Dieter Wicher

Olfactory sensory neurons (OSNs) detect odours at a wide range of intensities. In Drosophila, volatile compounds bind to specific odorant receptors (ORs), which tune the sensitivity of chemoreception. To test whether additional mechanisms underlie odour-specific neuronal processing, we analysed the spatial distribution of ORs in dendrites and investigated OSN synapses in the antennal lobe, the first relay station of the olfactory pathway. Here, we studied the molecular structure and plasticity of the presynaptic active zone (AZ), the specialized site of neurotransmitter release. We focused on a highly sensitive OSN type that expresses the receptor Or56a and is exclusively activated by geosmin, an odorant signalling ecologically harmful microorganisms. Our results uncover a differential arrangement of dendritic ORs and core AZ proteins in alarm odour-detecting Or56a compared to conventional food-odour detecting OSNs. Interestingly, the data also show that Or56a OSNs display a limited capacity for homeostatic plasticity in response to a genetic reduction of presynaptic release probability. We hypothesise that this feature reflects the basal tuning of geosmin-sensing neurons towards maximum levels of performance.

V-ATPases are ubiquitous and evolutionarily conserved rotatory proton pumps, which are crucial for maintaining various biological functions. Previous investigations have shown that a V-ATPase is present in the support cells of moth trichoid sensilla and influences their olfactory sensory neuron performance. Generally, V-ATPases are thought to regulate the pH value within the sensillum lymph, and aid K⁺ homeostasis within the sensillum. This, in turn, could influence various mechanisms involved within the support cells, like maintaining the receptor membrane potential (receptor current), nutrient and ion transport, odorant solubility, and various signaling mechanisms. In this study, we identify V-ATPase expression and localization in the Drosophila melanogaster antenna using bioinformatics and immunohistochemistry. Elucidating an olfactory V-ATPase function will improve our current understanding of how support cells contribute to Drosophila’s sense of smell.

V-ATPases are ubiquitous and evolutionarily conserved rotatory proton pumps, which are crucial for maintaining various biological functions. Previous investigations have shown that a V-ATPase is present in the support cells of moth trichoid sensilla and influences their olfactory sensory neuron performance. Generally, V-ATPases are thought to regulate the pH value within the sensillum lymph, and aid K+ homeostasis within the sensillum. This, in turn, could influence various mechanisms involved within the support cells like maintaining the receptor membrane potential (receptor current), nutrient and ion transport, odorant solubility and various signaling mechanisms. In this study, we identify V-ATPase expression and localization in the Drosophila melanogaster antenna using bioinformatics and immunohistochemistry. Elucidating an olfactory V-ATPase function will improve our current understanding of how support cells contribute to Drosophila's sense of smell.

Odor detection in insects is largely mediated by structures on antennae called sensilla, which feature a strongly conserved architecture and repertoire of olfactory sensory neurons (OSNs) and various support cell types. In Drosophila, OSNs are tightly apposed to supporting cells, whose connection with neurons and functional roles in odor detection remain unclear. Coupling mechanisms between these neuronal and non-neuronal cell types have been suggested based on morphological observations, concomitant physiological activity during odor stimulation, and known interactions that occur in other chemosensory systems. For instance, it is not known whether cell–cell coupling via gap junctions between OSNs and neighboring cells exists, or whether hemichannels interconnect cellular and extracellular sensillum compartments. Here, we show that innexins, which form hemichannels and gap junctions in invertebrates, are abundantly expressed in adult drosophilid antennae. By surveying antennal transcriptomes and performing various immunohistochemical stainings in antennal tissues, we discover innexin-specific patterns of expression and localization, with a majority of innexins strongly localizing to glial and non-neuronal cells, likely support and epithelial cells. Finally, by injecting gap junction-permeable dye into a pre-identified sensillum, we observe no dye coupling between neuronal and non-neuronal cells. Together with evidence of non-neuronal innexin localization, we conclude that innexins likely do not conjoin neurons to support cells, but that junctions and hemichannels may instead couple support cells among each other or to their shared sensillum lymph to achieve synchronous activity. We discuss how coupling of sensillum microenvironments or compartments may potentially contribute to facilitate chemosensory functions of odor sensing and sensillum homeostasis.

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.

Olfaction is a crucial sensory modality in insects and is underpinned by odor-sensitive sensory neurons expressing odorant receptors that function in the dendrites as odorant-gated ion channels. Along with expression, trafficking, and receptor complexing, the regulation of odorant receptor function is paramount to ensure the extraordinary sensory abilities of insects. However, the full extent of regulation of sensory neuron activity remains to be elucidated. For instance, our understanding of the intracellular effectors that mediate signaling pathways within antennal cells is incomplete within the context of olfaction in vivo. Here, with the use of optical and electrophysiological techniques in live antennal tissue, we investigate whether nitric oxide signaling occurs in the sensory periphery of Drosophila. To answer this, we first query antennal transcriptomic datasets to demonstrate the presence of nitric oxide signaling machinery in antennal tissue. Next, by applying various modulators of the NO-cGMP pathway in open antennal preparations, we show that olfactory responses are unaffected by a wide panel of NO-cGMP pathway inhibitors and activators over short and long timescales. We further examine the action of cAMP and cGMP, cyclic nucleotides previously linked to olfactory processes as intracellular potentiators of receptor functioning, and find that both long-term and short-term applications or microinjections of cGMP have no effect on olfactory responses in vivo as measured by calcium imaging and single sensillum recording. The absence of the effect of cGMP is shown in contrast to cAMP, which elicits increased responses when perfused shortly before olfactory responses in OSNs. Taken together, the apparent absence of nitric oxide signaling in olfactory neurons indicates that this gaseous messenger may play no role as a regulator of olfactory transduction in insects, though may play other physiological roles at the sensory periphery of the antenna.

Insects perceive countless odors with the help of odorant receptors (ORs) expressed by olfactory sensory neurons (OSNs). OR complexes are composed of odor-specific proteins OrX and olfactory co-receptor proteins Orco, with both extracellular C-terminus and intracellular N-terminus. Here, we generated a genetically modified Drosophila melanogaster fly line that expresses Orco proteins with an N-terminally fused green fluorescent protein (GFP). We generated four genotypes (homozygous and heterozygous) for which we performed antennal cryosectioning and immunolabeling to determine which genotype would show high GFP labeling intensity in the dendritic areas of OSNs. We found that fluorescent intensity was the highest in homozygous flies. As Orco is known to guide OrX proteins to the OSN dendrites, we checked whether the fusion of GFP to the Orco N-terminus may affect such trafficking and thereby OR function. Using single sensillum recording (SSR), we found no difference in the olfactory capability of homozygous flies compared with wild-type flies. As future application, we want to use this transgenic fly line to resolve the structure of OR complexes using Cryo electron microscopy (cryo-EM).

Insects detect volatile chemosignals with olfactory sensory neurons (OSNs) that express olfactory receptors. Among them, the most sensitive receptors are the odorant receptors (ORs), which form cation channels passing Ca²⁺. OSNs expressing different groups of ORs show varying optimal odor concentration ranges according to environmental needs. Certain types of OSNs, usually attuned to high odor concentrations, allow for the detection of even low signals through the process of sensitization. By increasing the sensitivity of OSNs upon repetitive subthreshold odor stimulation, Drosophila melanogaster can detect even faint and turbulent odor traces during flight. While the influx of extracellular Ca²⁺ has been previously shown to be a cue for sensitization, our study investigates the importance of intracellular Ca²⁺ management. Using an open antenna preparation that allows observation and pharmacological manipulation of OSNs, we performed Ca²⁺ imaging to determine the role of Ca²⁺ storage in mitochondria. By disturbing the mitochondrial resting potential and induction of the mitochondrial permeability transition pore (mPTP), we show that effective storage of Ca²⁺ in the mitochondria is vital for sensitization to occur, and release of Ca²⁺ from the mitochondria to the cytoplasm promptly abolishes sensitization. Our study shows the importance of cellular Ca²⁺ management for sensitization in an effort to better understand the underlying mechanics of OSN modulation.

Insects decode volatile chemical signals from its surrounding environment with the help of its olfactory system, in a fast and reliable manner for its survival. In order to accomplish this task, odorant receptors (ORs) expressed in olfactory sensory neurons (OSNs) in the fly’s antenna process such odor information. In order to study such a sophisticated process, we require access to the sensory neurons to perform functional imaging. In this article, we present different preparations to monitor odor information processing in Drosophila melanogaster OSNs using functional imaging of their Ca²⁺ dynamics. First, we established an in vivo preparation to image specific OSN population expressing the fluorescent Ca²⁺ reporter GCaMP3 during OR activation with airborne odors. Next, we developed a method to extract and to embed OSNs in a silica hydrogel with OR activation by dissolved odors. The odor response dynamics under these different conditions was qualitatively similar which indicates that the reduction of complexity did not affect the concentration dependence of odor responses at OSN level.

Insects detect volatile chemicals using antennae, which house a vast variety of olfactory sensory neurons (OSNs) that innervate hair-like structures called sensilla where odor detection takes place. In addition to OSNs, the antenna also hosts various support cell types. These include the triad of trichogen, tormogen, and thecogen support cells that lie adjacent to their respective OSNs. The arrangement of OSN supporting cells occurs stereotypically for all sensilla and is widely conserved in evolution. While insect chemosensory neurons have received considerable attention, little is known about the functional significance of the cells that support them. For instance, it remains unknown whether support cells play an active role in odor detection, or only passively contribute to homeostasis, e.g., by maintaining sensillum lymph composition. To investigate the functional interaction between OSNs and support cells, we used optical and electrophysiological approaches in Drosophila. First, we characterized the distribution of various supporting cells using genetic markers. By means of an ex vivo antennal preparation and genetically-encoded Ca²⁺ and K⁺ indicators, we then studied the activation of these auxiliary cells during odor presentation in adult flies. We observed acute responses and distinct differences in Ca²⁺ and K⁺ fluxes between support cell types. Finally, we observed alterations in OSN responses upon thecogen cell ablation in mature adults. Upon inducible ablation of thecogen cells, we notice a gain in mechanical responsiveness to mechanical stimulations during single-sensillum recording, but a lack of change to the neuronal resting activity. Taken together, these results demonstrate that support cells play a more active and responsive role during odor processing than previously thought. Our observations thus reveal that support cells functionally interact with OSNs and may be important for the extraordinary ability of insect olfactory systems to dynamically and sensitively discriminate between odors in the turbulent sensory landscape of insect flight.