Olfactory receptor neurons are sensitive to stimulus onset asynchrony: Implications for odour source discrimination.

In insects, olfactory receptor neurons (ORNs) are localized in sensilla. Within a sensillum, different ORN types are typically co-localized and exhibit non-synaptic reciprocal inhibition through ephaptic coupling. This inhibition is hypothesized to aid odour 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 odour 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 odour source discrimination.

Characterisation and automated quantification of induced seizure-related behaviours in Xenopus laevis tadpoles.

Epilepsy, a clinical diagnosis characterised by paroxysmal episodes known as seizures, affects 1% of people worldwide. Safe and patient-specific treatment is vital and can be achieved by the development of rapid pre-clinical models of for identified epilepsy genes. Epilepsy can result from either brain injury or gene mutations, and can also be induced chemically. Xenopus laevis tadpoles could be a useful model for confirmation of variants of unknown significance found in epilepsy patients, and for drug re-purposing screens that could eventually lead to benefits for patients. Here, we characterise and quantify seizure-related behaviours in X. laevis tadpoles arrayed in 24-well plates. To provoke acute seizure behaviours, tadpoles were chemically induced with either pentylenetetrazole (PTZ) or 4-aminopyridine (4-AP). To test the capacity to adapt this method for drug testing, we also exposed induced tadpoles to the anti-seizure drug valproate (VPA). Four induced seizure-like behaviours were described and manually quantified, and two of these (darting, circling) could be accurately detected automatically, using the video analysis software TopScan. Additionally, we recorded swimming trajectories and mean swimming velocity. Automatic detection showed that either PTZ or 4-AP induced darting behaviour and increased mean swimming velocity compared to untreated controls. Both parameters were significantly reduced in the presence of VPA. In particular, darting behaviour was a shown to be a sensitive measure of epileptic seizure activity. While we could not automatically detect the full range of seizure behaviours, this method shows promise for future studies since X. laevis is a well-characterised and genetically tractable model organism.

Alpha oscillations govern interhemispheric spike timing coordination in the honey bee brain.

In 1929 Hans Berger discovered the alpha oscillations: prominent, ongoing oscillations around 10 Hz in the electroencephalogram of the human brain. These alpha oscillations are among the most widely studied brain signals, related to cognitive phenomena such as attention, memory and consciousness. However, the mechanisms by which alpha oscillations affect human cognition await demonstration. Here, we suggest the honey bee brain as an experimentally more accessible model system for investigating the functional role of alpha oscillations. We found a prominent spontaneous oscillation around 18 Hz that is reduced in amplitude upon olfactory stimulation. Similar to alpha oscillations in primates, the phase of this oscillation biased both timing of neuronal spikes and amplitude of high-frequency gamma activity (40-450 Hz). These results suggest a common role of alpha oscillations across phyla and provide an unprecedented new venue for causal studies on the relationship between neuronal spikes, brain oscillations and cognition.

Social foraging extends associative odor-food memory expression in an automated learning assay for Drosophila melanogaster.

Animals socially interact during foraging and share information about the quality and location of food sources. The mechanisms of social information transfer during foraging have been mostly studied at the behavioral level, and its underlying neural mechanisms are largely unknown. Fruit flies have become a model for studying the neural bases of social information transfer, because they provide a large genetic toolbox to monitor and manipulate neuronal activity, and they show a rich repertoire of social behaviors. Fruit flies aggregate, they use social information for choosing a suitable mating partner and oviposition site, and they show better aversive learning when in groups. However, the effects of social interactions on associative odor-food learning have not yet been investigated. Here, we present an automated learning and memory assay for walking flies that allows the study of the effect of group size on social interactions and on the formation and expression of associative odor-food memories. We found that both inter-fly attraction and the duration of odor-food memory expression increase with group size. This study opens up opportunities to investigate how social interactions during foraging are relayed in the neural circuitry of learning and memory expression.

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.

The Role of the Sucrose-Responsive IR60b Neuron for Drosophila melanogaster: A Hypothesis.

In a recent paper, Joseph and colleagues (Joseph et al. 2017) have characterized an IR60b receptor-expressing neuron in Drosophila. They showed that it responds to sucrose and serves to limit sucrose consumption, and proposed that it may thereby act to prevent overfeeding. Here, we propose an alternative hypothesis for the functional role of sucrose feeding control, and for how this limitation of sucrose uptake is accomplished. Adult fruit flies feed by excreting saliva onto the food, and imbibing the predigested liquefied food, or by filling the crop, where the food is predigested. Enzymes in the saliva hydrolyze starch and disaccharides into absorbable monosaccharides. Premature ingestion into the midgut would not give the enzymes in the saliva enough time to predigest the food. Thus, IR60b neurons might serve as a sensor to monitor the digestive state of external food or crop content: when disaccharides (sucrose) concentration is high, ingestion to the gut is inhibited, keeping a low concentration of starch and disaccharides in the midgut.

A High-Bandwidth Dual-Channel Olfactory Stimulator for Studying Temporal Sensitivity of Olfactory Processing.

Animals encounter fine-scale temporal patterns of odorant mixtures that contain information about the distance and number of odorant sources. To study the role of such temporal cues for odorant detection and source localization, one needs odorant delivery devices that are capable of mimicking the temporal stimulus statistics of natural odor plumes. However, current odorant delivery devices either lack temporal resolution or are limited to a single odorant channel. Here, we present an olfactory stimulator that features precise control of high-bandwidth stimulus dynamics, which allows generating arbitrary fluctuating binary odorant mixtures. We provide a comprehensive characterization of the stimulator's performance and use it to demonstrate that odor background affects the temporal resolution of insect olfactory receptor neurons, and we present a hitherto unknown odor pulse-tracking capability of up to 60 Hz in Kenyon cells, which are higher order olfactory neurons of the insect brain. This stimulator might help investigating whether and how animals use temporal stimulus cues for odor detection and source localization. Because the stimulator is easy to replicate it can facilitate generating the same odor stimulus dynamics at different experimental setups and across different labs.

High-speed odor transduction and pulse tracking by insect olfactory receptor neurons.

Sensory systems encode both the static quality of a stimulus (e.g., color or shape) and its kinetics (e.g., speed and direction). The limits with which stimulus kinetics can be resolved are well understood in vision, audition, and somatosensation. However, the maximum temporal resolution of olfactory systems has not been accurately determined. Here, we probe the limits of temporal resolution in insect olfaction by delivering high frequency odor pulses and measuring sensory responses in the antennae. We show that transduction times and pulse tracking capabilities of olfactory receptor neurons are faster than previously reported. Once an odorant arrives at the boundary layer of the antenna, odor transduction can occur within less than 2 ms and fluctuating odor stimuli can be resolved at frequencies more than 100 Hz. Thus, insect olfactory receptor neurons can track stimuli of very short duration, as occur when their antennae encounter narrow filaments in an odor plume. These results provide a new upper bound to the kinetics of odor tracking in insect olfactory receptor neurons and to the latency of initial transduction events in olfaction.

Asymmetric neural coding revealed by in vivo calcium imaging in the honey bee brain.

Left-right asymmetries are common properties of nervous systems. Although lateralized sensory processing has been well studied, information is lacking about how asymmetries are represented at the level of neural coding. Using in vivo functional imaging, we identified a population-level left-right asymmetry in the honey bee's primary olfactory centre, the antennal lobe (AL). When both antennae were stimulated via a frontal odour source, the inter-odour distances between neural response patterns were higher in the right than in the left AL. Behavioural data correlated with the brain imaging results: bees with only their right antenna were better in discriminating a target odour in a cross-adaptation paradigm. We hypothesize that the differences in neural odour representations in the two brain sides serve to increase coding capacity by parallel processing.

Millisecond stimulus onset-asynchrony enhances information about components in an odor mixture.

Airborne odorants rarely occur as pure, isolated stimuli. In a natural environment, odorants that intermingle from multiple sources create mixtures in which the onset and offset of odor components are asynchronous. Odor mixtures are known to elicit interactions in both behavioral and physiological responses, changing the perceptive quality of mixtures compared with the components. However, relevant odors need to be segregated from a distractive background. Honeybees (Apis mellifera) can use stimulus onset asynchrony of as little as 6 ms to segregate learned odor components within a mixture. Using in vivo calcium imaging of projection neurons in the honeybee, we studied neuronal mechanisms of odor-background segregation based on stimulus onset asynchrony in the antennal lobe. We found that asynchronous mixtures elicit response patterns that are different from their synchronous counterpart: the responses to asynchronous mixtures contain more information about the constituent components. With longer onset shifts, more features of the components were present in the mixture response patterns. Moreover, we found that the processing of asynchronous mixtures activated more inhibitory interactions than the processing of synchronous mixtures. This study provides evidence of neuronal mechanisms that underlie odor-object segregation on a timescale much faster than found for mammals.

Effect of GABAergic inhibition on odorant concentration coding in mushroom body intrinsic neurons of the honeybee.

Kenyon cells, the intrinsic neurons of the insect mushroom body, have the intriguing property of responding in a sparse way to odorants. Sparse neuronal codes are often invariant to changes in stimulus intensity and duration, and sparse coding often depends on global inhibition. We tested if this is the case for honeybees' Kenyon cells, too, and used in vivo Ca²âº imaging to record their responses to different odorant concentrations. Kenyon cells responded not only to the onset of odorant stimuli (ON responses), but also to their termination (OFF responses). Both, ON and OFF responses increased with increasing odorant concentration. ON responses were phasic and invariant to the duration of odorant stimuli, while OFF responses increased with increasing odorant duration. Pharmacological blocking of GABA receptors in the brain revealed that ionotropic GABA(A) and metabotropic GABA(B) receptors attenuate Kenyon cells' ON responses without changing their OFF responses. Ionotropic GABA(A) receptors attenuated Kenyon cell ON responses more strongly than metabotropic GABA(B) receptors. However, the response dynamic, temporal resolution and paired-pulse depression did not depend on GABA(A) transmission. These data are discussed in the context of mechanisms leading to sparse coding in Kenyon cells.

Knockdown of NeuroD2 leads to seizure-like behavior, brain neuronal hyperactivity and a leaky blood-brain barrier in a Xenopus laevis tadpole model of DEE75.

Developmental and Epileptic Encephalopathies (DEE) are a genetically diverse group of severe, early onset seizure disorders. DEE are normally identified clinically in the first six months of life by the presence of frequent, difficult to control seizures and accompanying stalling or regression of development. DEE75 results from de novo mutations of the NEUROD2 gene that result in loss of activity of the encoded transcription factor, and the seizure phenotype was shown to be recapitulated in Xenopus tropicalis tadpoles. We used CRISPR/Cas9 to make a DEE75 model in Xenopus laevis, to further investigate the developmental etiology. NeuroD2.S CRISPR/Cas9 edited tadpoles were more active, swam faster on average, and had more seizures (C-shaped contractions resembling unprovoked C-start escape responses) than their sibling controls. Live imaging of Ca2+ signaling revealed prolongued, strong signals sweeping through the brain, indicative of neuronal hyperactivity. While the resulting tadpole brain appeared grossly normal, the blood-brain barrier (BBB) was found to be leakier than that of controls. Additionally, the TGFß antagonist Losartan was shown to have a short-term protective effect, reducing neuronal hyperactivity and reducing permeability of the BBB. Treatment of NeuroD2 CRISPant tadpoles with 5 mM Losartan decreased seizure events by more than 4-fold compared to the baseline. Our results support a model of DEE75 resulting from reduced NeuroD2 activity during vertebrate brain development, and indicate that a leaky BBB contributes to epileptogenesis.

The looks of an odour--visualising neural odour response patterns in real time.

BACKGROUND: Calcium imaging in insects reveals the neural response to odours, both at the receptor level on the antenna and in the antennal lobe, the first stage of olfactory information processing in the brain. Changes of intracellular calcium concentration in response to odour presentations can be observed by employing calcium-sensitive, fluorescent dyes. The response pattern across all recorded units is characteristic for the odour. METHOD: Previously, extraction of odour response patterns from calcium imaging movies was performed offline, after the experiment. We developed software to extract and to visualise odour response patterns in real time. An adaptive algorithm in combination with an implementation for the graphics processing unit enables fast processing of movie streams. Relying on correlations between pixels in the temporal domain, the calcium imaging movie can be segmented into regions that correspond to the neural units. RESULTS: We applied our software to calcium imaging data recorded from the antennal lobe of the honeybee Apis mellifera and from the antenna of the fruit fly Drosophila melanogaster. Evaluation on reference data showed results comparable to those obtained by previous offline methods while computation time was significantly lower. Demonstrating practical applicability, we employed the software in a real-time experiment, performing segmentation of glomeruli--the functional units of the honeybee antennal lobe--and visualisation of glomerular activity patterns. CONCLUSIONS: Real-time visualisation of odour response patterns expands the experimental repertoire targeted at understanding information processing in the honeybee antennal lobe. In interactive experiments, glomeruli can be selected for manipulation based on their present or past activity, or based on their anatomical position. Apart from supporting neurobiology, the software allows for utilising the insect antenna as a chemosensor, e.g. to detect or to classify odours.

Extracting spatial information from temporal odor patterns: insights from insects.

Extracting spatial information from temporal stimulus patterns is essential for sensory perception (e.g. visual motion direction detection or concurrent sound segregation), but this process remains understudied in olfaction. Animals rely on olfaction to locate resources and dangers. In open environments, where odors are dispersed by turbulent wind, detection of wind direction seems crucial for odor source localization. However, recent studies showed that insects can extract spatial information from the odor stimulus itself, independently from sensing wind direction. This remarkable ability is achieved by detecting the fine-scale temporal pattern of odor encounters, which contains information about the location and size of an odor source, and the distance between different odor sources.

Olfactory trace conditioning in Drosophila.

The neural representation of a sensory stimulus evolves with time, and animals keep that representation even after stimulus cessation (i.e., a stimulus "trace"). To contrast the memories of an odor and an odor trace, we here establish a rigorous trace conditioning paradigm in the fruit fly, Drosophila melanogaster. We modify the olfactory associative learning paradigm, in which the odor and electric shock are presented with a temporal overlap (delay conditioning). Given a few-second temporal gap between the presentations of the odor and the shock in trace conditioning, the odor trace must be kept until the arrival of electric shock to form associative memory. We found that memories after trace and delay conditioning have striking similarities: both reached the same asymptotic learning level, although at different rates, and both kinds of memory have similar decay kinetics and highly correlated generalization profiles across odors. In search of the physiological correlate of the odor trace, we used in vivo calcium imaging to characterize the odor-evoked activity of the olfactory receptor neurons in the antennal lobe. After the offset of odor presentation, the receptor neurons showed persistent, odor-specific response patterns that lasted for a few seconds and were fundamentally different from the response patterns during the stimulation. Weak correlation between the behavioral odor generalization profile in trace conditioning and the physiological odor similarity profiles in the antennal lobe suggest that the odor trace used for associative learning may be encoded downstream of the olfactory receptor neurons.

Mind the gap: olfactory trace conditioning in honeybees.

Trace conditioning is a form of classical conditioning, where a neutral stimulus (conditioned stimulus, CS) is associated with a following appetitive or aversive stimulus (unconditioned stimulus, US). Unlike classical delay conditioning, in trace conditioning there is a stimulus-free gap between CS and US, and thus a poststimulus neural representation (trace) of the CS is required to bridge the gap until its association with the US. The properties of such stimulus traces are not well understood, nor are their underlying physiological mechanisms. Using behavioral and physiological approaches, we studied appetitive olfactory trace conditioning in honeybees. We found that single-odor presentation created a trace containing information about odor identity. This trace conveyed odor information about the initial stimulus and was robust against interference by other odors. Memory acquisition decreased with increasing CS-US gap length. The maximum learnable CS-US gap length could be extended by previous trace-conditioning experience. Furthermore, acquisition improved when an additional odor was presented during the CS-US gap. Using calcium imaging, we tested whether projection neurons in the primary olfactory brain area, the antennal lobe, contain a CS trace. We found odor-specific persistent responses after stimulus offset. These post-odor responses, however, did not encode the CS trace, and perceived odor quality could be predicted by the initial but not by the post-odor response. Our data suggest that olfactory trace conditioning is a less reflexive form of learning than classical delay conditioning, indicating that odor traces might involve higher-level cognitive processes.

Sugar alcohols have the potential as bee-safe baits for the common wasp.

BACKGROUND: Pest insects are often baited with poisoned feeding stimulants, the most common of which are sugars. However, sugars are attractive for most animal species, which makes it difficult to target only a specific pest insect species. Here, we assessed different sugar alcohols for their potential as more species-selective feeding stimulants for pest insects. RESULTS: We tested the attractiveness of the sugar alcohols sorbitol, xylitol and erythritol with a capillary feeder assay in wasps (as potential pest insects, because introduced wasps are a pest in many regions) and bees (as non-target insects). For the common wasp (Vespula vulgaris), sorbitol and xylitol acted as nutritive feeding stimulants, and erythritol acted as a non-nutritive feeding stimulant. For the buff-tailed bumble bee (Bombus terrestris), sorbitol acted as a feeding stimulant, while for the honey bee (Apis mellifera), none of the sugar alcohols acted as feeding stimulant. CONCLUSION: The species-specific preferences for sugar alcohols suggest their potential as species-selective insect baits. The wasp-specific preference for xylitol suggests its potential as a bee-safe alternative to sugar-containing bait for controlling the common wasp. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Reduced olfactory acuity in recently flightless insects suggests rapid regressive evolution.

BACKGROUND: Insects have exceptionally fast smelling capabilities, and some can track the temporal structure of odour plumes at rates above 100 Hz. It has been hypothesized that this fast smelling capability is an adaptation for flying. We test this hypothesis by comparing the olfactory acuity of sympatric flighted versus flightless lineages within a wing-polymorphic stonefly species. RESULTS: Our analyses of olfactory receptor neuron responses reveal that recently-evolved flightless lineages have reduced olfactory acuity. By comparing flighted versus flightless ecotypes with similar genetic backgrounds, we eliminate other confounding factors that might have affected the evolution of their olfactory reception mechanisms. Our detection of different patterns of reduced olfactory response strength and speed in independently wing-reduced lineages suggests parallel evolution of reduced olfactory acuity. CONCLUSIONS: These reductions in olfactory acuity echo the rapid reduction of wings themselves, and represent an olfactory parallel to the convergent phenotypic shifts seen under selective gradients in other sensory systems (e.g. parallel loss of vision in cave fauna). Our study provides evidence for the hypothesis that flight poses a selective pressure on the speed and strength of olfactory receptor neuron responses and emphasizes the energetic costs of rapid olfaction.

Multiple memory traces after associative learning in the honey bee antennal lobe.

We investigated the effect of associative learning on early sensory processing, by combining classical conditioning with in vivo calcium-imaging of secondary olfactory neurons, the projection neurons (PNs) in the honey bee antennal lobe (AL). We trained bees in a differential conditioning paradigm in which one odour (A+) was paired with a reward, while another odour (B-) was presented without a reward. Two to five hours after differential conditioning, the two odour-response patterns became more different in bees that learned to discriminate between A and B, but not in bees that did not discriminate. This learning-related change in neural odour representations can be traced back to glomerulus-specific neural plasticity, which depended on the response profile of the glomerulus before training. (i) Glomeruli responding to A but not to B generally increased in response strength. (ii) Glomeruli responding to B but not to A did not change in response strength. (iii) Glomeruli responding to A and B decreased in response strength. (iv) Glomeruli not responding to A or B increased in response strength. The data are consistent with a neural network model of the AL, which we based on two plastic synapse types and two well-known learning rules: associative, reinforcer-dependent Hebbian plasticity at synapses between olfactory receptor neurons (ORNs) and PNs; and reinforcer-independent Hebbian plasticity at synapses between local interneurons and ORNs. The observed changes strengthen the idea that odour learning optimizes odour representations, and facilitates the detection and discrimination of learned odours.

Segregation of Unknown Odors From Mixtures Based on Stimulus Onset Asynchrony in Honey Bees.

Animals use olfaction to search for distant objects. Unlike vision, where objects are spaced out, olfactory information mixes when it reaches olfactory organs. Therefore, efficient olfactory search requires segregating odors that are mixed with background odors. Animals can segregate known odors by detecting short differences in the arrival of mixed odorants (stimulus onset asynchrony). However, it is unclear whether animals can also use stimulus onset asynchrony to segregate odorants that they had no previous experience with and which have no innate or learned relevance (unknown odorants). Using behavioral experiments in honey bees, we here show that stimulus onset asynchrony also improves segregation of those unknown odorants. The stimulus onset asynchrony necessary to segregate unknown odorants is in the range of seconds, which is two orders of magnitude larger than the previously reported stimulus asynchrony sufficient for segregating known odorants. We propose that for unknown odorants, segregating odorant A from a mixture with B requires sensory adaptation to B.