The Interactive Fly
Genes regulating behavior
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).