Wir bedanken uns bei Aylin Rodan für die kritische Durchsicht des Manuskripts und die Mitglieder des Labors für hilfreiche Diskussionen. Diese Forschung wird durch die NIH, R01AA019526 unterstützt.

1. Erstellen der Bestrahlungskammer <ol><li> Schneiden Sie ein Fläschchen Stecker in zwei Hälften mit einer Rasierklinge. Alternativ können Sie auch Wattebäusche. </li><li> Schieben Sie das Fläschchen Plug Hälfte oder die Watte, auf den Boden eines Lebensmittels Fläschchen um eine glatte Oberfläche, dass Fliegen nicht in. stecken machen </li><li> Wenn Sie mehrere Exposition Kammern, stellen Sie sicher, dass die Fläschchen Plug-and-Food bis zu dem gleichen Volumen in jeder Kammer. Eine Markierung auf der Seite der Flasche kann dies erleichtern. </li></ol> 2. Offenlegen der Fliegen <ol><li> Sammelt 8 fliegt von einem Geschlecht, Alter 1-5 Tage, dem Tag vor der Bestrahlung in regelmäßigen Essen Fläschchen ohne Hefe. </li><li> Label sie im Code, so dass der Experimentator den tatsächlichen Genotyp / Stamm ist geblendet. </li><li> Übertragung der acht fliegt in die Exposition Kammern durch Antippen. Nicht zu betäuben. </li><li> Fügen Sie eine kleine Menge der Nahrung Farbstoff, zB Acid Blue 9, bis 200 Proof Ethanol, damit es gut sichtbar. </li><li> Streichen Sie die Unterseite einer Flasche Stecker, oder Wattebausch, mit 0,5 ml Ethanol. </li><li> Legen Sie die Ethanol-Stecker in die Exposition Durchstechflasche. Stellen Sie sicher, dass der Alkohol in das Fläschchen, und nicht gegen die Wand der Durchstechflasche nach. </li><li> Starten Sie einen Timer auf. </li></ol> 3. Rekord Ethanol-induzierten Rausch <ol><li> Jede Minute, nachdem der Timer gestartet wird, tippen Sie auf die Exposition Kammer auf der Bank, um die Fliegen zu erschrecken und zu beobachten sie für 10 Sekunden. </li><li> Notieren Sie die Anzahl der stationären Fliegen für jede Minute. Fliegen sind stationär, wenn sie nicht aussteigen dem Rücken, an einem Ort bleiben, oder schnell zu vibrieren ihre Flügel über den gesamten Zehn-Sekunden-Beobachtung-Periode. Letzterer fliegt gelten als stationäre, weil die überwiegende Mehrheit dieser fliegt stationär zwischen den Perioden des schnellen Flügel Bewegung sind. Fliegen sind nicht stationär, wenn sie aussteigen dem Rücken und zu Fuß entfernt und / oder bis das Fläschchen Wand. </li><li> Eine Exposition Studie ist abgeschlossen, wenn 4 oder mehr Fliegen als stationäre aufgezeichnet. </li><li> Entsorgen Sie fliegt und lassen Sie die Belichtung Kammer Luft für die spätere Wiederverwendung. </li></ol> 4. Bestimmen Sie die Zeit auf 50% Sedierung Die Zeit bis zu 50% Sedierung (ST50) ist die Zeit nach dem Start der Ethanol-Exposition, wenn vier der acht fliegt stationär bleiben. Dieser wird wie folgt bestimmt: Entweder: die erste Messung Minute, wenn genau vier fliegt stationär sind. Oder: wenn weniger als vier Fliegen (Y1) sind an einem Zeitpunkt (Minute X), und mehr als vier (Y2) bei der nächsten Messung Zeitpunkt (Minute X +1), dann linear interpoliert die ST50 mit Hilfe der Formel stationär: ST50 = (4 - Y1) / (Y2 - Y1) + X 5. Wiederholen Sie die Exposition gegenüber Toleranz zu bestimmen Offenlegen der Fliegen <ol><li> Bestimmen Sie die ST50 (wt) für die Wildtyp-Kontrollstamm in Ihrem Experiment verwendet. </li><li> Setzen Sie den experimentellen Gruppen von Fliegen zu Ethanol wie oben beschrieben. </li><li> Messen Sie ihre ST50 (1), aber weiterhin aussetzen, bis Sie die Zeit, 2x ST50 (wt) zu erreichen. </li><li> Übertragen Sie die fliegt zurück in ein leeres Fläschchen Nahrung, und halten Sie für 4 Stunden. </li><li> Re-Setzen Sie die Fliegen, und bestimmen Sie die ST50 (2) der Exposition 2. </li></ol> Bestimmen Toleranz Berechnen Toleranz in% (ST50 (2) / ST50 (1) - 1) x100. 6. Repräsentative Ergebnisse: Sehen Sie hier sind einige repräsentative Ergebnisse, die wir mit 6-8 Fläschchen Fliegen pro Genotyp und Dosis erhalten. Wenn Ihr Standardfehler ist wesentlich größer, dann ein systematisches Problem wahrscheinlich aufgetreten. Stellen Sie sicher, dass das Ethanol in die Exposition Kammern Gesichter, und dass die Kammern das gleiche Volumen sind. <img src="/files/ftp_upload/2541/2541fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Dosis-Wirkungs-Kurve von Wildtyp-Fliegen auf steigende Prozentzahlen von Ethanol ausgesetzt. Acht Fläschchen Fliegen wurden pro Dosis und Geschlecht ausgesetzt. Männliche und weibliche Fliegen zeigen die gleichen ST50 bei jeder Dosis (p&gt; 0,22, 2-Wege-ANOVA), während die ST50 über Dosierungen sinkt signifikant mit steigenden Ethanol-Dosis (p &lt;0,001). Beachten Sie, dass aus überfüllten Flaschen fliegen kann wesentlich kleiner als jene von gesunden Flaschen, und dies Größenunterschied kann die ST50 beeinflussen. Seed jede Flasche mit 15-20 Frauen über 1-2 Tage, um eine optimale Larven Dichte pro Flasche zu bekommen. <img src="/files/ftp_upload/2541/2541fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Exposing fliegt entweder 90% oder 100% Ethanol gibt den zuvor beschriebenen Ethanol-Widerstand durch den weißen rabbit1 mutation2 (p &lt;0,001, 2-Wege-ANOVA) verursacht. <img src="/files/ftp_upload/2541/2541fig3.jpg" alt="Abbildung 3" /> Abbildung 3. Repeat Exposition gegenüber 100% Ethanol, im Abstand von 4 Stunden recovery interval, führt zu einem deutlichen Anstieg der ST50 nach dem zweiten Exposition (p &lt;0,001, gepaarter t-Test). Beachten Sie, dass die Fliegen für 24 Minuten wurden während der ersten Exposition (entsprechend auf das Doppelte ihrer na ve ST50) ausgesetzt. Exposure für einen kürzeren Zeitraum (12 Minuten) nicht zu einem signifikanten Anstieg in ST50 bei wiederholter Exposition (Daten nicht gezeigt).

<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>

In dieser Präsentation haben wir eine einfache Möglichkeit, Ethanol-induzierten Rausch auf der Grundlage früherer Untersuchungen durch Bainton et al bestimmen skizziert. 2000 1 und Rothenfluh et al. 2006 2. Da Alkohol-induzierte Sedierung mit Alkohol trinken bevorzugt 3 korreliert ist, ist dieser Test relevant für das Studium der durch Alkoholkonsum bedingte Störungen. Der beschriebene Assay basiert auf erschrecken-induzierten negativen Geotaxis basiert, und wie ein Alkohol Exposition stört diese angeborene Reaktion. Vor Beginn der Aufnahmen sicher, dass alle Ihre Fliegen zeigen eine normale Reaktion auf eine solche erschrecken, dh nach dem Klopfen Sie die Fliegen in der Lebensmittel-Fläschchen, sie sollten alle nach oben aus dem Boden in einem 10 Sekunden Beobachtungszeit. Beispiele von Fliegen mit einer verringerten negativen Geotaxis gehören Knall-und Kleinschreibung Beschlagnahme Mutanten 5, und Lokomotion-defizienten Fliegen durch die Degeneration von Dopamin-Neuronen 6 verursacht. Auch wenn die beschriebene Assay ist relativ einfach, verdienen zwei Variablen die größte Aufmerksamkeit, um reproduzierbare Ergebnisse zu gewährleisten. Erstens sollten alle Exposition Fläschchen die gleiche Menge sein. Dazu gehört auch, dass der Boden des Fläschchens die gleiche Menge an Nahrung + Fläschchen Stecker / Wattebausch, sowie das Einlegen der Ethanol-haltigen Stecker die gleiche Strecke für jede Flasche enthält. Eine komplett leere Flasche kann für diesen Test verwendet werden, aber es führt zu längeren ST50-Werte, die Verringerung der Durchsatz des Assays. Dieser Test eignet sich auch zur Höhe der Toleranz fliegt bestimmen zu entwickeln, auf Wiederholung Ethanol Exposition. Eine Reihe von Variablen sollte bei der Wiederholung Belichtungen werden. Erstens ist die inter-Belichtungsintervall hier als 4 Stunden beschrieben. Diese Variable kann geändert werden, aber wir empfehlen es nicht weniger als 2 Stunden, um Abstand von Ethanol aus der ersten Exposition zu gewährleisten. Siehe Scholz et al. 2000 4 für die Kinetik der Ethanol-Toleranz Entwicklung und Verfall. Die zweite Variable zu prüfen, ist die Dauer der ersten Exposition. Dies hat natürlich das gleiche für die Kontrolle und die experimentellen Fliegen werden. Dies kann jedoch nicht in der gleichen Menge Ethanol geliefert, diese beiden fliegen Stämme führen. Sedated fliegt absorbieren mehr Ethanol als aktive Fliegen 2, also eine Sorte, die früher möglicherweise eine höhere Dosis als zu einem späteren sedierenden Belastung erhalten sediert, und entwickeln so mehr Toleranz durch eine höhere Anfangsdosis. Es wird dringend warnen die Leser beim Vergleich Toleranz Entwicklung der Stämme, die Unterschiede in den Anfangsbedingungen ST50 (1) zu zeigen. Der Test wir hier beschreiben, ist ein nützliches Werkzeug in leicht und reproduzierbar zu ermitteln, fliegt Verhaltensreaktionen auf Alkohol.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Exposure vials</td>
<td>&nbsp;</td>
<td>www.flystuff.com</td>
<td>32-116</td>
<td>&nbsp;</td>
<tr>
<td>Plugs</td>
<td>&nbsp;</td>
<td>www.flystuff.com</td>
<td>49-102</td>
<td>&nbsp;</td>
<tr>
<td>Acid Blue 9</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>861146</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

a simple way to measure ethanol sensitivity in flies

Niedrige Dosen von Ethanol verursachen fliegt hyperaktiv werden, während hohe Dosen sedierende werden. Die Empfindlichkeit gegenüber Ethanol-induzierten Sedierung eines bestimmten fliegen Stamm mit, dass der Stamm s Ethanol Präferenz korreliert und daher Sedierung ist eine sehr relevante Maßnahme, um die Genetik der Alkohol Antworten und trinken zu studieren. Wir zeigen einen einfachen Weg, fliegt aussetzen Ethanol und messen ihre berauschende Wirkung. Der Test beschreiben wir können bestimmen, akute Empfindlichkeit sowie Ethanol-Toleranz durch wiederholte Exposition induziert. Es erfordert keine technisch aufwendige Setup, und kann daher in jedem Labor mit grundlegenden fliegen Kultur-Tools angewendet werden.

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

Ein einfacher Test, um die sedierende Wirkung von Ethanol auf Maß<em&gt; Drosophila</em&gt; Fliegen, über den Verlust des Gleichgewichtsreflexes basiert, wird beschrieben.

Ein einfacher Weg zu Ethanol Empfindlichkeit in Flies Measure

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 ...

a-simple-way-to-measure-ethanol-sensitivity-in-flies

Research

JoVE Journal

Neuroscience

23.1K Aufrufe.  University of Texas Southwestern Medical Center. Ein einfacher Test, um die sedierende Wirkung von Ethanol auf Maß Drosophila Fliegen, über den Verlust des Gleichgewichtsreflexes basiert, wird beschrieben.

Serie: 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: A Measure of Anxiety in der Maus

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 ...

Forschung

Neurowissenschaften

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.

Microdialysis Ethanol während operante Ethanol Selbstverwaltung und Ethanol Bestimmung durch Gaschromatographie

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.

Paddelstange: Verwaltung Alkohol zu Flies

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.

Hirnschnitt Biotinylierung: Eine<em&gt; Ex Vivo</em&gt; Ansatz zur Region spezifische Plasma Membrane Protein Handel Erwachsene Neuronen messen

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.

Eine einfache und kostengünstige Methode zur Bestimmung der Überempfindlichkeit auf Kälte und Anpassung an der Maus

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.

Eine preiswerte, skalierbare Behavioral Assay zur Messung von Ethanol Sedierung Empfindlichkeit und Rapid Toleranz in<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.

Eine einfache Alternative zur stereotaktische Injektion für Hirnspezifische Knockdown von miRNA

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.

Ein einfacher Weg zu messen Veränderungen in Reward-seeking Verhalten verwenden<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.

Eine einfache Ein-Schritt-Dissection-Protokoll für Whole-mount Vorbereitung von Adult<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).