Chronic exposure to odors at naturally occurring concentrations triggers limited plasticity in early stages of Drosophila olfactory processing

ABSTRACTIn insects and mammals, chronic exposure to odors at high concentrations in early life alters olfactory function, but the role of odor experience-dependent plasticity in more naturalistic contexts is less clear. We investigated olfactory plasticity in the Drosophila antennal lobe by exposing flies to odors at concentrations that are typically encountered in natural odor sources. These stimuli also strongly and selectively activated only a single class of olfactory receptor neuron (ORN) input, facilitating the investigation of input-specific plasticity. Overall, chronic exposure to three such odors elicited limited plasticity in the odor responses of second-order projection neurons (PNs). Exposure to some odors elicited mild increases in PN responses to weak stimuli, extending the lower bound of the dynamic range of PN signaling. When present, plasticity was observed broadly in multiple PN types and thus was not selective for PNs receiving direct input from the chronically active ORNs. Chronic E2-hexenal exposure did not affect PN intrinsic properties, local inhibitory innervation, ORN responses, or ORN-PN synaptic strength, but modestly increased broad lateral excitation evoked by some odors. These results show that PN odor coding is only mildly affected by strong persistent activation of a single olfactory input and highlight the stability of early stages of insect olfactory processing to significant perturbations in the sensory environment.

Automated control of odor dynamics for neurophysiology and behavior

Animals use their olfactory systems to avoid predators, forage for food, and identify mates. Olfactory systems detect and distinguish odors by responding to the concentration, temporal dynamics, and identities of odorant molecules. Studying the temporal neural processing of odors carried in air has been difficult because of the inherent challenge in precisely controlling odorized airflows over time. Odorized airflows interact with surfaces and other air currents, leading to a complex transformation from the odorized airflow that is desired to the olfactory stimulus that is delivered. Here, we present a method that achieves precise and automated control of the amplitude, baseline, and temporal structure of olfactory stimuli. We use this technique to analyze the temporal processing of olfactory stimuli in the early olfactory circuits and navigational behavior of larval Drosophila. Precise odor control and calcium measurements in the axon terminal of an Olfactory Receptor Neuron (ORN-Or42b) revealed dynamic adaptation properties: as in vertebrate photoreceptor neurons, Or42b-ORNs display simultaneous gain-suppression and speedup of their neural response. Furthermore, we found that ORN sensitivity to changes in odor concentration decreases with odor background, but the sensitivity to odor contrast is invariant -- this causes odor-evoked ORN activity to follow the Weber-Fechner Law. Using precise olfactory stimulus control with freely-moving animals, we uncovered correlations between the temporal dynamics of larval navigation motor programs and the neural response dynamics of second-order olfactory neurons. The correspondence between neural and behavioral dynamics highlights the potential of precise odor temporal dynamics control in dissecting the sensorimotor circuits for olfactory behaviors.

Squamous and Respiratory Metaplasia After Olfactory Mucosal Resection

Resection of the olfactory mucosa (OM) is sometimes unavoidable during surgery; however, it is not known whether the OM can completely recover thereafter. The aim of this study was to uncover whether the OM fully recovers after mucosal resection and describe the process of OM regeneration. 8-week-old male Sprague–Dawley rats (n = 18) were subjected to OM resection at the nasal septum; six rats were euthanized for histological examination 0, 30, and 90 days after surgery. Immunohistochemistry was performed to identify olfactory receptor neuron (ORN) lineage cells [mature and immature ORNs and ORN progenitors, and olfactory ensheathing cells (OECs)], as well as dividing and apoptotic cells. Squamous and respiratory metaplasia and inflammatory cell infiltration were also assessed. On day 30 after resection, the mucosa had regenerated, and mainly contained thin nerve bundles, basal cells, and immature ORNs, with a few mature ORNs and OECs. On day 90, the repaired nasal mucosa had degenerated into stratified squamous or ciliated pseudostratified columnar epithelia, with reducing ORNs. The lamina propria contained numerous macrophages. Partial regeneration was observed within 1 month after OM resection, whereas subsequent degeneration into squamous and respiratory epithelia occurred within 3 months. Given the poor persistence of ORNs and OECs, OM resection is likely to result in olfactory impairment. Overall, surgeons should be cautious not to injure the OM during surgery.

Transcriptome Profiling of Starvation in the Peripheral Chemosensory Organs of the Crop Pest Spodoptera littoralis Caterpillars

Starvation is frequently encountered by animals under fluctuating food conditions in nature, and response to it is vital for life span. Many studies have investigated the behavioral and physiological responses to starvation. In particular, starvation is known to induce changes in olfactory behaviors and olfactory sensitivity to food odorants, but the underlying mechanisms are not well understood. Here, we investigated the transcriptional changes induced by starvation in the chemosensory tissues of the caterpillar Spodoptera littoralis, using Illumina RNA sequencing. Gene expression profiling revealed 81 regulated transcripts associated with several biological processes, such as glucose metabolism, immune defense, response to stress, foraging activity, and olfaction. Focusing on the olfactory process, we observed changes in transcripts encoding proteins putatively involved in the peri-receptor events, namely, chemosensory proteins and odorant-degrading enzymes. Such modulation of their expression may drive fluctuations in the dynamics and the sensitivity of the olfactory receptor neuron response. In combination with the enhanced presynaptic activity mediated via the short neuropeptide F expressed during fasting periods, this could explain an enhanced olfactory detection process. Our observations suggest that a coordinated transcriptional response of peripheral chemosensory organs participates in the regulation of olfactory signal reception and olfactory-driven behaviors upon starvation.

Most primary olfactory neurons have individually neutral effects on behavior

Animals use olfactory receptors to navigate mates, food, and danger. However, for complex olfactory systems, it is unknown what proportion of primary olfactory sensory neurons can individually drive avoidance or attraction. Similarly, the rules that govern behavioral responses to receptor combinations are unclear. We used optogenetic analysis in Drosophila to map the behavior elicited by olfactory-receptor neuron (ORN) classes: just one-fifth of ORN-types drove either avoidance or attraction. Although wind and hunger are closely linked to olfaction, neither had much effect on single-class responses. Several pooling rules have been invoked to explain how ORN types combine their behavioral influences; we activated two-way combinations and compared patterns of single- and double-ORN responses: these comparisons were inconsistent with simple pooling. We conclude that the majority of primary olfactory sensory neurons have neutral behavioral effects individually, but participate in broad, odor-elicited ensembles with potent behavioral effects arising from complex interactions.

An olfactory pattern generator for on-demand combinatorial control of receptor activities

Olfactory systems employ combinatorial receptor codes for odors. Systematically generating stimuli that address the combinatorial possibilities of an olfactory code poses unique challenges. Here, we present a stimulus method to probe the combinatorial code, demonstrated using the Drosophila larva. This method leverages a set of primary odorants, each of which targets the activity of one olfactory receptor neuron (ORN) type at an optimal concentration. Our setup uses microfluidics to mix any combination of primary odorants on demand to activate any desired combination of ORNs. We use this olfactory pattern generator to demonstrate a spatially distributed olfactory representation in the dendrites of a single interneuron in the antennal lobe, the first olfactory neuropil of the larva. In the larval mushroom body, the next processing layer, we characterize diverse receptive fields of a population of Kenyon cells. The precision and flexibility of the olfactory pattern generator will facilitate systematic studies of processing and transformation of the olfactory code.

Odor sampling strategies in mice with genetically altered olfactory responses

Peripheral sensory cells and the central neuronal circuits that monitor environmental changes to drive behaviors should be adapted to match the behaviorally relevant kinetics of incoming stimuli, be it the detection of sound frequencies, the speed of moving objects or local temperature changes. Detection of odorants begins with the activation of olfactory receptor neurons in the nasal cavity following inhalation of air and airborne odorants carried therein. Thus, olfactory receptor neurons are stimulated in a rhythmic and repeated fashion that is determined by the breathing or sniffing frequency that can be controlled and altered by the animal. This raises the question of how the response kinetics of olfactory receptor neurons are matched to the imposed stimulation frequency and if, vice versa, the kinetics of olfactory receptor neuron responses determine the sniffing frequency. We addressed this question by using a mouse model that lacks the K+-dependent Na+/Ca2+ exchanger 4 (NCKX4), which results in markedly slowed response termination of olfactory receptor neuron responses and hence changes the temporal response kinetics of these neurons. We monitored sniffing behaviors of freely moving wildtype and NCKX4 knockout mice while they performed olfactory Go/NoGo discrimination tasks. Knockout mice performed with similar or, surprisingly, better accuracy compared to wildtype mice, but chose, depending on the task, different odorant sampling durations depending on the behavioral demands of the odorant identification task. Similarly, depending on the demands of the behavioral task, knockout mice displayed a lower basal breathing frequency prior to odorant sampling, a possible mechanism to increase the dynamic range for changes in sniffing frequency during odorant sampling. Overall, changes in sniffing behavior between wildtype and NCKX4 knockout mice were subtle, suggesting that, at least for the particular odorant-driven task we used, slowed response termination of the odorant-induced receptor neuron response either has a limited detrimental effect on odorant-driven behavior or mice are able to compensate via an as yet unknown mechanism.

Synaptic control of temporal processing in the Drosophila olfactory system

Temporal filtering of sensory stimuli is a key neural computation, but the way such filters are implemented within the brain is unclear. One potential mechanism for implementing temporal filters is short-term synaptic plasticity, which is governed in part by the expression of pre-synaptic proteins that position synaptic vesicles at different distances to calcium channels. Here we leveraged the Drosophila olfactory system to directly test the hypothesis that short-term synaptic plasticity shapes temporal filtering of sensory stimuli. We used optogenetic activation to drive olfactory receptor neuron (ORN) activity with high temporal precision and knocked down the presynaptic priming factor unc13A specifically in ORNs. We found that this manipulation specifically decreases and delays transmission of high frequencies, leading to poorer encoding of distant plume filaments. We replicate this effect using a previously-developed model of transmission at this synapse, which features two components with different depression kinetics. Finally, we show that upwind running, a key component of odor source localization, is preferentially driven by high-frequency stimulus fluctuations, and this response is reduced by unc13A knock-down in ORNs. Our work links the extraction of particular temporal features of a sensory stimulus to the expression of particular presynaptic molecules.

Multi-glomerular projection of single olfactory receptor neurons is conserved among amphibians

Individual receptor neurons in the peripheral olfactory organ extend long axons into the olfactory bulb forming synapses with projection neurons in spherical neuropil regions, called glomeruli. Generally, odor map formation and odor processing in all vertebrates is based on the assumption that receptor neuron axons exclusively connect to a single glomerulus without any axonal branching. We comparatively tested this hypothesis in multiple fish and amphibian species by applying sparse cell electroporation to trace single olfactory receptor neuron axons. Sea lamprey (jawless fish) and zebrafish (bony fish) support the unbranched axon concept, with 94% of axons terminating in single glomeruli. Contrastingly, axonal projections of the axolotl (salamander) branch extensively before entering up to six distinct glomeruli. Receptor neuron axons labeled in frog species (Pipidae, Bufonidae, Hylidae and Dendrobatidae) predominantly bifurcate before entering a glomerulus and 59% and 50% connect to multiple glomeruli in larval and post-metamorphotic animals, respectively. Independent of developmental stage, lifestyle and adaptations to specific habitats, it seems to be a common feature of amphibian olfactory receptor neuron axons to frequently bifurcate and connect to multiple glomeruli. Our study challenges the unbranched axon concept as a universal vertebrate feature and it is conceivable that also later diverging vertebrates deviate from it. We propose that this unusual wiring logic evolved around the divergence of the terrestrial tetrapod lineage from its aquatic ancestors and could be the basis of an alternative way of odor processing.Abstract Figure