Cette étude a été soutenue par FMMU nouvelle fondation et trouver le financement partiel du NIH.

1. Culture cellulaire Glia Les cellules gliales peuvent être cultivées à partir de différentes régions du système nerveux central. L&#39;ensemble du processus est montré dans la figure processus. <img src="/files/ftp_upload/2111/2111process.jpg" alt="Processus 1" /> Jour 1 plaque de culture de revêtement et des lamelles <ol><li> Dry rondes stérilisés lamelles de microscope en verre dans un autoclave. </li><li> Plaque les lamelles stérilisés dans la plaque de culture stérilisé de 24 puits. </li><li> Enduire les lamelles avec de la poly-lysine et incuber pendant 2 heures sous une température racine. </li><li> Manteau plaques de culture de 6 puits de la même manière qu&#39;à l&#39;étape 3. </li><li> Lavez les lamelles et la plaque de culture de 6 puits à deux reprises avec de l&#39;eau distillée et sécher à l&#39;air dans le capot de la culture. </li><li> Ajouter 200 pl de milieu DMEM (10% de FBS) dans chaque puits de la plaque de 24 puits et 2 ml dans chaque puits de la plaque à 6 puits et les mettre en étuve à 37 degrés avec 5% de CO 2 </li></ol> Jour 2 du cortex isolation et de culture de cellules gliales <ol><li> Stériliser la hotte à flux positifs de dissection. </li><li> Allumez la lumière UV pendant 20 min. </li><li> Pulvériser sur toutes les surfaces avec de l&#39;éthanol 70% et attendre 15 minutes avant utilisation. </li><li> changement de tension: les ratons Anesthetize (P2-P6) par l&#39;hypothermie, et décapiter à la base du magnum framen l&#39;aide de ciseaux d&#39;exploitation. </li><li> Ouvrez la boîte crânienne le long de la suture sagittale en utilisant un ciseau d&#39;iris et de décoller le crâne. </li><li> Enlever le cerveau et les mettre en réfrigérée milieu L15, sous stéréomicroscope, nettoyer soigneusement la membrane-mère et pie avec des vaisseaux sanguins, d&#39;isoler le cortex et laver plusieurs fois avec du milieu L15. </li><li> Couper le cortex en petits morceaux avec des ciseaux de microchirurgie. </li><li> Ajouter 0,125% de trypsine-EDTA préparé avec du milieu L15 en morceaux le cortex et incuber à 37 degrés pendant 15 min. </li><li> Transfert des blocs de tissus dans un milieu DMEM 20mL avec du FBS 20% en tube 50ml, ajouter à la solution mère de DNase 10ug/mL concentration finale, aspirer la solution des tissus haut et en bas pendant 20 heures avec le feu en verre poli Pasteure pipette, de recueillir la suspension seule cellule. </li><li> Laver les cellules en suspension une fois avec du milieu DMEM, rétablir la suspension des cellules dans du DMEM avec 10% de FBS, compter les cellules sous le microscope des cellules hémocytomètre une graine, à 5000-10000/cm 2. Maintenir les cellules dans un 37 degrés incubateur à 5%, le changement de la moitié des 2 moyennes fois par semaine. </li></ol> 2. Ganglions de la racine dorsale Neurones isolement, de la Culture et de purification Jour 1 Préparer le matériel de la culture <ol><li> Enduire les lamelles stérilisés à la poly-lysine lamelles de lavage, deux fois avec distiller l&#39;eau et l&#39;air sec, les mettre dans des plaques 24 puits. </li><li> Ajouter 100 ul milieu Neurobasal avec 2% B27 et 2.5S NGF (50 ng / ml), mettre la plaque en 37 CO degré 5% 2. </li></ol> Jour 2 Isoler les GHM à partir d&#39;embryons <ol><li> Stérilisé la hotte à flux de la même manière que la culture de cellules gliales. </li><li> Euthanasier un rat enceintes (E15) par overdose avec du CO 2, de l&#39;abdomen stérilisés par 70% d&#39;éthanol, les embryons ont été isolées de l&#39;utérus et de mettre en réfrigérée milieu L15. </li><li> Isoler avec des moelles épinières ganglions de la racine dorsale connecté sous stéréomicroscope et transfert à 35mm avec des plats refroidis milieu L15. </li><li> Recueillir suspension cellulaire unique et se laver une fois avec la FBN (milieu Neurobasal contenant 2% de B27 et 2.5S NGF (50 ng / ml)). </li></ol> Jour 3 Purifier neurones DRG <ol><li> 18h-24h après le semis neurone, ajouter une solution concentrée à 1 mM de stock FUDR à la culture de neurones à une concentration finale de 20 pM et un volume final de 200 pl. </li><li> 72h plus tard, de remplacer la moitié de la moyenne avec milieu Neurobasal contenant 2% de B27 et 2.5S NGF (50ng/mL) sans FUDR. Après cela, changer le milieu tous les autres jours. </li></ol> 3. Coculture avec des neurones DRG cellules gliales <ol><li> Lorsque la culture de cellules gliales atteint la confluence (environ 20 jours après le semis), ils étaient prêts à co-culture avec les neurones. </li><li> 24 heures avant d&#39;ajouter les neurones purifiés, gliales cellules moyennes ont été changées à moyen Neurobasal contenant 2% de B27 et 2.5S NGF (50 ng / ml). </li><li> Neurones purifiés ont été recueillies à partir des puits de culture, seule la suspension des cellules ont été obtenues par le passage mécanique grâce à la pipette Pasteure polies au feu. </li><li> Après avoir compté le nombre, les neurones ont été ensemencées à confluence 500/cm 2 sur les cellules gliales dans le milieu de Neurobasal contenant 2% de B27 et 2.5S NGF (50ng/mL). </li><li> L&#39;adhérence et la croissance neuritique des neurones sur les cellules gliales pourraient être enregistrées et analysées à différents moments par des logiciels d&#39;analyse d&#39;image et l&#39;immunocytochimie utilisant des anticorps de type cellulaire spécifique. </li></ol> 4. Les résultats représentatifs <ol><li> Culture de cellules gliales: Après l&#39;ensemencement, les cellules gliales deviennent confluentes environ 20 jours. Sous microsc contraste de phaseOPE, il a été facilement identifiés que les différentes sous-populations de cellules gliales morphologiques différentes sous-structures formées modèle de croissance et les astrocytes GFAP positives représentaient plus de 90% comme indiqué par la technique immuocytochemisty (Figure 1, Figure 2). </li><li> DRG neurones de la culture: Dans notre méthode, après 72 heures de traitement FUDR, la pureté des neurones DRG atteindra aussi élevé que 99% dans les 6 jours, les neurones DRG ont montré leurs morphologies et unique avec une croissance de densité des neurites élevé (figure 3, figure 4). </li><li> Gliales et neurones coculture de cellules: les neurones et l&#39;adhérence DRG neurites survenue facilement sur ​​les cellules gliales dans les 4 heures après l&#39;ensemencement, l&#39;observation minutieuse a montré que l&#39;adhésion des neurones et la croissance des neurites ont été influencés par les sous-populations de cellules gliales qui forment spéciale soubassements modèle de croissance, ce qui pourrait être facilement identifiés sous le microscope à contraste de phase et par immunocytochimie (Figure 5, Figure 6). </li></ol><img src="/files/ftp_upload/2111/2111fig1.jpg" alt="Figure 1" /> Figure 1. Morphologie des cellules gliales confluentes. Corticales cellules gliales ont été plaqués sur la lamelle revêtement polylysine et cultivées pendant 20 jours, notez le modèle de croissance différents de cellules gliales, les cellules sur le côté gauche disposés de manière rayonnée. <img src="/files/ftp_upload/2111/2111fig2.jpg" alt="Figure 2" /> Figure 2. Confluent cellules gliales étiquetés par la protéine fibrillaire gliale acide (GFAP) anticorps. Notez la disposition rayonnée de la GFAP (en rouge) des cellules positives sur le côté droit. <img src="/files/ftp_upload/2111/2111fig3.jpg" alt="Figure 3" /> Figure 3. Dorsale neurones ganglions de la racine a grandi in vitro, sans traitement FUDR. Les cellules contaminantes formé DRG fond neurones ». <img src="/files/ftp_upload/2111/2111fig4.jpg" alt="Figure 4" /> Figure 4. Dorsale neurones ganglions de la racine a progressé après un traitement in vitro de FUDR. Les cellules de fond contaminant avait été complètement éliminé. <img src="/files/ftp_upload/2111/2111fig5.jpg" alt="Figure 5" /> Figure 5. Dorsale neurones ganglions de la racine cultivée sur cellules gliales. Adhérence neurones et la croissance des neurites étaient inhibés sur les cellules rayonnée arrangé et limitée sur les cellules gliales côté droit. <img src="/files/ftp_upload/2111/2111fig6.jpg" alt="Figure 6" /> Figure 6. Dorsale neurones ganglions de la racine de croissance sur les cellules gliales étiqueté par l&#39;anticorps neurofilaments. Les neurites (vert) ont été inhibés sur le rayonnée disposées les cellules gliales côté gauche et limité sur le côté droit, toutes les cellules gliales ont été marqués par des anticorps GFAP (en rouge).

<ol>
	<li>Rossignol, S., Schwab, M., Schwartz, M., Fehlings, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+cord+injury:+time+to+move?.">Spinal cord injury: time to move?.</a> J Neurosci. 27 (44), 11782-11792 (2007).</li><li>Yiu, G., He, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+inhibition+of+CNS+axon+regeneration.">Glial inhibition of CNS axon regeneration.</a> Nat Rev Neurosci. 7 (8), 617-627 (2006).</li><li>Barres, B. 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+mystery+and+magic+of+glia:+a+perspective+on+their+roles+in+health+and+disease.">The mystery and magic of glia: a perspective on their roles in health and disease.</a> Neuron. 60 (3), 430-440 (2008).</li><li>Matyash, V., Kettenmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+in+astrocyte+morphology+and+physiology.">Heterogeneity in astrocyte morphology and physiology.</a> Brain Res Rev. , (2009).</li><li>Wanner, I. B., Deik, A., Torres, M., Rosendahl, A., Neary, J. T., Lemmon, V. P., Bixby, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+in+vitro+model+of+the+glial+scar+inhibits+axon+growth.+Glia.">A new in vitro model of the glial scar inhibits axon growth. Glia.</a> 56 (15), 1691-1709 (2008).</li><li>Kuffler, D. P., Sosa, I. J., Reyes, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schwann+cell+chondroitin+sulfate+proteoglycan+inhibits+dorsal+root+ganglion+neuron+neurite+outgrowth+and+substrate+specificity+via+a+soma+and+not+a+growth+cone+mechanism.">Schwann cell chondroitin sulfate proteoglycan inhibits dorsal root ganglion neuron neurite outgrowth and substrate specificity via a soma and not a growth cone mechanism.</a> J Neurosci Res. 87 (13), 2863-2871 (2009).</li><li>Kleitman, N., Wood, P. M., Bunge, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+culture+method+for+the+study+of+myelination.">Tissue culture method for the study of myelination.</a> Culturing nerve cells. , P559-P559 (1998).</li></ol>

Ce protocole expérimental a été conçu pour atteindre deux objectifs d&#39;étudier les cellules gliales, l&#39;influence en particulier l&#39;hétérogénéité des astrocytes sur l&#39;adhésion des neurones et la croissance des neurites. Le premier objectif était de maintenir l&#39;hétérogénéité des astrocytes autant que possible, dans cette expérience, la confluence des cellules gliales de la culture des astrocytes a été mélangé enrichi la culture primaire sans aucun traitement chimique et de la propag...

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		<div>
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		<thead>
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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>DMEM(high glucose)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10313-039</td>
<td>&nbsp;</td>
<tr>
<td>L15 medium</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11415-114</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10437-077</td>
<td>&nbsp;</td>
<tr>
<td>Neurobasal Medium</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>21103-049</td>
<td>&nbsp;</td>
<tr>
<td>poly-lysine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P4832</td>
<td>culture grade</td>
</tr>
<tr>
<td>NGF(2.5S)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>13257-019</td>
<td>&nbsp;</td>
<tr>
<td>B27 supplyment</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17504-044</td>
<td>&nbsp;</td>
<tr>
<td>0.25% trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25200-056</td>
<td>&nbsp;</td>
<tr>
<td>FUDR</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F0503</td>
<td>&nbsp;</td>
<tr>
<td>neurofilament antibody</td>
<td>&nbsp;</td>
<td>Abcam</td>
<td>ab24575</td>
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study glial cell heterogeneity influence on axon growth using a new coculture method

Dans le système nerveux central de tous les mammifères, les axones sectionnés après une blessure sont incapables de se régénérer à leurs objectifs initiaux et la récupération fonctionnelle est très pauvre 1. L&#39;échec de la régénération des axones est le résultat combiné de plusieurs facteurs, notamment l&#39;environnement des cellules gliales hostile, inhibition des molécules liées et une diminution de la myéline des neurones intrinsèques de régénération capacité 2. Les astrocytes sont les cellules les plus prédominantes gliales dans le système nerveux central et de jouer un rôle important dans les fonctions axone sous la physiologie et la pathologie des conditions 3. Contraste pour les oligodendrocytes homologues, les astrocytes sont une population cellulaire hétérogène composé par différentes sous-populations d&#39;astrocytes avec des morphologies différentes et 4 l&#39;expression des gènes. La signification fonctionnelle de cette hétérogénéité, comme leurs influences sur la croissance des axones, est largement inconnue. Pour étudier les cellules gliales, en particulier la fonction de l&#39;hétérogénéité des astrocytes dans le comportement des neurones, nous avons établi une nouvelle méthode de co-culture de haute purifiée dorsale neurones ganglions de la racine avec des cellules gliales obtenu à partir du cortex de rat. Par cette technique, nous avons pu comparer directement l&#39;adhérence et la croissance des axones des neurones sur les sous-populations différentes astrocytes dans les mêmes conditions. Dans ce rapport, nous donnons le protocole détaillé de cette méthode pour l&#39;isolement et la culture des astrocytes, neurones ganglions de la racine dorsale isolement et de purification, et la co-culture de neurones avec GHM astrocytes. Cette méthode pourrait également être étendu aux autres régions du cerveau à l&#39;étude cellulaire ou régionales interaction spécifique entre neurones et cellules gliales.

Watch this Scientific Journal Video about Study Glial Cell Heterogeneity Influence on Axon Growth Using a New Coculture Method at JoVE.com

Study Glial Cell Heterogeneity Influence on Axon Growth Using a New Coculture Method

Dans ce protocole, nous avons décrit une nouvelle méthode pour étudier l&#39;influence de l&#39;hétérogénéité des cellules gliales sur la croissance des axones avec un<em&gt; In vitro</em&gt; Système de co-culture. Corticaux de rat les cellules gliales ont été cultivées jusqu&#39;à confluence et co-cultivées avec hautement purifiées du rat dorsale neurones ganglions de la racine. Différents une influence sur l&#39;adhérence des cellules gliales et neurones croissance axonale a été comparé directement à la même culture. Cette méthode offre une nouvelle façon d&#39;étudier directement l&#39;influence des cellules gliales hétérogénéité sur l&#39;adhérence et la croissance des neurones axone.

Étudier l&#39;influence gliales hétérogénéité cellulaire sur la croissance axonale aide d&#39;une méthode coculture Nouveau

In the central nervous system of all mammals, severed axons after injury are unable to regenerate to their original targets and functional recovery is ...

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Neuroscience

17.2K vues.  Cedars Sinai Medical Center, UCLA. Dans ce protocole, nous avons décrit une nouvelle méthode pour étudier l'influence de l'hétérogénéité des cellules gliales sur la croissance des axones avec un In vitro Système de co-culture. Corticaux de rat les cellules gliales ont été cultivées jusqu'à confluence et co-cultivées avec hautement purifiées du rat dorsale neurones ganglions de la racine. Différents une influence sur l'adhérence des cellules gliales et neurone...

Vidéo: Study Glial Cell Heterogeneity Influence on Axon Growth Using a New Coculture Method

A Caenorhabditis elegans Model System for Amylopathy Study

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of Alzheimer's disease (AD) and is found in Lewy body dementia, inclusion body myositis and cerebral amyloid angiopathy 1-4. Amylopathies progressively develop with time. For this reason simple organisms with short lifespans may help to elucidate molecular aspects of these conditions. Here, we describe experimental protocols to study A&beta;-mediated neurodegeneration using the worm Caenorhabditis elegans. Thus, we construct transgenic worms by injecting DNA encoding human A&beta;42 into the syncytial gonads of adult hermaphrodites. Transformant lines are stabilized by a mutagenesis-induced integration. Nematodes are age synchronized by collecting and seeding their eggs. The function of neurons expressing A&beta;42 is tested in opportune behavioral assays (chemotaxis assays). Primary neuronal cultures obtained from embryos are used to complement behavioral data and to test the neuroprotective effects of anti-apoptotic compounds.

A Caenorhabditis elegans Model System for Amylopathy Study

We describe methods to study aspects of amylopathies in the worm C. elegans. We show how to construct worms expressing human A&beta;42 in neurons and how to test their function in behavioral assays. We further show how to obtain primary neuronal cultures that can be used for pharmacological testing.

A<em&gt; Caenorhabditis elegans</em&gt; Système modèle pour l&#39;étude de Amylopathy

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of ...

Recherche

Neurosciences

A Method to Make a Craniotomy on the Ventral Skull of Neonate Rodents

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain in adulthood and during development. Although most in vivo approaches use a craniotomy to study brain regions located on the dorsal side, brainstem regions such as the pons, located on the ventral side remain relatively understudied. The main goal of this protocol is to facilitate access to ventral brainstem structures so that they can be studied in vivo using electrophysiological and imaging methods. This approach allows study of structural changes in long-range axons, patterns of electrical activity in single and ensembles of cells, and changes in blood brain barrier permeability in neonate animals. Although this protocol has been used mostly to study the auditory brainstem in neonate rats, it can easily be adapted for studies in other rodent species such as neonate mice, adult rodents and other brainstem regions.

A surgical method is described to expose the ventral skull in neonate rats. Using this approach it is possible to open a craniotomy to perform acute electrophysiology and two-photon microscopy experiments in the brainstem of anesthetized pups.

Une méthode pour faire un Craniotomie sur le crâne ventrale de nouveaux-nés Rongeurs

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain ...

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 ...

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco&#39;s modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d&#160;(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how&#160;to establish primary mixed glial cultures from adult mouse spinal cord tissue.

Préparation de cultures mixtes gliales primaires de souris adultes Spinal Cord Tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

Utilisation Agarose linéaire Canaux pour étudier<em&gt; Drosophila</em&gt; Larvaire Ramper Comportement

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

Augmentation de la réponse du réseau à la stimulation des cultures cellulaires neuronales sur les tableaux de microélectrodes

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

New Methods to Study Gustatory Coding

The sense of taste allows animals to detect chemicals in the environment, giving rise to behaviors critical for survival. When Gustatory Receptor Neurons (GRNs) detect tastant molecules, they encode information about the identity and concentration of the tastant as patterns of electrical activity that then propagate to follower neurons in the brain. These patterns constitute internal representations of the tastant, which then allow the animal to select actions and form memories. The use of relatively simple animal models has been a powerful tool to study basic principles in sensory coding. Here, we propose three new methods to study gustatory coding using the moth Manduca sexta. First, we present a dissection procedure for exposing the maxillary nerves and the subesophageal zone (SEZ), allowing recording of the activity of GRNs from their axons. Second, we describe the use of extracellular electrodes to record the activity of multiple GRNs by placing tetrode wires directly into the maxillary nerve. Third, we present a new system for delivering and monitoring, with high temporal precision, pulses of different tastants. These methods allow the characterization of neuronal responses in vivo directly from GRNs before, during and after tastants are delivered. We provide examples of voltage traces recorded from multiple GRNs, and present an example of how a spike sorting technique can be applied to the data to identify the responses of individual neurons. Finally, to validate our recording approach, we compare extracellular recordings obtained from GRNs with tetrodes to intracellular recordings obtained with sharp glass electrodes.

We present three new methods to study gustatory coding. Using a simple animal, the moth Manduca sexta (Manduca), we describe a dissection protocol, the use of extracellular tetrodes to record the activity of multiple gustatory receptor neurons, and a system for delivering and monitoring precisely timed pulses of tastants.

Nouvelles méthodes pour étudier le codage désintoxiqué

The sense of taste allows animals to detect chemicals in the environment, giving rise to behaviors critical for survival. When Gustatory Receptor Neurons ...

Visualization of Motor Axon Navigation and Quantification of Axon Arborization In Mouse Embryos Using Light Sheet Fluorescence Microscopy

Spinal motor neurons (MNs) extend their axons to communicate with their innervating targets, thereby controlling movement and complex tasks in vertebrates. Thus, it is critical to uncover the molecular mechanisms of how motor axons navigate to, arborize, and innervate their peripheral muscle targets during development and degeneration. Although transgenic Hb9::GFP mouse lines have long served to visualize motor axon trajectories during embryonic development, detailed descriptions of the full spectrum of axon terminal arborization remain incomplete due to the pattern complexity and limitations of current optical microscopy. Here, we describe an improved protocol that combines light sheet fluorescence microscopy (LSFM) and robust image analysis to qualitatively and quantitatively visualize developing motor axons. This system can be easily adopted to cross genetic mutants or MN disease models with Hb9::GFP lines, revealing novel molecular mechanisms that lead to defects in motor axon navigation and arborization.

Here, we describe a protocol for visualizing motor neuron projection and axon arborization in transgenic Hb9::GFP mouse embryos. After immunostaining for motor neurons, we used light sheet fluorescence microscopy to image embryos for subsequent quantitative analysis. This protocol is applicable to other neuron navigation processes in the central nervous system.

Visualisation de l’axone moteur Navigation et la Quantification de ces axones dans des embryons de souris à l’aide de la microscopie de Fluorescence nappe de lumière

Spinal motor neurons (MNs) extend their axons to communicate with their innervating targets, thereby controlling movement and complex tasks in ...

Using Optical Coherence Tomography and Optokinetic Response As Structural and Functional Visual System Readouts in Mice and Rats

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for the investigation of processes of neurodegeneration, neuroprotection and neuro-repair involving the visual system, as these often correlate well with retinal changes. As a functional readout, visually evoked compensatory eye and head movements are commonly used in experimental models involving the visual function. Combining both techniques allows a quantitative in vivo investigation of structure and function, which can be used to investigate the pathological conditions or to evaluate the potential of novel therapeutics. A great benefit of the presented techniques is the possibility to perform longitudinal analyses allowing the investigation of dynamic processes, reducing variability and cuts down the number of animals needed for the experiments. The protocol described aims to provide a manual for acquisition and analysis of high quality retinal scans of mice and rats using a low cost customized holder with an option to deliver inhalational anesthesia. Additionally, the proposed guide is intended as an instructional manual for researchers using optokinetic response (OKR) analysis in rodents, which can be adapted to their specific needs and interests.

A detailed protocol for the assessment of structural and visual readouts in rodents by optical coherence tomography and optokinetic response is presented. The results provide valuable insights for ophthalmologic as well as neurologic research.

Utilisant la tomographie par cohérence optique et réponse optocinétique comme lectures structurale et fonctionnelle de système visuel chez les souris et les Rats

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for ...

A Method for 3D Reconstruction and Virtual Reality Analysis of Glial and Neuronal Cells

Serial sectioning and subsequent high-resolution imaging of biological tissue using electron microscopy (EM) allow for the segmentation and reconstruction of high-resolution imaged stacks to reveal ultrastructural patterns that could not be resolved using 2D images. Indeed, the latter might lead to a misinterpretation of morphologies, like in the case of mitochondria; the use of 3D models is, therefore, more and more common and applied to the formulation of morphology-based functional hypotheses. To date, the use of 3D models generated from light or electron image stacks makes qualitative, visual assessments, as well as quantification, more convenient to be performed directly in 3D. As these models are often extremely complex, a virtual reality environment is also important to be set up to overcome occlusion and to take full advantage of the 3D structure. Here, a step-by-step guide from image segmentation to reconstruction and analysis is described in detail.

The described pipeline is designed for the segmentation of electron microscopy datasets larger than gigabytes, to extract whole-cell morphologies. Once the cells are reconstructed in 3D, customized software designed around individual needs can be used to perform a qualitative and quantitative analysis directly in 3D, also using virtual reality to overcome view occlusion.

Une méthode pour la reconstruction 3D et l'analyse de la réalité virtuelle des cellules gliales et neuronales

Serial sectioning and subsequent high-resolution imaging of biological tissue using electron microscopy (EM) allow for the segmentation and reconstruction ...