Los autores no tienen nada que revelar.

1. Montaje del Dispositivo de Huelga ( Figura 1 ) <ol><li> Retire el émbolo de una jeringa de tuberculina de 1 ml. Corte el barril en la marca de 1 mL. </li><li> Retire una barrera de aerosol (3 mm de altura x 4 mm de diámetro) de una punta de pipeta de 200 μL y úsela como un impactador. Coloque el impactador dentro del cilindro de la jeringa. Golpee suavemente el cañón para mover el impactador al extremo de la punta, con el lado plano que cubre la abertura de la boquilla. </li><li> Conecte el extremo de la punta del cañón a un tubo de plástico que esté conectado al regulador de flujo de dióxido de carbono (CO 2 ) de una estación de anestesia Drosophila . </li><li> Sujete el cañón verticalmente y sujételo a un soporte de sujeción estándar para que el impactador permanezca en la parte inferior del cañón. </li><li> Modifique una punta de pipeta de 200 μL para hacer el soporte de la mosca. <ol><li> Corte 4 mm de la punta para hacer una abertura de 0.8 mm de diámetro, permitiendo que sólo la cabeza de la mosca para ser expuestos. NOTA: El thoRax y todas las otras partes del cuerpo de la mosca se mantendrá dentro de la punta de la pipeta. </li></ol></li><li> Modifique una punta de pipeta de 1000 μL y una tapa de aguja de 1 ml para hacer el conector. <ol><li> Cortar 44 mm de la abertura de la punta. Tome una longitud de 6 mm de una tapa de la aguja de la jeringa de 1 ml y empuje firmemente en el segmento restante de la punta de la pipeta de 1000 μL. </li></ol></li></ol> 2. Funcionamiento del dispositivo Strike <ol><li> Anestesiar una sola hembra de 2 días de edad que usa CO 2 en una almohadilla de mosca. </li><li> Suavemente transferirlo al sostenedor de la mosca usando un cepillo fino. Toque suavemente el soporte para que el cabezal volador se ve fuera del extremo de la punta. Si la probóscide de la mosca está expuesta fuera de la punta, suavemente doblarla de nuevo dentro de la punta con una aguja de jeringa de 1 ml embotada. NOTA: Asegúrese de mantener la probóscide mosca en el interior del soporte. De lo contrario, la mosca puede morir de una lesión de probóscide de succión. </li><li> Apriete la mosca hoHacia el cilindro de la jeringa con el conector de manera que el cabezal volador esté hacia abajo. </li><li> Ajuste la presión del gas a 100 kPa. Ajuste el caudal según el diseño del experimento. </li><li> Encienda y apague rápidamente el interruptor del interruptor del regulador de flujo para que el impactador golpee la cabeza de la mosca una vez. </li><li> Levante el porta mosquitera y muévalo sobre una almohadilla. Invertir el sostenedor de la mosca y golpee suavemente el lado para dejar la mosca para fuera. Dejar la mosca en un frasco vacío para recuperarse. </li></ol> 3. Seguimiento de Movimiento Video-asistido <ol><li> Llenar una placa de Petri de 6 cm de diámetro con elastómero de silicio transparente para hacer la arena de seguimiento. Deje un espacio de 3 mm entre el silicio y la tapa del plato para permitir que las moscas caminen libremente pero no vuelen. </li><li> Anestesiar cuatro moscas de la farsa o grupo tratado cada vez y colocarlos en la arena. Dejar las moscas a 22 ° C durante 1 h. </li><li> Coloque una cámara de dispositivo de carga acoplada (CCD) por encima de las arenas y grabe durante 5 min.</li><li> Analizar las trayectorias de movimiento registradas utilizando el software Ctrax (disponible gratuitamente en Caltech) 16 . Exportar los datos de seguimiento en un formato de programación (por ejemplo, Matlab) y analizar los datos basados ​​en la distancia recorrida por cada cuadro 17 . Calcular la distancia media de marcha de cada mosca y combinarla con todas las demás moscas registradas / grupo para obtener una distancia media acumulada recorrida por la población de archivos en el mismo grupo. </li></ol>

<ol>
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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+traumatic+encephalopathy:+where+are+we+and+where+are+we+going?.">Chronic traumatic encephalopathy: where are we and where are we going?.</a> Curr Neurol Neurosci Rep. 13 (12), 407 (2013).</li><li>McKee, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+spectrum+of+disease+in+chronic+traumatic+encephalopathy.">The spectrum of disease in chronic traumatic encephalopathy.</a> Brain. 136 (Pt 1), 43-64 (2013).</li><li>Petraglia, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+spectrum+of+neurobehavioral+sequelae+after+repetitive+mild+traumatic+brain+injury:+a+novel+mouse+model+of+chronic+traumatic+encephalopathy.">The spectrum of neurobehavioral sequelae after repetitive mild traumatic brain injury: a novel mouse model of chronic traumatic encephalopathy.</a> J Neurotrauma. 31 (13), 1211-1224 (2014).</li><li>Hirth, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+melanogaster+in+the+study+of+human+neurodegeneration.">Drosophila melanogaster in the study of human neurodegeneration.</a> CNS Neurol Disord Drug Targets. 9 (4), 504-523 (2010).</li><li>Littleton, J. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Method+to+Inflict+Closed+Head+Traumatic+Brain+Injury+in+Drosophila.">A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila.</a> J Vis Exp. (e52905), (2015).</li><li>Branson, K., Robie, A. A., Bender, J., Perona, P., Dickinson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+ethomics+in+large+groups+of+Drosophila.">High-throughput ethomics in large groups of Drosophila.</a> Nat Methods. 6 (6), 451-457 (2009).</li><li>Straw, A. D., Dickinson, M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motmot,+an+open-source+toolkit+for+realtime+video+acquisition+and+analysis.">Motmot, an open-source toolkit for realtime video acquisition and analysis.</a> Source Code Biol Med. 4 (5), 1-10 (2009).</li><li>Talavage, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functionally-detected+cognitive+impairment+in+high+school+football+players+without+clinically-diagnosed+concussion.">Functionally-detected cognitive impairment in high school football players without clinically-diagnosed concussion.</a> J Neurotrauma. 31 (4), 327-338 (2014).</li><li>Theadom, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequency+and+impact+of+recurrent+traumatic+brain+injury+in+a+population-based+sample.">Frequency and impact of recurrent traumatic brain injury in a population-based sample.</a> J Neurotrauma. 32 (10), 674-681 (2015).</li><li>Drobysheva, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+method+for+histological+detection+of+dopaminergic+neurons+in+Drosophila+melanogaster.">An optimized method for histological detection of dopaminergic neurons in Drosophila melanogaster.</a> J Histochem Cytochem. 56 (12), 1049-1063 (2008).</li></ol>

Este trabajo fue apoyado por la Facultad de Medicina de la Universidad Johns Hopkins fondo de inicio de facultad a LC

Los modelos animales que modelan fielmente las características del CTE, incluidas las alteraciones neurofisiológicas, los signos neuropatológicos y los déficits neuroconductuales, son esenciales para descubrir los mecanismos de la enfermedad y para desarrollar objetivos diagnósticos y terapéuticos. Es comprensible que ningún modelo animal de una enfermedad humana sea perfecto para imitar todos los puntos finales clínicamente relevantes. Sin embargo, creemos que un modelo de CTE robusto debe satisfacer los siguie...

La encefalopatía traumática crónica (CTE) ha sido reconocida recientemente como un trastorno neurodegenerativo distinto, separado de otras tauopatías como la enfermedad de Alzheimer 1 . A diferencia de la enfermedad de Alzheimer y otras tauopatías comunes-cuyos factores de riesgo más importantes son la edad avanzada y una historia familiar de demencia, CTE, como se indica por su nombre, implica una estrecha asociación con una historia de trauma cerebral, Como boxeadores y futbolistas, así como en veteranos militares 2 , 3 , 4 , 5 . Se cree que es iniciado por repetidos golpes concussive y subconcussive a la cabeza. Los pacientes pueden presentar síntomas y signos tales como déficits cognitivos, cambios de humor y comportamiento, y disfunción del movimiento, que se superponen significativamente con la enfermedad de Alzheimer, frontotemporalDemencia, demencia de cuerpos de Lewy y enfermedad de Parkinson 6 . En cambio, los exámenes post-mortem del tejido cerebral revelan un patrón distinto de acumulación de tau hiperfosforilada que rodea pequeños vasos sanguíneos en las profundidades de los surcos corticales, característica patognomónica que no se observa en otras condiciones degenerativas 7 . Sin embargo, hasta ahora, se sabe muy poco acerca de la patogénesis que conduce a la manifestación de la enfermedad. Esto se debe en gran parte a la falta de un modelo animal fiel - sólo recientemente se han generado modelos de roedores 5 , 8 . Estos organismos modelo tienen las desventajas de la atención costo-intensiva y una vida relativamente larga, que no son bien-adaptados para los estudios de la enfermedad neurodegenerative. En comparación con los homólogos de mamíferos, los animales invertebrados como Drosophila son una excelente alternativa, con su mantenimiento rentable,Extensas herramientas para diseccionar determinantes genéticos y una vida relativamente corta 9 . Sorprendentemente, la mosca y el cerebro humano comparten las vías moleculares y celulares evolutivamente conservadas, así como las similitudes anatómicas 10 , 11 , 12 . Dos ingeniosos modelos de Drosophila para estudiar la lesión cerebral traumática han sido reportados previamente 13 , 14 . El primer dispositivo &quot;High Impact Trauma&quot; (HIT) diseñado por Katzenberger y sus colegas contenía moscas de movimiento libre en un vial de plástico que estaba atado al extremo libre de un resorte metálico 13 , 15 . Cuando el vial de plástico se inclinó verticalmente y se soltó, golpeó una almohadilla de poliuretano e impartió un traumatismo a las moscas cuando rebotaron en la pared del vial y se recuperaron. En contraste, Barekat y sus colegas diseñaron un método de entrega diferenteLa plataforma de homogeneización Omni Bead Ruptor-24 14 . Las moscas fueron incapacitadas con CO 2 y colocadas en un tubo de tornillo de 2 mL que se aseguró al homogeneizador y se sometió a condiciones de agitación preprogramadas. Un beneficio de usar el sistema homogeneizador de tejidos es que el experimentador podría modular la intensidad de la lesión, la duración de la lesión y el número de episodios de lesiones. Sin embargo, ambos regímenes sufren el mismo inconveniente: las lesiones primarias en la cabeza se infligen aleatoriamente en términos de localización y fuerza del impacto. Además, ambos métodos dieron como resultado una mortalidad considerable, causada por daños colaterales inevitables a otras partes del cuerpo y órganos internos. Aquí, describimos un nuevo método para inducir mTBI en las moscas de la fruta. Nuestro aparato consiste en un impactador balístico propulsado por gas. En comparación con los modelos 14 , 15 de Drosophila existentes, nuestro método tiene la ventaja, Dirigido solamente al cabezal de mosca libre, permitiendo así el control preciso de varios factores, tales como la severidad del impacto, el intervalo de tiempo entre impactos y el número total de impactos sostenidos.

<table><tbody><tr><td>Aerosol Barrier</td><td>USA Scientific</td><td>1120-8810</td><td>Used as an impactor</td></tr><tr><td>200 &amp;#956;L Pipette Tip</td><td>USA Scientific</td><td>1111-0706</td><td>Used as a fly head holder</td></tr><tr><td>1000 &amp;#956;L Pipette Tip</td><td>USA Scientific</td><td>1122-1830</td><td>Used as a connector</td></tr><tr><td>1 mL Tuberculin Syringe</td><td>Becton Dickinson</td><td>309625</td><td></td></tr><tr><td>60 mm Petri Dishes</td><td>Fisher Scientific</td><td>FB0875713A</td><td>Used as a tracking arenas</td></tr><tr><td>Flow Regulator</td><td>Genesee Scientific</td><td>59-122WC</td><td></td></tr><tr><td>Standard Clamp Holder/stand</td><td>EISCO Scientific</td><td>CH0688</td><td></td></tr><tr><td>Fine Brush</td><td>Genesee Scientific</td><td>59-204</td><td></td></tr><tr><td>Flypad</td><td>Genesee Scientific</td><td>59-114</td><td></td></tr><tr><td>Sylgard Silicone Elastomer</td><td>Dow Corning</td><td>4019862</td><td></td></tr><tr><td>CCD Camera</td><td>Microsoft&amp;nbsp;</td><td>HD-5000</td><td></td></tr><tr><td>Ctrax Walking Fly Tracker</td><td>Caltech</td><td>Ctrax 0.2.11</td><td></td></tr><tr><td>MATLAB Image Processing Toolbox</td><td>MATLAB</td><td>R2015b</td><td></td></tr></tbody></table>

a novel method to model chronic traumatic encephalopathy in drosophila

La encefalopatía traumática crónica (CTE) es una enfermedad neurodegenerativa establecida que está estrechamente asociada con la exposición a lesiones traumáticas cerebrales leves y repetitivas (mTBI). Los mecanismos responsables de sus complejos cambios patológicos siguen siendo en gran medida evasivos, a pesar de un reciente consenso para definir los criterios neuropatológicos. En este trabajo se describe un método novedoso para desarrollar un modelo de CTE en Drosophila melanogaster ( Drosophila ) en un intento de identificar los genes clave y las vías que conducen a la acumulación de tau hiperfosforilada característica y la muerte neuronal en el cerebro. Los impactos de fuerza ajustable para infligir heridas cerradas leves se entregan directamente a la cabeza de mosca, sometiendo la cabeza a aceleración y deceleración rápidas. Nuestro método elimina los problemas potenciales inherentes con otros modelos de mTBI de Drosophila ( por ejemplo, la muerte animal podría ser inducida por daño aOtras partes del cuerpo oa los órganos internos). Cuanto menor sea el trabajo y el costo de cuidados intensivos de los animales, la vida corta y herramientas genéticas extensas hacen la mosca de la fruta ideal para estudiar la patogénesis CTE y que sea posible realizar a gran escala, genoma amplia genética y pantallas farmacológicas. Anticipamos que la caracterización en curso del modelo generará ideas mecanicistas importantes sobre la prevención de enfermedades y enfoques terapéuticos.

Para establecer un modelo de CTE utilizando Drosophila para adultos, determinamos la eficacia de nuestro dispositivo al infligir una sola lesión de cabeza cerrada. Para eliminar las variaciones relacionadas con el genotipo, el sexo o la edad, se utilizaron 2 días de edad Canton-S WT moscas hembra en el experimento. Podríamos controlar fácilmente la resistencia del impactador regulando el caudal de CO 2 a una presión de gas constante de 100 kPa. Las moscas expuest...

Watch this Scientific Journal Video about A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila at JoVE.com

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

Aquí, describimos un nuevo enfoque para infligir la lesión cerebral traumática de cabeza cerrada en Drosophila melanogaster . Nuestro método tiene la ventaja de entregar directamente impactos repetitivos con fuerza ajustable a la cabeza sola. Una mayor exploración del sistema de invertebrados ayudará a iluminar la patogénesis de la encefalopatía traumática crónica.

Un nuevo método para modelar la encefalopatía traumática crónica<em&gt; Drosophila</em

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

a-novel-method-to-model-chronic-traumatic-encephalopathy-in-drosophila

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

8.2K vistas.  Johns Hopkins University School of Medicine.  Aquí, describimos un nuevo enfoque para infligir la lesión cerebral traumática de cabeza cerrada en Drosophila melanogaster . Nuestro método tiene la ventaja de entregar directamente impactos repetitivos con fuerza ajustable a la cabeza sola. Una mayor exploración del sistema de invertebrados ayudará a iluminar la patogénesis de la encefalopatía traumática crónica. 

Video: A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

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

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Disección y tinción inmunofluorescente de seta del cuerpo y las neuronas fotorreceptoras en cerebros de adultos de Drosophila melanogaster

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Biología del Desarrollo

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 ...

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

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and thus current therapeutic treatment strategies are not curative. Through the use of whole exome sequencing, an increasing number of genes have been identified to play a role in human locomotion. Despite identifying these genes, it is not known how these genes are crucial to normal locomotive functioning. Therefore, a reliable assay, which utilizes model organisms to elucidate the role of these genes in order to identify novel targets of therapeutic interest, is needed more than ever. We have designed a sensitized version of the negative geotaxis assay that allows for the detection of milder defects earlier and has the ability to evaluate these defects over time. The assay is performed in a glass graduated cylinder, which is sealed with a wax barrier film. By increasing the threshold distance to be climbed to 17.5 cm and increasing the experiment duration to 2 min&#160;we have observed a greater sensitivity in detecting mild mobility dysfunctions. The assay is cost effective and does not require extensive training to obtain highly reproducible results. This makes it an excellent technique for screening candidate drugs in Drosophila mutants with locomotion defects.

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 ...

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies using Drosophila have contributed important breakthroughs in understanding neurological processes. Thus, with the goal of understanding the cellular and molecular basis of TBI pathologies in humans, we developed the High Impact Trauma (HIT) device to inflict closed head TBI in flies. Flies subjected to the HIT device display phenotypes consistent with human TBI such as temporary incapacitation and progressive neurodegeneration. The HIT device uses a spring-based mechanism to propel flies against the wall of a vial, causing mechanical damage to the fly brain. The device is inexpensive and easy to construct, its operation is simple and rapid, and it produces reproducible results. Consequently, the HIT device can be combined with existing experimental tools and techniques for flies to address fundamental questions about TBI that can lead to the development of diagnostics and treatments for TBI. In particular, the HIT device can be used to perform large-scale genetic screens to understand the genetic basis of TBI pathologies.

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Here we describe a method to inflict closed head traumatic brain injury (TBI) in Drosophila. This method provides a gateway to investigate the cellular and molecular mechanisms that underlie TBI pathologies using the vast array of experimental tools and techniques available for flies.

Un método para infligir Cerrado Jefe lesión cerebral traumática en<em&gt; Drosophila</em

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies ...

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 ...

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.

Purificación de las transcripciones y los metabolitos de<em&gt; Drosophila</em&gt; Cabezales

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

Biología

Morphological and Functional Evaluation of Axons and their Synapses during Axon Death in Drosophila melanogaster

Axon degeneration is a shared feature in neurodegenerative disease and when nervous systems are challenged by mechanical or chemical forces. However, our understanding of the molecular mechanisms underlying axon degeneration remains limited. Injury-induced axon degeneration serves as a simple model to study how severed axons execute their own disassembly (axon death). Over recent years, an evolutionarily conserved axon death signaling cascade has been identified from flies to mammals, which is required for the separated axon to degenerate after injury. Conversely, attenuated axon death signaling results in morphological and functional preservation of severed axons and their synapses. Here, we present three simple and recently developed protocols that allow for the observation of axonal morphology, or axonal and synaptic function of severed axons that have been cut-off from the neuronal cell body, in the fruit fly Drosophila. Morphology can be observed in the wing, where a partial injury results in axon death side-by-side of uninjured control axons within the same nerve bundle. Alternatively, axonal morphology can also be observed in the brain, where the whole nerve bundle undergoes axon death triggered by antennal ablation. Functional preservation of severed axons and their synapses can be assessed by a simple optogenetic approach coupled with a post-synaptic grooming behavior. We present examples using a highwire loss-of-function mutation and by over-expressing dnmnat, both capable of delaying axon death for weeks to months. Importantly, these protocols can be used beyond injury; they facilitate the characterization of neuronal maintenance factors, axonal transport, and axonal mitochondria.

Morphological and Functional Evaluation of Axons and their Synapses during Axon Death in Drosophila melanogaster

Here, we provide protocols to perform three simple injury-induced axon degeneration (axon death) assays in Drosophila melanogaster to evaluate the morphological and functional preservation of severed axons and their synapses.

Evaluación morfológica y funcional de los axónyis y sus sinapsis durante la muerte de Axon en Drosophila melanogaster

Axon degeneration is a shared feature in neurodegenerative disease and when nervous systems are challenged by mechanical or chemical forces. However, our ...

Tracking Freely Walking Flies: A Method to Assess Locomotion in Drosophila

Source: Sun, M. and Chen, L. L. <a href="https://jove.unicartagenaproxy.elogim.com/video/55602/" target="_blank">A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila</a>. J. Vis. Exp. (2017).
This video describes how to track freely walking flies to assess their general locomotion ability. The featured protocol demonstrates the assay&#39;s setup and data analysis using video tracking software.

Seguimiento de moscas que caminan libremente: un método para evaluar la locomoción en Drosophila

Source: Sun, M. and Chen, L. L. A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila. J. Vis. Exp. (2017).
This video describes how to ...

JoVE Encyclopedia of Experiments

Drosophila melanogaster (mosca de la fruta)

Adulto

A Technique to Inflict Closed-Head Traumatic Brain Injury in Drosophila Flies

Source: Sun, M., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/55602">A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila.</a> J. Vis. Exp. (2017).
This video demonstrates a procedure to inflict a closed-head traumatic injury in anesthetized Drosophila flies using a strike device with a gas-propelled impactor. The injury affects neuronal function and impairs walking ability.

Una técnica para infligir una lesión cerebral traumática de cabeza cerrada en moscas Drosophila

1. Assembly of the Strike Device (Figure 1)

	Remove the plunger from a 1 mL tuberculin syringe. Cut the barrel at the 1 mL mark.
	Remove an aerosol ...

Un nuevo método para modelar la encefalopatía traumática crónica

Resumen

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