Name: mass histology to quantify neurodegeneration in drosophila
Uploaded: 2016-12-15T12:30:00
Description: Watch this Scientific Journal Video about Mass Histology to Quantify Neurodegeneration in Drosophila at JoVE.com

This work was supported by grants to D.K. from the Medical Research Foundation of Oregon and from NIH/NINDS (NS047663). E.S. was supported by a training grant from the NIH (T32AG023477).

1. La fijación de la cabeza sobre los cuellos y inclusión en parafina Nota: Todos los pasos en el proceso de fijación debe hacerse en una campana de humos. Metilbenzoato, mientras que no plantean un riesgo para la salud, tiene un olor muy distinto, que puede ser abrumador si no se manejan en una campana de humos. <ol><li> Antes de anestesiar las moscas, conforman 50 ml de solución de Carnoy mediante la adición de 15 ml de cloroformo y 5 ml de ácido acético glacial y 30 ml de etanol al 99% (no mezcle el ...

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El método descrito proporciona un medio para cuantificar la neurodegeneración en el cerebro de Drosophila. Mientras que otros métodos, como contar un tipo celular específico, se pueden utilizar para identificar la neurodegeneración, la ventaja de este método es que se puede aplicar de forma más general. Contando las células requiere que estas células pueden ser identificados de forma fiable utilizando un anticuerpo específico o la expresión de un marcador específico de célula, lo que no siempre est...

Con el aumento de la esperanza de vida, las enfermedades neurodegenerativas como el Alzheimer o el Parkinson se han convertido en una amenaza creciente para la salud de la población en general. De acuerdo con los Institutos Nacionales de la Salud, 115 millones de personas en todo el mundo se prevé que se afectados por la demencia en 2050. Aunque se han hecho progresos significativos en la identificación de los genes y los factores de riesgo implicados en al menos algunas de estas enfermedades, para muchos de ellos, el subyacente mecanismos moleculares aún no se conocen o no se entiende bien. Simples organismos modelo de invertebrados como Caenorhabditis elegans y Drosophila melanogaster ofrecen una variedad de ventajas experimentales para estudiar los mecanismos de las enfermedades neurodegenerativas, incluyendo un ciclo de corta vida, gran número de progenie, y la disponibilidad de métodos genéticos y moleculares bien establecidos y, a veces únicos 1 -12. Además, estos organismos son susceptibles de imparcialpantallas de interacción que pueden identificar los factores que contribuyen a estas enfermedades por sus efectos agravantes o mejorar en fenotipos neurodegenerativos. El análisis de estas interacciones genéticas y la evaluación de los efectos del envejecimiento requiere protocolos cuantitativos para detectar la neurodegeneración y para medir su gravedad. Esta evaluación puede hacerse con relativa facilidad en la medición de los aspectos de comportamiento en Drosophila, como el aprendizaje olfativo, geotaxis negativos, o fototaxis rápido, que proporcionan un valor numérico rendimiento 13-21. También es posible determinar los efectos sobre la supervivencia neuronal contando las neuronas. Sin embargo, esto sólo es posible cuando se centra en una población específica que se pueda identificar claramente, al igual que las neuronas dopaminérgicas que se ven afectados en la enfermedad, e incluso entonces, los resultados han sido controvertidos 22-24. El protocolo descrito aquí utiliza el método de cuello para realizar secciones en serie de parafina, un métodoque fue desarrollado originalmente por Heisenberg y Böhl, que lo utilizó para aislar mutantes anatómicas cerebrales en Drosophila 25. El uso del método de cuello posteriormente se ha adaptado, incluso en criosecciones, vibratome secciones y secciones de plástico de 26-28. Aquí, se emplea este método para obtener secciones en serie de toda la cabeza de la mosca, que luego se puede utilizar para medir las vacuolas que se desarrollan en las moscas con fenotipos neurodegenerativas 16,21,29-32. Estas mediciones se pueden hacer en áreas específicas del cerebro o pueden cubrir todo el cerebro; el último enfoque permite identificar incluso débiles fenotipos degenerativas, como se observa durante el envejecimiento. Por último, cuando se utilizan los collares, hasta 20 moscas pueden ser procesados ​​como una preparación, que no sólo es menos tiempo, sino que también permite el análisis de control y las moscas experimentales en la misma preparación, minimizando artefactos debido a ligeros cambios en la preparación.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Name of the Reagent/Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td></td>
		</tr>
		<tr>
			<td>Collar</td>
			<td>Genesee Scientific</td>
			<td>TS 48-100</td>
			<td>We are using custom made collars that are made from one piece of metal instead of layers as the ones by Genesee. A discription to make collars can be found at http://flybrain.neurobio.arizona.edu/Flybrain/html/atlas/fluorescent/index.html&#160;</td>
		</tr>
		<tr>
			<td>Rubber ice cube tray for embedding</td>
			<td>Household store</td>
			<td></td>
			<td>The size can be made to fit by glueing in additional walls&#160;</td>
		</tr>
		<tr>
			<td>Crystallizing dish</td>
			<td>Fisher Scientific company</td>
			<td>08-762-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Ether</td>
			<td>Fisher Scientific Company</td>
			<td>E138-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Decon Laboratories Inc.</td>
			<td>2701</td>
			<td></td>
		</tr>
		<tr>
			<td>Choloroform</td>
			<td>Fisher Scientific Company</td>
			<td>C298-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial Acetic Acid</td>
			<td>Fisher Scientific Company</td>
			<td>A38-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylbenzoate</td>
			<td>Fisher Scientific Company</td>
			<td>M205-500</td>
			<td>Distinct Odor</td>
		</tr>
		<tr>
			<td>Use in fume hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Low Melting Point Paraffin Wax</td>
			<td>Fisher Scientific Company</td>
			<td>T565</td>
			<td>Make sure to keep extra melted in a 65&#176;C waterbath</td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Leica Biosystems</td>
			<td>Reichert Jung 2040 Autocut</td>
			<td></td>
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			<td>Microscope Slide</td>
			<td>Fisher Scientific Company</td>
			<td>12-550D</td>
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			<td>Microscope Cover Glass</td>
			<td>Fisher Scientific Company</td>
			<td>12-545-M</td>
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			<td>SafeClear</td>
			<td>Fisher Scientific Company</td>
			<td>314-629</td>
			<td>Three different containers for washes</td>
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			<td>Vertical Staining Jar with Cover</td>
			<td>Ted Pella Inc.&#160;</td>
			<td>432-1</td>
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			<td>Permount</td>
			<td>Fisher Scientific Company</td>
			<td>SP15-500</td>
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			<td>Poly-L-lysine Solution</td>
			<td>Sigma Life Science</td>
			<td>P8290-500</td>
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mass histology to quantify neurodegeneration in drosophila

enfermedades neurodegenerativas progresivas, como la enfermedad de Alzheimer (EA) o la enfermedad de Parkinson (EP) son una amenaza creciente para la salud humana en todo el mundo. Aunque los modelos de mamíferos han aportado importantes conocimientos sobre los mecanismos subyacentes de la patogenicidad, la complejidad de los sistemas de mamíferos, junto con sus altos costos están limitando su uso. Por lo tanto, la simple pero bien establecido modelo de sistema de Drosophila ofrece una alternativa para la investigación de las vías moleculares que se ven afectados en estas enfermedades. Además de anomalías de comportamiento, enfermedades neurodegenerativas se caracterizan por fenotipos histológicos tales como la muerte neuronal y axonopatía. Para cuantificar la degeneración neuronal y para determinar cómo se ve afectada por factores genéticos y ambientales, usamos un enfoque histológico que se basa en la medición de las vacuolas en el cerebro de moscas adultas. Para minimizar los efectos del error sistemático y la comparación directa de las secciones de control y experimental moscas en una preparación, que utilizan el método de &quot;cuello&quot; de las secciones de parafina. La neurodegeneración se evalúa a continuación, midiendo el tamaño y / o número de vacuolas que se han desarrollado en el cerebro de la mosca. Esto se puede hacer ya sea centrándose en una región específica de interés o mediante el análisis de todo el cerebro mediante la obtención de secciones en serie que abarcan la cabeza completa. Por lo tanto, este método permite medir no sólo la degeneración severa, pero también fenotipos relativamente suaves que sólo son detectables en algunas secciones, como ocurre durante el envejecimiento normal.

Utilizando los resultados método descrito en secciones seriadas teñidas por el pigmento del ojo 33 que abarcan toda la cabeza de la mosca. A parte de esto se muestra en la Figura 1B, donde las secciones de una cabeza individuales se muestran de arriba a abajo. Las secciones de diferentes moscas son vistos de izquierda a derecha en este ejemplo. Para facilitar la orientación y la identificación de las moscas, una mosca sin ojos (oculis sinusoidales)</em...

Watch this Scientific Journal Video about Mass Histology to Quantify Neurodegeneration in Drosophila at JoVE.com

Mass Histology to Quantify Neurodegeneration in Drosophila

Drosophila se utiliza ampliamente como un sistema modelo para estudiar la neurodegeneración. Este protocolo describe un método por el cual la degeneración, como se determina por la formación de vacuolas en el cerebro, se puede cuantificar. También minimiza los efectos debido al procedimiento experimental por el procesamiento y control de seccionamiento y moscas experimentales como una muestra.

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 ...

The overall goal of this procedure is to quantify Neurodegeneration in the Drosophila Brain, caused by a variety of factors including age, genetic modifications, or environmental influences, without the need for specific staining. This method is very useful in analyzing key questions in the field of neurodegenerative diseases. Such as what genes or environmental factors cause or contribute to these diseases?The main advantage of this method is that it avoids or minimizes systematic errors because control flies and experimental flies are treated as one sample. Generally, individuals new to this method may struggle, because it takes practice to ensure that the correct area of the brain is on the slide and to prevent damage that may occur during cutting. To begin, first use forceps to thread the subject flies by the neck into the collars.Align all the heads in the same orientation and ensure no damage occurs to the head or eyes. Include Eyeless Senoculus Flies at random, known positions so that the order of the flies can be easily identified in the sections. Additionally, if the fly strain of interest has light or white eyes, thread red eyed flies at intervals along the strip, to ensure sufficient pigment is present to stain the slide.Record the order of the flies, together with the collar number if more than one is used. Once a collar is finished, place it into Carnoy's solution for three point five to four hours. Perform a series of Ethanol, Methyl Benzoate, and Paraffin washes to prepare the fly tissue for embedding.Next, place the collars into a rubber ice cube tray, on per section. Gently pour molten paraffin over the collars, avoiding air bubbles and allow it to harden overnight. Remove the paraffin blocks containing the collars from the tray.Separate the paraffin block from the collar, using a razor blade. Gently breaking off the collar. The bodies will remain in the collar, while the heads will stay in the paraffin block.To section the tissue, first warm a heating plate to 50 degrees Celsius. Place any tools that will contact the wax, such as razor blades or metal mounting blocks onto the plate to warm. Determine the desired orientation for sectioning and then attach the paraffin segment to the mounting block by briefly melting it at the contact side.With a razor blade, trim the excess paraffin away from the fly heads, so that only a small row containing the embedded heads remains. Place the mounting block into the object holder of the Microtome, aligning the heads parallel to the edge of the blade. Cut seven micrometer sections and transfer the ribbon of sections to microscope slides.Place the slides on a heated plate at 37 degrees Celsius for about one minute, to allow the ribbon to expand. Remove excess water and then set the slides to dry further overnight. Once dry, remove the paraffin wax by placing the slides vertically in a tall slide staining jar, filled with Deparaffinization agent for 30 minutes to one hour.Repeat this wash three times. Finally, remove the slides from the jar and place two drops of embedding media onto each one. Cover the sections with a large cover slip and allow slides to dry for one to two days.Under low magnification on a fluorescence microscope, determine the orientation of the brain and focus on the region of interest. Once this region is identified, take the required images. Using imaging software, count the number of vacuoles per head.Measure vacuole size by selecting the vacuole of interest and determining the number of pixels covered. This image shows sections from different fly heads oriented left to right. From top to bottom, serial sections from the same fly head are visualized.The Eyeless Senoculus control fly is indicated by the arrow. Sections are stained by the naturally present fluorescent eye pigment which washes over the sections during cutting. Paraffin head sections from seven, 14, and 21 day old Swiss Cheese One flies, show a progressive age-related increase in vacuole number and area.This degeneration is quantifiable by counting the vacuoles and calculating the combined area and found to be significant. Similarly, in Wild Type flies, neurodegeneration was shown to occur with age. In examination of head sections of 10, 30, and 60 day Wild Type flies few to no vacuoles were seen to have formed in the 10 day condition.In the brains of the 30 day old individuals vacuoles were beginning to form. In the 60 day individuals, significantly more and larger area vacuoles were present. Once mastered, sections can be available for analysis within a week.When using this procedure, it's important to carefully record the order of the flies in the collar. To ensure you can identify them later on, after analysis. After watching this video, you should have a good understanding of how to prepare the sections and to quantify Neurodegeneration found in the Drosophila brain.

Research

JoVE Journal

Neuroscience

Mass Histology to Quantify Neurodegeneration in Drosophila

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