Nous tenons à remercier Yue (Pauline) Hu, Rachel Sinit pour leur assistance technique. L&#39;étude est soutenue par de subvention du NIH (PN2 EY016525) et par le financement de la recherche et du syndrome de Down Treatment Foundation et la Fondation Larry L. Hillblom.

Les interventions chirurgicales et animales sont réalisées dans le strict respect de la NIH Guide pour le soin et l&#39;utilisation des animaux de laboratoire. Toutes les expériences impliquant l&#39;utilisation d&#39;animaux sont approuvés par l&#39;UCSD institutionnel de protection des animaux et l&#39;utilisation Comité. 1. plasmide de clonage, expression et purification de Mono-biotinylé BDNF (mBtBDNF) REMARQUE: construire pré-proBDNFavi et BirA ADNc dans le vecteur pcDNA3.1 et coexprimer dans les cellules HEK293FT 10. Purifiez mBtBDNF utilisant des billes de Ni-NTA selon la méthode précédemment publiée de la production du facteur de croissance nerveuse monobiotinylé mature et biologiquement active (mBtNGF) 10. 2 Préparation de microfluidiques Chambers Dispositif microfluidique neurone de culture permet d&#39;isoler fluidiquement axones des corps cellulaires des neurones. Assemblez chambres avec lamelles fraîchement enduites juste avant chaque dissection. Chambres microfluidiques utilisésdans ce protocole sont achetés dans le commerce (voir matériaux et la table de l&#39;équipement). Lavage à la main et réutilisés chambres achetés dans le commerce jusqu&#39;à 5-6x. <ol><li> Lavage à la main des chambres microfluidiques en 1% Alconox. Rincer trois fois avec de l&#39;eau Milli-Q pendant 30 min chacun. chambres à nouveau dans 70% d&#39;éthanol de lavage des mains. </li><li> Disposez chambres à sécher sur du parafilm dans la hotte à flux laminaire. Irradier et stériliser les deux côtés des chambres de 20 minutes sous UV. Magasin chambres stérilisée en 15 cm plat stérile scellée avec du parafilm à la température ambiante. </li><li> Placer 24 x 40 mm n ° 1 des lamelles de verre dans un récipient en verre. Tremper les lamelles de 35% de l&#39;acide chlorhydrique nuit sur un agitateur. Le jour suivant, les lamelles rincer trois fois pendant 30 minutes chacune avec de l&#39;eau. </li><li> Stériliser les lamelles couvre-objet, un par un en trempant chaque lamelle couvre-objet dans de l&#39;éthanol à 100% et plus de flammes d&#39;un brûleur Bunsen. Rangez les lamelles sèches dans une boîte de Pétri stérile et le laisser à température ambiante jusqu&#39;à ce que le revêtement. </li><li> Poser oust les lamelles dans une boîte de culture de 15 cm. Manteau de chaque lamelle avec 0,7 ml de 0,01% de poly-L-lysine (PLL) et incuber à la hotte à température ambiante. </li><li> Après 1 heure, rincez les lamelles à trois reprises avec de l&#39;eau stérile. Lamelles sèches avec vide et le lieu de chaque lamelle dans une boîte de culture de 6 cm. </li><li> Pour assembler la chambre microfluidique, placez la chambre à côté microsillon en bas sur la lamelle enduit PLL avec prudence pour ne pas toucher les microsillons. Doucement pressé vers le bas dans la chambre avec une pointe de pipette afin d&#39;assurer la chambre est hermétiquement scellée. </li></ol> 3. Dissection neuronale Culture et plaque sur les chambres <ol><li> Placez deux hippocampes E17-E18 rat (un cerveau) disséqués dans un tube conique de 15 ml contenant 2 ml de tampon de dissection (HBSS, sans calcium, pas de magnésium, avec 1% stylo / angine et HEPES 10 mM. Rincer les tissus 3x avec 5 ml dissection tampon à chaque fois. Retirez le tampon de dissection autant que possible et ajouter 900 ul de di fraisssection tampon. </li><li> Pour digérer le tissu, ajouter 100 pl de 10x la trypsine (2,5%) dans le tampon de dissection à faire 1x concentration de travail. Placer le tube conique dans un bain d&#39;eau à 37 C °. Après 10 minutes de digestion, ajouter 100 ul de 10 mg / ml de DNase I à une concentration finale d&#39;environ 1 mg / ml. </li><li> En utilisant une pipette en verre de Pasteur polie au feu, triturer doucement les tissus par aspiration et refoulement 5-10x. Juste après trituration, étancher la trypsine avec 2 ml de placage milieu Neurobasal (avec 10% de FBS, 2 mM de Glutamax, 2% de B27). </li><li> Laisser l&#39;échantillon dans la hotte pendant 5 minutes pour permettre à tous les débris des tissus de s&#39;installer au fond. Retirez délicatement 2 ml de surnageant dans un tube de 15 ml conique stérile et centrifuger à 200 g pendant 5 min pour sédimenter les cellules. Reprendre le culot dans 50 ul placage médias </li><li> Compter les cellules avec un hémocytomètre. Charge de 15 à 20 ul de suspension cellulaire (~ 40 000 cellules) dans un compartiment de la chambre microfluidique. Placer la chambredans l&#39;incubateur pendant 10 min pour permettre aux cellules de se fixer à la lamelle. Après 10 min, ajouter des médias plus placage de remplir les deux compartiments de la chambre. </li><li> Le deuxième jour de la dissection, remplacer complètement les milieux d&#39;étalement avec support de maintien (Neurobasal avec Glutamax 2 mM, 2% de B27) à la fois dans le corps de la cellule et le compartiment de l&#39;axone. Les axones des neurones de l&#39;hippocampe commencent à traverser les microsillons dans les jours 3 et atteindre le compartiment axone entre le jour 5-7. Au cours de cette période de temps, remplacer la moitié du milieu de culture avec des milieux d&#39;entretien frais tous les 24 ~ 48 h. </li></ol> 4. axonale Transport de QD-BDNF <ol><li> Avant de vivre l&#39;imagerie de QD-BDNF transport axonal, épuiser BDNF à la fois du corps cellulaire et l&#39;axone compartiments de la chambre microfluidique par rincer soigneusement les deux compartiments avec BDNF-libres, du milieu sans sérum Neurobasal toutes les 30 min pendant 2 h. </li><li> Au cours de BDNF épuisement, préparer les conjugués QD-BDNF. Mélanger 50 nM de mono-biotinylé BDNF dimère avec 50 nM conjugués QD655-streptavidine dans les médias Neurobasal et incuber sur de la glace pendant 60 min. </li><li> Retirez le support dans le compartiment axone et ajouter 300 ul QD-BDNF avec une concentration finale de 0,25 nM pendant 4 heures à 37 ° C. Pour réduire au minimum le diffuseur de QD-BDNF dans le compartiment du corps de la cellule, il est très important de maintenir toujours un niveau supérieur de support dans le compartiment du corps de la cellule que dans le compartiment de l&#39;axone. Laver non lié QD-BDNF après incubation avant l&#39;imagerie en direct. </li><li> Faire de l&#39;imagerie en temps réel des transports QD-BDNF l&#39;aide d&#39;un microscope inversé équipé d&#39;une huile 100X objectif. Chauffer le cadre et la chambre de l&#39;environnement qui s&#39;y rattachent à une température constante (37 ° C) et CO 2 (5%). Utilisez un ensemble de Texas cubes rouge d&#39;excitation / émission de visualiser le signal de QD655. </li><li> Acquérir et capturer des images en accéléré dans les axones moyen à la vitesse de 1 image / s pour un total de 2 min à l&#39;aide d&#39;une caméra CCD. Utilisez microsillons sans axones that n&#39;ont pas QD et donc aucun signal de contrôle pour l&#39;infiltration. </li><li> Analyser le transport du BDNF utilisant n&#39;importe quel logiciel d&#39;analyse d&#39;image ou NIH ImageJ. </li></ol>

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Aucun conflit d&#39;intérêt déclaré.

Dans cette étude, nous rapportons le développement d&#39;une nouvelle technique pour produire biologiquement active, monobiotinylé BDNF (mBtBDNF) qui peut être utilisé pour tracer le transport axonal de BDNF. En conjuguant la protéine de quantum dot de la streptavidine, et en utilisant une chambre microfluidique, le procédé permet de détecter le transport axonal de BDNF dans les neurones primaires avec sensibilité molécule unique, en temps réel et avec une résolution spatiale et temporelle. Les outils utili...

Les neurones sont des cellules dont la très processus long et souvent très élaborés sont fondamentales pour établir et maintenir la structure et la fonction des circuits neuronaux polarisés. L&#39;axone joue un rôle essentiel dans le transport de cargaisons à destination et à partir de synapses. Les protéines et organites synthétisés dans le corps cellulaire doivent être transportés à travers les axones pour atteindre la terminaison présynaptique pour soutenir la fonction neuronale. De même, les signaux reçus à axones distaux doivent être transduites et transmis au soma. Ces processus sont essentiels pour la survie neuronale, la différenciation et la maintenance. Dans ce transport axonal dans certains neurones doit être effectuée sur des distances de plus de 1 000 fois le diamètre du corps de la cellule, la possibilité est envisagée facilement que même de petites déficits pourraient avoir une incidence nettement la fonction neuronale et circuit. Brain-derived neurotrophic factor (BDNF), un membre de la famille des neurotrophines de facteurs de croissance, est présent en marégions du cerveau ny, y compris l&#39;hippocampe, le cortex cérébral, et cerveau antérieur basal. BDNF joue un rôle crucial dans la cognition et la mémoire formation en soutenant la survie, la différenciation et la fonction des neurones qui participent à des circuits cognitifs. BDNF se lie à son récepteur, le TrkB tyrosine kinase, à la terminaison axonale où il active les voies de signalisation TrkB médiée dont le mitogène protéine kinase activée / extracellulaire de la protéine kinase régulée par un signal (MAPK / ERK), le phosphatidylinositol-3-kinase (PI3K) et la phospholipase C-gamma (PLCy). Les protéines qui participent à ces voies de signalisation sont conditionnés sur des structures vésiculaires endocytose pour former l&#39;endosome 1-6, qui sont ensuite transportées de façon rétrograde vers le soma neuronal signalisation BDNF / TrkB. La chambre de culture microfluidique est une plate-forme très utile pour étudier la biologie des axones dans les conditions normales, ainsi que dans le cadre des lésions et des maladies 7,8. Par isoler les axonesde corps cellulaires, le dispositif a permis d&#39;étudier un transport spécifiquement dans les axones 8-10. Les plates-formes microfluidiques PDMS base avec 450 um barrières microsillons utilisées dans cette étude ont été achetés dans le commerce (voir le tableau des matériaux). Pour d&#39;étudier le transport du BDNF, nous avons développé une nouvelle technologie pour produire monobiotinylé BDNF (mBtBDNF). Nous avons profité du peptide biotine accepteur, AP (également connu sous le nom AviTag). Il s&#39;agit d&#39;une séquence d&#39;acides 15 aminés qui contient un résidu de lysine qui peut être spécifiquement lié à un par la biotine ligase coli Escherichia enzyme biotine, BirA. Nous avons fusionné la AviTag à l&#39;extrémité C-terminale de la souris pré-proBDNF ADNc par PCR (figure 1A). La construction a été clonée dans le vecteur d&#39;expression de mammifère, pcDNA3.1-myc son vecteur. Nous avons également cloné l&#39;ADN BirA bactérienne dans le pCDNA3.1 myc son vecteur. Les deux plasmides ont été transitoirement co-transfectées dans des cellules HEK293FT d&#39;exprimer les deux protéines. BirA catalysé la ligature de Bioten particulier de la lysine résident dans le AviTag à l&#39;extrémité C-terminale de BDNF dans un rapport de 1: 1 pour produire monobiotinylé monomère BDNF. Biotinylé, BDNF matures avec une masse moléculaire de ~ 18 kDa a été récupéré et purifié à partir du support à l&#39;aide de résine Ni-(Figure 1C). La biotinylation du BDNF a été terminée, comme jugé par l&#39;incapacité à détecter BDNF non modifié par immunoblot (Figure 1D). Streptavidine points quantiques conjugués, QD 655, ont été utilisés pour étiqueter mBtBDNF faire QD-BDNF. La présence de la AviTag n&#39;interfère pas avec l&#39;activité de BDNF comme le mBtBDNF était capable d&#39;activer TrkB phosphorylée (figure 1E) et de stimuler la croissance des neurites (figure 1F), dans la mesure du BDNF humain recombinant (rhBDNF). Immunocoloration montré que QD-BDNF colocalisé avec TrkB dans les axones de l&#39;hippocampe, ce qui indique que QD-BDNF est bioactif (figure 1G). Pour étudier le transport du BDNF, QD-BDNF a été ajouté à distale compartiment axone d&#39;cultures microfluidiques contenant rat E18 neurones de l&#39;hippocampe (figure 2A). QD-BDNF transport rétrograde dans les axones a été capturé par imagerie en temps réel en direct de l&#39;étiquette fluorescente rouge (Soutien vidéos de S1, S2). En analysant le produit kymographe, QD-BDNF a été observée pour être transportée de manière rétrograde, à une vitesse de l&#39;ordre de 1,06 um / sec (figure 3A) en mouvement. GFP ou mCherry étiqueté BDNF ont été utilisés pour suivre le mouvement des axones de BDNF. Les principaux inconvénients sont qu&#39;ils ne sont pas assez brillant pour les études de molécules uniques. En outre, la présence à la fois antérograde et rétrograde BDNF mouvements, il est difficile d&#39;évaluer si oui ou non le BDNF a été transportée de manière rétrograde dans un complexe neurotrophine / récepteur. Dans cette vidéo, nous démontrons les techniques utilisées pour examiner le trafic en direct de QD-BDNF dans des chambres microfluidiques utilisant neurones primaires. Le ultrabrightness et une excellente photostabilité de points quantiques makes permettant d&#39;effectuer un suivi à long terme du transport du BDNF. Ces techniques peuvent être exploitées pour améliorer les études de la fonction axonale dans AD, HD, et d&#39;autres maladies neurodégénératives.

<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 joue un rôle important dans plusieurs aspects de la survie neuronale, la différenciation et la fonction. Les déficits structurels et fonctionnels dans les axones sont de plus en plus considérés comme une caractéristique précoce des maladies neurodégénératives, dont la maladie d&#39;Alzheimer (MA) et la maladie de Huntington (HD). Ne sait pas encore, c&#39;est le mécanisme (s) par lequel des lésions axonales est induite. Nous avons signalé le développement d&#39;une nouvelle technique pour produire biologiquement active, monobiotinylé BDNF (mBtBDNF) qui peut être utilisé pour tracer le transport axonal de BDNF. Quantum dot marqué BDNF (QD-BDNF) a été produit par la conjugaison de points quantiques 655 à mBtBDNF. Un dispositif microfluidique a été utilisé pour isoler les axones des corps cellulaires des neurones. Ajout de QD-BDNF dans le compartiment axonal permis imagerie en temps réel du transport du BDNF dans les axones. Nous avons démontré que QD-BDNF déplacé essentiellement exclusivement rétrograde, avec très peu de pauses, à une vitesse de l&#39;ordre de 1,06 um / s en mouvement. Ce système peut être utilisé pour enquêter sur moicanismes de la fonction axonale perturbé dans AD ou HD, ainsi que d&#39;autres maladies dégénératives.

Production et purification de biologiquement active BDNF Mono-biotinylé Le vecteur d&#39;expression de BDNF fusionnée avec une séquence AviTag (GGGLNDIFEAQKIEWHE) a été réalisé selon un protocole précédemment publié 10. La masse moléculaire de la protéine de fusion pleine longueur a été prévue pour être ~ 32 kDa ( <a href="http://ca.expasy.org/tools/pi_tool.html" target="_blank">http://ca.expasy.org/tools/pi_tool.html</a> ) monobiotinylé BDNF mûr avec une masse m...

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

Le transport axonal de BDNF, un facteur neurotrophique, est essentiel pour la survie et la fonction de plusieurs populations neuronales. Certaines maladies dégénératives sont marqués par la rupture de la structure et de la fonction axonale. Nous avons démontré les techniques utilisées pour examiner le trafic en direct de QD-BDNF dans des chambres microfluidiques utilisant neurones primaires.

Imagerie en temps réel des axonale Transport de Quantum Dot marqué BDNF dans les neurones primaires

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 vues.  University of California, San Diego.  Le transport axonal de BDNF, un facteur neurotrophique, est essentiel pour la survie et la fonction de plusieurs populations neuronales. Certaines maladies dégénératives sont marqués par la rupture de la structure et de la fonction axonale. Nous avons démontré les techniques utilisées pour examiner le trafic en direct de QD-BDNF dans des chambres microfluidiques utilisant neurones primaires. 

Vidéo: 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.

Manipulation mécanique des neurones à contrôler le développement axonale

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

Recherche

Neurosciences

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.

Neuromodulation et mitochondriale de transport: Live in Imaging neurones de l&#39;hippocampe sur des durées longues

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.

Une<em&gt; Ex Vivo</em&gt; Induite par laser Spinal Cord Injury modèle pour évaluer les mécanismes de la dégénérescence axonale dans en temps réel

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 ...

A Protocol for the Administration of Real-Time fMRI Neurofeedback Training

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control neuroplastic processes and correct abnormal function, or even shift functions from damaged tissue to physiologically healthy brain regions, hold the potential to dramatically improve overall health. Of the current neuroplastic interventions in development, neurofeedback training (NFT) from functional Magnetic Resonance Imaging (fMRI) has the advantages of being completely non-invasive, non-pharmacologic, and spatially localized to target brain regions, as well as having no known side effects. Furthermore, NFT techniques, initially developed using fMRI, can often be translated to exercises that can be performed outside of the scanner without the aid of medical professionals or sophisticated medical equipment. In fMRI NFT, the fMRI signal is measured from specific regions of the brain, processed, and presented to the participant in real-time. Through training, self-directed mental processing techniques, that regulate this signal and its underlying neurophysiologic correlates, are developed. FMRI NFT has been used to train volitional control over a wide range of brain regions with implications for several different cognitive, behavioral, and motor systems. Additionally, fMRI NFT has shown promise in a broad range of applications such as the treatment of neurologic disorders and the augmentation of baseline human performance. In this article, we present an fMRI NFT protocol developed at our institution for modulation of both healthy and abnormal brain function, as well as examples of using the method to target both cognitive and auditory regions of the brain.

The ability to induce and/or control neural plasticity may be critical in future treatments for neurologic disorders and the recovery from brain injury. In this paper, we present a protocol on the use of neurofeedback training with functional magnetic resonance imaging to modulate human brain function.

Un protocole pour l’Administration de la Real-Time IRMf formation Neurofeedback

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control ...

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.

Essai pour mesurer le transport des nucléocytoplasmes en temps réel dans les cellules NSC-34 de type Neuron moteur

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.

Visualisation du modèle de projection axonale des neurones du moteur embryonnaire dans<em&gt; Drosophila</em

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

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.

Transport axonal des Organlles dans les cultures de neurones moteurs à l’aide du système de chambres microfluidiques

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.

Cartographie cérébrale IRMf en temps réel chez les animaux

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.

Élargir la boîte à outils pour l’imagerie in vivo du transport axonal

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 Imagerie calcique d’ensembles neuronaux dans des réseaux de neurones sensoriels primaires dans des ganglions radiculaires dorsaux intacts

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