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).

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).

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).