NIH K08 EB003996

1. Poli-L-lattico (PLLA) Spinning Soluzione <ol><li> Sciogliere 0,4 g PLLA in 9 ml di cloroformio, mescolando a fuoco basso. </li><li> Aggiungere 1 ml di dimetilformammide alla soluzione, portando la concentrazione finale della soluzione al 4% PLLA (w / v) in cloroformio: DMF 9:1 (v / v). </li><li> Mettere la soluzione in un polipropilene o siringa di vetro con una punta metallica smussata 23ga. </li></ol> 2. Filatura di preparazione del substrato 1 <ol><li> Preparare una soluzione 8% (w / v) di 85:15 PLGA (poli-lattico-co-glicolico acido) in cloroformio, agitando a fuoco basso. </li><li> Cappotto pulito copertura in vetro scivola in PLGA coprendo la superficie di ogni vetrino con la soluzione di PLGA. Lasciare che il PLGA asciugare per un film sottile (ca. 30 min). </li></ol> 3. Electrospinning 2 <ol><li> Assicurare copertura in vetro rivestito PLGA scivola al collettore con nastro di carbonio conduttivo. Per le fibre allineate, il collettore è un motore ruota. Per le fibre di casuale, il collettore è un piatto fisso. </li><li> Luogo in pompa siringa con punta 20 cm dalla ruota collettore. Set pompa per circa 2 ml / ora e se si utilizza una ruota, impostare il motore a 300-400 giri al minuto. Se possibile, applicare un -2 kV polarizzazione CC al collettore e 15 kV a punta in metallo. In assenza di accesso ad una alimentazione bipolare, a terra il collettore. </li><li> Le fibre si getto dalla punta della siringa e raccogliere sulla ruota in movimento. Continuare a girare solo quando la densità desiderata delle fibre si ottiene. Strisciare la punta di metallo con un tovagliolo di carta, apposto su un non conduttivo asta periodicamente per evitare l&#39;intasamento sulla punta. </li></ol> 4. Moating 1 <ol><li> A seguito di filatura, &#39;fossato&#39; i campioni elettrofilate pipettando una linea di PLGA intorno al perimetro dello slip cover. Lasciare che il PLGA ad asciugare. Esso deve mantenere l&#39;allineamento delle fibre durante cultura. </li></ol> 5. Proteina di rivestimento <ol><li> Elettrofilate fibre di vetro e controlli devono essere rivestiti di proteine ​​prima di colture cellulari. Coprire i substrati in una soluzione proteica per almeno un&#39;ora e poi aspirare la soluzione in eccesso e proseguire fino a quando i substrati aspirazione sono a secco. Le soluzioni possibili sono le proteine: PLL (poli-L-lisina) (100 mg / ml), laminina (25 mg / ml) o fibronectina (50 mg / mL). Se il PLL è usato, i substrati devono essere risciacquati in acqua sterile e asciugati due volte dopo il rivestimento iniziale. </li></ol> 6. E15 ottenere embrioni di ratto 3 * Tutti gli esperimenti sono stati fatti in conformità con la Guida NIH per la cura e dell&#39;uso degli animali da laboratorio come approvato dalla University of Michigan Commissione Uso e cura degli animali. <ol><li> Preparazione: Verificare se l&#39;animale è incinta. Scongelare 3 mL tripsina, 3 FBS ml e una bottiglia di caldo L15. Fuoco lucidare una pipetta Pasteur. Disinfettare tutti gli strumenti chirurgici in etanolo al 70% per 30 min. </li><li> Preparare e caldo terreni in piastra (vedi tabella 1). </li><li> Eutanasia: eseguire un&#39;iniezione IP di ketamina / xylazina. Attendere 10-15 minuti e controllare per mancanza di riflessi corneale e pedale ritiro. Una volta che i riflessi sono assenti, eseguire una iniezione di pentobarbital IC (FatalPlus). </li><li> Tenda la pelle dell&#39;addome. Tagliare attraverso la pelle e dei muscoli per esporre la cavità addominale. Individuare l&#39;utero e staccarlo al collo dell&#39;utero e delle ovaie. </li><li> Rimuovere gli embrioni dall&#39;utero con le forbici e metterli immediatamente in caldo L15. (Tutte le procedure che seguono sono maturati nell&#39;ambito di un microscopio da dissezione e gli embrioni ei cavi sezionato deve essere immerso in acqua calda L15 in ogni momento.) Decapitare il embrioni chiudendo pinza intorno al mento e sotto l&#39;osso occipitale del cranio. </li></ol> 7. Dissezione 3 <ol><li> Posizionare l&#39;embrione su un lato. Proteggere il midollo spinale con un set di pinze mentre si utilizza l&#39;altro insieme a spogliare arti e organi addominali. </li><li> Girare l&#39;embrione e tenerla ferma con un set di pinze. Snip per tutta la lunghezza dell&#39;embrione, dal collo alla coda, fino a quando il cavo tutto è esposto. </li><li> Afferrare l&#39;estremità del cavo con attenzione e tirarlo su se stesso verso la coda da rimuovere. Il cavo tirerà libero con membrana e dei gangli spinali (DRG) allegato. </li><li> Se DRG espianto o dissociati cultura neuroni sensoriali deve essere eseguita, snip DRG il cordone e trasferirli in un piatto fresco di caldo L15. <ol><li> Per i DRG cultura espianto, passare alla fase 10,2 </li><li> Per dissociato cultura neuroni sensoriali, passare al punto 9 </li><li> Per la cultura del motoneurone, il DRG verrà rimosso con la membrana al passo 7,5 </li></ol></li><li> Rimuovere la membrana dal midollo spinale afferrandolo all&#39;estremità superiore del cavo e peeling verso il basso, simile a rimuovere un calzino lungo. Ripetere l&#39;operazione per tutti gli embrioni e poi tagliare le corde in piccoli (2-3 mm) pezzi. </li></ol><pclass = &quot;jove_title&quot;&gt; 8. Motor Neuron (MN) Elaborazione 5,6,7 <ol><li> Versare i cavi tritato e L15 in una forma conica e far girare a 1000 RPM per 2-3 minuti per far sedimentare i pezzi. </li><li> Eliminare il sopranatante ed aggiungere 3 mL di tripsina alle corde pellet. Incubare in un bagno d&#39;acqua a 37 ° C per 20 min. </li><li> Aggiungere 3 ml di FBS conica e far girare a 1000 RPM per 2-3 min a ri-pellet. </li><li> Eliminare il sopranatante e un cappotto lucido fuoco pipetta Pasteur con FBS. </li><li> Aggiungere una pipetta Pasteur-pieno di L15 alla conica e poi triturare delicatamente fino a quando la soluzione è omogeneo senza grumi visibili. Evitare bolle. </li><li> Preparare una soluzione al 9% di Optiprep in L15. Preparare un tubo con 3 ml di soluzione Optiprep per ogni due embrioni (se c&#39;è un numero dispari di embrioni, arrotondare per eccesso). </li><li> Goccia la soluzione omogeneizzato sulla soluzione Optiprep, divide equamente tra tutti i tubi. Spin le provette a 2000 RPM per 15 minuti. </li><li> Raccogliere accuratamente la parte superiore da 2 ml ciascuna provetta e piscina in una conica da 50 ml. Riempire il conico con L15 e far girare a 1000 RPM per 5 minuti per sciacquare le cellule del Optiprep. </li><li> Eliminare il sopranatante e risospendere le cellule in una piccola quantità di terreni in piastra (250-500 mL). Contare le cellule utilizzando esclusione blu trypan per identificare le cellule vive. Passare al punto 10,1 </li></ol> 9. Sensoriale Neuron (SN) Elaborazione 7 <ol><li> Versare il DRG rimosso dal midollo spinale e L15 in un tubo conico e girare a 1000 RPM per 2-3 minuti per far sedimentare i pezzi. </li><li> Eliminare il sopranatante ed aggiungere 3 mL di tripsina al DRG pellet. Incubare in un bagno d&#39;acqua a 37 ° C per 10 min. </li><li> Aggiungere 3 ml di FBS conica e far girare a 1000 RPM per 2-3 min a ri-pellet. </li><li> Eliminare il sopranatante e un cappotto lucido fuoco pipetta Pasteur con FBS. </li><li> Aggiungere una pipetta Pasteur-pieno di L15 alla conica e poi triturare delicatamente fino a quando la soluzione è omogeneo senza grumi visibili. Evitare bolle. </li><li> Spin l&#39;omogeneizzato a 1000 rpm per 5 min. Eliminare il sopranatante e risospendere le cellule in una piccola quantità di terreni in piastra SN (250-500 mL). Contare le cellule utilizzando esclusione blu trypan per identificare le cellule vive. Continuare a passo 10,1 </li></ol> 10. Placcatura e Cultura <ol><li> Per dissociato SN o MN cultura: <ol><li> Coprire i substrati con alcune gocce di media quindi aggiungere un volume adeguato di sospensione cellulare. Cellule dissociate dovrebbe essere placcato a bassa densità (25-50 cellule / mm 2). Consentire alle cellule di aderire per almeno un&#39;ora prima di allagare i pozzetti con i media. </li></ol></li><li> Per i DRG cultura espianto: <ol><li> Coprire i substrati con alcune gocce di media quindi aggiungere DRG ai media. Disporre i DRG in modo che siano distribuiti uniformemente in tutto il substrato (circa 4 DRG si inserisce su un foglio 22x22 copertina mm). Lasciare che il DRG di aderire per almeno 4 ore prima di allagare i pozzetti con i media. </li></ol></li><li> Culture può essere mantenuta per un massimo di 4 giorni senza cambiare supporto. Per i più culture, la metà cambiamenti dei media dovrebbe essere fatto con mezzi di feed. Alimentazione dei supporti è la stessa composizione di terreni in piastra meno la L-glutammina. </li></ol> 11. Immunocitochimica 1,5,8 <ol><li> Fissaggio delle cellule: i media Aspirare e sostituirlo con caldo al 4% PFA (paraformaldeide) per 30 minuti. Quindi eseguire 3X 5 min 1x lavaggi PBS. </li><li> Blocco / permeabilizzazione: Preparare blocco / perm soluzione (1,25% siero albumina bovina in PBS 1X, 0,05% Triton X-100 e il 2,0% di siero normale di capra). Aspirare il PBS e applicare blocco / perm soluzione ai campioni per 30 minuti. Quindi eseguire 1X 5 min 1x lavaggi PBS. </li><li> Anticorpo primario: Preparare la soluzione primaria di anticorpi di lavoro (10% siero di capra normale, 1,25% di sieroalbumina bovina, 0,1% Na azide, 0,05% Triton X-100 e a 10 ml con PBS 1x). Aggiungere la quantità appropriata di anticorpo primario (vedi tabella 2). Linea di piatti diversi con parafilm. Mettere una goccia 200μl di soluzione dell&#39;anticorpo primario sul parafilm per ciascun substrato. Invertire la substrati, galleggiante loro lato della cella verso il basso la soluzione di anticorpo primario di lavoro durante la notte (se la stanza è dotata di aria condizionata o particolarmente secchi, un pezzo di carta saturi d&#39;acqua può essere aggiunta in un angolo del piatto per evitare l&#39;evaporazione della soluzione di lavoro) . </li><li> Anticorpo secondario: Rimuovere i substrati dal primario (la soluzione di lavoro può essere raccolto, conservato a 4 ° C e riutilizzati) ed eseguire 2X 5 min 1x lavaggi PBS. Applicare 200 microlitri anticorpo secondario diluito 1:200 in PBS (anche rovesciato su un piatto rivestito parafilm) per 4 ore. </li><li> Togliere dal secondario quindi eseguire 2X 5 min 1x lavaggi PBS. Substrati montaggio su vetrini con Prolungare oro contenente DAPI o un altro contatore adatto nucleare macchia. </li></ol> 12. Rappresentante dei risultati: La morfologia tipica della allineate e casuale elfibre ectrospun sono mostrati in Figura 1. Noi di solito ottenere la purezza MN di&gt; neuroni del 90% 7,8 con questo protocollo e abbiamo mantenuto DRG culture per tutto il tempo una settimana, senza alcun significativa crescita dei fibroblasti. La figura 2 mostra l&#39;aspetto tipico MN su vetro e fibre dopo 24 ore di cultura e immunocolorazione. DRG immunostained colto per tre giorni su vetro e fibre sono rappresentate nella figura 3. <img src="/files/ftp_upload/2389/2389fig1.jpg" alt="Figura 1" /> Figura 1. Allineamento fibra viene manipolata per scelta del collezionista. A.) Allineati fibre filate su un collettore ruota in movimento e B.) fibre casuale filato su un collettore piano fisso. Barre di scala = 20 micron. <img src="/files/ftp_upload/2389/2389fig2.jpg" alt="Figura 2" /> Figura 2. Motoneuroni rappresentante colorate per TuJ1 (verde) e DAPI (blu) dopo 24 ore di cultura sul PLL A. patinata) fibre e B.) di vetro. Barre di scala = 20 micron. <img src="/files/ftp_upload/2389/2389fig3.jpg" alt="Figura 3" /> Figura 3. DRG macchiato rappresentante per neurofilamenti (verde) dopo 3 giorni di coltura su PLL rivestito A.), vetro e B.) fibre. Barre di scala = 200 micron. <table border="1"><tbody><tr><td> Soluzione: </td><td> MN media: </td><td> DRG / SN media: </td></tr><tr><td> 10 mg ml -1 albumina </td><td> 12,5 microlitri </td><td> - </td></tr><tr><td> 10 mg ml -1 apo-transferrina </td><td> 50 microlitri </td><td> 40 microlitri </td></tr><tr><td> 50 mg ml -1 β-estradiolo </td><td> 2,7 microlitri </td><td> 2,2 microlitri </td></tr><tr><td> 0,1 mg ml -1 biotina </td><td> 50 microlitri </td><td> - </td></tr><tr><td> 16 mg ml -1 catalasi </td><td> 8 microlitri </td><td> - </td></tr><tr><td> 15 mg ml -1 D-galattosio </td><td> 50 microlitri </td><td> - </td></tr><tr><td> 50 ml di idrocortisone mcg -1 </td><td> 3,7 microlitri </td><td> 2,9 microlitri </td></tr><tr><td> 0,63 mg ml -1 progesterone </td><td> 0,5 microlitri </td><td> - </td></tr><tr><td> 16 mg ml -1 putrescina </td><td> 50 microlitri </td><td> - </td></tr><tr><td> 50 mg ml -1 selenio </td><td> 3,4 microlitri </td><td> 4,1 microlitri </td></tr><tr><td> 2,5 mg ml -1 superossido dismutasi </td><td> 50 microlitri </td><td> - </td></tr><tr><td> * 500 ng mL -1 fattore di crescita nervosa </td><td> - </td><td> 1 ml </td></tr><tr><td> * 100X PSN </td><td> 0,5 ml </td><td> 0,5 ml </td></tr><tr><td> * B27 </td><td> 1 ml </td><td> 1 ml </td></tr><tr><td> * 2 mM L-glutammina </td><td> 35 microlitri </td><td> 35 microlitri </td></tr><tr><td> * Neurobasal </td><td> a 50 mL </td><td> a 50 mL </td></tr></tbody></table> Tabella 1. Aggiungi protagonista (*) i componenti per i media al momento dell&#39;uso. I restanti componenti possono essere preparati come una soluzione di riserva e conservato a -20 ° C fino al momento 6,7. <table border="1"><tbody><tr><td> Primaria (concentrazione) </td><td> Neuroni: </td><td> Glia: </td><td> Fibroblasti: </td></tr><tr><td> anti-β-tubulina (TuJ1) (1:1000) </td><td> ✓ </td><td> X </td><td> X </td></tr><tr><td> anti-neurofilamenti (1:1000) </td><td> ✓ </td><td> X </td><td> X </td></tr><tr><td> anti-S-100 (1:250) </td><td> X </td><td> ✓ </td><td> ✓ </td></tr><tr><td> NGFR anti-p75 (1:500) </td><td> ✓ </td><td> ✓ </td><td> X </td></tr></tbody></table> Tabella 2. La selezione dei anticorpo primario dipende gli obiettivi del ricercatore. Quanto sopra sono anticorpi diversi e le concentrazioni che abbiamo usato con successo nel nostro laboratorio. S-100 è particolarmente utile per identificare le cellule di Schwann e / o verificare la presenza di contaminanti fibroblasti, ma nota che esso deve essere usato in combinazione con NGFR p75 di distinguere tra cellule gliali e fibroblasts4. Abbiamo anche notato che NGFR p75 macchie neuriti molto leggermente, mentre neurofilamenti o TuJ1 sono una ottima scelta, se l&#39;obiettivo è quello di visualizzare neuriti individuali.

<ol>
	<li>Corey, J. M., Gertz, C. C., Wang, B. S., Birrell, L. K., Johnson, S. L., Martin, D. C., Feldman, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+design+of+electrospun+PLLA+nanofiber+scaffolds+compatible+with+serum-free+growth+of+primary+motor+and+sensory+neurons.">The design of electrospun PLLA nanofiber scaffolds compatible with serum-free growth of primary motor and sensory neurons.</a> Acta. Biomater. 4, 863-875 (2008).</li><li>Lin, D. Y., Johnson, M. A., Vohden, R. A., Chen, D., Martin, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tailored+nanofiber+morphologies+using+modulated+electrospinning+for+biomedical+applications.">Tailored nanofiber morphologies using modulated electrospinning for biomedical applications.</a> Mat. Res. Soc. Symp. Proc. 736, D3.8.1-D3.8.6 (2003).</li><li>Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culturing Nerve Cells. , (1998).</li><li>Kameda, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+glial+progenitor+markers+p75NTR+and+S100+protein+in+the+developing+mouse+parathyroid+gland.">Expression of glial progenitor markers p75NTR and S100 protein in the developing mouse parathyroid gland.</a> Cell Tissue Res. 327, 15-23 (2007).</li><li>Corey, J. M., Lin, D. Y., Mycek, K. B., Chen, Q., Samuel, S., Feldman, E. L., Martin, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aligned+electrospun+nanofibers+specify+the+direction+of+dorsal+root+ganglia+neurite+growth.">Aligned electrospun nanofibers specify the direction of dorsal root ganglia neurite growth.</a> J. Biomed. Mater. Res. A. 83, 636-645 (2007).</li><li>Vincent, A. M., Mobley, B. C., Hiller, A., Feldman, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IGF-I+prevents+glutamate-induced+motor+neuron+programmed+cell+death.">IGF-I prevents glutamate-induced motor neuron programmed cell death.</a> Neurobiol. Dis. 16, 407-416 (2004).</li><li>Russell, J. W., Windebank, A. J., Schenone, A., Feldman, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin-like+growth+factor-I+prevents+apoptosis+in+neurons+after+nerve+growth+factor+withdrawal.">Insulin-like growth factor-I prevents apoptosis in neurons after nerve growth factor withdrawal.</a> J. Neurobiol. 36, 455-467 (1998).</li><li>Gertz, C. C., Leach, M. K., Birrel, L. K., Martin, D. C., Feldman, E. L., Corey, 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=Accelerated+neuritogenesis+and+maturation+of+primary+spinal+motor+neurons+in+response+to+nanofibers.">Accelerated neuritogenesis and maturation of primary spinal motor neurons in response to nanofibers.</a> Dev. Neurobiol. 70, 589-603 (2010).</li><li>Tan, S. -. H., Kotaki, M., Ramakrishna, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+parameter+study+for+ultra-fine+fiber+fabrication+via+electrospinning+process.">Systematic parameter study for ultra-fine fiber fabrication via electrospinning process.</a> Polymer. 46, 6128-6134 (2005).</li><li>Wang, H. B., Mullins, M. E., Cregg, J. M., Hurtado, A., Oudega, M., Trombley, M. T., Gilbert, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creation+of+highly+aligned+electrospun+poly-L-lactic+acid+fibers+for+nerve+regeneration+applications.">Creation of highly aligned electrospun poly-L-lactic acid fibers for nerve regeneration applications.</a> J. Neural Eng. 6, (2009).</li></ol>

Nessun conflitto di interessi dichiarati.

Questo protocollo ha diversi punti critici. La prima riguarda la corretta produzione di substrati in fibra di elettrofilate. Nei liquidi, le fibre PLLA, PLGA film e fossato PLGA separerà dal coupon vetro di copertura come una singola unità. Se il PLGA viene omesso, le fibre PLLA non rimarrà un foglio - che si arricciano in un groviglio inutilizzabile. Così, il film PLGA è incluso come un substrato adatto per mantenere l&#39;allineamento delle fibre durante cultura. Il fossato viene aggiunto dopo spinning sia per garantire che le fibre siano saldamente fissate al film PLGA e di aggiungere la rigidità strutturale del film. Il fossato funge anche da manico utile quando i substrati sono manipolati durante il fissaggio, colorazione e montaggio. Triturazione è il secondo passo fondamentale. Ogni sforzo dovrebbe essere fatto per evitare la formazione di bolle. Inoltre, abbiamo ottenuto rendimenti molto più alti quando un incendio lucidato pipetta viene utilizzata in questa fase. Al fuoco lucido, la luce un becco Bunsen e allegare una lampadina per la pipetta. Passo la punta della pipetta rapidamente attraverso la fiamma 2-3 volte, mentre continua spremitura e rilasciando la lampadina - flusso d&#39;aria attraverso la pipetta aiuterà a prevenire la punta di chiudere completamente. Esaminare la punta della pipetta assicurando così il buco è ancora aperto e che i bordi appaiono leggermente arrotondati prima dell&#39;uso. Electrospinning è un processo molto versatile; i parametri electrospinning può essere modificata per produrre fibre con una varietà di morfologie. Tan et al. (2005) i dettagli di uno studio sistematico di parametri che influenzano il diametro del PLLA fibre elettrofilate. Wang et al. (2009) presenta uno studio dettagliato dei parametri che influenzano l&#39;allineamento delle fibre elettrofilate PLLA.

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>PLLA</td>
<td>&nbsp;</td>
<td>Boehringer Ingelheim</td>
<td>Resomer L210</td>
<td>&nbsp;</td>
<tr>
<td>PLGA 85:15</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>43471</td>
<td>&nbsp;</td>
<tr>
<td>L15</td>
<td>&nbsp;</td>
<td>Gibco </td>
<td>11415</td>
<td>&nbsp;</td>
<tr>
<td>Albumin</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>A2289 
</td>
<td>&nbsp;</td>
<tr>
<td>Apo-transferrin</td>
<td>&nbsp;</td>
<td>Akron </td>
<td>AK8227</td>
<td>&nbsp;</td>
<tr>
<td>Biotin</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>AC 23009-0010</td>
<td>&nbsp;</td>
<tr>
<td>Galactose</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>G0625</td>
<td>&nbsp;</td>
<tr>
<td>Progesterone</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P7556</td>
<td>&nbsp;</td>
<tr>
<td>Putrescine </td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P5780</td>
<td>&nbsp;</td>
<tr>
<td>Selenium </td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>S9133</td>
<td>&nbsp;</td>
<tr>
<td>&beta;-estradiol</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>E 1132 </td>
<td>&nbsp;</td>
<tr>
<td>Hydrocortisone </td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>H0396</td>
<td>&nbsp;</td>
<tr>
<td>Catalase </td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>C40 </td>
<td>&nbsp;</td>
<tr>
<td>Superoxide dismutase</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>S4636</td>
<td>&nbsp;</td>
<tr>
<td>Neurobasal</td>
<td>&nbsp;</td>
<td>Gibco </td>
<td>21103-049</td>
<td>&nbsp;</td>
<tr>
<td>PSN</td>
<td>&nbsp;</td>
<td>Gibco </td>
<td>15640 </td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine</td>
<td>&nbsp;</td>
<td>Nalgene </td>
<td>1680149</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin</td>
<td>&nbsp;</td>
<td>Nalgene </td>
<td>1689149</td>
<td>&nbsp;</td>
<tr>
<td>Fetal bovine serum</td>
<td>&nbsp;</td>
<td>Gibco </td>
<td>26140</td>
<td>&nbsp;</td>
<tr>
<td>Optiprep</td>
<td>&nbsp;</td>
<td>Accurate </td>
<td>2011</td>
<td>&nbsp;</td>
<tr>
<td>PLL</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>P4832 </td>
<td>&nbsp;</td>
<tr>
<td>Fibronectin</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>F 4759 </td>
<td>&nbsp;</td>
<tr>
<td>Laminin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>23017-015</td>
<td>&nbsp;</td>
<tr>
<td>B27</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>17504-044</td>
<td>&nbsp;</td>
<tr>
<td>BSA</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A7906</td>
<td>&nbsp;</td>
<tr>
<td>Anti-TuJ1</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>556321</td>
<td>&nbsp;</td>
<tr>
<td>Anti-neurofilament</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>AB1987</td>
<td>&nbsp;</td>
<tr>
<td>Anti-S100</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S2532</td>
<td>&nbsp;</td>
<tr>
<td>Anti-NGFR p75</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>N3908</td>
<td>&nbsp;</td>
<tr>
<td>Normal goat serum</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G6767</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T9284</td>
<td>&nbsp;</td>
<tr>
<td>Sodium azide</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S8032</td>
<td>&nbsp;</td>
<tr>
<td>Carbon tape</td>
<td>&nbsp;</td>
<td>Ted Pella</td>
<td>13073-1</td>
<td>&nbsp;</td>
<tr>
<td>Prolong Gold +DAPI</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>P36931</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

the culture of primary motor and sensory neurons in defined media on electrospun poly-l-lactide nanofiber scaffolds

Electrospinning è una tecnica per la produzione di micro-a nano-scala fibre. Fibre possono essere elettrofilate con vari gradi di allineamento, da altamente allineati a completamente casuale. Inoltre, le fibre possono essere filata da una varietà di materiali, compresi i polimeri biodegradabili come il poli-L-lattico (PLLA). Queste caratteristiche rendono le fibre elettrofilate adatto per una varietà di applicazioni di ponteggi in ingegneria dei tessuti. Il nostro obiettivo è l&#39;uso di fibre elettrofilate allineati per la rigenerazione dei nervi. Abbiamo precedentemente dimostrato che allineare le fibre elettrofilate PLLA la conseguenza diretta di entrambi i neuroni sensoriali e motorie primarie in vitro. Riteniamo che l&#39;uso di un sistema primario di coltura cellulare è essenziale per la valutazione dei biomateriali ai neuroni modello reale trovato in vivo il più possibile. Qui, descriviamo le tecniche utilizzate nel nostro laboratorio di electrospin ponteggi fibroso e cultura espianti gangli dorsali, così come dissociata neuroni sensoriali e motori, su ponteggi elettrofilate. Tuttavia, il electrospinning e / o tecniche di coltura qui presentate possono essere facilmente adattati per essere utilizzati in altre applicazioni.

Watch this Scientific Journal Video about The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds at JoVE.com

The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds

Allineati fibre elettrofilate diretta la crescita dei neuroni in vitro e sono una componente potenziale di ponteggi rigenerazione nervosa. Abbiamo descritto una procedura per la preparazione di substrati di fibre elettrofilate e il siero senza cultura di primaria ratto E15 sensoriali (DRG) e neuroni motori. Visualizzazione dei neuroni mediante immunocitochimica è anche incluso.

La cultura del motore principale e neuroni sensoriali in Media definiti in elettrofilate Poli-L-lactide Ponteggi nanofibre

Electrospinning is a technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned ...

the-culture-primary-motor-sensory-neurons-defined-media-on

Research

JoVE Journal

Neuroscience

19.3K Views.  University of Michigan. Allineati fibre elettrofilate diretta la crescita dei neuroni in vitro e sono una componente potenziale di ponteggi rigenerazione nervosa. Abbiamo descritto una procedura per la preparazione di substrati di fibre elettrofilate e il siero senza cultura di primaria ratto E15 sensoriali (DRG) e neuroni motori. Visualizzazione dei neuroni mediante immunocitochimica è anche incluso.

Video: The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Dissezione e la cultura di neuroni embrionali commissurali da midollo spinale

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Ricerca

Neuroscienze

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Nucleofection e cultura primaria di neuroni embrionali di topo ippocampale e corticale

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory  Neurons

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. Caged compounds are molecules made physiologically inactive by a chemical cage that can be broken by a flash of ultraviolet light. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged compounds for the study of olfactory transduction in dissociated mouse olfactory sensory neurons. The process of olfactory transduction (Figure 1) takes place in the cilia of olfactory sensory neurons, where odorant binding to receptors leads to the increase of cAMP that opens cyclic nucleotide-gated (CNG) channels2. Ca entry through CNG channels activates Ca-activated Cl channels. We show how to dissociate neurons from the mouse olfactory epithelium3 and how to activate CNG channels or Ca-activated Cl channels by photolysis of caged cAMP4 or caged Ca5. We use a flash lamp6,7 to apply ultraviolet flashes to the ciliary region to uncage cAMP or Ca while patch-clamp recordings are taken to measure the current in the whole-cell voltage-clamp configuration8-11.

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged cAMP or caged Ca for the study of olfactory transduction in dissociated mouse olfactory sensory neurons.

Fotolisi lampo di composti Caged nel Cilia di neuroni sensoriali olfattivi

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. ...

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 ...

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 ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Tutta Monte immunomarcatura di recettore olfattivo neuroni nel<em&gt; Drosophila</em&gt; Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

Cultura primario di topo neuroni dopaminergici

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. Studying neurons in vivo can be challenging due to their complexity, their varied and dynamic environments, and technical limitations. For these reasons, studying neurons in vitro can prove beneficial to unravel the complex mysteries of neurons. The well-defined nature of cell culture models provides detailed control over environmental conditions and variables. Here we describe how to isolate, dissociate, and culture primary neurons from chick embryos. This technique is rapid, inexpensive, and generates robustly growing sensory neurons. The procedure consistently produces cultures that are highly enriched for neurons and has very few non-neuronal cells (less than 5%). Primary neurons do not adhere well to untreated glass or tissue culture plastic, therefore detailed procedures to create two distinct, well-defined laminin-containing substrata for neuronal plating are described. Cultured neurons are highly amenable to multiple cellular and molecular techniques, including co-immunoprecipitation, live cell imagining, RNAi, and immunocytochemistry. Procedures for double immunocytochemistry on these cultured neurons have been optimized and described here.

Cell culture models provide detailed control over environmental conditions and thus provide a powerful platform to elucidate numerous aspects of neuronal cell biology. We describe a rapid, inexpensive, and reliable method to isolate, dissociate, and culture sensory neurons from chick embryos. Details of substrata preparation and immunocytochemistry are also provided.

Isolamento e Cultura della dissociato neuroni sensoriali da embrioni di gallina

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. ...

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

zebrafish<em&gt; In Situ</em&gt; Spinal Cord Preparazione per elettrofisiologiche Le registrazioni da spinale sensoriali e motori neuroni

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

Nel calcio in Vivo Imaging delle cellule ciliate linea laterale in Zebrafish larvale

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...