Name: quantitative analysis of climbing defects in a drosophila model of neurodegenerative disorders
Uploaded: 2015-06-13T15:00:00.000+00:00
Duration: 6 min 30 s
Description: Watch this Scientific Journal Video about Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders at JoVE.com

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.

Forschung an Drosophila melanogaster wurde in Übereinstimmung mit der Universität Forschungs Richtlinien Albertas. 1. Fly-Sammlung <ol><li> Sammeln Sie 20 Fliegen mit CO 2 (g) Betäubung und in eine 25 mm x 95 mm Sammelgefäß haltige Speisen. </li><li> Shop Stechflaschen mit Fliegen horizontal zu vermeiden Trapping Fliegen in allen Flüssigkeiten, die in den Boden anreichern kann das Fläschchen. </li><li> Entfernt inkubiere für mindestens 21 Stunden bei 22 ° C bei 45% Luftfeuchtigkeit in einem Brutschrank für ca. 15 Stunden. Stellen Sie den Inkubator mit einem 12 Stunden Licht: Dunkel-Zyklus. </li></ol> 2. Climbing Assay <ol><li> Am nächsten Morgen, Transfer 20 Fliegen aus einer Ampulle in einen 250-ml-Glasmesszylinder. Markieren Sie die Position des Zylinders, um sie konstant zu halten jeden Tag. Verwenden Sie einen Glaszylinder pro Genotyp um eine Kreuzkontamination zwischen den Genotypen zu verhindern. Waschen Sie am Ende jedes Experiments und rotate sie zwischen den Genotypen. <ol><li> Führen die Versuche im Umgebungslicht (oder rotes Licht, wenn es einen potentiellen Defekt in Vision) bei der Temperatur und Feuchtigkeit von 22 ° C bzw. 40%. Um zirkadianen Rhythmus verwechseln zu vermeiden, führen Sie immer Experimente zur gleichen Tageszeit. </li></ol></li><li> Verschließen Sie die Oberseite des Zylinders mit einer Sperrfolie (Wachsfilm), um das Entweichen jeglicher Fliegen zu verhindern (Abbildung 2). </li><li> Richten Sie die Videokamera auf einem Stativ. Fokus-Kamera auf der Linie 190 ml der 250 ml-Messzylinder (17,5 cm). </li><li> Die Anzahl der toten Fliegen am Boden des Zylinders und in der Lebensmittelfläschchen. Tragen Sie diese Nummer als die Sterblichkeit. </li><li> Sehr leicht den Zylinder gegen einen geschlossenzelligen Schaumpolster wiederholt tippen mit genügend Kraft, um die Fliegen an der inneren Bodenfläche zu verdrängen. Tippen Sie auf 5 bis 10 mal, während mit der anderen Hand auf Rekord auf der Kamera drücken. </li><li> Drücken Sie die &quot;Record&quot; Taste an der Kamera. </li><li> Starten Sie das video Kameraaufnahme und auf den Zylinder, sechs Mal in einem ausgeprägten nicht-rhythmische Muster. </li><li> Leiten jeden Versuch für 2 min von der Zeit die Fliegen letzten abgegriffen und notieren Sie die Anzahl der Fliegen der Höhe von 17,5 cm (190 ml) zu jedem gewählten Zeitpunkt (zu quantifizieren alle 10 sec) überqueren. Hinweis: Das ml Markierung auf dem Zylinder wird von einem Zylinder-Modell zum anderen variieren je nach Durchmesser. Um Fehler zu vermeiden, messen Sie die Höhe an jedem Zylinder verwendet. </li><li> Sobald die Studie beendet ist, entsorgen Sie fliegt in 95% Ethanol. </li><li> Wiederholen Sie die Schritte 2,1-2,9, bis alle Wiederholungen wurden mit frischen Fliegen jedes Mal getestet. Hinweis: Obwohl 5 Wiederholungen können genug mit einer Mutation, die eine starke Wirkung auf die Fortbewegung ist, wird 10 biologischen Replikate von 20 Fliegen (200 entfernt) empfohlen, um kleinere Unterschiede zu erkennen. </li><li> Bei Beendigung des Versuchs, wäscht die Zylinder im Labor Spül- und Trocken O / N wiederverwendet werden. </li></ol> 3. Analyse <ol><li> Analyze Videos von jeder fliegen Studie. Jede 10 Sekunden, notieren Sie die Gesamtzahl der Fliegen, die die Ziellinie passieren. <ol><li> Wenn eine Fliege klettert wieder nach unten und fällt, Datensatz, der als -1 fliegen und zählen Sie die nächste Fliege, um die Ziellinie als die gleiche Anzahl wie die Fliege, die wieder nach unten stieg oder fiel überqueren. Zum Beispiel, wenn der 15. fly unter die Ziellinie, die nächste Fliege, die Linie (16. Fliegen) gilt als der 15. Fliege und nicht der 16. überqueren. </li></ol></li><li> Subtrahiere die Mortalität aus der Gesamtzahl von Fliegen (20), um die Anzahl der Fliegen, die in der Studie bleiben erhalten. Zu jedem Zeitpunkt, erhalten die Fraktion von Fliegen über der Ziellinie. </li><li> Zeichnen Sie jeden Prozentsatz zu jedem Zeitpunkt (siehe Abbildung 3). </li><li> Analysieren Sie die Leistung am 120 s Datenpunkt und führen Sie Student t-Test bei 2 Gruppen vorhanden sind oder ANOVA und einen Post-Test für multiple Vergleiche (mit Bonferroni Modifikation für geplante und ungeplante Tukey für Zusammenarbeitmparisons). Der Kolmogorov-Smirnov-Tests 17 wird auch durchgeführt, um Normalität und gleiche Varianz zu ermitteln, sondern auch, um die Verteilungen des Mutanten-Gruppe an die Steuerung zu vergleichen. </li><li> Um die Daten über Alterung zu präsentieren, zeichnen den Prozentsatz von Fliegen Klettern bei 120 sec mit Fliegen unterschiedlichen Alters (2 Tage, 1 Woche, 2 Wochen), um zu sehen, ob es eine fortschreitende Defizit (Abbildung 4). </li></ol>

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The authors have nothing to disclose.

Drosophila hat bereits bewiesen, dass ein ausgezeichnetes Modell der Parkinson-Krankheit 14 und andere neurodegenerative Erkrankungen 1,2 sein. Zusätzlich zu den genetischen Werkzeugen von Drosophila, wird dessen Genom sehr für Gene in neurologischen Erkrankungen beteiligten 19 konserviert. Das Aufkommen der genomweite genetische Screening-Methoden (einschließlich ganze Exoms Sequenzierung) wird sich wahrscheinlich fortsetzen, um eine größere Liste der Kandidatenge...

Verheerenden neurodegenerativen Erkrankungen wie Parkinson-Krankheit, amyotrophe Lateralsklerose und Spastische Paraplegie werden zunehmend anerkannt. Leider sind die meisten dieser neurodegenerativen Erkrankungen noch ohne Behandlungen. Die weit verbreitete klinische Anwendung von genomweiten, unvoreingenommene Gentests wie Voll Exom-Sequenzierung hat zu einer wachsenden Anzahl von Genen geführt, dass in der menschlichen Bewegungsapparates verwickelt. Trotz dieser Fortschritte die pathologische Progression von früh bis spät Bühnen, in weiter Ferne in dieser Erkrankungen. Drosophila stellt ein mit den genetischen Werkzeuge zum Studium Gen Anforderung in einer kontrollierten Art und Weise räumlich und zeitlich. Darüber hinaus hat Drosophila nützlich beim Screening Medikamenten für neurologische Erkrankungen wie der Parkinson-1, Alzheimer-2, geistiger Behinderung und Epilepsie 3,4 5,6 ua bewährt. Unser Ziel war es, eine kostengünstige Entwicklungund zuverlässigen Assay, der Analyse mit hohem Durchsatz, die noch ausreichend empfindlich gegenüber kleinen Änderungen in der Motorleistung zu detektieren würde ermöglichen würde. Es gibt verschiedene Tests verwendet, um die Effekte der genetischen Mutation und / oder Umgebungsbedingung auf Drosophila Kletterverhalten zu quantifizieren. Die meisten der Tests zu profitieren auf die natürliche Tendenz von Fliegen zu klettern, als negative Geotaxis oder Kletter Assay bekannt. Benzer 7 vorgeschlagen 1967, dass der Gegenstrom-Vorrichtung für die Untersuchung der Phototaxis verwendet könnte auch zur Gravitaxis untersuchen. Seitdem haben Ganetzki 8 und viele andere 9 -12 auf der ersten Test gebaut. Das Prinzip ist, um eine bekannte Anzahl der Fliegen in einem Fläschchen zu stellen und tippen Sie auf die Fläschchen kräftig gegen eine harte Oberfläche, wodurch die Fliegen an den Boden des Fläschchens fällt. Da es ein angeborenes Verhalten, werden die Fliegen versuchen, auf der Oberseite des Fläschchens, der Schwerkraft entgegengesetzt zu klettern. Dieser Test ist quantitativ und measures, wie viele Fliegen haben während einer vorgegebenen Zeit an einer Markierung auf dem Fläschchen kletterte. Messung der Geschwindigkeit anstatt der gesamten Anzahl der Fliegen Steigen zu einem verlässlichen Parameter und in Fällen, wo die Anzahl der Fliegen Kriterien nicht signifikant war 13 gezeigt Defekten. Die Steig Assay in der Studie von vielen neurodegenerativen Erkrankungen einschließlich Parkinson-Krankheit 14 bewährt. Allerdings haben wir festgestellt, dass Lok Mängel möglicherweise nicht nachweisbar sein zum Zeitpunkt, wo Neurodegeneration ist bereits in pathologischen Studien 14 zu sehen. Somit kann die Verwendung des herkömmlichen Assay die Fähigkeit, die frühen Stadien der Krankheit zu untersuchen Pathogenese begrenzen. Das Aussehen der Lokomotive Defekte während späteren Phasen der Pathologie kann eine Krankheit, deren Progression zu fortgeschritten für komplette Rettungs reflektieren. Dies wirft ein mögliches Problem mit der Empfindlichkeit des traditionellen Kletter Assay. Der potenzielle Unfähigkeit des tradinalen Klettern Assay zu mild Lokomotive Mängel erkennen kann, um die Höhe, auf die die Fliegen sind erforderlich, um zu klettern zurückzuführen. Die herkömmlichen Assay 15,16 misst die Anzahl der Fliegen, um erfolgreich über eine Höhe von 2 bis 5 cm steigen in 10 bis 20 Sekunden.

<table><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 Mängel von neurodegenerativen Erkrankungen resultiert, kann ein später Beginn Symptom der Krankheit, nach Jahren der subklinischen Degeneration und damit aktuelle therapeutische Behandlungsstrategien sind nicht kurativ. Durch die Verwendung von Voll Exoms Sequenzierung, eine zunehmende Anzahl von Genen identifiziert worden, eine Rolle in menschlichen Fortbewegung spielt. Trotz der Identifizierung dieser Gene, ist es nicht bekannt, wie diese Gene sind entscheidend für die normale Bewegungsfunktion. Daher ist ein zuverlässiger Test, der Modellorganismen verwendet, um die Rolle dieser Gene, um neue Targets von therapeutischem Interesse zu identifizieren aufzuklären, ist mehr denn je erforderlich. Wir haben eine sensibilisierte Version der negativen geotaxis Assay, der zum Nachweis von Mängeln milder früheren ermöglicht, und die Fähigkeit hat, diese Mängel über der Zeit erfasst gestaltet. Der Test wird in einem Glas durchgeführt Meßzylinder, der mit einem Wachsbarriere-Film abgedichtet ist. Durch die Erhöhung der Schwellenabstand um 17,5 c bestiegen werdenm und die Erhöhung der Versuchsdauer bis 2 Minuten haben wir eine größere Empfindlichkeit beim Nachweis von mild Mobilitätsstörungen beobachtet. Der Test wird kostengünstig und erfordert keine umfangreiche Ausbildung zu hoch reproduzierbare Ergebnisse zu erhalten. Dies macht es eine ausgezeichnete Technik für das Screening von Wirkstoffkandidaten in Drosophila-Mutanten mit Bewegungsfehler.

Klettern ist eine starke und reproduzierbare Verhalten. Tatsächlich erreichen einen Tag alt Wildtyp-Fliegen die Zielentfernung Kletterleistung schnell (25-30 sec). Mutierten Fliegen präsentieren ein Leistungsbereich von mild (oder verzögert) die Unfähigkeit, die dem Ziel klettern abzuschließen. Wir veranschaulichen dies hier mit zwei verschiedenen mutierten Allelen. Die erste ist eine schwere Allel des Gens Spastin durch eine vollständige Löschung des Spastin Gen (Spas 5,75) 18 verursacht. In dieser Ze...

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.

Quantitative Analyse der Kletter Defekte in einem Drosophila Modell von neurodegenerativen Erkrankungen

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

quantitative-analysis-climbing-defects-drosophila-model

Research

JoVE Journal

Neuroscience

18.5K Aufrufe.  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. 

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

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of these diseases, including Alzheimer's, Parkinson's and Huntington's disease are characterized by age-dependent deterioration in learning and memory functions and movement coordination2. Here we use behavioral assays, including the negative geotaxis assay3 and the aversive phototaxic suppression assay (APS assay)4,5, to show that some of the behavior characteristics associated with human neurodegeneration can be recapitulated in flies. In the negative geotaxis assay, the natural tendency of flies to move against gravity when agitated is utilized to study genes or conditions that may hinder locomotor capacities. In the APS assay, the learning and memory functions are tested in positively-phototactic flies trained to associate light with aversive bitter taste and hence avoid this otherwise natural tendency to move toward light. Testing these trained flies 6 hours post-training is used to assess memory functions. Using these assays, the contribution of any genetic or environmental factors toward developing neurodegeneration can be easily studied in flies.

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Behavior assays for measuring locomotor functions, learning, and memory abilities in Drosophila.

Assaying Bewegungsapparat, Lernen und Gedächtnis Defizite in Drosophila Modelle von Neurodegeneration

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of ...

Forschung

Neurowissenschaften

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic screening using chemical mutagens is a useful strategy for mapping mutant phenotypes to genes among Drosophila and other model organisms that share conserved cellular pathways with humans. If the mutated gene of interest is not lethal in early developmental stages of flies, a climbing assay can be conducted to screen for phenotypic indicators of decreased brain functioning, such as low climbing rates. Subsequently, secondary histological analysis of brain tissue can be performed in order to verify the neuroprotective function of the gene by scoring neurodegeneration phenotypes. Gene mapping strategies include meiotic and deficiency mapping that rely on these same assays can be followed by DNA sequencing to identify possible nucleotide changes in the gene of interest.

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

We present a protocol using a forward genetic approach to screen for mutants exhibiting neurodegeneration in Drosophila melanogaster. It incorporates a climbing assay, histology analysis, gene mapping and DNA sequencing to ultimately identify novel genes related to the process of neuroprotection.

In Vivo Forward Genetic Screen zur Identifizierung neuartiger neuroprotektive gene in Drosophila melanogaster

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic ...

Genetik

An Automated Rapid Iterative Negative Geotaxis Assay for Analyzing Adult Climbing Behavior in a Drosophila Model of Neurodegeneration

Neurodegenerative diseases are frequently associated with a progressive loss of movement ability, reduced life span, and age-dependent neurodegeneration. To understand the mechanism of these cellular events, and their causal relationships with each other, Drosophila melanogaster, with its sophisticated genetic tools and diverse behavioral features, are used as disease models for assessing neurodegenerative phenotypes. Here we describe a high-throughput method to analyze Drosophila adult negative geotaxis behavior, as an indication for possible motor defects associated with neurodegeneration. An automated machine is designed and developed to drive fly synchronization using an initial electric impulse, later allowing the recording of negative geotaxis behavior over a course of secs to mins. Images from the digitally recorded video are then processed with the self-designed RflyDetection software for statistical data manipulation. Different from the manually controlled negative geotaxis assay based on single fly, this precise, fast, and high-throughput protocol allows data acquisition from more than hundreds of flies simultaneously, providing an efficient approach to advance our understanding in the underlying mechanism of locomotor deficits associated with neurodegeneration.

An Automated Rapid Iterative Negative Geotaxis Assay for Analyzing Adult Climbing Behavior in a Drosophila Model of Neurodegeneration

This step-by-step protocol analyzes Drosophila negative geotaxis behavior using an automated multi-cylinder system that hosts hundreds of flies and synchronizes their action by an electric motor. Upon synchronization, fly negative geotaxis behavior is assayed, digitally recorded, and analyzed using the self-designed RflyDetection software.

Eine automatisierte rasche Iterative Negative Geotaxis Assay für die Analyse von Erwachsenen Klettern Verhalten in einem Drosophila -Modell der Neurodegeneration

Neurodegenerative diseases are frequently associated with a progressive loss of movement ability, reduced life span, and age-dependent neurodegeneration. ...

Verhalten

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.

Beurteilung Neurodegenerative Phänotypen in<em&gt; Drosophila</em&gt; Dopaminergen Neuronen durch Klettern Assays und 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 ...

A Simple Technique to Assay Locomotor Activity in Drosophila

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and diverse behavioral features. Identifying behavioral deficiency in animal models is a crucial measure of disease severity, for example, in neurodegenerative diseases where patients often experience impairments in motor function. However, with the availability of various systems to track and assess motor deficits in fly models, such as drug-treated or transgenic individuals, an economical and user-friendly system for precise evaluation from multiple angles is still lacking. A method based on the AnimalTracker application programming interface (API) is developed here, which is compatible with the Fiji image processing program, to systematically evaluate the movement activities of both adult and larval individuals from recorded video, thus allowing for the analysis of their tracking behavior. This method requires only a high-definition camera and a computer peripheral hardware integration to record and analyze behavior, making it an affordable and effective approach for screening fly models with transgenic or environmental behavioral deficiencies. Examples of behavioral tests using pharmacologically treated flies are given to show how the techniques can detect behavioral changes in both adult flies and larvae in a highly repeatable manner.

A Simple Technique to Assay Locomotor Activity in Drosophila

The present protocol assesses the locomotor activity of Drosophila by tracking and analyzing the movement of flies in a hand-made arena using open-source software Fiji, compatible with plugins to segment pixels of each frame based on high-definition video recording to calculate parameters of speed, distance, etc.

Eine einfache Technik zur Bestimmung der lokomotorischen Aktivität in Drosophila

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and ...

Mass Histology to Quantify Neurodegeneration in Drosophila

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health worldwide. Although mammalian models have provided important insights into the underlying mechanisms of pathogenicity, the complexity of mammalian systems together with their high costs are limiting their use. Therefore, the simple but well-established Drosophila model-system&#160;provides an alternative for investigating the molecular pathways that are affected in these diseases. Besides behavioral deficits, neurodegenerative diseases are characterized by histological phenotypes such as neuronal death and axonopathy. To quantify neuronal degeneration and to determine how it is affected by genetic and environmental factors, we use a histological approach that is based on measuring the vacuoles in adult fly brains. To minimize the effects of systematic error and to directly compare sections from control and experimental flies in one preparation, we use the &#39;collar&#39; method for paraffin sections. Neurodegeneration is then assessed by measuring the size and/or number of vacuoles that have developed in the fly brain. This can either be done by focusing on a specific region of interest or by analyzing the entire brain by obtaining serial sections that span the complete head. Therefore, this method allows one to measure not only severe degeneration but also relatively mild phenotypes that are only detectable in a few sections, as occurs during normal aging.

Mass Histology to Quantify Neurodegeneration in Drosophila

Drosophila is widely used as a model system to study neurodegeneration. This protocol describes a method by which degeneration, as determined by vacuole formation in the brain, can be quantified. It also minimizes effects due to the experimental procedure by processing and sectioning control and experimental flies as one sample.

Massen Histologie zu Quantifizieren Neurodegenerative Erkrankungen in<em&gt; Drosophila</em

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health ...

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.

Quantitative Analyse der neuronalen dendritischen Arborization Komplexität in Drosophila

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.

Quantitative Zellbiologie der Neurodegeneration in Drosophila durch unvoreingenommene Analyse der Gewebekulturen Tagged Proteine mit ImageJ

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

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

Reinigung von Transcripts und Metaboliten aus<em&gt; Drosophila</em&gt; Köpfe

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

Biologie

Visualizing Synaptic Degeneration in Adult Drosophila in Association with Neurodegeneration

Drosophila serves as a useful model for assessing synaptic structure and function associated with neurodegenerative diseases. While much work has focused on neuromuscular junctions (NMJs) in Drosophila larvae, assessing synaptic integrity in adult Drosophila has received much less attention. Here we provide a straightforward method for dissection of the dorsal longitudinal muscles (DLMs), which are required for flight ability. In addition to flight as a behavioral readout, this dissection allows for the both DLM synapses and muscle tissue to be amenable to structural analysis using fluorescently labeled antibodies for synaptic markers or proteins of interest. This protocol allows for the evaluation of the structural integrity of synapses in adult Drosophila during aging to model the progressive, age-dependent nature of most neurodegenerative diseases.

Visualizing Synaptic Degeneration in Adult Drosophila in Association with Neurodegeneration

The goal of this procedure is to dissect the dorsal longitudinal muscle (DLM) tissue to assess the structural integrity of DLM neuromuscular junctions (NMJs) in neurodegenerative disease models using Drosophila melanogaster.

Visualisierung der synaptischen Degeneration bei erwachsenen Drosophila in Verbindung mit Neurodegeneration

Drosophila serves as a useful model for assessing synaptic structure and function associated with neurodegenerative diseases. While much work has focused ...

Quantitative Analyse der Kletter Defekte in einem Drosophila Modell von neurodegenerativen Erkrankungen

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