Primary culture of lobster (Homarus americanus) olfactory sensory neurons

Lobster olfactory sensory neurons have contributed to a number of advances in our understanding of olfactory physiology. To facilitate further study of their function, we have developed conditions allowing primary culture of the olfactory sensory neurons in a defined medium. The most common cells in the culture were round cell bodies with diameters of 10-15 micro m that often extended fine processes, features resembling olfactory sensory neurons. We discovered that acetylcholinesterase acted as a growth factor for these cells, improving their survival in culture. We also confirmed previous evidence from spiny lobsters that poly-D-lysine was a superior substrate for olfactory cells of this size and morphology. We then identified olfactory sensory neurons in the culture in two ways. Almost half the cells tested responded to application of a complex odorant with an inward current. An even more rigorous test was made possible by the development of an antiserum to OET-07, an ionotropic glutamate receptor homolog specifically expressed by Homarus americanus olfactory sensory neurons. It labeled a majority of the round cells in the culture, unequivocally identifying them as olfactory sensory neurons.     

Pheromone-binding proteins contribute to the activation of olfactory receptor neurons in the silkmoths antheraea polyphemus and Bombyx mori

The sensilla trichodea of the silkmoth Antheraea polyphemus are innervated by three types of receptor neurons each responding specifically to one of three pheromone components. The sensillum lymph of these sensilla surrounding the sensory dendrites contains three different types of pheromone-binding proteins (PBPs) in high concentrations. The sensilla trichodea of the silkmoth Bombyx mori are supplied by two receptor neurons each tuned specifically to one of the two pheromone components bombykol and bombykal, but only one type of PBP has been found so far in these sensilla. Recombinant PBPs of both silkmoth species in various combinations with pheromone components were applied to the receptor neurons via tip-opened sensilla during electrophysiological recordings. Over a fairly broad range of pheromone concentrations the responses of the receptor neurons depended on both, the pheromone component and the type of the PBP. Therefore, the PBPs appear to contribute to the excitation of the receptor neurons. Furthermore, bombykal in combination with the expressed PBP of B. mori failed to activate the corresponding receptor neuron of B. mori, but did so if combined with one of the PBPs of A. polyphemus. Therefore, a still unknown binding protein involved in bombykal transport might be present in B. mori.     

Effects of perceived and imagined odors on taste detection

We assessed the influence of different odors on detection of a sweet tastant, and the ability of imagined odors to elicit the same effects as perceived odors on taste perception. The tastant used was sucrose, and the two odorants were strawberry and ham. In the first experiment, participants either smelled or imagined one of two odors during taste detection tasks (between-subject design), whereas in the second one, subjects completed both the odor imagery and perception conditions with taste detection tasks (within-subject design). The effect was odorant-specific: detection of sucrose was significantly better when subjects smelled strawberry than when they smelled ham. Furthermore, imagined odors influenced taste perception in the same way as did perceived odors. We concluded that the odor-specific effect on taste perception is an authentic perceptual phenomenon. Our results also support the notion that odor-induced changes in taste perception are mediated centrally. Finally, our findings are in agreement with reports supporting the existence of odor imagery.     

Analysis of the functional maturation of olfactory neurons in chicks before and after birth

There has been indirect evidence that the olfactory system of mammals could be functional shortly before birth. Taking advantage of the accessibility of bird embryos, we studied the functional maturation of the olfactory mucosa during embryonic development in birds. Using the combination of electrophysiological EOG recordings and immunohistochemical studies, it was possible to directly demonstrate for the first time that the olfactory system is functional during embryogenesis from embryonic day (ED) 13 and that the beginning of olfactory function coincides with the first localization of the calcium dependent calmodulin kinase II (CaMKIIalpha) in the dendrites of the olfactory receptor neurons. CaMKII and olfactory receptor genes are expressed much earlier in olfactory neurons, both involved in the sensory transduction, but the pattern of expression of CaMKIIalpha changes during the ontogenesis. The increase of EOG amplitude between ED13 and ED15 also coincides with the increase of the number of neurons presenting the dendritic localization of CaMKIIalpha. These results suggest that the enzyme CaMKII might play a role in the functional maturation of the olfactory mucosa.     

Monosodium glutamate and sweet taste: generalization of conditioned taste aversion between glutamate and sweet stimuli in rats

Even though monosodium glutamate (MSG) is a prototypical umami substance, previous studies reported that a conditioned taste aversion (CTA) to MSG, mixed with amiloride to block the taste of sodium, generalizes to sucrose. These findings suggest that the taste of glutamate mimics the taste of sucrose and raise the question of whether glutamate has a broadly tuned sweet taste component. To test this hypothesis, CTA experiments were conducted to test for generalization between MSG and several sweet stimuli: sucrose, glucose, maltose, saccharin and SC-45647. Strong bidirectional generalization was seen between MSG mixed with amiloride and sucrose, glucose, saccharin and SC-45647. Weak generalization was seen between MSG and maltose, and sucrose and maltose. None of the CTAs generalized to NMDA. These findings support the hypothesis that the taste of MSG has broadly tuned, sweet-like characteristics, possibly due to the convergence of afferent signals for MSG, natural sugars and artificial sweeteners.     

Influences of age and sex on a microencapsulated odor memory test

While the influences of such variables as age and sex are well established for most standardized tests of odor identification and detection, this is not the case for tests of odor memory. In this study, 231 non-smoking men and women, ranging in age from 10 to 68 years, were administered a standardized 12-item match-to-sample microencapsulated odor memory test (OMT). Anosmics were excluded from the study. Each participant was asked to smell a target odorant after its release from a microencapsulated odorant pad and then, after a delay interval of 10, 30 or 60 s, to pick the target from a similarly presented set of four odors, three of which were foils. Backward counting by threes was required during the delay intervals in an effort to minimize semantic rehearsal. Overall OMT scores were higher for women than for men, and decreased, in each sex, as a function of age in a manner similar to the age-related decline observed in tests of odor identification and detection. Performance did not change as a function of delay interval. A significant correlation between the overall OMT test scores and scores on the University of Pennsylvania Smell Identification Test was observed for women, but not for men, in accord with the notion that women may be more likely to employ semantic cues in their strategies to remember odors. The findings are discussed in light of the complexities of the construct of odor memory.     

The influence of training on chemosensory event-related potentials and interactions between the olfactory and trigeminal systems

It is not possible to accurately predict the perceptual response to odorants and odorant mixtures without understanding patterns of suppression and facilitation that result from interactions between the olfactory and trigeminal systems. The current study extends previous findings by exploring the effect of intensive training on the interaction between these systems and also by using a different mixed chemosensory stimulus to examine whether the principles established in earlier studies generalize to different odorants. Stimuli were chosen so as to selectively activate the olfactory (H2S) and trigeminal (CO2) nerves. In addition, linalool was included as a stimulus that activated both systems. Thirty-five participants (19 men, 16 women) rated the intensity of each stimulus when presented both alone and in binary mixtures (linalool + H2S, and linalool + CO2). Chemosensory event-related potentials were obtained from three recording positions. Analysis of intensity ratings showed that linalool was significantly less intense than the other stimuli when presented alone. In binary mixtures, H2S was strongly suppressed by linalool. One week of intensive odor training produced significant and specific reductions in the intensity of linalool and H2S, both alone and in their mixture. Training with a different odor (champignol) had no effect. Chemosensory event-related potential data confirmed previous findings showing changes in topographical distribution that reflected the degree of trigeminal activity. Binary mixtures generally produced larger amplitudes than single stimuli. Latencies clearly differentiated between the three single stimuli and the binary mixtures. Changes were observed in event-related potentials that reflected those obtained for intensity ratings in that they were observed for linalool and H2S in the linalool trained group only. The amplitude of the late 'endogenous' component (P3) was significantly decreased for these odors at frontal recording sites. In summary, strong and specific training effects were observed in intensity ratings for participants trained with the test odor (linalool), but not for those trained with a different odor. This was supported by a significant decrease of amplitudes of the event-related potentials at frontal recording sites following training with the test odor only     

The influence of auditory and visual experience on the phonotactic behavior of the cricket,Gryllus bimaculatus

Female crickets lacking experience with phonotaxis to conspecific calling song respond to trains of continuously repeated sound pulses (trill), whereas experienced females do not. In the present study such inexperienced crickets were tested for their responsiveness to trills of pulse repetition periods from 30 to 70 ms on a Y- shaped maze. Stimulation with a repetition period of 30 ms led to unexpectedly low phonotactic and exploratory activity. Initial stimulation with trills of 30- ms repetition period drastically reduced the responsiveness of inexperienced animals to conspecific calling song and other attractive stimuli. Effects of visual stimulation on the phonotactic behavior of female crickets are demonstrated. Threatening visual stimuli changed the behavior of experienced animals to a state that resembles that of inexperienced animals. The relevance of these observations is discussed with respect to the development of the auditory pattern recognition mechanism in crickets.     

Role of visual and olfactory stimuli in host finding of adult cabbage root flies, Delia radicum

Visual host finding and form recognition by the cabbage root fly, Delia radicum, were studied using four shapes of yellow sticky traps as plant models. Cross, disc, and Y traps caught similar numbers of flies. Cross traps caught more females than vertical or horizontal rectangular traps. Black borders did not increase rectangle trap catches. Discs (8 cm diam.) at ground level caught more females than discs at 40 cm above ground. Larger discs (11.2 and 13.8 cm diam.) caught more females per trap than 8 cm diam. discs, but not as many per unit area.Volatile mustard oils were added to sticky cross traps as single and multiple-component baits. Traps with multiple-component baits did not consistently catch more females than single baited traps. Yellow traps baited with isothiocyanates caught 4–7 times as many females as clear plastic allylisothiocyanate (ANCS)-baited traps.Results indicated yellow discs or crosses at ground level beited with ANCS effectively monitored female D. radicum. Main factors affecting landing of female D. radicum are suggested to be color of substrate, height above ground, presence of host volatiles in vicinity, visual prominence, and area of attractive color. The view that host finding in some insects is mediated by complex responses to multichannel stimuli was supported.

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Résumé L'étude de la perception visuelle et de la reconnaissance des formes par les D. radicum adultes a été réalisée en utilisant des pièges jaunes gluants de quatre formes différentes comme leurres de plantes. Des pièges en croix, en disque et en Y ont permis la capture des mêmes nombres de mouches. Plus de femelles ont été capturées avec des pièges en croix qu'avec des pièges rectangulaires verticaux ou horizontaux. Les captures n'ont pas augmenté avec des pièges bordés de noir. Des disques de 8 cm de diamètre disposés au niveau du sol ont permis la capture de plus de femelles que des disques à 40 cm au-dessus du sol. La capture par piège a été supérieure avec des pièges de 11,2 et 13,8 cm de diamètre qu'avec ceux de 8 cm, mais moindre par unité de surface.Des essences volatiles de moutarde ont été ajoutées aux pièges en croix comme appâts simples ou composés. Les pièges avec appâtes composés n'ont pas été significativement plus efficaces que les pièges à appâts simples. Les pièges jaunes appâts avec de l'isothiocyanata ont entraîné la capture de 4 à 7 fois plus de mouches que des pièges en plastique transparent appâtés avec de l'allylisothiocyanate (ANCS). Les résultats ont montré que des disques ou des croix jaunes appâtés avec de l'ANCS au niveau du sol permettent d'organiser un avertissement agricole contre D. radicum. Les principaux facteurs intervenant dans l'atterrissage des femelles de D. radicum semblent être la couleur du substrat, l'élévation audessus du sol, le contraste visuel, la présence de substance de l'hôte dans le voisinage, et l'aire d'attractivité de la couleur. En conclusion, il est exact de considérer que la perception de l'hôte est provoquée chez quelques insectes par une réponse complexe à des stimuli multicanaux.

     

Orientation responses of the grasshopper, Melanoplus sanguinipes, to visual, olfactory and wind stimuli and their combinations

Prereproductive adults of the grasshopper, Melanoplus sanguinipes (F.) (Orthoptera, Acrididae), demonstrated orientation and movement towards both visual and olfactory stimulus sources in a still-air chamber. Visual stimuli (wheat and lima bean foliage, vertical black or yellow-green stripes, and a yellow-green broad leaf pattern) were approached more frequently than the control white background surface. Olfactory stimuli (chopped wheat foliage and a four-component, synthetic, grass odor blend of volatiles) elicited an even greater positive response than the visual stimuli. Changing the proportions of the four volatiles in the blend significantly reduced positive orientation responses to the stimulus source. Visual cues of wheat foliage and olfactory cues of either chopped wheat odor or the grass odor blend gave greater responses when combined than when presented separately.In flowing air or wind, nearly all insects demonstrated a rapid positive response to odors of chopped wheat and the grass odor blend, significantly greater than the response to the same stimuli in still air. However, positive responses to visual cues were not significantly greater in wind than in still air. When combined with the olfactory stimuli in flowing air, visual cues did not increase the incidence of response. Grasshoppers responding to grass odors in wind moved more rapidly and directly toward the source, and stopped less often and for shorter durations than insects responding to odor in still air or to visual cues.We conclude from these studies that M. sanguinipes adults show orientation behavior to both visual and olfactory stimuli from food plant sources, although leaf odors elicit a stronger positive response particularly when carried by wind.     

Genetic analysis of auditory neuropathy spectrum disorder in the Korean population

Auditory neuropathy spectrum disorder (ANSD) is caused by dys-synchronous auditory neural response as a result of impairment of the functions of the auditory nerve or inner hair cells, or synapses between inner hair cells and the auditory nerve. To identify a causative gene causing ANSD in the Korean population, we conducted gene screening of the OTOF, DIAPH3, and PJVK genes in 19 unrelated Korean patients with ANSD. A novel nonsense mutation (p.Y1064X) and a known pathogenic mutation (p.R1939Q) of the OTOF gene were identified in a patient as compound heterozygote. Pedigree analysis for these mutations showed co-segregation of mutation genotype and the disease in the family, and it supported that the p.Y1064X might be a novel genetic cause of autosomal recessive ANSD. A novel missense variant p.K1017R (c.3050A>G) in the DIAPH3 gene was also identified in the heterozygous state. In contrast, no mutation was detected in the PJVK gene. These results indicate that no major causative gene has been reported to date in the Korean population and that pathogenic mutations in undiscovered candidate genes may have an effect on ANSD.     

Odorant binding and conformational changes of a rat odorant-binding protein

Odorant-binding proteins (OBPs) are lipocalins secreted in the nasal mucus of vertebrates, which convey odorants to their neuronal receptors. We compared the binding properties of a recombinant rat OBP (OBP-1F) using a set of six odorants of various chemical structures. We examined the binding properties by both fluorescent probe competition and isothermal titration calorimetry. OBP-1F affinity constants, in the micromolar range, varied by more than one order of magnitude and were roughly correlated to the odorant size. The observed binding stoichiometry was found to be around one odorant per dimer. Using tyrosine differential spectroscopy, the binding of ligand was shown to induce local conformational changes. A three-dimensional structure of OBP-1F, modelled using the known structure of aphrodisin as template, allowed us to suggest the location of the observed structural changes outside of the binding pocket. These results are consistent with one binding site located in one of the two beta-barrels of the OBP-1F dimer and a subtle conformational change correlated with binding of an odorant molecule, which hampers uptake of a second odorant by the other hydrophobic pocket.     

Host location and discrimination mediated through olfactory stimuli in two species of Encyrtidae

The role of olfactory stimuli in host detection and evaluation was studied in two encyrtid Hymenoptera. The first, Epidinocarsis lopezi De Santis, is a monophagous parasitoid of the cassava mealybug Phenacoccus manihoti Matile-Ferrero, itself feeding exclusively on cassava, Manihot esculenta Crantz. The second, Leptomastix dactylopii Howard, is a monophagous parasitoid of the Citrus mealybug, Planococcus citri Risso, but this latter is highly polyphagous. The behaviour of females of both parasitoids (attaction and locomotion) was compared in a tubular olfactometer for the odours of their respective hosts on cassava and poinsettia. Tests were made using: 1) healthy host-plant alone; 2) host-plant infested with unparasitized mealybugs; 3) unparasitized mealybugs only; 4) host-plant infested with parasitized mealybugs and 5) parasitized mealybugs only. Only E. lopezi was attracted by the odour of the host-plant alone, but both species were attracted by the odour of an infested host-plant and that of unparasitized mealybugs. The odour of parasitized mealybugs, alone or on host-plant, induced an undirected activity. The attraction of E. lopezi to the odour of the host-plant alone could be linked to the monophagous diet of its host, whereas the attraction of the two species of parasitoids to the odours of infested host-plants and unparasitized mealybugs could be due to the fact that both parasitoids are specialists. The behavioural response of both species to the odour of parasitized mealybugs revealed a new aspect in host discrimination: the identification of parasitized hosts could be partly mediated through olfactory stimuli, and not only through gustatory stimuli.     

Detection of coloured stimuli by honeybees: minimum visual angles and receptor specific contrasts

Honeybees Apis mellifera were trained to distinguish between the presence and the absence of a rewarded coloured spot, presented on a vertical, achromatic plane in a Y-maze. They were subsequently tested with different subtended visual angles of that spot, generated by different disk diameters and different distances from the decision point in the device. Bees were trained easily to detect bee-chromatic colours, but not an achromatic one. Chromatic contrast was not the only parameter allowing learning and, therefore, detection: _min, the subtended visual angle at which the bees detect a given stimulus with a probability P ₀ = 0.6, was 5° for stimuli presenting both chromatic contrast and contrast for the green photoreceptors [i.e. excitation difference in the green photoreceptors, between target and background (green contrast)], and 15° for stimuli presenting chromatic but no green contrast. Our results suggest that green contrast can be utilized for target detection if target recognition has been established by means of the colour vision system. The green-contrast signal would be used as a far-distance signal for flower detection. This signal would always be detected before chromatic contrast during an approach flight and would be learned in compound with chromatic contrast, in a facilitation-like process.     

Volatile release from liquids: a comparison of in vivo APCI-MS, in-mouth headspace trapping and in vitro mouth model data

In-mouth volatile release from flavoured water was followed using atmospheric pressure chemical ionization-mass spectrometry (APCI-MS) or using a hand-held, computer-controlled device based on sequential trapping of flavours on Tenax traps. The present results verify recent in vitro data obtained with a sophisticated, fully computerized mouth model apparatus and confirm its validity for the simulation of in-mouth dynamic volatile release. In-nose APCI-MS measurements showed considerable person-to-person variability in non-trained individuals during drinking due to subconscious control of muscles during swallowing and subsequent breathing. Data showed a 'swallow breath' volume reaching the nasal cavity from the throat, not from the mouth cavity. Flavour enriched air from the mouth was shown to be transported to the nose (via exhalation) immediately after the swallowing event, but the dynamic process of volatile equilibration between residuals of the swallowed liquid and the exhaled air predominantly determined volatile in-nose concentration. Owing to its dynamic character, the process of volatile equilibration and release in the throat upon exhalation should be similar to the in-mouth process studied in the present work. A full mechanical simulation of retronasal volatile transport, however, will remain difficult.     

Age and rearing environment interact in the retention of early olfactory memories in honeybees

Due to the changing behavioral contexts at which social insects are exposed during the adult lifespan, they are ideal models to analyze the effect of particular sensory stimuli during young adulthood on later behavior. Specifically, our goal is to understand early influences on later foraging behavior. For that, olfactory memories were established by worker honeybees to different pre-foraging ages using either (1) classical conditioning in the proboscis extension response (PER) paradigm or (2) the offering of scented-sugar solution under different rearing conditions. By testing long-term memories (LTM) through a single PER test in workers of foraging ages (17-25 days), we found that retention of the early olfactory memories in honey bees is age-dependent and not time-dependent. Independently of the environmental conditions in which they were reared (laboratory cages or hives), bees were able to retain food-odor association from 5 days after emergence, but rarely before. In most experiments we observed a bi-modal pattern of response: bees exposed to scented-food at 5-8 and 13-16 days showed better retention than those exposed at 9-12 days. These differences disappeared for bees reared in hives. Retrieval of LTMs depending on the timing and the continuous inputs of appropriate sensory stimuli are discussed.     

Inward currents and increases in cytosolic Ca2+ concentration induced by cyclic ADP-ribose in turtle olfactory receptor cells

In olfactory receptor cells, it is well established that cyclic AMP (cAMP) and inositol-1,4,5-trisphosphate (IP(3)) act as second messengers during odor responses. In previous studies, we have shown that cAMP-increasing odorants induce odor responses even after complete desensitization of the cAMP-mediated pathway. These results suggest that at least one cAMP-independent pathway contributes to the generation of odor responses. In an attempt to identify a novel second messenger, we investigated the possible role of cyclic ADP-ribose (cADPR) in olfactory transduction. Turtle olfactory receptor cells were isolated using an enzyme-free procedure and loaded with fura-2/AM. The cells responded to dialysis with cADPR with an inward current and an increase of the intracellular Ca(2+) concentration, [Ca(2+)](i). Flooding of cells with 100 microM cADPR from the pipette also induced an inward current without changes in [Ca(2+)](i) in Na(+)-containing and Ca(2+)-free Ringer solution. In an Na(+)-free and Ca(2+)-containing Ringer solution, cADPR induced only a small inward current with a concomitant increase in [Ca(2+)](i). Inward currents and increases in [Ca(2+)](i) induced by cADPR were completely inhibited by removal of both Na(+) and Ca(2+) from the outer solution. The experiments suggest that cADPR activates a cation channel at the plasma membrane, allowing inflow of Na(+) and Ca(2+) ions. The magnitudes of the inward current responses to cAMP-increasing odorants were greatly reduced by prior dialyses of a high concentration of cADPR or 8-bromo-cyclic ADP-ribose (8-Br-cADPR), an antagonist. It is possible that the cADPR-dependent pathway contributes to the generation of olfactory responses.