Molecular Cloning and Characterization of a Lobster Gαs Protein Expressed in Neurons of Olfactory Organ and Brain

Abstract: We have isolated from an American lobster ( Homarus americanus ) olfactory organ cDNA library a clone, lobGα_s, with >70% identity to mammalian and arthropod Gα_s sequences. In genomic Southern blots, a fragment of lobGα_s detected only one band, suggesting the lobsters have a single Gα_s gene. In brain and olfactory organ, lobGα_s mRNA was expressed predominantly in neurons, including many of the neuronal cell body clusters of the brain. Gα_s protein was also expressed broadly, appearing on western blots as a band of 51.8 kDa in brain, eyestalk, pereiopod, dactyl, tail muscle, olfactory organ, and aesthetasc hairs. These results suggest that lobGα_s plays a role in a wide variety of signal transduction events. Its presence in the olfactory aesthetasc hairs, which are almost pure preparations of the outer dendrites of the olfactory receptor neurons, and the expression of lobGα_s mRNA in the olfactory receptor neurons of the olfactory organ indicate that lobGα_s may mediate olfactory transduction. That virtually all ORNs express lobGα_s mRNA equally predicts that hyperpolarizing odor responses mediated by cyclic AMP are a property of all lobster olfactory receptor neurons.     

Calcium Regulation of Cyclic Nucleotide Signaling in Lobster Olfactory Receptor Neurons

An elevated free Ca2+ concentration reduces odor-stimulated production of cyclic AMP (cAMP) in the outer dendritic membranes of lobster olfactory receptor neurons in vitro. This effect can occur within 50 ms of odor stimulation. The effect is concentration-dependent at submicromolar concentrations of free Ca2+. An elevated free Ca2+ concentration also reduces basal and forskolin-stimulated cAMP levels in a concentration-dependent manner, suggesting that Ca2+ is not targeting the activation of the odor receptor/G protein complex. The degradation of synthetic cAMP by phosphodiesterases is not enhanced by an increased free Ca2+ concentration, suggesting that Ca2+ acts by down-regulating the olfactory adenylyl cyclase. Western blot analysis of the lobster olfactory sensilla that contain the outer dendrites reveals a protein in the transduction zone with a molecular mass of approximately 138 kDa that is immunoreactive to an antiserum against adenylyl cyclase type III. Given earlier evidence that Ca2+ potentially enters the receptor cell through odor-activated inositol 1,4,5-trisphosphate-gated channels, our results suggest a possible route for cross talk between the cyclic nucleotide and the inositol phospholipid signaling pathways in lobster olfactory receptor neurons.     

Chemical communication in heliothine moths

The chemical and temporal features of the sex-pheromone emitted by Heliothis virescens females are encoded by a diverse array of output pathways from the male-specific macroglomerular complex (MGC) in the antennal lobe. Most output neurons (29 out of 32) were activated by antennal stimulation with the principal component of the sex-pheromone blend of this species, (Z)-11-hexadecenal. Six neurons were excited solely by this component, 8 neurons also responded to the second essential blend component, (Z)-9-tetradecenal, and 14 neurons displayed equivalent responses to the two. Many neurons also effectively encoded the onset and duration of the stimulus. In one additional neuron, a prolonged excitatory response (synergism) was evoked only by the blend of the two components, indicating that some MGC neurons function as blend detectors.In contrast to the situation in Helicoverpa zea, none of the MGC neurons in H. virescens responded selectively to (Z)-9-tetradecenal, suggesting that these two noctuid species employ different neural strategies to encode information about their respective pheromone blends.Three MGC-output neurons responded selectively to (Z)-11-hexadecenyl acetate, an odorant released by some sympatric species that disrupts normal upwind flight to pheromones. Thus, changes in the attractant and deterrent chemical signals, as well as the physical features of these odor plumes, are encoded in the MGC across a diverse parallel array of output pathways to the protocerebrum.Abbreviations AL antennal lobe - AN antennal nerve - 16:AL hexadecanal - MGC macroglomerular complex - 14:AL tetradecanal - Z11-16:AL (Z) 11-hexadecenal - Z11-16:AC (Z) 11-hexadecenyl acetate - Z9-14:AL (Z) 9-tetradecenal - Z9-14:FO (Z) 9-tetradecenyl formate     

The Molecular Phylogeny of a Nematode-Specific Clade of Heterotrimeric G-Protein α-Subunit Genes

In animal olfactory systems, odorant molecules are detected by olfactory receptors (ORs). ORs are part of the G-protein-coupled receptor (GPCR) superfamily. Heterotrimeric guanine nucleotide binding G-proteins (G-proteins) relay signals from GPCRs to intracellular effectors. G-proteins are comprised of three peptides. The G-protein α subunit confers functional specificity to G-proteins. Vertebrate and insect Gα-subunit genes are divided into four subfamilies based on functional and sequence attributes. The nematode Caenorhabditis elegans contains 21 Gα genes, 14 of which are exclusively expressed in sensory neurons. Most individual mammalian cells express multiple distinct GPCR gene products, however, individual mammalian and insect olfactory neurons express only one functional odorant OR. By contrast C. elegans expresses multiple ORs and multiple Gα subunits within each olfactory neuron. Here we show that, in addition to having at least one member of each of the four mammalian Gα gene classes, C. elegans and other nematodes also possess two lineage-specific Gα gene expansions, homologues of which are not found in any other organisms examined. We hypothesize that these novel nematode-specific Gα genes increase the functional complexity of individual chemosensory neurons, enabling them to integrate odor signals from the multiple distinct ORs expressed on their membranes. This neuronal gene expansion most likely occurred in nematodes to enable them to compensate for the small number of chemosensory cells and the limited emphasis on cephalization during nematode evolution. [Reviewing Editor: Dr. John Oakeshott] Damien M. O’Halloran and David A. Fitzpatrick contributed equally to this work.     

Brain and behaviour of living and extinct echidnas

The Tachyglossidae (long- and short-beaked echidnas) are a family of monotremes, confined to Australia and New Guinea, that exhibit striking trigeminal, olfactory and cortical specialisations. Several species of long-beaked echidna (Zaglossus robusta, Zaglossus hacketti, Megalibgwilia ramsayi) were part of the large-bodied (10 kg or more) fauna of Pleistocene Australasia, but only the diminutive (2–7 kg) Tachyglossus aculeatus is widespread today on the Australian mainland. We used high-resolution CT scanning and other osteological techniques to determine whether the remarkable neurological specialisations of modern echidnas were also present in Pleistocene forms or have undergone modification as the Australian climate changed in the transition from the Pleistocene to the Holocene. All the living and extinct echidnas studied have a similar pattern of cortical gyrification that suggests comparable functional topography to the modern short-beaked form. Osteological features related to olfactory, trigeminal, auditory and vestibular specialisation (e.g., foramina and cribriform plate area, osseous labyrinth topography) are also similar in living and extinct species. Our findings indicate that despite differences in diet, habitat and body size, the suite of neurological specialisations in the Tachyglossidae has been remarkably constant: encephalisation, sensory anatomy and specialisation (olfactory, trigeminal, auditory and vestibular), hypoglossal nerve size and cortical topography have all been stable neurological features of the group for at least 300,000 years.     

Decyl-thio-trifluoropropanone, a competitive inhibitor of moth pheromone receptors

An earlier study (Pophof 1998) showed that the esterase inhibitor decyl-thio-trifluoropropanone inhibited the responses of two receptor neurons of the moth Antheraea polyphemus tuned to straight-chain pheromone components, an acetate and an aldehyde, respectively. Here we report that decyl-thio-trifluoropropanone also inhibited the responses of two pheromone receptor neurons of Bombyx mori to bombykol and bombykal. In contrast, decyl-thio-trifluoropropanone activated receptor neurons of the moth Imbrasia cytherea tuned to the pheromone component (Z)-5-decenyl 3-methyl-butanoate. However, decyl-thio-trifluoropropanone did not affect the responses of two receptor neurons of B. mori females specialized to the plant volatiles benzoic acid and linalool, respectively. These results indicate that decyl-thio-trifluoropropanone, besides inhibiting the sensillar esterase, interferes with proteins involved specifically in the excitation of pheromone receptor neurons. In binding studies with radiolabelled decyl-thio-trifluoropropanone, the inhibitor was bound by the pheromone-binding protein of A. polyphemus. However, the amount of decyl-thio-trifluoropropanone causing response inhibition was 300 times lower than the amount of pheromone-binding protein present in the sensilla. Since the amount of decyl-thio-trifluoropropanone adsorbed corresponded to about the maximum number of receptor molecules calculated per sensillum, we expect that decyl-thio-trifluoropropanone, probably in complex with pheromone-binding protein, competitively inhibits the pheromone receptor molecules. Accepted: 8 January 2000     

Mutational Analysis of Cysteine Residues of the Insect Odorant Co-receptor (Orco) from Drosophila melanogaster Reveals Differential Effects on Agonist- and Odorant-tuning Receptor-dependent Activation

Insect odorant receptors are heteromeric odorant-gated cation channels comprising a conventional odorant-sensitive tuning receptor (ORx) and a highly conserved co-receptor known as Orco. Orco is found only in insects, and very little is known about its structure and the mechanism leading to channel activation. In the absence of an ORx, Orco forms homomeric channels that can be activated by a synthetic agonist, VUAA1. Drosophila melanogaster Orco (DmelOrco) contains eight cysteine amino acid residues, six of which are highly conserved. In this study, we replaced individual cysteine residues with serine or alanine and expressed Orco mutants in Flp-In 293 T-Rex cells. Changes in intracellular Ca²⁺ levels were used to determine responses to VUAA1. Replacement of two cysteines (Cys-429 and Cys-449) in a predicted intracellular loop (ICL3), individually or together, gave variants that all showed similar increases in the rate of response and sensitivity to VUAA1 compared with wild-type DmelOrco. Kinetic modeling indicated that the response of the Orco mutants to VUAA1 was faster than wild-type Orco. The enhanced sensitivity and faster response of the Cys mutants was confirmed by whole-cell voltage clamp electrophysiology. In contrast to the results from direct agonist activation of Orco, the two cysteine replacement mutants when co-expressed with a tuning receptor (DmelOR22a) showed an ∼10-fold decrease in potency for activation by 2-methyl hexanoate. Our work has shown that intracellular loop 3 is important for Orco channel activation. Importantly, this study also suggests differences in the structural requirements for the activation of homomeric and heteromeric Orco channel complexes.     

Olfactory Discrimination and Transient Mood Change in Young Men and Women: Variation by Season,Mood State,and Time of Day

Odor performance varies by clinical state and gender, though little is known about its variation by season or time of day. Many odors, including lavender, induce transient mood changes. This study explored discrimination differences between various lavender oil blends and their effects on transient mood in the morning and evening in depressed and nondepressed adults. We also determined seasonal influences on these measures. A total of 169 subjects, 98 women and 71 men (mean age ± SD, 19.3 ± 1.6 y) participated, with different subjects studied at different times of the year. The Beck Depression Inventory (BDI) classified subjects as depressed (score ≥10; N = 57) or nondepressed (score <10; N = 112). In the discrimination test, subjects compared pairs of two different lavender oil blends or a control. Transient change in mood was assessed by the Profile of Mood States (POMS) after each trial of five lavender blends interspersed by three control odors. Tests were conducted in the morning (08:00–10:00 h) and evening (18:00–20:00 h). In all subjects, discrimination was significantly better for some odor pairs than for others, thus demonstrating test specificity. Discrimination was better overall in the fall than winter/spring and better in depressed than nondepressed subjects for specific odor pairs. No significant gender or time-of-day differences in discriminability were detected. There were, however, significant group differences in transient mood profiles. Current depressed state affected mood response, with lavender increasing anger in depressed subjects only. In addition, depressed subjects and men, whether or not depressed, exhibited diurnal mood variation, with better mood in the evening; the former group also showed more evening energy. All subjects were more confused in the morning than evening. Season also affected transient mood; winter/spring-tested subjects reported more vigor than fall-tested subjects. In addition, summer-tested subjects showed more tension in the morning, whereas fall-tested subjects showed the opposite pattern in the evening. In all subjects, lavender increased fatigue, tension, confusion, and total mood disturbance, and it decreased vigor. The study showed that both chronobiological (seasonal and time-of-day) and clinical factors modify discrimination and mood response to odors. Brief lavender odor presentation may serve as a nonphotic method for altering mood in young depressed and nondepressed adults particularly during the fall, a time of heightened discriminability.     

The effect of environmental temperature on olfactory perception in Drosophila melanogaster

Olfaction provides chemical information to an animal about its environment. When environmental conditions change, individuals should be able to adequately maintain function. Temperature may influence olfaction in a double manner, as it modifies the concentrations of gaseous compounds and affects biological processes. Here, we address acclimatization to environmental temperature in the olfactory system of Drosophila melanogaster using heat and cold treatments. Because the consequences of temperature shifts persist for some time after the treatment's end, comparison of olfactory behaviors at the same temperature in treated and untreated flies allows us to infer the biological effects of temperature in olfaction.At intermediate odorant concentrations heat always generates a reduction of olfactory sensitivity, as they would be expected to compensate for the increase of volatiles in the air. Cold produces the opposite effect. These changes are observed in both sexes and in natural populations as well as in standard laboratory stocks.Short applications suffice to cause detectable olfactory perception changes, but even prolonged temperature treatments have only a transitory effect. Together, these results suggest that olfaction in Drosophila underlies acclimatization to environmental temperature. However, sensitivity changes are not immediate and may cause imperfect adjustment of olfactory function for short time periods.