This work is supported by a Canadian Health Research Institute (CIHR) Team Grant to Dr. Bolduc (co-PI) and Dr. Guy Rouleau (PI). We would like to thank Dr. Oksana Suchowersky, Dr. Kathryn Todd, the members of the University of Alberta fly club and Dr. Clayton Dickson for help in development of the method and statistical analysis.

Recherche sur Drosophila melanogaster était en conformité avec l&#39;Université des lignes directrices de recherche de l&#39;Alberta. 1. Fly Collection <ol><li> Recueillir 20 mouches utilisant le CO 2 (g) l&#39;anesthésie et le placer dans un flacon de collection 25 mm x 95 mm contenant de la nourriture. </li><li> Entreposer les flacons contenant des mouches horizontalement pour éviter le piégeage des mouches dans les liquides qui peuvent accumuler dans le fond du flacon. </li><li> Incuber mouches pendant au moins 21 heures à 22 ° C à 45% d&#39;humidité dans un incubateur pendant environ pendant 15 heures. Régler l&#39;incubateur avec une lumière de 12 h: cycle d&#39;obscurité. </li></ol> 2. Escalade Assay <ol><li> Le lendemain matin, transférer 20 mouches d&#39;un seul flacon en verre de 250 ml graduée. Marquer la position du cylindre pour le maintenir constant de tous les jours. Utilisez un cylindre de verre par génotype pour éviter la contamination croisée entre les génotypes. Laver à la fin de chaque expérience et roliter eux entre les génotypes. <ol><li> Mener des expériences à la lumière ambiante (ou la lumière rouge si il ya un défaut potentiel de la vision) à la température et l&#39;humidité de 22 ° C et 40% respectivement. Pour éviter rythme circadien confondre, toujours effectuer des expériences dans le même temps de la journée. </li></ol></li><li> Sceller la partie supérieure du cylindre avec un film barrière (film de cire) pour empêcher la fuite de tout vol (figure 2). </li><li> Mettre en place la caméra vidéo sur un trépied. appareil photo Focus sur la ligne 190 ml de 250 ml graduée (17,5 cm). </li><li> Compter le nombre de mouches mortes au fond du cylindre et dans les flacons de produits alimentaires. Inscrire ce numéro que le taux de mortalité. </li><li> Très tapoter légèrement le cylindre contre un coussin en mousse à cellules fermées à plusieurs reprises avec une force suffisante pour déplacer les mouches à la surface inférieure intérieure. Appuyez sur 5 - 10 fois tout en utilisant l&#39;autre main pour appuyer dossier sur l&#39;appareil photo. </li><li> Appuyez sur le bouton &quot;Enregistrer&quot; sur la caméra. </li><li> Lancer la video enregistrement de la caméra et tapez sur le cylindre à six reprises dans un modèle non-rythmique distincte. </li><li> Mener chaque essai pendant 2 min à partir du moment les mouches sont exploitées dernière et enregistrer le nombre de mouches qui traversent la hauteur de 17,5 cm (190 ml) à chaque point de temps choisi (de quantifier toutes les 10 secondes). Remarque: Le marquage sur le cylindre ml variera d&#39;un modèle de cylindre à l&#39;autre en fonction du diamètre. Pour éviter toute erreur, mesurer la hauteur sur chaque bouteille utilisée. </li><li> Une fois le procès terminé, disposer de mouches dans 95% d&#39;éthanol. </li><li> Répétez les étapes 2.1 à 2.9 jusqu&#39;à ce que toutes les répétitions ont été testés avec des mouches frais à chaque fois. Note: Bien que 5 répétitions peuvent être assez avec une mutation ayant un effet important sur la locomotion, 10 répétitions biologiques de 20 200 mouches (mouches) est recommandée pour détecter des différences plus petites. </li><li> À la fin de l&#39;expérience, se laver les cylindres dans le lave-vaisselle de laboratoire et O sec / N pour être réutilisé. </li></ol> 3. Analyse <ol><li> Analyze vidéos de chaque volent procès. Chaque 10 secondes, enregistrer le nombre total de mouches qui passent la ligne de cible. <ol><li> Si une mouche redescend ou tombe, fiche qui volent comme -1 et compter la prochaine volée de franchir la ligne de cible que le même numéro que la mouche qui est remonté vers le bas ou est tombé. Par exemple, si le 15 ème mouche tombe en dessous de la ligne de cible, la prochaine volée à franchir la ligne (16 e volent) est considéré comme le 15 ème volée et non pas le 16 ème. </li></ol></li><li> Soustraire la mortalité à partir du nombre total de mouches (20) pour obtenir le nombre de mouches qui restent dans le procès. A chaque point de temps, obtenir la fraction de mouches au-dessus de la ligne de cible. </li><li> Complot chaque pourcentage à chaque point de temps (voir Figure 3). </li><li> Analyser la performance au point de données 120 sec et effectuer test t de Student lorsque 2 groupes sont présents ou ANOVA et un post-test pour les comparaisons multiples (avec modification de Bonferroni pour planifiées et non planifiées Tukey pour comparisons). Le Kolmogorov-Smirnov teste 17 est également effectuée pour vérifier la normalité et variance égale, mais aussi de comparer les distributions du groupe mutant à la commande. </li><li> Pour présenter les données sur le vieillissement, tracer le pourcentage de mouches grimper à 120 sec avec des mouches de différents âges (2 jours, 1 semaine, 2 semaines) pour voir si il ya un déficit progressif (Figure 4). </li></ol>

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The authors have nothing to disclose.

Drosophila a déjà prouvé être un excellent modèle dans la maladie 14 et d&#39;autres conditions neurodégénératives de 1,2 de Parkinson. En plus des outils génétiques disponibles chez la drosophile, son génome est hautement conservé de gènes impliqués dans des troubles neurologiques 19. L&#39;avènement des méthodes de dépistage génétique larges génome (y compris le séquençage de l&#39;exome ensemble) est susceptible de continuer à fournir une plus ...

Maladies neurodégénératives dévastatrices telles que la maladie de Parkinson, la sclérose latérale amyotrophique, et paraplégie spastique familiale sont de plus en plus reconnus. Malheureusement, la plupart de ces maladies neurodégénératives sont encore sans traitements. L&#39;utilisation clinique généralisée de tests génétiques impartiales pangénomiques comme le séquençage de l&#39;exome ensemble a conduit à un nombre croissant de gènes étant impliqué dans les troubles locomoteurs humaines. Malgré ces progrès, la progression pathologique du début à la fin des étapes, reste insaisissable dans ces troubles. Drosophila fournit un avec les outils génétiques pour étudier exigence de gènes d&#39;une manière spatio-temporelle contrôlée. En outre, la drosophile est avéré utile pour le criblage de médicaments pour les troubles neurologiques comme la maladie de Parkinson 1, la maladie d&#39;Alzheimer 2, déficience intellectuelle et l&#39;épilepsie 3,4 5,6 entre autres. Notre objectif était de développer une solution rentableet le dosage fiable qui permettrait analyse à haut débit qui serait encore assez sensible pour détecter de petits changements dans les performances du moteur. Il existe plusieurs tests utilisés pour quantifier les effets de la mutation génétique et / ou de l&#39;état de l&#39;environnement sur ​​le comportement d&#39;escalade drosophile. La plupart des essais capitaliser sur la tendance naturelle de mouches à grimper, connus sous le nom géotaxie négative, ou le dosage de l&#39;escalade. 7 Benzer suggéré en 1967 que l&#39;appareil à contre-courant utilisé pour l&#39;étude de phototaxie pourrait également être utilisé pour étudier gravitaxis. Depuis lors, Ganetsky 8 et beaucoup d&#39;autres 9 -12 ont construit sur ​​le dosage initial. Le principe est de placer un nombre connu de mouches dans un flacon et appuyez sur le flacon fermement contre une surface dure, provoquant les mouches tombent au fond du flacon. Comme il est un comportement inné, vol tenteront de grimper au sommet de la fiole, par opposition à la gravité. Cet essai est quantitative et mesures combien de mouches ont grimpé passé un marqueur sur le flacon pendant une période de temps allouée. Mesure de la vitesse à la place du nombre total de mouches escalade est devenue un paramètre fiable et montré des défauts dans les cas où le nombre de critères de mouches était non significatif 13. Le test d&#39;escalade se sont révélées utiles dans l&#39;étude de nombreux troubles neurodégénératifs, y compris la maladie de Parkinson 14. Cependant, nous avons constaté que les défauts de locomotive peuvent ne pas être détectable au moment où la neurodégénérescence est déjà considérée dans les études pathologiques 14. Ainsi, l&#39;utilisation du dosage traditionnel peut limiter la capacité d&#39;étudier les stades précoces de la pathogenèse de la maladie. L&#39;apparition de défauts de locomotives durant les derniers stades de la pathologie peut refléter une maladie dont la progression est trop avancé pour le sauvetage complète. Cela soulève un problème potentiel avec la sensibilité de l&#39;essai d&#39;escalade traditionnelle. L&#39;incapacité potentielle de la traditiontional escalade test pour détecter les défauts de locomotives doux peut être attribuée à la hauteur à laquelle les mouches sont tenus de grimper. Les traditionnels dosage 15,16 mesures le nombre de mouches à monter avec succès sur une hauteur de 2 à 5 cm dans 10 à 20 sec.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Drosophila stocks</td>
			<td></td>
			<td></td>
			<td>The stocks are selected depending on the experiments. The temperature and humidity in the room and in the incubator must be controled and consistent to avoid flies being too staticky or too wet.</td>
		</tr>
		<tr>
			<td>Video camera</td>
			<td></td>
			<td></td>
			<td>Any digital camcorder will do. Make sure they can focus on close object.</td>
		</tr>
		<tr>
			<td>Graduated cylinder</td>
			<td>Kimble</td>
			<td>20028W</td>
			<td>Different models of graduated cylinder may have different diameter. It is therefore imporant to measure the height.</td>
		</tr>
		<tr>
			<td>computer</td>
			<td></td>
			<td></td>
			<td>Any model will do. We used the computer to monitor the climbing of the flies and record the number of flies at each time point</td>
		</tr>
	</tbody>
</table>

quantitative analysis of climbing defects in a drosophila model of neurodegenerative disorders

Locomotive défauts résultant de maladies neurodégénératives peut être un symptôme d&#39;apparition tardive de la maladie, après des années de dégénérescence infraclinique, et des stratégies ainsi actuels de traitement thérapeutique ne sont pas curatifs. Grâce à l&#39;utilisation de séquençage d&#39;exome ensemble, un nombre croissant de gènes ont été identifiés à jouer un rôle dans la locomotion humaine. Malgré l&#39;identification de ces gènes, on ne sait pas comment ces gènes sont essentiels pour le fonctionnement de la locomotive normale. Par conséquent, un test fiable, qui utilise des organismes modèles pour élucider le rôle de ces gènes afin d&#39;identifier de nouvelles cibles d&#39;intérêt thérapeutique, est plus que jamais nécessaire. Nous avons conçu une version sensibilisée de l&#39;essai de géotaxie négatifs qui permet la détection de défauts plus douces antérieure et a la capacité d&#39;évaluer ces défauts au cours du temps. Le dosage est réalisé dans un cylindre gradué de verre, qui est scellée avec un film de barrière cire. En augmentant la distance de seuil pour être grimpé à 17,5 cm et en augmentant la durée de l&#39;expérience à 2 min, nous avons observé une plus grande sensibilité dans la détection des dysfonctionnements de mobilité douces. Le dosage est rentable et ne nécessite pas une formation approfondie pour obtenir des résultats hautement reproductibles. Cela en fait une excellente technique pour le criblage de médicaments candidats dans des mutants de drosophile avec des défauts de locomotion.

Escalade est un comportement solide et reproductible. En effet, un jour de vieilles mouches de type sauvage atteindre la performance d&#39;escalade de distance de la cible rapidement (25 - 30 sec). Mouches mutantes présentent une gamme de performances de légère (ou retardée) pour compléter l&#39;incapacité de monter à la cible. Nous illustrons cette ici avec deux allèles mutants différents. Le premier est un allèle sévère de la spastin génique provoquée par une deletion complète du gène de spas spastin <...

Watch this Scientific Journal Video about Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders at JoVE.com

Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders

We present an optimized inexpensive and reliable negative geotaxis assay in Drosophila melanogaster as a model for neurodegenerative disorders. Being more sensitive to mild locomotor defects, this assay will help screen for potential genetic interactions and drug targets.

Analyse quantitative des défauts d&#39;escalade dans un modèle drosophile de maladies neurodégénératives

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and ...

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18.4K vues.  University of Alberta. We present an optimized inexpensive and reliable negative geotaxis assay in Drosophila melanogaster as a model for neurodegenerative disorders. Being more sensitive to mild locomotor defects, this assay will help screen for potential genetic interactions and drug targets. 

Vidéo: Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the fly as an experimental system include its genetic tractability, short life span and the possibility to observe and quantitatively analyze complex behaviors. The expression of disease-linked genes in specific neuronal populations of the Drosophila brain, can be used to model human neurodegenerative diseases such as Parkinson's and Alzheimer's 5.
Dopaminergic (DA) neurons are among the most vulnerable neuronal populations in the aging human brain. In Parkinson's disease (PD), the most common neurodegenerative movement disorder, the accelerated loss of DA neurons leads to a progressive and irreversible decline in locomotor function. In addition to age and exposure to environmental toxins, loss of DA neurons is exacerbated by specific mutations in the coding or promoter regions of several genes. The identification of such PD-associated alleles provides the experimental basis for the use of Drosophila as a model to study neurodegeneration of DA neurons in vivo. For example, the expression of the PD-linked human &alpha;-synuclein gene in Drosophila DA neurons recapitulates some features of the human disease, e.g. progressive loss of DA neurons and declining locomotor function 2. Accordingly, this model has been successfully used to identify potential therapeutic targets in PD 8.
Here we describe two assays that have commonly been used to study age-dependent neurodegeneration of DA neurons in Drosophila: a climbing assay based on the startle-induced negative geotaxis response and tyrosine hydroxylase immunostaining of whole adult brain mounts to monitor the number of DA neurons at different ages. In both cases, in vivo expression of UAS transgenes specifically in DA neurons can be achieved by using a tyrosine hydroxylase (TH) promoter-Gal4 driver line 3, 10.

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Here we describe two assays that have been established to study age-dependent neurodegeneration of dopaminergic (DA) neurons in Drosophila: the climbing/startle-induced negative geotaxis assay which allows to study the functional effects of DA neurons degeneration and the tyrosine hydroxylase immunostaining which is used to identify and count DA neurons in whole brain mounts.

Évaluer phénotypes neurodégénératives en<em&gt; Drosophila</em&gt; Neurones dopaminergiques par des tests d&#39;escalade et IMMUNOCOLORATION du cerveau entier

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the ...

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

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

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

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

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

Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the membrane with the co-transport of 3 Na+ and 1 H+ and the counter-transport of 1 K+. The stoichiometric current generated by the transport process can be monitored with whole-cell patch-clamp recordings from astrocytes. The time course of the recorded current is shaped by the time course of the glutamate concentration profile to which astrocytes are exposed, the kinetics of glutamate transporters, and the passive electrotonic properties of astrocytic membranes. Here we describe the experimental and analytical methods that can be used to record glutamate transporter currents in astrocytes and isolate the time course of glutamate clearance from all other factors that shape the waveform of astrocytic transporter currents. The methods described here can be used to estimate the lifetime of flash-uncaged and synaptically-released glutamate at astrocytic membranes in any region of the central nervous system during health and disease.

We describe an analytical method to estimate the lifetime of glutamate at astrocytic membranes from electrophysiological recordings of glutamate transporter currents in astrocytes.

Issu l&#39;évolution temporelle du glutamate Dégagement avec une analyse de déconvolution des courants de Transporteur ASTROCYTIQUES

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the ...

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.

The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

Haute résolution quantitative immunomarquage Analyse des Récepteurs Membranaires au rétiniennes Synapses ruban

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at the synaptic level. The Active Zone (AZ) is the region at the presynaptic membrane that mediates neurotransmitter release and is composed of a dense collection of scaffold proteins. AZs of Drosophila melanogaster (Drosophila) photoreceptors undergo molecular remodeling after prolonged exposure to natural ambient light. Thus the level of neuronal activity can rearrange the molecular composition of the AZ and contribute to the regulation of the functional output.
Starting from the light exposure set-up preparation to the immunohistochemistry, this protocol details how to quantify the number, the spatial distribution, and the delocalization level of synaptic molecules at AZs in Drosophila photoreceptors. Using image analysis software, clusters of the GFP-fused AZ component Bruchpilot were identified for each R8 photoreceptor (R8) axon terminal. Detected Bruchpilot spots were automatically assigned to individual R8 axons. To calculate the distribution of spot frequency along the axon, we implemented a customized software plugin. Each axon&#39;s start-point and end-point were manually defined and the position of each Bruchpilot spot was projected onto the connecting line between start and end-point. Besides the number of Bruchpilot clusters, we also quantified the delocalization level of Bruchpilot-GFP within the clusters. These measurements reflect in detail the spatially resolved synaptic dynamics in a single neuron under different environmental conditions to stimuli.

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Here we show how to quantify the number and spatial distribution of synaptic active zones in Drosophila melanogaster&#160;photoreceptors, highlighted with a genetically encoded molecular marker, and their modulation after prolonged exposure to light.

Analyse Synaptic Modulation de<em&gt; Drosophila melanogaster</em&gt; Photorécepteurs après exposition à la lumière prolongée

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

L’utilisation de Trace Eyeblink conditionnement de classique pour évaluer le dysfonctionnement hippocampique dans un modèle de Rat de Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay between perturbed BBB homeostasis and the pathogenesis of brain disorders needs further investigation, the development and validation of a reliable procedure to accurately detect BBB alterations may be crucial and represent a useful tool for potentially predicting disease progression and developing targeted therapeutic strategies.
Here, we present an easy and efficient procedure for evaluating BBB leakage in a neurodegenerative condition like that occurring in a preclinical mouse model of Huntington disease, in which defects in the permeability of BBB are clearly detectable precociously in the disease. Specifically, the high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin, which is able to cross the BBB only when the latter is impaired, is acutely infused into a mouse jugular vein and its distribution in the vascular or parenchymal districts is then determined by fluorescence microscopy.
Accumulation of green fluorescent-albumin in the brain parenchyma functions as an index of aberrant BBB permeability and, when quantitated by using Image J processing software, is reported as Green Fluorescence Intensity.

In this study, we present an easy and efficient procedure for evaluating the disruption of the blood-brain barrier under neurodegenerative conditions. To achieve our objective, we infused high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin into mouse jugular vein and evaluated its leakage into the brain parenchyma by fluorescence microscopy.

Évaluation de la perméabilité de la barrière hémato - encéphalique par perfusion intraveineuse d’albumine marquée FITC dans un modèle murin de la maladie neurodégénérative

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay ...

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. Drosophila larval neuronal dendritic arborization (da) is an ideal model for studying morphogenesis of neural dendrites and gene function in the development of nervous system. There are four classes of da neurons. Class IV is the most complex with a branching pattern that covers almost the entire area of the larval body wall. We have previously characterized the effect of silencing the Drosophila ortholog of SOX5 on class IV neuronal dendritic arborization complexity (NDAC) using four parameters: the length of dendrites, the surface area of dendrite coverage, the total number of branches, and the branching structure. This protocol presents the workflow of NDAC quantitative analysis, consisting of larval dissection, confocal microscopy, and image analysis procedures using ImageJ software. Further insight into da neuronal development and its underlying mechanisms will improve the understanding of neuronal function and provide clues about the fundamental causes of neurological and neurodevelopmental disorders.

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

This protocol focuses on quantitative analysis of neuronal dendritic arborization complexity (NDAC) in Drosophila, which can be used for studies of dendritic morphogenesis.

Analyse quantitative de la complexité d’arborisation dendritique neuronale chez la drosophile

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. ...

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal dysfunction and loss. Fluorescence-based imaging tools and technologies enable unprecedented analysis of subcellular neurobiological processes, yet there is still a need for unbiased, reproducible, and accessible approaches for extracting quantifiable data from imaging studies. We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-based imaging studies using Drosophila models of neurodegeneration. Specifically, we describe an easy-to-follow, semi-automated approach using Fiji/ImageJ to analyze two cellular processes: first, we quantify protein aggregate content and profile in the Drosophila optic lobe using fluorescent-tagged mutant huntingtin proteins; and second, we assess autophagy-lysosome flux in the Drosophila visual system with ratiometric-based quantification of a tandem fluorescent reporter of autophagy. Importantly, the protocol outlined here includes a semi-automated segmentation step to ensure all fluorescent structures are analyzed to minimize selection bias and to increase resolution of subtle comparisons. This approach can be extended for the analysis of other cell biological structures and processes implicated in neurodegeneration, such as proteinaceous puncta (stress granules and synaptic complexes), as well as membrane-bound compartments (mitochondria and membrane trafficking vesicles). This method provides a standardized, yet adaptable reference point for image analysis and quantification, and could facilitate reliability and reproducibility across the field, and ultimately enhance mechanistic understanding of neurodegeneration.

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-imaging-based cell biological studies of protein aggregation and autophagic flux in the central nervous system of Drosophila models of neurodegeneration.

Biologie cellulaire quantitative de neurodégénération chez la drosophile grâce à l’analyse impartiale des protéines fluorescent étiquetées à l’aide de ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a powerful tool for studying transcriptional profiles of cells, particularly in heterogeneous tissues such as the central nervous system. However, dissociation methods required for single cell sequencing can lead to experimental changes in the gene expression and cell death. Furthermore, these methods are generally restricted to fresh tissue, thus limiting studies on archival and bio-bank material. Single nucleus RNA sequencing (snRNA-Seq) is an appealing alternative for transcriptional studies, given that it accurately identifies cell types, permits the study of tissue that is frozen or difficult to dissociate, and reduces dissociation-induced transcription. Here, we present a high-throughput protocol for rapid isolation of nuclei for downstream snRNA-Seq. This method enables isolation of nuclei from fresh or frozen spinal cord samples and can be combined with two massively parallel droplet encapsulation platforms.

Here, we present a protocol to rapidly isolate high-quality nuclei from the fresh or frozen tissue for downstream massively parallel RNA sequencing. We include detergent-mechanical and hypotonic-mechanical tissue disruption and cell lysis options, both of which can be used for isolation of nuclei.

Isolement des noyaux d’adultes de la moelle épinière pour séquençage massivement parallèle ARN simple-noyau

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a ...