Visually mediated motor planning in the escape response of Drosophila

A key feature of reactive behaviors is the ability to spatially localize a salient stimulus and act accordingly. Such sensory-motor transformations must be particularly fast and well tuned in escape behaviors, in which both the speed and accuracy of the evasive response determine whether an animal successfully avoids predation [1]. We studied the escape behavior of the fruit fly, Drosophila, and found that flies can use visual information to plan a jump directly away from a looming threat. This is surprising, given the architecture of the pathway thought to mediate escape [2, 3]. Using high-speed videography, we found that approximately 200 ms before takeoff, flies begin a series of postural adjustments that determine the direction of their escape. These movements position their center of mass so that leg extension will push them away from the expanding visual stimulus. These preflight movements are not the result of a simple feed-forward motor program because their magnitude and direction depend on the flies' initial postural state. Furthermore, flies plan a takeoff direction even in instances when they choose not to jump. This sophisticated motor program is evidence for a form of rapid, visually mediated motor planning in a genetically accessible model organism.     

Preparing for escape: an examination of the role of the DCMD neuron in locust escape jumps

Many animals begin to escape by moving away from a threat the instant it is detected. However, the escape jumps of locusts take several hundred milliseconds to produce and the locust must therefore be prepared for escape before the jumping movement can be triggered. In this study we investigate a locust’s preparations to escape a looming stimulus and concurrent spiking activity in its pair of uniquely identifiable looming-detector neurons (the descending contralateral movement detectors; DCMDs). We find that hindleg flexion in preparation for a jump occurs at the same time as high frequency DCMD spikes. However, spikes in a DCMD are not necessary for triggering hindleg flexion, since this hindleg flexion still occurs when the connective containing a DCMD axon is severed or in response to stimuli that cause no high frequency DCMD spikes. Such severing of the connective containing a DCMD axon does, however, increase the variability in flexion timing. We therefore propose that the DCMD contributes to hindleg flexion in preparation for an escape jump, but that its activity affects only flexion timing and is not necessary for the occurrence of hindleg flexion.     

From neuron to behavior: Sensory‐motor coordination of zebrafish turning behavior

Recent development of optogenetics brought non‐invasive neural activation in living organisms. Transparent zebrafish larva is one of the suitable animal models for this technique, which enables us to investigate neural circuits for behaviors based on a whole individual nervous system. In this article we review our recent finding that suggests sensory‐motor coordination in larval zebrafish escape behavior. When water vibration stimulates mechanosensory Rohon‐Beard (RB) neurons, intra‐spinal reflex circuit launches contralateral trunk muscle contraction that makes rapid body curvature for turning. In addition, positional information of the stimulus is conveyed to supra‐spinal circuits, and then regulates the curvature strength for appropriate escape pathway from the threat. Sensory‐motor coordination is a fundamental feature to adapt behaviors to environment, and zebrafish larvae would be an excellent model for elucidating its neural backbones.     

Behavioral analyses of wind-evoked escape of the cricket, Gryllodes sigillatus

The wind-evoked escape behavior of the cricket Gryllodes sigillatus was investigated using an air puff stimulus. A high velocity air puff elicited the escape behavior in many crickets. The crickets tended to escape away from the stimulus source, but the direction was not accurately oriented 180 degrees from the stimulus. After bilateral cercal ablation, only a few crickets showed wind-evoked escape behavior, and their response rates did not increase even 19 days after ablation. Therefore, information on air motion detected by cercal filiform hairs is essential for triggering wind-evoked behavior. After unilateral cercal ablation, the 81.3% response rate of intact crickets decreased to 16.5%, that is, it decreased to almost 20% that of intact crickets. One week after unilateral cercal ablation, the response rate recovered to more than 60% that of intact crickets. However, the accuracy rate of the escape direction of G. sigillatus showed no change even immediately after the unilateral cercal ablation. Therefore, both cerci are not necessarily required to determine the escape direction. The behavioral characteristics of wind-evoked escape of G. sigillatus are compared with those of another species of cricket, Gryllus bimaculatus. The two species of cricket employ different strategies for wind-evoked escape.     

The fight and flight responses of crickets depleted of biogenic amines

Aggressive and escape behaviors were analysed in crickets (Orthoptera) treated with either reserpine, a nonspecific depleter of biogenic amines, or the synthesis inhibitors alpha-methyltryptophan (AMTP) and alpha-methyl-p-tyrosine (AMT) to specifically deplete serotonin, respectively dopamine and octopamine. Standard immunocytochemical techniques were used to verify depletion from central nervous tissue, and determine the effective dosages. Reserpinized crickets became exceedingly lethargic and had severely depressed escape responses. However, they were still able to express all the major elements of the escalating sequences of stereotype motor performances that typifies normal aggressive behavior in the cricket. AMT and AMTP treatment had opposing influences on escape behavior, being enhanced by serotonin depletion, but depressed by dopamine/octopamine depletion. AMTP-induced serotonin depletion had no influence on aggressive or submissive behaviors. AMT-treated crickets could normally only be brought to fight by coaxing. Though capable of expressing aggressive behavior per se, agonistic encounters between AMT-treated crickets were shorter, and rarely involved actual physical interactions. Hence, although amines seem to have similar actions on escape behavior in insects and crustaceans, the aminergic control of aggression seems to be fundamentally different in these arthropods groups. We conclude that amines are not in principle required for the initiation and operation of the motor circuits underlying aggression in the cricket. However, octopamine and/or dopamine seem necessary for establishing a level of excitability sufficient for aggressive behavior to become overt in response to appropriate natural releasing stimuli.     

Rearing conditions required for behavioral compensation after unilateral cercal ablation in the cricket Gryllus bimaculatus

The rearing condition necessary for behavioral compensation after sensory deprivation was investigated in the cricket Gryllus bimaculatus. The right-cercus-ablated cricket was reared in a glass vial with a slightly larger diameter than the body length of the cricket. After two weeks of rearing in the vial, the air-puff-evoked escape behavior of the cricket was investigated. The response rate (relative occurrence of the escape behavior after a standard air puff) obtained was identical with that of crickets reared in a large cage. On the other hand, unlike crickets reared in a large cage, the distorted escape directional property of the cricket reared in the vial was not compensated at all. Control experiments proved that the restraint in the vial did not affect the motor system, and the air motion from environments was not essential for the compensational recovery of the escape direction. Therefore, the ablated crickets required spontaneous walking in order to compensate the directionality of their escape. A self-generated wind caused by spontaneous walking appears necessary for the crickets to realize the defect of their sensory system and to compensate the related escape behavior. A hypothesis for the compensation mechanism based on the efference copy signal is proposed.     

Escape behavior in response to mechanical stimulation of hindwing in cricket, Gryllus bimaculatus

We studied behavioral responses of the cricket, Gryllus bimaculatus, to mechanical stimulation of the hindwing tip using three different kinds of stimuli: touching, bending and pinching. The most characteristic was a sequence of initial jump-like movements and subsequent running steps, that is referred to as escape behavior in this study. Touching stimulus elicited the escape behavior in 52% of resting animals tethered on a treadmill, whereas bending elicited the same behavior in 94% or 98% depending on the bend direction. Pinching was effective in all tested animals. The effectiveness of pinching stimulus in eliciting the escape behavior depends on the ongoing activity in the animal. Video and electromyographic recordings have revealed that, in the initial jump-like movements, forelegs and hindlegs move simultaneously on both sides while midlegs remain on the ground, followed by simultaneous movements of bilateral midlegs. The subsequent stepping was characterized by out-of-phase rhythmical activities of the leg muscles. Touching stimulus evoked tonic afferent responses of small amplitude in the second nerve root of the metathoracic ganglion. Bending stimulus evoked tonic responses of large units that showed rapid habituation and medium units that persisted during repeated stimulation. Pinching stimulus elicited only phasic responses of large and medium amplitude in the R2 afferents. The results suggest that touching, bending and pinching stimuli are transmitted to the metathoracic ganglion via different sensory systems having different effectiveness in activating the escape motor system.     

Prey Escape Direction is Influenced by the Pivoting Displays of Flush-Pursuing Birds

Painted redstart, Myioborus pictus, and its congeners in Central and South America, belong to a small fraction of insectivorous flush‐pursuing birds. Unlike most of the small insectivorous birds, which glean prey from substrates, the flush pursuers spread and pivot their conspicuously patterned tails and wings. This display triggers prey escape flights which are hypothesized to occur through visual stimulation of prey escape circuits [giant descending neuron cluster (GDNC) in Diptera] sensitive to the looming motion of an approaching bird, translational motion of a pivoting body with widely spread tail and contrast of the white‐black plumage pattern. In this paper, data from field observations of redstarts and experiments with bird models show an increase in the frequency of prey escapes away from the strong visual stimulation of an open tail, and in the direction opposite to that of the horizontal translational motion present in the pivots. We discuss how the effect on prey escape direction may enhance prey interception capabilities of redstarts during aerial pursuits. Combined with an earlier study the results show that, unlike the movements of typical gleaner–foragers, the flush displays by redstarts affect prey escape direction in a manner that may facilitate prey tracking and capture by birds. Because the GDNs, which mediate escape initiation, are not sensitive to motion direction, we hypothesize that other neurons, in addition to the GDNs, are involved in influencing the direction of escape responses.     

A look into the cockpit of the developing locust: Looming detectors and predator avoidance

For many animals, the visual detection of looming stimuli is crucial at any stage of their lives. For example, human babies of only 6 days old display evasive responses to looming stimuli (Bower et al. [1971]: Percept Psychophys 9: 193–196). This means the neuronal pathways involved in looming detection should mature early in life. Locusts have been used extensively to examine the neural circuits and mechanisms involved in sensing looming stimuli and triggering visually evoked evasive actions, making them ideal subjects in which to investigate the development of looming sensitivity. Two lobula giant movement detectors (LGMD) neurons have been identified in the lobula region of the locust visual system: the LGMD1 neuron responds selectively to looming stimuli and provides information that contributes to evasive responses such as jumping and emergency glides. The LGMD2 responds to looming stimuli and shares many response properties with the LGMD1. Both neurons have only been described in the adult. In this study, we describe a practical method combining classical staining techniques and 3D neuronal reconstructions that can be used, even in small insects, to reveal detailed anatomy of individual neurons. We have used it to analyze the anatomy of the fan‐shaped dendritic tree of the LGMD1 and the LGMD2 neurons in all stages of the post‐embryonic development of Locusta migratoria. We also analyze changes seen during the ontogeny of escape behaviors triggered by looming stimuli, specially the hiding response. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 1078–1095, 2014     

Thermal nociception in adult Drosophila: behavioral characterization and the role of the painless gene

Nociception, warning of injury that should be avoided, serves an important protective function in animals. In this study, we show that adult Drosophila avoids noxious heat by a jump response. To quantitatively analyze this nociceptive behavior, we developed two assays. In the CO2 laser beam assay, flies exhibit this behavior when a laser beam heats their abdomens. The consistency of the jump latency in this assay meets an important criterion for a good nociceptive assay. In the hot plate assay, flies jump quickly to escape from a hot copper plate (>45 degrees C). Our results demonstrate that, as in mammals, the latency of the jump response is inversely related to stimulus intensity, and innoxious thermosensation does not elicit this nociceptive behavior. To explore the genetic mechanisms of nociception, we examined several mutants in both assays. Abnormal nociceptive behavior of a mutant, painless, indicates that painless, a gene essential for nociception in Drosophila larvae, is also required for thermal nociception in adult flies. painless is expressed in certain neurons of the peripheral nervous system and thoracic ganglia, as well as in the definite brain structures, the mushroom bodies. However, chemical or genetic insults to the mushroom bodies do not influence the nociceptive behavior, suggesting that different painless-expressing neurons play diverse roles in thermal nociception. Additionally, no-bridge(KS49), a mutant that has a structural defect in the protocerebral bridge, shows defective response to noxious heat. Thus, our results validate adult Drosophila as a useful model to study the genetic mechanisms of thermal nociception.     

Targeted gene delivery in the cricket brain,using in vivo electroporation

The cricket (Gryllus bimaculatus) is a hemimetabolous insect that is emerging as a model organism for the study of neural and molecular mechanisms of behavioral traits. However, research strategies have been limited by a lack of genetic manipulation techniques that target the nervous system of the cricket. The development of a new method for efficient gene delivery into cricket brains, using in vivo electroporation, is described here. Plasmid DNA, which contained an enhanced green fluorescent protein (eGFP) gene, under the control of a G. bimaculatus actin (Gb′-act) promoter, was injected into adult cricket brains. Injection was followed by electroporation at a sufficient voltage. Expression of eGFP was observed within the brain tissue. Localized gene expression, targeted to specific regions of the brain, was also achieved using a combination of local DNA injection and fine arrangement of the electroporation electrodes. Further studies using this technique will lead to a better understanding of the neural and molecular mechanisms that underlie cricket behaviors.     

Topographical organization of the auditory pathway within the prothoracic ganglion of the cricket Gryllus campestris L.

Summary The topographical organization of the prothoracic ganglion of the cricket, Gryllus campestris L., is described from horizontal, transverse, and sagittal sections of preparations specially treated to elucidate longitudinal tracts, commissures, and areas of neuropil. These structures were compared to those reported from other insect thoracic ganglia, resulting in still further evidence for a common basic morphological pattern among insect central nervous systems.Six types of auditory interneurons, all existing as mirrorimage pairs, were identified through intracellular application of the dye Lucifer yellow, and then related to several morphological patterns. Two intrasegmental neurons (ON1, ON2) are similar in location of cell bodies and course of neurites and axons; three intersegmental neurons (AN1, AN2, TN1) are likewise similar to one another. The axons of the two intrasegmental neurons cross the midline of the ganglion in the newly described omega commissure. Axons of the other four types all course within the median portion of the ventral intermediate tract and project intersegmentally.All six neuron types arborize within the ventral portion of the ring tract, the same neuropilar region in which auditory sensory neurons terminate. The ring tract is therefore considered the most important region for auditory information processing within the cricket prothoracic ganglion.     

Motor activity and trajectory control during escape jumping in the locust <Emphasis Type="Italic">Locusta migratoria</Emphasis>

We investigated the escape jumps that locusts produce in response to approaching objects. Hindleg muscular activity during an escape jump is similar to that during a defensive kick. Locusts can direct their escape jumps up to 50° either side of the direction of their long axis at the time of hindleg flexion, allowing them to consistently jump away from the side towards which an object is approaching. Variation in jump trajectory is achieved by rolling and yawing movements of the body that are controlled by the fore- and mesothoracic legs. During hindleg flexion, a locust flexes the foreleg ipsilateral to its eventual jump trajectory and then extends the contralateral foreleg. These foreleg movements continue throughout co-contraction of the hindleg tibial muscles, pivoting the locust’s long axis towards its eventual jump trajectory. However, there are no bilateral differences in the motor programs of the left and right hindlegs that correlate with jump trajectory. Foreleg movements enable a locust to control its jump trajectory independent of the hindleg motor program, allowing a decision on jump trajectory to be made after the hindlegs have been cocked in preparation for a jump.     

Compensation of escape direction in unilaterally cercus-ablated crickets, Gryllus bimaculatus, is associated with the distance walked during recovery period

In response to an air puff stimulus, intact crickets, Gryllus bimaculatus, make an escape almost 180 degrees opposite to the stimulus source. In order to verify our previous hypothesis that a self-stimulation of the wind-sensory system is necessary for a compensational recovery of the escape direction (behavioral compensation) in unilaterally cercus-ablated crickets, we investigated the relationship between the conditions of rearing after a unilateral cercal ablation and the degree of behavioral compensation. A unilaterally cercus-ablated cricket reared in a large cage to permit free locomotion showed a significantly higher degree of recovery of escape direction compared with those reared under restrained conditions in a small glass vial. However, the degree of behavioral compensation in a cricket reared alone in a large cage was smaller than that of crickets reared in a cage of the same size with 5-6 other cercus-ablated crickets. Mutual stimulation possibly increased the extent of locomotion of crickets reared in a group and improved the degree of compensational recovery of the escape direction. To ascertain this, the distance a cricket moved during the recovery period was associated with the degree of compensational change of the escape direction. The result suggests that the degree of compensation of the escape direction clearly depended on the distance walked by the crickets. The compensation seemed not to be caused by other factors such as chemical ones in the case of group rearing because forced locomotion induced by touch stimulation on the body surface was solely effective in improving the escape direction.     

Understanding the regulation and function of adult neurogenesis: contribution from an insect model, the house cricket

Since the discovery of adult neurogenesis, a major issue is the role of newborn neurons and the function-dependent regulation of adult neurogenesis. We decided to use an animal model with a relatively simple brain to address these questions. In the adult cricket brain as in mammals, new neurons are produced throughout life. This neurogenesis occurs in the main integrative centers of the insect brain, the mushroom bodies (MBs), where the neuroblasts responsible for their formation persist after the imaginal molt. The rate of production of new neurons is controlled not only by internal cues such as morphogenetic hormones but also by external environmental cues. Adult crickets reared in an enriched sensory environment experienced an increase in neuroblast proliferation as compared with crickets reared in an impoverished environment. In addition, unilateral sensory deprivation led to reduced neurogenesis in the MB ipsilateral to the lesion. In search of a functional role for the new cells, we specifically ablated MB neuroblasts in young adults using brain-focused gamma ray irradiation. We developed a learning paradigm adapted to the cricket, which we call the "escape paradigm." Using this operant associative learning test, we showed that crickets lacking neurogenesis exhibited delayed learning and reduced memory retention of the task when olfactory cues were used. Our results suggest that environmental cues are able to influence adult neurogenesis and that, in turn, newly generated neurons participate in olfactory integration, optimizing learning abilities of the animal, and thus its adaptation to its environment. Nevertheless, odor learning in adult insects cannot always be attributed to newly born neurons because neurogenesis is completed earlier in development in many insect species. In addition, many of the irradiated crickets performed significantly better than chance on the operant learning task.     

Pigment-dispersing factor sets the night state of the medulla bilateral neurons in the optic lobe of the cricket,Gryllus bimaculatus

Pigment-dispersing factor (PDF) is an octadeca-neuropeptide widely distributed in the insect brain and suggested to be involved in the insect circadian systems. We have examined its effects on the neuronal activity of the brain efferents in the optic stalk including medulla bilateral neurons (MBNs) in the cricket, Gryllus bimaculatus. The MBNs are visually responding interneurons connecting the bilateral medulla, which show a clear day/night change in their light responsiveness that is greater during the night. Microinjection of PDF into the optic lobe induced a significant increase in the spontaneous activity of the brain efferents and the photo-responsiveness of the MBNs during the day, while little change was induced during the night. The enhancing effects began to occur about 20 min after the injection and another 10 min was necessary to reach the maximal level. The effects of PDF were dose-dependent. When 22 nl of anti-Gryllus-PDF (1:200) IgG was injected into the medulla, the photo-responsiveness of the MBNs was suppressed in both the day and the night with greater magnitude during the night. No significant suppression was induced by injection of the same amount of IgG from normal rabbit serum. These results suggest that in the cricket optic lobe, PDF is released during the night and enhances MBNs' photo-responsiveness to set their night state.     

Responses of descending neurons to looming stimuli in the praying mantis <Emphasis Type="Italic">Tenodera aridifolia</Emphasis>

Responses to visual stimuli of some neurons that descend the nerve cord from the brain were recorded extracellularly in the mantis Tenodera aridifolia. Most of the recorded neurons showed their largest responses to looming stimuli that simulated a black circle approaching towards the mantis. The neurons showed a transient excitatory response to a gradually darkening or receding circle. The neurons showed sustained excitation to the linearly expanding stimuli, but the spike frequency decreased rapidly. The responses of the neurons were affected by both the diameter and the speed of looming stimuli. Faster or smaller looming stimuli elicited a higher peak frequency. These responses were observed in both recordings from the connective between suboesophageal and prothoracic ganglia and the connective between prothoracic and mesothoracic ganglia. There was a one-to-one correspondence of spike firing between these two recordings with a fixed delay. The neurons had the receptive field on ipsilateral side to its axon at the cervical connective. These results suggest that there is a looming-sensitive descending neuron, with an axon projecting over prothoracic ganglion, in the mantis nervous system.     

Parasitoid wasp uses a venom cocktail injected into the brain to manipulate the behavior and metabolism of its cockroach prey

Unlike other venomous predators, the parasitoid wasp Ampulex compressa incapacitates its prey, the cockroach Periplaneta americana, to provide a fresh food supply for its offspring. We first established that the wasp larval development, from egg laying to pupation, lasts about 8 days during which the cockroach must remain alive but immobile. To this end, the wasp injects a cocktail of neurotoxins to manipulate the behavior of the cockroach. The cocktail is injected directly into the head ganglia using biosensors located on the stinger. The head sting induces first 30 min of intense grooming followed by hypokinesia during which the cockroach is unable to generate an escape response. In addition, stung cockroaches survive longer, lose less water, and consume less oxygen. Dopamine contained in the venom appears to be responsible for inducing grooming behavior. For the hypokinesia, our hypothesis is that the injected venom affects neurons located in the head ganglia, which send descending tonic input to bioaminergic neurons. These, in turn, control the thoracic premotor circuitry for locomotion. We show that the activity of identified octopaminergic neurons from the thoracic ganglia is altered in stung animals. The alteration in the octopaminergic neurons' activity could be one of the mechanisms by which the venom modulates the escape circuit in the cockroach's central nervous system and metabolism in the peripheral system.     

Neural Circuit Recording from an Intact Cockroach Nervous System

The cockroach ventral nerve cord preparation is a tractable system for neuroethology experiments, neural network modeling, and testing the physiological effects of insecticides. This article describes the scope of cockroach sensory modalities that can be used to assay how an insect nervous system responds to environmental perturbations. Emphasis here is on the escape behavior mediated by cerci to giant fiber transmission in Periplaneta americana. This in situ preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of neuronal activity. Peptides or other chemical reagents can then be applied directly to the nervous system in solution with the physiological saline. Insecticides could also be administered prior to dissection and the escape circuit can serve as a proxy for the excitable state of the central nervous system. In this context the assays described herein would also be useful to researchers interested in limb regeneration and the evolution of nervous system development for which P. americana is an established model organism.