Plasticity in inhibitory networks improves pattern separation in early olfactory processing.

Distinguishing between nectar and non-nectar odors presents a challenge for animals due to shared compounds in complex mixtures, where changing ratios often signify differences in reward. Changes in nectar production throughout the day and potentially many times within a forager's lifetime add to the complexity. The honeybee olfactory system, containing less than a 1000 of principal neurons in the early olfactory relay, the antennal lobe (AL), must learn to associate diverse volatile blends with rewards. We used a computational network model and live imaging of the honeybee's AL to explore the neural mechanisms and functions of the AL plasticity. Our findings revealed that when trained with a set of rewarded and unrewarded odors, the AL inhibitory network suppresses shared chemical compounds while enhancing responses to distinct compounds. This results in improved pattern separation and a more concise and efficient neural code. Our Ca2+ imaging data support our model's predictions. Furthermore, we applied these contrast enhancement principles to a Graph Convolutional Network (GCN) and found that similar mechanisms could enhance the performance of artificial neural networks. Our model provides insights into how plasticity at the inhibitory network level reshapes coding for efficient learning of complex odors.

Experience-dependent tuning of the olfactory system.

Insects rely on their sense of smell to navigate complex environments and make decisions regarding food and reproduction. However, in natural settings, the odors that convey this information may come mixed with environmental odors that can obscure their perception. Therefore, recognizing the presence of informative odors involves generalization and discrimination processes, which can be facilitated when there is a high contrast between stimuli, or the internal representation of the odors of interest outcompetes that of concurrent ones. The first two layers of the olfactory system, which involve the detection of odorants by olfactory receptor neurons and their encoding by the first postsynaptic partners in the antennal lobe, are critical for achieving such optimal representation. In this review, we summarize evidence indicating that experience-dependent changes adjust these two levels of the olfactory system. These changes are discussed in the context of the advantages they provide for detection of informative odors.

Gain modulation and odor concentration invariance in early olfactory networks.

The broad receptive field of the olfactory receptors constitutes the basis of a combinatorial code that allows animals to detect and discriminate many more odorants than the actual number of receptor types that they express. One drawback is that high odor concentrations recruit lower affinity receptors which can lead to the perception of qualitatively different odors. Here we addressed the contribution that signal-processing in the antennal lobe makes to reduce concentration dependence in odor representation. By means of calcium imaging and pharmacological approach we describe the contribution that GABA receptors play in terms of the amplitude and temporal profiles of the signals that convey odor information from the antennal lobes to higher brain centers. We found that GABA reduces the amplitude of odor elicited signals and the number of glomeruli that are recruited in an odor-concentration-dependent manner. Blocking GABA receptors decreases the correlation among glomerular activity patterns elicited by different concentrations of the same odor. In addition, we built a realistic mathematical model of the antennal lobe that was used to test the viability of the proposed mechanisms and to evaluate the processing properties of the AL network under conditions that cannot be achieved in physiology experiments. Interestingly, even though based on a rather simple topology and cell interactions solely mediated by GABAergic lateral inhibitions, the AL model reproduced key features of the AL response upon different odor concentrations and provides plausible solutions for concentration invariant recognition of odors by artificial sensors.

Antennal movements can be used as behavioral readout of odor valence in honey bees.

The fact that honey bees have a relatively simple nervous system that allows complex behaviors has made them an outstanding model for studying neurobiological processes. Studies on learning and memory routinely use appetitive and aversive learning paradigms that involve recording of the proboscis or the sting extension. However, these protocols are based on all-or-none responses, which has the disadvantage of occluding intermediate and more elaborated behaviors. Nowadays, the great advances in tracking software and data analysis, combined with affordable video recording systems, have made it possible to extract very detailed information about animal behavior. Here we describe antennal movements that are elicited by odor that have no, positive or negative valence. We show that animals orient their antennae towards the source of the odor when it is positive, and orient them in the opposite direction when the odor is negative. Moreover, we found that this behavior was modified between animals that had been trained based on protocols of different strength. Since this procedure allows a more accurate description of the behavioral outcome using a relatively small number of animals, it represents a great tool for studying different cognitive processes and olfactory perception.

Honey bees can store and retrieve independent memory traces after complex experiences that combine appetitive and aversive associations.

Real-world experiences often mix appetitive and aversive events. Understanding the ability of animals to extract, store and use this information is an important issue in neurobiology. We used honey bees as model organism to study learning and memory after a differential conditioning paradigm that combines appetitive and aversive training trials. First, we used an aversive conditioning paradigm that constitutes a clear opposite of the well-known appetitive olfactory conditioning of the proboscis extension response. A neutral odour is presented paired with the bitter substance quinine. Aversive memory is evidenced later as an odour-specific impairment in appetitive conditioning. Then, we tested the effect of mixing appetitive and aversive conditioning trials distributed along the same training session. Differential conditioning protocols like this were used previously to study the ability to discriminate odours; however, they were not focused on whether appetitive and aversive memories are formed. We found that after differential conditioning, honey bees establish independent appetitive and aversive memories that do not interfere with each other during acquisition or storage. Finally, we moved the question forward to retrieval and memory expression to evaluate what happens when appetitive and the aversive learned odours are mixed during a test. Interestingly, opposite memories compete in such a way that they do not cancel each other out. Honey bees showed the ability to switch from expressing appetitive to aversive memory depending on their satiation level.

Learning-dependent plasticity in the antennal lobe improves discrimination and recognition of odors in the honeybee.

Honeybees are extensively used to study olfactory learning and memory processes thanks to their ability to discriminate and remember odors and because of their advantages for optophysiological recordings of the circuits involved in memory and odor perception. There are evidences that the encoding of odors in areas of primary sensory processing is not rigid, but undergoes changes caused by olfactory experience. The biological meaning of these changes is focus of intense discussions. Along this review, we present evidences of plasticity related to different forms of learning and discuss its function in the context of olfactory challenges that honeybees have to solve. So far, results in honeybees are consistent with a model in which changes in early olfactory processing contributes to the ability of an animal to recognize the presence of relevant odors and facilitates the discrimination of odors in a way adjusted to its own experience.

Experience-dependent tuning of early olfactory processing in the adult honey bee, Apis mellifera.

Experience-dependent plasticity in the central nervous system allows an animal to adapt its responses to stimuli over different time scales. In this study, we explored the impacts of adult foraging experience on early olfactory processing by comparing naturally foraging honey bees, Apis mellifera, with those that experienced a chronic reduction in adult foraging experience. We placed age-matched sets of sister honey bees into two different olfactory conditions, in which animals were allowed to forage ad libitum In one condition, we restricted foraging experience by placing honey bees in a tent in which both sucrose and pollen resources were associated with a single odor. In the second condition, honey bees were allowed to forage freely and therefore encounter a diversity of naturally occurring resource-associated olfactory experiences. We found that honey bees with restricted foraging experiences had altered antennal lobe development. We measured the glomerular responses to odors using calcium imaging in the antennal lobe, and found that natural olfactory experience also enhanced the inter-individual variation in glomerular response profiles to odors. Additionally, we found that honey bees with adult restricted foraging experience did not distinguish relevant components of an odor mixture in a behavioral assay as did their freely foraging siblings. This study highlights the impacts of individual experience on early olfactory processing at multiple levels.

Odorant mixtures elicit less variable and faster responses than pure odorants.

In natural environments, odors are typically mixtures of several different chemical compounds. However, the implications of mixtures for odor processing have not been fully investigated. We have extended a standard olfactory receptor model to mixtures and found through its mathematical analysis that odorant-evoked activity patterns are more stable across concentrations and first-spike latencies of receptor neurons are shorter for mixtures than for pure odorants. Shorter first-spike latencies arise from the nonlinear dependence of binding rate on odorant concentration, commonly described by the Hill coefficient, while the more stable activity patterns result from the competition between different ligands for receptor sites. These results are consistent with observations from numerical simulations and physiological recordings in the olfactory system of insects. Our results suggest that mixtures allow faster and more reliable olfactory coding, which could be one of the reasons why animals often use mixtures in chemical signaling.

Parallel memory traces are built after an experience containing aversive and appetitive components in the crab Neohelice.

The neurobiology of learning and memory has been mainly studied by focusing on pure aversive or appetitive experiences. Here, we challenged this approach considering that real-life stimuli come normally associated with competing aversive and appetitive consequences and that interaction between conflicting information must be intrinsic part of the memory processes. We used Neohelice crabs, taking advantage of two well-described appetitive and aversive learning paradigms and combining them in a single training session to evaluate how this affects memory. We found that crabs build separate appetitive and aversive memories that compete during retrieval but not during acquisition. Which memory prevails depends on the balance between the strength of the unconditioned stimuli and on the motivational state of the animals. The results indicate that after a mix experience with appetitive and aversive consequences, parallel memories are established in a way that appetitive and aversive information is stored to be retrieved in an opportunistic manner.

Context-dependent memory traces in the crab's mushroom bodies: Functional support for a common origin of high-order memory centers.

The hypothesis of a common origin for the high-order memory centers in bilateral animals is based on the evidence that several key features, including gene expression and neuronal network patterns, are shared across several phyla. Central to this hypothesis is the assumption that the arthropods' higher order neuropils of the forebrain [the mushroom bodies (MBs) of insects and the hemiellipsoid bodies (HBs) of crustaceans] are homologous structures. However, even though involvement in memory processes has been repeatedly demonstrated for the MBs, direct proof of such a role in HBs is lacking. Here, through neuroanatomical and immunohistochemical analysis, we identified, in the crab Neohelice granulata, HBs that resemble the calyxless MBs found in several insects. Using in vivo calcium imaging, we revealed training-dependent changes in neuronal responses of vertical and medial lobes of the HBs. These changes were stimulus-specific, and, like in the hippocampus and MBs, the changes reflected the context attribute of the memory trace, which has been envisioned as an essential feature for the HBs. The present study constitutes functional evidence in favor of a role for the HBs in memory processes, and provides key physiological evidence supporting a common origin of the arthropods' high-order memory centers.

Sickness Behavior in Honey Bees.

During an infection, animals suffer several changes in their normal physiology and behavior which may include lethargy, appetite loss, and reduction in grooming and general movements. This set of alterations is known as sickness behavior and although it has been extensively believed to be orchestrated primarily by the immune system, a relevant role for the central nervous system has also been established. The aim of the present work is to develop a simple animal model to allow studying how the immune and the nervous systems interact coordinately during an infection. We administered a bacterial lipopolysaccharide (LPS) into the thorax of honey bees to mimic a bacterial infection, and then we evaluated a set of stereotyped behaviors of the animals that might be indicative of sickness behavior. First, we show that this immune challenge reduces the locomotor activity of the animals in a narrow time window after LPS injection. Furthermore, bees exhibit a loss of appetite 60 and 90 min after injection, but not 15 h later. We also demonstrate that LPS injection reduces spontaneous antennal movements in harnessed animals, which suggests a reduction in the motivational state of the bees. Finally, we show that the LPS injection diminishes the interaction between animals, a crucial behavior in social insects. To our knowledge these results represent the first systematic description of sickness behavior in honey bees and provide important groundwork for the study of the interaction between the immune and the neural systems in an insect model.

Learning about natural variation of odor mixtures enhances categorization in early olfactory processing.

Natural odors are typically mixtures of several chemical components. Mixtures vary in composition among odor objects that have the same meaning. Therefore a central 'categorization' problem for an animal as it makes decisions about odors in natural contexts is to correctly identify odor variants that have the same meaning and avoid variants that have a different meaning. We propose that identified mechanisms of associative and non-associative plasticity in early sensory processing in the insect antennal lobe and mammalian olfactory bulb are central to solving this problem. Accordingly, this plasticity should work to improve categorization of odors that have the opposite meanings in relation to important events. Using synthetic mixtures designed to mimic natural odor variation among flowers, we studied how honey bees learn about and generalize among floral odors associated with food. We behaviorally conditioned honey bees on a difficult odor discrimination problem using synthetic mixtures that mimic natural variation among snapdragon flowers. We then used calcium imaging to measure responses of projection neurons of the antennal lobe, which is the first synaptic relay of olfactory sensory information in the brain, to study how ensembles of projection neurons change as a result of behavioral conditioning. We show how these ensembles become 'tuned' through plasticity to improve categorization of odors that have the different meanings. We argue that this tuning allows more efficient use of the immense coding space of the antennal lobe and olfactory bulb to solve the categorization problem. Our data point to the need for a better understanding of the 'statistics' of the odor space.

Learning modifies odor mixture processing to improve detection of relevant components.

Honey bees have a rich repertoire of olfactory learning behaviors, and they therefore are an excellent model to study plasticity in olfactory circuits. Recent behavioral, physiological, and molecular evidence suggested that the antennal lobe, the first relay of the olfactory system in insects and analog to the olfactory bulb in vertebrates, is involved in associative and nonassociative olfactory learning. Here we use calcium imaging to reveal how responses across antennal lobe projection neurons change after association of an input odor with appetitive reinforcement. After appetitive conditioning to 1-hexanol, the representation of an odor mixture containing 1-hexanol becomes more similar to this odor and less similar to the background odor acetophenone. We then apply computational modeling to investigate how changes in synaptic connectivity can account for the observed plasticity. Our study suggests that experience-dependent modulation of inhibitory interactions in the antennal lobe aids perception of salient odor components mixed with behaviorally irrelevant background odors.

Mosaic activity patterns and their relation to perceptual similarity: open discussions on the molecular basis and circuitry of odor recognition.

Enormous advances have been made in the recent years in regard to the mechanisms and neural circuits by which odors are sensed and perceived. Part of this understanding has been gained from parallel studies in insects and rodents that show striking similarity in the mechanisms they use to sense, encode, and perceive odors. In this review, we provide a short introduction to the functioning of olfactory systems from transduction of odorant stimuli into electrical signals in sensory neurons to the anatomical and functional organization of the networks involved in neural representation of odors in the central nervous system. We make emphasis on the functional and anatomical architecture of the first synaptic relay of the olfactory circuit, the olfactory bulb in vertebrates and the antennal lobe in insects. We discuss how the exquisite and conserved architecture of this structure is established and how different odors are encoded in mosaic activity patterns. Finally, we discuss the validity of methods used to compare activation patterns in relation to perceptual similarity.

Apis mellifera octopamine receptor 1 (AmOA1) expression in antennal lobe networks of the honey bee (Apis mellifera) and fruit fly (Drosophila melanogaster).

Octopamine (OA) underlies reinforcement during appetitive conditioning in the honey bee and fruit fly, acting via different subtypes of receptors. Recently, antibodies raised against a peptide sequence of one honey bee OA receptor, AmOA1, were used to study the distribution of these receptors in the honey bee brain (Sinakevitch et al., 2011). These antibodies also recognize an isoform of the AmOA1 ortholog in the fruit fly (OAMB, mushroom body OA receptor). Here we describe in detail the distribution of AmOA1 receptors in different types of neurons in the honey bee and fruit fly antennal lobes. We integrate this information into a detailed anatomical analysis of olfactory receptor neurons (ORNs), uni- and multi-glomerular projection neurons (uPNs, and mPNs) and local interneurons (LNs) in glomeruli of the antennal lobe. These neurons were revealed by dye injection into the antennal nerve, antennal lobe, medial and lateral antenno-protocerbral tracts (m-APT and l-APT), and lateral protocerebral lobe (LPL) by use of labeled cell lines in the fruit fly or by staining with anti-GABA. We found that ORN receptor terminals and uPNs largely do not show immunostaining for AmOA1. About seventeen GABAergic mPNs leave the antennal lobe through the ml-APT and branch into the LPL. Many, but not all, mPNs show staining for AmOA1. AmOA1 receptors are also in glomeruli on GABAergic processes associated with LNs. The data suggest that in both species one important action of OA in the antennal lobe involves modulation of different types of inhibitory neurons via AmOA1 receptors. We integrated this new information into a model of circuitry within glomeruli of the antennal lobes of these species.

Dopamine interferes with appetitive long-term memory formation in honey bees.

Studies in vertebrates and invertebrates have proved the instructive role that different biogenic amines play in the neural representation of rewards and punishments during associative learning. Results from diverse arthropods and using different learning paradigms initially agreed that dopamine (DA) is needed for aversive learning and octopamine (OA) is needed for appetitive learning. However, the notion that both amines constitute separate pathways for appetitive and aversive learning is changing. Here, we asked whether DA, so far only involved in aversive memory formation in honey bees, does also modulate appetitive memory. Using the well characterized appetitive olfactory conditioning of the proboscis extension reflex (PER), we show that DA impairs appetitive memory consolidation. In addition, we found that blocking DA receptors enhances appetitive memory. These results are consistent with the view that aversive and appetitive components interact during learning and memory formation to ensure adaptive behavior.

Nonassociative plasticity alters competitive interactions among mixture components in early olfactory processing.

Experience-related plasticity is an essential component of networks involved in early olfactory processing. However, the mechanisms and functions of plasticity in these neural networks are not well understood. We studied nonassociative plasticity by evaluating responses to two pure odors (A and X) and their binary mixture using calcium imaging of odor-elicited activity in output neurons of the honey bee antennal lobe. Unreinforced exposure to A or X produced no change in the neural response elicited by the pure odors. However, exposure to one odor (e.g. A) caused the response to the mixture to become more similar to that of the other component (X). We also show in behavioral analyses that unreinforced exposure to A caused the mixture to become perceptually more similar to X. These results suggest that nonassociative plasticity modifies neural networks in such a way that it affects local competitive interactions among mixture components. We used a computational model to evaluate the most likely targets for modification. Hebbian modification of synapses from inhibitory local interneurons to projection neurons most reliably produced the observed shift in response to the mixture. These results are consistent with a model in which the antennal lobe acts to filter olfactory information according to its relevance for performing a particular task.

Opposite actions of dopamine on aversive and appetitive memories in the crab.

The understanding of how the reinforcement is represented in the central nervous system during memory formation is a current issue in neurobiology. Several studies in insects provide evidence of the instructive role of biogenic amines during the learning and memory process. In insects it was widely accepted that dopamine (DA) mediates aversive reinforcements. However, the idea of DA being exclusively involved in aversive memory has been challenged in recent studies. Here, we study the involvement of DA during aversive and appetitive memories in the crab Chasmagnathus. We found that DA-receptor antagonists impair aversive memory consolidation, in agreement with previous reports in insects, while administration of DA facilitates memory formation after a weak training protocol. In contrast, DA treatment during appetitive training was found to impair formation of long-term appetitive memory. In addition, as a first step in elucidating the neuroanatomical correlates of DA action on memory, we mapped dopaminergic neurons in the central nervous system of the crab. Results of the current study, together with those obtained in a previous work about the role of octopamine (OA), suggest that both amines (DA and OA) play a dual action in memory processes. On the one hand, DA and OA mediate the aversive and the appetitive signals, respectively, throughout training, while on the other hand, they interfere with the formation of memory of the opposite sign (DA in appetitive and OA in aversive). Our results support a new understanding about the way appetitive and aversive stimuli are processed during memory formation to ensure adaptive behavior.

Associative conditioning tunes transient dynamics of early olfactory processing.

Odors evoke complex spatiotemporal responses in the insect antennal lobe (AL) and mammalian olfactory bulb. However, the behavioral relevance of spatiotemporal coding remains unclear. In the present work we combined behavioral analyses with calcium imaging of odor induced activity in the honeybee AL to evaluate the relevance of this temporal dimension in the olfactory code. We used a new way for evaluation of odor similarity of binary mixtures in behavioral studies, which involved testing whether a match of odor-sampling time is necessary between training and testing conditions for odor recognition during associative learning. Using graded changes in the similarity of the mixture ratios, we found high correlations between the behavioral generalization across those mixtures and a gradient of activation in AL output. Furthermore, short odor stimuli of 500 ms or less affected how well odors were matched with a memory template, and this time corresponded to a shift from a sampling-time-dependent to a sampling-time-independent memory. Accordingly, 375 ms corresponded to the time required for spatiotemporal AL activity patterns to reach maximal separation according to imaging studies. Finally, we compared spatiotemporal representations of binary mixtures in trained and untrained animals. AL activity was modified by conditioning to improve separation of odor representations. These data suggest that one role of reinforcement is to "tune" the AL such that relevant odors become more discriminable.