Questo lavoro è stato sostenuto da NIH concedere EY 012.031 (ET-A), un premio dalla Fondazione FM Kirby (ET-A), ricerca per la Prevenzione della Cecità, Inc. (MAZ), il New Jersey Lions Eye Research Foundation (MAZ), L&#39;Istituto Eye of New Jersey (MAZ), il J. Giuseppe e Margherita DiSepio Retina Research Fund (MAZ), ed il Janice Mitchell Vassar e Ashby Mitchell Fellowship (AMK).

1. Fare una misura stand per inserti in colture cellulari La base di una pipetta da 5 ml di punta (ISC BioExpress, # P-3250-19) ha un diametro interno di 13 mm, che corrisponde perfettamente alla dimensione inferiore (12,6 mm, diametro esterno) di mm 10 (diametro interno) di cellule NUNC inserto cultura (8 micron, membrana in policarbonato, termica, # 137443). Per rendere lo stand, la pipetta è stata tagliata vicino alla base per la produzione da 6 mm lungo cilindro cavo. Poi, i pezzi sono stati rimossi dal lato del cilindro usando una fiamma riscaldato lama di creare 3 gambe (4 mm di altezza) con un anello (2 mm di altezza), che ha sollevato l&#39;altezza degli inserti di circa 5 mm. 2. Preparazione di carta da filtro sterile per il fissaggio e la cultura della retina suina Carta da filtro Whatman 4M (Cat. No. 1004) è stato tagliato in una forma circolare (6,3 mm di diametro) con una piccola impugnatura triangolare che è stato utilizzato per spostare la carta da filtro in un tubo di vetro per la sterilizzazione o una volta che la retina è stata allegata (come mostrato nel video). 3. Preparazione di espianti di retina Occhi da 5 - a 9 mesi di età, 150-230 lbs, americani maiali Yorkshire sono stati ottenuti da un mattatoio locale entro tre ore di enucleazione (trasportati in ghiaccio). All&#39;arrivo, gli occhi sono stati puliti del tessuto estraneo, immerso in soluzione betadine (10% Povidone-iodio, la Società Purdue Frederique, Stamford, CT) e lavato due volte in Dulbecco modificato (DMEM con 1 g / L di glucosio, L-glutamina, e piruvato di sodio, Cellgro-Mediatech Inc., Manasse, VA) integrato con 2,5 mg / ml di amfotericina B (Gibco-Invitrogen, Carlsbad, CA). Il segmento anteriore è stato rimosso, esponendo la retina neurale. Sei mm lame trapano (Storz oftalmica-Bausch &amp; Lomb, Manchester, MO) sono stati utilizzati per tagliare equatoriale, a tutto spessore espianti segmento posteriore. La retina è stata successivamente staccate delicatamente applicando un pezzo di carta asciutta da filtro sterile sullo strato di cellule gangliari, alzando il piede della retina neurale, e mettendo la carta da filtro con retina attaccato sul inserto cultura, fotorecettori verso l&#39;alto. L&#39;inserto è stato poi inserito in terreno di coltura, avendo cura di coprire completamente la retina, in un piatto ben 12-cultura (Fisher). Espianti multiple sono state ottenute regolarmente e colto da un solo occhio. 4. Tessuto cultura Il tessuto retinico è stato coltivato in Medio minimi essenziali di Eagle (MEM, Invitrogen-Gibco-Life Technologies Inc., Rockville, MD), pH 7.4, integrato con 0,2 mg / ml glutammina, 10 mg / ml di insulina suina, 1 piruvato mM, 0,1 mM di acido L-ascorbico, 100 U / ml di penicillina, 100 mg / ml di streptomicina, e 250 ng / ml di amfotericina B (antibiotico Antimicotici: Sigma, St. Louis, MO), e aerato con umidificata al 5% di CO 2, aria equilibrio, a 37 ° C. Medio è stato scambiato ogni due giorni. Tessuti possono essere utilizzati, trattati con una varietà di reagenti o sonde, o non trattata per le successive analisi biochimiche o morfologiche. La sezione seguente descrive le procedure per creare coerente a tutto spessore della retina sezioni. 5. Sezionamento <ol><li> Utilizzando un criostato <ol><li> Fissare tessuto retinico nel 4% parafomaldehyde notte a 4 ° C; </li><li> Sciacquare brevemente tessuto con tampone fosfato (PBS); </li><li> Rimuovere con attenzione il tessuto dalla carta da filtro mentre è in PBS in un piatto da 35 mm utilizzando un microscopio a dissezione; </li><li> Trasferimento di tessuto al 30% di saccarosio e mantenere una notte a 4 ° C; </li><li> Aspirazione via la soluzione in eccesso intorno al tessuto, aggiungere sufficiente Tissue-Tek medio (OCT Compound 4583) per coprire i tessuti, e lasciare in ammollo a temperatura ambiente per 2 ore di permeare il tessuto; </li><li> Costruire una cornice quadrata (10 mm x 10 mm, 5 mm di altezza) con impronte dentarie e mettetelo in tutto il tessuto; aggiungere più Tissue-Tek medio per riempire il fotogramma (assicurarsi che il campione di tessuto è piatto) e mettere in un freezer (- 20 ° C) per almeno 1 ora; </li><li> Trasferire il blocco congelato campione ad una piattaforma campione (assicurarsi che il blocco del campione è verticale alla piattaforma); </li><li> Montare il campione in una piattaforma pre-raffreddata criostato (-20 ° C, Leica, CM1900) e assicurarsi che il blocco è verticale per la lama; </li><li> Rimuovere la cera e il blocco di frame prima di sezionare il tessuto di spessore desiderato; </li><li> Posizionare le sezioni in gelatina trattati con diapositive. </li></ol></li><li> Utilizzando un Vibratome <ol><li> Fix e lavare il tessuto come sopra descritto per il criostato; </li><li> Posizionare il tessuto in pre-riscaldato, agar fuso 4% in un piccolo contenitore e conservare a 4 ° C fino a quando il rapprende agar completamente; </li><li> Utilizzare una lama affilata per tagliare l&#39;agar in un piccolo blocco con il campione di tessuto interno; </li><li> Incollare il blocco campione su un portacampioni vibratome con Krazy colla (assicurarsi che il pezzo di campione di tessuto è verticale alla piattaforma di secoloe titolare del campione, cioè perpendicolare alla lama) e montare il supporto su un sistema vibratome sezionamento (Leica, serie 1000) con il blocco del campione completamente immersi in acqua ghiacciata; </li><li> Tagliare il blocco campione in 50 sezioni micron e disporli sulla gelatina trattati con le diapositive con una spazzola morbida. </li></ol></li></ol> 6. Immunoistochimica Utilizza un qualsiasi protocollo standard. Immunolabeled sezioni spesse possono essere visualizzati con un microscopio confocale. 7. Rappresentante dei risultati: Morfologia è stata preservata durante il periodo di sette giorni di cultura. Le immagini della retina prima e dopo la coltura sono disponibili nel 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>

Nessun conflitto di interessi dichiarati.

I ricercatori hanno stabilito una visione varietà di sistemi di coltura della retina, compresi i sistemi organotipica, per studiare una serie di questioni, come il trapianto di cellule staminali della retina 1, la rigenerazione della retina 2, e l&#39;espressione genica esogeni 3-5. Tuttavia, retina dei mammiferi adulti è difficile da mantenere in vitro, principalmente a causa della alte energie richieste dei fotorecettori 6. Koizumi et al. Descritto un con...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> MEM </td><td> Invitrogen-Gibco </td><td> 10370-021 </td><td></td></tr><tr><td> 5 ml puntale </td><td> ISC BioExpress </td><td> P-3250-19 </td><td></td></tr><tr><td> NUNC cellula inserto cultura </td><td> Termico </td><td> 137443 </td><td> 8μm, Policarbonato </td></tr><tr><td> 4M filtro di carta Whatman </td><td> Whatman </td><td> 1004 </td><td></td></tr><tr><td> DMEM </td><td> Cellgro-Mediatech Inc </td><td> 10-013-CV </td><td></td></tr><tr><td> Agar </td><td> Fluka </td><td> 05040 </td><td></td></tr><tr><td> Fungizone amfotericina B </td><td> Invitrogen-Gibco </td><td> 15290018 </td><td></td></tr><tr><td> Trapano lame </td><td> Bausch and Lomb </td><td> T3096 </td><td> 6,00 millimetri </td></tr><tr><td> Ottobre Compound </td><td> Electron Microscopy Sciences </td><td> 62550-01 </td><td> 4583 </td></tr><tr><td></td><td> Scienze </td><td></td><td></td></tr><tr><td> Criostato </td><td> Leica </td><td> CM1900 </td><td></td></tr><tr><td> Vibratome </td><td> Leica </td><td> Serie 1000 </td><td></td></tr><tr><td> Microscopia confocale; </td><td> Carl Zeiss </td><td> LSM510 </td><td></td></tr><tr><td> Anti-proteina delle vescicole sinaptiche 2 (SV2) anticorpo monoclonale di topo </td><td> DSHB </td><td> N / A </td><td></td></tr><tr><td> Anti-proteina acidica fibrillare gliale (GFAP) anticorpo policlonale di coniglio </td><td> DAKO </td><td> DAKO </td><td></td></tr><tr><td> Propidio ioduro (PI) </td><td> Sigma </td><td> P4170 </td><td></td></tr></tbody></table>

organotypic culture of full-thickness adult porcine retina

Vi è una domanda riconosciuto per modelli in vitro che può sostituire o ridurre gli esperimenti sugli animali. Retina suina ha una struttura simile a neuronale retina umana ed è quindi una specie preziosa per lo studio dei meccanismi di lesione della retina umana e le malattie degenerative. Qui si descrive una soluzione economica per la cultura tecnica organotipica di retina suini adulti isolati da occhi ottenuti da un mattatoio. Dopo aver rimosso il segmento anteriore, una lama trapano è stato utilizzato per creare più neurali retina-membrana di Bruch-RPE-coroide, sclera espianti dal segmento posteriore degli occhi suini adulti. Un pezzo di carta da filtro sterile è stato utilizzato per sollevare la retina neurale fuori da ogni espianto. Il filtro di carta retina complesso è stato colto (lato fotorecettori verso l&#39;alto) in cima a un inserto, che si è tenuto lontano dal fondo del piatto la cultura da una misura stand. Il supporto permette una buona circolazione del terreno di coltura su entrambi i lati della retina. Nel complesso, questa procedura è semplice, riproducibile, e permette la conservazione dei nativi struttura della retina per almeno sette giorni, che lo rende un modello utile per una varietà di studi morfologici, farmacologico, biochimico e sulla retina dei mammiferi.

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

Qui si descrive una soluzione economica per la cultura tecnica organotipica di retina suini adulti per sette giorni. In breve, una carta sterile filtro è stato usato per sollevare la retina neurale fuori dal RPE e fotorecettori lato posto su un inserto sollevata da una misura stand.

Cultura organotipica di tutto spessore Retina suina adulti

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 Views.  University of Medicine and Dentistry of New Jersey - UMDNJ. Qui si descrive una soluzione economica per la cultura tecnica organotipica di retina suini adulti per sette giorni. In breve, una carta sterile filtro è stato usato per sollevare la retina neurale fuori dal RPE e fotorecettori lato posto su un inserto sollevata da una misura stand.

Video: 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.

Il organotipica ippocampale Cultura Modello Slice per l&#39;esame di danno neuronale

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

Ricerca

Neuroscienze

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.

Slice Cultura organotipica di GFP che esprimono embrioni di topo per imaging in tempo reale della crescita degli nervi periferici

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 elettroporazione in vivo di Retina topo in via di sviluppo

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.

Isolamento e la coltura di adulti neurali cellule staminali dal Subcallosal Zona mouse

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.

Preparazione di orizzontali Fette di topo adulto Retina per elettrofisiologiche Studi

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.

Impressione confocale dei migratori dei neuroni nella cultura di fetta organotipica del cervello embrionale del mouse usando<em&gt; In Utero</em&gt; Elettroporazione

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.

Semplice generazione di una cultura ad alto rendimento dei neuroni indotti da fibroblasti umani della pelle adulta

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.

Isolamento e cultura dei neuroni primari e degli glia dalla vescica urinaria del ratto adulto

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.

Cultura delle Neurosfere Derivate dalle Nicchie Neurogeniche nelle Arvicole Della Prateria Adulta

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.

Elettroporazione a cella singola attraverso diverse culture di fette organotipiche di neuroni eccitatori ippocampali del topo e specifici della classe

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

Methods Article

Cultura organotipica di tutto spessore Retina suina adulti