Shannon B. Olsson

Phosphorylation via PKC Regulates the Function of the Drosophila Odorant Co-Receptor

Insect odorant receptors (ORs) have a unique design of heterodimers formed by an olfactory receptor protein and the ion channel Orco. Heterologously expressed insect ORs are activated via an ionotropic and a metabotropic pathway that leads to cAMP production and activates the Orco channel. The contribution of metabotropic signaling to the insect odor response remains to be elucidated. Disruption of the Gq protein signaling cascade reduces the odor response (Kain et al., 2008). We investigated this phenomenon in HEK293 cells expressing Drosophila Orco and found that phospholipase C (PLC) inhibition reduced the sensitivity of Orco to cAMP. A similar effect was seen upon inhibition of protein kinase C (PKC), whereas PKC stimulation activated Orco even in the absence of cAMP. Mutation of the five PKC phosphorylation sites in Orco almost completely eliminated sensitivity to cAMP. To test the impact of PKC activity in vivo we combined single sensillum electrophysiological recordings with microinjection of agents affecting PLC and PKC function and observed an altered response of olfactory sensory neurons (OSNs) to odorant stimulation. Injection of the PLC inhibitor U73122 or the PKC inhibitor Gö6976 into sensilla reduced the OSN response to odor pulses. Conversely, injection of the PKC activators OAG, a diacylglycerol analog, or phorbol myristate acetate (PMA) enhanced the odor response. We conclude that metabotropic pathways affecting the phosphorylation state of Orco regulate OR function and thereby shape the OSN odor response.

Insect odorant response sensitivity is tuned by metabotropically autoregulated olfactory receptors

Insects possess one of the most exquisitely sensitive olfactory systems in the animal kingdom, consisting of three different types of chemosensory receptors: ionotropic glutamate-like receptors (IRs), gustatory receptors (GRs) and odorant receptors (ORs). Both insect ORs and IRs are ligand-gated ion channels, but ORs possess a unique configuration composed of an odorant-specific protein OrX and a ubiquitous coreceptor (Orco). In addition, these two ionotropic receptors confer different tuning properties for the neurons in which they are expressed. Unlike IRs, neurons expressing ORs are more sensitive and can also be sensitized by sub-threshold concentrations of stimuli. What is the mechanistic basis for these differences in tuning? We show that intrinsic regulation of Orco enhances neuronal response to odorants and sensitizes the ORs. We also demonstrate that inhibition of metabotropic regulation prevents receptor sensitization. Our results indicate that Orco-mediated regulation of OR sensitivity provides tunable ionotropic receptors capable of detecting odors over a wider range of concentrations, providing broadened sensitivity over IRs themselves.

The chemosensory basis for behavioral divergence involved in sympatric host shifts. I. Characterizing olfactory receptor neuron classes responding to key host volatiles.

The recent shift of Rhagoletis pomonella from its native host hawthorn to introduced, domestic apple has been implicated as an example of sympatric speciation. Recent studies suggest that host volatile preference might play a fundamental role in host shifts and subsequent speciation in this group. Single sensillum electrophysiology was used to test a proposed hypothesis that differences in R. pomonella olfactory preference are due to changes in the number or odor specificity of olfactory receptor neurons. Individuals were analyzed from apple, hawthorn, and flowering dogwood-origin populations, as well as from the blueberry maggot, Rhagoletis mendax Curran (an outgroup). Eleven compounds were selected as biologically relevant stimuli from previous electroantennographic/behavioral studies of the three R. pomonella populations to host fruit volatiles. Cluster analysis of 99 neuron responses showed that cells from all tested populations could be grouped into the same five classes, ranging from those responding to one or two volatiles to those responding to several host volatiles. Topographical mapping also indicated that antennal neuron locations did not differ by class or fly taxa. Our results do not support the hypothesis that differences in host preference among Rhagoletis populations are a result of alterations in the number or class of receptor neurons responding to host volatiles.

Plant species- and status-specific odorant blends guide oviposition choice in the moth Manduca sexta

The reproductive success of herbivorous insects largely depends on the mothers oviposition preference. In nocturnal insects, olfaction is arguably the most important sensory modality mediating mate finding, foraging, and host location. In most habitats, gravid females select among a number of plants of varying suitability, yet assessment of the neuroethological mechanisms underlying odor-guided choice between host plants is rare. Using a series of behavioral, electrophysiological, and chromatographic analyses in the Hawk moth, Manduca sexta, we show that gravid females perform a hierarchical choice among host plants of different species and qualities using olfactory cues. Both relevant plant species and qualities can be distinguished by volatile profiles collected from the headspace of these plants, and olfactory sensilla on female antennae detect more than half of the about 120 analytically detected volatiles in host plant headspace samples. Although olfactory sensory neurons present in antennal sensilla are mainly broadly tuned to multiple host compounds, some sensilla exhibit species and condition-specific responses. In fact, species and quality can be distinguished by the physiologically active components alone. Our findings thus suggest that distinguishing characteristics of both host species and quality are already represented at the sensory periphery.

Neuronal processing of complex mixtures establishes a unique odor representation in the moth antennal lobe.

Animals typically perceive natural odor cues in their olfactory environment as a complex mixture of chemically diverse components. In insects, the initial representation of an odor mixture occurs in the first olfactory center of the brain, the antennal lobe (AL). The contribution of single neurons to the processing of complex mixtures in insects, and in particular moths, is still largely unknown. Using a novel multicomponent stimulus system to equilibrate component and mixture concentrations according to vapor pressure, we performed intracellular recordings of projection and interneurons in an attempt to quantitatively characterize mixture representation and integration properties of single AL neurons in the moth. We found that the fine spatiotemporal representation of 2–7 component mixtures among single neurons in the AL revealed a highly combinatorial, non-linear process for coding host mixtures presumably shaped by the AL network: 82% of mixture responding projection neurons and local interneurons showed non-linear spike frequencies in response to a defined host odor mixture, exhibiting an array of interactions including suppression, hypoadditivity, and synergism. Our results indicate that odor mixtures are represented by each cell as a unique combinatorial representation, and there is no general rule by which the network computes the mixture in comparison to single components. On the single neuron level, we show that those differences manifest in a variety of parameters, including the spatial location, frequency, latency, and temporal pattern of the response kinetics.

Olfaction and Identification of Unrelated Individuals: Examination of the Mysteries of Human Odor Recognition

Although several studies have examined the effect of human odor on kin recognition and mate choice, few have focused on the impact of familiarity on recognition of nonrelatives by olfactory cues. As part of a program designed to engage students in scientific research, 53 high school students researched, planned, and implemented a project to analyze the effect of odor on human recognition of, and preference for, friends, sex, and self. A total of 37 students, including friends of their choosing, wore T-shirts for three consecutive nights. During that time, subjects were controlled for exposure to extraneous perfumes, household odors, and other humans. The students were then asked to smell a series of five shirts and evaluate them with respect to pleasantness. Students were also asked to identify the shirts belonging to themselves and their friend, and determine the sex of the person who wore each shirt. Although subjects were unable to distinguish sex by olfactory cues alone, a significant percentage of subjects were able to identify their own odor (51.6%), as well as distinguish the odor cue of their friend (38.7%). Additionally, subjects who could not identify their friends cue were apt to choose the odor of a member of the opposite sex as their friend. This result was not believed to rely on odor preference as neither individual, friend, nor stranger odors were rated significantly different with respect to odor pleasantness. The ability to recognize friends via odor cues lends credence to the hypothesis that association and familiarity are important aspects of conspecific olfactory recognition in humans. Furthermore, this study augments evidence that olfaction may supplement visual and auditory cues used in human conspecific and kin recognition.

Temporal response dynamics of Drosophila olfactory sensory neurons depends on receptor type and response polarity

Insect olfactory sensory neurons (OSN) express a diverse array of receptors from different protein families, i.e. ionotropic receptors (IR), gustatory receptors (GR) and odorant receptors (OR). It is well known that insects are exposed to a plethora of odor molecules that vary widely in both space and time under turbulent natural conditions. In addition to divergent ligand specificities, these different receptors might also provide an increased range of temporal dynamics and sensitivities for the olfactory system. To test this, we challenged different Drosophila OSNs with both varying stimulus durations (10–2000 ms), and repeated stimulus pulses of key ligands at various frequencies (1–10 Hz). Our results show that OR-expressing OSNs responded faster and with higher sensitivity to short stimulations as compared to IR- and Gr21a-expressing OSNs. In addition, OR-expressing OSNs could respond to repeated stimulations of excitatory ligands up to 5 Hz, while IR-expressing OSNs required ~5x longer stimulations and/or higher concentrations to respond to similar stimulus durations and frequencies. Nevertheless, IR-expressing OSNs did not exhibit adaptation to longer stimulations, unlike OR- and Gr21a-OSNs. Both OR- and IR-expressing OSNs were also unable to resolve repeated pulses of inhibitory ligands as fast as excitatory ligands. These differences were independent of the peri-receptor environment in which the receptors were expressed and suggest that the receptor expressed by a given OSN affects both its sensitivity and its response to transient, intermittent chemical stimuli. OR-expressing OSNs are better at resolving low dose, intermittent stimuli, while IR-expressing OSNs respond more accurately to long-lasting odor pulses. This diversity increases the capacity of the insect olfactory system to respond to the diverse spatiotemporal signals in the natural environment.

Antennal lobe processing correlates to moth olfactory behavior.

Animals typically perceive their olfactory environment as a complex blend of natural odor cues. In insects, the initial processing of odors occurs in the antennal lobe (AL). Afferent peripheral input from olfactory sensory neurons (OSNs) is modified via mostly inhibitory local interneurons (LNs) and transferred by projection neurons (PNs) to higher brain centers. Here we performed optophysiological studies in the AL of the moth, Manduca sexta, and recorded odor-evoked calcium changes in response to antennal stimulation with five monomolecular host volatiles and their artificial mixture. In a double staining approach, we simultaneously measured OSN network input in concert with PN output across the glomerular array. By comparing odor-evoked activity patterns and response intensities between the two processing levels, we show that host mixtures could generally be predicted from the linear summation of their components at the input of the AL, but output neurons established a unique, nonlinear spatial pattern separate from individual component identities. We then assessed whether particularly high levels of signal modulation correspond to behavioral relevance. One of our mixture components, phenyl acetaldehyde, evoked significant levels of nonlinear input-output modulation in observed spatiotemporal activation patterns that were unique from the other individual odorants tested. This compound also accelerated behavioral activity in subsequent wind tunnel tests, whereas another compound that did not exhibit high levels of modulation also did not affect behavior. These results suggest that the high degree of input-output modulation exhibited by the AL for specific odors can correlate to behavioral output.

A novel multicomponent stimulus device for use in olfactory experiments

Olfactory studies have expanded beyond the study of single compound odor perception to explore the processing of complex mixtures and blends. The spatiotemporal presentation of blend stimuli is a challenging task requiring volatiles with diverse chemical and physical properties to be presented as a unified stimulus. This not only necessitates accurate control of the timing and homogeneity of the odor stream, but requires attention to the concentration of each blend component presented. We have developed a novel, multicomponent stimulus system for use in olfactory experiments that is capable of presenting up to 8 different odors simultaneously or in sequence at defined concentrations and time scales. Each odor is separated to minimize physical or chemical interactions, and stimulations are performed from a saturated headspace of the odor solution. Stimulus concentrations can be measured empirically or estimated using common gas laws. Photoionization detector measurements show that stimuli could be presented as cohesive blends or single components at frequencies of at least 10Hz without leakage or contamination. Solid phase microextraction measurements also show that the concentration of each component could be equilibrated through regulation of each component lines flow rate based on the different partial vapor pressures of the odorants. This device provides a unique method for introducing complex volatile mixtures for olfactory studies in a variety of animal taxa and allows for accurate control of odor intensities in both time and space.

Receptor expression and sympatric speciation: unique olfactory receptor neuron responses in F1 hybrid Rhagoletis populations.

SUMMARY The Rhagoletis pomonella species complex is one of the foremost examples supporting the occurrence of sympatric speciation. A recent study found that reciprocal F1 hybrid offspring from different host plant-infesting populations in the complex displayed significantly reduced olfactory host preference in flight-tunnel assays. Behavioral and electrophysiological studies indicate that olfactory cues from host fruit are important chemosensory signals for flies to locate fruit for mating and oviposition. The reduced olfactory abilities of hybrids could therefore constitute a significant post-mating barrier to gene flow among fly populations. The present study investigated the source of changes in the hybrid olfactory system by examining peripheral chemoreception in F1 hybrid flies, using behaviorally relevant volatiles from the parent host fruit. Single-sensillum electrophysiological analyses revealed significant changes in olfactory receptor neuron (ORN) response specificities in hybrid flies when compared to parent ORN responses. We report that flies from F1 crosses of apple-, hawthorn- and flowering dogwood-origin populations of R. pomonella exhibited distinct ORN response profiles absent from any parent population. These peripheral alterations in ORN response profiles could result from misexpression of multiple receptors in hybrid neurons as a function of genomic incompatibilities in receptor-gene pathways in parent populations. We conclude that these changes in peripheral chemoreception could impact olfactory host preference and contribute directly to reproductive isolation in the Rhagoletis complex, or could be genetically coupled to other host-associated traits.