Model organisms and systems in neuroethology: one hundred years of history and a look into the future.

The Journal of Comparative Physiology lived up to its name in the last 100 years by including more than 1500 different taxa in almost 10,000 publications. Seventeen phyla of the animal kingdom were represented. The honeybee (Apis mellifera) is the taxon with most publications, followed by locust (Locusta migratoria), crayfishes (Cambarus spp.), and fruitfly (Drosophila melanogaster). The representation of species in this journal in the past, thus, differs much from the 13 model systems as named by the National Institutes of Health (USA). We mention major accomplishments of research on species with specific adaptations, specialist animals, for example, the quantitative description of the processes underlying the axon potential in squid (Loligo forbesii) and the isolation of the first receptor channel in the electric eel (Electrophorus electricus) and electric ray (Torpedo spp.). Future neuroethological work should make the recent genetic and technological developments available for specialist animals. There are many research questions left that may be answered with high yield in specialists and some questions that can only be answered in specialists. Moreover, the adaptations of animals that occupy specific ecological niches often lend themselves to biomimetic applications. We go into some depth in explaining our thoughts in the research of motion vision in insects, sound localization in barn owls, and electroreception in weakly electric fish.

Abstract Encoding of Categorical Decisions in Medial Superior Temporal and Lateral Intraparietal Cortices.

Categorization is an essential cognitive and perceptual process for decision-making and recognition. The posterior parietal cortex, particularly the lateral intraparietal (LIP) area has been suggested to transform visual feature encoding into abstract categorical representations. By contrast, areas closer to sensory input, such as the middle temporal (MT) area, encode stimulus features but not more abstract categorical information during categorization tasks. Here, we compare the contributions of the medial superior temporal (MST) and LIP areas in category computation by recording neuronal activity in both areas from two male rhesus macaques trained to perform a visual motion categorization task. MST is a core motion-processing region interconnected with MT and is often considered an intermediate processing stage between MT and LIP. We show that MST exhibits robust decision-correlated motion category encoding and working memory encoding similar to LIP, suggesting that MST plays a substantial role in cognitive computation, extending beyond its widely recognized role in visual motion processing.SIGNIFICANCE STATEMENT Categorization requires assigning incoming sensory stimuli into behaviorally relevant groups. Previous work found that parietal area LIP shows a strong encoding of the learned category membership of visual motion stimuli, while visual area MT shows strong direction tuning but not category tuning during a motion direction categorization task. Here we show that the medial superior temporal (MST) area, a visual motion-processing region interconnected with both LIP and MT, shows strong visual category encoding similar to that observed in LIP. This suggests that MST plays a greater role in abstract cognitive functions, extending beyond its well known role in visual motion processing.

A combinatorial code of transcription factors specifies subtypes of visual motion-sensing neurons in Drosophila.

Direction-selective T4/T5 neurons exist in four subtypes, each tuned to visual motion along one of the four cardinal directions. Along with their directional tuning, neurons of each T4/T5 subtype orient their dendrites and project their axons in a subtype-specific manner. Directional tuning, thus, appears strictly linked to morphology in T4/T5 neurons. How the four T4/T5 subtypes acquire their distinct morphologies during development remains largely unknown. Here, we investigated when and how the dendrites of the four T4/T5 subtypes acquire their specific orientations, and profiled the transcriptomes of all T4/T5 neurons during this process. This revealed a simple and stable combinatorial code of transcription factors defining the four T4/T5 subtypes during their development. Changing the combination of transcription factors of specific T4/T5 subtypes resulted in predictable and complete conversions of subtype-specific properties, i.e. dendrite orientation and matching axon projection pattern. Therefore, a combinatorial code of transcription factors coordinates the development of dendrite and axon morphologies to generate anatomical specializations that differentiate subtypes of T4/T5 motion-sensing neurons.

Parallel motion vision pathways in the brain of a tropical bee.

Spatial orientation is a prerequisite for most behaviors. In insects, the underlying neural computations take place in the central complex (CX), the brain's navigational center. In this region different streams of sensory information converge to enable context-dependent navigational decisions. Accordingly, a variety of CX input neurons deliver information about different navigation-relevant cues. In bees, direction encoding polarized light signals converge with translational optic flow signals that are suited to encode the flight speed of the animals. The continuous integration of speed and directions in the CX can be used to generate a vector memory of the bee's current position in space in relation to its nest, i.e., perform path integration. This process depends on specific, complex features of the optic flow encoding CX input neurons, but it is unknown how this information is derived from the visual periphery. Here, we thus aimed at gaining insight into how simple motion signals are reshaped upstream of the speed encoding CX input neurons to generate their complex features. Using electrophysiology and anatomical analyses of the halictic bees Megalopta genalis and Megalopta centralis, we identified a wide range of motion-sensitive neurons connecting the optic lobes with the central brain. While most neurons formed pathways with characteristics incompatible with CX speed neurons, we showed that one group of lobula projection neurons possess some physiological and anatomical features required to generate the visual responses of CX optic-flow encoding neurons. However, as these neurons cannot explain all features of CX speed cells, local interneurons of the central brain or alternative input cells from the optic lobe are additionally required to construct inputs with sufficient complexity to deliver speed signals suited for path integration in bees.

Hoverfly (Eristalis tenax) pursuit of artificial targets.

The ability to visualize small moving objects is vital for the survival of many animals, as these could represent predators or prey. For example, predatory insects, including dragonflies, robber flies and killer flies, perform elegant, high-speed pursuits of both biological and artificial targets. Many non-predatory insects, including male hoverflies and blowflies, also pursue targets during territorial or courtship interactions. To date, most hoverfly pursuits have been studied outdoors. To investigate hoverfly (Eristalis tenax) pursuits under more controlled settings, we constructed an indoor arena that was large enough to encourage naturalistic behavior. We presented artificial beads of different sizes, moving at different speeds, and filmed pursuits with two cameras, allowing subsequent 3D reconstruction of the hoverfly and bead position as a function of time. We show that male E. tenax hoverflies are unlikely to use strict heuristic rules based on angular size or speed to determine when to start pursuit, at least in our indoor setting. We found that hoverflies pursued faster beads when the trajectory involved flying downwards towards the bead. Furthermore, we show that target pursuit behavior can be broken down into two stages. In the first stage, the hoverfly attempts to rapidly decreases the distance to the target by intercepting it at high speed. During the second stage, the hoverfly's forward speed is correlated with the speed of the bead, so that the hoverfly remains close, but without catching it. This may be similar to dragonfly shadowing behavior, previously coined 'motion camouflage'.

Floor and ceiling mirror configurations to study altitude control in honeybees.

To investigate altitude control in honeybees, an optical configuration was designed to manipulate or cancel the optic flow. It has been widely accepted that honeybees rely on the optic flow generated by the ground to control their altitude. Here, we create an optical configuration enabling a better understanding of the mechanism of altitude control in honeybees. This optical configuration aims to mimic some of the conditions that honeybees experience over a natural water body. An optical manipulation, based on a pair of opposed horizontal mirrors, was designed to remove any visual information coming from the floor and ceiling. Such an optical manipulation allowed us to get closer to the seminal experiment of Heran & Lindauer 1963. Zeitschrift für vergleichende Physiologie47, 39-55. (doi:10.1007/BF00342890). Our results confirmed that a reduction or an absence of ventral optic flow in honeybees leads to a loss in altitude, and eventually a collision with the floor.

Temporal vision: measures, mechanisms and meaning.

Time is largely a hidden variable in vision. It is the condition for seeing interesting things such as spatial forms and patterns, colours and movements in the external world, and yet is not meant to be noticed in itself. Temporal aspects of visual processing have received comparatively little attention in research. Temporal properties have been made explicit mainly in measurements of resolution and integration in simple tasks such as detection of spatially homogeneous flicker or light pulses of varying duration. Only through a mechanistic understanding of their basis in retinal photoreceptors and circuits can such measures guide modelling of natural vision in different species and illuminate functional and evolutionary trade-offs. Temporal vision research would benefit from bridging traditions that speak different languages. Towards that goal, I here review studies from the fields of human psychophysics, retinal physiology and neuroethology, with a focus on fundamental constraints set by early vision.

Aerial course stabilization is impaired in motion-blind flies.

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

Behavioral state modulates the ON visual motion pathway of Drosophila.

The behavioral state of an animal can dynamically modulate visual processing. In flies, the behavioral state is known to alter the temporal tuning of neurons that carry visual motion information into the central brain. However, where this modulation occurs and how it tunes the properties of this neural circuit are not well understood. Here, we show that the behavioral state alters the baseline activity levels and the temporal tuning of the first directionally selective neuron in the ON motion pathway (T4) as well as its primary input neurons (Mi1, Tm3, Mi4, Mi9). These effects are especially prominent in the inhibitory neuron Mi4, and we show that central octopaminergic neurons provide input to Mi4 and increase its excitability. We further show that octopamine neurons are required for sustained behavioral responses to fast-moving, but not slow-moving, visual stimuli in walking flies. These results indicate that behavioral-state modulation acts directly on the inputs to the directionally selective neurons and supports efficient neural coding of motion stimuli.

Differential Tuning to Visual Motion Allows Robust Encoding of Optic Flow in the Dragonfly.

Visual cues provide an important means for aerial creatures to ascertain their self-motion through the environment. In many insects, including flies, moths, and bees, wide-field motion-sensitive neurons in the third optic ganglion are thought to underlie such motion encoding; however, these neurons can only respond robustly over limited speed ranges. The task is more complicated for some species of dragonflies that switch between extended periods of hovering flight and fast-moving pursuit of prey and conspecifics, requiring motion detection over a broad range of velocities. Since little is known about motion processing in these insects, we performed intracellular recordings from hawking, emerald dragonflies (Hemicordulia spp.) and identified a diverse group of motion-sensitive neurons that we named lobula tangential cells (LTCs). Following prolonged visual stimulation with drifting gratings, we observed significant differences in both temporal and spatial tuning of LTCs. Cluster analysis of these changes confirmed several groups of LTCs with distinctive spatiotemporal tuning. These differences were associated with variation in velocity tuning in response to translated, natural scenes. LTCs with differences in velocity tuning ranges and optima may underlie how a broad range of motion velocities are encoded. In the hawking dragonfly, changes in LTC tuning over time are therefore likely to support their extensive range of behaviors, from hovering to fast-speed pursuits.SIGNIFICANCE STATEMENT Understanding how animals navigate the world is an inherently difficult and interesting problem. Insects are useful models for understanding neuronal mechanisms underlying these activities, with neurons that encode wide-field motion previously identified in insects, such as flies, hawkmoths, and butterflies. Like some Dipteran flies, dragonflies exhibit complex aerobatic behaviors, such as hovering, patrolling, and aerial combat. However, dragonflies lack halteres that support such diverse behavior in flies. To understand how dragonflies might address this problem using only visual cues, we recorded from their wide-field motion-sensitive neurons. We found these differ strongly in the ways they respond to sustained motion, allowing them collectively to encode the very broad range of velocities experienced during diverse behavior.

The diversity of lobula plate tangential cells (LPTCs) in the Drosophila motion vision system.

To navigate through the environment, animals rely on visual feedback to control their movements relative to their surroundings. In dipteran flies, visual feedback is provided by the wide-field motion-sensitive neurons in the visual system called lobula plate tangential cells (LPTCs). Understanding the role of LPTCs in fly behaviors can address many fundamental questions on how sensory circuits guide behaviors. The blowfly was estimated to have ~ 60 LPTCs, but only a few have been identified in Drosophila. We conducted a Gal4 driver screen and identified five LPTC subtypes in Drosophila, based on their morphological characteristics: LPTCs have large arborizations in the lobula plate and project to the central brain. We compared their morphologies to the blowfly LPTCs and named them after the most similar blowfly cells: CH, H1, H2, FD1 and FD3, and V1. We further characterized their pre- and post-synaptic organizations, as well as their neurotransmitter profiles. These anatomical features largely agree with the anatomy and function of their likely blowfly counterparts. Nevertheless, several anatomical details indicate the Drosophila LPTCs may have more complex functions. Our characterization of these five LPTCs in Drosophila will facilitate further functional studies to understand their roles in the visual circuits that instruct fly behaviors.

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

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

Modelling Drosophila motion vision pathways for decoding the direction of translating objects against cluttered moving backgrounds.

Decoding the direction of translating objects in front of cluttered moving backgrounds, accurately and efficiently, is still a challenging problem. In nature, lightweight and low-powered flying insects apply motion vision to detect a moving target in highly variable environments during flight, which are excellent paradigms to learn motion perception strategies. This paper investigates the fruit fly Drosophila motion vision pathways and presents computational modelling based on cutting-edge physiological researches. The proposed visual system model features bio-plausible ON and OFF pathways, wide-field horizontal-sensitive (HS) and vertical-sensitive (VS) systems. The main contributions of this research are on two aspects: (1) the proposed model articulates the forming of both direction-selective and direction-opponent responses, revealed as principal features of motion perception neural circuits, in a feed-forward manner; (2) it also shows robust direction selectivity to translating objects in front of cluttered moving backgrounds, via the modelling of spatiotemporal dynamics including combination of motion pre-filtering mechanisms and ensembles of local correlators inside both the ON and OFF pathways, which works effectively to suppress irrelevant background motion or distractors, and to improve the dynamic response. Accordingly, the direction of translating objects is decoded as global responses of both the HS and VS systems with positive or negative output indicating preferred-direction or null-direction translation. The experiments have verified the effectiveness of the proposed neural system model, and demonstrated its responsive preference to faster-moving, higher-contrast and larger-size targets embedded in cluttered moving backgrounds.

Integration of Small- and Wide-Field Visual Features in Target-Selective Descending Neurons of both Predatory and Nonpredatory Dipterans.

For many animals, target motion carries high ecological significance as this may be generated by a predator, prey, or potential mate. Indeed, animals whose survival depends on early target detection are often equipped with a sharply tuned visual system, yielding robust performance in challenging conditions. For example, many fast-flying insects use visual cues for identifying targets, such as prey (e.g., predatory dragonflies and robberflies) or conspecifics (e.g., nonpredatory hoverflies), and can often do so against self-generated background optic flow. Supporting these behaviors, the optic lobes of insects that pursue targets harbor neurons that respond robustly to the motion of small moving objects, even when displayed against syn-directional background clutter. However, in diptera, the encoding of target information by the descending neurons, which are more directly involved in generating the behavioral output, has received less attention. We characterized target-selective neurons by recording in the ventral nerve cord of male and female predatory Holcocephala fusca robberflies and of male nonpredatory Eristalis tenax hoverflies. We show that both species have dipteran target-selective descending neurons that only respond to target motion if the background is stationary or moving slowly, moves in the opposite direction, or has un-naturalistic spatial characteristics. The response to the target is suppressed when background and target move at similar velocities, which is strikingly different to the response of target neurons in the optic lobes. As the neurons we recorded from are premotor, our findings affect our interpretation of the neurophysiology underlying target-tracking behaviors.SIGNIFICANCE STATEMENT Many animals use sensory cues to detect moving targets that may represent predators, prey, or conspecifics. For example, birds of prey show superb sensitivity to the motion of small prey, and intercept these at high speeds. In a similar manner, predatory insects visually track moving prey, often against cluttered backgrounds. Accompanying this behavior, the brains of insects that pursue targets contain neurons that respond exclusively to target motion. We here show that dipteran insects also have target-selective descending neurons in the part of their nervous system that corresponds to the vertebrate spinal cord. Surprisingly, and in contrast to the neurons in the brain, these premotor neurons are inhibited by background patterns moving in the same direction as the target.

Monopolatic motion vision in the butterfly Papilio xuthus.

The swallowtail butterfly Papilio xuthus can perceive the linear polarization of light. Using a novel polarization projection system, we recently demonstrated that P. xuthus can detect visual motion based on polarization contrast. In the present study, we attempt to infer via behavioural experiments the mechanism underlying this polarization-based motion vision. Papilio xuthus do not perceive contrast between unpolarized and diagonally polarized light, implying that they cannot unambiguously estimate angle and degree of polarization, at least as far as motion detection is concerned. Furthermore, they conflate brightness and polarization cues, such that bright vertically polarized light resembles dim unpolarized light. These observations are consistent with a one-channel 'monopolatic' detector mechanism. We extend our existing model of motion vision in P. xuthus to incorporate these polarization findings, and conclude that the photoreceptors likely to form the basis for the putative monopolatic polarization detector are R3 and R4, which respond maximally to horizontally polarized green light. R5-R8, we propose, form a polarization-insensitive secondary channel tuned to longer wavelengths of light. Consistent with this account, we see greater sensitivity to polarization for green-light stimuli than for subjectively equiluminant red ones. Somewhat counter-intuitively, our model predicts greatest sensitivity to vertically polarized light; owing to the non-linearity of photoreceptor responses, light polarized to an angle orthogonal to a monopolatic detector's orientation offers the greatest contrast with unpolarized light.

Object features and T4/T5 motion detectors modulate the dynamics of bar tracking by Drosophila.

Visual objects can be discriminated by static spatial features such as luminance or dynamic features such as relative movement. Flies track a solid dark vertical bar moving on a bright background, a behavioral reaction so strong that for a rigidly tethered fly, the steering trajectory is phase advanced relative to the moving bar, apparently in anticipation of its future position. By contrast, flickering bars that generate no coherent motion or have a surface texture that moves in the direction opposite to the bar generate steering responses that lag behind the stimulus. It remains unclear how the spatial properties of a bar influence behavioral response dynamics. Here,  we show that a dark bar defined by its luminance contrast to the uniform background drives a co-directional steering response that is phase advanced relative to the response to a textured bar defined only by its motion relative to a stationary textured background. The textured bar drives an initial contra-directional turn and phase-locked tracking. The qualitatively distinct response dynamics could indicate parallel visual processing of a luminance versus motion-defined object. Calcium imaging shows that T4/T5 motion-detecting neurons are more responsive to a solid dark bar than a motion-defined bar. Genetically blocking T4/T5 neurons eliminates the phase-advanced co-directional response to the luminance-defined bar, leaving the orientation response largely intact. We conclude that T4/T5 neurons mediate a co-directional optomotor response to a luminance-defined bar, thereby driving phase-advanced wing kinematics, whereas separate unknown visual pathways elicit the contra-directional orientation response.

Parameters of motion vision in low light in the hawkmoth Manduca sexta.

The hawkmoth Manduca sexta is nocturnally active, beginning its flight activity at sunset, and executing rapid controlled maneuvers to search for food and mates in dim light conditions. The visual system of this moth has been shown to trade off spatial and temporal resolution for increased sensitivity in these conditions. The study presented here uses tethered flying moths to characterize the flight performance envelope of the wide-field-motion-triggered steering response of M. sexta in low light conditions by measuring attempted turning in response to wide-field visual motion. Moths were challenged with a horizontally oscillating sinusoidal grating at a range of luminance, from daylight to starlight conditions. The impact of luminance on response to a range of temporal frequencies and spatial wavelengths was assessed across a range of pattern contrasts. The optomotor response decreased as a function of decreasing luminance, and the lower limit of the moth's contrast sensitivity was found to be between 1 and 5%. The preferred spatial frequency for M. sexta increased from 0.06 to 0.3 cycles deg-1 as the luminance decreased, but the preferred temporal frequency remained stable at 4.5 Hz across all conditions. The relationship between the optomotor response time to the temporal frequency of the pattern movement did not vary significantly with luminance levels. Taken together, these results suggest that the behavioral response to wide-field visual input in M. sexta is adapted to operate during crepuscular to nocturnal luminance levels, and the decreasing light levels experienced during that period changes visual acuity and does not affect their response time significantly.

Visual approach computation in feeding hoverflies.

On warm sunny days, female hoverflies are often observed feeding from a wide range of wild and cultivated flowers. In doing so, hoverflies serve a vital role as alternative pollinators, and are suggested to be the most important pollinators after bees and bumblebees. Unless the flower hoverflies are feeding from is large, they do not readily share the space with other insects, but instead opt to leave if another insect approaches. We used high-speed videography followed by 3D reconstruction of flight trajectories to quantify how female Eristalis hoverflies respond to approaching bees, wasps and two different hoverfly species. We found that, in 94% of the interactions, the occupant female left the flower when approached by another insect. We found that compared with spontaneous take-offs, the occupant hoverfly's escape response was performed at ∼3 times higher speed (spontaneous take-off at 0.2±0.05 m s-1 compared with 0.55±0.08 m s-1 when approached by another Eristalis). The hoverflies tended to take off upward and forward, while taking the incomer's approach angle into account. Intriguingly, we found that, when approached by wasps, the occupant Eristalis took off at a higher speed and when the wasp was further away. This suggests that feeding hoverflies may be able to distinguish these predators, demanding impressive visual capabilities. Our results, including quantification of the visual information available before occupant take-off, provide important insight into how freely behaving hoverflies perform escape responses from competitors and predators (e.g. wasps) in the wild.

Head movements quadruple the range of speeds encoded by the insect motion vision system in hawkmoths.

Flying insects use compensatory head movements to stabilize gaze. Like other optokinetic responses, these movements can reduce image displacement, motion and misalignment, and simplify the optic flow field. Because gaze is imperfectly stabilized in insects, we hypothesized that compensatory head movements serve to extend the range of velocities of self-motion that the visual system encodes. We tested this by measuring head movements in hawkmoths Hyles lineata responding to full-field visual stimuli of differing oscillation amplitudes, oscillation frequencies and spatial frequencies. We used frequency-domain system identification techniques to characterize the head's roll response, and simulated how this would have affected the output of the motion vision system, modelled as a computational array of Reichardt detectors. The moths' head movements were modulated to allow encoding of both fast and slow self-motion, effectively quadrupling the working range of the visual system for flight control. By using its own output to drive compensatory head movements, the motion vision system thereby works as an adaptive sensor, which will be especially beneficial in nocturnal species with inherently slow vision. Studies of the ecology of motion vision must therefore consider the tuning of motion-sensitive interneurons in the context of the closed-loop systems in which they function.

Transgenic line for the identification of cholinergic release sites in Drosophila melanogaster.

The identification of neurotransmitter type used by a neuron is important for the functional dissection of neuronal circuits. In the model organism Drosophila melanogaster, several methods for discerning the neurotransmitter systems are available. Here, we expanded the toolbox for the identification of cholinergic neurons by generating a new line FRT-STOP-FRT-VAChT::HA that is a conditional tagged knock-in of the vesicular acetylcholine transporter (VAChT) gene in its endogenous locus. Importantly, in comparison to already available tools for the detection of cholinergic neurons, the FRT-STOP-FRT-VAChT::HA allele also allows for identification of the subcellular localization of the cholinergic presynaptic release sites in a cell-specific manner. We used the newly generated FRT-STOP-FRT-VAChT::HA line to characterize the Mi1 and Tm3 neurons in the fly visual system and found that VAChT is present in the axons of both cell types, suggesting that Mi1 and Tm3 neurons provide cholinergic input to the elementary motion detectors, the T4 neurons.