides Collection <ol><li> Sélectionnez 10-15 pupes des côtés du flacon sous un microscope à dissection. </li><li> Pupes place dans le couvercle d&#39;une boîte de Pétri de 35 mm à sec. </li></ol> Note: Nous utilisons généralement le cerveau à partir de pupes entre 55-78 heures après la formation puparium (CSA). Il est légèrement plus difficile à disséquer le cerveau de la jeune nymphe depuis le capsules céphaliques ont tendance à s&#39;effondrer, tandis que le niveau global de la croissance des neurites est légèrement inférieure de la plus ancienne des pupes. Dans la souche Canton-S de type sauvage, ces étapes sont reconnus par les yeux qui sont pigmentées brun clair à légèrement rougeâtre, avec des pigments peu ailleurs. IV. Décapitation au microscope disséquant <ol><li> Placez deux gouttes de solution de dissection stériles dans le couvercle de la boîte de Pétri, à côté de pupes. </li><li> Maintenez une seule nymphe avec une pince fine dans la main gauche et l&#39;utilisation d&#39;une aiguille de calibre 27 seringue fixée à une seringue de 1 cc dans la main droite pour enlever la cuticule à l&#39;extrémité antérieure de la chrysalide. </li><li> Presser légèrement avec une pince nymphe et en tant que tête émerge utilisez une aiguille de calibre 27 pour couper le cou. </li><li> Avec la pointe de l&#39;aiguille ou la pince, le transfert à la tête goutte de dissection solution. </li><li> Répétez jusqu&#39;à ce que vous avez de 10 à 15 têtes dans une seule goutte. </li></ol> Retrait V. le cerveau de la tête sous loupe binoculaire <ol><li> Dans chaque main tenir une seringue de 1 cc avec une aiguille de calibre 27. </li><li> Utilisez-les pour positionner la tête de volée dans la goutte de dissection solution si la trompe est tournée vers vous et la surface dorsale de la tête est au nord. </li><li> Insérez l&#39;aiguille à gauche dans la trompe à la broche de la tête en bas et utilisez l&#39;aiguille de droite pour faire une fente dans l&#39;œil droit qui va de la ventrale à la surface dorsale. </li><li> Insérez l&#39;aiguille droite près de la gauche dans la région de la trompe, puis utilisez l&#39;aiguille de gauche à enfoncer délicatement la cuticule sur l&#39;oeil gauche, pousser le cerveau vers la fente de l&#39;oeil droit. Le cerveau doit sortir de la fente sur la droite, relativement intacte, souvent avec deux optiques lobes encore attaché et parfois les tissus oculaires pigmentés ainsi. </li><li> Poussez le cerveau sur le dos de l&#39;aiguille et le transfert d&#39;une goutte de solution saline stérile dans la dissection d&#39;un nouveau stérile, plat de 35 mm de Pétri. </li><li> Recueillir 10-15 cerveaux pour chaque génotype. </li></ol> VI. Suppression des lobes optiques et un traitement enzymatique <ol><li> Dans chaque main tenir une seringue de 1 cc avec une aiguille de calibre 27 et de les employer pour recueillir tous les cerveaux dans une petite zone de la goutte de solution de dissection </li><li> Utiliser des seringues que les outils de coupe pour enlever les lobes optiques de chaque cerveau de sorte que le tissu restant de la culture est la région du cerveau central. </li><li> Utilisez un Pipetman 20 pl, fixé à 5 pl, pour transférer tous les cerveaux d&#39;un génotype unique à un tube Eppendorf contenant 1 ml de solution de papaïne stérile. </li><li> Cerveaux incubés dans la papaïne pendant 10-15 minutes sur un rotateur réglé à 60 rpm. </li><li> Centrifuger à 3000 rpm pendant 3 minutes. </li></ol> A l&#39;intérieur capuche à flux laminaire stérile - toutes les étapes où le tissu est exposé à l&#39;air doit être fait dans une hotte à flux laminaire pour les étapes restantes. VII. Lavage <ol><li> Retirer et remplacer la solution d&#39;enzyme avec une solution stérile de dissection. </li><li> Centrifuger à 3000 rpm pendant 3 minutes. </li><li> Laver avec une solution stérile de disséquer un total de 3 fois. </li><li> Retirer salines dissection et la remplacer par DDM2. </li><li> Centrifuger et laver un total de 2 fois avec DDM2. </li><li> Transfert des cerveaux et des médias dans un plat stériles de 35 mm de Pétri. </li></ol> VIII. Trituration et de placage sous loupe binoculaire <ol><li> Placer 5 ul d&#39;DDM2 dans le centre d&#39;une lamelle. </li><li> Transférer 1 cerveau pour cette goutte d&#39;DDM2 avec une pointe de jaune. </li><li> Sous le microscope, l&#39;utilisation d&#39;aiguilles de calibre 27 à la place du cerveau dés en petits morceaux (devrait prendre &lt;1 minute). </li><li> Sous guidage visuel, casser la pointe d&#39;une pipette en verre qui a été tirée en une fine pointe de sorte que les plus gros morceaux peuvent être doucement aspiré dans la pointe de la pipette et expulsés de nouveau dans le milieu. Nous utilisons des tubes d&#39;aspiration bouche qui sont livrés en standard avec 100 ul de verre hématocrite capillaire. Remarque: Prenez soin de ne pas obtenir des bulles dans les médias et vous aurez besoin de déterminer empiriquement la pipette meilleure taille à utiliser. Les bons vous permettent de dissocier le cerveau avec des cellules individuelles et encore quelques grosses touffes, en environ 30 secondes / cerveau. Lorsque vous regardez ces à haute puissance, environ 10-20% des neurones devrait encore avoir quelques procédés restants et il y aura encore quelques grosses touffes. Si vous dissocier trop, alors toutes les cellules seront rondes et ils ont subi des dommages tant qu&#39;ils ne seront pas survivre. Cette étape nécessite de la pratique et doit être fait rapidement car il ya une grande surface à volume élevé et de l&#39;osmolalité et le pH de la goutte d&#39;DDM2 peuvent changer rapidement. Tissus doit être placé dans incubateur à CO 2 aussi vite que possible. </li><li> Écrire sur le couvercle boîte de Pétri: «génotype, la date et l&#39;heure&quot;. </li><li> Mettez la boîte de Petri de 35 mm dans un plat de Pétri de 100 mm (avec Kimwipe humide) et laissez s&#39;installer dans l&#39;incubateur CO 2 pendant 30 min. </li><li> Recouvrir le plat de 35 mm avec 1,5 ml de DDM2. </li><li> Gardez les cultures dans l&#39;incubateur à 5% de CO 2 incubateur (23 ° C). Remarque: Si vous n&#39;avez pas accès à un refroidissement incubateur à CO 2, utiliser une norme de mammifères incubateur de culture tissulaire et soit le mettre dans une pièce fraîche et chauffer à 23 ° C, ou mis dans le laboratoire, éteignez temp, et l&#39;utilisation glace dans un bac en bas de l&#39;incubateur pour réguler température ambiante autour. </li></ol> IX. Cellules nourricières <ol><li> Le Jour 1 (le lendemain), ajoutez 0,5 ml de CNBM/B27 (milieux conditionnés) de faire DDM2 03h01: CNBM/B27. </li><li> Le Jour 5 remplacer 1 ml de médias pour 03h01 DDM2: CNBM/B27. </li><li> Gardez les cellules d&#39;alimentation avec 3:01 DDM2: CNBM/B27 tous les 4-5 jours. Note: Les cultures peuvent être cultivées sans que les médias non-neuronales conditionné mais les neurones apparaissent un peu plus fragiles et sont plus difficiles à utiliser dans des expériences d&#39;électrophysiologie. </li></ol>

<ol>
	<li>Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culturing Nerve Cells. , (1991).</li><li>Rohrbough, J., O'Dowd, D. K., Baines, R. A., Broadie, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+bases+of+behavioral+plasticity:+Establishing+and+Modifying+synaptic+circuits+in+the+Drosophila+Genetic+System.">Cellular bases of behavioral plasticity: Establishing and Modifying synaptic circuits in the Drosophila Genetic System.</a> J. Neurobiol. 54, 254-271 (2003).</li><li>O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-gated+currents+and+firing+properties+of+embryonic+Drosophila+neurons+grown+in+a+chemically+defined+medium.">Voltage-gated currents and firing properties of embryonic Drosophila neurons grown in a chemically defined medium.</a> J. Neurobiol. 27, 113-126 (1995).</li><li>Lee, D., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+excitatory+synaptic+transmission+mediated+by+nAChR+in+cultured+Drosophila+neurons.">Fast excitatory synaptic transmission mediated by nAChR in cultured Drosophila neurons.</a> J. Neurosci. 19, 5311-5321 (1999).</li><li>Su, H., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+synaptic+currents+in+Drosophila+mushroom+body+Kenyon+cells+are+mediated+by+alpha-bungarotoxin-sensitive+nAChRs+and+picrotoxin-sensitive+GABA+receptors.">Fast synaptic currents in Drosophila mushroom body Kenyon cells are mediated by alpha-bungarotoxin-sensitive nAChRs and picrotoxin-sensitive GABA receptors.</a> J. Neurosci. 23, 9246-9253 (2003).</li><li>Jiang, S. A., Campusano, J. M., Su, H., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+mushroom+body+Kenyon+cells+generate+spontaneous+calcium+transients+mediated+by+PLTX-sensitive+calcium+channels.">Drosophila mushroom body Kenyon cells generate spontaneous calcium transients mediated by PLTX-sensitive calcium channels.</a> J. Neurophysiol. 94, 491-500 (2005).</li><li>Campusano, J. M., Su, H., Jiang, S. A., Sicaeros, B., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=nAChR-mediated+calcium+responses+and+plasticity+in+Drosophila+Kenyon+cells.">nAChR-mediated calcium responses and plasticity in Drosophila Kenyon cells.</a> Develop Neurobiol. 67, 1520-1532 (2007).</li><li>Oh, H. -. W., Campusano, J. M., Hilgenberg, L. G. W., Sun, X., Smith, M. A., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+analysis+of+chemical+synapses+and+gap+junctions+between+Drosophila+brain+neurons+in+culture.">Ultrastructural analysis of chemical synapses and gap junctions between Drosophila brain neurons in culture.</a> Develop Neurobiol. , (2007).</li></ol>

Neurones récoltés à partir de cerveaux de rongeurs embryonnaire / postnatale peut être cultivé en culture cellulaire primaire, où elles s&#39;étendent neurites et la forme fonctionnelle des connexions synaptiques. Méthodes de préparation de ces cultures sont bien établies et d&#39;études dans les cultures neuronales de rongeurs ont joué un rôle crucial dans l&#39;identification des gènes et des facteurs environnementaux impliqués dans la régulation de la formation des synapses et de la fonction (Banker et Goslin, 1991). Alor...

Lamelles de revêtement: Mettre des lamelles autoclavés dans un plat de Pétri de 60 mm. Pipeter 5 ul de Con A mix / laminine au centre de chaque lamelle. Placer dans 37 C incubateur pendant 2 heures. Rincez lamelles 3x avec 100 ul d&#39;eau autoclavée chacun. Utilisez l&#39;aspirateur attaché à une pipette Pasteur stérile. Au cours du dernier rinçage, ramasser la lamelle avec une pince et sec des deux côtés. Transférer la lamelle d&#39;un plat de Pétri de 35mm. Stocker à température ambiante pendant jusqu&#39;à un mois.

primary neuronal cultures from the brains of late stage drosophila pupae

Dans cette vidéo, nous démontrons la préparation des cultures primaires de neurones du cerveau des drosophiles stade avancé pupes. La procédure commence par l&#39;enlèvement des cerveaux des animaux à 70-78 heures après la formation puparium. Les cerveaux isolés sont présentés après une brève incubation de la papaïne suivie de plusieurs lavages en milieu de croissance sans sérum. Le processus de dissociation mécanique de chaque cerveau dans une baisse de 5 ul des médias sur une lamelle est illustré. Les axones et des dendrites des neurones post-mitotiques sont fait une embardée hors près du soma au cours de dissociation, mais les neurones commencent à se régénérer les processus au sein de quelques heures de placage. Les images montrent des cultures vivantes moins 2 jours. Neurones continuer à élaborer des processus pendant la première semaine de la culture. Populations neuronales spécifiques peuvent être identifiées en culture en utilisant GAL4 lignes pour conduire l&#39;expression tissu spécifique de marqueurs fluorescents tels que la GFP ou DP. Enregistrements de cellules entières ont démontré les neurones cultivés forme fonctionnelle, spontanément actifs synapses cholinergiques et GABAergiques. Un segment courte vidéo illustre la dynamique du calcium dans les neurones cultivés en utilisant Fura-2 comme un colorant indicateur de calcium pour contrôler transitoires calciques spontanées et la nicotine réponses de calcium évoquée dans un plat de neurones en culture. Ces cultures du cerveau pupe sont un système modèle utile dans lesquels des outils génétiques et pharmacologiques peuvent être utilisés pour identifier les facteurs intrinsèques et extrinsèques qui influencent la formation et la fonction des synapses centrales.

Watch this Scientific Journal Video about Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae at JoVE.com

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

Cette vidéo montre la préparation de cultures primaires de neurones du cerveau des drosophiles stade avancé pupes. Vues des cultures vivantes montrent neurites et l&#39;imagerie des niveaux de calcium en utilisant Fura-2.

Cultures neuronales primaires du cerveau des drosophiles stade tardif pupes

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

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15.0K vues.  University of California, Irvine (UCI). Cette vidéo montre la préparation de cultures primaires de neurones du cerveau des drosophiles stade avancé pupes. Vues des cultures vivantes montrent neurites et l'imagerie des niveaux de calcium en utilisant Fura-2.

Vidéo: Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

Préparation des cultures de neurones d&#39;embryons de drosophile Midgastrula scène

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

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Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information into directional movement towards the appropriate target cell. To fully understand how guidance information is transmitted from the cell surface to the underlying dynamic cytoskeletal networks, one needs a model system suitable for live cell imaging of protein dynamics at high temporal and spatial resolution. Typical vertebrate growth cones are too small to quantitatively analyze F-actin and microtubule dynamics. Neurons from the sea hare Aplysia californica are 5-10 times larger than vertebrate neurons, can easily be kept at room temperature and are very robust cells for micromanipulation and biophysical measurements. Their growth cones have very defined cytoplasmic regions and a well-described cytoskeletal system. The neuronal cell bodies can be microinjected with a variety of probes for studying growth cone motility and guidance. In the present protocol we demonstrate a procedure for dissection of the abdominal ganglion, culture of bag cell neurons and setting up an imaging chamber for live cell imaging of growth cones.

Aplysia californica neurons develop large growth cones in culture that are excellent for high-resolution imaging of growth cone motility and guidance. Here, we present a protocol for dissection and plating of Aplysia bag cell neurons as well as for setting up a chamber for live cell imaging.

Cultures de cellules neuronales de l&#39;aplysie pour l&#39;imagerie haute résolution des cônes de croissance

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information ...

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

Primaire Dissocié cultures Mésencéphale dopamine cellulaire du nouveau-né contre les rongeurs

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

La préparation des cultures primaires de cellules hématopoïétiques de la moelle osseuse murine pour l&#39;électroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons. The neurons are large (up to 1 mm in diameter) and identifiable, with distinct sizes, shapes, positions and pigmentations, and the cell bodies are externally exposed in five paired ganglia distributed throughout the body of the animal. These properties have allowed investigators to delineate the circuitry underlying specific behaviors in the animal1. The monosynaptic connection between sensory and motor neurons is a central component of the gill-withdrawal reflex in the animal, a simple defensive reflex in which the animal withdraws its gill in response to tactile stimulation of the siphon. This reflex undergoes forms of non-associative and associative learning, including sensitization, habituation and classical conditioning. Of particular benefit to the study of synaptic plasticity, the sensory-motor synapse can be reconstituted in culture, where well-characterized stimuli elicit forms of plasticity that have direct correlates in the behavior of the animal2,3. Specifically, application of serotonin produces a synaptic strengthening that, depending on the application protocol, lasts for minutes (short-term facilitation), hours (intermediate-term facilitation) or days (long-term facilitation). In contrast, application of the peptide transmitter FMRFamide produces a synaptic weakening or depression that, depending on the application protocol, can last from minutes to days (long-term depression). The large size of the neurons allows for repeated sharp electrode recording of synaptic strength over periods of days together with microinjection of expression vectors, siRNAs and other compounds to target specific signaling cascades and molecules and thereby identify the molecular and cell biological steps that underlie the changes in synaptic efficacy.
An additional advantage of the Aplysia culture system comes from the fact that the neurons demonstrate synapse-specificity in culture4,5. Thus, sensory neurons do not form synapses with themselves (autapses) or with other sensory neurons, nor do they form synapses with non-target identified motor neurons in culture. The varicosities, sites of synaptic contact between sensory and motor neurons, are large enough (2-7 microns in diameter) to allow synapse formation (as well as changes in synaptic morphology) with target motor neurons to be studied at the light microscopic level.
In this video, we demonstrate each step of preparing sensory-motor neuron cultures, including anesthetizing adult and juvenile Aplysia, dissecting their ganglia, protease digestion of the ganglia, removal of the connective tissue by microdissection, identification of both sensory and motor neurons and removal of each cell type by microdissection, plating of the motor neuron, addition of the sensory neuron and manipulation of the sensory neurite to form contact with the cultured motor neuron.

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

Primary cultures of Aplysia sensory-motor neurons provide a model preparation for studying synapse formation and synaptic plasticity in vitro. This video demonstrates the identification and microdissection of sensory and motor neurons from Aplysia ganglia as well as the methods for establishing and maintaining sensory-motor neurons in culture.

Préparation des Aplysie Sensori-motrice cultures de cellules neuronales

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons.  The neurons are large (up ...

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

Préparation de développement et de l&#39;adulte Drosophile Cerveaux et la rétine d&#39;imagerie en temps réel

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

Establishing Primary Adult Fibroblast Cultures From Rodents

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.

This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.

Établir des cultures primaires de fibroblastes adultes à partir de rongeurs

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

Cultures de cellules primaires à partir Drosophile Embryons Gastrula

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

Naïve cellules souches adultes Isolation de cultures de fibroblastes humains primaires

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This methods paper describes how to isolate mid-to-late stage follicles (Stage 10B-14) and utilize them to provide new insights into the molecular and morphologic events occurring during tight windows of developmental time. Isolated follicles can be used for a variety of experimental techniques, including in vitro development assays, live imaging, mRNA expression analysis and western blot analysis of proteins. Follicles at Stage 10B (S10B) or later will complete development in culture; this allows one to combine genetic or pharmacologic perturbations with in vitro development to define the effects of such manipulations on the processes occurring during specific periods of development. Additionally, because these follicles develop in culture, they are ideally suited for live imaging studies, which often reveal new mechanisms that mediate morphological events. Isolated follicles can also be used for molecular analyses. For example, changes in gene expression that result from genetic perturbations can be defined for specific developmental windows. Additionally, protein level, stability, and/or posttranslational modification state during a particular stage of follicle development can be examined through western blot analyses. Thus, stage-specific isolation of Drosophila follicles provides a rich source of information into widely conserved processes of development and morphogenesis.

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Stage-specific isolation of mid-to-late Drosophila follicles is useful for a variety of purposes. Such follicles develop in culture, which allows for genetic and/or pharmacologic manipulations to be coupled with in vitro development assays and live imaging. Additionally, follicles can be used for molecular studies, such as isolating mRNA and protein.

L&#39;utilitaire de la phase spécifique-mi-à la fin<em&gt; Drosophila</em&gt; Folliculo Isolation

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This ...