Vorremmo ringraziare Yue (Pauline) Hu, Rachel Sinit per la loro assistenza tecnica. Lo studio è supportato da NIH concedere (PN2 EY016525) e da un finanziamento di Down Syndrome Research Foundation e il trattamento e la Larry L. Hillblom Fondazione.

Le procedure chirurgiche e animali sono eseguite rigorosamente secondo la guida NIH per la cura e l&#39;uso di animali da laboratorio. Tutti gli esperimenti che comportano l&#39;uso di animali sono approvati dal UCSD Institutional Animal Care and Use Committee. 1 plasmidi clonazione, espressione e purificazione di Mono-biotinilato BDNF (mBtBDNF) NOTA: Construct pre-proBDNFavi e Bira cDNA in pcDNA3.1 vettoriale e coesprimere nelle cellule HEK293FT 10. Purificare mBtBDNF utilizzando Ni-NTA perline secondo il metodo precedentemente pubblicata di produrre fattore maturo e biologicamente attiva di crescita nervoso monobiotinylated (mBtNGF) 10. 2 Preparazione di microfluidici Chambers Microfluidic dispositivo neurone coltura rende possibile isolare fluidicamente assoni provenienti da corpi cellulari dei neuroni. Montare camere con vetrini rivestiti di fresco giusto prima di ogni dissezione. Camere di microfluidica usatein questo protocollo sono regolarmente acquistati (vedi Materiali e tabella di attrezzature). Lavare a mano e riutilizzati camere di commercio acquistati fino a 5-6x. <ol><li> Lavaggio a mano camere di microfluidica in 1% Alconox. Risciacquare tre volte con acqua Milli-Q per 30 minuti ciascuno. Camere di lavaggio a mano di nuovo in etanolo al 70%. </li><li> Stendere camere ad asciugare su Parafilm in cappa a flusso laminare. Irradiare e sterilizzare entrambi i lati delle camere per 20 minuti sotto UV. Conservare sterilizzato camere in un piatto 15 centimetri sterile sigillato con Parafilm a temperatura ambiente. </li><li> Mettere 24 x 40 mm N. 1 vetrini in un contenitore di vetro. Mettere a bagno i coprioggetti in 35% acido cloridrico durante la notte su un rotatore. Il giorno seguente, sciacquare i vetrini tre volte per 30 minuti ciascuno con acqua. </li><li> Sterilizzare i coprioggetti uno per uno per immersione ogni coprioggetto in 100% etanolo e fiammeggiante su un becco Bunsen. Conservare coprioggetto secco in una capsula di Petri sterile e tenerlo a temperatura ambiente fino al rivestimento. </li><li> Lay out i coprioggetti in un piatto di coltura 15 cm. Coat ogni coprioggetto con 0,7 ml di 0,01% di poli-L-lisina (PLL) e incubare nel cappuccio a temperatura ambiente. </li><li> Dopo 1 ora, sciacquare i vetrini tre volte con acqua sterile. Coprioggetto secco con vuoto e posto ogni coprioggetto in un piatto di cultura 6 cm. </li><li> Per assemblare la camera microfluidica, posizionare la camera con il lato microsolchi in basso sul coprioggetto rivestito PLL con cautela per non toccare i microsolchi. Premuto delicatamente verso il basso la camera con un puntale per garantire la camera è stata ermeticamente chiusi. </li></ol> 3. Dissezione di Cultura neuronale e piastra su Chambers <ol><li> Posizionare due ippocampi E17-E18 ratto (un cervello) sezionato in un tubo da 15 ml contenente 2 ml di tampone di dissezione (HBSS, no calcio, no magnesio, con 1% pen / strep e HEPES 10 mM. Sciacquare i tessuti 3x con 5 ml dissezione tampone ogni volta. Rimuovere il buffer dissezione per quanto possibile e aggiungere 900 ml di di frescotampone ssection. </li><li> Per digerire il tessuto, aggiungere 100 ml di tripsina 10x (2,5%) nel buffer dissezione per rendere concentrazione di lavoro 1x. Posizionare il tubo conico in un bagno di 37 ° C Acque. Dopo 10 min di digestione, aggiungere 100 ml di 10 mg / ml di DNasi I a una concentrazione finale di circa 1 mg / ml. </li><li> Usando una pipetta Pasteur di vetro lucidati a fuoco, triturare delicatamente i tessuti pipettando su e giù 5-10x. Subito dopo triturazione, placare la tripsina con 2 ml di placcatura medio (Neurobasal con 10% FBS, 2 Glutamax mM, 2% B27). </li><li> Lasciare il campione nella cappa per 5 minuti per consentire eventuali detriti dei tessuti di stabilirsi sul fondo. Rimuovere con cautela 2 ml di surnatante in una sterile tubo da 15 ml pulito e centrifugare a 200 xg per 5 minuti per far sedimentare le cellule. Risospendere il pellet in 50 microlitri mezzi placcatura </li><li> Contare le cellule con emocitometro. Carico 15-20 microlitri di sospensione cellulare (~ 40.000 cellule) in uno scomparto della camera microfluidica. Posizionare la camerain incubatrice per 10 minuti per consentire alle cellule di allegare alla coprioggetto. Dopo 10 minuti, aggiungere i media più in piastra per riempire entrambi i comparti della camera. </li><li> Il secondo giorno della dissezione, sostituire completamente i terreni in piastra con i media manutenzione (Neurobasal con 2 Glutamax mM, 2% B27), sia nel corpo cellulare e il vano assone. Gli assoni dei neuroni dell&#39;ippocampo iniziano a varcare i microsolchi in giorno 3 e raggiungere il vano assone tra il giorno 5-7. Durante questo periodo di tempo, sostituire la metà dei mezzi di coltura con terreno di mantenimento fresco ogni 24 ~ 48 ore. </li></ol> 4 assonale Trasporto di QD-BDNF <ol><li> Prima di vivere l&#39;imaging di QD-BDNF trasporto assonale, esaurire BDNF sia dal corpo cellulare e assone compartimenti della camera microfluidica da sciacquare entrambi i comparti con BDNF-liberi, siero media liberi Neurobasal ogni 30 min per 2 ore. </li><li> Durante BDNF esaurimento, preparare i coniugati QD-BDNF. Mescolare 50 nM di mono-biotinilato BDNF dimero con 50 Nm coniugati QD655-streptavidina nei media Neurobasal e incubare in ghiaccio per 60 min. </li><li> Rimuovere il supporto nel vano assone e aggiungere 300 microlitri QD-BDNF con una concentrazione finale di 0,25 nM per 4 ore a 37 ° C. Per ridurre al minimo la diffusione del QD-BDNF nel vano corpo cellulare, è molto importante mantenere sempre un livello superiore di supporto nel vano corpo cellulare che nel vano assone. Lavare non legato QD-BDNF dopo incubazione prima di imaging dal vivo. </li><li> Eseguire l&#39;imaging dal vivo di trasporto QD-BDNF utilizzando un microscopio invertito dotato di un olio lente obiettivo 100X. Scaldare la portata e la camera ambientale collegato ad esso per una temperatura costante (37 ° C) e CO 2 (5%). Utilizzare un set di Texas cubi rossi di eccitazione / emissione di visualizzare il segnale QD655. </li><li> Acquisire e catturare immagini time-lapse all&#39;interno dei assoni centrali alla velocità di 1 fotogramma / sec per un totale di 2 min con una camera CCD. Utilizzare microscanalature senza assoni that non hanno QD e quindi nessun segnale di come controllo per l&#39;infiltrazione. </li><li> Analizzare il trasporto BDNF utilizzando qualsiasi software di analisi dell&#39;immagine o NIH ImageJ. </li></ol>

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Nessun conflitto di interessi dichiarati.

In questo studio, riportiamo lo sviluppo di una nuova tecnica per produrre biologicamente attivo, monobiotinylated BDNF (mBtBDNF) che può essere utilizzato per tracciare il trasporto assonale di BDNF. Con coniugando la proteina di quantum dot streptavidina, e l&#39;utilizzo di una camera microfluidica, il metodo permette di rilevare il trasporto assonale di BDNF in neuroni primari con sensibilità singola molecola, in tempo reale e con risoluzioni spaziali e temporali. Gli strumenti utilizzati in questo documento forni...

I neuroni sono cellule altamente polarizzate i cui processi lunghi e spesso altamente elaborati sono fondamentali per stabilire e mantenere la struttura e la funzione dei circuiti neurali. L&#39;assone svolge un ruolo fondamentale nel portare carichi da e sinapsi. Le proteine ​​e organelli sintetizzati nel soma cellulare devono essere trasportati attraverso gli assoni per raggiungere il terminale presinaptico per supportare la funzione neuronale. Di conseguenza, i segnali ricevuti assoni distali devono essere trasdotte e convogliati al soma. Questi processi sono essenziali per la sopravvivenza neuronale, la differenziazione e la manutenzione. In tale trasporto assonale in alcuni neuroni deve essere condotta attraverso distanze più di 1.000 volte il diametro del corpo cellulare, la possibilità è facilmente previsto che anche i piccoli deficit potrebbero avere un impatto marcatamente funzione neuronale e del circuito. Brain-derived neurotrophic factor (BDNF), un membro della famiglia dei fattori di crescita neurotrofina, è presente in maregioni cerebrali ny, tra cui l&#39;ippocampo, la corteccia cerebrale, e prosencefalo basale. BDNF svolge un ruolo cruciale nella formazione conoscenza e la memoria sostenendo la sopravvivenza, la differenziazione e la funzione dei neuroni che partecipano a circuiti cognitivi. BDNF si lega al suo recettore, il TrkB tirosina chinasi, al terminale dell&#39;assone dove attiva vie di segnalazione TrkB-mediata, tra cui la proteina chinasi attivata da mitogeni / extracellulare proteina chinasi segnale-regolata (MAPK / ERK), fosfatidilinositolo-3-chinasi (PI3K) e fosfolipasi C-gamma (PLCγ). Le proteine ​​che partecipano a queste vie di segnalazione sono confezionati su strutture vescicolari endocitiche per formare il BDNF / TrkB segnalazione endosome 1-6 che vengono poi retrograda trasportato al soma neuronale. La camera di coltura microfluidica è una piattaforma molto utile per studiare biologia assonale in condizioni normali sia in ambito di lesioni e malattia 7,8. Con assoni isolantidai corpi cellulari, il dispositivo ha permesso di studiare un trasporto specificatamente assoni 8-10. Le piattaforme basate microfluidica PDMS con 450 micron barriere microsolchi utilizzati in questo studio sono stati acquistati in commercio (vedi tabella Materials). Per esaminare i trasporti BDNF, abbiamo sviluppato una nuova tecnologia per la produzione di monobiotinylated BDNF (mBtBDNF). Abbiamo approfittato del peptide biotina accettore, AP (noto anche come AviTag). Si tratta di una sequenza di acido 15 aminoacidi che contiene un residuo di lisina che possono essere specificamente ligato ad un biotina dal coli ligasi enzima biotina Escherichia, Bira. Abbiamo fuso il AviTag al C-terminale del mouse pre-proBDNF cDNA mediante PCR (Figura 1A). Il costrutto è stato clonato nel vettore di espressione di mammifero, pcDNA3.1-myc suo vettore. Abbiamo anche clonato il DNA batterico Bira nel pcDNA3.1-myc suo vettore. I due plasmidi sono stati transitoriamente co-trasfettate in cellule HEK293FT di esprimere entrambe le proteine. Bira catalizzata la legatura di Biotin particolare per la lisina risiedere nel AviTag al C-terminale di BDNF in un rapporto 1: 1 per produrre monobiotinylated BDNF monomero. Biotinilato, maturo BDNF con una massa molecolare di circa 18 kDa è stato recuperato e purificato dal supporto utilizzando Ni-resina (Figura 1C). La biotinilazione di BDNF era completa, come giudicato dalla incapacità di rilevare non modificato BDNF mediante immunoblotting (Figura 1D). Streptavidina punti quantici coniugati QD 655, sono stati usati per etichettare mBtBDNF per fare QD-BDNF. La presenza del AviTag non interferisce con l&#39;attività di BDNF come mBtBDNF era in grado di attivare TrkB fosforilata (Figura 1E) e stimolare la crescita dei neuriti (Figura 1F) nella misura del BDNF umano ricombinante (rhBDNF). Immunostaining dimostrato che QD-BDNF colocalizzava con TrkB in assoni ippocampali, indicando che QD-BDNF è bioattivo (Figura 1G). Per studiare il trasporto BDNF, QD-BDNF è stato aggiunto al compartimento assonale distale diculture microfluidica contenenti ratto E18 neuroni dell&#39;ippocampo (Figura 2A). QD-BDNF trasporto retrogrado all&#39;interno di assoni è stato catturato da in tempo reale di immagini dal vivo del tag fluorescente rossa (supporto video S1, S2). Analizzando il chimografo generato, QD-BDNF è stato osservato per essere trasportato retrograda ad una velocità di movimento intorno 1,06 micron / sec (Figura 3A). GFP o mCherry-tagged BDNF sono stati utilizzati per tracciare il movimento assonale di BDNF. I principali svantaggi sono che non sono abbastanza luminose per gli studi di singola molecola. Inoltre, la presenza di entrambi anterograda e movimenti retrogradi BDNF rende difficile valutare se il BDNF retrograda trasportato era in un complesso neurotrofina / recettore. In questo video, dimostriamo le tecniche utilizzate per esaminare il traffico in tempo reale di QD-BDNF in camere di microfluidica con neuroni primari. Il ultrabrightness ed eccellente fotostabilità dei punti quantici makes &#39;possibile effettuare il monitoraggio a lungo termine del trasporto BDNF. Queste tecniche possono essere sfruttati per migliorare gli studi di funzione assonale in AD, HD, e altre patologie neurodegenerative.

<table border="0" cellpadding="0" cellspacing="0" width="641">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:261px;">Name</td>
			<td style="width:205px;">Company</td>
			<td style="width:175px;">Catalog Number</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Platinum pfx DNA polymerase&#160;</td>
			<td>Invitrogen</td>
			<td>11708021</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EcoRI&#160;</td>
			<td>Fermentas</td>
			<td>FD0274</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BamHI&#160;</td>
			<td>Fermentas</td>
			<td>FD0054</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEK293FT cells</td>
			<td>Invitrogen</td>
			<td>R70007</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM-high glucose media</td>
			<td>Mediatech</td>
			<td>10-013-CV</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">d-biotin&#160;</td>
			<td>Sigma</td>
			<td>B4639</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TurboFect&#160;</td>
			<td>Fermentas</td>
			<td>R0531</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PMSF&#160;&#160;</td>
			<td>Sigma</td>
			<td>P7626</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">aprotinin</td>
			<td>Sigma</td>
			<td>A6279</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ni-NTA resins</td>
			<td>Qiagen</td>
			<td>30250</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">protease inhibitors cocktail</td>
			<td>Sigma</td>
			<td>&#160;S8820</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">silver staining kit&#160;</td>
			<td>G-Biosciences</td>
			<td>786-30</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">human recombinant BDNF</td>
			<td>Genentech</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microfluidic chambers</td>
			<td>Xona</td>
			<td>SND450</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">24x40 mm No. 1 glass coverslips&#160;</td>
			<td>VWR</td>
			<td>48393-060</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">poly-L-Lysine&#160;</td>
			<td>Cultrex</td>
			<td>3438-100-01</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HBSS</td>
			<td>Gibco</td>
			<td>14185-052</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNase I</td>
			<td>Roche</td>
			<td>10104159001</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin</td>
			<td>Gibco</td>
			<td>15090-046</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Neurobasal&#160;</td>
			<td>Gibco</td>
			<td>21103-049</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FBS&#160;</td>
			<td>Invitrogen</td>
			<td>16000-044</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GlutaMax&#160;</td>
			<td>Invitrogen</td>
			<td>35050-061</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B27&#160;&#160;</td>
			<td>Gibco</td>
			<td>17504-044</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QD655-streptavidin conjugates</td>
			<td>Invitrogen</td>
			<td>&#160;Q10121MP</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-Avi tag antibody</td>
			<td>GenScript</td>
			<td>A00674</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">streptavidin-agarose beads&#160;</td>
			<td>Life Technology</td>
			<td>&#160;SA100-04</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">trichloroacetic acid</td>
			<td>Sigma</td>
			<td>T6399</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HRP-streptavidin&#160;</td>
			<td>Thermo Scientific</td>
			<td>N100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-pTrkB antibody</td>
			<td colspan="2">a generous gift from Dr M. Chao of NYU</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-TrkB antibody</td>
			<td>BD Science</td>
			<td>610101</td>
		</tr>
	</tbody>
</table>

real-time imaging of axonal transport of quantum dot-labeled bdnf in primary neurons

BDNF svolge un ruolo importante in molti aspetti della sopravvivenza neuronale, la differenziazione e la funzione. Deficit strutturali e funzionali in assoni sono visti sempre più come una caratteristica precoce di malattie neurodegenerative, tra cui il morbo di Alzheimer (AD) e la malattia di Huntington (HD). Per quanto ancora poco chiaro è il meccanismo (s) di cui danno assonale è indotta. Abbiamo riportato lo sviluppo di una nuova tecnica per produrre biologicamente attivo, monobiotinylated BDNF (mBtBDNF) che può essere utilizzato per tracciare il trasporto assonale di BDNF. Quantum dot-marcato BDNF (QD-BDNF) è stato prodotto da coniugando quantum dot 655 a mBtBDNF. Un dispositivo di microfluidica è stato utilizzato per isolare gli assoni da corpi cellulari dei neuroni. L&#39;aggiunta di QD-BDNF al compartimento assonale consentito l&#39;imaging dal vivo di trasporto BDNF negli assoni. Abbiamo dimostrato che QD-BDNF spostato in sostanza esclusivamente retrograda, con pochissime pause, ad una velocità di spostamento di circa 1,06 micron / sec. Questo sistema può essere usato per studiare mechanisms di perturbato funzione assonale in AD o HD, così come altre malattie degenerative.

Produzione e purificazione di biologicamente attivi BDNF Mono-biotinilato Il vettore di espressione di BDNF fuso con una sequenza AviTag (GGGLNDIFEAQKIEWHE) è stato creato secondo un protocollo già pubblicato 10. La massa molecolare della proteina di lunghezza completa di fusione è stato previsto per essere ~ 32 kDa ( <a href="http://ca.expasy.org/tools/pi_tool.html" target="_blank">http://ca.expasy.org/tools/pi_tool.html</a> ) Monobiotinylated BDNF maturo con una massa molecol...

Watch this Scientific Journal Video about Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons at JoVE.com

Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons

Il trasporto assonale di BDNF, un fattore neurotrofico, è fondamentale per la sopravvivenza e la funzione di diverse popolazioni neuronali. Alcune malattie degenerative sono contrassegnate dalla rottura della struttura e funzione assonale. Abbiamo dimostrato le tecniche utilizzate per esaminare il traffico in tempo reale di QD-BDNF in camere di microfluidica con neuroni primari.

In tempo reale Imaging del trasporto assonale di Quantum Dot-etichettato BDNF in neuroni primari

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are ...

real-time-imaging-axonal-transport-quantum-dot-labeled-bdnf-primary

Research

JoVE Journal

Neuroscience

15.0K Views.  University of California, San Diego.  Il trasporto assonale di BDNF, un fattore neurotrofico, è fondamentale per la sopravvivenza e la funzione di diverse popolazioni neuronali. Alcune malattie degenerative sono contrassegnate dalla rottura della struttura e funzione assonale. Abbiamo dimostrato le tecniche utilizzate per esaminare il traffico in tempo reale di QD-BDNF in camere di microfluidica con neuroni primari. 

Video: Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons

Mechanical Manipulation of Neurons to Control Axonal Development

Cell manipulations and extension of neuronal axons can be accomplished with calibrated glass micro-fibers capable of measuring and applying forces in the 10-1000 &mu;dyne range1,2. Force measurements are obtained through observation of the Hookean bending of the glass needles, which are calibrated by a direct and empirical method3. Equipment requirements and procedures for fabricating, calibrating, treating, and using the needles on cells are fully described. The force regimes previously used and different cell types to which these techniques have been applied demonstrate the flexibility of the methodology and are given as examples for future investigation4-6. The technical advantages are the continuous 'visualization' of the forces produced by the manipulations and the ability to directly intervene in a variety of cellular events. These include direct stimulation and regulation of axonal growth and retraction7; as well as detachment and mechanical measurements on any type of cultured cell8.

Application and direct measurements of forces on neurons in the 2-1000 microdyne range are achieved with high precision using calibrated glass needles. This methodology can be used to control and measure several aspects of axonal development, including axonal initiation, axonal tension, velocity of axonal elongation, and force vectors.

Manipolazione meccanica di neuroni di controllo per lo sviluppo assonale

Cell manipulations and extension of neuronal axons can be accomplished with calibrated glass micro-fibers capable of measuring and applying forces in the ...

Ricerca

Neuroscienze

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

Neuromodulazione e mitocondriale di trasporto: Imaging Live in neuroni dell&#39;ippocampo oltre durate a lungo

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

Un<em&gt; Ex Vivo</em&gt; Laser-indotta Spinal Cord Injury modello per valutare i meccanismi di degenerazione assonale in tempo reale

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a connection between amyotrophic lateral sclerosis (ALS) and impairments in the nucleocytoplasmic pathway. ALS is a neurodegenerative disease affecting the motor neurons and resulting in paralysis and ultimately in death, within 2-5 years on average. Most cases of ALS are sporadic, lacking any apparent genetic linkage, but 10% are inherited in a dominant manner. Recently, hexanucleotide repeat expansions (HREs) in the chromosome 9 open reading frame 72 (C9orf72) gene were identified as a genetic cause of ALS and frontotemporal dementia (FTD). Importantly, different groups have recently proposed that these mutants affect nucleocytoplasmic transport. These studies have mostly shown the final outcome and manifestations caused by HREs on nucleocytoplasmic transport, but they do not demonstrate nuclear transport dysfunction in real time. As a result, only severe nucleocytoplasmic transport deficiency can be determined, mostly due to high overexpression or exogenous protein insertion. 
This protocol describes a new and very sensitive assay to evaluate and quantify nucleocytoplasmic transport dysfunction in real time. The rate of import of a NLS-NES-GFP protein (shuttle-GFP) can be quantified in real time using fluorescent microscopy. This is performed by using an exportin inhibitor, thus allowing the shuttle GFP only to enter the nucleus. To validate the assay, the C9orf72 HRE translated dipeptide repeats, poly(GR) and poly(PR), which have been previously shown to disrupt nucleocytoplasmic transport, were used. Using the described assay, a 50% decrease in the nuclear import rate was observed compared to the control. Using this system, minute changes in nucleocytoplasmic transport can be examined and the ability of different factors to rescue (even partially) a nucleocytoplasmic transport defect can be determined.

This protocol describes a method to sensitively measure the nucleocytoplasmic transport rate within motor neuron-like NSC-34 cells by quantifying the real-time change in the nuclear import of a NLS-NES-GFP protein.

Analisi per misurare il trasporto nucleocitoplasmatico in tempo reale all&#39;interno di motori neuronici come le cellule NSC-34

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a ...

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons extend growth cones to navigate along specific pathways by responding to a large number of axon guidance cues that emanate from the surrounding extracellular environment. Growth cone target recognition also plays a critical role in neuromuscular specificity. This work presents a standard immunohistochemistry protocol to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. This protocol includes a few key steps, including a genotyping procedure, to sort the desired mutant embryos; an immunostaining procedure, to tag embryos with fasciclin II (FasII) antibody; and a dissection procedure, to generate filleted preparations from fixed embryos. Motor axon projections and muscle patterns in the periphery are much better visualized in flat preparations of filleted embryos than in whole-mount embryos. Therefore, the filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition, and it can also be applied to both loss-of-function and gain-of-function genetic screens.

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

This work details a standard immunohistochemistry method to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. The filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition during neural development.

Visualizzazione del modello di proiezione Axonal dei motoni embrionali in<em&gt; Drosophila</em

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

Etichettatura balistica dei neuroni piramidali nelle sezioni cerebrali e nella cultura cellulare primaria

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...

Axonal Transport of Organelles in Motor Neuron Cultures using Microfluidic Chambers System

Motor neurons (MNs) are highly polarized cells with very long axons. Axonal transport is a crucial mechanism for MN health, contributing to neuronal growth, development, and survival. We describe a detailed method for the use of microfluidic chambers (MFCs) for tracking axonal transport of fluorescently labeled organelles in MN axons. This method is rapid, relatively inexpensive, and allows for the monitoring of intracellular cues in space and time. We describe a step by step protocol for: 1) Fabrication of polydimethylsiloxane (PDMS) MFCs; 2) Plating of ventral spinal cord explants and MN dissociated culture in MFCs; 3) Labeling of mitochondria and acidic compartments followed by live confocal imagining; 4) Manual and semiautomated axonal transport analysis. Lastly, we demonstrate a difference in the transport of mitochondria and acidic compartments of HB9::GFP ventral spinal cord explant axons as a proof of the system validity. Altogether, this protocol provides an efficient tool for studying the axonal transport of various axonal components, as well as a simplified manual for MFC usage to help discover spatial experimental possibilities.

Axonal transport is a crucial mechanism for motor neuron health. In this protocol we provide a detailed method for tracking the axonal transport of acidic compartments and mitochondria in motor neuron axons using microfluidic chambers.

Trasporto assonale di Organanelle nelle colture del motoneurone utilizzando il sistema di camere microfluidiche

Motor neurons (MNs) are highly polarized cells with very long axons. Axonal transport is a crucial mechanism for MN health, contributing to neuronal ...

Real-Time fMRI Brain Mapping in Animals

Dynamic fMRI responses vary largely according to the physiological conditions of animals either under anesthesia or in awake states. We developed a real-time fMRI platform to guide experimenters to monitor fMRI responses instantaneously during acquisition, which can be used to modify the physiology of animals to achieve desired hemodynamic responses in animal brains. The real-time fMRI set-up is based on a 14.1T preclinical MRI system, enabling the real-time mapping of dynamic fMRI responses in the primary forepaw somatosensory cortex (FP-S1) of anesthetized rats. Instead of a retrospective analysis to investigate confounding sources leading to the variability of fMRI signals, the real-time fMRI platform provides a more effective scheme to identify dynamic fMRI responses using customized macro-functions and a common neuroimage analysis software in the MRI system. Also, it provides immediate troubleshooting feasibility and a real-time biofeedback stimulation paradigm for brain functional studies in animals.

Animal brain functional mapping can benefit from the real-time functional magnetic resonance imaging (fMRI) experimental set-up. Using the latest software implemented in the animal MRI system, we established a real-time monitoring platform for small animal fMRI.

Mappatura cerebrale fMRI in tempo reale negli animali

Dynamic fMRI responses vary largely according to the physiological conditions of animals either under anesthesia or in awake states. We developed a ...

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Axonal transport maintains neuronal homeostasis by enabling the bidirectional trafficking of diverse organelles and cargoes. Disruptions in axonal transport have devastating consequences for individual neurons and their networks, and contribute to a plethora of neurological disorders. As many of these conditions involve both cell autonomous and non-autonomous mechanisms, and often display a spectrum of pathology across neuronal subtypes, methods to accurately identify and analyze neuronal subsets are imperative.
This paper details protocols to assess in vivo axonal transport of signaling endosomes and mitochondria in sciatic nerves of anesthetized mice. Stepwise instructions are provided to 1) distinguish motor from sensory neurons in vivo, in situ, and ex vivo by using mice that selectively express fluorescent proteins within cholinergic motor neurons; and 2) separately or concurrently assess in vivo axonal transport of signaling endosomes and mitochondria. These complementary intravital approaches facilitate the simultaneous imaging of different cargoes in distinct peripheral nerve axons to quantitatively monitor axonal transport in health and disease.

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Using transgenic fluorescent mice, detailed protocols are described to assess in vivo axonal transport of signaling endosomes and mitochondria within motor and sensory axons of the intact sciatic nerve in live animals.

Espansione del toolkit per l'imaging in vivo del trasporto assonale

Axonal transport maintains neuronal homeostasis by enabling the bidirectional trafficking of diverse organelles and cargoes. Disruptions in axonal ...

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the cytoplasm or the release of intracellular Ca2+ stores. Pirt-GCaMP3-based Ca2+ imaging of primary sensory neurons of the dorsal root ganglion (DRG) in mice offers the advantage of simultaneous measurement of a large number of cells. Up to 1,800 neurons can be monitored, allowing neuronal networks and somatosensory processes to be studied as an ensemble in their normal physiological context at a populational level in vivo. The large number of neurons monitored allows the detection of activity patterns that would be challenging to detect using other methods. Stimuli can be applied to the mouse hindpaw, allowing the direct effects of stimuli on the DRG neuron ensemble to be studied. The number of neurons producing Ca2+ transients as well as the amplitude of Ca2+ transients indicates sensitivity to specific sensory modalities. The diameter of neurons provides evidence of activated fiber types (non-noxious mechano vs. noxious pain fibers, A&#946;, A&#948;, and C fibers). Neurons expressing specific receptors can be genetically labeled with td-Tomato and specific Cre recombinases together with Pirt-GCaMP. Therefore, Pirt-GCaMP3 Ca2+ imaging of DRG provides a powerful tool and model for the analysis of specific sensory modalities and neuron subtypes acting as an ensemble at the populational level to study pain, itch, touch, and other somatosensory signals.

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

This protocol describes the surgical exposure of the dorsal root ganglion (DRG) followed by GCaMP3 (genetically-encoded Ca2+ indicator; Green Fluorescent Protein-Calmodulin-M13 Protein 3) Ca2+ imaging of the neuronal ensembles using Pirt-GCaMP3 mice while applying a variety of stimuli to the ipsilateral hind paw.

In vivo Imaging del calcio di insiemi neuronali in reti di neuroni sensoriali primari nei gangli della radice dorsale intatti

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the ...