The overall goal of this procedure is to measure ethanol self-administration in fruit flies as a proxy for changes in rewards states. This method can help answer key questions in neuroscience, such as, how experience is encoded in the brain, and leads to modification of behavior. The main advantage of this technique is that it is relatively simple, and does not require a complicated setup.
The protocol is composed of two discrete parts. The first is exposing the flies to rewarding and non-rewarding experiences, and the second is assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts of the protocol 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. Demonstrating the procedure will be Shir Zer, a master's student, and Julia Ryvkin, a graduate student, both from my lab. To begin the construction of the capillary feeder, first take a standard fly vial with small holes along the side of the vial.
Use a razor blade to cut the plug of the vial in half, crosswise. Mark four points onto the surface of the plug, creating a square shape to position the capillary holders. With an 18-gauge needle, make four holes through the plug at the position of the marks.
And make the holes wider using a micropipette. Using a razor blade, cut four micropipettes so that they firmly hold five microliter glass capillaries. Insert these into the holes in the prepared plug.
Mark the position on the plug of the two capillaries that will contain ethanol. Next, arrange glass vials in microcentrifuge racks. Melt fly food, and then pour two milliliters into each of the vials.
Allow the food to harden, and then cover the vials with plastic wrap. Begin by taking male flies that have been housed separately in smaller vials in seclusion. Assign these to two groups randomly.
Those who will undergo rewarding, versus non-rewarding mating experiences. Beginning with the non-rewarding group, add a previously mated female to each vial. Moving on to the reward group, add a virgin female to each vial.
Watch the mating encounters to ensure that males interacting with virgin females mate successfully within the first hour. And that males interacting with previously mated females do not mate. It is essential to observe carefully, to make sure that the mated cohort do indeed undergo mating, and that the rejected cohort do not manage to mate.
After one hour, remove from the experiment any males from the rejected cohort that managed to mate, and males from the mated cohort that did not end up mating by the end of the first training session. Conclude the conditioning session by aspirating out the previously mated females from the non-rewarding condition, keeping these individuals for later trials. Finally, remove and discard the females from the mated male group.
Let the flies rest for one hour, and then repeat the hour long conditioning assay two further times with an hour rest in between. These training sessions should be repeated again each day for three more days. After the final conditioning session on day four, aspirate the single males and place them in groups of six to eight into the capillary feeder vials, keeping flies in the same reward group together.
Use a soft plug to keep the males in place during transfer. Once all flies are inserted, replace the soft plug with the capillary feeder plug. Wet the plugs by gently adding five milliliters of water to the top of each plug, avoiding spillage into the vial.
Prepare the ethanol and water food solutions, and then place five microliter drops of these onto a strip of laboratory film. Allow the solution to fill the capillary until it reaches the five microliter mark. And then tap them gently on the strip to position the solution exactly at the premarked line.
Seal the ends of the capillaries by dipping them into small droplets of mineral oil. Finally, insert the two ethanol containing and non-ethanol containing capillaries into the adapters in the plug. The exposed sides should protrude one millimeter or less from the plug surface.
Keeping the opening for the capillaries close to the wet plug reduces the evaporation and allows easy access to the food, as flies normally sit on the plugs and climb down on the exposed capillary to feed. Additionally, prepare one mock vial without flies, which will be used as a control to assess natural evaporation. Finally, record the relative position of the liquid in each of the capillaries in vials with respect to the black line in millimeters.
Set the vials into the experimental incubator for 24 hours. Measure and record the levels of the liquid in each removed capillary with respect to the black line. Once the experiment is in progress, replace the capillary at a set time, each day.
Re-dampen the plugs with two milliliters of water, and then set the vials back into the incubator. Wild type Canton S strain male fruit flies collected upon eclosion and aged until four days old, were assayed over the course of four days for their innate preference to consume alcohol. Analysis of volumes consumed from ethanol and non-ethanol containing solutions, show that the flies exhibited a significant preference for the 15%ethanol food source.
The CAFE experiment was repeated with mated versus rejected males. Both groups had been previously housed singly, and exposed to females that either accepted or rejected mating. Upon introduction in groups to the CAFE vials, the rejected group consumed more from the ethanol capillaries.
This suggested that perceived lack of reward led to an increase in reward-seeking behavior. Conversely, successful mating increases internal reward levels, which in turn lowers ethanol consumption. While attempting this procedure, it's important to maintain a well-humidified environment for the entire experiment, with extra emphasis on the consumption part, watering the plugs every day, and keeping the capillary ends in close proximity to the wet plugs.
