Nous remercions Aylin Rodan pour la lecture critique du manuscrit, et des membres du laboratoire de discussion utile. Cette recherche est soutenue par le NIH, R01AA019526.

1. Créer la chambre d&#39;exposition <ol><li> Couper un bouchon de flacon de moitié l&#39;aide d&#39;une lame de rasoir. Sinon, utiliser des balles de coton. </li><li> Poussez la moitié prise flacon ou la boule de coton, au fond d&#39;un flacon de nourriture pour faire une surface lisse qui vole sera pas coincé po </li><li> Si vous faites des chambres d&#39;exposition multiple, assurez-vous que la fiche du flacon et de la nourriture prennent le même volume dans chaque chambre. Une marque sur le côté du flacon peut faciliter ce processus. </li></ol> 2. Exposer les mouches <ol><li> Collecter 8 mouches d&#39;un sexe, d&#39;âge 1-5 jours, la veille de l&#39;exposition dans des flacons nourriture régulière, sans levure. </li><li> Les étiqueter dans le code, tels que l&#39;expérimentateur est aveuglé au génotype réel / souche utilisée. </li><li> Transfert des huit mouches dans les chambres de l&#39;exposition en appuyant sur. Ne pas anesthésier. </li><li> Ajouter une petite quantité de colorant alimentaire, par exemple l&#39;acide bleu 9, à 200 d&#39;éthanol la preuve pour le rendre facilement visible. </li><li> Napper le fond d&#39;un plug flacon, ou boule de coton, avec 0,5 ml d&#39;éthanol. </li><li> Insérez la fiche d&#39;éthanol dans le flacon de l&#39;exposition. Assurez-vous que l&#39;alcool est confronté dans le flacon, et non pas vers la paroi du flacon. </li><li> Démarrer un compte à rebours jusqu&#39;à. </li></ol> 3. Enregistrement d&#39;éthanol-intoxication <ol><li> Chaque minute après le chronomètre est lancé, appuyez sur la chambre d&#39;exposition sur le banc pour faire sursauter les mouches et les observer pendant dix secondes. </li><li> Notez le nombre de mouches stationnaires pour chaque minute. Les mouches sont stationnaires si elles ne peuvent pas sortir leur dos, rester dans un endroit, ou rapidement vibrer leurs ailes à travers l&#39;ensemble de dix secondes d&#39;observation période. Les mouches derniers sont considérés comme stationnaires car la grande majorité de ces mouches sont stationnaires entre les périodes de mouvement d&#39;ailes rapides. Les mouches ne sont pas stationnaires si elles peuvent obtenir sur leur dos et de pied et / ou sur le mur du flacon. </li><li> Un essai d&#39;exposition est terminée lorsque 4 ou plus de mouches sont enregistrés comme stationnaire. </li><li> Jeter les mouches et laissez l&#39;air chambre d&#39;exposition à pour une réutilisation ultérieure. </li></ol> 4. Déterminer le temps de la sédation de 50% Le temps d&#39;une sédation de 50% (ST50) est le temps après le début de l&#39;exposition à l&#39;éthanol, alors que quatre des huit mouches restent stationnaires. Ceci est déterminé comme suit: Soit: la minute la première mesure où exactement quatre mouches sont stationnaires. Ou: si moins de quatre mouches (Y1) sont stationnaires à un moment donné du temps (minute X), et plus de quatre (Y2) au point de mesure du temps suivant (minute X +1), puis interpoler linéairement du ST50 en utilisant la formule: ST50 = (4 - Y1) / (Y2 - Y1) + X 5. Répétez l&#39;exposition afin de déterminer la tolérance Exposer les mouches <ol><li> Déterminer le ST50 (en poids) pour la souche contrôle sauvage utilisé dans votre expérience. </li><li> Exposer les groupes expérimentaux de mouches à l&#39;éthanol comme décrit ci-dessus. </li><li> Mesurer leur ST50 (1), mais continuer à exposer jusqu&#39;à l&#39;époque 2x ST50 (en poids). </li><li> Transférer les mouches de nouveau dans une fiole de nourriture vides, et tenir pendant 4 heures. </li><li> Re-exposer les mouches, et de déterminer le ST50 (2) de l&#39;exposition 2. </li></ol> Déterminer la tolérance Calculer la tolérance en% en (ST50 (2) / ST50 (1) - 1) x100. 6. Les résultats représentatifs: Montré ici, quelques résultats représentatifs, nous avons obtenu avec 6-8 flacons de mouches par le génotype et la dose. Si votre erreur standard est sensiblement plus grand, puis un problème systématique s&#39;est probablement produite. Assurez-vous que l&#39;éthanol visages dans les chambres de l&#39;exposition, et que les chambres sont au même volume. <img src="/files/ftp_upload/2541/2541fig1.jpg" alt="Figure 1" /> Figure 1. Courbe dose-réponse de type sauvage mouches exposées à des pourcentages croissants d&#39;éthanol. Huit flacons de mouches ont été exposées par dose et le sexe. Mouches mâles et femelles montrent le ST50 même à chaque dose (p&gt; 0,22, 2-way ANOVA), tandis que le ST50 travers doses diminue considérablement avec la dose d&#39;éthanol augmente (p &lt;0,001). Notez que les mouches à partir surpeuplés bouteilles peuvent être sensiblement plus petits que ceux à partir de bouteilles en bonne santé, et cette différence de taille peut affecter le ST50. Semences chaque bouteille avec des femelles de 15 à 20 de pose de 1-2 jours pour obtenir la densité larvaire optimale par bouteille. <img src="/files/ftp_upload/2541/2541fig2.jpg" alt="Figure 2" /> Figure 2. Exposer mouches soit de l&#39;éthanol à 90%, ou 100% reproduit le décrit précédemment éthanol-résistance provoquée par le blanc rabbit1 mutation2 (p &lt;0,001, 2-way ANOVA). <img src="/files/ftp_upload/2541/2541fig3.jpg" alt="Figure 3" /> Figure 3. Répétez l&#39;exposition à l&#39;éthanol 100%, espacées par un intervalle de récupération de 4 heures, provoque une augmentation significative de la ST50 après la seconde exposition (p &lt;0,001, t apparié-Test). Notez que les mouches ont été exposées pendant 24 minutes pendant la première exposition (correspondant à deux fois leur ai na ST50). D&#39;exposition pour un court laps de temps (12 minutes) n&#39;a pas provoqué une augmentation significative du ST50 sur la ré-exposition (données non présentées).

<ol>
	<li>Bainton, R. J., Tsai, L. T. -. Y., Singh, C. S., Moore, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Curr. Biol. 10, 187-194 (2000).</li><li>Rothenfluh, A., Threlkeld, R. J., Bainton, R. J., Tsai, L. T. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell. 127, 199-211 (2006).</li><li>Devineni, A. V., Heberlein, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Curr. Biol. 19, 2126-2132 (2009).</li><li>Scholz, H., Ramond, J., Singh, C. S., Heberlein, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neuron. 28, 261-271 (2000).</li><li>Allen, M. J., Godenschwege, T. A., Tanouye, M. A., Phelan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Semin. Cell Dev. Biol. 17, 31-41 (2006).</li><li>Feany, M. B., Bender, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature. 404, 394-398 (2000).</li></ol>

Dans cette présentation, nous avons défini une méthode simple pour déterminer l&#39;éthanol intoxication volontaire basée sur des essais antérieurs décrits par Bainton et al. 2000 1, et Rothenfluh et al. 2006 2. Puisque l&#39;alcool sédation induite est corrélé avec la préférence boire de l&#39;alcool 3, ce test est pertinent pour l&#39;étude des troubles liés à l&#39;alcool. Le test décrit est basé sur sursaut induite géotaxie négative, et comment une exposition à l&#39;alcool interfère avec cette réponse innée. Avant de commencer l&#39;exposition, vérifiez que tous vos mouches montrent une réaction normale à un tel sursaut, c&#39;est à dire après avoir appuyé sur le bas les mouches dans le flacon alimentaires, ils devraient tous passer du fond dans un délai de 10 secondes d&#39;observation. Exemples de mouches ayant réduit géotaxie négatifs comprennent la saisie des mutants sensibles bang-5, et la locomotion déficientes mouches causée par la dégénérescence des neurones dopaminergiques 6. Même si le dosage décrit est relativement simple, deux variables méritent le plus d&#39;attention afin d&#39;assurer des résultats reproductibles. D&#39;abord, tous les flacons de l&#39;exposition devrait être le même volume. Cela comprend en s&#39;assurant que le fond du flacon contenant le même volume de nourriture + flacon Plug / coton balle, ainsi que d&#39;insérer la fiche contenant de l&#39;éthanol à la même distance pour chaque flacon. Un flacon complètement vide peut être utilisé pour ce test, mais elle conduit à des valeurs plus ST50, en diminuant le débit de l&#39;essai. Ce test est également approprié pour déterminer la quantité de mouches de tolérance se développer après exposition à l&#39;éthanol répéter. Un certain nombre de variables doivent être considérés lorsque vous faites des expositions répétées. Tout d&#39;abord, l&#39;intervalle entre l&#39;exposition est ici décrit comme 4 heures. Cette variable peut être changé, mais nous recommandons de ne pas être inférieure à 2 heures, pour assurer un dégagement d&#39;éthanol à partir de la première exposition. Voir Scholz et al. 2000 4 pour la cinétique de développement tolérance à l&#39;éthanol et de décadence. La seconde variable à considérer est la durée de la première exposition. Ceci a évidemment être le même pour le contrôle et les mouches expérimental. Toutefois, cela peut ne pas aboutir à la même quantité d&#39;éthanol livré à ces deux souches voler. Mouches Sedated absorber plus d&#39;éthanol que les mouches actif 2, donc une souche qui endort tôt peuvent recevoir une dose supérieure à une souche plus tard, sédatifs, et ainsi développer plus de tolérance en raison d&#39;une dose initiale plus élevée. Nous fermement en garde le lecteur lorsque l&#39;on compare le développement de souches de la tolérance qui montrent des différences dans le ST50 initiale (1). Le test que nous décrivons ici est un outil utile pour facilement et de façon reproductible la détermination des réponses comportementales des mouches à l&#39;alcool.

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a simple way to measure ethanol sensitivity in flies

De faibles doses d&#39;éthanol provoquer des mouches pour devenir hyperactif, tandis que des doses élevées sont sédatifs. La sensibilité à l&#39;éthanol induit une sédation d&#39;une souche donnée volée est corrélée avec la préférence d&#39;éthanol que la souche S, et donc la sédation est une mesure très pertinente pour étudier la génétique des réponses d&#39;alcool et de boire. Nous démontrons un moyen simple d&#39;exposer des mouches à l&#39;éthanol et mesurer ses effets enivrants. Le test que nous décrivons peut déterminer la sensibilité aiguë, ainsi que la tolérance éthanol induite par une exposition répétée. Il ne nécessite pas une configuration technique impliqués, et peuvent donc être appliquées dans n&#39;importe quel laboratoire avec les outils de base de la culture voler.

Watch this Scientific Journal Video about A Simple Way to Measure Ethanol Sensitivity in Flies at JoVE.com

A Simple Way to Measure Ethanol Sensitivity in Flies

Un test simple pour mesurer les effets sédatifs de l&#39;éthanol sur<em&gt; Drosophile</em&gt; Mouches, fondée sur la perte du réflexe de redressement, est décrite.

Une façon simple de mesurer la sensibilité d&#39;éthanol dans les mouches

Low doses of ethanol cause flies to become hyperactive, while high doses are sedating. The sensitivity to ethanol-induced sedation of a given fly strain ...

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Neuroscience

23.1K vues.  University of Texas Southwestern Medical Center. Un test simple pour mesurer les effets sédatifs de l'éthanol sur Drosophile Mouches, fondée sur la perte du réflexe de redressement, est décrite.

Vidéo: A Simple Way to Measure Ethanol Sensitivity in Flies

Hyponeophagia: A Measure of Anxiety in the Mouse

Before the present day, when fast-acting and potent rodenticides such as alpha-chloralose were not yet in use, the work of pest controllers was often hampered by a phenomenon known as "bait shyness". Mice and rats cannot vomit, due to the tightness of the cardiac sphincter of the stomach, so to overcome the problem of potential food toxicity they have evolved a strategy of first ingesting only very small amounts of novel substances. The amounts ingested then gradually increase until the animal has determined whether the substance is safe and nutritious. So the old rat-catchers would first put a palatable substance such as oatmeal, which was to be the vehicle for the toxin, in the infested area. Only when large amounts were being readily consumed would they then add the poison, in amounts calculated not to affect the taste of the vehicle. The poisoned bait, which the animals were now readily eating in large amounts, would then swiftly perform its function.
Bait shyness is now used in the behavioural laboratory as a way of measuring anxiety. A highly palatable but novel substance, such as sweet corn, nuts or sweetened condensed milk, is offered to the mice (or rats) in a novel situation, such as a new cage. The latency to consume a defined amount of the new food is then measured.
Robert M.J. Deacon can be reach at <a href="mailto:robert.deacon@psy.ox.ac.uk">robert.deacon@psy.ox.ac.uk</a>

Mice and rats, due to their innate cautiousness, are initially slow in consuming a novel food, particularly in a novel place. This hyponeophagia can readily be measured in the laboratory, even though laboratory animals are much less anxious than their wild counterparts

Hyponeophagia: Une mesure de l&#39;anxiété chez la souris

Before the present day, when fast-acting and potent rodenticides such as alpha-chloralose were not yet in use, the work of pest controllers was often ...

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Neurosciences

Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol. However, ethanol is typically self-administered orally, rather than intravenously like many other drugs of abuse. The pharmacokinetics of orally administered drugs are more complex than intravenously administered drugs. Because understanding the relationship between the pharmacological and behavioral effects of ethanol requires knowledge of the time course of ethanol reaching the brain during and after drinking, we use in vivo microdialysis and gas chromatography with flame ionization detection to monitor brain dialysate ethanol concentrations over time. 
Combined microdialysis-behavioral experiments involve the use of several techniques. In this article, stereotaxic surgery, behavioral training and microdialysis, which can be adapted to test a multitude of self-administration and neurochemical centered hypotheses, are included only to illustrate how they relate to the subsequent phases of sample collection and dialysate ethanol analysis. Dialysate ethanol concentration analysis via gas chromatography with flame-ionization detection, which is specific to ethanol studies, is described in detail. Data produced by these methods reveal the pattern of ethanol reaching the brain during the self-administration procedure, and when paired with neurochemical analysis of the same dialysate samples, allows conclusions to be made regarding the pharmacological and behavioral effects of ethanol.

A method to determine the time course of ethanol concentration in the brains of rats during operant ethanol self-administration is described. Gas chromatography with flame ionization detection is used to quantify ethanol in the dialysate samples, because it has the sensitivity required for the small volumes that are generated.

Microdialyse d&#39;éthanol au cours de l&#39;éthanol détermination opérant auto-administration et d&#39;éthanol par chromatographie en phase gazeuse

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol.  ...

The FlyBar: Administering Alcohol to Flies

Fruit flies (Drosophila melanogaster) are an established model for both alcohol research and circadian biology. Recently, we showed that the circadian clock modulates alcohol sensitivity, but not the formation of tolerance. Here, we describe our protocol in detail. Alcohol is administered to the flies using the FlyBar. In this setup, saturated alcohol vapor is mixed with humidified air in set proportions, and administered to the flies in four tubes simultaneously. Flies are reared under standardized conditions in order to minimize variation between the replicates. Three-day old flies of different genotypes or treatments are used for the experiments, preferably by matching flies of two different time points (e.g., CT 5 and CT 17) making direct comparisons possible. During the experiment, flies are exposed for 1 hr&#160;to the pre-determined percentage of alcohol vapor and the number of flies that exhibit the Loss of Righting reflex (LoRR) or sedation are counted every 5 min. The data can be analyzed using three different statistical approaches. The first is to determine the time at which 50% of the flies have lost their righting reflex and use an Analysis of the Variance (ANOVA) to determine whether significant differences exist between time points. The second is to determine the percentage flies that show LoRR after a specified number of minutes, followed by an ANOVA analysis. The last method is to analyze the whole times series using multivariate statistics. The protocol can also be used for non-circadian experiments or comparisons between genotypes.

Drosophila has emerged as a significant model system for dissecting the cellular and molecular underpinnings of behavioral responses to alcohol. Here we present a protocol for the collection of alcohol sensitivity data in a circadian context that can be easily applied to other experiments and is well-suited for undergraduate research.

La barre stabilisatrice: Administration de l&#39;alcool à mouches

Fruit flies (Drosophila melanogaster) are an established model for both alcohol research and circadian biology. Recently, we showed that the circadian ...

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

Tranche de cerveau Biotinylation: Une<em&gt; Ex Vivo</em&gt; Approche de Mesure région spécifique membrane de protéines plasmatiques traite des adultes neurones

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Une méthode simple et peu coûteuse pour déterminer Sensibilité au froid et adaptation chez la souris

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in their etiology. The fruit fly, Drosophila melanogaster, is a powerful model for exploring molecular-genetic mechanisms underlying alcohol-related behaviors and therefore holds great promise for identifying and understanding the function of genes that influence AUD. The use of the Drosophila model for these types of studies depends on the availability of assays that reliably measure behavioral responses to ethanol. This report describes an assay suitable for assessing ethanol sensitivity and rapid tolerance in flies. Ethanol sensitivity measured in this assay is influenced by the volume and concentration of ethanol used, a variety of previously reported genetic manipulations, and also the length of time the flies are housed without food immediately prior to testing. In contrast, ethanol sensitivity measured in this assay is not affected by the vigor of fly handling, sex of the flies, and supplementation of growth medium with antibiotics or live yeast. Three different methods for quantitating ethanol sensitivity are described, all leading to essentially indistinguishable ethanol sensitivity results. The scalable nature of this assay, combined with its overall simplicity to set-up and relatively low expense, make it suitable for small and large scale genetic analysis of ethanol sensitivity and rapid tolerance in Drosophila.

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Straightforward assays for measuring ethanol sensitivity and rapid tolerance in Drosophila facilitate the use of this model organism for investigating these important ethanol-related behaviors. Here, a relatively simple, scalable assay for measuring ethanol sensitivity and rapid tolerance in flies is described.

Un, test de comportement évolutives et économiques pour mesurer l&#39;éthanol sédation sensibilité et la tolérance rapide dans<em&gt; Drosophila</em

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in ...

A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA

MicroRNAs (miRNAs) are key regulators of gene expression. In the brain, vital processes like neurodevelopment and neuronal functions depend on the correct expression of microRNAs. Perturbation of microRNAs in the brain can be used to model neurodegenerative diseases by modulating neuronal cell death. Currently, stereotactic injection is used to deliver miRNA knockdown agents to specific location in the brain. Here, we discuss strategies to design antagomirs against miRNA with locked nucleotide modifications (LNA). Subsequently describe a method for brain specific delivery of antagomirs, uniformly across different regions of the brain. This method is simple and widely applicable since it overcomes the surgery, associated injury and limitation of local delivery in stereotactic injections. We prepared a complex of neurotropic, cell-penetrating peptide Rabies Virus Glycoprotein (RVG) with antagomir against miRNA-29 and injected through tail vein, to specifically deliver in the brain. The antagomir design incorporated features that allow specific targeting of the miRNA and formation of non-covalent complexes with the peptide. The knock-down of the miRNA in neuronal cells, resulted in apoptotic cell death and associated behavioural defects. Thus, the method can be used for acute models of neuro-degeneration through the perturbation of miRNAs.

MicroRNAs play crucial roles in the brain and are potential targets for modeling neuro-degeneration. However, perturbing miRNA levels is challenging due to the short length of miRNA and inaccessibility of the brain tissue. This video presents a method for antagomir design and brain specific delivery using a neuropeptide in mice.

Une alternative simple à injection stéréotaxique pour Brain Knockdown spécifique de miARN

MicroRNAs (miRNAs) are key regulators of gene expression.  In the brain, vital processes like neurodevelopment and neuronal functions depend on the ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

Un moyen simple pour mesurer Transformations en récompense le comportement de recherche Utilisation<em&gt; Drosophila melanogaster</em

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

Une seule étape simple protocole Dissection pour Whole-mount Préparation des adultes<em&gt; Drosophila</em&gt; Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying Alzheimer's disease, Parkinson's disease, depression, and anxiety. Traditionally, microdialysis has been the most common (lauded) technique to examine neurotransmitter changes that occur in these disorders. But because microdialysis has the ability to measure slow 1-20 minute changes across large areas of tissue, it has the disadvantage of invasiveness, potentially destroying intrinsic connections within the brain and a slow sampling capability. A relatively newer technique, the microelectrode array (MEA), has numerous advantages for measuring specific neurotransmitter changes within discrete brain regions as they occur, making for a spatially and temporally precise approach. In addition, using MEAs is minimally invasive, allowing for measurement of neurotransmitter alterations in vivo. In our laboratory, we have been specifically interested in changes in the neurotransmitter, glutamate, related to Alzheimer's disease pathology. As such, the method described here has been used to assess potential hippocampal disruptions in glutamate in a transgenic mouse model of Alzheimer's disease. Briefly, the method used involves coating a multi-site microelectrode with an enzyme very selective for the neurotransmitter of interest and using self-referencing sites to subtract out background noise and interferents. After plating and calibration, the MEA can be constructed with a micropipette and lowered into the brain region of interest using a stereotaxic device. Here, the method described involves anesthetizing rTg(TauP301L)4510 mice and using a stereotaxic device to precisely target sub-regions (DG, CA1, and CA3) of the hippocampus.

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer&#039;s Mouse Models

Here, we describe the setup, software navigation, and data analysis for a spatially and temporally precise method of measuring tonic and phasic extracellular glutamate changes in vivo using enzyme-linked microelectrode arrays (MEA).