Le Pedro Barrie de la Maza Fondation a soutenu ce travail.

1) Administration intragastrique de la roténone <ol><li> Peser l&#39;animal. </li><li> Calculer le volume total de la roténone / véhicule solution nécessaire. Dans ce cas 0,01 ml par gramme de poids de l&#39;animal. </li><li> Chargez une seringue de 1 mL avec le volume calculé précédemment d&#39;une solution de roténone (0,625 mg / mL roténone, la carboxyméthylcellulose de 4% et 1,25% de chloroforme) ou la solution du véhicule (carboxyméthylcellulose 4% et 1,25% de chloroforme). Dans ce cas, elle correspond à une dose de 5 roténone mg / kg. </li><li> Ramassez la souris et, tenant la queue de la souris avec une main, étirer la peau du cou en le tirant avec les deux premiers doigts de l&#39;autre main. </li><li> Une fois que la tête est fixée entre les deux doigts, il s&#39;appuyer contre la paume de la main et le tourner à l&#39;envers. Fixer la queue en utilisant les quatrième et cinquième doigts. </li><li> Garder la tête vers l&#39;arrière, appuyez sur le palais d&#39;ouvrir la bouche de la souris. Introduire délicatement le gavage dans la bouche. Il est important d&#39;utiliser une aiguille de gavage et non une aiguille régulière. Lentement, déplacez le gavage dans le sens de l&#39;estomac assez loin. Cela permettra d&#39;éviter matériels obtenir dans les poumons. </li><li> Administrer la solution de roténone ou d&#39;un véhicule en appuyant doucement sur l&#39;embolie. Une fois terminé, lentement extraire le gavage. </li><li> Placez le dos de l&#39;animal dans une autre cage jusqu&#39;à ce que tous les animaux d&#39;une cage sont faites. </li></ol> Procédures Immunocoloration 2) <ol><li> Après le traitement, les souris sont anesthésiées à l&#39;aide Kétamine 100 mg / kg und Xylazin 10 mg / kg ip et intracardiaque perfusés avec du paraformaldéhyde 4% dans du PBS. </li><li> Les tripes sont ensuite extraits par la dissection, et placés dans du saccharose 15% pendant la nuit. Le lendemain, le tissu est transféré à 30% de sucrose et incubé à nouveau la nuit à 4 ° C. </li><li> Le tissu est ensuite placé dans Tissue-Tek et stockés dans un congélateur à -80 ° C jusqu&#39;à utilisation. </li><li> L&#39;utilisation d&#39;un cryostat, 20 microns intestin sections sont transférés à des diapositives Superfrost. </li><li> Blocage et immunomarquage procédures utilisant des anticorps anti-βIII-tubuline et anti-alpha-synucléine sont ensuite effectuée comme décrit précédemment 9. </li></ol> 3) Image Analysis L&#39;alpha-synucléine Inclusion Il est recommandé qu&#39;une personne externe ignore l&#39;origine des images &#39;effectue l&#39;analyse. Par conséquent, les données doivent être codifiées. L&#39;analyse des images a été réalisée en utilisant le logiciel FIDJI. <ol><li> Ouvrez le fichier image confocale. Ensuite, diviser les canaux de sorte qu&#39;il est possible de travailler sur chaque canal indépendamment. </li><li> Fermer les canaux qui ne vont pas être utilisé et sélectionnez Analyser&gt; Définir l&#39;échelle et définir la taille des pixels pour 1. Réglez la distance connue à 0,13 (cela dépendra de l&#39;objectif et la résolution utilisée) et cliquez sur mondiaux. La taille de l&#39;image sera désormais indiqué dans microns. </li><li> Sélectionnez l&#39;alpha-synucléine canaux et sélectionnez ganglionnaires, assurez-vous que la sélection englobe le ganglion dans toutes les tranches. </li><li> Image Press&gt; Dupliquer pour dupliquer l&#39;image. Avec le ganglion sélectionnés, seulement une image de la taille du ganglion sera dupliqué. </li><li> Appuyez sur Modifier&gt; Effacer extérieur. La partie de l&#39;image en dehors de la région sélectionnée va devenir noire. </li><li> Dupliquer l&#39;image à nouveau pour déterminer dans les étapes ultérieures à quel point la procédure est allé. Les images qui ont un signal élevé au bruit ratio de fond ne devrait pas être utilisé pour l&#39;analyse d&#39;image. </li><li> Sélectionnez Image&gt; Réglages&gt; Seuil puis sélectionnez MaxEntropy. Puis déplacez sur les tranches de l&#39;image recadrée et celui d&#39;origine afin de s&#39;assurer que la procédure s&#39;est bien déroulée. Le seuil entropique sélectionne un niveau de signal d&#39;intensité basée sur l&#39;histogramme d&#39;image et que les pixels avec des intensités supérieures à ce seuil sera affichée dans un blanc sur l&#39;image noire. </li><li> Sélectionnez Processus, puis cliquez sur lisse. Cette étape transforme les bords pixeled des images en bords lisses. </li><li> Sélectionnez processus, puis cliquez sur le binaire, et choisissez Créer un binaire. L&#39;image sera affichée en tant que noir sur l&#39;image blanche. </li><li> Dupliquer l&#39;image pour effectuer l&#39;étape suivante sur l&#39;image dupliquée. </li><li> Cliquez sur Analyser&gt; Analyse des particules. Ensuite, sélectionnez Afficher: Contours et cliquez sur Résumer. Le reste des paramètres doivent être feuillus en défaut. Cette fonction reconnaît le nombre total de pixels avec un signal et ceux qui sont regroupés. La surface totale des pixels avec un signal plus le nombre de groupes de pixels et la taille moyenne sont signalés. </li><li> Comparez et choisissez le plus grand morceau qui ajuste bien. La tranche sera marqué. </li><li> Copiez le tableau de données avec le nombre total d&#39;alpha-synucléine surface, le nombre d&#39;inclusions et la taille moyenne des particules. Collez les données dans un tableur Excel ou annotés ailleurs. </li><li> Retour à Fidji et à trouver la tranche préalablement sélectionné dans le canal de β-tubuline, et le sélectionner. </li><li> Sélectionnez la zone ganglionnaires et cliquez sur Analyser puis sélectionnez Mesure. Un autre tableau apparaît montrant lasurface totale du ganglion. </li><li> Extraire les données de la table à la feuille Excel à proximité de la mesure alpha-synucléine ou le recopier ailleurs. </li><li> Utilisation d&#39;Excel, divisez les différents paramètres obtenus dans la mesure alpha-synucléine par la surface ganglionnaires pour obtenir le total d&#39;alpha-synucléine certain nombre de surface et l&#39;inclusion normalisée par la taille ganglionnaires. </li></ol> Résultats Représentant 4) Lorsque le protocole gavage est effectué correctement les inconvénients pour l&#39;animal sont minimes. Traitement à l&#39;aide de cette concentration de roténone permet un effet local de la roténone sur les ENS, sans niveaux roténone dans le sang et aucune inhibition du complexe de muscles ou le cerveau mitochondriale J&#39;ai même après 1,5 mois de traitement (voir Figure 1). Si l&#39;analyse d&#39;images est effectuée correctement, une analyse rigoureuse de l&#39;alpha-synucléine modèle devrait être possible. Il fournit des données fiables sur le montant total de l&#39;alpha-synucléine (surface totale), l&#39;alpha-synucléine motif dans la cellule (nombre d&#39;inclusions) et la présence d&#39;alpha-synucléine inclusions (taille inclusion) (figure 2). <img src="/files/ftp_upload/2123/2123fig1.jpg" alt="Figure 1" /> Figure 1. Dysfonction motrice de la roténone souris traitées sans détection de la roténone dans le sang ou le SNC. Une norme, (50 ng / ml) et chromatogramme des échantillons de cerveau de 20, 10 et 5 mg / kg chez les souris traitées. B et C, la quantification des niveaux roténone dans le sang (B) et du SNC (C). B, les taux sanguins 1 , 2 et 3 heures après le traitement. Les souris ont été divisées en trois groupes (n = 3) et traités avec 2,5, 5, 10 et 20 mg / kg de roténone (n = 3), 300 ul de sang a été extrait heures 1, 2 et 3 après l&#39;administration de roténone et regroupées ensemble pour L&#39;analyse par HPLC. C, les souris ont été traitées pendant une semaine une fois par jour avec 5 (n = 3), 10 (n = 3) et 20 (n = 1) mg / kg de roténone, le cerveau et le tronc cérébral ont été extraits 1 et 2 heures après dernière administration et préparés pour l&#39;analyse HPLC. D, complexe mitochondrial I activité dans des échantillons de muscle et le cerveau de 1,5 mois chez les souris traitées. <img src="/files/ftp_upload/2123/2123fig2.jpg" alt="Figure 2" /> Figure 2. Roténone administrés localement induit la phosphorylation d&#39;alpha-synucléine, l&#39;accumulation et l&#39;agrégation avec gliose dans les ganglions de l&#39;ENS. (Barres d&#39;échelle 20 mm). A, B, C, anti-tubuline βIII, l&#39;alpha-synucléine et la coloration DAPI dans le duodénum (B) et de l&#39;iléon ( A, C) les articles. Flèche en B, 1,5 mois de traitement induit une augmentation de l&#39;alpha-synucléine modèle ponctuée intérieur de ganglions système nerveux entérique, comparativement à 3 mois de contrôle (A). Flèche en C, la formation de 3 mois de traitement induit des grandes alpha-synucléine inclusions (ø&gt; 6 m). D, immunofluorescence utilisant des anticorps anti-alpha-synucléine, Thioflavine S et DAPI. Flèche dans D, seulement trois mois chez les souris traitées ont montré l&#39;agrégation de ces grandes accumulations d&#39;alpha-synucléine. E, E &#39;, E&#39;&#39;, les étapes d&#39;analyse d&#39;images de quantification. E, par exemple de l&#39;image utilisée dans la quantification montrant un ganglion ENS immunocolorés contre les alpha-synucléine montrant inclusions différentes. E &#39;, par exemple d&#39;image obtenue après le protocole d&#39;analyse d&#39;image. E&#39;&#39;, le chevauchement entre certaines des inclusions d&#39;origine (vert) et la représentation obtenue après l&#39;exécution du protocole (lignes jaunes). FG, la quantification de l&#39;expérience présentée dans le AC a été faite en utilisant une segmentation automatique et les méthodes de seuillage basée sur l&#39;entropie. Single-astérisque, P &lt;0,05, et double-astérisque, P &lt;0,01. F, chaque colonne représente le total de l&#39;alpha-synucléine surface / ganglionnaires surface. G, chaque colonne représente le nombre total d&#39;alpha-synucléine inclusions / surface de ganglion. Tous les graphiques montrent moyenne ± SEM

<ol>
	<li>Braak, H., de Vos, R. A., Bohl, J., Del Tredici, K., K, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastric+alpha-synuclein+immunoreactive+inclusions+in+Meissner's+and+Auerbach's+plexuses+in+cases+staged+for+Parkinson's+disease-related+brain+pathology.">Gastric alpha-synuclein immunoreactive inclusions in Meissner's and Auerbach's plexuses in cases staged for Parkinson's disease-related brain pathology.</a> Neurosci Lett. 396, 67-72 (2006).</li><li>Wakabayashi, K., Takahashi, H., Ohama, E., Ikuta, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkinson's+disease:+an+immunohistochemical+study+of+Lewy+body-containing+neurons+in+the+enteric+nervous+system.">Parkinson's disease: an immunohistochemical study of Lewy body-containing neurons in the enteric nervous system.</a> Acta Neuropathol. 79, 581-583 (1990).</li><li>Wakabayashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restricted+occurrence+of+Lewy+bodies+in+the+dorsal+vagal+nucleus+in+a+patient+with+late-onset+parkinsonism.">Restricted occurrence of Lewy bodies in the dorsal vagal nucleus in a patient with late-onset parkinsonism.</a> J Neurol Sci. 165, 188-191 (1999).</li><li>Braak, H., Sastre, M., Bohl, J. R., de Vos, R. A., Del Tredici, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkinson's+disease:+lesions+in+dorsal+horn+layer+I,+involvement+of+parasympathetic+and+sympathetic+pre-+and+postganglionic+neurons.">Parkinson's disease: lesions in dorsal horn layer I, involvement of parasympathetic and sympathetic pre- and postganglionic neurons.</a> Acta Neuropathol. 113, 421-429 (2007).</li><li>Braak, H., Ghebremedhin, E., Rub, U., Bratzke, H., Del Tredici, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+in+the+development+of+Parkinson's+disease-related+pathology.">Stages in the development of Parkinson's disease-related pathology.</a> Cell Tissue Res. 318, 121-134 (2004).</li><li>Gorell, J. M., Johnson, C. C., Rybicki, B. A., Peterson, E. L., Richardson, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+risk+of+Parkinson's+disease+with+exposure+to+pesticides,+farming,+well+water,+and+rural+living.">The risk of Parkinson's disease with exposure to pesticides, farming, well water, and rural living.</a> Neurology. 50, 1346-1350 (1998).</li><li>Semchuk, K. M., Love, E. J., Lee, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkinson's+disease+and+exposure+to+agricultural+work+and+pesticide+chemicals.">Parkinson's disease and exposure to agricultural work and pesticide chemicals.</a> Neurology. 42, 1328-1335 (1992).</li><li>Butterfield, P. G., Valanis, B. G., Spencer, P. S., Lindeman, C. A., Nutt, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+antecedents+of+young-onset+Parkinson's+disease.">Environmental antecedents of young-onset Parkinson's disease.</a> Neurology. 43, 1150-1158 (1993).</li><li>Pan-Montojo, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progression+of+Parkinson's+disease+pathology+is+reproduced+by+intragastric+administration+of+rotenone+in+mice.">Progression of Parkinson's disease pathology is reproduced by intragastric administration of rotenone in mice.</a> PLoS One. 5, e8762-e8762 (2010).</li><li>Sherer, T. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+toxicity+in+rotenone+models+of+Parkinson's+disease.">Mechanism of toxicity in rotenone models of Parkinson's disease.</a> J Neurosci. 23, 10756-10764 (2003).</li><li>Bove, J., Prou, D., Perier, C., Przedborski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxin-induced+models+of+Parkinson's+disease.">Toxin-induced models of Parkinson's disease.</a> NeuroRx. 2, 484-494 (2005).</li><li>Inden, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurodegeneration+of+mouse+nigrostriatal+dopaminergic+system+induced+by+repeated+oral+administration+of+rotenone+is+prevented+by+4-phenylbutyrate,+a+chemical+chaperone.">Neurodegeneration of mouse nigrostriatal dopaminergic system induced by repeated oral administration of rotenone is prevented by 4-phenylbutyrate, a chemical chaperone.</a> J Neurochem. 101, 1491-1504 (2007).</li></ol>

Francisco Pan-Montojo a une demande de brevet en attente pour ce modèle animal (numéro de demande PCT / EP 2009/005688).

L&#39;administration intragastrique a déjà été réalisé 12. Toutefois, selon le manuscrit de Inden, le roténone a été administré dissous dans le sel de carboxyméthylcellulose de sodium à 0,5% (CMSS) seul. La roténone est une substance de haute liphophylic. Par conséquent, la roténone ne peut pas être dissous dans CMSS 0,5% seul et va précipiter, si fait. L&#39;utilisation de chloroforme crée une suspension roténone répartie uniformément en évitant la précipitation. Par ailleurs, en raiso...

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Rotenone</td>
<td>&nbsp;</td>
<td>SIGMA-Aldrich</td>
<td>R8875</td>
<td>Germany</td>
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<td>Chloroform</td>
<td>&nbsp;</td>
<td>Carl Roth</td>
<td>3313.1</td>
<td>Germany</td>
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<td>Carboxymethylcellulose</td>
<td>&nbsp;</td>
<td>SIGMA-Aldrich</td>
<td>C5678</td>
<td>Germany</td>
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<tr>
<td>Gavage 1,2 mm x 60 mm</td>
<td>&nbsp;</td>
<td>Unimed</td>
<td>&nbsp;</td>
<td>Switzerland</td>
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<td>LSM 510</td>
<td>&nbsp;</td>
<td>Zeiss Microscopes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>FIJI</td>
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oral administration of rotenone using a gavage and image analysis of alpha-synuclein inclusions in the enteric nervous system

Dans la maladie de Parkinson (MP) des patients, la pathologie associée suit un modèle caractéristique impliquant notamment le système nerveux entérique (ENS) 1,2, le bulbe olfactif (OB), le noyau moteur dorsal du nerf vague (DMV) 3, le intermédiolatérale noyau de la moelle épinière et 4 de la substantia nigra, fournissant la base pour la stadification neuropathologiques de la maladie 4,5. L&#39;ENS et l&#39;OB sont les structures les plus exposées nerveux et les premières à être touchées. Fait intéressant, PD a été liée à 6-8 exposition aux pesticides. Ici, nous montrons en détail deux méthodes utilisées dans notre étude précédente 9. Afin d&#39;analyser les effets de la roténone agit localement sur l&#39;ENS, nous avons administré la roténone en utilisant un gavage à un an C57/BL6 souris. La roténone est un pesticide largement utilisé qui inhibe fortement complexe mitochondrial I 10. Il est fortement lipophile et mal absorbé dans le tractus gastro-intestinal 11. Nos résultats ont montré que l&#39;administration de 5 mg / kg de roténone n&#39;a pas inhibé l&#39;activité complexe mitochondrial I dans le muscle ou le cerveau. Ainsi, ce qui suggère que la roténone en utilisant notre méthode d&#39;administration n&#39;a pas traversé le système hépatoportale et agissait uniquement sur l&#39;ENS. Ici, nous montrons une méthode pour administrer les pesticides en utilisant un gavage et le protocole d&#39;analyse d&#39;images utilisés pour analyser les effets des pesticides dans l&#39;alpha-synucléine accumulation dans l&#39;ENS. La première partie montre une méthode qui permet l&#39;administration intragastrique de pesticides (roténone) à une concentration désirée précis. La seconde méthode montre un protocole semi-automatique d&#39;analyse d&#39;images à analyser l&#39;accumulation d&#39;alpha-synucléine dans les ENS en utilisant un logiciel d&#39;analyse d&#39;image.

Watch this Scientific Journal Video about Oral Administration of Rotenone using a Gavage and Image Analysis of Alpha-synuclein Inclusions in the Enteric Nervous System at JoVE.com

Oral Administration of Rotenone using a Gavage and Image Analysis of Alpha-synuclein Inclusions in the Enteric Nervous System

La maladie de Parkinson a été lié à l&#39;exposition aux pesticides. Ici, nous montrons une méthode pour fournir des pesticides à l&#39;aide d&#39;une sonde gastrique à la concentration désirée et une méthode pour analyser leur effet dans l&#39;accumulation d&#39;alpha-synucléine dans le système nerveux entérique.

Administration orale de la roténone en utilisant un Gavage et analyse d&#39;images d&#39;alpha-synucléine inclusions dans le système nerveux entérique

In Parkinson's disease (PD) patients, the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS) 1,2, ...

oral-administration-rotenone-using-gavage-image-analysis-alpha

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JoVE Journal

Neuroscience

16.9K vues.  Technische Universität Dresden. La maladie de Parkinson a été lié à l'exposition aux pesticides. Ici, nous montrons une méthode pour fournir des pesticides à l'aide d'une sonde gastrique à la concentration désirée et une méthode pour analyser leur effet dans l'accumulation d'alpha-synucléine dans le système nerveux entérique.

Vidéo: Oral Administration of Rotenone using a Gavage and Image Analysis of Alpha-synuclein Inclusions in the Enteric Nervous System

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

Visualisation et la manipulation génétique des dendrites et des épines dans le cortex cérébral et l&#39;hippocampe de souris à l&#39;aide<em&gt; In utero</em&gt; Électroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

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Neurosciences

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

Livraison intraventriculaire directe des médicaments pour le système nerveux central rongeurs

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

In vivo optogénétiques stimulation du système nerveux central pour rongeurs

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

In Situ Ca2+ Imaging of the Enteric Nervous System

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ganglionated plexuses housed in the gut wall. Enteric neurons and enteric glia are the only cell types within the enteric ganglia. The activity of enteric neurons and glia is responsible for coordinating intestinal functions. This protocol describes methods for observing the activity of neurons and glia within the intact ENS by imaging intracellular calcium (Ca2+) transients with fluorescent indicator dyes. Our technical discussion focuses on methods for Ca2+ imaging in whole-mount preparations of the myenteric plexus from the rodent bowel. Bulk loading of ENS whole-mounts with a high-affinity Ca2+ indicator such as Fluo-4 permits measurements of Ca2+ responses in individual neurons or glial cells. These responses can be evoked repeatedly and reliably, which permits quantitative studies using pharmacological tools. Ca2+ responses in cells of the ENS are recorded using a fluorescence microscope equipped with a cooled charge-coupled device (CCD) camera. Fluorescence measurements obtained using Ca2+ imaging in whole-mount preparations offer a straightforward means of characterizing the mechanisms and potential functional consequences of Ca2+ responses in enteric neurons and glial cells.

In Situ Ca2+ Imaging of the Enteric Nervous System

The enteric nervous system (ENS) is a network of neurons and glia located in the gut wall that controls intestinal reflexes. This protocol describes methods for recording the activity of enteric neurons and glia in live preparations of ENS using Ca2+ imaging.

De l&#39;imagerie in situ de Ca du système nerveux entérique

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ...

Sequential Extraction of Soluble and Insoluble Alpha-Synuclein from Parkinsonian Brains

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, oligomers and fibrils. During disease, &#945;-syn undergoes conformational changes to form oligomers and high molecular weight aggregates that tend to make the protein more insoluble. Abnormally aggregated &#945;-syn is a neuropathological feature of Parkinson&#39;s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Biochemical characterization and analysis of insoluble &#945;-syn using buffers with increasing detergent strength and high-speed ultracentrifugation provides a powerful tool to determine the development of &#945;-syn pathology associated with disease progression. This protocol describes the isolation of increasingly insoluble/aggregated &#945;-syn from post-mortem human brain tissue. This methodology can be adapted with modifications to studies of normal and abnormal &#945;-syn biology in transgenic animal models harbouring different &#945;-syn mutations as well as in other neurodegenerative diseases that feature aberrant fibrillar deposits of proteins related to their respective pathologies.

Here, we present a protocol for the isolation of increasingly insoluble/aggregated alpha-synuclein (&#945;-syn) from post-mortem human brain tissue. Through the utilization of buffers with increasing detergent strength and high-speed ultracentrifugation techniques, the variable properties of &#945;-syn aggregation in diseased and non-diseased tissue can be examined.

Extraction séquentielle de soluble et insoluble alpha-synucléine de Brains parkinsoniens

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, ...

Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils

To study the prion-like behavior of misfolded alpha-synuclein, mouse models are needed that allow fast and simple transmission of alpha-synuclein prionoids, which cause neuropathology within the central nervous system (CNS). Here we describe that intraglossal or intraperitoneal injection of alpha-synuclein fibrils into bigenic Tg(M83+/-:Gfap-luc+/-) mice, which overexpress human alpha-synuclein with the A53T mutation from the prion protein promoter and firefly luciferase from the promoter for glial fibrillary acidic protein (Gfap), is sufficient to induce neuropathologic disease. In comparison to homozygous Tg(M83+/+) mice that develop severe neurologic symptoms beginning at an age of 8 months, heterozygous Tg(M83+/-:Gfap-luc+/-) animals remain free of spontaneous disease until they reach an age of 22 months. Interestingly, injection of alpha-synuclein fibrils via the intraperitoneal route induced neurologic disease with paralysis in four of five Tg(M83+/-:Gfap-luc+/-) mice with a median incubation time of 229 &#177;17 days. Diseased animals showed severe deposits of phosphorylated alpha-synuclein in their brains and spinal cords. Accumulations of alpha-synuclein were sarkosyl-insoluble and colocalized with ubiquitin and p62, and were accompanied by an inflammatory response resulting in astrocytic gliosis and microgliosis. Surprisingly, inoculation of alpha-synuclein fibrils into the tongue was less effective in causing disease with only one of five injected animals showing alpha-synuclein pathology after 285 days. Our findings show that inoculation via the intraglossal route and more so via the intraperitoneal route is suitable to induce neurologic illness with relevant hallmarks of synucleinopathies in Tg(M83+/-:Gfap-luc+/-) mice. This provides a new model for studying prion-like pathogenesis induced by alpha-synuclein prionoids in greater detail.

Peripheral injection of alpha-synuclein fibrils into the peritoneum or tongue of Tg(M83+/-:Gfap-luc+/-) mice, which express human alpha-synuclein with the familial A53T mutation and firefly luciferase, can induce neuropathology, including neuroinflammation, in their central nervous system.

Bioluminescence imagerie de la neuro-inflammation chez des souris transgéniques Après périphériques Ensemencement de l&#39;alpha-synucléine Fibrilles

To study the prion-like behavior of misfolded alpha-synuclein, mouse models are needed that allow fast and simple transmission of alpha-synuclein ...

Methods and Tips for Intravenous Administration of Adeno-associated Virus to Rats and Evaluation of Central Nervous System Transduction

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), optogenetics, cre-lox targeting, etc. The purpose of this manuscript is to aid the investigator attempting expansive central nervous system (CNS) gene transfer in the rat via tail vein injection of AAV. Wide-scale expression is relevant for conditions with widespread pathology, and a rat model is significant due to its greater size and physiologic similarities to humans compared to mice. In this example application, a wide-scale neuronal transduction is used to mimic a neurodegenerative disease that affects the entire spinal cord, amyotrophic lateral sclerosis (ALS). The efficient wide-scale CNS transduction can also be used to deliver therapeutic protein factors in pre-clinical studies. After a post-injection expression interval of several weeks, the effects of the transduction are evaluated. For a green fluorescent protein (GFP) control vector, the amount of GFP in the cerebellum is estimated quickly and reliably by a basic imaging program. For motor disease phenotypes that are induced by the ALS related protein transactive response DNA-binding protein of 43 kDa (TDP-43), the deficits are scored by escape reflex and rotarod. Beyond disease modeling and gene therapy, there are diverse potential applications for the wide-scale gene targeting described here. The expanded use of this method will aid in expediting hypothesis testing in the neurosciences and neurogenetics.

Methods for a wide-scale central nervous system gene delivery in the rat are covered. In this example, the purpose is to mimic a disease that affects the entire spinal cord. The widespread transduction can be used to deliver a therapeutic protein to the CNS from a one-time, peripheral administration.

Méthodes et conseils pour l’Administration intraveineuse de Virus Adeno-associé à des Rats et d’évaluation de la Transduction du système nerveux Central

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), ...

Exogenous Administration of Microsomes-associated Alpha-synuclein Aggregates to Primary Neurons As a Powerful Cell Model of Fibrils Formation

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the study of &#945;S aggregation in Parkinson&#39;s Disease (PD). Recently, the development of new animal models through the exogenous inoculation of brain extracts from diseased &#945;S transgenic mice or PD patients has given new hopes to the possibility of creating more adequate cell models of &#945;S aggregation. Unfortunately, when it comes to cells in cultures, administration of raw brain extracts has not proven as successful as in mice and the source of choice of exogenous aggregates is still in vitro preformed &#945;S fibrils.
We have developed a method to induce the formation of intracellular &#945;S inclusions in primary neurons through the exogenous administration of native microsomes-associated &#945;S aggregates, a highly toxic &#945;S species isolated from diseased areas of transgenic mice. This fraction of &#945;S aggregates that is associated with the microsomes vesicles, is efficiently internalized and induces the formation of intracellular inclusions positive for aggregated and phosphorylated &#945;S. Compared to in vitro-preformed fibrils which are made from recombinant &#945;S, our method is faster and guarantees that the pathogenic seeding is made with authentic &#945;S aggregates extracted from diseased animal models of PD, mimicking more closely the type of inclusions obtained in vivo. As a result, availability of tissues rich in &#945;S inclusions is mandatory.
We believe that this method will provide a versatile cell-based model to study the microscopic aspects of &#945;S aggregation and the related cellular pathophysiology in vivo and will be a starting point for the creation of more accurate and sophisticated cell paradigm of PD.

The goal of this protocol is to provide a cell-based system that replicates the formation of alpha-synuclein aggregates in vivo. Intracellular alpha-synuclein inclusions are seeded in primary neurons by the internalization and propagation of exogenous administered native microsomes-associated alpha-synuclein aggregates isolated from diseased alpha-synuclein transgenic mice.

Administration exogène d’Alpha-synucléine Microsomes associées aux agrégats de neurones primaires comme un modèle de cellulaire puissant de la Formation de fibrilles

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the ...

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is a complicated task, which is particularly difficult in regards to the development of &#947;-aminobutyric acid (GABA)ergic cortical interneurons (CIs). CIs are the main inhibitory neurons in the cerebral cortex, and they play key roles in neuronal networks, by regulating both the activity of individual pyramidal neurons, as well as the oscillatory behavior of neuronal ensembles. They are generated in transient embryonic structures (medial and caudal ganglionic eminences -&#160;MGE and CGE) that are very difficult to efficiently target using in utero electroporation approaches. Interneuron progenitors migrate long distances during normal embryonic development, before they integrate in the cortical circuit. This remarkable ability to disperse and integrate into a developing network can be hijacked by transplanting embryonic interneuron precursors into early post-natal host cortices. Here, we present a protocol that allows genetic modification of embryonic interneuron progenitors using focal ex vivo electroporation. These engineered interneuron precursors are then transplanted into early post-natal host cortices, where they will mature into easily identifiable CIs. This protocol allows the use of multiple genetically encoded tools, or the ability to regulate the expression of specific genes in interneuron progenitors, in order to investigate the impact of either genetic or environmental variables on the maturation and integration of CIs.

Here we present a protocol, designed to use chemogenetic tools to manipulate the activity of cortical interneuron progenitors transplanted into the cortex of early postnatal mice.

Transplantation de progéniteurs interneuron corticales génétiquement modifiés dans les cerveaux de souris postnatales précoces

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is ...

Generation of Alpha-Synuclein Preformed Fibrils from Monomers and Use In Vivo

Use of the in vivo alpha-synuclein preformed fibril (&#945;-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model Parkinson&#39;s disease synucleinopathy and nigrostriatal degeneration. The standardization of &#945;-syn PFF generation and in vivo application is critical in order to ensure consistent, robust &#945;-syn pathology. Here, we present a detailed protocol for the generation of fibrils from monomeric &#945;-syn, post-fibrilization quality control steps, and suggested parameters for successful neurosurgical injection of &#945;-syn PFFs into rats or mice. Starting with monomeric &#945;-syn, fibrilization occurs over a 7-day incubation period while shaking at optimal buffer conditions, concentration, and temperature. Post-fibrilization quality control is assessed by the presence of pelletable fibrils via sedimentation assay, the formation of amyloid conformation in the fibrils with a thioflavin T assay, and electron microscopic visualization of the fibrils. Whereas successful validation using these assays is necessary for success, they are not sufficient to guarantee PFFs will seed &#945;-syn inclusions in neurons, as such aggregation activity of each PFF batch should be tested in cell culture or in pilot animal cohorts. Prior to use, PFFs must be sonicated under precisely standardized conditions, followed by examination using electron microscopy or dynamic light scattering to confirm fibril lengths are within optimal size range, with an average length of 50 nm. PFFs can then be added to cell culture media or used in animals. Pathology detectable by immunostaining for phosphorylated &#945;-syn (psyn; serine 129) is apparent days or weeks later in cell culture and rodent models, respectively.&#160;

The goal of this article is to outline the steps required for the generation of fibrils from monomeric alpha-synuclein, subsequent quality control, and use of the preformed fibrils in vivo.

Génération de fibrilles préformées alpha-synucléine à partir de monomères et utilisation in vivo

Use of the in vivo alpha-synuclein preformed fibril (&#945;-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model ...