Biomedical Engineering Reference
In-Depth Information
syringes and set a piece of fi lter paper (10 × 30 mm) inside each of them. Soak one
of the fi lter papers with 5
L of
1-nonanol (syringe 2). The syringe method underlines again the importance of using
two odors in any absolute-conditioning experiment. The mechanic stimulation of
the air puff could act as a confounding CS in conditioning trials. The test with the
novel odor is therefore of fundamental importance to verify that such mechanic
stimulation is not driving the bee's responses, in which case PER will be specifi c to
the odor learned.
μ
L of 2-hexanol (syringe 1) and the other with 5
μ
2.2.8
Sucrose Solution (Reward US)
For US sucrose solution (usually 50 %, weight/weight, i.e., 1.80 M), dissolve
sucrose in a vial of distilled water by 50 % weight/weight. The choice of sucrose
concentration is crucial for the experimental success. Indeed, diluted sucrose con-
centrations (below 20 %) are suboptimal for the appetitive motivation of the bees,
while highly concentrated solutions are also suboptimal due to their high viscosity,
which renders diffi cult ingestion through the proboscis (Farina and Núñez
1991
).
Thus, concentration in the range of 30-50 % should be used. Dispense 1 mL sucrose
solution into Eppendorf tubes and keep them in the freezer until needed. Defrost a
tube to room temperature before each experiment.
2.3
Protocol for Absolute Classical Conditioning
2.3.1
Catching and Harnessing Bees in Harness Tubes
1. Capture honeybee foragers outside the hives with a pyramid (see above, or a
sweep net if not available) in the morning of the day of the experiment or in the
afternoon of the day before the experiment.
2. Put one to fi ve honeybees in a screw vial immediately after capture, and cool the
vial in ice water for about 3 min to anesthetize the bees. Cooling time should be
kept to a minimum as extended cooling could impair learning performances
(e.g., Frost et al.
2011
). As soon as the bees are immobilized, place each bee in
a harness tube using a piece of tape behind the head. In this position, mouthparts
and antennae should be able to move freely. Take care of not leaving the forelegs
free to move and reach the head once the bee has been harnessed.
How many bees have to be used per experiment is a critical question as one ide-
ally wants large sample sizes that ensure statistical power. Low sample sizes (e.g.,
around ten individuals per group) have to be avoided; if possible, use 40-50 bees
per experiment (see below, Data Analysis).
