Ringraziamo il Dott Toshio Terashima di Università di Kobe, in Giappone, per l&#39;insegnamento della tecnica di chirurgia laringea, e il dottor Lynn Enquist della Princeton University per la fornitura di PRV-Bartha. La ricerca è stata sostenuta da premio pioniere NIH DP1 OD000448 Erich D. Jarvis e un premio NSF Graduate Research Fellowship a Gustavo Arriaga. Le cifre precedente lavoro opportunamente accreditati sono utilizzati sotto la PLoS ONE accesso aperto licenza Creative Commons (CC-BY) in accordo con le politiche editoriali della rivista.

NOTA: Tutte le procedure sugli animali sono stati esaminati e approvati dal Istituzionale Cura e uso di animali Comitato Duke University. 1. Memorizzazione Pseudorabies Virus <ol><li> Otteniamo virus vivo (PRV152) dal laboratorio del Dr. Lynn Enquist alla Princeton University in un titolo di 1 x 10 9 pfu / m. Il protocollo per generare il virus è stato pubblicato 2. </li><li> Aliquota il virus a 20 ml per tubo all&#39;interno di un armadietto BSL-2 biosicurezza e conservare a -80 ° C in adeguate condizioni di biosicurezza. </li><li> Scongelare una aliquota di PRV immediatamente prima di iniettare. </li></ol> 2. Preparazione chirurgica per iniezioni nel muscolo <ol><li> Un&#39;anestesia generale per iniezione intramuscolare di ketamina-xilazina (100 mg / kg ketamina; 10 mg / kg xilazina) e mantenere un piano anestetico appropriato utilizzando isofluorano. </li><li> Proteggere le cornee con un unguento oftalmico. </li><li> Preparare tegli sito chirurgico secondo la tecnica asettica da taglio capelli e disinfezione del sito chirurgico con alternanza di macchie di Betadine e il 70% di alcol (almeno 3 cicli). Assicurarsi di utilizzare teli sterili per coprire aree chirurgiche. Assicurati di rispettare tecniche sterili durante tutta la procedura. </li><li> Fare un incisione cutanea e rivelare il muscolo di interesse. Ad esempio, per accedere al muscolo laringea cricotiroidea è necessario prima rimuovere la parte sovrastante del muscolo sternoioideo. </li><li> Sigillare eventuali muscoli sezionati con adesivo tessuto Vetbond. </li></ol> 3. Iniezione di PRV in muscolare <ol><li> Caricare il 10 microlitri sistema Microsiringa NanoFil con una soluzione PRV appena scongelato, inserire un ago in acciaio inossidabile 34 G, e montare con cautela sul dispositivo stereotassico. </li><li> Lentamente liberare lo spazio morto e verificare che la soluzione esce dalla punta microsiringa. Smaltire come rifiuto liquido bioharzard. </li><li> Utilizzando un microP stereotassicadispositivo ositioning posizionare con cura la punta microsiringa nel muscolo di interesse e lentamente riempire il muscolo fino a leggero gonfiore è visibile. La velocità di iniezione varierà a seconda della dimensione del muscolo e del volume da iniettare. Ad esempio, cinque iniezioni di 200 nl (1 ml totale) ad una velocità di 4 nl / sec devono essere effettuate 1 min a parte nello stesso sito per riempire il muscolo cricotiroideo. Spostare la siringa al prossimo muscolo di interesse (il cricoarytenoid laterale in questo caso) e ripetere la procedura di iniezione. Solo puntura ogni muscolo una volta. </li><li> Ritrarre la microsiringa dopo cinque minuti. </li><li> Sigillare la pausa nella fascia con adesivo tessuto Vetbond. </li><li> Dopo che tutte le iniezioni sono state completate, chiudere la ferita con adesivo tessuto Vetbond. A seconda delle linee guida locali institional uso animale, suture o clip ferita può essere utilizzato. </li><li> Monitorare l&#39;animale fino decubito sternale e fornire l&#39;analgesia, cibo, acqua, e la cura come richiesto da yle nostre linee guida sull&#39;uso degli animali istituzionali. </li><li> Dopo il tempo di sopravvivenza determinato sperimentalmente (in questo caso, 90 hr etichettare 2 ° ordine dei neuroni corticali), sacrificare l&#39;animale da sovradosaggio pentobarbital e nei profumi transcardially con soluzione fisiologica 0,9% seguita da 4% di formaldeide in PBS 0,1 M. </li><li> Rimuovere e post-fissare il cervello in 4% di formaldeide per 24 ore. </li><li> Cryoprotect cervello in tampone fosfato contenente il 30% di saccarosio per almeno 48 ore. </li></ol> 4. immunochimica Rilevamento di eGFP <ol><li> Tagliare sezioni a 40 micron su un microtomo a slitta e salvare sezioni galleggianti in 0.1M PBS. Sezioni più sottili, ad esempio 30 micron, possono essere utilizzate quando colorazione sezioni montate. </li><li> Placare le sezioni per 30 min a 0,3% H 2 O 2 in PBS al riparo dalla luce. </li><li> Bloccare antigeni non specifici nelle sezioni per 30 min in PBS contenente 0,3% Tween 20 con siero normale di capra dal Kit Vectastain Elite (VE kit). </li><li&gt; Incubare bloccato sezioni per 3,5 ore in coniglio anti-eGFP (1: 1.000) a temperatura ambiente. </li><li> Lavare sezioni tre volte in PBS per 5 minuti, poi incubare per 1 ora in anticorpo secondario di capra anti-coniglio dalla VE kit a RT. </li><li> Preparare la soluzione ABC dal VE kit secondo le istruzioni del produttore. </li><li> Lavare sezioni tre volte in PBS per 5 minuti, poi reagire per 1 ora in soluzione ABC dal VE kit a RT. </li><li> Lavare sezioni tre volte in tampone fosfato per 10 min, e sviluppare per 8 min in tampone fosfato, pH 7,4, contenente lo 0,05% DAB (3, 3&#39;-diaminobenzidina) e 0,015% H 2 O 2. </li><li> Montare sezioni su vetrini SuperFrost Plus e li disidratano attraverso una serie graduata alcol (70%, 95%, 100% e 100% per 5 minuti ciascuno). </li><li> Cancellare sezioni attraverso due lavaggi xilene (5 min ciascuno) e applicare il coprioggetto i vetrini con mezzo di montaggio Permount. </li><li> Immagine sezioni montati su un microscopio. Il prodotto di reazione DAB inside le cellule dovrebbero apparire marrone. Immagini digitalizzate possono essere colore invertito per evidenziare i processi sottili. </li></ol> 5. Preparazione chirurgica per iniezione di traccianti in regioni cerebrali Scoperto da PRV Tracing <ol><li> Un&#39;anestesia generale per iniezione intramuscolare di ketamina-xilazina (100 mg / kg ketamina; 10 mg / kg xilazina) e mantenere un piano anestetico appropriato utilizzando isofluorano. </li><li> Proteggere le cornee con un unguento oftalmico. </li><li> Fissare la testa in un telaio stereotassico appropriata. </li><li> Preparare il sito chirurgico secondo la tecnica asettica da taglio capelli e disinfezione del sito con alternanza macchie di Betadine e il 70% di alcol (almeno 3 cicli). </li><li> Effettuare una incisione del cuoio capelluto e ritrarre la pelle sopra la regione del cervello di interesse. </li><li> Eseguire una piccola craniotomia alle opportune coordinate stereotassica. Ad esempio, le coordinate per corteccia motoria laryngeally collegato individuato da PRV tracciando in unn topo adulto sono 1,2 millimetri laterale e 0,2 mm rostrale a Bregma. </li></ol> 6. L&#39;iniezione di Biotinylated Destrano ammine in Cervello <ol><li> Preparare 7,5% ammine destrano biotinilati (BDA) sciogliendo 25 mg di BDA (10.000 MW) in 333 ml di soluzione fisiologica sterile. </li><li> Caricare il sistema micropipetta Nanoject II con la soluzione BDA sufficiente per le iniezioni pianificate. </li><li> Lentamente liberare lo spazio morto e verificare che la soluzione sta per uscire dal punta micropipetta. </li><li> Utilizzando un dispositivo microposizionamento stereotassico abbassare con attenzione la punta micropipetta nella regione del cervello di interesse e iniettare lentamente BDA. Regolare la velocità di iniezione in funzione della dimensione della regione da etichettare. Ad esempio, 12 iniezioni di 4,6 nl dovrebbero essere fatto in quattro sedi diverse 0,2 millimetri a parte (rostro-caudale direzione) per coprire la corteccia motoria laryngeally collegato in topi adulti. Il volume di iniezione finale dovrebbe essere regolata in base alle dimensioni della regione del cervello di interesse, Tenendo presente che l&#39;etichetta può diffondersi dal nucleo di iniezione per diffusione attraverso i tessuti cerebrali e lungo i processi di neuroni marcati. </li><li> Sigillare la craniotomia con cemento dentale, e chiudere la ferita del cuoio capelluto con adesivo tessuto Vetbond. </li><li> Monitorare l&#39;animale fino decubito sternale e fornire l&#39;analgesia, cibo, acqua, e alle cure richieste dalle vostre linee guida sull&#39;uso degli animali istituzionali. </li></ol> 7. L&#39;iniezione di tossina colerica subunità b in muscolare <ol><li> Preparare 1% tossina colerica subunità b (CTB) sciogliendo 1 mg di CTB in 100 ml di soluzione fisiologica sterile. </li><li> Sei giorni dopo l&#39;iniezione BDA nel cervello, eseguire la preparazione chirurgica come descritto sopra per iniezioni PRV nel muscolo. </li><li> Caricare il 10 microlitri sistema Microsiringa NanoFil con la soluzione CTB, inserire un ago in acciaio inossidabile 34 G, e con attenzione montare sul dispositivo stereotassico. </li><li> Eseguire le microiniezioni nel muscolo (s) di interesse come precedentemente dedescritto per PRV. </li><li> Monitorare l&#39;animale fino decubito sternale e fornire l&#39;analgesia, cibo, acqua, e alle cure richieste dalle vostre linee guida sull&#39;uso degli animali istituzionali. </li><li> Tre giorni dopo l&#39;iniezione CTB, sacrifica l&#39;animale per overdose pentobarbital e profumato transcardiaca con soluzione fisiologica 0,9% seguito dal 4% di formaldeide in 0,1 M PBS. </li><li> Rimuovere e post-fissare il cervello in 4% di formaldeide per 24 ore. </li><li> Cryoprotect cervello in tampone fosfato contenente il 30% di saccarosio per almeno 48 ore. </li></ol> 8. Individuazione di BDA e CTB nelle stesse sezioni <ol><li> Tagliare sezioni a 40 micron su un microtomo e salvare sezioni galleggianti in 0.1M PBS. </li><li> Placare le sezioni per 30 min a 0,3% H 2 O 2 in PBS al riparo dalla luce. </li><li> Preparare la soluzione ABC dal VE kit secondo le istruzioni del produttore. </li><li> Lavare sezioni tre volte in PBS per 5 minuti, poi reagire per 1 ora in soluzione ABC a RT. </li&gt; <li> Lavare sezioni tre volte in tampone fosfato per 10 min, e sviluppare per 8 min in tampone fosfato, pH 7,4, contenente lo 0,05% DAB (3, 3&#39;-diaminobenzidina), 0,015% H 2 O 2, e 0,05% di cloruro di nichel. Il prodotto di reazione DAB all&#39;interno delle cellule dovrebbe apparire nero. </li><li> Sezioni di blocco per 30 minuti in PBS contenente 0,3% Tween 20 con siero normale di coniglio dal Kit Vectastain Elite. </li><li> Incubare bloccato sezioni per 2 ore in capra anti-CTB (1: 10.000) a temperatura ambiente. </li><li> Lavare sezioni tre volte in PBS per 5 minuti, poi incubare per 1 ora in anticorpo secondario di coniglio anti-capra dalla VE kit a RT. </li><li> Preparare la soluzione ABC dal VE kit secondo le istruzioni del produttore. </li><li> Lavare sezioni tre volte in PBS per 5 minuti, poi reagire per 30 min in soluzione ABC a RT. </li><li> Lavare sezioni tre volte in tampone fosfato per 10 min, e sviluppare fino a 8 min in tampone fosfato, pH 7,4, contenente lo 0,05% DAB (3, 3&#39;-diaminobenzidine) e 0,015% H 2 O 2. Il prodotto di reazione DAB all&#39;interno delle cellule dovrebbe apparire marrone. </li><li> Montare sezioni su vetrini SuperFrost Plus e li disidratano attraverso una serie graduata alcol (70%, 95%, 100% e 100% per 5 minuti ciascuno). </li><li> Cancellare sezioni attraverso due lavaggi xilene (5 min ciascuno) e applicare il coprioggetto i vetrini con mezzo di montaggio Permount. </li><li> Immagine sezioni montati su un microscopio. </li></ol>

<ol>
	<li>Card, J. P., Enquist, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Transneuronal circuit analysis with pseudorabies viruses.Multiple values selected. Unit 1.5, 1.51-1.5.28 (2001).</li><li>Smith, B. N., Banfield, B. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudorabies+virus+expressing+enhanced+green+fluorescent+protein:+a+tool+for+in+vitro+electrophysiological+analysis+of+transsynaptically+labeled+neurons+in+identified+central+nervous+system+circuits.">Pseudorabies virus expressing enhanced green fluorescent protein: a tool for in vitro electrophysiological analysis of transsynaptically labeled neurons in identified central nervous system circuits.</a> Proceedings of the National Academy of Sciences of the United States of America. 97 (16), 9264-9269 (2000).</li><li>Aston Jones, G., Card, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+pseudorabies+virus+to+delineate+multisynaptic+circuits+in+brain+opportunities+and+limitations.">Use of pseudorabies virus to delineate multisynaptic circuits in brain opportunities and limitations.</a> Journal of Neuroscience Methods. 103 (1), 51-61 (2000).</li><li>Pomeranz, L. E., Reynolds, A. E., Hengartner, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+biology+of+pseudorabies+virus+impact+on+neurovirology+and+veterinary+medicine.">Molecular biology of pseudorabies virus impact on neurovirology and veterinary medicine.</a> Microbiology and Molecular Biology Reviews. 69 (3), 462-500 (2005).</li><li>Brittle, E. E., Reynolds, A. E., Enquist, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+modes+of+pseudorabies+virus+neuroinvasion+and+lethality+in+mice.">Two modes of pseudorabies virus neuroinvasion and lethality in mice.</a> Journal of Virology. 78 (23), 12951-12963 (2004).</li><li>Reiner, A., Veenman, C. L., Medina, L., Jiao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathway+tracing+using+biotinylated+dextran+amines.">Pathway tracing using biotinylated dextran amines.</a> Journal of neuroscience. 103, 23-37 (2000).</li><li>Reiner, A., Honig, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroanatomical+tract-tracing+3+(Chapter+10).">Neuroanatomical tract-tracing 3 (Chapter 10).</a> Dextran Amines Versatile Tools for Anterograde and Retrograde Studies of Nervous System Connectivity. 10, 304-335 (2006).</li><li>Veenman, C. L., Reiner, A., Honig, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biotinylated+dextran+amine+as+an+anterograde+tracer+for+single+and+double+labeling+studies.">Biotinylated dextran amine as an anterograde tracer for single and double labeling studies.</a> Journal of Neuroscience Methods. 41 (3), 239-254 (1992).</li><li>Rajakumar, N., Elisevich, K., Flumerfelt, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biotinylated+dextran+a+versatile+anterograde+and+retrograde+neuronal+tracer.">Biotinylated dextran a versatile anterograde and retrograde neuronal tracer.</a> Brain Research. 607 (1-2), 47-53 (1993).</li><li>Dederen, P. J. W. C., Gribnau, A. A. M., Curfs, M. H. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrograde+neuronal+tracing+with+cholera+toxin+B+subunit:+comparison+of+three+different+visualization+methods.">Retrograde neuronal tracing with cholera toxin B subunit: comparison of three different visualization methods.</a> Histochemical Journal. 26 (11), 856-862 (1994).</li><li>Hattox, A. M., Priest, C. A., Keller, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+circuitry+involved+in+the+regulation+of+whisker+movements.">Functional circuitry involved in the regulation of whisker movements.</a> The Journal of Comparative Neurology. 442 (3), 266-276 (2002).</li><li>Bartha, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+reduction+of+virulence+of+Aujeszkys+disease+virus.">Experimental reduction of virulence of Aujeszkys disease virus.</a> Magy Allatorv Lapja. 16, 42-45 (1961).</li><li>Kuypers, H., Ugolini, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viruses+as+transneuronal+tracers.">Viruses as transneuronal tracers.</a> Trends in Neurosciences. 13 (2), 71-75 (1990).</li><li>Grinevich, V., Brecht, M., Osten, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monosynaptic+pathway+from+rat+vibrissa+motor+cortex+to+facial+motor+neurons+revealed+by+lentivirus-based+axonal+tracing.">Monosynaptic pathway from rat vibrissa motor cortex to facial motor neurons revealed by lentivirus-based axonal tracing.</a> The Journal of Neuroscience. 25 (36), 8250-8258 (2005).</li><li>Miyashita, E., Keller, A., Asanuma, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Input+output+organization+of+the+rat+vibrissal+motor+cortex.">Input output organization of the rat vibrissal motor cortex.</a> Experimental Brain Research. 99 (2), 223-232 (1994).</li><li>Hsu, S. M., Soban, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Color+modification+of+diaminobenzidine+(DAB)+precipitation+by+metallic+ions+and+its+application+for+double+immunohistochemistry.">Color modification of diaminobenzidine (DAB) precipitation by metallic ions and its application for double immunohistochemistry.</a> Journal of Histochemistry and Cytochemistry. 30 (10), 1079-1082 (1982).</li><li>Hsu, S. M., Raine, L., Fanger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+avidin+biotin-peroxidase+complex+(ABC)+in+immunoperoxidase+techniques+a+comparison+between+ABC+and+unlabeled+antibody+(PAP)+procedures.">Use of avidin biotin-peroxidase complex (ABC) in immunoperoxidase techniques a comparison between ABC and unlabeled antibody (PAP) procedures.</a> Journal of Histochemistry and Cytochemistry. 29 (4), 577-580 (1981).</li><li>Wouterlood, F. G., Jorritsma Byham, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+anterograde+neuroanatomical+tracer+biotinylated+dextran+amine+comparison+with+the+tracer+Phaseolus+vulgaris+leucoagglutinin+in+preparations+for+electron+microscopy.">The anterograde neuroanatomical tracer biotinylated dextran amine comparison with the tracer Phaseolus vulgaris leucoagglutinin in preparations for electron microscopy.</a> Journal of Neuroscience Methods. 48 (1-2), 75-87 (1993).</li><li>Altschuler, S. M., Bao, X. M., Miselis, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+architecture+of+nucleus+ambiguus+motoneurons+projecting+to+the+upper+alimentary+tract+in+the+rat.">Dendritic architecture of nucleus ambiguus motoneurons projecting to the upper alimentary tract in the rat.</a> The Journal of Comparative Neurology. 309 (3), 402-414 (1991).</li><li>Arriaga, G., Zhou, E. P., Jarvis, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Of+mice+birds+and+men+the+mouse+ultrasonic+song+system+has+some+features+similar+to+humans+and+songlearning+birds.">Of mice birds and men the mouse ultrasonic song system has some features similar to humans and songlearning birds.</a> PLoS ONE. 7 (10), e46610 (2012).</li><li>Card, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+considerations+for+the+use+of+pseudorabies+virus+in+transneuronal+studies+of+neural+circuitry.">Practical considerations for the use of pseudorabies virus in transneuronal studies of neural circuitry.</a> Neuroscience and Biobehavioral Reviews. 22 (6), 685-694 (1998).</li><li>Zuckerman, F. A., Zsak, L., Mettenleiter, T. C., Ben Porat, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudorabies+virus+glycoprotein+gIII+is+a+major+target+antigen+for+murine+and+swine+virus-specific+cytotoxic+T+lymphocytes.">Pseudorabies virus glycoprotein gIII is a major target antigen for murine and swine virus-specific cytotoxic T lymphocytes.</a> Journal of Virology. 64 (2), 802-812 (1990).</li><li>Card, J. P., Enquist, L. W., Moore, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroinvasiveness+of+pseudorabies+virus+injected+intracerebrally+is+dependent+on+viral+concentration+and+terminal+field+density.">Neuroinvasiveness of pseudorabies virus injected intracerebrally is dependent on viral concentration and terminal field density.</a> The Journal of Comparative Neurology. 407 (3), 438-452 (1999).</li><li>Pickard, G. E., Smeraski, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravitreal+injection+of+the+attenuated+pseudorabies+virus+PRV+Bartha+results+in+infection+of+the+hamster+suprachiasmatic+nucleus+only+by+retrograde+transsynaptic+transport+via+autonomic+circuits.">Intravitreal injection of the attenuated pseudorabies virus PRV Bartha results in infection of the hamster suprachiasmatic nucleus only by retrograde transsynaptic transport via autonomic circuits.</a> The Journal of Neuroscience. 22 (7), 2701-2710 (2002).</li><li>Smith, G. A., Gross, S. P., Enquist, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpesviruses+use+bidirectional+fast+axonal+transport+to+spread+in+sensory+neurons.">Herpesviruses use bidirectional fast axonal transport to spread in sensory neurons.</a> Proceedings of the National Academy of Sciences of the United States of America. 98 (6), 3466-3470 (2001).</li></ol>

No conflicts of interest declared.

Ci sono una serie di questioni che devono essere presi in considerazione al momento di pianificare un esperimento utilizzando PRV152 4,21. Ancora più importante, il virus della pseudorabbia è letale. Come accennato in precedenza, le grandi scimmie, inclusi gli esseri umani non sono suscettibili alle infezioni, ma cure appropriate devono essere esercitati per proteggere gli altri animali. Topi adulti in genere sopravvivono cinque a sette giorni dopo l&#39;inoculazione con il ceppo PRV152 attenuato. Pertanto,...

Tracing Transsynaptic è diventato un potente strumento utilizzato per analizzare efferenze centrali che regolano obiettivi periferici attraverso circuiti multi-sinaptici. Questo approccio è stato più ampiamente utilizzato nel cervello dei roditori utilizzando virus pseudorabbia patogeno suina (PRV), in particolare il ceppo attenuato PRV-Bartha descritta per la prima nel 1961 12. Qui, vi presentiamo un protocollo per l&#39;identificazione della rappresentazione corticale motore dei muscoli specifici o gruppi muscolari con un ceppo virale pseudorabbia ricombinanti (PRV152) che esprime la proteina fluorescente verde (eGFP) gene reporter 2. Il metodo descritto sfrutta il comportamento dei virus neurotropi, che producono progenie infettiva che sinapsi trasversali ad infettare altri neuroni all&#39;interno di un circuito funzionale 3,4,13. PRV152, che è isogenic con PRV-Bartha, attraversa solo sinapsi retrograda attraverso la sequenza gerarchica delle connessioni sinaptiche di distanza dal sito di infezione 3,5 &lt;/ sup&gt;. Controllando il proprio sito periferico di infezione è possibile limitare l&#39;ingresso del virus nel cervello attraverso uno specifico sottoinsieme di neuroni motori. Come sequenzialmente virus infetta catene di neuroni collegati, il modello risultante segnale di eGFP in tutto il cervello si risolverà la rete di neuroni che sono collegati alle cellule infette inizialmente. Un ulteriore vantaggio di utilizzare virus per tracciamento neurale è l&#39;amplificazione della proteina reporter (eGFP in questo caso) all&#39;interno delle cellule infette. Questa amplificazione del segnale fornisce un livello di sensibilità che consente di rilevare anche proiezioni sparse. Ad esempio, una proiezione sparse dalla corteccia motoria vibrissa ai motoneuroni facciali che controllano i baffi è stato trovato in ratti utilizzando viralmente espresso proteina fluorescente verde 14; studi precedenti non sono riusciti a trovare questo proiezione utilizzando traccianti classici senza giornalista amplificazione del gene 11,15. purtroppo, Molti vettori virali tracing, come quello usato nello studio citato, non attraversano sinapsi, limitando così il loro uso per tracciare i circuiti multi-sinaptici. Pur presentando notevoli vantaggi per identificare la rete di cellule che partecipano a un circuito del motore, la natura distribuita transsynaptic tracciare con PRV-152 rende interpretazione collegamenti specifici all&#39;interno del circuito difficile. Pertanto, vi presentiamo un metodo semplice per la convalida collegamenti specifici all&#39;interno dei circuiti identificate utilizzando PRV-152 per doppia etichettatura con ammine biotinilate destrano (BDA) e tossina del colera subunità b (CTB). L&#39;uso combinato di BDA e CTB è un approccio consolidato per tracciare le connessioni tra specifici gruppi di neuroni 6-8,11. Quando usati insieme, queste due rivelatori possono essere visualizzati nella stessa sezione con un DAB due colori (3, 3&#39;-diaminobenzidina) Procedura 16. Alto peso molecolare BDA (BDA10kDa) è stato scelto per questo protocollo perché yields etichettatura dettagliata dei processi neuronali 6,7,9. Ulteriori vantaggi BDA10kDa includono quanto segue: è preferenzialmente trasportato nella direzione anterograda 6-8; può essere trasportato da iontoforetica o pressione di iniezione 6-8; può essere visualizzato da un semplice avidina-HRP biotinilato (ABC) Procedura 17; e può essere ripreso dalla luce o microscopia elettronica 6,7,18. Rilevazione immunochimica di CTB con perossidasi e DAB è stato scelto per l&#39;etichettatura retrograda dei motoneuroni, perché è efficace nel rivelare i processi cellulari tra cui dendriti distali 10,19. Recentemente abbiamo usato questo approccio per identificare il percorso del motore vocale nei topi e per rivelare un collegamento sparse da corteccia motoria primaria per i motoneuroni laringei, che in precedenza era assume siano assenti 20.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>NanoFil Microinjection System</td>
			<td>World Precision Instruments</td>
			<td>IO-Kit</td>
			<td>34 gauge option</td>
		</tr>
		<tr>
			<td>Stereotaxic frame</td>
			<td>David Kopf Instruments</td>
			<td>Model 900</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanoject II Auto-Nanoliter Injector</td>
			<td>Drummond Scientific Company</td>
			<td>3-000-204</td>
			<td></td>
		</tr>
		<tr>
			<td>Sliding microtome</td>
			<td>Leica</td>
			<td>SM2010 R</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td>VetBond</td>
			<td>3M</td>
			<td>1469SB</td>
			<td></td>
		</tr>
		<tr>
			<td>Isofluorane (Forane)</td>
			<td>Baxter&#160;</td>
			<td>1001936060</td>
			<td></td>
		</tr>
		<tr>
			<td>Betadine Swab Stick</td>
			<td>Cardinal Health</td>
			<td>2130-01</td>
			<td>200 count</td>
		</tr>
		<tr>
			<td>Permount Mounting Medium</td>
			<td>Fisher Scientific</td>
			<td>SP15-500</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperFrost Plus slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated dextran amines</td>
			<td>Invitrogen</td>
			<td>D-1956</td>
			<td>10,000 MW</td>
		</tr>
		<tr>
			<td>Pseudorabies virus</td>
			<td>Laboratory of Dr. Lynn Enquist (Princeton University)</td>
			<td>PRV152</td>
			<td>Titer &#62; 1 x 107</td>
		</tr>
		<tr>
			<td>Anti-Cholera Toxin B Subunit (Goat)</td>
			<td>List Biological Laboratories</td>
			<td>703</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholera Toxin B Subunit</td>
			<td>List Biological Laboratories</td>
			<td>103B</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-eGFP</td>
			<td>Open Biosystems</td>
			<td>ABS4528</td>
			<td></td>
		</tr>
		<tr>
			<td>3, 3&#39;-diaminobenzidine</td>
			<td>Sigma-Aldrich</td>
			<td>D5905</td>
			<td>10 mg tablets</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>E7023</td>
			<td>200 proof</td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>F8775</td>
			<td>Dilute to 4%</td>
		</tr>
		<tr>
			<td>Hydrogen peroxide</td>
			<td>Sigma-Aldrich</td>
			<td>H3410</td>
			<td>30%</td>
		</tr>
		<tr>
			<td>Ketamine HCl &#38; Xylazine HCl</td>
			<td>Sigma-Aldrich</td>
			<td>K4138</td>
			<td>80 mg/mL &#38; 6 mg/mL</td>
		</tr>
		<tr>
			<td>Nickel chloride</td>
			<td>Sigma-Aldrich</td>
			<td>339350</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffer</td>
			<td>Sigma-Aldrich</td>
			<td>P3619</td>
			<td>1.0 M; pH 7.4</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Sigma-Aldrich</td>
			<td>P5493</td>
			<td>10X; pH 7.4</td>
		</tr>
		<tr>
			<td>Sodium Pentobarbital</td>
			<td>Sigma-Aldrich</td>
			<td>P3761</td>
			<td>50 mg/mL dose</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S9378</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylenes</td>
			<td>Sigma-Aldrich</td>
			<td>534056</td>
			<td>Histological grade</td>
		</tr>
		<tr>
			<td>VECTASTAIN Elite ABC Kit</td>
			<td>Vector Laboratories</td>
			<td>PK-6101 (rabbit); PK-6105 (goat)</td>
			<td></td>
		</tr>
		<tr>
			<td>Optixcare opthalmic ointment</td>
			<td>Vet Depot</td>
			<td>1017992</td>
			<td></td>
		</tr>
	</tbody>
</table>

transsynaptic tracing from peripheral targets with pseudorabies virus followed by cholera toxin and biotinylated dextran amines double labeling

Tracing Transsynaptic è diventato un potente strumento utilizzato per analizzare efferenze centrali che regolano obiettivi periferici attraverso circuiti multi-sinaptici. Questo approccio è stato più ampiamente utilizzato nel cervello utilizzando virus pseudorabbia patogeno suina (PRV) 1. PRV non infetta grandi scimmie, inclusi gli esseri umani, per cui è più comunemente usato negli studi di piccoli mammiferi, in particolare roditori. Il PRV152 pseudorabbia ceppo esprime la maggiore proteina fluorescente verde (eGFP) gene reporter e attraversa solo sinapsi funzionali retrograda attraverso la sequenza gerarchica delle connessioni sinaptiche di distanza dal sito di infezione 2,3. Altri ceppi PRV sono distinte proprietà microbiologiche e possono essere trasportati in entrambe le direzioni (PRV-Becker e PRV-Kaplan) 4,5. Questo protocollo si occuperà esclusivamente di PRV152. Fornendo il virus in un sito periferico, come i muscoli, è possibile limitare l&#39;ingresso del virus in tha cervello attraverso un insieme specifico di neuroni. Il modello risultante segnale di eGFP in tutto il cervello quindi risolve i neuroni che sono collegati alle cellule infette inizialmente. Come la natura distribuita del tracciato transsynaptic con virus pseudorabbia fa interpretare i collegamenti specifici all&#39;interno di una rete identificata difficile, vi presentiamo un metodo sensibile e affidabile utilizzando ammine biotinilate destrano (BDA) e tossina del colera subunità b (CTB) per confermare le connessioni tra le cellule identificate utilizzando PRV152. Rilevazione immunochimica di BDA e CTB con perossidasi e DAB (3, 3&#39;-diaminobenzidina) è stato scelto perché sono efficaci a rivelare i processi cellulari tra cui dendriti distali 6-11.

Colorazione per eGFP dovrebbe cominciare mostrando segnale debole in neuroni motori primari di circa 72 ore dopo l&#39;iniezione PRV152 nel muscolo. La replica e il trasporto transsynaptic del virus sono titer- e dipendenti dal tempo 4. Circa 90 ore dopo l&#39;iniezione, eGFP colorazione rivelerà segnale più forte nelle cellule infettate ordine 2 nd. Tempi di sopravvivenza più lunghi rivelerà 3 ° e cellule di ordine superiore, ma i tempi di sopravvivenza sono limitati dalla letalit?...

Watch this Scientific Journal Video about Transsynaptic Tracing from Peripheral Targets with Pseudorabies Virus Followed by Cholera Toxin and Biotinylated Dextran Amines Double Labeling at JoVE.com

Transsynaptic Tracing from Peripheral Targets with Pseudorabies Virus Followed by Cholera Toxin and Biotinylated Dextran Amines Double Labeling

Transsynaptic tracing has become a powerful tool for analyzing central efferents regulating peripheral targets through multi-synaptic circuits. Here we present a protocol that exploits the transsynaptic pseudorabies virus to identify and localize a functional brain circuit, followed by classical tract tracing techniques to validate specific connections in the circuit between identified groups of neurons.

Transsynaptic Tracing da obiettivi periferiche con Pseudorabies virus seguito da tossina colerica e Biotinilato Dextran ammine doppia etichettatura

Transsynaptic tracing has become a powerful tool used to analyze central efferents that regulate peripheral targets through multi-synaptic circuits. This ...

transsynaptic-tracing-from-peripheral-targets-with-pseudorabies-virus

Research

JoVE Journal

Neuroscience

12.6K Views.  Duke University Medical Center. Transsynaptic tracing has become a powerful tool for analyzing central efferents regulating peripheral targets through multi-synaptic circuits. Here we present a protocol that exploits the transsynaptic pseudorabies virus to identify and localize a functional brain circuit, followed by classical tract tracing techniques to validate specific connections in the circuit between identified groups of neurons.

Video: Transsynaptic Tracing from Peripheral Targets with Pseudorabies Virus Followed by Cholera Toxin and Biotinylated Dextran Amines Double Labeling

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

In utero Elettroporazione seguita da primaria Cultura neuronale per studiare la funzione dei geni in sottoinsieme di neuroni corticali

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

Ricerca

Neuroscienze

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

Etichettatura retrograda di cellule gangliari della retina in Zebrafish adulti con coloranti fluorescenti

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge in the field of virology; in particular for nuclear-replicating persistent DNA viruses that involve animal models for their study. Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in peripheral neurons. Latent virus serves as reservoir, from which it reactivates and induces a new herpetic episode. The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ in animal models. We describe a DNA-fluorescent in situ hybridization (FISH) approach efficiently detecting low-copy viral genomes within sections of neuronal tissues from infected animal models. The method relies on heat-based antigen unmasking, and directly labeled home-made DNA probes, or commercially available probes. We developed a triple staining approach, combining DNA-FISH with RNA-FISH&#160;and immunofluorescence, using peroxidase based signal amplification to accommodate each staining requirement. A major improvement is the ability to obtain, within 10 &#181;m tissue sections, low-background signals that can be imaged at high resolution by confocal microscopy and wide-field conventional epifluorescence. Additionally, the triple staining worked with a wide range of antibodies directed against cellular and viral proteins. The complete protocol takes 2.5 days to accommodate antibody and probe penetration within the tissue.

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

We established a fluorescent in situ hybridization protocol for the detection of a persistent DNA virus genome within tissue sections of animal models. This protocol enables studying infection process by codetection of the viral genome, its RNA products, and viral or cellular proteins within single cells.

La rilevazione del genoma e Trascrizioni di un virus DNA persistente in tessuti neuronali da fluorescente In situ Ibridazione In combinazione con Immunostaining

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge ...

Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis of primary cultures of neurons. We describe a set of procedures for: labeling newborn mice to be genotyped, rapid genotyping, and establishing low-density cultures of brain neurons from these mice. Individual mice are labeled by tattooing, which allows for long-term identification lasting into adulthood. Genotyping by the described protocol is fast and efficient, and allows for automated extraction of nucleic acid with good reliability. This is useful under circumstances where sufficient time for conventional genotyping is not available, e.g., in mice that suffer from neonatal lethality. Primary neuronal cultures are generated at low density, which enables imaging experiments at high spatial resolution. This culture method requires the preparation of glial feeder layers prior to neuronal plating. The protocol is applied in its entirety to a mouse model of the movement disorder DYT1 dystonia (&#916;E-torsinA knock-in mice), and neuronal cultures are prepared from the hippocampus, cerebral cortex and striatum of these mice. This protocol can be applied to mice with other genetic mutations, as well as to animals of other species. Furthermore, individual components of the protocol can be used for isolated sub-projects. Thus this protocol will have wide applications, not only in neuroscience but also in other fields of biological and medical sciences.

We describe procedures for labeling and genotyping newborn mice and generating primary neuronal cultures from them. The genotyping is rapid, efficient and reliable, and allows for automated nucleic-acid extraction. This is especially useful for neonatally lethal mice and their cultures that require prior completion of genotyping.

Rapida genotipizzazione degli animali Seguito da Stabilire colture primarie di neuroni del cervello

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis ...

Conditional Genetic Transsynaptic Tracing in the Embryonic Mouse Brain

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits during embryonic maturation of the brain however is technically challenging because most transsynaptic tracing methods developed to date depend on stereotaxic tracer injection. To overcome this problem, we developed a binary genetic strategy for conditional genetic transsynaptic tracing in the mouse brain. Towards this end we generated two complementary knock-in mouse strains to selectively express the bidirectional transsynaptic tracer barley lectin (BL) and the retrograde transsynaptic tracer Tetanus Toxin fragment C from the ROSA26 locus after Cre-mediated recombination. Cell-specific tracer production in these mice is genetically encoded and does not depend on mechanical tracer injection. Therefore our experimental approach is suitable to study neural circuit formation in the embryonic murine brain. Furthermore, because tracer transfer across synapses depends on synaptic activity, these mouse strains can be used to analyze the communication between genetically defined neuronal populations during brain development at a single cell resolution. Here we provide a detailed protocol for transsynaptic tracing in mouse embryos using the novel recombinant ROSA26 alleles. We have utilized this experimental technique in order to delineate the neural circuitry underlying maturation of the reproductive axis in the developing female mouse brain.

Capitalizing on a binary genetic strategy we provide a detailed protocol for neural circuit tracing in mice that express complementary transsynaptic tracers after Cre-mediated recombination. Because cell-specific tracer production is genetically encoded, our experimental approach is suitable to study the formation and maturation of neural circuitry during murine embryonic brain development at a single cell resolution.

Condizionale genetica Transsynaptic Tracing nel cervello embrionali di topo

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits ...

Immunohistochemistry and Multiple Labeling with Antibodies from the Same Host Species to Study Adult Hippocampal Neurogenesis

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly committed intermediate progenitor subtypes. Each of these subtypes expresses a set of specific molecular markers that, together with specific morphological criteria, can be used for their identification. Typically, immunofluorescent techniques are applied involving subtype-specific antibodies in combination with exo- or endogenous proliferation markers. We herein describe immunolabeling methods for the detection and quantification of all stages of adult hippocampal neurogenesis. These comprise the application of thymidine analogs, transcardial perfusion, tissue processing, heat-induced epitope retrieval, ABC immunohistochemistry, multiple indirect immunofluorescence, confocal microscopy and cell quantification. Furthermore we present&#160;a sequential multiple immunofluorescence protocol which circumvents problems usually arising from the need of using primary antibodies raised in the same host species. It allows an accurate identification of all hippocampal progenitor subtypes together with a proliferation marker within a single section. These techniques are a powerful tool to study the regulation of different progenitor subtypes in parallel, their involvement in brain pathologies and their role in specific brain functions.

This video article illustrates a comprehensive protocol to detect and quantify all stages of adult hippocampal neurogenesis within the same tissue section. We elaborated a method to overcome the limitations of indirect multiple immunofluorescence that arise when suitable antibodies from different host species are unavailable.

Immunoistochimica e multiplo etichettatura con anticorpi delle specie ospite stessi allo Studio adulti ippocampale neurogenesi

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly ...

Laser-guided Neuronal Tracing In Brain Explants

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake through electroporation in brain explants with targeted laser illumination and matching filter goggles during the procedure. The described technique of in vitro electroporation by itself yields relatively good visual control for targetting certain areas of the brain. By combining it with laser excitation of fluorescent genetic markers and their read-out through band-passing filter goggles, which can pick up the emissions of the genetically labeled cells/nuclei and the fluorescent tracing dye, a researcher can substantially increase the accuracy of injections by finding the area of interest and controlling for the dye-spread/uptake in the injection area much more efficiently. In addition, the laser illumination technique allows to study the functionality of a given neurocircuit by providing information about the type of neurons projecting to a certain area in cases where the GFP expression is linked to the type of transmitter expressed by a subpopulation of neurons.

We describe a technique to label neurons and their processes via anterograde or retrograde tracer injections into brain nuclei using an in vitro preparation. We modified an existing method of in vitro tracer electroporation by taking advantage of fluorescently labeled mouse mutants and basic optical equipment in order to increase labeling accuracy.

Neuronal Tracing negli espianti cerebrali a guida laser

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake ...

Improving the Application of High Molecular Weight Biotinylated Dextran Amine for Thalamocortical Projection Tracing in the Rat

High molecular weight biotinylated dextran amine (BDA) has been used as a highly sensitive neuroanatomical tracer for many decades. Since the quality of its labeling was affected by various factors, here, we provide a refined protocol for the application of high molecular weight BDA for studying optimal neural labeling in the central nervous system. After stereotactic injection of BDA into the ventral posteromedial nucleus (VPM) of the thalamus in the rat through a delicate glass pipette, BDA was stained with fluorescent streptavidin-Alexa (AF) 594 and counterstained with fluorescent Nissl stain AF500/525. On the background of green Nissl staining, the red BDA labeling, including neuronal cell bodies and axonal terminals, was more distinctly demonstrated in the somatosensory cortex. Furthermore, double fluorescent staining for BDA and the calcium-binding protein parvalbumin (PV) was carried out to observe the correlation of BDA labeling and PV-positive interneurons in the cortical target, providing the opportunity to study the local neural circuits and their chemical characteristics. Thus, this refined method is not only suitable for visualizing high quality neural labeling with the high molecular weight BDA through reciprocal neural pathways between the thalamus and cerebral cortex, but also will permit the simultaneous demonstration of other neural markers with fluorescent histochemistry or immunochemistry.

Here, we present a refined protocol to effectively reveal biotinylated dextran amine (BDA) labeling with a fluorescent staining method through a reciprocal neural pathway. It is suitable for analyzing the fine structure of BDA labeling and distinguishing it from other neural elements under a confocal laser scanning microscope.

Migliorare l'applicazione di alto peso molecolare del dextrano Biotinylated ammina per proiezione Thalamocortical traccia nel ratto

High molecular weight biotinylated dextran amine (BDA) has been used as a highly sensitive neuroanatomical tracer for many decades. Since the quality of ...

Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate decisions, will be crucial to design therapeutic strategies for many diseases affecting the nervous system. Current methods to track cell populations rely on their final outcomes in still images and they generally fail to provide sufficient temporal resolution to identify behavioral features in single cells. Moreover, variations in cell death, behavioral heterogeneity within a cell population, dilution, spreading, or the low efficiency of the markers used to analyze cells are all important handicaps that will lead to incomplete or incorrect read-outs of the results. Conversely, performing live imaging and single cell tracking under appropriate conditions represents a powerful tool to monitor each of these events. Here, a time-lapse video-microscopy protocol, followed by post-processing, is described to track neural populations with single cell resolution, employing specific software. The methods described enable researchers to address essential questions regarding the cell biology and lineage progression of distinct neural populations.

A robust protocol to monitor neural populations by time-lapse video-microscopy followed by software-based post-processing is described. This method represents a powerful tool to identify biological events in a selected population during live imaging experiments.

Seguita dalla singola cellula di rilevamento a Monitor biologia cellulare e la progressione di lignaggio di popolazioni neurali Multiple immagini dal vivo

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate ...

Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. Additionally, epigenetic chromatin modifications, like histone methylation, have an important role for establishing and maintaining specific cell fates within this process. The vast majority of histone methylation occurs on the flexible histone tail, which is accessible to histone modifiers, erasers, and histone reader proteins. In contrast, H3K79 methylation is located in the globular domain of histone 3 and is implicated in different developmental functions. H3K79 methylation is evolutionarily conserved and can be found in a wide range of species from Homo sapiens to Saccharomyces cerevisiae. The modification occurs in different cell populations within organisms, including neural progenitors. The location of H3K79 methylation in the globular domain of histone 3 makes it difficult to assess. Here, we present methods to isolate and culture cortical progenitor cells (CPCs) from embryonic cortical brain tissue (E11.5-E14.5) or cerebellar granular neuron progenitors (CGNPs) from postnatal tissue (P5-P7), and to efficiently immunoprecipitate H3K79me2 for quantitative PCR (qPCR) and genome-wide sequencing.

We present an effective and reproducible method to isolate and culture neural progenitor cells from embryonic and postnatal brain tissue for chromatin immunoprecipitation (ChIP) of histone 3 lysine 79 dimethylation (H3K79me2) - a histone mark located within the globular domain of histone 3.

Isolamento e coltura dei progenitori neurali seguito da immunoprecipitazione della cromatina di istone 3 lisina 79 Dimethylation Mark

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. ...