Ce travail a été soutenu par le prix IMPACT du Département de radio-oncologie de l&#39;Université d&#39;Alabama-Birmingham Comprehensive Cancer Center, la Fondation du cancer de l&#39;enfance de combat, et l&#39;Ange Gabrielle Fondation (à ESY.).

1. Culture cellulaire <ol><li> Cellules neuronales Culture et les traitent selon les besoins. </li><li> Récolte des cellules dans des tubes de 15 ml: médias aspirés, rincer avec du tampon phosphate salin (PBS, de calcium et de magnésium libre), ajouter la trypsine, la collecte en tubes de 15 ml et neutraliser la trypsine avec appropriées milieux contenant du sérum. </li><li> Centrifuger à 1000 xg pendant 5 min. </li><li> Aspirer les médias. </li><li> Remettre en suspension les cellules dans du PBS. </li><li> Centrifuger à 1000 xg pendant 5 min. </li><li> Aspirer les médias et remettre les cellules en PBS frais. </li><li> Compter les cellules à l&#39;aide d&#39;un hématimètre ou votre compteur de cellules préféré. </li><li> Des échantillons de cellules doit être préparée immédiatement avant de commencer le dosage. </li><li> Tous les échantillons doivent être manipulés à la lumière noire ou jaune pour éviter d&#39;endommager l&#39;ADN de la lumière ultraviolette. </li></ol> 2. Comet Assay 1. Préparation de la lame <ol><li> Faire fondre 1% (1 g / 100 ml 1X Tris Base, acide borique, de l&#39;EDTA, la tuberculose agaroseE) dans un four micro-ondes pendant 3 min jusqu&#39;à ce que tous les granules disparaissent. </li><li> Tremper les lames dans l&#39;agarose fondu et essuyez un côté nettoyer avec un Kimwipe. Laisser l&#39;agarose sécher à l&#39;air d&#39;un film transparent. Cela peut être fait à l&#39;avance et les glissières peuvent être stockés. </li></ol> 2. Comet Assay neutre <ol><li> Une solution de lyse froid (2,5 M NaCl, 100 mM d&#39;EDTA pH 10, 10 mM Tris Base, 1% de sodium lauryl sarcosinate, et 1% de Triton X-100, pH 10), à 4 ° C pendant au moins 1 heure avant utilisation. </li><li> 2.2.2. Faire fondre 1% d&#39;agarose à bas point de fusion (1 g / 100 ml dans 1X Tris Base, de l&#39;acide borique, de l&#39;EDTA) dans un micro-ondes pendant 3 minutes jusqu&#39;à ce que tous les granules disparaissent. L&#39;agarose a besoin d&#39;être refroidi à 37 ° C dans un bain d&#39;eau pour éviter l&#39;induction artificielle de la queue de la comète. Idéalement, l&#39;agarose doit être refroidie pendant une demi-heure avant utilisation. </li><li> 2.2.3. Diluer la suspension de cellules de sorte qu&#39;il ya 100.000 cellules par ml. Mélanger la suspension cellulaire avec le faible point de fusion d&#39;agarose (à 37 ° C)un ratio de 1:10 (v / v), homogénéiser brièvement au vortex, et immédiatement pipette 50 ul sur la diapositive Comet. Utiliser le côté de la pointe de pipette pour répandre la suspension de cellules uniformément sur toute la surface de l&#39;échantillon. Chaque groupe de traitement doit être au moins en triple exemplaire. </li><li> Lieu glisse à plat au réfrigérateur pendant 30 min jusqu&#39;à ce que le cercle apparaît dans la périphérie de la lame. </li><li> Prérefroidissement la solution de lyse et les diapositives plonger dans cette solution pendant 30 min à l&#39;obscurité à 4 ° C (ou au réfrigérateur). </li><li> Décanter ou aspirer la solution de lyse et ajouter 1X tampon d&#39;électrophorèse neutre (Tris base, l&#39;acide borique, de l&#39;EDTA, 1X TBE). Laisser les lames dans cette mémoire tampon pendant une demi-heure au réfrigérateur. </li><li> Ajouter prérefroidi tampon 1X électrophorèse neutre (TBE) dans la chambre d&#39;électrophorèse, placer les lames dans plateau coulissant électrophorèse. Aligner lames afin qu&#39;elles soient équidistantes à partir d&#39;électrodes. </li><li> Verser 1X tampon d&#39;électrophorèse neutre jusqu&#39;à 0,2 cm au-dessus des diapositives. Excès de tampon sera interFère par électrophorèse. </li><li> Régler la tension d&#39;alimentation de 1 V par cm (mesurée électrode à l&#39;autre) et tourner pendant 30 min à 4 ° C (ou la chambre froide) dans l&#39;obscurité. </li></ol> 3. Comet Assay alcalin <ol><li> Une solution de lyse froid (2,5 M NaCl, 100 mM d&#39;EDTA pH 10, 10 mM Tris Base, 1% de sodium lauryl sarcosinate, et 1% de Triton X-100, pH 10), à 4 ° C pendant au moins 1 heure avant utilisation. </li><li> Faire fondre 1% d&#39;agarose à faible point de fusion (1 g / 100 ml 1X Tris Base, de l&#39;acide borique, de l&#39;EDTA) dans un micro-ondes pendant 3 minutes jusqu&#39;à ce que tous les granules disparaissent. Puis refroidir dans un bain à 37 ° C pendant au moins 30 min. </li><li> Combiner des cellules à 1 x 105 / ml avec fondu à faible point de fusion d&#39;agarose (à 37 ° C) dans un rapport de 1:10 (v / v), homogénéiser brièvement au vortex, et immédiatement pipette 50 ul sur la diapositive Comet. Utiliser le côté de la pointe de pipette pour répandre la suspension de cellules uniformément sur toute la surface de l&#39;échantillon. Chaque groupe de traitement doit être au moins en triple exemplaire. </li><li> Lieu glissées plat au réfrigérateur pendant 30 min jusqu&#39;à ce que le cercle apparaît dans la périphérie de la lame. </li><li> Immerger les lames dans une solution de lyse refroidies et laisser à 4 ° C pendant 1 heure ou toute la nuit dans l&#39;obscurité. </li><li> Sortir les lames de la solution de lyse, les lames de vidange et de rinçage une fois avec du tampon de neutralisation froid pendant 5 minutes pour éliminer le détergent résiduel et les sels avant l&#39;étape de déroulement en milieu alcalin. </li><li> Placer les lames dans une chambre remplie d&#39;électrophorèse sur gel d&#39;électrophorèse tampon prérefroidi fraîchement préparé (300 mM NaOH, 1 mM EDTA,&gt; 13 pH) ne doit pas dépasser 0,5 cm au-dessus des diapositives. Aligner lames afin qu&#39;elles soient équidistantes à partir d&#39;électrodes. </li><li> Laissez diapositives s&#39;asseoir dans le tampon alcalin pendant 30 min dans l&#39;obscurité pour permettre déroulement de l&#39;ADN et l&#39;expression de l&#39;alcali-responsable des dégâts. </li><li> Régler la tension d&#39;alimentation de 1 V par cm (mesurée électrode à l&#39;autre) et tourner pendant 30 min à 4 ° C (ou la chambre froide). </li></ol> 4. Fixation et StaLes cellules sable de l&#39;examen <ol><li> Égoutter l&#39;excès de tampon d&#39;électrophorèse </li><li> Plonger les lames dans prérefroidi de l&#39;eau distillée pendant 5 min à température ambiante. </li><li> Immerger les lames dans prérefroidi l&#39;éthanol à 70% pendant 5 min à température ambiante. </li><li> Des échantillons secs pendant la nuit. Ne pas exposer à la lumière des diapositives. Échantillons peuvent être conservés pendant des mois à la température ambiante avant de marquer à ce stade. </li><li> Colorer les lames en les immergeant dans SYBR Green (dilué dans du PBS 1X) pendant 20 min au réfrigérateur. </li><li> Retirer les lames et laissez-les sécher complètement dans le noir. L&#39;agarose devient transparent lorsqu&#39;il est complètement sec. </li></ol> 4. Acquisition et analyse d&#39;images <ol><li> Acquérir des images à l&#39;aide microscope à fluorescence réglé sur le filtre vert (Zeiss AxioVision) et d&#39;analyser l&#39;aide du logiciel Comet Assay (Perceptive Instruments). </li><li> Cliquez sur &quot;tête de comète&quot; (le noyau) et le logiciel calcule les paramètres, y compris le moment et le montant moyen de la queue de l&#39;ADN dans lenoyau. </li><li> Analyser au moins 200 cellules par traitement. </li><li> Exporter des données vers Microsoft Excel. </li><li> Calculer la moyenne instant queue pour chaque groupe de traitement et les données des placettes, le cas échéant. </li></ol> 5. Les résultats représentatifs Un exemple d&#39;analyse test des comètes sur des cellules neuronales est illustré à la figure 2 et la figure 3. Dans ce cas, l&#39;irradiation des cellules neuronales induit dommages à l&#39;ADN. Que les cellules sont soumises au champ électrique, l&#39;ADN migre à des vitesses différentes en raison des différences de taille, qui est ensuite analysées en utilisant le logiciel essai de comète. Plus le dommage à l&#39;ADN, l&#39;ADN migre plus loin la hors du noyau. Cela diminue l&#39;intensité de fluorescence dans le noyau qui est ensuite capté par le logiciel et les résultats dans le moment plus de queue. Tableau 1 présente un tableau représentant de l&#39;analyse et de test des comètes figure 4 montre une représentationgraphe comparant sentant dommages à l&#39;ADN dans les cellules neuronales après une radiothérapie, telle que mesurée par le test des comètes. <img src="/files/ftp_upload/50049/50049fig1.jpg" alt="Figure 1"/> Figure 1. Schéma du test des comètes. <a href="https://www.jove.com/files/ftp_upload/50049/50049fig1large.jpg" target="_blank">Cliquez ici pour agrandir la figure</a> . <img src="/files/ftp_upload/50049/50049fig2.jpg" alt="Figure 2"/> Figure 2. Images représentatifs de cellules neuronales (A) et sans (B) avec queue de comète. <img src="/files/ftp_upload/50049/50049fig3.jpg" alt="Figure 3"/> Figure 3. Analyse test des comètes en utilisant le logiciel Comet Assay. (A) des captures d&#39;écran représentatifs de l&#39;image acquise avec Carl Zeiss microscope à fluorescence et analysées à l&#39;aide CometLogiciel de test. (B) En cliquant sur ​​le noyau ou la «tête de comète&quot; (entouré en vert) génère une carte graphique fluorescent et (entouré en jaune) et une table (C) de données (entouré en rouge). <a href="https://www.jove.com/files/ftp_upload/50049/50049fig3large.jpg" target="_blank">Cliquez ici pour agrandir la figure</a> . <img src="/files/ftp_upload/50049/50049fig4.jpg" alt="Figure 4"/> Figure 4. Graphe représentant obtenue en reportant le moment queue moyenne obtenue par l&#39;analyse de dommages à l&#39;ADN dans irradiés et non irradiés en utilisant des cellules neuronales test des comètes neutre. Comme prévu, le rayonnement 3Gy (rayons X) induit dommages à l&#39;ADN tel que représenté par le moment queue moyenne plus élevée dans les cellules irradiées neuronales. Montré est le moment queue moyenne (+ / - erreur standard), ** P &lt;0,01. <img src="/files/ftp_upload/50049/50049table1.jpg" alt="Tableau 1"/> Tableau 1. </strong&gt; tableau représentant obtenue par analyse test des comètes sur irradiés et non irradiés cellules neuronales. <a href="https://www.jove.com/files/ftp_upload/50049/50049table1large.jpg" target="_blank">Cliquez ici pour agrandir la figure</a> .

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Aucun conflit d&#39;intérêt déclaré.

Le test des comètes a la capacité unique d&#39;analyse de cellules individuelles. Ceci est avantageux pour l&#39;identification des sous-populations de cellules qui démontrent une réponse différentielle aux agents cytotoxiques. A quelques limitations pratiques doivent être prises en compte. Le nombre de cellules qui peuvent être évalués individuellement peuvent varier selon les individus. La taille de l&#39;échantillon doit être augmenté s&#39;il ya variation des dommages à l&#39;ADN dans une population. Vi...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires (optionnel) </td></tr><tr><td> Tubes de 1,5 ml </td><td> Santa Cruz Biotechnology, Inc </td><td> Sc-200271 </td><td></td></tr><tr><td> 10X TBE (Tris base, l&#39;acide borique, de l&#39;EDTA) </td><td> Fisher Scientific </td><td> BP13331 </td><td></td></tr><tr><td> Tube de 15 ml </td><td> Fisher Scientific </td><td> 0553851 </td><td></td></tr><tr><td> Agarose </td><td> Sigma </td><td> A5093 </td><td></td></tr><tr><td> Le papier d&#39;aluminium </td><td> Fisher Scientific </td><td> 01213101 </td><td></td></tr><tr><td> Gobelet </td><td> Fisher Scientific </td><td> FB102300 </td><td></td></tr><tr><td> Centrifuger </td><td> Thermo Scientific </td><td> 75004261PR </td><td></td></tr><tr><td> Logiciel Comet Assay </td><td> Comet Assay IV Système d&#39;analyse d&#39;images </td><td></td><td></td></tr><tr><td> Cylindre </td><td> Fisher Scientific </td><td> 08555F </td><td></td></tr><tr><td> EDTA </td><td> GIBCO </td><td> 15575 </td><td></td></tr><tr><td> Chambre d&#39;électrophorèse </td><td> Thermo Scientific </td><td> 09528101 </td><td></td></tr><tr><td> L&#39;éthanol </td><td> Fisher Scientific </td><td> A407P4 </td><td></td></tr><tr><td> Microscope à fluorescence </td><td> Zeiss Axio Vision </td><td></td><td> Toute microscope à fluorescence avec un filtre vert suffira </td></tr><tr><td> Hémacytomètre </td><td> Fisher Scientific </td><td> 0267152 </td><td></td></tr><tr><td> D&#39;agarose à faible point de fusion </td><td> Promega </td><td> V2111 </td><td></td></tr><tr><td> Micro-onde </td><td> Sears </td><td></td><td></td></tr><tr><td> Saline tamponnée au phosphate, calcium libre, magnésium libre </td><td> HyClone </td><td> SH3025601 </td><td></td></tr><tr><td> Alimentation </td><td> BioRad </td><td> 1645050 </td><td></td></tr><tr><td> Réfrigérateur </td><td> Sears </td><td></td><td></td></tr><tr><td> Règle </td><td> Staples </td><td></td><td></td></tr><tr><td> Glissement </td><td> Fisher Scientific </td><td> 12550143 </td><td></td></tr><tr><td> De chlorure de sodium </td><td> Sigma </td><td> S7653 </td><td></td></tr><tr><td> L&#39;hydroxyde de sodium </td><td> Sigma </td><td> S5881 </td><td></td></tr><tr><td> Sodium lauryl sarcosinate </td><td> Fisher Scientific </td><td> S529 </td><td></td></tr><tr><td> SYBR Green </td><td> Invitrogen </td><td> S7585 </td><td></td></tr><tr><td> Plateau </td><td> Fissa science </td><td> 15242B </td><td></td></tr><tr><td> Base de tris </td><td> Sigma </td><td> T6066 </td><td></td></tr><tr><td> Triton-X 100 </td><td> Sigma </td><td> T8787 </td><td></td></tr><tr><td> Trypsine </td><td> HyClone </td><td> SH3023601 </td><td></td></tr><tr><td> Vortex </td><td> Fisher Scientific </td><td> 02216108 </td><td></td></tr><tr><td> Bain d&#39;eau </td><td> Fisher Scientific </td><td> 154622Q </td><td></td></tr></tbody></table>

assaying dna damage in hippocampal neurons using the comet assay

Un certain nombre de médicaments ciblent les voies de réparation de l&#39;ADN et induire des cellules tuer en créant des lésions de l&#39;ADN. Ainsi, des procédés pour mesurer directement lésions de l&#39;ADN ont été largement évalués. Les méthodes traditionnelles sont longs, coûteux, et nécessitent beaucoup de ressources de réplication des cellules. En revanche, le test des comètes, un essai de cellule unique électrophorèse sur gel, est plus rapide, non invasif, peu coûteux, mesure directe et sensible de dommages à l&#39;ADN et la réparation. Toutes les formes de dommages à l&#39;ADN ainsi que réparation de l&#39;ADN peut être visualisé au niveau de la cellule unique à l&#39;aide de cette technique puissante. Le principe qui sous-tend le test des comètes est que l&#39;ADN intact est très ordonnée alors dommages à l&#39;ADN perturbe cette organisation. Les suintements d&#39;ADN endommagés dans la matrice d&#39;agarose et lorsqu&#39;il est soumis à un champ électrique, l&#39;ADN chargé négativement migre vers la cathode qui est chargée positivement. Les brins d&#39;ADN endommagés grands ne sont pas en mesure de migrer loin du noyau. Dommages à l&#39;ADNcrée des fragments d&#39;ADN plus petits qui voyagent plus loin que l&#39;ADN intact. Comet Assay, un logiciel d&#39;analyse d&#39;images, mesures et compare l&#39;intensité globale fluorescent de l&#39;ADN dans le noyau avec l&#39;ADN qui a migré hors du noyau. Signal fluorescent de l&#39;ADN migré est proportionnelle à endommager l&#39;ADN. ADN queue plus claire signifie lésions de l&#39;ADN accrue. Certains des paramètres qui sont mesurés sont actuellement queue qui est à la fois une mesure de la quantité d&#39;ADN et de la distribution de l&#39;ADN dans la queue de longueur de queue, et le pourcentage d&#39;ADN dans la queue. Ce test permet de mesurer réparation de l&#39;ADN ainsi car la résolution des lésions de l&#39;ADN signifie la réparation a eu lieu. La limite de sensibilité est d&#39;environ 50 cassures par 1,2 cellule diploïde mammalien. Les cellules traitées avec des agents endommageant l&#39;ADN, tels que l&#39;étoposide, peut être utilisé en tant que contrôle positif. Ainsi, le test des comètes est une procédure rapide et efficace pour mesurer dommages à l&#39;ADN.

Watch this Scientific Journal Video about Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay at JoVE.com

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

Le test des comètes est un moyen efficace de détection de ruptures simple et double brin, y compris alcali-labiles sites et DNA-DNA/DNA-protein liens croisés sur l&#39;ADN dans toutes les cellules, y compris les neurones hippocampiques. La méthode tire parti de la migration différentielle de l&#39;ADN dans un champ électrique dû à des différences de quantité de dommages de l&#39;ADN.

Le dosage de dommages de l&#39;ADN dans les neurones hippocampiques Utilisation de l&#39;essai de comète

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been ...

assaying-dna-damage-in-hippocampal-neurons-using-the-comet-assay

Research

JoVE Journal

Neuroscience

27.1K vues.  University of Alabama-Birmingham. Le test des comètes est un moyen efficace de détection de ruptures simple et double brin, y compris alcali-labiles sites et DNA-DNA/DNA-protein liens croisés sur l'ADN dans toutes les cellules, y compris les neurones hippocampiques. La méthode tire parti de la migration différentielle de l'ADN dans un champ électrique dû à des différences de quantité de dommages de l'ADN.

Vidéo: Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

Assaying &beta;-amyloid Toxicity using a Transgenic C. elegans Model

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant mechanism(s) of toxicity are still unclear. A&beta; is also deposited intramuscularly in Inclusion Body Myositis, a severe human myopathy. The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express human A&beta;. Depending on the tissue or timing of A&beta; expression, transgenic worms can have readily measurable phenotypes that serve as a read-out of A&beta; toxicity. For example, transgenic worms with pan-neuronal A&beta; expression have defects is associative learning (Dosanjh et al. 2009), while transgenic worms with constitutive muscle-specific expression show a progressive, age-dependent paralysis phenotype (Link, 1995; Cohen et al. 2006). One particularly useful C. elegans model employs a temperature-sensitive mutation in the mRNA surveillance system to engineer temperature-inducible muscle expression of an A&beta; transgene, resulting in a reproducible paralysis phenotype upon temperature upshift (Link et al. 2003). Treatments that counter A&beta; toxicity in this model [e.g., expression of a protective transgene (Hassan et al. 2009) or exposure to Ginkgo biloba extracts (Wu et al. 2006)] reproducibly alter the rate of paralysis induced by temperature upshift of these transgenic worms. Here we describe our protocol for measuring the rate of paralysis in this transgenic C. elegans model, with particular attention to experimental variables that can influence this measurement.

Assaying &amp;#946;-amyloid Toxicity using a Transgenic C. elegans Model

The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express the human &beta;-amyloid peptide (A&beta;). Induced expression of A&beta; in C. elegans muscle leads to a rapid, reproducible paralysis phenotype that can be used to monitor treatments that modulate A&beta; toxicity.

Dosage β-amyloïde toxicité utilisant un transgéniques C. elegans</emModèle>

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant ...

Recherche

Neurosciences

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

Neuromodulation et mitochondriale de transport: Live in Imaging neurones de l&#39;hippocampe sur des durées longues

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

L&#39;isolement et la culture de neurones de l&#39;hippocampe de souris pendant la grossesse

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Calcium Phosphate Transfection of Primary Hippocampal Neurons

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.

Phosphate de calcium transfection de primaire neurones de l&#39;hippocampe

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.
Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.

This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved detail.

Imagerie épines dendritiques des neurones de l&#39;hippocampe de rat primaire utilisant structuré Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activation of glutamate receptors that promotes Ca2+ influx and mitochondrial Ca2+ overload. A substantial change in intracellular Ca2+ homeostasis or remodeling of intracellular Ca2+ homeostasis may favor neuron damage in old neurons. For investigating Ca2+ remodeling in aging we have used live cell imaging in long-term cultures of rat hippocampal neurons that resemble in some aspects aged neurons in vivo. For this end, hippocampal cells are, in first place, freshly dispersed from new born rat hippocampi and plated on poli-D-lysine coated, glass coverslips. Then cultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, cultured neurons are loaded with fura2 and subjected to measurements of cytosolic Ca2+ concentration using digital fluorescence ratio imaging. Third, cultured neurons are transfected with plasmids expressing a tandem of low-affinity aequorin and GFP targeted to mitochondria. After 24 hr, aequorin inside cells is reconstituted with coelenterazine and neurons are subjected to bioluminescence imaging for monitoring of mitochondrial Ca2+ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca2+ responses to relevant stimuli as for example the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure may yield new insights as to how aging influence cytosolic and mitochondrial Ca2+ responses to selected stimuli as well as the testing of selected drugs aimed at preventing neuron cell death in age-related diseases.

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Intracellular Ca2+ remodeling in aging may contribute to excitotoxicity and neuron damage, processes mediated by Ca2+ overload. We aimed at investigating Ca2+ remodeling in the aging brain using fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca2+ in long-term cultures of rat hippocampal neurons, a model of neuronal aging.

Fluorescence et bioluminescence imagerie des Subcellular Ca<sup&gt; 2+</sup&gt; In Aged neurones de l&#39;hippocampe

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...

Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to navigate axonal growth. Netrin, slit, semaphorin, and ephrins are known guidance molecules that can attract or repel axons upon binding to receptors and co-receptors on the axon. The activated receptors initiate various signaling molecules in the growth cone that alter the structure and movement of the neuron. Here, we describe the detailed protocol for a stripe assay to assess the ability of a guidance molecule to attract or repel neurons. In this method, dissociated hippocampal neurons from E15.5 mice are cultured on laminin-coated dishes processed with alternating stripes of ectodomain of fibronectin and leucine-rich transmembrane protein-2 (FLRT2) and control immunoglobulin G (IgG) fragment crystallizable region (Fc) protein. Both axons and cell bodies were strongly repelled from the FLRT2-coated stripe regions after 24 h of culture. Immunostaining with tau1 showed that ~90% of the neurons were distributed on the Fc-coated stripes compared to the FLRT2-Fc-coated stripes (~10%). This result indicates that FLRT2 has a strong repulsive effect on these neurons. This powerful method is applicable not only for primary cultured neurons but also for a variety of other cells, such as neuroblasts.

Axon guidance molecules regulate neuronal migration and targeted growth-cone navigation. We present a powerful method, the stripe assay, to assess the ability of guidance molecules to attract or repulse neurons. In this protocol, we demonstrate the stripe assay by showing FLRT2's ability to repel cultured hippocampal neurons.

Stripe Assay pour étudier l&#39;activité attractive ou répulsive d&#39;un substrat protéique utilisant dissociées hippocampique Neurones

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to ...

Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching variations of the neuronal dendrites within the hippocampus are implicated in cognition and memory formation. There are several approaches to Golgi staining, all of which have been useful for determining the morphological characteristics of dendritic arbors and produce a clear background. The present Golgi-Cox method, (a slight variation of the protocol that is provided with a commercial Golgi staining kit), was designed to assess how a relatively low dose of the chemotherapeutic drug 5-flurouracil (5-Fu) would affect dendritic morphology, the number of spines, and the complexity of arborization within the hippocampus. The 5-Fu significantly modulated the dendritic complexity and decreased the spine density throughout the hippocampus in a region-specific manner. The data presented show that the Golgi staining method effectively stained the mature neurons in the CA1, the CA3, and the dentate gyrus (DG) of the hippocampus. This protocol reports the details for each step so that other researchers can reliably stain tissue throughout the brain with high quality results and minimal troubleshooting.

Here we present a Golgi-Cox protocol in extensive detail. This reliable tissue stain method allows for a high-quality assessment of the cytoarchitecture in the hippocampus, and throughout the entire brain, with minimal troubleshooting.

Évaluation de la complexité dendritique de l&#39;hippocampe chez les souris vieillies utilisant la méthode de Golgi-Cox

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

Mesurer l’endocytose de la vésicule synaptique dans des neurones Hippocampal cultivés

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

Assaying Circuit Specific Regulation of Adult Hippocampal Neural Precursor Cells

Adult neurogenesis is a dynamic process by which newly activated neural stem cells (NSCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate new neurons, which integrate into an existing neural circuit and contribute to specific hippocampal functions. Importantly, adult neurogenesis is highly susceptible to environmental stimuli, which allows for activity-dependent regulation of various cognitive functions. A vast range of neural circuits from various brain regions orchestrates these complex cognitive functions. It is therefore important to understand how specific neural circuits regulate adult neurogenesis. Here, we describe a protocol to manipulate neural circuit activity using designer receptor exclusively activated by designer drugs (DREADDs) technology that regulates NSCs and newborn progeny in rodents. This comprehensive protocol includes stereotaxic injection of viral particles, chemogenetic stimulation of specific neural circuits, thymidine analog administration, tissue processing, immunofluorescence labeling, confocal imaging, and imaging analysis of various stages of neural precursor cells. This protocol provides detailed instructions on antigen retrieval techniques used to visualize NSCs and their progeny and describes a simple, yet effective way to modulate brain circuits using clozapine N-oxide (CNO) or CNO-containing drinking water and DREADDs-expressing viruses. The strength of this protocol lies in its adaptability to study a diverse range of neural circuits that influence adult neurogenesis derived from NSCs.

The goal of this protocol is to describe an approach for analyzing behavior of adult neural stem/progenitor cells in response to chemogenetic manipulation of a specific local neural circuit.

Régulation spécifique du circuit d'analyse des cellules neuronales neuronales hippocampales adultes

Adult neurogenesis is a dynamic process by which newly activated neural stem cells (NSCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate ...