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Trace conditioning is a form of Pavlovian learning in which the conditioned stimulus (CS) and the unconditioned stimulus (US) are separated by a temporal gap. Insects learn trace associations of variable nature (appetitive, aversive) and involving CSs of different sensory modalities (olfactory, visual). The accessibility of the insect neural system in behaving animals allowed identifying neural processes driving trace conditioning: the existence of prolonged neural responses to the CS after stimulus offset and the anticipation of US responses during the free-stimulus gap. Specific brain structures, such as the mushroom bodies seem to be allocated to this learning form. Here, we posit that a further component facilitating trace conditioning in insects relates to neuromodulatory mechanisms underlying enhanced attention. We thus propose a model based on different types of mushroom-body neurons, which provides a cognitive account of trace conditioning in insects., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Interview with Martin Giurfa, who studies learning and memory in insects at Paul Sabatier University., (Copyright © 2022.)

Honey bees exhibit remarkable visual learning capacities, which can be studied using virtual reality (VR) landscapes in laboratory conditions. Existing VR environments for bees are imperfect as they provide either open-loop conditions or 2D displays. Here we achieved a true 3D environment in which walking bees learned to discriminate a rewarded from a punished virtual stimulus based on color differences. We included ventral or frontal background cues, which were also subjected to 3D updating based on the bee movements. We thus studied if and how the presence of such motion cues affected visual discrimination in our VR landscape. Our results showed that the presence of frontal, and to a lesser extent, of ventral background motion cues impaired the bees' performance. Whenever these cues were suppressed, color discrimination learning became possible. We analyzed the specific contribution of foreground and background cues and discussed the role of attentional interference and differences in stimulus salience in the VR environment to account for these results. Overall, we show how background and target cues may interact at the perceptual level and influence associative learning in bees. In addition, we identify issues that may affect decision-making in VR landscapes, which require specific control by experimenters., (© 2021. The Author(s).)

The capacity to process numbers can be found in many vertebrate species, which share similar behavioral and neural mechanisms for number estimation. Honeybees possess a miniature brain but exhibit remarkable cognitive abilities, including the capacity to perform numerosity judgments in a foraging context. Honeybee foragers can count up to four landmarks en route to the goal and use this information to decide where to land. They also learn to match visual images based on item number and may use absolute or relative numerosity. They can be trained to choose item arrays based on rules such as 'smaller than', in which case their performance reveals the existence of zero as a conceptual low end of a succession of positive numbers. Moreover, in symbolic-matching protocols, they learn to master visual discriminations based on subtracting or adding one item to a perceived sample, depending on the color of the sample array. Here I review the works on numeric processing in bees and discuss them from a cognitive and ecological perspective. I conclude that these abilities exhibit distinctive features of the vertebrates' numeric systems, and argue in favor of an evolutionary convergence of numeric abilities in many animal species, including vertebrates, and invertebrates.

Taste perception allows discriminating edible from non-edible items and is crucial for survival. In the honey bee, the gustatory sense has remained largely unexplored, as tastants have been traditionally used as reinforcements rather than as stimuli to be learned and discriminated. Here we provide the first characterization of antennal gustatory perception in this insect using a novel conditioning protocol in which tastants are dissociated from their traditional food-reinforcement role to be learned as predictors of punishment. We found that bees have a limited gustatory repertoire via their antennae: they discriminate between broad gustatory modalities but not within modalities, and are unable to differentiate bitter substances from water. Coupling gustatory conditioning with blockade of aminergic pathways in the bee brain revealed that these pathways are not restricted to encode reinforcements but may also encode conditioned stimuli. Our results reveal unknown aspects of honey bee gustation, and bring new elements for comparative analyses of gustatory perception in animals.

Iridescence-change of colour with changes in the angle of view or of illumination-is widespread in the living world, but its functions remain poorly understood. The presence of iridescence has been suggested in flowers where diffraction gratings generate iridescent colours. Such colours have been suggested to serve plant-pollinator communication. Here we tested whether a higher iridescence relative to corolla pigmentation would facilitate discrimination, learning and retention of iridescent visual targets. We conditioned bumblebees ( Bombus terrestris ) to discriminate iridescent from non-iridescent artificial flowers and we varied iridescence detectability by varying target iridescent relative to pigment optical effect. We show that bees rewarded on targets with higher iridescent relative to pigment effect required fewer choices to complete learning, showed faster generalization to novel targets exhibiting the same iridescence-to-pigment level and had better long-term memory retention. Along with optical measurements, behavioural results thus demonstrate that bees can learn iridescence-related cues as bona fide signals for flower reward. They also suggest that floral advertising may be shaped by competition between iridescence and corolla pigmentation, a fact that has important evolutionary implications for pollinators. Optical measurements narrow down the type of cues that bees may have used for learning. Beyond pollinator-plant communication, our experiments help understanding how receivers influence the evolution of iridescence signals generated by gratings., (© 2017 The Author(s).)

Free-flying honeybees exhibit remarkable cognitive capacities but the neural underpinnings of these capacities cannot be studied in flying insects. Conversely, immobilized bees are accessible to neurobiological investigation but display poor visual learning. To overcome this limitation, we aimed at establishing a controlled visual environment in which tethered bees walking on a spherical treadmill learn to discriminate visual stimuli video projected in front of them. Freely flying bees trained to walk into a miniature Y-maze displaying these stimuli in a dark environment learned the visual discrimination efficiently when one of them (CS+) was paired with sucrose and the other with quinine solution (CS-). Adapting this discrimination to the treadmill paradigm with a tethered, walking bee was successful as bees exhibited robust discrimination and preferred the CS+ to the CS- after training. As learning was better in the maze, movement freedom, active vision and behavioral context might be important for visual learning. The nature of the punishment associated with the CS- also affects learning as quinine and distilled water enhanced the proportion of learners. Thus, visual learning is amenable to a controlled environment in which tethered bees learn visual stimuli, a result that is important for future neurobiological studies in virtual reality.

Pheromones are chemical messengers that trigger stereotyped behaviors and/or physiological processes in individuals of the same species. Recent reports suggest that pheromones can modulate behaviors not directly related to the pheromonal message itself and contribute, in this way, to behavioral plasticity. We tested this hypothesis by studying the effect of pheromones on sucrose responsiveness and habituation in honey bees. We exposed workers to three pheromone components: geraniol, which in nature is used in an appetitive context, and isopentyl acetate (IPA) and 2-heptanone (2H), which signal aversive situations. Pheromones associated with an aversive context induced a significant decrease of sucrose responsiveness as 40% and 60% of bees exposed to IPA and 2H, respectively, did not respond to any sucrose concentration. In bees that responded to sucrose, geraniol enhanced sucrose responsiveness while 2H, but not IPA, had the opposite effect. Geraniol and IPA had no effect on habituation while 2H induced faster habituation than controls. Overall, our results demonstrate that pheromones modulate reward responsiveness and to a lower degree habituation. Through their effect on sucrose responsiveness they could also affect appetitive associative learning. Thus, besides conveying stereotyped messages, pheromones may contribute to individual and colony-level plasticity by modulating motivational state and learning performances.

The honeybee olfactory system is a well-established model for understanding functional mechanisms of learning and memory. Olfactory stimuli are first processed in the antennal lobe, and then transferred to the mushroom body and lateral horn through dual pathways termed medial and lateral antennal lobe tracts (m-ALT and l-ALT). Recent studies reported that honeybees can perform elemental learning by associating an odour with a reward signal even after lesions in m-ALT or blocking the mushroom bodies. To test the hypothesis that the lateral pathway (l-ALT) is sufficient for elemental learning, we modelled local computation within glomeruli in antennal lobes with axons of projection neurons connecting to a decision neuron (LHN) in the lateral horn. We show that inhibitory spike-timing dependent plasticity (modelling non-associative plasticity by exposure to different stimuli) in the synapses from local neurons to projection neurons decorrelates the projection neurons' outputs. The strength of the decorrelations is regulated by global inhibitory feedback within antennal lobes to the projection neurons. By additionally modelling octopaminergic modification of synaptic plasticity among local neurons in the antennal lobes and projection neurons to LHN connections, the model can discriminate and generalize olfactory stimuli. Although positive patterning can be accounted for by the l-ALT model, negative patterning requires further processing and mushroom body circuits. Thus, our model explains several-but not all-types of associative olfactory learning and generalization by a few neural layers of odour processing in the l-ALT. As an outcome of the combination between non-associative and associative learning, the modelling approach allows us to link changes in structural organization of honeybees' antennal lobes with their behavioural performances over the course of their life.

Learning theories distinguish elemental from configural learning based on their different complexity. Although the former relies on simple and unambiguous links between the learned events, the latter deals with ambiguous discriminations in which conjunctive representations of events are learned as being different from their elements. In mammals, configural learning is mediated by brain areas that are either dispensable or partially involved in elemental learning. We studied whether the insect brain follows the same principles and addressed this question in the honey bee, the only insect in which configural learning has been demonstrated. We used a combination of conditioning protocols, disruption of neural activity, and optophysiological recording of olfactory circuits in the bee brain to determine whether mushroom bodies (MBs), brain structures that are essential for memory storage and retrieval, are equally necessary for configural and elemental olfactory learning. We show that bees with anesthetized MBs distinguish odors and learn elemental olfactory discriminations but not configural ones, such as positive and negative patterning. Inhibition of GABAergic signaling in the MB calyces, but not in the lobes, impairs patterning discrimination, thus suggesting a requirement of GABAergic feedback neurons from the lobes to the calyces for nonelemental learning. These results uncover a previously unidentified role for MBs besides memory storage and retrieval: namely, their implication in the acquisition of ambiguous discrimination problems. Thus, in insects as in mammals, specific brain regions are recruited when the ambiguity of learning tasks increases, a fact that reveals similarities in the neural processes underlying the elucidation of ambiguous tasks across species.

Insects possess small brains but exhibit sophisticated behavioral performances. Recent works have reported the existence of unsuspected cognitive capabilities in various insect species, which go beyond the traditional studied framework of simple associative learning. In this study, I focus on capabilities such as attention, social learning, individual recognition, concept learning, and metacognition, and discuss their presence and mechanistic bases in insects. I analyze whether these behaviors can be explained on the basis of elemental associative learning or, on the contrary, require higher-order explanations. In doing this, I highlight experimental challenges and suggest future directions for investigating the neurobiology of higher-order learning in insects, with the goal of uncovering l architectures underlying cognitive processing., (© 2015 John Wiley & Sons, Ltd.)

Honeybees constitute established model organisms for the study of appetitive learning and memory. In recent years, the establishment of the technique of olfactory conditioning of the sting extension response (SER) has yielded new insights into the rules and mechanisms of aversive learning in insects. In olfactory SER conditioning, a harnessed bee learns to associate an olfactory stimulus as the conditioned stimulus with the noxious stimulation of an electric shock as the unconditioned stimulus. Here, we review the multiple aspects of honeybee aversive learning that have been uncovered using Pavlovian conditioning of the SER. From its behavioral principles and sensory variants to its cellular bases and implications for understanding social organization, we present the latest advancements in the study of punishment learning in bees and discuss its perspectives in order to define future research avenues and necessary improvements. The studies presented here underline the importance of studying honeybee learning not only from an appetitive but also from an aversive perspective, in order to uncover behavioral and cellular mechanisms of individual and social plasticity.

The antennal lobe (AL) is the first olfactory center of the insect brain and is constituted of different functional units, the glomeruli. In the AL, odors are coded as spatiotemporal patterns of glomerular activity. In honeybees, olfactory learning during early adulthood modifies neural activity in the AL on a long-term scale and also enhances later memory retention. By means of behavioral experiments, we first verified that olfactory learning between the fifth and eighth day of adulthood induces better retention performances at a late adult stage than the same experience acquired before or after this period. We checked that the specificity of memory for the odorants used was improved. We then studied whether such early olfactory learning also induces long-term structural changes in the AL consistent with the formation of long-term olfactory memories. We also measured the volume of 15 identified glomeruli in the ALs of 17-day-old honeybees that either experienced an odor associated with sucrose solution between the fifth and eighth day of adulthood or were left untreated. We found that early olfactory experience induces glomerulus-selective increases in volume that were specific to the learned odor. By comparing our volumetric measures with calcium-imaging recordings from a previous study, performed in 17-day-old bees subjected to the same treatment and experimental conditions, we found that glomeruli that showed structural changes after early learning were those that exhibited a significant increase in neural activity. Our results make evident a correlation between structural and functional changes in the AL following early olfactory learning., (© 2012 The Authors. European Journal of Neuroscience © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

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, Takeda's 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.

Visual learning admits different levels of complexity, from the formation of a simple associative link between a visual stimulus and its outcome, to more sophisticated performances, such as object categorization or rules learning, that allow flexible responses beyond simple forms of learning. Not surprisingly, higher-order forms of visual learning have been studied primarily in vertebrates with larger brains, while simple visual learning has been the focus in animals with small brains such as insects. This dichotomy has recently changed as studies on visual learning in social insects have shown that these animals can master extremely sophisticated tasks. Here we review a spectrum of visual learning forms in social insects, from color and pattern learning, visual attention, and top-down image recognition, to interindividual recognition, conditional discrimination, category learning, and rule extraction. We analyze the necessity and sufficiency of simple associations to account for complex visual learning in Hymenoptera and discuss possible neural mechanisms underlying these visual performances.

The formation of a stable olfactory memory requires activity within several brain regions. The honeybee provides a valuable model to map complex olfactory learning tasks onto certain brain areas. To this end, we used injections of the local anaesthetic procaine to reversibly block spike activity in a specific brain region, the mushroom body (MB). We first investigated the physiological effects of procaine on cultured MB neurons from adult honeybee brains. Using the whole-cell configuration of the patch-clamp technique, we show that procaine blocks voltage-gated Na+ and K+ currents in a dose-dependent manner between 0.1 and 10 mm. The effects are reversible within a few minutes of wash. Lidocaine acts similarly, but is less effective at the tested concentrations. We then studied the role of the MBs during reversal learning by blocking the neural activity within these structures by injecting procaine. During reversal learning bees learn to revert their responses to two odorants, one rewarded (A+) and one unrewarded (B-), if their contingencies are changed (A- vs B+). Injecting procaine into the MBs impaired reversal learning. Procaine treatment during acquisition left the later retention of the initial learning (A+ vs B-) intact. Similarly, a differential conditioning task involving novel odorants (C+ vs D-) was intact under procaine treatment. Our experiments show that local injections of procaine can be used to map learning tasks onto specific regions of the insect brain. We conclude that intact MB activity is required for the acquisition of reversal learning, but not for simple differential learning tasks.

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.