Name: bilaminar co-culture of primary rat cortical neurons and glia
Uploaded: 2011-11-12T14:00:00
Description: Watch this Scientific Journal Video about Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia at JoVE.com

Les auteurs remercient les membres de laboratoire antérieurs qui ont contribué au raffinement de ce protocole et le NIH pour le soutien au fil des ans (et DA19808 DA15014 à l&#39;OM). Anna Abt 1 est un membre de la &quot;formation à la recherche interdisciplinaire et translationnelle NeuroSIDA&quot; (T32-MH078795); donc, ce travail a été soutenu en partie par les Instituts nationaux de santé en vertu de Ruth L. Kirschstein National Research Service Award 5T32MH079785.

1. Dissection GLIA (~ 2 semaines avant neurones placage) <ol><li> Pour se préparer à la place des outils de dissection stériles dans 70% d&#39;éthanol, ajouter 4 ml de dissection moyennes froide pour boîtes de 60 mm (un seul plat par cerveau), et au lieu de 2,5% de trypsine et de DNase sur la glace à dégeler (voir détails sur les outils chirurgicaux dans le tableau VI ). </li><li> Utilisez des ciseaux grande pour décapiter 2-4 jours chiots anciens et les chefs placer dans un plat de 100 mm stérile. </li><li>...

<ol>
	<li>Cook, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+chemokines:+regulation+of+fractalkine/CX3CL1+homeostasis+by+SDF/CXCL12+in+cortical+neurons.">Interactions between chemokines: regulation of fractalkine/CX3CL1 homeostasis by SDF/CXCL12 in cortical neurons.</a> J. Biol. Chem. 285, 10563-10563 (2010).</li><li>Nicolai, J., Burbassi, S., Rubin, J., Meucci, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CXCL12+inhibits+expression+of+the+NMDA+receptor's+NR2B+subunit+through+a+histone+deacetylase-dependent+pathway+contributing+to+neuronal+survival.">CXCL12 inhibits expression of the NMDA receptor's NR2B subunit through a histone deacetylase-dependent pathway contributing to neuronal survival.</a> Cell. Death. Dis. 1, e33-e33 (2010).</li><li>Sengupta, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphine+increases+brain+levels+of+ferritin+heavy+chain+leading+to+inhibition+of+CXCR4-mediated+survival+signaling+in+neurons.">Morphine increases brain levels of ferritin heavy chain leading to inhibition of CXCR4-mediated survival signaling in neurons.</a> J. Neurosci. 29, 2534-2544 (2009).</li><li>Meucci, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokines+regulate+hippocampal+neuronal+signaling+and+gp120+neurotoxicity.">Chemokines regulate hippocampal neuronal signaling and gp120 neurotoxicity.</a> Proc. Natl. Acad. Sci. U. S. A. 95, 14500-14500 (1998).</li><li>Khan, M. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+neuronal+P53+activity+by+CXCR+4.">Regulation of neuronal P53 activity by CXCR 4.</a> Mol. Cell. Neurosci. 30, 58-66 (2005).</li><li>Patel, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+neuronal+CXCR4+by+the+micro-opioid+agonist+DAMGO.">Modulation of neuronal CXCR4 by the micro-opioid agonist DAMGO.</a> J. Neurovirol. 12, 492-500 (2006).</li><li>Shimizu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+transcription+factor+E2F1+in+CXCR4-mediated+neurotoxicity+and+HIV+neuropathology.">Role of the transcription factor E2F1 in CXCR4-mediated neurotoxicity and HIV neuropathology.</a> Neurobiol. Dis. 25, 17-26 (2007).</li><li>Khan, M. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemokine+receptor+CXCR4+regulates+cell-cycle+proteins+in+neurons.">The chemokine receptor CXCR4 regulates cell-cycle proteins in neurons.</a> J. Neurovirol. 9, 300-314 (2003).</li><li>Khan, M. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemokine+CXCL12+promotes+survival+of+postmitotic+neurons+by+regulating+Rb+protein.">The chemokine CXCL12 promotes survival of postmitotic neurons by regulating Rb protein.</a> Cell. Death. Differ. 15, 1663-1672 (2008).</li><li>Khan, M. Z., Vaidya, A., Meucci, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CXCL12-mediated+regulation+of+ANP32A/Lanp,+a+component+of+the+inhibitor+of+histone+acetyl+transferase+(INHAT)+complex,+in+cortical+neurons.">CXCL12-mediated regulation of ANP32A/Lanp, a component of the inhibitor of histone acetyl transferase (INHAT) complex, in cortical neurons.</a> J. Neuroimmune. Pharmacol. 6, 163-170 (2011).</li><li>Dotti, C. G., Sullivan, C. A., Banker, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+establishment+of+polarity+by+hippocampal+neurons+in+culture.">The establishment of polarity by hippocampal neurons in culture.</a> J. Neurosci. 8, 1454-1468 (1988).</li><li>Kaech, S., Banker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+hippocampal+neurons.">Culturing hippocampal neurons.</a> Nat. Protoc. 1, 2406-2415 (2006).</li><li>Goslin, K., Banker, G., Goslin, K., Banker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culturing Nerve Cells. , 339-370 (1998).</li><li>D'Ambrosio, J., Fatatis, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoblasts+modulate+Ca2++signaling+in+bone-metastatic+prostate+and+breast+cancer+cells.">Osteoblasts modulate Ca2+ signaling in bone-metastatic prostate and breast cancer cells.</a> Clin. Exp. Metastasis. 26, 955-964 (2009).</li></ol>

Ce protocole prévoit un procédé de culture primaires de rat neurones corticaux, en présence de cellules gliales, tout en permettant aux neurones pour être facilement isolé pour analyse expérimentale. L&#39;appui au développement d&#39;un phénotype glie santé neuronale, tout en modulant les réponses neuronales à des traitements expérimentaux de manière physiologiquement pertinents. De plus, par passage de la glie primaire avant la culture avec les neurones de cette population cellulaire devient sélective e...

<table border="1">
	<tbody>
		<tr>
			<th>
				Reagent</th>
			<th>
				Concentration</th>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>16 mM</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>22 mM</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>135 mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>0.22 mM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>pH</td>
			<td>7.4</td>
		</tr>
		<tr>
			<td>Osmolarity</td>
			<td>310&plusmn;10 mOsm</td>
		</tr>
	</tbody>
</table>
Table 1. Dissection Medium.
<table border="1">
	<tbody>
		<tr>
			<th>
				Reagent</th>
			<th>
				Concentration</th>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>90%</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>10%</td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>50 &mu;g/mL</td>
		</tr>
	</tbody>
</table>
Tabe 2. Glia Plating Medium.
<table border="1">
	<tbody>
		<tr>
			<th>
				Reagent</th>
			<th>
				Concentration</th>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>90%</td>
		</tr>
		<tr>
			<td>Horse Serum</td>
			<td>10%</td>
		</tr>
	</tbody>
</table>
Table 3. Neuron Plating Medium.
<table>
	<tbody>
		<tr>
			<th>
				Reagent</th>
			<th>
				Concentration</th>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>98%</td>
		</tr>
		<tr>
			<td>N2 Supplement</td>
			<td>1%</td>
		</tr>
		<tr>
			<td>1M HEPES</td>
			<td>1%</td>
		</tr>
		<tr>
			<td>Ovalbumin</td>
			<td>50 mg/100mL</td>
		</tr>
	</tbody>
</table>
Table 4. N2 Medium.
<table border="1">
	<tbody>
		<tr>
			<th>
				Reagent</th>
			<th>
				Concentration</th>
		</tr>
		<tr>
			<td>Boric Acid</td>
			<td>50 mM</td>
		</tr>
		<tr>
			<td>Sodium Borate</td>
			<td>12.5 mM</td>
		</tr>
	</tbody>
</table>
Table 5. Borate Buffer (for poly-lysine).
<table border="1">
	<tbody>
		<tr>
			<th>
				Reagent</th>
			<th>
				Company</th>
			<th>
				Catalogue number</th>
		</tr>
		<tr>
			<td>High glucose Dulbecco's Modified Eagle 
			Medium (DMEM)</td>
			<td>Invitrogen</td>
			<td>11995-073</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS), Heat-inactivated</td>
			<td>Hyclone</td>
			<td>26400-044</td>
		</tr>
		<tr>
			<td>Horse Serum, Heat-inactivated</td>
			<td>Hyclone</td>
			<td>H1138</td>
		</tr>
		<tr>
			<td>Gentamicin (50mg/mL)</td>
			<td>Invitrogen</td>
			<td>15750-060</td>
		</tr>
		<tr>
			<td>N2 Supplement (100x)</td>
			<td>Invitrogen</td>
			<td>17502-048</td>
		</tr>
		<tr>
			<td>HEPES buffer solution</td>
			<td>Invitrogen</td>
			<td>15630-080</td>
		</tr>
		<tr>
			<td>Albumin from chicken egg white, Grade VI 
			(Ovalbumin)</td>
			<td>Sigma-Aldrich</td>
			<td>A2512</td>
		</tr>
		<tr>
			<td>2.5% Trypsin</td>
			<td>Invitrogen</td>
			<td>15090-046</td>
		</tr>
		<tr>
			<td>0.5% Trypsin-EDTA (10X)</td>
			<td>Invitrogen</td>
			<td>15400-054</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I from bovine pancreas 
			(DNase)</td>
			<td>Sigma-Aldrich</td>
			<td>D-5025</td>
		</tr>
		<tr>
			<td>Paraplast</td>
			<td>Fisher</td>
			<td>12-646-106</td>
		</tr>
		<tr>
			<td>Poly-L-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P1274</td>
		</tr>
		<tr>
			<td>Cytosine-&beta;-D-arabinofuranoside hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>C6645</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Leica</td>
			<td>Leica ZOOM 2000</td>
		</tr>
		<tr>
			<td>Cover glasses, Circles, 15 mm, Thickness 
			0.13-0.17 mm</td>
			<td>Carolina</td>
			<td>633031</td>
		</tr>
		<tr>
			<td>Thermanox sheets</td>
			<td>Grace BioLabs</td>
			<td>HS4550</td>
		</tr>
		<tr>
			<td>Large forceps</td>
			<td>Biomedical 
			Research 
			Instruments</td>
			<td>70-4000</td>
		</tr>
		<tr>
			<td>Fine-tipped No.5 forceps</td>
			<td>Fine Science 
			Tools</td>
			<td>91150-20</td>
		</tr>
		<tr>
			<td>Pattern No.1 forceps</td>
			<td>Biomedical 
			Research</td>
			<td>10-1400</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Instruments</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Scissors, straight, sharp-blunt</td>
			<td>Biomedical 
			Research 
			Instruments</td>
			<td>28-1435</td>
		</tr>
		<tr>
			<td>Micro Dissecting scissors</td>
			<td>Biomedical 
			Research 
			Instruments</td>
			<td>11-2070</td>
		</tr>
		<tr>
			<td>Micro Dissecting Curved scissors</td>
			<td>Biomedical 
			Research 
			Instruments</td>
			<td>11-1395</td>
		</tr>
	</tbody>
</table>

bilaminar co-culture of primary rat cortical neurons and glia

Cette vidéo va vous guider dans le processus de neurones de rat en culture corticaux, en présence d&#39;une couche nourricière gliales, un système connu comme un bilaminaire ou co-culture de modèle. Ce système est approprié pour une variété de besoins expérimentaux nécessitant soit un verre ou de plastique et substrat de croissance peut également être utilisé pour la culture des autres types de neurones. Neurones corticaux de rat obtenu à partir de la phase tardive embryonnaires (E17) sont étalées sur des lamelles de verre ou de boîtes de culture tissulaire face à une couche nourricière de cellules gliales cultivées sur des lamelles en plastique ou des plats (connu sous le nom Thermanox), respectivement. Le choix entre les deux configurations dépend de la technique expérimentale utilisée spécifiques, qui peuvent exiger, ou non, que les neurones sont cultivés sur le verre (par exemple l&#39;imagerie calcique par rapport Western blot). La couche nourricière gliales, les astrocytes une culture enrichie de cellules gliales secondaires mixtes, est préparé séparément à partir du cortex des ratons nouveau-nés (P2-4) avant le neuronaledissection. Un avantage majeur de ce système de culture par rapport à une culture de neurones est seulement le soutien de la croissance neuronale, la survie et la différenciation fournies par des facteurs trophiques sécrétés de la couche nourricière gliales, qui ressemble plus précisément l&#39;environnement du cerveau in vivo. Par ailleurs, la co-culture peut être utilisée pour étudier les interactions neuronales-gliales 1. Dans le même temps, la contamination glie dans la couche de neurones est empêchée par des moyens différents (culture à faible densité, l&#39;ajout d&#39;inhibiteurs mitotiques, le manque de sérum et de l&#39;utilisation de milieu de culture optimisé) conduisant à une couche pratiquement pure neuronale, comparable à d&#39;autres méthodes établies 1 -3. Les neurones peuvent être facilement séparées de la couche gliales à tout moment pendant la culture et utilisés pour différentes applications allant de l&#39;électrophysiologie expérimentale 4, biologie cellulaire et moléculaire 5-8, 5 biochimie, l&#39;imagerie et le microroscopy 4,6,7,9,10. Les neurones primaires s&#39;étendent axones et dendrites de former des synapses fonctionnelles 11, un processus qui n&#39;est pas observée dans les lignées cellulaires neuronales, même si certaines lignées cellulaires ne s&#39;étendent processus. Un protocole détaillé de neurones de rat en culture hippocampe en utilisant ce système de co-culture a été décrit précédemment 4,12,13. Ici, nous détaillons un protocole modifié adapté pour les neurones corticaux. Comme environ 20x10 6 cellules sont récupérées à partir de chaque embryon de rat, cette méthode est particulièrement utile pour des expériences nécessitant un grand nombre de neurones (mais pas préoccupé par une population très homogène neuronale). La préparation des neurones et cellules gliales doit être planifiée dans des délais spécifiques. Nous allons fournir le protocole étape par étape pour la culture de neurones de rat corticale ainsi que la culture de cellules gliales pour soutenir les neurones.

Watch this Scientific Journal Video about Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia at JoVE.com

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

Ici nous fournissons un protocole de neurones de rat en culture corticaux, en présence d&#39;une couche nourricière gliales. Les neurones cultivés en place de polarité et de créer des synapses, et peut être séparée de la glie pour une utilisation dans diverses applications, telles que l&#39;électrophysiologie, imagerie calcique, tests de survie cellulaire, immunocytochimie, et de l&#39;ARN / ADN / isolement des protéines.

Didermique co-culture de neurones de rat corticale primaire et des cellules gliales

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

bilaminar-co-culture-of-primary-rat-cortical-neurons-and-glia

Research

JoVE Journal

Neuroscience

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

In utero électroporation suivie par la culture neuronale primaire pour étudier la fonction des gènes dans un sous-ensemble des neurones corticaux

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

Recherche

Neurosciences

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Nucléofection et de la culture primaire de neurones embryonnaires de souris hippocampe et cortex

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP. A significant loss of &Delta;&Psi;m renders cells depleted of energy with subsequent death. Reactive oxygen species (ROS) are important signaling molecules, but their accumulation in pathological conditions leads to oxidative stress. The two major sources of ROS in cells are environmental toxins and the process of oxidative phosphorylation. Mitochondrial dysfunction and oxidative stress have been implicated in the pathophysiology of many diseases; therefore, the ability to determine &Delta;&Psi;m and ROS can provide important clues about the physiological status of the cell and the function of the mitochondria. 
Several fluorescent probes (Rhodamine 123, TMRM, TMRE, JC-1) can be used to determine &Delta;&psi;m in a variety of cell types, and many fluorescence indicators (Dihydroethidium, Dihydrorhodamine 123, H2DCF-DA) can be used to determine ROS. Nearly all of the available fluorescence probes used to assess &Delta;&Psi;m or ROS are single-wavelength indicators, which increase or decrease their fluorescence intensity proportional to a stimulus that increases or decreases the levels of &Delta;&Psi;m or ROS. Thus, it is imperative to measure the fluorescence intensity of these probes at the baseline level and after the application of a specific stimulus. This allows one to determine the percentage of change in fluorescence intensity between the baseline level and a stimulus. This change in fluorescence intensity reflects the change in relative levels of &Delta;&Psi;m or ROS. In this video, we demonstrate how to apply the fluorescence indicator, TMRM, in rat cortical neurons to determine the percentage change in TMRM fluorescence intensity between the baseline level and after applying FCCP, a mitochondrial uncoupler. The lower levels of TMRM fluorescence resulting from FCCP treatment reflect the depolarization of mitochondrial membrane potential. We also show how to apply the fluorescence probe H2DCF-DA to assess the level of ROS in cortical neurons, first at baseline and then after application of H2O2. This protocol (with minor modifications) can be also used to determine changes in &#x2206;&#x03A8;m and ROS in different cell types and in neurons isolated from other brain regions.

We demonstrate application of the fluorescence indicator, TMRM, in cortical neurons to determine the relative changes in TMRM fluorescence intensity before and after application of a specific stimulus. We also show application of the fluorescence probe H2DCF-DA to assess the relative level of reactive oxygen species in cortical neurons.

Détermination des espèces d&#39;oxygène Potentiel membrane mitochondriale et réactive dans les neurones corticaux de rat en direct

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP.  A ...

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture System

Proper neuron to glia interaction is critical to physiological function of the central nervous system (CNS). This bidirectional communication is sophisticatedly mediated by specific signaling pathways between neuron and glia1,2 . Identification and characterization of these signaling pathways is essential to the understanding of how neuron to glia interaction shapes CNS physiology. Previously, neuron and glia mixed cultures have been widely utilized for testing and characterizing signaling pathways between neuron and glia. What we have learned from these preparations and other in vivo tools, however, has suggested that mutual signaling between neuron and glia often occurred in specific compartments within neurons (i.e., axon, dendrite, or soma)3. This makes it important to develop a new culture system that allows separation of neuronal compartments and specifically examines the interaction between glia and neuronal axons/dendrites. In addition, the conventional mixed culture system is not capable of differentiating the soluble factors and direct membrane contact signals between neuron and glia. Furthermore, the large quantity of neurons and glial cells in the conventional co-culture system lacks the resolution necessary to observe the interaction between a single axon and a glial cell.
	In this study, we describe a novel axon and glia co-culture system with the use of a microfluidic culture platform (MCP). In this co-culture system, neurons and glial cells are cultured in two separate chambers that are connected through multiple central channels. In this microfluidic culture platform, only neuronal processes (especially axons) can enter the glial side through the central channels. In combination with powerful fluorescent protein labeling, this system allows direct examination of signaling pathways between axonal/dendritic and glial interactions, such as axon-mediated transcriptional regulation in glia, glia-mediated receptor trafficking in neuronal terminals, and glia-mediated axon growth. The narrow diameter of the chamber also significantly prohibits the flow of the neuron-enriched medium into the glial chamber, facilitating probing of the direct membrane-protein interaction between axons/dendrites and glial surfaces.

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System

This study describes the procedures of setting up a novel neuronal axon and (astro)glia co-culture platform. In this co-culture system, manipulation of direct interaction between a single axon (and single glial cell) becomes feasible, allowing mechanistic analysis of the mutual neuron to glial signaling.

Analyse en Imagerie du Neuron à l&#39;interaction Glia dans la plate-forme microfluidique Culture (MCP) à base neuronale et Axon Glia co-culture système

Proper  neuron to glia interaction is critical to physiological function of the central  nervous system (CNS). This bidirectional communication is ...

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.
Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.

This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved detail.

Imagerie épines dendritiques des neurones de l&#39;hippocampe de rat primaire utilisant structuré Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich structures suggest alterations in synaptic integrity and connectivity. Here we provide a detailed protocol for culturing primary rat cortical neurons, Phalloidin staining for F-actin puncta, and subsequent quantification techniques. First, the frontal cortex of E18 rat embryos are dissociated into low-density cell culture, then the neurons grown in vitro for at least 12-14 days. Following experimental treatment, the cortical neurons are stained with AlexaFluor 488 Phalloidin (to label the dendritic F-actin puncta) and microtubule-associated protein 2 (MAP2; to validate the neuronal cells and dendritic integrity). Finally, specialized software is used to analyze and quantify randomly selected neuronal dendrites. F-actin rich structures are identified on second order dendritic branches (length range 25-75 &#181;m) with continuous MAP2 immunofluorescence. The protocol presented here will be a useful method for investigating changes in dendritic synapse structures subsequent to experimental treatments.

Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons

Filamentous actin (F-actin) plays an important role in spinogenesis, synaptic plasticity, and synaptic stability. Quantification of F-actin puncta is therefore a useful tool to study the integrity of synaptic structures. This protocol describes the procedures of quantifying F-actin puncta labeled with Phalloidin in low-density primary cortical neuronal cultures.

Quantification des filamenteuses Actine (F-actine) punctas chez le rat neurones corticaux

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich ...

Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have a robust in vitro model that can be exploited for various neurobiology studies. Though primary neuron cultures (i.e., long-term hippocampal cultures) have provided scientists with models, it does not yet represent the complexity of brain network completely. In the wake of these limitations, a new model has emerged using neurospheres, which bears a closer resemblance to the brain tissue. The present protocol describes the plating of high and low densities of mixed cortical and hippocampal neurons isolated from the embryo of embryonic day 14-16 Sprague Dawley rats. This allows for the generation of neurospheres and long-term primary neuron culture as two independent platforms to conduct further studies. This process is extremely simple and cost-effective, as it minimizes several steps and reagents previously deemed essential for neuron culture. This is a robust protocol with minimal requirements that can be performed with achievable results and further used for a diversity of studies related to neuroscience.

Presented here is a protocol for the spontaneous generation of neurospheres enriched in neural progenitor cells from high density plated neurons. During the same experiment, when neurons are plated at a lower density, the protocol also results in prolonged primary rat neuron cultures.

Génération de neurosphères à partir de neurones hippocampes et corticales primaires mixtes isolées de l'embryon de rat Sprague Dawley E14-E16

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have ...

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have identified a special population of vascular cells, the periventricular endothelial cells, that play a critical role in the migration and distribution of forebrain GABAergic interneurons during embryonic development. This, coupled with their cell-autonomous functions, alludes to novel roles of periventricular endothelial cells in the pathology of neuropsychiatric disorders like schizophrenia, epilepsy, and autism. Here, we have described three different in vitro assays that collectively evaluate the functions of periventricular endothelial cells and their interaction with GABAergic interneurons. Use of these assays, particularly in a human context, will allow us to identify the link between periventricular endothelial cells and brain disorders. These assays are simple, low cost, and reproducible, and can be easily adapted to any adherent cell type.

We present three simple in vitro assays-the long-distance migration assay, the co-culture migration assay, and chemo-attraction assay-that collectively evaluate the functions of human stem cell derived periventricular endothelial cells and their interaction with GABAergic interneurons.

Migrations, Chemo-Attraction et Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons (en anglais)

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have ...

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.

Isolement et culture des neurones primaires et glia de la vessie urinaire adulte de rat

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

Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory bulb. Throughout adult life, they are key for axonal growing of newly generated olfactory neurons, from the lamina propria to the ONL. Due to their pro-regenerative properties, these cells have been used to foster axonal regeneration in spinal cord or optic nerve injury models.
We present an in vitro model to assay and measure OEG neuroregenerative capacity after neural injury. In this model, reversibly immortalized human OEG (ihOEG) is cultured as a monolayer, retinas are extracted from adult rats and retinal ganglion neurons (RGN) are cocultured onto the OEG monolayer. After 96 h, axonal and somatodendritic markers in RGNs are analyzed by immunofluorescence and the number of RGNs with axon and the mean axonal length/neuron are quantified.
This protocol has the advantage over other in vitro assays that rely on embryonic or postnatal neurons, that it evaluates OEG neuroregenerative properties in adult tissue. Also, it is not only useful for assessing the neuroregenerative potential of ihOEG but can be extended to different sources of OEG or other glial cells.

We present an in vitro model to assess olfactory ensheathing glia (OEG) neuroregenerative capacity, after neural injury. It is based on a coculture of axotomized adult retinal ganglion neurons (RGN) on OEG monolayers and subsequent study of axonal regeneration, by analyzing RGN axonal and somatodendritic markers.

Coculture de neurones rétiniens axotomisés de ganglion de rat avec glia ensheathing olfactif, comme modèle in vitro de régénération axonale adulte

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory ...