Financial support for the experiments presented in the study was provided by the Marie Heim-V&#246;gtlin program of the Swiss National Science Foundation.
We thank Ms. Johanna Nyffeler for developing a set of modified MATLAB programs for screening for statistically significant differences in real-time cell analyzer&#8217;s data and Dr. Yama Abassi for helpful discussion.

Préparation 1 <ol><li> Placez la station de la temps réel analyseur de cellules dans un incubateur de culture de tissu fixée à 37 ° C et 5% de CO 2. Effectuer toutes les manipulations de culture cellulaire et de traitements pharmacologiques dans une hotte de culture de tissu dans des conditions stériles. </li><li> NOTE: neuronale lignées cellulaires nécessitant différents réglages de température par rapport à des conditions normales de culture, doivent être ajustés en conséquence. Pour les lignées cellulaires faiblement adhérentes, utiliser les agents de revêtement pour faciliter l&#39;interaction des cellules avec les microélectrodes d&#39;or dans le fond des puits de la plaque E-96. </li><li> Préparer 1,000X solutions stock des composés pharmacologiques pour le traitement de culture cellulaire (agents neuroprotecteurs ou deuxième inhibiteurs de voies de messagerie dans le solvant approprié - diméthylsulfoxyde ou de l&#39;eau stérile) et de les stocker à -20 ° C. Utilisez le solvant comme véhicule dans les traitements suivants. </li><li> Culture des cellules de neuroblastome humain SK-N-SH dans du DMEM / F1Deux milieu supplémenté avec 10% de sérum bovin fœtal (FBS) (milieu de prolifération) dans des flacons de culture cellulaire avec 175 cm 2 de surface de densité d&#39;environ 75%. Garder nombre de passage de cellules dans une plage (environ 10 passages) pour vérifier la cohérence entre les expériences en termes de taux de prolifération cellulaire et de la réponse de cytotoxicité. Les nombres inférieurs de passage sont préférés. </li><li> Laver la couche adhérente de cellules SK-N-SH avec du PBS, et trypsiniser avec une solution de trypsine-EDTA à 0,05% pendant 5 min à 37 ° C. Centrifuger les cellules à 170 xg pendant 5 min pour éliminer la trypsine. Remettre en suspension les cellules dans du milieu de prolifération de 10 ml. Compter les cellules avec Sceptre compteur de cellules et d&#39;ajuster le nombre de cellules à 300 000 cellules / ml en diluant les cellules avec un milieu de prolifération. </li></ol> 2. placage et la prolifération des cellules SK-N-SH <ol><li> Ajouter milieu de prolifération de 100 ul dans chaque puits de la plaque de E-96 et le laisser pendant 30 min dans la hotte de culture de tissu à la température ambiante pour équilibrer. Insérez le E-Plate 96 dans la station d&#39;analyse de cellules en temps réel dans l&#39;incubateur à CO2 à 37 ° C. </li><li> Démarrez le logiciel de l&#39;analyseur de cellules en temps réel. Sur la page Mise sélectionner les puits inclus dans l&#39;expérience et entrent dans les boîtes de saisie des informations sur le type de cellule, le nombre de cellules, les noms et les concentrations de composés chimiques utilisés pour le traitement des cellules. NOTE: Pour les fins du présent protocole au moins 4 répétitions sont recommandés pour chaque traitement. </li><li> Sur la page du logiciel de l&#39;annexe déterminer &quot;Steps&quot; inclus dans l&#39;expérience, en sélectionnant le nombre de balayages de mesure l&#39;indice cellulaire et l&#39;intervalle entre les balayages pour chaque étape. Depuis l&#39;étape 1 est prédéterminée pour la mesure de fond, sélectionnez «Ajouter une étape» et réglez l&#39;étape 2 pour mesurer l&#39;indice cellulaire toutes les 15 minutes pendant 96 heures. NOTE: Pour les traitements, dans lequel les effets avec une cinétique rapide sont attendus, mis balayages à intervalles plus rapprochés. </li><li> Cliquez sur Démarrer pour lancer l&#39;étape prédéterminée automatiquement 1et mesurer l&#39;impédance des supports de fond. Cette valeur est soustraite automatiquement par le logiciel au niveau de chaque point de données une fois que les cellules sont ajoutées aux puits. </li><li> Utilisation préparé précédemment dans l&#39;étape 1.5), suspension de cellules de 300 000 cellules / ml dans du milieu de prolifération. 30 000 cellules / puits pour la toxicité cellulaire / études de neuroprotection et 15 000 cellules / puits pour des études de prolifération de cellules. Sortez le E-Plate et ajouter 100 suspension cellulaire par puits pour la toxicité cellulaire / études de neuroprotection et ajouter 50 ul suspension de cellules et milieu de prolifération de 50 pi par puits pour les études de la prolifération cellulaire. Le nombre de cellules étalées par puits doit être optimisée pour chaque lignée cellulaire de façon empirique. </li><li> Agitez doucement le E-Plate 96, même pour le placage des cellules. Laisser la plaque E-96 à 30 min dans la hotte de culture de tissu à la température ambiante pour permettre aux cellules de se déposent uniformément sur le fond des puits. </li><li> Insérez le E-Plate 96 dans la station d&#39;analyse cellulaire en temps réel et commencer à l&#39;étape 2 de l&#39;annexe pâge. Surveiller les valeurs de l&#39;indice de la cellule à travers l&#39;expérience en regardant les courbes de cellules sur la page de terrain et les données de l&#39;indice cellulaire premières sur la page Index portable du logiciel. </li></ol> 3. la privation de sérum <ol><li> 24 heures après l&#39;étalement des cellules, mettre en pause l&#39;expérience, et retirez le E-Plate 96 de la station d&#39;analyse cellulaire en temps réel. Diluer deuxième inhibiteurs de messager des stocks 1,000X avec un milieu sans sérum / DMEM F12 - 200 × la concentration finale. Traiter les cellules avec 1 pi inhibiteurs de la seconde piste de messagerie pour être testés pour leur implication dans la neurotoxicité / neuroprotection 30 min avant de commencer la cytotoxicité, pour inhiber les voies respectives. Retour le E-Plate 96 de la gare et reprendre les mesures expérimentales de l&#39;indice de la cellule. </li><li> Après 30 min de pause à nouveau l&#39;expérience. Retirez délicatement milieu de prolifération pleinement de l&#39;E-Plate 96 puits avec une pipette (ou multi-canal pipette), en prenant soin de ne pas perturber la couche de cellules adhérentesen orientant le bord de la pointe de la pipette à l&#39;angle du puits. Ajouter 200 pl / puits de DMEM sans sérum / milieu F12 (milieu de privation de sérum). Assurer un échange rapide d&#39;un milieu de culture cellulaire pour réduire au minimum l&#39;agitation mécanique des cellules. </li><li> Diluer les composés à tester pour leurs effets neuroprotecteurs de 1,000X actions avec le milieu DMEM / F12 sans sérum à 200 × la concentration finale. Ajouter 1 composés de pi à tester leurs effets neuroprotecteurs et 1 pi inhibiteurs de la seconde piste de messagerie dans les puits individuels selon le plan expérimental. </li><li> Dans les cellules pour des études de prolifération ne changez pas moyen. Diluer les composés à tester de 1,000X actions à 200 × la concentration finale dans le milieu de la prolifération, de traiter avec 1 l par puits et continuer à la culture en milieu de prolifération. </li><li> Reprendre l&#39;expérience pour poursuivre Index cellulaires mesures toutes les 15 minutes pendant 96 heures comme indiqué précédemment. </li></ol> 4. données Analyse <ol><li> Pour neurotoxicité / données de neuroprotection normaliser l&#39;indice cellulaire au dernier point de temps avant le traitement pharmacologique ou changement de milieu pour réduire les variations entre les expériences en sélectionnant &quot;Cell normalisée Index&quot; et &quot;normaliser Time&quot; sur la page de la parcelle. </li><li> Mettez en surbrillance les puits sur la page de la parcelle pour les conditions expérimentales d&#39;intérêt et cliquez sur &quot;Ajouter&quot; pour tracer les courbes normalisées de l&#39;indice de la cellule. Observer la cinétique de l&#39;effet neurotoxique / neuroprotecteur, ou de messagers deuxième inhibition que les courbes de l&#39;indice cellulaire normalisées en fonction du temps. </li><li> Respecter les courbes de l&#39;indice de portable pour les traitements médicamenteux testés en milieu de prolifération en fonction du temps d&#39;évaluer si un effet sur la prolifération est impliqué dans l&#39;effet neuroprotecteur / neurotoxique. </li><li> Exporter l&#39;info expérimental pour tous les points Cell temps Indice de la page du logiciel Indice de cellules dans un fichier Excel pour lancer l&#39;évaluation statistique des résultats <br/&gt; NOTE: Dans une première étape dans l&#39;analyse statistique, un ensemble de programmes MATLAB modifiés qui utilisent le test t de Welch, avec le p-valeur tracée semilogarithmically, utiliser axe inversé sur l&#39;échelle de temps (à condition que les fichiers de code supplémentaires). Ces programmes permettent la détection des valeurs de p au-dessus de l&#39;évolution temporelle de l&#39;ensemble de l&#39;expérience en temps réel de l&#39;analyseur de la cellule, et ainsi faciliter la sélection de points dans le temps pour l&#39;analyse statistique plus détaillée. Voir le matériel complémentaire pour des informations détaillées sur les programmes. </li><li> Pour l&#39;analyse statistique des différences cellulaires valeurs de l&#39;indice sous différentes conditions de traitement à des moments spécifiques, sélectionnez des valeurs de l&#39;indice cellulaire à des moments respectifs à partir du fichier Excel exporté. Analyser les valeurs de l&#39;indice de la cellule pour les points de temps choisis avec un sens ou dans les deux sens l&#39;analyse de variance (Anova) suivie d&#39;un test post-hoc en utilisant un logiciel statistique. </li></ol>

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Les frais de publication de la vidéo-article en cours ont été parrainés par &quot;ACEA Biosciences&quot;.

Le protocole actuel présente l&#39;utilité d&#39;une cellule d&#39;analyse en temps réel pour évaluer de façon continue et dans des conditions sans étiquette / les effets neuroprotecteurs des composés neurotoxiques dans des lignées cellulaires neuronales et pour mieux comprendre les secondes voies de messagers impliqués dans l&#39;effet. Même si l&#39;utilité du temps réel analyseur de cellules pour étudier la cytotoxicité et les effets des médicaments sur la prolifération ce...

La mort cellulaire neuronale joue un rôle critique dans la pathophysiologie de nombreux troubles liés au cerveau 1. La disponibilité d&#39;informations fiables et à haut débit dans des tests de toxicité in vitro est essentiel d&#39;avoir un meilleur aperçu des mécanismes de la neurotoxicité et d&#39;aider à sélectionner des molécules neuroprotectrices comme candidats thérapeutiques dans le développement de médicaments 2. Cependant, il ya beaucoup de limitations à la plus largement utilisée in vitro neurotoxicité assays.They évaluer la neurotoxicité / neuroprotection à un seul point dans le temps ne permet pas la résolution cinétique; utilisent souvent l&#39;étiquette ou de la sonde qui peut interférer avec les voies de signalisation et de limiter d&#39;autres études dans la même population de cellules, et sont souvent de main-d&#39;œuvre, et dans de nombreux cas ne fournit pas une approche mécanistique. Dans la présente étude, nous avons démontré l&#39;utilité d&#39;une cellule d&#39;analyse en fonction de l&#39;impédance en temps réel afin de déterminer la neurotoxicité et de neuroprotection dans une lignée cellulaire neuronale, en temps réel et sans marqueur sousconditions et permettent de mieux comprendre les mécanismes en aval à travers l&#39;analyse de la seconde piste de messagerie impliqués dans l&#39;effet. Des études antérieures ont confirmé la validité de la cellule d&#39;analyse en temps réel afin de déterminer la cytotoxicité, ainsi que les effets sur la prolifération cellulaire dans les lignées cellulaires par comparaison avec des techniques classiques 3,4,5,6. Par exemple, une bonne corrélation a été observée entre les lectures de la norme viabilité cellulaire WST-1 essai et cellulaires valeurs de l&#39;indice à plusieurs points dans le temps dans des conditions de prolifération basale et après deux paradigmes différents toxiques dans les cellules HeLa 3. Dans A549 et MDA-MB-231 cellules prolifération et la cytotoxicité provoquée par le paclitaxel de stabilisation des microtubules ont montré des valeurs très similaires lors de l&#39;évaluation par la mesure de l&#39;indice cellulaire et la sulforhodamine utilisé en standard B (SRB) essai 4. Dans la lignée cellulaire neuronale de neurones de l&#39;hippocampe immortalisés Index HT-22 cellulaires mesures ont été validés pour leur capacité to détecter la prolifération cellulaire, la cytotoxicité et la glutamate cytoprotection contre le 3-(4,5-diméthylthiazol-2-yl) largement utilisé du 2,5-diphényl-bromure (MTT) 5. Dans la même étude, les résultats d&#39;analyse de MTT et les mesures de l&#39;indice cellulaire aussi bien corrélées à la mesure neuronale des cellules progénitrices prolifération, cytotoxicité après les facteurs de croissance privation et de sauvetage de la cytotoxicité par le pan-caspase inhibiteur QVD 5. La cytotoxicité induite dans des cellules NIH 3T3 par Vandetanib (récepteur du facteur de croissance de l&#39;endothélium vasculaire et d&#39;un inhibiteur du récepteur du facteur de croissance épidermique) a montré des résultats similaires avec des valeurs mesurées de l&#39;indice cellulaire ou neutre essai d&#39;absorption rouge 6. Récemment, nous avons utilisé le système d&#39;analyse de cellules en temps réel pour évaluer les effets neuroprotecteurs de la figure 2A de la sérotonine (5-HT 2A) agoniste du récepteur de chlorhydrate de (±) -2,5-diméthoxy-4-iodoamphétamine (DOI) dans une lignée cellulaire neuronale ( cellules SK-N-SH) et criblés pour la participation du second voies de messagerie par le biais de la surveillance de l&#39;effet de leur inhibition chimique sur la neuroprotection observée 7. Fait intéressant, le récepteur 5-HT 2A a agonistes fois hallucinogènes et nonhallucinogenic (comme DOI et lisuride, respectivement), ce qui peut activer les deux secondes voies de messagerie communs et distincts 8. Les avantages de la technique présentée est qu&#39;il permet de recueillir des informations en temps réel sur la survie des cellules au cours de jours, pour délimiter les voies de seconds messagers impliqués, afin d&#39;évaluer la contribution possible des effets de prolifération à la neuroprotection, et pour sélectionner un temps optimal des études supplémentaires de point final sur la même population de cellules. Un diagramme schématique du flux de travail dans le protocole actuel est présenté sur la figure 1.

<table border="0" cellpadding="0" cellspacing="0" width="1015">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Name of Material/ Equipment</td>
			<td style="width:161px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:457px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">xCELLigence&#160; RTCA SP system bundle</td>
			<td style="width:161px;">ACEA Biosciences</td>
			<td style="width:119px;">No: 00380601030</td>
			<td>consists of RTCA Analyzer, RTCA SP Station and RTCA Control Unit</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">E-plate 96</td>
			<td style="width:161px;">ACEA Biosciences</td>
			<td style="width:119px;">No: 05232368001</td>
			<td>for culturing the cells, inserted in the RTCA SP Station</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">SK-N-SH cells</td>
			<td style="width:161px;">ATCC - (in partnership with LGC Standards)</td>
			<td style="width:119px;">HTB-11</td>
			<td>can be replaced by another adherent neuronal cell line of interest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">DMEM/F12&#160;</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8437</td>
			<td>cell culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">fetal bovine serum</td>
			<td style="width:161px;">Life Technologies</td>
			<td style="width:119px;">16140-063</td>
			<td>supplements proliferation, but not serum deprivation medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">tisue culture flask T175</td>
			<td style="width:161px;">Sarstedt</td>
			<td style="width:119px;">83.1812.302&#160;</td>
			<td>for culturing cells, which will be later plated on the E-Plate 96</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">0.05% Trypsin-EDTA</td>
			<td style="width:161px;">Life Technologies</td>
			<td style="width:119px;">25300-054</td>
			<td>for trypsinization of cells cultured in tissue culture flasks</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Scepter 2.0 cell counter</td>
			<td style="width:161px;">Merck Millipore</td>
			<td style="width:119px;">PHCC20060</td>
			<td>automated cell counter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">phosphate-buffered saline</td>
			<td style="width:161px;">Life Technologies</td>
			<td style="width:119px;">10010-015</td>
			<td>for washing the cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">(&#177;)-DOI hydrochloride</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:119px;">D101</td>
			<td>5-HT2A agonist for cell culture treatment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">LY-294,002 hydrochloride</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:119px;">L9908</td>
			<td>PI3-K inhibitor for cell culture treatment&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">lisuride maleate</td>
			<td style="width:161px;">Tocris Bioscience</td>
			<td align="right" style="width:119px;">4052</td>
			<td>compound with 5-HT2A agonistic activity for cell culture treatment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">dimethyl sulfoxide</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:119px;">D4540</td>
			<td>for dissolving LY-294,002 and lisuride maleate</td>
		</tr>
	</tbody>
</table>

real-time impedance-based cell analyzer as a tool to delineate molecular pathways involved in neurotoxicity and neuroprotection in a neuronal cell line

De nombreux troubles liés au cerveau ont mort des cellules neuronales impliquées dans leur physiopathologie. Amélioration des modèles in vitro pour étudier les effets neuroprotecteurs ou neurotoxiques de drogues et les voies en aval concernés aiderait mieux comprendre les mécanismes moléculaires de neuroprotection / neurotoxicité et pourrait faciliter le développement de médicaments. Cependant, de nombreux tests in vitro existantes sur la toxicité comportent des limites importantes - plus évaluer la neurotoxicité et la neuroprotection à un seul point du temps, ne permettant pas d&#39;observer l&#39;évolution temporelle et la cinétique de l&#39;effet. En outre, la possibilité de recueillir des informations sur les voies de signalisation impliquées dans la neuroprotection aval en temps réel serait d&#39;une grande importance. Dans le protocole en vigueur, nous décrivons l&#39;utilisation d&#39;une cellule d&#39;analyse en fonction de l&#39;impédance en temps réel pour déterminer les effets neuroprotecteurs de la serotonine 2A (5-HT 2A) des agonistes du récepteur dans une lignée de cellules neuronales dans le cadre sans marquage et en temps réel conditions à l&#39;aide de mesures d&#39;impédance. De plus, nous démontrons que les inhibiteurs de la seconde piste de messagerie peuvent être utilisés pour délimiter les molécules en aval impliqués dans l&#39;effet neuroprotecteur. Nous décrivons également l&#39;utilité de cette technique pour déterminer si un effet sur la prolifération cellulaire contribue à un effet neuroprotecteur observé. Le système utilise des plaques spéciales microélectroniques dénommé E-plaques qui contiennent alternance réseaux de microélectrodes d&#39;or sur la surface inférieure des puits, servant de capteurs cellulaires. L&#39;affichage de l&#39;impédance est modifiée par le nombre de cellules adhérentes, la viabilité des cellules, la morphologie, et l&#39;adhérence. Un paramètre sans dimension appelé l&#39;indice cellulaire est dérivé à partir des mesures d&#39;impédance électrique et est utilisée pour représenter l&#39;état de la cellule. Dans l&#39;ensemble, le temps réel analyseur de cellules à base d&#39;impédance permet en temps réel, l&#39;évaluation sans étiquette de la neuroprotection et la neurotoxicité, et l&#39;évaluation de la deuxième participation de voies de messagerie, contribuant à plusévaluation détaillée et à haut débit de composés neuroprotecteurs potentiels in vitro, pour la sélection des candidats thérapeutiques.

<html> Privation de sérum conduit à diminuer dans les piles valeurs de l&#39;indice, qui peuvent être surveillés en permanence avec la cellule d&#39;analyse en temps réel Stimuli neurotoxiques aux cellules conduisent à une diminution de cellulaires valeurs de l&#39;indice, qui peut être surveillé en temps réel avec la technique présentée et dont la dynamique dépend de la relance neurotoxique spécifique et le type de cellule étudiée. Figure 2 mo...

Watch this Scientific Journal Video about Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line at JoV

Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

Improved in vitro neurotoxicity assays would aid the identification of new neuroprotective compounds. The utility of a real-time impedance-based cell analyzer to determine cytotoxicity and cytoprotection in neuronal cell lines and to delineate the involvement of second messenger pathways, thus gaining insight in the mechanism of neuroprotection is presented.

Analyseur portable à base d&#39;impédance en temps réel comme un outil pour délimiter voies moléculaires impliquées dans la neurotoxicité et la neuroprotection dans une lignée cellulaire neuronale

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic ...

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Neuroscience

11.3K vues.  University of Zürich. Improved in vitro neurotoxicity assays would aid the identification of new neuroprotective compounds. The utility of a real-time impedance-based cell analyzer to determine cytotoxicity and cytoprotection in neuronal cell lines and to delineate the involvement of second messenger pathways, thus gaining insight in the mechanism of neuroprotection is presented.

Vidéo: Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3. Specifically, it affects the ability of neurons to receive inputs from other cells2 and contributes to the propagation of action potentials4,5. The morphology of the neurites also affects how information is processed. The diversity of dendrite morphologies facilitate local and long range signaling and allow individual neurons or groups of neurons to carry out specialized functions within the neuronal network6,7. Alterations in dendrite morphology, including fragmentation of dendrites and changes in branching patterns, have been observed in a number of disease states, including Alzheimer's disease8, schizophrenia9,10, and mental retardation11. The ability to both understand the factors that shape dendrite morphologies and to identify changes in dendrite morphologies is essential in the understanding of nervous system function and dysfunction.
Neurite morphology is often analyzed by Sholl analysis and by counting the number of neurites and the number of branch tips. This analysis is generally applied to dendrites, but it can also be applied to axons. Performing this analysis by hand is both time consuming and inevitably introduces variability due to experimenter bias and inconsistency. The Bonfire program is a semi-automated approach to the analysis of dendrite and axon morphology that builds upon available open-source morphological analysis tools. Our program enables the detection of local changes in dendrite and axon branching behaviors by performing Sholl analysis on subregions of the neuritic arbor. For example, Sholl analysis is performed on both the neuron as a whole as well as on each subset of processes (primary, secondary, terminal, root, etc.) Dendrite and axon patterning is influenced by a number of intracellular and extracellular factors, many acting locally. Thus, the resulting arbor morphology is a result of specific processes acting on specific neurites, making it necessary to perform morphological analysis on a smaller scale in order to observe these local variations12.
The Bonfire program requires the use of two open-source analysis tools, the NeuronJ plugin to ImageJ and NeuronStudio. Neurons are traced in ImageJ, and NeuronStudio is used to define the connectivity between neurites. Bonfire contains a number of custom scripts written in MATLAB (MathWorks) that are used to convert the data into the appropriate format for further analysis, check for user errors, and ultimately perform Sholl analysis. Finally, data are exported into Excel for statistical analysis. A flow chart of the Bonfire program is shown in Figure 1.

We have developed a computer program to analyze neuronal morphology. In combination with two existing open source analysis tools, our program performs Sholl analysis and determines the number of neurites, branch points, and neurite tips. The analyses are performed so that local changes in neurite morphology can be observed.

Analyse automatisée des Sholl morphologie neuronale numérisés à de multiples échelles

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3.  Specifically, it affects the ability of neurons to ...

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Neurosciences

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

Dérivation efficace des ressources humaines progéniteurs neuronaux et des neurones à partir pluripotentes cellules souches embryonnaires humaines à l&#39;induction de petites molécules

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Although the brain represents less than 5% of the body by mass, it utilizes approximately one quarter of the glucose used by the body at rest1. The function of non rapid eye movement sleep (NREMS), the largest portion of sleep by time, is uncertain. However, one salient feature of NREMS is a significant reduction in the rate of cerebral glucose utilization relative to wakefulness2-4. This and other findings have led to the widely held belief that sleep serves a function related to cerebral metabolism. Yet, the mechanisms underlying the reduction in cerebral glucose metabolism during NREMS remain to be elucidated.
	One phenomenon associated with NREMS that might impact cerebral metabolic rate is the occurrence of slow waves, oscillations at frequencies less than 4 Hz, in the electroencephalogram5,6. These slow waves detected at the level of the skull or cerebral cortical surface reflect the oscillations of underlying neurons between a depolarized/up state and a hyperpolarized/down state7. During the down state, cells do not undergo action potentials for intervals of up to several hundred milliseconds. Restoration of ionic concentration gradients subsequent to action potentials represents a significant metabolic load on the cell8; absence of action potentials during down states associated with NREMS may contribute to reduced metabolism relative to wake.
	Two technical challenges had to be addressed in order for this hypothetical relationship to be tested. First, it was necessary to measure cerebral glycolytic metabolism with a temporal resolution reflective of the dynamics of the cerebral EEG (that is, over seconds rather than minutes). To do so, we measured the concentration of lactate, the product of aerobic glycolysis, and therefore a readout of the rate of glucose metabolism in the brains of mice. Lactate was measured using a lactate oxidase based real time sensor embedded in the frontal cortex. The sensing mechanism consists of a platinum-iridium electrode surrounded by a layer of lactate oxidase molecules. Metabolism of lactate by lactate oxidase produces hydrogen peroxide, which produces a current in the platinum-iridium electrode. So a ramping up of cerebral glycolysis provides an increase in the concentration of substrate for lactate oxidase, which then is reflected in increased current at the sensing electrode. It was additionally necessary to measure these variables while manipulating the excitability of the cerebral cortex, in order to isolate this variable from other facets of NREMS.
	We devised an experimental system for simultaneous measurement of neuronal activity via the elecetroencephalogram, measurement of glycolytic flux via a lactate biosensor, and manipulation of cerebral cortical neuronal activity via optogenetic activation of pyramidal neurons. We have utilized this system to document the relationship between sleep-related electroencephalographic waveforms and the moment-to-moment dynamics of lactate concentration in the cerebral cortex. The protocol may be useful for any individual interested in studying, in freely behaving rodents, the relationship between neuronal activity measured at the electroencephalographic level and cellular energetics within the brain.

A procedure is described for manipulating the activity of cerebral cortical pyramidal neurons optogenetically while the electroencephalogram, electromyogram, and cerebral lactate concentration are monitored. Experimental recordings are performed on cable-tethered mice while they undergo spontaneous sleep/wake cycles. Optogenetic equipment is assembled in our laboratory; recording equipment is commercially available.

Électroencéphalographie simultanée, en temps réel Mesure de la concentration de lactate et manipulation optogenetic de l&#39;activité neuronale dans le cortex cérébral des rongeurs

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

Rapide Micro-iontophorèse de glutamate et le GABA: un outil utile d&#39;étudier l&#39;intégration synaptique

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Sex Stratified Neuronal Cultures to Study Ischemic Cell Death Pathways

Sex differences in neuronal susceptibility to ischemic injury and neurodegenerative disease have long been observed, but the signaling mechanisms responsible for those differences remain unclear. Primary disassociated embryonic neuronal culture provides a simplified experimental model with which to investigate the neuronal cell signaling involved in cell death as a result of ischemia or disease; however, most neuronal cultures used in research today are mixed sex. Researchers can and do test the effects of sex steroid treatment in mixed sex neuronal cultures in models of neuronal injury and disease, but accumulating evidence suggests that the female brain responds to androgens, estrogens, and progesterone differently than the male brain. Furthermore, neonate male and female rodents respond differently to ischemic injury, with males experiencing greater injury following cerebral ischemia than females. Thus, mixed sex neuronal cultures might obscure and confound the experimental results; important information might be missed. For this reason, the Herson Lab at the University of Colorado School of Medicine routinely prepares sex-stratified primary disassociated embryonic neuronal cultures from both hippocampus and cortex. Embryos are sexed before harvesting of brain tissue and male and female tissue are disassociated separately, plated separately, and maintained separately. Using this method, the Herson Lab has demonstrated a male-specific role for the ion channel TRPM2 in ischemic cell death. In this manuscript, we share and discuss our protocol for sexing embryonic mice and preparing sex-stratified hippocampal primary disassociated neuron cultures. This method can be adapted to prepare sex-stratified cortical cultures and the method for embryo sexing can be used in conjunction with other protocols for any study in which sex is thought to be an important determinant of outcome.

Primary disassociated embryonic hippocampal neuronal cultures are useful for investigating the signaling mechanisms involved in neuron death. Sexing the embryos before the isolation and dissociation of the hippocampus allows the preparation of separate male and female cultures, which enables the researcher to identify and investigate sex-specific cell signaling.

Sex differences in neuronal susceptibility to ischemic injury and neurodegenerative disease have long been observed, but the signaling mechanisms ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

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

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

Induire la plasticité de Astrocytic récepteurs neuronaux par manipulation des taux de combustion

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

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

Utilisant Méthodologies combinés pour définir le rôle du Plasma Membrane Livraison Pendant Axon Branching et Neuronal morphogénèse

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

Microglia as a Surrogate Biosensor to Determine Nanoparticle Neurotoxicity

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay described here will aid in elucidating the role of microglia in neuroinflammation and neurodegenerative diseases. The use of microglia, resident immune cells of the brain, as a surrogate biosensor provides novel insight into how inflammatory responses mediate neuronal insults. Here, we utilize an immortalized murine microglial cell line, designated BV2, and describe a method for nanoparticle exposure using silver nanoparticles (AgNPs) as a standard. We describe how to expose microglia to nanoparticles, how to remove nanoparticles from supernatant, and how to use supernatant from activated microglia to determine toxicity, using hypothalamic cell survival as a measure. Following AgNP exposure, BV2 microglial activation was validated using a tumor necrosis factor alpha (TNF-&#945;) enzyme linked immunosorbent assay (ELISA). The supernatant was filtered to remove the AgNP and to allow cytokines and other secreted factors to remain in the conditioned media. Hypothalamic cells were then exposed to supernatant from AgNP activated microglia and survival of neurons was determined using a resazurin-based fluorescent assay. This technique is useful for utilizing microglia as a surrogate biomarker of neuroinflammation and determining the effect of neuroinflammation on other cell types.

Microglia (immune cells of the brain), are used as a surrogate biosensor to determine how nanoparticles influence neurotoxicity. We describe a series of experiments designed to assay microglial response to nanoparticles and exposure of hypothalamic neurons to supernatant from activated microglia to determine neurotoxicity.

Microglies comme un biocapteur Surrogate pour déterminer Nanoparticules neurotoxicité

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.
Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

In Utero électroporation approches pour étudier l&#39;excitabilité des neurones sous - populations et connectivité unicellulaire

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

Hippocampe organotypiques comme modèle d’étude Neuroprotection et son caractère invasif des cellules tumorales

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...