Morphofunctional experience-dependent plasticity in the honeybee brain

Abstract

Repeated or prolonged exposure to an odorant without any positive or negative reinforcement produces experience-dependent plasticity, which results in habituation and latent inhibition. In the honeybee (Apis mellifera), it has been demonstrated that, even if the absolute neural representation of an odor in the primary olfactory center, the antennal lobe (AL), is not changed by repeated presentations, its relative representation with respect to unfamiliar stimuli is modified. In particular, the representation of a stimulus composed of a 50:50 mixture of a familiar and a novel odorant becomes more similar to that of the novel stimulus after repeated stimulus preexposure. In a calcium-imaging study, we found that the same functional effect develops following prolonged odor exposure. By analyzing the brains of the animals subjected to this procedure, we found that such functional changes are accompanied by morphological changes in the AL (i.e., a decrease in volume in specific glomeruli). The AL glomeruli that exhibited structural plasticity also modified their functional responses to the three stimuli (familiar odor, novel odor, binary mixture). We suggest a model in which rebalancing inhibition within the AL glomeruli may be sufficient to elicit structural and functional correlates of experience-dependent plasticity.

Figure 1.

Fluorescence response maps of single glomeruli that were identified in at least one subject per group, averaged over subjects. Shown are background subtracted and normalized signals as a function of time from stimulus onset (−ΔF/F). The colormap reaches from inhibitive −0.1 (blue) to activated +0.2 (red). Figures within rows are responses to different odors within the same preexposed group. Columns show responses to the same odors in differently preexposed groups. The right y-axis shows in blue the number of subjects in which the glomerulus was identified and which therefore contributed to the averaging.

Figure 2.

Activation dynamics during the presentation of three odor stimuli in the principal component coordinate system (1-sec stimulus and 1-sec post-stimulus) for the three groups: (A) control (CTR) bees, (B) hexanol (HEX) preexposed bees, and (C) nonanol (NNL) preexposed bees. Activation vectors represent all measured glomeruli in a given treatment group. HEX activation vector is shown in blue, NNL in cyan, and their binary mixture (MIX) in light blue. The origin, marked by a yellow circle, corresponds to baseline activation. Arrows show the temporal axis; numbers indicate time after stimulus onset in seconds. Dashed gray lines connect the 300 msec time-points, which are the points of maximal odor separation. A dotted and dashed orange line connects the origin with the binary mixture 300 msec time-point.

Figure 3.

(A) Euclidean distances between representations of MIX and pure compounds within each group, normalized to the ED between the two pure compounds. Error bars represent standard deviations obtained via bootstrap resampling with replacement of glomeruli (N = 1000). The difference was significant in the NNL preexposed group (z-test, (*) P = 0.019). (B) Ratios of normalized EDs [(ED_{HEX_MIX} − ED_{NON_MIX})/(ED_{HEX_MIX} + ED_{NON_MIX})] within each group. Error bars represent standard deviations obtained by bootstrapping. The shift between the HEX and the NNL preexposed groups was significant (z-test, (*) P = 0.041).

Figure 4.

The average volumes of the six glomeruli in the three treatment groups (CTR bees, HEX preexposed, and NNL preexposed bees). An example of segmented and 3D-reconstructed glomeruli T1-17, 33, 42, 28, 38, 47 from an immunolabeled left AL image stack is shown in the inset. Volume data were obtained by left–right averaging (see Materials and Methods). Error bars represent standard errors of the mean (n_CTR = 13, n_HEX = 15, n_NNL = 15). Groups with significantly different means are indicated ((*) P < 0.05, (**) P < 0.01).

Figure 5.

Odor-evoked activity of glomeruli T1-17 (A) and T1-33 (B) in the three treatment groups, averaged over single bees. Error bars represent standard errors of the mean (n_CTR = 5, n_HEX = 5, n_NNL = 4). The left histograms show the responses to 1-hexanol of all three treatment groups; the middle histograms, to 1-nonanol; and the right histograms, to the 50:50 mixture. The treatment effect was found to be significant via one-way ANOVA, and group means were confronted via two-sample t-tests ((*) P < 0.05, (**) P < 0.01).