Spatial perception mediated by insect antennal mechanosensory system.

Animals perceive their surroundings using various modalities of sensory inputs to guide their locomotion. Nocturnal insects such as crickets use mechanosensory inputs mediated by their antennae to orient in darkness. Spatial information is acquired via voluntary antennal contacts with surrounding objects, but it remains unclear whether the insects modulate behaviors mediated by other sensory organs based on that information. Crickets exhibit escape behavior in response to a short air puff, which is detected by the abdominal mechanosensory organs called cerci and is perceived as a 'predator approach' signal. We placed objects of different shapes at different locations with which the cricket actively made contact using its antennae. We then examined the effects on wind-elicited escape behavior. The crickets changed their movement trajectory in response to nearby objects such as walls so that they could avoid collision with these obstacles even during the cercal-mediated behavior. For instance, when a wall was placed in front of the crickets so that it was detected by one antenna, the escape trajectory in response to a stimulus from behind was significantly biased toward the side opposite the wall. Even when the antenna on the free side without the wall was ablated, this collision avoidance was also observed, suggesting that the mechanosensory inputs from one antennae detecting an object edge would be sufficient to perceive the location of obstacle in front. This study demonstrated that crickets were able to use the spatial information acquired with their antennal system to modify their behavior mediated by other sensory organs.

Internal state transition to switch behavioral strategies in cricket phonotaxis.

Animals employ multiple behavioral strategies for exploring food and mating partners based on both their internal state and external environment. Here, we examined how cricket phonotaxis, which was considered an innate reactive behavior of females to approach the calling song of conspecific males, depended on these internal and external conditions. Our observation revealed that the phonotaxis process consisted of two distinctive phases: wandering and approaching. In the latter phase, crickets moved directly towards the sound source. The transition into this phase, referred to as the 'approach phase', was based on changes in the animal's internal state. Moreover, retention of the approach phase required recognition of the calling song, while song loss downregulated cricket mobility and induced frequent stopping. This is a typical movement in local search behaviors. Our results indicate that phonotaxis is not only a reactive response but a complicated process including multiple behavioral strategies.

Multisensory enhancement of burst activity in an insect auditory neuron.

Detecting predators is crucial for survival. In insects, a few sensory interneurons receiving sensory input from a distinct receptive organ extract specific features informing the animal about approaching predators and mediate avoidance behaviors. Although integration of multiple sensory cues relevant to the predator enhances sensitivity and precision, it has not been established whether the sensory interneurons that act as predator detectors integrate multiple modalities of sensory inputs elicited by predators. Using intracellular recording techniques, we found that the cricket auditory neuron AN2, which is sensitive to the ultrasound-like echolocation calls of bats, responds to airflow stimuli transduced by the cercal organ, a mechanoreceptor in the abdomen. AN2 enhanced spike outputs in response to cross-modal stimuli combining sound with airflow, and the linearity of the summation of multisensory integration depended on the magnitude of the evoked response. The enhanced AN2 activity contained bursts, triggering avoidance behavior. Moreover, cross-modal stimuli elicited larger and longer lasting excitatory postsynaptic potentials (EPSP) than unimodal stimuli, which would result from a sublinear summation of EPSPs evoked respectively by sound or airflow. The persistence of EPSPs was correlated with the occurrence and structure of burst activity. Our findings indicate that AN2 integrates bimodal signals and that multisensory integration rather than unimodal stimulation alone more reliably generates bursting activity. NEW & NOTEWORTHY Crickets detect ultrasound with their tympanum and airflow with their cercal organ and process them as alert signals of predators. These sensory signals are integrated by auditory neuron AN2 in the early stages of sensory processing. Multisensory inputs from different sensory channels enhanced excitatory postsynaptic potentials to facilitate burst firing, which could trigger avoidance steering in flying crickets. Our results highlight the cellular basis of multisensory integration in AN2 and possible effects on escape behavior.

Direction-Specific Adaptation in Neuronal and Behavioral Responses of an Insect Mechanosensory System.

Stimulus-specific adaptation (SSA) is considered to be the neural underpinning of habituation to frequent stimuli and novelty detection. However, neither the cellular mechanism underlying SSA nor the link between SSA-like neuronal plasticity and behavioral modulation is well understood. The wind-detection system in crickets is one of the best models for investigating the neural basis of SSA. We found that crickets exhibit stimulus-direction-specific adaptation in wind-elicited avoidance behavior. Repetitive air currents inducing this behavioral adaptation reduced firings to the stimulus and the amplitude of excitatory synaptic potentials in wind-sensitive giant interneurons (GIs) related to the avoidance behavior. Injection of a Ca(2+) chelator into GIs diminished both the attenuation of firings and the synaptic depression induced by the repetitive stimulation, suggesting that adaptation of GIs induced by this stimulation results in Ca(2+)-mediated modulation of postsynaptic responses, including postsynaptic short-term depression. Some types of GIs showed specific adaptation to the direction of repetitive stimuli, resulting in an alteration of their directional tuning curves. The types of GIs for which directional tuning was altered displayed heterogeneous direction selectivity in their Ca(2+) dynamics that was restricted to a specific area of dendrites. In contrast, other types of GIs with constant directionality exhibited direction-independent global Ca(2+) elevation throughout the dendritic arbor. These results suggest that depression induced by local Ca(2+) accumulation at repetitively activated synapses of key neurons underlies direction-specific behavioral adaptation. This input-selective depression mediated by heterogeneous Ca(2+) dynamics could confer the ability to detect novelty at the earliest stages of sensory processing in crickets. SIGNIFICANCE STATEMENT: Stimulus-specific adaptation (SSA) is considered to be the neural underpinning of habituation and novelty detection. We found that crickets exhibit stimulus-direction-specific adaptation in wind-elicited avoidance behavior. Repetitive air currents inducing this behavioral adaptation altered the directional selectivity of wind-sensitive giant interneurons (GIs) via direction-specific adaptation mediated by dendritic Ca(2+) elevation. The GIs for which directional tuning was altered displayed heterogeneous direction selectivity in their Ca(2+) dynamics and the transient increase in Ca(2+) evoked by the repeated puffs was restricted to a specific area of dendrites. These results suggest that depression induced by local Ca(2+) accumulation at repetitively activated synapses of key neurons underlies direction-specific behavioral adaptation. Our findings elucidate the subcellular mechanism underlying SSA-like neuronal plasticity related to behavioral adaptation.

Spatial dynamics of action potentials estimated by dendritic Ca(2+) signals in insect projection neurons.

The spatial dynamics of action potentials, including their propagation and the location of spike initiation zone (SIZ), are crucial for the computation of a single neuron. Compared with mammalian central neurons, the spike dynamics of invertebrate neurons remain relatively unknown. Thus, we examined the spike dynamics based on single spike-induced Ca(2+) signals in the dendrites of cricket mechanosensory projection neurons, known as giant interneurons (GIs). The Ca(2+) transients induced by a synaptically evoked single spike were larger than those induced by an antidromic spike, whereas subthreshold synaptic potentials caused no elevation of Ca(2+). These results indicate that synaptic activity enhances the dendritic Ca(2+) influx through voltage-gated Ca(2+) channels. Stimulation of the presynaptic sensory afferents ipsilateral to the recording site evoked a dendritic spike with higher amplitude than contralateral stimulation, thereby suggesting that alteration of the spike waveform resulted in synaptic enhancement of the dendritic Ca(2+) transients. The SIZ estimated from the spatial distribution of the difference in the Ca(2+) amplitude was distributed throughout the right and left dendritic branches across the primary neurite connecting them in GIs.

Auditory modulation of wind-elicited walking behavior in the cricket Gryllus bimaculatus.

Animals flexibly change their locomotion triggered by an identical stimulus depending on the environmental context and behavioral state. This indicates that additional sensory inputs in different modality from the stimulus triggering the escape response affect the neuronal circuit governing that behavior. However, how the spatio-temporal relationships between these two stimuli effect a behavioral change remains unknown. We studied this question, using crickets, which respond to a short air-puff by oriented walking activity mediated by the cercal sensory system. In addition, an acoustic stimulus, such as conspecific 'song' received by the tympanal organ, elicits a distinct oriented locomotion termed phonotaxis. In this study, we examined the cross-modal effects on wind-elicited walking when an acoustic stimulus was preceded by an air-puff and tested whether the auditory modulation depends on the coincidence of the direction of both stimuli. A preceding 10 kHz pure tone biased the wind-elicited walking in a backward direction and elevated a threshold of the wind-elicited response, whereas other movement parameters, including turn angle, reaction time, walking speed and distance were unaffected. The auditory modulations, however, did not depend on the coincidence of the stimulus directions. A preceding sound consistently altered the wind-elicited walking direction and response probability throughout the experimental sessions, meaning that the auditory modulation did not result from previous experience or associative learning. These results suggest that the cricket nervous system is able to integrate auditory and air-puff stimuli, and modulate the wind-elicited escape behavior depending on the acoustic context.

Persistence of auditory modulation of wind-induced escape behavior in crickets.

Animals, including insects, change their innate escape behavior triggered by a specific threat stimulus depending on the environmental context to survive adaptively the predators' attack. This indicates that additional inputs from sensory organs of different modalities indicating surrounding conditions could affect the neuronal circuit responsible for the escape behavior. Field crickets, Gryllus bimaculatus, exhibit an oriented running or jumping escape in response to short air puff detected by the abdominal mechanosensory organ called cerci. Crickets also receive a high-frequency acoustic stimulus by their tympanal organs on their frontal legs, which suggests approaching bats as a predator. We have reported that the crickets modulate their wind-elicited escape running in the moving direction when they are exposed to an acoustic stimulus preceded by the air puff. However, it remains unclear how long the effects of auditory inputs indicating surrounding contexts last after the sound is terminated. In this study, we applied a short pulse (200 ms) of 15-kHz pure tone to the crickets in various intervals before the air-puff stimulus. The sound given 200 or 1000 ms before the air puff biased the wind-elicited escape running backward, like the previous studies using the longer and overlapped sound. But the sounds that started 2000 ms before and simultaneously with the air puff had little effect. In addition, the jumping probability was higher only when the delay of air puff to the sound was 1000 ms. These results suggest that the cricket could retain the auditory memory for at least one second and alter the motion choice and direction of the wind-elicited escape behavior.

Motor state changes escape behavior of crickets.

Animals change their behavior depending on external circumstances, internal factors, and their interactions. Locomotion state is a crucial internal factor that profoundly affects sensory perception and behavior. However, studying the behavioral impacts of locomotion state in free-moving animals has been challenging due to difficulty in reproducing quantitatively identical stimuli in freely moving animals. We utilized a closed-loop controlled servosphere treadmill system, enabling unrestricted confinement and orientation of small animals, and investigated wind-induced escape behavior in freely moving crickets. When stimulated during locomotion, the crickets quickly stopped before initiating escape behavior. Moving crickets exhibited a higher probability of escape response compared to stationary crickets. The threshold for pausing response in moving crickets was also much lower than the escape response threshold. Moving crickets had delayed reaction times for escape and greater variance in movement direction compared to stationary crickets. The locomotion-related response delay may be compensated by an elevated sensitivity to airflow.

Central projections of cercal giant interneurons in the adult field cricket, Gryllus bimaculatus.

The structures of neurons, such as dendrites and axonal projections, are closely related to their response properties and their specific functions in neural circuits. Identified neurons, having genetically determined morphological features and pre- and postsynaptic partners, play significant roles in specific behaviors. Giant interneurons (GIs) are identified in the terminal abdominal ganglion of the cricket as mechanosensory projection neurons and are sensitive to airflow stimulation of the cerci. GIs are classified into ventral GIs (vGIs) or dorsal GIs (dGIs) depending on the location of their axons running within the connective nerve cord. Based on their response properties to airflow, vGIs are presumed to be involved in triggering the wind-elicited escape response, whereas dGIs are thought to be airflow direction-encoding neurons. The previous findings regarding airflow sensitivity point to possible differences in the morphology of the central projections that may correspond to their neural functions. However, the detailed morphologies of the GIs in the cephalic and thoracic ganglia of adult crickets remain unclear. In this study, we stained six GIs, namely, GI 8-1 (medial giant interneuron, MGI), 9-1 (lateral giant interneuron, LGI), 9-2, 9-3, 10-2, and 10-3, using intracellular iontophoretic or pressure injection of dyes. Staining revealed remarkable differences in the axonal branching patterns between vGIs and dGIs. The dGIs were further divided into subgroups based on the profiles of their axon collaterals and projection sites in the brain. The anatomical differences between the GIs' central projections seemed to be related to their information encodement and behavioral functions.

Roles of neural communication between the brain and thoracic ganglia in the selection and regulation of the cricket escape behavior.

To survive a predator's attack, prey animals must exhibit escape responses that are appropriately regulated in terms of their moving speed, distance, and direction. Insect locomotion is considered to be controlled by an interaction between the brain, which is involved in behavioral decision-making, and the thoracic ganglia (TG), which are primary motor centers. However, it remains unknown which descending and ascending signals between these neural centers are involved in the regulation of the escape behavior. We addressed the distinct roles of the brain and TG in the wind-elicited escape behavior of crickets by assessing the effects of partial ablation of the intersegmental communications on escape responses. We unilaterally cut the ventral nerve cord (VNC) at different locations, between the brain and TG, or between the TG and terminal abdominal ganglion (TAG), a primary sensory center of the cercal system. The partial ablation of ascending signals to the brain greatly reduced the jumping response rather than running, indicating that sensory information processing in the brain is essential for the choice of escape responses. The ablation of descending signals from the brain to the TG impaired locomotor performance and directional control of the escape responses, suggesting that locomotion in the escape behavior largely depends on the descending signals from the brain. Finally, the extracellular recording from the cervical VNC indicated a difference in the descending activities preceding the escape responses between running and jumping. Our results demonstrated that the brain sends the descending signals encoding the behavioral choice and locomotor regulation to the TG, while the TG seem to have other specific roles, such as in the preparation of escape movement.

Action selection based on multiple-stimulus aspects in wind-elicited escape behavior of crickets.

Escape behavior is essential for animals to avoid attacks by predators. In some species, multiple escape responses could be employed. However, it remains unknown what aspects of threat stimuli affect the choice of an escape response. We focused on two distinct escape responses (running and jumping) to short airflow in crickets and examined the effects of multiple stimulus aspects including the angle, velocity, and duration on the choice between these responses. The faster and longer the airflow, the more frequently the crickets jumped. This meant that the choice of an escape response depends on both the velocity and duration of the stimulus and suggests that the neural basis for choosing an escape response includes the integration process of multiple stimulus parameters. In addition, the moving speed and distance changed depending on the stimulus velocity and duration for running but not for jumping. Running away would be more adaptive escape behavior.

Sexual dimorphism in shape and distribution of GABA-like immuno-reactive neurons in cricket terminal abdominal ganglion.

We studied age-related sexual dimorphism of the terminal abdominal ganglion in the cricket, Gryllus bimaculatus using osmium/ethyl-gallate staining and GABA immunohistochemistry. We determinedthat the volume of the terminal abdominal ganglion increases with increasing body size. The growth ratio between longitude and width in the terminal abdominal ganglion is the same in all nymph stages and sexes. However, the longitudinal growth of the male terminal abdominal ganglion ceases in the 8th instar and the adult stage. Therefore, in the adult stage, sexual dimorphism is observed in ganglion morphology. Additionally, sexual dimorphism is also observed in numbers of neuronal somata and GABA-like immunoreactive neurons in the terminal abdominal ganglion. At the beginning of the 8th instar, the number of somata is greater in female crickets than in males. GABA-like immunoreactive neurons, which were classified into eight clusters, increased between the 8th instar and adult in both sexes, but their numbers were sexually dimorphic in the 7th and 8th instars, and in the adult. Females had a greater number of GABA-like immunoreactive neurons than males. These differences occurred mainly in the 8th neuromere cluster, as well as the anterior region of the 9-11th neuromeres.

Deep learning-assisted comparative analysis of animal trajectories with DeepHL.

A comparative analysis of animal behavior (e.g., male vs. female groups) has been widely used to elucidate behavior specific to one group since pre-Darwinian times. However, big data generated by new sensing technologies, e.g., GPS, makes it difficult for them to contrast group differences manually. This study introduces DeepHL, a deep learning-assisted platform for the comparative analysis of animal movement data, i.e., trajectories. This software uses a deep neural network based on an attention mechanism to automatically detect segments in trajectories that are characteristic of one group. It then highlights these segments in visualized trajectories, enabling biologists to focus on these segments, and helps them reveal the underlying meaning of the highlighted segments to facilitate formulating new hypotheses. We tested the platform on a variety of trajectories of worms, insects, mice, bears, and seabirds across a scale from millimeters to hundreds of kilometers, revealing new movement features of these animals.

Dendritic design implements algorithm for synaptic extraction of sensory information.

While sensory information is encoded by firing patterns of individual sensory neurons, it is also represented by spatiotemporal patterns of activity in populations of the neurons. Postsynaptic interneurons decode the population response and extract specific sensory information. This extraction of information represented by presynaptic activities is a process critical to defining the input-output function of postsynaptic neuron. To understand the "algorithm" for the extraction, we examined directional sensitivities of presynaptic and postsynaptic Ca(2+) responses in dendrites of two types of wind-sensitive interneurons (INs) with different dendritic geometries in the cricket cercal sensory system. In IN 10-3, whose dendrites arborize with various electrotonic distances to the spike-initiating zone (SIZ), the directional sensitivity of dendritic Ca(2+) responses corresponded to those indicated by Ca(2+) signals in presynaptic afferents arborizing on that dendrite. The directional tuning properties of individual dendrites varied from each other, and the directional sensitivity of the nearest dendrite to the SIZ dominates the tuning properties of the spiking response. In IN 10-2 with dendrites isometric to the SIZ, directional tuning properties of different dendrites were similar to each other, and each response property could be explained by the directional profile of the spatial overlap between that dendrite and Ca(2+)-elevated presynaptic terminals. For IN 10-2, the directional sensitivities extracted by the different dendritic-branches would contribute equally to the overall tuning. It is possible that the differences in the distribution of synaptic weights because of the dendritic geometry are related to the algorithm for extraction of sensory information in the postsynaptic interneurons.

Trade-off between motor performance and behavioural flexibility in the action selection of cricket escape behaviour.

To survive a predator's attack successfully, animals choose appropriate actions from multiple escape responses. The motor performance of escape response governs successful survival, which implies that the action selection in escape behaviour is based on the trade-off between competing behavioural benefits. Thus, quantitative assessment of motor performance will shed light on the biological basis of decision-making. To explore the trade-off underlying the action selection, we focused on two distinct wind-elicited escape responses of crickets, running and jumping. We first hypothesized a trade-off between speed and directional accuracy. This hypothesis was rejected because crickets could control the escape direction in jumping as precisely as in running; further, jumping had advantages with regard to escape speed. Next, we assumed behavioural flexibility, including responsiveness to additional predator's attacks, as a benefit of running. The double stimulus experiment revealed that crickets running in the first response could respond more frequently to a second stimulus and control the movement direction more precisely compared to when they chose jumping for the first response. These data suggest that not only the motor performance but also the future adaptability of subsequent behaviours are considered as behavioural benefits, which may be used for choosing appropriate escape reactions.

Crickets alter wind-elicited escape strategies depending on acoustic context.

Acoustic signals trigger various behaviours in insects such as courtship or escape from predators. However, it remains unknown whether insects utilize acoustic signals to recognize environmental contexts. The cricket is a prominent model insect for neuroethological studies on acoustic behaviour because female crickets exhibit positive phonotaxis in response to male calling songs, and flying crickets display avoidance behaviour for high-frequency sounds such as echolocation call of bats. The carrier frequency of these sounds is a major factor in determining whether they initiate these acoustic behaviours. Here, we examined the impacts of different frequencies of tone sounds on cercal-mediated escape behaviour, using a 5-kHz tone corresponding to the calling song and a 15-kHz tone serving as a trigger of avoidance behaviours. Neither frequency elicited a response in the standing cricket by itself, but they had different impacts on walking responses to airflow stimuli. While the 15-kHz tone reduced response probability, extended moving distance, and enhanced turn-angle variability, the 5-kHz tone had no effect. Although both frequencies of tones facilitated walking backward, the 15-kHz tone had a larger effect than the 5-kHz tone. These frequency dependencies of behavioural modulation suggest that crickets can recognize acoustic contexts and alter their escape strategy accordingly.

Post-molting development of wind-elicited escape behavior in the cricket.

Arthropods including insects grow through several developmental stages by molting. The abrupt changes in their body size and morphology accompanying the molting are responsible for the developmental changes in behavior. While in holometabolous insects, larval behaviors are transformed into adult-specific behaviors with drastic changes in nervous system during the pupal stage, hemimetabolous insects preserve most innate behaviors whole life long, which allow us to trace the maturation process of preserved behaviors after the changes in body. Wind-elicited escape behavior is one of these behaviors and mediated by cercal system, which is a mechanosensory organ equipped by all stages of nymph in orthopteran insects like crickets. However, the maturation process of the escape behavior after the molt is unclear. In this study, we examined time-series of changes in the wind-elicited escape behavior just after the imaginal molt in the cricket. The locomotor activities are developed over the elapsed time, and matured 24h after the molt. In contrast, a stimulus-angle dependency of moving direction was unchanged over time, meaning that the cercal sensory system detecting airflow direction was workable immediately after the molt, independent from the behavioral maturation. The post-molting development of the wind-elicited behavior was considered to result not simply from maturation of the exoskeleton or musculature because the escape response to heat-shock stimulus did not change after the molt. No effect of a temporal immobilization after the imaginal molt on the maturation of the wind-elicited behavior also implies that the maturation may be innately programmed without experience of locomotion.

Mitochondria are intracellular magnesium stores: investigation by simultaneous fluorescent imagings in PC12 cells.

To determine the nature of intracellular Mg2+ stores and Mg2+ release mechanisms in differentiated PC12 cells, Mg2+ and Ca2+ mobilizations were measured simultaneously in living cells with KMG-104, a fluorescent Mg2+ indicator, and fura-2, respectively. Treatment with the mitochondrial uncoupler, carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), increased both the intracellular Mg2+ concentration ([Mg2+]i) and the [Ca2+]i in these cells. Possible candidates as intracellular Mg2+ stores under these conditions include intracellular divalent cation binding sites, endoplasmic reticulum (ER), Mg-ATP and mitochondria. Given that no change in [Mg2+]i was induced by caffeine application, intracellular IP3 or Ca2+ liberated by photolysis, it appears that no Mg2+ release mechanism thus exists that is mediated via the action of Ca2+ on membrane-bound receptors in the ER or via the offloading of Mg2+ from binding sites as a result of the increased [Ca2+]i. FCCP treatment for 2 min did not alter the intracellular ATP content, indicating that Mg2+ was not released from Mg-ATP, at least in the first 2 min following exposure to FCCP. FCCP-induced [Mg2+]i increase was observed at mitochondria localized area, and vice versa. These results suggest that the mitochondria serve as the intracellular Mg2+ store in PC12 cell. Simultaneous measurements of [Ca2+]i and mitochondrial membrane potential, and also of [Ca2+]i and [Mg2+]i, revealed that the initial rise in [Mg2+]i followed that of mitochondrial depolarization for several seconds. These findings show that the source of Mg2+ in the FCCP-induced [Mg2+]i increase in PC12 cells is mitochondria, and that mitochondrial depolarization triggers the Mg2+ release.

Numerical simulation of the twist-grain-boundary phase of chiral liquid crystals.

We study the structure of the twist-grain-boundary phase of chiral liquid crystals by numerically minimizing the Landau-de Gennes free energy. We analyze the morphology of layers at the grain boundary, to better understand the mechanism of frustration between the smectic layer order and chirality. As the chirality increases, the layer compression energy strongly increases while the effective layer bending rigidity is reduced due to unlocking of the layer orientation and the director. This results in large deviation of the layer morphology from that of Scherk's first minimal surface and linear stack of screw dislocations.

Boundary conditions for equilibrating incommensurate periodic patterns.

Simulation of periodic patterns often suffer from artifacts due to incommensurability of the intrinsic length scale and the system size. We introduce a simple numerical scheme to avoid this problem in finding equilibrium domain morphologies from a Ginzburg-Landau-type free energy. In this scheme, the boundary values are determined only by the local equilibrium condition at the adjacent bulk sites. The scheme is especially advantageous in equilibrating patterns that have two or more characteristic lengths. We demonstrate it using a model of lamellar-lamellar coexistence in block copolymer blends.