Les auteurs tiennent à remercier Qiuli Wang, Centre de ressources de langues, Colby College, de l&#39;assistance technique avec le traitement vidéo et Eric Thomas, département de musique, Colby College, pour fournir la musique de fond. Ce projet a été financé par des subventions du Centre national des ressources de recherche, INBRE (P20RR016463-12), l&#39;Institut national des sciences médicales générales (P20 de GM103423-12), les ressortissants Institutes of Health et la Division des sciences Grant, Colby College (STA). JL et LWM ont été pris en charge par des subventions du Fonds Scholar été, Colby College.

1. Drosophila sursaut induite par dosage de locomotion <ol><li> Traitement de la toxicomanie <ol><li> Calme pour immobiliser nombre désiré (environ 8-12) de 1-3 jour-vieux mâle vole en utilisant le CO 2 et les transporter dans des flacons contenant de la nourriture de la drogue complété. Note: Un autre anesthésique par exemple, l&#39;éther ou de la glace peut être utilisé pour endormir les mouches pour permettre le comptage et la manipulation. </li><li> Autoriser les mouches de se remettre de la sédation pendant 20 minutes (ou jusqu&#39;à ce que la récupération) avec le flacon en position horizontale (pour empêcher les mouches de se retrouver sur la nourriture) et puis placez le flacon en position verticale dans un endroit sombre de 12 h, la lumière incubateur de 12 h à 25 ° C pour le reste de l&#39;expérience. </li></ol></li><li> Dispositif expérimental <ol><li> Diviser cette configuration à double flacon en trois parties égales de 6,33 cm par marquage des cercles autour des flacons avec un marqueur permanent. </li><li> Après 3 jours d&#39;exposition au médicament, transfert vole sans anesthésie dans le flacon fond et placer rapidement le flacon hautsur l&#39;ouverture. Collez les deux flacons avec du ruban adhésif transparent. </li><li> Autoriser les mouches à s&#39;acclimater à leur nouvel environnement pendant 15 min. </li><li> Placer les flacons sur un fond blanc et mis en place un appareil photo numérique à une distance appropriée de l&#39;appareil à double flacon avec une minuterie en vue. S&#39;assurer que la totalité de l&#39;appareil est visible dans un cadre photo unique et que toutes les mouches sont au point. Pour maintenir des cadres cohérents entre les essais, marquer l&#39;emplacement de la caméra et le flacon. </li></ol></li><li> Mobilité Assay <ol><li> Afficher clairement le numéro de l&#39;essai, traitement de la toxicomanie, et la minuterie en vue de la caméra. </li><li> Taper fermement l&#39;appareil à double flacon contre le comptoir 3 fois et veiller à ce que toutes les mouches tombent au fond du flacon. Commencer simultanément la minuterie. </li><li> Toutes les 5 secondes pendant 1 min, prendre une photo de l&#39;appareil. Remarque: vous pouvez, une vidéo peut être capturé et mis en pause à des intervalles appropriés pour les mesures. </li><li> Permettre de récupérer vol au repos pendant 1 min. </li><li&gt; Répétez 2 fois plus avec 1 min de temps de récupération entre chaque essai. Note: Chaque appareil doit prendre 5 minutes pour compléter la collecte de données. Maintenir la même force de taper entre les essais. Multiple (au moins 3) appareil peut être facilement manipulé simultanément. </li></ol></li><li> Analyse des données <ol><li> Passez en revue les images et enregistrer le nombre de mouches dans chaque section au fil du temps. Calculer le pourcentage de mouches dans chaque section au fil du temps. Notes: Répétez cette procédure entière avec les mêmes mouches à 2 ou 3 points de temps d&#39;intérêt, par exemple, jour 3, 5, et 7 Si trop de mouches meurent pendant l&#39;expérience, il est possible d&#39;intensifier le nombre de procès en première instance pour compenser pour la mortalité. Utiliser l&#39;analyse statistique appropriée pour comparer les données. </li></ol></li></ol> 2. Drosophila spontanée Locomotion Assay <ol><li> Préparation des aliments <ol><li> Reconstituer 3 g de milieu Drosophila instantanée avec 15 ml d&#39;eau désionisée et la roténone souhaitée(Ou un autre médicament d&#39;intérêt) dosage. </li><li> Une fois le mélange alimentaire est devenue ferme (environ 5 min), charger attentivement la nourriture à environ 1 cm de hauteur en fabricant fournis tubes transparents (5 mm X 65 mm). Ajouter le médicament perfusé nourriture pour les tubes en plaçant soigneusement les tubes verticalement dans la nourriture et les tordre jusqu&#39;à ce qu&#39;ils puissent être enlevés avec de la nourriture à l&#39;intérieur du tube. Remarque: Il est utile de placer un doigt sur l&#39;ouverture du tube pour créer un vide. Aliments ne doivent pas contenir de bulles d&#39;air ou avoir une surface inégale que les mouches peuvent se coincer. </li></ol></li><li> Configuration expérimentale <ol><li> Placer un capuchon en plastique sur l&#39;extrémité du tube la plus proche de la nourriture. Poussez le couvercle en plastique sur le tube aussi peu que possible, car il peut créer une bulle d&#39;air dans le flacon si on le pousse à force. </li><li> Sedate 1 jour-vieux mâle vole en utilisant le CO 2 et insérer délicatement 1 mâle mouche dans chaque tube avec un pinceau. Répéter en fonction du nombre d&#39;essais souhaités. </li><li> Branchez l&#39;partie la plus éloignée du tube de la nourriture avec une petite boule de coton, qui peut être roulé à la main de grand magasin a acheté des boules de coton. </li><li> Autoriser les mouches de récupérer les tubes en position horizontale pendant 15 minutes et faire en sorte que toutes les mouches sont vivants et actifs. Insérer les tubes dans DAM et assurez-vous que tous les tubes sont dans la même position par rapport au barrage. Remarque: il est possible de les placer à la zone de surveillance au moyen de l&#39;ampoule, ou pour pousser tous les flacons sur le côté, de sorte que l&#39;extrémité du tube est surveillée. Remarque: Voir la discussion des variations sur cette méthode. </li></ol></li><li> Collecte des données <ol><li> Placez le DAM dans un endroit sombre 12 h, incubateur de lumière de 12 h réglé à 25 ° C. Connectez le DAM pour le système de collecte de données. Ouvrez le logiciel de DAM et dans les préférences, sélectionnez la longueur du bac à 10 min. Lancer la collecte des données et permettre au programme de collecte de données pendant 7 jours. Remarque: longueur Bin peut être ajustée si nécessaire. </li><li> Analyse des données Remarque: Processobtenir les données de coups par minute en tant que mesure de la locomotion spontanée à long terme. <ol><li> Ouvrir le fichier DAM programme et l&#39;accès de surveiller les données de numérisation, cliquez données de sélection d&#39;entrée. </li><li> Sélectionnez la plage de surveillance approprié et sélectionnez la longueur du bac à intervalles de 10 minutes. </li><li> Dans le type de fichier de sortie choisir les fichiers de canal. Laissez toutes les autres options par défaut. </li><li> Cliquez données de numérisation et enregistrer dans un dossier désigné. </li><li> Importer des données dans un logiciel d&#39;analyse de données circadien d&#39;obtenir coups par min. Remarque: Pour l&#39;analyse des données des logiciels Clocklab est couramment utilisé. D&#39;autres options sont également disponibles. </li></ol></li></ol></li></ol>

<ol>
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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+glutathione+S-transferase+activity+rescues+dopaminergic+neuron+loss+in+a+Drosophila+model+of+Parkinson's+disease.">Increased glutathione S-transferase activity rescues dopaminergic neuron loss in a Drosophila model of Parkinson's disease.</a> Proceedings of the National Academy of Sciences of the United States of America. 102, 8024-809 (2005).</li><li>Ahmad, S. T., Steinmetz, S. B., Bussey, H. M., Possidente, B., Seggio, J. 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Les auteurs n&#39;ont rien à divulguer.

Dans cette étude, nous décrivons deux procédures pour mesurer à la fois la locomotion spontanée à long terme et de la locomotion induite sursaut court-terme dans un modèle drosophile induit roténone de la maladie de Parkinson. On peut aussi mesurer ces caractéristiques de la locomotion chez les mouches exposés à d&#39;autres agents pharmacologiques connus pour modéliser la maladie de par exemple de la maladie de Parkinson, le paraquat 14, les modèles génétiqu...

les irrégularités de locomotion sont un symptôme majeur de la maladie de Parkinson et sont en grande partie due à une dégradation des neurones dopaminergiques de la substantia nigra 1. La roténone est un insecticide cétoniques qui a été largement étudié pour modéliser les déficits moteurs parkinsoniens chez la drosophile 2-6. La roténone provoque des dommages oxydatifs en bloquant la voie de la phosphorylation oxydative, ce qui entraîne finalement la mort de la cellule 7. Les neurones dopaminergiques sont plus sensibles à la toxicité de la roténone, ce qui rend les effets de la substance chimique principalement basée 2,7 moteur. En induisant les symptômes de la maladie de Parkinson chez les mouches, nous pouvons mieux comprendre la maladie et ses symptômes remédier 6,8-11. Drosophila fournit un bon modèle pour étudier cet effet parce qu&#39;ils sont génétiquement docile, facile à entretenir, et ont un cycle de vie rapide. Plusieurs études ont montré que la roténone provoque à court terme sursaut induitedéfauts de locomotion chez la drosophile de les mouches sont maintenus sur l&#39;alimentation de la roténone complété, ils montrent une réponse plus lente geotactic négatif après sursaut 2-6. Leur incapacité à monter vers le haut dans un appareil flacon aussi rapidement que les essais de contrôle indique des défauts de locomotion induite sursaut. L&#39;effet de la roténone sur le long terme, le mouvement spontané n&#39;est pas bien décrite. Surveillance de l&#39;activité chez la drosophile (barrages) ont été utilisés avec succès pour surveiller les mouvements chez la drosophile rythme circadien étudie 12,13. Les mouches sont placés dans des tubes individuels, qui sont chargés dans la DAM. Cet appareil est équipé d&#39;un capteur infrarouge, qui compte le nombre de fois où une mouche rompt le faisceau infrarouge. Ces chiffres peuvent être utilisés comme mesure de la locomotion et 12,13 activité intacte. En plaçant les mouches dans un DAM, l&#39;effet de la roténone sur leur locomotion à long terme peut être caractérisé. Cette étude décrit les méthodes à AEMlocomotion et la locomotion spontanée à long terme ure à court terme sursaut induite afin de mieux comprendre les effets de la roténone médiation déficiences motrices. Caractérisation des carences de locomotion mimant la maladie de Parkinson sont importants car ils permettent l&#39;étude d&#39;autres composés qui peuvent inverser ces défauts de locomotion.

<table border="0" cellpadding="0" cellspacing="0" width="628">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Standard narrow vials</td>
			<td style="width:127px;">Genesee Scientific</td>
			<td style="width:119px;">32-120</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Rotenone</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:119px;">R8875</td>
			<td>Store in freezer, make fresh for each experiment</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Dimethyl Sulfoxide (DMSO)</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:119px;">D8418</td>
			<td>Solvent for rotenone</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Instant Drosophila medium</td>
			<td style="width:127px;">Carolina Biological&#160;</td>
			<td style="width:119px;">Formula 4-24</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Drosophila activity monitor (DAM)</td>
			<td style="width:127px;">Trikinetics</td>
			<td style="width:119px;">DAM2</td>
			<td>trikinetics.com</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">DAM tubes</td>
			<td style="width:127px;">Trikinetics</td>
			<td style="width:119px;">Tubes 5X65 mm</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;"></td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Recipe for Rotenone +food (125 mM dose)</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td>Make 62.5 mM rotenone stock solution in DMSO by dissolving 25 mg rotenone in 1 ml DMSO&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td>For 125 mM dose, add 10 mM rotenone stock in DMSO to 5 ml water.</td>
		</tr>
	</tbody>
</table>

methods to characterize spontaneous and startle-induced locomotion in a rotenone-induced parkinson's disease model of drosophila

La maladie de Parkinson est une maladie neurodégénérative qui résulte de la dégénérescence des neurones dopaminergiques dans le système nerveux central, principalement dans la substance noire. La maladie provoque des déficiences motrices, qui présentent comme la rigidité, des tremblements et de la démence chez les humains. La roténone est un insecticide qui provoque des dommages oxydatifs en inhibant la fonction de la chaîne de transport des électrons dans les mitochondries. Il est également utilisé pour modéliser la maladie de Parkinson dans la drosophile. Les mouches ont une réponse négative geotactic inhérente, ce qui les obligent à monter vers le haut après avoir été surpris. Il a été établi que la roténone provoque des anomalies de mortalité et de locomotion début qui perturbent la capacité des mouches à grimper après qu&#39;ils ont été exploitées vers le bas. Cependant, l&#39;effet de la roténone sur le mouvement spontané n&#39;est pas bien documenté. Cette étude décrit deux essais de débit sensibles, reproductibles, et élevés pour caractériser les carences induites roténone enlocomotion et la locomotion spontanée à long terme à court terme sursaut induite chez la drosophile. Ces dosages peuvent être facilement adaptés pour caractériser d&#39;autres modèles de Drosophila de défauts de locomotion et de l&#39;efficacité d&#39;agents thérapeutiques.

Drosophila sursaut induite par dosage de locomotion De type sauvage, Canton-S, les mouches ont montré une réponse négative geotactic robuste avec seulement environ 88% et 5% de mouches dans les parties supérieure et inférieure, respectivement, de l&#39;appareil à double flacon après 30 secondes (Figure 1). Mouches exposées à 125 pM et 250 pM roténone pendant 3 jours ont montré une légère diminution du nombre de mouches dans la ...

Watch this Scientific Journal Video about Methods to Characterize Spontaneous and Startle-induced Locomotion in a Rotenone-induced Parkinson's Disease Model of Drosophila at JoVE.com

Methods to Characterize Spontaneous and Startle-induced Locomotion in a Rotenone-induced Parkinson&#039;s Disease Model of Drosophila

La maladie de Parkinson est une maladie neurodégénérative qui résulte de la dégénérescence des neurones dopaminergiques dans le système nerveux central, ce qui provoque des défauts de locomotion. modèles de roténone la maladie de Parkinson chez la drosophile. Le présent document décrit deux essais qui caractérisent les lacunes à la fois spontanées et surprendre induites locomotion causés par la roténone.

Méthodes pour caractériser spontanée et induite par Locomotion sursaut dans un modèle de la maladie de Parkinson induite de la roténone-de<em&gt; Drosophila</em

Parkinson&#8217;s disease is a neurodegenerative disorder that results from the degeneration of dopaminergic neurons in the central nervous system, ...

methods-to-characterize-spontaneous-startle-induced-locomotion

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JoVE Journal

Neuroscience

Methods to Characterize Spontaneous and Startle-induced Locomotion in a Rotenone-induced Parkinson's Disease Model of Drosophila

10.0K vues.  Colby College.  La maladie de Parkinson est une maladie neurodégénérative qui résulte de la dégénérescence des neurones dopaminergiques dans le système nerveux central, ce qui provoque des défauts de locomotion. modèles de roténone la maladie de Parkinson chez la drosophile. Le présent document décrit deux essais qui caractérisent les lacunes à la fois spontanées et surprendre induites locomotion causés par la roténone. 

Vidéo: Methods to Characterize Spontaneous and Startle-induced Locomotion in a Rotenone-induced Parkinson's Disease Model of Drosophila

Autologous Blood Injection to Model Spontaneous Intracerebral Hemorrhage in Mice

Investigation of the pathophysiology of injury after intracerebral
hemorrhage (ICH) requires a reproducible animal model. While ICH
accounts for 10-15% of all strokes, there remains no specific
effective therapy. The autologous blood injection model in mice
involves the stereotaxic injection of arterial blood into the basal
ganglia mimicking a spontaneous hypertensive hemorrhage in man. The
response to hemorrhage can then be studied in vivo and the
neurobehavioral deficits quantified, allowing for description of the
ensuing pathology and the testing of potential therapeutic agents. The
procedure described in this protocol uses the double injection
technique to minimize risk of blood reflux up the needle track, no
anticoagulants in the pumping system, and eliminates all dead space
and expandable tubing in the system.

The autologous blood injection model of intracerebral hemorrhage in mice described in this protocol uses the double injection technique to minimize risk of blood reflux up the needle track, no anticoagulants in the pumping system, and eliminates all dead space and expandable tubing in the system.

Injection de sang autologue au modèle spontané Hémorragie intracérébrale chez la souris

Investigation of the pathophysiology of injury after intracerebral
hemorrhage (ICH) requires a reproducible animal model.  While ICH
accounts for 10-15% ...

Recherche

Neurosciences

Methods to Assay Drosophila Behavior

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases1. We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials2-4. The rapid iterative negative geotaxis (RING) assay5 has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously using large number of animals, with the high-throughput approach making it more amenable for screening experiments.

Methods to Assay Drosophila Behavior

Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.

Méthodes de dosage à Drosophila Comportement

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular ...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

Méthodes d&#39;enregistrement multi-unités pour caractériser l&#39;activité neuronale dans le Criquet (<em&gt; Schistocerca americana</em&gt; Circuits olfactifs)

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such behavior is spontaneous locomotor activity. Here we describe our protocol that utilizes Drosophila population monitors and a tracking system that allows continuous monitoring of the spontaneous locomotor activity of flies for several days at a time. This method is simple, reliable, and objective and can be used to examine the effects of aging, sex, changes in caloric content of food, addition of drugs, or genetic manipulations that mimic human diseases.

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster are useful in studying genetic or environmental manipulations that affect behaviors such as spontaneous locomotor activity.&#160; Here we describe a protocol that utilizes monitors with infrared beams and data analysis software to quantify spontaneous locomotor activity.&#160;

Détermination de l&#39;activité locomotrice spontanée,<em&gt; Drosophila melanogaster</em

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such ...

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

Chronic Traumatic Encephalopathy (CTE) is an established neurodegenerative disease that is closely associated with exposure to repetitive mild Traumatic Brain Injury (mTBI). The mechanisms responsible for its complex pathological changes remain largely elusive, despite a recent consensus to define the neuropathological criteria. Here, we describe a novel method to develop a model of CTE in Drosophila melanogaster&#160;(Drosophila&#160;)&#160;in an attempt to identify the key genes and pathways that lead to the characteristic hyperphosphorylated tau accumulation and neuronal death in the brain. Adjustable-strength impacts to inflict mild closed injury are delivered directly to the fly head, subjecting the head to rapid acceleration and deceleration. Our method eliminates the potential problems inherent with other Drosophila mTBI models (e.g.,animal death might be induced by damage to other parts of the body or to internal organs). The less labor- and cost-intensive animal care, short life span, and extensive genetic tools make the fruit fly ideal to study CTE pathogenesis and make it possible to perform large-scale, genome-wide forward genetic and pharmacological screens. We anticipate that the ongoing characterization of the model will generate important mechanistic insights on disease prevention and therapeutic approaches.

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

Here, we describe a new approach to inflict closed-head traumatic brain injury in Drosophila melanogaster. Our method has the advantage of directly delivering repetitive impacts with adjustable strength to the head alone. Further exploration of the invertebrate system will help to illuminate the pathogenesis of chronic traumatic encephalopathy.

Une nouvelle méthode pour modéliser l&#39;encéphalopathie traumatique chronique dans<em&gt; Drosophila</em

Chronic Traumatic Encephalopathy (CTE) is an established neurodegenerative disease that is closely associated with exposure to repetitive mild Traumatic ...

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx due to the abnormal activation of glutamate receptors results in DA excitotoxicity and has been identified as an important mechanism for DA neuron loss. In this study, we isolate, dissociate, and culture midbrain neurons from the mouse ventral mesencephalon (VM) of ED14 mouse embryos. We then infect the long-term primary mouse midbrain cultures with an adeno-associated virus (AAV) expressing a genetically encoded calcium indicator, GCaMP6f under control of the human neuron-specific synapsin promoter, hSyn. Using live confocal imaging, we show that cultured mouse midbrain neurons display spontaneous Ca2+ fluxes detected by AAV-hSyn-GCaMP6f. Bath application of glutamate to midbrain cultures causes abnormal elevations in intracellular Ca2+ within neurons and this is accompanied by caspase-3 activation in DA neurons, as demonstrated by immunostaining. The techniques to identify glutamate-mediated apoptosis in primary mouse DA neurons have important applications for the high content screening of drugs that preserve DA neuron health.

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Here we present a protocol to measure in vitro Ca2+ fluxes in midbrain neurons and their downstream effects on caspase-3 using primary mouse midbrain cultures. This model can be employed to study pathophysiologic changes related to abnormal Ca2+ activity in midbrain neurons, and to screen novel therapeutics for anti-apoptotic properties.

Quantification spontanée Ca2+ Fluxes et leurs effets en aval dans les neurones midbrains de souris primaires

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx ...

Applying the RatWalker System for Gait Analysis in a Genetic Rat Model of Parkinson's Disease

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta. Gait abnormalities, including decreased arm swing, slower walking speed, and shorter steps are common in PD patients and appear early in the course of disease. Thus, the quantification of motor patterns in animal models of PD will be important for phenotypic characterization during disease course and upon therapeutic treatment. Most cases of PD are idiopathic; however, the identification of hereditary forms of PD uncovered gene mutations and variants, such as loss-of-function mutations in Pink1 and Parkin, two proteins involved in mitochondrial quality control that could be harnessed to create animal models. While mice are resistant to neurodegeneration upon loss of Pink1 and Parkin (single and combined deletion), in rats, Pink1 but not Parkin deficiency leads to nigral DA neuron loss and motor impairment. Here, we report the utility of FTIR imaging to uncover gait changes in freely walking young (2 months of age) male rats with combined loss of Pink1 and Parkin prior to the development of gross visually apparent motor abnormality as these rats age (observed at 4-6 months), characterized by hindlimb dragging as previously reported in Pink1 knockout (KO) rats.

Applying the RatWalker System for Gait Analysis in a Genetic Rat Model of Parkinson&#039;s Disease

Here we describe the RatWalker system, built by redesigning the MouseWalker apparatus to accommodate for the increased size and weight of rats. This system uses frustrated total internal reflection (FTIR), high-speed video capture, and open-access analysis software to track and quantify gait parameters.

Application du système RatWalker pour l’analyse de la marche dans un modèle génétique de la maladie de Parkinson chez le rat

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars ...

The 6-hydroxydopamine Rat Model of Parkinson's Disease

Motor symptoms of Parkinson's disease (PD)-bradykinesia, akinesia, and tremor at rest-are consequences of the neurodegeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc) and dopaminergic striatal deficit. Animal models have been widely used to simulate human pathology in the laboratory. Rodents are the most used animal models for PD due to their ease of handling and maintenance. Moreover, the anatomy and molecular, cellular, and pharmacological mechanisms of PD are similar in rodents and humans. The infusion of the neurotoxin, 6-hydroxydopamine (6-OHDA), into a medial forebrain bundle (MFB) of rats reproduces the severe destruction of dopaminergic neurons and simulates PD symptoms. This protocol demonstrates how to perform the unilateral microinjection of 6-OHDA in the MFB in a rat model of PD and shows the motor deficits induced by 6-OHDA and predicted dopaminergic lesions through the stepping test. The 6-OHDA causes significant impairment in the number of steps performed with the contralateral forelimb.

The 6-hydroxydopamine Rat Model of Parkinson&#039;s Disease

The 6-hydroxydopamine (6-OHDA) model has been used for decades to advance the understanding of Parkinson's Disease. In this protocol, we demonstrate how to perform unilateral nigrostriatal lesions in the rat by injecting 6-OHDA in the medial forebrain bundle, assess motor deficits, and predict lesions using the stepping test.

Le modèle de rat 6-hydroxydopamine de la maladie de Parkinson

Motor symptoms of Parkinson's disease (PD)-bradykinesia, akinesia, and tremor at rest-are consequences of the neurodegeneration of dopaminergic neurons in ...

Induction and Assessment of Levodopa-induced Dyskinesias in a Rat Model of Parkinson's Disease

Levodopa (L-DOPA) remains the gold-standard therapy used to treat Parkinson&#39;s disease (PD) motor symptoms. However, unwanted involuntary movements known as L-DOPA-induced dyskinesias (LIDs) develop with prolonged use of this dopamine precursor.&#160;It is estimated that the incidence of LIDs escalates to approximately 90% of individuals with PD within 10&#8211;15 years of treatment. Understanding the mechanisms of this malady and developing both novel and effective anti-dyskinesia treatments requires consistent and accurate modeling for pre-clinical testing of therapeutic interventions. A detailed method for reliable induction and comprehensive rating of LIDs following 6-OHDA-induced nigral lesioning in a rat model of PD is presented here. Dependable LID assessment in rats provides a powerful tool that can be readily utilized across laboratories to test emerging therapies focused on reducing or eliminating this common treatment-induced burden for individuals with PD.

Induction and Assessment of Levodopa-induced Dyskinesias in a Rat Model of Parkinson&#039;s Disease

This article describes methods to induce and evaluate levodopa-induced dyskinesias in a rat model of Parkinson&#39;s disease. The protocol offers detailed information regarding the intensity and frequency of a range of dyskinetic behaviors, both dystonic and hyperkinetic, providing a reliable tool to test treatments targeting this unmet medical need.

Induction et évaluation des dyskinésies induites par la lévodopa dans un modèle rat de la maladie de Parkinson

Levodopa (L-DOPA) remains the gold-standard therapy used to treat Parkinson&#39;s disease (PD) motor symptoms. However, unwanted involuntary movements ...

Analyzing the Parkinson's Disease Mouse Model Induced by Adeno-associated Viral Vectors Encoding Human &#945;-Synuclein

Parkinson&#39;s disease is a neurodegenerative disorder that involves the death of the dopaminergic neurons of the nigrostriatal pathway and, consequently, the progressive loss of control of voluntary movements. This neurodegenerative process is triggered by the deposition of protein aggregates in the brain, which are mainly constituted of &#945;-synuclein. Several studies have indicated that neuroinflammation is required to develop the neurodegeneration associated with Parkinson&#39;s disease. Notably, the neuroinflammatory process involves microglial activation as well as the infiltration of peripheral T cells into the substantia nigra (SN). This work analyzes a mouse model of Parkinson&#39;s disease that recapitulates microglial activation, T-cell infiltration into the SN, the neurodegeneration of nigral dopaminergic neurons, and motor impairment. This mouse model of Parkinson&#39;s disease is induced by the stereotaxic delivery of adeno-associated viral vectors encoding the human wild-type &#945;-synuclein (AAV-h&#945;Syn) into the SN. The correct delivery of viral vectors into the SN was confirmed using control vectors encoding green fluorescent protein (GFP). Afterward, how the dose of AAV-h&#945;Syn administered in the SN affected the extent of h&#945;Syn expression, the loss of nigral dopaminergic neurons, and motor impairment were evaluated. Moreover, the dynamics of h&#945;Syn expression, microglial activation, and T-cell infiltration were determined throughout the time course of disease development. Thus, this study provides critical time points that may be useful for targeting synuclein pathology and neuroinflammation in this preclinical model of Parkinson&#39;s disease.

Analyzing the Parkinson&#039;s Disease Mouse Model Induced by Adeno-associated Viral Vectors Encoding Human &amp;#945;-Synuclein

This work analyzes the vector dose and exposure time required to induce neuroinflammation, neurodegeneration, and motor impairment in this preclinical model of Parkinson&#39;s disease. These vectors encoding the human &#945;-synuclein are delivered into the substantia nigra&#160;to recapitulate the synuclein pathology associated with Parkinson&#39;s disease.