Vorremmo ringraziare i Proff. Huibert Mansvelder e Bert Sakmann per il supporto esteso, il dottor Marcel Oberlaender per le discussioni fruttuose e di fornire tracciamento neuronale, e Brendan Lodder per l&#39;assistenza tecnica. Dati è stata acquisita utilizzando il ntrode VI per LabView, generosamente offerto da R. Bruno (Columbia Univ., NY, USA). Questa ricerca è stata sostenuta dalla Max Planck Society e il Centro Bernstein for Computational Neuroscience, Tuebingen (finanziato dal ministero federale tedesco dell&#39;Istruzione e della ricerca (BMBF; FKZ: 01GQ1002)) (RTN), Centro per Neurogenomics e Cognitive Research (CNCR) , Neuroscienze Campus Amsterdam (NCA), finanziamenti per CPJdK (NWO-ALW # 822.02.013 e ENC-Network # p3-c3) e la VU University Amsterdam.

1. Preparazione del animale Tutte le procedure sperimentali sono svolte in conformità con la legge olandese e previa valutazione da parte di un comitato etico locale presso la VU University Amsterdam, Paesi Bassi. <ol><li> Anestetizzare un ratto Wistar (P25-P45, ♂ / ♀) con isoflurano (2-3% di ossigeno) e successivamente con uretano (20% in 0,9% NaCl, 1.6-1.7 g / kg) mediante iniezione intraperitoneale. Valutare la profondità dell&#39;anestesia monitorando il ritiro pizzico, riflessi palpebra, e movimenti vibrisse. </li><li> Somministrare una dose aggiuntiva di uretano (10% della dose iniziale) nel caso l&#39;animale non raggiunge sufficiente profondità dell&#39;anestesia o quando si osservano contrazioni Vibrissae durante il corso dell&#39;esperimento. </li><li> Posto l&#39;animale anestetizzato sul telaio stereotassico dotato di una piastra elettrica, inserire sonda di temperatura rettale e mantenere la temperatura corporea dell&#39;animale a 37,5 ± 0,5 ° C da una piastra elettrica. </li><li&gt; posizione della testa dell&#39;animale nel frame stereotassico, tagliare i capelli sulla sommità della testa con le forbici per rendere la pelle visibile. </li><li> Iniettare 100 ml 1% lidocaina (in 0.9% NaCl) per via sottocutanea per l&#39;anestesia locale nel sito dell&#39;operazione e aspettare 3-5 minuti. Rimuovere la pelle che copre la superficie del cranio tagliando in direzione caudale-to-rostrale e pulire il tessuto rimanente. </li><li> Pulire il cranio ampiamente con 0,9% di NaCl e tamponi assorbenti. </li><li> Determinare le coordinate della zona di destinazione sulla emisfero sinistro (per la corteccia somatosensoriale primaria (S1): 2.5 mm posteriore e 5,5 millimetri lateralmente bregma). Segnare la posizione sul cranio con un pennarello pelle chirurgico. </li><li> Raschiare l&#39;osso delicatamente da un trapano dentistico dalla regione di interesse finché l&#39;osso diventa trasparente e vasi sanguigni sono chiaramente visibili. </li><li> Fare un 0,5 millimetri x 0,5 millimetri craniotomia tagliando una finestra nel cranio assottigliata con un bisturi (# 11), evitando di danneggiare la dura madree vasi sanguigni. Optional: segnare i bordi della craniotomia con un pennarello pelle chirurgico per migliorare la visibilità. </li><li> Applicare un sottile strato di colla sul cranio secco e poi cemento dentale per costruire una vasca (cioè camera di registrazione) che racchiude la craniotomia. </li><li> Tagliare i baffi a lato controlaterale esattamente a 5 mm dal follicolo baffo. Facoltativo: evidenziare i baffi tagliati con mascara nero. </li></ol> 2. Juxtasomal Recordings e biocitina Etichettatura <ol><li> Fai pipette di patch di vetro borosilicato con diametro interno di punta di circa 1 micron di ottenere elettrodi con 3-5 resistenza mW. Nota: La morfologia pipetta ideale per la registrazione juxtasomal è un cono graduale esile, un angolo di cono basso, e un ~ 1 micron punta (Figura 1). </li><li> A seconda della profondità di registrazione (rispetto alla superficie piale), le dimensioni del cono elettrodo di registrazione devono essere regolati. Criticamente importante è che tegli cono diametro dovrebbe essere inferiore a 75 micron al punto di entrata nel cervello per evitare danni meccanici o stress per l&#39;area di registrazione. Ciò indica che per le registrazioni superficiali corticali, un cono di 300-500 micron, con un diametro esterno massimo di 75 micron è necessaria (Figura 1A) che registrazioni da strati corticali relativamente profondi richiedono una conicità più di 1,500-1,800 micron, di nuovo con esterno massimo diametro di 75 micron (a 1.800 micron dalla punta dell&#39;elettrodo). </li><li> Caricare la pipetta di patch con la normale suoneria di ratto (NRR) integrato con il 2% biocitina (vedi Tabella 1 per le soluzioni e reagenti) e montare la pipetta di patch su un palco testa fissata ad un micromanipolatore. </li><li> Impostare l&#39;angolo della porta elettrodo a 34 ° rispetto al piano sagittale per indirizzare specificamente colonna D2 di ratto S1. </li><li> Collegare il palcoscenico testa ad un amplificatore in bridge-mode (cioè pinza corrente). </li><li> Posizionare la patchpipetta in prossimità della craniotomia. Riempite la vasca con 0,9% di NaCl e determinare la resistenza degli elettrodi, che dovrebbe essere tra 3-5 mW, valutato applicando un impulso quadrato con iniezione di corrente positiva (1 nA, 200 msec on / off). </li><li> Stabilire sovrapressione di 100-150 mbar e far avanzare la pipetta di patch a 1 micron passi durante l&#39;applicazione corrente positiva come impulsi quadrati (1 nA, 200 msec on / off). Monitorare la variazione di resistenza robusta momento stabilire un contatto con la dura madre. A questo punto, impostare le coordinate del micromanipolatore a &#39;zero&#39; per consentire misurazioni precise di profondità. </li><li> Anticipo in 1 micron step-mode fino a quando la pipetta di patch penetra la dura madre indicata da un improvviso calo di resistenza dell&#39;elettrodo. Rimuovere la pressione della pipetta azienda. </li><li> Ricerca di singole unità, mentre avanza in 1 micron passi. Monitorare la resistenza di continuo mediante l&#39;applicazione di 200 msec on / off impulsi. Un aumento della resistenza dell&#39;elettrodo typicalleato indica la vicinanza di un singolo neurone. Far avanzare l&#39;elettrodo fino potenziale d&#39;azione positiva (AP) forme d&#39;onda di ~ 2 mV sono registrati (Figure 2A e 2B). </li><li> Facoltativo: registrare il pattern spiking spontanea del neurone e determinare baffo-spiking evocata da, per esempio, caudalmente deviando basette singole per 200 msec ad un angolo di 3,3 ° utilizzando un dispositivo piezoelettrico 18. </li><li> Far avanzare l&#39;elettrodo fino a quando la resistenza è 25-35 mW e picchi hanno ampiezze di 3-8 mV per ottenere condizioni ottimali di riempimento juxtasomal. Avviare il ripieno juxtasomal applicando impulsi quadrati di corrente positiva (1 nA, 200 msec on / off). Lentamente e gradualmente aumentare la corrente a passi di 0,1 nA monitorando la forma d&#39;onda AP e frequenza (Figure 2A e 2B). </li><li> Controllare l&#39;apertura membrana come un chiaro aumento della frequenza AP durante la su-fase dell&#39;impulso blocco (Figura 2C </strong&gt;). La forma d&#39;onda picco durante il riempimento mostra un aumento della larghezza e ridotto (Figure 2C e 2D), dopo-iperpolarizzazione. Parametri supplementari includono un aumento del rumore o un piccolo (1-5 mV) spostamento DC negativo. </li><li> Aumentare o diminuire la corrente (1 nA), mentre il riempimento di mantenere stabile biocitina infusione. Ridurre o interrompere impulsi di corrente al momento improvviso aumento della frequenza AP per evitare la tossicità da eccesso afflusso di ioni extracellulari, come ioni sodio. Nota: ogni neurone risponderà in modo diverso per l&#39;iniezione e juxtasomal corrente devono essere regolati in base alle condizioni di registrazione singoli parametri di etichettatura. </li><li> Strettamente monitorare il segnale dopo l&#39;arresto della iniezione di corrente. La forma d&#39;onda picco dopo una sessione di riempimento è di solito allarga e mostra una forte riduzione dopo iperpolarizzazione. Attendere recupero del neurone, che è evidente quando la forma d&#39;onda di picco ritorna alle sue proprietà originali (ossia presenza dinormale, Figura dopo-iperpolarizzazione 2). </li><li> Ripetere biocitina riempimento sessioni dopo la completa ripresa del neurone per aumentare il carico biocitina per migliorare la qualità della colorazione. </li><li> Ritrarre la pipetta di patch a passi di 1 micron fino a quando l&#39;ampiezza picco scende a ridurre ogni stress meccanico alla cella. Attendere almeno 1 ora per il biocitina di diffondere intracellulare mentre strettamente controllo della temperatura corporea dell&#39;animale e la respirazione. </li></ol> 3. Perfusione animali e rimozione del cervello <ol><li> Preparare il setup perfusione, lavare, e precaricare il tubo con il 0,9% di NaCl. </li><li> Posizionare l&#39;animale su un vassoio chirurgico e fissare con nastro adesivo etichettatura standard. Garantire una sufficiente profondità dell&#39;anestesia; pizzico piede e riflessi palpebrali dovrebbero essere assenti. </li><li> Effettuare una mediale a laterale incisione (5-6 cm) attraverso la parete addominale appena sotto la gabbia toracica e procedere in direzione antero-posteriore per esporre lo sterno.Tirare sterno anteriormente e separare accuratamente il fegato dal diaframma. </li><li> Fai una piccola incisione nella membrana, tagliare le costole inferiori e continuare l&#39;incisione per tutta la lunghezza della cavità addominale per esporre il cuore. </li><li> Rimuovere il pericardio. </li><li> Inserire l&#39;ago nel ventricolo sinistro e fare un&#39;incisione nell&#39;atrio destro. Profumato con 0,9% di NaCl (~ 8 ml / min) fino a decolorazione del fegato è completa. </li><li> Passare l&#39;infusione al 4% paraformaldeide (PFA) fino rigidità della zampa anteriore e la mascella inferiore è evidente. </li><li> Decapitare il ratto usando un paio di forbici </li><li> Rimuovere i restanti muscoli del collo ed esporre completamente il cranio. </li><li> Posizionare le forbici nel tronco cerebrale sul lato dorsale e tagliare l&#39;osso attentamente lungo la sutura sagittale, mantenendo la posizione dorsale. </li><li> Rimuovere le ossa da entrambi i lati della sutura sagittale per esporre il cervello con pinze. Rimuovere con attenzione la dura per evitare dAmage. </li><li> Inserire delicatamente una spatola smussato al lato ventrale del cervello e rimuovere il cervello delicatamente. </li><li> Post-fissare l&#39;intera notte cervello in PFA a 4 ° C. Accendere il cervello per 0,05 M tampone fosfato (PB) e conservare a 4 ° C. </li></ol> 4. Affettare il cervello nelle sezioni tangenziali <ol><li> Prendete il cervello fuori dal 0,05 M PB e metterlo su una carta da filtro fronte anteriore. Utilizzare una lama di rasoio affilata per tagliare il cervelletto lungo il piano coronale e separare gli emisferi tagliando lungo il piano sagittale intermedio. </li><li> Applicare supercolla sulla piattaforma di montaggio e montare l&#39;emisfero sinistro sul suo piano sagittale con anteriore rivolto verso destra. Fissare la piattaforma di montaggio ad un angolo di 45 ° su un vibratome e immergere il cervello in 0,05 M PB. </li><li> Assicura una lama di rasoio sul vibratome e assicurarsi che il primo contatto con la superficie del cervello è al centro del piano antero-posteriore dell&#39;emisfero. Tagliare 24 100 micronsezioni e raccoglierle in una piastra a 24 pozzetti contenenti 0,05 M PB. </li></ol> 5. Procedure istologiche <ol><li> Eseguire protocolli istologici per la citocromo ossidasi colorazione 24 e il-biotina-perossidasi avidina metodo secondo metodi precedentemente descritti 25. Optional: visualizza biocitina utilizzando coniugato avidina fluorescente / streptavidina-Alexa. Questo permette inoltre a doppio colorazione con tecniche di tracciatura retrograda o anterograda. </li><li> Lavare le sezioni 5 x 5 min con 0,05 M PB e preparare il tetraidrocloride 3-3&#39;-diaminobenzidina (DAB) contenente soluzione per la colorazione citocromo ossidasi per visualizzare barili nel layer 4 (L4) di S1 (vedi Tabella 2 per le soluzioni e reagenti). Incubare le sezioni 6-12 della pia nella soluzione preriscaldato per 30-45 minuti a 37 ° C. </li><li> Sciacquare le sezioni con 0,05 M PB per 6 x 5 min e dissetare attività della perossidasi endogena incubando tutte le sezioni nel 3% H 2 </sub&gt; O 2 in 0,05 M PB per 20 minuti a temperatura ambiente (RT). </li><li> Sciacquare le sezioni con 0,05 M PB per 5 x 10 min. Incubare sezioni ABC-soluzione (vedi Tabella 3 per le soluzioni e reagenti) notte a 4 ° C. </li><li> Sciacquare le sezioni con 0,05 M PB per 5 x 10 min e preparare la DAB-soluzione per visualizzare il neurone biocitina-riempita (vedi Tabella 4 per le soluzioni e reagenti). Incubare le sezioni in soluzione filtrata per 45-60 minuti a temperatura ambiente. </li><li> Sciacquare le sezioni con 0,05 M PB per 5 x 10 min. Sezioni Mount su vetrini da microscopio e copertura antiscivolo con Mowiol (cfr. tabella 5 per le soluzioni e reagenti). </li><li> Determinare la qualità di etichettatura utilizzando microscopio ottico. </li></ol>

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P., Huber, D., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+activity+in+barrel+cortex+underlying+vibrissa-based+object+localization+in+mice.">Neural activity in barrel cortex underlying vibrissa-based object localization in mice.</a> Neuron. 67 (6), 1048-1061 (2010).</li><li>Veinante, P., Deschenes, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+study+of+motor+cortex+projections+to+the+barrel+field+in+rats.">Single-cell study of motor cortex projections to the barrel field in rats.</a> J. Comp. Neurol. 464 (1), 98-103 (2003).</li><li>Boudewijns, Z. S., 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=Layer-specific+high-frequency+action+potential+spiking+in+the+prefrontal+cortex+of+awake+rats.">Layer-specific high-frequency action potential spiking in the prefrontal cortex of awake rats.</a> Front. Cell. Neurosci. 7, 99 (2013).</li><li>Oberlaender, M., Bruno, R. M., Sakmann, B., Broser, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmitted+light+brightfield+mosaic+microscopy+for+three-dimensional+tracing+of+single+neuron+morphology.">Transmitted light brightfield mosaic microscopy for three-dimensional tracing of single neuron morphology.</a> J. Biomed. Opt. 12 (6), 064029 (2007).</li><li>Boudewijns, Z. S., 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=Semi-automated+three-dimensional+reconstructions+of+individual+neurons+reveal+cell+type-specific+circuits+in+cortex.">Semi-automated three-dimensional reconstructions of individual neurons reveal cell type-specific circuits in cortex.</a> Commun. Integr. Biol. 4 (4), 486-488 (2011).</li><li>Bruno, R. M., Hahn, T. T., Wallace, D. J., de Kock, C. P., Sakmann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+experience+alters+specific+branches+of+individual+corticocortical+axons+during+development.">Sensory experience alters specific branches of individual corticocortical axons during development.</a> J. Neurosci. 29 (10), 3172-3181 (2009).</li><li>Schubert, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observing+without+disturbing:+how+different+cortical+neuron+classes+represent+tactile+stimuli.">Observing without disturbing: how different cortical neuron classes represent tactile stimuli.</a> J. Physiol. 581 (1), 5 (2007).</li><li>Neumann, E., Kakorin, S., Toensing, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fundamentals+of+electroporative+delivery+of+drugs+and+genes).">Fundamentals of electroporative delivery of drugs and genes).</a> Bioelectrochem. Bioenerg. 48 (1), 3-16 (1999).</li><li>Haas, K., Sin, W. C., Javaherian, A., Li, Z., Cline, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+electroporation+for+gene+transfer+in+vivo.">Single-cell electroporation for gene transfer in vivo.</a> Neuron. 29 (3), 583-591 (2001).</li></ol>

I authros hanno nulla da rivelare.

Il metodo juxtasomal permette la registrazione in azione vivo potenziale chiodare da singole unità le varie patologie comportamentali (anestetizzati, sveglio testa fissa o muoversi liberamente) con l&#39;opzione di-biocitina etichettatura del neurone registrato per la post hoc tipo di cellula di classificazione e / o ricostruzione 3D. Il principale vantaggio è quello di ottenere parametri fisiologici nella configurazione extracellulare (quindi non invasiva), essendo ancora in grado di etiche...

Reti neuronali consistono di tipi cellulari diversi, caratterizzati da altamente specifiche proprietà morfologiche e fisiologiche 1-7. Di conseguenza, tipi di cellule individuali eseguono compiti specializzati all&#39;interno della rete (si veda ad esempio Gentet et al. 8 e Burgalossi et al. 9). Stiamo solo cominciando a capire le funzioni di tipo specifico di cellule attraverso le reti neuronali e molto è ancora da scoprire. A tal fine, molti laboratori stanno attuando approcci sperimentali che consentono l&#39;analisi delle proprietà morfologiche della stessa popolazione neuronale da cui sono stati ottenuti i parametri fisiologici 1,10-15. Qui, dimostriamo l&#39;etichettatura tecnica juxtasomal 16,17 che comporta registrazioni elettrofisiologiche utilizzando pipette di patch convenzionali nella configurazione extracellulare (quindi non invasiva) in combinazione con elettroporazione del neurone registrato con biocitina. Ilgrande vantaggio di questo approccio è che la natura non invasiva assicura che l&#39;azione potenziale spiking dei singoli neuroni viene registrato senza alterarne (es. dialisi) il contenuto intracellulare della cellula. Seguito da elettroporazione, l&#39;approccio juxtasomal offre la possibilità di identificazione del post hoc delle cellule e la ricostruzione di collegare la funzione (fisiologia) alla struttura (morfologia). In genere, la ricostruzione morfologica prevede la ricostruzione della morfologia dendritica e assonale che può essere estesa a quantificazione di colonna vertebrale e / o bouton densità o anche la ricostruzione della morfologia neuronale a risoluzione nanometrica usando la microscopia elettronica. La tecnica di registrazione juxtasomal può essere utilizzata per le registrazioni in vivo di vari tipi di cellule attraverso strati corticali o in aree sub-corticali in una vasta gamma di specie, anche se la maggior parte degli studi hanno applicato la tecnica a piccoli roditori come topi o ratti. La nostra ricerca è focalizzata sulla registrazione e l&#39;etichettatura dei neuronidi ratto corteccia somatosensoriale primaria (S1) e coinvolge l&#39;identificazione visiva dei neuroni registrati 18, ricostruzioni dendritiche in combinazione con la registrazione precisa in un sistema di riferimento standardizzato per decodificare le reti corticali 4,19 e la ricostruzione dettagliata di architettura assonale per caratterizzare locale specifico tipo di cellule e lungo raggio proiezione obiettivi 20. Rispetto al vivo di tecniche alternative di registrazione (intracellulari o cellule intere), le registrazioni juxtasomal sono relativamente stabili e possono quindi essere applicate attraverso gli stati comportamentali tra cui anestetizzato 21,22, sedati 14, sveglio testa fissa 23, o anche liberamente in movimento animali 9 . Qui vi mostriamo etichettatura juxtasomal in S1 di un ratto uretano-anestetizzato, anche se sottolineiamo l&#39;applicabilità generale di questa tecnica per molte preparazioni di scelta.

<table border="0" cellpadding="0" cellspacing="0" width="784">
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:224px;">SM-6 control system</td>
			<td style="width:275px;">Luigs &#38; Neumann</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:224px;">LN- Mini 23 XYZ</td>
			<td style="width:275px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">LN- Mini 55 Manipulatorblock X2</td>
			<td style="width:275px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Lynx-8 amplifier</td>
			<td style="width:275px;">Neuralynx</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Axoclamp-2B amplifier</td>
			<td style="width:275px;">Axon Instruments</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Osada model EXL-M40</td>
			<td style="width:275px;">Osada, inc.</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Piezoelectric device</td>
			<td style="width:275px;">Physik Instrumente</td>
			<td style="width:119px;">PL140.10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Labview</td>
			<td>National Instruments, Austin, TX, USA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Ntrode Virtual Instrument&#160;</td>
			<td>R. Bruno, Columbia Univ., NY, USA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">(Labview acq. software)</td>
			<td style="width:275px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Sugi absorbent swabs</td>
			<td style="width:275px;">Kettenbach</td>
			<td style="width:119px;">30601</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Cytochrome C from equine heart</td>
			<td style="width:275px;">Sigma</td>
			<td style="width:119px;">C2506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Catalase from bovine liver</td>
			<td style="width:275px;">Sigma</td>
			<td style="width:119px;">C9322</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">DAB</td>
			<td style="width:275px;">Sigma</td>
			<td style="width:119px;">D5637</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">H2O2</td>
			<td style="width:275px;">Boom</td>
			<td style="width:119px;">7047</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Vectastain standard ABC-kit</td>
			<td style="width:275px;">Vector</td>
			<td style="width:119px;">PK6100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Triton X100</td>
			<td style="width:275px;">Sigma</td>
			<td style="width:119px;">T9284</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Urethane</td>
			<td style="width:275px;">Sigma</td>
			<td style="width:119px;">U2500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Isoflurane</td>
			<td style="width:275px;">Pharmachemie</td>
			<td style="width:119px;">45.112.110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Lidocaine</td>
			<td style="width:275px;">Sigma</td>
			<td style="width:119px;">L5647</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Simplex rapid dental cement</td>
			<td style="width:275px;">Kemdent</td>
			<td>ACR308/ACR924</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Biocytin</td>
			<td style="width:275px;">Molekula</td>
			<td style="width:119px;">36219518</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">PFA</td>
			<td style="width:275px;">Merck Millipore</td>
			<td style="width:119px;">8187151000&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Trizma base</td>
			<td style="width:275px;">Sigma</td>
			<td style="width:119px;">T4661</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Mowiol 4-88</td>
			<td style="width:275px;">Aldrich</td>
			<td style="width:119px;">81381</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Analytical grade glycerol</td>
			<td style="width:275px;">Fluka</td>
			<td style="width:119px;">49767</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">HEPES</td>
			<td style="width:275px;">Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">NaCl</td>
			<td style="width:275px;">Sigma Aldrich</td>
			<td style="width:119px;">31434</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">KCl</td>
			<td style="width:275px;">Sigma Aldrich</td>
			<td style="width:119px;">60130</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">CaCl</td>
			<td style="width:275px;">Sigma Aldrich</td>
			<td style="width:119px;">22,350-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">MgCl2</td>
			<td style="width:275px;">Fluka</td>
			<td style="width:119px;">63072</td>
			<td></td>
		</tr>
	</tbody>
</table>

juxtasomal biocytin labeling to study the structure-function relationship of individual cortical neurons

La corteccia cerebrale è caratterizzata da molteplici strati e molti tipi di cellule distinte che insieme come rete sono responsabili per molte funzioni cognitive superiori, tra cui il processo decisionale, il comportamento sensoriale-guidata o memoria. Per comprendere come tali reti neuronali intricate eseguire operazioni, un passo fondamentale è quello di determinare la funzione (o attività elettrica) di tipi di cellule individuali all&#39;interno della rete, preferibilmente quando l&#39;animale sta eseguendo un compito cognitivo rilevante. Inoltre, è altrettanto importante determinare la struttura anatomica della rete e l&#39;architettura morfologica dei singoli neuroni per consentire reverse engineering rete corticale. Innovazioni tecniche oggi disponibili consentono di registrare l&#39;attività cellulare in sveglio, comportandosi animali con la possibilità preziosa di post hoc identificare i neuroni registrati. Qui, dimostriamo la tecnica di etichettatura juxtasomal biocitina, che coinvolge l&#39;azione di registrazione potenziometroal chiodare nella configurazione extracellulare (o loose-patch) con pipette di patch convenzionali. La configurazione di registrazione juxtasomal è relativamente stabile e applicabile a tutte le condizioni comportamentali, tra cui anestetizzato, sedato, sveglio testa fissa, e anche in animali liberi di muoversi. Pertanto, questo metodo consente il collegamento cellulare-tipo di azione specifico potenziale chiodare nel comportamento animale alla ricostruzione dei singoli neuroni e, in definitiva, l&#39;intera microcircuito corticale. In questo video manoscritto, mostriamo come singoli neuroni nella configurazione juxtasomal possono essere etichettati con biocitina nel ratto uretano-anestetizzato per l&#39;identificazione post hoc e la ricostruzione morfologica.

La conoscenza dettagliata sulla struttura 3D dei singoli neuroni è fondamentale per chiarire i principi organizzativi delle reti neuronali. Il nostro metodo prevede un gasdotto per ottenere l&#39;etichettatura biocitina alta qualità da una preparazione in vivo, consentendo in tal modo la classificazione post hoc neuronale e la ricostruzione dettagliata di dendritica e l&#39;architettura assonale di singoli neuroni ad alta risoluzione. A seconda della qualità di etichettatura juxtasomal, i neuroni so...

Watch this Scientific Journal Video about Juxtasomal Biocytin Labeling to Study the Structure-function Relationship of Individual Cortical Neurons at JoVE.com

Juxtasomal Biocytin Labeling to Study the Structure-function Relationship of Individual Cortical Neurons

Per comprendere la struttura delle reti neuronali, caratterizzazione funzionale e morfologica dei singoli neuroni è una necessità. Qui, dimostriamo etichettatura juxtasomal biocitina, che permette registrazioni elettrofisiologiche nella configurazione extracellulare, pur mantenendo la capacità di etichettare intracellulare neurone per la ricostruzione post-hoc di dendritiche e dell&#39;architettura assonale.

Juxtasomal biocitina Etichettatura per studiare la struttura-funzione relazioni dei singoli neuroni corticali

The cerebral cortex is characterized by multiple layers and many distinct cell-types that together as a network are responsible for many higher cognitive ...

juxtasomal-biocytin-labeling-to-study-structure-function-relationship

Research

JoVE Journal

Neuroscience

13.5K Views.  VU University Amsterdam.  Per comprendere la struttura delle reti neuronali, caratterizzazione funzionale e morfologica dei singoli neuroni è una necessità. Qui, dimostriamo etichettatura juxtasomal biocitina, che permette registrazioni elettrofisiologiche nella configurazione extracellulare, pur mantenendo la capacità di etichettare intracellulare neurone per la ricostruzione post-hoc di dendritiche e dell'architettura assonale. 

Video: Juxtasomal Biocytin Labeling to Study the Structure-function Relationship of Individual Cortical Neurons

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.

Laser Microdissezione Cattura di Drosophila Neuroni periferici

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

Ricerca

Neuroscienze

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

Bilaminar co-coltura di neuroni corticali primari di ratto e Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord

Here we present a technique to label the trajectories of small groups of DRG neurons into the embryonic spinal cord by diffusive staining using the lipophilic tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)1. The comparison of axonal pathways of wild-type with those of mouse lines in which genes are mutated allows testing for a functional role of candidate proteins in the control of axonal branching which is an essential mechanism in the wiring of the nervous system. Axonal branching enables an individual neuron to connect with multiple targets, thereby providing the physical basis for the parallel processing of information. Ramifications at intermediate target regions of axonal growth may be distinguished from terminal arborization. Furthermore, different modes of axonal branch formation may be classified depending on whether branching results from the activities of the growth cone (splitting or delayed branching) or from the budding of collaterals from the axon shaft in a process called interstitial branching2 (Fig. 1).
The central projections of neurons from the DRG offer a useful experimental system to study both types of axonal branching: when their afferent axons reach the dorsal root entry zone (DREZ) of the spinal cord between embryonic days 10 to 13 (E10 - E13) they display a stereotyped pattern of T- or Y-shaped bifurcation. The two resulting daughter axons then proceed in rostral or caudal directions, respectively, at the dorsolateral margin of the cord and only after a waiting period collaterals sprout from these stem axons to penetrate the gray matter (interstitial branching) and project to relay neurons in specific laminae of the spinal cord where they further arborize (terminal branching)3. DiI tracings have revealed growth cones at the dorsal root entry zone of the spinal cord that appeared to be in the process of splitting suggesting that bifurcation is caused by splitting of the growth cone itself4 (Fig. 2), however, other options have been discussed as well5.
This video demonstrates first how to dissect the spinal cord of E12.5 mice leaving the DRG attached. Following fixation of the specimen tiny amounts of DiI are applied to DRG using glass needles pulled from capillary tubes. After an incubation step, the labeled spinal cord is mounted as an inverted open-book preparation to analyze individual axons using fluorescence microscopy.

The stereotyped projections of sensory afferents into the rodent spinal cord offer an easily accessible experimental system to study axonal branching through the tracing of single axons.

DII-Etichettatura di neuroni DRG per studiare Branching assonale in un monte intero Preparazione di Spinal Cord embrionali di topo

Here we  present a technique to label the trajectories of small groups of DRG neurons  into the embryonic spinal cord by diffusive staining using the ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

In Vivo a due fotoni di imaging dei cambi molecolari esperienza-dipendenti nei neuroni corticali

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

Optogenetic methods have emerged as a powerful tool for elucidating neural circuit activity underlying a diverse set of behaviors across a broad range of species. Optogenetic tools of microbial origin consist of light-sensitive membrane proteins that are able to activate (e.g., channelrhodopsin-2, ChR2) or silence (e.g., halorhodopsin, NpHR) neural activity ingenetically-defined cell types over behaviorally-relevant timescales. We first demonstrate a simple approach for adeno-associated virus-mediated delivery of ChR2 and NpHR transgenes to the dorsal subiculum and prelimbic region of the prefrontal cortex in rat. Because ChR2 and NpHR are genetically targetable, we describe the use of this technology to control the electrical activity of specific populations of neurons (i.e., pyramidal neurons) embedded in heterogeneous tissue with high temporal precision. We describe herein the hardware, custom software user interface, and procedures that allow for simultaneous light delivery and electrical recording from transduced pyramidal neurons in an anesthetized in vivo preparation. These light-responsive tools provide the opportunity for identifying the causal contributions of different cell types to information processing and behavior.

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

The recent development of neuroscience tools that combine genetics and optics, termed "optogenetics", enables control over neural circuit activity with an unprecedented level of spatial and temporal resolution. Here we provide a protocol for integrating in vivo recording with optogenetic manipulation of genetically-defined subsets of prefrontal cortical and subicular pyramidal neurons.

Un metodo per High Fidelity optogenetic controllo dei singoli Piramidale neuroni<em&gt; In vivo</em

Optogenetic methods  have emerged as a powerful tool for elucidating neural circuit activity  underlying a diverse set of behaviors across a broad range ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

Manipolazione genetica delle cerebellare granuli neuroni<em&gt; In Vitro</em&gt; E<em&gt; In Vivo</em&gt; Per studiare neuronale Morfologia e migrazione

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of live neurons with a specific primary antibody raised against an extracellular portion of the protein. Given that receptors are trafficked to and from the surface, if cells are permeabilized after fixation then both cell-surface and internal protein will be detected by the same labeled secondary antibody. Here, we adapted a method used to study protein trafficking (&#8220;antibody feeding&#8221;) to differentially label protein that had been internalized by endocytosis during the antibody incubation step and protein that either remained on the cell surface or was trafficked to the surface during this period. The ability to distinguish these two pools of protein was made possible through the incorporation of an overnight blocking step with highly-concentrated unlabeled secondary antibody after an initial incubation of unpermeabilized neurons with a fluorescently-labeled secondary antibody. After the blocking step, permeabilization of the neurons allowed detection of the internalized pool with a fluorescent secondary antibody labeled with a different fluorophore. Using this technique we were able to obtain important information about the subcellular location of this putative receptor, revealing that it was, indeed, trafficked to the cell-surface in neurons. This technique is broadly applicable to a range of cell types and cell-surface proteins, providing a suitable antibody to an extracellular epitope is available.

We describe a method to label protein on the surface of living neurons using a specific polyclonal antibody to extracellular epitopes. Protein bound by the antibody on the cell surface and subsequently internalized via endocytosis can be distinguished from protein remaining on, or trafficked to, the surface during the incubation.

Etichettatura differenziale di Cell-superficie e interiorizzato Proteine ​​dopo Antibody Alimentazione dal vivo colture di neuroni

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of ...

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with&#160;tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.

The isolation of individual dopamine neurons or the ventral tegmental area with direct or indirect immunohistochemistry is demonstrated using laser capture microdissection. Parameters for isolation of tissue from a glass slide using an infrared laser and from membrane slides using the combination of an infrared and ultraviolet laser are discussed.

Laser Capture Microdissezione - Una dimostrazione dell&#39;isolamento individuale dopamina neuroni e l&#39;intera area ventrale tegmentale

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue ...

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 ...

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich structures suggest alterations in synaptic integrity and connectivity. Here we provide a detailed protocol for culturing primary rat cortical neurons, Phalloidin staining for F-actin puncta, and subsequent quantification techniques. First, the frontal cortex of E18 rat embryos are dissociated into low-density cell culture, then the neurons grown in vitro for at least 12-14 days. Following experimental treatment, the cortical neurons are stained with AlexaFluor 488 Phalloidin (to label the dendritic F-actin puncta) and microtubule-associated protein 2 (MAP2; to validate the neuronal cells and dendritic integrity). Finally, specialized software is used to analyze and quantify randomly selected neuronal dendrites. F-actin rich structures are identified on second order dendritic branches (length range 25-75 &#181;m) with continuous MAP2 immunofluorescence. The protocol presented here will be a useful method for investigating changes in dendritic synapse structures subsequent to experimental treatments.

Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons

Filamentous actin (F-actin) plays an important role in spinogenesis, synaptic plasticity, and synaptic stability. Quantification of F-actin puncta is therefore a useful tool to study the integrity of synaptic structures. This protocol describes the procedures of quantifying F-actin puncta labeled with Phalloidin in low-density primary cortical neuronal cultures.

La quantificazione di filamentosa Actina (F-actina) Puncta nel ratto neuroni corticali

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich ...