We thank Mark G. Perkins, Pearce Laboratory, Department of Anesthesiology, University of Wisconsin-Madison, for the construction of the SAA prototype. The work is supported by the National Institute of General Medical Sciences (NIGMS) with R01GM134107 and by the R&#38;D fund of the Department of Anesthesiology, University of Wisconsin-Madison.

NOTE: See the Table of Materials for details about all the materials used in the protocol.
1. Construction of the SAA
<ol>
	<li>Make the frame by cutting wood and assembling the frame using the dimensions in Figure 1A.</li>
	<li>Modify 50 mL conical tube caps.
	<ol>
		<li>Drill two holes in each cap with a 9/32 in drill bit. Sand the holes to clean up the ragged plastic. Sand the top of the cap to roughen the surface (this helps with glue adhesion).</li>
		<li>Cut 5 mL serological pipettes to size (3 in for inflow and 1.5 in for outflow) by scoring the plastic and then breaking it clean at the scored line. Sand the ends of the cut/broken pipettes.</li>
		<li>Glue netting to the tubes (allow proper drying time for the adhesive). Cut the netting to the size of the tube after the adhesive dries.</li>
		<li>Insert the tubes into the holes of the conical caps with both tubes extending (3/4 in) above the cap; ensure that the inflow tube extends longer into the tube than the outflow (Figure 1B).</li>
		<li>Apply glue to the tops of the caps around the tubes to secure the parts together (allow proper drying time for the adhesive before continuing).</li>
	</ol>
	</li>
	<li>Attach the caps to the frame, and route the tubing (Figure 1C).
	<ol>
		<li>Attach adhesive cable tie-downs to the frame (3.25 in apart, center to center).</li>
		<li>Affix the caps to the frame using zip ties; cut the zip tie tag ends short.</li>
		<li>Cut and connect lengths (9 in) of Tygon tubing to the inflow/outflow tubes on each modified cap (Figure 1D). Starting at the upstream end, attach first to the inflow, and then subsequently attach tubing from the outflow to the inflow of the next position.</li>
		<li>Add a flow indicator to the most downstream &#34;inflow&#34; (position 10, Figure 1E).</li>
		<li>Put a 50 mL conical tube on the first position, and fill it with water to just below the inflow tube (Figure 1F).</li>
	</ol>
	</li>
	<li>Prepare the interfacing for the vaporizer. Remove the plungers, cut notches out of two 10 mL dispensing syringes (1/2 in deep x 1/4 in wide, Figure 1G), and insert them into the vaporizer inflow and outflow with the notches facing directly toward the front of the vaporizer to align with the holes (Figure 1H). Optional: Glue the modified syringes into place. If affordable, use a commercial manifold (see the Table of Materials for one option).</li>
	<li>Hook up the entire system. Use Tygon tubing to attach the components together in the following order: carrier gas tank with regulator &#62; gas specific flowmeter &#62; vaporizer &#62; SAA (Figure 1C).</li>
	<li>Fill empty positions on the array with empty 50 mL conical tubes. Turn on the gas tank, open the flowmeter to ~2 L/min, and turn on the vaporizer to 0%. Confirm gas flow through the system by checking the flowmeter upstream of the vaporizer and the flow indicator downstream of the last chamber of the SAA for flow. Alternatively, insert the downstream tubing end into water, and look for bubbles. 
	&#8203;NOTE: As the system is not pressurized, a water column higher than a couple of centimeters will stop the flow. If there is no flow at the downstream end of the array, check the following: the vaporizer needs to be on to allow flow; check that the tank regulator and flow meters are allowing flow; check the array positions to make sure the tubes are screwed on tightly; and check for leaks around the adhesive on the modified caps.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65144/65144fig01.jpg" /> 
Figure 1: Construction of the SAA. (A) Schematic, with measurements, of the wooden frame that supports the SAA. (B) Schematized cross-section, with measurements, of a modified cap with inflow and outflow tubes made of 5 mL serological pipettes. (C) Assembled SAA (reproduced from Olufs et al.5) (D) Details of a modified 50 mL conical cap showing inflow and outflow tubes. (E) Downstream (position 10) outflow with the flow indicator. (F) Upstream (position 1) water-filled tube to humidify the carrier gas. The red arrow indicates the water level. (G) Modified 10 mL dispensing syringe for the makeshift manifold. The red circle highlights the cut-out notch located between the 8 mL and 10 mL marks (or 1/2 in x 1/4 in). (H) Rear view of the Tec7 vaporizer showing the insertion and orientation of the modified syringes. Only one syringe is in place in this view to show, on the left, the hole (red arrow) that needs to be aligned with the notch of the modified syringe. Note: Misalignment of this cut-out notch and the outflow opening will disrupt anesthetic administration. This part is a potential weak spot in this custom-made system. If funds are available, a commercial manifold should be used. Abbreviation: SAA = serial anesthesia array. <a href="https://www.jove.com/files/ftp_upload/65144/65144fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Prior to anesthetic exposure
<ol>
	<li>Twenty-four hours or more prior to the anesthetic exposure, sort the fly cohorts as needed for the experiment using the preferred method (e.g., CO2 or ether).</li>
</ol>
3. Operation of the SAA
<ol>
	<li>Transfer flies from food vials into empty 50 mL conical tubes (without CO2).
	<ol>
		<li>Count and record any dead flies prior to exposure.</li>
	</ol>
	</li>
	<li>Uncap and screw 50 mL conical tubes with flies onto the SAA.</li>
	<li>Turn on the carrier gas, and set to the desired flow rate. 
	NOTE: We typically use 1-2 L/min.</li>
	<li>Set the anesthetic vaporizer to the desired concentration. 
	NOTE: We typically use 2% for isoflurane and 3.5% for sevoflurane, which are equipotent doses in mammals10.</li>
	<li>Expose the flies for the desired duration (min: 15 min). 
	NOTE: A minimum exposure time of 15 min is recommended to avoid possible variability in equilibration across the positions of the SAA. In this system, it takes 2-3 min for the anesthetics to equilibrate across all the positions.</li>
	<li>At the end of the exposure, flush the system with fresh gas flow (vaporizer set to 0%) at 1.5 L/min for 5 min corresponding to approximately 10x volumes of the total SAA volume.</li>
</ol>
4. Checklist prior to starting an experiment
<ol>
	<li>Open the high-pressure regulator (on top of the air tank) completely, and then close it half a turn to ensure carrier gas flow.</li>
	<li>Follow tubing for each line to the i) flowmeters and ii) vaporizer (make sure the inflow/outflow are connected correctly), and iii) check the anesthetic level in the vaporizers.</li>
	<li>After loading the chambers with subjects, check that the air/gas is flowing with the bubble test or the flow indicator. 
	NOTE: Some vaporizers do not allow air flow when the dial is in the off position.</li>
	<li>When gas is flowing, confirm that both the flowmeter and the downstream flow indicator indicate flow.</li>
	<li>At the end of the experiment, allow for 4-5 min of airflow to wash out the anesthetic.</li>
</ol>

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A., Perouansky, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+variability+affects+absolute+and+relative+potencies+and+kinetics+of+the+anesthetics+isoflurane+and+sevoflurane+in+Drosophila+melanogaster.">Genetic variability affects absolute and relative potencies and kinetics of the anesthetics isoflurane and sevoflurane in Drosophila melanogaster.</a> Scientific Reports. 8, 2348 (2018).</li><li>Stollings, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+modulation+by+volatile+anesthetics.">Immune modulation by volatile anesthetics.</a> Anesthesiology. 125 (2), 399-411 (2016).</li><li>Yamaguchi, M., Yoshida, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila as a model organism. In Drosophila Models for Human Diseases., edited by. , 1-10 (2018).</li><li>Hoffmann, J. 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A., Perouansky, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+among+genetic+background,+anesthetic+agent,+and+oxygen+concentration+shape+blunt+traumatic+brain+injury+outcomes+in+Drosophila+melanogaster.">Interactions among genetic background, anesthetic agent, and oxygen concentration shape blunt traumatic brain injury outcomes in Drosophila melanogaster.</a> International Journal of Molecular Sciences. 21 (18), 6926 (2020).</li><li>Fischer, J. A., Olufs, Z. P. G., Katzenberger, R. J., Wassarman, D. A., Perouansky, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthetics+influence+mortality+in+a+Drosophila+model+of+blunt+trauma+with+traumatic+brain+injury.">Anesthetics influence mortality in a Drosophila model of blunt trauma with traumatic brain injury.</a> Anesthesia &amp; Analgesia. 126 (6), 1979-1986 (2018).</li><li>Katzenberger, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Drosophila+model+of+closed+head+traumatic+brain+injury.">A Drosophila model of closed head traumatic brain injury.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (44), E4152-E4159 (2013).</li><li>Katzenberger, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+to+inflict+closed+head+traumatic+brain+injury+in+Drosophila.">A method to inflict closed head traumatic brain injury in Drosophila.</a> Journal of Visualized Experiments. (100), e52905 (2015).</li><li>Loewen, C. A., Ganetzky, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mito-nuclear+interactions+affecting+lifespan+and+neurodegeneration+in+a+Drosophila+model+of+Leigh+syndrome.">Mito-nuclear interactions affecting lifespan and neurodegeneration in a Drosophila model of Leigh syndrome.</a> Genetics. 208 (4), 1535-1552 (2018).</li><li>Johnson-Schlitz, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthetic+preconditioning+of+traumatic+brain+injury+is+ineffective+in+a+Drosophila+model+of+obesity.">Anesthetic preconditioning of traumatic brain injury is ineffective in a Drosophila model of obesity.</a> Journal of Pharmacology and Experimental Therapeutics. 381 (3), 229-235 (2022).</li><li>Li, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoflurane+postconditioning+reduces+ischemia-induced+nuclear+factor-kappaB+activation+and+interleukin+1beta+production+to+provide+neuroprotection+in+rats+and+mice.">Isoflurane postconditioning reduces ischemia-induced nuclear factor-kappaB activation and interleukin 1beta production to provide neuroprotection in rats and mice.</a> Neurobiology of Disease. 54, 216-224 (2013).</li><li>Leibovitch, B. A., Campbell, D. B., Krishnan, K. S., Nash, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+that+affect+ion+channels+change+the+sensitivity+of+Drosophila+melanogaster+to+volatile+anesthetics.">Mutations that affect ion channels change the sensitivity of Drosophila melanogaster to volatile anesthetics.</a> Journal of Neurogenetics. 10 (1), 1-13 (1995).</li><li>Tinklenberg, J. A., Segal, I. S., Guo, T. 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The authors have no conflicts of interest to declare.

Critical steps in the construction of the SAA include ensuring tight fittings to avoid leakage of the anesthetic mixture of gases. The SAA must be housed in a fume hood to avoid contamination of the laboratory space. All the elements from the carrier gas cylinders to the flow indicator downstream of the SAA should be checked as outlined in the checklist.
Other methods of administering VGAs to flies are complicated to operate (the inebriometer)21, have low throughput22, do not allow the simultaneous exposure of multiple populations23, do not allow precise control of the anesthetic concentration21, or have a readout that is difficult to translate into clinically accepted terms24.
The current version of the SAA relies on a commercial vaporizer, and hence, toxicologic studies are limited to volatile anesthetics. If used with other volatile substances, a vaporizer could be used &#34;off label&#34; after calibrating the output. Alternatively, a different method of vaporizing the volatile substances could be applied, which would require dedicated measurements to titrate the drug concentrations, as described previously25.
Apart from the flow indicators, there are no alarms (i.e., if the tanks empty, the flow through the SAA will be interrupted). Depending on the intensity of the use, the SAA may need cleaning, tightening, and possibly replacement of the Tygon tubing. We have performed &#34;maintenance&#34; on our original SAA twice in 7 years of use.
This method for anesthetizing fruit flies allows the use of the genetic toolbox available to Drosophila researchers in a high-throughput system. Multiple cohorts of flies of different populations (e.g., genotype, age, sex) can be simultaneously exposed to identical anesthetic concentrations and the desired combination of carrier gas (air, O2, N2O, noble gases) suitable to the research question at hand.
We show here that the SAA has been useful for revealing unexpected changes in resilience to isoflurane toxicity in the ND2360114 fly line and that standard laboratory fly lines differ in their responsiveness to AP.&#160;Identifying these findings was possible because of the tight control of the experimental conditions and the high throughput of the SAA.
The SAA can be adapted to study the effects of other volatile organic compounds (VOCs) on insects (e.g., honeybees). For VOCs with vapor pressures close to those of volatile anesthetics (isoflurane: 240 mmHg at 20 &#176;C), conventional vaporizers could be used, but the output would have to be calibrated. The commercial vaporizer for desflurane is heated, potentially offering additional flexibility.

The existence of collateral anesthetic effects (i.e., effects that are not immediately observable but may have delayed behavioral consequences) is generally accepted, but the understanding of their mechanisms and risk factors remains rudimentary1,2. Their delayed manifestation and subtleness limit the number of potentially important variables that can be investigated in mammalian models within reasonable time frames and at an acceptable cost. The fruit fly (Drosophila melanogaster) offers unique advantages in the context of neurodegenerative disease3 and for toxicologic screening4 that have, to date, not been applied to the study of anesthetic collateral effects. 
We developed the serial anesthesia array (SAA) to facilitate the use of fruit flies in the study of anesthetic pharmacodynamics and pharmacogenetics. A key advantage of the SAA is the simultaneous exposure to identical experimental conditions of multiple cohorts. When paired with the experimental flexibility of fruit flies, the high throughput of the SAA allows the exploration of biological and environmental variables on a scale impossible in mammalian models. 
In principle, the SAA is simply a series of connected anesthetizing locations (chambers made of 50 mL vials) through which a carrier gas delivers volatile agents. The system's first chamber contains distilled water through which the carrier gas is humidified (flies are sensitive to dehydration), and it terminates with a simple flow indicator that indicates the gas flow through the system. Fine nets placed on the openings of the connecting tubing separate the chambers to prevent the migration of flies between the chambers. The number of locations "in series" is limited by the resistance to the unpressurized gas flow (tubing, nets). 
We characterized the kinetics of this SAA prototype in a previous publication5. Although the exact pharmacokinetic properties will vary between SAAs, the relevant basics that have been tested experimentally are as follows: (i) an initial flow of 1.5-2 L/min equilibrates all the chambers (total volume of &plusmn;550 mL) with the desired concentration of anesthetic within 2 min; (ii) the concentration of anesthetic vapor delivered to the chambers does not change appreciably between the first and the last location because the amount of anesthetic contained in the volume of gas in an individual chamber (50 mL) far exceeds the amount taken up by any number of flies; and (iii) once the chambers have equilibrated, the carrier gas flow can be reduced (50-100 mL/min or less) to avoid waste and contamination of the environment (volatile anesthetics have greenhouse gas properties). The minimal flow necessary to maintain a steady-state concentration of vapor depends primarily on the leakiness of the SAA, as the uptake of vapor by the flies is negligible. Under these standard conditions (2% isoflurane and 1.5 L/min carrier gas flow), flies are anesthetized (i.e., immobile) in all positions of the array within 3-4 min, with unnoticeable differences between positions. VGAs can be administered for minutes to hours, and our typical exposure paradigms are in the range of 15 min to 2 h. To flush the system, the vaporizer is turned off, and flow is maintained to exchange approximately 10x volumes of the array (1.5 L/min for 5 min). The speed of anesthetic elimination will vary with the set rate of flow.
Volatile anesthetic agents interact with numerous still unidentified targets, including the immune-inflammatory system6. The contribution of individual molecular targets to primary versus collateral outcomes (the "anesthetic state" vs. long- and short-term "side effects") is poorly understood. Therefore, a sensitive, high-throughput fly system is valuable to inform experiments in higher animals, despite the obvious differences between flies and mammals7. Some differences can, in fact, be advantageous; for example, the fly's immune system differs from that of higher animals in that it lacks the adaptive arm of the response8. While this may seem like a limitation for understanding disease in humans, it offers a unique opportunity to study the interaction of VGAs with the innate immune-inflammatory response in isolation from the adaptive response9. This allows studies of the pharmacologic effects of VGA on inflammation and their modulation by the varied genetic backgrounds present in a population.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Serial Anesthesia Array:&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Serological Pipettes</td>
			<td>Fisher Scientific</td>
			<td>13-676-10C</td>
			<td>Polystyrene, 5mL serological pipette</td>
		</tr>
		<tr>
			<td>50 mL Conical Tubes</td>
			<td>Fisher Scientific</td>
			<td>1495949A</td>
			<td>Polypropylene, 50 mL</td>
		</tr>
		<tr>
			<td>Cable Tie Mounting Pad</td>
			<td>Grainger</td>
			<td>6EEE6</td>
			<td>1.25 inch L x 1 inch W x 0.28 inch H</td>
		</tr>
		<tr>
			<td>Dispensing Syringe</td>
			<td>Grainger</td>
			<td>5FVE0</td>
			<td>10 mL with Luer-Lock Connection</td>
		</tr>
		<tr>
			<td>Fabric Mesh Netting</td>
			<td></td>
			<td></td>
			<td>1 mm mesh</td>
		</tr>
		<tr>
			<td>Flow Indicator</td>
			<td>Grainger</td>
			<td>8RH52</td>
			<td>5/16 to 1/2 inch connection size, paddle wheel style</td>
		</tr>
		<tr>
			<td>Tygon Tubing</td>
			<td>Tygon</td>
			<td>E-3603</td>
			<td>ID: 5/16, OD: 7/16, wall: 1/16</td>
		</tr>
		<tr>
			<td>Wood Frame</td>
			<td></td>
			<td></td>
			<td>10 feet of 2 inch x 3/4 inch</td>
		</tr>
		<tr>
			<td>Zip Tie</td>
			<td></td>
			<td></td>
			<td>&#62;5inch</td>
		</tr>
		<tr>
			<td>Vaporizer Interface (Budget Alternative to Manifold):</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dispensing Syringe</td>
			<td>Grainger</td>
			<td>5FVE0</td>
			<td>10 mL with Luer-Lock Connection</td>
		</tr>
		<tr>
			<td>Commercial Manifold and Vaporizers:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1/4 inch Equal Barbed Y Connector</td>
			<td>Somni Scientific</td>
			<td>BF-9000</td>
			<td></td>
		</tr>
		<tr>
			<td>1/8 inch NPT to 1/4 inch Barbed Elbow (Plastic)</td>
			<td>Somni Scientific</td>
			<td>BF-9004</td>
			<td></td>
		</tr>
		<tr>
			<td>AIR 0-4 LPM Flowmeter w/ black knob</td>
			<td>Somni Scientific</td>
			<td>FP-4002</td>
			<td></td>
		</tr>
		<tr>
			<td>Flowmeter auxiliary mounting bracket</td>
			<td>Somni Scientific</td>
			<td>NonInvPart</td>
			<td></td>
		</tr>
		<tr>
			<td>Medical Air, 1/8 inch NPT Male x DISS Male</td>
			<td>Somni Scientific</td>
			<td>GF-11012</td>
			<td></td>
		</tr>
		<tr>
			<td>TT-2 Table Top Anesthesia System, built in dual diverter valve system. Includes 6&#39; color coded tubing X2. (Vaporizer not Included)</td>
			<td>Somni Scientific</td>
			<td>TT-17000</td>
			<td></td>
		</tr>
		<tr>
			<td>Tec 7 Isoflurane Vaporizer</td>
			<td>GE Datex-Ohmeda</td>
			<td>1175-9101-000</td>
			<td>Agent-specific vaporizer (Isoflurane)</td>
		</tr>
		<tr>
			<td>Tec 7 Sevoflurane Vaporizer</td>
			<td>GE Datex-Ohmeda</td>
			<td>1175-9301-000</td>
			<td>Agent-specific vaporizer (Sevoflurane)</td>
		</tr>
	</tbody>
</table>

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).

An SAA video link is provided here: Perouansky Research Methods - Department of Anesthesiology - UW-Madison (wisc.edu) (https://anesthesia.wisc.edu/research/researchers/perouansky-laboratory/perouansky-research-methods/)&#160;Our lab has used the SAA to (i) study the effect of genotype on behavioral sensitivity to anesthetics5; (ii) screen mitochondrial mutants for the collateral effects of anesthetics11; and (iii) investigate the pharmacodynamics of isoflurane and sevoflurane on outcomes in traumatic brain injury (TBI)12,13,14,15,16,17. The published results clearly demonstrate that the genetic background influences the pharmacodynamics of clinically used VGAs with respect to both the conventional phenotype of anesthesia and the collateral effects of anesthetic toxicity, as well as tissue protection5,11,13,14,15.
Representative example 1 (Figure 2):Genetic drift in resilience to isoflurane toxicity detected by reliably reproducible experimental conditions 
The discovery of a gradual quantitative change in VGA-induced mortality among separately cultured ND2360114 flies is an example of the usefulness of reliable comparisons of anesthetic pharmacodynamics across experimental groups using the SAA. ND23 is a gene encoding a subunit in the core of Complex I of the mETC (analogous to Ndufs8 in mammals)18. Mutations in this subunit are a cause of Leigh syndrome, a lethal mitochondrial disease. We observed a gradual weakening of the isoflurane-induced mortality phenotype over time in various homozygous ND2360114 stocks cultured simultaneously under standard laboratory conditions (i.e., without exposure to VGAs). This evolutionary adaptation to isoflurane toxicity occurred in the absence of any exposure to VGAs and is probably a collateral effect of &#34;survival of the fittest&#34; within the mutant stocks. This gradual change in isoflurane sensitivity would have remained unrecognized without our confidence that the experimental conditions were identical across the assays and over time. We conclude that selection favors modifiers of the effects of ND2360114, with coincidental increased resilience to isoflurane toxicity. As inflammation in the central nervous system plays an important role in the pathogenesis of Leigh syndrome, the witnessed evolution of resistance may be due to adaptive changes in the innate immune-inflammatory response, with resistance to isoflurane toxicity being an accidental byproduct.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65144/65144fig02.jpg" /> 
Figure 2: Variation in isoflurane toxicity-induced mortality as a result of evolutionary pressure in ND2360114 flies. Seven lines (A-G) isolated from a single population through single-pair matings, expanded, and tested for 24 h mortality (PM24) following a 2 h exposure to 2% isoflurane (at 10-13 days old) show variability in the phenotype arising from a single population. Data shown as box and whisker plots. The boxes represent the second and third quartiles of the data, with the whiskers extending to the minimum and maximum data points. The mean and median are indicated by &#34;+&#34; and horizontal lines, respectively. The percent mortalities of the individual replicates (N) are shown as circles. N = 3-4 vials of 20-50 flies/vial. P-value for an ordinary one-way ANOVA; p = 0.012 indicates a significant difference among the means. <a href="https://www.jove.com/files/ftp_upload/65144/65144fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Representative example 2 (Figure 3): Illustration of a high-throughput application of the SAA to reveal genetic background effects on isoflurane pharmacodynamics 
As an example of the high throughput of the system, Figure 3 illustrates the effects of identical exposures to isoflurane (15 min of 2% isoflurane) prior to traumatic brain injury (TBI)16, a protocol testing anesthetic preconditioning (AP) in this fly model13,15,19. The readout is mortality 24 h after TBI corrected for natural attrition (MI24). In this model, all the flies regained mobility (i.e., were alive) within 30 min after TBI, and the mortality recorded in the MI24 was a result of secondary brain injury (sBI). In the four fly lines, AP with isoflurane reduced the MI24 to various degrees, indicating that responsiveness to AP is a quantitative trait. As the inflammatory response is an important factor in morbidity from sBI, AP may involve modulation of the immune system20.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65144/65144fig03.jpg" /> 
Figure 3: Influence of genetic background on the suppression of mortality (MI24) by preconditioning with isoflurane. Preconditioning flies with 15 min of 2% isoflurane (purple) reduced the mortality index at 24 h (MI24) in w1118 and y1w1118 strains (p &#60; 0.0001 and p = 0.036, respectively). The MI24 was not significantly lower in the preconditioned Oregon R (OR) and Canton S (CS) lines (p = 0.16 and p = 0.27, respectively). Data shown as box and whisker plots. The boxes represent the second and third quartiles of the data, with the whiskers extending to the minimum and maximum data points. The mean and median are indicated by &#34;+&#34; and horizontal lines, respectively. The MI24 values of the individual replicates (N) are shown as circles. N = 15-33 vials of 30-40 flies/vial for TBI-treated flies. N = 2-15 vials of 30-40 flies/vial for untreated controls. P-values from an unpaired, two-tailed Student&#39;s t-test. <a href="https://www.jove.com/files/ftp_upload/65144/65144fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about The Serial Anesthesia Array for the High-Throughput Investigation of Volatile Agents Using Drosophila melanogaster at JoVE.com

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.

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 ...

the-serial-anesthesia-array-for-high-throughput-investigation

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JoVE Journal

Genetics

625 Views.  University of Wisconsin-Madison. 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. 

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

High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii

Systematic identification and characterization of genetic perturbations have proven useful to decipher gene function and cellular pathways. However, the conventional approaches of permanent gene deletion cannot be applied to essential genes. We have pioneered a unique collection of ~70 temperature-sensitive (ts) lethal mutants for studying cell cycle regulation in the unicellular green algae Chlamydomonas reinhardtii1. These mutations identify essential genes, and the ts alleles can be conditionally inactivated by temperature shift, providing valuable tools to identify and analyze essential functions. Mutant collections are much more valuable if they are close to comprehensive, since scattershot collections can miss important components. However, this requires the efficient collection of a large number of mutants, especially in a wide-target screen. Here, we describe a robotics-based pipeline for generating ts lethal mutants and analyzing their phenotype in Chlamydomonas. This technique can be applied to any microorganism that grows on agar. We have collected over 3000 ts mutants, probably including mutations in most or all cell-essential pathways, including about 200 new candidate cell cycle mutations. Subsequent molecular and cellular characterization of these mutants should provide new insights in plant cell biology; a comprehensive mutant collection is an essential prerequisite to ensure coverage of a broad range of biological pathways. These methods are integrated with downstream genetics and bioinformatics procedures for efficient mapping and identification of the causative mutations that are beyond the scope of this manuscript.

High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii

Temperature-sensitive (ts) lethal mutants are valuable tools to identify and analyze essential functions. Here we describe methods to generate and classify ts lethal mutants in high throughput.

Systematic identification and characterization of genetic perturbations have proven useful to decipher gene function and cellular pathways. However, the ...

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in humans, but more tools and research efforts are needed to better elucidate the mechanisms involved. For over a century, the genetically highly tractable model of Drosophila has been instrumental in the discovery of key genes and molecular pathways that proved to be highly conserved across species. Many biological processes and disease mechanisms are functionally conserved in the fly, such as development (e.g., body plan, heart), cancer, and neurodegenerative disease. Recently, the study of obesity and secondary pathologies, such as heart disease in model organisms, has played a highly critical role in the identification of key regulators involved in metabolic syndrome in humans.
Here, we propose to use this model organism as an efficient tool to induce obesity, i.e., excessive fat accumulation, and develop an efficient protocol to monitor fat content in the form of TAGs accumulation. In addition to the highly conserved, but less complex genome, the fly also has a short lifespan for rapid experimentation, combined with cost-effectiveness. This paper provides a detailed protocol for High Fat Diet (HFD) feeding in Drosophila to induce obesity and a high throughput triacylglyceride (TAG) assay for measuring the associated increase in fat content, with the aim to be highly reproducible and efficient for large-scale genetic or chemical screening. These protocols offer new opportunities to efficiently investigate regulatory mechanisms involved in obesity, as well as provide a standardized platform for drug discovery research for rapid testing of the effect of drug candidates on the development or prevention of obesity, diabetes and related metabolic diseases.

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

This is a high fat diet feeding protocol to induce obesity in Drosophila, a model for understanding fundamental molecular mechanisms implicated in lipotoxicity. It also provides a high throughput triacylglyceride assay for measuring fat accumulation in Drosophila and potentially other (insect) models under various dietary, environmental, genetic or physiological conditions.

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in ...

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution based analysis methodology (nucleosome density ChIP-seq; ndChIP-seq) that enables the generation of combined measurements of micrococcal nuclease (MNase) accessibility with histone modification genome-wide. Nucleosome position and local density, and the posttranslational modification of their histone subunits, act in concert to regulate local transcription states. Combinatorial measurements of nucleosome accessibility with histone modification generated by ndChIP-seq allows for the simultaneous interrogation of these features. The ndChIP-seq methodology is applicable to small numbers of primary cells inaccessible to cross-linking based ChIP-seq protocols. Taken together, ndChIP-seq enables the measurement of histone modification in combination with local nucleosome density to obtain new insights into shared mechanisms that regulate RNA transcription within rare primary cell populations.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) methodology for the generation of sequence datasets suitable for a nucleosome density ChIP-seq analytical framework integrating micrococcal nuclease (MNase) accessibility with histone modification measurements.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have been directed toward elucidation of the location and timing of initiation of genome duplication rather than its completion. However, it is critical that we understand regions of the genome that are last to complete replication, because these regions suffer elevated levels of chromosomal breakage and mutation, and they have been associated with both disease and aging. Here we describe how we have extended a technique that has been used to monitor replication initiation to instead identify those regions of the genome last to complete replication. This approach is based on a combination of flow cytometry and high throughput sequencing. Although this report focuses on the application of this technique to yeast, the approach can be used with any cells that can be sorted in a flow cytometer according to DNA content.

We describe a technique for combining flow cytometry and high throughput sequencing to identify late replicating regions of the genome.

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have ...

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with a number of challenges including sample preservation quality, degraded DNA, and destructive sampling of rare specimens. In order to more effectively use herbarium material in large sequencing projects, a dependable and scalable method of DNA isolation and library preparation is needed. This paper demonstrates a robust, beginning-to-end protocol for DNA isolation and high-throughput library construction from herbarium specimens that does not require modification for individual samples. This protocol is tailored for low quality dried plant material and takes advantage of existing methods by optimizing tissue grinding, modifying library size selection, and introducing an optional reamplification step for low yield libraries. Reamplification of low yield DNA libraries can rescue samples derived from irreplaceable and potentially valuable herbarium specimens, negating the need for additional destructive sampling and without introducing discernible sequencing bias for common phylogenetic applications. The protocol has been tested on hundreds of grass species, but is expected to be adaptable for use in other plant lineages after verification. This protocol can be limited by extremely degraded DNA, where fragments do not exist in the desired size range, and by secondary metabolites present in some plant material that inhibit clean DNA isolation. Overall, this protocol introduces a fast and comprehensive method that allows for DNA isolation and library preparation of 24 samples in less than 13 h, with only 8 h of active hands-on time with minimal modifications.

This article demonstrates a detailed protocol for DNA isolation and high-throughput sequencing library construction from herbarium material including rescue of exceptionally poor-quality DNA.

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with ...

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our entire society's ability to treat systemic infections. In the United States alone, antibiotic-resistant infections kill approximately 23,000 people a year and cost around 20 billion USD in additional healthcare. One approach to combat antimicrobial resistance is combination therapy, which is particularly useful in the critical early stage of infection, before the infecting organism and its drug resistance profile have been identified. Many antimicrobial treatments use combination therapies. However, most of these combinations are additive, meaning that the combined efficacy is the same as the sum of the individual antibiotic efficacy. Some combination therapies are synergistic: the combined efficacy is much greater than additive. Synergistic combinations are particularly useful because they can inhibit the growth of antimicrobial drug resistant strains. However, these combinations are rare and difficult to identify. This is due to the sheer number of molecules needed to be tested in a pairwise manner: a library of 1,000 molecules has 1 million potential combinations. Thus, efforts have been made to predict molecules for synergy. This article describes our high-throughput method for predicting synergistic small molecule pairs known as the Overlap2 Method (O2M). O2M uses patterns from chemical-genetic datasets to identify mutants that are hypersensitive to each molecule in a synergistic pair but not to other molecules. The Brown lab exploits this growth difference by performing a high-throughput screen for molecules that inhibit the growth of mutant but not wild-type cells. The lab's work previously identified molecules that synergize with the antibiotic trimethoprim and the antifungal drug fluconazole using this strategy. Here, the authors present a method to screen for novel synergistic combinations, which can be altered for multiple microorganisms.

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

Synergistic drug combinations are difficult and time-consuming to identify empirically. Here, we describe a method for identifying and validating synergistic small molecules.

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our ...

Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System (REQS)

Drosophila melanogaster is a new model organism for studies in exercise biology. To date, two main exercise systems, the Power Tower and the Treadwheel have been described. However, a method to measure the amount of additional animal activity induced through the exercise treatment has been lacking. The Rotating Exercise Quantification System (REQS) fills this need, providing a measure of animal activity for animals experiencing rotational exercise. This protocol details how to use the REQS to assess animal activity during rotational exercise and illustrates the type of data that can be generated. Here, we demonstrate how the REQS is used to measure sex- and strain-specific differences in exercise induced activity. The REQS can also be used to evaluate the impact of various other experimental parameters such as age, diet, or population size on exercise induced activity. In addition, it can be used to compare the efficacy of different exercise training protocols. Importantly, it provides an opportunity to standardize exercise treatments between strains, allowing the researcher to achieve equal amounts of activity between groups if needed. Thus, the REQS is a notable new resource for exercise biologists working with the Drosophila model system and complements existing exercise systems.

Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System REQS

The Rotating Exercise Quantification System (REQS) can induce exercise in Drosophila melanogaster through rotation while simultaneously measuring the amount of activity performed by the animals. Here, we present a point-by-point protocol detailing how to measure activity levels of animals experiencing rotational exercise treatments using the REQS.

Drosophila melanogaster is a new model organism for studies in exercise biology. To date, two main exercise systems, the Power Tower and the Treadwheel ...

The TreadWheel: Interval Training Protocol for Gently Induced Exercise in Drosophila melanogaster

The incidence of complex metabolic diseases has increased as a result of a widespread transition towards lifestyles of increased caloric intake and lowered activity levels. These multifactorial diseases arise from a combination of genetic, environmental, and behavioral factors. One such complex disease is Metabolic Syndrome (MetS), which is a cluster of metabolic disorders, including hypertension, hyperglycemia, and abdominal obesity. Exercise and dietary intervention are the primary treatments recommended by doctors to mitigate obesity and its subsequent metabolic diseases. Exercise intervention, in particular aerobic interval training, stimulates favorable changes in the common risk factors for Type 2 Diabetes Mellitus (T2DM), Cardiovascular Disease (CVD), and other conditions. With the influx of evidence describing the therapeutic effect exercise has on metabolic health, establishing a system that models exercise in a controlled setting provides a valuable tool for assessing the effects of exercise in an experimental context. Drosophila melanogaster is a great tool for investigating the physiological and molecular changes that result from exercise intervention. The flies have short lifespans and similar mechanisms of metabolizing nutrients when compared to humans. To induce exercise in Drosophila, we developed a machine called the TreadWheel, which utilizes the fly&#39;s innate, negative geotaxis tendency to gently induce climbing. This enables researchers to perform experiments on large cohorts of genetically diverse flies to better understand the genotype-by-environment interactions underlying the effects of exercise on metabolic health.

The TreadWheel: Interval Training Protocol for Gently Induced Exercise in Drosophila melanogaster

The TreadWheel uses rotational motion to gently induce exercise in adult Drosophila melanogaster by exploiting flies&#39; innate, negative geotaxis. It allows for analysis of the interactions between exercise and factors, such as genotype, sex, and diet, and their effect on physiological and molecular assays to assess metabolic health.

The incidence of complex metabolic diseases has increased as a result of a widespread transition towards lifestyles of increased caloric intake and ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

Designing Automated, High-throughput, Continuous Cell Growth Experiments Using eVOLVER

Continuous culture methods enable cells to be grown under quantitatively controlled environmental conditions, and are thus broadly useful for measuring fitness phenotypes and improving our understanding of how genotypes are shaped by selection. Extensive recent efforts to develop and apply niche continuous culture devices have revealed the benefits of conducting new forms of cell culture control. This includes defining custom selection pressures and increasing throughput for studies ranging from long-term experimental evolution to genome-wide library selections and synthetic gene circuit characterization. The eVOLVER platform was recently developed to meet this growing demand: a continuous culture platform with a high degree of scalability, flexibility, and automation. eVOLVER provides a single standardizing platform that can be (re)-configured and scaled with minimal effort to perform many different types of high-throughput or multi-dimensional growth selection experiments. Here, a protocol is presented to provide users of the eVOLVER framework a description for configuring the system to conduct a custom, large-scale continuous growth experiment. Specifically, the protocol guides users on how to program the system to multiplex two selection pressures &#8212; temperature and osmolarity &#8212; across many eVOLVER vials in order to quantify fitness landscapes of Saccharomyces cerevisiae mutants at fine resolution. We show how the device can be configured both programmatically, through its open-source web-based software, and physically, by arranging fluidic and hardware layouts. The process of physically setting up the device, programming the culture routine, monitoring and interacting with the experiment in real-time over the internet, sampling vials for subsequent offline analysis, and post experiment data analysis are detailed. This should serve as a starting point for researchers across diverse disciplines to apply eVOLVER in the design of their own complex and high-throughput cell growth experiments to study and manipulate biological systems.

The eVOLVER framework enables high-throughput continuous microbial culture with high resolution and dynamic control over experimental parameters. This protocol demonstrates how to apply the system to conduct a complex fitness experiment, guiding users on programming automated control over many individual cultures, measuring, collecting, and interacting with experimental data in real-time.

Continuous culture methods enable cells to be grown under quantitatively controlled environmental conditions, and are thus broadly useful for measuring ...