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Butterflies, like many insects, use gustatory and olfactory cues innately to assess the suitability of an oviposition site and are able to associate colours and leaf shapes with an oviposition reward. Studies on other insects have demonstrated that the quality of the reward is a crucial factor in forming associative memory. We set out to investigate whether the large cabbage white Pieris brassicae (Linnaeus) has the ability to associate an oviposition experience with a neutral olfactory cue. In addition, we tested whether the strength of this association is dependent on the gustatory response to the glucosinolate sinigrin, which is a known oviposition stimulus for P. brassicae. Female butterflies were able to associate a neutral odour with an oviposition experience after a single oviposition experience, both in a greenhouse and in a semi-natural outdoor setting. Moreover, butterflies performed best when trained with concentrations of sinigrin that showed the strongest response by specific gustatory neurons on the forelegs. Our study provides novel insight into the role of both gustatory and olfactory cues during oviposition learning in lepidopterans and contributes to a better understanding of how these insects might be able to adapt to a rapidly changing environment. [ABSTRACT FROM AUTHOR]

The amygdala has long held the center seat in the neural basis of threat conditioning. However, a rapidly growing literature has elucidated extra-amygdala circuits in this process, highlighting the sensory cortex for its critical role in the mnemonic aspect of the process. While this literature is largely focused on the auditory system, substantial human and rodent findings on the olfactory system have emerged. The unique nature of the olfactory neuroanatomy and its intimate association with emotion compels a review of this recent literature to illuminate its special contribution to threat memory. Here, integrating recent evidence in humans and animal models, we posit that the olfactory (piriform) cortex is a primary and necessary component of the distributed threat memory network, supporting mnemonic ensemble coding of acquired threat. We further highlight the basic circuit architecture of the piriform cortex characterized by distributed, auto-associative connections, which is prime for highly efficient content-addressable memory computing to support threat memory. Given the primordial role of the piriform cortex in cortical evolution and its simple, well-defined circuits, we propose that olfaction can be a model system for understanding (transmodal) sensory cortical mechanisms underlying threat memory. [ABSTRACT FROM AUTHOR]

The health of insect pollinators, particularly the honey bee, Apis mellifera (Linnaeus, 1758), is a major concern for agriculture and ecosystem health. In response to mounting evidence supporting the detrimental effects of neonicotinoid pesticides on pollinators, a novel 'bee safe' butenolide compound, flupyradifurone (FPF) has been registered for use in agricultural use. Although FPF is not a neonicotinoid, like neonicotinoids, it is an excitotoxic nicotinic acetylcholine receptor agonist. In addition, A. mellifera faces threats from pathogens, such as the microsporidian endoparasite, Nosema ceranae (Fries et al. 1996). We therefore sought 1) to increase our understanding of the potential effects of FPF on honey bees by focusing on a crucial behavior, the ability to learn and remember an odor associated with a food reward, and 2) to test for a potential synergistic effect on such learning by exposure to FPF and infection with N. ceranae. We found little evidence that FPF significantly alters learning and memory at short-term field-realistic doses. However, at high doses and at chronic, field-realistic exposure, FPF did reduce learning and memory in an olfactory conditioning task. Infection with N. ceranae also reduced learning, but there was no synergy (no significant interaction) between N. ceranae and exposure to FPF. These results suggest the importance of continued studies on the chronic effects of FPF.

Early detection of cancer is critical in medical sciences, as the sooner a cancer is diagnosed, the higher are the chances of recovery. Tumour cells are characterized by specific volatile organic compounds (VOCs) that can be used as cancer biomarkers. Through olfactory associative learning, animals can be trained to detect these VOCs. Insects such as ants have a refined sense of smell, and can be easily and rapidly trained with olfactory conditioning. Using urine from patient-derived xenograft mice as stimulus, we demonstrate that individual ants can learn to discriminate the odour of healthy mice from that of tumour-bearing mice and do so after only three conditioning trials. After training, they spend approximately 20% more time in the vicinity of the learned odour than beside the other stimulus. Chemical analyses confirmed that the presence of the tumour changed the urine odour, supporting the behavioural results. Our study demonstrates that ants reliably detect tumour cues in mice urine and have the potential to act as efficient and inexpensive cancer bio-detectors. [ABSTRACT FROM AUTHOR]

In the present study, we tested associative olfactory learning performance of the ant Formica clara in laboratory conditions based on their MaLER responses. Ants were harnessed with the help of a paper-made holder and conditioned in two groups by forward pairing either limonene or eugenol (conditioned stimuli – CS) with a reward (unconditioned stimulus – US). After conditioning, memory tests were performed for each CS 15, 60 and 90 minutes following the conditioning in the absence of US. A further test was performed for each group with the CS of the other group to see if the ants showed an odor generalization. The frequencies of MaLER-positive responses during tests were used as indication of learning performance. Ants learned the association between the CS and US during conditioning for both odors and showed initially significant and gradually decreasing MaLER-positive responses during tests. The results showed that conditioning to the odors was absolute and no generalization was observed. We conclude that associative learning is a universal strategy for ants to adjust their behaviors to environmental changes around them and the MaLER technique is a powerful and easy to apply technique to test learning and memory capacities in ants. [ABSTRACT FROM AUTHOR]

Malnutrition in vertebrates, including humans, is known to affect brain development. Protein deprivation in the early stages of life dramatically impairs cognitive abilities. In insects, dietary protein deficiency influences several physiological and behavioural traits, but it is unknown how protein to carbohydrate (P:C) ratio simultaneously affects survival and cognitive performance.We used the geometric framework approach to examine the effects of P:C ratio in the diet of newly emerged honey bees on their survival, sucrose sensitivity, and learning and memory by conditioning of the proboscis extension response.The P:C ratio in the diet did not affect sucrose sensitivity, whereas it affected lifespan and cognition differently. While lifespan peaked at P:C ratio of 1:19, learning and short‐term memory peaked at P:C ratios of 1:9 and 1:4. Protein deprivation slightly reduced bee lifespan but highly impaired learning and short‐term memory. On the other hand, an excess of protein dramatically reduced bee lifespan with lesser effects on learning and short‐term memory.Bees were only able to form an early long‐term memory when they had been eating a protein diet (P:C ratio of 1:9) during 24 hr before the test, regardless of the diet eaten previously.Our study shows that nutrition affects honey bee cognitive abilities, but that these effects, unlike in vertebrates, can be quickly reversed with an appropriate diet. Read the free Plain Language Summary for this article on the Journal blog. [ABSTRACT FROM AUTHOR]

Memory consolidation is a time-dependent process occurring over hours, days, or longer in different species and requires protein synthesis. An apparent exception is a memory type in Drosophila elicited by a single olfactory conditioning episode, which ostensibly consolidates quickly, rendering it resistant to disruption by cold anesthesia a few hours post-training. This anesthesia-resistant memory (ARM), is independent of protein synthesis. Protein synthesis independent memory can also be elicited in Drosophila by multiple massed cycles of olfactory conditioning, and this led to the prevailing notion that both of these operationally distinct training regimes yield ARM. Significantly, we show that, unlike bona fide ARM, massed conditioning-elicited memory remains sensitive to the amnestic treatment two hours post-training and hence it is not ARM. Therefore, there are two protein synthesis-independent memory types in Drosophila. [ABSTRACT FROM AUTHOR]

Insects adapt their response to stimuli, such as odours, according to their pairing with positive or negative reinforcements, such as sugar or shock. Recent electrophysiological and imaging findings in Drosophila melanogaster allow detailed examination of the neural mechanisms supporting the acquisition, forgetting, and assimilation of memories. We propose that this data can be explained by the combination of a dopaminergic plasticity rule that supports a variety of synaptic strength change phenomena, and a circuit structure (derived from neuroanatomy) between dopaminergic and output neurons that creates different roles for specific neurons. Computational modelling shows that this circuit allows for rapid memory acquisition, transfer from short term to long term, and exploration/exploitation trade-off. The model can reproduce the observed changes in the activity of each of the identified neurons in conditioning paradigms and can be used for flexible behavioural control.

Social insects live in communities where cooperative actions heavily rely on the individual cognitive abilities of their members. In the honey bee (Apis mellifera), the specialization in nectar or pollen collection is associated with variations in gustatory sensitivity, affecting both associative and non-associative learning. Gustatory sensitivity fluctuates as a function of changes in motivation for the specific floral resource throughout the foraging cycle, yet differences in learning between nectar and pollen foragers at the onset of food collection remain unexplored. Here, we examined nectar and pollen foragers captured upon arrival at food sources. We subjected them to an olfactory proboscis extension reflex (PER) conditioning using a 10% sucrose solution paired (S10%+P) or unpaired (S10%) with pollen as a co-reinforcement. For non-associative learning, we habituated foragers with S10%+P or S10%, followed by dishabituation tests with either a 50% sucrose solution paired (S50%+P) or unpaired (S50%) with pollen. Our results indicate that pollen foragers show lower performance than nectar foragers when conditioned with S10%. Interestingly, performance improves to levels similar to those of nectar foragers when pollen is included as a rewarding stimulus (S10%+P). In non-associative learning, pollen foragers tested with S10%+P displayed a lower degree of habituation than nectar foragers and a higher degree of dishabituation when pollen was used as the dishabituating stimulus (S10%+P). Altogether, our results support the idea that pollen and nectar honey bee foragers differ in their responsiveness to rewards, leading to inter-individual differences in learning that contribute to foraging specialization., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Social avoidance is a common component of neuropsychiatric disorders that confers substantial functional impairment. An unbiased approach to identify brain regions and neuronal circuits that regulate social avoidance might enable development of novel therapeutics. However, most paradigms that alter social avoidance are irreversible and accompanied by multiple behavioral confounds. Here we report a straightforward behavioral paradigm in male mice enabling the reversible induction of social avoidance or approach with temporal control. C57BL/6J mice repeatedly participated in both negative and positive social experiences. Negative social experience was induced by brief social defeat by an aggressive male CD-1 mouse, while positive social experience was induced by exposure to a female mouse, each conducted daily for five days. Each social experience valence was conducted in a specific odorant context (i.e. negative experience in odorant A, positive experience in odorant B). Odorants were equally preferred pre-conditioning. However, after conditioning, mice sniffed positive experience-paired odorants more than negative experience-paired odorants. Furthermore, positive- or negative-conditioned odorant contexts increased or decreased, respectively, the approach behavior of conditioned mice toward conspecifics. Because individual mice undergo both positive and negative conditioning, this paradigm may be useful to examine neural representations of social approach or avoidance within the same subject. [ABSTRACT FROM AUTHOR]

Associative learning is key to how bees recognize and return to rewarding floral resources. It thus plays a major role in pollinator floral constancy and plant gene flow. Honeybees are the primary model for pollinator associative learning, but bumblebees play an important ecological role in a wider range of habitats, and their associative learning abilities are less well understood. We assayed learning with the proboscis extension reflex (PER), using a novel method for restraining bees (capsules) designed to improve bumblebee learning. We present the first results demonstrating that bumblebees exhibit the memory spacing effect. They improve their associative learning of odor and nectar reward by exhibiting increased memory acquisition, a component of long-term memory formation, when the time interval between rewarding trials is increased. Bombus impatiens forager memory acquisition (average discrimination index values) improved by 129% and 65% at inter-trial intervals (ITI) of 5 and 3 min, respectively, as compared to an ITI of 1 min. Memory acquisition rate also increased with increasing ITI. Encapsulation significantly increases olfactory memory acquisition. Ten times more foragers exhibited at least one PER response during training in capsules as compared to traditional PER harnesses. Thus, a novel conditioning assay, encapsulation, enabled us to improve bumblebee-learning acquisition and demonstrate that spaced learning results in better memory consolidation. Such spaced learning likely plays a role in forming long-term memories of rewarding floral resources.

Apis dorsata is an open-nesting, undomesticated, giant honey bee found in southern Asia. We characterized a number of aspects of olfactory system of Apis dorsata and compared it with the well-characterized, western honeybee, Apis mellifera, a domesticated, cavity-nesting species. A. dorsata differs from A. mellifera in nesting behavior, foraging activity, and defense mechanisms. Hence, there can be different demands on its olfactory system. We elucidated the glomerular organization of A. dorsata by creating a digital atlas for the antennal lobe and visualized the antennal lobe tracts and localized their innervations. We showed that the neurites of Kenyon cells with cell bodies located in a neighborhood in calyx retain their relative neighborhoods in the pedunculus and the vertical lobe forming a columnar organization in the mushroom body. The vertical lobe and the calyx of the mushroom body were found to be innervated by extrinsic neurons with cell bodies in the lateral protocerebrum. We found that the species was amenable to olfactory conditioning and showed good learning and memory retention at 24 h after training. It was also amenable to massed and spaced conditioning and could distinguish trained odor from an untrained novel odor. We found that all the above mentioned features in A. dorsata are very similar to those in A. mellifera. We thereby establish A. dorsata as a good model system, strikingly similar to A. mellifera despite the differences in their nesting and foraging behavior. [ABSTRACT FROM AUTHOR]

The α′β′ subtype of Drosophila mushroom body neurons (MBn) is required for memory acquisition, consolidation and early memory retrieval after aversive olfactory conditioning. However, in vivo functional imaging studies have failed to detect an early forming memory trace in these neurons as reflected by an enhanced G-CaMP signal in response to presentation of the learned odor. Moreover, whether cellular memory traces form early after conditioning in the mushroom body output neurons (MBOn) downstream of the α′β′ MBn remains unknown. Here, we show that aversive olfactory conditioning suppresses the calcium responses to the learned odor in both α′3 and α′2 axon segments of α′β′ MBn and in the dendrites of α′3 MBOn immediately after conditioning using female flies. Notably, the cellular memory traces in both α′3 MBn and α′3 MBOn are short-lived and persist for <30 min. The suppressed response in α′3 MBn is accompanied by a reduction of acetylcholine (ACh) release, suggesting that the memory trace in postsynaptic α′3 MBOn may simply reflect the suppression in presynaptic α′3 MBn. Furthermore, we show that the α′3 MBn memory trace does not occur from the inhibition of GABAergic neurons via GABAA receptor activation. Because activation of the α′3 MBOn drives approach behavior of adult flies, our results demonstrate that aversive conditioning promotes avoidance behavior through suppression of the α′3 MBn–MBOn circuit. [ABSTRACT FROM AUTHOR]

Honey bees transfer different informational components of the discovered feeding source to their nestmates during the waggle dance. To decode the multicomponent information of this complex behavior, dance followers have to attend to the most relevant signal elements while filtering out less relevant ones. To achieve that, dance followers should present improved abilities to acquire information compared with those bees not engaged in this behavior. Through proboscis extension response assays, sensory and cognitive abilities were tested in follower and non-follower bees. Individuals were captured within the hive, immediately after following waggle runs or a bit further from the dancer. Both behavioral categories present low and similar spontaneous odor responses (SORs). However, followers exhibit differences in responsiveness to sucrose and odor discrimination: followers showed increased gustatory responsiveness and, after olfactory differential conditioning, better memory retention than non-followers. Thus, the abilities of the dance followers related to appetitive behavior would allow them to improve the acquisition of the dance surrounding information.

The aim of the present study was to investigate whether repetitive aversive odor conditioning induced changes at the level of the peripheral olfactory system in humans. A total of 51 volunteers participated. A pair of indistinguishable odor enantiomers [(+)-rose oxide and (−)-rose oxide] were used as stimuli. During the pre-conditioning, participants’ ability to discriminate between the two odors was assessed using a three-alternative, forced-choice discrimination test. In addition, electro-olfactograms (EOG) from the olfactory epithelium were recorded. Participants underwent three conditioning sessions on consecutive days. The experimental group received an electrical stimulus to the forearm only following (+)-rose oxide presentation, whereas its enantiomer sibling was never paired with the aversive stimulus; the control group did not receive any electrical stimulation. During the post-conditioning session, their ability to discriminate the two enantiomers was assessed again using the discrimination test and EOG recordings were obtained similarly to the pre-conditioning session. Results showed significant differences in the peripheral electrophysiological responses between the conditioned and the unconditioned stimulus, demonstrating contextually induced changes at the level of the first neuron in the olfactory system. [ABSTRACT FROM AUTHOR]

Because of the relative simplicity of its nervous system, Caenorhabditis elegans is a useful model organism to study learning and memory at cellular and molecular levels. For appetitive conditioning in C. elegans, food has exclusively been used as an unconditioned stimulus (US). It may be difficult to analyze neuronal circuits for associative memory since food is a multimodal combination of olfactory, gustatory, and mechanical stimuli. Here, we report classical appetitive conditioning and associative memory in C. elegans, using 1-nonanol as a conditioned stimulus (CS), and potassium chloride (KCl) as a US. Before conditioning, C. elegans innately avoided 1-nonanol, an aversive olfactory stimulus, and was attracted by KCl, an appetitive gustatory stimulus, on assay agar plates. Both massed training without an intertrial interval (ITI) and spaced training with a 10-min ITI induced significant levels of memory of association regarding the two chemicals. Memory induced by massed training decayed within 6 h, while that induced by spaced training was retained for more than 6 h. Animals treated with inhibitors of transcription or translation formed the memory induced by spaced training less efficiently than untreated animals, whereas the memory induced by massed training was not significantly affected by such treatments. By definition, therefore, memories induced by massed training and spaced training are classified as short-term memory (STM) and long-term memory (LTM), respectively. When animals conditioned by spaced training were exposed to 1-nonanol alone, their learning index was lower than that of untreated animals, suggesting that extinction learning occurs in C. elegans. In support of these results, C. elegans mutants defective in nmr-1, encoding an NMDA receptor subunit, formed both STM and LTM less efficiently than wild-type animals, while mutations in crh-1, encoding a ubiquitous transcription factor CREB required for memory consolidation, affected LTM, but not STM. The paradigm established in the present study should allow us to elucidate neuronal circuit plasticity for appetitive learning and memory in C. elegans.

Neprilysins are Type II metalloproteinases known to degrade and inactivate a number of small peptides, in particular the mammalian amyloid-β peptide (Aβ). In Drosophila, several neprilysins expressed in the brain are required for middle-term (MTM) and long-term memory (LTM) in the dorsal paired medial (DPM) neurons, a pair of large neurons that broadly innervate the mushroom bodies (MB), the center of olfactory memory. These data indicate that one or several peptides need to be degraded for MTM and LTM. We have previously shown that the fly amyloid precursor protein (APPL) is required for memory in the MB. We show here that APPL is also required in adult DPM neurons for MTM and LTM formation. This finding prompted us to search for an interaction between neprilysins and Drosophila Aβ (dAβ), a cleavage product of APPL. To find out whether dAβ was a neprilysin's target, we used inducible drivers to modulate neprilysin 1 (Nep1) and dAβ expression in adult DPM neurons. Experiments were conducted either in both sexes or in females. We show that Nep1 inhibition makes dAβ expression detrimental to both MTM and LTM. Conversely, memory deficits displayed by dAβ-expressing flies are rescued by Nep1 overexpression. Consistent with behavioral data, biochemical analyses confirmed that Nep1 degrades dAβ. Together, our findings establish that Nep1 and dAβ expressed in DPM neurons are functionally linked for memory processes, suggesting that dAβ is a physiological target for Nep1. [ABSTRACT FROM AUTHOR]

Once associating another person with an unpleasant smell, how do we perceive and judge this person from that moment on? Here, we used aversive olfactory conditioning followed by a social attribution task during functional magnetic resonance imaging to address this question. After conditioning, where one of two faces was repeatedly paired with an aversive smell, the participants reported negative affect when viewing the smell-conditioned but not the neutral face. When subsequently confronted with the smell-conditioned face (without any smell), the participants tended to judge both positive and negative behaviors as indicative of personality traits rather than related to the situation. This effect was predicted by the degree of the preceding olfactory evaluative conditioning. Whole brain analysis of stimulus by stage interaction indicated differential activation of the ventromedial prefrontal cortex and right angular gyrus to the conditioned versus the neutral person during the attribution phase only. These results suggest that negative smell associations do not simply induce a negative perception of the target person but rather bias the attribution style towards trait attributions. The fact that this bias was evident regardless of behavior valence suggests it may reflect enhanced psychological distance. Thus, the known observation of social rejection triggered by aversive smell may be driven by a shift in social attribution style. [ABSTRACT FROM AUTHOR]

Because of the relative simplicity of its nervous system, Caenorhabditis elegans is a useful model organism to study learning and memory at cellular and molecular levels. For appetitive conditioning in C. elegans, food has exclusively been used as an unconditioned stimulus (US). It may be difficult to analyze neuronal circuits for associative memory since food is a multimodal combination of olfactory, gustatory, and mechanical stimuli. Here, we report classical appetitive conditioning and associative memory in C. elegans, using 1-nonanol as a conditioned stimulus (CS), and potassium chloride (KCl) as a US. Before conditioning, C. elegans innately avoided 1-nonanol, an aversive olfactory stimulus, and was attracted by KCl, an appetitive gustatory stimulus, on assay agar plates. Both massed training without an intertrial interval (ITI) and spaced training with a 10-min ITI induced significant levels of memory of association regarding the two chemicals. Memory induced by massed training decayed within 6 h, while that induced by spaced training was retained for more than 6 h. Animals treated with inhibitors of transcription or translation formed the memory induced by spaced training less efficiently than untreated animals, whereas the memory induced by massed training was not significantly affected by such treatments. By definition, therefore, memories induced by massed training and spaced training are classified as short-term memory (STM) and long-term memory (LTM), respectively. When animals conditioned by spaced training were exposed to 1-nonanol alone, their learning index was lower than that of untreated animals, suggesting that extinction learning occurs in C. elegans. In support of these results, C. elegans mutants defective in nmr-1, encoding an NMDA receptor subunit, formed both STM and LTM less efficiently than wild-type animals, while mutations in crh-1, encoding a ubiquitous transcription factor CREB required for memory consolidation, affected LTM, but not STM. The paradigm established in the present study should allow us to elucidate neuronal circuit plasticity for appetitive learning and memory in C. elegans. [ABSTRACT FROM AUTHOR]

Honey bees ( Apis mellifera) are prone to judge an ambiguous stimulus negatively if they had been agitated through shaking which simulates a predator attack. Such a cognitive bias has been suggested to reflect an internal emotional state analogous to humans who judge more pessimistically when they do not feel well. In order to test cognitive bias experimentally, an animal is conditioned to respond to two different stimuli, where one is punished while the other is rewarded. Subsequently a third, ambiguous stimulus is presented and it is measured whether the subject responds as if it expects a reward or a punishment. Generally, it is assumed that negative experiences lower future expectations, rendering the animals more pessimistic. Here we tested whether a most likely negatively experienced formic acid treatment against the parasitic mite Varroa destructor also affects future expectations of honey bees. We applied an olfactory learning paradigm (i.e., conditioned proboscis extension response) using two odorants and blends of these odorants as the ambiguous stimuli. Unlike agitating honey bees, exposure to formic acid did not significantly change the response to the ambiguous stimuli in comparison with untreated bees. Overall evidence suggests that the commonest treatment against one of the most harmful bee pests has no detrimental effects on cognitive bias in honey bees. [ABSTRACT FROM AUTHOR]

Restless Legs Syndrome (RLS) is a prevalent but poorly understood disorder that ischaracterized by uncontrollable movements during sleep, resulting in sleep disturbance.Olfactory memory in Drosophila melanogaster has proven to be a useful tool for the study ofcognitive deficits caused by sleep disturbances, such as those seen in RLS. A recently generatedDrosophila model of RLS exhibited disturbed sleep patterns similar to those seen in humans withRLS. This research seeks to improve understanding of the relationship between cognitivefunctioning and sleep disturbances in a new model for RLS. Here, we tested learning andmemory in wild type and dBTBD9 mutant flies by Pavlovian olfactory conditioning, duringwhich a shock was paired with one of two odors. Flies were then placed in a T-maze with oneodor on either side, and successful associative learning was recorded when the flies chose theside with the unpaired odor. We hypothesized that due to disrupted sleep patterns, dBTBD9mutant flies would be unable to learn the shock-odor association. However, the current studyreports that the recently generated Drosophila model of RLS shows successful olfactorylearning, despite disturbed sleep patterns, with learning performance levels matching or betterthan wild type flies.

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.

Even with the aide of modern technology, the search for cadaver or human remains underwater is still assisted by sniffer dogs mainly because of their superior sense of olfaction. However, dogs rely on volatile organic compounds in the air and that this may constraint their ability when searching for submerged cadavers. On the other hand, it has long been recognized that fishes use olfaction to sample odors from their surroundings to accomplish a task and are capable of acquiring new skills through training or conditioning. Despite decades of experimental and observational studies of the olfactory sensitivities of fishes, its potential application to forensic sciences has never been truly explored. In this pioneering research, we explore the possibility of using fish olfaction in detecting cadaver odors (porcine origin), using common carps Cyprinus carpio as model species in a series of experiments under laboratory conditions. We first observed the innate behavior of carps towards cadaver odors. Afterwards, the carps were trained in two-choice chamber experimental tanks by appetitive olfactory conditioning and odor masking methods. We also experimented on the effects of cadaver odors by early exposure using eggs and larval impregnation techniques, and observing the behaviors when they develop to early juveniles. In general, we found out that common carps are naturally repelled to cadaver odors. However using our devised conditioning protocol, results show that the conditioned carps were able to learn to be attracted to cadaver odors despite their innate aversion. The development of fish for cadaver detection is a simple but innovative idea and that it may present a cost-effective and reliable solution for the shortcomings of the existing methods in underwater cadaver search. We anticipate that this research will open up a variety of different studies in pursuit of developing fishes as biosensors and its application to forensic sciences.

Neprilysins are type II metalloproteinases known to degrade and inactivate a number of small peptides. Neprilysins in particular are the major amyloid-β peptide-degrading enzymes. In mouse models of Alzheimer's disease, neprilysin overexpression improves learning and memory deficits, whereas neprilysin deficiency aggravates the behavioral phenotypes. However, whether these enzymes are involved in memory in nonpathological conditions is an open question. Drosophila melanogaster is a well suited model system with which to address this issue. Several memory phases have been characterized in this organism and the neuronal circuits involved are well described. The fly genome contains five neprilysin-encoding genes, four of which are expressed in the adult. Using conditional RNA interference, we show here that all four neprilysins are involved in middle-term and long-term memory. Strikingly, all four are required in a single pair of neurons, the dorsal paired medial (DPM) neurons that broadly innervate the mushroom bodies (MBs), the center of olfactory memory. Neprilysins are also required in the MB, reflecting the functional relationship between the DPM neurons and the MB, a circuit believed to stabilize memories. Together, our data establish a role for neprilysins in two specific memory phases and further show that DPM neurons play a critical role in the proper targeting of neuropeptides involved in these processes. [ABSTRACT FROM AUTHOR]

Octopamine- and dopamine-based neuromodulatory systems play a critical role in learning and learning-related behaviour in insects. To further our understanding of these systems and resulting phenotypes, we quantified DNA sequence variations at six loci coding octopamine-and dopamine-receptors and their association with aversive and appetitive learning traits in a population of honeybees. We identified 79 polymorphic sequence markers (mostly SNPs and a few insertions/deletions) located within or close to six candidate genes. Intriguingly, we found that levels of sequence variation in the protein-coding regions studied were low, indicating that sequence variation in the coding regions of receptor genes critical to learning and memory is strongly selected against. Non-coding and upstream regions of the same genes, however, were less conserved and sequence variations in these regions were weakly associated with between-individual differences in learning-related traits. While these associations do not directly imply a specific molecular mechanism, they suggest that the cross-talk between dopamine and octopamine signalling pathways may influence olfactory learning and memory in the honeybee. [ABSTRACT FROM AUTHOR]

In this brief review, I argue that the impact of a stimulus on behavioral control increase as the distance of the stimulus to the body decreases. Habituation, i.e., decrement in response intensity repetition of the triggering stimulus, is the default state for sensory processing, and the likelihood of habituation is higher for distal stimuli. Sensitization, i.e., increment in response intensity upon stimulus repetition, occurs in a state dependent manner for proximal stimuli that make direct contact with the body. In Pavlovian conditioning paradigms, the US is always a more proximal stimulus than the CS. The mechanisms of associative and non-associative learning are not independent. CS-US pairings lead to formation of associations if sensitizing modulation from a proximal US prevents the habituation for a distal anticipatory CS.

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. [ABSTRACT FROM AUTHOR]

Repeated exposures to physical stressors cross-sensitize to the locomotor activating effects of psychostimulants in rodents. In the present study, we examined the effect of a conditioned stressor on expression of cocaine-induced sensitization in rats. We determined whether a mint odor cue previously paired with footshock stress (FS) would elicit a sensitized locomotor response in cocaine pre-exposed rats. Rats were given once daily injections of cocaine (30 mg/kg, i.p.) or saline for 6 days in activity monitoring chambers. Subsequently, and in a different and distinct context, equal numbers of rats in each drug condition were exposed to 10 min of brief, intermittent FS or no FS, either in the presence or absence of the mint odor cue. Upon re-exposure to the activity chambers (in which cocaine exposures had been given), all rats previously exposed to cocaine showed robust conditioned locomotion. In response to a cocaine challenge (10 mg/kg, i.p.), cocaine relative to saline pre-exposed rats showed a sensitized locomotor response. Finally, in those cocaine pre-exposed rats that had been given prior odor-FS pairings, concurrent delivery of the cocaine challenge and presentation of the odor cue markedly potentiated the expression of sensitization. To our knowledge, this is the first report of a facilitation of cocaine-induced locomotor sensitization by a conditioned stressor. [ABSTRACT FROM AUTHOR]

Essential oils are used by beekeepers to control the Varroa mites that infest honeybee colonies. So, bees can be exposed to thymol formulations in the hive. The effects of the monoterpenoid thymol were explored on olfactory memory and gene expression in the brain of the honeybee. In bees previously exposed to thymol (10 or 100 ng/bee), the specificity of the response to the conditioned stimulus (CS) was lost 24 h after learning. Besides, the octopamine receptor OA1 gene Amoa1 showed a significant decrease of expression 3 h after exposure with 10 or 100 ng/bee of thymol. With the same doses, expression of Rdl gene, coding for a GABA receptor subunit, was not significantly modified but the trpl gene was upregulated 1 and 24 h after exposure to thymol. These data indicated that the genes coding for the cellular targets of thymol could be rapidly regulated after exposure to this molecule. Memory and sensory processes should be investigated in bees after chronic exposure in the hive to thymol-based preparations. [ABSTRACT FROM AUTHOR]

The neurotransmitters receptor gene are multifunctional modulators of behaviors in social insects, with significant effects among species and several dominant roles within the same species. Multiple amines receptors affect diverse behavioral and physiological processes in the honeybee. Apis mellifera ligustica is excellent bee species due to its remarkable learning abilities. Here we analyze whether changes in expression of neurotransmitters receptor genes are related to olfaction learning behavior. We present a behavioral experiment on honeybees' appetitive olfactory conditioning and examine the relationship between learning success and neurotransmitters receptor gene expression. Bees were trained using proboscis extension response to discriminate learner and failed learner bees and examine the expression of neurotransmitters receptors genes in the brains of learner and failed-learner bees. Real-time quantitative PCR was performed to assess neurotransmitter expression levels. Our finding showed significant results in differentiating the learner and failed-learner bee. The expression level of AmDop1, AmDop2, AmDop3, AmOa1, AmTyr1 and 5-HT showed significantly higher expression in learner and failed-learner bees. Our finding suggests the critical role of neurotransmitters receptors in the olfactory learning behavior of A. mellifera. These significant neurotransmitters receptors could thus act together to define optimal motivational or attentional levels and improve learning success. This study revealed the dynamic modulation of neurotransmitters expression in the brain of learner and failed learner bees and suggested an essential role for neurotransmitters receptors gene in regulating the olfactory learning behavior in honeybees. We suggested that neurotransmitters receptors are expressed in the brain of bees and are likely to be directly influential in regulating olfactory learning success in honeybees. • The olfactory learning success was examined using the PER of Apis mellifera ligustica. • Olfactory learning trials were performed to differentiate the learner and failed learner bees. • The expression level of neurotransmitters receptors was examined in the learner's brain and fail-learner of A. mellifera bees. • Neurotransmitters play a vital role in the olfactory learning performance of bees. [ABSTRACT FROM AUTHOR]

In this brief review, I argue that the impact of a stimulus on behavioral control increase as the distance of the stimulus to the body decreases. Habituation, i.e., decrement in response intensity repetition of the triggering stimulus, is the default state for sensory processing, and the likelihood of habituation is higher for distal stimuli. Sensitization, i.e., increment in response intensity upon stimulus repetition, occurs in a state dependent manner for proximal stimuli that make direct contact with the body. In Pavlovian conditioning paradigms, the unconditioned stimulus (US) is always a more proximal stimulus than the conditioned stimulus (CS). The mechanisms of associative and non-associative learning are not independent. CS-US pairings lead to formation of associations if sensitizing modulation from a proximal US prevents the habituation for a distal anticipatory CS. [ABSTRACT FROM AUTHOR]

Animals are often immersed in a chemical world consisting of mixtures of many compounds rather than of single substances, and they constantly face the challenge of extracting relevant information out of the chemical landscape. To this purpose, the ability to discriminate among different stimuli with different valence is essential, but it is also important to be able to generalise, i.e. to treat different but similar stimuli as equivalent, as natural variation does not necessarily affect stimulus valence. Animals can thus extract regularities in their environment and make predictions, for instance about distribution of food resources. We studied perceptual similarity of different plant odours by conditioning individual carpenter ants to one odour, and subsequently testing their response to another, structurally different odour. We found that asymmetry in generalisation, where ants generalise from odour A to B, but not from B to A, is dependent on both chain length and functional group. By conditioning ants to a binary mixture, and testing their reaction to the individual components of the mixture, we show that overshadowing, where parts of a mixture are learned better than others, is rare. Additionally, generalisation is dependent not only on the structural similarity of odorants, but also on their functional value, which might play a crucial role. Our results provide insight into how ants make sense of the complex chemical world around them, for example in a foraging context, and provide a basis with which to investigate the neural mechanisms behind perceptual similarity. [ABSTRACT FROM AUTHOR]

Abstract: Task-partitioning is an important organisational principle in insect colonies and is thought to increase colony efficiency. In task-partitioning, tasks such as the collection of resources are divided into subtasks in which the material is passed from one worker to another. Previous models have assumed that worker–worker interactions are random, but experimental evidence suggests that receivers can have preferences to handle familiar materials. We used an agent-based simulation model to explore how non-random interactions during task-partitioning with direct transfer affect colony work efficiency. Because task-partitioning also allows receivers and donors to acquire foraging related information we analysed the effect of non-random interactions on informative interaction patterns. When receivers non-randomly rejected donors offering certain materials, donors overall experienced increased time delays, hive stay durations and a decreased number of transfer partners. However, the number of transfers was slightly increased, which can improve the acquisition and quality of information for donors. When receivers were non-randomly attracted to donors offering certain materials, donors experienced reduced transfer delays, hive stay durations and an increased number of simultaneous receivers. The number of transfers is slightly decreased. The effects of the two mechanisms “non-random rejection” and “non-random attraction” are biggest if the number of foragers and receivers is balanced. In summary, our results show that colony ergonomics are improved if receivers do not reject donors and if mechanisms exist that help receivers detect potential donors, such as learning the odour of the transferred food. Finally, our simulations suggest that non-random interactions can potentially affect the foraging patterns of colonies in changing environments. [Copyright &y& Elsevier]

Abstract: In the honeybee, we investigated the role of transmissions mediated by GABA-gated chloride channels and glutamate-gated chloride channels (GluCls) of the mushroom bodies (MBs) on olfactory learning using a single-trial olfactory conditioning paradigm. The GABAergic antagonist picrotoxin (PTX) or the GluCl antagonist l-trans-pyrrolidine-2,4-dicarboxylic acid (l-trans-PDC) was injected alone or in combination into the α-lobes of MBs. PTX impaired early long-term olfactory memory when injected before conditioning or before testing. l-trans-PDC alone induced no significant effect on learning and memory but induced a less specific response to the conditioned odor. When injected before PTX, l-trans-PDC was able to modulate PTX effects. These results emphasize the role of MB GABA-gated chloride channels in consolidation processes and strongly support that GluCls are involved in the perception of the conditioned stimulus. [Copyright &y& Elsevier]

Abstract: 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 50years 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. [Copyright &y& Elsevier]

Abstract: It has been largely assumed that the individual experience of disease vector insects may affect host choice and, as a consequence, have an important influence on parasite transmission. In particular, it is speculated that vector insects should be able to learn and remember the most and/or less defensive hosts, shifting their preference accordingly. Nevertheless, despite the invested efforts in testing the capacity to learn and remember information of blood-sucking insects, only little conclusive information has been obtained hitherto. Recently, the ability of Rhodnius prolixus to associate a behaviourally neutral odour to the perspective of either obtaining a blood-meal or being punished has been demonstrated, the same odour becoming attractant or repellent for the bugs, respectively, according to the individual previous experience. The present work represents a step forward in the study of the cognitive abilities of Chagas disease vectors and their influence on host choice. We tested whether or not bugs bias their choice for a host based on the association of its odour with a negative experience. Our results show that whereas naïve bugs presented no preference when confronted to the odour of two different hosts, bugs previously exposed to the contingency of the odour of one host and a mechanical perturbation mimicking defensive behaviour, biased their preference towards the other host. This constitutes the first evidence of olfactory conditioning to host odours in triatomine bugs, vectors of Chagas disease and one of the few available up to date on haematophagous insects. The epidemiological significance of this finding is discussed. [Copyright &y& Elsevier]

Honey bees (Apis melfifera) are well known for their excellent learning abilities. Although most age groups learn quickly to associate an odor with a sucrose reward, newly emerged bees and old foragers often perform poorly. For a long time, the reason for the poor learning performance of these age groups was unclear. We show that reduced sensitivity for sucrose is the cause for poor associative learning In newly emerged bees but not in old foragers. By increasing the sensitivity for sucrose through octopamine, we selectively improved the learning performance of insensitive newly emerged bees. Interestingly, the learning performance of foragers experiencing the same treatment remained low, despite the observed Increase In sensitivity for the reward. We thus demonstrate that increasing sensitivity for the reward can improve the associative learning performance of bees when they are young but not when they had foraged for a long time. Importantly, octopamine can have very different effects on bees, depending on their initial sensory sensitivity. These differential effects of octopamine have important consequences for interpreting the action of biogenic amines on Insect behavior. [ABSTRACT FROM AUTHOR]

Honeybees (Apis mellifera) senesce within 2 weeks after they discontinue nest tasks in favour of foraging. Foraging involves metabolically demanding flight, which in houseflies (Musca domestica) and fruit flies (Drosophila melanogastei) is associated with markers of ageing such as increased mortality and accumulation of oxidative damage. The role of flight in honeybee ageing is incompletely understood. We assessed relationships between honeybee flight activity and ageing by simulating rain that confined foragers to their colonies most of the day. After 15 days on average, flight-restricted foragers were compared with bees with normal (free) flight: one group that foraged for -l5days and two additional control groups, for flight duration and chronological age, that foraged for -5days. Free flight over l5days on average resulted in impaired associative learning ability. In contrast, flight-restricted foragers did as well in learning as bees that foraged for 5 days on average. This negative effect of flight activity was not influenced by chronological age or gustatory responsiveness, a measure of the bees' motivation to learn. Contrasting their intact learning ability, flight-restricted bees accrued the most oxidative brain damage as indicated by malondialdehyde protein adduct levels in crude cytosolic fractions. Concentrations of mono- and poly-ubiquitinated brain proteins were equal between the groups, whereas differences in total protein amounts suggested changes in brain protein metabolism connected to forager age, but not flight. We propose that intense flight is causal to brain deficits in aged bees, and that oxidative protein damage is unlikely to be the underlying mechanism. [ABSTRACT FROM AUTHOR]

Abstract: Defining learning at the molecular and physiological level has been one of the greatest challenges in biology. Recent research suggests that by studying fruit fly (Drosophila melanogaster) brain organization we can now begin to unravel some of these mysteries. The fruit fly brain is organized into executive centers that regulate anatomically separate behavioral systems. The mushroom body is an example of an executive center which is modified by olfactory conditioning. During this simple form of learning, an odor is paired with either food or shock. Either experience alters distinguishable specific circuitry within the mushroom body. Results suggest that after conditioning an odor to food, the mushroom body will activate a feeding system via a subset of its circuitry. After conditioning an odor to shock, the mushroom body will instead activate an avoidance system with other subsets of mushroom body neurons. The results of these experiments demonstrate a mechanism for flies to display anticipation of their environment after olfactory conditioning has occurred. However, these results fail to provide evidence for reinforcement, a consequence of action, as part of this mechanism. Instead, specific subsets of dopaminergic and octopaminergic neurons provide a simple pairing signal, in contrast to a reinforcement signal, which allows for prediction of the environment after experience. This view has implications for models of conditioning. [Copyright &y& Elsevier]

The brain mechanisms by which animals deal with multiple experiences to predict outcomes are not yet fully understood. We explored the choice strategies that flies use to assess degrees of disadvantage, as well as how flies weigh past and recent experiences to guide decisions. Drosophila were exposed to two conditioning events in a T-maze: an odor paired with an electric shock followed by a second odor paired with an electric shock of a different intensity. Subsequently, flies were forced to choose between the two odors. We found that flies chose to avoid the more ‘dangerous’ odor by a linear subtraction mechanism that was based on two coexisting memories. We also found that flies weighed experiences of the same danger level (60 V electric shocks) according to the times when the experiences had occurred. More recent experiences had a greater impact and past experiences gradually became ‘overlooked’ during decisions as the time delay between the two events lengthened. However, the past memory was not so much disrupted as it was overshadowed by recent memories during decisions. Finally, when a past experience was more disadvantageous, wild-type flies were able to coordinate both the temporal factor and the degree of disadvantage into their decisions. By contrast, amnesiac mutant flies made choices completely according to the temporal factor, ignoring the degree of disadvantage. Taken together, wild-type flies are able to store multiple olfactory memories and can coherently evaluate learned experiences to guide their decisions according to the degree of disadvantage and/or the temporal factor. [ABSTRACT FROM AUTHOR]