Diese Arbeit wurde vom NIH EY 012031 (ET-A), eine Auszeichnung von der FM Kirby Foundation (ET-A), Forschung zur Erblindung, Inc. (MAZ), The New Jersey Lions Eye Research Foundation (MAZ) Prevent unterstützt, The Eye Institute of New Jersey (MAZ), die Joseph J. und Marguerite DiSepio Retina Research Fund (MAZ) und die Janice Mitchell Vassar und Ashby Mitchell Fellowship (AMK).

1. Einen maßgeschneiderten stehen für Zellkultur-Einsätzen Die Basis eines 5 ml Pipettenspitze (ISC BioExpress, # P-3250 bis 19) hat einen Innendurchmesser von 13 mm, das entspricht genau dem Boden Größe (12,6 mm, Außendurchmesser) einer 10 mm (Innendurchmesser) NUNC Zelle Kultur einzufügen (8 um, Polycarbonat-Membran, Thermal, # 137443). Um den Stand, wurde die Pipette nahe der Basis geschnitten, um einen 6 mm langen Hohlzylinder erzeugt. Dann wurden Stücke aus der Seite des Zylinders entfernt mit einer Flamme erwärmt Klinge bis 3 Beine (4 mm in der Höhe) unter einem Ring (2 mm Höhe), die die Höhe der Einsätze um etwa 5 mm angehoben erstellen. 2. Herstellung von sterilen Filterpapier für den Anbau und die Kultur der Schweine Netzhaut Whatman 4M Filterpapier (Kat. Nr. 1004) wurde in eine runde Form (6,3 mm im Durchmesser) mit einem kleinen, dreieckigen Griff, der für die Bewegung der Filterpapier in einem Glasrohr zur Sterilisation oder wenn die Netzhaut angebracht wurde verwendet wurde geschnitten (wie gezeigt, in dem Video). 3. Vorbereitung der Netzhaut Explantate Eyes aus 5 - bis 9-Monate alten, 150-230 lbs, waren amerikanische Yorkshire Schweine aus einem örtlichen Schlachthof innerhalb von drei Stunden Enukleation (Transport auf Eis) gewonnen. Nach der Ankunft, die Augen von fremden Gewebe gereinigt wurden, tauchte in betadine Lösung (10% Povidon-Iod, der Purdue Frederique Company, Stamford, CT) und zweimal in Dulbecco Modified Eagle Medium (DMEM mit 1g / l Glucose, L-Glutamin, und Natriumpyruvat, CellGro-mediatech Inc., Manasse, VA) mit 2,5 g / ml Amphotericin B (Gibco-Invitrogen, Carlsbad, CA) ergänzt. Das vordere Segment wurde entfernt, so dass die neuronalen Netzhaut. Six-mm Trepan Klingen (Storz Ophthalmic-Bausch und Lomb, Manchester, MO) wurden verwendet, um äquatorialen, voller Dicke hinteren Segment Explantate abgeschnitten. Die Netzhaut wurde anschließend durch leichtes Anlegen ein Stück trockenes steriles Filterpapier auf die Ganglienzellschicht, Abheben der neuralen Netzhaut, und indem das Filterpapier mit angeschlossenem Netzhaut auf die Kultur legen, Photorezeptoren nach oben abgezogen. Der Einsatz wurde dann in Kulturmedium gebracht, so dass Sie sicher, dass zur Deckung der Netzhaut, in einer 12-well Kulturschale (Fisher). Mehrere Explantate wurden routinemäßig und erhielt kultivierte aus einem einzigen Auge. 4. Gewebekultur Die Retina wurde in Minimum Essential Eagle-Medium (MEM, Invitrogen-Gibco-Life Technologies Inc., Rockville, MD), pH 7,4, ergänzt mit 0,2 mg / ml Glutamin, 10 pg / ml Insulin vom Schwein, 1 mM Pyruvat, 0,1 mM L-Ascorbinsäure, 100 U / ml Penicillin, 100 ug / ml Streptomycin und 250 ng / ml Amphotericin B (Antibiotika antimykotische: Sigma, St. Louis, MO), und kohlensäurehaltiges mit befeuchteten 5% CO 2, Balance Luft, bei 37 ° C. Das Medium wurde alle 2 Tage gewechselt. Tissue kann verwendet werden, behandelt mit einer Vielzahl von Reagenzien oder Sonden oder links für nachfolgende biochemische oder morphologische Analysen unbehandelt. Der folgende Abschnitt beschreibt Verfahren, um konsistente voller Dicke der Netzhaut Querschnitte erstellen. 5. Sectioning <ol><li> Mit einem Kryostat <ol><li> Fix Netzhautgewebe in 4% parafomaldehyde über Nacht bei 4 ° C; </li><li> Das Gewebe spülen kurz mit Phosphat-gepufferter Kochsalzlösung (PBS); </li><li> Entfernen Sie vorsichtig das Gewebe aus dem Filterpapier, während es in PBS in einer 35 mm Schale mit einem Dissektionsmikroskop; </li><li> Transfer-Gewebe zu 30% Saccharose und halten über Nacht bei 4 ° C; </li><li> Saug entfernt die überschüssige Lösung um das Gewebe zu erhalten, ausreichend Tissue-Tek Medium (OCT Compound 4583), um Gewebe decken, und lassen Sie es bei Raumtemperatur für 2 Stunden einweichen, um das Gewebe durchdringen; </li><li> Baue einen quadratischen Rahmen (10 mm x 10 mm, 5 mm Höhe) mit Dentalwachs und legen Sie sie in das Gewebe, fügen Sie mehr Tissue-Tek Medium mit dem Rahmen (sicherstellen, dass die Gewebeprobe ist flach) und in einen Gefrierschrank zu füllen (- 20 ° C) für mindestens 1 Stunde; </li><li> Übertragen Sie die gefrorene Probe blockieren, um eine Probe-Plattform (sicherstellen, dass die Sample-Block steht senkrecht auf der Plattform); </li><li> Montieren Sie die Probe-Plattform in einem vorgekühlten Kryostaten (-20 ° C, Leica, CM1900) und stellen Sie sicher, dass der Block steht senkrecht auf der Klinge; </li><li> Entfernen Sie das Wachs und den Rahmen zu blockieren, bevor Schneiden des Gewebes in der gewünschten Dicke; </li><li> Setzen Sie die Teile auf Gelatine-behandelten Objektträgern. </li></ol></li><li> Mit einem Vibratom <ol><li> Fix und spülen Sie das Gewebe wie oben für den Kryostat beschrieben; </li><li> Legen Sie das Gewebe in vorgewärmte, geschmolzen 4% Agar in einen kleinen Behälter und halten bei 4 ° C, bis der Agar erstarrt vollständig; </li><li> Mit einem scharfen Messer auf die Agar in einen kleinen Block mit der Gewebeprobe Innenverkleidung; </li><li> Kleben Sie die Sample-Block auf einer Vibratom Probenhalter mit Krazy Kleber (stellen Sie sicher, das Stück Gewebeprobe steht senkrecht auf der Plattform von the Probenhalter, dh senkrecht zur Klinge) und montieren Sie die Halterung auf einem Vibratom Schneidesystem (Leica, Series 1000) mit der Probe Block vollständig im eisigen Wasser getaucht; </li><li> Schneiden Sie die Sample-Block in 50 Abschnitte um und legen Sie sie auf Gelatine-behandelten Folien mit einer weichen Bürste. </li></ol></li></ol> 6. Immunhistochemie Verwenden Sie Standard-Protokoll. Immunolabeled dicke Schnitte können mit einem konfokalen Mikroskop betrachtet werden. 7. Repräsentative Ergebnisse: Morphologie wurde während des Zeitraums von sieben Tagen der Kultur erhalten. Bilder der Netzhaut vor und nach der Kultivierung sind in dem Video.

<ol>
	<li>Johnson, T. V., Martin, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+an+adult+retinal+explant+organotypic+tissue+culture+system+as+an+in+vitro+intraocular+stem+cell+transplantation+model.">Development and characterization of an adult retinal explant organotypic tissue culture system as an in vitro intraocular stem cell transplantation model.</a> Invest Ophthalmol Vis Sci. 49, 3503-3512 (2008).</li><li>Kretz, A., Marticke, J. K., Happold, C. J., Schmeer, C., Isenmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+primary+culture+technique+of+adult+retina+for+regeneration+studies+on+adult+CNS+neurons.">A primary culture technique of adult retina for regeneration studies on adult CNS neurons.</a> Nat Protoc. 2, 131-140 (2007).</li><li>Donovan, S. L., Dyer, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+square+wave+electroporation+of+retinal+explant+cultures.">Preparation and square wave electroporation of retinal explant cultures.</a> Nat Protoc. 1, 2710-2718 (2006).</li><li>Koizumi, A., Zeck, G., Ben, Y., Masland, R. H., Jakobs, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+culture+of+physiologically+functional+adult+mammalian+retinas.">Organotypic culture of physiologically functional adult mammalian retinas.</a> PLoS One. 2, e221-e221 (2007).</li><li>Lye, M. H., Jakobs, T. C., Masland, R. H., Koizumi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+culture+of+adult+rabbit+retina.">Organotypic culture of adult rabbit retina.</a> J Vis Exp. , (2007).</li><li>Ames, A., Li, Y. Y., Heher, E. C., Kimble, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energy+metabolism+of+rabbit+retina+as+related+to+function:+high+cost+of+Na++transport.">Energy metabolism of rabbit retina as related to function: high cost of Na+ transport.</a> J Neurosci. 12, 840-853 (1992).</li><li>Kobuch, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+adult+porcine+retina+and+retinal+pigment+epithelium+in+perfusion+culture:+characterisation+of+an+organotypic+in+vitro+model.">Maintenance of adult porcine retina and retinal pigment epithelium in perfusion culture: characterisation of an organotypic in vitro model.</a> Exp Eye Res. 86, 661-668 (2008).</li><li>Kaempf, S., Walter, P., Salz, A. K., Thumann, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+organotypic+culture+model+of+adult+mammalian+neurosensory+retina+in+co-culture+with+retinal+pigment+epithelium.">Novel organotypic culture model of adult mammalian neurosensory retina in co-culture with retinal pigment epithelium.</a> J Neurosci Methods. 173, 47-58 (2008).</li><li>Guduric-Fuchs, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+study+of+pig+retinal+development.">Immunohistochemical study of pig retinal development.</a> Mol Vis. 15, 1915-1928 (2009).</li><li>Chandler, M. J., Smith, P. J., Samuelson, D. A., MacKay, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoreceptor+density+of+the+domestic+pig+retina.">Photoreceptor density of the domestic pig retina.</a> Vet Ophthalmol. 2, 179-184 (1999).</li></ol>

Vision-Forscher haben eine Vielzahl von Netzhaut-Kultur etabliert, darunter organotypischen Systeme, um eine Vielzahl von Themen, wie Retina Stammzelltransplantation 1, retinale Regeneration 2 und exogene Genexpression 3-5 studieren. Allerdings ist erwachsener Säugetiere Netzhaut schwer aufrecht zu erhalten in vitro, vor allem aufgrund der hohen Energiebedarf der Photorezeptoren 6. Koizumi et al. Beschrieben ein Kaninchen Netzhaut organotypischen Kultur, in ...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> MEM </td><td> Invitrogen-Gibco </td><td> 10370-021 </td><td></td></tr><tr><td> 5 ml Pipettenspitze </td><td> ISC BioExpress </td><td> P-3250 bis 19 </td><td></td></tr><tr><td> NUNC Zellkultureinsatzes </td><td> Thermisch </td><td> 137443 </td><td> 8μm, Polycarbonat </td></tr><tr><td> Whatman 4M Filterpapier </td><td> Whatman </td><td> 1004 </td><td></td></tr><tr><td> DMEM </td><td> CellGro-mediatech Inc </td><td> 10 bis 013-CV </td><td></td></tr><tr><td> Agar </td><td> Fluka </td><td> 05040 </td><td></td></tr><tr><td> Fungizone Amphotericin B </td><td> Invitrogen-Gibco </td><td> 15290018 </td><td></td></tr><tr><td> Trephine Klingen </td><td> Bausch and Lomb </td><td> T3096 </td><td> 6.00mm </td></tr><tr><td> OCT-Verbindung </td><td> Electron Microscopy Sciences </td><td> 62550-01 </td><td> 4583 </td></tr><tr><td></td><td> Sciences </td><td></td><td></td></tr><tr><td> Kryostat </td><td> Leica </td><td> CM1900 </td><td></td></tr><tr><td> Vibratom </td><td> Leica </td><td> Serie 1000 </td><td></td></tr><tr><td> Die konfokale Mikroskopie; </td><td> Carl Zeiss </td><td> LSM510 </td><td></td></tr><tr><td> Anti-synaptischen Vesikel Protein 2 (SV2) monoklonalen Maus-Antikörper </td><td> DSHB </td><td> N / A </td><td></td></tr><tr><td> Anti-sauren Gliafaserproteins (GFAP) polyklonale Kaninchen-Antikörper </td><td> DAKO </td><td> DAKO </td><td></td></tr><tr><td> Propidiumiodid (PI) </td><td> Sigma </td><td> P4170 </td><td></td></tr></tbody></table>

organotypic culture of full-thickness adult porcine retina

Es ist ein anerkannter Bedarf an In-vitro-Modellen, zu ersetzen oder reduzieren kann Tierversuch. Porcine Netzhaut hat eine ähnliche neuronale Struktur der menschlichen Netzhaut und ist daher eine wertvolle Arten für das Studium Mechanismen der menschlichen Netzhaut Verletzungen und degenerativen Erkrankungen. Hier beschreiben wir eine kostengünstige Methode zur organotypischen Kultur von ausgewachsenen Schweinen Netzhaut vor Augen von einem Schlachthof erhalten isoliert. Nach dem Entfernen der vorderen Segment wurde ein Trepan Klinge verwendet, um mehrere neurale Retina-Bruch-Membran-RPE-Aderhaut-Sklera Explantate aus dem hinteren Bereich des ausgewachsenen Schweinen Augen zu schaffen. Ein Stück sterile Filterpapier wurde verwendet, um die neuronalen Netzhaut abhebt von jedem Explantat. Das Filterpapier-Retina-Komplex wurde kultiviert (Photorezeptor Seite nach oben) auf dem Gipfel eines Einsatzes, die vom Boden der Kulturschale mit einem maßgeschneiderten Stand gehalten wurde. Der Stand ermöglicht eine gute Zirkulation des Kulturmediums auf beiden Seiten der Netzhaut. Insgesamt ist dieses Verfahren einfach, reproduzierbar und erlaubt Erhaltung der einheimischen retinalen Struktur für mindestens sieben Tage, so dass es ein nützliches Modell für eine Vielzahl von morphologischen, pharmakologische und biochemische Untersuchungen an Säugetieren Netzhaut.

Watch this Scientific Journal Video about Organotypic Culture of Full-thickness Adult Porcine Retina at JoVE.com

Organotypic Culture of Full-thickness Adult Porcine Retina

Hier beschreiben wir eine kostengünstige Methode zur organotypischen Kultur von ausgewachsenen Schweinen Netzhaut für sieben Tage. Kurz gesagt, wurde ein steriles Filterpapier verwendet, um die neuronalen Netzhaut abheben von der RPE und Ort Photorezeptor Seite nach oben auf einem Einsatz von einem maßgeschneiderten Stand angehoben.

Organotypischen Kultur der Full-Dicke ausgewachsenen Schweinen Retina

There is a recognized demand for in vitro models that can replace or reduce animal experiments. Porcine retina has a similar neuronal structure to human ...

organotypic-culture-of-full-thickness-adult-porcine-retina

Research

JoVE Journal

Neuroscience

14.9K Aufrufe.  University of Medicine and Dentistry of New Jersey - UMDNJ. Hier beschreiben wir eine kostengünstige Methode zur organotypischen Kultur von ausgewachsenen Schweinen Netzhaut für sieben Tage. Kurz gesagt, wurde ein steriles Filterpapier verwendet, um die neuronalen Netzhaut abheben von der RPE und Ort Photorezeptor Seite nach oben auf einem Einsatz von einem maßgeschneiderten Stand angehoben.

Serie: Organotypic Culture of Full-thickness Adult Porcine Retina

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of the hippocampus is relatively preserved 1. The organotypic culture system allows for the examination of neuronal, astrocytic and microglial effects, but as an ex vivo preparation, does not address effects of blood flow, or recruitment of peripheral inflammatory cells. To that end, this culture method is frequently used to examine excitotoxic and hypoxic injury to pyramidal neurons of the hippocampus, but has also been used to examine the inflammatory response. Herein we describe the methods for generating hippocampal slice cultures from postnatal rodent brain, administering toxic stimuli to induce neuronal injury, and assaying and quantifying hippocampal neuronal death.

The organoptypic hippocampal slice culture model is an in vitro model used to examine neuronal injury in a variety of paradigms. In this article, we describe the methods for generating slice cultures and quantifying neuronal injury.

Die Organotypische hippokampalen Slice-Kultur-Modell für die Prüfung neuronaler Schädigung

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

Forschung

Neurowissenschaften

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in which the enhanced version of the green fluorescent protein (EGFP) is exclusively expressed in all neurons of the developing central and peripheral nervous system1, allowing the possibility to both film the innervation of the forelimb and to manipulate this process with pharmacological and genetic techniques2. The most critical parameter in the successful cultivation of such slice cultures is the method by which the slices are prepared. After extensive testing of a variety of methods, we have found that a vibratome is the best possible device to slice the embryos such that they routinely result in a culture that demonstrates viability over a period of several days, and most importantly, develops in an age-specific manner. For mid-gestation embryos, this includes the normal outgrowth of spinal nerves from the spinal cord and the dorsal root ganglia to their targets in the periphery and the proper determination of skeletal and muscle tissue. 
In this work, we present a method for processing whole embryos of embryonic day (E) E10 to E12 into 300 - 400 micrometer slices for cultivation in a standard tissue culture incubator, which can be studied for up to two days after slice preparation. Critical for the success of this approach is the use of a vibratome to slice each agarose-embedded embryo. This is followed by the cultivation of the slices upon Millicell culture membrane inserts placed upon a small volume of medium, resulting in an interface culture technique. One litter with an average of 7 embryos routinely produces at least 14 slices (2-3 slices of the forelimb region per embryo), which varies slightly due to the age of the embryos as well as to the thickness of the slices. About 80% of the cultured slices show nerve outgrowth, which can be measured througout the culturing period2. Representative results using the tauGFP mouse line are demonstrated.

We present a method to prepare organotypic slices of mid-gestation mouse embryos for the cultivation and time-lapse imaging of peripheral nerve outgrowth.

Organotypischen Kultur der GFP-exprimierenden Maus Embryonen für Real-time Imaging der peripheren Nerven Outgrowth

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in ...

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

In-vivo-Elektroporation von Entwicklung Maus Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

Isolierung und Kultur von adulten neuralen Stammzellen aus der Maus subcallosa Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in these preparations is perpendicular to the retinal surface, making it ideal for the study of radially oriented neurons like photoreceptors and bipolar cells. However, the large dendritic arbors of horizontal cells, wide-field amacrine cells, and ganglion cells are mostly truncated, leaving markedly reduced synaptic activity in these cells. Whereas ganglion cells and displaced amacrine cells can be studied in a whole-mounted preparation of the retina, horizontal cells and amacrine cells located in the inner nuclear layer are only poorly accessible for electrodes in whole retina tissue.
To achieve maximum accessibility and synaptic integrity, we developed a horizontal slice preparation of the mouse retina, and studied signal transmission at the synapse between photoreceptors and horizontal cells. Horizontal sectioning allows&#160;(1) easy and unambiguous visual identification of horizontal cell bodies for electrode targeting, and (2) preservation of the extended horizontal cell dendritic fields, as a prerequisite for intact and functional cone synaptic input to horizontal cell dendrites.
Horizontal cells from horizontal slices exhibited tonic synaptic activity in the dark, and they responded to brief flashes of light with a reduction of inward current and diminished synaptic activity. Immunocytochemical evidence indicates that almost all cones within the dendritic field of a horizontal cell establish synapses with its peripheral dendrites. The horizontal slice preparation is therefore well suited to study the physiological properties of horizontally extended retinal neurons as well as sensory signal transmission and integration across selected synapses.

We developed a horizontal slice preparation of the adult mammalian retina with the plane of sectioning parallel to the retinal surface. Dendritic fields of nonradially oriented retinal neurons were not truncated, allowing the study of signal processing with patch-clamp and imaging techniques.

Vorbereitung von Horizontal Scheiben der erwachsenen Maus Retina für elektrophysiologische Untersuchungen

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in ...

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It has helped to describe the different steps of radial migration and characterize the molecular mechanisms controlling this process. To directly and dynamically analyze migrating neurons they have to be traced over time. This protocol describes a workflow that combines in utero electroporation with organotypic slice culture and time-lapse confocal imaging, which allows for a direct examination and dynamic analysis of radially migrating cortical neurons. Furthermore, detailed characterization of migrating neurons, such as migration speed, speed profiles, as well as radial orientation changes, is possible. The method can easily be adapted to perform functional analyses of genes of interest in radially migrating cortical neurons by loss and gain of function as well as rescue experiments. Time-lapse imaging of migrating neurons is a state-of-the-art technique that once established is a potent tool to study the development of the cerebral cortex in mouse models of neuronal migration disorders.

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

This protocol provides instructions for direct observation of radially migrating cortical neurons. In utero electroporation, organotypic slice culture, and time-lapse confocal imaging are combined to directly and dynamically study the effects of overexpression or downregulation of genes of interest in migrating neurons and to analyze their differentiation during development.

Zeitraffer-konfokale Bildgebung der Migration von Neuronen in der organotypischen Scheibe Kultur der embryonalen Maus Gehirn mit<em&gt; In Utero</em&gt; Elektroporation

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

Einfache Erstellung einer High-Yield-Kultur der induzierten Neuronen aus Menschenhaut Erwachsenen Fibroblasten

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and peripheral neuroregulation. Although extensive research on the lower urinary tract nervous system has been conducted, most studies have focused on primary culture. This protocol introduces a method for the isolation and culture of bladder neurons and glia from Sprague&#8211;Dawley rats. In this method, the neurons and glia were incubated in a 37 &#176;C, 5% CO2 incubator for 5&#8211;7 days. As a result, they grew into mature shapes suitable for related subsequent immunofluorescence experiments. Cells were morphologically observed using an optical microscope. Neurons, synaptic vesicles, and glia were identified by &#946;-III-tubulin and MAP-2, Synapsin-1, and GFAP staining, respectively. Meanwhile, immunocytochemistry was performed on several neurotransmitter-related proteins, such as choline acetyltransferase, DYNLL2, and SLC17A9.

This protocol attempts to establish a repeatable protocol for primary neurons and glia isolation from rat bladder for further cellular experiments.

Isolation und Kultur der primär neuronen und Glia von Adult Ratte Harnblase

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and ...

Culture of Neurospheres Derived from the Neurogenic Niches in Adult Prairie Voles

Neurospheres are primary cell aggregates that comprise neural stem cells and progenitor cells. These 3D structures are an excellent tool to determine the differentiation and proliferation potential of neural stem cells, as well as to generate cell lines than can be assayed over time. Also, neurospheres can create a niche (in vitro) that allows the modeling of the dynamic changing environment, such as varying growth factors, hormones, neurotransmitters, among others. Microtus ochrogaster (prairie vole) is a unique model for understanding the neurobiological basis of socio-sexual behaviors and social cognition. However, the cellular mechanisms involved in these behaviors are not well known. The protocol aims to obtain neural progenitor cells from the neurogenic niches of the adult prairie vole, which are cultured under non-adherent conditions, to generate neurospheres. The size and number of neurospheres depend on the region (subventricular zone or dentate gyrus) and sex of the prairie vole. This method is a remarkable tool to study sex-dependent differences in neurogenic niches in vitro and the neuroplasticity changes associated with social behaviors such as pair bonding and biparental care. Also, cognitive conditions that entail deficits in social interactions (autism spectrum disorders and schizophrenia) could be examined.

We established the conditions to culture neural progenitor cells from the subventricular zone and dentate gyrus of the adult brain of prairie voles, as a complementary in vitro study, to analyze the sex-dependent differences between neurogenic niches that could be part of functional plastic changes associated with social behaviors.

Kultur der Neurosphären Abgeleitet von den neurogenen Nischen in Adult Prairie Voles

Neurospheres are primary cell aggregates that comprise neural stem cells and progenitor cells. These 3D structures are an excellent tool to determine the ...

Single-Cell Electroporation across Different Organotypic Slice Culture of Mouse Hippocampal Excitatory and Class-Specific Inhibitory Neurons

Electroporation has established itself as a critical method for transferring specific genes into cells to understand their function. Here, we describe a single-cell electroporation technique that maximizes the efficiency (~80%) of in vitro gene transfection in excitatory and class-specific inhibitory neurons in mouse organotypic hippocampal slice culture. Using large glass electrodes, tetrodotoxin-containing artificial cerebrospinal fluid and mild electrical pulses, we delivered a gene of interest into cultured hippocampal CA1 pyramidal neurons and inhibitory interneurons. Moreover, electroporation could be carried out in cultured hippocampal slices up to 21 days in vitro with no reduction in transfection efficiency, allowing for the study of varying slice culture developmental stages. With interest growing in examining the molecular functions of genes across a diverse range of cell types, our method demonstrates a reliable and straightforward approach to in vitro gene transfection in mouse brain tissue that can be performed with existing electrophysiology equipment and techniques.

Presented here is a protocol for single-cell electroporation that can deliver genes in both excitatory and inhibitory neurons across a range of in vitro hippocampal slice culture ages. Our approach provides precise and efficient expression of genes in individual cells, which can be used to examine cell-autonomous and intercellular functions.

Einzelzell-Elektroporation über verschiedene organotypische Slice-Kulturen von Hippocampus-Exzitatorischen und klassenspezifischen inhibitorischen Neuronen der Maus

Electroporation has established itself as a critical method for transferring specific genes into cells to understand their function. Here, we describe a ...