Active Tracking of Temporally Changing Gas-Phase Odor Mixture Using an Array of Cells Expressing Olfactory Receptors.

A cell expressing an olfactory receptor (OR) exhibits excellent odorant detection ability and thus is widely applied in odor biosensors. Most of those biosensors, however, could detect only liquid-phase nonchanging single-component odorants. In this paper, we raised up an odor biosensor for the active tracking of temporally changing gas-phase odor mixture by an array of cells expressing ORs. A thin stable liquid film covered the cell, thus allowing gas-phase odorants to penetrate. The online image processing generated individual cell brightness data which were used to compute the biosensor response. Based on the obtained responses, we adjusted the known odor components to be similar with the unknown odor. The function of our biosensor was validated by tracking the variable single-component odorant or the binary odor mixture. The influence from the sensor drift could be overcome by comparing the adjacent unknown and known odor responses. In the odor mixture quantification, adding the OR label to mixed cells and then quantifying separately (named as the pre-label method) was more efficient, while directly using the cell response pattern (named as the label-free method) was still capable even if the OR odor had cross-sensitivity.

Xenopus laevis Oocyte Array Fluidic Device Integrated with Microelectrodes for A Compact Two-Electrode Voltage Clamping System.

We report on a compact two-electrode voltage clamping system composed of microfabricated electrodes and a fluidic device for Xenopus laevis oocytes. The device was fabricated by assembling Si-based electrode chips and acrylic frames to form fluidic channels. After the installation of Xenopus oocytes into the fluidic channels, the device can be separated in order to measure changes in oocyte plasma membrane potential in each channel using an external amplifier. Using fluid simulations and experiments, we investigated the success rates of Xenopus oocyte arrays and electrode insertion with respect to the flow rate. We successfully located each oocyte in the array and detected oocyte responses to chemical stimuli using our device.

Use of Visual Information by Ant Species Occurring in Similar Urban Anthropogenic Environments.

Many insects, including ants, are known to respond visually to conspicuous objects. In this study, we compared orientation in an arena containing only a black target beacon as local information in six species of ants of widely varying degree of phylogenic relatedness, foraging strategy, and eye morphology (Aphaenogaster, Brachyponera, Camponotus, Formica, and two Lasius spp.), often found associated in similar urban anthropogenic habitats. Four species of ants displayed orientation toward the beacon, with two orienting toward it directly, while the other two approached it via convoluted paths. The two remaining species did not show any orientation with respect to the beacon. The results did not correlate with morphological parameters of the visual systems and could not be fully interpreted in terms of the species' ecology, although convoluted paths are linked to higher significance of chemical signals. Beacon aiming was shown to be an innate behavior in visually naive Formica workers, which, however, were less strongly attracted to the beacon than older foragers. Thus, despite sharing the same habitats and supposedly having similar neural circuits, even a very simple stimulus-related behavior in the absence of other information can differ widely in ants but is likely an ancestral trait retained especially in species with smaller eyes. The comparative analysis of nervous systems opens the possibility of determining general features of circuits responsible for innate and possibly learned attraction toward particular stimuli.

3D-Printed Bubble-Free Perfusion Cartridge System for Live-Cell Imaging.

The advent of 3D-printing technologies has had a significant effect on the development of medical and biological devices. Perfusion chambers are widely used for live-cell imaging in cell biology research; however, air-bubble invasion is a pervasive problem in perfusion systems. Although 3D printing allows the rapid fabrication of millifluidic and microfluidic devices with high resolution, little has been reported on 3D-printed fluidic devices with bubble trapping systems. Herein, we present a 3D-printed millifluidic cartridge system with bent and flat tapered flow channels for preventing air-bubble invasion, irrespective of bubble volume and without the need for additional bubble-removing devices. This system realizes bubble-free perfusion with a user-friendly interface and no-time-penalty manufacturing processes. We demonstrated the bubble removal capability of the cartridge by continually introducing air bubbles with different volumes during the calcium imaging of Sf21 cells expressing insect odorant receptors. Calcium imaging was conducted using a low-magnification objective lens to show the versatility of the cartridge for wide-area observation. We verified that the cartridge could be used as a chemical reaction chamber by conducting protein staining experiments. Our cartridge system is advantageous for a wide range of cell-based bioassays and bioanalytical studies, and can be easily integrated into portable biosensors.

Dropping Counter: A Detection Algorithm for Identifying Odour-Evoked Responses from Noisy Electroantennograms Measured by a Flying Robot.

The electroantennogram (EAG) is a technique used for measuring electrical signals from the antenna of an insect. Its rapid response time, quick recovery speed, and high sensitivity make it suitable for odour-tracking tasks employing mobile robots. However, its application to flying robots has not been extensively studied owing to the electrical and mechanical noises generated. In this study, we investigated the characteristics of the EAG mounted on a tethered flying quadcopter and developed a special counter-based algorithm for detecting the odour-generated responses. As the EAG response is negative, the algorithm creates a window and compares the values inside it. Once a value is smaller than the first one, the counter will increase by one and finally turns the whole signal into a clearer odour stimulated result. By experimental evaluation, the new algorithm gives a higher cross-correlation coefficient when compared with the fixed-threshold method. The result shows that the accuracy of this novel algorithm for recognising odour-evoked EAG signals from noise exceeds that of the traditional method; furthermore, the use of insect antennae as odour sensors for flying robots is demonstrated to be feasible.

Morphology of visual projection neurons supplying premotor area in the brain of the silkmoth Bombyx mori.

Sex pheromones orient male moths toward conspecific female moths; the presence of visual information modulates this behavior. In the current study, we explore candidate neuronal pathways for the interaction between vision and the locomotor signal for pheromone orientation. We describe the connectivity between visual neuropils and brain premotor centers, the posterior slope (PS) and the lateral accessory lobe (LAL), in the silkmoth Bombyx mori. Using a single-cell labeling technique, we analyze visual projection neurons supplying these areas. Neurons from both the medulla and lobula complex projected to the PS but only the neurons originating in the lobula complex had additional processes to the LAL. Further, we identified populations of putative feedback neurons from the premotor centers to the optic lobe. Neurons originating in the PS were likely to project to the medulla, whereas those originating in the LAL were likely to project to the lobula complex. The anatomical study contributes to further understanding of integration of visual information on the locomotor control in the insect brain.

Flexibility and control of thorax deformation during hawkmoth flight.

The interaction between neuromuscular systems and body mechanics plays an important role in the production of coordinated movements in animals. Lepidopteran insects move their wings by distortion of the thorax structure via the indirect flight muscles (IFMs), which are activated by neural signals at every stroke. However, how the action of these muscles affects thorax deformation and wing kinematics is poorly understood. We measured the deformation of the dorsal thorax (mesonotum) of tethered flying hawkmoths, Agrius convolvuli, using a high-speed laser profilometer combined with simultaneous recordings of electromyograms and wing kinematics. We observed that locally amplified mesonotum deformation near the wing hinges ensures sufficient wing movement. Furthermore, phase asymmetry in IFM activity leads to phase asymmetry in mesonotum oscillations and wingbeats. Our results revealed the flexibility and controllability of the single structure of the mesonotum by neurogenic action of the IFMs.

Cell-Based Odorant Sensor Array for Odor Discrimination Based on Insect Odorant Receptors.

The olfactory system of living organisms can accurately discriminate numerous odors by recognizing the pattern of activation of several odorant receptors (ORs). Thus, development of an odorant sensor array based on multiple ORs presents the possibility of mimicking biological odor discrimination mechanisms. Recently, we developed novel odorant sensor elements with high sensitivity and selectivity based on insect OR-expressing Sf21 cells that respond to target odorants by displaying increased fluorescence intensity. Here we introduce the development of an odorant sensor array composed of several Sf21 cell lines expressing different ORs. In this study, an array pattern of four cell lines expressing Or13a, Or56a, BmOR1, and BmOR3 was successfully created using a patterned polydimethylsiloxane film template and cell-immobilizing reagents, termed biocompatible anchor for membrane (BAM). We demonstrated that BAM could create a clear pattern of Sf21 sensor cells without impacting their odorant-sensing performance. Our sensor array showed odorant-specific response patterns toward both odorant mixtures and single odorant stimuli, allowing us to visualize the presence of 1-octen-3-ol, geosmin, bombykol, and bombykal as an increased fluorescence intensity in the region of Or13a, Or56a, BmOR1, and BmOR3 cell lines, respectively. Therefore, we successfully developed a new methodology for creating a cell-based odorant sensor array that enables us to discriminate multiple target odorants. Our method might be expanded into the development of an odorant sensor capable of detecting a large range of environmental odorants that might become a promising tool used in various applications including the study of insect semiochemicals and food contamination.

Pheromone responsiveness threshold depends on temporal integration by antennal lobe projection neurons.

The olfactory system of male moths has an extreme sensitivity with the capability to detect and recognize conspecific pheromones dispersed and greatly diluted in the air. Just 170 molecules of the silkmoth (Bombyx mori) sex pheromone bombykol are sufficient to induce sexual behavior in the male. However, it is still unclear how the sensitivity of olfactory receptor neurons (ORNs) is relayed through the brain to generate high behavioral responsiveness. Here, we show that ORN activity that is subthreshold in terms of behavior can be amplified to suprathreshold levels by temporal integration in antennal lobe projection neurons (PNs) if occurring within a specific time window. To control ORN inputs with high temporal resolution, channelrhodopsin-2 was genetically introduced into bombykol-responsive ORNs. Temporal integration in PNs was only observed for weak inputs, but not for strong inputs. Pharmacological dissection revealed that GABAergic mechanisms inhibit temporal integration of strong inputs, showing that GABA signaling regulates PN responses in a stimulus-dependent fashion. Our results show that boosting of the PNs' responses by temporal integration of olfactory information occurs specifically near the behavioral threshold, effectively defining the lower bound for behavioral responsiveness.

Identification of repellent odorants to the body louse, Pediculus humanus corporis, in clove essential oil.

The control of body lice is an important issue for human health and welfare because lice act as vectors of disease such as typhus, relapsing fever, and trench fever. Body lice exhibit avoidance behavior to some essential oils, including clove essential oil. Therefore, odorants containing clove essential oil components may potentially be useful in the development of repellents to body lice. However, such odorants that induce avoidance behavior in body lice have not yet been identified from clove essential oil. Here, we established an analysis method to evaluate the avoidance behavior of body lice to specific odorants. The behavioral analysis of the body lice in response to clove essential oil and its constituents revealed that eugenol, a major component of clove essential oil, has strong repellent effect on body lice, whereas the other components failed to induce obvious avoidance behavior. A comparison of the repellent effects of eugenol with those of other structurally related odorants revealed possible moieties that are important for the avoidance effects to body lice. The repellent effect of eugenol to body lice was enhanced by combining it with the other major component of clove essential oil, ß-caryophyllene. We conclude that a synthetic blend of eugenol and ß-caryophyllene is the most effective repellent to body lice. This finding will be valuable as the potential use of eugenol as body lice repellent.

Odorant concentration differentiator for intermittent olfactory signals.

Animals need to discriminate differences in spatiotemporally distributed sensory signals in terms of quality as well as quantity for generating adaptive behavior. Olfactory signals characterized by odor identity and concentration are intermittently distributed in the environment. From these intervals of stimulation, animals process odorant concentration to localize partners or food sources. Although concentration-response characteristics in olfactory neurons have traditionally been investigated using single stimulus pulses, their behavior under intermittent stimulus regimens remains largely elusive. Using the silkmoth (Bombyx mori) pheromone processing system, a simple and behaviorally well-defined model for olfaction, we investigated the neuronal representation of odorant concentration upon intermittent stimulation in the naturally occurring range. To the first stimulus in a series, the responses of antennal lobe (AL) projection neurons (PNs) showed a concentration dependence as previously shown in many olfactory systems. However, PN response amplitudes dynamically changed upon exposure to intermittent stimuli of the same odorant concentration and settled to a constant, largely concentration-independent level. As a result, PN responses emphasized odorant concentration changes rather than encoding absolute concentration in pulse trains of stimuli. Olfactory receptor neurons did not contribute to this response transformation which was due to long-lasting inhibition affecting PNs in the AL. Simulations confirmed that inhibition also provides advantages when stimuli have naturalistic properties. The primary olfactory center thus functions as an odorant concentration differentiator to efficiently detect concentration changes, thereby improving odorant source orientation over a wide concentration range.

Two types of local interneurons are distinguished by morphology, intrinsic membrane properties, and functional connectivity in the moth antennal lobe.

Neurons in the silkmoth antennal lobe (AL) are well characterized in terms of their morphology and odor-evoked firing activity. However, their intrinsic electrical properties including voltage-gated ionic currents and synaptic connectivity remain unclear. To address this, whole cell current- and voltage-clamp recordings were made from second-order projection neurons (PNs) and two morphological types of local interneurons (LNs) in the silkmoth AL. The two morphological types of LNs exhibited distinct physiological properties. One morphological type of LN showed a spiking response with a voltage-gated sodium channel gene expression, whereas the other type of LN was nonspiking without a voltage-gated sodium channel gene expression. Voltage-clamp experiments also revealed that both of two types of LNs as well as PNs possessed two types of voltage-gated potassium channels and calcium channels. In dual whole cell recordings of spiking LNs and PNs, activation of the PN elicited depolarization responses in the paired spiking LN, whereas activation of the spiking LN induced no substantial responses in the paired PN. However, simultaneous recording of a nonspiking LN and a PN showed that activation of the nonspiking LN induced hyperpolarization responses in the PN. We also observed bidirectional synaptic transmission via both chemical and electrical coupling in the pairs of spiking LNs. Thus our results indicate that there were two distinct types of LNs in the silkmoth AL, and their functional connectivity to PNs was substantially different. We propose distinct functional roles for these two different types of LNs in shaping odor-evoked firing activity in PNs.

A simple behaviour provides accuracy and flexibility in odour plume tracking--the robotic control of sensory-motor coupling in silkmoths.

Odour plume tracking is an essential behaviour for animal survival. A fundamental strategy for this is to move upstream and then across-stream. Male silkmoths, Bombyx mori, display this strategy as a pre-programmed sequential behaviour. They walk forward (surge) in response to the female sex pheromone and perform a zigzagging 'mating dance'. Though pre-programmed, the surge direction is modulated by bilateral olfactory input and optic flow. However, the nature of the interaction between these two sensory modalities and contribution of the resultant motor command to localizing an odour source are still unknown. We evaluated the ability of the silkmoth to localize an odour source under conditions of disturbed sensory-motor coupling, using a silkmoth-driven mobile robot. The significance of the bilateral olfaction of the moth was confirmed by inverting the olfactory input to the antennae, or its motor output. Inversion of the motor output induced consecutive circling, which was inhibited by covering the visual field of the moth. This suggests that the corollary discharge from the motor command and the reafference of self-generated optic flow generate compensatory signals to guide the surge accurately. Additionally, after inverting the olfactory input, the robot successfully tracked the odour plume by using a combination of behaviours. These results indicate that accurate guidance of the reflexive surge by integrating bilateral olfactory and visual information with innate pre-programmed behaviours increases the flexibility to track an odour plume even under disturbed circumstances.

Learning-stage-dependent plasticity of temporal coherence in the auditory cortex of rats.

Temporal coherence among neural populations may contribute importantly to signal encoding, specifically by providing an optimal tradeoff between encoding reliability and efficiency. Here, we considered the possibility that learning modulates the temporal coherence among neural populations in association with well-characterized map plasticity. We previously demonstrated that, in appetitive operant conditioning tasks, the tone-responsive area globally expanded during the early stage of learning, but shrank during the late stage. The present study further showed that phase locking of the first spike to band-specific oscillations of local field potentials (LFPs) significantly increased during the early stage of learning but decreased during the late stage, suggesting that neurons in A1 were more synchronously activated during early learning, whereas they were more asynchronously activated once learning was completed. Furthermore, LFP amplitudes increased during early learning but decreased during later learning. These results suggest that, compared to naïve encoding, early-stage encoding is more reliable but energy-consumptive, whereas late-stage encoding is more energetically efficient. Such a learning-stage-dependent encoding strategy may underlie learning-induced, non-monotonic map plasticity. Accumulating evidence indicates that the cholinergic system is likely to be a shared neural substrate of the processes for perceptual learning and attention, both of which modulate neural encoding in an adaptive manner. Thus, a better understanding of the links between map plasticity and modulation of temporal coherence will likely lead to a more integrated view of learning and attention.

Dynamic use of optic flow during pheromone tracking by the male silkmoth, Bombyx mori.

Several insects require both olfactory and visual cues during odour-source localisation to successfully locate an odour source. In the male silkmoth, Bombyx mori, detection of the female sex pheromone triggers a programmed walking pattern, starting from a surge (straight-line walking) followed by zigzag walking. Although pheromone-triggered behaviour in silkmoths is well understood, the role of visual cues remains obscure. To address this question, we performed behavioural experiments on tethered-walking moths by recording their locomotion during stimulation with a pheromone and a visual motion pattern (optic flow). The experiments were conducted under open- and closed-loop visual stimuli. We found that the use of optic flow input was determined by the behavioural state of surge and zigzagging. Silkmoths exhibited an optomotor response, which is a behavioural visual response, by turning towards the same direction as optic flow stimuli only during surge, but not during zigzagging. In addition, modulation of the zigzag walking pattern was observed when the moths were presented with biased closed-loop visual stimuli (visual feedback with biased constant optic flow); however, the directional preference mechanism was different from that of the optomotor response. Based on these findings, we suggest that the optomotor response is utilised for course control during straight-line walking, whereas the absence of optomotor response during zigzagging is used to effectively perform the programmed walking pattern. Considering the neural basis of programmed behaviour, we speculate that at least two visual pathways are involved in the state-dependent use of optic flow during odour tracking behaviour in silkmoths.

Antennal lobe organization and pheromone usage in bombycid moths.

We investigated the neuroanatomy of the macroglomerular complex (MGC), which is involved in sex pheromone processing, in five species in the subfamily Bombycinae, including Ernolatia moorei, Trilocha varians, Rondotia menciana, Bombyx mandarina and Bombyx mori. The glomerulus located at the dorsal-most part of the olfactory centre shows the largest volume in moth species examined to date. Such normal glomerular organization has been observed in E. moorei and T. varians, which use a two-component mixture and includes the compound bombykal as a mating signal. By contrast, the other three species, which use another component as a single attractant, exhibited a modified arrangement of the MGC. This correlation between pheromone usage and neural organization may be useful for understanding the process of speciation.

A single sex pheromone receptor determines chemical response specificity of sexual behavior in the silkmoth Bombyx mori.

In insects and other animals, intraspecific communication between individuals of the opposite sex is mediated in part by chemical signals called sex pheromones. In most moth species, male moths rely heavily on species-specific sex pheromones emitted by female moths to identify and orient towards an appropriate mating partner among a large number of sympatric insect species. The silkmoth, Bombyx mori, utilizes the simplest possible pheromone system, in which a single pheromone component, (E, Z)-10,12-hexadecadienol (bombykol), is sufficient to elicit full sexual behavior. We have previously shown that the sex pheromone receptor BmOR1 mediates specific detection of bombykol in the antennae of male silkmoths. However, it is unclear whether the sex pheromone receptor is the minimally sufficient determination factor that triggers initiation of orientation behavior towards a potential mate. Using transgenic silkmoths expressing the sex pheromone receptor PxOR1 of the diamondback moth Plutella xylostella in BmOR1-expressing neurons, we show that the selectivity of the sex pheromone receptor determines the chemical response specificity of sexual behavior in the silkmoth. Bombykol receptor neurons expressing PxOR1 responded to its specific ligand, (Z)-11-hexadecenal (Z11-16:Ald), in a dose-dependent manner. Male moths expressing PxOR1 exhibited typical pheromone orientation behavior and copulation attempts in response to Z11-16:Ald and to females of P. xylostella. Transformation of the bombykol receptor neurons had no effect on their projections in the antennal lobe. These results indicate that activation of bombykol receptor neurons alone is sufficient to trigger full sexual behavior. Thus, a single gene defines behavioral selectivity in sex pheromone communication in the silkmoth. Our findings show that a single molecular determinant can not only function as a modulator of behavior but also as an all-or-nothing initiator of a complex species-specific behavioral sequence.

Sex-linked transcription factor involved in a shift of sex-pheromone preference in the silkmoth Bombyx mori.

In the sex-pheromone communication systems of moths, odorant receptor (Or) specificity as well as higher olfactory information processing in males should be finely tuned to the pheromone of conspecific females. Accordingly, male sex-pheromone preference should have diversified along with the diversification of female sex pheromones; however, the genetic mechanisms that facilitated the diversification of male preference are not well understood. Here, we explored the mechanisms involved in a drastic shift in sex-pheromone preference in the silkmoth Bombyx mori using spli mutants in which the genomic structure of the gene Bmacj6, which encodes a class IV POU domain transcription factor, is disrupted or its expression is repressed. B. mori females secrete an ∼11:1 mixture of bombykol and bombykal. Bombykol alone elicits full male courtship behavior, whereas bombykal alone shows no apparent activity. In the spli mutants, the behavioral responsiveness of males to bombykol was markedly reduced, whereas bombykal alone evoked full courtship behavior. The reduced response of spli males to bombykol was explained by the paucity of bombykol receptors on the male antennae. It was also found that, in the spli males, neurons projecting into the toroid, a compartment in the brain where bombykol receptor neurons normally project, responded strongly to bombykal. The present study highlights a POU domain transcription factor, Bmacj6, which may have caused a shift of sex-pheromone preference in B. mori through Or gene choice and/or axon targeting.

Insect-machine hybrid system for understanding and evaluating sensory-motor control by sex pheromone in Bombyx mori.

To elucidate the dynamic information processing in a brain underlying adaptive behavior, it is necessary to understand the behavior and corresponding neural activities. This requires animals which have clear relationships between behavior and corresponding neural activities. Insects are precisely such animals and one of the adaptive behaviors of insects is high-accuracy odor source orientation. The most direct way to know the relationships between neural activity and behavior is by recording neural activities in a brain from freely behaving insects. There is also a method to give stimuli mimicking the natural environment to tethered insects allowing insects to walk or fly at the same position. In addition to these methods an 'insect-machine hybrid system' is proposed, which is another experimental system meeting the conditions necessary for approaching the dynamic processing in the brain of insects for generating adaptive behavior. This insect-machine hybrid system is an experimental system which has a mobile robot as its body. The robot is controlled by the insect through its behavior or the neural activities recorded from the brain. As we can arbitrarily control the motor output of the robot, we can intervene at the relationship between the insect and the environmental conditions.

Highly sensitive and selective odorant sensor using living cells expressing insect olfactory receptors.

This paper describes a highly sensitive and selective chemical sensor using living cells (Xenopus laevis oocytes) within a portable fluidic device. We constructed an odorant sensor whose sensitivity is a few parts per billion in solution and can simultaneously distinguish different types of chemicals that have only a slight difference in double bond isomerism or functional group such as -OH, -CHO and -C(=O)-. We developed a semiautomatic method to install cells to the fluidic device and achieved stable and reproducible odorant sensing. In addition, we found that the sensor worked for multiple-target chemicals and can be integrated with a robotic system without any noise reduction systems. Our developed sensor is compact and easy to replace in the system. We believe that the sensor can potentially be incorporated into a portable system for monitoring environmental and physical conditions.