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

The SAA allows high throughput processing for investigating the pharmacology of volatile agents in invertebrate model organisms that are endowed with a sophisticated brain and a vast repertoire of behaviors. In combination with the genetic toolbox available in Drosophila melanogaster, this technique allows the pharmacogenetics of volatile agents to be studied rapidly and affordably. Many people are exposed to volatile agents as pollutants or therapeutic agents.

In addition to anesthesia, volatile anesthetics have numerous toxic and beneficial collateral effects. The degree of effect is partly determined by genetics, which has remained largely unexplored. After making the wooden frame, modify 50 milliliter chronicle tube caps by drilling two holes in each cap with a 9/32 inch drill bit.

Sand the holes to clean up the ragged plastic. Also sand the top of the cap to roughen the surface. Then cut five milliliter serological pipettes to size by scoring the plastic and breaking it clean at the scored line.

Sand the ends of the cut or broken pipettes. Afterward, glue netting to the tubes and cut the netting to the tube size once the adhesive dries. Insert the tubes into the holes of the conical caps with both tubes extending above the cap.

Ensure that the inflow tube extends longer into the tube than the outflow. Apply glue to the tops of the caps around the tubes to secure the parts together and allow them to dry. Attach adhesive cable tie downs to the frame.

Affix the caps to the frame using zip ties and cut the zip tie tag ends short. Cut and connect the length of the tygon tubing to the inflow and outflow tubes on each modified cap. Starting at the upstream end, attach the tubing first to the inflow, then from the outflow to the inflow of the subsequent position.

Next, add a flow indicator to the most downstream inflow. Put a 50 milliliter conical tube in the first position and mark the water fill mark. Remove the tube.

Fill it with water below the inflow tube and place it back in position. Prepare the interfacing for the vaporizer. Remove the plungers, cut notches out of two 10 milliliter dispensing syringes, 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.

Hook up the entire system. Use tygon tubing to attach the carrier gas tank with the regulator, gas specific flow meter, vaporizer and SAA. Fill empty positions on the array with empty 50 milliliter chronicle tubes.

Now turn on the gas tank. Open the flow meter to two liters per minute and turn on the vaporizer to 0%Confirm gas flow through the system by checking the flow meter 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 the water and look for bubbles.

24 hours or more prior to the anesthetic exposure sort the fly cohorts as needed for the experiment using the preferred method. Transfer flies from food vials into empty 50 milliliter conical tubes. Count and record any dead flies prior to exposure.

Uncap and screw 50 milliliter conical tubes with flies onto the SAA. Turn on the carrier gas and set it to the desired flow rate. Set the anesthetic vaporizer to the desired concentration and expose the flies for the desired duration.

At the end of the exposure flush the system with the fresh gas flow at 1.5 liters per minute for five minutes, corresponding to approximately 10x volumes of the total SAA volume. Open the high pressure regulator completely then close it half a turn to ensure carrier gas flow. Follow tubing for each line to the flow meters and vaporizer, and check the anesthetic level in the vaporizers.

Confirm that when gas flows, it indicates flow on the flow meter and on the downstream flow indicator. At the end of the experiment, allow four to five minutes of airflow to wash out the anesthetic. Seven lines isolated from a single population through single pair meetings showed variation in iso fluorine toxicity-induced mortality due to evolutionary pressure in ND23 60114 flies.

Pre-conditioning flies with 15 minutes of 2%iso fluorine prior to traumatic brain injury reduced the mortality index at 24 hours in white 1118 and yellow/white strains. The mortality index at 24 hours was not significantly lower in the pre-conditioned Oregon R and Canton S lines. During assembly the notches in the modified syringes must align with the holes of the vaporizer ports.

During exposure, periodically check that there is flow through the system. This procedure can be followed for a wide variety of behavioral, survival, and molecular studies that could lead to insights into the pharmacology of volatile agents.