Los autores desean agradecer a Qiuli Wang, Centro de Recursos de Idiomas, Colby College, para la asistencia técnica con el procesamiento de vídeo y Eric Thomas, departamento de música, Colby College, para proporcionar la música de fondo. Este proyecto fue apoyado por subvenciones del Centro Nacional para Recursos de Investigación, INBRE (P20RR016463-12), el Instituto Nacional de Ciencias Médicas Generales (GM103423-12 P20), los nacionales de los Institutos de la Salud y la división de ciencia de Grant, Colby College (STA). JL y LWM fueron apoyados por becas de Fondo Académico de Verano, Colby College.

1.-Ensayo de locomoción inducida sobresalto Drosophila <ol><li> Tratamiento de Drogas <ol><li> Sedate para inmovilizar el número deseado (aproximadamente 8-12) de 1-3 días de edad moscas macho usando CO 2 y transportarlos a los viales que contengan el alimento suplementado con las drogas. Nota: Otro anestésico por ejemplo, el éter o el hielo puede ser utilizado para sedar a las moscas para permitir el recuento y la manipulación. </li><li> Permitir que las moscas para recuperarse de la sedación durante 20 minutos (o hasta que la recuperación) con el vial en posición horizontal (para evitar que las moscas se queda pegada en la comida) y luego coloque el vial en posición vertical en una oscuridad de 12 h, 12 h luz incubadora a 25 ° C para el resto del experimento. </li></ol></li><li> Experimental Set Up <ol><li> Divida esta configuración doble vial en tres secciones iguales de 6,33 cm, marcando círculos alrededor de los viales con un marcador permanente. </li><li> Después de 3 días de exposición al fármaco, transferencia vuela sin anestesiar en el vial inferior y colocar rápidamente el vial superiorsobre la abertura. Pegue las dos viales junto con cinta adhesiva transparente. </li><li> Permitir que las moscas se aclimaten al nuevo ambiente durante 15 min. </li><li> Coloque los viales en un fondo blanco y establecieron una cámara digital a una distancia apropiada del aparato vial doble con un contador de tiempo en la vista. Asegúrese de que el conjunto quede visible en un único marco de imagen y que todas las moscas están en foco. Para mantener marcos consistentes entre los ensayos, marque la ubicación de la cámara y el vial. </li></ol></li><li> Ensayo de Movilidad <ol><li> Claramente mostrar el número de prueba, el tratamiento farmacológico y el cronómetro en vista de la cámara. </li><li> Pulse firmemente el aparato vial doble contra la encimera 3 veces y asegurarse de que todas las moscas caen al fondo del vial. Simultáneamente iniciar el temporizador. </li><li> Cada 5 segundos durante 1 minuto, toma una foto del aparato. Nota: Como alternativa, un video podría ser capturado y se detuvo a intervalos apropiados para las mediciones. </li><li> Permitir que las moscas se recuperen en reposo durante 1 min. </li><li&gt; Repetir 2 veces más con el tiempo de recuperación de 1 minuto entre cada ensayo. Nota: Cada aparato debe tomar 5 minutos para completar la recopilación de datos. Mantener fuerza similar de tapping entre los ensayos. Múltiple (al menos 3) aparato puede ser manejado fácilmente de forma simultánea. </li></ol></li><li> Análisis de Datos <ol><li> Revise las imágenes y grabar el número de moscas en cada sección a través del tiempo. Calcular el porcentaje de moscas en cada sección con el tiempo. Notas: repetir todo este procedimiento con las mismas moscas a los 2 o 3 puntos de tiempo de interés, por ejemplo, los días 3, 5, y 7 Si demasiadas moscas mueren durante el experimento es posible ampliar el número juicio original para compensar para la mortalidad. Utilice el análisis estadístico apropiado para comparar los datos. </li></ol></li></ol> Ensayo 2. Drosophila espontánea Locomotion <ol><li> Preparación de Alimentos <ol><li> Reconstituir 3 g de medio Drosophila instantánea con 15 ml de agua desionizada y rotenona deseada(U otro fármaco de interés) dosis. </li><li> Una vez que la mezcla de los alimentos se ha convertido en firme (aproximadamente 5 minutos), cargue con cuidado la comida que es de aproximadamente 1 cm de alto en fabricante suministra tubos transparentes (5 mm x 65 mm). Añadir el fármaco infundido alimentos a los tubos colocando cuidadosamente los tubos verticalmente en la comida y torcer hasta que puedan ser retirados con la comida en el interior del tubo. Nota: Es útil colocar un dedo en la abertura del tubo para crear un vacío. Los alimentos no deben contener burbujas de aire o tienen una superficie irregular como las moscas se pueden atascar. </li></ol></li><li> Configuración Experimental <ol><li> Colocar una tapa de plástico sobre el extremo del tubo más próximo a la comida. Empuje la tapa de plástico en el tubo de tan poco como sea posible, ya que puede crear una burbuja de aire en el vial, si se empuja a la fuerza. </li><li> Sedate 1 días de edad moscas macho usando CO 2 e inserte con cuidado 1 macho vuela en cada tubo con un pincel. Repita dependiendo del número de ensayos deseados. </li><li> Enchufe elfinal del tubo más alejado de la comida con una pequeña bola de algodón, que se puede enrollar la mano más grande comprados en la tienda de bolas de algodón. </li><li> Permitir que las moscas se recuperen con los tubos en posición horizontal durante 15 minutos y asegurarse de que todas las moscas están vivos y activos. Inserte los tubos en DAM y asegúrese de que todos los tubos están en la misma posición respecto a la represa. Nota: Es posible colocar con el área de vigilancia en el centro del vial, o para empujar todos los viales a un lado, de manera que se está supervisando el extremo del tubo. Nota: Véase la discusión para variantes de este método. </li></ol></li><li> Recogida de datos <ol><li> Coloque el DAM en una oscuridad de 12 h, 12 h incubadora luz ajustado a 25 ° C. Conecte el DAM al sistema de recolección de datos. Abra el software DAM y en las preferencias de selección de longitud bin a 10 min. Comienza la recogida de datos y permitir que el programa para recoger datos durante 7 días. Nota: la longitud Bin se puede ajustar si es necesario. </li><li> Análisis de Datos Nota: Procesolos datos para obtener recuentos por min como una medida para la locomoción espontánea a largo plazo. <ol><li> Abrir archivos DAM programar y supervisar el acceso de exploración de datos haciendo clic en Seleccionar datos de entrada. </li><li> Seleccionar rango apropiado de monitor y seleccione la longitud bin a intervalos de 10 min. </li><li> En salida de tipo de archivo, elija Archivos de canal. Deje el resto de opciones por defecto. </li><li> Haga clic en los datos de exploración y guardar en una carpeta designada. </li><li> Importar datos en un circadiano software de análisis de datos para obtener los recuentos por min. Nota: Para el análisis de datos de software Clocklab es de uso general. Otras opciones están también disponibles. </li></ol></li></ol></li></ol>

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Los autores no tienen nada que revelar.

En este estudio, se describen dos procedimientos para medir tanto la locomoción espontánea a largo plazo y la locomoción inducida de sobresalto-a corto plazo en un modelo de Drosophila inducida por rotenona de la enfermedad de Parkinson. También se puede medir estas características de locomoción en las moscas expuestas a otros agentes farmacológicos conocidos para modelar la enfermedad de Parkinson, por ejemplo, paraquat 14, modelos genéticos de la enfermedad de Parkinson, por ej...

Deficiencias de locomoción son el principal síntoma de la enfermedad de Parkinson y son causadas en gran parte por el deterioro de las neuronas dopaminérgicas de la sustancia negra 1. Rotenona es un insecticida cetónico que se ha estudiado ampliamente para modelar los déficits motores de Parkinson en Drosophila 2-6. Rotenona causa daño oxidativo mediante el bloqueo de la vía de la fosforilación oxidativa, lo que provoca en última instancia, la muerte celular 7. Las neuronas dopaminérgicas son más propensos a la toxicidad rotenona, por lo que los efectos de la sustancia química principalmente motor basa 2,7. Mediante la inducción de síntomas de la enfermedad de Parkinson en las moscas, podemos comprender mejor la enfermedad y remediar sus síntomas 6,8-11. Drosophila proporciona un buen modelo para el estudio de este efecto, ya que son genéticamente manejable, fácil de mantener, y tienen un ciclo de vida rápido. Varios estudios han demostrado que la rotenona causa inducida por sobresalto corto plazodefectos de locomoción en la mosca Drosophila -cuando se mantienen en los alimentos suplementados con rotenona, que muestran una respuesta más lenta geotáctico negativo después de sobresalto 2-6. Su incapacidad para subir hacia arriba en un aparato vial lo más rápidamente pruebas de control es indicativo de defectos de locomoción inducida de sobresalto-. El efecto de la rotenona en a largo plazo, movimiento espontáneo no está bien descrita. Monitores de actividad Drosophila (presas) se han utilizado con éxito para controlar el movimiento en el ritmo circadiano de Drosophila estudia 12,13. Las moscas se colocan en tubos individuales, que se cargan en el DAM. Este aparato está equipado con un sensor de infrarrojos, que cuenta el número de veces que una mosca rompe el haz de luz infrarroja. Estos recuentos se pueden utilizar como una medida de la locomoción y la actividad 12,13 sin molestias. Mediante la colocación de las moscas en un DAM, el efecto de la rotenona en su locomoción a largo plazo puede ser caracterizado. Este estudio describe los métodos de medición alocomoción y la locomoción espontánea a largo plazo ure corto plazo de sobresalto inducida con el fin de comprender mejor los efectos de la rotenona mediada por deficiencias motoras. Caracterización de las deficiencias de locomoción que imitan la enfermedad de Parkinson son importantes porque permiten el estudio de otros compuestos que pueden revertir estos defectos de locomoción.

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

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

La enfermedad de Parkinson es un trastorno neurodegenerativo que resulta de la degeneración de las neuronas dopaminérgicas en el sistema nervioso central, principalmente en la sustancia negra. La enfermedad provoca deficiencias motoras, que presentan como rigidez, temblores y la demencia en los seres humanos. Rotenona es un insecticida que causa el daño oxidativo mediante la inhibición de la función de la cadena de transporte de electrones en la mitocondria. También se utiliza para modelar la enfermedad de Parkinson en la Drosophila. Las moscas tienen una respuesta geotáctico negativo inherente, que los obliga a subir hacia arriba al ser sorprendido. Se ha establecido que la rotenona causa defectos de mortalidad y de locomoción principios que alteran la capacidad de las moscas para subir después de haber sido golpeado ligeramente hacia abajo. Sin embargo, el efecto de la rotenona en movimiento espontáneo no está bien documentada. Este estudio describe dos ensayos de rendimiento sensibles, reproducibles y de alta para caracterizar deficiencias inducida rotenona-inlocomoción y la locomoción espontánea a largo plazo a corto plazo de sobresalto-inducida en Drosophila. Estos ensayos pueden ser convenientemente adaptados para caracterizar otros modelos de Drosophila de defectos de la locomoción y la eficacia de los agentes terapéuticos.

Ensayo de locomoción inducida por sobresalto Drosophila Wildtype, Cantón-S, las moscas mostraron una respuesta negativa geotáctico robusto con sólo aproximadamente el 88% y el 5% de las moscas en las secciones superior e inferior, respectivamente, del aparato de doble vial después de 30 segundos (Figura 1). Las moscas expuestas a 125 M y 250 M rotenona durante 3 días mostraron una ligera disminución en el número de moscas en la secc...

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

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

La enfermedad de Parkinson es un trastorno neurodegenerativo que resulta de la degeneración de las neuronas dopaminérgicas en el sistema nervioso central, causando defectos de locomoción. Rotenona modelos de la enfermedad de Parkinson en Drosophila. Este documento describe dos ensayos que caracterizan a las deficiencias de locomoción tanto espontáneos como inducidos por el sobresalto causado por la rotenona.

Métodos para caracterizar espontánea y de sobresalto-inducida Locomotion en un modelo de enfermedad de Parkinson inducida por rotenona-de<em&gt; Drosophila</em

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

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

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JoVE Journal

Neuroscience

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

10.0K vistas.  Colby College.  La enfermedad de Parkinson es un trastorno neurodegenerativo que resulta de la degeneración de las neuronas dopaminérgicas en el sistema nervioso central, causando defectos de locomoción. Rotenona modelos de la enfermedad de Parkinson en Drosophila. Este documento describe dos ensayos que caracterizan a las deficiencias de locomoción tanto espontáneos como inducidos por el sobresalto causado por la rotenona. 

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

Autologous Blood Injection to Model Spontaneous Intracerebral Hemorrhage in Mice

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

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

Inyección de sangre autóloga al modelo hemorragia intracerebral espontánea en ratones

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

Investigación

Neurociencias

Methods to Assay Drosophila Behavior

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

Methods to Assay Drosophila Behavior

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

Métodos de ensayo a Drosophila Comportamiento

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

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

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

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

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

Varias unidades Métodos de grabación para caracterizar la actividad neuronal en el Locust (<em&gt; Schistocerca americana</em&gt;) Circuitos olfativas

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

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

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

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

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

Determinación de la actividad locomotora espontánea en<em&gt; Drosophila melanogaster</em

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

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

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

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

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

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 ...

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

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

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

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

Cuantificación de los flujos espontáneos de Ca2+ y sus efectos aguas abajo en las neuronas primarias de cerebro medio de ratón

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

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

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

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

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

Aplicación del sistema RatWalker para el análisis de la marcha en un modelo genético de ratas de la enfermedad de Parkinson

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

The 6-hydroxydopamine Rat Model of Parkinson's Disease

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

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

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

El modelo de rata 6-hidroxidopamina de la enfermedad de Parkinson

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

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

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

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

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

Inducción y evaluación de discinesias inducidas por levodopa en un modelo de rata de la enfermedad de Parkinson

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

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

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

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

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