Pheromone components affect motivation and induce persistent modulation of associative learning and memory in honey bees

Abstract

Since their discovery in insects, pheromones are considered as ubiquitous and stereotyped chemical messengers acting in intraspecific animal communication. Here we studied the effect of pheromones in a different context as we investigated their capacity to induce persistent modulations of associative learning and memory. We used honey bees, Apis mellifera, and combined olfactory conditioning and pheromone preexposure with disruption of neural activity and two-photon imaging of olfactory brain circuits, to characterize the effect of pheromones on olfactory learning and memory. Geraniol, an attractive pheromone component, and 2-heptanone, an aversive pheromone, improved and impaired, respectively, olfactory learning and memory via a durable modulation of appetitive motivation, which left odor processing unaffected. Consistently, interfering with aminergic circuits mediating appetitive motivation rescued or diminished the cognitive effects induced by pheromone components. We thus show that these chemical messengers act as important modulators of motivational processes and influence thereby animal cognition.

Fig. 1. Pheromone components modulate associative olfactory learning and memory retention in honey bees.

a–c Pheromone components modulate associative olfactory learning and memory according to their valence. a Experimental protocol used. b Associative olfactory conditioning of PER in honey bees exposed to geraniol (GER), 2-heptanone (2H), or mineral oil 15 min before training. Proportion of bees showing a conditioned response (PER) to the rewarded (solid lines) and unrewarded (dotted lines) odorants during successive conditioning trials. GER-preexposed bees (n = 75 independent bees) performed better than control bees exposed to mineral oil (n = 129 independent bees), while 2H preexposed bees (n = 73 independent bees) performed worse than controls. (*) p < 0.05; (**) p < 0.001. c Retention tests performed 2, 24, or 72 h post conditioning. Preexposure to GER (blue), 2H (orange), or mineral oil (gray) was performed prior to conditioning. The figure shows the proportions of bees showing a specific memory (i.e., responding to the CS+ and not to the CS−) in the retention tests. The sample size is reported above each bar and refers to independent bees. Retention was better in bees exposed to GER compared with control bees. The opposite was observed in bees exposed to 2H. (*) p < 0.05; (**) p < 0.01; (***) p < 0.001.

Fig. 2. Effect of pheromone components on neural odor processing.

a–f Pheromone components do not affect olfactory coding and odorant similarity in the antennal lobe. a Experimental protocol used. b Two-photon microscopy image of the left antennal lobe (AL) stained with a fluorescent calcium-sensitive dye injected in the projection neurons (PN) of the medial and the lateral antennal protocerebral tracts. Activity in PN dendrites in AL glomeruli can be visualized in this way. c Signal intensity was recorded along a line crossing ten glomeruli identified using the antennal lobe atlas of the honey bee (AN antennal nerve, v ventral, l lateral, m medial, d dorsal. Numbers refer to identified glomeruli) and averaged between the identified borders of each glomerulus (white circles). Scale bars = 200 µm. d Signal intensity of each identified glomerulus over time after background subtraction and normalization (−ΔF/F). Red lines represent the onset and offset of limonene (Lim, above) and eugenol (Eug, below) presentation to the bee. e Change in normalized fluorescence during the first 600 ms of odor stimulation with Lim and Eug 15 min before (“−15 min”) mineral oil (“Oil”; n = 8 independent bees) or pheromone- component (GER, 2H; n = 8 independent bees for both components) exposure, as well as 15 min (“+15 min”) and 2 h (“+2 h”) after exposure. No significant variation in activity was found for each odorant over time for both pheromonal treatments. f Euclidian distance—a measure of odor distinguishability—in the odor-coding space defined by the activity recorded for the ten identified glomeruli between the odor representations of Lim and Eug 15 min before, as well as 15 min and 2 h after the exposure to Oil, GER, and 2H. The circles constitute individual data, and the horizontal bars represent the medians of each distribution. Odor discrimination did not change over time after exposure to pheromone components.

Fig. 3. Pheromone components modulate sucrose responsiveness according to their valence.

Bees were preexposed to geraniol (GER, n = 112 independent bees), 2-heptanone (2H, n = 156 independent bees), or mineral oil (n = 172 independent bees). They were then stimulated with a series of six increasing concentrations of sucrose solution (0.1, 0.3, 1, 3, 10, and 30, w/w). For each bee that responded at least to the highest sucrose concentration (30%), we calculated an individual sucrose-responsiveness score (SRS) as the number of sucrose concentrations to which a bee responded. The figure shows the median, quartiles, and max and min (upper and lower whiskers) SRS values of bees preexposed to GER, 2H, or oil, and retained in the analyses. Individual bees are indicated by the dots. Preexposure to GER and to 2H induced a significant increase and decrease of SRS, respectively, with respect to bees exposed to mineral oil. (*) p = 0.0007; (**) p < 0.0001.

Fig. 4. Effect of pheromone components on aminergic signaling in the bee brain.

a–e Pharmacological treatment with agonists/antagonists of the dopaminergic and octopaminergic system of honey bees exposed to either GER or 2H counteracted totally or partially the effects of pheromone components on learning and memory. a Experimental protocol used. b Proportion of conditioned responses (PER) to the rewarded (solid lines) and nonrewarded odors (dotted lines) during five CS+ and CS− trials, and proportion of bees with specific memory (i.e., the proportion of bees responding to the CS+ and not to the CS−) in retention tests performed 2, 24, or 72 h after conditioning (bar diagram) in the case of bees injected either with PBS (n = 43 independent bees, controls) or epinastine (OA-receptor antagonist) [0.4 µM (n = 42 independent bees), 4 mM (n = 45 independent bees)] and preexposed to geraniol (GER). (*) p < 0.05; (**) p ≤ 0.001. c Same as in b but for bees injected either with PBS (n = 45 independent bees) or flupentixol (DA-receptor antagonist) [0.2 µM (n = 40 independent bees), 2 mM (n = 43 independent bees)] and exposed to geraniol (GER). Both the OA and the DA-receptor antagonist counteracted the enhancing effect of GER on learning and memory formation. (*) p < 0.05. d Same as in b but for bees injected either with PBS (n = 53 independent bees) or with octopamine [20 µM (n = 53 independent bees) and 2 mM (n = 49 independent bees)] and preexposed to 2-heptanone (2H). OA treatment counteracted the decrement of learning and memory induced by 2H. (*) p < 0.05; (**) p ≤ 0.001. e Same as in b but for bees injected either with PBS (n = 55 independent bees) or with flupentixol (DA-receptor antagonist) [0.2 µM (n = 61 independent bees) and 2 mM (n = 62 independent bees)] and exposed to 2-heptanone (2H). The DA-receptor antagonist had no effect on learning but rescued memory retention. (**) p ≤ 0.001.