This research was support by an Aims2Cure, UK and a UCL Impact Award Ph.D. studentship to JMP and an MRC Capacity Building Ph.D. studentship in Dementia to JMP.

Alle hierin beschriebenen Experimente wurden in Übereinstimmung mit den United Kingdom Animals (Scientific Procedures) Act von 1986 durchgeführt. 1. Herstellung des Instruments, Kultur, Medien und Gerichte <ol><li> Bereiten Sie zwei Edelstahl Labor Präparierscheren und zwei Edelstahl-Laborzangen. Autoklaven alle Instrumente und in eine Kultur Haube O / N unter ultraviolettem Licht (UV) Licht für die Sterilisation. </li><li> Bilden 500 ml Minimum Essential Medium (MEM) Kulturmedium (10% Fötales Rinderserum [FBS], 20 mM KCl, 25 mM NaHCO 3, 30 mM D-Glucose, 2 mM L-Glutamin, 100 U / ml Penicillin, 100 ug / ml Streptomycin und 6 &amp; mgr; g / ml Ampicillin) und Filter zu sterilisieren. Lagerung bei 4 ° C für Monate. </li><li> Bereiten Sie 24 Well-Kulturplatten mit 14 mm, die Nummer 1 Dicke Deckgläser. Autoklaven werden die Deckgläser und Mantel in 100 mg / l Poly-D-Lysin (PDL), wie vorstehend beschrieben 3. UV-Licht sterilisiert O / N. </li></ol> 2. Herstellung von CGC Kulturlösungen <ol><li> Vorbereiten &quot;Lösung A&quot; (100 ml) in autoklavierten, UV-Licht behandelt, steriles dH &amp; sub2; O, enthaltend 250 mg Glukose, 300 mg Rinderserumalbumin [BSA], 955 mg phosphatgepufferte Kochsalzlösung [PBS] (Ca 2+ und Mg + free), und 1 ml 3,8% MgSO 4 · 7 H 2 O. </li><li> Vorbereiten &quot;Lösung B&quot; (10 ml), die 1 ml 5 mg / ml Trypsin in 9 ml Lösung A verdünnt </li><li> Vorbereiten &quot;Lösung C&quot; (10 ml), enthaltend 1.000 Einheiten DNase, 0,5 mg Sojabohnentrypsininhibitor, 100 &amp; mgr; l 3,8% MgSO 4 · 7H 2 O, 10 ml mit Lösung A. verdünnt </li><li> Vorbereiten &quot;Lösung D&quot; (20 ml), die 3,2 ml der Lösung C auf 20 ml mit Lösung A. verdünnt </li><li> Vorbereitung Balanced Salt Solution (EBSS) Lösung BSA / Earles enthaltende EBSS, 25 mM NaHCO3, 3 mM MgSO 4 · 7H 2 O, und4% BSA, pH 7,4, wie zuvor beschrieben, 3. </li><li> Bereiten LME durch Zugabe von reinem LME in 5 ml MEM Kulturmedium, um eine Endkonzentration von 150 mM LME zu geben, zurück die Lösung auf pH 7,4 mit einem pH-Meter Tisch Modell und Filter-sterilisiert unter Verwendung eines 28 mm Polyethersulfon (PES) 0,2 um Spritzen Filter. </li></ol> 3. Kultivierung der BVKA <ol><li> Sammeln cerebella 4-7 Tage alten Rattenjungen, wie zuvor beschrieben 3. Platzieren Sie sofort in Petrischale mit 5 ml Lösung A auf Eis. </li><li> Gießen Sie überschüssige Lösung von Kleinhirne und Ort Gewebe in der Petrischale Deckel. </li><li> Zerhacken Gewebe fein mit einer gut geflammt Rasierklinge in mindestens drei verschiedenen Richtungen. </li><li> Gehackte Gewebe zu Lösung B (rinse Petrischale mit Lösung begrenzte Gewebe sicherzustellen geht verloren) und im Wasserbad bei 37 ° C für 5 min. Vorsichtig schütteln alle paar min. </li><li> In Lösung D (20 ml) zu dem Rohr, um das Trypsin, schütteln und Cent zu neutralisierenrifuge bei 65 × g für 5 min. </li><li> Überstand abgießen. </li><li> Resuspendieren in 4 ml Lösung C Man reibt gut (ca. 10 mal) mit je drei geflammten Glaspipetten mit abnehmendem Durchmesser, bis die Suspension so homogen wie möglich ist. </li><li> Langsam und vorsichtig ein paar Tropfen dieses Homogenats auf dem BSA / EBSS. Wenn es beginnt, durch die BSA / EBSS Spüle, verreiben mehr und / oder fügen Sie ein wenig mehr Lösung C. Das Homogenat sollten in einer Schicht auf der Oberseite des BSA / EBSS sitzen. Nicht schütteln. Spin bei 100 xg für 5 min. </li><li> Überstand entfernen und Pellet (weich) in 1 ml MEM-Medium. </li><li> Zählen von Zellen unter Verwendung eines Hämozytometers und Platte bei 800.000 Zellen / Deckglas in 500 &amp; mgr; l MEM-Medium. In einem Brutschrank bei 37 ° C unter 6% CO 2. </li><li> Ändern Sie den folgenden Tag (24 bis 36 Stunden nach der prep) des Mediums. Stellen eine Lösung von MEM-Medium mit 10 &amp; mgr; M des Zellzyklus-Inhibitors arabinofuranosyl Cytidin (AraC). <ol><li> Für jedenDeckglas, behalten 250 ul des alten Mediums in einem frischen 24-Well-Platte, saugen Sie den Rest, fügen Sie 250 ul normalen MEM-Medium und saugen Sie diese, um die Zellen zu waschen, dann die 250 ul alte Medium zurück in den Zellen und füllen mit 250 ul AraC enthaltenden Medium. Hinweis: Zellen kann von 6 Tagen in vitro (DIV) verwendet werden. </li></ol></li></ol> 4. Wahlweise zum Abbau der Mikroglia (Durchgeführt bei 6 DIV) <ol><li> Hinzuzufügen reinen LME bis 5 ml eines separaten Aliquot MEM-Medium auf eine Endkonzentration von 150 mM LME ergeben. </li><li> Rückkehr der Lösung auf pH 7,4 und mit einem Filter sterilisiert 28 mm Polyethersulfon (PES), 0,2 &amp; mgr; m Spritzenfilter filtriert. </li><li> Verdünnte Lösung auf eine Konzentration von 50 mM (2 x LME) in MEM-Medium und in einem Wasserbad bei 37 ° C für 10 min mit dem normalen MEM Medium für Kontrollkulturen. </li><li> Entfernen Sie die Hälfte (250 ul) der MEM-Medium von CGC Kulturen und bei 37 ° C zu halten. </li><li> Treat-Zellenmit MEM-Medium, das 2 x LME (250 ul), das heißt 1: 1 verdünnt hat ein schönes Konzentration von 25 mM oder mit vorgewärmter MEM-Medium ohne LME (250 ul) für Kontrollkulturen. </li><li> Zellen bei 37 ° C inkubieren in einer befeuchteten Atmosphäre mit 6% CO 2 für 1 Stunde. </li><li> Wash Zellen zweimal in frischem vorgewärmten MEM-Medium zu LME haltigen Medien zu entfernen, und ersetzen Sie dann den gehaltenen Kulturmedium und ein gleiches Volumen an frischem vorgewärmten MEM-Medium in die entsprechenden Wells. </li><li> Zurück Kulturen in den Inkubator (mit 6% CO 2 bei 37 ° C befeuchtet) ruhen lassen für 24 h vor der weiteren Behandlung. </li></ol>

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+rat+cerebellar+granule+neurons+and+application+to+identify+neuroprotective+agents.">Culture of rat cerebellar granule neurons and application to identify neuroprotective agents.</a> Methods Mol Biol. 846, 23-37 (2012).</li><li>Thiele, D. L., Kurosaka, M., Lipsky, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotype+of+the+accessory+cell+necessary+for+mitogen-stimulated+T+and+B+cell+responses+in+human+peripheral+blood:+delineation+by+its+sensitivity+to+the+lysosomotropic+agent,+L-leucine+methyl+ester.">Phenotype of the accessory cell necessary for mitogen-stimulated T and B cell responses in human peripheral blood: delineation by its sensitivity to the lysosomotropic agent, L-leucine methyl ester.</a> J Immunology. 131 (5), 2282-2290 (1983).</li><li>Giulian, D., Vaca, K., Corpuz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+glia+release+factors+with+opposing+actions+upon+neuronal+survival.">Brain glia release factors with opposing actions upon neuronal survival.</a> J Neurosci. 13 (1), 29-37 (1993).</li><li>Guillemin, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obtention+and+characterization+of+primary+astrocyte+and+microglial+cultures+from+adult+monkey+brains.">Obtention and characterization of primary astrocyte and microglial cultures from adult monkey brains.</a> J Neurosci Res. 49 (5), 576-591 (1997).</li><li>Hewett, J. A., Hewett SJ, ., Winkler, S., Pfeiffer SE, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+nitric+oxide+synthase+expression+in+cultures+enriched+for+mature+oligodendrocytes+is+due+to+microglia.">Inducible nitric oxide synthase expression in cultures enriched for mature oligodendrocytes is due to microglia.</a> J Neurosci Res. 56 (2), 189-198 (1999).</li><li>Jebelli, J., Hooper, C., &amp;Pocock, J. 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The authors declare that they have no competing financial interests.

Die wichtigsten Schritte, um die erfolgreiche selektive Elimination von Mikroglia CGC sichern und / oder Mischkulturen sind: 1) die Aufrechterhaltung einer sterilen und gesunde CGC Kultur; 2) Sterilfiltrieren der LME haltigen Medium und Rückführen der Lösung auf pH 7,4; 3) halten die beibehalten CGC Medien und LME haltigen Medien bei 37 ° C, um Hitzeschock zu vermeiden; und 4) schnell arbeiten, um die Zeit-Zellen werden außerhalb des Inkubators gehalten zu reduzieren. Wir h...

Das menschliche Gehirn enthält schätzungsweise 85 Milliarden Neuronen und eine weitere 85 Milliarden nicht-neuronalen Zellen, sowie Glia 1. Daher der griechischen Etymologie &quot;Glia&quot; übersetzt in englisch als - zum größten Teil von den letzten 100 Jahren haben Neurowissenschaftler überwiegend auf der neuronalen Zellpopulation konzentriert, zu glauben, Gliazellen auf wenig mehr als passive Stützzellen, die strukturelle Unterstützung für den Neuronen vorgesehen sein &quot;Leim&quot;. In jüngster Zeit wurde es jedoch zunehmend offensichtlich geworden, dass neuronale-glial Wechselwirkungen können weitaus Grund grundlegende Aspekte der Neurobiologie, Neurophysiologie und der Entstehung und Progression von vielen neurodegenerativen Krankheiten. Kleinhirn-Körnerzellen (BVKA), die am häufigsten vorkommende homogene neuronalen Population im menschlichen Gehirn, dominieren das Kleinhirn und machen mehr als 90% der Zellbestandteile. Folglich wurden diese Zellen ausgiebig in vitro als Modellsystem für T verwendeter studieren der neuronalen Entwicklung, Funktion und Pathologie 2-6. Allerdings enthalten CGC Kulturen noch Mikroglia und anderen Glia in wohl signifikante Anteile. Als Ergebnis CGC Daten mutmaßlich Anzeige direkten neuronalen Reaktionen auf verschiedene Zellen Behandlungen können in der Tat ergeben - teilweise oder insgesamt - aus dem indirekten sekundäre Antwort benachbarter Glia in der Kultur. Um dies zu beurteilen, haben wir selektiv abgespalten Mikroglia von CGC neuronalen Kulturen mit Hilfe von L-Leucinmethylester (LME). LME ist ein lysomotropic Mittel ursprünglich benutzt, um selektiv Makrophagen 7 zerstört und seit verwendet worden, um auch selektiv abzureichern Mikroglia aus neuralen, Astrozyten und gemischten Gliakulturen 8.9.10. LME wird von Makrophagen und Mikroglia verinnerlicht, bei dem es lysosomalen Unterbrechung und anschließender Apoptose 13,14 bewirkt. Makrophagen und Mikrogliazellen sind charakteristischerweise reich an Lysosomen, wodurch sie particula seinrly anfällig bei Exposition gegenüber LME Behandlung. Dieses Protokoll bietet eine leistungsstarke und dennoch einfache und einfache Möglichkeit, den Beitrag der Mikroglia in Experimenten unter Verwendung von CGC und andere neuronale / Gliazellen Kultursystemen zu ermitteln.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Forceps&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>F4142</td>
			<td>The curved end facilitates removal of the cerebellum&#160;</td>
		</tr>
		<tr>
			<td>Micro-dissecting scissors</td>
			<td>Sigma-Aldrich</td>
			<td>S3146</td>
			<td>Straight, sharp point facilitates rodent P4-7 dissection&#160;</td>
		</tr>
		<tr>
			<td>L-leucine methyl ester hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>7517-19-3</td>
			<td></td>
		</tr>
		<tr>
			<td>EBSS solution&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>E7510-500 ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>27964-99-4</td>
			<td>Coat coverslips 1 day before use&#160;</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase</td>
			<td>Sigma-Aldrich</td>
			<td>D5025</td>
			<td></td>
		</tr>
		<tr>
			<td>Soybean trypsin inhibitor&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T6414</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-ED1 antibody&#160;</td>
			<td>Abcam</td>
			<td>ab31630</td>
			<td></td>
		</tr>
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</table>

selective depletion of microglia from cerebellar granule cell cultures using l-leucine methyl ester

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating innate immunity. Primary rodent cell culture models have greatly advanced our understanding of neuronal-glial interactions, but only recently have methods to specifically eliminate microglia from mixed cultures been utilized. One such technique &#8211; described here &#8211; is the use of L-leucine methyl ester, a lysomotropic agent that is internalized by macrophages and microglia, wherein it causes lysosomal disruption and subsequent apoptosis13,14. Experiments using L-leucine methyl ester have the power to identify the contribution of microglia to the surrounding cellular environment under diverse culture conditions. Using a protocol optimized in our laboratory, we describe how to eliminate microglia from P5 rodent cerebellar granule cell culture. This approach allows one to assess the relative impact of microglia on experimental data, as well as determine whether microglia are playing a neuroprotective or neurotoxic role in culture models of neurological conditions, such as stroke, Alzheimer&#8217;s or Parkinson&#8217;s disease.

Die Fähigkeit dieser Technik selektiv zu eliminieren Mikroglia CGC und / oder Mischkulturen beruht auf der nachfolgenden Fähigkeit des Untersuchers genau zu identifizieren und zu differenzieren Mikroglia aus ihrer umgebenden Zellen. Dies kann mit einer Mikroglia-spezifischen Zellmacher, wie isolectin-B4 erreicht werden, wie in 1 dargestellt. Wie in Figur 2 gezeigt, wurden keine beobachtbaren Änderungen in Astrozyten und neuronalen Dichte und Morphologie in Bezug auf jede Hauptbehandl...

Watch this Scientific Journal Video about Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester at JoVE.com

Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester

Microglia can influence neurons and other glia in culture by various non-cell autonomous mechanisms. Here, we present a protocol to selectively deplete microglia from primary neuronal cultures. This method has the potential to elucidate the role of microglial-neuronal interactions, with implications for neurodegenerative conditions where neuroinflammation is a hallmark feature.

Selektive Depletion von Mikroglia von Kleinhirn-Körnerzellkulturen Mit L-Leucin-Methylester

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating ...

selective-depletion-microglia-from-cerebellar-granule-cell-cultures

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JoVE Journal

Neuroscience

8.6K Aufrufe.  University of Washington. Microglia can influence neurons and other glia in culture by various non-cell autonomous mechanisms. Here, we present a protocol to selectively deplete microglia from primary neuronal cultures. This method has the potential to elucidate the role of microglial-neuronal interactions, with implications for neurodegenerative conditions where neuroinflammation is a hallmark feature. 

Serie: Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. Since then, the technique was developed further and currently there are many different ways to prepare organotypic cultures. The method presented here was adapted from the one described by Stoppini et al. for the preparation of the slices and from Gogolla et al. for the staining procedure 3,4.
A unique feature of this technique is that it allows you to study different parts of the brain such as hippocampus or cerebellum in their original structure, providing a big advantage over dissociated cultures in which all the cellular organization and neuronal networks are disrupted. In the case of the cerebellum it is even more advantageous because it allows the study of Purkinje cells, extremely difficult to obtain as dissociated primary culture. This method can be used to study certain developmental features of the cerebellum in vitro, as well as for electrophysiological and pharmacological experiments in both wild type and mutant mice.
The method described here was designed to study the effect of apoptotic stimuli such as Fas ligand in the developing cerebellum, using TUNEL staining to measure apoptotic cell death. If TUNEL staining is combined with cell type specific markers, such as Calbindin for Purkinje cells, it is possible to evaluate cell death in a cell population specific manner. The Calbindin staining also serves the purpose of evaluating the quality of the cerebellar cultures.

This method describes the generation of organotypic cerebellar cultures and the effect of certain apoptotic stimuli on the viability of different cerebellar cell types.

Organotypische Cerebellar Cultures: Apoptotic Challenges and Detection

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. ...

Forschung

Neurowissenschaften

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

Quantitative Analyse der synaptischen Vesikel Pool Replenishment in Cultured Cerebellar Nervenzellen Granulat mit FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

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 ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

Genetische Manipulation von Kleinhirnkörnerzellen<em&gt; In-vitro-</em&gt; Und<em&gt; In Vivo</em&gt;, Um neuronale Morphologie und Migration Studieren

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then radial migration in the molecular layer and the Purkinje cell layer to reach the internal granular layer of the cerebellar cortex. Default in migratory processes induces either cell death or misplacement of the neurons, leading to deficits in diverse cerebellar functions. Centripetal granule cell migration involves several mechanisms, such as chemotaxis and extracellular matrix degradation, to guide the cells towards their final position, but the factors that regulate cell migration in each cortical layer are only partially known. In our method, acute cerebellar slices are prepared from P10 rats, granule cells are labeled with a fluorescent cytoplasmic marker and tissues are cultured on membrane inserts from 4 to 10 hr before starting real-time monitoring of cell migration by confocal macroscopy at 37 &#176;C in the presence of CO2. During their migration in the different cortical layers of the cerebellum, granule cells can be exposed to neuropeptide agonists or antagonists, protease inhibitors, blockers of intracellular effectors or even toxic substances such as alcohol or methylmercury to investigate their possible role in the regulation of neuronal migration.

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal cerebellum development, immature granule cells originating from the germinal zone exhibit distinct modalities of migration to reach their final destination and to establish neuronal networks. This protocol describes the preparation of cerebellar slices and the confocal macroscopic approach used to investigate the factors that regulate neuronal migration.

Ex-vivo-Bildgebung von Postnatale Kleinhirn-Körnerzellmigration mittels konfokaler Makros

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

Vorbereitung der akuten Gehirnscheiben mit einem optimierten N-Methyl-D-Glucamine-schützende Recovery-Methode

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the co-existence of glial cells and neurons in CGN culture might lead to biases in the accurate assessment of neuronal viability. Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining has been used to measure cell viability by simultaneously evaluating the viable and dead cells. We used FDA-PI double staining to improve the sensitivities of the colorimetric assays and to evaluate neuronal viability in CGNs. Furthermore, we added blue fluorescent DNA stains (e.g., Hoechst) to improve the accuracy. This protocol describes how to improve the accuracy of assessment of neuronal viability by using these methods in CGN culture. Using this protocol, the number of glial cells can be excluded by using fluorescence microscopy. A similar strategy can be applied to distinguish the unwanted glial cells from neurons in various mixed cell cultures, such as primary cortical culture and hippocampal culture.

This protocol describes how to accurately measure neuronal viability using Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining in cultured cerebellar granule neurons, a primary neuronal culture used as an in vitro model in neuroscience and neuropharmacology research.

Beurteilung der neuronalen Lebensfähigkeit unter Verwendung von Fluorescein-Diacetat-Propidium-Iodid-Doppelfärbung in Cerebellar-Granulat-Neuron-Kultur

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

Modellierung von neuronalen Tod und Degeneration in Maus primären zerebelläre Granulat Neuronen

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

Quantifying Microglia Morphology from Photomicrographs of Immunohistochemistry Prepared Tissue Using ImageJ

Microglia are brain phagocytes that participate in brain homeostasis and continuously survey their environment for dysfunction, injury, and disease. As the first responders, microglia have important functions to mitigate neuron and glia dysfunction, and in this process, they undergo a broad range of morphologic changes. Microglia morphologies can be categorized descriptively or, alternatively, can be quantified as a continuous variable for parameters such as cell ramification, complexity, and shape. While methods for quantifying microglia are applied to single cells, few techniques apply to multiple microglia in an entire photomicrograph. The purpose of this method is to quantify multiple and single cells using readily available ImageJ protocols. This protocol is a summary of the steps and ImageJ plugins recommended to convert fluorescence and bright-field photomicrographs into representative binary and skeletonized images and to analyze them using software plugins AnalyzeSkeleton (2D/3D) and FracLac for morphology data collection. The outputs of these plugins summarize cell morphology in terms of process endpoints, junctions, and length as well as complexity, cell shape, and size descriptors. The skeleton analysis protocol described herein is well suited for a regional analysis of multiple microglia within an entire photomicrograph or region of interest (ROI) whereas FracLac provides a complementary individual cell analysis. Combined, the protocol provides an objective, sensitive, and comprehensive assessment tool that can be used to stratify between diverse microglia morphologies present in the healthy and injured brain.

Microglia are brain immune cells that survey and react to altered brain physiology through morphologic changes which may be evaluated quantitatively. This protocol outlines an ImageJ based analysis protocol to represent microglia morphology as continuous data according to metrics such as cell ramification, complexity, and shape.

Quantifizierung der Mikroglia Morphologie von Mikrofotos von Immunohistochemistry vorbereitet Gewebe mit ImageJ

Microglia are brain phagocytes that participate in brain homeostasis and continuously survey their environment for dysfunction, injury, and disease. As ...

Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical conduction. Myelin alteration has been shown to occur in various neurological diseases, where it is associated with functional deficits. Here, we provide a detailed description of an ex vivo model consisting of mouse organotypic cerebellar slices, which can be maintained in culture for several weeks and further be labeled to visualize myelin.

Here we present a method to prepare organotypic slice cultures from mouse cerebellum and myelin sheath staining by immunohistochemistry suitable for investigating mechanisms of myelination and remyelination in the central nervous system.

Vorbereitung und Immunostaining Myelinating organotypischen zerebelläre Slice Kulturen

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical ...