This work was supported by grants DA041613 and GM128974 from the National Institutes of Health to TFCM and RRHA.

1. Construction of the testing apparatus
<ol>
	<li>Create a cardboard template the size of a 24-well cell culture plate by tracing around the plate on cardboard and cutting out the designated area.</li>
	<li>Cut a piece of small insect screen mesh the size of the cell culture plate using the cardboard template from step 1.1.</li>
	<li>Prepare a 24-well cell culture plate by placing a small line of hot glue around the perimeter of the top of the plate using a hot glue gun and affixing the screen mesh on top of the open wells.</li>
	<li>Secure a wooden craft stick to each of three sides of the same cell culture plate from step 1.3 using a hot glue gun. The modified cell culture plate should now resemble the plate diagram shown in Figure 1A and the experimental setup shown in Figure 2. 
	NOTE: Prepare at least as many cell culture plates as will fit in the filming chambers (see below).</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61108/61108fig1.jpg" /> 
Figure 1: Diagram of the testing apparatus and filming chamber. (A) Upper Diagrams. The top, side, and front views of the testing apparatus are shown, respectively. A screen mesh lays flat on top of a 24-well cell culture plate. The wooden craft sticks, represented by the arrowheads, are attached to three adjacent sides for stability and alignment aid, two on the side of the well plate with six wells and one on the side of the plate with four wells. All attachments are hot glued onto the apparatus. (B) Lower Diagrams. The top, side, and front views of the assay set-up are shown, respectively. A slit is cut in the right side of the box, from the opening for the lid to the back of the opening, with the bottom of the slit level to the inner surface. The hole on the top of the box, the surface parallel to the ground, is centered for maximum video exposure. The shaded box represents the video camera. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61108/61108fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61108/61108fig2.jpg" /> 
Figure 2: Photograph of the assay system. The video camera is placed on top of the polystyrene chamber, with the lens inserted in the cut-out hole, illustrated in the diagrams of Figure 1B. Two sets of modified 24-well cell culture plates rest on top of an illumination pad that is inserted in a slit through the side of the chamber. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61108/61108fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Construction of the filming chamber
<ol>
	<li>Create a filming chamber by cutting a hole the size of the video camera lens on the side of a polystyrene box. Cut an additional slit the width of the illumination pad in the opposite side of the polystyrene box. The filming chamber should resemble the filming chamber shown in Figure 1B and Figure 2.</li>
	<li>Prepare the filming chamber for use by inserting the illumination pad into the slit and positioning the camera in the lens hole above the illumination pad.</li>
	<li>Place all materials and perform all subsequent testing in a controlled environment, preferably a behavioral chamber with approximately 30% humidity, 25 &#176;C temperature, uniform airflow, and noise levels less than 65 dB.</li>
</ol>
3. Preparation of the testing apparatus and flies
<ol>
	<li>Pipette 1 mL of 100% ethanol through the screen mesh into each well.</li>
	<li>Dry the screen mesh with a piece of cheesecloth.</li>
	<li>Cut two pieces of cheesecloth the dimensions of the cell culture plate using the cardboard template created in step 1.1. Place them on top of the dry screen mesh of the modified cell culture plate containing ethanol from step 3.2.</li>
	<li>Create a small piece of thin, flexible plastic cutting board by tracing around the cardboard template created in step 1.1 as a general guide and expanding the traced area by 1&#8211;2 cm on one of the short sides. Cut out the expanded traced area from the thin, flexible plastic cutting board. After cutting, ensure that the plastic still fits between the three wooden craft sticks on the testing apparatus, but hangs off one end by 1&#8211;2 cm.</li>
	<li>(Optional) If an aspirator needs to be created, assemble an aspirator like the one shown in Figure 3 by first cutting a P1000 pipette tip in half. Insert the piece with a larger diameter into one end of a ~30 cm piece of flexible tubing to serve as a mouthpiece.</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61108/61108fig3.jpg" /> 
Figure 3: A fly aspirator in which flies are collected with an interchangeable mouthpiece attached to flexible tubing and a wide bore serological pipette with a cotton gauze stopper. The operator can aspirate a single fly into the pipette for transfer without anesthesia. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61108/61108fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="6">
	<li>(Optional) To complete the aspirator assembly, cover the wide end of a 10 cm piece of serological pipette with gauze to prevent flies from getting into the tubing and insert the pipette, gauze first, into the open end of the tubing to serve as a fly chamber. The aspirator should resemble that shown in Figure 3.</li>
	<li>Using an aspirator (Figure 3, steps 3.5 and 3.6), aspirate one fly per well into a separate 24-well cell culture plate. Use the flexible plastic to cover any wells containing previously aspirated flies. Record the well position and any relevant genotype or phenotype information of each fly.</li>
	<li>Hold the flexible plastic flush with the top of the cell culture plate containing the flies to prevent their escape and invert the plate onto the top of the modified cell culture plate with the ethanol. The sheet of flexible plastic should be resting on top of the sheets of cheesecloth. Align the inverted cell culture plate containing flies using the craft sticks to ensure each well with ethanol aligns with each well containing a fly.</li>
	<li>The experimental setup should resemble Figure 2.</li>
</ol>
4. Testing the flies
<ol>
	<li>Ensure the illumination pad is lit at full brightness for maximum visual contrast. Start recording with the video camera.</li>
	<li>To expose the flies to ethanol, carefully remove the plastic from between the well plate and testing apparatus, taking care not to dislodge the cheesecloth.</li>
	<li>Terminate the video recording once all flies have lost postural control. Once it is suspected that all flies have lost postural control, tap firmly in the center of the plate to ensure that all flies have complete loss of postural control. If there is movement, continue to record. Continue to tap periodically (every 1&#8211;2 min) until no movement occurs.</li>
	<li>(Optional) To quickly recover the flies, remove only the top plate from the testing apparatus, revealing sedated flies resting on the cheesecloth. Aspirate individual flies into chosen containers for recovery.</li>
	<li>Replace the ethanol in the modified cell culture plates with 1 mL of fresh 100% ethanol at least 1x every hour to control for evaporation and humidification of the ethanol and to maintain consistent ethanol exposure throughout the assay. Dry the screen mesh with cheesecloth.</li>
	<li>Repeat for as many samples as desired. 
	NOTE: For highest throughput, aspirate the next round of flies into new cell culture plates during the video recording. The protocol can be paused here, as the video recording can be reviewed later.</li>
</ol>
5. Determination of fly sedation time
<ol>
	<li>Record sedation time for each individual fly by watching the video recording. Sedation time is defined as the moment a fly loses complete postural control and locomotor ability. It is recommended to watch the film in reverse and record the time that the fly begins to move to ensure accuracy.</li>
</ol>

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The authors have nothing to disclose.

Here, we present a simple, inexpensive, and high-throughput method for assessing sedation time due to ethanol exposure in Drosophila melanogaster. Unlike many current methods, which require group analyses, this assay enables a single person to collect individual sedation time data for ~2,000 flies within an 8 h work period. We found that a single person can score 48 flies for sedation time in about 5 min. At this rate, 2,000 flies can be scored in approximately 4 h, though scoring can be conducted later. With our assay, the recorded sedation time for most flies ranges from 5&#8211;15 min at an exposure to 1 mL of 100% ethanol. Lower concentrations of ethanol or smaller delivery volumes will result in longer sedation times.
Current methods for assessing sedation time require testing large numbers of flies without readily enabling measurements on single individuals15,16,17,18,19,20,21,22,23,24,25,26. Many current sedation and sensitivity assays rely upon ST5022,23,24, the timepoint at which 50% of the flies are sedated as a result of ethanol exposure. Although obtaining the ST50 for groups of flies was not the primary motivation for developing this assay, the video recordings demonstrate higher utility compared to current methods, as the recordings can be used to ascertain the ST50 for groups of individually tested flies and to measure the percentage of flies that satisfy a given criterion (e.g., loss of postural control) at any time point. It should be noted that such video analyses would require additional time.
Unlike current inebriometer assays, the method we describe does not require specialized tools to set up and can be performed in any laboratory using common materials. Using this method, we have obtained reliable and consistent sedation times for individual flies. The assay can, in principle, be extended to assess the effects of exposure to any volatile substance. The assay can also be applied to measure effects of acute toxicity of volatiles on other insects, including other fly species. Individual sedation time data can be used to assess the extent of phenotypic variation within a population, such as the DGRP.
We used small insect screen mesh to prevent direct contact with the ethanol solution while allowing adequate quantities of ethanol vapors to reach the fly. The layer of white cheesecloth on top of the screen mesh provides visual contrast between the fly and the surface below and ensures that flies do not get caught in the screen mesh, which could lead to ambiguous determination of loss of postural control. Commercially available membranes that are porous to water and air gave inconsistent results and were insufficiently penetrable to ethanol vapors. We intentionally used small insect screen mesh because it is a uniformly porous material that minimizes variation in ethanol exposure as a result of fly position within a well. Modifications can be made to this protocol based on available materials, although we recommend a controlled behavioral chamber, access to 90%&#8211;100% ethanol close to the fly, and uniform ethanol exposure.
Fly position within the cell culture plates should be randomized between replicates to avoid positional bias. For larger experiments that require use of this assay across multiple days and are therefore subject to environmental variation that could influence assay results (e.g., changes in barometric pressure)27, we strongly recommend that flies be tested at the same time each day and randomized both within and across days, especially if different lines and/or sexes are to be compared against one another.
The method we developed is best suited for measuring the effect of acute alcohol exposure but is not suitable for obtaining consumption data or modeling addiction. Alcohol sedation sensitivity data obtained from this assay can, however, be integrated with other measures of alcohol-related phenotypes. One limitation of the system is that the vertical height of standard cell culture plates allows for vertical fly movement that cannot be readily tracked by video for detailed assessment of overall activity or locomotion. However, this limitation does not affect accurate assessment of sedation time. When using flies of different genotypes (e.g., in DGRP-derived outbred populations28), this assay also enables retrieval of individual flies to collect pools of flies with contrasting phenotypes for bulk DNA sequencing and extreme QTL mapping29,30. Overall, this assay permits rapid, inexpensive collection of alcohol sedation data on large numbers of single flies.

The National Institute on Alcohol Abuse and Alcoholism reports that in 2015 excessive alcohol consumption, designated as &#34;alcohol use disorder&#34;, affected an estimated 16 million people in the United States. Alcohol abuse causes a wide range of adverse physiological effects and is a major cause of death in the U.S. In humans, decreased sensitivity, or a low level of response to alcohol, has a strong genetic component and is associated with a higher risk of developing alcohol use disorders1,2,3,4. Genetic risk studies on human populations are challenging because of population admixture, diverse developmental histories and environmental exposures, and reliance on self-reported questionnaires to quantify alcohol-related phenotypes, which are often confounded with other neuropsychiatric conditions.
Drosophila melanogaster provides an excellent model to study the genetic underpinnings of alcohol sensitivity5,6,7,8. The Drosophila model allows strict control over genetic background, and virtually unlimited numbers of individuals of the same genotype can be reared rapidly under well-controlled environmental conditions without regulatory restrictions and at relatively low cost. In addition to publicly available mutations and RNAi lines that target a majority of genes in the genome, the availability of the Drosophila melanogaster Genetic Reference Panel (DGRP), a population of 205 inbred wild-derived lines with complete genome sequences, has enabled genome-wide association studies9,10. Such studies have identified genetic networks associated with effects on development time and viability upon developmental exposure to ethanol11,12. Evolutionary conservation of fundamental biological processes enables translational inferences to be drawn by superimposing human orthologs on their fly counterparts.
Flies exposed to ethanol undergo physiological and behavioral changes that resemble human alcohol intoxication, including loss of postural control8, sedation, and development of tolerance13,14,15. Alcohol induced sedation in Drosophila can be quantified using inebriometers. These are 122 cm long vertical glass columns with slanted mesh partitions to which flies can attach16,17,18. A group of at least 50 flies (sexes can be analyzed separately) are introduced in the top of the column and exposed to ethanol vapors. Flies that lose postural control fall through the column and are collected at 1 min intervals. The mean elution time serves as a measure of sensitivity to alcohol intoxication. When flies are exposed to alcohol a second time after recovering from the first exposure, they can develop tolerance, as evident from a shift in mean elution time13,15,19,20. Whereas inebriometer assays have led to identification of genes, genetic networks, and cellular pathways associated with alcohol sedation sensitivity and development of tolerance12,13,14,21, the assay is time consuming, low-throughput, and ineffective for measuring alcohol sensitivity in single flies.
Alternative ethanol sedation assays that do not require the elaborate inebriometer set-up allow for more convenient measurements but are still limited in throughput and generally require analyses of groups of flies rather than individuals21,22,23,24,25. Assessing single flies minimizes the potential for confounding effects due to group interactions, such as those stemming from social behaviors. Here, we present a simple, low-cost, high-throughput assay for assessing alcohol sedation sensitivity in large numbers of single flies.

<table><tbody><tr><td>24-well Cell Culture Plates</td><td>Corning</td><td>3526</td><td>Flat-bottomed; will house flies throughout assay</td></tr><tr><td>Aspirator</td><td></td><td></td><td></td></tr><tr><td>Cheesecloth</td><td>Genesee Scientific</td><td>53-100</td><td>Widely available.</td></tr><tr><td>Ethanol</td><td>Decon Labs</td><td>V1001</td><td>Widely available.</td></tr><tr><td>Flexible Plastic Cutting Board (Plate Cover)</td><td>Walmart</td><td>550098612</td><td>Any flat plastic that can slide easily and cover a 24-well plate completely. Flexible plastic cutting board works well.</td></tr><tr><td>Gauze (for aspirator)</td><td>Honeywell North</td><td>67622</td><td>Widely available.</td></tr><tr><td>Illumination Pad</td><td>Amazon (AGPtek)</td><td>ASIN B00YA9GP0G</td><td>Any light pad to provide contrast is suitable.</td></tr><tr><td>Jumbo Craft Sticks</td><td>Michaels</td><td>10334892</td><td>Any craft stick at least 7 cm long is suitable.</td></tr><tr><td>P1000 Pipette Tip (for aspirator)</td><td>Genesee Scientific</td><td>24-165RL</td><td>Any P1000 pipette tip is suitable.</td></tr><tr><td>Serological Pipette (for aspirator)</td><td>Genesee Scientific</td><td>12-104</td><td></td></tr><tr><td>Small Insect Screen Mesh</td><td>Lowe&#039;s (Saint-Gobain ADFORS)</td><td>89322</td><td>Any small insect screen mesh is suitable.</td></tr><tr><td>Testing Chamber</td><td></td><td></td><td>Interior space dimension big enough to encompass light pad. Can be constructed from a polystyrene box.</td></tr><tr><td>Tygon Tubing (for aspirator)</td><td>Grainger</td><td>9CUG7</td><td>Widely available.</td></tr><tr><td>Video Camera</td><td>Canon</td><td>1959C001AA</td><td>Any video camera is suitable.</td></tr></tbody></table>

high-throughput method for measuring alcohol sedation time of individual drosophila melanogaster

Drosophila melanogaster provides an excellent model to study the genetic underpinnings of alcohol sensitivity. In contrast to studies in human populations, the Drosophila model allows strict control over genetic background, and virtually unlimited numbers of individuals of the same genotype can be reared rapidly under well-controlled environmental conditions without regulatory restrictions and at relatively low cost. Flies exposed to ethanol undergo physiological and behavioral changes that resemble human alcohol intoxication, including loss of postural control, sedation, and development of tolerance. Here, we describe a simple, low-cost, high-throughput assay for assessing alcohol sedation sensitivity in large numbers of single flies. The assay is based on video recording of single flies introduced without anesthesia in 24-well cell culture plates in a set-up that enables synchronous initiation of alcohol exposure. The system enables a single person to collect individual ethanol sedation data on as many as 2,000 flies within an 8 h work period. The assay can, in principle, be extended to assess the effects of exposure to any volatile substance and applied to measure effects of acute toxicity of volatiles on other insects, including other fly species.

Two 24-well microtiter plates could generate data simultaneously on 48 individual flies within as little as 10 min. Table 1 lists measurements of ethanol sedation times for 48 individual flies, males and females separately, of two DGRP lines with different sensitivities to alcohol exposure on development time and viability13. Flies of line RAL_555 were less sensitive than line RAL_177 (Figure 4, Table 2; p &#60; 0.0001, ANOVA). Males and females of RAL_177 showed no sexually dimorphic effect (Figure 4, Table 2; p &#62; 0.1, ANOVA), whereas females of line RAL_555 were less sensitive to ethanol exposure than the males (Figure 4, Table 2; p &#60; 0.006, ANOVA). The large number of flies that can be measured simultaneously and the ability to measure sexes and different lines contemporaneously can increase accuracy by reducing error due to environmental variation.
<table border="1" fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>A.</td>
			<td colspan="6">Ethanol Sedation Time (s)</td>
			<td>B.</td>
			<td colspan="6">Ethanol Sedation Time (s)</td>
		</tr>
		<tr>
			<td></td>
			<td colspan="3">Females</td>
			<td colspan="3">Males</td>
			<td></td>
			<td colspan="3">Females</td>
			<td colspan="3">Males</td>
		</tr>
		<tr>
			<td></td>
			<td>414</td>
			<td>365</td>
			<td>477</td>
			<td>423</td>
			<td>568</td>
			<td>309</td>
			<td></td>
			<td>937</td>
			<td>742</td>
			<td>622</td>
			<td>460</td>
			<td>331</td>
			<td>498</td>
		</tr>
		<tr>
			<td></td>
			<td>201</td>
			<td>384</td>
			<td>498</td>
			<td>411</td>
			<td>523</td>
			<td>626</td>
			<td></td>
			<td>791</td>
			<td>619</td>
			<td>197</td>
			<td>467</td>
			<td>455</td>
			<td>562</td>
		</tr>
		<tr>
			<td></td>
			<td>228</td>
			<td>364</td>
			<td>333</td>
			<td>440</td>
			<td>403</td>
			<td>267</td>
			<td></td>
			<td>504</td>
			<td>744</td>
			<td>513</td>
			<td>570</td>
			<td>582</td>
			<td>506</td>
		</tr>
		<tr>
			<td></td>
			<td>440</td>
			<td>416</td>
			<td>404</td>
			<td>408</td>
			<td>422</td>
			<td>384</td>
			<td></td>
			<td>970</td>
			<td>540</td>
			<td>369</td>
			<td>865</td>
			<td>533</td>
			<td>492</td>
		</tr>
		<tr>
			<td></td>
			<td>888</td>
			<td>283</td>
			<td>285</td>
			<td>322</td>
			<td>369</td>
			<td>287</td>
			<td></td>
			<td>595</td>
			<td>550</td>
			<td>606</td>
			<td>392</td>
			<td>544</td>
			<td>345</td>
		</tr>
		<tr>
			<td></td>
			<td>1079</td>
			<td>519</td>
			<td>315</td>
			<td>393</td>
			<td>376</td>
			<td>284</td>
			<td></td>
			<td>418</td>
			<td>709</td>
			<td>553</td>
			<td>308</td>
			<td>477</td>
			<td>388</td>
		</tr>
		<tr>
			<td></td>
			<td>718</td>
			<td>287</td>
			<td>432</td>
			<td>275</td>
			<td>206</td>
			<td>411</td>
			<td></td>
			<td>366</td>
			<td>564</td>
			<td>558</td>
			<td>385</td>
			<td>576</td>
			<td>377</td>
		</tr>
		<tr>
			<td></td>
			<td>598</td>
			<td>337</td>
			<td>398</td>
			<td>279</td>
			<td>631</td>
			<td>372</td>
			<td></td>
			<td>437</td>
			<td>692</td>
			<td>578</td>
			<td>460</td>
			<td>511</td>
			<td>412</td>
		</tr>
		<tr>
			<td></td>
			<td>241</td>
			<td>398</td>
			<td>364</td>
			<td>347</td>
			<td>374</td>
			<td>808</td>
			<td></td>
			<td>665</td>
			<td>729</td>
			<td>484</td>
			<td>532</td>
			<td>425</td>
			<td>354</td>
		</tr>
		<tr>
			<td></td>
			<td>229</td>
			<td>423</td>
			<td>534</td>
			<td>386</td>
			<td>396</td>
			<td>628</td>
			<td></td>
			<td>312</td>
			<td>576</td>
			<td>305</td>
			<td>334</td>
			<td>531</td>
			<td>506</td>
		</tr>
		<tr>
			<td></td>
			<td>388</td>
			<td>488</td>
			<td>451</td>
			<td>523</td>
			<td>322</td>
			<td>533</td>
			<td></td>
			<td>682</td>
			<td>638</td>
			<td>420</td>
			<td>560</td>
			<td>548</td>
			<td>379</td>
		</tr>
		<tr>
			<td></td>
			<td>252</td>
			<td>529</td>
			<td>375</td>
			<td>427</td>
			<td>330</td>
			<td>540</td>
			<td></td>
			<td>1045</td>
			<td>741</td>
			<td>708</td>
			<td>832</td>
			<td>509</td>
			<td>472</td>
		</tr>
		<tr>
			<td></td>
			<td>674</td>
			<td>401</td>
			<td>303</td>
			<td>401</td>
			<td>307</td>
			<td>311</td>
			<td></td>
			<td>394</td>
			<td>675</td>
			<td>381</td>
			<td>477</td>
			<td>449</td>
			<td>784</td>
		</tr>
		<tr>
			<td></td>
			<td>303</td>
			<td>453</td>
			<td>351</td>
			<td>429</td>
			<td>525</td>
			<td>262</td>
			<td></td>
			<td>540</td>
			<td>690</td>
			<td>520</td>
			<td>556</td>
			<td>495</td>
			<td>226</td>
		</tr>
		<tr>
			<td></td>
			<td>258</td>
			<td>483</td>
			<td>302</td>
			<td>389</td>
			<td>562</td>
			<td>319</td>
			<td></td>
			<td>356</td>
			<td>615</td>
			<td>336</td>
			<td>454</td>
			<td>524</td>
			<td>590</td>
		</tr>
		<tr>
			<td></td>
			<td>346</td>
			<td>426</td>
			<td>385</td>
			<td>416</td>
			<td>596</td>
			<td>287</td>
			<td></td>
			<td>626</td>
			<td>678</td>
			<td>840</td>
			<td>634</td>
			<td>677</td>
			<td>509</td>
		</tr>
	</tbody>
</table>
Table 1: Measurements of ethanol sedation times (s) of individual flies of (A) DGRP lines RAL_177 and (B) RAL_555 for separate sexes (n = 48). See also Table 2, Figure 4.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61108/61108fig4.jpg" /> 
Figure 4: Alcohol sedation times of DGRP lines RAL_177 and RAL_555. The bars represent means and the error bars SEM (n = 48). Sedation times for RAL_177 flies were less than those for RAL_55 flies (p &#60; 0.0001, ANOVA). Individual data points are indicated in Table 1. Additional statistically significant differences between sexes and lines are indicated in the text and in Table 2. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61108/61108fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Analysis</td>
			<td>Source of Variation</td>
			<td>df</td>
			<td>SS</td>
			<td>F-Value</td>
			<td>P-value</td>
		</tr>
		<tr>
			<td rowspan="4">Full Model Pooled</td>
			<td>Line</td>
			<td>1</td>
			<td>769627</td>
			<td>34.869</td>
			<td>&#60;0.0001</td>
		</tr>
		<tr>
			<td>Sex</td>
			<td>1</td>
			<td>105001</td>
			<td>4.757</td>
			<td>0.0304</td>
		</tr>
		<tr>
			<td>Line x Sex</td>
			<td>1</td>
			<td>86021</td>
			<td>3.897</td>
			<td>0.0498</td>
		</tr>
		<tr>
			<td>Error</td>
			<td>188</td>
			<td>4149491</td>
			<td colspan="2"></td>
		</tr>
		<tr>
			<td rowspan="2">Reduced Model Females</td>
			<td>Line</td>
			<td>1</td>
			<td>685126</td>
			<td>23.58</td>
			<td>&#60;0.0001</td>
		</tr>
		<tr>
			<td>Error</td>
			<td>94</td>
			<td>2730718</td>
			<td colspan="2"></td>
		</tr>
		<tr>
			<td rowspan="2">Reduced Model Males</td>
			<td>Line</td>
			<td>1</td>
			<td>170522</td>
			<td>11.3</td>
			<td>0.0011</td>
		</tr>
		<tr>
			<td>Error</td>
			<td>94</td>
			<td>1418774</td>
			<td colspan="2"></td>
		</tr>
		<tr>
			<td rowspan="2">Reduced Model RAL_177</td>
			<td>Sex</td>
			<td>1</td>
			<td>473</td>
			<td>0.023</td>
			<td>0.8800</td>
		</tr>
		<tr>
			<td>Error</td>
			<td>94</td>
			<td>1943741</td>
			<td colspan="2"></td>
		</tr>
		<tr>
			<td rowspan="2">Reduced Model RAL_555</td>
			<td>Sex</td>
			<td>1</td>
			<td>190549</td>
			<td>8.12</td>
			<td>0.0054</td>
		</tr>
		<tr>
			<td>Error</td>
			<td>94</td>
			<td>2205751</td>
			<td colspan="2"></td>
		</tr>
	</tbody>
</table>
Table 2: Analyses of variance for sedation time across sex and DGRP line. The model used was Y = &#181; + L + S + LxS + &#949;, where &#181; is the overall mean, L is the fixed effect of the DGRP line (RAL_177, RAL_555), S is the fixed effect of sex (male, female), LxS is the interaction term (fixed), and &#949; is the error term. The models Y = &#181; + L + &#949; and Y = &#181; + S + &#949; were used for the reduced models. Line, Sex, and the Line x Sex interaction term were all significant in the full model at &#945; &#60; 0.05. Reduced models by sex and DGRP line RAL_555 were also significant at &#945; &#60; 0.01. See also Table 1, Figure 4. df = degrees of freedom, SS = Type I Sums of Squares.

Watch this Scientific Journal Video about High-Throughput Method for Measuring Alcohol Sedation Time of Individual Drosophila melanogaster at JoVE.com

High-Throughput Method for Measuring Alcohol Sedation Time of Individual Drosophila melanogaster

Current methods to measure alcohol sensitivity in Drosophila are designed to test groups of flies. We present a simple, low-cost, high-throughput assay for assessing alcohol sedation sensitivity in large numbers of single flies. The method does not require specialized tools and can be performed in any laboratory using common materials.

High-Throughput Method for Measuring Alcohol Sedation Time of Individual Drosophila melanogaster

Drosophila melanogaster provides an excellent model to study the genetic underpinnings of alcohol sensitivity. In contrast to studies in human ...

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Anxiolytics, Sedatives and Hypnotics

SCIENTISTS IN ACTION

7.1K Views.  Clemson University. Drosophila biedt een uitstekend model om de genetische onderbouwing van alcoholgevoeligheid te bestuderen, omdat u alcoholgevoeligheid nauwkeurig kwantificeren onder gecontroleerde genetische achtergronden en omgevingsomstandigheden. Huidige methoden om alcoholgevoeligheid te meten in Drosophila-testgroepen van vliegen. We presenteren een eenvoudige, goedkope, high-throughput methode om alcoholgevoeligheid in grote aantallen enkele vliegen te beoordelen.Variatie in alcoholgevoeligheid ...

Video: High-Throughput Method for Measuring Alcohol Sedation Time of Individual Drosophila melanogaster

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.

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 ...

Neuroscience

Methods to Assay Drosophila Behavior

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases1. We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials2-4. The rapid iterative negative geotaxis (RING) assay5 has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously using large number of animals, with the high-throughput approach making it more amenable for screening experiments.

Methods to Assay Drosophila Behavior

Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular ...

The Serial Anesthesia Array for the High-Throughput Investigation of Volatile Agents Using Drosophila melanogaster

Volatile general anesthetics (VGAs) are used worldwide on millions of people of all ages and medical conditions. High concentrations of VGAs (hundreds of micromolar to low millimolar) are necessary to achieve a profound and unphysiological suppression of brain function presenting as "anesthesia" to the observer. The full spectrum of the collateral effects triggered by such high concentrations of lipophilic agents is not known, but interactions with the immune-inflammatory system have been noted, although their biological significance is not understood.
To investigate the biological effects of VGAs in animals, we developed a system termed the serial anesthesia array (SAA) to exploit the experimental advantages offered by the fruit fly (Drosophila melanogaster). The SAA consists of eight chambers arranged in series and connected to a common inflow. Some parts are available in the lab, and others can be easily fabricated or purchased. A vaporizer, which is necessary for the calibrated administration of VGAs, is the only commercially manufactured component. VGAs constitute only a small percentage of the atmosphere flowing through the SAA during operation, as the bulk (typically over 95%) is carrier gas; the default carrier is air. However, oxygen and any other gases can be investigated. 
The SAA's principal advantage over prior systems is that it allows the simultaneous exposure of multiple cohorts of flies to exactly titrable doses of VGAs. Identical concentrations of VGAs are achieved within minutes in all the chambers, thus providing indistinguishable experimental conditions. Each chamber can contain from a single fly to hundreds of flies. For example, the SAA can simultaneously examine eight different genotypes or four genotypes with different biological variables (e.g., male vs. female, old vs. young). We have used the SAA to investigate the pharmacodynamics of VGAs and their pharmacogenetic interactions in two experimental fly models associated with neuroinflammation-mitochondrial mutants and traumatic brain injury (TBI).

The Serial Anesthesia Array for the High-Throughput Investigation of Volatile Agents Using Drosophila melanogaster

The fruit fly (Drosophila melanogaster) is widely used for biological and toxicological research. To expand the utility of flies, we developed an instrument, the serial anesthesia array, that simultaneously exposes multiple fly samples to volatile general anesthetics (VGAs), making it possible to investigate the collateral effects (toxic and protective) of VGAs.

Volatile general anesthetics (VGAs) are used worldwide on millions of people of all ages and medical conditions. High concentrations of VGAs (hundreds of ...

Genetics

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.

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

Flypub To Study Ethanol Induced Behavioral Disinhibition and Sensitization

Alcohol use disorder (AUD) remains a serious problem in our society. To develop effective interventions for addiction, it is important to understand the underlying neurobiological mechanisms, for which diverse experimental approaches and model systems are needed. The main ingredient of alcoholic beverages is ethanol, which causes adaptive changes in the central nervous system and behavior upon chronic intake. Behavioral sensitization (i.e., escalated responses) in particular represents a key adaptive change underlying addiction. Most ethanol-induced behavioral sensitization studies in animal models have been conducted on the locomotor activating effect of ethanol. A prominent effect of ethanol is behavioral disinhibition. Behavioral sensitization to the disinhibition effect of ethanol, however, is underrepresented. To address this issue, we developed the Flypub assay that allows measuring the escalated increase in disinhibited courtship activities upon recurring ethanol exposure in Drosophila melanogaster.&#160;Here, we report the step-by-step Flypub assay including assembly of ethanol exposure chambers, setup of the assay station, criteria for fly care and collection, ethanol delivery, quantification of disinhibited courtship activities, data processing and statistical analysis. Also provided are how to troubleshoot critical steps, overcome limitations and expand its utility to assess additional ethanol-induced behaviors. The Flypub assay in combination with powerful genetic tools in Drosophila melanogaster will facilitate the task of discovering the mechanism underlying ethanol-induced behavioral sensitization.

The Flypub assay measures the behaviors that the fruit fly Drosophila melanogaster displays under the influence of ethanol. The assay can be readily mastered by experimenters at all levels and applied to various vaporized stimuli, facilitating substance abuse and addiction studies.

Alcohol use disorder (AUD) remains a serious problem in our society. To develop effective interventions for addiction, it is important to understand the ...

Advancing Sleep Disorder Treatments: A Drosophila-Based Screening Approach

High-Throughput Small Molecule Drug Screening For Age-Related Sleep Disorders Using Drosophila melanogaster

Sleep, an essential component of health and overall well-being, often presents challenges for older individuals who frequently experience sleep disorders characterized by shortened sleep duration and fragmented patterns. These sleep disruptions also correlate with an increased risk of various illnesses in the elderly, including diabetes, cardiovascular diseases, and psychological disorders. Unfortunately, existing drugs for sleep disorders are associated with significant side effects such as cognitive impairment and addiction. Consequently, the development of new, safer, and more effective sleep disorder medications is urgently needed. However, the high cost and lengthy experimental duration of current drug screening methods remain limiting factors.
This protocol describes a cost-effective and high-throughput screening method that utilizes Drosophila melanogaster, a species with a highly conserved sleep regulation mechanism compared to mammals, making it an ideal model for studying sleep disorders in the elderly. By administering various small compounds to aged flies, we can assess their effects on sleep disorders. The sleep behaviors of these flies are recorded using an infrared monitoring device and analyzed with the open-source data package Sleep and Circadian Analysis MATLAB Program 2020 (SCAMP2020). This protocol offers a low-cost, reproducible, and efficient screening approach for sleep regulation. Fruit flies, due to their short life cycle, low husbandry cost, and ease of handling, serve as excellent subjects for this method. As an illustration, Reserpine, one of the tested drugs, demonstrated the ability to promote sleep duration in elderly flies, highlighting the effectiveness of this protocol.

Author Spotlight: Overcoming Challenges in Drosophila Sleep Measurement Using DAM System 

Presented is a protocol for high-throughput drug screening to improve sleep by monitoring the sleep behavior of fruit flies in an elderly Drosophila model.

Sleep, an essential component of health and overall well-being, often presents challenges for older individuals who frequently experience sleep disorders ...

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by stressed conspecifics due to an odorant released during stress known as the Drosophila stress odorant (dSO). Through the use of the T-maze apparatus, one can quantify the avoidance of the dSO by responder flies in a very affordable and robust assay. Conditions necessary to obtain a strong performance are presented here. A stressful experience is necessary for the flies to emit dSO, as well as enough emitter flies to cause a robust avoidance response to the presence of dSO. Genetic background, but not their group size, strongly altered the avoidance of the dSO by the responder flies. Canton-S and Elwood display a higher performance in avoiding the dSO than Oregon and Samarkand strains. This behavioral assay will allow identification of mechanisms underlying this social behavior, and the assessment of the influence of genes and environmental conditions on both emission and avoidance of the dSO. Such an assay can be included in batteries of simple diagnostic tests used to identify social deficiencies of mutants or environmental conditions of interest.

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Here, we present a protocol to quantify the avoidance of stressed individuals. This paradigm is powerful yet user-friendly and can be used to assess the influence of genes and environment on one kind of social interaction in Drosophila melanogaster.

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by ...

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.

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 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.

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

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community&#39;s investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer&#39;s two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

We present a protocol for measuring oxygen consumption and extracellular acidification in Drosophila melanogaster larval and adult brains. A metabolic analyzer is utilized with an adapted and optimized protocol. Micro-tissue restraints are a critical component of this protocol and were designed and created specifically for their use in this analysis.

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...

High-Throughput Method for Measuring Alcohol Sedation Time of Individual Drosophila melanogaster

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