These studies were supported by grants from the National Institutes of Health, National Institute for Alcoholism and Alcohol Abuse to M.G. (P20AA017828, R01AA020634, P50 AA022537). The authors thank Jill Bettinger for helpful discussions and Jacqueline DeLoyht for technical assistance.

1. Giorno Prima Assay <ol><li> Raccogliere mosche in fiale alimenti freschi in gruppi di 11 (sesso unico) in breve (1-5 minuti) CO 2. </li><li> Consentire mosche di recuperare O / N in fiale alimentari in uno spazio ambiente controllato (tipicamente 25 ° C, 60% umidità relativa, 12 hr luce / buio ciclo). </li><li> Preparare la soluzione di etanolo (s) diluendo puro (100%) di etanolo in purificato (≥18 MW) di acqua a concentrazione finale (s) appropriato per l&#39;esperimento previsto. Lasciare la soluzione (s) per tornare alla RT O / N. Nota: La diluizione di etanolo è esotermica. </li></ol> 2. Giornata di test <ol><li> Per ogni flacone di mosche da testare, preparare (i) un ambiente pulito, vuoto alimentare flacone; Per esempio, il test flacone, (ii) un nuovo plug acetato di cellulosa, (iii) un tappo silicone e (iv) 1 ml di soluzione di etanolo (vedi Tabella 3). </li><li> Preparare sala prove regolando la temperatura a 20-25 ° C e umidità relativa al 55-65%. </li><li> Hanno un altro lavoratoreassegnare un codice univoco a ciascun gruppo di flaconi e registrare il codice per dopo. Posizionare fiale codificati con le mosche in sala prove per ambientarsi per pochi minuti. </li><li> Etichetta fiale test vuoti per abbinare i codici di fiale vola da 2.3. </li><li> Costruire un registro test cartaceo inserendo i codici in colonne (una colonna / flaconcino) in un foglio di calcolo simile alla Tabella 1. </li><li> Utilizzo del registro di test come una guida, organizzare fiale alimentari codificati con mosche e fiale di test vuoti in corrispondenza array in sala prove. Nota: Un ragionevole numero massimo di fiale di test è 24 (cioè, 6 set di 4 flaconcini ciascuna). </li><li> Trasferimento vola da flaconcini alimentari in abbinati / etichettati fiale test vuoto e inserire immediatamente spine in acetato di cellulosa in fiale di prova fino a quando le spine di acetato di cellulosa sono 2 cm sotto le cime delle fiale. </li><li> Hereafter, gestire ogni fila di quattro fiale come insieme ad sfalsati min intervalli di 1. </li><li> Per la valutazione tempo 0, cogliere ogni flaconcino individualmente with pollice e l&#39;indice, toccare delicatamente sul tavolo tre volte per battere mosche al fondo della fiala, attendere 30 sec e poi contare il numero di mosche che sono immobili / morti. Registrare il numero di mosche immobili / morte per ogni flacone al tempo 0 min nel registro test cartaceo. </li><li> Inizia timer conteggio continuamente al tempo 0 e iniziare immediatamente aggiungendo 1 ml di etanolo a tappi acetato di cellulosa in fiale per la prima riga / set di 4 fiale. Aggiungere 1 ml di etanolo per i tappi di acetato di cellulosa in fiale a intervalli di 5 sec nell&#39;ordine in cui saranno testati. Aggiungere etanolo per i tappi di acetato di cellulosa in un movimento circolare in modo che l&#39;etanolo viene assorbita in modo uniforme in tutto i tappi di acetato di cellulosa. Quando l&#39;etanolo è stato aggiunto a tutti i 4 flaconcini di test nel set, inserire un tappo di silicone in ogni fiala per sigillarlo. </li><li> A volte 1, 2, 3, 4 e 5 min, aggiungere 1 ml di etanolo al secondo, terzo, quarto, quinto e sesto set di 4 flaconcini, rispettivamente. Continuare inserendo tappi in silicone dopoaggiungendo etanolo per ciascun set di 4 fiale. </li><li> Al momento 6 min, testare la prima serie di 4 flaconi afferrando ogni flacone con il pollice e l&#39;indice, picchiettando delicatamente sul tavolo tre volte per battere mosche al fondo del flacone, in attesa 30 sec e poi contare e registrare il numero totale di mosche che sono sedati. Score vola come sedato se (i) stanno sul pavimento del flacone, ma non camminano o (ii) si trovano sulla schiena, con o senza sbattere le ali. </li><li> Maneggiare ogni flacone all&#39;interno del set ad intervalli di 5 sec utilizzando il programma in tabella 2. </li><li> A volte 7, 8, 9, 10 e 11 min, testare il secondo, terzo, quarto, quinto e sesto set di fiale, rispettivamente, come fatto per la prima serie. </li><li> Al tempo 12 min, testare la prima serie di 4 provette nuovamente come descritto in 2.12 e continuare il test secondo, terzo, quarto, quinto e sesto serie di flaconi a 13, 14, 15, 16 e 17 minuti, rispettivamente. </li><li> Continuare mosche test come descritto in 2.12 fino a quando tutte le mosche sono sedated. </li><li> Inserire il numero totale di mosche in ogni fiala nel registro test cartaceo. Censore mosche immobile / morti al tempo 0 dal numero totale di mosche. </li><li> Calcolare i cento mosche attivi ad ogni valutazione di time-point e dati plot come% mosche attivi (asse y) in funzione del tempo (asse x). Quantificare etanolo sedazione interpolando sedazione Tempo 50 valori (ST50, tempo al 50% sedazione) dal terzo ordine curva polinomiale o sigmoidale adatta o calcolare l&#39;area sotto la curva. </li><li> Compilare i dati provenienti da flaconi decodificati e effettuare analisi statistiche (ad esempio, una via ANOVA con Bonferroni test di confronto multiplo), secondo il disegno sperimentale. </li></ol>

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The authors have nothing to disclose.

Test semplice che riproducibile quantificare fenotipi significativi sono di grande valore per l&#39;analisi del comportamento. Il lavoro qui descritto affronta diversi aspetti pratici di un test per misurare la sensibilità etanolo sedazione e tolleranza rapida in Drosophila. Sebbene non sia un obiettivo di questo lavoro, analisi comportamentali sono facilitate mantenendo l&#39;ambiente e lo sfondo costante genetico per soggetti di prova all&#39;interno di uno studio. Inoltre, i confronti dovrebbero generalment...

L&#39;uso di alcol disturbo (AUD) è un problema di salute enorme in tutto il mondo (rivisto in 1). Sebbene i meccanismi di guida lo sviluppo di AUD sono complesse, questi disturbi hanno una notevole componente genetica (ad esempio, 2). La grande ereditabilità di AUD e le risposte comportamentali conservati in etanolo in molte specie (recensito in 3,4) hanno generato un forte interesse ad utilizzare organismi modello genetici per studiare il coinvolgimento di specifici geni nei comportamenti etanolo legate verso una migliore comprensione delle basi molecolari di AUD. Il moscerino della frutta, Drosophila melanogaster, è emerso come organismo modello principale per esplorare i meccanismi molecolari-genetica dei comportamenti etanolo correlati (recensito in 3,4). Studi in mosche hanno evidenziato i ruoli per diverse vie di segnalazione nelle risposte comportamentali a etanolo (recensito in 5). Curiosamente, alcuni dei geni e dei percorsi che influenzare respons comportamentalies a etanolo in linea sono stati implicati in roditori comportamenti etanolo correlati e / o AUD umana (ad esempio, 6-14). La conservazione di meccanismi di guida comportamenti etanolo legate tra le specie, insieme con la suite di strumenti genetici disponibili nel sistema modello Drosophila, sottolineano l&#39;utilità del modello di mosca della frutta per studiare la genetica di risposte comportamentali a etanolo. Sensibilità 15,16 e tolleranza (rivisto in 17) di etanolo negli esseri umani è legato allo sviluppo di AUD. Entrambe queste risposte comportamentali a etanolo può essere modellato in linea attraverso una serie di test di laboratorio (rivisto in 3,4). Tutti i saggi mosca noti gli autori sono basati su uno dipendente dal tempo etanolo-indotta sedazione / incoordinazione o di recupero in funzione del tempo da etanolo sedazione. In un precedente articolo dal nostro gruppo sulla genetica della sensibilità etanolo e rtolleranza APID in Drosophila, un test comportamentale basato su etanolo sedazione vapore indotta di mosche è stato utilizzato 18. Test in questo test è stato avviato con il trasferimento dal vivo adulti vola senza anestesia svuotare fiale alimentari, intrappolando le mosche nelle fiale con una spina di acetato di cellulosa, aggiungendo etanolo verso l&#39;alto (cioè, lato non volo) del tappo di acetato di cellulosa, e sigillare la fiala contenente mosche, spina acetato di cellulosa e di etanolo con un tappo di silicone (vedi schema in Figura S3, riferimento 18). Più fiale che rappresentano diversi gruppi di mosche sono state valutate in parallelo, aumentando la velocità di questo test. Fiale sono stati dati un codice anonimo e sperimentatori sono stati accecati per gruppo di trattamento per prevenire la distorsione intenzionale nella valutazione della sedazione. In un esperimento standard mosche in fiale erano picchiettare leggermente ad intervalli di 6 min e, dopo un recupero di 30 secondi, il numero di mosche sedati in ogni flacone è stato contato e converted per cento mosche attivi. Mosche assorbiti vapori di etanolo dal tappo di acetato di cellulosa in modo dipendente dal tempo, causando un aumento progressivo in etanolo interno 18 e sedazione (riferimento cf 18 e Figure 1A e 1B in questo rapporto). La sedazione in questo test è stato operativamente definita come mosche (i) in piedi in assenza di camminare o (ii) che giace sulla schiena, con o senza sbattere le ali. Qui, questo dosaggio etanolo sedazione è descritta in dettaglio, ulteriore ottimizzazione operativa che riguardano l&#39;uso è previsto, e il test viene utilizzato per indirizzare il contributo delle opzioni integrazione alimentare sulla sensibilità fly sedazione.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>food vials</td>
			<td>VWR</td>
			<td>89092-772</td>
			<td>narrow</td>
		</tr>
		<tr>
			<td>Flugs</td>
			<td>Genesee/flystuff.com</td>
			<td>49-102</td>
			<td>narrow</td>
		</tr>
		<tr>
			<td>silicone stopper</td>
			<td>Fisher Scientific</td>
			<td>09-704-1l</td>
			<td>#4</td>
		</tr>
		<tr>
			<td>ethanol</td>
			<td>Pharmaco-Aaper</td>
			<td>111000200</td>
			<td>200 proof</td>
		</tr>
	</tbody>
</table>

an inexpensive, scalable behavioral assay for measuring ethanol sedation sensitivity and rapid tolerance in drosophila

L&#39;uso di alcol disturbo (AUD) è una sfida di salute gravi. Nonostante una grande componente ereditaria per AUD, pochi geni sono stati inequivocabilmente implicati nella loro eziologia. Il moscerino della frutta, Drosophila melanogaster, è un modello potente per esplorare i meccanismi molecolari alla base genetica comportamenti alcol-correlati e detiene quindi una grande promessa per l&#39;identificazione e la comprensione della funzione dei geni che influenzano AUD. L&#39;uso del modello Drosophila per questi tipi di studi dipende dalla disponibilità di saggi che misurano affidabile risposte comportamentali a etanolo. Questo rapporto descrive un test adatto per valutare la sensibilità etanolo e tolleranza rapida in mosche. Etanolo sensibilità misurata in questo saggio è influenzata dal volume e la concentrazione di etanolo utilizzato, una varietà di manipolazioni genetiche precedentemente riportati, e anche la lunghezza di tempo in linea sono alloggiati senza cibo immediatamente prima del test. Al contrario, etanolo sensitivity misurata in questo dosaggio non è influenzato dal vigore di movimentazione mosca, sesso in linea, e la supplementazione di mezzo di crescita con antibiotici o lieviti vivi. Tre metodi diversi per quantificazione sensibilità etanolo sono descritte, tutto conduce a risultati sostanzialmente indistinguibili sensibilità etanolo. La natura scalabile di questo test, in combinazione con la sua semplicità complessiva di set-up e relativamente a basso costo, lo rendono adatto per l&#39;analisi piccola e grande scala genetica della sensibilità etanolo e tolleranza rapida in Drosophila.

I dati grezzi di questa analisi etanolo sedazione sono i numeri di mosche che vengono sedati in funzione del tempo di esposizione al vapore etanolo. I dati grezzi sono convertiti in linea attivi cento in funzione del tempo (dati primario, figure 1A, B, D - F). La sensibilità all&#39;etanolo sedazione dai dati primari può essere quantificata come sedazione Tempo 50 (ST50), il tempo necessario per il 50% di mosche per diventare sedato o aea sotto la curva (AUC), via interpolazione curva...

Watch this Scientific Journal Video about An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila at JoVE.com

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 ...

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JoVE Journal

Neuroscience

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

10.2K Views.  Virginia Commonwealth University. 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.

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

Combining Behavioral Endocrinology and Experimental Economics: Testosterone and Social Decision Making

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in rodents have shown a direct link between testosterone and aggressive behavior1 and folk wisdom adapts these findings to humans, suggesting that testosterone induces antisocial, egoistic or even aggressive behavior2. However, many researchers doubt a direct testosterone-aggression link in humans, arguing instead that testosterone is primarily involved in status-related behavior3,4. As a high status can also be achieved by aggressive and antisocial means it can be difficult to distinguish between anti-social and status seeking behavior.
We therefore set up an experimental environment, in which status can only be achieved by prosocial means. In a double-blind and placebo-controlled experiment, we administered a single sublingual dose of 0.5 mg of testosterone (with a hydroxypropyl-&beta;-cyclodextrin carrier) to 121 women and investigated their social interaction behavior in an economic bargaining paradigm. Real monetary incentives are at stake in this paradigm; every player A receives a certain amount of money and has to make an offer to another player B on how to share the money. If B accepts, she gets what was offered and player A keeps the rest. If B refuses the offer, nobody gets anything. A status seeking player A is expected to avoid being rejected by behaving in a prosocial way, i.e. by making higher offers.
The results show that if expectations about the hormone are controlled for, testosterone administration leads to a significant increase in fair bargaining offers compared to placebo. The role of expectations is reflected in the fact that subjects who report that they believe to have received testosterone make lower offers than those who say they believe that they were treated with a placebo. These findings suggest that the experimental economics approach is sensitive for detecting neurobiological effects as subtle as those achieved by administration of hormones. Moreover, the findings point towards the importance of both psychosocial as well as neuroendocrine factors in determining the influence of testosterone on human social behavior.

The procedure described in this protocol shows that testosterone administration and folk beliefs about testosterone may be associated with directly opposed social behaviors.

Combinando comportamentali Endocrinologia ed Economia Sperimentale: Decisione testosterone e sociale Fare

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in ...

Ricerca

Neuroscienze

A Simple Way to Measure Ethanol Sensitivity 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 is correlated with that strain s ethanol preference, and therefore sedation is a highly relevant measure to study the genetics of alcohol responses and drinking. We demonstrate a simple way to expose flies to ethanol and measure its intoxicating effects. The assay we describe can determine acute sensitivity, as well as ethanol tolerance induced by repeat exposure. It does not require a technically involved setup, and can therefore be applied in any laboratory with basic fly culture tools.

A simple assay to measure the sedating effects of ethanol on Drosophila flies, based on the loss of righting reflex, is described.

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 General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

Un metodo generale per la valutazione di incubazione della brama di saccarosio in ratti

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Measuring Changes in Tactile Sensitivity in the Hind Paw of Mice Using an Electronic von Frey Apparatus

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain perception in rodents. Withdrawal thresholds can be measured first at baseline and then following drug, venom, injury, allergen,&#160;or otherwise evoked inflammation by applying an accurate force on very specific areas of the skin. An electronic von Frey apparatus allows precise assessment of mouse hind paw withdrawal thresholds that are not limited by the available filament sizes in contrast to classical von Frey measurements. The ease and rapidity of measurements allow for incorporation of assessment of tactile sensitivity outcomes in diverse models of rapid-onset inflammatory and neuropathic pain as multiple measurements can be taken within a short time period. Experimental measurements for individual rodent subjects can be internally controlled against individual baseline responses and exclusion criteria easily established to standardize baseline responses within and across experimental groups. Thus, measurements using an electronic von Frey apparatus represent a useful modification of the well-established classical von Frey filament-based assays for rodent mechanical allodynia that may also be applied to other nonhuman mammalian models.

Electronic von Frey measurements are an effective and improved alternative to classical von Frey filaments, as they allow rapid and precise measurement of changes in rodent mechanical withdrawal thresholds to continuously applied pressure stimuli before and after an inflammatory event.

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain ...

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 Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the development of more effective treatments is the relative lack of understanding of the molecular underpinnings of the effects of ethanol on behavior. The nematode, Caenorhabditis elegans (C.&#160;elegans), provides a useful model in which to generate and test hypotheses about the molecular effects of ethanol. Here, we describe an assay that has been developed and used to examine the roles of particular genes and environmental factors in behavioral responses to ethanol, in which locomotion is the behavioral output. Ethanol dose-dependently causes an acute depression of crawling on an agar surface. The effects are dynamic; animals exposed to a high concentration demonstrate an initial strong depression of crawling, referred to here as initial sensitivity, and then partially recover locomotion speed despite the continued presence of the drug. This ethanol-induced behavioral plasticity is referred to here as the development of acute functional tolerance. This assay has been used to demonstrate that these two phenotypes are distinct and genetically separable. The straightforward locomotion assay described here is suitable for examining the effects of both genetic and environmental manipulations on these acute behavioral responses to ethanol in C.&#160;elegans.

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

C. elegans is a useful model for studying the effects of ethanol on behavior. We present a behavioral assay that quantifies the effects of ethanol on the locomotion speed of crawling worms; both initial sensitivity and the development of acute functional tolerance to ethanol can be measured with this assay.

Un test per misurare gli effetti di etanolo sulla velocità di Locomotion<em&gt; Caenorhabditis elegans</em

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the ...

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

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

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

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

Un test comportamentale per Mechanosensation di cloni in marcm-based<em&gt; Drosophila melanogaster</em

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

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

Metodo elettrofisiologiche per la registrazione intracellulare di tensione Risposte di<em&gt; Drosophila</em&gt; Fotorecettori e Interneuroni a luce stimoli<em&gt; In Vivo</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Measuring and Altering Mating Drive in Male Drosophila melanogaster

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

Measuring and Altering Mating Drive in Male Drosophila melanogaster

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

Misurazione e l&#39;alterazione di accoppiamento Drive a Maschio<em&gt; Drosophila melanogaster</em

Despite decades of investigation, the neuronal and molecular bases of motivational states remain mysterious. We have recently developed a novel, ...

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in regulating neuronal growth and development. However, the mechanisms underlying Drd1&#945; receptor abnormalities mediating behavioral responses and modulating working memory function are still unclear. Using a novel RNA in situ hybridization assay, the current study identified dopamine Drd1&#945; receptor and tyrosine hydroxylase (TH) RNA expression from DA-related circuitry in the nucleus accumbens (NAc) area and substantia nigra region (SNR), respectively. Drd1&#945; expression in the NAc shows a &#34;discrete dot&#34; staining pattern. Clear sex differences in Drd1&#945; expression were observed. In contrast, TH shows a &#34;clustered&#34; staining pattern. Regarding TH expression, female rats displayed a higher signal expression per cell relative to male animals. The methods presented here provide a novel in situ hybridization technique for investigating changes in dopamine system dysfunction during the progression of central nervous system diseases.

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of dopamine D1-alpha receptor in the nucleus accumbens is critical for clarifying D1 receptor dysfunction during a central nervous system disease. We performed a novel RNA in situ hybridization assay to visualize single RNA molecules in a specific brain area.

Identificazione della dopamina D1-alfa del ricevitore all'interno del roditore Nucleus Accumbens da un innovativo RNA In Situ Detection Technology