The Interactive Fly

Genes regulating behavior

Behavioral paradigms

Analysis of Locomotor Behavior
Computations underlying Drosophila photo-taxis, odor-taxis, and multi-sensory integration
Dynamical feature extraction at the sensory periphery guides chemotaxis
The effect of stress on motor function in Drosophila
Characterization of the decision network for wing expansion in Drosophila using targeted expression of the TRPM8 channel
The nicotinic acetylcholine receptor Dα7 is required for an escape behavior in Drosophila
A defensive kicking behavior in response to mechanical stimuli mediated by Drosophila wing margin bristles
Mechanosensory interactions drive collective behaviour in Drosophila
A multilevel multimodal circuit enhances action selection in Drosophila
Fluctuation-driven neural dynamics reproduce Drosophila locomotor patterns
Coordination and fine motor control depend on Drosophila TRPγ
The nutritional and hedonic value of food modulate sexual receptivity in Drosophila melanogaster females
Recovery of locomotion after injury in Drosophila depends on proprioception
Ancient anxiety pathways influence Drosophila defense behaviors
Mushroom body signaling is required for locomotor activity rhythms in Drosophila
A single dopamine pathway underlies progressive locomotor deficits in a Drosophila model of Parkinson disease
Generative rules of Drosophila locomotor behavior as a candidate homology across phyla
Behavior reveals selective summation and max pooling among olfactory processing channels
Selective inhibition mediates the sequential recruitment of motor pools
Predictability and hierarchy in Drosophila behavior
Simultaneous activation of parallel sensory pathways promotes a grooming sequence in Drosophila
Social effects for locomotion vary between environments in Drosophila melanogaster females
Mutations in the Drosophila homolog of human PLA2G6 give rise to age-dependent loss of psychomotor activity and neurodegeneration
High throughput measurement of locomotor sensitization to volatilized cocaine in Drosophila melanogaster
Thermosensory perception regulates speed of movement in response to temperature changes in Drosophila melanogaster
Optogenetic dissection of descending behavioral control in Drosophila
Neural control of startle-induced locomotion by the mushroom bodies and associated neurons in Drosophila

Larval Motion
A Model of Drosophila Larva Chemotaxis
Neuroendocrine control of Drosophila larval light preference
Transmembrane channel-like (tmc) gene regulates Drosophila larval locomotion
Bolwig's organ and the Sensorimotor structure of Drosophila larva phototaxis
Nociceptive neurons protect Drosophila larvae from parasitoid wasps
Two alternating motor programs drive navigation in Drosophila larva
Developmental timing of a sensory-mediated larval surfacing behavior correlates with cessation of feeding and determination of final adult size
Inhibition of fatty acid desaturases in Drosophila melanogaster larvae blocks feeding and developmental progression
Identification of inhibitory premotor interneurons activated at a late phase in a motor cycle during Drosophila larval locomotion
Imaging fictive locomotor patterns in larval Drosophila
Drosophila food-associated pheromones: Effect of experience, genotype and antibiotics on larval behavior
Functional genetic screen to identify interneurons governing behaviorally distinct aspects of Drosophila larval motor programs
Interactions among Drosophila larvae before and during collision
Continuous lateral oscillations as a core mechanism for taxis in Drosophila larvae
High-content behavioral profiling reveals neuronal genetic network modulating Drosophila larval locomotor program
Turns with multiple and single head cast mediate Drosophila larval light avoidance
Dendritic and axonal L-type calcium channels cooperate to enhance motoneuron firing output during Drosophila larval locomotion
Divergent connectivity of homologous command-like neurons mediates segment-specific touch responses in Drosophila

Vision and Locomotion
Dissection of the peripheral motion channel in the visual system of Drosophila melanogaster
A simple vision-based algorithm for decision making in flying Drosophila
How Ants Use Vision When Homing Backward
Multi-stability with ambiguous visual stimuli in Drosophila orientation behavior
Feature integration drives probabilistic behavior in the Drosophila escape response
Asymmetric processing of visual motion for simultaneous object and background responses
Visual place learning in Drosophila melanogaster
The relative roles of vision and chemosensation in mate recognition of Drosophila
On the encoding of panoramic visual scenes in navigating wood ants
Pulsed light stimulation increases boundary preference and periodicity of episodic motor activity in Drosophila melanogaster
Quantitative predictions orchestrate visual signaling in Drosophila
Moonwalker descending neurons mediate visually evoked retreat in Drosophila
Visual projection neurons in the Drosophila lobula link feature detection to distinct behavioral programs
A neural circuit architecture for angular integration in Drosophila

Flying
Free flight odor tracking in Drosophila: Effect of wing chemosensors, sex and pheromonal gene regulation
Crossmodal visual input for odor tracking during fly flight
Flying Drosophila maintain arbitrary but stable headings relative to the angle of polarized light
Plume-tracking behavior of flying Drosophila emerges from a set of distinct sensory-motor reflexes
Visual control of altitude in flying Drosophila
A descending neuron correlated with the rapid steering maneuvers of flying Drosophila
Flying Drosophila orient to sky polarization
Drosophila tracks carbon dioxide in flight
Flies evade looming targets by executing rapid visually directed banked turns
Wing-pitch modulation in maneuvering fruit flies is explained by an interplay between aerodynamics and a torsional spring
The Function and Organization of the Motor System Controlling Flight Maneuvers in Flies
Flies compensate for unilateral wing damage through modular adjustments of wing and body kinematics
A visual horizon affects steering responses during flight in fruit flies
Forewings match the formation of leading-edge vortices and dominate aerodynamic force production in revolving insect wings
An array of descending visual interneurons encoding self-motion in Drosophila

Walking
A screen for constituents of motor control and decision making in Drosophila reveals visual distance-estimation neurons
Saccadic body turns in walking Drosophila
Kinematic responses to changes in walking orientation and gravitational load in Drosophila melanogaster
GABAergic inhibition of leg motoneurons is required for normal walking behavior in freely moving Drosophila
Walking modulates speed sensitivity in Drosophila motion vision
Speed-dependent interplay between local pattern-generating activity and sensory signals during walking in Drosophila
A simple strategy for detecting moving objects during locomotion revealed by animal-robot interactions
A biphasic locomotor response to acute unsignaled high temperature exposure in Drosophila
Data-driven analysis of motor activity implicates 5-HT2A neurons in backward locomotion of larval Drosophila


Dissection of the peripheral motion channel in the visual system of Drosophila melanogaster
In the eye, visual information is segregated into modalities such as color and motion, these being transferred to the central brain through separate channels. This study genetically dissected the achromatic motion channel in the fly Drosophila melanogaster at the level of the first relay station in the brain, the lamina, where it is split into four parallel pathways (L1-L3, amc/T1). The functional relevance of this divergence is little understood. It was shown that the two most prominent pathways, L1 and L2, together are necessary and largely sufficient for motion-dependent behavior. At high pattern contrast, the two pathways are redundant. At intermediate contrast, they mediate motion stimuli of opposite polarity, L2 front-to-back, L1 back-to-front motion. At low contrast, L1 and L2 depend upon each other for motion processing. Of the two minor pathways, amc/T1 specifically enhances the L1 pathway at intermediate contrast. L3 appears not to contribute to motion but to orientation behavior (Rister, 2007).

Visual systems process the information from the environment in parallel neuronal subsystems. In higher vertebrates, for instance, the visual modalities of color, form, and motion are segregated at the level of the retina into separate channels. Similarly, insects have distinct sets of photoreceptors for motion and color. Investigating the motion channel in the fly Drosophila, this study shows that at the next level below the eye, the lamina, the motion channel is again split into several functionally distinct parallel pathways (Rister, 2007).

Directional responses to visual motion have been intensely studied, predominantly in dipteran flies. They are provided by arrays of elementary movement detectors (EMDs), the smallest motion-sensitive units that temporally compare the intensity fluctuations in neighboring visual elements (sampling units). Their neuronal implementation in flies is still unknown. In the rabbit retina, a candidate interneuron computing directional motion has recently been identified. The present study is confined to the input side of the movement-detection circuitry (Rister, 2007).

The compound eye of Drosophila is composed of about 750 ommatidia. Each of these contains eight photoreceptors (R1-8) that can be structurally and functionally grouped into two subsystems: six large photoreceptors (R1-6) mediate the detection of motion , whereas two small ones (R7, R8), together forming one rhabdomere in the center of the ommatidium, are required for color vision (Rister, 2007).

The lamina consists of corresponding units called neuro-ommatidia, or cartridges. These are the sampling units of the motion channel, whereas the color channel (R7, R8) bypasses the lamina cartridge to terminate in the second neuropil, the medulla. The lamina is anatomically and ultrastructurally known in exquisite detail. Its functional significance, however, is little understood (Rister, 2007).

In the lamina, the motion channel is split into four parallel pathways. In each cartridge, the photoreceptor terminals are connected by tetradic synapses to four neurons, L1, L2, L3, and the amacrine cell α (amc; connecting to the medulla via the basket cell T1). The most prominent of these are the large monopolar cells L1 and L2. Their position in the center and their radially distributed dendrites throughout the depth of the cartridge suggest a key role in peripheral processing. This can be visualized by 3H-deoxyglucose activity labeling. Single-unit recordings of L1 and L2 in large flies so far have revealed only subtle differences between them. Their specific functional contribution to behavior is largely unknown (Rister, 2007).

Several hypotheses have been advanced over the last 40 years. The loss of L1 and L2 and concomitantly of optomotor responses in the mutant Vacuolar medullaKS74 had prompted a proposal that these cells were involved in motion detection. If indeed L1 and L2 mediate motion vision, are they functionally specialized or redundant? The latter is unlikely to be the whole answer, considering the differing synaptic relationships of the two neurons. For one, they have their terminals in separate layers of the medulla. Second, L2, but not L1, has feedback synapses onto R1-6. These might play a role in neuronal adaptation and could exert a modulatory influence on the photoreceptor output (Rister, 2007).

In Drosophila, L2 innervates and reciprocally receives input from a second-order interneuron, L4, that has two conspicuous backward oriented collaterals connecting its own cartridge to the neighboring ones along the x and y axes of the hexagonal array. In this network, the L2 neurons are connected to the L4 neurons of two adjacent cartridges, and the L4 neurons are directly connected to all six neighboring L4s. The significance of this circuitry is not yet understood (Rister, 2007).

Using the two-component UAS/GAL4 system for targeted transgene expression, single interneurons or combinations of them were manipulated, in all lamina cartridges. To study whether a particular pathway was necessary for a given behavioral task, their synaptic output was blocked using the temperature-sensitive allele of shibire, shits1. In addition, the inverse strategy was adopted, studying whether single lamina pathways were sufficient for mediating the behavior in the same experimental context. Using a mutant of the histamine receptor gene outer rhabdomeres transientless (ort) that has all lamina pathways impaired, the wild-type ort-cDNA was expressed in chosen types of lamina interneurons known to receive histaminergic input from R1-6. Testing necessity and sufficiency it is not possible to relate the structural organization of the lamina to visually guided behavior (Rister, 2007).

A simple vision-based algorithm for decision making in flying Drosophila

Animals must quickly recognize objects in their environment and act accordingly. Previous studies indicate that looming visual objects trigger avoidance reflexes in many species; however, such reflexes operate over a close range and might not detect a threatening stimulus at a safe distance. This study analyzed how fruit flies respond to simple visual stimuli both in free flight and in a tethered-flight simulator. Whereas Drosophila, like many other insects, are attracted toward long vertical objects, smaller visual stimuli elicit not weak attraction but rather strong repulsion. Because aversion to small spots depends on the vertical size of a moving object, and not on looming, it can function at a much greater distance than expansion-dependent reflexes. The opposing responses to long stripes and small spots reflect a simple but effective object classification system. Attraction toward long stripes would lead flies toward vegetative perches or feeding sites, whereas repulsion from small spots would help them avoid aerial predators or collisions with other insects. The motion of flying Drosophila depends on a balance of these two systems, providing a foundation for studying the neural basis of behavioral choice in a genetic model organism (Maimon, 2008).

Observations on free-flying flies together with experiments on tethered flies in closed and open loop demonstrate that Drosophila possess two opposing visuomotor reflexes that explain salient features of the animal's flight behavior. Animals are attracted to long vertical objects, whereas they are repulsed by small objects. The visually guided behaviors detailed in this paper are most probably mediated by motion-sensitive neurons downstream of photoreceptors R1-R6. Specifically, in the lobula plate of blowflies, 'feature-detecting' cells respond vigorously to elongated vertical contours. The homologs of these neurons might mediate fixation of long stripes in Drosophila. In houseflies and hoverflies, other neurons in the lobula plate and lobula respond best to small stimuli, and the homologs of these cells might underlie small-object aversion in Drosophila. Because the chromaticity of the stimuli was not varied, the contribution of color as an additional cue that flies use in triggering attractive and aversive flight responses cannot be excluded (Maimon, 2008).

Males of many fly species, including houseflies (Musca domestica, Fania cunnicularis), flesh flies (Sarcophaga bullata), and hoverflies (Syritta pipiens), chase females as part of courtship. Long-legged flies (Dolichopodidae) and robber flies (Asilidae) prey upon small insects on the wing. Thus, at least in certain behavioral contexts, some flying dipterans are attracted toward small spots, not repelled. Drosophila, however, do not prey on other insects, and courtship does not involve flight. From an ethological point of view, Drosophila would do well to avoid any small object in midair, even static objects, because these could only signify a hazard. In contrast, dipterans that chase conspecifics, or hunt while flying, require a more sophisticated algorithm that may, for example, rely on more complex features of object motion or color to differentiate repulsive predators from attractive mates and prey (Maimon, 2008).

The results indicate that flying flies use a rather simple vision-based algorithm to avoid potentially harmful objects. Might walking flies use a similar strategy? The visual-motor behaviors of walking Drosophila are likely to be more complicated because these flies exhibit social behaviors such as courtship and aggression while on the ground. For example, male flies could not chase and orient toward female flies if small objects were aversive to them. It has been suggested that walking Drosophila might exhibit a similar behavior as the one reported here for flying Drosophila, i.e., attraction to tall stripes and aversion to small spots. However, this behavior is not consistent with studies using 'Buridan's paradigm,' which show that walking flies respond equivalently to long and short visual objects, or another study that did not report either clear attraction to or repulsion by small objects. Collectively, these studies do not present a simple picture of comparable reflexes in walking Drosophila, as might be expected from the more complicated suite of behaviors that occur on the ground (Maimon, 2008).

This study describes a new visuomotor reflex: small-object repulsion, which has a measurable influence on free-flight behavior. Whereas the neural mechanisms of this reflex remain unknown, the differing responses to long and short objects suggest that the two behaviors may be, at least partly, mediated by different neural circuits (although it is likely that many of the same cells are activated in both behaviors, especially near the sensory and motor periphery). An intriguing possibility is that the visual control of flight in Drosophila arises from a handful of partly nonoverlapping sensorimotor neural pathways, including long-object fixation, small-object repulsion, expansion avoidance, optomotor equilibrium, and landing responses. These innate behaviors, and potentially others yet to be discovered, could additionally be modified by learning. The molecular tools available in Drosophila should allow for a rich, mechanistic description of each individual pathway. More significantly, however, elementary rules governing the interaction of these putative sensorimotor modules may come into sharper focus, thereby allowing for the formulation of a bottom-up, biologically driven theory of behavior (Maimon, 2008).

How Ants Use Vision When Homing Backward

Ants can navigate over long distances between their nest and food sites using visual cues. Can ants use their visual memories of the terrestrial cues when going backward? The results suggest that ants do not adjust their direction of travel based on the perceived scene while going backward. Instead, they maintain a straight direction using their celestial compass. This direction can be dictated by their path integrator but can also be set using terrestrial visual cues after a forward peek. If the food item is too heavy to enable body rotations, ants moving backward drop their food on occasion, rotate and walk a few steps forward, return to the food, and drag it backward in a now-corrected direction defined by terrestrial cues. Furthermore, it was shown that ants can maintain their direction of travel independently of their body orientation. It thus appears that egocentric retinal alignment is required for visual scene recognition, but ants can translate this acquired directional information into a holonomic frame of reference, which enables them to decouple their travel direction from their body orientation and hence navigate backward. This reveals substantial flexibility and communication between different types of navigational information: from terrestrial to celestial cues and from egocentric to holonomic directional memories (Schwarz, 2017).

Crossmodal visual input for odor tracking during fly flight

Flies generate robust and high-performance olfactory and visual behaviors. Adult fruit flies can distinguish small differences in odor concentration across antennae separated by less than 1 mm, and a single olfactory sensory neuron is sufficient for near-normal gradient tracking in larvae. During flight a male housefly chasing a female executes a corrective turn within 40 ms after a course deviation by its target. The challenges imposed by flying apparently benefit from the tight integration of unimodal sensory cues. Crossmodal interactions reduce the discrimination threshold for unimodal memory retrieval by enhancing stimulus salience, and dynamic crossmodal processing is required for odor search during free flight because animals fail to locate an odor source in the absence of rich visual feedback. The visual requirements for odor localization are unknown. In this study, a hungry fly was tethered in a magnetic field, allowing it to yaw freely, odor plumes were presented, and how visual cues influence odor tracking was examined. Flies were found to be unable to use a small-field object or landmark to assist plume tracking, whereas odor activates wide-field optomotor course control to enable accurate orientation toward an attractive food odor (Duistermars, 2008).

This study investigated the motor control of active plume tracking by adapting a magnetic tether system into a 'virtual plume simulator' in which a fly is free to steer into and out of a spatially discrete plume of vinegar odor while simultaneously receiving visual feedback from a stationary wraparound electronic display. Flight behavior on the magnetic tether, like in free flight, is characterized by segments of straight flight interspersed with transient 'spikes' in angular velocity called 'body saccades' for their functional analogy with human gaze-stabilizing eye movements. Within a visual arena composed of equally spaced, high-contrast vertical stripes, a pattern that generates strong, spatially homogeneous optic-flow signals when the animal rotates on its pivot, the vinegar plume was periodically switched between 0° and 180° positions in the circular arena and the fly's heading was tracked. Under these conditions, the animal periodically encounters the plume by steering into it. Upon plume contact, identified by the animal's heading with respect to the odor port, the interval between saccades increases, whereas subsequently deviating from the plume results in a return of the typical saccadic rhythm. Thus by presenting only the water vapor control, flies iterate saccades with little apparent preferred orientation, resulting in an even distribution of heading within the arena. By contrast, encountering an odor plume results in stabilized flight heading directed toward the plume at either side of the arena. These results confirm that the two odor plumes were the most attractive features of the arena, that both locations could reliably and reversibly elicit stable odor tracking, and that the plume itself is narrow, as reflected by the 18° width of the heading histograms at half-maximum (Duistermars, 2008).

When compared to the high-contrast panorama in the absence of an odor plume, the visually uniform arena itself elicits smaller angle saccades, with shorter intervals between them. In an odor plume, these two responses would work against one another for stable tracking; smaller amplitude saccades would keep the animal close to the plume, but shorter saccade intervals (increased rate) would not. Upon encountering the odor plume within the high-contrast visual panorama, flies show decreased saccade amplitude and increased intersaccade interval (ISI) in comparison to the same flight trajectories oriented outside the plume. These two responses combine to facilitate plume tracking because saccades that would move the fly out of the plume are fewer and smaller. Under uniform featureless visual conditions, there are no such changes in saccade frequency or amplitude upon plume contact, indicating a crossmodal influence on saccade motor commands. Furthermore, only within the high-contrast visual treatment are saccade amplitude and ISI outside the attractive odor plume lower than during the no-odor experiment. It would appear, therefore, that like the casting dynamics of free flight, saccade amplitude and ISI are influenced both by plume acquisition and subsequent plume loss but in a visual-context-dependent manner. It seems reasonable to postulate that visual feedback provides a directional cue that enables a fly to correct a deviation from the plume during a saccade (Duistermars, 2008).

Once initiated, saccade dynamics are coordinated entirely by mechanosensory feedback, whereas flying straight and avoiding collisions require well-studied optomotor equilibrium reflexes. Saccades in the odor plume are fewer and smaller, but not altogether absent. Is the visually dependent quantitative reduction in saccade rate and amplitude fully sufficient to enable stable plume tracking, or do odor cues activate optomotor responses in order to stabilize flight heading between saccades? There is no a priori reason to suspect the latter, particularly because a walking fruit fly is capable of orienting toward a static concentration gradient delivered across the antennae in the absence of visual cues. Yet, for accurate odor tracking during free flight, Drosophila require visual feedback from the lateral panorama (Duistermars, 2008).

To address this issue, the accuracy of plume tracking was measured for animals exposed to a sequence of different visual conditions. Taking advantage of a well-known and powerful object-orientation reflex in which flies actively fixate a high-contrast, vertical stripe within their forward field of view, a narrow stripe was slowly oscillated to visually 'drag' flies into the odor plume at the 180° arena position before instantly replacing the stripe with a stationary visual panorama. Therefore, for each experimental treatment, animals started from the same heading within either the water or vinegar plume and were subsequently exposed to either a high-contrast pattern of evenly spaced stripes or a featureless grayscale panorama of identical mean luminance, which provides light levels necessary to sustain active flight but provides minimal visual motion cues. To quantify the accuracy of plume tracking for each flight trajectory, the cumulative deviation from the odor plume was derived by subtracting 180 from the heading values, taking the absolute value and integrating it over time. The resultant cumulative time series has units of degree seconds and represents the flies' ability to stabilize the plume such that low values represent accurate plume tracking, and high values correspond to orientation 'error'(turning away from the plume into other regions of the arena). Note that cumulative deviation generally cannot remain at zero because flies continuously make fine-scale, back-and-forth adjustments to their heading, which results in an ever increasing cumulative deviation from the 180° arena position. Therefore, by design, mean cumulative plume deviation is a conservative estimate of a fly's ability to actively track a plume (Duistermars, 2008).

For the water vapor plume set against the uniform grayscale visual panorama, flies executed the usual rhythm of saccades and thus deviated from the plume within a few seconds after the start of the trial. Switching the visual panorama to high-contrast stripes did not significantly change flies' cumulative plume deviation. Predictably, for the water vapor plume and both visual treatments, flies oriented randomly throughout the arena. However, upon activating the odor plume against the high-contrast stripe background, the same animals remained tightly centered within the plume for the duration of the trial, resulting in 75% reduction in cumulative plume deviation. Remarkably, replacing the high-contrast visual scene with the featureless uniform panorama resulted in decreased tracking accuracy because animals quickly deviated from the odor plume in a manner similar to the no-odor control. The cumulative plume deviation was not significantly different between the uniform visual arena with odor and the striped arena without odor, indicating that in the absence of rich visual feedback the flies essentially behave as if there were no odor, highlighting the crossmodal requirements for odor tracking (Duistermars, 2008).

The visual influence on odor-tracking accuracy was further examined by activating the odor plume continuously while presenting a sequence of three visual treatments including high-contrast stripes, uniform grayscale, and a second high-contrast treatment. Each fly therefore started within the vinegar plume and was exposed to the three visual stimuli at 20 second intervals. When the striped panorama appeared at the start of the trial, flies maintained their heading into the plume. But once the stripes disappeared, the flies steered out of the plume and began generating saccades. Whereas occasionally they reencounter the plume within the uniform visual panorama, they generally are unable to remain there until the high-contrast pattern reappears, at which point accurate plume tracking resumes. Mean cumulative plume deviation increased significantly between the first high-contrast treatment and the uniform arena and then recovered to the initial value for the second high-contrast treatment (Duistermars, 2008).

For the visual manipulation experiments, the order of experimental treatments followed a predetermined sequence and each fly was presented with the sequence once and only once. To examine whether treatment order influenced the results, the entire experiment was repeated with a random block design in which the set of visual and olfactory treatments were randomly shuffled for each individual fly. The randomized experiment disclosed the same results as the ordered experiment; the high-contrast visual panorama significantly reduces the cumulative deviation from the odor plume by comparison to a uniform grayscale panorama (Duistermars, 2008).

Anatomical, physiological, and behavioral analyses suggest that the fly optomotor system is segregated into two parallel channels: one processes wide-field visual motion and the other processes small-field visual and object motion. It is thought that these two separate systems contribute to figure-ground discrimination and enable animals to see and track moving objects against a cluttered visual background. Additionally, active stripe fixation during flight may represent the fly's attempt to approach a suitable landing site, such as a plant stalk. Another remarkable use of small-field vision is demonstrated by home-base foragers, such as ants, that use objects located some distance from food resources or nests as landmarks to navigate return paths to those sites. This study shows that crossmodal feedback generated by the fly's movement within a homogeneous wide-field visual landscape enables active plume tracking. Is the synergistic crossmodal influence on odor tracking specific to wide-field visual signals, or can flies also use small-field visual cues, such as spatial landmarks, to maintain their heading in an odor plume? To examine this idea, flies were subjected to a stationary vertical stripe offset 90° from the odor plume. Flies starting within a control water plume veered out of the plume within several seconds and instead fixated the visual object, thus resulting in a rapidly increasing mean cumulative plume deviation. Starting a new trial with the vinegar-odor stimulus, the same flies showed a stronger tendency to stay in or near the plume, resulting in a roughly 50% reduction in cumulative plume deviation. At first glance, it might appear that the small-field stripe enhances odor tracking by comparison to the no-odor control. However, the critical question is whether a laterally displaced small-field object reduces the cumulative plume deviation by comparison to a uniform panorama, and it does not. The mean cumulative plume deviation for the small-field stripe is equivalent to the measurement for the uniform grayscale panorama. Accurate plume tracking requires wide-field visual input (Duistermars, 2008).

Unlike the propagation of visual or acoustic stimuli, an olfactory signal contains no intrinsic directional information. Therefore, animals often rely on ambient wind cues to determine the route to an odor source. Odor tracking by upwind flight requires visual feedback generated by background motion because an animal cannot easily distinguish ambient wind direction from self-induced airflow during flight. As such, in the absence of wind cues, tethered Drosophila provided with a visual stimulus analogous to being carried downwind steer so as to maintain an upwind heading. This response persists whether the animal views the moving ground below or the visual landscape laterally. In addition to directional control, when faced with headwinds, insects such as flies, beetles, bees, and moths regulate their airspeed and altitude by the use of visual cues, the combination of which enables accurate navigation of a female pheromone plume by a male moth. In previous free-flight experiments, it has not been possible to determine whether optomotor stabilization is triggered directly by odor cues or indirectly by wind-driven ground motion. This study shows that rotational stabilization reflexes are directly activated by odor cues independent of ambient wind cues (Duistermars, 2008).

The crossmodal influence of visual feedback on odor tracking in flies provides insight into how complex behaviors are controlled within environments containing nondirectional, weak, noisy, or subthreshold sensory stimuli. Fruit flies have 700 times lower visual spatial resolution than humans, and they have five times fewer olfactory receptor types. Yet their ability to find smelly things in visual landscapes as diverse as forests, deserts, and backyard patios would suggest behavioral performance greater than might be predicted by the sum of the salient sensory inputs. The results presented in this study show that odor signals activate powerful visual stabilization reflexes to accurately track an appetitive odor plume. The requisite visual feedback cues emerge from the wide-field visual-processing centers of the brain, not the small-field object-tracking centers, thus hinting at possible neuroanatomical substrates. Furthermore, the functional interaction of crossmodal integration for plume tracking in flies is reminiscent of multisensory enhancement (MSE) exhibited by single neurons within the cat superior colliculus. Here, neurons with overlapping receptive fields generally obey a principle known as 'inverse effectiveness,' whereby smaller, modality-specific responses are associated with larger, multisensory responses. As such, MSE results in cell excitability that is greater than the mathematical sum of the individual inputs, especially when unimodal input is weak. The superior colliculus forms a tissue map registering spatial information from two sensory modalities. It seems unlikely that visual-olfactory integration in the fly brain occurs with a structurally analogous system but, rather, a functionally analogous one. In gypsy moths, spiking responses within visually selective premotor interneurons are enhanced by sex pheromones. Similarly, the rattlesnake optic tectum contains individual neurons that exhibit nonlinear crossmodal enhancement of visual and thermal responses, presumably to guide prey capture in near darkness. It would appear that crossmodal integration at the behavioral and cellular level represents a functional adaptation for distinguishing and responding to critically important features of a complex sensory environment (Duistermars, 2008).

Free flight odor tracking in Drosophila: Effect of wing chemosensors, sex and pheromonal gene regulation

The evolution of powered flight in insects had major consequences for global biodiversity and involved the acquisition of adaptive processes allowing individuals to disperse to new ecological niches. Flies use both vision and olfactory input from their antennae to guide their flight; chemosensors on fly wings have been described, but their function remains mysterious. This study examineed Drosophila flight in a wind tunnel. By genetically manipulating wing chemosensors, it was shown that these structures play an essential role in flight performance with a sex-specific effect. Pheromonal systems are also involved in Drosophila flight guidance: transgenic expression of the pheromone production and detection gene, desat1, produced low, rapid flight that was absent in control flies. This study suggests that the sex-specific modulation of free-flight odor tracking depends on gene expression in various fly tissues including wings and pheromonal-related tissues (Houot, 2017).

A screen for constituents of motor control and decision making in Drosophila reveals visual distance-estimation neurons

Climbing over chasms larger than step size is vital to fruit flies, since foraging and mating are achieved while walking. Flies avoid futile climbing attempts by processing parallax-motion vision to estimate gap width. To identify neuronal substrates of climbing control, a large collection of fly lines with temporarily inactivated neuronal populations were screened in a novel high-throughput assay. The observed climbing phenotypes were classified; lines in each group are reported. Selected lines were further analysed by high-resolution video cinematography. One striking class of flies attempts to climb chasms of unsurmountable width; expression analysis led to C2 optic-lobe interneurons. C2 columnar feedback neurons project from the second visual neuropil, the medulla, to the most peripheral optic-lobe region, the lamina. Inactivation of C2 or the closely related C3 neurons with highly specific intersectional driver lines consistently reproduced hyperactive climbing whereas strong or weak artificial depolarization of C2/C3 neurons strongly or mildly decreased climbing frequency. Contrast-manipulation experiments support conclusion that C2/C3 neurons are part of the distance-evaluation system (Triphan, 2016).

Flying Drosophila maintain arbitrary but stable headings relative to the angle of polarized light

Animals must use external cues to maintain a straight course over long distances. This study investigated how the fruit fly, Drosophila melanogaster, selects and maintains a flight heading relative to the axis of linearly polarized light, a visual cue produced by the atmospheric scattering of sunlight. To track flies' headings over extended periods, a flight simulator was used that coupled the angular velocity of dorsally presented polarized light to the stroke amplitude difference of the animal's wings. In the simulator, most flies actively maintained a stable heading relative to the axis of polarized light for the duration of 15 minute flights. Individuals selected arbitrary, unpredictable headings relative to the polarization axis, which demonstrates that Drosophila can perform proportional navigation using a polarized light pattern. When flies flew in two consecutive bouts separated by a 5 minute gap, the two flight headings were correlated, suggesting individuals retain a memory of their chosen heading. Adding a polarized light pattern to a light intensity gradient was found to enhance flies' orientation ability, suggesting Drosophila use a combination of cues to navigate. For both polarized light and intensity cues, flies' capacity to maintain a stable heading gradually increased over several minutes from the onset of flight. These findings are consistent with a model in which each individual initially orients haphazardly but then settles on a heading which is maintained via a self-reinforcing process. This may be a general dispersal strategy for animals with no target destination (Warren, 2081).

Multi-stability with ambiguous visual stimuli in Drosophila orientation behavior

It is widely accepted for humans and higher animals that vision is an active process in which the organism interprets the stimulus. To find out whether this also holds for lower animals, an ambiguous motion stimulus was designed that serves as something like a multi-stable perception paradigm in Drosophila behavior. Confronted with a uniform panoramic texture in a closed-loop situation in stationary flight, the flies adjust their yaw torque to stabilize their virtual self-rotation. To make the visual input ambiguous, a second texture was examined. Both textures got a rotatory bias to move into opposite directions at a constant relative angular velocity. The results indicate that the fly now had three possible frames of reference for self-rotation: either of the two motion components as well as the integrated motion vector of the two. In this ambiguous stimulus situation, the flies generated a continuous sequence of behaviors, each one adjusted to one or another of the three references (Toepfer, 2018).

A screen for constituents of motor control and decision making in Drosophila reveals visual distance-estimation neurons

Climbing over chasms larger than step size is vital to fruit flies, since foraging and mating are achieved while walking. Flies avoid futile climbing attempts by processing parallax-motion vision to estimate gap width. To identify neuronal substrates of climbing control, a large collection of fly lines with temporarily inactivated neuronal populations were screened in a novel high-throughput assay. The observed climbing phenotypes were classified; lines in each group are reported. Selected lines were further analysed by high-resolution video cinematography. One striking class of flies attempts to climb chasms of unsurmountable width; expression analysis led to C2 optic-lobe interneurons. C2 columnar feedback neurons project from the second visual neuropil, the medulla, to the most peripheral optic-lobe region, the lamina. Inactivation of C2 or the closely related C3 neurons with highly specific intersectional driver lines consistently reproduced hyperactive climbing whereas strong or weak artificial depolarization of C2/C3 neurons strongly or mildly decreased climbing frequency. Contrast-manipulation experiments support conclusion that C2/C3 neurons are part of the distance-evaluation system (Triphan, 2016).

Plume-tracking behavior of flying Drosophila emerges from a set of distinct sensory-motor reflexes

For a fruit fly, locating fermenting fruit where it can feed, find mates, and lay eggs is an essential and difficult task requiring the integration of olfactory and visual cues. An approach has been developed to correlate flies' free-flight behavior with their olfactory experience under different wind and visual conditions, yielding new insight into plume tracking based on over 70 hr of data. To localize an odor source, flies exhibit three iterative, independent, reflex-driven behaviors, which remain constant through repeated encounters of the same stimulus: (1) 190 +/- 75 ms after encountering a plume, flies increase their flight speed and turn upwind, using visual cues to determine wind direction. Due to this substantial response delay, flies pass through the plume shortly after entering it. (2) 450 +/- 165 ms after losing the plume, flies initiate a series of vertical and horizontal casts, using visual cues to maintain a crosswind heading. (3) After sensing an attractive odor, flies exhibit an enhanced attraction to small visual features, which increases their probability of finding the plume's source. Due to plume structure and sensory-motor delays, a fly's olfactory experience during foraging flights consists of short bursts of odor stimulation. As a consequence, delays in the onset of crosswind casting and the increased attraction to visual features are necessary behavioral components for efficiently locating an odor source. These results provide a quantitative behavioral background for elucidating the neural basis of plume tracking using genetic and physiological approaches (van Breugel, 2014).

Computations underlying Drosophila photo-taxis, odor-taxis, and multi-sensory integration

To better understand how organisms make decisions on the basis of temporally varying multi-sensory input, this study identified computations made by Drosophila larvae responding to visual and optogenetically induced fictive olfactory stimuli. The larva's navigational decision was modeled to initiate turns as the output of a Linear-Nonlinear-Poisson cascade. Reverse-correlation was used to fit parameters to this model; the parameterized model predicted larvae's responses to novel stimulus patterns. For multi-modal inputs, it was found that larvae linearly combine olfactory and visual signals upstream of the decision to turn. This prediction was verified by measuring larvae's responses to coordinated changes in odor and light. Other navigational decisions were studied, and larvae were found to integrate odor and light according to the same rule in all cases. These results suggest that photo-taxis and odor-taxis are mediated by a shared computational pathway (Gepner, 2015).

A key step in 'cracking' neural circuits is defining the computations carried out by those circuits. Recent work has refined the measurements of circuits' behavioral outputs, for example, from simply counting animals accumulating near an odor source to specifying the sequence of motor outputs that allow odor gradient ascent. This study carried on this refinement, quantifying the transformation from sensory activity to motor decision with sub-second temporal resolution. Reverse-correlation analysis captured the essential features of the larva's navigational decision making, including the time scales and stimulus features associated with various decisions (Gepner, 2015).

The results are consistent with the understanding of how larvae navigate natural environments previously developed by observing behavior in structured environments of light or gaseous odors. For instance, when placed in environments with spatially varying Ethyl Butyrate or Ethyl Acetate oncentrations, larvae initiate turns more frequently when headed in directions of decreasing concentrations of these attractive odors. The current study has shown that larvae initiate turns in response to a decrease in activity in Or42a or Or42b receptor neurons, the primary receptors for Ethyl Acetate and Ethyl Butyrate. Additionally, this study showed that larvae mainly use only the previous two seconds of receptor activity to decide whether to turn. This detail cannot be resolved from experiments in natural odor gradients, nor can the fact that larvae integrate changes in odor receptor activity over a much longer time period to decide the size of their turns (Gepner, 2015).

When larvae move their heads through a spatially heterogeneous environment, they generate changes in sensory input that could be used to decode local spatial gradients. It has been directly shown that warming a cold larva during a head-sweep causes the larva to accept that head-sweep, beginning a new run. For light and odor, a strong circumstantial case has been made that larvae use information gathered during head-sweeps to bias turn direction: the first head-sweep of a turn is unbiased but larvae are more likely to begin a run following a sweep in a direction of higher concentration of attractive odor, lower concentration of carbon dioxide, or lower luminosity and larvae with only a single functional odor receptor can still bias turn direction via head-sweeping. This work has directly shown that larvae do in fact use changes in odor receptor activity and light level measured during head-sweeps to determine whether to begin a new run or initiate a second head-sweep (Gepner, 2015).

The experiments with a single stimulus also found a previously unknown difference in how larvae use CO2 receptor activity to modulate turn size and head-sweep acceptance compared to visual stimuli and to attractive olfactory receptor activity. This could be related to a difference in how larvae modulate their forward motion in response to changes in CO2 concentration compared to changes in light intensity or EtAc concentration. Previous studies have measured larvae's responses to linear temporal ramps of light intensity, and EtAc and CO2 concentrations (Gershow, 2012). For light and EtAc, larvae changed their rate of turning and the size of their turns in response to changing environmental conditions, but when they were actually engaged in forward movement, their speed of progress was the same whether conditions were improving or declining. In contrast, larvae dramatically decreased their forward run speed in response to increases in the concentration of CO2. Larvae with non-functional CO2 receptors did not change their speed at all in response to CO2, so this modulation was due to a sensory-motor transformation and not to metabolic effects (Gepner, 2015).

Larvae move forward through a series of tail to head peristaltic waves of muscle contraction and modulate their forward speed by changing the frequency with which they initiate these waves. The observed CO2 dependent speed modulation might therefore reflect the presence of a pathway by which Gr21a receptor neuron activity can down-regulate the probability of initiating forward peristaltic waves. In order to accept a head-sweep, that is, transition from head-sweeping to forward movement, larvae must initiate a new peristaltic wave (Lahiri, 2011). If an increase in Gr21a activity decreases the probability of initiating such a wave, this would explain why an increase in Gr21a activity prior to head-sweep initiation results in an increased probability of head-sweep rejection. Similarly, an increase in Gr21a activity might bias the larva towards larger reorientations by decreasing the probability of quick, small course corrections (Gepner, 2015).

In addition to defining the computations by which larvae navigate environments of varying light or varying odor, a quantitative model of odor-light integration was developed for this study. Previously, it has proven difficult to establish even a qualitative understanding of odor and light integration using static combinations of the two cues. Consider a simple experiment where a petri dish is divided into light and dark halves and a droplet of attractive odor is placed on the lighted half. If a larva moves towards the odor at the expense of moving out of darkness, is this because the larva naturally places more importance on odor than light regardless of intensity, because the particular concentrations of odor and intensities of light in the experiment favor a move towards odor, or because behavior is variable and larvae often make idiosyncratic choices? In the reverse-correlation experiments, hundreds of larvae were presented with thousands of combinations of light and odor variation and it was thus possible to resolve these ambiguities. The study determined not just how larvae balance an overall attraction to odor and aversion to light, but how they combine transient odor and light signals to make individual navigational decisions (Gepner, 2015).

This study has demonstrated the power of reverse-correlation analysis of larvae's behavioral responses to white-noise visual and fictive olfactory stimuli to decode the computations underlying the Drosophila larva's navigation of natural environments. This analysis could be used to decode the rules by which the larva integrates signals from distinct sensory organs. Larvae appear to use a single linear combination of odor and light inputs to make all navigational decisions, suggesting these signals are combined at early stages of the navigational circuitry (Gepner, 2015).

This work used optogenetics to explore how perturbations in the activities of identified neurons are interpreted behaviorally. CsChrimson was expressed in specific neurons to relate patterns of activity in these neurons to decisions regulating the frequency, size, and direction of turns. Using model parameters extracted from reverse-correlation experiments, it was possible to predict how larvae would respond to novel perturbations of these neurons' activities. How activity in one particular neuron type modulated the larva's responses to a natural light stimulus was explored and predictions were made of how the larva's natural response to blue light stepsv would be altered by simultaneous perturbation of this neuron. This study addressed sensory neurons, but the approach can be used generally to identify computations carried out on activities of interneurons, to determine whether activity in a neuron is interpreted as attractive or aversive, to measure how that activity combines with other sources of information to produce decisions, and to find neurons most responsible for making navigational decisions (Gepner, 2015).

An array of descending visual interneurons encoding self-motion in Drosophila

The means by which brains transform sensory information into coherent motor actions is poorly understood. In flies, a relatively small set of descending interneurons are responsible for conveying sensory information and higher-order commands from the brain to motor circuits in the ventral nerve cord. This study describes three pairs of genetically identified descending interneurons that integrate information from wide-field visual interneurons and project directly to motor centers controlling flight behavior. The physiological responses of these three cells were measured during flight, and they were found to respond maximally to visual movement corresponding to rotation around three distinct body axes. After characterizing the tuning properties of an array of nine putative upstream visual interneurons, it was shown that simple linear combinations of their outputs can predict the responses of the three descending cells. Last, a machine vision-tracking system was developed that allows monitoring of multiple motor systems simultaneously, and each visual descending interneuron class was found to correlate with a discrete set of motor programs (Suver, 2016).

This study has identified three pairs of descending neurons that integrate input from an array of visual interneurons in Drosophila. DNOVS1 and DNOVS2 are likely the homologs of neurons in larger flies, whereas DNHS1 is a previously undescribed cell. The tuning properties of these neurons during flight were measured, and they were found to encode three distinct axes of rotation, which are predicted by a linear summation of the responses of presynaptic vertical system (VS) and horizontal system (HS) cells (lobula plate tangential cells). The descending neurons project to nonoverlapping regions of dorsal neuropil within the ventral nerve cord (VNC), suggesting that they control different motor programs associated with flight. Indeed, it was found that the neurons were most strongly correlated with various body movements in response to visual motion, although this by no means indicates a causal relationship (Suver, 2016).

The large axonal diameter and abrupt terminals of DNOVS1 in the neck neuropil suggest that this cell is specialized for regulating rapid movements of the head. Neck motor neurons innervate 21 pairs of neck muscles and their activation elicits rotations of the head about specific axes. The neck motor neurons respond strongly to visual motion. In Calliphora, the haltere nerve innervates the neck motor neuropil and contralateral haltere interneurons are electrically coupled with both neck motor neurons and DNOVS1. This convergence of visual and mechanosensory feedback could enable precise compensatory movements of the head to reduce motion blur. Although the halteres provide self-motion information more rapidly than the visual system, DNOVS1 likely represents the fastest pathway by which visual information influences the fly's motor responses (Suver, 2016).

DNOVS2 and DNHS1 also have terminals in the neck motor region, although they do not appear to be electrically coupled to any motor neurons. Unlike DNOVS1, however, these cells also project to the wing and haltere neuropils. Visual input to the haltere motor system has been documented physiologically in blowflies and behaviorally in Drosophila, a phenomenon that might be mediated by DNHS1. The tuning curves for both head yaw and abdominal ruddering were quite similar to that of DNHS1, a correlation that should be explored in future studies (Suver, 2016).

Drosophila exhibit flight patterns in which straight sequences are interspersed with rapid stereotyped turns called body saccades. Flies execute saccades by generating a rapid banked turn in which they first roll to reorient their flight force and then counter roll to regain an upright pose. In Drosophila hydei, these roll and counter-roll axes are oriented at ~36° and 8°, respectively, relative to the longitudinal axis of the fly. The orientation of the initial roll axis is remarkably similar to the axis that elicits a peak response in the DNOVS2 cell (~35.7°), suggesting a possible role for this neuron during body saccades (Suver, 2016).

The representation of self-motion appears to be similar between Drosophila and blowflies despite the fact that fruit flies possess approximately half the number of VS cells. Theoretically, two cells would be sufficient to encode any arbitrary rotation in the azimuthal plane. However, the receptive fields of the VS cells cover different sectors of the visual world and no two cells extend over the entire field of view. Therefore, more than two cells are required to accurately encode rotation in a sparse visual scene. In addition, the large number of VS cells likely increases the speed and accuracy of self-motion estimation. The reduction in the number of VS cells in Drosophila may reflect a much lower number of ommatidia covering the azimuth compared with larger flies. It is not known, however, if the reduced number of VS cells translates into a smaller number of downstream interneurons. This study identified two descending neurons downstream of the VS cells, whereas four have been described in blowflies. However, it is premature to draw conclusions until more comprehensive maps of the descending neurons have been compiled for both species (Suver, 2016).

This study found that the visually evoked responses of LPTCs during flight were, on average, a few millivolts higher than during quiescence, consistent with prior studies. In contrast, this study observed much more substantial increases in the spike rate response of DNOVS2 and DNHS1 to visual motion during flight, suggesting that the relatively small flight-dependent increases observed in LPTCs are amplified in the descending neurons. With the exception of VS2, none of the descending interneurons or LPTCs displayed a statistically significant shift in their orientation tuning during flight compared with quiescence (Suver, 2016).

Collectively, the three cells that are described in this study encode body rotation around three approximately orthogonal axes (two in the azimuthal plane and one in the sagittal plane). Therefore, the projections of the three cells in the VNC might constitute a map from which any arbitrary rotation could be reconstructed. For example, all three descending neurons that were characterized in this study project to the neck neuropil, where local circuits could use the map to compute any arbitrary rotation. In general, this representation scheme would be analogous to the vestibulocerebellum system in rabbits, in which distinct classes of visually sensitive neurons respond to three axes of rotation and translation oriented at 45° and 135° on the azimuth and the vertical axis. Pigeons possess a similar system, with neurons in the accessory optic system and vestibulocerebellum encoding translation and rotation. The orientation of the three axes encoded in the vestibulocerebellum is quite similar to those encoded by the descending neurons in Drosophila; two cells are tuned to rotation on either side of the midline and one is tuned to yaw. The three axes identified in this study may not be the only ones encoded by descending neurons, but the similarities between mammals and flies suggest that animals may use a simple encoding scheme that avoids redundancy (Suver, 2016).

An alternate interpretation of these descending neurons is that they do not constitute a general map of body rotation, but rather are simply elements in parallel pathways that control distinct sensory motor reflexes. In this view, the outputs of the three cells may never be combined by downstream circuits to calculate an arbitrary angle of body rotation, but each cell may drive motor reflexes that rely solely on estimates of rotation about the individual axes of rotation. The fact that the projection patterns of the three cells are somewhat distinct (e.g., DNOVS1 only projects to the first thoracic neuropil and DNHS1 skips the second thoracic neuropil) supports this view. However, it is possible that the information conveyed by the three cells is functionally combined via their synergistic effects on distinct motor reflexes. For example, a reflex mediated by DNHS1 on the haltere motor system might combine with the effects of a reflex mediated by DNOVS2 on the wing motor system that would be behaviorally appropriate for a compensatory reflex regulating body motion about an axis not encoded directly by the two cells. In addition, a combination of these two encoding schemes might be implemented in which some systems (e.g., the neck motor system) compute arbitrary rotation angles from the three neurons and others (e.g., the wing motor system) rely on the direct influence of only one rotation axis and possibly indirect action from others (Suver, 2016).

This study reveals the neural circuitry responsible for encoding rotation along three body axes during flight in Drosophila. The descending interneurons that were characterized are involved in the transformation from visual to motor output and this takes place in as few as six synapses, making it a relatively tractable circuit. In this system, behaviorally relevant visual information is delivered to multiple motor systems and provides a basis for further investigations of cellular mechanisms of sensorimotor processing in a behaving animal (Suver, 2016).

Dynamical feature extraction at the sensory periphery guides chemotaxis

Behavioral strategies employed for chemotaxis have been described across phyla, but the sensorimotor basis of this phenomenon has seldom been studied in naturalistic contexts. This study examined how signals experienced during free olfactory behaviors are processed by first-order olfactory sensory neurons (OSNs) of the Drosophila larva. OSNs can act as differentiators that transiently normalize stimulus intensity-a property potentially derived from a combination of integral feedback and feed-forward regulation of olfactory transduction. In olfactory virtual reality experiments, this study reports that high activity levels of the OSN suppress turning, whereas low activity levels facilitate turning. Using a generalized linear model, how peripheral encoding of olfactory stimuli modulates the probability of switching from a run to a turn is explained. This work clarifies the link between computations carried out at the sensory periphery and action selection underlying navigation in odor gradients (Schultz, 2015).

Most primary sensory neurons operate differently from proportional counters. Individual OSNs of C. elegans and cockroaches function as bipolar detectors that selectively respond to either increases or decreases in stimulus intensity. A similar specialization into ON-OFF detection pathways has been observed for thermotaxis in C. elegans and motion perception in adult flies. In contrast with these binary sensory responses, this study discovered that a single larval OSN is sensitive to both the stimulus intensity and its first derivative. The enhanced information-processing capacity of primary olfactory neurons in the larva is consistent with the response characteristics of OSNs in adult flies, which encode complex dynamical features of airborne odorant stimuli (Schultz, 2015).

To describe the input-output response properties of single larval OSNs, the study set out to build a biophysical model of the olfactory transduction pathway. IFB motifs constitute the core mechanism of chemoreception in bacteria, olfactory transduction, and phototransduction. In adult flies, wheather the potential involvement of negative feedback on the olfactory transduction cascade could account for dynamical and adaptive features of OSN response was invsestigated. On the other hand, IFF motifs are implicated in the regulation of numerous cellular and developmental processes, and their contribution to sensory processing has been documented in recent work. These results led this study to conjecture that two regulatory motifs might be involved in larval olfactory transduction: an IFB and an IFF featuring direct excitation and indirect inhibition. Using a parameter optimization approach, it was found that a pure IFF motif is sufficient to approximate the response properties of the OSN. Combining the IFF and IFB motifs was nonetheless necessary to recapitulate the richness of OSN dynamics elicited by naturalistic olfactory stimuli. Consistent with the model proposed by Nagel (2011), the numerical simulations indicate that the integral feedback applies to the signaling pathway specific to the odorant receptor (OR). Nagel suggested that a diffusible effector-potentially intracellular calcium-inhibits the activity of the OR, thereby affecting the onset and offset kinetics of the OSN response. By contrast, the IFF motif would describe a regulatory mechanism acting on components of the transduction pathway downstream from the OR. It is plausible that the IFF regulation is also mediated by intracellular calcium (Schultz, 2015).

What features of the olfactory stimulus are encoded in the spiking dynamics of single larval OSNs? The biophysical model of the olfactory transduction cascade shows that the spiking activity of the OSN follows a standard hyperbolic dose-response when stimulated by prolonged pulses of odor. In this regime, the maximum OSN firing rate observed for IAA is modest. Changes in odor concentration occurring on a timescale relevant to the behavior-a second or shorter-can produce significantly higher (or lower) firing rates. This sensitivity to positive and negative changes in stimulus intensity can be explained by the mathematical solution that was derived for the OSN dynamics. Upon changes in odor concentration, the dose-response function describing the OSN spiking activity is transiently rescaled (or 'normalized') by the short-term history of the stimulus derivative ('memory' on characteristic time scale of 1 s). As a result, positive derivatives in stimulus intensity excite the OSN. Negative derivatives can inhibit the OSN firing rate in a manner consistent with the stimulus-offset inhibitions observed in adult-fly OSNs. This model indicates that a single Or42a OSN combines the function of a slope (ON) detector in response to positive gradients and an OFF detector in response to negative gradients. When larvae ascend Gaussian odor gradients originating from single odor sources, high OSN firing rates were expected. Robust inhibition of OSN spiking activity would result from motion that takes larvae down the odor gradient (Schultz, 2015).

How relevant are the features encoded by the spiking activity of the Or42a OSN to the behavioral dynamics directing chemotaxis? To address this question, the odor stimulation was substituted with optogenetics-based light stimulation and unprecedented control was gained over the spiking activity evoked in a genetically targeted OSN. Under the conditions of open-loop light stimulation, it was found that OFF responses (offset inhibition of the OSN firing) promote turning, whereas ON responses (sustained high firing) suppress turning. A Generalized Linear Model (GLM) was applied to describe the link between the OSN spiking dynamics and the probability of switching from a run to a turn. The accuracy of the model's output showed a striking dependence on the nonlinear transformation achieved by the olfactory transduction cascade. Ultimately, the biophysical model for the OSN spiking dynamics was combined with the GLM to make robust predictions about closed-loop behavior in virtual and in real odor gradients (Schultz, 2015).

The integrated stimulus-to-behavior GLM clarifies how features encoded in the activity pattern of individual primary olfactory neurons influence behavioral dynamics. The information transmitted by a single larval OSN is sufficient to represent positive and negative odor gradients through the excitation and inhibition of spiking activity. Unlike for chemotaxis and thermotaxis in C. elegans where the ON and OFF pathways are associated with different cellular substrates, the same larval OSN is capable of controlling up-gradient and down-gradient sensorimotor programs. This observation echoes findings recently made for thermotaxis in the Drosophila larva. Furthermore, it corroborates the idea that sensory representations are rapidly transformed into motor representations in the circuit controlling chemotaxis (Schultz, 2015).

In the future, it will be important to define whether the sensorimotor principles proposed for the Or42a OSN can be generalized to OSNs expressing other odorant receptors. In addition, the network of interneurons located in the larval antennal lobe is expected to participate in the processing of olfactory information arising from the OSNs. Although this work suggests that the computations achieved by the antennal lobe are not strictly necessary to guide robust chemotaxis, the function of the transformation carried out by the synapse between the Or42a OSN and its cognate projection neuron (PN) remains to be elucidated in the larva. As adult-fly PNs encode the second derivative of olfactory stimuli (Kim, 2015) including circuit elements downstream of the OSNs in the present multilevel model are expected to improve the accuracy of the behavioral predictions of the model (Schultz, 2015).

The aim of this study was to clarify the relationships between the peripheral encoding of naturalistic olfactory stimuli and gradient ascent toward an odor source. By exploiting the sufficiency of a single OSN to direct larval chemotaxis, a mathematical model was developed accounting for the transformation of time-varying stimuli into the firing rate of an OSN and the conversion of dynamical patterns of OSN activity into the selection between two basic types of action-running and turning. It will be interesting to examine the validity of the present model for the sensorimotor control of other aspects of larval chemotaxis such as turn orientation through lateral head casts (casting-to-turn transitions). In adult flies, turn orientation is determined by the crossing of the boundaries of odor plumes: upon encountering of an odor plume, flies veer upwind whereas exiting the plume initiates lateral and vertical casting-an orientation strategy related to the surge-and-cast response of moths. To orient in a rapidly changing olfactory landscape, the OSNs of various flying insects are capable of tracking rapid odor pulses on sub-second timescales and differentiating these signals. Whether the processing of turbulent olfactory inputs involves more temporal integration than that described by the sensorimotor model proposed in this study remains to be elucidated. Finally, the Drosophila larva offers a unique opportunity to delineate the neural circuit basis of behavior. Interdisciplinary approaches combining behavioral screens, functional imaging, and circuit reconstruction on the one hand, and computational modeling and robotics on the other hand, should improve understanding of how brains with reduced numerical complexity exploit streams of sensory information to direct action selection (Schultz, 2015).

A Model of Drosophila Larva Chemotaxis

Detailed observations of larval Drosophila chemotaxis have characterised the relationship between the odour gradient and the runs, head casts and turns made by the animal. This study used a computational model to test whether hypothesised sensorimotor control mechanisms are sufficient to account for larval behaviour. The model combines three mechanisms based on simple transformations of the recent history of odour intensity at the head location. The first is an increased probability of terminating runs in response to gradually decreasing concentration, the second an increased probability of terminating head casts in response to rapidly increasing concentration, and the third a biasing of run directions up concentration gradients through modulation of small head casts. This model can be tuned to produce behavioural statistics comparable to those reported for the larva, and this tuning results in similar chemotaxis performance to the larva. Each mechanism can be demonstrated to enable odour approach, but the combination of mechanisms is most effective. How these low-level control mechanisms relate to behavioural measures such as the preference indices was used to investigate larval learning behaviour in group assays (Davies, 2015).

Visual control of altitude in flying Drosophila

Unlike creatures that walk, flying animals need to control their horizontal motion as well as their height above the ground. Research on insects, the first animals to evolve flight, has revealed several visual reflexes that are used to govern horizontal course. For example, insects orient toward prominent vertical features in their environment and generate compensatory reactions to both rotations and translations of the visual world. Insects also avoid impending collisions by veering away from visual expansion. In contrast to this extensive understanding of the visual reflexes that regulate horizontal course, the sensory-motor mechanisms that animals use to control altitude are poorly understood. Using a 3D virtual reality environment, this study found that Drosophila utilize three reflexes -- edge tracking, wide-field stabilization, and expansion avoidance -- to control altitude. By implementing a dynamic visual clamp, it was found that flies do not regulate altitude by maintaining a fixed value of optic flow beneath them, as suggested by a recent model (Franceschini, 2007). The results identify a means by which insects determine their absolute height above the ground and uncover a remarkable correspondence between the sensory-motor algorithms used to regulate motion in the horizontal and vertical domains (Straw, 2010).

This study shows that Drosophila flies establish an altitude set point on the basis of nearby horizontal edges and tend to fly at the same height as such features. This reflex is invariant to contrast sign, such that a light-to-dark edge is roughly as attractive as a dark-to-light edge. Flies respond to wide-field motion with syndirectional velocity changes such that vertical, forward, and lateral visual motions elicit movement in the same direction. Finally, flies also avoid strong ventral expansion, flying upward away from the stimulus in what may be interpreted as a collision avoidance reflex. Thus, flies use a combination of at least three sensory-motor reflexes to control their vertical motion during flight. For each of these components, a similar response is involved in the azimuthal control of steering, suggesting close parallels for the reflexes used to control altitude and those used to control horizontal steering. The steering reflexes used in walking and flying are similar (e.g., the stripe fixation and wide-field stabilization behaviors of walking Drosophila are similar to those described in flight), and presumably they evolved prior to those required to regulate altitude. The similarity of the sensory-motor algorithms in the horizontal and vertical domains suggests that the neural substrates for altitude control either converged upon or were co-opted from those that underlie steering (Straw, 2010).

What is the relevance of these reflexes for animals flying in natural environments? On the basis of experiments with extended horizontal edges, it is speculated that flies approach and fly level with nearby objects, such as vegetation, using the visual edge created by the top of such objects with the background. This reflex might simply provide a convenient local set point for altitude, but it might also increase the probability of landing near the top of such objects. Indeed, when small solid cylinders were placed in the middle of the flight tunnel, it was observed that flies landed almost exclusively near the top. Recent experiments have demonstrated that when walking flies explore a 3D landscape, they also show a strong preference for the tops of objects, suggesting that there may be some ethological advantage to elevated perches. In many situations, numerous horizontal edges of various sizes, contrasts, and distances will be visible to a flying fly, and these experiments do not offer insight into how flies choose among them, although comparison with object fixation by walking flies would suggest that animals may attend to the closest, fastest-moving edge. Furthermore, the edge-tracking behavior is only one of the altitude responses described in this study, and it is unclear how it would interact with the other visual-motor altitude control pathways that presumably operate in parallel (Straw, 2010).

Given the complex evolutionary history of insects and their diverse natural histories, it is expected that other species may employ different algorithms for flight control. For example, although no evidence was found for ventral optic flow regulation of altitude in flies, honeybees do descend when presented with ventral regressive optic flow and thus may employ such an algorithm. Furthermore, many species of insects appear to fly at a level altitude without use of any obvious nearby horizontal edges or to travel hundreds of meters above ground during migration, suggesting mechanisms beyond those described in this study. It will be interesting to discover how the rules used for altitude control vary with the particular life history of different species. Nevertheless, the identification of a large suite of algorithms in Drosophila will make possible their study at the cellular and behavioral levels using convenient genetic tools (Straw, 2010).

A descending neuron correlated with the rapid steering maneuvers of flying Drosophila

To navigate through the world, animals must stabilize their path against disturbances and change direction to avoid obstacles and to search for resources. Locomotion is thus guided by sensory cues but also depends on intrinsic processes, such as motivation and physiological state. Flies, for example, turn with the direction of large-field rotatory motion, an optomotor reflex that is thought to help them fly straight. Occasionally, however, they execute fast turns, called body saccades, either spontaneously or in response to patterns of visual motion such as expansion. These turns can be measured in tethered flying Drosophila, which facilitates the study of underlying neural mechanisms. Whereas there is evidence for an efference copy input to visual interneurons during saccades, the circuits that control spontaneous and visually elicited saccades are not well known. Using two-photon calcium imaging and electrophysiological recordings in tethered flying Drosophila, this study identified a descending neuron whose activity is correlated with both spontaneous and visually elicited turns during tethered flight. The cell's activity in open- and closed-loop experiments suggests that it does not underlie slower compensatory responses to horizontal motion but rather controls rapid changes in flight path. The activity of this neuron can explain some of the behavioral variability observed in response to visual motion and appears sufficient for eliciting turns when artificially activated. This work provides an entry point into studying the circuits underlying the control of rapid steering maneuvers in the fly brain (Schnell, 2017).

Flying Drosophila orient to sky polarization

Insects maintain a constant bearing across a wide range of spatial scales. Monarch butterflies and locusts traverse continents, and foraging bees and ants travel hundreds of meters to return to their nests, whereas many other insects fly straight for only a few centimeters before changing direction. Despite this variation in spatial scale, the brain region thought to underlie long-distance navigation is remarkably conserved, suggesting that the use of a celestial compass is a general and perhaps ancient capability of insects. Laboratory studies of Drosophila have identified a local search mode in which short, straight segments are interspersed with rapid turns. However, this flight mode is inconsistent with measured gene flow between geographically separated populations, and individual Drosophila can travel 10 km across desert terrain in a single night - a feat that would be impossible without prolonged periods of straight flight. To directly examine orientation behavior under outdoor conditions, a portable flight arena was build in which a fly viewed the natural sky through a liquid crystal device that could experimentally rotate the polarization angle. The findings indicate that Drosophila actively orient using the sky's natural polarization pattern (Weir, 2012).

Collectively, these results indicate that Drosophila possess the optic and neural machinery to navigate, if in a rudimentary fashion, using the pattern of skylight polarization. They can hold a straighter course when provided with a natural polarization pattern than they can when this signal is scrambled by a circular polarizer. When an artificial pattern of linear polarization (but naturalistic in terms of color and intensity) was shifted instantaneously by 90, flies changed course accordingly. When the unaltered polarization pattern of skylight was shifted by 90 without changing its other features, flies also responded with course adjustments (Weir, 2012).

Central place foragers such as bees and desert ants have been the subject of intensive investigation into the role of a celestial compass in insect navigation. Among other topics, the important concepts of time compensation, path integration, and multisensory integration have been examined in detail in these organisms. A small specialized region of the eye called the dorsal rim area is thought to be critical for these behaviors in many species, although the evidence in flies is somewhat contradictory. Flies possess a dorsal rim area, which has been implicated in polarization responses, but prior experiments using a tethered flight arena suggest that the rest of the eye may play a role in responses to polarized light. The current results do not bear directly on this discrepancy, because the sky stimulus was visible to ommatidia both within and outside the dorsal rim area. Within the dorsal rim area, photoreceptors R7 and R8, which have been proposed to underlie polarization vision, both express an opsin with a peak sensitivity in the ultraviolet. Thus, the observation of polarization dependent responses to wavelengths longer than 400 nm provides further indirect evidence for the role of other photoreceptors besides R7 and R8 within the dorsal rim. Their involvement cannot however be ruled out because it is possible that they exhibit some small but functional sensitivity to the wavelengths used in these experiments. The possible existence of alternate, spectrally distinct pathways for detecting polarized light may have contributed to the variability that was measured in experiments in which UV light was attenuated by filters (Weir, 2012).

Through studies of migratory insects such as monarch butterflies and locusts, the neural circuitry that underlies polarization vision and its influence on motor behavior has begun to be elucidated. Researchers have traced the polarization vision pathway from the eye to the central brain to neurons arborizing in the thoracic ganglion. This electrophysiological evidence suggests that the central complex, a series of unpaired neuropils of the central brain, plays a key role in processing polarized light. The ubiquity of this brain region along with the relevance of polarization vision to the life history of a variety of species suggests that orientation responses using polarized light may represent a rather ancient component of insect behavior. At first glance, the fruit fly, which is neither a central place forager nor known as a seasonal migrant, seems to be a strange choice of species in which to study polarization vision. Because long-distance directed flights, either for migration or homing, have not been directly observed in flies, one cannot rely on innate motivation to navigate to a specific location when designing experiments. Nonetheless, a fly (or any insect for that matter) that finds itself in a resource-poor area, without observable attractive cues, faces a critical challenge. Maintaining a straight path ensures that it does not waste limited resources repeatedly traversing the same ground. Indeed, evidence suggests that several species of fruit flies, including Drosophila melanogaster, could fly over 10 km across a desert without access to food or water. Given the energy resources of even a well-fed fly, this feat would only be possible by maintaining a straight heading. Because the sun is often obscured by clouds, masked by local features, or below the horizon, an alternative source of compass information - such as that available from sky light polarization - would be extremely useful for animals attempting to maintain a heading relative to global coordinates. An intrinsic compass preference would not be necessary, simply the ability to choose a heading and maintain it. The current experiments were designed to mimic this situation, and it was observed that flies did indeed use skylight polarization to help maintain a steady course. The fruit fly, too often thought of without reference to its evolutionary history, thus displays another of the almost implausibly complex behaviors found in the insect world. The wealth of behavioral, physiological, and genetic tools available in Drosophila make it an ideal system in which to examine the open questions surrounding this behavior. Observation of flies using celestial polarization to hold a course is a step in this direction (Weir, 2012).

A simple strategy for detecting moving objects during locomotion revealed by animal-robot interactions

An important role of visual systems is to detect nearby predators, prey, and potential mates, which may be distinguished in part by their motion. When an animal is at rest, an object moving in any direction may easily be detected by motion-sensitive visual circuits. During locomotion, however, this strategy is compromised because the observer must detect a moving object within the pattern of optic flow created by its own motion through the stationary background. However, objects that move creating back-to-front (regressive) motion may be unambiguously distinguished from stationary objects because forward locomotion creates only front-to-back (progressive) optic flow. Thus, moving animals should exhibit an enhanced sensitivity to regressively moving objects. This study explicitly tested this hypothesis by constructing a simple fly-sized robot that was programmed to interact with a real fly. The measurements indicate that whereas walking female flies freeze in response to a regressively moving object, they ignore a progressively moving one. Regressive motion salience also explains observations of behaviors exhibited by pairs of walking flies. Because the assumptions underlying the regressive motion salience hypothesis are general, it is suspected that the behavior observed in Drosophila may be widespread among eyed, motile organisms (Zabala, 2012).

Experiments using a computer-controlled robot indicate that walking female flies respond to a regressively moving fly-sized object with much greater probability than to a progressively moving target. Under the assumption that the freezing behavior of the fly is a proxy for its ability to detect the small target, the results provide strong support for the regressive motion saliency hypothesis and confirm anecdotal observations of interactions between pairs of flies. These experiments provide further evidence for the utility of behavioral robotics as a method for analyzing the sensory basis of social interactions. The results might alternatively be taken as evidence for progressive motion blindness, but either way, the experiments suggest that the ability of flies to detect small moving objects during locomotion is strongly constrained by the optic flow patterns created by self-motion and that they can use regressive motion as a simple rule of thumb that does not require sophisticated compensation for self-motion. This is the first time that this hypothesis has been proposed or tested. However, the phenomenon may be related to the principle of motion camouflage, a mechanism for crypsis that has been proposed for flying insect. To implement motion camouflage, an animal must move in a way so that it appears stationary (to another moving animal) relative to a distant background. The current results would suggest that such camouflage might not require perfect compensation, but might be effective as long as an animal's own motion did not create regressive motion on another's retina (Zabala, 2012).

This analysis depends critically on the interpretation that walking females reflexively freeze when detecting a nearby moving object. This is believed to be justified since many animals freeze upon detecting a nearby animal, a simple behavioral reflex that has several advantages. By stopping an animal's visual performance is no longer compromised by the optic flow created by self-motion, a stationary animal is harder to detect, and once stopped, an animal can better prepare for an action such as an escape. An alternative interpretation of the data is that progressively moving objects are as detectable, but female flies simply 'decide' not to stop. According to this view, the reflex might serve some useful function such as to establish rights of way when animals are on a collision course, similar to rules used by boat captains. However, it is not easy to imagine a selective scenario by which such etiquette would evolve, and given the distance at which these reflexes operates, the interpretation is favored that the difference in behavioral responses to progressive and regressive motion indicates a limitation of visual processing rather than a behavioral choice. Another interesting question is whether this reflex evolved in flies specifically for mediating interactions between conspecifics, or alternatively, represents a general reaction to nearby organisms (Zabala, 2012).

Sensitivity to regressive motion is not a general feature of visual reflexes in flies. Indeed, studies of object orientation behavior in tethered flying animals indicate that flies are more sensitive to progressively moving objects than to regressively moving ones, and this asymmetry has been proposed to explain the stable fixation of vertical stripes. In contrast, studies of object orientation and optomotor equilibriumin walking flies suggests that the sensitivity to regressive and progressive motion is comparable. However, these prior studies focus on a fly's ability to orient toward visual landmarks and walk straight using large-field optomotor cues and may not be relevant to the behavior describe in this study, which are interpreted as a reflex used to detect the presence of a nearby organism. In particular, the female flies responded to the robot's motion by freezing, not by steering toward the stimulus as would be expected in an object orientation behavior. The neurons responsible for the behavior are not known, and the evidence precludes the involvement of looming detector neurons that have recently been described in this species (de Vries, 2012; Fotowat, 2009). Large-field neurons might be capable of detecting small targets provided the contrast was sufficient. However, the results indicating that the response to regressive motion may be triggered by targets smaller than an ommatidial acceptance angle implicate the classes of cells in insects termed figure detectors (FDs) and small target movement detectors (STMDs). The fact that the behavior degrades if the animal is rotating at the onset of stimulus motion suggests that the underlying circuits cannot detect a regressively moving object when superimposed on the reafferent large field rotatory optic flow generated by self-motion. Nevertheless, the detection threshold of approximately one-third an ommatidial acceptance angle is impressive for an animal (Drosophila) that is not behaviorally specialized for detecting small prey as are dragonflies or detecting mates and territorial interlopers as are hoverflies and houseflies. Further exploration of these hypotheses will require identification of the underlying visual interneurons and recordings of their visual responses during locomotion, an approach that may be possible in Drosophila due to recent methodological advances (Zabala, 2012).

A biphasic locomotor response to acute unsignaled high temperature exposure in Drosophila

Unsignaled stress can have profound effects on animal behavior. While most investigation of stress-effects on behavior follows chronic exposures, less is understood about acute exposures and potential after-effects. This study examined walking activity in Drosophila following acute exposure to high temperature or electric shock. Compared to initial walking activity, flies first increase walking with exposure to high temperatures then have a strong reduction in activity. These effects are related to the intensity of the high temperature and number of exposures. The reduction in walking activity following high temperature and electric shock exposures survives context changes and lasts at least five hours. Reduction in the function of the biogenic amines octopamine / tyramine and serotonin both strongly blunt the increase in locomotor activity with high temperature exposure. However, neither set of biogenic amines alter the long lasting depression in walking activity after exposure (Ostrowski, 2018).

Data-driven analysis of motor activity implicates 5-HT2A neurons in backward locomotion of larval Drosophila

Rhythmic animal behaviors are regulated in part by neural circuits called the central pattern generators (CPGs). Classifying neural population activities correlated with body movements and identifying the associated component neurons are critical steps in understanding CPGs. Previous methods that classify neural dynamics obtained by dimension reduction algorithms often require manual optimization which could be laborious and preparation-specific. This study presents a simpler and more flexible method that is based on the pre-trained convolutional neural network model VGG-16 and unsupervised learning, and successfully classifies the fictive motor patterns in Drosophila larvae under various imaging conditions. Voxel-wise correlation mapping was also used to identify neurons associated with motor patterns. By applying these methods to neurons targeted by 5-HT2A-GAL4, which was generated by the CRISPR/Cas9-system, two classes of interneurons were identified, termed Seta and Leta, which are specifically active during backward but not forward fictive locomotion. Optogenetic activation of Seta and Leta neurons increased backward locomotion. Conversely, thermogenetic inhibition of 5-HT2A-GAL4 neurons or application of a 5-HT2 antagonist decreased backward locomotion induced by noxious light stimuli. This study establishes an accelerated pipeline for activity profiling and cell identification in larval Drosophila and implicates the serotonergic system in the modulation of backward locomotion (Park, 2018).

Flies evade looming targets by executing rapid visually directed banked turns

Avoiding predators is an essential behavior in which animals must quickly transform sensory cues into evasive actions. Sensory reflexes are particularly fast in flying insects such as flies, but the means by which they evade aerial predators is not known. Using high-speed videography and automated tracking of flies in combination with aerodynamic measurements on flapping robots, this study showed that flying flies react to looming stimuli with directed banked turns. The maneuver consists of a rapid body rotation followed immediately by an active counter-rotation and is enacted by remarkably subtle changes in wing motion. These evasive maneuvers of flies are substantially faster than steering maneuvers measured previously and indicate the existence of sensory-motor circuitry that can reorient the fly's flight path within a few wingbeats (Muijres, 2014).

Previous studies on free-flying flies suggested that yaw torque is generated by a regulation of wing rotation, effectively changing the relative aerodynamic angle of attack during the upstroke and downstroke. In this study of banked turns, it was also found that wing rotation was an important control parameter, although its contribution was relatively minor. In addition, the primary changes this study measured in wing rotation were associated with shifts in timing, possibly modulating rotational lift rather than angle of attack during upstroke and downstroke. This discrepancy with previous studies might reflect an interesting difference in the control of pitch and roll compared with yaw. It is also noteworthy that stroke angle exerts a stronger influence over forces and moments than either wing rotation or stroke deviation. This finding is consistent with many previous studies in tethered flight which show that flies robustly modulate stroke amplitude in response to sensory signals that elicit changes in flight force, as well as roll, pitch, and yaw (Muijres, 2014).

These results indicate that flies escape from looming objects by exhibiting a rapid banked turn. The motor basis of these rapid maneuvers are quite distinct from those previously described in that the change in direction is generated by a combination of pitch and roll, requiring active torque and countertorque generated by a fine-scaled, coordinated change in all aspects of wing motion. The changes in heading during these maneuvers are roughly 5 times as fast (5300o s-1) as those measured during voluntary saccadic turns (1000o s-1), suggesting that this strategy provides the animals with the fastest possible means for altering direction. Using the genetic and physiological approaches available in the closely related species D. melanogaster, it should be possible to elucidate the neural circuitry and muscle physiology that underlies these rapid behaviors (Muijres, 2014).

Wing-pitch modulation in maneuvering fruit flies is explained by an interplay between aerodynamics and a torsional spring

While the wing kinematics of many flapping insects have been well characterized, understanding the underlying sensory, neural, and physiological mechanisms that determine these kinematics is still a challenge. Two main difficulties in understanding the physiological mechanisms arise from the complexity of the interaction between a flapping wing and its own unsteady flow, as well as the intricate mechanics of the insect wing hinge, which is among the most complicated joints in the animal kingdom. These difficulties call for the application of reduced-order approaches. This strategy was used to model the torques exerted by the wing hinge along the wing-pitch axis of maneuvering fruit flies as a damped torsional spring with elastic and damping coefficients as well as a rest angle. Furthermore, the air flows were modeled using simplified quasistatic aerodynamics. The findings suggest that flies take advantage of the passive coupling between aerodynamics and the damped torsional spring to indirectly control their wing-pitch kinematics by modulating the spring parameters. The damped torsional-spring model explains the changes measured in wing-pitch kinematics during roll correction maneuvers through modulation of the spring damping and elastic coefficients. These results, in conjunction with the previous literature, indicate that flies can accurately control their wing-pitch kinematics on a sub-wing-beat time scale by modulating all three effective spring parameters on longer time scales (Beatus, 2015).

A visual horizon affects steering responses during flight in fruit flies

To navigate well through three-dimensional environments, animals must in some way gauge the distances to objects and features around them. Humans use a variety of visual cues to do this, but insects, with their small size and rigid eyes, are constrained to a more limited range of possible depth cues. For example, insects attend to relative image motion when they move, but cannot change the optical power of their eyes to estimate distance. On clear days, the horizon is one of the most salient visual features in nature, offering clues about orientation, altitude and, for humans, distance to objects. This study set out to determine whether flying fruit flies treat moving features as farther off when they are near the horizon. Tethered flies respond strongly to moving images they perceive as close. The strength of steering responses was measured while independently varying the elevation of moving stimuli and the elevation of a virtual horizon. Responses to vertical bars were found to be increased by negative elevations of their bases relative to the horizon, closely correlated with the inverse of apparent distance. In other words, a bar that dips far below the horizon elicits a strong response, consistent with using the horizon as a depth cue. Wide-field motion also had an enhanced effect below the horizon, but this was only prevalent when flies were additionally motivated with hunger. These responses may help flies tune behaviors to nearby objects and features when they are too far off for motion parallax (Caballero, 2016).

Neuroendocrine control of Drosophila larval light preference

Animal development is coupled with innate behaviors that maximize chances of survival. This study shows that the prothoracicotropic hormone (PTTH), a neuropeptide that controls the developmental transition from juvenile stage to sexual maturation, also regulates light avoidance in Drosophila melanogaster larvae. PTTH, through its receptor Torso, acts on two light sensors (the Bolwig's organ and the peripheral class IV dendritic arborization neurons) to regulate light avoidance. PTTH was found to concomitantly promote steroidogenesis and light avoidance at the end of larval stage, driving animals toward a darker environment to initiate the immobile maturation phase. Thus, PTTH controls the decisions of when and where animals undergo metamorphosis, optimizing conditions for adult development (Yamanaka, 2013)

Animal development is associated with multiple primitive, innate behaviors, allowing inexperienced juveniles to choose an environment that maximizes their survival fitness before the transition to adulthood. In insects, this transition is timed by a peak of ecdysone production induced by the prothoracicotropic hormone (PTTH). In the larval brain of Drosophila, PTTH is produced by two pairs of neurosecretory cells projecting their axons onto the prothoracic gland (PG), where ecdysone is produced. Transition to adulthood is associated with drastic changes in larval behavior: Feeding larvae remain buried in the food, whereas wandering larvae (at the end of larval development) crawl out and find a spot where they immobilize and pupariate. Mechanisms allowing proper coordination of these behavioral changes with the developmental program remain elusive (Yamanaka, 2013)

Two pairs of neurons in the central brain were recently reported to control larval light avoidance. Using specific antibodies to PTTH, this study established that these neurons labeled by the NP0394-Gal4 and NP0423-Gal4 lines correspond to the PTTH-expressing neurons. Moreover, silencing the ptth gene by using NP0423-Gal4 or a ubiquitous driver (tub-Gal4) impaired light avoidance, indicating that PTTH itself controls this behavior. PTTH activates Torso, a receptor tyrosine kinase whose knockdown in the PG prevents ecdysone production and induces a developmental delay. In contrast, knocking down torso in the PG did not cause any change in light avoidance, indicating that the role of PTTH in ecdysteroidogenesis is functionally distinct from its role in light avoidance behavior (Yamanaka, 2013)

Because in Drosophila the PTTH-producing neurons only innervate the PG, it was reasoned that PTTH is secreted into the hemolymph and reaches the cells or organs involved in light avoidance. Consistent with this, inactivation of PTTH-expressing neurons affects light avoidance with 8 to 10 hours delay, arguing against PTTH neurons projecting directly on their target cells to control light avoidance. PTTH peptide is present in the PTTH-expressing neurons throughout larval development and shows a marked increase before wandering, correlating with the rapid increase of ecdysteroidogenesis at this stage. Using an enzyme-linked immunosorbent assay (ELISA), it was found that PTTH is readily detected in the hemolymph with a fluctuation pattern similar to that of its accumulation in the PTTH-expressing neurons. Furthermore, hemolymph PTTH levels were significantly decreased upon RNA interference (RNAi)–mediated knockdown of ptth in the PTTH-expressing neurons, suggesting that in addition to the paracrine control of ecdysteroidogenesis in the PG, PTTH also carries endocrine function (Yamanaka, 2013)

Pan-neuronal knockdown of torso (elav>torso-RNAiGD) recapitulates the loss of light avoidance observed upon torso ubiquitous knockdown (tub>torso-RNAiGD), suggesting that PTTH acts on neuronal cells to control light avoidance. The potential role of torso was specifically tested in two neuronal populations previously identified as light sensors in Drosophila larvae: (1) the Bolwig's organ (BO) and (2) the class IV dendritic arborization (da) neurons tiling the larval body wall. An enhancer trap analysis of torso, as well as in situ hybridization using a torso antisense probe, confirmed torso expression in class IV da neurons. In parallel, torso transcripts were detected by means of quantitative reverse transcription polymerase chain reaction in larval anterior tips containing the BO, and their levels were efficiently knocked down by using the BO-specific drivers Kr5.1-Gal4 and Rh5-Gal4, demonstrating torso expression in the BO. The knockdown of torso in the BO (Kr5.1>torso-RNAiGD and GMR>torso RNAiGD) or in the class IV da neurons (ppk>torso-RNAiGD) abolished larval light avoidance (motoneurons serve as a negative control: OK6>torso-RNAiGD). Knocking down torso in both neuronal populations (ppk>, GMR>torso-RNAiGD) mimicked the effect observed with the BO driver or class IV da neuron driver alone. A similar loss of light avoidance was observed when these neurons were separately inactivated by expressing the hyperpolarizing channel Kir2.1 (GMR>Kir2.1 and ppk>Kir2.1), suggesting that both of these light sensors are necessary for light avoidance behavior. Down-regulation of PTTH/Torso signaling did not lead to any neuronal morphology or locomotion defect, further indicating its direct effect on light sensing. The knockdown of torso in class IV da neurons or in the BO had no effect on the pupariation timing. Taken together, these results indicate that PTTH/Torso signaling is required for light avoidance behavior in two distinct populations of light-sensing neurons and that this function is separate from its role in controlling developmental progression (Yamanaka, 2013)

Drosophila light-sensing cells use photosensitive opsins that upon exposure to light, activate transient receptor potential (TRP) cation channels, thus depolarizing the membrane and triggering neural activation. Although the BO and class IV da neurons use different photosensitive molecules and TRP channels, one can assume that PTTH/Torso signaling regulates the phototransduction pathway through a similar mechanism in both types of neurons. Immunohistochemical detection of Rh5, the opsin involved in light avoidance behavior in the BO, showed no difference in protein level in torso mutant background. PTTH/Torso signaling knockdown did not change the expression level of Gr28b, a gustatory receptor family gene that plays an opsin-like role in class IV da neurons. These results strongly suggest that PTTH affects signaling components downstream of the photoreceptors (Yamanaka, 2013)

The neural activity of the light sensors was investigated using the calcium indicator GCaMP3 for live calcium imaging. torso mutant class IV da neurons showed a 25% reduction of their response to light as compared with that of control. This was accompanied by a loss of light avoidance, indicating that such partial reduction of the GCaMP3 signal corresponds to a reduction of neural activity strong enough to exert a behavioral effect. Indeed, blocking the firing of class IV da neurons by using TrpA1-RNAi caused a similar 25% reduction of the GCaMP3 signal and behavioral effect. This suggests that in da neurons, PTTH/Torso signaling exerts its action upstream of TrpA1 channel activation. Accordingly, a strong genetic interaction was observed between torso and TrpA1 mutants for light preference. A genetic interaction between torso and Rh5 mutants was also detected, further supporting that PTTH/Torso signaling affects a step in phototransduction between the photoreceptor molecule and the TRP channel. Collectively, these data are consistent with the notion that PTTH/Torso signaling acts to facilitate TRP activation downstream of photoreceptor-dependent light sensing (Yamanaka, 2013)

A previous study suggested that larval photophobic behavior diminishes at the end of larval development, perhaps facilitating larval food exit and entry into the wandering phase. The present finding and the increase of PTTH at the beginning of the wandering stage appear to contradict such a hypothesis. Indeed, a sustained larval light avoidance mediated by PTTH was detected that persisted through the wandering stage. These results imply that wandering behavior is triggered by a signal distinct from light preference. Consistent with this notion, the timing of wandering initiation in ppk>torso-RNAiGD or Kr5.1>torso-RNAiGD larvae was found comparable with that of control animals, despite the fact that these animals are not photophobic (Yamanaka, 2013)

As found in other insects, wandering is either directly or indirectly triggered by PTTH- induced ecdysone production. Therefore, concomitant PTTH-mediated photophobicity could ensure that wandering larvae maintain a dark preference for pupariation site, providing better protection from predators and dehydration during the immobile pupal stage. To test this hypothesis, a light/dark preference assay was developed for pupariation. When exposed to a light/dark choice, larvae indeed showed a strong preference to pupariate in the dark. This behavior was abolished either by inactivating PTTH-expressing neurons (ptth>Kir2.1), by silencing ptth in the PTTH-expressing neurons (NP0423>ptth-RNAi, dicer2), or by introducing a torso mutant background (torso[e00150]/[1]). Dark site preference for pupariation was observed in Drosophila populations collected in the wild, confirming that this innate behavior was selected in a natural environment (Yamanaka, 2013)

This work illustrates the use of a single biochemical messenger, PTTH, for the concomitant control of two major functions during larval development. PTTH establishes a neuroendocrine link between distinct neuronal components previously shown to be involved in light avoidance. In contrast to previous interpretations but consistent with another study, this study showed that wandering is independent of light preference and that PTTH maintains a strong light avoidance response through to the time of pupariation. High levels of circulating PTTH during the wandering stage could reinforce the robustness of light avoidance, which might otherwise be compromised by active roaming. This eventually promotes larvae to pupariate in the dark, a trait potentially beneficial for ecological selection. PTTH is thus at the core of a neuroendocrine network, promoting developmental progression and appropriate innate behavioral decisions to optimize fitness and survival (Yamanaka, 2013)

Transmembrane channel-like (tmc) gene regulates Drosophila larval locomotion

Drosophila larval locomotion, which entails rhythmic body contractions, is controlled by sensory feedback from proprioceptors. The molecular mechanisms mediating this feedback are little understood. By using genetic knock-in and immunostaining, this study found that the Drosophila melanogaster transmembrane channel-like (tmc) gene is expressed in the larval class I and class II dendritic arborization (da) neurons and bipolar dendrite (bd) neurons, both of which are known to provide sensory feedback for larval locomotion. Larvae with knockdown or loss of tmc function displayed reduced crawling speeds, increased head cast frequencies, and enhanced backward locomotion. Expressing Drosophila TMC or mammalian TMC1 and/or TMC2 in the tmc-positive neurons rescued these mutant phenotypes. Bending of the larval body activated the tmc-positive neurons, and in tmc mutants this bending response was impaired. This implicates TMC's roles in Drosophila proprioception and the sensory control of larval locomotion. It also provides evidence for a functional conservation between Drosophila and mammalian TMCs (Guo, 2016).

Drosophila tracks carbon dioxide in flight

Carbon dioxide (CO2) elicits an attractive host-seeking response from mosquitos yet is innately aversive to Drosophila melanogaster despite being a plentiful byproduct of attractive fermenting food sources. Prior studies used walking flies exclusively, yet adults track distant food sources on the wing. This study shows that a fly tethered within a magnetic field allowing free rotation about the yaw axis actively seeks a narrow CO2 plume during flight. Genetic disruption of the canonical CO2-sensing olfactory neurons does not alter in-flight attraction to CO2; however, antennal ablation and genetic disruption of the Ir64a acid sensor do. Surprisingly, mutation of the obligate olfactory coreceptor (Orco; Or83b) does not abolish CO2 aversion during walking yet eliminates CO2 tracking in flight. The biogenic amine octopamine regulates critical physiological processes during flight, and blocking synaptic output from octopamine neurons inverts the valence assigned to CO2 and elicits an aversive response in flight. Combined, these results suggest that a novel Orco-mediated olfactory pathway that gains sensitivity to CO2 in flight via changes in octopamine levels, along with Ir64a, quickly switches the valence of a key environmental stimulus in a behavioral-state-dependent manner (Wasserman, 2013).

These results show that a single molecule can carry both negative and positive hedonic valence depending on the behavioral state of the animal. It is posited that flight behavior is accompanied by neuromodulatory activation of the olfactory system by octopamine that rapidly shifts the function of olfactory sensory pathways in a manner similar to the operational gain and frequency response shifts triggered by locomotor activity in fly visual interneurons. Recent work in other organisms has identified similar roles for neuromodulators that serve to alter the state of neuronal circuits in a behaviorally contextual manner, thereby enabling computational flexibility and behavioral robustness to ever-changing internal and external environmental conditions. These findings unravel the paradox of why D. melanogaster would find an environmental signal indicating a potential food source repellent instead of attractive; for Drosophila gathered on the ground, under crowded social conditions, CO2 secreted as part of a stress pheromone releases an innate avoidance response. Taking flight appears to fully and rapidly switch the valence of this stimulus, triggering CO2 attraction consistent with the search for sugar-rich food resources undergoing fermentation that robustly attract D. melanogaster vinegar flies. These findings lay the groundwork for further exploring the neural substrate for a rapid and robust switch in hedonic valence (Wasserman, 2013).

Nociceptive neurons protect Drosophila larvae from parasitoid wasps

Natural selection has resulted in a complex and fascinating repertoire of innate behaviors that are produced by insects. One puzzling example occurs in fruit fly larvae that have been subjected to a noxious mechanical or thermal sensory input. In response, the larvae 'roll' with a motor pattern that is completely distinct from the style of locomotion that is used for foraging. The sensory neurons that are used by the Drosophila larvae to detect nociceptive stimuli have been precisely mapped. By using complementary optogenetic activation and targeted silencing of sensory neurons, it has demonstrated that a single class of neuron (class IV multidendritic neuron) is sufficient and necessary for triggering the unusual rolling behavior. In addition, it was found that larvae have an innately encoded preference in the directionality of rolling. Surprisingly, the initial direction of rolling locomotion is toward the side of the body that has been stimulated. It is proposed that directional rolling might provide a selective advantage in escape from parasitoid wasps that are ubiquitously present in the natural environment of Drosophila. Consistent with this hypothesis, it has been documented that larvae can escape the attack of Leptopilina boulardi parasitoid wasps by rolling, occasionally flipping the attacker onto its back. It is concluded that the class IV multidendritic neurons of Drosophila larvae are nociceptive. The nociception behavior of Drosophila melanagaster larvae includes an innately encoded directional preference. Nociception behavior is elicited by the ecologically relevant sensory stimulus of parasitoid wasp attack (Hwang, 2007).

The Drosophila GAL4/UAS (UAS: upstream activation sequence) system allows for the targeting of gene expression to precise cells of the animal. Several GAL4 drivers have been identified that allow for the targeting of gene expression to arborizing md neurons. These drivers were examined to determine whether they would be useful for targeting specific subsets of md neurons in behavioral experiments. It was planned to cross the drivers to UAS-tetanus toxin light chain (UAS-TnT-E) lines and then to test animals that were transheterozygous for the GAL4 driver and UAS-TnT-E in nociception behavioral assays. Because the tetanus toxin light chain cleaves the v-snare synaptobrevin, it reduces evoked synaptic vesicle release in the neurons that express the GAL4 driver, effectively silencing them (Hwang, 2007).

Four GAL4 drivers were identified that would be useful for these experiments. The GAL4109(2)80 driver (md-GAL4) was expressed in all four classes of md neurons whose projections decorated the entire epidermis. The class I and II neurons, whose relatively unbranched dendrites tile only a subset of the entire epidermis, were strongly targeted by the c161-GAL4 driver. On the basis of central projections, and behavioral evidence, the class I neurons have been previously proposed to function as proprioceptors. The function of class II md neurons is not known. The class III and IV md neurons possess more complex dendrites that tile the entire epidermis. There is no region that lacks endings from these cells, a feature that would be expected for nociceptive neurons. The class III neurons (as well as class II neurons) were targeted by the recently described 1003.3-GAL4 driver. Finally, the pickpocket1.9-GAL4 (ppk-GAL4) driver targets class IV md neurons (Hwang, 2007).

When md-GAL4 was used to drive the expression of UAS-TnT-E in all four classes of md neurons, the behavioral response to noxious heat was completely abrogated. The c161-GAL4 driver, which targeted to all neurons of the class I and II subtypes, was assayed. Compared to the control (UAS-TnT-E/+), larvae expressing UAS-TnT-E under the control of c161-GAL4 showed only a slight (although statistically significant) delay in their initiation of nocifensive responses to the noxious heat probe, contrasting with the results seen with md-GAL4. However, the rolling behavioral output did not appear to be as coordinated as in the wild-type, and it took these larvae longer than the control lines to achieve a complete roll. This result is consistent with previous studies that implicated the class I md neurons in proprioceptive feedback that plays a role in peristaltic locomotion. The results might indicate that the class I (or possibly the class II) md neurons also provide proprioceptive feedback necessary for the completion of rolling behavior (Hwang, 2007).

The class II and class III neurons with the 1003.3-GAL4 driver to drive UAS-TnT-E. These larvae appeared normal in their initiation of the behavioral response compared to the control UAS-TnT-E/+. The rolling response of larvae with silenced class II and class III neurons appeared to be coordinated, and no obvious defects were observed in the quality of the behavior. This result also suggests that the coordination defects seen with the c161-GAL4 driver are unlikely to be due to the inhibition of class II neurons (Hwang, 2007).

These data indicate that the blocking of the output of class I and class II or of class II and class III neurons did not strongly affect thermal nociception responses. This suggested that the class IV neurons might be the relevant neurons involved in the thermal nociception response. To test this, the class IV neurons were specifically inactivated with the ppk-GAL4 driver. Indeed, larvae of the ppk-GAL4/UAS-TnT-E genotype showed a dramatically impaired thermal nociception response compared to the control UAS-TnT-E/+. The frequency of larvae that failed to perform nocifensive behavior even after 10 s of stimulation was significantly increased relative to control genotypes. The rare larvae of this genotype that did produce the rolling behavior appeared to be coordinated. Although the blocking of the class IV neurons did not completely eliminate thermal nociception, the remaining response could be due to an incomplete block of the synaptic output in these cells by tetanus toxin light chain. Alternatively, parallel processing might occur through sensory neurons that have yet to be identified as nociceptive (Hwang, 2007).

Prior studies indicated that strong mechanical stimuli elicited the same nocifensive behavior that can be elicited by noxious heat. Thus, which of the various subtypes of md neurons were required for mechanical responses was tested. The blocking of the class I and class II neurons did impair the behavioral responses to mechanical stimuli. However, this result is difficult to interpret given that these larvae also appeared to be uncoordinated. The blocking of the output of classes II and III had a milder effect on mechanical nociception. Finally, as with thermal nociception, the blocking of class IV neurons most strongly diminished the response to mechanical stimuli suggesting the possibility that these cells have polymodal nociceptive functions (Hwang, 2007).

Combined, these results suggested that the synaptic output of class IV md neurons plays a major role in the initiation of nocifensive behavioral responses to noxious heat or mechanical stimulation. However, there are caveats to this interpretation of the data. For example, although the data suggested that the class IV md neurons are required for the nocifensive behavior, they did not prove that these neurons are nociceptors. Instead, it was possible that these neurons were required for the central nervous system to efficiently control the behavior. For example, they could merely provide proprioceptive feedback (Hwang, 2007).

Therefore, whether the activation of md neurons would be sufficient to elicit nocifensive behaviors was tested. If the activation of these neurons could be shown to trigger the nocifensive behavior, it would place them upstream of the rolling behavioral output in this neuronal pathway (Hwang, 2007).

Targeted photoactivation of neurons has been achieved with Channelrhodopsin-2 (ChR2), a light-activated cation channel from green algae. Importantly, ChR2 has been shown to be capable of causing light-induced action potentials in Drosophila motor neurons. Further, GAL4 lines were used for the driving of UAS-Channelrhodopsin-2 in either dopaminergic or octopaminergic neurons. Remarkably, illumination with blue light resulted in specific associative learning effects in larvae that were dependent on the feeding of larvae all-trans retinal (which forms the chromophore for Channelrhodopsin-2) (Hwang, 2007).

Flies were generated that express fluorescently tagged Channelrhodopsin-2 (ChR2-YFP [YFP: yellow fluorescent protein] or ChR2-mCherry) under control of the GAL4/UAS system (UAS-ChR2-YFP, UAS-ChR2-mCherry). The fluorescent tags allowed identification, a priori, of lines that generated detectable expression levels of ChR2 in the md neurons. The expression of ChR2-YFP and ChR2-mCherry proteins has been shown to render rat hippocampal neurons light sensitive (Hwang, 2007).

Several of the YFP-tagged lines were selected for behavioral analysis. The lines selected showed detectable fluorescence in peripheral sensory neurons when they were crossed to appropriate GAL4 drivers. A more strongly expressing line (AB) with UAS insertions on both the second and third chromosomes was chosen for behavioral analysis, and it was crossed to the GAL4 strains described above. The larval progeny from these crosses were raised to third-instar larvae fed on yeast paste that either contained all-trans retinal (atr+) or on yeast paste that did not contain all-trans retinal (atr-); the latter contained the diluent alone (0.5% EtOH). Individual third-instar larvae were then transferred to a small droplet of water in a Petri dish so that they could be viewed on a fluorescent stereomicroscope that was equipped with a video recorder (allowing the behavioral response to the illumination of blue light to be recorded) (Hwang, 2007).

The atr+ yeast paste had no effect in control larvae that lacked a GAL4 driver (UAS-ChrR2-YFP/+). However, in the presence of GAL4 drivers, behaviors to pulses of blue light (460-500 nm) were observed that were strongly dependent on the feeding of all-trans retinal. Even in the presence of GAL4 drivers, ChR2-YFP-expressing larvae that were fed the atr- yeast paste only rarely produced behaviors in response to blue light (Hwang, 2007).

The effects were tested of the expression of ChR2-YFP with the class I-IV (md-GAL4) driver. It was found that in response to blue light pulses, the larvae performed one of two distinct behavioral responses. In the most prevalent response, the blue light caused the larvae to simultaneously contract the muscles of all body segments and to scrunch like a compressed accordion. In a more rarely occurring response, the larvae rolled with a motor pattern that appeared to be quite similar to the nocifensive rolling behavior. However, in these larvae, the light-induced rolling was eventually followed by the accordion-like muscle contraction behavior. The latter behavior is never seen in response to noxious heat or mechanical stimuli (Hwang, 2007).

It was hypothesized that the accordion-like behavior and the rolling behavior might reflect the activation of distinct behavioral pathways as a consequence of the activation multiple neurons with distinct functions. For example, it seemed possible that some md neurons might trigger segmental muscle contractions, whereas others might trigger nocifensive rolling. If this were the case, then the behaviors described above could be the result of competition for these two pathways by light-induced activation of the relevant triggering sensory inputs. Thus whether the activation of the two behavioral pathways could be separated by more precise targeting of the ChR2-YFP to distinct md-neuron subsets was further tested (Hwang, 2007).

Indeed, when ChR2-YFP was expressed in the class I and II neurons (c161-GAL4) or in the class II and III neurons (1003.3-GAL4), rolling behavior was never observed in response to illumination with blue light. Instead, the blue light caused the larvae to perform the accordion response with high penetrance. No rolling behavior was observed in response to the activation of the class I, II, or III neurons; this is consistent with the inactivation studies described above because no evidence was found that the synaptic output of these neuronal types is strongly required for the initial steps of thermal nociception behavior (Hwang, 2007).

It is hypothesized that the accordion phenotype reflects a role for the class II and/or class III md neurons in propagating the wave of muscle contraction during peristaltic locomotion. During normal locomotion, the activation of the class II or the class III neurons might occur via muscle contraction within a segment. This might produce a signal coordinating the contraction of muscles in the next segment. The accordion phenotype likely represents a manifestation of this process but in an abnormal situation in which the signal is sent to all segments simultaneously via optogenetic activation (Hwang, 2007).

In contrast, optogenetic activation of class IV md neurons (ppk-GAL4/UAS-ChR2-YFP) caused robust nocifensive-like rolling behavior and never resulted in the accordion-like behavior. The penetrance of the nocifensive response to the blue light pulse was impressive, with 87% of the larvae responding with rolling behavior in lines strongly expressing ChR2-YFP. With lines that expressed ChR2-YFP more weakly, the same behavior was observed but with a reduced frequency (Hwang, 2007).

Qualitatively, this light-induced nocifensive behavior appeared to be very similar to thermally and mechanically induced rolling behavior. However, the light-induced behavior was initiated very rapidly (<100 ms after the light was turned on). This was more rapid than the rolling induced with standard nociception stimulus of 47°C, where the larvae often require thermal stimulation of several seconds to elicit the response. The rapid behavioral responses with ChR2-YFP likely reflect the extremely rapid kinetics of a light-activated channel relative to slower kinetics of thermal nociception at 47°C. Indeed, very rapid thermal nociception responses can also be observed (rolling that initiates < 100 ms after contact with the thermal probe), but these require a stronger thermal nociception stimulus (53°C probe) (Hwang, 2007).

Combined, these data conclusively demonstrate that the activation of class IV md neurons of the third-instar Drosophila larva is sufficient to trigger a nocifensive-like motor output. In addition, it was found that the output of the class IV neurons is necessary for triggering normal nocifensive behavior in thermal and mechanical nociception assays. It is therefore proposed that the class IV multidendritic neurons function as nociceptors (Hwang, 2007).

A thermal stimulus was used as a convenient method for the identification of neurons and mutants that affect the nociception pathway. To perform the rolling escape response, the larva uses its muscles to move in a highly coordinated fashion that is distinct from peristaltic locomotion. This suggests the existence of multiple central pattern generators in the larval brain. Rolling locomotion causes larvae to move at a significantly higher velocity (3-5 mm/s) than typical peristaltic locomotion (1 mm/s). This increased velocity presumably provides a selective advantage in a rapid escape from a potentially damaging thermal insult. However, it is unlikely that the rolling behavior evolved solely for thermal nociception because strong mechanical stimuli also elicit the rolling response (Hwang, 2007).

Indeed, the following observations suggest that thermal nociception is unlikely to be the only selective pressure that drove the evolution of this behavior. Surprisingly, it was found that Drosophila larvae have a genetically encoded tendency to initially roll toward the noxious heat probe rather than away from it. When animals were stimulated on the right side of the body, the preferred initial direction of rolling was to the right (i.e., rolling clockwise). When they were stimulated on the left, the preferred direction of rolling was toward the left (i.e., rolling counterclockwise). The result was that the animals tended to initially roll in a direction that pushed them against the thermal probe rather than in a direction that carried them away. After failing to escape after several revolutions, the larvae did reverse the direction of rolling. The choice of roll direction was not random, indicating that the larval brain was capable of sensing the direction from which the stimulus was coming. Yet, paradoxically, the circuitry that controlled rolling was strongly biased to produce the initial direction of motor output toward the side of the body that had been stimulated (Hwang, 2007).

In natural populations of Drosophila, then, there must be a strong selective advantage in directional rolling toward the side of the body in which the nociceptors have been activated. It is not obvious to how this would be the case if noxious heat were the sole selective pressure driving the evolution of this behavior (Hwang, 2007).

An explanation was sought for this paradox in the natural ecology of Drosophila. Diverse species of parasitoid wasps require a Drosophila host in order to complete their life cycle. For example, the figitid wasp, Leptopilina boulardi, is an obligate and ubiquitous parasitoid of Drosophila. Female Leptopilina lay their eggs within the Drosophila by penetrating the larval cuticle and epidermis with a sharp ovipositor. It has been reported that larval hosts of sufficient size (third instar) can behaviorally defend from wasp attack with vigorous movements. Inexperienced wasps have been reported to be more susceptible to larval defenses, whereas experienced wasps eventually learn to attack smaller, more defenseless, animals. Thus, it was of interest to observe larval behavioral responses to wasp attack in order to determine whether the defensive behaviors triggered by wasps was similar to the responses that were triggered by activation of class IV neurons via Channelrhodopsin-2YFP (Hwang, 2007).

It was reasoned that the very fine and highly branched dendrites of the class IV multidendritic neurons might be capable of detecting attacks from the wasp ovipositor. If this were the case, wasp attack should elicit the rolling response. It was further reasoned that the bias in rolling direction that was observed experimentally might be of benefit in evading a wasp attack. Rolling in the direction counter to the side of the attack might actually assist the wasp in its attempts to penetrate the cuticle. In contrast, rolling toward the direction of wasp attack might deflect the ability of the wasp ovipositor to penetrate the cuticle and allow the larva to escape. It was hypothesized that such a mechanism would favor the evolution of neuronal circuits that encoded a behavioral tendency to roll toward the side of the body in which nociceptors had been activated rather than away (Hwang, 2007).

As predicted, it was found that third-instar Drosophila larvae did respond to attack by Leptopilina boulardi by producing the rolling response. The wasp-triggered rolling resembled both mechanical- and optogenetically triggered rolling. Also, as expected, larvae were observed that rolled toward the side of the body from which the wasp attack had come. However, the overall outcome of the larval responses was somewhat unexpected. Surprisingly, as a consequence of the larva rolling toward the wasp, the thin threadlike ovipositor of the wasp became wrapped around the larva. The rolling behavior of the larva thus acted like a spool winding a thread. After the larva had performed several revolutions, the wasp ovipositor eventually reached the limits of its length. The larva continued to roll and finally carried the wasp through the air and onto its back. At this point the wasp, clearly in danger of getting stuck in the medium, appeared to prematurely break off its attack. The larva then quickly left the area as the female wasp retracted its ovipositor (Hwang, 2007).

In conclusion, this study provided strong evidence that at least one of the classes of Drosophila multidendritic neuron is nociceptive. These are the class IV neurons that express the pickpocket gene. First, it was shown that the blocking of the synaptic output of class IV md neurons significantly impairs thermal and mechanical nociception behavior. Second, it was shown that optogenetic activation of these neurons is sufficient to trigger the stereotyped rolling response (Hwang, 2007).

It is important to note that these data do not rule out the possibility that the class IV neurons might have polymodal functions. It is possible, for example, that the firing rate triggered by nociceptive stimuli exceeds a critical threshold that triggers rolling, whereas lower firing rates could be used to regulate turning or rate of locomotion. The latter possibility would be consistent with prior studies that proposed that the class IV md neurons function as proprioceptors. This hypothesis was based on the fact that larvae mutant for pickpocket move rapidly and turn less frequently than wild-type larvae and by the fact that pickpocket is solely expressed in class IV neurons. It will be interesting to further investigate the pickpocket mutant phenotypes in light of the nociceptive function for the class IV neurons that were presented in this study (Hwang, 2007).

The differences in the central projections of distinct types of md neurons are notable in light of the functions that were observed. Of the four classes of multidendritic neuron, only the class IV md neurons have projections that cross the midline to innervate contralateral postsynaptic targets. This has interesting similarity to pain processing in vertebrate nervous systems, in which ascending tracts of the contralateral spinal chord carry painful sensory information to higher-order neurons of the brain. Future studies will allow determination of whether the brain of the larva is involved in the perception of the noxious stimulus or whether lower-level processing in the abdominal or thoracic ganglion plays a role (Hwang, 2007).

Perhaps the most interesting question related to this issue is whether input from the class IV neurons of larvae, or of adult flies, has negative hedonic value. In larvae, this might allow the avoidance of regions of fruits whose odor is associated with the presences of wasps. Interestingly, the class IV neurons persist through metamorphosis and are present in adult flies, where they are unlikely to activate a rolling central pattern generator. Furthermore, noxious heat is known to be an effective unconditioned stimulus in adult Drosophila operant learning paradigms. Can the optogenetic activation of the class IV md neurons be used as a substitute for an unconditioned stimulus in operant negative associative learning paradigms? Does electric shock used in olfactory learning paradigms activate overlapping neuronal circuits (Hwang, 2007)?

The unambiguous identification of the class IV md neurons as nociceptors opens the door to further analysis of these interesting questions and will allow dissection of this neuronal circuit, from the molecule to the behavior. In addition, identification of parasitoid wasp attack as an ecologically relevant stimulus that elicits rolling demonstrates an evolutionarily important adaptive function for this fascinating behavior (Hwang, 2007).

Two alternating motor programs drive navigation in Drosophila larva

When placed on a temperature gradient, a Drosophila larva navigates away from excessive cold or heat by regulating the size, frequency, and direction of reorientation maneuvers between successive periods of forward movement. Forward movement is driven by peristalsis waves that travel from tail to head. During each reorientation maneuver, the larva pauses and sweeps its head from side to side until it picks a new direction for forward movement. This study characterized the motor programs that underlie the initiation, execution, and completion of reorientation maneuvers by measuring body segment dynamics of freely moving larvae with fluorescent muscle fibers as they were exposed to temporal changes in temperature. Reorientation maneuvers were found to be characterized by highly stereotyped spatiotemporal patterns of segment dynamics. Reorientation maneuvers are initiated with head sweeping movement driven by asymmetric contraction of a portion of anterior body segments. The larva attains a new direction for forward movement after head sweeping movement by using peristalsis waves that gradually push posterior body segments out of alignment with the tail (i.e., the previous direction of forward movement) into alignment with the head. Thus, reorientation maneuvers during thermotaxis are carried out by two alternating motor programs: (1) peristalsis for driving forward movement and (2) asymmetric contraction of anterior body segments for driving head sweeping movement (Lahiri, 2011).

Animals execute complex behaviors by breaking tasks into discrete steps that can be accomplished through the deployment of motor programs. Understanding orientation behavior, how an animal senses and moves with respect to stimulus gradients in its environment, has long been a classic problem in neuroethology. Classic studies in the phototactic behavior of maggot larvae, for example, identified and explored how movement patterns like head sweeping allow these small animals to change their direction of movement with respect to light sources. However, comprehensive understanding of the neural basis of orientation behavior necessitates using genetically and physiologically tractable model organisms like Drosophila (Lahiri, 2011).

In an earlier study, it was shown that decision-making during larva thermotaxis occurs during the reorientation maneuvers that separate successive periods of forward movement (Luo, 2010). This study used the direct visualization of segment dynamics with high-resolution fluorescence microscopy to characterize the motor programs that drive thermotaxis in transgenic Drosophila larvae with fluorescently labeled muscle fibers. Initiation, execution, and completion of these reorientation maneuvers were shown to be carried out by alternating deployment of two types of motor programs: one for asymmetric contraction that drives anterior bending (head sweeping) and one for peristalsis that drives forward movement. The high degree of stereotypy in each motor program suggests that navigational strategy can be executed with a relatively small set of commands that emanate from the larval brain. First, the brain uses thermosensory input to trigger the transition from peristaltic forward movement to head sweeping movement that signifies the onset of each reorientation maneuver. During the initial bending movements of a head sweep, the brain specifies the amplitude of each head sweep to be carried out by one group of body segments. Finally, to complete the reorientation maneuver, the larva returns to the motor program for peristalsis to straighten the body along the new direction for forward movement (Lahiri, 2011).

To complete a turn, peristalsis can resume from different positions depending on the size of the head sweep. It is unclear why this happens, but these observations do demonstrate that peristalsis can be initiated at different points along the body. One reason might be to avoid disrupting the angle of reorientation defined by the angular size of large head sweeps: if peristalsis were initiated at the tail after a large head sweep, the position of the pivot might be pushed forward when the peristalsis reaches the pivot, perhaps increasing the angle of the reorientation beyond the size of the original head sweep. The ability to initiate peristalsis in different segments depending on the degree of body bend might point to a role for sensory feedback in the motor circuit. Proprioceptive feedback has recently been shown to play an essential role in generating peristalsis in the Drosophila larva (Lahiri, 2011 and references therein).

Drosophila larvae also exhibit phototaxis and chemotaxis, but the navigational strategies have yet to be defined in the same detail as larval thermotaxis. Comparative analysis of navigational strategies and motor programs during different modes of navigation would illuminate whether shared sensorimotor pathways are used during different navigational modes. This study focused on the two motor programs that build thermotaxis, but other motor programs appear to be used during other types of behavioral response, such as reverse crawling, hunching, and rolling movements that are used during nociceptive and rapid avoidance responses. Elucidating the complete set of motor programs that can be carried out by the Drosophila larva will yield the building-blocks of its total behavioral repertoire. Elucidating the pathways by which these motor programs are triggered by environmental stimuli will yield a complete understanding of brain and behavior in this small animal (Lahiri, 2011).

It is noted that the progression of work on thermotactic navigation in the Drosophila larva parallels earlier studies of bacterial chemotactic navigation. The biased-random walk strategy of bacterial chemotaxis was originally established by using a tracking microscope to follow the movements of individual bacteria as they navigated chemical gradients: E. coli swims by alternating periods of forward movement (runs) with erratic reorientation movements (tumbles), and the bacterium postpones tumbles when it senses increasing amounts of chemoattractant in its surroundings. A tracking microscope was also used to establish that the larva navigates by alternating periods of forward movement with turning decisions. A recent study of bacterial chemotaxis, which parallels this study, used advances in fluorescent labeling and video microscopy to directly visualize how the detailed movements of individual bacterial flagella contribute to the initiation, execution, and conclusion of tumbles. Both bacteria and Drosophila larva use essentially two motor programs to navigate. By regulating the transition from forward movement to reorientation movement in response to sensory conditions, both organisms lengthen runs that happen to be pointed in favorable directions. The head sweeping that characterizes navigational decisions during larval thermotaxis allows the animal to explore an additional axis during reorientation, enabling it to point more runs towards favorable directions. The Drosophila larva does not improve its navigation beyond bacterial navigation by using a larger repertoire of motor programs, but by using both motor programs to more efficiently assess preferred directions in its environment (Lahiri, 2011).

Saccadic body turns in walking Drosophila

Drosophila melanogaster structures its optic flow during flight by interspersing translational movements with abrupt body rotations. Whether these 'body saccades' are accompanied by steering movements of the head is a matter of debate. By tracking single flies moving freely in an arena, it was discovered that walking Drosophila also perform saccades. Movement analysis revealed that the flies separate rotational from translational movements by quickly turning their bodies by 15 degrees within a tenth of a second. Although walking flies moved their heads by up to 20 degrees about their bodies, their heads moved with the bodies during saccadic turns. This saccadic strategy contrasts with the head saccades reported for e.g., blowflies and honeybees, presumably reflecting optical constraints: modeling revealed that head saccades as described for these latter insects would hardly affect the retinal input in Drosophila because of the lower acuity of its compound eye. The absence of head saccades in Drosophila was associated with the absence of haltere oscillations, which seem to guide head movements in other flies. In addition to adding new twists to Drosophila walking behavior, this analysis shows that Drosophila does not turn its head relative to its body when turning during walking (Geurten, 2014; PubMed).

Kinematic responses to changes in walking orientation and gravitational load in Drosophila melanogaster

Walking behavior is context-dependent, resulting from the integration of internal and external influences by specialized motor and pre-motor centers. Neuronal programs must be sufficiently flexible to the locomotive challenges inherent in different environments. Although insect studies have contributed substantially to the identification of the components and rules that determine locomotion, understanding of how multi-jointed walking insects respond to changes in walking orientation and direction and strength of the gravitational force is still not understood. In order to answer these questions the kinematic properties of untethered Drosophila was measured with high temporal and spatial resolution during inverted and vertical walking. In addition, the kinematic responses to increases in gravitational load were measured. Animals were found to be capable of shifting their step, spatial and inter-leg parameters in order to cope with more challenging walking conditions. For example, flies walking in an inverted orientation decreased the duration of their swing phase leading to increased contact with the substrate and, as a result, greater stability. It was also found that when flies carry additional weight, thereby increasing their gravitational load, some changes in step parameters vary over time, providing evidence for adaptation. However, above a threshold that is between 1 and 2 times their body weight flies display locomotion parameters that suggest they are no longer capable of walking in a coordinated manner. Finally, it was found that functional chordotonal organs are required for flies to cope with additional weight, as animals deficient in these proprioceptors display increased sensitivity to load bearing as well as other locomotive defects (Mendes, 2014: PubMed).

GABAergic inhibition of leg motoneurons is required for normal walking behavior in freely moving Drosophila

Walking is a complex rhythmic locomotor behavior generated by sequential and periodical contraction of muscles essential for coordinated control of movements of legs and leg joints. Studies of walking in vertebrates and invertebrates have revealed that premotor neural circuitry generates a basic rhythmic pattern that is sculpted by sensory feedback and ultimately controls the amplitude and phase of the motor output to leg muscles. However, the identity and functional roles of the premotor interneurons that directly control leg motoneuron activity are poorly understood. This study took advantage of the powerful genetic methodology available in Drosophila to investigate the role of premotor inhibition in walking by genetically suppressing inhibitory input to leg motoneurons. For this, an algorithm was developed for automated analysis of leg motion to characterize the walking parameters of wild-type flies from high-speed video recordings. Further, genetic reagents were used for targeted RNAi knockdown of inhibitory neurotransmitter receptors in leg motoneurons together with quantitative analysis of resulting changes in leg movement parameters in freely walking Drosophila. The findings indicate that targeted down-regulation of the GABAA receptor Rdl (Resistance to Dieldrin) in leg motoneurons results in a dramatic reduction of walking speed and step length without the loss of general leg coordination during locomotion. Genetically restricting the knockdown to the adult stage and subsets of motoneurons yields qualitatively identical results. Taken together, these findings identify GABAergic premotor inhibition of motoneurons as an important determinant of correctly coordinated leg movements and speed of walking in freely behaving Drosophila (Gowda, 2018).

The effect of stress on motor function in Drosophila

Exposure to unpredictable and uncontrollable conditions causes animals to perceive stress and change their behavior. It is unclear how the perception of stress modifies the motor components of behavior and which molecular pathways affect the behavioral change. In order to understand how stress affects motor function, an experimental platform was developed that quantifies walking motions in Drosophila. Stress induction using electrical shock was found to result in backwards motions of the forelegs at the end of walking strides. These leg retrogressions persisted during repeated stimulation, although they habituated substantially. The motions also continued for several strides after the end of the shock, indicating that stress induces a behavioral aftereffect. Such aftereffect could also be induced by restricting the motion of the flies via wing suspension. Further, the long-term effects could be amplified by combining either immobilization or electric shock with additional stressors. Thus, retrogression is a lingering form of response to a broad range of stressful conditions, which cause the fly to search for a foothold when it faces extreme and unexpected challenges. Mutants in the cAMP signaling pathway enhanced the stress response, indicating that this pathway regulates the behavioral response to stress. These findings identify the effect of stress on a specific motor component of behavior and define the role of cAMP signaling in this stress response (Chadha, 2014: PubMed).

Feature integration drives probabilistic behavior in the Drosophila escape response

Animals rely on dedicated sensory circuits to extract and encode environmental features. How individual neurons integrate and translate these features into behavioral responses remains a major question. This study has identified a visual projection neuron type that conveys predator approach information to the Drosophila giant fiber (GF) escape circuit. Genetic removal of this input during looming stimuli reveals that it encodes angular expansion velocity, whereas other input cell type(s) encode angular size. Motor program selection and timing emerge from linear integration of these two features within the GF. Linear integration improves size detection invariance over prior models and appropriately biases motor selection to rapid, GF-mediated escapes during fast looms. These findings suggest feature integration, and motor control may occur as simultaneous operations within the same neuron and establish the Drosophila escape circuit as a model system in which these computations may be further dissected at the circuit level (von Reyn, 2017).

Characterization of the decision network for wing expansion in Drosophila using targeted expression of the TRPM8 channel

After emergence, adult flies and other insects select a suitable perch and expand their wings. Wing expansion is governed by the hormone Bursicon and can be delayed under adverse environmental conditions. How environmental factors delay Bursicon release and alter perch selection and expansion behaviors has not been investigated in detail. This study provides evidence that in Drosophila the motor programs underlying perch selection and wing expansion have different environmental dependencies. Using physical manipulations, it was demonstrated that the decision to perch is based primarily on environmental valuations and is incrementally delayed under conditions of increasing perturbation and confinement. In contrast, the all-or-none motor patterns underlying wing expansion are relatively invariant in length regardless of environmental conditions. Using a novel technique for targeted activation of neurons, this study shows that the highly stereotyped wing expansion motor patterns can be initiated by stimulation of NCCAP, a small network of central neurons that regulates the release of Bursicon. Activation of this network using the cold-sensitive rat TRPM8 channel is sufficient to trigger all essential behavioral and somatic processes required for wing expansion. The delay of wing expansion under adverse circumstances thus couples an environmentally sensitive decision network to a command-like network that initiates a fixed action pattern. Because NCCAP mediates environmentally insensitive ecdysis-related behaviors in Drosophila development before adult emergence, the study of wing expansion promises insights not only into how networks mediate behavioral choices, but also into how decision networks develop (Peabody, 2009).

The introduction of genetically encoded effectors, which permit the targeted manipulation of neuronal activity in vivo, is increasingly facilitating neurobiological studies of behavioral choice in vivo, as exemplified by the work presented in this study. Using a new method for stimulating neurons in live animals, it was demonstrated that the NCCAP network, first implicated in governing ecdysis, effects the fly's decision to expand its wings after eclosion. This decision is normally coupled to the decision to perch, which this study demonstrates to be based on evaluation of environmental variables. The postponement of wing expansion by the fly under adverse circumstances is thus a consequence of a value-based choice to prolong search behavior and the delayed activation of a command network responsible for the execution of the wing expansion decision (Peabody, 2009).

The results support a model in which the motor programs underlying perch selection respond primarily to environmental input. Although the environmental features monitored by the fly and the sensory channels that process them remain to be determined, it is clear from the experiments that flies assess conditions during phase I and assign longer search periods to more adverse environments. The observation that expansion is not deferred indefinitely, even under the high-perturbation condition, suggests that the benefits of continued searching are weighed against the risks of further delay. How these risks, which may include predation and desiccation, are represented physiologically to encode the 'value' of different environments is still unclear, but the work described in this study should facilitate their investigation (Peabody, 2009).

Elucidating which neurons mediate the decision to perch will also require further investigation. Although perching can be induced by the activation of NCCAP using UAS-TRPM8, NCCAP is clearly not normally required for terminating phase I, since its suppression does not affect perching. Also, the perching that does occur when NCCAP is stimulated by UAS-TRPM8 is more tightly linked temporally with the initiation of expansion (i.e., phase III) than with the onset of NCCAP stimulation (i.e., the shift to 18°C), suggesting that neurons downstream of NCCAP mediate phase I termination, most likely at the level of the motor networks underlying perch selection and expansion. Inhibition of one motor system by another has been demonstrated to explain such behavioral hierarchies in other cases, as in the dominance of feeding over withdrawal in mollusks (Peabody, 2009).

In contrast to perch selection, the expansion program shows no obvious environmental dependence, but it is strongly dependent on levels of NCCAP activity for its initiation. This is consistent with the known effects of Bursicon, which is released by a subset of NCCAP and is required for both wing extensibility and phase III behaviors. The probability of expansion occurring at all is likely to depend on whether the Bursicon released into the CNS reaches a critical threshold, while the initiation of expansion most likely depends on the timing and/or rate of Bursicon release. The rate of release should decline with NCCAP suppression, which will reduce the excitability of the Bursicon-expressing neurons, and may account for both the prolongation of phase II under this condition and the appearance of graded wing expansion deficits. It is also possible that the timing of Bursicon release was modulated by manipulations of NCCAP if delay circuits intrinsic to NCCAP are responsible for initiating its secretion (Peabody, 2009).

Although it remains to be determined whether NCCAP participates in the decision to expand, it is clear that its activation drives all the behavioral and somatic processes necessary for wing expansion, placing NCCAP neurons high in the execution pathway of the decision and indicating that their function in wing expansion is command-like. Because this command-like function relies on Bursicon and not direct synaptic activation, it differs from familiar command systems that mediate fast behavioral switches, such as those involved in defensive escape, and more closely resembles systems in which hormones or neuromodulators elicit profound behavioral transitions such as those responsible for egg-laying in mollusks, stomatogastric ganglion modulation in crustacea, and ecdysis in insects. Because of the technical difficulty involved, the ability of such systems to drive behavioral programs upon activation has rarely been demonstrated. However, as illustrated by the work presented in this study, the introduction of genetic techniques for in vivo neuronal activation should make such demonstrations increasingly possible (Peabody, 2009).

The work presented in this study argues that the motor programs for perch selection and wing expansion have distinct regulatory mechanisms, but leaves unanswered the question of how the decisions to perch and expand are coupled: Specifically, the mechanism by which NCCAP is activated following perching remains to be clarified. NCCAP could, in principle, receive input from either the perch selection motor network or from sensory processing pathways. Such input is likely to be indirect since perching and expansion are separated by a delay (i.e., phase II), which varies considerably in duration between individual animals and different conditions. This delay period, during which Bursicon secretion is initiated, may act, at least in part, to permit Bursicon to plasticize the cuticle of the wing and body before expansion (Peabody, 2009).

Behavioral transitions during ecdysis in both Drosophila and the hawkmoth Manduca sexta, are known to be regulated by inhibitory delay circuits. In the case of eclosion hormone, the delay circuit temporally separates the somatic and behavioral aspects of the hormone's action and has been proposed to act as a control point for environmental modulation by light, which accelerates eclosion, perhaps by suppressing the inhibitory delay circuit. Interestingly, the modulation of eclosion by light is eliminated in animals lacking NCCAP, supporting a role for these neurons in the inhibitory pathway. Because ablation of the EH-expressing neurons causes wing expansion deficits, it is possible that in addition to modulating eclosion, EH may similarly modulate wing expansion by simultaneously upregulating excitability in both the Bursicon-expressing neurons and in a delay circuit that inhibits them. By analogy to the action of EH on ecdysis, the latter circuit, which could include non-Bursicon-expressing NCCAP neurons, would inhibit Bursicon release until inhibition is alleviated by an environmentally mediated signal. Such a model might explain not only the wing expansion deficits observed in animals lacking EH-expressing neurons, but also the eventual wing expansion of animals kept in the high-perturbation condition. In these animals, run down in the delay circuit may trigger Bursicon release before the decision to perch, as happens when NCCAP is activated by UAS-TRPM8, and thus also account for their abbreviated phase II (Peabody, 2009).

The merits of this model, and others that invoke delay circuits, remain to be tested. It is interesting to note, however, that the mechanism(s) coupling environmental input to NCCAP is likely to be added during metamorphosis, because the function of this network changes from an intrinsically regulated mediator of pupal ecdysis to an extrinsically modulated mediator of wing expansion. Despite its broad phylogenetic conservation, this network also appears to be differentially regulated in different species, with CCAP-expressing neurons being direct targets of EH in many insects, but not Drosophila. Such differential regulation may underlie, at least in part, species-specific differences in the coordination of wing expansion with adult emergence: In some insects, expansion is initiated at emergence without benefit of environmental input, and in others, like honeybees, it is initiated and completed before emergence. Investigation of the basis of these differences, and of the neural mechanisms that adapt NCCAP function to the individual developmental needs and life histories of different insects, should provide insight into general mechanisms of behavioral adaptation (Peabody, 2009).

In general, the work presented in this study demonstrates that the behavioral programs used by Drosophila to achieve wing expansion can serve as a simple and fruitful model for investigating networks underlying behavioral choice. Elucidation of the neuronal pathways that mediate the decisions to perch and expand the wings will shed light on the development and evolution of behavioral networks as well as their architecture and function. Indeed, the decision-making architecture outlined here for wing expansion may prove relevant to the type of instinctive behaviors in which an environmentally sensitive 'appetitive' phase is coupled to a 'consummatory' phase consisting of a stereotyped, motor pattern. Finally, tools that permit one to manipulate decision-making in living, behaving animals, such as UAS-TRPM8, which is introduced in this study, clearly will play an essential role in mapping the neural networks underlying behavior in Drosophila and other animals (Peabody, 2009).

A defensive kicking behavior in response to mechanical stimuli mediated by Drosophila wing margin bristles

Mechanosensation, one of the fastest sensory modalities, mediates diverse behaviors including those pertinent for survival. It is important to understand how mechanical stimuli trigger defensive behaviors. This study reports that Drosophila melanogaster adult flies exhibit a kicking response against invading parasitic mites over their wing margin with ultrafast speed and high spatial precision. Mechanical stimuli that mimic the mites' movement evoke a similar kicking behavior. Further, a TRPV channel, Nanchung, and a specific Nanchung-expressing neuron under each recurved bristle that forms an array along the wing margin were identified as being essential sensory components for this behavior. Electrophysiological recordings demonstrate that the mechanosensitivity of recurved bristles requires Nanchung and Nanchung-expressing neurons. Together, these results reveal a novel neural mechanism for innate defensive behavior through mechanosensation (Li, 2016).

Walking modulates speed sensitivity in Drosophila motion vision

Changes in behavioral state modify neural activity in many systems. In some vertebrates such modulation has been observed and interpreted in the context of attention and sensorimotor coordinate transformations. This study reports state-dependent activity modulations during walking in a visual-motor pathway of Drosophila. Two-photon imaging was used to monitor intracellular calcium activity in motion-sensitive lobula plate tangential cells (LPTCs) in head-fixed Drosophila walking on an air-supported ball. Cells of the horizontal system (HS)--a subgroup of LPTCs--showed stronger calcium transients in response to visual motion when flies were walking rather than resting. The amplified responses were also correlated with walking speed. Moreover, HS neurons showed a relatively higher gain in response strength at higher temporal frequencies, and their optimum temporal frequency was shifted toward higher motion speeds. Walking-dependent modulation of HS neurons in the Drosophila visual system may constitute a mechanism to facilitate processing of higher image speeds in behavioral contexts where these speeds of visual motion are relevant for course stabilization (Chiappe, 2010).

Moving animals analyze optic flow (the patterns of retinal image shift caused by relative motion between the eyes and visual contrasts in the surroundings) to stabilize their locomotor path, balance, and gaze. Horizontal system (HS) neurons are necessary elements of the optic-flow-processing and optomotor pathways in flies and have been thoroughly studied in blowflies and in Drosophila. HS neuron receptive fields consist of large overlapping areas, stacked along the dorsal-ventral axis; the three HS neurons in each optic lobe are accordingly referred to as HS-North (HSN), HS-Equatorial, and HS-South. HS-North and HS-Equatorial respond best to simultaneous horizontal front-to-back motion on the ipsilateral eye and back-to-front optic flow on the contralateral eye, a pattern of image motion that occurs during yaw rotations of the body and head. The neurons are also stimulated during translation of the fly (Chiappe, 2010).

A tethered fly-on-a-ball imaging setup was used to record Drosophila HSN neuron calcium transients in response to motion stimuli. Walking-dependent gain modulation was found in HS neurons, similar to recent results in vertical system (VS) lobula plate tangential cells (LPTCs) during tethered flight. Such state-dependent regulation of neural sensitivity has been suggested to reduce energy consumption, increasing the system's gain only when the animal requires it most. In studies in flying flies, LPTC membrane potentials were more depolarized and their synaptic responses stronger during flight. Assuming that voltage-dependent calcium currents underlie the calcium transients that were observe, the changes seen in flying flies likely apply to the walking fly as well. However, other sources of calcium transients may include release from internal stores (Chiappe, 2010).

The origin of behavior-dependent gain modulation of HS neurons remains unclear. Flies with fixed legs have HSN responses that closely resemble those from stationary flies. This does not support the idea that response amplification is initiated by a walking command signal alone and instead suggests that active proprioceptive feedback produced by leg movement is also required to increase HS neuron response. Numerous neuronal mechanisms can account for gain modulation, including changes in background synaptic activity, inhibition, and release of neuromodulators such as octopamine, and additional experiments will be required to identify the specific mechanisms involved (Chiappe, 2010).

When presented with constant spatial frequency grating patterns over a range of temporal frequencies, insects display optomotor turning reactions that give rise to a bell-shaped tuning curve, featuring a maximum response at some intermediate, temporal frequency optima (TFO) with reduced amplitude responses for lower and higher temporal frequencies. The published TFO measured for Drosophila optomotor turning behavior in response to rotatory motion by walking tethered flies is in the range of 1-4 Hz, and in tethered flight it is reported to peak within the range of 5-10 Hz. However, the electrophysiological responses of LPTCs to moving gratings show a lower temporal frequency peak at approximately 1 Hz in restrained Drosophila (for VS neurons and HS neurons). The results may partly resolve this paradox. In the calcium imaging data, the TFO measured in HS neurons for quiescent flies parallels the electrophysiological measurements, peaking at approximately 1 Hz, while the responses for the actively walking flies feature a TFO that is shifted toward higher speeds, closer to the 3 Hz peak previously reported for walking flies. It remains to be seen whether this result can be generalized to other LPTCs and to other motion-sensitive neurons that have not yet been recorded from. However, given the strong agreement of fixed-leg data with recent electrophysiological results and several recent results demonstrating gain modulation, it appears quite likely that the behavioral state of the fly will, in general, affect the speed tuning of motion-sensitive neurons. Perhaps if these experiments were repeated in a flying fly, the TFO would be shown to shift even further toward the behaviorally observed range for flight (Chiappe, 2010).

Changes in the tuning of visual neurons have been observed across species during adaptation to repeated presentations of the same stimulus. In the current experiments, flies are head-fixed and are presented with identical visual stimuli independent of behavioral state -- a very different situation. However, some of the cellular mechanisms underlying the tuning shift that were observe during walking may be shared with those involved in adaptation-related tuning changes. In fly motion vision, it has been proposed that time constants of filters that make up elementary motion detectors might be modified during adaptation, but evidence for this idea is mixed (Chiappe, 2010).

The gain modulation and TFO shift that this study reports is also reminiscent of modulations that have been observed in visual responses under increased temperatures. In the behaviorally relevant temperature range, blowfly photoreceptors have been shown to be more sensitive at elevated temperatures, with an amplitude increase of over 50% and temporal responses that are more than twice as fast. Additionally, the responses of motion-sensitive H1 neurons in the blowfly lobula plate were shown to exhibit an increase in TFO from 2 to 4 Hz. The authors of the H1 study concluded that the increased sensitivity of photoreceptors is the most likely source of the increased sensitivity in the H1 neuron. In these studies, the dramatic changes required an increase of 10°C or more. However, in the current experiments, the brain was under perfusion that was temperature controlled to 21°C, and so any potential heating of the brain was clamped by the bath.

The data demonstrate that when flies are actively walking, HS neurons show both an upward shift in response amplitude and a prominent increase in gain at higher temporal frequencies. What role might the enhanced sensitivity to higher image motion speeds serve? When flies are stationary, responses of motion-sensitive neurons to high image speeds are attenuated, as seen in temporal frequency tuning curves. This may be a strategy that stabilizes optomotor responses, whereby flies do not respond with a high behavioral gain to motion that they cannot ultimately track. However, when animals walk, the retina is exposed to a higher range of image speeds because of self motion. The increased response gain to high temporal frequencies that were observed may enable the detection of the faster retinal image shifts that the fly experiences when walking through its visual environment (Chiappe, 2010).

Visual place learning in Drosophila melanogaster

The ability of insects to learn and navigate to specific locations in the environment has fascinated naturalists for decades. The impressive navigational abilities of ants, bees, wasps and other insects demonstrate that insects are capable of visual place learning but little is known about the underlying neural circuits that mediate these behaviours. Drosophila is a powerful model organism for dissecting the neural circuitry underlying complex behaviours, from sensory perception to learning and memory. Drosophila can identify and remember visual features such as size, colour and contour orientation. However, the extent to which they use vision to recall specific locations remains unclear. This study describes a visual place learning platform and demonstrate that Drosophila are capable of forming and retaining visual place memories to guide selective navigation. By targeted genetic silencing of small subsets of cells in the Drosophila brain, it was shown that neurons in the ellipsoid body, but not in the mushroom bodies, are necessary for visual place learning. Together, these studies reveal distinct neuroanatomical substrates for spatial versus non-spatial learning, and establish Drosophila as a powerful model for the study of spatial memories (Ofstad, 2011).

Vision provides the richest source of information about the external world, and most seeing organisms devote enormous neural resources to visual processing. In addition to visual reflexes, many animals use visual features to recall specific routes and locations, such as the placement of a nest or food source. When leaving the nest, bees perform structured 'orientation flights' to learn visual landmarks. If subsequently displaced from their outbound flight, bees take direct paths back to their nests using these learned visual cues. However, it is not clear how insects, which have relatively compact nervous systems, perform these navigational feats. In mammals, the identification of place, grid and head direction cells suggests the existence of a 'cognitive map'. Unfortunately, little is known about the cellular basis of invertebrate visual place learning. To identify the neurons and dissect the circuits that underlie navigation, place learning was studied in Drosophila (Ofstad, 2011).

To test explicitly for visual place learning in Drosophila, a thermal-visual arena inspired by the Morris water maze and a heat maze, used with cockroaches and crickets, was developed. In the Drosophila place learning assay, flies must find a hidden 'safe' target (that is, a cool tile) in an otherwise unappealing warm environment. Notably, there are no local cues that identify the cool tile. Rather, the only available spatial cues are provided by the surrounding electronic panorama that displays a pattern of evenly spaced bars in three orientations. To assay spatial navigation and visual place memory, fifteen adult flies are introduced in the arena and confined to the array surface by placing a glass disk on top of a 3-mm-high aluminium ring. During the first 5-min trial, nearly all flies (94%) eventually succeed in locating the cool target. In subsequent trials, the cool tile and the corresponding visual panorama are rapidly shifted to a new location (rotated by either 90° clockwise or 90° anticlockwise, chosen at random). Importantly, the target and visual panorama are coupled so that although the absolute position of the cool tile changes, its location relative to the visual panorama remains constant. The results demonstrate that over the course of ten training trials flies improve dramatically in the time they require to locate the cool tile. This improvement is accomplished by taking a shorter, more direct route to the target, without noteworthy changes in the mean walking speed. To ensure that social interactions between flies were not influencing place learning (for example flies following each other to the safe spot), single flies were also trained, and it was found that flies tested individually show equivalent place learning. As would be predicted for bona fide visual place learning, the improvement in place memory is critically dependent on the visual panorama. Flies tested in the dark show no improvement in the time, path length or directness of their routes to the target (Ofstad, 2011).

To verify that flies are using the spatially distinct features of the visual panorama to direct navigation, flies were also tested using an uncoupled condition whereby the cool tile was still randomly relocated for each trial but the display remained stationary throughout. With this training regime, the visual panorama provides no consistent location cues, but idiothetic and weaker spatial cues such as the distance and local orientation of the arena wall are still available to the flies. The results demonstrate that flies trained with the uncoupled visual panorama show little improvement in the time taken to find the cool tile and no improvement in the directness of their approaches. Thus, spatially relevant visual cues are required for flies to learn the location of the target (Ofstad, 2011).

As a further test of visual place memory, flies were challenged immediately after training with a probe trial in which the visual landscape is relocated as usual but no cool tile is provided (to determine whether the flies will go to the non-existent safe spot). It was proposed that if the flies learned to locate the cool tile by using the peripheral visual landmarks, then they should bias their searches to the area of the arena where the visual landscape indicates the cool tile should be, even when the target is absent. Indeed, flies preferentially search in the arena quadrant where they have been trained to locate the now 'imaginary' cool tile. In contrast, if flies were trained in the dark or with an uncoupled visual landscape, conditions that contain no specific information about the location of the cool tile, the flies instead searched the arena uniformly during the probe trial. Together, these results demonstrate that fruit flies can learn spatial locations on the basis of distal visual cues and use this memory to guide navigation. By varying the time between the end of a single round of training (ten trials) and testing during a probe trial, it was also shown that flies retain these visual place memories for at least 2h (Ofstad, 2011).

Next it was considered where spatial memories are processed (or stored) in the Drosophila brain. It was reasoned that specific regions of the fly brain would function as the neuroanatomical substrate for visual place learning, and therefore animals were tested in which different brain areas were selectively inactivated using the GAL4/UAS expression system. In essence, small subsets of neurons were conditionally silenced in adult flies by targeting expression of the inward-rectifying potassium channel Kir2.1 to limit potential side-effects of Kir2.1 expression during development, a temperature-sensitive GAL80ts was used that blocks Kir2.1 expression when flies are reared at 18°C but allows expression when the temperature is raised to 30°C before testing. GAL4 driver lines were selected for expression in two areas: the mushroom bodies and the central complex. The mushroom bodies have been the subject of extensive studies of learning and memory in Drosophila , and have been shown to be essential for associative olfactory conditioning but not for some other forms of learning, such as tactile, motor and non-visually guided place learning. The central complex is thought to be a site of orientation behaviour, multisensory integration and other 'high-order' processes. In some social insects, the mushroom bodies have been implicated in visual place learning, and in the cockroach bilateral surgical lesions to these structures abolish spatial learning. However, no evidence was seen for involvement of the mushroom bodies in the assay. In fact, silencing mushroom body intrinsic neurons using the GAL4 drivers R9A11, R10B08, R67B04, had no significant effect on the performance of flies in visual place learning. The differing requirement for the mushroom bodies between Drosophila and other species may be explained by the observations that mushroom body inputs in Drosophila are predominantly olfactory. In sharp contrast, silencing subsets of neurons with projections to the central complex ellipsoid body did have a significant effect. Notably, silencing a different subset of ring neurons with line R38H02 leaves visual place learning intact. Thus, specific circuits within the ellipsoid body (but not the entire structure) are necessary for visual place learning (Ofstad, 2011).

To confirm that silencing the ellipsoid body neurons in lines R15B07 and R28D01 produces a specific impairment in visual place memory, these flies were tested in a series of behavioural paradigms and shown to display normal locomotor, optomotor, thermosensory and visual pattern discrimination behaviours. In addition, it was reasoned that if these flies have a general defect in memory (or in processing thermally driven learned behaviours), then they should show impairment in multiple types of learning (or in using thermal signals to drive learning and memory). Thus, a novel olfactory conditioning paradigm was developed using temperature (rather than electric as the unconditioned stimulus. As expected, silencing the mushroom bodies leads to a total loss of odour learning. In contrast, silencing subsets of neurons in the ellipsoid body has no effect on olfactory learning yet ablates visual place learning. Taken together, these results demonstrate that subsets of cells in the ellipsoid body are specifically required for visual place learning and substantiate the presence of distinct neuroanatomical substrates for visually guided spatial (place) versus non-spatial (olfactory) learning in Drosophila (Ofstad, 2011).

Mammals probably use place, grid and head direction cells to solve and perform navigational tasks. The tight correlation between place cell activity and an animal's position in space has established the hippocampus as the substrate for a cognitive map. This map is probably informed by head direction cells (indicating an animal's orientation) and grid cells that tile the surrounding environment and could support path integration. Although it is not known whether there are direct correlates to these cells in flies, invertebrates are capable of solving similarly challenging navigational feats and do so using significantly smaller brains. Indeed, flies are able to use idiothetic cues, and path integration, to aid navigation. The current studies demonstrate that Drosophila can learn and recall spatial locations in a complex visual arena and do so with remarkable efficacy (Ofstad, 2011).

It was also shown that subsets of neurons in the fly brain (ring neurons of the ellipsoid body) are critical for visual place learning, probably by implementing, storing, or reading spatial information. Strikingly, flies in which ellipsoid body neurons were silenced have a basic 'circling' search routine that is reminiscent of the behaviour displayed by rats with hippocampal lesions. Imaging of neuronal activity in the fly brain while the animal is executing a navigation task should help further define the role of the central complex, and ellipsoid body neurons in particular, in spatial memory (for example in a head-fixed preparation with a virtual-reality arena. Ultimately, elucidating the cellular basis for place learning in Drosophila will help uncover fundamental principles in the organization and implementation of spatial memories in general (Ofstad, 2011).

Asymmetric processing of visual motion for simultaneous object and background responses

Visual object fixation and figure-ground discrimination in Drosophila are robust behaviors requiring sophisticated computation by the visual system, yet the neural substrates remain unknown. Recent experiments in walking flies revealed object fixation behavior mediated by circuitry independent from the motion-sensitive T4-T5 cells required for wide-field motion responses. In tethered flight experiments under closed-loop conditions, this study found similar results for one feedback gain, whereas intact T4-T5 cells were necessary for robust object fixation at a higher feedback gain and in figure-ground discrimination tasks. Dynamical models were implemented based on neurons downstream of T4-T5 cells-one a simple phenomenological model and another, physiologically more realistic model-and found that both predict key features of stripe fixation and figure-ground discrimination and are consistent with a classical formulation. Fundamental to both models is motion asymmetry in the responses of model neurons, whereby front-to-back motion elicits stronger responses than back-to-front motion. When a bilateral pair of such model neurons, based on well-understood horizontal system cells, downstream of T4-T5, is coupled to turning behavior, asymmetry leads to object fixation and figure-ground discrimination in the presence of noise. Furthermore, the models also predict fixation in front of a moving background, a behavior previously suggested to require an additional pathway. Thus, the models predict several aspects of object responses on the basis of neurons that are also thought to serve a key role in background stabilization (Fenk, 2014).

The relative roles of vision and chemosensation in mate recognition of Drosophila

Animals rely on sensory cues to classify objects in their environment and respond appropriately. However, the spatial structure of those sensory cues can greatly impact when, where, and how they are perceived. This study examined the relative roles of visual and chemosensory cues in the mate recognition behavior of fruit flies (Drosophila melanogaster) by using a robotic fly dummy that was programmed to interact with individual males. By pairing male flies with dummies of various shapes, sizes, and speeds, or coated with different pheromones, it was determined that visual and chemical cues play specific roles at different points in the courtship sequence. Vision is essential for determining whether to approach a moving object and initiate courtship, and males were more likely to begin chasing objects with the same approximate dimensions as another fly. However, whereas males were less likely to begin chasing larger dummies, once started, they would continue chasing for a similar length of time regardless of the dummy's shape. The presence of female pheromones on the moving dummy did not affect the probability that males would initiate a chase, but it did influence how long they would continue chasing. Male pheromone both inhibits chase initiation and shortens chase duration. Collectively, these results suggest that male Drosophila use different sensory cues to progress through the courtship sequence: visual cues are dominant when deciding whether to approach an object whereas chemosensory cues determine how long the male pursues its target (Agrawal, 2014).

Developmental timing of a sensory-mediated larval surfacing behavior correlates with cessation of feeding and determination of final adult size

Controlled organismal growth to an appropriate adult size requires a regulated balance between nutrient resources, feeding behavior and growth rate. Defects can result in decreased survival and/or reproductive capability. Since Drosophila adults do not grow larger after eclosion, timing of feeding cessation during the third and final larval instar is critical to final size. This study demonstrates that larval food exit is preceded by a period of increased larval surfacing behavior termed the Intermediate Surfacing Transition (IST) that correlates with the end of larval feeding. This behavioral transition occurred during the larval Terminal Growth Period (TGP), a period of constant feeding and exponential growth of the animal. IST behavior is dependent upon function of a subset of peripheral sensory neurons expressing the Degenerin/Epithelial sodium channel (DEG/ENaC) subunit, Pickpocket1 (PPK1). PPK1 neuron inactivation or loss of PPK1 function caused an absence of IST behavior. Transgenic PPK1 neuron hyperactivation caused premature IST behavior with no significant change in timing of larval food exit resulting in decreased final adult size. These results suggest a peripheral sensory mechanism functioning to alter the relationship between the animal and its environment thereby contributing to the length of the larval TGP and determination of final adult size (Wegman, 2010).

The primary job description for insect larvae could be simplistically summarized as eating and growing. This sustained activity during their first week or so of life is necessary to fulfill their most critical function as a safe container nurturing the essential imaginal disks that will eventually be transformed into adult structures during morphogenesis. Since maintained association with an adequate food source is key to survival, larvae display numerous food-associated behaviors in response to a variety of environmental cues. Significant changes in larval responses to these cues during the third and final larval instar result in the major transition from foraging to wandering behavior. Results presented in this study and elsewhere (Ainsley, 2008), suggest that these two major larval stages, foraging (in the food) and wandering (out of the food) actually consist of a series of separable innate behaviors meant to alter the relationship between the animal and its environment (Wegman, 2010).

Midway through the third and final instar, larvae stop feeding and enter 'wandering stage' when they exit the food source completely in search of an appropriate pupation site. During this transition period, larvae display striking changes in body position and the spatial relationship between larvae and the food source. The constant feeding observed in foraging stage animals is associated with maintenance of a vertical anterior mouthhooks-down larval body position with only the posterior spiracles, the external openings to the larval tracheal system, protruding from the surface of the food. This body position allows constant contact with the food source for feeding and also maintains exposure of the posterior spiracle openings to the atmosphere to allow respiratory exchange. Full body immersion in the moist food source also prevents potential larval dessication resulting from excessive exposure to a drying external atmosphere. However, pupariation within the moist food source often results in lethality caused by suffocation due to occlusion of spiracular air exchange or by failure of adult eclosion due to inability to exit the pupal case. As a consequence, although this head-down full body immersion position is beneficial during larval foraging stages, it is essential for the animal to move to the surface of the food prior to pupariation. Results presented in this study show that changes in the spatial relationship between larvae and the food source during the final instar is modulated by activity of a subset of peripheral sensory neurons expressing the DEG/ENaC subunit PPK1 (Wegman, 2010).

Evolved differences between insect species also suggest that food surfacing behavior and complete food exit are genetically separable. Of the many Drosophila species that have been characterized, D. melanogaster is one of the few that actually fully exits the food source for pupariation. Entry into 'wandering' behavior in D. melanogaster is normally associated with complete larval exit from the food and movement up the sides of culture vials in search of a moderately dry pupation site. Many closely related Drosophila species choose to simply move to the food surface where they pupate. In these species, the change in body position from the vertical head-down foraging position to the food surface appears to be the key behavioral transition necessary for survival through metamorphosis. Although it is likely that this striking difference in larval behavior represents an adaptive response to environmental or food conditions, genetic and/or physiological explanations for this difference are not understood (Wegman, 2010).

The developmental transition in larval thermotactic preference reflected as dispersal vs. ARS behavior was initiated early in the third instar correlating with the previously characterized critical period for PPK1 neuron function from 80 to 90 h AEL (Ainsley, 2008). This timing coincides with the appearance of IST behavior suggesting that these two behaviors reflect the same developmental and behavioral transition preceding larval food exit. As further evidence of the functional correlation of these two intermediate behaviors, IST behavior was also dependent upon PPK1 function and activity of the PPK1 sensory neurons. IST behavior was absent in ppk1 null mutant larvae and sharply suppressed in transgenic animals with electrically silenced PPK1 neurons. In addition, hyperactivation of PPK1 neurons caused a premature and enhanced appearance of IST behavior paralleling the previously observed alterations in larval thermotactic behavior (Wegman, 2010).

The key role of PPK1 protein for normal IST behavior suggests that the temporal timing of PPK1 expression may function as a central regulatory switch for the developmental timing of this behavioral transition. However, previous studies have shown that PPK1 expression first appears in mdIV neurons in late embryos and is sustained into late third instar stages (Ainsley, 2008). These results indicate that developmentally regulated control of PPK1 expression is likely not a potential mechanism for control of IST behavior (Wegman, 2010).

Induction of premature and enhanced IST behavior did not lead to premature final food exit but caused a significant decrease in final adult size. This result is consistent with premature feeding termination caused by IST behavior. Therefore, these innate behaviors are genetically and functionally separable and the observed IST behavior is not simply the beginning step of final food exit and wandering behavior (Wegman, 2010).

Both the IST and ARS behavioral transitions correlate roughly with the proposed timing of larval critical size assessment early in the third instar. As discussed earlier, the developmental period between critical size assessment and larval food exit known as the TGP is essential for growth to final maximum size. If disrupted either behaviorally or metabolically, a decrease in TGP duration should result in a decrease in final adult size. This is consistent with the observed effect of PPK1 neuron hyperactivation on final adult size (Wegman, 2010).

Expression of the PPK1 DEG/ENaC subunit is tightly restricted to the mdIV neurons in the larval body wall and two bipolar neurons innervating the posterior spiracles (ps) (Ainsley, 2008). Although possible functional relationships between mdIV neurons and ps neurons are not clear, both morphological and anatomical differences suggest distinct physiological functions. The md/da sensory neurons within the larval body wall have been spatially and morphologically divided into four subclasses based upon complexity of their dendritic arbors and their relative location within the larval PNS. The mdIV neurons are just one subclass of the larger collection of md/da neurons present within the larval body wall. Although experimental characterization of the md/da neurons has been extremely useful in studies aimed at understanding the development of dendritic fields, the physiological functions and/or any interactions between the md/da subgroups remain poorly understood (Wegman, 2010).

The mdIV neurons have been implicated as nociceptive neurons involved in activating a writhing escape response when exposed to noxious heat. This escape response has been functionally attributed to the necessity for Drosophila larvae to protect themselves from parasitoid wasps.The mdIV neurons and PPK1 have been shown to be required for a larval nocifensive response to noxious mechanical stimuli (Zhong, 2010). The relevance of these responses to harsh thermal or mechanical stimulation within the normal endogenous larval environment remains uncertain. In experimental conditions reported in this study, hyperactivation of the mdIV neurons using either the constitutively-active PPK1[S511V] isoform or the low-threshold voltage-gated sodium channel, NaChBaceGFP, did not result in induction of the previously reported 'nocifensive' response (Wegman, 2010).

PPK1 neurons appear to play a much more prominent role in the normal larval response to its environment than simply a response to harsh stimuli. This supports the possibility of a polymodal role for this class of sensory neurons that may depend upon more than one source of activation signal. It must also be noted that the ppk1GAL4 driver transposon used in these studies and in previous studies demonstrating a nociceptive mdIV function does not distinguish between the mdIV neurons and the single PPK1-expressing bipolar neuron innervating each posterior spiracle (ps) (Ainsley, 2008). In addition, there are distinct morphological differences between the three mdIV neurons (ddaC, v'ada and vdaB) within each larval hemisegment. The mdIV ddaC neuron displays a more extensive and symmetrical dendritic arbor than the v'ada and vdaB neurons. Their relative dorsal/ventral locations within the body wall may also expose each mdIV subtype to a different range of stimuli whether mechanical or chemical. Therefore, results produced using the ppk1GAL4 transposon would not detect any differences in the relative contributions of mdIV subtypes or the ps neurons to any of the proposed functional roles (Wegman, 2010).

Feeding larvae detect and respond to multiple sensory inputs providing information about food resources and their immediate environment. Responses can be separated into distinct innate behaviors meant to coordinate internal growth signals with changing environmental conditions. The current findings demonstrate that the PPK1 neurons contribute to the regulation of larval developmental timing and feeding behavior transitions within normal environmental parameters. Results presented in this study have focused upon the larval PPK1-expressing neurons and their role in larval feeding behavior, however, other recent work has also described a role for PPK1-expressing neurons in regulation of adult feeding behavior (Ribeiro, 2010). Although a clear functional connection between the physiological roles of larval and adult PPK1 neurons is not yet apparent, both appear to contribute in some way to monitoring of external sensory information from food sources as input to help modulate internal nutrient homeostasis (Wegman, 2010).

Inhibition of fatty acid desaturases in Drosophila melanogaster larvae blocks feeding and developmental progression

Fatty acid desaturases are metabolic setscrews. To study their systemic impact on growth in Drosophila melanogaster, fatty acid desaturases were inhibited using the inhibitor CAY10566. As expected, the amount of desaturated lipids is reduced in larvae fed with CAY10566. These animals cease feeding soon after hatching, and their growth is strongly attenuated. A starvation program is not launched, but the expression of distinct metabolic genes is activated, possibly to mobilize storage material. Without attaining the normal size, inhibitor-fed larvae molt to the next stage indicating that the steroid hormone ecdysone triggers molting correctly. Nevertheless, after molting, expression of ecdysone-dependent regulators is not induced. While control larvae molt a second time, these larvae fail to do so and die after few days of straying. These effects are similar to those observed in experiments using larvae deficient for the fatty acid Desaturase1 gene. Based on these data, it is proposed that the ratio of saturated to unsaturated fatty acids adjusts a sensor system that directs feeding behavior. It is also hypothesized that loss of fatty acid desaturase activity leads to a block of the genetic program of development progression indirectly by switching on a metabolic compensation program (Wang, 2016).

Mechanosensory interactions drive collective behaviour in Drosophila

Collective behaviour enhances environmental sensing and decision-making in groups of animals. Experimental and theoretical investigations of schooling fish, flocking birds and human crowds have demonstrated that simple interactions between individuals can explain emergent group dynamics. These findings indicate the existence of neural circuits that support distributed behaviours, but the molecular and cellular identities of relevant sensory pathways are unknown. This study shows that Drosophila melanogaster exhibits collective responses to an aversive odour: individual flies weakly avoid the stimulus, but groups show enhanced escape reactions. Using high-resolution behavioural tracking, computational simulations, genetic perturbations, neural silencing and optogenetic activation it was demonstrated that this collective odour avoidance arises from cascades of appendage touch interactions between pairs of flies. Inter-fly touch sensing and collective behaviour require the activity of distal leg mechanosensory sensilla neurons and the mechanosensory channel NOMPC. Remarkably, through these inter-fly encounters, wild-type flies can elicit avoidance behaviour in mutant animals that cannot sense the odour—a basic form of communication. These data highlight the unexpected importance of social context in the sensory responses of a solitary species and open the door to a neural-circuit-level understanding of collective behaviour in animal groups (Ramdya, 2014).

Drosophila melanogaster is classified as a solitary species but flies aggregate at high densities (~1 fly per cm2) to feed, providing opportunities for collective interactions. Although groups affect circadian rhythms and dispersal in Drosophila, how social context influences individual sensory behaviours is unknown. To study this question, an automated behavioural assay was developed to track responses of freely-walking flies to laminar flow of air or an aversive odorant, 5% carbon dioxide (CO2). Odour was presented to one half of a planar arena for 2 min. Avoidance behaviour was quantified as the percentage of time a fly spent in the air zone during the second minute of a trial. Unexpectedly, isolated flies spent very little time avoiding this odour, despite the aversion to CO2 observed in other assays. However, increasing the number of flies was associated with substantial increases in odour avoidance. This effect peaked at 1.13 flies per cm2, a density typical for fly aggregates and was only apparent for flies in the odour zone. Time-course analysis revealed that, within only a few seconds after odour onset, a larger proportion of flies in high-density groups had left the odour zone compared to isolated individuals. Additionally, the motion of flies after odour onset was coherent at higher densities, with flies moving in the same direction, out of the odour zone; this effect was not observed for flies in the air zone (Ramdya, 2014).

To determine the basis of these global behavioural differences, the locomotion of individual flies was examined. Single animals are typically sedentary but walk more when exposed to CO2. In groups, however, it was discovered that 63% of the time, the first walking response of a fly after odour onset coincided with proximity to a neighbouring fly. These Encounters were more frequent with increasing group density. Moreover, walking bouts initiated during an Encounter ('Encounter Responses') were significantly longer than those spontaneously initiated in isolation. These observations indicated that inter-fly interactions might contribute to the enhanced odour avoidance of groups of flies (Ramdya, 2014).

This possibility was examined initially by computational simulation of the olfactory assay. The dynamics of the simulation were driven by three phenomena observed in behavioural assays. First, flies initiate more spontaneous bouts of walking in odour than in air. Second, flies are more likely to turn and retreat after entering the odour zone from the air zone. Third, close proximity to another fly elicits Encounter Responses in stationary flies. Importantly, these elements could reproduce collective behaviour: higher numbers of simulated flies exhibited greater avoidance. While changing the olfactory parameters preserved stronger responses in groups than isolated individuals, diminishing the Encounter Response probability could abolish and even reverse collective behaviour. These results suggested that Encounter Responses are a crucial component of Drosophila group dynamics. To experimentally test the role of inter-fly interactions in collective behaviour, attempts were made to explain the mechanistic basis of Encounter Responses. Although the olfactory experiments were performed in the dark, the presence of light did not diminish Encounter Response frequency. Volatile chemicals are known modulators of many social behaviours, but putative anosmic flies (lacking known olfactory co-receptors) did not reduce Encounter Responses. By contrast, disruption of the mechanosensory channel NOMPC significantly diminished Encounter Response frequency. These data suggested that mechanosensing is required for Encounter Responses (Ramdya, 2014).

By observing groups of flies at high spatiotemporal resolution, it was found that active flies elicited motion in stationary animals through gentle touch of peripheral appendages (legs and wings). Leg touches took place exclusively on distal segments and resulted in spatially stereotyped walking reactions. These reactions were kinematically indistinguishable from Encounter Responses. This analysis indicates that appendage touch is the stimulus that elicits Encounter Responses. The precise stereotypy of these locomotor responses, similar to cockroach escape reactions, implies their dependence upon somatotopic neural circuits linking touch with movement (Ramdya, 2014).

As fly appendages also house taste receptors, whether mechanical stimulation was sufficient to elicit Encounter Responses was tested by tracking stationary flies following touch of appendages with a metallic disc. A stereotyped relationship was observed between the location of mechanical touch and subsequent walking trajectories, whose associated kinematics were indistinguishable from those of Encounter Responses. Thus, mechanical touch alone can elicit Encounter Responses. Consistently, genetic ablation of flies' oenocytes, to remove cuticular hydrocarbon contact chemosensory signals, had no effect on the ability of these animals to elicit Encounter Responses in wild-type flies. These data imply that Encounter Responses are mediated solely by mechanosensory stimulation (Ramdya, 2014).

Next, mechanosensory neurons required for touch- evoked Encounter Responses were identified by driving tetanus toxin (Tnt) expression with a panel of candidate mechanosensory Gal4 lines. R55B01-Gal4/UAS-Tnt flies exhibited significantly diminished Encounter Responses compared to a gustatory neuron driver line, without reduced ability to produce sustained high-velocity walking bouts. R55B01-Gal4- driven expression of a UAS-CD4:tdGFP reporter was detected in neurons innervating leg and wing neuropils of the thoracic ganglia. Consistently, green fluorescent protein (GFP) labelled neurons in several leg mechanosensory structures: the femoral and tibial chordotonal organs, and distal leg mechanosensory sensilla neurons. Notably, among the screened lines only R55B01- Gal4 drove expression in leg mechanosensory sensilla (Ramdya, 2014).

To ascertain the contribution to Encounter Responses of leg mecha- nosensory sensilla and/or chordotonal structures (which can also sense touch), additional Gal4 driver lines were identified that drove expression in subsets of these neuron classes. By intersecting piezo-Gal4 with cha3-Gal80, a Gal4 suppression line, it was possible to limit leg expression to mechanosensory sensilla neurons (termed 'Mechanosensory Sensilla driver' line). Importantly, silencing neurons with this driver significantly diminished Encounter Response frequency. By contrast, silencing leg chordotonal organs alone had no effect on Encounter Response frequency (Ramdya, 2014).

The sufficiency of leg mechanosensory sensilla neuron activity to elicit Encounter Response-like walking was tested by expressing channelrhodopsin-2 (ChR2) in each class of leg mechanosensory neurons and recording behavioural responses to blue light pulses. Optogenetic stimulation of flies expressing ChR2 in leg mechanosensory sensilla neurons, but not chordotonal organs, resulted in Encounter Response- like walking, consistent with natural elicitation of Encounter Responses by inter-fly touch of distal leg segments (Ramdya, 2014).

Identification of a neuronal basis for Encounter Responses allowed testing of the model's prediction that inter-fly interactions are required for collective odour avoidance. First, leg mechanosensory sensilla neurons were silenced by expressing Tnt with R55B01-Gal4 or the Mechanosensory Sensilla driver. Second, nompC mutants were tested. Each of these perturbations abolished collective odour avoidance, supporting the link between mechanosensation and group behaviour (Ramdya, 2014).

Touch may enhance odour avoidance by increasing awareness of the stimulus. Alternatively, touch may produce an odour-independent Encounter Response reaction that initiates departure from the odour zone. To distinguish between these possibilities, it was asked if odour-insensitive flies displayed increased avoidance in the presence of odour-sensitive animals. Indeed, both in simulations and in real flies, increasing the number of odour-sensitive individuals led to greater avoidance behaviour of odour-insensitive individuals. Thus, in this context, touch-mediated modulation of odour awareness plays little, if any, role in collective avoidance (Ramdya, 2014).

Combining systems-level and neurogenetic approaches, this study has uncovered a hierarchy of mechanisms that drive collective motion in Drosophila. Active flies elicit spatially stereotyped walking responses in stationary flies through appendage touch interactions, requiring the NOMPC mechanosensory channel and distal leg mechanosensory sensilla neurons. Through Encounter Responses, odour reactions of sensitive flies spark cascades of directed locomotion of less sensitive (or even insensitive) individuals, causing a coherent departure from the odour zone. This behavioural positive feedback and group motion are absent among flies in the non-odour zone since they are less likely to initiate walking and, consequently, have a reduced frequency of Encounters. Additionally, flies retreat when encountering the odour while transiting from the air zone. Together these behavioural phenomena cause flies to escape the odour zone and then remain in the air zone, resulting in higher odour avoidance for groups compared to isolated animals. When distal appendage mechanosensory touch detection is impaired, groups of flies cannot produce Encounter Responses, are less likely leave the odour zone, and instead behave like isolated flies. Encounters are likely to have widespread influence on sensory-evoked actions of individuals in groups. For example, movement of flies towards areas of high elevation is also increased in higher density groups (Ramdya, 2014).

Behaviour in animal groups arises from the detection and response to intentional and unintentional signals of conspecifics. While neural circuits controlling pairwise interactions, such as courtship, are increasingly well-understood, little is known about those orchestrating group- level behaviours. The identification of sensory pathways that mediate collective behaviour in Drosophila opens the possibility to understand the neural basis by which an individual's actions may influence—and be influenced by group dynamics (Ramdya, 2014).

A multilevel multimodal circuit enhances action selection in Drosophila

Natural events present multiple types of sensory cues, each detected by a specialized sensory modality. Combining information from several modalities is essential for the selection of appropriate actions. Key to understanding multimodal computations is determining the structural patterns of multimodal convergence and how these patterns contribute to behaviour. Modalities could converge early, late or at multiple levels in the sensory processing hierarchy. This study shows that combining mechanosensory and nociceptive cues synergistically enhances the selection of the fastest mode of escape locomotion in Drosophila larvae. In an electron microscopy volume that spans the entire insect nervous system, the multisensory circuit was reconstructed supporting the synergy and spanning multiple levels of the sensory processing hierarchy. The wiring diagram revealed a complex multilevel multimodal convergence architecture. Using behavioural and physiological studies, functionally connected circuit nodes were identified that trigger the fastest locomotor mode, and others were identified that facilitate it. Evidence is provided evidence that multiple levels of multimodal integration contribute to escape mode selection. It is proposed that the multilevel multimodal convergence architecture may be a general feature of multisensory circuits enabling complex input-output functions and selective tuning to ecologically relevant combinations of cues (Ohyama, 2015).

Different combinations of nociceptive and mechanosensory stimulation induced different likelihoods of the key escape sequences: rolling followed by fast crawling versus fast crawling alone. Nociceptor activation alone evoked a relatively low likelihood of rolling and a high likelihood of fast crawling. Vibration alone evoked only fast crawling and essentially no rolling. Combined with nociceptor activation, vibration increased the likelihood of rolling; the effect is dose-dependent and super-additive (synergistic). This vibration-induced facilitation of rolling is mediated through the mechanosensory chordotonal neurons (Ohyama, 2015).

It is suspected that the information from the two modalities converges onto central neurons involved in the selection of rolling. To identify such neurons and thus determine where in the sensory processing hierarchy multisensory convergence occurs, a was performed behavioural screen for neurons whose thermogenetic activation triggers rolling. A 'hit' was identified in the R72F11 Drosophila line, that drove GAL4 expression in neurons potentially early in the sensory processing hierarchy. Activating the neurons in R72F11 triggered rolling in a significant fraction of animals, and inhibiting them significantly decreased rolling in response to bimodal stimulation (Ohyama, 2015).

R72F11 drives expression selectively in four lineage-related, segmentally repeated projection neurons with basin-shaped arbors in the ventral, sensory domain of the nerve cord; therefore they were named Basins-1-4. The dendrites of Basin-1 and Basin-3 span a ventrolateral domain of the nerve cord, where the mechanosensory chordotonal terminals are located. The dendrites of Basin-2 and Basin-4 span both the ventrolateral chordotonal domain and a ventromedial domain where the nociceptive MD IV terminals are located. It was therefore asked whether the mechanosensory chordotonal and the nociceptive MD IV neurons directly converge on Basin-2 and Basin-4 (Ohyama, 2015).

In an electron microscopy volume that spans 1.5 nerve cord segments, the chordotonal and MD IV arbors were scanned. The left and right Basin-1, -2, -3 and -4 among the reconstructed neurons (Ohyama, 2015).

Basin-1 and Basin-3 received many inputs (each >25 synapses and >15% of total input, on average) from chordotonal neurons, but very few (no more than 1% of total input synapses) from MD IV neurons. Basin-2 and Basin-4 received many inputs from both chordotonal neurons (on average >20 synapses and >10% total input) and MD IV neurons (on average >20 synapses and >10% total input), each on distinct dendritic branchesl Of all the 301 partners downstream of MD IV and chordotonal neurons, only Basin-2 and Basin-4 reproducibly received >5 synapses from both chordotonal and MD IV neurons, suggesting that they are probably key integrators of chordotonal and MD IV inputs (Ohyama, 2015).

To investigate whether the observed anatomical inputs from the sensory neurons onto Basins were functional and excitatory, calcium transients were imaged in response to MD IV or chordotonal activation collectively in all Basins or in individual Basin types, using lines that drive expression selectively in Basin-1 or Basin-4 (Ohyama, 2015).

In Basin-1, calcium transients were observed in response to vibration, but not in response to MD IV activation, consistent with the large number of synapses it receives from chordotonal neurons and the relatively few from MD IV neurons. In Basin-4, calcium transients were observed in response to both vibration and MD IV activation, consistent with the large number of synapses it receives from both sensory types. Basin-4 integrated the inputs from the two modalities, responding significantly more to bimodal than to unimodal (Ohyama, 2015).

Next, it was asked whether the multisensory Basin-4 interneurons contribute to rolling selection. Silencing Basin-4 neurons significantly decreased rolling in response to bimodal stimulation, indicating these neurons are involved in triggering rolling. Selective activation of the multisensory Basin-4 interneurons triggered rolling in a dose-dependent way, with strongest activation triggering rolling in 45% of animals (Ohyama, 2015).

It was also asked whether a second level of multimodal integration (that is, integration of information from distinct Basin types, that receive distinct combinations of chordotonal and MD IV inputs), enhances the selection of rolling. Indeed, co-activation of Basin-1 with the bimodal Basin-4 facilitated rolling, resulting in a significantly higher likelihood of rolling compared to activation of Basin-4 alone (70% versus 45%). Thus, information from distinct Basin types may converge again onto downstream neurons involved in triggering rolling (Ohyama, 2015).

To identify potential sites of convergence of information from the different types of first-order Basin interneurons a 'hit' was examined from the thermogenetic activation screen, R69F06, a Drosophila line that drove GAL4 expression in neurons that project far from the early sensory processing centres. Thermogenetic activation of neurons in R69F06 triggered rolling in a high fraction of larvae, and inhibiting them significantly decreased rolling in response to bimodal stimulation (Ohyama, 2015).

R69F06 drives expression in a few neurons in the brain, in the sub-oesophageal zone (SEZ) and in a pair of thoracic neurons whose axons descend through the dorsal, motor domain of the nerve cord. Selectively activating the single pair of thoracic neurons triggered rolling in 76% of larvae. These command-like neurons were named Goro (a romanization of the Japanese for rolling) (Ohyama, 2015).

Activation of Basins evoked strong calcium transients in the Goro neurons, indicating that these cell types involved in the same behaviour are functionally connected. To identify the shortest anatomical pathways from Basins to Goro that might support the observed functional connectivity and to determine whether the information from distinct Basin types converges onto Goro, electron microscopy reconstruction was again used (Ohyama, 2015).

A second electron microscopy volume (from a second larva) was used that spans the entire larval nervous system and therefore also includes Goro neurons. In the new volume, chordotonal, MD IV and Basin neurons were reconstructed from segment A1, as well as the Goro neurons (Ohyama, 2015).

To find putative pathways from distinct Basin types to Goro neurons, all neurons downstream of all axonal outputs were reconstructed from the four left and right Basin homologues from segment A1. Thirty-one pairs of reproducible downstream partners were identified. Among these second-order nerve cord interneurons were identified that constitute the shortest pathways from Basins to Goro neurons (called A05q and A23g where 'A' stands for abdominal neuron). They receive inputs from distinct Basin types and synapse onto Goro neurons. Thus, information from distinct Basin types, that receive distinct combinations of MD IV and chordotonal inputs, converges onto Goro neurons -providing a second level of multimodal convergence (Ohyama, 2015).

Ten distinct second-order projection neuron types downstream of Basins ascend to the brain. Some of these integrate Basin information across multiple distal segments of the body, either exclusively from a single Basin type, or from distinct Basin types (that receive distinct combinations of sensory inputs; for example, A00c-a4 and A00c-a5). Then, distinct second-order PNs, that receive distinct combinations of Basin inputs (and therefore distinct combinations of mechanosensory and nociceptive inputs), re-converge again on third-order interneurons in the brain. Thus, following convergence of local mechanosensory and nociceptive information from a single segment onto multisensory Basins, global mechanosensory and multisensory information from multiple segments is integrated within the brain pathway (Ohyama, 2015).

By tracing upstream of Goro dendritic inputs, brain neurons were identified that send descending axons that synapse onto Goro neurons. Tracing downstream of a multisensory second-order ascending projection neuron (A00c-a4), third-order projection neurons were identified connecting the ascending pathways from Basins to a descending path onto Goro neurons. Thus, the activity of the command-like Goro neurons may be modulated by the more local multisensory and unisensory information via the nerve cord Basin-Goro pathway and by the global body-wide nociceptive and mechanosensory multisensory information via the brain Basin-Goro (Ohyama, 2015).

A third-order SEZ feedback neuron was identified that receives convergent body-wide mechanosensory and multisensory information and descends through the nerve cord sensory domain. The SEZ feedback neuron synapses onto the first-order (Basins) and second-order neurons from both the nerve cord (A05q) and brain (A00c) Basin-Goro pathways. Both the nerve cord and brain Basin-Goro pathways may therefore be jointly regulated based on integrated global multisensory information (Ohyama, 2015).

Next, the functional role of the nerve cord and brain Basin-Goro pathways was explored. Basin activation could activate Goro neurons in the absence of the brain, suggesting the nerve cord Basin-Goro pathway is excitatory and sufficient for activating Goro neurons and triggering rolling. Consistent with this idea, Basin activation evoked calcium transients in their nerve-cord targets, the A05q neurons, and A05q activation evoked calcium transients in Goro neurons. Furthermore, thermogenetic activation of the neurons in a line that drives expression, among others, in the A05q neurons triggered (Ohyama, 2015).

Calcium imaging in the terminals of three Basin-target neurons that ascend to the brain (A00c-a6, A00c-a5 and A00c-a4)) revealed that, collectively, they respond to vibration, to MD IV activation, and to Basin activation, suggesting that this connection is also excitatory (Ohyama, 2015).

Silencing the A00c neurons decreased rolling in response to bimodal stimulation, and their co-activation with Basin-4 facilitated rolling. Therefore, downstream from early local multisensory integration by Basin-4, additional levels of integration of global mechanosensory and multisensory information appear to further facilitate the transition to rolling behaviour (Ohyama, 2015).

The rolling response triggered by multisensory cues (or by strong nociceptive cues alone) is followed by fast crawling. Similarly, optogenetic activation of the first-order multisensory Basin-4 neurons triggered both locomotor modes; rolling followed by fast crawling. However, optogenetic activation of the Goro neurons triggered only rolling, but not fast crawling, suggesting that they act as dedicated command-like neurons for rolling. This also suggests that the act of rolling itself is insufficient to trigger fast crawling. In the future it will be interesting to determine how all of the 31 novel neuron types directly downstream of the Basins identified in the electron microscopy reconstruction contribute to the selection of the two locomotor modes, rolling and crawling, in a defined sequence (Ohyama, 2015).

By combining behavioural and physiological studies with large-scale electron microscopy reconstruction this study has mapped a multisensory circuit that mediates the selection of the fastest mode of escape locomotion (rolling) in Drosophila larva. Mechanosensory and nociceptive sensory neurons were found to converge on specific types of first-order multisensory interneurons that integrate their inputs. Then, interneurons that receive distinct combinations of mechanosensory and nociceptive inputs converge again at multiple levels downstream, all the way to command-like neurons in the nerve cord. Activating just a single type of first-order multisensory interneuron triggers rolling probabilistically. Co-activation of first-order interneurons that receive distinct combinations of mechanosensory and nociceptive inputs increases rolling probability. Thus, action selection starts at the first-order multisensory interneurons and multiple stages of multimodal integration in the distributed network enhance this selection (Ohyama, 2015).

Given that spurious firing from distinct sensors is uncorrelated, whereas event-derived signals will be temporally correlated across the sensory channels, multimodal integration even at a single level improves the signal-to-noise ratio. Multilevel multimodal integration can offer additional advantages. Theoretical studies show that a multilevel convergence architecture enables more complex input-output relationships. Similarly, the multilevel multimodal convergence architecture described in this study could offer better discrimination between different kinds of multisensory events. The weights in such networks could be tuned either through experience or through evolution to respond selectively to highly specific combinations of two cues. Using a simple model, it can be demonstrated that compared to early-convergence a multilevel architecture could specifically enhance the selection of the fastest escape mode in the most threatening situations, either in response to weak multimodal or strong unimodal nociceptive cues (Ohyama, 2015).

The multilevel multimodal convergence architecture may be a general feature in multisensory integration circuits, enabling complex response profiles tunable to specific ecological needs. For example, physiological studies in mammals have identified multisensory neurons that integrate the same cues at several stages in the sensory processing hierarchy, although it is unclear whether the multisensory neurons at distinct levels are causally related to the same behaviour. Due to the size of networks involved, synaptic-level resolution studies of the underlying convergence architecture across multiple levels were unattainable (Ohyama, 2015).

In addition to the multilevel multimodal feed-forward convergence motif, electron microscopy reconstruction revealed higher-order and local feedback neurons. Recent theoretical models of multisensory integration suggest that the output of individual multisensory neurons is normalized by the activity of other multisensory neurons in that population, but the anatomical implementation of such feedback has not been identified. Some of the feedback neurons in the multisensory circuit described in this study may have roles in such normalization computations (Ohyama, 2015).

Another circuit motif revealed by the study is the divergence of sensory information into nerve cord and ascending brain pathways and subsequent re-convergence of the shorter and the longer pathway onto the same command-like neurons in motor nerve cord (Goro). The nerve cord pathway integrates nociceptive and mechanosensory information from a local region of the body (few segments), whereas the ascending brain pathway integrates the information across all body segments and provides a means of modulating command-like neuron activity based on global body-wide nociceptive and mechanosensory information. The multisensory circuit described in this study in a genetically tractable model system provides a resource for investigating in detail how multiple brain and nerve cord pathways interact with each other and contribute to the selection of different modes of locomotion (rolling and crawling) in a defined sequence (Ohyama, 2015).

The electron microscopy volume spanning the entire insect nervous system acquired for this study can be used to map circuits that mediate many different behaviours. Combining information from a complete wiring diagram with functional studies has been very fruitful in the 302-neuron nervous system of C. elegans. Recently, similar approaches have been applied to microcircuits in smaller regions of larger nervous systems. This study has demonstrated that relating local and global structure to function in a complete nervous system is now possible for the larger and more elaborate nervous system of an insect (Ohyama, 2015).

Identification of inhibitory premotor interneurons activated at a late phase in a motor cycle during Drosophila larval locomotion
Rhythmic motor patterns underlying many types of locomotion are thought to be produced by central pattern generators (CPGs). This study used the motor circuitry underlying crawling in larval Drosophila as a model to try to understand how segmentally coordinated rhythmic motor patterns are generated. Whereas muscles, motoneurons and sensory neurons have been well investigated in this system, far less is known about the identities and function of interneurons. A recent study identified a class of glutamatergic premotor interneurons, PMSIs (period-positive median segmental interneurons), that regulate the speed of locomotion. This study reports on the identification of a distinct class of glutamatergic premotor interneurons called Glutamatergic Ventro-Lateral Interneurons (GVLIs). Calcium imaging was used to search for interneurons that show rhythmic activity, and GVLIs were identified as interneurons showing wave-like activity during peristalsis. Paired GVLIs were present in each abdominal segment A1-A7 and locally extended an axon towards a dorsal neuropile region, where they formed GRASP-positive putative synaptic contacts with motoneurons. The interneurons expressed vesicular glutamate transporter (vGluT) and thus likely secrete glutamate, a neurotransmitter known to inhibit motoneurons. These anatomical results suggest that GVLIs are premotor interneurons that locally inhibit motoneurons in the same segment. Consistent with this, optogenetic activation of GVLIs with the red-shifted channelrhodopsin, CsChrimson ceased ongoing peristalsis in crawling larvae. Simultaneous calcium imaging of the activity of GVLIs and motoneurons showed that GVLIs' wave-like activity lagged behind that of motoneurons by several segments. Thus, GVLIs are activated when the front of a forward motor wave reaches the second or third anterior segment. It is proposed that GVLIs are part of the feedback inhibition system that terminates motor activity once the front of the motor wave proceeds to anterior segments (Itakura, 2015).

Imaging fictive locomotor patterns in larval Drosophila
A preparation has been established in larval Drosophila to monitor fictive locomotion simultaneously across abdominal and thoracic segments of the isolated CNS using genetically encoded Ca2+ indicators. The Ca2+ signals closely followed spiking activity measured electrophysiologically in nerve roots. Three motor patterns are analyzed. Two comprise waves of Ca2+ signals which progress along the longitudinal body axis in a posterior-to-anterior or anterior-to-posterior direction. These waves had statistically indistinguishable inter-segmental phase delays compared to segmental contractions during forward and backward crawling behavior, despite being around 10 times slower. During these waves, motor neurons of the dorsal longitudinal and transverse muscles were active in the same order as the muscle groups are recruited during crawling behavior. A third fictive motor pattern exhibits a left-right asymmetry across segments and bears similarities with turning behavior in intact larvae, occurring equally frequently and involving asymmetry in the same segments. Ablation of the segments in which forward and backward waves of Ca2+ signals were normally initiated did not eliminate production of Ca2+ waves. When the brain and SOG were removed, the remaining ganglia retained the ability to produce both forward and backward waves of motor activity, although the speed and frequency of waves changed. Bilateral asymmetry of activity was reduced when the brain was removed, and abolished when the SOG was removed. This work paves the way to study the neural and genetic underpinnings of segmentally coordinated motor pattern generation in Drosophila using imaging techniques (Pulver, 2015).

Fluctuation-driven neural dynamics reproduce Drosophila locomotor patterns

The neural mechanisms determining the timing of even simple actions, such as when to walk or rest, are largely mysterious. One intriguing, but untested, hypothesis posits a role for ongoing activity fluctuations in neurons of central action selection circuits that drive animal behavior from moment to moment. To examine how fluctuating activity can contribute to action timing, high-resolution measurements of freely walking Drosophila melanogaster were paired with data-driven neural network modeling and dynamical systems analysis. Dynamical models that best reproduced both Drosophila basal and odor-evoked locomotor patterns exhibited specific characteristics. First, ongoing fluctuations were required. In a stochastic resonance-like manner, these fluctuations allowed neural activity to escape stable equilibria and to exceed a threshold for locomotion. Second, odor-induced shifts of equilibria in these models caused a depression in locomotor frequency following olfactory stimulation. These models predict that activity fluctuations in action selection circuits cause behavioral output to more closely match sensory drive and may therefore enhance navigation in complex sensory environments. Together these data reveal how simple neural dynamics, when coupled with activity fluctuations, can give rise to complex patterns of animal behavior.

The nutritional and hedonic value of food modulate sexual receptivity in Drosophila melanogaster females

Food and sex often go hand in hand because of the nutritional cost of reproduction. For Drosophila melanogaster females, this relationship is especially intimate because their offspring develop on food. Since yeast and sugars are important nutritional pillars for Drosophila, availability of these foods should inform female reproductive behaviours. Yet mechanisms coupling food and sex are poorly understood. This study shows that yeast increases female sexual receptivity through interaction between its protein content and its odorous fermentation product acetic acid, sensed by the Ionotropic odorant receptor neuron Ir75a. A similar interaction between nutritional and hedonic value applies to sugars where taste and caloric value only increase sexual receptivity when combined. Integration of nutritional and sensory values would ensure that there are sufficient internal nutrients for egg production as well as sufficient environmental nutrients for offspring survival. These findings provide mechanisms through which females may maximize reproductive output in changing environments (Gorter, 2016).

Recovery of locomotion after injury in Drosophila depends on proprioception

Locomotion is necessary for survival in most animal species. However, injuries to the appendages mediating locomotion are common. This study assessed the recovery of walking in Drosophila melanogaster following leg amputation. Whereas flies pre-amputation explore open arenas in a symmetric fashion, foreleg amputation induces a strong turning bias away from the side of the amputation. However, unbiased walking behavior was found to return over time in wild type flies, while recovery is significantly impaired in proprioceptive mutants. To identify the biomechanical basis of this locomotor impairment and recovery, individual leg motion (gait) were examined at a fine scale. A minimal mathematical model that links neurodynamics to body mechanics during walking shows that redistributing leg forces between the right and left side enables the observed recovery. Altogether, this study suggests that proprioceptive input from the intact limbs plays a critical role in the behavioral plasticity associated with locomotor recovery after injury (Isakov, 2026).

Ancient anxiety pathways influence Drosophila defense behaviors

Anxiety helps us anticipate and assess potential danger in ambiguous situations; however, the anxiety disorders are the most prevalent class of psychiatric illness. Anxiety research makes wide use of three rodent behavioral assays-elevated plus maze, open field, and light/dark box-that present a choice between sheltered and exposed regions. Exposure avoidance in anxiety-related defense behaviors has been confirmed to be a correlate of rodent anxiety by treatment with known anxiety-altering agents and is now used to characterize anxiety systems. Modeling anxiety with a small neurogenetic animal would further aid the elucidation of its neuronal and molecular bases. Drosophila neurogenetics research has elucidated the mechanisms of fundamental behaviors and implicated genes that are often orthologous across species. In an enclosed arena, flies stay close to the walls during spontaneous locomotion, a behavior proposed to be related to anxiety. This study tested this hypothesis with manipulations of the GABA receptor, serotonin signaling, and stress. The effects of these interventions were strikingly concordant with rodent anxiety, verifying that these behaviors report on an anxiety-like state. Application of this method was able to identify several new fly anxiety genes. The presence of conserved neurogenetic pathways in the insect brain identifies Drosophila as an attractive genetic model for the study of anxiety and anxiety-related disorders, complementing existing rodent systems (Mohammad, 2016).

Drosophila food-associated pheromones: Effect of experience, genotype and antibiotics on larval behavior

Animals ubiquitously use chemical signals to communicate many aspects of their social life. These chemical signals often consist of environmental cues mixed with species-specific signals-pheromones-emitted by conspecifics. During their life, insects can use pheromones to aggregate, disperse, choose a mate, or find the most suitable food source on which to lay eggs. Before pupariation, larvae of several Drosophila species migrate to food sources depending on their composition and the presence of pheromones. Some pheromones derive from microbiota gut activity and these food-associated cues can enhance larval attraction or repulsion. To explore the mechanisms underlying the preference (attraction/repulsion) to these cues and clarify their effect, this study manipulated factors potentially involved in larval response. In particular, it was found that the (1) early exposure to conspecifics, (2) genotype, and (3) antibiotic treatment changed D. melanogaster larval behavior. Generally, larvae-tested either individually or in groups-strongly avoided food processed by other larvae. Compared to previous reports on larval attractive pheromones, the data suggest that such attractive effects are largely masked by food-associated compounds eliciting larval aversion. The antagonistic effect of attractive vs. aversive compounds could modulate larval choice of a pupariation site and impact the dispersion of individuals in nature.

Functional genetic screen to identify interneurons governing behaviorally distinct aspects of Drosophila larval motor programs

Drosophila larval crawling is an attractive system to study patterned motor output at the level of animal behavior. Larval crawling consists of waves of muscle contractions generating forward or reverse locomotion. In addition, larvae undergo additional behaviors including head casts, turning, and feeding. It is likely that some neurons are used in all these behaviors (e.g. motor neurons), but the identity (or even existence) of neurons dedicated to specific aspects of behavior is unclear. To identify neurons that regulate specific aspects of larval locomotion, a genetic screen was performed to identify neurons that, when activated, could elicit distinct motor programs. 165 Janelia CRM-Gal4 lines--chosen for sparse neuronal expression--were used to express the warmth-inducible neuronal activator TrpA1, and a screen was carried out for locomotor defects. The primary screen measured forward locomotion velocity, and 63 lines were identified that had locomotion velocities significantly slower than controls following TrpA1 activation (28 ° C). A secondary screen was performed on these lines, revealing multiple discrete behavioral phenotypes including slow forward locomotion, excessive reverse locomotion, excessive turning, excessive feeding, immobile, rigid paralysis, and delayed paralysis. While many of the Gal4 lines had motor, sensory, or muscle expression that may account for some or all of the phenotype, some lines showed specific expression in a sparse pattern of interneurons. These results show that distinct motor programs utilize distinct subsets of interneurons, and provide an entry point for characterizing interneurons governing different elements of the larval motor program (Clark, 2016).

Mushroom body signaling is required for locomotor activity rhythms in Drosophila

In the fruitfly Drosophila melanogaster, circadian rhythms of locomotor activity under constant darkness are controlled by pacemaker neurons. To understand how behavioral rhythmicity is generated by the nervous system, it is essential to identify the output circuits from the pacemaker neurons. The importance of mushroom bodies (MBs) in generating behavioral rhythmicity remains controversial because contradicting results have been reported as follows: (1) locomotor activity in MB-ablated flies is substantially rhythmic, but (2) activation of restricted neuronal populations including MB neurons induces arrhythmic locomotor activity. This study reports that neurotransmission in MBs is required for behavioral rhythmicity. For adult-specific disruption of neurotransmission in MBs, the GAL80/GAL4/UAS ternary gene expression system was used in combination with the temperature-sensitive dynamin mutation shibirets1. Blocking of neurotransmission in GAL4-positive neurons including MB neurons induced arrhythmic locomotor activity, whereas this arrhythmicity was rescued by the MB-specific expression of GAL80. These results indicate that MB signaling plays a key role in locomotor activity rhythms in Drosophila (Mabuchi, 2016).

A single dopamine pathway underlies progressive locomotor deficits in a Drosophila model of Parkinson disease

Expression of the human Parkinson-disease-associated protein α-synuclein in all Drosophila neurons induces progressive locomotor deficits. This study identify a group of 15 dopaminergic neurons per hemisphere in the anterior medial region of the brain whose disruption correlates with climbing impairments in this model. These neurons selectively innervate the horizontal β and β' lobes of the mushroom bodies and their connections to the Kenyon cells are markedly reduced when they express α-synuclein. Using selective mushroom body drivers, it was shown that blocking or overstimulating neuronal activity in the β' lobe, but not the β or γ lobes, significantly inhibits negative geotaxis behavior. This suggests that modulation of the mushroom body β' lobes by this dopaminergic pathway is specifically required for an efficient control of startle-induced locomotion in flies (Riemensperger, 2013).

These results demonstrate that an α-synuclein model of Parkinson-disease primarily derives from gradual dysfunction of a subset of dopamine neurons in the PAM cluster without overt cell death. Progressive disruption of synaptic structure or activity in these cells by sustained α-synA30P or tyrosine hydroxylase-dsRNA expression, or by dTRPA1 activation, all impaired the fly's climbing ability. Strikingly, the expression of α-synA30P in these 2 x 15 dopamine neurons progressively altered locomotion to the same extent as the expression of the pathogenic protein in the ~100,000 neurons of the Drosophila brain. These PAM DNs and the MBb β lobe Kenyon cells they innervate form a neuronal circuit involved in control of startle-induced negative geotaxis (SING) behavior. These DNs are very susceptible to α-syn toxicity and they play an important role in locomotion, comparable to the midbrain DNs in humans whose degeneration causes the motor symptoms of PD. Thus, in flies, as in humans, motor impairments in PD conditions correlate to the degeneration of a specific subset of brain DNs located in the substantia nigra pars compacta in humans and in the PAM cluster in Drosophila. This opens the way for future studies in a genetically tractable organism to decipher the pathological pathways activated by α-syn that cause disruption of these dopaminergic projections as well as the cellular interaction mechanisms leading from dopaminergic terminal loss to progressive locomotor dysfunction (Riemensperger, 2013).

Generative rules of Drosophila locomotor behavior as a candidate homology across phyla
The discovery of shared behavioral processes across phyla is a significant step in the establishment of a comparative study of behavior. This study used immobility as an origin and reference for the measurement of fly locomotor behavior; speed, walking direction and trunk orientation as the degrees of freedom shaping this behavior; and cocaine as the parameter inducing progressive transitions in and out of immobility. Using these, the generative rules that shape Drosophila locomotor behavior, bringing about a gradual buildup of kinematic degrees of freedom during the transition from immobility to normal behavior, and the opposite narrowing down into immobility, were characterized. Transitions into immobility unfold via sequential enhancement and then elimination of translation, curvature and finally rotation. Transitions out of immobility unfold by progressive addition of these degrees of freedom in the opposite order. The same generative rules have been found in vertebrate locomotor behavior in several contexts (pharmacological manipulations, ontogeny, social interactions) involving transitions in-and-out of immobility. Recent claims for deep homology between arthropod central complex and vertebrate basal ganglia provide an opportunity to examine whether the rules reported in this study also share common descent. This approach prompts the discovery of behavioral homologies, contributing to the elusive problem of behavioral evolution (Gomez-Marin, 2016). 

Behavior reveals selective summation and max pooling among olfactory processing channels

The olfactory system is divided into processing channels (glomeruli), each receiving input from a different type of olfactory receptor neuron (ORN). This study investigated how glomeruli combine to control behavior in freely walking Drosophila. Optogenetically activating single ORN types were shown to typically produced attraction, although some ORN types produced repulsion. Attraction consisted largely of a behavioral program with the following rules: at fictive odor onset, flies walked upwind, and at fictive odor offset, they reversed. When certain pairs of attractive ORN types were co-activated, the level of the behavioral response resembled the sum of the component responses. However, other pairs of attractive ORN types produced a response resembling the larger component (max pooling). Although activation of different ORN combinations produced different levels of behavior, the rules of the behavioral program were consistent. The results illustrate a general method for inferring how groups of neurons work together to modulate behavioral programs (Bell, 2016).

Interactions among Drosophila larvae before and during collision
In populations of Drosophila larvae, both, an aggregation and a dispersal behavior can be observed. However, the mechanisms coordinating larval locomotion in respect to other animals, especially in close proximity and during/after physical contacts are currently only little understood. This study tested whether relevant information is perceived before or during larva-larva contacts, analyze its influence on behavior and ask whether larvae avoid or pursue collisions. Employing frustrated total internal reflection-based imaging (FIM) it was found that larvae visually detect other moving larvae in a narrow perceptive field and respond with characteristic escape reactions. To decipher larval locomotion not only before but also during the collision, a two color FIM approach (FIM2c) was utilized, allowing faithful extraction of the posture and motion of colliding animals. It was found that during collision, larval locomotion freezes and sensory information is sampled during a KISS phase (german: Kollisions Induziertes Stopp Syndrom or english: collision induced stop syndrome). Interestingly, larvae react differently to living, dead or artificial larvae, discriminate other Drosophila species and have an increased bending probability for a short period after the collision terminates. Thus, Drosophila larvae evolved means to specify behaviors in response to other larvae (Otto, 2016). 

On the encoding of panoramic visual scenes in navigating wood ants
A natural visual panorama is a complex stimulus formed of many component shapes. It gives an animal a sense of place and supplies guiding signals for controlling the animal's direction of travel. Insects with their economical neural processing are good subjects for analyzing the encoding and memory of such scenes. Honeybees and ants foraging from their nest can follow habitual routes guided only by visual cues within a natural panorama. This study analyzed the headings that ants adopt when a familiar panorama composed of two or three shapes is manipulated by removing a shape or by replacing training shapes with unfamiliar ones. Ants were shown to recognize a component shape not only through its particular visual features, but also by its spatial relation to other shapes in the scene, and that (2) each segmented shape contributes its own directional signal to generating the ant's chosen heading. It was found earlier that ants trained to a feeder placed to one side of a single shape and tested with shapes of different widths learn the retinal position of the training shape's center of mass (CoM) when heading toward the feeder. They then guide themselves by placing the shape's CoM in the remembered retinal position. This use of CoM in a one-shape panorama combined with the results here suggests that the ants' memory of a multi-shape panorama comprises the retinal positions of the horizontal CoMs of each major component shape within the scene, bolstered by local descriptors of that shape (Buehlmann, 2016).

Selective inhibition mediates the sequential recruitment of motor pools

Locomotor systems> generate diverse motor patterns to produce the movements underlying behavior, requiring that motor neurons be recruited at various phases of the locomotor cycle. Reciprocal inhibition produces alternating motor patterns; however, the mechanisms that generate other phasic relationships between intrasegmental motor pools, all of the motor neurons that innervate single muscles, are unknown. This study investigated one such motor pattern in the Drosophila larva, using a multidisciplinary approach including electrophysiology and ssTEM-based circuit reconstruction. It was found that two motor pools that are sequentially recruited during locomotion have identical excitable properties. In contrast, they receive input from divergent premotor circuits. It was also found that this motor pattern is not orchestrated by differential excitatory input but by a GABAergic interneuron acting as a delay line to the later-recruited motor pool. These findings show how a motor pattern is generated as a function of the modular organization of locomotor networks through segregation of inhibition, a potentially general mechanism for sequential motor patterns (Zwart, 2016).

Speed-dependent interplay between local pattern-generating activity and sensory signals during walking in Drosophila

In insects, the coordinated motor output required for walking is based on the interaction between local pattern-generating networks providing basic rhythmicity and leg sensory signals which modulate this output on a cycle-to-cycle basis. How this interplay changes speed-dependently and thereby gives rise to the different coordination patterns observed at different speeds is understood insufficiently. This study used amputation to reduce sensory signals in single legs and decouple them mechanically during
walking in Drosophila. This allows for the dissociation between locally-generated motor output in the stump and coordinating influences from intact legs. Leg stumps are still rhythmically active during walking. While the oscillatory frequency in intact legs is dependent on walking speed, stumps show a high and relatively constant oscillation frequency at all walking speeds. At low walking speeds strict cycle-to-cycle coupling between stumps and intact legs is not present. In contrast, at high walking speeds stump oscillations are strongly coupled to the movement of intact legs on a 1-to-1 basis. While during slow walking there is no preferred phase between stumps and intact legs, a preferred time interval between touch-down or lift-off events in intact legs and levation or depression of stumps is present. Based on these findings, the study hypothesizes that, as in other insects, walking speed in Drosophila is predominantly controlled by indirect mechanisms and that direct modulation of basic pattern-generating circuits plays a subsidiary role. Furthermore, inter-leg coordination strength seems to be speed-dependent and greater coordination is evident at higher walking speeds (Berendes, 2016).

Pulsed light stimulation increases boundary preference and periodicity of episodic motor activity in Drosophila melanogaster

There is considerable interest in the therapeutic benefits of long-term sensory stimulation for improving cognitive abilities and motor performance of stroke patients. In this study, the effect of chronic sensory stimulation (pulsed light stimulation) on motor activity in w1118 flies was investigated. Flies were exposed to a chronic pulsed light stimulation protocol prior to testing their performance in a standard locomotion assay. Flies responded to pulsed light stimulation with increased boundary preference and travel distance in a circular arena. In addition, pulsed light stimulation increased the power of extracellular electrical activity, leading to the enhancement of periodic electrical activity which was associated with a centrally-generated motor pattern (struggling behavior). In contrast, such periodic events were largely missing in w1118 flies without pulsed light treatment. These data suggest that the sensory stimulation induced a response in motor activity associated with the modifications of electrical activity in the central nervous system (CNS). Finally, without pulsed light treatment, the wild-type genetic background was associated with the occurrence of the periodic activity in wild-type Canton S (CS) flies, and w+ modulated the consistency of periodicity. It is concluded that pulsed light stimulation modifies behavioral and electrophysiological activities in w1118 flies. These data provide a foundation for future research on the genetic mechanisms of neural plasticity underlying such behavioral modification (Qui, 2016).

Continuous lateral oscillations as a core mechanism for taxis in Drosophila larvae

Taxis behaviour, chemotaxis for example, in Drosophila larva is thought to consist of distinct control mechanisms triggering specific actions. This study supports a simpler hypothesis: that taxis results from direct sensory modulation of continuous lateral oscillations of the anterior body, sparing the need for 'action selection'. Analysis of larvae motion reveals a rhythmic, continuous lateral oscillation of the anterior body, encompassing all head-sweeps, small or large, without breaking the oscillatory rhythm. Further, it was show that an agent-model that embeds this hypothesis reproduces a surprising number of taxis signatures observed in larvae. Also, by coupling the sensory input to a neural oscillator in continuous time, it was shown that the mechanism is robust and biologically plausible. The mechanism provides a simple architecture for combining information across modalities, and explaining how learnt associations modulate taxis. The results in the light of larval neural circuitry are discussed, and testable predictions are made (Wystrach , 2016).

Predictability and hierarchy in Drosophila behavior

Even the simplest of animals exhibit behavioral sequences with complex temporal dynamics. Prominent among the proposed organizing principles for these dynamics has been the idea of a hierarchy, wherein the movements an animal makes can be understood as a set of nested subclusters. Although this type of organization holds potential advantages in terms of motion control and neural circuitry, measurements demonstrating this for an animal's entire behavioral repertoire have been limited in scope and temporal complexity. This study use a recently developed unsupervised technique to discover and track the occurrence of all stereotyped behaviors, including locomotion and grooming, performed by fruit flies moving in a shallow arena. Calculating the optimally predictive representation of the fly's future behaviors, it was shown that fly behavior exhibits multiple time scales and is organized into a hierarchical structure that is indicative of its underlying behavioral programs and its changing internal states (Berman, 2016).

Simultaneous activation of parallel sensory pathways promotes a grooming sequence in Drosophila

A central model that describes how behavioral sequences are produced features a neural architecture that readies different movements simultaneously, and a mechanism where prioritized suppression between the movements determines their sequential performance. A previous paper described a model whereby suppression drives a Drosophila grooming sequence that is induced by simultaneous activation of different sensory pathways that each elicit a distinct movement. The current study has confirm this model using transgenic expression to identify and optogenetically activate sensory neurons that elicit specific grooming movements. Simultaneous activation of different sensory pathways elicits a grooming sequence that resembles the naturally induced sequence. Moreover, the sequence proceeds after the sensory excitation is terminated, indicating that a persistent trace of this excitation induces the next grooming movement once the previous one is performed. This reveals a mechanism whereby parallel sensory inputs can be integrated and stored to elicit a delayed and sequential grooming response (Hampel, 2017).

Quantitative predictions orchestrate visual signaling in Drosophila

Vision influences behavior, but ongoing behavior also modulates vision in animals ranging from insects to primates. The function and biophysical mechanisms of most such modulations remain unresolved. This study combine behavioral genetics, electrophysiology, and high-speed videography to advance a function for behavioral modulations of visual processing in Drosophila. It was argued that a set of motion-sensitive visual neurons regulate gaze-stabilizing head movements. During flight turns, Drosophila perform a set of head movements that require silencing their gaze-stability reflexes along the primary rotation axis of the turn. Consistent with this behavioral requirement, pervasive motor-related inputs to the visual neurons were found, that quantitatively silence their predicted visual responses to rotations around the relevant axis while preserving sensitivity around other axes. This work proposes a function for a behavioral modulation of visual processing and illustrates how the brain can remove one sensory signal from a circuit carrying multiple related signals (Kim, 2017).

The Function and Organization of the Motor System Controlling Flight Maneuvers in Flies

Animals face the daunting task of controlling their limbs using a small set of highly constrained actuators. This problem is particularly demanding for insects such as Drosophila, which must adjust wing motion for both quick voluntary maneuvers and slow compensatory reflexes using only a dozen pairs of muscles. To identify strategies by which animals execute precise actions using sparse motor networks, the activity was imagined of a complete ensemble of wing control muscles in intact, flying flies. The experiments uncovered a remarkably efficient logic in which each of the four skeletal elements at the base of the wing are equipped with both large phasically active muscles capable of executing large changes and smaller tonically active muscles specialized for continuous fine-scaled adjustments. Based on the responses to a broad panel of visual motion stimuli, a model is developed by which the motor array regulates aerodynamically functional features of wing motion (Lindsay, 2017).

Flies compensate for unilateral wing damage through modular adjustments of wing and body kinematics

Using high-speed videography, this study investigated how fruit flies compensate for unilateral wing damage, in which loss of area on one wing compromises both weight support and roll torque equilibrium. The results show that flies control for unilateral damage by rolling their body towards the damaged wing and by adjusting the kinematics of both the intact and damaged wings. To compensate for the reduction in vertical lift force due to damage, flies elevate wingbeat frequency. Because this rise in frequency increases the flapping velocity of both wings, it has the undesired consequence of further increasing roll torque. To compensate for this effect, flies increase the stroke amplitude and advance the timing of pronation and supination of the damaged wing, while making the opposite adjustments on the intact wing. The resulting increase in force on the damaged wing and decrease in force on the intact wing function to maintain zero net roll torque. However, the bilaterally asymmetrical pattern of wing motion generates a finite lateral force, which flies balance by maintaining a constant body roll angle. Based on these results and additional experiments using a dynamically scaled robotic fly, a simple bioinspired control algorithm is proposed for asymmetric wing damage (Muijres, 2017).

Moonwalker descending neurons mediate visually evoked retreat in Drosophila

Insects, like most animals, tend to steer away from imminent threats. Drosophila melanogaster, for example, generally initiate an escape take-off in response to a looming visual stimulus, mimicking a potential predator. The escape response to a visual threat is, however, flexible and can alternatively consist of walking backward away from the perceived threat, which may be a more effective response to ambush predators such as nymphal praying mantids. Flexibility in escape behavior may also add an element of unpredictability that makes it difficult for predators to anticipate or learn the prey's likely response. Whereas the fly's escape jump has been well studied, the neuronal underpinnings of evasive walking remain largely unexplored. A cluster of descending neurons-the moonwalker descending neurons (MDNs)-the activity of which is necessary and sufficient to trigger backward walking, as well as a population of visual projection neurons-the lobula columnar 16 (LC16) cells-that respond to looming visual stimuli and elicit backward walking and turning has been previously reported. Given the similarity of their activation phenotypes, this study hypothesized that LC16 neurons induce backward walking via MDNs and that turning while walking backward might reflect asymmetric activation of the left and right MDNs. Data from functional imaging, behavioral epistasis, and unilateral activation experiments that support these hypotheses. The study conclude that LC16 and MDNs are critical components of the neural circuit that transduces threatening visual stimuli into directional locomotor output (Sen, 2017).

Visual projection neurons in the Drosophila lobula link feature detection to distinct behavioral programs

Visual projection neurons (VPNs) provide an anatomical connection between early visual processing and higher brain regions. This study characterized lobula columnar (LC) cells, a class of Drosophila VPNs that project to distinct central brain structures called optic glomeruli. This study anatomically describes 22 different LC types and show that, for several types, optogenetic activation in freely moving flies evokes specific behaviors. The activation phenotypes of two LC types closely resemble natural avoidance behaviors triggered by a visual loom. In vivo two-photon calcium imaging reveals that these LC types respond to looming stimuli, while another type does not, but instead responds to the motion of a small object. Activation of LC neurons on only one side of the brain can result in attractive or aversive turning behaviors depending on the cell type. These results indicate that LC neurons convey information on the presence and location of visual features relevant for specific behaviors (Wu, 2016).

This report presents anatomical and functional studies of lobula columnar (LC) cells, prominent visual projection neurons from the lobula to target regions in the central brain called optic glomeruli. Comprehensive anatomical analyses of the dendritic arbors and central brain projections of LC neurons support the notion that these cells encode diverse visual stimuli, distinct for each LC cell type, and convey this information to cell-type specific downstream circuits. Precise genetic tools that target individual LC cell types allowed exploration of the behavioral consequences of optogenetic activation of these cell types. Activating cells of single LC neuron types was often sufficient to evoke a range of coordinated behaviors in freely behaving flies. Using two-photon calcium imaging from head-fixed flies, two LC cell types with activation phenotypes similar to avoidance responses, were shown to selectively encode visual looming, a stimulus that also evokes similar avoidance behaviors, while a third cell type responded strongly to a small moving object. These results suggest that LC cell types encode visual features that are relevant for specific behaviors. Activation of LC cells in only one brain hemisphere can result in either an attractive or repulsive directional turning response, depending on cell type. Thus which LC neuron channel is activated determines the valence of the behavior, whereas comparison across the brain by two such channels of the same type provides information about the location of relevant visual features (Wu, 2016).

Anatomical properties of LC neurons have been previously described both in Drosophila and other Diptera. This work extends these studies by providing a comprehensive description of LC neurons in Drosophila, including the identification of several previously unreported cell types. Further, these anatomical analyses with were combined the generation of highly specific genetic markers (split-GAL4 lines) for each cell type. Each of the 22 LC types described has morphologically distinct dendritic arbors in the lobula with stereotyped arbor stratification, size and shape. As observed in the medulla, where synapse-level connectomics data are available for many cell types, different layer patterns and arbor shapes are likely to reflect differences in synaptic connectivity and neuronal computation. Arbors of LC neurons are found in all lobula strata, though with large differences between layers. Only LC4 (and perhaps LPLC1 and LPLC2) cells are potentially postsynaptic to neurons in the most distal lobula layer, Lo1, while other strata such as Lo4 and Lo5B include processes of more than half of the LC types. The presence of at least some LC dendrites in each lobula layer implies that all of the about 50 different interneuron types that convey visual information from the medulla, and to a lesser extent from the lobula plate, to the lobula, are potentially presynaptic to some LC cells, although a far smaller number is likely presynaptic to any single LC cell type. The predicted differences in the synaptic inputs to different LC cell types also suggest that they will differ in their responses to visual stimuli. Thus, individual LC neuron types are expected to encode specific visual stimuli, while the population of all LC cell types together should signal a wide range of behaviorally relevant visual features (Wu, 2016).

The visual responses of several LC cell types measured using two-photon calcium imaging support the expectation that different types selectively respond to different visual features. The three LC neuron types examined preferentially responded to distinct stimuli, with either a dark looming stimulus (LC6 and LC16) or a small moving object (LC11) evoking the strongest measured responses. LC6 and LC16 showed stronger responses to a dark expanding disc than to related stimuli such as an expanding bright disk or a darkening stimulus that lacks the expanding motion. The reduction in the LC6 and LC16 responses when the edge motion is removed from the stimulus is precisely what is expected of loom-sensitive neurons and is reminiscent of behavioral studies in houseflies showing that darkening contrast combined with edge motion is the most effective stimulus for triggering takeoffs. Consistent with their similar responses in the imaging experiments, LC6 and LC16 have very similar lobula layer patterns while LC11 has a different arbor stratification indicating that LC11 receives inputs from a different set of medulla cell types than LC6 and LC16 (Wu, 2016).

It is likely that the selectivity for visual stimuli observed in LC neuron responses is both a property of the stimulus selectivity of their inputs-some selectivity was seen while imaging in the dendrites of a few LC cell types-and specific computations implemented by individual LC neuron types. In addition, cells post-synaptic to the LC cells may integrate the responses of several individual LC neurons of the same type to provide more robust detection of specific visual features. For example, while LC6 and LC16 cells as populations are strongly excited by dark looming stimuli, it is currently unknown whether individual LC6 and LC16 neurons, which have dendritic extents well below the maximum size of the looming stimuli, and also well below the size known to elicit maximal behavioral responses, show the same response properties. The anatomical data and genetic reagents provide a starting point for the additional functional and ultra-structural studies that will be required to elucidate the circuit mechanisms that produce the response properties of these and other LC cell types (Wu, 2016).

The suggestion that LC cells are feature-responsive neurons has been partly based on the apparent dramatic reduction in retinotopy between LC neuron dendrites, which have a retinotopic arrangement in the lobula, and their axons, which appear to discard this spatial information as they converge onto optic glomeruli, the cell-type specific target regions in the central brain. This study extended previous analyses of LC neuron arbor convergence by directly visualizing multiple single LC cells in a glomerulus in the same fly. These experiments revealed no detectable retinotopy of LC cell processes in most glomeruli even at this cellular level of resolution. It is possible that the responses of individual LC cells carry information about retinotopic position; given the comparatively small size of LC dendrites (the lateral spread of even the largest LC cells covers less than 20% of visual columns) and the retinotopic distribution of these dendrites in the lobula it would be surprising if they did not. Such retinotopic responses could for example be relevant for those LC cell types that appear to have presynaptic sites in the lobula and are thus likely to provide input to retinotopically organized circuits. However, with the caveat that synapse-level connectivity was not examined, for most LCs the available anatomical information appears to support the view that much retinotopic information is discarded at the glomerulus level. Consistent with this anatomical observation, the calcium imaging experiments from single LC cell types revealed visual responses to localized stimuli that could be measured throughout a cross-section of the glomerulus without clear retinotopic arrangement of the responding axons. Because of the columnar nature and apparently restricted visual field of the dendrites of LC neurons, the features computed by individual LC neurons are likely to be well defined in subregions of the eye, with perhaps downstream circuits required to integrate these locally-extracted features, as discussed above for looming. There is currently little insight into how these computations are initiated in the optic glomeruli and this remains an exciting area for future investigation (Wu, 2016).

Unlike the other LC neurons, it was found that LC10, and to a lesser extent LC9, cells retain some retinotopic information in the arrangement of their axon terminals indicating that the loss of retinotopy is not a necessary consequence of axonal convergence onto a glomerular target region. More specifically, it was observed that the order of LC10 axonal terminals in the anterior optic tubercle (AOTu) along the DV axis matches the sequence of AP positions of the corresponding dendrites in the lobula. This organization could facilitate synaptic interactions of LC10 cells corresponding to different azimuthal positions in the visual field with distinct target cells. Consistent with a possible general role of the AOTu in the processing or the relaying of retinotopic information, retinotopic responses have recently been observed in the dendrites of central complex neurons that, mainly based on work in other insects, are thought to be synaptic targets of output neurons of the lateral zone of the AOTu (Wu, 2016).

It was found that, independent of the presence or absence of retinotopy at the glomerulus level, positional information can be extracted from the differential activity of LC cells between the two optic lobes. It was directly demonstrated this capability by genetically restricting optogenetic LC neuron activation to only one optic lobe. This unilateral activation evoked directional turning responses relative to the activated brain side. Thus, LC neuron signaling appears to convey information on both different visual features and their location. This may further extend the similarities to the antennal lobes where differences in odorant receptor neuron activity between the left and right antennal lobes may contribute to odorant tracking (Wu, 2016).

Activation of different types of LC neurons can induce distinct behaviors including jumping, reaching, wing extension, forward walking, backward walking and turning. While specific activation phenotypes have been reported for a variety of cell types and behaviors, many of these studies have focused on command-like neurons thought to orchestrate specific motor programs. By contrast, the activation phenotypes reported in this study result from the optogenetic stimulation of different types of related visual projection neurons. A plausible interpretation of these results is that activation of LC neurons can mimic the presence of the visual features that these neurons normally respond to and thus elicits behavioral responses associated with these fictive stimuli. This possibility is supported by several lines of evidence from studies of LC6 and LC16. First, optogenetic depolarization of each of these cell types evokes a specific behavioral response-backward walking for LC16 and jumping for LC6-that resembles a similar natural avoidance or escape behavior. Second, backward walking and jumping can both also be elicited by presentation of a predator-mimicking visual loom and, third, in calcium imaging experiments both LC16 and LC6 showed a preferential response to a similar looming stimulus compared to a number of related stimuli. Although this study did not explore LC10 response properties, it is noted that LC10-activation phenotypes also show similarities to natural behaviors: movements resembling the directed foreleg extension displayed during activation-evoked reaching occur, for example, during gap-climbing behavior and in aggressive fly-fly interactions (Wu, 2016).

Overall, the LC neuron activation phenotypes that were observed suggest that the encoding of visual information at the level of LC neurons is sufficiently specialized to contribute to distinct behavioral responses in a cell-type dependent fashion. However, patterns of LC neuron activation that produce more refined fictive stimuli than were employed in the current work will be required to fully explore the LC neuron behavioral repertoire. Likewise, more comprehensive physiological studies of the response properties of the LC cell types will be needed (Wu, 2016).

How does LC cell activation evoke specific behavioral responses? In the simplest scenario, LC neuron depolarization could directly activate a single postsynaptic premotor descending interneuron that would then in turn trigger the observed behavior. This appears plausible in some cases: for example, activation of LC4 neurons (called ColA cells in larger flies) might evoke a jumping response via activation of the Giant Fiber (GF) cells, a pair of large descending neurons known to be postsynaptic to ColA and LC4 and which have a known role in escape behavior. For other LC cell types, there is currently no evidence suggesting a direct connection to descending neurons. For example, candidate descending neurons for the LC16 backward walking response, the moon-walker descending interneurons, do not have dendrites in or near the LC16 glomerulus. Responses to diverse visual stimuli, some of which may derive from LC neuron activity, have also been observed in higher order brain centers without direct connections to LC neurons such as the central complex (Wu, 2016).

The activation experiments also provide several indications that the signaling downstream of LC neurons is likely to be more complex; for example, activation of a single LC cell type can give rise to multiple behaviors such as reaching, wing extension and turning for LC10, or backward walking and turning for LC16. Changes of the spatial pattern of LC neuron activation, as in the stochastic labeling experiments of this study, can further modify activation phenotypes. For example, unilateral LC16 activation primarily evokes turning away from the location of LC16 activation, not backward walking, suggesting that the relative differences in LC16 activity between the two eyes can guide the direction of motor output through downstream signaling. Furthermore, several different LC neuron types may contribute to the same or similar behaviors, as suggested by the jumping phenotypes of LC4, LC6, LC15, LPLC1 and LPLC2. Presumably, visual signals and other information downstream of LC neurons are integrated to select appropriate behavioral actions. Such additional processing is also suggested by the cases of neurons with overlapping response properties but distinct activation phenotypes such as LC6 and LC16. It is also noted that some responses to LC neuron activation appear to be context dependent; for example, reduced forward walking was observed for several LC cell types on the platform of the single-fly assay that is much smaller than the arena used in the arena assay (Wu, 2016).

In addition, this study examined onoy the behavior of standing or walking flies and LC neuron signaling might have different consequences depending on the behavioral state. For example, looming stimuli can also elicit avoidance responses in flying flies, but these responses differ from the takeoff or retreat behaviors of walking animals. Therefore, while LC cell activity appears to convey visual information that is specialized for sets of related behavioral responses, LC neurons do not appear to instruct a single behavioral output (Wu, 2016).

The most common activation phenotypes observed in the screen were apparent avoidance responses. Furthermore, in addition to the LC cells examined in this study, other VPNs may also contribute to avoidance behaviors. This predominance of avoidance phenotypes is perhaps not unexpected. Since escape responses have to be fast and reliably executed under many different conditions, neurons that signal features that can evoke escape may be particularly likely to show phenotypes in an activation screen. Given the importance of predator avoidance for fly survival, it appears plausible that a considerable fraction of visual output neurons might be utilized for the detection of visual threats ranging from looming to small objects. Furthermore, it is likely that CsChrimson-mediated depolarization of an entire population of LC neurons is more similar to the pattern of neuronal activity induced by an imminent collision, and thus responses of many individual loom-sensitive neurons, so it is not surprising that the activation screen revealed at least two looming-sensitive neuron types (Wu, 2016).

The escape-inducing neurons that were identifed in this study could provide inputs to different escape response pathways, such as long- and short-mode escape, or act as multiple inputs to the same downstream circuits. Interestingly, neurons with avoidance-like activation phenotypes project to two separate groups of adjacent glomeruli, one in the dorsal Posterior Ventrolateral Protocerebrum (PVLP; LC6, LC16 and also LC15) and one more ventral and medial (LC4, LPLC1 and LPLC2), the latter two with dendrites in the lobula and lobula plate. This spatial organization may facilitate synaptic interactions of functionally related LC neuron types with common downstream pathways for a specific behavior. The second group is close to dendritic branches of the GF, large descending neurons required for short-mode responses in Drosophila and a postsynaptic partner of LC4/ColA and possibly also the two LPLC cell types. LC6 terminals do not overlap with GF dendrites and LC6 cells may play a role in the GF-independent escape pathways that have been proposed in both Drosophila and housefly. Parallel neuronal pathways involved in escape behaviors have been identified or postulated in both vertebrates and invertebrates, but a contribution of several identified visual projection neurons to such pathways, as suggested by the activation screen, has not been previously reported. Different visual output neurons with distinct tuning of their response properties to looming parameters such as speed, size, luminance change or edge detection might have evolved to ensure robust responses to avoid predators or collisions. It is, however, currently not known whether LPLC1, LPLC2, LC4 and LC15 are indeed sensitive to looming stimuli and if so, whether their response details differ from LC16, LC6 and each other. Nevertheless, the identification of these neurons opens the possibility to examine the potential contribution of several visual pathways to avoidance behaviors (Wu, 2016).

LC neurons are a subset of the about a hundred VPN cell types that relay the output of optic lobe circuits to targets in the central brain. These data strongly support existing proposals for LC cell types as feature-detecting neurons, which have been mainly based on the distinct anatomical properties of LC cells. While these anatomical features distinguish LC neurons from many other VPNs, an association of VPN pathways with specific behaviors is not unique to LC cell types. The notion that individual neuronal pathways are tuned for specific behavioral requirements is a prominent theme in invertebrate neuroethology, with these neurons described as 'matched filters' for behaviorally relevant features of the external world. A number of previously studied VPN pathways, outside of the LC subgroup, have been described as encoding specific behaviorally related visual stimuli. In particular, very similar to the current results for LC6 and LC16, a group of tangential cells of the lobula and lobula plate (Foma-1 neurons) were found to respond to looming visual stimuli and, upon optogenetic activation, trigger escape responses. And perhaps most famously, the long-studied lobula plate tangential cells (LPTCs), such as the HS and VS cells, integrate local motion signals so as to preferentially respond to global optic flow patterns that are remarkably similar to visual motion encountered during specific behavioral movements. These findings are consistent with the idea that, at the outputs of the fly visual system, VPN pathways are found whose encoding properties are already well matched to particular fly behaviors or groups of behaviors. Matching the response properties of these deep sensory circuits to behavioral needs may be a general evolutionary solution to the challenge of dealing with the complexity of the visual world with limited resources (Wu, 2016).

LC neurons have long been recognized as a potential entry point for the circuit-level study of visual responses outside of the canonical motion detection pathways. This study has provided a comprehensive anatomical description of LC cell types and genetic reagents to facilitate such further investigations. It was also shown that activation of several LC cell types results in avoidance behaviors and that some of these same LC types respond to stimuli that can elicit such behaviors. Other LC neurons appear to mediate attractive behavioral responses. This work provides a starting point for exploring the circuit mechanisms both upstream and downstream of LC neurons (Wu, 2016).

A neural circuit architecture for angular integration in Drosophila

Many animals keep track of their angular heading over time while navigating through their environment. However, a neural-circuit architecture for computing heading has not been experimentally defined in any species. This study describes a set of clockwise- and anticlockwise-shifting neurons in the Drosophila central complex whose wiring and physiology provide a means to rotate an angular heading estimate based on the fly's angular velocity. Each class of shifting neurons exists in two subtypes, with spatiotemporal activity profiles that suggest different roles for each subtype at the start and end of tethered-walking turns. Shifting neurons are required for the heading system to properly track the fly's heading in the dark, and stimulation of these neurons induces predictable shifts in the heading signal. The central features of this biological circuit are analogous to those of computational models proposed for head-direction cells in rodents and may shed light on how neural systems, in general, perform integration (Green, 2017).

High-content behavioral profiling reveals neuronal genetic network modulating Drosophila larval locomotor program

Two key questions in understanding the genetic control of behaviors are: what genes are involved and how these genes interact. To answer these questions at a systems level, a high-content profiling of Drosophila larval locomotor behaviors was conducted for over 100 genotypes. 69 genes were studied whose C. elegans orthologs were neuronal signalling genes with significant locomotor phenotypes, and RNAi was conducted with ubiquitous, pan-neuronal, or motor-neuronal Gal4 drivers. Inactivation of 42 genes, including the nicotinic acetylcholine receptors nAChRalpha1 and nAChRalpha3, in the neurons caused significant movement defects. Bioinformatic analysis suggested 81 interactions among these genes based on phenotypic pattern similarities. Comparing the worm and fly data sets, this study found that these genes were highly conserved in having neuronal expressions and locomotor phenotypes. However, the genetic interactions were not conserved for ubiquitous profiles, and may be mildly conserved for the neuronal profiles. Unexpectedly, the data also revealed a possible motor-neuronal control of body size, because inactivation of Rdl and Galphao in the motor neurons reduced the larval body size. Overall, these data established a framework for further exploring the genetic control of Drosophila larval locomotion. In conclusion, high content, quantitative phenotyping of larval locomotor behaviours provides a framework for system-level understanding of the gene networks underlying such behaviours (Aleman-Meza, 2017).

Social effects for locomotion vary between environments in Drosophila melanogaster females

Despite strong purifying or directional selection, variation is ubiquitous in populations. One mechanism for the maintenance of variation is indirect genetic effects (IGEs), as the fitness of a given genotype will depend somewhat on the genes of its social partners. IGEs describe the effect of genes in social partners on the expression of the phenotype of a focal individual. Here, it is asked what effect IGEs, and variation in IGEs between abiotic environments, has on locomotion in Drosophila. This trait is known to be subject to intralocus sexually antagonistic selection. The coefficient of interaction, Psi, was estimated using six inbred lines of Drosophila. It was found that Psi varied between abiotic environments, and that it may vary across among male genotypes in an abiotic environment specific manner. Evidence was found that social effects of males alter the value of a sexually dimorphic trait in females, highlighting an interesting avenue for future research into sexual antagonism. It is concluded that IGEs are an important component of social and sexual interactions and that they vary between individuals and abiotic environments in complex ways, with the potential to promote the maintenance of phenotypic variation (Signor, 2017).

Mapping the neural substrates of behavior

Assigning behavioral functions to neural structures has long been a central goal in neuroscience and is a necessary first step toward a circuit-level understanding of how the brain generates behavior. This study mapped the neural substrates of locomotion and social behaviors for Drosophila melanogaster using automated machine-vision and machine-learning techniques. From videos of 400,000 flies, the behavioral effects were quantified of activating 2,204 genetically targeted populations of neurons. A novel quantification of anatomy was combined with behavioral analysis to create brain-behavior correlation maps, which are shared as browsable web pages and interactive software. Based on these maps, hypotheses were generated of regions of the brain causally related to sensory processing, locomotor control, courtship, aggression, and sleep. The maps directly specify genetic tools to target these regions, which were used to identify a small population of neurons with a role in the control of walking (Robie, 2017).

Turns with multiple and single head cast mediate Drosophila larval light avoidance

Drosophila larvae exhibit klinotaxis (directional orientation involving turning toward a stimulus) when placed in a gradient of temperature, chemicals, or light. The larva samples environmental stimuli by casting its head from side to side. By comparing the results of two consecutive samples, it decides the direction of movement, appearing as a turn proceeded by one or more head casts. By analyzing larval behavior in a light-spot-based phototaxis assay, this study showed that, in addition to turns with a single cast (1-cast), turns with multiple head casts (n-cast) helped to improve the success of light avoidance. Upon entering the light spot, the probability of escape from light after the first head cast was only ~30%. As the number of head casts increased, the chance of successful light avoidance increased and the overall chance of escaping from light increased to >70%. The amplitudes of first head casts that failed in light avoidance were significantly smaller in n-cast turns than those in 1-cast events, indicating that n-cast turns might be planned before completion of the first head cast. In n-casts, the amplitude of the second head cast was generally larger than that of the first head cast, suggesting that larvae tried harder in later attempts to improve the efficacy of light avoidance. It is proposed that both 1-cast turns and n-cast turns contribute to successful larval light avoidance, and both can be initiated at the first head cast (Zhao, 2017).

Forewings match the formation of leading-edge vortices and dominate aerodynamic force production in revolving insect wings

In many flying insects, forewings and hindwings are coupled mechanically to achieve flapping flight synchronously while being driven by action of the forewings. How the forewings and hindwings as well as their morphologies contribute to aerodynamic force production and flight control remains unclear. This study demonstrates that the forewings can produce most of the aerodynamic forces even with the hindwings removed through a computational fluid dynamic study of three revolving insect wing models, which are identical to the wing morphologies and Reynolds numbers of hawkmoth (Manduca sexta), bumblebee (Bombus ignitus) and fruitfly (Drosophila melanogaster). The forewing morphologies match the formation of leading-edge vortices (LEV) and are responsible for generating sufficient lift forces at the mean angles of attack and the Reynolds numbers where the three representative insects fly. The LEV formation and pressure loading keep almost unchanged with the hindwing removed, and even lead to some improvement in power factor and aerodynamic efficiency. Moreover, the results indicate that the size and strength of the LEVs can be well quantified with introduction of a conical LEV angle, which varies remarkably with angles of attack and Reynolds numbers but within the forewing region while showing less sensitivity to the wing morphologies. This implies that the forewing morphology very likely plays a dominant role in achieving low-Reynolds number aerodynamic performance in natural flyers as well as in revolving and/or flapping micro air vehicles (Chen, 2017).

Dendritic and axonal L-type calcium channels cooperate to enhance motoneuron firing output during Drosophila larval locomotion

Behaviorally adequate neuronal firing patterns are critically dependent on the specific types of ion channel expressed and on their subcellular localization. This study combines in situ electrophysiology with genetic and pharmacological intervention in larval Drosophila melanogaster of both sexes to address localization and function of L-type like calcium channels in motoneurons. Dmca1D (Cav1 homolog) L-type like calcium channels localize to both the somatodendritic and the axonal compartment of larval crawling motoneurons. In situ patch-clamp recordings in genetic mosaics reveal that Dmca1D channels increase burst duration and maximum intraburst firing frequencies during crawling-like motor patterns in semi-intact animals. Genetic and acute pharmacological manipulations suggest that prolonged burst durations are caused by dendritically localized Dmca1D channels, which activate upon cholinergic synaptic input and amplify EPSPs, thus indicating a conserved function of dendritic L-type channels from Drosophila to vertebrates. By contrast, maximum intraburst firing rates require axonal calcium influx through Dmca1D channels, likely to enhance sodium channel de-inactivation via a fast afterhyperpolarization through BK channel activation. Therefore, in unmyelinated Drosophila motoneurons different functions of axonal and dendritic L-type like calcium channels likely operate synergistically to maximize firing output during locomotion (Kadas, 2017).

Divergent connectivity of homologous command-like neurons mediates segment-specific touch responses in Drosophila

Animals adaptively respond to a tactile stimulus by choosing an ethologically relevant behavior depending on the location of the stimuli. This study investigated how somatosensory inputs on different body segments are linked to distinct motor outputs in Drosophila larvae. Larvae escape by backward locomotion when touched on the head, while they crawl forward when touched on the tail. A class of segmentally repeated second-order somatosensory interneurons, that was named Wave, was identified whose activation in anterior and posterior segments elicit backward and forward locomotion, respectively. Anterior and posterior Wave neurons extend their dendrites in opposite directions to receive somatosensory inputs from the head and tail, respectively. Downstream of anterior Wave neurons, premotor circuits were identified including the neuron A03a5, which together with Wave, is necessary for the backward locomotion touch response. Thus, Wave neurons match their receptive field to appropriate motor programs by participating in different circuits in different segments (Takagi, 2017).

GABAergic inhibition of leg motoneurons is required for normal walking behavior in freely moving Drosophila

Walking is a complex rhythmic locomotor behavior generated by sequential and periodical contraction of muscles essential for coordinated control of movements of legs and leg joints. Studies of walking in vertebrates and invertebrates have revealed that premotor neural circuitry generates a basic rhythmic pattern that is sculpted by sensory feedback and ultimately controls the amplitude and phase of the motor output to leg muscles. However, the identity and functional roles of the premotor interneurons that directly control leg motoneuron activity are poorly understood. This study took advantage of the powerful genetic methodology available in Drosophila to investigate the role of premotor inhibition in walking by genetically suppressing inhibitory input to leg motoneurons. For this, an algorithm was developed for automated analysis of leg motion to characterize the walking parameters of wild-type flies from high-speed video recordings. Further, genetic reagents were used for targeted RNAi knockdown of inhibitory neurotransmitter receptors in leg motoneurons together with quantitative analysis of resulting changes in leg movement parameters in freely walking Drosophila. The findings indicate that targeted down-regulation of the GABAA receptor Rdl (Resistance to Dieldrin) in leg motoneurons results in a dramatic reduction of walking speed and step length without the loss of general leg coordination during locomotion. Genetically restricting the knockdown to the adult stage and subsets of motoneurons yields qualitatively identical results. Taken together, these findings identify GABAergic premotor inhibition of motoneurons as an important determinant of correctly coordinated leg movements and speed of walking in freely behaving Drosophila (Gowda, 2018).

This report investigated the role of inhibitory premotor input to leg motoneurons in the walking behavior of freely moving Drosophila by suppression of GABAergic inhibitory input to leg motoneurons. The findings indicate that the reduction of inhibitory GABAergic input to leg motoneurons caused by targeted Rdl down-regulation has marked effects on walking behavior. Thus, walking speed was markedly slower compared with controls, and correlated with this, the amplitudes of the leg protraction (swing) and retraction (stance) phases were significantly smaller and the durations of protraction (swing) and retraction (stance) phases were significantly higher than in controls. Moreover, the concurrency state, in which three legs swing together in a tripod gait, was proportionally reduced compared with controls. These prominent effects on walking parameters are similar regardless of whether Rdl down-regulation occurs throughout development or whether it is restricted to the adult stage. Taken together, these findings reveal a prominent, albeit highly specific role of GABAergic premotor inhibitory input to leg motoneurons in the control of normal walking behavior (Gowda, 2018).

The insight into the role of premotor inhibition in walking control reported in this stuyd is the result of two key experimental methodologies. The first is the highly specific genetic access to identified neuronal populations that can now be attained in the Drosophila model system. This is made possible through remarkable targeted expression systems, which together with the availability of libraries of genetically encoded drivers and reporters for molecular manipulation, make it possible to selectively up or down-regulate gene expression in highly specific neuronal populations in intact and freely behaving animals. This study used the Gal4/UAS expression system to achieve targeted genetic access to leg motoneurons and down-regulate GABAergic premotor input to these motoneurons by Rdl RNAi expression. In addition different forms of the Gal80 repressor were used to limit Gal4/UAS targeted expression to adult stages or to specific regions of the central nervous system and, hence, refine the spatiotemporal specificity of the resulting genetic access to motoneurons. Given the wealth of Gal4 drivers and UAS-RNAi reporters currently available, it will be possible to use similar transgenic technology to manipulate other inhibitory and excitatory neurotransmitter receptors in future studies of interneuronal components of the walking circuitry (Gowda, 2018).

The second key method is the development of an advanced automated high-speed video recording and analysis technique that makes it possible to record the protraction (swing) and retraction (stance) phases at high spatiotemporal resolution for each leg in freely walking flies. With this technique, a quantitative assessment of leg motion parameters in freely walking animals can be carried out that can reveal subtle differences in amplitude and phase of movements of individual legs. This quantitative assessment has been critical for uncovering the role of premotor inhibition in walking behavior. Indeed, since the overall leg coordination during walking is unaffected by the reduction of GABAergic input to leg motoneurons, a more conventional qualitative behavioral analysis is unlikely to discern differences in walking between experimental and control animals. Recently, comparable high-resolution recording and analysis methods have been used to quantify leg movement parameters in freely walking flies. These studies have provided important information on walking speed, interleg coordination, and other locomotor parameters and have also documented a role of sensory proprioceptive input to step precision during walking. The fact that these methods for high-resolution recording and analysis are currently available for studying leg movement parameters in freely walking flies should accelerate understanding of walking behavior and the neuronal circuitry involved in its control in Drosophila (Gowda, 2018).

Given the prominent role of inhibitory premotor input to leg motoneurons reported in this study, it will now be important to identify and genetically access the premotor interneurons that provide this inhibitory input. While there is currently little information on the identity of the premotor interneurons that control the activity of leg motoneurons in adult flies, insight into inhibitory premotor interneurons has recently been obtained in larval stages. Thus, in Drosophila larva, a set of inhibitory local interneurons, termed PMSI neurons, have been identified that control the speed of axial locomotion by limiting the burst duration of motoneurons involved in peristaltic locomotion (Kohsaka, 2014). Moreover, a second set of inhibitory premotor interneurons called GVLI neurons have been reported that may be part of a feedback inhibition system involved in terminating each of the waves of motor activity that underlie larval peristalsis (Itakura, 2015). Finally, a pair of segmentally repeated GABAergic interneurons termed GDL neurons have been identified that are necessary for the coordinated propagation of peristaltic motor waves during both forward and backward crawling movements of larvae (Gjorgjieva, 2013). Whether or not these inhibitory premotor interneurons persist into the adult stage and act in the control of walking behavior is not known. Glutamate has previously been suggested to act as an inhibitory neurotransmitter in the Drosophila CNS. To investigate the likelihood that centrally labeled OK371 nonmotoneurons might also play any role in the leg motoneuron inhibition a set of preliminary experiments were carried out involving knockdown of GluCl channels in motoneurons. These experiments reveal enhanced defects in walking behavior with loss of coordination, suggesting there could be a possible role of OK371-labeled glutamatergic interneurons in leg motoneuron inhibition (Gowda, 2018).

In general terms, there are fundamental similarities in the principle mechanisms of locomotion in insects and vertebrates. These mechanistic similarities might also reflect similar motor circuit properties. For example, much like the PMSI neurons in Drosophila, which control the speed of locomotion by limiting motoneuron burst duration, the premotor V1 spinal interneurons in mammals are involved in the regulation of leg motoneuron burst and step cycle duration and thus also likely control the speed of walking movements. Hence, a characterization of the behavioral effects of inhibitory input to leg motoneurons in Drosophila, notably in freely walking flies, is likely to provide useful comparative information for understanding the functional role, and possibly the evolutionary origin, of premotor inhibition in vertebrate locomotory circuitry (Gowda, 2018).

Mutations in the Drosophila homolog of human PLA2G6 give rise to age-dependent loss of psychomotor activity and neurodegeneration

Infantile neuroaxonal dystrophy (INAD) is a fatal neurodegenerative disorder that typically begins within the first few years of life and leads to progressive impairment of movement and cognition. Several years ago, it was shown that >80% of patients with INAD have mutations in the phospholipase gene, PLA2G6. Interestingly, mutations in PLA2G6 are also causative in two other related neurodegenerative diseases, atypical neuroaxonal dystrophy and Dystonia-parkinsonism. While all three disorders give rise to similar defects in movement and cognition, some defects are unique to a specific disorder. At present, the cellular mechanisms underlying PLA2G6-associated neuropathology are poorly understood and there is no cure or treatment that can delay disease progression. This study shows that loss of iPLA2-VIA, the Drosophila homolog of PLA2G6, gives rise to age-dependent defects in climbing and spontaneous locomotion. Moreover, using a newly developed assay, this study shows that iPLA2-VIA mutants also display impairments in fine-tune motor movements, motor coordination and psychomotor learning, which are distinct features of PLA2G6-associated disease in humans. Finally, iPLA2-VIA mutants were shown to exhibit increased sensitivity to oxidative stress, progressive neurodegeneration and a severely reduced lifespan. Altogether, these data demonstrate that Drosophila iPLA2-VIA mutants provide a useful model to study human PLA2G6-associated neurodegeneration (Iliadi, 2018).

Thermosensory perception regulates speed of movement in response to temperature changes in Drosophila melanogaster

Temperature influences physiology and behavior of all organisms. For ectotherms, which lack central temperature regulation, temperature adaptation requires sheltering from or moving to a heat source. As temperature constrains the rate of metabolic reactions, it can directly affect ectotherm physiology and thus behavioral performance. This direct effect is particularly relevant for insects whose small body readily equilibrates with ambient temperature. In fact, models of enzyme kinetics applied to insect behavior predict performance at different temperatures, suggesting that thermal physiology governs behavior. However, insects also possess thermosensory neurons critical for locating preferred temperatures, showing cognitive control. This suggests that temperature-related behavior can emerge directly from a physiological effect, indirectly as consequence of thermosensory processing, or through both. To separate the roles of thermal physiology and cognitive control, this study developed an arena that allows fast temperature changes in time and space, and in which animals' movements are automatically quantified. Wild-type and thermosensory receptor mutants Drosophila melanogaster were exposed to a dynamic temperature environment, and their movements were tracked. The locomotor speed of wild-type flies closely matched models of enzyme kinetics, but the behavior of thermosensory mutants did not. Mutations in thermosensory receptor dTrpA1 (Transient receptor potential) expressed in the brain resulted in a complete lack of response to temperature changes, while mutation in peripheral thermosensory receptor Gr28b(D) resulted in diminished response. It is concluded that flies react to temperature through cognitive control, informed by interactions between various thermosensory neurons, whose behavioral output resembles that of enzyme kinetics (Soto-Padilla, 2018).

Optogenetic dissection of descending behavioral control in Drosophila

In most animals, the brain makes behavioral decisions that are transmitted by descending neurons to the nerve cord circuitry that produces behaviors. In insects, only a few descending neurons have been associated with specific behaviors. To explore how descending neurons control an insect's movements, this study developed a novel method to systematically assay the behavioral effects of activating individual neurons on freely behaving terrestrial D. melanogaster. A two-dimensional representation was calculated of the entire behavior space explored by these flies, and descending neurons with specific behaviors were associated by identifying regions of this space that were visited with increased frequency during optogenetic activation. Applying this approach across a large collection of descending neurons, it was found that (1) activation of most of the descending neurons drove stereotyped behaviors, (2) in many cases multiple descending neurons activated similar behaviors, and (3) optogenetically-activated behaviors were often dependent on the behavioral state prior to activation (Cande, 2018).

Neural control of startle-induced locomotion by the mushroom bodies and associated neurons in Drosophila

Startle-induced locomotion is commonly used in Drosophila research to monitor locomotor reactivity and its progressive decline with age or under various neuropathological conditions. A widely used paradigm is startle-induced negative geotaxis (SING), in which flies entrapped in a narrow column react to a gentle mechanical shock by climbing rapidly upwards. This study combined in vivo manipulation of neuronal activity and splitGFP reconstitution across cells to search for brain neurons and putative circuits that regulate this behavior. The activity of specific clusters of dopaminergic neurons (DANs) afferent to the mushroom bodies (MBs) modulates SING, and DAN-mediated SING regulation requires expression of the DA receptor Dop1R1/Dumb, but not Dop1R2/Damb, in intrinsic MB Kenyon cells (KCs). Previous observations were confirmed that activating the MB α'β', but not αβ, KCs decreased the SING response, and further MB neurons implicated in SING control were identified, including KCs of the γ lobe and two subtypes of MB output neurons (MBONs). Co-activating the αβ KCs antagonizes α'β' and γ KC-mediated SING modulation, suggesting the existence of subtle regulation mechanisms between the different MB lobes in locomotion control. Overall, this study contributes to an emerging picture of the brain circuits modulating locomotor reactivity in Drosophila that appear both to overlap and differ from those underlying associative learning and memory, sleep/wake state and stress-induced hyperactivity (Sun, 2018).

This study has identified MB afferent, intrinsic and efferent neurons that underlie modulation of startle-induced locomotion in the Drosophila brain. Using in vivo activation or silencing of synaptic transmission in neuronal subsets, specific compartments of the MBs were shown to be central to this modulation. Implicated neurons include α'β' and γ KCs, subsets of PAM and PPL1 DANs, and the MBONs V2 and M4/M6. Some of the potential synaptic connections between these elements were characterized using splitGFP reconstitution across cells. Although the picture is not complete, these results led to a proposal of a scheme of the neuronal circuits underlying the control of locomotor reactivity in an insect brain (Sun, 2018).

It has been previously reported that the degeneration of DANs afferent to the MBs in the PAM and PPL1 clusters is associated with accelerated decline of SING performance in aging flies. This study has specifically addressed the role of these and other DANs in SING modulation. The initial observation was that thermoactivation of TH-Gal4-targeted DANs consistently led to decreased locomotor reactivity, while silencing synaptic output from these neurons had no effect. This result was verified by rapid optogenetic photostimulation, indicating that indeed DAN activation affects locomotor reactivity during the execution of the behavior. In contrast, blocking selectively synaptic output of the PAM DANs neurons resulted in a slight increase in SING performance, suggesting that a subset of spontaneously active neurons in the PAM inhibits SING. It should be noted, however, that this effect appeared small probably in part because SING performance was already very high for the control flies in the assay condition. This issue may have prevented detection of other modulatory neurons in the course of this study. Interestingly, the data suggest that those PAM neurons that inhibit SING are targeted by NP6510-Gal4, a driver that expresses in 15 PAM DANs that project to the MB β1 and β'2 compartments. The degeneration of these neurons also appears to be largely responsible for α-synuclein-induced decline in SING performance in a Parkinson disease model. Moreover, one observation is provided in this study, using DAN co-activation with TH-Gal4 and R58E02-Gal4, suggesting that other subsets of the PAM cluster may modulate locomotor reactivity with opposite effects, i.e., increase SING when they are stimulated (Sun, 2018).

This study further indicated that thermoactivation of two DANs of the PPL1 cluster, either MB-MP1 that projects to the γ1 peduncle in the MB horizontal lobes or MB-V1 that projects to the α2 and α'2 compartments of the MB vertical lobes, was sufficient to significantly decrease SING performance. This suggests that the MB-afferent DANs of the PPL1 cluster are also implicated in SING modulation. Other DAN subsets could play a role and are still to be identified. However, inactivation of a DA receptor, Dop1R1/Dumb, in MB KCs precluded DAN-mediated SING modulation, strongly suggesting that DANs afferent to the MBs play a prominent role in the neuronal network controlling fly's locomotor reactivity. In contrast, inactivating Dop1R2/Dumb in KCs did not show any effect on DA-induced SING control (Sun, 2018).

Therefore, these results suggest that DA input to the MBs can inhibit or increase the reflexive locomotor response to a mechanical startle, allowing the animal to react to an instant, sudden stimulus. In accordance with this interpretation, previous reports have shown that the MB is not only a site for associative olfactory learning, but that it can also regulate innate behaviors. By combining synaptic imaging and electrophysiology, a previous study demonstrated that dopaminergic inputs to the MB intrinsic KCs play a central role in this function by exquisitely modulating the synapses that control MB output activity, thereby enabling the activation of different behavioral circuits according to contextual cues (Sun, 2018).

A decrease in SING performance has been previously reported when KCs in the α'β' lobes, but not in the αβ and γ lobes, were thermogenetically stimulated or their synaptic output silenced. Using a set of specific drivers, the contribution of the various MB lobes in the modulation of this innate reflex was precisely studied. It was confirmed that the α'β' KCs down-regulate SING when they are activated but not when their output is inhibited. Other unidentified neurons, targeted by the rather non-selective c305a-Gal4 and G0050-Gal4 drivers, trigger a decrease in SING performance when they are inhibited by Shits1, and are therefore potential SING-activating neurons. It was further found that the MB γ lobes contain KCs that strongly inhibit SING when activated, both by thermogenetic and optogenetic stimulation, as shown with the γ-lobe specific driver R16A06-Gal4. However, thermoactivation of γ neurons with other drivers, like mb247-Gal4, which express both in the αβ and γ lobe, did not decrease SING. This could result from an inhibitory effect of αβ neuron activation on SING modulation by γ neurons. To test this hypothesis, a double-driver was generated by recombining mb247-Gal4 with R16A06-Gal4. Because both drivers express in the γ lobes, one would expect a stronger effect on SING modulation after thermoactivation with the double-driver than with R16A06-Gal4 alone. The opposite was observed, i.e., that SING was decreased to a lesser extent with the double-driver than with R16A06-Gal4 alone. Activation of mb247-Gal4 αβ neurons therefore likely counterbalanced the effect of γ neuron activation with R16A06-Gal4 on SING modulation. A similar and even more obvious result was obtained when mb247-Gal4 was recombined with the α'β' driver R35B12-Gal4: co-activation of the neurons targeted by these two drivers prevented the strong SING modulation normally induced by R35B12-Gal4 alone. These results suggest the existence of an inter-compartmental communication process for locomotor reactivity control in the Drosophila MB. Comparably, it was recently suggested, in the case of memory retrieval, that MB output channels are ultimately pooled such that blockade (or activation) of all the outputs from a given population of KCs may have no apparent effect on odor-driven behavior, while such behavior can be changed by blocking a single output. Such a transfer of information could occur, as was previously reported, through connections involving the MBONs within the lobes or outside the MB (Sun, 2018).

Finally, the activation of two sets of MB efferent neurons, cholinergic MBON-V2 and glutamatergic MBON-M4/M6, consistently decreased SING performance of the flies. In contrast, silencing these neurons had no effect on locomotor behavior. The dendrites of these MBONs arborize in the medial part of the vertical lobes (α2, α'3) and the tips of the horizontal lobes (β'2 and γ5), respectively, as further evidence that the prime and γ lobes, and DANs efferent to these compartments, are involved in SING modulation. Results are also shown from GRASP observations suggesting that the PAM DANs either lay very close or in some other manner make potential synaptic connections with the MBON-M4/M6 neurons in their MB compartments, as well as the M4/M6 with the PAM in the SMP. The results also provide evidence that the PPL1 DANs and MBON-V2 contact each other in the vertical lobes and that axo-axonic synaptic contacts may occur between the MBON-V2 and M4/M6 neurons in their common projection region in the SMP (Sun, 2018).

These MBONs are known to be involved in opposite ways in olfactory memory: DAN-induced synaptic repression of cholinergic or glutamatergic MBONs would result in the expression of aversive or attractive memory, respectively. This study finds, in contrast, that the activation of these two sets of MBONs had similar depressing effects on SING behavior. Interestingly, it has been recently reported that the glutamatergic MBONs and PAM neurons that project to the MB β'2 compartment are also required for modulation of another innate reflex, CO2 avoidance (Lewis, 2015). CO2 exposure, like mechanical startle, represents a potential danger for the flies, thus triggering an avoidance behavior that can be suppressed by silencing these MBONs in specific environmental conditions. However, it is the activation of glutamatergic MBONs that inhibits SING. This apparent discrepancy might be explained if the downstream circuits were different for these two escape behaviors (CO2 avoidance and fast climbing). Overall, the current results further support the hypothesis of a primary role of the MB as a higher brain center for adapting innate sensory-driven reflex to a specific behavioral context (Cohn, 2015; Lewis, 2015; Sun, 2018 and references therein).

Even though the model remains to be confirmed and elaborated, a proposed scheme summarizing the current working hypothesis is presented of the neural components underlying SING control. Sensory information from mechanical stimulation triggers an innate climbing reflex (negative geotaxis) that can be regulated by signals transmitted from MB-afferent DANs (in the PAM and PPL1 cluster) to select KCs and two sets of MBONs (V2 and M4/M6) in specific MB compartments. Processing of this information could occur through synergistic or antagonistic interactions between the MB compartments and, finally, the MBON neurons would convey the resulting modulatory signal to downstream motor circuits controlling the climbing reflex. It was observed that the axonal projections of these MBONs make synaptic contacts with each other and converge together to the SMP where the dendrites of DANs lie, suggesting that these projections might form feedback loops to control DA signaling in the circuits (Sun, 2018).

DA signals for SING modulation originate from PAM neuron subsets and neurons inside the PPL1 cluster (MB-MP1 and MB-V1) that project to the MB lobes (see Schematic representation of MB-associated neural components modulating startle-induced locomotion). Axon of MB-V1 is shown as a dashed line because a driver specific for this neuron could not be tested in this study. The α'β' and γ KCs appear to be the main information integration center in this network, while their effect on SING modulation is opposed by the activity of αβ lobe KCs. Processed SING modulation signals are then transferred by two subtypes of MB efferent neurons, MBON-V2 and M4/M6, to the downstream SING reflex motor circuits. These two MBON subtypes have their axons converging together in the SMP where they may form axo-axonic synaptic connections, in which MBON-V2 would be presynaptic to MBON-M4/M6. The SMP also contains dendrites of the PAM and PPL1 DANs, thereby potentially forming instructive feedback loops on DA-mediated SING modulation. Most neurons identified here downregulated SING performance when they were activated, except for a subset of the PAM clusters that appeared constitutively inhibitory (represented as darker neurons in the figure) and the αβ lobe KCs that seem to antagonize SING modulation by other MB neurons. The different MB lobes are shown in various shades of green as indicated. The PAM DANs, PPL1 DANs and MBONs are drawn in magenta, light blue and dark gray, respectively. PAM: protocerebral anterior medial; PPL1: protocerebral posterior lateral; MBON: mushroom body output neuron; SMP superior medial protocerebrum; ped: peduncle; pre: presynaptic; pos: postsynaptic (Sun, 2018).

SING performance can be affected by a collection of factors including the arousal threshold of the fly, the ability to sense gravity and also climbing ability. 'Arousal' is defined as a state characterized by increased motor activity, sensitivity to sensory stimuli, and certain patterns of brain activity. A distinction can be made between endogenous arousal (i.e., wakefulness as opposed to sleep) and exogenous arousal (i.e., behavioral responsiveness). In Drosophila, DA level and signaling control all known forms of arousal. Because the MB plays an important role in sleep regulation, sleep- or wake-promoting networks might indeed in part interact or overlap with those controlling locomotor reactivity. However, this study observed that thermoactivation with various drivers had in a number of cases opposite effects on sleep/wake state and SING. First, neuronal thermoactivation with TH-Gal4 suppresses sleep but decreases the SING response. Second, extensive thermogenetic activation screen revealed that α′β′ and γm KCs are wake-promoting and γd KCs are sleep-promoting. In the current experiments, neuronal activation of α′β′ or γ KCs both led to strongly decreased locomotor reactivity. Third, stimulating MBON-M4 and M6, which are wake-promoting, decreased SING performance (Sun, 2018).

Another brain structure, the EB, plays important roles in the control of locomotor patterns and is also sleep-promoting. Furthermore, the EB is involved in the dopaminergic control of stress- or ethanol-induced hyperactivity, which can be considered as forms of exogenously-generated arousal. Several drivers labeling diverse EB neuronal layers were used, and no noticeable effects of thermoactivation of these neurons on the SING response was found. It is concluded that the circuits responsible for SING modulation, although they apparently share some similarities, are globally different from those controlling sleep/wake state and environmentally-induced hyperactivity (Sun, 2018).

Overall, this work identified elements of the neuronal networks controlling startle-induced locomotion in Drosophila and confirmed the central role of the MBs in this important function. Future studies are required to complete this scheme and explore the intriguing interactions between the different MB compartments in SING neuromodulation (Sun, 2018).

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