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
Tyramine action on motoneuron excitability and adaptable tyramine/octopamine ratios adjust Drosophila locomotion to nutritional state
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
Thermoresponsive motor behavior is mediated by ring neuron circuits in the central complex of Drosophila
Online learning for orientation estimation during translation in an insect ring attractor network
Under warm ambient conditions, Drosophila melanogaster suppresses nighttime activity via the neuropeptide pigment dispersing factor
Optogenetic dissection of descending behavioral control in Drosophila
Neural control of startle-induced locomotion by the mushroom bodies and associated neurons in Drosophila
Contribution of non-circadian neurons to the temporal organization of locomotor activity
State-dependent decoupling of sensory and motor circuits underlies behavioral flexibility in Drosophila
DeepFly3D, a deep learning-based approach for 3D limb and appendage tracking in tethered, adult Drosophila
Sex-specific among-individual covariation in locomotor activity and resting metabolic rate in Drosophila melanogaster
Deficits in the vesicular acetylcholine transporter alter lifespan and behavior in adult Drosophila melanogaster
Frequency-specific modification of locomotor components by the white gene in Drosophila melanogaster adult flies
Mating experience modifies locomotor performance and promotes episodic motor activity in Drosophila melanogaster
Reconstruction of motor control circuits in adult Drosophila using automated transmission electron microscopy
Allatostatin-C/AstC-R2 is a novel pathway to modulate the circadian activity pattern in Drosophila
Daily rewiring of a neural circuit generates a predictive model of environmental light
Developmental temperature affects thermal dependence of locomotor activity in Drosophila
A behavioral screen for mediators of age-dependent TDP-43 neurodegeneration identifies SF2/SRSF1 among a group of potent suppressors in both neurons and glia
Integration of sleep homeostasis and navigation in Drosophila

Larval and Pupal 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
A group of segmental premotor interneurons regulates the speed of axial locomotion in Drosophila larvae
Identification of excitatory premotor interneurons which regulate local muscle contraction during Drosophila larval locomotion
Sensorimotor pathway controlling stopping behavior during chemotaxis in the Drosophila melanogaster larva
Piezo-like gene regulates locomotion in Drosophila larvae
Regulation of forward and backward locomotion through intersegmental feedback circuits in Drosophila larvae
System level analysis of motor-related neural activities in larval Drosophila
Optimal searching behaviour generated intrinsically by the central pattern generator for locomotion
A multilayer circuit architecture for the generation of distinct locomotor behaviors in Drosophila
A Drosophila larval premotor/motor neuron connectome generating two behaviors via distinct spatio-temporal muscle activity
Characterization of proprioceptive system dynamics in behaving Drosophila larvae using high-speed volumetric microscopy
Circadian and Genetic Modulation of Visually-Guided Navigation in Drosophila Larvae
Interspecies variation of larval locomotion kinematics in the genus Drosophila and its relation to habitat temperature
Pupal behavior emerges from unstructured muscle activity in response to neuromodulation in Drosophila
Nitric oxide mediates activity-dependent change to synaptic excitation during a critical period in Drosophila
Localization of muscarinic acetylcholine receptor-dependent rhythm-generating modules in the Drosophila larval locomotor network

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
Two pairs of Drosophila central brain neurons mediate larval navigational strategies based on temporal light information processing
Modular assays for the quantitative study of visually guided navigation in both flying and walking flies
Fly eyes are not still: a motion illusion in Drosophila flight supports parallel visual processing
Stabilizing responses to sideslip disturbances in Drosophila melanogaster are modulated by the density of moving elements on the ground
Aerial course stabilization is impaired in motion-blind flies
Mechanisms of punctuated vision in fly flight

Free flight odor tracking in Drosophila: Effect of wing chemosensors, sex and pheromonal gene regulation
Crossmodal visual input for odor tracking during fly flight
Context-dependent representations of movement in Drosophila dopaminergic reinforcement pathways
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
Sensing complementary temporal features of odor signals enhances navigation of diverse turbulent plumes
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
FMRFa receptor stimulated Ca2+ signals alter the activity of flight modulating central dopaminergic neurons in Drosophila melanogaster
A balance between aerodynamic and olfactory performance during flight in Drosophila
Extended flight bouts require disinhibition from GABAergic mushroom body neurons
Exploring thermal flight responses as predictors of flight ability and geographic range size in Drosophila
Flies regulate wing motion via active control of a dual-Function gyroscope
Heading choices of flying Drosophila under changing angles of polarized light
A Multi-regional Network Encoding Heading and Steering Maneuvers in Drosophila
Small eyes in dim light: Implications to spatio-temporal visual abilities in Drosophila melanogaster
Flight activity and glycogen depletion on a low-carbohydrate diet
Unc-4 acts to promote neuronal identity and development of the take-off circuit in the Drosophila CNS
A Neural Network for Wind-Guided Compass Navigation Active vision shapes and coordinates flight motor responses in flies Haltere and visual inputs sum linearly to predict wing (but not gaze) motor output in tethered flying Drosophila Haltere and visual inputs sum linearly to predict wing (but not gaze) motor output in tethered flying Drosophila Oxygen Dependence of Flight Performance in Ageing Drosophila melanogaster The long-distance flight behavior of Drosophila supports an agent-based model for wind-assisted dispersal in insects Natural variation in the regulation of neurodevelopmental genes modifies flight performance in Drosophila Thermal and Oxygen Flight Sensitivity in Ageing Drosophila melanogaster Flies: Links to Rapamycin-Induced Cell Size Changes Suppression of motion vision during course-changing, but not course-stabilizing, navigational turns

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
Static stability predicts the continuum of interleg coordination patterns in Drosophila
Visual control of walking speed in Drosophila
Speed dependent descending control of freezing behavior in Drosophila melanogaster
Neural coding of leg proprioception in Drosophila
Fast near-whole-brain imaging in adult Drosophila during responses to stimuli and behavior
The manifold structure of limb coordination in walking Drosophila
Random walk revisited: Quantification and comparative analysis of Drosophila walking trajectories
TwoLumps ascending neurons mediate touch-evoked reversal of walking direction in Drosophila
Serotonergic Modulation of Walking in Drosophila
Adult Movement Defects Associated with a CORL Mutation in Drosophila Display Behavioral Plasticity
Spatiotemporally precise optogenetic activation of sensory neurons in freely walking Drosophila
Decentralized control of insect walking: A simple neural network explains a wide range of behavioral and neurophysiological results
Mechanisms underlying attraction to odors in walking Drosophila
A size principle for recruitment of Drosophila leg motor neurons
Neural coding of leg proprioception in Drosophila
Variation in the response to exercise stimulation in Drosophila: marathon runner versus sprinter genotypes
Functional advantages of Levy walks emerging near a critical point
Two Brain Pathways Initiate Distinct Forward Walking Programs in Drosophila
Walking Drosophila navigate complex plumes using stochastic decisions biased by the timing of odor encounters
A pair of ascending neurons in the subesophageal zone mediates aversive sensory inputs-evoked backward locomotion in Drosophila larvae
Distributed control of motor circuits for backward walking in Drosophila
Drosophila uses a tripod gait across all walking speeds, and the geometry of the tripod is important for speed control
Navigation of a Freely Walking Fruit Fly in Infinite Space Using a Transparent Omnidirectional Locomotion Compensator (TOLC)
The Panopticon-Assessing the Effect of Starvation on Prolonged Fly Activity and Place Preference
Identification of FoxP circuits involved in locomotion and object fixation in Drosophila
Fast tuning of posture control by visual feedback underlies gaze stabilization in walking Drosophila
Behavioral signatures of structured feature detection during courtship in Drosophila
Building an allocentric travelling direction signal via vector computation

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).

Context-dependent representations of movement in Drosophila dopaminergic reinforcement pathways

Dopamine plays a central role in motivating and modifying behavior, serving to invigorate current behavioral performance and guide future actions through learning. This study examined how this single neuromodulator can contribute to such diverse forms of behavioral modulation. By recording from the dopaminergic reinforcement pathways of the Drosophila mushroom body during active odor navigation, this study reveals how their ongoing motor-associated activity relates to goal-directed behavior. Dopaminergic neurons were found to correlate with different behavioral variables depending on the specific navigational strategy of an animal, such that the activity of these neurons preferentially reflects the actions most relevant to odor pursuit. Furthermore, this study shows that these motor correlates are translated to ongoing dopamine release, and acutely perturbing dopaminergic signaling alters the strength of odor tracking. Context-dependent representations of movement and reinforcement cues are thus multiplexed within the mushroom body dopaminergic pathways, enabling them to coordinately influence both ongoing and future behavior (Zolin, 2021).

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).

Sensing complementary temporal features of odor signals enhances navigation of diverse turbulent plumes

It has been shown that during odor plume navigation, walking Drosophila melanogaster bias their motion upwind in response to both the frequency of their encounters with the odor, and the intermittency of the odor signal, which this study defines to be the fraction of time the signal is above a detection threshold. This study combined and simplified previous mathematical models that recapitulated these data to investigate the benefits of sensing both of these temporal features, and how these benefits depend on the spatiotemporal statistics of the odor plume. Through agent-based simulations, this study found that navigators that only use frequency or intermittency perform well in some environments - achieving maximal performance when gains are near those inferred from experiment - but fail in others. Robust performance across diverse environments requires both temporal modalities. However, a steep tradeoff was found when using both sensors simultaneously, suggesting a strong benefit to modulating how much each sensor is weighted, rather than using both in a fixed combination across plumes. Finally, it was shown that the circuitry of the Drosophila olfactory periphery naturally enables simultaneous intermittency and frequency sensing, enhancing robust navigation through a diversity of odor environments. Together, these results suggest that the first stage of olfactory processing selects and encodes temporal features of odor signals critical to real-world navigation tasks (Jayaram, 2022).

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).

FMRFa receptor stimulated Ca2+ signals alter the activity of flight modulating central dopaminergic neurons in Drosophila melanogaster

Neuropeptide signaling influences animal behavior by modulating neuronal activity and thus altering circuit dynamics. Insect flight is a key innate behavior that very likely requires robust neuromodulation. Cellular and molecular components that help modulate flight behavior are therefore of interest and require investigation. In a genetic RNAi screen for G-protein coupled receptors that regulate flight bout durations, several receptors have been identified, including the receptor for the neuropeptide FMRFa (FMRFaR). To further investigate modulation of insect flight by FMRFa CRISPR-Cas9 mutants were generated in the gene encoding the Drosophila FMRFaR. The mutants exhibit significant flight deficits with a focus in dopaminergic cells. Expression of a receptor specific RNAi in adult central dopaminergic neurons resulted in progressive loss of sustained flight. Further, genetic and cellular assays demonstrated that FMRFaR stimulates intracellular calcium signaling through the IP3R and helps maintain neuronal excitability in a subset of dopaminergic neurons for positive modulation of flight bout durations (Ravi, 2018).

A balance between aerodynamic and olfactory performance during flight in Drosophila

The ability to track odor plumes to their source (food, mate, etc.) is key to the survival of many insects. During this odor-guided navigation, flapping wings could actively draw odorants to the antennae to enhance olfactory sensitivity, but it is unclear if improving olfactory function comes at a cost to aerodynamic performance. This study computationally quantified the odor plume features around a fruit fly in forward flight and confirmed that the antenna is well positioned to receive a significant increase of odor mass flux (peak 1.8 times), induced by wing flapping, vertically from below the body but not horizontally. This anisotropic odor spatial sampling may have important implications for behavior and the algorithm during plume tracking. Further analysis also suggests that, because both aerodynamic and olfactory functions are indispensable during odor-guided navigation, the wing shape and size may be a balance between the two functions (Li, 2018).

Extended flight bouts require disinhibition from GABAergic mushroom body neurons

Insect flight is a complex behavior that requires the integration of multiple sensory inputs with flight motor output. Although previous genetic studies identified central brain monoaminergic neurons that modulate Drosophila flight, neuro-modulatory circuits underlying sustained flight bouts remain unexplored. Certain classes of dopaminergic and octopaminergic neurons that project to the mushroom body, a higher integrating center in the insect brain, are known to modify neuronal output based on contextual cues and thereby organismal behavior. This study focuses on how monoaminergic modulation of mushroom body GABAergic output neurons (MBONs) regulates the duration of flight bouts. Octopaminergic neurons in the sub-esophageal zone stimulate central dopaminergic neurons (protocerebral anterior medial, PAM) that project to GABAergic MBONs. Either inhibition of octopaminergic and dopaminergic neurons or activation of GABAergic MBONs reduces the duration of flight bouts. Moreover, activity in the PAM neurons inhibits the GABAergic MBONs. These data suggest that disinhibition of the identified neural circuit very likely occurs after flight initiation and is required to maintain the "flight state" when searching for distant sites, possibly related to food sources, mating partners, or a suitable egg-laying site (Manjila, 2018).

Exploring thermal flight responses as predictors of flight ability and geographic range size in Drosophila

Thermal flight performance curves (TFPCs) may be a useful proxy for determining dispersal on daily timescales in winged insect species. To better understand how flight performance may be correlated with geographic range extent and potential latitudinal climate variability, the thermal performance curves of flight ability was estimated in 11 Drosophilidae species (in 4 degrees C increments across 16-28 degrees C) after standard laboratory rearing for two generations. Whether key morphological, evolutionary or ecological factors (e.g. species identity, sex, body mass, wing loading, geographic range size) predicted traits of TFPCs (including optimum temperature, maximum performance, thermal breadth of performance) or flight ability (success/failure to fly) was tested. Although several parameters of TFPCs varied among species, these were typically not statistically significant probably owing to the relatively small pool of species assessed and the limited trait variation detected. The best explanatory model of these flight responses across species included significant positive effects of test temperature and wing area. However, the rank of geographic distribution breadth and phylogeny failed to explain significant variation in most of the traits, except for thermal performance breadth, of thermal flight performance curves among these 11 species. Future studies that employ a wider range of Drosophilidae species, especially if coupled with fine-scale estimates of species' environmental niches, would be useful (De Araujo, 2019).

Flies regulate wing motion via active control of a dual-function gyroscope

Flies execute their remarkable aerial maneuvers using a set of wing steering muscles, which are activated at specific phases of the stroke cycle. The activation phase of these muscles-which determines their biomechanical output - arises via feedback from mechanoreceptors at the base of the wings and structures unique to flies called halteres. Evolved from the hindwings, the tiny halteres oscillate at the same frequency as the wings, although they serve no aerodynamic function and are thought to act as gyroscopes. Like the wings, halteres possess minute control muscles whose activity is modified by descending visual input, raising the possibility that flies control wing motion by adjusting the motor output of their halteres, although this hypothesis has never been directly tested. Using genetic techniques possible in Drosophila melanogaster, this study tested the hypothesis that visual input during flight modulates haltere muscle activity and that this, in turn, alters the mechanosensory feedback that regulates the wing steering muscles. The results suggest that rather than acting solely as a gyroscope to detect body rotation, halteres also function as an adjustable clock to set the spike timing of wing motor neurons, a specialized capability that evolved from the generic flight circuitry of their four-winged ancestors. In addition to demonstrating how the efferent control loop of a sensory structure regulates wing motion, these results provide insight into the selective scenario that gave rise to the evolution of halteres (Dickerson, 2019).

Heading choices of flying Drosophila under changing angles of polarized light

Many navigating insects include the celestial polarization pattern as an additional visual cue to orient their travels. Spontaneous orientation responses of both walking and flying fruit flies (Drosophila melanogaster) to linearly polarized light have previously been demonstrated. Using newly designed modular flight arenas consisting entirely of off-the-shelf parts and 3D-printed components this study presented individual flying flies with a slow and continuous rotational change in the incident angle of linear polarization. Under such open-loop conditions, single flies choose arbitrary headings with respect to the angle of polarized light and show a clear tendency to maintain those chosen headings for several minutes, thereby adjusting their course to the slow rotation of the incident stimulus. Importantly, flies show the tendency to maintain a chosen heading even when two individual test periods under a linearly polarized stimulus are interrupted by an epoch of unpolarized light lasting several minutes. Finally, it was shown that these behavioral responses are wavelength-specific, existing under polarized UV stimulus while being absent under polarized green light. Taken together, these findings provide further evidence supporting Drosophila's abilities to use celestial cues for visually guided navigation and course correction (Mathejczyk, 2019).

A Multi-regional Network Encoding Heading and Steering Maneuvers in Drosophila

An internal sense of heading direction is computed from various cues, including steering maneuvers of the animal. Although neurons encoding heading and steering have been found in multiple brain regions, it is unclear whether and how they are organized into neural circuits. This study shows that, in flying Drosophila, heading and turning behaviors are encoded by population dynamics of specific cell types connecting the subregions of the central complex (CX), a brain structure implicated in navigation. Columnar neurons in the fan-shaped body (FB) of the CX exhibit circular dynamics that multiplex information about turning behavior and heading. These dynamics are coordinated with those in the ellipsoid body, another CX subregion containing a heading representation, although only FB neurons flip turn preference depending on the visual environment. Thus, the navigational system spans multiple subregions of the CX, where specific cell types show coordinated but distinct context-dependent dynamics (Shiozaki, 2020).

Small eyes in dim light: Implications to spatio-temporal visual abilities in Drosophila melanogaster

Fruit flies, Drosophila melanogaster, are active over a range of light intensities in the wild, but lab-reared flies are often tested only in bright light. Similarly, scarce feeding during larval stages-common in nature-generates smaller adults, and a wide range of eye sizes not found in well-fed lab colonies. Both dimmer light and smaller eyes limit light capture and have undetermined effects on visual behaviors such as flight. This study used moving sinusoidal gratings to test spatial acuity, temporal acuity, and contrast threshold of female flies of varying eye sizes at different light intensities. Vision was also tested in the smaller and often neglected male fruit flies. As light intensity drops from 50.1 lx to 0.3 lx, flies have a reduced spatial acuity (females: from 0.1 to 0.06 cycles per degree, CPD, males: 0.1 to 0.04 CPD) and temporal acuity (females: from 50 Hz to 10 Hz, males: 25 Hz to 10 Hz), and an increased contrast detection threshold (females: from 10% to 29%, males: 19% to 48%). No major sex-specific differences were found after accounting for eye size. Visual abilities in both small (eye area of 0.1-0.17 mm(2)) and large flies (0.17-0.23 mm(2)) suffer at 0.3 lx compared to 50.1 lx, but small flies suffer more (spatial acuity: 0.03 vs 0.06 CPD, contrast threshold: 76% vs 57%, temporal acuity: 5 Hz vs 10 Hz). These results suggest visual abilities of small flies suffer more than large flies at low light levels, possibly leading to size- and light intensity-dependent effects on foraging, navigation, and flight (Palavalli-Nettimi, 2020).

Flight activity and glycogen depletion on a low-carbohydrate diet

Glycogen is a critical store for locomotion. Depleted glycogen stores are associated with increased fatigue during exercise. The reduced effectiveness of low-carbohydrate diets for weight loss over longer time periods may arise because such diets reduce glycogen stores and thereby physical activity energy expenditure. To explore the effect of a low-carbohydrate diet on activity and glycogen utilisation, adult Drosophila melanogaster were fed a standard or low-carbohydrate diet for nine days, and patterns of flight activity and rates of glycogen depletion were measured. It was hypothesised that flight activity and rates of glycogen depletion would be reduced on a low-carbohydrate diet. Flight activity was elevated in the low-carbohydrate group but glycogen depletion rates were unchanged. It is concluded that increased activity is likely a foraging response to carbohydrate deficiency and it is speculated that the previously demonstrated metabolic depression that occurs on a low-carbohydrate diet in this species may allow for increased flight activity without increased glycogen depletion (Winwood-Smith, 2020).

Unc-4 acts to promote neuronal identity and development of the take-off circuit in the Drosophila CNS

The Drosophila ventral nerve cord (VNC) is composed of thousands of neurons born from a set of individually identifiable stem cells. The VNC harbors neuronal circuits required to execute key behaviors, such as flying and walking. Leveraging the lineage-based functional organization of the VNC, this study investigated the developmental and molecular basis of behavior by focusing on lineage-specific functions of the homeodomain transcription factor, Unc-4. Unc-4 was found to function in lineage 11A to promote cholinergic neurotransmitter identity and suppress the GABA fate. In lineage 7B, Unc-4 promotes proper neuronal projections to the leg neuropil and a specific flight-related take-off behavior. It was also uncovered that Unc-4 acts peripherally to promote proprioceptive sensory organ development and the execution of specific leg-related behaviors. Through time-dependent conditional knock-out of Unc-4, it was found that its function is required during development, but not in the adult, to regulate the above events (Lacin, 2020).hn 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 Neural Network for Wind-Guided Compass Navigation

Spatial maps in the brain are most accurate when they are linked to external sensory cues. This study shows that the compass in the Drosophila brain is linked to the direction of the wind. Shifting the wind rightward rotates the compass as if the fly were turning leftward, and vice versa. The mechanisms are described of several computations that integrate wind information into the compass. First, an intensity-invariant representation of wind direction is computed by comparing left-right mechanosensory signals. Then, signals are reformatted to reduce the coding biases inherent in peripheral mechanics, and wind cues are brought into the same circular coordinate system that represents visual cues and self-motion signals. Because the compass incorporates both mechanosensory and visual cues, it should enable navigation under conditions where no single cue is consistently reliable. These results show how local sensory signals can be transformed into a global, multimodal, abstract representation of space (Okubo, 2020).

Active vision shapes and coordinates flight motor responses in flies

Animals use active sensing to respond to sensory inputs and guide future motor decisions. In flight, flies generate a pattern of head and body movements to stabilize gaze. How the brain relays visual information to control head and body movements and how active head movements influence downstream motor control remains elusive. Using a control theoretic framework, the optomotor gaze stabilization reflex was studied in tethered flight and quantified how head movements stabilize visual motion and shape wing steering efforts in fruit flies (Drosophila). By shaping visual inputs, head movements increased the gain of wing steering responses and coordination between stimulus and wings, pointing to a tight coupling between head and wing movements. Head movements followed the visual stimulus in as little as 10 ms-a delay similar to the human vestibulo-ocular reflex-whereas wing steering responses lagged by more than 40 ms. This timing difference suggests a temporal order in the flow of visual information such that the head filters visual information eliciting downstream wing steering responses. Head fixation significantly decreased the mechanical power generated by the flight motor by reducing wingbeat frequency and overall thrust. By simulating an elementary motion detector array, this study showd that head movements shift the effective visual input dynamic range onto the sensitivity optimum of the motion vision pathway. Taken together, these results reveal a transformative influence of active vision on flight motor responses in flies. This work provides a framework for understanding how to coordinate moving sensors on a moving body (Cellini, 2020).

Haltere and visual inputs sum linearly to predict wing (but not gaze) motor output in tethered flying Drosophila

In the true flies (Diptera), the hind wings have evolved into specialized mechanosensory organs known as halteres, which are sensitive to gyroscopic and other inertial forces. Together with the fly's visual system, the halteres direct head and wing movements through a suite of equilibrium reflexes that are crucial to the fly's ability to maintain stable flight. As in other animals (including humans), this presents challenges to the nervous system as equilibrium reflexes driven by the inertial sensory system must be integrated with those driven by the visual system in order to control an overlapping pool of motor outputs shared between the two of them. This study introduced an experimental paradigm for reproducibly altering haltere stroke kinematics and used it to quantify multisensory integration of wing and gaze equilibrium reflexes. Multisensory wing-steering responses reflect a linear superposition of haltere-driven and visually driven responses, but multisensory gaze responses are not well predicted by this framework. These models, based on populations, extend also to the responses of individual flies (Rauscher, 2021).

Odour boosts visual object approach in flies

Multisensory integration is synergistic-input from one sensory modality might modulate the behavioural response to another. Work in flies has shown that a small visual object presented in the periphery elicits innate aversive steering responses in flight, likely representing an approaching threat. Object aversion is switched to approach when paired with a plume of food odour. The 'open-loop' design of prior work facilitated the observation of changing valence. How does odour influence visual object responses when an animal has naturally active control over its visual experience? This study used closed-loop feedback conditions, in which a fly's steering effort is coupled to the angular velocity of the visual stimulus, to confirm that flies steer toward or 'fixate' a long vertical stripe on the visual midline. They tend either to steer away from or 'antifixate' a small object or to disengage active visual control, which manifests as uncontrolled object 'spinning' within this experimental paradigm. Adding a plume of apple cider vinegar decreases the probability of both antifixation and spinning, while increasing the probability of frontal fixation for objects of any size, including a normally typically aversive small object (Cheng, 2021).

Oxygen Dependence of Flight Performance in Ageing Drosophila melanogaster

Similar to humans, insects lose their physical and physiological capacities with age, which makes them a convenient study system for human ageing. Although insects have an efficient oxygen-transport system, little is known about how their flight capacity changes with age and environmental oxygen conditions. Two types of locomotor performance in ageing Drosophila melanogaster flies: the frequency of wing beats and the capacity to climb vertical surfaces. Flight performance was measured under normoxia and hypoxia. As anticipated, ageing flies showed systematic deterioration of climbing performance, and low oxygen impeded flight performance. Against predictions, flight performance did not deteriorate with age, and younger and older flies showed similar levels of tolerance to low oxygen during flight. It is suggested that among different insect locomotory activities, flight performance deteriorates slowly with age, which is surprising, given that insect flight is one of the most energy-demanding activities in animals. Apparently, the superior capacity of insects to rapidly deliver oxygen to flight muscles remains little altered by ageing, but this study showed that insects can become oxygen limited in habitats with a poor oxygen supply (e.g., those at high elevations) during highly oxygen-demanding activities such as flight (Privalova, 2021).

The long-distance flight behavior of Drosophila supports an agent-based model for wind-assisted dispersal in insects

Despite the ecological importance of long-distance dispersal in insects, its mechanistic basis is poorly understood in genetic model species, in which advanced molecular tools are readily available. One critical question is how insects interact with the wind to detect attractive odor plumes and increase their travel distance as they disperse. To gain insight into dispersal, release-and-recapture experiments were conducted in the Mojave Desert using the fruit fly, Drosophila melanogaster. Chemically baited traps were deployed in a 1 km radius ring around the release site, equipped with cameras that captured the arrival times of flies as they landed. In each experiment, between 30,000 and 200,000 flies were released. By repeating the experiments under a variety of conditions, it was possible to quantify the influence of wind on flies' dispersal behavior. The results confirm that even tiny fruit flies could disperse ∼12 km in a single flight in still air and might travel many times that distance in a moderate wind. The dispersal behavior of the flies is well explained by an agent-based model in which animals maintain a fixed body orientation relative to celestial cues, actively regulate groundspeed along their body axis, and allow the wind to advect them sideways. The model accounts for the observation that flies actively fan out in all directions in still air but are increasingly advected downwind as winds intensify. The results suggest that dispersing insects may strike a balance between the need to cover large distances while still maintaining the chance of intercepting odor plumes from upwind sources (Leitch, 2021).

Natural variation in the regulation of neurodevelopmental genes modifies flight performance in Drosophila

The winged insects of the order Diptera are colloquially named for their most recognizable phenotype: flight. These insects rely on flight for a number of important life history traits, such as dispersal, foraging, and courtship. Despite the importance of flight, relatively little is known about the genetic architecture of flight performance. Accordingly, this study sought to uncover the genetic modifiers of flight using a measure of flies' reaction and response to an abrupt drop in a vertical flight column. A genome wide association study (GWAS) was conducted using 197 of the Drosophila Genetic Reference Panel (DGRP) lines, and a combination was identified of additive and marginal variants, epistatic interactions, whole genes, and enrichment across interaction networks. Egfr, a highly pleiotropic developmental gene, was among the most significant additive variants identified. 13 of the additive candidate genes (Adgf-A/Adgf-A2/CG32181, bru1, CadN, flapper (CG11073), CG15236, flippy (CG9766), CREG, Dscam4, form3, fry, Lasp/CG9692, Pde6, Snoo) were functionally validated, and a novel approach was introduced to whole gene significance screens: PEGASUS_flies. Additionally, ppk23, an Acid Sensing Ion Channel (ASIC) homolog, was identified as an important hub for epistatic interactions. A model is proposed that suggests genetic modifiers of wing and muscle morphology, nervous system development and function, BMP signaling, sexually dimorphic neural wiring, and gene regulation are all important for the observed differences flight performance in a natural population. Additionally, these results represent a snapshot of the genetic modifiers affecting drop-response flight performance in Drosophila, with implications for other insects (Spierer, 2021).

Thermal and Oxygen Flight Sensitivity in Ageing Drosophila melanogaster Flies: Links to Rapamycin-Induced Cell Size Changes

Ectotherms can become physiologically challenged when performing oxygen-demanding activities (e.g., flight) across differing environmental conditions, specifically temperature and oxygen levels. Achieving a balance between oxygen supply and demand can also depend on the cellular composition of organs, which either evolves or changes plastically in nature; however, this hypothesis has rarely been examined, especially in tracheated flying insects. The relatively large cell membrane area of small cells should increase the rates of oxygen and nutrient fluxes in cells; however, it does also increase the costs of cell membrane maintenance. To address the effects of cell size on flying insects, the wing-beat frequency was measured in two cell-size phenotypes of Drosophila melanogaster when flies were exposed to two temperatures (warm/hot) combined with two oxygen conditions (normoxia/hypoxia). The cell-size phenotypes were induced by rearing 15 isolines on either standard food (large cells) or rapamycin-enriched food (small cells). Rapamycin supplementation (downregulation of TOR activity) produced smaller flies with smaller wing epidermal cells. Flies generally flapped their wings at a slower rate in cooler (warm treatment) and less-oxygenated (hypoxia) conditions, but the small-cell-phenotype flies were less prone to oxygen limitation than the large-cell-phenotype flies and did not respond to the different oxygen conditions under the warm treatment. It is suggested that ectotherms with small-cell life strategies can maintain physiologically demanding activities (e.g., flight) when challenged by oxygen-poor conditions, but this advantage may depend on the correspondence among body temperatures, acclimation temperatures and physiological thermal limits (Szlachcic, 2021).

Suppression of motion vision during course-changing, but not course-stabilizing, navigational turns

From mammals to insects, locomotion has been shown to strongly modulate visual-system physiology. Does the manner in which a locomotor act is initiated change the modulation observed? Patch-clamp recordings were performed from motion-sensitive visual neurons in tethered, flying Drosophila. This study observed motor-related signals in flies performing flight turns in rapid response to looming discs and also during spontaneous turns, but motor-related signals were weak or non-existent in the context of turns made in response to brief pulses of unidirectional visual motion (i.e., optomotor responses). Thus, the act of a locomotor turn is variably associated with modulation of visual processing. These results can be understood via the following principle: suppress visual responses during course-changing, but not course-stabilizing, navigational turns. This principle is likely to apply broadly-even to mammals-whenever visual cells whose activity helps to stabilize a locomotor trajectory or the visual gaze angle are targeted for motor modulation (Fenk, 2021).

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).

Static stability predicts the continuum of interleg coordination patterns in Drosophila

During walking, insects must coordinate the movements of their six legs for efficient locomotion. This interleg coordination is speed-dependent; fast walking in insects is associated with tripod coordination patterns, while slow walking is associated with more variable, tetrapod-like patterns. To date, however, there has been no comprehensive explanation as to why these speed-dependent shifts in interleg coordination should occur in insects. Tripod coordination would be sufficient at low walking speeds. The fact that insects use a different interleg coordination pattern at lower speeds suggests that it is more optimal or advantageous at these speeds. Furthermore, previous studies focused on discrete tripod and tetrapod coordination patterns. Experimental data, however, suggest that changes observed in interleg coordination are part of a speed-dependent spectrum. This study explored these issues in relation to static stability as an important aspect for interleg coordination in Drosophila. A model was created that uses basic experimentally measured parameters in fruit flies to find the interleg phase relationships that maximize stability for a given walking speed. The model predicted a continuum of interleg coordination patterns spanning the complete range of walking speeds as well as an anteriorly directed swing phase progression. Furthermore, for low walking speeds the model predicted tetrapod-like patterns to be most stable, while at high walking speeds tripod coordination emerged as most optimal. Finally, the basic assumption of a continuum of interleg coordination patterns was validated in a large set of experimental data from walking fruit flies and these data were compared with the model-based predictions (Szczecinski, 2018).

Visual control of walking speed in Drosophila
An animal's self-motion generates optic flow across its retina, and it can use this visual signal to regulate its orientation and speed through the world. While orientation control has been studied extensively in Drosophila and other insects, much less is known about the visual cues and circuits that regulate translational speed. This study shows that flies regulate walking speed with an algorithm that is tuned to the speed of visual motion, causing them to slow when visual objects are nearby. This regulation does not depend strongly on the spatial structure or the direction of visual stimuli, making it algorithmically distinct from the classic computation that controls orientation. Despite the different algorithms, the visual circuits that regulate walking speed overlap with those that regulate orientation. Taken together, these findings suggest that walking speed is controlled by a hierarchical computation that combines multiple motion detectors with distinct tunings (Creamer, 2018).

Speed dependent descending control of freezing behavior in Drosophila melanogaster

The most fundamental choice an animal has to make when it detects a threat is whether to freeze, reducing its chances of being noticed, or to flee to safety. This study shows that Drosophila melanogaster exposed to looming stimuli in a confined arena either freeze or flee. The probability of freezing versus fleeing is modulated by the fly's walking speed at the time of threat, demonstrating that freeze/flee decisions depend on behavioral state. A pair of descending neurons crucially implicated in freezing is described. Genetic silencing of DNp09 descending neurons disrupts freezing yet does not prevent fleeing. Optogenetic activation of both DNp09 neurons induces running and freezing in a state-dependent manner. These findings establish walking speed as a key factor in defensive response choices and reveal a pair of descending neurons as a critical component in the circuitry mediating selection and execution of freezing or fleeing behaviors (Zacarias, 2018).

Neural coding of leg proprioception in Drosophila

Animals rely on an internal sense of body position and movement to effectively control motor behavior. This sense of proprioception is mediated by diverse populations of mechanosensory neurons distributed throughout the body. This study investigated neural coding of leg proprioception in Drosophila, using in vivo two-photon calcium imaging of proprioceptive sensory neurons during controlled movements of the fly tibia. The axons of leg proprioceptors are organized into distinct functional projections that contain topographic representations of specific kinematic features. Using subclass-specific genetic driver lines, this study shows that one group of axons encodes tibia position (flexion/extension), another encodes movement direction, and a third encodes bidirectional movement and vibration frequency. Overall, these findings reveal how proprioceptive stimuli from a single leg joint are encoded by a diverse population of sensory neurons, and provide a framework for understanding how proprioceptive feedback signals are used by motor circuits to coordinate the body (Mamiya, 2018).

Variation in the response to exercise stimulation in Drosophila: marathon runner versus sprinter genotypes

Animals' behaviors vary in response to their environment, both biotic and abiotic. These behavioral responses have significant impacts on animal survival and fitness, and thus, many behavioral responses are at least partially under genetic control. In Drosophila for example, genes impacting aggression, courtship behavior, circadian rhythms, and sleep have been identified. Animal activity also is influenced strongly by genetics. Previous work used the Drosophila melanogaster Genetics Reference Panel (DGRP) to investigate activity levels and identified over 100 genes linked to activity. This study re-examine these data to determine if Drosophila strains differ in their response to rotational exercise stimulation, not simply in the amount of activity, but in activity patterns and timing of activity. Specifically, it was asked if there are fly strains exhibiting either a "marathoner" pattern of activity, i.e. remaining active throughout the two-hour exercise period, or a "sprinter" pattern, i.e. carrying out most of the activity early in the exercise period. The DGRP strains examined differ significantly in how much activity is carried out at the beginning of the exercise period, and this pattern is influenced by both sex and genotype. Interestingly, there is no clear link between the activity response pattern and lifespan of the animals. Using GWASs, ten high confidence candidate genes were identified that control to which degree Drosophila exercise behaviors fit a marathoner or sprinter activity pattern. This finding suggests that, similar to other aspects of locomotor behavior, timing of activity patterns in response to exercise stimulation is under genetic control (Riddle, 2020).

Functional advantages of Levy walks emerging near a critical point

A special class of random walks, so-called Lévy walks, has been observed in a variety of organisms ranging from cells, insects, fishes, and birds to mammals, including humans. Although their prevalence is considered to be a consequence of natural selection for higher search efficiency, some findings suggest that Lévy walks might also be epiphenomena that arise from interactions with the environment. Therefore, why they are common in biological movements remains an open question. Based on some evidence that Lévy walks are spontaneously generated in the brain and the fact that power-law distributions in Lévy walks can emerge at a critical point, it was hypothesized that the advantages of Lévy walks might be enhanced by criticality. However, the functional advantages of Lévy walks are poorly understood. Here, we modeled nonlinear systems for the generation of locomotion and showed that Lévy walks emerging near a critical point had optimal dynamic ranges for coding information. This discovery suggested that Lévy walks could change movement trajectories based on the magnitude of environmental stimuli. This study then showed that the high flexibility of Lévy walks enabled switching exploitation/exploration based on the nature of external cues. Finally, this study analyzed the movement trajectories of freely moving Drosophila larvae and showed empirically that the Lévy walks may emerge near a critical point and have large dynamic range and high flexibility. The results suggest that the commonly observed Lévy walks emerge near a critical point and could be explained on the basis of these functional advantages (Abe, 2020).

Two Brain Pathways Initiate Distinct Forward Walking Programs in Drosophila

An animal at rest or engaged in stationary behaviors can instantaneously initiate goal-directed walking. How descending brain inputs trigger rapid transitions from a non-walking state to an appropriate walking state is unclear. This study identify two neuronal types, P9 and BPN, in the Drosophila brain that, upon activation, initiate and maintain two distinct coordinated walking patterns. P9 drives forward walking with ipsilateral turning, receives inputs from central courtship-promoting neurons and visual projection neurons, and is necessary for a male to pursue a female during courtship. In contrast, BPN drives straight, forward walking and is not required during courtship. BPN is instead recruited during and required for fast, straight, forward walking bouts. Thus, this study reveals separate brain pathways for object-directed walking and fast, straight, forward walking, providing insight into how the brain initiates context-appropriate walking programs (Bidaye, 2020).

In insects, the small number of ~300-500 descending neurons (DNs) that project to the ventral nerve cord (VNC) provides the opportunity to finely probe how locomotor control is encoded in descending pathways. Although specific DNs have been associated with specific aspects of walking, including walking initiation, in different insect species, lack of reproducible access to these neurons makes it difficult to examine their function. In addition, little is known about how higher brain neurons control context-specific walking initiation in insects. The sophisticated genetic tools in Drosophila melanogaster allow reproducible access to specific neurons, including DNs, and greatly facilitate functional characterization. Recent work revealed that many DNs can modify an ongoing walking program. In addition, the activity of a few DNs has been shown to correlate with locomotor patterns. However, it is still unclear if specific brain inputs, at the level of both DNs and higher brain areas, can initiate a coordinated walking pattern and how distinct walking initiations are manifested in different behavioral contexts (Bidaye, 2020).

This study leveraged recently developed genetic tools and coupled them with novel behavioral assays and functional imaging to examine walking initiation neurons in Drosophila. From a targeted optogenetic screen for walking initiation neurons, this study identified two neuronal types that initiate two distinct modes of forward walking: P9 DN induces forward walking with an ipsilateral turning component, whereas novel higher brain neurons, which this study names Bolt protocerebral neurons (BPNs), induce straight forward walking. Functional connectivity and neural silencing studies revealed that the ipsilateral turning walking program driven by P9 contributes to object-directed walking in the context of courtship. In contrast, BPNs were dispensable for courtship. Instead, in vivo imaging and behavioral experiments showed that BPNs are specifically necessary for fast, long-duration, straight forward walking. These studies show that the activity of specific brain neurons is sufficient to switch downstream motor control circuits from a non-walking state into a walking state and provide evidence for distinct descending pathways for walking initiations in different contexts (Bidaye, 2020).

P9 receives inputs from courtship-promoting neurons and visual projection neurons and drives forward walking with an ipsilateral turning component. Moreover, P9 is required during object-directed walking in the context of courtship, whereas BPN is not required for object-directed walking in this context. Instead, BPN activity is correlated with straight, long, fast walking bouts, and manipulating activity of these neurons modifies walking speed and duration without affecting turning. Thus, the characterization of these neurons reveals two distinct pathways that initiate two different forward walking programs in different behavioral contexts (Bidaye, 2020).

P9 and BPN uncover separate brain pathways for object-directed forward steering and fast, straight, forward walking, respectively. The existence of these independent walking initiation pathways suggests that specific brain inputs can drive distinct and complete walking programs in a context-specific manner. This may be achieved by driving a common downstream walking circuit that is modulated to generate specific walking modes, as suggested by neuromechanical models for directional control of walking. Alternatively, these pathways may recruit distinct downstream premotor circuits, each generating a distinct motor pattern. Examining the downstream circuits of these walking initiation pathways will help unravel the mechanisms of these distinct walking initiations (Bidaye, 2020).

Complex motor patterns observed in natural behaviors likely result from combined activity of populations of descending commands from the brain. Interactions among DNs may bring about a concerted change in the functional state of nerve cord circuits, as seen in crawling circuits in C. elegans and Drosophila larvae (Carreira-Rosario, 2018). Therefore, in natural behaviors, P9 and BPN pathways may be active in parallel with other descending signals. An interesting question is whether a population of descending commands generates a motor output more complex than the sum of its constituents. Genetic access to characterized walking initiation neurons (P9 and BPN) will now enable examination of this question and will guide future experiments aimed at elucidating how brain neurons influence walking contro (Bidaye, 2020).

From an activation screen of approximately half of all DNs in the fly (Namiki, 2018), the P9 DNs were the only DNs to initiate walking. These results suggest that very few single DNs encode forward walking initiation. Similarly, a previous unbiased behavioral screen revealed MDN as the only DN sufficient to drive backward walking (Bidaye, 2014). Although DN combinations likely elicit and modulate walking, these studies suggest that P9 neurons have a privileged role as a DN class triggering a complete forward walking program (Bidaye, 2020).

Previous studies have shown the strong importance of vision in pursuing a potential mate during courtship. Male flies initiate walking toward any object with visual characteristics that grossly match those of a potential mate, indicating that visual information may directly influence walking control neurons. This study has shown that P9 receives direct inputs from LC9 that serve as feature detectors, suggesting that P9 participates in a shallow pathway from visual detection to motor command. Direct connections between visual projection neurons and DNs are also seen in the giant fiber escape pathway and in visually guided flight control, suggesting a common circuit motif to minimize response times when rapid action is required (Bidaye, 2020).

In addition to visual inputs, P9 is also activated by pC1 neurons (Koganezawa, 2016), master regulators of courtship behavior that integrate olfactory, pheromonal, and auditory cues and drive a complex courtship sequence. pC1-activated flies show enhanced object pursuit behavior that persists for several minutes, suggesting strong potentiation of the downstream locomotor control circuit. The findings of this study reveal a circuit configuration in which pC1 may gate/potentiate the LC-P9 sensorimotor loop, leading to context-specific object-directed walking. Consistent with this, recent studies demonstrated that LC10 neurons participate in object tracking during courtship and proposed that LC10 and pC1 converge on downstream targets to drive object-directed walking (Ribeiro, 2018). Although LC10 did not activate P9, it is possible that its effect on P9 is gated by pC1 activity. Similar to the P9 pathway, state-dependent gating of sensorimotor loops in Drosophila was recently demonstrated for a visually evoked landing circuit (Ache, 2019), suggesting a general circuit architecture for context-dependent, modular regulation of sensory-driven responses (Bidaye, 2020).

Although this study defined a role for P9 in object pursuit during courtship behavior, it is unlikely that P9 neurons are the only DNs involved in walking control during courtship, as males with P9 transmission blocked are able to track females for short periods. In addition, this study has not explored the role of P9 in other steering behaviors. Indeed, cricket DNs implicated in walking control have been shown to be responsive to multiple sensory stimuli in a state-dependent fashion (Bidaye, 2020).

The ability of BPNs to drive high-speed, straight forward walks (or 'sprints') inspired the name 'Bolt' neurons, given Usain Bolt's unmatched sprinting records. Unilateral BPN activation induced straight forward walking, in contrast to turning phenotypes elicited by unilateral activation of other walking initiation neurons (P9, MDN, and cricket DNs). BPNs also likely function in speed control, as increasing activity increased walking speed. The activation, silencing, and neural activity recording studies suggest BPNs are important when animals execute long, straight, fast walking (i.e., when the fly needs to cover a large distance in a short time). This suggests a potential role in exploratory or escape-like behaviors. However, their widespread dendritic fibers in the higher brain suggest that they might integrate multiple sensory inputs and may represent higher order regulators of walking, akin to pC1 neurons for courtship control. This anatomy makes BPNs unlikely to be involved in reflexive escape behaviors (e.g., like the jumping response encoded by the giant fiber neurons). Instead, the hypothesis is favored that BPN is involved in sustained walking during certain types of exploratory behaviors (e.g., exploring new environments) or other forms of escape that require moving out of an unpleasant environment. As not all BPNs showed detectable responses during spontaneous walking (Figure S6; STAR Methods), there may be functional subclasses within the BPN population (Bidaye, 2020).

The BPN pathway shows remarkable similarity to a recently described mouse MLR-caudal brainstem circuit that promotes high-speed walking: in both cases, unilateral activation induces walking initiation without a turning bias, activation strength correlates with walking speed, and reduced activity causes specific defects in high-speed walking. Moreover, this study demonstrate that BPN is active during high-speed straight forward walks, when the animal is spontaneously walking. Taken together, these studies suggest that high-speed walking is executed by specialized walking circuits that serve an essential function across different organisms (Bidaye, 2020).

Although BPNs are necessary for fast walking, the ability of BPNs to drive different walking speeds at different stimulation frequencies provides an opportunity to examine downstream mechanisms for speed control. Recent studies in zebrafish showed a gradient of recruitment of distinct premotor circuits at increasing swimming speeds. In Drosophila, recent work (Azevedo, 2020) has characterized distinct motor neurons recruited in a similar manner as leg movements accelerate. Examining how BPNs recruit these motor programs in an intensity-dependent manner will help illuminate the mechanism of walking speed control in Drosophila (Bidaye, 2020).

Unlike the mammalian locomotor system, which has been explored at multiple hierarchical levels such as the cortex, basal ganglia, cerebellum, brain stem, and spinal cord, the invertebrate walking control has been investigated mainly at the level of the nerve cord circuits and DNs. The only higher brain structure analyzed in the context of locomotion is the central complex (CC), which has been implicated as the site for generation of an internal heading signal. These heading signals are thought to directly influence CC output neurons involved in turning and speed control. It is unclear how CC or other higher brain areas control downstream locomotor circuits (Bidaye, 2020).

The P9 and BPN pathways elucidate genetically defined, specific brain neurons that drive coordinated walking behaviors. These neurons are upstream of nerve cord circuits and likely downstream or independent of CC navigation circuits. As BPNs are higher brain neurons located outside the CC and are not involved in turning behaviors, BPNs could constitute a CC-independent pathway involved in non-directed high-speed forward walking, providing an important landmark in examining higher brain regions for walking control. In addition, the LC9-P9-pC1 neural circuit motif provides a model of how context impinges on locomotor decisions directly at the level of DNs. Furthermore, because BPN and P9 activation elicits complete, distinct walking programs, they are critical nodes that will enable evaluation of how downstream locomotor circuits in the VNC execute different walking patterns. Thus, P9 and BPN pathways reveal organization of the invertebrate walking control circuits across different hierarchical levels and provide an important advance in understanding of how brain pathways switch on downstream walking control circuits (Bidaye, 2020).

Walking Drosophila navigate complex plumes using stochastic decisions biased by the timing of odor encounters

How insects navigate complex odor plumes, where the location and timing of odor packets are uncertain, remains unclear. This study imaged complex odor plumes simultaneously with freely-walking flies, quantifying how behavior is shaped by encounters with individual odor packets. Navigation was stochastic and did not rely on the continuous modulation of speed or orientation. Instead, flies turned stochastically with stereotyped saccades, whose direction was biased upwind by the timing of prior odor encounters, while the magnitude and rate of saccades remained constant. Further, flies used the timing of odor encounters to modulate the transition rates between walks and stops. In more regular environments, flies continuously modulate speed and orientation, even though encounters can still occur randomly due to animal motion. In less predictable environments, where encounters are random in both space and time, walking flies navigate with random walks biased by encounter timing (Demir, 2020).

A pair of ascending neurons in the subesophageal zone mediates aversive sensory inputs-evoked backward locomotion in Drosophila larvae

Animals typically avoid unwanted situations with stereotyped escape behavior. For instance, Drosophila larvae often escape from aversive stimuli to the head, such as mechanical stimuli and blue light irradiation, by backward locomotion. Responses to these aversive stimuli are mediated by a variety of sensory neurons including mechanosensory class III da (C3da) sensory neurons and blue-light responsive class IV da (C4da) sensory neurons and Bolwig's organ (BO). How these distinct sensory pathways evoke backward locomotion at the circuit level is still incompletely understood. This study shows that a pair of cholinergic neurons in the subesophageal zone, designated AMBs, evoke robust backward locomotion upon optogenetic activation. Anatomical and functional analysis shows that AMBs act upstream of MDNs, the command-like neurons for backward locomotion. Further functional analysis indicates that AMBs preferentially convey aversive blue light information from C4da neurons to MDNs to elicit backward locomotion, whereas aversive information from BO converges on MDNs through AMB-independent pathways. This study also found that, unlike in adult flies, MDNs are dispensable for the dead end-evoked backward locomotion in larvae. These findings thus reveal the neural circuits by which two distinct blue light-sensing pathways converge on the command-like neurons to evoke robust backward locomotion, and suggest that distinct but partially redundant neural circuits including the command-like neurons might be utilized to drive backward locomotion in response to different sensory stimuli as well as in adults and larvae (Omamiuda-Ishikawa, 2020).

Distributed control of motor circuits for backward walking in Drosophila

How do descending inputs from the brain control leg motor circuits to change how an animal walks? Conceptually, descending neurons are thought to function either as command-type neurons, in which a single type of descending neuron exerts a high-level control to elicit a coordinated change in motor output, or through a population coding mechanism, whereby a group of neurons, each with local effects, act in combination to elicit a global motor response. The Drosophila Moonwalker Descending Neurons (MDNs), which alter leg motor circuit dynamics so that the fly walks backwards, exemplify the command-type mechanism. This study identified several dozen MDN target neurons within the leg motor circuits showed that two of them mediate distinct and highly-specific changes in leg muscle activity during backward walking: LBL40 neurons provide the hindleg power stroke during stance phase; LUL130 neurons lift the legs at the end of stance to initiate swing. Through these two effector neurons, MDN directly controls both the stance and swing phases of the backward stepping cycle. These findings suggest that command-type descending neurons can also operate through the distributed control of local motor circuits (Feng, 2020).

Drosophila uses a tripod gait across all walking speeds, and the geometry of the tripod is important for speed control

Changes in walking speed are characterized by changes in both the animal's gait and the mechanics of its interaction with the ground. This study examined these changes in walking Drosophila. he fly's center of mass movement with high spatial resolution and the position of its footprints were measured. Flies predominantly employ a modified tripod gait that only changes marginally with speed. The mechanics of a tripod gait can be approximated with a simple model - angular and radial spring-loaded inverted pendulum (ARSLIP) - which is characterized by two springs of an effective leg that become stiffer as the speed increases. Surprisingly, the change in the stiffness of the spring is mediated by the change in tripod shape rather than a change in stiffness of individual legs. The effect of tripod shape on mechanics can also explain the large variation in kinematics among insects, and ARSLIP can model these variations (Chun, 2021).

Navigation of a Freely Walking Fruit Fly in Infinite Space Using a Transparent Omnidirectional Locomotion Compensator (TOLC)

Animal behavior is an essential element in behavioral neuroscience study. However, most behavior studies in small animals such as fruit flies (Drosophila melanogaster) have been performed in a limited spatial chamber or by tethering the fly's body on a fixture, which restricts its natural behavior. This study has developed the Transparent Omnidirectional Locomotion Compensator (TOLC) for a freely walking fruit fly without tethering, which enables its navigation in infinite space. The TOLC maintains a position of a fruit fly by compensating its motion using the transparent sphere. The TOLC is capable of maintaining the position error < 1 mm for 90.3% of the time and the heading error < 5° for 80.2% of the time. The inverted imaging system with a transparent sphere secures the space for an additional experimental apparatus. Because the proposed TOLC allows observation of a freely walking fly without physical tethering, there is no potential injury during the experiment. Thus, the TOLC will offer a unique opportunity to investigate longitudinal studies of a wide range of behavior in an unrestricted walking Drosophila (Pun, 2021).

The Panopticon-Assessing the Effect of Starvation on Prolonged Fly Activity and Place Preference

Animal behaviours are demonstrably governed by sensory stimulation, previous experience and internal states like hunger. With increasing hunger, priorities shift towards foraging and feeding. During foraging, flies are known to employ efficient path integration strategies. However, general long-term activity patterns for both hungry and satiated flies in conditions of foraging remain to be better understood. Similarly, little is known about how permanent contact chemosensory stimulation affects locomotion. To address these questions, a novel, simplistic fly activity tracking setup, the Panopticon, was developed. Using a 3D-printed Petri dish inset, this assay allows recording of walking behaviour, of several flies in parallel, with all arena surfaces covered by a uniform substrate layer. Two constellations of providing food were tested: (i) in single patches and (ii) omnipresent within the substrate layer. Fly tracking is done with FIJI, further assessment, analysis and presentation is done with a custom-built MATLAB analysis framework. This study found that starvation history leads to a long-lasting reduction in locomotion, as well as a delayed place preference for food patches which seems to be not driven by immediate hunger motivation (Mahishi, 2021).

Identification of FoxP circuits involved in locomotion and object fixation in Drosophila

The FoxP family of transcription factors is necessary for operant self-learning, an evolutionary conserved form of motor learning. The expression pattern, molecular function and mechanisms of action of the Drosophila FoxP orthologue remain to be elucidated. By editing the genomic locus of FoxP with CRISPR/Cas9, this study found that the three different FoxP isoforms are expressed in neurons, but not in glia and that not all neurons express all isoforms. Furthermore, FoxP expression was found in the protocerebral bridge, the fan-shaped body and in motor neurons, but not in the mushroom bodies. Finally, this study discovered that FoxP expression during development, but not adulthood, is required for normal locomotion and landmark fixation in walking flies. While FoxP expression in the protocerebral bridge and motor neurons is involved in locomotion and landmark fixation, the FoxP gene can be excised from dorsal cluster neurons and mushroom-body Kenyon cells without affecting these behaviours (Palazzo, 2020).

The genomic locus of the Drosophila FoxP gene was edited in order to better understand the expression patterns of the FoxP isoforms and their involvement in behaviour. The isoforms differ with respect to their expression in neuronal tissue. For instance, isoform B (FoxP-iB) expression was found in neuropil areas such as the superior medial protocerebrum, the protocerebral bridge, the noduli, the vest, the saddle, the gnathal ganglia and the medulla, while areas such as the antennal lobes, the fan-shaped body, the lobula and a glomerulus of the posterior ventrolateral protocerebrum contain other FoxP isoforms but not isoform B. Previous results that FoxP is expressed in a large variety of neuronal cell types was corroborated. Genomic manipulations created several new alleles of the FoxP gene which had a number of behavioural consequences that mimicked other, previously published alleles. Specifically, it was found that constitutive knock-out of either FoxP-IB alone or of all FoxP isoforms affects several parameters of locomotor behaviour, such as walking speed, the straightness of walking trajectories or landmark fixation. Mutating the FoxP gene only in particular neurons can have different effects. For instance, knocking FoxP out in neurons of the dorsal cluster (where FoxP is expressed) or in MB Kenyon cells (where no FoxP expression was detected) had no effect in Buridan's paradigm, despite these neurons being required for normal locomotion in Buridan's paradigm. By contrast, without FoxP in the protocerebral bridge or motor neurons, flies show similar locomotor impairments as flies with constitutive knock-outs. These impairments appear to be due to developmental action of the FoxP gene during larval development, as no such effects can be found if the gene is knocked out in all cells in the early pupal or adult stages (Palazzo, 2020).

The exact expression pattern of FoxP has been under debate for quite some time now. Initial work combined traditional reporter gene expression with immunohistochemistry. A previous study created a FoxP-Gal4 line where a 1.5 kb fragment of genomic DNA upstream of the FoxP coding region was used to drive Gal4 expression. These the resulting expression pattern was validated with the staining of a commercial polyclonal antibody against FoxP. The same antibody was used in the current work and observed perfect co-expression with the reporter. The previous description of the FoxP expression pattern as a small number of neurons distributed in various areas of the brain, particularly in the protocerebral bridge, matches the current results (Palazzo, 2020).

Subsequent reports on FoxP expression patterns also used putative FoxP promoter fragments to direct the expression of Gal4. One study used a 1.4 kb sequence upstream of the FoxP transcription start site, while another used 1.9 kb. The larger fragment contained the sequences of the two previously used fragments. The latest study reporting on FoxP expression in Drosophila avoided the problematic promoter fragment method and instead tagged FoxP within a genomic segment contained in a fosmid, intended to ensure expression of GFP-tagged FoxP under the control of its own, endogenous regulatory elements. This study was the first to circumvent the potential for artifacts created either by selection of the wrong promoter fragment or by choosing an inappropriate basal promoter with the fragment. However, since they also used insertion of a transgene, their expression pattern, analogous to that of a promoter fragment Gal4 line, may potentially be subject to local effects where the fosmid with the tagged FoxP was inserted (Palazzo, 2020).

In an attempt to eliminate, the last source of error for determining the expression pattern of FoxP in Drosophila, CRISPR/Cas9 with homology-directed repair was used to tag FoxP in situ, avoiding both the potential local insertion effects of the previous approaches and without disrupting the complex regulation that may occur from more distant parts in the genome. For instance, in human cells, there are at least 18 different genomic regions that are in physical contact with the FOXP2 promoter, some of which act as enhancers. The effects of these regions may be disrupted even if the entire genomic FoxP locus were inserted in a different genomic region as in. Interestingly, the first promoter fragment approach and the fosmid approach agree both with the most artefact-avoiding genome editing approach and the immunohistochemistry with an antibody validated by at least three different FoxP-KO approaches. This converging evidence from four different methods used in three different laboratories suggests that FoxP is expressed in about 1800 neurons in the fly nervous system, of which about 500 are located in the ventral nerve cord. Expression in the brain is widespread with both localized clusters and individual neurons across a variety of neuronal cell types. Notably, the four methods also agree that there is no detectable FoxP expression in the adult or larval MBs. By contrast, in honey bees, there is converging evidence of FoxP expression in the MBs (Palazzo, 2020).

This comparison of the current data with the literature prompts the question why two different promoter fragment approaches suggested FoxP expression in the MBs (confirmed by a ribosome-based approach) when there is no FoxP protein detectable there (Palazzo, 2020).

A first observation used the classic hsp70-based pGaTB vector to create a Gal4 line, while two other studies used the more modern Drosophila synthetic core promoter (DSCP)-based pBPGUw vector. The two vectors differ with regard to their effects on gene expression. In addition to carrying two different basal promoters, the modern pBPGUw sports a 3'UTR that is designed to increase the longevity and stability of the mRNA over the pGaTB vector, which can result in twofold higher Gal4 levels (Palazzo, 2020).

This observation is complemented by single-cell transcriptome data. FoxP RNA can be detected in more than 4100 brain cells, likely overcounting the actual FoxP expression more than threefold. For instance, FoxP RNA is detected in over 1000 glial cells where none of the published studies has ever detected any FoxP expression (Palazzo, 2020).

Taking these two observations together, it becomes plausible that there may be transient, low-level FoxP transcription in some MB neurons (and likely thousands of other cells as well), which in wild-type animals rarely leads to any physiologically relevant FoxP protein levels in these cells. Only when gene expression is enhanced by combining some arbitrary promoter fragments with genetically engineered constructs designed to maximize Gal4 yield such as the pBPGUw vector, such transient, low-abundance mRNAs may be amplified to a detectable level (Palazzo, 2020).

These considerations may also help explain why the ribosome-based method of was able to detect FoxP RNA in MB Kenyon cells: the transcript that was detected may have been present and occupied by ribosomes, but ribosomal occupancy does not automatically entail translation. It remains unexplained, however, how a previous study failed to detect all those much more strongly expressing and numerous neurons outside of the MBs. All of the above is consistent with other insect species showing FoxP expression on the protein level in their MBs, as only limited genetic alterations would be needed for such minor changes in gene expression (Palazzo, 2020).

The stochasticity of gene expression is a well-known fact and known to arise from the transcription machinery. Post-transcriptional gene regulation is similarly well-known. It is thus not surprising if it is observed that many cells often express transcripts that rarely, if ever, are translated into proteins. The final arbiter of gene expression must therefore remain the protein level, which is why this study validated the expression analysis with the appropriate antibody. On this decisive level, FoxP has not been detected in the MBs at this point (Palazzo, 2020).

The genome editing approach allowed distinguishing of differences in the expression patterns of different FoxP isoforms. The isoform specifically involved in operant self-learning, FoxP-iB, is only expressed in about 65% of all FoxP-positive neurons. The remainder express either FoxP-iA or FoxP-iIR or both. Neurons expressing only non-iB isoforms are localized in the antennal lobes, the fan-shaped body, the lobula and a glomerulus of the posterior ventrolateral protocerebrum. Combined with all three isoforms differing in their DNA-binding FH box, the different expression patterns for the different isoforms adds to the emerging picture that the different isoforms may serve very different functions (Palazzo, 2020).

Alterations of FoxP family genes universally result in various motor deficits on a broad scale in humans and mice for both learned and innate behaviours. Also in flies, manipulations of the FoxP locus by mutation or RNA interference have revealed that FoxP is involved in flight performance and other, presumably inborn, locomotor behaviours as well as in motor learning tasks (Palazzo, 2020).

The locomotor phenotypes described so far largely concerned the temporal aspects of locomotion, such as initiation, speed or duration of locomotor behaviours. Using Buridan's paradigm, this study reports that manipulations of FoxP can also alter spatial aspects of locomotion, such as landmark fixation or the straightness of trajectories. The results further exemplify the old insight that coarse assaults on gene function such as constitutive knock-outs of entire genes or isoforms very rarely yield useful, specific phenotypes. Rather, it is often the most delicate of manipulations that reveal the involvement of a particular gene in a specific behaviour. This fact is likely most often due to the pleiotropy of genes, often paired with differential dominance which renders coarse neurogenetic approaches useless in most instances, as so many different behaviours are affected that the specific contribution of a gene to a behavioural phenotype becomes impossible to dissect (Palazzo, 2020).

In the case of FoxP, it was already known, for instance, that the different isoforms affect flight performance to differing degrees and that a variety of different FoxP manipulations affected general locomotor activity. This study shows that a complete knock-out of either FoxP-iB or all isoforms affected both spatial and temporal parameters of locomotion, but the insertion mutation FoxP3955 did not alter stripe fixation. Remarkably, despite the ubiquitous and substantial locomotor impairments after nearly any kind of FoxP manipulation be it genomic or via RNAi reported in the published literature failed to detect the locomotor defects of these flies (Palazzo, 2020).

While some of the manipulations used in this study did not affect locomotion significantly (e.g. knock-out in MBs or DCNs), most of them affected both spatial and temporal locomotion parameters, despite these parameters commonly not co-varying. Thus, while one would expect these behaviours to be biologically separable, the manipulations carried out in this study did not succeed in this separation (Palazzo, 2020).

Taken together, the results available to-date reveal FoxP to be a highly pleiotropic gene with phenotypes that span both temporal and spatial domains of locomotion in several behavioural modalities, lifespan, motor learning, social behaviour and habituation. It is straightforward to conclude that only precise, cell-type-specific FoxP manipulations of specific isoforms will be capable of elucidating the function this gene serves in each phenotype. With RNAi generally yielding varying levels of knock-down and, specifically, with currently available FoxP RNAi lines showing only little, if any, detectable knock-down with RT-qPCR, CRISPR/Cas9-mediated genome editing lends itself as the method of choice for this task. Practical considerations when designing multi-target gRNAs for FoxP prompted testing of the CRISPR/Cas9 system as an alternative to RNAi with an isoform-unspecific approach first, keeping the isoform-specific approach for a time when more experience in this technique has been collected. In a first proof-of-principle, CRISPR/Cas9 was used to remove FoxP from MB Kenyon cells, DCNs, motor neurons and the protocerebral bridge (Palazzo, 2020).

MBs have been shown to affect both spatial and temporal aspects of locomotion reported a subtle structural phenotype in a subset of MB Kenyon cells that did not express FoxP. As detailed above, two groups have reported FoxP expression in the MBs and it appears that some transcript can be found in MB Kenyon cells. With a substantial walking defect both in FoxP3955 mutant flies (which primarily affects FoxP-iB expression) and in flies without any FoxP, together with the MBs being critical for normal walking behaviour, the MBs were a straightforward candidate for a cell-type-specific FoxP-KO. However, flies without FoxP in the MBs walk perfectly normally. There are two possible reasons for this lack of an effect of this manipulation: either FoxP protein is not present in MBs or it is not important in MBs for walking. While at this point it is not possible to decide between these two options, the expression data concurring with those from previous studies suggest the former explanation may be the more likely one. Remarkably, a publication that did report FoxP expression in the MBs did not detect the walking deficits in FoxP3955 mutant flies despite testing for such effects. Motor aberrations as those described here and in other FoxP manipulations constitute a potential alternative to the decision-making impairments ascribed to these flies (Palazzo, 2020).

DCNs were recently shown to be involved in the spatial component (landmark fixation) of walking in Buridan's paradigm, but removing FoxP from DCNs showed no effect, despite abundant FoxP expression in DCNs. It is possible that a potential effect in stripe fixation may have been masked by already somewhat low fixation in both control strains. On the other hand, even at such control fixation levels, significant increases in stripe deviation can be obtained. Before this is resolved, one explanation is that FoxP is not required in these neurons for landmark fixation in Buridan's paradigm, while the neurons themselves are required (Palazzo, 2020).

Motor neurons are involved in all aspects of behaviour and have been shown to be important for operant self-learning. With abundant expression of FoxP in motor neurons, these neurons are considered a prime candidate for a clear FoxP-cKO phenotype. Indeed, removing FoxP specifically from motor neurons only, mimicked the effects of removing the gene constitutively from all cells. It is noteworthy that this manipulation alone was sufficient to affect both temporal and spatial parameters, albeit only one of the two driver lines showed clear-cut results. One would not necessarily expect motor neurons to affect purportedly 'higher-order' functions such as landmark fixation. It is possible that the higher tortuosity in the trajectories of the flies where D42 was used to drive the UAS-gRNA construct is largely responsible for the greater angular deviation from the landmarks in these flies and that this tortuosity, in turn is caused by the missing FoxP in motor neurons. Alternatively, D42 is also driving in non-motor neurons where FoxP is responsible for landmark fixation. The driver line C380 showed similar trends, albeit not quite statistically significant at an alpha value of 0.5%, suggesting that potentially the increased meander parameter may be caused by motor neurons lacking FoxP (Palazzo, 2020).

The protocerebral bridge is not only the arguably most conspicuous FoxP-positive neuropil, it has also been reported to be involved in temporal aspects of walking. Moreover, the protocerebral bridge provides input to other components of the central complex involved in angular orientation. Similar to the results in motor neurons, removing FoxP from a small group of brain neurons innervating the protocerebral bridge, phenocopies constitutive FoxP mutants (Palazzo, 2020).

Taken together, the motor neuron and protocerebral bridge results suggest that both sets of neurons serve their locomotor function in sequence. At this point, it is unclear which set of neurons precedes the other in this sequence (Palazzo, 2020).

There is ample evidence that the FoxP family of transcription factors acts during development in a variety of tissues. What is less well known is if adult FoxP expression serves any specific function. A recent study in transgenic mice in operant conditioning and motor learning tasks showed postnatal knock-out of FOXP2 in cerebellar and striatal neurons affected leverpressing and cerebellar knock-out also affected motor-learning. At least for these tasks in mammals, a FoxP family member does serve a postnatal function that is independent of brain development (brain morphology was unaltered in these experiments). Also in birds, evidence has been accumulating that adult FoxP expression serves a song plasticity function. The temporally controlled experiments in this study suggest that at least locomotion in Buridan's paradigm can function normally in the absence of FoxP expression in the adult, as long as FoxP expression remains unaltered during larval development. Future research on the role of FoxP in locomotion and landmark fixation hence needs to focus on the larval development before pupation (Palazzo, 2020).

Fast tuning of posture control by visual feedback underlies gaze stabilization in walking Drosophila

Locomotion requires a balance between mechanical stability and movement flexibility to achieve behavioral goals despite noisy neuromuscular systems, but rarely is it considered how this balance is orchestrated. This study combined virtual reality tools with quantitative analysis of behavior to examine how Drosophila uses self-generated visual information (reafferent visual feedback) to control gaze during exploratory walking. It was found that flies execute distinct motor programs coordinated across the body to maximize gaze stability. However, the presence of inherent variability in leg placement relative to the body jeopardizes fine control of gaze due to posture-stabilizing adjustments that lead to unintended changes in course direction. Surprisingly, whereas visual feedback is dispensable for head-body coordination, studies found that self-generated visual signals tune postural reflexes to rapidly prevent turns rather than to promote compensatory rotations, a long-standing idea for visually guided course control. Together, these findings support a model in which visual feedback orchestrates the interplay between posture and gaze stability in a manner that is both goal dependent and motor-context specific (Cruz, 2021).

Behavioral signatures of structured feature detection during courtship in Drosophila

Many animals detect other individuals effortlessly. In Drosophila, previous studies have examined sensory processing during social interactions using simple blobs as visual stimulation; however, whether and how flies extract higher-order features from conspecifics to guide behavior remains elusive. Arguing that this should be reflected in sensorimotor relations, this study developed unbiased machine learning tools for natural behavior quantification and applied these tools, which may prove broadly useful, to study interacting pairs. By transforming motor patterns with female-centered reference frames, this study established circling, where heading and traveling directions intersect, as a unique pattern of social interaction during courtship. Circling was found to be highly visual, with males exhibiting view-tuned motor patterns. Interestingly, males select specific wing and leg actions based on the positions and motions of the females' heads and tails. Using system identification, visuomotor transformation functions were derived indicating history-dependent action selection, with distance predicting action initiation and angular position predicting wing choices and locomotion directions. Integration of vision with somatosensation further boosts these sensorimotor relations. Essentially comprised of orchestrated wing and leg maneuvers that are more variable in the light, circling induces mutually synchronized conspecific responses stronger than wing extension alone. Finally, this study found that actions depend on integrating spatiotemporally structured features with goals. Altogether, we identified a series of sensorimotor relations during circling, implying that during courtship, flies detect complex spatiotemporally structured features of conspecifics, laying the foundation for a mechanistic understanding of conspecific recognition in Drosophila (Ninf, 2022).

Building an allocentric travelling direction signal via vector computation

Many behavioural tasks require the manipulation of mathematical vectors, but, outside of computational models, it is not known how brains perform vector operations. This study shows how the Drosophila central complex, a region implicated in goal-directed navigation, performs vector arithmetic. First, a neural signal in the fan-shaped body is described that explicitly tracks the allocentric travelling angle of a fly, that is, the travelling angle in reference to external cues. Past work has identified neurons in Drosophila and mammals that track the heading angle of an animal referenced to external cues (for example, head direction cells), but this new signal illuminates how the sense of space is properly updated when travelling and heading angles differ (for example, when walking sideways). A neuronal circuit was characterized that performs an egocentric-to-allocentric (that is, body-centred to world-centred) coordinate transformation and vector addition to compute the allocentric travelling direction. This circuit operates by mapping two-dimensional vectors onto sinusoidal patterns of activity across distinct neuronal populations, with the amplitude of the sinusoid representing the length of the vector and its phase representing the angle of the vector. The principles of this circuit may generalize to other brains and to domains beyond navigation where vector operations or reference-frame transformations are required (Lyu 2022).

Whether mammalian brains have neurons that are tuned to the allocentric travelling direction of an animal as in Drosophila is still unknown. Although a defined population of neurons tuned to travelling direction has yet to be highlighted in mammals, such cells could have been missed because their activity would loosely resemble that of the head-direction cells outside a task in which the animal is required to sidestep or walk backwards (Lyu 2022).

Neurons are often modelled as summing their synaptic inputs, but the heading inputs that PFN cells receive from the EPG system appear to be multiplied by the self-motion (for example, optic flow) input, resulting in an amplitude or gain modulation. Multiplicative or gain-modulated responses appear in classic computational models for how neurons in area 7a of the primate parietal cortex might implement a coordinate transformation, alongside similar proposals in mammalian navigation. The Drosophila circuit described in this study strongly resembles aspects of the classic models of the parietal cortex. Units that multiply their inputs are also at the core of the 'attention' mechanism used, for example, in machine-based language processing. The experimental evidence for input multiplication in a biological network may indicate that real neural circuits have greater potential for computation than is generally appreciated (Lyu 2022).

This study describes a travelling direction signal and how it is built; related results and conclusions appear in a parallel study. The mechanisms described for calculating the travelling direction are robust to left-right rotations of the head and to the possibility of the allocentric projection vectors being non-orthogonal. It is possible that the travelling signal of hΔB cells is compared with a goal-travelling direction to drive turns that keep a fly along a desired trajectory. Augmented with an appropriate speed signal (or if the fly generally travels forward relative to its body), the hΔB signal could also be integrated over time to form a spatial-vector memory via path integration. There are hundreds more PFN cells beyond the 40 PFNd and 20 PFNv cells examined in this study, and thus the central complex could readily convert other angular variables from egocentric to allocentric coordinates via the algorithm described in this study. Because many sensory, motor and cognitive processes can be formalized in the language of linear algebra and vector spaces, defining a neuronal circuit for vector computation may open the door to better understanding of several previously enigmatic circuits and neuronal activity patterns across multiple nervous systems (Lyu 2022).

Fast near-whole-brain imaging in adult Drosophila during responses to stimuli and behavior

Whole-brain recordings give a global perspective of the brain in action. This study describes a method using light field microscopy to record near-whole brain calcium and voltage activity at high speed in behaving adult flies. Global activity maps were first obtained for various stimuli and behaviors. Notably, brain activity was found to increase on a global scale when the fly walked but not when it groomed. This global increase with walking was particularly strong in dopamine neurons, which showed little activity otherwise. Second, maps were extracted of spatially distinct sources of activity as well as their time series using principal component analysis and independent component analysis. The characteristic shapes in the maps matched the anatomy of subneuropil regions and, in some cases, a specific neuron type. Brain structures that responded to light and odor were consistent with previous reports, confirming the new technique's validity. Previously uncharacterized behavior-related activity wee also observed as well as patterns of spontaneous voltage activity (Aimon, 2019).

The manifold structure of limb coordination in walking Drosophila

Terrestrial locomotion requires animals to coordinate their limb movements to efficiently traverse their environment. While previous studies in hexapods have reported that limb coordination patterns can vary substantially, the structure of this variability is not yet well understood. This study characterized the symmetric and asymmetric components of variation in walking kinematics in the genetic model organism Drosophila. Drosophila were found to use a single continuum of coordination patterns without evidence for preferred configurations. Spontaneous symmetric variability was associated with modulation of a single control parameter-stance duration-while asymmetric variability consisted of small, limb-specific modulations along multiple dimensions of the underlying symmetric pattern. Commands that modulated walking speed, originating from artificial neural activation or from the visual system, evoked modulations consistent with spontaneous behavior. These findings suggest that Drosophila employ a low-dimensional control architecture, which provides a framework for understanding the neural circuits that regulate hexapod legged locomotion (DeAngelis, 2019).

Decentralized control of insect walking: A simple neural network explains a wide range of behavioral and neurophysiological results
Controlling the six legs of an insect walking in an unpredictable environment is a challenging task. Solutions proposed to deal with this task are usually based on the highly influential concept that (sensory-modulated) central pattern generators (CPG) are required to control the rhythmic movements of walking legs. This study investigated a different view. To this end, a sensor based controller operating on artificial neurons was introduced, being applied to a (simulated) insectoid robot required to exploit the 'loop through the world' allowing for simplification of neural computation. Such a decentralized solution is shown to lead to adaptive behavior when facing uncertain environments which are demonstrated for a broad range of behaviors never dealt with in a single system by earlier approaches. These patterns are found to be stable against disturbances and when starting from various leg configurations. This neuronal architecture easily allows for starting or interrupting a walk, all being difficult for CPG controlled solutions. This approach can as well account for the neurophysiological results usually interpreted to support the idea that CPGs form the basis of walking, although this approach is not relying on explicit CPG-like structures. Application of CPGs may however be required for very fast walking. This neuronal structure allows to pinpoint specific neurons known from various insect studies. Interestingly, specific common properties observed in both insects and crustaceans suggest a significance of the controller beyond the realm of insects (Schilling, 2020).

Mechanisms underlying attraction to odors in walking Drosophila

Mechanisms that control movements range from navigational mechanisms, in which the animal employs directional cues to reach a specific destination, to search movements during which there are little or no environmental cues. Even though most real-world movements result from an interplay between these mechanisms, an experimental system and theoretical framework for the study of interplay of these mechanisms is not available. This study rectifies this deficit. A new method is created to stimulate the olfactory system in Drosophila. As flies explore a circular arena, their olfactory receptor neurons (ORNs) are optogenetically activated within a central region making this region attractive to the flies without emitting any clear directional signals outside this central region. In the absence of ORN activation, the fly's locomotion can be described by a random walk model where a fly's movement is described by its speed and turn-rate (or kinematics). Upon optogenetic stimulation, the fly's behavior changes dramatically in two respects. First, there are large kinematic changes. Second, there are more turns at the border between light-zone and no-light-zone and these turns have an inward bias. Surprisingly, there is no increase in turn-rate, rather a large decrease in speed that makes it appear that the flies are turning at the border. Similarly, the inward bias of the turns is a result of the increase in turn angle. These two mechanisms entirely account for the change in a fly's locomotion. No complex mechanisms such as path-integration or a careful evaluation of gradients are necessary (Tao, 2020).

A size principle for recruitment of Drosophila leg motor neurons

To move the body, the brain must precisely coordinate patterns of activity among diverse populations of motor neurons. This study used in vivo calcium imaging, electrophysiology, and behavior to understand how genetically-identified motor neurons control flexion of the fruit fly tibia. Leg motor neurons exhibit a coordinated gradient of anatomical, physiological, and functional properties. Large, fast motor neurons control high force, ballistic movements while small, slow motor neurons control low force, postural movements. Intermediate neurons fall between these two extremes. This hierarchical organization resembles the size principle, first proposed as a mechanism for establishing recruitment order among vertebrate motor neurons. Recordings in behaving flies confirmed that motor neurons are typically recruited in order from slow to fast. However, this study also found that fast, intermediate, and slow motor neurons receive distinct proprioceptive feedback signals, suggesting that the size principle is not the only mechanism that dictates motor neuron recruitment. Overall, this work reveals the functional organization of the fly leg motor system and establishes Drosophila as a tractable system for investigating neural mechanisms of limb motor control (Azevedo, 2020).

Neural coding of leg proprioception in Drosophila

Animals rely on an internal sense of body position and movement to effectively control motor behavior. This sense of proprioception is mediated by diverse populations of mechanosensory neurons distributed throughout the body. This study investigated neural coding of leg proprioception in Drosophila, using in vivo two-photon calcium imaging of proprioceptive sensory neurons during controlled movements of the fly tibia. The axons of leg proprioceptors are organized into distinct functional projections that contain topographic representations of specific kinematic features. Using subclass-specific genetic driver lines, this study shows that one group of axons encodes tibia position (flexion/extension), another encodes movement direction, and a third encodes bidirectional movement and vibration frequency. Overall, these findings reveal how proprioceptive stimuli from a single leg joint are encoded by a diverse population of sensory neurons, and provide a framework for understanding how proprioceptive feedback signals are used by motor circuits to coordinate the body (Mamiya, 2018).

This study used in vivo calcium imaging to investigate the population coding of leg proprioception in the femoral chordotonal organ (FeCO) of Drosophila. The results reveal a basic logic for proprioceptive sensory coding: genetically distinct proprioceptor subclasses detect and encode distinct kinematic features, including tibia position, directional movement, and vibration. The cell bodies of each proprioceptor subclass reside in separate parts of the FeCO in the leg, and their axons project to distinct regions of the fly VNC. This organization suggests that different kinematic features may be processed by separate downstream circuits, and function as parallel feedback channels for the neural control of leg movement and behavior (Mamiya, 2018).

Claw neurons encode the position of the tibia relative to the femur, club neurons encode bidirectional tibia movement, and hook neurons encode birectional tibia movement. Specifically, each branch of a claw neuron consists of two sub-branches, whose calcium signals increase when the tibia is flexed or extended. Imaging from single claw neurons revealed that individual cells can be narrowly tuned to even more specific tibia angles. These data are consistent with previous reports of angular range fractionation in the locust FeCO. Interestingly, minimal activity in claw axons was observed when the tibia was close to 90°, and no single claw neuron was found tuned to this range in a limited sample. Similar tuning distributions have been observed in multiunit recordings from the FeCO of locusts and stick insects. However, single-unit recordings from these species also revealed the existence of a small number of position-tuned cells with peak activity in this middle range. It is possible that the driver lines that were used did not label the FeCO neurons tuned to this range. It is also possible that this represents a real difference between Drosophila and other insects. The fly FeCO has about half as many neurons as that of the stick insect and locust, and the biomechanics of the organ may also differ between species (Mamiya, 2018).

How does the position tuning of claw neurons relate to natural leg kinematics? When a fly is standing still, the tibia of the front leg rests ~90° relative to the femur; during straight walking, the tibia flexes to ~40° and extends to 120°. Thus, it is predicted that claw neurons are largely silent in a stationary fly, while extension- and flexion-tuned neurons are rhythmically active during walking. Encoding deviations from the natural resting position may reflect an adaptive strategy to minimize metabolic cost (Mamiya, 2018).

Position-encoding claw neurons exhibit response hysteresis (a lag between input and output): both flexion- and extension-tuned sub-branches of the claw showed larger steady-state activity when the tibia is moved in a direction that increases their activity. This response asymmetry is notable because it presents a problem for downstream circuits and computations that rely on a stable readout of tibia angle. Proprioceptive hysteresis has also been described in vertebrate muscle spindles and FeCO neurons of other insects. One possible solution for solving the ambiguities created by hysteresis would be to combine the tonic activity of claw neurons with signals from directionally selective hook neurons. This could allow a neuron to decode tibia position based on past history of tibia movement. However, it is also possible that tibia angle hysteresis is a useful feature of the proprioceptive system, rather than a bug. For example, it has been proposed that hysteresis could compensate for the nonlinear properties of muscle activation in short sensorimotor loops (Mamiya, 2018).

This study identified two functional subclasses of FeCO neurons that respond phasically to tibia movement. Club neurons respond to both flexion and the extension of the tibia, while hook neurons respond only to flexion. In both population and single neuron imaging experiments, directionally selective responses to tibia extension were observe, although it was not possible to identify a specific Gal4 line for this response subclass. The movement sensitivity of the club and hook neurons resembles that of other phasic proprioceptors, including primary muscle spindle afferents, and movement-tuned FeCO neurons recorded in the locust and stick insect. Although the slow temporal dynamics of GCaMP6f did not permit a detailed analysis of velocity tuning, the results indicate that FeCO neurons respond to the natural range of leg speeds encountered during walking. In the future, it will be interesting to investigate how FeCO neurons encode leg movements during walking, and how active movements may be encoded differently from passive movements, for example through presynaptic inhibition of FeCO axon terminals (Mamiya, 2018).

In addition to their directional tuning, this sudy found that club and hook neurons differ in their sensitivity to fast (100-2,000 Hz), low-amplitude (0.9-0.054 μm) tibia vibration. Club neurons are strongly activated by vibration stimuli, but hook neurons are not. This difference in vibration sensitivity is not likely to be caused by a difference in velocity tuning because these differences are relatively small at the range of the speeds experienced during tibia vibration. Rather, it seems that the club neurons have a lower mechanical threshold and/or may be more sensitive to the constant acceleration produced by vibration (Mamiya, 2018).

The functional role of vibration-sensitive FeCO neurons is not entirely clear. Previous studies in stick insects and locusts have found that vibration-tuned FeCO neurons do not contribute to postural reflexes in the same manner as FeCO neurons tuned to joint position and directional movement. This raises the possibility that vibration-tuned chordotonal neurons sense external mechanosensory stimuli. For example, club neurons could monitor substrate vibrations in the environment, which serve as an important communication signal for many insect species. Abdominal vibrations produced during courtship by male Drosophila coincide with pausing behavior in females, and hence increased receptivity to copulation. These vibrations occur at frequencies that match the sensitivity of club neurons (200-2,000 Hz). Therefore, club neurons are well-positioned to mediate intraspecific vibratory communication during courtship or other behaviors (Mamiya, 2018).

Using genetic driver lines for specific FeCO neuron subclasses, this study provides the first detailed anatomical characterization of Drosophila leg proprioceptors. The anatomy and imaging experiments revealed a systematic relationship between the functional tuning of proprioceptor subclasses and their anatomical structure. The cell bodies of the three proprioceptor subclasses are clustered in different regions of the femur, an organization that may reflect biomechanical specialization for detecting position, movement, and vibration. Proprioceptor axons then converge within the leg nerve, before branching within the VNC to form subclass-specific projections that are called the club, claw, and hook. This organization was found to be highly stereotyped across flies (Mamiya, 2018).

The axons of claw neurons split into three symmetric branches, resembling a claw. This unique arborization pattern is suggestive of a Cartesian coordinate system; for example, each branch could represent a different spatial axis. However, this study found that each claw neuron innervates all three branches, and that the X, Y, and Z branches all encode the same stimuli. Specifically, calcium imaging experiments revealed that each claw branch is divided into two sub-branches that are specialized for encoding flexion or extension of the tibia. If each claw branch is functionally similar, what is the purpose of this tri-partite structure? Each branch may target different downstream neurons, or could be independently modulated by presynaptic inhibition. Interestingly, the axons of directionally tuned hook neurons arborized alongside the claw but did not innervate all three of the claw branches. Thus, the X, Y, and Z branches may facilitate integration of positional information with directionally tuned movement signals (Mamiya, 2018).

It was surprising to discover a topographic map of leg vibration frequency within the axon terminals of club neurons. This structure has not previously been described in flies, but resembles the tonotopic map of sensory afferents in the cricket auditory system or the cochlear nucleus in vertebrates. Interestingly, the spatial layout of the frequency map in club axons was consistent across different vibration amplitudes, despite a shift in the peak frequency tuning curve. Recordings from single club neurons suggest that this frequency map is comprised of individual axons that are each tuned to a narrow frequency band. An orderly map of vibration frequency could facilitate feature identification in downstream circuits, for example through lateral inhibition between neighboring axons with shared tuning (Mamiya, 2018).

Neurons in the FeCO population can be generally classified as either tonic (position-encoding) or phasic (movement-encoding). This division has been observed among proprioceptors of many animals, including other insects, crustaceans, and mammals. For example, mammalian muscle spindles are innervated by both phasic (Group 1a) and tonic (Group II) afferents. The same has been found in other primary mechanosensory neurons, including touch, hearing, and vestibular afferents. The ubiquity of tonic and phasic neurons suggests that these two parallel information channels are essential building blocks of sensory circuits. Now that this study has identified genetic tools that delineate tonic and phasic neurons in the proprioceptive system of Drosophila, these circuits have the potential to provide general insights into the utility of this sensory coding strategy (Mamiya, 2018).

Flies possess other chordotonal organs: the most well-studied is the Johnston's organ (JO), which detects antennal movements produced by near-field sound, wind, gravity, and touch. Unlike the FeCO, the JO monitors rotation of a body segment that is not actively controlled by muscles or coupled to the substrate. The JO is also much larger (~500 versus ~135 neurons). Despite these differences, the coding schemes of the two mechanosensory organs share some key similarities. JO neurons can be classified into tonic and phasic classes, some exhibit direction selectivity, and their axon terminals form a rough tonotopic map of frequency. The FeCO and JO share genetic and developmental homology, which suggests that mechanosensory specialization in these organs could arise through similar molecular or biomechanical mechanisms (Mamiya, 2018).

With the advent of new methods for simultaneously monitoring the activity of hundreds or thousands of neurons, a critical challenge has been to link the activity of large neuronal populations to the underlying diversity of specific cell types. Previous efforts have used statistical methods to compare the responses of single neurons to simultaneous optical or electrophysiological population recordings. This study took a different approach, which took advantage of the fact that neurons in the fly can be reliably identified across individuals. Two-photon imaging was first used to monitor activity across a population of proprioceptive sensory neurons during controlled leg movements. From this population data, spatially distinct axon branches were identified that encode specific proprioceptive stimulus features.Genetic driver lines were sought that specifically labeled each axon branch and further characterized their functional tuning with targeted calcium imaging. With this approach, it was possible to identify and characterize the major neuronal subclasses in a key proprioceptive organ (Mamiya, 2018).

With a genetic handle on position, movement, and direction pathways, it should now be possible to trace the flow of proprioceptive signals into downstream circuits and to identify the functional role of specific proprioceptor subclasses within the broader context of motor control and behavior. It is anticipated that Drosophila will provide a useful complement to other model organisms in dissecting fundamental mechanisms of proprioception and deepening understanding of this mysterious 'sixth sense' (Mamiya, 2018).

Random walk revisited: Quantification and comparative analysis of Drosophila walking trajectories

Walking trajectory is frequently measured to assess animal behavior. Air-supported spherical treadmills have been developed for real-time monitoring of animal walking trajectories. However, current systems for mice mainly employ computer mouse microcameras (chip-on-board sensors) to monitor ball motion, and these detectors exhibit technical issues with focus and rotation scale. In addition, computational methods to analyze and quantify the "random walk" of organisms are under-developed. This work has overcome the hurdle of frame-to-signal translation to develop a treadmill system with camera-based detection. Moreover, a package of mathematical methods were generated to quantify distinct aspects of Drosophila walking trajectories. By extracting and quantifying certain features of walking dynamics with high temporal resolution, it was found that depending on their internal state, flies employ different walking strategies to approach environmental cues. This camera-based treadmill system and method package may also be applicable to monitor the walking trajectories of other diverse animal species (Tsai, 2019).

TwoLumps ascending neurons mediate touch-evoked reversal of walking direction in Drosophila

External cues, including touch, enable walking animals to flexibly maneuver around obstacles and extricate themselves from dead-ends. In a screen for neurons that enable Drosophila melanogaster to retreat when it encounters a dead-end, this study identified a pair of ascending neurons, the TwoLumps Ascending (TLA) neurons. Silencing TLA activity impairs backward locomotion, whereas optogenetic activation triggers backward walking. TLA-induced reversal is mediated in part by the Moonwalker Descending Neurons (MDNs), which receive excitatory input from the TLAs. Silencing the TLAs decreases the extent to which freely walking flies back up upon encountering a physical barrier in the dark, and TLAs show calcium responses to optogenetic activation of neurons expressing the mechanosensory channel NOMPC. It is infered that TLAs convey feedforward mechanosensory stimuli to transiently activate MDNs in response to anterior body touch (Sen, 2019)

Serotonergic Modulation of Walking in Drosophila

To navigate complex environments, animals must generate highly robust, yet flexible, locomotor behaviors. For example, walking speed must be tailored to the needs of a particular environment. Not only must animals choose the correct speed and gait, they must also adapt to changing conditions and quickly respond to sudden and surprising new stimuli. Neuromodulators, particularly the small biogenic amine neurotransmitters, have the ability to rapidly alter the functional outputs of motor circuits. This study shows that the serotonergic system in the vinegar fly, Drosophila melanogaster, can modulate walking speed in a variety of contexts and also change how flies respond to sudden changes in the environment. These multifaceted roles of serotonin in locomotion are differentially mediated by a family of serotonergic receptors with distinct activities and expression patterns (Howard, 2019).

Adult Movement Defects Associated with a CORL Mutation in Drosophila Display Behavioral Plasticity

The CORL family of CNS-specific proteins share a Smad-binding region with mammalian SnoN and c-Ski protooncogenes. In this family Drosophila CORL has two mouse and two human relatives. Roles for the mouse and human CORL proteins are largely unknown. Based on genome-wide association studies linking the human CORL proteins Fussel15 and Fussel18 with ataxia, this study tested the hypothesis that dCORL mutations will cause adult movement disorders. For initial tests, side by side studies were conducted of adults with the small deletion Df(4)dCORL and eight control strains. Deletion mutants exhibit three types of behavioral plasticity. First, significant climbing defects attributable to loss of dCORL are eliminated by age. Second, significant phototaxis defects due to loss of dCORL are partially ameliorated by age and are not due to faulty photoreceptors. Third, Df(4)dCORL males raised in groups have a lower courtship index than males raised as singles though this defect is not due to loss of dCORL Subsequent tests showed that the climbing and phototaxis defects were phenocpied by dCORL(21B) and dCORL(23C) two CRISPR generated mutations. Overall, the finding that adult movement defects due to loss of dCORL are subject to age-dependent plasticity suggests new hypotheses for CORL functions in flies and mammals (Dimitriadou, 2020).

Spatiotemporally precise optogenetic activation of sensory neurons in freely walking Drosophila

Previous work has characterized how walking Drosophila coordinate the movements of individual limbs. To understand the circuit basis of this coordination, one must characterize how sensory feedback from each limb affects walking behavior. However, it has remained difficult to manipulate neural activity in individual limbs of freely moving animals. This study demonstrates a simple method for optogenetic stimulation with body side-, body segment-, and limb-specificity that does not require real-time tracking. Instead, precise locations were activated at random in time and space and use post hoc analysis to determine behavioral responses to specific activations. Using this method, this study has characterized limb coordination and walking behavior in response to transient activation of mechanosensitive bristle neurons and sweet-sensing chemoreceptor neurons. these findings reveal that activating these neurons has opposite effects on turning, and that activations in different limbs and body regions produce distinct behaviors (DeAngelis, 2020).

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).

Proprioception is vital for animals to control their locomotion behavior, although the underlying mechanisms remain to be worked out in Drosophila and other animals. This study reports that the tmc gene contributes to proprioception and sensory feedback for normal forward crawling behavior in Drosophila larvae. tmc is expressed in Drosophila larval sensory neurons. Behavioral and calcium imaging studies indicate that Drosophila TMC plays an important role in proprioception and regulation of crawling behavior. Moreover, behavioral defects due to loss of tmc function in Drosophila were rescued by expressing mammalian TMC proteins, indicative of an evolutionarily conserved function (Guo, 2016).

Several types of body-wall sensory neurons appear to play a role in the larval locomotion regulation. Silencing chordotonal cho) neurons results in increased frequency and duration of turning and reduced duration of linear locomotion, a phenotype similar to that caused by tmc mutation, suggesting that the Cho neurons and the tmc-expressing neurons might converge to the same motor output pathway. Interestingly, blocking class IV da neurons produces an opposite phenotype: fewer turns. Given that the central projection of class IV da neurons in the VNC is distinct from that of class I da neurons and bd neurons, it will be interesting to see how they regulate the same behavior in opposing manners (Guo, 2016).

Different neurons might use different mechanosensitive ion channels in coordinating proprioceptive cues, similar to what has been found in the touch-sensitive neurons. The TRPN channel NOMPC functions in class III da neurons to mediate gentle touch sensation whereas the DEG/ENaC ion channels PPK and PPK26, the TRP channel Painless, and Piezo function in class IV da neurons to mediate mechanical nociception. As to proprioception, chordotonal organs, class I and class IV da neurons and bd neurons may all contribute to proprioception to regulate larval locomotion behavior. It is reported that NOMPC is expressed in class I da neurons and bd neurons, and mutations of NOMPC cause prolonged stride duration and reduced crawling speed of mutant larvae. In contrast, the DEG/ENaC ion channels PPK and PPK26 function in class IV da neurons to modulate the extent of linear locomotion; reduction of these channel functions leads to decreased turning frequency and enhanced directional crawling (Guo, 2016).

Drosophila TMC protein exhibits sequence conservation with TMC family members in other species in the putative transmembrane domains, although it is much larger than its mouse or human homologs. It is of interest to determine whether the Drosophila TMC functions encompass a combination of functions of its mammalian homologs (Guo, 2016).

Among eight tmc genes in human and mice, tmc1 and tmc2 are found to be required for sound transduction in the hair cells of the inner ear. However, these genes are very broadly expressed, so it is possible that they might also function in other tissues. In light of the finding that Drosophila TMC functions in sensory neurons to regulate locomotion and mouse TMC1 or TMC2 functionally rescue the fly mutant phenotype, it will be interesting to test whether TMC1 and TMC2 have similar functions in addition to their involvement in hearing. This work indicates that the Drosophila tmc gene participates in proprioception. Whether mammalian tmc genes, including tmc1 and tmc2, participate in proprioception is an interesting open question (Guo, 2016).

In contrast to tmc1 and tmc2 in mammals and the Drosophila tmc gene, the tmc-1 gene of Caenorhabditis elegans was reported to contribute to high sodium sensation in ASH polymodal avoidance neurons, in which TMC-1 ion channels could be activated by high concentrations of extracellular sodium salts and permeate cations. It will be of interest to explore the potential roles of tmc genes in various species in mechanosensation or osmosensation (Guo, 2016).

How mammalian TMC1 and TMC2 function in sound transduction is still not fully understood, and whether they are the pore-forming channel subunits is under debate. It remains to be shown whether TMC1 and TMC2 can yield channel activities in heterologous expression systems, and they likely require other proteins for their function in mechanotransduction. Attempts were made to ectopically express the Drosophila tmc gene product in a variety of heterologous systems. However, no obvious mechanosensitive currents could be detected when these cells are exposed to mechanical stimuli. One possibility is that the Drosophila TMC protein fails to be trafficked to plasma membrane in the expression system that was used. Alternatively, additional components are required to form a mechanosensitive complex as gating of certain mechanogated ion channels such as NOMPC might require interactions of ion channels with extracellular matrix and/or intracellular cytoskeleton. Analyses of Drosophila tmc gene functions in larval locomotion regulation in this study, and in other future behavioral studies, may provide an opportunity to search for additional components that are necessary for the function of TMC proteins (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).

Tyramine action on motoneuron excitability and adaptable tyramine/octopamine ratios adjust Drosophila locomotion to nutritional state

Adrenergic signaling profoundly modulates animal behavior. For example, the invertebrate counterpart of norepinephrine, octopamine, and its biological precursor and functional antagonist, tyramine, adjust motor behavior to different nutritional states. In Drosophila larvae, food deprivation increases locomotor speed via octopamine-mediated structural plasticity of neuromuscular synapses, whereas tyramine reduces locomotor speed, but the underlying cellular and molecular mechanisms remain unknown. This study shows that tyramine is released into the CNS to reduce motoneuron intrinsic excitability and responses to excitatory cholinergic input, both by tyramine(honoka) receptor activation and by downstream decrease of L-type calcium current. This central effect of tyramine on motoneurons is required for the adaptive reduction of locomotor activity after feeding. Similarly, peripheral octopamine action on motoneurons has been reported to be required for increasing locomotion upon starvation. It was further shown that the level of tyramine-beta-hydroxylase (TBH), the enzyme that converts tyramine into octopamine in aminergic neurons, is increased by food deprivation, thus selecting between antagonistic amine actions on motoneurons. Therefore, octopamine and tyramine provide global but distinctly different mechanisms to regulate motoneuron excitability and behavioral plasticity, and their antagonistic actions are balanced within a dynamic range by nutritional effects on TBH (Schutzler, 2019).

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).

Two pairs of Drosophila central brain neurons mediate larval navigational strategies based on temporal light information processing

Some animals are attracted by sun light, others are highly repulsed by it. Especially for slowly moving animals, such as Drosophila larvae, direct sunlight may be perceived as noxious stimulus as it increases the risk of desiccation, DNA-damaging by UV-light and exposure to predators. For several reasons, model organisms like Drosophila larvae are well-suited for investigating how light cues are translated into an appropriate behavioral output. First, many of the genetic tools, which were created for use in adult fruit flies, work also in larvae. Second, the lower number of cells in Drosophila larvae compared to adults makes this system adequate for reconstructing neural circuits. Third, the relatively simple behavioral repertoire of larvae facilitates the study of basic functions like navigation with regards to light. Larvae navigate robustly away from a light source by the use of several sophisticated behavioral strategies which are based on temporal or spatial information processing. Two central brain neurons, the NP394-neurons, are highly important for larval light avoidance. It was even reported that these cells seem to play a functional role in a putative larval light preference switch right before pupation. However, the exact function of the NP394-neurons in light navigation remains unknown. This study shows that the functional role of NP394-neurons in larval light navigation is restricted to behaviors based on temporal information processing, but not for spatial navigation (Humberg, 2108).

Modular assays for the quantitative study of visually guided navigation in both flying and walking flies

The quantitative study of behavioral responses to visual stimuli provides crucial information about the computations executed by neural circuits. Insects have long served as powerful model systems, either when walking on air suspended balls (spherical treadmill), or flying while glued to a needle (virtual flight arena). This study presents detailed instructions for 3D-printing and assembly of arenas optimized for visually guided navigation, including codes for presenting both celestial and panorama cues. These modular arenas can be used either as virtual flight arenas, or as spherical treadmills and consist entirely of commercial and 3D-printed components placed in a temperature and humidity controlled environment. Robust optomotor responses are induced in flying Drosophila by displaying moving stripes in a cylinder surrounding the magnetically tethered fly. Similarly, changes in flight heading are induced by presenting changes in the orientation of linearly polarized UV light presented from above. Finally, responses to moving patterns are induced when individual flies are walking on an air-suspended ball. These modular assays allow for the investigation of a diverse combination navigational cues (sky and panorama) in both flying and walking flies. They can be used for the molecular dissection of neural circuitry in Drosophila and can easily be rescaled for accommodating other insects (Mathejczyk, 2020).

Fly eyes are not still: a motion illusion in Drosophila flight supports parallel visual processing

Most animals shift gaze by a 'fixate and saccade' strategy, where the fixation phase stabilizes background motion. A logical prerequisite for robust detection and tracking of moving foreground objects, therefore, is to suppress the perception of background motion. In a virtual reality magnetic tether system enabling free yaw movement, Drosophila implemented a fixate and saccade strategy in the presence of a static panorama. When the spatial wavelength of a vertical grating was below the Nyquist wavelength of the compound eyes, flies drifted continuously- and gaze could not be maintained at a single location. Because the drift occurs from a motionless stimulus-thus any perceived motion stimuli are generated by the fly itself-it is illusory, driven by perceptual aliasing. Notably, the drift speed was significantly faster than under a uniform panorama suggesting perceptual enhancement due to aliasing. Under the same visual conditions in a rigid tether paradigm, wing steering responses to the unresolvable static panorama were not distinguishable from a resolvable static pattern, suggesting visual aliasing is induced by ego motion. It is hypothesized that obstructing the control of gaze fixation also disrupts detection and tracking of objects. Using the illusory motion stimulus, it was shown that magnetically tethered Drosophila track objects robustly in flight even when gaze is not fixated as flies continuously drift. Taken together, this study provides further support for parallel visual motion processing and reveals the critical influence of body motion on visuomotor processing. Motion illusions can reveal important shared principles of information processing across taxa (Salem, 2020).

Stabilizing responses to sideslip disturbances in Drosophila melanogaster are modulated by the density of moving elements on the ground

Stabilizing responses to sideslip disturbances are a critical part of the flight control system in flies. While strongly mediated by mechanoreception, much of the final response results from the wide-field motion detection system associated with vision. In order to be effective, these responses must match the disturbance they are aimed to correct. To do this, flies must estimate the velocity of the disturbance, although it is not known how they accomplish this task when presented with natural images or dot fields. The recent finding, that motion parallax in dot fields can modulate stabilizing responses only if perceived below the fly, raises the question of whether other image statistics are also processed differently between eye regions. One such parameter is the density of elements moving in translational optic flow. Depending on the habitat, there might be strong differences in the density of elements providing information about self-motion above and below the fly, which in turn could act as selective pressures tuning the visual system to process this parameter on a regional basis. By presenting laterally moving dot fields of different densities this study found that, in Drosophila melanogaster, the amplitude of the stabilizing response is significantly affected by the number of elements in the field of view. Flies countersteer strongly within a relatively low and narrow range of element densities. But this effect is exclusive to the ventral region of the eye, and dorsal stimuli elicit an unaltered and stereotypical response regardless of the density of elements in the flow. This highlights local specialization of the eye and suggests the lower region may play a more critical role in translational flight stabilization (Ruiz, 2021).

Aerial course stabilization is impaired in motion-blind flies

Visual motion detection is among the best understood neuronal computations. As extensively investigated in tethered flies, visual motion signals are assumed to be crucial to detect and counteract involuntary course deviations. During free flight, however, course changes are also signalled by other sensory systems. Therefore, it is yet unclear to what extent motion vision contributes to course control. To address this question, flies were genetically rendered motion-blind by blocking their primary motion-sensitive neurons, and their free-flight performance was quantified. Such flies were found to have difficulties maintaining a straight flight trajectory, much like unimpaired flies in the dark. By unilateral wing clipping, an asymmetry was generated in propulsive force, and the ability of flies to compensate for this perturbation was tested. While wild-type flies showed a remarkable level of compensation, motion-blind animals exhibited pronounced circling behaviour. These results therefore directly confirm that motion vision is necessary to fly straight under realistic conditions (Aerial course stabilization is impaired in motion-blind flies (Leonte, 2021).

Mechanisms of punctuated vision in fly flight

To guide locomotion, animals control gaze via movements of their eyes, head, and/or body, but how the nervous system controls gaze during complex motor tasks remains elusive. In many animals, shifts in gaze consist of periods of smooth movement punctuated by rapid eye saccades. Notably, eye movements are constrained by anatomical limits, which requires resetting eye position. By studying tethered, flying fruit flies (Drosophila), this study showed that flies perform stereotyped head saccades to reset gaze, analogous to optokinetic nystagmus in primates. Head-reset saccades interrupted head smooth movement for as little as 50 ms-representing less than 5% of the total flight time-thereby enabling punctuated gaze stabilization. By revealing the passive mechanics of the neck joint, it was shown that head-reset saccades leverage the neck's natural elastic recoil, enabling mechanically assisted redirection of gaze. The consistent head orientation at saccade initiation, the influence of the head's angular position on saccade rate, the decrease in wing saccade frequency in head-fixed flies, and the decrease in head-reset saccade rate in flies with their head range of motion restricted together implicate proprioception as the primary trigger of head-reset saccades. Wing-reset saccades were influenced by head orientation, establishing a causal link between neck sensory signals and the execution of body saccades. Head-reset saccades were abolished when flies switched to a landing state, demonstrating that head movements are gated by behavioral state. A control architecture is proposed for active vision systems with limits in sensor range of motion (Cellini, 2021).

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).

A group of segmental premotor interneurons regulates the speed of axial locomotion in Drosophila larvae

Animals control the speed of motion to meet behavioral demands. Yet, the underlying neuronal mechanisms remain poorly understood. This study shows that a class of segmentally arrayed local interneurons (period-positive median segmental interneurons, or PMSIs) regulates the speed of peristaltic locomotion in Drosophila larvae. PMSIs formed glutamatergic synapses on motor neurons and, when optogenetically activated, inhibited motor activity, indicating that they are inhibitory premotor interneurons. Calcium imaging showed that PMSIs are rhythmically active during peristalsis with a short time delay in relation to motor neurons. Optogenetic silencing of these neurons elongated the duration of motor bursting and greatly reduced the speed of larval locomotion. These results suggest that PMSIs control the speed of axial locomotion by limiting, via inhibition, the duration of motor outputs in each segment. Similar mechanisms are found in the regulation of mammalian limb locomotion, suggesting that common strategies may be used to control the speed of animal movements in a diversity of species (Kohsaka, 2014).

PMSIs are the first interneuronal population shown to be involved in Drosophila larval locomotion. Anatomical and functional analyses strongly suggest that PMSIs are premotor local interneurons that inhibit motor neurons in the same or a neighboring segment. Previous electrophysiological analyses showed that GABA or glutamate application elicits inhibitory responses in motor neurons that reverse at near resting potential and are blocked by the chloride channel blocker picrotoxin. Based on these observations, it has been suggested that motor neurons express Cl−-permeable GABA and glutamate receptors. Glutamate-gated inhibitory channels have been identified and well characterized in arthropods and other invertebrates including C. elegans. Although no such receptors are known in vertebrates, previous structural and pharmacological analyses suggest that invertebrate glutamate-gated chloride channels are orthologous to vertebrate glycine channels. Drosophila homologs of the receptors have been cloned and shown to produce a glutamate-gated chloride current when expressed in Xenopus oocytes (Cully, 1996) and exhibit inhibitory action in Drosophila adult brain (Liu, 2013). Thus, it is likely that PMSIs inhibit motor neurons through glutamate-gated chloride channels. The motor neurons are also glutamatergic but send excitatory input to the muscles. Previous studies report that there are 40 putative vGluT-positive glutamatergic neurons in each hemisegment, of which 34 are motor neurons and six are interneurons. Since the number of PMSIs is comparable to that of the estimated glutamatagic interneurons, PMSIs most likely represent a majority of the glutamatergic interneurons in the ventral nerve cord (Kohsaka, 2014).

This study demonstrated that the duration of motor bursting and segmental muscle contraction is elongated when PMSIs are inhibited. The results indicate that PMSIs regulate the duration of motor output in each segment by terminating motor bursting. Consistent with this idea, dual-color Ca2+ imaging showed that activation of PMSIs is delayed with respect to that of the postsynaptic motor neurons. This temporal pattern allows PMSIs to regulate the time window of motor firing via inhibition. Thus, a main function of PMSIs seems to be to limit the duration of motor output (Kohsaka, 2014).

Similar roles in shaping motor outputs have been proposed for V1 neurons in mice and aIN neurons in Xenopus, both of which are inhibitory interneurons expressing Engrailed and have been proposed to share evolutionarily conserved roles. Loss or acute inactivation of V1 neurons elongates the duration of motor bursting during fictive locomotion in isolated mouse spinal cord. Xenopus aIN neurons provide early-cycle inhibition to motor neurons and other CPG interneurons during swimming. Thus, regulation by on-cycle inhibition seems to be a common mechanism for shaping the duration of motor outputs in vertebrates and in Drosophila larvae. Interestingly, PMSIs share several cellular properties with vertebrate V1 and aIN neurons. The three classes of neurons are all inhibitory premotor interneurons that are rhythmically activated during motor cycles. They are unipolar and send their axons first toward motor neurons and then extend an ascending ipsilateral axon longitudinally. Whereas V1 and aIN use glycine as the inhibitory neurotransmitter, PMSIs use glutamate, which is considered to be the invertebrate counterpart of glycine. These shared features may underlie the common function in motor control (Kohsaka, 2014).

Several mechanisms have been proposed for speed control of animal locomotion, including the recruitment of different motor neurons and change in electrophysiological properties of motor and other CPG neurons. The current results on PMSIs and previous studies on V1 and aIN neurons suggest that limiting the duration of motor firing by inhibition might be a phylogenetically conserved mechanism for speed control. In mice lacking V1 neurons, not only the duration of motor firing but also that of motor cycles is elongated, and thus the speed of locomotion is reduced. Although the role of aIN neurons in speed control has not been directly examined, close correlations have been observed between the activity of these neurons and the frequency of the tadpole swimming. This study demonstrates that blocking activities of PMSIs elongates the duration of motor bursting and reduces the speed of axial locomotion in Drosophila larvae. Taken together, these results suggest that evolutionarily distant organisms with anatomically and functionally distinct motor systems may adopt similar strategies for speed control of locomotion. It is important to note that both activation and inhibition of PMSIs activity lead to a decrease in locomotor speed (paralysis upon activation with ChR2 and slowed locomotion upon inhibition with Shits or NpHR). Thus, these neurons need to be activated at an optimum level and timing to output locomotion with appropriate speed (Kohsaka, 2014).

It still remains to be determined how the change in the duration of motor bursting affects the speed of locomotion. A simple model would be that since motor bursting in each segment is elongated in the absence of PMSI activity, it takes longer for the motor wave to propagate along the segments. In many undulatory movements, such as lamprey and leech swimming and Drosophila larval crawling, intersegmental phase lag (not intersegmental time lag) remains constant at different speeds. This is because the phase of muscle contraction in different segments must remain constant in order to maintain the same motor output pattern (e.g., forming approximately one full wave at a given time during lamprey swimming). Because of this intersegmental coordination, segmental lag of motor activity may have to be prolonged in the absence of PMSI activity to match up with the elongation of segmental motor bursting; otherwise, too many muscle segments would contract at the same time during peristalsis. Indeed, electrophysiological recordings showed that intersegmental time lag of motor firing was prolonged to a similar extent as the motor bursting (~2 fold) when PMSI activity was silenced. Likewise, in mice lacking V1 neurons, while the left-right and flexor-extensor coordination is maintained, both motor bursting and step cycles are elongated to a similar extent (2- to 3-fold). Thus, a common strategy, limiting the duration of motor bursting, may be used to regulate the speed of diverse animal locomotion such as larval locomotion and mammalian limb movements because it leads to changes in the most critical parameters of the speed, intersegmental time delay in axial locomotion, and left-right/flexor-extensor step cycle in limb locomotion. Understanding how intersegmental coordination is regulated in Drosophila larvae is an important future goal (Kohsaka, 2014).

It is also important to explore what might be the upstream neural circuits that activate PMSIs. Good candidates are multidendritic neurons, which are known to be required for fast larval locomotion and believed to feedback muscle contraction status. Another interesting possibility is that PMSIs control the speed of locomotion in response to environmental changes such as temperature or to meet internal demands such as hunger. Preliminary data using the GRASP technique suggest that PMSIs indeed receive afferent projections from sensory neurons. Once the upstream neurons are identified, the input-output relationship between these neurons and PMSIs can be systematically studied using optogenetics and other methods. It is anticipated that such analyses will not only clarify the roles of PMSIs in local neural circuits, but also shed light on conserved mechanisms by which inhibitory interneurons regulate animal locomotion (Kohsaka, 2014).

Identification of excitatory premotor interneurons which regulate local muscle contraction during Drosophila larval locomotion

Drosophila larval locomotion was used as a model to elucidate the working principles of motor circuits. Larval locomotion is generated by rhythmic and sequential contractions of body-wall muscles from the posterior to anterior segments, which in turn are regulated by motor neurons present in the corresponding neuromeres. Motor neurons are known to receive both excitatory and inhibitory inputs, combined action of which likely regulates patterned motor activity during locomotion. Although recent studies identified candidate inhibitory premotor interneurons, the identity of premotor interneurons that provide excitatory drive to motor neurons during locomotion remains unknown. This study searched for and identified two putative excitatory premotor interneurons in this system, termed CLI1 and CLI2 (cholinergic lateral interneuron 1 and 2). These neurons were segmentally arrayed and activated sequentially from the posterior to anterior segments during peristalsis. Consistent with their being excitatory premotor interneurons, the CLIs formed (GFP Reconstruction Across Synaptic Partners) (GRASP)- and ChAT-positive putative synapses with motoneurons and were active just prior to motoneuronal firing in each segment. Moreover, local activation of CLI1s induced contraction of muscles in the corresponding body segments. Taken together, these results suggest that the CLIs directly activate motoneurons sequentially along the segments during larval locomotion (Hasegawa, 2016).

Animals perform various types of rhythmic movements such as respiration, chewing and locomotion for their survival. These rhythmic movements are thought to be regulated by neuronal circuits termed central pattern generators (CPGs). CPGs consist of interneurons and motoneurons whose rhythmic activities induce coordinated patterns of muscle contraction. Although CPGs are regulated by descending and sensory inputs, rhythms very similar to those seen in the intact animal can be generated without these inputs. Because CPGs of invertebrates and vertebrates share many characteristics, CPGs in one animal could be a model for other animals. Moreover, because CPGs show many characteristics common to other neuronal systems, CPGs could be a general model linking neuronal circuits to behaviour. Despite the efforts to elucidate the function of CPGs, their identities and functional mechanisms are not completely understood, in particular in animals with a large central nervous system (CNS). This is partly because manipulating the function of specific neurons in the neural circuits is often difficult, especially in animals with vast numbers of neurons such as mammals (Hasegawa, 2016).

The Drosophila larva is emerging as an excellent model system for studies of CPGs because one can use sophisticated genetic methods, such as the Gal4-UAS system, to manipulate and visualize the activity of specific component neurons in a moderately sized CNS consisting of ~10,000 neurons. Larval forward locomotion is executed by the sequential contraction of muscles from the posterior to the anterior segments. Motoneurons in the ventral nerve cord (VNC) actualize the sequential muscle contraction by being activated from the posterior to the anterior segments during forward locomotion. CPGs responsible for the locomotion seem to be present in the VNC, since neuronal circuits in the thoracic and abdominal segments have been shown to be sufficient for generating the behavior (Berni, 2012; Berni, 2015). Calcium imaging of the entire CNS has visualized neurons that are active during larval locomotion including those in the brain, sub-oesophageal zone (SEZ), and the VNC16. However, the identities of these neurons are only beginning to be characterized (Hasegawa, 2016).

Previous studies showed that motor neurons in the VNC receive both excitatory and inhibitory inputs. It is therefore likely that specific patterns of motoneuron activation are regulated by the balance and the timing of excitatory and inhibitory inputs as shown in other systems. Recently, two types of inhibitory premotor interneurons that regulate larval locomotion have been identified. PMSIs (period-positive median segmental interneurons) are glutamatergic inhibitory premotor interneurons that regulate the speed of larval locomotion (Kohsaka, 2014). Another glutamatergic interneuron, GVLIs (glutamatergic ventro-lateral interneurons) seem to function as premotor inhibitory neurons to terminate motor bursting (Itakura, 2015). In contrast, premotor interneurons that provide excitatory inputs to motor neurons during locomotion remain to be identified, although they are known to be cholinergic. A recent study identified two cholinergic descending interneurons that form putative synaptic contacts with segmental motoneurons (Couton, 2015). However, whether they are active and play roles during locomotion remains unknown (Hasegawa, 2016).

This study sought and identified putative excitatory premotor interneurons that activate motoneurons during locomotion. These neurons, termed CLI1 and CLI2 (cholinergic lateral interneuron), are segmental interneurons that show wave-like activity during locomotion concurrent with the activity propagation of motoneurons. Consistent with CLIs being excitatory premotor neurons, these neurons form GRASP- and ChAT-positive synaptic contacts with motor neurons and are activated just before the activation of motoneurons in each segment. In addition, forced activation of these neurons locally induces the contraction of muscles. These results suggest that wave-like activity of CLIs activates motoneurons sequentially along the segments during forward locomotion (Hasegawa, 2016).

What are the circuit mechanisms that regulate Drosophila larval locomotion? To answer this question, it is necessary first to identify the neuronal components of the circuits. Excitatory inputs are critical for the generation of locomotor rhythms in various animals. However, identities and roles of excitatory interneurons that regulate Drosophila larval locomotion are unknown. The present study sought such excitatory interneurons using calcium imaging and identified CLI1s and CLI2s as candidate interneurons that excite motor neurons. Anatomical and behavioural studies suggest that these neurons directly activate motoneurons locally in each segment during larval locomotion (Hasegawa, 2016).

The following four lines of evidence suggest that CLIs are excitatory premotor interneurons: (i) CLIs are activated just before the activation of motoneurons in each segment during fictive locomotion, consistent with their providing excitatory drive to motoneurons. (ii) CLIs express ChAT, which synthesizes acetylcholine, a neurotransmitter known to excite motor neurons in this system. (iii) CLIs form GRASP-positive contacts with motoneurons. (iv) Local activation of CLIs results in the contraction of muscles in the corresponding body segments. Although these data are consistent with direct connection between CLIs and motoneurons, it remains possible that CLIs also excite motor neurons indirectly via other interneurons (Hasegawa, 2016).

CLI1s and CLI2s share many morphological and functional characteristics. i) They are neighboring neurons that send axons along a common path to reach the neuropile. This suggests that they are sibling neurons derived from the same neuroblast. Consistent with this notion, they also share the expression of R47E12-Gal4. ii) They both project axons along the same fascicle in the anterior commissure and locally innervate motor neurons in the contralateral side of the CNS. iii) They both are cholinergic premotor interneurons and are activated simultaneously during forward locomotion. iv) Activation of these neurons elicits muscle contraction. Taken together, these observations suggest that CLI1s and CLI2s belong to a class of interneurons that fulfill common function(s). There are also distinct features between these two neurons. i) CLI1s innervate the medial neuropile while CLI2s innervate a lateral region, suggesting that they target distinct neurons. ii) CLI1s but not CLI2s project to the next anterior segment. iii) CLI2s are active both during forward and backward locomotion, whereas CLI1s are active only during forward locomotion. Thus, CLI1s only participate in forward locomotion and may activate motor neurons not only in the same segment but also in the next anterior segment, and thus contribute to feed-forward propagation of motor excitation. In contrast, CLI2s may act locally to excite motoneurons only in the same segment and do so both during forward and backward locomotion (Hasegawa, 2016).

It is currently unknown what motor neurons are the targets of CLI1/2s. Dendrites of motoneurons that innervate different muscle domains form myotopic map along both antero-posterior and medio-lateral axes. The axon terminals of CLI1s are located in the medial neuropile, a region occupied by the dendrites of motoneurons innervating ventral muscles. Thus, CLI1s may form synaptic contacts with the ventral motoneurons. Similarly, candidate targets of CLI2s are dorsal motoneurons, since axon terminals of CLI2s are located in a lateral region occupied by these motoneurons. Consistent with this, it was observed that lifting of the tail, which is likely caused by dorsal muscle contraction when CLI2s but not CLI1s is activated. Moreover, CLI1s and CLI2s are activated at a similar timing as aCC in the same segment, a motor neuron that innervates a dorsal muscle and is activated simultaneously with other motor neurons innervating dorsal/ventral internal muscles. Future studies such as connectomic analyses using serial EM will determine more precisely the downstream circuits of the CLIs (Hasegawa, 2016).

It is also important to determine in the future the upstream circuits of the CLIs. Since dendritic region of CLI1s and CLI2s partially overlap, these neurons may share common upstream neurons. In particular, because the wave-like activity of CLIs was observed in the isolated CNS that receives no sensory inputs, the activity of CLIs must be regulated by the central circuits that generate a rhythm in an autonomous manner. However, it is also possible that CLIs are activated in response to specific sensory stimulation. Recently, neuronal circuits regulating larval behavior in response to specific sensory stimuli have been identified. It will be interesting to study the link between these circuits and CLIs (Hasegawa, 2016).

The wave-like activity of CLIs that occurs concomitant with motor activation strongly suggests that these neurons contribute to sequential activation of motor neurons along the segments during locomotion. Since these neurons are commissural neurons, they may also play a role in left-right coordination, as has been proposed for Dbx1-positive neurons in vertebrates and recently identified EL neurons in Drosophila. However, loss-of-function analyses thus far failed to reveal roles of CLIs in larval behaviors. Shibirets, tetanus toxin light chain, Kir2.1, hid and reaper, and ChAT-RNAi were used to inhibit the function of CLIs but no obvious phenotypes were observed. This could be due to insufficient silencing of these neurons by the activity manipulations. It could also be due to the redundancy in the circuit function. It should be noted in this regard that there are likely more CLIs-like neurons present in each segment. The axon terminals of CLI1 and CLI2 only cover part of the motor dendritic region, suggesting other neurons excite motor neurons not targeted by CLIs. Indeed, preliminary results obtained by the ongoing EM reconstruction of the larval CNS suggest that about 10 neurons, in the same neuroblast lineage as CLIs, send their axons locally and contralaterally to the motor region along the common path as CLI1s and CLI2s. It is likely that a group of CLIs-like neurons function in a similar manner and together excite the entire motor system. Unfortunately, direct testing of this possibility is not currently feasible due to the unavailability of Gal4 lines specific to this lineage (Hasegawa, 2016).

Recently, research has identified two classes of segmental premotor inhibitory interneurons PMSIs and GVLIs. These neurons are activated slightly later than the motor neurons and appear to inhibit the activity of motoneurons at distinct timings during the motor cycle: PMSIs at an early phase and GVLIs at a final phase of motoneuronal activation (Kohsaka, 2014; Itakura, 2015). This study identified CLIs that are activated prior to motor neurons and appear to provide an excitatory drive to the motoneurons. These three classes of premotor interneurons likely help shape the pattern of motor activity by providing excitatory and inhibitory inputs to motoneurons at distinct phases of the motor cycle. Since there are only ~400 interneurons per hemisegment in the larval ventral nerve cord, whose connectivity is being reconstructed, it is hoped that all major classes of premotor interneurons in this system will be identified in the near future. Systematic analyses of CLIs, PMSIs, GVLIs and other premotor neurons will elucidate how the motor patterns generating distinct behaviors are shaped by the combinatorial action of premotor interneurons (Hasegawa, 2016).

Sensorimotor pathway controlling stopping behavior during chemotaxis in the Drosophila melanogaster larva

Sensory navigation results from coordinated transitions between distinct behavioral programs. During chemotaxis in the Drosophila melanogaster larva, the detection of positive odor gradients extends runs while negative gradients promote stops and turns. This algorithm represents a foundation for the control of sensory navigation across phyla. The present work identified an olfactory descending neuron, PDM-DN, which plays a pivotal role in the organization of stops and turns in response to the detection of graded changes in odor concentrations. Artificial activation of this descending neuron induces deterministic stops followed by the initiation of turning maneuvers through head casts. Using electron microscopy, the main pathway was reconstructed that connects the PDM-DN neuron to the peripheral olfactory system and to the pre-motor circuit responsible for the actuation of forward peristalsis. The results set the stage for a detailed mechanistic analysis of the sensorimotor conversion of graded olfactory inputs into action selection to perform goal-oriented navigation (Tastekin, 2018).

Piezo-like gene regulates locomotion in Drosophila larvae

To maintain proper locomotive patterns, animals constantly monitor body posture with their proprioceptive receptors. In Drosophila, the chordotonal organs (Cho) are especially important in the regulation of locomotion pattern. However, how Cho neurons that are normally activated with sound (vibration) transduce static displacement caused by body position change remains unclear. This study reports that piezo-like (pzl), a homolog for mammalian piezo1 and 2, is essential for Cho's function in locomotion. The mutant allele of pzl showed severe defects in crawling pattern and body gesture control, which were rescued by expressing Pzl specifically in Cho neurons. The ability of Cho neurons to respond to micrometer-scale body wall displacement requires pzl. Intriguingly, human or mouse Piezo1 can rescue pzl-mutant phenotypes, suggesting a conserved role of the Piezo-family proteins in locomotion (Hu, 2019).

'Proprioception' refers to the sensory input and feedback by which animals keep track of and control different parts of their bodies for balance and correct locomotive patterns. Selective loss of function of proprioceptors results in movement defects in human. Proprioception is thought to be mediated with mechanosensitive proprioceptors. In insects, some chordotonal organs (Cho) serve proprioceptive roles. Perturbation of Cho neurons in Drosophila results in defective locomotion and posture control (Hu, 2019).

Despite Cho's roles in locomotion, the mechanism underlying their mechanosensation to static displacement remains largely unknown. Mechanosensation that mediates the detection of touch, nociception, hearing, and proprioception is an important sensory modality. In many circumstances, especially proprioception, the identity of the mechanosensitive neurons or the channels is largely unknown. In Drosophila, the mechanosensitive channel NompC and other putative channels are crucial for larval crawling. Humans with dominant mutations of Piezo2 suffer from different forms of distal arthrogryposis. The other member of the Piezo family, Piezo1, however, has broader roles. Structures of the mouse Piezo1 protein were recently solved, revealing a trimeric propeller-like structure. Unlike most animals that have two piezo genes, only one ortholog was reported in Drosophila (Dmpiezo). This study reports a gene named piezo-like (pzl; CG45783), a homolog of piezo gene families, and explores its roles in locomotion regulation in Drosophila (Hu, 2019).

In the fruit fly, Cho neurons of the Johnston organ in the antenna are the major sensors for airborne sound, gravity, and wind. Moreover, larval Cho was reported to sense low temperature. Previous work and the current study suggest that Cho neurons are required for Drosophila locomotion. These studies raise the possibility that Cho is capable of integrating multiple sensory cues to facilitate the animals' survival in a complex environment with cross-modal information (Hu, 2019).

It appears that different roles of Cho neurons rely on distinct mechanotransduction channels. This study observed only a mild defect in the pzl-mutant larvae to low-frequency vibration but not to the stimuli to which larval Cho neurons are optimally tuned. Considering that low-frequency vibration may cause stronger displacement at the same sound level, the defect of pzl mutant may result from lower sensitivity to static displacement. Alternatively, it is possible that pzl contributes to sound sensing at certain frequencies. Nevertheless, it appears that pzl plays a more important role in sensing static displacement (Hu, 2019).

In Drosophila, multiple types of proprioceptive neurons were found to participate in the locomotion regulation. Blocking nociceptive class IV da neurons causes the animals to move relatively straight on a plane surface, while silencing class I da neurons and bd neurons resulted in an opposite phenotype-increased number and duration of turning and reduced linear locomotion. Larvae with loss of function of Cho neurons showed more turning and backward movement. It seems that the Cho neurons and class I da/bd neurons converge at least partially onto the same downstream motor pathway (Hu, 2019).

All these proprioceptors may use different mechanotransduction channels to coordinate mechanical cues. Class IV da neurons modulate the extent of linear locomotion via the DEG/ENaC ion channels. In contrast, NompC and Dmtmc function in class I da neurons and bd neurons to regulate stride duration and crawling speed. The present study identified a gene, pzl, and its function in Cho, adding new knowledge to transduction mechanisms in proprioceptive neurons. Notably, RNAi knockdown of pzl appeared to have more head lifting compared with pzl knockout. The mRNA levels of Dmpiezo and nompC were slightly increased in the pzl knockout, suggesting that the mechanotransduction channels may have compensatory roles in regulating animal behaviors (Hu, 2019).

It has been demonstrated that mammalian piezo1 and piezo2 as well as fly DmPiezo are pore-forming channel subunits. Given its conservation with mammalian Piezo, attempts were made to record Pzl'’s channel activity by ectopically expressing pzl in a variety of heterologous systems. However, no channel activity of Pzl was detected in these experimental settings. It is very likely that the Drosophila Pzl cannot achieve a detectable level of plasma membrane proteins, because immunostaining for the protein tags fused to Pzl failed to show any signal (Hu, 2019).

In an in vivo ectopic expression system, however, fluorescence was observed for the Pzl-GFP fusion protein. Still no mechano-gated current was recorded. It is possible that Pzl fails to be trafficked to the plasma membrane at all, because of a lack of necessary molecular partners. Alternatively, additional components may be required for Pzl to form a functional channel. These results revealed an interesting feature of Pzl that distinguishes it from other Piezo proteins: Pzl is more dependent on other partners or naive environments to be fully functional. Besides, although Dmpiezo in flies has been reported to be involved only in nociception, mammalian Piezo proteins, especially Piezo1, are found to be essential in many aspects of mechanotransduction functions. This study showed that pzl has very broad expression in adult flies, suggesting diverse roles of the pzl gene (Hu, 2019).

Regulation of forward and backward locomotion through intersegmental feedback circuits in Drosophila larvae

Animal locomotion requires spatiotemporally coordinated contraction of muscles throughout the body. This study investigate how contractions of antagonistic groups of muscles are intersegmentally coordinated during bidirectional crawling of Drosophila larvae. Two pairs of higher-order premotor excitatory interneurons present in each abdominal neuromere were identified that intersegmentally provide feedback to the adjacent neuromere during motor propagation. The two feedback neuron pairs are differentially active during either forward or backward locomotion but commonly target a group of premotor interneurons that together provide excitatory inputs to transverse muscles and inhibitory inputs to the antagonistic longitudinal muscles. Inhibition of either feedback neuron pair compromises contraction of transverse muscles in a direction-specific manner. These results suggest that the intersegmental feedback neurons coordinate contraction of synergistic muscles by acting as delay circuits representing the phase lag between segments. The identified circuit architecture also shows how bidirectional motor networks could be economically embedded in the nervous system (Kohsaka, 2019).

System level analysis of motor-related neural activities in larval Drosophila

The way in which the central nervous system (CNS) governs animal movement is complex and difficult to solve solely by the analyses of muscle movement patterns. This problem is tackled by observing the activity of a large population of neurons in the CNS of larval Drosophila. Focus was placed on two major behaviors of the larvae - forward and backward locomotion - and the neuronal activity related to these behaviors during the fictive locomotion that occurs spontaneously in the isolated CNS. A genetically-encoded calcium indicator, GCaMP, and a nuclear marker were analyzed in all neurons, and then digitally scanned light-sheet microscopy was used to record (at a fast frame rate) neural activities in the entire ventral nerve cord (VNC). The experimental procedures and computational pipeline enabled systematic identification of neurons that showed characteristic motor activities in larval Drosophila. Cells were found whose activity was biased toward forward locomotion and others biased toward backward locomotion. In particular, neurons near the boundary of the subesophageal zone (SEZ) and thoracic neuromeres were identified that were strongly active during an early phase of backward but not forward fictive locomotion (Yoon, 2019).

Optimal searching behaviour generated intrinsically by the central pattern generator for locomotion

Efficient searching for resources such as food by animals is key to their survival. It has been proposed that diverse animals from insects to sharks and humans adopt searching patterns that resemble a simple Levy random walk, which is theoretically optimal for 'blind foragers' to locate sparse, patchy resources. To test if such patterns are generated intrinsically, or arise via environmental interactions, free-moving Drosophila larvae were tracked with (and without) blocked synaptic activity in the brain, suboesophageal ganglion (SOG) and sensory neurons. In brain-blocked larvae, extended substrate exploration was found to emerge as multi-scale movement paths similar to truncated Levy walks. Strikingly, power-law exponents of brain/SOG/sensory-blocked larvae averaged 1.96, close to a theoretical optimum (micro congruent with 2.0) for locating sparse resources. Thus, efficient spatial exploration can emerge from autonomous patterns in neural activity. These results provide the strongest evidence so far for the intrinsic generation of Levy-like movement patterns (Sims, 2019).

A multilayer circuit architecture for the generation of distinct locomotor behaviors in Drosophila

Animals generate diverse motor behaviors, yet how the same motor neurons (MNs) generate two distinct or antagonistic behaviors remains an open question. This study has characterized Drosophila larval muscle activity patterns and premotor/motor circuits to understand how they generate forward and backward locomotion. All body wall MNs were shown to be activated during both behaviors, but a subset of MNs change recruitment timing for each behavior. TEM was used to reconstruct a full segment of all 60 MNs and 236 premotor neurons (PMNs), including differentially-recruited MNs. Analysis of this comprehensive connectome identified PMN-MN 'labeled line' connectivity; PMN-MN combinatorial connectivity; asymmetric neuronal morphology; and PMN-MN circuit motifs that could all contribute to generating distinct behaviors. A recurrent network model was generated that reproduced the observed behaviors, and functional optogenetics was used to validate selected model predictions. This PMN-MN connectome will provide a foundation for analyzing the full suite of larval behaviors (Zarin, 2019).

This study reports a comprehensive larval proprioceptor-PMN-MN connectome and describes individual muscle/MN phase activity during both forward and backward locomotor behaviors. PMN-MN connectivity motifs were identified that could generate muscle activity phase relationships, and selected experimental validation was performed. Proprioceptor-PMN connectivity was identified that provides an anatomical explanation for the role of proprioception in promoting locomotor velocity, and it identifies a new candidate escape motor circuit. Finally, a recurrent network model was generated that produces the observed sequence of motor activity, showing that the identified pool of premotor neurons is sufficient to generate two distinct larval behaviors. It is concluded that different locomotor behaviors can be generated by a specific group of premotor neurons generating behavior-specific motor rhythms (Zarin, 2019a and b).

Locomotion is a rhythmic and flexible motor behavior that enables animals to explore and interact with their environment. Birds and insects fly, fish swim, limbed animals walk and run, and soft-body invertebrates crawl. In all cases, locomotion results from coordinated activity of muscles with different biomechanical output. This precisely regulated task is mediated by neural circuits composed of motor neurons (MNs), premotor interneurons (PMNs), proprioceptors, and descending command-like neurons. A partial map of neurons and circuits regulating rhythmic locomotion have been made in mouse, cat, fish, tadpole, lamprey, leech, crayfish, and worm. These pioneering studies have provided a wealth of information on motor circuits, but with the exception of C. elegans, there has been no system where all MNs and PMNs have been identified and characterized. Thus, a comprehensive picture of how an ensemble of interconnected neurons generate diverse locomotor behaviors is missing (Zarin, 2019a and b).

How does the Drosophila larva executes multiple behaviors, in particular forward versus backward locomotion (Carreira-Rosario, 2018). Are there different motor neurons used in each behavior? Are the same motor neurons used but with distinct patterns of activity determined by premotor inputs? How does the ensemble of premotor and motor neurons generate additional behaviors, such as escape behavior via lateral rolling? A rigorous answer to these questions requires both comprehensive anatomical information -- i.e., a premotor/motor neuron connectome -- and the ability to measure rhythmic neuronal activity and perform functional experiments. All of these tools are currently available in Drosophila, and this study used them to characterize the neuronal circuitry used to generate forward and backward locomotion, and how proprioception is integrated by the PMN ensemble (Zarin, 2019a and b).

The Drosophila larva is composed of 3 thoracic (T1-T3) and 9 abdominal segments (A1-A9), with sensory neurons extending from the periphery into the CNS, and motor neurons extending out of the CNS to innervate body wall muscles. Most segments contain 30 bilateral body wall muscles that are grouped by spatial location and orientation: dorsal longitudinal (DL; includes previously described DA and some DO muscles), dorsal oblique (DO), ventral longitudinal (VL), ventral oblique (VO), ventral acute (VA) and lateral transverse (LT). Using these muscles, the larval nervous system can generate forward locomotion, backward locomotion, turning, hunching, digging, self-righting, and escape. This study focused on forward and backward locomotion. Forward crawling behavior in larvae involves a peristaltic contraction wave from posterior to anterior segments; backward crawling entails a posterior propagation of the contraction wave (Zarin, 2019a and b).

Body wall muscles are innervated by approximately 60 MNs per segment, consisting of 28 left/right pairs that typically each innervate one muscle, and whose neuromuscular junctions have big boutons, therefore also called type-Ib MNs; two pairs of type-Is (small bouton) MNs that innervate large groups of dorsal or ventral muscles; three type II ventral unpaired median MNs that provide octopaminergic innervation to most muscles; and one or two type III insulinergic MNs innervating muscle 12. All MNs in segment A1 have been identified by backfills from their target muscles, and several have been shown to be rhythmically active during larval locomotion, but only a few of their premotor inputs have been described. Some excitatory PMNs are involved in initiating activity in their target MNs, while some inhibitory PMNs limit the duration of MN activity or produce intrasegmental activity offsets. Interestingly, some PMNs are active specifically during forward locomotion or backward locomotion. In addition, there are six pair of proprioceptor neurons in each abdominal segment (ddaE, ddaD, vpda, dmd1, dbd and vbd). They are important for promoting locomotor velocity and posture, and some of their CNS targets have been identified, but to date little is known about how or if they are directly connected to the PMN/MN circuits (Zarin, 2019a and b).

It is a major goal of neuroscience to comprehensively reconstruct neuronal circuits that generate specific behaviors, but to date this has been done only in C. elegans. Recent studies in mice and zebrafish have shed light on the overall distribution of PMNs and their connections to several well-defined MN pools. However, it remains unknown if there are additional PMNs that have yet to be characterized, nor are their any insights into potential connections between PMNs themselves, which would be important for understanding the network properties that produce coordinated motor output. In the locomotor central pattern generator circuitry of leech, lamprey, and crayfish, the synaptic connectivity between PMNs or between PMNs and other interneurons are known to play critical roles in regulating the swimming behavior. However, it is difficult to be certain that all the neural components and connections of these circuits have been identified. Thus, the comprehensive anatomical circuitry reconstructed in this study provides an anatomical constraint on the functional connectivity used to drive larval locomotion; all synaptically-connected neurons may not be relevant, but at least no highly connected local PMN is absent from this analysis (Zarin, 2019a and b).

The current results confirm and significantly extend previous studies of Drosophila larval locomotion. For example, a recent study has shown that the GABAergic A14a inhibitory PMN (also called iIN1) selectively inhibits MNs innervating muscle 22/LT2 (CMuG F4; CMuG refers to Co-active Muscle Group), thereby delaying muscle contraction relative to muscle 5/LO1 (CMuG F2). This study was extended by showing that A14a also disinhibits MNs in early CMuGs F1/2 via the inhibitory PMN A02e. Thus, A14a both inhibits late CMuGs and disinhibits early CMuGs. In addition, previous work has suggested that all MNs receive simultaneous excitatory inputs from different cholinergic PMNs. However, dual calcium imaging data of the A27h excitatory PMN shows that it is active during CMuG F4 and not earlier. Therefore, MNs may receive temporally distinct excitatory inputs, in addition to the previously reported temporally distinct inhibitory inputs. This study has identified dozens of new PMNs that are candidates for regulating motor rhythms; functional analysis of all of these PMNs is beyond the scope of this paper, particularly due to the additional work required to screen and identify Gal4/LexA lines selectively targeting these PMNs, but the predictions of this paper are clear and testable when reagents become available (Zarin, 2019a and b).

MNs innervating a single Spatial Muscle Group (SMuG) belong to more than one CMuG, therefore SMuGs do not generally match CMuGs. This could be due to the several reasons: (1) MNs in each SMuGs receive inputs from overlapping but not identical array of PMNs. (2) Different MNs in the same SMuG receive a different number of synapses from the same PMN. (3) MNs in the same SMuG vary in overall dendritic size and total number of post-synapses, thereby resulting in MNs of the same SMuGs fall into different CMuGs (Zarin, 2019a and b).

This study demonstrates that during both forward and backward crawling, most of longitudinal and transverse muscles of a given segment contract as early and late groups, respectively. In contrast, muscles with oblique or acute orientation often show different phase relationships during forward and backward crawling. Future studies will be needed to provide a biomechanical explanation for why oblique muscles -- but not longitudinal or transverse muscles -- need to be recruited differentially during forward or backward crawling. Also, it will be interesting to determine which spatial muscle groups (e.g., either or both VOs and VLs) are responsible for elevating cuticular denticles during propagation of the peristaltic wave in forward and backward crawling; if the VOs, it would mean that lifting the denticles occurs at different phases of the crawl cycle in forward and backward locomotion. Finally, understanding how the premotor-motor circuits described in this study are used to generate diverse larval motor behaviors will shed light on mechanisms underlying the multi-functionality of neuronal circuits (Zarin, 2019a and b).

A recent study has reported that proprioceptive sensory neurons (dbd, vbd, vpda, dmd1, ddaE, and ddaD) show sequential activity during forward crawling. dbd responds to stretching and whereas the other five classes are activated by muscle contraction (Vaadia, 2019). All proprioceptors show connectivity to the tier of PMNs described in this study, and this study has identified circuit motifs that are consistent with the observed timing and excitatory function of each proprioceptor neuron. It will be of great interest perform functional experiments to test these anatomical circuit motifs for functional relevance (Zarin, 2019a and b).

A recurrent network model accurately predicts the order of activation of specific PMNs, yet many of its parameters remain unconstrained, and some PMNs may have biological activity inconsistent with activity predicted by this model. Sources of uncertainty in the model include incomplete reconstruction of inter-segmental connectivity and descending command inputs, the potential role of gap junctions (which are not resolved in the TEM reconstruction), as well as incomplete characterization of PMN and MN biophysical properties. Recent studies have suggested that models constrained by TEM reconstructions of neuronal connectivity are capable of predicting features of neuronal activity and function in the Drosophila olfactory and visual systems, despite the unavoidable uncertainty in some model parameters. Similarly, for the locomotor circuit described in this study, it is anticipated that the addition of model constraints from future experiments will lead to progressively more accurate models of PMN and MN dynamics. Despite it's limitations, the ability for the PMN network to generate appropriate muscle timing for two distinct behaviors in the absence of any third-layer or command-like interneurons suggests that a single layer of recurrent circuitry is sufficient to generate multiple behavioral outputs, and provides insight into the network architecture of multifunctional pattern generating circuits (Zarin, 2019a and b).

Previous work in other animal models have identified multifunctional muscles involved in more than one motor behavior: swimming and crawling in C. elegans and leech; walking and flight in locust; respiratory and non-respiratory functions of mammalian diaphragm muscle unifunctional muscles which are only active in one specific behavior in the lobster Homarus americanus; swimming in the marine mollusk Tritonia diomedea; and muscles in different regions of crab and lobster stomach. Single-muscle calcium imaging data indicates that all imaged larval body wall muscles are bifunctional and are activated during both forward and backward locomotion. It will be interesting to determine if all imaged muscles are also involved in other larval behaviors, such as escape rolling, self-righting, turning, or digging. It is likely that there are different CMuGs for each behavior, as this study has seen for forward and backward locomotion, raising the question of how different CMuGs are generated for each distinct behavior (Zarin, 2019a and b).

Circadian and Genetic Modulation of Visually-Guided Navigation in Drosophila Larvae

Organisms possess an endogenous molecular clock which enables them to adapt to environmental rhythms and to synchronize their metabolism and behavior accordingly. Circadian rhythms govern daily oscillations in numerous physiological processes. Drosophila larvae have relatively simple nervous system compared to their adult counterparts, yet they both share a homologous molecular clock with mammals, governed by interlocking transcriptional feedback loops with highly conserved constituents. Larvae exhibit a robust light avoidance behavior, presumably enabling them to avoid predators and desiccation, and DNA-damage by exposure to ultraviolet light, hence are crucial for survival. Circadian rhythm has been shown to alter light-dark preference, however it remains unclear how distinct behavioral strategies are modulated by circadian time. To address this question, this study investigate the larval visual navigation at different time-points of the day employing a computer-based tracking system, which allows detailed evaluation of distinct navigation strategies. The results show that due to circadian modulation specific to light information processing, larvae avoid light most efficiently at dawn, and a functioning clock mechanism at both molecular and neuro-signaling level is necessary to conduct this modulation (Asirim, 2020).

Interspecies variation of larval locomotion kinematics in the genus Drosophila and its relation to habitat temperature

Speed and trajectory of locomotion are the characteristic traits of individual species. Locomotion kinematics may have been shaped during evolution towards increased survival in the habitats of each species. Although kinematics of locomotion is thought to be influenced by habitats, the quantitative relation between the kinematics and environmental factors has not been fully revealed. Comparative analyses of larval locomotion was performed in 11 Drosophila species. Larval locomotion kinematics was found to be divergent among the species. The diversity is not correlated to the body length but is correlated instead to the habitat temperature of the species. Phylogenetic analyses using Bayesian inference suggest that the evolutionary rate of the kinematics is diverse among phylogenetic tree branches. The results of this study imply that the kinematics of larval locomotion has diverged in the evolutionary history of the genus Drosophila and evolved under the effects of the ambient temperature of habitats (Matsuo, 2021).

Characterization of proprioceptive system dynamics in behaving Drosophila larvae using high-speed volumetric microscopy

Proprioceptors provide feedback about body position that is essential for coordinated movement. Proprioceptive sensing of the position of rigid joints has been described in detail in several systems; however, it is not known how animals with a flexible skeleton encode their body positions. Understanding how diverse larval body positions are dynamically encoded requires knowledge of proprioceptor activity patterns in vivo during natural movement. This study used high-speed volumetric swept confocally aligned planar excitation (SCAPE) microscopy in crawling Drosophila larvae to simultaneously track the position, deformation, and intracellular calcium activity of their multidendritic proprioceptors. Most proprioceptive neurons were found to activate during segment contraction, although one subtype was activated by extension. During cycles of segment contraction and extension, different proprioceptor types exhibited sequential activity, providing a continuum of position encoding during all phases of crawling. This sequential activity was related to the dynamics of each neuron's terminal processes, and could endow each proprioceptor with a specific role in monitoring different aspects of body-wall deformation. This study demonstrates this deformation encoding both during progression of contraction waves during locomotion as well as during less stereotyped, asymmetric exploration behavior. The results provide powerful new insights into the body-wide neuronal dynamics of the proprioceptive system in crawling Drosophila, and demonstrate the utility of the SCAPE microscopy approach for characterization of neural encoding throughout the nervous system of a freely behaving animal (Vaadia, 2019).

This study demonstrates a new approach for live volumetric imaging of sensory activity in behaving animals, leveraging an optimized form of high-speed SCAPE microscopy. This methodology was used to examine the activity patterns of a heterogeneous collection of proprioceptive neurons during crawling, as well as during more complex movements such as head turning and retraction, to determine how larvae sense body-shape dynamics. Imaging revealed 3D distortion of proprioceptive dendrites during movement and GCaMP activity that occurred coincident with dendritic deformations. It is noted that the results are consistent with a complementary study (He, 2019), which examined ddaD and ddaE dorsal proprioceptors and also demonstrated increased activity during dendrite folding. The He study elucidated that this deformation-dependent signaling is reliant on the mechanosensory channel TMC (Vaadia, 2019).

This survey of the full set of hypothesized multidendritic proprioceptors in behaving larvae revealed that most neurons (all class I neurons, dmd1, and vbd) increase activity during segment contraction. By contrast, dbd neurons showed increased activity during segment stretch, which is consistent with previous electrophysiological recordings of dbd in a dissected preparation. The temporal precision afforded by high-speed SCAPE microscopy further revealed that different proprioceptors exhibit sequential onset of activity during forward crawling. Timing of activity was associated with distinct dendrite morphologies and movement dynamics, suggesting that proprioceptors monitor different features of segment deformation. The complementary sensing of segment contraction versus stretch in class I, dmd1, and vbd versus dbd neurons provides an additional measure of movement that is conceptually similar to the responses of Golgi tendon organs versus muscle spindles in mammals. Combined, these results indicate that this set of proprioceptors function together to provide a continuum of sensory feedback describing the diverse 3D dynamics of the larval body (Vaadia, 2019).

Prior work suggested that the proprioceptors analyzed in this study have partially redundant functions during forward crawling because silencing different subsets caused similar behavioral deficits, namely slower crawling, whereas silencing both subsets had a more severe effect. Slow locomotion may be a common outcome in a larva that is lacking in part of its sensory feedback circuit, yet the results suggest that each cell type has a unique role. The demonstration of the varying activity dynamics of proprioceptors during crawling and more complex movements indicates that diverse sensory information is available to the larva, and suggests that feedback from a combination of these sensors could be used to infer aspects of speed, angle, restraint, and overall body deformation. This feedback system is likely to be important for a wide range of complex behaviors, such as body bending and nociceptive escape (Vaadia, 2019).

How can an understanding of proprioceptor activity patterns inform models of sensory feedback during locomotion? Electron microscopic reconstruction has shown that ddaD, vbd, and dmd1 proprioceptors synapse onto inhibitory premotor neurons (period-positive median segmental interneurons, A02b) (Schneider-Mizell, 2016), which promote segment relaxation and anterior wave propagation (Kohsaka, 2014). Thus, activity of these sensory neurons may signal successful segment contraction and promote forward locomotion, in part by promoting segment relaxation. Furthermore, vpda neurons provide input onto excitatory premotor neurons A27h, which acts through GABAergic dorsolateral (GDL) interneurons to inhibit contraction in neighboring anterior segments, thereby preventing premature wave propagation (Fushiki, 2016). In this way, vpda feedback could contribute to proper timing of contraction in anterior segments during forward crawling. In contrast to other proprioceptors, dbd neurons are active during segment stretch. Their connectivity also tends to segregate from contraction-sensing neurons, and understanding how the timing of this input promotes wave propagation is an important future question. This study's dynamic recordings of the function of these neurons during not just crawling but also exploration behavior provide essential new boundary data for testing putative network models derived from this anatomical roadmap (Vaadia, 2019).

SCAPE's high-speed 3D imaging capabilities enabled 10 VPS imaging of larvae during rapid locomotion. Fast volumetric imaging not only prevented motion artifacts but also revealed both the 3D motion dynamics and cellular activity associated with crawling behavior. SCAPE's large, 1-mm-wide field of view allowed multiple cells along the larva to be monitored at once, while providing sufficient resolution to identify individual dendrite branches. Because SCAPE data are truly 3D, dynamics could be examined in any section or view. Additionally, fast two-color imaging enabled simultaneous 3D tracking of cells, monitoring of GCaMP activity, and correction for motion-related intensity effects. The demonstration that larvae that are compressed during crawling exhibit altered dendrite deformation, and thus altered proprioceptive signaling, underscores the benefit of being able to image unconstrained larvae, volumetrically, in real time. Furthermore, rapid volumetric imaging allowed for the analysis of sensory responses during non-stereotyped, exploratory head movements in 3 dimensions, revealing activity patterns that could be utilized for encoding of complex, simultaneous movements. This finding also demonstrates the quantitative nature of SCAPE data and its high signal to noise, which enabled real-time imaging of neural responses without averaging from multiple neurons (Vaadia, 2019).

This study provides an example of how high-resolution, high-speed volumetric imaging enabled investigation of the previously intractable question of how different types of proprioceptive neurons encode forward locomotion and exploration behavior during naturalistic movement. Imaging could readily be extended to explore a wider range of locomotor behaviors such as escape behavior, in addition to other sensory modalities such as gustation and olfaction. Detectable signals reveal rich details including the firing dynamics of dendrites and axonal projections during crawling. Waves of activity in central neurons within the ventral nerve cord can also be observed. It is expected that the in vivo SCAPE microscopy platform utilized in this study could ultimately allow complete activity mapping of sensory activity during naturalistic behaviors throughout the larval CNS. Using SCAPE, it is conceivable to assess how activity from proprioceptive neurons modulates central circuits that execute motor outputs, which will provide critical information for a dissection of the neural control of behavior with whole-animal resolution (Vaadia, 2019).

Pupal behavior emerges from unstructured muscle activity in response to neuromodulation in Drosophila

Identifying neural substrates of behavior requires defining actions in terms that map onto brain activity. Brain and muscle activity naturally correlate via the output of motor neurons, but apart from simple movements it has been difficult to define behavior in terms of muscle contractions. By mapping the musculature of the pupal fruit fly and comprehensively imaging muscle activation at single cell resolution, this study describes a multiphasic behavioral sequence in Drosophila. This characterization identifies a previously undescribed behavioral phase and permits extraction of major movements by a convolutional neural network. Movements were deconstructed into a syllabary of co-active muscles and specific syllables were identified that are sensitive to neuromodulatory manipulations. Muscle activity shows considerable variability, with sequential increases in stereotypy dependent upon neuromodulation. This work provides a platform for studying whole-animal behavior, quantifying its variability across multiple spatiotemporal scales, and analyzing its neuromodulatory regulation at cellular resolution (Elliott, 2021).

The behavioral hallmark of pupal development is the Drosophila pupal ecdysis sequence, which is one of a general class of behavioral sequences used by insects to molt. These sequences typically divide into three principal phases during which the animal first loosens and then sheds its old exoskeleton before expanding its newly secreted one. Ecdysis sequences are strongly dependent on the action of hormones for their initiation and progression, and together with vertebrate reproductive behaviors, they have long served as a useful model of hormone-behavior interactions. Neural control of ecdysis behaviors is typically exercised by a conserved complement of hormones including eclosion hormone, ecdysis triggering hormone (ETH), crustacean cardioactive peptide (CCAP), and Bursicon. The sites of action of these hormones within the nervous system have been principally studied in Drosophila, but detailed neuroethological studies have been undertaken in larger insects, such as the locust, Schistocerca gregaria, and cricket, Teleogryllus oceanicus. Motor program organization of the adult ecdysis sequences in these insects is quite stereotyped, suggesting central control of motor execution, but sensory feedback can also modulate behavioral performance. While characterization of central nervous system (CNS) activity underlying ecdysis sequences has remained limited in larger insects, progress has been made in Drosophila where a fictive ecdysis sequence can be elicited in an excised pupal brain by exposure to ETH. Fictive activity imaged using Ca++ indicators grossly correlates with the motor patterns executed during pupal ecdysis, but comprehensively interpreting CNS activity remains a challenge (Elliott, 2021).

The Drosophila pupal ecdysis sequence occurs at the onset of metamorphosis and has been adapted to initiate transformation of the body plan. Although it occurs in the context of the pupal molt and has three principal phases characterized by distinct motor programs, its function in molting is limited to casting off the cuticular linings of the gut and trachea. Its primary function is, instead, to create internal pressure at points along the body to evert adult parts such as the head, legs, and wings. ETH initiates the pupal ecdysis sequence after a long period of behavioral quiescence and targets neurons that express its two receptor isoforms, ETHRA and ETHRB. Neurons expressing ETHRB are largely dispensable during larval life, but are essential for pupal ecdysis. Neurons expressing ETHRA include the neuroendocrine cells that secrete CCAP and Bursicon. These cells are likewise essential at pupal, but not larval, ecdysis. They become active approximately 10 min after the onset of pupal ecdysis, and their activation mediates the transition to the next behavioral phase. Understanding how the nervous system transforms such hormonal signals into temporally ordered behavioral sequences will require a more complete description of the motor neuron activity that dictates the behavioral sequences themselves (Elliott, 2021).

This study use body-wide fluorescence imaging from the dorsal, lateral, and ventral views to characterize the pupal ecdysis sequence at single-cell resolution. Using improved imaging methods-including a new pan-muscle LexA driver that permits dual imaging of muscle and neuron activity-this study identified novel elements of the pupal ecdysis sequence, including previously undescribed movements and a phase of stochastic muscle activity preceding ecdysis. Muscle activity was found to exhibit a high degree of variability, with individual muscles recruited stochastically into repeating small ensembles, termed called syllables. Syllable activity is synchronized over anatomical compartments to form movements, which are sufficiently stereotyped to be learned by a convolutional neural network (CNN). It is possible to prevent synchronization at specific motor program transitions by suppressing neuromodulatory neurons, which is lethal. The suppression of proprioceptive neurons, which blocks initiation of pupal ecdysis, is also unexpectedly lethal. Overall, this analysis at single-cell resolution reveals a dynamic system in which movements are not rigidly specified but form from variable components subject to neuromodulatory reorganization (Elliott, 2021).

Behavior is linked to neural mechanisms by the muscle activity that governs movement. To gain insight into how nervous systems specify behavior, muscle activity was examined during the Drosophila pupal ecdysis sequence at single-cell resolution using genetic Ca++ indicators. The pupal ecdysis sequence consists of multiple motor programs, dependent for their execution on hormonal cues. Hormonal signaling was found to coordinate muscle activity across individual muscle ensembles and anatomical compartments to ensure stereotypy of behavioral execution. Although stereotypy is evident at the level of phases, the recruitment of muscles into movements is not stereotyped and some muscle activity is not correlated with movement. Importantly, a phase of stochastic muscle Ca++ activity precedes the onset of behavior, indicating that prior to the action of ETH, nervous system activity exhibits intrinsic variability. This variability is reduced, but not eliminated, by the action of neuromodulators, which incrementally increase behavioral coherence (Elliott, 2021).

The results significantly extend previous descriptions of pupal ecdysis and illustrate the power of pan-muscle Ca++ imaging. Behavioral fine-mapping at single-cell resolution permits the definition and automated detection of elemental movements, the identification of a syllabary of movement-associated muscles and muscle ensembles, and the analysis of their sensitivity to neuronal manipulations. Importantly, single-cell analysis permits the identification of muscle activity that is not consistently associated with movements. The most salient example of such idiosyncratic activity occurs in P0, a previously undescribed phase of muscle activity lacking coordinated movement. Variability persists in phases that exhibit idiosyncratic muscle activation comingled with stereotyped movement syllables. Furthermore, muscle recruitment into syllables, and recruitment of syllables into movements, exhibits considerable variability both within and across animals. All observations suggest that variability in the order of recruitment of behavioral elements is a pervasive feature of the pupal ecdysis sequence with stereotypy emerging only at higher levels of behavioral description (Elliott, 2021).

Variability in pupal ecdysis behavior may arise from the need to adjust movement to changing forces on the body wall, both from inside and outside. Inside the animal, hydrostatic pressure varies globally in response to local contractions of the body wall. This pressure may need to be countered to maintain control of movement. Outside the animal, the wall of the puparium may form an inhomogeneous substrate as the distribution of molting fluid and air at different places varies. A notable feature of pupal ecdysis is that it is heralded by the appearance of a large air bubble in the abdomen that is expelled into the puparium by the movements executed during P1. After expulsion from the body, air is displaced by the animal's movements, first posteriorly and then anteriorly. In the presence of residual molting fluid, pockets of air likely cause fluctuations in surface tension between the body wall and puparium. Forces exerted both by internal pressure and by substrate interactions within the puparium may thus require that motor output be dynamically adjusted, presumably by sensory cues (Elliott, 2021).

The importance of sensory cues to pupal ecdysis is evident from the finding that animals lacking proprioceptive input die at P0 without initiating the behavioral sequence. The cause of death remains to be determined, but its timing suggests that proprioceptive feedback may signal muscle responsiveness to neural input during the period of muscle reactivation and thus provide a readiness signal for ecdysis initiation. Alternatively, pressure on the body wall due to air bubble growth may trigger pupal ecdysis. Sensory cues have been shown to gate behavioral transitions in the adult ecdysis sequences of crickets and also to adjust motor program execution when extrication from parts of the old cuticle fails (Elliott, 2021).

For the pupal ecdysis sequence, more work will be required to determine the sources of the observed variability. Notably, sources that arise frequently in the context of other behaviors, such as external environmental stimuli (i.e., stimuli outside the puparium) and competing physiological needs, are absent for pupal ecdysis. In addition, proprioceptive cues, while they may tune ecdysis behavior, are not essential for generating it in that a fictive sequence is generated by an excised pupal brain treated with ETH. Finally, the evidence suggests that at least some behavioral variability derives from the operation of the ecdysis neural network itself since stochasticity is clearly evident at P0 and then appears to extend to other phases (Elliott, 2021).

The variability of P0 muscle bouts is reminiscent in some ways of the neurogenic bursts of muscle activity observed in Drosophila embryos prior to hatching. Initial embryonic bursts consist of uncoordinated muscle activity that becomes increasingly organized over several hours as the locomotor networks mature. By the time of hatching, complete peristaltic motor sequences are regularly performed. Similar precocious network activity is also found in a variety of other systems, including pupal moths where the developing circuitry for flight drives low-threshold neuromuscular responses in muscles that fail to elicit contractions. Such activity has also been proposed to support network maturation. P0 motor activity may share this function as patterns of muscle activity become increasingly complex during P0 and recognizable syllables emerge. However, in contrast to other systems, fully coordinated activity does not appear until the phase ends with the first P1 Lift, and even after this muscle and syllable recruitment remain irregular, suggesting continued network variability (Elliott, 2021).

This apparently intrinsic variability may relate to the tradeoffs required of any multifunctional system. Pupal ecdysis, like most behaviors, depends on the integration of signals from CPGs, proprioceptors, neuromodulators, and possibly higher-order command systems-all of which are likely used in the context of larval behavior. For example, circuits that generate waves of activity along the anteroposterior axis are required for both larval locomotion and pupal ecdysis, but while they generate coordinated bilateral activity in locomotion, they generate mostly alternating activity during ecdysis. This degree of flexibility may be bought at the cost of reproducibility of execution. Indeed, precise reproducibility may not be prioritized by nervous systems. This may instead favor solutions that are 'good enough' as has been generally proposed for biological control systems. According to this view, further optimization of pupal ecdysis, in the form of behavioral stereotypy,may incur costs on performance in the execution of other behaviors that rely on the same circuit elements (Elliott, 2021).

Variability in pupal motor output is substantially altered by the neuromodulatory action of the ecdysis hormones ETH, CCAP, and Bursicon. Previous evidence indicates that these hormones induce the motor activity characteristic of P1 and P2, even in an isolated pupal nervous system. Consistent with the ability of neuromodulators to reconfigure motor networks, this induction likely represents reorganization and possibly stabilization of network activity, and also increased network coherence. P1 is distinguished from P0 by the presence of coherent movements and P2 is more coherent than P1 in recruitment of muscles, syllables, and compartmental activity. The reduced stereotypy in P3 may result from waning neuromodulatory action. The ecdysis hormones thus reorganize motor output and increase the stereotypy of its execution. This conclusion is supported by suppression of neuromodulatory signaling, which disrupts muscle activity at multiple levels, as expected from the broad distribution of ecdysis hormone receptors (Elliott, 2021).

What causes the release of Bursicon, CCAP, and ETH at pupal ecdysis is unknown, but the results suggest that ecdysis activity itself may be a factor. Release of ETH occurs only after muscle responsiveness to neural stimulation is ensured, suggesting that the latter may represent a checkpoint for release. Similarly, the aborted swing-like activity occurring in the absence of NCCAP and NBurs activity indicates that entry into P2 has a hormone-independent component that may act as a checkpoint for hormone release, which then sustains P2 network activity. One possible mechanism might be that as the animal pulls back during P1 and creates an anterior space, sensory feedback from the head signals the readiness for P2. Similar checkpoint control mechanisms have been proposed to operate in the adult ecdysis sequence of locusts and crickets (Elliott, 2021).

The granular analysis of muscle activity presented in this study also reveals how neuromodulators may serve to coordinate action across anatomically and functionally distinct compartmental boundaries. ETHRB neuron suppression blocks the Lift, a movement of the posterior compartment, while suppressing CCAP neurons prematurely terminates the first (and only) swing-like movement by blocking its progression into the anterior compartment. Additionally, the distribution of CCAP-R appears to reflect mechanisms for selectively regulating distinct compartments across the D-V axis. CCAP-R is expressed selectively in motor neurons that innervate dorsal and ventral muscles. These motor neurons have dendrites that occupy similar positions on the myotopic map and share similar synaptic inputs. This distinguishes them from motor neurons that innervate the lateral transverse muscles, which do not express CCAP-R. However, a subset of lateral transverse muscles (i.e., M21-23) themselves express CCAP-R, and the pattern of CCAP-R expression may thus represent a mechanism for synchronizing activity across the D-V axis during the swing movements of P2 when CCAP is released. Notably, muscle synchrony across compartments in posterior segments is lost in the one, partially executed Swing when CCAP-expressing neurons are suppressed and no further Swings are executed (Elliott, 2021).

The putative source of CCAP for the muscles of PME2 are the type III terminals on muscle M12, which are selectively retained in the anterior compartment (i.e., segments 1-4). CCAP-R-expressing muscles in the anterior compartment may thus receive CCAP modulation prior to those in the posterior compartment. This may facilitate the delayed appearance of the contralateral brace, which is formed principally by segmental PME2s and which changes the character of the Swing after head eversion. Interestingly, the anatomical asymmetry in the distribution of M12 is one of several observed in pupal muscles across the D-V and A-P body axes. These have only been partially described previously and result from the selective degradation of larval muscles in the ventral and posterior compartments. The asymmetries in muscle distribution correlate with several pupal movements that are physically partitioned across the A-P axis, including the Lift, which occurs only in the posterior compartment during P1, and the AntComp, which is restricted to the anterior compartment during P3. Synaptic mechanisms for controlling movements across the A-P boundary in the larva have been described previously, and similar mechanisms may synergize with anatomical asymmetries and neuromodulatory mechanisms in the pupa to spatially and temporally constrain muscle activity to specific compartments (Elliott, 2021).

It is worth noting that the expression of CCAP-R by the lateral transverse muscles indicates the potential importance of muscle modulation in shaping behavior. Whether modulation of muscle properties also underlies the generalized failure of muscles to respond to synaptic input at the onset of P0 is unclear. Animals stop moving shortly after pupariation and do not resume until pupal ecdysis, but neuromuscular activity may be maintained throughout this period. This study observed neuromuscular activity without muscle responses at the earliest pre-pupal stages examined (approximately stage P2). However, more detailed imaging would be required to determine the extent, continuity, and pattern of the input. Drosophila muscles are targets of a variety of neuropeptides including myoinhibitory peptides, which have also been implicated in pupal ecdysis behavior, and it is possible that these play a role in suppressing muscle responses to neural input. Alternatively, neuromuscular signaling may be progressively potentiated during P0. Such a mechanism has been proposed to operate in pupal moth flight muscles, which respond electrically, but fail to contract, to synaptic input corresponding to flight motor patterns. As animals approach eclosion, rising octopamine levels upregulate neuromuscular efficacy so that flight muscles activate in response to input (Elliott, 2021).

A goal of computational neuroethology is to describe behavior at a level of resolution that permits the identification of its neural determinants. The muscle-level description provided in this study lends itself naturally to this purpose. For example, the activity patterns of the muscles comprising PME2 have likely neural correlates within the synaptic and neuromodulatory networks that govern pupal ecdysis behavior. At the neuromodulatory level, the NCCAP cells that terminate on M12 are likely sources of CCAP modulation of PME2 muscles, perhaps to help reverse their P-to-A activation at P2. At the synaptic level, the regular, intersegmental pattern of activation of PME2 muscles in nearly all pupal movements and behavioral bouts suggests that PME2 motor neurons are driven by CPG neurons that generate anteroposterior rhythms. The results thus provide testable predictions about the patterns of neuromodulatory and synaptic connectivity between muscles, motor neurons, and premotor interneurons of various types (Elliott, 2021).

Testing these predictions will be facilitated by data emerging from reconstruction of the larval CNS at synaptic resolution. Although pupal behaviors differ from those of the larva, broad similarities suggest that at least some neural substrates are shared. The initial P-to-A activity flow of the pupal ecdysis sequence is reminiscent of larval forward peristalsis, and its reversal after alternating bilateral Swing movements resembles the switch to backward peristalsis following sensory stimuli. An important difference, however, is that the basic features of larval locomotion are identifiable in the default activity of the excised larval brain, whereas the pupal nervous system produces patterned activity resembling pupal ecdysis only in response to ETH. Pupal ecdysis will thus permit investigation of the mechanisms by which intrinsic neuronal activity is organized by neuromodulatory control. Analyzing behavior at single-muscle resolution, as demonstrated in this study, will facilitate this investigation and should be applicable to other translucent preparations of neuroscientific interest, such as larval fruit flies, roundworms, and larval zebrafish (Elliott, 2021).

Nitric oxide mediates activity-dependent change to synaptic excitation during a critical period in Drosophila

The emergence of coordinated network function during nervous system development is often associated with critical periods. These phases are sensitive to activity perturbations during, but not outside, of the critical period, that can lead to permanently altered network function for reasons that are not well understood. In particular, the mechanisms that transduce neuronal activity to regulating changes in neuronal physiology or structure are not known. This study take advantage of a recently identified invertebrate model for studying critical periods, the Drosophila larval locomotor system. Manipulation of neuronal activity during this critical period is sufficient to increase synaptic excitation and to permanently leave the locomotor network prone to induced seizures. Using genetics and pharmacological manipulations, this study identifies nitric oxide (NO)-signaling as a key mediator of activity. Transiently increasing or decreasing NO-signaling during the critical period mimics the effects of activity manipulations, causing the same lasting changes in synaptic transmission and susceptibility to seizure induction. Moreover, the effects of increased activity on the developing network are suppressed by concomitant reduction in NO-signaling and enhanced by additional NO-signaling. These data identify NO signaling as a downstream effector, providing new mechanistic insight into how activity during a critical period tunes a developing network (Giachello, 2021).

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).

Thermoresponsive motor behavior is mediated by ring neuron circuits in the central complex of Drosophila

Insects are ectothermal animals that are constrained in their survival and reproduction by external temperature fluctuations which require either active avoidance of or movement towards a given heat source. In Drosophila, different thermoreceptors and neurons have been identified that mediate temperature sensation to maintain the animal's thermal preference. However, less is known how thermosensory information is integrated to gate thermoresponsive motor behavior. This study used transsynaptic tracing together with calcium imaging, electrophysiology and thermogenetic manipulations in freely moving Drosophila exposed to elevated temperature and identify different functions of ellipsoid body ring neurons, R1-R4, in thermoresponsive motor behavior. The results show that warming of the external surroundings elicits calcium influx specifically in R2-R4 but not in R1, which evokes threshold-dependent neural activity in the outer layer ring neurons. In contrast to R2, R3 and R4d neurons, thermogenetic inactivation of R4m and R1 neurons expressing the temperature-sensitive mutant allele of dynamin, shibire(TS), results in impaired thermoresponsive motor behavior at elevated 31 °C. trans-Tango mediated transsynaptic tracing together with physiological and behavioral analyses indicate that integrated sensory information of warming is registered by neural activity of R4m as input layer of the ellipsoid body ring neuropil and relayed on to R1 output neurons that gate an adaptive motor response. Together these findings imply that segregated activities of central complex ring neurons mediate sensory-motor transformation of external temperature changes and gate thermoresponsive motor behavior in Drosophila (Buhl, 2021).

Online learning for orientation estimation during translation in an insect ring attractor network

Insect neural systems are a promising source of inspiration for new navigation algorithms, especially on low size, weight, and power platforms. There have been unprecedented recent neuroscience breakthroughs with Drosophila in behavioral and neural imaging experiments as well as the mapping of detailed connectivity of neural structures. General mechanisms for learning orientation in the central complex (CX) of Drosophila have been investigated previously; however, it is unclear how these underlying mechanisms extend to cases where there is translation through an environment (beyond only rotation), which is critical for navigation in robotic systems. This study developed a CX neural connectivity-constrained model that performs sensor fusion, as well as unsupervised learning of visual features for path integration; the viability of this circuit was demonstrated for use in robotic systems in simulated and physical environments. Furthermore, a theoretical understanding is demonstrated of how distributed online unsupervised network weight modification can be leveraged for learning in a trajectory through an environment by minimizing orientation estimation error. Overall, these results may enable a new class of CX-derived low power robotic navigation algorithms and lead to testable predictions to inform future neuroscience experiments (Robinson, 2022).

Under warm ambient conditions, Drosophila melanogaster suppresses nighttime activity via the neuropeptide pigment dispersing factor

Rhythmic locomotor behaviour of flies is controlled by an endogenous time-keeping mechanism, the circadian clock, and is influenced by environmental temperatures. Flies inherently prefer cool temperatures around 25°C, and under such conditions, time their locomotor activity to occur at dawn and dusk. Under relatively warmer conditions such as 30°C, flies shift their activity into the night, advancing their morning activity bout into the early morning, before lights-ON, and delaying their evening activity into early night. The molecular basis for such temperature-dependent behavioural modulation has been associated with core circadian clock genes, but the neuronal basis is not yet clear. Under relatively cool temperatures such as 25°C, the role of the circadian pacemaker ventrolateral neurons (LNvs), along with a major neuropeptide secreted by them, pigment dispersing factor (PDF), has been showed in regulating various aspects of locomotor activity rhythms. However, the role of the LNvs and PDF in warm temperature-mediated behavioural modulation has not been explored. This study shows that flies lacking proper PDF signalling or the LNvs altogether, cannot suppress their locomotor activity resulting in loss of sleep during the middle of the night, and thus describe a novel role for PDF signalling and the LNvs in behavioural modulation under warm ambient conditions. In a rapidly warming world, such behavioural plasticity may enable organisms to respond to harsh temperatures in the environment (Iyengar, 2022).

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).

Contribution of non-circadian neurons to the temporal organization of locomotor activity

In the fruit fly, Drosophila melanogaster, the daily cycle of rest and activity is a rhythmic behavior that relies on the activity of a small number of neurons. The small Lateral Neurons ventral (sLNvs) are considered key in the control of locomotor rhythmicity. Previous work has shown that these neurons undergo structural remodeling on its axonal projections on a daily basis. Such remodeling endows sLNvs with the possibility to make synaptic contacts with different partners at different times along the day as has been previously described. By using different genetic tools to alter membrane excitability of the sLNv putative postsynaptic partners, their functional role on the control of locomotor activity was tested. Optical imaging was used to test the functionality of these contacts. These different neuronal groups affect the consolidation of rhythmic activity, suggesting that non-circadian cells are part of the circuit that controls locomotor activity. The results suggest that new neuronal groups, in addition to the well-characterized clock neurons, contribute to the operations of the circadian network that controls locomotor activity in Drosophila melanogaster (Pirez, 2018).

State-dependent decoupling of sensory and motor circuits underlies behavioral flexibility in Drosophila

An approaching predator and self-motion toward an object can generate similar looming patterns on the retina, but these situations demand different rapid responses. How central circuits flexibly process visual cues to activate appropriate, fast motor pathways remains unclear. This study identified two descending neuron (DN) types that control landing and contribute to visuomotor flexibility in Drosophila. For each, silencing impairs visually evoked landing, activation drives landing, and spike rate determines leg extension amplitude. Critically, visual responses of both DNs are severely attenuated during non-flight periods, effectively decoupling visual stimuli from the landing motor pathway when landing is inappropriate. The flight-dependence mechanism differs between DN types. Octopamine exposure mimics flight effects in one, whereas the other probably receives neuronal feedback from flight motor circuits. Thus, this sensorimotor flexibility arises from distinct mechanisms for gating action-specific descending pathways, such that sensory and motor networks are coupled or decoupled according to the behavioral state (Ache, 2019).

DeepFly3D, a deep learning-based approach for 3D limb and appendage tracking in tethered, adult Drosophila

Studying how neural circuits orchestrate limbed behaviors requires the precise measurement of the positions of each appendage in 3-dimensional (3D) space. Deep neural networks can estimate 2-dimensional (2D) pose in freely behaving and tethered animals. However, the unique challenges associated with transforming these 2D measurements into reliable and precise 3D poses have not been addressed for small animals including the fly, Drosophila melanogaster. This study presents DeepFly3D, a software that infers the 3D pose of tethered, adult Drosophila using multiple camera images. DeepFly3D does not require manual calibration, uses pictorial structures to automatically detect and correct pose estimation errors, and uses active learning to iteratively improve performance.More accurate unsupervised behavioral embedding was demonstrated using 3D joint angles rather than commonly used 2D pose data. Thus, DeepFly3D enables the automated acquisition of Drosophila behavioral measurements at an unprecedented level of detail for a variety of biological applications (Gunel, 2019).

Sex-specific among-individual covariation in locomotor activity and resting metabolic rate in Drosophila melanogaster

A key endeavor in evolutionary physiology is to identify sources of among- and within-individual variation in resting metabolic rate (RMR). Although males and females often differ in whole-organism RMR due to sexual size dimorphism, sex differences in RMR sometimes persist after conditioning on body mass, suggesting phenotypic differences between males and females in energy-expensive activities contributing to RMR. One potential difference is locomotor activity, yet its relationship with RMR is unclear and different energy budget models predict different associations. This study quantified locomotor activity (walking) over 24 h and RMR (overnight) in 232 male and 245 female Drosophila melanogaster that were either mated or maintained as virgins between two sets of measurements. Accounting for body mass, sex, and reproductive status, RMR and activity were significantly and moderately repeatable. RMR and activity were positively correlated among but not within individuals. Moreover, activity varied throughout the day and between the sexes. Partitioning the analysis by sex and activity by time of day revealed that all among-individual correlations were positive and significant in males but nonsignificant or even significantly negative in females. Such differences in the RMR-activity covariance suggest fundamental differences in how the sexes manage their energy budget (Videlier, 2019).

Deficits in the vesicular acetylcholine transporter alter lifespan and behavior in adult Drosophila melanogaster

The neurotransmitter acetylcholine (ACh) is involved in critical organismal functions that include locomotion and cognition. Importantly, alterations in the cholinergic system are a key underlying factor in cognitive defects associated with aging. One essential component of cholinergic synaptic transmission is the vesicular ACh transporter (VAChT), which regulates the packaging of ACh into synaptic vesicles for extracellular release. Mutations that cause a reduction in either protein level or activity lead to diminished locomotion ability whereas complete loss of function of VAChT is lethal. While much is known about the function of VAChT, the direct role of altered ACh release and its association with either an impairment or an enhancement of cognitive function are still not fully understood. It was hypothesized that point mutations in Vacht cause age-related deficits in cholinergic-mediated behaviors such as locomotion, and learning and memory. Using Drosophila melanogaster as a model system, several mutations within Vacht were studied and their effect on survivability and locomotive behavior were observed. A weak hypomorphic Vacht allele was found that shows a differential effect on ACh-linked behaviors. It was also demonstrated that partially rescued Vacht point mutations cause an allele-dependent deficit in lifespan and defects in locomotion ability. Moreover, using a thorough data analytics strategy to identify exploratory behavioral patterns, new paradigms were introduced for measuring locomotion-related activities that could not be revealed or detected by a simple measure of the average speed alone. Together, these data indicate a role for VAChT in the maintenance of longevity and locomotion abilities in Drosophila and additional measurements of locomotion are provided that can be useful in determining subtle changes in Vacht function on locomotion-related behaviors (White, 2020).

Frequency-specific modification of locomotor components by the white gene in Drosophila melanogaster adult flies

The classic eye-color gene white (w) in Drosophila melanogaster (fruitfly) has unexpected behavioral consequences. How w affects locomotion of adult flies is largely unknown. This study shows that a mutant allele (w(1118)) selectively increases locomotor components at relatively high frequencies (> 0.1 Hz). The (w(1118)) flies had reduced transcripts of w(+) from the 5' end of the gene. Male flies of (w(1118)) walked continuously in circular arenas while the wildtype Canton-S walked intermittently. Through careful control of genetic and cytoplasmic backgrounds, this study found that the (w(1118)) locus was associated with continuous walking. w(1118) -carrying male flies showed increased median values of path length per second (PPS) and 5-min path length compared with w(+) -carrying males. Additionally, flies carrying 2-4 genomic copies of mini-white(+) (mw(+)) in the w(1118) background showed suppressed median PPSs and decreased 5-min path length compared with controls, and the suppression was dependent on the copy number of mw(+). Analysis of the time-series (i.e. PPSs over time) by Fourier transform indicated that w(1118) was associated with increased locomotor components at relatively high frequencies (> 0.1 Hz). The addition of multiple genomic copies of mw(+) (2-4 copies) suppressed the high-frequency components. Lastly, the downregulation of w(+) in neurons but not glial cells resulted in increased high-frequency components. It is concluded that mutation of w modified the locomotion in adult flies by selectively increasing high-frequency locomotor components (Xiao, 2020).

Mating experience modifies locomotor performance and promotes episodic motor activity in Drosophila melanogaster

Sexual behavior is a routine among animal species. Sexual experience has several behavioral consequences in insects, but its physiological basis is less well-understood. The episodic motor activity with a periodicity around 19 s was unintentionally observed in the wildtype Canton-S flies and was greatly reduced in the white-eyed mutant w(1118) flies. Episodic motor activity co-exists with several consistent locomotor performances in Canton-S flies whereas reduced episodic motor activity is accompanied by neural or behavioral abnormalities in w(1118) flies. The improvements of both episodic motor activity and locomotor performance are co-inducible by a pulsed light illumination in w(1118). This study shows that mating experience of w(1118) males promoted fast and consistent locomotor activities and increased the power of episodic motor activities. Compared with virgin males, mated ones showed significant increases of boundary preference, travel distance over 60 s, and increased path increments per 0.2 s. In contrast, mated males of Canton-S showed decreased boundary preference, increased travel distance over 60 s, and increased path increments per 0.2 s. Additionally, mated males of w(1118) displayed increased power amplitude of periodic motor activities at 0.03-0.1 Hz. These data indicated that mating experience promoted fast and consistent locomotion and improved episodic motor activities in w(1118) male flies (Qiu, 2020).

Reconstruction of motor control circuits in adult Drosophila using automated transmission electron microscopy

To investigate circuit mechanisms underlying locomotor behavior, this study used serial-section electron microscopy (EM) to acquire a synapse-resolution dataset containing the ventral nerve cord (VNC) of an adult female Drosophila melanogaster. To generate this dataset, GridTape, a technology that combines automated serial-section collection with automated high-throughput transmission EM, was developed. Using this dataset, neuronal networks were studied that control leg and wing movements by reconstructing all 507 motor neurons that control the limbs. A specific class of leg sensory neurons was shown to synapse directly onto motor neurons with the largest-caliber axons on both sides of the body, representing a unique pathway for fast limb control. Open access is provided to the dataset and reconstructions registered to a standard atlas to permit matching of cells between EM and light microscopy data. GridTape instrumentation designs and software are provided to make large-scale EM more accessible and affordable to the scientific community (Phelps, 2021).

Large-scale neuronal wiring diagrams at synapse resolution will be a crucial element of future progress in neuroscience. This paper presents GridTape, a technology for accelerating large-scale electron microscopy (EM) data acquisition. The power of this approach is demonstrated by acquiring a dataset encompassing an adult female Drosophila ventral nerve cord (VNC). This dataset was used to identify a monosynaptic circuit that directly links a specialized proprioceptive cell type, the bilateral campaniform sensillum (bCS) neurons, with specific motor neurons (MNs). The results highlight how EM datasets can be used to characterize cell types and guide development of cell type-specific driver lines. The public release of this dataset provides a resource for studying the circuit connectivity underlying motor control and demonstrates the rapid advances that can be powered by the GridTape approach (Phelps, 2021).

Data acquisition remains a rate-limiting step in generating EM connectomics datasets. Manual sectioning for TEM is slow, imprecise, and unreliable. Meanwhile, SEM approaches that circumvent the need for manual sectioning have slow imaging speeds or require massive parallelization of expensive electron optics to acquire comparable datasets. GridTape builds on previous efforts toward TEM parallelization and automation, but overcomes the need for manual sectioning, allowing faster and more consistent section collection and imaging. Because imaging is nondestructive, GridTape is compatible with enhancement by post-section labeling and allows for re-imaging. By eliminating the need to separately handle thousands of fragile sections, GridTape reduces data loss and artifact frequency. This results in better alignment of sections into a coherent, high signal-to-noise image volume, leading to efficient and accurate reconstructions (Phelps, 2021).

GridTape is also less expensive than high-throughput SEM platforms. For the current price of one commercial multi-beam SEM system, ten TEMCA-GTs can be built, and samples collected on GridTape can be distributed across microscopes for simultaneous imaging. The fixed microscope hardware costs are accompanied by consumable costs associated with support film coating (∼USD$4 per slot, or ∼$18,000 for this study), but this cost is expected to decrease due to technological improvements and economies of scale (Phelps, 2021).

In the future, GridTape acquisition rates will increase as cameras and imaging sensors improve. Because TEM imaging is a widefield technique, imaging throughput can be increased by using larger camera arrays and brighter electron sources. Moreover, sections larger than current slot dimensions could be accommodated with wider tape and larger slots, although custom microscopes may be necessary for very large samples and slot size will depend on material properties of the support film (Phelps, 2021).

The EM dataset presented in this study provides a public resource for understanding how the Drosophila nervous system generates behavior. An adult Drosophila VNC was chosen because it is an ideal test case for generating and validating a connectomic dataset. The circuit is genetically and electrophysiologically accessible and neurons are identifiable across individuals. The VNC is compact, containing approximately a third of the neurons in the adult CNS, but contains neuronal networks for executing complex motor behaviors. Because the brain controls behavior via descending projections to the VNC, it is critical to be able to study neuronal circuits in both the brain and the VNC at synaptic resolution. Notably, this VNC dataset complements the recent release of an EM dataset comprising the complete adult female Drosophila brain (Phelps, 2021).

The VNC dataset was validated by automatically mapping its synapses with high accuracy, successfully registering the predicted synapse density map to a standard atlas and finding a high degree of similarity between EM and LM reconstructed neurons. A pipeline is demonstrated for identifying cells of interest in the dataset by comparing EM reconstructions to LM data. Finally, as a foundation for future work, >1,000 neuron reconstructions and their connectivity are made publicly available. Although these reconstructions were generated manually, advances in automated segmentation approaches are dramatically accelerating analysis of serial-section TEM data (Phelps, 2021).

Flexible motor control relies heavily on feedback from proprioceptors, a class of sensory neurons that measure body position, velocity, and load. In both vertebrates and invertebrates, proprioceptive feedback is processed by the central nervous system to tune motor output. In insects, morphologically distinct subclasses of chordotonal neurons encode different features of leg movement such as position, velocity, and vibration. Campaniform sensilla encode load signals similar to mammalian Golgi tendon organs. Althoughthe main proprioceptor types are known and the signals they encode, it is now an opportune time to understand how motor circuits integrate proprioceptive inputs to control the body by mapping the complete wiring diagram of an adult Drosophila VNC (Phelps, 2021).

EM datasets also enable the discovery of cell types and synaptic connections that may be overlooked by other methods. For instance, targeted reconstruction of sensory afferents revealed that the leg sensory neurons with the largest-caliber axons are the bCS neurons, which make direct synapses onto large-caliber leg MNs. This connection is monosynaptic and bCS inputs are specifically located near the putative MN spike initiation zone, suggesting that speed and reliability are essential for the function of these connections (Phelps, 2021).

The unique bilateral and intersegmental projections of bCS neurons suggests that they directly influence multiple limbs on both sides of the body. This leads to several hypotheses about their function. Prior work suggested that campaniform sensilla encode information about step timing that could drive the transition between stance and swing phases of walking. However, this study observed that bCS neurons synapse onto the same MNs on both sides of the bod, suggesting they drive symmetric movements of left and right legs. This makes it unlikely that bCS neurons contribute to walking, which involves antiphase movement of contralateral legs. Instead, bCS neurons may underlie a fast reflex where multiple legs flex in response to bCS activation. CS can signal either increases or decreases in load, depending on the sensillum's placement and orientation on the leg. Therefore, bCS neuron activation could forcefully stabilize posture in response to additional weight (e.g., to prevent the body from being crushed) or to grip a surface in response to a loss of load (e.g., to prevent being blown away by a gust of wind). The genetic tools that were created to target bCS neurons will enable future analyses of their function (Phelps, 2021).

Monosynaptic sensory-to-motor neuron connectivity is infrequent in larval Drosophila, but has been observed in other adult insects. Direct sensory feedback may be key in adults for precise control of their segmented limbs. The absence of such connections in larvae may indicate that controlling a limbless body relies less on sensory feedback and more on feedforward processing. As adult flies move much faster than larvae, another possibility is that fast monosynaptic sensory feedback is crucial for fast-moving animals. Indeed, research on escape responses has demonstrated that high-velocity movements are often controlled by the fastest neuronal pathways (Phelps, 2021).

MNs have diverse but stereotyped functions, reflecting the array of muscles and muscle fibers they innervate. Some MNs have unique and reproducible transcription factor signatures that underlie their physiological properties and axonal morphology. These unique transcription factor patterns specify morphologies that are fairly stereotyped across animals. The results extend these findings by quantitatively demonstrating that most dendritic arborizations of leg MNs are sufficiently stereotyped to be individually identifiable by structure alone. Because the complete population of MNs controlling the two front legs was reconstructed, it was possible to show that mirror symmetry in primary neurite number and position is a systematic principle of MN populations. In contrast, sensory neurons have more redundant copies and variable copy numbers (Phelps, 2021).

Previously, comprehensive neuronal connectivity maps were acquired for the nerve cords of other organisms including C. elegans, leeches, lampreys, and Drosophila larvae. These maps enabled a more complete understanding of how the nervous system controls locomotor rhythms underlying swimming and crawling. Less is known about the connectivity underlying motor control in limbed animals. The EM dataset presented in this study as a public resource will enable complete connectivity mapping for the circuits that control the legs and wings of an adult Drosophila. Combined with recent advances in recording activity from genetically identified VNC neurons during behavior, adult Drosophila is emerging as a powerful system for studying motor control. With these tools, it is expected that a deeper understanding of the circuit basis for complex motor control is within reach (Phelps, 2021).

Allatostatin-C/AstC-R2 is a novel pathway to modulate the circadian activity pattern in Drosophila

Seven neuropeptides are expressed within the Drosophila brain circadian network. Previous mRNA profiling suggested that Allatostatin-C (AstC) is an eighth neuropeptide and specifically expressed in dorsal clock neurons (DN1s). The results of this study show that AstC is, indeed, expressed in DN1s, where it oscillates. AstC is also expressed in two less well-characterized circadian neuronal clusters, the DN3s and lateral-posterior neurons (LPNs). Behavioral experiments indicate that clock-neuron-derived AstC is required to mediate evening locomotor activity under short (winter-like) and long (summer-like) photoperiods. The AstC-Receptor 2 (AstC-R2) is expressed in LNds, the clock neurons that drive evening locomotor activity, and AstC-R2 is required in these neurons to modulate the same short photoperiod evening phenotype. Ex vivo calcium imaging indicates that AstC directly inhibits a single LNd. The results suggest that a novel AstC/AstC-R2 signaling pathway, from dorsal circadian neurons to an LNd, regulates the evening phase in Drosophila (Diaz, 2019).

To learn more about how the ~150 clock neurons within the adult fly brain communicate, RNA-sequencing data were examined from the LNds, LNvs, and DN1s for neuropeptides not yet associated with this circuitry. AstC was a promising candidate, because mRNAs encoding both the peptide and one of its receptors (AstC-R2) were identified within the three clock neuron clusters; these data suggested a novel intra-clock circuitry signaling pathway. AstC transcripts as well as the neuropeptide are, indeed, well expressed in the DN1s, and the neuropeptide signal undergoes strong cycling in DD as well as LD conditions. Moreover, AstC is also expressed in two other circadian neuron subgroups, the DN3s and the LPNs, and it is the first neuropeptide identified in these circadian clusters. Behavioral data after RNAi knockdown indicate that the AstC binds to AstC-R2 expressed in E-cells to modulate the timing of evening locomotor activity. Ex vivo calcium imaging indicates that AstC directly inhibits a single LNd (Diaz, 2019).

AstC is required in the clock neurons to regulate the evening locomotor activity phase in short and long photoperiods, suggesting that 'masking' effects in 12:12 LD obscured a phenotypic effect. This shift in the timing of the E-peak occurs when AstC is reduced in all circadian neurons (tim-GAL4 driver) (Diaz, 2019).

AstC cycles in the DN1s, not only under standard 12:12 LD conditions but also under DD conditions and the short photoperiod condition of 6:18 LD. In all cases, AstC staining intensity in the DN1 soma is lowest during the early day. It was hypothesizef that the AstC staining is reduced at this time, because the peptide is being transported from the soma to the dendritic arbors for secretion. Indeed, this early-day timing correlates with DN1 firing, as DN calcium and firing frequency are highest at this time and, thus, may be associated with activity-dependent peptide release. This temporal regulation could coincide with the timing of the AstC effect on the phase of the evening locomotor peak. If this is the case, however, residual AstC remaining after knockdown in the DN1s with the clk4.1M-GAL4 driver must still be sufficient to promote a wild-type phenotype (Diaz, 2019).

In addition, the LPNs are probably not a key circadian source of AstC: their AstC levels were also dramatically reduced in the clk856-GAL4-mediated knockdown without any phenotypic effect. All AstC-expressing DN3s are targeted by the tim-GAL4 driver, yet most of these DN3s are not included in the clk856-GAL4 driver. Although a tim-GAL4 source from outside the circadian network cannot be excluded, these results suggest that the DN3s are the key source of AstC. This tentative conclusion is based on negative data, and the lack of a DN3-specific GAL4 driver makes it impossible to test this model directly. Therefore, three possible models are proposed: (1) the DN3s are the primary source of AstC within the circadian circuit; (2) the DN1s, DN3s, and LPNs, or some combination, are functionally redundant; or (3) a small amount of residual AstC within DN1s is sufficient for its behavioral role in the evening activity peak assay. Although neuron-specific deletion of AstC might contribute to distinguishing between these three possibilities, no accurate and efficient CRISPR-based strategy is available for achieving temporal and spatial specificity (Diaz, 2019).

Once released by dorsal circadian neurons, AstC signals to the LNds via binding to its receptor, AstC-R2. This is because AstC-R2 knockdown in the LNds and knockdown of AstC in the entire circadian circuit give rise to the identical delayed E-peak phenotype. Moreover, the DNs and the LNds both extend projections to the dorsal protocerebrum region (near the pars intercerebralis), where they come in close proximity. In summary, this study adds a new player to the neuronal circuitry governing E-peak modulation. It is suggested that the LNd neuronal activity is modulated not only by signaling from M-cells but also by AstC signaling from the dorsal region of the brain (Diaz, 2019).

The functional imaging strongly indicates that AstC binding to LNd-localized AstC-R2 leads to neuronal inhibition. The effect is consistent with several previous electrophysiology experiments and contributes to an emerging theme of LNd inhibition. It is also one of the first pieces of evidence indicating that the DNs can be a source of inhibition onto the LNds. Interestingly, only a single LNd is directly AstC sensitive, further attesting to LNd heterogeneity and suggesting that behavioral regulation of the evening phase arises from this signaling to a single LNd. It is suggested that this response is communicated directly to the rest of the LNds - for example, via gap junctions - but a more indirect and circuitous route of communication cannot be excluded (Diaz, 2019).

It is noted that there are several caveats to the current model. The AstC and AstC-R2 knockdown experiments were conducted with only a single RNAi line each, due to the lack of additional functional RNAi lines. However, the two experiments show essentially identical phenotypes, suggesting that off-target effects are unlikely to pose a problem. It was not possible to rule out that the AstC knockdown phenotypes are not due to a developmental requirement. Experiments to address this point are challenging, because when the temperature is raised to reduce tubgal80ts repression and allow for an adult-only knockdown, the heat itself dramatically changes the E-peak timing. Lastly, lack of LPN- and DN3-specific drivers precluded addressing whether DN3-derived AstC is required for this evening activity peak modulation and whether the DN3s can directly inhibit the LNds (Diaz, 2019).

Although the AstC peptide sequence is highly conserved among insect species, only the AstC-R2 receptor has a mammalian homolog, the somatostatin-galanin-opioid receptor family. Inhibitory somatostatin (SST) interneurons are present in the mammalian equivalent of a central core clock, the suprachiasmatic nucleus (SCN). SST interneurons are also known to affect sleep and circadian behaviors. Interestingly, SST is associated with proper adaptation under photoperiod conditions for both diurnal and nocturnal mammals, suggesting a highly conserved function with AstC/AstC-R2 for adaptation under different equinox environments. It will be interesting to see whether the SCN-resident SST interneurons are important for this adaptation, like the AstC-containing clock neurons described in this study (Diaz, 2019).

Daily rewiring of a neural circuit generates a predictive model of environmental light

Behavioral responsiveness to external stimulation is shaped by context. This study examined how sensory information can be contextualized, by examining light-evoked locomotor responsiveness of Drosophila relative to time of day. Light elicits an acute increase in locomotion (startle) that is modulated in a time-of-day-dependent manner: startle is potentiated during the nighttime, when light is unexpected, but is suppressed during the daytime. The internal daytime-nighttime context is generated by two interconnected and functionally opposing populations of circadian neurons-LNvs generating the daytime state and DN1 as generating the nighttime state. Switching between the two states requires daily remodeling of LNv and DN1a axons such that the maximum presynaptic area in one population coincides with the minimum in the other. It is proposed that a dynamic model of environmental light resides in the shifting connectivities of the LNv-DN1a circuit, which helps animals evaluate ongoing conditions and choose a behavioral response (Song, 2021).

Alterations of neuronal activity, rather than morphology, are usually considered the cause of cognitive flexibility. The mechanism that this study describes relies on physical cellular restructuring. What are the advantages of a system like this? While near-instantaneous electrical activity is the basic language of neurons, many behaviors and internal states occur on much longer time scales. Morphological remodeling is a slow process, aligning with functions that change over the course of hours. In support of this view, changes in neuronal morphology have been found to underlie appetite, sexual experience, and foraging history. Although seemingly wasteful, physical remodeling may be particularly useful for encoding relatively stable states, due to presumably high energetic barrier (Song, 2021).

Understanding the mechanisms of circuit state transitions may help clarify the etiology of mood disorders like depression or bipolar disorder. These disorders are characterized by excessive or insufficient transitions between extreme states and, as such, may reflect a collapse of organizational principles that permit flexible circuit function. It is generally unknown how behavioral states can be stable across long time scales while also being able to undergo flexible transitions. Motifs from the LNv-DN1a circuit illustrate one solution to this apparent contradiction. LNvs and DN1as are arranged in a mutually inhibitory system, which may help ensure stability, consistency, and accuracy over long time scales. In the absence of external influences, reciprocal inhibition can stabilize a winner-take-all steady state. Structural plasticity is a potential way to overcome rigidity by providing a molecular mechanism to surmount electrical inhibition (Song, 2021).

In this model, the molecular oscillations of the circadian clock direct oscillations in cell shape through molecular effectors such as Rho1, ultimately leading to changes in behavior. Rho1 overexpression experiments suggest that neuronal morphology causally influences behavior. However, it cannot be ruled out that high levels of Rho1 cause off-target effects that have not been accounted for. Previous work used conditional methods to determine that Rho1 regulates daily LNv remodeling rhythms, but this study used constitutive Rho1 overexpression (to avoid using high temperatures required for the conditional experiment), and thus cannot exclude the possibility of developmental confounds (Song, 2021).

It is possible, if not likely, that other circadian neuronal populations also contribute to generating predictions about light. In this study, the optogenetic silencing phenotypes of LNvs and DN1as together recapitulate the phenotype produced by network-wide loss of clock function. However, the native circuit signal may be built by the cooperative action of multiple subpopulations with overlapping tuning. Hints of subpopulation cooperativity are apparent in the data: there was a stronger phenotype when PDF was knocked down in both small and large LNvs, compared to small LNvs alone. Glycine was recently found to be a fast, inhibitory neurotransmitter that is co-released from s-LNvs in addition to the neuropeptide PDF. While a necessary and sufficient role for the neuropeptide PDF in the behavioral assay, it cannot be ruled out that glycine instead mediates the fast inhibition seen during ex vivo calcium imaging. Furthermore, this study focused on two time points, but other populations may be more influential during other times of day. In support of this theory, it was recently found that lateral dorsal neurons also show axon remodeling rhythms centered around the evening, a time point that this study did not address. Because the other clock neuron subpopulations are active at different times, and recurrent inhibition is prevalent in the fly circadian network, it is likely that LNv-DN1a interactions are necessary, but not sufficient, for circadian network operations in the context described in this study (Song, 2021).

What this study calls 'startle' is commonly referred to as 'masking' because acute reactivity to lights-on and lights-off can distort measurements of circadian rhythmicity. For this reason, most assays of circadian function are done in unchanging environmental conditions (e.g., constant darkness). This study showed that startle to lights-on can also be an informative indicator of internal timekeeping. The conditional experiments using optogenetics suggest that clock neuron activity contextualizes light on an ongoing basis. However, because the experimental animals were exposed to daytime light during the habituation period, the possibility that LNv and DN1a activity is also required during entrainment cannot be excluded. Another consideration is that light resets the molecular clock. One hour of nighttime light can advance or delay circadian rhythms, which is apparent in the timing of rest and activity on subsequent days. It is unknown if this phase-shifting phenomenon affects the acute reactivity observed in this study (Song, 2021).

Circadian clocks have evolved in the context of consistent light schedules, and this predictability has been relatively unchallenged across evolutionary history. Ubiquitous artificial lighting introduces new strains on the circadian system. Misalignment between internal rhythms and the external world can have profound consequences on health and cognition. The feeling of jetlag demonstrates the acute physical and mental burden of when internal clocks are in conflict with the external world. Chronic misalignment, such as in the case of night-shift workers, causes increased rates of cancer. The use of electronic devices before bedtime has been linked to delays in sleep onset and reductions in sleep quality. The system this study described in Drosophila presents a model to understand the acute consequences of circadian misalignment (Song, 2021).

A predictive nervous system enables continual evaluation of reality relative to context. One result of this is that a fixed stimulus can evoke a multitude of behaviors depending on an animal's history, needs, and external context. This study shows how the Drosophila circadian system creates a dynamic internal reference for what environmental conditions should be. Many of the motifs observed are conserved: Mammalian circadian clock neuron subpopulations are also active at different times and are linked by recurrent inhibition, suggesting that mammalian temporal estimation may operate using similar principles. The paradigm that this study developed offers opportunities to understand the interface between internal models and sensory evidence. Circadian neurons are sensitive to environmental inputs: can they autonomously compute prediction error? They communicate with downstream dopaminergic populations: are those analogous to mammalian midbrain dopaminergic neurons whose activities reflect prediction error? It is proposed that flies assign valence to experienced environmental conditions, a computation that uses an internal model generated through circuit remodeling (Song, 2021).

Developmental temperature affects thermal dependence of locomotor activity in Drosophila

In their natural environments, animals have to cope with fluctuations in numerous abiotic and biotic factors, and phenotypic plasticity can facilitate survival under such variable conditions. However, organisms may differ substantially in the ability to adjust their phenotypes in response to external factors. This study investigated how developmental temperature affects the thermal performance curve for locomotor activity in adult fruit flies (Drosophila melanogaster). The thermal dependence was examined of spontaneous activity in individuals originating from two natural populations (from tropical (India) and temperate climate zone (Slovakia)) that developed at three different temperatures (19 °C, 25 °C, and 29 °C). Firstly, developmental temperature was found to have a significant impact on overall activity - flies that developed at high temperature (29 °C) were, on average, less active than individuals that developed at lower temperatures. Secondly, developmental acclimation had a population-specific effect on the thermal optimum for activity. Whereas the optimal temperature was not affected by thermal conditions experienced during development in flies from India, developmental temperature shifted thermal optimum in flies from Slovakia. Thirdly, high developmental temperature broadened performance breadth in flies from the Indian population but narrowed it in individuals from the Slovak population. Finally, no consistent effect of acclimation temperature was detected on circadian rhythms of spontaneous activity. Altogether, these results demonstrate that developmental temperature can alter different parameters (maximum performance, thermal optimum, performance breadth) of the thermal performance curve for spontaneous activity. Since adult fruit flies are highly vagile, this sensitivity of locomotion to developmental conditions may be an important factor affecting fitness in changing environments (Klepsatel, 2022).

A behavioral screen for mediators of age-dependent TDP-43 neurodegeneration identifies SF2/SRSF1 among a group of potent suppressors in both neurons and glia

Cytoplasmic aggregation of Tar-DNA/RNA binding protein 43 (TDP-43) occurs in 97 percent of amyotrophic lateral sclerosis (ALS), ~40% of frontotemporal dementia (FTD) and in many cases of Alzheimer's disease (AD). Cytoplasmic TDP-43 inclusions are seen in both sporadic and familial forms of these disorders, including those cases that are caused by repeat expansion mutations in the C9orf72 gene. To identify downstream mediators of TDP-43 toxicity, This study expressed human TDP-43 in a subset of Drosophila motor neurons. Such expression causes age-dependent deficits in negative geotaxis behavior. Using this behavioral readout of locomotion, this study conducted an shRNA suppressor screen and identified 32 transcripts whose knockdown was sufficient to ameliorate the neurological phenotype. The majority of these suppressors also substantially suppressed the negative effects on lifespan seen with glial TDP-43 expression. In addition to identification of a number of genes whose roles in neurodegeneration were not previously known, this screen also yielded genes involved in chromatin regulation and nuclear/import export - pathways that were previously identified in the context of cell based or neurodevelopmental suppressor screens. A notable example is SF2, a conserved orthologue of mammalian SRSF1, an RNA binding protein with roles in splicing and nuclear export. The identification SF2/SRSF1 as a potent suppressor of both neuronal and glial TDP-43 toxicity also provides a convergence with C9orf72 expansion repeat mediated neurodegeneration, where this gene also acts as a downstream mediator (Azpurua, 2021).

Integration of sleep homeostasis and navigation in Drosophila

During sleep, the brain undergoes dynamic and structural changes. In Drosophila, such changes have been observed in the central complex, a brain area important for sleep control and navigation. The connectivity of the central complex raises the question about how navigation, and specifically the head direction system, can operate in the face of sleep related plasticity. To address this question, this study develop a model that integrates sleep homeostasis and head direction. By introducing plasticity, the head direction system was shown to function in a stable way by balancing plasticity in connected circuits that encode sleep pressure. With increasing sleep pressure, the head direction system nevertheless becomes unstable and a sleep phase with a different plasticity mechanism is introduced to reset network connectivity. The proposed integration of sleep homeostasis and head direction circuits captures features of their neural dynamics observed in flies and mice (Flores-Valle, 2021).

Localization of muscarinic acetylcholine receptor-dependent rhythm-generating modules in the Drosophila larval locomotor network

This study explored how muscarinic acetylcholine receptor (mAChR)-modulated rhythm-generating networks are distributed in the central nervous system (CNS) of soft-bodied Drosophila larvae. Fictive motor patterns were measured in isolated CNS preparations, using a combination of Ca(2+) imaging and electrophysiology while manipulating mAChR signaling pharmacologically. Bath application of the mAChR agonist oxotremorine potentiated bilaterally asymmetric activity in anterior thoracic regions and promoted bursting in posterior abdominal regions. Application of the mAChR antagonist scopolamine suppressed rhythm generation in these regions and blocked the effects of oxotremorine. Oxotremorine triggered fictive forward crawling in preparations without brain lobes. Oxotremorine also potentiated rhythmic activity in isolated posterior abdominal CNS segments as well as isolated anterior brain and thoracic regions, but it did not induce rhythmic activity in isolated anterior abdominal segments. Bath application of scopolamine to reduced preparations lowered baseline Ca(2+) levels and abolished rhythmic activity. Overall, these results suggest that mAChR signaling plays a role in enabling rhythm generation at multiple sites in the larval CNS (Jonaitis, 2022).


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