Name: a simple way to measure ethanol sensitivity in flies
Uploaded: 2011-02-19T17:00:00
Duration: 11 min 15 s
Description: Watch this Scientific Journal Video about A Simple Way to Measure Ethanol Sensitivity in Flies at JoVE.com

Ringraziamo Aylin Rodan per la lettura critica del manoscritto, ed i membri del laboratorio di discussione utile. Questa ricerca è sostenuta dal NIH, R01AA019526.

1. Creazione della camera di esposizione <ol><li> Tagliare una spina fiala a metà con una lama di rasoio. In alternativa, utilizzare batuffoli di cotone. </li><li> Spingere la metà spina fiala, o il batuffolo di cotone, sul fondo del flacone cibo per fare una superficie liscia che vola non si blocca in </li><li> Se rendendo camere di esposizione multipla, accertarsi che la spina flaconcino e cibo occupano lo stesso volume in ogni camera. Un segno sul lato del flaconcino può facilitare questo. </li></ol> 2. Esponendo le mosche <ol><li> Colleziona 8 vola di un sesso, età 1-5 giorni, il giorno prima dell&#39;esposizione in flaconi cibo normale, senza lievito. </li><li> Etichettarli in codice, in modo che lo sperimentatore è cieco al genotipo reale / ceppo usato. </li><li> Trasferire le otto vola nelle camere di esposizione toccando. Non anestetizzare. </li><li> Aggiungere una piccola quantità di colorante alimentare, ad esempio acido blu 9, a 200 etanolo prova per renderlo facilmente visibile. </li><li> Rivestire il fondo di una spina fiala, o batuffolo di cotone, con 0,5 ml di etanolo. </li><li> Inserire la spina di etanolo nel flaconcino di esposizione. Assicurarsi che l&#39;alcol è rivolto nel flaconcino, e non verso la parete del flaconcino. </li><li> Avviare un timer che conta su. </li></ol> 3. Record etanolo-indotta intossicazione <ol><li> Ogni minuto dopo che il timer viene avviato, toccare la camera di esposizione in panchina far trasalire le mosche e osservare per dieci secondi. </li><li> Registrare il numero di mosche stazionaria per ogni minuto. Le mosche sono stazionari se non possono scendere le spalle, rimangono in una posizione, o in rapida vibrare le ali durante l&#39;intero dieci secondi periodo di osservazione. Le mosche ultimi sono considerati fissi, perché la stragrande maggioranza di queste mosche sono fermi tra i periodi di rapido movimento dell&#39;ala. Le mosche non sono stazionari se possono scendere le spalle e piedi e / o sulla parete flaconcino. </li><li> Una prova di esposizione è terminata quando 4 o più mosche sono registrate come stazionaria. </li><li> Gettare le mosche e lasciare che l&#39;aria camera di esposizione per poi riutilizzare. </li></ol> 4. Determinare il tempo al 50% sedazione Il tempo per il 50% sedazione (ST50) è il tempo dopo l&#39;inizio dell&#39;esposizione etanolo quando quattro degli otto mosche rimangono stazionari. Ciò è determinato come segue: In entrambi i casi: il minuto prima misurazione quando esattamente quattro mosche sono stazionarie. Oppure: se meno di quattro mosche (Y1) sono fermi ad un certo punto il tempo (minuti X), e più di quattro (Y2) al punto successivo tempo di misura (minuti X +1), poi linearmente interpolare i ST50 con la formula: ST50 = (4 - Y1) / (Y2 - Y1) + X 5. Ripetere l&#39;esposizione di determinare tolleranza Esponendo le mosche <ol><li> Determinare la ST50 (peso) per la wild-type di controllo utilizzati nell&#39;esperimento. </li><li> Esporre i gruppi sperimentali di mosche in etanolo come descritto sopra. </li><li> Misurare la loro ST50 (1), ma continua a esporre fino a raggiungere il tempo 2x ST50 (in peso). </li><li> Trasferire le mosche nuovamente dentro una fiala vuota cibo, e tenere premuto per 4 ore. </li><li> Ri-esporre le mosche, e determinare il ST50 (2) di esposizione 2. </li></ol> Determinare tolleranza Calcolare la tolleranza in% (ST50 (2) / ST50 (1) - 1) x100. 6. Rappresentante dei risultati: Qui, ci sono alcuni risultati rappresentativi abbiamo ottenuto con 6-8 fiale di mosche per genotipo e la dose. Se l&#39;errore standard è sostanzialmente più grande, poi un problema sistematico probabilmente si è verificato. Assicurarsi che l&#39;etanolo si affaccia nelle camere di esposizione, e che le camere sono lo stesso volume. <img src="/files/ftp_upload/2541/2541fig1.jpg" alt="Figura 1" /> Figura 1. Curva dose-risposta di tipo selvatico vola esposti a percentuali crescenti di etanolo. Otto fiale di mosche sono state esposte per dose e sesso. Moscerini maschi e femmine mostrano la ST50 stesso ad ogni dose (p&gt; 0,22, 2-way ANOVA), mentre l&#39;ST50 attraverso le dosi riduce significativamente con l&#39;aumento della dose di etanolo (p &lt;0,001). Si noti che vola da sovraffollati bottiglie possono essere notevolmente più piccoli di quelli da bottiglie sano, e questa differenza di dimensioni può influenzare la ST50. Semi di ogni bottiglia con le femmine 15-20 che per 1-2 giorni per ottenere la densità ottimale larvale a bottiglia. <img src="/files/ftp_upload/2541/2541fig2.jpg" alt="Figura 2" /> Figura 2. Esporre vola a uno etanolo al 90%, o 100% riproduce descritti in precedenza etanolo-resistenza causata dal bianco rabbit1 mutation2 (p &lt;0,001, 2-way ANOVA). <img src="/files/ftp_upload/2541/2541fig3.jpg" alt="Figura 3" /> Figura 3. Ripetete l&#39;esposizione al 100% di etanolo, distanziate da un intervallo di recupero di 4 ore, causa un significativo aumento ST50 dopo la seconda esposizione (p &lt;0,001, paired t-Test). Si noti che le mosche sono stati esposti per 24 minuti durante la prima esposizione (corrispondente al doppio della loro ve na ST50). Esposizione per un tempo più corto (12 minuti) non ha causato un aumento significativo ST50 su ri-esposizione (dati non riportati).

<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>

Nessun conflitto di interessi dichiarati.

In questa presentazione, abbiamo delineato un modo semplice per determinare l&#39;etanolo-indotta intossicazione sulla base di precedenti saggi descritto da Bainton et al. 2000 1, e Rothenfluh et al. 2006 2. Dal momento che l&#39;alcol-indotta sedazione è correlato con la preferenza di bere alcolici 3, questo saggio è rilevante per lo studio dei disturbi da uso di alcol. Il test descritto è basato sulla geotassi negativo sussultare-indotta, e come un&#39;esposizione di alcol interferisce con questa risposta innata. Prima di iniziare le esposizioni, assicurarsi che tutti i moscerini mostrano una normale risposta a un tale spavento, cioè dopo aver premuto verso il basso le mosche nel flacone cibo, esse dovrebbero salire dal basso in una volta 10 seconda osservazione. Esempi di mosche aver ridotto geotassi negativi includono bang-sensibili mutanti sequestro 5, e locomozione con deficit vola causati dalla degenerazione dei neuroni dopaminergici 6. Anche se il test descritto è relativamente semplice, due variabili meritano la massima attenzione per garantire risultati riproducibili. In primo luogo, tutti i flaconcini esposizione dovrebbe essere lo stesso volume. Ciò include fare in modo che la parte inferiore della fiala contiene la stessa quantità di cibo + fiala spina / cotone palla, così come l&#39;inserimento della spina etanolo contenenti la stessa distanza per ogni flacone. Un flaconcino completamente vuoto può essere utilizzato per questa analisi, ma porta a valori più ST50, diminuendo la velocità del test. Questo test è adatto anche per determinare la quantità di mosche sviluppare tolleranza in caso di esposizione ripetuta etanolo. Un certo numero di variabili devono essere considerati quando si fa esposizioni ripetute. In primo luogo, l&#39;inter-esposizione intervallo è qui descritta come 4 ore. Questa variabile può essere cambiato, ma si raccomanda di non essere inferiore a 2 ore, per garantire uno spazio di etanolo dalla prima esposizione. Vedi Scholz et al. 2000 4 per la cinetica di sviluppo dell&#39;etanolo tolleranza e di degrado. La seconda variabile da considerare è la durata della prima esposizione. Questo è ovvio che essere lo stesso per il controllo e le mosche sperimentale. Tuttavia, questo non può comportare la stessa quantità di etanolo consegnato ad entrambi questi ceppi volare. Vola sedato assorbono più etanolo che vola attivo 2, quindi un ceppo che seda anteriore può ricevere una dose maggiore di un sedativo dopo ceppo e, quindi, sviluppare una maggiore tolleranza a causa di una dose iniziale più elevato. Siamo fermamente avvisare il lettore durante il confronto lo sviluppo di tolleranza di ceppi che mostrano le differenze nei primi ST50 (1). Il test che descriviamo qui è uno strumento utile in modo semplice e riproducibile per determinare le risposte comportamentali vola ad alcol.

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a simple way to measure ethanol sensitivity in flies

Basse dosi di etanolo causa vola a diventare iperattivi, mentre alte dosi sono sedativi. La sensibilità all&#39;etanolo sedazione indotta di un ceppo di volare dato è correlato con la preferenza etanolo che il ceppo s, e quindi la sedazione è una misura molto rilevante per studiare la genetica di risposte e di bere alcolici. Si dimostra un modo semplice di esporre vola a etanolo e misurare i suoi effetti inebrianti. Il test che descriviamo in grado di determinare la sensibilità acuta, così come la tolleranza etanolo indotta da esposizione ripetuta. Non richiede una configurazione tecnica in gioco e possono quindi essere applicato in qualsiasi laboratorio con strumenti di base della cultura volare.

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 semplice per misurare gli effetti sedativi di etanolo<em&gt; Drosophila</em&gt; Mosche, basato sulla perdita del riflesso di raddrizzamento, è descritto.

Un modo semplice per misurare sensibilità etanolo in Flies

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

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: una misura di ansia nel 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 ...

Ricerca

Neuroscienze

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.

Microdialisi di etanolo durante la determinazione operante auto-amministrazione ed etanolo etanolo mediante gascromatografia

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.

Flybar: Amministrazione alcol a 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.

Slice Cervello biotinilazione: An<em&gt; Ex Vivo</em&gt; Approccio alla Misura Regione-specifica proteina Tratta plasma membrana in neuroni adulti

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.

Un metodo semplice e poco costoso per la determinazione fredda sensibilità e adattamento nei topi

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 poco costoso, test comportamentale scalabile per la misurazione etanolo sedazione sensibilità e tolleranza Rapid 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.

Una alternativa semplice per iniezione stereotassica per Brain Knockdown specifico 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.

Un modo semplice per misurare alterazioni in ricompensa il comportamento di ricerca Utilizzando<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.

Un semplice passo-dissezione Protocollo per tutto il montaggio Preparazione per adulti<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.

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).