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).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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 (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).

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

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

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

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

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

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

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

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

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

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).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

A decrease in SING performance has been previously reported when KCs in the α'β' lobes, but not in the αβ and γ lobes, were thermogenetically stimulated or their synaptic output silenced. Using a set of specific drivers, the contribution of the various MB lobes in the modulation of this innate reflex was precisely studied. It was confirmed that the α'β' KCs down-regulate SING when they are activated but not when their output is inhibited. Other unidentified neurons, targeted by the rather non-selective c305a-Gal4 and G0050-Gal4 drivers, trigger a decrease in SING performance when they are inhibited by Shi^ts1, and are therefore potential SING-activating neurons. It was further found that the MB γ lobes contain KCs that strongly inhibit SING when activated, both by thermogenetic and optogenetic stimulation, as shown with the γ-lobe specific driver R16A06-Gal4. However, thermoactivation of γ neurons with other drivers, like mb247-Gal4, which express both in the αβ and γ lobe, did not decrease SING. This could result from an inhibitory effect of αβ neuron activation on SING modulation by γ neurons. To test this hypothesis, a double-driver was generated by recombining mb247-Gal4 with R16A06-Gal4. Because both drivers express in the γ lobes, one would expect a stronger effect on SING modulation after thermoactivation with the double-driver than with R16A06-Gal4 alone. The opposite was observed, i.e., that SING was decreased to a lesser extent with the double-driver than with R16A06-Gal4 alone. Activation of mb247-Gal4 αβ neurons therefore likely counterbalanced the effect of γ neuron activation with R16A06-Gal4 on SING modulation. A similar and even more obvious result was obtained when mb247-Gal4 was recombined with the α'β' driver R35B12-Gal4: co-activation of the neurons targeted by these two drivers prevented the strong SING modulation normally induced by R35B12-Gal4 alone. These results suggest the existence of an inter-compartmental communication process for locomotor reactivity control in the Drosophila MB. Comparably, it was recently suggested, in the case of memory retrieval, that MB output channels are ultimately pooled such that blockade (or activation) of all the outputs from a given population of KCs may have no apparent effect on odor-driven behavior, while such behavior can be changed by blocking a single output. Such a transfer of information could occur, as was previously reported, through connections involving the MBONs within the lobes or outside the MB (Sun, 2018).

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

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

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

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