Wir möchten Ryan Maneri (Oystercatcher Productions, LLC) für die Dreharbeiten unsere Verfahren und Rodney McPhail (Department of Biological Sciences, Purdue University) für die Unterstützung bei der Bearbeitung der Dissektion Video danken. Die Forschung im Labor Suter wird durch Zuschüsse aus dem NIH (R01 NS049233) und die Bindley Bioscience-Center an der Purdue University, um DMS unterstützt

Lösungen <ul><li> L15-ASW Zellkulturmedium (1l) <ul><li> 1 Beutel Pulver L15 </li><li> Add 800 ml H 2 O Reinstwasser </li><li> NaCl 400 mM </li><li> MgSO 4 27 mM </li><li> MgCl 2 28 mM </li><li> L-Glutamin 4 mM </li><li> Gentamicin 50 pg / ml </li><li> HEPES 5 mM </li><li> Passen Sie auf pH 7,9 </li><li> Add Tropfen für Tropfen CaCl 2 9,3 mm (Stopp, wenn Niederschläge) </li><li> Add H 2 O Reinstwasser auf 1 l </li><li> Überprüfen Sie Osmolarität (950-1000 mmol / kg) mit Osmometer (Dampfdruck, Wescor # 5520) </li><li> Filter 0,22 um mit einer positiven Druckfiltration Einheit (Filter: Millipore SVGV010RS) </li><li> Lagerung bei 4 ° C für bis zu 1 Monat </li></ul></li><li> Poly-L-Lysin (70-150 kD) 200 pg / ml Stammlösung in sterilem H 2 O Reinstwasser bei -20 o C </li><li> 0,5 M MgCl 2-Lösung </li></ul> Tag 1: Präparation Aplysia Abdominalganglion Vorbereitung der enzymatischen Spaltung Lösung <ol><li> Wiegen 9-10 mg der Dispase Enzym (Worthington, Cat: LS02111) in ein Eppendorf-Röhrchen </li><li> Add 900 ul L15-ASW Kulturmedium und 100 ul sterilem H 2 O Reinstwasser </li><li> Mix und bei Raumtemperatur (RT) </li></ol> Präparation Abdominalganglion <ol><li> Füllen Sie eine 50 ml Spritze mit 0,5 M MgCl 2-Lösung. </li><li> Nehmen Sie ein Tier aus dem Tank, sanft behandeln, um Tieres Farbwerk Abwehrreaktion zu vermeiden. </li><li> Legen Sie die Aplysia auf seiner Seite auf eine Dissektion Bord (Styropor board), rostral Seite auf die rechte, kaudalen Seite nach links. </li><li> Legen Sie die Nadel in Tier unmittelbar hinter dem Kopf und injizieren alle MgCl 2-Lösung in die Körperhöhle. </li><li> Reiben Sie das Tier auf die MgCl 2-Lösung im gesamten Körperhöhle zerstreuen, für ein paar Minuten für das Tier warten, um vollständig betäubt. </li><li> Pin den Kopf und Schwanz des Tieres, um die Zerlegung Board mit zwei Nadeln. </li><li> Verwenden einer Pinzette die Haut anheben, Schnitt durch Haut und Muskel-Schicht mit großen Scheren auf den Kopf und Schwanz, um eine große Öffnung an der Seite zu machen. </li><li> Finden Sie die Abdominalganglion unter der Bürste wie Eierstock. Verwenden einer Pinzette, um den Anschluss Nerven ca. 1 cm vor dem Abdominalganglion halten, schneiden Sie die Nerven mit Dissektion Schere vor der Zange Halteposition und hinter dem Abdominalganglion. </li><li> Legen Sie Ganglion in die Dispase-Lösung und Inkubation für den 15./16 h bei 22 ° C mit einer Temperatur-kontrollierten Wasserbad. </li></ol> Tag 2: Beschichtung von Aplysia Tasche Zellen Herstellung von Poly-Lysin-beschichtete Deckgläschen <ol><li> In einem Fluss Bank, bereiten drei 35mm Petrischalen als Arbeitszeit Gerichte für die Präparation des Ganglion, die sie mit 4 ml L15-ASW Medium jeder. Übertragen Sie die Abdominalganglion aus der Dispase Lösung für eine der Arbeitssprachen Gerichte nach 15,5 Stunden der Verdauung. </li><li> Mit einer Pinzette zu nehmen Säure gereinigt # 1.5 Deckgläser (22x22 mm) in Ethanol gelagert, entfernen Sie Alkohol und sterilisieren Deckgläser durch Abflammen sie kurz über Bunsenbrenner. Lassen Sie sie kurz abkühlen, bevor Sie die Deckgläser in den Kulturschalen. </li><li> Bereiten Sie eine gebogene Spitze Beschichtung durch Biegen einer gelben Spitze über dem Bunsenbrenner (Flamme nur kurz zum Schmelzen von Kunststoff zu vermeiden!). </li><li> Bereiten Sie entsprechende Menge von 20 ug / ml Poly-Lysin-Lösung durch Verdünnung 200 ug / ml Stammlösung mit sterilem H 2 O Reinstwasser. </li><li> Cover jede Deckglas mit 500 ul 20 pg / ml Poly-Lysin und inkubieren für 20 Minuten bei RT. </li><li> Waschen Sie jeden Deckglas 3-5 mal mit 500 ul sterilem H 2 O Reinstwasser jeder. </li><li> Entfernen von Wasser aus Deckgläser; füllen jedes Gericht mit 4 ml L15-ASW Kulturmedium. </li></ol> Die Dissoziation von bag-Zellen <ol><li> Unter einem Dissektionsmikroskop, schneiden Sie die Abdominalganglion in Hälfte mit Dissektion Schere und Pinzette zuvor mit 95% igem Ethanol (Abbildung 1 Zeile 1) gereinigt. </li><li> Auf der einen Hälfte des Ganglion, zwischen Sack Zellverband und die hemiganglion Zellen (Abbildung 1 Zeile 2) geschnitten, und schneiden übrigen Nerven-Erweiterungen auf der anderen Seite der Tasche Zellen (Abbildung 1 Zeile 3). Die Arbeit auf der einen Hälfte des Ganglion zu einer Zeit, lassen Sie die andere Hälfte in die Schüssel, während Zellen, die aus ersten Cluster sind vergoldet werden. </li><li> Mit zwei Zangen (oder einer Pinzette und Schere Dissektion), Release Bag Zellverband, indem Sie neben dem Bindegewebe um die Tasche Zellverband. Cut entfernt Bindegewebe mit Dissektion Schere, wenn nötig. Vorsicht nicht zu quetschen und berühren Sie die Tasche Zellverband zu viel während dieses Prozesses. </li><li> Wenn die meisten der Bindegewebe entfernt ist, übertragen Sie die bag Zellklumpen in einer neuen Arbeitsgruppe Schale mit dem gebogenen gelben Spitze vorbereitet früher. Seien Sie vorsichtig, dass keine der Tasche Zellhaufen innerhalb der gelben Spitze eingefangen werden oder stecken in der Luft / Medium-Schnittstelle. </li><li> Man reibt den Cluster mit gelber Spitze, um einzelne Tüte Zellen zu entfernen. Tun Sie dies durch Pipettieren der Cluster in und aus der Spitze. Beginnen Sie mit geringen Scherkräften und erhöhen Kraft nach Bedarf. Verwenden Sie immer den niedrigsten Kraft benötigt, um Neuronen, ohne töten zu viele Zellen zu entfernen. </li><li> Sammeln Sie 3-5 gesunde Tasche Zellen und übertragen Sie sie auf einen Teller mit einem Poly-Lysin-beschichtete Deckglas. Vermeiden Abholung toten Zellen (sie sind schwarz / transparent in der Mitte) und Bindegewebe Schutt. </li><li> Wiederholen Sie Schritt 6. Ort und ca. 15-25 Live-Neuronen in der Mitte jedes Deckglas. Wenn tote Zellen oder Schmutz sowie transferiert werden, entfernen Sie die unerwünschten Material, indem man es nach oben oder bläst es weg von den gesunden Zellen. Dieser Vorgang dauert ca. 2-3 Stunden für beide Cluster. </li><li> Sobald Sie fertig sind halten die Kulturschalen auf der Flow Bench für mindestens 2 Stunden bei RT, damit Zellen zu befestigen (keep Flow Bench Gebläse ausschalten, um unerwünschte Schwingungen zu vermeiden). </li><li> Die Schalen werden in einem Brutschrank bei 14 ° C über Nacht oder bis zur weiteren Verwendung. Wachstum Kegel kann in 4-10 Stunden zu entwickeln. </li></ol><img src="/files/ftp_upload/old/662_2008_2_10_0/image001.jpg" alt="Abbildung 1. Schematische Darstellung der Aplysia californica Abdominalganglion. Farbe Linien zeigen, wo zu schneiden, um bag Zellhaufen zu erhalten." /> Abbildung 1. Schematische Darstellung der Aplysia californica Abdominalganglion. Farbe Linien zeigen, wo zu schneiden, um bag Zellhaufen zu erhalten. Tag 3: Einrichten Kammer für die Live-Darstellung von Aplysia Tasche Zellen Versammlung der Bildgebung Kammer <ol><li> Vor der Verwendung Tasche Zellen für Live Cell Imaging Experimenten untersuchen Zellen auf einem Low-Power-Mikroskop. Wenn Zellen nicht ein gesundes Wachstum Kegel entwickelt haben, liefern frisches Medium und lassen bei RT für 1-2 Stunden. </li><li> Zur Versorgung Platten mit frischem L15-ASW Medium, entfernen Sie die Hälfte (~ 2 ml) des alten Mediums mit einer Vakuum-Linie, und 2 ml der neuen L15-ASW Medium pro Petrischale. Es ist wichtig, nicht vollständig entfernen Medium aus der Schale auf einmal, da diese Zellen aus dem Deckglas entfernt werden. </li><li> Für Beutel cell imaging, montieren wir zwei Deckgläsern und zwei Abstandhalter aus Kunststoff in einer Sandwich-Imaging-Kammer. Die obere Deckglas in das Sandwich ist kürzer geschnitten, um den einfachen Austausch von Medium und Reagenzien ermöglichen. Bei der Montage sollte Deckgläser mit Tasche Zellen befestigt in L15-ASW Medium jederzeit getaucht werden, um Zellen aus Verdrängen zu verhindern. </li><li> Zur Vorbereitung der Kammer, abgeschnitten ~ 2mm von der rechten Seite eines # 1 Deckgläser 22x22 mm mit Diamant-Stift (Glasschneider) und der Herrscher. </li><li> Mit einer 1 ml Spritze, gelten Hochvakuumfett zu beiden Seiten der kürzeren Deckglas, und befestigen Sie Abstandhalter aus Kunststoff auf beiden Seiten. Bewerben Vakuumfett an die Spitze der beiden Abstandshalter. </li><li> Kehren Sie die Deckglas und spacer Montage, und setzen in Petrischale mit Tasche Zellen auf einem Deckglas. Richten und sanft nach unten drücken, um Top-Deckglas nach unten Deckglas-Stick mit dem hinteren Ende von zwei Q-Tips. </li><li> Mit einer Pinzette vorsichtig aus Sandwich-Montage aus der Petrischale, ohne das Medium austritt. Richten Sie die beiden Deckgläser, falls erforderlich, und wischen Sie überschüssiges Medium mit einem Kimwipe. Legen Sie das Sandwich auf den unteren Teil der Objektträger Halter und befestigen Sie den Deckel vorsichtig mit Clips. </li><li> Reinigen Sie die Mitte der oberen Seite des Sandwich mit Q-Tips abgetupft in Glasreiniger (Sparkle ist als Glasreiniger verwendet). Trocknen schnell aus dem Glasreiniger mit einem zweiten, trockenen Q-Tip, der Streifen Linien zu vermeiden. Machen Sie dasselbe für die untere Deckglas. Saubere Deckgläser frei von Streifen Linien sind für hohe Bildqualität wichtig. </li></ol> Bag-Zellen unter dem Mikroskop Da Aplysia Tasche Zelle neuronalen Kulturen mit geringer Dichte sind, ist es am besten, wenn Ihr Mikroskop Setup Tischpositionierarbeitsweg Funktionen zur Erleichterung der Wechsel zwischen den Zellen. Bag-Zellen können leicht bei RT für mehrere Stunden auf der Bühne gehalten während der Bildgebung Experimente. Austausch des Mediums in regelmäßigen Abständen, um Verdunstung zu vermeiden.

<ol>
	<li>Forscher, P., Kaczmarek, L. K., Buchanan, J. A., Stephen, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+AMP+induces+changes+in+distribution+and+transport+of+organelles+within+growth+cones+of+Aplysia+bag+cell+neurons.">Cyclic AMP induces changes in distribution and transport of organelles within growth cones of Aplysia bag cell neurons.</a> J. Neurosci. 7, 3600-3611 (1987).</li><li>Suter, D. M., Errante, L. D., Belotserkovsky, V., Forscher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Ig+superfamily+cell+adhesion+molecule,+apCAM,+mediates+growth+cone+steering+by+substrate-cytoskeletal+coupling.">The Ig superfamily cell adhesion molecule, apCAM, mediates growth cone steering by substrate-cytoskeletal coupling.</a> J. Cell. Biol. 141, 227-240 (1998).</li><li>Schaefer, A. W., Kabir, N., Forscher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filopodia+and+actin+arcs+guide+the+assembly+and+transport+of+two+populations+of+microtubules+with+unique+dynamic+parameters+in+neuronal+growth+cones.">Filopodia and actin arcs guide the assembly and transport of two populations of microtubules with unique dynamic parameters in neuronal growth cones.</a> J. Cell. Biol. 158, 139-152 (2002).</li><li>Suter, D. M., Schaefer, A. W., Forscher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+dynamics+are+necessary+for+SRC+family+kinase-dependent+growth+cone+steering.">Microtubule dynamics are necessary for SRC family kinase-dependent growth cone steering.</a> Curr. Biol. 14, 1194-1199 (2004).</li></ol>

Aplysia Tasche Zelle Neuronen eine serumfreien neuronalen Zellkultur-System mit sehr wenigen nicht-neuronalen Zellen. Diese Neuronen bilden sehr große Wachstum Kegel geeignet, um eine Reihe von wichtigen zellbiologischen Fragestellungen zu beantworten. Bag Zelle Neuronen können leicht manipuliert werden und abgebildet bei Raumtemperatur über mehrere Stunden. Unter Verwendung von fluoreszierenden Speckle Microscopy (FSM) kann man quantitativ zu analysieren die verschiedenen Parameter des F-Aktin und Mikrotubuli-Polymerisation und Translok...

<table><tbody><tr><td>#1 glass coverslips 22x22 mm</td><td>Tool</td><td>VWR international</td><td>48366067</td><td></td></tr><tr><td>#1.5 glass coverslips 22x22 mm</td><td>Tool</td><td>Corning</td><td>2870-22</td><td></td></tr><tr><td>35 mm Petri dishes</td><td>Tool</td><td>Falcon BD</td><td>353001</td><td></td></tr><tr><td>Filters for medium filtration</td><td>Tool</td><td>EMD Millipore</td><td>SVGV010RS</td><td></td></tr><tr><td>High vacuum grease</td><td>Tool</td><td>Dow Corning</td><td>1597418</td><td></td></tr><tr><td>Osmometer</td><td>Tool</td><td>Wescor</td><td>5520</td><td></td></tr><tr><td>Plastic shims</td><td>Tool</td><td>Small Parts, Inc.</td><td>SHSP-200</td><td></td></tr><tr><td>Calcium chloride</td><td>Reagent</td><td>Fisher Scientific</td><td>C79-500</td><td></td></tr><tr><td>Gentamicin</td><td>Reagent</td><td>Invitrogen</td><td>15750-060</td><td></td></tr><tr><td>HEPES</td><td>Reagent</td><td>Sigma-Aldrich</td><td>H4030</td><td></td></tr><tr><td>L15 medium</td><td>Reagent</td><td>Invitrogen</td><td>41300-039</td><td></td></tr><tr><td>L-glutamine</td><td>Reagent</td><td>Sigma-Aldrich</td><td>G8540</td><td></td></tr><tr><td>Magnesium chloride</td><td>Reagent</td><td>Mallinckrodt Baker Inc.</td><td>5958-04</td><td></td></tr><tr><td>Magnesium sulfate</td><td>Reagent</td><td>Mallinckrodt Baker Inc.</td><td>6070-12</td><td></td></tr><tr><td>Poly-L-lysine (70-150 kD) </td><td>Reagent</td><td>Sigma-Aldrich</td><td>P6282</td><td></td></tr><tr><td>Sodium chloride</td><td>Reagent</td><td>Mallinckrodt Baker Inc.</td><td>7581</td><td></td></tr><tr><td>Neutral Protease (Dispase)</td><td>Reagent</td><td>Worthington Biochemical</td><td>LS02111</td><td></td></tr></tbody></table>

neuronal cell cultures from aplysia for high-resolution imaging of growth cones

Neuronale Wachstum Kegel sind die sehr beweglichen Strukturen an der Spitze der Axone, die Führung cues in der Umgebung zu erkennen und kann transduzieren diese Informationen in gerichtete Bewegung in Richtung der entsprechenden Zielzelle. Um vollständig zu verstehen, wie Führung Informationen von der Zelloberfläche an die zugrunde liegende Dynamik des Zytoskeletts Netzwerke übertragen, benötigt man ein Modell-System eignet sich für Live Cell Imaging von Protein Dynamik bei hoher zeitlicher und räumlicher Auflösung. Typische Wirbeltiere Wachstum Kegel sind zu klein, um quantitativ zu analysieren F-Aktin und Mikrotubuli-Dynamik. Neuronen aus dem Seehasen Aplysia californica sind 5-10 mal größer als Wirbeltiere Neuronen, kann leicht bei Raumtemperatur aufbewahrt werden und sind sehr robust Zellen für Mikromanipulation und biophysikalische Messungen. Ihr Wachstum Kegel sehr zytoplasmatischen Regionen und eine gut beschriebene Zytoskelett-System definiert. Die neuronalen Zellkörpern können mit einer Vielzahl von Sonden zur Untersuchung von Wachstum Kegel Motilität und Führung mikroinjiziert werden. In der vorliegenden Protokoll zeigen wir ein Verfahren für die Präparation des Abdominalganglion, Kultur bag Zelle Neuronen und die Einrichtung eines bildgebenden Kammer für Live Cell Imaging Wachstum Kegel.

Watch this Scientific Journal Video about Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones at JoVE.com

Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Aplysia californica Neuronen entwickeln große Wachstum Kegel in Kultur, die sich hervorragend für hochauflösende Bildgebung des Wachstums Kegel Motilität und Führung sind. Hier präsentieren wir ein Protokoll für die Präparation und Beschichtung von Aplysia Tasche Zelle Neuronen sowie für die Einrichtung einer Kammer für Live Cell Imaging.

Neuronalen Zellkulturen von Aplysia für High-Resolution Imaging of Growth Cones

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information ...

neuronal-cell-cultures-from-aplysia-for-high-resolution-imaging

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

11.4K Aufrufe.  Purdue University. Aplysia californica Neuronen entwickeln große Wachstum Kegel in Kultur, die sich hervorragend für hochauflösende Bildgebung des Wachstums Kegel Motilität und Führung sind. Hier präsentieren wir ein Protokoll für die Präparation und Beschichtung von Aplysia Tasche Zelle Neuronen sowie für die Einrichtung einer Kammer für Live Cell Imaging.

Serie: Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and learning and memory. There are four known families of PKC isoforms in vertebrates: classical PKCs (&alpha;, &beta;I, &beta;II and &gamma;), novel type I PKCs (&#949; and &eta;), novel type II PKCs (&delta; and &theta;), and atypical PKCs (&zeta; and &iota;). The classical PKCs are activated by Ca2+ and diacylclycerol (DAG), while the novel PKCs are activated by DAG, but are Ca2+-independent. The atypical PKCs are activated by neither Ca2+ nor DAG. In Aplysia californica, our model system to study memory formation, there are three nervous system specific PKC isoforms one from each major class, namely the conventional PKC Apl I, the novel type I PKC Apl II and the atypical PKC Apl III. PKCs are lipid-activated kinases and thus activation of classical and novel PKCs in response to extracellular signals has been frequently correlated with PKC translocation from the cytoplasm to the plasma membrane. Therefore, visualizing PKC translocation in real time in live cells has become an invaluable tool for elucidating the signal transduction pathways that lead to PKC activation. For instance, this technique has allowed for us to establish that different isoforms of PKC translocate under different conditions to mediate distinct types of synaptic plasticity and that serotonin (5HT) activation of PKC Apl II requires production of both DAG and phosphatidic acid (PA) for translocation 1-2. Importantly, the ability to visualize the same neuron repeatedly has allowed us, for example, to measure desensitization of the PKC response in exquisite detail 3. In this video, we demonstrate each step of preparing Sf9 cell cultures, cultures of Aplysia sensory neurons have been described in another video article 4, expressing fluorescently tagged PKCs in Sf9 cells and in Aplysia sensory neurons and live-imaging of PKC translocation in response to different activators using laser-scanning microscopy.

In this video, we demonstrate visualization of PKC translocation in living cells using fluorescently tagged PKCs.

Live-Darstellung von PKC-Translokation in Sf9-Zellen und in Aplysia sensorischen Neuronen

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and ...

Forschung

Neurowissenschaften

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific molecular mechanisms are identified, new therapeutic strategies can be developed to treat abnormalities in specific behaviors caused by degenerative diseases or aging of the nervous system. The marine snail Aplysia californica is well suited for the investigations of cellular and molecular basis of behavior because neural circuitry underlying a specific behavior could be easily determined and the individual components of the circuitry could be easily manipulated. These advantages of Aplysia have led to several fundamental discoveries of neurobiology of learning and memory. Here we describe a preparation of the Aplysia nervous system for the electrophysiological and molecular analyses of individual neurons. Briefly, ganglion dissected from the nervous system is exposed to protease to remove the ganglion sheath such that neurons are exposed but retain neuronal activity as in the intact animal. This preparation is used to carry out electrophysiological measurements of single or multiple neurons. Importantly, following the recording using a simple methodology, the neurons could be isolated directly from the ganglia for gene expression analysis. These protocols were used to carry out simultaneous electrophysiological recordings from L7 and R15 neurons, study their response to acetylcholine and quantitating expression of CREB1 gene in isolated single L7, L11, R15, and R2 neurons of Aplysia.

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

Marine snail Aplysia californica has been widely used as a neurobiology model for the studies on cellular and molecular basis of behavior. Here a methodology is described for exploring the nervous system of Aplysia for the electrophysiological and molecular analyses of single neurons of identified neural circuitry.

Aplysia Ganglien-Vorbereitung für elektrophysiologische und molekulare Analysen einzelner Neuronen

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific ...

Biotechnik

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., homosynaptic facilitation) 1. These hypotheses can be tested by simultaneously monitoring changes in intracellular calcium and alterations in synaptic efficacy. We demonstrate how this can be accomplished by combining calcium imaging with intracellular recording techniques. Our experiments are conducted in a buccal ganglion of the mollusc Aplysia californica. This preparation has a number of experimentally advantageous features: Ganglia can be easily removed from Aplysia and experiments use adult neurons that make normal synaptic connections and have a normal ion channel distribution. Due to the low metabolic rate of the animal and the relatively low temperatures (14-16 &deg;C) that are natural for Aplysia, preparations are stable for long periods of time. 
To detect changes in intracellular free calcium we will use the cell impermeant version of Calcium Orange 2 which is easily 'loaded' into a neuron via iontophoresis. When this long wavelength fluorescent dye binds to calcium, fluorescence intensity increases. Calcium Orange has fast kinetic properties 3 and, unlike ratiometric dyes (e.g., Fura 2), requires no filter wheel for imaging. It is fairly photo stable and less phototoxic than other dyes (e.g., fluo-3) 2,4. Like all non-ratiometric dyes, Calcium Orange indicates relative changes in calcium concentration. But, because it is not possible to account for changes in dye concentration due to loading and diffusion, it can not be calibrated to provide absolute calcium concentrations. 
An upright, fixed stage, compound microscope was used to image neurons with a CCD camera capable of recording around 30 frames per second. In Aplysia this temporal resolution is more than adequate to detect even a single spike induced alteration in the intracellular calcium concentration. Sharp electrodes are simultaneously used to induce and record synaptic transmission in identified pre- and postsynaptic neurons. At the conclusion of each trial, a custom script combines electrophysiology and imaging data. To ensure proper synchronization we use a light pulse from a LED mounted in the camera port of the microscope. Manipulation of presynaptic calcium levels (e.g. via intracellular EGTA injection) allows us to test specific hypotheses, concerning the role of intracellular calcium in mediating various forms of plasticity.

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

We demonstrate how changes in the intracellular free calcium concentration and synaptic efficacy can be simultaneously monitored in a ganglion preparation of Aplysia. We image intracellular calcium using a fluorescent dye, Calcium Orange, and induce and monitor synaptic transmission with sharp (intracellular) electrodes.

Überwachen von Änderungen in der intrazellulären Calcium-Konzentration und Synaptic Wirksamkeit in der Muschel<em&gt; Aplysia</em

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., ...

Neural Explant Cultures from Xenopus laevis

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During axon outgrowth, the growth cone must integrate multiple sources of guidance cue information to modulate its cytoskeleton in order to propel the growth cone forward and accurately navigate to find its specific targets1. How this integration occurs at the cytoskeletal level is still emerging, and examination of cytoskeletal protein and effector dynamics within the growth cone can allow the elucidation of these mechanisms. Xenopus laevis growth cones are large enough (10-30 microns in diameter) to perform high-resolution live imaging of cytoskeletal dynamics (e.g.2-4 ) and are easy to isolate and manipulate in a lab setting compared to other vertebrates. The frog is a classic model system for developmental neurobiology studies, and important early insights into growth cone microtubule dynamics were initially found using this system5-7 . In this method8, eggs are collected and fertilized in vitro, injected with RNA encoding fluorescently tagged cytoskeletal fusion proteins or other constructs to manipulate gene expression, and then allowed to develop to the neural tube stage. Neural tubes are isolated by dissection and then are cultured, and growth cones on outgrowing neurites are imaged. In this article, we describe how to perform this method, the goal of which is to culture Xenopus laevis growth cones for subsequent high-resolution image analysis. While we provide the example of +TIP fusion protein EB1-GFP, this method can be applied to any number of proteins to elucidate their behaviors within the growth cone.

Neural Explant Cultures from Xenopus laevis

Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.

Neural Explantatkulturen aus<em&gt; Xenopus laevis</em

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons. The neurons are large (up to 1 mm in diameter) and identifiable, with distinct sizes, shapes, positions and pigmentations, and the cell bodies are externally exposed in five paired ganglia distributed throughout the body of the animal. These properties have allowed investigators to delineate the circuitry underlying specific behaviors in the animal1. The monosynaptic connection between sensory and motor neurons is a central component of the gill-withdrawal reflex in the animal, a simple defensive reflex in which the animal withdraws its gill in response to tactile stimulation of the siphon. This reflex undergoes forms of non-associative and associative learning, including sensitization, habituation and classical conditioning. Of particular benefit to the study of synaptic plasticity, the sensory-motor synapse can be reconstituted in culture, where well-characterized stimuli elicit forms of plasticity that have direct correlates in the behavior of the animal2,3. Specifically, application of serotonin produces a synaptic strengthening that, depending on the application protocol, lasts for minutes (short-term facilitation), hours (intermediate-term facilitation) or days (long-term facilitation). In contrast, application of the peptide transmitter FMRFamide produces a synaptic weakening or depression that, depending on the application protocol, can last from minutes to days (long-term depression). The large size of the neurons allows for repeated sharp electrode recording of synaptic strength over periods of days together with microinjection of expression vectors, siRNAs and other compounds to target specific signaling cascades and molecules and thereby identify the molecular and cell biological steps that underlie the changes in synaptic efficacy.
An additional advantage of the Aplysia culture system comes from the fact that the neurons demonstrate synapse-specificity in culture4,5. Thus, sensory neurons do not form synapses with themselves (autapses) or with other sensory neurons, nor do they form synapses with non-target identified motor neurons in culture. The varicosities, sites of synaptic contact between sensory and motor neurons, are large enough (2-7 microns in diameter) to allow synapse formation (as well as changes in synaptic morphology) with target motor neurons to be studied at the light microscopic level.
In this video, we demonstrate each step of preparing sensory-motor neuron cultures, including anesthetizing adult and juvenile Aplysia, dissecting their ganglia, protease digestion of the ganglia, removal of the connective tissue by microdissection, identification of both sensory and motor neurons and removal of each cell type by microdissection, plating of the motor neuron, addition of the sensory neuron and manipulation of the sensory neurite to form contact with the cultured motor neuron.

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

Primary cultures of Aplysia sensory-motor neurons provide a model preparation for studying synapse formation and synaptic plasticity in vitro. This video demonstrates the identification and microdissection of sensory and motor neurons from Aplysia ganglia as well as the methods for establishing and maintaining sensory-motor neurons in culture.

Vorbereitung der Aplysia Sensory-Motor neuronalen Zellkulturen

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons.  The neurons are large (up ...

Biologie

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

Isolation von sensorischen Neuronen von<em&gt; Aplysia californica</em&gt; Für Patch-Clamp-Recordings von Glutamaterge Currents

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis of primary cultures of neurons. We describe a set of procedures for: labeling newborn mice to be genotyped, rapid genotyping, and establishing low-density cultures of brain neurons from these mice. Individual mice are labeled by tattooing, which allows for long-term identification lasting into adulthood. Genotyping by the described protocol is fast and efficient, and allows for automated extraction of nucleic acid with good reliability. This is useful under circumstances where sufficient time for conventional genotyping is not available, e.g., in mice that suffer from neonatal lethality. Primary neuronal cultures are generated at low density, which enables imaging experiments at high spatial resolution. This culture method requires the preparation of glial feeder layers prior to neuronal plating. The protocol is applied in its entirety to a mouse model of the movement disorder DYT1 dystonia (&#916;E-torsinA knock-in mice), and neuronal cultures are prepared from the hippocampus, cerebral cortex and striatum of these mice. This protocol can be applied to mice with other genetic mutations, as well as to animals of other species. Furthermore, individual components of the protocol can be used for isolated sub-projects. Thus this protocol will have wide applications, not only in neuroscience but also in other fields of biological and medical sciences.

We describe procedures for labeling and genotyping newborn mice and generating primary neuronal cultures from them. The genotyping is rapid, efficient and reliable, and allows for automated nucleic-acid extraction. This is especially useful for neonatally lethal mice and their cultures that require prior completion of genotyping.

Schnelle Genotypisierung der Tiere nach Aufbau Primärkulturen von Neuronen des Gehirns Gefolgt

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis ...

Rewiring Neuronal Circuits: A New Method for Fast Neurite Extension and Functional Neuronal Connection

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and accurately reconnect them with an appropriate target. Here a procedure is described to rapidly initiate, elongate, and precisely connect new functional neuronal circuits over long distances. The extension rates achieved reach over 1.2 mm/h, 30-60 times faster than the in vivo rates of the fastest growing axons from the peripheral nervous system (0.02 to 0.04 mm/h)28 and 10 times faster than previously reported for the same neuronal type at an earlier stage of development4. First, isolated populations of rat hippocampal neurons are grown for 2-3 weeks in microfluidic devices to precisely position the cells, enabling easy micromanipulation and experimental reproducibility. Next, beads coated with poly-D-lysine (PDL) are placed on neurites to form adhesive contacts and pipette micromanipulation is used to move the resulting bead-neurite complex. As the bead is moved, it pulls out a new neurite that can be extended over hundreds of micrometers and functionally connected to a target cell in less than 1 h. This process enables experimental reproducibility and ease of manipulation while bypassing slower chemical strategies to induce neurite growth. Preliminary measurements presented here demonstrate a neuronal growth rate far exceeding physiological ones. Combining these innovations allows for the precise establishment of neuronal networks in culture with an unprecedented degree of control. It is a novel method that opens the door to a plethora of information and insights into signal transmission and communication within the neuronal network as well as being a playground in which to explore the limits of neuronal growth. The potential applications and experiments are widespread with direct implications for therapies that aim to reconnect neuronal circuits after trauma or in neurodegenerative diseases.

This procedure describes how to rapidly initiate, extend and connect neurites organized in microfluidic chambers using poly-D-lysine-coated beads fixed to micropipettes that guide neurite elongation.

Neuronale Schaltungen neu verdrahten: Eine neue Methode zur schnellen Neuritenverlängerung und funktionellen neuronalen Verbindung

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and ...

Post-differentiation Replating of Human Pluripotent Stem Cell-derived Neurons for High-content Screening of Neuritogenesis and Synapse Maturation

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model system to explore disease mechanisms and carry out high content screening (HCS) to interrogate compound libraries or identify gene mutation phenotypes. However, with human cells the transition from NPC to functional, mature neuron requires several weeks. Synapses typically start to form after 3 weeks of differentiation in monolayer culture, and several neuron-specific proteins, for example the later expressing pan-neuronal marker NeuN, or the layer 5/6 cerebral cortical neuron marker CTIP2, begin to express around 4-5 weeks post-differentiation. This lengthy differentiation time can be incompatible with optimal culture conditions used for small volume, multi-well HCS platforms. Among the many challenges are the need for well-adhered, uniformly distributed cells with minimal cell clustering, and culture procedures that foster long-term viability and functional synapse maturation. One approach is to differentiate neurons in a large volume format, then replate them at a later time point in HCS-compatible multi-wells. Some main challenges when using this replating approach concern reproducibility and cell viability, due to the stressful disruption of the dendritic and axonal network. Here we demonstrate a detailed and reliable procedure for enzymatically resuspending human induced pluripotent stem cell (hiPSC)-derived neurons after their differentiation for 4-8 weeks in a large-volume format, transferring them to 384-well microtiter plates, and culturing them for a further 1-3 weeks with excellent cell survival. This replating of human neurons not only allows the study of synapse assembly and maturation within two weeks from replating, but also enables studies of neurite regeneration and growth cone characteristics. We provide examples of scalable assays for neuritogenesis and synaptogenesis using a 384-well platform.

This protocol describes a detailed procedure for resuspending and culturing human stem cell derived neurons that were previously differentiated from neural progenitors in vitro for multiple weeks. The procedure facilitates imaging-based assays of neurites, synapses, and late-expressing neuronal markers in a format compatible with light microscopy and high-content screening.

Post-Differenzierung Replating von Human Pluripotent Stem Cell abgeleiteten Neuronen für High-Content Screening von Neuritogenese und Synapse Reifung

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model ...

Use of Primary Cultured Hippocampal Neurons to Study the Assembly of Axon Initial Segments

Neuronal axon initial segments (AIS) are sites of initiation of action potentials and have been extensively studied for their molecular structure, assembly and activity-dependent plasticity. Giant ankyrin-G, the master organizer of AIS, directly associates with membrane-spanning voltage gated sodium (VSVG) and potassium channels (KCNQ2/3), as well as 186 kDa neurofascin, a L1CAM cell adhesion molecule. Giant ankyrin-G also binds to and recruits cytoplasmic AIS molecules including beta-4-spectrin, and the microtubule-binding proteins, EB1/EB3 and Ndel1. Giant ankyrin-G is sufficient to rescue AIS formation in ankyrin-G deficient neurons. Ankyrin-G also includes a smaller 190 kDa isoform located at dendritic spines instead of the AIS, which is incapable of targeting to the AIS or rescuing the AIS in ankyrin-G-deficient neurons. Here, we described a protocol using cultured hippocampal neurons from ANK3-E22/23-flox mice, which, when transfected with Cre-BFP exhibit loss of all isoform of ankyrin-G and impair the formation of AIS. Combined a modified Banker glia/neuron co-culture system, we developed a method to transfect ankyrin-G null neurons with a 480 kDa ankyrin-G-GFP plasmid, which is sufficient to rescue the formation of AIS. We further employ a quantification method, developed by Salzer and colleagues to deal with variation in AIS distance from the neuronal cell bodies that occurs in hippocampal neuron cultures. This protocol allows quantitative studies of the de novo assembly and dynamic behavior of AIS.

Here, we described a protocol to quantitatively study the assembly and structure of the axon initial segments (AIS) of hippocampal neurons that lack pre-assembled AIS due to the absence of a giant ankyrin-G.

Neuronalen Zellkulturen von Aplysia für High-Resolution Imaging of Growth Cones

Zusammenfassung

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