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Down syndrome cell adhesion molecule ( Dscam ) gene encodes a cell adhesion molecule required for neuronal wiring. A remarkable feature of arthropod Dscam is massive alternative splicing generating thousands of different isoforms from three variable clusters of alternative exons. Dscam expression and diversity arising from alternative splicing have been studied during development, but whether they exert functions in adult brains has not been determined. Here, using honey bees, we find that Dscam expression is critically linked to memory retention as reducing expression by RNAi enhances memory after reward learning in adult worker honey bees. Moreover, alternative splicing of Dscam is altered in all three variable clusters after learning. Since identical Dscam isoforms engage in homophilic interactions, these results suggest a mechanism to alter inclusion of variable exons during memory consolidation to modify neuronal connections for memory retention., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Ustaoglu, McQuarrie, Rochet, Dix, Haussmann, Arnold, Devaud and Soller.)

Analysing large numbers of brain samples can reveal minor, but statistically and biologically relevant variations in brain morphology that provide critical insights into animal behaviour, ecology and evolution. So far, however, such analyses have required extensive manual effort, which considerably limits the scope for comparative research. Here we used micro-CT imaging and deep learning to perform automated analyses of 3D image data from 187 honey bee and bumblebee brains. We revealed strong inter-individual variations in total brain size that are consistent across colonies and species, and may underpin behavioural variability central to complex social organisations. In addition, the bumblebee dataset showed a significant level of lateralization in optic and antennal lobes, providing a potential explanation for reported variations in visual and olfactory learning. Our fast, robust and user-friendly approach holds considerable promises for carrying out large-scale quantitative neuroanatomical comparisons across a wider range of animals. Ultimately, this will help address fundamental unresolved questions related to the evolution of animal brains and cognition., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Lösel et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Honey bees are of great economic and ecological importance, but are facing multiple stressors that can jeopardize their pollination efficiency and survival. Therefore, understanding the physiological bases of their stress response may help defining treatments to improve their resilience. We took an original approach to design molecules with this objective. We took advantage of the previous identified neuropeptide allatostatin A (ASTA) and its receptor (ASTA-R) as likely mediators of the honey bee response to a biologically relevant stressor, exposure to an alarm pheromone compound. A first series of ASTA-R ligands were identified through in silico screening using a homology 3D model of the receptor and in vitro binding experiments. One of these (A8) proved also efficient in vivo, as it could counteract two behavioral effects of pheromone exposure, albeit only in the millimolar range. This putative antagonist was used as a template for the chemical synthesis of a second generation of potential ligands. Among these, two compounds showed improved efficiency in vivo (in the micromolar range) as compared to A8 despite no major improvement in their affinity for the receptor in vitro. These new ligands are thus promising candidates for alleviating stress in honey bees., (© 2022. The Author(s).)

Whether animals can actively avoid food contaminated with harmful compounds through taste is key to assess their ecotoxicological risks. Here, we investigated the ability of honey bees to perceive and avoid food resources contaminated with common metal pollutants known to impair behaviour at low concentrations. In laboratory assays, bees did not discriminate food contaminated with arsenic, lead or zinc and ingested it readily, up to estimated doses of 929.1 μg g -1 As, 6.45 mg g -1 Pb and 72.46 mg g -1 Zn. A decrease of intake and appetitive responses indicating metal detection was only observed at the highest concentrations of lead (3.6 mM) and zinc (122.3 mM) through contact with the antennae and the proboscis. Electrophysiological analyses confirmed that only high concentrations of the three metals in a sucrose solution induced a consistently reduced neural response to sucrose in antennal taste receptors (As: >0.1 μM, Pb: >1 mM; Zn: >100 mM). Overall, cellular and behavioural responses did not provide evidence for specific mechanisms that would support selective detection of toxic metals (arsenic, lead), as compared to zinc, which has important biological functions. Our results thus show that honey bees can avoid metal pollutants in their food only at high concentrations unlikely to be encountered in the environment. By contrast, they appear to be unable to detect low, yet harmful, concentrations found in flowers. Metal pollution at trace levels is therefore a major threat for pollinators., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Changes in gene expression are a hallmark of learning and memory consolidation. Little is known about how alternative mRNA processing, particularly abundant in neuron-specific genes, contributes to these processes. Prototype RNA binding proteins of the neuronally expressed ELAV/Hu family are candidates for roles in learning and memory, but their capacity to cross-regulate and take over each other's functions complicate substantiation of such links. Honey bees Apis mellifera have only one elav/Hu family gene elavl2, that has functionally diversified by increasing alternative splicing including an evolutionary conserved microexon. RNAi knockdown demonstrates that ELAVL2 is required for learning and memory in bees. ELAVL2 is dynamically expressed with altered alternative splicing and subcellular localization in mushroom bodies, but not in other brain regions. Expression and alternative splicing of elavl2 change during memory consolidation illustrating an alternative mRNA processing program as part of a local gene expression response underlying memory consolidation., (© 2021. The Author(s).)

Environmental pollutants can exert sublethal deleterious effects on animals. These include disruption of cognitive functions underlying crucial behaviours. While agrochemicals have been identified as a major threat to pollinators, metal pollutants, which are often found in complex mixtures, have so far been overlooked. Here, we assessed the impact of acute exposure to field-realistic concentrations of three common metal pollutants, lead, copper and arsenic, and their combinations, on honey bee appetitive learning and memory. All treatments involving single metals slowed down learning and disrupted memory retrieval at 24 h. Combinations of these metals had additive negative effects on both processes, suggesting common pathways of toxicity. Our results highlight the need to further assess the risks of metal pollution on invertebrates., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Pollutants can have severe detrimental effects on insects, even at sublethal doses, damaging developmental and cognitive processes involved in crucial behaviours. Agrochemicals have been identified as important causes of pollinator declines, but the impacts of other anthropogenic compounds, such as metallic trace elements in soils and waters, have received considerably less attention. Here, we exposed colonies of the European honey bee Apis mellifera to chronic field-realistic concentrations of lead in food and demonstrated that consumption of this trace element impaired bee cognition and morphological development. Honey bees exposed to the highest of these low concentrations had reduced olfactory learning performances. These honey bees also developed smaller heads, which may have constrained their cognitive functions as we show a general relationship between head size and learning performance. Our results demonstrate that lead pollutants, even at trace levels, can have dramatic effects on honey bee cognitive abilities, potentially altering key colony functions and the pollination service., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

Securing food supply for a growing population is a major challenge and heavily relies on the use of agrochemicals to maximize crop yield. It is increasingly recognized, that some neonicotinoid insecticides have a negative impact on non-target organisms, including important pollinators such as the European honeybee Apis mellifera. Toxicity of neonicotinoids may be enhanced through simultaneous exposure with additional pesticides, which could help explain, in part, the global decline of honeybee colonies. Here we examined whether exposure effects of the neonicotinoid thiamethoxam on bee viability are enhanced by the commonly used fungicide carbendazim and the herbicide glyphosate. We also analysed alternative splicing changes upon pesticide exposure in the honeybee. In particular, we examined transcripts of three genes: (i) the stress sensor gene X box binding protein-1 (Xbp1), (ii) the Down Syndrome Cell Adhesion Molecule (Dscam) gene and iii) the embryonic lethal/abnormal visual system (elav) gene, which are important for neuronal function. Our results showed that acute thiamethoxam exposure is not enhanced by carbendazim, nor glyphosate. Toxicity of the compounds did not trigger stress-induced, alternative splicing in the analysed mRNAs, thereby leaving dormant a cellular response pathway to these man-made environmental perturbations.

Honey bee foragers must supply their colony with a balance of pollen and nectar to sustain optimal colony development. Inter-individual behavioural variability among foragers is observed in terms of activity levels and nectar vs. pollen collection, however the causes of such variation are still open questions. Here we explored the relationship between foraging activity and foraging performance in honey bees (Apis mellifera) by using an automated behaviour monitoring system to record mass on departing the hive, trip duration, presence of pollen on the hind legs and mass upon return to the hive, during the lifelong foraging career of individual bees. In our colonies, only a subset of foragers collected pollen, and no bee exclusively foraged for pollen. A minority of very active bees (19% of the foragers) performed 50% of the colony's total foraging trips, contributing to both pollen and nectar collection. Foraging performance (amount and rate of food collection) depended on bees' individual experience (amount of foraging trips completed). We argue that this reveals an important vulnerability for these social bees since environmental stressors that alter the activity and reduce the lifespan of foragers may prevent bees ever achieving maximal performance, thereby seriously compromising the effectiveness of the colony foraging force.

Behavioural and cognitive processes play important roles in mediating an individual's interactions with its environment. Yet, while there is a vast literature on repeatable individual differences in behaviour, relatively little is known about the repeatability of cognitive performance. To further our understanding of the evolution of cognition, we gathered 44 studies on individual performance of 25 species across six animal classes and used meta-analysis to assess whether cognitive performance is repeatable. We compared repeatability ( R ) in performance (1) on the same task presented at different times (temporal repeatability), and (2) on different tasks that measured the same putative cognitive ability (contextual repeatability). We also addressed whether R estimates were influenced by seven extrinsic factors (moderators): type of cognitive performance measurement, type of cognitive task, delay between tests, origin of the subjects, experimental context, taxonomic class and publication status. We found support for both temporal and contextual repeatability of cognitive performance, with mean R estimates ranging between 0.15 and 0.28. Repeatability estimates were mostly influenced by the type of cognitive performance measures and publication status. Our findings highlight the widespread occurrence of consistent inter-individual variation in cognition across a range of taxa which, like behaviour, may be associated with fitness outcomes.This article is part of the theme issue 'Causes and consequences of individual differences in cognitive abilities'., (© 2018 The Author(s).)

Brain structure and learning capacities both vary with experience, but the mechanistic link between them is unclear. Here, we investigated whether experience-dependent variability in learning performance can be explained by neuroplasticity in foraging honey bees. The mushroom bodies (MBs) are a brain center necessary for ambiguous olfactory learning tasks such as reversal learning. Using radio frequency identification technology, we assessed the effects of natural variation in foraging activity, and the age when first foraging, on both performance in reversal learning and on synaptic connectivity in the MBs. We found that reversal learning performance improved at foraging onset and could decline with greater foraging experience. If bees started foraging before the normal age, as a result of a stress applied to the colony, the decline in learning performance with foraging experience was more severe. Analyses of brain structure in the same bees showed that the total number of synaptic boutons at the MB input decreased when bees started foraging, and then increased with greater foraging intensity. At foraging onset MB structure is therefore optimized for bees to update learned information, but optimization of MB connectivity deteriorates with foraging effort. In a computational model of the MBs sparser coding of information at the MB input improved reversal learning performance. We propose, therefore, a plausible mechanistic relationship between experience, neuroplasticity, and cognitive performance in a natural and ecological context.

Ants have recently emerged as useful models for the study of olfactory learning. In this framework, the development of a protocol for the appetitive conditioning of the maxilla-labium extension response (MaLER) provided the possibility of studying Pavlovian odor-food learning in a controlled environment. Here we extend these studies by introducing the first Pavlovian aversive learning protocol for harnessed ants in the laboratory. We worked with carpenter ants Camponotus aethiops and first determined the capacity of different temperatures applied to the body surface to elicit the typical aversive mandible opening response (MOR). We determined that 75°C is the optimal temperature to induce MOR and chose the hind legs as the stimulated body region because of their high sensitivity. We then studied the ability of ants to learn and remember odor-heat associations using 75°C as the unconditioned stimulus. We studied learning and short-term retention after absolute (one odor paired with heat) and differential conditioning (a punished odor versus an unpunished odor). Our results show that ants successfully learn the odor-heat association under a differential-conditioning regime and thus exhibit a conditioned MOR to the punished odor. Yet, their performance under an absolute-conditioning regime is poor. These results demonstrate that ants are capable of aversive learning and confirm previous findings about the different attentional resources solicited by differential and absolute conditioning in general., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

The honey bee mushroom bodies (MBs) are brain centers required for specific learning tasks. Here, we show that environmental conditions experienced as young adults affect the maturation of MB neuropil and performance in a MB-dependent learning task. Specifically, olfactory reversal learning was selectively impaired following early exposure to an impoverished environment lacking some of the sensory and social interactions present in the hive. In parallel, the overall number of synaptic boutons increased within the MB olfactory neuropil, whose volume remained unaffected. This suggests that experience of the rich in-hive environment promotes MB maturation and the development of MB-dependent learning capacities., (© 2017 Cabirol et al.; Published by Cold Spring Harbor Laboratory Press.)

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.

Workers of social insects, such as bees, ants and wasps, show some degree of inter-individual variability in decision-making, learning and memory. Whether these natural cognitive differences translate into distinct adaptive behavioural strategies is virtually unknown. Here we examined variability in the movement patterns of bumblebee foragers establishing routes between artificial flowers. We recorded all flower visitation sequences performed by 29 bees tested for 20 consecutive foraging bouts in three experimental arrays, each characterised by a unique spatial configuration of artificial flowers and three-dimensional landmarks. All bees started to develop efficient routes as they accumulated foraging experience in each array, and showed consistent inter-individual differences in their levels of route fidelity and foraging performance, as measured by travel speed and the frequency of revisits to flowers. While the tendency of bees to repeat the same route was influenced by their colony origin, foraging performance was correlated to body size. The largest foragers travelled faster and made less revisits to empty flowers. We discuss the possible adaptive value of such inter-individual variability within the forager caste for optimisation of colony-level foraging performances in social pollinators.

As honey bee populations worldwide are declining there is an urgent need for a deeper understanding of stress reactivity in these important insects. Our data indicate that stress responses in bees (Apis mellifera L.) may be mediated by neuropeptides identified, on the basis of sequence similarities, as allatostatins (ASTA, ASTC and ASTCC). Effects of allatostatin injection are compared with stress-related changes in learning performance induced by the honeybee alarm pheromone, isopentylacetate (IPA). We find that bees can exhibit two markedly different responses to IPA, with opposing effects on learning behaviour and memory generalisation, and that strikingly similar responses can be elicited by allatostatins, in particular ASTCC. These findings lend support to the hypothesis that allatostatins mediate stress reactivity in honey bees and suggest responses to stress in these insects are state dependent.

Sequencing of the honeybee genome revealed many neuropeptides and putative neuropeptide receptors, yet functional characterization of these peptidic systems is scarce. In this study, we focus on allatostatins, which were first identified as inhibitors of juvenile hormone synthesis, but whose role in the adult honey bee (Apis mellifera) brain remains to be determined. We characterize the bee allatostatin system, represented by two families: allatostatin A (Apime-ASTA) and its receptor (Apime-ASTA-R); and C-type allatostatins (Apime-ASTC and Apime-ASTCC) and their common receptor (Apime-ASTC-R). Apime-ASTA-R and Apime-ASTC-R are the receptors in bees most closely related to vertebrate galanin and somatostatin receptors, respectively. We examine the functional properties of the two honeybee receptors and show that they are transcriptionally expressed in the adult brain, including in brain centers known to be important for learning and memory processes. Thus we investigated the effects of exogenously applied allatostatins on appetitive olfactory learning in the bee. Our results show that allatostatins modulate learning in this insect, and provide important insights into the evolution of somatostatin/allatostatin signaling.

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.

In reversal learning, subjects first learn to respond to a reinforced stimulus A and not to a non-reinforced stimulus B (A(+) vs. B(-)) and then have to learn the opposite when stimulus contingencies are reversed (A(-) vs. B(+)). This change in stimulus valence generates a transitory ambiguity at the level of stimulus outcome that needs to be overcome to solve the second discrimination. Honey bees (Apis mellifera) efficiently master reversal learning in the olfactory domain. The mushroom bodies (MBs), higher-order structures of the insect brain, are required to solve this task. Here we aimed at uncovering the neural circuits facilitating reversal learning in honey bees. We trained bees using the olfactory conditioning of the proboscis extension reflex (PER) coupled with localized pharmacological inhibition of Gamma-AminoButyric Acid (GABA)ergic signaling in the MBs. We show that inhibition of ionotropic but not metabotropic GABAergic signaling into the MB calyces impairs reversal learning, but leaves intact the capacity to perform two consecutive elemental olfactory discriminations with ambiguity of stimulus valence. On the contrary, inhibition of ionotropic GABAergic signaling into the MB lobes had no effect on reversal learning. Our results are thus consistent with a specific requirement of the feedback neurons (FNs) providing ionotropic GABAergic signaling from the MB lobes to the calyces for counteracting ambiguity of stimulus valence in reversal learning.

Memory is a dynamic process that allows encoding, storage, and retrieval of information acquired through individual experience. In the honeybee Apis mellifera, olfactory conditioning of the proboscis extension response (PER) has shown that besides short-term memory (STM) and mid-term memory (MTM), two phases of long-term memory (LTM) are formed upon multiple-trial conditioning: an early phase (e-LTM) which depends on translation from already available mRNA, and a late phase (l-LTM) which requires de novo transcription and translation. Here we combined olfactory PER conditioning and neuropharmacological inhibition and studied the involvement of the NO-cGMP pathway, and of specific molecules, such as cyclic nucleotide-gated channels (CNG), calmodulin (CaM), adenylyl cyclase (AC), and Ca(2+)/calmodulin-dependent protein kinase (CaMKII), in the formation of olfactory LTM in bees. We show that in addition to NO-cGMP and cAMP-PKA, CNG channels, CaM, AC, and CaMKII also participate in the formation of a l-LTM (72-h post-conditioning) that is specific for the learned odor. Importantly, the same molecules are dispensable for olfactory learning and for the formation of both MTM (in the minute and hour range) and e-LTM (24-h post-conditioning), thus suggesting that the signaling pathways leading to l-LTM or e-LTM involve different molecular actors.

Storage of information into long-term memory (LTM) usually requires at least two waves of transcription in many species. However, there is no clear evidence of this phenomenon in insects, which are influential models for memory studies. We measured retention in honeybees after injecting a transcription inhibitor at different times before and after conditioning. We identified two separate time windows during which the transcription blockade impairs memory quantitatively and qualitatively, suggesting the occurrence of an early transcription wave (triggered during conditioning) and a later one (starting several hours after learning). Hence insects, like other species, would require two transcription waves for LTM formation.

The biological concept of stress originated in mammals, where a "General Adaptation Syndrome" describes a set of common integrated physiological responses to diverse noxious agents. Physiological mechanisms of stress in mammals have been extensively investigated through diverse behavioral and physiological studies. One of the main elements of the stress response pathway is the endocrine hypothalamo-pituitary-adrenal (HPA) axis, which underlies the "fight-or-flight" response via a hormonal cascade of catecholamines and corticoid hormones. Physiological responses to stress have been studied more recently in insects: they involve biogenic amines (octopamine, dopamine), neuropeptides (allatostatin, corazonin) and metabolic hormones (adipokinetic hormone, diuretic hormone). Here, we review elements of the physiological stress response that are or may be specific to honey bees, given the economical and ecological impact of this species. This review proposes a hypothetical integrated honey bee stress pathway somewhat analogous to the mammalian HPA, involving the brain and, particularly, the neurohemal organ corpora cardiaca and peripheral targets, including energy storage organs (fat body and crop). We discuss how this system can organize rapid coordinated changes in metabolic activity and arousal, in response to adverse environmental stimuli. We highlight physiological elements of the general stress responses that are specific to honey bees, and the areas in which we lack information to stimulate more research into how this fascinating and vital insect responds to stress.

Latent inhibition (LI) is a decrement in learning performance that results from the nonreinforced pre-exposure of the to-be-conditioned stimulus, in both vertebrates and invertebrates. In vertebrates, LI development involves dopamine and serotonin; in invertebrates there is yet no information. We studied differential olfactory conditioning of the proboscis extension response in the honeybee Apis mellifera, and we compared LI in individuals treated with antagonists of biogenic amines (dopamine, octopamine, and serotonin). An antagonist of octopamine receptors and two antagonists of serotonin receptors showed LI disruption. We thus provide evidence that serotonin would participate in the regulation of LI in honeybees.

Sex pheromones are intraspecific olfactory signals emitted by one sex to attract a potential mating partner. Behavioural responses to sex pheromones are generally highly stereotyped. However, they can be modulated by experience, as male moths previously exposed to female sex pheromone respond with a lower threshold upon further detection, even after long delays. Here, we address the question of the neural mechanisms underlying such long-term modulation. As previous work has shown increased responses to pheromone in central olfactory neurons, we asked whether brief exposure to the pheromone increases input activity from olfactory receptor neurons. Males pre-exposed to sex pheromone exhibited increased peripheral sensitivity to the main pheromone component. Among nine antennal genes targeted as putatively involved in pheromone reception, one encoding a pheromone-binding protein showed significant upregulation upon exposure. In the primary olfactory centre (antennal lobe), the neural compartment processing the main pheromone component was enlarged after a brief pheromone exposure, thus suggesting enduring structural changes. We hypothesise that higher peripheral sensitivity following pre-exposure leads to increased input to the antennal lobe, thus contributing to the structural and functional reorganization underlying a stable change in behaviour.

We have previously shown that driving PI3K levels up or down leads to increases or reductions in the number of synapses, respectively. Using these tools to assay their behavioral effects in Drosophila melanogaster, we showed that a loss of synapses in two sets of local interneurons, GH298 and krasavietz, leads to olfaction changes toward attraction or repulsion, while the simultaneous manipulation of both sets of neurons restored normal olfactory indexes. We show here that olfactory central adaptation also requires the equilibrated changes in both sets of local interneurons. The same genetic manipulations directed to projection (GH146) or mushroom body (201Y, MB247) neurons did not affect adaptation. Also, we show that the equilibrium is a requirement for the glomerulus-specific size changes which are a morphological signature of central adaptation. Since the two sets of local neurons are mostly, although not exclusively, inhibitory (GH298) and excitatory (krasavietz), we interpret that the normal phenomena of sensory perception, measured as an olfactory index, and central adaptation rely on an inhibition/excitation ratio.

Ants exhibit impressive olfactory learning abilities. Operant protocols in which ants freely choose between rewarded and non-rewarded odours have been used to characterise associative olfactory learning and memory. Yet, this approach precludes the use of invasive methods allowing the dissection of molecular bases of learning and memory. An open question is whether the memories formed upon olfactory learning that are retrievable several days after training are indeed based on de novo protein synthesis. Here, we addressed this question in the ant Camponotus fellah using a conditioning protocol in which individually harnessed ants learn an association between odour and reward. When the antennae of an ant are stimulated with sucrose solution, the insect extends its maxilla-labium to absorb the solution (maxilla-labium extension response). We differentially conditioned ants to discriminate between two long-chain hydrocarbons, one paired with sucrose and the other with quinine solution. Differential conditioning leads to the formation of a long-term memory retrievable at least 72 h after training. Long-term memory consolidation was impaired by the ingestion of cycloheximide, a protein synthesis blocker, prior to conditioning. Cycloheximide did not impair acquisition of either short-term memory (10 min) or early and late mid-term memories (1 or 12 h). These results show that, upon olfactory learning, ants form different memories with variable molecular bases. While short- and mid-term memories do not require protein synthesis, long-term memories are stabilised via protein synthesis. Our behavioural protocol opens interesting research avenues to explore the cellular and molecular bases of olfactory learning and memory in ants.

In honeybees, associative learning is embedded in a social context as bees possess a highly complex social organization in which communication among individuals is mediated by dance behavior informing about food sources, and by a high variety of pheromones that maintain the social links between individuals of a hive. Proboscis extension response conditioning is a case of appetitive learning, in which harnessed bees learn to associate odor stimuli with sucrose reward in the laboratory. Despite its recurrent use as a tool for uncovering the behavioral, cellular, and molecular bases underlying associative learning, the question of whether social signals (pheromones) affect appetitive learning has not been addressed in this experimental framework. This situation contrasts with reports underlining that foraging activity of bees is modulated by alarm pheromones released in the presence of a potential danger. Here, we show that appetitive learning is impaired by the sting alarm pheromone (SAP) which, when released by guards, recruits foragers to defend the hive. This effect is mimicked by the main component of SAP, isopentyl acetate, is dose-dependent and lasts up to 24 h. Learning impairment is specific to alarm signal exposure and is independent of the odorant used for conditioning. Our results suggest that learning impairment may be a response to the biological significance of SAP as an alarm signal, which would detract bees from responding to any appetitive stimuli in a situation in which such responses would be of secondary importance.

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.

The storage of stable memories is generally considered to rely on changes in the functional properties and/or the synaptic connectivity of neural networks. However, these changes are not easily tractable given the complexity of the learning procedures and brain circuits studied. Such a search can be narrowed down by studying memories of specific stimuli in a given sensory modality and by working on networks with a modular and relatively simple organization. We have therefore focused on associative memories of individual odors and the possible related changes in the honeybee primary olfactory center, the antennal lobe (AL). As this brain structure is organized in well-identified morpho-functional units, the glomeruli, we looked for evidence of structural and functional plasticity in these units in relation with the bees' ability to store long-term memories (LTMs) of specific odors. Restrained bees were trained to form an odor-specific LTM in an appetitive Pavlovian conditioning protocol. The stability and specificity of this memory was tested behaviorally 3 d after conditioning. At that time, we performed both a structural and a functional analysis on a subset of 17 identified glomeruli by measuring glomerular volume under confocal microscopy, and odor-evoked activity, using in vivo calcium imaging. We show that long-term olfactory memory for a given odor is associated with volume increases in a subset of glomeruli. Independent of these structural changes, odor-evoked activity was not modified. Lastly, we show that structural glomerular plasticity can be predicted based on a putative model of interglomerular connections.

It is generally accepted that, after learning, memories stabilize over time and integrate into long-term memory (LTM) through the process of consolidation, which depends on de novo protein synthesis. Besides, studies on several species have shown that reactivation of already stabilized LTM can either make this memory labile and then restabilize it (a process called reconsolidation) or inhibit it (extinction). However, the identity of both processes and their interactions with consolidation are still under debate. Regarding memory stabilization, Drosophila offers a striking exception since, in this species, LTM is not the sole stable form of memory. Under specific learning conditions, anesthesia-resistant memory (ARM) can be formed through processes yet unknown but that are resistant to cycloheximide, a classical protein synthesis inhibitor that impairs LTM. Here, we took advantage of this dichotomy to ask whether both ARM and LTM could be extinguished and/or reconsolidated. We also studied whether two forms of memory extinction and reconsolidation exist in flies, as for memory stabilization. We show that either reconsolidation or extinction can be induced after olfactory conditioning in Drosophila, depending on the number of reactivations as in other species. Furthermore, regarding the effect of cycloheximide, the ARM/LTM dichotomy for stabilization does not apply to extinction and reconsolidation. Blocking protein synthesis interfered with both processes regardless of whether initial stabilization was sensitive (LTM) or not (ARM) to cycloheximide. These results thus show that Drosophila is a useful model to tackle these questions and that reconsolidation is not necessarily a mere repetition of consolidation.

Complex external stimuli such as odorants are believed to be internally represented in the brain by spatiotemporal activity patterns of extensive neuronal ensembles. These activity patterns can be recorded by optical imaging techniques. However, optical imaging with conventional fluorescence dyes usually does not allow for resolving the activity of biologically defined groups of neurons. Therefore, specifically targeting reporter molecules to neuron populations of common genetic identity is an important goal. We report the use of the genetically encoded calcium-sensitive fluorescence protein cameleon 2.1 in the Drosophila brain. We visualized odorant-evoked intracellular calcium concentration changes in selectively labeled olfactory projection neurons both postsynaptically in the antennal lobe, the primary olfactory neuropil, and presynaptically in the mushroom body calyx, a structure involved in olfactory learning and memory. As a technical achievement, we show that calcium imaging with a genetically encoded fluorescence probe is feasible in a brain in vivo. This will allow one to combine Drosophila's advanced genetic tools with the physiological analysis of brain function. Moreover, we report for the first time optical imaging recordings in synaptic regions of the Drosophila mushroom body calyx and antennal lobe. This provides an important step for the use of Drosophila as a model system in olfaction.

In an attempt to correlate behavioral and neuronal changes, we examined the structural and functional effects of odor exposure in Drosophila. Young adult flies were exposed to a high concentration of the selected odor, usually benzaldehyde or isoamyl acetate, for 4 d and subsequently tested for their olfactory response to a variety of odorants and concentrations. The behavioral response showed specific adaptation to the exposed odor. By contrast, olfactory transduction, as measured in electroantennograms, remained normal. In vivo volume measurements were performed on olfactory glomeruli, the anatomical and functional units involved in odor processing. Pre-exposed flies exhibited volume reduction of certain glomeruli, in an odor-selective manner. Of a sample of eight glomeruli measured, dorsal medial (DM) 2 and ventral (V) were affected by benzaldehyde exposure, whereas DM6 was affected by isoamyl acetate. Estimation of the number of synapses indicates that volume reduction involves synapse loss that can reach 30% in the V glomerulus of flies adapted to benzaldehyde. Additional features of odorant-induced adaptation, including concentration dependence and perdurance, also show correlation, because both effects are elicited by high odor concentrations and are long-lasting (>1 week). Finally, the dunce mutant fails to develop behavioral adaptation as well as morphological changes in the olfactory glomeruli after exposure. These neural changes thus appear to require the cAMP signaling pathway.