Parallel Olfactory Processing in the Honey Bee Brain: Odor Learning and Generalization under Selective Lesion of a Projection Neuron Tract

Abstract

The function of parallel neural processing is a fundamental problem in Neuroscience, as it is found across sensory modalities and evolutionary lineages, from insects to humans. Recently, parallel processing has attracted increased attention in the olfactory domain, with the demonstration in both insects and mammals that different populations of second-order neurons encode and/or process odorant information differently. Among insects, Hymenoptera present a striking olfactory system with a clear neural dichotomy from the periphery to higher-order centers, based on two main tracts of second-order (projection) neurons: the medial and lateral antennal lobe tracts (m-ALT and l-ALT). To unravel the functional role of these two pathways, we combined specific lesions of the m-ALT tract with behavioral experiments, using the classical conditioning of the proboscis extension response (PER conditioning). Lesioned and intact bees had to learn to associate an odorant (1-nonanol) with sucrose. Then the bees were subjected to a generalization procedure with a range of odorants differing in terms of their carbon chain length or functional group. We show that m-ALT lesion strongly affects acquisition of an odor-sucrose association. However, lesioned bees that still learned the association showed a normal gradient of decreasing generalization responses to increasingly dissimilar odorants. Generalization responses could be predicted to some extent by in vivo calcium imaging recordings of l-ALT neurons. The m-ALT pathway therefore seems necessary for normal classical olfactory conditioning performance.

Keywords: insect; olfaction; olfactory conditioning; parallel processing; projection neurons; specific lesion.

Figure 1

Dual olfactory pathway of the honey bee brain. (A) Schematic overview of the dual olfactory pathway of the honey bee brain (adapted from Carcaud et al., 2012, 2015). Odorant molecules are detected by olfactory receptor neurons (ORN) on the antenna which project to the antennal lobe (AL). Then, projection neurons (PN) convey information to the mushroom bodies (MB) and the lateral horn (LH) via two main tracts, the medial antennal lobe tract (m-ALT, magenta) and the lateral antennal lobe tract (l-ALT, green). Lesion site of the m-ALT and of the optic lobe (OL) are indicated (lesion 1 and lesion 2 respectively). (B) Mass staining in the AL, showing the course of l-ALT and m-ALT PNs from the AL to LH and the MB calyces. Abbreviations: m-ca, median calyx; l-ca, lateral calyx.

Figure 2

Antenna cover efficiency. (A) Photograph of a harnessed honey bee prepared for unilateral PER conditioning, with one antenna covered with latex.(B)Test of cover efficiency. Conditioning of the proboscis extension response (PER) in one group of bees with both antennae covered (n = 20) and in another group without any cover (n = 17). The percentage of PER increases in the course of training for the group without cover, whereas it remains almost null for the group with both antennae covered (^***p < 0.001, Mann-Whitney test).

Figure 3

Associative olfactory conditioning and generalization tests on intact honey bees. (A) Intact bees learn to associate the odorant CS (1-nonanol, C9-ol) with the sucrose US, as shown by the increase in the PER percentage along trials (n = 42; ^***p < 0.001, Cochran's Q test). (B) Generalization tests after PER conditioning, using 5 new odorants which differed from the CS by their chain length (C8-ol and C7-ol), by their functional group (C9-one) or both (C8-one, C7-one). Intact bees respond differentially to the tested odorants (^***p < 0.001, Cochran's Q test), depending on their chemical properties, and show no extinction. Abbreviations: C8-ol, 1-octanol; C7-ol, 1-heptanol; C9-one, 2-nonanone; C8-one, 2-octanone; C7-one, 2-heptanone.

Figure 4

PER conditioning and generalization tests after an optic lobe lesion. (A) Staining during the OL lesion using tetramethylrhodamine and counter-staining with Alexa488 coupled to phalloidin. Abbreviations: MB, mushroom body; me, medulla; lo, lobula; r, rostral; c, caudal; l, lateral; m, medial. The black and white arrowhead points to the lesion site. (B) PER conditioning in bees with an OL lesion. OL-lesioned bees manage to associate the odor CS (1-nonanol, C9-ol) with the sucrose reward (US), as shown by the increase in the PER percentage along trials (n = 30; ^***p < 0.001, Cochran's Q test). (C) Generalization tests after PER conditioning, using five new odorants differing from the CS by their chain length and/or by their functional group. Bees with an OL lesion respond differentially to the tested odorants (^***p < 0.001, Cochran's Q test). (D) Highly significant correlation between the responses of bees with an OL lesion and the responses of intact bees, during generalization tests (^***p < 0.001, R² = 0.97).

Figure 5

PER conditioning and generalization tests after an m-ALT lesion. (A) Staining with tetramethylrhodamine induced by the m-ALT lesion and Alexa488 coupled to phalloidin as counter-staining. The black and white arrowhead points to the lesion site. A stained m-ALT tract (left) or the stained somata clusters of m-ALT PNs (mSC, right) were used as indicators for a successful lesion. Abbreviations: r, rostral; c, caudal; l, lateral; m, medial. (B) PER conditioning in bees with an m-ALT lesion. Only a few m-ALT lesioned bees managed to associate the odor CS (1-nonanol, C9-ol) with the sucrose reward (US), as shown by the weak increase in PER percentage with trials (n = 59; ^***p < 0.001, Cochran's Q test). (C) Generalization tests after PER conditioning, using five new odorants, shows that bees with an m-ALT lesion respond differentially to the different odorants (^*p < 0.05, Cochran's Q test). (D) Significant correlation between the responses of bees with an m-ALT lesion and intact bees during generalization tests (^*p < 0.05, R² = 0.71).

Figure 6

PER conditioning performances in the three experimental groups. Acquisition performances are significantly lower in bees with an OL lesion than in intact bees, showing an effect of surgery (^***p < 0.001, Mann-Whitney test). PER conditioning performances are much weaker in bees with an m-ALT lesion than in bees with an OL lesion (^***p < 0.001, Mann-Whitney test), demonstrating a specific effect induced by the m-ALT PN lesion.

Figure 7

Comparison of neural coding in l-ALT neurons with behavioral performances. (A) Retrograde staining of l-ALT PNs innervating ventral AL glomeruli (adapted from Carcaud et al., 2015). Z-projection of optical slices performed at the indicated depths (d). Abbreviations: r, rostral; c, caudal; l, lateral; m, medial; lSC, l-ALT PN somata cluster; AN, antennal nerve. (B) Odor-induced calcium signals in l-ALT PNs in the AL to the six different odorants, differing in their functional group (alcohol and ketone) and their carbon chain length (7, 8, and 9 carbons) and to the air control. The CS in the behavioral experiment (1-nonanol; C9-ol) is framed in red. (C) Euclidian distances (dissimilarity measure between activity maps) calculated between the CS (C9-ol) and the five new tested odorants are significantly different (^***p < 0.001, repeated measure ANOVA). (D–F) Significant and similar correlations between l-ALT Euclidian distances and the responses in the generalization tests of: (D) intact bees (R² = 0.81, ^*p < 0.05); (E) bees with an OL lesion (R² = 0.71, ^*p < 0.05); and (F) bees with m-ALT lesion (R² = 0.61, (^*)p = 0.068).