Ce travail est soutenu par le Bureau de la recherche et du développement, de la recherche médicale Service, Department of Veterans Affairs. Cette étude a été financée par une bourse de l&#39;examen du mérite VA (à MCL) et à l&#39;Université du Tennessee Health Science Center de la sclérose multiple Fonds de recherche.

1. Marquage de l&#39;anticorps (figure 1) <ol><li> Assurez 0,1 M de tampon NaHCO 3: <ul type="circle"><li> 8,4 g de NaHCO 3 </li><li> 29,2 g de NaCl </li><li> 1 litre H 2 O </li></ul></li><li> Amener le tampon à un pH de 8,4 avec cette solution (10,6 g Na 2 CO 3, 29,2 g de NaCl, 1 litre H 2 0). </li><li> Ajouter 35 ul Atto550 NHS à 70 ul anti-hnRNPA1 et 500 pi de tampon NaHCO 3 et tourner pendant 1 heure à température ambiante. Après la rotation d&#39;une heure, l&#39;injection du mélange dans un appareil de dialyse et dialyse dans 2 litres de PBS pendant une nuit. </li><li> Le lendemain, concentrer les Atto550 dialysés NHS anti-hnRNPA1 l&#39;aide d&#39;une colonne de centrifugation (Amicon Ultra 0,5). </li><li> Après NHS Atto550 les anticorps anti-anticorps hnRNPA1 a été concentré, nano-goutte d&#39;échantillon pour déterminer la concentration. </li></ol> 2. La transfection d&#39;anticorps (figure 2) <ol><li> Seed 10 5 cellules / puits dans 500 ul de Dulbecco Eagle modifié Moyen (DMEM/F12 +10% SVF + 1% d&#39;antibiotiques) dans un 8 diapositive 24 h et avant la transfection. Confluence cellulaire doit être d&#39;au moins 70%. </li><li> Vingt-quatre heures après l&#39;ensemencement, ajouter 2 pi de Ab-DeliverIN réactif dans un tube Eppendorf. </li><li> Ensuite, ajoutez 2 pg de Atto550 NHS des anticorps anti-anticorps hnRNPA1 (0,5 pg / pl) dans le même tube Eppendorf et incuber 10-15 min à température ambiante. </li><li> Pendant l&#39;incubation, aspirer les médias et ajouter 394 ul de DMEM frais de support pour les cellules. </li></ol> Remarque: Ajouter 500 ul de DMEM à une chambre de cellules intactes pour agir à titre de témoin. <ol start="5"><li> Après incubation, ajouter 100 ul de DMEM le mélange d&#39;anticorps, mélanger par pipetage de haut en bas, et d&#39;ajouter aux cellules dans du DMEM pour un volume total de 500 ul. </li><li> Changer les médias après 48 heures de la livraison d&#39;anticorps. </li></ol> Remarque: le protocole a été répété avec marqué au FITC Rigg comme témoin positif. 3. Imagerie en direct et fixes pour déterminer l&#39;efficacité <ol><li> Cellules d&#39;image en direct à 48 h à l&#39;aide des Cy3 filtre sur un microscope à fluorescence à l&#39;image des Atto550 transfectées NHS anticorps marqués. </li><li> Après imagerie en temps réel a été achevée, aspirer les médias et les cellules fixes. </li><li> Après médias aspiration, traiter les cellules avec du paraformaldéhyde 0,4% pendant 15 min à température ambiante. Laver les cellules 4 x 5 min à chaque fois avec du PBS. Ensuite, montez les cellules avec un milieu de montage contenant réactif DAPI et lamelle fixe. Mesurer l&#39;efficacité de transfection des cellules fixes avec AxioVision logiciel. Si AxioVision logiciel n&#39;est pas à portée de main, il suffit de compter les événements de votre image en main les résultats et le brancher sur le calcul en 3.7. Sinon, le logiciel ImageJ peut être utilisée pour ces calculs. </li><li> Tout d&#39;abord, utiliser le &quot;Evénements&quot; de mesure de compter le nombre total de cellules dans votre image de son choix à un grossissement de 20x. </li><li> Ensuite, utilisez la &quot;Evénements&quot; de mesure de compter les cellules qui ne sont pastransfecté avec succès avec des anticorps dans le même 20x de grossissement d&#39;image. </li><li> Pour accéder aux mesurées &quot;Evénements&quot; dans un format de style Excel, cliquez sur &quot;Propriétés&quot;, cliquez sur &quot;Mesure&quot; onglet, puis cliquez sur pour afficher «Comme le tableau.&quot; </li><li> Enfin, faites le calcul suivant pour déterminer votre efficacité de la transfection en comparant les deux événements précédemment mesurés: ((cellules totales - Cellules non transfectées) / cellules totales) x 100 = efficacité de la transfection. </li></ol> 4. Les résultats représentatifs Les images en direct de non transfectées (figure 3A) par rapport aux transfectées (figure 3B) neurones montrent que le marquage des anticorps (rouge ou vert dans chaque figure) est clairement observé dans les cellules (figure 3B, C, D). Suite à l&#39;aspiration et de lavage avec support pour éliminer les anticorps ou entre adhérentes à la surface des cellules, des anticorps marqués sont encore présents dans les neurones (figure 3C, D).<html> Pour calculer l&#39;efficacité de la transfection, utilisez le logiciel AxioVision pour calculer nombre total de cellules par rapport aux cellules qui n&#39;ont pas été transfectées avec succès. Ces valeurs ont été entrées dans une formule d&#39;efficacité de transfection pour acquérir l&#39;efficacité de transfection en pourcentage du nombre total de cellules (figure 4). <img alt="Figure 1" src="/files/ftp_upload/4154/4154fig1.jpg" /> Figure 1. Schéma des procédures utilisées à des anticorps étiquette avec l&#39;ester NHS 550 Atto suivie par des étapes nécessaires pour concentrer les anticorps marqués. <a href="http://www.jove.com/files/ftp_upload/4154/4154fig1large.jpg" target="_blank">Cliquez ici pour agrandir la figure</a> . <img alt="Figure 2" src="/files/ftp_upload/4154/4154fig2.jpg" /> Figure 2. Schéma des procédures utilisées pour transfecter des anticorps marqués dans les cellules vivantes en culture.arge.jpg &quot;target =&quot; _blank &quot;&gt; Cliquez ici pour agrandir la figure. <img alt="Figure 3" src="/files/ftp_upload/4154/4154fig3.jpg" /> Figure 3. Les images en direct des neurones transfectés 48 heures après la transfection. <img alt="Figure 4" src="/files/ftp_upload/4154/4154fig4.jpg" /> Figure 4. Correction d&#39;images et rincés avec les événements en compte afin de déterminer l&#39;efficacité de transfection.

<ol>
	<li>Burkhardt, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epitope-specific+recognition+of+type+II+collagen+by+rheumatoid+arthritis+antibodies+is+shared+with+recognition+by+antibodies+that+are+arthritogenic+in+collagen-induced+arthritis+in+the+mouse.">Epitope-specific recognition of type II collagen by rheumatoid arthritis antibodies is shared with recognition by antibodies that are arthritogenic in collagen-induced arthritis in the mouse.</a> Arthritis Rheum. 46, 2339-2348 (2002).</li><li>Chester, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lambert-Eaton+syndrome+antibodies:+reaction+with+membranes+from+a+small+cell+lung+cancer+xenograft.">Lambert-Eaton syndrome antibodies: reaction with membranes from a small cell lung cancer xenograft.</a> J. Neuroimmunol. 18, 97-104 (1988).</li><li>Lee, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HTLV-1+induced+molecular+mimicry+in+neurological+disease.">HTLV-1 induced molecular mimicry in neurological disease.</a> Curr. Top Microbiol. Immunol. 29, 125-136 (2005).</li><li>Reeves, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+lupus+erythematosus.+Antibodies+to+DNA,+DNA-binding+proteins,+and+histones.">Systemic lupus erythematosus. Antibodies to DNA, DNA-binding proteins, and histones.</a> Rheum. Dis. Clin. North Am. 20, 1-28 (1994).</li><li>Levin, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoimmunity+due+to+molecular+mimicry+as+a+cause+of+neurological+disease.">Autoimmunity due to molecular mimicry as a cause of neurological disease.</a> Nat. Med. 8, 509-513 (2002).</li><li>Lee, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoantibodies+that+recognize+functional+domains+of+hnRNPA1+implicate+molecular+mimicry+in+the+pathogenesis+of+neurological+disease.">Autoantibodies that recognize functional domains of hnRNPA1 implicate molecular mimicry in the pathogenesis of neurological disease.</a> Neurosci. Lett. 401, 188-193 (2006).</li><li>Kalume, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mimicry:+cross-reactive+antibodies+from+patients+with+immune-mediated+neurologic+disease+inhibit+neuronal+firing.">Molecular mimicry: cross-reactive antibodies from patients with immune-mediated neurologic disease inhibit neuronal firing.</a> J. Neurosci. Res. 77, 82-89 (2004).</li><li>Levin, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-reactivity+between+immunodominant+human+T+lymphotropic+virus+type+I+tax+and+neurons:+implications+for+molecular+mimicry.">Cross-reactivity between immunodominant human T lymphotropic virus type I tax and neurons: implications for molecular mimicry.</a> J. Infect. Dis. 186, 1514-1517 (2002).</li><li>Lee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+potential+link+between+autoimmunity+and+neurodegeneration+in+immune-mediated+neurological+disease.">A potential link between autoimmunity and neurodegeneration in immune-mediated neurological disease.</a> J. Neuroimmunol. 235, 56-69 (2011).</li><li>Smeenk, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antinuclear+antibodies:+cause+of+disease+or+caused+by+disease.">Antinuclear antibodies: cause of disease or caused by disease.</a> Rheumatology (Oxford). 39, 581-584 (2000).</li><li>Lee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-translational+glycosylation+of+target+proteins+implicate+molecular+mimicry+in+the+pathogenesis+of+HTLV-1+associated+neurological+disease.">Post-translational glycosylation of target proteins implicate molecular mimicry in the pathogenesis of HTLV-1 associated neurological disease.</a> J. Neuroimmunol. 204, 140-148 (2008).</li><li>Reisinger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum-free+transfection+of+CHO+cells+with+chemically+defined+transfection+systems+and+investigation+of+their+potential+for+transient+and+stable+transfection.">Serum-free transfection of CHO cells with chemically defined transfection systems and investigation of their potential for transient and stable transfection.</a> Cytotechnology. 60, 115-123 (2009).</li><li>Weill, C. O., Biri, S., Erbacher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cationic+lipid-mediated+intracellular+delivery+of+antibodies+into+live+cells.">Cationic lipid-mediated intracellular delivery of antibodies into live cells.</a> Biotechniques. 44, Pvii-Pxi (2008).</li><li>Radic, M. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneous+nuclear+ribonucleoprotein+P2+is+an+autoantibody+target+in+mice+deficient+for+Mer,+Axl,+and+Tyro3+receptor+tyrosine+kinases.">Heterogeneous nuclear ribonucleoprotein P2 is an autoantibody target in mice deficient for Mer, Axl, and Tyro3 receptor tyrosine kinases.</a> J. Immunol. 176, 68-74 (2006).</li><li>Vincent, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+'passive+transfer'+of+paraneoplastic+stiff+person+syndrome+with+antibodies+to+an+intracellular+antigen.">Successful 'passive transfer' of paraneoplastic stiff person syndrome with antibodies to an intracellular antigen.</a> Brain. 133, 3164-3165 (2010).</li><li>Vincent, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stiff,+twitchy+or+wobbly:+are+GAD+antibodies+pathogenic.">Stiff, twitchy or wobbly: are GAD antibodies pathogenic.</a> Brain. 131 (Pt 10), 2536-2537 (2008).</li><li>Magrys, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+anti-alpha-enolase+autoantibodies+in+pathogenicity+of+autoimmune-mediated+retinopathy.">The role of anti-alpha-enolase autoantibodies in pathogenicity of autoimmune-mediated retinopathy.</a> J. Clin. Immunol. 27, 181-192 (2007).</li><li>Geis, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stiff+person+syndrome-associated+autoantibodies+to+amphiphysin+mediate+reduced+GABAergic+inhibition.">Stiff person syndrome-associated autoantibodies to amphiphysin mediate reduced GABAergic inhibition.</a> Brain. 133, 3166-3180 (2010).</li></ol>

Aucun conflit d&#39;intérêt déclaré.

Afin de tester des hypothèses sur des molécules spécifiques et de leur expression, des gènes et d&#39;autres vecteurs sont couramment transfecté dans des cellules. L&#39;aspect unique de cette procédure est la possibilité de facilement transfecter des anticorps dans les cellules cibles. Nous avons effectué cette méthode dans cinq expériences séparées, chacune en double. Résultats d&#39;efficacité de transfection étaient similaires chez toutes les expériences. Surtout, le procédé permet l&#39;entrée d&...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td></tr><tr><td> Fluorescent Microscope AxioObserver A1 </td><td> Zeiss </td><td></td></tr><tr><td> AxioVision version 4.2 </td><td> Zeiss </td><td></td></tr><tr><td> Anti-rhnRNPA1 </td><td> Abcam </td><td> Ab4791 </td></tr><tr><td> Marqué au FITC anti-Rigg </td><td> OZ Biosciences </td><td> AI20100 </td></tr><tr><td> Atto550 ester de NHS </td><td> Sigma </td><td> 92835 </td></tr><tr><td> Ab-DeliverIN </td><td> OZ Biosciences </td><td> AI20100 </td></tr><tr><td> DMEM/F12 +10% FBS antibiotiques +1% </td><td> Gibco </td><td> 11320-033 </td></tr><tr><td> Poly-D-Lysine diapositives Culture de 8 puits </td><td> BD </td><td> 354632 </td></tr><tr><td> Multi-Purpose Rotator </td><td> Scientifique Industries Inc </td><td> Modèle 151 </td></tr><tr><td> DAPI Montage des médias </td><td> Millipore </td><td> S7113 </td></tr><tr><td> PBS </td><td> Ambion </td><td> 9626 </td></tr><tr><td> Dialyseur </td><td> Thermo Fisher Scientific </td><td> 66380 </td></tr><tr><td> Amicon Ultra-0.5 </td><td> Millipore </td><td> UFC501096 </td></tr><tr><td> Paraformaldéhyde (solution à 4%) </td><td> Sciences microscopie électronique </td><td></td></tr><tr><td> Nano-Drop </td><td> Thermo Fisher Scientific </td><td></td></tr></tbody></table>

antibody transfection into neurons as a tool to study disease pathogenesis

Anticorps offrir la possibilité d&#39;acquérir un nouvel aperçu de divers événements qui se déroulent dans les systèmes vivants. La capacité à produire des anticorps hautement spécifiques à des protéines cibles a permis de très précis des questions biologiques à traiter. Surtout, les anticorps ont été impliqués dans la pathogenèse de plusieurs maladies humaines, notamment le lupus érythémateux systémique (LES), la polyarthrite rhumatoïde (PR), les syndromes paranéoplasiques, la sclérose en plaques (MS) et humaine de type virus T-lymphotrope 1 (HTLV-1) myélopathie associée / paraparésie spastique tropicale (TSP / HAM) 1-9. Comment anticorps provoquent la maladie est un domaine de recherche en cours et les données suggèrent que les interactions entre les anticorps et les différentes molécules intracellulaires résultats dans l&#39;inflammation, modifié cellulaire de messagerie, et l&#39;apoptose 10. Il a été montré que les patients atteints de SEP et TSP / HAM produire des anticorps contre la protéine intracellulaire d&#39;ARN hétérogène protéine de liaison ribonuclear A1 (hnRNP A1) 3, 5-7, 9, 11. Des données récentes indiquent que les anticorps à la fois intra-neuronale et antigènes de surface sont pathogènes 3, 5-9, 11. Ainsi, une procédure qui permet l&#39;étude des anticorps intracellulaire: les interactions entre protéines donnerait une grande perspicacité dans la pathogenèse des maladies. Les gènes sont souvent transfecté dans des cellules primaires et les lignées cellulaires en culture, mais la transfection d&#39;anticorps dans les cellules a été entravée par la modification de la structure d&#39;anticorps ou de l&#39;efficacité de transfection pauvres 12. D&#39;autres méthodes de transfection comprennent la transfection d&#39;anticorps à base de liposomes cationiques (composés de DOTAP / DOPE) et polyéthylèneimines (PEI), tous deux ce qui a entraîné une diminution de dix fois dans la transfection d&#39;anticorps par rapport aux témoins 12. Le procédé mis en oeuvre dans notre étude est similaire à lipide cationique à médiation méthodes et utilise un mécanisme à base de lipides pour former des complexes non covalents avec des anticorps à travers électrostatique et hydrinteractions ophobic 13. On utilise Ac-DeliverIN réactif, qui est une formulation de lipide capable de capturer des anticorps par des interactions non covalentes électrostatiques et hydrophobes et les délivrer à l&#39;intérieur des cellules. Ainsi, chimiques et génétiques raccords ne sont pas nécessaires pour la livraison des anticorps fonctionnels dans les cellules vivantes. Cette méthode nous a permis d&#39;effectuer des anticorps différents traçage et expériences de localisation de protéines, ainsi que les analyses des conséquences moléculaires de l&#39;anticorps intracellulaire: interactions protéiques 9. Dans ce protocole, nous allons montrer comment pour transfecter des anticorps dans les neurones rapide, reproductible et avec un haut degré d&#39;efficacité de la transfection. A titre d&#39;exemple, nous allons utiliser des anticorps anti-hnRNP A1 et anti-IgG. Pour la quantification facile d&#39;efficacité de la transfection, nous avons utilisé des anticorps anti-hnRNP A1 étiquetés avec Atto-550-NHS et marqué au FITC IgG. Atto550 NHS est un nouveau label avec absorption moléculaire élevé et quantique yield. Source d&#39;excitation et des filtres à fluorescence pour Atto550 sont similaires à Cy3 (Ex. 556 Em. 578). En outre, Atto550 a photostabilité élevée. Marqué au FITC IgG ont été utilisés comme contrôle pour montrer que cette méthode est polyvalent et se teignait pas dépendant. Cette approche et les données qui sont générées aidera à comprendre le rôle que les anticorps à des antigènes cibles intracellulaires pourrait jouer dans la pathogenèse des maladies humaines.

Watch this Scientific Journal Video about Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis at JoVE.com

Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis

Une réponse rapide pour étudier les interactions et les effets sur les mécanismes moléculaires liés à la présence d&#39;anticorps dans un environnement intracellulaire est décrite. Le procédé comprend la transfection d&#39;anticorps dans des cellules vivantes à l&#39;aide d&#39;une formation de complexe non covalent sur la base d&#39;une formulation de lipide. La technique est adaptable à des lignées de cellules immortalisées et des cellules primaires.

La transfection d&#39;anticorps dans les neurones comme outil pour étudier la pathogenèse des maladies

Antibodies provide the ability to gain novel insight into various events taking place in living systems.  The ability to produce highly specific ...

antibody-transfection-into-neurons-as-tool-to-study-disease

Research

JoVE Journal

Neuroscience

10.7K vues.  Veterans Administration Medical Center, Memphis, TN. Une réponse rapide pour étudier les interactions et les effets sur les mécanismes moléculaires liés à la présence d'anticorps dans un environnement intracellulaire est décrite. Le procédé comprend la transfection d'anticorps dans des cellules vivantes à l'aide d'une formation de complexe non covalent sur la base d'une formulation de lipide. La technique est adaptable à des lignées de cellules immortalisées et des cellules primaires.

Vidéo: Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

Microdissection laser des Drosophile Neurones périphériques

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

Recherche

Neurosciences

Cut-loading: A Useful Tool for Examining the Extent of Gap Junction Tracer Coupling Between Retinal Neurons

In addition to chemical synaptic transmission, neurons that are connected by gap junctions can also communicate rapidly via electrical synaptic transmission. Increasing evidence indicates that gap junctions not only permit electrical current flow and synchronous activity between interconnected or coupled cells, but that the strength or effectiveness of electrical communication between coupled cells can be modulated to a great extent1,2. In addition, the large internal diameter (~1.2 nm) of many gap junction channels permits not only electric current flow, but also the diffusion of intracellular signaling molecules and small metabolites between interconnected cells, so that gap junctions may also mediate metabolic and chemical communication. The strength of gap junctional communication between neurons and its modulation by neurotransmitters and other factors can be studied by simultaneously electrically recording from coupled cells and by determining the extent of diffusion of tracer molecules, which are gap junction permeable, but not membrane permeable, following iontophoretic injection into single cells. However, these procedures can be extremely difficult to perform on neurons with small somata in intact neural tissue.
Numerous studies on electrical synapses and the modulation of electrical communication have been conducted in the vertebrate retina, since each of the five retinal neuron types is electrically connected by gap junctions3,4. Increasing evidence has shown that the circadian (24-hour) clock in the retina and changes in light stimulation regulate gap junction coupling3-8. For example, recent work has demonstrated that the retinal circadian clock decreases gap junction coupling between rod and cone photoreceptor cells during the day by increasing dopamine D2 receptor activation, and dramatically increases rod-cone coupling at night by reducing D2 receptor activation7,8. However, not only are these studies extremely difficult to perform on neurons with small somata in intact neural retinal tissue, but it can be difficult to adequately control the illumination conditions during the electrophysiological study of single retinal neurons to avoid light-induced changes in gap junction conductance.
Here, we present a straightforward method of determining the extent of gap junction tracer coupling between retinal neurons under different illumination conditions and at different times of the day and night. This cut-loading technique is a modification of scrape loading9-12, which is based on dye loading and diffusion through open gap junction channels. Scrape loading works well in cultured cells, but not in thick slices such as intact retinas. The cut-loading technique has been used to study photoreceptor coupling in intact fish and mammalian retinas7, 8,13, and can be used to study coupling between other retinal neurons, as described here.

An easy and convenient method to determine the extent of gap junction tracer coupling between retinal neurons is described. This technique enables one to investigate the function of the electrical synapses between neurons in the intact retina under different illumination conditions and at different times of the day and night.

Coupez-chargement: Un outil utile pour examiner l&#39;étendue du couplage Tracer Gap jonction entre neurones rétiniens

In addition to chemical synaptic transmission, neurons that are connected by gap junctions can also communicate rapidly via electrical synaptic ...

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 ...

Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic effects of drugs and downstream pathways involved would help gain insight into the molecular mechanisms of neuroprotection/neurotoxicity and could potentially facilitate drug development. However, many existing in vitro toxicity assays have major limitations &#8211; most assess neurotoxicity and neuroprotection at a single time point, not allowing to observe the time-course and kinetics of the effect. Furthermore, the opportunity to collect information about downstream signaling pathways involved in neuroprotection in real-time would be of great importance. In the current protocol we describe the use of a real-time impedance-based cell analyzer to determine neuroprotective effects of serotonin 2A (5-HT2A) receptor agonists in a neuronal cell line under label-free and real-time conditions using impedance measurements. Furthermore, we demonstrate that inhibitors of second messenger pathways can be used to delineate downstream molecules involved in the neuroprotective effect. We also describe the utility of this technique to determine whether an effect on cell proliferation contributes to an observed neuroprotective effect. The system utilizes special microelectronic plates referred to as E-Plates which contain alternating gold microelectrode arrays on the bottom surface of the wells, serving as cell sensors. The impedance readout is modified by the number of adherent cells, cell viability, morphology, and adhesion. A dimensionless parameter called Cell Index is derived from the electrical impedance measurements and is used to represent the cell status. Overall, the real-time impedance-based cell analyzer allows for real-time, label-free assessment of neuroprotection and neurotoxicity, and the evaluation of second messenger pathways involvement, contributing to more detailed and high-throughput assessment of potential neuroprotective compounds in vitro, for selecting therapeutic candidates.

Improved in vitro neurotoxicity assays would aid the identification of new neuroprotective compounds. The utility of a real-time impedance-based cell analyzer to determine cytotoxicity and cytoprotection in neuronal cell lines and to delineate the involvement of second messenger pathways, thus gaining insight in the mechanism of neuroprotection is presented.

Analyseur portable à base d&#39;impédance en temps réel comme un outil pour délimiter voies moléculaires impliquées dans la neurotoxicité et la neuroprotection dans une lignée cellulaire neuronale

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

Le système olfactif comme un modèle pour étudier des modèles axonale croissance et la morphologie<em&gt; In Vivo</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

Intramuscular Injections Along the Motor End Plates: A Minimally Invasive Approach to Shuttle Tracers Directly into Motor Neurons

Diseases affecting the integrity of spinal cord motor neurons are amongst the most debilitating neurological conditions. Over the last decades, the development of several animal models of these neuromuscular disorders has provided the scientific community with different therapeutic scenarios aimed at delaying or reversing the progression of these conditions. By taking advantage of the retrograde machinery of neurons, one of these approaches has been to target skeletal muscles in order to shuttle therapeutic genes into corresponding spinal cord motor neurons. Although once promising, the success of such gene delivery approach has been hampered by the sub-optimal number of transduced motor neurons it has so far shown to yield. Motor end plates (MEPs) are highly specialized regions on the skeletal musculature that are in direct synaptic contact to the spinal cord &#945; motor neurons. In this regard, it is important to note that, so far, the efforts to retrogradely transfer genes into motor neurons were made without reference to the location of the MEP region in the targeted muscles. Here, we describe a simple protocol 1) to reveal the exact location of the MEPs on the surface of skeletal muscles and 2) to use this information to guide the intramuscular delivery and subsequent optimal retrograde transport of retrograde tracers into motor neurons. We hope to utilize the results from these tracing experiments in further studies into investigating retrograde transport of therapeutic genes to spinal cord motor neurons through the targeting of MEPs.

The efficacy of intramuscular uptake and retrograde transport of molecules to corresponding motor neurons depends on the location of the injection sites with respect to the motor end plates (MEPs). Here, we describe how to locate MEPs on skeletal muscles to optimise retrograde transport of tracers into motor neurons.

Les injections intramusculaires le long des plaques motrices de fin: une approche minimalement invasive à la navette Tracers Directement dans motoneurones

Diseases affecting the integrity of spinal cord motor neurons are amongst the most debilitating neurological conditions. Over the last decades, the ...

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 ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

Hippocampe organotypiques comme modèle d’étude Neuroprotection et son caractère invasif des cellules tumorales

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

Exogenous Administration of Microsomes-associated Alpha-synuclein Aggregates to Primary Neurons As a Powerful Cell Model of Fibrils Formation

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the study of &#945;S aggregation in Parkinson&#39;s Disease (PD). Recently, the development of new animal models through the exogenous inoculation of brain extracts from diseased &#945;S transgenic mice or PD patients has given new hopes to the possibility of creating more adequate cell models of &#945;S aggregation. Unfortunately, when it comes to cells in cultures, administration of raw brain extracts has not proven as successful as in mice and the source of choice of exogenous aggregates is still in vitro preformed &#945;S fibrils.
We have developed a method to induce the formation of intracellular &#945;S inclusions in primary neurons through the exogenous administration of native microsomes-associated &#945;S aggregates, a highly toxic &#945;S species isolated from diseased areas of transgenic mice. This fraction of &#945;S aggregates that is associated with the microsomes vesicles, is efficiently internalized and induces the formation of intracellular inclusions positive for aggregated and phosphorylated &#945;S. Compared to in vitro-preformed fibrils which are made from recombinant &#945;S, our method is faster and guarantees that the pathogenic seeding is made with authentic &#945;S aggregates extracted from diseased animal models of PD, mimicking more closely the type of inclusions obtained in vivo. As a result, availability of tissues rich in &#945;S inclusions is mandatory.
We believe that this method will provide a versatile cell-based model to study the microscopic aspects of &#945;S aggregation and the related cellular pathophysiology in vivo and will be a starting point for the creation of more accurate and sophisticated cell paradigm of PD.

The goal of this protocol is to provide a cell-based system that replicates the formation of alpha-synuclein aggregates in vivo. Intracellular alpha-synuclein inclusions are seeded in primary neurons by the internalization and propagation of exogenous administered native microsomes-associated alpha-synuclein aggregates isolated from diseased alpha-synuclein transgenic mice.

Administration exogène d’Alpha-synucléine Microsomes associées aux agrégats de neurones primaires comme un modèle de cellulaire puissant de la Formation de fibrilles

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the ...

Cell-based Assay to Study Antibody-mediated Tau Clearance by Microglia

Alzheimer&#39;s disease (AD) is a progressive neurodegenerative condition in which aggregated tau and amyloid proteins accumulate in the brain causing neuronal dysfunction which eventually leads to cognitive decline. Hyperphosphorylated tau aggregates in the neuron are believed to cause most of the pathology associated with AD. These aggregates are assumed to be released into the extracellular compartment and taken up by adjacent healthy neurons where they induce further tau aggregation. This &#34;prion-like&#34; spreading can be interrupted by antibodies capable of binding and &#34;neutralizing&#34; extracellular tau aggregates as shown in preclinical mouse models of AD. One of the proposed mechanisms by which therapeutic antibodies reduce pathology is antibody-mediated uptake and clearance of pathological aggregated forms of tau by microglia. Here, we describe a quantitative cell-based assay to assess tau uptake by microglia. This assay uses the mouse microglial cell line BV-2, allows for high specificity, low variability and medium throughput. Data generated with this assay can contribute to a better characterization of anti-tau antibody effector functions.

Here we describe a cell-based assay to quantitatively assess tau uptake by microglia with the aim of creating an investigational tool to better characterize the mechanisms of action of anti-tau antibodies.

Cell-based Assay pour étudier la clairance Tau humoral de la microglie

Alzheimer&#39;s disease (AD) is a progressive neurodegenerative condition in which aggregated tau and amyloid proteins accumulate in the brain causing ...