Ce travail a été financé par NINDS et l&#39;American Heart Association à S.Ge. Cette approche a été initialement créé et développé par l&#39;auteur dans le laboratoire du Dr Hongjun Song à l&#39;Université Johns Hopkins. Nous tenons à remercier le Dr Hongjun chanson pour ses conseils et de soutien.

1. Injection du virus Haute-titrage des rétrovirus modifiés (1 × 10 9 unités / ml) est produite par co-transfection des vecteurs rétroviraux et VSVG en cellules HEK293T, suivie par ultracentrifugation du surnageant viral. Pour les sources et méthodes de production, voir la démonstration JoVE excellente (3). Remarque: Les jeunes adultes (4-6 semaines) femelle souris C57Bl / 6 (Charles River) sont logés dans des conditions standard. Toutes les procédures à suivre le Guide du Conseil national de recherches pour le soin et l&#39;utilisation des animaux de laboratoire selon un protocole approuvé par le IACUC l&#39;Université Stony Brook. <ol><li> 2UL Dégel des rétrovirus gelés par animal sur la glace. </li><li> Anesthésier (100 mg de kétamine + 10 xylazine ug par gramme de poids corporel) et monter l&#39;animal sur un cadre stéréotaxique (Steolting). Enlever les cheveux sur la tête et essuyer la peau avec de l&#39;éthanol à 70%. </li><li> Exposer le crâne et percez quatre trous peu profonds à l&#39;aide d&#39;une fraise dentaire (0,6 mm foret) aux coordonnées suivantes: <ul><li> anterioposterior = -2 mm du bregma; latérale = ± 1,6 mm; ventrale = 2,5 mm; anterioposterior = -3 mm de bregma; latérale = ± 2,6 mm; ventrale = 3,2 mm. </li><li> anterioposterior = -2 mm du bregma; latérale = ± 1,6 mm; ventrale = 2,5 mm; anterioposterior = -3 mm de bregma; latérale = ± 2,6 mm; ventrale = 3,2 mm. </li></ul></li><li> Monter une Hamilton 1 microlitre, plate-bout de la seringue et injecter 0,5 rétrovirus ul par site à un taux de 0,25 l / min dans le gyrus denté. (Voir les coordonnées ci-dessus pour la profondeur ventrale.) Pause 2 minutes après chaque injection avant retirant lentement la seringue pour empêcher le retour du liquide. Entre les injections, brièvement rincer la surface externe de l&#39;extrémité de la seringue avec du PBS stérile et un applicateur pour enlever les traces de sang. </li><li> Fermez la plaie avec du matériel de suture chirurgical stérile (type P-1; Taille 6-0) et le retour de l&#39;animal aux conditions de logement standard jusqu&#39;à l&#39;heure désirée stades après l&#39;injection. </li></ol> 2. Préparation Trancher Pour obtenir de haute qualité à partir des tranches aiguë chez la souris adulte, nous utilisons une solution de découpe modifiés pour la préparation de tranche (voir ci-dessous). <ol><li> Solution de coupe pré-froid à 0-4 ° C sur la glace et la bulle avec 95% d&#39;O 2 -5% CO 2 pendant au moins 30 minutes. </li><li> Anesthésier et transcardiaque perfuser un animal avec glacée saturée en oxygène solution de découpe. </li><li> Enlever rapidement le cerveau dans une boîte de Petri avec du papier-filtre pré-mouillé avec le froid, la solution de découpe oxygénée. Couper la tranche de cervelet et une surface de montage d&#39;au moins 1mm antérieure à l&#39;injection de coordonnées (visibles). Collez le cerveau sur une scène vibrotome et le monter dans la chambre de coupe remplie de la solution de découpe à froid et oxygéné. </li><li> Préparer les tranches coronales ou horizontale (300-350μm) et les transférer avec une pipette de gros calibre à une chambre d&#39;incubation à température ambiante contenant ACSF barbotage avec 95% d&#39;O 2 / 5% CO 2. </li><li> Incuber les coupes, bouillonne en permanence, à 32 ° C pendant 30-60 minutes. Retour dans la chambre à température ambiante pendant la durée de l&#39;enregistrement. </li></ol> 3. Des expériences électrophysiologiques <ol><li> Les tranches sont transférées dans la chambre d&#39;enregistrement qui est perfusée en continu avec de l&#39;oxygène saturé ACSF à 32 ° C - 34 ° C. </li><li> Une électrode de stimulation bipolaire de tungstène est placé dans la couche moléculaire du gyrus denté en utilisant un objectif de microscope à faible grossissement (10X). </li><li> DGCS nouveau-né dans la zone sous-granulaire / couche de cellules granulaires sont identifiés par l&#39;expression de la GFP ou dTomato. L&#39;enregistrement patchclamp cellule entière est effectuée sur les nouveaux neurones à fort grossissement (40x). </li><li> Propriétés de cuisson des cellules enregistrées sont obtenues par injection d&#39;une série de courants sous-courants pince mode. </li><li> Stimuli électriques sont livrés par l&#39;électrode de stimulation par l&#39;intermédiaire d&#39;un isolateur stimulation contrôlée par le logiciel d&#39;enregistrement, et évoqué les courants postsynaptiques dans la DGCS-nés sont enregistrés. </li></ol> 4. Analyse des données Amplitudes des réponses évoquées postsynaptiques sont analysés à différents stades de développement de l&#39;adulte née neurones. 5. Les résultats représentatifs En utilisant le protocole ci-dessus, l&#39;expression de GFP dans les neurones nés gyrus denté semblable à la figure 1 est commune. Notez que les nouveaux neurones sont facilement visualisés et deux dendrites et les axones sont solidement étiquetés. Expression en fines axones GCR et de petites épines dépend de la qualité et le titre du virus, le promoteur utilisé et la longueur de l&#39;expression in vivo. Dans nos mains, les cellules-nés et sub-cellulaire des organites sont habituellement visibles en quelques jours après l&#39;injection, la colonne vertébrale et d&#39;expression peut être tracée dès les premiers stades de développement. Wtrou de cellules à partir d&#39;enregistrements nouveaux neurones à des stades différents du temps permettent l&#39;étude des propriétés uniques de cellules nouveau-né, par exemple, des potentiels d&#39;action (figure 2a), ainsi que le processus d&#39;intégration synaptique dans les circuits neuronaux existants lors de la maturation (Figure 2b). <img src="/files/ftp_upload/2548/2548fig1.jpg" alt="Figure 1" /> Figure 1 à 2 photons image confocale de nouveaux neurones dans une section horizontale gyrus denté d&#39;une souris adulte. Cellules vertes sont 21dpi (jours après l&#39;injection) exprimant la GFP DGCS nés étiquetés avec Pux-GFP rétrovirus. <img src="/files/ftp_upload/2548/2548fig2.jpg" alt="Figure 2" /> Figure 2 a, b) du potentiel d&#39;action tirs propriétés d&#39;un nouveau-né à 21 DGC dpi.) Représentant a évoqué excitateurs courants postsynaptiques (EPSCs) enregistrées sur une GCR-né à 21 dpi.

<ol>
	<li>Zhao, C., Deng, W., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+and+functional+implications+of+adult+neurogenesis.">Mechanisms and functional implications of adult neurogenesis.</a> Cell. 132, 645-660 (2008).</li><li>Ge, S., Goh, E. L., Sailor, K. A., Kitabatake, Y., Ming, G. L., Song, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA+regulates+synaptic+integration+of+newly+generated+neurons+in+the+adult+brain.">GABA regulates synaptic integration of newly generated neurons in the adult brain.</a> Nature. 439, 589-593 (2006).</li><li>Gavrilescu, L. C., Van Etten, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+replication-defective+retrovirus+by+transient+transfection+of+293T+cells.">Production of replication-defective retrovirus by transient transfection of 293T cells.</a> J Vis Exp. , (2007).</li></ol>

Neurogenèse dans l&#39;hippocampe continue des adultes cerveau des mammifères fournit un système modèle expérimental unique pour étudier le développement et l&#39;intégration neuronale dans le cerveau mature. Le protocole présenté ici associe l&#39;étiquetage rétroviraux et aux méthodes électrophysiologiques pour étudier l&#39;intégration des nouveaux-nés neurones granulaires denté dans les cerveaux de souris adultes. Afin de s&#39;assurer que vos expériences sont réussies, nous recommandons ce qui suit au cours des étapes critiques de la procédure: Pour éviter l&#39;infection et l&#39;inflammation indésirables, tous les outils doivent être stérilisés (par autoclavage, tout type de stérilisateur, ou d&#39;éthanol à 70%) avant la chirurgie. Utiliser du PBS stérile pour laver la pointe de l&#39;aiguille de la seringue avant l&#39;injection. Pour l&#39;injection du virus, les animaux doivent être bien monté sur le dispositif de stéréotaxie et les sources de mouvements inutiles doivent être minimisés pendant l&#39;injection. Assurez-vous que la tête est bien fixe et ferme, et ajuster la position du nez de la souris au niveau de bregma et lambda avant l&#39;injection du calcul des coordonnées. Extrait du virus dans la seringue rapidement pour minimiser l&#39;exposition à des températures ambiantes - les rétrovirus sont particulièrement sensibles à la température. Injecter lentement le virus à la dose recommandée pour minimiser les dégâts de pression. Utiliser le recommande à bout plat seringue (vs commune embout biseauté) assurera une répartie secteur denté infection gyrus. Avant de préparer les tranches, solution de découpe et l&#39;incubation doit être ACSF barbotage avec 95% de CO 2 O 2 -5% pendant au moins 30 minutes pour permettre à l&#39;oxygène pour saturer les solutions. Les animaux doivent être perfusés rapidement et tissus maintenus dans le froid, saturée en oxygène solution pour la meilleure qualité tranche. Pour l&#39;enregistrement, l&#39;augmentation de l&#39;intensité du stimulus progressivement jusqu&#39;à ce qu&#39;il y est une réponse postsynaptique. Changer l&#39;emplacement de l&#39;électrode de stimulation dans la couche moléculaire du gyrus denté, si nécessaire. En résumé, l&#39;approche recommandée offre un moyen d&#39;explorer les propriétés distinctes et possible des rôles fonctionnels des adultes-né neurones au cours du stade précoce intégration active, circuits matures.

<ul><li> Solution de découpe (en mm): 110 chlorure de choline, 2,5 KCl, 1,3 KH 2 PO 4, 25,0 NaHCO 3, 0,5 CaCl2, MgCl2 7, 10 dextrose, ascorbate de sodium 1,3, 0,6 pyruvate de sodium, l&#39;acide 5,0 kynurénique. </li><li> Artificial liquide céphalo-rachidien (ACSF, en mm): 125,0 NaCl, 25,0 NaHCO 3, 2,5 KCl, CaCl 2 2, 1,3 KH 2 PO 4, 1,3 MgCl 2, 1,3 ascorbate de sodium, 0,6 pyruvate de sodium, 10 dextrose. </li><li> Solution interne pour la cellule entière patchclamp (en mm): 120 gluconate de potassium, KCl 15, 4 MgCl 2, 0,1 EGTA, HEPES 10,0, 4 MgATP, 0,3 Na 3 GTP, 7 phosphocréatine (pH 7,4, à 300 mOsm). </li></ul>

studying the integration of adult-born neurons

Neurogenèse adulte se produit dans le cerveau des mammifères dans la zone sous-ventriculaire (SVZ) des ventricules latéraux et dans la zone sous-granulaire (SGZ) du gyrus dentelé hippocampique long de la vie. Des rapports antérieurs ont montré que la neurogenèse hippocampique adulte est associée à des troubles cérébraux divers, y compris l&#39;épilepsie, la schizophrénie, la dépression et l&#39;anxiété (1). Décrypter le processus d&#39;intégration des neurones normaux et aberrants pour adultes-né peut faire la lumière sur l&#39;étiologie de ces maladies et d&#39;informer le développement de nouvelles thérapies. Neurogenèse adulte SGZ miroirs embryonnaire et post-natal du développement neuronal, y compris les étapes de la spécification destin, la migration, l&#39;intégration synaptique, et la maturation. Cependant, l&#39;intégration complète se produit sur une période prolongée, de 6 semaines période. Initial entrée synaptique à l&#39;adulte-né cellules granulaires SGZ denté (DGCS) est GABAergique, suivie par l&#39;entrée glutamatergique moins 14 jours (2). Les facteurs spécifiques qui réglementent formation des circuits de l&#39;adulte-né neurones dans le gyrus denté sont actuellement inconnus. Notre laboratoire utilise une réplication déficiente vecteur rétroviral basé sur le virus de la leucémie murine de Moloney pour livrer des protéines fluorescentes et de l&#39;hypothèse des gènes de régulation de ces cellules proliférantes. Cette technique fournit une haute spécificité virale et la résolution pour l&#39;analyse de la date de naissance de cellules, le lignage, la morphologie et la synaptogenèse. Une expérience typique emploie souvent deux ou trois des virus contenant étiquette unique, transgène, et les éléments de promoteur pour analyse unicellulaire d&#39;un processus de développement souhaité in vivo. Le protocole suivant décrit une méthode d&#39;analyse fonctionnelle d&#39;intégration neurone né avec un seul verte (GFP) ou rouge (dTomato) rétrovirus protéine fluorescente et de patch-clamp électrophysiologie.

Watch this Scientific Journal Video about Studying the Integration of Adult-born Neurons at JoVE.com

Studying the Integration of Adult-born Neurons

Une façon d&#39;étudier l&#39;intégration des nouveaux-nés des cellules granulaires denté chez les animaux adultes est décrite. Cette technique utilise un rétrovirus conçu pour étiqueter les nouveaux neurones, suivie par des enregistrements électrophysiologiques de déterminer à l&#39;intégration fonctionnelle in vivo.

Etudier l&#39;intégration des adultes-né Neurones

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the ...

studying-the-integration-of-adult-born-neurons

Research

JoVE Journal

Neuroscience

13.7K vues.  State University of New York at Stony Brook. Une façon d'étudier l'intégration des nouveaux-nés des cellules granulaires denté chez les animaux adultes est décrite. Cette technique utilise un rétrovirus conçu pour étiqueter les nouveaux neurones, suivie par des enregistrements électrophysiologiques de déterminer à l'intégration fonctionnelle in vivo.

Vidéo: Studying the Integration of Adult-born Neurons

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

In utero électroporation suivie par la culture neuronale primaire pour étudier la fonction des gènes dans un sous-ensemble des neurones corticaux

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

Recherche

Neurosciences

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons4,5. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

Sélective transduction virale des adultes-né pour les neurones olfactifs chronique In vivo Stimulation optogénétique

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The differentiation process is initiated by breaking the human PSCs into clumps which round up to form aggregates when the cells are placed in a suspension culture. The aggregates are then grown in hESC medium from days 1-4 to allow for spontaneous differentiation. During this time, the cells have the capacity to become any of the three germ layers. From days 5-8, the cells are placed in a neural induction medium to push them into the neural lineage. Around day 8, the cells are allowed to attach onto 6 well plates and differentiate during which time the neuroepithelial cells form. These neuroepithelial cells can be isolated at day 17. The cells can then be kept as neurospheres until they are ready to be plated onto coverslips. Using a basic medium without any caudalizing factors, neuroepithelial cells are specified into telencephalic precursors, which can then be further differentiated into dorsal telencephalic progenitors and glutamatergic neurons efficiently. Overall, our system provides a tool to generate human glutamatergic neurons for researchers to study the development of these neurons and the diseases which affect them.

This procedure yields telencephalic neurons by going through checkpoints which are similar to those observed during human development. The cells are allowed to spontaneously differentiate, are exposed to factors which push them towards the neural lineage, are isolated, and are plated onto coverslips to allow for terminal differentiation and maturation.

La spécification des neurones glutamatergiques télencéphaliques de cellules souches pluripotentes humaines

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

Rapide Micro-iontophorèse de glutamate et le GABA: un outil utile d&#39;étudier l&#39;intégration synaptique

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of live neurons with a specific primary antibody raised against an extracellular portion of the protein. Given that receptors are trafficked to and from the surface, if cells are permeabilized after fixation then both cell-surface and internal protein will be detected by the same labeled secondary antibody. Here, we adapted a method used to study protein trafficking (&#8220;antibody feeding&#8221;) to differentially label protein that had been internalized by endocytosis during the antibody incubation step and protein that either remained on the cell surface or was trafficked to the surface during this period. The ability to distinguish these two pools of protein was made possible through the incorporation of an overnight blocking step with highly-concentrated unlabeled secondary antibody after an initial incubation of unpermeabilized neurons with a fluorescently-labeled secondary antibody. After the blocking step, permeabilization of the neurons allowed detection of the internalized pool with a fluorescent secondary antibody labeled with a different fluorophore. Using this technique we were able to obtain important information about the subcellular location of this putative receptor, revealing that it was, indeed, trafficked to the cell-surface in neurons. This technique is broadly applicable to a range of cell types and cell-surface proteins, providing a suitable antibody to an extracellular epitope is available.

We describe a method to label protein on the surface of living neurons using a specific polyclonal antibody to extracellular epitopes. Protein bound by the antibody on the cell surface and subsequently internalized via endocytosis can be distinguished from protein remaining on, or trafficked to, the surface during the incubation.

Étiquetage différentiel de surface cellulaire et intériorisé protéines après Anticorps alimentation de Live de culture neurones

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of ...

A Microbiomechanical System for Studying Varicosity Formation and Recovery in Central Neuron Axons

Axonal varicosities are enlarged structures along the shafts of axons with a high degree of heterogeneity. They are present not only in brains with neurodegenerative diseases or injuries, but also in the normal brain. Here, we describe a newly-established micromechanical system to rapidly, reliably, and reversibly induce axonal varicosities, allowing us to understand the mechanisms governing varicosity formation and heterogeneous protein composition. This system represents a novel means to evaluate the effects of compression and shear stress on different subcellular compartments of neurons, different from other in vitro systems that mainly focus on the effect of stretching. Importantly, owing to the unique features of our system, we recently made a novel discovery showing that the application of pressurized fluid can rapidly and reversibly induce axonal varicosities through the activation of a transient receptor potential channel. Our biomechanical system can be utilized conveniently in combination with drug perfusion, live cell imaging, calcium imaging, and patch clamp recording. Therefore, this method can be adopted for studying mechanosensitive ion channels, axonal transport regulation, axonal cytoskeleton dynamics, calcium signaling, and morphological changes related to traumatic brain injury.

This protocol describes a physiologically relevant, pressurized fluid approach for rapid and reversible induction of varicosities in neurons.

Un système de Microbiomechanical pour l’étude de la Formation de varicosité et récupération dans les axones des neurones centraux

Axonal varicosities are enlarged structures along the shafts of axons with a high degree of heterogeneity. They are present not only in brains with ...

An In Vitro Model for Studying Tau Aggregation Using Lentiviral-mediated Transduction of Human Neurons

Aberrant aggregation of the protein tau is pathogenically involved in a number of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD). Although mouse models of tauopathy have provided a valuable resource for investigating the neurotoxic mechanisms of aggregated tau, it is becoming increasingly apparent that, due to interspecies differences in neurophysiology, the mouse brain is unsuitable for modeling the human condition. Advances in cell culture methods have made human neuronal cultures accessible for experimental use in vitro and have aided in the development of neurotherapeutics. However, despite the adaptation of human neuronal cell cultures, in vitro models of human tauopathy are not yet widely available. This protocol describes a cellular model of tau aggregation in which human neurons are transduced with lentiviral-derived vectors that code for pathogenically mutated tau fused to a yellow fluorescent protein (YFP) reporter. Transduced cultures produce tau aggregates that stain positively for thioflavin and display markers of neurotoxicity, such as decreased axonal length and increased lysosomal volume. This procedure may be a useful and cost-effective model for studying human tauopathies.

This protocol details a procedure in which human neuronal cultures are transduced with lentiviral constructs coding for mutant human tau. Transduced cultures display tau aggregates and associated pathologies.

Un modèle in vitro pour étudier l’agrégation de Tau à l’aide de la transduction à médiation Lentivirale des neurones humains

Aberrant aggregation of the protein tau is pathogenically involved in a number of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD). ...

Wireless Electrophysiological Recording of Neurons by Movable Tetrodes in Freely Swimming Fish

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain activity from freely moving fish would advance research on the neural basis of fish behavior considerably. Moreover, precise control of the recording location in the brain is critical to studying coordinated neural activity across regions in fish brain. Here, we present a technique that records wirelessly from the brain of freely swimming fish while controlling for the depth of the recording location. The system is based on a neural logger associated with a novel water-compatible implant that can adjust the recording location by microdrive-controlled tetrodes. The capabilities of the system are illustrated through recordings from the telencephalon of goldfish.

A novel wireless technique for recording extracellular neural signals from the brain of freely swimming goldfish is presented. The recording device is composed of two tetrodes, a microdrive, a neural data logger, and a waterproof case. All parts are custom-made except for the data logger and its connector.

Enregistrement électrophysiologique sans fil des neurones par les tetrodes mobiles dans les poissons nageant librement

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain ...

Studying Pre-formed Fibril Induced &#945;-Synuclein Accumulation in Primary Embryonic Mouse Midbrain Dopamine Neurons

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with immunostaining and unbiased automated image analysis, this model allows for the analysis of the effects of drugs and genetic manipulations on &#945;-synuclein aggregation in neuronal cultures. Primary midbrain cultures provide a reliable source of bona fide embryonic dopamine neurons. In this protocol, the hallmark histopathology of Parkinson&#8217;s disease, Lewy bodies (LB), is mimicked by the addition of &#945;-synuclein pre-formed fibrils (PFFs) directly to neuronal culture media. Accumulation of endogenous phosphorylated &#945;-synuclein in the soma of dopamine neurons is detected by immunostaining already at 7 days after the PFF addition. In vitro cell culture conditions are also suitable for the application and evaluation of treatments preventing &#945;-synuclein accumulation, such as small molecule drugs and neurotrophic factors, as well as lentivirus vectors for genetic manipulation (e.g., with CRISPR/Cas9). Culturing the neurons in 96 well plates increases the robustness and power of the experimental setups. At the end of the experiment, the cells are fixed with paraformaldehyde for immunocytochemistry and fluorescence microscopy imaging. Multispectral fluorescence images are obtained via automated microscopy of 96 well plates. These data are quantified (e.g., counting the number of phospho-&#945;-synuclein-containing dopamine neurons per well) with the use of free software that provides a platform for unbiased high-content phenotype analysis. PFF-induced modeling of phosphorylated &#945;-synuclein accumulation in primary dopamine neurons provides a reliable tool to study the underlying mechanisms mediating formation and elimination of &#945;-synuclein inclusions, with the opportunity for high-throughput drug screening and cellular phenotype analysis.

Here, we present a detailed protocol to study neuronal &#945;-synuclein accumulation in primary mouse dopamine neurons. Phosphorylated &#945;-synuclein aggregates in neurons are induced with pre-formed &#945;-synuclein fibrils. Automated imaging of immunofluorescently labeled cells and unbiased image analysis make this robust protocol suitable for medium-to-high throughput screening of drugs that inhibit &#945;-synuclein accumulation.

Étude de l’accumulation préforme induite d’α-synucléine par fibrile dans les neurones dopaminergiques de la souris embryonnaire primaire

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with ...

Activity of Posterior Lateral Line Afferent Neurons during Swimming in Zebrafish

Sensory systems gather cues essential for directing behavior, but animals must decipher what information is biologically relevant. Locomotion generates reafferent cues that animals must disentangle from relevant sensory cues of the surrounding environment. For example, when a fish swims, flow generated from body undulations is detected by the mechanoreceptive neuromasts, comprising hair cells, that compose the lateral line system. The hair cells then transmit fluid motion information from the sensor to the brain via the sensory afferent neurons. Concurrently, corollary discharge of the motor command is relayed to hair cells to prevent sensory overload. Accounting for the inhibitory effect of predictive motor signals during locomotion is, therefore, critical when evaluating the sensitivity of the lateral line system. We have developed an in vivo electrophysiological approach to simultaneously monitor posterior lateral line afferent neuron and ventral motor root activity in zebrafish larvae (4-7 days post fertilization) that can last for several hours. Extracellular recordings of afferent neurons are achieved using the loose patch clamp technique, which can detect activity from single or multiple neurons. Ventral root recordings are performed through the skin with glass electrodes to detect motor neuron activity. Our experimental protocol provides the potential to monitor endogenous or evoked changes in sensory input across motor behaviors in an intact, behaving vertebrate.

We describe a protocol to monitor changes in the afferent neuron activity during motor commands in a model vertebrate hair cell system.

Activité des neurones afférents de la ligne latérale postérieure pendant la baignade chez le poisson zèbre

Sensory systems gather cues essential for directing behavior, but animals must decipher what information is biologically relevant. Locomotion generates ...