The overall goal of this procedure is to study the molecular mechanisms underlying learning and memory formation. In honeybees, the first step is to collect free flying bees from their hive. Next, the bees are harnessed to plastic tubes.
The third step of the procedure is to perform classical conditioning. Drugs can be injected at certain time points around conditioning in order to study their effect on learning and memory formation. The final step of the procedure is to test memory retention.
Ultimately, changes in behavior after drug treatment may reveal the molecular mechanisms underlying learning and memory formation. In honeybees. This method can help answering key questions in the field of the honing and memory by uncovering the molecular mechanisms of memory formation.
In general, researchers new to this method will have difficulties because the timing of the stimuli presentation during conditioning and memory retrieval must be exact. Also, the feeding regime is critical. The procedure will be demonstrated by cutting gearing and Johannes faberg to graduate students from my laboratory On the day before the experiment, collect bees between two and 4:00 PM using a UV light permeable plexiglass pyramid.
Once back in the lab, place the pyramid on its base and darken the walls with a towel leaving the apex uncovered. Open the apex and hold a glass vial over the opening, allowing a single bee to travel into the vial before reclosing. Continue this procedure until all bees have been transferred into a glass vial in autumn and winter.
When bees are not flying outside, a beehive is kept inside a greenhouse in a net, single bees are directly caught from the net. By using one glass vial per b immobilize a bee by cooling the vial on ice for two and a half to three and a half minutes. Monitor the bee carefully and remove it from the ice as soon as it stops moving.
Harness the immobilize bee in a small plastic tube with sticky tape. Be sure that the bee is able to move its osis freely, but not its head, thorax, or legs. It is also important to not compress the neck.
Once a bee is prepared. Place the tube into a numbered borehole on a rack. For better handling and identification.
The tubes are always returned to the same borehole. After handling, feed the bees between four and 6:00 PM in a location distinct from the conditioning site. Elicit the Probos extension response or PER by touching the bees and antennae.
With a sucrose solution, allow the bee to consume one droplet of about four microliters of SRAs solution. Making sure not to smear the bee's an antennae, pro bois, or the plastic tube. Feed each bee until they no longer.
Show a fast and reliable PER when their an antennae touched with the solution. On subsequent evenings of the experiment, feed each bee four times with one droplet of sucrose solution. Fill a plastic bowl approximately 0.5 to one centimeter high with tap water.
Place the rack of harnessed bees in the bowl using a lysa plates as a platform. Cover the bowl with cardboard and store at room temperature.Overnight. Bees undergo olfactory conditioning around 10:00 AM on the morning after collection.
30 minutes before the start of the experiment. Place the rack of harness bees near the conditioning site, but away from the opening of the exhaust pipe where the bees will be trained. Next, prepare the condition stimulus working in a hood.
Use a filter tip to pipette four microliters of clove oil onto a round filter paper. Place the scented filter paper into a 20 milliliter syringe and insert the plunger. Set the audio player that will deliver an acoustic signal to the experimenter.
This ensures the precise timing of stimulus, onset stimulus, offset stimulus, duration, and placing. Prepare to record the bee's behavior throughout conditioning. A positive score is given if the bee extends its SSIS between the onset of the condition stimulus and the presentation of the unconditioned stimulus.
Crossing a virtual line between the open mandibles To begin an acquisition trial, select the bee to be trained and position it in front of the exhaust for 10 seconds. Shortly before the end of this ten second interval, place the odor containing syringe three centimeters from the bees and antennae. Present the odor over five seconds by pushing 20 milliliters of air through the syringe.
After three seconds, introduce the unconditioned stimulus by touching the ventral part of the distal flagella of both an antennae with a sucrose solution, moist and toothpick. Being careful not to deposit solution on the bees and antennae, the bee is then allowed to lick the toothpick. This compound stimulus lasts four seconds be bees are removed from the training context and placed back into the rack.
13 seconds after the presentation of the condition stimulus one training trial lasts 28 seconds. Return the bee to the training site after an inter trial interval of 10 minutes. Conduct the second of three trials as shown previously, and wait an additional 10 minutes before performing the final pairing.
After training is complete for the day, place the racks back into the bowl. Remember to feed each bee four times with one four microliter droplet of sucrose solution between four and 6:00 PM As demonstrated earlier, retention tests can be performed at any interval from minutes to days after training. Prepare for the memory test by placing the rack of bees near the conditioning site for 30 minutes.
Initiate testing by placing a bee in front of the exhaust for 10 seconds. Present the condition stimulus for five seconds and note the occurrence of a PER. After testing is complete for the day, place the racks back into the bowl.
Remember to feed each bee four times with one four microliter droplet of sucrose solution between four and 6:00 PM as demonstrated earlier at the end of the entire experiment, verify that the bee is still able to extend its probos by touching the an antennae with SRAs solution to be included in the analysis, bees must survive the entire experiment and have an intact PER. In some studies, systemic injections are used to study the effect of pharmacological interventions on olfactory memory. These injections can be delivered at different times depending on the design of the experiment.
To begin, make a small hole in the cuticular of the posterior part of the S scootin near the S scootle fissure above the flight muscle, using a disposable hypodermic needle. Next, fill a glass capillary tube with one microliter of the solution to be tested. Insert the capillary into the hole in the cuticular and inject the solution into the flight muscle.
Trained experimenters are able to inject one B every 30 seconds, allowing a precise timing of injection and conditioning. Two experiments are presented here. The first experiment examines the impact of a systemic injection of saline delivered before training on learning a memory.
There are three groups in this study, A group receiving no treatment, a one microliter systemic injection of phosphate buffered saline, or a sham group. These treatments were administered 30 minutes before training shown here on the left. The percentage of animals showing the conditioned response during acquisition increased across the three training trials with no differences existing between groups.
The bar graphs to the right show that neither the PBS injection nor the sham lesion affected memory retention. 24 hours after training in the second experiment, injections were given 40 minutes After training, bees were divided into one group receiving saline and another receiving one microliter of the protein synthesis inhibitor and ESO mycin the bar graphs to the right show a clear decline in memory retention in bees receiving Anisa mycin. These results highlight the importance of protein synthesis in the formation of olfactory memory.
Following this procedure, electrophysiological and electrophysiological recordings can be made in order to answer additional questions like what is the neurophysiological basis of learning and memory formation After its development? This technique paved the way for researchers in the field of learning and memory research to explore the molecular and neurophysiological mechanism in memory formation in the honeybee.
