Il nostro lavoro è sostenuto dal Fondo Nazionale Svizzero (FNS), l&#39;iniziativa svizzera in Systems Biology (SystemsX.ch), la Distrofia Muscolare USA Association (MDA), la Association Française contre les Myopathies (AFM), il Regno Unito mitocondriale Disease Foundation (UMDF), la Fondazione Gebert-Rüf Programma Malattie Rare (GRF), la Società svizzera per la ricerca delle malattie muscolari (SSEM / FSRMM), la Swiss Life &quot;Jubiläumsstiftung Volksgesundheit für Forschung und medizinische&quot;, la Roche Research Foundation e l&#39;Università di Basilea.

1. Preparazione della coltura principale umano delle cellule muscolari <ol><li> Stabilire le colture di cellule muscolari umane secondo il espianto-re-tecnica di espianto 4. In primo luogo, rimuovere non-tessuto muscolare dalle biopsie. Poi, incorporare 1 mm 3 espianti muscolari in un coagulo di plasma, che consente di fibroblasti di emergere dalle espianti. </li><li> Gli espianti sono incorporati trasferita su un piatto di plasma-gelatina rivestita e farli crescere in mezzo di crescita normale (MEM supplementato con 25% di media 199, 10% siero bovino fetale [FBS], 1% di penicillina / streptomicina, 10 ug / ml di insulina, 10 ng / ml di fattore di crescita epidermico umano [EGF], 12,5 ng / ml di fattore di crescita dei fibroblasti umani [FGF]) in un monostrato. Le cellule che emergono dalle espianti in queste condizioni sono cellule muscolari. Queste cellule continuerà a crescere in un monostrato dopo la rimozione degli espianti. </li><li> Coltivare le cellule aneurally sul 0,1% di gelatina rivestite con lastre in terreno di crescita normali. Le cellule possono essereaneurally coltivate e amplificato come necessario. È preferibile usare cellule muscolari a un passaggio tra 3 e 8 per eseguire i co-culture. E &#39;fondamentale per innervare miotubi giovani, dopo questo punto, i co-culture hanno un alto rischio di fallire. Inoltre, miotubi non deve essere troppo grande e troppo spessa. </li><li> Due giorni prima della innervazione è previsto, piatto le cellule muscolari su piastre da 35 mm di coltura tissutale (o piastre da 6 pozzetti) ad una densità di 500.000 cellule per piatto in mezzo una crescita normale. A tale densità, le cellule sono a confluenza corretto per avviare la fusione il giorno seguente, e la successiva innervazione il giorno dopo. Utilizzando queste condizioni, i miotubi sono allo stadio giusto per innervazione ottimale e quindi la successiva contrazione della fibra. </li><li> Il giorno successivo, sostituire il mezzo di crescita normale con un mezzo fusione composta MEM supplementato con 25% di media 199, 5% FBS, 10 ug / ml di insulina e 1% di penicillina / streptomicina. </li></ol> 2. Intercettaz<html> Uno degli Rat Espianti embrionali del Midollo Spinale L&#39;intera procedura di isolare gli espianti midollo spinale viene eseguita al microscopio binoculare, in soluzione salina bilanciata di Hank (HBSS, Gibco / ref 14.170 Invitrogen) contenente 10% siero fetale bovino (FBS). <ol><li> E &#39;fondamentale che gli embrioni di ratto sono tra ED 13 e 14, come uno stadio di sviluppo diverso metterebbe a repentaglio l&#39;intera procedura. Sacrifica una femmina gravida di CO 2 e raccogliere l&#39;intera catena embrione in media HBSS. Isolare ogni embrione e decapitare per loro l&#39;eutanasia. </li><li> Sezionare il midollo spinale embrione dal resto del corpo in un unico pezzo e rimuovere muscolo circostante e tessuto connettivo. Fate molta attenzione a mantenere i gangli della radice dorsale (DRG) collegati al midollo spinale per assicurare innervazione funzionale. </li><li> Tagliare ogni midollo spinale trasversalmente in modo che ogni espianto contiene almeno 2 DRG collegato ad esso (espianti di 1 mm 3). </li></ol><html> ove_title &quot;&gt; 3. innervazione delle cellule muscolari embrionali umane con Espianti Spinal Cord Rat <ol><li> Rimuovere il mezzo fusione sulla coltura cellula muscolare, lasciando uno strato sottile di supporto sul monostrato. </li><li> Porre gli espianti attenzione sul livello delle cellule muscolari, cinque espianti equidistanti per piastra da 35 mm. </li><li> Aggiungere molto lentamente, goccia a goccia, la fusione di media su ogni espianto. È necessario aggiungere mezzo per le cellule muscolari per sopravvivere, ma non troppo per consentire gli espianti di aderire prima e in contatto con le cellule. Se il mezzo non viene aggiunto correttamente (troppo forte o media troppo), gli espianti galleggia e non riescono a innervare le cellule muscolari. </li><li> Una volta che gli espianti iniziano ad aderire, all&#39;inizio del mezzo di fusione a 2 ml per piastra da 35 mm, e mettere i piatti con molta attenzione nuovamente dentro l&#39;incubatrice. </li></ol> 4. Manutenzione delle eterologhe nervo-muscolo Co-culture <ol><li> Cambia il mezzo di fusione due volte a settimana. </li></ol>_title &quot;&gt; 5. rappresentativi Risultati Appena due giorni dopo l&#39;innervazione, è possibile vedere le neuriti emergono da ciascun espianto e stabilire contatti con le cellule muscolari, rappresentati dai pulsante strutture simili (Fig. 1). Già nel 4-6 giorni dopo l&#39;innervazione, poche fibre singole inizierà a contrarsi. Le contrazioni persistono per molti mesi, mantenuto semplicemente cambiando il terreno di coltura due volte a settimana. Dopo circa 2 settimane, l&#39;espianto del midollo spinale si degenera, ma l&#39;innervazione è conservata. Questo è normale. Uno strato sottile di componente nervo rimane sulla superficie dello strato di cellule muscolari. Questa rete nervosa è sufficiente a mantenere la funzionalità del gruppo motore. Se gli espianti galleggiano nel mezzo il giorno dopo innervazione, non potranno mai aderire. Ciò accade quando lo strato cellula muscolare ènon sufficientemente confluenti, o quando il mezzo viene aggiunto troppo grosso. A volte, aderisce espianti, ma non neuriti emergerà. Ciò è probabilmente dovuto alla mancanza di DRG attaccata ad essa. In questo caso anche l&#39;innervazione non verrà stabilita. Manipolare i piatti con molta attenzione la prima settimana, come gli espianti può ancora staccare facilmente. Per scambiare i mezzi di comunicazione di fusione, aggiungere il liquido goccia a goccia con una velocità minima per evitare il distacco delle cellule. <img alt="Figura 1" src="/files/ftp_upload/3616/3616fig1.jpg" /> Figura 1. Midollo spinale espianti-cellule muscolari co-colture dal giorno 1 al giorno 15.

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	<li>Delaporte, C., Dautreaux, B., Fardeau, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+myotube+differentiation+in+vitro+in+different+culture+conditions.">Human myotube differentiation in vitro in different culture conditions.</a> Biol. Cell. 57, 17-22 (1986).</li><li>Kobayashi, T., Askanas, V., Engel, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+muscle+cultured+in+monolayer+and+cocultured+with+fetal+rat+spinal+cord:+importance+of+dorsal+root+ganglia+for+achieving+successful+functional+innervation.">Human muscle cultured in monolayer and cocultured with fetal rat spinal cord: importance of dorsal root ganglia for achieving successful functional innervation.</a> J. Neurosci. 7, 3131-3141 (1987).</li><li>Braun, S., Croizat, B., Lagrange, M. C., Warter, J. 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M., Poindron, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+types+required+to+efficiently+innervate+human+muscle+cells+in+vitro.">Cell types required to efficiently innervate human muscle cells in vitro.</a> Exp. Cell Res. 259, 204-212 (2000).</li><li>Lefebvre, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+a+spinal+muscular+atrophy-determining+gene.">Identification and characterization of a spinal muscular atrophy-determining gene.</a> Cell. 80, 155-165 (1995).</li><li>Schrank, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+the+survival+motor+neuron+gene,+a+candidate+gene+for+human+spinal+muscular+atrophy,+leads+to+massive+cell+death+in+early+mouse+embryos.">Inactivation of the survival motor neuron gene, a candidate gene for human spinal muscular atrophy, leads to massive cell death in early mouse embryos.</a> Proc. Natl. Acad. Sci. U.S.A. 94, 9920-9925 (1997).</li><li>Hsieh-Li, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+for+spinal+muscular+atrophy.">A mouse model for spinal muscular atrophy.</a> Nat. Genet. 24, 66-70 (2000).</li><li>Monani, U. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+centromeric+survival+motor+neuron+gene+(SMN2)+rescues+embryonic+lethality+in+Smn(-/-)+mice+and+results+in+a+mouse+with+spinal+muscular+atrophy.">The human centromeric survival motor neuron gene (SMN2) rescues embryonic lethality in Smn(-/-) mice and results in a mouse with spinal muscular atrophy.</a> Hum. Mol. Genet. 9, 333-339 (2000).</li><li>Sleigh, J. N., Gillingwater, T. 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Uno strumento fisiologico in vitro per studiare la funzionalità delle cellule muscolari in un contesto normale e patologico è di maggior interesse per i myologists, perché le colture di cellule muscolari di solito non ricapitolare l&#39;importanza delle cellule multiple e delle cellule di tipo connessioni. L&#39;aggiunta di purificato motoneuroni di cellule muscolari non è sufficiente per ottenere un&#39;unità funzionale motore in quanto la presenza di cellule di Schwann è richiesto per l&#39;innervazione sia efficiente e 5 non è topologicamente (3D + distribuzione speciale) pertinente Il protocollo qui descritto ha stati utilizzati con successo per stabilire cellule muscolari innervati in vitro ed i risultati di una completa maturazione NMJ che può essere facilmente valutare. Modelli murini sono ora disponibili per molte malattie neuromuscolari. Purtroppo, molti di questi modelli sono limitate in termini di traduzione della malattia in pazienti umani, ad esempio i modelli del mouse per l&#39;atrofia muscolare spinale, Duchennedistrofia muscolare o la sclerosi laterale amiotrofica. L&#39;atrofia muscolare spinale è causata da una mutazione nel gene SMN1, che in parte può essere mitigata dalla presenza di un gene SMN secondo nel genoma umano 6. Al contrario, il genoma topi contiene solo un gene per SMN, e la sua soppressione è embrionalmente letale 7. Pertanto, al fine di sviluppare modelli di topo atrofia muscolare spinale, un essere umano SMN2 copia è stata introdotta artificialmente nei topi knockout smn 8,9. Questi animali rappresentano ancora più vicino al modello genetico patologia umana, ma il fenotipo osservato nei topi è dovuta alla espressione di un gene SMN2 topi che normalmente non esprimono 10. Nel caso di distrofia muscolare di Duchenne, i topi mdx hanno un gene mutato distrofina simile a pazienti umani, ma il risultato è molto meno grave rispetto ai sintomi osservati nei pazienti 11. Il modello di topo più utilizzato per amyotrophic sclerosi laterale, i topi SOD1, finora deluso intuizioni rivelatrici sui meccanismi della malattia e gli approcci terapeutici 12,13. Pertanto, midollo spinale, delle cellule muscolari espianti co-colture per studiare la funzione, le caratteristiche di genomica e proteomica del muscolo provenienti da pazienti, in un ambiente completamente fisiologico, rappresentano uno strumento prezioso per studiare queste malattie complesse. Inoltre questo etero-specie modello aiuta a discriminare tra i meccanismi patologici neurogene e miogenico. 3,14,15 La co-coltura qui presentato ha anche alcune limitazioni. Ad esempio, gli esperimenti immunoistochimica sulle cellule muscolari sono difficili da eseguire su questa struttura, poiché il terminale nervoso incarnata dalla espianto copre l&#39;apparato NMJ postsinaptico. Questo sistema può anche essere utilizzato per valutare gli effetti neurotrofici o miotonica di farmaci in un sistema integrato di cultura e relativamente maturo, ma non consente ad elevata capacitàscreening, come il volume sarebbe troppo grande. Curiosamente, la omologa nervo-muscolo co-coltura con sia il nervo e componenti muscolari proveniente da solo topo o ratto non è funzionale e non riesce a causare contrazioni muscolari. Recentemente, tuttavia, omologo innervazione tra le cellule satelliti muscolari di topo e topo espianti del midollo spinale è stata eseguita con successo dalla modifica della data di raccolta degli embrioni (Callizot, Steinschneider e colleghi, risultati non pubblicati). Ulteriori progressi nella creazione di protocolli di routine per omologa co-colture potrebbe aiutare a superare la limitazione delle specie che esiste per questa tecnica di oggi. Nonostante questi limiti, si descrive qui un metodo che consente lo studio di molti aspetti del muscolo e motoneurone fisiologia, una tecnica che non è difficile da eseguire, non richiede alcun materiale speciale e che i risultati in stabile midollo spinale espianti muscolo-co- culture che possono essere facilmente curati e Studied per molti mesi. Alcuni aspetti del protocollo sono di particolare importanza e di bisogno di cure particolari per garantire una corretta innervazione. In primo luogo, gli embrioni di ratto devono essere tra 13 e 14 ED di età. In secondo luogo, 500.000 cellule per 35-mm piatto assicura il corretto livello di confluenza cella per facilitare l&#39;adesione degli espianti che vengono aggiunti alle cellule muscolari immediatamente quando inizia la fusione cellulare. Infine, come sottolineato nel protocollo, l&#39;aggiunta del mezzo dopo il deposito degli espianti sulle cellule muscolari è la parte più difficile di questa procedura deve essere eseguita con estrema cura.

<table border="1"><tbody><tr><td> Nome di reagente </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> HBSS </td><td> Gibco / Invitrogen </td><td> 14170 </td></tr><tr><td> MEM </td><td> Gibco / Invitrogen </td><td> 31095 </td></tr><tr><td> Medium 199 </td><td> Gibco / Invitrogen </td><td> 31153 </td></tr><tr><td> Siero fetale bovino </td><td> Fetale Clone Perbio </td><td> SH30066.03 </td></tr><tr><td> Insulina </td><td> Sigma </td><td> I9278 </td></tr><tr><td> EGF umano </td><td> Sigma </td><td> E9644 </td></tr><tr><td> Umana FGF </td><td> Sigma </td><td> F0291 </td></tr><tr><td> Penicillina / streptomicina soluzione </td><td> Gibco </td><td> 15140 </td></tr></tbody></table>

a functional motor unit in the culture dish: co-culture of spinal cord explants and muscle cells

Le cellule muscolari primarie aneurally coltivate in monostrato raramente contratto spontaneamente perché, in assenza di un componente nervo, la differenziazione cellulare è limitata e la stimolazione motore neurone manca 1. Queste limitazioni ostacolano lo studio in vitro di molte patologie neuromuscolari in cellule muscolari coltivate. È importante sottolineare che i vincoli sperimentali monostrato di coltura, le cellule muscolari possono essere superate innervazione funzionale di miofibre con espianti midollo spinale in co-culture. Qui, si mostrano le diverse fasi necessarie per ottenere un efficiente, innervazione corretto di cellule muscolari primarie, portando a completare contrazione differenziazione e fibra secondo il metodo sviluppato da Askanas 2. Per fare ciò, le cellule muscolari sono co-coltura con espianti del midollo spinale di embrioni di ratto a 13,5 ED, con gangli delle radici dorsali ancora attaccato le fette del midollo spinale. Dopo pochi giorni, il muscolo fibri iniziare a contratto e alla fine diventare cross-striata attraverso innervazione da neuriti funzionali sporgenti dalle espianti del midollo spinale che collegano alle cellule muscolari. Questa struttura può essere mantenuta per molti mesi, semplicemente regolare scambio del terreno di coltura. Le applicazioni di questo prezioso strumento sono numerosi, in quanto rappresenta un modello funzionale per l&#39;analisi multidisciplinari di sviluppo umano e innervazione del muscolo. In realtà, un completo de novo installazione giunzione neuromuscolare avviene in un piatto di coltura, consentendo una misurazione di facile molti parametri ad ogni passo, in un contesto fondamentale e fisiologico. Solo per citare alcuni esempi, genomici e / o studi di proteomica può essere effettuata direttamente sulle co-culture. Inoltre, gli effetti pre-e post-sinaptica può essere specificamente e separatamente valutati a livello della giunzione neuromuscolare, perché entrambi i componenti provengono da diverse specie,ratto e umano, rispettivamente. Il nervo-muscolo co-coltura può essere eseguita anche con cellule muscolari umane isolate da pazienti affetti da patologie neuromuscolari muscolo o 3, e quindi può essere utilizzato come strumento di screening per farmaci candidati. Infine, nessuna attrezzatura speciale, ma un normale BSL2 struttura è necessaria per riprodurre un unità funzionale motore in un piatto di coltura. Questo metodo è quindi utile sia per il muscolo, nonché le comunità di ricerca neuromuscolari per studi fisiologici e meccanicistica della funzione neuromuscolare, in un contesto normale e malattia.

Watch this Scientific Journal Video about A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells at JoVE.com

A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

Cellule muscolari coltivate sono un modello inadeguato per ricapitolare muscolo innervato<em&gt; In vivo</em&gt;. Un&#39;unità motore funzionale può essere riprodotto<em&gt; In vitro</em&gt; Dalla innervazione del muscolo differenziate cellule umane primarie di ratto utilizzando espianti di midollo spinale degli embrioni. Questo articolo descrive come co-colture di espianti di midollo spinale e delle cellule muscolari sono stabiliti.

Un gruppo motore funzionale nella piastra di coltura: Co-coltura degli espianti midollo spinale e le cellule muscolari

Human primary muscle cells cultured  aneurally in monolayer rarely contract spontaneously because, in the absence of  a nerve component, cell ...

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Nanyang Technological University

Research

JoVE Journal

Neuroscience

15.2K Views.  University of Basel. Cellule muscolari coltivate sono un modello inadeguato per ricapitolare muscolo innervato In vivo. Un'unità motore funzionale può essere riprodotto In vitro Dalla innervazione del muscolo differenziate cellule umane primarie di ratto utilizzando espianti di midollo spinale degli embrioni. Questo articolo descrive come co-colture di espianti di midollo spinale e delle cellule muscolari sono stabiliti.

Video: A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

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

Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation depends on a combination of axon guidance cues1 and survival factors2 located along the trajectory of growing axons and/or within their sensory organ targets. For example, functional interference with a classic axon guidance signaling pathway, semaphorin-neuropilin, generated misrouting of otic axons3. Also, several growth factors expressed in the sensory targets of the inner ear, including Neurotrophin-3 (NT-3) and Brain Derived Neurotrophic Factor (BDNF), have been manipulated in transgenic animals, again leading to misrouting of SAG axons4. These same molecules promote both survival and neurite outgrowth of chick SAG neurons in vitro5,6.
Here, we describe and demonstrate the in vitro method we are currently using to test the responsiveness of chick SAG neurites to soluble proteins, including known morphogens such as the Wnts, as well as growth factors that are important for promoting SAG neurite outgrowth and neuron survival. Using this model system, we hope to draw conclusions about the effects that secreted ligands can exert on SAG neuron survival and neurite outgrowth. 
SAG explants are dissected on embryonic day 4 (E4) and cultured in three-dimensional collagen gels under serum-free conditions for 24 hours. First, neurite responsiveness is tested by culturing explants with protein-supplemented medium. Then, to ask whether point sources of secreted ligands can have directional effects on neurite outgrowth, explants are co-cultured with protein-coated beads and assayed for the ability of the bead to locally promote or inhibit outgrowth. We also include a demonstration of the dissection (modified protocol7) and culture of E6 spinal cord explants. We routinely use spinal cord explants to confirm bioactivity of the proteins and protein-soaked beads, and to verify species cross-reactivity with chick tissue, under the same culture conditions as SAG explants. These in vitro assays are convenient for quickly screening for molecules that exert trophic (survival) or tropic (directional) effects on SAG neurons, especially before performing studies in vivo. Moreover, this method permits the testing of individual molecules under serum-free conditions, with high neuron survival8.

We demonstrate how to dissect and culture chick E4 statoacoustic ganglion and E6 spinal cord explants. Explants are cultured under serum-free conditions in 3D collagen gels for 24 hours. Neurite responsiveness is tested with growth factor-supplemented medium and with protein-coated beads.

Dissezione e Cultura della gangliari Chick Statoacoustic Espianti e del midollo spinale in gel di collagene per saggi crescita dei neuriti

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation ...

Chicken Embryo Spinal Cord Slice Culture Protocol

Slice cultures can facilitate the manipulation of embryo development both pharmacologically and through gene manipulations. In this reduced system, potential lethal side effects due to systemic drug applications can be overcome. However, culture conditions must ensure that normal development proceeds within the reduced environment of the slice. We have focused on the development of the spinal cord, particularly that of spinal motor neurons. We systematically varied culture conditions of chicken embryo slices from the point at which most spinal motor neurons had been born. We assayed the number and type of motor neurons that survived during the culture period and the position of those motor neurons compared to that in vivo. We found that serum type and neurotrophic factors were required during the culture period and were able to keep motor neurons alive for at least 24 hr and allow those motor neurons to migrate to appropriate positions in the spinal cord. We present these culture conditions and the methodology of preparing the embryo slice cultures using eviscerated chicken embryos embedded in agarose and sliced using a vibratome.

Slice cultures facilitate the manipulation of embryo development by gene and pharmacological perturbations. However, culture conditions must ensure that normal development can proceed within the reduced environment of the slice. We illustrate a protocol that facilitates normal spinal cord development to proceed for at least 24 hr.

Embrione di pollo midollo spinale Fetta protocollo Cultura

Slice cultures can facilitate the manipulation of  embryo development both pharmacologically and through gene manipulations. In  this reduced system, ...

Analysis of Developing Tooth Germ Innervation Using Microfluidic Co-culture Devices

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena are not well understood yet. In particular, the role of innervation in tooth development and regeneration is neglected.
Several in vivo studies have provided important information about the patterns of innervation of dental tissues during development and repair processes of various animal models. However, most of these approaches are not optimal to highlight the molecular basis of the interactions between nerve fibres and target organs and tissues.
Co-cultures constitute a valuable method to investigate and manipulate the interactions between nerve fibres and teeth in a controlled and isolated environment. In the last decades, conventional co-cultures using the same culture medium have been performed for very short periods (e.g., two days) to investigate the attractive or repulsive effects of developing oral and dental tissues on sensory nerve fibres. However, extension of the culture period is required to investigate the effects of innervation on tooth morphogenesis and cytodifferentiation.
Microfluidics systems allow co-cultures of neurons and different cell types in their appropriate culture media. We have recently demonstrated that trigeminal ganglia (TG) and teeth are able to survive for a long period of time when co-cultured in microfluidic devices, and that they maintain in these conditions the same innervation pattern that they show in vivo.
On this basis, we describe how to isolate and co-culture developing trigeminal ganglia and tooth germs in a microfluidic co-culture system.This protocol describes a simple and flexible way to co-culture ganglia/nerves and target tissues and to study the roles of specific molecules on such interactions in a controlled and isolated environment.

Co-cultures represent a valuable method to study the interactions between nerves and target tissues and organs. Microfluidic systems allow co-culturing ganglia and whole developing organs or tissues in different culture media, thus representing a valuable tool for the in vitro study of the crosstalk between neurons and their targets.

Analisi di sviluppo di denti Germ innervazione Uso di dispositivi Microfluidic Co-cultura

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena ...

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 ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 106 neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

Isolamento e cultura dei neuroni del midollo spinale da topi neonatali

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

Laminectomia e impianto della finestra del cavo spinale nel mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

Identificazione dei marcatori di superficie cellulare delle cellule staminali primarie e delle cellule progenitrici mediante l'etichettatura metabolica di Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...

Direct Injection of a Lentiviral Vector Highlights Multiple Motor Pathways in the Rat Spinal Cord

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. Injection directly into spinal cord tissue bypasses these barriers, providing access to cell bodies or synapses where molecules can be incorporated. Combining viral vector technology with this method allows for introduction of target genes into nervous tissue for the purpose of gene therapy or tract tracing. Here a virus engineered for highly efficient retrograde transport (HiRet) is introduced at the synapses of propriospinal interneurons (PNs) to encourage specific transport to neurons in the spinal cord and brainstem nuclei. Targeting PNs takes advantage of the numerous connections they receive from motor pathways such as the rubrospinal and reticulospinal tracts, as well as their interconnection with each other throughout spinal cord segments. Representative tracing using the HiRet vector with constitutively active green fluorescent protein (GFP) shows high fidelity details of cell bodies, axons and dendritic arbors in thoracic PNs and in reticulospinal neurons in the pontine reticular formation. HiRet incorporates well into brainstem pathways and PNs but shows age dependent integration into corticospinal tract neurons. In summary, spinal cord injection using viral vectors is a suitable method for introduction of proteins of interest into neurons of targeted tracts.

This protocol demonstrates injection of a retrogradely transportable viral vector into rat spinal cord tissue. The vector is taken up at the synapse and transported to the cell body of target neurons. This model is suitable for retrograde tracing of important spinal pathways or targeting cells for gene therapy applications.

Iniezione diretta di un vettore lentivirale evidenzia più vie motorie del midollo spinale del ratto

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. ...

Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into motor neuron is obtained by ectopic expression of alternative modules of transcription factors, namely Ngn2, Isl1 and Lhx3 (NIL) or Ngn2, Isl1 and Phox2a (NIP). NIL and NIP specify, respectively, spinal and cranial motor neuron identity. Our protocol starts with the generation of modified iPSC lines in which NIL or NIP are stably integrated in the genome via a piggyBac transposon vector. Expression of the transgenes is then induced by doxycycline and leads, in 5 days, to the conversion of iPSCs into MN progenitors. Subsequent maturation, for 7 days, leads to homogeneous populations of spinal or cranial MNs. Our method holds several advantages over previous protocols: it is extremely rapid and simplified; it does not require viral infection or further MN isolation; it allows generating different MN subpopulations (spinal and cranial) with a remarkable degree of maturation, as demonstrated by the ability to fire trains of action potentials. Moreover, a large number of motor neurons can be obtained without purification from mixed populations. iPSC-derived spinal and cranial motor neurons can be used for in vitro modeling of Amyotrophic Lateral Sclerosis and other neurodegenerative diseases of the motor neuron. Homogeneous motor neuron populations might represent an important resource for cell type specific drug screenings.

Conversion of Human Induced Pluripotent Stem Cells iPSCs into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

This protocol allows rapid and efficient conversion of induced pluripotent stem cells into motor neurons with a spinal or cranial identity, by ectopic expression of transcription factors from inducible piggyBac vectors.

Conversione di cellule staminali pluripotenti indotte dall'uomo (IPSC) in neuroni motore spinale e cranico funzionale utilizzando vettori PiggyBac

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into ...