Diese Arbeit wurde von der IMPACT Award vom Department of Radiation Oncology, University of Alabama-Birmingham Comprehensive Cancer Center, The Fighting Kinderkrebsstiftung und der Gabrielle &#39;s Angel Foundation (zum ESY.) Unterstützt.

Ein. Cell Culture <ol><li> Kultur neuronalen Zellen und behandeln Sie sie nach Bedarf. </li><li> Ernte Zellen in 15 ml Röhrchen: Aspirat Medien, mit Phosphat spülen Kochsalzlösung (PBS, Calcium und Magnesium befreit), fügen Trypsin, sammeln sich in 15 ml Röhrchen und neutralisieren Trypsin mit geeigneten serumhaltigen Medien. </li><li> Spin bei 1.000 xg für 5 min. </li><li> Absaugen Medien. </li><li> Die Zellen in PBS. </li><li> Spin bei 1.000 xg für 5 min. </li><li> Absaugen Medien und resuspendieren Zellen in frischem PBS. </li><li> Zählen von Zellen unter Verwendung eines Hämacytometers oder Ihre bevorzugte Zelle Zähler. </li><li> Zellproben sollte unmittelbar vor Beginn des Tests vorbereitet werden. </li><li> Alle Proben sollten im Dunkeln oder gelbem Licht gehandhabt werden, um DNA-Schäden durch UV-Licht zu verhindern. </li></ol> 2. Comet Assay Ein. Slide Vorbereitung <ol><li> Schmelze 1% Agarose (1 g / 100 ml 1X Tris Base, Borsäure, EDTA, TBE) in einer Mikrowelle für 3 min bis alle Granulate verschwinden. </li><li> Objektträger in das geschmolzene Agarose und Wischen einer Seite mit einer Kimwipe reinigen. Lassen Sie die Agarose an der Luft trocknen, um eine transparente Folie. Dies kann im Voraus und Folien erfolgen kann gespeichert werden. </li></ol> 2. Neutral Comet Assay <ol><li> Chill Lyse-Lösung (2,5 M NaCl, 100 mM EDTA, pH 10, 10 mM Tris-Base, 1% Natriumlaurylsarcosinat und 1% Triton X-100, pH 10) bei 4 ° C für mindestens 1 Stunde vor der Verwendung. </li><li> 2.2.2. Melt 1% Agarose mit niedrigem Schmelzpunkt (1 g / 100ml in 1X Tris Base, Borsäure, EDTA) in einer Mikrowelle für 3 min, bis alle Körner verschwinden. Die Agarose muss auf 37 ° C in einem Wasserbad abgekühlt werden, um künstliche Induktion Kometenschweif vermeiden. Idealerweise muß die Agarose eine halbe Stunde vor der Verwendung gekühlt werden. </li><li> 2.2.3. Verdünnen der Zellsuspension, so daß es 100.000 Zellen pro ml sind. Kombinieren der Zellsuspension mit der Agarose mit niedrigem Schmelzpunkt (bei 37 ° C) beiein Verhältnis von 1:10 (v / v), kurz vortexen und sofort pipettieren 50 ul auf die Comet Slide. Verwenden Sie die Seite der Pipettenspitze die Zellsuspension gleichmäßig über den Probenraum. Jede Behandlungsgruppe sollte mindestens in dreifacher Ausfertigung. </li><li> Objektträger flach im Kühlschrank für 30 Minuten, bis ein Kreis erscheint in der Peripherie der Folie. </li><li> Prechill die Lyse-Lösung und tauchen Folien in dieser Lösung für 30 min im Dunkeln bei 4 ° C (oder im Kühlschrank). </li><li> Abgießen oder absaugen die Lyse-Lösung und fügen 1X neutral Elektrophorese-Puffer (Tris-Base, Borsäure, EDTA, 1X TBE). Lassen Sie Folien in diesem Puffer für eine halbe Stunde in den Kühlschrank stellen. </li><li> Fügen vorgekühlte 1X Neutral Electrophoresis Buffer (FSME) in Elektrophoresekammer, die Objektträger in der Elektrophorese Diamagazin. Richten gleitet, so daß sie äquidistant von Elektroden sind. </li><li> Gießen 1X neutral Elektrophoresepuffer bis zu 0,2 Zentimeter über Rutschen. Überschüssiger Puffer wird unterFere mit Elektrophorese. </li><li> Set Stromversorgungsspannung bis 1 V pro cm (gemessen Elektrode zu Elektrode) und führen für 30 min bei 4 ° C (oder der Kühlraum) im Dunkeln. </li></ol> 3. Alkaline Comet Assay <ol><li> Chill Lyse-Lösung (2,5 M NaCl, 100 mM EDTA, pH 10, 10 mM Tris-Base, 1% Natriumlaurylsarcosinat und 1% Triton X-100, pH 10) bei 4 ° C für mindestens 1 Stunde vor der Verwendung. </li><li> Melt 1% Agarose mit niedrigem Schmelzpunkt (1 g / 100 ml in 1X Tris Base, Borsäure, EDTA) in einer Mikrowelle für 3 min, bis alle Körner verschwinden. Dann in einem 37 ° C Wasserbad für mindestens 30 min. </li><li> Kombinieren Zellen mit 1 x 105 / ml mit geschmolzenen Agarose mit niedrigem Schmelzpunkt (37 ° C) in einem Verhältnis von 1:10 (v / v), Vortex kurz und unmittelbar pipettieren 50 ul auf das Slide Comet. Verwenden Sie die Seite der Pipettenspitze die Zellsuspension gleichmäßig über den Probenraum. Jede Behandlungsgruppe sollte mindestens in dreifacher Ausfertigung. </li><li> Aufschiebenes flach im Kühlschrank für 30 Minuten, bis ein Kreis erscheint in der Peripherie der Folie. </li><li> Objektträger in vorgekühlte Lyselösung und lassen bei 4 ° C für 1 h bis über Nacht im Dunkeln. </li><li> Die Objektträger aus der Lyselösung, abtropfen die Folien und spülen einmal mit kaltem Neutralisationspuffer für 5 min, um restliches Detergens und Salzen vor der Alkali-Abwickeln Schritt zu entfernen. </li><li> Die Objektträger in eine Gelelektrophorese Kammer mit vorgekühltem frisch zubereiteten Elektrophorese-Puffer (300 mM NaOH, 1 mM EDTA, pH&gt; 13) nicht bis 0,5 cm über Rutschen überschreiten gefüllt. Richten gleitet, so daß sie äquidistant von Elektroden sind. </li><li> Lassen Folien im alkalischen Puffer sitzen für 30 min im Dunkeln zum Abwickeln der DNA und der Expression von Alkali-ersatzpflichtigen ermöglichen. </li><li> Set Stromversorgungsspannung bis 1 V pro cm (gemessen Elektrode zu Elektrode) und führen für 30 min bei 4 ° C (oder der Kühlraum). </li></ol> 4. Befestigungs-und Staining Cells <ol><li> Überschüssiges Electrophoresis Buffer </li><li> Objektträger in vorgekühlte destilliertem Wasser für 5 min bei RT. </li><li> Objektträger in vorgekühlte 70% Ethanol 5 min bei RT. </li><li> Dry Proben über Nacht. Setzen Sie Folien helles Licht. Die Proben können für Monate bei Raumtemperatur vor Scoring zu diesem Zeitpunkt gespeichert werden. </li><li> Stain Objektträger durch Eintauchen in Sybr grün (1X in PBS verdünnt) für 20 min in den Kühlschrank stellen. </li><li> Objektträger und ihnen erlauben, vollständig trocknen im Dunkeln. Die Agarose wird transparent, wenn sie vollständig trocken. </li></ol> 4. Bild-Akquisition und Analyse <ol><li> Erwerben Sie Bilder mit Fluoreszenzmikroskop auf den grünen Filter (Zeiss AxioVision) und analysieren mit Comet Assay Software (Perceptive Instruments). </li><li> Klicken Sie auf den &quot;Kometen Kopf&quot; (der Kern) und die Software berechnet die Parameter einschließlich der mittleren Schwanz Moment und DNA-Menge in derKern. </li><li> Analysieren mindestens 200 Zellen pro Behandlung. </li><li> Exportieren von Daten in Microsoft Excel. </li><li> Berechnung der mittleren tail moment für jede Behandlungsgruppe und Plot-Daten als angemessen. </li></ol> 5. Repräsentative Ergebnisse Ein Beispiel für Comet Assay Analyse neuronaler Zellen ist in Abbildung 2 und Abbildung 3 dargestellt. In diesem Fall induziert Bestrahlung der neuronalen Zellen DNA-Schäden. Da die Zellen mit dem elektrischen Feld ausgesetzt werden, wandert die DNA mit unterschiedlichen Geschwindigkeiten aufgrund der Unterschiede in der Größe, die anschließend analysiert wird unter Verwendung des Kometentest Software. Je mehr die DNA-Schäden, je weiter die DNA wandert aus dem Zellkern. Dies verringert die Fluoreszenzintensität in den Zellkern, die anschließend durch den Software und führt zu höheren Leitwerksmoment abgeholt. Tabelle 1 stellt eine Tabelle von repräsentativen Kometenassay Analyse und Abbildung 4 zeigt einen repräsentativentative Graph DNA-Schäden in neuronalen Zellen nach der Bestrahlung durch den Comet Assay gemessen. <img src="/files/ftp_upload/50049/50049fig1.jpg" alt="Abbildung 1"/> Abbildung 1. Ablaufdiagramm des Comet Assay. <a href="https://www.jove.com/files/ftp_upload/50049/50049fig1large.jpg" target="_blank">Klicken Sie hier für eine größere Abbildung zu sehen</a> . <img src="/files/ftp_upload/50049/50049fig2.jpg" alt="Abbildung 2"/> Abbildung 2. Repräsentative Bilder von neuronalen Zellen (A) ohne und (B) mit Kometenschweif. <img src="/files/ftp_upload/50049/50049fig3.jpg" alt="Abbildung 3"/> Abbildung 3. Comet Assay-Analyse mit Comet Assay Software. (A) Repräsentative Schirmschüsse Bild aufgenommen mit Carl Zeiss Fluoreszenzmikroskop und mittels CometAssay Software. (B) Klick auf den Kern oder die &quot;Kometen Kopf&quot; (eingekreist in grün) erzeugt eine fluoreszierende Karte und Grafik (eingekreist in gelb) und eine (C) Datentabelle (rot eingekreist). <a href="https://www.jove.com/files/ftp_upload/50049/50049fig3large.jpg" target="_blank">Klicken Sie hier für eine größere Abbildung zu sehen</a> . <img src="/files/ftp_upload/50049/50049fig4.jpg" alt="Abbildung 4"/> Abbildung 4. Repräsentative Graph durch Auftragen der mittleren Leitwerksmoment durch Analysieren DNA-Schädigung in bestrahlten und unbestrahlten neuronalen Zellen mit neutralen Kometenassay erhalten wird. Wie erwartet, induziert 3Gy Strahlung (Röntgenstrahlen) DNA-Schädigung, wie durch die höhere mittlere Leitwerksmoment in den bestrahlten neuronalen Zellen dargestellt. Dargestellt ist die mittlere tail moment (+ / - Standardfehler), ** P &lt;0,01. <img src="/files/ftp_upload/50049/50049table1.jpg" alt="Tabelle 1"/> Tabelle 1. </strong&gt; Vertreter Tisch Comet Assay Analyse bestrahlten und nicht-bestrahlten neuronalen Zellen erhalten. <a href="https://www.jove.com/files/ftp_upload/50049/50049table1large.jpg" target="_blank">Klicken Sie hier für eine größere Abbildung zu sehen</a> .

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Keine Interessenskonflikte erklärt.

Der Comet Assay hat die einzigartige Fähigkeit zur Analyse von einzelnen Zellen. Dies ist vorteilhaft bei der Identifizierung von Subpopulationen von Zellen, die differenzielle Reaktion auf zytotoxischen Mitteln demonstrieren. Einige praktische Einschränkungen zu berücksichtigen. Die Anzahl der Zellen, die einzeln ausgewertet werden kann je nach dem Individuum. Der Stichprobenumfang erhöht werden muss, wenn es Varianz in DNA-Schädigung innerhalb einer Population. Lebensfähigen Einzelzell-Suspension ist für diesen...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalognummer </td><td> Kommentare (optional) </td></tr><tr><td> 1,5 ml Röhrchen </td><td> Santa Cruz Biotechnology, Inc. </td><td> Sc-200.271 </td><td></td></tr><tr><td> 10X TBE (Tris-Base, Borsäure, EDTA) </td><td> Fisher Scientific </td><td> BP13331 </td><td></td></tr><tr><td> 15 ml Tube </td><td> Fisher Scientific </td><td> 0553851 </td><td></td></tr><tr><td> Agarose </td><td> Sigma </td><td> A5093 </td><td></td></tr><tr><td> Aluminiumfolie </td><td> Fisher Scientific </td><td> 01213101 </td><td></td></tr><tr><td> Becher </td><td> Fisher Scientific </td><td> FB102300 </td><td></td></tr><tr><td> Zentrifugieren </td><td> Thermo Scientific </td><td> 75004261PR </td><td></td></tr><tr><td> Comet Assay-Software </td><td> Comet Assay IV Image Analysis System </td><td></td><td></td></tr><tr><td> Zylinder </td><td> Fisher Scientific </td><td> 08555F </td><td></td></tr><tr><td> EDTA </td><td> GIBCO </td><td> 15575 </td><td></td></tr><tr><td> Electrophoresis Kammer </td><td> Thermo Scientific </td><td> 09528101 </td><td></td></tr><tr><td> Ethanol </td><td> Fisher Scientific </td><td> A407P4 </td><td></td></tr><tr><td> Fluoreszenzmikroskop </td><td> Zeiss Axio Vision- </td><td></td><td> Jede Fluoreszenzmikroskop mit Grünfilter genügt </td></tr><tr><td> Hämazytometer </td><td> Fisher Scientific </td><td> 0267152 </td><td></td></tr><tr><td> Agarose mit niedrigem Schmelzpunkt </td><td> Promega </td><td> V2111 </td><td></td></tr><tr><td> Mikrowelle </td><td> Sears </td><td></td><td></td></tr><tr><td> Phosphatgepufferter Kochsalzlösung, Calcium-freien, Magnesium kostenlos </td><td> HyClone </td><td> SH3025601 </td><td></td></tr><tr><td> Stromversorgung </td><td> BioRad </td><td> 1645050 </td><td></td></tr><tr><td> Kühlschrank </td><td> Sears </td><td></td><td></td></tr><tr><td> Herrscher </td><td> Staples </td><td></td><td></td></tr><tr><td> Gleiten </td><td> Fisher Scientific </td><td> 12550143 </td><td></td></tr><tr><td> Kochsalz </td><td> Sigma </td><td> S7653 </td><td></td></tr><tr><td> Natriumhydroxid </td><td> Sigma </td><td> S5881 </td><td></td></tr><tr><td> Natriumlaurylsarcosinat </td><td> Fisher Scientific </td><td> S529 </td><td></td></tr><tr><td> SYBR Green </td><td> Invitrogen </td><td> S7585 </td><td></td></tr><tr><td> Tablett </td><td> Fisihrem Scientific </td><td> 15242B </td><td></td></tr><tr><td> Tris Base </td><td> Sigma </td><td> T6066 </td><td></td></tr><tr><td> Triton-X 100 </td><td> Sigma </td><td> T8787 </td><td></td></tr><tr><td> Trypsin </td><td> HyClone </td><td> SH3023601 </td><td></td></tr><tr><td> Wirbel </td><td> Fisher Scientific </td><td> 02216108 </td><td></td></tr><tr><td> Wasserbad </td><td> Fisher Scientific </td><td> 154622Q </td><td></td></tr></tbody></table>

assaying dna damage in hippocampal neurons using the comet assay

Eine Reihe von Medikamenten gezielt die DNA Reparaturmechanismen und induzieren Zelle, indem sie DNA-Schäden zu töten. So wurden Prozesse direkt zu messen DNA-Schäden wurde ausgiebig evaluiert. Herkömmliche Methoden sind zeitaufwendig, teuer und erfordern ressourcenintensiv replizierende Zellen. Im Gegensatz dazu ist die Comet-Assay, eine einzelne Zelle Gelelektrophorese-Assay, ein schneller, nicht-invasiven, kostengünstigen, direkt und empfindliches Maß von DNA-Schäden und Reparatur. Alle Formen von DNA-Schäden sowie DNA-Reparatur kann an der einzelnen Zelle mit diesem leistungsstarken Technik visualisiert werden. Das Prinzip des Comet Assay ist, dass intakte DNA ist hoch geordnete wohingegen DNA Schädigung unterbricht diese Organisation. Die beschädigten DNA sickert in der Agarose-Matrix und, wenn sie einem elektrischen Feld ausgesetzt wird, wandert der negativ geladenen DNA zur Kathode, das positiv geladen ist. Die großen unbeschädigten DNA-Stränge sind nicht in der Lage zu weit vom Kern migrieren. DNA-Schädenentstehen kleinere DNA-Fragmente, die weiter als die intakte DNA reisen. Kometentest eine Bildanalyse-Software, misst und vergleicht die gesamte Fluoreszenzintensität der DNA im Zellkern mit DNA, die aus dem Nukleus migriert ist. Fluoreszenzsignal aus der migrierten DNA ist proportional zur DNA-Schäden. Längere heller DNA tail bedeutet erhöhte DNA-Schäden. Einige der Parameter, die gemessen werden Leitwerksmoment die ein Maß für sowohl der Menge an DNA und Verteilung der DNA im Schwanz, Schwanzlänge und Prozentsatz der DNA in dem Endstück ist. Dieser Test ermöglicht es, DNA-Reparatur als auch messen, da Auflösung von DNA-Schäden bezeichnet Reparatur stattgefunden hat. Die Grenze der Empfindlichkeit liegt bei etwa 50 Strangbrüche pro diploid Säugetierzellen 1,2. Zellen mit irgendwelchen DNA-schädigende Mittel, wie zum Beispiel Etoposid behandelt wird, kann als eine positive Kontrolle verwendet werden. So ist die Comet-Assay ist eine schnelle und wirksame Verfahren, um DNA-Schäden zu messen.

Watch this Scientific Journal Video about Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay at JoVE.com

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

Der Comet Assay ist ein effizientes Mittel zur Feststellung von Einzel-und Doppelstrang-Brüche, einschließlich Alkali-labile Websites und DNA-DNA/DNA-protein Cross-Links auf der DNA in allen Zellen, einschließlich Neuronen im Hippocampus. Die Methode nimmt den Vorteil dieser differentielle Migration von DNA in einem elektrischen Feld aufgrund von Unterschieden in Menge an DNA-Schäden.

Testen DNA Damage in Hippocampus-Neuronen mit dem Comet Assay

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been ...

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Research

JoVE Journal

Neuroscience

27.1K Aufrufe.  University of Alabama-Birmingham. Der Comet Assay ist ein effizientes Mittel zur Feststellung von Einzel-und Doppelstrang-Brüche, einschließlich Alkali-labile Websites und DNA-DNA/DNA-protein Cross-Links auf der DNA in allen Zellen, einschließlich Neuronen im Hippocampus. Die Methode nimmt den Vorteil dieser differentielle Migration von DNA in einem elektrischen Feld aufgrund von Unterschieden in Menge an DNA-Schäden.

Serie: Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

Assaying &beta;-amyloid Toxicity using a Transgenic C. elegans Model

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant mechanism(s) of toxicity are still unclear. A&beta; is also deposited intramuscularly in Inclusion Body Myositis, a severe human myopathy. The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express human A&beta;. Depending on the tissue or timing of A&beta; expression, transgenic worms can have readily measurable phenotypes that serve as a read-out of A&beta; toxicity. For example, transgenic worms with pan-neuronal A&beta; expression have defects is associative learning (Dosanjh et al. 2009), while transgenic worms with constitutive muscle-specific expression show a progressive, age-dependent paralysis phenotype (Link, 1995; Cohen et al. 2006). One particularly useful C. elegans model employs a temperature-sensitive mutation in the mRNA surveillance system to engineer temperature-inducible muscle expression of an A&beta; transgene, resulting in a reproducible paralysis phenotype upon temperature upshift (Link et al. 2003). Treatments that counter A&beta; toxicity in this model [e.g., expression of a protective transgene (Hassan et al. 2009) or exposure to Ginkgo biloba extracts (Wu et al. 2006)] reproducibly alter the rate of paralysis induced by temperature upshift of these transgenic worms. Here we describe our protocol for measuring the rate of paralysis in this transgenic C. elegans model, with particular attention to experimental variables that can influence this measurement.

Assaying &amp;#946;-amyloid Toxicity using a Transgenic C. elegans Model

The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express the human &beta;-amyloid peptide (A&beta;). Induced expression of A&beta; in C. elegans muscle leads to a rapid, reproducible paralysis phenotype that can be used to monitor treatments that modulate A&beta; toxicity.

Assaying β-Amyloid-Toxizität mit Hilfe eines transgenen C. elegans Modell

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant ...

Forschung

Neurowissenschaften

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 und Mitochondriale Transport: Live Imaging in Hippocampus-Neuronen über lange Zeiträume

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

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

Isolierung und Kultivierung von Neuronen im Hippocampus von Mäusen Pränatale

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Calcium Phosphate Transfection of Primary Hippocampal Neurons

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.

Calciumphosphat-Transfektion von primären Hippocampus-Neuronen

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.
Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.

This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved detail.

Imaging dendritischen Dornen von Rat Primär Hippocampus Neuronen mit Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activation of glutamate receptors that promotes Ca2+ influx and mitochondrial Ca2+ overload. A substantial change in intracellular Ca2+ homeostasis or remodeling of intracellular Ca2+ homeostasis may favor neuron damage in old neurons. For investigating Ca2+ remodeling in aging we have used live cell imaging in long-term cultures of rat hippocampal neurons that resemble in some aspects aged neurons in vivo. For this end, hippocampal cells are, in first place, freshly dispersed from new born rat hippocampi and plated on poli-D-lysine coated, glass coverslips. Then cultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, cultured neurons are loaded with fura2 and subjected to measurements of cytosolic Ca2+ concentration using digital fluorescence ratio imaging. Third, cultured neurons are transfected with plasmids expressing a tandem of low-affinity aequorin and GFP targeted to mitochondria. After 24 hr, aequorin inside cells is reconstituted with coelenterazine and neurons are subjected to bioluminescence imaging for monitoring of mitochondrial Ca2+ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca2+ responses to relevant stimuli as for example the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure may yield new insights as to how aging influence cytosolic and mitochondrial Ca2+ responses to selected stimuli as well as the testing of selected drugs aimed at preventing neuron cell death in age-related diseases.

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Intracellular Ca2+ remodeling in aging may contribute to excitotoxicity and neuron damage, processes mediated by Ca2+ overload. We aimed at investigating Ca2+ remodeling in the aging brain using fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca2+ in long-term cultures of rat hippocampal neurons, a model of neuronal aging.

Fluoreszenz- und Biolumineszenz-Bildgebung subzellulärer Ca<sup&gt; 2+</sup&gt; Im Alter von Neuronen im Hippocampus

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...

Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to navigate axonal growth. Netrin, slit, semaphorin, and ephrins are known guidance molecules that can attract or repel axons upon binding to receptors and co-receptors on the axon. The activated receptors initiate various signaling molecules in the growth cone that alter the structure and movement of the neuron. Here, we describe the detailed protocol for a stripe assay to assess the ability of a guidance molecule to attract or repel neurons. In this method, dissociated hippocampal neurons from E15.5 mice are cultured on laminin-coated dishes processed with alternating stripes of ectodomain of fibronectin and leucine-rich transmembrane protein-2 (FLRT2) and control immunoglobulin G (IgG) fragment crystallizable region (Fc) protein. Both axons and cell bodies were strongly repelled from the FLRT2-coated stripe regions after 24 h of culture. Immunostaining with tau1 showed that ~90% of the neurons were distributed on the Fc-coated stripes compared to the FLRT2-Fc-coated stripes (~10%). This result indicates that FLRT2 has a strong repulsive effect on these neurons. This powerful method is applicable not only for primary cultured neurons but also for a variety of other cells, such as neuroblasts.

Axon guidance molecules regulate neuronal migration and targeted growth-cone navigation. We present a powerful method, the stripe assay, to assess the ability of guidance molecules to attract or repulse neurons. In this protocol, we demonstrate the stripe assay by showing FLRT2's ability to repel cultured hippocampal neurons.

Stripe Assay die anziehende oder abstoßende Aktivität eines Proteins Substrat unter Verwendung Dissociated Neuronen im Hippocampus zu studieren

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to ...

Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching variations of the neuronal dendrites within the hippocampus are implicated in cognition and memory formation. There are several approaches to Golgi staining, all of which have been useful for determining the morphological characteristics of dendritic arbors and produce a clear background. The present Golgi-Cox method, (a slight variation of the protocol that is provided with a commercial Golgi staining kit), was designed to assess how a relatively low dose of the chemotherapeutic drug 5-flurouracil (5-Fu) would affect dendritic morphology, the number of spines, and the complexity of arborization within the hippocampus. The 5-Fu significantly modulated the dendritic complexity and decreased the spine density throughout the hippocampus in a region-specific manner. The data presented show that the Golgi staining method effectively stained the mature neurons in the CA1, the CA3, and the dentate gyrus (DG) of the hippocampus. This protocol reports the details for each step so that other researchers can reliably stain tissue throughout the brain with high quality results and minimal troubleshooting.

Here we present a Golgi-Cox protocol in extensive detail. This reliable tissue stain method allows for a high-quality assessment of the cytoarchitecture in the hippocampus, and throughout the entire brain, with minimal troubleshooting.

Bewertung der Hippocampalen Dendritischen Komplexität bei gealterten Mäusen unter Verwendung der Golgi-Cox-Methode

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

Messung der synaptischen Vesikel Endozytose in kultivierten Hippocampal Neuronen

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

Assaying Circuit Specific Regulation of Adult Hippocampal Neural Precursor Cells

Adult neurogenesis is a dynamic process by which newly activated neural stem cells (NSCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate new neurons, which integrate into an existing neural circuit and contribute to specific hippocampal functions. Importantly, adult neurogenesis is highly susceptible to environmental stimuli, which allows for activity-dependent regulation of various cognitive functions. A vast range of neural circuits from various brain regions orchestrates these complex cognitive functions. It is therefore important to understand how specific neural circuits regulate adult neurogenesis. Here, we describe a protocol to manipulate neural circuit activity using designer receptor exclusively activated by designer drugs (DREADDs) technology that regulates NSCs and newborn progeny in rodents. This comprehensive protocol includes stereotaxic injection of viral particles, chemogenetic stimulation of specific neural circuits, thymidine analog administration, tissue processing, immunofluorescence labeling, confocal imaging, and imaging analysis of various stages of neural precursor cells. This protocol provides detailed instructions on antigen retrieval techniques used to visualize NSCs and their progeny and describes a simple, yet effective way to modulate brain circuits using clozapine N-oxide (CNO) or CNO-containing drinking water and DREADDs-expressing viruses. The strength of this protocol lies in its adaptability to study a diverse range of neural circuits that influence adult neurogenesis derived from NSCs.

The goal of this protocol is to describe an approach for analyzing behavior of adult neural stem/progenitor cells in response to chemogenetic manipulation of a specific local neural circuit.

Assaying Circuit Spezifische Regulation von adulthippocampal neuralen Vorläuferzellen

Adult neurogenesis is a dynamic process by which newly activated neural stem cells (NSCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate ...