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Physicochemical properties of odors partly correlate with AL odor representation. (a) Hierarchical cluster analysis for the 16 odors (except for geosmin, due to no data available) based on the Euclidean distances between physicochemical properties of the 16 odors. Glomerular names are colored according to the three clusters found in Fig. 4a, to facilitate comparison between these two analyses. (b) A complete distance matrix measured with Euclidean distances for 16 odors based on physicochemical properties. Each axis of the matrix is ordered as in (a). (c) Principal component analyses for the 16 odors based on physicochemical properties. The percentages of variance accounted by each PC component are shown on each axis. (d) Scatterplot of physicochemical distance versus PN response distance for all 120 pairwise combinations of the 16 odors. Pairwise distance of odors based on PN odor responses was calculated as in Fig. 4b. (PDF 762 kb)

Similar spatial odor representation patterns between PNs and OSNs in the AL. (a) Spatial response patterns for each odor are reconstructed on a template AL using odor response intensity of the 31 PN classes. (b) Spatial response patterns for each odor are reconstructed on a template AL using odor response intensity of the 29 OSN classes. In each map, the AL is viewed from anterior (top) and posterior (bottom). Each glomerulus name is indicated on the template AL (bottom right). Scale bars = 20 μm. (PDF 105472 kb)

Axonal projections of PNs in the LH and in the MB. (a) The position of glomeruli are mapped on a template AL. (b) Reconstructed axonal projections in the MB calyx and LH. The PNs are labeled with the same color scheme as in (a). (c) Individual traces of reconstructed axonal projections for the 28 PN classes after registration to the template MB calyx and LH. PNs and glomeruli are colored according to the clusters in Fig. 4c, except for VM2 (magenta), which is included in all the three clusters, and D, DM6 (cyan), which are included in the second and third clusters. Anterior view (top) and dorsal view (bottom). Scale bars = 50 μm. (PDF 43110 kb)

Odor list. (DOCX 63 kb)

Sequence similarity of odorant receptors (ORs) is not the main factor to determine odor response similarity in the AL glomerular clusters. (a) Phylogenetic tree of D. melanogaster 60 ORs. The alignment data of the 60 ORs were obtained from the Database of Olfactory Receptors (DOR; http://caps.ncbs.res.in/DOR/index.html ), and the phylogenetic tree of the 60 ORs was calculated according to [75]. The percentages of replicate trees in which the associated ORs clustered together in the bootstrap test (1000 replicates) are shown next to the branches. Glomerular name is indicated after the OR name if it is identified in the established OR-glomerulus map [5]. Glomerular names are colored according to the three glomerular clusters detected in Fig. 4c. (b) A complete distance matrix measured with Euclidean distances for 25 OR sequence similarities. (c) Scatterplot of sequence distance versus anatomical glomerular distance for all 300 pairwise combinations of the 25 ORs and the 25 glomeruli. (d) Scatterplot of sequence distance versus PN response distance for all 300 pairwise combinations of the 25 ORs and the 25 glomeruli. Pairwise distance of PN response distance was calculated based on odor response of 25 PN classes using cosine distances for 17 odors. Multiple receptors co-expressed in the same OSN class and project to the same glomerulus (i.e., DL3 and DL4) have been excluded in these analyses (c, d). (PDF 842 kb)

Correlation between glomerular distance and PN odor response similarity. Scatterplot of anatomical glomerular distance versus PN response distance for all 465 pairwise combinations of the 31 glomeruli. Pairwise distance of PN response distance was calculated based on odor response of 31 PN classes using cosine distances for 17 odors. (PDF 740 kb)

Odor response intensity of all individual PNs. Odor response intensity of each PN to 17 odors for each glomerulus, calculated by using spike frequencies during a 1-s odor stimulation period. n indicates the number of PNs recorded for each glomerulus. Odor responses of different PNs within the same PN class are indicated by different colors. The order of the 17 odors is arranged as in the inset on the bottom right for all graphs. (PDF 822 kb)

Odor representation analyzed with Euclidean distances. (a) Hierarchical cluster analysis for 17 odors based on the Euclidean distances between odor response intensities of 31 PN classes. The cut-off threshold is set at 70% of the maximum linkage distance, detecting three groups of separately clustered odors colored in red, blue, and green. (b) A complete distance matrix using Euclidean distances for 17 odors based on odor response intensities of the 31 PN classes. Each axis of the matrix is ordered as in (a). (PDF 486 kb)

Flying insects have developed a remarkably sensitive olfactory system to detect faint and turbulent odor traces. This ability is linked to the olfactory receptors class of odorant receptors (ORs), occurring exclusively in winged insects. ORs form heteromeric complexes of an odorant specific receptor protein (OrX) and a highly conserved co-receptor protein (Orco). The ORs form ligand gated ion channels that are tuned by intracellular signaling systems. Repetitive subthreshold odor stimulation of olfactory sensory neurons sensitizes insect ORs. This OR sensitization process requires Orco activity. In the present study we first asked whether OR sensitization can be monitored with heterologously expressed OR proteins. Using electrophysiological and calcium imaging methods we demonstrate that D. melanogaster OR proteins expressed in CHO cells show sensitization upon repeated weak stimulation. This was found for OR channels formed by Orco as well as by Or22a or Or56a and Orco. Moreover, we show that inhibition of calmodulin (CaM) action on OR proteins, expressed in CHO cells, abolishes any sensitization. Finally, we investigated the sensitization phenomenon using an ex vivo preparation of olfactory sensory neurons (OSNs) expressing Or22a inside the fly's antenna. Using calcium imaging, we observed sensitization in the dendrites as well as in the soma. Inhibition of calmodulin with W7 disrupted the sensitization within the outer dendritic shaft, whereas the sensitization remained in the other OSN compartments. Taken together, our results suggest that CaM action is involved in sensitizing the OR complex and that this mechanisms accounts for the sensitization in the outer dendrites, whereas further mechanisms contribute to the sensitization observed in the other OSN compartments. The use of heterologously expressed OR proteins appears to be suitable for further investigations on the mechanistic basis of OR sensitization, while investigations on native neurons are required to study the presently unknown additional mechanisms involved in OSN sensitization.

Odorant receptors (ORs) detect volatile molecules and transform this external information into an intracellular signal. Insect ORs are heteromers composed of two seven transmembrane proteins, an odor-specific OrX and a coreceptor (Orco) protein. These ORs form ligand gated cation channels that conduct also calcium. The sensitivity of the ORs is regulated by intracellular signaling cascades. Heterologously expressed Orco proteins form also non-selective cation channels that cannot be activated by odors but by synthetic agonists such as VUAA1. The stoichiometry of OR or Orco channels is unknown. In this study we engineered the simplest oligomeric construct, the Orco dimer (Orco di) and investigated its functional properties. Two Orco proteins were coupled via a 1-transmembrane protein to grant for proper orientation of both parts. The Orco di construct and Orco wild type (Orco wt) proteins were stably expressed in CHO (Chinese Hamster Ovary) cells. Their functional properties were investigated and compared by performing calcium imaging and patch clamp experiments. With calcium imaging experiments using allosteric agonist VUAA1 we demonstrate that the Orco di construct—similar to Orco wt—forms functional calcium conducting ion channel. This was supported by patch clamp experiments. The function of Orco di was seen to be modulated by CaM in a similar manner as the function of Orco wt. In addition, Orco di interacts with the OrX protein, Or22a. The properties of this complex are comparable to Or22a/Orco wt couples. Taken together, the properties of the Orco di construct are similar to those of channels formed by Orco wt proteins. Our results are thus compatible with the view that Orco wt channels are dimeric assemblies.

The perception and processing of chemical signals from the environment is essential for any living systems and is most probably the first sense developed in life. This perspective discusses the physical limits of chemoreception and gives an overview on the receptor types developed during evolution to detect chemical signals from the outside world of an organism. It discusses the interaction of chemoreceptors with downstream signaling elements, especially the interaction between electrical and chemical signaling. It is further considered how the primary chemosignal is appropriately amplified. Three examples of chemosensory systems illustrate different strategies of such amplification.