Side-specificity of olfactory learning in the honeybee: generalization between odors and sides

Abstract

Honeybees (Apis mellifera) can be trained to associate an odor stimulus with a sucrose reward. The neural structures involved in the detection and integration of olfactory stimuli are represented bilaterally in the brain. Little is known about the respective roles of the two sides of the brain in olfactory learning. Does each side learn independently of the other, or do they communicate, and if so, to what extent and at what level of neural integration? We addressed these questions using the proboscis extension response (PER) conditioning paradigm applied in a preparation that allows the separation of the two input sides during olfactory stimulations. Bees conditioned to two odorants A and B, one being learned on each side (A+/B+ training), showed in extinction tests rather unspecific responses: They responded to both odorants on both sides. This could be attributable to either a transfer of the learned information between sides, or to a generalization between odorants on each side. By subjecting bees to conditioning on one side only (A+/0 training), we found that the learned information is indeed transferred between sides. However, when bees were trained explicitly to give opposite values to the two odorants on the two sides (A+B-/B+A- training), they showed clear side-specific response patterns to these odorants. These results are used in the elaboration of a functional model of laterality of olfactory learning and memory processing in the honeybee brain.

Figure 1

(A) Proboscis extension responses by honeybees during conditioning to two different odors on the two sides (A+/B+ training). Responses developed identically on both sides, eventually reaching ∼75% responses (NS: nonsignificant, log-linear analysis). (B) Bees' performances during the test phase after A+/B+ training. Bees show an unspecific response pattern, responding with a high rate (> 70%) to both odors on both sides. Different letters indicate significantly different levels of responses (pairwise comparisons after Cochran's Q test).

Figure 2

Bees' performances (A) in a unilateral olfactory conditioning procedure (A+/0 training); (B) in the test phase after 3 h retention. Bees responded most strongly to odor A on the trained side and the least to odor B (novel odor) on the untrained side. Note that bees responded more to odor A on the untrained side than to odor B on the trained side. This shows that both transfer of information between sides and generalization between odors on one given side take place. However, transfer appears to have a higher impact than generalization, because responses to A on the contralateral side were higher than responses to B on the conditioning side. Different letters indicate significantly different levels of responses (pairwise comparisons after Cochran's Q test).

Figure 3

Bees' performances (A) in a unilateral differential conditioning procedure (A+B−/0 training); (B) in the test phase after 3 h retention. As before, honeybees transferred information about odor A to the opposite side. Responses to odor B were low on both sides. Comparing these results to those of Figure 2 (A+/0 training, see dotted line), responses to B appear to be inhibited on both sides, suggesting a transfer of inhibitory information between sides. Different letters indicate significantly different levels of responses (pairwise comparisons after Cochran's Q test).

Figure 4

Bees' performances (A) in a bilateral differential conditioning procedure (A+/B− training). In this case, conditioning performances were significantly higher on the left than on the right side, so acquisition curves are shown for each side (broken lines); (B) in the test phase after 3 h retention. As before, honeybees transfer information about odor A to the opposite side. Responses to odor B are on both sides lower than in Figure 2 (A+/0 training, see dotted line) although bees responded more on Side 1 (A+ side) than on Side 2 (B− side). Different letters indicate significantly different levels of responses (pairwise comparisons after Cochran's Q test).

Figure 5

Performances of bees in an inverted differential conditioning procedure (A+B−/B+A− training). (A) During the conditioning phase, responses to each odor increase on the side where it is rewarded, and decrease on the side where it is explicitly unrewarded (***: P< 0.001, log-linear analysis comparing responses to CS+ and CS−). (B) During the test phase, bees show a clear side-specific pattern of responses. Different letters indicate significantly different levels of responses (pairwise comparisons after Cochran's Q test).

Figure 6

Separation of olfactory input sides using thin plastic walls waxed onto the head. (A) Honeybee placed in a holder with a wall between the two antennae. The wall is placed slightly to one side to allow the proboscis to move freely. (B) Odor stimulations are provided on one side of the wall. An exhaust vent placed behind the bee ensures a laminar flow along the wall.