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 medulla^KS74 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).

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).

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

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

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

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)?

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

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

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

To complete a turn, peristalsis can resume from different positions depending on the size of the head sweep. It is unclear why this happens, but these observations do demonstrate that peristalsis can be initiated at different points along the body. One reason might be to avoid disrupting the angle of reorientation defined by the angular size of large head sweeps: if peristalsis were initiated at the tail after a large head sweep, the position of the pivot might be pushed forward when the peristalsis reaches the pivot, perhaps increasing the angle of the reorientation beyond the size of the original head sweep. The ability to initiate peristalsis in different segments depending on the degree of body bend might point to a role for sensory feedback in the motor circuit. Proprioceptive feedback has recently been shown to play an essential role in generating peristalsis in the Drosophila larva (Hughes, 2007; Song, 2007; Cheng, 2010; Lahiri, 2011 and references therein).

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

Arousal is fundamental to many behaviors, but whether it is unitary or whether there are different types of behavior-specific arousal has not been clear. In Drosophila, dopamine promotes sleep-wake arousal. However, there is conflicting evidence regarding its influence on environmentally stimulated arousal. This study shows that loss-of-function mutations in the D1 dopamine receptor DopR enhance repetitive startle-induced arousal while decreasing sleep-wake arousal (i.e., increasing sleep). These two types of arousal are also inversely influenced by cocaine, whose effects in each case are opposite to, and abrogated by, the DopR mutation. Selective restoration of DopR function in the central complex rescues the enhanced stimulated arousal but not the increased sleep phenotype of DopR mutants. These data provide evidence for at least two different forms of arousal, which are independently regulated by dopamine in opposite directions, via distinct neural circuits (Lebestky, 2009).

'Arousal', a state characterized by increased activity, sensitivity to sensory stimuli, and certain patterns of brain activity, accompanies many different behaviors, including circadian rhythms, escape, aggression, courtship, and emotional responses in higher vertebrates. A key unanswered question is whether arousal is a unidimensional, generalized state. Biogenic amines, such as dopamine (DA), norepinephrine (NE), serotonin (5-HT), and histamine, as well as cholinergic systems, have all been implicated in arousal in numerous behavioral settings. However, it is not clear whether these different neuromodulators act on a common 'generalized arousal' pathway or rather control distinct arousal pathways or circuits that independently regulate different behaviors. Resolving this issue requires identifying the receptors and circuits on which these neuromodulators act, in different behavioral settings of arousal (Lebestky, 2009).

Most studies of arousal in Drosophila have focused on locomotor activity reflecting sleep-wake transitions, a form of 'endogenously generated' arousal. Several lines of evidence point to a role for DA in enhancing this form of arousal in Drosophila. Drug-feeding experiments, as well as genetic silencing of dopaminergic neurons, have indicated that DA promotes waking during the subjective night phase of the circadian cycle. Similar conclusions were drawn from studying mutations in the Drosophila DA transporter (dDAT). Consistent with these data, overexpression of the vesicular monoamine transporter (dVMAT-A), promoted hyperactivity in this species, as did activation of DA neurons in quiescent flies (Lebestky, 2009).

Evidence regarding the nature of DA effects on 'exogenously generated' or environmentally stimulated arousal, such as that elicited by startle, is less consistent. Classical genetic studies and quantitative trait locus (QTL) analyses have suggested that differences in DA levels may underlie genetic variation in startle-induced locomotor activity (see Carbone, 2006 and Jordan, 2006). Fmn (dDAT; Dopamine transporter) mutants displayed hyperactivity in response to mechanical shocks, implying a positive-acting role for DA in controlling environmentally induced arousal (Kume, 2005). In contrast, other data imply a negative-acting role for DA in controlling stimulated arousal. Mutants in Tyr-1, which exhibit a reduction in dopamine levels, show an increase in stimulated but not spontaneous levels of locomotor activity. Genetic inhibition of tyrosine hydroxylase-expressing neurons caused hyperactivity in response to mechanical startle (Friggi-Grelin, 2003). Finally, transient activation of DA neurons in hyperactive flies inhibited locomotion (Lima, 2005). Whether these differing results reflect differences in behavioral assays, the involvement of different types of DA receptors, or an 'inverted U'-like dosage sensitivity to DA (Birman, 2005), is unclear (Lebestky, 2009).

This investigation has developed a novel behavioral paradigm for environmentally stimulated arousal, using repetitive mechanical startle as a stimulus, and a screen was carried out for mutations that potentiate this response. One such mutation is a hypomorphic allele of the D1 receptor ortholog, DopR. This same mutation caused decreased spontaneous activity during the night phase of the circadian cycle, due to increased rest bout duration. In both assays, cocaine influenced behavior in the opposite direction as the DopR mutation, and the effect of cocaine was abolished in DopR mutant flies, supporting the idea that DA inversely regulates these two forms of arousal. Genetic rescue experiments, using Gal4 drivers with restricted CNS expression, indicate that these independent and opposite influences of DopR are exerted in different neural circuits. These data suggest the existence of different types of arousal states mediated by distinct neural circuits in Drosophila, which can be oppositely regulated by DA acting via the same receptor subtype (Lebestky, 2009).

Previous studies of arousal in Drosophila have focused on sleep-wake transitions, a form of 'endogenous' arousal. This study has introduced and characterized a quantitative behavioral assay for repetitive startle-induced hyperactivity, which displays properties consistent with an environmentally triggered ('exogenous') arousal state. A screen was conducted for mutations affecting this behavior, the phenotype of one such mutation (DopR) was analyzed, and the neural substrates of its action was mapped by cell-specific genetic rescue experiments. The results reveal that DopR independently regulates Repetitive Startle-induced Hyperactivity (ReSH) and sleep in opposite directions by acting on distinct neural substrates. Negative regulation of the ReSH response requires DopR function in the ellipsoid body (EB) of the central complex (CC), while positive regulation of waking reflects a function in other populations of neurons, including PDF-expressing circadian pacemaker cells. Both of these functions, moreover, are independent of the function of DopR in learning and memory, which is required in the mushroom body. These data suggest that ReSH behavior and sleep-wake transitions reflect distinct forms of arousal that are genetically, anatomically, and behaviorally separable. This conclusion is consistent with earlier suggestions, based on classical genetic studies, that spontaneous and environmentally stimulated locomotor activity reflect 'distinct behavioral systems' in Drosophila (Lebestky, 2009).

Several lines of evidence suggest that ReSH behavior represents a form of environmentally stimulated arousal. First, hyperactivity is an evolutionarily conserved expression of increased arousal. Although not all arousal is necessarily expressed as hyperactivity, electrophysiological studies indicate that mechanical startle, the type of stimulus used in this study, evokes increases in 20-30 Hz and 80-90 Hz brain activity, which have been suggested to reflect a neural correlate of arousal in flies (Nitz, 2002; van Swinderen, 2004). Second, ReSH does not immediately dissipate following termination of the stimulus, as would be expected for a simple reflexive stimulus-response behavior, but rather persists for an extended period of time, suggesting that it reflects a change in internal state. Third, this state, like arousal, is scalable: more puffs, or more intense puffs, produce a stronger and/or longer-lasting state of hyperactivity. Fourth, this state exhibits sensitization: even after overt locomotor activity has recovered to prepuff levels, flies remain hypersensitive to a single puff for several minutes. Fifth, this sensitization state generalizes to a startle stimulus of at least one other sensory modality (olfactory). In Aplysia, sensitization of the gill/siphon withdrawal reflex has been likened to behavioral arousal. Taken together, these features strongly suggest that ReSH represents an example of environmentally stimulated ('exogenous') arousal in Drosophila (Lebestky, 2009).

DopR mutant flies exhibited longer rest periods during their subjective night phase, suggesting that DopR normally promotes sleep-wake transitions. These data are consistent with earlier studies indicating that DA promotes arousal by inhibiting sleep (Andretic, 2005, Kume, 2005; Wu, 2008). In contrast, prior evidence regarding the role of DA in startle-induced arousal is conflicting. Some studies have suggested that DA negatively regulates locomotor reactivity to environmental stimuli, consistent with the current observations, while others have suggested that it positively regulates this response. Even within the same study, light-stimulated activation of TH⁺ neurons produced opposite effects on locomotion, depending on the prestimulus level of locomotor activity (Lima, 2005; Lebestky, 2009 and references therein).

This study has found that DA and DopR negatively regulate environmentally stimulated arousal: the DopR mutation enhanced the ReSH response, while cocaine suppressed it. Furthermore, the effect of cocaine in the ReSH assay was eliminated in the DopR mutant but could be rescued by Gal4-driven DopR expression, confirming that the effect of the drug is mediated by DA. Taken together, these results reconcile apparently conflicting data on the role of DA in 'arousal' in Drosophila by identifying two different forms of arousal -- repetitive startle-induced arousal and sleep-wake arousal -- that are regulated by DA in an inverse manner (Lebestky, 2009).

The finding that DopR negatively regulates one form of environmentally stimulated arousal leaves open the question of whether this is true for all types of exogenous arousing stimuli. The 'sign' of the influence of DA on exogenously generated arousal states may vary depending on the type or strength of the stimulus used, the initial state of the system prior to exposure to the arousing stimulus (Birman, 2005; Lima and Miesenbock, 2005), or the precise neural circuitry that is engaged. Future studies using arousing stimuli of different sensory modalities or associated with different behaviors should shed light on this question (Lebestky, 2009).

Several lines of evidence suggest that endogenous DopR likely acts in the ellipsoid body (EB) of the central complex (CC) to regulate repetitive startle-induced arousal. First, multiple Gal4 lines that drive expression in the EB rescued the ReSH phenotype of DopR mutants. Second, endogenous DopR is expressed in EB neurons, including those in which the rescuing Gal4 drivers are expressed. Third, the domain of DopR expression in the EB overlaps the varicosities of TH⁺ fibers. In an independent study of dopaminergic inputs required for regulating EtOH-stimulated hyperactivity TH⁺ neurons were identified that are a likely source of these projections to the EB. Fourth, rescue of the ReSH phenotype is associated with re-expression of DopR in EB neurons. Finally, rescue is observed using conditional DopR expression in adults. Taken together, these data argue that rescue of the ReSH phenotype by the Gal4 lines tested reflects their common expression in the EB and that this is a normal site of DopR action in adult flies (Lebestky, 2009).

A requirement for DopR in the EB in regulating ReSH behavior is consistent with the fact that the CC is involved in the control of walking activity. However, the mushroom body has also been implicated in the control of locomotor behavior, and DopR is strongly expressed in this structure as well. Rescue data argue against the MB and in favor of the CC as a neural substrate for the ReSH phenotype of DopR mutants. Unexpectedly, the nocturnal hypoactivity phenotype of DopR mutants was not rescued by restoration of DopR expression to the CC. Thus, not all locomotor activity phenotypes of the DopR mutant necessarily reflect a function for the gene in the CC (Lebestky, 2009).

Interestingly, Gal4 line c547 expresses in R2/R4m neurons of the EB, while lines 189y and c761 express in R3 neurons, yet both rescued the ReSH phenotype of DopR mutants. Similar results have been obtained in experiments to rescue the deficit in ethanol-induced behavior exhibited by the DopR mutant. Double-labeling experiments suggest that endogenous DopR is expressed in all of these EB neuronal subpopulations. Perhaps the receptor functions in parallel or in series in R4m and R3 neurons, so that restoration of DopR expression in either population can rescue the ReSH phenotype. Whether these DopR-expressing EB subpopulations are synaptically interconnected is an interesting question for future investigation (Lebestky, 2009).

Despite its power as a system for studying neural development, function, and behavior, Drosophila has not been extensively used in affective neuroscience, in part due to uncertainty about whether this insect exhibits emotion-like states or behaviors. Increased arousal is a key component of many emotional or affective behaviors. The data presented in this study indicate that Drosophila can express a persistent arousal state in response to repetitive stress. ReSH behavior exhibits several features that distinguish it from simple, reflexive stimulus-response behaviors: scalability, persistence following stimulus termination, and sensitization. In addition, the observation that mechanical trauma promotes release from Drosophila of an odorant that repels other flies suggests that the arousal state underlying ReSH behavior may have a negative 'affective valence' as well. These considerations, taken together with the fact that ReSH is influenced by genetic and pharmacologic manipulations of DA, a biogenic amine implicated in emotional behavior in humans, support the idea that the ReSH response may represent a primitive 'emotion-like' behavior in Drosophila (Lebestky, 2009).

The phenotype of DopR flies is reminiscent of attention-deficit hyperactivity disorder (ADHD), an affective disorder linked to dopamine, whose symptoms include hyper-reactivity to environmental stimuli. If humans, like flies, have distinct circuits for different forms of arousal, then the current data suggest that ADHD may specifically involve dopaminergic dysfunction in those circuits mediating environmentally stimulated, rather than endogenous (sleep-wake), arousal. Given that DA negatively regulates environmentally stimulated arousal circuits in Drosophila, such a view would be consistent with the fact that treatment with drugs that increase synaptic levels of DA, such as methylphenidate (ritalin), can ameliorate symptoms of ADHD (Lebestky, 2009).

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

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

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

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

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

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

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

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

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

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

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

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

Next it was considered where spatial memories are processed (or stored) in the Drosophila brain. It was reasoned that specific regions of the fly brain would function as the neuroanatomical substrate for visual place learning, and therefore animals were tested in which different brain areas were selectively inactivated using the GAL4/UAS expression system. In essence, small subsets of neurons were conditionally silenced in adult flies by targeting expression of the inward-rectifying potassium channel Kir2.1 to limit potential side-effects of Kir2.1 expression during development, a temperature-sensitive GAL80^ts was used that blocks Kir2.1 expression when flies are reared at 18°C but allows expression when the temperature is raised to 30°C before testing. GAL4 driver lines were selected for expression in two areas: the mushroom bodies and the central complex. The mushroom bodies have been the subject of extensive studies of learning and memory in Drosophila , and have been shown to be essential for associative olfactory conditioning but not for some other forms of learning, such as tactile, motor and non-visually guided place learning. The central complex is thought to be a site of orientation behaviour, multisensory integration and other 'high-order' processes. In some social insects, the mushroom bodies have been implicated in visual place learning, and in the cockroach bilateral surgical lesions to these structures abolish spatial learning. However, no evidence was seen for involvement of the mushroom bodies in the assay. In fact, silencing mushroom body intrinsic neurons using the GAL4 drivers R9A11, R10B08, R67B04, had no significant effect on the performance of flies in visual place learning. The differing requirement for the mushroom bodies between Drosophila and other species may be explained by the observations that mushroom body inputs in Drosophila are predominantly olfactory. In sharp contrast, silencing subsets of neurons with projections to the central complex ellipsoid body did have a significant effect. Notably, silencing a different subset of ring neurons with line R38H02 leaves visual place learning intact. Thus, specific circuits within the ellipsoid body (but not the entire structure) are necessary for visual place learning (Ofstad, 2011).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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