Diese Studie wurde von FMMU neue Erkenntnis Stiftung und teilweise NIH finanziell unterstützt.

1. Glia Cell Culture Gliazellen können aus verschiedenen Regionen des zentralen Nervensystems gezüchtet werden. Der gesamte Prozess ist in Bearbeitung Abbildung dargestellt. <img src="/files/ftp_upload/2111/2111process.jpg" alt="Prozess 1" /> Tag 1 Coating Kultur Platte und Deckgläser <ol><li> Dry sterilisiert Glas Mikroskop runde Deckgläser in einem Autoklaven. </li><li> Platte der sterilisierten Deckgläschen in sterilisierte 24-well Platten. </li><li> Bestreichen Sie die Deckgläser mit Poly-Lysin und inkubieren für 2 Stunden unter root Temperatur. </li><li> Coat 6-well-Platten in der gleichen Weise wie in Schritt 3. </li><li> Waschen Sie die Deckgläser und 6-Loch-Kulturplatte zweimal mit destilliertem Wasser ab und trocknen in Kultur Kapuze. </li><li> Geben Sie 200 ul DMEM Medium (mit 10% FBS) in jedes Well in 24-Well-Platte und 2 ml in jedes Well in 6-Well-Platte und setzte sie in Inkubator unter 37 Grad mit 5% CO 2 </li></ol> Tag 2 Isolating Kortex und Gliazellen Kultur <ol><li> Sterilisieren Sie die positive flow Dissektion Kapuze. </li><li> Schalten Sie UV-Licht für 20 min. </li><li> Spray alle Oberflächen mit 70% Ethanol und warten Sie 15 min vor dem Gebrauch. </li><li> Wechsel angespannt: Anesthetize Rattenjungen (P2-P6) durch Hypothermie und enthaupten an der Basis der framen magnum mit Betriebssystem Schere. </li><li> Öffnen der Schädeldecke entlang der Sagittalnaht mit einem Iris-Schere und ziehen Sie den Schädel. </li><li> Entfernen Sie das Vorderhirn und steckte sie in ein gekühltes L15 Medium unter Stereomikroskop, sorgfältig reinigen Sie die Dura und Pia Membran mit Blutgefäßen, isolieren die Hirnrinde und waschen mehrmals mit L15-Medium. </li><li> Schneiden Sie die Hirnrinde in kleine Stücke mit mikrochirurgischen Schere. </li><li> Add 0,125% Trypsin-EDTA mit L15 Medium in Cortex Stücke und inkubieren vorbereitet in 37 Grad für 15 min. </li><li> Transfer-Gewebe-Blöcke in 20 mL DMEM-Medium mit 20% FBS in 50ml Tube, fügen DNase-Stammlösung zu einer Endkonzentration 10ug/mL absaugen Gewebe Lösung nach oben und unten für 20-mal mit dem Feuer aus poliertem Glas Pasteure Pipette, sammeln die einzelnen Zellen Suspension. </li><li> Waschen Sie die Zellsuspension einmal mit DMEM-Medium, resuspendieren Zellen in DMEM mit 10% FBS, count-Zellen unter dem Mikroskop mit einer Zählkammer, Samenzellen zu 5000-10000/cm 2. Pflegen Sie Zellen in einer 37 Grad 5% Inkubator, ändern die Hälfte des Mediums 2 mal pro Woche. </li></ol> 2. Spinalganglien Neuronen Isolation, Kultur und Reinigung Tag 1 Bereiten Kultur Werkstoff <ol><li> Bestreichen Sie die sterilisierten Deckgläschen mit Poly-Lysin, waschen Deckgläser zweimal mit destillieren Wasser und der Luft trocknen, steckte sie in 24-Well-Platten. </li><li> Add 100 &amp;mgr; Neurobasal Medium mit 2% B27 und 2.5S NGF (50 ng / mL), legte Platte in 37 Grad 5% CO 2. </li></ol> Tag 2 Isolate DRGs aus Embryonen <ol><li> Sterilisiert die Abzugshaube in der gleichen Weise wie Gliazellen Kultur. </li><li> Euthanize eine schwangere Ratte (E15) von Überdosierung mit CO 2, sterilisiert Bauch von 70% Ethanol, wurden Embryonen aus der Gebärmutter isoliert und in gekühlten L15 Medium. </li><li> Isolieren Rückenmark mit angeschlossenem Spinalganglien unter Stereomikroskop und Transfer zum 35mm Gerichte mit gekühltem L15 Medium. </li><li> Sammeln Sie einzelne Zellsuspension und waschen einmal mit NBF Medium (Neurobasal Medium mit 2% B27 und 2.5S NGF (50 ng / mL)). </li></ol> Tag 3 Purify DRG-Neuronen <ol><li> 18h-24h nach Neuron Aussaat, fügen Sie eine 1 mM konzentriert FUDR Stammlösung auf das Neuron Kultur bis zu einer Endkonzentration von 20 uM und ein Endvolumen von 200 ul. </li><li> 72h später ersetzen die Hälfte des Mediums mit Neurobasal Medium mit 2% B27 und 2.5S NGF (50ng/mL) ohne FUDR. Danach wechseln das Medium jeden zweiten Tag. </li></ol> 3. Cokultur DRG-Neuronen mit Gliazellen <ol><li> Wenn Gliazellen Kultur Konfluenz (ca. 20 Tage nach der Aussaat) erreichten, waren sie bereit für Kokultur mit Neuronen. </li><li> 24 Stunden vor der Zugabe des gereinigten Neuronen wurden Gliazellen mittel-bis Neurobasal Medium mit 2% B27 und 2.5S NGF (50 ng / mL) geändert. </li><li> Gereinigtes Neuronen aus Kultur-Wells wurden gesammelt, wurden einzelne Zellen durch mechanische, die durch Feuer poliert Pasteure Pipette erhalten. </li><li> Nach Auszählung der Zahl, wurden Neuronen bei 500/cm 2 auf konfluenten Gliazellen in Neurobasal Medium mit 2% B27 und 2.5S NGF (50ng/mL) ausgesät. </li><li> Neurons Haftung und Neuritenwachstum auf Gliazellen konnte aufgezeichnet und analysiert werden zu verschiedenen Zeitpunkten durch Bildanalyse-Software und Immunzytochemie mit Zelltyp spezifischen Antikörpern. </li></ol> 4. Repräsentative Ergebnisse <ol><li> Gliazelle Kultur: Nach dem Impfen wird Gliazellen konfluieren rund 20 Tage. Unter Phasenkontrast Mikroskopopa, es war leicht zu erkennen, dass verschiedene morphologische Gliazellen Subpopulationen unterschiedliche Wachstumsmuster Unterkonstruktionen gebildet und die GFAP positive Astrozyten einen Anteil von mehr als 90%, wie durch immuocytochemisty Technik (Bild 1, Bild 2) gezeigt. </li><li> DRG-Neuronen Kultur: In unserer Methode, nach 72 Stunden FUDR Behandlung, die Reinheit des DRG-Neuronen wird so hoch wie 99% innerhalb von 6 Tagen, zeigte DRG-Neuronen ihrer einzigartigen Morphologie und mit hoher Dichte Neuritenwachstum (Abbildung 3, Abbildung 4). </li><li> Gliazelle und Neuronen Kokultur: DRG-Neuronen Haftung und Neuritenwachstum aufgetreten leicht auf Gliazellen innerhalb von 4 Stunden nach der Aussaat, sorgfältige Beobachtungen haben gezeigt, dass Neuronen Haftung und Neuritenwachstum von Glia-Zell-Subpopulationen, die spezielle Wachstumsmuster Unterkonstruktionen entstanden beeinflusst, könnte dies leicht identifiziert werden unter Phasenkontrast-Mikroskop und durch Immunzytochemie (Abbildung 5, Abbildung 6). </li></ol><img src="/files/ftp_upload/2111/2111fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Morphologie der konfluenten Gliazellen. Kortikale Gliazellen auf Polylysin beschichteten Deckglas wurden ausplattiert und kultiviert für 20 Tage, beachten Sie die unterschiedlichen Wachstumsmuster von Gliazellen, Zellen auf der linken Seite in einer Strahlungsleistung Weise angeordnet. <img src="/files/ftp_upload/2111/2111fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Confluent Gliazellen durch sauren Gliafaserproteins (GFAP)-Antikörper markiert. Beachten Sie die abgestrahlte Anordnung von GFAP (rot) positive Zellen auf der rechten Seite. <img src="/files/ftp_upload/2111/2111fig3.jpg" alt="Abbildung 3" /> Abbildung 3. Spinalganglien Neuronen wuchs in vitro ohne FUDR Behandlung. Kontaminierenden Zellen gebildet DRG-Neuronen &quot;Hintergrund. <img src="/files/ftp_upload/2111/2111fig4.jpg" alt="Abbildung 4" /> Abbildung 4. Spinalganglien Neuronen wuchs in vitro nach FUDR Behandlung. Der Hintergrund kontaminierenden Zellen waren vollständig eliminiert. <img src="/files/ftp_upload/2111/2111fig5.jpg" alt="Abbildung 5" /> Abbildung 5. Spinalganglien Neuronen auf Glia-Zellen gezüchtet. Neurons Haftung und Neuritenwachstum wurden auf die abgestrahlte angeordneten Zellen gehemmt und begrenzt auf der rechten Seite Gliazellen. <img src="/files/ftp_upload/2111/2111fig6.jpg" alt="Abbildung 6" /> Abbildung 6. Spinalganglien Neuronen wachsen auf Gliazellen durch Neurofilament Antikörper markiert. Die Neuriten (grün) auf die abgestrahlte angeordnet linken Seite Gliazellen waren gehemmt und begrenzt auf der rechten Seite, waren alle Gliazellen durch GFAP Antikörper (rot) gekennzeichnet.

<ol>
	<li>Rossignol, S., Schwab, M., Schwartz, M., Fehlings, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+cord+injury:+time+to+move?.">Spinal cord injury: time to move?.</a> J Neurosci. 27 (44), 11782-11792 (2007).</li><li>Yiu, G., He, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+inhibition+of+CNS+axon+regeneration.">Glial inhibition of CNS axon regeneration.</a> Nat Rev Neurosci. 7 (8), 617-627 (2006).</li><li>Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mystery+and+magic+of+glia:+a+perspective+on+their+roles+in+health+and+disease.">The mystery and magic of glia: a perspective on their roles in health and disease.</a> Neuron. 60 (3), 430-440 (2008).</li><li>Matyash, V., Kettenmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+in+astrocyte+morphology+and+physiology.">Heterogeneity in astrocyte morphology and physiology.</a> Brain Res Rev. , (2009).</li><li>Wanner, I. B., Deik, A., Torres, M., Rosendahl, A., Neary, J. T., Lemmon, V. P., Bixby, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+in+vitro+model+of+the+glial+scar+inhibits+axon+growth.+Glia.">A new in vitro model of the glial scar inhibits axon growth. Glia.</a> 56 (15), 1691-1709 (2008).</li><li>Kuffler, D. P., Sosa, I. J., Reyes, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schwann+cell+chondroitin+sulfate+proteoglycan+inhibits+dorsal+root+ganglion+neuron+neurite+outgrowth+and+substrate+specificity+via+a+soma+and+not+a+growth+cone+mechanism.">Schwann cell chondroitin sulfate proteoglycan inhibits dorsal root ganglion neuron neurite outgrowth and substrate specificity via a soma and not a growth cone mechanism.</a> J Neurosci Res. 87 (13), 2863-2871 (2009).</li><li>Kleitman, N., Wood, P. M., Bunge, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+culture+method+for+the+study+of+myelination.">Tissue culture method for the study of myelination.</a> Culturing nerve cells. , P559-P559 (1998).</li></ol>

This experiment protocol was designed to reach two goals to study glial cells, especially astrocyte heterogeneity's influence on neuron adhesion and neurite growth. The first goal was to maintain astrocyte heterogeneity as much as possible, in this experiment, the confluent glial cell culture enriched astrocytes was mixed primary culture without any chemical treatment and digestion propagation, which may further damage the cell and induce injury response of astrocytes. The final astrocyte purity is more than 90% GFAP pos...

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>DMEM(high glucose)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10313-039</td>
<td>&nbsp;</td>
<tr>
<td>L15 medium</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11415-114</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10437-077</td>
<td>&nbsp;</td>
<tr>
<td>Neurobasal Medium</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>21103-049</td>
<td>&nbsp;</td>
<tr>
<td>poly-lysine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P4832</td>
<td>culture grade</td>
</tr>
<tr>
<td>NGF(2.5S)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>13257-019</td>
<td>&nbsp;</td>
<tr>
<td>B27 supplyment</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17504-044</td>
<td>&nbsp;</td>
<tr>
<td>0.25% trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25200-056</td>
<td>&nbsp;</td>
<tr>
<td>FUDR</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F0503</td>
<td>&nbsp;</td>
<tr>
<td>neurofilament antibody</td>
<td>&nbsp;</td>
<td>Abcam</td>
<td>ab24575</td>
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study glial cell heterogeneity influence on axon growth using a new coculture method

In dem zentralen Nervensystem von Säugetieren, sind abgetrennte Axone nach einer Verletzung nicht in ihre ursprünglichen Ziele und funktionelle Wiederherstellung zu regenerieren ist sehr schlecht 1. Das Scheitern der Axonregeneration ist ein kombiniertes Ergebnis von mehreren Faktoren ab, einschließlich der feindlichen Gliazellen Umwelt, hemmende Myelin verwandten Molekülen und verminderte innere Neuron regenerative Kapazität 2. Astrozyten sind der vorherrschende Typ Gliazellen im zentralen Nervensystem und spielen eine wichtige Rolle bei der Axon-Funktionen unter Physiologie und Pathologie Bedingungen 3. Gegensatz zu den homologen Oligodendrozyten, Astrozyten sind eine heterogene Zellpopulation durch verschiedene Astrozyten Subpopulationen mit unterschiedlichen Morphologien und Genexpression 4 zusammen. Die funktionelle Bedeutung dieser Heterogenität, wie ihre Einflüsse auf die Axon-Wachstum, ist weitgehend unbekannt. Zur Untersuchung der Gliazellen, vor allem die Funktion der Astrozyten Heterogenität in Neuron Verhalten, haben wir eine neue Methode durch Co-Kultivierung hoch gereinigten Spinalganglien Neuronen mit Gliazellen aus der Ratte Cortex erhalten. Mit dieser Technik konnten wir den direkten Vergleich Neuron Haftung und Axon-Wachstum auf verschiedenen Astrozyten Subpopulationen unter den gleichen Bedingungen. In diesem Bericht geben wir die ausführliche Protokoll dieser Methode für Astrozyten Isolation und Kultur, Spinalganglien Neuronen Isolierung und Aufreinigung, und die Co-Kultur von DRG-Neuronen mit Astrozyten. Diese Methode könnte auch auf andere Hirnregionen erweitert werden, um zelluläre oder regional spezifische Wechselwirkung zwischen Neuronen und Gliazellen zu studieren.

Watch this Scientific Journal Video about Study Glial Cell Heterogeneity Influence on Axon Growth Using a New Coculture Method at JoVE.com

Study Glial Cell Heterogeneity Influence on Axon Growth Using a New Coculture Method

In diesem Protokoll beschrieben wir eine neue Methode, um den Einfluss von Gliazellen Heterogenität auf Axon Wachstum mit einer Studie<em&gt; In-vitro-</em&gt; Co-Kultur-System. Rat kortikalen Gliazellen wurden bis zur Konfluenz kultiviert und kokultiviert mit hoch gereinigtem Ratten Spinalganglien Neuronen. Verschiedene Gliazellen Einfluss auf Neuronen Haftung und Axon-Wachstum wurde direkt in der gleichen Kultur verglichen. Diese Methode bietet eine neue Möglichkeit, direkt Studie der Gliazellen Heterogenität Einfluss auf Neuron Adhäsion und Wachstum der Axone.

Study Gliazellen Heterogenität Einfluss auf Axon Wachstum Mit einer neuen Methode Cokultur

In the central nervous system of all mammals, severed axons after injury are unable to regenerate to their original targets and functional recovery is ...

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Research

JoVE Journal

Neuroscience

17.2K Aufrufe.  Cedars Sinai Medical Center, UCLA. In diesem Protokoll beschrieben wir eine neue Methode, um den Einfluss von Gliazellen Heterogenität auf Axon Wachstum mit einer Studie In-vitro- Co-Kultur-System. Rat kortikalen Gliazellen wurden bis zur Konfluenz kultiviert und kokultiviert mit hoch gereinigtem Ratten Spinalganglien Neuronen. Verschiedene Gliazellen Einfluss auf Neuronen Haftung und Axon-Wachstum wurde direkt in der gleichen Kultur verglichen. Diese Methode bietet eine neue Möglichkeit, ...

Serie: Study Glial Cell Heterogeneity Influence on Axon Growth Using a New Coculture Method

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved4. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth5-7.
Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons9.

An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.

Genetic Study of Axonregeneration mit kultivierten adulten Spinalganglienneuronen

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

Forschung

Neurowissenschaften

A Caenorhabditis elegans Model System for Amylopathy Study

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of Alzheimer's disease (AD) and is found in Lewy body dementia, inclusion body myositis and cerebral amyloid angiopathy 1-4. Amylopathies progressively develop with time. For this reason simple organisms with short lifespans may help to elucidate molecular aspects of these conditions. Here, we describe experimental protocols to study A&beta;-mediated neurodegeneration using the worm Caenorhabditis elegans. Thus, we construct transgenic worms by injecting DNA encoding human A&beta;42 into the syncytial gonads of adult hermaphrodites. Transformant lines are stabilized by a mutagenesis-induced integration. Nematodes are age synchronized by collecting and seeding their eggs. The function of neurons expressing A&beta;42 is tested in opportune behavioral assays (chemotaxis assays). Primary neuronal cultures obtained from embryos are used to complement behavioral data and to test the neuroprotective effects of anti-apoptotic compounds.

A Caenorhabditis elegans Model System for Amylopathy Study

We describe methods to study aspects of amylopathies in the worm C. elegans. We show how to construct worms expressing human A&beta;42 in neurons and how to test their function in behavioral assays. We further show how to obtain primary neuronal cultures that can be used for pharmacological testing.

A<em&gt; Caenorhabditis elegans</em&gt; Modell-System für Amylopathy Study

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of ...

A Method to Make a Craniotomy on the Ventral Skull of Neonate Rodents

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain in adulthood and during development. Although most in vivo approaches use a craniotomy to study brain regions located on the dorsal side, brainstem regions such as the pons, located on the ventral side remain relatively understudied. The main goal of this protocol is to facilitate access to ventral brainstem structures so that they can be studied in vivo using electrophysiological and imaging methods. This approach allows study of structural changes in long-range axons, patterns of electrical activity in single and ensembles of cells, and changes in blood brain barrier permeability in neonate animals. Although this protocol has been used mostly to study the auditory brainstem in neonate rats, it can easily be adapted for studies in other rodent species such as neonate mice, adult rodents and other brainstem regions.

A surgical method is described to expose the ventral skull in neonate rats. Using this approach it is possible to open a craniotomy to perform acute electrophysiology and two-photon microscopy experiments in the brainstem of anesthetized pups.

Eine Methode, um eine Kraniotomie auf der Bauch Schädel des Neugeborenen Nagetiere Stellen

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

Induktion von Plastizität Astrozytäre Rezeptoren durch Manipulation der neuronalen Feuerraten

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco&#39;s modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d&#160;(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how&#160;to establish primary mixed glial cultures from adult mouse spinal cord tissue.

Herstellung von primären gemischten Gliazellen Kulturen von erwachsenen Maus Rückenmarksgewebe

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

Linear Agarose Kanäle zu studieren<em&gt; Drosophila</em&gt; Larval Krabbeln Verhalten

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

New Methods to Study Gustatory Coding

The sense of taste allows animals to detect chemicals in the environment, giving rise to behaviors critical for survival. When Gustatory Receptor Neurons (GRNs) detect tastant molecules, they encode information about the identity and concentration of the tastant as patterns of electrical activity that then propagate to follower neurons in the brain. These patterns constitute internal representations of the tastant, which then allow the animal to select actions and form memories. The use of relatively simple animal models has been a powerful tool to study basic principles in sensory coding. Here, we propose three new methods to study gustatory coding using the moth Manduca sexta. First, we present a dissection procedure for exposing the maxillary nerves and the subesophageal zone (SEZ), allowing recording of the activity of GRNs from their axons. Second, we describe the use of extracellular electrodes to record the activity of multiple GRNs by placing tetrode wires directly into the maxillary nerve. Third, we present a new system for delivering and monitoring, with high temporal precision, pulses of different tastants. These methods allow the characterization of neuronal responses in vivo directly from GRNs before, during and after tastants are delivered. We provide examples of voltage traces recorded from multiple GRNs, and present an example of how a spike sorting technique can be applied to the data to identify the responses of individual neurons. Finally, to validate our recording approach, we compare extracellular recordings obtained from GRNs with tetrodes to intracellular recordings obtained with sharp glass electrodes.

We present three new methods to study gustatory coding. Using a simple animal, the moth Manduca sexta (Manduca), we describe a dissection protocol, the use of extracellular tetrodes to record the activity of multiple gustatory receptor neurons, and a system for delivering and monitoring precisely timed pulses of tastants.

Neue Methoden zum Studieren der Gustatory Coding

The sense of taste allows animals to detect chemicals in the environment, giving rise to behaviors critical for survival. When Gustatory Receptor Neurons ...

Visualization of Motor Axon Navigation and Quantification of Axon Arborization In Mouse Embryos Using Light Sheet Fluorescence Microscopy

Spinal motor neurons (MNs) extend their axons to communicate with their innervating targets, thereby controlling movement and complex tasks in vertebrates. Thus, it is critical to uncover the molecular mechanisms of how motor axons navigate to, arborize, and innervate their peripheral muscle targets during development and degeneration. Although transgenic Hb9::GFP mouse lines have long served to visualize motor axon trajectories during embryonic development, detailed descriptions of the full spectrum of axon terminal arborization remain incomplete due to the pattern complexity and limitations of current optical microscopy. Here, we describe an improved protocol that combines light sheet fluorescence microscopy (LSFM) and robust image analysis to qualitatively and quantitatively visualize developing motor axons. This system can be easily adopted to cross genetic mutants or MN disease models with Hb9::GFP lines, revealing novel molecular mechanisms that lead to defects in motor axon navigation and arborization.

Here, we describe a protocol for visualizing motor neuron projection and axon arborization in transgenic Hb9::GFP mouse embryos. After immunostaining for motor neurons, we used light sheet fluorescence microscopy to image embryos for subsequent quantitative analysis. This protocol is applicable to other neuron navigation processes in the central nervous system.

Visualisierung von motorischen Axon Navigation und Quantifizierung der Axon Arborization In Mausembryonen mit Fluoreszenz-Lichtblattmikroskopie

Spinal motor neurons (MNs) extend their axons to communicate with their innervating targets, thereby controlling movement and complex tasks in ...

Using Optical Coherence Tomography and Optokinetic Response As Structural and Functional Visual System Readouts in Mice and Rats

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for the investigation of processes of neurodegeneration, neuroprotection and neuro-repair involving the visual system, as these often correlate well with retinal changes. As a functional readout, visually evoked compensatory eye and head movements are commonly used in experimental models involving the visual function. Combining both techniques allows a quantitative in vivo investigation of structure and function, which can be used to investigate the pathological conditions or to evaluate the potential of novel therapeutics. A great benefit of the presented techniques is the possibility to perform longitudinal analyses allowing the investigation of dynamic processes, reducing variability and cuts down the number of animals needed for the experiments. The protocol described aims to provide a manual for acquisition and analysis of high quality retinal scans of mice and rats using a low cost customized holder with an option to deliver inhalational anesthesia. Additionally, the proposed guide is intended as an instructional manual for researchers using optokinetic response (OKR) analysis in rodents, which can be adapted to their specific needs and interests.

A detailed protocol for the assessment of structural and visual readouts in rodents by optical coherence tomography and optokinetic response is presented. The results provide valuable insights for ophthalmologic as well as neurologic research.

Mit optischen Kohärenztomographie und optokinetischen Antwort als strukturelle und funktionelle visuelles System auslesen bei Mäusen und Ratten

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for ...

A Method for 3D Reconstruction and Virtual Reality Analysis of Glial and Neuronal Cells

Serial sectioning and subsequent high-resolution imaging of biological tissue using electron microscopy (EM) allow for the segmentation and reconstruction of high-resolution imaged stacks to reveal ultrastructural patterns that could not be resolved using 2D images. Indeed, the latter might lead to a misinterpretation of morphologies, like in the case of mitochondria; the use of 3D models is, therefore, more and more common and applied to the formulation of morphology-based functional hypotheses. To date, the use of 3D models generated from light or electron image stacks makes qualitative, visual assessments, as well as quantification, more convenient to be performed directly in 3D. As these models are often extremely complex, a virtual reality environment is also important to be set up to overcome occlusion and to take full advantage of the 3D structure. Here, a step-by-step guide from image segmentation to reconstruction and analysis is described in detail.

The described pipeline is designed for the segmentation of electron microscopy datasets larger than gigabytes, to extract whole-cell morphologies. Once the cells are reconstructed in 3D, customized software designed around individual needs can be used to perform a qualitative and quantitative analysis directly in 3D, also using virtual reality to overcome view occlusion.

Eine Methode zur 3D-Rekonstruktion und Virtual Reality-Analyse von Glia- und Neuronalzellen

Serial sectioning and subsequent high-resolution imaging of biological tissue using electron microscopy (EM) allow for the segmentation and reconstruction ...