Jean-Christophe Sandoz

Perceptual and Neural Olfactory Similarity in Honeybees

The question of whether or not neural activity patterns recorded in the olfactory centres of the brain correspond to olfactory perceptual measures remains unanswered. To address this question, we studied olfaction in honeybees Apis mellifera using the olfactory conditioning of the proboscis extension response. We conditioned bees to odours and tested generalisation responses to different odours. Sixteen odours were used, which varied both in their functional group (primary and secondary alcohols, aldehydes and ketones) and in their carbon-chain length (from six to nine carbons).The results obtained by presentation of a total of 16 × 16 odour pairs show that (i) all odorants presented could be learned, although acquisition was lower for short-chain ketones; (ii) generalisation varied depending both on the functional group and the carbon-chain length of odours trained; higher generalisation was found between long-chain than between short-chain molecules and between groups such as primary and secondary alcohols; (iii) for some odour pairs, cross-generalisation between odorants was asymmetric; (iv) a putative olfactory space could be defined for the honeybee with functional group and carbon-chain length as inner dimensions; (v) perceptual distances in such a space correlate well with physiological distances determined from optophysiological recordings of antennal lobe activity. We conclude that functional group and carbon-chain length are inner dimensions of the honeybee olfactory space and that neural activity in the antennal lobe reflects the perceptual quality of odours.

Aversive Learning in Honeybees Revealed by the Olfactory Conditioning of the Sting Extension Reflex

Invertebrates have contributed greatly to our understanding of associative learning because they allow learning protocols to be combined with experimental access to the nervous system. The honeybee Apis mellifera constitutes a standard model for the study of appetitive learning and memory since it was shown, almost a century ago, that bees learn to associate different sensory cues with a reward of sugar solution. However, up to now, no study has explored aversive learning in bees in such a way that simultaneous access to its neural bases is granted. Using odorants paired with electric shocks, we conditioned the sting extension reflex, which is exhibited by harnessed bees when subjected to a noxious stimulation. We show that this response can be conditioned so that bees learn to extend their sting in response to the odorant previously punished. Bees also learn to extend the proboscis to one odorant paired with sugar solution and the sting to a different odorant paired with electric shock, thus showing that they can master both appetitive and aversive associations simultaneously. Responding to the appropriate odorant with the appropriate response is possible because two different biogenic amines, octopamine and dopamine subserve appetitive and aversive reinforcement, respectively. While octopamine has been previously shown to substitute for appetitive reinforcement, we demonstrate that blocking of dopaminergic, but not octopaminergic, receptors suppresses aversive learning. Therefore, aversive learning in honeybees can now be accessed both at the behavioral and neural levels, thus opening new research avenues for understanding basic mechanisms of learning and memory.

Invertebrate learning and memory: Fifty years of olfactory conditioning of the proboscis extension response in honeybees

The honeybee Apis mellifera has emerged as a robust and influential model for the study of classical conditioning, thanks to the existence of a powerful Pavlovian conditioning protocol, the olfactory conditioning of the proboscis extension response (PER). In 2011, the olfactory PER conditioning protocol celebrates 50 years since it was first introduced by Kimihisa Takeda in 1961. Here, we review its origins, developments, and perspectives in order to define future research avenues and necessary methodological and conceptual evolutions. We show that olfactory PER conditioning has become a versatile tool for the study of questions in extremely diverse fields in addition to the study of learning and memory and that it has allowed behavioral characterizations, not only of honeybees, but also of other insect species, for which the protocol was adapted. We celebrate, therefore, Takedas original work and prompt colleagues to conceive and establish further robust behavioral tools for an accurate characterization of insect learning and memory at multiple levels of analysis.

Neural representation of olfactory mixtures in the honeybee antennal lobe

Natural olfactory stimuli occur as mixtures of many single odors. We studied whether the representation of a mixture in the brain retains single‐odor information and how much mixture‐specific information it includes. To understand mixture representation in the honeybee brain, we used in vivo calcium imaging at the level of the antennal lobe, and systematically measured odor‐evoked activity in 24 identified glomeruli in response to four single odorants and all their possible binary, ternary and quaternary mixtures. Qualitatively, mixture‐induced activity patterns always contained glomeruli belonging to the pattern of at least one of the components, suggesting a high conservation of component information in olfactory mixtures. Quantitatively, glomerular activity saturated quickly and increasing the number of components resulted in an increase of cases in which the response of a glomerulus to the mixture was lower than that to the strongest component (‘suppression’). This shows global inhibition in the antennal lobe, probably acting as overall gain control. Single components were not equally salient (in terms of number of active glomeruli) and mixture activity patterns were always more similar to the more salient components, in a way that could be predicted linearly. Thus, although a gain control system in the honeybee antennal lobe prevents saturation of the olfactory system, mixture representation follows essentially elemental rules.

Long-Term Memory Leads to Synaptic Reorganization in the Mushroom Bodies: A Memory Trace in the Insect Brain?

The insect mushroom bodies (MBs) are paired brain centers which, like the mammalian hippocampus, have a prominent function in learning and memory. Despite convergent evidence for their crucial role in the formation and storage of associative memories, little is known about the mechanisms underlying such storage. In mammals and other species, the consolidation of stable memories is accompanied by structural plasticity involving variations in synapse number and/or size. Here, we address the question of whether the formation of olfactory long-term memory (LTM) could be associated with changes in the synaptic architecture of the MB networks. For this, we took advantage of the modular architecture of the honeybee MB neuropil, where synaptic contacts between olfactory input and MB neurons are segregated into discrete units (microglomeruli) which can be easily visualized and counted. We show that the density in microglomeruli increases as a specific olfactory LTM is formed, while the volume of the neuropil remains constant. Such variation is reproducible and is clearly correlated with memory consolidation, as it requires gene transcription. Thus stable structural synaptic rearrangements, including the growth of new synapses, seem to be a common property of insect and mammalian brain networks involved in the storage of stable memory traces.

Revisiting olfactory classical conditioning of the proboscis extension response in honey bees: A step toward standardized procedures

The honey bee Apis mellifera has emerged as a robust and influential model for the study of classical conditioning thanks to the existence of a powerful Pavlovian conditioning protocol, the olfactory conditioning of the proboscis extension response (PER). In 2011, the olfactory PER conditioning protocol celebrated its 50 years since it was first introduced by Kimihisa Takeda in 1961. In this protocol, individually harnessed honey bees are trained to associate an odor with sucrose solution. The resulting olfactory learning is fast and induces robust olfactory memories that have been characterized at the behavioral, neuronal and molecular levels. Despite the success of this protocol for studying the bases of learning and memory at these different levels, innumerable procedural variants have arisen throughout the years, which render comparative analyses of behavioral performances difficult. Moreover, because even slight variations in conditioning procedures may introduce significant differences in acquisition and retention performances, we revisit olfactory PER conditioning and define here a standardized framework for experiments using this behavioral protocol. To this end, we present and discuss all the methodological steps and details necessary for successful implementation of olfactory PER conditioning.

Antennal Lobe Processing Increases Separability of Odor Mixture Representations in the Honeybee

Local networks within the primary olfactory centers reformat odor representations from olfactory receptor neurons to second-order neurons. By studying the rules underlying mixture representation at the input to the antennal lobe (AL), the primary olfactory center of the insect brain, we recently found that mixture representation follows a strict elemental rule in honeybees: the more a component activates the AL when presented alone, the more it is represented in a mixture. We now studied mixture representation at the output of the AL by imaging a population of second-order neurons, which convey AL processed odor information to higher brain centers. We systematically measured odor-evoked activity in 22 identified glomeruli in response to four single odorants and all their possible binary, ternary and quaternary mixtures. By comparing input and output responses, we determined how the AL network reformats mixture representation and what advantage this confers for odor discrimination. We show that increased inhibition within the AL leads to more synthetic, less elemental, mixture representation at the output level than that at the input level. As a result, mixture representations become more separable in the olfactory space, thus allowing better differentiation among floral blends in nature.

SIDE-SPECIFIC OLFACTORY CONDITIONING LEADS TO MORE SPECIFIC ODOR REPRESENTATION BETWEEN SIDES BUT NOT WITHIN SIDES IN THE HONEYBEE ANTENNAL LOBES

Honeybees can be trained to associate odorants to sucrose reward by conditioning the proboscis extension response. Using this paradigm, we have recently shown that bees can solve a side-specific task: they learn simultaneously to discriminate a reinforced odor A from a non-reinforced odor B at one antenna (A+B-) and the reversed problem at the other antenna (A-B+). Side-specific (A+B-/B+A-) conditioning is an interesting tool to measure neurophysiological changes due to olfactory learning because the same odorant is excitatory (CS+) on one brain side and inhibitory (CS-) on the opposite side. In the bee brain, the antennal lobe (AL) is the first olfactory relay where the olfactory memory is established. Using calcium imaging, we compared odor-evoked activity in the functional units, the glomeruli, of the two ALs, both in naive and conditioned individuals. Each odor evoked a different pattern of glomerular activity, which was symmetrical between sides and highly conserved among naive animals. In conditioned bees, response patterns were overall symmetrical but showed more active glomeruli and topical differences between sides. By representing odor vectors in a virtual olfactory space whose dimensions are the responses of 23 identified glomeruli, we found that distances between odor representations on each brain side were significantly higher in conditioned than in naive bees, but only for CS+ and CS-. However, the distance between CS+ and CS- representations was equal to that of naive individuals. Our work suggests that side-specific conditioning decorrelates odor representations between AL sides but not between CS+ and CS- within one AL.

Individual olfactory learning in Camponotus ants

We studied olfactory learning in two ant species, Camponotus mus from Argentina and Camponotus fellah from Israel. To this end, we established an experimental laboratory protocol in which individual ants were trained to associate odours with gustatory reinforcers. Ants were trained individually to forage in a Y-maze in which two odours had to be discriminated. One odour was positively reinforced with sucrose solution and the other was negatively reinforced with quinine solution. After a training session of 24 trials, ants of both species learned to differentiate the two odour pairs, the structurally dissimilar limonene and octanal, and the structurally similar heptanal and 2-heptanone. In nonreinforced tests, ants consistently chose the odour previously reinforced with sucrose solution and spent more time searching in the arm of the maze presenting this odour. Learning performances were more robust in the case of limonene versus heptanal. These results thus show for the first time that individual ants perceive and learn odours in controlled laboratory conditions.

Olfactory conditioning of the proboscis extension in bumble bees

The foraging behaviour of bumble bees is well documented for nectar and/or pollen gathering, but little is known about the learning processes underlying such behaviour. We report olfactory conditioning in worker bumble bees Bombus terrestris L. (Hymenoptera: Apidae) obtained under laboratory conditions on restrained individuals. The protocol was adapted from the proboscis extension conditioning previously described in the honey bee Apis mellifera L. Bumble bees were found to be able to learn a pure odorant when it was presented in paired association with a sugar reward, but not when odour and reward were presented in an explicitly unpaired procedure. This suggests an associative basis for this olfactory learning. Bumble bees showed similar conditioning abilities when stimulated with two different floral odours. An effect of the sugar reward concentration on the learning performances was found.