Stimulus duration encoding occurs early in the moth olfactory pathway.

Pheromones convey rich ethological information and guide insects' search behavior. Insects navigating in turbulent environments are tasked with the challenge of coding the temporal structure of an odor plume, obliging recognition of the onset and offset of whiffs of odor. The coding mechanisms that shape odor offset recognition remain elusive. We designed a device to deliver sharp pheromone pulses and simultaneously measured the response dynamics from pheromone-tuned olfactory receptor neurons (ORNs) in male moths and Drosophila. We show that concentration-invariant stimulus duration encoding is implemented in moth ORNs by spike frequency adaptation at two time scales. A linear-nonlinear model fully captures the underlying neural computations and offers an insight into their biophysical mechanisms. Drosophila use pheromone cis-vaccenyl acetate (cVA) only for very short distance communication and are not faced with the need to encode the statistics of the cVA plume. Their cVA-sensitive ORNs are indeed unable to encode odor-off events. Expression of moth pheromone receptors in Drosophila cVA-sensitive ORNs indicates that stimulus-offset coding is receptor independent. In moth ORNs, stimulus-offset coding breaks down for short ( < 200 ms) whiffs. This physiological constraint matches the behavioral latency of switching from the upwind surge to crosswind cast flight upon losing contact with the pheromone.

Olfactory cilia, regulation and control of olfaction.

The sense of smell is still considered a fuzzy sensation. Softly wafting aromas can stimulate the appetite and trigger memories; however, there are many unexplored aspects of its underlying mechanisms, and not all of these have been elucidated. Although the final sense of smell takes place in the brain, it is greatly affected during the preliminary stage, when odorants are converted into electrical signals. After signal conversion through ion channels in olfactory cilia, action potentials are generated through other types of ion channels located in the cell body. Spike trains through axons transmit this information as digital signals to the brain, however, before odorants are converted into digital electric signals, such as an action potential, modification of the transduction signal has already occurred. This review focuses on the early stages of olfactory signaling. Modification of signal transduction mechanisms and their effect on the human sense of smell through three characteristics (signal amplification, olfactory adaptation, and olfactory masking) produced by olfactory cilia, which is the site of signal transduction are being addressed in this review.

Mechanism of sensory perception unveiled by simultaneous measurement of membrane voltage and intracellular calcium.

Measuring neuronal activity is important for understanding neuronal function. Ca2+ imaging by genetically encoded calcium indicators (GECIs) is a powerful way to measure neuronal activity. Although it revealed important aspects of neuronal function, measuring the neuronal membrane voltage is important to understand neuronal function as it triggers neuronal activation. Recent progress of genetically encoded voltage indicators (GEVIs) enabled us fast and precise measurements of neuronal membrane voltage. To clarify the relation of the membrane voltage and intracellular Ca2+, we analyzed neuronal activities of olfactory neuron AWA in Caenorhabditis elegans by GCaMP6f (GECI) and paQuasAr3 (GEVI) responding to odorants. We found that the membrane voltage encodes the stimuli change by the timing and the duration by the weak semi-stable depolarization. However, the change of the intracellular Ca2+ encodes the strength of the stimuli. Furthermore, ODR-3, a G-protein alpha subunit, was shown to be important for stabilizing the membrane voltage. These results suggest that the combination of calcium and voltage imaging provides a deeper understanding of the information in neural circuits.

The primacy model and the structure of olfactory space.

Understanding sensory processing involves relating the stimulus space, its neural representation, and perceptual quality. In olfaction, the difficulty in establishing these links lies partly in the complexity of the underlying odor input space and perceptual responses. Based on the recently proposed primacy model for concentration invariant odor identity representation and a few assumptions, we have developed a theoretical framework for mapping the odor input space to the response properties of olfactory receptors. We analyze a geometrical structure containing odor representations in a multidimensional space of receptor affinities and describe its low-dimensional implementation, the primacy hull. We propose the implications of the primacy hull for the structure of feedforward connectivity in early olfactory networks. We test the predictions of our theory by comparing the existing receptor-ligand affinity and connectivity data obtained in the fruit fly olfactory system. We find that the Kenyon cells of the insect mushroom body integrate inputs from the high-affinity (primacy) sets of olfactory receptors in agreement with the primacy theory.

Dense and Persistent Odor Representations in the Olfactory Bulb of Awake Mice.

Recording and analysis of neural activity are often biased toward detecting sparse subsets of highly active neurons, masking important signals carried in low-magnitude and variable responses. To investigate the contribution of seemingly noisy activity to odor encoding, we used mesoscale calcium imaging from mice of both sexes to record odor responses from the dorsal surface of bilateral olfactory bulbs (OBs). The outer layer of the mouse OB is comprised of dendrites organized into discrete "glomeruli," which are defined by odor receptor-specific sensory neuron input. We extracted activity from a large population of glomeruli and used logistic regression to classify odors from individual trials with high accuracy. We then used add-in and dropout analyses to determine subsets of glomeruli necessary and sufficient for odor classification. Classifiers successfully predicted odor identity even after excluding sparse, highly active glomeruli, indicating that odor information is redundantly represented across a large population of glomeruli. Additionally, we found that random forest (RF) feature selection informed by Gini inequality (RF Gini impurity, RFGI) reliably ranked glomeruli by their contribution to overall odor classification. RFGI provided a measure of "feature importance" for each glomerulus that correlated with intuitive features like response magnitude. Finally, in agreement with previous work, we found that odor information persists in glomerular activity after the odor offset. Together, our findings support a model of OB odor coding where sparse activity is sufficient for odor identification, but information is widely, redundantly available across a large population of glomeruli, with each glomerulus representing information about more than one odor.

Olfactory receptor neurons are sensitive to stimulus onset asynchrony: implications for odor source discrimination.

In insects, olfactory receptor neurons (ORNs) are localized in sensilla. Within a sensillum, different ORN types are typically co-localized and exhibit nonsynaptic reciprocal inhibition through ephaptic coupling. This inhibition is hypothesized to aid odor source discrimination in environments where odor molecules (odorants) are dispersed by wind, resulting in turbulent plumes. Under these conditions, odorants from a single source arrive at the ORNs synchronously, while those from separate sources arrive asynchronously. Ephaptic inhibition is expected to be weaker for asynchronous arriving odorants from separate sources, thereby enhancing their discrimination. Previous studies have focused on ephaptic inhibition of sustained ORN responses to constant odor stimuli. This begs the question of whether ephaptic inhibition also affects transient ORN responses and if this inhibition is modulated by the temporal arrival patterns of different odorants. To address this, we recorded co-localized ORNs in the fruit fly Drosophila melanogaster and exposed them to dynamic odorant mixtures. We found reciprocal inhibition, strongly suggesting the presence of ephaptic coupling. This reciprocal inhibition does indeed modulate transient ORN responses and is sensitive to the relative timing of odor stimuli. Notably, the strength of inhibition decreases as the synchrony and correlation between arriving odorants decrease. These results support the hypothesis that ephaptic inhibition aids odor source discrimination.

Cell type- and layer-specific plasticity of olfactory bulb interneurons following olfactory sensory neuron ablation.

Lifelong neurogenesis endows the mouse olfactory system with a capacity for regeneration that is unique in the mammalian nervous system. Throughout life, olfactory sensory neurons (OSNs) are generated from olfactory epithelium (OE) stem cells in the nose, while the subventricular zone generates neuroblasts that migrate to the olfactory bulb (OB) and differentiate into multiple populations of inhibitory interneurons. Methimazole (MMZ) selectively ablates OSNs, but OE neurogenesis enables OSN repopulation and gradual recovery of OSN input to the OB within 6 weeks. However, it is not known how OB interneurons are affected by this loss and subsequent regeneration of OSN input following MMZ treatment. We found that dopaminergic neuron density was significantly reduced 7-14 days post-MMZ but recovered substantially at 35 days. The density of parvalbumin-expressing interneurons was unaffected by MMZ; however, their soma size was significantly reduced at 7-14 days post-MMZ, recovering by 35 days. Surprisingly, we found a transient increase in the density of calretinin-expressing neurons in the glomerular and external plexiform layers, but not the granule cell layer, 7 days post-MMZ. This could not be accounted for by increased neurogenesis but may result from increased calretinin expression. Together, our data demonstrate cell type- and layer-specific changes in OB interneuron density and morphology after MMZ treatment, providing new insight into the range of plasticity mechanisms employed by OB circuits during loss and regeneration of sensory input.

The Ca2+-activated Cl- channel TMEM16B shapes the response time course of olfactory sensory neurons.

Mammalian olfactory sensory neurons (OSNs) generate an odorant-induced response by sequentially activating two ion channels, which are in their ciliary membranes. First, a cationic, Ca2+-permeable cyclic nucleotide-gated channel is opened following odorant stimulation via a G protein-coupled transduction cascade and an ensuing rise in cAMP. Second, the increase in ciliary Ca2+ opens the excitatory Ca2+-activated Cl- channel TMEM16B, which carries most of the odorant-induced receptor current. While the role of TMEM16B in amplifying the response has been well established, it is less understood how this secondary ion channel contributes to response kinetics and action potential generation during single as well as repeated stimulation and, on the other hand, which response properties the cyclic nucleotide-gated (CNG) channel determines. We first demonstrate that basic membrane properties such as input resistance, resting potential and voltage-gated currents remained unchanged in OSNs that lack TMEM16B. The CNG channel predominantly determines the response delay and adaptation during odorant exposure, while the absence of the Cl- channels shortens both the time the response requires to reach its maximum and the time to terminate after odorant stimulation. This faster response termination in Tmem16b knockout OSNs allows them, somewhat counterintuitively despite the large reduction in receptor current, to fire action potentials more reliably when stimulated repeatedly in rapid succession, a phenomenon that occurs both in isolated OSNs and in OSNs within epithelial slices. Thus, while the two olfactory ion channels act in concert to generate the overall response, each one controls specific aspects of the odorant-induced response. KEY POINTS: Mammalian olfactory sensory neurons (OSNs) generate odorant-induced responses by activating two ion channels sequentially in their ciliary membranes: a Na+, Ca2⁺-permeable cyclic nucleotide-gated (CNG) channel and the Ca2⁺-activated Cl⁻ channel TMEM16B. The CNG channel controls response delay and adaptation during odorant exposure, while TMEM16B amplifies the response and influences the time required for the response to reach its peak and terminate. OSNs lacking TMEM16B display faster response termination, allowing them to fire action potentials more reliably during rapid repeated stimulation. The CNG and TMEM16B channels have distinct and complementary roles in shaping the kinetics and reliability of odorant-induced responses in OSNs.

Olfactory sensory neuron population expansions influence projection neuron adaptation and enhance odour tracking.

The evolutionary expansion of sensory neuron populations detecting important environmental cues is widespread, but functionally enigmatic. We investigated this phenomenon through comparison of homologous olfactory pathways of Drosophila melanogaster and its close relative Drosophila sechellia, an extreme specialist for Morinda citrifolia noni fruit. D. sechellia has evolved species-specific expansions in select, noni-detecting olfactory sensory neuron (OSN) populations, through multigenic changes. Activation and inhibition of defined proportions of neurons demonstrate that OSN number increases contribute to stronger, more persistent, noni-odour tracking behaviour. These expansions result in increased synaptic connections of sensory neurons with their projection neuron (PN) partners, which are conserved in number between species. Surprisingly, having more OSNs does not lead to greater odour-evoked PN sensitivity or reliability. Rather, pathways with increased sensory pooling exhibit reduced PN adaptation, likely through weakened lateral inhibition. Our work reveals an unexpected functional impact of sensory neuron population expansions to explain ecologically-relevant, species-specific behaviour.

Distinct olfactory mucosal macrophage populations mediate neuronal maintenance and pathogen defense.

The olfactory mucosa is important for both the sense of smell and as a mucosal immune barrier to the upper airway and brain. However, little is known about how the immune system mediates the conflicting goals of neuronal maintenance and inflammation in this tissue. A number of immune cell populations reside within the olfactory mucosa and yet we have little understanding of how these resident olfactory immune cells functionally interact with the chemosensory environment. Identifying these interactions will allow therapeutic manipulations that treat disorders such as post-viral olfactory dysfunction. Macrophages are the most prevalent immune cell type in the uninflamed olfactory mucosa and here, we identify two distinct tissue macrophage populations in murine olfactory mucosa. P2ry12hi macrophages are transcriptionally specialized for neuron interactions, closely associated with olfactory neuron cell bodies, long-term tissue residents, and functionally specialized to phagocytose cells and debris, including olfactory neurons. Conversely, MHC Class IIhi macrophages are transcriptionally dedicated to cytokine production and antigen presentation, localized primarily within the olfactory lamina propria, more rapidly replaced by blood monocytes, and rapidly produce chemokines in response to viral infection. We further show that these macrophage signatures are present in human olfactory biopsies, and P2ry12-like olfactory macrophages are reduced in patients with long-term smell loss following COVID-19. Together, these data show that two olfactory macrophage populations regulate neurons and initiate the immune response, contributing to our understanding of both olfactory immunity and tissue-resident macrophage biology.

Response Plasticity of Drosophila Olfactory Sensory Neurons.

In insect olfaction, sensitization refers to the amplification of a weak olfactory signal when the stimulus is repeated within a specific time window. In the vinegar fly, Drosophila melanogaster, this occurs already at the periphery, at the level of olfactory sensory neurons (OSNs) located in the antenna. In our study, we investigate whether sensitization is a widespread property in a set of seven types of OSNs, as well as the mechanisms involved. First, we characterize and compare the differences in spontaneous activity, response velocity and response dynamics, among the selected OSN types. These express different receptors with distinct tuning properties and behavioral relevance. Second, we show that sensitization is not a general property. Among our selected OSN types, it occurs in those responding to more general food odors, while OSNs involved in very specific detection of highly specific ecological cues like pheromones and warning signals show no sensitization. Moreover, we show that mitochondria play an active role in sensitization by contributing to the increase in intracellular Ca2+ upon weak receptor activation. Thus, by using a combination of single sensillum recordings (SSRs), calcium imaging and pharmacology, we widen the understanding of how the olfactory signal is processed at the periphery.

The influence of olfactory experience on the birthrates of olfactory sensory neurons with specific odorant receptor identities.

Olfactory sensory neurons (OSNs) are one of a few neuron types that are generated continuously throughout life in mammals. The persistence of olfactory sensory neurogenesis beyond early development has long been thought to function simply to replace neurons that are lost or damaged through exposure to environmental insults. The possibility that olfactory sensory neurogenesis may also serve an adaptive function has received relatively little consideration, largely due to the assumption that the generation of new OSNs is stochastic with respect to OSN subtype, as defined by the single odorant receptor gene that each neural precursor stochastically chooses for expression out of hundreds of possibilities. Accordingly, the relative birthrates of different OSN subtypes are predicted to be constant and impervious to olfactory experience. This assumption has been called into question, however, by evidence that the birthrates of specific OSN subtypes can be selectively altered by manipulating olfactory experience through olfactory deprivation, enrichment, and conditioning paradigms. Moreover, studies of recovery of the OSN population following injury provide further evidence that olfactory sensory neurogenesis may not be strictly stochastic with respect to subtype. Here we review this evidence and consider mechanistic and functional implications of the prospect that specific olfactory experiences can regulate olfactory sensory neurogenesis rates in a subtype-selective manner.

Structures and functions of the normal and injured human olfactory epithelium.

The olfactory epithelium (OE) is directly exposed to environmental agents entering the nasal cavity, leaving OSNs prone to injury and degeneration. The causes of olfactory dysfunction are diverse and include head trauma, neurodegenerative diseases, and aging, but the main causes are chronic rhinosinusitis (CRS) and viral infections. In CRS and viral infections, reduced airflow due to local inflammation, inflammatory cytokine production, release of degranulated proteins from eosinophils, and cell injury lead to decreased olfactory function. It is well known that injury-induced loss of mature OSNs in the adult OE causes massive regeneration of new OSNs within a few months through the proliferation and differentiation of progenitor basal cells that are subsequently incorporated into olfactory neural circuits. Although normal olfactory function returns after injury in most cases, prolonged olfactory impairment and lack of improvement in olfactory function in some cases poses a major clinical problem. Persistent inflammation or severe injury in the OE results in morphological changes in the OE and respiratory epithelium and decreases the number of mature OSNs, resulting in irreversible loss of olfactory function. In this review, we discuss the histological structure and distribution of the human OE, and the pathogenesis of olfactory dysfunction associated with CRS and viral infection.

Peripheral preprocessing in Drosophila facilitates odor classification.

The mammalian brain implements sophisticated sensory processing algorithms along multilayered ("deep") neural networks. Strategies that insects use to meet similar computational demands, while relying on smaller nervous systems with shallow architectures, remain elusive. Using Drosophila as a model, we uncover the algorithmic role of odor preprocessing by a shallow network of compartmentalized olfactory receptor neurons. Each compartment operates as a ratiometric unit for specific odor-mixtures. This computation arises from a simple mechanism: electrical coupling between two differently sized neurons. We demonstrate that downstream synaptic connectivity is shaped to optimally leverage amplification of a hedonic value signal in the periphery. Furthermore, peripheral preprocessing is shown to markedly improve novel odor classification in a higher brain center. Together, our work highlights a far-reaching functional role of the sensory periphery for downstream processing. By elucidating the implementation of powerful computations by a shallow network, we provide insights into general principles of efficient sensory processing algorithms.

Predation cues induce predator specific changes in olfactory neurons encoding defensive responses in agile frog tadpoles.

Although behavioural defensive responses have been recorded several times in both laboratory and natural habitats, their neural mechanisms have seldom been investigated. To explore how chemical, water-borne cues are conveyed to the forebrain and instruct behavioural responses in anuran larvae, we conditioned newly hatched agile frog tadpoles using predator olfactory cues, specifically either native odonate larvae or alien crayfish kairomones. We expected chronic treatments to influence the basal neuronal activity of the tadpoles' mitral cells and alter their sensory neuronal connections, thereby impacting information processing. Subsequently, these neurons were acutely perfused, and their responses were compared with the defensive behaviour of tadpoles previously conditioned and exposed to the same cues. Tadpoles conditioned with odonate cues differed in both passive and active cell properties compared to those exposed to water (controls) or crayfish cues. The observed upregulation of membrane conductance and increase in both the number of active synapses and receptor density at the postsynaptic site are believed to have enhanced their responsiveness to external stimuli. Odonate cues also affected the resting membrane potential and firing rate of mitral cells during electrophysiological patch-clamp recordings, suggesting a rearrangement of the repertoire of voltage-dependent conductances expressed in cell membranes. These recorded neural changes may modulate the induction of an action potential and transmission of information. Furthermore, the recording of neural activity indicated that the lack of defensive responses towards non-native predators is due to the non-recognition of their olfactory cues.

The olfactory network of larval Xenopus laevis regenerates accurately after olfactory nerve transection.

Across vertebrate species, the olfactory epithelium (OE) exhibits the uncommon feature of lifelong neuronal turnover. Epithelial stem cells give rise to new neurons that can adequately replace dying olfactory receptor neurons (ORNs) during developmental and adult phases and after lesions. To relay olfactory information from the environment to the brain, the axons of the renewed ORNs must reconnect with the olfactory bulb (OB). In Xenopus laevis larvae, we have previously shown that this process occurs between 3 and 7 weeks after olfactory nerve (ON) transection. In the present study, we show that after 7 weeks of recovery from ON transection, two functionally and spatially distinct glomerular clusters are reformed in the OB, akin to those found in non-transected larvae. We also show that the same odourant response tuning profiles observed in the OB of non-transected larvae are again present after 7 weeks of recovery. Next, we show that characteristic odour-guided behaviour disappears after ON transection but recovers after 7-9 weeks of recovery. Together, our findings demonstrate that the olfactory system of larval X. laevis regenerates with high accuracy after ON transection, leading to the recovery of odour-guided behaviour.

A pattern recognition artificial olfactory system based on human olfactory receptors and organic synaptic devices.

Neuromorphic sensors, designed to emulate natural sensory systems, hold the promise of revolutionizing data extraction by facilitating rapid and energy-efficient analysis of extensive datasets. However, a challenge lies in accurately distinguishing specific analytes within mixtures of chemically similar compounds using existing neuromorphic chemical sensors. In this study, we present an artificial olfactory system (AOS), developed through the integration of human olfactory receptors (hORs) and artificial synapses. This AOS is engineered by interfacing an hOR-functionalized extended gate with an organic synaptic device. The AOS generates distinct patterns for odorants and mixtures thereof, at the molecular chain length level, attributed to specific hOR-odorant binding affinities. This approach enables precise pattern recognition via training and inference simulations. These findings establish a foundation for the development of high-performance sensor platforms and artificial sensory systems, which are ideal for applications in wearable and implantable devices.

Distinct information conveyed to the olfactory bulb by feedforward input from the nose and feedback from the cortex.

Sensory systems are organized hierarchically, but feedback projections frequently disrupt this order. In the olfactory bulb (OB), cortical feedback projections numerically match sensory inputs. To unravel information carried by these two streams, we imaged the activity of olfactory sensory neurons (OSNs) and cortical axons in the mouse OB using calcium indicators, multiphoton microscopy, and diverse olfactory stimuli. Here, we show that odorant mixtures of increasing complexity evoke progressively denser OSN activity, yet cortical feedback activity is of similar sparsity for all stimuli. Also, representations of complex mixtures are similar in OSNs but are decorrelated in cortical axons. While OSN responses to increasing odorant concentrations exhibit a sigmoidal relationship, cortical axonal responses are complex and nonmonotonic, which can be explained by a model with activity-dependent feedback inhibition in the cortex. Our study indicates that early-stage olfactory circuits have access to local feedforward signals and global, efficiently formatted information about odor scenes through cortical feedback.

Phasic/tonic glial GABA differentially transduce for olfactory adaptation and neuronal aging.

Phasic (fast) and tonic (sustained) inhibition of γ-aminobutyric acid (GABA) are fundamental for regulating day-to-day activities, neuronal excitability, and plasticity. However, the mechanisms and physiological functions of glial GABA transductions remain poorly understood. Here, we report that the AMsh glia in Caenorhabditis elegans exhibit both phasic and tonic GABAergic signaling, which distinctively regulate olfactory adaptation and neuronal aging. Through genetic screening, we find that GABA permeates through bestrophin-9/-13/-14 anion channels from AMsh glia, which primarily activate the metabolic GABAB receptor GBB-1 in the neighboring ASH sensory neurons. This tonic action of glial GABA regulates the age-associated changes of ASH neurons and olfactory responses via a conserved signaling pathway, inducing neuroprotection. In addition, the calcium-evoked, vesicular glial GABA release acts upon the ionotropic GABAA receptor LGC-38 in ASH neurons to regulate olfactory adaptation. These findings underscore the fundamental significance of glial GABA in maintaining healthy aging and neuronal stability.