Sparse and stereotyped encoding implicates a core glomerulus for ant alarm behavior.

Ants communicate via large arrays of pheromones and possess expanded, highly complex olfactory systems, with antennal lobes in the brain comprising up to ∼500 glomeruli. This expansion implies that odors could activate hundreds of glomeruli, which would pose challenges for higher-order processing. To study this problem, we generated transgenic ants expressing the genetically encoded calcium indicator GCaMP in olfactory sensory neurons. Using two-photon imaging, we mapped complete glomerular responses to four ant alarm pheromones. Alarm pheromones robustly activated ≤6 glomeruli, and activity maps for the three pheromones inducing panic alarm in our study species converged on a single glomerulus. These results demonstrate that, rather than using broadly tuned combinatorial encoding, ants employ precise, narrowly tuned, and stereotyped representations of alarm pheromones. The identification of a central sensory hub glomerulus for alarm behavior suggests that a simple neural architecture is sufficient to translate pheromone perception into behavioral outputs.

Non-canonical odor coding in the mosquito.

Aedes aegypti mosquitoes are a persistent human foe, transmitting arboviruses including dengue when they feed on human blood. Mosquitoes are intensely attracted to body odor and carbon dioxide, which they detect using ionotropic chemosensory receptors encoded by three large multi-gene families. Genetic mutations that disrupt the olfactory system have modest effects on human attraction, suggesting redundancy in odor coding. The canonical view is that olfactory sensory neurons each express a single chemosensory receptor that defines its ligand selectivity. We discovered that Ae. aegypti uses a different organizational principle, with many neurons co-expressing multiple chemosensory receptor genes. In vivo electrophysiology demonstrates that the broad ligand-sensitivity of mosquito olfactory neurons depends on this non-canonical co-expression. The redundancy afforded by an olfactory system in which neurons co-express multiple chemosensory receptors may increase the robustness of the mosquito olfactory system and explain our long-standing inability to disrupt the detection of humans by mosquitoes.

A transcriptional rheostat couples past activity to future sensory responses.

Animals traversing different environments encounter both stable background stimuli and novel cues, which are thought to be detected by primary sensory neurons and then distinguished by downstream brain circuits. Here, we show that each of the ∼1,000 olfactory sensory neuron (OSN) subtypes in the mouse harbors a distinct transcriptome whose content is precisely determined by interactions between its odorant receptor and the environment. This transcriptional variation is systematically organized to support sensory adaptation: expression levels of more than 70 genes relevant to transforming odors into spikes continuously vary across OSN subtypes, dynamically adjust to new environments over hours, and accurately predict acute OSN-specific odor responses. The sensory periphery therefore separates salient signals from predictable background via a transcriptional rheostat whose moment-to-moment state reflects the past and constrains the future; these findings suggest a general model in which structured transcriptional variation within a cell type reflects individual experience.

Olfactory sensitivity differentiates morphologically distinct worker castes in Camponotus floridanus.

BACKGROUND: Camponotus floridanus ant colonies are comprised of a single reproductive queen and thousands of sterile female offspring that consist of two morphologically distinct castes: smaller minors and larger majors. Minors perform most of the tasks within the colony, including brood care and food collection, whereas majors have fewer clear roles and have been hypothesized to act as a specialized solider caste associated with colony defense. The allocation of workers to these different tasks depends, in part, on the detection and processing of local information including pheromones and other chemical blends such as cuticular hydrocarbons. However, the role peripheral olfactory sensitivity plays in establishing and maintaining morphologically distinct worker castes and their associated behaviors remains largely unexplored. RESULTS: We examined the electrophysiological responses to general odorants, cuticular extracts, and a trail pheromone in adult minor and major C. floridanus workers, revealing that the repertoire of social behaviors is positively correlated with olfactory sensitivity. Minors in particular display primarily excitatory responses to olfactory stimuli, whereas major workers primarily manifest suppressed, sub-solvent responses. The notable exception to this paradigm is that both minors and majors display robust, dose-dependent excitatory responses to conspecific, non-nestmate cuticular extracts. Moreover, while both minors and majors actively aggress non-nestmate foes, the larger and physiologically distinct majors display significantly enhanced capabilities to rapidly subdue and kill their adversaries. CONCLUSIONS: Our studies reveal the behavioral repertoire of minors and majors aligns with profound shifts in peripheral olfactory sensitivity and odor coding. The data reported here support the hypothesis that minors are multipotential workers with broad excitatory sensitivity, and majors are dedicated soldiers with a highly specialized olfactory system for distinguishing non-nestmate foes. Overall, we conclude that C. floridanus majors do indeed represent a physiologically and behaviorally specialized soldier caste in which caste-specific olfactory sensitivity plays an important role in task allocation and the regulation of social behavior in ant colonies.

Odor coding in the mammalian olfactory epithelium.

Noses are extremely sophisticated chemical detectors allowing animals to use scents to interpret and navigate their environments. Odor detection starts with the activation of odorant receptors (ORs), expressed in mature olfactory sensory neurons (OSNs) populating the olfactory mucosa. Different odorants, or different concentrations of the same odorant, activate unique ensembles of ORs. This mechanism of combinatorial receptor coding provided a possible explanation as to why different odorants are perceived as having distinct odors. Aided by new technologies, several recent studies have found that antagonist interactions also play an important role in the formation of the combinatorial receptor code. These findings mark the start of a new era in the study of odorant-receptor interactions and add a new level of complexity to odor coding in mammals.

Conformational Sensing by a Mammalian Olfactory Receptor.

To identify odors, the mammalian nose deploys hundreds of olfactory receptors (ORs) from the rhodopsin-like class of the G protein-coupled receptor superfamily. Odorants having multiple rotatable bonds present a problem for the stereochemical shape-based matching process assumed to govern the sense of smell through OR-odorant recognition. We conformationally restricted the carbon chain of the odorant octanal to ask whether an OR can respond differently to different odorant conformations. By using calcium imaging to monitor signal transduction in sensory neurons expressing the mouse aldehyde OR, Olfr2, we found that the spatial position of the C7 and C8 carbon atoms of octanal, in relation to its -CHO group, determines whether an aliphatic aldehyde functions as an agonist, partial agonist or antagonist. Our experiments provide evidence that an odorant can manipulate an OR through its intrinsic conformational repertoire, in unexpected analogy to the photon-controlled aldehyde manipulation observed in rhodopsin.

Functional characterization of odorant receptors in the ponerine ant, Harpegnathos saltator.

Animals use a variety of sensory modalities-including visual, acoustic, and chemical-to sense their environment and interact with both conspecifics and other species. Such communication is especially critical in eusocial insects such as honey bees and ants, where cooperation is critical for survival and reproductive success. Various classes of chemoreceptors have been hypothesized to play essential roles in the origin and evolution of eusociality in ants, through their functional roles in pheromone detection that characterizes reproductive status and colony membership. To better understand the molecular mechanisms by which chemoreceptors regulate social behaviors, we investigated the roles of a critical class of chemoreceptors, the odorant receptors (ORs), from the ponerine ant Harpegnathos saltator in detecting cuticular hydrocarbon pheromones. In light of the massive OR expansion in ants (∼400 genes per species), a representative survey based on phylogenetic and transcriptomic criteria was carried out across discrete odorant receptor subfamilies. Responses to several classes of semiochemicals are described, including cuticular hydrocarbons and mandibular gland components that act as H. saltator pheromones, and a range of more traditional general odorants. When viewed through the prism of caste-specific OR enrichment and distinctive OR subfamily odorant response profiles, our findings suggest that whereas individual HsOrs appear to be narrowly tuned, there is no apparent segregation of tuning responses within any discrete HsOr subfamily. Instead, the HsOR gene family as a whole responds to a broad array of compounds, including both cuticular hydrocarbons and general odorants that are likely to mediate distinct behaviors.

Neuronal Response Latencies Encode First Odor Identity Information across Subjects.

Odorants are coded in the primary olfactory processing centers by spatially and temporally distributed patterns of glomerular activity. Whereas the spatial distribution of odorant-induced responses is known to be conserved across individuals, the universality of its temporal structure is still debated. Via fast two-photon calcium imaging, we analyzed the early phase of neuronal responses in the form of the activity onset latencies in the antennal lobe projection neurons of honeybee foragers. We show that each odorant evokes a stimulus-specific response latency pattern across the glomerular coding space. Moreover, we investigate these early response features for the first time across animals, revealing that the order of glomerular firing onsets is conserved across individuals and allows them to reliably predict odorant identity, but not concentration. These results suggest that the neuronal response latencies provide the first available code for fast odor identification.SIGNIFICANCE STATEMENT Here, we studied early temporal coding in the primary olfactory processing centers of the honeybee brain by fast imaging of glomerular responses to different odorants across glomeruli and across individuals. Regarding the elusive role of rapid response dynamics in olfactory coding, we were able to clarify the following aspects: (1) the rank of glomerular activation is conserved across individuals, (2) its stimulus prediction accuracy is equal to that of the response amplitude code, and (3) it contains complementary information. Our findings suggest a substantial role of response latencies in odor identification, anticipating the static response amplitude code.

Antennal Olfactory Physiology and Behavior of Males of the Ponerine Ant Harpegnathos saltator.

In comparison to the large amount of study on the communication abilities of females in ant societies and their associated chemical ecology and sensory physiology, such study of male ants has been largely ignored; accordingly, little is known about their olfactory sensory capabilities. To address this, we explored peripheral odor sensitivities in male Harpegnathos saltator by measuring the electrophysiological activity of olfactory sensory neurons within antennal trichoid and coeloconic sensilla using an extracellular recording technique. In an initial trial of 46 compounds, sensilla trichodea responded strongly to two alarm pheromone components, while a limited number of non-hydrocarbon odorants elicited strong responses in sensilla coeloconica. Both sensillar types responded indifferently to 31 cuticular hydrocarbons (CHCs) and synthetic long-chain hydrocarbons (HCs) typically found on insect cuticle. In a search for sensilla responding to CHCs and other compounds, we found some sensilla that responded to synthetic HCs and CHCs from virgin queen postpharyngeal glands that are potentially used in close range mate recognition. Olfactometer bioassays of male ants to 15 non-HCs correlated sensory responsiveness to the respective behavioral responses. Comparing olfactory responses between H. saltator males and females, we found that sensilla coeloconica and basiconica of workers showed greater responses and broader selectivity to all compounds. The rarity of CHC-responding trichoid sensilla in Harpegnathos males suggests a more specific role in sexual communication compared to that in females, which use CHCs in a broader communication context.

Synaptic clusters function as odor operators in the olfactory bulb.

How the olfactory bulb organizes and processes odor inputs through fundamental operations of its microcircuits is largely unknown. To gain new insight we focus on odor-activated synaptic clusters related to individual glomeruli, which we call glomerular units. Using a 3D model of mitral and granule cell interactions supported by experimental findings, combined with a matrix-based representation of glomerular operations, we identify the mechanisms for forming one or more glomerular units in response to a given odor, how and to what extent the glomerular units interfere or interact with each other during learning, their computational role within the olfactory bulb microcircuit, and how their actions can be formalized into a theoretical framework in which the olfactory bulb can be considered to contain "odor operators" unique to each individual. The results provide new and specific theoretical and experimentally testable predictions.

Cholinergic inputs from Basal forebrain add an excitatory bias to odor coding in the olfactory bulb.

Cholinergic modulation of central circuits is associated with active sensation, attention, and learning, yet the neural circuits and temporal dynamics underlying cholinergic effects on sensory processing remain unclear. Understanding the effects of cholinergic modulation on particular circuits is complicated by the widespread projections of cholinergic neurons to telencephalic structures that themselves are highly interconnected. Here we examined how cholinergic projections from basal forebrain to the olfactory bulb (OB) modulate output from the first stage of sensory processing in the mouse olfactory system. By optogenetically activating their axons directly in the OB, we found that cholinergic projections from basal forebrain regulate OB output by increasing the spike output of presumptive mitral/tufted cells. Cholinergic stimulation increased mitral/tufted cell spiking in the absence of inhalation-driven sensory input and further increased spiking responses to inhalation of odorless air and to odorants. This modulation was rapid and transient, was dependent on local cholinergic signaling in the OB, and differed from modulation by optogenetic activation of cholinergic neurons in basal forebrain, which led to a mixture of mitral/tufted cell excitation and suppression. Finally, bulbar cholinergic enhancement of mitral/tufted cell odorant responses was robust and occurred independent of the strength or even polarity of the odorant-evoked response, indicating that cholinergic modulation adds an excitatory bias to mitral/tufted cells as opposed to increasing response gain or sharpening response spectra. These results are consistent with a role for the basal forebrain cholinergic system in dynamically regulating the sensitivity to or salience of odors during active sensing of the olfactory environment.

Sparse coding and lateral inhibition arising from balanced and unbalanced dendrodendritic excitation and inhibition.

The precise mechanism by which synaptic excitation and inhibition interact with each other in odor coding through the unique dendrodendritic synaptic microcircuits present in olfactory bulb is unknown. Here a scaled-up model of the mitral-granule cell network in the rodent olfactory bulb is used to analyze dendrodendritic processing of experimentally determined odor patterns. We found that the interaction between excitation and inhibition is responsible for two fundamental computational mechanisms: (1) a balanced excitation/inhibition in strongly activated mitral cells, leading to a sparse representation of odorant input, and (2) an unbalanced excitation/inhibition (inhibition dominated) in surrounding weakly activated mitral cells, leading to lateral inhibition. These results suggest how both mechanisms can carry information about the input patterns, with optimal level of synaptic excitation and inhibition producing the highest level of sparseness and decorrelation in the network response. The results suggest how the learning process, through the emergent development of these mechanisms, can enhance odor representation of olfactory bulb.

An arterially perfused nose-olfactory bulb preparation of the rat.

A main feature of the mammalian olfactory bulb network is the presence of various rhythmic activities, in particular, gamma, beta, and theta oscillations, with the latter coupled to the respiratory rhythm. Interactions between those oscillations as well as the spatial distribution of network activation are likely to determine olfactory coding. Here, we describe a novel semi-intact perfused nose-olfactory bulb-brain stem preparation in rats with both a preserved olfactory epithelium and brain stem, which could be particularly suitable for the study of oscillatory activity and spatial odor mapping within the olfactory bulb, in particular, in hitherto inaccessible locations. In the perfused olfactory bulb, we observed robust spontaneous oscillations, mostly in the theta range. Odor application resulted in an increase in oscillatory power in higher frequency ranges, stimulus-locked local field potentials, and excitation or inhibition of individual bulbar neurons, similar to odor responses reported from in vivo recordings. Thus our method constitutes the first viable in situ preparation of a mammalian system that uses airborne odor stimuli and preserves these characteristic features of odor processing. This preparation will allow the use of highly invasive experimental procedures and the application of techniques such as patch-clamp recording, high-resolution imaging, and optogenetics within the entire olfactory bulb.

Physiological organization and topographic mapping of the antennal olfactory sensory neurons in female hawkmoths, Manduca sexta.

The hawkmoth, Manduca sexta, has been a keystone system for developmental, neurobiological, and ecological studies for several decades. Because many of its behaviors are driven by olfactory cues, a thorough understanding of the Manduca olfactory system is essential to studying its biology. With the aim of functionally characterizing single antennal olfactory sensory neurons (OSNs) and determining their detailed topographic location, we performed systematic single-sensillum recordings on 4 morphological types of olfactory sensilla: trichoid-A and -B and basiconic-A and -B. We were able to unambiguously differentiate the colocalized cells associated with single sensilla based on their spike amplitudes. Using a panel of 61 biologically relevant compounds established in behavioral and gas chromatography-electrophysiology experiments, we made 223 recordings from these sensilla. Based on the response spectra of 187 responding OSNs, the sensilla fell into 12 distinct functional classes encompassing 29 OSNs. Selectivity of the 25 responding OSNs varied from narrowly tuned (responding to only one or a subset of compounds), to very broadly tuned (responding to multiple compounds), in a concentration-dependent manner. Four OSNs, however, did not respond to the tested components. Topographic mapping of the sensilla revealed that some physiological sensillum types are confined to particular locations on the antennal surface while other classes are more or less irregularly scattered all over the antennal annuli. Such information will prove beneficial for future receptor deorphanization, in situ hybridization, and molecular manipulation experiments.

Pheromone representation in the ant antennal lobe changes with age.

While the neural basis of age-related decline has been extensively studied,1,2,3 less is known about changes in neural function during the pre-senescent stages of adulthood. Adult neural plasticity is likely a key factor in social insect age polyethism, where individuals perform different tasks as they age and divide labor in an age-dependent manner.4,5,6,7,8,9 Primarily, workers transition from nursing to foraging tasks,5,10 become more aggressive, and more readily display alarm behavior11,12,13,14,15,16 as they get older. While it is unknown how these behavioral dynamics are neurally regulated, they could partially be generated by altered salience of behaviorally relevant stimuli.4,6,7 Here, we investigated how odor coding in the antennal lobe (AL) changes with age in the context of alarm pheromone communication in the clonal raider ant (Ooceraea biroi).17 Similar to other social insects,11,12,16 older ants responded more rapidly to alarm pheromones, the chemical signals for danger. Using whole-AL calcium imaging,18 we then mapped odor representations for five general odorants and two alarm pheromones in young and old ants. Alarm pheromones were represented sparsely at all ages. However, alarm pheromone responses within individual glomeruli changed with age, either increasing or decreasing. Only two glomeruli became sensitized to alarm pheromones with age, while at the same time becoming desensitized to general odorants. Our results suggest that the heightened response to alarm pheromones in older ants occurs via increased sensitivity in these two core glomeruli, illustrating the importance of sensory modulation in social insect division of labor and age-associated behavioral plasticity.

Pheromone representation in the ant antennal lobe changes with age.

While the neural basis of age-related decline has been extensively studied (1-3), less is known about changes in neural function during the pre-senescent stages of adulthood. Adult neural plasticity is likely a key factor in social insect age polyethism, where individuals perform different tasks as they age and divide labor in an age-dependent manner (4-9). Primarily, workers transition from nursing to foraging tasks (5, 10), become more aggressive, and more readily display alarm behavior (11-16) as they get older. While it is unknown how these behavioral dynamics are neurally regulated, they could partially be generated by altered salience of behaviorally relevant stimuli (4, 6, 7). Here, we investigated how odor coding in the antennal lobe (AL) changes with age in the context of alarm pheromone communication in the clonal raider ant (Ooceraea biroi) (17). Similar to other social insects (11, 12, 16), older ants responded more rapidly to alarm pheromones, the chemical signals for danger. Using whole-AL calcium imaging (18), we then mapped odor representations for five general odorants and two alarm pheromones in young and old ants. Alarm pheromones were represented sparsely at all ages. However, alarm pheromone responses within individual glomeruli changed with age, either increasing or decreasing. Only two glomeruli became sensitized to alarm pheromones with age, while at the same time becoming desensitized to general odorants. Our results suggest that the heightened response to alarm pheromones in older ants occurs via increased sensitivity in these two core glomeruli, illustrating the importance of sensory modulation in social insect division of labor and age-associated behavioral plasticity.

Presynaptic inhibition of olfactory sensory neurons: new mechanisms and potential functions.

Presynaptic inhibition is the suppression of neurotransmitter release from a neuron by inhibitory input onto its presynaptic terminal. In the olfactory system, the primary sensory afferents from the olfactory neuroepithelium to the brain's olfactory bulb are strongly modulated by a presynaptic inhibition that has been studied extensively in brain slices and in vivo. In rodents, this inhibition is mediated by γ-amino butyric acid (GABA) and dopamine released from bulbar interneurons. The specialized GABAergic circuit is now well understood to include a specific subset of GAD65-expressing periglomerular interneurons that stimulate presynaptic GABAB receptors to reduce presynaptic calcium conductance. This inhibition is organized to permit the selective modulation of neurotransmitter release from specific populations of olfactory sensory neurons based on their odorant receptor expression, includes specialized microcircuits to create a tonically active inhibition and a separate feedback inhibition evoked by sensory input, and can be modulated by centrifugal projections from other brain regions. Olfactory nerve output can also be modulated by dopaminergic circuitry, but this literature is more difficult to interpret. Presynaptic inhibition of olfactory afferents may extend their dynamic range but could also create state-dependent or odorant-specific sensory filters on primary sensory representations. New directions exploring this circuit's role in olfactory processing are discussed.

Age and Task Modulate Olfactory Sensitivity in the Florida Carpenter Ant Camponotus floridanus.

Age-related changes in behavior and sensory perception have been observed in a wide variety of animal species. In ants and other eusocial insects, workers often progress through an ordered sequence of olfactory-driven behavioral tasks. Notably, these behaviors are plastic, and workers adapt and rapidly switch tasks in response to changing environmental conditions. In the Florida carpenter ant, smaller minors typically perform most of the work needed to maintain the colony, while the larger majors are specialized for nest defense and rarely engage in these routine tasks. Here, we investigate the effects of age and task group on olfactory responses to a series of odorant blends in minor and major worker castes. Consistent with their respective roles within the colony, we observed significant age-associated shifts in the olfactory responses of minors as they transitioned between behavioral states, whereas the responses of majors remained consistently low regardless of age. Furthermore, we have identified a unitary compound, 3-methylindole, which elicited significantly higher responses and behavioral aversion in minor nurses than in similarly aged foragers suggesting that this compound may play an important role in brood care. Taken together, our results suggest that age- and task-associated shifts in olfactory physiology may play a critical role in the social organization of ant colonies.

Recruitment of grid-like responses in human entorhinal and piriform cortices by odor landmark-based navigation.

Olfactory navigation is universal across the animal kingdom. Humans, however, have rarely been considered in this context. Here, we combined olfactometry techniques, virtual reality (VR) software, and neuroimaging methods to investigate whether humans can navigate an olfactory landscape by learning the spatial relationships among discrete odor cues and integrating this knowledge into a spatial map. Our data show that over time, participants improved their performance on the odor navigation task by taking more direct paths toward targets and completing more trials within a given time period. This suggests that humans can successfully navigate a complex odorous environment, reinforcing the notion of human olfactory navigation. fMRI data collected during the olfactory navigation task revealed the emergence of grid-like responses in entorhinal and piriform cortices that were attuned to the same grid orientation. This result implies the existence of a specialized olfactory grid network tasked with guiding spatial navigation based on odor landmarks.

Higher-order olfactory neurons in the lateral horn support odor valence and odor identity coding in Drosophila.

Understanding neuronal representations of odor-evoked activities and their progressive transformation from the sensory level to higher brain centers features one of the major aims in olfactory neuroscience. Here, we investigated how odor information is transformed and represented in higher-order neurons of the lateral horn, one of the higher olfactory centers implicated in determining innate behavior, using Drosophila melanogaster. We focused on a subset of third-order glutamatergic lateral horn neurons (LHNs) and characterized their odor coding properties in relation to their presynaptic partner neurons, the projection neurons (PNs) by two-photon functional imaging. We show that odors evoke reproducible, stereotypic, and odor-specific response patterns in LHNs. Notably, odor-evoked responses in these neurons are valence-specific in a way that their response amplitude is positively correlated with innate odor preferences. We postulate that this valence-specific activity is the result of integrating inputs from multiple olfactory channels through second-order neurons. GRASP and micro-lesioning experiments provide evidence that glutamatergic LHNs obtain their major excitatory input from uniglomerular PNs, while they receive an odor-specific inhibition through inhibitory multiglomerular PNs. In summary, our study indicates that odor representations in glutamatergic LHNs encode hedonic valence and odor identity and primarily retain the odor coding properties of second-order neurons.