Computational exploration of molecular receptive fields in the olfactory bulb reveals a glomerulus-centric chemical map

© The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.Progress in olfactory research is currently hampered by incomplete knowledge about chemical receptive ranges of primary receptors. Moreover, the chemical logic underlying the arrangement of computational units in the olfactory bulb has still not been resolved. We undertook a large-scale approach at characterising molecular receptive ranges (MRRs) of glomeruli in the dorsal olfactory bulb (dOB) innervated by the MOR18-2 olfactory receptor, also known as Olfr78, with human ortholog OR51E2. Guided by an iterative approach that combined biological screening and machine learning, we selected 214 odorants to characterise the response of MOR18-2 and its neighbouring glomeruli. We found that a combination of conventional physico-chemical and vibrational molecular descriptors performed best in predicting glomerular responses using nonlinear Support-Vector Regression. We also discovered several previously unknown odorants activating MOR18-2 glomeruli, and obtained detailed MRRs of MOR18-2 glomeruli and their neighbours. Our results confirm earlier findings that demonstrated tunotopy, that is, glomeruli with similar tuning curves tend to be located in spatial proximity in the dOB. In addition, our results indicate chemotopy, that is, a preference for glomeruli with similar physico-chemical MRR descriptions being located in spatial proximity. Together, these findings suggest the existence of a partial chemical map underlying glomerular arrangement in the dOB. Our methodology that combines machine learning and physiological measurements lights the way towards future high-throughput studies to deorphanise and characterise structure-activity relationships in olfaction.Peer reviewe

Neuromediators in the developing olfactory system: 3D-reconstruction towards a functional understanding

During ontogenesis of holometabolic insects like butterflies (Lepidoptera) life circumstances change radically. While larval life is dominated by eating and growing, adult animals need to cope with quite different properties of sensory stimuli, especially during flight. This asks for a complete rebuilding of the brain during metamorphosis to fulfill adult-specific requirements. One well-established model system to examine these develomental processes is the brain of the sphinx moth Manduca sexta. Within about three weeks of pupal development the whole brain increases about ten times in size, several larval cells die, other adult-specific neuroblasts start to proliferate while other neuronal cells are restructured to deal with adult-specific tasks. Many of the underlying ontogenetic processes of outgrowing and differentiation of neural cells seem to be conserved between insects and vertebrates. On the molecular level as well as on the level of neuropilar organization exist parallels between both, which usually are more accessible in the insect model. One example would be target selection of sensory receptor neurons, which is steered by similar contact receptors. Another example is the analogous organization of olfactory neuropils into spherical glomeruli which exhibit odor-dependent activation. The gaseous signal molecule nitric oxide (NO) and its influence on neurogenesis represents another example of a conserved concept during individual development of various systems, including antennal lobe development in Manduca. The modes of action of NO are manyfold: it may influence proteins by ADP-ribosylation or S-nitrosylation, or may stimulate further intracellular signalling cascades. Most prominent example in Manduca would be the transient cGMP synthesis by NO-dependent soluble guanylyl cyclase. This cGMP in turn exhibits its own broad spectrum of downstream activation, reaching from directly dependent ion channels (cyclic nucleotide gated channels, CNGs) to protein kinases and phosphodiesterases. Within the framework of our working hypothesis the release of neuropeptides in developing local interneurons is mediated by selective cGMP production during a defined time window. This allows for specific refinement of synaptic contacts coming from a previous unspecific developmental signal. Our existing neuropeptide data was extended by anatomical and pharmacological examinations regarding allatotropin and further characterization of the lateral cell group by MALDI-TOF mass spectrometry. The emphasis of this dissertation however was laid on further characterization of the NO/cGMP signalling pathway during antennal lobe develoment of Manduca sexta. First, the cell group in question was examined towards its ability to extend the NO signal by Cu,Zn superoxide dismutase expression, followed by a test for cell death during antennal lobe development. Since no dying cells were found throughout antennal lobe development, a stable cell population could be assumed. Now, the NO/cGMP signalling pathway was further examined with respect to temporal occurrence, specificity, and number of cells involved; additionally, its possible mode of action on neuronal development was pointed out by integrating previous work. Within this framework, 3D-reconstruction based volumetry was used, which proved to be a powerful tool to answer other questions during antennal lobe development as well. Prerequisite herefore was, however, the establishment of a suitable protocol, together with the definition of easily and reproducably identifyable neuropilar regions in the antennal lobe during development. After introduction of this protocol, an extention onto whole brain neuropils was obvious, which was done in a supervised diploma work. In this context, a Manduca standard brain was created and used to volumetrically examine brain sex dimorphism. In a cooperation project, anatomical expertise on the antennal lobe system was applied by identification and classification of intracellulary recorded projection neurons, combined wtih a 4D representation of the data obtained

Connectomics Analysis Reveals First-, Second-, and Third-Order Thermosensory and Hygrosensory Neurons in the Adult Drosophila Brain.

Animals exhibit innate and learned preferences for temperature and humidity-conditions critical for their survival and reproduction. Leveraging a whole-brain electron microscopy volume, we studied the adult Drosophila melanogaster circuitry associated with antennal thermo- and hygrosensory neurons. We have identified two new target glomeruli in the antennal lobe, in addition to the five known ones, and the ventroposterior projection neurons (VP PNs) that relay thermo- and hygrosensory information to higher brain centers, including the mushroom body and lateral horn, seats of learned and innate behavior. We present the first connectome of a thermo- and hygrosensory neuropil, the lateral accessory calyx (lACA), by reconstructing neurons downstream of heating- and cooling-responsive VP PNs. A few mushroom body-intrinsic neurons solely receive thermosensory input from the lACA, while most receive additional olfactory and thermo- and/or hygrosensory PN inputs. Furthermore, several classes of lACA-associated neurons form a local network with outputs to other brain neuropils, suggesting that the lACA serves as a hub for thermo- and hygrosensory circuitry. For example, DN1a neurons link thermosensory PNs in the lACA to the circadian clock via the accessory medulla. Finally, we survey strongly connected downstream partners of VP PNs across the protocerebrum; these include a descending neuron targeted by dry-responsive VP PNs, meaning that just two synapses might separate hygrosensory inputs from motor circuits. These data provide a comprehensive first- and second-order layer analysis of Drosophila thermo- and hygrosensory systems and an initial survey of third-order neurons that could directly modulate behavior.MRC LMB Graduate Studentship to M.W.P. Boehringer Ingelheim Fonds PhD Fellowship and a Herchel Smith Studentship to A.S.B. Cambridge Neuroscience-PSL collaborative grant supported by the Embassy of France in London to G.S.X.E.J

Odor guided behavior and its modulation in the fruit fly Drosophila melanogaster

Geosmin is produced by a group of microbes, is highly aversive to Drosophila. We furthermore showed that Drosophila has one type of sensory neurons expressing an odorant receptor (OR 56a) that is extremely sensitive and fully dedicated to detect this compound. The Geosmin detection System is conserved across the genus Drosophila. Exploiting aversivespecific circuits could be an efficient method for manipulating disease vectors as well as agricultural pests. Next I showed that flies exhibit increased attraction to ethyl acetate, to another food odor, even to a repellent and pheromone after starvation. All odors evoked stronger physiological responses in starved flies with the effects being most pronounced at low odor concentrations. I investigated, which molecular players could be involved in such modulation. Conducting RNAi knockdown experiments with some the identified genes coupled with a quantitative olfactory assay to identify regulators of such modulation. Only silencing the CCHamide1 receptor resulted in an abolished starvation effect, suggesting a major role in starvation-induced modulation. This study shows that the fly’s antennal olfactory sensing neurons (OSNs) seem to be constantly tuned to current needs. There are Inhibitory projection neurons (iPNs) target the LH exclusively and bypass the Mushroom body (MBc). By using a specific driver line (MZ699) we were able to investigate the impact of this inhibitory pathway on olfactory processing within the LH. We performed calcium imaging experiments and found three domains with different highly stereotypic response patterns in lateral horn (LH). The posterior medial domain (LH PM domain) was strongly activated by innately attractive odorants, while the anterior lateral domain was mainly activated by repellent odorants. The third anterior medial domain (LH AM domain) was valence independent and mainly coded for odorant intensity. Do these iPNS decode odor features and govern decision making? By selectively silencing iPNs (via interrupting GABA synthesis) we were able to reduce the flies’ attraction to almost all attractive odorants

Leucine-rich repeat transmembrane proteins instruct discrete dendrite targeting in an olfactory map

Olfactory systems utilize discrete neural pathways to process and integrate odorant information. In Drosophila, axons of first-order olfactory receptor neurons (ORNs) and dendrites of second-order projection neurons (PNs) form class-specific synaptic connections at ~50 glomeruli. The mechanisms underlying PN dendrite targeting to distinct glomeruli in a three-dimensional discrete neural map are unclear. We found that the leucine-rich repeat (LRR) transmembrane protein Capricious (Caps) was differentially expressed in different classes of PNs. Loss-of-function and gain-of-function studies indicated that Caps instructs the segregation of Caps-positive and Caps-negative PN dendrites to discrete glomerular targets. Moreover, Caps-mediated PN dendrite targeting was independent of presynaptic ORNs and did not involve homophilic interactions. The closely related protein Tartan was partially redundant with Caps. These LRR proteins are probably part of a combinatorial cell-surface code that instructs discrete olfactory map formation

The Role of Drosophila Odorant Receptors in Odor Coding

Drosophila melanogaster is a powerful genetic model organism, and a promising model system in olfaction. At the onset of my thesis research, the expression patterns of fly’s 62 odorant receptors (ORs) were largely unknown. I set out to understand the rules of connectivity of olfactory sensory neurons and the resulting properties of olfactory circuit. Consequently, we assembled maps of the olfactory neuron projections in the fly brain and characterized the contribution of several ORs to olfactory-guided behavior. We compiled near-complete maps of OR-specific neuronal projections to the antennal lobe glomeruli of adult and larval fly brains. We analyzed expression profiles of 42 ORs, 31 of which are expressed in the adult and 21 in the larva, with an overlap of 10 ORs between the two developmental stages. Our results show surprising complexity in organization of the fly’s olfactory circuit. Four adult olfactory neuron populations co-express two ORs each and another olfactory neuron population expresses one odorant and one gustatory receptor. One glomerulus receives co-convergent input from two separate populations of olfactory neurons. Three ORs label sexually dimorphic glomeruli implicated in sexual courtship, and are thus candidate Drosophila pheromone receptors. The organization of larval antennal lobe is remarkably similar to that of adult flies and mammals; each glomerulus occupies a unique stereotyped position in the antennal lobe. Unlike in adults, each OR is expressed in only one neuron, forming glomeruli with single afferents. The olfactory sensory maps provide experimental framework for relating ORs to olfactory neuroanatomy, and ultimately, to output of the olfactory system. The Drosophila larval olfactory system shows great promise as a behavioral model. Larvae exhibit robust chemotaxis to odors and have a simple olfactory system. We utilized larvae to study response properties of three olfactory neurons to a large panel of odors. Behavioral assays of larvae with single olfactory neurons ablated, showed minimal effects on chemotaxis response, and thus great redundancy in function of olfactory neuron populations. Larvae with only Or42a olfactory neurons functional are able to chemotax robustly, demonstrating that chemotaxis is possible in the absence of the remaining elements of the olfactory circuit

Brain architecture in the terrestrial hermit crab Coenobita clypeatus (Anomura, Coenobitidae), a crustacean with a good aerial sense of smell

<p>Abstract</p> <p>Background</p> <p>During the evolutionary radiation of Crustacea, several lineages in this taxon convergently succeeded in meeting the physiological challenges connected to establishing a fully terrestrial life style. These physiological adaptations include the need for sensory organs of terrestrial species to function in air rather than in water. Previous behavioral and neuroethological studies have provided solid evidence that the land hermit crabs (Coenobitidae, Anomura) are a group of crustaceans that have evolved a good sense of aerial olfaction during the conquest of land. We wanted to study the central olfactory processing areas in the brains of these organisms and to that end analyzed the brain of <it>Coenobita clypeatus </it>(Herbst, 1791; Anomura, Coenobitidae), a fully terrestrial tropical hermit crab, by immunohistochemistry against synaptic proteins, serotonin, FMRFamide-related peptides, and glutamine synthetase.</p> <p>Results</p> <p>The primary olfactory centers in this species dominate the brain and are composed of many elongate olfactory glomeruli. The secondary olfactory centers that receive an input from olfactory projection neurons are almost equally large as the olfactory lobes and are organized into parallel neuropil lamellae. The architecture of the optic neuropils and those areas associated with antenna two suggest that <it>C. clypeatus </it>has visual and mechanosensory skills that are comparable to those of marine Crustacea.</p> <p>Conclusion</p> <p>In parallel to previous behavioral findings of a good sense of aerial olfaction in C. clypeatus, our results indicate that in fact their central olfactory pathway is most prominent, indicating that olfaction is a major sensory modality that these brains process. Interestingly, the secondary olfactory neuropils of insects, the mushroom bodies, also display a layered structure (vertical and medial lobes), superficially similar to the lamellae in the secondary olfactory centers of <it>C. clypeatus</it>. More detailed analyses with additional markers will be necessary to explore the question if these similarities have evolved convergently with the establishment of superb aerial olfactory abilities or if this design goes back to a shared principle in the common ancestor of Crustacea and Hexapoda.</p

Odor representations in the mammalian olfactory bulb

A first key step in studying a sensory modality is to define how the brain represents the features of the sensory stimulus. This has proven to be a challenge in olfaction, where even the stimulus features have been a matter of considerable debate. In this review, we focus on olfactory representations in the first stage of the olfactory pathway, the olfactory bulb (OB). We examine the diverging viewpoints on spatially organized versus distributed representations. We then consider how odor sampling through respiration is a key part of the odorant code. Finally, we ask how the bulb handles the challenging task of representing mixtures. We suggest that current evidence points toward a representation that is spatially organized at the inputs but later distributed, with the spatial organization not being used for much computation. Nevertheless, this is a simple representation that effectively represents multiple individual odorants, as well as odor mixtures

Individual Neurons Confined to Distinct Antennal-Lobe Tracts in the Heliothine Moth: Morphological Characteristics and Global Projection Patterns

To explore fundamental principles characterizing chemosensory information processing, we have identified antennal-lobe projection neurons in the heliothine moth, including several neuron types not previously described. Generally, odor information is conveyed from the primary olfactory center of the moth brain, the antennal lobe, to higher brain centers via projection neuron axons passing along several parallel pathways, of which the medial, mediolateral, and lateral antennal-lobe tract are considered the classical ones. Recent data have revealed the projections of the individual tracts more in detail demonstrating three main target regions in the protocerebrum; the calyces are innervated mainly by the medial tract, the superior intermediate protocerebrum by the lateral tract exclusively, and the lateral horn by all tracts. In the present study, we have identified, via iontophoretic intracellular staining combined with confocal microscopy, individual projection neurons confined to the tracts mentioned above, plus two additional ones. Further, using the visualization software AMIRA, we reconstructed the stained neurons and registered the models into a standard brain atlas, which allowed us to compare the termination areas of individual projection neurons both across and within distinct tracts. The data demonstrate a morphological diversity of the projection neurons within distinct tracts. Comparison of the output areas of the neurons confined to the three main tracts in the lateral horn showed overlapping terminal regions for the medial and mediolateral tracts; the lateral tract neurons, on the contrary, targeted mostly other output areas in the protocerebrum

Metamorphic development of the olfactory system in the red flour beetle (Tribolium castaneum, HERBST)

Background: Insects depend on their olfactory sense as a vital system. Olfactory cues are processed by a rather complex system and translated into various types of behavior. In holometabolous insects like the red flour beetle Tribolium castaneum, the nervous system typically undergoes considerable remodeling during metamorphosis. This process includes the integration of new neurons, as well as remodeling and elimination of larval neurons. Results: We find that the sensory neurons of the larval antennae are reused in the adult antennae. Further, the larval antennal lobe gets transformed into its adult version. The beetle’s larval antennal lobe is already glomerularly structured, but its glomeruli dissolve in the last larval stage. However, the axons of the olfactory sensory neurons remain within the antennal lobe volume. The glomeruli of the adult antennal lobe then form from mid-metamorphosis independently of the presence of a functional OR/Orco complex but mature dependent on the latter during a postmetamorphic phase. Conclusions: We provide insights into the metamorphic development of the red flour beetle’s olfactory system and compared it to data on Drosophila melanogaster, Manduca sexta, and Apis mellifera. The comparison revealed that some aspects, such as the formation of the antennal lobe’s adult glomeruli at mid-metamorphosis, are common, while others like the development of sensory appendages or the role of Orco seemingly differ