The work was funded by the University of Zurich. The authors would like to thank Estrela Neto and Dr. Meriem Lamghari for helping in establishing the co-culture conditions.

Tutti i topi sono stati mantenuti e gestiti secondo la Legge benessere degli animali svizzera e nel rispetto delle norme del ufficio cantonale di veterinaria, Zurigo. 1. Preparazione di dissezione Materiale, Cultura Media, dispositivi microfluidici <ol><li> Autoclave pinze micro-dissezione e forbici (121 ° C, tempo di sterilizzazione: 20 min) e conservarle in un contenitore sterile. </li><li> Sterilizzare coprioggetto di vetro (24 mm x 24 mm) da loro incubazione in 1 M HCl per 24 ore in un agitatore orbitale a 37 ° C. Lavare per tre volte con una soluzione sterile, H 2 O distillata e tre volte con etanolo 99%. Secco poi i coprioggetti a 37 ° C o sotto cappa a flusso sterile. Infine, autoclave o esporre i coprioggetti alla luce UV (30 min) per completare la sterilizzazione. Coprioggetto possono essere conservati in etanolo al 70%. </li><li> Rimuovere accuratamente i dispositivi di isolamento AXIS Axon dal pacchetto con pinza sterile e metterli in una piastra di Petri sterile. </li&gt; <li> Utilizzando una biopsia sterile (ø: 1mm) creare un foro per ogni campione da coltivate (figura 1) in corrispondenza delle camere di coltura. NOTA: Non pugno troppo vicino alle microscanalature, in quanto potrebbero essere danneggiati dalla pressione applicata. </li><li> Sterilizzare i dispositivi di isolamento AXIS Axon da loro immergendo in etanolo al 70%. Asciutto, quindi AXIS Axon dispositivi di isolamento e coprioggetti completamente sotto una cappa a flusso sterili. Attendere almeno 3 ore prima di procedere. NOTA: essiccazione incompleta provocherà assemblaggio difettoso dei dispositivi microfluidici. </li><li> Posizionare ogni vetrino in una capsula di Petri da 35 mm o in un pozzo all&#39;interno di un 6-pozzetti. </li><li> Posizionare il dispositivo di isolamento AXIS Axon sul vetrino e premere delicatamente ma con fermezza, con una pinza con estremità piegate in modo da consentire la piena adesione tra il dispositivo di isolamento e il vetrino di vetro. </li><li> In ciascuna camera di coltura, pipetta 150 ml di poli-D-lisina (0,1 mg / ml in sterile, distillata H 2O). Posizionare i dispositivi microfluidici sotto vuoto per 5 minuti, per eliminare tutta l&#39;aria dalle camere di coltura. </li><li> Se l&#39;aria è ancora visibile all&#39;interno delle camere, ri-pipetta la soluzione poli-D-lisina nelle camere. </li><li> Incubare i dispositivi con poli-D-lisina O / N a 37 ° C. </li><li> Lavare camere tre volte con sterili, H 2 O. distillata </li><li> Riempire camere con 150 microlitri soluzione laminina lavoro (Sigma-Aldrich, 5 mcg / ml, in PBS o terreno privo di siero), e incubare O / N a 37 ° C. </li><li> Preparare 50 ml di terreno per le colture gangli trigeminali 37, così composto: 48 ml Neurobasal medio, 1 ml di B-27, 100 U / ml di penicillina / streptomicina, 2 mM L-glutammina, 5 ng / ml fattore di crescita nervoso (NGF) , 12:25 citosina arabinoside. </li><li> Preparare 50 ml di mezzo di crescita per dente germinali 37, così composto: 40 ml DMEM-F12, 10 ml di siero bovino fetale (FBS, concentrazione finale: 20%), 100 U / ml di penicillina / streptomicina, 2 mM L-glutammina, 150 mg / ml di acido ascorbico. </li></ol> Embrione Generazione ed Dissection 2. mouse <ol><li> Determinare l&#39;età embrionale secondo tappo vaginale (spina vaginale: embrionale giorno di sviluppo 0.5, E0.5) e confermare tramite criteri morfologici. Per questo protocollo, generalmente usiamo gli embrioni E14.5 E17.5-mouse. </li><li> Pulire l&#39;area dissezione e lo stereoscopio con etanolo al 70%. </li><li> Sacrifica la madre incinta tramite dislocazione cervicale. Bloccare il collo del mouse con il primo e secondo dito su una griglia, e tirare con decisione la coda. </li><li> Sezionare la pelle intorno basso addome, e aprire l&#39;addome con le forbici. Individuare l&#39;utero: durante queste ultime fasi della gravidanza, l&#39;utero abbondantemente riempire la cavità addominale. </li><li> Sezionare l&#39;utero e posto in un tubo riempito con PBS sul ghiaccio. Quando sul ghiaccio, il tessuto può essere lasciato per vari hr. Eliminare il cadavere della madre in base alle linee guida istituzionali </li> &lt;li&gt; sezionare gli embrioni dall&#39;utero e liberarli dai loro tessuti extraembrionali. Mettere gli embrioni in PBS sul ghiaccio. </li><li> Decapitare gli embrioni con forbici, e separare la mascella inferiore dal resto della testa con le forbici micro-dissezione (Figura 2A). Rimuovere proprio la mascella inferiore, senza danneggiare i gangli del trigemino; come quest&#39;ultimo sono localizzate in prossimità della mascella inferiore, il loro danneggiamento accidentale è possibile. Conservare la mandibola e il resto della testa in PBS freddo, sul ghiaccio. </li><li> Per sezionare TG, prendere la testa e metterlo su un piatto di vetro Petri dissezione, già pieno di PBS freddo. Utilizzando le pinze, togliere la pelle e il cranio. Rimuovere poi il telencefalo e il cervelletto ponendo forcipe sotto telencefalo e ascensore; telencefalo e cervelletto si capovolgere insieme, lasciando il fondo del cranio esposto. </li><li> Localizzare gangli del trigemino (Figura 2B). Utilizzare le pinzeper separare il TG dai nervi trigemino. Eliminare i resti delle proiezioni trigeminali con gli aghi di dissezione come coltelli. Posizionare il TG sezionato in una capsula di Petri riempita con PBS freddo e tenerli su ghiaccio. </li><li> Per sezionare denti embrionali, posizionare la mascella inferiore, precedentemente separato dal cranio, sulla piastra di Petri di vetro dissezione, pieno di PBS freddo. Usando aghi dissezione come coltelli, rimuovere la lingua e la pelle che circonda la mascella. Separare la destra emi-griffe sinistra e tagliando lungo la linea mediana della mandibola. I germi dei denti sono facilmente visibili, come mostrato nella Figura 1C. Isolare i germi dei denti utilizzando aghi dissezione e rimuovere l&#39;eccesso di tessuti non-dentali. Posizionare i germi dente sezionato in una capsula di Petri riempita con PBS freddo e tenerli su ghiaccio. </li></ol> 3. Microfluidic co-culture <ol><li> Dopo la dissezione, rimuovere laminina dai dispositivi microfluidica. Riempire le camere con 200 microlitri della respective media. </li><li> Con pinze, trasferire delicatamente i TG e dei denti germi sezionato nei fori creati dalla punzonatura (Figura 1D). Assicurarsi che i germi dei denti non galleggiano e che affondano fino a quando entrano in contatto con le lamelle. </li><li> Cultura campioni in incubatore a 37 ° C, 5% di CO 2. </li><li> Modificare il terreno di coltura ogni 48 ore. Non svuotare le camere completamente, e non pipettare direttamente nelle camere di coltura. Completo svuotamento delle camere comporterebbe la formazione di bolle d&#39;aria all&#39;interno delle camere; pipettaggio diretto nella camera potrebbe causare un danno assonale. Per evitare questi problemi, rimuovere il supporto punta della pipetta verso il lato esterno dei pozzetti; analogamente, pipettare il mezzo fresco sulla parete del pozzetto situato di fronte alle camere. </li><li> Durante il periodo di coltura, co-culture possono essere facilmente ripreso al microscopio time-lapse. Co-culture possono essere mantenuti per più di 10 giorni. </li><li> Dopo il perio culturad, lavare le camere pipettando 150 ml di soluzione in un pozzo per ogni camera, e lasciando PBS fluire attraverso le camere per tre volte. </li><li> Rimuovere la PBS e fissare i campioni pipettando 150 ml di paraformaldeide 4% (in PBS) in un pozzetto per camera; incubare a temperatura ambiente per 15 min. </li><li> Lavare le camere due volte con PBS come descritto in 3.6. </li><li> Procedere con ulteriori analisi. </li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics+co-culture+systems+for+studying+tooth+innervation.">Microfluidics co-culture systems for studying tooth innervation.</a> Frontiers in physiology. 5 (August), (2014).</li><li>Connor, R., Tessier-Lavigne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+maxillary+factor,+a+maxillary+process-derived+chemoattractant+for+developing+trigeminal+sensory+axons.">Identification of maxillary factor, a maxillary process-derived chemoattractant for developing trigeminal sensory axons.</a> Neuron. 24, 165-178 (1999).</li></ol>

The authors declare that they have no competing financial interests.

Precedente in studi in vitro di dente innervazione erano basati su co-colture convenzionali di gangli trigeminali e tessuti dentali o cellule 26,28,29. Sono stati condotti Questi studi per investigare principalmente gli effetti interessanti di queste cellule o tessuti di assoni sensoriali 38. Anche se portare significativi progressi nel campo, alcuni problemi tecnici sono state sollevate. Germi denti cominciano a degenerare dopo pochi giorni di coltura 37. Sulla base di...

Innervazione svolge un ruolo chiave nello sviluppo, omeostasi e la rigenerazione di organi e tessuti 1,2. Inoltre, innervazione è coinvolto nella regolazione della proliferazione delle cellule staminali, la mobilitazione e la differenziazione 3-5. Infatti, recenti studi realizzati in tessuti del complesso orofacciale hanno dimostrato che i nervi parasimpatici sono necessari per la funzione epiteliali cellule progenitrici durante lo sviluppo e la rigenerazione delle ghiandole salivari 6,7. Analogamente, è stato dimostrato che innervazione è necessario per lo sviluppo e il mantenimento delle papille gustative 8 - 11. Pertanto, è importante analizzare i ruoli ancora trascurati innervazione nello sviluppo di altri importanti organi e tessuti orofacciali come i denti. Nonostante la ricca innervazione dei denti adulti, e in contrasto con tutti gli altri organi e tessuti del corpo, svidenti ping cominciano ad essere innervato nelle fasi postnatali prime. Denti si sviluppano a causa di interazioni sequenziali e reciproche tra l&#39;ectoderma orale e cranio neurale mesenchima crest-derivati. Queste interazioni danno luogo a ameloblasts epiteliali derivate e odontoblasti mesenchima-derivati ​​che sono responsabili della formazione di smalto e dentina, rispettivamente 12. Nervi sensoriali del gangli trigeminali e nervi simpatici dai gangli cervicale superiore innervano i denti adulti 13 - 15. Durante l&#39;embriogenesi, fibre nervose che emana dal progetto gangli trigeminali verso i germi di sviluppo dei denti e progressivamente li circondano, ma non penetrare nel dentale papilla mesenchima 13. Fibre nervose entrano la polpa dentale mesenchima a stadi di sviluppo più avanzati che correlano con la differenziazione odontoblast e matrice dentina deposizione 16. Dental polpa innervazione è completed subito dopo l&#39;eruzione dei denti nel cavo orale 13. Studi precedenti hanno rivelato che diversi semaforine e neurotrofine sono coinvolti nella regolazione della innervazione durante l&#39;odontogenesi 16-19. Studi precedenti hanno chiaramente dimostrato che innervazione è un prerequisito per la formazione dei denti nei pesci 20. Studi più recenti hanno dimostrato che l&#39;omeostasi delle cellule staminali mesenchima dentale in incisivi mouse è regolata da nervi sensoriali attraverso la secrezione di sonic hedgehog (Shh) 21. Tuttavia, il ruolo di innervazione in dente iniziazione, lo sviluppo e la rigenerazione è ancora molto controversa nei mammiferi 22 - 24. Una pletora di studi in vivo hanno fornito importanti informazioni sui modelli di innervazione di tessuti dentali durante lo sviluppo e riparazione processi di vari modelli animali 13,25,26. Tuttavia, la maggior parte di questi Approdolori non sono ottimali per evidenziare le basi molecolari delle interazioni tra le fibre nervose e organi bersaglio e dei tessuti. Co-culture costituiscono un valido metodo per indagare e manipolare le interazioni tra le fibre nervose e denti in un ambiente controllato e isolato 26 a 29 -. Allo stesso tempo, co-coltura è soggetta a varie regolazioni tecniche. Ad esempio, nervi e tessuti dentali specifici (ad esempio, pasta dentale, follicolo dentale, epitelio dentale) spesso richiedono terreni di coltura diversa per garantire la sopravvivenza del tessuto per lunghi periodi di tempo 30-32. Negli ultimi decenni, co-colture convenzionali che utilizzano lo stesso mezzo di coltura sono stati effettuati per periodi molto brevi (ad esempio, due giorni) per studiare gli effetti di attrazione o repulsione di sviluppare tessuti orali e dentali sulle fibre nervose sensoriali 27-29.Tuttavia, l&#39;estensione del periodo di coltura è necessario per studiare gli effetti di innervazione sul dente morfogenesi e citodifferenziazione, e per studiare le dinamiche di fibre nervose si diramano all&#39;interno organi bersaglio. Pertanto, co-colture non contigui sarebbe più adatto per realizzare studi su tessuti interazioni neuronali-dentale. Sistemi di microfluidica permettono co-colture di neuroni e diversi tipi di cellule nella loro terreni di coltura appropriati. In questi dispositivi, tessuti dentali e neuroni sono separati in differenti compartimenti, pur consentendo la crescita degli assoni dai corpi cellulari neuronali attraverso microcanali verso il compartimento contenente il tessuto bersaglio 33. Dispositivi microfluidici sono già stati utilizzati per studiare le interazioni tra neuroni e microglia 34,35, così come cellule alle interazioni cellulari nel cancro e neovascolarizzazione 35. Inoltre, questi sistemi sono stati utilizzati per studiare le interazioni tra dorsal gangli e osteoblasti 36. Abbiamo recentemente dimostrato che gangli del trigemino (TG) ed i denti sono in grado di sopravvivere per lunghi periodi di tempo quando co-coltivate in dispositivi microfluidici 37. Inoltre, abbiamo dimostrato che i denti da diverse fasi di sviluppo di mantenere in queste condizioni in vitro gli stessi effetti ripugnanti o attraenti innervazione del trigemino che mostrano in vivo 37. Questo protocollo fornisce informazioni su un modo semplice, potente e flessibile di co-coltura gangli / nervi e tessuti bersaglio e per studiare il ruolo di molecole specifiche su tali interazioni in un ambiente controllato e isolato.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>AXIS Axon Isolation Devices</td>
			<td>Millipore</td>
			<td>AX15010-TC</td>
			<td>Microchannels of different lenght are available</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma Aldrich</td>
			<td>L2020</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco</td>
			<td>17504</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse beta-NGF</td>
			<td>R&#38;D Systems</td>
			<td>1156-NG-100</td>
			<td>Human and Rat beta-NGF (R&#38;D Systems) are equivalent</td>
		</tr>
		<tr>
			<td>DMEM-F12</td>
			<td>Gibco</td>
			<td>11320-033</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of developing tooth germ innervation using microfluidic co-culture devices

Innervazione svolge un ruolo chiave nello sviluppo, omeostasi e la rigenerazione di organi e tessuti. Tuttavia, i meccanismi alla base di questi fenomeni non sono ancora ben compresi. In particolare, il ruolo di innervazione in sviluppo dentale e rigenerazione è trascurato. Diversi studi in vivo hanno fornito importanti informazioni sui modelli di innervazione di tessuti dentali durante lo sviluppo e riparazione processi di vari modelli animali. Tuttavia, la maggior parte di questi approcci non sono ottimali per evidenziare le basi molecolari delle interazioni tra le fibre nervose e organi bersaglio e dei tessuti. Co-culture costituiscono un valido metodo per indagare e manipolare le interazioni tra le fibre nervose e denti in un ambiente controllato e isolato. Negli ultimi decenni, co-colture convenzionali utilizzando lo stesso terreno di coltura sono stati effettuati per periodi molto brevi (ad esempio, due giorni)per studiare gli effetti di attrazione o repulsione di sviluppare tessuti orali e dentali su fibre nervose sensoriali. Tuttavia, l&#39;estensione del periodo di coltura è necessario per studiare gli effetti del innervazione sul dente morfogenesi e citodifferenziazione. Sistemi di microfluidica permettono co-colture di neuroni e diversi tipi di cellule nella loro terreni di coltura appropriati. Abbiamo recentemente dimostrato che gangli del trigemino (TG) ed i denti sono in grado di sopravvivere per un lungo periodo di tempo in cui co-coltivate in dispositivi microfluidici, e che mantengono in queste condizioni lo stesso modello innervazione che mostrano in vivo. Su questa base, si descrive come isolare e co-coltura di sviluppo del trigemino gangli e dei denti germi in un protocollo di co-coltura microfluidica system.This descrive un modo semplice e flessibile di co-coltura gangli / nervi e tessuti bersaglio e per studiare i ruoli di molecole specifiche su tali interazioni in un controlled e ambiente isolato.

Questi risultati mostrano che isolati gangli trigemini possono crescere in un compartimento del dispositivo microfluidico e, inoltre, che lo sviluppo dei germi isolati dente è mantenuta per un lungo periodo di tempo in altro compartimento del dispositivo microfluidico. Diversi terreni di coltura sono utilizzati nei due scomparti, e le microscolpiture tra i due compartimenti consentono estensione dell&#39;assone dal ganglio trigeminale verso i germi di sviluppo dei denti. Figura 3 rappresenta una visual...

Watch this Scientific Journal Video about Analysis of Developing Tooth Germ Innervation Using Microfluidic Co-culture Devices at JoVE.com

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

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 ...

analysis-developing-tooth-germ-innervation-using-microfluidic-co

Research

JoVE Journal

Neuroscience

8.2K Views.  University of Zurich. 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.

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

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

In elettroporazione in vivo di Retina topo in via di sviluppo

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

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 ...

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

Human primary muscle cells cultured aneurally in monolayer rarely contract spontaneously because, in the absence of a nerve component, cell differentiation is limited and motor neuron stimulation is missing1. These limitations hamper the in vitro study of many neuromuscular diseases in cultured muscle cells. Importantly, the experimental constraints of monolayered, cultured muscle cells can be overcome by functional innervation of myofibers with spinal cord explants in co-cultures.
Here, we show the different steps required to achieve an efficient, proper innervation of human primary muscle cells, leading to complete differentiation and fiber contraction according to the method developed by Askanas2. To do so, muscle cells are co-cultured with spinal cord explants of rat embryos at ED 13.5, with the dorsal root ganglia still attached to the spinal cord slices. After a few days, the muscle fibers start to contract and eventually become cross-striated through innervation by functional neurites projecting from the spinal cord explants that connecting to the muscle cells. This structure can be maintained for many months, simply by regular exchange of the culture medium.
The applications of this invaluable tool are numerous, as it represents a functional model for multidisciplinary analyses of human muscle development and innervation. In fact, a complete de novo neuromuscular junction installation occurs in a culture dish, allowing an easy measurement of many parameters at each step, in a fundamental and physiological context.
Just to cite a few examples, genomic and/or proteomic studies can be performed directly on the co-cultures. Furthermore, pre- and post-synaptic effects can be specifically and separately assessed at the neuromuscular junction, because both components come from different species, rat and human, respectively. The nerve-muscle co-culture can also be performed with human muscle cells isolated from patients suffering from muscle or neuromuscular diseases3, and thus can be used as a screening tool for candidate drugs. Finally, no special equipment but a regular BSL2 facility is needed to reproduce a functional motor unit in a culture dish. This method thus is valuable for both the muscle as well as the neuromuscular research communities for physiological and mechanistic studies of neuromuscular function, in a normal and disease context.

Cultured muscle cells are an inadequate model to recapitulate innervated muscle in vivo. A functional motor unit can be reproduced in vitro by innervation of differentiated human primary muscle cells using rat embryo spinal cord explants. This article describes how co-cultures of spinal cord explants and muscle cells are established.

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 ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

Visualizzazione e manipolazione genetica di dendriti e spine nella corteccia cerebrale e nell&#39;ippocampo mouse utilizzando<em&gt; In utero</em&gt; Elettroporazione

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture System

Proper neuron to glia interaction is critical to physiological function of the central nervous system (CNS). This bidirectional communication is sophisticatedly mediated by specific signaling pathways between neuron and glia1,2 . Identification and characterization of these signaling pathways is essential to the understanding of how neuron to glia interaction shapes CNS physiology. Previously, neuron and glia mixed cultures have been widely utilized for testing and characterizing signaling pathways between neuron and glia. What we have learned from these preparations and other in vivo tools, however, has suggested that mutual signaling between neuron and glia often occurred in specific compartments within neurons (i.e., axon, dendrite, or soma)3. This makes it important to develop a new culture system that allows separation of neuronal compartments and specifically examines the interaction between glia and neuronal axons/dendrites. In addition, the conventional mixed culture system is not capable of differentiating the soluble factors and direct membrane contact signals between neuron and glia. Furthermore, the large quantity of neurons and glial cells in the conventional co-culture system lacks the resolution necessary to observe the interaction between a single axon and a glial cell.
	In this study, we describe a novel axon and glia co-culture system with the use of a microfluidic culture platform (MCP). In this co-culture system, neurons and glial cells are cultured in two separate chambers that are connected through multiple central channels. In this microfluidic culture platform, only neuronal processes (especially axons) can enter the glial side through the central channels. In combination with powerful fluorescent protein labeling, this system allows direct examination of signaling pathways between axonal/dendritic and glial interactions, such as axon-mediated transcriptional regulation in glia, glia-mediated receptor trafficking in neuronal terminals, and glia-mediated axon growth. The narrow diameter of the chamber also significantly prohibits the flow of the neuron-enriched medium into the glial chamber, facilitating probing of the direct membrane-protein interaction between axons/dendrites and glial surfaces.

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System

This study describes the procedures of setting up a novel neuronal axon and (astro)glia co-culture platform. In this co-culture system, manipulation of direct interaction between a single axon (and single glial cell) becomes feasible, allowing mechanistic analysis of the mutual neuron to glial signaling.

Analisi Imaging di Neuron Interaction Glia in Platform Cultura Microfluidic (MCP) a base di Axon neurone e glia co-cultura di sistema

Proper  neuron to glia interaction is critical to physiological function of the central  nervous system (CNS). This bidirectional communication is ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Immagini dal vivo della mitosi in via di sviluppo embrionali di topo Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and morphological levels. Compound muscle action potential (CMAP) recording following phrenic nerve stimulation can be used to quantitatively assess functional diaphragm innervation by PhMNs of the cervical spinal cord in vivo in anesthetized rats and mice. Because CMAPs represent simultaneous recording of all myofibers of the whole hemi-diaphragm, it is useful to also examine the phenotypes of individual motor axons and myofibers at the diaphragm NMJ in order to track disease- and therapy-relevant morphological changes such as partial and complete denervation, regenerative sprouting and reinnervation. This can be accomplished via whole-mount immunohistochemistry (IHC) of the diaphragm, followed by detailed morphological assessment of individual NMJs throughout the muscle. Combining CMAPs and NMJ analysis provides a powerful approach for quantitatively studying diaphragmatic innervation in rodent models of CNS and PNS disease.

Compound muscle action potential recording quantitatively assesses functional diaphragm innervation by phrenic motor neurons. Whole-mount diaphragm immunohistochemistry assesses morphological innervation at individual neuromuscular junctions. The goal of this protocol is to demonstrate how these two powerful methodologies can be used in various rodent models of spinal cord disease.

Funzionale e morfologica Valutazione della membrana innervazione da Phrenic Motor Neurons

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and ...

High-resolution Volume Imaging of Neurons by the Use of Fluorescence eXclusion Method and Dedicated Microfluidic Devices

Volume is an important parameter regarding physiological and pathological characteristics of neurons at different time scales. Neurons are quite unique cells regarding their extended ramified morphologies and consequently raise several methodological challenges for volume measurement. In the particular case of in vitro neuronal growth, the chosen methodology should include sub-micrometric axial resolution combined with full-field observation on time scales from minutes to hours or days. Unlike other methods like cell shape reconstruction using confocal imaging, electrically-based measurements or Atomic Force Microscopy, the recently developed Fluorescence eXclusion method (FXm) has the potential to fulfill these challenges. However, although being simple in its principle, implementation of a high-resolution FXm for neurons requires multiple adjustments and a dedicated methodology. We present here a method based on the combination of fluorescence exclusion, low-roughness multi-compartments microfluidic devices, and finally micropatterning to achieve in vitro measurements of local neuronal volume. The high resolution provided by the device allowed us to measure the local volume of neuronal processes (neurites) and the volume of some specific structures involved in neuronal growth, such as growth cones (GCs).

Volume is an important parameter regarding physiological and pathological characteristics of cells. We describe a fluorescent exclusion method allowing full-field measurement of in vitro neuronal volume with sub-micrometric axial resolution required for the analysis of neurites and dynamic structures implied in neuronal growth.

Alta risoluzione Volume Imaging di neuroni nell'esclusione di uso di fluorescenza metodo e dedicato dispositivi microfluidici

Volume is an important parameter regarding physiological and pathological characteristics of neurons at different time scales. Neurons are quite unique ...

Data Acquisition and Analysis In Brainstem Evoked Response Audiometry In Mice

Brainstem evoked response audiometry (BERA) is of central relevance in the clinical neurophysiology. As other evoked potential (EP) techniques, such as visually evoked potentials (VEPs) or somatosensory evoked potentials (SEPs), the auditory evoked potentials (AEPs) are triggered by the repetitive presentation of identical stimuli, the electroencephalographic (EEG) response of which is subsequently averaged resulting in distinct positive (p) and negative (n) deflections. In humans, both the amplitude and the latency of individual peaks can be used to characterize alterations in synchronization and conduction velocity in the underlying neuronal circuitries. Importantly, AEPs are also applied in basic and preclinical science to identify and characterize the auditory function in pharmacological and genetic animal models. Even more, animal models in combination with pharmacological testing are utilized to investigate for potential benefits in the treatment of sensorineural hearing loss (e.g., age- or noise-induced hearing deficits). Here we provide a detailed and integrative description of how to record auditory brainstem-evoked responses (ABRs) in mice using click and tone-burst application. A specific focus of this protocol is on pre-experimental animal housing, anesthesia, ABR recording, ABR filtering processes, automated wavelet-based amplitude growth function analysis, and latency detection.

Brainstem evoked response audiometry is an important tool in clinical neurophysiology. Nowadays, brainstem evoked response audiometry is also applied in the basic science and preclinical studies involving both pharmacological and genetic animal models. Here we provide a detailed description of how auditory brainstem responses can be successfully recorded and analyzed in mice.

Acquisizione e analisi dei dati nel cervello evocato Audiometria di risposta nei topi

Brainstem evoked response audiometry (BERA) is of central relevance in the clinical neurophysiology. As other evoked potential (EP) techniques, such as ...

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have identified a special population of vascular cells, the periventricular endothelial cells, that play a critical role in the migration and distribution of forebrain GABAergic interneurons during embryonic development. This, coupled with their cell-autonomous functions, alludes to novel roles of periventricular endothelial cells in the pathology of neuropsychiatric disorders like schizophrenia, epilepsy, and autism. Here, we have described three different in vitro assays that collectively evaluate the functions of periventricular endothelial cells and their interaction with GABAergic interneurons. Use of these assays, particularly in a human context, will allow us to identify the link between periventricular endothelial cells and brain disorders. These assays are simple, low cost, and reproducible, and can be easily adapted to any adherent cell type.

We present three simple in vitro assays-the long-distance migration assay, the co-culture migration assay, and chemo-attraction assay-that collectively evaluate the functions of human stem cell derived periventricular endothelial cells and their interaction with GABAergic interneurons.

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

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