Questo lavoro è sostenuto da fondi di avvio di The Jackson Laboratory.

1. Preparazione Glia Cultura del mouse per Condizionamento Cultura neuronale <ol><li> T75 beute sono rivestiti con una soluzione di collagene (1:3 diluizione Purecol in DDH 2 O). Le beute sono in posizione verticale e lasciato asciugare per una notte in una cappa di coltura tissutale. </li><li> Dissezione buffer (aCSF: 119 mM NaCl, 5 mM KCl, 1 mM MgCl 2, 30 mM glucosio, 25 mM HEPES, pH 7,4, senza calcio) e supporti glia (DMEM supplementato con 10% FBS, 10 unità / ml di penicillina, 10 ug / ml di streptomicina, e Glutamax) sono preparati e conservati a 4 ° C fino al momento dell&#39;uso. </li><li> Il giorno della cultura glia, i media glia viene riscaldato per almeno 30 minuti in un bagno d&#39;acqua a 37 ° C prima della dissezione del cervello di topo. Topi embrionali o neonatale sono sacrificati per decapitazione rapida. Cortecce cerebrali sono sezionato sotto un microscopio dissezione. </li><li> Tessuti cerebrali sezionati vengono digeriti in soluzione di papaina (100 ml papaina e 20 microlitri DNasi I 1% in 2 ml di tampone dissezione) a 37 ° C for 20 min. </li><li> Dopo la digestione papaina, tessuti cerebrali vengono sciacquati con 10 ml di pre-riscaldate supporti glia. Supporti glia freschi (2 ml) viene quindi aggiunta ai tessuti cerebrali digeriti, ei tessuti vengono triturato con un fuoco lucidato vetro pipetta Pasteur. </li><li> Supporti glia freschi (8 ml) viene aggiunto ai tessuti dissociate. A 70 micron cella-filtro viene utilizzato per filtrare la sospensione dissociato tessuto. Le cellule vengono quindi seminate in fiasche T75 (solitamente 2-3 embrioni possono seme uno beuta T75) e posta in un 37 ° C incubatore per colture cellulari. </li><li> Il giorno dopo, i media dei palloni T75 viene aspirato. Le beute vengono risciacquati con 10 ml di PBS. Supporti glia fresco (10-15 ml) viene poi aggiunto a ciascun contenitore. </li><li> Entro 10-12 giorni, glia nelle T75 flaconi devono essere confluenti. Cellule gliali sono lavate con PBS, e 5 ml di 0,05% tripsina viene aggiunto a ciascuna beuta T75. </li><li> Dopo incubazione tripsina a 37 ° C per 5 minuti, le cellule gliali sono cellule dissociate e gliali da ciascuna beuta T75 sono divisi in duediversi collagene rivestite T75 palloni. </li><li> Inserti Cultura (Millipore PICM03050) sono preparati ponendoli in piastre da 6 pozzetti, rivestimento con collagene, e aria di essiccazione durante la notte. Supporti glia (2 ml ciascuna) si trova sotto ogni inserto cultura. </li><li> Glia da confluenti T75 recipienti vengono tripsinizzate e seminate su gli inserti culturali (uno T75 pallone per piastra da 6 pozzetti, 2 ml ogni inserto). Questi inserti glia cultura saranno utilizzati per il condizionamento delle colture neuronali. </li></ol> 2. Cultura neuronale <ol><li> I vetrini per colture neuronali vengono puliti mediante immersione nel 70% HNO3 almeno una notte. Vetrini vengono quindi lavati con DDH 2 O almeno 3 volte, ed essiccato in un forno Isotemp (&gt; 200 ° C) per una notte. </li><li> Coprioggetti puliti sono posti in una piastra da 6 pozzetti, uno coprioggetto per pozzetto. Coprioggetti sono rivestite con poli-L-lisina soluzione (2 ml, 0,1% in 100 mM di acido borico, pH = 8,5) durante la notte in un incubatore a 37 ° C. </li><li> Ilpoli-L-lisina soluzione viene rimossa dai vetrini coprioggetto e vengono lavate 3 volte con autoclavato DDH 2 O. Coprioggetti vengono poi incubate a 37 ° C per una notte in terreni in piastra (Neurobasal supplementato con 5% di siero siero di cavallo, 10 unità / ml di penicillina, 10 mg / ml di streptomicina, Glutamax incubatore; 2% B27 supplemento viene aggiunto al mezzo di placcatura il giorno in cui viene utilizzato). </li><li> Il giorno seguente, i mezzi di placcatura viene rimosso e 2 ml di terreni in piastra freschi con B-27 viene aggiunto a ciascun pozzetto che ha un vetrino coprioggetto. </li><li> Topi in gravidanza (E15-18) sono sacrificati con CO 2, e topi embrionali sono eutanasia per decapitazione. L&#39;ippocampo o striato è sezionato dal tessuto cerebrale e posta in un tubo da 15 ml con tampone ghiacciato dissezione, per la coltura di neuroni ippocampali o neuroni striatali medium spiny, rispettivamente. </li><li> Tessuti cerebrali sezionati sono dissociate con papaina come descritto ai punti 1.4) e 1.5). </li><li> Fopo dissociazione, il numero di cellule in ciascuna sospensione neuronale è conteggiato con un emocitometro. Neuroni vengono quindi seminate su ciascun vetrino ad una densità di 0,4 x 10 6 cellule per pozzetto di una piastra da 6 pozzetti e coltivate in un incubatore a 37 ° C overnight (neuroni sono DIV 0). </li><li> Per preparare inserti per coltura glia condizionamento le colture neuronali, il supporto viene rimosso dal glia inserti di coltura glia e medie placcatura fresco è aggiunto (2 ml nell&#39;inserto e 2 ml sotto l&#39;inserto). </li><li> Il giorno successivo (DIV 1), coprioggetto con i neuroni sono posti sotto inserti di coltura glia, un vetrino in ciascun pozzetto. </li><li> I neuroni sono alimentati con la metà del volume di nuove pre-riscaldate supporti di alimentazione (con B-27) ogni 3-4 giorni fino a quando non sono pronti per esperimenti di trasfezione e di imaging. </li></ol> 3. Transfection <ol><li> Trasfezione neuronale viene eseguita utilizzando Lipofectamine 2000. Per ogni cultura neuronale (una cultura per vetrino), 2 microlitriLipofectamine 2000 e 1 pg di DNA vengono utilizzati. Fresco media Neurobasal senza supplemento viene utilizzato per diluire Lipofectamine e DNA. </li><li> DNA e Lipofectamine sono incubate per 20 minuti a temperatura ambiente dopo la miscelazione. </li><li> Salva 1 ml di mezzi di comunicazione da sotto ogni inserimento cultura glia, e 1 ml da dentro ogni inserimento, e trasferire i media a un tubo conico. Aggiungere lo stesso volume di mezzi di alimentazione + B27 al mezzo salvato e incubare a 37 ° C. Questo mezzo è utilizzato per colture neuronali dopo trasfezione. </li><li> Seguendo il 20-min Lipofectamine / DNA incubazione, inserti di coltura glia vengono rimossi momentaneamente dai piastre da 6 pozzetti. Il 200 pl Lipofectamine / DNA miscela viene aggiunta a ciascun coprioggetto con i neuroni. Gli inserti glia coltura vengono poi restituiti a ciascun pozzetto, sopra i neuroni. Generazione di bolle sotto la membrana di inserti di coltura deve essere evitato. Neuroni sono incubate con l&#39;Lipofectamine / DNA miscela a 37 ° C per 2-4 ore. </li><li> Dopo the 2-4 ore periodo di incubazione, inserti glia vengono rimossi di nuovo. I terreni di coltura con il Lipofectamine / DNA miscela viene rimosso da ogni pozzetto. I media salvati al passo 3.3) è aggiunto neuroni (2 ml per pozzetto). Inserti di coltura glia vengono restituiti a ciascun pozzetto, e il supporto glia di ogni inserto viene rimosso, e 2 ml del terreno di passo 3,3) viene aggiunto in ogni inserto. </li><li> I neuroni sono coltivate per ulteriori 24-72 ore per permettere l&#39;espressione di cDNA trasfettate prima TIRF immagini viene eseguita su questi neuroni. </li></ol> 4. TIRF Imaging <ol><li> Artificiale liquido cerebrospinale (aCSF: 119 mM NaCl, 2,5 mM KCl, 30 mM di glucosio, 2 mM CaCl 2, 1 mM MgCl 2, 25 mM HEPES, pH = 7,4) sarà utilizzato per vivo-imaging esperimenti. Il nostro sistema di imaging TIRF è personalizzato set-up con 100 mW Ciano laser per l&#39;eccitazione, e una carica dell&#39;elettrone Moltiplicando Coupled Device (EMCCD) fotocamera per un rivelatore. </li><li> Prima di esperimenti di imaging, il aCSF è avvertito in un 37 &amp; dad esempio, l&#39;acqua bagno C per almeno 30 minuti. L&#39;inserto di riscaldamento per l&#39;imaging in tempo reale viene posto sul piatto microscopio, e l&#39;inserto di riscaldamento, laser, e la fotocamera sono riscaldati per almeno 30 minuti prima dell&#39;inizio degli esperimenti di imaging. </li><li> Un coprioggetto con i neuroni trasfettati viene trasformata in live-imaging camera. Preriscaldata aCSF viene collocato nella camera dopo la camera è assemblato; questo passo dovrebbe essere completata il più rapidamente possibile. </li><li> Una volta che la camera è posizionato sull&#39;inserto riscaldamento sul palco microscopio, neuroni trasfettati sono identificati visivamente sotto epi-fluorescenza modalità di imaging. </li><li> Dopo sani neuroni trasfettati sono identificati (vedi figure 1 e 2), di solito effettuare 1 min di foto-candeggina per eliminare preesistente fluorescenza pHluorin sulla membrana plasmatica, come pHluorin recettori sulla membrana plasmatica hanno livelli di fluorescenza molto elevato quando TIRF immagini è appena partito, che interferisce con la visualizzazione dei singoli componentiertion eventi. </li><li> Dopo fotosbiancante, l&#39;impostazione del guadagno del moltiplicatore di elettroni sulla fotocamera EMCCD è impostato al massimo, e la registrazione viene eseguita per 1 min a 10 Hz. </li><li> Ogni registrazione conterrà 600 immagini. I singoli eventi di inserzione sono identificati giocando dietro registrazione con ImageJ. Eventi di inserimento individuali sono analizzati manualmente utilizzando ImageJ. </li></ol> 5. Risultati rappresentativi Coerentemente cultura neuronale è la chiave del successo dal vivo-imaging esperimenti. Figura 1 mostra i neuroni dell&#39;ippocampo di topo coltivate utilizzando il nostro protocollo da 11 a DIV DIV 18. E &#39;chiaro dalle immagini che i neuroni hanno sviluppato processi estensivi e che i processi dendritici sono coperte densamente di spine dendritiche, le indicazioni che questi neuroni sono sani di cultura. Nostro protocollo cultura potrebbe essere utilizzato anche per altri tipi di neuroni nel cervello. Ad esempio, abbiamo recentemente utilizzato questo protocollo alla culturastriatali neuroni spinosi medi in uno studio per esaminare recettore della dopamina D2 (DRD2) inserimento in colture di neuroni striatali medio del mouse spinosi 9. Figura 2 mostra il mouse medie neuroni spinosi striatali coltivate utilizzando il nostro protocollo a DIV 8. Abbiamo anche eseguito con successo TIRF imaging superecliptic DRD2s pHluorin-etichetta in questo tipo di neurone. Figura 3 mostra l&#39;espressione di pHluorin-etichettato DRD2 (pH-DRD2) in un neurone spinoso medio e visualizzazione di pH-DRD2 inserimento in questo neurone. <img alt="Figura 1" src="/files/ftp_upload/4450/4450fig1.jpg" /> Figura 1. Mouse cultura hippocampal utilizzando il metodo cultura dell&#39;interfaccia. DIV: giorni in vitro, il giorno della cultura è considerato come DIV 0. Dalle immagini, è evidente che i neuroni ippocampali maturi in questo tipo di coltura tra 11 e DIV DIV 15. A DIV 11 e 13, dendriti neuronali sono coperti di spine filipodia e immaturi. A 15 e DIV18, dendriti neuronali sono coperti di spine di grandi dimensioni, l&#39;indicazione di più neuroni maturi. I pannelli a sinistra: bassa ingrandimento immagini di neuroni ippocampali di topo di diverse età. Pannelli di destra: le immagini ad alto ingrandimento (zoom su dendriti neuronali del pannello di sinistra) di processi neuronali dendritiche con spine dendritiche di diverse età. <img alt="Figura 2" src="/files/ftp_upload/4450/4450fig2.jpg" /> Figura 2. Mouse striatale terreno spinoso neurone cultura utilizzando il metodo cultura dell&#39;interfaccia. I neuroni delle immagini sono in DIV 8. <img alt="Figura 3" src="/files/ftp_upload/4450/4450fig3.jpg" /> Figura 3. Un neurone striatale topo medio spinoso trasfettate con super eclittica pHluorin-tag del recettore D2 della dopamina (pH-DRD2). Immagine a sinistra è la proiezione massima intensità di un time-lapse (600 immagini, 100 msec per immagine). Frecce bianche indicano individuale inserimento vescicolare del pH-DRD2. Questa immagine conserva le informazioni di inserimento nella dimensione xy ma perde le informazioni sull&#39;ora. Immagine a destra mostra yt sporgenza massima intensità, in cui la pila XYT è ruotato di 90 gradi intorno all&#39;asse y, e il pixel massimo su ciascun asse x linea viene proiettata su un unico asse y pixel. Questa immagine mantiene informazioni inserimento lungo l&#39;asse yt ma perde l&#39;asse x informazioni.

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Per ragioni sconosciute, i neuroni di topo sono sempre più difficili da coltivare di neuroni di ratto. Nella nostra esperienza, una coltura mista di neuroni e glia funziona bene per primari neuroni di topo in coltura. Tuttavia, una tale coltura mista non è adatto per esperimenti di imaging TIRF, come in questo tipo di neuroni cultura ei processi tendono a crescere sopra cellule gliali, situando il somata neuronale e processi dendritici fuori dalla portata della microscopia TIRF. Pertanto, una densità minore colture n...

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imaging phluorin-tagged receptor insertion to the plasma membrane in primary cultured mouse neurons

Una migliore comprensione dei meccanismi che regolano il traffico tra il recettore di membrana plasmatica (PM) e compartimenti intracellulari richiede un approccio sperimentale con ottime risoluzioni spaziali e temporali. Inoltre, tale approccio deve inoltre essere in grado di distinguere recettori localizzati sulla PM da quelli in compartimenti intracellulari. Soprattutto, rilevando recettori in una vescicola singola richiede spiccata sensibilità di rilevamento, poiché ogni vescicola trasporta soltanto un piccolo numero di recettori. Approcci standard per l&#39;esame di traffico recettore includono biotinilazione superficie seguita da rilevamento biochimiche, che manca sia le necessarie risoluzioni spaziali e temporali, e l&#39;esame al microscopio a fluorescenza di recettori di superficie immunolabeled, che richiede la fissazione chimica delle cellule e quindi manca di sufficiente risoluzione temporale 1-6. Per superare questi limiti, noi e altri hanno sviluppato e utilizzato una nuova strategia che consente la visualizzazione di inserimento dinamico di recettori in PM con eccellenti risoluzioni spaziali e temporali 7-17. Il metodo comprende la codifica di un pH-sensibile GFP, la superecliptic pHluorin 18, al N-terminale del dominio extracellulare del recettore. Superecliptic pHluorin ha la proprietà unica di essere fluorescente a pH neutro e non fluorescente a pH acido (pH &lt;6.0). Pertanto, i recettori sono contrassegnati non fluorescente quando all&#39;interno del lume di vescicole acide traffico intracellulare o compartimenti endosomali, e diventano facilmente visualizzato solo quando esposta all&#39;ambiente extracellulare pH neutro, sulla superficie esterna del PM. La nostra strategia permette quindi di distinguere recettori di superficie PM da quelle che, nell&#39;ambito del traffico vescicole intracellulari. Per raggiungere sufficienti risoluzioni spaziali e temporali, nonché la sensibilità necessaria per studiare il traffico dinamica dei recettori, abbiamo impiegato interna totale rifletteremicroscopia a fluorescenza di ioni (TIRFM), che ci ha permesso di ottenere la risoluzione ottimale spaziale di imaging ottico (~ 170 nm), la risoluzione temporale di video-tasso di microscopia (30 fotogrammi / sec), e la sensibilità per rilevare la fluorescenza di GFP unico molecola. Con immagini pHluorin-tagged recettori sotto TIRFM, siamo stati in grado di visualizzare direttamente i singoli eventi recettore inserimento nel PM in neuroni in coltura. Questo approccio di imaging può potenzialmente essere applicato a qualsiasi proteina di membrana con un dominio extracellulare che potrebbe essere etichettato con pHluorin superecliptic, e consentirà dissezione dei meccanismi principali modalità per l&#39;inserimento di differenti proteine ​​di membrana (recettori, canali ionici, trasportatori, ecc) per il PM.

Watch this Scientific Journal Video about Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons at JoVE.com

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

Etichettando i domini extracellulari dei recettori di membrana con pHluorin superecliptic, e da imaging questi recettori di fusione in neuroni di topo in coltura, si può visualizzare direttamente i singoli eventi di inserzione vescicolari dei recettori della membrana plasmatica. Questa tecnica sarà determinante per chiarire i meccanismi molecolari che regolano l&#39;inserimento recettore alla membrana plasmatica.

Imaging pHluorin-tagged inserimento recettore alla membrana plasmatica nella Primaria neuroni mouse coltivate

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

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Neuroscience

13.1K Views.  The Jackson Laboratory. Etichettando i domini extracellulari dei recettori di membrana con pHluorin superecliptic, e da imaging questi recettori di fusione in neuroni di topo in coltura, si può visualizzare direttamente i singoli eventi di inserzione vescicolari dei recettori della membrana plasmatica. Questa tecnica sarà determinante per chiarire i meccanismi molecolari che regolano l'inserimento recettore alla membrana plasmatica.

Video: Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

Multiple-topo Imaging neuroanatomici Risonanza Magnetica

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Ricerca

Neuroscienze

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

Analisi quantitativa di Replenishment pool delle vescicole sinaptiche in colture di neuroni granuli cerebellari utilizzando coloranti FM

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Inducing Dendritic Growth in Cultured Sympathetic Neurons

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs 4-6. Altered patterns of dendritic growth and plasticity are associated with impaired neurobehavioral function in experimental models 7, and are thought to contribute to clinical symptoms observed in both neurodevelopmental disorders 8-10 and neurodegenerative diseases 11-13. Such observations underscore the functional importance of precisely regulating dendritic morphology, and suggest that identifying mechanisms that control dendritic growth will not only advance understanding of how neuronal connectivity is regulated during normal development, but may also provide insight on novel therapeutic strategies for diverse neurological diseases.
Mechanistic studies of dendritic growth would be greatly facilitated by the availability of a model system that allows neurons to be experimentally switched from a state in which they do not extend dendrites to one in which they elaborate a dendritic arbor comparable to that of their in vivo counterparts. Primary cultures of sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rodents provide such a model. When cultured in defined medium in the absence of serum and ganglionic glial cells, sympathetic neurons extend a single process which is axonal, and this unipolar state persists for weeks to months in culture 14,15. However, the addition of either bone morphogenetic protein-7 (BMP-7) 16,17 or Matrigel 18 to the culture medium triggers these neurons to extend multiple processes that meet the morphologic, biochemical and functional criteria for dendrites. Sympathetic neurons dissociated from the SCG of perinatal rodents and grown under defined conditions are a homogenous population of neurons 19 that respond uniformly to the dendrite-promoting activity of Matrigel, BMP-7 and other BMPs of the decapentaplegic (dpp) and 60A subfamilies 17,18,20,21. Importantly, Matrigel- and BMP-induced dendrite formation occurs in the absence of changes in cell survival or axonal growth 17,18.
Here, we describe how to set up dissociated cultures of sympathetic neurons derived from the SCG of perinatal rats so that they are responsive to the selective dendrite-promoting activity of Matrigel or BMPs.

We describe a protocol for using bone morphogenetic protein-7 (BMP-7) or Matrigel to selectively induce dendritic growth in primary sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rats.

Indurre crescita dendritica in colture di neuroni simpatici

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.
Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination6-10. 
FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application8,10. 
We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This 'FRAP-FLIP' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines10, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

Diffusione laterale e esocitosi delle proteine ​​di membrana in neuroni in coltura valutata utilizzando Recovery fluorescenza e fluorescenza-loss photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

Slice Cervello biotinilazione: An<em&gt; Ex Vivo</em&gt; Approccio alla Misura Regione-specifica proteina Tratta plasma membrana in neuroni adulti

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

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 ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

Utilizzando metodologie combinati per definire il ruolo della membrana plasmatica di consegna Durante Axon ramificazione e neuronale morfogenesi

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

Modellazione di morte neuronale e degenerazione in neuroni cerebellari del granello primario del Mouse

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus in the central nervous system (CNS) is the control center for energy and insulin signal response regulation. Chronic inflammation in peripheral tissues and imbalances of certain chemokines (such as CCL5, TNF&#945;, and IL-6) contribute to diabetes and obesity. However, the functional mechanism(s) connecting chemokines and hypothalamic insulin signal regulation still remain unclear.
In vitro primary neuron culture models are convenient and simple models which can be used to investigate insulin signal regulation in hypothalamic neurons. In this study, we introduced exogeneous GLUT4 protein conjugated with GFP (GFP-GLUT4) into primary hypothalamic neurons to track GLUT4 membrane translocation upon insulin stimulation. Time-lapse images of GFP-GLUT4 protein trafficking were recorded by deconvolution microscopy, which allowed users to generate high-speed, high-resolution images without damaging the neurons significantly while conducting the experiment. The contribution of CCR5 in insulin regulated GLUT4 translocation was observed in CCR5 deficient hypothalamic neurons, which were isolated and cultured from CCR5 knockout mice. Our results demonstrated that the GLUT4 membrane translocation efficiency was reduced in CCR5 deficient hypothalamic neurons after insulin stimulation.

This protocol describes a technique for observation of real-time Green Fluorescence Protein (GFP) tagged Glucose Transporter 4 (GLUT4) protein trafficking upon insulin stimulation and characterization of the biological role of CCR5 in the insulin&#8211;GLUT4 signaling pathway with Deconvolution Microscopy.

Immagini in diretta della proteina GLUT4 tratta di neuroni ipotalamici primario del Mouse usando la microscopia di Deconvolution

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model system is to provide a controlled microenvironment and maintain the high survival rate and the natural features of dissociated neuronal and nonneuronal cells as much as possible under in vitro conditions. In this article, we demonstrate a method of isolating primary neurons from the developing mouse cerebellum, placing them in an in vitro environment, establishing their growth, and monitoring their viability and differentiation for several weeks. This method is applicable to embryonic neurons dissociated from cerebellum between embryonic days 12&ndash;18.

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

Cultura primaria dei neuroni isolata dal cerbellello del topo embrionale

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model ...