Nous remercions les membres des laboratoires Kopito, Luo et Pearce pour les nombreuses discussions utiles au cours du développement de ces méthodes. Nous remercions également Brian Temsamrit pour une lecture critique de ce manuscrit. Ce travail a été soutenu par le Fonds de l’Université des Sciences et des fiducies de bienfaisance W.W. Smith.

Les auteurs n’ont rien à divulguer.

Comme le nombre de patients souffrant de maladies neurodégénératives ne cesse d’augmenter, il y a un besoin urgent d’augmenter la compréhension de la pathogenèse moléculaire de ces maladies, afin que les meilleurs traitements peuvent être développées. Nous décrivons ici des méthodes qui permettent de surveiller la transmission de prion-comme des agrégats de protéine pathogène entre différents types de cellules dans l’organisme modèle, Drosophila melanogaster. Nous avons récemment utilisé ...

L’hypothèse de prion stipule que l’agent infectieux responsable pour les encéphalopathies spongiformes transmissibles (p. ex., maladie de Creutzfeldt - Jakob chez les humains, la tremblante du mouton, maladie du dépérissement chronique chez les cerfs et de wapitis et « maladie de la vache folle » chez les bovins ) est uniquement composé de protéines et dépourvu d’acides nucléiques1. Dans les maladies de prion, la protéine prion cellulaire (PrPC) suppose un pli non indigènes, stable (PrP-Sc) qui est hautement bêta feuille riche et peut se propager autonome en convertissant et recruter des molécules monomères de PrPC pour amyloïde stable agrégats. PrPSc agrégats utilisent ce mécanisme auto-répliquant de répartir entre les différentes cellules dans l’organisme et même entre des organismes individuels2.
Repliement et agrégation est également un élément central de la plupart des maladies neurodégénératives (maladie d’Alzheimer (ma), maladie de Parkinson (MP), maladie de Huntington (MH) et la sclérose latérale amyotrophique (SLA))3. Formation des assemblées intra - ou extra-cellulaires protéines agrégées dans ces maladies est étroitement associée à la cytotoxicité4 et progresse le long de chemins très reproductibles et propres à la maladie à travers le cerveau au cours du temps5, 6. ces schémas de propagation suggèrent que des agrégats pathogènes associés à ces troubles ont des propriétés de type prion. Fort soutien existe maintenant pour la transmission de prion-comme des agrégats associés à annonce, PD, HD et ALS - elles s’étendent de cellule-cellule et modèle le changement conformationnel de formes monomères de la même protéine dans les cellules affectées précédemment7, 8.
La plupart des études portant sur la propagation du prion comme des agrégats de protéine à ce jour ont été effectuée à l’aide de modèles de culture de cellules de mammifères, où agrégats transvaser dans le cytoplasme des cellules naïves de l’espace extracellulaire ou d’une autre cellule cytoplasme9,10,11,12,13,14,15, ou en injectant des matériaux contenant de l’agrégat dans le cerveau de souris et surveillance agréger l’apparence à l’extérieur de l’injection site16,17,18,19,20,21,22, 23. plus récemment, les animaux transgéniques ont été utilisés pour démontrer que des agrégats intracellulaires se propager à d’autres cellules dans le cerveau intact24,25,26,27, 28,29,30. Nous décrivons ici une méthode pour une visualisation directe de cession globale entre différentes cellules dans le cerveau intact de Drosophila melanogaster. Modèles de drosophile de HD/polyglutamine (polyQ) maladies ont été développés tout d’abord il y a près de vingt ans31,32 et ont fourni beaucoup de précieux conseils sur les mécanismes pathogènes qui sous-tendent ces troubles 33. HD est une maladie neurodégénérative héréditaire causée par une mutation autosomique dominante du gène codant pour la protéine huntingtine (Htt)34. Cette mutation se traduit par l’élargissement d’un tronçon de polyQ près aminée de Htt au-delà d’un seuil pathogène de ~ 37 glutamate, causant les protéines à repliement et agrégation35,36. Protéines de Htt sauvage contenant < 37 glutamate dans ce tronçon atteindre leur giron natif, mais peuvent être induites à global sur le contact physique direct avec un Htt globale « graine »12,27,37. Nous exploitons cette homotypique, nucléée agrégation de Htt sauvage comme une lecture pour le transfert de type prion et entrée cytoplasmique de mutant Htt agrégats originaires d’autres cellules.
Détermination des mécanismes de quels agrégats prion comme voyage entre les cellules peut conduire à l’identification de nouvelles cibles thérapeutiques pour les maladies neurodégénératives incurables. Nous prenons l’avantage du cycle de vie rapid, facilité d’utilisation et la traçabilité génétique de Drosophila melanogaster de définir des mécanismes moléculaires de la cellule-cellule propagation du mutant Htt agrégats. Notre stratégie expérimentale utilise deux systèmes d’expression binaire disponibles dans drosophile, le bien établi Gal4 spécifiques en amont activant séquence (Gal4-UAS) système38 et le récemment développés QF-QUAS système39. Ces deux systèmes indépendants de couplage permet de restreindre l’expression de transgènes Htt mutant et sauvage aux populations cellulaires distincts au sein de la même mouche40. En utilisant cette approche, nous examinons prion comme propagation du mutant Htt en surveillant la redistribution des cytoplasmique Htt sauvage de son état normalement diffuse, soluble à un état agrégé, conséquence directe d’un contact physique avec un mutant pré-formé Htt agrégat « semence ». Conversion de type sauvage Htt par mutant Htt peut être confirmée en utilisant biochimiques ou de transfert de techniques biophysiques qui signalent des interactions protéine-protéine, tels que l’énergie de résonance de fluorescence (FRET)9,27,41 .
Ce qui est important, nous pouvons également accéder à un grand nombre d’outils génétiques chez la drosophile pour identifier des gènes et/ou des voies qui véhiculent la propagation du prion comme des agrégats de protéine. Nous avons récemment utilisé cette approche pour dévoiler un rôle clé pour le récepteur scavenger surface des cellules,42,Draper43, en transférant mutant Htt agrégats d’axones neurones à proximité glia phagocytaire chez la drosophile central système nerveux (SNC)27. Ainsi, l’approche génétique et d’imagerie dotés que nous décrivons ici peut révéler des informations biologiques base importantes concernant un phénomène maladie pertinents dans le simple à utiliser mais puissant organisme, drosophile.

<table border="0" cellpadding="0" cellspacing="0" style="width:849px;" width="849">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Phosphate buffered saline (PBS), 10X, pH 7.4</td>
			<td style="width:123px;">ThermoFisher Scientific</td>
			<td style="width:344px;">AM9625</td>
			<td style="width:167px;">Dilute to 1X</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Triton X-100</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">T9284-1L</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Kimwipes</td>
			<td style="width:123px;">Thomas Scientific</td>
			<td style="width:344px;">2904F24</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">20% paraformaldehyde (PFA)</td>
			<td style="width:123px;">Electron Microscopy Sciences</td>
			<td style="width:344px;">15713-S</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Normal Goat Serum (NGS), filtered</td>
			<td style="width:123px;">Lampire Biological Laboratories</td>
			<td style="width:344px;">7332500</td>
			<td style="width:167px;">Aliquot and freeze upon receipt</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Chicken anti-GFP</td>
			<td style="width:123px;">Aves Labs</td>
			<td style="width:344px;">GFP-1020</td>
			<td style="width:167px;">Use at 1:500 dilution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Rabbit anti-DsRed</td>
			<td style="width:123px;">Clontech</td>
			<td style="width:344px;">632496</td>
			<td style="width:167px;">Use at 1:2000 dilution; can recognize DsRed-based fluorescent proteins (e.g. mCherry, mStrawberry, tdTomato, etc.)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Mouse anti-Bruchpilot</td>
			<td style="width:123px;">Developmental Studies Hybridoma Bank</td>
			<td style="width:344px;">nc82</td>
			<td style="width:167px;">Use at 1:100 dilution; will label active pre-synaptic structures thoughout the fly brain</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">FITC anti-chicken</td>
			<td style="width:123px;">ThermoFisher Scientific</td>
			<td style="width:344px;">SA1-7200</td>
			<td style="width:167px;">Use at 1:250 dilution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Alexa Fluor 568 anti-rabbit</td>
			<td style="width:123px;">Life Technologies</td>
			<td style="width:344px;">A11011</td>
			<td style="width:167px;">Use at 1:250 dilution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Alexa Fluor 647 anti-mouse antibody</td>
			<td style="width:123px;">Life Technologies</td>
			<td style="width:344px;">A21235</td>
			<td style="width:167px;">Use at 1:250 dilution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Slowfade Gold Antifade Reagent</td>
			<td style="width:123px;">Life Technologies</td>
			<td style="width:344px;">S36936</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Microscope Slides (25 x 75 x 1.0 mm)</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td style="width:344px;">12-550-143</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Cover Glass (22 x 22 mm)</td>
			<td style="width:123px;">Globe Scientific</td>
			<td style="width:344px;">1404-15</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Dumont Biology Grade Forceps, Style 3</td>
			<td style="width:123px;">Ted Pella</td>
			<td style="width:344px;">503</td>
			<td style="width:167px;">use in non-dominant hand</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Dumont Biology Grade Forceps, Style 5</td>
			<td style="width:123px;">Ted Pella</td>
			<td style="width:344px;">505</td>
			<td style="width:167px;">use in dominant hand</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">LAS X image analysis software</td>
			<td style="width:123px;">Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Imaris image analysis software</td>
			<td style="width:123px;">Bitplane</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

monitoring cell-to-cell transmission of prion-like protein aggregates in drosophila melanogaster

Agrégation des protéines est un élément central de la plupart des maladies neurodégénératives, y compris la sclérose latérale amyotrophique (SLA), maladie de Huntington (MH), la maladie d’Alzheimer (ma) et maladie de Parkinson (MP). Agrégats de protéines sont étroitement associées à la neuropathologie dans ces maladies, bien qu’on ne connaît pas le mécanisme exact par lequel agrégation de protéines aberrantes perturbe l’homéostasie cellulaire normale. Nouvelles données fort appuyer l’hypothèse qu’agrégats pathogènes en AD, PD, HD, et ALS ont beaucoup de similitudes aux prions, qui sont des protéines uniquement les agents infectieux responsables des encéphalopathies spongiformes transmissibles. Prions répliquent par la création de modèles la conversion des versions nativement plié de la même protéine, entraînant la propagation du phénotype agrégation. Comment prions et protéines de type prion dans AD, PD, HD et ALS déplacer d’une cellule à l’autre est actuellement un domaine de recherche intense. Ici, un modèle de Drosophila melanogaster qui permet une surveillance de transmission de prion-like, cellule-cellule des agrégats de la huntingtine mutante (Htt) associé à HD est décrite. Ce modèle tire parti des outils puissants pour manipuler l’expression du transgène dans de nombreux tissus différents de drosophile et utilise une protéine cytoplasmique fluorescent-étiquetées directement rapport à prions comme transfert de mutant Htt agrégats. Ce qui est important, l’approche que nous décrivons ici peut servir à identifier de nouveaux gènes et voies qui médient propagation d’agrégats de protéines entre diverses cellules types in vivo. Information obtenue grâce à ces études étendra la compréhension limitée des mécanismes pathogènes qui sous-tendent les maladies neurodégénératives et révéler de nouvelles possibilités d’intervention thérapeutique.

Les méthodes décrites ici produisent des données robustes témoignant de prion comme transfert des agrégats de protéine Htt de population d’une cellule à l’autre chez la mouche intacte CNS. Conversion de type sauvage Htt du diffus au ponctuée observe par fluorescence directe de cette protéine de fusion YFP glia bénéficiaire à la suite de HttQ91-mCherry expression dans donateurs Orn (A-C de laFigure 2et Figu...

Watch this Scientific Journal Video about Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster at JoVE.com

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Accumulation de preuves souscrit l’idée qu’agrégats de protéine pathogène est associée aux maladies neurodégénératives répartis entre les cellules ayant des propriétés prion. Nous décrivons ici une méthode qui permet la visualisation de cellule à cellule propagation des agrégats de type prion dans l’organisme modèle, Drosophila melanogaster.

Surveillance de cellule à cellule Transmission des agrégats de protéine Prion chez Drosophila Melanogaster

Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), ...

monitoring-cell-to-cell-transmission-prion-like-protein-aggregates

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Neuroscience

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

7.9K vues.  University of the Sciences. Accumulation de preuves souscrit l’idée qu’agrégats de protéine pathogène est associée aux maladies neurodégénératives répartis entre les cellules ayant des propriétés prion. Nous décrivons ici une méthode qui permet la visualisation de cellule à cellule propagation des agrégats de type prion dans l’organisme modèle, Drosophila melanogaster.

Vidéo: Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed in sensory hairs called sensilla, which cover the surface of olfactory organs. The surface of each sensillum is covered with tiny pores, through which odorants pass and dissolve in a fluid called sensillum lymph, which bathes the sensory dendrites of the OSNs housed in a given sensillum. The OSN dendrites express odorant receptor (OR) proteins, which in insects function as odor-gated ion channels4, 5. The interaction of odorants with ORs either increases or decreases the basal firing rate of the OSN. This neuronal activity in the form of action potentials embodies the first representation of the quality, intensity, and temporal characteristics of the odorant6, 7.
Given the easy access to these sensory hairs, it is possible to perform extracellular recordings from single OSNs by introducing a recording electrode into the sensillum lymph, while the reference electrode is placed in the lymph of the eye or body of the insect. In Drosophila, sensilla house between one and four OSNs, but each OSN typically displays a characteristic spike amplitude. Spike sorting techniques make it possible to assign spiking responses to individual OSNs. This single sensillum recording (SSR) technique monitors the difference in potential between the sensillum lymph and the reference electrode as electrical spikes that are generated by the receptor activity on OSNs1, 2, 8. Changes in the number of spikes in response to the odorant represent the cellular basis of odor coding in insects. Here, we describe the preparation method currently used in our lab to perform SSR on Drosophila melanogaster and Anopheles gambiae, and show representative traces induced by the odorants in a sensillum-specific manner.

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Electrophysiological responses of olfactory sensory neurons to odorants can be measured in insects using single sensillum recordings. In this video article we will demonstrate how to perform single sensillum recordings in the antennae of the vinegar fly (Drosophila melanogaster) and the maxillary palps of the malaria mosquito (Anopheles gambiae).

Enregistrements Sensillum simple chez les insectes Drosophila melanogaster Et Anopheles gambiae</em

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed ...

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Neurosciences

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

Étiquetage des cellules isolées dans le système nerveux central des<em&gt; Drosophila melanogaster</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

A growing number of genetically encoded tools are becoming available that allow non-invasive manipulation of the neural activity of specific neurons in Drosophila melanogaster1. Chief among these are optogenetic tools, which enable the activation or silencing of specific neurons in the intact and freely moving animal using bright light. Channelrhodopsin (ChR2) is a light-activated cation channel that, when activated by blue light, causes depolarization of neurons that express it. ChR2 has been effective for identifying neurons critical for specific behaviors, such as CO2 avoidance, proboscis extension and giant-fiber mediated startle response2-4. However, as the intense light sources used to stimulate ChR2 also stimulate photoreceptors, these optogenetic techniques have not previously been used in the visual system. Here, we combine an optogenetic approach with a mutation that impairs phototransduction to demonstrate that activation of a cluster of loom-sensitive neurons in the fly's optic lobe, Foma-1 neurons, can drive an escape behavior used to avoid collision. We used a null allele of a critical component of the phototransduction cascade, phospholipase C-&beta;, encoded by the norpA gene, to render the flies blind and also use the Gal4-UAS transcriptional activator system to drive expression of ChR2 in the Foma-1 neurons. Individual flies are placed on a small platform surrounded by blue LEDs. When the LEDs are illuminated, the flies quickly take-off into flight, in a manner similar to visually driven loom-escape behavior. We believe that this technique can be easily adapted to examine other behaviors in freely moving flies.

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Genetically encoded optogenetic tools enable noninvasive manipulation of specific neurons in the Drosophila brain. Such tools can identify neurons whose activation is sufficient to elicit or suppress particular behaviors. Here we present a method for activating Channelrhodopsin2 that is expressed in targeted neurons in freely walking flies.

Stimulation optogenetic de comportement de fuite en<em&gt; Drosophila melanogaster</em

A  growing number of genetically encoded tools are becoming available that allow non-invasive  manipulation of the neural activity of specific neurons in ...

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;

Détermination de l&#39;activité locomotrice spontanée,<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 ...

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by stressed conspecifics due to an odorant released during stress known as the Drosophila stress odorant (dSO). Through the use of the T-maze apparatus, one can quantify the avoidance of the dSO by responder flies in a very affordable and robust assay. Conditions necessary to obtain a strong performance are presented here. A stressful experience is necessary for the flies to emit dSO, as well as enough emitter flies to cause a robust avoidance response to the presence of dSO. Genetic background, but not their group size, strongly altered the avoidance of the dSO by the responder flies. Canton-S and Elwood display a higher performance in avoiding the dSO than Oregon and Samarkand strains. This behavioral assay will allow identification of mechanisms underlying this social behavior, and the assessment of the influence of genes and environmental conditions on both emission and avoidance of the dSO. Such an assay can be included in batteries of simple diagnostic tests used to identify social deficiencies of mutants or environmental conditions of interest.

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Here, we present a protocol to quantify the avoidance of stressed individuals. This paradigm is powerful yet user-friendly and can be used to assess the influence of genes and environment on one kind of social interaction in Drosophila melanogaster.

Essai simple pour la quantification de l&#39;évitement social en<em&gt; Drosophila melanogaster</em

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by ...

Conditions Affecting Social Space in Drosophila melanogaster

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a straightforward manner. As we previously demonstrated 1, in a two-dimensional chamber, we first force the flies to form a tight group, subsequently allowing them to take their preferred distance from each other. After the flies have settled, we measure the distance to the closest neighbor (or social space), processing a static picture with free online software (ImageJ). The analysis of the distance to the closest neighbor allows researchers to determine the effects of genetic and environmental factors on social interaction, while controlling for potential confounding factors. Diverse factors such as climbing ability, time of day, sex, and number of flies, can modify social spacing of flies. We thus propose a series of experimental controls to mitigate these confounding effects. This assay can be used for at least two purposes. First, researchers can determine how their favorite environmental shift (such as isolation, temperature, stress or toxins) will impact social spacing 1,2. Second, researchers can dissect the genetic and neural underpinnings of this basic form of social behavior 1,3. Specifically, we used it as a diagnostic tool to study the role of orthologous genes thought to be involved in social behavior in other organisms, such as candidate genes for autism in humans 4.

Conditions Affecting Social Space in Drosophila melanogaster

The effect of genes and environment on social space of Drosophila melanogaster can be quantified through a powerful but straightforward analytical paradigm. We show here different factors that can affect this social space, and thus need to be taken into consideration when designing experiments in this paradigm.

Conditions affectant espace social<em&gt; Drosophila melanogaster</em

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a ...

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense organs provide an excellent model system for the study of mechanosensation. This protocol describes a simple touch behavior in fruit flies which can be used to identify mutations that interfere with mechanosensation. The tactile stimulation of a macrochaete bristle on the thorax of flies elicits a grooming reflex from either the first or third leg. Mutations that interfere with mechanotransduction (such as NOMPC), or with other aspects of the reflex arc, can inhibit the grooming response. A traditional screen of adult behaviors would have missed mutants that have essential roles during development. Instead, this protocol combines the touch screen with mosaic analysis with a repressible cell marker (MARCM) to allow for only limited regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP). By testing MARCM clones for abnormal behavioral responses, it is possible to screen a collection of lethal p-element mutations to search for new genes involved in mechanosensation that would have been missed by more traditional methods.

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

A Behavioral Essai pour mécanosensibilité de marcm-fondé clones dans<em&gt; Drosophila melanogaster</em

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense ...

Foraging Path-length Protocol for Drosophila melanogaster Larvae

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral variation. The larval path-length assay was developed to measure individual differences in foraging behavior that were later linked to the foraging gene. Larval path-length is an easily scored trait that facilitates the collection of large sample sizes, at minimal cost, for genetic screens. Here we provide a detailed description of the current protocol for the larval path-length assay first used by Sokolowski. The protocol details how to reproducibly handle test animals, perform the behavioral assay and analyze the data. An example of how the assay can be used to measure behavioral plasticity in response to environmental change, by manipulating feeding environment prior to performing the assay, is also provided. Finally, appropriate test design as well as environmental factors that can modify larval path-length such as food quality, developmental age and day effects are discussed.

Foraging Path-length Protocol for Drosophila melanogaster Larvae

We provide a detailed protocol for a Drosophila melanogaster foraging path-length assay. We discuss the preparation and handling of test animals, how to perform the assay and analyze the data.

Foraging Protocole Chemin de longueur pour<em&gt; Drosophila melanogaster</em&gt; larves

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral ...

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of the right type and quantity of food therefore has a major influence on quality of life. Research on feeding behavior focuses on the underlying processes that ensure actual feeding and unravels the role of factors regulating internal energy homeostasis and the neuronal bases of decision-making. The model organism Drosophila melanogaster, with its great variety of genetically traceable tools for labeling and manipulating single neurons, allows mapping of neuronal networks and identification of molecular signaling cascades involved in the regulation of food intake. This report demonstrates the CApillary FEeder assay (CAFE) and shows how to measure food intake in a group of flies for time spans ranging from hours to days. This easy-to-use assay consists of glass capillaries filled with liquid food that flies can freely access and feed on. Food consumption in the assay is accurately determined using simple measurement tools. Herein we describe step-by-step the method from setup to successful execution of the CAFE assay, and provide practical examples to analyze the food intake of a group of flies under controlled conditions. The reader is guided through possible limitations of the assay, and advantages and disadvantages of the method compared to other feeding assays in D. melanogaster are evaluated.

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

The CApillary FEeder (CAFE) assay is a simple, budget-friendly, highly reliable method for investigating mechanisms underlying food intake. Used with the highly versatile genetic model organism Drosophila melanogaster, it provides a powerful means of gaining new insights into regulatory mechanisms of food intake.

Capillaire Feeder Assay mesures prise alimentaire dans<em&gt; Drosophila melanogaster</em

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of ...

Measuring and Altering Mating Drive in Male Drosophila melanogaster

Despite decades of investigation, the neuronal and molecular bases of motivational states remain mysterious. We have recently developed a novel, reductionist, and scalable system for in-depth investigation of motivation using the mating drive of male Drosophila&#160;melanogaster (Drosophila), the methods for which we detail here. The behavioral paradigm centers on the finding that male mating drive decreases alongside fertility over the course of repeated copulations and recovers over ~3 d. In this system, the powerful neurogenetic tools available in the fly converge with the genetic accessibility and putative wiring diagram available for sexual behavior. This convergence allows rapid isolation and interrogation of small neuronal populations with specific motivational functions. Here we detail the design and execution of the satiety assay that is used to measure and alter courtship motivation in the male fly. Using this assay, we also demonstrate that low male mating drive can be overcome by stimulating dopaminergic neurons. The satiety assay is simple, affordable, and robust to influences of genetic background. We expect the satiety assay to generate many new insights into the neurobiology of motivational states.

Measuring and Altering Mating Drive in Male Drosophila melanogaster

This article describes a behavioral assay that uses male mating drive in Drosophila melanogaster to study motivation. Using this method, researchers can utilize advanced fly neurogenetic techniques to uncover the genetic, molecular, and cellular mechanisms that underlie this motivation.