The growing field of Joof olfaction has motivated increasingly sophisticated behavioral assays, such as the olfactory magnetic tether apparatus or OMT. This procedure involves gluing the dorsal thorax of the fly to a mnuchin pin. The fly is suspended at a 30 degree angle with the pin at a proper pitch, then aligned between two rare earth magnets.
When done correctly, this will result in a free range of motion. An odor port is then aligned to produce a plume that passes smoothly from the delivery nozzle over the fly's head into the vacuum chamber. The ability of the fly to locate odorant is then tested in the presence of visual stimuli.
Hi, I'm Brian DeMars from the laboratory of Dr.Mark Fry in the Department of Physiological Science at the University of California Los Angeles. Today we will show you how to tether flies for use in an olfactory magnetic tether apparatus. We will also show you how to align the magnets and odor ports and set the mass flow rates for the odor delivery system.
We use this procedure in our laboratory to study visual and olfactory mediated flight control and fruit flies. So let's get started. To begin tethering collect flies four to six days post ELOs, starve them for four to six hours.
In a food list vial containing a moist kim wipe to prevent desiccation Next transfer. A batch of starred flies to a small vial inserted into a brass block on a peltier cooling stage set at four degrees Celsius. At this temperature, the flies will anesthetize within approximately 30 seconds.
Place a Kim wipe on top of the cooling stage to absorb moisture. Next place anesthetized flies on top of the stage for sorting, select the largest females for tethering. Using a fine brush, push a single fly into position in the custom-built sarcophagus shaped to accommodate a single wild type female fruit fly.
Now use the microscope to ensure that the fly is correctly positioned with its dorsal side. Up the anterior of the fly should be facing you and the posterior away from you. Next position a minuchin pin with a magnetic rod mounted on a microm manipulator.
The pin should be aligned with the blunt end facing the dorsal thorax of the fly. This is just behind the head between the two wings. The fly should be positioned on the pin correctly along three axes.
The pitch, the roll, and the ya the pin should be perfectly vertical when viewed, head on toward the eyes, even a small amount of roll between the fly and the pin cannot be tolerated. The side view angle between the pin and the long axis of the body or pitch should be at a nose up angle of 30 degrees relative to the horizon. Keep in mind that the pin may need to be trimmed for optimal tether operation.
This will depend on the distance between the suspension magnets. Once the mnuchin pin is aligned, use a thin steel wire to apply a small drop of UV activated glue to the blunt end of the mnuchin pin. Now manipulate the pin so the glue droplet touches the flies thorax just above the head.
Be careful not to use too much glue. With the proper drop size, the glue should a kneel and flow from the pin onto the thorax. Next, cure the glue with two 22nd bursts of UV light.
Once tethering is complete hand, a small square Kim wipe to each fly using forceps due to an instinctive landing reflex. The flies will grasp the Kim wipes. After one hour of recovery, the tethered flies are ready for experiments.
To achieve a smooth 360 degree range of motion, it is critical to align the magnets properly. To do this, hold the lower ring magnets horizontally with a plastic sleeve and place them directly on top of a clear vacuum chamber. The upper rod magnet is fixed vertically approximately three quarters of an inch above the lower magnets, a V dual bearing is attached with epoxy to the bottom surface of the upper magnet.
This will standardize fly positioning and minimize rotational friction. Keep in mind that these magnets can be dangerous and misuse can result in damaged equipment or minor bodily injury. Next, roughly align the upper and lower magnets visually.
By placing the upper rod magnet directly above the center point of the lower ring magnets, it will be useful to attach the upper magnet to a micro manipulator for fine scale adjustment. Now, place a fly in the arena and adjust the horizontal and vertical position of the upper magnet until the fly can smoothly and steadily rotate 360 degrees at the start of every experiment, each fly should be checked for smooth rotation to ensure that the magnets are properly aligned and that the mnuchin pins are not damaged. If the setup is incorrect, the fly may not spin at all.
May have restricted range of motion or may rotate at inconsistent velocities. This could be a problem with gluing, which can be tested by rotating a striped grid in front of the fly to elicit an omo response. If the fly does not spin or spins inconsistently, then replace it with another fly.
If this fly does not spin correctly and these issues exist for every fly, then the problem is likely due to the alignment of the magnets and further adjustment will be required to achieve stable odor. Tracking the initial setup of the odor system will require running narrow gauge Teflon tubing from the gas multiplexer to the custom built water odor vials. The outlet of these vials should connect directly to the hypodermic port tubes using the same Teflon tubing.
Next, a clear acrylic vacuum chamber is positioned under the arena to support the magnets. Use a four millimeter glass tube mounted below the fly and connected to the vacuum chamber to bring the vacuum opening closer to the fly and reduce the diameter of the opening. Now place a tethered fly into the arena using forceps.
Once the fly is in place, turn on an attractive odor. You can visually verify if the fly heads toward the position of the odor port when the odor is turned on. If the fly appears to have no preference for the odor, use trial and error to optimize the OMT.
To do this first, adjust the position of the odor port until the fly appears to track the odor. Here the odor ports are mounted on microm manipulators and positioned four millimeters dorsal and three millimeters anterior to the fly's head. If changing the position of the odor port does not improve odor tracking, adjust the mass flow rate until the fly appears to track the odor.
We use settings as seven milliliters per minute. If adjusting the mass flow rate does not improve odor tracking, adjust the flow rate of the vacuum until the fly appears to track the odor. We set the vacuum flow rate at 13 liters per minute using a flow regulator.
Once the position of the port and the flow rates are set, periodically switch between delivering water vapor and odor vapor using a switchable gas multiplexer. If you have set up your apparatus correctly, you should observe two things. First, under constant rotating visual stimuli, a tethered fly placed in the OMT should rotate smoothly 360 degrees in the vertical or ya axis jerky turns called kos are characteristic of spontaneous turning behavior in the absence of constant visual stimuli.
If flies cannot rotate smoothly, improper gluing and misaligned magnets may be to blame. Second, you should also see that when the odor is turned on, the fly should head in the direction of the odor port. When the odor is turned off, the fly should secod spontaneously with no preference for any position in the arena.
If flies do not actively track the odor plume, this may mean that you need to adjust the position of the odor port, the airflow rate, and or the vacuum flow rate. We've just shown you how to tether a fly for use in an olfactory magnetic tether apparatus. We've also shown you how to properly align the magnets and odor ports and how to set the mass flow rates for the odor delivery system.
So that's it. Thanks for watching and good luck with your experiments.
