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.

La investigación sobre Drosophila melanogaster se encontraba en cumplimiento con la Universidad de las pautas de investigación de Alberta. 1. Fly Colección <ol><li> Suma 20 moscas utilizando CO 2 (g) anestesia y colocar en un vial de recogida 25 mm x 95 mm que contiene alimentos. </li><li> Tienda viales que contienen moscas horizontalmente para evitar atrapar moscas en cualquier líquido que pueda acumularse en la parte inferior del vial. </li><li> Incubar las moscas durante al menos 21 horas a 22 ° C a 45% de humedad en una incubadora durante aproximadamente durante 15 horas. Establezca la incubadora con una luz de 12 horas: oscuridad ciclo. </li></ol> Ensayo 2. Escalada <ol><li> A la mañana siguiente, la transferencia de 20 moscas de un solo vial en un vaso de 250 ml cilindro graduado. Marque la posición del cilindro para mantenerla constante todos los días. Utilice un cilindro de vidrio por genotipo para evitar la contaminación cruzada entre los genotipos. Lavar al final de cada experimento y roTate los entre los genotipos. <ol><li> Llevar a cabo los experimentos en la luz ambiental (o luz roja si hay un posible defecto en la visión) a la temperatura y la humedad de 22 ° C y 40% respectivamente. Para evitar confundir ritmo circadiano, siempre realizar experimentos en el mismo momento del día. </li></ol></li><li> Selle la parte superior del cilindro con una película de barrera (película de cera) para evitar cualquier escape de las moscas (Figura 2). </li><li> Configure la cámara de vídeo en un trípode. Cámara Enfoque en la línea de 190 ml de los 250 ml cilindro graduado (17,5 cm). </li><li> Contar el número de moscas muertas en la parte inferior del cilindro y en los viales de alimentos. Anote este número como la mortalidad. </li><li> Muy golpee suavemente el cilindro contra una almohadilla de espuma de célula cerrada en varias ocasiones con la fuerza suficiente para desplazar a las moscas a la superficie inferior interna. Toque 5-10 veces, mientras con la otra mano para presionar registro en la cámara. </li><li> Pulse el botón &quot;Grabar&quot; en la cámara. </li><li> Inicie la video grabación de la cámara y toque el cilindro de seis veces en un patrón no-rítmica distinta. </li><li> Llevar a cabo cada ensayo durante 2 minutos desde el momento en que las moscas están última intervenidos y registre el número de moscas que cruzan la altura de 17,5 cm (190 ml) en cada punto de tiempo elegido (cuantificar cada 10 seg). Nota: La ml marcado en el cilindro variará de un modelo de cilindro a otro dependiendo de diámetro. Para evitar errores, medir la altura de cada cilindro utilizado. </li><li> Una vez que el juicio ha terminado, a disponer de moscas en etanol al 95%. </li><li> Repita los pasos 2.1 a 2.9 hasta que todas las réplicas se han probado con moscas frescos en todo momento. Nota: Aunque 5 repeticiones pueden ser lo suficientemente con una mutación que tiene un fuerte efecto sobre la locomoción, 10 réplicas biológicas de 20 moscas (moscas) 200 se recomienda para detectar diferencias más pequeñas. </li><li> Al finalizar el experimento, lavar los cilindros en el lavavajillas de laboratorio y O seco N para ser reutilizado /. </li></ol> 3. Análisis <ol><li> Analyvídeos ze de cada vuelan juicio. Cada 10 segundos, registra el número total de moscas que pasan la línea de meta. <ol><li> Si una mosca sube de nuevo hacia abajo o se cae, ficha que vuelan como -1 y contar la próxima marcha para cruzar la línea de meta como el mismo número que la mosca que subió de nuevo hacia abajo o cayó. Por ejemplo, si el 15 de mosca cae por debajo de la línea de meta, la próxima marcha para cruzar la línea (los 16 º vuelan) se considera la marcha 15 y no el 16. </li></ol></li><li> Reste la mortalidad por el número total de moscas (20) para obtener el número de moscas que permanecen en el juicio. En cada punto de tiempo, obtener la fracción de moscas por encima de la línea de meta. </li><li> Sinopsis cada porcentaje en cada momento (ver Figura 3). </li><li> Analizar el rendimiento en el punto de datos 120 seg y realice estudiante t-test cuando 2 grupos están presentes o ANOVA y un post-test para comparaciones múltiples (con modificación de Bonferroni para planificarse y Tukey para co no planificadomparisons). La prueba de Kolmogorov-Smirnov 17 también se lleva a cabo para determinar la normalidad y la igualdad de varianza, sino también para comparar las distribuciones del grupo mutante con el control. </li><li> Para presentar los datos sobre el envejecimiento, trazar el porcentaje de moscas subiendo a 120 seg con moscas de diferentes edades (2 días, 1 semana, 2 semanas) para ver si hay un déficit progresivo (Figura 4). </li></ol>

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The authors have nothing to disclose.

Drosophila ya ha demostrado ser un excelente modelo en la enfermedad 14 y otras condiciones neurodegenerativas 1,2 de Parkinson. Además de las herramientas genéticas disponibles en Drosophila, su genoma está altamente conservada de genes implicados en los trastornos neurológicos 19. El advenimiento de los métodos de detección genética del genoma de ancho (incluyendo la secuenciación del exoma) es probable que continúe para proporcionar una lista más grande de ...

Trastornos neurodegenerativos devastadores como la enfermedad de Parkinson, la esclerosis lateral amiotrófica, y la paraplejía espástica hereditaria se reconocen cada vez más. Desafortunadamente, la mayoría de estos trastornos neurodegenerativos siguen sin tratamientos. El uso clínico generalizado de las pruebas genéticas, imparciales en todo el genoma como la secuenciación del exoma conjunto ha llevado a un creciente número de genes están implicados en los trastornos locomotores humanos. A pesar de este progreso, la progresión patológica desde temprano a las etapas finales de los años, sigue siendo difícil de alcanzar en estos trastornos. Drosophila ofrece uno con las herramientas genéticas para el estudio de requerimiento de genes de una manera espacial y temporal controlada. Además, Drosophila ha demostrado ser útil en el cribado de fármacos para enfermedades neurológicas como el Parkinson 1, 2 Alzheimer, discapacidad intelectual y epilepsia 3,4 5,6 entre otros. Nuestro objetivo era desarrollar un rentabley ensayo fiable que permita análisis de alto rendimiento que todavía sería lo suficientemente sensible para detectar pequeños cambios en el rendimiento del motor. Hay varios ensayos utilizados para cuantificar los efectos de la mutación genética y / o condición ambiental en el comportamiento de escalada Drosophila. La mayoría de los ensayos de sacar provecho de la tendencia natural de las moscas para subir, conocidos como geotaxis negativos, o el ensayo de escalada. Benzer 7 sugirió en 1967 que el aparato de contracorriente utilizado para el estudio de phototaxis también podría ser utilizado para estudiar gravitaxis. Desde entonces, Ganetsky 8 y muchos otros 9 -12 se han basado en el ensayo inicial. El principio es colocar un número conocido de moscas en un vial y toque el vial firmemente contra una superficie dura, haciendo que las moscas que caen al fondo del vial. Como se trata de un comportamiento innato, las moscas se intentan subir a la parte superior del vial, se opuso a la gravedad. Este ensayo es cuantitativo y measures cuántas moscas han subido más allá de un marcador en el vial durante un período de tiempo asignado. Medida de la velocidad en lugar del número total de moscas escalada se ha convertido en un parámetro fiable y se muestra defectos en los casos en que el número de criterios moscas no fue significativa 13. El ensayo de escalada ha demostrado ser útil en el estudio de muchos trastornos neurodegenerativos incluyendo la enfermedad de Parkinson 14. Sin embargo, hemos observado que los defectos de locomotoras pueden no ser detectables en el momento en que la neurodegeneración ya se ve en los estudios patológicos 14. Así, el uso del ensayo tradicional puede limitar la capacidad de estudiar las primeras etapas de la patogénesis de la enfermedad. La aparición de defectos de locomotoras durante las etapas posteriores de la patología puede reflejar una enfermedad cuya progresión es demasiado avanzado para el rescate completo. Esto plantea un problema potencial con la sensibilidad del ensayo tradicional de escalada. La incapacidad potencial de la tradicióncional escalada ensayo para detectar defectos leves de la locomotora se puede atribuir a la altura a la que se requieren las moscas a subir. Los tradicionales ensayo de 15,16 mide el número de moscas que subir con éxito más de una altura de 2 a 5 cm de 10 a 20 seg.

<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

Defectos locomotores resultantes de trastornos neurodegenerativos pueden ser un síntoma de aparición tardía de la enfermedad, después de años de degeneración subclínica, y por lo tanto las estrategias actuales de tratamiento terapéutico no son curativos. Mediante el uso de toda la secuenciación del exoma, un número creciente de genes han sido identificados para jugar un papel en la locomoción humana. A pesar de la identificación de estos genes, no se sabe cómo estos genes son cruciales para el funcionamiento normal locomotora. Por lo tanto, un ensayo fiable, que utiliza organismos modelo para dilucidar el papel de estos genes con el fin de identificar nuevas dianas de interés terapéutico, se necesita más que nunca. Hemos diseñado una versión sensibilizada del ensayo geotaxis negativas que permite la detección de defectos más leves anteriormente y tiene la capacidad para evaluar estos defectos en el tiempo. El ensayo se realiza en un cilindro de vidrio, que se sella con una película de barrera de cera se graduó. Al aumentar la distancia de umbral para ser subido a 17,5 cm y el aumento de la duración de la prueba a 2 min hemos observado una mayor sensibilidad en la detección de disfunciones de movilidad suaves. El ensayo es rentable y no requiere de una amplia formación para obtener resultados altamente reproducibles. Esto hace que sea una excelente técnica para el cribado de fármacos candidatos en los mutantes de Drosophila con defectos de locomoción.

La escalada es un comportamiento fuerte y reproducible. De hecho, un día de edad de tipo salvaje moscas alcanzan el rendimiento escalada distancia objetivo rápidamente (25-30 seg). Moscas mutantes presentan una gama de rendimiento de leves (o retardada) para completar la imposibilidad de subir a la meta. Ilustramos esta aquí con dos alelos mutantes diferentes. El primero es un alelo severa de la Spastin gen causado por una deleción completa del gen Spastin (balnearios 5,75) 18. En esta línea (balnearios ...

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.

Análisis cuantitativo de los Defectos de escalada en un modelo de Drosophila de enfermedades neurodegenerativas

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

18.5K vistas.  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. 

Video: 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.

Locomotor ensayo, aprendizaje, memoria y déficit en Drosophila Los modelos de neurodegeneración

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

Investigación

Neurociencias

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.

En Vivo Forward Genetic Screen para Identificar Nuevos Genes Neuroprotectores en Drosophila melanogaster

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

Genética

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.

Un ensayo Geotaxis negativa iterativo rápido automatizado para analizar comportamiento escalar adulto en un modelo de Drosophila de la neurodegeneración

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

Comportamiento

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.

La evaluación de fenotipos neurodegenerativas en<em&gt; Drosophila</em&gt; Las neuronas dopaminérgicas mediante ensayos de Escalada y inmunotinción de todo el cerebro

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.

Una técnica simple para evaluar la actividad locomotora en Drosophila

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

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic pathways. Quantitative descriptions are rarely performed, although genetic manipulations produce a range of phenotypic effects and variations are observed even among individuals within control groups.&#160;Emerging evidence shows that morphology, size and location of organelles play a previously underappreciated, yet fundamental role in cell function and survival. Here we provide step-by-step instructions for performing quantitative analyses of phenotypes at the Drosophila larval neuromuscular junction (NMJ). We use several reliable immuno-histochemical markers combined with bio-imaging techniques and morphometric analyses to examine the effects of genetic mutations on specific cellular processes. In particular, we focus on the quantitative analysis of phenotypes affecting morphology, size and position of nuclei within the striated muscles of Drosophila larvae. The Drosophila larval NMJ is a valuable experimental model to investigate the molecular mechanisms underlying the structure and the function of the neuromuscular system, both in health and disease. However, the methodologies we describe here can be extended to other systems as well.

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Morphology, size and location of intracellular organelles are evolutionarily conserved and appear to directly affect their function. Understanding the molecular mechanisms underlying these processes has become an important goal of modern biology. Here we show how these studies can be facilitated by the application of quantitative techniques.

¿Por qué importa la cuantificación: Caracterización de fenotipos en el<em&gt; Drosophila</em&gt; Larval unión neuromuscular

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

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.

La histología de masas para cuantificar la neurodegeneración en<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.

Análisis cuantitativo de la complejidad de la arborización dendrítica Neuronal en 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.

Biología celular cuantitativa de neurodegeneración en Drosophila mediante análisis imparcial de fluorescencia etiquetas proteínas con ImageJ

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

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.

Visualización de la degeneración sináptica en Drosophila adulta en asociación con neurodegeneración

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

Análisis cuantitativo de los Defectos de escalada en un modelo de Drosophila de enfermedades neurodegenerativas

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