Diese Arbeit wurde von NINDS und American Heart Association zu S.Ge. finanziert Dieser Ansatz wurde ursprünglich gegründet, entwickelt und vom Autor in Dr. Hongjun Lied-Labor in der Johns Hopkins University. Wir möchten Dr. Hongjun Lied für seine Hilfe und Unterstützung bedanken.

1. Virus Injection High-Titer entwickelt Retrovirus (1 × 10 9 Einheiten / ml) wird durch Co-Transfektion von retroviralen Vektoren hergestellt und VSVG in HEK293T Zellen durch Ultrazentrifugation von viralen Überstand gefolgt. Für Herkunft und Produktionsmethoden, siehe den ausgezeichneten JoVE Demonstration (3). Hinweis: Young adult (4-6 Wochen alt) weibliche C57BL / 6 Mäuse (Charles River) wurden unter den Standardbedingungen untergebracht. Alle Verfahren folgen dem National Research Council Guide für die Pflege und Verwendung von Labortieren unter einem Protokoll, das von der Stony Brook University IACUC genehmigt. <ol><li> Thaw 2UL von gefrorenen Retrovirus pro Tier auf dem Eis. </li><li> Betäuben (100 ug Ketamin + 10 pg Xylazin pro Gramm Körpergewicht) und montieren Sie das Tier auf einem stereotaktischen Rahmen (Steolting). Entfernen Sie die Haare auf dem Kopf, und wischen Sie die Haut mit 70% Ethanol. </li><li> Expose des Schädels und bohren Sie vier flachen Löchern mit einem zahnärztlichen Bohrer (0,6 mm-Bohrer) an den folgenden Koordinaten: <ul><li> anterioposterior = -2 mm von Bregma; lateral = ± 1,6 mm; ventral = 2,5 mm; anterioposterior = -3 mm vom Bregma; lateral = ± 2,6 mm; ventral = 3,2 mm. </li><li> anterioposterior = -2 mm von Bregma; lateral = ± 1,6 mm; ventral = 2,5 mm; anterioposterior = -3 mm vom Bregma; lateral = ± 2,6 mm; ventral = 3,2 mm. </li></ul></li><li> Montieren Sie einen 1μl Hamilton, Flachbild-Spritze und injizieren 0,5 ul Retrovirus pro Standort mit einer Rate von 0,25 ml / min in den Gyrus dentatus. (Siehe oben die Koordinaten für ventralen Tiefe.) Pause von 2 Minuten nach jeder Injektion, bevor langsam Zurückziehen der Spritze Flüssigkeit Rückfluss zu verhindern. Zwischen den Injektionen, kurz abspülen der äußeren Oberfläche der Spritze mit sterilem PBS und einem Applikator, um Spuren von Blut zu entfernen. </li><li> Schließen Sie die Wunde mit sterilen chirurgischen Nahtmaterial (Typ P-1, Größe 6-0) und kehren das Tier auf Standard Wohnbedingungen, bis die gewünschte Zeit Stadien nach der Injektion. </li></ol> 2. Slice-Vorbereitung Um qualitativ hochwertige akuten Scheiben von ausgewachsenen Mäusen, verwenden wir eine modifizierte Schneiden Lösung für Stück Vorbereitung (siehe unten). <ol><li> Pre-chill Schneiden Lösung auf 0-4 ° C auf Eis und Blase mit 95% O 2 -5% CO 2 für mindestens 30 Minuten. </li><li> Betäuben und transkardial perfuse ein Tier mit eiskaltem sauerstoffgesättigten Schneiden Lösung. </li><li> Schnelles Entfernen des Gehirns in eine Petrischale mit Filter-Papier mit kaltem, mit Sauerstoff Zuschnittlösung vorbenetzt. Cut off des Kleinhirns und in Scheiben schneiden einer Montagefläche mindestens 1 mm anterior der (sichtbare) Injektion Koordinaten. Kleben Sie die Gehirn-auf eine vibrotome Bühne und montieren Sie ihn in den Mahlraum mit Kalt-und sauerstoffreiches Schneid-Lösung gefüllt. </li><li> Bereiten koronalen oder horizontale Schnitte (300-350μm) und übertragen Sie sie mit einer großen Bohrung Pipette eine Inkubation Kammer mit Raumtemperatur ACSF sprudelte mit 95% O 2 / 5% CO 2. </li><li> Inkubieren Sie die Schnitte, sprudelnde kontinuierlich, bei 32 ° C für 30-60 Minuten. Return der Kammer auf Raumtemperatur für die Dauer der Aufnahme. </li></ol> 3. Elektrophysiologischen Experimenten <ol><li> 34 ° C - Scheiben sind in die Aufnahme Kammer, die kontinuierlich mit Sauerstoff gesättigt ACSF ist bei 32 ° C perfundiert übertragen </li><li> Eine bipolare Wolfram Stimulationselektrode ist in der molekularen Schicht des Gyrus dentatus mit geringer Vergrößerung Mikroskop-Objektiv (10-fach) platziert. </li><li> Newborn DGCs in der Sub-granular zone / Granularzelltumoren Schicht werden durch die Expression von GFP oder dTomato identifiziert. Whole-cell patchclamp Aufnahme befindet sich auf neugeborene Neuronen unter hoher Vergrößerung (40X) durchgeführt. </li><li> Firing Eigenschaften der aufgenommenen Zellen werden durch Injektion von einer Reihe von Strömen unter Current-Clamp-Modus erhalten. </li><li> Elektrische Reize werden durch die Stimulationselektrode über eine Stimulation Isolator durch die Recording-Software gesteuert geliefert, und evozierte postsynaptische Ströme in den neugeborenen DGCs werden aufgezeichnet. </li></ol> 4. Data Analysis Amplituden der evozierten postsynaptischen Antworten sind in unterschiedlichen Entwicklungsstadien der Erwachsenen-geboren Neuronen analysiert. 5. Repräsentative Ergebnisse Mit dem obigen Protokoll ist die GFP-Expression in Neugeborenen Gyrus dentatus Neuronen ähnlich Abbildung 1 gemeinsam. Beachten Sie, dass neugeborene Neuronen einfach visualisiert werden und sowohl Dendriten und Axone sind robust bezeichnet. Expression in dünne DGC Axonen und kleine Stacheln, hängt von der Qualität und der Titer des Virus, der Promotor verwendet und die Länge der in-vivo-Expression. In unseren Händen, sind neugeborene Zellen und subzellulären Organellen in der Regel innerhalb weniger Tage nach der Injektion sichtbar, und der Wirbelsäule Ausdruck kann von den frühesten Stadien der Entwicklung zurückgeführt werden. WLoch-Zell-Aufnahmen von neugeborenen Neuronen in verschiedenen Zeitstufen ermöglichen die Untersuchung einzigartig Neugeborenen Zelle Eigenschaften, zB Aktionspotentiale (Abbildung 2a), sowie den Prozess der synaptischen Integration in bestehende neuronale Schaltkreise während der Reifung (Abb. 2b). <img src="/files/ftp_upload/2548/2548fig1.jpg" alt="Abbildung 1" /> Abbildung 1 2-Photonen-konfokale Abbildung von neugeborenen Neuronen in einer horizontalen Gyrus dentatus Abschnitt einer erwachsenen Maus. Grüne Zellen sind 21dpi (Tage nach der Injektion) GFP-exprimierenden Neugeborenen DGCs mit PUX-GFP Retrovirus bezeichnet. <img src="/files/ftp_upload/2548/2548fig2.jpg" alt="Abbildung 2" /> Abbildung 2 a) Aktionspotential feuern Eigenschaften eines Neugeborenen DGC bei 21 dpi. B) repräsentativ evozierten exzitatorischen postsynaptischen Ströme (EPSCs) auf ein neugeborenes DGC bei 21 dpi erfasst.

<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>

Kontinuierliche Neurogenese im Hippocampus von erwachsenen Säugetieren Gehirn bietet eine einzigartige experimentelle Modellsystem, um die neuronale Entwicklung und Integration in das reife Gehirn zu studieren. Das Protokoll hier vorgestellten verbindet retroviralen Kennzeichnung und elektrophysiologische Methoden, um die Integration von Neugeborenen dentatus Granulat Neuronen im Gehirn von erwachsenen Mäusen untersucht werden. Um sicherzustellen, dass Ihre Experimente erfolgreich sind, empfehlen wir die folgenden während der kritischen Schritte des Verfahrens: Zur Vermeidung von unerwünschten Infektion und Entzündung, sollten alle Instrumente sterilisiert (durch Autoklavieren, jede Art von Sterilisator oder 70% Ethanol) vor dem Eingriff werden. Verwenden Sie sterile PBS auf die Spitze der Spritze Nadel vor der Injektion zu waschen. Für Virus Injektion, müssen die Tiere gut auf der stereotaktischen Vorrichtung montiert sein und die Quellen der unnötige Bewegung muss während der Injektion minimiert werden. Vergewissern Sie sich, der Kopf ist gut fixiert und fest, und passen Sie die Nase Position der Maus auf Stufe Bregma und Lambda vor Berechnung Injektion Koordinaten. Auszug Virus in die Spritze schnell um die Exposition gegenüber Raumtemperaturen zu minimieren - Retroviren sind besonders temperaturempfindlich. Inject Virus langsam in der empfohlenen Rate, um den Druck zu minimieren. Die Anwendung der empfohlenen flacher Spitze Spritze (vs. gemeinsamen abgeschrägte Spitze) wird dafür sorgen, eine gleichmäßig verteilte Gyrus dentatus Infektion Bereich. Vor der Zubereitung Scheiben, müssen Schneid-Lösung und Inkubation ACSF mit 95% O 2 -5% CO 2 für mindestens 30 Minuten eingeleitet werden, damit Sauerstoff in die Lösung zu sättigen. Die Tiere sollten schnell durchströmt werden und Gewebe in kaltem, mit Sauerstoff gesättigte Lösung für die besten schneiden die Qualität erhalten bleibt. Für die Aufnahme, erhöhen Sie die Reizstärke allmählich, bis es eine postsynaptische Antwort. Ort wechseln der Stimulationselektrode in der molekularen Schicht des Gyrus dentatus wie nötig. Zusammengefasst bietet der empfohlene Ansatz einen Weg, um verschiedene Eigenschaften und mögliche funktionelle Rolle von Erwachsenen geboren Neuronen während Early-Stage-Integration zu erforschen in aktive, reife Schaltung.

<ul><li> Cutting-Lösung (in mM): 110 Cholinchlorid, 2,5 KCl, 1,3 KH 2 PO 4, 25,0 NaHCO 3, 0,5 CaCl 2, 7 MgCl 2, 10 Dextrose, 1,3 Natriumascorbat, 0,6 Natriumpyruvat, 5,0 Kynurensäure. </li><li> Künstliche Cerebrospinalflüssigkeit (ACSF, in mM): 125.0 NaCl, 25,0 NaHCO 3, 2,5 KCl, 2 CaCl 2, 1,3 KH 2 PO 4, 1,3 MgCl 2, 1,3 Natriumascorbat, 0,6 Natriumpyruvat, 10 Dextrose. </li><li> Interne Lösung für whole-cell patchclamp (in mm): 120 Kaliumgluconat, 15 KCl, 4 MgCl 2, 0,1 EGTA, 10,0 HEPES, 4 MgATP, 0,3 Na 3 GTP, 7 Phosphokreatin (pH 7,4, 300 mOsm). </li></ul>

studying the integration of adult-born neurons

Neurogenese tritt bei erwachsenen Säugetieren Gehirn in der Sub-ventrikuläre Zone (SVZ) der lateralen Ventrikel und in der Sub-körnige Zone (SGZ) der hippocampalen des ganzen Lebens. Frühere Berichte haben gezeigt, dass adulte Neurogenese mit vielfältigen Störungen des Gehirns, einschließlich Epilepsie, Schizophrenie, Depression und Angst (1) verbunden ist. Die Entzifferung der Prozess der normalen und aberranten Erwachsene geboren Neuron Integration kann Licht auf die Genese dieser Erkrankungen Schuppen und informieren die Entwicklung neuer Therapien. SGZ adulte Neurogenese Spiegel embryonale und postnatale neuronale Entwicklung, einschließlich der Stufen des Schicksals Spezifikation, Migration, Integration synaptischer und Reifung. Allerdings tritt eine vollständige Integration über einen längeren, 6-Wochen-Frist. Initial synaptischen Eingang zum Erwachsenen-geboren SGZ dentatus Körnerzellen (DGCs) ist GABAergen, gefolgt von glutamatergen Eingang bei 14 Tagen (2). Die spezifischen Faktoren, die Schaltung Bildung von Erwachsenen geboren Neuronen im Gyrus dentatus zu regulieren sind derzeit nicht bekannt. Unser Labor verwendet eine replikationsdefizient retroviralen Vektor basierend auf dem Moloney Maus-Leukämie-Virus, fluoreszierende Proteine ​​und die Hypothese auf regulatorische Gene, um diese wuchernden Zellen zu liefern. Diese virale Technik bietet eine hohe Spezifität und Auflösung für die Analyse von Zell-Geburtsdatum, Geschlecht, Morphologie und Synaptogenese. Ein typisches Experiment oft beschäftigt zwei oder drei Viren mit einzigartigen Label, Transgen und Promotor-Elemente für die Single-Cell-Analyse eines gewünschten Entwicklungsprozess in vivo. Das folgende Protokoll beschreibt eine Methode für die Analyse von funktionalen neugeborenen Neuronen Integration mit einem einzigen grünen (GFP) oder rot (dTomato) fluoreszierendes Protein Retrovirus-und Patch-Clamp-Elektrophysiologie.

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

Studying the Integration of Adult-born Neurons

Ein Weg, um die Integration von Neugeborenen dentatus Körnerzellen im erwachsenen Tieren Studie beschrieben wird. Diese Technik verwendet eine technisch Retrovirus zu neugeborenen Neuronen, durch elektrophysiologische Aufnahmen folgten, um in vivo funktionale Integration bestimmen Etikett.

Die Untersuchung der Integration von Adult-geboren Neurons

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 ...

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Research

JoVE Journal

Neuroscience

13.7K Aufrufe.  State University of New York at Stony Brook. Ein Weg, um die Integration von Neugeborenen dentatus Körnerzellen im erwachsenen Tieren Studie beschrieben wird. Diese Technik verwendet eine technisch Retrovirus zu neugeborenen Neuronen, durch elektrophysiologische Aufnahmen folgten, um in vivo funktionale Integration bestimmen Etikett.

Serie: 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 Elektroporation gefolgt von primären neuronalen Kultur für ein Studium Gene Function in Subset von kortikalen Neuronen

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

Forschung

Neurowissenschaften

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.

Selektive Viral Transduktion von Adult-geboren olfaktorischen Neuronen für chronische In vivo Optogenetische Stimulation

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.

Die Spezifikation der telencephalen glutamatergen Neuronen von menschlichen pluripotenten Stammzellen

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.

Schnelle Micro-Iontophorese von Glutamat und GABA: Ein nützliches Tool, um Synaptic Integration Untersuchen

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.

Differential Markierung von Zelloberflächen und internalisierten Proteine ​​Antikörper nach Fütterung der Live-kultivierten Neuronen

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.

Ein Microbiomechanical-System für das Studium Varizen-Bildung und Erholung in zentralen Neuronen Axone

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.

Ein In-Vitro-Modell für das Studium der Tau Aggregation mit Lentiviral-vermittelter Transkription menschlicher Neuronen

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.

Drahtlose elektrophysiologische Aufzeichnung von Neuronen durch bewegliche Tetrodes in frei schwimmenden Fischen

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.

Studium der vorgeformten Fibril-induzierten Synuclein-Akkumulation in primären embryonalen Maus-Midbrain-Neuronen

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.

Aktivität von posterioren Lateral Line Afferent Neuronen beim Schwimmen im Zebrafisch

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