Funding for this research was provided by a Program in Neuroscience Award from the Florida State University College of Medicine and support from the Department of Biological Science at FSU.&#160; Additional Funding was provided by a Grant-in-Aid from the Alcohol Beverage Manufacturer&#8217;s Research Fund.

1. Assembly of the FlyBar
Rationale and overview: The system is designed to administer controlled percentages of alcohol vapor to flies. Note: Figure 1 provides a schematic overview of the FlyBar set-up as described below in three stages (assembly of the air flow, set-up of the alcohol and water bottles, and assembly of the observation vials). In short, a steady airflow is split into two fractions that are bubbled through alcohol and water, respectively, mixed, and administered ...

<ol>
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The costs of alcohol abuse and alcoholism for society is tremendous, both in terms of human29 and economic costs30,31. Drosophila as a model offers a fast and versatile system to quickly examine the behavioral responses of a large number of individuals and as such has been extensively used for both alcohol5,7,32-34 and circadian research35-37.
Here, we described a straightforward protocol for the controlled administration of alcohol vapor to...

Drosophila melanogaster demonstrate biphasic behavioral responses to alcohol1 that are analogous to human responses to this drug2,3. Upon initial exposure to low concentrations of alcohol, flies exhibit increased locomotor activity, replaced by a lack of motor coordination, the loss of postural control and righting reflexes (Loss of Righting Reflex: LoRR), and sedation (complete lack of motor activity in response to mechanical stimulation) as exposure to alcohol progresses4-9. The endogenous circadian clock is a strong modulator of alcohol sensitivity and toxicity as observed in mice10,11, rats12, and humans13. Recent advances in Drosophila research have shown the circadian clock modulates acute alcohol sensitivity but not alcohol tolerance1. The powerful genetic approaches available in Drosophila through mutant studies and transgenic manipulations of spatial and temporal gene expression provide a system that allows rapid progress in identifying the underlying cellular and molecular mechanisms for complex behaviors. The use of Drosophila as an investigative tool has permitted substantive advances in understanding alcohol neurobiology that can be rapidly translated to mammals14-16. In order to facilitate understanding of the molecular mechanisms through which the circadian clock modulates alcohol sensitivity and to uniformly measure behavioral responses across circadian time points, an alcohol administration protocol suitable for use in dim red light conditions is required. For Drosophila, alcohol may be administered through food supplementation for chronic exposure or reliably through administrating alcohol in the form of vapor for acute exposures. Here, we describe an alcohol administration protocol suitable for the assessment of the circadian modulation of Loss-of-Righting Reflex (LoRR)1 as well as sedation.

Flies are entrained with 12 hr:12 hr LD cycles at constant temperature and then transferred to a controlled light regime for 2-5&#160;days depending on the experimental question. Flies are exposed to ethanol vapor in a device known as the FlyBar.&#160; In this device, controlled amounts of air are bubbled through water and alcohol; the vapors are then mixed and directed into a vial housing the flies. Every 5&#160;min&#160;the flies are scored for the number that fails to display righting reflexes or have become sedated. LoRR percentages for each time point are calculated and compared among circadian time points or between strains of flies. The simplicity and reliability of alcohol delivery using the FlyBar alcohol delivery combined with behavioral analysis options provides a significant benefit for circadian experiments conducted under dark conditions.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Alcohol 190 proof</td>
			<td style="width:115px;">Various</td>
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			<td style="width:296px;">We use Whisper 60</td>
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			<td height="21" style="height:21px;width:285px;">Silicone tubing 1/8&#8221;</td>
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			<td style="width:296px;">Either this or previous</td>
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			<td style="width:119px;">1722A43</td>
			<td style="width:296px;">To hold flow meters</td>
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			<td height="21" style="height:21px;width:285px;">Vial</td>
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			<td height="21" style="height:21px;width:285px;">5 mm Pyrex Glass tubes</td>
			<td style="width:115px;">Trikinetics</td>
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			<td style="width:296px;">Fits best in previous stopper.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Teflon tape</td>
			<td style="width:115px;">Hardware store</td>
			<td style="width:119px;"></td>
			<td style="width:296px;">To achieve snug fit in stoppers if necessary</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Rubber stopper with two holes</td>
			<td style="width:115px;">VWR</td>
			<td style="width:119px;">59582-122</td>
			<td style="width:296px;">Fits our bottles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Disposable glass pipets</td>
			<td style="width:115px;">VWR</td>
			<td style="width:119px;">53283-768</td>
			<td style="width:296px;">Cut to length and bend by heating</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Very fine nylon netting</td>
			<td style="width:115px;">VWR</td>
			<td style="width:119px;">Various</td>
			<td style="width:296px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">15 watt bulbs</td>
			<td style="width:115px;">Hardware store</td>
			<td style="width:119px;"></td>
			<td style="width:296px;">Overhead red light</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Photographic red safe light filters</td>
			<td style="width:115px;"></td>
			<td style="width:119px;"></td>
			<td style="width:296px;">Overhead red light</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Mini Flashlights with red filters</td>
			<td style="width:115px;">Mag-light</td>
			<td style="width:119px;"></td>
			<td style="width:296px;"></td>
		</tr>
	</tbody>
</table>

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.

Circadian Modulation of Alcohol Sensitivity using the 50% LoRR as a Marker.
A representative example showing circadian modulation in alcohol sensitivity during the day is presented in Figure 2.&#160; LoRR was measured at six time points during the 2nd day of DD in Canton-S and the 50% LoRR was determined for each time point. Analysis showed a significant effect of time of day (ANOVA: F5,45=7.39, p&#60;0.001, N=6-10 per time point). The Fisher LSD...

Watch this Scientific Journal Video about The FlyBar: Administering Alcohol to Flies at JoVE.com

The FlyBar: Administering Alcohol to Flies

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.

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

the-flybar-administering-alcohol-to-flies

Research

JoVE Journal

Neuroscience

10.6K visualizações.  Florida State University.  Drosophila surgiu como um sistema modelo importante para dissecar as bases celulares e moleculares de respostas comportamentais ao álcool. Aqui é apresentado um protocolo para a recolha de dados de sensibilidade do álcool em um contexto circadiano que pode ser facilmente aplicado a outros ensaios e é bem adequada para a pesquisa de graduação. 